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WO1999066322A1 - Capteur de detection d'analytes - Google Patents

Capteur de detection d'analytes Download PDF

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Publication number
WO1999066322A1
WO1999066322A1 PCT/IL1999/000309 IL9900309W WO9966322A1 WO 1999066322 A1 WO1999066322 A1 WO 1999066322A1 IL 9900309 W IL9900309 W IL 9900309W WO 9966322 A1 WO9966322 A1 WO 9966322A1
Authority
WO
WIPO (PCT)
Prior art keywords
base member
analyte
sensor
macromolecules
macromolecule
Prior art date
Application number
PCT/IL1999/000309
Other languages
English (en)
Inventor
Alan Joseph Bauer
Original Assignee
Biosensor Systems Design, Inc. (1998)
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 claimed from US09/110,686 external-priority patent/US6096497A/en
Application filed by Biosensor Systems Design, Inc. (1998) filed Critical Biosensor Systems Design, Inc. (1998)
Priority to AU41628/99A priority Critical patent/AU4162899A/en
Priority to EP99925262A priority patent/EP1093583A1/fr
Priority to US09/701,906 priority patent/US6322963B1/en
Publication of WO1999066322A1 publication Critical patent/WO1999066322A1/fr

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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
    • 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

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 induced electromagnetic effects that are produced as a result of such interactions.
  • 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 enzymes, 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 determining 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 placed into contact with 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 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.
  • an a voltage or current is applied to the sensor surface or an associated medium.
  • 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 electrochemically 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 actually 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 electrochemically active species whose presence alter the system applied voltage as recorded at a given sensor electrode. Not all 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 electrochemically 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 electrochemically-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-analyte 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 all 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 applied 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 all 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 make use of any applied electrical or electromagnetic signal.
  • a sensor which has an electrically conductive or semiconductive base member, and at least one macromolecular entity disposed proximate the base member and interactive at a level of specificity with at least one predetermined analyte.
  • An electrical signal is induced in the base member responsive to the interaction of the macromolecular entity with the analyte.
  • a detection unit detects the induced electrical signal.
  • a self-assembled monolayer is bound to the base member, proximate the macromolecular entity.
  • Macromolecular entities are arranged in a monolayer or multilayer.
  • a plurality of macromolecular entities is employed for the detection of at least one analyte.
  • electrical leads of the detection unit are coupled to the base member at no more than two positions of the base member.
  • the coupling may be passive.
  • the electrical connections may be coupled to a load for delivery of power thereto.
  • the base member is a conducting foil, coating, thin-film, ink, or solid piece.
  • a plurality of base members is employed in detection of at least one analyte.
  • a packaging layer is disposed above the macromolecular entity, the packaging layer being soluble in a medium that contains the analyte
  • the invention provides a method for detecting an analyte, having the following steps: providing an electrically 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; exposing analyte to the macromolecule; and detecting an electrical signal induced in or about the base member, wherein the detected electrical signal is responsive to analyte presence or interaction with the macromolecule.
  • a self-assembled monolayer is bound to the base member, the macromolecules are immobilized proximate to the self-assembled monolayer.
  • a plurality of macromolecules having different specificity of interaction are immobilized for the detection of at least one analyte.
  • the step of detecting is performed by coupling electrical 5 leads of a detection unit to the base member at no more than two positions of the base member.
  • the coupling may be passive.
  • a plurality of base members is provided in the detection of at least one analyte.
  • the macromolecular o entities are arranged in a monolayer or multilayer proximate the base member.
  • a packaging layer is disposed above the macromolecules, the packaging layer being soluble in a medium that contains the analyte.
  • a further step5 comprises coupling the electrical connections to a load for delivery of power thereto.
  • Fig. 1 is a schematic view of an embodiment of a sensor (100) in accordance with the invention, wherein macromolecular entities (140) are immobilized above a self assembled monolayer (130) and covered by a packaging layer (150), and having a base member (120) that is a solid conductive piece;
  • Fig. 2 is a schematic view of a first alternate embodiment of a sensor (200) in accordance with the invention which is similar to the embodiment shown in Fig. 1 except that the self assembled monolayer has been omitted and macromolecules (240) are physically absorbed proximate the base member (220);
  • Fig. 3 is a schematic view of a second alternate embodiment of a sensor (300) which is similar to the embodiment shown in Fig.
  • Fig. 4 is a schematic view of a third alternate embodiment of a sensor (400) which is similar to the embodiment shown in Fig. 3 except that one type of macromolecule (440) occupies the multilayer;
  • Fig. 5 is a schematic view of a fourth alternate embodiment of a sensor (500) in accordance with the invention wherein a base member (520) is a o conductive coating, thin film or ink on a non-conducting solid support ( 15);
  • Fig. 6 is a schematic view of a fifth alternate embodiment of a sensor (600) in accordance with the invention which is similar to the embodiment of Fig. 4, in which a packaging layer (650) is present prior to analyte delivery (left side of figure) and absent (right side of figure) when analyte (655) is present and interacts 5 (657) with the macromolecular binding agents (640);
  • a packaging layer (650) is present prior to analyte delivery (left side of figure) and absent (right side of figure) when analyte (655) is present and interacts 5 (657) with the macromolecular binding agents (640);
  • Fig. 7 is a schematic view of a sixth alternate embodiment of a sensor (700) in accordance with the invention in which induced current is measured or stored over a resistor (775) placed between electrodes (760) of a detection unit (770);
  • Fig. 8 is a schematic view of a seventh alternate embodiment of a sensor0 (800) in accordance with the invention in which a "multiplexed" plurality of base members (820-823) with different macromolecules (840-843) and optional SAM
  • a computer control unit (880) monitors output from a detection unit (870);
  • Fig. 9 is a schematic representation of a macromolecular species illustrating5 electrostatic fields (92, 94) associated therewith;
  • Fig.10 is a plot of current induced in a base member (measured as voltage over a 150-kilo-ohm resistor) versus time for the reaction of the macromolecule lysozyme with its substrate, bacteria
  • Fig.ll is a plot of current induced in a base member versus time for the reaction of the macromolecule lysozyme with its substrate, bacteria
  • Fig.12 is a plot of signal frequency versus time for the reaction of the macromolecule lysozyme with its substrate, bacteria;
  • Fig. 13 is a plot of current induced in a base member versus time for the interaction of sodium chloride, then lithium chloride with the binding agent 12-crown-4;
  • Fig. 14 is a schematic view of a seventh alternate embodiment of a sensor (1400) in which an optional source of an electrical signal (1490) is integrated into o the sensor system.
  • the sensor design disclosed herein is based on electromagnetic induction of electrons in conducting materials when said electrons are exposed to fluctuating macromolecular electrostatic fields.
  • the sensor utilizes a novel method of detecting an analyte wherein macromolecular binding agents are first immobilized proximate an electrically conductive base member.
  • the bound macromolecules are always moving; the motion of the electrostatic fields associated with the macromolecules serve to generate an induced electrical signal in the base member.
  • the fluctuating electrostatic fields0 generate fluctuating magnetic fields, and these fluctuating magnetic fields induce electron motion in the base member.
  • changes in induced current as recorded across a resistor (775) by a voltmeter-based detection unit (770) is indicative of the presence of analyte in a sample exposed to a base-member (720) proximate a binding agent (740) that interacts at a level of specificity for the target analyte.
  • a typical sensor comprises (i) a multilayer substrate comprising a conducting base member or layer and an optional self-assembled monolayer (or other chemical entity that rests between the base member and the macromolecules); (ii) at least one macromolecule that displays a level of affinity of interaction toward a predetermined analyte or group of analytes; and (iii) a detection unit for measuring an electromotive force (emf), current or other electrical feature electromagnetically induced in or about the base member and responsive to the presence of analyte.
  • emf electromotive force
  • the sensor exploits the phenomenon of electromagnetic induction, the process by which fluctuating magnetic fields can induce electron motion in nearby electrically conducting materials. Since the sensor works on physical properties shared by nearly all macromolecular binding agents, the methodology is equally appropriate for a large variety of analyte classes, utilizing various macromolecular binding agents such as nucleic acids, enzymes, antibodies, antigens, peptides, or receptors. Additionally, charged or polar synthetic binding agents are appropriate for use in the present invention. The one requirement for binding agent is that it demonstrates a level of specificity of interaction with a predetermined analyte or group of analytes.
  • a change in motional behavior of the binding agent or addition of electrostatic material associated with the analyte causes an increased electromagnetic induction in the base member and thus signals binding of analyte.
  • the amount of change in induced emf above background readings may be correlated to the concentration of analyte present in the sample of interest.
  • Experience of the inventor has shown that non-specific interactions of sensor and sample do not produce a significant induced signal.
  • Such conclusions are based on studies in complex matrices such as blood plasma, milk, stool, and ground beef homogenized in phosphate buffer.
  • the present sensor method has been successfully employed in numerous sensor systems (see Examples) and opens the door to the fast, sensitive, and inexpensive detection of a wide range analytes.
  • a sensor as described in the present application has excellent sensitivity in detection (parts-per-billion or better) and, in short, has the properties and production features necessary for use in numerous0 domestic, law-enforcement, medical, pharmaceutical, food, hygiene and industrial applications.
  • the present invention is distinguished from the prior art in several ways. Firstly, the methodology is applicable to nearly all binding agents and not simply to those that produce or interact with electrons or electrochemically active5 compounds. Secondly, the method requires neither passage of electrical signal into a system nor detection of an electrical signal from a component of the material in contact with the binding agents. There is no requirement in the present invention for application of electromagnetic radiation, voltage, or electrical current. Additionally, the present invention does not require use of sample-contacting transistor devices or any active electrode species. It should be noted that the medium containing the analyte need not be electrically conductive, nor is the base member required to have a redox potential different from that of the analyte.
  • An “analyte” is a material that is the subject of detection or quantification.
  • a “base member” or base layer is a solid or liquid element on or near which macromolecules can be physically or chemically immobilized for the purpose of sensor construction.
  • 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” 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.
  • a self-assembled monolayer is formed from molecules that bond with the surface upon their direct contact from solvent, vapor, or spray.
  • a self-assembled monolayer possesses a molecular thickness, i.e., it is ideally 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.
  • Electrode or “lead” is a wire, electrical lead, connection, 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” of base member and associated macromolecules.
  • Contact is generally electrically passive in nature and occurs at two positions.
  • “Induced” and “induction” have their normal meaning in the electrical arts with respect to electromagnetism. Specifically by these terms it is intended to exclude oxidation-reduction chemistries and applied electrical signals.
  • the sensor according to the invention exploits the fact that macromolecular binding agents and most relevant analytes are electrically-polar species. Such polarity is due to full charges, fixed dipoles, and induced dipoles. Proteins (enzymes, antibodies, receptors) and peptides are made up of amino acid building blocks, and several of the amino acid side chains are positively or negatively charged in solution. Lysine, arginine, and histidine can display net positive charge on their side chains, while aspartic acid and glutamic acid have negatively-charged side chains except at very low pH values. Nucleic acids have negatively charged phosphate groups, and polymers of nucleic acids are thus highly negatively-charged.
  • the macromolecular binding agent specifically sequesters a target analyte in proximity to the base member; the altered electrostatic fields associated with the binding agent-analyte complex increase induced electron motion in the base member relative to the situation prior to analyte presence.
  • the signal that is induced in the base member is generally recorded within fifteen to thirty seconds. At very low analyte concentrations, there may be latency due to delays in analyte reaching the immobilized binding agent.
  • Salmonella enteriditis at 10 cells per milliliter of ground beef-buffer solution required approximately 90 seconds. This high sensitivity may be related to the charged protein flagella on the bacteria; these flagella will continue to move— and thus add to the induced effects — when a bacterium is bound to an antibody binding agent proximate base member.
  • an analyte (655) is disposed proximate a sensor (600).
  • the analyte (655) 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, cellular metabolites, enzyme inhibitors, receptor ligands, nerve agents, peptides, proteins, fatty acids, steroids, hormones, narcotic agents, synthetic molecules, medications, nucleic acid single-stranded or double-stranded polymers.
  • the analyte (655) can be present in a solid, liquid, gas or aerosol. As will be seen below in the discussion of alternate embodiments, the analyte (655) could even be a group of different analytes, that is, a collection of distinct molecules, macromolecules, ions, organic compounds, viruses, spores, cells or the like that are the subject of detection or quantification. Some of the analyte (657) physically interacts with the sensor and causes an increase in electromagnetic induction in the conductive base member (620).
  • the sensor (100) as shown as a preferred embodiment in Fig. 1 includes a base member (120), which may be in the form of a solid piece, a chip, a foil, a wire, a coating, or a liquid.
  • the base member (120) may be any conducting material such as metals, conducting organic polymers, conductive "inks", conductive liquids, or graphite. Low-resistivity semiconductor materials may also serve as the base member (120). Insulating and dielectric materials are not appropriate for the role of base member, although a base member may be coated onto an insulating material such as plastic. This is the situation in Fig. 5 in which a conducting coating, ink, or film (520) has been deposited on an electrically-insulating support (515).
  • the insulating material (515) is then referred to as a "support” or “solid support” for the base member (520).
  • Macroscopic properties of the base member include its electrically-conductive nature and possible reactivity with chemicals to form self-assembled monolayers. Microscopic properties of the base layer include free electrons that can move within the lattice of the atoms or ions that compose the base member.
  • a base member For use in the invention, a base member must show a level of electrical conductivity.
  • a plastic container that is coated with a silver thin film has silver as the base member on a plastic support.
  • "conductive inks" coated or painted onto plastic disposable members are particularly useful as base members in the present invention.
  • a conductive ink is defined as a liquid that contains, in part, electrically-conducting material. Organic volatiles evaporate over time and a conducting surface remains. o The preferred embodiments have the macromolecular binding agents immobilized proximate a conducting foil or deposited thin film base member.
  • Preferred base member materials include but are not limited to aluminum, gold, silver, copper, conducting organic polymers, platinum, iridium palladium, rhodium, mercury, osmium, ruthenium, gallium arsenide, indium phosphide, mercury5 cadmium telluride, and the like.
  • Aluminum foil and silver conductive inks are particularly preferred as they are conductive, inexpensive, food-safe, and amenable to treatment with self-assembled monolayers.
  • a natural oxide layer can provide electrical insulation between the conductive metal and the macromolecular electrostatic fields (to avoid short-out). Copper, gold, silver, ando uminum are particularly preferred as base member materials, especially foils or thin films of these metals on plastic supports.
  • the macromolecules can be chemically or physically immobilized in proximity to the base member. The macromolecules can be immobilized directly to the base member or can interact with a chemical layer deposited on the base member. 5 Referring again to Fig. 6, a self-assembled monolayer (SAM) (630) is attached to the base member (620) by any appropriate method.
  • the SAM ideally possesses molecular thickness, that is, that the entire chemical layer is only one molecule thick.
  • the SAM (630) may serve to chemically or physically bind macromolecules (640), to aid in their long-term stabilization, to transduce signal resulting from interaction of macromolecules (640) with analyte (657) to the base member (620), or to provide differential levels of electrical connectivity between the macromolecules (640) and the base member (620).
  • macromolecules (240) may also be physically absorbed to the base member (220). If a SAM is applied, it may be a mixed SAM formed from several chemical components.
  • chemicals (not shown) other than SAM's may be employed in certain applications, and serve the same role as defined for SAM's, namely binding, stabilization, or signal transduction.
  • the SAM (630) generally forms on the base member by direct deposition from solution or through vapor deposition.
  • SAM layers are generally formed from chemicals with formulae R-COOH, R-SH, R-S-S-R', R-S-R', R-SiCl 3 , or R-Si-(OR") 3 .
  • R and R' are each composed of at least one organic functionality, while R" is either a methyl or ethyl group.
  • the specific SAM (630) selected is a function of the base member material (620) employed.
  • the SAM (630) usually has surfactant properties.
  • SAM's are formed from chemicals with generally formula R-COOH when aluminum foil or silver oxide serves as base member (Ulman, A.
  • R-group of the SAM (630) may be any organic group and might contain an aromatic moiety in order to aid in amplification of the signal from the fluctuating macromolecular electromagnetic fields to the base member.
  • a mixed SAM in which two different chemicals are deposited on the base member (620) may be employed. One chemical may serve to bind the macromolecules to the base member, while the other might aid in the physical stabilization of the macromolecules during dry storage of sensor "strips".
  • R-COOH if R is an aliphatic group, then the SAM is termed a fatty acid.
  • Fatty acids are particularly appealing as they can form good insulation between the macromolecules and conducting members and are available from natural sources such as vegetable oils. Fatty acids have been shown to form SAM's on aluminum and copper (oxide) samples. R can also be aromatic in nature, and a SAM with a phenyl moiety offers significant electrical connectivity between the fluctuating macromolecular electrostatic fields and the
  • Organic alcohols readily (R-OH) form self-assembled monolayers.
  • SAM's for gold and silver include a wealth of sulfur containing compounds. Notable among them are organic thiols, sulfides, and disulfides. 5
  • Organic thiols correspond to the general formula RSH where R represents an organic moiety. R can be aryl, alkyl, a combination of the two, long- or short-chain.
  • the pendant functional group is chosen from those functional groups that are reactive with amino (-NH 2 ) or other reactive groups on proteins, DNA, or synthetic binding agents, or that can be adapted to be reactive0 with said groups.
  • the pendant group may be selected to provide stability to the macromolecules (640) by serving as either a source of hydrophilic or hydrophobic stabilization.
  • Functional pendant groups that are reactive with amino groups or that are easily adapted to be reactive with amino groups include carboxylic acids that can be converted to more reactive functionalities.
  • Functional5 pendant groups that stabilize enzymes by providing a source of hydrophilic interaction include hydroxyl and carboxylic acid moieties.
  • Hydrophobic stabilization can be provided by pendant methyl or other appropriate alkyl and aryl groups.
  • Organic sulfides correspond to the general formula RSR' where R and R' represent organic moieties. The nature of R and R' can be varied to balance insulation and electrical connectivity between the macromolecules and the conductive base member.
  • the terminal functional groups can be chosen
  • Organic disulfides correspond to the general formula RS-SR' where R and o R' have the meanings given above.
  • macromolecular entities suitable for use in the sensor (600) 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 polymers5 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 well as synthetic macromolecules in the role of macromolecules (640). Natural, synthetic,0 semi-synthetic, over-expressed and genetically-altered macromolecules may be employed as binding agents.
  • the macromolecules (640) may form monolayers as in
  • Fig. 1 multilayers as in Fig. 4, or mixed layers of several distinct binding agents as in Fig. 3.
  • a monolayer of mixed binding agents may also be employed (not shown).
  • the macromolecule component is neither limited in type or number.5 Enzymes, peptides, receptors, receptor ligands, antibodies, catalytic antibodies, antigens, cells, fatty acids, synthetic molecules, and nucleic acids are possible macromolecular binding agents in the present invention.
  • the sensor method may be applied to nearly any macromolecule because it relies on the following properties shared by substantially all macromolecular binding agents: (1) that the macromolecules chosen as binding agents are highly specific molecules designed to bind only with a selected analyte or group of analytes;
  • an induced current may be measured in a closed electrical circuit that contains the base member.
  • a background induced current is present in the circuit due to fluctuations of the macromolecular electrostatic fields. Presence of analyte causes an increased electromotive force in the base member and thus a larger induced current as measured by a detection device that is electrically contacted to the base member at two positions.
  • the broad and generally applicable nature of the present invention is preserved during binding of macromolecules (140) 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 electrical effect in the base member that is associated with analyte presence does not rely on any specific enzyme chemistries, optical effects, fluorescence, chenriluminescence, oxidation/reduction phenomena or applied electrical signals. This feature distinguishes the present invention with all known prior art. Additionally, according to the operation of the invention current is actually generated, and the generated electricity may be of use in powering devices such as the sensor itself.
  • the macromolecules (140) preferably rest within a distance not much larger than 50 nanometers (nm) from the base member (120) so as to allow for easy measurement of the electrical effects induced by fluctuating macromolecular electrostatic fields. A larger separation between macromolecules and base member is also possible. In such a case, a conductor, dielectric, or insulator may be disposed between the base member and the macromolecules.
  • proximate with respect to macromolecule disposition relative the base member refers to any distance that allows for macromolecule-related induction of an electrical signal in the base member, said induced electrical signal being responsive to the presence of analyte.
  • a type of commercial aluminum foil that is factory-coated with mineral oil has also proven acceptable for use as base member.
  • the detection unit (170) is any device that can detect one or more electrical signals induced in a conducting element as a function of analyte interaction with at least one macromolecular entity. Examples of such signals include but are not limited to induced current; induced electromotive force; voltage; impedance; signal sign, frequency component or noise signature of a predetermined electrical signal propagated into a base member at a first location and received at a second location.
  • a voltmeter-based detection unit (770) as shown in Fig. 7 may have one or more additional resistors (775) built into its electronics so as to allow for measurement of the flow of electrons when the base member is part of an electrical circuit that includes the detection unit (770).
  • a detection unit may be based on a digital electrical metering device, it may also have additional functions that include but are not limited to data storage, data transfer, alert signaling, command/control functions, and process control. Detection units may be contacted through "leads" or “electrodes” (860 - 863) to one or more base members (820-823, Fig. 8). Referring again to Fig. 1, contacts between base member (120) and detection unit (170) are generally at two positions (165, 167) on the base member (120).
  • the detection unit (570) is a voltmeter device with a very high internal impedance, one can measure the induced emf directly through passive contact of leads (560) to the base member (520). The size of the induced effect is proportional to the rate of fluctuation or size of the magnetic fields, while the direction of electron flow is such that it generates magnetic fields to opposes the causative fluctuating magnetic fields (Lenz's Law). If a detection unit contains an ammeter or voltmeter with an in-parallel resistor (775) (Fig. 7), the induced electromotive force can be measured as an induced current or voltage over the resistor (775).
  • the induced electrical signal is measured in the sensor strip in response to the continual motions of the charged macromolecules (140) prior to and during their interaction with analyte; changes in the measured electrical signal implies that analyte is present.
  • Baseline readings are recorded for sample that lacks target analyte or analytes (Figs. 10 - 13).
  • Figs. 10 - 13 Reference is now made to Fig. 6.
  • Lower absolute induced electromotive force (induced emf) readings could theoretically result from reduced motion of macromolecules (640) in the presence of analyte (655, 657), while higher readings (in absolute terms) generally occur in response to greater enzyme motion or additional electrostatic material of the analyte (657) bound up with the macromolecules (640).
  • the detection unit may be integrated into a computer (880) as shown in Fig. 8 or other solid-state electronic device for easier signal processing and data storage.
  • a computer 880
  • electrical leads (760) of a digital voltmeter-based detection unit (770) are contacted to a sensor strip base-member (720), at two points (765, 767) away from the region directly in contact with analyte (so as to keep all of the electrical connections dry and to protect the user).
  • Induced electromotive force in the electrically conductive base member is recorded as a voltage differential across a resistor (775) attached to the leads, and the specific change in voltage values prior to and during analyte exposure may be correlated to relative analyte concentration.
  • an optional packaging layer (650) for the sensor (600) is a layer of water-soluble chemicals deposited above the immobilized macromolecules (640).
  • the packaging layer (650) is deposited by soaking or spraying methods.
  • the packaging layer (650) serves to stabilize the macromolecules (640) during prolonged storage. In the absence of a packaging layer, oil and dirt may build up on the macromolecules (640) and may interfere with the rapid action of the sensor system.
  • Glucose and a salt, such as NaCl are typically used for the packaging layer (650) so as to guarantee their dissolution in aqueous samples, and thus facilitate interaction between macromolecular binding agent (640) and analytes (657). Other hydrophilic chemicals may be chosen.
  • the packaging layer dissolves (650), the binding agents are free to immediately interact with analyte (655, 657). Water-soluble polymers, sugars, salts, organic, and inorganic compounds are all appropriate for use in preparation of the packaging layer.
  • electrostatic fields (92, 94) associated with a macromolecular binding agent fluctuate prior to and during interaction with analytes (657).
  • the fluctuating fields (92, 94) increase in strength in the presence of analyte due to increased motion of the binding agent and/or increased electrostatic material contributed by the analyte (657).
  • Fluctuating electrostatic fields give rise to fluctuating magnetic fields (not shown); these magnetic fields in turn induce motions of free electrons in a conductive base member (620). These electron motions induce an electrical signal in the base member.
  • This induced electrical signal may be detected as an induced emf or current, impedance or resistance change, alteration in noise signature, sign change in a signal, alteration in electrical frequency or the like. Induced electrical signals are recorded through contact of appropriate electrical leads (660) of a detection unit (670). Referring to Fig. 1, contact between the leads (160) and the base member (120) usually occurs passively at two positions (165, 167). It will be appreciated by those skilled in the art that the electrical characteristics of the induced currents flowing within the base member (620) should vary when changes occur in the amplitude or frequency of motion of the electrostatic fields of the macromolecules (640).
  • the sensor makes use of fluctuations and changes in electrostatic fields (92, 94) associated with macromolecular binding agents.
  • electrostatic fields are shown schematically as positive (field (92)) and negative (field (94)) regions in Fig. 9.
  • Some of the atoms (96) of a hypothetical binding agent are shown.
  • the electrostatic fields (represented as horizontal and vertical lines) are calculated according to the location of charged and polar moieties in the macromolecule. Real three-dimensional structures are generally determined from macromolecular x-ray crystallographic or nuclear magnetic resonance studies.
  • the electrostatic fields (92, 94) of any particular macromolecule depend specifically on the spatial positions of charged/polar atoms or groups in the macromolecule.
  • the atoms (96) in a macromolecule move when the macromolecule is tethered to a solid support; the associated electrostatic fields (92, 94) fluctuate with changing atomic positions. Analyte presence leads to either altered atomic motion of the macromolecule or additional electrostatic material contributed by the analyte.
  • the binding agent the macromolecules (640)
  • analyte causes significantly increased lateral and horizontal motions of the charged enzyme macromolecules.
  • the added motions of enzyme-associated electrostatic fields induce a greater electromotive force in the base member.
  • Such analyte-related increases in enzyme motion have been previously described by Atomic Force Microscope studies (Radmacher, et al. Science 265:1577-1579 (1994)).
  • introduction of analyte (655) results in significantly greater enzyme motions and resultant induced electromotive force.
  • the base member (620) has been part of a closed electrical circuit, induced current values of nearly 10 microamperes have been recorded.
  • the detection unit (770) may be calibrated for the lower background readings when target analyte or analytes are absent.
  • the sensor employs either a digital voltmeter or ammeter as part of the detection unit.
  • an appropriate resistor (775) may be placed between the electrode leads (760); voltage readings are made across the resistor in a complete circuit that includes the resistor-contaijiing electrodes (760), the detection unit (770) and the base member (720).
  • An alternative ammeter-based detection unit (170) as shown in Fig. 1 is placed in series in a circuit that contains the base member (120), the leads (160) and the detection unit (170).
  • a change in induced current as measured in a circuit that contains the base member (120) signals the presence of analyte, while the magnitude of the change may give information on analyte concentration.
  • a detection unit may measure several different signals at once, such as induced current and signal frequency.
  • the signal (induced emf. induced current, voltage, and the like) recorded by the detection unit generally fluctuates rapidly in value, as there is no coordination between the individual macromolecular entities. Thus, rapid changes in signal value or sign may serve to identify presence of analyte.
  • a detection unit can be designed to produce an audible, visual, digital, or analog signal, alarm, or situation response due to analyte presence.
  • Conducting materials are normally at a single electrical potential (voltage) at all points along their surfaces.
  • variations in macromolecular electrostatic fields (92, 94) near a conductive base member (120) lead to the establishment of an induced emf in the base member (120).
  • the induced emf results from electron motion in the base
  • sensing can take place far away from the point of macromolecule-analyte contact, as the effects of free electron motion are propagated throughout the electrically-conductive base member. This fact allows for closed-package "food sensing” or the sensing of potentially hazardous samples (blood) in closed containers.
  • One portion of the sensor contacts the material of interest, while the leads (160) of a detection unit (170) are contacted to two positions (165, 167) elsewhere along the sensor strip. This is an important feature of the present sensor.
  • any material that can be recognized at a level of specificity by a peptide, protein, antibody, enzyme, nucleic acid single strand, or a synthetic binding agent can be detected safely in food, body fluids, air or other samples quickly, cheaply, and with high sensitivity.
  • Response is very rapid, generally less than 30 seconds, cost is minimal, and experience of the inventor has shown that the sensors are very sensitive to dilute solutions of analyte (parts-per-billion or lower of antibiotics: 10 and fewer cells per milliliter of bacterial pathogens).
  • Recent experiments by the inventor with an antibody-antigen system showed high induced current readings when the theoretical number of target analyte cells (E. coli 0157:H7) was one cell per milliliter of sample.
  • an appropriate resistor (775) (Fig. 7) is placed in series with the base member (720), and the base member is part of a complete electrical circuit.
  • Typical current produced by a functioning sensor may be between 0.1 and 10 microamperes; these values can be converted to several hundred millivolts with an appropriate resistor.
  • a first alternate embodiment of the invention is shown in Fig. 2. This is similar to the first embodiment, except that the SAM has been omitted. This arrangement is useful in applications where low cost or quick production is a driving factor.
  • a second alternate embodiment of the invention is shown in Fig. 3. wherein macromolecules of different specificities of interaction with at least one analyte (340, 345) are arranged in a plurality of layers (341, 342, 343), The macromolecules (340, 345) may be selected to bind with the same or different
  • the different macromolecular binding agents are indicated by hatched ellipses (340) and un-hatched ellipses (345), respectively.
  • This feature of multiple binding agents on a single base member (320) enables multiple binding opportunities for a single analyte or multi-targeting of a group of analytes.
  • the base member (320) may be a solid o foil, or other suitable material as may be advantageous for a particular application.
  • FIG. 4 A third alternate embodiment of the invention is shown in Fig. 4, which is similar to the embodiment of Fig. 3, except that the macromolecule multilayer is composed entirely of one type of binding agent (440).
  • the base member (420) may be a solid foil, or other suitable 5 material as may be advantageous for a particular application.
  • the output of a sensor as in Fig. 7 (700) is optionally connected across a load resistance (775).
  • the load resistance (775) may be applied as power in a device that requires electrical energy for operation.
  • a fourth alternate embodiment, shown in Fig. 5, has a conductive ink, thin0 film, or coating serve as the base member (520).
  • the conductive material (520) is deposited on a non-conductive support (515) such as the plastic of a disposable container or element.
  • An optional SAM (530) may be deposited, and then macromolecules (540) and packaging layer (550) are deposited to yield a functional sensor strip that may then be contacted to the leads (560) of an appropriate5 detection unit (570).
  • the base member itself does not necessarily have to provide this continuity , although it may be deposited so as to allow for full electrical connectivity.
  • the device and methodology described here lend themselves to "multitasking" in which one or several analytes are detected by the use of one or several distinct binding agents (840- 843) either on a single base member or on separate base members (820- 823).
  • base members are either monitored for induced emf simultaneously by a detection unit (870), or each base member is interrogated in a pre-determined order by the detection unit (870).
  • Analyte presence is determined by the behavior of each sensor strip in the multiplexed unit.
  • a sensor (800) might be constructed from four separate base members (820-823).
  • a carboxylic acid-based SAM (830-833), antibodies specific for different food pathogens (840-843), and a water-soluble "packaging layer" (850-853) are deposited separately on each base member.
  • the distinct macromolecular entities are shown as clear ellipses (840), ellipses with vertical lines (841), horizontal lines (842), and crossed lines (843).
  • a multiplexed detection unit (870) has two electrical leads (860-863) contacted to each base member, and a computer processor (880) monitors induced emf for all four sensor "strips" when a food sample is brought into contact with the sensor's multiplexed strips.
  • Altered induced current (versus a predetermined background for each strip) associated with particular base members signals the presence of specific pathogens in the sample.
  • the pathogens detected are directly correlated to the specific antibodies associated with each base member that displays an increased induced emf or current. For example, if the base member (823) that supports a particular antibody (843) shows a higher induced emf, then one may conclude that the pathogen target which the antibody (843) recognizes is present in the food sample.
  • Multiplexing is not limited in number of base member or types of macromolecules simultaneously employed. Base member size can be micron-scale for large-scale sensing or detection operations. Screening of combinatorial libraries may require hundreds of separate base members with associated macromolecular entities for the detection of potential pharmaceutical lead compounds.
  • a simple food sensor may have an enzyme or a group of antibodies (for E. coli 0157:H7, Listeria, Salmonella, Vibrio and other pathogens) absorbed to a piece of aluminum foil.
  • a more complex medical sensor might require the use of a solid-state electronic device that converts the signal into an action such as release of a drug in response to changes in induced emf.
  • Some semiconductors are of use in the present invention.
  • An appropriate semiconductor has a low resistivity and is electrically responsive to fluctuating magnetic fields. While a single sensor strip may recognizes a particular analyte; multiplexed devices have multiple strips for detection of several target analytes or for redundancy to reduce false readings.
  • the invention allows for the creation of very small sensors due to the ability to attach macromolecules in high concentration.
  • the device independent of electrical connections, has been made as small as millimeter dimensions and should be appropriate for IC fabrication in micron dimensions for the sensor strips of base member and associated macromolecules.
  • the senor is inexpensive and quick to assemble.
  • a fully functional sensor strip can be made in less than one-half an hour. All of the requisite materials are generally non-toxic, and the completed sensor strips can be sterilized.
  • the senor exhibits high detection selectivity. Enzymes are quite specific for their substrates, as antibodies are for antigens. Nucleic acid double-strand interactions can be quite specific as can be receptor-ligand interactions. Synthetic binding agents may also be used for specific detection of target analytes Thus, the sensor in different possible embodiments has high selectivity and sensitivity for the desired application. Parts-per-billion detection have been routinely performed with antibiotic-detecting sensors prepared with the enzyme penicillinase (see Examples below).
  • the sensors exhibit long-term stability.
  • this fact is related to the simplicity of the design that significantly reduces the number of materials used in sensor strip preparation (base member, optional SAM layer, macromolecules, optional packaging layer).
  • base member optional SAM layer
  • macromolecules optional packaging layer
  • this feature is due to the stability of the materials used to make the device.
  • Immobilized macromolecules have been found at times to be more stable than their solution counterparts (for example, Dekker, RF, Applied Biochemistry and Biotechnology 22:289-310 (1989)), as the support material can offer protection from extreme environmental conditions.
  • SAM's and packaging layers can also be selected to offer physical and chemical stabilization of 5 bound macromolecules in order to further enhance their long-term stability.
  • the sensor provides a near-instantaneous readout.
  • This speed is a major benefit as most sensor systems in the prior art have time-delays of minutes to hours due to detection response. Speed is absolutely critical in such applications as poison gas detection or food pathogen identification.
  • Altered macromolecular motions and changed electrostatic fields occur5 instantaneously with the arrival of analyte, and the response is generally recorded within 5 to 15 seconds-depending on the conductive properties of the base member.
  • the sample can be delivered to the sensor via solid, liquid, gas, or aerosol media.
  • the design is highly flexible. For instance, if one wishes to detect several analytes at once, appropriate macromolecules are selected and placed on separately-monitored strips of conductor base member in a multiplexed format. In addition, if one wishes to adjust the sensitivity of the device a number of means are available.
  • the number, type or chemical composition of the macromolecule component can be changed.
  • the length or nature of the organic groups in the SAM layer can be altered.
  • the conductive member can be chosen from a variety of materials having different resistivities. Appropriate semiconductor materials may be used for further modulation of the signal strength relative to analyte concentration.
  • the orientation of the macromolecules on the device can be manipulated so as to allow for specific orientation of all macromolecules. Seventh, the sensor is simple to use.
  • the present sensor in contrast, relies on electrostatic properties exhibited by all charged macromolecules.
  • any macromolecule that can interact at a level of specificity with a given analyte or group of analytes should fin use in the present methodology.
  • This development significantly increases the number of target analyte molecules that can be routinely detected. This is a major consideration in food safety sensing in which speed, cost, sensitivity, and flexibility of target selection are of primary importance.
  • the sensor strips actually produce energy. Typical sensor strips produce microampere quantities of current. Such electrical energy from a multiplexed system could be used to power the detector unit or find use in battery applications. Thousands of such strips lithographically produced on a chip could produce significant amounts of energy at low cost and in an environmentally-friendly manner.
  • a conductive film can be deposited on a solid support by any means, including electroless deposition, spin coating, sputtering, vapor deposition, "printing" or dip-coating.
  • Figs. 10-13 show plots of sensing experiments performed with the present invention.
  • the enzyme lysozyme Sigma Chemical Company, St. Louis, USA, catalogue number L-6876
  • SAM formed from an aqueous solution of parahydroxybenzoic acid, Aldrich Chemical Company, Milwaukee, USA, catalogue number H-5376
  • a packaging layer of sodium chloride and glucose was deposited above the enzyme layer. Sensor strips were cut from a larger sheet (approximately 10 cm x 10 cm). In the experiments recorded in Figs.
  • the sensor strips were partially inserted into a disposable plastic 1.5 milliliter tube; readings were made by contacting the leads of a5 commercially-available digital multimeter (Radio Shack, catalogue number 22- 168 A, supplied with PC port connection and data display software) to two points of the base member on the outside of the disposable tube.
  • a5 commercially-available digital multimeter Radio Shack, catalogue number 22- 168 A, supplied with PC port connection and data display software
  • a larger "strip” approximately 3 cm x 6 cm
  • Water, then bacteria were placed on the strip, which was then folded over upono itself. Contact with water/bacteria occurred on the "inside”, while contact with the same multimeter leads was performed on the dry, exposed "outside”. In all measurements, background against boiled water was measured for approximately 90 seconds.
  • Fig. 10 shows the induced current read as a voltage over a 150 kilo-ohm resistor that was connected to the multimeter leads.
  • Fig. 11 shows the current induced in the base member-containing circuit, while Fig. 12 shows the electrical frequency recorded during lysozyme action. In all three systems, significant signals appear specifically when bacteria were present.
  • Fig. 13 shows data for a chemical sensor, according to the embodiment of Fig. 2, except that the packaging layer was omitted.
  • Crown ethers are known to coordinate metals. Specifically, 12-crown-4 coordinates lithium, but does not bind the larger sodium ion. 12-crown-4 (Aldrich, 19,490-5) was immobilized on aluminum foil from an aqueous solution of the crown either. The foil was next rinsed in water. Sensor strips were cut from the prepared sheets. The strips were placed in disposable tubes as described above for the lysozyme system. For one minute, a background was recorded against water. During the next minute, the sensor strip was challenged with sodium chloride. Afterwards, lithium chloride (Aldrich, 31,046-8) was added.
  • the plot shows induced emf in the base member (recorded as an induced current) during the experiment.
  • the crown ether is a relatively rigid molecule, binding of positive lithium ions led to relatively large induced currents as measured between two points of the exposed sensor strip.
  • the senor has been presented as a system that does not require the application of an external electrical signal.
  • the electrical connections between the detection unit and the base member have been completely passive, that is to say, not requiring application of any external power, and utilizing no more than two points of connection.
  • This presentation emphasizes a point of distinction between the present invention and prior art that makes use of applied signals and changes in exit signal as a function of analyte presence.
  • the sensor (1400) is similar to other embodiments with the addition of a power source (1490) in the electrical circuit.
  • the induced electromotive force in the base member alters the exit signal and the size of change in signal is used to determine if analyte is present.
  • the changes in applied signal result from electromagnetic induction in the base member (1420) and not from any electrochemical phenomena or specific electrical properties associated with either the analytes, medium, binding agents, or associated cofactors. If one measures resistance between two points (1465, 1467) of a base member (1420) with appropriate electrical leads (1460), the resistance fluctuates during sensor (1400) function.
  • a power source (1490) applies a current through contact of an electrode (1460) to the base member (at position 1465), and the induced emf generated in the base member (1420) as a function of macromolecule (1440) interaction with target analyte (1457) causes fluctuations in the resistance reading that is calculated from voltage that reaches the contact point (1467) of the second electrode (1460) and then is measured by the detection unit (1470).
  • This variation is correlated to changes in induced emf in the base member and its effect on the applied current that is used to measure resistance in the base member.
  • the phenomenon may be due to the Hall effect, in which magnetic fields are applied to a current flowing in a conductor.
  • the moving macromolecular entities (1440) act as the source of the magnetic fields.
  • Example 1 A piece of commercially-available aliiminum foil (10 cm by 10 cm) is soaked for ten minutes in a dilute ethanolic solution of a long-chain organic compound, HS-(CH2) 10 -COOH (Aldrich, 45,056-1) and then allowed to air-dry.
  • the organic acid forms a SAM insulating layer between the conducting aluminum foil and an enzyme layer is formed by soaking the SAM-coated foil in a 50 microgram/milliliter solution of lysozyme for twenty minutes.
  • the foil is soaked briefly in a glucose/ NaCl solution and allowed to air dry.
  • the foil is cut into 0.25 cm x 4 cm pieces and inserted into UHT milk container packaging so as to allow one side to contact the milk, while the other side remains exposed to the outside.
  • the induced emf generated in the foil by the moving lysozyme molecules is measured by contacting the outside of the sensor with two probes of a digital voltmeter that has 1000 mega-ohm resistance between the two contacting electrodes (MDMVOIOO, Red Lion, York, Pennsylvania, USA). A background value is recorded for fresh milk.
  • Sterile milk is tested after insertion into its packaging, and when the absolute value of the voltage increases (it can become more positive or more negative) in response to increased motion of lysozyme, the milk is rejected as not being sterile (for lysozyme motions in the presence of substrate, see Radmacher, et al. Science 265:1577-1579 (1994)).
  • Example 2 Aluminum foil was soaked in an aqueous solution of parahydroxybenzoic acid, rinsed in water, and then soaked in a dilute solution of three distinct antibodies (Biodesign, Kennebunk, USA, catalogue numbers C11016M, C65310M, and C11018M) that recognize the pathogenic E. coli strain 0157:H7.
  • the completed sensor sheet was cut into strips, and sensor strips were contacted to food samples (ground beef homogenized in phosphate buffer)so that one end contacted the ground beef solution while the other end was available for contact by the passive leads of a detection unit.
  • food samples ground beef homogenized in phosphate buffer
  • Example 3 Aluminum foil (Reynolds) was soaked in a water solution of parahydroxybenzoic acid, rinsed in water, and then soaked for an hour in an antibody specific for the human pathogen, Helicobacter pylori (Biodesign, B65660R). The completed sensor foils were dried and then cut into strips 0.25 centimeter in width. Stomach biopsies from thirty-seven patients were placed in separate Eppendorf 1.5 milliliter tubes that contained approximately 0.5 rnilliliter of sterile saline solution. Sensor strips were cut to approximately 5 centimeters in length. Half of a strip was placed inside the Eppendorf tube, and the tube was closed.
  • the DNA single strand is bound to the base member in a manner that leaves it parallel to the base member (due to Fe-phosphate interactions).
  • a sample is placed in a 1.5 milliliter Eppendorf tube and half of a 5 centimeter long sensor strip (0.25 cm diameter) is placed inside the Eppendorf tube. The tube is closed and the solution shaken in order to contact solution to the sensor strip.
  • induced current readings taken by contacting the leads of a voltmeter-based detection unit (as per the embodiment in Fig. 7) to the exposed strip are less than 20 millivolts (absolute value). If DNA is present and there is interaction with the binding agent, then the readings are in the hundreds of millivolts (absolute value). The specific score depends on the size of the resistor (775) placed between the electrodes connected to the detection unit.
  • a sensor was prepared as in Example 3, but the enzyme acetylcholine esterase (Sigma, C-2629) served as the macromolecular binding agent. Sensor strips were again placed in Eppendorf tubes. A solution of nerve agent was placed at the base of the Eppendorf tube, and there was no contact between the strip and the agent. Vapors from the chemical nerve agent reached the strip and induced emf values as high as 700 millivolts were recorded by a digital multimeter (with high internal impedance) contacting the strips at two points on the outside of the sealed Eppendorf tube. Detection of the nerve agent in gas form demonstrates the efficacy of the sensor as a poison gas monitor; binding of organic phosphate molecules ostensibly caused inhibition of the immobilized enzyme.
  • acetylcholine esterase Sigma, C-2629
  • Example 6 A sensor was prepared for use in antibiotic residue testing.
  • Penicillinase (Sigma, P0389) was the macromolecular binding agent and was physically absorbed onto aluminum foil coated with a SAM layer (parahydroxybenzoic acid . The foil was soaked in sodium chloride and glucose in order to further stabilize the bound enzyme molecules and render the enzyme available for sensing in milk. Sensor strips were cut into 15 cm x 0.25 cm pieces. Strips were further cut down to 5 centimeter length prior to use.
  • Antibiotics penicillin G; amoxicillin; cloxicillin; dicloxicillin; cephapirin; cephalexin; ampicillin; cephasporin C — all purchased from Sigma
  • penicillin G amoxicillin
  • cloxicillin cloxicillin
  • dicloxicillin cephapirin
  • cephalexin ampicillin
  • cephasporin C ampicillin
  • Samples of milk containing a given antibiotic were placed in Eppendorf tubes or disposable plastic vials. Sensor strips were contacted to the milk samples, and the leads of a digital multimeter with high internal impedance were contacted at two exposed (dry)positions on the sensor. Scores were recorded. Milk without antibiotics gave readings of 13 millivolts; samples tainted with one of the eight antibiotics showed readings from 150-250 millivolts (at FDA-mandated parts-per-billion cut-off values for the antibiotics in question).
  • Aluminum foil was soaked in an ethanolic solution parahydroxybenzoic acid, rinsed in water, and then soaked in an aqueous solution of antibodies, each capable of binding one of the following food-related pathogens: Salmonella, Listeria, E. coli 0157:H7, and Campylobacter.
  • the foil was soaked in an aqueous solution of sucrose and sodium chloride and then allowed to air-dry.
  • Sensor strips were cut to 0.5 cm x 1.5 cm and attached to disposable containers such that a portion of the strip was inside the container and the remainder was exposed.
  • a sensor strip may be exposed to water in the closed container, and a background reading may be measured by passively contacting exposed sensor strip at two positions with leads of a digital multimeter (Radio Shack, described previously).
  • Induced current may be recorded as a voltage measured over a 150 kilo-ohm resistor placed in parallel with respect to the sensor strip. Food samples may be added to the water, the mixture shaken, and then new readings taken. If one or mere of the target pathogens is present, a significant increase in voltage will be recorded. The food sample will be known to have a dangerous pathogen present.
  • Example 8 Commercially-available aluminum foil (Reynolds, 10 cm x 10 cm) is sprayed with an aqueous solution of parahydroxybenzoic acid (1 mM). The foil is rinsed in water, cut into several pieces and then soaked in separate aqueous solutions that each contain an antigen associated with one of the following viruses: HTV I, HIV II, HTLV I, HTLV II, Hepatitis B, Hepatitis C. After a twenty minute soak, the sensor sheets (foil-SAM-antigen) are separately soaked for ten minutes ino solutions of glucose/NaCl and then allowed to dry in air. Sensor strips are cut to 0.25 cm x 1.5 cm size and used in analyzing whole blood samples for the presence of antibodies for the aforementioned viruses.
  • the sensor strips (one for each antigen) are placed partly inside a 1.5 milliliter disposable tube and contacted to saline. Background readings are recorded for each strip by a detection unit. A drop5 of blood is added to the tube, the sample shaken in order to afford contact between the blood and the strips, and levels of induced electromotive force are determined for each base member. Significant changes in signal frequency in a given sensor strip signals the presence of antibodies to the viral antigen associated with that particular strip. Alternatively, all of the viral antigens may be immobilized on ao single sheet of SAM-coated foil. The foil (after packaging coat application's cut into strips and single strips are placed between the barrel and needle of a disposable syringe.
  • the syringe-sensor units are sterilized and then used to analyze blood taken up in the syringe.
  • Changes (relative to a predetermined background) in induced current, emf, impedance, resistance, signal sign, frequency, or the like5 indicate that antibodies have bound to immobilized antigens.
  • the implication is that antibodies to at least one of the relevant viruses are present in the blood.
  • Example 9 A piece of polyethylene (PE) is placed in an oxygen plasma or a UV-ozone cleaner. The effect is to oxidize the hydrophobic, inert PE surface and to make it amenable to further chemical manipulations.
  • the oxidized PE is exposed to the vapors of silicon tetrachloride (SiCl ) and then water vapors. This process is repeated until the sample is shown to be fully hydrophilic ("wetted" as measured by a near-zero contact angle value of water on the modified PE surface).
  • SiCl 4 H 2 0 treatments may be required for preparation of PE with a chemically-bonded Si0 2 layer on its surface.
  • the modified PE sample is then exposed to the vapors of a boiling solution of isopropanol, water, and the compound (CH 3 0) 3 -Si-(CH 2 ) 3 -SH.
  • This compound will create a "siloxane" network (-Si-O-Si-) between the modified surface of the PE and the compound.
  • the PE emerging from this solution is cured in an oven.
  • the process of subjecting the PE to the compound (CH 3 0) 3 -Si-(CH 2 ) 3 -SH and heating is repeated twice again.
  • the resultant product is a PE with a thiol (-SH) terminated SAM surface wherein the thiol groups face upwards and away from the PE.
  • the miol-terminated PE is placed in a commercially-available electroless silvering solution and removed. A 40 nm silver (Ag) layer is thereby deposited on the PE. (Note that silver binds very tightly to the thiols on the surface of the PE).
  • This PE-Ag is immediately placed in an ethanolic solution of a compound with the general formula HS-R-OH and 11-mercaptoundecanoic acid (11-MUA) in a 10:1 (HS-R-OH: 11-MUA) ratio.
  • the former compound serves the following two functions:
  • the pendant hydroxyl group (-OH) serves to hydrate the macromolecules on the sensor surface and provide stability to the macromolecules in a non-aqueous environment because the hydroxyl group is very similar to water; and (2) the R group is chosen to balance insulation and electrical connectivity between the silver and the macromolecular binding agents.
  • R is chosen to be a phenyl group or the (CH 2 ) 6 moiety when 11-MUA is used.
  • the latter compound, 11-MUA allows for enzyme immobilization.
  • the 11-MUA is further modified in an aqueous solution of l-ethyl-3- ⁇ 3-(dimethylamino)propyl ⁇ carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). These compounds modify the 11-MUA and make it quite reactive to neutral amino (NH 2 ) groups that are present on all proteins or 5 phosphate groups on the termini of nucleic acids.
  • EDC l-ethyl-3- ⁇ 3-(dimethylamino)propyl ⁇ carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • the sensor sheet in this Example is then soaked in the appropriate dilute glucokinase solution at neutral to slightly basic pH (one-half hour) to effect enzyme coupling to the modified PE-Ag surface through binding of lysine amino groups o and the functionalized MUA.
  • the sensor sheet is removed from the macromolecule solution and sonicated in an aqueous sodium chloride and sucrose solution to remove loosely-bound enzyme molecules.
  • the sensor now has the following arrangement:
  • sensor strips are cut to size and contacted to the leads of a detection unit appropriate for measuring the current induced by the motion of glucokinase in0 the presence of glucose.
  • the electrodes will be contacted at two different positions on the silver surface. The device is now ready for use in glucose monitoring for diabetic patients.
  • Example 10 Commercial aluminum foil is treated with a mixture (ratio of 1:10) of5 4-carboxy-benzoic acid and parahydroxybenzoic acid.
  • the SAM layer is next treated with l-ethyl-3- ⁇ 3-(dime ylamino)propyl ⁇ carbodiimide hydrochloride (EDC, Sigma E-1769) and N-hydroxysuccinimide (NHS, Aldrich, 13067-2) ) in order to convert the exposed carboxy group of 4-carboxy-benzoic acid to a functionality (NHS ester) that is very reactive to protein amine groups and nucleic acid terminal phosphate groups.
  • EDC l-ethyl-3- ⁇ 3-(dime ylamino)propyl ⁇ carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • a packaging layer is deposited by spraying the foil with an aqueous solution of sodium chloride and sucrose.
  • the aluminum foil sheets are allowed to dried, and then they are cut into sensor strips for use in analyte detection.
  • a strip is placed between the barrel and needle of a disposable syringe.
  • the packaging layer is dissolved and the immobilized binding agents are brought into direct contact with the blood sample. If target analytes are present (possibly bacterial toxins), they interact with the bound macromolecules, and there is an increase in the amount of electrostatic material near the base member.
  • Fluctuations of larger electrostatic fields implies fluctuation of larger magnetic fields, and there is a corresponding increase in induced electron motion in the base member.
  • a voltmeter-based detection unit is selected (as in the embodiment of Fig. 7), it may be contacted via electrodes at two exposed points on the aluminum foil base member.
  • An appropriate resistor is placed between the electrodes within the detection unit so as to allow for an easily measurable signal.
  • the detection of signals above a predetermined baseline value in the digital readout of the voltmeter signals that an analyte of interest is present. In the case when the analyte is a dangerous material or pathogen, an alarm is activated to warn of the potential health hazard.
  • Several such foil strips each one specific for a different pathogen, can be grouped to make a multiplexed system for multiple analyte detection or system redundancy.
  • Example 11 A deoxyribonucleic acid single strand polymer of 200 bases is physically absorbed to silver-coated plastic disposable container.
  • the silver is deposited on the plastic disposable tube by coating the plastic with silver ink (Jelt, Jeltargent). Silver ink is deposited so as to guarantee electrical connectivity between the silver on the inside and outside of the tube.
  • the deposited silver acts as base member in this example.
  • a blood sample taken from a patient is exposed to the immobilized nucleic acid binding agent of the sensor strip.
  • the binding of viral RNA to the immobilized complementary single-strand DNA is detected by a detection unit that detects an induced current flowing in a circuit that contains the exposed portion of the silver base member.
  • the detection unit displays a visual alarm that virus has been detected when the current reading (in microamperes) passes a predetermined threshold.
  • Example 12 Human receptor molecules are immobilized along the electrically-conductive lanes of a highly-parallel microfluidic analytic chip.
  • the receptors are the macromolecular entities, while the lanes serve as multiple base members.
  • Chemical products of a combinatorial library are sequentially exposed to the immobilized macromolecules (receptor), and a computer (880 in Fig. 8) monitors for interaction between the chemicals and the receptor molecules. Interactions that lead to altered induced emf readings (relative to background in absence of chemicals) in one or more base member units (820-823 in Fig. 8) are noted, and the corresponding chemicals are selected as lead compounds in the synthesis of ligands for this target receptor.
  • a 200-lane system that allows for ninety seconds of interaction for each chemical with macromolecules allows for screening of 192,000 chemicals per twenty-four hours. While the sensing strip architecture of "base material-SAM-macromolecule" has been described previously, analyte detection based on electromagnetic induction in a conducting base member is new in the art. Prior-art sensors require applied electrical signals and generally 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 an induced emf or induced current in a conducting base member.
  • any sensor strip designed for the detection of an induced current between two points of a conducting or low-resistivity semiconducting base member as a function of analyte interaction with the macromolecular binding agents immobilized on said strip would be produced and used with the express purpose of violating the invention described in this patent application.
  • Such strips would find application in food pathogen detection, medical diagnostics, poison gas detection, and the like, while the detection units would constitute separate components.

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Abstract

Cette invention se rapporte à un capteur servant à détecter des analytes recherchés. On détermine la présence ou la concentration d'analytes en mesurant la variation de la force électromotrice induite, du courant ou d'une autre propriété électrique dans un élément de base pendant l'exposition de l'analyte au capteur. Selon une classe de réalisations, le dispositif faisant l'objet de cette invention immobilise les macromolécules naturelles ou synthétiques suffisamment proches d'un élément de base électroconducteur pour s'assurer que toute modification du mouvement et/ou des champs électrostatiques des macromolécules pendant l'interaction avec un analyte prédéterminé entraîne une modification de la force électromotrice dans l'élément de base.
PCT/IL1999/000309 1998-06-15 1999-06-10 Capteur de detection d'analytes WO1999066322A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU41628/99A AU4162899A (en) 1998-06-15 1999-06-10 A sensor for analyte detection
EP99925262A EP1093583A1 (fr) 1998-06-15 1999-06-10 Capteur de detection d'analytes
US09/701,906 US6322963B1 (en) 1998-06-15 1999-06-10 Sensor for analyte detection

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
IL124903 1998-06-15
US09/110,686 1998-07-07
US09/110,686 US6096497A (en) 1998-06-15 1998-07-07 Electrostatic enzyme biosensor
IL125720 1998-08-11
IL127019 1998-11-12
IL129754 1999-05-04

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077522A1 (fr) * 1999-06-15 2000-12-21 Biosensor Systems Design, Inc. Dispositif et procede servant a detecter des substances a analyser
WO2002012891A1 (fr) * 2000-08-08 2002-02-14 Toyo Kohan Co., Ltd. Coffret d'activation de substrat et procede de detection d'adn ou similaire a l'aide dudit coffret
WO2002031504A1 (fr) * 2000-10-12 2002-04-18 Biosensor Systems Design, Inc. Systeme servant a detecter des substances a analyser
WO2005090981A3 (fr) * 2004-03-24 2005-10-27 Technion Res & Dev Foundation Recepteurs artificiels
EP1609411A1 (fr) 2000-06-11 2005-12-28 Orsense Ltd. Procédé et dispositif de mesure de la concentration de glucose ou d'autre substance dans le sang
US8354066B2 (en) 2004-03-24 2013-01-15 Technion Research & Development Foundation Ltd. Artificial receptors
US8883417B2 (en) * 2000-11-27 2014-11-11 Intelligent Medical Devices, Inc. Clinically intelligent diagnostic methods utilizing micromixers disposed in wells

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EP0441120A2 (fr) * 1990-01-09 1991-08-14 Yeda Research And Development Co. Ltd. Biosenseurs
WO1997001092A1 (fr) * 1995-06-20 1997-01-09 Australian Membrane And Biotechnology Research Institute Auto-assemblage de membranes de detection
US5605662A (en) * 1993-11-01 1997-02-25 Nanogen, Inc. Active programmable electronic devices for molecular biological analysis and diagnostics
WO1997022875A1 (fr) * 1995-12-19 1997-06-26 Ecole Polytechnique Federale De Lausanne Membranes composites micro-usinees, permeables aux ions pour la detection ionique amperometrique
WO1997041425A1 (fr) * 1996-04-25 1997-11-06 Pence, Inc. Biocapteur et methode afferente

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Publication number Priority date Publication date Assignee Title
EP0441120A2 (fr) * 1990-01-09 1991-08-14 Yeda Research And Development Co. Ltd. Biosenseurs
US5605662A (en) * 1993-11-01 1997-02-25 Nanogen, Inc. Active programmable electronic devices for molecular biological analysis and diagnostics
WO1997001092A1 (fr) * 1995-06-20 1997-01-09 Australian Membrane And Biotechnology Research Institute Auto-assemblage de membranes de detection
WO1997022875A1 (fr) * 1995-12-19 1997-06-26 Ecole Polytechnique Federale De Lausanne Membranes composites micro-usinees, permeables aux ions pour la detection ionique amperometrique
WO1997041425A1 (fr) * 1996-04-25 1997-11-06 Pence, Inc. Biocapteur et methode afferente

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SOUTEYRAND E ET AL: "DIRECT DETECTION OF BIOMOLECULES BY ELECTROCHEMICAL IMPEDANCE MEASUREMENTS", SENSORS AND ACTUATORS B, vol. B20, no. 1, 1 May 1994 (1994-05-01), pages 63 - 69, XP000455277, ISSN: 0925-4005 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077522A1 (fr) * 1999-06-15 2000-12-21 Biosensor Systems Design, Inc. Dispositif et procede servant a detecter des substances a analyser
EP1609411A1 (fr) 2000-06-11 2005-12-28 Orsense Ltd. Procédé et dispositif de mesure de la concentration de glucose ou d'autre substance dans le sang
WO2002012891A1 (fr) * 2000-08-08 2002-02-14 Toyo Kohan Co., Ltd. Coffret d'activation de substrat et procede de detection d'adn ou similaire a l'aide dudit coffret
JPWO2002012891A1 (ja) * 2000-08-08 2004-01-15 東洋鋼鈑株式会社 基板の活性化キット及びこれを用いたｄｎａ等の検出方法
US7078172B1 (en) 2000-08-08 2006-07-18 Toyo Kohan Co., Ltd. Substrate activation kit and method for detecting DNA and the like using the same
JP4730808B2 (ja) * 2000-08-08 2011-07-20 東洋鋼鈑株式会社 基板の活性化キット及びこれを用いたｄｎａ等の検出方法
WO2002031504A1 (fr) * 2000-10-12 2002-04-18 Biosensor Systems Design, Inc. Systeme servant a detecter des substances a analyser
US8883417B2 (en) * 2000-11-27 2014-11-11 Intelligent Medical Devices, Inc. Clinically intelligent diagnostic methods utilizing micromixers disposed in wells
WO2005090981A3 (fr) * 2004-03-24 2005-10-27 Technion Res & Dev Foundation Recepteurs artificiels
US8354066B2 (en) 2004-03-24 2013-01-15 Technion Research & Development Foundation Ltd. Artificial receptors

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