HK1012195A - Multi-array, multi-specific electrochemiluminescence testing - Google Patents

Description

Multi-array, multi-specific electrochemiluminescence assays

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The present invention is a co-pending application having application number 08/402,076 filed on 3.10.1995 and a partial continuation of the co-pending application having application number 08/402,277 filed on 3.10.1995, the entire contents of each of which are incorporated herein by reference. 1. Introduction to

The present invention provides patterned (patterned) multi-array, multi-specific surfaces (PMAMS) for electrochemiluminescence-based assays, and methods of making and using PMAMS. 2. Background of the invention 2.1. diagnostic assays

From an economic standpoint, there is a strong need for rapid, sensitive diagnostic techniques. Diagnostic techniques are important in a wide variety of economic markets, including health care, research, agricultural, veterinary, and industrial markets. Improvements in sensitivity, time required, ease of use, durability or cost may open up a completely new diagnostic market where there is no technology available to meet the needs of the market. Some diagnostic techniques may have high sensitivity, but are too expensive to meet market needs. Other technologies may be cost effective but not durable enough for various markets. Entirely new diagnostic techniques that can integrate these features are a significant advancement and opportunity in the diagnostic industry.

There are a number of different analytical techniques for diagnostic use. These techniques include radiolabelling, enzyme-linked immunoassays, chemocolorimetric assays, fluorescent labels, chemiluminescent labels, and electrochemiluminescent labels. Each of these techniques uniquely combines several factors, sensitivity level, ease of use, durability, speed, and cost, which determine and limit their application in different diagnostic markets. These differences arise in part from the physical constraints inherent to each technology. For example, radioactive labels are inherently not durable because the label itself decays and for many applications the disposal of the radioactive waste produced also incurs economic, safety and environmental expenses.

Many of the sensitive diagnostic techniques currently available are limited by the market place, primarily because of the need for skilled technicians to perform the tests. For example, the currently used electrochemiluminescence methods not only require skilled technicians, but also repeated washing and preparation steps. This adds both cost and further requires disposal of waste. Novel diagnostic methods that simplify the testing steps and reduce the cost per test are extremely important and useful for developing new markets and improving operations in existing markets. 2.2. Electrochemiluminescence assay

Electrochemiluminescence ("ECL") is a phenomenon in which electrically excited substances emit photons (see, for example, Leland Powell, 1990, journal of the electrochemical society (j. electrochem Soc.)137 (10): 3127-3131). Such substances are called ECL labels and herein also TAG (label). Commonly used ECL labels include organometallic compounds in which the metal is, for example, a noble metal selected from group VIII, including ruthenium-containing and osmium-containing organometallic compounds, for example, the ruthenium (2, 2' -bipyridine) 3+ moiety (also known as "rubby"), as disclosed, for example, by Bard et al (U.S. patent US5,238,808). The light generated by ECL markers can be used as an information signal (reporter signal) in diagnostic methods (U.S. patent US5,221,605 to Bard et al). For example, ECL labels may be covalently bound to a binding agent such as an antibody or nucleic acid probe. ECL label/binding agent complexes can be used to assay for a variety of substances (U.S. patent US5,238,808 to Bard et al). For ECL-based detection systems, it is of paramount importance that an electrical potential is required to excite the ECL labels to emit photons. The potential waveform is applied across an electrode surface (typically a metal surface) and a counter electrode to an ECL assay solution (see, e.g., U.S. patent No. US5,068,088; US5,093,268; US5,061,445; US5,238,808; US5,147,806; US5,247,243; US5,296,191; US5,310,687; US5,221,605).

Various devices known in the art can be used to carry out and detect ECL reactions. For example, Zhang et al (U.S. Pat. No. 5,324,457 discloses an exemplary electrode for an electrochemical cell for performing ECL. Levantis et al (U.S. Pat. No. 5,093,268) discloses an electrochemical cell for performing an ECL reaction.Kamin et al (U.S. Pat. No. 5,147,806) discloses an apparatus for performing and detecting an ECL reaction, including a voltage control apparatus.Zoski et al (U.S. Pat. No. 5,061,445) discloses an apparatus for performing and detecting an ECL reaction, including a potential waveform diagram for initiating an ECL reaction, a digital/analog converter, a control apparatus, a detection apparatus, and a method for detecting the current generated by an ECL reaction at a working electrode to provide feedback information to an electronic control apparatus.

For example, US patent US 5,093,268 reviews ECL technology in detail. Briefly, ECL technology is a method of detecting an analyte of interest present in a sample at a relatively small concentration in the sample.

The ECL moiety, referred to as TAG in the above-referenced issued patents, may or may not bind to the analyte, but in each case is promoted to an excited state as a result of a series of chemical reactions triggered by the electrical energy received from the working electrode. It is advantageous to provide a molecule that promotes ECL of TAG, for example, oxalate, or more preferably tripropylamine (see US 5,310,687). 2.3. Commercial ECL assay

To date, all commercial ECL reactions have been carried out on centimeter-scale electrode surfaces. Centimeter-scale electrodes unify between the desire for increased intensity of ECL signal from larger electrodes and a reduction in the total sample volume required for each assay. However, electrodes on the order of a centimeter or more do not achieve the sensitivity required for many assays. To address this problem, all commercial ECL systems also utilize coated magnetic beads to capture ECL analytes or reagents to increase sensitivity. The beads are then brought close to the working electrode to increase sensitivity.

However, there are many limitations to using magnetic beads. The beads themselves are coated with proteins that flake and degrade over time, causing a change in signal. Due to the complexity in handling and formatting bead-based assays, commercial ECL diagnostics require a complex, sequential procedure for each assay performed with a given sample, which adds time and cost to each assay to be performed. The 5 micron size beads prevent the majority of bead-bound ECL TAGs from reaching the thin film adjacent to the working electrode, which results in inefficient excitation of ECL TAGs.

Leventis et al (US 5,093,268) propose a method of simultaneously measuring more than one different analyte by using a different ECL label for each analyte, each ECL label emitting photons at a different wavelength for each different analyte in a single measurement. However, this technique has limitations, such as the inability to utilize sufficient quantities of effective ECL labels radiating at different wavelengths, and the need to optimize the chemistry of each ECL label. These practical limitations have prevented commercialization of such multi-wavelength, multi-analyte ECL detection systems.

Another approach to increasing ECL sensitivity is to improve electrode technology. Zhang et al (US patent 5,324,457) have deposited films of ECL materials directly on various metal and semiconductor surfaces. The technique of Zhang and Bard, which exploits the global saturation of the electrode surface, results (as described by the authors) in inhomogeneous irregular deposits that are not suitable for high sensitivity assays.

The aforementioned methods for performing ECL assays also require that the assay components, including the electrodes, must be cleaned by any of a number of methods, including the use of dilute acids, dilute bases, wash solutions, and the like, for example, as disclosed in U.S. patent No. 5,147,806.

It is therefore an object of the present invention to provide a new, cost-effective assay for conducting a plurality of ECL reactions sequentially or simultaneously, and, in a preferred embodiment, to provide an internally mounted control standard for improved accuracy.

It is also an object of the present invention to provide a cartridge comprising one or more carriers suitable for carrying out a plurality of ECL reactions carried out simultaneously or sequentially, said cartridge also being easy to handle.

A further related object of the present invention is to reduce the time and cost of performing an individual assay for an analyte of interest in a biological sample.

It is yet a further related object of the present invention to provide methods and devices for simultaneously performing a plurality of assays on a plurality of analytes of interest in a single biological sample. 3. Summary of the invention

The present invention relates to a cartridge for performing ECL reactions and assays, the cartridge comprising a plurality of discrete binding domains immobilized on a support, the discrete binding domains being spatially aligned with pairs of one or more electrodes and one or more counter electrodes. The cartridge preferably includes a first carrier having a plurality of discrete binding domains immobilized on a surface. The cartridge may have a pair of one or more electrodes and one or more counter electrodes. The pair of electrodes and counter electrode can be individually accessible by a source of electrical energy in the form of a voltage waveform effective to trigger electrochemiluminescence. The cartridge may further comprise a second carrier capable of being placed adjacent to the first carrier so as to provide a sample-containing device therebetween and/or to act as an electrode. The binding domains are patterned on the surface of the support and prepared to bind the analyte or reagent of interest.

The invention also relates to a device for measuring electrochemiluminescence of a sample, which device is provided with a carrier or cartridge handling means, a voltage control means adapted to apply a control voltage waveform effective to trigger electrochemiluminescence, a photon detector means for detecting electrochemiluminescence from the sample, and a sample handling means.

The invention also relates to methods of using these cartridges for measuring electrochemiluminescence of a sample: contacting a plurality of binding domains of a cartridge with a sample containing a plurality of analytes of interest under ECL assay conditions, then applying a voltage waveform effective to trigger electrochemiluminescence at each of the plurality of electrode and counter electrode pairs, and detecting or measuring the triggered electrochemiluminescence. Broadly, the present invention provides ECL assay methods in which the sample is not in contact with the electrodes. Furthermore, as an alternative to using a pair of electrodes and counter electrodes, the present invention provides for scanning the electrodes and counter electrodes over the entire bonding area.

The invention also provides kits comprising components comprising cartridges adapted to simultaneously measure a plurality of electrochemiluminescence reactions, a support surface having a plurality of domains immobilized thereon, and an assay medium for performing an ECL assay of a chemical reaction.

The present invention also provides an electrode prepared from a graphitic nanotube (graphite nanotube). 4. Description of the drawings

FIG. 1 illustrates two carriers forming a cartridge of the present invention, wherein there are a plurality of binding domains 14 on carrier 10 and a plurality of corresponding electrodes 16 on carrier 12, such that when the two carriers are brought into close proximity, a counter electrode pair is positioned adjacent each binding domain.

FIG. 1A illustrates two carriers forming a cartridge of the present invention, wherein there are a plurality of binding domains 14 on carrier 10 and a plurality of corresponding electrodes 16 on carrier 12, such that when the two carriers are brought into proximity, a counter electrode pair is positioned adjacent each binding domain.

Fig. 2 illustrates two carriers forming the cartridge of the present invention, wherein a plurality of bonding areas 30 on the carrier 26 abut one of the individual electrodes 32, such that when the carriers 26 and 28 are brought into close proximity, a counter electrode 38 is disposed adjacent each bonding area 30.

Fig. 3 illustrates two carriers forming a cartridge of the present invention, wherein a number of the binding domains 48 have pairs 50 of electrodes and counter electrodes on the carriers 44 adjacent thereto. Carrier 46 may optionally be placed adjacent to carrier 44 such that carrier 46 provides a sample-containing device adjacent to binding region 48 and electrodes 50.

Fig. 4 illustrates two carriers forming a cartridge of the present invention, wherein a plurality of binding domains 64 on carrier 60 are contacted with a sample believed to contain an analyte. The carrier 62 has a region 66, the region 66 containing a reaction medium for detecting or measuring an analyte of interest or for carrying out a desired reaction, such that the proximity of the carrier 60 and the carrier 62 brings the binding region 64 and the region 66 into contact with each other.

FIG. 5A shows a top view of patterned binding regions for a multi-array, multi-specific binding surface. The geometric shapes, triangles, squares and circles represent binding regions specific for different analytes. These binding domains may be hydrophobic or hydrophilic. The surrounding surface may have the opposite properties (hydrophilic or hydrophobic) of the binding domains to minimize the extent to which the binding reagents or analytes spread from the binding domains.

Figure 5B shows a top view of the microfluidic conduits for delivering binding reagents and/or analytes to those discrete binding sites. Each dot shows a cross-section of one microfluidic conduit (e.g., a capillary).

FIG. 5C shows a side view of a microfluidic conduit showing the delivery of binding reagents and/or analytes to one or more arrays of patterned binding domains when aligned or aligned microfluidic conduits are in proximity. Each microfluidic conduit may carry a different binding agent to a discrete binding region.

FIG. 6A shows a situation where the multi-array electrode is closely aligned to a surface having patterned multi-array, multi-specific binding regions. A removable electrode protective barrier is shown between the electrode array and the array of bonding surfaces. The entire apparatus includes one cartridge for performing a variety of ECL reactions.

Figure 6B shows the proximity of the addressable working array and counter electrode in alignment. The electrodes may be shaped to complement the binding regions, or may be other shapes (e.g., interdigitated).

Figure 7 shows a side view of an array of aligned or aligned addressable working and counter electrodes and complementary binding surfaces in proximity, wherein a conductive polymer is grown from the electrode surface across the gap between the electrode array and the binding region to extend the potential field around ECL labels of the sample, thereby increasing the efficiency of the ECL reaction.

Figure 8 shows a side view of an array with aligned or aligned addressable working and counter electrodes and complementary binding surfaces in close proximity, with conductive particles interspersed between the two components to extend the potential field. The efficiency of the ECL reaction is increased by extending the potential field around the ECL label of the sample. Those conductive particles may be magnetic for ease of control.

Figure 9 shows a side view of an array of aligned or aligned addressable working and counter electrodes and complementary binding surfaces in close proximity, wherein the electrodes have thin projections that extend into the gap between the electrode surface and the binding region in order to extend the potential field around the ECL labels of the sample in order to increase the efficiency of the ECL reaction.

FIG. 10 shows a side view of an array of aligned or aligned addressable working and counter electrodes and complementary binding surfaces when brought into close proximity, where the surfaces are not parallel, but fit into each other in a complementary fashion.

Fig. 11 shows a side view of a carrier with a metal layer thereon to provide a single electrode and bonding surface assembly in the form of a cassette. An array of self-assembled monolayers ("SAMs") is disposed in a pattern on the metal layer.

Fig. 12 shows a side view of a carrier with a metal layer thereon to provide a single electrode and bonding surface assembly in the form of a cassette. An array of SAMs is patterned on the metal layer and conductive particles are interspersed among the patterned SAMs to extend the potential field around the ECL label of the sample to improve the efficiency of the ECL reaction.

Fig. 13 shows a side view of a carrier with a metal layer on it to provide a single electrode and bonding surface assembly in the form of a cassette. An array of self-assembled monolayers, SAMs, is patterned on the above-mentioned metal layer and shows that a conducting polymer and/or fiber grows from the ECL marker in order to extend the potential field around the ECL marker of the sample, thereby improving the efficiency of the ECL reaction.

FIG. 14 is a diagram of a support having an array of electrode pairs controlled by a computer.

FIG. 15 is a diagram of a support having an array of electrode pairs.

FIG. 16 is a diagram of a support having an array of electrode pairs and a computer system for controlling the process of firing each electrode pair.

FIG. 17 is a diagram of a carrier with an array of electrode pairs and a computer system with multiple voltage sources and a multiplexer for controlling the process of firing each electrode pair.

FIG. 18 is a diagram of a carrier having an array of electrode pairs and a computer system having a plurality of commutating voltage sources for controlling the process of firing each electrode pair.

Fig. 19(a) to 19(e) are plan views regarding several alternative electrode-counter electrode pair combinations.

FIG. 20 shows the support with the sandwich assay intact.

Fig. 21 shows two opposing PMAMS surfaces on a support.

Fig. 22A shows an array of microfluidic conduits (2201) and a fibril mat (2200).

Fig. 22B shows a bonding area (2202).

Figure 23A shows an apparatus for forming a fibril mat by vacuum filtration.

Fig. 23B shows a fibril mat (2304) on a filtrate membrane (2303).

Figure 24 shows the use of a roller to produce a fibril mat.

Figure 25 shows a schematic of a multi-layered fibril mat in which the upper layer has binding regions for the assay.

FIG. 26 shows a schematic of fibrils derivatized with moieties that enhance non-specific binding, and several biological and non-biological substances bound to the surface.

Figure 27 shows a schematic representation of fibrils derivatized with moieties that enhance non-specific binding, and several substances bound to the derivatized fibrils, and some substances bound to ligands.

Figure 28 shows several substances covalently attached to fibrils, while still others are bound to additional entities.

Fig. 29 shows the use of a multi-layered fibril mat as a light filter, which can pass light and/or can absorb and/or scatter light, depending on the location of the light source on or within the mat.

Figure 30A shows periodic voltammetric recordings from electrochemical measurements on carbon fibril pad electrodes.

Figure 30B shows periodic voltammetric recordings from electrochemical measurements on gold foil electrodes.

Figure 31 compares the electrochemical properties of a fibril mat as a function of the thickness of the mat and the scan rate.

Figure 32 illustrates that as the concentration of fibrils in protein solution increases, the non-specific binding on fibrils generally increases.

FIG. 33 illustrates that non-specific binding between ECL-TAG 1-labeled protein and carbon fibrils can be attenuated using a surfactant.

Figure 34 shows a top view of an experimental element used to measure electrochemical properties and ECL on fibril mat electrodes.

Figure 35 shows ECL signal obtained with fibril pads as electrodes and 1000pM of TAG1 (solid line) in solution and signal from assay buffer (no TAG1) (dashed line).

FIG. 36 shows a schematic of a two surface PMAMS device in which two supported electrode arrays are separated by a patterned dielectric layer.

Fig. 37 shows a device with a number of bonding areas (3702) on a carrier and electrodes and counter electrodes on another carrier.

FIG. 38 shows a cartridge in which the binding domains are provided on the surface of different objects supported on a counter electrode.

Figure 39 shows the gel in contact with the working and counter electrodes.

Figure 40 shows ECL intensities and periodic voltammetric recordings from gels labeled with ECL in contact with working and counter electrodes.

Figure 41 shows ECL intensity and periodic voltammetric recordings from non-ECL labeled gels in contact with working and counter electrodes.

Figure 42 shows a schematic of a two-surface cartridge for ECL.

Figure 43 illustrates that the fibril pad can be used as an electrode for ECL of antibody-TAG 1 absorbed onto the pad.

Fig. 44A shows ECL intensity of TAG 1-labeled proteins immobilized on electrodes.

FIG. 44B shows a periodic voltammetric recording of the coated electrode.

Figure 45A shows quasi-reversible repeat generation of ECL signal from immobilized ECL TAG 1-labeled protein.

Fig. 45B shows a periodic voltammetric recording of the coated electrode with the coating partially retained.

Figure 46A shows the irreversible generation of ECL signal from a protein labeled with immobilized ECL TAG 1.

Fig. 46B shows a periodic voltammetric recording of a coated electrode with substantial loss of coating.

FIG. 47 shows a multi-array ECL apparatus and a microprocessor containing controller means for generating and analyzing ECL signals. 5. Detailed description of the invention

Accordingly, the present invention broadly comprises cartridges for performing a variety of electrochemical assays. These cartridges are composed of carriers having a plurality of binding domains that specifically bind one or more analytes of interest. These binding domains are fabricated as patterned multi-array multispecific surfaces ("PMAMS") on the support. PMAMS provides a significant improvement over previously known ECL assay methods by, for example, greatly increasing the density of assays that can be performed, and by enabling a number of different assays to be performed quickly and simultaneously. The cartridge may include a plurality of electrodes capable of selectively triggering the emission of light from ECL labeled reagents bound to the binding region. Fig. 47 shows a multi-array ECL device having electrodes 4700, 4704, a substrate 4702 with binding regions 4706, and a microprocessor containing controller device 4720, controller device 4720 for generating and analyzing ECL signals communicated via leads 4710-4716.

In the embodiment of the invention shown in fig. 1, the cartridge comprises two supports 10, 12, wherein there are a plurality of binding domains 14 on the surface of the first support 10 and a plurality of electrode/counter electrode pairs 16 on the surface of the second support 12. The binding regions and electrode/counter electrode pairs are aligned such that when the first and second supports 10, 12 are brought together, each of the plurality of electrode/counter electrode pairs 16 is positioned adjacent a different one of the plurality of binding regions 14. The first support 10 underlying the bonding region 14 is preferably a PMAMS with a gold film surface and a transparent bonding region. The second support 12 is preferably a transparent flat plastic sheet having a transparent electrode/counter electrode pair 16 thereon. The bonding areas 14 are preferably produced by micro-embossing an organic substance onto the surface of the carrier. Self-assembled monolayers (composed of various individual monomers) with biotin or a linker moiety are patterned. Avidin or streptavidin is then bound to the exposed biotin (see, for example, U.S. patent US 5,093,268). Thereafter, a binding reagent is applied by applying discrete amounts of a suitable biomarker binding reagent, such as a biotin-labeled antibody, to the locations on the support surface where the monolayer has been imprinted, which binding reagent is capable of selectively binding to the analyte of interest. Fig. 1A illustrates a system comprising a cartridge (fig. 1) contained in a housing (11).

In certain embodiments of the invention, it is desirable to reproducibly immobilize a specified or predetermined amount of one or more reagents on a surface. Immobilization is used broadly in any method by which reagents are attached to a surface, including but not limited to: covalent chemical binding, non-specific adsorption, drying of reagents on a surface, electrostatic interactions, hydrophobic and/or hydrophilic interactions, blocking or entrapment in a liquid or gel, biospecific binding (e.g., ligand/receptor interactions or inclusion of oligonucleotides), metal/ligand binding, chelation, and/or entanglement in a polymer.

The amount of reagent immobilized on the surface can be predetermined in several ways. For example, the amount of reagent on the surface may be defined by one or more reservoir components and/or surface components with reagents stored therein. The amount of reagent can also be specified by the number of individual molecules of reagent immobilized on the surface. Can be used forThe amount of reagent is specified in terms of the density of a particular reagent in a given area. The reagent amount may be measured as the percentage of the total area of a surface with a particular reagent, or relative to the amount of other reagents on that surface. The amount of reagent may also be defined as the amount of reagent necessary to give sufficient ECL intensity on a particular surface to achieve the desired specificity of the assay. In a specific example, 1cm ²The gold surface area of (a) may be coated with a single layer of alkanethiol.

Reagents may also be reproducibly immobilized on the coated surface. The coating may be used to enhance the immobilization of certain agents and/or to reduce or inhibit the immobilization of other agents. The surface may be fully coated or the surface may be partially coated (i.e., a patterned coating). The composition of the coating may be uniform or may contain elements of different compositions. In a specific example, the coating may be provided as a patterned monolayer film that immobilizes immunoglobulin G at certain regions by covalent chemical bonds and prevents it from being immobilized at other regions.

The coating may also be used to predetermine the amount of one or more reagents that are immobilized on the surface in a subsequent step or process. The amount of a particular agent can also be controlled by limiting the amount of agent deposited.

The surface has a reagent (or coating) immobilized thereon in a quantitative, repeatable manner, which enables repeatable and quantifiable measurement of ECL signals from the sample, thus enabling calibration.

Preferably, the electrode/counter electrode pairs 16 are sub-centimeter sized and can be fabricated with the electrode leads 20 (e.g., from a transparent metal film) by well known methods for fabricating liquid crystal displays and electrochromic displays. The electrode leads 20 are connected to the shape signal generator 18 by electrical connection wires 19. Advantageously, the electrode/counter electrode pair is individually addressable under computer control so that potentials can be selectively applied to those discrete binding domains. The light detector means 22 and the digital computer means 24 are configured to record and analyze the results when an appropriate label bound to the binding area has excited ECL luminescence.

Fig. 1A shows an apparatus comprising a case, a plurality of electrical leads, a shape-setting signal generator, a light detecting device and a digital computer, which are contained in a housing (11) as shown in fig. 1. The cartridge is inserted into the housing through an opening (15).

In another embodiment, one working electrode is used to generate ECL signals simultaneously at multiple binding domains. In this embodiment, ECL signals from each binding region are identified by using a light imaging device.

In summary, the advantage of assays performed using the cartridge of the present invention is that a number of discrete binding domains are used. For example, the use of such cartridges enables the rapid and/or simultaneous detection or measurement of a wide variety of analytes of interest. In a preferred embodiment, the assays of the invention are also assays that benefit from the use of reagents, analytes or binding surfaces labeled with ECL. The ECL assay of the invention comprises contacting a plurality of binding domains with a sample suspected of containing an analyte of interest and triggering ECL luminescence from bound ECL labels on the analyte or a competitor for the analyte, on a reagent that binds to the analyte, or on the plurality of binding domains.

The present invention also provides an ECL assay for detecting or determining an analyte of interest comprising: (a) contacting one or more of a plurality of discrete binding domains immobilized on the surface of one or more supports, wherein said contacting is with a sample comprising molecules bound to an electrochemiluminescent label, wherein said sample is not in contact with any electrode or counter electrode during said contacting step; (b) bringing an electrode into proximity with one or more of the plurality of binding domains; (c) applying a voltage waveform effective to trigger ECL at one or more of the plurality of bonding areas; and detecting or measuring ECL.

In another embodiment, the invention provides a method of determining ECL, the method comprising: (a) contacting one or more of a plurality of discrete binding domains (i) immobilized on the surface of one or more supports, and (ii) spatially aligned with and in proximity to a plurality of electrode and counter electrode pairs, wherein said contacting is with a sample comprising a molecule that binds to an electrochemiluminescent label; (b) bringing an electrode and a counter electrode into proximity with one or more of the plurality of binding domains; (c) applying a voltage waveform effective to trigger electrochemiluminescence at one or more of the plurality of binding domains; and (d) detecting or measuring electrochemiluminescence.

The plurality of binding domains on the support are capable of interacting with the sample to be assayed. PMAMS may be further contacted with a solution containing the reagents necessary to complete the assay. The binding surface is then brought into contact (e.g., pressed together) with the surface of a complementary electrode (preferably a clean, unused electrode), and an electrical potential is then applied with the electrode to excite ECL.

In a preferred method of performing an assay using the device of FIG. 1, a sample suspected of containing an analyte of interest is applied to a plurality of binding domains 14, along with an ECL-labeled reagent suitable for detecting the analyte. Thereafter, the support 11 and the support 12 are brought together so that each of the plurality of binding domains 14 is between a different pair of electrodes and counter electrodes of the plurality of electrode/counter electrode pairs 16 with the sample contained therebetween. It should be noted that it is not necessary to have the electrode and counter electrode pair in mechanical contact with the binding region in order to excite the ECL when a suitable potential is applied between the electrode and counter electrode pair. A potential waveform suitable for triggering ECL light emission is applied to a plurality of electrode/counter electrode pairs 16 from a waveform signal generator 18 through electrical connection lines 19. Any signal emitted by ECL labels on the plurality of binding domains 14 is detected by the light detection means 22 and analyzed by recording by the digital computer means 24.

The present invention provides methods for detecting multiple analytes of interest in a volume of a multicomponent liquid sample, where the multiple analytes of interest can be present in various concentrations.

In general, less than 10 can be detected from multicomponent samples^-3Molarity of a plurality of analytes. Preferably, less than 10 can be detected from a multicomponent sample^-12Molarity of a plurality of analytes.

The present invention provides methods for performing assays from multicomponent samples that can be performed in a heterogeneous assay, that is, an assay in which a plurality of unbound labeled reagents are separated from a plurality of bound labeled reagents before the plurality of bound labeled reagents are subjected to electrochemical energy testing conditions, and a homogeneous assay, that is, an assay in which a plurality of unbound labeled reagents are subjected to electrochemical energy testing conditions with bound labeled reagents.

In the assays of the invention, the electromagnetic radiation used to detect a particular analyte is distinguishable from the electromagnetic radiation relative to other analytes by identifying its location and/or location as one or more features of a pattern corresponding to the pattern of binding domains in the PMAMS.

In the homogeneous assay of the invention, the amount of electromagnetic radiation emitted by the bound labelled reagent is either increased or decreased compared to the unbound reagent, or by detecting the electromagnetic radiation emitted by a source spatially corresponding to one or more features of a pattern corresponding to the pattern of binding domains in the PMAMS.

In one particular example of the method of the invention, shown in fig. 20, a sandwich assay is performed on a support (5), the sandwich (5) having on its surface a plurality of Binding Domains (BD) which are specific for binding to a particular analyte (An). When a sample suspected of containing an analyte is applied to the binding area, the analyte binds to the binding area. An antibody (Ab) adapted to selectively bind to the analyte (An) and which has been labelled with An ECL moiety (TAG) to form An Ab-TAG is then applied to the analyte at the binding zone. After overdosing, unbound Ab-TAG is washed off the binding area and a potential waveform suitable to trigger electrochemiluminescence is applied to the TAG via electrodes (not shown) to trigger ECL luminescence from any TAG on the binding area. The ECL signal is detected by an optical detection device and recorded by a digital computer device (e.g., shown at 22 and 24 in fig. 1).

Further embodiments, features and some variations of the present invention are provided below. 5.1. Preparation of the bonding surface

For a better understanding of the invention, a more detailed description of the binding domains prepared on the support is provided. A patterned array of binding domains on a surface that are specific for a plurality of analytes is referred to herein as a patterned, multi-array, multi-specific surface or PMAMS. For example, PMAMS on a support is prepared by forming a self-assembled monolayer ("SAMs") pattern (see Ferguson et al, 1993, Macromolecules, 26 (22): 5870-. Methods of forming surface patterns also include the use of physical etching (e.g., micromachining) (Abbott et al, 1992, Science, 257: 1380-. Other methods of patterning the surface include spatially controlled methods of dispensing fluids or particles (e.g., Micropen dispensing (e.g., with a microfluidic conduit, delivered to the surface using X-Y translation)), microcapillary filling (Kim et al, 1995, Nature, 376: 581), ink jet technology, or syringe dispensers. A combination of these techniques can be used to obtain complex surface patterns. In fig. 5A, a carrier 600 is shown with independently shaped binding regions, which are indicated as geometric shapes 602 for illustrative purposes only, to indicate that different binding specificities may be present on a single carrier. The surface 604 between the binding regions may be hydrophobic or hydrophilic so as to restrict the deposition of binding agent, forming binding regions. The binding regions and/or surfaces between the binding regions may be susceptible and resistant to non-specific binding, and/or they may be susceptible or resistant to attachment of the binding agent by covalent or non-covalent interactions. In the case where nonspecific binding by hydrophobic interaction is not the desired method for attaching the binding chemistry to the surface, a detergent may be added to prevent accidental nonspecific binding.

In general, the width or diameter or widest dimension of the bonding region is from 0.1 μm to 1mm, depending on the geometry of the region. The surface is selectively derivatized with components that have specific binding, for example, to an ECL assay solution. In addition, non-specific interactions at the binding domains are reduced while maintaining a specific binding moiety by binding a moiety such as polyethylene glycol to the exposed surface of the discrete binding domains (Prime et al, 1993, J.cheem. Soc.115: 10714-10721; Prime et al, 1991, Science, 252: 1164-1167; Page-Grosdemange et al, 1991, J.Am.chem. Soc.113: 12-20).

In general terms, PMAMS may contain from 2 to 10⁸And a binding region. The preferred number of binding domains is from 50 to 500. While in other embodiments the number of bonding areas is 25 to 100.

The carrier may be a variety of materials including, but not limited to, glass, plastic, polymer, elastomeric, metal, ceramic, alloy, composite foil, semiconductor, insulator, silicon, and/or layered materials, and the like. Derivatized elastic carriers can be prepared, for example, as described by Ferguson et al (1993, Macromolecules, 26: 5870-.

The surface of the support on which the PMAMS is prepared may comprise various materials, for example, mesh, felt, fibrous material, gel, solid (e.g., comprised of metal) elastomer, and the like. The carrier surface may have various structural, chemical and/or optical properties. For example, the surface may be rigid or flexible, flat or deformed, transparent, translucent, partially or fully reflective, or opaque, and may have some composite properties, some regions with different properties, and may be a composite of more than one material. The surfaces may have patterned surface binding areas and/or patterned areas on one or more surfaces where catalytic reactions may occur according to the present invention, and/or an array of addressable electrodes on one or more surfaces. The surface of the carrier may be formed into any suitable shape including planar, spherical, cubic, and cylindrical. In a particular embodiment, the PMAMS-loaded vector is a probe. In another embodiment, the PMAMS-loaded support contains carbon, such as graphite, glassy carbon, or carbon black.

In one embodiment, the PMAMS-loaded support comprises one or more carbon fibers. These fibers may be amorphous carbon or graphitic carbon. They may also be part of the family of carbon nanotubes, buckeytube or fullerene cage carbon molecules.

In a preferred embodiment, the PMAMS loaded carrier comprises one or more carbon fibrils (chenopodium fibrils)^TM) (U.S. Pat. No. 4,663,230). The individual carbon fibrils (as disclosed in U.S. Pat. Nos. 4,663,230; 5,165,909 and 5, 5,171,560) may have a diameter in the range of about 3.5nm to 70nm and a length greater than that diameter10 of²And has a plurality of substantially continuous layers of ordered carbon atoms in an outer region and a distinct inner core region. For illustrative purposes only, a typical diameter of a carbon fibril may be between about 7nm and 25nm, while a typical length may range between 1 μm and 10 μm.

The carbon material may be formed into an aggregate. As disclosed in US patent US 5,110,693 and its references, two or more individual carbon fibrils may constitute microscopic aggregates of entangled fibrils. The size of these aggregates can be from 5nm to several centimeters. For illustrative purposes only, one type of microscopic aggregate ("cotton candy or CC") is similar to a spindle or entangled fiber rod, which may be from 5nm to 20 μm in diameter and from 0.1 μm to 1000 μm in length. Also for illustrative purposes, microscopic aggregates of another type of fibrils ("bird nests, or BN") may be approximately spherical, and may range in diameter from 0.1 μm to 1000 μm. Larger aggregates of each type (CC and/or BN) or mixtures of each type may be constructed.

Fibrils that may be used in the carrier include, but are not limited to, individual fibrils, aggregates of one or more fibrils, suspensions of one or more fibrils, dispersions of fibrils, mixtures of fibrils and other materials (e.g., oils, waxes, polymers, gels, plastics, adhesives, epoxies, polytetrafluoroethylene, metals, organic liquids, organic solids, inorganic solids, acids, bases, ceramics, glass, rubber, elastomers, biomolecules, media, and the like), as well as combinations thereof.

In some cases the fibrils may be magnetic and in other cases non-magnetic. The extent to which the fibrils can be made magnetic or non-magnetic is controlled by the amount of catalyst that is in the fibrils as a result of the fibril production process, U.S. Pat. No. 4,663,230; such processes are disclosed in US5,165,909 and US5,171,560. The PMAMS may be located on, in, or near the vector.

PMAMS can be generated from different types of surface binding groups. Self-assembled monolayers that can be used to form a monolayer on the surface to which they are bound include, but are not limited to, alkanethiols (which bind gold and other metals), alkyltrichlorosilane (e.g., which bind silicone/silica), alkanecarboxylic acids (e.g., which bind alumina), and combinations thereof. The monolayer may be formed first and then the chemistry used to attach the binding agent is attached. The derivatization process after self-assembly produces a more perfect two-dimensional crystal coating of a monolayer on the surface of the support with fewer pinholes or defects. The monolayer can be derivatized with a binding agent before or after self-assembly. Regular defects in the monolayer may be desirable and may be obtained by derivatization of the monolayer or support surface prior to self-assembly. If the space occupied by the derivatized group (e.g., exposed binding group) on the binding agent is large, it may produce a closed-packed surface at the exposed end, but with some regular gaps at the metal surface. This facilitates the flow of feed through these regular slits to the ECL-labeled moiety, which binds to the moiety that is in contact with the sample solution.

The preparation of incomplete monolayers is known in the art. Other methods for preparing incomplete monolayers include, but are not limited to: forming a monolayer with a dilute solution of the binding reagent, terminating the reaction that forms the monolayer before completion, damaging a more complete monolayer (e.g., ionic particles) with radiation, light, or a chemical reagent. In one embodiment, repeated imprinting without re-inking the stamp can give a range of defective monolayers (Wilbur et al, 1995, Langmuir, 11: 825).

PMAMS can be generated on the surface of the substrate. The substrate may be highly conductive, e.g., a metal electrode or a conductive polymer film; alternatively, the substrate may be an insulator; alternatively, the matrix may be semiconducting and/or have moderate conductivity. The matrix material may be an ion conductor or a porous material. Such porous materials may be used as support materials and/or electrically conductive materials and/or filter materials and/or channeling materials (e.g., to pass fluids, ionic species, etc.).

The porous material may be combined with some other material. For example, a composite structure of porous material may be fabricated with additional porous materials, conductive materials, semiconductor materials, channel structures, and/or solutions (e.g., ionic fluids). Such composite structures may be layered structures, sandwich structures and/or interspersed composites. A solid matrix of porous material supported on a metal electrode may be used. The porous material may also be sandwiched between conductive, semiconductive, or a combination of semiconductive and conductive materials. The one or more binding domains may be contained on a continuous sheet of porous material and/or may be located on a number of discrete objects on a carrier, each object having one or more binding domains. The surface of the porous material (e.g., gel) can be flat, hemispherical, or any regular or irregular shape, and/or can have various physical (e.g., elastic, rigid, low density, high density, density gradient, dry, wet, etc.) and/or optical (e.g., transparent, translucent, opaque, reflective, refractive, etc.) and/or electrical (e.g., conductive, semi-conductive, insulating variable conductivity, e.g., wet versus dry, etc.) properties.

A pattern of channels can be formed in the matrix. The thickness of the layer of porous material may be from 5 microns to 2000 microns. The layer of porous material may also be greater than 2mm thick.

The pores may extend partially and/or completely through the material, or may be part of a network of pores. These pores can range in size from as wide as 50 angstroms to 10000 μm. In a preferred embodiment, the material has pores ranging in size from 200 angstroms to 500 angstroms and pores ranging in size from 0.5 μm to 100 μm.

The porosity of the material may be continuous throughout the material, or may increase or decrease as a function of different locations in the material. The material may have a wide variety of different sized pores distributed in a random and/or random manner.

The porous material may be a composite of more than one material.

For example, some of the pores in the above materials may be large enough to pass through objects as large as biological cells, some may pass through biological media as large as proteins or antibodies, some may only pass through small (< 1000 molecular weight) organic molecules, and/or combinations of all of those above.

The porosity of the material may be such that one or more molecules, liquids, solids, emulsions, suspensions, gases, gels and/or dispersions can diffuse into, within and/or through the material. The porosity of the material allows the biological medium to diffuse (actively or passively) or be forced into, within and/or through the material by some means. Examples of biological media include, but are not limited to, whole blood, partial blood, plasma, serum, urine, protein solutions, antibodies or fragments thereof, cells, subcellular particles, viruses, nucleic acids, antigens, lipoproteins, lipids, glycoproteins, carbohydrates, peptides, hormones, or pharmaceutical agents. The porous material may have one or more layers of different porosity, which allows the biomedia to pass through one or more layers, but not through other layers.

The porous material is capable of carrying an electrical current generated by the flow of ionic species. In a further refinement, the porous material is a porous water-swellable gel, for example, polyacrylamide or agar. Various other gel compositions are known (see, for example, Soane, D.S., polymer applications in biotechnology; Soane, D.S., Ed; Simon & Schuster: Englewood Cliffs, NJ, 1992, or hydrogels in medicine and pharmacy, Vol.I-III; Peppas, N.A.Ed.; CRC Press: Boca, Raton, FL, 1987). The binding domains can be attached to the substrate by covalent and non-covalent bonds. (numerous reviews and books on this subject have been written; some examples are Tampion J. and Tampion M.D., immobilized cells: principles and applications, Cambridge university Press: NY, 1987; solid phase biochemistry: aspects of analysis and synthesis, Scouten, W.H.Ed., John Wiley and Sons: NY, 1983; methods in enzymology, immobilized enzymes and cells, Pt.BMOSbach, K.Ed., Elsevier Applied Science: London, 1988; methods in enzymology, immobilized enzymes and cells, Pt.C Mosbackh, K.Ed, Elsevier Applied Science: London, 1987; methods in enzymology, immobilized enzymes and cells, Pt.CMOSbach, K.Ed., K.E.E.E.J., Elsevier Applied Science: London, 1987; see also the above-cited medical hydrogels). For example, proteins can be attached to crosslinked copolymers of polyacrylamide and N-acryloyl succinimide by treatment with a protein solution. The binding domains may also be incorporated into the porous matrix prior to polymerization or gel formation. In one embodiment, various coupling chemistries may be used to attach the binding regions to the uncrosslinked polymer. The polymer may then be crosslinked (e.g., using chemical methods including amide linkages, disulfides, nucleophilic attack on epoxy compounds, etc.) (see, e.g., Pollack et al, 1980, J.Am.chem.Soc.102 (20): 6324-36). The binding domains can be attached to the monomer species and then incorporated into the polymer chain during polymerization (see Adalsteinson, O., 1979, J.mol. Catal.6 (3): 199-. In yet another embodiment, the binding domains may be incorporated into the gel by trapping the binding domains in the pores during polymerization/gel formation, or by infiltration of the binding domains into the porous matrix and/or membrane. In addition, the binding domains can be adsorbed on the surface of porous matrices (e.g., polymer gels and membranes) by non-specific adsorption caused by hydrophobic and/or ionic interactions. Biotin may advantageously be used as a cross-linking or binding agent. Avidin, streptavidin, or other biotin binding reagents may be bound into the binding region.

PMAMS can be generated on porous materials (e.g., gels) with varying pore sizes and solvent content. For example, polyacrylamide gels with varying pore sizes can be made by varying the concentration of acrylamide and the degree of crosslinking.

On such PMAMS, where the pore size is smaller than the analyte, the binding reaction proceeds essentially on the surface of the gel. In this case, filtration through the gel and/or electrophoresis may be used to concentrate the analyte at the gel surface and to adjust the reaction kinetics (e.g., increase the rate) of the binding reaction. Faster reaction kinetics are beneficial in rapid assays (e.g., results are obtained in a short time) and can result in high sensitivity in a shorter time.

On PMAMS, where the pore size is larger than the analyte, the binding reaction can take place on the gel surface as well as in its bulk. In this case, filtration and/or electrophoresis may be used to improve the reaction kinetics of binding and to remove unbound material from the surface.

PMAMS formed on the gel can be stored wet and/or can be stored in a dry state and reconstituted during the assay. The reagents necessary for ECL assays may be incorporated into the gel prior to storage (either by permeation into the gel, or by addition during formation of the gel) and/or may be added during the assay.

Patterned binding domains of PMAMS can be generated by applying droplets or microdroplets containing each binding domain in a matrix to a substrate in liquid form. Solidification and/or gelation of the liquid is then induced by various well-known techniques (polymerization, cross-linking, cooling below the gelation phase transition temperature, heating). An agent that causes solidification or gel formation may be included in the droplets so that at some point after spreading the droplets solidify and/or gel. Subsequent treatment (e.g., exposure to light, radiation, and/or redox potential) can be used to cause curing and/or gel formation. In other embodiments, such droplets or microdroplets may also be slurries, pre-polymerization mixtures, clusters of particles, and/or substantially solid droplets. In addition, vapor deposition may be utilized.

The pattern may also be formed by forming a layered structure of the substrate, each layer containing one or more binding domains. For example, agarose linked to antibodies (using standard chemical methods) can be poured into a container and allowed to gel by cooling. The subsequent layer containing the other antibody can then be subsequently poured onto the first layer and allowed to gel. The cross-section of such a layered structure gives a continuous surface with many different binding areas. Such profiles may be stacked and another profile may be cut to produce a PMAMS surface with an even greater density of bonded regions. It is also possible to arrange the lines of the matrix containing the given binding element next to each other and/or to superpose these lines. Such structures can also be cut in cross-section and used as PMAMS surfaces.

Certain substrates may also be patterned with the ability to be separated. For example, a mixture of nucleic acid probes can be separated by electrophoresis in a polyacrylamide sheet that creates a surface with many different binding domains.

The PMAMS binding domains can also be prepared on the support using microfluidic conduits. A partial list of microfluidic conduits includes hollow capillaries, capillaries made with and/or filled with a matrix (e.g., porous or solvent-swollen media), solid supports that can carry thin films or droplets. The capillary may be solid and the reagent flowing along the outer surface of the capillary may allow the reagent fluid reservoir to be exposed to the porous substrate tip in contact with the PMAMS surface. For example, the reagent reservoirs may be continuously or periodically replenished so that the tips of a given porous matrix may reproducibly deposit reagents (e.g., alkanethiols to form a monolayer; and/or binding reagents, etc.) multiple times. In addition, varying the porosity of the tip controls the flow of reagents to the surface. Different or the same binding agent may be present in a number of capillaries, and/or a plurality of different binding agents may be present in a given capillary. The capillaries are contacted with the surface of a PMAMS (e.g., a patterned SAM) such that certain regions are exposed to the binding reagent, resulting in discrete binding domains. Each of the different binding reagents is present in a different microfluidic conduit and is carried from the array of fluidic conduits to a metal surface, SAM, or the like as desired. The microfluidic conduit may also be used to coat the microscopic imprint with the desired molecule prior to application to the surface of the carrier. For example, a separate microfluidic conduit may be used to apply different binding reagents attached to a moiety that promotes adsorption to the surface of the support (e.g., free thiols on a hydrocarbon linker that promotes gold adsorption) to form the PMAMS. Thus, for example, microscopic stamps smeared with microfluidic conduits with antibodies of different specificity incorporating a linker with free thiol can be used to apply such antibodies in desired areas on the gold surface to form discrete binding regions of PMAMS.

Another method of patterned fluid delivery involves the use of microprinting devices that deliver fluid droplets by injecting a fluid drop through a small orifice (e.g., an inkjet printer). The injection of drops of fluid in these devices may be caused by different mechanisms including heat, electrostatic charging, and/or the application of pressure from a piezoelectric device. Patterns of more than one liquid may be formed by using a plurality of holes and/or one hole and fitting appropriate valves.

In one method of making PMAMS, droplets containing the desired binding agent are delivered directly (preferably simultaneously) by microfluidic conduits onto discrete areas on a surface to form discrete binding domains. The binding agent may contain a functional chemical group that forms a bond with a chemical group on the surface to which it is to be applied. In another variation, the binding agent in the droplet non-specifically adsorbs or binds to the surface (e.g., dries on the surface).

Alternatively, the droplets deposited on the surface contain a matrix-forming agent. Such a matrix may be a solid, polymer or gel that can be formed by evaporation of the solvent. It can be formed by polymerization of monomeric species. It may be formed by cross-linking preformed polymers. It may be formed by adjusting the temperature (e.g., cooling and/or heating). It may be formed by other methods. For example, the polymeric substance may be cooled by cooling or by addition of an agent that causes gelation. The formation of the solid matrix can be induced by generating reactive species at the electrodes (including the substrate), by light (or other radiation), by the addition of agents that induce curing or gelation, by cooling or heating. In addition, the surface may contain a catalyst capable of initiating matrix formation (e.g., gelation or polymerization).

In a preferred technique, patterned hydrophilic/hydrophobic regions may be employed to prevent spreading of the applied fluid or gel. Such fluids or gels may contain binding agents to be attached to the surface of the support to form binding domains of the PMAMS. In this case, the use of such hydrophilic/hydrophobic boundaries helps to confine the resulting binding domains to discrete regions. Alternatively, the fluid contains a reagent that forms a matrix on the surface, and the binding reagent is contained in a defined zone when deposited on the surface. For example, the droplets may be confined to a defined area using hydrophilic/hydrophobic boundary aids. Furthermore, the hydrophilic or hydrophobic regions may be given groups which can bind (e.g., covalently or non-covalently) into the matrix, allowing the matrix to more stably adhere to the substrate (Itaya and Bard, 1978, anal. chem.50 (11): 1487-1489). In another technique, the applied fluid or gel is a sample containing the analyte of interest, which is applied to the prepared PMAMS. In a preferred embodiment, a solution may be deposited on discrete areas using capillaries containing a hydrophobic solution, resulting in hydrophilic areas surrounded by hydrophobic areas. Alternatively, a hydrophobic binding region surrounded by a hydrophilic region may be used with a hydrophobic fluid containing a binding reagent or analyte. Hydrophobic and hydrophilic are relative terms with respect to each other and/or with respect to the applied sample, that is to say that this enables the spreading or wetting of the fluid or gel sample applied to the binding area to be controlled. In turn, controlled solution deposition from microfluidic arrays can be accomplished with physical surface properties, such as wells (wells) or channeling at the surface. Microfluidic conduits may be included in a cartridge or, more preferably, used to apply specific reagents to the carrier prior to use.

More than one attachment chemistry may be applied to the same support surface and/or multiple imprints may be used to create a surface with hydrophilic and hydrophobic binding domains. For example, a region in which a hydrophilic binding region is desired at position 1 and a hydrophobic binding region is desired at position 2 can be prepared as follows. A first hydrophilic imprint is made having a disc at position 1 and a larger ring at position 2. A second hydrophobic imprint is made, the disc of which at position 2 fits within the annular monolayer left by the imprint 1. Finally, the surface is cleaned by a hydrophobic solution of the monolayer composition.

Specifically, PMAMS is produced by microcontact printing (i.e., embossing). The monolayer thus applied consists of surface binding groups, for example for gold surfaces, preferably with alkanes (e.g., (CH)₂)_n) A thiol group of a spacer. There is a spacer group attached (preferably covalently bonded) to the linking group a. "A" may be, for example, avidin, streptavidin, or biotin, or any other suitable binding agent having an available complementary binding pair "B". A: b-linkages may be covalent or non-covalent, and Bard et al disclose certain linkage chemistries known in the art to be useful (US 5,221,605 and US5,310,687). "B" is further linked to a binding reagent, e.g., an antibody, antigen, nucleic acid, drug, or other substance suitable for forming a binding region capable of binding to one or more analytes of interest in the sample to be tested. B may also be attached to an ECL TAG or label. The linker group B can be delivered to the SAM by means of a capillary or microfluidic conduit array (fig. 5A-5C) that can place a number of "B" reagents with different binding surface specificities on a single layer of "a" linkages. A and B may also be linked prior to or prior to attachment to the monolayer. As illustrated, in fig. 5A, the independently shaped binding regions are represented as geometric shapes 602 for simplicity of illustration, to indicate that different binding specificities can exist on a single support 600. Fig. 5B provides a top view of the array of microfluidic conduits (e.g., capillaries) 606. Dot 610 is a cross-section of the catheter. Fig. 5C provides a side view of the microfluidic conduit array 608. The lines extending from the top and bottom are individual microfluidic conduits 610. The following geometric figure 612 represents one Specific binding domains are formed upon delivery of the binding reagents from each individual capillary.

By way of example, after the first imprint, the bare surface (e.g., gold) region may be reacted with a second alkanethiol that does not have chemical species a attached and that has a hydrophobic/hydrophilic nature opposite that of the first monolayer. Specific binding domains are prepared on the surface in this manner.

A binding reagent specific for or intended for the analyte of interest may be employed for each binding zone, or a binding reagent that specifically binds to a plurality of analytes of interest may be employed.

In yet another variation, as shown in FIG. 5A above, the support surface may be imprinted multiple times with materials (e.g., binding reagents, ECL labels, SAMs) having different attachment chemistries and/or binding moieties.

The patterned binding agents may be stable and/or robust chemical groups (e.g., to withstand the conditions to which they are exposed), which are then subsequently attached to less stable or durable binding groups. A variety of linkages may be utilized to optimize the conditions at each step in the preparation of the PMAMS surface and/or to simplify the preparation of the PMAMS surface. For example, a first PMAMS surface can be prepared in a conventional manner and then modified to produce a different PMAMS surface. In another example, a conventional PMAMS surface may be reacted with a solution mixture of binding agents that themselves contain binding domains that direct them to specific domains (e.g., binding domains) on the PMAMS surface. For example, a pattern of binding regions, each giving a different oligonucleotide sequence, is attached to the surface. The surface is then treated with a solution containing a mixture of auxiliary binding reagents, each of which is attached to an oligonucleotide sequence complementary to a sequence on the surface. In this way the purpose of patterning these auxiliary bonding portions is achieved. Preferably, the oligonucleotide sequence is a 6 to 30 mer of DNA. Certain groups of segments 6 to 30 may contain substantially similar sequence complementarity such that the approximate binding constants for hybridization are similar within a given group and are identifiably different from less complementary sequences. In another embodiment, the auxiliary binding moiety is a protein (e.g., an antibody).

The method described for inhibiting wetting or spreading of reagents or samples applied to a surface as described in sections 5 and 13 below may also be used for preparing PMAMS (and/or applying samples). The applied potential (e.g., from an electrode/counter electrode pair) can be used to further control the deposition and/or spreading of reagents and/or samples (see, e.g., Abbott et al, 1994, Langmuir, 10 (5): 1493-.

The PMAMS binding agent may be located on a carbon-containing material. They may also be located on individual carbon fibrils, or the PMAMS binding agent may be located on aggregates of one or more fibrils. In many embodiments, the PMAMS binding agent can be located on one or more of a fibril suspension, a fibril dispersion, a mixture of fibrils with other materials (as described in the examples above), mixtures thereof, and the like.

The PMAMS binding agent may be located on a plurality of individual fibrils and/or aggregates of fibrils which are located on, in or near the support. In one example, the binding agent is located on discrete individual fibrils or aggregates of fibrils. These fibrils, or aggregates of fibrils, may occupy positions within several different binding domains on the carrier and may constitute binding domains as described herein.

In another example, individual such binding domains or a plurality of such binding domains are located in separate distinct regions of the support. By way of non-limiting example, individual such binding domains or collections of binding domains may be located in recesses, pits, and/or holes in the carrier. In yet another example, the single, constant binding domains or a plurality of binding domains may be located within a drop of water, gel, elastomer, plastic, oil, etc., that is positioned on the surface of the carrier. In yet another example, individual binding domains can be positioned on the support by a coating (which can be patterned) having different binding affinities for different binding agents and/or assemblies of binding agents/fibrils.

The binding domains are desirably located on a plurality of individual fibrils and/or aggregates of fibrils are prepared on a support by means of one or more microfluidic conduits (e.g., capillaries). Different or equivalent binding agents may be present in or on a number of microfluidic conduits, and/or different binding agents may be present in or on a given microfluidic conduit. The capillary may be brought into contact with the carrier (spotting), and/or the capillary may deliver the reagent while the microfluidic conduit and/or surface is scanned or translated relative to another (i.e., a pen-like method of writing). The microfluidic conduit may deliver the binding agent on the fibrils to the carrier, thereby exposing certain regions of the carrier to fibril-binding agent complexes to create discrete binding domains. In a preferred case, the different binding reagents are each present in a different microfluidic conduit, which are simultaneously delivered from the conduit array to the carrier. In one example, the binding agents and/or the fibrils to which they are localized are derivatized with a chemical functional group that forms a bond (e.g., covalent or non-covalent interaction) with the surface of the support. In certain embodiments, the binding agent and fibrils bind or adsorb to the surface non-specifically. While for the other case the binding agent positioned on the fibrils can be delivered into recesses, pits and/or holes in the surface of the carrier. In another example, the binding agents are delivered to a surface coated with a material that has a stronger or weaker binding affinity for certain binding agents or binding agent/fibril aggregates, and thus creates regions of the agent that occupy spatial positions that are different from those of other binding agents.

The binding agent is localized on one or more magnetic individual fibrils or aggregates of fibrils. In such a case, the magnetic carrier may attract the binding agent located on the magnetic fibrils to the carrier.

The carrier may contain several different regions which are magnetic and surrounded by some non-magnetic regions. The binding agent located on the magnetic fibrils can be located on the magnetic region of the support. In one example, the carrier may contain one or more different regions which are magnetic and surrounded by non-magnetic regions, and the strength of the magnetic field in the magnetic regions may be adjusted or varied. In this regard, the use of such an adjustable or changeable magnetic field facilitates attachment or release of the fibril-localized binding agent to or from the surface of the support, and thus may serve to agitate or mix the regions.

The number of binding domains ranges from approximately 2 to 10⁸And preferably 25 to 500 regions.

The binding region may be located on the working electrode and/or the counter electrode.

The various embodiments described herein for the different types of PMAMS, carriers, and electrodes and their structures can also be implemented in combination with each other.

PMAMS carriers can be preserved (e.g., by protective surface coatings, dry surfaces, durable packaging under vacuum or inert atmosphere, freezing, and related methods) for future use. 5.2 binding reagents

The binding regions of the invention are prepared to contain a binding reagent that specifically binds to at least one analyte (ligand) of interest. The binding reagents are selected in discrete binding regions such that the binding regions have the desired binding specificity. The binding agent may be selected from any molecule known in the art that is capable of, or is predicted to be capable of, specifically binding to a certain analyte of interest. Can be obtained from the following section 5.10 "ECL measurement availableThe analyte of interest is selected from those mentioned above. Thus, binding agents include, but are not limited to, receptors, ligands for receptors, antibodies or binding portions thereof (e.g., Fab, (Fab) ')'₂) Proteins or fragments thereof, nucleic acids, oligonucleotides, glycoproteins, polysaccharides, antigens, epitopes, cells and cellular components, subcellular particles, carbohydrate moieties, enzymes, enzyme substrates, lectins, protein a, protein G, organic compounds, metallo-organic compounds, viruses, saw 40561b, viroids, lipids, fatty acids, lipopolysaccharides, peptides, cellular metabolites, hormones, drugs, sedatives, barbiturates, alkaloids, steroids, vitamins, amino acids, sugars, non-biological polymers, biotin, avidin, streptavidin, organic linking compounds such as polymeric resins, lipoproteins, cytokinins, lymphokines, hormones, synthetic polymers, organic and inorganic molecules, and the like. Nucleic acids and oligonucleotides may refer to analogs of DNA, RNA and/or oligonucleotides including, but not limited to, the following: oligonucleotides containing modified bases or modified sugars, oligonucleotides containing backbone chemicals other than phosphodiester linkages (see, e.g., Nielsen, p.e., 1995, Annu rev. biophysis. biomcl. street.24167-183) and/or oligonucleotides that have been synthesized or modified to give chemical groups (here, we define a nucleic acid or oligonucleotide containing two or more nucleobases and/or derivatives of nucleobases) that can be used to form bonds (covalent or non-covalent) with other molecules.

PMAMS of the invention can have a plurality of discrete binding domains, including at least one binding domain comprising binding agents that are identical to each other and have different specificities than those binding agents contained in other binding domains, so as to provide binding to different analytes of interest with different binding domains. By way of example, such a PMAMS comprises a binding region comprising an antibody to Thyroid Stimulating Hormone (TSH), a binding region comprising an oligonucleotide that hybridizes to Hepatitis C Virus (HCV), a binding region comprising an oligonucleotide that hybridizes to HIV, a binding region comprising an antibody to an HIV protein or glycoprotein, a binding region comprising an antibody to Prostate Specific Antigen (PSA), and a binding region comprising an antibody to Hepatitis B Virus (HBV), or subregions comprising those binding regions previously described.

PMAMS may have a plurality of discrete binding domains, including at least one binding domain having a plurality of binding reagents therein, which bind with different specificities, such that a single binding domain can bind to multiple analytes of interest. By way of example, such a PMAMS comprises a binding region comprising antibodies to a T cell antigen receptor and antibodies to a T cell surface antigen such as CD 4.

PMAMS may have a plurality of discrete binding domains comprising (i) at least one binding domain comprising binding agents that are the same as each other and differ in specificity from at least one of those binding agents contained in other binding domains; and (ii) at least one binding domain having a plurality of binding agents contained therein, the binding agents having different binding specificities. By way of example, PMAMS is made to have (a) at least one binding domain that contains a single property of a binding agent (e.g., an antibody to a T cell antigen receptor, e.g., an α, β T cell antigen receptor or a γ, δ T cell antigen receptor) such that the at least one binding domain binds all cells expressing such T cell antigen receptor; and (b) at least one binding domain comprising two different binding reagents, e.g., an antibody to a T cell antigen receptor and an antibody to CD4, such that the at least one binding domain binds to CD4 expressing that T cell antigen receptor⁺T lymphocytes (that is, a subpopulation of T lymphocytes).

In another embodiment, at least one binding region contains binding agents that are different molecules but have the same binding specificity (e.g., binding agents such as epidermal growth factor and antibodies to epidermal growth factor receptor).

A plurality of binding reagents may be selected such that, although the binding reagents are different and have different binding specificities, they recognize the same analyte (in an alternative embodiment, recognize different analytes). For example, where the analyte is an analyte having a plurality of binding moieties (e.g., a cell having different cell surface antigens), different binding reagents that bind to different binding moieties will recognize the same analyte. As another example, antibodies to different cell surface antigens on a single cell will recognize the same cell. As yet another example, antibodies to different epitopes of a single antigen may be used as binding agents for recognizing the antigen.

In yet another embodiment, only binding reagents that specifically bind to a single analyte of interest are present in one or more binding zones. Alternatively, a binding reagent that specifically binds to more than one analyte of interest is present in one or more binding regions (e.g., a cross-reactive antibody). In a particular embodiment, a binding reagent that binds to a class of analytes, for example, having similar characteristics, may be used.

The binding region may also be loaded with PMAMS containing binding reagents that are specific for the desired standard analyte and are used as an internal standard (e.g., a binding region that can be contacted with a sample containing a defined amount of analyte to which the binding reagents bind). Multiple binding domains containing binding reagents specific for the same analyte may also be loaded into the PMAMS to enable statistical averaging of assay results. Those binding reagents are not only specific for the same analyte, but may be identical, recognizing the same binding on the analyteAnd (4) partial. Thus, a plurality of binding domains (e.g., between 2 and 10) can be prepared that specifically bind to the same binding moiety⁸Within a range of pixels) so that ECL measurement records can be statistically averaged to control variation and improve accuracy. Many of the binding domains on PMAMS may be specific for either a control analyte or an analyte of interest, or both, on a single support.

As another example, one or more discrete binding domains with known initial concentrations of ECL label can be prepared. The internally mounted ECL layer serves as a control to monitor, for example, label degradation and temperature effects.

A binding reagent that is an enzyme specific for a substrate that is the analyte of interest, wherein the enzyme reaction product on the substrate is an information reagent (a detectable reagent), e.g., a product that triggers an ECL reaction, a fluorescent molecule, a substance that changes color upon contact with a suitable enzyme (e.g., a chromogenic substrate for horseradish peroxidase), and the like, can be employed. In one example of such an embodiment, the enzyme used as the binding reagent is Glucose Dehydrogenase (GDH), which can be used to detect or measure glucose in a sample. The ECL label is located within or near the binding region containing GDH. NADH is produced by enzymatic reaction on glucose, and can react with ECL markers to promote ECL (Martin et al, 1993, anal. Chim. acta 281: 475).

The signal to noise ratio during detection and measurement of electrochemiluminescence can be increased by a binding zone containing a binding reagent that enhances background binding, that is, binds to binding components present on the analyte of interest as well as on other analytes in the sample. As an example, the analyte of interest herein is a specific subpopulation of cells (e.g., CD 4) ⁺Cells) and the sample is a fluid sample (e.g., blood) containing patient cells, then antibodies to sialic acid can be used as binding reagents to enhance binding to almost all of the fines in the sampleBackground binding of cells (since sialic acid is a component of almost all cell surface glycoproteins), then antibodies to cell surface antigens specific to a subpopulation of cells (e.g., antibodies to CD 4) can be used as binding agents (in the same or different binding regions as those containing antibodies to sialic acid). 5.3 Voltage waveform

The voltage waveforms (potential/time change) applied across the electrode and counter electrode pairs of the plurality of ECL cells must be sufficient to trigger an ECL reaction. The voltage waveform is generally a uniform voltage variation that starts at a first voltage, steadily moves to a second voltage, moves back to the first voltage to a third voltage, and then returns to the first voltage. For example, the waveform may start at a first voltage in the range of-0.5 volts to 0.5 volts, ramp up to a second voltage in the range of 1 volt to 2.5 volts, move back to the first voltage to a third voltage in the range of 0.0 volts to-1 volt. As another example, in a simpler wave, the voltage may be modified from 0.0 to +3.5 to 0.0. The voltage waveform may have a linear ramp, a step function, and/or other functions. The voltage waveform may have a time period when the voltage remains fixed at a potential. The applied potential can be controlled relative to one or more reference electrodes, or no reference electrode is available. Further, a negative potential may be used. Thus, the voltage used to induce ECL luminescence from the cartridges of the invention is readily selected for optimal ECL signal intensity and specificity for ECL labels and assay media.

In some applications, it is preferred to change the voltage when measuring the light emitted from the binding area. This is particularly important for determining the threshold of the electric field that causes the binding region to emit light. In this case, the potential applied at the binding region starts from a value which is considered to be below the threshold required for luminescence and the emitted light is measured for the first time. If no light is measured, or if the light is below a predetermined threshold, the potential applied to the electrode pair is increased under computer control, e.g., by a computer controlled voltage source, and measured again. This process may be repeated until a predetermined amount of light is received. In this way, the applied voltage can be used as a measurement signal.

The ECL signal may be generated from an alternating voltage applied across the pair of electrodes.

For example, as disclosed in U.S. patents US 5,324,457 and US 5,068,088, one of ordinary skill in the art of voltage and current setting can readily select the optimum voltage and voltage sweep for triggering ECL emission.

If the working electrode is a semiconductor or contains another composition that produces an electrical current in response to light, the desired electrical potential for producing ECL can be produced by illuminating the surface of the working electrode with light. 5.4. Addressable electrode and method of using the same

A number of electrode/counter electrode pairs can be addressed in a number of ways. Several exemplary such techniques are shown in fig. 14 through 18. Shown by way of example in these figures are four electrode/counter electrode pairs 101, 102, 103, 104 and a waveform signal generator, typically a digital computer and preferably the same computer used to process ECL detected by the detection means.

In fig. 14, each electrode/counter electrode pair 101 to 104 is individually addressed by a pair of lines connected to a waveform signal generator. By way of example, lines 105, 106 access (access) electrode/counter electrode pair 101. A waveform signal generator may be used to apply the appropriate waveform to any one or more pairs of lines connected to each electrode/counter electrode pair at any given time.

To reduce the number of connections required to address the counter electrode pair, an alternative to the direct connection scheme of fig. 14 may be provided. For example, a row and column access scheme is shown in FIG. 15 for electrically exciting some or all of the electrodes. In this arrangement, one of the electrodes 201, 202 in each column of a number of electrode/counter electrode pairs is connected to a common electrical conductor 205 on the carrier 200, and each counter electrode in each row of the plurality of electrode/counter electrode pairs is connected to a conductor 207, 208 on the carrier 200. Conductors 205, 206 are connected to connections C1, C2, respectively, at the edge of carrier 200, while conductors 207, 208 are connected to connections R1, R2, respectively. Each of these connections is then connected to a waveform signal generator by a separate line. As a result, in the configuration of fig. 15, the number of required connections and the number of signal lines from the waveform signal generator have been reduced from 8 to 4.

To enable rapid and sequential firing of each electrode pair, it is advantageous to employ a computer-controlled switching device. The configuration of fig. 16 shows a number of electrodes connected to a first multiplexer 310. A number of counter electrodes are connected to the second multiplexer 320. The first multiplexer is also connected to a first pole of a voltage source 330, the voltage source 330 typically providing a time-varying potential as described below. The second multiplexer is also connected to a second pole of the voltage source. Using addressing lines a0-A3 connected to each multiplexer and to register 340, computer processor 350 may direct the multiplexer to selectively connect any or all of the first electrodes to a first pole of a voltage source and any or all of the second electrodes to a second pole of the voltage source.

As shown in fig. 17, a number of voltage sources are connected to each electrode through separate sets of multiplexers. If a first potential or range of potentials is desired at a particular electrode pair, the multiplexers 410, 420 in combination with the voltage source 430 supplying that potential are addressed by the computer processor 350, typically via the register 340, thereby connecting that particular voltage source to the electrode pair. If a different potential or range of potentials is required for another electrode pair, the multiplexers 440, 450 in combination with a different voltage source 460 are addressed by the computer processor, whereby that voltage source is connected to the electrode pair by the combined multiplexers 440, 450.

If at least a portion of the electrode pairs in the electrode array of this embodiment are independently energizable, as shown in fig. 14 or 15, for example, one electrode pair may be energized by one voltage source while another electrode pair is simultaneously energized by another voltage source. Alternatively, the two voltage sources of fig. 17 could be replaced by a single voltage source connected to both sets of multiplexers connected in parallel, which would enable both electrode pairs to be excited with the same voltage source.

Instead of configuring a set of two multiplexers for each voltage source as shown in fig. 17, a number of voltage sources 520, 530 may be configured as shown in fig. 18. These voltage sources may be connected to a set of individual multiplexers 310, 320 through a computer controlled electrical switch 510 or switches. As shown in fig. 18, the computer will instruct the switch 510 to connect a particular voltage source to the multiplexer and will also instruct the multiplexer (signaled via their access lines a 0-A3) to connect the selected voltage source to the particular electrode pair desired.

Alternatively, the potential applied to each electrode pair may be varied in any of the embodiments. This is particularly advantageous when cassettes with many different binding areas are used. Such a cartridge may require different ranges of electrical potential to be applied at different binding regions. Several different embodiments are contemplated which can vary the potential applied to each electrode.

A computer controlled voltage source may advantageously be used. The computer controlled voltage source is a voltage source that can be accessed by a computer to select a particular potential to be provided. Alternatively, the calculation may be programmed to sequentially apply a certain range of potentials over a predetermined time range. In such a system, an access line electrically connected to the computer and the voltage source allows the computer to program the voltage source to generate a specific potential to be applied to the electrode pair to which the voltage is to be applied.

Other methods for accessing many electrode pairs may also be employed. For example, a number of reference electrodes may be placed adjacent to each of a number of electrode and counter electrode pairs in order to sense the voltage applied thereto. In this way additional control of the voltage waveform can be maintained.

FIG. 36 shows another embodiment of the present invention; the electrode arrays (3600, 3601) are supported on each of two surfaces (3602, 3603) separated by a pattern of apertures in an insulator 3604 (e.g., a piece of plastic sheet perforated with holes 3605). Each electrode passes through a number of slits. If a potential is applied to one electrode on each surface, current can only pass through the gap in contact with the two electrodes, thus limiting the location of any electrochemical or ECL that may occur. In the preferred embodiment shown in this figure, the electrodes (3600, 3601) are an array of wires on a carrier. The two sets of electrodes on the two surfaces are oriented perpendicular to each other. The slits in the insulating sheet are located only at the intersections of the electrodes on each surface.

An advantage of this embodiment over those electrode pairs that are individually addressed is that fewer electrical leads are required.

In an alternative embodiment, insulator 3604 is omitted and the surfaces are placed in close proximity so that only a slit exists between the two surfaces. In this embodiment, the potential applied between the electrodes at each surface will preferably cause a current to pass through the intersection of the electrodes (i.e., where the distance between the two electrodes is minimal), thus limiting the location of any possible electrochemical or ECL events. 5.5. Light detection

The light produced by the triggered ECL luminescence is detected with a suitable light detector or detector placed adjacent to the device of the present invention. The light detector may be, for example, a thin film, a photomultiplier tube, a photodiode, an avalanche photodiode, a charge coupled device ("CCD"), or other light detector or camera. The light detector may be a single detector to detect the sequential emission of light, or may be a number of detectors to detect and spatially resolve the simultaneous emission of light at a single wavelength or multiple wavelengths of emitted light. The emitted and detected light may be visible light or may be emitted as non-visible radiation such as infrared or ultraviolet radiation. The detector may be stationary or may be movable. The emitted light or other radiation may be directed to the detector by means of lenses, mirrors and fibre-optic light guides or pipes (single, multiple, fixed or movable) positioned on or adjacent to the binding surface of the cassette or the detector may receive the light directly. In addition, the carrier, PMAMS, and the electrode surface itself can be used to direct or transmit light.

The PMAMS may be formed on the surface of the photodetector array such that each detector receives light from only one binding region. The light detecting array may be a CCD chip and the binding regions may be attached (with standard attachment chemistry) to the semiconductor device surface.

The droplets deposited on the binding area or on a nearby second surface may act as microlenses to direct or control the emitted light. Alternatively, the light detector may be oriented directly in front of the cartridge, and instead of using a light pipe, light from any one of a number of binding regions may be directed to the detector using various light focusing means such as parabolic reflectors or lenses. Light emanating from at least two discrete binding domains may be measured simultaneously or sequentially.

A "chopper" device between the light measuring device and the bonding area being measured can be used to control errors due to thermal drift, aging of the device, or electrical noise inherent in the light detector. The shutter may be any of those common mechanical shutters well known to those of ordinary skill in the art, such as a rotating disk having a slit or opening through which light can pass. Alternatively, the light may be blocked by an LCD aperture, an array of LCD apertures, a solid light valve, or the like. Alternatively, a planar array of LCD shutters or solid light valves may be employed, such as are known in the optical computing arts. These means are preferably located between the plane of the cartridge and a light guide or light focusing means which directs light from the binding area to the light detector. In one embodiment, a shutter is located over each binding region. When using an LCD shutter and light valve, the shutter may be modulated at different frequencies to provide different light blocking rates simultaneously for different light emitting combined areas. With this technique, many different optical signals can be overlapped and simultaneously measured with a single optical detector. An electronic bandpass filter, electrically connected to the photodetector, may then be used to split the single electrical signal into several electrical components, each corresponding to one of a number of individual optical components. By blocking the light as described above, or by employing other mechanisms well known in the art, an AC light waveform is generated that allows the DC noise component to be electrically filtered out.

The ECL signal can also be calibrated by comparing the results of the previous determinations to standard reagents for signal modulation for reagent depletion. ECL Signal analysis

The signal produced by a given binding region may have a range of values, and these values are correlated with quantitative measurements, providing an "analog" signal. In another technique, a "digital" signal is obtained from each zone indicating the presence or absence of an analyte.

Statistical analysis is used for both techniques and is particularly useful for converting many digital signals to provide quantitative results. However, certain analytes require a digital signal indicating the presence/absence of a threshold concentration. The 'analog' and/or 'digital' formats may be utilized alone or in combination. Other statistical methods may be used for PMAMS. For example, a PMAMS concentration gradient can be generated on a surface (Chaudhury et al, 1992, science, 256: 1539-. This technique is used to determine concentration by statistical analysis of binding events across a concentration gradient. Multiple linear arrays of PMAMS with concentration gradients can be generated with multiple different specific binding reagents. The concentration gradient may consist of a number of discrete binding domains giving those different concentrations of binding agent.

It is also important to have a control assay system on the binding surface of the cartridge to ensure consistency of each assay to control signal variations (e.g., due to degradation, fluctuations in the cartridge and other components, aging, thermal drift, noise in the electronics, and noise in the light detection device, etc.). For example, multiple redundant binding domains for the same analyte (containing the same binding reagents or different binding reagents specific for the same analyte) may be utilized. In another example, a known concentration of analyte is utilized, or the control zone of the PMAMS is covalently linked to a known amount of ECL label, or a known amount of ECL label in solution is used.

Assays performed in accordance with the present invention will quickly and efficiently collect large amounts of data, which may be stored, for example, in the form of a database, comprised of a collection of clinical or research data. The collected data may also be used for rapid forensic or personal identification. For example, the use of a number of nucleic acid probes affected by human DNA samples can be used for DNA fingerprinting and can be readily used to identify clinical or research samples. 5.7. Preparation of a Multi-electrode array

The width or diameter of the electrodes may be approximately 0.001mm to 10 mm. A preferred range of electrode pair sizes (width or diameter or widest dimension depending on the electrode pair geometry) is 0.01mm to 1 mm.

The electrodes are preferably made of a suitable conductive material, such as a transparent metal film or a semiconductor (e.g., gold or indium tin oxide, respectively), as is well known in the art, for example, for making liquid crystal displays and the like. In the assembled state of the cartridge, sufficient space remains between the first and second carrier to accommodate an analytical sample, for example in the form of a film or a wetted surface.

The electrodes may be made of materials containing carbon, carbon fibers, carbon nanotubes (nanotubes) and/or aggregates of these materials.

The electrodes can be made from carbon fibrils. One or more individual fibrils and/or aggregates of one or more fibrils may be processed to form larger aggregates (U.S. patent US5,124,075).

This larger aggregate is a mat or mesh (hereinafter "fibril mat") in which fibrils may be intertwined or interwoven. The fibril mat generally has a 50M²G to 400M²Surface area in g.

By way of example, the fibril mat may be used as a working, counter or reference electrode in analysis and/or preparation electrochemistry. In one example, the fibril mat is used as an electrode for Electrochemiluminescence (ECL).

A mat of fibrils may be used to support the binding regions of PMAMS. PMAMS of the invention have a number of discrete binding domains, where two or more of the discrete binding domains may be identical to each other, or may be different. The fibril mat carries one or more binding domains.

A plurality of bonding areas can be made on the fibril mat using one or more microfluidic conduits. Different or equivalent binding agents may be present in a number of microfluidic conduits, and/or a plurality of different binding agents may be present in one microfluidic conduit.

In fig. 22A and 22B, droplets containing those binding agents that are desired are preferably delivered simultaneously onto regions of the fibril mat 2200 to form discrete binding regions 2202, using a plurality of microfluidic conduits, preferably in an array. Those binding agents form bonds with the portion present in the fibril mat described above. Those binding reagents may be non-specifically adsorbed onto the pad or dried on the surface.

The desired binding agent is delivered to the pad of fibrils while suction filtration is applied to the pad. In this case, the suction filtration does not draw, or draws some or all of the binding agent into or through the pad, and in doing so, reduces the amount of lateral spreading of the binding agent on the surface of the pad during the patterning process.

A mat of fibrils is prepared by pressing a suspension of carbon fibrils on a substrate through which the liquid of the suspension can pass (e.g., a filtrate). Examples of filters that can be used to form the fibril mat include filter paper, filters formed from polymeric (e.g., nylon) membranes, metal microsieves, ceramic filters, glass filters, elastomeric filters, glass fiber filters, and/or combinations of two or more such filter materials. Those of ordinary skill in the filtration art will recognize that these materials are but a few examples of the many possible materials suitable for filtering solid suspensions.

Fig. 23A and 23B show an embodiment in which a fibril mat can be made by suction filtration. The dispersion and/or suspension of carbon fibrils 2301 is filtered with a filter 2300, the filter 2300 optionally being provided with a filter membrane 2303 and/or a filter support 2302. The suspension is filtered by suction applied to the filter by a vacuum source 2305, for example, through a filter flask 2306. The fibril mat 2304 is gathered on the filter membrane 2303 and/or the filter support 2302. The fibril mat 2304 with or without the filter membrane 2302 can be removed from the filtrate.

In another embodiment, the suspension of fibrils is forced through a filter with pressure. In one example, pressure is applied to the suspension of constrained fibrils by pressing a layer of constrained air and/or liquid against the suspension with a piston. In one particular example, the fibril filter is confined to a syringe, the piston is a syringe plunger, and the filter is a disposable syringe filter (many such filters are well known to those of ordinary skill in the art).

The suspension of fibrils is forced through a filter by capillary action or filtered by wicking the suspension into or through a filter.

In another example, individual fibrils or aggregates of fibrils are covalently crosslinked into a mat, and the fibrils derivatized with a photosensitive moiety are irradiated with light, and the photosensitive moiety polymerizes upon exposure to light.

The fibrils can be entrapped in their pores by a filter, which acts as a carrier, thus constituting a mat of the complex. In fig. 24, a fibril mat 2400 can be prepared by passing a slurry of fibrils 2401 sent from a source 2402 between two large rollers 2403. In this process, the rollers press the liquid of the suspension and produce a large, continuous mat of fibrils from which smaller mats can be cut, similar to those seen in the process of making paper or polymer sheets.

The mat of fibrils may be freestanding or may be supported.

The filtration rate can be varied to achieve the desired pad properties. For example, those properties that can be altered include uniformity or non-uniformity of structure, degree of entanglement of fibrils or fibril aggregates, thickness, porosity of the mat, and/or combinations thereof.

The suspension of carbon fibrils is restrained and the liquid suspending the fibrils is removed. In one example, the liquid in which the fibrils are suspended is evaporated. In another example, the liquid is removed by heating. In yet another example, the suspension is centrifuged and the resulting liquid (e.g., supernatant) is removed. In another example, the liquid is removed by suction.

The suspension may be placed on one or more of the above-mentioned filtrates and the suspension dried by evaporation. The suspension may be dried by heating or baking in an oven, or the liquid may be removed by freezing and extracting the liquid. In yet another example, the liquid is removed by pumping. Many other methods well known to those of ordinary skill in the art may be used to remove liquid from the suspension.

A fibril suspension suitable for forming a fibril mat by filtration may be formed by dispersing one or more carbon fibrils in a suitable liquid, quasi-solid or gel. Examples of suitable liquids include, but are not limited to, water, ethanol, methanol, hexane, methylene chloride, buffered solutions, surfactants, organic solvents, solutions containing biological media (e.g., proteins, antibodies or fragments thereof, cells, subcellular particles, viruses, nucleic acids, antigens, lipoproteins, liposugars, lipids, glycoproteins, carbohydrates, peptides, hormones or drugs, solutions of small molecules, polymer precursors, solutions of acids or bases, oils, and/or combinations thereof).

A suspension of fibrils can be prepared by dispersing carbon fibrils in an aqueous solution by sonication. In another embodiment, a surfactant or detergent may be added.

The thickness of the fibril mat may be generally 0.01 μm to 10,000 μm.

In some preferred embodiments, the thickness of the fibril mat is between 1 μm and 100 μm. In some particularly preferred embodiments, the width or diameter of the fibril mat ranges from 10mm to 200 mm.

The fibril mat can be repeatedly rinsed and re-filtered to remove residual material remaining from the suspension.

The fibril mat prepared by filtration or evaporation is heated (e.g., in an oven) to remove the residual liquid of the suspension that is not removed by filtration.

Successive filtration steps can be used to form a mat of fibrils consisting of one or more distinct layers either in contact with or in close proximity to one or more other layers. The layers may be distinguished by several properties including, but not limited to, differences in porosity, density, thickness, size distribution of individual fibrils and/or microscopic aggregates of fibrils, type, number and/or size of fibril aggregates, chemical derivatization of fibrils (see below), and/or other substances attached to fibrils.

Fig. 25, a multi-layered fibril mat 2500 is prepared by successive filtration steps. A 0.5 μm to 100 μm thick layer 2501 of plain fibers constituting the first layer; a 0.5 μm to 10 μm thick layer of fibrils 2502 constituting the second layer, the layer of fibrils 2502 having moieties such as poly (ethylene glycol) that prevent absorption of proteins and other molecules; a 0.5 to 5 μm thick layer 2503 with one or more bonding regions (see above) constitutes the third layer. The binding region contains one or more antibodies 2504 that bind to analyte 2505. The antibody/analyte complex can bind to a labeled antibody 2506. The label may be an ECL label. In still other embodiments, the marker may be one or more of a number of markers described elsewhere in this application. Such a multi-layer mat may be freestanding or may be supported on one of many possible carriers as described above.

A multi-layer pad may be formed having a multi-layer combination, wherein some or all of the layers may be different.

The filtrate, fibrils and/or fibril mat used to construct the fibril mat may be coated. In some particular embodiments, the coating is a metal. These coatings may be patterned so that some portions are coated and some other portions are uncoated. In one example, the coating is applied by electrodeposition. In another example, the coating is applied using electroless plating.

The filtrate is coated with a metal and the fibrils are derivatized with a chemical functional group that bonds to the metal. The filter is a metal net or a metal sheet.

The fibril mat may be flat or may be deformed, regular or irregular, round, oval, rectangular, or one of many shapes, rigid or soft, transparent, translucent, partially or wholly opaque, and may have areas of composite or some different individual or composite properties.

The pad may be a disc or a small piece taken from a plate.

A plurality of fibril mats are preferably made simultaneously, and preferably in an array. In one example, an array of microfluidic conduits forms a plurality of fibril mats on the support. In another array of filters, or a patterned filter (e.g., regions of different porosity) is used to prepare an array of fibril mats.

A mask (e.g., a screen) with an array of holes is used to cover certain portions of the filter or support and a number of discrete fibrous mats are made simultaneously by filtration and/or evaporation.

The density of the fibril mat is from 0.1 to 3.0g/cm ². The pad may have a variable density. For example, mechanical force or pressure may be applied to the pad from time to time in order to increase or decrease the density.

The fibril mat may have some pores. The apertures may extend partially and/or completely through the pad, or may be part of a network or some number of apertures. These pores range in size from approximately 50 angstroms to 1000 μm. In a preferred embodiment, the fibril mat has pores ranging in size from 200 angstroms to 500 angstroms. The porosity of the pad depends among other factors primarily on the density of the pad.

The porosity of the pad described above is throughout the entire pad, or may increase or decrease with position in the pad. The fibril mat may have a variety of pores of different sizes distributed in a random and/or random manner.

The fibril mat may contain several different regions of different porosity. For example, the fibril mat may have one or more layers, each layer having a different porosity. The fibril mat may have a number of columns of different porosity extending through the mat.

The porosity of the pad can be varied by including different numbers of carbon fibril aggregates having different sizes, shapes, compositions or combinations. In one particular example, the pad is prepared from individual fibrils, CC fibrils (described above) and BN fibrils (described below), or some different combination. For example, in a fibril mat, some pores may be large enough to pass objects as large as biological cells, some may pass biological media as large as proteins or antibodies, some may pass only small (molecular weight < 1000) organic molecules, and/or there may be a combination of these pores.

The porosity of the pad is such that one or more molecules, liquids, solids, emulsions, suspensions, gases, gels and/or dispersions can diffuse into, within and/or through the pad. The porosity of the fibril mat is such that the biological medium can diffuse (actively or passively) or be forced into, within and/or through the mat by some means. Examples of biological media include, but are not limited to, whole blood, fractionated blood, plasma, serum, urine, protein solutions, antibodies or fragments thereof, cells, subcellular particles, viruses, nucleic acids, antigens, lipoproteins, lipids, glycoproteins, carbohydrates, peptides, hormones, or pharmaceutical agents. The fibril mat may have one or more layers of different porosity so that the material may pass through one or more layers but not through other of those layers.

The mat of fibrils is carried or supported on another material. By way of example, the support material may be metal, plastic, polymer, elastomer, gel, paper, ceramic, glass, liquid, wax, oil, paraffin, organic solids, carbon, or a mixture of two or more of the foregoing. The material may be solid or liquid. If it is solid, it may contain one or many pores or pores. In a specific example, the support may be a metal mesh, a nylon filter membrane or a filter paper. The carrier may be a conductor, a semiconductor and/or an insulator.

In one embodiment disclosed in US 5,304,326 and US 5,098,771, the fibrils may be dispersed in another material. For example, the fibrils can be dispersed in oil, wax, paraffin, plastic (e.g., ABS, polystyrene, polyethylene, acrylonitrile, etc.), ceramic, polytetrafluoroethylene, polymer, elastomer, gel, and/or combinations thereof. Dispersions of fibrils in other materials are electrically conductive. Dispersions of fibrils in other materials can be molded, pressed, shaped, cast, spun, braided, and/or sprayed to form objects of desired shape and/or form. The fibril mat may be provided with additional material, such as fine fibers, chips or metal spheres, in order to increase the electrical conductivity of the mat. In another example, the fibril mat may be provided with other carbon, glass and/or metal fibers of varying size, shape and density to create porosity not attainable with fibrils alone. In another aspect, the pad can be provided with magnetic beads (e.g., DYNAL beads). In the latter case, the beads may be used to modify the porosity of the pad, or the beads themselves may be used as a carrier to immobilize the binding domains.

Other carbon fibers (e.g., carbon nanostructures, carbon nanotubes, buckminster fullerene molecules, bucky tubes, fullerene cage molecules, or combinations thereof) may be used in place of carbon fibrils.

The carbon fibrils can be prepared with chemical functional groups covalently attached to their surface. As described in International patent WO 90/14221, these chemical functional groups include, but are not limited to COOH, OH, NH₂N-hydroxysuccinimide (NHS) -ester, poly- (ethylene glycol), thiol, alkyl ((CH)₂)_n) Groups and/or combinations thereof. These and other chemical functional groups can be used to attach other materials to the surface of the fibril.

Certain chemical functional groups (e.g. COOH, NH)₂SH, NHS-esters) can be used to couple other small molecules to the fibrils. There are many possibilities for such combinations of chemical functional groups and small molecules.

In many embodiments, the NHS-ester group is used to link other molecules or materials that carry nucleophilic chemical functional groups (e.g., amines). In a preferred embodiment, the nucleophilic chemical functional group is present on and/or in a biomolecule, which is native and/or chemically derivatized. Examples of suitable biomolecules include, but are not limited to, amino acids, proteins and functional fragments thereof, antibodies, binding fragments of antibodies, enzymes, nucleic acids, and combinations thereof. This is one of many such possible techniques and is generally applicable to those examples given herein and many other similar materials and/or biomolecules. In a preferred embodiment, the agent useful for ECL can be attached to the fibril via a NHS-group.

Antibodies useful for ECL assays may be attached to one or more fibrils or a fibril pad by covalent bonding (e.g., reaction with NHS-ester groups), by reaction with a suitable linker (see above), by non-specific binding, and/or by binding by these methods. Nucleic acids and/or cells may be attached to fibrils or fibrous mats by covalent attachment to NHS-esters attached to the fibrils.

It may be desirable to control the degree of non-specific binding of the material to the fibrils and/or fibril mat. By way of non-limiting example only, it may be desirable to reduce or prevent non-specific uptake of a protein, antibody fragment, cell, subcellular particle, virus, serum, and/or one or more components thereof, ECL marker (e.g., Ru^II(bpy)₃And Ru^III(bpy)₃Derivatives), oxalates, trialkylamines, antigens, analytes, and/or combinations of these substances. In another example, it may be desirable to enhance binding of biomolecules.

One or more chemical moieties that reduce or prevent non-specific binding may be present in, on or near one or more fibrils, one or more fibril aggregates and/or fibril mats. Non-specific binding is controlled by covalently attaching a PEG moiety to one or more fibrils and/or a pad of fibrils. Charged residues (e.g., phosphate, ammonium ions) can be covalently attached to one or more fibrils or fibril mats.

Materials for the carrier, electrode and/or binding region may be treated with a surfactant to reduce non-specific binding. For example, the fibrils or fibril mat may be treated with surfactants and/or detergents (e.g., tween series, Triton, span, Brij) well known to those of ordinary skill in the art. Washing with a solution of a surfactant and/or detergent, soaking, incubating, sonicating and/or treating the fibril or fibril mat in combination with these methods. Solutions of PEGs and/or molecules that behave in a manner similar to PEG (e.g., oligo-or polysaccharides, other hydrophilic oligomers or polymers) can be used ("polyethylene glycol chemistry: biotechnology and biomedical applications", Harris, j.m. editions, 1992, plenium press) surfactants and/or detergents, and/or used together.

Undesired non-specific absorption of certain substances, such as those listed above, can be blocked by competitive non-specific absorption. Such competitive binding substances may be Bovine Serum Albumin (BSA) immunoglobulin g (igg).

Non-specific binding of ECL-TAG can be reduced by chemical modification of TAG. For example, TAG may be modified to increase its hydrophilicity (e.g., by imparting Ru (bPy) ₃) The bipyridyl ligand in (b) adds hydrophilic polar hydrogen bonds and/or charged functional groups) and thus reduces non-specific binding of TAG to other surfaces.

It may be desirable to immobilize biomolecules or other mediators onto the fibrils or fibril mat. Antibodies, antibody fragments, proteins, enzymes, enzyme substrates, inhibitors, cofactors, antigens, incomplete antigens, lipoproteins, lipid sugars, cells, subcellular components, cellular receptors, viruses, nucleic acids, antigens, lipids, glycoproteins, carbohydrates, peptides, amino acids, hormones, protein-binding ligands, agents, and/or combinations thereof may be attached.

It may also be desirable to attach non-biological substances to the fibrils such as, but not limited to, polymers, elastomers, gels, coatings, ECL markers, redox active substances (e.g., tripropylamine, oxalate), inorganic materials, chelating agents, linkers, and the like.

One or more or many substances may become non-specifically bound (e.g. absorbed) to the surface of the fibrils or fibril mat.

Biomolecules or other mediators can be attached to the fibrils or fibril mat by non-specific absorption. The extent of non-specific absorption for any given fibril, fibril mat and/or biomolecule is determined by certain properties of each of the substances. Certain chemical functional groups or biological components present on the fibrils can reduce or enhance non-specific binding. The presence of hydrophobic and/or hydrophilic spots (patches) on the surface of the protein may enhance or reduce the non-specific binding of the protein to the fibrils or fibril mat. Hydrophilic and/or hydrophobic spots are used to control non-specific binding in the controlled areas.

Alkyl Chains (CH) may be used₂) And/or carboxylic acid groups derivatize the fibrils to enhance non-specific binding of biomolecules or mediators or other materials.

Figure 26 schematically illustrates the example for the case of individual fibrils. Fibrils 2600 are derivatized with alkyl chains 2601. Biomolecules 2602, 2603, and 2604 bind non-specifically to the alkyl chain. Polymer/elastomer 2605 is also bonded.

Underivatized fibrils, fibril aggregates, and/or fibril mats are used to immobilize biomolecules, biological mediators, and other materials by non-specific binding.

ECL TAG contains charged residues. ECL TAGs were selectively attracted to the support and/or the electrodes. For example, a derivatized ECL TAG having a net negative charge has a lower affinity for electrodes at greater reduction potentials, however, has a higher affinity for electrodes when the electrode potential becomes more oxidizing. The affinity of ECLtag and/or binding reagent for the electrode is adjusted (e.g., decreasing the affinity during the binding and/or washing step and increasing the affinity of ECL TAG and/or binding reagent during ECL measurement recording to increase the effective potential sensed by ECL TAG).

In fig. 28, molecules (biological and non-biological) can be attached to fibrils via covalent bonds. Fibrils 2800 bearing a NHS-ester chemical functionality can form covalent bonds 2801 with biomolecules or biological media 2802, 2803. These biomedia may use amino groups to form covalent bonds by reaction with NHS-ester groups. A fixing polymer 2800. One of ordinary skill in the art will recognize the versatility of the NHS-ester group as a coupling agent for molecules, and will be able to select the appropriate biomolecule and the appropriate reaction conditions to achieve immobilization.

One or more of a pair of components and/or molecules "M1" and "S1" are attached to the fibril and exhibit mutual affinity or binding capacity. M1/S1 may be antibody/antigen, antibody/partial antigen, enzyme/substrate, enzyme/cofactor, enzyme/inhibitor, lectin/carbohydrate, receptor/hormone, receptor/effector, nucleic acid/nucleic acid, protein/nucleic acid, virus/ligand, cell/cellular receptor, and the like. There are many combinations of "binding pairs" M1/S1, and some combination can be selected as appropriate to achieve the desired binding. Either M1 or S1 or both M1 and S1 may be attached to one or more fibrils.

Fig. 27 and 28 illustrate some of the many possible configurations that are possible for this embodiment. In fig. 27, fibrils 2700 derivatized with alkyl chains 2701 non-specifically bind molecules 2702, molecules 2702 having mutual affinity or binding ability to another molecule 2703. Molecule 2703 is also attached to another molecule 2704. Blocking molecules 2705 can non-specifically adsorb onto fibrils. Blocking polymer 2706 and/or polymer 2707 with ligand (2708) are non-specifically absorbed, wherein ligand (2708) has affinity for molecule 2709.

In fig. 28, a fibril 2800 is covalently linked to biomolecules 2802 and 2803 and a linker molecule (linker group)2804 by 2801. The linker molecule 2804 has a mutual affinity or binding ability for another biomolecule 2805. The biomolecule 2803 has a mutual affinity or binding ability to another linker molecule 2806, wherein the linker molecules 2806 and 2807 are covalently linked. A polymer 2808 with a ligand 2812 specific for the binding pair 2809 is covalently attached to the fibril. A blocking molecule (e.g., BSA)2811 and a blocking polymer 2810 are covalently linked.

The fibrils may be derivatized with a biotin and/or biotinylated linker to which avidin and/or streptavidin may be bound. Avidin and/or streptavidin may bind to the fibrils, while biotinylated antibodies and/or proteins may bind. Avidin and/or streptavidin may be immobilized on the fibrils by non-specific binding, covalent bonding, additional or identical coupling pairs, or a combination thereof. The use of (strept) avidin and biotin as a "binding pair" is a widely used method of attaching biomolecules or biological mediators to other materials and is well known to those of ordinary skill in the art (Spinke et al, 1993, Langmuir, 9: 1821).

A binding pair may be a monoclonal antibody and an antigen that binds to the antibody.

Multiple binding pairs (e.g., M1/S1/M2) can be constructed. M1 is a monoclonal antibody, S1 is the antigen for M1, and M2 is an antibody that binds to S1. Such a complex may constitute an antibody/antigen/antibody "sandwich" complex (such an antibody may or may not be monoclonal). M2 may be an antibody labeled with an ECL-active label (see above), a fluorescent label, a radioactive label, an enzymatic label, and/or combinations thereof.

M1 may be a component capable of complexing with a metal, metal ion or metal organic compound (a "chelator"), whereas S1 is a metal, metal ion or metal organic compound (a "chelator") that forms a complex with M1, whereas M2 is a component on a biomolecule that binds to the M1/S1 complex (Gershon and Khilko, 1995, journal of the immunological methods, 7371).

The fabrication of metal electrode patterns and conductive elements of such electrodes for distributing current over a surface is carried out by methods well known in the art (see, for example, US 5,189,549). A metal film is prepared on a transparent surface to produce a liquid crystal display, and is well suited for preparing the electrode of the present invention. See: haneko, 1987, lcd tv displays, principles and applications of lcd displays, KTK science press, tokyo, d. The transparent electrode surface can also be prepared, for example, according to the method of DiMilla et al (J.Am.chem.Soc.116 (5): 2225-2226). 0.5nm titanium and 5nm gold were deposited on a transparent substrate (glass or plastic). The transparent electrode structure prepared by the method of Kumar described above can be prepared using a thin gold layer prepared by the method of dimila described above. It would be desirable to improve the process to increase the thickness of the conductive layer to improve current carrying capability while preferably maintaining transparency, as will be apparent to one of ordinary skill. Such techniques may be used to prepare electrode surfaces aligned with or adjacent to discrete bonding regions of pmams.

Furthermore, the membrane and/or monolayer may be comprised of compositions that facilitate the transfer of an electrical potential from the electrode surface to the ECL label, rather than employing insulating compositions (e.g., alkyl chains) as taught by Zhang and Bard. For example, Pi orbital overlap in a wide conjugate system can be employed for electron transfer. Such Pi orbital electron transfer is provided by polypyrrole or other conjugated ring or double bond structures.

Oligonucleotides may be used to modulate electron transfer. For example, overlapping Pi bonds in double-stranded DNA can be used to increase the electron transfer rate. Oligonucleotides bound to the surface of the electrode can be used as binding reagents for the binding region. Once the complementary oligonucleotide sequences are bound, a double strand with organized overlapping Pi bonds is formed. In a particular embodiment, an initial or originally immobilized (e.g., covalently bound to a support) oligonucleotide is ECL labeled. In another embodiment, an auxiliary complementary oligonucleotide or an oligonucleotide that is partially complementary to the original oligonucleotide is ECL-labeled. Tertiary oligonucleotides complementary or partially complementary to the auxiliary oligonucleotides are labeled (e.g., a sandwich assay). Branched oligonucleotide chains may also be used. A wide variety of oligonucleotides and/or copies of oligonucleotides (e.g., oligonucleotides with modified bases and/or with modified backbones containing, for example, chlorine and/or sulfur) can be utilized. Studies on the differences can be performed. The variable stability of the Pi overlap in the oligonucleotide and/or the complex of oligonucleotides can be monitored by modulating the electron transfer. The signal generated by the Pi-bond stabilized pair of ECL-labeled duplex oligonucleotides (e.g., ECL light generated and/or impedance measurements) can be correlated against the signal from the more disordered single-stranded oligonucleotide and/or the desired signal. The change in ECL signal between a fully complementary ECL-labeled double-stranded oligonucleotide and a partially complementary ECL-labeled double-stranded oligonucleotide can be correlated. In addition, oligonucleotide complexes of multiple oligonucleotides can be utilized. For example, a triple helix may be employed.

Adjustment of the electron transfer rate can be measured with ECL detection and electronics. ECL labels may be covalently attached to some strands of the oligonucleotide and/or non-specifically bound (e.g., inserted). The DNA can be bound to the electrode without a linker (e.g., 5' thiolated DNA uptake on gold), or with a short (< 10 atoms) linker to ensure low resistance to electron transport from the DNA to the electrode. A linker chain that efficiently transports electrons from the electrode to the DNA strand (e.g., a polyacetylene strand) can be used.

A hybrid monolayer and/or film may be used, wherein at least one constituent of the monolayer or film facilitates the transfer of an electrical potential as the case may be. Alternatively, a molecule or particle that facilitates potential transfer is adsorbed to the monolayer or membrane. As in the previous examples, Pi-conjugated monolayers and/or adsorbed branches and/or conductive microparticles adjacent to the electrode surface may be used. Patterned regular gaps are created in a single layer or film. By using controlled patterns of slits in an ordered substantially vertical SAM comprised of (i.e., insulating) long chain alkanethiols to which ECL labels have been sequentially attached, the effective potential applied to the ECL labels can be controlled. For example, fig. 11 shows a cartridge 1200, the cartridge 1200 being formed from a single carrier having a metal layer 1204, a SAM pattern 1206, and gaps 1208 between the SAM patterns.

ECL-labeled proteins may be non-covalently attached to the monolayer surface. ECL-labeled proteins can be adsorbed onto the surface of gold surfaces derivatized with methyl-terminated alkanethiols. The gold surface functions as a working or counter electrode. As shown in fig. 11 to 13, a plurality of binding domains may be bound to a single carrier. In a preferred embodiment, the binding region comprises a labeled and/or unlabeled protein and/or nucleic acid and/or cell and/or chemical substance.

Alternatively, the lengths of those components of the monolayer (e.g., the lengths of the alkyl chains in the alkanethiol monolayer) may be varied to control the effective potential at the exposed surface of the monolayer.

In general, alkanethiols can have a carbon chain length between 1 carbon and 100 carbons. In a preferred embodiment, the carbon length of the alkanethiol contains from 2 to 24 carbons. The carbon chain length of alkanethiols is between 2 and 18 carbons. The carbon chain length is between 7 and 11 carbons. Such alkanethiols can have various head groups exposed to the assay medium, including methyl, hydroxyl, amine, carboxylic acid, oligo (ethylene glycol), phosphate, phosphoryl, biotin, nitrilotriacetic acid, glutathione, epoxide, dinitrophenyl, and/or NHS esters. Other headgroups include ligands commonly used to purify and immobilize recombinant fusion protein tags (e.g., Sassenfeld, 1990, TIBTECH, 8: 88-93). The binding domains can be derivatized to varying degrees to achieve various densities of binding agent. For example, different densities of activatable chemicals may be employed, and/or derivatization may be carried out to varying degrees. Mixed chemistries can be used to produce the desired binding density. The mixed monolayers can be used to control the density of activatable groups and/or binding agents. The density of the binding groups is controlled to optimize the signal-to-noise ratio of the ECL. The total number of binding sites within a binding domain is controlled to optimize the intensity of ECL light signals relative to other ECL light signals from other binding domains, whether such ECL light signals are detected sequentially or simultaneously, and/or detected with a light detection device.

A voltage waveform may be applied to excite the ECL labels one or more times in combination with the binding region in the pmams. The same alkanethiol-derivatized surface with bound ECL label can be subjected to a potential sufficient to excite ECL luminescence multiple times to generate an ECL light signal multiple times. The potential is applied sufficiently to reversibly generate ECL. An electrical potential is applied to quasi-reversibly generate ECL. In a quasi-reversible sequence of voltage waveforms, the binding region with ECL labels inside (e.g., bound to) can be chemically and/or physically altered. The sequence of applied voltage waveforms may irreversibly cause ECL to emit light.

Further, a potential sufficient to release the components of the monolayer may be applied. It may be desirable to release some monolayer components where the volume on the electrode surface is small (e.g., another support or plate located on the electrode surface). Thus, even some ECL labels that are not efficiently excited can be excited by the electrode surface to produce an electrochemiluminescent signal due to the monolayer being destroyed, while ECL labels are confined to a small volume, limiting diffusion from the electrode. Various monolayer compositions can be used to control the degree of monolayer damage for a given potential. Wherein the monolayer has a strong affinity between the components to prevent further destruction of the monolayer. Longer chain alkanethiols are more effective against damage than shorter chain alkanethiols. By varying the length of the chain, the desired stability can be obtained.

Modification of the binding region within PMAMS can be used to modulate ECL signaling. A series of voltage waveforms are applied to generate a number of ECL signals. Additional and/or better results may be obtained with the plurality of ECL signals. Statistical analysis of the rate of modulation of ECL signals can be correlated to the overall quality of one or more binding domains. Further, the number of ECL signals may be utilized to improve signal-to-noise ratio, for example, by filtering certain ECL signals in a sequence. Multiple potential waveform pulses may then be utilized to reduce unwanted signal conditioning due to non-specific binding. A potential may be applied to prevent non-specific binding of certain charged species. In addition, an electrical potential can be applied to facilitate localization of the analyte or chemical of interest in the vicinity of the binding region. The applied voltage waveform provides a large overpotential (e.g., a higher Potential than necessary to generate ECL). The ECL signal in a sequence of voltage waveforms or in a single voltage waveform pulse may be overpotential adjusted. Furthermore, overpotential can be used to modulate ECL reaction kinetics and/or chemically and/or physically modulate binding potential. Further, data regarding the mass and/or electronic properties of the electrodes may be evaluated and/or correlated and/or extrapolated using one or more voltage waveforms and/or other electronic probes known to those of ordinary skill in the art.

Preferably, the efficiency of the ECL reaction can be increased by extending the working electrode surface area by providing additional conductive means in contact with the electrode. The electrode surface area can be increased by protrusions or extensions from the electrode (e.g., wire or metal whiskers) of conductive material or conductive particles to bring the electric field closer to the ECL marker. Alternatively, grooves or wells in the electrode structure may be used for the same purpose.

In particular, the conductive particles may fill gaps on the electrode surface and/or cover the support or monolayer, increasing the absolute value of the electric field around the ECL marker, as shown in fig. 12. These conductive particles extend the electrode surface area and thus increase the efficiency of the ECL reaction. Fig. 12 shows a cell 1300 with a carrier 1302 carrying a patterned SAM 1306 on a metal layer 1304 and indicating that conductive microparticles are filled in the gaps (e.g., 1208 in fig. 11) and extend above the metal surface, between the two SAM patterns. For magnetic conductive particles, a magnet or magnetic field may be used to attract the particles to the surface. The potential between the electrode surface and the binding region of the PMAMS with two close supports can also be extended with conductive particles as described above. In fig. 8, the cell 900 is comprised of a first carrier 902 with multiple arrays of electrodes and a second carrier 904 with PMAMS. The conductive microparticles 906 are positioned between two opposing surfaces to extend the potential towards the ECL label on the binding zone (not shown).

Alternatively, a conductive polymer is grown from the exposed gap on the electrode surface, as shown in fig. 13, in order to extend the potential around the ECL marker of the sample. Fig. 13 shows a cartridge 1400 having a carrier 1402, the carrier 1402 carrying a metal layer 1404 on a patterned SAM surface 1406. A conductive polymer 1408 is grown across the SAM surface to spread the electric field provided by the multi-array electrodes (not shown) to the binding regions (not shown) on the SAM surface. It is also possible to extend the potential between the electrode surface and the binding region of the PMAMS of two adjacent carriers with a conducting polymer as described above and shown in figure 7. In fig. 7, cassette 800 is made up of carriers 802 and 804 in close proximity. A conductive polymer 806 is grown between the two opposing surfaces in order to spread the potential towards the ECL labels on the binding region (not shown).

Fig. 9 shows a cartridge 1000 comprised of a first carrier 1002 with a multi-electrode array and a second carrier 1004 with a PMAMS binding surface, where the conductive projections (1006) of the working electrodes (e.g., thin wires or other protrusions) extend the electric field around the ECL labels in the PMAMS binding region.

The electrode pairs can be made in various configurations. The simplest structure drawn in the figures of this specification is made of a metal and/or metal oxide film and/or a semiconductor film applied on a non-conductive planar surface. The electrodes of the electrode pairs preferably define a region of relatively constant width therebetween, thereby providing a relatively constant electric field.

Other configurations of the electrodes are provided. Plan views of several such structures are shown in fig. 19(a) to 19 (e). Fig. 19(a) shows a staggered interdigitated comb-like electrode pair. In this structure, each electrode has a plurality of fingers extending from a conductor formed in a comb shape. The electrode and counter electrode pair may be positioned adjacent to the binding region, or the binding region may be positioned between the electrode and counter electrode. Fig. 19(b) shows a pair of concentric electrodes, one circular and one semicircular. Fig. 19(c) shows two semicircular electrodes whose straight sides face each other. Fig. 19(d) shows a pair of rectangular electrodes. Fig. 19(e) shows a pair of interdigitated electrodes having complementary opposing curved surfaces to form a curved gap therebetween.

The electrode/counter electrode pairs may also be formed into specific shapes that are complementary to the shapes on the PMAMS binding surface for alignment purposes. An example shape is shown in fig. 6B. A carrier 712 carrying electrode pairs 714 and 720 is shown. The electrode pairs may be, for example, circular 714, interdigitated 716, triangular interdigitated 718, or multiple electrode interdigitated 720.

In the embodiments shown in fig. 14 to 19 described above, the electrode pairs are located on a single support. Alternatively, as shown in fig. 2, the electrode pairs are located on first and second opposing supports. 5.8, Box

The cassette contains one or more vectors of the invention. The cartridge may include a number of binding regions and one or more working electrodes.

Fig. 2 shows a cartridge in which a plurality of bonding areas 30 on the carrier 26 each adjoin a different one of a plurality of electrodes 32. The counter electrode 38 is formed on a second carrier 28. ECL measurements are performed by placing a sample on the binding areas 30 and then moving the carriers 26 and 28 together so that each of the counter electrodes 38 abuts each binding area 30 as previously described, and ECL reactions are triggered via leads 34 by the waveform signal generator means 39, as previously described, and ECL signals are detected and recorded by the light detection means 40, leads 41 and digital computer means 42.

Fig. 3 shows a cartridge in which each of a plurality of binding domains 48 has a different one of a plurality of electrode/counter electrode pairs 50 adjacent thereto on support 44. Optionally, carrier 46 may be positioned adjacent to carrier 44 such that carrier 46 forms a sample-containing device adjacent to a plurality of binding regions 48 and a plurality of electrodes 50. Thus, an ECL reaction can be triggered by the waveform signal generator means 54 via the electrical connection means 52, and the ECL signal is detected by the light detection means 56 and recorded and analyzed by the digital computer means 58.

As shown in fig. 21, a cartridge is provided containing one or more pairs of carriers, each pair of carriers being positioned such that the surface of one first carrier 1501 containing binding regions faces the surface of a second carrier containing binding regions, wherein each surface contains an electrode 1504 and a binding region 1506; this results in each bonding area on the first carrier facing and being aligned with one electrode on the second carrier and each bonding area on the second carrier facing and being aligned with one electrode on the first carrier.

Figure 4 shows a cartridge in which ECL electrodes are optional. The binding area 64 on the support 64 is contacted with a sample suspected of containing an analyte. The area 66 on the support 62 contains a reaction medium for detecting or measuring the analyte of interest, or for carrying out the desired reaction. The support 60 and the support 62 are brought together so that the binding zones 64 and 66 are in contact and the presence of the analyte or reaction product is determined by an indicator system, e.g., by colorimetric chemiluminescence or fluorescent signal that can be detected by a photodetector device 68 and recorded and analyzed by a digital computer device 70.

In a preferred embodiment, the cartridge or device of the present invention includes a means for delivering a sample to a plurality of discrete binding sites (see, e.g., element 1 of FIG. 1 of U.S. patent No. 5,147,806; element 1 of FIG. 1 of U.S. patent No. 5,068,088; the entire contents of both U.S. patents are incorporated herein by reference). The means for sending a sample may be stationary or movable and may be any means for sending a sample known in the art including, but not limited to, one or more inlets, holes, wells, channels, tubes, microfluidic conduits (e.g., capillaries), tubes, sleeves, and the like. The fluid may be moved through the system by a variety of well-known methods, such as by pump, pipette, syringe, gravity flow, capillary action, wicking, electrophoresis, pressure, vacuum, and the like. The means for fluid movement may be located on the cartridge or on a separate unit. The sample can be placed on all binding regions together. Alternatively, capillary fluid transport devices may be used to place samples on specific binding regions. Alternatively, the sample may be placed on the carrier by a self-aspirating tube for sending the fluid sample directly to the PMAMS on the carrier, or into a reservoir of the cartridge or holder of the cartridge for later direct sending to the binding surface.

The support may be made from a variety of materials including, but not limited to, glass, plastic, ceramic, polymeric materials, elastomeric materials, metals, carbon or carbon containing materials, alloys, composite foils, silicon and/or layered materials. The carrier may have various structural, chemical and/or optical properties. They may be rigid or flexible, flat or deformed, transparent, translucent, partially or totally reflective or opaque, and may have composite properties, have regions of different properties, and may be composites of more than one material.

Reagents for performing the assay may be stored in the cartridge and/or in a separate container. The reagents may be stored in a dry and/or wet state. In one embodiment, the dry reagents in the cartridge are rehydrated by the addition of the test sample. In a different embodiment, the reagent is stored in a solution in a "blister pack" that bursts open due to pressure from a movable roller or piston. The cartridge may contain a waste chamber or sponge for storing liquid waste after the assay is completed. In one embodiment, the cartridge includes a means for preparing a biological sample to be tested. A filter may be included for removing cells from the blood. In another example, the cartridge may include a device such as a precision capillary for metering the sample.

The plurality of bonding areas on the carriers and the plurality of electrode/counter electrodes are typically placed in adjacent alignment with each other by mechanical means, for example, by using guide posts, guide pins, hinges (between each carrier) or guide edges. The carrier and the electronic device using the optical guidance marks defined on the carrier can be positioned with optical guidance means. Other systems employing electrical or magnetic alignment means may also be utilized.

The carrier of the cartridge is constructed so that the electrode pair does not come into contact with the sample until it is desired to trigger the ECL reaction. For example, the electrodes may be held apart from the surface of the binding region by various mechanical means, such as removable electrode protection means, until contact of the electrodes with the sample is desired.

The cartridge or device of the present invention includes a reference electrode, for example, an Ag/AgCl or Saturated Calomel Electrode (SCE).

The carriers may be held together by clamps, adhesives, rivets, pins, or any other suitable mechanical attachment means. Those carriers can also be held together by the surface tension of the liquid sample, or by a holding-down device that is removably placed on opposite sides of the two carriers.

The cartridge may also comprise more than two supports, e.g. with alternating layers of binding regions and electrodes, or a plurality of supports comprising binding surfaces and electrode surfaces on a single support. This will form a three-dimensional array of ECL analysis elements. Optionally, all of the foregoing components of the cartridge are transparent except for certain areas between the two binding regions. For example, a plurality of transparent binding surfaces, electrode surfaces and carriers may be stacked.

The first and second carriers may be flat and opposed so as to define a sample holding volume therebetween. Alternatively, the first and second carrier layers may be structured in other suitable shapes, including spherical, cubic, cylindrical, provided that the two carriers and any other components thereof conform in shape. For example, fig. 10 shows a cassette 1100 formed of two adjacent non-planar carriers 1102 and 1104. Each support has a surface complementary to the morphology of the other supports. Each support may have a PMAMS surface or a multi-electrode array or both. One or both of the carriers may be resilient so as to conform to the shape of the other carrier. It is also possible to prepare the carrier or cassette in a pre-cut form or to dispense a suitable length of carrier or cassette from a roll dispenser. The cartridge may also include sample containment means, e.g., sample holding volumes, as well as sample dispersion wells, channels, recesses, and the like.

Fig. 37 shows a cartridge in which a bonding area (3702) in and/or on a matrix (3703) is present on a surface (3701). A second surface carrying a working electrode (3704) and a counter electrode (3705) is placed so that the bonding area is in close proximity to the working electrode. Light may be detected through one or both surfaces under conditions that result in the generation of light from ECL labels bound to the binding domains. A number of optical signals from those binding regions are measured simultaneously using an array of photodetectors 3706, e.g., a CCD array, an enhanced CCD array, or an avalanche photodiode array. The photodetector array images light generated from the binding region. Lenses, reflectors and/or optical waveguides may be used to enhance imaging. In other examples, light detected from a light detector segment or region (e.g., a light detection pixel) is associated with a binding region. Image analysis can be used to assist in the correlation of the detected light with the binding region. In a preferred embodiment, the surface is elastic or compliant, thus enabling intimate contact with the electrode surface. The binding region is linked to polymers that are capable of carrying an ion stream from the counter electrode to the working electrode. In a more preferred embodiment, the object is a water swellable polymer capable of carrying an ion stream from the counter electrode to the working electrode.

Fig. 38 shows a cartridge in which the binding regions (3805, 3806, 3807) are placed on the surface of different objects (3808, 3809, 3810), the objects (3808, 3809, 3810) being carried on the counter electrode (3800). The working electrode (3801) is placed in proximity to the surface of those objects. In the case of ECL, which results in label-labeled groups bound to the binding domains, light may be detected through one electrode or both electrodes (if each or both of the electrodes is transparent or translucent) and/or from the side. A number of optical signals from each binding region are measured simultaneously with an array of optical detectors (3802). Such objects may be elastic and/or compliant so as to be in intimate contact with the working electrode. The objects may be polymers capable of carrying an ion stream from the counter electrode to the working electrode. The objects may be water swellable polymers capable of carrying an ion stream from the counter electrode to the working electrode.

A transparent support containing one or more binding domains is brought into contact with a fibril pad electrode. The agent can be made to flow between the carrier/binding region and the fibril mat, or through the mat to the binding region. Light can pass from the binding area, through the transparent carrier to the detector.

In another preferred embodiment, the electrode is coated with a semi-transparent or transparent layer of carbon fibrils to increase the effective surface area of the electrode.

The PMAMS vectors and/or cassettes of the invention can advantageously be packaged into kits. The kit includes one or more PMAMS vectors prepared according to the invention for performing ECL reactions including assays, controls, and the like. Reagents may optionally be included in the kit, including control reagents, ECL assay and calibration reagents, and the like. A reagent mixture may be included that contains a plurality of binding reagents specific for a plurality of different analytes. 5.9 performing the plant reaction of ECL

In one embodiment, the PMAMS on the carrier and the cartridge containing the carrier are designed to be inserted into a device containing means for applying one or more test samples to the PMAMS binding region and initiating a plurality of ECL reactions. Such a device may be suitably modified in accordance with the present invention from conventional devices to perform a number of carrier or cartridge based ECL assays. The present invention provides various devices suitable for performing ECL measurements using each of the specific examples of PMAMS described in the sections above. Zoski et al disclose an apparatus for carrying out ECL reactions (U.S. Pat. No. 5, 5,061,445). The improvements needed include providing for the manipulation of the carrier and/or cartridge, the delivery of multiple samples, the accessing of multiple electrodes with a voltage waveform source, and the acquisition and processing of multiple ECL signals.

Fig. 6A shows some components of an illustrative device of the invention. Such a device 700 includes upper and lower carriers 702, 704 and an electrode guard 710. The upper support carries a number of electrode/counter electrode pairs (not shown). The lower carrier carries a binding region 706. The device enables removal of the electrode guard from the cassette and positioning of the electrode/counter electrode to contact the bound analyte on the binding area. The reagent or fluid flow space 708 is adjacent to the carrier carrying the binding domains. The above-described device can also send an identical or separately determined voltage wave to each of a number of electrode/counter electrode pairs simultaneously or sequentially to initiate an ECL reaction in the cartridge, and then measure the emitted ECL radiation with a photon detector, e.g., a photodetector device. The apparatus may also include temperature control means for maintaining the temperature of the support and/or cartridge, or the ambient temperature thereon, and adjusting the temperature as required to optimize ECL reaction conditions. The temperature control device is preferably a heating and cooling device, such as a resistive heating element, a cooling fan, a freezer, and any other suitable heating or cooling source. The temperature control means may also include a temperature sensor, for example a thermostat or thermocouple device, and means for activating or deactivating the heating or cooling means in response to a detected change in temperature.

The above-described apparatus also provides means for holding, moving and manipulating one or more carriers or cassettes to perform ECL reactions. The apparatus also includes stationary or movable sample delivery means, such as those described above for the cartridge, for placing the sample on the PMAMS binding area.

The apparatus also includes an electrode contact means which enables the array of individually accessible electrode connection means of the cartridge to be electrically connected to an electronic waveform signal generator means (e.g. potentiostat) (see, for example, figure 5 of US patent US 5,068,088). The waveform signal generator means sends signals sequentially or simultaneously to independently trigger multiple ECL reactions in the cartridge.

During ECL assays, the ion stream between the working electrode and the counter electrode may flow through a liquid capable of conducting ions (e.g., water containing ionic salts), through a membrane of such liquid, and/or through a solid matrix capable of conducting ions.

Thus, a device for measuring electrochemiluminescence in a sample may comprise a plurality of chambers for holding at least one sample, wherein a chamber may be formed by one or more electrodes and one or more counter electrodes and a first carrier comprising a plurality of discrete binding domains. The electrode and the counter electrode may be arranged on a surface of the first carrier or on a surface of a second carrier, wherein the second carrier is in close proximity to the binding area on the first carrier. The electrodes and counter electrodes may be present in pairs. The chamber may also include a plurality of sensing electrodes to sense voltages adjacent the working electrode. The cartridge may also include a chamber containing a reference electrode.

The device also includes a light detection means which can detect, for example, ECL reactions carried out in the cartridge by one or more detector means. By way of example only, such a detector device includes a fibre channel array aligned with and positioned adjacent to an electrode array, connected to an array of light detector devices, or connected to a single light detector device capable of scanning the array of emitted ECL signals.

The above devices may optionally include a digital computer or microprocessor to control the operation of the various components of the device.

The device also comprises a signal processing device. By way of example only, in one embodiment, the signal processing device comprises a digital computer for transmitting, recording, analyzing and/or displaying the results of each ECL measurement.

Optionally, the apparatus includes an electrode translation device to, for example, scan one or more electrode/counter electrode pairs across the binding surface to sequentially trigger ECL.

Filters of selected sizes can be used in parallel arrays of PMAMS. 5.10 feasible ECL assay

ECL markers for use in the present invention may be selected from those known in the art (see section 2.2 above and U.S. patent No. 5,310,687). ECL labels, for example, may include metal-containing organic compounds in which the metal is selected from ruthenium, osmium, rhenium, iridium, rhodium, platinum, palladium, molybdenum, technetium, and tungsten. Suitable linking chemistries for preparing ECL TAG reagents are well known and have been disclosed, for example, by Bard et al (U.S. patents US 5,310,687 and US 5,221,605). The method of binding the ECL label to the binding reagent may be covalent and/or non-covalent. ECL labels may be non-covalently bound to the binding agent (e.g., by hydrophobic effect or ionic interaction). In another example of non-covalent attachment, the ECL label is bound (covalently or non-covalently) to a complex, which in turn is non-covalently attached to a binding agent. A more specific example is Ru (bpy) ₃Covalently attached to the Ni (II) -tris-nitrilotriacetic acid complex by a linker. This molecule will be attached to binding agents that include peptide sequences containing a number of histidines. Some other receptor ligand pairs that can be used in a similar manner are known in the art (Sassenfeld, 1990, TIBTECH 8: 88-93). In addition, ECL labels containing a plurality of metal-organic compounds (e.g., containing Ru) that are structurally branched networks (e.g., networks through a hydrocarbon linker) can be used. Such branched networks containing a variety of organometallic moieties capable of ECL can be attached at one or more sites to a molecule to be labelled by ECL. In another embodiment, the ECL label comprising a plurality of metal organic compounds is a linear polymer, with the metal organic groups attached at a plurality of positions along the polymer chain (e.g., linear, branched, or cyclic polymers).

Various binding domains can be used in various other ECL assays well known in the art. In a quantitative assay, a known amount of ECL-labeled reagent is used and the measured amount of ECL is correlated with a known standard in order to calculate the amount of analyte present. Forward, reverse, competitive and sandwich assays can be performed using methods well known to those of ordinary skill in the art. For example, in a competitive assay, a method of quantitatively determining the amount of an analyte of interest in a multi-component liquid sample is performed as follows. Simultaneously contacting the binding surface with (a) a known amount of ECL-labeled ligand that competes with the analyte of interest when bound to a binding reagent present on the binding zone, and (b) a sample suspected of containing the analyte of interest; the contacting is effected under conditions such that the analyte of interest and the ligand competitively bind to the binding agent. In the presence of analyte in the sample, the amount of competing ECL-labeled ligand bound to the binding zone is reduced, thereby reducing (relative to the amount of ECL obtained in the absence of analyte in the sample). The ECL in the resulting binding domain is initiated and the amount of light emitted, and thus the amount of analyte of interest present in the sample, is quantitatively determined. Alternatively, the sample may be contacted with the binding surface prior to contacting the binding surface with ECL-labeled ligand; the ECL-labeled ligand then competes with and replaces some of the previously bound analyte from the sample on the PMAMS surface. In an alternative embodiment, the sample may be treated to contain ECL-labeled substances/molecules and a standard amount of unlabeled analyte of interest may be contacted with the binding surface prior to or simultaneously with contacting the binding surface with the sample to perform a competitive assay.

In sandwich assays, the ECL-labeled ligand is a binding partner that specifically binds to a second binding moiety on the analyte of interest. Thus, when there is analyte in the sample that specifically binds to the binding reagent in the binding region of the PMAMS, a "sandwich" is thus formed, which consists of the binding reagent on the binding region that binds to the analyte from the sample, and binds to the ECL-labelled binding partner. In another competitive sandwich assay, a replica of the analyte itself is attached to the binding domains of a multi-array binding surface prior to exposure to the sample. The sample is then contacted with the binding surface. An ECL-labeled binding partner that specifically binds to the analyte will bind to the analyte in the absence of free analyte (from the sample) in the assay solution, but will be competitively inhibited in the presence of free analyte (from the sample) in the assay solution.

In an alternative embodiment, the sequence marking is performed. For example, in one particular embodiment of a sandwich assay, an analyte bound to a binding zone is sequentially contacted with a plurality of ECL-labeled, analyte binding partners. ECL measurements and optional washing steps were performed between two contacts with each different binding pair. In this manner, ECL measurements of a plurality of different binding moieties of an analyte (e.g., CD 8) may be performed ⁺A, b T cell antigen receptor positive T cells). In addition, each of a plurality of ECL labels, each emitting at a different wavelength, can be linked to a different binding reagent that is specific for a different component on the analyte. Further, for example, a number of different reporter methods (e.g., ECL labels, fluorescent labels, and enzyme-linked labels) can be used, each of which is attached to a different binding reagent specific for a different binding moiety for the analyte, e.g., to distinguish CD4⁺A, b T cell antigen receptor-positive cells and CD8⁺A, b T cell antigen receptor-positive cells.

In some preferred embodiments, the binding region contains a labeled protein and/or nucleic acid and/or cell and/or chemical substance. Such labeled components (e.g., ECL labels) can be added to the binding region during fabrication, before the start of the assay, during the assay, and/or at the end of the assay. For example, multiple labeled components can be added at any time, and measurement records can be taken sequentially. Such measurement records may provide cumulative information. In another embodiment, the binding region of the PMAMS can be reused many times. After completion of a given assay, the surface is washed under conditions to restore the activity of one or more binding domains of the PMAMS surface. By way of example, certain binding reactions can be reversed by varying the ionic strength of the reaction solution. Alternatively, the bound complex may be decomposed thermally. Certain binding domains may themselves be self-renewing. Binding regions containing catalytic (e.g., enzymatic) functionality can be used more than once. The binding region may be used continuously and thus may be used for biosensor applications.

In addition, the assay can be formatted such that binding reagents attached to the multi-array multispecific, patterned surface are labeled with ECL. Upon binding to certain analytes of interest in the sample, ECL signal is quantitatively modulated. For example, ECL-labeled binding reagents attached to the surface may be specific for analytes on the cell surface, e.g., antigens such as alpha and beta T cell antigen receptor antigens or CD4 or CD8 antigens. Upon exposure to the mixture of cells, the cells bound to the surface, when the electrode surface is in proximity to the multi-array multispecific surface, can sterically hinder the ability of the electrode surface to excite ECL-labeled binding reagents, thereby attenuating ECL signal.

Homogeneous assays and heterogeneous assays can be performed. In a heterogeneous assay, unbound labeled reagent is separated from bound labeled reagent (e.g., by a washing step) before the bound or unbound labeled reagent is exposed to an electric potential. In a homogeneous assay, unbound labelled reagent and bound labelled reagent are exposed to a potential effect together. In homogeneous assays, the intensity or spectral characteristics of the signal emitted by the bound labeled reagent is greater than or less than the intensity of the signal emitted by the unbound labeled reagent. The presence or absence of each of the bound and unbound components can be determined by measuring the difference in intensity.

Once the required step of contacting the binding reagent with the analyte and its competitor and any competing partner for the analyte is complete, the ECL label is ensured to be in an environment that results in ECL. Suitable ECL assay media are known in the art. Such assay media advantageously include molecules of ECL that promote ECL labeling, including but not limited to oxalate, NADH, and most preferably tripropylamine. Such "promoter" molecules may be provided freely in solution, or may be provided by previous attachment to or may be provided by production (e.g., as a chemical reaction product) at: PMAMS, monolayers on said surfaces, binding regions, electrode surfaces, binding reagents and/or ECL labels, etc. If the medium surrounding the ECL labels bound to the binding domains obtained in the contacting step results in ECL, no modification of the medium is required. Alternatively, the media may be adjusted or replaced to provide the media that results in ECL. The electrode and counter electrode have been brought into proximity of, or contact with, the binding area, a voltage waveform is applied, and ECL is detected or measured.

In a preferred embodiment of the invention, the above step of contacting the binding reagent with the analyte or a competitor for the analyte and any competing partner for the analyte is carried out in the absence of an electrode and a counter electrode, that is, so that the sample does not contact the electrode or the counter electrode. These steps are followed by bringing the electrode and the counter electrode sufficiently close to the ECL label bound to the binding zone to initiate an ECL reaction.

The nucleic acid strands can be sequenced using vectors with PMAMS. For example, PMAMS with many binding regions are prepared using different oligonucleotide probes of known nucleotide sequences as binding reagents in different binding regions. That is, different binding regions will comprise different binding agents of known nucleotide sequences. The nucleotide strand or strands or fragments to be sequenced are then bound (hybridized) to the PMAMS binding region. The nucleic acids to be sequenced are labeled with ECL. Binding assays were performed on PMAMS and oligonucleotide strands were ordered with ECL signal distribution from discrete binding regions on PMAMS.

The above methods are based on the ability of short nucleotides to hybridize to their complementary or substantially complementary sequences in another nucleic acid molecule (see, e.g., Strezoska et al, 1991, Proc Natl. Acad. Sci. USA 88: 1089-. Conditions may be chosen such that the desired degree of sequence complementarity is necessary for successful hybridization. Hybridization of a DNA molecule of unknown sequence with a probe of predetermined sequence detects the presence of a complementary sequence in the DNA molecule. The method is preferably carried out such that the hybridization reaction is performed with oligonucleotide probes that bind to the binding region and the sample DNA in solution.

PMAMS can also be used to isolate, screen, and/or select for new molecules or complexes that function (e.g., bind or catalyze) as desired. PMAMS can be used to isolate and/or target compounds for therapeutic purposes. The methods of the invention can be used to prepare PMAMS containing a number of peptides, nucleic acids, viral vectors or polymers synthesized using a wide variety of combinatorial chemistries. A wide variety of vectors treated with PMAMS, for example, can be used to rapidly screen for binding to ECL-labeled cell receptors, for example. In one approach, a first PMAMS with highly divergent, unrelated peptide sequences was used to isolate the leader binding peptide sequence. PMAMS with peptides having a related sequence that correlates with a sequence that shows binding to a molecule of interest (e.g., a cellular receptor) on the first PMAMS are then used. This process is repeated until a peptide having the desired binding properties is found.

The analyte of interest may be, for example, whole cells, subcellular particles, viruses, prions, viroids, nucleic acids, proteins, antigens, lipoproteins, lipopolysaccharides, lipids, glycoproteins, carbohydrate moieties, cellulose derivatives, antibodies or fragments thereof, peptides, hormones, medicaments, cell or cell components, organic compounds, non-biological polymers, synthetic organic molecules, metallo-organic compounds or inorganic molecules present in the sample.

For example, the sample may be derived from a solid, emulsion, suspension, liquid or gas. Furthermore, the sample may be derived from, for example, body fluids or tissues, water, food, blood, serum, plasma, urine, feces, tissues, saliva, oil, organic solvents or air. The sample may include a reducing agent or an oxidizing agent.

With the present invention, assays for detecting or measuring the following substances can be performed by binding a binding reagent specific for these substances into the binding region of the binding surface of the present invention: albumin, alkaline phosphate, alt/SGPT, ammonia, liquefying enzyme, AST/SGOT, whole methionine, nitrogen used in blood, calcium, carbon dioxide, chloride, whole cholesterol, creatinine, GGT, glucose, HDL cholesterol, iron, LDH, magnesium, phosphorus, potassium, whole protein, sodium, triglyceride, uric acid, drugs of abuse, hormones, cardiovascular system regulators, tumor markers, infectious disease antigens, antigens inducing allergy, immune proteins, markers of cytokine anemia/metabolism, carbamoylbenzene , digoxin, gentamicin, lithium, phenobarbital, phenytoin, procainamide, quinidine, theophylline, tobramycin, valproic acid, vancomycin hydrochloride, amphetamine, barbiturates, benzodiazepines , cannabinoids, cocaine, LSD, methamphetamine, methaqualone, opiates, opium, calcium, carbon dioxide, chloride, total cholesterol, creatinine, GGT, glucose, HDL cholesterol, glucose, phenylindine, phroppxy-phene, ethanol, salicylate, paracetamol, estradiol, progesterone, testosterone, hcG/bhCG, hormone that stimulates lymph nodes, luteinizing hormone, prolactin, thyroid hormone such as that which stimulates thyroid hormone, T4, TUP, whole T3, free T4, free T3, cortisol, creatinine kinase-MB, whole creatinine kinase, PT, APTT/PTT, ISOs, creatinine kinase ISOs, myoglobin, light chain of muscle, troponin 1, troponin T, Chlamydia, gonorrhea virus, herpes virus, Lyme disease virus, Epstein Barr virus, IgE, rubella G virus, rubella M virus, CMV-G, CMV-M, toxin-G, toxin-M, HBsAg (hepatitis B virus surface antigen), HIV1, HIV2, anti-HBc, anti-HBs, anti-HCV, anti-HAV, anti-IgM, anti-HBc, HBeAg, anti-HBeAg, TB, prostate specific antigen, CEA, AFP, PAP, CA125, CA15-3, CA19-9, B2 microglobulin, hemoglobin, red blood cells, HBcAb, HTLV, ALT, STS syphilis, ABO blood group antigens and other blood group antigens, cytomegalovirus, ferritin, B-12, folate, glycated hemoglobin, amphetamine, antidepressants, and other psychotropic drugs.

ECL may be measured at several different binding domains, sequentially or simultaneously.

PMAMS specific for an analyte of interest that is a cell surface protein is first exposed to a sample containing cells in which it is desired to count the cells in the sample. In a preferred embodiment, a known sample volume and/or diluted sample is exposed to a PMAMS having a plurality of binding regions specific for at least one cell surface antigen. Bound cells are then quantified by attaching an auxiliary binding group linked to an ECL label. This is a group that interacts with a wide range of cell types, for example, an ECL label linked to a hydrophobic group that can insert into the cell membrane or to a lectin directed against the cell surface sugars. The ECL label is linked to an auxiliary antibody directed against the cell surface antibody. In a more specific example, several cell types that bind to the same region can be distinguished by using a plurality of ECL marker labeled helper antibodies. It is preferred to ensure that the number of discrete binding domains specific for a given analyte on the cell surface exceeds the average number of cells present in the sample that will bind. Statistical techniques can then be used to determine the number of cells per sample volume. Other particles such as viruses may also be counted using this technique, for example, where the binding agent recognizes an antigen on the virus. The regions may be small compared to the size of the cells so that only one cell can bind to each region, thus resulting in a digital signal for each region, which can then be statistically analyzed for the sum of those regions. The area may be large compared to the size of the cells, so that multiple cells can bind to one area. In this case, the signal from each region may be corrected to give the number of cells in a unit volume of sample. Image analysis using an array of photodetectors (e.g., a CCD camera or an array of avalanche photodiodes) can be employed to count cells and determine their morphology.

The present invention preferably also provides a method for performing ECL reactions, for example, a method that measures at a rate of 1000 ECL reactions in the range of 5 minutes to 15 minutes. 5.11 PMAMS for use with other analytical methods and/or ECL

The above-described techniques for ECL-based detection may be used, for example, as an area where catalysts and other chemical reactions may occur, along with other assay techniques. The discrete binding domains of the invention can be used in other assay techniques, for example, in clinical chemistry assays, such as electrolyte assays, clinical enzyme assays, blood protein assays, assays for glucose, urea, and creatinine, and the like. Other assay techniques that may be used in conjunction with ECL assays and/or alone with PMAMS of the present invention include chemiluminescent-based labels, fluorescence-based assays, enzyme-linked assay systems, electrochemical assays (see, e.g., Hickman, 1991, science, 252: 688-.

PMAMS carriers with droplets in which there are many different chemistries in the droplet array can be utilized. Each droplet may contain a different binding reagent and/or a different chemical assay (i.e., for the same assay reaction medium). For example, the droplets may be hydrophilic, on a hydrophilic surface-binding region surrounded by a hydrophobic surface region. The droplets are protected with a hydrophobic solution that covers the surface. The hydrophilic solution to be determined is deposited on the second PMAMS with a hydrophilic binding region surrounded by a hydrophobic region. The two surfaces are brought into close proximity so that the hydrophilic regions on the opposing surfaces are brought into contact and subjected to spectroscopic analysis to detect the reaction products of the chemical assay.

The fibril mat may be patterned so that there are a number of discrete hydrophobic and/or hydrophilic regions surrounded by hydrophilic and/or hydrophobic regions. The droplets of aqueous solution containing the binding agent may be located on a hydrophilic region and defined by a surrounding hydrophobic region. These droplets may contain, for example, fibrils, aggregates of fibrils, binding reagents, ECL reagents, reagents for assays, surfactants, PEGs, detergents, many of the biomolecules mentioned above as examples, and/or combinations thereof.

The hydrophobic solution covering the first PMAMS is controllably removed (e.g., evaporated, wicked) so that only a portion of the droplets on top are exposed to the environment. The hydrophilic solution to be assayed for photochemical reactions is then exposed to the PMAMS surface-mixing and analysis (e.g., spectroscopic analysis) of the hydrophilic microdroplets and the solution to be assayed is performed.

The PMAMS binding region can also be used as a prefilter or filter. For example, cell-specific PMAMS may be used in some cases as a filter for certain cell types alone, as well as in conjunction with a filter of screening size. The resulting analyte solution is then exposed to PMAMS specific for subcellular particulate matter (e.g., viruses). Particulate subcellular PMAMS and/or filter of screening size are used to generate small molecule (e.g., protein, small chemical entity) analyte solutions. By using a tandem PMAMS assay system, analyte solutions can be sequentially purified in order to reduce non-specific analyte interactions.

The opacity of the material used for the carrier, the electrodes and/or the bonding areas can be varied in order to obtain the desired properties. Such materials may be translucent, transparent, or substantially opaque, depending on the thickness, composition, and/or optical density of the material.

The opacity of the fibril mat increases with the thickness of the mat. Very thin pads are semi-transparent. The thicker pad is substantially opaque. In some examples, a pad having a thickness in the range of 0.01 μm to 0.5 μm is substantially translucent. In other examples, pads greater than 20 μm thick are substantially opaque. Pads with a thickness between 0.5 and 20 μm have a moderate opacity, which increases with increasing thickness of the fibril pad. The opacity of a pad of a particular thickness depends on the composition, density, derivatization, number of layers, type and amount of those materials dispersed in the pad, and/or combinations thereof. It also depends on the wavelength of the light used.

If a material is substantially translucent at a given thickness and substantially opaque at another thickness, light emitted from the material at one depth may pass out of the material while light emitted from another depth (e.g., deeper) may be substantially absorbed or scattered by the material. In one example, the variable opacity of the material allows the material to be used as an optical filter.

Light emitted from a certain depth in the fibril mat may substantially pass out of the mat and may be observed with a detector placed on or near the surface of the fibril mat. Light emanating from another depth may be substantially absorbed and/or scattered by the pad and not observed by a detector on or near the surface of the fibril pad described above. This property of the fibril mat (and/or optically similar material) can be used to distinguish between bound and unbound reagents in ECL assays.

Certain agents may diffuse (active or passive), be pulled (e.g., by suction filtration and/or capillary action), be wicked, or be pushed by pressure into the porous material to a sufficient depth so that light emitted by these agents is substantially or entirely absorbed or scattered by the pad. In one example, the fibril mat functions as a physical filter or optical filter through which some agents pass, some agents are entrained, and/or some agents are associated with a very thin layer at or near the surface of the mat. Agents that bind to the binding region or regions and/or substances that bind to the binding region or regions (which are located on the surface of the fibril mat or in a very thin layer near the surface of the mat on the PMAMS) are prevented from diffusing or being pulled into or through the mat. The agent and/or other solution is flowed or suspended over and/or distributed throughout the surface of the fibril mat so that the agent is only associated with a very thin layer on the surface of the mat. The wash reagent may be passed through the pad one or more times in one or more directions. The agent can be associated with the fibril mat, one or more binding domains, other or the same agent bound to one or more binding domains, entrained in the mat, passed through the mat, or a combination thereof.

The porous material used for the carrier and/or electrodes may have more than one layer, with the upper layer having bonded areas and the other layers in the pad having no bonded areas. In one example, the thickness of the fibril mat (shown schematically in fig. 29), the upper layer 2900, is sufficient to prevent light from passing through that light originating from the layers 2901, 2902 of the mat below that layer. Light originating from sources 2904, 2905 associated with this upper layer may be detected with detector 2906 located on or near the surface of the pad. Light originating from sources 2907, 2908, 2909 in lower layers 2901, 2902 may be absorbed or scattered by any or all of the layers and may not be detected with detectors 2906, 2910.

A pre-filtration step may be used to select a particular size, type, derivative of fibrils and/or aggregates of fibrils prior to making the mat. The filter medium used to filter the fibril suspension is a fibril mat of certain porosity or porosities.

Porous materials (e.g., fibril mats) may serve as supports for binding domains, electrodes for ECL or other electrochemical uses, filters to control reagent delivery, and/or filters that may transmit, absorb, and/or scatter light to various degrees. 5.12 electrochromic ECL display Panel

The invention also provides a method of producing isolated electrochemical pixels for flat panel displays. Lithography has been proposed for use with electrochemical and electrochemiluminescence based flat panel displays to produce pixels that have limited impact (i.e., limited cross-talk) on adjacent pixels when subjected to electronic access (see U.S. patent 5,189,549). A limitation of lithography for reducing such cross talk is that the electrolytic material must be able to change its conductivity when exposed to light. It is a feature of the present invention to reduce cross-talk between pixels without the need to use materials capable of photo-induced conductivity modulation, thereby enabling the use of a wide range of different solutions, gels or films.

In the sandwich structure, two electrode surfaces as active areas of the pixels are on two surfaces facing each other. The electrode surfaces are for example coated with a complementary electrochromic material. To reduce cross talk, a conductive electrolyte film is placed between the electrode surfaces, while there are non-conductive areas between different pairs of electrodes (i.e., between pixel elements). If the coated electrode surface is hydrophilic, the surface area surrounding the electrode is made hydrophobic (e.g., by stamping or deposition through a mask), and hydrophilic conductive droplets are placed on the electrode on the first surface (e.g., by a fluidic array), and then the second surface is aligned by a machine and brought into contact with the first surface, thereby aligning the electrodes. In this way, the droplets of electrolyte can be confined to an area within one pixel without conductive material between pixels. The electrode pairs of the pixels are in close proximity side-by-side on the same surface. If the coated electrode pair is hydrophilic, the area surrounding both electrodes is made hydrophilic, while a hydrophobic ring is made surrounding the hydrophilic electrode area (e.g., by embossing or deposition through a mask). The droplets described in the two examples above were stabilized with a hydrophobic solution. The viscosity of the solution can be increased to increase the stability of the array of small droplets. The hydrophilicity and hydrophobicity can be reversed. In other embodiments, the droplets may contain a solution that is polymerizable to improve the stability and/or conductivity of the membrane (e.g., a conductive polymer) between or over the electrode pair. Furthermore, structural characteristics can be exploited to limit cross-talk between pixels. For example, an elastomeric stamp (e.g., poly (dimethylsiloxane)) having ring-shaped embossed protrusion features that can wrap around pairs of electrode pixels on a surface side-by-side can be used to isolate the electrolyte, gel, or film between pixels. Alternatively, side-by-side pairs of electrode pixels may be placed on the surface in an electrically insulating structure similar to a well, and an electrolyte, gel or film placed in the well over the electrodes and covering or coating the entire surface to isolate and contain those electrolytic components of each pixel. 5.13 PMAMS for other chemical reactions

The PMAMS of the present invention can also be used to perform those chemical reactions that do not bind to ECL. For example, all of the techniques and non-ECL assays described in section 5.11 above may be employed.

There is provided a cartridge for detecting or measuring an analyte of interest in a sample, the cartridge comprising: (a) a first support having a plurality of discrete binding domains on a surface thereof, so as to form at least one binding surface, at least some of the discrete binding domains having a different binding specificity to other binding domains, each of said plurality of discrete binding domains being hydrophilic and surrounded by a hydrophobic region, and (b) a second support having a plurality of hydrophilic regions comprising a plurality of reaction media suitable for carrying out a chemical assay thereon, so as to form an assay surface, wherein said plurality of discrete binding domains and plurality of reaction media are capable of coming into contact, thereby bringing a sample to be analyzed present on each binding domain into contact with the reaction media to detect or measure an analyte of interest. Alternatively, the binding region may be hydrophobic and the second support has a plurality of hydrophobic regions containing the reaction medium.

The present invention also provides a method for detecting or measuring an analyte of interest in a sample, the method comprising: (a) placing a sample droplet containing the analyte to be detected or measured on a plurality of discrete binding domains on the surface of a support, wherein the plurality of discrete binding domains comprises at least one binding domain containing binding reagents that are the same as one another and that differ in specificity from those contained in other binding domains, each of said plurality of discrete binding domains being characterized as being hydrophobic or hydrophilic, provided that the region of the support surface surrounding each binding domain is (i) hydrophobic if the binding domain is hydrophilic and (ii) hydrophilic if the binding domain is hydrophobic so that the analyte or analytes of interest in said sample can bind to said binding domains, and (b) contacting the droplet on a first support with a second support surface having a plurality of discrete hydrophilic domains, the plurality of discrete hydrophilic regions comprise a reaction medium suitable for carrying out a chemical assay thereon, and (c) determining the presence of an analyte of interest bound to the binding regions.

There is also provided a method for detecting or measuring an analyte of interest in a sample, the method comprising (a) placing a droplet of the sample containing the analyte to be detected or measured on a plurality of discrete binding domains on a support surface, wherein the plurality of discrete binding domains comprises at least one binding domain containing reagents that are identical to each other and that differ in specificity from the binding reagents contained in the other binding domains, each of the discrete binding domains being characterized as hydrophobic or hydrophilic, provided that the area of the support surface surrounding each of the binding domains is (i) hydrophobic if the binding domain is hydrophilic and (ii) hydrophilic if the binding domain is hydrophobic, so as to allow one or more analytes of interest in the sample to bind to the binding domains, and (b) placing a droplet of the reaction medium on the droplet of the sample; and (c) determining the presence of an analyte of interest bound to the binding area.

In a particular example of this aspect of the invention, each of a number of binding domains has bound thereto a different enzyme having as substrate a sequential intermediate in a chemical reaction, the binding domains being located on the surface of the PMAMS such that the product of a given enzymatic reaction, which is a reactant of a subsequent enzyme, flows to the next enzyme in the reaction pathway. The invention also provides for the use of the above method for the monolithic immobilization of enzymes on self-assembled monolayers, e.g., for industrial applications.

For example, sheets with such immobilized enzymes on one or both sides may be stacked in order to obtain a high ratio of surface area to solution volume. Alternatively, the enzyme so immobilized may be attached to a porous material. Furthermore, the enzyme thus immobilized may be on the probe, on the stirring agent, on the wall of a tube or capillary, or on the wall of a vessel such as a incubator.

In an alternative aspect of the invention, non-ECL assays such as those described above may be performed on PMAMS analogues which differ from the PMAMS analogues described above in that they contain discrete regions for carrying out non-ECL reactions, which discrete regions do not necessarily already carry a binding agent and thus are not necessarily binding regions. Such PMAMS analogs have discrete regions for reaction and are prepared to inhibit the spread and/or diffusion of fluid applied to those discrete regions. In one embodiment, the regions are hydrophobic or hydrophilic relative to surrounding regions on the surface of the support to help confine the reaction medium and/or sample to the discrete regions. Spreading or diffusion can be inhibited by a method using a well, a method of depositing a reaction medium or sample on a felt pad or a porous material, a method of depositing and drying a reaction medium or sample on a gel, a film, or the like. The diameter or width of each of such discrete regions is less than 1mm, preferably in the range of 50nm to 1mm, most preferably in the range of 1 micron to 1 mm. The same or different reaction medium may be deposited on each of the discrete binding domains described above prior to application of the sample, or application of the sample may precede deposition of the reaction medium.

In a preferred case of non-ECL assays using PMAMS analogs, droplets of the reaction medium are placed on a plurality of discrete zones, preferably simultaneously delivered from an array of microfluidic conduits, and then, optionally, a more viscous solution (e.g., oil) is placed on top of the reaction medium, or, alternatively, between two discrete zones; a sample containing the analyte to be detected or measured is then applied to each zone, either discretely to each discrete zone, or in blocks exposing the entire surface of the PMAMS analogue containing those zones to the fluid sample. The exposure to the resulting reaction in those binding regions is allowed to proceed and the results observed by employing an indicator and detection system selected from those known in the art.

The invention is further illustrated in the following examples, which are not intended to limit the scope of the invention in any way. 6. EXAMPLE 6.1 preparation of MAB PMAMS surface by microscopic imprint

The exposed and affected photoresist substrate, 1 to 2 microns thick, is prepared in a square array pattern according to well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 (poly (dimethylsiloxane) available from Dow Corning) and the corresponding curing agent for SYLGARD184 was poured onto the base plate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. By exposure to a hydrophilic, OH-terminated alkanethiol, SH (CH) in an ethanol solution (1 to 10mM) ₂)₁₁-(OCH₂CH₂)₆OH, "inking" the resulting elastomeric stamp, contacting the resulting elastomeric stamp with an aligned gold surface using a machine in alignment with the pins, and removing the resulting elastomeric stamp. Then, using hydrophobic CH₃Is a terminal alkanethiol, SH (CH)₂)₁₀CH₃(1 to 10mM in ethanol) for several seconds (e.g., 2 seconds to 10 seconds) (Kumar et al, supra and Prime et al, science, 252: 1164-7). The resulting surface was then dried gently with a stream of nitrogen. Thereafter, the capillary array containing the hydrophilic solution is brought into aligned contact with the aligned surface of the capillary with the SH (CH) using a machine with pins₂)₁₁-(OCH₂CH₂)₆The OH region is aligned. Each capillary in the capillary array contains monoclonal antibodiesThe bodies (MABs), specific for the analyte of interest, can be covalently bound to the active OH groups on the hydrophilic region via amide bonds. 6.2 preparation of MAB and nucleic acid PMAMS surfaces by microscopic imprinting

The exposed and developed photoresist substrate, 1 to 2 microns thick, is prepared in a square array pattern according to well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding curing agent for SYLGARD184 is poured onto the substrate and cured. The polymerized SYLGARD184 was carefully removed from the silicon substrate. By exposure to a hydrophilic, OH-terminated alkanethiol, SH (CH) in an ethanol solution (1 to 10mM) ₂)₁₁-(OCH₂CH₂)₆OH, "inking" the resulting elastomeric stamp, contacting the resulting elastomeric stamp with an aligned gold surface using a machine in alignment with the pins, and removing the resulting elastomeric stamp. Then, using hydrophobic CH₃Is a terminal alkanethiol, SH (CH)₂)₁₀CH₃(1 to 10mM in ethanol) for several seconds (e.g., 2 seconds to 10 seconds) (Kumar et al, supra and Prime et al, science, 252: 1164-7). The resulting surface was then dried slowly with a stream of nitrogen. Thereafter, the capillary array containing the hydrophilic solution is brought into aligned contact with the aligned surface of the capillary with the SH (CH) using a machine with pins₂)₁₁-(OCH₂CH₂)₆The OH region is aligned. Each capillary in the capillary array contains an antibody or modified nucleic acid, specific for the analyte of interest, that can be covalently bound to a reactive OH group on the hydrophilic region via an amide bond. 6.3 preparation of PMAMS surface by etching

Exposing the cleaned gold surface to a hydrophilic, OH-terminated alkanethiol, SH (CH) in an ethanol solution (1-10mM)₂)₁₁-(OCH₂CH₂)₆OH (Prime et al, science 252: 1164-. Optically aligning a linear array of fine-tipped etching implements with a machine to contact an aligned gold surface and aligning the X and X of said surface with the linear array Etching in Y direction to generate SH (CH)₂)₁₁-(OCH₂CH₂)₆A two-dimensional grid array of OH regions. Then, using hydrophobic SH (CH)₂)₁₁-(OCH₂CH₂)₆CH₃The solution (1 to 10mM in ethanol) washes the negative for a few seconds (e.g., 2 to 10 seconds). The resulting surface was then dried slowly with a stream of nitrogen. Thereafter, the capillary array containing the hydrophilic solution was brought into aligned contact with the above-mentioned surface pins using a machine, said surface bringing the capillaries into contact with SH (CH)₂)₁₁-(OCH₂CH₂)₆The OH region is aligned to the contact. Each capillary in the capillary array contains an antibody or nucleic acid, specific for the analyte of interest, that can covalently bind to a reactive OH group on the hydrophilic region. 6.4 Sandwich assay on PMAMS surface

Transparent PMAMS surfaces were prepared as described above, which were substantially transparent and had patterned multispecific arrays of primary antibodies (primary antibodies) attached to the surface. A transparent carrier, an electrode array and a single-layer surface are selected. The PMAMS surface is then exposed to a sample of solution suspected of containing the analyte of interest to be determined. The sample is then washed away, leaving the antibody-bound analyte on the surface. The PMAMS surface is then exposed to a solution containing secondary ECL-labeled antibodies specific for the analyte bound to the surface. This solution was then washed off the PMAMS surface, leaving the ECL-labeled secondary antibody bound to the area where the analyte was present.

The electrode is protected against detection by a removable barrier to prevent premature contact of the sample with the electrode surface and avoid contamination effects. The barrier was then removed and the PMAMS surface was brought into aligned contact with the electrode array wetted with assay buffer. The electrode array is connected to an electronic waveform signal generator and an electrical potential is applied to the working electrode/counter electrode pair. The emitted light is then interpreted by the CCD and the signal is sent to a microprocessor which converts the signal into the desired measurement output form.

The measurement output is compared to a measurement output obtained against a known amount of the analyte of interest to calculate the actual amount of analyte. 6.5 measurement on the surface of the first and second PMAMS

Transparent PMAMS surfaces were prepared as described above, with patterned multispecific primary antibody arrays attached to the surface. The PMAMS surface is then exposed to a sample of solution suspected of containing the analyte of interest to be determined. The sample is then washed away, leaving the antibody-bound analyte on the surface.

The second PMAMS under the protective cover was configured with an alternating hydrophobic/hydrophilic pattern with a patterned number of microdroplets of secondary antibodies labeled with ECL label on the pattern.

The barrier protecting the second PMAMS aligned with the first PMAMS was removed and the microdroplets were aligned with the primary antibody binding region on the first PMAMS. The second PMAMS was lifted and the electrode array was brought into surface aligned contact with the first PMAMS. The electrode array is connected to a potential waveform signal generator and an electrical potential is applied to the working/counter electrode pair. The photomultiplier tube then interprets the emitted light and the signal is sent to a microprocessor which converts the signal into the desired measurement output form.

The measurement output is compared to a measurement output obtained against a known amount of the analyte of interest to calculate the actual amount of analyte. 6.6 nucleic acid determination on the surface of PMAMS

A transparent PMAMS surface was fabricated as described above, with a patterned multispecific array of single-stranded nucleic acid probes attached to the surface. The probes are complementary to the 5' region of the nucleic acid analyte of interest. The PMAMS surface is then exposed to a sample of solution suspected of containing the hybridisable nucleic acid analyte of interest to be determined, which has been previously denatured, i.e. treated, to render the analyte of interest single stranded. The sample is then washed away, leaving the hybridized analyte on the surface. Thereafter, the PMAMS surface is exposed to a solution containing secondary ECL-labeled nucleic acid probes specific for the 3' end of the nucleic acid analyte bound to the surface. This solution is then washed off the PMAMS surface, leaving ECL-labeled nucleic acid probes bound to the areas where the analyte is present.

The barrier protecting the second PMAMS aligned with the first PMAMS was removed and the microdroplets were aligned to the primary antibody binding region on the first PMAMS. The second PMAMS was lifted and the electrode array was brought into aligned contact with the surface of the first PMAMS.

The electrode array is connected to a potential waveform signal generator and an electrical potential is applied to the working/counter electrode pair. The emitted light is then interpreted by the CCD and the signal is sent to a microprocessor which converts the signal into the desired measurement output form.

The measurement output is compared to a measurement output obtained against a known amount of the analyte of interest to calculate the actual amount of analyte. 6.7 competitive assay on PMAMS surfaces with photomultiplier Detector

Transparent PMAMS surfaces were prepared as described above, with patterned multispecific arrays of primary antibodies specific for the analyte of interest attached to the surface. The PMAMS surface is then exposed to a solution sample to be assayed, which is a mixture of the sample suspected of containing the analyte of interest and a known amount of ECL-labeled molecules that compete with the analyte of interest for binding to the antibody. Thereafter, the sample is washed away, leaving the antibody-bound analyte and/or labeled competitive binding substance on the surface.

The electrode array is protected with a removable barrier to prevent the sample from contacting the electrode surface and from contaminating effects. The barrier was then removed and the electrode array wetted with assay buffer was brought into contact alignment with the PMAMS surface. The electrode array is connected to a potential waveform signal generator and an electrical potential is applied to the working/counter electrode pair. The emitted light is then interpreted by a photomultiplier tube and the signal is sent to a microprocessor which converts the signal into the desired measurement output form.

The measurement output is compared to a measurement output obtained against a known amount of the analyte of interest to calculate the actual amount of analyte. 6.8 competitive assay on PMAMS surfaces with CCD Detector

A transparent PMAMS surface was prepared as described above, with a patterned multispecific array of primary antibodies attached to the surface. The PMAMS surface is then exposed to a sample of solution suspected of containing the analyte of interest to be determined. The sample is then washed away, leaving the antibody-bound analyte on the surface.

The second PMAMS under the protective cover is configured with an alternating hydrophobic/hydrophilic pattern on which are patterned microdroplets of a number of known amounts of ECL-labeled molecules that compete with the analyte of interest.

The barrier protecting the second PMAMS aligned with the first PMAMS was removed and the microdroplets were aligned to the primary antibody binding region on the first PMAMS. The second PMAMS was lifted and the electrode array was aligned to contact the PMAMS surface. The electrode array is connected to a potential waveform signal generator and an electrical potential is applied to the working/counter electrode pair. The emitted light is then interpreted by a CCD and the signal is sent to a microprocessor which converts the signal into the desired measurement output form.

The measurement output is compared to a measurement output obtained against a known amount of the analyte of interest to calculate the actual amount of analyte. 6.9 by using SH (CH)₂)₁₀CH₃Preparation of MAB PMAMS by microscopic imprinting of alkanethiols

Surface of

The exposed and developed photoresist substrate, 1 to 2 microns thick, is prepared in a square array pattern according to well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 (poly (dimethylsilane) available from Dow Corning) and the corresponding SYLGARD184 curing agent was poured onto the base plate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. By exposure to a hydrophilic, OH-terminated alkanethiol, SH (CH) in an ethanol solution (1 to 10mM) ₂)₁₁OH, "inking" the resulting elastomeric stamp, bringing the resulting elastomeric stamp into register contact with the aligned gold surface with a pin using a machine, and removing the resulting elastomeric stamp. Then, using hydrophobic CH₃Is a terminal alkanethiol, SH (CH)₂)₁₀CH₃(1 to 10mM in ethanol) for a few seconds (e.g., 2 seconds to 10 seconds) (Kumar et al, supra). The resulting surface was then dried slowly with a stream of nitrogen. Thereafter, the capillary array containing the hydrophilic solution is brought into aligned contact with the aligned surface of the capillary with the SH (CH) using a machine with pins₂)₁₁The OH regions are aligned so as to place a specific antibody at each region. Each capillary in the capillary array contains monoclonal antibodies specific for the analyte of interest that can covalently bind to the reactive OH groups on the hydrophilic regions. 6.10 by using SH (CH)₂)₁₀CH₃Preparation of MAB and nucleic acid by microscopic imprinting with alkanethiol

PMAMS surface

The exposed and developed photoresist substrate, 1 to 2 microns thick, is prepared in a square array pattern according to well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding curing agent for SYLGARD184 is poured onto the silicon substrate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. By exposure to a hydrophilic, OH-terminated alkanethiol, SH (CH) in an ethanol solution (1 to 10mM) ₂)₁₁OH, inking the resulting elastomeric stamp", the resulting elastomeric stamp was pinned in registration with an aligned gold surface using a machine and the resulting elastomeric stamp was removed. Then, using hydrophobic CH₃Is terminal alkanethiol, SH (CH)₂)₁₀CH₃A solution of (1 to 10mM in ethanol) washes the negative for several seconds (e.g., 2 seconds to 10 seconds) (Kumar et al, supra). The resulting surface was then dried slowly with a stream of nitrogen. Thereafter, an array of capillaries containing a hydrophilic solution is brought into aligned contact with the aligned surface of the capillary with some SH (CH) using a machine with pins₂)₁₁The OH regions are aligned so that some specific antibodies and/or hybridizable nucleic acids are placed on each region. Each capillary in the capillary array contains monoclonal antibodies or modified nucleic acids specific for the analyte of interest that are covalently bound to the active OH groups on the hydrophilic regions by amide bonds. 6.11 preparation of PMAMS surfaces with streptavidin-biotin linkers

The exposed and developed photoresist substrate of 1 mm to 2 mm thickness is prepared in a square array pattern according to a well-known method. A10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding curing agent for SYLGARD184 is poured onto the silicon substrate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. The resulting elastogram was "inked" by exposure to a mixture of mercaptoundecanol and 12-mercapto (8-biotinimido-3, 6-dioxaoctyl) dodecanamide, where the molar fraction of biotinylated thiol was 0.1 (see Spinke et al, 1993, Langmuir, 9: 1821-5, and Spinke et al, 1993, j. chem. phys.99 (9): 7012-9). Then, using hydrophobic CH ₃Is a terminal alkanethiol, HS (CH)₂)₁₀CH₃Solutions of alkanethiols (1 to 10mM in ethanol) clean the negative for a few seconds (e.g., 2 seconds to 10 seconds) (see Kumar et al, supra, biebauck, Whitesides). The resulting surface was then dried slowly with a stream of nitrogen. The machine is then used to make the hair containing the streptavidin solution in each capillaryThe capillary array is aligned with the aligned surface using pins. Each capillary in the capillary array is aligned and contacted to the biotinylated region and the capillary array is removed, washing the surface. A second capillary array containing a plurality of biotinylated antibodies and biotinylated nucleic acid solutions is then mechanically brought into aligned contact with the aligned surfaces using pins to place some specific antibodies and nucleic acids on each area. 6.12 preparation of MAB Individual surfaces

An electrode array of interleaved working and counter electrode pairs on gold on a silicon surface is fabricated by methods known in the art (see, e.g., Kumar et al, supra). In this example, the array of electrodes and the array of discrete binding regions are present on the same surface of the carrier. The exposed and developed photoresist substrate, 1 to 2 microns thick, is prepared in a square array pattern according to well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 (poly (dimethylsiloxane (PDMS)) available from Dow Corning) and the corresponding curing agent for SYLGARD184 was poured onto a silicon substrate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. By exposure to a hydrophilic, OH-terminated alkanethiol, SH (CH) in an ethanol solution (1 to 10mM) ₂)₁₁-(OCH₂CH₂)₆OH, "inking" the resulting elastic stamp, bringing the resulting elastic stamp into aligned contact with aligned working electrodes on the surface of the gold electrode array with pins using a machine, and then removing the resulting elastic stamp. Then, by contacting the capillary with SH (CH) on the surface of the electrode array₂)₁₁-(OCH₂CH₂)₆The OH regions are aligned and the capillary array containing the hydrophilic solution is brought into aligned contact with the pins by a machine to place some specific antibody on each region. Each capillary in the capillary array contains monoclonal antibodies specific for the analyte of interest that can be covalently bound to the active OH groups on the hydrophilic regions by amide bonds. 6.13 measurements on MAB-alone surfaces

The vector described in section 6.12 above was made. As previously described, PDMS stamps were made from a photoresist substrate in a circular pattern, each of these rings independently defining the extent of one working/counter electrode pair. Thereafter, the electrode array surface is exposed to the sample to be analyzed, washed with a mixture of ECL-labeled secondary antibodies, and then washed with an assay buffer solution containing tripropylamine. The PDMS stamps were then aligned and brought into alignment contact, aligning the rings of the PDMS stamps, to define and define separate volumes of assay buffer over each electrode pair. An overpotential is applied to the pair of electrodes to release the monolayer from the surface, exposing the working electrode to ECL-labeled secondary antibody. The photomultiplier tube then interprets the light emitted through the transparent PDMS and the signal is sent to a microprocessor which converts the signal into the desired measurement output form.

The measurement output is compared to a measurement output obtained against a known amount of the analyte of interest to calculate the actual amount of analyte. 6.14 preparation of separate surfaces with working and counter electrodes

An electrode array of interdigitated working gold and gold counter-electrode pairs with gold binding regions between two interdigitated electrodes on gold of a silicon carrier is fabricated by methods known in the art (see, e.g., Kumar et al, supra). In this example, the array of electrodes and the array of discrete binding regions are present on the same surface. A 1-2 micron thick photoresist cliche that has been exposed and developed is patterned into bonding areas between interdigitated electrode pairs in accordance with well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 (poly (dimethylsiloxane (PDMS)) available from Dow Corning) and the corresponding SYLGARD184 curing agent was poured onto a silicon substrate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. By exposure to a hydrophilic, OH-terminated alkanethiol, SH (CH) in an ethanol solution (1 to 10mM) ₂)₁₁-(OCH₂CH₂)₆OH, "inking" the resulting elastomeric stamp, bringing the resulting elastomeric stamp into aligned contact with aligned gold bonding areas on the surface of the electrode array with pins using a machine, and thereafter removing the resulting elastomeric stamp. Then, the capillary array containing the hydrophilic solution was brought into aligned contact with pins by a machine to bring the capillaries into contact with SH (CH) on the surface of the electrode array₂)₁₁-(OCH₂CH₂)₆The OH regions are aligned so that some specific antibody is placed on each region. Each capillary in the capillary array contains monoclonal antibodies specific for the analyte of interest that can be covalently bound to the active OH groups on the hydrophilic regions by amide bonds. 6.15 measurements on separate surfaces with working and counter electrodes

The carrier surface described in section 6.14 above was made using the method described above. The prepared surface is exposed to the sample to be analyzed, washed with a mixture of ECL-labeled secondary antibodies, and then washed with an assay buffer solution containing tripropylamine. The electrode array is connected to a potential waveform signal generator and an electrical potential is applied to the working/counter electrode pair. The photomultiplier tube then interprets the emitted light and the signal is sent to a microprocessor which converts the signal into the desired measurement output form.

The measurement output is compared to a measurement output obtained against a known amount of the analyte of interest to calculate the actual amount of analyte. 6.16 preparation of the surface with counter electrode

The exposed and developed photoresist substrate, 1 to 2 microns thick, is prepared in a square array pattern according to well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding curing agent for SYLGARD184 is poured onto the silicon substrate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. By exposure to a hydrophilic, OH-terminated alkanethiol, SH (CH) in an ethanol solution (1 to 10mM)₂)₁₁-(OCH₂CH₂)₆OH, "inking" the resulting elastomeric stamp, contacting the resulting elastomeric stamp with a machine in registration with aligned patterned counter electrodes and square bonding areas on the gold surface with pins, and removing the resulting elastomeric stamp. The patterned gold surface is formed of accessible annular counter electrodes defining a pattern of stamped SH (CH)₂)₁₁-(OCH₂CH₂)₆Range of some binding regions for OH. There is a gap or separate space between each gold counter electrode and each square gold backing plate for each single layer bonding area. Thereafter, an array of capillaries containing a hydrophilic solution is brought into aligned contact with the aligned surface of the capillary with some SH (CH) using a machine with pins ₂)₁₁The OH regions are aligned so that some specific antibodies and nucleic acids are placed on each region. Each capillary in the capillary array contains antibodies or nucleic acids specific for the analyte of interest that can covalently bind to the reactive OH groups on the hydrophilic regions. 6.17 measurements on separate surfaces with working and counter electrodes on different surfaces

The carrier surface described in the example of section 6.16 above is exposed to the sample solution to be analyzed. The support surface is then washed and exposed to a solution containing a number of ECL-labelled monoclonal antibodies or ECL-labelled nucleic acids of different specificities, followed by washing with assay buffer containing tripropylamine. A transparent, accessible array of working electrodes is fabricated such that each working electrode in the array corresponds to a discrete binding/counter electrode area on the support as described above in section 6.16. Both supports were wetted with assay buffer and brought into conforming contact with the alignment shape by machine. The electrode array is connected to a potential waveform signal generator and potentials are applied to aligned working/counter electrode pairs which generate a potential field between the two supports. The CCD then interprets the light emitted through the transparent working electrode and the signal is sent to a microprocessor which converts the signal into the desired measurement output form.

The measurement output is compared to a measurement output obtained against a known amount of the analyte of interest to calculate the actual amount of analyte. 6.18 preparation of CC (dispersed) fibril Mat by vacuum filtration

An aqueous slurry of CC fibrils was prepared of 1 mg fibrils per ml of solution by mixing 0.1% w/w CC fibrils/deionized water. CC fibrils (larger, micron-scale aggregates dispersed into small aggregates or individual fibers) were dispersed in the slurry by immersing a 400 watt Sonication apparatus (Sonication horn) in the slurry for between 10 minutes and 1 hour. The extent of dispersion was monitored with an optical microscope.

A nylon filter membrane (pore size 0.47 μm, diameter 25mm) was placed on a sintered glass filter with a diameter of 25 mm. The dispersed fibril slurry was filtered by using a suction filtration membrane/filtrate device (fig. 23A). An aliquot of the slurry (5ml) was diluted with 20ml of deionized water, after which it was filtered through the membrane/filter device described above. For a typical pad of about 0.25 to 0.33g/cc, 6 aliquots are required for a pad of about 100 μm.

Suction filtration was continued until all water from the dispersion was removed from the pad (by visual inspection). The pad was peeled (by hand) directly from the filter membrane.

The mat is dried in an oven at 60 c for approximately 10 to 15 minutes. The mat is cut, punched or otherwise cut for use. 6.19 preparation of fibril mats by evaporation on Metal mesh supports

An aqueous slurry of CC fibrils was prepared of 1 mg fibrils per ml of solution by mixing 0.1% w/w CC fibrils/deionized water. The CC fibrils were dispersed (larger, micron-scale aggregates were dispersed into small aggregates or individual fibers) by immersing a 400 watt sonication apparatus in the slurry for between 10 minutes and 1 hour. The extent of dispersion was monitored with an optical microscope.

A block of 1cm²The stainless steel mesh (400 pieces) was placed on a filter paper having a diameter of 25 mm. A 5ml aliquot of the slurry was pipetted onto the surface of the entire screen/filter paper. The water in the slurry is evaporated, either at room temperature and pressure or in a furnace.

Once the fibrils were dried, additional aliquots were added. The fibrils and screen were peeled off the filter paper as a single unit.

The pad is cut, perforated or otherwise cut for use. 6.20 immobilization of Biotin on fibrils carrying NHS-ester functionality

The COOH-derived fibrils (supplied by Hyperion Catalysts) were suspended in-10 mg/ml anhydrous dioxane with constant agitation. More than 20 times by mole of N-hydroxysuccinimide was added and dissolved. Next, more than 20 times the mole of ethyl-diamino-propyl-carbodiimide (EDAC) was added and the mixture was stirred at room temperature for 2 hours.

After agitation, the supernatant was aspirated, and the solid was washed three times with anhydrous dioxane, once with anhydrous methanol, and filtered on a 0.45 μm polysulfone membrane. The filtrate was washed with additional methanol and placed in a glass vial under vacuum until no further weight loss was observed.

10.4mg of NHS-ester fibrils (ORIGEN reagent 402-01, pH7.8, IGEN Co.) were washed with PBS-1 (. about.70 mM phosphate, 150mM NaCl). The washed fibrils were suspended in 2.3ml of avidin solution (8.3mg avidin per ml PBS-1).

The suspension was allowed to stand at room temperature for 1.5 hours, and the flask was continuously rotated for agitation.

After 1.5 hours, the suspension was stored at 4 ℃ for 16 hours, then the suspension was left at room temperature and washed with PBS-1 and the suspension was stored as a suspension in PBS-1. 6.21 immobilization of monoclonal antibody (anti-AFP) on carbon fibrils

Carbon fibrils functionalized with NHS esters were prepared as described in the example in section 6.20.

14mg fibril-NHS ester was mixed with 500ml PBS-1 buffer. The mixture was sonicated for 20 minutes until it became a viscous slurry. An additional 500ml PBS-1 buffer was added.

anti-AFP (. alpha. -fetoprotein) antibody in 80ml of PBS-1 was added to the above slurry. The reaction was allowed to proceed at room temperature for 2.5 hours.

6ml of PBS-1 buffer was added and the reaction mixture was centrifuged at 4 ℃ for 5 minutes. Remove the supernatant with a pipette. This step was repeated 9 times.

After the final wash, the supernatant was removed and the fibril-anti-AFP product was stored at 4 ℃. 6.22 periodic voltammetric recording of fibril mat: comparison of fibril mats with gold foil electrodes

Measuring K at 0.5M₂SO₄6mM Fe in (1)^3+/2+(CN)₆Periodic voltammetric recordings of (a). In FIG. 30A, pad CVs of plain fibers of CC (dispersed) were measured at 0.10mA/cm at 10, 25 and 50 mV/sec. The pad is made as described in the example of section 6.18. In FIG. 30B, the CV is measured at 0.05mA/cm, at 10, 25 and 50 mV/sec for gold foil electrodes. All potentials are in volts for Ag/AgCl. 6.23 electrochemical properties of fibril mat electrodes: comparison of anodic peak current with thickness of the pad

For the same geometric area (0.20 cm)²) But different thickness of fibril mat, measured at 0.5M K₂SO₄6mM Fe in (1)^3+/2+(CN)₆Periodic voltammetric recordings of. For thicknesses in the range of 24 μm to 42.5 μm, the anodic peak current (fig. 31) increased as the thickness of the pad increased. The anodic peak current also increases with increasing scan rate (e.g., rate in the range of 10 mV/sec to 150 mV/sec) for each thickness. The rate of increase of the anodic peak current also increases with increasing thickness as a function of thickness. A fibril mat with a thickness of 24 μm worked comparable to a gold foil electrode. 6.24 non-protein and fibril Specific binding

Nonspecific binding of protein to carbon fibrils (CC) was measured as follows: i) make Ru (bipy)₃ ²⁺("TAG 1") the labeled protein solution was exposed to a known amount of carbon fibrils until equilibrium was reached; ii) centrifuging the labelled protein/fibril solution and collecting the supernatant, and iii) measuring the amount of labelled protein remaining in the supernatant using Electrochemiluminescence (ECL).

To generate the curve shown in figure 32, 3 μ g/ml of anti-CEA antibody attached to derivatized TAG1 (antibody to carcinoembryonic antigen attached to one derivatized TAG1 ECL label) was added to serial dilutions of CC (flat) fibrils in 0.1M potassium phosphate buffer at pH 7. After 20 minutes of rotation, the fibrils were removed by centrifugation. In the analyzer of ORIGEN1.5 (IGEN), an aliquot of the reaction mixture supernatant diluted 5-fold with ORIGEN assay buffer was subjected to ECL assay and the amount of protein (unbound) remaining in the supernatant was determined. When higher concentrations of carbon fibrils are present, increased binding of the protein labeled with derivatized TAG1 results in a decrease in ECL signal (relative to the ECL signal of the subject of the reaction mixture not exposed to fibrils). 6.25 reduction of nonspecific binding of proteins to fibrils with detergent/surfactant

The effect of the supernatant on fibril-bound protein was analyzed as described in the example of section 6.24. Triton X-100 was added to the anti-CEA attached to the derivatized TAG 1/fibril mixture, the solution was incubated for 20 minutes, the tube was centrifuged, and an aliquot of the supernatant diluted 5-fold with ORIGEN assay buffer was analyzed with ECL. The results are shown in the following table and in fig. 33.

Test tube number	[T-X100]，ppm	Peak intensity	protein-TAG 1. mu.g/ml	[GF]，ppm
					1918171615141312	1674837418209105522613	16111634169715831772146362723	2.652.652.652.652.652.652.652.65	5252525252525252

The curve obtained by plotting the ECL intensity of the protein labeled with derivatized TAG1 in solution against the concentration of triton X-100 is shown in fig. 33. A stronger ECL signal corresponds to more derivatized-TAG 1 labeled protein in the supernatant, which in turn corresponds to less derivatized-TAG 1 labeled, fibril bound protein. Concentrations of triton X-100 ranging from 10ppm to 100ppm attenuated the extent of binding, increasing the concentration from 100ppm to 200ppm did not further attenuate the extent of binding. 6.26 ECL of free TAG in solution with fibril pad electrode

The fibril mat prepared as in the example of section 6.18 was placed in the mounting area 3403 of the electrode clamp 3401 of the working electrode of the "fibrill cell" clamp shown in fig. 34. The electrode clamp 3401 is inserted into the bottom of the electrochemical cell chamber 3400. A3M Ag/AgCl reference electrode (Cyprus # EE008) was loaded into the cell chamber through the aperture 3402 of the reference cell. The elements were filled with assay buffer (IGEN #402-005-01 batch #5298) and attached to PMT clamp 3404. The potential was swept from 0 volts to 3 volts at 100 mV/sec for Ag/AgCl using an EG & G PARC 175 model Universal programmer and an EG & G175 model potentiostat/galvanostat. ECL was measured using a Hamamatsu R5600U-01, which was powered at 900V using a Pacfic Instruments model 126 photometer. Analog data was digitized by a CIO-DAS-1601A/D board driven by a HEM Snap-Master at 10 Hz. The fibre Cell was drained and rinsed with 1000pM of TAG1(IGEN # 402-. The potential is swept as with assay buffer. The ECL trace (measured at 24.0 ± 0.2 ℃) for assay buffer 3501 and 1000pM TAG13502 is shown in figure 35. The ECL peak area corrected for dark areas was 22.10nAs for assay buffer and 46.40nAs for 1000pM of TAG 1. 6.27 ECL of labeled antibody adsorbed with fibril pad electrode

A fibril mat having a thickness of 0.0035 inches was made from flat CC dispersed fibrils in the manner described in the example of section 6.18. The dried pad was then punched into a 3mm disk and mounted onto a carrier. The carrier used in this experiment was made from a 0.030 inch polyester plate that was patterned by screen printed conductive gold ink. The conductive gold ink forms the counter electrode, the reference electrode, and provides leads for the working electrode and other electrodes. Two discs of fibril mats were mounted on each patterned support using conductive tape (Aahesives Research) containing double-sided carbon. After mounting, those disks were spotted with 0.5. mu.l of 10. mu.g/ml anti-TSH antibody (Ru-TSH mono 1: 226 JUN95, IGEN Co.) attached to derivatized TAG1 in deionized water or 0.5. mu.l of capture antibody in deionized water, 10. mu.g/ml anti-TSH not labeled with TAG1, and allowed to dry. After drying, the pads were filled with IGEN assay buffer. Those pads filled on the support were placed on an IGEN origin 1.5 based instrument and the ECL was interpreted with a scan rate of 500 mV/sec from 0mV to 4500 mV. FIG. 43 compares the signals from a mat 4301 containing TAG 1-antibody and from a mat 4302 containing capture antibody not labeled with TAG 1. 6.28 ECL with fibril pad electrode for sandwich assay

anti-AFP capture antibodies were immobilized on the fibrils as described above. anti-AFP fibrils were washed into deionized water (dI) and resuspended to a density of 1 mg/ml. A four-layer fibril mat was produced using vacuum filtration as described in example 6.18. 2mg of anti-AFP fibrils was added to 3mg of smooth, CC-dispersed fibrils and the mixture was diluted to a total volume of 20ml in deionized water. The diluted mixture was filtered over a 0.45 μm nylon filter. After this initial mat layer there were two core layers each consisting of 5mg of smooth, CC dispersed fibrils. The core was then covered with a layer of mixed fibrils equivalent to the initial layer described above. This results in a fibril mat with about 40% of the anti-AFP fibrils on the upper and lower surfaces and about 100% of the smooth fibrils in the core. The mixed pad was allowed to air dry in vacuum and the mixed pad was punched into 3mm circular discs. The discs are then loaded onto a carrier as described in example 6.27. The dry, supported, AFP-resistant pad was filled with AFP calibrant A, C and F (IGEN corporation) and incubated at room temperature for 15 minutes on top of the bench. After incubation, the supported electrodes were rinsed with a stream of deionized water for 10 seconds, after which they were blotted dry with a lint-free swab. The fibril pad was then filled with anti-AFP antibody attached to an antibody labeled with derivatized TAG1(IGEN corporation) and incubated at room temperature on top of the test stand for 15 minutes. After incubation, the supported electrode was rinsed with deionized water and dried with a swab. Thereafter, the fibril mat was filled with IGEN assay buffer and interpreted as described in example 6.27. 629. ECL detection of TAG 1-labeled avidin on polyacrylamide surfaces

Crosslinked polyacrylamide gels containing covalently bound biotin were prepared by copolymerizing acrylamide, bis-acrylamide and N-acryloyl-N' -biotinyl-3, 6-dioxaoctane-1, 9-diamine (biotin is attached to the acrylamide moiety through a tri (ethylene glycol) linker) using well-known conditions (initiated with ammonium persulfate and TEMED). In this experiment, the concentrations of the three monomeric species were 2.6M, 0.065M and 0.023M, respectively (these concentrations of acrylamide and bis-acrylamide were reported to result in gels with pore sizes smaller than most proteins). The polymerization of a solution containing those monomers described above between two glass plates held apart by a distance of about 0.7mm results in the formation of slab gels of the same thickness. After completion of the polymerization reaction, the gel was soaked in PBS with four changes and washed free of unbound biotin. Avidin labeled with derivatized TAG1 (here, avidin refers to NeutrAvidin, used in this experiment is a modified avidin designed to display reduced NSB) was allowed to bind to the surface of the gel by soaking the gel for 20 minutes in a solution containing protein at a concentration of 50 μ g/mL in PBS. Thereafter, the gel was washed free of excess TAG 1-labeled avidin by soaking in four changes of ECL assay buffer (200mM sodium phosphate, 100mM tripropylamine, 0.02% (w/v) Tween 20, pH 7.2). As shown in fig. 39, the gel (3900) is then placed in contact with gold working electrode (3901) and counter electrode (3902) patterned on a glass carrier (3903). The voltage between the two electrodes was ramped from 0.0V to 3.0V and back to 0.0V at a rate of 500 mV/sec, which resulted in an ECL light signal measured from a PMT (3904) placed on top of the gel (fig. 40). Gels prepared without the inclusion of biotin containing an acrylamide derivative had no ECL signal (figure 41). The signal obtained with the biotin-containing polymer indicates that an almost complete monolayer of protein is present on the surface of the gel. 6.30 ECL Sandwich immunoassay on Polyacrylamide surfaces

Crosslinked polyacrylamide gels containing covalently bound biotin were prepared as described in the example of section 6.29. Streptavidin is absorbed onto the surface of the gel to form a substance capable of capturing the biotin label. Treating the surface with a solution comprising tripropylamine, an analyte of unknown concentration, a biotin-labelled antibody against the analyte and a different ECL TAG 1-labelled antibody against the analyte. The presence of the analyte results in the formation of a complex of the analyte and the two antibodies, which are then captured on the streptavidin surface. ECL label bound to secondary antibodies present on the surface was measured as described in the example of section 6.29. 631. Multi-ECL sandwich immunoassay on polyacrylamide surface supported on electrodes

A 1-2 micron thick photoresist master plate, exposed and developed, is prepared in a well-known manner in a pattern of circular depressions arranged in an array. A10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding curing agent for SYLGARD184 is poured onto the base plate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. The resulting elastomeric stamp was prepared by exposure to a solution containing a hydroxyl terminated thiol HS- (CH) in ethanol ₂)₁₁-(OCH₂CH₂)₃A solution of-OH (1 to 10mM) is "inked" and brought into contact with an aligned gold plate and then removed. With a composition containing a thiol HS- (CH)₂)₁₀-CH₃The negative was washed for a few seconds with a solution (1 to 10mM in ethanol). The resulting surface was then rinsed with ethanol and dried with a stream of nitrogen. Treatment of the surface with a solution containing acryloyl chloride and triethylamine in dioxane results in functionalization of the hydroxyl terminated regions with acrylic acid groups. Thereafter, a capillary array containing a mixture of acrylamide, bis-acrylamide, N-acryloylsuccinimide, azo-bis-cyanovaleric acid, and an antibody having an amino group is brought into contact with an aligned surface which aligns the capillary with acrylic acid-terminated regions to place a prepolymer solution containing specific antibodies at each region. Each capillary in the capillary array contains an antibody specific for a different analyte of interest. Exposing the patterned prepolymer droplets to ultraviolet radiation results in the formation of crosslinked gels on the substrate, each crosslinked gel giving a bonding area on the surface. The assay is performed by treating the substrate with a mixture of analytes which are capable of binding at one or more binding regions present on the gel surface in a buffered solution comprising tripropylamine and secondary antibodies labelled with ECL-TAG 1. Then, as shown in FIGS. 42A to 42B, the binding regions (4200, 4201, 4202) (on the polyacrylamide droplet (4203) on the gold electrode (4232)) were placed Is placed against the ITO working electrode (4204). The light emitted from each of those binding regions described above was quantified with a CCD camera (4205) and compared with the binding regions with an internal standard contained in the sample solution. 6.32 multiple ECL competitive immunoassays on Polyacrylamide surfaces Supported on electrodes

A 1-2 micron thick photoresist base plate, which has been exposed and developed, is prepared in a pattern of circular depressions arranged in an array according to well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding curing agent for SYLGARD184 is poured onto the base plate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. The resulting elastomeric stamp was prepared by exposure to a solution containing a hydroxyl terminated thiol HS- (CH) in ethanol₂)₁₁-(OCH₂CH₂)₃A solution of-OH (1 to 10mM) is "inked" and brought into contact with an aligned gold plate and then removed. With a composition containing a thiol HS- (CH)₂)₁₀-CH₃The negative was washed for a few seconds with a solution (1 to 10mM in ethanol). The resulting surface was then rinsed with ethanol and dried with a stream of nitrogen. Treatment of the surface with a solution containing acryloyl chloride and triethylamine in dioxane results in functionalization of the hydroxyl terminated regions with acrylic acid groups. Thereafter, an array of capillaries containing a mixture of acrylamide, bisacrylamide, N-acryloylsuccinimide, azo-bis-cyanovaleric acid, and an antibody was brought into contact with an aligned surface which aligned the capillaries with acrylic acid-terminated regions to place a prepolymer solution containing specific antibodies at each region. Those capillaries in the capillary array described above contain antibodies specific for different analytes of interest. Exposing the patterned prepolymer droplets to ultraviolet radiation results in the formation of crosslinked gels on the substrate, each crosslinked gel giving a bonding area on the surface. The assay is performed by treating the negative with a mixture of analytes capable of binding in a buffered solution at one or more binding domains present on the gel surface The solution contains tripropylamine and ECL-TAG1 labelled analogues of the above-mentioned analyte (that is, competition for binding to those binding regions is established for ECL-TAG1 labelled and unlabelled analyte). Then, as shown in fig. 42, the bonding areas (4200, 4201 and 4202) (on the polyacrylamide droplet (4203) on the gold electrode (4232)) were placed next to the ITO working electrode (4204). The light emitted from each of those binding regions is quantified with a CCD camera (4205) and compared with the binding regions for the internal standard contained in the sample solution. 6.33 multiple ECL assay for cells bound on Polyacrylamide surfaces carried on electrodes

Stator

A 1-2 micron thick photoresist base plate, which has been exposed and developed, is prepared in a pattern of circular depressions arranged in an array according to well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding curing agent for SYLGARD184 is poured onto the base plate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. The resulting elastomeric stamp was prepared by exposure to a solution containing a hydroxyl terminated thiol HS- (CH) in ethanol₂)₁₁-(OCH₂CH₂)₃A solution of-OH (1 to 10mM) is "inked" and brought into contact with an aligned gold plate and then removed. With a composition containing a thiol HS- (CH) ₂)₁₀-CH₃The negative was washed for a few seconds with a solution (1 to 10mM in ethanol). The resulting surface was then rinsed with ethanol and dried with a stream of nitrogen. Treatment of the surface with a solution containing acryloyl chloride and triethylamine in dioxane results in functionalization of the hydroxyl terminated regions with acrylic acid groups. Thereafter, a capillary array containing a mixture of acrylamide, bisacrylamide, N-acryloylsuccinimide, azo-bis-cyanovaleric acid, and an antibody against the cell surface was brought into contact with an alignment surface that aligned the capillary with acrylic acid-terminated regions to place the prepolymer solution at each region. Subjecting the patterned prepolymer droplets to ultraviolet light, resulting in the formation of crosslinked gels on said substrate, eachThe bulk cross-linked gel gives a binding domain on the surface. The assay is performed by first treating the binding region with a suspension of cells, followed by treating the binding region with a mixture of binding reagents that are capable of binding to one or more cells bound to the gel surface in a buffered solution containing tripropylamine and an ECL-TAG 1-labelled secondary antibody and/or other binding reagent specific for the analyte. Then, as shown in fig. 42, the bonding areas (4200, 4201, and 4202) (on the polyacrylamide droplet (4203) on the gold electrode (4232)) were placed next to the ITO working electrode (4204). The light emitted from each of those binding regions described above was quantified with a CCD camera (4205) and compared with the light emitted from the internal standard for inclusion in the sample solution. 6.34 fine particles for binding analyte to Polyacrylamide surface carried on electrode

Multiple ECL assay of cells

A 1-2 micron thick photoresist base plate, which has been exposed and developed, is prepared in a pattern of circular depressions arranged in an array according to well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding curing agent for SYLGARD184 is poured onto the base plate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. The resulting elastomeric stamp was prepared by exposure to a solution containing a hydroxyl terminated thiol HS- (CH) in ethanol₂)₁₁-(OCH₂CH₂)₃A solution of-OH (1 to 10mM) is "inked" and brought into contact with an aligned gold plate and then removed. With a composition containing a thiol HS- (CH)₂)₁₀-CH₃The negative was washed for a few seconds with a solution (1 to 10mM in ethanol). The resulting surface was then rinsed with ethanol and dried with a stream of nitrogen. Treatment of the surface with a solution containing acryloyl chloride and triethylamine in dioxane results in functionalization of the hydroxyl terminated regions with acrylic acid groups. Thereafter, a capillary array containing a mixture of acrylamide, bisacrylamide, N-acryloylsuccinimide, azo-bis-cyanovaleric acid, and cells is aligned withThe surfaces are brought into contact, the aligned surfaces aligning the capillary with the acrylic terminated areas so that a prepolymer solution containing specific cell types is placed at each area. Exposing the patterned prepolymer droplets to ultraviolet light results in the formation of cross-linked gels on said substrate, each cross-linked gel giving a bonding area on the surface. The assay is performed by treating the gel with a sample containing a mixture of analytes capable of binding to one or more binding domains present on the surface of said gel in a buffered solution containing tripropylamine and ECL-TAG1 labelled antibodies and/or other binding reagents specific for said analytes. Then, as shown in fig. 42, the bonding areas (4200, 4201, and 4202) (on the polyacrylamide droplet (4203) on the gold electrode (4232)) were placed next to the ITO working electrode (4204). The light emitted from each of those binding regions is quantified using a CCD camera (4205) and compared to the light emitted from the binding regions for the internal standard contained in the sample solution. 6.35 multiple ECL competitive hybridization assay on Polyacrylamide surfaces Supported on electrodes

A 1-2 micron thick photoresist base plate, which has been exposed and developed, is prepared in a pattern of circular depressions arranged in an array according to well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding curing agent for SYLGARD184 is poured onto the base plate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. The resulting elastomeric stamp was prepared by exposure to a solution containing a hydroxyl terminated thiol HS- (CH) in ethanol₂)₁₁-(OCH₂CH₂)₃A solution of-OH (1 to 10mM) is "inked" and brought into contact with an aligned gold plate and then removed. With a composition containing a thiol HS- (CH)₂)₁₀-CH₃The negative was washed for a few seconds with a solution (1 to 10mM in ethanol). The resulting surface was then rinsed with ethanol and dried with a stream of nitrogen. Treatment of the surface with a solution containing acryloyl chloride and triethylamine in dioxane results in functionalization of the hydroxyl terminated regions with acrylic acid groups. After thatAn array of capillaries containing a mixture of acrylamide, bisacrylamide, N-acryloylsuccinimide, azo-bis-cyanovaleric acid, and nucleic acid probes functionalized with an amino group is brought into contact with an alignment surface which aligns the capillaries with acrylic-terminated regions to place a prepolymer solution containing specific probes at each region. The capillaries in the capillary array described above contain probes specific for the nucleic acid sequence of interest. Exposing the patterned prepolymer droplets to ultraviolet light results in the formation of crosslinked gels on said substrate, each crosslinked gel giving a bonding area on the surface. The assay is performed by treating the backsheet with a sample mixture which may contain sequences capable of binding at one or more binding regions present on the surface of the gel in a buffered solution containing tripropylamine and sequences labelled with ECL-TAG1 which compete with the analyte of interest for binding to the surface. Then, as shown in fig. 42, the bonding areas (4200, 4201, and 4202) (on the polyacrylamide droplet (4203) on the gold electrode (4232)) were placed next to the ITO working electrode (4204). The light emitted from each of those binding regions described above was quantified with a CCD camera (4205) and compared with the light emitted from the binding region for the internal standard contained in the sample solution. 6.36 multiple ECL hybridization Sandwich assay on Polyacrylamide surface Supported on electrodes

A 1-2 micron thick photoresist base plate, which has been exposed and developed, is prepared in a pattern of circular depressions arranged in an array according to well known methods. A10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding curing agent for SYLGARD184 is poured onto the base plate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. The resulting elastomeric stamp was prepared by exposure to a solution containing a hydroxyl terminated thiol HS- (CH) in ethanol₂)₁₁-(OCH₂CH₂)₃A solution of-OH (1 to 10mM) is "inked" and brought into contact with an aligned gold plate and then removed. With a composition containing a thiol HS- (CH)₂)₁₀-CH₃The negative was washed for a few seconds with a solution (1 to 10mM in ethanol). The resulting surface was then rinsed with ethanol and dried with a stream of nitrogen. Treatment of the surface with a solution containing acryloyl chloride and triethylamine in dioxane results in functionalization of the hydroxyl terminated regions with acrylic acid groups. Thereafter, a capillary array containing a mixture of acrylamide, bisacrylamide, N-acryloylsuccinimide, azo-bis-cyanovaleric acid, and a nucleic acid probe functionalized with an amino group was brought into contact with an aligned surface which aligned the capillary with acrylic-terminated regions to place a prepolymer solution containing a specific probe at each region. The capillaries of the capillary array contain probes specific for the nucleic acid sequence of interest. Exposing the patterned prepolymer droplets to ultraviolet light results in the formation of crosslinked gels on said substrate, each crosslinked gel giving a bonding area on the surface. The assay is performed by treating the substrate with a sample mixture which may contain sequences capable of binding to one or more binding domains present on the surface of the gel in a buffered solution containing tripropylamine and ECL-TAG1 labelled sequences which bind to the analyte at sequences which are not complementary to those bound to the surface. Then, as shown in fig. 22, the bonding areas (4200, 4201, and 4202) (on the polyacrylamide droplet (4203) on the gold electrode (4232)) were placed next to the ITO working electrode (4204). The light emitted from each of those binding regions described above was quantified with a CCD camera (4205) and compared with the light emitted from the binding region for the internal standard contained in the sample solution. 6.37 multiple assays of different types on Polyacrylamide surfaces carried on electrodes

A 1-2 micron thick photoresist base plate, which has been exposed and developed, is prepared in a pattern of circular depressions arranged in an array according to well known methods. Pouring a 10: 1 mixture of SYLGARD silicone elastomer 184 and a corresponding curing agent for SYLGARD184 onto a substrate andand (5) curing. The polymerized SYLGARD184 is carefully removed from the silicon substrate. The resulting elastomeric stamp was prepared by exposure to a solution containing a hydroxyl terminated thiol HS- (CH) in ethanol₂)₁₁-(OCH₂CH₂)₃A solution of-OH (1 to 10mM) is "inked" and brought into contact with an aligned gold plate and then removed. With a composition containing a thiol HS- (CH)₂)₁₀-CH₃The negative was washed for a few seconds with a solution (1 to 10mM in ethanol). The resulting surface was then rinsed with ethanol and dried with a stream of nitrogen. Treatment of the surface with a solution containing acryloyl chloride and triethylamine in dioxane results in functionalization of the hydroxyl terminated regions with acrylic acid groups. Thereafter, an array of capillaries containing a mixture of acrylamide, bisacrylamide, N-acryloylsuccinimide, azo-bis-cyanovaleric acid, and any of the binding reagents described in those examples in sections 6.31 to 6.36 is brought into contact with an aligned surface that aligns the capillaries with acrylic-terminated regions to place a prepolymer solution containing specific probes at each region. Each capillary of the capillary array contains binding domains specific for the analyte of interest. Exposing the patterned prepolymer droplets to ultraviolet light results in the formation of crosslinked gels on said substrate, each crosslinked gel giving a bonding area on the surface. The assay is performed by treating the backsheet with a sample mixture which may contain an analyte capable of binding at one or more binding domains present on the surface of the gel in a buffered solution containing tripropylamine and an analogue of ECL-TAG1 labelled analyte which competes with the analyte for binding to the binding domains and/or an ECL-TAG1 labelled secondary binding reagent for the analyte of interest. Then, as shown in FIG. 22, the binding regions (4200, 4201, and 4202) (on the polyalkylene amide droplet (4203) on the gold electrode (4232)) were placed in close proximity to the ITO working electrode (4204). The light emitted from each of those binding regions is quantified with a CCD camera (4205) and combined with the light emitted from the light source for inclusion in the sample solution The light emitted from the binding region of the standard was compared. 6.38 highly reversible ECL

Polishing with 0.5 μm and 0.03 μm corundum paste by hand sequentially, followed by application of 1: 3H₂O₂/H₂SO₄Chemical etching and etching at H₂SO₄Between 0.2V and 1.7V for Ag/AgCl to clean polycrystalline gold electrodes (available from Bio-analytical services, 2 mm)²). Thereafter, the cleaned electrode was immersed in octyl mercaptan (C) dissolved in ethanol₈SH) overnight. C was covered by 20. mu.l of TAG 1-labeled Bovine Serum Albumin (BSA) dissolved in phosphate-buffered saline (PBS, 0.15M NaCl/0.1M NaPi, pH 7.2)₈SH modified electrodes and after 10 minutes incubation the surface was thoroughly washed with the same buffer for protein uptake.

ECL was performed in a three-electrode cell with an Ag/AgCl reference electrode, a platinum wire counter electrode, and an EG & G283 potentiostat. The light intensity was measured with a photometer from Pacific instruments and a Hamamatsu photomultiplier tube placed at the bottom of the electrochemical cell. The electrode with the adsorbed proteins was immersed in a solution of 0.1M TPA and 0.2M phosphate, pH 7.2. As shown in fig. 44A, a highly reversible ECL response (substantially similar to the intensity of the forward and backward scans) was observed when the potential was varied periodically between 0.0V and 1.2V, indicating the reversibility of the ECL process and the stability of the thiol and protein layers on the electrodes.

Periodic voltammetric experiments were performed on the same instrument as used for ECL, but without PMT and photometer. In this experiment, one C₈SH-covered electrodes (no protein) were placed in a 1mM solution of potassium ferricyanide (in PBS) and swept back and forth across the electrodes from +0.5V to 1.2V, followed by another cycle between +0.5V and-0.3V. The results show that the monolayer was intact and did not absorb at 1.2V, due to only capacitive current and no ferricyanide induced current in the voltammetric recordings between +0.5V and-0.3V (fig. 44B). 6.39 quasi-reversible ECL

Electrode modification and protein adsorption were performed in the same manner as described above. In ECL experiments, the potential was swept between 0.0V and 1.5V and the corresponding light intensity was recorded. As shown in fig. 45A, there is some ECL loss between the forward and backward scans of the same cycle, and also between the different two cycles. Periodic voltammetric recordings of thiol/gold in ferricyanide after oxidation at 1.5V showed significant amounts of faradaic current, indicating at least local absorption of the thiol monolayer at 1.5V (fig. 45B). 6.40, irreversible ECL

In these experiments, electrode modification and protein adsorption were performed in the same manner as in the example of section 6.38. To measure ECL, the potential was swept through to 2.0V and back to 0.0V. Intense light was observed for the forward scan (more light than was observed in the reversible case in the example of section 6.38), but it dropped onto the background of the reverse scan as shown in fig. 46A. Periodic voltammetric recordings of the modified electrode in ferricyanide after oxidation at 2.0V indicated that the vast majority of the thiol monolayer was desorbed. 6.41 ECL Sandwich immunoassay with Primary antibodies immobilized on patterned gold electrodes

In this example, antibodies to the failure specific antigen (PSA) were immobilized on patterned gold electrodes for immunoassay of PSA.

The 1 to 2 micron thick exposed and developed photoresist cliche is prepared in a well known manner as a layer of photoresist on a silicon carrier having a 1mm by 1mm patch with the photoresist removed. A10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding curing agent for SYLGARD184 is poured onto the base plate and cured. The polymerized SYLGARD184 is carefully removed from the silicon substrate. The resulting elastic "stamp" was obtained by exposing it to a solution containing a hydroxyl-terminated HS- (CH) in ethanol₂)₁₁(OCH₂CH₂)₃-OH and nitrilotriacetic acid (NTA) -terminated thiol HS- (CH)₂)₁₁(OCH₂CH₂)₃OC(O)NH(CH₂)₄CH(CO₂H)N(CH₂CO₂H)₂Is "inked". The "inked" stamp was brought into contact with the gold plate and removed to form a 1mm by 1mm SAM. The plate was washed for a few seconds with a solution containing only hydroxyl terminated thiols in ethanol to prevent non-specific binding of proteins to the areas outside the imprinted features. Thereafter, the resulting surface was rinsed with ethanol and dried with a stream of nitrogen. With NiCl ₂The surface is treated with a solution containing a peptide (His) giving anti-PSA mouse monoclonal antibody₆The solution of the fusion protein tag of the binding site of (a) treats the surface, which results in the immobilization of the above-mentioned fusion protein tag on the surface in a controlled manner. This process produces reproducible and predetermined amounts of protein immobilized on the surface. Through handle (His)₆The sequence is located on the original structure of the above-described fusion protein tag, controlling the orientation of the protein on the surface. The absolute amount of immobilized protein was controlled by the ratio of NEA-terminated thiol to hydroxyl-terminated thiol in the imprinted SAM and by the surface area of the imprinted features. A calibration curve for PSA was determined by preparing solutions containing known concentrations of PSA in serum (concentrations ranging from 1fM to 1 μ M). A large number of surfaces prepared as described above were treated with a PSA calibration standard and then with a solution containing the optimal concentration of secondary antibodies against PSA (labeled with a derivative of label 1). The calibration curve was determined by immersing those surfaces in a solution containing 0.1M TPA and 0.2M phosphate (pH 7.2) and measuring the peak intensity of the emitted light as the potential at the gold surface was periodically varied between 0.0V and 2.0V at a scan rate of 0.5V/sec. The work to determine the unknown concentration of PSA in serum in a sample was performed in the same procedure, except that the concentration of PSA was calculated from the peak ECL signal by reference to the calibration curve described above. 7. Incorporated reference

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the claims. The disclosures of the various publications cited herein are incorporated by reference in their entirety.

Claims (204)

1. A device for detecting an analyte by electrochemiluminescence, the device comprising an electrode and a counter electrode, the electrode or counter electrode having immobilised on its surface a predetermined amount of a reagent which is capable of binding an electrochemiluminescence labelled species or a binding partner of an electrochemiluminescence labelled species.

2. A device for detecting an analyte by electrochemiluminescence, the device comprising an electrode or a counter electrode having immobilised on its surface a predetermined amount of a reagent capable of binding directly or indirectly to an electrochemiluminescence labelled species.

3. A device for detecting an analyte by electrochemiluminescence comprising an electrode having immobilized on its surface a predetermined amount of a reagent that binds to a component of a binding electrochemiluminescence assay.

4. A device according to claim 3, wherein said electrode is a working electrode.

5. A device as claimed in claim 3, characterized in that the electrode is a counter electrode.

6. A device according to claim 3, wherein the complex of immobilised reagent and electrode substantially allows electron transfer between the electrode and the electrochemiluminescent label.

7. A device as claimed in claim 3, characterized in that the electrodes are made of a porous material.

8. A device for detecting an analyte by electrochemiluminescence, the device comprising a porous electrode and a counter electrode.

9. A device for detecting an analyte by electrochemiluminescence, the device comprising a working electrode and a counter electrode, the working electrode having immobilized on its surface and in electrochemical contact with a predetermined amount of a reagent that binds to a component of a binding electrochemiluminescence assay.

10. A device for detecting an analyte by electrochemiluminescence, the device comprising a working electrode and a counter electrode, the counter electrode having immobilized on its surface a predetermined amount of a reagent and being in electrochemical contact with the reagent, the reagent being capable of binding a component of a binding electrochemiluminescence assay.

11. A device for detecting an analyte by electrochemiluminescence, the device comprising an electrode, a counter electrode and a porous matrix in electrochemical contact with the electrode, the porous matrix comprising on its surface a predetermined amount of a reagent capable of binding a component of a binding electrochemiluminescence assay.

12. A device for detecting an analyte by electrochemiluminescence, the device comprising an electrode and a plurality of discrete binding domains capable of binding a component of a binding electrochemiluminescence assay.

13. A device for detecting an analyte by electrochemiluminescence, the device comprising a working electrode and a plurality of discrete binding domains, each of the binding domains containing a predetermined amount of a reagent that binds to a component of a binding electrochemiluminescence assay.

14. A device as claimed in claim 13, wherein the device further comprises a counter electrode.

15. A device as claimed in claim 13, further comprising a counter electrode, sample delivery means and light detection means.

16. A device for detecting an analyte by electrochemiluminescence, the device comprising:

(a) one of the electrodes is provided with a conductive layer,

(b) a plurality of discrete binding domains, each of the binding domains containing a predetermined amount of a reagent capable of binding a component of a binding electrochemiluminescence assay,

(c) a counter electrode, which is arranged on the back side of the substrate,

(d) means for delivering a sample to said binding regions,

(e) Means for inducing electrochemiluminescence, and

(f) a device for electrochemiluminescence detection.

17. A device for detecting an analyte by electrochemiluminescence, the device comprising:

(a) one of the electrodes is provided with a conductive layer,

(b) a support having immobilized thereon a plurality of discrete binding domains, each of the binding domains containing a predetermined amount of a reagent that binds a component of a binding electrochemiluminescence assay,

(c) a counter electrode, which is arranged on the back side of the substrate,

(d) means for delivering a sample to said binding regions,

(e) means for inducing electrochemiluminescence, and

(f) a device for electrochemiluminescence detection.

18. A device according to claim 17, wherein said plurality of regions are in electrochemical contact with said electrode.

19. A device as claimed in claim 17, wherein said electrodes are porous.

20. A device as claimed in claim 19, wherein said electrode is formed from a carbon-containing material.

21. A device according to claim 17, wherein said electrodes are comprised of fibrils.

22. A device according to claim 17, wherein said electrochemiluminescence detection means detects electrochemiluminescence from one or more of said discrete binding domains.

23. A device according to claim 17, wherein said support is a porous matrix.

24. A device as claimed in claim 17 wherein said support is a porous substrate and said electrodes are porous.

25. A device as claimed in claim 17 wherein said support is a porous matrix, said matrix being positioned between said electrode and a counter electrode.

26. A device for detecting an analyte by electrochemiluminescence, the device comprising:

(a) one of the electrodes is provided with a conductive layer,

(b) a plurality of discrete binding domains, each of the binding domains containing a predetermined amount of a reagent capable of binding a component of a binding electrochemiluminescence assay,

(c) a counter electrode, which is arranged on the back side of the substrate,

(d) means for delivering a sample to said binding regions,

(e) means for inducing electrochemiluminescence, and

(f) many devices are used for electrochemiluminescence detection.

27. A device as claimed in claim 26 wherein said plurality of means for electrochemiluminescence detection is used to image one or more of said binding domains.

28. A device according to claim 27, wherein at least one of said plurality of detection means detects electrochemiluminescence from a binding region.

29. A device according to claim 27, wherein said plurality of means for electrochemiluminescence detection is a CCD array or a diode array.

30. A device for detecting an analyte by electrochemiluminescence comprising an electrode having immobilized on its surface a plurality of discrete binding domains capable of binding a component of a binding electrochemiluminescence assay.

31. A device for detecting an analyte by electrochemiluminescence, the device comprising an electrode having immobilized on its surface a plurality of discrete binding domains, each of the binding domains comprising a predetermined amount of a reagent that binds to a component of a binding electrochemical assay.

32. A device for detecting an analyte by electrochemiluminescence, the device comprising an electrode having a plurality of discrete binding domains immobilized on the surface of the electrode, the binding domains comprising an electrochemiluminescence label.

33. A device for detecting an analyte by electrochemiluminescence, the device comprising an electrode having a surface to which a plurality of discrete binding domains are immobilised, and a counter electrode, the binding domains being capable of generating a plurality of electrochemiluminescence signals.

34. A device for detecting an analyte by electrochemiluminescence, the device comprising a support having immobilised on its surface a plurality of discrete areas, each of said areas containing a predetermined amount of a reagent capable of binding a component of a binding electrochemiluminescence assay.

35. A device for detecting an analyte by electrochemiluminescence, the device comprising an electrode and a support having a plurality of discrete binding domains immobilized on a surface thereof, each binding domain comprising a predetermined amount of a reagent that binds to a component of a binding electrochemiluminescence assay, the electrode being in electrochemical contact with the binding domain.

36. A device as claimed in claim 35 wherein said electrode is a working electrode.

37. A device as claimed in claim 35, wherein said electrode is a counter electrode.

38. A device for detecting an analyte by electrochemiluminescence, the device comprising:

(a) an electrochemical cell having a plurality of cells, each cell having a plurality of cells,

(b) a first surface comprising an electrode, a second surface comprising an electrode,

(c) a second surface having immobilised thereon a plurality of binding domains, each of the binding domains comprising a binding reagent capable of binding a component of a binding electrochemical assay, said first and second surfaces being spatially aligned so as to allow electron transfer between said electrode and an electrochemiluminescent label which is directly or indirectly attached to said binding reagent.

39. A device for detecting an analyte by electrochemiluminescence, the device comprising an electrochemical cell and an electrode having immobilized on a surface thereof a plurality of binding domains, each of the binding domains comprising a binding reagent capable of binding a component of a binding electrochemiluminescence assay.

40. A method for performing a plurality of electrochemiluminescence assays, the method comprising the steps of:

(a) contacting the plurality of discrete binding domains with a sample containing one or a plurality of analytes of interest under assay conditions;

(b) causing one or more of said plurality of regions to electrochemiluminesce; and

(c) detecting electrochemiluminescence at a plurality of said binding domains.

41. A method for performing a plurality of electrochemiluminescence assays on a plurality of different analytes of interest, the method comprising the steps of:

(a) contacting the plurality of discrete binding domains with a sample containing a plurality of analytes of interest under assay conditions;

(b) causing the plurality of discrete binding domains to electrochemiluminesce simultaneously; and

(c) simultaneously detecting electrochemiluminescence from each of the discrete binding domains.

42. An article comprising a plurality of discrete binding domains on a support, said binding domains having a spatial configuration relative to each other and different binding specificities for binding a plurality of different analytes of interest in an electrochemiluminescence assay.

43. An article comprising a plurality of discrete binding domains on a support, each of said domains having a spatial configuration with respect to each other and each of said domains having a different binding specificity for an analyte of interest in an electrochemiluminescence assay, each domain comprising a different binding component that binds to a different analyte of interest.

44. An article comprising a plurality of discrete binding domains on a support, said domains having a spatial configuration with respect to each other and each of said domains having a different binding specificity for an analyte of interest in an electrochemiluminescence assay, each domain binding, directly or indirectly, to a different analyte of interest and to an electrochemiluminescence-enabling composition.

45. An article comprising a plurality of discrete binding domains on a support, the support comprising a porous material, each of said domains having a spatial configuration with respect to each other and each of said domains having a different binding specificity for an analyte of interest in an electrochemiluminescence assay, each domain comprising a different binding component that binds to a different analyte of interest.

46. The article of claim 45, wherein said porous material comprises carbon.

47. An article according to claim 45 wherein said porous material comprises fibrils.

48. An article of manufacture comprising a plurality of discrete binding domains on a support, the support comprising functionalised fibrils, said domains having a spatial configuration relative to each other and each of said domains having a different binding specificity for an analyte of interest in an electrochemiluminescence assay, each domain binding, directly or indirectly, a different analyte of interest and binding an electrochemiluminescence-enabling composition.

49. An article comprising a plurality of discrete binding domains on a support, the support comprising one or more polymeric matrices, said domains having a spatial configuration relative to each other and each of said domains having a different binding specificity for an analyte of interest in an electrochemiluminescence assay, each domain binding, directly or indirectly, a different analyte of interest and binding an electrochemiluminescence-enabling composition.

50. A cartridge for detecting an analyte in a sample by electrochemiluminescence, the cartridge comprising:

(a) a plurality of discrete binding domains on a support; and

(b) one or more electrode and counter electrode pairs.

51. A cartridge for detecting an analyte in a sample by electrochemiluminescence, the cartridge comprising:

(a) a plurality of discrete binding domains on a support;

(b) one or more pairs of electrodes and counter electrodes spatially aligned with those discrete binding regions; and

(c) means for delivering a sample to the plurality of discrete binding domains.

52. The cartridge of claim 49, wherein said plurality of discrete binding domains form at least one surface capable of binding a component of a binding electrochemiluminescence assay.

53. A cartridge according to claim 50, wherein the plurality of binding domains comprises binding domains having different binding specificities for simultaneously binding a plurality of different analytes of interest present in the sample.

54. A cartridge for detecting or measuring electrochemiluminescence, the cartridge comprising:

(a) a first support having a plurality of discrete binding domains thereon;

(b) A plurality of electrode and counter electrode pairs, each of said plurality of discrete binding domains being closely aligned with one of said plurality of electrode and counter electrode pairs, said discrete binding domains and electrode and counter electrode pairs forming a plurality of cells for detecting or making measurements of electrochemiluminescence, said electrode/counter electrode pairs being accessible by a source of electrical energy in the form of a voltage waveform effective to initiate electrochemiluminescence; and

(c) means for delivering a sample to the plurality of discrete binding domains.

55. A cartridge according to claim 53, wherein the plurality of binding domains comprises binding domains having different binding specificities for simultaneously binding a plurality of different analytes of interest present in the sample.

56. A cartridge for detecting or measuring an analyte of interest in a sample, the cartridge comprising:

(a) a first support having a plurality of discrete binding domains on a surface of the first support, at least one of the discrete binding domains having a binding specificity different from that of the other binding domains, each of the plurality of discrete binding domains being hydrophilic and surrounded by hydrophobic domains;

(b) A second support having a plurality of hydrophilic regions, the hydrophilic regions comprising a reaction medium suitable for carrying out a chemical assay thereon; and

(c) means for contacting said plurality of discrete binding areas with said plurality of reaction media, whereby a sample to be analyzed present on each binding area is contacted with the reaction media.

57. The cassette of claim 50, wherein said discrete binding domains further comprise an internal control.

58. A method for preparing a plurality of discrete binding domains on a support, the support comprising binding reagents capable of binding an analyte of interest, comprising the steps of:

(a) forming a self-assembled monolayer on a support, said monolayer comprising linking groups a on a surface of the monolayer not adjacent to said support, said first linking groups a being capable of specifically binding a linking group B; and

(b) contacting the first linking group a with a binding reagent capable of binding to the analyte of interest, the binding reagent binding to the linking group B, such that the binding reagent binds to the analyte of interest via a: the B bonds are attached to the monolayer to form a bonding surface that is configured as a plurality of discrete bonding regions.

59. A method according to claim 57, wherein a plurality of different binding reagents are bound to a plurality of discrete binding domains, and wherein said contacting step is carried out by delivering a plurality of fluid samples onto said monolayer from a plurality of fluid conduits, each fluid sample comprising a different binding reagent.

60. A method for detecting or measuring an analyte in an electrochemiluminescence binding assay, comprising the steps of:

(a) contacting a plurality of discrete binding domains immobilized on the surface of one or more supports with a sample comprising a plurality of analytes and a component of said assay linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to induce electrochemiluminescence at one or more of said regions in the presence of a reaction medium suitable for performing an electrochemiluminescence assay; and

(c) detecting or measuring electrochemiluminescence from the plurality of regions.

61. A kit for performing a plurality of electrochemiluminescence assays for a plurality of analytes of interest, comprising:

(a) an article comprising a plurality of discrete binding domains on a support, said binding domains having a spatial configuration relative to each other and different binding specificities for a plurality of different analytes of interest in an electrochemiluminescence assay; and

(b) A container containing a reagent necessary for performing said assay.

62. A kit according to claim 60, further comprising a means for performing said assay.

63. A kit for performing a plurality of electrochemiluminescence assays for a plurality of analytes of interest, comprising:

a container containing binding components specific for a plurality of different analytes for use in a plurality of electrochemiluminescence assays.

64. A kit according to claim 62, further comprising one or more containers containing components that are not bound for a plurality of electrochemical assays.

65. A kit according to claim 62 further comprising an article of manufacture comprising a plurality of discrete binding domains on a support, said binding domains having a spatial configuration relative to each other and having different binding specificities for a plurality of different analytes of interest in an electrochemiluminescence assay.

66. A cartridge, the cartridge comprising:

(a) a plurality of discrete binding domains on a support forming at least one binding surface;

(b) a plurality of electrode and counter electrode pairs, wherein the discrete binding domains are spatially aligned with the plurality of electrode and counter electrode pairs; and

(c) Means for delivering a sample to the plurality of discrete binding domains.

67. A cartridge, the cartridge comprising:

(a) a plurality of discrete binding domains on a support forming at least one binding surface; and

(b) a plurality of electrode and counter electrode pairs, wherein the discrete binding domains are spatially aligned with and can be adjacent to the plurality of electrode and counter electrode pairs, wherein the binding domains are hydrophilic or hydrophobic with respect to the surface of the support.

68. A cartridge, the cartridge comprising:

(a) a plurality of discrete binding domains on a support forming at least one binding surface;

(b) a plurality of electrode and counter electrode pairs, wherein the discrete binding domains are spatially aligned with the plurality of electrode and counter electrode pairs, the plurality of electrodes being on the binding surface, each electrode abutting a binding domain, and the counter electrode being on a second support; and

(c) a means for delivering the sample to the plurality of discrete binding domains.

69. The cassette according to claim 68, wherein the binding domains are hydrophilic or hydrophobic with respect to the surface of the carrier.

70. A cassette according to claim 66, wherein said carrier comprises from 2 to 500 binding domains.

71. A cartridge according to claim 66, wherein the plurality of binding domains comprises binding domains with different binding specificities for simultaneously binding a plurality of different analytes of interest present in the sample.

72. A cartridge for detecting or measuring electrochemiluminescence, the cartridge comprising:

(a) a first support having a plurality of discrete binding domains thereon;

(b) a plurality of electrode and counter electrode pairs, said plurality of discrete binding domains aligned with and adjacent to said plurality of electrode and counter electrode pairs, said discrete binding domains and said plurality of electrode and counter electrode pairs forming a plurality of elements for detecting or performing an electrochemiluminescence measurement, said electrode/counter electrode pairs being accessible by a source of electrical energy in the form of a voltage waveform effective to trigger electrochemiluminescence; and

(c) a means for delivering the sample to the plurality of discrete binding domains.

73. The cartridge of claim 72 further comprising a second carrier positionable adjacent said first carrier to provide a sample-containing device between the two carriers, wherein said plurality of electrode and counter electrode pairs are affixed to said second carrier.

74. The cassette according to claim 72, wherein said plurality of electrode and counter electrode pairs are secured to said first carrier.

75. The cartridge of claim 72 further comprising a second carrier capable of being positioned adjacent to said first carrier to provide a sample-containing device between the two carriers, wherein a plurality of electrodes are positioned on said first carrier and a plurality of counter electrodes are positioned on said second carrier such that said plurality of electrodes and counter electrodes are positioned adjacent to each other.

76. The cassette according to claim 72, wherein said plurality of discrete binding domains comprises between 5 and 1000 binding domains.

77. The cartridge of claim 72 wherein the plurality of discrete binding domains comprises at least one binding domain comprising binding reagents that are identical to each other and that have a different specificity than the binding reagents contained in other binding domains for binding a plurality of different analytes of interest.

78. The cassette of claim 72, wherein said plurality of discrete binding domains comprises at least one binding domain comprising binding reagents having different binding specificities.

79. The cartridge of claim 78 further comprising a reflective surface adjacent said first or said second carrier.

80. The cartridge of claim 78 wherein said first and second supports, said plurality of discrete binding domains, and said plurality of electrode and counter electrode pairs are substantially transparent.

81. The cassette according to claim 72, wherein the pair of electrodes and counter-electrodes have a size range of: the diameter or width is from 0.001mm to 10 mm.

82. The cassette according to claim 80, wherein said bonding area has a size range of: the diameter or width is from 0.001mm to 10 mm.

83. The cartridge according to claim 72, wherein each of said pair of electrodes and counter electrode is individually accessible by at least one source of electrical energy.

84. The cassette of claim 72, wherein said binding region is hydrophilic and is surrounded by a hydrophobic surface.

85. The cassette of claim 72, wherein said conjugate region is hydrophobic and is surrounded by a hydrophilic surface.

86. The cassette of claim 72, wherein said bonding surface comprises a patterned, self-assembled monolayer.

87. The cassette according to claim 86, wherein the patterned, self-assembled monolayer comprises an alkanethiol.

88. The cartridge of claim 87, wherein the binding domain is capable of binding an analyte selected from the group consisting of: proteins, nucleic acids, carbohydrate moieties, antibodies, antigens, cells, organic compounds, and metal organic compounds.

89. The cartridge of claim 72, wherein said binding region comprises functional reagents in an assay selected from the group consisting of a clinical chemistry assay, a chemiluminescence assay, an immunoassay, and a nucleic acid probe assay.

90. The cartridge of claim 73 further comprising a removable electrode protective barrier interposed between said second support and said discrete binding areas, said electrode protective barrier being removable prior to triggering said electrochemiluminescence.

91. The cartridge of claim 72 further comprising a conductive material on or adjacent to a plurality of electrodes of the pair of electrodes, the conductive material positioned to enable an electrical potential to extend away from the electrode surface into an assay medium.

92. An apparatus for measuring electrochemiluminescence of a sample, the apparatus comprising:

(a) A plurality of elements for retaining at least one sample, said plurality of elements being comprised of a plurality of electrode and counter electrode pairs and a first support containing a plurality of discrete binding domains aligned with and adjacent to said plurality of electrode and counter electrode pairs, said electrode and counter electrode pairs being individually accessible, said elements being adapted to perform electrochemiluminescence measurements,

(b) voltage control means adapted to apply a controllable voltage waveform to said pair of electrodes and counter-electrode, said voltage waveform being effective to trigger electrochemiluminescence in said plurality of elements, and

(c) a photodetector means for detecting electrochemiluminescence from said sample.

93. A kit suitable for measuring electrochemiluminescence, the kit comprising in one or more containers:

(a) a plurality of discrete binding domains on a support forming at least one binding surface; and

(b) a plurality of electrode and counter electrode pairs spatially aligned with and capable of being brought into proximity with the plurality of discrete binding domains.

94. The kit of claim 93, further comprising at least one second carrier capable of being placed adjacent to the binding surface carrier to provide a device containing a sample.

95. The kit of claim 94, wherein said second carrier carries said plurality of electrode and counter electrode pairs, said plurality of electrode and counter electrode pairs being aligned with and brought adjacent to said plurality of discrete binding domains.

96. The kit of claim 93, further comprising reagents suitable for performing an electrochemiluminescence assay and a predetermined amount of a purified analyte of interest.

97. A cartridge for detecting or measuring an analyte in a sample, the cartridge comprising:

(a) a first support having a plurality of discrete binding domains on a surface thereof so as to form at least one binding surface, at least some of said discrete binding domains having a binding specificity different from that of other binding domains, each of said plurality of discrete binding domains being hydrophilic and surrounded by hydrophobic domains, and

(b) a second support having a plurality of hydrophilic regions, the hydrophilic regions comprising a plurality of reaction media suitable for carrying out a chemical assay thereon so as to form an assay surface,

the plurality of discrete binding areas can be contacted with the plurality of reaction media such that a sample to be analyzed present on each binding area is contacted with one of the reaction media to detect or measure an analyte of interest.

98. The cartridge of claim 72, further comprising a temperature control device.

99. A cartridge for carrying out a reaction of interest, the cartridge comprising:

(a) a first support having a plurality of discrete regions on a surface of said support, each of said regions being hydrophilic and surrounded by a hydrophobic region on a surface of said first support; and

(b) a second support having a plurality of discrete regions on the surface of the second support, each of said regions (i) being hydrophilic and surrounded by a hydrophobic region on the surface of said second support, (ii) comprising a number of reaction media suitable for carrying out a reaction of interest, and (iii) being spatially aligned with said regions on the surface of said first support, whereby said second support is positioned such that each of said regions on the surface of said second support is in contact with an aligned region on the surface of said first support.

100. The cassette of claim 66, wherein said discrete binding domains further comprise an internal control.

101. The device of claim 92 wherein said plurality of discrete bond areas comprises at least two identical bond areas.

102. A binding surface which is the product of the following process:

(a) forming a self-assembled monolayer on a support, said monolayer comprising a first linking group a on a surface of the monolayer not adjacent to said support, said first linking group a being capable of specifically binding to a second linking group B, said monolayer being applied to at least one region on said support; and

(b) contacting said first linking group a with a binding reagent capable of binding an analyte of interest, said binding reagent being linked to said linking group B, such that said binding reagent binds to said analyte via a binding site on said first linking group a: b bonds to the monolayer to form a bonding surface,

the binding surface is configured as a plurality of discrete binding domains containing the binding reagent that binds to an analyte of interest.

103. A device comprising the bonding surface of claim 22.

104. The cassette according to claim 66, wherein said carrier comprises an elastomeric material.

105. A method for preparing a plurality of discrete binding domains on a support, the method comprising the steps of:

(a) Forming a self-assembled monolayer on a support, said monolayer comprising a first attachment group a on a surface of said monolayer not adjacent to said support, said first attachment group a being capable of specifically binding a second attachment group B, applying said monolayer to at least one binding region on said support; and

(b) contacting the first linking group a with a binding reagent that binds the analyte of interest, linking the binding reagent to the linking group B, such that the binding reagent binds to the analyte of interest via a binding site on the first linking group a: b bonds to the monolayer, forming a bonding surface,

the binding surface is configured as a plurality of discrete binding domains containing a binding reagent that binds to the analyte of interest.

106. The method of claim 105, wherein in step (a), said monolayer is patterned on said support by a method selected from the group consisting of: microetching, micro-pen deposition, and micro-embossing.

107. The method of claim 105, wherein said binding agent is selected from the group consisting of proteins and fragments and derivatives thereof, and nucleic acids and fragments and derivatives thereof.

108. The method of claim 105, wherein said binding agent is selected from the group consisting of antibodies and binding fragments thereof, antigens, epitopes, cells and cellular components, enzymes, enzyme substrates, lectins, protein a, protein B, organic compounds, metallo-organic compounds, and carbohydrates.

109. The method of claim 105, wherein said binding reagent comprises a plurality of different binding reagents and said contacting step is performed by delivering a plurality of fluid samples from a multi-array of fluid conduits onto said monolayer, each fluid sample comprising a different binding reagent, such that discrete binding domains of a binding surface on said monolayer have different binding reagents attached thereto.

110. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting a plurality of discrete binding domains on the surface of one or more supports with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at each of a plurality of electrode and counter electrode pairs spatially aligned with and adjacent to the plurality of discrete binding domains, and

(c) Detecting or measuring said electrochemiluminescence.

111. The method of claim 110, wherein said plurality of discrete bonded regions ranges from 5 to 1000 bonded regions.

112. The method of claim 110 wherein the plurality of discrete binding domains includes at least one binding domain comprising binding reagents that are identical to each other and that have a different specificity than the binding reagents contained in other binding domains for binding a plurality of different analytes of interest.

113. The method of claim 110 wherein said carrier comprises a reflective surface.

114. The method of claim 110 wherein the support, binding surface, and pair of electrodes and counter electrode are substantially transparent.

115. The method as claimed in claim 110, wherein the size range of the bonding areas is: the diameter or width is from 0.001mm to 10 mm.

116. The method of claim 110, wherein said pair of electrodes and counter electrode are individually accessible by at least one source of electrical energy.

117. The method of claim 110, wherein said binding domain is hydrophilic and is surrounded by a hydrophobic surface.

118. The method of claim 110, wherein said binding domain is hydrophobic and is surrounded by a hydrophilic surface.

119. The method of claim 110 wherein the bonding region is a patterned, self-assembled monolayer.

120. The method of claim 119, wherein the binding domain specifically binds to an analyte selected from the group consisting of: proteins, nucleic acids, carbohydrate components, antibodies, antigens, cells and cellular components, organic compounds, and metal organic compounds.

121. The method of claim 110, further comprising contacting said binding region with a reaction medium suitable for performing an assay selected from the group consisting of: clinical chemistry assays, immunoassays, and nucleic acid probe assays.

122. The method of claim 110 wherein said one or more supports is a plurality of supports positioned one above the other and at least a portion of said supports is substantially transparent to light generated by electrochemiluminescence such that each of said plurality of supports is positioned adjacent another of said plurality of supports.

123. A method for detecting or measuring electrochemiluminescence in a sample, the method comprising:

(a) Contacting one or more of a plurality of discrete binding domains with a sample comprising molecules linked to electrochemiluminescent labels, said plurality of binding domains being located on the surface of one or more supports, characterized in that a plurality of electrode and counter electrode pairs are spatially aligned with said plurality of discrete binding domains, wherein said plurality of electrode and counter electrode pairs are present on the surface of a second support,

(b) positioning said second carrier surface adjacent to said binding surface such that each said electrode and counter electrode pair is adjacent to a different binding area,

(c) applying a voltage waveform effective to trigger electrochemiluminescence at each of said plurality of electrode and counter electrode pairs, an

(d) Detecting or measuring said electrochemiluminescence.

124. The method of claim 110, wherein during step (a) said plurality of electrode and counter electrode pairs are protected from contact with said sample by a removable electrode protective barrier, and said method further comprises removing said barrier prior to applying said voltage waveform.

125. A method for detecting or measuring an analyte of interest in a sample, the method comprising:

(a) Placing a droplet of a sample containing an analyte to be detected or measured on a plurality of discrete binding domains on a support surface, said plurality of discrete binding domains including at least one binding domain containing binding reagents that are the same as each other and that differ in specificity from the binding reagents contained in other binding domains, each of said discrete binding domains being characterized as being hydrophobic or hydrophilic, provided that the region of said support surface surrounding each of said binding domains is (i) hydrophobic if said binding domain is hydrophilic and (ii) hydrophilic if said binding domain is hydrophobic so as to allow said one or more analytes of interest in said sample to bind to said binding domain, and

(b) contacting said droplets on said first support with a surface of a second support having a plurality of discrete hydrophilic regions comprising thereon a reaction medium suitable for performing a chemical assay, and

(c) determining the presence of the analyte of interest bound to the binding area.

126. A method for detecting or measuring an analyte of interest in a sample, the method comprising:

(a) placing a droplet of a sample containing an analyte to be detected or measured on a plurality of discrete binding zones on a support surface, said plurality of discrete binding zones comprising at least one binding zone containing binding reagents that are the same as one another and that differ in specificity from the binding reagents contained in other binding zones, each of said discrete binding zones being characterized as being hydrophobic or hydrophilic, provided that the area of said support surface surrounding each of said binding zones is (i) hydrophobic if said binding zone is hydrophilic and (ii) hydrophilic if said binding zone is hydrophobic so as to allow said one or more analytes of interest in said sample to bind to said binding zones, and

(b) placing a droplet of a reaction medium on the droplet of the sample; and

(c) determining the presence of the analyte of interest bound to the binding area.

127. A method for detecting or measuring electrochemiluminescence in a sample, the method comprising the following steps performed in the order recited:

(a) Contacting the sample with a surface of a carrier, said surface comprising a plurality of discrete binding domains, said binding domains being spatially aligned with and capable of being brought into proximity with a plurality of electrode and counter electrode pairs;

(b) bringing said binding region adjacent to said plurality of electrode and counter electrode pairs;

(c) applying a voltage waveform effective to trigger electrochemiluminescence; and

(d) detecting or measuring electrochemiluminescence.

128. A method for detecting or measuring electrochemiluminescence in a sample, the method comprising:

(a) contacting the sample with a surface of a carrier, said surface comprising a plurality of discrete binding domains;

(b) scanning a pair of electrodes and counter-electrodes adjacent said binding region over said surface of said support and simultaneously applying a voltage waveform effective to trigger electrochemiluminescence; and

(c) detecting or measuring electrochemiluminescence.

129. The method of claim 109, wherein each of said binding domains is prepared such that it is spatially aligned with and adjacent to a plurality of electrode and counter electrode pairs.

130. A method of detecting or measuring an analyte of interest in a sample, the method comprising:

(a) Contacting the sample with a surface of a support, said surface comprising a plurality of discrete binding domains, said binding domains being spatially aligned with and capable of being brought into proximity with a plurality of electrode and counter electrode pairs, said binding domains (i) containing an electrochemiluminescent label and (ii) capable of binding an analyte of interest, wherein said contacting is under conditions such that binding of any analyte in said sample to said binding domains occurs;

(b) bringing said binding region adjacent to said plurality of electrode and counter electrode pairs;

(c) applying a voltage waveform effective to trigger electrochemiluminescence; and

(d) detecting or measuring electrochemiluminescence, wherein a decrease in electrochemiluminescence relative to electrochemiluminescence observed in the absence of the contacting step or in the absence of any analyte of interest in the sample indicates the presence or amount of the analyte in the sample.

131. A method of detecting or measuring an analyte of interest in a sample, the method comprising:

(a) Contacting a sample with a surface of a support, said surface comprising a plurality of discrete binding domains, said binding domains being spatially aligned with and capable of being brought into proximity with a plurality of electrode and counter electrode pairs, said binding domains being capable of binding an analyte of interest, wherein said contacting is under conditions such that binding of any analyte in said sample to said binding domains occurs, and wherein said sample comprises molecules attached to an electrochemiluminescent label;

(b) bringing said binding region adjacent to said plurality of electrode and counter electrode pairs;

(c) applying a voltage waveform effective to trigger electrochemiluminescence; and

(d) detecting or measuring electrochemiluminescence, wherein an increase in electrochemiluminescence at a background level indicates the presence or amount of the analyte in the sample.

132. A method of detecting or measuring an analyte of interest in a sample, the method comprising:

(a) contacting the sample with a surface of a support, said surface comprising a plurality of discrete binding domains, said binding domains being spatially aligned with and capable of being brought into proximity with a plurality of electrode and counter electrode pairs, said binding domains being capable of binding an analyte of interest, wherein said contacting is under conditions such that binding of any analyte in said sample to said binding domains occurs;

(b) Contacting the binding region with a binding partner for the analyte of interest, wherein the binding partner is linked to an electrochemiluminescent label;

(c) bringing said binding region adjacent to said plurality of electrode and counter electrode pairs;

(d) applying a voltage waveform effective to trigger electrochemiluminescence; and

(e) detecting or measuring electrochemiluminescence, wherein an increase in electrochemiluminescence at a background level indicates the presence or amount of the analyte in the sample.

133. The method of claim 131 wherein steps (a) and (b) are performed simultaneously.

134. The method of claim 131 wherein step (a) is performed prior to step (b); further, the binding region is washed after step (a) and before step (b) to remove unbound analyte.

135. A method of detecting or measuring an analyte of interest in a sample, the method comprising:

(a) contacting a first sample with a surface of a support, said surface comprising a plurality of discrete binding domains, said binding domains being spatially aligned with and capable of being brought into proximity with a plurality of electrode and counter electrode pairs, wherein said sample comprises an analyte of interest linked to an electrochemiluminescent label, wherein said contacting is under conditions such that binding of said analyte in said sample to said binding domains occurs;

(b) Contacting a second sample with said binding region under conditions that allow binding of any analyte in said second sample to said binding region, wherein an electrochemiluminescent label is not attached to any analyte in said second sample;

(c) bringing said binding region adjacent to said plurality of electrode and counter electrode pairs;

(d) applying a voltage waveform effective to trigger electrochemiluminescence; and

(e) detecting or measuring electrochemiluminescence, wherein a decrease in electrochemiluminescence relative to the electrochemiluminescence observed when step (b) is omitted, or when the analyte is absent from said second sample, indicates the presence or amount of said analyte in said sample.

136. The method of claim 131, wherein said sample is derived from a mammal, and wherein said method is used to determine or confirm the identity of said mammal.

137. The method of claim 131, wherein said sample comprises cells from a mammal and said method is used to determine the number of cells in said sample.

138. A method for performing a reaction of interest, the method comprising:

(a) applying a sample to each of a plurality of discrete areas on the surface of a first support, each of said areas being hydrophilic and surrounded by a hydrophobic area on the surface of said first support; and

(b) positioning a second support having a plurality of discrete regions on a surface thereof, each of said regions (i) being hydrophilic and surrounded by a hydrophobic region on said second support surface; (ii) (ii) comprises a reaction medium adapted to carry out a reaction of interest, and (iii) is spatially aligned with the aforementioned regions on the first carrier surface so that each of the regions on the second carrier surface is brought into contact with an aligned region on the first carrier surface, thereby bringing the sample into contact with the reaction medium.

139. The method of claim 109, wherein said plurality of fluid samples are delivered to said monolayer simultaneously.

140. The method of claim 110 wherein said carrier comprises an elastomeric material.

141. The method of claim 119, wherein the patterned self-assembled monolayer comprises an alkanethiol.

142. A method for detecting or measuring an analyte of interest, the method comprising:

(a) contacting one or more of a plurality of discrete binding domains on the surface of one or more supports, wherein said contacting is with a sample comprising molecules linked to an electrochemiluminescent label, characterized in that during said contacting step, said sample does not contact any electrodes or counter electrodes;

(b) bringing an electrode adjacent to one or more of said plurality of binding domains;

(c) applying a voltage waveform effective to trigger electrochemiluminescence at one or more of the plurality of binding domains; and

(d) detecting or measuring electrochemiluminescence.

143. A method for detecting or measuring an analyte of interest, the method comprising:

(a) contacting one or more of a plurality of discrete binding domains, said plurality of binding domains (i) located on the surface of one or more supports, and (ii) spatially aligned and adjacent to a plurality of electrode and counter electrode pairs, wherein said contacting is with a sample comprising a molecule linked to an electrochemiluminescent label;

(b) Bringing one electrode and a counter electrode adjacent to one or more of said plurality of binding domains;

(c) applying a voltage waveform effective to trigger electrochemiluminescence at one or more of the plurality of binding domains; and

(d) detecting or measuring electrochemiluminescence.

144. A cartridge, the cartridge comprising:

(a) an electrode comprising a predetermined amount of material on its surface, the material comprising a binding reagent configured as one or more discrete binding domains, said binding reagent being capable of specifically binding an electrochemiluminescent labeled molecule or a binding pair of an electrochemiluminescent labeled molecule, and said material (i) allowing electron transfer between said electrode and an electrochemiluminescent label in said electrochemiluminescent labeled molecule, said electrochemiluminescent labeled molecule being bound to said binding reagent or to said binding pair when said binding pair is bound to said binding reagent, or (ii) allowing said electrode to generate photons from said electrochemiluminescent label; and

(b) a counter electrode.

145. A cartridge comprising an electrode comprising a predetermined amount of material on a surface thereof, the material comprising binding reagents configured as one or more discrete binding domains, said binding reagents being labeled electrochemiluminescence, and said material allowing transfer of electrons between said electrode and said electrochemiluminescence label.

146. The cartridge of claim 143, wherein the electrodes are porous.

147. The cassette recited in claim 144 wherein said electrodes are porous.

148. A cartridge, the cartridge comprising:

(a) a carrier having one or more discrete binding domains, the binding domains comprising a plurality of binding agents;

(b) a porous ion-permeable substrate, said binding region being attached to said porous substrate;

(c) an electrode in electrochemical contact with said binding region; and

(d) a counter electrode.

149. The cartridge of claim 147, wherein said binding reagent is labeled electrochemiluminescence.

150. A cartridge, the cartridge comprising:

(a) a binding region comprising a binding agent; and

(b) a porous support, said binding region being attached to said porous support, said porous support being an electrode in electrochemical contact with said binding region.

151. The cartridge of claim 149, further comprising a counter electrode.

152. The cartridge of claim 150, wherein said binding reagent is covalently bound to said electrode.

153. A cartridge, the cartridge comprising:

(a) a first support having a first surface, the first surface comprising an electrode; and

(b) A second support having a second surface, the second surface comprising a binding region, said first and second surfaces being spatially aligned for electrochemical contact.

154. A kit, such kit comprising:

(a) a first support having a first surface, the first surface comprising an electrode;

(b) a second support having a second surface, the second surface comprising a binding region, said binding region comprising a predetermined amount of a reagent, said first and second surfaces being spatially aligned for electrochemical contact; and

(c) a vial containing an electrochemiluminescent labeled substance.

155. A cartridge, the cartridge comprising:

(a) a first support having a first surface, the first surface comprising an electrode; and

(b) a second support having a second surface comprising one or more discrete binding domains, said first and second surfaces being spatially aligned so as to enable electron transfer between an electrochemiluminescent label bound to said binding domains and said electrode.

156. The cassette of claim 154, wherein said second surface is a porous material and said cassette further comprises a counter electrode.

157. A cartridge, the cartridge comprising:

(a) an electrode having a first surface;

(b) a counter electrode having a second surface;

(c) a porous material between said first and second surfaces, said porous material containing binding reagents capable of binding an electrochemiluminescent labelled molecule, said porous material and said first surface being spatially aligned so as to allow electron transfer between an electrochemiluminescent label on a molecule bound to one or more of said binding reagents and said first surface.

158. A cartridge comprising

(a) A porous material;

(b) a counter electrode;

(c) a support having a plurality of discrete binding domains thereon, said binding domains being spatially aligned so as to enable electron transfer between an electrochemiluminescent labelled species and said porous electrode, said electrochemiluminescent labelled species being attached to said binding domains.

159. A cartridge, the cartridge comprising:

(a) a porous electrode; and

(b) a counter electrode; the porous electrode comprises a material on its surface, which material comprises binding reagents, said material allowing the transfer of electrons between the electrode and an electrochemiluminescent label, which electrochemiluminescent label is bound to one or more of the binding reagents.

160. A cartridge, the cartridge comprising:

(a) a carrier having a plurality of discrete binding domains thereon;

(b) a compartment for transporting fluid toward and away from the binding region; and

(c) an electrode and a counter electrode spatially aligned to generate an electrochemiluminescent signal from an electrochemiluminescent label bound to the plurality of discrete binding domains.

161. A cartridge, the cartridge comprising:

(a) an electrode comprising a plurality of discrete binding domains on a surface thereof; and

(b) a counter electrode.

162. A cartridge, the cartridge comprising:

(a) a plurality of discrete binding domains on a support;

(b) an electrode; and

(c) a counter electrode capable of generating a plurality of electrochemiluminescent signals from a plurality of electrochemiluminescent labels attached to the plurality of discrete binding domains.

163. The cartridge of claim 161, wherein the plurality of discrete binding domains contain an electrochemiluminescent label, and further comprising means for delivering a sample to the plurality of discrete binding domains.

164. The cartridge of claim 161 further comprising means for delivering a sample to the plurality of discrete binding domains.

165. The cartridge of claim 160, wherein said electrode is capable of generating an electrochemiluminescent signal from an electrochemiluminescent label, said electrochemiluminescent label being bound to at least one of said binding regions.

166. A cartridge, the cartridge comprising:

(a) a carrier having a plurality of discrete binding domains thereon; and

(b) one or more pairs of electrodes and counter electrodes.

167. The cartridge of claim 165 further comprising means for delivering a sample to said plurality of discrete binding domains.

168. A kit, such kit comprising:

(a) an electrode comprising a plurality of discrete bonding areas patterned on a surface thereof;

(b) a compartment for transporting fluid toward and away from said binding regions;

(c) a counter electrode; and

(d) a vial containing a quantity of an electrochemiluminescent labeled molecule.

169. A cartridge, the cartridge comprising:

(a) a plurality of discrete binding domains connected to an electrode, said electrode being adjacent to a porous ion-permeable substrate; and

(b) a counter electrode.

170. A cartridge, the cartridge comprising:

(a) A plurality of discrete binding domains, said binding domains being associated with a porous ion-permeable substrate;

(b) an electrode adjacent to said ion-permeable porous matrix; and

(c) a counter electrode.

171. A cartridge, the cartridge comprising:

(a) a plurality of discrete binding domains, said binding domains being associated with a porous ion-permeable substrate; and

(b) an electrode in electrochemical contact with a plurality of said binding domains.

172. The cartridge of claim 170 further comprising a counter electrode.

173. A cartridge, the cartridge comprising:

(a) a plurality of discrete binding domains, said plurality of binding domains being linked to a porous support; and

(b) an electrode in electrochemical contact with a plurality of said binding domains.

174. A cartridge, the cartridge comprising:

(a) an electrochemical cell comprising an electrode, a counter electrode and an ionic solution in contact with said electrode; and

(b) a support having a plurality of discrete binding domains thereon, said plurality of binding domains being in electrochemical contact with said electrode.

175. The cartridge of claim 143, 144 or 160, wherein said electrodes comprise carbon fibrils.

176. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding reagents in the cartridge of claim 143 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

177. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding reagents in the cartridge of claim 144 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

178. The method of claim 175, wherein the electrodes are porous.

179. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding reagents of the cartridge of claim 147 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) Applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

180. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding reagents of the cartridge of claim 150 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

181. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting a binding region contained on a first surface with a sample comprising molecules linked to an electrochemiluminescent label, said first surface being aligned with a second surface comprising an electrode, said first and second surfaces being spatially aligned for electrochemical contact;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

182. The method of claim 180, wherein said first surface is a porous material.

183. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding reagents of the cartridge of claim 156 with a sample comprising a molecule linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the first surface and the second surface; and

(c) detecting or measuring said electrochemiluminescence.

184. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding regions of the cartridge of claim 157 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

185. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding reagents of the cartridge of claim 158 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode surface and the counter electrode surface; and

(c) Detecting or measuring said electrochemiluminescence.

186. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding regions in the cartridge of claim 159 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode surface and the counter electrode surface; and

(c) detecting or measuring said electrochemiluminescence.

187. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding regions in the cartridge of claim 160 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

188. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding regions in the cartridge of claim 161 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) Applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

189. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting a plurality of discrete binding domains on a support with a sample comprising molecules linked to an electrochemiluminescent label by delivering a sample comprising said molecules to said binding domains, said sample being capable of modulating an electrochemiluminescent signal, wherein said modulation is correlated with the concentration of an analyte of interest in said sample;

(b) applying a voltage waveform at one electrode and the counter electrode effective to trigger electrochemiluminescence from an electrochemiluminescent label, said electrochemiluminescent label being attached to a molecule that binds to at least one of said binding domains;

(c) measuring the electrochemiluminescence; and

(d) determining the concentration of the analyte of interest.

190. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) Contacting those binding reagents of the cartridge of claim 143 with a sample comprising a first molecule and a second molecule linked to an electrochemiluminescent label, wherein the second molecule is a specific binding pair for said first molecule;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

191. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding regions in the cartridge of claim 165 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at least one of the pair of electrodes and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

192. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding regions in the cartridge of claim 168 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) Detecting or measuring said electrochemiluminescence.

193. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding regions in the cartridge of claim 169 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

194. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding regions in the cartridge of claim 171 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) detecting or measuring said electrochemiluminescence.

195. A method for detecting or measuring electrochemiluminescence, the method comprising:

(a) contacting those binding regions of the cartridge of claim 173 with a sample comprising molecules linked to an electrochemiluminescent label;

(b) applying a voltage waveform effective to trigger electrochemiluminescence at the electrode and counter electrode; and

(c) Detecting or measuring said electrochemiluminescence.

196. The method of claim 175, 188 or 189, wherein said electrodes comprise carbon fibrils.

197. A method for performing a plurality of electrochemiluminescence assays, the method comprising the steps of:

(a) contacting a plurality of discrete polymeric matrices with a sample containing one or a plurality of analytes of interest, each polymeric matrix comprising one or more binding domains;

(b) causing one or more of said plurality of regions to electrochemiluminesce;

(c) and detecting electrochemiluminescence.

198. A method according to claim 197, wherein the electrochemiluminescence is detected using a photographic negative.

199. A method for performing a plurality of electrochemiluminescence assays, the method comprising the steps of:

(a) contacting a plurality of binding domains comprising oligonucleotides with a sample containing one or a plurality of analytes of interest;

(b) causing one or more regions of said plurality of regions to electrochemiluminesce; and

(c) electrochemiluminescence is detected at a plurality of the binding regions.

200. The method of claim 199, wherein said plurality of binding regions comprise oligonucleotides linked to electrochemiluminescent labels.

201. The method of claim 199, wherein said plurality of binding domains comprise oligonucleotides linked to electrochemiluminescent labels and one or more of said analytes, which when bound to said oligonucleotides, cause modulation of an electrochemiluminescent signal from one or more binding domains.

202. A method for performing a plurality of electrochemiluminescence assays, the method comprising detecting the presence of less than about 10 concentrations in a multi-component sample^-3The method comprising the steps of:

(a) contacting the plurality of discrete binding domains with a sample containing one or a plurality of analytes of interest;

(b) causing one or more of said plurality of regions to electrochemiluminesce; and

(c) electrochemiluminescence is detected at a plurality of the binding regions.

203. A system for performing a plurality of electrochemiluminescence assays, comprising:

(a) a plurality of binding domains specific for a plurality of different analytes;

(b) a voltage waveform signal generator; and

(c) a photon detector device.

204. A system for performing a plurality of electrochemiluminescence assays, comprising:

(a) An electrode;

(b) a counter electrode;

(c) a plurality of binding domains specific for a plurality of different analytes;

(d) means for triggering electrochemiluminescence; and

(e) a device for electrochemiluminescence detection.