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EP1366353A2 - Procede d'identification d'une matiere biochimique - Google Patents

Procede d'identification d'une matiere biochimique

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Publication number
EP1366353A2
EP1366353A2 EP00979828A EP00979828A EP1366353A2 EP 1366353 A2 EP1366353 A2 EP 1366353A2 EP 00979828 A EP00979828 A EP 00979828A EP 00979828 A EP00979828 A EP 00979828A EP 1366353 A2 EP1366353 A2 EP 1366353A2
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EP
European Patent Office
Prior art keywords
cellular material
cells
detected
cellular
biosensor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP00979828A
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German (de)
English (en)
Inventor
Spiridon Kintzios
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Individual
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Individual
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Filing date
Publication date
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Publication of EP1366353A2 publication Critical patent/EP1366353A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means

Definitions

  • 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.
  • 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).
  • 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 " ).
  • 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).
  • PCR reverse transcriptase reverse transcriptase
  • RIA radioimmunoassay
  • 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.
  • 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.
  • 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.
  • 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.
  • 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):
  • Biosensor applications based on electrophysiological effects are known in the art. Examples of such biosensors and applications of them are:
  • 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).
  • 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.
  • 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.
  • 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.
  • the cellular material includes at least one of: (a) a multiplicity of cells and (b) portions of cells.
  • the multiplicity of cells is a multiplicity of cells which have been cultured.
  • the multiplicity of cells is in the form of tissue.
  • the detection region containing cellular material comprises a conductive matrix containing the cell material.
  • the presence of said material to be detected causes a change in conductivity across at least some of the cellular material.
  • the change in conductivity is an increase in conductivity.
  • the cellular material is immobilized.
  • the cellular material includes at least some specialized cellular material which is specifically responsive to the material to be detected.
  • 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.
  • 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.
  • the container contains cellular material.
  • the container is adapatcd to enable the fluid to flow through the predetermined region.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • biosensor material 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 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.
  • the detectant-cell interaction can be evaluated in a direct and rapid way.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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)
  • artificial phospolipid bilayer membranes such as liposomes
  • bearing receptor molecules or other cell components which react specifically with the detectants
  • Liposome construction and receptor incorporation may be effected as is well known in the art.
  • 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.
  • the cellular material comprises whole T-cells.
  • 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.
  • the cellular material is immobilized in an appropriate matrix or on a suitable substrate.
  • 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.
  • a suitable matrix material is a 0.8-5% (w/v) solution of agarose. calcium alginate or poly(carbamoyl) sulfonic acid.
  • 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.
  • 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.
  • 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).
  • FIGS. 1A-1C 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a vessel having an '"open " configuration may in principle be re-used, even if the cellular material is dispersed throughout the container.
  • vessel 10 may be constructed to detect detectants in gaseous or liquid states.
  • 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.
  • 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.
  • the support/matrix and the cellular material are dispersed homogeneously and continuously throughout the vessel.
  • 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).
  • the electrophysiological property measured is the change in electromotive force (emf) prior to, during and following application of a sample containing detectant.
  • 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.
  • the biosensor 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 ⁇ l).
  • 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.
  • 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:
  • 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.
  • CPW medium described by Reinert and Yoeman. Plant Cell and Tissue Culture. Springer-Verlag, Berlin, 1982
  • pectinase from Aspergilus niger
  • cellulase from Trichoderma viridae
  • 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.
  • control solution phosphate buffer pH 7.4
  • sample buffer containing 1 ⁇ g/ml virus
  • 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.
  • 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.
  • 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.
  • human epithelial cells a cell line from endometrium/vagina
  • HCV Hepatitis C virus
  • 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.
  • control blood free of the Hepatitis C virus, irrespective of the presence of other kinds of viruses
  • sample solution blood infected with the Hepatitis C virus
  • 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.
  • CPW solution CPW medium described by Reinert and Yoeman, Plant Cell and Tissue Culture. Springer-Verlag. Berlin, 1982
  • pectinase from Aspergilus niger
  • cellulase from Trichoderma viridae
  • 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.
  • 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.
  • 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.
  • the sensitivity of the biosensor constructed and operative in accordance with the present invention can be increased.
  • the present biosensors may be reusable.
  • 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.
  • the present invention enables the detection of viruses and other microorganisms before the host develops antibodies against such viruses.
  • antibodies such as anti-HIV antibodies
  • standard diagnostic methods such as ELISA and immunohistochemical methods.
  • ELISA and immunohistochemical methods rely on the high specificity of antibody/antigen and antibody/antibody interactions.
  • 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.
  • the present invention can be used to screen new vaccines, pharmaceuticals and other bioactive compounds, expediting the detection of novel, improved molecules.
  • the present biosensor can be used to help elucidate whether certain compounds act on the cell membrane surface or inside target cells.
  • 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.
  • 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.
  • the cell immobilization matrix may be made of various materials (e.g. calcium alginate.
  • 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).

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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un procédé permettant d'indiquer la présence dans un liquide d'une matière à déceler, lequel procédé consiste: à mettre le liquide contenant la matière à déceler en contact avec une région de détection contenant une matière cellulaire; à surveiller une propriété électrique dans la région de détection; et à détecter la présence de la matière en détectant au moins une modification prédéterminée de la propriété électrique dans la région de détection. L'invention se rapporte également à un appareil permettant d'indiquer la présence dans un liquide d'une matière à déceler, qui comprend: un conteneur capable de contenir une matière cellulaire placée à l'intérieur d'une région prédéterminée dudit conteneur, ledit conteneur étant apte à mettre le liquide en contact avec une matière cellulaire contenue dans le conteneur; et un détecteur capable de détecter au moins une modification prédéterminée d'une propriété électrique dans la région prédéterminée.
EP00979828A 1999-11-19 2000-11-16 Procede d'identification d'une matiere biochimique Withdrawn EP1366353A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GR99100398 1999-11-19
GR99100398 1999-11-19
PCT/IB2000/001685 WO2001036965A2 (fr) 1999-11-19 2000-11-16 Procede d'identification d'une matiere biochimique

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EP1366353A2 true EP1366353A2 (fr) 2003-12-03

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EP (1) EP1366353A2 (fr)
JP (1) JP2003514539A (fr)
AU (1) AU1721001A (fr)
GR (1) GR1003489B (fr)
IL (1) IL149728A0 (fr)
WO (1) WO2001036965A2 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6248542B1 (en) 1997-12-09 2001-06-19 Massachusetts Institute Of Technology Optoelectronic sensor
US8216797B2 (en) 2001-02-07 2012-07-10 Massachusetts Institute Of Technology Pathogen detection biosensor
US7422860B2 (en) 2001-02-07 2008-09-09 Massachusetts Institute Of Technology Optoelectronic detection system
MXPA03007070A (es) 2001-02-07 2004-10-15 Massachusetts Inst Technology Sistema de deteccion optoelectronico.
GR1005420B (el) * 2006-01-20 2007-01-31 Σπυριδων Ευαγγελου Κιντζιος Μιμητικα κυτταρα
KR20170124616A (ko) * 2008-07-16 2017-11-10 칠드런'즈 메디컬 센터 코포레이션 마이크로채널을 갖는 기관 모방 장치 및 그 사용 및 제조 방법

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4343782A (en) * 1978-04-20 1982-08-10 Shapiro Howard M Cytological assay procedure
DE19540098C2 (de) * 1995-10-27 2001-05-17 Inst Chemo Biosensorik Verfahren und Mehrkanalbiosensor zur Mehrkomponentenanalyse von Mischungen und/oder Gemischen
AU1059997A (en) * 1995-11-08 1997-05-29 Trustees Of Boston University Cellular physiology workstations for automated data acquisition and perfusion control
EP0950184A1 (fr) * 1996-06-27 1999-10-20 Cellstat Technologies, Inc. Procede et dispositif de criblage a haut rendement
ATE322010T1 (de) * 1996-11-29 2006-04-15 Anordnungen von unabhängig voneinander ansteuerbaren, gestützten flüssigbilayer- membranen und ihre anwendungsverfahren
US5981268A (en) * 1997-05-30 1999-11-09 Board Of Trustees, Leland Stanford, Jr. University Hybrid biosensors
SE9702112D0 (sv) * 1997-06-04 1997-06-04 Holdingbolaget Vid Goeteborgs Method and apparatus for detection of a receptor antagonist

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AMARITA F. ET AL: "Hybrid biosensors to estimate lactose in milk", ANALYTICA CHIMICA ACTA, vol. 349, 1997, pages 153 - 158 *
SEMENCHUK I.N. ET AL: "Effect of various methods of immobilization on the stability of a microbial biosensor for surfactants based on Pseudomonas rathonis", PRIKLADNAYA BIOKHIMIYA I MIKROBIOLOGIYA, vol. 36, no. 1, January 2000 (2000-01-01) - February 2000 (2000-02-01), pages 80 - 84 *
ZIEGLER W. ET AL: "Agar-supported lipid bilayers - basic structures for biosensor design. electircal and mechanical properties", COLLOIDS AND SURFACES, vol. 140, 1998, pages 357 - 367 *

Also Published As

Publication number Publication date
WO2001036965A3 (fr) 2002-01-17
WO2001036965A2 (fr) 2001-05-25
JP2003514539A (ja) 2003-04-22
GR1003489B (el) 2000-11-30
AU1721001A (en) 2001-05-30
IL149728A0 (en) 2002-11-10

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