WO2000077522A1 - Analytic sensor apparatus and method - Google Patents

Analytic sensor apparatus and method Download PDF

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Publication number
WO2000077522A1
WO2000077522A1 PCT/US2000/015400 US0015400W WO0077522A1 WO 2000077522 A1 WO2000077522 A1 WO 2000077522A1 US 0015400 W US0015400 W US 0015400W WO 0077522 A1 WO0077522 A1 WO 0077522A1
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WO
WIPO (PCT)
Prior art keywords
sensor
analyte
base member
sensor strip
detection unit
Prior art date
Application number
PCT/US2000/015400
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French (fr)
Inventor
Alan Joseph Bauer
Original Assignee
Biosensor Systems Design, Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority to IL130478 priority Critical
Priority to IL13047899A priority patent/IL130478D0/en
Priority to IL131193 priority
Priority to IL13119399A priority patent/IL131193D0/en
Priority to IL13198399A priority patent/IL131983D0/en
Priority to IL132491 priority
Priority to IL131983 priority
Priority to IL13249199A priority patent/IL132491D0/en
Priority to US09/426,564 priority
Priority to US09/426,564 priority patent/US6342347B1/en
Priority to IL13305999A priority patent/IL133059D0/en
Priority to IL133059 priority
Priority to IL13332399 priority
Priority to IL133323 priority
Application filed by Biosensor Systems Design, Inc. filed Critical Biosensor Systems Design, Inc.
Priority claimed from US09/979,354 external-priority patent/US6503701B1/en
Publication of WO2000077522A1 publication Critical patent/WO2000077522A1/en

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00Nanotechnology for materials or surface science, e.g. nanocomposites
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001Enzyme electrodes
    • C12Q1/002Electrode membranes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54373Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438Electrodes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/205Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/914Hydrolases (3)
    • G01N2333/924Hydrolases (3) acting on glycosyl compounds (3.2)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/914Hydrolases (3)
    • G01N2333/978Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G01N2333/98Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/914Hydrolases (3)
    • G01N2333/978Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G01N2333/986Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides (3.5.2), e.g. beta-lactamase (penicillinase, 3.5.2.6), creatinine amidohydrolase (creatininase, EC 3.5.2.10), N-methylhydantoinase (3.5.2.6)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/99Isomerases (5.)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2415/00Assays, e.g. immunoassays or enzyme assays, involving penicillins or cephalosporins

Abstract

A sensor for detecting analytes of interest is described. Analyte presence or concentration is determined through measurement of changes in induced electromotive force, current or other electrical property in a sensor strip during analyte exposure to the sensor. According to one class of embodiments, the present device immobilizes natural or synthetic macromolecules sufficiently close to an electrically-conductive base member to insure that interaction of analyte with the macromolecules will lead to altered de novo electrical signals in the sensor strip of base member and macromolecules. Performance of the sensor is enhanced by the use of a resistance-modifying element in a circuit that includes the sensor strip, and by an adhesive agent disposed between the base member and at least one electrical lead of a detection unit.

Description

ANALYTIC SENSOR APPARATUS AND METHOD

Background of the Invention 1. Field of the Invention

This invention pertains to a sensor and method for detecting or quantifying analytes. More particularly the present invention is directed to the detection of analytes by interaction thereof with an immobilized macromolecular entity and the analysis of certain de novo electrical effects that are produced as a result of such interactions. The invention is an extension of the sensor and method described in PCT application PCT/IL99/00309 of common assignee herewith. 2. Description of the Related Art

Chemical and biological sensors are devices that can detect or quantify analytes by virtue of interactions between targeted analytes and macromolecular binding agents such as en^mes, receptors, DNA strands, heavy metal chelators, or antibodies. Such sensors have practical applications in many areas of human endeavor. For example, biological and chemical sensors have potential utility in fields as diverse as blood glucose monitoring for diabetics, detection of pathogens commonly associated with spoiled or contaminated food, genetic screening, and environmental testing.

Chemical and biological sensors are commonly categorized according to two features, namely, the type of material utilized as binding agent and the means for detecting an interaction between binding agent and targeted analyte or analytes. Major classes of biosensors include enzyme (or catalytic) biosensors, immunosensors and DNA biosensors. Chemical sensors make use of synthetic macromolecules for detection of target analytes. Some common methods of detection are based on electron transfer, generation of chromophores, or fluorophores, changes in optical or acoustical properties, or alterations in electric properties when an electrical signal is applied to the sensing system.

Enzyme (or catalytic) biosensors utilize one or more enzyme types as the macromolecular binding agents and take advantage of the complementary shape of the selected enzyme and the targeted analyte. Enzymes are proteins that perform most of the catalytic work in biological systems and are known for highly specific catalysis. The shape and reactivity of a given enzyme limit its catalytic activity to a very small number of possible substrates. Enzymes are also known for speed, working at rates as high as 10,000 conversions per second per enzyme molecule. Enzyme biosensors rely on the specific chemical changes related to the enzyme/analyte interaction as the means for deteπnining the presence of the targeted analyte. For example, upon interaction with an analyte, an enzyme may generate electrons, a colored chromophore or a change in pH (due to release of protons) as the result of the relevant catalytic enzymatic reaction. Alternatively, upon interaction with an analyte, an enzyme may cause a change in a fluorescent or chemiluminescent signal that can be recorded by an appropriate detection system. Immunosensors utilize antibodies as binding agents. Antibodies are protein molecules that bind with specific foreign entities, called antigens, that can be associated with disease states. Antibodies attach to antigens and either remove the antigens from a host and/or trigger an immune response. Antibodies are quite specific in their interactions and, unlike enzymes, they are capable of recognizing and selectively binding to very large bodies such as single cells. Thus, antibody- based biosensors allow for the identification of certain pathogens such as dangerous bacterial strains. As antibodies generally do not perform catalytic reactions, there is a need for special methods to record the moment of interaction between target analyte and recognition agent antibody. Changes in mass (surface plasmon resonance, acoustic sensing) are often recorded; other systems rely on fluorescent probes that give signals responsive to interaction between antibody and antigen. Alternatively, an enzyme bound to an antibody can be used to deliver the signal through the generation of color or electrons; the ELISA (Enzyme-Linked ImmunoSorbent Assay) is based on such a methodology.

DNA biosensors utilize the complimentary nature of the nucleic acid double-strands and are designed for the detection of DNA or RNA sequences usually associated with certain bacteria, viruses or given medical conditions. A sensor generally uses single-strands from a DNA double helix as the binding agent. The nucleic acid material in a given test sample is then denatured and exposed to the binding agent. If the strands in the test sample are complementary to the strands used as binding agent, the two interact. The interaction can be monitored by various means such as a change in mass at the sensor surface or the presence of a fluorescent or radioactive signal. Alternative arrangements have binding of the sample of interest to the sensor and subsequent treatment with labeled nucleic acid probes to allow for identification of the sequence(s) of interest.

Chemical sensors make use of non-biological macromolecules as binding agents. The binding agents show specificity to targeted analytes by virtue of the appropriate chemical functionalities in the macromolecules themselves. Typical applications include gas monitoring or heavy metal detection; the binding of analyte may change the conductivity of the sensor surface or lead to changes in charge that can be recorded by an appropriate field-effect transistor (FET). Several synthetic macromolecules have been used successfully for the selective chelation of heavy metals such as lead. The present invention has applicability to all of the above noted binding agent classes.

Known methods of detecting interaction of analyte and binding agent can be grouped into several general categories: chemical, optical, acoustical, and electrical. In the last, a voltage or current is applied to the sensor surface or an associated medium. As binding events occur on the sensor surface, there are changes in electrical properties of the system. The leaving signal is altered as function of analyte presence.

The most relevant prior art to the present invention involves sensors that are based on electrical means for analyte detection. There are several classes of sensors that make use of applied electrical signals for determination of analyte presence. "Amperometric" sensors make use of oxidation-reduction chemistries in which electrons or electrochemicaUy active species are generated or transferred due to analyte presence. An enzyme that interacts with an analyte may produce electrons that are delivered to an appropriate electrode; alternatively, an amperometric sensor may employ two or more enzyme species, one interacting with analyte, while the other generates electrons as a function of the action of the first enzyme (a "coupled" enzyme system). Glucose oxidase has been used frequently in amperometric biosensors for glucose quantification for diabetics. Other amperometric sensors make use of electrochemicaUy active species whose presence alters the system applied voltage as recorded at a given sensor electrode. Not aU sensing systems can be adapted for electron generation or transfer, and thus many sensing needs cannot be met by amperometric methods alone. The general amperometric method makes use of an applied voltage and effects of electrochemicaUy active species on said voltage. An example of an amperometric sensor is described in U.S. Patent No. 5,593,852, in which Heller and Pishko disclose a glucose sensor that relies on electron transfer effected by a redox enzyme and electrochemicaUy-active enzyme cofactor species. The present invention does not require application of an external voltage, oxidation/reduction chemistry, or electron generation/transfer. An additional class of electrical sensing systems includes those sensors that make use primarily of changes in an electrical response of the sensor as a function of analyte presence. Some systems pass an electric current through a given medium; if analyte is present, there is a corresponding change in exit electrical signal, and this change implies that analyte is present. In some cases, the binding agent-a alyte complex causes an altered signal, while in other systems, the bound analyte itself is the source of changed electrical response. Such sensors are distinguished from amperometric devices in that they do not necessarily require the transfer of electrons to an active electrode. Sensors based on the application of an electrical signal are not universal, in that they depend on alteration of voltage or current as a function of analyte presence; not aU sensing systems can meet such a requirement. An example of this class of sensors is U.S. Patent No. 5,698,089, in which Lewis and Freund disclose a chemical sensor in which analyte detection is determined by change of an apphed electrical signal. Binding of analyte to chemical moieties arranged in an array alters the conductivity of the array points; unique analytes can be determined by the overall changes in conductivity of aU of the array points. The present invention does not rely on arrays or changes of applied electrical signal as a function of analyte presence. The present sensor does not require any applied electrical or electromagnetic signal.

Several other publications that do not fall into the preceding categories are worthy of mention in the prior art. Radmacher, et al. (Science 265:1577-1579 (1994)) noted the existence of augmented spatial fluctuations in enzymes interacting with substrates, but did not apply this phenomenon to analyte detection. Holzrichter, et al., U.S. Patent No. 5,620,854, did make use of macromolecule motion to detect analyte; their system relies specificaUy on atomic force or scanning tunneling microscopes for detection of said motion. An additional patent is that of Stanbro, et al., U.S. Patent No. 5,H4,674, which discloses a sensor that is based on the interference of apphed electrical fields. Interaction of target analyte with a binding agent alters the interference of the applied electrical field. The present invention does not make use of applied electrical fields, currents, or voltages.

Other prior-art voltage-based sensors require the use of semiconducting field-effect transistors (FET's) and rely on the chemical generation or physical trapping of charged species near the sensor surface. The method has found widespread use in the detection of positively- charged heavy metals as weU as analytes that are involved in proton (H ) generating enzyme reactions. Sato, et al. ("Endoscopic Urease Sensor System for Detecting Helicobacter pylori on Gastric Mucosa", Gastrointestinal Endoscopy 49: 32-38 (1999)) describe a pH-sensitive FET for the detection of the enzyme urease associated with the pathogenic bacteria H. pylori. The present invention does not rely on the use of a FET in the sensor element contacted to analyte-containing samples.

While hundreds of sensors have been described in patents and in the scientific literature, actual commercial use of such sensors remains limited. In particular, virtually aU sensor designs set forth in the prior art contain one or more inherent weaknesses. Some lack the sensitivity and/or speed of detection necessary to accomplish certain tasks. Other sensors lack long-term stability. Still others cannot be sufficiently miniaturized to be commercially viable or are prohibitively expensive to produce. Some sensors must be pre-treated with salts and/or enzyme cofactors, a practice that is inefficient and bothersome. To date, virtuaUy aU sensors are limited by the known methods of determining that contact has occurred between an immobilized binding agent and targeted analytes. Use of fluorescent or other external detection probes adds to sensor production requirements and reduces lifetimes of such sensor systems. AdditionaUy, the inventor believes that there is no sensor method disclosed in the prior art that is generaUy applicable to the vast majority of macromolecular binding agents, including enzymes, antibodies, antigens, nucleic acids, receptors, and synthetic binding agents. Summary of the Invention

It is therefore a primary object of the present invention to provide an improved analyte detection system, in which a detection unit is electrically connected to a sensor strip so as to allow for detection of internally-generated electrical signals in the sensor strip that are responsive to analyte presence;

It is a further object of the invention to describe an electrical circuit that includes sensor strip and detection unit to aUow for rapid, sensitive, and inexpensive analyte detection; and,

It is an additional object of the invention to improve the consistency and ease of use in detection of an analyte in a sensor system by inclusion of resistance-modifying elements in the detector unit-sensor strip electrical circuit.

These and other objects of the present invention are attained by a sensor that includes a base member having a conductive electrical property; a macromolecular entity bound to the base member, wherein the macromolecular entity and the base member define a sensor strip, the macromolecular entity being interactive at a level of specificity with a predetermined analyte, and an electrical signal is internaUy-generated in the sensor strip responsive to presence of the analyte; and, a resistance-modifying element disposed in a circuit between the base member and a detection unit for detection of the electrical signal.

According to still another aspect of the invention, the resistance-modifying element is a self-assembled monolayer.

According to yet another aspect of the invention, the resistance-modifying element is a chemical entity.

According to another aspect of the invention, the internaUy-generated electrical signal is further processed for determination of analyte presence or concentration. According to yet another aspect of the invention, a serial dilution unit is included for the determination of analyte concentration.

The invention provides a method for detecting analyte, including the following steps: providing an electricaUy conductive base member; immobilizing at least one macromolecule in proximity to the base member, wherein the macromolecule is capable of interacting at a level of specificity with a predetermined analyte, wherein the base member and the macromolecule define a sensor strip; exposing predetermined analyte to the macromolecule; and, detecting an electrical signal internaUy-generated in the sensor strip, the electrical signal being responsive to a presence of the predetermined analyte, wherein the step of detecting is performed with an electrical circuit that includes the base member and a resistance modifying element.

According to an aspect of the invention, contact between equipotential electrodes of a detection unit and the sensor strip is facilitated by presence of an adhesive agent between the sensor strip and detection unit electrodes. According to an additional aspect of the invention, an additional step includes processing the electrical signals for determination of analyte presence.

According to stiU another aspect of the invention, the method includes the steps of serially diluting a sample and exposing the serial dilutions to a plurality of sensor strips for the determination of analyte concentration range. Brief Description of the Drawing

For a better understanding of these and other objectives of the present invention, reference is made to the foUowing detailed description of the invention, by way of example, which is to be read in conjunction with the following drawings, wherein:

Fig. 1 is a schematic view of a sensor detection system 100 in accordance with the invention in which a sensor strip 122 comprised of base member 120, self-assembled monolayer 130, macromolecular layer 140 and packaging layer 150 forms a closed electrical circuit with electrodes 160 and 161, and detection unit 170;

Fig. 2 is a schematic view of a first alternate embodiment of a sensor system detection 200 in accordance with the invention in which a resistive element 299 is placed in the closed sensor circuit of sensor strip 222, electrodes 260 and 261 and detection unit 270;

Fig. 3 is a schematic view of a second alternate embodiment of a sensor detection system 300 in accordance with the invention in which a semiconductive element 398 is included in the closed sensor circuit of sensor strip 322, electrodes 360 and 361 and detection unit 370;

Fig. 4 is a schematic view of a third alternate embodiment of a sensor detection system 400 in accordance with the invention in which a computer 480 for data processing is attached to detection unit 470;

Fig. 5 is a schematic view of a fourth alternate embodiment of a sensor detection system 500 in accordance with the invention in which an adhesive agent 533 is present on a sensor strip 522 and aids in contact with detection unit 570 electrode 560 and resistance modified electrode 561;

Fig. 6 is a schematic view of a fifth alternate embodiment of a sensor detection system 600 in accordance with the invention in which resistive elements 699 and 697 are incorporated into detector unit 670 leads 660 and 661. Adhesive agent 633 attaches each resistive element to one location on sensor strip 622;

Fig. 7 is a graph of results from a sensing experiment performed with an antibiotic sensor based on the present invention;

Fig. 8 is a graph of results from a sensing experiment performed with a lactose sensor based on the present invention;

Fig. 9 is a graph of results from a sensing experiment performed with a control sensor in the presence of lactose.

Fig. 10 is a schematic view of an electrical generator based on de novo electrical signals resulting from interaction of macromolecules with cognate molecules; Fig. 11 is a schematic view of a sensor system for the detection/quantification of an analyte in which a serial dilution unit 1190 dilutes a sample and presents serial dilutions to multiple sensor strips 1121-1124;

Fig. 12 is a schematic view of a sensor system for the detection of analyte through the non-contact monitoring of magnetic field flux resulting from the interaction of macromolecule with target analyte; and

Fig. 13 is a graph of detection of lactose based on a sensor system embodiment described in Fig. 12. Description of the Preferred Embodiment

Without being bound by any particular theory, the foUowing discussion is offered to facilitate understanding of the invention. The sensor design disclosed herein is based on de novo electrical signals generated in a sensor strip as a function of analyte presence. The sensor utilizes a novel method of detecting an analyte wherein macromolecular binding agents are first immobilized proximate an electrically conductive base member. De novo electrical signals such as current, magnetic field strength, induced electromotive force, alternating voltage or changes in impedance or resistance, signal sign switching, signal frequency, electrical noise and components thereof can be monitored for change during exposure of the macromolecular binding agents to a sample that may contain target analyte. In the aforementioned PCT application, certain aspects of electrode-sensor strip contact were not fuUy described. In the present invention, the advantages of particular forms of sensor strip contact are disclosed more fuUy. SpecificaUy, a resistive or semiconductive element may be integrated into a sensor strip, a detection unit or its associated electrodes in order to facilitate signal measurement.

Referring now to Figs. 1 - 4, a typical sensor detection system 100 comprises (i) a sensor strip 122; (ϋ) a detection unit 170 for the detection of one or more electrical signals generated internally in the sensor strip 122; (iii) electricaUy-passive electrodes 160, 161 to provide contact between the sensor strip 122 and the detection unit 170. In some preferred embodiments, there can be (iv) a resistive element 299 (Fig. 2) or semiconductive element 398 (FigJ) in the sensor circuit comprised of the sensor strip, electrodes, and detection unit. The detection unit 170 may also serve to ground the sensor strip 122 prior to measurement, so that stray signals are removed prior to exposure of sample to the sensor strip 122. Such grounding may be performed either through an electrode or a separate contact between the detection unit 170 and the sensor strip 122 (not shown). Grounding may also be performed at times during sensor action so as to enhance signal quality and/or increase signal redundancy. In other preferred embodiments of the invention a computer 480 for processing the induced signal (Fig. 4) or a component thereof may be included. AdditionaUy, the computer 480 may be used for controlling sample handling, serial dilution and monitoring of the unprocessed signal or the processed signal. Alternatively, an external electrical signal may be apphed to the sensor strip, and the exit signal monitored for the presence and magnitude of the internaUy-generated electrical signal.

According to a method of the invention, one contacts equipotential passive leads of a detection unit to a sensor strip and then measures an electromotive force (emf), current, or other electrical signal internaUy-generated in the sensor strip as a function of analyte interaction with the sensor strip. A resistive or semiconductive element placed in the closed electrical circuit formed by the sensor strip, two passive electrodes, and detection unit aids in signal detection. Placement of the resistive or semiconductive element between at least one electrode and the sensor strip (see Figs. 2, 3 and 6) is optimal.

As described in the noted PCT application, the methodology of detection is very sensitive. Specific detection of pathogenic bacteria in a complex meat matrix was performed within two minutes at 1-10 ceUs per milliliter of sample (concentration determined afterwards by plating). Measurement of internaUy-generated (de novo) current or voltage in a sensor strip according to the present invention allows for rapid, specific and sensitive determination of analyte presence. In the foUowing description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It wiU be apparent, however, to one skilled in the art that the present invention may be practiced without these specific details. In other instances weU-known circuits and control logic have not been shown in detail in order not to unnecessarily obscure the present invention.

Certain terms are now defined in order to facilitate better understanding of the present invention. An "analyte" is a material that is the subject of detection or quantification. A "base member" or base layer is a sohd or liquid element on or near which macromolecules can be physicaUy or chemicaUy immobilized for the purpose of sensor strip construction. Conducting and semiconducting foils, coatings, thin-films, inks, and sohd pieces are particularly preferred for the role of base member.

"Macromolecules", "macromolecular binding agents", "binding agents" or "macromolecular entities" can be any natural, mutated, synthetic, or semi-synthetic molecules that are capable of interacting with a predetermined analyte or group of analytes at a level of specificity.

A self-assembled monolayer ("SAM") is herein defined as a class of chemicals that bind or interact spontaneously or otherwise with a metal, metal oxide, glass, quartz or modified polymer surface in order to form a chemisorbed monolayer. As the phrase "self-assembled" imphes, a self-assembled monolayer is formed from molecules that bond with the surface upon their direct contact from solvent, vapor, or spray. As the word "monolayer" implies, a self- assembled monolayer possesses a molecular thickness, i.e., it is ideaUy no thicker than the length of the longest molecule used therein. In practice, this may not be the case, but a thicker chemical layer between macromolecules and base member is acceptable for sensor construction. A "chemical entity" is a layer other than a SAM that is disposed proximate the base member. It may serve to partiaUy insulate the base member from direct contact from a detection unit and as such, the chemical entity may serve as a resistance-modifying element as defined below. A chemical entity may be deposited on or near one or both sides of a base member by any means and may also serve in the role of resistance-modifying element disposed between base member and detection unit.

A "packaging layer" is defined as a chemical layer disposed above the macromolecules. The packaging layer may aid in long term stability of the macromolecules, and in the presence of a sample that may contain analyte of interest, the packaging layer may dissolve to aUow for rapid interaction of analyte and binding agents. The packaging layer may also serve in conjunction with the charged macromolecules in the role of a resistance-modifying element. Such may be the case when a sensor is coated equaUy on both sides with SAM's (or chemical entities), macromolecules, and packaging layers. A "sensor strip" is defined as a minimum of a single base member and associated macromolecule or macromolecules. If multiple macromolecular entities, SAM, chemical entity, packaging layer or other layers are physicaUy associated with the base member, then they are included in the term "sensor strip". In some preferred embodiments of the invention sensors strips are un-powered, that is, no electrical signal is applied to them. In other preferred embodiments the sensor strip may be powered through apphcation of voltage, current, or other electrical signal to the sensor strip. A "sensor element" is defined minimaUy as a base unit and a macromolecule. A "base unit" is a solid or liquid element on or near which macromolecules can be immobilized for direct detection of analyte-responsive magnetic flux, as described below. An "electrode" or "lead" is a wire, electrical lead, connection, electrical contact or the like that is attached at one end to a detection unit and contacted at the other end directly or indirectly to a sensor strip. Contact to sensor strip is generally electrically passive in nature and occurs at two positions. One of the electrodes may serve as an electron sink or electrical ground. The electrodes may be prepared from either conducting or semiconducting materials or a combination thereof. The electrodes are generaUy equipotential. In preferred embodiments employing electrically passive electrode contact with the sensor strip, neither electrode is used to deliver an electrical signal to the un-powered sensor strip.

The terms "internally-generated" and "de novo" electrical signals are used with respect to the electrical arts. SpecificaUy by these terms it is intended to exclude oxidation-reduction chemistries and electrical phenomena resulting directly or otherwise from the obhgate apphcation of an external electrical or electromagnetic signal. An internaUy-generated or de novo electrical signal is one that is produced in the sensor strip without any required apphcation to the sensor strip of electrical or electromagnetic signal. AdditionaUy, there is no oxidative transfer of electrons between the base member and binding agent, analyte, or medium.

A "detection unit" is any device or material that aUows for the detection of one or more electrical signals internaUy-generated in the sensor strip. The detection unit is generally contacted to a sensor strip at two positions through passive contact of equipotential electrodes. The detection unit may simultaneously measure more than one type of signal and it may be contacted to a plurality of sensor strips. AdditionaUy, it may further process the signal or a component thereof for the purpose of analyte detection and concentration range determination For example voltage sensitive dyes or materials could serve as the detection unit.. "Resistive element" and "semiconductive element" refer to resistance-modifying elements that are included in a "sensor circuit" that minimaUy includes one such element in addition to a base member and a detection unit. The purpose of such element is to aid in facUe signal capture. The resistive element may be a resistive thin coating or other material whose presence between a base member and a detection unit facUitates measurement of de novo electrical signals in a sensor strip. A "semiconductive element" is a semiconductor that serves the role of a resistance-modifying element between the base member and the detector unit. The presence of analyte leads to augmented internaUy-generated electrical signals in a sensor strip. The de novo electrical signal is most easily measured if there is a resistive or semiconductive element or layer between the base member and the detection unit. Examples of appropriate resistive and conductive elements include, but are not limited to, non-conductive or dielectric coatings, organic and inorganic semiconductors, and the like. Semiconducting, doped-sihcon is particularly preferred and can be placed between one or both detection unit electrodes and a sensor strip. Contact between a sensor strip and a resistive or semiconductive element may be facilitated by the presence of an adhesive agent between the two components. Resistive or semiconductive elements may be incorporated directly into detection unit, associated electrodes or sensor strips and are shown as distinct elements in the accompanying diagrams for the purpose of convenience only.

Referring again to Fig. 1, which schematically illustrates a preferred embodiment of the invention, free analyte 155 is disposed proximate a sensor strip 122 prior to (left side of figure, labeled "L") and after (right side of figure, labeled "R") dissolution of packaging layer 150. The analyte (shown as free analyte 155, and analyte 157 interacting with the macromolecular layer 140) can be a member of any of the following categories, listed herein without limitation: cells, organic compounds, antibodies, antigens, virus particles, pathogenic bacteria, metals, metal complexes, ions, spores, yeasts, molds, ceUular metabolites, enzyme inhibitors, receptor ligands, nerve agents, peptides, proteins, fatty acids, steroids, hormones, narcotic agents, synthetic molecules, medications, enzymes, nucleic acid single-stranded or double-stranded polymers. The analyte 155 can be present in a solid, liquid, gas or aerosol. The analyte 155 could even be a group of different analytes, that is, a coUection of distinct molecules, macromolecules, ions, organic compounds, viruses, spores, ceUs or the like that are the subject of detection or quantification. Some of the analyte 157 physically interacts with the sensor strip 122 after dissolution of the packaging layer 150 and causes an increase in internaUy-generated electrical signals measured in the sensor strip 122. Contact of electrodes 160 and 161 to sensor strip 122 aUows for measurement of such a de novo electrical signal that is responsive to analyte presence. There is no requirement for apphcation of a voltage or other electrical signal to the sensor strip 122 prior to or during measurement of internaUy-generated electrical signals by the detection unit 170. In some preferred embodiments, one may apply such an external signal, in which case the de novo electrical signal generated in the sensor system and responsive to analyte presence wiU alter the exit signal in such a system.

Examples of macromolecular entities suitable for use in the sensor detection system 100 include but are not limited to enzymes that recognize substrates and inhibitors; antibodies that bind antigens, antigens that recognize target antibodies, receptors that bind ligands, ligands that bind receptors, nucleic acid single-strand polymers that can bind to form DNA-DNA, RNA- RNA, or DNA-RNA double strands, and synthetic molecules that interact with targeted analytes. The present invention can thus make use of enzymes, peptides, proteins, antibodies, antigens, catalytic antibodies, fatty acids, receptors, receptor ligands, nucleic acid strands, as weU as synthetic macromolecules in the role of the macromolecular layer 140. Natural, synthetic, semi-synthetic, over-expressed and genetically-altered macromolecules may be employed as binding agents. The macromolecular layer 140 may form monolayers as in Fig. 1, multilayers or mixed layers of several distinct binding agents (not shown). A monolayer of mixed binding agents may also be employed (not shown).

The macromolecule component is neither limited in type nor number. Enzymes, peptides, receptors, receptor ligands, antibodies, catalytic antibodies, antigens, ceUs, fatty acids, synthetic molecules, and nucleic acids are possible macromolecular binding agents in the present invention. The sensor method may be applied to detection of many classes of analyte because it relies on the following properties shared by substantiaUy all sensor detection systems:

(1) that the macromolecules chosen as binding agents are highly specific entities designed to bind only with a selected analyte or group of analytes;

(2) that a sensor strip including a macromolecular binding agent and an electrically- conductive base member can exhibit internaUy-generated electrical signals;

(3) that the internaUy-generated electrical signals are responsive in magnitude, sign, and or frequency to the presence of analyte in a sample exposed to sensor strip; and

(4) that the internaUy-generated electrical signals can be detected in a closed electrical circuit comprised of sensor strip, a detection unit, associated electrodes, and a resistive or semiconductive element. For example, in the alternate embodiment of Fig. 2, which is generally similar to the first embodiment, an induced current may be measured in a closed electrical circuit that contains a sensor strip 222, electrodes 260 and 261, detection unit 270, and a resistive element 298. A relatively small background induced current is present in the circuit due to the macromolecules present on the sensor strip. The background current may optionaUy be zeroed by grounding of the sensor strip prior to sample exposure. Presence of analyte in sample causes increased induced current as measured by detection device 270 that is passively contacted through electrodes 260 and 261 to the sensor strip 222. The resistive element need not contribute significant resistance to the overall detection circuit, but its presence between sensor strip and detection electrodes is of importance for successful signal capture. It is possible to optionally apply a current to the sensor system shown in Fig. 2, in which case the exit current would be the sum or difference between the apphed current and the analyte-responsive internaUy-generated current.

Referring again to Fig. 1, the macromolecular layer 140 and the packaging layer 150 may serve the role of a resistance-modifying element. Since the packaging layer 150 and the macromolecular layer 140 are uneven in their surface properties, excessive electrode pressure during fabrication or from an ad hoc apphcation of the electrodes 160, 161 during a sensing operation can short out the circuit. This short occurs as a consequence of both electrodes 160, 161 being in a nonresistive mode of contact with the base member 120, which under certain non-grounded, conditions, can lead to a condition of no signal. Thus, a resistance-modifying element, such as semiconductive element 398 (FigJ) or a resistive element 299 (Fig. 2) is included in the sensor circuit to obviate this problem and additionally to remove the need for deposition of an additional conductive layer on the sensor strip. In many applications, the sensor is disposable, and is intended be provided without the detection unit 170, in which case the detection unit 170 is external to the sensor, and the resistance-modifying element is accessible to the detection unit 170 using electrical contact points, pin-outs, or the like.

The ohmic resistance of the resistance-modifying element does not have to be very high and is preferably between about 1 and 20 ohms. Values of 1 - 12 ohms have been found to work weU in prototypes. For example in the embodiment shown in Fig. 6, the adhesive agent 633 has been realized as Scotch brand glue stick (product number 6008), which has been apphed to sensor strips and has served the role of both adhesive agent and resistive element. Alternatively, one or more resistors may be placed between base element and detection unit in order to effect resistance modification. The broad and generally applicable nature of the present invention is preserved during binding of the macromolecular layer 140 (Fig. 1) in proximity to the base member 120 because binding can be effected by either specific covalent attachment or general physical absorption. It is to be emphasized that the change in de novo signal that is associated with analyte presence does not depend on any specific enzyme chemistries, optical effects, fluorescence, cheπuluminescence, oxidation/reduction phenomena or apphed electrical signals. AdditionaUy, there are no reference electrodes, and the two detection unit electrodes are generaUy equipotential prior to measurement of signal internaUy-generated in the sensor strip. These features are important advantages of the present invention. Additionally, during operation of the sensor according to the invention, current is actually generated, and the generated electricity may be of use in powering devices such as certain components of the sensor system itself.

With respect to electricity generation, the generator design disclosed herein is based on electromagnetic induction of electrons in conducting materials when said electrons are exposed to fluctuating macromolecular electrostatic fields. The generator utilizes a novel method of generating electricity wherein macromolecular binding agents are first immobilized proximate an electrically conductive base element. The bound macromolecules are always moving; the motion of the electrostatic fields associated with the macromolecules serves to generate an induced electrical signal in the base element. The fluctuating electrostatic fields generate fluctuating magnetic fields, and these fluctuating magnetic fields induce electron motion in the base member. When the base member is part of an electric circuit, induced current wiU flow. The process of electromagnetic induction by mechanical means was first described by Faraday in 1831, and in the present invention, biological and synthetic macromolecules serve to generate the fluctuating magnetic fields required to induce current flow in a conductive base element. A typical generator strip comprises (i) a multilayer substrate comprising a conducting base element or layer and an optional chemical layer;(ii) at least one macromolecule that displays a level of affinity of interaction toward a predetermined cognate molecule or group of cognate molecules; and (iii) electrical leads contacted to the base element generally at two positions. In Fig. 10, four such generator strips 1021-1024 are shown attached through independent sets of leads to a rectifier-type device 1077 for the production and utilization of electrical energy.

The generator exploits the phenomenon of electromagnetic induction, the process by which fluctuating magnetic fields can induce electron motion in nearby electrically conducting materials. Since the device works on physical properties shared by nearly aU macromolecules, the methodology is appropriate for a large variety of macromolecule classes. AdditionaUy, charged or polar synthetic binding agents are appropriate for use in electricity generation. The one requirement for a macromolecular moiety is that it demonstrates a level of specificity of interaction with a predetermined cognate molecule or group of cognate molecules. The physical motion of macromolecules (proteins and DNA) immobilized in proximity to sohd supports is of importance to the present generator methodology and has been described in the scientific literature (Proteins on insulating mica: Thomson, NH, et al., Biophysics Journal 70:2421-2431 (1996); DNA on insulating mica: BezaniUa, M, et al, Biophysics Journal 67: 2454-2459 (1994)).

According to preferred embodiments of the generator, in order to generate electricity , one first immobilizes one or more biological or synthetic macromolecules in proximity to an appropriately conductive base element. A change in motional behavior of the binding agent or addition of electrostatic material associated with the cognate molecule causes an increased electromagnetic induction in the base element and thus allows for triggered increases in current production by the device. Experience of the inventor has shown that non-specific interactions of macromolecules and sample do not produce a significant induced current. Such conclusions are based on studies in complex matrices such as blood plasma, milk, stool, and ground beef homogenized in phosphate buffer. This point is significant as the generator may be employed in waste streams or industrial run-offs, converting waste products into electricity.

The present invention provides for the generation of electrical energy by the mechanical motions of charged/polar macromolecules. The amount of energy can be fixed by the specific macromolecule/molecule system selected as weU as the number of base elements employed in a multiplexed device. The number of macromolecules bound in proximity to a conducting base element is dependent on the size of the specific macromolecules as well as packing efficiencies. For a macromolecule such as an enzyme that is 100 Angstroms on a side, and at high packing efficiencies, 10 " macromolecules can be immobilized per square centimeter of base element. Paul Hansma and his coUeagues (Radmacher, et al. cited previously and public seminar, Jerusalem, Israel 1996) have shown by atomic force microscopy (AFM) that the enzyme lysozyme experienced approximately 1.5 nanometer vertical and 10 nanometer horizontal displacements during its catalytic cycle. The enzyme performed its reaction at a rate of 20 turnovers per second. Thus, one could expect the theoretical net motion of 1012 macromolecules bound in proximity to a square centimeter of base element to be 100 kUometers per second. This value assumes a net motion of 5 nanometers per cycle, 20 cycles per second, and that aU of the macromolecules are engaged in catalysis. In reality, the density of macromolecules may be lower, the amount of motion per cycle could be less, less than 100% of aU enzyme molecules may be functioning, and enzyme motions are not coordinated so a real "net" distance would be less. StiU, the exercise is instructive in demonstrating the tremendous amount of mechanical motion of charged bodies, and the potential for converting those motions into electrical energy. The motion of macromolecule-associated electrostatic fields creates fluctuating magnetic fields that induce current flow in the base element. Enzymes, due to their substrate-responsive motional behavior, are preferred macromolecules for use in the macromolecular layer according to the present invention. Enzyme turnover rates as high as of 10,000 conversions per second have been documented.

Referring again to Fig. 1, the macromolecular layer 140 used in the present sensor invention is located proximate base member 120. A chemical entity or SAM 130 (shown at the left side of Fig. 1) may optionally be disposed between the base member 120 and the macromolecular layer 140, or the macromolecular layer 140 could be positioned on an element (not shown) that is separate from the base member 120 itself. For the purposes of this invention, "proximate" with respect to macromolecule disposition relative the base member is defined as any distance that aUows for analyte-responsive generation of a de novo electrical signal in a sensor strip comprised of the macromolecules of the macromolecular layer 140 and the base member 120.

The detection unit 170 is any device or material that can detect one or more de novo electrical signals in a sensor strip 122 as a result of the latter's exposure to a sample that contains analyte 155. Examples of such signals include but are not limited to current; magnetic field strength; induced electromotive force; voltage; impedance; signal sign; frequency component or noise signature of a predetermined electrical signal propagated into a sensor strip at a first location and received at a second location. While the detection unit 170 may be a digital electrical metering device, it may also have additional functions that include, but are not limited to sensor strip grounding, data storage, data transfer, data processing, alert signaling, command/control functions, and process control. Detection units may be contacted through "leads", realized as electrodes 160 and 161 to one or a plurality of sensor strips 122. Contacts between the sensor strip 122 and detection unit 170 are generaUy at two positions 165, 167 on the sensor strip. Referring to Fig. 5, if the detection unit 570 is a voltmeter device with very high internal impedance, one can measure an internaUy-generated emf directly through passive contact of electrodes 560 and 561 to the sensor strip. A semiconductive element 598 incorporated into electrode 561 aUows for measurement of the induced emf in sensor strip 522. Adhesive agent 533 aids in good contact between the sensor strip 522 and detection unit 570 electrodes 560 and 561.

The internally-generated electrical signal is measured in a sensor circuit that includes at least one resistive element 299 (Figs. 2, 6) or a semiconductive element 398 (Fig. 3). Baseline readings may be determined from a sample that lacks target analyte or analytes or for a grounded sensor strip prior to sample exposure. For example, milk that lacked any antibiotics, registered internaUy-generated (de novo) voltage readings of 8 miUivolts in a sensor strip composed of aluminum foil, carboxyhc acid-based SAM's, peniciUinase (an enzyme that recognizes the analyte, penicillin), and the packaging layer of sodium chloride and glucose. Milk spiked with penicillin at 4J parts-per-billion (weight-to-volume) yielded an internaUy- generated signal of 371 millivolts. The specific design of a detection unit depends on what quantity or quantities (current, magnetic field flux, frequency, impedance, etc.) are being observed. The detection unit may be integrated into a computer 480 as shown in Fig. 4 or other solid-state electronic device for easier signal processing and data storage. The same or a different computer may be used to control sample apphcation or sample serial dϋution in order to monitor both sample manipulation as weU as the internaUy-generated electrical responses in a single or multiplexed sensor strip arrangement. The detection unit may also be a voltage-sensitive dye or colored material.

With respect to quantification of analyte presence, one may employ serial dUution of sample with analyte detection as described in the present apphcation. Specifically, a sample of interest is serially diluted in a serial dilution unit 1190 (Fig 11), and each dilution is exposed either to one sensor strip of base member plus macromolecules or to independent sensor strips 1121-1124. As all of the strips are generaUy of identical embodiment, they exhibit identical sensitivity. One determines the limit of sensitivity for sensor strips prior to an experiment. Thus, when one exposes the serial dilutions to sensor strips, there wiU be a response m those samples that have analyte present at or above the lower limit of sensitivity One may thus calculate the concentration of analyte as a function of the known sensitivity of the system and the highest level of dilution that StiU yields a signal One may alternatively use various embodiments for the sensor strips (not shown) In such a case, the sensitivity of each sensor stπp must be determined pπor to use

As descπbed in the patent apphcation PCT/IL99/00309, the methodology of detection is very sensitive Knowledge of absolute sensitivity of a given embodiment is cπtical for seπal dilution-based quantification, as one may calculate an analyte concentration as a function of seπal dilution Seπal dilution can be controUed by a seπal dilution unit, (SDU), 1190 (Fig 11) and pre-determined sensor stπp sensitivity Thus, if one dilutes a sample one thousand fold, and the known sensitivity of a sensor stπp is 1 ceU per millihter, then a positive score would imply that at least 1000 cells per miUihter were present m the oπgmal sample If a further tenfold dilution yields no score, then one knows that the concentration of cells in the initial sample was between one thousand and ten thousand ceUs per milhliter A finer seπal dilution may yield a more precise value

Referring again to Fig 1, an optional packagmg layer 150 for the sensor detection system 100 is a layer of water-soluble chemicals deposited above the immobilized macromolecules of the macromolecular layer 140 The packagmg layer 150 is deposited by soakmg or spraymg methods The packagmg layer 150 serves to stabihze the macromolecular layer 140 during prolonged storage In the absence of a packagmg layer, oil and dirt may build up on the macromolecular layer 140 and may mterfere with the rapid action of the sensor system Glucose and a salt, such as sodium chloπde, are typically used for the packagmg layer 150 so as to guarantee their dissolution m aqueous samples, and thus facilitate direct mteraction between macromolecular bmdmg agent (macromolecular layer 140) and analytes 157 Additionally, for sensor stπps that are coated equally on both sides, the packagmg layer may also serve as part of a resistive element 299 (Fig 2), as electrons may pass through nonconducting mateπal contained in the packagmg layer Electrode 161 contacts this layer at position 167 Other hydrophihc chemicals may be chosen for this role When the packagmg layer 150 dissolves, the bmdmg agents are free to immediately mteract with analyte 155 and 157, as shown on the πght side of Fig 1 Water-soluble polymers, sugars, salts, organic, and inorganic compounds are aU appropπate for use m preparation of the packagmg layer 150

Specific features of the detection unit such as arrangements of electrodes and modes of response to analyte presence may be found in the PCT patent apphcation noted previously. Leads may be contacted to end or internal regions of a sensor strip. Contact of at least one lead to an end of a strip appears to aid in signal acquisition. The detection unit may also include a mixing element or chamber in order to aid in bringing analyte to macromolecules. There are several points to note in regards to the method of detection of analyte as performed by the present invention. Conducting materials are normaUy at a single electrical potential (voltage) at aU points along their surfaces. In the present invention, interactions of macromolecular binding agents with target analytes aUow for internal generation of electrical signals in a sensor strip composed minimaUy of a conductive base member and an associated macromolecular binding agent. A sensor circuit that includes sensor strip, a detection unit, detection unit electrodes and a resistance-modifying element aUows for facUe detection of the electrical signals generated in the sensor strip. Readings as high as 500 millivolts or 10 microamperes have been routinely recorded in functioning analyte detection systems according to the invention, employing enzyme, nucleic acid, receptor, antibody, and synthetic binding agents.

The implications of the analyte detection methodology are significant. Firstly, detection can take place far away from the point of macromolecule-analyte contact, as the internaUy- generated electrical signals are propagated throughout the conductive portions of a sensor strip. This fact aUows for closed-package "food sensing" or the sensing of potentiaUy hazardous samples, e.g. blood in closed containers. One portion of the sensor contacts the material of interest, while detection of analyte-responsive de novo electrical signals occurs between two points on the exposed portion of the sensor strip. This remote detection capability is an important feature of the present sensor. The implications are that nearly any material that can be recognized at a level of specificity by a peptide, protein, antibody, enzyme, nucleic acid single strand, synthetic binding agent, or the like can be detected and quantified safely in food, body fluids, air or other samples quickly, cheaply, and with high sensitivity. Response is very rapid, generaUy less than 90 seconds. Cost of manufacture is low, and sensitivity has been shown to be very high.

It is beheved that the internaUy-generated electrical signals result specificaUy from changes in magnetic fields associated with the binding elements. According to a method of magnetic flux detection, one may provide a non-conducting "base unit", possibly an inexpensive organic polymer. Macromolecules are immobilized in proximity to the base unit. As shown in Fig. 12, a sample that may contain a target analyte is contacted to the sensor element 1222 of base unit and macromolecules and a detection unit 1270 in a non-contact mode of operation detects magnetic fields or magnetic field flux that is responsive to analyte presence.

Whereas the invention described in the noted PCT apphcation PCT/IL99/00309 made use of specifically conductive base members and detection of de novo electrical signals, the sensor element does not necessitate use of a conducting base element, and direct detection of magnetic fields may alternatively be performed in the absence of contact between a detection unit and a sensor element minimaUy composed of macromolecules and a base unit. A base unit may not be necessary if the macromolecules are present in a solution that contains the analyte of interest.

Magnetic field flux generated during the interaction of macromolecules and analyte may be detected without recourse to a base unit.

The present invention has several advantages compared to the known prior art:

While the sensor method disclosed in the noted co-pending PCT apphcation that is based on detection of de novo electrical signals has allowed for rapid determination of analyte presence in complex sample matrices, the issues of electrode contact to sensor strip and sensor circuit components have heretofore not been fully described. While some embodiments, particularly involving alterations in SAM or base member have allowed for analyte detection, a goal of the present invention is to optimize the specific relationship of sensor strip to the other components of the sensor circuit. Thus, the present invention aUows for analyte detection by virtue of monitoring of electrical signals internaUy-generated in a sensor strip and measured in a sensor circuit that includes a resistive or semiconductive element as described. Multiple base members may be employed in a single sensor strip so as to increase system detection redundancy and/or multiple analyte detection capabUities. Each sensor strip is monitored on its own sensor circuit.

In an alternate preferred embodiment, an adhesive agent 633 (Fig. 6) may be apphed to a sensor strip 622 in order to facilitate strong electrical contact between it and the detection unit 670 the electrodes 660 and 661 that have been modified to include resistive elements 697, 699. The adhesive agent may also serve in the role of resistance-modifying element . Table 1 lists some of the possible components, detectable de novo electrical or magnetic signals and target apphcation markets relevant to the present invention. Each grouping is independent of the others and one may combine a base member, a macromolecule, and a signal for an apphcation area of choice. The table is in no way meant to be limiting in scope or spirit of the present invention.

Table 1

Base Member Macromolecule Signal Application

Metal Enzyme Magnetic Flux Food Safety

Conductive Film* Antibody Induced Current Chemicals Organic Conductor Nucleic Acids Impedance Biologicals Conductive Liquid Fatty Acid Resistance Environment Conductive Ink Receptor Sign Switching Hygiene Graphite Synthetic- Frequency Internet Semiconductor Molecule Noise Signature Genetic Testing Protein Electromagnetic- Diagnostics Peptide Induction Process Control CeU Capacitance Drug Screening

Catalytic- Fourier Transform Drug Release Antibody Band-Pass FUtered Glucose Testing

Synthetic Receptor Magnetic Fields Forensics Receptor Ligand Voltage Veterinary- Antigen Testing

*A conductive film can be deposited on a sohd support by any means, including electroless deposition, spin coating, sputtering, vapor deposition, plating, "printing" or dip- coating. Example 1

Aluminum foil sheets were soaked overnight in an aqueous solution of parahydroxybenzoic acid and then rinsed in water. The foU sheets were then soaked in a dilute solution of penicillinase (approximately 40 minutes) and transferred to a solution of sodium chloride and sucrose (packaging layer) prior to drying under ambient room conditions. Sensor strips were cut from the sheets and used in the detection of ampicillin. The detector used was a Radio Shack multimeter (Catalogue Number 22-168A) that comes fitted with a computer cable and PC-appropriate software. The supphed leads were replaced with two banana leads that were modified for detection unit specifications. To one lead was soldered a thin metal rod (approximately 5 centimeters in length; gold-coated at the end) and to the other was soldered a gold-coated sihcon wafer. The vapor-deposited gold was used to facilitate soldering of the silicon piece (approximately l x l cm") to the lead. The exposed side of the silicon chip was coated with UHU stick glue, and a sensor strip was pressed onto the glue-coated sihcon chip. The metal rod was placed flush on top of the strip. In this example, based on the alternate embodiment shown in Fig. 3, there are two banana leads, a semiconductive sihcon element, the sensor strip, and the multimeter-based detection unit. No power was apphed to the sensor strip. De novo voltages were measured for a strip in air and in the presence of saline (data not shown). As the readings did not exceed 0.4 mV, the system was considered to be free of contamination and ready for analyte detection. The same saline solution was employed for dissolution of ampiciUin; the ampicUlin solution was used in testing the penicillinase (macromolecular binding agent)-based sensor strips. As shown in Fig. 7, a strip was tested first in air and then in the presence of the saline-ampi llin solution. The x-axis shows seconds for the experiment, while the y-axis is in miUivolts. The ampiciUin solution was added at approximately sixty seconds into the experiment. Significant de novo signal was recorded only and specificaUy when ampiciUin was present in the sample. Example 2 Aluminum foil (Reynolds) sheet is soaked in an ethanohc solution of stearic acid. The

SAM-coated foil is washed in water and then soaked in an aqueous solution of lysozyme. The sensor sheet is soaked briefly in sodium chloride and glucose and then aUowed to dry. The sheet is cut into strips and the strips are packaged. For use, each strip is placed into a 1.5 miUihter Eppendorf tube. Sample that may contain bacteria, the lysozyme substrate, is added to the Eppendorf tube and the tobe is closed. Two leads of a detection unit are contacted to the exposed portion of the sensor strip, and through one of the leads, one lead having a sihcon semiconductor placed at its end such that the sihcon chip is in direct contact with the sensor strip. When the detection unit reads a low voltage background for the strip, the sample is contacted to the strip on the inside of the Eppendorf tube, whUe voltage measurements are made between the two contact points between the sensor strip and the detection unit electrodes. Signals significantly above pre-determined background values imply the presence of bacteria in sample. The sensor strip may be grounded during the course of a sensing experiment; return of signal after grounding suggests that the signal is due to the action of the lysozyme macromolecules associated with the aluminum foU base member. Example 3

Semiconductor-grade sihcon was cut into chips. The chips were coated with strips of conducting silver paint. SpecificaUy, a coat of Jeltargent conducting sUver paint was applied either in a straight line or in an "L" shape. The paint was allowed to air-dry. Approximately half of the coated chips were soaked in an aqueous solution of the enzyme, lactase (beta- galactosidase) for fifty-minutes while half were left as control chips. Those that were soaked in enzyme solution were further soaked for ten minutes in a sodium chloride solution and then aUowed to air dry. Thus, the sensor strip included a semiconductive layer (sihcon), a base member (silver paint), macromolecular entities (lactase), and a packaging layer (sodium chloride). The detection unit, electrodes, and semiconductive element were the same as described in Example 2. AdditionaUy, UHU glue was apphed to the sihcon chips (opposite the side with silver) and aided in contact of sensor strip to the semiconductive element, as shown in Fig. 5. Chips, both with and without enzyme, were exposed to saline and then to a solution known to contain lactose, the enzyme substrate. Fig. 8 shows results for a lactase-coated chip, while Fig. 9 shows the data for a control chip that lacked enzyme. In both cases, the chips were exposed initiaUy to air, then to saline until a steady baseline had been established, and finally to a solution that contained lactose. In the experiment detailed in Fig. 8, saline was placed on the chip after approximately thirty seconds, and lactose-containing solution was then added approximately thirty seconds later (approximately one minute into the experiment). In the control experiment (Fig. 9), saline was placed on the chip after approximately one minute, while lactose-containing solution was added one minute later (two minutes into the experiment). Such experiments were performed three separate times on different chips for lactase-containing and control chips (six experiments total). The data displayed in Figs. 8 and 9 show that the maximum readout of the control experiment lacking macromolecular binding agent was around 6 millivolts (Fig. 9), whϋe the sensor strip that contained the enzyme lactase showed readings in excess of negative 100 miUivolts (Fig. 8). Similar results were seen for the other four experiments (data not shown). No external electrical signal was apphed to the un- powered sensor strip and lactase does not perform oxidation-reduction chemistry. Example 4

Semiconductor-grade sihcon (1 cm x 1 cm) is photolithographicaUy modified to yield a structure of 5 x 104 aluminum wires (0J micron width) with 0J micron spacing between them. The wires are coated with a SAM prepared from an ethanolic solution of parahydroxybenzoic acid. The enzyme triosephosphate isomerase (TIM) is physicaUy absorbed to the SAM layer, and a packaging layer of sodium chloride/glucose is deposited by soaking. The final generator "chip" has 5 x 104 sensor strip "lanes" of enzyme-coated conducting wires. The wires are coated at their ends with a dielectric resistance-modifying element and then contacted by electrodes that lead to a rectifier and ultimately to a load. The chip is exposed to an aqueous solution of 1 millimolar glyceraldehyde 3-phosphate (GAP), a TIM substrate. TIM interconverts GAP and product dihydroxyacetone phosphate (DHAP). The chip is sealed in plastic so that a few microhters of solution remain above the enzyme macromolecules. Current generated by the interaction of TIM with molecules of GAP and DHAP is directed by electrodes to a rectifier 1077 (Fig. 10). DC current is used to power a smart card (not shown) on which the chip is physicaUy fastened. WO 00/77522 PCTYUSOO/15400

24

Example 5

Long graphite rods are soaked in an aqueous solution of protease enzymes. Proteases digest proteins by breaking amide linkages between amino acids. Several hundred thousand of the graphite rods are connected in paraUel to a rectifier, and the graphite rods are exposed to agricultural waste streams that are rich in protein debris. Protease action on the protein (the substrate of the proteases) leads to increased motion of the charged protease molecules and thus heightened electromagnetic induction in the graphite. Current induced in the graphite is directed to a rectifier and then on to a device that processes the wastes. New proteins are always delivered to the macromolecules and the system continues to produce electricity until either the enzymes are no longer functional or fouling prevents delivery of proteins to the protease enzymes. At that time, the rods are replaced for fresh generator rods. Example 6

The concentration of bacteria in milk is to be determined. A sample of milk is diluted from ten to one million fold in a serial dUution unit 1190 as shown in Fig. 11. Each dilution is apphed to a separate sensor strip (sensor strips 1121-1124) that is prepared from aluminum foU, parahydroxybenzoic acid (the SAM), the enzyme lysozyme, and a packaging layer of NaCl and glucose. The sensitivity for a given strip is determined to be 3 ceUs per milhhter for the present embodiment. A computer 1180 dehvers portions of each serial dilution to independent sensor strips and then measures for an induced current in each strip. The increased induced current is measured in aU samples from ten-fold to ten-thousand fold dUution. One hundred thousand-fold and one million-fold dUutions show no activity. The concentration of cells in the original milk sample is therefore calculated to be between 30,000 and 300,000 ceUs per miUihter (absolute strip sensitivity times levels of dilution). A finer serial dilution screen is performed in order to further narrow down the range of ceU concentration values. Example 7

1.5-milhliter plastic "Eppendorf' tubes were soaked in an aqueous solution of the lactose-hydrolyzing enzyme, lactase (beta-galactosidase). For the experiment, a detection unit was prepared that aUowed for detection of magnetic flux by measurement of an induced electromotive force as per the sensor system embodiment of Fig. 12. Clean aluminum foU was contacted to an electrode of a commercially avaUable digital multimeter. The foU was additionally contacted to a semiconductor-grade sihcon chip that was coated with gold on one side, and this side was soldered to the second multimeter lead. Magnetic field flux impinging on the foU caused an induced emf to be produced in the foU. The foU was exposed to air for one minute, and then to an Eppendorf tube lacking lactase that contained goat milk. At approximately 2.5 minutes into the experiment, an Eppendorf tube that had been soaked in lactase and that also contained goat milk was placed in physical proximity to the aluminum foil in the detection unit. There was a significant increase in the induced emf (read in millivolts) due to the interaction of lactase with its substrates, water and milk-sugar, lactose. For this example, lactase enzyme molecules served as the macromolecules (not shown), wh e the Eppendorf tube served as the base unit of sensor element 1222, and the target analyte was lactose. The detection unit 1270 consisted of the multimeter, leads, foU, and semiconductor. The sensor element was composed of one Eppendorf tube and the physicaUy absorbed lactase molecules. The data for the experiment are summarized in Fig. 13. Example 8

A comparative clinical study was performed in order to determine the efficacy of the present invention in the detection of Hehcobacter pylori, the causative agent of gastric ulcers and other gastrointestinal ailments. Single gastric biopsies were removed during gastrointestinal endoscopies. Biopsies were soaked in a buffered solution prepared for the detection of the enzyme urease. Urease is externaUy associated with H. pylori and its enzymatic degradation of urea can be linked to pH sensitive dyes in order to detect a color change if the enzyme (and by implication, the bacteria) is present. Each biopsy was briefly removed from the urease detection solution, agitated in sterile saline in order to remove any associated H. pylori ceUs, and then returned to the urease detection solution. The saline solutions were then challenged with sensor strips according to the embodiment shown in Fig. 1, modified by the addition of resistive elements as shown in Fig. 6. The deta s of the sensor are aluminum foU (base member 120), parahydroxybenzoic acid SAM (130), antibodies specific for H. pylori (macromolecular layer 140), sodium chloride, and glucose (packaging layer 150). The strips were exposed to saline solutions and simultaneously contacted to leads (electrodes 160, 161) of a digital voltmeter (detection unit 170). Resistive elements 697 and 699 as shown in Fig. 6 were generaUy realized in the form of a layer of Scotch brand glue stick that aids in electrode-sensor strip contact wltile also serving the role of providing a resistive element between the strip and the leads. The glue thus served both as the adhesive agent 633 as well as the resistive elements 697, 699. The overall agreement between the present invention and the traditional urease test is currently 84% (Table I). Patient 32 was positive by the present invention in six different tests, aU of which were complete within two minutes; the same patient was found positive by urease testing after 24 hours. It is believed that the sample contained a low concentration of H. pylori. The urease test is considered to be 90% accurate, with poor performance at low concentrations of ceUs. It may be noted that aU disagreements between the results according to the present invention and the urease test reflect positive results detected by the present invention and negative results according to urease detection. In the case of patient 35, two strips of the present invention were read as positive whUe one was negative — a case for multiplexed systems based on the present invention.

Table 2

Patient Present Invention Urease Test Comment

1 Positive Negative Large Ulcer

2 Negative Negative Match

3 Negative Negative Match

4 Positive Positive Match

5 Negative Negative Match

6 Positive Positive Match

7 Positive Negative

8 Positive Positive Match

9 Negative Negative Match

10 Negative Negative Match

11 Positive Negative

12 Positive Positive Match

13 Positive Negative

14 Negative Negative Match

15 Negative Negative Match

16 Positive Negative

17 Positive Positive Match

18 Positive Positive Match

19 Negative Negative Match

20 Positive Positive Match

21 Negative Negative Match

22 Positive Positive Match

23 Positive Positive Match

24 Positive Positive Match

25 Positive Positive Match

26 Positive Positive Match

27 Negative Negative Match

28 Negative Negative Match

29 Negative Negative Match

30 Positive Positive Match

31 Positive Positive Match

32 Positive Positive Match (urease: 24 hours)

33 Positive Positive Match

34 Positive Positive Match

35 Positive Positive Match

36 Positive Negative

37 Positive Positive Match 38 Positive Positive Match

While the sensing strip architecture of "base material-SAM-macromolecule" has been described previously, analyte detection and quantification based on electrical signals generated de novo in a sensor strip is beheved to be new in the art. Prior-art sensors require apphed electrical signals and generaUy rely on the chemical generation of charged species near the sensor surface. Today, there are neither aluminum foil-based sensor strips (as described in the Examples above) nor sensor strips designed exclusively for the detection of a de novo emf or current in a sensor strip. Thus, any sensor strip designed for the detection of a de novo electrical signal between two points of a sensor strip as a function of analyte presence would be produced and used with the express purpose of violating the invention described in this patent apphcation. Such strips would find application in food pathogen detection, medical diagnostics, and the like, whUe the detection and serial dilution units would constitute separate components.

The present invention has been described with a certain degree of particularity, however those versed in the art will readuy appreciate that various modifications and alterations may be carried out without departing from the spirit and scope of the foUowing claims: Therefore, the embodiments and examples described here are in no means intended to limit the scope or spirit of the methodology and associated devices related to the present invention.

Claims

Claims
1. A sensor (100) for detecting an analyte, comprising: abase member (120) having a conductive electrical property; a macromolecular entity (140) bound to said base member (120);
CHARACTERIZED IN THAT said macromolecular entity (140) and said base member (120) define a sensor strip, said macromolecular entity (140) being interactive at a level of specificity with a predetermined analyte (155, 157), wherein an electrical signal is internaUy-generated in said sensor strip responsive to presence of the analyte (155, 157); and a resistance-modifying element (299, 398) disposed in a circuit between said base member and a detection unit (170) for detection of said electrical signal.
2. The sensor according to claim 1, further comprising a self-assembled monolayer (130) bound to said base member ( 120) and proximate said macromolecular entity ( 140).
3. The sensor according to any of claims 1 - 2, further comprising two equipotential leads (160, 161) coupling said sensor strip to said detection unit (170), wherein at least one of said equipotential leads (160, 161) is connected to said resistance-modifying element (299, 398).
4. The sensor according to claim 3, further comprising an adhesive agent (533) disposed between said base member, and at least one of said equipotential leads (560, 561).
5. The sensor according to claim 4, wherein said adhesive agent (533) acts as a resistance- modifying element.
6. The sensor according to any of claims 1 - 2, wherein the base member (120) is a conducting foU, coating, thin-film, ink, or sohd piece.
7. The sensor according to any of claims 1 - 2, further comprising a packaging layer (150) disposed above said macromolecular entity (140), said packaging layer (150) being soluble in a medium that contains the analyte. (155, 157).
8. The sensor according to any of claims 1 - 2, wherein said resistance-modifying element is realized as a self-assembled monolayer (130).
9. The sensor according to any of claims 1 - 2, wherein said resistance-modifying element is realized as a chemical entity.
10. The sensor according to any of claims 1 - 2, further comprising a serial dilution unit (1190) for presenting a plurahty of dflutions of a sample containing said analyte to said sensor stπp.
11. The sensor according to claim 10, wherein said sensor strip comprises a plurahty of sensor strips, each of said sensor strips accepting a different dilution of said analyte from said serial dilution unit.
12. A method for detecting a predetermined analyte, comprising the steps of: providing an electrically conductive base member (120); immobilizing at least one macromolecule (140) in proximity to said base member (120), wherein said macromolecule (140) is capable of interacting at a level of specificity with said predetermined analyte (155, 157), CHARACTERIZED IN THAT said base member (120) and said macromolecule ( 140) define a sensor strip; exposing said predetermined analyte to said macromolecule (140); and, detecting an electrical signal that is internaUy-generated in said sensor strip, said electrical signal being responsive to a presence of said predetermined analyte (155, 157), wherein said step of detecting is performed with an electrical circuit that includes said base member (120) and at least one resistance-modifying element (298, 399).
13. The method according to claim 12, further comprising the steps of: binding a self-assembled monolayer (130) to said base member (120); and immobilizing said macromolecule (140) proximate said self-assembled monolayer (130).
14. The method according to any of claims 12 - 13, wherein said step of detecting is performed by equipotentially coupling a detection unit (170) to said sensor strip at no more man two positions (165, 167) of said sensor strip.
15. The method according to claim 14, wherein said step of coupling said detection unit
(170) is performed by passively contacting electrical leads (160, 161) to said sensor strip.
16. The method according to claim 15 further comprising the step of providing an adhesive agent in said circuit in contact with said sensor strip and at least one of said electrical leads.
17. The method according to claim 15, further comprising the step of coupling said electrical leads (160, 161) to a load for delivery of power thereto.
18. The method according to any of claims 12 - 13, further comprising the step of disposing a packaging layer (150) above said macromolecule (140), said packaging layer (150) being soluble in a medium that contains said predetermined analyte (155, 157).
19. The method according to any of claims 12 - 13, wherein said resistance-modifying element is realized as a self-assembled monolayer.
20. The method according to any of claims 12 - 13, wherein said resistance-modifying element is realized as a chemical entity.
21. The method according to any of claims 12 - 13, further comprising the steps of: seriaUy diluting said predetermined analyte; to obtain a plurahty of serial dilutions; and presenting said serial dilutions separately to said sensor strip.
22. The method according to claim 21, wherein said sensor strip comprises a plurality of sensor strips, and said step of presenting said serial dflutions is performed by presenting each of said serial dilutions to a respective one of said sensor strips. AMENDED CLAIMS
[received by the International Bureau on 12 November 2000 (12 1 1 00) , original claims 1-22 replaced by new claims 1-18 (3 pages)]
1 A sensor (100) for detecting an analyte, compπsmg a base member ( 120) having a conductive electπcal property, a macromolecular layer ( 140), CHARACTERIZED IN THAT said macromolecular layer (140) and said base member (120) define a sensor stπp, (122) macromolecules of the macromolecular layer (140) bemg mteractive at a level of specificity with a predetermmed analyte (155, 157), wherem electπcal current is generated responsive to presence of the analyte (155, 157), and a semiconductive element (398) is disposed in a sensor circuit (370, 398. 322) and situated between said base member (120) and a detection umt ( 170) for detection of said electπcal current
2 The sensor according to claim 1, further compnsing a chemical entity disposed between said base member ( 120) and said macromolecular layer ( 140)
3 The sensor according to any of claims 1 - 2, further compπsmg two equipotential electrodes
(160, 161) coupling said sensor stπp (122) to said detection unit (170)
4 The sensor according to any of claims 1 - 2 wherem said semiconductive element (398) is physicaUy incorporated mto the sensor stπp (322)
5 The sensor according to any of claims 1 - 2, wherem said semiconductive element (398) is physically incorporated mto at least one of the detection unit (370) electrodes (360, 361)
6 The sensor according to any of claims 1 - 2, wherem the semiconductive element (398) is physicaUy incorporated mto the detection unit (370)
7. The sensor according to any of claims 1 - 2, wherein the base member (120) is an electricaUy conducting foil, coating, thin-film, ink. or sohd piece.
8. The sensor according to any of claims 1 - 2, further comprising a packaging layer (150) disposed above said macromolecular layer (140), said packaging layer (150) being soluble in a medium that contains the analyte. (155, 157).
9. The sensor according to any of claims 1 - 2 wherein a plurahty of sensor strips (1121 - 1124) is employed in the detection of at least one predetermined analyte.
10. A method for detecting a predetermined analyte, comprising the steps of: providing an electricaUy conductive base member (120); forming a macromolecular layer (140) in proximity to said base member (120), wherein macromolecules of said macromolecular layer (140) are capable of interacting at a level of specificity with said predetermined analyte (155, 157), CHARACTERIZED IN THAT the base member ( 120) and the macromolecular layer ( 140) define a sensor strip ( 122); exposing said predetermined analyte (155, 157) to said macromolecular layer ( 140); and, detecting an electrical current generated in a closed electrical circuit , said current being responsive to presence of said predetermined analyte (155, 157), wherein said closed electrical circuit -minimally includes said base member (120), a detection unit (170) and at least one semiconductive element (398).
11. The method according to claim 10, further comprising the steps of: disposing a chemical entity proximate said base member ( 120); and forming said macromolecular layer ( 140) proximate said chemical entity.
12. The method according to any of claims 10 - 11, wherein said step of detecting is performed by equipotentiaUy coupling electrodes (160, 161) of a detection unit (170) to said sensor strip (322).
13. The method according to claim 12, wherein said step of coupling said detection unit (170) is performed by passively contacting said electrodes ( 160, 161 ) to said sensor strip ( 122 ).
14. The method according to any of claims 10 - 11, wherein the semiconductive element (398) is realized as an organic semiconductor physicaUy incorporated into the sensor strip (322).
15. The method according to any of claims 10 - 11, wherein the semiconductive element (398) is realized as an organic semiconductor physically incorporated into one of the detection unit (370) electrodes (360, 361).
16. The method according to any of claims 10 - 11, further comprising the step of disposing a packaging layer (150) above said macromolecular layer (140), said packaging layer (150) being soluble in a medium that contains said predetermined analyte ( 1 5, 157).
17. The method according to any of claims 10 - 11 wherein a plurahty of sensor strips (1121 - 1124) is employed in the detection of at least one predetermined analyte.
18. The method according to any of claims 10 - 11 further comprising the step of providing a signal to a device.
PCT/US2000/015400 1999-05-04 2000-06-05 Analytic sensor apparatus and method WO2000077522A1 (en)

Priority Applications (14)

Application Number Priority Date Filing Date Title
IL130478 1999-06-15
IL13047899A IL130478D0 (en) 1999-06-15 1999-06-15 An electric generator
IL131193 1999-08-01
IL13119399A IL131193D0 (en) 1999-08-01 1999-08-01 Sensor and method for determining a concentration range of an analyte in a sample
IL132491 1999-09-21
IL13198399A IL131983D0 (en) 1999-05-04 1999-09-21 A sensor for detecting the presence of an analye in a sample
IL131983 1999-09-21
IL13249199A IL132491D0 (en) 1999-10-21 1999-10-21 Sensor and method for identifying an analyte in a sample
US09/426,564 1999-10-22
US09/426,564 US6342347B1 (en) 1999-10-22 1999-10-22 Electromagnetic sensor
IL13305999A IL133059D0 (en) 1999-11-21 1999-11-21 Sensor for identifying and analyte in a sample
IL133059 1999-11-22
IL133323 1999-12-06
IL13332399 1999-12-06

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EP20000944617 EP1185868A1 (en) 1999-06-15 2000-06-05 Analytic sensor apparatus and method
AU58687/00A AU5868700A (en) 1999-06-15 2000-06-05 Analytic sensor apparatus and method

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