EP1549785A2 - Detection d'agents de menace biologique - Google Patents

Detection d'agents de menace biologique

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Publication number
EP1549785A2
EP1549785A2 EP03796339A EP03796339A EP1549785A2 EP 1549785 A2 EP1549785 A2 EP 1549785A2 EP 03796339 A EP03796339 A EP 03796339A EP 03796339 A EP03796339 A EP 03796339A EP 1549785 A2 EP1549785 A2 EP 1549785A2
Authority
EP
European Patent Office
Prior art keywords
affinity reagent
analyte
probe
complex
conditions
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03796339A
Other languages
German (de)
English (en)
Inventor
Phillip D. Barnes
Leonard Brillson
Stephen C. Lee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ohio State University Research Foundation
Original Assignee
Ohio State University Research Foundation
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Filing date
Publication date
Application filed by Ohio State University Research Foundation filed Critical Ohio State University Research Foundation
Publication of EP1549785A2 publication Critical patent/EP1549785A2/fr
Withdrawn legal-status Critical Current

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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • 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

Definitions

  • the present invention relates to methods and devices for the detection of fluid-borne and airborne analytes. More particularly, the invention relates to a method of analysis which can be used in combination with a portable sensor device to recognize and confirm the presence of analytes such as chemical and biological toxins in the air and in fluids.
  • Detection of threat agents can be achieved using a variety of conventional devices and techniques.
  • the most sensitive and accurate systems for detecting threat agents are generally used only in controlled laboratory environments.
  • Such devices are often large and immobile, and require proximity to a multitude of reagents, technicians and power sources. Thus, they have limited portability and resistance under field conditions.
  • these devices often require a high level of operator skill and training.
  • demand has increased for small, portable devices that are capable of rapid and reliable detection of threat agents and require minimal operator skill.
  • Such devices generally comprise small or miniaturized semiconductor chips or other suitable substrates to which are bound affinity reagents such as polymers, oligonucleotides, proteins and other molecules. Analytes bind to the affinity reagents resulting in generation and transmission of a visual, audible or other signal to the device operator.
  • affinity reagents such as polymers, oligonucleotides, proteins and other molecules.
  • the method comprises providing an affinity reagent having a known charge density in a medium which is capable of conducting electrical current. Application of a potential to a source electrode in the medium results in a flow of current between the source electrode and a drain electrode.
  • the medium is a solid or semi-solid substrate to which the affinity reagent is bound.
  • the substrate-bound affinity reagent is exposed under a first set of conditions to a sample which potentially comprises threat agents. Interaction between an analyte in the sample and the affinity reagent changes the charge density of the affinity reagent.
  • This change is manifested in a change in the flow of current between two electrical terminals in the medium.
  • Identity of the bound analyte as a target threat agent is confirmed by exposure of the analyte-affinity reagent complex to a second set of conditions which comprise a probe which confers a change in the net charge density of the complex.
  • This second change in charge density of the complex results in an alteration in the flow of current through the electrically conductive medium between the source and drain electrodes.
  • Positive confirmation of the presence of a threat agent is achieved by determining the change in charge density of the analyte-affinity reagent complex caused by application of the probe conditions.
  • Analyte identity is probed by controlled manipulation of the charge density of the affinity reagent-analyte complex using a variety of methods.
  • local pH surrounding the complex is modulated and the resultant change in charge density is detected based on the resultant alteration in current flow in the electrically conductive medium.
  • a voltage is applied to the complex to produce a measurable change in net charge density that is manifested in an alteration in current flow.
  • a second affinity reagent is applied to the complex, wherein binding of this second affinity reagent produces a change in the charge density of the complex that alters the flow of current in the electrically conductive medium.
  • Confirmation of analyte identity as a threat agent is achieved when the charge density of the probed complex and the resultant altered current flow matches a charge reference profile specific for a threat agent.
  • Charge reference profiles are predetermined based charge densities of target analyte/affinity reagent complexes and probed complexes, that have been determined under controlled conditions, or that have been predicted based on known charge properties of the complexes.
  • a match of a bound analyte to a threat agent may be achieved by correlation of the charge densities on the complexes, or by correlation of the resultant current flows.
  • Affinity reagents include, for example: proteins, such as specific biological receptors, antibodies or antibody fragments; peptides; bio- and non-biopolymers; oligonucleotides such as DNA and RNA; and other molecules which are specific for and bind or interact with specific threat agents.
  • Target analytes are threat agents that include proteins, peptides, oligonucleotides such as DNA and RNA, and other molecules that pose a particular threat to human or animal safety.
  • Positive detection of threat agents in the environment provides a warning of the presence of biological pathogens such as infectious species of bacteria, protozoa, fungi, and viruses, as well as prions, and dangerous chemical agents in the environment.
  • the sensor devices comprise one or more modified three terminal transistors, each of which comprises a semiconductor chip, preferably a metal oxide chip, a source terminal, a drain terminal, and a gate terminal.
  • terminal refers to a contact on an electrical device at which electric current enters or leaves.
  • gate terminal refers to a contact on a device as described herein wherein the contact is an exposed resistor, preferably a metal oxide resistor, on the surface of which is disposed one or more affinity reagents specific for a threat agent.
  • the source and drain terminals comprise contacts that are each attached to the end of wires that serves as electrodes used for making connections in the electrical circuitry of the devices described herein. In contrast, in various embodiments the gate electrode does not comprise an electrode.
  • the gate may comprise one or more nanostructures, such as nanotubes and other fullerene-like structures, constructed of carbon or other elements.
  • Each of the source and drain terminals comprise electrodes which are attached to a power source for delivery of a voltage to the source for creation of a potential between the source and the drain. The charge potential of the gate modulates the flow of current between the source and the drain.
  • the gate terminal may also comprise an electrode which is connected to a power source to enable the application of an electrical potential to the gate terminal to affect modulation of the flow of current through the transistor.
  • a protective coating or blocking layer is disposed on the surface of the transistor, preferably on the surface of the resistor at the gate terminal.
  • One of the functions of the coating layer is to minimize non-specific binding or interaction of non-targeted analytes with the surface of each transistor, particularly the gate portion of the transistor.
  • the blocking layer may comprise a biomolecule such as a peptide or a protein, a non-biological polymer such as PEG polyethylene glycol, a surfactant or lipid monolayer or multilayer, or other amphipathic molecules.
  • the blocking layer may comprise surface-exposed hydrophobic or hydrophilic elements, or other moieties or structures which interact with, respond to or amplify the response to target analytes.
  • the sensor further comprises a probe module which delivers a confirmatory test probe to the gate terminal and an alert capability such as an audible, visual or other alarm.
  • the confirmatory test probe interacts with the bound analyte and results in a change in charge density.
  • the current which flows between the source and the drain terminals is a function of the charge density of the bound affinity reagent and the voltage that is applied to the source electrode.
  • Analyte molecule interaction with the affinity reagent alters the charge density at the gate terminal which is manifested in an alteration in the flow of current between the source and drain terminals.
  • the charge density of the complex is altered inducing an alteration in the flow of current.
  • a charge reference profile is obtained for each threat analyte of interest which establishes the charge densities and effective currents through a sensor device each set of conditions. Confirmation of the identity of a bound analyte is achieved by correlation of the charge reference profile for a specific threat agent with the charge densities and/or current flow obtained using the sensor to detect a threat agent in a sample.
  • a match between the probed charge density and a charge reference profile is conveyed by activation of the alarm.
  • detection is achieved using a preset trigger in the sensor device which is actuated and stimulates an alarm when the flow of current induced by a probed complex matches the threshold current that correlates with the charge reference profile for the threat agent under specific conditions.
  • the sensor device may optionally comprise a processor which is used to capture charge density data for later analysis.
  • the sensor device is optionally integrated into a larger device, alone or together with one or more additional transistors, that comprises one or more of the following: power sources, wired or wireless data transmission capabilities, alarm capabilities, and control capabilities.
  • FIG 1 depicts a standard, prior art MOSFET.
  • FIG 2 (a) depicts a transistor portion of a sensor according to the present disclosure
  • panel (b) shows the same transistor in side view.
  • FIG 3 shows various different surface treatments for silicon dioxide chips.
  • FIG 4 shows in panel (a) the layout of a BioFET sensor according to the present disclosure. Dashed lines indicate boundary between reusable and disposable sections.
  • Two 1.5 V button batteries (Bl and B2) provide 3 N power to the transistor array (checkerboard) and the light emitting diode (L);
  • panel (b) shows an alternate embodiment of a BioFET specifically depicting six transistor cartridges which are connected to three fluid dispensing chambers.
  • FIG 5 shows a gas sensor according to the present invention.
  • Blocks A1-E4 represent individual transistors.
  • FIG 6 shows in panels (a) and (b) BioFET transistors according to the present invention which is set up to detect liquid samples converted to vapor phase in a chamber that is continually gas-purged.
  • FIG 7 shows in panels (a) - (c) results using a BioFET sensor for gas detection.
  • FIG 8 shows in panel (a) a biomaterial sensor according to the present invention.
  • Blocks Al- E4 represent individual transistors;
  • panel (b) shows a BioFET transistor according to the present invention which is set up to detect biological agents (affinity reagent is streptavidin and target threat agent analyte is biotin-hrp), maintained under ambient nitrogen gas.
  • biological agents affinity reagent is streptavidin and target threat agent analyte is biotin-hrp
  • FIG 9 shows in panels (a) - (c) test results using a BioFET sensor for biological agent detection.
  • Panel (a) shows current-voltage characteristics for Si transistor with gate metal removed before biotreatment, with bioreceptor and blocking agent (BSA) and with subsequent target (biotin-HRP) molecule applied. Streptavidin-biotin binding at the channel produces a large current response.
  • BSA bioreceptor and blocking agent
  • biotin-HRP target molecule
  • Affinity reagents include but are not limited to proteins, such as specific biological receptors, antibodies or antibody fragments, peptides, bio- and non-biopolymers, fatty acids, oligonucleotides such as DNA and RNA, natural and synthetic macromolucles and other molecules which are specific for and bind or interact with specific threat agents.
  • affinity reagents include 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.
  • Affinity reagents may be prepared using a variety of known techniques, including macromolecule synthesis, recombinant gene expression of biological macromolecules, antibody production and phage display.
  • Phage display is a commonly used method for displaying a high number of macromolecules such as peptides and proteins, including human antibodies and enzymes, on the surface of a small bacterial virus called a phage. This method permits screening of a large number of potential ligands in order to identify those compounds that bind with high affinity and high specificity to the phage-bound molecules.
  • affinity reagents which are specific for target analytes, specifically known threat agents are identified by generating one or more phage libraries which express the target analyte on the phage surface, screening the phage display libraries to select binding compounds with high affinity and high specificity to the target analyte, and producing and evaluating the selected binding compounds.
  • Phage display can be used in combination with one or more of the known methods for making affinity reagents in order to select and confirm the most specific reagents which have the highest affinity for the target analyte.
  • Antibody reagents for detection of many pathogens of interest are commercially available, or can be conveniently produced from available hybridomas.
  • some specific peptidic affinity reagents are also known for some of the threat agents and can either be purchased or produced by chemical synthesis.
  • specific antibody or peptide affinity reagents can be produced de novo using phage display or other protem engineering and expression technologies.
  • Affinity reagents may be used in native form.
  • affinity reagents may be in the form of chimeric molecules which posses portions of one or more molecules, each of which has a different specificity.
  • an affinity reagent may be in the form of a bifunctional molecule which has in one portion binding specificity for the surface of a substrate used for presenting the reagent, and has in one or more additional portions binding specificity for a particular target threat agent.
  • an affinity reagent may comprise a target analyte-specific portion and a dendremer portion which contributes a high charge character to the affinity reagent.
  • An example of such an affinity reagent is a monoclonal antibody or antibody fragment which is specific for a target threat agent covalently linked to a dendremer.
  • Target analytes are molecules of interest which may be present in a sample, and include molecules such as threat agents.
  • Threat agents include proteins, peptides, oligonucleotides such as DNA and RNA, and other molecules that pose a threat to human or animal safety.
  • Threat agents may be evidence of the presence of biological pathogens such as various species of bacteria, protozoa, fungi, and viruses, prions, and dangerous chemical agents in the environment. There are many examples of known threat agents.
  • Some specific examples include: anthrax toxin, bubonic plague, smallpox, tularemia, botulininum toxin, ricin, hemorrhagic fever or Q toxin, and Venezuelan equine encephalitis toxin, nerve agents such as Tabun (code name GA), Sarin (Code name BG), Soman (code name GD), and NX.
  • Affinity reagents may be targeted to specific sites on the threat agent pathogens, or to one or more molecules found in the pathogens, or to one or more reactive moieties or epitopes on the surfaces of molecules found in the pathogens. Charge properties of Proteins
  • the methods and devices disclosed herein operate based on changes in the charge density of charged molecules upon the binding of one charged molecule to another, or upon the application to a charged molecule of a probe which affects charge density.
  • biological molecules particularly proteinaceous molecules such as proteins, protein fragments, and peptides, and small molecules that interact with charged polymers or proteinaceous molecules.
  • the charge properties of proteinaceous molecules are well understood. Net charge on protein analytes is a consequence of the identities and number of charged amino acids in the primary amino acid sequence and varies with pH in a predictable fashion.
  • N P N L [H + ]/([H + ] +K N )
  • Np the number of protonated residues
  • K N the dissociation constant for that aa species
  • Proteins contain many ionizable groups on the side chains of their amino acids as well as their amino - and carboxyl - termini. These include basic groups on the side chains of lysine, arginine and histidine and acidic groups on the side chains or glutamate, aspartate, cysteine and tyrosine.
  • the pH of the solution, the pK of the side chain, and the side chain's environment influence the charge on each side chain.
  • the H-H equation can be used to calculate the fractional (i.e.
  • the isoelectric point (pi) of a protein is the pH at which the protein has a zero net charge.
  • pH > pi a protein has a net negative charge.
  • pH ⁇ pi a protein has a net positive charge. The pi varies for different proteins.
  • Lysine, arginine, histidine and the n-terminus will confer a charge that is between (and including) +1 or 0.
  • Tyrosine, cystiene, glutamic acid, aspartic acid and the c-terminus will confer a charge between (and including) -1 and 0.
  • Tyrosine is a weak acid (donates a proton) and has a pKa of 10. At any pH below 8 (2 units away from pKa) it is going to be 100% protonated and therefore uncharged, giving an average charge of 0.
  • the charges on such other molecules can be determined or predicted.
  • the presence of a target analyte is determined using an affinity reagent for a particular target analyte, wherein the affinity reagent is presented for contact with a sample in an electrically conductive medium.
  • Samples represent the mix of analytes present in the environment that are either airborne, surface borne or fluid borne. Samples for analytes may be passively assessed for presence of a target analyte by the passage of air or fluids over the device. Samples may also be assessed by the deliberate passage of captured air or fluids over the device. In one embodiment, surface borne analytes may be captured and suspended in a fluid medium for passage over the sensing surface of the sensor device.
  • affinity reagents are bound to an electrically conductive substrate.
  • affinity reagents are capacitatively coupled to thin insulators on electrically semi-conductive substrates.
  • insulators include: insulators such as silicon dioxide, silicon nitride, aluminum oxide, hafnium oxide, zirconium oxide, lanthanum aluminate, strontium titanate, barium titanate, lead titanate and other metal oxides or combinations thereof.
  • semiconductor substrates include semiconductor chips such as III-N compounds, II-NI compounds, or a combination of one or more Group IN elements, including silicon, germanium, gallium arsenide, gallium phosphide, gallium antimonide, indium arsenide, indium phosphide, indium antimonide, gallium nitride, aluminum nitride, titanium dioxide, zinc oxide, zinc sulfide, zinc selenide, zinc telmride, cadmium sulfide, cadmium selenide, cadmium telluride, diamond, silicon carbide, lead sulfide, lead selenide, lead telluride, combinations and alloys thereof.
  • Group IN elements including silicon, germanium, gallium arsenide, gallium phosphide, gallium antimonide, indium arsenide, indium phosphide, indium antimonide, gallium nitride, aluminum nitride, titanium dioxide, zinc oxide, zinc sulfide,
  • Initial detection of target analytes in a sample is achieved through detection of a change in affinity reagent charge density which occurs upon interaction of an analyte in the sample with the affinity reagent. This change in charge density is manifested in a change in the flow of current through the medium. Positive identification of binding of a threat analyte can be achieved by detecting either the change in the charge density or a change in the current flow in the medium. After formation of the analyte/affinity reagent complex, analyte identity is confirmed by subsequent controlled manipulation of the charge density of the analyte/affinity reagent complex. These manipulations of charge are described herein as probes.
  • Probes comprise application of one or more materials or forces upon a complex between an analyte and an affinity reagent.
  • a probe is any manipulation that alters the charge density of such a complex such that the change in charge density or the induced change in current flow can be detected or measured.
  • the charge reference profile for a particular threat agent is obtained by measuring or calculating the charge of the analyte complexed with an affinity reagent at one, two or preferably three or more probe conditions.
  • a reference current flow for a particular threat agent is obtained by measuring or calculating the induced current flow in a substrate or medium under known conditions in the presence of a target analyte/affinity reagent complex.
  • the charge profile and reference current flow for a target analyte is determined for the target analyte/affinity reagent complex which is bound to an electrically conductive substrate at one, two or preferably three or more probe conditions.
  • the unique charge profile and reference current flow for an analyte/affinity reagent complex differs from the charge density and current flow of a non-target analyte that interacts nonspecifically with an affinity reagent.
  • charge detection means such as the current-voltage response (i.e., current measured between source and drain at a specified source-drain voltage of the semiconductor channel in the sensor device)
  • the charge density and current flow of an analyte/affinity reagent complex is interrogated under one or more probe conditions.
  • target analyte identity can be confirmed using varied pH conditions as a probe, wherein the changed pH conditions results in a change in the charge density of the analyte/affinity reagent complex. This method is useful for any charged species, particularly proteinaceous molecules. The charge on a protein varies based upon the pH surrounding the protein.
  • the charge on a protein can be either measured or predicted.
  • a protein will have a specific charge profile wherein the charge of the protein can be known with high predictability at a range of pHs.
  • complexes between molecules such as between an analyte and a cognate affinity reagent have predictable charge densities at a given pH.
  • an analyte/affinity reagent complex in an electrically conductive medium is exposed serially or in parallel to a first pH, and then to a second pH. The charge density at each pH condition is compared to a charge reference profile for a particular threat agent to confirm the identity of the complexed analyte.
  • the charge density and induced current flow is determined for the target analyte/affinity reagent complex at one, two or preferably three or more pH conditions.
  • target analyte identity can be confirmed by applying one or more different voltages to the analyte/affinity reagent complex. Because of the unique charge character of each target threat agent and its cognate affinity reagent, application of voltage to a complex between the two will result in a unique charge density for the complex under controlled conditions.
  • the charge density and induced alteration in current flow is determined for the target analyte/affinity reagent complex at one, two or preferably three or more voltage conditions.
  • target analyte identity can be confirmed by contacting the analyte/affinity reagent complex with one or more secondary affinity reagents.
  • Such reagents are by definition specific for a target threat agent, therefore interaction between the two increases the confidence that the analyte bound to the first affinity reagent is specific.
  • Interaction of the secondary affinity reagent confers a net change in the charge character of the analyte/affinity reagent complex.
  • the charge density of the target analyte/affinity reagent complex bound to the secondary affinity reagent is further probed by application of a voltage at one, two or preferably three or more voltage conditions.
  • affinity agents are partially recessed into the blocking layer and target analyte identity is confirmed by contacting the analyte/affinity reagent complex with one or more secondary affinity reagents.
  • the degree to which the affinity agent is recessed into the insulating layer governs the degree to which the secondary affinity reagent can interact with the analyte/primary affinity reagent complex. Recessing primary affinity agents into the insulating layer also reduces undesirable interactions between moieties on the primary and secondary affinity reagents.
  • Analyte/affinity reagent complexes may be probed according to the methods described herein, either serially or in parallel. Various combinations of probes may be used to achieve greater confidence regarding the identity of a target analyte.
  • probes using varied pH are conducted using two or more identical transistors with affinity agents specific for a particular threat agent. All of the transistors are maintained at the same pH (a first set of conditions). Binding of an analyte at that pH results in a change in charge density that is approximately identical for each transistor. Thereafter, each transistor is exposed to a different pH which is achieved by deployment to each transistor of discrete buffer solutions.
  • the pH solutions are deployed by the operator prior to contact with an analyte, such that the total number of pH probe conditions is one fewer than in the previously described embodiment.
  • the probes are deployed serially to one or more transistors, wherein more than one probe is first used, then a second probe is deployed to provide additional information for determining the identity of a bound analyte.
  • a first probe condition such as application of a pH buffer.
  • a second probe in the form of a secondary affinity reagent is deployed to each transistor.
  • the sensors detect the formation of complexes between analytes, preferably protein or peptide analytes such as, for example, toxins, pathogens, and the like, and specific biological affinity reagents, such as, for example, antibodies and peptides.
  • analytes preferably protein or peptide analytes such as, for example, toxins, pathogens, and the like
  • specific biological affinity reagents such as, for example, antibodies and peptides.
  • analyte/affinity reagent complex enables confirmation of the identity of an analyte as a threat agent. Secondary probes permit discrimination between threat agents ant other analytes that nonspecifically interact with an affinity reagent.
  • analyte binding is the sole basis of detection.
  • existing systems are susceptible to artifacts that result from immunological cross-reactivity between affinity reagent sand analytes in the sample, and non-specific binding of analytes to sensing surfaces. These artifacts engender erroneous responses.
  • the methods and devices of the present invention permit analysis of additional properties of the analyte/affinity reagent complex.
  • Sensors as described herein have many desirable practical attributes. They are small, light, and easily packaged into a wearable sensor and can be mass-produced a t low cost using standard microelectronic fabrication from standard semiconductor and biomaterials. The devices consume very low power, and only draw power when an analyte binds to the affinity reagent to induce a current change between the source and drain terminals. As a result, a tiny battery can operate the sensors for months. Portions of the sensors, for example, the battery and LED, can be made re-usable.
  • the sensors are used by operators to detect the presence of threat agents in the local environment.
  • the operator preferably uses a sensor passively in the form of a badge which is attached to the outer portion of the operator's gear or clothing.
  • a sensor may be used actively as a testing or sampling device.
  • the sensor when deployed in the field, the sensor is in a powered mode and ready to receive samples in gaseous or fluid form.
  • the operator deploys a probe for analyte identity confirmation either before or after initial analyte binding.
  • the operator may deploy a probe, such as a set of pH buffers, to two or more transistors prior to entering the field.
  • the first and second conditions used to confirm analyte identity comprise a combination of two of the probed pH conditions.
  • the operator may be in the field and receive a signal from the sensor that an analyte has bound. Thereafter, the operator may deploy a secondary affinity reagent probe. Binding of this secondary affinity reagent to the analyte/first affinity reagent complex provides the second set of conditions used for confirming the identity of the bound analyte as a threat agent.
  • the operator is first alerted to the presence of possible target analytes by an audible or visual signal. Thereafter, the operator activates one or more probes to confirm the identity of an analyte as one or more possible threat agents.
  • pre-established gates or triggers are set in the sensor based on the charge reference profile for a particular threat agent.
  • the trigger settings represent the predetermined charge flow (current) for a threat agent/affinity reagent complex under the probed conditions.
  • current the charge flow for a threat agent/affinity reagent complex under the probed conditions.
  • a Metal Oxide Semiconductor Field Effect Transistor is a semiconductive device which has three terminals designated as the source, the gate and the drain.
  • FIG 1 shows one form of a standard metal oxide semiconductor field effect transistor (“MOSFET”) chip.
  • MOSFET (1) is made of a semiconducting material (2), examples of which are described herein, and comprise three key functional portions: a drain terminal (3), a source terminal (4), and a gate terminal (5).
  • the source and drain terminals are regions on the surface of the semiconductive device that are spaced apart from each other with the gate located in between. The source and the drain regions share the same polarity and comprise metal contacts for attachment to electrodes.
  • an electrode is attached to the source terminal (9) and an electrode is attached to the drain terminal (8), with a battery located in between for application of an electrical potential between the source and drain terminals.
  • the gate region usually comprises an insulative material (7), usually made of an oxide such as silicon dioxide, with a metal contact on the surface (6) for connection to an electrode (10).
  • a voltage is applied from the battery to the source terminal via the source electrode, creating a current that flows from the source to the drain via a conducting channel (11) that is induced under the insulative oxide material of the gate. Current flow in the channel is modulated by application of electrical potential to the gate, usually via the gate electrode.
  • a fourth electrode (12) may be located on the base of the substrate to further modulate the potential applied to the channel, thus further modulating current flow from the source to the drain.
  • FIG 2 (a) shows a modified MOSFET device (13) according to the present invention.
  • the source (14) and drain (15) terminals are depicted at the ends of the top surface of the substrate semiconductor material (16), with the gate terminal (17) disposed therebetween.
  • the depiction shows one embodiment of a sensor according to the present invention, in which the gate comprises a recessed area (18) in the substrate that comprises the oxide resistor of the gate terminal. Positioned over the recessed area is depicted an affinity reagent layer (19) which is to be deposited on the gate resistor area. Depicted above the affinity reagent layer is a layer that represents an analyte (20).
  • a modified MOSFET Operation of a modified MOSFET is essentially the same as a standard MOSFET, except that the surface of the oxide resistor is available for interaction with a range of charges species.
  • current flow in the channel is modulated by build up of charge density in the form of one or more charged species such as charged molecules (e.g., proteins) on the gate resistor.
  • charged molecules e.g., proteins
  • current flow between the source and drain is fixed at a given voltage between those two terminals.
  • Contact of the gate with a charged species alters the electrical potential in the gate region, thereby altering the current flow in the channel between the source and the drain.
  • Modified MOSFETs are used in one embodiment of the present invention to provide the electrically conductive substrate used according to the methods described herein.
  • the standard MOSFET forms the basis for the sensor devices provided in one embodiment of the present invention.
  • standard MOSFET is modified by removal of the metal portion at the gate terminal of the transistor to expose the oxide resistor of the gate. All other aspects of a standard MOSFET are unchanged.
  • the surface of the transistor, particularly the surface of the exposed oxide resistor at the gate may be altered using standard treatment techniques.
  • FIG 3 shows some options for surface treatments for chips used according to the present invention.
  • the chip surface may be plasma treated or grooved. Surface treatment of chips enables optimization of the surface to enhance adsorption of blocking and affinity reagents and to enhance charge transfer between affinity reagents and the chip.
  • sensor devices are made using modified MOSFETs.
  • the gate metal is removed to expose the oxide resistor at the gate terminal.
  • These devices comprise one or more modified transistors.
  • Each transistor comprises source, drain, and gate terminals.
  • the gate region comprises a resistor, preferably a metal oxide resistor, and a coating disposed on the surface of the resistor comprising one or more affinity reagents which are specific for a threat agent.
  • the gate comprises channels or other surface structure for receiving the affinity reagent, i one embodiment, the affinity regent is in a dried or lyophilized form.
  • the affinity reagent is in an aqueous solution, preferably a buffer that enhances stability of the affinity reagent, and is satisfactory for permitting complex formation between the affinity regent and its cognate ligand.
  • the affinity reagent layer is selective for one a known targeted threat agent.
  • Biological affinity reagents exhibit high affinity & selectivity in binding to their cognate ligands.
  • the interactions are extraordinarly specific: the range of substitutions allowed in the cognate epitopes of antibodies is extremely narrow and highly conservative of epitope structure.
  • Specific interaction or binding is due to one or more multiple non-covalent interactions such as ionic, hydrophobic, van der Waals, etc., between affinity reagents and their cognate ligands.
  • Peptides are smaller than antibodies (as much as 100 fold) and typically make fewer contacts to their cognate ligands. Their ligand recognition is thus more promiscuous, but still can be highly specific.
  • Affinity reagents are bound directly to the transistor surface.
  • affinity reagents are bound to the surface of the resistor through an intermediate molecule.
  • the intermediate molecule is attached at one end to the gate region of the transistor and at the other end to one or more affinity reagents.
  • the role of the intermediate molecule is to allow for an extension of the affinity reagent from the surface.
  • Intermediate molecules are conductive and may contribute additional charge character to the affinity reagent.
  • Intermediate molecules optionally permit the attachment of multiple affinity reagents to provide an amplification effect; they may also serve as blocking agents that block access to the transistor surface, thereby preventing non-specific binding of agents that would interfere with BioFET function.
  • intermediate molecules include amine groups, polyaromatic molecules, lipophilic, liposoluble, or hydrophobic groups, single- or poly- saturated aliphatic chains, unsaturated aliphatic chains, and aromatic chains.
  • Intermediate molecules may be and can be branched or linear in form, and are bound to the transistor surface by covalent bonding, or non-covalent bonding.
  • Non-covalent bonds include hydrophobic bonds, ionic bonds, and .pi.-bond overlaps.
  • a transistor also comprises a blocking reagent layer disposed on the transistor surface, preferably on the surface of the oxide resistor of the gate terminal.
  • the blocking layer inhibits or prevents non-specific binding of non-targeted molecules to the transistor substrate, thus enhancing the sensing specificity and reducing the likliehood of false positive readings with the sensor.
  • the blocking layer may comprise a biomolecule such as a peptide or a protein, a non-biological polymer such as PEG polyethylene glycol, a surfactant or lipid monolayer or multilayer, or other amphipathic molecules, a hybrid surfactant bilayer, a surfactant multilayer, or a self-assembled monolayer of biological or non-biological material.
  • the blocking layer may comprise surface-exposed hydrophobic or hydrophilic elements, or other moieties or structures which interact with, respond to or amplify the response to target analytes.
  • the blocking layer is applied to the surface of the gate after the affinity reagent is bound, so as to block any reactive groups that remain on the surface of the resistor.
  • the blocking layer comprises nanotubes or other nanostractures which are coated with antibodies. Antibodies to carbon nanotubes are available the crystal structures of the antibody-carbon nanostructure have been determined. At appropriate concentrations, the antibodies are known to encrust and fully obscure the surface of the carbon nanotubes. As such, these antibodies constitute an attractive blocking reagent for carbon nanotubes, absolutely prohibiting access to the nanotube surface by interfering materials in the analytical environment.
  • the blocking layer comprises antibodies, wherein the antigen binding domains of the antibodies comprise isolated, single polypeptides which are prepared by methods well known to those skilled in the art. These isolated polypeptides can be engineered to provide bi-functional affinity reagents which comprise a first domain that is specific for and binds to the transistor substrate, and a second domain with target analyte specificity.
  • a blocking/analyte affinity reagent layer may be used to self assemble on nanotube components or other sensor surfaces to provide a device with extraordinar sensitivity that is highly resistant to nonspecific analyte binding.
  • Other bi-functional molecules can be designed using affinity reagents that comprise portions of one or more specific molecules such as antibodies, membrane receptors, and other ligand-specific molecules.
  • the gate region further comprises one or more nanostructures, such as nanotubes and other fullerene-like structures, constructed of carbon or other elements.
  • Nanostructures include any one or any subset of carbon based nanostructures that include nanofibers, single-walled nanotubes, and multi-walled nanotubes, or other fullerene type structures.
  • Variants of nanostructures include both closed, cage-like forms and extended linear forms, such as those listed above, as well as those with other atoms included.
  • Examples of atoms other than carbon known to be included in such nanostructures include boron, boron nitride, and carbon boron nitride, silicon, germanium, gallium nitride, zinc oxide, indium phosphide, molybdenum disulphide, and silver.
  • the sensor further comprises a probe module which delivers a confirmatory test probe to the gate region.
  • the confirmatory test probe interacts with the bound analyte and results in a charge transfer that allows confirmation of the identity of the bound analyte by comparison with a threat charge reference profile.
  • the probe module is used to apply a specific probe to one or more transistors.
  • FIG 4 (a) shows a sensor device according to the present invention.
  • the power sources (batteries, 24) and the LED (designated "L", 25) form a reusable portion of the depicted device.
  • the bulb (26), delivery tubes (27) and probe chambers (28) comprise the probe module portion of the sensor.
  • the chip (29) comprising an array of discrete transistors (30) is operably connected to the probe module to enable operator controlled deployment of the probe reagents to the transistor array.
  • a sensor comprises an array of threat agent specific transistors such that combinations of one or more transistors can be subjected to different probe conditions.
  • a sensor (31) may comprise one or more arrays (32) of transistors wherein each array is specific for a different threat agent.
  • the sensor is configured with a probe unit that provides one or more different pH solutions, a charge, or a secondary affinity reagent, or combinations thereof.
  • the device shown in FIG 4 (b) depicts a probe unit that is capable of delivering three different probes from three probe dispensers (33) via microfluidic or other delivery processes (34).
  • Probe modules are connected to these transistor cartridges or transistor arrays and may be operator controlled for delivery of probe conditions to each transistor.
  • one or more, and preferably three different pH solutions are maintained in the probe module for operator controlled delivery to a transistor cartridge.
  • the solutions may be maintained for example in separate blister packs.
  • the pH buffer components may be maintained in dry form in small tubes which are connected by a gated channel to a liquid reservoir.
  • An actuator such as a compression bulb, directs the flow of the liquid in the reservoir through the channels and directs the solutions to one or more of the transistors, such that each of three transistors receives a specific pH buffer probe.
  • Three pHs low, neutral, high pH, specific buffers and pHs used are optimized for each analyte) are likely to provide sufficient data for unambiguous threat identification, but additional pH conditions can provide adjustable levels of discrimination.
  • the probe module comprises a screen which is positioned over the gate region and is connected to an electrode.
  • the screen is capable of applying different voltages to the analyte/affinity reagent complex.
  • the probe module comprises one or more secondary affinity reagents.
  • secondary affinity reagents are not identical to the substrate-bound affinity reagent, and are preferably specific for a moiety on the target analyte that is different from the moiety recognized by the bound affinity reagent.
  • Secondary affinity reagents may be maintained for example in a separate blister packs, in liquid, semi-dry or lyophilized form.
  • An actuator such as a compression bulb, directs the flow of the secondary affinity reagent to one or more of the transistors.
  • more than one secondary affinity reagent may be used, such that each of two or more transistors receives a different secondary affinity reagent probe.
  • Sensors also comprises a power source and an alarm capability for notifying the operator of the presence of a threat.
  • Sensors also comprise circuitry for connecting the electrodes at each terminal to a power source and to each other to create a circuit. Circuitry or micro circuitry is positioned at the transistor or in some contiguous area or microcircuit board of the sensor device. Electrodes are coupled or connected to circuitry. Changes in current flow caused by charge transfer through bound affinity reagents are transferred via the circuitry and through the electrodes to enable detection of threat agents. Sufficient power is supplied by conventional button batteries. Typically, 3, 6, or 9 volt batteries are sufficient to produce target-specific responses.
  • the power source forms part of the sensor.
  • the power source may be part of a larger device into which the sensor is integrated.
  • the sensor device is optionally integrated into a larger device, alone or together with one or more additional transistors, that comprises one or more of the following: power sources, wired or wireless data transmission capabilities, processor capabilities, alarm capabilities, and control capabilities.
  • Sensors may further comprise a processing unit, which may include a computer or computational device that is used with any of the embodiments described herein, including appropriate software or hardware configured to provide near real-time and historical analysis of changes in conductivity of single sensors or arrays of sensors according to the invention.
  • a processing unit which may include a computer or computational device that is used with any of the embodiments described herein, including appropriate software or hardware configured to provide near real-time and historical analysis of changes in conductivity of single sensors or arrays of sensors according to the invention.
  • the sensor it is housed in an instrumentation unit which includes a computer-controlled signal control and processing unit, and a transmitter that sends data by wire or wireless methods to remote sites. Recording devices may also be included in the instrumentation, either at the site of the sensing device or at remote sites, to maintain a historical record of signal activity, and to provide immediate alarming under certain conditions.
  • the data recording software includes a pre-set signal threshold, which if exceeded by sensing signal output, triggers an alarm system that can be situated at either the site housing
  • Sensors according to the present invention operate on a principle of current modulation in the region of semiconductor termed the channel that connects source and drain electrodes.
  • Conventional metal oxide semiconductor field effect transistors operate by controlling voltage applied to a metal-on-insulator-on-semiconductor channel.
  • the sensors according to the present disclosure operate by sensing charge at the bare insulating oxide surface of the semiconductor channel. Depending on the charge residing at the free surface of this channel, the charge density within this volume of semiconductor increases or decreases. Therefore, the magnitude of current that flows between source and drain for a given source to drain voltage depends on the charge applied to the channel region.
  • Formation of analyte/affinity reagent complexes on the gate result in a change in charge density that induces a change in the current flow between the source and drain terminals. Moving a standard MOSFET from depletion to strong inversion (i.e., shifting the
  • Field effect transistors exhibit a monotonically increasing current with voltage applied between source and drain that saturates at a characteristic current value. Changes in charge that increase (decrease) charge density in the channel produce current - voltage curves with similar shape that shift to higher (lower) saturation current values.
  • the sensor can be operated in depletion mode, i.e., the channel region is depleted of charge before any changes in surface charge, by appropriate semiconductor doping. Only when surface layer induces charge in the channel does it begin to conduct significant current.
  • threshold and ambient source to drain current values are tailored for a particular analyte.
  • These thresholds are set in the device such that a transistor has a memory that represents the preset threshold corresponding to the potential for a particular analyte/affinity reagent complex.
  • Charge reference profiles for specific analyte/affinity reagent complexes are used to establish thresholds for each threat specific transistor.
  • multiple transistors are used in a sensor, each with a different present threshold that corresponds to the predetermined charge reference profile for a given threat agent complexed with its affinity reagent under specific probe conditions.
  • Arrays of multiple transistors specific for one or more threat agents are used to scan a range of different charge conditions to obtain a high degree of confidence of analyte identification as a threat agent.
  • Threshold voltage of FETs can be adjusted for maximum conductance at specific conditions. When all of the transistors in an array are at maximum conductance, the bound analyte is confirmed to be a true target threat agent, and an LED is illuminated to notify the operator.
  • This configuration requires little operator expertise: a match to previously determined (i.e., during sensor development) analyte fingerprints allows current to flow through the channel to a transducer, producing an audible or visual signal.
  • the thresholds may be digitally controlled or tuned to allow for refined adjustment by the operator.
  • changes in source to drain current are assessed and measured at initial analyte binding and upon application of a probe for a given source to drain voltage.
  • Results with devices described herein show sizable (40 microamp) changes in source to drain current for a source to drain voltage of 5 V when an analyte binds to an affinity reagent.
  • the changes in current caused by the change in charge density on the gate resistor may be measured and captured by the processor unit for further analtsis. Determination of a match between the measured charge and current parameters and the predeterimined charge reference profile for a target anlatye results in the triggering of an alarm to alert the operator of ( the presence of the treat agent.
  • FIG 6 panels (a) and (b) show gas sensor experimental set up.
  • the FET was maintained with a controlled atmosphere over the SiO2 gate sensing region to isolate environmental effects, prevent contamination, and verify sensor stability.
  • Characterization was performed with probe station and sensitive current vs voltage (I-V) testing using: HP4145B I-V analyzer, Keithley 6517A Electrometer.
  • I-V current vs voltage
  • To test sensor sensitivity gas-phase samples are introduced on a "carrier” gas to provide controlled gas flow. Characterization was performed as a function of time and temperature. Selectivity and surface charge properties were assessed using I-V characteristic. Room temperature was used for water and HC1 tests, while isopropanol was heated to 50 deg C to achieve accelerated relaxation.
  • EXAMPLE 2 Sensing of Streptavidin cognate ligand, biotin: Charge Exchange at the Streptavidin-SiO /Si Interface
  • a model protein for use with the sensor device is the streptavidin molecule.
  • Streptavidin is a tetrameric protein with four protein lobes having topological dimensions of several nanometers both laterally and perpendicular to an adsorbed surface. Streptavidin binds strongly to the small molecule biotin, providing a receptor-target testbed. Streptavidin- biotin binding has numerous applications, including targeting in radioimmunodiagnosis and radioimmunotherapy of cancer. The nature of this strong binding is of considerable interest itself for the design of new drugs and ligands for proteins and nucleic acids. This interface can be probed using a combination of surface science techniques.
  • Atomic force microscopy is capable of detecting individual streptavidin molecules and their distribution across the surface.
  • a Kelvin probe can provide surface potentials and measure the dipole changes due to adsorption.
  • X-ray photoemission spectroscopy XPS
  • XPS X-ray photoemission spectroscopy
  • LEEN Low energy electron-excited nano-scale luminescence
  • FIG 8 (a) shows biosensor chip layout.
  • Blocks A, B and C 1-4 were treated with BSA as a blocking agent and streptavidin as the affinity reagent.
  • Blocks Dl-2 and El-2 were treated with BSA blocking agent alone and blocks D3-4 and E3-4 were treated with streptavidin alone. All blocks were subsequently exposed to biotin HRP.
  • Biomaterial samples were applied in a two-step process: first, an application of bioreceptor sites, streptavidin, and second, application of "target" biomaterials (Biotin-horseradish peroxidase). Characterization was performed before and after each biomaterial application. Selectivity and surface charge properties are assessed using I-V characteristic.
  • EXAMPLE 3 Optimization of Sensor using Streptavidin
  • a picoliter ultrapump dispenses protein in phosphate-buffered saline solution on the transistor gate channel with source and drain contacts electrically isolated. To maximize the effect of charge transfer, the charged protein coverage should be high and the distance from the transistor channel should be small (i.e., high capacitance) with no intervening layer.
  • Streptavidin has been successfully applied to silicon (and other inorganic surfaces) by direct adso ⁇ tion. Planar silicon binds streptavidin, though much less efficiently than porous silicon.
  • the sensor channel is fabricated from non-porous silicon, the surface area and protein loading of the sensing channel is increased by texturing the channel surface.
  • Metal-oxide FETs are prepared by conventional microelectronic techniques in a class 100/1000 clean room, then stripped of their gate metallization.
  • the biomolecule interactions with the Si/SiO 2 can be controlled using materials science processing techniques.
  • Plasma and wet chemical treatments of the Si surface can be used to prepare SiO 2 surface layers with hydrophilic/phobic properties that increase surface bonding and coverage of the adsorbate.
  • Increasing surface roughness or patterning the surface with ion or electron beams can increase bonding and coverage as well. However, these can introduce charged defects that mitigate charge transfer. Control of FET threshold voltage to within +0.1 N is achieved using ion implantation to tune threshold voltages.
  • Extruded fluid channels outside +0.01 cm diameter tolerance is achieved by etching grooves in plastic substrate, cover with plastic plate. Negative relationship between affinity reagent size and signal magnitude is mitigated by optimizing the performance of affinity reagents from peptide size (0.25-2 nM diameter) to scFv (7nM diameter) to antibody size (15nM diameter) using intermediate molecules.
  • Atomic force microscopy is used to monitor surface morphology and protein adso ⁇ tion in air with ⁇ 5 nm lateral resolution, depending on tip condition and protein thickness. This resolution permits detection of individual molecules, for example streptavidin, with tetramer diameters of ⁇ 10 nm.
  • Kelvin probe measurements of contact potential are available both in air and UHV.
  • UHV-compatible, dried protein-Si/SiO 2 surface are examined by XPS to confirm surface coverage of protein, C, O, and N Is core level shifts associated with the SiO 2 surface, rigid energy level shifts associated with adsorbate charging, and ambient contamination. Denatured protein is expected to retain its original charge.
  • SIMS mapping with - 100 nm spatial resolution to provide additional interface bonding information about the oxide and adsorbed surfaces.
  • Prevention of contamination or degradation of affinity reagents prior to field operation is achieved by use of protease inhibitors, and by lyophilization and sealing prior to use with disposable tape, similar to long-term storage technique for ink jet printer cartridges.
  • Photoresist masking is used to prevent shorting of source and drain by pH solution and other fluids such as water or secondary affinity reagents.

Abstract

L'invention concerne des procédés et des dispositifs de détection et de confirmation de l'identité d'analytes tels que des agents de menace, en particulier des analytes portés dans l'air ou dans un fluide. Les procédés consistent à fournir un réactif d'affinité ayant une densité de charge connue dans un milieu capable de conduire le courant électrique. L'identité de l'analyte est sondée par une manipulation régulée de la densité de charge du complexe réactif d'affinité-analyte, à l'aide d'une variété de procédés.
EP03796339A 2002-09-20 2003-09-22 Detection d'agents de menace biologique Withdrawn EP1549785A2 (fr)

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