WO2001036965A2

WO2001036965A2 - Electric substance detectors comprising cellular material

Info

Publication number: WO2001036965A2
Application number: PCT/IB2000/001685
Authority: WO
Inventors: Spiridon Kintzios
Original assignee: Spiridon Kintzios
Priority date: 1999-11-19
Filing date: 2000-11-16
Publication date: 2001-05-25
Also published as: WO2001036965A3; AU1721001A; IL149728A0; GR1003489B; JP2003514539A; EP1366353A2

Abstract

There is provided a method for indicating the presence of a material to be detected in a fluid comprising: bringing the fluid containing the material to be detected into contact with a detection region containing cellular material; monitoring an electrical property across the detection region; and detecting the presence of the material by sensing at least a predetermined change in the electrical property across the detection region. Also provided is an apparatus for indicating the presence of a material to be detected in a fluid, comprising: a container capable of holding cellular material located within a predetermined region of said container, said container being adapted to enable a fluid to be brought into contact with cellular material held in said container; and a detector capable of sensing at least a predetermined change in an electric property across said predetermined region.

Description

TITLE

METHOD FOR IDENTIFYING BIOCHEMICAL MATERIAL

FIELD OF THE INVENTION The present invention relates to a method for the qualitative and/or quantitative determination of the presence of biological or chemical material in a sample.

BACKGROUND OF THE INVENTION In many laboratory or other situations, it is desired or necessary to detect the presence of various molecules or other chemical or biological substances in a liquid or gas sample. Such chemical or biological substances include, for example, viruses, hormones and toxins in the blood of humans and animals, toxic contaminants in the atmosphere, and herbicide residues in water. The molecule(s) or other substance(s), the identity of which is being determined, may belong to various chemical groups, and have entirely different properties and biological functions. The molecules or other substances may also exist in a free form or bound to other molecules or even cells (e.g. bound to bacteria).

Conventional methods for molecule detection and identification rely on measuring differences in the physical and/or chemical properties of various molecules, such as molecular weight, solubility, charge, etc. These methods have the advantage of being able to separate with high efficiency the sought-after molecule from the remaining molecules in the sample. Frequently, however, the combined application of more than one detection method is required for the complete qualitative and quantitative determination of a sample composition. For example, various chromatographic methods (gas and liquid), mass spectrometry and nuclear magnetic resonance spectrometry often are used in combination to separate and identify the components of mixtures, even when such mixtures contain a large number of such components. These methods are often used to elucidate the structure of many or all of said components as well, without exposing workers to any considerable risk.

Another class of analytical methods relies on the selective and specific recognition of various molecules (ligands) in a sample by antibodies or the F_ab or F_v fragments thereof (e.g. enzyme-linked immunosorbent assay, or "ELISA^"). However, these methods have lower reliability and sensitivity (e.g. sensitivity that only enables detection of the molecule of interest when it is present at a concentration of more than 100 molecules of interest per billion molecules of sample, i.e. sensitivity of greater than 100 ppb) in comparison to methods for molecular structure detection, such as the molecular analysis of DNA and RNA molecules. The latter techniques typically include amplification of nucleic acid sequences by the polymerase chain reaction (PCR). followed by detection of the of the amplified sequences by methods such as radioimmunoassay (RIA). The techniques which utilize PCR are typically very sensitive (> 0.1 - 1.0 parts per billion) and selective, although they can be associated with risks for the persons working with them, in that these latter methods frequently utilize radioactive labels.

Typically, the equipment required for conducting qualitative and quantitative analysis of a sample by conventional methods is expensive and space-consuming. Such equipment must often be operated by specially trained personnel, and such equipment often demands special laboratory infrastructure. Moreover, the time needed for running a complete analysis varies between a few hours to several weeks. Therefore, conventional methods of determination have considerable disadvantages as far as the issues of practicality, time and cost of each analysis are concerned. Often, the molecules or other biological or chemical substance of interest can be recognized by specialized ''receptors^", which as known in the art are proteins or protein-based biomolecules found on the surface of or otherwise associated with living cells. Each receptor selectively and non-covalently bind molecules, or portions thereof, of a particular structural configuration. Such receptors can be cell membrane proteins, antibody proteins or other molecules. In other cases, instead of selectively interacting with a certain receptor, the molecule(s) under determination interact with structural or functional cell components, such as membrane parts, microtubules, enzymes and genes. This interaction often causes complicated changes in cell metabolism, but not necessarily in a specific fashion. In recent years there has been a rapid increase in the number of diagnostic applications based on biological sensors (biosensors). The reason for this is the desire, in the case of routine analysis, to avoid bulky and heavy analytical instruments (e.g. liquid and gas chromatographs) with their concomitant high demands of trained personnel and specialized laboratory infrastructure. In such cases (e.g. blood tests or the monitoring of environmental pollution) the use of portable, easy-to-apply equipment is often indicated as the method of choice. Biosensors utilize the specific interaction(s) of the molecule(s) of interest with a biological compound, such as a receptor or an enzyme, to detect the presence of such molecule(s) in a sample. Biosensors are typically constructed with a small amount of biosensor material mounted on a substrate or support. A sample to be tested is then brought into contact with the biosensor material, and the interaction of the molecule(s) of interest with the biosensor material is detected as a function of some measurable physical parameter (e.g. a change in the index of refraction of the substrate or support as the molecule(s) of interest binds to the biosensor material).

Biosensors must fulfill a number of target performance requirements, some of which are (as explained in Eggins, Biosensors - An Introduction. Wiley & Teubner, Chichester. 1996):

• Sensitivity in the range of parts-per-million (ppm) to parts-per-billion (ppb)

• Specificity of detection of a single molecule (or as few molecules as possible from amongst a number of molecules of similar structure)

• Assay time 1 -60 minutes • Minimal sample volume

• Satisfactory shelf-life (at least a few days)

• Risk-free application

• Minimal personnel training requirements

• Relatively low cost The overwhelming majority of existing biosensors operate by indirectly measuring patterns of physical chemical properties of enzymes or antibody molecules. These methods are often characterized by a long response time (>90 min), a short shelf life for the unit containing the detecting molecules (one day to one week maximum), and a relatively low sensitivity (e.g. detection of molecules of interest only at concentrations of 100 ppb or higher). In addition, when biomolecules other than antibodies are used as the biosensing material, the selectivity of existing biosensors (i.e. the ability of a biosensor to distinguish the molecules of interest from other molecules, especially other molecules of similar structure, which may be found in a sample) is often inadequate. In the case where antibodies serve as the biosensing material, the low sensitivity does not allow for these methods to successfully replace more sensitive DNA and RNA molecular analysis methods (as for example methods including application of the polymerase chain reaction (PCR)).

Biosensor applications based on electrophysiological effects are known in the art. Examples of such biosensors and applications of them are:

• Separation of E. coli bacteria from a blood mixture via dielectrophoresis and subsequent electronic lysis on a single microfabricated bioelectronic chip. Bacteria identification is conducted through electronically enhanced hybridization of the bacterial DNA/RNA (Cheng et al.. Nature Biotechnology 16:541 -546). Moreover, a modification of this method is applied for the detection of various DNA/RNA molecules in a solution (see U.S. Patents Nos. 5.653.939. 5,728.532, 5,858.666). Such applications are highly complex and demand sophisticated equipment and trained personnel, while being able to detect, at least currently, only biological polymers, such as polynucleotides and proteins, and not smaller molecules, such as environmental pollutants.

• The bananatrode. consisting of a mixture of banana with graphite powder liquid paraffin in an electrode cup, is used to determine the presence of dopamine in a sample, by monitoring the conversion of dopamine to quinone by the enzyme polyphenolase.

• Piezoelectric sensors consisting of antibodies immobilized on crystals are known in the art, and several applications are reported including microgravimetric immunoassays for viruses, microbial toxins and other contaminants (Suleiman et al., Analyst 119 (l l):2279-82). The use of such piezoelectric sensors is relatively time-consuming (> 90 minutes).

• Some recent applications rely on the use of whole, intact organs (in particular insect antennae) for the detection of specific volatile compounds by recording electric signals produced thereof (Schroth et al., Biosensors and Bioelectronics 14:303-308 (1999): Schutz et al.. Biosensors and Bioelectronics 14:221-228

( 1999)). The handling and storage of such biosensors remains quite problematic. The following is a list of other art believed to be relevant to the present invention: Cevc. Biochemica et Biophysica Ada 1031-3: 31 1-382 (1990); Goldsworthy et al., Plant Cell Tissue and Organ Cult. 30:221-226 (1992); U.S. Patent No. 5.653,939; Ogata et al.. Ami. Plant Physiol. 10:339-351 (1983); Smith, Aust. J. Plant Physiol. 10:329-337 ( 1983); Tsong et al., Ann. Rev. Physiol. 50:273-290; Vigh et al., TIBS 23:369-374 ( 1998): WO 98/54294; WO 98/55870; WO 98/23948; Iwata et al., Brit. J. Pharmacol. 126: 1691-1698 (1999); Wang et al., Virology 205(1 ): 133-140 (1994); DE 19540098; US 4,343,782; Marty et al., Analusis 26:M144-M149 (1998); Rawson et al.. Biosensors 4:299-312 (1989).

SUMMARY OF THE INVENTION

It would be useful to have a biosensor and biosensor method which can detect biomolecules with greater sensitivity and greater selectivity than biosensors and biosensor methods which are presently available, which do not require large amounts of laboratory space, are safe to use and do not require highly trained personnel for their operation, which yield results more quickly than presently known biosensor methods, and which biosensor can in principle be used over a longer period of time than biosensors presently known in the art. There is thus provided, in accordance with a preferred embodiment of the invention, a method for indicating the presence of a material to be detected in a fluid, comprising: bringing the fluid containing the material to be detected into contact with a detection region containing cellular material; monitoring an electrical property across the detection region; and detecting the presence of the material by sensing at least a predetermined change in the electrical property across the detection region.

In accordance with one preferred embodiment of the invention, the electrical property is an electric potential. In accordance with another preferred embodiment of the invention, the electrical property is an electrical conductance. In accordance with another preferred embodiment of the invention, the electrical property is resistance. In accordance with a preferred embodiment of the invention, the fluid containing the material to be detected is flowed through the detection region.

In accordance with a preferred embodiment of the invention, the cellular material includes at least one of: (a) a multiplicity of cells and (b) portions of cells. In accordance with one preferred embodiment of the invention, the multiplicity of cells is a multiplicity of cells which have been cultured. In accordance with another preferred embodiment of the invention, the multiplicity of cells is in the form of tissue.

In accordance with a preferred embodiment of the invention, the detection region containing cellular material comprises a conductive matrix containing the cell material.

In accordance with one preferred embodiment of the invention, the presence of said material to be detected causes a change in conductivity across at least some of the cellular material. In accordance with a preferred embodiment of the invention, the change in conductivity is an increase in conductivity.

In accordance with a preferred embodiment of the invention, the cellular material is immobilized.

In accordance with a preferred embodiment of the invention, the cellular material includes at least some specialized cellular material which is specifically responsive to the material to be detected.

In accordance with a preferred embodiment of the invention, the cellular material includes at least two species of specialized cellular material which species are each specifically responsive to a different material to be detected. In accordance with a preferred embodiment of the invention, the cellular material includes at least two species of specialized cellular material which species are each responsive in a different manner to said material to be detected.

There is also provided, in accordance with a preferred embodiment of the invention, an apparatus for indicating the presence of a material to be detected in a fluid, comprising: a container capable of holding cellular material located within a predetermined region of said container, said container being adapted to enable a fluid to be brought into contact with cellular material held in said container; and a detector capable of sensing at least a predetermined change in an electric property across said predetermined region.

In accordance with a preferred embodiment of the invention, the container contains cellular material.

In accordance with a preferred embodiment of the invention, the container is adapatcd to enable the fluid to flow through the predetermined region.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be understood and appreciated more fully from the following detailed description, taken in conjunction with the drawings in which:

FIGS. 1A-1C depict several possible configurations of the detection portion of a biosensor constructed and operative in accordance with the present invention;

FIG. 2 briefly outlines the procedure for constructing and operating a biosensor in accordance with the present invention;

FIG. 3 is a diagram illustrating the results of a qualitative determination of plant pathogenic viruses;

FIG. 4 is a diagram illustrating the results of a quantitative determination of plant pathogenic viruses (tobacco viruses); FIG. 5 is another diagram illustrating the results of a quantitative determination of plant pathogenic viruses (tobacco viruses);

FIG. 6 is a diagram illustrating the results of a qualitative determination of the Hepatitis C virus in a human blood sample;

FIG. 7 is a diagram illustrating the results of the quantitative and qualitative determination of the herbicide glyphosate and the phenylalanine analogue compound, p-flourophenylalanine; and FIG. 8 is another diagram illustrating the results of the quantitative and qualitative determination of the herbicide glyphosate and the phenylalanine analogue compound. p-Ilourophenylalanine.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

The present invention utilizes cellular material — e.g. immobilized cells (or cell components), tissues containing many cells or even organs or portions thereof — as the biosensor material. The present invention is based on observing the electrical response of this cellular material to various molecules or other chemical or biological substances.

Because, in principle, different cells having different responses to different chemical and/or biological material, or cell lines exhibiting a differential response to different chemical and/or biological material, can be found and isolated, the present invention can in principle be utilized to construct biosensors to detect a nearly limitless number of materials to be detected (hereinafter "detectants^"). Once cells displaying the desired response characteristics have been found and isolated, such cells may then be proliferated in vitro. Thus, in a preferred embodiment, the present invention may be utilized on a large scale, e.g. by growing cells having the desired response characteristics, immobilizing said cells by loading them in a column, and then detecting the change in electrical response across the region of the column containing the cells as a sample containing detectant(s) is flowed through said region. As immobilized cells can retain their properties unaltered for a period of 2-3 months, the present invention thus provides a biosensor which contains biosensor material that is utilizable for a longer period of time than most biosensor material presently in use (e.g. biosensors employing antibodies or enzymes). Furthermore, unlike in some bioassays presently in use. e.g. ELISA. the measured response of biosensors constructed and operative in accordance with the present invention to a given detectant is believed to correlate more closely to the actual mode of action of the detectant. particularly when whole, intact cells are used as the biosensor material.

Throughout the remainder of the description, when the biosensor material is referred to as "cellular material'', it will be understood that portions of cells, e.g. membranes, organelles and the like, are also contemplated, as are larger collections of cells such as tissues and organs.

The cellular response to the material to be detected (the detectant) can be evaluated by measuring changes in the cellular electric properties, e.g. electric potential, conductance, or resistance, upon binding of the detectant to the cells (or portions thereof) of the cellular material. Using this approach, the detectant-cell interaction can be evaluated in a direct and rapid way. In an especially preferred embodiment of the present invention, the changes measured are changes in electric potential. The biosensor of the present invention is also in principle potentially reusable, in that it is possible that only a finite percentage of all the receptors present in the cellular material (e.g. on the surface of immobilized cells) will be utilized to detect the detectant(s) in a given sample, leaving the remaining receptors available for detection of detectant(s) in another sample. Furthermore, the biosensor of the present invention is usable even in cases where the mechanism(s) through which cellular electrophysiological properties are changed is unknown or not fully elucidated. For example, when whole cells are used as the cellular material, in some cases the binding of detectant molecules to the receptors on the surfaces the cells making up the cellular material of the biosensor will affect the ions channels in those cells, and in so doing affect the electrophysiological properties (e.g. electric potential at the cell surface, conductance, resistance). However, the mechanism through which detectant binding affects the electrophysiological properties of the cellular material may be other than ion-channel based, e.g. detectant binding may give rise to structural or conformational changes in the membranes or other portions of the cells of cellular material, and indeed the mechanism by which detectant binding has an affect on the electrophysiological properties of the cellular material may be extremely complicated. In the practice of the present invention, the practitioner need not know the mechanism of action (e.g. via ion channels or structural changes in cells). Rather, in the practice of the present invention, it is sufficient to know that under a given set of conditions, a given detectant affects the electrophysiological properties of the cellular material in a particular way (e.g.. the conductance of a given quantity of the cellular material in a arranged in a particular configuration changes by about a certain amount upon binding of a particular detectant).

The present invention thus present advantages over prior art methods, for example patch clamp techniques, which have commonly been used to study the electrophysiological aspects of interactions between ligands and membranes. The measurement of the membrane potential, membrane conductance and membrane electromotive force across a cell surface using patch clamp techniques is usually complicated, due the zoning effects and the 'cable property' (Smith, Aust. J. Plant Physiol. 10:329-337). The present invention is simpler to implement, in part because it enables measurement of electrophysiological properties across a large number of entire cells or portions thereof, e.g. membranes.

The biosensor of the present invention may be conceived of as having two parts: a detection (biosensor) unit, and a recording unit. Preferably, the detection (biosensor) unit comprises cellular material (i.e. whole cells, or aggregations of cells such as tissues or organs, or cell components, or lyophilized cells) immobilized in a matrix or on a substrate of appropriate material, such as agar. Preferably, the cellular material is immobilized in such a way that the functional integrity of the cellular material, particularly its specific mode of interaction with the detectant(s), is preserved. Therefore the detection unit of the biosensor preferably comprises at least one type of cellular material capable of interacting specifically with one or more detectants. which detectants may either (i) be recognized by receptors located on the cell surface and/or anchored in the cell membrane or (ii) react with structural or functional cell components, thus affecting cell metabolism and function and inducing a change in the electrical potential or other electrical properties of the cellular material.

Thus, for example, suitable cellular material for use in the practice of the present invention may consist of animal or plant cells specifically susceptible or resistant to various biotic or abiotic stress factors (such as viruses and toxins). In certain instances, cells that are the primary natural in vivo targets of the detectants may be used, enabling the biosensor to obtain a high degree of specificity. The cellular material which is immobilized in the construction of the biosensor according to the present invention may be isolated from natural sources or may be clonally proliferated by in vitro culture. The latter procedure may include methods such as selection (i.e. selection in vitro) to create cellular material with a desired specific response for a particular detectant (i.e. for a certain stress factor)

In another preferred embodiment of the invention, artificial phospolipid bilayer membranes (such as liposomes), bearing receptor molecules or other cell components (which react specifically with the detectants), may be used as the cellular material. Liposome construction and receptor incorporation may be effected as is well known in the art.

In another preferred embodiment of the invention, the cellular material comprises whole membranes which have been isolated from lyophilized cells. Other cell components isolated following lyophilization may also be present along with the isolated membranes. Such membranes preferably contain at least some of the transmembrane proteins or other receptors normally present in the membranes in vivo.

In another preferred embodiment of the invention, the cellular material comprises whole T-cells. As is well known in the art, T-cells often have biomolecules of high selectivity associated therewith, e.g. antibodies and MHC molecules, and therefore the use of a collection T-cells. especially when developed as a cell line producing a particular receptor molecule (e.g. hybridoma cells producing a single antibody) may be used in a preferred embodiment of the present invention. As stated, in accordance with the present invention, the cellular material is immobilized in an appropriate matrix or on a suitable substrate. Preferably, the matrix or substrate (a) is not toxic to the immobilized cells or other cellular material, (b) enables the viability of the cellular material and its specific mode of interaction with the detectant(s) to be preserved, for at least enough time for an assay to take place, and more preferably during biosensor storage, (c) does not change during sample application, and (d) in the case of a matrix, is sufficiently porous (i.e. has a sufficient number of pores of large enough diameter) to enable the detectant(s) to reach the immobilized cellular material relatively unimpeded.

Thus, for example, in accordance with a preferred embodiment of the invention, a suitable matrix material is a 0.8-5% (w/v) solution of agarose. calcium alginate or poly(carbamoyl) sulfonic acid. When the cellular material comprises whole cells, cell immobilization into the matrix may be done as is well known in the art. The immobilization of cell aggregates, tissues or portions thereof, cell membranes, antibodies etc., may be achieved in a similar fashion. Increasing the density of immobilized cells, cell aggregates/tissues, and portions of cells may be used to increase sensor sensitivity.

Preferably, the biosensor is configured with appropriate electrodes for the measurement of the electric potential (or other electric properties) of the immobilized cellular material. The electrodes may be made of various electroconductive materials, such as silver (Ag/AgCl electrodes), platinum, as are known in the art. Preferably, the electrodes are constructed of a material which does not affect the viability of the cellular material or affect its specific mode of interaction with the detectant(s).

Reference is now made to FIGS. 1A-1C, which depict schematically three different configurations according to which the detection portion of a biosensor constructed and operative in accordance with the present invention may be constructed and operative. FIGS. 1A-1 C each show a vessel 10. As shown in FIGS. 1 A and I B. in a preferred embodiment of the invention, vessel 10 may be a column, similar in structure to a column as is commonly used for column chromatography, although as shown in FIG. 1 C, this need not be the case. Disposed within the vessel is a matrix or substrate 12. e.g. a mixture of agarose and calcium alginate as described above, in w hich cellular material, e.g. cells 14, has been immobilized. The immobilized cells 14 or other cellular material may be located primarily near one end of vessel 10. or the cellular material may be spread substantially homogeneously throughout vessel 10. Also disposed within the vessel 10 are electrodes 16 and 18. In operation, a sample 20 (represented schematically by a collection of dots), which is a fluid (liquid or gas) sample containing one or more detectants, preferably dissolved in a solvent, is applied at one end of vessel 10. As shown in FIGS. 1A and I B. electrodes 16 and 18 are preferably positioned so that one electrode is in the vicinity of sample application, and the other electrode is surrounded by matrix or substrate but is not initially in contact with the applied sample (although, if the immobilized cellular material is dispersed throughout the matrix/substrate, the second electrode will also be in contact with the cellular material). In such a configuration, the electrode in the vicinity of the applied sample will be the measuring electrode and the electrode which is not initially in contact with the sample will be reference electrode.

It will be appreciated that in contrast to columns used in column chromatography, in which the chromatographic substrate (e.g. silica gel) must be kept "wet^*" with solvent, matrix/substrate 12 need not and preferably is not perfused with solvent. Instead, in a preferred embodiment of the invention, the sample will move through the region containing the immobilized cellular material via gravity, capillary action, forced flow, or a combination thereof. The matrix or substrate should be electrically conductive, in order to enable measurement of the change in at least one electrical property in the region between the electrodes.

In FIG. 1 A. vessel 10 is open at end 22 (at which end sample 20 is initially deposited) and is further provided with an opening 24 at the end distal to end 22. For this reason. FIG. 1 A is said to depict an "open" configuration. In the open configuration, sample 20 is allowed to flow through vessel 10. The electric potential (or other electrophysiological property) between the electrodes is monitored from before application of sample 20 until its passage through at least the region of the vessel in which the cellular material is immobilized. Changes in the electric potential as the sample passes throught the detection zone are noted. If the sample contains the detectant of interest, the cellular material immobilized on the matrix or substrate 12 will bind or otherwise interact with the detectant to give a characteristic change in the potential (or other electrophysiological quantity, e.g. resistance across the length of the vessel between the two electrodes). This characteristic change is predetermined, and the cellular material is chosen on this basis, in accordance with the detectant which it is desired to detect.

Detectant in the sample will interact with the cellular material 14 located in the vicinity of end 22. Thus the electrical properties of the cells near end 22 will change shortly after the application of a sample containing detectant. As the sample moves through vessel 10. more and more cells of the cellular material will interact with detectant. Eventually the sample will elute through vessel 10, and for this reason a vessel having an '"open^" configuration may in principle be re-used, even if the cellular material is dispersed throughout the container. Depending on the nature of the sample, vessel 10 may be constructed to detect detectants in gaseous or liquid states.

In the "closed^" configuration depicted in FIG. IB, which is analogous to the set-up shown in FIG. 1 A but which lacks an opening 24, the sample flows through the vessel by gravity and/or capillary action, but does not flow as fast as in the "open" configuration. As in FIG. 1A, the change in electrophysiological response is monitored as the sample passes through the detection zone. If small volumes of sample are used relative to the volume matrix/substrate in the container, and if the cellular material is located only near the "open" end of the container depicted in FIG. I B. then a "closed^" container as depicted in FIG. 1 B may in principle be reused.

FIG. I C also depicts a "closed" configuration in which sample is applied to the open end of the vessel, as in FIG. I B. In the vessel shown in FIG. IC, however, the support/matrix and the cellular material are dispersed homogeneously and continuously throughout the vessel. Thus both electrodes are located in a portion of the vessel containing cellular material, and thus the vessel depicted in FIG. IC can function as a galvanic couple (battery). In this case, the electrophysiological property measured is the change in electromotive force (emf) prior to, during and following application of a sample containing detectant.

As will be explained more hereinbelow with reference to FIGS. 3. 4 and 6, it has been observed empirically that the electrophysiological property being measured changes noticeably upon application of a sample containing detectant. but that when a multiplicity of whole cells are used as the cellular material of the biosensor, the value of the electrophysiological property often reverts partly or fully to the baseline value of this property, which baseline value was obtained prior to application of the sample. Without wishing to be bound by any particular theory, it is believed that this observation is due to the fact that living cells tend to maintain an equilibrium with respect to the potential difference across the cell membrane, and thus, while initial contact with a detectant may bring about a change in the electrophysiological properties of the cell so contacted, after a period of time each cell so affected will reestablish equilibrium insofar as possible. It is believed that in some cases, e.g. detectants hich are especially pathogenic viruses, the functioning of the cell may be so disrupted by contact with the detectant that the initial equilibrium cannot be re-established.

Thus, the detection of a detectant is made possible, provided that the pattern of the potential (or another electrical property) of the biosensor in response to various concentrations of the detectant is known, relative to other detectants of similar structure or function and in comparison to biosensors having a different detectant specificity and response and, particularly for the purpose of a quantitative determination, by correlating the concentration of the detectant with the total pattern area of the electric property from the rest value.

When the biosensor is used as a battery (galvanic couple), the qualitative and quantitative pattern of its electromotive force before and after sample application may be correlated with the concentration and structure of the detectant.

The recording unit may be any device connected via the electrodes to the biosensor and appropriate for measuring the electric potential or other electric property of the immobilized cellular material, including devices for the analog-to-digital conversion of signals and suitable equipment and software for processing these signals.

Sample application can be done in any suitable way depending on the liquid or gas phase of the sample solution. The sample volume can be very small (< 5 μl). A solvent free of the detectant can be used as the reference solution (control). The procedure for biosensor construction and operation is briefly outlined in

FIG. 2. It must be emphasized that biosensor construction is preferably done under sterile conditions in order to avoid the contamination of the cellular material.

The invention will be better understood through the following illustrative and non-limitative examples of preferred embodiments thereof. Example 1

Qualitative and Quantitative Determination of Plant Pathogenic Viruses

A biosensor was constructed under sterile conditions by immobilizing protoplasts of a tobacco (Nicotiana tabacum L.) cultivar, which has a differential response against the three different viruses, as indicated in Table I:

Table I Virus Cultivar response

CMV (cucumber mosaic virus) Susceptible

TRV (tobacco rattle virus) Susceptible + hypersensitive reaction

CGMM V (cucumber green mottle Resistant

Mosaic virus)

Protoplasts were isolated by proplasmolysing 0.5 g of tobacco leaves in 20 ml of CPW solution supplemented with 0.7 M mannitol for one hour, and then incubating the leaves in 20 ml of a CPW solution (CPW medium described by Reinert and Yoeman. Plant Cell and Tissue Culture. Springer-Verlag, Berlin, 1982) supplemented with 0.7 M mannitol. 3 mg pectinase (from Aspergilus niger) and 2 mg cellulase (from Trichoderma viridae) for 20 hours. One ml of protoplast and single cell solution (at a density of 40 X 10⁴ cells/ml) was centrifuged at 14.000 RPM for 20 minutes at 20°C. The pellet obtained was resolved and mixed with a 1% (w/v) solution of low-melting point agarose in distilled water at 40°C (in order to avoid agarose solidification). The cell-agarose mixture was transferred into an appropriate!) configured box of approximately 15 cm volume equipped with Ag/AgCl electrodes (i.e. an example of a "closed^" biosensor construction, as depicted in 1 (b)). In this manner. 45 biosensors were constructed for each concentration or group tested. Thirty-nine of these were used the same day they were produced, and the remaining six were stored at -5°C, two were stored for 2 weeks, two for 4 weeks and two for six weeks prior to use. No difference in response between the stored and unstored biosensors was observed, and therefore the data obtained from all the biosensors was pooled. Prior to each assay, the electrodes of each sensor were connected to the recording device, which comprised an Advantec PCL-71 1TM PC I/O card. The analog-to-digital Converter (ADC) of this card, which was a 16-bit, 5-scale unipolar/bipolar converter, recorded the signal (voltage), with an accuracy of -0.0 lmV. The software responsible for the recording of the signal and processing of data was a modified version of the Advantec GenieTM v2.0.

For the qualitative detection of the viruses, 20 μl of control solution (phosphate buffer pH 7.4) or sample (buffer containing 1 μg/ml virus) were applied to the biosensor apparatus, as indicated in Table II:

Table II

For the quantitative determination of the CGMMV virus in each assay, gradually increasing volumes of the sample (buffer containing 1-2 μg/ml virus) and the control solution (phosphate buffer pH 7.4) were applied, as indicated in Table III:

Table III

The results of the qualitative virus determination are presented in FIG. 3 and the results of the quantitative determination in FIG. 4.

Referring to FIG. 3. there is a clear difference between the sensor response to different viruses and to the control. In each case, the biosensor response to each virus solution is expressed as a deviation of the potential from the rest value. The pathogenic strains CMV and TRV elicit a rapid response, whereas the response to the non-pathogenic strain CGMMV is delayed in comparison. CMV elicits a partially irreversible response, while TRV elicits a fully irreversible response. This effect can be recognized by the pattern of the biosensor response, wherein the measured potential did not revert to the initial rest value but to a new, 'modified^* steady-state level. This may indicate consumption of the cellular material during cell-virus interaction, e.g. destruction of some of the cellular material, or irreversible binding of some virus particles to some cells. In contrast, the biosensor response against CGMMV and the control solution was fully reversible. With respect to quantitative determination of the CGMMV strain, there is a roughly linear correlation between the biosensor response (area under the voltage curve) and the virus concentration for virus quantities greater than 20 ng (see also FIG. 5).

The storage of biosensors for two months at a temperature below 0°C did not affect measurements taken at regular intervals.

Example 2 Detection of Human Pathogenic Viruses (Hepatitis C Virus)

A biosensor was constructed under sterile conditions by immobilizing human epithelial cells (a cell line from endometrium/vagina) which have a unique response to the Hepatitis C virus (HCV) which is distinguishably different from the response of these cells to other types of viruses or other detectants.

After cell detachment from the culture vessel by adding trypsine/EDTA for 10 minutes at 37°C. and concentration of cells by centrifugation (6 minutes at 1200 rpm and 20°C), cells (at a density of 4 X 10⁶/ml) were mixed with a 1% (w/v) solution of low-melting point agarose in distilled water at 37°C (in order to avoid agarose solidification). The cell-agarose mixture was transferred into an appropriately configured box of approximately 15 cm^J volume equipped with Ag/AgCl electrodes, as demonstrated in 1 (c) of FIG. 1 (a "closed^", "galvanic couple" biosensor). In this manner. 45 biosensors were constructed for each concentration or group tested. Thirty-nine of these were used the same day they were produced, and the remaining six were stored at -5°C. two were stored for 2 weeks, two for 4 weeks and two for six weeks prior to use. No difference in response between the stored and unstored biosensors was observed, and therefore the data obtained from all the biosensors was pooled. Prior to each assay, the electrodes of each sensor were connected to the recording device, which comprised an Advantec PCL-71 1TM PC I/O card. The analog-to-digital Converter (ADC) of this card, which was a 16-bit, 5-scale unipolar/bipolar converter, recorded the signal (voltage), with an accuracy of -0.0 l mV. The software responsible for the recording of the signal and processing of data was a modified version of the Advantec GenieTM v2.0.

For virus detection, 20 μl of control (blood free of the Hepatitis C virus, irrespective of the presence of other kinds of viruses) or sample solution (blood infected with the Hepatitis C virus) were assayed. Assays were conducted after the solution had been entirely dispersed into the gel matrix. The verification of the presence or absence of the virus in each sample was done by a standard method (ELISA). The results of the assays are presented in FIG. 6, which shows a clear difference between the sensor response to the virus-infected sample (vir) and the control (co). The virus elicits a deviation of the biosensor potential ("electromotive force^") from the average rest value of +5 mV (control) to - 5 mV (sample).

Example 3

Qualitative and Quantitative Determination of Chemical Herbicides

A biosensor was constructed under sterile conditions by immobilizing protoplasts of two johnsongrass (Sorghum halepense L.) biotypes. which have a differential response against the herbicide glyphosate and / fluoro-L-phenyalanine, which is a structural analogue of L-phenylalanine.

Protoplasts were isolated by proplasmolysing 0.5 g of johnsongrass leaves in 20 ml of CPW solution (CPW medium described by Reinert and Yoeman, Plant Cell and Tissue Culture. Springer-Verlag. Berlin, 1982) supplemented with 0.7 M mannitol for one hour, and then incubating the leaves in 20 ml of CPW solution supplemented with 0.7 M mannitol. 3 mg pectinase (from Aspergilus niger) and 2 mg cellulase (from Trichoderma viridae) for 20 hours. One ml of protoplast and single cell solution (at a density of 40 X 10⁴ cells/ml) was centrifuged at 14,000 RPM for 20 minutes at 20°C. The pellet obtained was resolved and mixed with a 1% (w/v) solution of low-melting point agarose in distilled water at 40°C (in order to avoid agarose solidification). The cell-agarose mixture was transferred into an appropriately configured box of approximately 15 cm³ volume equipped with Ag/AgCl electrodes, as shown in 1(b) in FIG. 1 ("closed" biosensor). In this manner, 45 biosensors were constructed for each concentration or group tested. Thirty-nine of these were used the same day they were produced, and the remaining six were stored at -5°C. two were stored for 2 weeks, two for 4 weeks and two for six weeks prior to use. No difference in response between the stored and unstored biosensors was observed, and therefore the data obtained from all the biosensors was pooled. Prior to each assay, the electrodes of each sensor were connected to the recording device, which comprised an Advantec PCL-71 1TM PC I/O card. The analog-to-digital Converter (ADC) of this card, which was a 16-bit, 5-scale unipolar/bipolar converter, recorded the signal (voltage), with an accuracy of -0.0 lmV. The software responsible for the recording of the signal and processing of data was a modified version of the Advantec GenieTM v2.0.

For the qualitative determination of the compounds, 20 μl of control solution (distilled water) or sample (10^"3 or 10^"4 M glyphosate or p-fluorophenylalanine in water) were applied to the biosensor. The results of the glyphosate determination are presented in FIG. 7 and the results of the /?-fluorophenylalanine determination in FIG. 8. It is clear from these results that, although both compounds inhibit plant cell growth in essentially the same manner (inhibition of biosynthesis of aromatic amino acids or their incorporation into proteins), these compounds nevertheless elicit a different, compound- and biotype-specific deviation of the potential from the rest value. This deviation is also concentration-dependent. Therefore, the detection (and quantification) of each compound is possible, provided that the pattern of the response of the biosensor potential to various concentrations of each compound of interest is known, relative to other compounds of similar structure or function.

It will be appreciated by persons skilled in the art that the present invention may be used in the detection, identification and quantification of molecules and other materials in biological and non-biological samples, such as the diagnosis of disease and infectious agents in medicine, veterinarian science and phytopathology, toxicology testing, analysis of metabolic products in living organisms, quality assurance through contaminant detection and monitoring of environmental pollution. These applications can be either commercial (in the sense of routine analyses) or serve pure research purposes.

Because the present invention may be employed using a virtually limitless variety of sources of cellular material in the biosensor of the present invention, the present invention enables the specific detection of thousands of different molecules and other chemical and/or biological material. Immobilization of artificial liposomes, bearing a single type of receptor will allow for the respective augmentation of the sensor sensitivity. The biosensor and method of the present invention may also be used to discriminate between different cell types or different developmental stages of a single cell/tissue, depending on which the molecules the cell (type) expresses on its surface. In this way, the early detection of disease development (such as cancer) may be facilitated. Furthermore, by increasing the density of the immobilized cells in the matrix or substrate, e.g. by using a gel matrix made of conductive material and/or by immobilizing artificial liposomes (bearing receptors) at increased densities, the sensitivity of the biosensor constructed and operative in accordance with the present invention can be increased. To the degree that the cell material used in construction of the present biosensors is not destroyed or irreversibly bound by detectants, the present biosensors may be reusable. Furthermore, unlike many biosensor methods known in the art, in the practice of the present invention no prior knowledge regarding the mechanism of interaction of between the compound of interest (the detectant) with a particular receptor or enzyme or other cell system need be available or utilized: the existence of cellular material capable of a specific response to a compound (expressed as a pattern of an electric property) is all that is required. In this way it is possible to construct biosensors for different applications in a short time. It will also be appreciated that the present invention enables the detection of viruses and other microorganisms before the host develops antibodies against such viruses. At present, it is the detection of antibodies, such as anti-HIV antibodies, that is the basis of standard diagnostic methods (such as ELISA and immunohistochemical methods). These standard diagnostic methods rely on the high specificity of antibody/antigen and antibody/antibody interactions. However, the time required for for an infected host to produce anti-virus antibodies in sufficient concentrations to enable detection by ELISA or other standard methods may be on the order of months, during which infection will remain undetected. The present invention offers the possiblity of detecting the virulent molecule itself. In the case of viruses, this can be currently done only by applying molecular analysis methods, which, as already mentioned, require highly sophisticated equipment and trained personnel, include risks and usually require a long assay time.

In the same sense, the present invention can be used to screen new vaccines, pharmaceuticals and other bioactive compounds, expediting the detection of novel, improved molecules. In addition, based on its working principle, the present biosensor can be used to help elucidate whether certain compounds act on the cell membrane surface or inside target cells.

Since the assay of the present invention is relatively rapid, the present invention may be used in the detection of compounds in real time. For example, a biosensor of the present invention may constitute part of a continuous monitoring system for monitoring environmental pollution, a chemical or biochemical reaction in vivo, or the development of a disease in a host.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. For example, the signal-recording device may be substituted by an integrated or other electronic circuit; the size of the signal recording device may be reduced, possibly omitting the necessity of using a personal computer; both the recording device and the Ag/AgCl electrodes may be substituted by another system for signal acquisition and processing, such as field effect transistors: and the signal recording device may be incorporated into the biosensor matrix. In these cases other electric properties (such as capacitance, current or resistance) may be evaluated. In addition, the cell immobilization matrix may be made of various materials (e.g. calcium alginate. ceramic, polypyrroles, ion-exchanging polymers, etc.) and cells may be immobilized in different ways (e.g. electrophoretically). The biosensor may be appropriately configured in order to facilitate the inlet of samples in liquid or gas phase (e.g. by attaching a micropump).

Claims

1 . A method for indicating the presence of a material to be detected in a fluid comprising: bringing the fluid containing the material to be detected into contact with a detection region containing cellular material; monitoring an electrical property across the detection region; and detecting the presence of the material by sensing at least a predetermined change in the electrical property across the detection region.

2. A method according to claim 1 , wherein said electrical property is an electric potential.

3. A method according to claim 1 , wherein said electrical property is an electrical conductance.

4. A method according to claim 1 , wherein said electrical property is resistance.

5. A method according to claim 1 , wherein said fluid containing the material to be detected is flowed through said detection region.

6. A method according to claim 1 and wherein said cellular material includes at least one of: a multiplicity of cells; and portions of cells.

7. A method according to claim 6 wherein said multiplicity of cells is a multiplicity of cells which have been cultured.

8. A method according to claim 6 wherein said multiplicity of cells is in the form of tissue.

9. A method according to claim 1 and wherein said detection region containing cellular material comprises a conductive matrix containing said cellular material.

10. A method according to claim 1 and wherein the presence of said material to be detected causes a change in conductivity across at least some of said cellular material.

1 1. A method according to claim 1 and wherein the presence of said material to be detected causes increased conductivity across at least some of said cellular material.

12. A method according to claim 1 and wherein said cellular material is immobilized.

13. A method according to claim 1 and wherein said cellular material includes at least some specialized cellular material which is specifically responsive to said material to be detected.

14. A method according to claim 1 and wherein said cellular material includes at least two species of specialized cellular material which species are each specifically responsive to a different material to be detected.

15. A method according to claim 1 and wherein said cellular material includes at least two species of specialized cellular material which species are each responsive in a different manner to said material to be detected.

16. An apparatus for indicating the presence of a material to be detected in a fluid, comprising: a container capable of holding cellular material located within a predetermined region of said container, said container being adapted to enable a fluid to be brought into contact with cellular material held in said container; and a detector capable of sensing at least a predetermined change in an electric property across said predetermined region.

17. An apparatus according to claim 16, wherein said container contains cellular material

1 8. An apparatus according to claim 16. wherein said container is adapated to enable said fluid to flow through said predetermined region.

19. An apparatus according to claim 16 wherein said property is an electric potential.

20. An apparatus according to claim 16 wherein said property is electrical conductance.

21. An apparatus according to claim 16 wherein said property is resistance.

22. An apparatus according to claim 16 and wherein said cellular material includes at least one of: a multiplicity of cells; and portions of cells.

23. An apparatus according to claim 22 wherein said multiplicity of cells is a multiplicity of cells which have been cultured.

24. An apparatus according to claim 22 wherein said multiplicity of cells is in the form of tissue.

25. An apparatus according to claim 16 and wherein said predetermined region containing cellular material comprises a conductive matrix containing said cell material.

26. An apparatus according to claim 16 and wherein the presence of said material to be detected causes a change in conductivity across at least some of said cellular material.

27. An apparatus according to claim 16 and wherein the presence of said material to be detected causes increased conductivity across at least some of said cellular material.

28. An apparatus according to claim 16 and wherein said cellular material is immobilized.

29. An apparatus according to claim 16 and wherein said cellular material includes at least some specialized cellular material which is specifically responsive to said material to be detected.

30. An apparatus according to claim 16 and wherein said cellular material includes at least two species of specialized cellular material which species are each specifically responsive to a different material to be detected.

31. An apparatus according to claim 16 and wherein said cellular material includes at least two species of specialized cellular material which species are each responsive in a different manner to said material to be detected.