WO2005108612A2 - Nanoscale biosensor device, system and technique - Google Patents

Abstract

The present invention provides biosensors for the detection of nucleic acids, such as double stranded DNA. The biosensors are electrodes on a solid support that have means for binding nucleic acids near the electrodes. The nucleic acids are captured such that they span the electrode pair, and the capture can be detected by electrical means.

Description

Nanoscale Biosensor Device, System and Technique

RELATED APPLICATION DATA

[0001] This application claims the benefit of United States provisional patent application number 60/525,697 filed on November 28, 2003, which is herein incorporated by reference in its entirety.

FIELD OF INVENTION

[0002] The present invention relates to biosensors for detecting and/or quantifying analytes.

BACKGROUND

[0003] Biosensors are devices that can detect and/or quantify analytes using known interactions between a targeted analyte and a binding agent that is typically a biological macromolecule such as an enzyme, receptor, nucleic acid, protein, lectin, or antibody. Biosensors are categorized by the type of macromolecule utilized and the means for detecting the contact between the binding agent and the analyte. Major classes of biosensors include enzyme (or catalytic) biosensors, immunosensors, and DNA biosensors. Biosensors have been used for monitoring blood glucose levels, for the detection of poisonous gases and explosive, for genetic screening, for environmental testing, and the like.

[0004] DNA biosensors typically utilize the complimentary nature of the DNA or RNA molecules, and are designed for the specific detection of the particular nucleic acids. A DNA biosensor generally uses a single-stranded DNA as the binding agent. The nucleic acid material in a test sample is placed into contact with the binding agent under conditions where the biosensor DNA and the target nucleic acid analyte can form a hybrid duplex if they are complementary to each other. The interaction can be monitored by mass spectroscopy, by optical means, such as fluorescence, by detecting a radioactive signal, or other means.

[0005] DNA biosensors have been used for the identification of single base differences in gene sequences between individuals (single nucleotide polymorphisms or "SNPs") and for gene expression analysis. SNPs play an important role in predisposing individuals to diseases, such as cancer, and in determining the efficacy and the toxicity of therapeutics. Gene expression analysis can provide indispensable insights into the biology of diseases and the genetic changes associated with disease progression. Gene expression patterns can also serve to identify new targets for therapeutic intervention. These types of information find use as diagnostic markers for diseases, and as prognostic markers of disease outcome and treatment response.

[0006] Similarly, expression of human and pathogen genes can identify potential markers of infection and targets for therapeutic intervention. Many of the most common biowarfare agents, such as smallpox and anthrax, elicit initial symptoms in humans that might be difficult to distinguish from those of other, non-lethal infections. However, early detection in the infection cycle is often critical to patient recovery and to managing the spread of the infection in the population, and a need exists for rapid and accurate techniques for detecting such agents.

[0007] A recent technique for SNP detection, gene expression analysis, detection of pathogens, and the like, is the DNA microarray. DNA microarrays are generally fabricated on solid supports, such as glass, plastic, gel, or silicon. On these solid supports, nucleic acid probes are selectively spotted or addressed to specific locations. The target nucleic acid is amplified and tagged with a fluorescent label and then applied to the micro array. The micro arrays are then imaged using optical detection tools. These existing technologies suffer three major drawbacks:

1) Extensive sample preparation and technical skill required; 2) Sample contamination 3) High cost of instrumentation and disposables; and 4) Royalty costs 5) Poor scalability of platform

[0008] Thus, there is a need for biosensors for the detection and/or quantification of analytes that are accurate, convenient, and inexpensive.

SUMMARY

[0009] The present invention provides methods for detecting an analyte in a sample.

The methods comprise providing a biosensor comprising a solid support and an electrode pair, wherein the electrode pair comprises a first electrode and a second electrode wherein at least one of the first electrode and the second electrode comprises a primary probe; contacting the biosensor with a sample suspected of comprising the analyte wherein, if present, the analyte specifically binds with the primary probe; spanning the electrode pair with a double stranded nucleic acid that completes an electric circuit between the electrode pair if the sample comprises the analyte; and measuring an electromagnetic property of the electrode pair, wherein the electromagnetic property indicates the presence of the analyte in the sample. The solid support can be plastic, glass, silicon, silicon oxide, silicon dioxide, mica, quartz, or combinations thereof. The first electrode and the second electrode can be independently selected from a material consisting of aluminum, cadmium, cesium, chromium, cobalt, copper, germanium, gold, indium tin oxide, iridium, iron, lithium, molybdenum, nickel, osmium, palladium, platinum, potassium, rhodium, rubidium, ruthenium, silicon, silver, sodium, titanium, tungsten, vanadium, or zinc. The primary probe can be DNA, PNA, RNA, or combinations thereof.

[0010] The invention also provides methods for detecting a double stranded nucleic acid in a sample. The methods comprise providing a biosensor comprising a solid support and an electrode pair, wherein the electrode pair comprises a first electrode and a second electrode wherein at least one of the first electrode and the second electrode comprises a means for attaching a double stranded nucleic acid; contacting the biosensor with a sample suspected of comprising the double stranded nucleic acid, wherein, if the sample comprises the double stranded nucleic acid, the double stranded nucleic acid attaches to the attachment means; spanning the electrode pair with the double stranded nucleic acid and completing an electric circuit between the electrode pair if the sample comprises the double stranded nucleic acid; and measuring an electromagnetic property across the electrode pair, wherein the electromagnetic property indicates the presence of the double stranded nucleic acids in the sample.

[0011] The invention also provides methods for detecting a double stranded nucleic acid in a sample. The methods comprise providing a biosensor comprising a solid support and an electrode pair, wherein the electrode pair comprises a first electrode and a second electrode wherein at least one of the first electrode and the second electrode comprises a primary probe; contacting the biosensor with a sample suspected of comprising the target nucleic acid and a hybridization solution wherein if the sample comprises the target nucleic acid, the target nucleic acid hybridizes to the probe; and providing the means for performing said target amplification.

[0012] These and other aspects of the present invention will become evident upon reference to the following detailed description. In addition, various references are set forth herein which describe in more detail certain procedures or compositions, and are therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 illustrates a schematic depiction of a nanoscale biochip for the electronic detection of nucleic acids.

[0014] Figure 2 illustrates a current-voltage curve of gold electrodes spaced 30-40 nm apart. The sigmoidal lines represent the current observed upon application of dithiolated double-stranded DNA. Horizontal lines represents small residual current observed after DNasel treatment of biochip.

[0015] Figure 3 illustrates an aspect of the device and its capacitance current. Figure 3 A illustrates a scanning electron microscope of a gold electrode nanogap structure, and Figure 3B illustrates a current- voltage curve of the blank chip. The current- voltage curve shown in panel B represents the capacitance current of the device.

[0016] Figure 4 illustrates atomic force microscope (AFM) images of human genomic

DNA bound to APTES-treated mica. In Figure 4A, the mica is without DNA, and in Figure 4B, genomic DNA is directionally applied to the mica. The images were obtained using the NanoR AFM from Pacific Nanotechnology Inc.

[0017] Figure 5 illustrates AFM images of nanogap devices and corresponding electrical signal. Figure 5 A is an AFM image of double-stranded DNA laying across nanogap. Figure 5B is an AFM image of the chip of Figure 5 A treated with DNase (notably, the arrow indicates reference landmarks). Figure 5C illustrates electrical data of chip before DNase treatment (as in Figure 5A) and after DNase treatment (as in Figure 5B).

[0018] Figure 6 is a structural diagram representation of Fmoc-aminoheaxanethiol. [0019] Figure 7 illustrates gold surfaces covered by an Fmoc layer and bound to DNA.

In Figure 7A, lambda DNA is bound, and in Figure 7B, 1.5kb DNA is bound to the Fmoc layer.

[0020] Figure 8 is a schematic representation of a DNA attachment via base stack intercalator/molecular wire complex.

[0021] Figure 9 is a structural diagram of ethidium molecular wire.

[0022] • Figure 10 provides AFM images of double-stranded DNA on an ethidium- molecular wire/hexanethiol mixed monolayer, where DNA is deposited from a solution containing 100 mM MgCl₂ (Figure 10A) and 500 mM MgCl₂ (Figure 10B).

[0023] Figure 11 is a schematic representation of vertical flow cell for the directional deposition of DNA solutions. The DNA solutions are pumped to the fluid delivery port by a syringe pump and form a receding meniscus as the pump withdraws the fluid. The receding meniscus is the force that stretches the DNA across the gold electrode nanogap.

[0024] Figure 12 illustrates the specific detection of the target molecule. The probe on the electrode hybridizes with the target DNA, a secondary probe hybridizes with the target DNA at a different location, and the secondary probe and ds DNA become connected through a connector molecule.

[0025] Figure 13 illustrates the probe on the electrode hybridizes with the target DNA, a secondary probe hybridizes with the target DNA at a different location, and the secondary probe can be connected with the ds DNA via a ligated general sequence that can hybridize with the secondary probe and with the ds DNA.

[0026] Figure 14 illustrates the assay where the electrical connection is completed by ds

DNA attached to nanoparticles.

[0027] Figure 15 illustrates the assay where the electrical connection is completed by ds

DNA attached to nanoparticles, and the connection at one of the electrodes is through the nanoparticles. [0028] Figure 16 illustrates the assay where the probe on the electrode hybridizes with the target DNA, and a polymerase is used to extend the probe to provide a ds DNA that can span the electrode pair to complete the electrical connection.

[0029] Figure 17 illustrate the assay where the electrical connection is completed by microspheres having oligonucleotides complementary to the target or the secondary probe.

[0030] Figure 18 illustrates the assay where the electrical connection is completed by two or more microspheres where the oligonucleotides on the second microsphere can only hybridize with the oligonucleotides on the first microsphere.

[0031] Figure 19 shows the assay specific for the target being accomplished off the biosensor, and the biosensor being used for detection only.

DETAILED DESCRIPTION

I. Definitions

[0032] Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Definition of standard chemistry terms may be found in reference works, including Carey and Sundberg (1992) "Advanced Organic Chemistry 3^rd Ed." Vols. A and B, Plenum Press, New York. The practice of the present invention will employ, unless otherwise indicated, conventional methods of synthetic organic chemistry, mass spectroscopy, preparative and analytical methods of chromatography, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art.

[0033] The term "biosensor" refers to a sensor that uses a biological macromolecule (e.g. nucleic acid, carbohydrate, protein, antibody, etc.) to specifically recognize/bind to a target analyte.

[0034] The term "nucleic acid" as used herein refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as compared to a reference nucleic acid. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36: 1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompasses by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, rRNA, tRNA, oligonucleotide primer, probe and amplification product.

[0035] As used herein, the terms "label," "detectable label," and "reporter molecule" refer to a molecule capable of being detected, including, but not limited to, radioactive isotopes, electrochemical tags, fluorescers, chemiluminescers, chromophores, magnetic resonance agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin, avidin, strepavidin or haptens) and the like. The term "fluorescer" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable wavelength range. Suitable fluorescers include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), rhodamine, 5-(2'- aminoethyl) aminonapthalene-1-sulfonic acid (EDANS), anthranilamide, coumarin, terbium chelate derivatives, Reactive Red 4, BODIPY dyes, cyanine dyes, Alexa 488, fluorescein iso- thiocyanate (FITC), Cy3, Cy5, PE, Texas Red, Cascade Blue, TMR and tetramethyl rhodamine isothiocyanate (TRITC).

[0036] The term "antibody," as used herein, includes, but is not limited to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). "Antibody" also includes, but is not limited to, a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an antigen. Examples include polyclonal, monoclonal, chimeric, humanized, and single chain antibodies, and the like. Fragments of immunoglobulins, include Fab fragments and fragments produced by an expression library, including phage display. See, e.g., Paul, Fundamental Immunology, 3^rd Ed., 1993, Raven Press, New York, for antibody structure and terminology.

[0037] The terms "specifically binds to" or "specifically immunoreactive with" refers to a binding reaction which is determinative of the presence of the target analyte in the presence or absence of a heterogeneous population of proteins and other biologies. Thus, under designated assay conditions, the specified binding moieties bind preferentially to a particular target analyte and do not bind in a significant amount to other components present in a test sample. Specific binding to a target analyte under such conditions may require a binding ' moiety that is selected for its specificity for a particular target analyte. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an analyte. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically a specific or selective reaction will provide a signal to noise ratio at least twice background and more typically more than 10 to 100 times background.

[0038] As used herein, a "solid support" refers to a surface such as a plastic plate, magnetic bead, latex bead, microtiter plate well, glass plate, nylon, agarose, acrylamide, SiO2, porous material matrix and the like.

[0039] "Specific" in reference to the binding of two molecules or a molecule and a complex of molecules refers to the specific recognition of one for the other and the formation of a stable complex as compared to substantially less recognition of other molecules and the lack of formation of stable complexes with such other molecules. Additional hallmarks of specific binding include saturability and competability. Exemplary of specific binding are antibody-antigen interactions, enzyme-substrate interactions, polynucleotide hybridizations and/or formation of duplexes, cellular receptor-ligand interactions, and so forth. [0040] The phrases "hybridizing specifically to" or "specific hybridization" or

"selectively hybridize to," refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[0041] The term "stringent conditions" refers to conditions under which a probe will hybridize preferentially to its target sequence, and to a lesser extent to, or not at all to, other sequences. "Stringent hybridization" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I chapter 2 Overview of principles of hybridization and the strategy of nucleic acid probe assays, Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5 °C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T_m for a particular probe.

[0042] An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42 °C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72 °C for about 15 minutes. An example of stringent wash conditions is a 0.2 X SSC wash at 65 °C for 15 minutes (see, Sambrook et al (1989) Molecular Cloning-A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.) supra for a description of SSC buffer). Often, a high stringency wash is preceded by a medium or a low stringency wash to remove background probe signal. An example of a medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1 X SSC at 45 °C for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6 X SSC at 40 °C for 15 minutes. In general, a signal to noise ratio of 2X (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypep tides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

[0043] In one particularly preferred embodiment, stringent conditions are characterized by hybridization in 1 M NaCl, 10 mM Tris-HCl, pH 8.0, 0.01% Triton X-100, 0.1 mg/ l fragmented herring sperm DNA with hybridization at 45 °C with rotation at 50 RPM followed by washing first in 0.9 M NaCl, 0.06 M NaH₂PO₄, 0.006 M EDTA, 0.01% Tween-20 at 45 °C for 1 hr, followed by 0.075 M NaCl, 0.005 M NaH₂PO₄, 0.5 mM EDTA at 45 °C for 15 minutes.

[0044] As used herein, the term "subject" encompasses mammals and non-mammals.

Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. The term does not denote a particular age or gender.

II. OVERVIEW

[0045] The present invention is directed to devices, systems and/or techniques for detecting analytes using the nanogap chips. The biosensors of the present invention can be used to detect analyte/molecule binding events, such as, for example, ligand/receptor, enzyme/substrate, protein/protein, DNA/DNA, DNA/RNA, RNA/RNA, nucleic acid/protein interactions. The nanogap chips are an important component of molecular analysis based on the electron transport ability of DNA which is a nanoscale phenomenon. In contrast to currently available DNA detection technologies, the present invention utilizes the electron transport properties of DNA molecules and the changes in these properties in response to analyte/molecule interaction. The present invention provides a level of sensitivity and specificity that is not economically attainable by existing DNA detection methods. Furthermore, the present invention requires reduced sample manipulation and elimination of optical detection technology thereby leading to substantial time and cost savings.

[0046] In one aspect, present invention is directed to a nanoscale biosensor, system and/or technique for the electronic detection of nucleic acids. The biosensor, system and/or technique of this aspect employs the electron transport properties of single DNA molecules to create nanomolecular circuits based on DNA hybridization. The biosensor, system and/or technique of the present invention may avoid the need for amplification or fluorescent detection (although such techniques may also be employed in addition to those of the present invention). As such, the present invention provides for broad deployment opportunities for the molecular diagnosis, genomic analysis and routine clinical management of patients in many areas of medicine.

[0047] The present invention allows for direct detection of molecular interactions on an array of nanogap electrodes. The molecular interactions can be detected electrically, and without the need for the labeling of the deposition material or of the target sample, although labeling may still be performed for use with the present invention.

III. NANOSCALE BIOSENSOR

[0048] The biosensor, system, and/or technique of the present invention may include one or more electrode pairs spaced at nanoscale distances (Figure 1). One embodiment of a basic biosensor (molecular sensing apparatus) of this invention, schematically illustrated in Figure 1, comprises a first electrode, a second electrode, and a double stranded DNA (ds DNA) spanning the gap between the two electrodes. The two electrodes can be separated by an air gap, preferably, the electrodes are separated by a spacer, such as an insulator, a dielectric, or a semiconductor. The ds DNA can be directly bound to the electrodes or it can be coupled to the first electrode and/or the second electrode through one or more linkers or functional groups. When a multiplicity of sensor elements are used, each electrode pair can be spanned by one or more ds DNA.

[0049] The biosensor can be made from a solid substrate, such as glass, alkali-free borosilicate glass, silicon, silicon oxide, silicon dioxide, silicon nitride, Teflon, alumina, mica, polyester, fiberglass, ceramics, plastic (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethanes, and the like), sapphire, nitrides, arsenides, carbides, oxides, phosphides, selinides and quartz. Thus, the solid substrate is preferably nonconductive or an insulator. The electrodes, such as gold electrodes, can be deposited on the surface of the solid substrate.

[0050] In one aspect of the invention, the solid substrate can be treated with a passivation agent such that the solid substrate has reduced ability to bind with the probe and/or the analyte, thereby increasing the signal to noise ratio. Thus, the solid substrate can be treated with a material such as silicon oxide, silicon dioxide, silicon nitride, silicon oxy- nitride, an organic film such as polyamide, an aminofunctional siloxane, such as an alkyl- silane compound such as proyl-, pentyl-, hexyl-silane, a fluoro-organosilane compound such as fluoromethyl silane, difluoromethyl silane, trifluoromethyl silane, or combinations thereof. In one aspect, PEG-silanes are used to create coatings on surface of the solid substrate (SiO₂, glass, silica, and others), where PEG molecules can optionally be derivatized with alkoxysilane or chlorosilane terminal reactive groups, and the hydrolysis and chemical condensation of the reactive silanes serves to anchor the PEG molecules to oxide substrates such as glass or silicon oxide with exposed surface silanol groups. In the PEG-silane, PEG can be polyethyleneglycol polymer, preferably of molecular weight 2000 to 5000 atomic mass units, and silane is a hydrolyzable silane precursor group, such as trialkoxy or trichlorosilane. Thus, the PEG-silanes can be PEG-silane, methoxy-PEG-silane, vinylsulfone-PEG-silane, NHS-PEG-silane, COOH-PEG-silane, and the like. Preferably, the substrate is treated with PEG-silane, fluoro-silane, or alkyl-silane for chip passivation.

[0051] The electrodes can be formed from essentially any conductive material.

Preferred conductive materials have resistivities of less than about 10^"3 ohm-meters, preferably less than about 10^"4 ohm meters, more preferably less than about 10^" ohm meters, and most preferably less than about 10^"7 ohm meters. The electrodes are formed from materials that include, but are not limited to ruthenium, osmium, cobalt, rhodium, rubidium, lithium, sodium, potassium, vanadium, cesium, beryllium, magnesium, calcium, chromium, molybdenum, silicon, germanium, aluminum, iridium, nickel, palladium, platinum, iron, copper, titanium, tungsten, silver, gold, zinc, cadmium, indium tin oxide, carbon or carbon nanotubes, semiconductor electrodes such as Si, Ge, ZnO, CdS, TiO₂ and GaAs and alloys or compounds of these materials. Preferably, the electrodes are gold lines. These electrodes may also be covered with conductive polymers to enhance the stability of the electrodes immobilized with probes. Monomolecular films may also be employed to cover the electrodes. Such films may be, for example, conductive polymers such as polyacethylene, polypyrrole, polythiophene and polyaniline as well as polyethylene, polypropylene, poly(vinylchloride), poly(vinyl alcohol), poly(methylmethacrylate), poly(vinyl-idene fluoride), cellulose and lipid membrane.

[0052] The electrodes comprising an electrode pair can be of any convenient dimension.

The electrodes can have a thickness of about 1 nm to about 1000 nm, preferably about 1 nm to about 100 nm. Thus, the electrodes can have a thickness of about 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm and the like. The electrode pair can be spaced such that the ds DNA can span the gap between the electrodes and complete the electric circuit thereby allowing for the measurement of an electromagnetic property. In certain embodiments, the electrodes are separated by distance ranging from about of 0.1 nm to about 1000 nm, preferably from about 0.5 nm to about 200 nm, more preferably from about 1 nm to about 100 nm, and most preferably from about 1 nm to about 50 nm. Thus, the interelectrode spacing can be less than about 200 nm, preferably less than about 150 nm, more preferably less than about 100 nm, and most preferably less than about 50 nm, about 40 nm, or about 30 nm, and can be 1 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, or any distance in between. The gap between the electrodes can be an air gap, filled with oxygen or with an inert gas (e.g. argon, etc.), a vacuum, or the gap can be filled with an insulator, semiconductor, or a dielectric. Preferably, the gap between the electrodes is filled with an insulator. Insulators for use in the invention include, elements, compounds or substances that have resistivity greater than about 10^"3, preferably greater than about 10^'2 ohm-meters, more preferably greater than about 10^"' ohm meters, and most preferably greater than about 10 ohm meters. Insulators for use in the invention include, but are not limited to SiO₂, TiO₂, ZrO₂, porcelain, ceramic, glass, clay, polystyrene, Teflon, plastics having a resistivity greater than 10^"3 ohm-meters, and other high resistivity plastics, insulating oxides or sulfides of the transition metals in the periodic table of the elements, and the like.

[0053] The electrodes on the solid substrate can generally be prepared in a wide variety of ways. For example, gold can be deposited on a surface via vacuum deposition processes (sputtering and evaporation) or solution deposition (electroplating or electroless processes). When electroplated metal is used, grain refining additives (brighteners) can optionally be added to alter surface deposition properties. Thus, the present invention provides methods of making a substrate comprising a plurality of gold electrodes. In one method, electroplating is used. The electrode metal, preferably gold, is then coated onto the substrate, and the patterns of the device, comprising the electrodes and their associated interconnections, are made using lithographic techniques, and wet chemical etching. Typically, a non-conductive chemically resistive insulating material such as solder mask or plastic is laid down using these photolithographic techniques, leaving only the electrodes and a connection point to the leads exposed. In another method, the electrodes, such as gold electrodes, can be sputtered onto the surfaces of the solid substrate. The ion beam sputtering of gold onto a surface is well known by those skilled in the art. Sputtering gold can produce electrodes upon which a variety of chemistry and molecular binding may be performed. Gold can be sputtered onto glass coverslips, smooth silicon, quartz or a similar flat surface. Preferably, an AFM may be used as the deposition tool to generate electrodes as small as 0.2 microns or smaller.

[0054] Thus, in one aspect, gold electrodes can be manufactured by an electron beam (e- beam) lithography process which carves out and/or forms the gaps between gold surfaces on each chip individually and in a serial manner. The gold electrodes can be spaced 30-40 nm apart with an air gap in between. In another method, metal can be directionally deposited onto the substrate at a pre-determined angle to the solid substrate. A step in the insulating substrate serves as a wedge that electrically isolates and physically separates the electrode materials in a controllable manner. In one embodiment, the gold electrodes of the "nanogap" structure can be separated by an approximately 20 nm wide insulating layer (Figure 3A) and allow for low leakage currents between the electrodes (usually < 10 pA), and provides voltage windows of 10 volts (Figure 3B). Optionally, the silicon dioxide portions of the chip can be coated with an PEG- silane , thereby increases the accessible voltage window, such as up to 14 volts, but can also render the chip more resistant to buffer solutions.

[0055] In one aspect of the invention, the surface of the electrode can be treated with a positively charged molecular monolayer on which the probe can be attached. Thus, the surface of the electrode (for example, a gold electrode) can be treated with an aminofunctional silane, such as N-(2-aminoethyl)-3-aminopropyltrimethoxy silane (AEAPTMS), 3- aminopropylmethyldiethoxysilane (APMDES), or aminopropyltriethoxysilane (APTES), for example, to facilitate amine-mediated DNA attachment. Preferably, the electrode is treated with APTES. The APTES placed on the electrode provides positively charged amino groups that can bind tightly to a negative charge. Materials such as DNA and RNA containing negatively charged groups can therefore bond to the electrodes after the APTES treatment. In another aspect, the surface of the electrode can be treated with molecules such as, alkylsiloxanes, alkanethiolates, and fatty acids. For example, the molecules can have the structure X— R— Y, where X is a sulfur group, e.g., SH, SPO₃— , OSO₃H, Z-S— S- (where Z is an alkyl group, such as an alkane), R comprises a linear or branched alkyl group, which is preferably an alkane, and Y may be selected from the group comprising hydrogen, alcohols, carboxylic acids, esters, alkenes, ketones, aldehydes, amines, sulfonic acids, halogens, and alkyl halogens. Molecules comprising a sulfur group, such as a thiol, a thioate, a sulfide, or alkylthiolate, are preferred where electrodes comprise a gold or platinum surface. In another aspect, the surface of the electrode can be treated with a self-assembled monolayer. The self- assembled monolayer (SAM) can be made from an alkanethiol, a phospholipids and an oligo (phenylenevinylene) .

[0056] As an example, the electrodes an array can be contacting with a solution comprising an alkylthiolate, such as mercaptohexanol, preferably under conditions suitable to associate a self-assembled monolayer of the alkylthiolate with electrodes. For example, the solution may be an aqueous solution comprising at least 250 μM, such as at least 500 μM of the alkylthiolate can be contacted with electrodes for a time sufficient to prepare a protective layer that inhibits other molecules from associating with electrodes having the protective layer. For example, electrodes may be exposed to solution for a sufficient period of time, such as at least 15 minutes, at least 30 minutes, at least 60 min up to about 150 minutes or up to about 300 minutes or overnight or up to about 24 hours. Following exposure to the solution, the electrodes of the array can be contacted with a liquid, such as ethanol or other solvent, to remove any molecules not covalently associated with an electrode. The electrode thus treated can be associated with one or more probe molecules by contacting it with a solution comprising the probe molecules.

[0057] In one aspect of the present invention, one or more probe molecules can be bound with the electrode. The probe can be selected from among any molecules known in the art to be capable of, or putatively capable of, specifically binding an analyte of interest. Further, the probe can comprise a linker or functional group through which the probe molecule can be coupled to the electrode. Binding preferably takes place through a covalent bond between the probe and the electrode. For example, a probe can be coupled to a gold or a platinum electrode by a bond comprising a sulfur group of the molecule and the gold or platinum electrode. Alternatively, or in combination with a covalent bond, binding may occur through an ionic bond or other physio-chemical interaction that retains the coupling between the molecule and the surface, preferably unless it is intended to dissociate the molecule from the surface.

[0058] The probe molecule can be selected based upon the targets that are to be identified/quantified, and can preferably bind with the first portion of the target molecule to be detected. Thus, for example, where the target is a nucleic acid the probe is preferably a nucleic acid or a nucleic acid binding protein or protein complex. Where the target is a protein, the probe is preferably a receptor, a ligand, another protein or an antibody that specifically binds that protein. Where the target is a sugar or glycoprotein, the probe is preferably a lectin, and so forth. Thus, the probe molecule can be an enzyme, an enzyme substrate, a receptor, ligand for a receptor, a nucleic acid, a polynucleotide, a protein or fragments thereof, a lectin, an antibody, binding portion of an antibody (e.g., Fab, (Fab)'₂), glycoproteins, polysaccharides, antigens, epitopes, carbohydrate moieties, protein A, protein G, an organic compound, lipids, fatty acids, lipopolysaccharides, cellular metabolites, hormones, pharmacological agents, tranquilizers, barbiturates, alkaloids, steroids, vitamins, amino acids, sugars, nonbiological polymers, biotin, avidin, streptavidin, organic linking compounds such as polymer resins, hpoproteins, cytokines, lymphokines, hormones, synthetic polymers, and the like. For example, a polynucleotide that can hybridize with a second, at least partially complementary polynucleotide, can be the probe molecule.

[0059] Methods of synthesizing or isolating the probes are well known to those of skill in the art. For example, nucleic acids for use as probes can be produced or isolated according to any of a number of methods well known to those of skill in the art. In one embodiment, the nucleic acid can be an isolated naturally occurring nucleic acid (e.g., genomic DNA, cDNA, mRNA, etc.), using methods well known to those of skill in the art. Alternatively, the nucleic acid can be chemically synthesized using, for example, the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981), Tetrahedron Letts., 22(20): 1859-1862, and a commercially avialable automated synthesizer. Purification of oligonucleotides, where necessary, can be typically performed by either native acrylamide gel electrophoresis or by anion-exchange HPLC. Antibodies or antibody fragments can be produces by a number of methods well known to those of skill in the art (see, e.g., Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory). For example, the antibodies can be produced by immunizing an animal (e.g. a rabbit) with an immunogen containing the epitope it is desired to recognize/capture. A number of immunogens may be used to produce specifically reactive antibodies. Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies. Naturally occurring protein may also be used either in pure or impure form. Synthetic peptides made as well using standard peptide synthesis chemistry.

[0060] In some aspects of the invention, a secondary probe can be utilized. The secondary probe can be selected based upon the targets that are to be identified/quantified, and preferably can bind with the second portion of the target molecule to be detected where the second portion is different from the first portion, and do not bind with the electrodes. Thus, for example, if the probe hybridizes with the 5 '-end of the target molecule, then the secondary probe can hybridize near the 3 '-end of the target molecule.

[0061] The analyte includes, but is not limited to a whole cell, a subcellular particle, virus, prion, viroid, nucleic acid, protein, antigen, lipoprotein, lipopolysaccharide, lipid, glycoprotein, carbohydrate moiety, cellulose derivative, antibody or fragment thereof, peptide, hormone, pharmacological agent, cell or cellular components, organic compounds, non- biological polymer, synthetic organic molecule, organo-metallic compounds, or an inorganic molecule present in the sample. The sample can be derived from, for example, a solid, emulsion, suspension, liquid or gas. Furthermore, the sample may be derived from, for example, body fluids or tissues, water, food, blood, serum, plasma, urine, feces, tissue, saliva, oils, organic solvents, earth, water, air, or food products. The sample may comprise a reducing agent or an oxidizing agent, solubilizer, diluent, preservative, or other suitable agents.

IV. ON-CHIP ASSAYS

[0062] The biosensor can be used to detect the specific binding of the probe with the analyte. The sample suspected of comprising the analyte can be contacted with the biosensor under conditions such that the probe binds with the analyte if present. Preferably, the probe binds with the first portion of the analyte. Thus, for example, when the probe is a nucleic acid, the contacting of the biosensor with the sample suspected of containing the target nucleic acid takes place under hybridization conditions. The hybridization conditions are well known to those of skill in the art, and can be readily optimized for the particular system employed. Preferably, stringent hybridization conditions are employed. Similarly, when the probe is a protein or an antibody, conditions suitable for antibody binding can be used.

[0063] The specific binding of the probe with the analyte can be detected by measuring an electromagnetic property of the electrode pair, wherein the electromagnetic property indicates the presence of the analyte in the sample. The measurement of the electromagnetic property can be by changes in resistivity or conductivity, amperometry, voltammetry, coulometry, and the like. Preferably, the electrode pair can be spanned with a double stranded nucleic acid that completes an electric circuit between the electrode pair if the sample comprises the analyte. Thus, the double stranded nucleic acid does not have to be specific for the target or the probe as it is used to complete the electric circuit.

[0064] The ds DNA can have a length that preferably is at least as long as the distance between the electrode pair, but can be longer. Thus, the ds DNA can be from about 5 base pairs (bp) to about 100,000 bp long, preferably less than 1000 bp long, more preferably less than 10,000 bp long, even more preferably less than 2000 bp long. Thus, the ds DNA can be from 5 bp to about 2000 bp long or any integer in between, such as 10 bp, 150 bp, 200 bp, 250 bp, 300 bp, 330 bp, 340, bp, 350 bp, 370 bp, 400 bp, 450 bp, 500 bp, and the like.

[0065] In one aspect of the invention, spanning the electrode is by a double stranded

DNA (ds DNA) capable of binding with a connector molecule. The ds DNA can be linked with a connector molecule, or can have a site to which a connector molecule can attach. The connector molecule can be any suitable substance which will enable the ds DNA to bind to the probe-target complex. Thus, the connector molecule can be streptavidin, avidin, neutravidin and the like, where the ds DNA can be linked with the probe-target complex through a streptavidin/biotin interaction. Avidin and streptavidin are large proteins (70 kD) which each contain 4 biotin binding sites. Biotin is a small molecule which attaches with very high affinity to the binding site (I j =10^"15 M), and can only be removed under extreme conditions. Other paired molecules, for example, can be digoxigenin & anti-digoxigenin or any other antibody & antigen pair. Alternatively, the avidin/biotin interaction can be replaced with small molecule interactors. These are small chemical chemical groups that have high affinity for each other, but greatly reduced non-specific binding. An example of a small molecule interacting pair is phenyl(di)boronic acid with salicylhydroxamic acid.

[0066] In one aspect, avidin or streptavidin can be adsorbed onto the electrode or can be covalently attached to a secondary probe. The ds DNA can be biotinylated wherein the ds DNA can be covalent attached to biotin or a biotin analog. The covalent attachment may be direct or through a linker. The biosensor, having the target bound to the probe, can be exposed to an aqueous solution of secondary probes and biotinylated double stranded nucleic acid. The secondary probe can bind with the target that is not bound to the probe, and the binding of avidin or streptavidin with biotin results in an avidin/streptavidin-biotin complex. The ds nucleic acid thus bound to the probe-target complex can be stretched across the electrode pair using a flow field and/or an electric field to complete the circuit (Figure 12). [0067] The ds DNA can be attached to the biosensor by one of the methods described above, and the ds DNA can be stretched by applying an external force. The ds DNA can be stretched across the electrode pair using any one of the methods known in the art, such as by using elongational forces exerted by fluid flow, gravitational streaming, forced air or fluid streaming and stretching induced by charged electric fields, although other related methods such as mechanical stretching of DNA may also be employed. Preferably, the stretching of the ds DNA molecule is by using an electric field or liquid flow as described by Zimmermann et al. (Nucl. Acid Resear. 22:492-497 (1994)).

[0068] In one aspect, the ds DNA can be stretched using an elongational flow, or by a steady shear flow. In another method, the ds DNA can be stretched using an electrical field (U.S. Patent No. 5,538,898) or a field of alternating current. Washizu and Kurosawa ((1990) IEEE Transactions on Industry Applications 26:1165-1172) have demonstrated that DNA will stretch to its full length in its B-DNA form in a field having strength 10⁶ V/m and a frequency of 400 kHz or more. At certain lower frequencies (around 10 kHz), the DNA will also stretch fully, but in a direction peφendicular to the field rather than parallel to it. Typically, an oscillating voltage or a steady voltage can be applied to the electrode pair to which the ds DNA is attached. A sinusoidal voltage of 200 v peak-to-peak (p-p), preferable a voltage of about 0.5 V to about 150 V, or any voltage in between can be applied. The electrode separation and geometry would determine the field strength and field gradient. These are important factors in the stretching of the DNA. The gravitational streaming (U.S. Patent No. 5,707,797) method involves tilting the supporting surface at an angle which will efficiently extend the DNA. Generally, the angle of tilt will be between about 20 to about 90 degrees from horizontal. The tilt angle employed will, of course, depend also on the surface employed for the support. Differences in surface material and surface tension of the DNA solution may affect the rate of streaming and the ultimate extension of the DNA molecule.

[0069] In another aspect, the secondary probe has a part that binds with the target and a part that binds with the ds DNA. The biosensor, having the target bound to the probe, can be exposed to an aqueous solution of secondary probe and ds nucleic acid. The secondary probe can bind with the target that is not bound to the probe, and the binding with the ds DNA (Figure 13). Thus, in one aspect, the ds DNA comprises a single-strand portion at either the 5'-end or the 3'-end of at least one of the strands. The secondary probe can be attached to the ds DNA via the single strand portion when the two are complementary or via a ligated general sequence that is complementary to both the secondary probe and the single-stranded portion of the ds DNA. The ds nucleic acid thus bound to the probe-target complex can be stretched across the electrode pair using a flow field and/or an electric field to complete the circuit.

[0070] In another aspect, the secondary probe has a part that binds with the target and a part that binds with oligonucleotides attached to a nanoparticle (Figure 14). In one aspect, the method comprises contacting with at least two types of nanoparticles having oligonucleotides attached thereto. Nanoparticles useful in the practice of the invention include metal (e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g., ferromagnetite) colloidal materials, including ZnS, ZnO, TiO₂, Agl, AgBr, Hgl₂, PbS, PbSe, ZnTe, CdTe, In₂S , InAs, and GaAs nanoparticles. The nanoparticles preferably have an average diameter from about 5 nm to about 150 nm, more preferably from about 5 to about 50 nm, most preferably from about 10 to about 30 nm. The nanoparticles can be made using methods known in the art or they can be obtained from commercial suppliers, such as Ted Pella, Inc. (gold), Amersham Coφoration (gold) and Nanoprobes, Inc. (gold). The nanoparticles can be attached to oligonucleotides.

[0071] In one aspect, each nanoparticle can have a plurality of oligonucleotides attached to it wherein each nanoparticle-oligonucleotide conjugate can bind to a plurality of oligonucleotides or nucleic acids having the complementary sequence. For example, the first nanoparticle can have oligonucleotides that can bind with the secondary probes, or can have a sequence complementary to a second portion of the target nucleic acid and with the oligonucleotides attached to a second nanoparticle. The second nanoparticle can have oligonucleotides that can bind with the oligonucleotides on the first nanoparticle and other nanoparticles, but not the secondary probes or with the target nucleic acid. Thus, the first nanoparticle can bind with the secondary probe or with the target, but the second nanoparticle can only bind with the first nanoparticle.

[0072] The probe attached to the electrode has a sequence complementary to a first portion of the sequence of a target nucleic acid to be detected. The target nucleic acid is contacted with the biosensor under conditions effective to allow hybridization of the probes with the nucleic acid. In this manner the target becomes bound to the biosensor, and any unbound target can be washed from the sensor before adding nanoparticle-oligonucleotide conjugates. Next, the target bound to the biosensor can be optionally contacted with a secondary probe and with a first type of nanoparticles having oligonucleotides attached thereto. The secondary probe has a sequence complementrary to a second of the target nucleic acid and the oligonucleotides on the nanoparticle have a sequence complementary to the secondary probe. The contacting takes place under conditions effective to allow hybridization of the oligonucleotides on the nanoparticles with the secondary probe. In this manner the first type of nanoparticles become bound to the biosensor. After the nanoparticle-oligonucleotide conjugates are bound to the biosensor, the biosensor can be washed to remove any unbound nanoparticle-oligonucleotide conjugates and nucleic acid. Finally, the first type of nanoparticle-oligonucleotide conjugate bound to the biosensor can be contacted with a second type of nanoparticles having oligonucleotides attached thereto. These oligonucleotides can have a sequence complementary to at least a portion of the sequence(s) of the oligonucleotides attached to the first type of nanoparticles, and the contacting takes place under conditions effective to allow hybridization of the oligonucleotides on the first type of nanoparticles with those on the second type of nanoparticles. After the nanoparticles are bound, the biosensor can be washed to remove any unbound nanoparticle-oligonucleotide conjugates. The nanoparticles thus bound can span the electrode pair to complete the circuit as shown in Figures 14 and 15. In one aspect, the probe-target complex can be present on both electrodes of the electrode pair, and the first type of nanoparticles becomes bound both of the electrodes (Figure 14). In another aspect, the probe-target complex can be present on only one of the electrodes of the electrode pair, and the circuit is completed when the nanoparticles, either the first type or the second type, contacts the second electrode (Figure 15).

[0073] In another aspect of the invention, the analyte to be detected is nucleic acid, and the nucleic acid bound to the probe can be amplified by the polymerase chain reaction where the probe can be used as a primer for the amplification reaction (Figure 16). Nucleic acids can be amplified using the polymerase chain reaction (PCR, U.S. Patent No. 5,498,392), by the ligase chain reaction (LCR, U.S. Patent No. 5,589,136), by isothermal amplification (U.S. Patent No. 6,379,929), and the like. For example, the nucleic acids can be amplified by strand displacement amplification (SDA, U.S. Patent No.5,270,184), transcription mediated amplification (TMA, U.S. Patent No.5,399,491), rolling circle amplification (RCA, U.S. Patent No.6,255,082), branched chain amplification (BCA), and isothermal nucleic acid sequence-based amplification (NASBA, U.S. Patent No. 5,409,818). In PCR™ amplification of the nucleic acid target, two probes act as the primer sequences. The biochip, with the target bound to the chip, can be exposed to an amplification solution. The amplification solution typically contains an excess of deoxyribonucleoside triphosphates along with a DNA polymerase, e.g., Taq polymerase, that facilitates template-dependent nucleic acid synthesis. If the probe-target complex has been formed, the polymerase will cause the probe to be extended along the target nucleic acid sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended probe will dissociate from the target(s) to form reaction products and the process can be repeated. These multiple rounds of amplification, referred to as "cycles", can be conducted until a sufficient amount of amplification product is produced. The final cycle produces a ds DNA bound to the biosensor can be stretched across the electrode pair using a flow field and/or an electric field to complete the circuit (Figure 16).

[0074] In yet another aspect of the invention, the circuit can be completed using microspheres functionalized with oligonucleotides complementarty to the target nucleic acid or the secondary probe (Figure 17). The microsphere typically comprise a core on which is plated a conductive metal. Thus, the core can be glass, mica, silica, silica dioxide, quartz, latex, copolymer, styrene, polystyrene, glycidyl metaacrylate (GMA), butylmetaacrylate (BMA), divinylbenzene (DVB), and the like. The core can then be plated with a substantially uniform thickness of a conductive metal such as gold, silver, platinum, palladium, copper, nickel, titanium, and combinations thereof. Preferably, the micropheres comprise gold. The microspheres are preferably about 0.1 μM to about 25 μM in diameter, more preferably about 0.5 μM to about 20 μM, or any diameter in between, such as, 1 μM, 2 μM, 5 μM, 7 μM, 8 μM, 10 μM, 12 μM in diameter.

[0075] Oligonucleotides complementary to the target nucleic acids or to the secondary probes can be immobilized on the microspheres. The biosensor having the probes hybridized with the target nucleic acid can be optionally contacted with a secondary probe and with a microsphere having oligonucleotides attached thereto. The contacting takes place under conditions effective to allow hybridization of the oligonucleotides on the microsphere with the target or the secondary probe. In this manner the first type of microspheres become bound to the biosensor. Some of the bound microspheres can span the electrode pair thereby completing the circuit, as shown in Figure 17.

[0076] In another aspect, multiple microsphere can be used (Figure 18). For example, the first microsphere can have oligonucleotides that can bind with the secondary probes, or can have a sequence complementary to a second portion of the target nucleic acid and with the oligonucleotides attached to a second microsphere. The second microsphere can have oligonucleotides that can bind with the oligonucleotides on the first microsphere and other microspheres, but not the secondary probes or with the target nucleic acid. Thus, the first microsphere can bind with the secondary probe or with the target, but the second microsphere can only bind with the first microsphere. The hybridization of the biosensor with the microspheres can complete the circuit either through the first microsphere, as shown in Figure 17, where the single microsphere can span the electrode pair, or the circuit can be completed through at least three microspheres, where the first microsphere binds to each of the electrodes in the electrode pair, and the oligonucleotides on the second microsphere hybridize with the oligonucleotides on the first microsphere thereby completing the circuit (Figure 18).

[0077] Upon the completion of the circuit, the sensors of this invention can be read using standard methods well known to those of skill in the art. In particular, the sensors of this invention provide a signal that is a change in conductivity (resistivity) of the sensor element(s). Thus, the sensors of this invention can be read using techniques including, but not limited to amperommetry, voltammetry, capacitance, and impedence. Suitable techniques include, but are not limited to, electro gravimetry; coulometry (including controlled potential coulometry and constant current coulometry); voltametry (cyclic voltametry, pulse voltametry (normal pulse voltametry, square wave voltametry, differential pulse voltametry, Osteryoung square wave voltametry, and coulostatic pulse techniques); stripping analysis (aniodic stripping analysis, cathiodic stripping analysis, square wave stripping voltammetry); conductance measurements (electrolytic conductance, direct analysis); time-dependent electrochemical analyses (chronoamperometry, chronopotentiometry, cyclic clironopotentiometry and amperometry, AC polography, chronogalvametry, and chronocoulometry); AC impedance measurement; capacitance measurement; and photoelectrochemistry. In preferred embodiments, devices for measuring electron transfer amperometrically involves sensitive (nanoamp to picoamp) current detection and include a means of controlling the voltage potential, usually a potentiostat.

V. OFF-CHIP ASSAY

[0078] In another aspect of the invention, the assay for the detection of the analyte can be conducted without the use of the biosensor, and the detection of the analyte can be accomplished using the biosensor of the invention. Thus, for example, if the analyte to be detected is a nucleic acid, sample suspected of containing the target nucleic acid can be exposed to a probe specific for the target, wherein the probe and/or target can be extended along a template by an enzyme or polymerase. Such reactions can produce long double stranded DNA. Alternatively the probe and target combination can be amplified by the polymerase chain reaction where the probe can be used as a primer for the amplification reaction (Figure 19). Nucleic acids can be amplified using the polymerase chain reaction (PCR, U.S. Patent No. 5,498,392), by the ligase chain reaction (LCR, U.S. Patent No. 5,589,136), by isothermal amplification (U.S. Patent No. 6,379,929), and the like, to produce a ds DNA. The ds DNA, if present, can be detected using the biosensor of the invention. Typically, the solution suspected of containing ds DNA can be exposed to the biosensor wherein at least one of the first electrode and the second electrode comprises a means for attaching a double stranded nucleic acid. The ds DNA thus attached to the electrode can be stretched across the electrode pair using a flow field and/or an electric field to complete the circuit (Figure 19).

ATTACHMENT OF DNA

[0079] In one aspect of the invention, the electrode is a gold electrode, and attachment of the ds DNA to the electrode is via a sulfur linkage. In general, the nucleic acid can be constructed with a thiol linker which can be used to directly complex to gold surfaces (Ito, et. al., Anal. Chim. Acta., 1996, 327, 29). The sulfhydryl groups can be introduced into the nucleic acid binding agent, and can be used to bind the binding agent to the gold electrodes due to the extremely strong gold-sulfur interaction. It is known that gold binds to sulfur with a binding force comparable to that of a covalent bond. Therefore, gold-sulfur interactions can be used to tether biomolecules to electrodes. Introduction of the functional group into the nucleic acid can be conducted by means of enzymatic reaction or by using DNA synthesizer. The enzymes useful in the enzymatic reaction may be, for example, terminal deoxynucleotidyl transferase, poly A polymerase, polynucleotide kinase, DNA polymerase, polynucleotide adenylyltransferase and RNA ligase. Polymerase chain reaction method (PCR method), nick translation method and random primer method may also be employed to introduce the functional group. The functional group may be introduced to any part of the nucleic acid, such as 3' or 5' terminal, as well as a site randomly selected.

[0080] In another aspect of the invention, the nucleic acid binding agents can be covalently attached to the electrode using a chemically reactive surface. Such reactive surfaces include, but are not limited to, surfaces with terminal succinimide groups, aldehyde groups, carboxyl groups, vinyl groups, and photoactivatable aryl azide groups, and the nucleic acids can be modified to contain primary amines, where the coupling is carried out by a catalyst such as the carbodiimide ED AC (l-ethyl-3-[3-dimethylaminopropyl]carbodiimide). Furthermore, the spontaneous coupling of succinimide, or in the alternative, aldehyde surface groups, to primary amines at a physiological pH may be incoφorated for attaching molecules to the electrodes.

(a) Attachment of DNA via amines

[0081] The thiol-nucleic acid described above may not produce a close-packed surface due to the large hydrophilic nucleic acid group. Further, DNA attachment via thiol linkage may not be efficient for attaching larger DNA molecules (larger than 50 bases). Accordingly, in order to more efficiently and reliably attach large DNA molecules to certain electrodes the surface of the electrode can be treated with a positively charged molecular monolayer on which the DNA can self-assemble. Thus, the surface of the electrode (for example, a gold electrode) can be treated with an aminofunctional silane, such as N-(2-aminoethyl)-3- aminopropyltrimethoxy silane (AEAPTMS), 3-aminopropylmethyldiethoxysilane (APMDES), or aminopropyltriethoxysilane (APTES) to facilitate amine-mediated DNA attachment. Preferably, the electrode is treated with APTES. The APTES placed on the electrode provides positively charged amino groups that can bind tightly to a negative charge. Materials such as DNA and RNA containing negatively charged groups can therefore bond to the electrodes after the APTES treatment.

[0082] In one aspect, the silane compounds are attached to the electrodes via an oxide layer formed on the electrode. An oxide layer can be formed, provided and/or created on gold by exposing the surface to UV-ozone in a UVO (for example, for 60 minutes in a Jelight UVO cleaner model 42 (Jelight, Irvine, CA)). The oxide layer may be a suitable substrate for covalent attachment of silane(s) and gold oxide trapped under a monolayer is typically stable.

[0083] An APTES-treated electrode, where a positively charged silane layer was formed on the gold oxide, efficiently traps DNA (Figure 4). In addition, the silane-linked amine monolayer introduces a linker between the DNA and the electrode that is three-carbon long and would thus be less insulative than the longer linkers presently used in the art.

(b) DNA Attachment via Fmoc [0084] For shorter DNA (for example, less than 2kb), it may be advantageous to employ a stable amino-alkanethiol surface such as, for example, 9-fluorenylmethoxycarbonyl (Fmoc) protected C6-amine thiol, which contains a six-carbon chain (Figure 6). hi general, a compound of structure HS-(CRιR₂)_n-NH-R₃ can be used, where n is an integer between 1 and 10, Ri and R₂ are independently selected to be hydrogen or lower alkyl, and R is an amine protecting group. "Lower alkyl" means the monovalent branched or unbranched saturated hydrocarbon, consisting of carbon and hydrogen atoms, having from one to twelve carbon atoms inclusive, unless otherwise indicated. Examples of lower alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, n- hexyl, octyl, dodecyl, and the like. Amine protective groups include dimethoxytrityl (DMT), tert-butylcarbamate (tBoc) and trifluoroacetyl (Tfa), Fmoc, benzoyl (Bz) and phenoxyacetyl (pac). Figure 7A shows gold surfaces covered by an Fmoc layer with lambda DNA, while Figure 7B shows 1.5kb DNA bound to an Fmoc layer. An advantage of attaching DNA to the electrodes via Fmoc is that the bound DNA is stretched out, a conformation that provides for good electrical measurements.

(c) DNA Attachment via Intercalation

[0085] In order to increase current conduction through bound double-stranded DNA, it may be advantageous to attach double-stranded DNA via direct coupling of its electron- transporting base stack to the electrode surface (rather than through the DNA backbone, as is the case in the amine-mediated attachment modes). One technique for direct electronic coupling by covalently attaching to the gold surface employs a "molecular wire" molecule that ends in an intercalator molecule that can immobilize double-stranded DNA via interaction with the DNA base stacks (Figure 8). Typically, molecular wire comprises a plurality of monomeric units which share conjugated π-orbitals, ie., comprises a plurality of interspersed double and/or triple bonds. Of course, suitable conductive oligomer may also contain one or more .sigma. bonds. Examples of conductive oligomers comprise oligo pheylene vinylene and poly pyrroles

[0086] Higashi et al. (1999) Langmuir 15:111-115 have shown that a structure consisting of a long chain alkyl disulfide attached to the intercalator acridine can self-assemble into a monolayer on a gold surface and that the intercalation interaction is of sufficient strength and stability to localize double-stranded DNA to the surface. Nakao et al. ,(2002) Nano Letters 3:475-479 disclose a glass surface spin-coated with an intercalator polymer provides a means for the even distribution of stretched and oriented DNA molecules by directional deposition, and the DNA does not form clumps as is common on untreated glass surfaces.

[0087] In one aspect of the invention, the nucleic acid binding agent is a compound of structure HS-L-I, where L is a linker and I is an intercalator or an intercalating dye. Preferably, the compound can intercalate into the base stack of DNA and simultaneously communicate electronically with the electrode. Thus, the linker (L) is selected such that it is conductive. L can have the structure -(-C≡C-)_n-, -(-C=C-)_n-, -(-C≡C-Ar), , -(-C=C-Ar)„-, and the like, where n is an integer between 0 and 10, and Ar is a substituted or unsubstituted aryl or heteroaryl group, such as for example phenyl acetylene. The intercalator can be selected to bind to the major groove or the minor grove. The intercalator for use in the invention is not limited, and can be bisintercalator, trisintercalator, and polyintercalator, such as Hoechst 33258, naphthalene diimide, rivanol, doxorubicin, daunorubicin, actinomycin D, methylene blue, quinacrine, daunomycin, metalointercalator, bisacridine, ethidium, ethidium bromide, an acridine derivative (such as acridine orange, acridine yellow, 9-aminoacridine, hydrochloride hydrate, 2-aminoacridone, 9,9'-biacridyl, 9-chloroacridine, 6,9-dichloro-2- methoxyacridine, n-(l-leucyl)-2-aminoacridone, and 10-octadecyl acridine orange), an intercalating nucleic acid stain, a cyanine dye, such as SYBR green, proflavin, propidium iodide, acriflavin, proflavin, actinomycin, anthracyclines, or nogalamycin.

[0088] In one aspect of the invention, the intercalator is naphthalene diimide.

Naphthalene diimide has a strong ability to discriminate between double and single-stranded DNA and is known to oxidize guanine bases, which demonstrates an electronic connection between the naphthalene diimide and the DNA bases. However, if the ability of naphthalene diimide to electronically couple the molecular wire to the DNA bases is inadequate, it may be advantageous to employ other intercalators with different electronic and redox properties such as daunomycin or methylene blue. Daunomycin and methylene blue have been used by others in electrochemical DNA detection systems that rely on intercalation.

[0089] A structural diagram of ethidium molecular wire is provided in Figure 9, where an ethidium head group is linked to a phenyl acetylene molecular wire portion which, terminates in a thiol group. "Molecular wire" refers to the phenyl acetylene portion of this molecule, but by definition, may be any combination of atoms that confers enhanced electron transport activity. Similarly, while ethidium is the intercalator used in this molecule, many others are known to those skilled in the art. Indeed there are also a number of intercalators that preferentially recognize double-stranded DNA, i.e., dsDNA versus single-stranded DNA, i.e., ssDNA. In some aspects of the invention, an intercalator is selected that preferentially recognize dsDNA versus ssDNA. For example, ethidium is an excellent intercalator that recognizes dsDNA as well as RNA/DNA hybrids.

[0090] In one aspect of the invention, the binding of DNA to the electrode via the molecular wire is in a stretched, linear orientation. The stretched, linear orientation can be achieved by selecting the binding surface and the conditions under which binding occurs. The affinity of the DNA for a positively charged surface, for example, can be attenuated by depositing the DNA in a salt solution. This diminished attraction for the surface allows the forces of fluid flow to stretch out the DNA. By contrast, if the DNA has too great an attraction for the surface, it will bind in a balled up conformation (Figure 10A).

[0091] These molecular wire molecules, however, can have a strong tendency to lay down on the surface due the affinity of the multiple aromatic rings for the gold electrode. In order for the molecular wire to stand up, where the multiple aromatic rings are not interacting with the gold electrodes but project the ethidium portion in a manner able to bind DNA, it may be advantageous to dilute the molecular wire on the surface using "filler" molecules. In one embodiment, the filler molecule also has a thiol moiety and competes for the gold surface, thereby limiting the available binding area for the molecular wire. In this way, the molecular wire may be forced to "stand up."

V. CAPTURING DNA

[0092] In one aspect of the invention, the biosensor can be exposed to a sample. A nucleic acid such as, e.g., ds DNA, DNA/RNA hybrid, ds PNA, ds RNA, in the sample can be captured at or near the electrode by one of the capture agents described above. The presence of double stranded DNA across the electrode pair completes the circuit thereby allowing for the detection by electrical means. The efficiency of capturing DNA at the electrode can be enhanced by employing a solution phase hybridization/extension system to generate double- stranded nucleic acid in solution, which would then be captured at the detection electrodes. In principle, any polymerase (for example, thermos thermophilus (Roche Diagnostics, Penzburg, Germany) may be used. Notably, the thermos thermophilus enzyme has both a DNA- dependent DNA polymerase activity and an RNA-dependent DNA polymerase activity. The optimal temperature for activity is 75 °C, which would minimize the effects of secondary structure of the single-stranded DNA or RNA template. Additional reaction conditions are known to those of skill in the art and are provided by the manufacturer. Double-stranded nucleic would then be captured at the detection electrodes. Such a method can be used to discriminate between single-nucleotide differences by appropriate selection of the probe.

[0093] An alternate embodiment of the biosensor for increasing the likelihood of localizing double stranded nucleic acid at the nanogap is as follows. In a first step, single stranded nucleic acid (probe DNA) on or near at least one of two closely spaced electrodes in an electrode pair is immobilized. Thereafter, a solution with an analyte which specifically binds to the immobilized probe DNA forming a probe/analyte complex is introduced. A polymerase enzyme, for example, thermos thermophilus (Roche Diagnostics, Penzburg, Germany), which extends the probe/analyte complex to generate double stranded DNA in solution is added. This generates double stranded DNA or extended DNA molecule, in a preferred embodiment, that contains a longer doubled-stranded region than the probe/analyte complex prior to the enzyme extension. In a preferred embodiment, the extended DNA molecule would be long enough to make simultaneous contact with both electrodes in a pair of closely space electrodes. In one aspect of the invention, contact between the DNA molecule and electrode pair can be further enhanced electronically by introducing molecular wires on the surface and/ or intercalators with molecular wires or other such non-insulative molecules that electronically couple the extended DNA molecule with the electrode.

[0094] In another aspect of the biosensor, system and/or technique for the electronic detection of nucleic acids disclosed herein, an alternating current can be applied between the two electrodes of the nanogap structure while the nanogap structure is exposed to a solution containing an analyte of interest.

[0095] One suitable technique acquires the electrical measurements dry. Following the deposition of the DNA, either in the flow cell described above or by pipet, the chips may be rinsed in water and dried in a stream of argon, nitrogen, purified air and the like prior to measurements.

[0096] ^~ Current-voltage curves for the biosensors can be determined by applying a changing voltage bias from about -7 to about +5 volts and by measuring the corresponding current. Alternatively, voltages can also range between -15 to +15 volts. Alternatively current can be measured at a single voltage or a specific set of voltages. Further, a current can be forced through the device pre and/or post biomolecule deposition where a corresponding voltage is measured, the differences in currents or voltages, respectively, indicating the presence of an analyte. In one aspect of the invention, a range of electromagnetic properties can be measured. These electromagnetic properties may include but are not necessarily limited to direct electric current, alternating electric current, permittivity, resistivity, electron transfer, electron tunneling, electron hopping, electron transport, electron conductance, voltage, electrical impedance, signal loss, dissipation factor, resistance, capacitance, inductance, magnetic field, electrical potential, charge or magnetic potential. Furthermore, a measured electromagnetic property or changes thereof, are not necessarily limited to the presences of an analyte but an indication of the amount or quantity of analyte present.

[0097] To verify that the measured current or electromagnetic property is mediated by the double-stranded DNA, it may be advantageous to retest the device after treatment with DNase enzyme or appropriate restriction enzymes.

[0098] To assess background noise, the biosensors can be tested without attached DNA and with attached single- stranded DNA. To gain a first measure of specificity, the nanogap structures may be tested with double-stranded DNAs containing a range of base pair mismatches. Given that electron transport through DNA is mediated by the base stack, the single-stranded DNA and mismatched double-stranded DNA do not enable electron transport and therefore produce little or no current.

[0099] In the event that single-stranded DNA yields more than a background signal, the chips can be treated with a single-strand-specific nuclease, such as SI nuclease. Treatment with a single strand-specific nuclease following hybridization destroys unhybridized single- stranded probes. Thus, observation of a single-stranded DNA signal does not prohibit the use of the technology as a biosensor.

SNP Detection

[00100] In one aspect of the invention, the biosensor is used in genetic diagnosis. For example, the biosensor can be used to detect target sequences such as the gene for nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which is a gene associated with a variety of cancers, the Apo E4 gene that indicates a greater risk of Alzheimer's disease, allowing for easy presymptomatic screening of patients, mutations in the cystic fibrosis gene, or any of the others well known in the art. In one aspect, the biosensor can be exposed to ds DNA that is the full length gene, part of the gene, or an EST of the gene of interest, and the current is measured. Then, the biosensor is exposed to a sample from a subject that may have the disease, and the current is measured. The two currents are compared. If the currents are similar, then a single nucleotide polymoφhism (SNP) for the disease is not present in the subject. However, if the current from the sample from the subject is lower, then the SNP for the disease is present in the subject. As one of skill in the art will recognize, the control sample and the test sample can be run at the same time, and more than one gene or fragment of a gene can be tested at the same time.

EXAMPLES

[00101] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative puφoses only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

EXAMPLE 1 [00102] The biosensor chip shown in Figure 1 was exposed to APTES. Then a solution containing bacteriophage lambda, double stranded DNA (dsDNA), where each DNA molecule was 16 um in length, and therefore, imagable by atomic force microscopy, was contacted with the biosensor chip, and the current was measured. The addition of dsDNA gave a signal (Figure 2).

[00103] The biosensor chip with the dsDNA attached to the electrodes was then exposed to a solution containing the enzyme Dnase 1. The enzyme digests the DNA. The treatment of the biosensor with the enzyme resulted in no current being observed (Figure 2). Further, the biosensor chip treated with APTES and exposed to the solution containing the dsDNA, and exposed to the enzyme were imaged by atomic force microscopy, and the images are in Figure 5A, and 5B respectively. Figures 5A and 5B show that the electrical signal is a DNA- dependent signal, as treatment of the chip with DNase destroys the DNA (as imaged by atomic force microscopy) and abrogates the electrical signal.

[00104] The solution containing the dsDNA was heated for 15 minutes at 95°C thereby denaturing the DNA. The solution containing the denatured DNA was then contacted with the biosensor chip, and current was measured. The addition of denatured DNA did not yield a signal.

EXAMPLE 2

[00105] The biosensor chip shown in Figure 1 having gold electrodes was exposed to hexanethiol for a short time thereby forming a molecular wire monolayer, followed by exposure to a solution containing the ethidium-molecular wire for an extended deposition. The biosensor chip thus treated was exposed to a solution containing dsDNA. AFM images show that DNA binds to the chip at a very high density (Figure 10 A), and in the balled up conformation. Figure 10A indicates that the surface has too great an affinity for DNA.

[00106] The DNA containing solution was prepared by varying concentrations of magnesium chloride, and these solutions were then contacted with the biosensor chip. AFM images show that at a salt concentration of lOOmM and above, DNA being bound at high density and in a stretched out configuration (Figure 10B). Thus, the affinity of the dsDNA for the ethidium molecular wire surface can be decreased by depositing the DNA from a solution containing a salt solution, such as magnesium chloride.

EXAMPLE 3 [00107] PEG-silane modification: The biosensor were modified with PEG-silane. The biosensor chips were immersed in acetone for 30 minutes, followed with 1 minute of sonication, and then washed with EtOH and dried using an Ar stream. The dried chips were then treated with 1 hour UVO, and then immersed immediately in EtOH for 25-30 minutes. The PEG-silane solutions were prepared by mixing 0.1 % (mol/mol) Peg-silane and 1% (v/v) Dl H₂O in dry EtOH. If the total solution is 5 ml, for instance, the H₂O volume is 50 μl and Peg-silane 11.8 μl. The silane solution was hydrolyzed for 10 minutes and the chips were then immersed into the solution. The solution was gelated for 1 hour during which the solution was shaken 2 or 3 times. The chips were washed throughout with EtOH, dried using Ar, and then heated at 90°C for 2 hours under vacuum in an oven. The electrical signal of the PEG-silane treated chips was measured, and the potential range is from 0 to -7 volts.

[00108] DNA probing: 20 μl of DTP A Staph probe DNA (25 bases) solution [1 μM in 1 M phosphate buffer (K₂HPO :KH₂PO = 1 :1)] was used to cover the 100 μM device region on the surface of each chip. The chips were immersed in the solution for 1 hour, washed Dl H O for about 5 seconds, and then immersed inside freshly prepared 1 mM 6-mercapto-l-hexanol solution overnight. The chips were washed with Dl H₂O and dried in an Ar stream.

[00109] Hybridization with targets: 40 μl of matched (25 bases CompStaphBiotin) and mismatched (BioTarg 28mer) target DNA solutions (1 μM in 10 mM phosphate buffer / 150 mM MgCl₂) was added to the chips prepared above covering the surfaces of different chips. Hybridization was carried out at 40°C inside a hybridization oven for 1 hour. The chips were washed with 5 mM phosphate buffer / 10 mM MgCl₂ solution (preheated to 40°C) for about 30 seconds and dried with Ar stream.

[00110] NeutrAvidin binding: 40 μl of freshly prepared neutrAvidin solution (0.5 mg/ml in 0.1 M phosphate buffer) was added onto the surface of each chip and allowed to stay for 30 minutes. The chips were washed for 30 seconds with 5 mM phosphate buffer / 10 M MgCl₂ solution and then immersed into this solution for 30 minutes, followed with another 40-second washing with the same salt solution. Finally, the chips were dried using an Ar stream.

[00111] 1.4 kb biotinylated DNA stretching: 25 μl (2 x 12.5 μl) of biotinylated DNA (in

Dl H₂O) was added onto the surface of each chip with a pipettor and the solution was left on the surface for 1 hour. The chips were washed for 40 seconds with 5 M phosphate buffer / 10 mM MgCl solution, and for 20 second with EtOH to remove any salt. The chips were dried using an Ar stream.

[00112] Electrical measurement of DNA conductivity: both the matched and the mismatched chips were measured on Keithley 4200 system.

[00113] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. All publications, patents and patent applications cited herein are hereby incoφorated by reference in their entirety.

Claims

We Claim:

1. A method for detecting an analyte in a sample, the method comprising: providing a biosensor comprising a solid support and an electrode pair, wherein the electrode pair comprises a first electrode and a second electrode wherein at least one of the first electrode and the second electrode comprises a primary probe; contacting the biosensor with a sample suspected of comprising the analyte wherein, if present, the analyte specifically binds with the primary probe; spanning the electrode pair with a double stranded nucleic acid that completes an electric circuit between the electrode pair if the sample comprises the analyte; and measuring an electromagnetic property of the electrode pair, wherein the electromagnetic property indicates the presence of the analyte in the sample.

2. The method of claim 1, wherein the solid support is selected from the group consisting of plastic, glass, silicon, silicon oxide, silicon dioxide, mica, and quartz, and combinations thereof

3. The method of claim 2, wherein the solid support is silicon dioxide.

4. The method of claim 2, wherein the solid support is glass.

5. The method of claim 1, wherein the first electrode and the second electrode independently comprise a material selected from the group consisting of aluminum, cadmium, cesium, chromium, cobalt, copper, germanium, gold, indium tin oxide, iridium, iron, lithium, molybdenum, nickel, osmium, palladium, platinum, potassium, rhodium, rubidium, ruthenium, silicon, silver, sodium, titanium, tungsten, vanadium, and zinc.

6. The method of claim 5, wherein the material is gold.

7. The method of claim 1, wherein the biosensor comprises a self-assembled monolayer (SAM) selected from the group consisting of an alkanethiol, a phospholipids and an oligo (phenyl ene vinylene) .

8. The method of claim 1, wherein the primary probe comprises a nucleic acid, a protein, a polysaccharide, a lectin, or a sugar, or combinations thereof.

9. The method of claim 8, wherein the primary probe comprises a nucleic acid.

10. The method of claim 8, wherein the primary probe comprises a PNA.

1 1. The method of claim 8, wherein the primary probe comprises a protein.

12. The method of claim 1, wherein the sample is body fluids or tissues.

13. The method of claim 12, wherein the body fluids are selected from the group consisting blood, serum, plasma, urine, and saliva.

14. The method of claim 1, wherein the sample is a pathogen.

15. The method of claim 1, wherein the analyte is selected from the group consisting of a nucleic acid, a protein, a polysaccharide, a lectin, and a sugar, or combinations thereof.

16. The method of claim 15, wherein the analyte is nucleic acid.

17. The method of claim 16, wherein the nucleic acid is DNA, PNA, RNA, or combinations thereof.

18. The method of claim 15, wherein the protein is a receptor, an antibody, a binding portion of an antibody, a glycoproteins, an antigens, an epitopes, a protein A, or a protein G.

19. The method of claim 1, wherein the electromagnetic property is selected from the group consisting of direct electric current, alternating electric current, permitivity, resistivity, electron transfer, voltage, electrical impedance, signal loss, dissipation factor, resistance, capacitance, inductance, and electrical potential.

20. The method of claim 1, further comprising contacting the biosensor with a secondary probe before spanning, wherein the secondary probe binds to the analyte at a location different from the binding location of the primary probe.

21. The method of claim 20, wherein the secondary probe comprises connector molecules.

22. The method of claim 21, wherein the connector molecules are streptavidin, avidin, or neutravidin.

23. The method of claim 22, wherein the double stranded nucleic acid comprises biotin.

24. The method of claim 20, wherein the secondary probe further binds to a nucleic acid comprising a first nanoparticle but not to nucleic acid comprising a second nanoparticle or a second microsphere.

25. The method of claim 24, wherein the first nanoparticle comprises nucleic acid that hybridizes to the target, the secondary probe, and the nucleic acid on the second nanoparticle or the second microsphere.

26. The method of claim 24, wherein the second nanoparticle comprises nucleic acid that hybridizes to the nucleic acid on the first nanoparticle.

27. The method of claim 20, wherein the secondary probe further binds to the double stranded nucleic acid that completes the electric circuit.

28. The method of claim 20, wherein the secondary probe further binds to an oligonucleotide that can bind to the double stranded nucleic acid that completes the electric circuit.

29. The method of claim 1, wherein the analyte bound with the primary probe is DNA.

30. The method of claim 29, wherein the DNA is replicated to provide a double stranded DNA that completes the electric circuit between the electrode pair.

31. The method of claim 1 , wherein the spanning comprises adding a first microphere comprising an oligonucleotide complementary to the analyte, the secondary probe, or both, and the binding of the oligonucleotide with the analyte, the secondary probe or both provides the double stranded nucleic acid that completes the electric circuit.

32. The method of claim 31, further comprising adding a second microsphere comprising an oligonucleotide complementary to the oligonucleotide comprising the first microsphere but not to the analyte or the secondary probe.

33. A method for detecting a double stranded nucleic acid in a sample, the method comprising: providing a biosensor comprising a solid support and an electrode pair, wherein the electrode pair comprises a first electrode and a second electrode wherein at least one of the first electrode and the second electrode comprises a means for attaching a double stranded nucleic acid; contacting the biosensor with a sample suspected of comprising the double stranded nucleic acid, wherein, if the sample comprises the double stranded nucleic acid, the double stranded nucleic acid attaches to the attachment means; spanning the electrode pair with the double stranded nucleic acid and completing an electric circuit between the electrode pair if the sample comprises the double stranded nucleic acid; and measuring an electromagnetic property across the electrode pair, wherein the electromagnetic property indicates the presence of the double stranded nucleic acids in the sample.

34. The method of claim 33, wherein the solid support is selected from the group consisting of plastic, glass, silicon, silicon oxide, silicon dioxide, mica, and quartz, and combinations thereof

35. The method of claim 34, wherein the solid support is silicon dioxide.

36. The method of claim 33, wherein the first electrode and the second electrode independently comprise a material selected from the group consisting of aluminum, cadmium, cesium, chromium, cobalt, copper, germanium, gold, indium tin oxide, iridium, iron, lithium, molybdenum, nickel, osmium, palladium, platinum, potassium, rhodium, rubidium, ruthenium, silicon, silver, sodium, titanium, tungsten, vanadium, and zinc.

37. The method of claim 36, wherein the material is gold.

38. The method of claim 33, wherein the biosensor further comprises a self-assembled monolayer (SAM) selected from the group consisting of an alkanethiol, a phospholipids and an oligo (phenylenevinylene).

39. The method of claim 38, wherein the means of attaching comprises a sulfur group, an amino group, an intercalator, or avidin streptavidin-biotin

40. The method of claim 39, wherein the means is a sulfur group and the sulfur group comprises the double stranded nucleic acid.

41. The method of claim 39, wherein the means comprises an amino group.

42. The method of claim 41, wherein the amino group is an amino functional silane or aminoalkane thiol.

43. The method of claim 41, wherein the means comprises an intercalator.

44. The method of claim 43, wherein the intercalator comprises a molecular wire having the structure HS-L-I, wherein L is a linker and I is an intercalating compound.

45. The method of claim 44, wherein L is-(-C≡C-)_n-, -(-C=C-)_n-, -(-C≡C- Ar)_n-, or -(- C=C-Ar)_n-, wherein n is an integer between 0 and 10, and Ar is a substituted or unsubstituted aryl or heteroaryl group.

46. The method of claim 45, wherein L is -(-C≡C-Ph)_n- wherein n is an integer between 0 and 10.

47. The method of claim 44, wherein I is selected from the group consisting of acridine orange, naphthalene diimide, methylene blue, daunomycin, bisacridine, and ethidium.

48. The method of claim 47, wherein I is ethidium.

49. The method of claim 43, further comprising a filler molecule.

50. The method of claim 49, wherein the filler molecule is an alkane thiol.

51. The method of claim 33, wherein the electromagnetic property is selected from the group consisting of direct electric current, alternating electric current, permitivity, resistivity, electron transfer, voltage, electrical impedance, signal loss, dissipation factor, resistance, capacitance, inductance, and electrical potential.

52. The method of claim 33, wherein the sample is body fluids or tissues.

53. The method of claim 52, wherein the body fluids are selected from the group consisting blood, serum, plasma, urine, and saliva.

54. The method of claim 33, wherein the sample is a pathogen.

55. A method for performing amplification of a target nucleic acid molecule on a biosensor, the method comprising: providing a biosensor comprising a solid support and an electrode pair, wherein the electrode pair comprises a first electrode and a second electrode wherein at least one of the first electrode and the second electrode comprises a primary probe; contacting the biosensor with a sample suspected of comprising the target nucleic acid and a hybridization solution wherein if the sample comprises the target nucleic acid, the target nucleic acid hybridizes to the probe; and providing the means for performing said target amplification.

56. The method of claim 55, wherein the solid support is selected from the group consisting of plastic, glass, silicon, silicon oxide, silicon dioxide, mica, and quartz, and combinations thereof

57. The method of claim 56, wherein the solid support is silicon dioxide.

58. The method of claim 55, wherein the first electrode and the second electrode independently comprise a material selected from the group consisting of aluminum, cadmium, cesium, chromium, cobalt, copper, germanium, gold, indium tin oxide, iridium, iron, lithium, molybdenum, nickel, osmium, palladium, platinum, potassium, rhodium, rubidium, ruthenium, silicon, silver, sodium, titanium, tungsten, vanadium, and zinc.

59. The method of claim 58, wherein the material is gold.

60. The method of claim 55, wherein the biosensor comprises a self-assembled monolayer (SAM) selected from the group consisting of an alkanethiol, a phospholipids and an oligo (phenylenevinylene).

61. The method of claim 55, wherein the primary probe comprises DNA, PNA, RNA, or combinations thereof.

62. The method of claim 61, wherein the primary probe comprises a DNA.

63. The method of claim 61, wherein the primary probe comprises a PNA.

64. The method of claim 55, wherein target amplification comprises a method selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA), rolling circle amplification (RCA), branched chain amplification (BCA), ligase chain reaction (LCR) and isothermal nucleic acid sequence-based amplification (NASBA).

65. The method of claim 55, wherein the sample is body fluids or tissues.

66. The method of claim 65, wherein the body fluids are selected from the group consisting blood, serum, plasma, urine, and saliva.

67. The method of claim 55, wherein the sample is a pathogen.