WO2004096986A2

WO2004096986A2 - Method for quantitative detection of nucleic acid molecules

Info

Publication number: WO2004096986A2
Application number: PCT/US2004/012161
Authority: WO
Inventors: Dennis M. Connolly; Charles D. Deboer
Original assignee: Integrated Nano-Technologies, Llc
Priority date: 2003-04-29
Filing date: 2004-04-20
Publication date: 2004-11-11
Also published as: WO2004096986A3

Abstract

The present invention is directed to a method of quantitatively detecting target nucleic acid molecules in a sample. In carrying out this method, one or more different groups of two or more electrically separated electrical conductors are provided with capture probes attached to the conductors such that a gap exists between the capture probes on the electrically separated conductors. The capture probes are contacted with a sample, potentially containing the target nucleic acid molecules, under conditions effective to permit any of the target nucleic acid molecule present in the sample to hybridize to the capture probes and thereby connect the capture probes. The presence of the target nucleic acid molecules is detected by determining whether electricity is conducted between the electrically separated conductors. The electrically separated conductors are then interrogated to quantify the concentration of target nucleic acid molecules present in the sample.

Description

METHOD FOR QUANTITATIVE DETECTION OF NUCLEIC ACID

MOLECULES

FIELD OF THE INVENTION

[0001] This invention relates to methods for the quantitative detection of nucleic acid molecules.

BACKGROUND OF THE INVENTION

[0002] Nucleic acids, such as DNA or RNA, have become of increasing interest as analytes for clinical or forensic uses. Powerful new molecular biology technologies enable one to detect congenital or infectious diseases. These same technologies can characterize DNA for use in settling factual issues in legal proceedings, such as paternity suits and criminal prosecutions. [0003] For the analysis and testing of nucleic acid molecules, amplification of a small amount of nucleic acid molecules, isolation of the amplified nucleic acid fragments, and other procedures are necessary. The science of amplifying small amounts of DNA have progressed rapidly and several methods now exist. These include linked linear amplification, ligation-based amplification, transcription-based amplification and linear isothermal amplification. Linked linear amplification is described in detail in U.S. Patent No. 6,027,923 to Wallace et al. Ligation-based amplification includes the ligation amplification reaction (LAR) described in detail in Wu et al., Genomics, 4:560 (1989) and the ligase chain reaction described in European Patent No. 0320308B1. Transcription-based amplification methods are described in detail in U.S. Patent Nos. 5,766,849 and 5,654,142, Kwoh et al., Proc. Natl. Acad. Sci.

U.S.A., 86:1173 (1989), and PCT Publication No. WO 88/10315 to Ginergeras et al. The more recent method of linear isothermal amplification is described in U.S. Patent No. 6,251,639 to Kurn. [0004] The most common method of amplifying DNA is by the polymerase chain reaction ("PCR"), described in detail by Mullis et al., Cold

Spring Harbor Quant. Biol.. 51:263-273 (1986), European Patent No. 201,184 to Mullis, U.S. Patent No. 4,582,788 to Mullis et al, European Patent Nos. 50,424, 84,796, 258017, and 237362 to Erlich et al., and U.S. Patent No. 4,683,194 to Saiki et al. The PCR reaction is based on multiple cycles of hybridization and nucleic acid synthesis and denaturation in which an extremely small number of nucleic acid molecules or fragments can be multiplied by several orders of magnitude to provide detectable amounts of material. One of ordinary skill in the art knows that the effectiveness and reproducibility of PCR amplification is dependent, in part, on the purity and amount of the DNA template. Certain molecules present in biological sources of nucleic acids are known to stop or inhibit PCR amplification (Belec et al., Muscle and Nerve, 21 (8): 1064 (1998);

Wiedbrauk et al., Journal of Clinical Microbiology, 33(10):2643-6 (1995); Deneer and Knight, Clinical Chemistry, 40(l):171-2 (1994)). For example, in whole blood, hemoglobin, lactoferrin, and immunoglobulin G are known to interfere with several DNA polymerases used to perform PCR reactions (Al-Soud and Radstrom, Journal of Clinical Microbiology. 39(2):485^193 (2001); Al-Soud et al., Journal of Clinical Microbiology. 38(l):345-50 (2000)). These inhibitory effects can be more or less overcome by the addition of certain protein agents, but these agents must be added in addition to the multiple components already used to perform the PCR. Thus, the removal or inactivation of such inhibitors is an important factor in amplifying DNA from select samples.

[0005] On the other hand, isolation and detection of particular nucleic acid molecules in a mixture requires a nucleic acid sequencer and fragment analyzer, in which gel electrophoresis and fluorescence detection are combined. Unfortunately, electrophoresis becomes very labor-intensive as the number of samples or test items increases.

[0006] For this reason, a simpler method of analysis using DNA oligonucleotide probes is becoming popular. New technology, called VLSIPS™, has enabled the production of chips smaller than a thumbnail where each chip contains hundreds of thousands or more different molecular probes. These techniques are described in U.S. Patent No. 5,143,854 to Pirrung et al., PCT

Publication No. WO 92/10092, and PCT WO 90/15070. These biological chips have molecular probes arranged in arrays where each probe ensemble is assigned a specific location. These molecular array chips have been produced in which each probe location has a center to center distance measured on the micron scale. Use of these array type chips has the advantage that only a small amount of sample is required, and a diverse number of probe sequences can be used simultaneously. Array chips have been useful in a number of different types of scientific applications, including measuring gene expression levels, identification of single nucleotide polymorphisms, and molecular diagnostics and sequencing as described in U.S. Patent No. 5,143,854 to Pirrung et al.

[0007] Array chips where the probes are nucleic acid molecules have been increasingly useful for detection for the presence of specific DNA sequences. Most technologies related to array chips involve the coupling of a probe of known sequence to a substrate that can either be structural or conductive in nature. Structural types of array chips usually involve providing a platform where probe molecules can be constructed base by base or covalently binding a completed molecule. Typical array chips involve amplification of the target nucleic acid followed by detection with a fluorescent label to determine whether target nucleic acid molecules hybridize with any of the oligonucleotide probes on the chip. After exposing the array to a sample containing target nucleic acid molecules under selected test conditions, scanning devices can examine each location in the array and quantitate the amount of hybridized material at that location. Alternatively, conductive types of array chips contain probe sequences linked to conductive materials such as metals. Hybridization of a target nucleic acid typically elicits an electrical signal that is carried to the conductive electrode and then analyzed. [0008] For most solid support or array technologies, small oligonucleotide capture probes are immobilized or synthesized on the support. The sequence of the capture probes imparts the specificity for the hybridization reaction. Several different chemical compositions exist currently for capture probe studies. The standard for many years has been straight deoxyribonucleic acids. The advantage of these short single stranded DNA molecules is that the technology has existed for many years and the synthesis reaction is relatively inexpensive. Furthermore, a large body of technical studies is available for quick reference for a variety of scientific techniques, including hybridization. However, many different types of DNA analogs are now being synthesized commercially that have advantages over DNA oligonucleotides for hybridization. Some of these include PNA (protein nucleic acid), LNA (locked nucleic acid) and methyl phosphonate chemistries. In general, all of the DNA analogs have higher melting temperatures than standard DNA oligonucleotides and can more easily distinguish between a fully complementary and single base mis-match target. This is possible because the DNA analogs do not have a negatively charged backbone, as is the case with standard DNA. This allows for the incoming strand of target DNA to bind tighter to the DNA analog because only one strand is negatively charged. The most studied of these analogs for hybridization techniques is the PNA analog, which is composed of a protein backbone with substituted nucleobases for the amino acid side chains (see www.appliedbiosystems.com or www.eurogentec.com). Indeed, PNAs have been used in place of standard DNA for almost all molecular biology techniques including DNA sequencing (Arlinghaus et al., Anal Chem., 69:3747- 53 (1997)), DNA fingerprinting (Guerasimova et al, Biotechniques, 31:490-495 (2001)), diagnostic biochips (Prix et al., Clin. Chem., 48:428-35 (2002); Feriotto et al., Lab Invest, 81:1415-1427 (2001)), and hybridization based microarray analysis (Weiler et al, Nucleic Acids Res. 25:2792-2799 (1997); Igloi, Genomics. 74:402-407 (2001)). [0009] Techniques for forming sequences on a substrate are known. For example, the sequences may be formed according to the techniques disclosed in U.S. Patent No. 5,143,854 to Pirrang et al., PCT Publication No. WO 92/10092, or U.S. Patent No. 5,571,639 to Hubbell et al. Although there are several references on the attachment of biologically useful molecules to electrically insulating surfaces such as glass (^ttp://www.piercenet.com/Teclmical/default.cfm?tmpl=../Lib/ViewDoc.cfm =3483; McGovern et al., Langmuir. 10:3607-3614 (1994)) or silicon oxide (Examples 4-6 of U.S. Patent No. 6,159,695 to McGovern et al.), there are few examples of effective molecular attachment to electrically conducting surfaces except for gold (Bain et al., Langmuir. 5:723-727 (1989)) and silver (Xia et al., Langmuir, 22:269, (1998)). In general, the problem of attaching biologically active molecules to the surface of a substrate, whether it is a metal electrical conductor or an electrical insulator such as glass, is more difficult than the simple chemical reaction of a reactive group on the biological molecule with a complementary reactive group on the substrate. For example, a metal electrical conductor has no reactive sites, in principle, except those that may be adventitiously or deliberately positioned on the surface of the metal. [0010] Hybridization of target DNAs to such surface bound capture probes poses difficulties not seen, if both species are soluble. Steric effects result from the solid support itself and from too high of a probe density. Studies have shown that hybridization efficiency can be altered by the insertion of a linker moiety that raises the complementary region of the probe away from the surface (Schepinov et al., Nucleic Acid Res.. 25:1155-1161 (1997); Day et al., Biochem J.. 278:735-740 (1991)), the density at which probes are deposited (Peterson et al., Nucleic Acids Res.. 29:5163-5168 (2001); Wilkins et al., Nucleic Acids Res.. 27:1719-1729)), and probe conformation (Riccelli et al., Nucleic Acids Res.. 29:996-1004 (2001)). Insertion of a linker moiety between the complementary region of a probe and its attachment point can increase hybridization efficiency and optimal hybridization efficiency has been reported for linkers between 30 and 60 atoms in length.

Likewise, studies of probe density suggest that there is an optimum probe density, and that this density is less than the total saturation of the surface (Schepinov et al., Nucleic Acid Res.. 25:1155-1161 (1997); Peterson et al, Nucleic Acids Res., 29:5163-5168 (2001); Steel et al., Anal. Chem., 70:4670-4677 (1998)). For example, Peterson et al. reported that hybridization efficiency decreased from 95% to 15% with probe densities of 2.0x10¹² molecules/cm² and 12.0x10¹² molecules/cm , respectively.

[0011] Quantitiation of hybridization events often depends on the type of signal generated from the hybridization reaction. The most common analysis technique is fluorescent emission from several different types of dyes and fluorophores. However, quantitating samples in this manner usually requires a large amount of the signaling molecule to be present to generate enough emission to be quantitated accurately. More importantly, quantitation of fluorescence generally requires expensive analysis equipment for linear response. Furthermore, the hybridization reactions take up to two hours, which for many uses, such as detecting biological warfare agents, is simply too long. Therefore, a need exists for a system which can rapidly detect and quantitate biological material in samples. [0012] The present invention is directed to achieving these objectives.

SUMMARY OF THE INVENTION

[0013] The present invention is directed to a method of quantitatively detecting target nucleic acid molecules in a sample. In carrying out this method, one or more different groups of two or more electrically separated electrical conductors are provided with capture probes attached to the conductors such that a gap exists between the capture probes on the electrically separated conductors. The capture probes are contacted with a sample, potentially containing the target nucleic acid molecules, under conditions effective to permit any of the target nucleic acid molecule present in the sample to hybridize to the capture probes and thereby connect the capture probes. The presence of the target nucleic acid molecules is detected by determining whether electricity is conducted between the electrically separated conductors. The electrically separated conductors are then interrogated to quantify the concentration of target nucleic acid molecules present in the sample.

[0014] The ability of the present invention to quantify the amount of a target nucleic acid molecule in a sample has many benefits. Some disease states are rated by the number of organisms or molecules present in a fixed quantity of fluid. For example, the severity of HJN diagnosis is often dependent on viral load, or the amount of HIV particles circulating in the bloodstream. Diagnosing any particular patient with HIV is relatively easy to perform, but it would be advantageous to obtain additional quantitative information. Likewise, the progression of the disease and the effectiveness of treatment are again judged by the individual's viral load. In other instances, it would be good to obtain quantitative measurements of the numbers of organisms present in a sample of interest. Not only can the present invention detect the presence of biowarfare agents, such as anthrax or smallpox, but it will allow for precise quantitation of the severity of attack. This is important when determining the level of exposure to personnel, who is to receive antibiotics first, and how best to cleanup and decontaminate after exposure. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 shows a preferred method of attaching oligomer DNA probes to a metal surface.

[0016] FIGS. 2 and 3 show a method of preparing two different gold surfaces, one unblocked and one blocked with cyanide ions.

[0017] FIG. 4 shows a method of directing different oligomer DNA probes to different electrodes by covering one of the gold electrodes with a blocking agent.

[0018] FIG. 5 shows a digital electron micrograph of gold wires deposited on DNA strands between electrodes.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention is directed to a method of quantitatively detecting target nucleic acid molecules in a sample. In carrying out this method, one or more different groups of two or more electrically separated electrical conductors are provided with capture probes attached to the conductors such that a gap exists between the capture probes on the electrically separated conductors. The capture probes are contacted with a sample, potentially containing the target nucleic acid molecules, under conditions effective to permit any of the target nucleic acid molecule present in the sample to hybridize to the capture probes and thereby connect the capture probes. The presence of the target nucleic acid molecules is detected by determining whether electricity is conducted between the electrically separated conductors. The electrically separated conductors are then interrogated to quantify the concentration of target nucleic acid molecules present in the sample.

[0020] One aspect of the present invention involves the detection of multiple DNA sequences from a plurality of DNA sequences based on hybridization techniques. This method involves a sample collection method whereby bacteria, viruses or other DNA containing species are collected and concentrated. This method also incorporates a sample preparation method that involves the liberation of the genetic components. After liberating the DNA, the sample is injected into a detection chip containing complementary DNA probes for the target of interest. In this manner, the device may contain multiple sets of probe molecules that each recognizes a single but different DNA sequence. This process ultimately involves the detection of hybridization products. [0021] In the collection phase, bacteria, viruses or other DNA containing samples are collected and concentrated. A plurality of collection methods will be used depending on the type of sample to be analyzed. Liquid samples will be collected by placing a constant volume of the liquid into a lysis buffer. Airborne samples can be collected by passing air over a filter for a constant time. The filter will be washed with lysis buffer. Alternatively, the filter can be placed directly into the lysis buffer. Waterborne samples can be collected by passing a constant amount of water over a filter. The filter can then be washed with lysis buffer or soaked directly in the lysis buffer. Dry samples can be directly deposited into lysis buffer for removal of the organism of interest. [0022] After sample collection and lysis, cell debris can be removed by precipitation or filtration. Ideally, the sample will be concentrated by filtration, which is more rapid and does not required special reagents. Samples will be forced through filters that will allow only the cellular material to pass through, trapping whole organisms and broken cell debris. [0023] Here, the electrical conductivity of nucleic acid molecules is relied upon to transmit the electrical signal. Hans- Werner Fink and Christian Schoenenberger reported in Nature (1999), which is hereby incorporated by reference in its entirety, that DNA conducts electricity like a semiconductor. This flow of current can be sufficient to construct a simple switch, which will indicate whether or not a target nucleic acid molecule is present within a sample.

Optionally, after hybridization of the target nucleic acid molecules to sets of capture probes, the nucleic acid molecules can be coated with a conductor, such as a metal. This technique is described in PCT Publication Nos. WO 99/04440, WO 99/57550, and WO 00/25136, U.S. Patent No. 6,399,303, U.S. Patent Application Serial Nos. 10/288,657 and 10/383,397, and U.S. Patent Publication Nos. US

20020182608 and US 20030040000, which are hereby incorporated by reference in their entirety. The coated nucleic acid molecule can then conduct electricity across the gap between the pair of probes, thus producing a detectable signal indicative of the presence of a target nucleic acid molecule. In order to increase the chances of capturing a target DNA of interest from a dilute and complex mixture of DNA sequences, multiple test structures can be placed within the test cartridge. These test structures can be used to detect the same target nucleic acid molecule, if present in a sample, a plurality of times or to detect different nucleic acid molecules, if present in a sample. In the latter case, the different probes can be designed to capture different target nucleic acid molecules from a single source (e.g. organism) to verify that that source is indeed present in a sample. Alternatively, the probe could be designed to capture target nucleic acid molecules from different sources (e.g. organisms) to permit a sample to be subjected to a battery of tests. These alternative strategies are particularly useful in analyzing a sample for pathogens. The advantage of this approach is to be able to overcome a low false positive rate by positively identifying the presence of any organism by use of statistics. [0024] In carrying out the method of the present invention, a sample collection phase is initially carried out where bacteria, viruses, or other species are collected and concentrated. The target nucleic acid molecule whose sequence is to be determined is usually isolated from a tissue sample. If the target nucleic acid molecule is genomic, the sample may be from any tissue (except exclusively red blood cells). For example, whole blood, peripheral blood lymphocytes or PBMC, skin, hair, or semen are convenient sources of clinical samples. These sources are also suitable if the target is RNA. Blood and other body fluids are also a convenient source for isolating viral nucleic acids. If the target nucleic acid molecule is mRNA, the sample is obtained from a tissue in which the mRNA is expressed. If the target nucleic acid molecule in the sample is RNA, it may be reverse transcribed to DNA, but need not be converted to DNA in the present invention.

[0025] Further details of how to carry out the process of the present invention are set forth in U.S. Patent No. 6,399,303 Bl to Connolly, which is hereby incorporated by reference in its entirety.

[0026] A plurality of collection methods can be used depending on the type of sample to be analyzed. Liquid samples can be collected by placing a constant volume of the liquid into a lysis buffer. Airborne samples can be collected by passing air over a filter for a constant time. The filter can be washed with lysis buffer. Alternatively, the filter can be placed directly into the lysis buffer. Waterborne samples can be collected by passing a constant amount of water over a filter. The filter can then be washed with lysis buffer or soaked directly in the lysis buffer. Dry samples can be directly deposited into lysis buffer for removal of the organism of interest.

[0027] When whole cells, viruses, or other tissue samples are being analyzed, it is typically necessary to extract the nucleic acids from the cells or viruses, prior to continuing with the various sample preparation operations. Accordingly, following sample collection, nucleic acids may be liberated from the collected cells, viral coat, etc., into a crude extract, followed by additional treatments to prepare the sample for subsequent operations such as denaturation of contaminating (DNA binding) proteins, purification, filtration, and desalting. [0028] Liberation of nucleic acids from the sample cells or viruses, and denaturation of DNA binding proteins may generally be performed by physical or chemical methods. For example, chemical methods generally employ lysing agents to disrupt the cells and extract the nucleic acids from the cells, followed by treatment of the extract with chaotropic salts such as guanidinium isothiocyanate or urea, to denature any contaminating and potentially interfering proteins. Generally, where chemical extraction and/or denaturation methods are used, the appropriate reagents may be incorporated within the extraction chamber, a separate accessible chamber, or externally introduced.

[0029] Alternatively, physical methods may be used to extract the nucleic acids and denature DNA binding proteins. U.S. Patent No. 5,304,487, which is hereby incorporated by reference in its entirety, discusses the use of physical protrusions within microchannels or sharp edged particles within a chamber or channel to pierce cell membranes and extract their contents. More traditional methods of cell extraction may also be used, e.g., employing a channel with restricted cross-sectional dimension which causes cell lysis when the sample is passed through the channel with sufficient flow pressure. Alternatively, cell extraction and denaturing of contaminating proteins may be carried out by applying an alternating electrical current to the sample. More specifically, the sample of cells is flowed through a microtubular array while an alternating electric current is applied across the fluid flow. A variety of other methods may be utilized within the device of the present invention to effect cell lysis/extraction, including, e.g., subjecting cells to ultrasonic agitation, or forcing cells through microgeometry apertures, thereby subjecting the cells to high shear stress resulting in rupture.

[0030] Following extraction, it is often desirable to separate the nucleic acids from other elements of the crude extract, e.g., denatured proteins, cell membrane particles, and the like. Removal of particulate matter is generally accomplished by filtration, flocculation, or the like. Ideally, the sample is concentrated by filtration, which is more rapid and does not require special reagents. A variety of filter types may be readily incorporated into the device. Samples can be forced through filters that will allow only the cellular material to pass through, trapping whole organisms and broken cell debris. Further, where chemical denaturing methods are used, it may be desirable to desalt the sample prior to proceeding to the next step. Desalting of the sample, and isolation of the nucleic acid may generally be carried out in a single step, e.g., by binding the nucleic acids to a solid phase and washing away the contaminating salts or performing gel filtration chromatography on the sample. Suitable solid supports for nucleic acid binding include, e.g., diatomaceous earth, silica, or the like. Suitable gel exclusion media is also well known in the art and is commercially available from, e.g., Pharmacia and Sigma Chemical. This isolation and/or gel filtration/desalting may be carried out in an additional chamber, or alternatively, the particular chromatographic media may be incorporated in a channel or fluid passage leading to a subsequent reaction chamber. [0031] The probes are preferably selected to bind with the target such that they have approximately the same melting temperature. This can be done by varying the lengths of the hybridization region. A-T rich regions may have longer target sequences, whereas G-C rich regions would have shorter target sequences. [0032] Hybridization assays on substrate-bound oligonucleotide arrays involve a hybridization step and a detection step. In the hybridization step, the sample potentially containing the target and an isostabilizing agent, denaturing agent, or renaturation accelerant is brought into contact with the probes of the array and incubated at a temperature and for a time appropriate to allow hybridization between the target and any complementary probes. [0033] Including a hybridization optimizing agent in the hybridization mixture significantly improves signal discrimination between perfectly matched targets and single-base mismatches. As used herein, the term "hybridization optimizing agent" refers to a composition that decreases hybridization between mismatched nucleic acid molecules, i.e., nucleic acid molecules whose sequences are not exactly complementary. [0034] An isostabilizing agent is a composition that reduces the base-pair composition dependence of DNA thermal melting transitions. More particularly, the term refers to compounds that, in proper concentration, result in a differential melting temperature of no more than about 1°C. for double stranded DNA oligonucleotides composed of AT or GC, respectively. Isostabilizing agents preferably are used at a concentration between 1 M and 10 M, more preferably between 2 M and 6 M, most preferably between 4 M and 6 M, between 4 M and 10 M, and, optimally, at about 5 M. For example, a 5 M agent in 2 x SSPE (Sodium Chloride/Sodium Phosphate/EDTA solution) is suitable. Betaines and lower tetraalkyl ammonium salts are examples of suitable isostabilizing agents. [0035] Betaine (N,N,N,-trimethylglycine; (Rees et al., Biochem., (1993) 32:137-144), which is hereby incorporated by reference in its entirety) can eliminate the base pair composition dependence of DNA thermal stability. Unlike tetramethylammonium chloride ("TMAC1"), betaine is zwitterionic at neutral pH and does not alter the polyelectrolyte behavior of nucleic acids while it does alter the composition-dependent stability of nucleic acids. Inclusion of betaine at about 5 M can lower the average hybridization signal, but increases the discrimination between matched and mismatched probes.

[0036] A denaturing agent is a compositions that lowers the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double-stranded nucleic acid or the hydration of nucleic acid molecules. Denaturing agents can be included in hybridization buffers at concentrations of about 1 M to about 6 M and, preferably, about 3 M to about 5.5 M. [0037] Denaturing agents include formamide, formaldehyde, dimethylsulfoxide ("DMSO"), tetraethyl acetate, urea, guanidine thiocyanate ("GuSCN"), glycerol and chaotropic salts. As used herein, the term "chaotropic salt" refers to salts that function to disrupt van der Waal's attractions between atoms in nucleic acid molecules. Chaotropic salts include, for example, sodium trifluoroacetate, sodium tricholoroacetate, sodium perchlorate, and potassium thiocyanate.

[0038] A renaturation accelerant is a compound that increases the speed of renaturation of nucleic acids by at least 100-fold. They generally have relatively unstructured polymeric domains that weakly associate with nucleic acid molecules. Accelerants include heterogenous nuclear ribonucleoprotein ("hnRP") Al and cationic detergents such as, preferably, cetyltrimethylammonium bromide ("CTAB") and dodecyl trimethylammonium bromide ("DTAB"), and, also, polylysine, spermine, spermidine, single stranded binding protein ("SSB"), phage T4 gene 32 protein, and a mixture of ammonium acetate and ethanol. Renaturation accelerants can be included in hybridization mixtures at concentrations of about 1 μM to about 10 mM and, preferably, 1 μM to about 1 mM. The CTAB buffers work well at concentrations as low as 0.1 mM. [0039] Addition of small amounts of ionic detergents (such as N-lauroyl- sarkosine) to the hybridization buffers can also be useful. LiCl is preferred to

NaCl. Hybridization can be at 20°-65°C, usually 37°C to 45°C for probes of about 14 nucleotides. Additional examples of hybridization conditions are provided in several sources, including: Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y. (1989); and Berger and Kimmel, "Guide to Molecular Cloning Techniques," Methods in Enzymology, Volume 152, Academic Press, Inc., San Diego, Calif. (1987); Young and Davis, Proc. Natl. Acad. Sci. USA, 80:1194 (1983), which are hereby incorporated by reference in their entirety. [0040] In addition to aqueous buffers, non-aqueous buffers may also be used. In particular, non-aqueous buffers which facilitate hybridization but have low electrical conductivity are preferred.

[0041] The sample and hybridization reagents are placed in contact with the array and incubated. Contact can take place in any suitable container, for example, a dish or a cell specially designed to hold the probe array and to allow introduction and removal of fluids. Generally, incubation will be at temperatures normally used for hybridization of nucleic acids, for example, between about 20°C and about 75°C, e.g., about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, or about 65°C. For probes longer than about 14 nucleotides, 37-45°C is preferred. For shorter probes, 55-65°C is preferred. More specific hybridization conditions can be calculated using formulae for determining the melting point of the hybridized region. Preferably, hybridization is carried out at a temperature at or between ten degrees below the melting temperature and the melting temperature. More preferred, hybridization is carried out at a temperature at or between five degrees below the melting temperature and the melting temperature. The target is incubated with the capture probes for a time sufficient to allow the desired level of hybridization between the target and any complementary capture probes. After incubation with the hybridization mixture, the electrically separated conductors are washed with the hybridization buffer, which also can include the hybridization optimizing agent. These agents can be included in the same range of amounts as for the hybridization step, or they can be eliminated altogether. [0042] Details on how capture probes are attached to electrical conductors are set forth in U.S. Patent Application Serial No. 10/288,657, which is hereby incorporated by reference in its entirety.

[0043] FIG. 1 illustrates the method of attaching oligomer probes to a gold surface. The short DNA oligomer is terminated with a six carbon chain ending in a thiol, or mercapto group. The oligomer is normally shipped from the manufacturer in the oxidized, or disulfide form. Before attachment to the gold surface, the oligomer is reduced to the thiol form by treatment with an excess of a reducing agent such as sodium borohydride or TCEP (Tris[2- Carboxyethylphosphine] hydrochloride). The gold surface is then immersed in a solution of the reduced oligomer for a period of approximately 10 minutes, whereupon a self-assembled monolayer of the oligomer covers the gold surface. [0044] For efficient detection of a low number of target nucleic acid molecules, it is advantageous for the probe oligomers to correspond to the beginning and end regions of the target nucleic acid molecule be attached to adjacent metal electrodes, and not be attached to different regions of a single electrodes, which could lead to target being hybridized along a single electrode, and thus the loss of an electrical signal.

[0045] In a preferred embodiment of the present invention, one set of electrically connected nickel electrodes is plated with gold, probe oligomers corresponding to one end of the target nucleic acid molecule are attached to the gold, any remaining open gold is blocked with an alkane thiol, the remaining second set of electrically connected nickel electrodes is plated with gold, and the second probe oligomer corresponding to the other end of the target nucleic acid molecule is attached to the gold.

[0046] In another preferred embodiment of the present invention, both nickel electrodes are plated with gold, the first from a gold cyanide solution and the second from a gold sulfite solution. It has been found that gold can be electroplated onto nickel electrodes from either a solution of gold cyanide or gold sulfite. Moreover, it has been found that gold which has been electroplated from a solution of gold cyanide ions on its surface, which will block the attachment of a thiol terminated nucleic acid probe molecule. Further confirmation of the blocking effect of cyanide ions is also found in view of the observation that a gold surface treated with cyanide ions will no longer cause evolution of oxygen bubbles from an ammonia solution of hydrogen peroxide. Therefore, gold is electroplated from a gold cyanide solution onto the left hand electrodes of a set of nickel electrodes as shown in FIG. 2. After rinsing with water, gold is electroplated from a solution of gold sulfite onto the right hand electrodes as shown in FIG. 3. The separation of the two electrodes in these structures is very small, on the order of about a micron, which is small enough so that a reasonable sized target nucleic acid molecule, about 3000 to 5000 base pairs, can reach from one electrode to the other and form a bridge. In addition, the width of the electrode wires is also small, about 2 microns, so that nucleic acid probes bound near the center of the wires can still form bridges with target nucleic acid molecules to the other electrode. This is important because the small size of the features of the structures in FIGS. 2 and 3 make it impossible to direct the nucleic acid probes to one electrode or the other by mechanical means such as, by placing different droplets of solution on one feature or the other. The surface tension of water, along with the small size of the features, makes mechanical placement methods practically impossible. Electroplating, on the other hand, allows us to put cyanide blocked gold on one electrode and sulfite (unblocked) gold on the other electrode without precision manipulations. If such a set of electrodes, shown in FIG. 3, is bathed with a solution of nucleic acid probes in neutral or basic pH buffer, the nucleic acid will only attach to the sulfite plated gold, which is not blocked. No significant amount of the nucleic acid goes to the cyanide blocked gold electrodes. When the sulfite gold electrodes are saturated with nucleic acid the electrodes are rinsed with water and then bathed in a solution of nucleic acid probes in acidic pH buffer. The acid removes the cyanide ion and the thiol terminated nucleic acid probes are attached to the now un-blocked gold surface. The final result is one kind of nucleic acid probe on the sulfite plated gold and another kind of nucleic acid probe on the cyanide plate gold. When such electrodes with directed probes are exposed to target nucleic acid molecules, having end base sequences complementary to the sequences of the probes, under hybridization conditions, the result of hybridization between the target nucleic acid molecules and the probes will be bridges of nucleic acids from one electrode to the other. No target nucleic acid molecules will be able to form unproductive bridges from one part on an electrode wire to another part of the same wire, because each wire will have only probes complementary to one end of the target nucleic acid molecules.

[0047] In practice, it has been found that the electrodeposition of gold onto metal electrodes depends on the type of metal used for the electrodes, the size and area of the electrodes, the voltage and current and time used in the electrodeposition, and the concentration and chemical makeup of the electrodeposition solution used. For example, it is difficult or impossible to electroplate gold onto chromium with good uniformity and adhesion, On the other hand, electroplated gold on nickel or cobalt has good uniformity and adhesion. When the electrodes consist of wire 2 microns wide by 250 microns long and spaced one micron apart, with a total number of 700 wires to a set, good quality gold electrodeposition is achieved by grounding the electrodes and passing 6 microamps of direct current through a gold anode placed about 1mm above the electrodes, using a commercial gold cyanide electroplating such as the SG-10 gold solution sold by the Transene Company, Danvers, MA 01923. On the other hand, when a solution of 4.7% sodium gold sulfite with 9% sodium sulfite, 2.4% sodium pyrophosphate, 3% disodiumphosphate and 0.5% antimony potassium tartrate was used as the electroplating solution, good results were obtained when 100 wires at a time were grounded and 5.7 microamps of direct current were passed through the same said gold anode for 2 seconds. The electrical current from the 12 volt power supply was passed through a 2 megaohm resistor before the gold anode. When the length of the electrode wires was increased from 250 to 300 microns, it was found necessary to increase the voltage to 16 volts to achieve similar gold depositions.

[0048] In another embodiment of the present invention, nucleic acid probes can be directed to different electrodes by blocking one of the gold electrodes by plating a thin layer of another metal over one of the gold electrodes, attaching the first nucleic acid probe, then etching off the blocking metal, either chemically or electrochemically, and then attaching the second probe DNA to the now exposed and un-blocked second gold electrode. Alternatively, the blocking metal can be overplated with gold to provide a new gold surface for the second to a probe. The procedure is diagramed in a side view in FIG. 4. It has been found that a thin layer of nickel can be plated onto one gold electrode from a nickel chloride solution, the first probe can be attached to the unplated gold electrode, and then the nickel can be etched from the gold with a mild acid solution without harming the first probe. The second probe can then be attached to the exposed electrode to produce the desired result of two electrodes with different probes on each electrode. Other metals can be electroplated over gold to provide a blocking layer, such as tin, indium, zinc, cobalt, titanium, chromium, copper, zirconium and iridium. The thickness of the plated layer can range from about 10 nm to about 1000 nm, more preferably from about 30 nm to 100 nm. The layer needs to be only thick enough to provide a continuous blocking layer; thinner layers are easier to remove by etching. [0049] Other blocking materials for gold can also be used. For example, an acrylic monomer such as 2-vinylpyridine can be polymerized on the surface of the gold by electrochemical initiation of the polymerization reaction. To uncover and unblock the gold surface, the gold can be washed wit a mild acid such as acetic acid which will dissolve the polyvinylpyridine. Other monomers can be used, such as vinyl imidiazole.

[0050] Various other methods exist for attaching the capture probes to the electrical conductors. For example, U.S. Patent Nos. 5,861,242, 5,861,242, 5,856,174, 5,856,101, and 5,837,832, which are hereby incorporated by reference in their entirety, disclose a method where light is shone through a mask to activate functional (for oligonucleotides, typically an -OH) groups protected with a photo- removable protecting group on a surface of a solid support. After light activation, a nucleoside building block, itself protected with a photo-removable protecting group (at the 5'-OH), is coupled to the activated areas of the support. The process can be repeated, using different masks or mask orientations and building blocks, to place probes on a substrate.

[0051] Alternatively, new methods for the combinatorial chemical synthesis of peptide, polycarbamate, and oligonucleotide arrays have recently been reported (see Fodor et al., Science, 251:767-773 (1991); Cho et al., Science, 261:1303-1305 (1993); and Southern et al, Genomics 13:1008-10017 (1992), which are hereby incorporated by reference in their entirety). These arrays (see Fodor et al., Nature, 364:555-556 (1993), which is hereby incorporated by reference in its entirety) harbor specific chemical compounds at precise locations in a high-density, information rich format, and are a powerful tool for the study of biological recognition processes.

[0052] Preferably, the probes are attached to the leads through spatially directed oligonucleotide synthesis. Spatially directed oligonucleotide synthesis maybe carried out by any method of directing the synthesis of an oligonucleotide to a specific location on a substrate. Methods for spatially directed oligonucleotide synthesis include, without limitation, light-directed oligonucleotide synthesis, microlithography, application by ink jet, microchannel deposition to specific locations and sequestration with physical barriers. In general, these methods involve generating active sites, usually by removing protective groups, and coupling to the active site a nucleotide which, itself, optionally has a protected active site if further nucleotide coupling is desired. [0053] In one embodiment, the lead-bound oligonucleotides are synthesized at specific locations by light-directed oligonucleotide synthesis which is disclosed in U.S. Patent No. 5,143,854, Published PCT Application Serial No. WO 92/10092, and Published PCT Application Serial No. WO 90/15070, which are hereby incorporated by reference in their entirety. In a basic strategy of this process, the surface of a solid support modified with linkers and photolabile protecting groups is illuminated through a photolithographic mask, yielding reactive hydroxyl groups in the illuminated regions. A 3'-O-phosphoramidite- activated deoxynucleoside (protected at the 5'-hydroxyl with a photolabile group) is then presented to the surface and coupling occurs at sites that were exposed to light. Following the optional capping of unreacted active sites and oxidation, the substrate is rinsed and the surface is illuminated through a second mask, to expose additional hydroxyl groups for coupling to the linker. A second 5 '-protected, 3'- O-phosphoramidite-activated deoxynucleoside (C-X) is presented to the surface. The selective photodeprotection and coupling cycles are repeated until the desired set of probes are obtained. Photolabile groups are then optionally removed, and the sequence is, thereafter, optionally capped. Side chain protective groups, if present, are also removed. Since photolithography is used, the process can be miniaturized to specifically target leads in high densities on the support. [0054] The protective groups can, themselves, be photolabile.

Alternatively, the protective groups can be labile under certain chemical conditions, e.g., acid. In this example, the surface of the solid support can contain a composition that generates acids upon exposure to light. Thus, exposure of a region of the substrate to light generates acids in that region that remove the protective groups in the exposed region. Also, the synthesis method can use 3'- protected 5'-O-phosphoramidite-activated deoxynucleoside. In this case, the oligonucleotide is synthesized in the 5' to 3' direction, which results in a free 5' end.

[0055] The general process of removing protective groups by exposure to light, coupling nucleotides (optionally competent for further coupling) to the exposed active sites, and optionally capping unreacted sites is referred to herein as "light-directed nucleotide coupling."

[0056] The probes may be targeted to the electrically separated conductors by using a chemical reaction for attaching the probe or nucleotide to the conductor which preferably binds the probe or nucleotide to the conductor rather than the support material. Alternatively, the probe or nucleotide may be targeted to the conductor by building up a charge on the conductor which electrostatically attracts the probe or nucleotide.

[0057] Nucleases can be used to remove probes which are attached to the wrong conductor. More particularly, a target nucleic acid molecule may be added to the probes. Targets which bind at both ends to probes, one end to each conductor, will have no free ends and will be resistant to exonuclease digestion. However, probes which are positioned so that the target cannot contact both conductors will be bound at only one end, leaving the molecule subject to digestion. Thus, improperly located probes can be removed while protecting the properly located probes. After the protease is removed or inactivated, the target nucleic acid molecule can be removed and the device is ready for use. [0058] The capture probes can be formed from natural nucleotides, chemically modified nucleotides, or nucleotide analogs, as long as they have activated hydroxyl groups compatible with the linking chemistry. Such RNA or DNA analogs comprise but are not limited to 2'-O-alkyl sugar modifications, methylphosphonate, phosphorothioate, phosphorodithioate, formacetal, 3'- thioformacetal, sulfone, sulfamate, and nitroxide backbone modifications, amides, and analogs, where the base moieties have been modified. In addition, analogs of oligomers may be polymers in which the sugar moiety has been modified or replaced by another suitable moiety, resulting in polymers which include, but are not limited to, polyvinyl backbones (Pitha et al., "Preparation and Properties of Poly (I-vinylcytosine)," Biochim. Biophys. Acta, 204:381-8 (1970); Pitha et al., "Poly(l-vinyluracil): The Preparation and Interactions with Adenosine Derivatives," Biochim. Biophys. Acta, 204:39-48 (1970), which are hereby incorporated by reference in their entirety), morpholino backbones (Summerton, et al., "Morpholino Antisense Oligomers: Design, Preparation, and Properties," Antisense Nucleic Acid Drug Dev., 7:187-9 (1997), which is hereby incorporated by reference in its entirety) and peptide nucleic acid (PNA) analogs (Stein et al., "A Specificity Comparison of Four Antisense Types: Morpholino, 2'-O-methyl RNA, DNA, and Phosphorothioate DNA," J. Antisense Nucleic Acid Drug Dev., 7:151-7 (1997); Egholm et al., "Peptide Nucleic Acids (PNA)-Oligonucleotide Analogues with an Achiral Peptide Backbone," (1992); Faruqi et al., "Peptide Nucleic Acid-Targeted Mutagenesis of a Chromosomal Gene in Mouse Cells," Proc. Natl. Acad. Sci. USA, 95:1398-403 (1998); Christensen et al., "Solid-Phase Synthesis of Peptide Nucleic Acids," J. Pent. Sci., 1:175-83 (1995); Nielsen et al., "Peptide Nucleic Acid (PNA). A DNA Mimic with a Peptide Backbone," Bioconjug. Chem., 5:3-7 (1994), which are hereby incorporated by reference in their entirety).

[0059] The capture probes can contain the following exemplary modifications: pendant moieties, such as, proteins (including, for example, nucleases, toxins, antibodies, signal peptides and poly-L-lysine); intercalators (e.g., acridine and psoralen), chelators (e.g., metals, radioactive metals, boron and oxidative metals), alkylators, and other modified linkages (e.g., alpha anomeric nucleic acids). Such analogs include various combinations of the above- mentioned modifications involving linkage groups and/or structural modifications of the sugar or base for the purpose of improving RNAseH-mediated destruction of the targeted RNA, binding affinity, nuclease resistance, and or target specificity.

[0060] The present invention can be used for numerous applications, such as detection of pathogens. For example, samples may be isolated from drinking water or food and rapidly screened for infectious organisms. The present invention may also be used for food and water testing. In recent times, there have been several large recalls of tainted meat products. The detection system of the present invention can be used for the in-process detection of pathogens in foods and the subsequent disposal of the contaminated materials. This could significantly improve food safety, prevent food borne illnesses and death, and avoid costly recalls. Capture probes that can identify common food borne pathogens, such as Salmonella and E. coli., could be designed for use within the food industry.

[0061] In yet another embodiment, the present invention can be used for real time detection of biological warfare agents. With the recent concerns of the use of biological weapons in a theater of war and in terrorist attacks, the device could be configured into a personal sensor for the combat soldier or into a remote sensor for advanced warnings of a biological threat. The devices which can be used to specifically identify the agent, can be coupled with a modem to send the information to another location. Mobile devices may also include a global positioning system to provide both location and pathogen information. [0062] In yet another embodiment, the present invention may be used to identify an individual. A series of probes, of sufficient number to distinguish individuals with a high degree of reliability, are placed within the device. Various polymorphism sites are used. Preferentially, the device can determine the identity to a specificity of greater than one in one million, more preferred is a specificity of greater than one in one billion, even more preferred is a specificity of greater than one in ten billion. [0063] It is a preferred feature of the present invention to coat the bridge between conductors formed by target nucleic acid molecules hybridized to capture probes. In doing so, the target nucleic acid is linked to a catalytic site for reducing metal from a metastable solution containing metal ions and a reducing agent. [0064] In a preferred embodiments of the present invention. The sodium counter ions in DNA are replaced with silver ions by flooding the sample with an excess of silver salt such as silver nitrate. The silver ion acts as the catalytic site for the deposition of silver or gold from a metastable solution of silver or gold salts along with a reducing agent One preferred reducing agent is 4- (methylamino)phenol sulfate, also known as Metol by those skilled in the art of photography. Other reducing agents such as hydroquinone can also be used. The activity and lifetime of the metastable solution of silver and reducing agent are influenced by the counter ions present, the temperature, the concentration of the reagents, and their ratios. Optimization of formulation for particular applications is done by factorial experimental design, as is well known by those skilled in the art of electroless metal plating.

[0065] In another embodiment of the present invention, the metal deposited is gold. A solution of potassium tetrachloroaurate is mixed with potassium thiocyanate and allowed to sit until the color fades. Then a reducing agent such as Metol is added, and the mixture is applied to the work. After sufficient time for deposition of gold onto the nucleic acid molecule, the reaction is stopped by rinsing away the solution with water.

[0066] In another embodiment of the present invention, the nucleic acid molecule is allowed to react with palladium ions, in the form of palladium acetate, either before or after hybridization. Unbound palladium ions are washed away from the hybrids and the remaining bound palladium acts as a catalytic site for the deposition of nickel from a commercial electroless nickel plating solution such as the Nickelex solution sold by the Transene Company, Danvers, MA. [0067] In another embodiment of the invention, the nucleic acid molecule is allowed to react with stannous ions, in the form of stannous chloride, either before or after hybridization. Unbound stannous ions are washed away from the hybrids, and the remaining bound stannous ions act as a catalytic site for the deposition of silver from a commercial electroless silver plating solution such as that sold by Peacock Labs, Philadelphia, PA.

[0068] After metal is deposited on the nucleic acid molecule bridges, communication between the electrodes is possible and can provide a detection signal. Typically, the communication is by electrical current, with a measurement of electrical resistance providing the detection signal. [0069] In a preferred embodiment of the present invention, the electrical resistance signal is proportional to the amount of nucleic acid molecules present in the target solution. The metal wires connecting two electrodes are very thin, only about 500 nm in diameter. From the electrical conductivity of bulk gold, resistance of a one micron long wire about one tenth micron in radius should have a resistance of approximately 10 ohms. However, a chemically deposited wire will never achieve the electrical conductance of bulk gold, because of higher resistance at the grain boundaries of the deposited gold particles. In practice, the resistances of such wires are in the range of several thousand ohms. When more than one wire connected the gold electrodes, the combined resistance follows the well known law: 1/Rtotai = 1 Rι + 1/R_- When there are less than about 5 wires connecting electrodes, the measured resistance provides a quantitative measure of the number of wires, and thus the number of DNA molecules present in the sample. Where there a more than about S wires connecting electrodes, the sample can be quantified by gradually increasing the voltage between the electrodes until wires begin to blow out, or melt. The total number of wires present can be quantified by measuring the current needed to blow out all the wires. [0070] In another embodiment of the present invention, the presence of a nucleic acid molecule can be determined by light scattered from the deposited metal specks on the nucleic acid molecule by scatter light. FIG. 5 shows a dark field micrograph of a spot of nucleic acid molecule decorated with deposited gold. The smooth background appears black, because it does not scatter light, while the gold on the nucleic acid molecule scatters light and gives the signal that the nucleic acid molecule is present. The amount of light is proportional to the amount of nucleic acid molecule present, allowing a quantitative reading of the amount of target nucleic acid molecule.

[0071] It is important to have the catalyst for the metal deposition strongly associated with the target nucleic acid molecule to prevent general and spontaneous deposition of metal everywhere, which leads to false positive signals. [0072] It is a feature of this invention that the catalyst for the reduction of metal on the nucleic acid molecule is a metal ion. This is advantageous compared to methods using reduced specks of metal as catalytic sites, because residual reducing agent can cause spontaneous deposition of metal. An unreduced metal ion is used as the catalyst for deposition of gold or other metal. Stringent washing conditions can be used to remove any catalyst that maybe accidentally deposited in background areas of the detector. A metal speck, on the other hand, cannot be removed by washing because metals are not soluble in washing solvents.

EXAMPLES

[0073] The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.

Example 1

[0074] A clean microscope slide was soaked in a solution of 1000 parts of toluene with 1 part of 3-aminoproplytriethoxysilane for 10 minutes, then blown dry in a stream of nitrogen. This provides a surface that is strongly adherent to DNA. Then a solution of .65 micrograms of DNA (D 1501 from Sigma, St.

Louis, MO) in 100 microliters of water was mixed with 1 microgram of palladium acetate in 1 microliter of acetone and allowed to rest for 10 minutes. One microliter of the mixture was spotted onto the silanized glass microscope slide and allowed to dry. A control spot of DNA without any palladium acetate was similarly placed near the palladium containing spot and also allowed to dry. The slide was then rinsed with a stream of pure water and then allowed to soak in a beaker of pure water for 10 minutes. The slide was then allowed to dry and placed on a 70 degree C. hot plate. The slide was covered with a solution of Nickelex (Transene Company, Danvers, MA) electroless nickel plating solution and allowed to sit for 4 minutes at 70 degrees C. The slide was rinsed with water and observed to have a bright mirror circle of nickel where the DNA spot containing DNA had been placed, but no nickel anywhere else.

Example 2

[0075] A microscope slide coated with 3-aminopropyltrimethoxysliane

(Coming Ultra-Gap slide, Coming Corporation, Coming, NY) was spotted with 1/2 microliter portions of a solution of 20 picograms of silver salt of DNA. (Prepared by mixing a solution of Calf Thymus DNA (Sigma Corporation, No. D1501) at 5 micrograms per ml with 1 microliter of 6% silver nitrate in water, then precipitating the DNA with ethanol, spinning down the pellet, decanting the solution, washing the pellet with 70% ethanol, then pure ethanol, drying the pellet and then redissolving the DNA pellet in water.) After drying the spot, the glass slide was rinsed with water and air dried. Then 200 microliters of a .25% solution of potassium tetrachloroaurate in water was placed in an ependorf tube. Then 140 microliters of a .65% solution of potassium thiocyanate was added to the tube. A bright orange color was generated. When the color had faded to colorless (about 10 seconds), 120 microliters of a .83% solution of p-(methylamino)phenol sulfate also containing .5% sodium sulfite was added to the tube and mixed. The mixed solution was flooded onto the glass slide and allowed to sit for 4 minutes at 60 degrees C. Then the slide was rinsed with water, air dried and observed by dark field optical microscopy. The background of the glass slide, being smooth and flat, shows as black under dark field microscopy. The gold enhanced DNA scatters light and shows as a white circle. Example 3

[0076] Silicon chips having gold electrodes 2 microns wide, spaced on 3 micron centers were cleaned by placing them in a clean, disposable Petri dish. The chips were washed at room temperature for 10 minutes in a solution that is 1 part ammonium hydroxide to 10 parts 30% hydrogen peroxide. The chips were then rinsed with several changes of E pure water, and then dried under nitrogen gas. PNA probes containing a cysteine group were then reduced with Reductacryl beads (CN Corporation, 858-) in 1% sodium dodecylsulfate in 100 mM sodium phosphate buffer at pH 7.8. The beads were removed by centrifugation. The reduced probe solution was placed on the gold electrodes at room temperature for 20 minutes, which causes the thiol group on the cysteine to attach to the gold surface via a dative bond. The chips with probes attached were then hybridized with target DNA in 100 mM phosphate with 0.1% sodium dodcylsulfate with 100 mM sodium chloride and 10% formamide buffer by incubating the target DNA solution for 10 minutes at 70 degrees C to denature the double stranded DNA. The denatured solution was then placed on the chip on a 60 degree hot block in a humidified chamber, the heat block was turned off and allowed to cool to room temperature for 20 minutes. The chips were then twice rinsed in 100 mM phosphate buffer with 0.1% sodium dodecylsulfate and once in pure water. The chip was then covered with 100 mM silver nitrate and allowed to sit at room temperature of 20 minutes. The chip was then rinsed with pure water. Then equal portions of the following solutions were added to an ependorf tube: 1) 0.33% potassium tetrachloroaurate in water 2) 0.60% potassium thiocyanate in water 3) 0.71% p-(methylaminophenol)sulfate and 0.43% sodium sulfite in water. When solutions 1 and 2 are mixed, a deep orange color forms and fades in a few seconds. The solution #3 was not added until the orange color had substantially faded. The mixture was immediately applied to the chip sitting in a petri dish on a 55 degree C. hot block. The petri dish was covered and allowed to sit on the hot block for 5 minutes, when the chip was removed and washed to stop the chemistry. The chip was examined by scanning electron microscopy and thin gold wires corresponding to the DNA were seen between the electrodes. FIG. 5 shows a micrograph of the some of the wires. [0077] Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

WHAT IS CLAIMED:

1. A method of quantitatively detecting target nucleic acid molecules in a sample, said method comprising: providing one or more different groups of two or more electrically separated electrical conductors with capture probes attached to the conductors such that a gap exists between the capture probes on the electrically separated conductors; contacting the capture probes with a sample, potentially containing the target nucleic acid molecules, under conditions effective to permit any of the target nucleic acid molecule present in the sample to hybridize to the capture probes and thereby connect the capture probes; detecting a presence of the target nucleic acid molecules by determining whether electricity is conducted between the electrically separated conductors; and interrogating the electrically separated conductors to quantify the concentration of target nucleic acid molecules present in the sample.

2. The method according to claim 1 , wherein the capture probes are oligonucleotides.

3. The method according to claim 1, wherein the capture probes are peptide nucleic acid analogs.

4. The method according to claim 1, wherein the capture probes are LNA.

5. The method according to claim 1, wherein the capture probes are methyl phosphate DNA short oligomers.

6. The method according to claim 1 , wherein the target nucleic acid molecules are DNA.

7. The method according to claim 1, wherein the target nucleic acid molecules are RNA.

8. The method according to claim 1, wherein the capture probes are complementary to target nucleic acid molecules from the genetic material of a pathogenic bacteria.

9. The method according to claim 8, wherein the pathogenic bacteria is a biowarfare agent.

10. The method according to claim 8, wherein the pathogenic bacteria is a foodborne pathogen.

11. The method according to claim 1 , wherein the capture probes are complementary to target nucleic acid molecules from the genetic material of a virus.

12. The method according to claim 1, wherein the capture probes are complementary to target nucleic acid molecules from the genetic material of a human.

13. The method according to claim 1 , wherein the capture probes are complementary to polymorphisms where the base or bases complementary to the polymorphism are located at an end of the capture probes.

14. The method according to claim 1 further comprising: coating the capture probes as well as any target nucleic acid molecule hybridized to the capture probe with a conductive material after said contacting.

15. The method according to claim 14, wherein the conductive material is silver. - SO -

lό. The method according to claim 14, wherein the conductive material is gold.

17. The method according to claim 14, wherein the conductive material is nickel.

18. The method according to claim 1 , wherein said interrogating comprises: increasing voltage conducted between the electrically separated conductors to a peak voltage level at which no electricity is conducted between the electrically separated conductors and quantifying the concentration of target nucleic acid molecules in the sample based on the peak voltage level.

19. The method according to claim 1, wherein said interrogating comprises: increasing voltage conducted between the electrically separated conductors in stepped intervals and quantifying the concentration of target nucleic acid molecules in the sample based on the stepped voltage intervals applied to the electrically separated conductors.

20. The method according to claim 1 , wherein said interrogating comprises: applying radiation between the electrically separated conductors and quantifying the concentration of target nucleic acid molecules in the sample based on scattering of radiation applied between the electrically separated conductors, wherein electrically spaced conductors having hybridized target nucleic acid molecules have a different radiation scattering pattern than electrically spaced conductors with no hybridized target nucleic acid molecules.

21. The method according to claim 20, wherein the target nucleic acid molecules hybridized to the capture probes are decorated with metallic particles to enhance the differences in radiation scattering pattern between electrically separated conductors having hybridized target nucleic acid molecules and electrically separated conductors where there are no hybridized target nucleic acid molecules.

22. The method according to claim 20, wherein the radiation is light.