WO2006067276A1 - Utilization of nanowires in chemical and biological analysis - Google Patents

Utilization of nanowires in chemical and biological analysis

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Publication number
WO2006067276A1
WO2006067276A1 PCT/FI2005/000550 FI2005000550W WO2006067276A1 WO 2006067276 A1 WO2006067276 A1 WO 2006067276A1 FI 2005000550 W FI2005000550 W FI 2005000550W WO 2006067276 A1 WO2006067276 A1 WO 2006067276A1
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Prior art keywords
nanowire
electrode
electrodes
nanowires
molecules
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PCT/FI2005/000550
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French (fr)
Inventor
Jorma Antero Virtanen
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Nanolab Systems Oy
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6816Hybridisation assays characterised by the detection means
    • C12Q1/6825Nucleic acid detection involving sensors
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54373Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438Electrodes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587Nanoparticles

Abstract

The invention provides devices and methods for ultrasensitive chemical and biological analysis. The method is based on end derivatized nanowires (104). After affinity or supramolecular binding these nanowires (104) can be detected either optically or electronically in a certain location that is specific for a given analyte (108). Nanowires can carry very large number of optically detectable groups. Alternatively, they can close an electronic circuitry (102, 103, 109), and the change in an electric property (110) of that circuitry is detected.

Description

UTILIZATION OF NANOWIRES IN CHEMICAL AND BIOLOGICAL ANALYSIS

BACKGROUND

1. Field of the invention

The present invention is directed to detecting a chemical or biological compound in a sample. More specifically, it will provide devices and methods to measure an affinity binding, including antigen-antibody interaction, DNA hybridization, ligand-receptor binding, enzyme-substrate, or enzyme-inhibitor interaction, by attaching a conductive nanowire between two electrodes by an affinity molecule(s) and measuring the change in the current.

2. Prior art and overall description

Numerous analytical methods have been developed. There is tendency to increase the speed of the analysis, reduce the size of the instrumentation and cost, decrease the detection limit, and increase the specificity and sensitivity. Despite of the enormous progress, there is still need for better methods and instrumentation. For example, the detection of viruses is too expensive and slow in order to screen each blood portion that has been donated. Instead, often several portions (up to hundred) are pooled, and the sample is taken from that pool. If the virus is found the whole pool is discarded. Another example is the detection of mad cow disease. The samples must be sent to specialized laboratories, instead of immediate analysis. There are several other examples that demonstrate the need for the better analytical methods.

Electronic measurements are inherently amenable to sensitive and fast detection. The measurement of small currents and voltages is presently facile process. Also the color or cloudiness of the sample is not problematic in electronic measurement. There are at least two possible principles in electronic measurement. The binding of the analyte may change the electrical properties, such as conductance or capacitance, of an existing circuitry, or the analyte may close otherwise open circuitry. Very powerful closing agents that are compatible with single molecule affinity binding are nano wires, especially carbon nanotubes (CNT). Methods to bind conductive particles between two electrodes by affinity binding are well known in the art. Also creating a nanowire by metallization of the double helical DNA is known in the art. An electric circuit can be modified by a binding reaction (Mroczkowski et al. US5284748). Two electrodes can be in a close proximity and a contact may be created between them by binding conductive particles in the intermediate area. In order to achieve this by one particle the separation of electrodes should be an order of one micrometer and preferably submicrometer. This requires extremely accurate lithography. Moreover, the structures should be stable in aqueous salt solutions. With larger separations many particles are required to close the gap, which will result into a lower limit of detection, because it is unlikely that two or more particles will bind side by side unless the number of bound particles is high. In a sharp contrast to the present invention, the binding molecules are attached onto a substrate between the electrodes in the device described by Mroczkowski et al.

In a molecular sensing apparatus described by Maracas the electrodes are so close that DNA or RNA double helix is able to bridge the gap. This approach is limited to conductive molecules, such as DNA double helix, which is conductive over short distances.

The existing problems can be alleviated by the methods of the present invention, which are based on the binding of a nanowire between two electrodes by affinity binding. However, there some obstacles to overcome. One is solubilization of the nano wires so that they still can be in electronic contact with the electrodes after the binding event. The second is the attachment of binding or recognition molecules so that the nanowires are effectively connecting two electrodes. If the both ends have the same binding molecules they may bind with the same electrode, and only some will connect two different electrodes. Accordingly, better result is obtained, if each end of the nanowire has a different binding molecule. This is a non- trivial task, because tens of affinity molecules may be bound with each end, and mixtures are easily created. The present invention provides methods to overcome these obstacles. The CNTs and gold nanowires are most extensively discussed, because they are currently preferred nanowires for the implementation of the present invention.

It would be highly preferable, if the sampling device, syringe or cassette, would be able to perform the actual assay immediately without any further transfer of a sample. Furthermore, it should be possible to perform tens or hundreds of different assays for various classes of biomolecules from the same sample. The device should be disposable and accordingly of very low cost.

An electric conductivity of DNA can be utilized in DNA testing (Meade et al. WO 95/15971 and 96/40712). Single stranded DNA does not conduct electricity, but hybridized DNA has a significant conductivity. A single stranded probe is attached on to a gold surface. The bare area is covered by an insulator. After the target that has an electroactive moiety hybridizes with the probe the electrons have a path via the doublex to get from a soluble reducing agent to the gold electrode. The current can be measured and is relative to the number of target molecules that have been bound with the probes. A single mismatch reduces the current significantly. This method is specific for DNA and can not be used for immunoassays. On the contrary, all affinity interactions, including antigen-antibody interactions, can be measured by the methods and devices of this invention.

The present invention truly enables a disposable self-contained device that is able to perform hundreds, or even thousands of tests quantitatively and fast from a very small amount of sample.

The present invention solves most problems associated with the prior art. The assay can be performed in the sample collection device. No transfer or aliquoting of the sample is required. Results are obtained fast and they are quantitative. The detection is very sensitive, because a single conductive nanowire can be detected and only one analyte molecule is necessary for the binding of that nanowire. Thus, in its ultimate form the present invention can count molecules. The electrode arrays allow multiple tests to be performed from each sample. The device can have a low cost processor and display. All this combined enables a self-contained disposable device that is able to analyze quantitatively, sensitively and fast hundreds of compounds. The device can also be modular, so that only the sample collection and microfluidic unit is disposable, while processing and display units are used repeatedly, and can be specially designed hand held devices, cell phone, or computer equipped with a adapter.

Electric or electrochemical detection methods have been utilized previously. Some examples are:

US4444892 4/1984 Malmros Analytical device having semiconductive organic polymeric element associated with analyte-binding substance US4218298 8/1980 Shimada et al. Selective chemical sensitive FET transducer

US4859306 /1989 Siddiqi et al. Selective ion-permeable dry electrodes for analyzing selected ions in aqueous solutions.

US4945045 7/1990 Forrest et al. Electrochemical methods of assay

US4920047 4/1990 Giaver et al. Electrical detection of the immune reaction

US5156810 10/1992 Ribi et al.Biosensors employing electrical, optical and mechanical signals

US5284748 2/1994 Mroczkowsld et al. Method for electrical detection of a binding reaction

US6060023 5/2000 Maracas Molecular sensing apparatus

US6100045 8/2000 Van Es et al. Detection of analytes using electrochemistry

J. Wang "Electroanalytical Techniques in Clinical Chemistry and Laboratory

Medicine", VCH, New York, 1988. QD115W246e 1988

W.F. Smyth, "Voltametric Determination of Molecules of Biological

Significance", John Wiley&Sons, New York, 1992.

Prior art of disposables and self-contained clinical chemistry devices can be found in the following patents:

US3799742 3/1974 Coleman Miniaturized integrated analytical test container

US4859421 8/1989 Apicella, Disposable antigen concentrator and detector

US4918025 4/1990 Grenner, Self contained immunoassay element

US5053197 10/1991 Bowen, Diagnostic assay module

US5133937 7/1992 Frackleton et al., Analysis system having a removable reaction cartridge and temperature control

US5167922 12/1992 Long, Assay cartridge

US5198368 3/1993 Khalil et al., Methods for performing a solid-phase immunoassay

US 5217905 6/1993 Marchand et al., Device and method for the rapid qualitative and quantitative determination of the presence of a reactive ligand in a fluid

US5223219 6/1993 Subramanian et al., Analytical cartridge and system for detecting analytes in liquid samples

US5503985 4/1996 Cathey et al., Disposable device for diagnostic assays

US5580794 12/1996 Allen, Disposable electronic assay device

US5981203 11/1999 Meyerhoff et al., Unitary sandwich enzyme immunoassay cassette, device and method of use US5714390 2/1998 Hallowitz et al., Cartridge test system for the collection and testing of blood in a single step

US5616467 4/1997 Olsen, Method and kit for analyte detection employing gold-sol bound antibodies.

Sample collection and filtering:

US4632901 12/1986 Valkirs et al., Method and apparatus for immunoassays

US4965187 10/1990 Tonelli, Method and apparatus for assaying whole blood

US5248303 9/1993 Margolin, Medical syringe with needle-retracting mechanism.

Substrates and patterning

US4357311 11/1982 Schutt, Substrate for immunoassay and means of preparing same

US4886761 12/1989 Gustafson et al., Polysilicon binding assay support and methods

US4959303 9/1990 Houts et al., Assay for antigens by binding immune complexes to solid supports free of protein and non-ionic binders

US5514501 5/1996 Tarlov, Process for UV-photopatterning of thiolate monolayers self-assembled on gold, silver and other substrates

US4302530 11/1981 Zemel, Method for making substance-sensitive electrical structures by processing substance sensitive photoresist material

US4562157 12/85 Lowe et al.,Diagnostic device incorporating a biochemical ligand

US5413732

Reagent patterning and stabilization:

US3572400 3/1971 Casner et al., Dispensing of fluids to small areas US4634027 1/1987 Kanarvogel, Liquid dispensing apparatus and an anti- drip valve cartridge therefore.

US4216245 8/1980 Johnson, Method of making printed reagent test devices US5001048 9/1990 Woodrum , Reversible immobilization of assay reagents in a multizone test device US5554339 9/1996 Cozzette et al., Process for the manufacture of wholly microfabricated biosensors

US4329317 5/1982 Detweiler et al.5 Method for stabilizing a specimen slide for testing occult blood

US5413732 5/1995 Buhl et al., Reagent compositions for analytical testing

J. Lipkowski and P.N. Ross, "Adsorption of Molecules at Metal Electrodes", VCH, New York, 1992.

Examples of microfluidic systems are:

US4426451 1/1984 Columbus, Multi-zoned reaction vessel having pressure-actuable control means between zones

US4753776 6/1998 Hillman et al., Blood separation device comprising a filter and a capillary flow pathway exiting the filter

US4855240 8/1989 Rosenstein et al.,Solid phase assay employing capillary flow

US4963498 10/1990 Hillman et al., Capillary flow device

US5304487 4/1994 Wilding, Fluid handling in mesoscale analytical devices

US5698406 12/1997 Cathey et al., Disposable device in diagnostic assays

US5798215 8/1998 Cathey et al., Device for use in analyte detection assays

US5714390 2/1998 Hallowitz et al., Cartridge test system for the collection and testing of blood in a single step.

Immunoassays and panels:

US5744358 4/1998 Jackowski, Method and device for diagnosing and distinguishing chest pain in early onset thereof

US5075220 12/1991 Pronovost, Determination of chlamydial or gonococcal antigen using a positivley-charged ionically binding support

US5030561 7/1991 Donahue et al., Chlamydia assay using amidine modified supports or particles

US4497900 2/1985 Abram, Immunoassay for Neisseria gonorrhoeae antigens

US4497899 2/1985 Armstrong, Immunoassay for Chlamydia trachomatis antigens

Kohler et al., "Antigen Detection to Diagnose Bacterial Infections", Boca

Raton, Florida, CRC Press, Inc. 1986, pp. 138-144. SUMMARY OF THE PRESENT INVENTION

The present method provides methods to close electronic circuitry with conducting nanowire, such as gold nanowire or CNT5 using affinity binding or other supramolecular or chemical interaction. The qualitative and quantitative analysis of DNA, proteins, glycoproteins, lipoproteins, carbohydrates, pollen, viruses, bacteria, and several other biological molecules and particles is possible with this method.

The present invention provides functionalized nanowires for the analysis of chemical and biological compounds.

It is a further object of the present invention provides devices for the home, point of care (POC) and clinical laboratory scale analysis of chemical and biological compounds.

One embodiment of this encompasses a disposable microfluidic cassette for the taking and processing of a sample, and performing the affinity binding reaction. This cassette will be inserted into a electronic measurement device that can be a hand held amperometer, or be a part some other consumer device, such as cell phone.

FIGURE CAPTIONS

Fig. 1. A. Schematic depiction of two electrodes and a nanowire that has been attached with the first electrode. B. A target DNA fragment is hybridized with the oligonucleotide probes, and the nanowire will be bound also with the second electode.

Fig. 2. A. Schematics of the nonspecific binding of the oligonucleotide probe with the first electode. B. Potential induced detachment of the nanowire from the second electrode.

Fig. 3. A. Schematics of the antibody mediated binding of the nanowire with the second electrode. In this specific example both electrodes and the nanowire are coated with molecules or particles that facilitate electron transfer. Fig. 4. A schematic depiction of a chain of nanowires between two electrodes. This arrangement corresponds capacitors that are in series.

Fig. 5. A. Schematic representation of a segment of a capillary that contains a comb-like electrodes.

Fig. 6. Schematic representation of one embodiment of the present invention. Sample is taken inside from the tip. Some reagents and filter are in the cavity 606. This specific device has eight pairs of electrodes 608, 609 and is able to perform eight assays from one sample.

Fig. 7. Schematic representation of another embodiment of the present invention that is able to perform electrophoretic sample processing. This device has T-shaped capillary that is separated with a thin membrane 704 from the analysis capillary 705. Some reagents 711 are in the sample intake capillary (702).

Fig. 8. Schematic depiction of a capillary that has parallel grids. Grids are conductively connected to outside of the capillary.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

DNA can be used to bind biomolecules, and nanoparticles also for electronic purposes (see Virtanen J and Virtanen S., USP 5,718,915) The present invention utilized these methods and methods that are presented in a co- owned patent application (Virtanen J, et al., Novel hybride materials and related methods and devices). In Fig. 1 is shown one example of the present invention for DNA analysis. A nanowire 104 is attached with the first electrode 103. The second electrode 102 and the nanowire have oligonucleotide probes 106, and 107, respectively. When a target DNA 108 is hybridized with both probes 106, and 107 (Fig. 1 B), the nanowire is bound with both electrodes 102, and 103. When a electric potential source 110 is connected with the electrodes using wire 109, a current can be measured. The current depends on the number and type of the nanowires. Nonspecific binding (Fig. 2 A) can be differentiated by reversing the potential. Negatively charged oligonucleotide probes will be repelled from the second electrode 102, and the whole nanowire will be detached. Thus, no current is observed.

Immunoassays and other affinity analysis can be performed analogously (Fig. 3). In this specific example the substrate 101 is coated with conductive particles 309, such as gold nanoparticles or cytochrome c, to increase the electric contact between the nanowire 104 and electrodes 102 and 103. The nanowire is further coated with molecules, such as tryptophan that promote the electron transfer.

Functionalization and solubilization of the CNTs

The DNA and other affinity analysis is preferably performed in water. Accordingly, the nanowires of this invention must be soluble on water, while maintaining their electrical properties. After affinity binding a good contact with the electrodes is desirable.

Well known class of solubilizing agents includes detergents, which are able to solubilize the CNTsand other nanowires into water in micellar form. Suitable detergents are sodium dodecylsulphonate (SDS), are sodium dodecylbenzenesulphonate (SDBS)3 Tween, Triton, and octadecyl trimethylammonium bromide. Because these form an electrically insulating layer around the nanowire, direct current (DC) measurements are difficult, and alternating current (AC) measurements are preferred with these detergents.

Gold nanowires can be selectively functionalized from both ends with disulfides, for example, biotin disulfide, that can further bind avidin or streptavidin (Caswell K.K., et al., J. Am. Chem. Soc. 125 (2003) 13914). Gold nanowires can be coupled into chains that are several micrometers long (ibid). If avidin is used as a coupling agent, these chains have 4 - 5 run gaps, and AC measurements must be used. Biotinylated oligonucleotides and other affinity molecules can be attached with the ends of these nanowires or chains..

We have found that the amino acid tryptophan is very good solubilizing agent that also provides good electrical contacts. Tryptophan will adsorb very strongly on the surface of the CNTs, and also gold nano wires. Tryptophan can be used in conjunction of many other chemicals, and its amino or carboxyl functionalities provide further possibilities for derivatization. Certain DNA sequences, especially (GT)n , in which n = 10- 45, solubilize CNTs very well (Zheng M, et al., Structure-Based Carbon Nanotube Sorting by Sequence-Dependent DNA Assembly, Science 302 (2003) 1545).

The currently preferred functionalization methods for CNTs are described in the co-owned patent application (Virtanen J., at al., Novel hybride materials and related methods and devices). Hydrazine will functionalize CNTs sonochemically. The reaction product can be conjugated with oligonucleotides and other affinity molecules.

The CNTs can be coupled with micro- or nanoparticles during sonication, or afterwards using the newly created functional groups. A new sonication will cut the CNTs and expose a new functionality that is determined by the reagents that are present during the cutting. Now a different micro-, nanoparticle, or molecules can be attached onto the exposed ends of the CNTs. These new moieties can be antibodies or oligonucleotidies, or some other affinity binding molecules. These polymer-CNT-biomolecule hybrids can have a wide utility in bioanalysis, because of their enhanced reactivity, and reduced nonspecific binding as compared with traditional particles.

Advantageous binding molecules that can be conjugated with the end functional groups are, for example biotin and admantane. Biotin will bind avidin or avidin analogs, and admantane binds very strongly with β- cyclodextrin. Avidin and with β-cyclodextrin in turn can be connected with a biomolecule, supramolecular component or particle, such as gold colloidal particle, polystyrene sphere, quantum dot, nanowire, or any surface, including electrode surface.

The substitutions at a given end of the CNTs can be amplified greatly by coupling first dendrimers or analogous molecules with the functional groups at that end. For example, amino functionalized end can be conjugated with carboxylic dendrimer. If the dendrimer is high enough generation, for example generation 5, only one dendrimer molecule might fit on each end. Still the number of carboxylic groups can be much bigger than the number of amino groups. The carboxylic groups might be reacted with relatively small amino dendrimer, for example generation 3. The end is now covered with very large number of amino groups, perhaps ten fold number relative to the original number of amino groups. Moreover, the purity is better, because the purity of dendrimers is easier to achieve than the purity of the CNTs.

One method of this invention involves attachment of two primers at the ends of the CNTs. Polymerase chain reaction (PCR) can be performed by standard protocols and the product observed in real time electronically.

Branching may be promoted by attaching two different affinity molecules, such as short oligonucleotides to the end of the first CNT. The other two CNTs will each have one kind of complementary oligonucleotide so that they both can bind the first CNT. This kind of use of oligonucleotides to create nanocircuitry has been described (J. Virtanen, USP 1994).

Highly sensitive, optical, or totally electronic affinity measurement of biomolecules is provided by the CNTs, which have matching pair of antibodies or oligonucleotides at opposite ends. Advantageously, the CNTs are so short that they do not form a circular structure, but instead bind on the adjacent electrodes that are 10 nm to 5000 nm apart. The affinity binding of even one CNT gives a strong electrical signal that is of the order of 1 μA, and can be easily detected.

Especially, when tryptophan, or analogous hetrocyclic molecules containing functionalities such as amino or carboxylic groups, have been solubilizing agents, it is possible to attach onto the side walls molecules, or particles. These molecules include fluorophors, polarizable molecules, affinity molecules, such as antibodies, receptors, ligands, enzymes, oligonucleotides, and oligonucleotide analogs including peptide nucleic acids. It is possible to attach thousands of molecules per micrometer of CNT. Thus, CNTs can serve as highly visible labels in bioanalysis. If biotin has been attached onto the walls of a CNT that is end conjugated with an oligonucleotide probe, the CNT can be made clearly detectable after the binding with the target and the second immobilized probe by adding avidin coated particles that are easily detectable. These particles can be colored or fluorescent particles, such as CdS quantum dots. Similarly, other affinity based analysis can be greatly amplified. Purification methods

The asymmetric CNT product contains often CNTs that have different lengths. It is desirable to fractionate the product so that the size distribution is minimized. Fractionation methods include centrifugation, electrophoresis, especially if one or both end groups are charged, dielectrophoresis, and size exclusion chromatography, or gel filtration. For electrophoresis purposes electrically charged molecules or particles can be attached onto one or both ends of the CNTs. These molecules include sulfonate, carboxylic, trimethylammonium, and other corresponding derivatives of aromatic hydrocarbons. Dendrimers provide a method to attach a large number of charged groups onto the ends of the CNTs.

Fabrication of the microfluidic system

Figs. 5 - 8 give examples of microfluidic systems that are advantageous for the present invention.

Although gold is currently preferred as an electrode material, many other conductive, semiconductive materials are possible. Examples include platinum, palladium, osmium, iridium, silver, chromium, vanadium, tungsten, copper, nickel, graphite, semiconductors, such as silicon, germanium, zinc sulfide and selenide and conductive compounds and plastics, such as polyaniline, polyacetylene, polythiophene, and polyphenylene, tetrathiofulvalene, tetracyanoquinodimethane and their derivatives. Corrosion of an electrode prevents the use of some metals like iron and aluminum. Amalgams and composite materials are often more corrosion resistant, durable, and/or conductive than any pure component alone. Example is boron doped silver, several semiconductors, and carbon composites. If an electrode is coated by a thin protective layer, such as fatty acid monolayer, almost any metal can be used including iron and aluminum. This layer can be also conductive, for example, it can be gold, amorphous carbon, graphite, fullerene, tantalum nitride, tetrathiophene carboxylic acid, redox protein, such as cytochrome c, cytochrome c oxidase, or horse radish peroxidase. Electric current can flow across thin insulating layers by tunneling, and accordingly conductivity is not mandatory.

Various geometries can be used to implement this method for bioanalysis. The simplest method involves two parallel electrodes on a flat substrate. The separation of the electrodes is slightly or clearly less than the average length of the CNTs. Currently preferred separation is 50 nm - 50 μm. Comb-like interleaved electrodes will give more surface area, and are sometimes advantageous.

The electrodes may be on two different surfaces, and form a capacitor-like structure. The sample flows between the capacitor plates. In this implementation the CNT will bridge the capacitor plates making the capacitor leaky. Either the current or the leakage may be measured.

The electrodes are preferably in a nano- or microfluidic system. They may be in a capillary or cavity. The electrodes may in a single capillary, but they may also be in an intersection, in which flow can be arranged in two different directions, for example, perpendicularly against each other (Fig. 13). First the sample flows into one direction, and then the wash solution flows into another direction. Loose CNTs will be washed away. The CNTs that are bound only from one end will change their direction with the flow. Only those CNTs, which are bound onto both electrodes will stay between two electrodes all the time, and give a constant current.

The device of the present invention can be manufactured by several means. In mass production molding is a preferred method. Machining, laser, and water jet cutting are sometimes advantageous. Photolithographic method, in which a resist layer defines microfluidic structures is sometimes preferred, especially for small scale production. Methods that are better for mass production are given in Examples 1 and 2. Electrodes can also printed by screen-printing technology. For example, commercially available carbon ink paste and silver/silver chloride ink are suitable materials for working, counter, and reference electrodes. Other printing or stamping techniques, such as ink jet printing can be used.

Attachment of recognition molecules onto the electrodes

Molecules can be bonded onto the surface by physical or chemical means. When a voltage, resistance, or current through a nanowire between two electrodes is to be measured, the nanowires should be as close as possible to the electrodes. Accordingly, the recognition molecules should be either bonded directly onto the surface or connected with relatively short spacers. Electric current can easily flow over a thin monolayer by tunneling mechanism. Longer spacers can be used, if they are electrically conductive. For instance, double helical oligonucleotides are electrically conductive and can be used as spacers. Compounds like tetrathiophene carboxylic acid are also conductive enough to be useful spacers. Small conductive particles, such as 1 - 100 nm gold spheres can be coated with recognition molecules and bound with the surface. The same or even better result can be achieved by having an electrode surface that has 1 - 100 nm roughness. Currently 10 - 20 nm surface roughness is preferred. Sputtered metal surface has typically this kind of roughness.

Although metal surfaces are often polycrystalline, single crystal surfaces may sometimes be preferable. For instance, pyridine has highest affinity for the gold (210) surface of all possible gold surfaces, while it has the lowest affinity for (111) surface. In general, positively charged surfaces have higher affinity for electrically neutral molecules than negatively charged surfaces. The problem with high positive potential is that some important classes of compounds, such as mercapto compounds, will be oxidized and detached from the surface.

Spacers, recognition and other molecules can be bonded onto the surface by dispersion forces, hydrophobic force, hydrogen bonds, charge transfer, ionic or covalent bonds. Covalent bonds are strongest and most stable in hydrated milieu. Dispersion forces, such as van der Waals force, can be significant if a molecule is large and has tens of interactions, which combined can be comparable to a covalent bond.

Molecules can be adsorbed either from gaseous or liquid phase onto the surface. Laser ablation allows the evaporation of quite large molecules, such as peptides and oligonucleotides. However, proteins and oligonucleotides are preferably adsorbed from a buffer. Mercapto, amino, isonitrilo, carbonyl and carboxylate groups form bonds with various metals. Sulfur atoms interact especially strongly with gold and also with other noble metals. Organic mercapto compounds can also be dissolved into organic solvents, such as ethanol, and they will spontaneously form a self-assembled monolayer. Also ether oxygen binds with gold, although much more weakly than sulfur. Polymers containing many ether oxygens, such as polyethylene glycol will still bind strongly enough. Amphiphilic molecules form a monolayer at water-air interface. This monolayer can be compressed in a controlled way so that molecules occupy a desired average area. The monolayer can be deposited onto a solid surface such as an electrode.

After the recognition and other molecules have been deposited, they may be surrounded by a liquid, liquid crystalline, or solid matrix to increase their stability. The matrix is preferably such that it will be dissolved by a sample. It is also possible to add a special buffer or some other solvent before or during the assay to expose the recognition molecules. Well known stabilizers are, for example, trehalose, glucose, glycine, glycerol, dextran, cyclodextran, starch, polyvinyl alcohol, and polyethylene glycol. Surfactants may be added to speed up the hydration. Surfactants include tween-20, octyl glucoside, sodium dodecyl sulfate, sodium palmitate, potassium oleate, sodium cholate, trimethyl octadecyl ammonium bromide, and phospholipids, such as dihexanoyl phosphatidyl choline, l-palmitoyl-2- oleoyl-sn-3 -phosphatidyl choline, and l,2-dilinolenoyl-sn-3-phosphatidyl ethanolamine. Various salts, such as sodium chloride, sodium phosphate, sodium acetate, potassium lactate, sodium citrate, calsium chloride, and magnesium chloride, may be a part of the formulation. Combinations of these and other compounds can be used to reduce crystallization. These compounds are often solubilized into water, and the water solution is applied by a pipette, ink jet printer, or by pins on to the surface. Water is best removed by lyophilization.

Similarly, nanowires may be enclosed inside a solid matrix that is soluble into water. If the nanowires are stored inside the microfluidic system, the matrix prevents the denaturation of the affinity molecules that are bound with the nanowires. It also prevents the sticking of the nanowires with the walls of the microfluidic system.

Oligonucleotides may be terminated by amino, mercapto, biotinyl, and several other groups. There may be several amino or other groups as well as combinations of various groups. To give more freedom for oligonucleotides these groups, which are intended to bind with a substrate, can be separated from the actual oligonucleotide sequence with a spacer. Spacer is preferably water soluble polymer, such as polyethylene glycol, polyvinyl alcohol, polyacrylic acid, or optionally a copolymer of two or more hydrophilic and hydrophobic monomers.

Sample preparation and fractionation

The analytes can be fractionated by various chromatographic methods, including, affinity, size exclusion, ion exchange, adsorption, and reverse phase chromatographies. Electrophoresis is another well known technique for the separation of biomolecules. All these methods are well known in the art.

Ionic and electronic current

Immediately after a potential is coupled between electrodes there will both ionic and electronic current. In an example described here, it is supposed that the electrodes form an isolated cell, and that the electrodes have a constant charge at the beginning and that this charge does not change during the ionic current. The Nernst-Planck equation gives the flux of any ionic or molecular species in the electric field.

ox RT ox

Each ionic species may have different diffusion constant Dh charge zh and concentration Cj(x,i). Each has an effect on the potential φ(x,t). Thus, an analytic solution is in most cases very difficult or even impossible and the equations must be solved numerically.

The diffusion coefficient is usually between 10"5 and 10"6 cm2 /s. This means that the molecules and ions will diffuse from one electrode to another in about one second or less, because of very small distance between the electrodes. The electric field induced transport is faster for any significant potentials. Based on this qualitative inspection, it is possible to deduct that the ionic current will last only a very short time and an equilibrium is established. This corresponds to a charging of a capacitor. After charging, no ionic current should flow, provided that no electrochemical reactions are happening on the electrode surfaces.

Bioanalysis

Electromagnetic field may be used to assist affinity binding. Electric potential may be constant or alternating. In many cases a combination of static and alternating field is preferred. For example, when DNA is hybridized with the probes on the electrodes, positive potential will pull DNA towards the electrode. However, the electrode will be saturated fast with negative ions, and an alternating field that is superimposed on the static field will help to release the fast moving smaller ions back into the bulk solution, and DNA will be concentrated onto the electrode surface. The flow near the surface is slow, and it is desirable that the DNA is pushed back into the solution after a short incubation time. Then the static potential will be changed negative.

The electrodes can be coated by many methods that are well known in the art. One currently preferred method is to use thiol-gold binding. Another currently preferred method is to coat the electrode with cytochrome C either by direct adsorption or by conjugating with a self-assembled monolayer, such as 6-mercaptohexylamine, or 11-mercaptoundecanoic acid layer using amide bond. Aliphatic amino or carboxyl functionalized biomolecules can be further conjugated with cytochrome C.

The immobilization of the CNT from one end is enough for the observation of the current, and especially fluorescence, or luminescence. If the target DNA is not amplified, or chemically modified, two probes are needed for the binding, one on the substrate (electrode), and one at the end of the CNT. These two binding events that must occur simultaneously give high fidelity. The conductance, and also optical detection, can be made independent of the direction of the flow, if both ends of the CNT are bound with the substrate. The tight immobilization may require four probes, and the fidelity is extremely high.

Also electric potential may be used to lift up or pull down the unbound end of the CNT. This kind of system works like a diode. The current will flow, when the potential under the unbound end of the CNT is positive, and it will not flow or is very weak, when the potential is negative, because the negative electrode will repel the oligonucleotide probes that are attached with the CNT. This method can be extended into the detection other kinds of affinity binding reactions. Either the binding molecules should be charged, or some other charged molecules or particles are attached at the end or near the end of the CNTs (Fig. 2).

One embodiment of the present method is to coat one electrode permanently with nanowires that can be detached form the other electrode by the perpendicular flow or negative electric potential. When the loose end is bound with the target onto the electrode, the flow or moderate electric potential will not have any disruptive effect. In still another embodiment the nanowires 404 that are bound with the electrode(s) 402 and 403 are not long enough to bridge the gap between the electrodes (Fig. 4). The loose ends, or an end and a wall, or wall and wall are bound together with a target 405 at least in one joint so that a conducting path is created. Some connections can be made by other means, for example, biotin-avidin binding.

A device construction that enables the analysis of large volumes, up to several milliliters, consists of a tube that has a diameter between 0.1 mm and 10 mm, and grids 802 and 803 that are perpendicular to the direction of the flow (Fig. 8). Grids can be transmission electron microscope grids without any membrane. The affinity molecules are attached onto the grids. The spacing of the grids is such that the nanowires 804 are able to create a contact between them. Currently, it is possible to fabricate CNTs that are several millimeters long. Thus, the spacing of the grids can be anything between 100 nm and 10 mm. The spacing requires spacers that can be dielectric nano- or microparticles, such as polystyrene spheres.

In another embodiment of the present invention the binding molecules(s) are located in the gap between two electrodes. Matching binding molecules are attached onto the side walls of the nanowires. When an analyte is present, it will bind with the pair of binding molecules and the nano wire will be immobilized between the electrodes. The nanowire must be long enough so that it can still be in contact with both electrodes. Thus, an analyte will induce the closing of the circuit.

Immunoassays

Antibody-antigen interaction can be used in many different ways either to bind or prevent the binding of nanowires onto a surface of an electrode. In a traditional sandwich type assay one member of a matching antibody pair is bound on to nanowire 307 and the other on to an electrode 306. The corresponding antigen 308 will form a bridge between two antibodies and bind the conductive particle on to the surface of the electrode as is depicted in Fig. 3. In this specific example the electrodes are coated also with conductive particles or particles that assist electron flow 309. The nanowire can similarly be coated with particles 310 that advantageously assist electron flow and solubilization of nanowires (see also Example 1 and 5). Most proteins can be assayed by sandwich assay. A cleavable spacer provides another way to perform a sandwich assay (Virtanen, 1996). The number of the bound conductive particles is directly proportional to the concentration of the antigen. For smaller molecules, such as steroids and several drugs, the competitive assay is the method of choice. The antibody is attached, for example, on to the nanowire, and the antigen or an analog of the antigen is attached onto the electrode. The antigens in the sample saturate the antibody molecules on the conductive particles in a concentration dependent manner. The competitive method is not equally sensitive as the sandwich assay, because zero and very small concentrations of the antigen gives maximum or near maximum binding. Small changes are very difficult to differentiate from the normal experimental error.

Currently the preferred electrode material is gold. The gold surface can be first coated with streptavidin, which will adsorb spontaneously from an aqueous solution. Biotinylated antibody will bind with steptavidin providing a good coating. Several other ways of attaching of antibodies onto the gold surface are well known in the art. These include forming a monolayer of polylysine or copolymer of lysine and cysteine on to the gold surface, and attaching periodate oxidized antibody in the presence of sodiumcyano- borohydride on to this monolayer. The simplest way is to reduce the antibody with dithiotreitol or with some other reductant and let the reduced antibody to chemisorb directly on to the gold surface. This is currently the preferred method, because the insulating organic layer is very thin, having a thickness of only half of an antibody molecule.

A wide variety of immunoassays can be performed with the present method. Nonlimiting examples include hCG (pregnancy test), prostate specific antigen (PSA) detection, insulin, proinsulin, glucagon, glycated hemoglobin, growth hormone, fetoprotein, TSH5 C-reactive protein, CK-MB, myoglobin, troponin, interferons, interleukins, ferritin, tumor negrosis factor, trypsin, plasminogen, cardiolipin, Cortisol, aldosterone, estradiol, digoxin, benzodiazepine, vancomycin, amphetamine, cocaine, morphine, tetrehydrocannabinol, phenobarbital, secobarbital, parathione, adenovirus, chlamydia, cytomegalovirus, hepatitis viruses, HIV, influenza, and parainfluenza.

DNA testing DNA tests are analogous to sandwich immunoassays as is depicted in Fig. 1. The present invention allows testing of tens or even hundreds of thousands of sequences simultaneously. The main large-scale application of oligonucleotide arrays is comparative expression analysis. Gene expression patterns in healthy as well as in diseased tissues and cells will greatly increase the understanding of the function of living organisms. The effect of drugs can be understood in much more detail than presently. Another application for the oligonucleotide arrays is the finding of single nucleotide polymorphisms (SNPs). It is estimated that in human genome one out of a thousand nucleotides is polymorphic. These SNPs are the main reason for human diversity. Once the SNPs have been characterized and correlated with certain disease states, SNPs can be used to predict an individuals tendency for many diseases including various cancers (Cole et al. (1999) The genetics of cancer-3D model, Nature Genetics Supplement 21: 38-41), heart disease (Lusis (2000) Atherosclerosis, Nature 407: 233-241), and alzheimers disease. Another example of DNA diagnostics is the detection of fetal DNA in maternal plasma (Lo (2000) Clinical Chemistry 46:1903- 1906). DNA and RNA studies are not limited to humans. Plant genomics has enormous economical importance. Knowledge of pathogen genes and gene expression can be used in diagnostics and for the design of new drugs.

Oligonucleotide probes can synthesized so that they contain aliphatic amino, mercapto, or other groups. They will bind spontaneously on to an electrode surface. Mercapto groups have a drawback that they tend to be oxidized by the positive electrode (anode). The positive charge is sometimes used to attract sample DNA to the close proximity of the electrode. According to the present invention, nanowires and not the analytes themselves are attracted to the electrode. The charge of the particles can be opposite to the charge of an analyte. Even, when nanowires are partially covered by negatively charged oligonucleotide probes, which further bind negatively charged targets, the nanowires can be positively charged or electrically neutral. Thus, it is possible to use a negative charge to attract these nanowires. In addition to stabilizing the sulfur-gold bond, the negative charge repels the oligonucleotide probes on the surface of the anode. The probes have 5 to 10,000 nucleotides, and preferably 10 - 80 nucleotides. Long probes have generally genomic origin. The target is often a PCR product. Other amplification methods are equally possible, including isothermal and ligation amplification. The probes should have preferably about the same length as the amplicons. Hybridization conditions are well known in the art. Temperature should preferably be 20 0C below the melting temperature of the dublex. The salt concentration has a very big effect on the melting temperature and kinetics of the hybridization. The melting temperature depends also on the guanidine and cytidine contents of the probes and the target. A higher salt concentration in the buffer and higher C/G-content will increase the melting temperature. The electric current will increase the temperature of the elecrolyte. The power input is P=UI, where U is the potential and I is the current. The increase in the temperature is ΔT=UIt/Cm, where t is the time and C is the thermal capacity of the medium of a mass m. A certain thin layer of the walls must be included into the mass m. The method of the present invention is less sensitive to the external conditions than the most currently used methods. This is due to the electric field that can be utilized to increase the rate of the hybridization as well as to test the stringency of the hybridization.

Polymerase and ligase chain reactions as well as isothermal amplification can be performed in the device of this invention. The temperature cycling (not for isothermal) can obtained by electric potential induced heating between the electrodes or by embedding heating elements near the electrochemical cells. The heating is more effective, if instead of a direct current, an alternating current is used.

Oligonucleotide analogs, such as peptidenucleotide acids (PNAs)5 and thionucleotide acids, offer often increased stability and/or stringency of hybridization.

Oligonucleotide probes may be prehybridized with complementary oligonucleotides. The purpose of this kind of array is to study interaction of biomolecules with double helical DNA.

Measurement of the electrical properties

Electrical power may be provided by a battery (Galvanic cell), solar cell, electromagnetic radiation, magnetic induction, direct contact with external power source, or by any other commonly known means.

Almost any electrical measurement device that is able to measure either voltage, current, capacitance, inductance, impedance, or phase shift can be a part of the present invention (AJ. Bard and L.R. Faulkner "Electrochemical Methods: Fundamentals and Applications", Wiley, New York, 1980).QD553.B37 The coupling of these devices to the processing units and to the networks is well known in the art. Examples can be found in several books, including JJ. Barbarello "PC Hardware Projects, Volume 3" (Prompt Publications, Indianapolis, 1998), WJ. Tompkins and J.G. Webster "Interfacing Sensors to the IBM PC" (Prentice Hall PTR, Englewood Cliffs, 1998), S. McDowell and M.D. Seyer "USB Explained" (Prentice Hall, Upper Saddle River, 1999), and J. Axelson "Serial Port Complete" (Lakeview Research, Madison, 1998). Networks include Local- Area- Networks, and Internet. Networks can utilize among others, metal cables, fiberoptics, or be wireless.

EXPERIMENTAL DETAILS

While this invention has been described in detail with reference to certain examples and illustrations of the invention, it should be appreciated that the present invention is not limited to the precise examples. Rather, in view of the present disclosure, many modifications and variations would present themselves to those skilled in the art without departing from the scope and spirit of this invention. The examples provided are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to limit in any way the present invention.

Example 1.

A glass slide was spin coated with 2 % 950 kD polymethyl methacrylate (PMMA) in chlorobenzene by spinning 500 rpm 40 seconds. The slide was baked at 200 0C for 2 minutes. Electrode patterns (Fig. 5) were written by 10 kV e-beam. The current was 200 μC/cm2. The pattern was developed with methyl i-butyl ketone/i-propanol (MEKyTPA)1 :3 mixture for 30 seconds, and rinsed with IPA for 20 seconds, and finally cleaned with oxygen plasma (30 W5 200 mT) for 15 seconds. A solution of thiol terminated oligonucleotide was placed on the wide part of one electrode. The solution spread spontaneously along the hydrophilic surface (after the plasma treatment). A solution of cytochrome c was placed similarly onto the other electrode.

The top piece was molded from silicone. The mold was made by gluing a 50 μm thick and 1 mm wide aluminum foil onto the slide. The slide was covered with silicone. When the silicone was hard, it was removed from the molding slide, and the glass slide that had the electrodes was placed on tip of it. The external wires were connected with conducting clue onto the exposed parts of the electrodes.

In this example the separation of the electrodes was 200 nm. The analogous procedure, in which photolithography was used, gave electrodes that were separate 2 μm.

Example 2.

CNTs (10 mg, purity about 80 %) were suspended with 10 ml of THF. Hydrazine solution (1ml, IM) in THF was added. The vial was closed under nitrogen. The contents were kept in ultrasonic bath for 6 h. The THF turned almost black, and most of the CNTs had dissolved. The magnetic impurities were removed by keeping the vial close to a permanent magnet, and pipetting the THF into another vial. Succinic anhydride (1 mmol, 100 mg) was added, and the mixture was stirred. The reaction mixture was added into 30 ml of 0.5 % tween in water (alternatively poly d(G/C)). The mixture was dialyzed againstθ.05 M 2-N-morpholino ethane sulfonic acid (MES) buffer. The 0.1 M solution of l-ethyl-3,3- dimethylaminopropyl carbodi- imide-HCl (EDC), and 0.1 μmoles of 20-mer aminoterminated oligonucleotide probe was added. The mixture was dialyzed against PBS buffer.

Example 3.

CNTs (10 mg, purity about 80 %) were suspended with 10 ml of THF. The solution was saturated with hydrogen sulfide and kept in ultrasonic bath for 4 hours. The solvent was evaporated and hydrazine solution (1ml, IM) in THF was added. The contents were sonicated for one hour with tip sonicator under nitrogen. The THF turned almost black, and most of the CNTs had dissolved. The magnetic impurities were removed by keeping the vial close to a permanent magnet, and pipetting the THF into another vial. Thiol-hydratzino-CNTs were separated by sentrifugation, and dispersed into THF. Vinylsulfone-PEGcarboxylic acid (2mg) was added. After 1 hour SMB-PEG-SMB (10 mg; Nektar Inc., Alabama, USA) and triethylamine (1 mg) were added. The product was separated by centrifugation, and suspended into 0.1 % tween in water that contained 10 μmoles of amino terminated oligonucleotide probe. After 24 hour dialysis EDC was added and the solution was applied onto the cytochrome c coated electrode of the device in Example 1. When femtomolar solution of complementary oligonucleotide was added into the capillary, the current was 1.2 μA.

Example 3.

The product from Example 2 was treated with 10 mg of bis(N- hydroxysuccinimide)polyethyleneglycoldicarboxylate (MW 5,000, Nektar, Inc., Alabama) in 2 ml of phosphate buffer, pH 7.5. After one hour the CNTs were washed with water/methanol. The free carboxylate groups were reactivated by 5 mg N-hydroxysuccinimide, and 10 mg EDC, and aminohexyl-T16 oligonucleotide was added. After one hour the mixture was washed with water/methanol, and centrifuged. Tween-60 (1 %) in water was added, and the mixture sonicated. The solution was used as such.

Example 4.

CNTs (10 mg, purity about 80 %), and L-tryptophan (20 mg) were suspended with 10 ml of water. The mixture kept in ultrasonic bath for 8 hours, and further treated with a tip sonifier (300 W) for 15 minutes. After centrifugation at 5000 rpm for 10 minutes the supernatant was pipetted into another test tube. Fluorescein isothiocyanate (40 mg) in 1 ml of ethanol was added. The mixture was dialyzed three times against 200 ml of water. Imaging with confocal microscope showed that the CNTs were coated with fluorescein.

Example 5.

Into hCG antibody solution (3 mg in 1 ml of PBS) was added 0.1 mg biotin- PEG-NHS (Nektar, Alabama, USA). This solution was added after one hour as such into excess of gold nanorods (20 mg) that were coated with avidin on both ends. The antibody-gold rod conjugates were separated by gel filtration. The elution was monitored by the absorption at 280 nm and 540 run. These nanorods were bound onto an electrode that was coated with biotinylated cytochrome c (see Example 1). Femtomolar solution of hCG was put into the capillary. The current was 2 μA.

Claims

Claims:
1. An analytical method known for the utilization of specific binding reaction between an analyte and a chemically derivatized nanowire and electrode to close an electronic circuitry so that a change in the electronic properties of the said circuitry depends on the presence of the said analyte.
2. A method of claim 1 , in which the said analyte is DNA5 and the said nanowire and electrode are derivatized with a first oligonucleotide and second oligonucleotide and the said specific binding reaction is hybridization of DNA with the first and second oligonucleotide.
3. A method of claim 1, in which the said analyte is an antigen, and the said nanowire and electrode are derivatized with a first antibody and second antibody and the said specific binding reaction is between antigen and the first and second antibody.
4. A method of claim 1 , in which the said nanowire is coated with tryptophan derivative, and the said electrode is coated with cytochrome c.
5. A device for the detection of specific binding reaction known for the closing of the electronic circuitry with nano wires using the said specific binding reaction of an analyte in a micro- or nanofluidic system.
6. A device of claim 1, in which the said nanowire is attached from the first end with the first electrode, while the second end is chemically connected to the first affinity molecule, and the second affinity molecule is chemically connected with the second electrode, and the first and second affinity molecules form a binding pair for the said analyte.
7. A chemically modified nanowire for the method of claim 1.
8. A nanowire of claim 6, which is a carbon nanotube that is chemically connected with an affinity molecule.
9. A nanowire of claim 6, which is a gold nanowire that is chemically connected with an affinity molecule.
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CN103950891A (en) * 2014-05-21 2014-07-30 江南大学 Preparing method of high conductivity chain-shaped photoelectric nanometer bridge
CN104075811A (en) * 2014-05-14 2014-10-01 电子科技大学 THz detection structure and manufacturing method of high-TCR absorption sensitive composite film

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CN104075811A (en) * 2014-05-14 2014-10-01 电子科技大学 THz detection structure and manufacturing method of high-TCR absorption sensitive composite film
CN103950891A (en) * 2014-05-21 2014-07-30 江南大学 Preparing method of high conductivity chain-shaped photoelectric nanometer bridge

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