Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING 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
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
  • C12Q1/002—Electrode membranes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
  • G01N33/5438—Electrodes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
  • G01N2333/98—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
  • G01N2333/986—Hydrolases (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)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/99—Isomerases (5.)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2415/00—Assays, e.g. immunoassays or enzyme assays, involving penicillins or cephalosporins

Definitions

• 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 dete ⁇ nining the presence of the targeted analyte.
• an enzyme 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.
• an enzyme 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.
• ELISA Enzyme-Linked ImmunoSorbent Assay
• 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).
• FET field-effect transistor
• 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.
• sensors that are based on electrical means for analyte detection.
• 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.
• 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.
• the binding agent-a alyte complex causes an altered signal
• the bound analyte itself is the source of changed electrical response.
• 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.
• 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.
• the resistance-modifying element is a self-assembled monolayer.
• the resistance-modifying element is a chemical entity.
• the internaUy-generated electrical signal is further processed for determination of analyte presence or concentration.
• 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.
• an additional step includes processing the electrical signals for determination of analyte presence.
• 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.
• 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;
• Fig. 13 is a graph of detection of lactose based on a sensor system embodiment described in Fig. 12. Description of the Preferred Embodiment
• 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.
• 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.
• a typical sensor detection system 100 comprises (i) a sensor strip 122; ( ⁇ ) 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.
• a resistive element 299 Fig. 2
• semiconductive element 398 FigJ
• 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.
• 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.
• 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.
• emf electromotive force
• 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.
• 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.
• Micromolecules 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.
• SAM self-assembled monolayer
• a self-assembled monolayer is formed from molecules that bond with the surface upon their direct contact from solvent, vapor, or spray.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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:
• the macromolecules chosen as binding agents are highly specific entities designed to bind only with a selected analyte or group of analytes;
• a sensor strip including a macromolecular binding agent and an electrically- conductive base member can exhibit internaUy-generated electrical signals
• internaUy-generated electrical signals are responsive in magnitude, sign, and or frequency to the presence of analyte in a sample exposed to sensor strip;
• 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.
• a closed electrical circuit comprised of sensor strip, a detection unit, associated electrodes, and a resistive or semiconductive element.
• 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.
• 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.
• a resistance-modifying element such as semiconductive element 398 (FigJ) or a resistive element 299 (Fig.
• 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.
• 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.
• 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.
• 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.
• induced current wiU flow When the base member is part of an electric circuit, induced current wiU flow.
• 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.
• a multilayer substrate comprising a conducting base element or layer and an optional chemical layer
• at least one macromolecule that displays a level of affinity of interaction toward a predetermined cognate molecule or group of cognate molecules
• electrical leads contacted to the base element generally at two positions.
• 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.
• 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.
• 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 (Radraum, et al.
• 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.
• the macromolecular layer 140 used in the present sensor invention is located proximate base member 120.
• a chemical entity or SAM 130 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.
• 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.
• 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 d ⁇ ution 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.
• serial dUution of sample with analyte detection as described in the present apphcation.
• 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.
• Fig 11 serial dilution unit 1190
• each dilution is exposed either to one sensor strip of base member plus macromolecules or to independent sensor strips 1121-1124.
• all of the strips are generaUy of identical embodiment, they exhibit identical sensitivity.
• 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
• 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 chlo ⁇ de 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
• the packagmg layer may also serve as part of a resistive element
• Electrodes 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.
• Conducting materials are normaUy at a single electrical potential (voltage) at aU points along their surfaces.
• 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.
• analyte detection methodology is 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 internaUy-generated electrical signals result specificaUy from changes in magnetic fields associated with the binding elements.
• 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.
• 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 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.
• an adhesive agent 633 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.
• 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.
• 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.
• 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.
• the detection unit 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.
• 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.
• 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
• Fig. 9 shows the data for a control chip that lacked enzyme.
• Semiconductor-grade sihcon (1 cm x 1 cm) is photolithographicaUy modified to yield a structure of 5 x 10 4 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 10 4 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.
• GAP millimolar glyceraldehyde 3-phosphate
• 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.
• 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.
• 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.
• 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.
• 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.

Applications Claiming Priority (15)

Publications (1)

Family

ID=27563025

Family Applications (1)

Country Status (3)

Families Citing this family (53)

Family Cites Families (6)

• 2000
  • 2000-06-05 WO PCT/US2000/015400 patent/WO2000077522A1/en not_active Application Discontinuation
  • 2000-06-05 EP EP00944617A patent/EP1185868A1/de not_active Withdrawn
  • 2000-06-05 AU AU58687/00A patent/AU5868700A/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events