HK1066059B - Apparatus for luminescence assays - Google Patents

Description

Device for luminescence measurement

The application is a divisional application with the application number of CN 92102540.8. The filing date of this parent application is 2/7/1992; the invention provides a method and apparatus for improved luminescence assays.

The present application is a continuation of the copending patent filed on 6.2.1991 and entitled "electrochemiluminescence assay" (application No.: 07/652427, inventor: Massey et al), which is a continuation of the 266882 application filed on 3.11.1988 and is now abandoned, and is also a continuation of the 539389 application filed on 18.6.1990 and is a continuation of the 266882 application. This application is filed concurrently with Massey et al, filed concurrently with the application entitled "method and apparatus for magnetic particle-based luminescence assay with composite magnet" (CMS accession number 370068-3401), which is a continuation of the application with application number 07/652427. All the above related applications are references of the present application.

Technical Field

The present invention relates generally to methods and devices for performing binding measurements, and more particularly to determining analytes of interest by measuring luminescence emitted by one or more labeled compounds in a measurement system, and more particularly to highly sensitive, accurate and reproducible homogeneous or heterogeneous specific binding measurements in which a luminescent component is concentrated in a measurement composition and collected on a detection system prior to the generation of electrochemiluminescence.

Background

Many methods and systems have been studied and developed for detecting and quantifying biochemical and biological analytes. Methods and systems that can measure trace amounts of microorganisms, drugs, hormones, viruses, antibodies, nucleic acids and other proteins are of great interest to researchers and clinical staff.

Using known binding reaction methods, a large number of substances, such as antigen-antibody reactions, nucleic acid hybridization techniques and protein-ligand systems, have been detected and analyzed. The high degree of specificity in many biochemical and biological binding systems has led to a number of measurement methods and systems of great significance for research and diagnostics. Specifically, the presence or absence of an observable amount of label on one or more binding materials indicates the presence or absence of an analyte of interest. Of particular interest are luminescent materials produced from the markers by photochemical, chemical and electrochemical methods. "photoluminescence" is a process by which a material induces luminescence when it absorbs electromagnetic radiation. Fluorescence and phosphorescence are typical examples of photoluminescence. The "chemiluminescence" method is a method of providing luminescence by chemical conversion of energy. "electrochemiluminescence" is the emission of light provided by electrochemical means.

A chemiluminescent measurement technique has been developed in which a sample containing an analyte of interest is mixed with a reagent labeled with a chemiluminescent label, and the reaction mixture is induced so that some portion of the labeled reagent binds to the analyte. After induction, the bound portion of the mixture is separated from the unbound portion, and the concentration of the label in one or both of these portions can be determined by chemiluminescence. The amount of chemiluminescence measured in either or both of these fractions may be indicative of the amount of analyte of interest in the biological sample.

The Electrochemiluminescence (ECL) measurement technique is an improvement over the chemiluminescence technique. The method provides a sensitive and precise measurement of the presence and concentration of an analyte of interest. Using such methods, an induced sample is passed through a voltammetric working electrode to excite luminescence. In a particular chemical environment, this electrochemiluminescence is excited by a voltage applied to the working electrode for a certain time and under certain conditions of the method. The light generated by the label is measured and thus may indicate the presence or amount of analyte. For a detailed description of electrochemiluminescence, see PCT published patent applications: US85/01253(WO86/02734), US87/00987 and US88/03947, which are also incorporated herein by reference.

In performing electrochemiluminescence measurements, it is desirable that no separation step be required in the measurement procedure and that signal modulation for different concentrations of analyte be maximized so that accurate and sensitive measurements can be obtained. In prior methods that do not separate the measurement, microparticles are suspended in the measurement sample and combined with one or more measurement binding components.

U.S. patent 4305925 relates to the detection and determination of clinically relevant proteins and peptides using nephelometry and turbidimetry, and discloses a method involving the binding of antigens or antibodies to latex particles having light scattering or light absorbing effects.

U.S. patent 4480042 relates to a technique using a particulate reagent consisting of a shell-core particle having a shell layer of particles containing functional groups to which biological compounds of interest can be covalently bound; and the high index of reactivity of the shell core creates sensitivity to light scattering measurements. The technology is based on agglutination reactions, which produce agglutinates from bivalent antibodies reacting with multivalent antigens that can be detected and/or measured by various methods.

U.S. patent 4419453 also relates to the use of colored latex agglutination assays that can be used to detect the presence of immunochemicals (e.g., antibodies and immunogens). According to this prior art, it does not appear possible to use microparticles for measuring luminescence phenomena. It is expected that the luminescence produced by free chemiluminescent or electrochemiluminescent moieties may be absorbed, scattered, and even affected by the particulate matter.

Unexpectedly, U.S. Pat. No. 539389(PCT application No. U.S.89/04919) discloses a sensitive specific binding assay for luminescence phenomena in which inert microparticles can specifically bind to a binding reagent in an assay system. The measurement can be carried out in heterogeneous phase (one or more separation steps) and advantageously in homogeneous phase (no separation steps).

Us patent 89/04919 relates to a composition for measuring binding reactions of luminescence phenomena, comprising composite suspended particles having a surface capable of binding to a component in a measurement mixture. In another aspect, a system for detecting or quantitatively analyzing an analyte in a sample is provided, which enables a measurement method using the measurement composition of the present invention. The system includes a means for inducing luminescence from the labeled compound in the measurement medium and a luminescence measurement means for detecting the analyte in the sample.

It has now been found that the combination of a component associated with an electrochemiluminescent moiety in a measurement system with suspended microparticles allows for a large modulation of the intensity of the luminescent signal generated by the electrochemiluminescent moiety associated with the component, thereby providing a means for monitoring specific binding reactions in the measurement system. It has further been surprisingly found that the suspended particles have little or no effect on the intensity of the luminescent signal generated by the electrochemiluminescent moiety which is linked to a component in the system which is not yet bound to the suspended microparticles.

Thus, the method for detecting an analyte in a sample described in U.S. patent 89/04919 includes the steps of:

(1) forming a composition comprising:

(a) a sample containing a sample in which a desired analyte may be present;

(b) a substance for measurement selected from: (i) an analyte or similar analyte to be measured; (ii) an analyte or analyte-like binding partner to be measured; and (iii) a reaction component capable of binding to (i) or (ii), wherein one of the substances for measurement is linked to a labeling compound having a chemical moiety capable of inducing luminescence; and

(c) a composite suspending particle capable of specifically binding to the analyte and/or the substance of (b) (i), (ii) or (iii);

(2) incubating the composition to form a complex comprising the particle and the labeled compound;

(3) inducing luminescence from the labeled compound; and

(4) measuring the luminescence emitted by the composition and detecting the analyte present in the sample.

The same type of method described above can be used to quantitatively analyze the amount of analyte in a sample by comparing the luminescence of a measured composition to the luminescence of a composition containing a known amount of analyte.

Similar analytes may be natural or synthetic, and are compounds having a comparable binding specificity to the analyte, but also include compounds with higher or lower binding capacity. Suitable binding partners for the present invention are well known to the public, e.g. antibodies, enzymes, nucleic acids, lectins, cofactors and receptors. The reactive component capable of binding to the analyte or an analogue thereof and/or to a binding partner thereof may be a secondary antibody or a protein such as protein a or protein G, but may also be avidin or biotin, or other components known in the art capable of participating in a binding reaction.

Luminescence is preferably luminescence generated by Electrochemiluminescence (ECL) induced by the action of a voltammetric working electrode by a labeled compound, whether it is bound or unbound to a specific binding partner. The electrochemiluminescent reaction mixture is controlled to emit light by applying a voltage to the working electrode for a time and in a manner that produces light. While emitting visible light is preferred, the composition or system may also emit other types of electromagnetic radiation, such as infrared or ultraviolet light, X-rays, microwaves, and the like. The terms "electrochemiluminescence", "luminescence", "luminescent" and "emitted light" as used herein all include emitted light and other forms of electromagnetic radiation.

The method described in us patent 89/04919 requires the detection and quantification of very small amounts of analyte in various measurements performed in research and clinical settings. The need for researchers and clinical personnel has made it very urgent to reduce the detection limitations of measurements made using these methods, to increase the sensitivity of these measurements, and to the speed at which they can make measurements.

Various methods are known for enriching various markers prior to their measurement, thereby enhancing the signal produced by them. In us patent 4652333, particles labeled with fluorescent, phosphorescent or atomic fluorescent labels may be concentrated by microfiltration prior to measurement.

Methods for enriching labeled immunochemicals prior to measurement are also known to the public in the art, e.g., magnetically sensitive labeling particles may be moved close to the surface of a measurement vessel. In us 4731337, 4777145 and 4115535, particles are first moved close to the wall of a container and then fluoresce with excitation light.

In us patent 4945045, the particles are concentrated on a magnetic electrode. The electrochemical reaction occurs at an electrode modified by labeling the chemical mediator. When binding occurs, the immunochemical binding reaction changes the efficiency of the mediator generating the modulating signal.

None of the prior art methods relate to the surface-selective excitation method of the present invention. Although surface excitation (e.g., electrochemiluminescence) is not limited by any mechanistic explanation, it is believed that the label on the solid phase complex is necessarily oxidized at the electrode, which requires electrons to move from the label to the electrode. It is also believed that this "jump" in electrons is due to the well-known phenomenon of tunneling, i.e., electrons do not have to "cross" a potential barrier to pass through space (a region of high potential, such as a solution). Electrons can move from one molecule to another or from one molecule to an electrode without additional input of energy through a potential barrier. But this tunneling can only occur over very short distances, with the probability of tunneling decreasing exponentially with the distance between the two increases. If the distance is less than 25A (2.5nm), the probability of a tunneling phenomenon occurring between the two is very high; if the distance is large, the probability is low. The distance of 25A is a rule of thumb used by a person skilled in the art and is not absolutely limiting.

Therefore, only those electrochemiluminescent labels having a 25A electrode surface are expected to participate in the electrochemiluminescence process. In general, the particle area in the surface area of the 25A electrode is very small.

Therefore, it cannot be expected that the electrochemiluminescence generated from the particle surface can be measured in any meaningful amount. But the light generated by the electrochemiluminescence method must pass through the particles to reach the photomultiplier. Since the particles are substantially opaque (a concentrated suspension of particles appears black), even if a large amount of light can be generated by electrochemiluminescence, it is not expected that the light will pass through the particles and be measured by a photomultiplier.

Disclosure of Invention

It is an object of the present invention to provide homogeneous (non-separating) and heterogeneous (separating) methods, reagents and devices for performing binding measurements.

It is a further object of the present invention to provide non-dissociating specific binding measurements, reagents and devices based on measuring the electrochemiluminescence emitted by a measurement composition containing a particulate material.

It is a further object of the present invention to provide such measurements, reagents and devices which have high sensitivity and accuracy, rapid measurement, high specificity and low detection limitations compared to the prior art.

The definition of terms used in this application is: "electrochemiluminescent moiety", "metal-containing electrochemiluminescent moiety", "label", "labeling compound", and "labeling substance" are used interchangeably. The "electrochemiluminescent moiety", "metal-containing electrochemiluminescent moiety", "organometallic compound", "metal chelate", "transition metal chelate", "rare earth metal chelate", "labeled compound", "labeled substance", and "label" are all linked to a molecule, such as an analyte or an analog thereof, a binding partner of the analyte or an analog thereof, and a binding partner of the binding partner, or a reactive component capable of binding to the analyte or an analog thereof or the binding partner, and all of the above are within the scope of the present invention. Each of the above classes can also be linked to one or more combinations of binding partners and/or one or more reactive components. In addition, each of the above classes can also be linked to an analyte or analog thereof that has been bound to a binding partner, a reactive component, or a combination of one or more binding partners and/or one or more reactive components. Also within the scope of the invention are the above classes of compounds that bind to an analyte or analog thereof, either directly or through other molecules as discussed above. In short, these ligands can be used as the measuring substance.

The terms "detection" and "quantitative analysis" both denote measurements, which can be understood as: quantitative analysis may require preparation of the composition of interest and calibration.

The terms "collecting and enriching the complexes" may be used interchangeably to describe the enrichment of the complexes in the measurement composition and the collection of the complexes on the electrode surface.

Description of the drawings:

FIG. 1 shows a measuring chamber for carrying out the non-separation and separation measurements of microparticles according to the invention.

Fig. 2 is a schematic view of a voltage control device in measurement using the measurement chamber of fig. 1.

FIG. 3 shows direct binding PCR using electrochemiluminescent labelled oligonucleotides and biotin electrochemiluminescent labelled oligonucleotides as primers.

FIG. 4 shows a standard PCR using biotin-containing primers to generate biotin-containing PCR products.

FIG. 5 shows asymmetric PRC measurements of biotin-containing single-stranded DNA generated for subsequent hybridization to electrochemiluminescent labeled oligonucleotides.

FIG. 6 shows the specificity study of direct incorporation of electrochemiluminescent labeled oligonucleotides into biotin-containing PCR products.

FIG. 7 shows a standard curve for direct incorporation of electrochemiluminescent labels and biotin-containing oligonucleotides into the HPV16 PCR product.

FIG. 8 shows Ha-ras oncogene point mutations.

FIG. 9 shows of Aa-ras oncogenes³²The specificity of the electrochemiluminescence labeled probe is identified by the P electrochemiluminescence labeled probe.

FIG. 10 shows the determination of point mutations in Ha-ras oncogene³²Determination of the relative value of the electrochemiluminescent marker in the P electrochemiluminescent marker.

FIG. 11 shows a standard curve for a rapid "no wash" hybridization measurement of HPV 18.

Fig. 12 shows a measurement chamber for measuring gravity-precipitated complexes.

FIG. 13 shows the settling distance of the complex as a function of time under the action of gravity, i.e., the settling rate of Dynal particles (Y ═ 0.28+ 0.48X; speed ═ 0.5 mm/min).

FIG. 14 shows the intensity of electrochemiluminescence as a function of time, i.e., the intensity versus time of the thickness of two shims, in a gravimetric chamber with the composition measured at different heights on the counter electrode, where the white dots represent the 0.015 "shim value and the black dots represent the 0.075" shim value.

FIG. 15 shows a comparison of the measurement of the electrochemiluminescence intensity of alpha fetal protein in a gravimetric chamber measuring the composition at different heights on the surface of the counter electrode, i.e., an AFP measurement chamber, where the white dots represent the value of a 0.015 "shim and the black dots represent the value of a 0.075" shim.

FIG. 16 shows a precipitation measurement chamber in which an electromagnet is used to precipitate a composite on the surface of an electrode.

FIG. 17 shows the relative rates of microparticle complex precipitation under the influence of magnetic field and gravity, i.e., the comparison of microparticle precipitation times between magnetic field precipitation and gravity precipitation, where the white dots represent the magnetic field precipitation values and the black dots represent the gravity precipitation values.

Figure 18 shows a collection chamber with permanent magnets.

FIG. 19 shows the increase in ECL intensity as a function of time measured using the collection chamber of FIG. 18, i.e., the effect of collection time on ECL intensity.

Fig. 20 shows the magnetic field lines at the electrode surface as a function of the orientation of the magnet under the electrode surface.

FIG. 21 shows a rotating flow cell in which a complex is centrifugally deposited on the surface of an electrode, a method and apparatus for centrifugation of a captured particle according to the present invention, and a centrifugal flow cell according to the present invention.

FIG. 22 shows detection of evanescent wave fluorescence.

Fig. 23 and 24 show that the measuring chamber and the composite magnet of the magnetic microparticle-based separation or non-separation measuring method of the present invention and the composite magnet of the magnet system generate magnetic lines of force approximately parallel to the electrode surface.

FIG. 25 shows a measurement cell for performing microparticle-based non-separation and separation measurements of the present invention using the working electrode and composite magnet of FIGS. 23 and 24.

In one of the broadest embodiments of the present invention, a method for binding measurement of an analyte in a sample, the measuring step comprising:

(a) a compositional composition comprising:

(i) the sample;

(ii) a measuring substance containing a component linked to a labeling compound capable of inducing luminescence, and

(iii) a composite particle capable of specific binding to an analyte or the measuring substance;

(b) incubating the composition to form a complex comprising the particle and the labeled compound;

(c) collecting the complex at a measurement zone;

(d) inducing luminescence from the labeled compound in the complex by surface-selective excitation; and

(e) measuring the emitted light, thereby measuring the analyte in the sample.

The complexes can be collected on the surface of the electrode where the electrochemiluminescence is excited and induced by applying a voltage across the electrode, or on the surface, and then fluorescence is induced by surface excitation as described below. Surface sensitive techniques for exciting and detecting fluorescent labels have been described for Total Internal Reflection Fluorescence (TIRF), and another surface sensitive technique for measuring labels employs total internal reflection for raman and infrared absorption. Surface plasmon resonance is an optical technique that can be used to measure surface markers according to the present invention. Therefore, the present invention is a method of exciting luminescence using a surface excitation technique.

While the invention is preferably practiced where the complexes are collected at the measurement zone, i.e., where the complexes are collected on a surface capable of producing luminescence, another method for use in the invention is to collect the complexes outside the measurement zone and then place them in the measurement zone or use other steps to induce and measure luminescence.

The collection of the complexes may also be performed by a number of different methods including gravity sedimentation, filtration, centrifugation, and magnetic attraction of magnetically sensitive particles forming part of the complexes. Several embodiments are described in further detail below.

Measuring electrochemiluminescence at the electrode surface using gravity includes:

(a) a compositional composition comprising:

(i) the sample;

(ii) a measuring substance containing a component linked to a labeling compound capable of inducing electrochemiluminescence, and

(iii) composite suspended particles having a density greater than the equilibrium amount of the composition and capable of specific binding to an analyte or the measurement substance;

(b) incubating the composition to form a complex comprising the particle and the labeled compound;

(c) introducing the composition into a measurement chamber;

(d) allowing the composition to reside in the measurement chamber for a sufficient time to allow particles to settle by gravity on the electrode surface, the complex being collected at least at the electrode surface below the major portion of the measurement chamber vessel;

(e) inducing luminescence of the labeled compound in the collected complex by applying a voltage to the electrode; and

(f) the emitted light is measured at the electrode surface, thereby measuring the analyte in the sample.

Continuous or semi-continuous measurements can be made in the flow chamber while batch measurements can be made. In the flow chamber, the solid phase remains in the measurement chamber, while the liquid flows through and out of the measurement chamber. If the solid phase (e.g., particles) is denser than water (greater than 1.0g/ml), gravity acting on the particles causes them to fall to the bottom of the chamber. The chamber may be configured such that the particles sink to the bottom and the liquid flows through the chamber, or the chamber may be configured such that a majority of the sample in the chamber is held in the column chamber above the working electrode of the ECL system. Sufficient residence time in the chamber allows the particles to settle on the electrode surface before ECL is induced.

In a second embodiment of the invention, a measurement composition comprising suspended particles having a density greater than the equilibrium amount of the measurement composition may be centrifuged to move the particles to a measurement area for contact with an electrode to induce electrochemiluminescence, or may be contacted directly with the electrode during the centrifugation step. In this embodiment, the measurement chamber may be provided with means for rapid rotation and encapsulation of the sample. Centrifugal forces cause particles in the sample to move outward from the axis of rotation of the sample housing and to collect on the outer surface of the sample housing. The outer surface of such a sample housing may constitute the working electrode of an ECL measurement system.

In a third embodiment, the particles may be removed from the measurement composition by filtration. In this embodiment, the density of the particles need not be greater than the equilibrium amount of the measured composition. According to the invention, the particles can be separated from the solution and concentrated by filtering the solution, for example by pumping the particles over a filter surface. The surface of the filter may be coated with a thin metal film that can be used as a working electrode in an ECL detection system.

In a preferred embodiment, the suspended particles are magnetically sensitive particles, such as paramagnetic or ferromagnetic particles, and are collected in the measurement zone, preferably directly on the electrode surface, by applying a magnetic field to the particles. The measurement chamber is equipped with a magnet whose magnetic field exerts a magnetic force on the particles as they remain in the batch chamber or flow through the flow chamber, separating them from the bulk of the solution on the surface of the chamber closest to the magnet. If the magnet is configured in a natural orientation and in close proximity to the working electrode of the ECL detection system, particles are concentrated on the surface of the working electrode.

Binding measurements on a variety of heterogeneous and homogeneous formations can be performed by collecting and concentrating the complex on the electrode surface using the methods described above. In heterogeneous binding measurements, the complex should be separated from the composition prior to measuring the luminescence generated by the label. In homogeneous measurements, there is no need to separate the bound (solid phase) and unbound labeling reagents.

In a multi-phase measurement, when the complex is concentrated on the working electrode surface, the measurement signal generated by the label is much larger than the signal without the collection step. In contrast, the signal generated by the uncomplexed labeling reagent is unchanged. Thus, despite the presence of uncomplexed labelled reagent in the measurement chamber, the signal generated by the collected complexes is stronger than the signal of the uncomplexed complexes at the time of measurement. The results of the collection step significantly improve the detection limit of the binding measurements.

In a preferred embodiment of the invention, the homogeneous binding measurement may comprise an in situ separation step. After the measurement composition (i.e. sample, measurement substance and particles) is pumped into the measurement chamber and the complex is trapped by the working electrode, another liquid is pumped into the measurement chamber which does not contain the label or labeling reagent, thereby allowing in situ washing or separation of the complex from unbound components of the measurement composition. The measurement procedure is technically a multiphase combined measurement, but the separation in the measurement chamber has the advantage that it does not require additional separation devices and, in general, the process is faster than the separation carried out externally.

Using the method of the present invention, heterogeneous binding measurements can be made by mixing the components of the measurement composition with each other and reacting them for a predetermined time. The measurement composition is then subjected to separation, which includes separation of the solution from the particles. The electrochemiluminescence in the complex or solution is then measured. Measuring the electrochemiluminescence of the complex after concentration compared to not concentrated may allow for higher accuracy and lower detection limits for the analyte being measured.

The present invention and its objects and features will be more fully understood from the following description of several preferred embodiments.

The present invention can be widely applied to various analytes that can participate in binding reactions. These reactions include: antigen-antibody, ligand receptor, DNA and RNA interactions and other known reactions. The present invention relates to methods and devices for the qualitative and quantitative detection of such analytes in multi-component samples.

Sample (I)

The analyte-containing sample may be a solid, emulsion, suspension, liquid or gas, and may be derived from cells and cell derivatives, water, food, blood, serum, hair, sweat, urine, feces, tissue, saliva, oils, organic solvents, or air. The sample may also be water, acetonitrile, dimethylsulfoxide, dimethylformamide, n-methylpyrrolidone or alcohols, and mixtures thereof.

Analyte

Typical analytes in a sample are whole cell or surface antigens, subcellular particles, viruses, prions, viroids, antibodies, antigens, haptens, fatty acids, nucleic acids, proteins, lipoproteins, polysaccharides, lipopolysaccharides, glycoproteins, peptides, polypeptides, cellular metabolites, hormones, drugs, synthetic organic molecules, organometallic molecules, tranquilizers, barbituric acids, alkaloids, steroids, vitamins, amino acids, sugars, lectins, recombinant or derivative proteins, biotin, avidin, streptavidin, or anomeric molecules. In general, the concentration of the analyte is 10^-3Molar or less than 10^-3Mols, e.g. 10^-12Molar or less.

Substance for measurement

The measuring substance to be combined with the analyte-containing sample contains at least one substance selected from the group consisting of: (i) adding an analyte or an analogue thereof as described above, (ii) a binding partner for the analyte or an analogue thereof, and (iii) a reaction component as described above capable of binding to (i) or (ii), one of the measuring substances being linked to a compound or moiety, such as an electrochemiluminescent moiety capable of inducing luminescence. The labeled substance may be a whole cell or surface antigen, a subcellular particle, a virus, a Prion, a viroid, an antibody, an antigen, a hapten, a lipid, a fatty acid, a nucleic acid, a protein, a lipoprotein, a polysaccharide, a lipopolysaccharide, a glycoprotein, a peptide, a polypeptide, a cellular metabolite, a hormone, a drug, a synthetic organic molecule, an organometallic molecule, a tranquilizer, barbituric acid, an alkaloid, a steroid, a vitamin, an amino acid, a sugar, a non-biological polymer (preferably soluble), a lectin, a recombinant or derivative protein, a biotin, avidin, streptavidin, or an inorganic molecule. In one embodiment, the agent is an electrochemiluminescent moiety that binds to an antibody, antigen, nucleic acid, hapten, short nucleotide sequence, oligomer, ligand, enzyme, or biotin, avidin, streptavidin, protein A, protein G, or complexes thereof, or other secondary binding partner capable of binding to the primary binding partner by protein interaction.

The analyte analog may be natural or synthetic. In general, such compounds should have binding properties comparable to the analyte, but may also be compounds with higher or lower binding capacity. Reactive components capable of reacting with the analyte or an analogue and/or a binding partner thereof and with the electrochemiluminescent moiety via which they are capable of binding to the analyte are preferably secondary antibodies or proteins, such as protein a or protein G, or avidin or biotin, or other components known in the art capable of participating in a binding reaction.

Marker substance

The electrochemiluminescent moiety is a metal chelate, and any metal can be used for the metal of the chelate, such as a metal chelate that is capable of emitting light under electrochemical conditions, i.e. under electrical conditions applied to the reaction system in question. Examples of the metal of such a metal chelate compound include transition metals and rare earth metals. Preferably, ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, chromium or tungsten is used as the metal, preferably ruthenium and osmium.

The ligands attached to the metal of the chelate are usually natural heterocycles or organic compounds which play an important role in determining whether the metal chelate is soluble in water, organic solvents or other non-aqueous solvents. The ligand may be a multidentate compound and may be substituted. The ligands for the bidentate compound include aromatic and aliphatic ligands. Suitable aromatic multidentate ligands include aromatic heterocyclic ligands. Preferred aromatic heterocyclic ligands are nitrogen-containing, such as bipyridine, bipyrazole, terpyridine and phenanthrol. Suitable substituents include alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate, formaldehyde, formamide, cyano, amino, hydroxyl, imino, hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureide, thio, phosphorus-containing, and N-hydroxysuccinimide carboxylate. The chelate may contain one or more monodentate compound ligands, a number of which are known. Suitable monodentate ligands include carbon monoxide, cyanides, isocyanides, halides and aliphatic, aromatic and heterocyclic phosphines, amines, stilbenes and arsines.

Suitable chelates may include: bis [ (4, 4 ' -carbomethoxy) -2, 2 ' -bipyridine 2- [ 3- (4-methyl-2, 2 ' -bipyridin-4-yl) propyl ] -1, 3-dioxolane ruthenium (II); bis (2, 2 ' -bipyridine) [ 4- (but-1-al) -4 ' -methyl-2, 2 ' -bipyridine ] ruthenium (II); bis (2, 2 '-bipyridine) [ 4- (4' -methyl-2, 2 '-bipyridin-4' -yl) -butyric acid ] ruthenium (II); tris (2, 2' -bipyridine) ruthenium (II); (2, 2 ' -bipyridine) [ bis (1, 2-diphenylphosphino) ethylene ] 2- [ 3- (4-methyl-2, 2 ' -bipyridin-4 ' -yl) propyl ] -1, 3-dioxolane osmium (II); bis (2, 2 ' -bipyridine) [ 4- (4 ' -methyl-2, 2 ' -bipyridine) -butylamine ] ruthenium (II); bis (2, 2 '-bipyridine) [ 1-bromo-4 (4' -methyl-2, 2 '-bipyridin-4-yl ] butane ] ruthenium (II), bis (2, 2' -bipyridine) maleimidocaproic acid, 4-methyl-2, 2 '-bipyridine-4' -butanamide ruthenium (II) other electro-luminescent moieties may be found in PCT applications US87/00987 and 88/0394, which are also incorporated herein by reference.

The electrochemiluminescent moiety functions to emit electromagnetic radiation so as to be introduced into the electrochemical energy reaction system. For this purpose, they must be capable of being excited into an excited state and also of emitting electromagnetic radiation, for example photons in light, converted from the excited state. Without wishing to be bound by a theoretical analysis of the mechanism by which the electrochemiluminescent moiety participates in the electrochemiluminescence reaction, we believe that oxidation occurs by the introduction of electrochemical energy into the reaction system and then transformation to an excited state by reaction with the reactants in the reaction system. This state is relatively unstable, and the metal chelate is rapidly converted to a more stable state. The chelate then emits electromagnetic radiation, such as photons in light, which can be detected.

The amount of metal chelate or other metal-containing electrochemiluminescent moiety used in the present invention varies with different systems. Generally, the moiety is used in an amount effective to produce a detectable (if desired, quantitative) emission of electromagnetic energy from the composition or system. In general, the detection and/or quantitative measurement of an analyte is obtained by comparing the luminescence of a sample containing the analyte and an electrochemiluminescent moiety with the luminescence calibrated with known amounts of the analyte and electrochemiluminescent moiety. This is a homogeneous measurement. For multiphase measurements, the measurements are performed after separation prior to electrochemiluminescence analysis, as described above.

It will be appreciated by those skilled in the art that the identity and amount of the metal electrochemiluminescent moiety will vary depending on the particular conditions in different systems. Those skilled in the art, with the benefit of the information provided herein, can empirically determine without undue experimentation suitable metal-containing electrochemiluminescent moieties and sufficient amounts thereof to achieve the desired results.

Granules

The particles have a particulate material with a diameter of 0.001-200 μm (e.g. 0.05-200 μm), preferably 0.1-100 μm, most preferably 0.5-10 μm, and a surface component capable of binding the analyte and/or one or more other substances as described above. The microparticulate material may be cross-linked starch, dextran, cellulose, protein, organic polymers, styrene copolymers such as styrene/butadiene copolymers, acrylonitrile/butadiene/styrene copolymers, vinyl acetate acrylate copolymers or vinyl chloride/acrylic acid copolymers, inert inorganic particles, chromium dioxide, oxides of iron, silicon and silicon mixtures, and proteinaceous materials, or mixtures thereof. Preferably, the particles are suspended in an electrochemiluminescent system. The particles may be or may comprise magnetically sensitive particles.

Measuring medium

In order for the system to be operable by the introduction of electrochemical energy from the electrodes, it is necessary to provide a dielectric which is capable of impregnating the electrodes and which contains an electrochemiluminescent moiety. The dielectric carries charge by ions. Generally, the dielectric is in the liquid phase and is a solution of one or more salts or other species in water, an organic liquid or mixture of organic liquids, or a mixture of water and one or more organic liquids. However, other forms of dielectric may be used in several embodiments of the invention. For example, the dielectric can be a dispersion of one or more substances in a fluid (e.g., a liquid, vapor, or supercritical fluid) or a solution of one or more substances in a solid, vapor, or supercritical fluid.

A suitable dielectric is an aqueous salt solution. Salts sodium or potassium salts may preferably be used, but in several embodiments it may be appropriate to add other cations, as long as the cations do not affect the sequence of the electrochemiluminescence reaction. The anion salt may be a phosphate salt, but other anions may be used in several embodiments of the invention, as long as the anion selected does not affect the sequence of the electrochemiluminescence reaction.

The composition may also be a non-aqueous composition. However, in several embodiments supercritical fluids may be used, and more specifically, dielectrics containing non-aqueous compositions of organic liquids may be used. Like aqueous media, non-aqueous media are also charged by ions. In general, this means that the salt is dissolved in the organic liquid medium. Suitable organic liquids are acetonitrile, dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), methanol, ethanol, or mixtures of two or more thereof. It is noted that tetraalkylammonium salts (e.g., tetrabutylammonium tetrafluoroborate) soluble in organic solvents are used, which together with the above constitute the nonaqueous electrolyte medium.

In some embodiments of the invention, the dielectric is a buffer system, and a commonly used phosphate buffer is desirable. Sodium phosphate-sodium chloride aqueous solution and sodium phosphate-sodium fluoride aqueous solution are examples in this regard.

Other measuring Components

As described in published PCT application US89/04859 (title of the invention: performing an electrochemiluminescence reaction using an amine-derived reducing agent), desirable reducing agents, typically amines or amine-containing moieties (of larger molecules), can be oxidized and converted to highly reducing agents upon spontaneous decomposition. This document is also incorporated by reference herein. It is believed that the amine or amine-containing moiety can also be oxidized by introducing electrochemical energy into the reaction system. The amine or amine-containing moiety loses one electron and then deprotonates or rearranges to convert to a strong reducing agent. The reducing agent reacts with the oxidized metal-containing electrochemiluminescent moiety to bring it to an excited state as described above. To this end, the amine or amine-containing moiety preferably has a residue centered on a carbon atom that contains an electron donated by the carbon atom and an α -carbon atom capable of acting as a proton donor during deprotonation to form a reducing agent. The amine-derived reducing agent provides the excitation source required to convert the metal-containing electrochemiluminescent moiety to its excited state, thereby emitting detectable electromagnetic radiation.

A wide variety of amines or corresponding amine-containing moieties can be used in the practice of the present invention. In general, the choice of amine or amine-containing moiety should be appropriate for the pH of the system in which the electrochemiluminescence analysis is to be performed. Another relevant factor is that the amine or amine-containing moiety should be compatible with the environment in which the effect must be produced when the assay is performed, i.e., compatible with a potentially aqueous or nonaqueous environment. Another factor to consider is the choice of amine or amine-containing moiety that should be formed under specific conditions to form an amine-derived reducing agent sufficient to reduce the oxidized metal-containing electrochemiluminescent moiety in the system.

The amines (and corresponding moieties derived therefrom) useful in the present invention are aliphatic amines and substituted aliphatic amines, such as primary, secondary and tertiary alkyl amines, wherein the alkyl groups each contain 1 to 3 carbon atoms. Tripropylamine, by contrast, is a particularly preferred amine because it emits electromagnetic radiation of particularly high intensity, as shown in several embodiments, to improve the sensitivity and accuracy of detection and quantitative measurements. Also suitable are diamines (e.g., hydrazine) and polyamines (e.g., polyethyleneimine). Examples of other amines suitable for the practice of the invention are: triethanolamine, triethylamine, 1, 4-diazabicyclo (2.2.2) -octane, 1-piperidineethanol, 1, 4-piperazine-bis (ethanesulfonic acid), triisopropylamine and polyethyleneimine.

Generally, the metal-containing electrochemiluminescent moieties used in the present invention are reaction-limiting components. Thus, more specifically, the amine or amine-containing moiety should be provided in excess of the stoichiometric amount. The amine or amine moiety is used in a concentration of 50-150mM, and in order to be able to use it at around pH7, a concentration of 100mM is often advantageous. In several embodiments, the upper concentration of the amine or amine-containing moiety is determined by the maximum solubility of the amine or amine-containing moiety in the use environment (e.g., in water). In general, the amine and amine-containing moieties are used in amounts sufficient to convert the oxidized metal-containing electrochemiluminescent moiety to its excited state, thereby producing luminescence. The amount of amine or amine-containing moiety to be used in a particular assay system can be determined by one of skill in the art based on his own experience and in light of the information provided herein without undue experimentation.

The measurements according to the invention are preferably carried out in the presence of an accelerator, in particular a compound of formula:

wherein R represents hydrogen or C_nH_2n+1R' represents C_nH_2nX represents 0 to 70 and n represents 1 to 20, wherein n is preferably 1 to 4. For example, a commercially available compound of the formula (wherein X represents 9-10) is available under the trade name Triton X-100:

and a compound of the formula (wherein X represents 40) under the trade name TritonN-401 (NPE-40):

generally, the promoter should be used in an amount sufficient to increase the emitted electromagnetic radiation to a desirable level. Specifically, the amount is 0.01% to 5.0%, more specifically 0.1% to 1.0% (V/V).

The electrochemiluminescent moieties used in the present invention induce the emission of electromagnetic radiation by exciting it to an excited state by acting on the system in which the electrochemiluminescent moiety is combined with electrochemical energy. The possibility that oxidation of the electrochemiluminescent moiety and the strong reductant-forming compound can occur depends on their precise chemical structure and factors such as the pH of the system and the nature of the electrode used to introduce the electrochemical energy. The person skilled in the art knows how to determine the best possible and the emission wavelength of an electrochemiluminescence system. Several preferred methods of exciting an electrochemiluminescence system are disclosed in published PCT patent application US89/01814, the contents of which are incorporated herein by reference.

Device for measuring electrochemiluminescence

The apparatus for carrying out the measurement of the invention is shown in figures 1 and 2. A preferred electrochemiluminescent device is disclosed in fig. 1, but the method of the invention is not limited to application of the device 10, and other types of electrochemiluminescent devices may be used that include a working electrode or other triggering surface that provides electrochemical energy to trigger the electrochemiluminescent portion to electrochemiluminescence. When the method of the present invention is performed in a static or flow mode, the device 10 includes a flow-through chamber, which provides significant advantages for a wide variety of samples, including binding measurement samples. Details of apparatus for carrying out the electrochemiluminescence measurements of the present invention are disclosed in PCT applications US89/04854 and US 90/01370.

The apparatus 10 includes an electrochemical chamber 12, a light detection measurement device 14 (which may be a photomultiplier tube), a photodiode, a charge coupled device, a photographic film or emulsion, or the like, and a pump 16 (which may be a peristaltic pump) that moves liquid through and from the chamber 12. Alternatively, a positive displacement pump may be used. An on-off mechanism 18 is provided between the chamber 12 and the photomultiplier tube 14 to control the on operation of the photomultiplier tube 14 only when the chamber 12 is exposed to the electrochemiluminescence measurement. The opening and closing mechanism may be closed (e.g., in operation). The apparatus 10 may also include a light-tight chamber (not shown in FIG. 1) that is comprised of various components that shield the photomultiplier tube 14 from any extraneous light when performing an electrochemiluminescence measurement.

The chamber 12 itself also includes a first assembly block 20 through which a feed tube 22 and a discharge tube 24 extend, preferably constructed of stainless steel. The assembly block 20 has a first outer surface 26 and a second inner surface 28 to define one side of a volume 30 of the chamber 12 holding a sample. Chamber 12 may be purged and/or inspected and/or measured for solution while apparatus 10 is in operation. Feed and drain tubes 22, 24 extend through assembly block 20 from outer surface 26 to inner surface 28 and open to sample chamber 30. The second assembly block 32, which is made of stainless steel, also has a first outer surface 34 and a second inner surface 36. The second assembly block 32 is separated from the first assembly block 20 by an annular gap 38 made of teflon or other non-contaminating material. Thus, the outer surface 34 of the assembly block 32 defines a portion of the other side of the sample chamber 30. Annular space 38 has a peripheral portion 40 and a central bore 42 with an inner edge 44 defining a sidewall of sample chamber 30. The peripheral portion 40 separates the inner surface 28 of the first assembly block 20 from the outer surface 34 of the second assembly block 32 to prevent the solution from flowing out of the sample chamber 30 between the two surfaces 28 and 34. The assembly block 32 also has a central aperture 46 in which a viewing window 48 is sealed to define the remainder of the other side of the sample chamber 30 (i.e., the continuation of the outer surface 34). The material of which the viewing window 48 is constructed should be substantially transparent at the wavelength of the electrochemiluminescence light emitted by the electrochemiluminescence portion. Therefore, the observation window 48 should be made of glass, plastic, quartz, or the like.

Feed tube 22 intersects sample chamber 30 at one end 50 of sample chamber 30 adjacent annular gap 38, and discharge tube 24 intersects sample chamber 30 at another end 52 of sample chamber 30 adjacent annular gap 38. Thus, the combination of feed conduit 22, sample chamber 30, and discharge conduit 24 define a narrow, substantially laminar, continuous flow path for the solution to and through chamber 12 and out of chamber 12. Arrows a and B indicate inflow and outflow of the feed pipe 22 and the discharge pipe 24, respectively.

In one embodiment, working electrode system 54 is disposed on inner surface 28 of first assembly block 20 and includes first and second working electrodes 56 and 58. In another embodiment, one working electrode may be provided, or only electrode 56 may be used as the working electrode. Electrochemical and electrochemiluminescent reactions are performed at the working electrodes 56, 58. The working electrodes 56, 58 are solid voltammetric electrodes and therefore may be made of platinum, gold, carbon, or other materials as desired. The terminal tubes 60, 62, which are connected to the working electrodes 56, 58, respectively, pass through the first assembly block 20.

The connection tubes 60, 62 are connected to a first "working electrode" end 64 of a voltage controller 66 (shown in FIG. 2). Thus, the voltage controller 66 operates as a voltage regulator to provide a voltage signal to the working electrodes 56, 58 and to measure the current drawn therefrom as needed for measuring electrochemiluminescence. In addition, the connection tubes 60, 62 may be connected to respective ends of a voltage controller 66 for respective operations.

The voltage regulator of the voltage controller 66 is also operative via the counter electrode 68 and the reference electrode 70. In one embodiment, the assembly block 32 is made of stainless steel and the counter electrode 68 is on the exposed surfaces 72, 74 of the assembly block 32. The interface formed by counter electrodes 72, 74 and working electrodes 56, 58 applies a voltage to the solution in sample chamber 30 to energize a chemical reaction, excite electrochemiluminescence in the sample, and/or provide energy for decontaminating and examining the surfaces of chamber 12. The counter electrodes 72, 74 are connected via a connecting line 76 to a further "counter electrode" end 78 of the voltage controller 66.

Reference electrode 70 provides a reference voltage from which the voltage applied by working electrodes 56, 58 is generated, e.g., +1.2V vs. Reference electrode 70 is disposed at a location 80 of discharge conduit 24 from chamber 12 and is connected to a third "reference electrode" end 84 of voltage controller 66 by a wiring conduit 82. In the case of three-electrode operation, current may not flow through the reference electrode 70. The reference electrode 70 can be used for three electrode operation to provide uniform, known and stable voltage, and thus is made of silver/silver chloride (Ag/AgCl) or a saturated calomel electrode. The voltage controller 66 can also be operated with only two electrodes, namely the working electrode 56 and the electrode 58 as a counter/reference electrode. In operation with these two electrodes, the counter/reference electrode 58 is electrically connected to the voltage controller terminals 78, 84 on the voltage controller 66. In this case, the voltage controller 66 basically operates as a battery. The voltage controller 66 supplies voltage signals to the working electrode 56 and the counter electrode 58, and may measure the current flowing through each electrode as desired. The reference electrode 70 may also be a so-called "quasi-reference" electrode made of platinum, gold, stainless steel or other material that provides a voltage of poor stability, but which is measurable in terms of solution. In operation with two and three electrodes, reference electrode 70 or 58 can provide a reference for measuring the voltage applied to working electrode 56. A stable voltage reference is now considered to be advantageous. The voltage controller 66, when operating with its voltage regulator, measures the current between the working electrodes 56, 58 and the counter electrodes 72, 74 by controlling the various electrodes based on the known voltage provided on the working electrodes 56, 58 by the reference electrode 70. Voltage regulators for this purpose are known and the internal structure of the voltage controller 66 may correspond to any conventional commercially available voltage regulator having the above-described functions and therefore does not form part of the present invention per se. The structure of the device 10 may also not include an internal voltage controller 66 and may be adapted to interface with an external voltage regulator so as to be separately controllable to provide the desired voltage signals to the electrodes 56, 58, 72, 74 and 70. These voltage signals (used in a manner described below) provide repeatable initial conditions for the surfaces of working electrodes 56, 58 and the surfaces of chamber 12, and this feature may significantly improve the accuracy of the electrochemiluminescence measurements as a whole.

The pump 16 is disposed at the position of the discharge pipe 24, and sucks the sample solution into the feed pipe 22 in the arrow direction a. The solution flows through feed tube 22, sample chamber 30, and discharge tube 24, and then through reference electrode 70, and exits in arrow direction B. In addition, pump 16 may be disposed on feed line 22 to provide for the flow of solution through apparatus 10. All solutions and fluids flowing through chamber 12 use the same flow path, i.e., through feed line 22, sample chamber 30, and discharge line 24, thereby allowing the fluid exiting chamber 12 to be subjected to hydrodynamic silencing. Pump 16 may be controlled to operate to maintain the solution in chamber 12 at any time.

The flow configuration of the device 10 allows the working electrode to receive a variable voltage or maintain a previous operating voltage while one or more solutions are continuously being processed without exposing the working electrodes 56, 58 (or the counter and reference electrodes 72, 74, 70) to air. Exposure to air can cause the circuit to be disturbed by the reference electrode 70, thereby causing unknown arbitrary changes in voltage and destroying the reproducibility of the surface condition of the working electrodes 56, 58. The flow configuration allows for rapid changes between the initial step of performing the cleaning and monitoring electrode system 54 and the measurement step of measuring one or more forms of waves, or for accelerated excitation of electrochemiluminescence.

Fig. 23 and 24 show a chamber housing a magnet system that produces magnetic field lines that are substantially parallel to the electrode surfaces 56, 58 and a magnet 27/37. The magnet system is composed of stacked and oriented permanent magnets or electromagnets with alternate north and south poles of magnets 27/37. The individual magnets of magnet system 27/37 are separated by air or any non-magnetic inductive material. The arrangement according to fig. 23 and 24 allows the magnetic field lines to act on the upper region of the working electrode, which is almost horizontal to the electrode surface. This will align the magnetically sensitive particles on the surface of the electrodes in a certain direction and make it easy to use the electrochemical energy supplied by the electrodes (see fig. 20).

The magnet system 27/37 of fig. 23 and 24 also has the advantage that the magnetic field lines do not extend a significant distance from the magnet structure (see fig. 20), so that the magnetic field generated by the magnet system is unlikely to induce permanent magnets and ferromagnetic material in the vicinity of the electrode assembly, and does not significantly affect the operation of the photomultiplier tubes in the vicinity of the flow cell assembly. The apparatus of fig. 25 is similar to that of fig. 1 and 2 and reference is made to the description of fig. 1 and 2 above, but the apparatus of fig. 25 is disposed vertically below the horizontally oriented electrode 56, or the electrodes 56, 58 are north-south composite magnet systems 27/37 as in fig. 23 and 24.

The invention also relates to reagent compositions. Broadly, the reagent composition may be any of the components of the measurement system of the present invention, namely (a) a dielectric, (b) a marker compound comprising an electrochemiluminescent moiety, (c) a particle comprising a magnetically sensitive particle, (d) an analyte or analogue thereof, (e) a binding partner for the analyte or analogue, (f) a reactive component capable of reacting with (d) or (e), (g) a reducing agent, or (h) an accelerator for the electrochemiluminescence reaction. The reagent may be combined with another commonly used reagent, i.e. a mixture of two, three and more components may be prepared, provided that these components do not react with the other components during storage, so as to impair their effect in the desired measurement. The reagent is preferably a two-component or a mixture of components comprising the particles and one or more other components.

The invention also relates to kits which may comprise a container containing one or more components of one of (a) to (h) above, or which may comprise a container containing one or more reagent compositions comprising a mixture of the above components, all of which may be used in the measurement methods and measurement systems of the invention.

Description of the preferred embodiments of the invention

In carrying out the particle measurement of the present invention, various particles can be used, and generally the particles have a density of 1.0 to 5.0g/mL, preferably a density of 1.1 to 2 g/mL. The choice of the optimum density is within the working reach of the expert in the field, and the sedimentation rate of gravimetric measurement can be suitably chosen between the measurement speed and the composite required to obtain a homogeneous layer on the electrode surface.

Various average diameter particles can be used, and particles having an average diameter of 0.001 to 200 μm (e.g., 0.05 to 200 μm), preferably 0.01 to 10 μm, can be used.

Various concentrations of particles may also be used in the measurement composition, for example a concentration range of 1-10000. mu.g/mL, preferably a concentration range of 5-1000. mu.g/mL may be used. The density, size and concentration of the particles are preferably selected so that the rate of particle precipitation is at least 0.5mm/min, preferably at a faster rate.

In the practice of the invention using filtration, the desired pore size of the filter apparatus is such that 0.01 to 90% of the particles, preferably 10 to 90% of the particles, have an average diameter.

The prior art has described a large number of magnetic particles which can be used for the measurements of the present invention. For example, U.S. patents 4628037, 4695392, 4695393, 4698302, 4554088, british patent 2005019A and european patent 0180384 (all of which are incorporated herein by reference) describe various magnetic particles that can be used successfully. The particles may be paramagnetic or ferromagnetic and may be coated with various materials capable of binding to the binding compound, enabling the magnetic particles to be used for immunoassays. The magnetic particles used in the present invention preferably have a magnetic susceptibility of at least 0.001cgs units, preferably at least 0.01cgs units. The density of the magnetic particles can be in a wide range, substantially lower than that of water, i.e. 0.01-5g/mL, preferably 0.5-2 g/mL. The particle size may be in the range 0.001 to 200. mu.m, for example 0.001 to 100 or 0.05 to 200. mu.m, preferably in the range 0.01 to 10 μm. The concentration of the particles is also very wide and may be in the range of 1-10000. mu.g/mL, preferably in the range of 5-1000. mu.g/mL.

As described in european patent 0180384, the magnetic particles used are preferably of low magnetic resonance, so that after removal of the magnetic field from the electrode surface, the particles are demagnetized and the measurement chamber can be purged. The density, concentration and size of the magnetic particles are selected so that the settling time is at least 0.5mm/min, preferably above this rate. In order not to interfere with the operation of the photomultiplier during operation of the magnetic cell, the magnet assembly is removed from the surface of the magnetic pole prior to inducing electrochemiluminescence.

Measuring

The method of the present invention can be used for various measurements.

A measurement was made as shown in figure 3. PCR products generated by the reaction Using Biotin and ECL marker (tris (2, 2' -bipyridine) Ru II, Ru (bpy)₃ ²⁺) And (6) marking. The bifunctional DNA is captured by streptavidin beads using a binding method with biotin (streptavidin), followed by washing, and the beads of bound product are then subjected to an assay for detection of ECL markers.

A measurement was made as shown in figure 4. Biotin-containing PCR products were captured on streptavidin beads and biotin-free chains were removed. The PCR product-bound beads were then contacted with ECL-labeled (Ru (bpy))₃ ²⁺) The oligonucleotide is hybridized, followed by ECL analysis to detect the label.

A measurement was made as shown in figure 5. The hybrids were captured on streptavidin beads and then analyzed for ECL without washing.

One measurement was performed and the results obtained are shown in fig. 6. This measurement is carried out using DNA samples isolated from SiHa and HeLa cell lines which are positive for the virus type and oligonucleotides specific for each virus type to detect the presence or absence of HPV16 and 18. Primers 2PV16 and 2PV18 contained biotin, while primers 3PV16 and 3PV18 were ECL-labeled oligonucleotides. 2/3PV16 and 2/3PV18 oligonucleotides are specific for HPV16 and 18, respectively. ECL in the resulting bead-captured ECL labels was analyzed using the analyzer shown in fig. 1. The results are shown graphically as the amount of ECL in each primer combination sample.

One measurement was performed and the results obtained are shown in fig. 7. The resulting beads were analyzed for ECL using the analyzer shown in fig. 1, with ECL labels bound. The peak of ECL photons is shown graphically in terms of increased HPV16DNA concentration (ratio of viral copies to total cellular DNA copies). Primers used for the analysis of HPV16 were 1PV16 (biotin marker) and 2PV16(ECL marker). The DNA used for each PCR was kept constant at 1. mu.g with calf thymus DNA.

One measurement was performed and the results obtained are shown in fig. 8. PCR measurements were performed with biotin-containing HRP2 (probes 1T24 and 1CHR) with unlabeled HRP1 and biotin-containing HRP1 (probes 2T24 and 2CHR) with unlabeled HRP2, yielding a bead-bound single-stranded target for hybridization. The DNA samples were normal (placental) Ha-ras gene and mutant (NIH3T3-T24) Ha-ras gene. The bead-bound DNA was then washed with TEMAC for hybridization with ECL-labeled 1T24(1T24), ECL-labeled 2T24(2T24), ECL-labeled 1CHR (1CHR) and ECL-labeled 2CHR (2CHR), and the resultant bead-bound ECL label was analyzed for ECL using the analyzer shown in FIG. 1. The results are shown graphically as the amount of ECL for each sample probe combination.

One measurement was performed and the results obtained are shown in fig. 9. PCR measurements were performed with biotin-containing HRP2 (probes 1T24 and 1CHR) with only unlabeled HRP1 as shown in FIG. 8. The probe used is a probe containing³²P1T 24 and 1CHR (1T24-P, 1CHR-P) served as controls. By containing³²1T24 and 1CHR for P and ECL markers the effect of ECL markers was determined. The samples were washed with TEMAC as previously described. Adding scintillation cocktail to scintillation counter and analyzing the resulting beads for binding³²And P. Results graphically represent the per second combination of sample probes³²P number.

One measurement was performed and the results obtained are shown in fig. 10. Measurements were made as described in figure 4. The sample used was placental DNA and was amplified with biotinylated HRP2 (probes 1T24 and 1CHR) with unlabelled HRP 1. Sampling the PCR products to obtain a set of samples containing different amounts of the products, and combining the samples³²P-labeled probes (1T23-P32 and 1CHR-P32) or with ECL markers (1T24-ECL and 1CHR-ECL) and then normalizing the results with the mean peak of each marker for 90. mu.l of sample, these normalization profiles allow for more effective comparison of signal to background and to the comparison reaction of the two methods. The lower amount of the dilution curve is shown in the inset to illustrate the response. The samples were processed as described above (fig. 6 and 8).

One measurement and the results obtained are carried out according to fig. 11. PCR measurements were performed with biotin-containing 2PV18 and unlabeled 1PV18, HeLaDNA (400 copies per cell) using the PCR described in FIG. 3. The resulting PCR reaction was then hybridized with the specific probe ECL-labeled 3PV 18. The hybridization mixture was applied to the avidin-coated beads and the resultant bead-bound ECL labels were directly analyzed by ECL analyzer as described in fig. 1. The results are graphically represented as a correlation between the amount of ECL and the copy number of HPV18 added to the PCR.

The following are non-limiting examples which are intended to illustrate, but not limit, the invention. It will be apparent that various changes may be made without departing from the spirit and scope of the invention.

Example (b): apparatus, materials and methods

(1) Instrument for measuring the position of a moving object

As in the flow direction device shown in fig. 1 and 2, three electrodes are used.

A working electrode: au flakes with a diameter of 3mm

A counter electrode: au flakes with a diameter of 3mm

Reference electrode: Ag/AgCl

Teflon gasket: 3.81mm (0.15') thick

Organic glass panel

Feeding a pipe: 1.0668mm (0.042'), polypropylene

Air extraction rate: 0.01-5 mL/min (variable)

Voltage stabilizer: a microprocessor-controlled luminometer using Hamamatsu R374 PMT (low gain red sensitive photocell); the variable voltage of PMT is 0-1400V

(2) Material

(a) Electrochemiluminescent markers: ru (bpy)₃ ²⁺

(b) Electrochemiluminescence buffer: 112mM KH₂PO₄，88mM K₂ HPO₄·3H₂O，50μM NaCl，6.5mM NaN₃0.8 μ M Triton X-100, 0.4mM Tween 20, 100mM tripropylamine in water

(c) Electrochemiluminescence diluent: 37.5mM KH₂PO₄，109.2mM K₂HPO₄·3H₂O，151.7mM NaCl，0.65mM NaN₃0.43mM bovine serum albumin in water

(d)Ru(bpy)₃ ²⁺-NHS: ru (2, 2 ' -bipyridyl) (4- [ 3- (1, 3-dioxolan-2-yl) propyl ] -4 ' -methyl-2, 2 ' -bipyridine)²⁺

(e) Dynal particles

(i) Dynal M-450 Dynabeads, superparamagnetic particles of 4.5 μ M diameter, 30mg/mL were supplied by Dynal (45 North Station Plaza, Great rock, NY 11021)

(ii) Dynal M-280 Dynabeads, 2.8. mu.M diameter superparamagnetic particles, 10mg/mL were supplied by Dynal (45 North Station Plaza, Great rock, NY 11021)

(3) Electrogenerated chemiluminescence cycle (three electrode chamber operation)

The electrochemiluminescence cycle consists of three steps: (1) pretreatment, (2) measurement, and (3) purification. The pre-treatment step includes changing the voltage triangle wave from 0.0V- +2.2V to-1.0V- +0.6V at a rate of 2.0V per second. The measuring step includes changing the triangular wave from +0.6V- +2.8V to +2.0V at 1.0V per second. The purification step comprises changing the voltage square wave from +0.0V- +3.0V to-0.5V-0.0V. All voltages were referenced to an Ag/AgCl reference electrode.

Example 1: apparatus and method for gravity collection of microparticles

Measurements were taken in the chamber shown in FIG. 12, and FIG. 12 illustrates a device for measuring using gravity comprising a transparent window 48, a spacer 222, a deposition chamber 20 (including a feed inlet 22, a working electrode 56/58, a counter electrode 72/74, and a discharge outlet 24). The deposition chamber was horizontal, i.e. perpendicular to the earth's gravitational field, and the labeled microparticles (Dynal) in ECL buffer were transported to the deposition chamber by a peristaltic pump, after the particles were transported to the chamber, the pump was turned off, the microparticles in the chamber settled on the surface of the working electrode, and the settling of the microparticles settled at a rate of about 10mm at 0.5mm/min (as shown in fig. 13). The amount of particle settling is a function of settling time and rate, and the ECL intensity is directly proportional to the amount of particles settling on the working electrode. The amount of particles falling to the surface and hence the ECL strength is limited by the height of the liquid sample relative to the working electrode. FIG. 14 shows ECL intensity as a function of deposition time for 2 chambers of different shim thickness (0.381 and 1.905mm [0.015 and 0.075 inches ]), with similar microparticle deposition rates for the 2 chambers, but with the maximum reading for the thicker shim being 5 times that for the thinner shim. The results of the AFP (alpha-fetoprotein) measurement for 2 chambers are shown in fig. 15, with the chambers additionally padded thicker yielding 5 times the ECL signal intensity.

Example 2: electrochemiluminescence apparatus and method for precipitating microparticles

Magnetic collection with deposition chamber

A deposition chamber for performing the measurement using the magnetically deposited microparticles of fig. 16. Reference numeral 48 in FIG. 16 denotes a transparent window, 122 denotes a spacer, 22 denotes a feed port in the deposition chamber, 56, 58 denotes a working electrode, 24 denotes a sample discharge port, 20 denotes the deposition chamber itself, and 27 denotes an electromagnet.

The plane of the deposition chamber was horizontal and the labeled microparticles (Dynal) in the electrochemiluminescence buffer were transported to the deposition chamber by a peristaltic pump, which was turned off after the microparticles were transported to the deposition chamber. The magnetic field generated by the electromagnet 27 is used to transport the microparticles in the deposition chamber to the working electrode, and the electromagnet can operate at 12V and 1.5A. Due to the use of electromagnets, the deposition rate of the microparticles far exceeded that of particles deposited by gravity alone (as shown in fig. 17).

Example 3: electrochemiluminescence apparatus and method for depositing microparticles

Magnetic collection with a Collection Chamber

The measurements were performed in a collection chamber as described in figure 18. Reference numeral 48 in FIG. 18 denotes a transparent window, 132 denotes a spacer, 22 denotes a feed port in the collection chamber, 56, 58 denote working electrodes, 20 denotes the collection chamber itself, 24 denotes a sample discharge port, and 37 denotes a permanent magnet.

The plane of the collection chamber was oriented horizontally and the labeled microparticles (Dynal) in electrochemiluminescence buffer were transported to the electrochemical chamber using a peristaltic pump, with permanent magnets 37 fixed directly below the working electrode/solution interface at 0.889mm (0.035 inch) intervals prior to introduction of the sample. When the sample is delivered to the electrochemical cell, the microparticles are deposited on the surface of the working electrode defined by the area of the magnet.

After all samples were deposited, the pump was turned off and the magnetic field was removed. The longer the collection time, the more particles are deposited. The intensity of electrochemiluminescence increases with increasing concentration of particles at the working electrode (as shown in figure 19).

Example 4: deposition of microparticles using magnets

Orientation of magnetic field

As shown in fig. 20, electric fields 98 and 98 ', microparticles 96, 96' attracted to magnet 27/37 (whether it be a permanent magnet or an electromagnet) are aligned with the direction of magnetic field 98, 98 ', resulting in particle alignments 96 and 96' parallel (a) and perpendicular (B) to, and near, the surface of working electrode 56/58.

Example 5: filtration to collect and enrich particles

Microparticles of a wide range of magnetic, non-magnetic and density can be collected by filtration through the surface of a membrane filter. In one embodiment of the invention, the particles are pumped through a filter membrane having a pore size smaller than, preferably substantially smaller than, the diameter of the particles and a sufficiently high surface density that particle collection does not block the pores of the membrane. The membrane filter preferably has a high transparency to allow the membrane filter to be placed on the surface of the working electrode after collection of the particles to induce electrochemiluminescence and measure luminescence from the particles, thereby quantitatively measuring ECL label on the particles.

In another embodiment, a membrane filter having the above-described pore sizes is attached or disposed on the surface of the adsorbent material such that capillary action naturally flows the liquid containing the microparticles through the membrane filter without any means for flowing the liquid through the filter.

In a preferred embodiment, a membrane filter having the above-described pore sizes is coated with a thin metal film or other conductive material, allowing the membrane surface to be used as a working electrode in an ECL device. The conductive film is readily applied to the film by conventional methods in the fabrication of microelectronic devices, such as thermal evaporation or spray coating. Such a filtering electrode is easily assembled in the flow chamber such that the flow path of the liquid passes through the filtering electrode. The particles in the liquid stream are captured by the filtering electrodes and are easily washed in situ, thereby providing a fast and simple method for performing multi-phase measurements without the need for any additional washing equipment.

Example 6: collecting and enriching particles by centrifugation

The rotating flow cell shown in FIG. 21 provides another method for luminescence measurements with complexes trapped on the surface of the working electrode. The measurement solution enters the device through the inlet 261 and is pumped into the chamber 262 through the rotary seal 263, with a rotary motion into the chamber in the direction indicated by arrow R. The denser particles of the composite are concentrated on the surface of working electrode 264. While the chamber continues to rotate, the solution flows out of the chamber through the discharge opening 268. The output light flowing through the transparent window of the chamber is measured by a photomultiplier tube 265. The output light is reflected by the vertically disposed working electrode surface 264 to a curved mirror surface 266 located at the center of the chamber, which is also shown as counter electrode 269. The chamber is then rinsed and cleaned for the next cycle. This operation can be performed while stopping or rotating the chamber. FIG. 21 shows a centrifugation method and apparatus for trapping particles of the present invention and a centrifugation flow cell of the present invention.

Example 7: coating particles with moderate surface concentrations of labeled non-specific proteins

30mg (1ml) of 4.5 μ M uncoated magnetically sensitive polystyrene M-450 DYNABEADS (DYNAL, Oslo, Norway) were washed with 2ml of solution per wash by magnetic separation with 150mM phosphate buffer (pH 7.5). 150 μ g Ru (bpy)₃ ²⁺Phosphate buffered saline (PBS, 1ml) labeled mouse IgG (Jackson immunochemicals) and 0.05% thimerosal were added to the particles, the mixture was allowed to incubate overnight with rotation at room temperature, and then the solution was magnetically separated from the particles and removed. To block unreacted sites, 1ml of 3% BSA/PBS and 0.05% sodium azide were added to the particles, and the resulting solution was incubated at room temperature for 2 hours, the particles were washed 5 times with 2ml each, and then suspended in 6ml of the same buffer for storage.

Example 8: measurement of electrochemiluminescence with magnetically sensitive particles

Homogeneous and heterogeneous, polymeric and non-polymeric magnetically sensitive particles were coated with the marker protein described in example 7 (Dynal, Oslo, Norway; Polysciences, Warrington, PA 18976; Cortex Biochem, San Leandro, CA 94577; Aldrich, Milwaukee, WI 53201). The coated particles were washed 3 times with electrochemiluminescence buffer and then 2ml of 300. mu.g/ml suspension was prepared. Mu.l of the particle suspension was delivered to the flow cell using a peristaltic pump (example 3). As the particles flow toward the working electrode, they are attracted and concentrated on the surface of the working electrode due to the magnetic action. The electrochemiluminescence of the magnetic particles was measured with a Hamamatsu R374 photomultiplier tube placed in the upper center of the flow cell (where the particles are concentrated on the surface of the working electrode). Table I gives the light emission of electrochemiluminescence obtained from the magnetically sensitive particles coated with the marker protein.

Table I: electrochemiluminescence measurements of different magnetically sensitive particles

Particle size diameter (. mu.m) Density (g/ml) raw Material electrochemiluminescence dose

Glass 8.02.4 soda lime glass 2200

2.02.4 soda lime glass 8500

0.3-3.52.5 SiO quartz₂ 1150

Gold 1.0-2.019.3 Au 1100

Example 9: measurement of electrochemiluminescence with non-magnetic particles

Homogeneous and heterogeneous, polymeric and non-polymeric non-magnetically sensitive particles were coated with the marker protein described in example 7 (Aldrich, Milwaukee, WI 53201 Duke Scientific, Palo Alto, CA 94303). The coated particles were washed 3 times with electrochemiluminescence buffer and then 2ml of 300. mu.g/ml suspension was prepared. 500 μ l of the particle suspension was delivered to the flow cell using a peristaltic pump. The coated particles were gravity-concentrated on the working electrode as described in example 1. Electrochemiluminescence of the non-magnetic particles was measured with a Hamamatsu R374 photomultiplier tube placed in the upper center of the flow cell (where the particles are concentrated on the surface of the working electrode). Table II gives the resulting light emission of electrochemiluminescence from the coated non-magnetic particles.

Table II: measurement of electrochemiluminescence from gravity-collected non-magnetically sensitive particles

Particle size diameter particle density feedstock electrogenerated chemistry

Luminescence (. mu.m) (g/ml)

Rhone-Poulenc 4.01.5 polystyrene divinylbenzene 1680

/Fe₃O₄

1.5-2.11.4 polystyrene/Fe₃O₄ 462

Polysciences 1.5-2.12.1 polystyrene/FeO₂ 504

Dynal 4.51.5 polystyrene/Fe₂O₃ 4200

Cortex 1.0-101.3 cellulose/Fe₃O₄ 125

1.0-101.8 polyacrolein/Fe₃O₄ 125

1.0-251.2 polyacrylamide/Fe₃O₄W 125

Activated carbon

Nickel 3.08.9 Ni 125

Example 10: preparation of physisorbed goat antithyroid hormone (TSH) -coated Dynal particles (reagent I)

1ml of 4.5 μ M uncoated magnetic polystyrene particles having-OH residues on the surface (DYNAL, DYNABEADS M-450, DYNAL A.S. Oslo, Norway) were washed with 2ml each time by magnetic separation with a 150mM sodium carbonate/sodium bicarbonate solution (pH 9.6). 0.5mg of affinity purified goat anti-TSH, HCG purified antibody (CIBA) in sodium carbonate/sodium bicarbonate solution (1ml) was added to the particles. The mixture was incubated at room temperature overnight, then the solution was separated from the particles by magnetic force and the solution was removed. 1ml of 3% BSA/PBS W/0.05% overlapping sodium nitrogen was added and incubated at room temperature for 2 hours with stirring to block unreacted sites. The particles were washed 5 times (2 ml each time) and then suspended in 1ml of the same buffer and stored until use. The final concentration of reagent I was 3% by weight.

Example 11: preparation of ouabain-BSA conjugate (reagent II)

Activation of Orthointusin

A solution of 60.4mg of ouabain octahydrate (Aldrich Cat #14, 193-3) in deionized water (6ml) (film-wrapped) was mixed with 87mg of sodium meta-periodate (Mallinckrodt Cat #1139) and the mixture incubated with rotation at room temperature for 2 hours. The reaction mixture was quenched by passing it through Dowex 1X 8-50 ion exchange resin (Aldrich Cat #21, 740-9) charged with deionized water. 200. mu.l of 1M sodium phosphate (pH7.2) was added to adjust the pH of the solution to 7.0.

Activated ouabain binding to BSA

50mg of activated ouabain (4.6ml) was added dropwise to a solution of 108mg of Bovine Serum Albumin (BSA) (Miles Fraction V) in PBS (5ml, 0.15M, pH7.8) in a ratio of ouabain to BSA of 40: 1. The reaction was incubated at room temperature for 2 hours. 30mg of sodium cyanoborohydride was added rapidly while mixing. Free ouabain and excess sodium cyanoborohydride were removed by dialysis at 4 ℃ in sodium diazoxide (0.15M PBS W/0.05%) at pH 7.8. ouabain-BSA binding reagent II was stored at 4 ℃.

Example 12: preparation of physically adsorbed ouabain-BSA coated Dynal particles (reagent III)

5mg of 4.5 μ M uncoated magnetic polystyrene particles having-OH residues on the surface (DYNAL, DYNABEADS M-450, DYNAL A.S. Oslo, Norway) were washed with 150mM sodium carbonate/sodium bicarbonate solution (pH9.6) by magnetic separation with 10ml per wash. A sodium carbonate/sodium bicarbonate solution (5ml) of 3mg of ouabain-BSA conjugate (binding agent II) was added to the particles. The mixture was incubated overnight at room temperature with rotation and the solution was removed after magnetic separation from the particles. 5ml of 3% BSA/PBS W/0.05% overlapping sodium nitrogen was added, incubated at room temperature for 2 hours, and spun to block unreacted parts. The particles were washed 5 times (10 ml each time) and finally suspended in 1ml of the same buffer and stored for further use. The final concentration of reagent III was 3% by weight.

Example 13: ru (bpy)₃ ²⁺Preparation of labeled murine anti-digoxin (reagent IV)

Using Ru (bpy)₃ ²⁺Labeled 1mg of mouse anti-digoxin (Cambridge medical technologies Cat #200-014 Lot A3575), buffer exchanged the monoclonal antibody (anti-digoxin antibody) into 0.15M potassium phosphate buffer (0.15M NaCl, pH7.8) using a Centricon 30 microconcentrator (Amicon) at a final volume of 0.5 ml. Dissolve 0.5mg Ru (bpy) in 125. mu.l of anhydrous dimethylsulfoxide (Aldrich)₃ ²⁺-NHS ready for use. 0.18mg of Ru (bpy)₃ ²⁺-NHS (45. mu.l) was vibrationally added to the protein solution to give 25: 1 molar ratios of Ru (bpy) of 1057 and 150000, respectively, based on molecular weight₃ ²⁺: a protein. The reaction tubes were incubated with shaking at room temperature in the dark for 30 minutes. The reaction was stopped by adding 25. mu.l of 1M glycine and incubated for a further 10 minutes. The reaction mixture was purified by Sephadex G-25 column (1X 20cm of 0.15M potassium phosphate, 0.15M NaCl and 0.05% sodium azide, pH 7.2). Collection and precipitation of Ru (bpy)₃ ²⁺The labeled murine anti-digoxigenin moiety, labeled protein (reagent IV), was assayed with 12 labels per protein molecule.

Example 14: ru (bpy)₃ ²⁺Preparation of labeled murine antithyroid hormone (TSH) (reagent V)

Using Ru (bpy)₃ ²⁺0.5mg of mouse anti-TSH (CIBA) was labeled, and the anti-TSH antibody was buffer-exchanged into 0.15M potassium phosphate buffer, 0.15M NaCl (pH7.8) using a Centricon 30 microconcentrator (Amicon) to give a final volume of 0.35 ml. Will be 0.5mg Ru(bpy)₃ ²⁺the-NHS was dissolved in 75. mu.l of anhydrous dimethylsulfoxide (Aldrich) and used. 0.176mg of Ru (bpy)₃ ²⁺-NHS (26.4. mu.l) was added to the protein solution with shaking to give 50: 1 molar ratio of Ru (bpy) based on molecular weight of 1057 and 150000 respectively₃ ²⁺A marker: a protein. The reaction tubes were incubated with shaking at room temperature in the dark for 30 minutes. The reaction was stopped by adding 25. mu.l of 1M glycine and incubated for a further 10 minutes. The reaction mixture was purified by Sephadex G-25 column (1X 20c M, 0.15M potassium phosphate, 0.15M NaCl and 0.05% sodium azide, pH7.2), and Ru (bpy) was collected and precipitated₃ ²⁺Labeled murine anti-TSH moieties. The labeled protein (reagent V) was assayed with 14 labels per protein molecule.

Example 15: one-step split sandwich assay for Thyroid Stimulating Hormone (TSH)

Mu.l of calibration serum (London Diagnostics TSH Lumi TAG Kit), 25. mu.l of Ru (bpy)₃ ²⁺After combining the ECL buffer solution of labeled mouse anti-TSH (reagent V) and 25. mu.l of ECL buffer solution of sheep anti-TSH-DYNAL particles (reagent I), the mixture was incubated in a polypropylene tube at room temperature for 15 minutes, and the particles were washed by magnetic separation, suspended in 500. mu.l of ECL buffer solution, and the washing step was repeated 2 more times. The particles were finally suspended in 1ml of ECL buffer and the electrochemiluminescence of each sample was read as described in example 3. The amount of electrochemiluminescence is proportional to the concentration of the analyte in the sample (the amount of electrochemiluminescence increases proportional to the concentration of the analyte). Table III gives the values of the measurement curves.

Table III: one-step separation interlayer measurement: detecting TSH

TSH concentration electrochemiluminescence dose

(mu IU/ml) (duplicate samples)

0.00 1918 1885

0.05 2584 2530

0.10 3365 3288

0.50 8733 8652

2.50 35688 35347

10.0 125316 136994

25.0 300248 288272

50.0 549034 564948

Example 16: one-step non-split sandwich assay for Thyroid Stimulating Hormone (TSH)

Mu.l of calibration serum (London Diagnostics TSH Lumi TAG Kit), 25. mu.l of Ru (bpy)₃ ²⁺After combining the ECL buffer solution of labeled mouse anti-TSH (reagent V) and 25. mu.l ECL buffer solution of goat anti-TSH-DYNAL particles (reagent I), the mixture was incubated in a polypropylene tube at room temperature for 15 minutes and mixed, before obtaining the result, 1ml ECL buffer solution was added, and then the number of electrochemiluminescence of each sample was read as described in example 3. The amount of electrochemiluminescence is proportional to the concentration of the analyte in the sample (the amount of electrochemiluminescence increases proportional to the concentration of the analyte). Table IV gives the values of the measurement curves.

Table IV: one-step measurement without interlayer separation: detecting TSH

TSH concentration electrochemiluminescence dose

(mu IU/ml) (duplicate samples)

0.00 2610 2769

0.05 2870 2894

0.10 2970 2950

0.50 3473 3403

2.50 5588 5495

10.0 13051 13139

25.0 26468 27306

50.0 47104 48664

Example 17: two-step separation and comparison determination of digoxin

Mu.l of calibration serum (TDx Assay, Abbott Labs, Chicago, IL) and 25. mu.l of Ru (bpy)₃ ²⁺After combining the labeled murine anti-digoxin (reagent IV) ECL buffer solutions, incubation for 20 minutes at room temperature for mixing, adding 25. mu.l ECL buffer solution of ouabain-BSA-DYNAL particles (reagent III), incubation for 20 minutes at room temperature for mixing, washing the particles by magnetic separation, suspending the particles in 500. mu.l ECL buffer solution, and repeating the washing step 2 more times. The particles were finally suspended in lml ECL buffer and the electrochemiluminescence number of each sample was read as described in example 3. The amount of electrochemiluminescence is inversely proportional to the concentration of the analyte in the sample (the amount of electrochemiluminescence decreases as the concentration of the analyte increases). Table V gives the values of the measurement curves.

Table V: two separate comparative measurements: detection of digoxin

Digoxin concentration electrochemiluminescence

(ng/ml) (double samples)

0.0 22031 21154

0.5 13367 13638

1.0 9506 9607

2.0 5244 5129

3.0 2959 2994

5.0 1581 1631

Example 18: two-step no-separation comparative determination of digoxin

Mu.l of calibration serum (TDx Assay, Abbott Labs, Chicago, IL) and 25. mu.l of Ru (bpy)₃ ²⁺After combining the labeled rat anti-digoxin (reagent IV) ECL buffer solutions, incubating for 20 minutes at room temperature for mixing, adding 25. mu.l ECL buffer solution of ouabain-BSA-DYNAL particles (reagent III), incubating for 20 minutes at room temperature for mixing, suspending the particles in 1ml ECL buffer solution before reading, and reading the electrochemiluminescence number of each sample according to the method described in example 3. The amount of electrochemiluminescence is inversely proportional to the concentration of the analyte in the sample (the amount of electrochemiluminescence decreases with increasing concentration of the analyte). Table VI gives the values of the measurement curves.

Table VI: two-step inseparable comparative measurements: detection of digoxin

Digoxin concentration electrochemiluminescence

(ng/ml) (double samples)

0.0 42051 39643

0.5 28721 28074

1.0 22190 21364

2.0 14660 14542

3.0 11315 11893

5.0 9161 8945

Example 19: washing of the final reaction sample on the electrode by a read cycle for digoxin two-step no-separation comparative assay

Mu.l of calibration serum (TDx Assay, Abbott Labs, Chicago, IL) and 25. mu.l of Ru (bpy)₃ ²⁺After combining the ECL buffer solutions of labeled murine anti-digoxin (reagent IV), incubating for 20 minutes at room temperature for mixing, adding 25. mu.1 ECL buffer solution of ouabain-BSA-DYNAL particles (reagent III), incubating for 20 minutes at room temperature for mixing, suspending the particles in 1ml of ECL buffer solution before reading, and reading the electrochemiluminescence number of each sample as described in example 3. The amount of electrochemiluminescence is inversely proportional to the concentration of the analyte in the sample (the amount of electrochemiluminescence decreases as the concentration of the analyte increases). Table VIII gives the values of the measurement curves.

Table VIII: two separate comparative measurements: detection of digoxin

Digoxin concentration electrochemiluminescence

(ng/ml) (double samples)

0.0 42613 35309

0.5 24211 24168

1.0 17561 17206

2.0 10491 9909

3.0 6712 7145

5.0 4680 4603

Example 20: synthesis of oligonucleotides

Oligonucleotides were synthesized using beta-cyanoethyl phosphoramidate (1) on an automated oligonucleotide synthesizer using biosystems, with modification of the 5 'and 3' ends of the oligonucleotide amino groups using a modified solid phase (controlled pore glass) in the final binding step. The amino group modifier was supplied by Clontech (San Diego CA) and the resulting 5 '-modified oligonucleotides, referred to as (C6, NH), all had a spacer segment of 6 carbon atoms for the amino groups and 3 carbon atoms for the 3' -modified oligonucleotide pair. The oligonucleotides constructed and their modification and use are described in detail below.

The HPV oligonucleotide has been described in the reference (2) as region E6.

The sequence of the oligonucleotides is as follows:

HPV 16；1PV16 5′(C6，NH2)TTAGTGAGTATAGACATTATTGTTATAGTT；

2PV16 5′(C6，NH2)CAGTTAATACACCTAATTAACAAATCACAC；

3PV16 5′(C6，NH2)ACAACATTAGAACAGCAATACAACAAACCG；

HPV18；1PV18 5′(C6，NH2)TTAGAGAATTAAGACATTATTCAGACT；

2PV18 5′(C6，NH2)CACCGCAGGCACCTTATTAATAAATTGTAT；

3PV18 5′(C6，NH2)GACACATTGGAAAAACTAACTAACACTGGG.

the oligonucleotides enable PCR to generate various fragments, 3PV16 or 3PV18 and 2PV16 or 2PV18 to form 62bp fragments; 1PV16 forms a 100bp fragment with 2PV 16; 1PV18 formed a 103bp fragment with 2PV 18. It is known that the 3PV16 and 3PV18 oligonucleotides can also be used as probes for hybridization to the products of the PCR reactions of 1PV16 and 2PV16 and 1PV18 and 2PV18, and as probes for hybridization to the strands generated by the oligonucleotides 2PV16 and 2PV18 within the PCR.

The oligonucleotides used for Ha-ras point mutation measurements were as follows:

HRP1 5′(C6，NH2)GCGATGACGGAATATAAGCTGGTGGTGGTG；

HRP2 5′(C6，NH2)TTCTGGATCAGCTGGATGGTCAGCGCACTC；

the above 2 oligonucleotide primers were PCR synthesized as 80bp fragments, and the sequence of the probe for the point mutation was as follows:

1T24 5′(C6，NH2)GGCGCCGTCGGTGTGGGCAA；

1CHR 5′(C6，NH2)GGCGCCGGCGGTGTGGGCAA；

2T24 5′(C6，NH2)TTGCCCACACCGACGGCGCC；

2CHR 5′(C6，NH2)TTGCCCACACCGCCGGCGCC.

in addition to the above sequences, we synthesized the above 1CHR and 2T24 sequences, which did not contain a5 ' amino modification but which carry a3 ' amino modification, and these 3 ' amino modified oligonucleotides were labeled with ECL labels and used for hybridization. The site of mutation/mismatch is clearly indicated by the nucleotide. Probes 1T24 and 1CHR hybridized to the strand produced by the oligonucleotide HRP2 in PCR, and probes 2T24 and 2CHR hybridized to the strand produced by the oligonucleotide HRP1 in PCR.

The 2 sequences of particle-bound oligonucleotides JK8 and JK8C were derived from aequorin sequences and were complementary to each other. The sequence of JK8 and JK8C is as follows:

JK8 5′(C6，NH2)GTCCAATCCATCTTGGCTTGTCGAAGTCTGA

JK8C 5′(C6，NH2)TCAGACTTCGACAACCCAAGATGGATTGGA1C.

oligonucleotide JK7 is an amino group modified with an amino modifier capable of undergoing amino modification within the sequence obtained from Clontech (San Diego CA). JK7 use Ru (bpy)₃ ²⁺Labeling with a labeling substance in the following order:

JK7 5′TCAGACTTCGACAA(NH2)CCCAAGATGGATTGGA：

oligonucleotide probe T35 for aequorin RNA generated by in vitro transcription Using Biotin and Ru (bpy)₃ ²⁺Labeling, sequence of T35 is as follows:

T35 5′(NH2)GATTTTTCCATTGTGGTTGACATCAAGGAA

for the detection of E.coli DNA, we synthesized oligonucleotides specific for the Trp E/D region of genome (3) as shown below:

TRP.Co3 5′(C6，NH2)GCCACGCAAGCGGGTGAGGAGTTCC(NH2)

the sequence is Ru (bpy)₃ ²⁺A marker, and

TRP.Co4 5′(C6，NH2)GTCCGAGGCAAATGCCAATAATGG

this sequence is labeled with biotin, as described in detail below.

Example 21: labeled oligonucleotides

All synthetic oligonucleotides were purified by gel filtration through a Biogel P6(BioRad Lans) column to remove the amino groups removed. Biotin was introduced through the 5' -amino group of the PCR primers using NHS-biotin (Clontech, San Diego CA) (4). Ru (bpy) introduction via amino group of modified oligonucleotide as described below₃ ²⁺-NHS. Oligonucleotides (0.1. mu. mole) in 100. mu.l PBS (pH7.4) and 5. mu. mole Ru (bpy) in DMSO at room temperature in the dark₃ ²⁺The label was reacted overnight. The oligonucleotides were recovered from these labeled reactions by ethanol precipitation. Recent studies have demonstrated that with 0.5. mu. mole Ru (bpy)₃ ²⁺The label was able to efficiently label (> 80%) (data not shown).

The labeled oligonucleotide was further purified by HPLC on a reverse Vydae C-18 semi-preparative column with mobile phases: (A)100mM tetraethylammonium acetate, pH 7.0; and (B) 50% of (a) and 50% acetonitrile, moving from a gradient of 20% to 40% B.

Probes 1CHR and 1T24 were also used³²P, T4 Polynucleotide kinase and a probe with a specific activity of 77000cpm/ng generated by established methods were labeled (5).

Example 22: preparation of nucleic acid magnetic particles

DynalM450 beads were activated with toluene-4-sulfonic acid 2-fluoro-1-methylpyridinium according to the conventional method (6), and then these activated beads were reacted with oligonucleotides J K8 and J K8C, and 650. mu.l of 33nmole oligonucleotide 0.1M Na HCO₃The solution was added to 100mg of activated Dynal particles, incubated for 3 hours for mixing, and 4ml of ethanolamine (0.1M) was added to block the particles. The bound particles were mixed with 0.5mg/ml of ECL buffer containing salmon sperm single-stranded DNA, washed 4 to 5 times in the ECL buffer, and resuspended in 10mg/ml of ECL buffer containing 100. mu.g/ml of salmon sperm single-stranded DNA.

Example 23: preparation of streptavidin magnetic particles I

DynalM450 particles were activated with toluene-4-sulfonic acid 2-fluoro-1-methylpyridinium according to the conventional method (6), and then the activated particles were reacted with streptavidin (Sigma Ltd). With 0.1M Na HCO₃The activated particles (50mg) were washed and 1.5mg streptavidin was added and the reaction was allowed to proceed overnight. 4ml ethanolamine (0.1M) was added to block the particles. The bound particles were mixed with 0.5mg/ml of ECL buffer solution of salmon sperm single-stranded DNA, washed 4 to 5 times in the ECL buffer solution, and suspended in 10mg/ml of ECL buffer solution containing 100. mu.g/ml of salmon sperm single-stranded DNA. Streptavidin from DynalM-280 also proved to be of great significance, but the signal was lower with the present measurement sequence. For the immunoassay, the particles used can be blocked with BSA after binding the antigen or antibody with the buffer for passive coating.

Example 24: preparation of streptavidin magnetic particle II

Mu.l of dimethylsulfoxide containing 50mg/ml biotin-x-NHS (Clontech, San Diego CA, 5002-1) was added to 15mg BSA (in 2-3ml PBS), mixed and incubated at room temperature for 30 minutes. The reaction was stopped by adding 30. mu.l of 1M glycine and incubated at room temperature for 10 minutes. The reaction mixture was purified by Gel filtration chromatography (Biorad, Bio-Gel P6). Biotin-BSA was filtered using a 0.2 μm syringe. 5mg biotin-BSA in 10ml of 0.2M sodium carbonate/sodium bicarbonate buffer (pH9.6) was added to 300mg Dynabead (Dynal 14002) washed with sodium carbonate/sodium bicarbonate buffer, and the mixture was vortexed and incubated at room temperature overnight to mix. After magnetic separation of the particles, 10ml of ECL diluent and 100. mu.l tRNA (10mg/ml) were added, mixed at room temperature for 3-4 hours, washed once with 10ml of ECL diluent, and then suspended in 10ml of ECL diluent and 100. mu.l tRNA (10 mg/ml). After mixing, the protein was immobilized on the particles by incubation at 2-6 ℃ overnight. After magnetic separation of the particles, they were suspended in 10ml PBS containing 15mg streptavidin (Scripps S1214) and mixed for 1 hour, and the particles were washed 4 times in 10ml of ECL dilution, each for 5 minutes. Finally, the particles were suspended in 29.7ml of ECL diluent and 300. mu.l tRNA (10mg/ml) to a final concentration of 10mg/ml particles + 100. mu.g/ml tRNA.

Example 25: detection of DNA immobilized on particles by hybridization with ECL DNA probes

By combining J K8 and J K8C pellets (example 22) with Ru (bpy)₃ ²⁺The labelled oligonucleotide J k7 was hybridized, demonstrating the ability to detect ECL following hybridization to the particles. A large number of particles (300. mu.g) in ECL buffer were mixed with 50. mu.l of ECL buffer containing 12.5, 6.3, 3.01 and 1.5fmole marker J K7, respectively, and after hybridization of these mixtures at 52 ℃ for 4 hours, the mixtures were washed with 1ml of ECL buffer and then suspended in 830. mu.l of ECL buffer. The above samples were analyzed as described in example 1, with probe J K7 being complementary to the J K8 sequence, but not complementary to the J K8C sequence.

TABLE VIII

Amount of particle Probe (fmole) ECL amount

J K8 12.5 5085

6.3 3035

3.01 1345

1.5 657

J K8 C 12.5 451

6.3 345

3.01 256

1.5 212

The results shown in Table VIII demonstrate that specific sequences present directly immobilized on the particle surface can be detected by ECL via specific hybridization.

Example 26: RNA measurement from bead-bound ECL

Dynal M450 particles were coated with an antibody (7) specific for RNA/DNA antibodies generated using a plasmid derived from our cloned aequorin gene (8) according to the conventional method (example 10). Briefly, plasmid pA 5' was cleaved with purified EcoRI and transcribed in vitro with T3 RNA polymerase producing T3-RI RNA (negative RNA). Plasmid pA 5' was also cleaved with purified BamHI and transcribed in vitro with T7 RNA polymerase which produced T7-Bam RNA (positive NRA). Thus, the two RNAs represent two complementary RNAs, and both are treated with equal volumes of phenol: extracting and purifying with chloroform (50: 50), extracting with chloroform and precipitating with 2.5 volume ethanol to obtain supernatant, and measuring the amount of RNA by gel electrophoresis and spectrophotometry. These methods are well established and well known to those skilled in the art (9). In a known manner, at a ratio of 25: 1(Ru (bpy))₃ ²⁺A marker: antibiotinsStreptocin) in molar excess from Ru (bpy)₃ ²⁺The marker marks streptavidin (example 13). The labeled streptavidin was purified by the known method (10) using an iminobiotin column. By assaying for streptavidin, each streptavidin tetramer contained 10 Ru (bpy)₃ ²⁺A label. The labeled streptavidin was then complexed with biotin-containing T35, the oligonucleotide and labeled streptavidin mixed at a ratio of 1: 1. Each 20pmole was mixed into a final volume of 15. mu.l of ECL buffer and incubated overnight at 4 ℃ to form labeled streptavidin-oligonucleotide (SA-T35) complexes. Samples of positive and negative RNA (10ng) were hybridized with 2. mu.l of SA-T35 complex (one-step measurement) or with 25ng of biotin-containing T35 (two-step measurement). Samples were prepared in 50. mu.l and hybridized at 50 ℃ for 3 hours, then 200. mu.g of anti-DNA/RNA antibody coated particles (in 20. mu.l PBS 0.1% BSA) were added. The mixture was mixed for 1 hour at room temperature and washed 2 times in ECL buffer. The sample after hybridization with SA-T35 complex was suspended in 530. mu.l of ECL buffer and analyzed as described in example 1. The sample hybridized only with biotin-containing T35 was incubated with 50pmole of labeled streptavidin mixed for 1 hour and washed 2 times in ECL buffer. The hybridized samples were suspended in 530. mu.l of ECL buffer and analyzed as described in example 1. The results are shown in Table IX.

TABLE IX

Measurement of the average amount of R NA ECL

One-step method is 815

Minus 91

Two-step positive 1123

Negative 194

Example 27: polymerase chain reaction

Polymerase chain reaction was carried out essentially as described in (11, 12, 13). Unless otherwise stated, 100. mu.l of each reaction is generally used. PCR is carried out asymmetrically by incorporating Ru (bpy)₃ ²⁺Labeling using 5pmole biotin-containing oligonucleotide and 50pmole Ru (bpy)₃ ²⁺A labeled oligonucleotide. Measurement of Ha-ras point mutation was performed under the same conditions, except that Ru (bpy) was not added₃ ²⁺A labeled oligonucleotide. Unseparated HPV measurements were performed in an asymmetric fashion, but using a 10-fold excess of biotin-containing oligonucleotide, typically 40 pmole. The conditions of the thermal cycle were as follows: direct incorporation of HPV18 and 16 was measured at 93 ℃ for 1 second, 50 ℃ for 1 second, and 60 ℃ for 2 minutes; ha-ras point mutation was measured at 93 ℃ for 1 second and 69 ℃ for 2 minutes; unseparated HPV was measured at 93 ℃ for 10 seconds, 50 ℃ for 30 seconds, and 60 ℃ for 2 minutes. Depending on the measurement and the required sensitivity, the number of cycles for carrying out the PCR is from 30 to 40.

Example 28: measurement of DNA Probe (I): detection and quantitative analysis of human papillomavirus PCR products by incorporation of enzymes

In the direct incorporation of Ru (bpy)₃ ²⁺After PCR of the labeled oligonucleotides, the entire reaction mixture (90-100. mu.l) was added to 600. mu.g streptavidin-conjugated magnetic particles I and incubated for 20 minutes at room temperature with shaking. The solid phase in the sample was separated with a magnetic strip, washed 2 times with ECL buffer, suspended in 530. mu.l ECL buffer and analyzed for electrochemiluminescence as described in example 1. This measurement is illustrated in fig. 3, the results of which are demonstrated with human papillomavirus samples (2, 14). Relating to Ru (bpy)₃ ²⁺Specific studies of the direct incorporation of labeled oligonucleotides into biotin-containing PCR products used closely related HPV16 and HPV18 type viruses, and DNA samples that are both positive for viral-type and virus-specific oligonucleotides for the presence or absence of HPV16 and 18. The primers were as follows: 2PV16, 2PV18 are biotin-containing, 3PV16, 3PV18 are Ru (bpy)₃ ²⁺A labeled oligonucleotide. 2/3PV16 and 2/3PV18 oligonucleotides are specific for HPV16 and 18, respectively. Generated beads-trapped Ru (bpy)₃ ²⁺The labels were analysed for ECL as described in example 1. The results are shown in FIG. 6 as the amount of ECL for each combination of sample primers.

To illustrate the nature of the quantities we make measurements, we present Ru (bpy)₃ ²⁺Standard Curve for direct incorporation of labels and biotin-containing oligonucleotides into the HPV16 PCR product, bead-bound Ru (bpy)₃ ²⁺The labels were analysed for ECL as described in example 1. The peak ECL photon is given as the ratio of viral copy number to total cellular DNA copy number compared to the increase in HPV16DNA concentration. The primers used for HPV16 analysis were 1PV16 (Biotin marker) and 2PV16(Ru (bpy)₃ ²⁺A marker). The DNA used for each PCR was kept at a constant amount of 1. mu.g using calf thymus. The results of this standard curve are shown in fig. 7. The results of the specificity and quantitative analysis on this measurement indicate that these ECL markers can be used to perform DNA assays easily and quickly. Also described are methods in which the label can be readily associated with an enzymatic reaction without affecting the enzyme.

Example 29: measurement of DNA Probe (II): detection and determination of point mutations in PCR amplification products of human Ha-ras oncogenes

We used the oligonucleotides HRP1 and HRP2 to perform the PCR reaction of the Ha-ras gene. Using biotin-containing HRP1 with unlabelled HRP2, the resulting PCR product was able to hybridize with Ru (bpy)₃ ²⁺Labeled probe 2CHR hybridized to 2T 24. In contrast, with the use of biotin-containing HRP2 with unlabeled HPR1, the PCR product generated was able to hybridize with Ru (bpy)₃ ²⁺Labeled Probe 1CHR hybridizes to 1T 24. The DNA used was human placental cell DNA (normal) and murine NIH3T3 cell DNA transfected with a mutant Ha-ras gene derived from bladder cancer T24 (15).

The measurement method is as follows: mu.l of the PCR reaction mixture was added to 600. mu.g streptavidin-conjugated magnetic particles I, incubated at room temperature for 30 minutes, the solid phase was separated from these samples using a magnetic strip, and 50mM NaOH followed by hybridization buffer (0.9M NaCl, 50mM NaPO)₄pH7.7, 5mM EDTA, 0.1% W/V Polysucrose, 0.1% W/V polyvinylpyrrolidone, 0.1% W/V bovine serum albumin) and then suspended in a suspension containing 10. mu.g/ml Ru (bpy)₃ ²⁺Labeled oligonucleotides in hybridization buffer. The above samples were hybridized at 66 ℃ for 15 minutes.

Separating the solid phase with magnetic strip, passing through 0.9M NaCl, 50mM NaPO₄pH7.7, 5mM EDTA washing 2 times, then 3M four methyl ammonium chloride, 50mM Tris-HCl pH8.0, 2mM EDTA, 0.025% Triton X-100 at room temperature washing 1 times, at 66 degrees C washing 2 times, each time 20 minutes. The solid phase was washed 3 times with ECL buffer, suspended in 530. mu.l of ECL buffer and detected for electrochemiluminescence as described in example 1. Fig. 4 illustrates this measurement.

By using³²Measurement of Ha-ras PCR products with P-labeled probes and Using Ru (bpy)₃ ²⁺The labeled phases were similar except that the solid phase was finally suspended in 250. mu.l of ECL buffer, and then these samples were transferred to 5ml of scintillation fluid and counted using a Beckman LS-100C liquid scintillation counter tube.

The data measured for Ha-ras oncogene point mutations can be seen in FIG. 8. As shown in fig. 4, PCR measurements used biotin-containing HRP2 with unlabeled HRP1 (for probes 1T24 and 1CHR) and biotin-containing HRP1 with unlabeled HRP2 (for probes 2T24 and 2CHR) to give bead-bound single-stranded targets for hybridization. The DNA samples were normal (placental) Ha-ras gene and mutant (NI H3T3-T24) Ha-ras gene. Bead-bound DNA with Ru (bpy)₃ ²⁺Markers 1T24(1T24), Ru (bpy)₃ ²⁺Markers 2T24(2T24), Ru (bpy)₃ ²⁺Labeled 1CHR (1CHR) and Ru (bpy)₃ ²⁺Labeled 2CHR (2CHR) after hybridization and TEMAC washing, resulting in bead-bound Ru (bpy)₃ ²⁺The labels were analyzed by ECL as described in example 1. The results are expressed as ECL amounts for various combinations of sample probes. As expected (shown in FIG. 8), the normal probe hybridized well with the normal DNA (see CHR probe), and the mutant probe hybridized well with the mutant gene (see T24 probe).It is significant that the performance of these probes is not exactly the same. To investigate this apparent anomaly, we used a mixture with and without Ru (bpy)₃ ²⁺Of a marker³²p-labeled probes were further investigated for the above probes. As described below, using³²P-labeled Probe Studies Ru (bpy)₃ ²⁺The marker probe is specific for the Ha-ras oncogene. As shown in FIG. 8, PCR measurements were performed using only the biotin-containing HRP2 (for probes 1T24 and 1CHR) with unlabeled HRP 1. The probes used were: to contain³²P1T 24 and 1CHR (1T24-P, 1CHR-P) as controls, with³²P and Ru (bpy)₃ ²⁺1T24 and 1CHR measurement of markers Ru (bpy)₃ ²⁺The role of the marker. Samples were washed with TEMAC as described above and the resulting beads bound³²p were analyzed in scintillation counting tubes with scintillation cocktail added. Results with various combinations of sample probes per second³²P number (see fig. 9). The results show that it is possible to determine,³²p Probe and Ru (bpy)₃ ²⁺The role of the labeled probes is the same, and problems with probe specificity arise from the various sequences of specific probes used. To further illustrate our Ru (bpy)₃ ²⁺Markers and³²p identity, we compared these labeled probes. Amplification was performed as described previously with placental DNA and biotin-containing HRP2 with unlabelled HRP1 (for probes 1T24 and 1 CHR). Samples were then taken from the resulting PCR products, resulting in a set of samples containing varying amounts of product. These samples were combined with³²P-labeled probes (1T24-P32 and 1CHR-P32), or with Ru (bpy)₃ ²⁺Marker (1T24-Ru (bpy)₃ ²⁺And 1CHR-Ru (bpy)₃ ²⁺) And (4) hybridizing. The results of the above studies were normalized by the mean of the individual markers from 90 μ l samples, these normalized plots allow for more efficient comparison of signal to background, and for comparison of the two methods. FIG. 10 illustrates the reaction at lower levels in the dilution curve. The samples were processed as described previously (fig. 8 and 9). The results in FIG. 10 show that two markers are associated with our Ru (bpy)₃ ²⁺Good reaction identity of the labeled probes. These studies demonstrated that Ru (bpy) distinguishes single base changes in sample DNA₃ ²⁺Labeled probe and³²the P-labeled probe can function in the same manner. This fact can be explained by the fact that Ru (bpy)₃ ²⁺The label has little effect on the properties of the labeled probe.

Example 30: measurement of DNA Probe (III): detection and quantitative analysis of human papillomavirus PCR products in non-separable measurements

For the non-isolation measurement of HPV18, we performed an asymmetric PCR reaction with an excess of biotin-containing primers. The PCR reaction can be carried out by using Ru (bpy)₃ ²⁺The labeled probe is directly hybridized to produce an excess of single-stranded biotin-containing DNA. For hybridization, after amplification was complete we will have a 1000ECL amount of Ru (bpy) specific for the amplified HPV gene₃ ²⁺Labeled oligonucleotide (. about.2 ng) was added to 15. mu.l PCR and incubated at 50 ℃ for 15 minutes. Mu.l of ECL buffer containing 600. mu.g of streptavidin-conjugated magnetic particles I was then added to the hybridization mixture and incubated with shaking at room temperature for 15 minutes. ECL buffer was added to increase the sample volume to 530. mu.l, and electrochemiluminescence was detected as described in example 1. Fig. 5 illustrates this measurement. To illustrate this non-isolation measurement, we plotted a standard curve of HPV18 DNA. PCR measurements were performed with biotin-containing 2PV18, unlabeled 1PV18, and HeLa DNA (14). Resulting PCR reaction with specific probe Ru (bpy)₃ ²⁺Label 3PV 18. The hybridization mix was then added to streptavidin-coated particles, bead-bound Ru (bpy) produced as described in example 1₃ ²⁺The label was directly subjected to ECL analysis. The results are expressed as the amount of ECL as a function of the copy number of HPV18 added to the PCR containing the control ras oligonucleotide probe (see FIG. 11). These results demonstrate that, depending on the nature of the ECL measurement system, it is possible to determine the nucleic acid sequence rapidly without separation.

Example 31: determination of specific genomic DNA sequences

The assay described in this example uses 2 oligonucleotides, all hybridized to the same DNA strand in close proximity, one captured and the other labeled complex (sandwich hybridization). The assay uses E.coli DNA specific for the trpE/D gene region and a probe. Coli DNA was prepared according to the conventional method (16), and salmon sperm control DNA was supplied from Sigma. Mu.l hybridization buffer (10 XPBS, 10mM EDTA and 0.7% SDS), 2ng biotin-labeled TRP, CO4 and 5ng Ru (bpy)₃ ²⁺CO3 to a DNA sample, the sample was prepared to 100. mu.l with water, heated to 97 ℃, incubated at 97 ℃ for 10 minutes, then cooled to 50 ℃ and hybridized for 2 hours. Mu.l of streptavidin-coated magnetic particles II were added to the sample, mixed for 2 hours at room temperature, and the particles were washed 4 times in ECL buffer, resuspended in 500. mu.l of ECL buffer and analyzed as described in example 3. Positive DNA was E.coli and negative DNA was salmon sperm, and the results are shown in Table X.

Table X

Mean electrochemiluminescence of DNA content

Positive 10184

25 257

50 266.5

Minus 1087

25 70

50 75

The above results show that the electrochemiluminescence measurement system can be used to detect genomic genes in E.coli on unamplified DNA using the sandwich hybridization measurement method. In this example, as with streptavidin-coated magnetic particles II, streptavidin-coated magnetic particles I can also be used.

Example 32: enrichment of particles on evanescent wave fluorescence detector

Enrichment of the labeled complexes on the detection surface using an evanescent wave detector can be used to improve the sensitivity of the measurement. Such detectors may use optical fibers or planar optical waveguides 300 to carry light 310 from the light source to the liquid environment. Due to incident beam striking the high refractive index dielectric (n)₁) And a low refractive index dielectric (n)₂) The interface therebetween creates Total Internal Reflection (TIR) 310' that reflects light through the waveguide or fiber. When the incident angle of the light beam is larger than the critical angle theta₀315 (the angle represents the angle between the perpendicular 300 'and the optical path 310'), θ₀＝Sin^-1(n₂/n₁) Light is 100% internally reflected at the interface. In optical waveguides and optical fibers, light propagates at angles of incidence greater than the critical angle and is reflected through the medium with total internal reflection. FIG. 22 shows propagation of TIR in a waveguide or fiber.

Although light can be totally reflected at each point of interaction with the interface, the electromagnetic field outside the medium is not equal to 0. As the electromagnetic field penetrates from the outside of the fiber or waveguide to the outside environment, the electromagnetic field needs to decay exponentially as required by the physics of the continuous transit interface. This electromagnetic field is called evanescent field 320 and can excite fluorophores into fluorescence. The attenuation rate of evanescent field is determined by the incident wavelength and the refractive index n₁And n₂And an angle of incidence. Using quartz waveguides and visible light in an aqueous environment, the evanescent field 320 decays by about 90% over a distance of 100nm from the waveguide/solution interface. In FIG. 22, the surrounding medium 330 has a refractive index n₂And an optical fiber or waveguide 300 and a refractive index n.

The same principle of light propagation in waveguides and fibers that produces evanescent fields also allows light generated when the fluorophore emits light to be efficiently absorbed into the optical element. In addition, any light generated outside of the evanescent zone 320 may be effectively returned by the entering optical element. Combining these effects enables the fiber or waveguide to be used as an effective optical element for measuring the presence and concentration of fluorophore labels in an aqueous environment at or near the surface of the fluorophore labels. U.S. patent 4447546 (incorporated herein by reference) describes a suitable method and apparatus for performing fluorescence immunoassay using optical fibers to excite and measure the evanescent zone fluorescence of labeled immunoreactive reagents.

The use of optical fibers or waveguides in the present invention can improve the sensitivity of fluorescence binding measurements that can be accomplished using reagents that are fluorescently labeled. After incubation of the particles, sample and reagents, the particles are concentrated on the surface of the waveguide or optical fiber. Since the surface area of the particles is larger than the geometric area of the waveguide or fiber, more fluorophore can be collected in the evanescent field around the optical element. Thus, the luminescence signal generated by the particles will be stronger, resulting in a higher sensitivity of the quantitative assay and thus an improvement of the various detection limits.

The foregoing preferred embodiments have been described in detail, but are not to be construed as limiting the invention, and it will be apparent from the description that various changes may be made therein without departing from the spirit of the invention, which is to be accorded the scope of the invention without departing from the spirit thereof.

Reference to the literature

1.Beaucage SL，Caruthers MH.Deoxynucleosidephosphoramidites，a new class of key intermediatesfor deoxypolynucleotide synthesis.Tetrahedron Lett1982；22：1859-62.

2.Shibata DK，Arnheim N，Martin JW.Detection of humanpapilloma virus in paraffin-embedded tissue usingthe polymerase chain reaction.J Exp Med1988；167：225-30.

3.Yanofsky，C.et al(1981)Nucleic Acids Res.24，6647-6668.

4.Updyke TV，Nicolson GL.Immunoaffinity isolation ofmembrane antigens with biotinylated monoclonalantibodies and streptavidin-agarose.Methods Enzymol1986；121：717-25.

5.Cardullo RA，Agrawal S，Flores C，Zamecnik DC，WolfDE.Detection of nucleic acid hybridization bynonradiative fluorescence resonance energy transfer.Proc Natl Acad Sci 1988；85：8790-4.

6.Ngo TT.Procedure for activating polymers withprimary and or secondary hydroxyl groups.MakromolChem Macromol Symp 1988；17：224-39.

7.Coutlee F，Bobo L，Mayur K，Yolken RH，Viscidi RP.Immunodetection of DNA with biotinylated RNA probes：A study of reactivity of a monoclonal antibody toDNA-RNA hybrids.Anal Biochem 1989；181：96-105.

8.Casadei J，Powell MJ，Kenten JH.Expression andsecretion of aequorin as a chimeric antibody usinga mammalian expression vector.Proc Natl Acad Sci1990；87：2047-51.

9.Molecular cloning，a laboratory manual 2nd EdSambrook，J.Cold Spring Harbor Laboratory New York

10.Heney，G.and Orr，G.A.(1981)Anal Biochem.114，92-96.

11.Mullis KB，Faloona FA.Specific synthesis of DNA invitro via a polymerase-catalyzed chain reaction.Methods Enzymol 1987；155：335-50.

12.Lyons J，Janssen JWG，Bartram C，Layton M，Mufti GJ.Mutation of Ki-ras and N-ras oncogenes inmyelodysplastic syndromes.Blood 1988；71：1707-12.

13.Saiki RK，Gelfand DH，Stoffel S，Scharf SJ，HiguchiR，Horn GT，Mullis KB，Erlich HA.Primer-directedenzymatic amplification of DNA with a thermostableDNA polymerase.Science 1988；239：487-91.

14.Yee C，Krishnan-Hewlett I，Baker CC，Schlegel R，Howley PM.Presence and Expression of HumanPapillomavirus sequences in human cervical carcinomacell lines.Am J Pathol 1985；119：361-6.

15.Reddy EP，Reynolds RK，Santo E，Barbacid M.A pointmutation is responsible for the acquisition of thetransforming properties by the T24 humanbladdercarcinoma oncogene.Nature 1982；300：149-52.

16.Marmur，J.(1961)J.Mol.Biol 3，208.

Claims (8)

1. A device for performing a binding measurement of an analyte of interest in a sample using electrochemiluminescence measurements on the surface of an electrode, the device comprising:

(a) a sample chamber defining a sample content and intersecting the feed inlet and the discharge outlet;

(b) an electrode, an electrode surface of which is exposed to and located in the vicinity of a portion of the sample content;

(c) voltage control means for applying electrochemical energy to said electrodes sufficient to produce luminescence;

(d) means for magnetically collecting particles on the surface of the electrode, wherein the collecting means comprises a magnet that generates a magnetic field in a region proximate to the surface of the electrode; and

(e) and a light detection means for measuring the light emission generated on the electrode.

2. The apparatus defined by claim 1 wherein said magnet is located below said electrode.

3. The apparatus defined by claim 1 wherein said magnet includes at least one north-south magnet disposed vertically below said electrode.

4. The apparatus defined by claim 1 wherein said magnets comprise at least one pair of first and second magnets separated by a non-magnetic material.

5. The apparatus defined in claim 4, wherein the magnets are arranged in an anti-parallel manner, wherein for each pair of magnets, a north pole of a first magnet is adjacent to a south pole of a second magnet and a south pole of the first magnet is adjacent to a north pole of the second magnet.

6. The apparatus defined in claim 1 wherein said magnet comprises a permanent magnet or an electromagnet.

7. The apparatus defined in claim 1 wherein said magnet is a permanent magnet.

8. The apparatus defined by claim 1 wherein said magnet is an electromagnet.