WO2002023185A2

WO2002023185A2 - Devices and methods for direct electronic readout of biomolecular interactions

Info

Publication number: WO2002023185A2
Application number: PCT/US2001/028821
Authority: WO
Inventors: John J. Schwartz; Joseph Jacobson
Original assignee: Engeneos, Inc.
Priority date: 2000-09-15
Filing date: 2001-09-14
Publication date: 2002-03-21
Also published as: AU2001290954A1; WO2002023185A3

Abstract

Disclosed herein are methods and devices for direct electronic readout of biomolecular interactions. The devices of the invention include substrates having a number of electrically conductive sites. The sites are arranged in a spatially-addressable array and are electrically isolated from each other. The devices also include a number of types of probes, each type of probe having a hybridization moiety capable of recognising and hybridizing to a different nucleic acid sequence. Generally, each site is in electrically conductive contact with one type of probe. Similarly, each type of probe is in electrically conductive contact with at least one electrically conductive site. The devices and methods of the invention permit detection of genetic lesions indicative of disease. Also, the devices and methods permit measurement of nucleic acid concentrations useful for predictive medicine as well as therapeutic drug monitoring, for example.

Description

DEVICES AND METHODS FOR DIRECT ELECTRONIC READOUT OF BIOMOLECULAR INTERACTIONS

BACKGROUND OF THE INVENTION

Recent years have seen an unprecedented explosion of knowledge in biology, molecular biology, and the medical sciences. We now know more about the genetic composition of humans, other animals, plants, fungi, bacteria, and viruses than we have ever known before, and we are acquiring more knowledge about their genetic sequences at a dizzying rate. As with other discoveries throughout human history, however, these advances have not sated our appetite for knowledge, hideed, the more that we learn about the sequences of our DNA, the more that our appetite grows. Clinicians demand more and better assays for genetic clues useful in diagnosis, prognosis, or treatment indications (or contraindications) for their patients. Researchers insist on faster machines for sequencing DNA, Courts insist on unequivocal DNA evidence for paternity suits and for "DNA fingerprinting" in criminal cases. Even the military wants a genetic database with genetic information from all military personnel for use as "dog tags." The military and civil protection authorities also need a tool for rapidly detecting any of the many etiologic agents that may be used in biowarfare.

The resources required to meet this need for DNA detection and sequence information are staggering. The Human Genome Project and private industry required years of work and very large sums of money to construct a human genomic DNA sequence that is admittedly incomplete and not error-free. The potential of this information to medical science, clinicians, private enterprise, the legal system, the government, and other interested parties cannot be unlocked until the means to rapidly detect and analyze nucleic acid information in a high-throughput manner are available. SUMMARY OF THE INVENTION Explication of the Invention

The present invention depends, in part, upon new applications of elements of electronics and nucleic acid hybridization arrays, governed by principles of genomics, biochemistry and molecular biology. The invention provides new methods and devices for the direct electronic readout of biomolecular interactions including interactions involving nucleic acids, hi particular, the invention allows for the detailed analysis of biomolecular interactions such as hybridization events in complex, highly diverse mixtures of nucleic acids. This is accomplished by exploiting the electrical conductivity of nucleic acids, particularly for the purpose of detecting and characterizing genetic lesions. For example, the present invention can detect differences in conductivity amongst oligonucleotides of different lengths, differences amongst single-stranded and double-stranded nucleic acids, and differences amongst double-stranded nucleic acids with varying numbers and types of mismatches. The discoveries described herein provide bioelectronic devices and methods useful for forensic medicine, predictive medicine, pharmacogenomics, including drug treatment monitoring, contaminant monitoring, and biowarfare monitoring, to name but a few.

In one aspect, the invention relates to a bioelectronic device that can be used to analyze a sample containing, or suspected of containing, nucleic acid. As contemplated herein, nucleic acid includes at least DNA and RNA. Such samples include, but are not limited to, tissue and bodily fluids. In some embodiments, the invention can be used to determine whether the nucleotide sequence of a nucleic acid in a test sample corresponds to a control or reference sequence. In another embodiment, the invention can be used to detect the presence of a genetic lesion. Genetic lesions are typified by a variety of perturbations or abnormalities in a gene which can involve multiple nucleotides or single nucleotides. Genetic lesions can include any mutation or polymorphism, including a single nucleotide polymorphism (SNP), insertion, deletion, inversion, or other alteration in a gene or nucleotide sequence, hi other embodiments, the device can be used to detect the presence of one or more specific nucleic acids in a sample, or to compare the amounts of two or more nucleic acids in a sample. For example, the device can be used to detect loss of heterozygosity of genomic DNA present in a genomic sample, or to analyze a distribution of expressed nucleic acids within a nucleic acid sample, or to determine the relative populations of various fauna and/or flora in a particular environment. According to the invention, a biomolecular assay device permits direct electronic read-out of the aforementioned hybridization events because it includes a substrate having a number of electrically conductive sites. The sites are arranged in a spatially-addressable array and are electrically isolated from each other. The device also includes a number of types of probes, each type of probe having a hybridization moiety capable of recognizing and hybridizing to a different nucleic acid sequence. Generally, each site is in electrically conductive contact with only one type of probe. Conversely, each type of probe is in electrically conductive contact with at least one electrically conductive site. The term "probe" is understood to mean any type of sequence- specific nucleic acid binding molecule. In a preferred embodiment of the invention, the electrically conductive sites are embedded in an electrically non-conductive material in or on the substrate. In another preferred embodiment, the electrically conductive sites include metallic surfaces, such as gold, silver, or platinum; electrically conductive polymer surfaces, such as polypyrrole or poly(3,4- ethylenedioxythiophene); or semiconductors such as silicon, hi the preferred embodiment wherein the electrically conductive sites include gold metallic surfaces, the surfaces are preferably linked to the probes by sulfur linkages, amine-phosphine linkages, or silane.

In one preferred embodiment of the invention, the probes include nucleic acids such as oligodeoxyribonucleic acids or oligoribonucleic acids. In another preferred embodiment, the probes include nucleic acid analogs such as peptide nucleic acids or thiodiester analogs of nucleic acids. It is understood that the probes may also include "mixed" polymers (e.g. an oligodeoxyribonucleic acid fused to an oligoribonucleic acid or to a peptide nucleic acid) and/or polymers incorporating one or more non-natural nucleotides.

In some embodiments, each type of probe corresponds to a different sequence from a genome of an organism. In a preferred embodiment, each different sequence is unique within the genome of the organism. In a more preferred embodiment, each different sequence corresponds to at least twelve consecutive nucleotides (more preferably at least fourteen consecutive nucleotides, and even more preferably at least fifteen consecutive nucleotides) from the genome of the organism.

In another preferred embodiment, the probes include crosslinking moieties capable of covalently binding the probe to a nucleic acid hybridized to the hybridization moiety of the probe, hi this preferred embodiment, the crosslinking moiety may optionally be interposed between the hybridization moiety and the electrically conductive site. In an alternative preferred embodiment, the hybridization moiety may be interposed between the crosslinking moiety and the electrically conductive site. In another alternative preferred embodiment, the crosslinking moiety may be located elsewhere with respect to the hybridization moiety. In one preferred embodiment, the probe includes a crosslinking moiety such as psoralen, azinomycin B; bispiperidines; cisplatin; 5-(aziridin-l-yl)-2,4-dinitrobenzamide; 2-amino-6-(2- phenylsulfoxyethyl)purine; 2-amino-6-(2-methylsulfinylethyl)purine; adriamycin; mechlorethamine and phosphoramide mustard; azidophenacyl photoreactive crosslinking agents; chloroethylnitrosoureas, disuccinimidyl suberate; bis[sulfosuccinimidyl] suberate; dimethyl suberimidate; dimethyl pimelimidate; dimethyl adipimidate; disuccinimidyl glutarate; methyl N- succinimidyl adipate; l,5-difluoro-2,4-dinitrobenzene; l-ethyl-3-[3-d memylaminopropyl]- carbodiimide; /?-azidophenyl glyoxal; bis-[j8-(4-azidosalicylamido)ethyl]disulfide; or derivatives thereof.

In a preferred embodiment of the invention, the device also includes calibration sites useful for analyzing the integrity of a nucleic acid, hi this preferred embodiment, the calibration sites are electrically isolated from each other and each calibration site is in electrically conductive contact with a different type of calibration probe. Pairs of calibration probes correspond to pairs of sequences from a genome of an organism. In the genome of the organism, the pairs of sequences are separated by known and varying distances. In some preferred embodiments, the distances are in increments of at least 1 kb.

In a preferred embodiment of the invention, the device includes or is treated with a blocking agent to prevent nonspecific binding of nucleic acids to the substrate, hi a more preferred embodiment, the blocking agent is a detergent or a polymer such as a protein, a nucleic acid, polypyrrolidone, or ethylene glycols. hi a highly preferred embodiment, the sites include gold metallic surfaces and the blocking agent has an alkanethiol or alkanephosphine group.

In certain preferred embodiments of the invention, the device includes a reference sample including reference fragments of nucleic acids. In these preferred embodiments, each reference fragment is hybridized to hybridization moieties of at least two different types of probes. In one preferred embodiment, the reference fragments are derived from a genome of a reference organism, hi another preferred embodiment, the reference fragments are complementary either to RNA molecules expressed by a reference organism, or to their complement (e.g., cDNA). In a preferred embodiment, the reference fragments are covalently bound to the probes.

In another embodiment of the invention, the device includes a substrate having a number of electrically conductive sites. The sites are arranged in a spatially-addressable array and are electrically isolated from each other. The device also includes a number of types of probes, each type of probe having a sequence-specific nucleic acid binding moiety capable of recognizing and hybridizing to a different nucleic acid sequence. Generally, each site is in electrically conductive contact with one type of probe. Similarly, each type of probe is in electrically conductive contact with at least one electrically conductive site. In a preferred embodiment, the sequence-specific nucleic acid binding moiety is a protein such as lambda repressor, TATA binding protein, DNA methylases, or RNA binding proteins. In other embodiments, other peptides or drugs that bind a nucleic acid in a sequence-specific manner may be used.

A significant aspect of the present invention relates to practical methodologies which exploit the aforementioned bioelectronic devices. As contemplated herein, bioelectronic devices for direct electronic readout can be used to: diagnose a disease; select a treatment regimen and/or identify a preferred drug treatment program; identify and characterize a genetic lesion or abnormality indicative of disease, as well as predictive of disease; detect an etiologic agent such as a biowarfare agent; detect contaminants in foods, the environment, and medical materials such as organs, for example; provide forensic identification of a likely source of a biological sample; construct a genetic database for identification; and, construct a physical map of an oliogonucleotide, for example.

In certain embodiments, the invention relates to qualitative methods of analyzing the nucleic acid sequence of a sample. The method includes exposing a biomolecular assay device to a sample under conditions permitting a nucleic acid therein to hybridize to a reference fragment hybridized to at least two different types of probes. The method further includes measuring a value of electrical current, electrical resistance, capacitance, inductance, or a voltage drop between the two sites in contact with the two different types of probes. If the nucleic acid in the sample differs from a control nucleic acid, then the measured value will differ by at least a predetermined amount from a control value obtained using the control nucleic acid. In certain other embodiments, the present invention provides devices and methods for measuring the concentration of a nucleic acid in a sample. As contemplated herein, the nucleic acid can be genomic DNA or RNA, for example, messenger RNA. For example, one preferred embodiment involves a method of measuring the concentration of a nucleic acid in a sample, the method comprising the steps of contacting a sample with anyone of the above-described devices, wherein a plurality of identical first probes are in electrically conductive contact with one or more first electrically conductive sites, a plurality of identical second probes are in electrically conductive contact with one or more second electrically conductive sites, and a plurality of reference fragments are hybridized to a hybridization moiety of a first probe and to a hybridization moiety of a second probe; and, measuring a value of electrical current, electrical resistance, capacitance, inductance, or voltage drop between said one or more first electrically conductive sites and said one or more second electrically conductive sites, thereby to quantitatively detect a number of hybridization events between a target nucleic acid and a reference fragment, the number of hybridization events being indicative of the concentration of the target nucleic acid in the sample. Additionally, other embodiments permit diagnosis of a disease or monitoring a disease state by measuring the concentration of a nucleic acid in a sample. For example, in a method of monitoring a course of treatment for a disease in an individual, the steps are, first, obtaining a first sample; second, obtaining, at a later time, a second sample; third, measuring the concentration of a nucleic acid in the first and second samples as described above; and, fourth, comparing the concentration of the nucleic acid in the first sample to the concentration of the nucleic acid in the second sample, wherein the concentration of the nucleic acid in the samples is indicative of the status of a disease in the individual. In yet other embodiments, the present invention permits selection of a drug treatment regimen for an individual similarly by measuring the concentration of a nucleic acid which corresponds to a disease state, or alternatively a gene or a pathway related to the disease state. Furthermore, use of the present invention to measure concentrations of nucleic acids in a sample permits contamination monitoring. DNA or RNA from a micro-organism could correlate with contamination and measuring concentrations could correlate with onset of, as well as extent of, contamination.

Because direct electronic readout can detect a mutation even in relatively large nucleic acids, the invention is particularly well suited to mapping mutations and other genetic differences between two nucleic acids. Once a difference is detected between two large nucleic acids, further comparisons can be done as described above using increasingly smaller subregions. In a preferred embodiment of the device, the aforementioned reference fragments include fragments of a reference genome and the sample includes fragments of a sample genome. In this preferred embodiment, the reference fragments are preferably substantially non-overlapping. In one preferred embodiment, the nucleotide sequence of the control nucleic acid is known, hi another preferred embodiment, the reference fragments include a contig. The contig represents a contiguous portion of a nucleic acid such as, for example, a chromosome. Alternatively, the contig may represent a portion of a chromosome, preferably containing a locus an allele of which is associated with a disease. As used herein, "contig" means a collection of overlapping or non- overlapping nucleic acid sequences which, when assembled into a continuous sequence, collectively correspond to any contiguous genomic sequence.

In one preferred embodiment, the nucleic acid content of the sample is preferably treated, before exposure to the device, by exposing the sample to a restriction enzyme, mechanical shearing, chemical digestion, or sonication. In another preferred embodiment, the nucleic acid content of the sample is treated to enrich the sample for one type of chromosome (e.g., chromosome 21) while depleting the sample of at least one other type of chromosome (e.g., chromosome 4, or all chromosomes other than chromosome 21). More preferably, the enriching step includes exposing the nucleic-acid sample to a chromosome-specific hybridization moiety which may be associated with a solid support or a magnetic or paramagnetic bead. Advantages of the Invention As will be appreciated from the description provided herein, the invention provides numerous advantages over materials and methods available currently. The invention permits rapid detection and analysis of complex and highly diverse preparations of nucleic acids while providing heightened sensitivity. Qualitative as well as quantitative analyses are possible using the present invention as is described elsewhere herein. For example, the entire genomes of two individuals can be rapidly compared and their genetic differences identified, mapped and sequenced. Moreover, the direct electronic readout of genetic information completely avoids laborious processes such as those involved in conventional chemical sequencing or screening technologies. For example, direct electronic readout eliminates the need for external reporter molecules such as florescent tags because the binding event generates its own reporter, namely an electronic signal. Additionally, samples can be rapidly screened for any number of genetic lesions such as oncogenes or other nucleic acids associated with disease, and/or screened for contaminants such as bacteria, viruses, and/or other pathogenic/etiologic agents. The high- throughput capabilities of the invention also greatly facilitate large-scale analyses, such as those required in pharmacogenomics studies or in compiling "DNA fingerprints", useful, for example, as a supplement to, or replacement for, military "dog tags." All of these advantages are provided while leveraging established chemical and electronic technologies to achieve inexpensive, rapid DNA analysis with nucleotide-level sensitivity.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 is a schematic depiction of a preferred device for direct electronic readout of biomolecular interactions. DETAILED DESCRIPTION OF THE INVENTION

The present invention depends, in part, upon new applications of elements of electronics and nucleic acid hybridization arrays, governed by principles of genomics, biochemistry and molecular biology, to provide new biomolecular assay devices and methods for the direct electronic readout of biomolecular interactions, including analysis of complex, highly diverse nucleic acids. In particular, the invention allows for the detailed analysis of complex, highly diverse mixtures of nucleic acids from a variety of sources such as tissues, bodily fluids, bacteria, virus, fungi, to name but a few. This is accomplished by exploiting the electrical conductivity of nucleic acids, particularly differences in conductivity amongst oligonucleotides of different lengths, differences amongst single-stranded and double-stranded nucleic acids, and differences amongst double-stranded nucleic acids with varying numbers and types of mismatches. Bioelectronic Devices for Direct Electronic Readout

The bioelectronic devices of the present invention permit direct electronic readout of a biomolecular interaction. The devices comprise a solid substrate including a multiplicity of electrically conductive sites arranged in a spatially addressable array and electrically isolated from each other by an electrically non-conductive material. The sites are sufficiently large, and the substrate material is sufficiently rigid, that the sites define an array which is sufficiently constant in space to allow a user or user-operated machine to address or deliver specific probes to specific sites, to make electrical contact with specific sites, and to measure electrical properties between sites. The devices further comprise a multiplicity of types of probes arranged on the spatially addressable array of sites such that each site is effectively in electrical contact with a single type of probe, and each type of probe is in electrically conductive contact with at least one site. In addition, although each site will be in electrically conductive contact with a single type of probe, there will be a large multiplicity (e.g., 10³-10⁷) of probes of that type per site. Furthermore, although it is preferred that each site shall be in contact with a single type of probe, it is inevitable that there will be some contamination during the manufacturing process, and therefore it is likely that each site will contain a small percentage of probes intended for a different site. Thus, if this percentage is kept sufficiently low so as not to render the device inoperative for its intended purpose, each site may be characterized as "effectively" in electrically conductive contact with a single type of probe. Thus, as used herein, a site that is "effectively in electrically conductive contact with a single type of probe" is one at which no other type of probe is present in an amount sufficient to substantially impair the functioning of the site or render it ineffective for its intended purpose.

The devices of the invention may be provided as described above, or may be provided with a reference sample of nucleic acids. Such a reference sample comprises a multiplicity of reference fragments which bind to the probes in a sequence specific manner and thereby form bridges or connections between sites. As described in detail below, the reference fragments may be covalently bound to the probes to which they have hybridized, and cross-linking agents may be provided as elements of the probes or reference fragments to achieve this cross-linking. Because nucleic acids are electrically conductive, the reference fragments will form electrically conductive connections between the sites to which they bind. Therefore, if a voltage is applied across two sites which are connected by a nucleic acid, the electrical current, resistance, capacitance, inductance, or voltage drop between the sites may be measured. Furthermore, because there are differences amongst the electrical conductivities of double-stranded nucleic acids and single-stranded nucleic acids, and differences amongst double-stranded nucleic acids having differing numbers and types of mismatches, the above-described device may be used to analyze a sample of nucleic acids to determine whether there are nucleic acids in the sample which are partially or completely complementary to the reference fragments.

As shown in the preferred embodiment of the invention depicted in Figure 1, direct electronic readout device 10 includes substrate 12 with electrically conductive sites 14 arranged in a spatially-addressable array. The electrically conductive sites 14 are electrically isolated from each other on substrate 12, but are in electrically conductive contact with one or more probes 16. Each probe 16 includes a hybridization moiety capable of hybridizing to a nucleic acid sequence 18. Each electrically conductive site 14 is preferably in electrically conductive contact with a multiplicity of probes 16, and each of the probes 16 at any particular electrically conductive site 14 is preferably identical. In contrast, different electrically conductive sites 14 generally are associated with different probes 16 having hybridization moieties recognizing different nucleic acid sequences 18 (or different portions of the same nucleic acid sequence 18).

In use, direct electronic readout device 10 is exposed to a sample to interrogate the sample for the presence of a target nucleic acid 20 complementary to a nucleic acid sequence 18 bridging two electrically conductive sites 14. If present, target nucleic acid 20 hybridizes with nucleic acid sequence 18, leading to the formation of a double-stranded nucleic acid bridging the two electrically conductive sites 14 as shown at position A. As previously discussed, a double- stranded nucleic acid as shown at A is a better electrical conductor than a single-stranded nucleic acid as shown at B. Thus, by assessing the electrical properties of a connection between two electrically conductive sites 14, the presence and concentration of a target nucleic acid 20 can be determined. Furthermore, because the conductivity of a double-stranded nucleic acid depends on the degree at complementarily, mismatches and other genetic mutating can be readily detected. Unlike DNA chips that rely on fluorescent detection schemes, the current invention permits direct electronic detection and analysis of nucleic acids. The invention therefore avoids the heterogeneity and potential run-to-run variability that the addition of fluorescent detection moieties necessarily introduces. Direct electronic readout also permits detection with very high sensitivity and a relatively low signal-to-noise ratio. Accordingly, the elements in the spatially- addressable array (electrically conductive sites, in the present invention) can be more densely packed in the present invention than with conventional fluorescent detection, permitting more nucleic acid analyses per exposure to a sample, and substantial cost savings.

Additional considerations in the manufacture and use of the devices of the invention are described below.

A. Substrates

The substrates of the invention may generally be formed of any materials which are sufficiently rigid to provide for a spatially addressable array, and which can form the multiplicity of electrically conductive sites arranged in a spatially addressable array, and electrically isolated from each other. Thus, the substrate must be formed of at least two materials, one of which is electrically conductive to form the sites, and one of which is essentially non-conductive to electrically isolate the sites from each other.

Preferred electrically conductive materials for the sites include gold, silver and platinum, but other metals may be substituted as well. Electrically conductive non-metallic materials may also be used to form the sites, such as silicon or silicon-containing compounds, or electrically conductive polymers such as polypyrrole or poly(3,4-ethylenedioxythiophene).

Preferred non-conductive materials for isolating the sites from each other on the substrate include non-conductive polymers (e.g., polyethylene, polypropylene, polyurethane), glasses or ceramics. The substrates may be of any arbitrary shape that allows the array of sites to be spatially addressable. For convenience of manipulation and automation, substantially flat and rectilinear substrates are preferred, but convex, concave, spherical, cylindrical, and other shapes are possible. Most preferably, the substrate comprises a multiplicity of metallic sites embedded in and passing through both major faces of a substantially flat and rectangular sheet of a non- conductive plastic polymeric material. By passing through the non-conductive sheet, the sites will be accessible on one surface for adhering probes and on one surface for contacting electrodes to apply and measure electrical voltage, current, resistance, capacitance, or inductance between sites. Alternatively, only one face of the metallic sites may be exposed and bound by probes, and electrodes may be contacted with that exposed surface or through a hole made through the non-conductive material for that purpose. Preferably, the sites are arranged on the substrate in a rectilinear array of rows and columns for ease of addressing.

The sizes of the sites and the distance between sites will be determined in part by the physical length of the reference fragments which are intended to bridge or connect them. Thus, the diameter of the sites should not be so large, nor the distance between sites so large, that a reference fragment bound to a probe on the site could not physically span the distance from that probe to another probe on an adjacent or nearby site. For purposes of the present invention, it is preferred that the sites be less than or equal to 1 mm in diameter and that the centers of adjacent sites be separated by less than 0.01-10 mm, preferably less than 0.1-5mm. B. Probes The probes of the present invention may comprise any type of sequence-specific nucleic acid binding molecule. Thus, the probes comprise a hybridization moiety which is capable of binding to or hybridizing with a specific nucleic acid sequence or specific set of nucleic acid sequences. It is the hybridization moiety, and the specific sequence(s) to which it binds hat determines the "type" of the probe. The hybridization moiety may itself comprise a nucleic acid, including a ribonucleic acid or deoxyribonucleic acid, or a nucleic acid analog or derivative with enhanced binding affinity or stability. Alternatively, the hybridization moiety may comprise a DNA or RNA-binding protein which is specific for a particular sequence or small subset of sequences. Examples of such proteins include the lambda repressor, TATA binding proteins, and DNA methylases. hi other embodiments, other peptides or drugs that bind a nucleic acid in a sequence-specific manner may be used. Particularly preferred hybridization moieties are nucleic acid analogs in which the phosphodiester backbone of a nucleic acid is replaced by thiodiester bonds or by peptide bonds (e.g., peptide nucleic acids or PNAs). PNAs are particularly preferred because they form duplexes with nucleic acids which are more stable than DNA-DNA duplexes and, therefore, will not be displaced from binding to a reference fragment by a sample nucleic acid. For hybridization moieties which comprise nucleic acid or nucleic acid analogs, it is preferred that the moiety comprise a sequence of at least 12 nucleotide residues (or nucleotide analogs) to ensure stable and sequence-specific hybridization. In addition, the sequences are preferably chosen to correspond to rare or unique sequences in the genome of an organism being analyzed. Thus, depending upon the size and complexity of the genome, the hybridization moieties may comprise at least 16, 18, or 20 consecutive nucleotides (or nucleotide analogs). The probes may be bound or adhered to the sites by any standard covalent or non- covalent chemistries which are appropriate to the chemical composition of the probe and the surface of the site. For gold surfaces, sulfur (e.g., between amines and NHS-esters or between thiols and maleimide) or amine-phosphine linkages are particularly preferred. C. Optional crosslinking moieties

The probes may optionally include one or more crosslinking moieties. Once a nucleic acid has hybridized to the hybridization moiety of a probe, the optional crosslinking moiety may crosslink the nucleic acid to the hybridization moiety. In one embodiment, a crosslinking moiety is incorporated within a hybridization moiety (e.g., as a reactive nucleotide analog), hi another embodiment, a crosslinking moiety is interposed between the hybridization moiety and the electrically conductive site. In an alternative embodiment, the hybridization moiety is interposed between a crosslinking moiety and the electrically conductive site. The crosslinking moiety, if present, and its placement with respect to the hybridization moiety are preferably selected to minimize interference with the electrical aspects of the assay. A wide variety of suitable crosslinking moieties are known in the art (see, e.g., the 2000 Pierce Chemical catalog, incorporated by reference herein). For example, suitable optional crosslinking moieties may include psoralen, azinomycin B; bispiperidines; cisplatin; 5-(aziridin-l-yl)-2,4-dinitrobenzamide; 2-amino-6-(2-phenylsulfoxyethyl)purine; 2-amino-6-(2-methylsulfinylethyl)purine; adriamycin; mechlorethamine and phosphoramide mustard; azidophenacyl photoreactive crosslinking agents; chloroethylnitrosoureas, disuccinimidyl suberate; bis[sulfosuccinimidyl] suberate; dimethyl suberimidate; dimethyl pimelimidate; dimethyl adipimidate; disuccinimidyl glutarate; methyl N- succinimidyl adipate; l,5-difluoro-2,4-dinitrobenzene; l-ethyl-3-[3-dimethylaminopropyl]- carbodiimide; ?-azidophenyl glyoxal; bis-[/3-(4-azidosalicylamido)ethyl]disulfide; and derivatives thereof. hi other embodiments, the probe does not comprise a crosslinking moiety. For example, the hybridization moiety of the probe may comprise a PNA, which has a particularly high affinity for nucleic acids. In another preferred embodiment, once a nucleic acid has hybridized to a hybridization moiety of a probe, the device maybe treated with an external crosslinking agent such as a multifunctional chemical crosslinking agent or ultraviolet light. Methods for analyzing a nucleic acid-containing sample The bioelectronic devices of the present invention can be used in a wide variety of assays to analyze a nucleic acid sample. These assays generally involve exposing a device to a sample comprising DNA or other nucleic acid moiety, measuring a value of an electrical current, electrical resistance, capacitance, inductance, or voltage drop between two sites of the device, and comparing the value to a control value. A. Preparation of the nucleic acid-containing sample

The nucleic acid content of a sample may include naturally-occurring nucleic acids such as deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or artificial nucleic acids. DNA molecules may include, for example, chromosomes, chromosomal fragments, cDNAs, episomes, plasmids, synthetic oligodeoxyribonucleic acids and other molecules having natural or non- natural bases. RNA molecules may include, for example, mRNA, tRNA, rRNA, hnRNA, exons, introns, or other forms of RNA. The sample may contain sample nucleic acids that have been synthesized artificially using chemical methods known in the art, or may be generated using recombinant DNA technology, hi some embodiments, the sample is preferably not subjected to amplification (e.g., polymerase chain reaction), to minimize the risk of introducing errors into the nucleotide sequences of the sample. The sample may contain nucleic acids from a patient or other organism. The sample may contain nucleic acids from a multiplicity of organisms. For example, the sample may be taken from an environment that may contain many organisms, such as a soil sample, a water sample, a laboratory culture, or a sample from a patient's body, such as a salivary sample or a stool sample. Alternatively, the presence or absence of nucleic acids in a sample may be unknown. For example, in one embodiment the invention maybe used to check the sterility of an environment. In this embodiment, the bioelectronic device can be used to screen for the presence of a nucleic acid, which, if present, would indicate the presence of a biological contaminant. Thus, the sample may or may not actually contain any nucleic acids in embodiments used to distinguish between those two conditions.

In a preferred embodiment, the sample is taken from a patient to analyze nucleic acids from the patient. In one preferred embodiment, the sample is taken non-invasively from a source such as saliva, stool, semen, vaginal secretions, prostatic secretions, urine, tears, sweat, sputum, or breast exudate. In another preferred embodiment, the sample is, or is derived from, tissue surgically removed from a patient, such as a biopsy sample, hi another preferred embodiment, the sample is, or is derived from, a blood, serum, or blood plasma sample.

In embodiments in which the sample is taken from a biological source, it is preferable to treat the sample to improve the accessibility of the nucleic acids in the sample to the device. Thus, for example, if the nucleic acids are within biological tissue, it maybe preferable to disaggregate the tissue and/or to lyse the cells to release the nucleic acids from the cells. Any lysis is preferably done in the presence of nuclease inhibitors by methods well known in the art. In some preferred embodiments, the nucleic acids are at least partially purified before exposure to the biomolecular assay device. In other embodiments, the acid sample is applied directly to the device without further purification. hi a preferred embodiment, samples containing long sample nucleic acids are treated to reduce the length of the nucleic acids prior to exposure to the biomolecular assay device. This treatment may include, for example, exposing the nucleic acid sample to a restriction enzyme, mechanical shearing, chemical digestion, sonication, and/or other methods known in the art for introducing breaks into a nucleic acid.

B. Exposing the device to the sample The nucleic acid content of a sample may be applied to the biomolecular assay device by any means permitting the nucleic acids to hybridize or otherwise associate in a sequence-specific manner with reference fragments hybridized to the hybridization moieties of the device. Thus, the nucleic acid may be present in a liquid phase that is applied to a surface of the device, as by a dropper or a flow cell, for example. Alternatively, the device, or a portion thereof, can be dipped or otherwise partially or completely submerged within a liquid phase containing the nucleic acid. It is important that the nucleic acid be exposed to the device under conditions permitting a nucleic acid to hybridize to a complementary reference fragment. The required conditions will vary according to, for example, the nucleotide composition of the reference fragment (e.g., the percentage of cytosine and guanine residues), the type of nucleic acid or nucleic acid analog used as the reference fragment (e.g., DNA, RNA, or PNA), and the type of sample nucleic acids to be hybridized with the reference fragment. The conditions for permitting hybridization may include, for example, temperature, pH, salt concentrations, and the concentrations of other chemical species known to influence the binding of a nucleic acid to a second nucleic acid or to a nucleic acid analog. Selecting appropriate conditions for hybridization is well known in the art and discussed in such works as, for example, Sambrook et al., Molecular Cloning: A Reference Manual herein incorporated by reference.

C. Measuring electrical current, electrical resistance, capacitance, inductance, or voltage drop

Once the nucleic acid has been exposed to the device, a value of electrical current, electrical resistance, capacitance, inductance, or voltage drop between two electrically conductive sites is measured. DNA has an inherent electrical conductivity, presumably through the π-bonds of the stacked bases. When the DNA is single stranded, the conductivity is low. When the DNA is collinear and double stranded, then the conductivity is high. Accordingly, when a nucleic acid hybridizes to a reference fragment connecting two electrically conductive sites, the electrical conductivity of the DNA is increased (i.e., the electrical resistance between the two sites is decreased). In a highly preferred embodiment, even a single base mismatch in the hybridization of the sample nucleic acid to the reference fragment can be sensed by this type of conductivity measurement.

Generally, the measurement is done in the presence of a voltage differential between two electrically conductive sites. The voltage differential may be applied before or after hybridization of the sample nucleic acid to the reference fragment. Because of the interdependence of electrical properties, an electrical current, electrical resistance, capacitance, inductance, voltage drop, or other relevant electrical property may be measured. Circuits permitting the measurement of these properties are well known in the art and need not be elaborated herein. D. Comparison to a control value

Comparing the measured value to a control value allows the detection of the hybridization of the nucleic acid with the reference fragment and reveals information about the nucleotide sequence of the sample. Generally, the control value corresponds to an expected value for a control nucleic acid hybridized to the reference fragment. The control nucleic acid is preferably the same type of nucleic acid as the sample nucleic acid (e.g., DNA, RNA, PNA, etc.) and is more preferably perfectly complementary to the reference fragment. Although the control value might be calculated based on known conductivity properties of nucleic acids, the control value is preferably determined experimentally, for example by the manufacturer of the device or by the end-user. E. Direct electronic measurement of nucleic acid concentration

Just as direct electronic readout is a powerful tool for detecting the presence of a nucleic acid and for analyzing its nucleotide sequence, it is similarly powerful for determining the concentration of a nucleic acid in a sample. A preferred device according to the invention has a large multiplicity (e.g. 10³-10⁷) of probes at each electrically conductive site. More preferred are devices incorporating more than 10 such electrically conductive sites (e.g. 10 such sites). The probes at a first site are preferably joined to the probes at a second site by large multiplicity of reference fragments to minimize any risk of saturating the detection system. Conductivity, and other electrical properties relating the first and second sites, will be a function of the number of reference fragments joining the sites and a function of the percentage of the reference fragments that are "occupied" — the percentage of the reference fragments that have hybridized with a sample nucleic acid, creating a double-stranded nucleic acid competent to conduct electricity. If the hybridization conditions are selected such that the reference fragments are only partially occupied upon exposure to a sample, then the percent occupancy will be a function of the concentration of the target nucleic acid in the sample. Thus, the invention provides a direct electronic readout of the concentration of a target nucleic acid. Algorithms (e.g. using Fourier transformation) for inferring percent occupancy based on the detected electrical property and for mferring concentration from percent occupancy are known in the art and need not be elaborated herein.

Accordingly, the device can be used to great advantage in detecting the concentration of a nucleic acid such as a messenger RNA. Messenger RNA levels are often correlated with particular disease states, such as cancers. Thus, just as the device is useful in detecting the presence of a messenger RNA that may indicate the presence, absence, or condition of a particular disease state, the sensitivity of the device permits the detection of messenger RNA concentration, which may often be more informative. Similarly, the device can be used to profile changes in messenger RNA expression over time, which maybe indicative of the status of a disease, the progression of a disease, or the efficacy (or side effects) of a treatment regimen. Indeed, the awareness this invention permits of ongoing changes in messenger RNA levels may in some cases be useful for predicting a future disease state (e.g. by detecting changes in a premalignant lesion).

Importantly, because the concentration is measured using direct electronic readout, the invention permits the monitoring of concentration changes in real-time. The electronics of the invention function nearly instantaneously; any time lags in detecting concentration changes are functions primarily of the kinetics of hybridization and dissociation, including effects of diffusion.

F. Whole-genome analysis The versatility of the device and its methods of use is underscored by the options available through judicious selection of reference fragments. For example, the reference fragments can collectively cover an entire genome of an organism. If a nucleic acid sample from a second organism is exposed to the device, the genetic differences between the organisms can be identified and characterized. In one embodiment, this provides useful information even where the nucleotide sequences of the genomes are unknown. For example, the method can be used to distinguish two strains of a pathogen differing in some property, such as virulence or drug resistance. If one strain (e.g., a less virulent strain) is used as the reference sample, the device will detect genetic differences if another strain (e.g., a more virulent strain) is exposed to the device, hi addition to the diagnostic value of the information, each reference fragment where the strains differ is identified by the device. Even if the full nucleotide sequence of each reference fragment is unknown, each reference fragment is hybridized to at least two probes. Standard molecular biological techniques can then be used, using the sequence information provided by one or both probes, to clone the fragment or otherwise further characterize the genetic differences within the genomic region identified by the reference fragment. In another embodiment, the nucleotide sequence of the reference fragment is mostly or fully known. For example, the reference fragments can correspond to a genome of one person and the sample nucleic acid can come from a second person. Both genomes would be identical at almost all positions to each other and the genomes of other humans as sequenced by, for example, the Human Genome Project. The biomolecular assay device can identify each reference fragment having a nucleotide sequence that differs from the nucleotide sequence of the sample nucleic acid to which it is hybridized. Because the location of every reference fragment in the human genome is known, the approximate positions of every genetic difference between two persons can be ascertained. The positions of the differences can be further refined to any extent desired by using progressively smaller reference fragments corresponding to the approximate location of the genetic difference. Thus, the precise location and identity of a genetic difference that may predispose an individual to a disease can be determined using this method. Similarly, a sample nucleic acid from a tumor of an individual can be compared to a reference nucleic acid from a normal tissue of the same individual to identify genetic changes in the tumor tissue. G. Forensic Science and DNA Typing

Establishing the identity of a genome from one individual with a reference genome also has powerful implications in forensic medicine, permitting rapid and thorough "DNA fingerprinting" to match crime-scene DNA against samples taken from victims and suspects. Forensic medicine is a scientific field employing, inter alia, genetic typing of biological evidence found at a crime scene as a means for positively identifying a perpetrator of a crime. To make such an identification, DNA is taken from even very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen, found at a crime scene. The DNA can then be analyzed using devices and methods of the invention. An exemplary forensic science method for identifying a likely source of a biological sample comprises the steps of obtaining at least a portion of a biological sample and also obtaining a test sample from an individual. The test sample is compared to at least a portion of the biological sample to detect identical nucleic acids using a device of the invention, wherein the presence of identical nucleic acids indicates that the individual is a likely source of the biological sample. If appropriate, PCR technology may be used to amplify DNA sequences prior to analysis of the DNA using the invention. The amplified sequence can then be compared to a reference fragment, e.g. a reference fragment derived from the DNA of a suspect, thereby allowing identification of the origin of the biological sample.

In a similar fashion, the invention facilitates paternity testing. The invention also provides a method for identifying a biological relative of an individual. An exemplary method involves obtaining a control sample from an individual and also obtaining a test sample from a potential biological relative. The control sample is then compared to the test sample to detect related nucleic acids using a device of the invention, wherein the presence of related nucleic acids is indicative of a biological relation between the individual and the potential biological relative.

Methods and devices according to the invention are useful in "DNA typing," in which DNA sequences between individuals are analyzed. For example, the United States military is considering the use of DNA typing, using techniques such as restriction fragment length polymorphism (RFLP) analysis, for identification of its personnel. Unfortunately, RFLP analysis and similar techniques are time-consuming and laborious. The present invention, which allows simultaneous screening of large numbers of polymorphic markers in the DNA of an individual, has tremendous advantages over RFLP analysis, and could permit positive identification of an individual, living or dead, from extremely small tissue samples.

On a related matter, the invention provides a method of constructing a genetic database for use in identifying individuals. For example, a preferred method comprises the steps of obtaining, samples from a plurality of individuals and, in each of the samples, detecting a plurality of nucleic acids using a device of the invention. Data indicative of the nucleic acids detected in each of the samples are then collected and stored in a database. H. Identification of Proprietary Genetic Materials and Organisms As valuable new plants and livestock are developed by genetic engineering, there will be a need for DNA typing to verify the source and ownership of agricultural products. The sequence information that has come from genome sequencing in humans, plants and animals will lead to increased application of genetic engineering techniques to develop pharmaceutical agents and create improved crops and livestock. Examples include strains that are more resistant to disease and harsh climates, as well as crops that have a greater yield or higher nutritive value. I. Environmental Monitoring The invention is also useful in detecting and characterizing mutagens or other contaminants. For example, the devices disclosed herein may be used to detect mutations induced by chronic exposure of cells to chemical agents. Similarly, the devices may be used for individual monitoring of employees who maybe exposed to chemicals or radiation in the workplace (e.g., through periodic screening for mutations in populations of circulating lymphocytes). Similarly, the invention can be used to detect an etiologic agent that may be a biowarfare agent; such a method comprises the step of detecting a nucleic acid indicative of the presence of an etiologic agent using a device of the invention. Also, the invention provides a method to assess a concentration of a biowarfare agent. Furthermore, the invention provides a method to detect contaminants, as well as a method to assess the concentration of a contaminant in a sample. J. Targeted nucleic acid analysis

In some embodiments, the reference fragments do not correspond to an entire genome of an organism. For example, in one preferred embodiment, the reference fragments correspond specifically to those nucleic acids that are expressed by an organism or by a particular cell or tissue type. Thus, the reference fragments may be complementary to RNA or to cDNA from a particular organism, tissue, or cell type, hi another preferred embodiment, the reference fragments form a "contig" representing, for example, a single chromosome or a portion thereof. As used herein, "contig" means a collection of overlapping or non-overlapping nucleic acid sequences which, when assembled into a continuous sequence, collectively correspond to a contiguous genomic sequence such as a chromosome or a contiguous portion thereof. In another preferred embodiment, the reference fragments correspond to nucleic acids associated with a health-related condition of an organism. For example, the reference fragments might be selected to include nucleic acids having diagnostic value in cardiovascular disease, cancer, or another condition. Similarly, the reference fragments might be selected to include nucleic acids suspected to play a causal role in a disease.

K. Predictive Medicine and Genetic Diseases The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to treat an individual prophylactically. For example, the invention may be used to monitor the influence of agents (e.g. drugs, compounds) on a multiplicity of marker genes indicative of a particular medical condition or patient outcome, hi one preferred embodiment, the invention is used in genetic diagnosis. There are estimated to be 4,000 to 5,000 genetic diseases in humans, in which a mutational change in a gene destroys or hinders the function of a gene product, leading to a serious medical condition. The affected genes and proteins have thus far been identified for a small fraction of human genetic diseases, although the number is increasing steadily. A few examples of human genetic diseases for which mutations associated with the disease have been identified include cystic fibrosis, phenylketonuria, Alzheimer's disease, cancer, Duchenne muscular dystrophy, and familial hypercholesterolemia. Although, in some cases, the disease is associated with one or very few specific mutations, it is becoming evident that many, if not most, genetic diseases can be caused by any of numerous mutations scattered along the affected gene. Detecting mutations within disease-linked genes is important both to screen for carriers of recessive genetic diseases and to make prenatal diagnoses enabling therapeutic intervention. By appropriate choice of reference fragments, the invention provides a new gene-targeted DNA sequencing procedure that rapidly detects any mutation within a target gene, facilitating the diagnosis of genetic diseases and identification of carriers, especially when a variety of different mutations may cause the defect. Perhaps even more important is the rapid, high-throughput nature of the procedure which promises to facilitate population studies aimed at discovering which mutations within a target gene are actually associated with a disease and which mutations represent harmless polymorphisms. This information is expected to lead to simplification of the technology for specific detection of disruptive mutations, and valuable structure-function relationships that facilitate the development of therapeutics. As will be appreciated from the teachings set forth herein, the invention provides devices and methods for diagnosing a disease in an individual comprising the steps of obtaining a sample and detecting the presence of a nucleic acid in the sample using a device of the invention wherein the presence of the nucleic acid in the sample is indicative of the presence of a disease in the individual. Similarly, the invention permits detecting a genetic lesion indicative of the genetic disease by contacting a sample with a device of the invention to detect the presence of a genetic lesion indicative of a genetic disease. As already explained herein, disease detection and disease monitoring can be accomplished by measuring the concentration of a nucleic acid in a sample wherein the concentration of the nucleic acid in the sample is indicative, for example, of onset of a disease in the individual or, is indicative of the status of a disease in the individual. L. Pharmacogenomics

Given the relationship between disease and genetic mutations, the invention is particularly useful in pharmacogenomics studies. "Pharmacogenomics," as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in development and on the market. More specifically, the term refers to the study of how a patient's genes determine his or her response to a drug. A particular patient's genes can affect drug responsiveness in at least two ways. First, the presence of a genetic lesion per se can render a patient more or less vulnerable to a disease and/or more or less susceptible to responding positively to a drug of choice. Using the present invention's devices and methods, it is possible to predict vulnerability and/or a pre-disposition t treatment by first detecting the presence of a genetic lesion at the DNA level. Second, a patient's ability to respond to a drug can be monitored and qualitatively assessed using the present invention's devices and methods. For example a drug's efficacy could be monitored via expression levels of messenger RNA corresponding to expression of a gene related to the disease state. For example, increased levels of messenger RNA over the first 24-72 hours post-administration of a drug could indicate positive responsiveness to the drug even though symptom abatement had not yet been evident.

Thus, pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects. For example, differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in pharmacogenomics studies in determining whether to administer a particular drug, and may tailor the dosage and/or therapeutic regimen of treatment based on an individual's genomic profile as determined using the present invention. Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally- occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

In brief, the invention provides methods for selecting a treatment regimen for an individual involving the steps of obtaining a sample from an individual which comprises nucleic acid and detecting a lesion in the nucleic acid using a device of the invention, wherein the lesion is indicative of a correlation between a disease and a preferred treatment regimen for the individual. The invention further provides a method of monitoring a disease in an individual comprising the step of measuring the concentration of a nucleic acid in the sample, wherein the concentration of the nucleic acid in the sample is indicative of the status of a disease in the individual. And, the invention also provides a method of monitoring a course of treatment for a disease in an individual which comprises the steps of obtaining serial samples and then measuring the concentration of a nucleic acid in the earlier and later samples followed by a comparison of the concentration of the nucleic acid in the earlier sample to the concentration of the nucleic acid in the later sample, wherein monitoring the relative concentration of the nucleic acid in the samples correlates with the status of disease in the individual. Another pharmacogenomics approach to identifying genes that predict drug response, known as a "genome-wide association," relies on comparing markers across the genomes of each of a statistically significant number of patients taking part in a Phase H/TR drug trial to identify those markers associated with a particular observed drug response or side effect. The present invention can be used to great advantage in such as study, facilitating the rapid comparison of genomes of participating patients. Alternatively, a method termed the "candidate gene approach" can be used to identify genes that predict drug response. This approach is designed to target a drug to a patient based upon the predictiveness of the patient's response to the drug. Ideally, a clinician's administration of a drug should take into account the variability of a patient population, and the possibility that certain patients may respond better to certain drugs than others. _^ Thus, a candidate gene could be used to differentiate sub-populations of responsiveness to a drug. Here, too, the invention may be used to provide rapid sequence comparison of a multiplicity of candidate genes, thereby to determine which alleles of which genes may be indicative of a particular drug response. M. Infectious Agent Identification and Bioremediation Generally, the invention may also be used for rapid, high-throughput identification of infectious agents. The invention is therefore valuable not only in analyzing body fluid (e.g. blood, serum, plasma, urine, sweat, tears, peritoneal fluid, lymph, vaginal secretions, prostatic secretions, semen, spinal fluid, ascitic fluid, saliva, sputum, breast exudate, digestive fluids, etc.) or tissue (e.g. cervical scrapings, tumor tissue, etc.), but also for testing of foods and pharmaceutical products, for sterility testing (e.g. of medical devices or medical facilities) and other environmental testing (e.g. monitoring of water supplies). For example, the methods and devices disclosed herein allow for direct screening of clinical samples to detect HIN nucleic acid sequences. Similarly, other viruses, such as HTLV-I and HTLV-IJ, both of which are associated with leukemia, or human papilloma viruses, which are associated with cervical cancer and other disorders, may be detected in this way. Bacterial infections, such as Helicobacter pylori (associated with chronic ulcers) and tuberculosis, may also be detected.

The invention is also particularly valuable in assessing the concentrations of contaminants or etiologic/biowarfare agents in a sample by measuring the concentration of a nucleic acid indicative of the contaminant or etiologic/biowarfare agent as described above.

In a preferred embodiment, the invention is used to detect the presence of contaminants, such as but not limited to, toxic bacteria in the screening of water and food samples. Moreover, the invention is used to measure the concentration of contaminants. This is especially useful in the screening of biological materials such as blood, donor organs, for example. Samples maybe treated to lyse bacteria to release their nucleic acids, and exposed to a device with reference fragments selected to detect one or more bacterial contaminants which may include, for example, pathogenic strains such as salmonella, campylobacter, Vibrio cholerae, enterotoxic strains of E. coli, and Legionnaire's disease bacteria or viral contaminants such as HIN, Hepatitis C, for example. Similarly, bioremediation strategies may be evaluated using the methods and devices of the invention.

Ν. Enriching a nucleic acid-containing sample for a particular nucleic acid hi one preferred embodiment, the sample is treated to enrich for the presence of a particular nucleic acid. This enriching technique may be useful, for example, when a contaminating nucleic acid may be present, or when only a particular subset of nucleic acids are being tested. Sorting the sample for a particular nucleic acid may be done by any means known in the art. hi one highly preferred embodiment, the nucleic acid is exposed to a specific hybridization moiety that can discriminate among the nucleic acids in the sample (e.g., a chromosome-specific hybridization moiety). The specific hybridization moiety can then be used to selectively purify the desired nucleic acid. For example, the hybridization moiety might be associated with a solid support or a magnetic or paramagnetic bead, allowing the easy separation of the hybridization moiety, and its associated nucleic acid, from other, contaminating nucleic acids. The invention is defined by the appended claims and all equivalents embraced therein.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

CLAΓMS

What is claimed is: 1. A device for direct electronic readout of a biomolecular interaction, the device

comprising:

a substrate comprising a multiplicity of electrically conductive sites arranged in a

spatially addressable array, each of said sites electrically isolated from each other; and

a multiplicity of types of probes, each type of probe comprising a hybridization

moiety capable of hybridizing to a different nucleic acid sequence;

wherein each of said sites is effectively in electrically conductive contact with a

single type of said multiplicity of types of probes, and each type of said multiplicity of

probes is in electrically conductive contact with at least one of said sites.

2. A device as in claim 1, wherein said substrate comprises a multiplicity of electrically

conductive sites embedded in an electrically non-conductive material.

3. A device as in either of claims 1-2, wherein each of said sites comprises a metallic

surface.

4. A device as in claim 3, wherein the metallic surfaces comprise gold.

5. A device as in claim 4, wherein the electrically conductive contacts between said gold metallic surfaces and said probes comprise sulfur linkages.

6. A device as in claim 4, wherein the electrically conductive contacts between said gold metallic surfaces and said probes comprise amine-phosphine linkages.

7. A device as in claim 3, wherein the metallic surfaces comprise silver.

8. A device as in claim 3, wherein the metallic surfaces comprise platinum.

9. A device as in either of claims 1-2, wherein each of said sites comprises an electrically conductive polymer.

10. A device as in claim 9, wherein the electrically conductive polymers comprise polypyrrole.

11. A device as in claim 9, wherein the electrically conductive polymers comprise poly(3,4- ethylenedioxythiophene).

12. A device as in either of claims 1 -2, wherein each of said sites comprises silicon.

13. A device as in any of the preceding claims, wherein the probes comprise nucleic acids.

14. A device as in claim 13, wherein the probes comprise oligodeoxyribonucleic acids.

15. A device as in claim 13, wherein the probes comprise oligoribonucleic acids.

16. A device as in any of claims 1-12, wherein the probes comprise nucleic acid analogs.

17. A device as in claim 16, wherein the probes comprise peptide nucleic acids.

18. A device as in claim 16, wherein the probes comprise thiodiester analogs of nucleic acids.

19. A device as in any of the preceding claims, wherein each of said types of probes corresponds to a different sequence from a genome of an organism.

20. A device as in claim 19, wherein each of said different sequences from the genome of said organism is a unique sequence in said genome.

21. A device as in claim 20, wherein said sequences comprise at least 12 consecutive

nucleotides from said genome.

22. A device as in any of the preceding claims, wherein each of said probes comprises a crosslinking moiety capable of covalently binding said probe to a nucleic acid in said sample hybridized to said hybridization moiety.

23. A device as in claim 22, wherein said crosslinking moiety is interposed between said hybridization moiety and the site to which said probe is in electrically conductive contact.

24. A device as in claim 22, wherein said hybridization moiety is interposed between said crosslinking moiety and the site to which said probe is in electrically conductive contact.

25. A device as in any of claims 22-24, wherein said crosslinking moiety is selected from the group consisting of psoralen; azinomycin B; bispiperidines; cisplatin; 5-(aziridin-l-yl)-2,4- dinitrobenzamide; 2-amino-6-(2-phenylsulfoxyethyl)purine; 2-amino-6-(2- methylsulfinylethyl)purine; adriamycin; mechlorethamine and phosphoramide mustard;

azidophenacyl photoreactive crosslinking agents; chloroethylnitrosoureas, disuccinimidyl suberate; bis[sulfosuccinimidyl] suberate; dimethyl suberimidate; dimethyl pimelimidate; dimethyl adipimidate; disuccinimidyl glutarate; methyl N-succinimidyl adipate; l,5-difluoro-2,4- dinitrobenzene; l-ethyl-3-[3-dimethylaminopropyl]-carbodiimide;j3-azidophenyl glyoxal; and bis-[/3-(4-azidosalicylamido)ethyl]disulfide.

26. A device as in any of the preceding claims, further comprising a multiplicity of calibration sites electrically isolated from each other, wherein each calibration site is in electrically conductive contact with a different type of calibration probe, wherein pairs of said calibration probes correspond to different sequences from a genome of an organism, and wherein each of said pairs of probes are separated by known and varying distances within said genome.

27. A device as in claim 26, wherein said distances are in increments of at least 1 kb.

28. A device as in any of the preceding claims, further comprising a blocking agent bound to said substrate to prevent nonspecific binding of a nucleic acid to said substrate.

29. A device as in claim 28, wherein said blocking agent is selected from the group consisting of detergents and polymers.

30. A device as in claim 29, wherein said polymer is selected from the group consisting of

proteins, nucleic acids, polypyrrolidone, or ethylene glycols.

31. A device as in any of claims 28-30, wherein said sites comprise gold metallic surfaces

and said blocking agent comprises an alkanethiol group or an alkanephosphine group.

32. A device as in any of the preceding claims, further comprising a reference sample

comprising a multiplicity of reference fragments of nucleic acids, each of said reference

fragments hybridized to said hybridization moieties of at least two different types of said probes.

33. A device as in any of the preceding claims, wherein said reference fragments of nucleic

acids are derived from a genome of a reference organism.

34. The device of claim 32, wherein said reference fragments of nucleic acids are

complementary to

(i) RNA molecules expressed by a reference organism or (ii) nucleic acids complementary thereto.

35. A device as in any of claims 32-34, wherein said reference fragments are covalently bound to said probes to which they are hybridized.

36. A device for direct electronic readout of a biomolecular interaction, the device

comprising:

a multiplicity of types of probes, each type of probe comprising a sequence-

specific nucleic acid binding protein capable of binding to a different nucleic acid

sequence;

probes is in electrically conductive contact with at least one of said sites.

37. The device of claim 36, wherein said sequence-specific nucleic acid binding protein is

selected from the group consisting of lambda repressor, TATA binding protein, and DNA

methylases.

38. A method of analyzing nucleic acid sequence differences indicative of a genetic lesion in

a sample comprising nucleic acid, the method comprising the steps of:

(a) exposing the device of any one of claims 32-35 to a sample comprising a

multiplicity of nucleic acids under conditions permitting at least one of said nucleic acids

to hybridize to at least one of said reference fragments hybridized to said hybridization

moieties of said at least two different types of said probes,

(b) measuring a value of electrical current, electrical resistance, capacitance, inductance, or voltage drop between said sites in electrically conductive contact with said

at least two different types of said probes, (c) comparing said measured value with a control value obtained using a control nucleic acid, wherein a difference between said measured value and said control value

above a predetermined amount indicates a difference between said sample nucleic acid

and said control nucleic acid.

39. A method as in claim 38, wherein said reference fragments comprise fragments of a

reference genome and wherein said sample comprises fragments of a sample genome.

40. A method as in claim 39, wherein said reference fragments are substantially non-

overlapping.

41. A method as in any of claims 38-40, wherein the nucleotide sequence of said control

nucleic acid is known.

42. A method as in claim 41, wherein said multiplicity of reference fragments comprise a

contig.

43. A method as in claim 42, wherein said contig represents a single chromosome.

44. A method as in claim 42, wherein said contig represents a portion of a single

chromosome, wherein an allele of said portion of said single chromosome is associated with a

disease.

45. A method as in any of claims 38-44, wherein said sample comprises a multiplicity of

types of chromosomes of an organism, the method further comprising the additional step, prior to

step (a), of simultaneously

enriching said sample for at least one of said multiplicity of said types of

chromosomes and

depleting said sample of at least one other of said multiplicity of said types of

chromosomes.

46. A method as in claim 45, wherein said enriching step comprises exposing said sample to

a chromosome-specific hybridization moiety.

47. A method as in claim 46, wherein said chromosome-specific hybridization moiety is

associated with a solid support.

48. A method as in claim 46, wherein said chromosome-specific hybridization moiety is

associated with a magnetic or paramagnetic bead.

49. A method as in any of claims 38-48, further comprising the additional step, prior to step

(a), of exposing said sample to a restriction enzyme.

50. A method as in any of claims 38-49, further comprising the additional step, prior to step

(a), of mechanically shearing said sample.

51. A method as in any of claims 38-50, further comprising the additional step, prior to step

(a), of chemically digesting said sample.

52. A method as in any of claims 38-51, further comprising the additional step, prior to step (a), of sonicating said sample.

53. A method of diagnosing a disease in an individual, the method comprising the steps of:

obtaining a sample; and

detecting the presence or absence of a nucleic acid in the sample using a device as

in any of claims 1-36,

wherein the presence or absence of the nucleic acid in the sample is indicative of

the disease in the individual.

54. A method of detecting a genetic lesion indicative of a genetic disease, the method

comprising the steps of:

providing a sample comprising genetic material;

contacting the sample with a device as in any of claims 1-36; and,

detecting the presence of a genetic lesion indicative of a genetic disease.

55. A method of selecting a treatment regimen for an individual, the method comprising the

steps of:

obtaining a sample from the individual, said sample comprising nucleic acid; and

detecting a lesion in the nucleic acid using a device as in any of claims 1-36, wherein the lesion is indicative of a correlation between a disease and a preferred

treatment regimen for the individual.

56. A method for identifying a biological relative of an individual, the method comprising the

steps of:

obtaining a control sample from the individual;

obtaining a test sample from a potential biological relative;

comparing the control sample to the test sample to detect related nucleic acids

using a device as in any of claims 1 -36,

wherein the presence of related nucleic acids is indicative of a biological relation

between the individual and the potential biological relative.

57. A forensic science method for identifying a likely source of a biological sample, the

method comprising the steps of:

obtaining at least a portion of the biological sample;

obtaining a test sample from an individual;

comparing the test sample to at least a portion of the biological sample to detect

identical nucleic acids using a device as in any of claims 1-36,

wherein the presence of identical nucleic acids indicates that the individual is a

likely source of the biological sample.

58. A method of detecting an etiologic agent that may be a biowarfare agent, the method

comprising the step of: detecting a nucleic acid indicative of the presence of an etiologic agent using a

device as in any of claims 1-36.

59. A method of constructing a genetic database for use in identifying an individual based on

a sample from the individual, the method comprising the steps of:

obtaining samples from a plurality of individuals;

in each of the samples, detecting a plurality of nucleic acids using a device as in

any of claims 1-36; and

recording data indicative of the nucleic acids detected in each of the samples

thereby to construct a genetic database.

60. A method of mapping a genetic difference between a test nucleic acid and a reference nucleic acid, the method comprising the steps of:

a) detecting a difference between a region of a test nucleic acid and a region of a

reference nucleic acid using a device as in any of claims 32-35; and

b) repeating step a) using smaller subregions of the test nucleic acid and the

reference nucleic acid, thereby to more precisely map the genetic difference.

61. A method of detecting contaminants, the method comprising the steps of:

providing a sample suspected of containing a contaminant;

contacting the sample with a device as in any of claims 1-36; and, detecting nucleotides in the sample indicative of contamination.

62. A method of measuring the concentration of a nucleic acid in a sample, the method comprising the steps of: contacting a sample with a device as in any of claims 32-35, wherein a plurality of identical first probes are in electrically conductive contact with one or more first electrically conductive sites, a plurality of identical second probes are in electrically conductive contact with one or more second electrically conductive sites, and a plurality of reference fragments are hybridized to a hybridization moiety of a first probe and to a hybridization moiety of a second probe; and, measuring a value of electrical current, electrical resistance, capacitance, inductance, or voltage drop between said one or more first electrically conductive sites and said one or more second electrically conductive sites, thereby to quantitatively detect a number of hybridization events between a target nucleic acid and a reference fragment, the number of hybridization events being indicative of the concentration of the target nucleic acid in the sample.

63. A method of diagnosing a disease in an individual, the method comprising the steps of: obtaining a sample; and measuring the concentration of a nucleic acid in the sample using the method of

claim 62, wherein the concentration of the nucleic acid in the sample is indicative of the presence or absence of a disease in the individual.

64. A method of monitoring a disease in an individual, the method comprising the steps of: obtaining a sample; and measuring the concentration of a nucleic acid in the sample using the method of claim 62,

wherein the concentration of the nucleic acid in the sample is indicative of the

status of a disease in the individual.

65. A method of monitoring a course of treatment for a disease in an individual, the method

comprising the steps of:

obtaining a first sample;

obtaining, at a later time, a second sample;

measuring the concentration of a nucleic acid in the first and second samples using the method of claim 62, and

comparing the concentration of the nucleic acid in the first sample to the

concentration of the nucleic acid in the second sample, wherein the concentration of the

nucleic acid in the samples is indicative of the status of a disease in the individual.

66. A method of selecting a treatment regimen for an individual, the method comprising the

steps of:

obtaining a sample; and

measuring the concentration of a nucleic acid in the sample using the method of

claim 62, wherein the concentration of the nucleic acid in the sample is indicative of a

preferred treatment regimen for the individual.

67. A method to assess a concentration of a biowarfare agent, the method comprising the step

of:

measuring the concentration of a nucleic acid indicative of the concentration of a

biowarfare agent using the method of claim 62.

68. A method to assess the concentration of a contaminant in a sample, the method

comprising the steps of:

measuring the concentration of a nucleic acid in a sample using the method of

claim 62,

concentration of a contaminant in the sample.