EP1831670A2

EP1831670A2 - Nanoelectronic devices for dna detection, and recognition of polynucleotide sequences

Info

Publication number: EP1831670A2
Application number: EP05855662A
Authority: EP
Inventors: Jean-Christophe P. Gabriel; Shripal Gandhi; George Gruner; Charles Steven Joiner, Jr.; Ron Sosnowski; Alexander Star; Eugene Tu; Christian Valcke; Joseph Niemann
Original assignee: Niemann Joseph; Nanomix Inc
Current assignee: Nanomix Inc
Priority date: 2004-12-28
Filing date: 2005-12-23
Publication date: 2007-09-12
Also published as: EP1831670A4; WO2006071895A2; JP2008525822A; WO2006071895A3

Abstract

A nanotube device is configured as an electronic sensor for a target DNA sequence. A film of nanotubes is deposited over electrodes on a substrate. A solution of single-strand DNA is prepared so as to be complementary to a target DNA sequence. The DNA solution is deposited over the electrodes, dried, and removed from the substrate except in a region between the electrodes. The resulting structure includes strands of the desired DNA sequence in direct contact with nanotubes between opposing electrodes, to form a sensor that is electrically responsive to the presence of target DNA strands. Alternative assay embodiments are described which employ linker groups to attach ssDNA probes to the nanotube sensor device.

Description

NANOELECTRONIC DEVICES FOR DNA DETECTION, AND RECOGNITION OF POLYNUCLEOTIDE SEQUENCES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present US Patent Application claims priority to each of the following US provisional and non-provisional patent applications pursuant to applicable US law, and the related con-currently filed International Patent Application claims priority to each of the following applications which were filed within the 12 month period provided by the Paris Convention. Each of these applications is specifically incorporated herein, in its entirety, by reference: [0002] This US Patent Application claims priority pursuant to 35 USC

§119(e) to each of the following US Provisional Patent Applications: [0003] No. 60/639954, filed December 28, 2004, entitled "Nanotube

Network-on-Top Architecture for Biosensor"; [0004] No. 60/657,275, filed February 28, 2005, entitled "Nanotube sensor devices for DNA detection";

[0005] No. 60/668,879, filed April 5, 2005, entitled "Nanoelectronic System

For Virus Detection and Identification";

[0006] No. 60/730,905, filed October 27, 2005, entitled "Nanoelectronic

Sensors And Analyzer System For Monitoring Anesthesia Agents And Carbon Dioxide In Breath";

[0007] No. 60/738,694, filed November 21 , 2005, entitled "Nanoelectronic

Sensor Devices For DNA Detection And Discrimination Of Human Hereditary Polymorphisms; and [0008] No. 60/748,834, filed December 9, 2005, entitled "Nanoelectronic Sensors Having Substrates With Pre-Pattemed Electrodes, And Environmental Ammonia Control System".

[0009] This application also claims priority as a continuation-in-part of US

Patent Application No. 11/274,747, filed November 14, 2005, entitled "Carbon Nanotube based Glucose sensing", which in turn claims priority to No. 60/627,743, filed November 13, 2004. [0010] This application also claims priority as a continuation-in-part of US

Application No. 11/212,026 filed August 24, 2005, entitled "Nanotube sensor devices for DNA detection", which in turn claims priority to US Provisional Application No. 60/604,293 filed August 24, 2004 and to US Provisional Application No. 60/629,604 filed November 19, 2004.

[0011] This application also claims priority as a continuation-in-part of US

Application No. 10/846,072 filed May 14, 2004, entitled "Flexible nanotube transistors", which in turn claims priority to US Provisional Application No. 60/471 ,243 filed May 16, 2003. [0012] This application also claims priority as a continuation-in-part of US

Application No. 10/773,631 filed February 6, 2004, entitled "Analyte Detection In Liquids With Carbon Nanotube Field Effect Transmission Devices", which in turn claims priority to US Provisional Application No. 60/445,654 filed February 6, 2003. [0013] This application also claims priority as a continuation-in-part of US

Application No. 10/704,066 filed November 7, 2003 entitled "Nanotube-Based Electronic Detection Of Biomolecules" (Publication 2004-0132,070); which in turn claims priority to US Provisional Application No. 60/424,892 filed November 8, 2002. [0014] This application also claims priority as a continuation-in-part of US

Application No. 10/656,898 filed September 5, 2003 entitled "Polymer Recognition Layers For Nanostructure Sensor Devices", which claims priority to Provisional Application No. 60/408,547 filed September 5, 2002. [0015] This application also claims priority as a continuation-in-part of US Application No. 10/345,783 filed January 16, 2003, entitled "Electronic sensing of biological and chemical agents using functionalized nanostructures" (Publication 2003-0134,433), which claims priority to US Provisional Application No. 60/349,670 filed January 16, 2002. [0016] All of the foregoing US provisional and non-provisional patent applications identified in this "Cross-Reference To Related Applications" section, together with any and all priority documents thereof, are specifically incorporated herein, in their entirety, by reference. FIELD OF THE INVENTION

[0017] The present invention relates to sensors for detecting polynucleotide sequences, and more particularly to sensors using nanotubes as electronic transducers of DNA hybridization.

BACKGROUND OF THE INVENTION

Description of Related Art

[0018] Because base sequences in polynucleotides encode genetic information, the ability to read these sequences has contributed to many advances in biotechnology. This work has identified many important sequences that are linked to medical conditions. For example, the BRCA gene is usually present in women who suffer from breast cancer. To take advantages of these linkages in medical testing, various techniques have been developed to scan tissue samples for the occurrence of specific important sequences. These techniques have shortcomings that make them expensive, slow, and complex, so that they are unlikely to be useful for routine medical testing.

[0019] These techniques universally rely on the tendency of polynucleotides to hybridize. A strand of single-strand DNA (ssDNA) in solution readily combines with a complementary strand (cDNA) that contains an opposite base to pair with each base in the ssDNA. The result of this combination is double-stranded DNA (dsDNA), which can be processed and separated from ssDNA. Thus, to scan for a particular target sequence, an experimenter provides the appropriate cDNA as a probe sequence. If the target sequence is present in a sample, the target ssDNA will hybridize with the probe ssDNA to produce dsDNA, and this hybridization can be detected in some way. [0020] Current methods have mainly focused on optical detection using fluorescence-labeled oligonucleotides with dyes, quantum dots or enhanced absorption of light by oligonucleotide-modified gold nanoparticles. For example, electrochemical detection of DNA hybridization may be performed using nanostructures as electrodes. However, electrochemical methods rely upon electrochemical behavior of the labels. [0021] A first shortcoming arises because many methods of detecting this hybridization involve modification of the sample ssDNA before hybridization. Often, a fluorescent molecule is attached to the ssDNA. This molecule, known as a label, causes the ssDNA to be detected by optical instruments such as microscopes and spectrometers. Labeling is used to detect sample DNA after a hybridization step. If the target sequence is present in a labeled sample, the labeled ssDNA will be incorporated in labeled dsDNA, and the dsDNA will thus be detectable with optical instruments. Although the use of optical detection makes this approach convenient, the chemical reaction by which the DNA is labeled is expensive and time-consuming. A detection method which did not require labeling would significantly increase the usefulness of DNA scanning for routine medical tests.

[0022] A second problem results from the low sensitivity of traditional detection methods. Although some of these methods are sensitive to low concentrations of DNA, they require large absolute numbers of DNA molecules. In a medical application, only a few cells are usually available, and consequently only a few DNA molecules of the target sequence will be present in a sample. This problem has been ameliorated by the use of the polymerase chain reaction (PCR), which can amplify the quantity of target DNA a million-fold. Like labeling, PCR is a complex chemical reaction, which makes tests expensive and slow. [0023] Thus, there is a clear need for a sensitive, fast, technique for detecting specific target DNA sequences. Such a technique should operate without the use of PCR or labeling. What is needed are devices and methods for label-free DNA detection, such as label-free electronic methods offering sensitivity, selectivity and low cost for the detection of DNA hybridization.

SUMMARY OF THE INVENTION

[0024] Nanostructures possess unique properties for sensor applications; in that they may be essentially one- dimensional so as to be extremely sensitive to electronic perturbations, are readily functionalized, and are compatible with many semiconducting manufacturing processes. Embodiments having aspects of the invention employ nanostructures which have properties heavily influence by the atoms are on the surface, thus providing a basis for sensitive electronic detection. [0025] As used herein, a " nanostructure" is any object which has at least one dimension smaller than 100 nm and comprises at least one sheet or other shape of crystalline material. In a number of particular examples, a nanostructure may comprise an elongate tube-like configuration including one or more rolled lattice sheets of atoms connected with graphite-like chemical bonds. Examples include, but are not limited to, nanowires, nanospheres, single-walled nanotubes, double-walled nanotubes, multi-walled nanotubes, fullerenes and fullerene-like "onions". Chemical constituents of the crystalline material include, but are not limited to, carbon, boron, nitrogen, oxygen, boron nitride, molybdenum disulfide, and tungsten disulfide. Exemplary embodiments preferably include one or more carbon nanotubes, and more preferably one or more single-walled carbon nanotubes (SWNTs). [0026] In the exemplary embodiments described in particular detail, the devices include SWNTs which have a size and form in which virtually all the atoms on their surface. These devices have operating characteristics which permit measurement of direct electron transfer between SWNTs and analyte species, such as DNA molecules. For simplicity, the nanostructures included in the examples described in detail may be referred to as "nanotubes", It is noted that alternative embodiments may include alternative nanostructures in nanostructured sensor elements without departing from the spirit of the invention. [0027] Nanoelectronic Detector Devices. A number of alternative embodiments of nanosensor detector or sensor devices having aspects of the invention may be employed for the electronic detection and/or identification of chemical and/or biomolecular analytes, such as polynucleotide species. The alternative device embodiments generally include an element including at least one nanostructure ("nanostructure element") whose electronic properties are highly sensitive to interaction with a target analyte. One or more conducting elements may communicate with the nanostructure element to provide signal(s) for measurement of one or more device electronic properties which are influenced by the response of the nanostructure element to exposure to an analyte medium. [0028] Generally the nanostructure element and conductors are disposed adjacent to a supporting substrate, which typically includes at least a dielectric surface (or surface coating) to provide electrical isolation of device elements. Substrates may be rigid or flexible, and may be generally planar or flat, or alternatively may have functional shapes, such as a tubular configuration.

Substrates have a chemical composition, of which examples include, but are not limited to, silicon oxide, silicon nitride, aluminum oxide, polyimide, and polycarbonate. In a number of examples described herein, the substrate includes one or more layers, films or coatings comprising such materials as silicon oxide, SIO2, Si3N4, and the like, upon the surface of a silicon wafer or chip. In further alternative embodiments substrates include flexible and/or porous materials. Additional alternative substrates having aspects of the invention are described in the Examples included herein. [0029] Nanotube network devices. In embodiments of nanosensor devices, the nanostructured element may comprise a collective structure which includes a plurality of nanostructures, such as SWNTs or other nanotubes arranged to form a collective structure. In certain examples, a plurality nanotubes may be arranged generally parallel to one another. Such nanostructure elements may be disposed generally parallel to a substrate or alternatively may be disposed generally perpendicularly to a substrate.

[0030] However, in a number of preferred embodiments of nanosensor devices i, the nanostructured element may advantageously comprise a random interconnected network of nanotubes ("nanotube network") disposed on or adjacent a substrate, and communicating with at least one electrical lead. [0031] A "nanotube network", as used herein, is a film of nanotubes disposed on a substrate in a defined area. A film of nanotubes comprises at least one nanotube disposed on a substrate in such a way that the nanotube is substantially parallel to the substrate. The film may comprise many nanotubes oriented parallel to each other. Alternatively, the film may comprise many nanotubes oriented randomly. The film may comprise few nanotubes in a selected area of substrate, or the film may comprise many nanotubes in a selected area of substrate. The number of nanotubes in an area of substrate is referred to as the density of a network. Preferably, the film comprises many nanotubes oriented randomly, with the density high enough that electric current may pass through the network from one side of the defined area to the other side, such as via nanotube-to-nanotube contact points. [0032] Nanotube networks may be made by such methods as chemical vapor deposition (CVD) with traditional lithography, by solvent suspension deposition, vacuum deposition, and the like. See for example, US Patent Application No. 10/177,929 (corresponding to WO2004-040.671); US Patent Application No. 10/280,265; US Patent Application No. 10/846,072; and L. Hu et al., Percolation in Transparent and Conducting Carbon Nanotube Networks, Nano Letters (2004), 4, 12, 2513-17, each of which applications and publication is incorporated herein by reference. Further alternative method embodiments for making nanotube networks having aspects of the invention are described in the Examples included herein. [0033] Properties of the nanostructure elements (e.g., nanotube network) may by measured using one or more contacts. A contact includes a conducting element disposed such that the conducting element is in electrical communication with the nanostructure element, such as a nanotube network. For example, contacts may be disposed directly on a substrate surface, or alternatively may by disposed over a nanotube network. Electric current flowing in the nanotube network may be measured by employing at least two contacts that are placed within the defined area of the nanotube network, such that each contact is in electrical communication with the network. [0034] Nanostructure conductance properties. In certain embodiment, a source and a drain electrode may be spaced apart in communication with the nanotube network where the interconnections between nanotubes provides a conduction path between source and drain.

[0035] In the particular examples of nanoelectronic devices having aspects of the invention employed in the following examples, the source and drain may be configured as a pattern of interdigitated electrodes electrically communicating with the nanotube network, and arranged upon a substrate to be approximately coextensive with the nanotube network. Conveniently, the electrodes may be deposited over the network, for example by employing photolithographic masking and metal vapor deposition, although alternative arrangements are possible, such as disposing the electrodes between the network and the substrate. [0036] Transistor embodiments. In some embodiments of the invention, an additional conducting element, referred to as a gate or counter electrode, is provided such that it is not in electrical communication with the nanostructured element (such as at least one nanotube), but such that there is an electrical capacitance between the gate electrode and the nanostructured element . [0037] Exemplary devices comprise field-effect transistors where the channel of the transistor comprises the nanotube(s), and the device may be referred to as a nanotube field effect transistors or NTFET. For example, the gate electrode is a conducting plane within the substrate beneath the silicon oxide. Examples of such nanotube electronic devices are provided, among other places, in patent application Serial Nos. 10/656,898, filed September 5, 2003 and 10/704,066, filed November 7, 2003 (published as US 2004-0132,070), both of which are incorporated herein, in their entirety, by reference. [0038] Alternatively, a gate or counter electrode may comprise a conductive layer disposed adjacent (e.g., under, above, beside), but electrically isolated from, the nanostructure element, such as a conductive polymeric material deposited on a flexible substrate. Resistance, impedance, transconductance or other properties of the nanotubes may be measured under the influence of a selected or variable gate voltage.

[0039] A transistor device arrangement lends itself to measurement of the channel transconductance as a function of gate voltage (e.g., G/Vg signal). A transistor has a maximum conductance, which is the greatest conductance measured with the gate voltage in a range, and a minimum conductance, which is the least conductance measured with the gate voltage in a range. A transistor has an on-off ratio, which is the ratio between the maximum conductance and the minimum conductance. To make a sensitive chemical sensors, a nanotube transistor has an on-off ratio preferably greater than 1.2, more preferably greater than 2, and most preferably greater than 10. [0040] For example, FIG. 1 shows an exemplary conductance curve as a function of gate voltage between +10 V and -10 V for a nanotube electronic device. Relatively high conductance in the "on" curve portion 101 occurs at gate voltages less than about -5 V; relatively low conductance in the "off' curve portion 102 occurs at gate voltages greater than about 0 V. For this device, the on/off ratio is about 100.

[0041] In another preferred embodiment, the gate electrode is a conducting element in contact with a conducting liquid, said liquid being in contact with the nanotube network. Examples of this embodiment are provided, among other places, in Bradley et al., Phys. Rev. Lett. 91 , 218301 (2003), which is incorporated herein, in its entirety, by reference.

[0042] Alternative contact arrangements. It should be understood that other additional or alternative measurements are useful with this device arrangement, including the measurement of the capacitance of the gate electrode (or other counter-electrode) relative to the source (and/or drain) electrode. For example, a voltage may be applied to one or more contacts to induce an electrical field in a nanotube network relative to a counter electrode or gate electrode, and the capacitance of the network may be measured. Conveniently, the source (and/or drain) and gate electrodes of a transistor having a nanostructured channel (e.g., nanotube network) may be employed using suitable circuitry to measure the capacitance of the channel relative to the gate, as an alternative or additional sensor signal to measurements of one or more channel transconductance properties. Alternative embodiments configured to optimize measurements of capacitance or other properties are possible without departing from the spirit of the invention.

[0043] The conducting elements provide for connecting to an electrical circuit for observing an electrical property of the nanotube sensor. Any suitable electrical property may provide the basis for sensor sensitivity, for example, electrical resistance, electrical conductance, current, voltage, capacitance, transistor on current, transistor off current, or transistor threshold voltage.

Alternatively, sensitivity may be based on measurements including a combination, relationship, pattern and/or ratios of properties and/or the variation of one or more properties over time.

[0044] For example, a transistor sensor may be controllably scanned through a selected range of gate voltages, the voltages compared to corresponding measured sensor current flow (generally referred to herein as an I- Vg curve or scan). Such an I-Vg scan may be through any selected gate voltage range and at one or more selected source-drain potentials. The Vg range is typically selected from at least device "on" voltage through at least the device "off' voltage. The scan can be either with increasing Vg, decreasing Vg, or both, and may be cycled +- at any selected frequency.

[0045] From such measurements, and from derived properties such as hysteresis, time constants, phase shifts, and/or scan rate/frequency dependence, and the like, correlations may be determined with target detection and/or concentration and the like. The electronic sensor device may include and/or be coupled with a suitable microprocessor or other computer device of known design, which may be suitably programmed to carry out the measurement methods and analyze the resultant signals. Those skilled in the art will appreciate that other electrical and/or magnetic properties, and the like may also be measured as a basis for sensitivity. Accordingly, this list is not meant to be restrictive of the types of device properties that can be measured.

[0046] Those skilled in the art will appreciate that other electrical properties may also readily be observed and measured. Accordingly, this list is not meant to be restrictive of the types of device properties that can be measured. [0047] Recognition of Analytes. Additional materials may be included in association with the nanostructure element (e.g., species or layers attached or absorbed upon one or more of the nanostructure element, the substrate, the conductor, and the like) to mediate the interaction of the device elements with the analyte medium, including target species, cross contaminants and the like. Such materials may include one or more of recognition layers or molecular transducers (such as the ssDNA oligomer probes in the following examples), catalyst materials, passivation materials, inhibition materials, protective materials, filters, analyte attractors, concentrators, binding species, and the like. Such materials and elements can function to improve selectivity, specificity and/or device service characteristics.

[0048] Polynucleotides Species. The invention provides an electronic sensor device with which to detect specific target sequences of polynucleotides. The sensor comprises nanostructured elements, (for example single and/or multiwalled carbon nanotubes and/or interconnecting networks comprising such nanotubes) which interact with polynucleotides so as to act as sensing elements. In the particular examples described in detail, the nanostructured elements comprise carbon nanotubes, and more particularly, randomly oriented networks of carbon nanotubes. In these examples, the nanotubes are modified before sensing by the adsorption of ssDNA probe sequences. No labeling of the DNA is required. Further, the invention provides a method for using the sensor device. [0049] As used herein, "DNA" means polynucleotides. Examples of polynucleotides include, but are not limited to, deoxyribonucleic acid, ribonucleic acid, messenger ribonucleic acid, transfer ribonucleic acid, and peptide nucleic acid. The defining characteristics of polynucleotides are a chain of nucleic acids and a sequence of bases, each base chemically bonded to a nucleic acid and each base capable of pairing with an appropriate base on a matching sequence. Those skilled in the art will appreciate that other variations of polynucleotides may be produced which share these defining characteristics. Accordingly, a "single- strand DNA", referred to hereafter as "ssDNA", may be a single strand of deoxyribonucleic acid, ribonucleic acid, or any other polynucleotide as described above. A "double-strand DNA", referred to hereafter as "dsDNA", may be a double strand of any polynucleotide described above. "Complimentary DNA", referred to hereafter as "cDNA", may be any strand of a polynucleotide described above which is a single-strand sequence complimentary to an already referenced single-strand sequence.

[0050] In certain embodiments, the invention provides a nanotube sensor device comprising a nanotube network, one or more contacts, and ssDNA contacting the nanotubes. Multiple methods are available for preparing the ssDNA contacting the nanotubes. In one embodiment, ssDNA in solution is mixed with nanotubes in suspension, as described in by Zheng, M. et al. in Nature Materials 2003, 2, 338-342. The resulting solution contains nanotubes around which are wrapped ssDNA strands. The solution is cast onto a substrate, so that ssDNA-wrapped nanotubes are disposed on the substrate. After the disposal of the nanotubes, contacts are made using standard techniques of lithography and metal deposition. In a preferred embodiment, a nanotube network is disposed on a substrate and contacts are made. The resulting electronic device is exposed to a solution containing ssDNA. When the solution is removed, it is found that ssDNA has coated the nanotube network, without coating the substrate. [0051] In certain embodiments, the invention provides devices in which ssDNA contacts the nanotubes directly, without the use of an intervening linker molecule. Further, the ssDNA contacts the nanotubes but does not contact the substrate in areas which are not contacted by nanotubes. [0052] The ssDNA in a particular sensor device may be selected to be cDNA for a particular target sequence. The target sequence is the sequence of bases that the sensor device is intended to detect. The cDNA for the target sequence is known as the probe sequence. Once a target sequence is specified, a quantity of DNA with the probe sequence must be obtained. A variety of techniques are known for synthesizing DNA with specified sequences and for synthesizing DNA complementary to a given sequence. Those skilled in the art will have knowledge of these techniques. Further, appropriate cDNA or other polynucleotide to make a probe specific to a desired target sequence can generally be obtained from known commercial suppliers serving the biotechnology industry. [0053] A sensor device may be used by exposing the nanotube network to a solution containing sample ssDNA. The network should be exposed to the solution for a period of time long enough for hybridization to occur. This period of time depends on the concentration of the sample DNA, the quantity of the solution, the temperature of the room, the pH of the solution, and other variables. Those skilled in the art are familiar with the effect of these variables on DNA hybridization and are capable of choosing an appropriate period of time, solution composition, temperature and other conditions of hybridization without undue experimentation. [0054] Multiple methods of using the sensor devices are disclosed.

[0055] In one embodiment, the sensor device is first measured by varying a gate voltage applied by a conducting plane beneath the insulator of the substrate. Then the network is exposed to a solution containing sample ssDNA for the period of time disclosed above. Next, the solution is removed, and a period of time is allowed to lapse sufficient for the substrate to become substantially dry. This period of time may be made briefer by taking actions which speed the drying process. For example, dry air may be blown over the substrate. After the substrate is dry, the sensor device is measured again by varying the gate voltage. The resulting measurement is compared to the first measurement to see if dsDNA is present.

[0056] In another embodiment, the network is exposed to pure water to obtain a baseline. The sensor device is first measured by varying a gate voltage applied by a conducting plane beneath the insulator of the substrate. Then the network is exposed to a solution of sample DNA in pure water. If the sample DNA contains target DNA, hybridization may occur over time, and the resulting measurement of the sensor device changes in comparison to the first measurement. [0057] In yet another embodiment, the network is exposed to pure water to obtain a baseline. The sensor device is first measured by varying a gate voltage applied by a conducting plane beneath the insulator of the substrate. Then the network is exposed to a solution of sample DNA in a buffer compounded (in terms of temperature, pH, dissolved species, and the like) to promote hybridization. Following a period of time for hybridization, the network may be washed to remove unhybridized DNA and other material. Following washing, the network is again exposed to pure water, and the measurement is repeated. If the sample DNA contains target DNA, hybridization of this DNA will result measurable changes in sensor device characteristics in comparison to the first measurement. [0058] In yet another embodiment, the baseline measurement is performed in the same buffer as is used for hybridization. Then the network is exposed to a solution of sample DNA in the hybridization buffer. Following a period of time for hybridization, the measurement is repeated. If the sample DNA contains target DNA, hybridization of this DNA will result measurable changes in sensor device characteristics in comparison to the first measurement. [0059] In another embodiment, the network is exposed to a conducting liquid. Preferably, the conducting liquid is a buffer appropriate for physiological fluids; most preferably, the conducting liquid is phosphate buffer solution (PBS). The sensor device is first measured by varying a gate voltage applied by a conducting element in contact with the conducting liquid. Then the network is exposed to a solution of sample DNA in a similar conducting liquid. While the network is exposed, the sensor device is measured by varying the gate voltage. If the sample DNA contains target DNA, hybridization occurs over time, and the resulting measurement of the sensor device changes in comparison to the first measurement.

[0060] It should be noted that, with respect to all the described sensor embodiments, that the occurrence, speed and specificity of polynucleotide hybridization depends on various conditions. In each of these hybridization schemes, the binding energy of the dsDNA can be challenged through stringency techniques. This can be done through temperature increases or buffer changes, for example sodium hydroxide. [0061] Additional stringency controls may include various ionic constituents of the hybridization medium, such as sodium or magnesium ions. Alternatively or additionally, a voltage may be applied to elements of the sensor (e.g, a nanotube network) before, during and/or after hybridization to influence polynucleotide behavior. For example, a polynucleotide such as cDNA has a phosphate-based backbone which typically is ionized in the hybridization medium so as to carry a localized negative charge. Selectively charged sensor elements may be used to provide an attractive or repulsive stringency factor, for example, to destabilize a SNP-mismatched probe hybrid relative to a corresponding fully-matched probe hybrid (e.g., during incubation or during a rinse process). [0062] Through variations in stringency, it is possible to differentiate binding of strands with complete or incomplete complementary base pairs. Changes in electrical properties of the nanotubes in response to the stringency process allow discrimination of single base mismatches (SNP), among other things. One of ordinary skill in the art will be able to vary the hybridization conditions so as to tune the operation of certain embodiments of the sensors of the invention to obtain a selected degree of sensitivity to complete and less-than-complete hybridization of the target sequence. [0063] For example, in an assay to discriminate between a DNA sample which is homozygous for a particular allele, on the one hand, and an otherwise comparable sample which is heterozygous for this allele, the stringency of the hybridization conditions may be adjusted (e.g. by variation in temperature) so as to produce a distinctly different device measurement response between the homozygous and heterozygous samples.

[0064] In the case of each of the sensor embodiments having aspects of the invention, these sensors may be constructed in arrays, e.g., arrays of transistor sensors functionalized for a plurality of different target DNA fragments. See Application No. 10/388,701 entitled "Modification Of Selectivity For Sensing For Nanostructure Device Arrays" (published as US 2003-0175,161), incorporated by reference above.

[0065] A more complete understanding of the nanotube sensor devices will be afforded to those skilled in the art, as well as a realization of additional advantages and objects thereof, by a consideration of the following detailed description of the preferred embodiment. Reference will be made to the appended sheets of drawings which will first be described briefly. [0066] Additional alternative embodiments of DNA detection devices and methods having aspects of the invention are described in Examples A through H below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG. 1 is a schematic diagram showing an exemplary conductance curve for a nanotube transistor device.

[0068] Example A:

[0069] FIG. 2 is a schematic diagram showing an exemplary design for a nanotube sensor using a random network of nanotubes. [0070] FIG. 3 is a schematic cross-sectional diagram showing the exemplary nanotube sensor of FIG. 2.

[0071] FIG. 4 is a flow chart showing exemplary steps of a method for making a nanoelectronic sensor according to the invention, and as described in Example A.

[0072] FIG. 5 is a flow chart showing exemplary steps of a method for sensing an polynucleotide according to the invention.

[0073] FIG. 6 is a chart showing conductance as a function of gate voltage for a nanotube electronic device in three circumstances, as described further in the detailed description of the preferred embodiment.

[0074] Example B:

[0075] FIG. 7A shows the device characteristics of the sensor of Example B after functionalization with the pyrene-DNA conjugate and treatment with cDNA.

[0076] FIG. 7B shows the device characteristics of the sensor of Example B after functionalization with the pyrene-DNA conjugate, treatment with SNP-DNA, and subsequent treatment with cDNA.

[0077] Example C:

[0078] FIG. 8A shows an exemplary DNA assay embodiment according to certain aspects of the invention, employing a detector probe linked to the sensor. [0079] FIGS. 8B-F shows an alternative DNA assay embodiment according to certain aspects of the invention, employing electroactive incalators.

[0080] FIGS. 8A-D shows an alternative DNA assay embodiment according to certain aspects of the invention, employing amplifier groups.

[0081] FIGS. 9A-B shows an alternative DNA assay embodiment according to certain aspects of the invention, employing antibody-antigen binding to link the detector probe to the sensor.

[0082] FIGS. 10A-D shows two alternative sensor architectures according to certain aspects of the invention, in which the detector probe is linked to nanostructures such as nanotubes. [0083] FIGS. 11A-C shows two alternative sensor architectures according to certain aspects, of the invention, in which the detector probe is linked to the sensor substrate. [0084] FIGS. 12A-B shows two alternative sensor architectures according to certain aspects of the invention, in which the detector probe is linked to the sensor electrodes.

[0085] Example D: [0086] FIGS. 13-14 illustrates schematically an exemplary nanotube and detection portion of an electroanalytical device having aspects of the invention, where in a target is measured by direct electrical interaction.

[0087] FIGS. 15A-B illustrates schematically an exemplary nanotube and detection portion of an electroanalytical device having aspects of the invention, where in a target is measured by differential blocking of the nanotube with respect to strong effector molecules.

[0088] FIGS. 16A-B illustrates schematically an exemplary nanotube and detection portion of an electroanalytical device having aspects of the invention, where in a target is measured by characteristic dissociation point with respect to a stringency parameter.

[0089] Example E:

[0090] TABLE 1. details the oligonucleotide structures for both capture probes and targets that are employed in Example E.

[0091] TABLE 2 describes the two capture probes and the target DNA that are employed in Example E.

[0092] FIGS. 17A-D illustrates a DNA assay device, wherein

[0093] FIG. 17A is a cross-sectional diagram which illustrates an exemplary electronic sensing device for detecting an analyte, conFIG.d in this example as a

NTFET; [0094] FIG. 17B are three views of a photomicrograph (SEM) of a sensor generally similar to that of FIG. 1A: View (a) showing the layout of interdigitated source and drain contacts; View (b) showing an enlarged detail of a nanotube network N and the contacts; and View (c) showing an enlarged detail of the margin of a nanotube network; [0095] FIG. 17C is a photograph of a sensor generally similar to that of

FIGS. 1A, 1 B, fabricated on a die, and mounted as a chip in a conventional

CERDIP package; and [0096] FIG. 17D is a photograph of a sensor generally similar to that of

FIGS. 1A and 1 B, packaged in the manner shown in FIG. 1C, and installed on an exemplary circuit board of an electronic sensor system.

[0097] FIGS. 18A-D illustrate the process of Autoprobe testing of wafers, wherein:

[0098] FIGS. 18A and 18B are examples of wafer maps of device maximum conductance and of device modulation respectively for an exemplary NTFET wafer; and

[0099] FIGS. 18C and 18D are examples device population plots of the conductance and of the modulation data shown in FIGS. 18A and 18B respectively.

[00100] FIGS. 19A-D illustrate fluorescent confirmation of probe attachment, wherein:

[00101] FIG. 19A shows the device after incubation with the unlabeled ssDNA capture probe, but before treatment with target DNA;

[00102] FIG. 19B shows the fluorescent image of a NTNFET device following incubation with Cy5-labeled target DNA;

[00103] FIG. 19C shows a comparable fluorescent image of a NTNFET device following incubation with Cy5-labeled target DNA; and [00104] FIG. 19D is a bar graph showing a quantitative comparison of target

DNA hybridization based on fluorescence signal from the devices of FIGS. 19A-C.

[00105] FIGS. 20A-20B illustrate NTNFET device electronic responses to

DNA, wherein:

[00106] FIG. 2OA illustrates the effect of the complementary probe hybridization; and

[00107] FIG. 2OA illustrates the effect of the non-complementary probe hybridization.

[00108] FIGS. 21 A though 21 E illustrate an allele-specific assay to detect single-nucleotide polymorphisms, wherein: [00109] FIG. 21A is a plot which shows the allele specific wild-type capture probe hybridized with wild-type synthetic HFE target; [00110] FIG. 21 B is a plot which shows the mutant capture probe exposed the same hybridization conditions with wild-type synthetic HFE target; [00111] FIG. 21 C is a plot which summarizes both electronic (1-G/G0) and fluorescent optical responses from the fluorescent target labels for hemochromatosis detection;

[00112] FIG. 21 D is a graph of electronic response in NTNFET devices to HFE single-nucleotide polymorphisms, as a function of electrode pitch; and [00113] FIG. 21 E is a graph illustrating the enhancement detection of single- nucleotide polymorphisms in the presence of obscuring nonhomologous DNA by means of "blocking" with triton X-100. [00114] Example F:

[00115] FIGS. 22A-22D illustrate NTNFET response to hybridization of unlabeled oligonucleotides at different concentrations of ionic species and target DNA, wherein: [00116] FIG. 22A is a plot of modulated conductance response for the exemplary NTNFET device in a sodium phosphate buffer for nM target DNA concentrations;

[00117] FIG. 22B is a plot of modulated conductance response for the exemplary NTNFET device under the influence of Mg+2 ions nanoM target DNA concentrations;

[00118] FIG. 22C is a plot of modulated conductance response for the exemplary NTNFET device with Mg+2 ions at picoMolar ranges of the DNA target species; and [00119] FIG. 22D is a plot of the data of FIGS. 22A-22C showing of the normalized conductance (G/G0) of the three NTNFET devices as function of target DNA concentrations. [00120] Example G:

[00121] FIGS. 23A through 23H illustrate the operation of a multiplex assay panel embodiment having aspects of the invention, wherein: [00122] FIG. 23A is a diagram showing the array as incubated with a mixture of different probes kinds which bind to the sensors of the array; [00123] FIG. 23B shows the array with excess and non-binding probes rinsed away;

[00124] FIG. 23C is a diagram showing the sensors as interrogated by measurement circuitry; [00125] FIG. 23D shows sensor signals correlated with probe binding configuration.;

[00126] FIG. 23E is a diagram showing the array incubated with sample target species;

[00127] FIG. 23F shows the array with excess sample rinsed away; [00128] FIG. 23G shows the sensors interrogated following sample exposure; and

[00129] FIG. 23H shows sensor signals correlated with probe-target binding status.

[00130] FIGS. 24A through 24D illustrate an example of employing multiple signals from a single sensor to characterized the probe-sensor configuration, wherein:

[00131] FIGS. 24A-B show the array embodiment exposed to incubation procedures generally as described above with respect to FIGS. 23A-B ; and

[00132] FIGS. 24C-D show measurements of both source-drain conductance (left hand axis of each sub-plot) and channel-gate capacitance (right hand axis of each sub-plot).

[00133] FIGS. 25A through 25E illustrate the use on enhancement groups to facilitate probe characterization.

[00134] Example H: [00135] FIG. 26 shows an exemplary field effect transistor sensor in which a biofunctional layer is deposited above a nanotube network.

[00136] FIG. 27 shows an exemplary "network-on-top" field effect transistor sensor having aspects of the invention in which a nanotube network is disposed above a biofunctional layer. [00137] FIG. 28 shows an alternative exemplary field effect transistor sensor having aspects of the invention having a subsequently deposited pattern of source and drain conductors. [00138] FIG. 29 shows an addition alternative exemplary sensor having aspects of the invention.

[00139] FIGS. 3OA and 3OB are top and side views of exemplary sensor having aspects of the invention, showing a plurality of recognition molecules. [00140] FIG. 31 is an alternative embodiment having aspects of the invention, generally similar to that of FIG. 8A in which the probe group is attached to a biofunctional layer.

[00141] Example I:

[00142] FIG. 32A is a diagram contrasting alternative configurations of a nanosensors having aspects of the invention, one providing for a transport-limited parallel or tangential flow of analyte medium with a sensor having a porous substrate and providing for a reaction-limited perpendicular or through-flow of analyte medium.

[00143] FIGS. 32B-32C are photomicrographs of two alternative micro- porous alumina membranes, such as may be employed in through-flow sensor embodiments having aspects of the invention.

[00144] FIGS. 33A-33D illustrate alternative exemplary embodiments of nanosensors having aspects of the invention and providing for flow of analyte medium through a porous substrate, and show an exemplary module for fluidic sample analysis.

[00145] FIGS. 34A-34B illustrate alternative exemplary embodiments of nanosensors having aspects of the invention and providing for electric field stringency to control binding of non-specific polynucleotides.

[00146] FIG. 35 illustrates alternative exemplary embodiments of nanosensors having aspects of the invention having capillary delivery of samples.

[00147] FIGS. 36A-36C illustrate alternative exemplary embodiments of nanosensors having aspects of the invention a having a network of nanotubes superficially applied to a porous membrane. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT [00148] In certain embodiments, the present invention provides a nanotube sensor device that detects a target DNA sequence. The device requires no labeling of the target DNA and responds electronically to the presence of the target DNA. In the detailed description that follows, like element numerals are used to indicate like elements appearing in one or more of the figures. [00149] It should be understood that the various embodiments and examples described herein should be read together, and one of ordinary skill in the art will see that alternative embodiments may be made by combining aspects of more than one example or embodiment, without departing from the spirit of the invention.

[00150] EXAMPLE A

[00151] DNA Functionalization of Carbon Nanotube Device [00152] Referring to FIGS. 2 and 3, a nanotube DNA sensor 100 according to the invention may comprise a suitable substrate 140, for example, a degenerately doped silicon wafer. Other substrates may include, for example, other semiconductors, or insulating substrates such as ceramics or polymers. Substrate 140 may be passivated with a silicon oxide film 180, as known in the art. [00153] Optionally, a gate electrode 170 may be formed in a lower layer of the substrate, and connected to a contact 176 via any suitable conductor 175. Alternatively, the substrate may comprise a conducting base material, such as doped silicon, covered by an insulating layer, such as SiO2, in which the conducting base material is connected to circuitry to serve as a gate or counter electrode.

[00154] In the example shown, a network of randomly oriented nanotubes 120 is disposed over a silicon substrate 140, and the device includes a pair of contacts 101 , 110 having interdigitated portions disposed over network 120, the network providing a conducting channel between the contact pair. The substrate 140 outside of the generally rectangular area 130 should be substantially free of the nanotube network 120. [00155] Alternative embodiments may comprise a single or a plurality of nanotubes disposed adjacent a substrate, in which the nanotubes are in electrical contact with one or more contacts. In some embodiments, most or all of the nanotubes may span to electrically conduct between a pair of adjacent contacts. [00156] In the randomly oriented interconnecting nanotube network 120 of the example shown, however, it is not necessary that all or a majority of the nanotube be in electrical contact with one or more electrodes. Inter-nanotube contacts may serve to provide a conductive path, permitting current or charge transmission through the network. [00157] In the example shown, the contacts 101 , 110 are deposited over network 120. Alternatively, contacts may be deposited upon substrate 140, and network 120 formed upon the contacts.

[00158] One or more of contacts 101 , 110 may be provided, and may optionally have a covering passivation layer 180, as known in the art. For example, contacts 101 , 110 may comprise one or more metal layers, such as titanium and gold.

[00159] Contacts 101 , 1 10 may comprise a plurality of interdigitated portions disposed over a generally rectangular region 130. The interdigitated configuration advantageously increases the surface area of the contacts that can be exposed to a nanotube film between the contacts. Other configurations of contacts may also be suitable, for example, parallel labyrinths of any desired shape, or any other configuration providing a sensor region between opposing contacts. The rectangular shape of region 130 is merely exemplary, and this region may comprise any desired shape. Contacts 101 , 110 may be configured as source and drain electrodes for a field-effect transistor device, or merely serve as connections to a resistive or capacitive sensor. In certain embodiments, a single contact (e.g., 101) may be employed to induce an electrical field or capacitance of the network 120 relative to gate electrode 170 or other counter electrode, so as to provide a sensor signal. [00160] Contacts 101 , 110 and portions of the substrate 140 that are not between the contacts may be protected by a barrier material 160. For example, an epoxy resin, or any other suitable polymer or resin material, may be deposited to form a barrier 160, and removed, such as by etching, from a region between the opposing contacts 101 , 110.

[00161] A plurality of single-strand DNA molecules 150 may be disposed over the nanotube film using any suitable method, for example as described herein below. The DNA molecules may be attached directly to nanotubes in the nanotube film 120, or may rest on the substrate 140 near nanotubes in the film. In the alternative, DNA molecules may be disposed over a material interposed between the nanotube film and the DNA. The DNA should, however, be disposed sufficiently close to the nanotube film so that a reaction between the ssDNA and complementary ssDNA strands influences a measured electrical property of sensor 100.

[00162] In one exemplary embodiment of the invention (see Example A), the ssDNA contacts the nanotubes directly, without the use of an intervening linker molecule . Further, the ssDNA contacts the nanotubes but does not contact the substrate in areas which are not contacted by nanotubes. The ssDNA molecule 150 may be removed from substrate 140 except from over the nanotube film 120. [00163] The ssDNA in a particular sensor device is selected to be cDNA for a particular target sequence. The target sequence is the sequence of bases that the sensor device is intended to detect. The cDNA for the target sequence is known as the probe sequence. Once a target sequence is specified, a quantity of DNA with the probe sequence must be obtained. A variety of techniques are known for synthesizing DNA with specified sequences and for synthesizing DNA complementary to a given sequence. Those skilled in the art will have knowledge of these techniques. Further, cDNA may often be obtained from commercial sources.

[00164] It should be appreciated that a plurality of nanotube sensors like sensor 100 may be formed in parallel on a single substrate, and later separated. Separated devices may be mounted in chip carriers as known in the art, and integrated with conventional electronics to provide useful sensing instrumentation that should be capable of sensing a targeted polynucleotide. Multiple sensors sensitive to different sequences may be combined in an electronic device to detect a variety of different polynucleotide sequences at once. One of ordinary skill may construct suitable electronics for a sensing instrument, using the disclosure herein.

[00165] FIG. 4 shows exemplary steps in a method 400 for making a nanoelectronic sensor for particular DNA sequences. Steps 410 through 490 may be performed in any operative order.

[00166] At step 410, a gate electrode may be formed on a substrate, for example a passivated silicon or other semiconducting substrate, or on a semiconducting substrate such as a ceramic or polymer material. The electrode may comprise a metal or other conducting material, and may be formed using photolithography and lift-off as known in the art, or any other suitable method. In certain embodiments, the gate electrode comprises bulk silicon substrate wafer material, connected to suitable circuitry.

[00167] At step 420, the substrate (and embedded gate electrode, if included) may be coated with a passivation or insulating layer, such as a silicon oxide layer, as known in the art.

[00168] At step 440 , one or more nanotubes is placed in the substrate in electrical communication with each of the opposing contacts. For example, the substrate 140 may be coated with carbon nanotubes in a random network, as described in the earlier-referenced United States patent application Serial No. 10/177,929. In the alternative, other methods as known in the art for forming nanotubes between contacts may be used. The resulting nanotubes may be oriented in a specified fashion, or randomly oriented. If randomly oriented, the nanotubes should provide a network of connected nanotubes that connects the opposing contacts via at least one pathway. Nanotubes should be removed from the substrate in areas other than between the opposing documents, using any suitable method, such as plasma etching.

[00169] At step 430 , a pair of opposing contacts, such as source and drain electrodes, may be formed on the substrate. The contacts may be above the nanotubes, or may be between the nanotubes and the substrate. For example, titanium contacts may be formed and covered with a gold layer using photolithography and lift-off to form opposing contacts. The contacts may comprise a plurality of interdigitated portions disposed over an intermediate region of any desired shape.

[00170] At step 450, an optional layer of barrier material may be deposited over the contacts. Various polymers and resins are known in the art, and may comprise a suitable barrier. In an embodiment of the invention, an epoxy coating may be used. The barrier may be applied only in certain areas of the substrate, or applied over the entire substrate and removed from operative areas of the sensor such as between the contacts. The barrier may provide for electrical insulation, preventing short-circuiting of the sensor when in contact with an conductive fluid, or otherwise protecting the sensor from exposure to the environment. The barrier may also be helpful in controlling the deposition of other materials, including but not limited to nanotubes and DNA molecules. Any number of barrier layers may be used. [00171] At step 460, a solution of oligonucleotide (ssDNA) may be prepared. The desired ssDNA ("probe sequence") may be obtained from a commercial source or synthesized as known in the art. A water or organic solution of the probe sequence may be prepared at a suitable concentration. For example, a solution of 10^"4 M concentration may be prepared by dissolving 100,000 p mole of the oligonucleotide in 1000 μl_ of pure (18 MΩ). Other solvents compatible with ssDNA may be used. Prior to depositing the ssDNA, the electrical properties of the sensor device may optionally be noted as a baseline. [00172] At step 470, the oligonucleotide solution may be applied over the active region 130 of the sensor device. For example, a drop of DNA solution may be placed on the chip over region 130. Then, the solution may be dried to evaporate the carrier and leave the ssDNA behind intact. For example, the may be placed in a humidified chamber at room temperature until dry. Then the chip may be removed from the chamber, rinsed with 18 MΩ water and blown dry with dry nitrogen. At step 490, excess ssDNA may be removed. This may occur by rinsing and blowing, as just described. More aggressive methods, e.g., etching, may be used if excess DNA is bonded to other areas of the substrate. In the alternative, excess DNA may be left in place if doing so does not disrupt sensor operation. [00173] The electrical properties of the sensor device may be again observed and compared to the baseline properties. To the extent ssDNA has been successfully deposited, a change in the electrical properties should be observable. Properties that may be observed may include, for example, sensor gate voltage, conductance, resistance, or any combination, curve or hysteresis involving these or other electrical properties.

[00174] FIG. 5 shows exemplary steps of a method 500 for using a sensor device according to the invention. Essentially, a sensor is used by exposing the nanotube network to a solution containing sample ssDNA, and observing changes in the electrical properties of the sensor. At step 510, the sample is prepared as known in the art. For example, DNA may be extracted from a patient's cells by dissolution. Double-stranded DNA should be reduced to ssDNA using a method as known in the art. If a sufficiently large sample of DNA is available, if may be possible to avoid use of a PCR method to increase DNA concentration. Since the sensor of the present invention may operate using an extremely small sample volume (e.g., less than 100 μl_), use of PCR may in some instances be avoided. [00175] At step 520, the sensor is exposed to the sample solution. The sensor should be left in the solution for a period of time long enough for hybridization to occur between at least one ssDNA molecule on the nanotube network and a complementary ssDNA molecule in solution. This period of time depends on the concentration of the sample DNA, the quantity of the solution, the temperature of the room, the pH of the solution, and other variables. Those skilled in the art are familiar with the effect of these variables on DNA hybridization and are capable of choosing an appropriate period of time. [00176] At step 530, an electrical response of the sensor is observed.

Various different properties may be useful, depending on the configuration of the sensor. In one embodiment, the sensor device is first measured by varying a gate voltage applied by a conducting plane beneath the insulator of the substrate. Then the network is exposed to a solution containing sample ssDNA for the period of time disclosed above. Next, the solution is removed, and a period of time is allowed to lapse sufficient for the substrate to become substantially dry. This period of time may be made briefer by taking actions which speed the drying process. For example, dry air may be blown over the substrate. After the substrate is dry, the sensor device is measured again by varying the gate voltage. The resulting measurement is compared to the first measurement to see if dsDNA is present. [00177] In another embodiment, the network is exposed to pure water. The sensor device is first measured by varying a gate voltage applied by a conducting plane beneath the insulator of the substrate. Then the network is exposed to a solution of sample DNA in pure water. While the network is exposed, the sensor device is measured by varying the gate voltage. If the sample DNA contains target DNA, hybridization occurs over time, and the resulting measurement of the sensor device changes in comparison to the first measurement. [00178] In another embodiment, the network is exposed to a conducting liquid. Preferably, the conducting liquid is a buffer appropriate for physiological fluids; most preferably, the conducting liquid is phosphate buffer solution (PBS). The sensor device is first measured by varying a gate voltage applied by a conducting element in contact with the conducting liquid. Then the network is exposed to a solution of sample DNA in a similar conducting liquid. While the network is exposed, the sensor device is measured by varying the gate voltage. If the sample DNA contains target DNA, hybridization occurs over time, and the resulting measurement of the sensor device changes in comparison to the first measurement.

[00179] At step 540, the observed electrical response should be correlated to the target species to determine a positive or negative result. For example, with gene testing, the target sequence is ether present, or it is not. Reaction between the sensor and the targeted gene sequence should produce results that are consistent and repeatable for sensors of a given type. Thus, a positive or negative result, and a confidence level, may be based on a comparison between a particular sensor response and statistical control data for sensors of the same type. Confidence in a result may be increased by performing multiple measurements using multiple sensors in parallel.

[00180] A degenerately doped silicon wafer with a silicon oxide film was coated with carbon nanotubes in a random network, as described in the earlier- referenced United States patent application Serial No. 10/177,929 and generally in accordance with the description hereinabove. Titanium contacts 30 nm thick covered with gold contacts 120 nm thick were deposited and patterned by photolithography and lift-off to form opposing contacts. The contacts each comprised a plurality of interdigitated portions disposed over a generally rectangular region. A network of randomly oriented nanotubes was disposed over the silicon substrate. Nanotubes in the network were in electrical contact with interdigitated portions of the contacts. After the deposition of the contacts, nanotubes outside of the generally rectangular area were removed by oxygen plasma etching, leaving nanotube network remaining. The use of interdigitated sets of metal electrodes with nanotube network interposed generally between the interdigitated contacts results in many nanotubes connected in parallel across the electrodes. [00181] A die was separated from the wafer and mounted in a standard 40- pin chip carrier, with wires connecting the interdigitated wires on the chip to the contacts on the chip carrier. The contact pads and wires on the packages were coated with epoxy resin, which was allowed to cure. Chips in packages thus prepared were rinsed with acetone, isopropanol, deionized water, and then 18 MΩ water. [00182] A solution of oligonucleotide 5'-CCT AAT AAC AAT-3' at concentration 10-4 M was prepared by dissolving 84500 pmole of the oligonucleotide in 845 μl_ of pure water (18 MΩ water from a NANOpure Infinity UV water system). A chip prepared as described above was measured by varying a gate voltage applied by a conducting plane underneath the insulator. The resulting curve is shown in FIG. 6 as item 600. Then a drop containing 20 μl_ of DNA solution was placed on the chip. The chip and solution were placed in a humidified chamber at room temperature for 12 hours. Then the chip was removed from the chamber and rinsed with 18 MΩ water and blown dry with dry nitrogen. The chip was measured by varying the gate voltage. The resulting curve is shown in FIG. 6 as item 610. This curve represents a sensor device prepared for use as a sensor. At this stage, the nanotube network is contacted by ssDNA with a probe sequence. The effect of the ssDNA coating on the electronic measurement is that the curve 610 is shifted to the left of curve 600. [00183] To demonstrate contact between the nanotubes and the probe ssDNA, one chip was prepared with a labeled ssDNA. Labeled ssDNA is not necessary for the preferred embodiment and is only described here for illustrative purposes. A solution of oligonucleotide 5'-HS-(CH2)6-CCT AAT AAC AAT- fluorescein -3' at concentration 10-5 M in 18 MΩ water was prepared as a receptor DNA sequence. A chip was exposed to this solution overnight, rinsed, and dried with nitrogen gas. An optical fluorescence micrograph of this chip was observed, and a green fluorescein label appeared as a bright area only in a defined area where the nanotube network was present, and not in other areas of the substrate. This demonstrated that the receptor DNA strand was attached to the nanotubes of the sensor. [00184] Next, a solution of target DNA, oligonucleotide 5'-ATT GTT ATT AGG-3 complementary to the receptor DNA strand, at concentration 10-4 M, was prepared by dissolving 132,000 pmole of the oligonucleotide in 1320 μL of 18 MΩ water. A diluted solution of target DNA at concentration 10-8 M was thereby prepared. The chip was exposed to a 20 μL drop of this solution in a humidified chamber at room temperature for one hour. The chip was then removed from the chamber and rinsed with 18 MΩ water and blown dry with dry nitrogen.

[00185] FIG. 6 shows the resulting curve FIG. as item 620. This curve represents the result of hybridization of the probe DNA with the target DNA. The effect of the target DNA hybridization on the electronic measurement is that the curve 620 is shifted to the right of curve 600. [00186] EXAMPLE B

[00187] Noncovalent Chemical Functionalization of Carbon Nanotube

Devices

[00188] for Single Base Mismatch DNA detection.

[00189] B-1. Summary: In one exemplary embodiment having aspects of the invention, a nanotube sensor device comprises a carbon nanotube network field effect transistor ("NTFET" or "NTNFET") device functionalized with single- stranded DNA (ssDNA). In certain embodiments, single-stranded DNA (ssDNA) may be immobilized on NTFET devices through polymer and polyaromatic molecules non-covalently attached to carbon nanotubes. The significant differences in the electronic response of functionalized NTFETs to complementary single-stranded DNA (cDNA) and single base mismatch single-stranded DNA (sbmDNA) may be measured. This exemplary sensor includes the following structure, elements and functions:

[00190] a) One or more carbon nanotube FET device comprising a single nanotube and/or a networks of nanotubes disposed to form a conducting channel between at least a source and a drain electrode. [00191] b) The FET geometry may include a bottom gate electrode and/or a liquid gate electrode.

[00192] c) Polymer and/or aromatic linker molecules attached non- covalently to the carbon nanotubes [00193] d) ssDNA is attached chemically to the linker molecule to create a probe.

[00194] e) In operation, when complementary cDNA is exposed to the sensor, it hybridizes with the probe, with a measurable effect on device electrical characteristics.

[00195] f) In operation, when single base mismatch sbmDNA is exposed to the sensor, it also hybridizes with the probe, but produces measurably distinct device characteristics.

[00196] NTNFET devices were prepared according to procedures further described, among other places, in US patent application Nos. 10/177,929, 10/656,898, and 10/704,066, each incorporated by reference above. Electric current is an electrical property that may be measured using contacts. A contact comprises a conducting element that may be disposed on the substrate, such that the conducting element is in electrical communication with the nanotube network. At least two contacts may be placed within the defined area of the nanotube network, such that each contact is in electrical communication with the network. [00197] In some embodiments of the invention, an additional conducting element, referred to as a gate electrode, is provided such that it is not in electrical communication with the at least one nanotube, but such that there is an electrical capacitance between the gate electrode and the at least one nanotube. In one exemplary preferred embodiment, the gate electrode is a conducting plane within the substrate beneath the silicon oxide. Examples of such nanotube electronic devices are provided, among other places, in the above incorporated patent applications Nos. 10/656,898 and 10/704,066.

[00198] The sensor NT devices may be made using standard photolithography techniques on, for example, 100 mm wafers. NTFET devices were fabricated using SWNTs grown by chemical vapor deposition (CVD) at 900⁰C using dispersed iron nanoparticles as growth promoter and a methane/hydrogen gas mixture. Electrical leads were patterned on top of the nanotubes from titanium films 30 nm thick capped with a gold layer 120 nm thick. After conducting initial electrical measurements to establish the device characteristic, the substrates were wire bonded and packaged in a 40-pin CERDIP package before conducting the DNA experiments. The contact pads and wires on the packages were coated with epoxy resin, which was allowed to cure. The DNA experiments were performed by putting a single drop of the DNA solution on the package, which is located in a sealed jar, containing a beaker with ~100 ml. of water to prevent the evaporation of the drop. [00199] Electronic measurements of NTFET devices, such as current flow between S/D electrodes as a function of applied gate voltage, were conducted using Parallel Measurement System (PMS). This system is capable of measuring device characteristics of up to 12 nanotube-based sensors simultaneously. A set of 32 independent analog switches are digitally controlled via PC and allow the user to select the junctions to be measured. Applied source-drain bias and gate voltage are both user defined (amplitude, frequency, function). The system can measure device conductance as both a function of time and gate-voltage. [00200] B-2 Preparation Procedures:

[00201] B-2.1 Preparation of Chips. Before each chip was used, it was packaged and the wires and contacts were coated with epoxy, which was allowed to cure. The chip was rinsed from a squirt bottle with acetone, isopropanol, deionized water, and finally was washed using the formalized washing procedure (Section B-2.2), after which initial I-Vg curves were taken. [00202] B-2.2 Washing Procedure. A packaged chip was briefly rinsed with a squirt of 18 MW water to remove any analyte on the surface. In a crystallizing dish, approximately 50ml of a 0.01 M Phosphate buffered saline solution (pH 7.4 @25°C) was poured over the chip. It was washed on an orbital shaker at speed setting 6 for 5 minutes. The solution was then discarded. The chip was then washed four times with 18 MW water in the same way. [00203] B-2.3 I-Vg Curves. While I-Vg curves (plots of NTFET current versus scanned gate voltage) were captured for all devices on the chip, only one for each chip is shown in this report. The curve that is shown in each case should be considered to be representative of all curves obtained for each chip. [00204] B-3 Pyrene-labeling and DNA:

[00205] B-3.1. Formation of Pyrene Monolayer. A packaged chip (in this case, W517 26:21) was cleaned and initial I-Vg measurements were taken. A 2.5 mg/mL solution of pyrene butanoic acid succinimidyl ester in N₁N- dimethylformamide (DMF) was prepared by dissolving 3.08 mg of the pyrene substance in 1.232 ml_ of DMF. 50 mL of this solution was placed on the surface of the chip, which was then sealed inside of a chamber for 2 hours at room temperature with an open container of DMF to prevent the drop from evaporating. The chip was then removed, was rinsed with DMF, acetone, and isopropanol, and was then cleaned (Section 2.2), and I-Vg curves were taken.

[00206] B-3.2 Covalent Attachment of DNA. 20 mL of the DNA-NH2 solution was placed on the surface of the chip, which was then sealed inside of a chamber overnight at room temperature with an open container of water to provide humidity and prevent the drop from evaporating. The chip was then removed, was washed according to the washing procedure, and I-Vg curves were taken.

[00207] B-4 Detecting DNA Hybridization:

[00208] B-4.1 Detection Procedures. A 10-6 M solution of the DNA oligonucleotide was prepared by diluting 10 mL of a 10-4 solution of the oligonucleotide with 990 mL of a 0.01 M Phosphate buffered saline solution (pH 7.4 @25°C). 20 mL of this DNA solution was placed on the surface of a chip, which had been functionalized with a DNA-pyrene layer according to Section B-3. The chip was then sealed inside of a chamber overnight at room temperature with an open container of water to provide humidity and prevent the drop from evaporating. The chip was then removed, was washed, and I-Vg curves were taken. [00209] B-4.2 cDNA. A chip (W517 26:21 ) was functionalized according to Section B-3, and was then treated with cDNA according to Section B-4.1. [00210] FIG. 7A shows the I-Vg curve, which reveals that the curve is shifted to the right, suggesting that the device can detect the hybridization of the covalently bound DNA with the cDNA. A shift to the right is consistent with shifts seen in previous experiments when double stranded DNA is present. [00211] B-4.3. SNP-DNA. A chip (W517 26:24) was functionalized according to Section B-3, and was then treated with SNP-DNA according to Section B-4.1. [00212] FIG. 7B shows the I-Vg curve, which reveals a fairly insignificant shift to the right. This may be due to partial (but incomplete) binding of the SNP- DNA to the DNA attached to the device, or it may be that the SNP-DNA is washed away during the washing procedure, as this magnitude of shift has also been shown to be associated with drift (possibly due to a very thin layer of water adsorbed to the nanotubes). Either way, it can be asserted that the devices are able to distinguish between cDNA and a SNP.

[00213] B-4.4. SNP-DNA + cDNA. The chip (W517 26:24) that had already been treated with SNP-DNA was then treated with cDNA according to Section B- 4.1. FIG. 7B shows the I-Vg curve and reveals a shift to the right, which is similar to the shift seen with cDNA in Section B-4.2. This indicates that the device can detect the cDNA after being exposed to the SNP-DNA. If the SNP-DNA was not washed away in Section B-4.3, then the cDNA can displace the SNP-DNA, producing a result that is consistent with the data seen for hybridization in Section B-4.2 and elsewhere. [00214] The nanoscale electronic devices, NTFETs, may be used for real time monitoring and detection of nucleic acids (RNA and DNA) in small quantities. For DNA oligonucleotide hybridization assays, the NTNFET devices can detect a small amount of single-stranded DNA (ssDNA). Such assays are faster and much more sensitive than existing methods and, for example, reduce the necessary number of DNA duplication cycles or even eliminate PCR.

[00215] When there is a single mismatched base between two DNA strands, hybridization can still occur but the hybridization complex with the "kink" due to the mismatch will be less stable. These mismatches are called single nucleotide polymorphism (SNP), and were discovered as a result of the Human Genome

Project. SNPs are the key target for commercial genetic tests and can be potentially identified by NTNFET devices.

[00216] EXAMPLE C: [00217] DNA assays using nanoelectronic devices.

[00218] A number of different exemplary DNA (or other polynucleotide) assay embodiments having aspects of the invention are shown in FIGS. 8-12.

The structure and methods shown are exemplary, and other alternative embodiments may use structures and methods described elsewhere in this application. Where the different embodiments include substantially similar elements, the same reference numbers are used to designate such elements in the description of each embodiment.

[00219] C-1 Structure:

[00220] As shown in FIGS. 8-9, and also in FIGS. 10-12, the sensor 10 comprises a platform having at least one nanostructure, such as nanotube 12 disposed adjacent substrate 14 and in electrical communication between at least a source electrode 16 and a drain electrode 18.

[00221] Optionally, the device may include at least one additional electrode, such as gate electrode 20 disposed adjacent nanotube 12. The gate electrode 20 is shown embedded in substrate 14, but alternative electrodes types and locations may be included (e.g., a bottom gate electrode , top gate and/or liquid gate electrode), as described above with respect to other NTFET sensor embodiments.

[00222] Although a single nanotube 12 is shown schematically in FIGS. 6-9 in simple end-contact with electrodes 16 and 18, alternative nanostructures arrangements may be employed as described above without departing from the spirit of the invention. [00223] FIGS. 10A and B show schematically two alternative configurations. FIG. 10A shows a plurality of conductor "islands" interconnected by nanotubes, and FIG. 10 B shows a nanotube network embodiment, in which plurality of nanotubes form an interconnecting network or film of nanotubes providing a conducting channel between source and drain electrodes. In such a nanotube film, individual nanotubes need not span between source and drain electrodes, and the conducting channel may comprise one or more channels or paths via a plurality of nanotubes connected to one another in series. Preferably, the density and/or composition of such a network of nanotubes is selected (by controlled formation and/or by post-formation modification) to provide a desired degree of conductivity and sensor sensitivity. Optionally, a plurality of source and/or a plurality of drain electrodes may be included, for example an interdigitating series of such electrodes. The nanotube 12 may be disposed to lie under, beside or above the electodes, or combinations thereof. [00224] Nanotube films may be made directly on the substrate, e.g. by nanodispersed-catalyst-mediated CVD, solution deposition and the like. Alternatively, a nanotube film may be made separately and deposited upon the substrate 14 as a separate step, either directly or including a film carrier layer. See Patent Application Nos. 10/177,929 and 10/846,072 incorporated above. Note that substrate 14 may be a rigid structure, e.g. a semiconductor wafer, monocrystalline silicon, polycrystalline silicon, or the like, or alternatively may be flexible, e.g. a polymer sheet, web, or the like. Portions of the nanotube film may be selectively removed from portions of the substrate so as to tailor the nanotube film in relation to the electrodes 16 and 18. Likewise, the contacts or electrodes 16 and 18 (and/or gate or additional electrodes) may be deposited or formed prior to the nanotubes 12 or afterwards. Optionally, additional electronic circuitry may be formed integrally with sensor 10 on substrate 14, e.g. for signal processing and the like. Known methods for constructing elements and layers of integrated electronic circuitry may be employed in the making of sensor 10 and optional elements, such as CVD, vacuum deposition, photolithography and masking, chemical etching, spin coating, substrate doping, substrate oxide formation, substrate nitride formation and the like. [00225] Likewise, the sensor shown may be included in an integrated array of sensors, as described above. Note that the enhancements and alternative elements describe above with respect to other sensor and NTFET embodiments, such as passivation of contacts, dielectric and/or catalyst containment layers covering the substrate, hydrophobic coatings on the nanostructures, and the like, may optionally be included in the embodiments described below. [00226] C-2 Detecting probes:

[00227] As shown in FIGS. 8-9 and FIG. 10, the sensor includes a detecting probe, such as probe 22, the probe includes a linker group, such as linker 26 which is associated (preferably non-covalently) with the nanotube 12, so as to bind the probe to the sensor 10. A cDNA 24 is bound to linker 26 (preferably covalently) at one portion of the cDNA, the cDNA also having an exposed complement base sequence extending outward from linker 26. The linker may be a molecule or group configured to non-covalently bind to nanotube 12 and to covalently bond to cDNA 24, e.g., an aromatic molecule such as pyrene and/or a polymer.

[00228] Note that a linker group may connect to more than one cDNA, and conversely a cDNA may connect to more than one linker group, depending on the nature and conformation of the linker. For example a liner group comprising a distributed polymer layer may have a plurality of cDNA molecules bonded at different points on the polymer layer. Note in this regard the definitions of dsDNA, ssDNA, cDNA and other nucleotide species set forth in the Summary of the Invention herein. [00229] Thus, in certain alternative embodiments, the "cDNA" is not necessarily a deoxyribose polynucleotide, but may include other target-specific polynucleotide species, such as RNA, a modified or substituted DNA, and the like, having a detector nucleotide sequence which provides for at least partial hybridization with a selected target sequence. [00230] Similarly, the target "ssDNA" molecule is not necessarily a discrete fully-denatured deoxyribose polynucliotide strand, but may include RNA, dsDNA, partially-denatured dsDNA, species with "sticky ends", and the like, wherein the target molecule includes a target nucleotide sequence which provides for at least a partial hybridization with the "cDNA" of the probe. [00231] In the embodiment shown in FIG. 8A, the probe 22 is shown detecting a single-stranded fragment of DNA 30 by hybridizing with target base sequence 32. Suitable sensor circuitry (not shown in FIGS. 8-12) is connected to sensor 10 so as to detect and/or quantify an electrical response of sensor 10 to the hybridization of DNA 30, in a manner described above with respect to other sensor embodiments. For example, the conductance between source 16 and drain 18 may change upon hybridization, the change being measured. Alternatively, in an NTFET DNA sensor embodiment, the hybridization of DNA 30 may cause a phase shift in the device characteristics of sensor 10 produced as the voltage of gate electrode 20 is varied through a selected voltage range. Additional or alternative properties of sensor 10 may be measured to detect hybridization. [00232] In certain applications according to aspects of the invention, the sensor 10 may be used to discriminate between a relatively complete hybrid match between cDNA 24 and selected target sequence 32 on the one hand, and a contrasting partial, discontinuous, and/or or looped hybridization of the target sequence on the other hand. The sensor 10 produces an electrical response to the hybridization event with signal characteristics reflecting the degree and/or character of hybridization of probe cDNA 24 to a target sequence 32. For example, the signal produced upon partial hybridization of a sequence which has a single base mismatch (sbmDNA) relative to the corresponding probe sequence can be distinguished from the hybridization of a completely matched sequence. This capability of sensor 10 provides for the characterization of single nucleotide polymorphisms (SNPs), among other things.

[00233] Note in this regard the ssDNA 30 may alternatively be a RNA polynucleotide, a hetero or modified polynucleotide, a plasmid, a viral fragment, a double stranded DNA fragment (e.g. having a "sticky end" or other exposed strand target portion available for hybridization with probe 22), a partially-annealed dsDNA fragment, an oligonucleotide, or the like. [00234] In a exemplary method of use according to aspects of the invention, the probe 24 may be prepared to suit a selected target sequence 32, the cDNA being obtained by known methods. Commercial sources exist for custom synthesis of oligonucleotides having a specified sequence, and sequences of interest may also be obtained, modified and/or amplified by a number of known methods, such as PCR, reverse transcription, plasmid amplification, and the like. Note in this regard that cDNA may contain nucleotides and/or hetero-groups in addition to a nucleotide sequence complementary to target sequence 32, for example, tail or head portions selected for binding to linker 26, selected for purification, amplification and/or other processing steps, optional labeling groups, and the like. The cDNA 24 may then be bonded to linker group 26 (e.g. pyrene) by known reactions and methods (e.g., formation of a DNA-5'-amine of pyrene) to create probe 22. [00235] Prefabricated sensor platforms 10 may then be functionalized, for example by treatment with a solution or suspension of probe 22 so as to bind linker 26 to nanotube 12 (e.g., by pi-pi stacking of pyrene molecules associated with the graphitic lattice of nanotube 12), followed by washing and drying. The functionalized sensor 10 may then be used for detection of an analyte ssDNA having target sequence 32, suspended in a sample medium. Suitable calibration procedures may be carried out, e.g. by exposing sensor 10 to an equivalent sample medium having ssDNA known to lack target sequence 32. [00236] In an alternative exemplary method of use, the prefabricated sensor platforms 10 may be pre-treated with a linker group material 26 (e.g., a polymer selected to react with or bind to a portion of cDNA 24). A target-specific cDNA 24 may be prepared, and the sensor 10 functionalized by binding with the cDNA 24 to create probe 22 in situ.

[00237] In an alternative sensor embodiment (not shown) according to aspects of the invention, an array sensor system comprises a space-apart plurality of individual sensors 10. The array may be prefabricated as described above, and the sensors 10 may be individually functionalized with one of a plurality of different probes, each having cDNA specific to a particular selected target sequence. For example, ink-jet type application methods may be used to treat the array in a predetermined pattern of functionality. Such a multi-functionality array may be employed so that a single analyte sample medium may be tested for a plurality of different target DNA sequences substantially simultaneously. Signal processing circuitry of known design may be used to process signals from the plurality of sensors 10 of the array serially, in parallel, or according to any selected pattern. Accessory elements, such as microfluidic reservoirs, channels, needle, valves, pumps, and/or injectors, and the like, may be included in the array embodiment, configured to provide controlled functionalization of the sensors, controlled sample delivery to the sensors, sample purging from the sensors, washing/reconditioning of the sensors, and/or controlled calibration of the sensors, and the like. [00238] C-3 Alternative Assay embodiments: [00239] FIGS. 8B and 9 illustrate a number of alternative assay embodiments according to the invention, one or more of which may be employed instead of or in combination with the embodiments described above. [00240] FIG. 8B shows schematically an alternative exemplary embodiment according to aspects of the invention, employing an electroactive intercalator 34, either in the sample medium and/separately introduced following hybridization. The intercalator 34 associates with the hybridized portion (double stranded region) of the probe 22-target sequence 32 complex, so as to amplify and/or modify the measured response of sensor 10, so as to facilitate measurement and/or detection of hybridization.

[00241] As shown in the structures of FIGS. 8C-F, examples include the use of electroactive intercalators such as daunomycin, methylene blue, lr(bpy)(phen)(phi)3+, and the like; groove binders, such as Ru(NH3)5CI2+, and the like; or combinations thereof.

[00242] FIG. 9A shows schematically an alternative exemplary assay embodiment according to aspects of the invention, employing an secondary or sandwich probe 40, configured to hybridize with a second portion of ssDNA 30, referred to as "sandwich sequence" 44. The sandwich probe 40 includes a second cDNA 42 having a portion including a sequence of bases complementary to sandwich sequence 44. The cDNA 42 includes a portion which is in turn bound to an amplifier group 46, preferably covalently. The amplifier group 46 serves to increase or modify the signal response of sensor 10 upon hybridization of target sequence 32 to detector probe 22. The amplifier group 46 may be a group or label which causes a detectable and/or a quantifiable signal of sensor 10 without further reactivity. Alternatively, amplifier group 46 may be a group which causes a detectable and/or a quantifiable signal of sensor 10 upon further reaction with another promoter material, such as a chemical or biochemical substrate. Examples of amplifier groups are shown in FIGS. 9A-C. [00243] There are a number of alternative methods of use embodiments according to aspects of the invention for the assay shown in FIG. 9A. For example, comprising:

[00244] a) bonding probe 22 to nanotube 12 of sensor 10; [00245] b) treating sensor 10 with a sample putatively containing analyte ssDNA 30 having target sequence 32, so as to bind ssDNA 30, if present, to probe 22, followed by washing; [00246] c) treating sensor 10 with a solution containing sandwich probe 40 having selected amplifier group 46, so as to bind probe 40 to ssDNA 30, if present, followed by washing;

[00247] d) if needed for-the selected amplifier 46, treating sensor with a solution including the further promoter material; [00248] e) acquiring a signal from sensor 10; and

[00249] f) analyzing the signal to determine the presence and/or concentration of analyte ssDNA 30.

[00250] The steps a-f above may be carried out in alternative order. For example, step (c) may be a pretreatment of treatment of the analyte sample, carried out prior to step (b). Likewise, additional calibration steps may be optionally included at various times. The washing steps are exemplary, as one of ordinary skill in the art will readily be able to tune or optimize the methods embodiments for particular applications to avoid cross contamination and other sources of error, without undue experimentation and without departing from the spirit of the invention.

[00251] Note that in certain embodiments, the sandwich sequence 44 may be a common sequence expected to be present in the sample DNA fragments, and target sequence 32 may be an analyte-specific sequence of unknown presence in the sample. Alternatively, probe 46 may be configured to undergo relatively non-specific binding to sample DNA in comparison to more highly target- specific binding of probe 22. In this regard, probe 46 optionally may include additional groups to promote binding to sample DNA and/or to prevent undesired blocking of probe 22.

[00252] In certain embodiments, amplifier group 46 may be comprise a promoter or catalyst, such as an enzyme, causing an oxidation/reduction or other reaction with a chemical or biochemical substrate thereby influencing sensor 10 to provide a detectable response. For example, amplifier group 46 may comprise urease. Step (d) above may comprise treating with a urea solution, to produce ammonia and carbon dioxide if bound probe 46 is present, so as to modify the pH of the solution and thereby detectably change the signal of sensor 10. Other examples of enzyme systems which may be employed are cholinesterase; peroxidase (e.g. HRP); glucose oxidase, and the like. Other examples of amplifier group 46 are ferrocene, metal nanoparticles, labels (nanoparticles, proteins, etc.), and the like.

[00253] FIG. 9E shows schematically an alternative exemplary assay embodiment according to aspects of the invention. In this alternative assay embodiment, the sandwich probe 40 and ssDNA 30 are generally similar to that shown and described with respect to FIG. 9A.

[00254] However, in the embodiments illustrated in FIG. 9E the detector probe 50 comprises a tether group 57 and a corresponding detector group 53, joined or mated to one another. Tether group 57 includes linker 58 connected to a tether species (in this case antibody 56). Detector group 53 includes cDNA 52 connected to a tether-mating species (in this case antigen 54 where the antigen is selected to have epitopes configured to bind to the receptors or binding sites of antibody 56). [00255] For example, as shown in FIG. 9E, the antigen 54 may comprise biotin and the antibody 56 may comprise streptavidin. In other alternative embodiments (not shown) the arrangement may be the reverse of that shown in FIG. 9E, e.g., an antigen may be connected to the linker, and the antibody connected to the cDNA. Other alternative combinations of tether and tether- mating species may be employed, where the tether and tether-mating species are selected to be readily joinable or mate-able to one another to form the self- assembled detector probe 50. [00256] Note that the tether group 57 and the detector group 53 may generally be prepared separately. For example, a partially-functionalized sensor platform including the tether group 57 may be prepared, and provided and stored without the detector group 53. Such a sub-assembly does not have vulnerability to substances or conditions that may specifically degrade polynucleotides (for example endonucleases, exonucleases and the like). Thus, this embodiment is especially suitable for applications in which it is desired to prefabricate a sensor assembly without a target-specific cDNA (i.e., a relatively generic sensor), and then introduce any one of a number of different target-specific detector groups which are conveniently joinable or mate-able to the tether group (self-assembly or simple reaction) at the time of, or shortly before, sample measurement. The rapid, robust, and selective antigen-antibody binding reaction is a preferred embodiment of the tether/tether-matching system.

[00257] The tether/tether-matching system illustrated in FIG. 9E may also be employed in conjunction with other embodiments having aspects of the invention, such as the exemplary embodiments shown in FIGS. 8-9D, providing similar advantages. Likewise, a particular sensor 10 may be functionalized employing more than one combination of tether/tether-matching system, wherein the detector groups each have the same selected cDNA type (in certain applications where a particular cross-reactivity is desired, more than one kind of cDNA may be employed in a particular sensor. However, more typically, the sensor will be configured to maximize target selectivity)

[00258] Additionally, the self assembling tether/tether-mating system is particularly useful in sensor array embodiments having aspects of the invention, in that a relatively generic sensor array can be pre-fabricated with tether groups bonded to the plurality of sensors. A selected pattern of different target-specific detector groups may then be applied by known methods to complete the patterned target-specific functionalization of the sensor array, e.g. by multiple or automated pipette systems or by "ink jet" methodology. [00259] C-4 Alternative sensor arrangements:

[00260] FIGS. 10A and 10B shows an "island" sensor embodiment 70 and a "nanotube network" sensor embodiment 72, respectively, each according to certain aspects of the invention. These embodiments are generally applicable to the assay embodiments shown in FIGS. 8-9. Nanostructures 12 (in this case, single wall carbon nanotubes, "SWCNT" or abbreviated "NT") communicate electrically with source electrode 16 and drain electrode 18. In the embodiment 72, a plurality of nanotubes 12 form an interconnecting network between source 16 and drain 18. Linker groups 76 may be seen to connect the cDNA strands 74 to nanotubes 12. The ssDNA strands 78 may be seen to be diffusing in the vicinity of cDNA strands 74. [00261] FIGS. 10C and 10D schematically show the connection of cDNA strands 74 with nanotubes 12 be action of linker groups 76, as exemplified by (FIG. 10C) an organic group 76 (e.g., pyrene) covalently bonded to the cDNA 74 or (FIG. 10D) a reactive polymer group 76' covalently bonded to cDNA 74, or the like or combinations thereof. [00262] FIGS. 11 A and 11 B show alternative sensor architectures 80 and 82 in which the cDNA 84 is attached to the surface of substrate 14' (e.g., a silicon dioxide layer covering a silicon wafer) by means of a chemical connection, such as a covalent bond. FIG. 11C shows the sequence of steps of an alternative linker method to bind the cDNA 84 to the substrate 14, employing known reactants and methods, as shown in the sequence of steps (steps 1-3). Hybridization of ssDNA 88 to cDMA 84 influences the electrical properties of sensor 80 or 82 respectively so as to produce a detection signal generally similar to that described above for the various assay embodiments. [00263] FIGS. 12A and 12B show alternative sensor architectures 90 and 92 in which the cDNA 94 is attached to the surface of electrodes or contacts 16' and 18' (electrodes may be bare, oxidized surface, or a coated surface) by means of a chemical connection, such as a covalent bond, e.g., by formation of DNA-5'-thiol at the cDNA 5' end, employing known reactants and methods. Alternatively, the cDNA 94 may be attached to contacts 16' or 18' by polymer linker groups, and the like. Hybridization of ssDNA 98 to cDMA 94 influences the electrical properties of sensor 90 or 92 respectively so as to produce a detection signal generally similar to that described above for the various assay embodiments. [00264] C-5 Alternative separation and purification:

[00265] Various label groups known in the art may be employed for separation of the target DNA from genomic DNA in the vicinity of the nanotube device 10. For example, labels (nanoparticles, proteins, etc.) may be used for separation of the target DNA from genomic DNA as an additional step. Alternatively magnetic beads and antibodies may be employed for such separation. In certain exemplary embodiments according to aspects of the invention, pre-measurement sample DNA purification and/or segregation, and the like are carried out adjacent the sensor (or adjacent the sensor array in array embodiments) as part of an integrated sample processing and measurement system, and may include magnetic controls, electrostatic controls, combinations of these, and the like. Optionally, a microprocessor or computer element is included to control and coordinate both sample DNA purification and/or segregation and sample detection and measurement. [00266] EXAMPLE D: [00267] DNA Analytical Nanostructured Devices For Human Identification. [00268] Several publications have described the use of silicon nanowire FETs for detecting DNA hybrids (see, for example, Nano Lett. 2004, 4, 245 and Nano Lett. 2004, 4, 51 , both of which references are incorporated by reference). Nanowires, however have diameters greater than nanotubes by orders of magnitude and are therefore much less sensitive to surface-generated perturbations in conductivity.

[00269] Advantages of carbon nanotube devices. As described herein, carbon nanotubes display unique structural and electrical properties. In an exemplary embodiment of a polynucliotide analysis system having aspects of the invention, the electrical features of nanotubes as they relate to the interaction with DNA may be employed for human identification. [00270] The inventors herein have published results detecting streptavidin attachment to biotinylated nanotubes, demonstrating a ten-fold increase in sensitivity over similar studies with nanowire FETs. See, for example Nano Lett. 2003, 3, 459, and also US Patent Application No. 10/704,066 filed November 7, 2003 entitled "Nanotube-Based Electronic Detection Of Biomolecules" (published as US 2004-0132070 on July 8, 2004), both of which publications are incorporated by reference. In addition to greater sensitivity, the use of nanotubes facilitates the biological functionalization through covalent and non-covalent attachment chemistry. [00271] Exemplary embodiments of electroanalytical nanotube devices having aspects of the invention are modified by biological molecules which enhance biosensing performance. These devices, which may be configured as ordered arrays of single-nanotube transistors, show efficient electrical communications and sensitivities required for applications in DNA hybridizations. Compared to conventional bio-detection methods such as fluorescence and luminescence, these devices offer homogeneous detection, no requirement for an assay 'label', small size, multiplexed detection, high sensitivity, low manufacturing cost and minimal drive electronics. Such NTFET devices also have characteristics that may be optimized for high-throughput applications. [00272] Embodiments of NTFET electroanalytical devices having aspects of the invention accomplish these advantages because DNA attaches to the single- atom thick wall of the nanotube to affect the flow of the electrons through the nanotube. By including the nanotube as the conductor of the transistor, very slight changes in the nature of the molecules attached to the nanotube may be distinguished. In certain device embodiments, current flows along several conducting channels within the nanotube, and the presence of a biomolecule binding event blocks the nearby channels, thus increasing the overall resistance. A change of the conducting path leads to the change of the resistance that is detected. [00273] Embodiments of NTFET electroanalytical devices having aspects of the invention can detect the difference between ssDNA capture molecules and the duplex molecule formed by hybridization of the complement without the need for a non-DNA label. All of these features enhance the performance of instruments for point-of-use applications for DNA identification.

[00274] Current DNA human identification practice and requirements. In the US₁ the use of DNA identification at the point of booking has long been debated. Strong arguments have been made for the effectiveness of such a tool in apprehending criminals, and serious crimes have already been solved through the use of incidental DNA samples (eg. from discarded coffee cups). Despite opposition, many states have passed legislation, contingent on federal funding, requiring DNA testing as part of the arrest recording procedure. An additional barrier to implementation of record of arrest DNA testing (RADT) is the lack of a testing system capable of handling the DNA tests from an anticipated 12-16 million annual arrests. In addition to accommodating this huge increase in DNA identity testing, an ideal system would need to provide results in less than 48 hours and maintain a simple chain of custody. [00275] Two options for meeting these needs would be: centralized test centers to which samples would be shipped, or distributed testing done at the site of arrest recording. Centralized testing labs with conventional sieving technology could meet the throughput requirements simply increasing the number and or size of existing facilities. But this approach would depend on highly skilled personal and well-managed sample handling. There would be challenges in reaching a 48- hour turn-around time, and the chain of custody would be complex. On the other hand, on-site RADT would be: 1) easy to use, 2) adaptable to a wide variation (~100 fold) of throughput requirements, and 3) small enough to be placed in police station crime labs. There are no products currently on the market that meet the needs of on-site RADT.

[00276] There is also a need for portable, easy to use instruments that provide very fast turn-around times and that can taken to crime scenes, disasters sites and meet security application requirements. Sieving technology, even with micro-channels in DNA chips is unlikely to meet those performance needs. [00277] DNA analytical targets. The current standard genetic variant used for human DNA identification is the short tandem repeat (STR) DNA sequence. The likelihood of supplanting STRs with the standard variant for medical applications - the single nucleotide polymorphism (SNP) - diminishes as the size of the STR databases grows. Due to predictions of large markets for SNPs in medical applications, nearly all new technology is being developed for SNPs while STRs are virtually ignored by technology development companies. [00278] Autosomal SNPs may be used to attain the same probabilistic resolution for human identification as STRs. In one example, this includes a panel of from about 35 to about 150 loci, depending on the particular human identification application. A SNP panel product is currently being offered by Orchid for paternity testing, an application which does not require access to databases. SNPs are also being investigated for their ability to predict physical characteristics of the individual presenting the sample. The physical traits being examined include: skin, hair and eye color.

[00279] NTFET devices having aspects of the invention provide a tremendous opportunity in the forensics arena. The ability to detect DNA without labels, the small size, low energy requirements and the integration of work flow steps permit NTFETs to be the enabling technology of portable and easy to use DNA identification instruments.

[00280] Certain embodiments having aspects of the inventions differentiate DNA fragments by size, and include a nanotube-based device having the capability of providing signals descriptive of the DNA attached to them.

Alternative embodiments are configured and optimized for determination of STR loci and the identification of SNPs.

[00281] To accommodate STR loci used in human identification, the DNA length may be determined at a resolution of four bases, and/or to differentiate among possible homozygote and heterozygote alleles of an STR locus. In certain embodiments, a single-nanotube field effect transistor having aspects of the invention is employed to make this determination. [00282] DNA fragment indentification. Nucleic acids, such as single- stranded DNAs, short double-stranded DNAs, and some total RNAs can attach directly to single-wall nanotubes (SWNT) in water-based media. As shown in FIG. 13, the DNA typically binds to the SWNT so that the hydrophilic sugar-phosphate backbone of the DNA is pointing to the exterior to achieve solubility in water. Molecular modeling has shown that the non-specific DNA-SWNT interactions in water are from the nucleic acid-base stacking on the nanotube surface. In addition to natural source DNA fragments, this allows attachment of any synthetic oligonucleotide as well. [00283] As described herein (see EXAMPLE B₁ above), it has been demonstrated that a 12-mer oligonucleotide capture probe may be attached to an NTFET and DNA hybridization in the proximity of nanotubes may be measured. The oligonucleotide probe in that example was attached to carbon nanotubes using pyrene derivative. Thus, the device can detect the hybridization of the immobilized capture DNA with its single strand complementary sequence. [00284] 1) "Counting" of nucleotide bases. Exemplary embodiments of electroanalytical devices having aspects of the invention include a platform comprising a photo-lithographically fabricated silicon chip, with locations for the addition of nanotubes to form FETs. [00285] As shown in FIGS. 13 and 14, capture oligonucleotides are designed so that overhanging ssDNA from the target is juxtaposed with the nanotube. In this example, the capture oligonucleotide is disposed on or adjacent a nanotube by suitable attachment chemistry, and contains a sequence complementary to a portion of the target oligonucleotide (e.g., a STR). Following capture of a target STR fragment, the fragment may interact and becomes associated with the nanotube. Targets of differing lengths have measurably distinct effects on the nanotube electrical properties.

[00286] One mechanism proposed for to explain the effect of DNA attachment on NTFET conductance is sharing of electron π orbitals (shown as "e- " on the figure) with carbon atoms in the nanotube. Sequences of different lengths have different numbers of electrons available for sharing with the nanotube. FIGS. 13 and 14 illustrate a relatively long STR and a relatively short STR respectively. The characteristics of the detected FET signal are correlated with DNA targets of different lengths. Differences in measured signals may subsequently be used to discriminate DNA samples of different lengths.

[00287] 2) Bind DNA, add strong transistor effectors. As an anternative to "counting nucleotides", a similar molecular arrangement may be used to measure the portion of the nanotube not bound to the DNA. Since the nanotube can be as narrow as 2 nm in diameter, a single-strand portion of DNA interacting with it can protect the CNT from further reactions.

[00288] As shown in FIGS. 15A and 15B, after DNA target binding, molecules that have a strong effect on NTFET conductance (strong effector moleculses or s.e.m.) can be reacted with the nanotube. The arrangement of this example is generally similar to that of FIGS. 14 A, B. Since NTFETs can be made with CNTs as short as 100nm, it is possible to measure the unprotected portion of the NT by counting the strong effector molecules that bind outside the DNA protected region. The length of the target DNA might then be calculated by the relative level of electrical deflection caused by the strong effector molecules. [00289] 3) "Denaturing" the single strand portion of target DNA from the nanotube. As shown in FIGS. 16A and 16B, real-time electrical readout of bound versus unbound states permit precise measurement of ssDNA dissociation. The arrangement of this example is generally similar to that of FIGS. 14 A, B. As an alternative to directly measuring the length of the DNA, length differences may be determined by measuring binding kinetics. The interaction of the DNA with the nanotube is dissociable. Stringency conditions may be selectively and/or controllably applied to wholly or partially "melt" the DNA away from the nanotube at a characteristic temperature or other stringency parameter (the event of "melting" or dissociating from the nanotube being electrically detectable), thus permitting increased selectivity and discrimination capability between different DNA samples. [00290] EXAMPLE E [00291] Discrimination of Human Hereditary Polymorphisms.

[00292] Certain embodiments having aspects of the invention employ interactions between ssDNA oligonucleotides probes and SWNTs, and the subsequent DNA hybridization processes that take place adjacent the device surface. In particular examples, NTNFETs are selectively functionalized with DNA oligonucleotides probes, which retain hybridization specificity. Thus, for example, NTNFETs with immobilized synthetic oligonucleotides selectively recognize target DNA sequences with single-nucleotide polymorphism (SNP). The exemplary embodiments can discriminate between oligonucleotides with single base mismatch, for example, a wild type versus H63D mutation in the HFE gene, responsible for hereditary hemochromatosis.

[00293] Exemplary Nanosensor Devices. The particular examples of nanoelectronic devices having aspects of the invention employed in the following examples comprise NTNFETs including a nanostructure element comprising a random interconnected network of carbon nanotubes, the network electrically communicating between interdigitated patterns of source and drain electrodes, and may be influenced by the bias field of a gate electrode comprising a dielectric- covered, doped silicon substrate.

[00294] FIG. 17A shows an exemplary electronic sensing device 100 having aspects of the invention, for detecting an analyte 101 , comprising a nanostructure sensor 102. Sensor 102 comprises a substrate 104, and a conducting channel or layer 106 comprising a nanostructure material, such as a nanotube or network of nanotubes, disposed on the substrate. The nanostructure material 106 may contact the substrate as shown, or in the alternative, may be spaced a distance away from the substrate, with or without a layer of intervening material. In an embodiment of the invention, conducting channel 106 may comprise one or more carbon nanotubes. For example, conducting channel 106 may comprise a plurality of nanotubes forming a mesh, film or network. Certain exemplary embodiments having aspects of the invention include nanostructure elements which may be made using chemical vapor deposition (CVD) and traditional lithography, or may be deposited by other methods, such as solvent suspension deposition, AFM manipulation, and the like. Certain embodiments include one or more discrete nanotubes in electrical contact with one or more metal electrodes. A number of different arrangements of active nanostructures may be included without departing from the spirit of the invention.

[00295] One or more conductive elements or contacts (two are shown, 1 10, 112) may be disposed over the substrate and electrically connected to conducting channel 106 comprising a nanostructure material. The conductive elements permit electrical charge and/or current to be applied to the nanostructured material of channel 106, and may be used in the measurement of an electrical property of the channel 106. For example, contacts 110, 112 may comprise source and drain electrodes, respectively, permitting application of a source-drain voltage Vsd, and inducing a current in channel 106. Elements 110, 112 may comprise metal electrodes in contact with conducting channel 106. In the alternative, a conductive or semi-conducting material (not shown) may be interposed between contacts 110, 112 and conducting channel 106.

[00296] In the example of FIG. 17A, the device 100 may be operated as a gate-controlled field effect transistor, with sensor 102 further comprising a gate electrode 114. Such a device is referred to herein as a nanotube field effect transistor or NTFET. Gate 114 may comprise a base portion of substrate 104, such as a doped-silicon wafer material isolated from contacts 110, 112 and channel 106 by a dielectric layer 116, so as to permit a capacitance to be created by an applied gate voltage Vg. For example, the substrate 104 may comprise a silicon back gate 114, isolated by a dielectric layer 116 comprising SiO2. Alternatively, the device 100 may be employed in other measurement modes. For example, device 100 may be employed as a capacitive or impedance sensor using known circuitry to create an electric field gradient between conducting channel 106 (e.g., via either of contacts 110, 112) and gate 114 and to measure the capacitance and/or impedance of this structure in relation to the influence of an analyte.

[00297] In the example of FIG. 17A, Sensor 102 may further comprise a layer of inhibiting or passivation material 118 covering regions adjacent to the connections between the conductive elements 110, 112 and conducting channel 106. The inhibiting material may be impermeable to at least one chemical species, such as to the analyte 101 or to environmental materials such as water or other solvents, oxygen, nitrogen, and the like. The inhibiting material 118 may comprise a passivation material as known in the art, such as silicon dioxide, aluminum oxide, silicon nitride, or other suitable material. Further details concerning the use of inhibiting materials in a NTFET are described in prior application Serial No. 10/280,265, filed October 26, 2002, entitled "Sensitivity Control For Nanotube Sensors" (now US Patent No. 6,894,359) which is incorporated by reference herein. [00298] The conducting channel 106 (e.g., a carbon nanotube layer) may be functionalized to produce a sensitivity to one or more target analytes 101. Although nanostructures such as carbon nanotubes may respond to a target analyte through charge transfer or other interaction between the device and the analyte, more generally a specific sensitivity can be achieved by employing a recognition material 120, also called a functionalization material, that induces a measurable change in the device characteristics upon interaction with a target analyte. The functionalization or a recognition material 120 shown in FIG. 17A is a generic representation. Further description in the Example F details the functionalization for DNA detection, and in particular, the discrimination of human hereditary polymorphisms.

[00299] FIG. 17B shows a scanning electron microscopy (SEM) image of the a single random network NTNFET device 10. View (a) of FIG. 17B shows a complete device with interdigitated source S and drain D electrodes with contact leads extending to pads at the substrate edge. Enlarged view (b) shows a portion of the device 10 including a portion of nanotube network N disposed adjacent the electrodes, the network margin being trimmed to limit it to the interdigitated electrode region. An additional enlarged view (c) shows the details of the lead/network pattern of a small portion of the device, showing a single digit of source S separated by spacing L from adjacent digit of drain D. In this example L is approximately 10μm.

[00300] The interconnected random carbon nanotube network N can be seen to collectively communicate between S and D, although it may be seen that, in general, the individual nanotubes do not lead across space L. In this example, the electrodes are deposited on top of the network N, although alternative configurations may be employed. Lying beneath the network N is the substrate, which in this example is a doped silicon wafer protected by a surface dielectric layer, and in which optionally the silicon base serves as a gate electrode. [00301] In certain sensor system embodiments, device 100 may be packaged in a conventional manner to conveniently permit connection to operating circuitry. FIG. 17C is a photograph of a sensor generally similar to that of FIGS. 17A and 17B, fabricated on a die of a wafer, and mounted as a chip in a conventional 40 pin CERDIP package using wire-bonding techniques. [00302] Device 100 may further comprise suitable circuitry in communication with sensor elements to perform electrical measurements. FIG. 17D is a photograph of a sensor generally similar to that of FIGS. 17A and 17B, packaged in the manner shown in FIG. 17C, and installed on an exemplary circuit board of an electronic sensor system. For example, a conventional power source may supply a source-drain voltage (Vsd) between contacts 110, 112. Measurements via the sensor device 100 may be carried out by circuitry represented schematically by meter 122 connected between contacts 110, 112. In embodiments including a gate electrode 114, a conventional power source 124 may be connected to provide a selected or controllable gate voltage (Vg). Device 100 may include one or more electrical supplies and/or a signal control and processing unit (not shown) as known in the art, in communication with the sensor 102.

[00303] Suitable elements may be included in the sensor system architecture to facilitate application of liquid phase biological analyte media, and the system may be readily adapted to make the NTNFET biochip detection platform suitable for whole blood samples. [00304] NTFET devices for DNA detection. A large plurality of such devices may be fabricated simultaneously at a wafer-level scale. In this example, each wafer consists of about one thousand dies with 2.54 x 2.54 mm2 dimensions, with several devices patterned onto each die, the devices having a size of about 210μm by 270μm. [00305] The devices were fabricated using SWNTs grown via chemical vapor deposition (CVD) at 900°C using dispersed iron nanoparticles as growth promoter and a methane/hydrogen gas mixture on doped Si 100 mm wafers with SiO2 at its surface. Nanotubes outside the intended network area N were removed using oxygen plasma to electrically isolate each device. [00306] Electrical leads or electrodes were patterned on top of the nanotubes from evaporated Ti-Au films (30 nm and 120 nm thick, respectively) using standard photolithography techniques. The electrode pattern comprises source S and drain D electrodes in an interdigitated with a separation L of about 10 μm. The electrode separation L may be selected to optimized device performance, and in this example devices with other dimensions (pitches ranging from 5 to 100 μm) were also present on the die. As seen in the enlarged image, the nanotubes are disposed in a random interconnecting arrangement, so as to provide an communication path between source S and drain D. [00307] Devices with other dimensions (e.g., pitches ranging from 5 to 100 μm) may be fabricated on the die, permitting adjacent devices to be optimized with differing functional geometry. Electronic characterization of NTNFET devices, such as current flow between source and drain electrodes as a function of applied gate voltage and bias voltage, were conducted using an autoprobe tester.

[00308] FIGS. 18A-D illustrate the process of testing of I-Vg data of NTFET wafers as measured by a conventional autoprobe instrument, for example the 100 mm wafer as described above with NTNFET devices having 10 μm pitch structure. FIGS. 18A and 18B are examples of wafer maps of device maximum conductance and of device modulation (modulation = (Gmax-Gmin)/Gmin), respectively for an exemplary NTFET wafer. FIGS. 18C and 18D are examples device population plots of the conductance and of the modulation data shown in FIGS. 18A and 18B respectively. Note that for device maximum channel conductance (18C), the population behavior is very narrowly defined, with on a small percentage of devices falling short of the plateau of maximum conductance. Note that for device modulation (18D), the behavior of the population is similarly narrowly defined, the bulk of the devices having modulation within a narrow range. [00309] Data Acquisition. For DNA detection, in this example chips with multiple NTNFET devices were wire bonded and packaged in a 40-pin CERDIP package and tested using a NTNFET custom electronic test fixture, which measures an array of up to 12 separate sensors from each Si chip. The housing of the test fixture comprises of a modified shielded I/O board (SCB-68, National Instruments) with a 40-pin ZIF socket. The I/O board was linked to a PC with a data acquisition card (PCI-6014, National Instruments). Programming to manage data acquisition was performed in LabVIEW (National Instruments). An analog output voltage was used to sweep the gate of the NTNFETs. Device characteristics such as source-drain voltage and current were calculated in LabVIEW from voltage measurements across sense resistors. Continuous I-VG measurements were taken with a gate voltage triangle wave sweep at frequency of 3 Hz from -10V to +10V.

[00310] DNA Immobilization. In this example, chemicals were purchased from Aldrich and used as received. Oligonucleotides unmodified and modified with Cy5 or FITC fluorescent labels at the 5'-end were synthesized by Alpha DNA. Allele-specific oligonucleotides for H63D polymorphism were synthesized by IDT. The oligonucleotide structures for both capture probes and targets are identified in TABLE !

[00311] All DNA solutions were prepared using 18MW water (NANOpure Infinity UV water system). In this example, packaged chips with NTNFET devices were cleaned in acid baths containing HNO3 (0.1M), HCI (0.1M), and 18MW water on the orbital shaker for 15 minutes in each bath. As a washing procedure, the packages were rinsed by hand with 40OmM phosphate (PB) buffer (pH 7.2) and then washed two times in 40OmM PB on the orbital shaker for 5 minutes. The packages are then rinsed with 5OmM PB and blown dry with nitrogen prior to electronic testing. [00312] For capture probe attachment to a NTNFET device, the chips were incubated in 5mM solutions of oligonucleotides in 20OmM PB buffer for one hour in a humid chamber. The standard washing procedure was then applied to remove excess and weakly bound DNA molecules prior to hybridization experiments. The hybridization experiments were performed by incubating the chips in 20OmM PB buffer solutions with complementary DNA (10μl_ at 5OnM, unless otherwise noted) for one hour in a humid chamber, followed by a standard washing procedure. All incubations were performed at room temperature (-22⁰C). [00313] Optical Imaging. Optical data were acquired by a Zeiss Axioskop 40 microscope, equipped with a TE cooled monochromatic CCD camera (DVC). Cy5 and FITC specific filter sets were obtained from Chroma. Images were captured using a Meteor ll/digital frame grabber board and lntellicam software (Matrox). ImageJ was used for image processing and quantitation. The chips were imaged in 0.1 M sodium bicarbonate buffer (pH 8.3) to maximize FITC fluorescence emission.

[00314] DNA immobilization and hybridization on NTNFETs. First we consider deposition of ssDNA on NTNFET devices. As a capture probe ssDNA: 5 -CCT AAT AAC AAT-3' (Alpha DNA) was selected. For fluorescent demonstration example embodiments, 50 nM of target DNA was used to allow integration of the fluorescence signal in 10-20 seconds to minimize photobleaching. For this purposed, A plurality of substantially identical interdigitated NTNFET devices were prepared with a distance between electrodes of 10 μm.

[00315] Probe Attachment. First, attachment of ssDNA capture probes to the devices were confirmed using Cy5-labeled ssDNA oligonucleotides, to demonstrate the functionalization of the device with a ssDNA capture probe. A bare NTNFET device without capture probe way photomicrographed, and should no measurable fluorescence signal. Next, an NTNFET device was treated with a target-matched probe comprising Cy5-labeled ssDNA: 5'-CCT AAT AAC AAT-3' (Alpha DNA), followed by thorough washings to remove excess and weakly bound DNA molecules. The device after incubation shows clear fluorescence, confirming attachment of the capture probe. [00316] The confirmation procedure was repeated with another NTNFET device, which was treated with a non-target-matched control capture probe, in this example a Cy5-labeled dA12 oligonucleotide. After thorough washings to remove excess and weakly bound DNA molecules, the device also showed clear fluorescence, confirming attachment of the capture probe. [00317] Interestingly, in both probe attachment confirmation demonstrations (matched and un-matched), fluorescence clearly comes from device areas covered with carbon nanotubes; there is no fluorescence from the bare silicon surface. This observation supports selective adsorption of ssDNA molecules on the sidewalls of carbon nanotubes. [00318] Hybridization Comparison. Additional substantially identical interdigitated NTNFET devices were treated as described below to demonstrate the hybridization of the capture probe with a target ssDNA. In each case the respective probe comprised an unlabeled ssDNA oligonucleotide of the same kind as used in the attachment examples, i.e., the target-matched probe comprising unlabeled 5'-CCT AAT AAC AAT-3' (Alpha DNA), and the non-target-matched probe comprising un-labeled dA12 oligonucleotide. In each example, the device were treated with the respective probe and washed thoroughly to remove excess and weakly bound probe molecules. Fluorescence microscopy images of the NTNFET devices were made after DNA incubations for 1 hour followed by removing unbound DNA oligomers. The fluorescent signals were measured as a difference between carbon nanotube device area and bare silicon wafer after 20 s integration.

[00319] FIGS. 19A, B and C show a series of fluorescent images of exemplary demonstration embodiments, the image covering a portion of the interdigitated electrode and network region of device 10 corresponding to approximately the left-hand portion of FIG. 17B, View (b). Note that although the original images were produced and analyzed as true color photomicrograph images, in this patent application the images are presented as processed negative images which are consistent with the originals for demonstration purposes, while allowing clear monochrome printing for patent publication purposes. [00320] FIG. 19A shows the device after incubation with the unlabeled ssDNA capture probe, but before treatment with target DNA, the device showing no significant fluorescence (the matched and un-matched probes showed essentially the same signal).

[00321] FIG. 19B shows the fluorescent image of a NTNFET device (pre- treated with target-matched probe 5'-CCT AAT AAC AAT-3' DNA as described above) following incubation with Cy5-labeled target DNA, and subsequent washing. Note that the changes in fluorescence between FIGS. 2A and 2B confirm that DNA hybridization takes place under these conditions. [00322] FIG. 19C shows a comparable fluorescent image of a NTNFET device (pre-treated with non-target-matched probe dA12 oligonucleotide as described above, see TABLE 1) following incubation with Cy5-labeled target DNA, and subsequent washing. The dA12 oligonucleotide control probe lacks a complementary sequence to the target probe, as there is only six base homology between dA12 and the target DNA sequence.

[00323] FIG. 19D is a bar graph showing a quantitative comparison of target DNA hybridization, calculated as a difference between fluorescence signal from carbon nanotube area versus bare Si surface after 20 sec. integration, have shown two orders of magnitude stronger signal for DNA hybridization system containing fully matched DNA (FIG. 2B) as opposed to system containing mismatched DNA oligonucleotides (FIG. 2C). [00324] Electronic Detection. FIGS. 20A-20B illustrate NTNFET device electronic responses for DNA immobilization and hybridization. Transistor characteristics such as G-Vg transfer curves (i.e., source-drain conductance (G) as a function of applied gate voltage (Vg)) are sensitive to changes in environment around carbon nanotubes, including molecular presence permitting sensor detection from signals of G-Vg characteristics such as modulation and threshold voltages, and the like.

[00325] As shown in both FIGS. 2OA and 2OB, the transfer characteristics of the bare NT network devices are consistent with p-type with positive threshold voltages (continuous line in each figure). Note that there is significant hysteresis with respect to increasing versus decreasing gate voltage Vg. [00326] Further, FIGS. 2OA and 2OB illustrates the effect of incubation of the device with ssDNA capture probe, which is immobilized on the device and results in a shift of G-Vg curve towards more negative gate voltage values (dashed line in each figure), showing a similar effect of shift of threshold voltage for the 5'-CCT AAT AAC AAT-3' DNA probe (FIG. 3A) and the dA12 oligonucleotide probe (FIG. 3B).

[00327] In each example, the ssDNA probe adsorption and continued attachment after washing was verified by fluorescent imaging. Similar shifts were observed for both unlabeled and fluorescence labeled (not shown) ssDNA sequences. Notably, the probe ssDNA is selectively absorbed on the nanotube network as compared to the peripheral substrate/dielectric surface. This behavior implies that the ssDNA is adsorbed on side-walls of carbon nanotubes and result in electron doping to the carbon nanotube semiconductor channels of NTNFET.. [00328] Finally, FIGS. 2OA and 2OB illustrates the effect of hybridization with target DNA on NTNFET electronic properties in each example (alternate dash-dot line in each figure), were investigated by measurements. FIG. 2OA illustrates the effect of the complementary probe hybridization with target DNA; and FIG. 2OA illustrates the effect of the non-complementary probe hybridization with the same target DNA. There is significantly smaller change in device conductance in system containing mismatched DNA oligonucleotides compared to fully matched DNA. [00329] Hemochromatosis SNP discrimination. The device embodiments having aspects of the invention demonstrate the practical utility of nanoelectronic detection methods. In another example, FIGS. 21 A though 21 C illustrate an allele-specific assay to detect the presence of single-nucleotide polymorphisms (SNP) using nanoelectronic detectors, such as NTNFETs. SNPs are the most abundant and highly conserved variations in the human genome and have been associated with a wide variety of diseases. The screening of large populations necessitates cost- effective and efficient high-throughput scanning, which will be facilitated by electronic and label-free techniques.

[00330] This example addresses the H63D polymorphism in the human HFE gene which is associated with hereditary hemochromatosis, a common, easily treated disease of iron metabolism. Hereditary hemochromatosis (HHC) is an autosomal recessive disorder of iron metabolism characterized by increased iron absorption and deposition in the liver, pancreas, heart, joints, and pituitary gland. [00331] The hereditary hemochromatosis gene (HFE or HLA-H) is located on Chromosome 6. HFE gene transcription and exon splicing results in an mRNA for about 2700 bp long. Translation of the gene results in hereditary hemochromatosis protein which has a 348-amino acid sequence. The HFE protein is a transmembrane protein expressed in intestinal and liver cells; it works in conjunction with another small protein called beta-2-microglobulin to regulate iron uptake. [00332] Several mutations have been identified in the HFE gene which can result in disease. One of these is the H63D mutation, a single nucleotide polymorphism (SNP)₁ and is related to clinical iron overload. In the H63D mutation, a G replaces C at nucleotide 187 of the gene (187CaG), causing aspartate to substitute for histidine at amino acid position 63 in the HFE protein. [00333] TABLE 2 describes the two capture probes and the target DNA used in this example. These include capture probes comprising wild-type (w-t) and mutant (mut) alleles. In the exemplary detection assays having aspects of the invention, NTNFET devices were functionalized by adsorption of capture probes including either the wild-type or mutant 17-mer alleles. As can also be seen in Table 2, the target is a 51-mer long allele which includes the wild-type (w-t) target sequence. [00334] FIGS. 21 A and 21 B depicts (the solid line) the transfer characteristic (G-Vg curve) of the device showing the effect of incubation of the device with the ssDNA capture probe, wild-type probe (FIG. 21A) and mutant probe (FIG. 21 B) respectively. Also FIGS. 21 A and 21 B, depicts the G-Vg curve following hybridization with the HFE target probe (the dashed line in each figure). [00335] Shown in FIG. 21 A, the allele specific wild-type capture probe is hybridized with wild-type synthetic HFE target (50 nM). The w-t/w-t hybrid matching combination was stable to the post-hybridization washing procedure, and resulted in significant decrease of the device conductance and a significant shift of G-Vg curve towards more negative gate voltage values. [00336] In contrast, FIG. 21 B, shows the mutant capture probe when exposed the same hybridization conditions with wild-type synthetic HFE target (50 nM). The single-base mismatch combination between mut capture probe and w-t target was not stable towards the washing conditions, and resulted in significantly smaller change in the device characteristics (no significant change in conductance and no curve shift towards negative Vg). This SNP discrimination was achieved at relative low stringency conditions: ~25°C below melting temperature for this sequence, Tm = 46°C. The electronic measurements of FIGS. 21 A and 4B were confirmed by fluorescent imaging of the fluorescently labeled target alleles. [00337] FIG. 21 C summarizes both electronic (1-G/G0) and fluorescent optical responses from the fluorescent target labels for hemochromatosis detection. In the plot in FIG. 21 C, data from three devices with similar geometry and 10 urn S/D pitch ware used, and mean normalized response values are shown. For electronic response, average of normalized signals for three NTFET devices were calculated; error bars equal one standard deviation. [00338] Other devices on the chip have demonstrated similar trends in their responses, as shown in FIG. 21D, with is a graph of electronic response (1-G/G0) to the presence of HFE single-nucleotide polymorphisms (SNP), in NTNFET devices with electrode pitch from 5 - 100 μm.. For electronic response, the average of normalized signals for nine NTNFET devices were calculated; error bars equal one standard deviation. On/Off modulation for devices hybridized with wt target plotted on right axis. The devices with 10 μm electrode pitch yields the best signal-to-noise ratio. Larger pitch devices trended toward increased noise or were below the percolation threshold whereas smaller pitch devices exhibited poor modulation

[00339] Assay reproducibility and selectivity was tested in the presence of nonhomologous DNA to increase sample complexity. Chips were prepared with allele specific capture probes as described above and hybridized with 100 pM wt target containing 5 μg/ml denatured (100oC, 10') salmon sperm DNA. [00340] FIG. 21 E is a plot of electronic (1-G/G0) responses in SNP detection assay in the presence of 5 μg/ml denatured salmon DNA. The plot compares the "no blocking" case with a "blocking" case: TX100 = 0.01% triton X-100 in PB buffer, 15', room temperature. This example indicated that the NTNFET response due to hybridization was obscured by the nonhomologous DNA suggesting nonspecific adsorption to the nanotubes or competitive displacement of the capture probes. The former mechanism is shown by blocking nonspecific binding sites (NSB) with 0.01% Triton X-100 in PB. Triton is a nonionic surfactant containing an aliphatic chain and a hydrophilic PEG group (n = 9-10) and has been shown to reduce NSB on nanotubes. The chips were incubated with the triton solution for 15' at room temperature, and washed as previously described. The triton blocking step enabled SNP discrimination at 100 pM target in the presence of 104 fold molar excess of nonhomologous DNA. This result demonstrates that adsorbed capture probes are able to withstand mild surfactants and are not readily displaced. [00341] Even at these target DNA concentrations (50 nM), the sensitivity of the NTNFET device is comparable to fluorescence measurements for SNP detection assays. Fluorescence techniques require measurement from all carbon nanotube network area (several micrometers square) to get reasonable fluorescence response in 10-20 s integration times. In contrast, since electronic measurement using NTNFET devices involves molecular interactions between DNA molecules and sidewalls of carbon nanotubes, carbon nanotubes themselves function as labels. An equally measurable electronic signal only requires that DNA molecules interact along isolated carbon nanotubes (typically few nanometers), due to the strong influence of DNA hybridization in the conducting path through the carbon nanotube network. [00342] These results clearly demonstrate that NTNFET device characteristics, such as maximum conductance or threshold voltage, provide a nanoelectronic sensor-based SNP detection assay which are comparable to state- of-the-art optical techniques. Label-free detection has several advantages including cost, time, and simplicity. Hand-held field-ready devices as opposed to laboratory methods using labor-intense labeling and sophisticated optical equipment are enabled by this approach. [00343] EXAMPLE F [00344] Use Of Counter Ions to Increase DNA Assay Sensitivity.

[00345] It has been demonstrated that deposition of a ssDNA capture probe on to the example NTNFET results in a similar change in the transfer characteristics (G-Vg) for deposition from pure water and for deposition from standard biologic buffers, such as conventional sodium phosphate buffer (PB). However, hybridization procedures performed in pure water (10 nM target DNA) did not demonstrate reproducible changes in G-Vg curve. For this reason, a buffered solution of salts is employed in the assay embodiments to facilitate hybridization and increase the change in NTNFET device characteristics. [00346] Alternative assays embodiments having aspects of the invention for electronic detection of biomolecules such as DNA take advantage of the properties of the molecules in response to variation ionic constituents and target concentrations in the analyte medium. [00347] The examples shown in FIGS. 22A-22D demonstrate the electronic response of the exemplary NTNFET device embodiment to hybridization of unlabeled oligonucleotides at different concentrations of ionic species and target DNA. The devices used are generally similar to those employed in Example E above. The oligonucleotides in this example are the unlabeled complementary 12-mer capture and target oligonucleotides describe in the forgoing examples and listed in TABLE 1.

[00348] Although NTNFET devices used in this demonstration had different base conductances (bare un-modulated device or GO), the G-Vg characteristics such as modulation and threshold voltages were quite similar. Plotting normalized values of maximum device conductance has demonstrated similar trends for different ranges of complementary DNA (target) concentrations shown as depicted in FIG. 22D. [00349] FIG. 22A is a plot of source-drain conductance (G) as function of gate voltage (Vg) for the exemplary NTNFET device. The demonstration was carried out in 200 mM phosphate buffer at a pH or 7.2. The upper-most curve shows the response of the bare device (both rising and falling Vg as described in FIG. 3A). The second curve shows the effect of the unlabeled ssDNA probe attachment without hybridization. The lower five curves show the effect of increasing unlabeled target DNA concentration on hybridization (incubation followed by washing) under the influence of sodium ions only, representing target DNA concentrations of 1 , 10, 50, 100, and 200 nM in descending order. [00350] FIGS. 22B and 22C are plots generally comparable to that of FIG. 22A, but with the demonstration carried out in buffer comprising 10 mM phosphate buffer with a magnesium ion source added, 20 mM MgCI2. Similarly, the uppermost curve shows the response of the bare device. The second curve shows the effect of the unlabeled ssDNA probe attachment without hybridization. [00351] In FIG. 22B, the lower four curves show the effect of increasing target DNA concentration on hybridization under the influence of the additional Mg+2 ions for the same nanoMolar range as in FIG. 5A, representing target DNA concentrations of 1 , 10, 50, 100, and 200 nM in descending order (the curves for 50 nM and 100 nM are superimposed and thus not distinct at the scale of plot). Note the dramatically increased effect of hybridization on device conductance under the influence to magnesium ions.

[00352] FIG. 22C demonstrates the sensitivity of the device and assay for hybridization at picoMolar ranges of the DNA target species, under the same hybridization conditions as FIG. 22B (Mg+2 ions). The lower four curves show the effect of increasing target DNA concentration in descending order, but in this figure, the curves represent target DNA concentrations of 1 , 10, and 50 pM and 1 nM respectively. [00353] FIG. 22D is a plot of the data of FIGS. 22A-22C showing of the normalized conductance (G/G0) of the three NTNFET devices as function of target DNA concentrations. The data show that the addition of Mg2+ during hybridization increased the sensitivity of DNA detection by 1000 fold, from 1 nM to 1 pM, or from 5x109 to 5x106 molecules, compared to Na+ alone. Furthermore, the dynamic range was increased from roughly 2.5 to 5 logs. Note also that the slope of the linear trend lines the Na+ hybridization data and the Mg2+ hybridization data differ by almost factor of two, i.e., -0.11 for Na+ data versus - 0.06 for the Mg2+ data.

[00354] Due to the assay methodology and in particular standardized buffer washes which were performed after each DNA incubation, NTNFET response to residual buffer or cation effects is negated. After hybridization, the NTNFET devices were washed with the same salt concentrations (standard washing procedure). This ensures that the observed changes in NTNFET device characteristics are not related to random changes in mobile charge concentrations on the device surface. [00355] Moreover, control procedures were carried out where the devices were hybridized by incubation in the Na+ containing medium, the unbound target DNA was washed away, and then the device was exposed to Mg2+ solution. Although replacement of Na+ with Mg2+ had small effects on the device response, the changes were significantly smaller than the changes demonstrated due to the presence of Mg2+ during hybridization incubation. It is believed that effect of the Mg2+ ions is predominantly to increase the extent and overall efficiency of DNA hybridization on nanotubes. The dominant mechanism for improved sensitivity is driving the formation of DNA duplexes rather than the direct effect of the ionic species on device characteristics.

[00356] The observed changes in NTNFET electronic characteristics can be correlated with DNA detection. The results were confirmed by using fluorescently labeled DNA compounds which verified DNA adsorption and hybridization were selective for nanotubes. Although sensors with only a few or a single carbon nanotube sensing elements can be fabricated, sensors used in this study contain a random network of nanotubes, covering a relatively large surface area between two metal electrodes (FIG. 17B). The random network geometry has several advantages: it eliminates the problems of nanotube alignment and assembly, conductivity variations due to chirality and geometry, and is tolerant to individual SWNT channel failure since the device characteristics are averaged over a large number of nanotubes. In addition, such devices can be developed on low-cost flexible and/or transparent polymer substrates by spray deposition or casting nanotubes from solution.

[00357] In terms of sensitivity, SWNTs may be superior if one considers it to be a true nanoscale sensor. As for intrinsic device characteristics, nanotubes exhibit surprisingly large electrical or 1/f noise. The magnitude of the 1/f noise is inversely proportional to the number of charge carriers in the device so a network with a large number of SWNTs reduces the 1/f noise by approximately (n)-1.3 , where (n) is the number of SWNTs in the channel of the device. Thus, where 1/f noise is a significant factor then large nanotube networks will have a distinct advantage over single nanotube channel devices. [00358] The following articles relate in some manner to aspects of the invention herein, and are incorporated by reference:

[00359] a. Star, A, Gabriel, J. -C P, Bradley, K & Grϋner, G (2003) Nano Lett. 3, 459^63.

[00360] b. Star, A, Han, T-R, Gabriel, J-C P, Bradley, K & Grϋner, G, 2003 NanoLett 3 1421-23. [00361] c. Star, A, Han, T-R, Joshi, V & Stetter, J R (2004) Electroanalysis 16, 108-112. [00362] d. Bradley, K₁ Gabriel, J-C P & Grϋner, G (2003) Nano Lett. 3, 1353-1355.

[00363] In summary, electronic signal output from the NTNFET biochips clearly differentiated between mutant and wild-type alleles of the HFE gene, responsible for hereditary hemochromatosis. A number of alternative embodiments are possible without departing from the spirit of the invention and without undue experimentation. The sensor architecture may be readily adapted to make the NTNFET biochip detection platform suitable for whole blood samples. Alternative embodiments, such as longer capture probes or the addition of a high affinity linker sequence (such as the (GT)20 oligonucleotide), can make the attachment more robust for more complex samples. Covalent attachment of DNA capture probes may also improve signal to noise ratio because then surfactants can be used to lower background. Moreover, microfluidics may be employed in order to facilitate the sample delivery and the manipulation of small volumes of DNA samples.

[00364] EXAMPLE G

[00365] Self-assembled Multiplex Assay Panel

[00366] Example G as set forth in the present application includes the subject matter of the corresponding "Example C" described in US Provisional Application No. 60/629,604, filed November 19, 2004, together with further illustration of the inventive concepts. US Provisional Application No. 60/629,604 is incorporated by reference in and claimed as priority by US Patent Application No. 11/212,026 filed August 24, 2005 entitled "Nanotube Sensor Devices For DNA Detection". Each of these applications is incorporated by reference herein. [00367] As noted above in EXAMPLE C herein, a tether-probe combination may comprise a pair of species with a highly specific mutual binding affinity, such as an antibody-antigen pair, an cell-surface receptor and a mating ligand, or any one of a broad class of biological species providing ligand pairs with specific mutual affinity. The self assembly property of such probe-tether pairs permits a sensor array having aspects of the invention to be pre-patterned with a plurality of different tether groups, and conveniently self assembled with corresponding specific probe groups at a convenient time, shortly before carrying out a DNA assay where probe groups are either perishable, or not previously determined. In many important bio-medical applications, assay targets are time variable, such as the constantly changing strains of influenza, HIV and the like. The inventive architecture described permits a pre-pattemed "generic" array device may be self- assembled as a completed target-specific assay array at or close to the time of use.

[00368] EXAMPLE G provides an important alternative approach to an self assembling array of target-specific detectors having aspects of the invention. Nanoelectronic devices having aspects of the invention provide the capability of molecular scale detection. Small devices with sensitivity levels that allow detection of a single target biomolecule enable novel array architectures providing embodiments with unique utility..

[00369] Molecular diagnostic tests generally consist of a panel of analytes to provide a health care provider with sufficient information to determine susceptibility to a disease state. One example of this is a test for mutations in the gene coding for the Cystic Fibrosis Transmembrane conductance Regulator protein that are associated with the Cystic Fibriosis disease. Over 900 mutations have been identified in that gene and 25 of them have been designated by the College of Obstetricians and Gynecologists as being associated with the disease at a high enough frequency to warrant testing. Therefore the CF panel consists of 25 tests for genetic variants. Many technologies have emerged to accommodate the need of analyzing several variables simultaneously (multiplexing). These range from the rudimentary and generally hand-crafted dot blot methods to the sophisticated photolithographically synthesized bio-polymers found in massively parallel micro-arrays. These methods are costly and time consuming. All current methods rely on placing known probes, capture molecules or ligands to a predetermined location. This is known as addressing. This process requires mechanisms capable of precise control and micron tolerances in many cases. It is concomitantly cumbersome and expensive. [00370] The following exemplary embodiments having aspects of the invention provide a novel method of configuring multiplex analysis without the need for predetermining the site of an interrogating molecule and subsequently restricting its placement to that site.

[00371] A plurality of different probe groups (e.g., each comprising a target- specific detector species with an affinity for a different target) may be distinguished by the signals that may be acquired by the various alternative embodiments on nanostructured sensor described herein. For example, different DNA probes, each with a distinct hybridization sequence, may be configured to have different overall sequence lengths. Different polynucleotide lengths with differing molecular masses can be distinguished by the difference in there electrical influence on a nanostructure such as a SWNT, for example producing a distinct signature (e.g., in nanotube conductance, capacitance or the like) with may be measured. Similarly, different probes, even if of the same or similar sequence length, may comprise distinct secondary structure or additional groups providing a probe-specific signal with may be measured. In other alternative [00372] FIGURES 23A through 23H illustrate the operation of a multiplex assay panel embodiment having aspects of the invention. The multiplex assay panel comprises an array nanosensors (5 are shown). The sensors may be substantially the same in configuration and properties. The array may conveniently comprise a large number of sensors, e.g., arranged in a two dimensional pattern on a chip, and packaged to permit individual sensors to be selectively interrogated by measurement circuitry. [00373] In certain examples, the array sensors comprise NTFETs as described elsewhere in this application, permitting convenient measurement of transconductance properties (source/drain), modulation characteristics (variable gate bias), and/or capacitance properties (capacitance or impedance of channel relative to gate electrode) through patterned source-drain-gate contacts of the transistor array. It should be understood that other kinds of nanoelectronic sensors may be employed without departing from the spirit of the invention, including capacitive sensors, breakdown voltage sensors, magnetic sensors and the like.

[00374] Alternative embodiments (not shown) may have more than one type or class of sensor, each class of sensor being represented by a plurality of same type. Such a multi-class array may permit measurements under differing conditions or dynamic ranges. Likewise, alternative embodiments may employ probes exploiting and detection schemes other than polynucleotide hybridization affinity, such as antigen-antibody affinity, receptor-ligand affinity, and the like. Multiplex array panels may thus be adapted to a wide range of applications, including molecular diagnostics (DNA, RNA, proteins); Organism detection (viral, bacterial); Chemical analysis (combinations of gases, water contaminants); pharmaceutical production and the like. [00375] As shown in FIGURE 23A, the array is incubated with a mixture of different probes kinds (represented by shapes A, B and C) which bind to the sensors of the array in a random fashion (or quasi-random or irregular fashion), so that a desired mixture of sensor functionalization is obtained. The dilution and/or stringency conditions may be adjusted to favor a single probe attachment per sensor. Following incubation, excess and non-binding probes may be rinse away, as shown in FIGURE 23B.

[00376] As shown in FIGURE 23C, the sensors of the array may be individually interrogated by measurement circuitry, and the signals may be conveniently stored in a processor for further operations. Note that the array may be maintained during measurement under conditions which are optimized for probe characterization measurement, and may be substantially different than those prevailing during probe incubation. For example, measurements may be made wet or dry, under a particular buffer composition, particular temperature, and the like. [00377] As shown in FIGURE 23D, the signal or signals from each sensor may be correlated with a specific probe binding configuration. In the example shown, particular signal magnitudes are correlated with the single binding of each of the three probe varieties "A", "B", and "C", and also with the configuration of no binding (empty) and multiple binding. A map of the inferred probe configuration of each sensor may be stored in processor memory. This provides a map of multiplex detectors that may not have a predictable spatial order, but whose detection potential is known. [00378] It should be understood that while the schematic example of FIGURE 23C indicates measurement of a single quantity, multiple or complex properties may be measured and employed in probe-sensor characterization. For example NTFET devices characteristics, hysteresis, on-off ratio, trigger threshold, and the like or combinations of these may be employed in this determination (see discussion re FIGURE 24).

[00379] In operation, as shown in FIGURE 23E, the assay panel array is incubated with a sample medium putatively containing target species. In the example shown, the corresponding targets "b" and "c" corresponding to probes "B" and "C are present in the sample, non-target species "d" is also present, but target species "a" corresponding to probe "A" is absent. Note that the buffer or media composition and stringency conditions for sample incubation may be adjusted to optimize specific binding while preserving probe-sensor attachment. Following incubation, excess sample may be rinse away, as shown in FIGURE 23F , leaving targets "b" and "c" bound to the corresponding probes.

[00380] As shown in FIGURE 23G, the sensors of the array may be individually interrogated by measurement circuitry, and the signals may be conveniently stored in a processor for further operations. As noted with respect to FIGURE 23G, that the array may be maintained during measurement under conditions which are optimized for the sample measurement, and may be substantially different than those prevailing during sample incubation. [00381] As shown in FIGURE 23H, the signal or signals from each sensor may be correlated with probe-target binding status. In certain embodiments, particular probe-target combinations may be distinguished from the measured signals, but this is not a necessary criteria. In the example shown, the process need only correlate a signal as "positive" or "negative" for any given sensor. Reference is then made to the stored probe-sensor map to determine which target species is present (or absent) for each sensor. Also, in this example, measurement of sensors with no probe or multiple probes is omitted. [00382] FIGURE 24A to 24D illustrate an example of employing multiple signals from a single sensor to characterized the probe-sensor configuration. The incubation procedures of FIGURES 24A-B may be generally as described above with respect to FIGURES 23A-B.

[00383] As shown in FIGURES 24C-D, this example includes obtaining measurements of both source-drain conductance (left hand axis of each sub-plot) and channel-gate capacitance (right hand axis of each sub-plot). One or more of the particular values of these properties, their ratios, or their sums or differences may be employed to characterize the probe status of the respective sensor (e.g., "A", "B", "C multiple or empty). Note that similar measurement strategies may be employed in target species determination (not shown). [00384] FIGURE 25A to 25E illustrate the use on enhancement groups to facilitate probe characterization. Reference is made to Example C herein (see FIGURES 8 and 9), and in particular the use of enhancement groups such as electroactive incalators, amplifier groups, and the like to increase and modify the sensor signal in response to a species interaction event. Likewise, such enhancement groups as magnetic microbeads, nanodots and the like may be employed to increase probe characterization signal strength and distinction. For example, a range of alternative enhancement groups may be employed to permit "single-molecule" probe characterization and mapping. [00385] The incubation procedures of FIGURES 25A-B may be generally as described above with respect to FIGURES 23A-B, with the exception that in the example shown, enhancement groups E1 , E2, and E3 are attached to probes "C", "A" and "B" respectively, with the probe attachment thus binding a combination of both probe and enhancement group to each sensor. [00386] In alternative embodiments (not shown) the enhancement group may be added and bound to the probe after attachment of the probe to the particular sensor. This may be either by a specific affinity between probe and enhancement group (e.g., antibody-antigen affinity). In other alternative assay embodiments, the enhancement group may be the same for each kind of probe, the same enhancement group, the influence of the enhancement group be sufficient to produce a distinct measurement when attached to the different kinds of probes. [00387] As shown in FIGURE 25C₁ the sensors of the array may be individually interrogated by measurement circuitry, for example as in the manner shown in FIGURES 23C and 24C. As shown in FIGURE 25D, the probe status for each sensor is characterized by the signal or signals generated by the sensor in response to the combination of probe and enhancement group.

[00388] As shown in FIGURE 25E, the enhancement group may be removed prior to assay panel use for sample target determinations, for example by particular reagents or buffers, variation in stringency conditions and the like. In alternative assay embodiments, the Alternatively, the enhancement group may remain attached during assay panel use for sample target determinations. [00389] It should be noted that the multiplex assay array embodiments described in EXAMPLE C and in EXAMPLE G may be used in combination without departing from the spirit of the invention. For example, specific portions of the array (each such portion comprising a plurality of sensors) may be "printed" with a tether group having a particular affinity for a class of probes in the manner described in Example C, while other portions of the array may be functionalized in the manner of EXAMPLE G. [00390] EXAMPLE H

[00391] Nanosensors With Overlaying Nanotube Network [00392] A number of embodiments of nanostructured sensor devices having aspects of the invention employ an architecture in which a network of nanostructured elements (such as single-walled carbon nanotubes or SWCNT) are formed or deposited directly on a substrate material, for example a silicon wafer coated with a dielectric layer. Various functionalization materials may then be deposited above the nanotube network, for example a polymer recognition layer sensitive to a selected analyte, or a coating of molecular transducers including probe molecules with an affinity for a selected target biomolecule, and the like. [00393] In alternative embodiments having aspect of the invention, the above described sensor architecture may be described as being reversed, i.e. as a "NT network-on-top" architecture. In such an embodiment, a functionalization layer may be deposited upon the substrate prior to formation of the nanotube network, and the NT network subsequently deposited or formed above the functionalization layer. This architecture permits a greater exposure of the nanotube network to the analyte medium, and also permits the substrate to be conveniently coated or isolated from the analyte medium without impairing the exposure of the nanotube network. The functionalization layer may comprise a composite structure, for example including a substrate passivation layer, a ligand layer, a bio-probe layer, a selectively permeable layer, and the like. The addition of a "NT network-on-top architecture increased the range of operational arrangements available to provide maximum flexibility in sensor functionalization strategies.

[00394] These embodiments may be made employing photolithographic, CVD and other wafer-level manufacturing technology common in the electronics industry to reduce the cost-per-measurement to a low level. In addition, the nanostructured conductive and semiconductor element precursors (e.g., carbon nanotubes in carrier fluid dispersion) may advantageously be deposited and/or applied to devices using ink-jet type "droplet on demand" technology. Optionally substrates other than monocrystaline silicon (e.g., polycrystalline semiconductors, polymer substrates, flexible substrates, polyimide, polycarbonate, PET and the like) may be employed to further reduce production costs. [00395] Nanostructured sensor platform. FIG. 26 an exemplary field NT network effect transistor sensor having aspects of the invention, including a interconnected nanotube network 11 deposited or formed upon a substrate 12. The network 11 spans and electrically communicates between a spaced-apart contact pair comprising source electrode 13 and drain electrode 14. In this transistor embodiment, there is an additional gate electrode 15 disposed beneath a dielectric portion of substrate 12. An optional contact passivation material 16 coats the electrodes 13 and 14 without interfering with electrical communication of electrodes 13 and 14 with network 11. At least one recognition or functionalization material, shown as a biofunctional layer 17, is disposed in contact with network 1 1 and arranged generally upon and/or diffused into the upper surface of network 11. [00396] The architecture shown in FIG. 26 may be referred to as a "NT network-on-bottom" arrangement, although the biofunctional layer 17 or other recognition material may optionally be disposed to penetrate downward to the substrate 12. Typically, the contacts 13, 14 may be formed first, then network 11 formed or deposited, and subsequently the biofunctional layer 17 or other recognition material deposited. One or more optional operative coatings (not shown) may be deposited either under, within, and/or over the biofunctional layer, such as a semi-permeable layer configured and composed to restrict exposure of network 11 and/or substrate 12 to particular species within an analyte medium. [00397] "Nanotube Network-On-Top" Sensor Architecture. FIG. 27 illustrates an exemplary "network-on-top" field effect transistor sensor 20 having aspects of the invention, in which a nanotube network 21 is disposed above a biofunctional layer 27 (and showing optional contact passivation coatings 26). [00398] At least one recognition or functionalization material, shown as a biofunctional layer 27, is disposed in contact with substrate 22. Nanotube network 21 is formed or deposited upon functionalization layer 27 and in electrical communication between spaced-apart source electrode 23 and drain electrode 24. In this transistor embodiment, there is an additional gate electrode 25 disposed beneath a dielectric portion of substrate 22. Optional contact passivation material 26 coats the electrodes 23 and 24 without interfering with electrical communication of electrodes 23 and 24 with network 21. [00399] It should be understood that the substrate 22 can comprise a dielectric material, an insulating polymer, a flexible substrate or a combination of these. For example, the substrate may include an insulating layer (not shown) covering the gate electrode 25, while other components of the substrate may comprise a flexible polymer and the like.

[00400] Likewise, it should be understood that the network may comprise other nano-structural elements in addition to or instead of SWCNTs, such as multiwall nanotubes, nanowires, and the like. [00401] The making of NTFET sensors such as shown in the examples herein may employ conventional methods to form the described elements, such as methods used silicon wafer processing in the electronic industry. Alternatively or additionally, the described elements may be formed employing printing and deposition methods of the type generally known as "ink jet" methods. This is particularly useful in the making of compact arrays having a plurality of sensors in a single package, or where for purposes of mass production of sensors it is desired to make a plurality of sensors "at the wafer level" for subsequent division and packaging. Additionally, ink jet droplet-on-demand methodology is suitable for the employment of flexible or sheet-like substrates. [00402] It should be noted that single wall nanotube networks may be formed by a number of methods. Among these are in-situ growth of nanotubes from dispersed nanoparticles of catalysts, as described, among other places in US Patent Application No. 10/177,929 filed June 21 , 2002 entitled "Dispersed Growth Of Nanotubes On A Substrate", incorporated above. Alternatively, nanotube networks may be made using deposition methods such as described in US Patent Application No. 10/846,072 filed May 14, 2004 entitled "Flexible nanotube transistors", incorporated above. A useful method of making nanotube networks is described in L. Hu, D. S. Hecht, and G. Grϋner, Percolation In Transparent And Conducting Carbon Nanotube Networks, Nano Lett. 2004, Vol. 4, No. 12, 2513- 2517, which publication is incorporated here by reference as if fully set forth herein. [00403] It may be seen in FIG. 27, when compared to FIG. 26, that the "network-on-top" permits a greater exposure of the nanotube network 21 to the analyte medium, and also permits the substrate 22 to be conveniently coated or isolated from the analyte medium without impairing the exposure of the nanotube network 21. The functionalization layer 27 may comprise a composite structure (not shown), for example including a substrate passivation layer, a ligand layer, a bio-probe layer, a selectively permeable layer, and the like. [00404] Alternative "Nanotube Network-On -Top" arrangements may be employed. For example, FIG. 28 shows an alternative structural arrangement of a field effect transistor sensor 30 having aspects of the invention generally similar to the embodiment of FIG. 27, and in which a nanotube network 31 is disposed above a biofunctionalization layer 37, but below a subsequently deposited pattern of source and drain conductors 33 and 34. [00405] FIG. 29 shows an addition alternative exemplary field effect transistor sensor 40 having aspects of the invention, having a nanotube network 41 disposed above both a biofunctionalization layer 47 and source and drain conductors 43 and 44, with the nanotube network 41 covering at least portions of one or both of the electrode elements.

[00406] Nanostructured Sensor Functionalization. The above described sensor embodiments, such as a preferred embodiment of a carbon nanotube network transistor, may be treated or engaged with many alternative functionalization materials, probes, molecular transducers, coatings and the like. As used herein, the term "functionalization" with respect to a nanostructured sensor device includes generally alternation or additions to the basic electronic device platform to produce or increase sensitivity to one or more target or analyte species, such that the sensitivity induces a measurable effect. [00407] In examples of functionalization of NTFET or similar devices having aspects of the invention, fuctionalization may include such things as altering or creating defects in the lattice structure of nanotubes, changing the overall composition of a nanotube network, covalently attaching groups to nanotubes, non-covalently attaching groups or materials to nanotubes, attaching groups to substrates or electrodes adjacent to nanotubes, or combinations of theses. In the case of examples of NT network FETs or related devices having aspects of the invention, such functionalization alterations or additions may be performed either before, during or after the formation of the NT network. [00408] A functionalization group may induce a measurable effect upon recognition of a target or analyte by measurable electron transfer effects or by alternations of the microenvironment adjacent the nanotube device so as to induce measurable electronic effects, or combinations of these. [00409] Functionalization may include more than one layer, coating or deposited material, e.g., an NTFET may include a recognition material which interacts with a target analyte combined with a protective material which restricts exposure or response to one or more non-target species, such as a selectively permeable layer. [00410] Similarly functionalization may include the interaction of more than one group or species in addition to the nanotubes of the NTFET or similar device, such as both a fixed recognition material (e.g., a probe biomolecule noncovalently bonded to the nanotubes) and a selected cofactor or substrate species introduced into an analyte medium prior to or coincident with measurement. For example, the above incorporated patent application No. 60/629, 604 describes the use of an intercollator species introduced into an analyte medium to increase the signal produced by an NTFET with a cDNA probe upon hybridization with a target sample DNA sequence (See discussion of FIG. 31 below). [00411] A biofunctional layer (e.g., 27 in FIG. 27) may be configured and composed to do one or more operational tasks, for example it may preventing nonspecific binding on non-target species, it may facilitating attachment of biomolecules and/or probe species, it may react to alter the electrical state or electro-chemical environment of the nanotubes when the sensor is exposed to a target species, or combinations of the above. Examples of particular biofunctional compositions or sublayers include those which avoid nonspecific binding of biomolecules, such as a polyethilene glycol (PEG); those that avoid nonspecific binding and to which a biomolecule can be attached, such as a PEG-polyethilene imine (PEI) blend; and/or those that include surfaces or biomolecules that lead to specific binding, such as polymer, and biomolecule coatings. See A. Star et al, Electronic Detection Of Specific Protein Binding Using Nanotube FET Devices, Nano Lett. 2003, Vol. 3, No. 4, 459-463, which publication is incorporated here by reference as if fully set forth herein. [00412] FIGs. 30A-30B shows an exemplary field effect transistor sensor 40 having an additional recognition species such as recognition molecule 58 deposited or ligated to biofunctional layer 57, which in turn is disposed in contact with substrate 52. Nanotube network 51 is formed or deposited upon functionalization layer 57 and in electrical communication between spaced-apart source electrode 53 and drain electrode 54. In this transistor embodiment, there is an additional gate electrode 55 disposed beneath a dielectric portion of substrate 52. Recognition molecules may include, for example, DNA probes, enzymes, antibodies and the like, configured and selected to bind, react with and/or hybridize with a target species.

[00413] Reference is made to the above described FIGS. 8A, 10C and 10D to illustrate particular examples of biofunctionalization having aspects of the invention. In the embodiment shown in FIG. 8A, the probe 70 is shown detecting a single-stranded fragment of DNA 84 by hybridizing with target base sequence 72. In this example linker molecule 67 (such as pyrene) may be covalently bound to cDNA 68 to form probe 70. Suitable sensor circuitry (not shown) is connected to sensor 60 so as to detect and/or quantify an electrical response of sensor 60 to the hybridization of DNA 74, in a manner described above with respect to other sensor embodiments. See FIGS. 10C and 10D. For example, the conductance between source 63 and drain 64 may change upon hybridization, the change being measured. Alternatively, in an NTFET DNA sensor embodiment, the hybridization of DNA 74 may cause a phase shift in the device characteristics of sensor 60 produced as the voltage of gate electrode 65 is varied through a selected voltage range. Additional or alternative properties of sensor 60 may be measured to detect hybridization.

[00414] FIG. 31 is an alternative embodiment having aspects of the invention, generally similar to that of FIG. 8A, in which the probe group is attached to a biofunctional layer adjacent the substrate. In the embodiment shown in FIG. 31 , the probe 90 is shown detecting a single-stranded fragment of DNA 94 by hybridizing with target base sequence 92. In this example, biofunctional layer 95 may be covalently bound to cDNA 88 to form probe 90 via bond 96 (e.g. layer 95 may contain groups configured to bond to the cDNA, such as the polymer shown in FIG. 10C). Suitable sensor circuitry (not shown) is connected to sensor 80 so as to detect and/or quantify an electrical response of sensor 80 to the hybridization of DNA 94, in a manner generally similar to the example of FIG. 8A. For example, the conductance between source 83 and drain 84 may change upon hybridization, the change being measured. Alternatively, in an NTFET DNA sensor embodiment, the hybridization of DNA 94 may cause a phase shift in the device characteristics of sensor 80 produced as the voltage of gate electrode 85 is varied through a selected voltage range. Additional or alternative properties of sensor 80 may be measured to detect hybridization.

[00415] Further examples of biofunctionalization are described in US patent application No. 10/431 ,963 filed 5/8/03, entitled "Electronic Sensing of Biomolecular Processes" (published as No. 2004-0067,530 on 4/8/04), which is incorporated herein by this reference. In addition, as described in the patent applications incorporated above, including No. 10/704,066 entitled "Nanotube- Based Electronic Detection Of Biomolecules"; and No. 60/627,743 entitled "Nanotube based Glucose sensing", carbon nanotubes have properties which allow them to be bound or tethered to various biomolecules or chemical species, so that the properties of the biomolecule or chemical species modifies the properties of the nanotube so as to provide useful functionalization capability for NTFETs and like sensors. [00416] Exemplary method embodiment for making NTFETS. [00417] 1. prepare substrate, array layout, etc.. [00418] 2. deposit contacts

[00419] 3. deposit biofunctional layer (e.g., recognition material, ligand material, protective coating (PEG, etc.), or combinations of these [00420] 4. deposit NT solution, and cure/dry to form a nanotube network spanning contacts.

[00421] 5. optionally attach additional bioprobe groups. [00422] 6. dice chips, package as sensor device, including circuitry encapsulation, and the like. [00423] EXAMPLE I [00424] Nanosensors With porous substrates

[00425] One advantage of disposing a nanosensor device upon a micro- porous membrane or substrate, is that detection chemistry may be accelerated, analyte molecules concentrated, and sensitivity improved. Reaction kinetics in conventional microfluidics, were a liquid sample media flows parallel to a sensor surface, are determined by Nernst diffusion layer (~5 μm thick). In contrast, in exemplary embodiments having aspects of the invention a sensor comprises a porous substrate, and a fluid (liquid or gas) sample may be controlled microfluidically to flow through the substrate. This arrangement makes binding reactions rate-limiting and may decrease assay time by 100 to 1000 fold. [00426] In addition, exemplary embodiments having aspects of the invention employ the fluid flow of a sample medium to concentrate a dilute target in proximity to the active sensor surface. Likewise, a greater sensitivity may be achieved by cumulative reaction with target species with the sensor as the sample media moves through the substrate.

[00427] As shown schematically in FIG. 32A₁ where analyte medium flows parallel to a nanosensor surface, the detection chemistry tends to be transport limited, depending on the diffusion of target molecules across a surface boundary layer to interact with sensitive elements, e.g., a CNT film and/or associated functionalization material. Where a micro-porous membrane permits flow of analyte medium perpendicularly through the nanosensor surface, the detection chemistry tends to be reaction limited, i.e., the rate at which the target molecules bind or otherwise interact with the sensitive elements. This effect can permit the porous substrate to respond more quickly.

[00428] Similarly, in certain embodiments, the micro-porous membrane can act as a filter, to concentrate or detain target molecules adjacent the sensitive elements, as solvent or suspension phase fluid (e.g., gas or liquid solvent) pass through the membrane relatively unimpeded. This can be particularly advantageous for target analytes in low concentration or traces, such as in forensics, explosive detection, and the like. Note that additional controls can be used to regulate membrane transport, such as electrophoretic effects, and the like, without departing from the spirit of the invention. [00429] FIG. 32B shows a SEM micrograph of a commercially available microporous anodic alumina membrane with a regular pore diameter of about 20 nm (Anopore® membrane, by Whatman pic), and FIG. 32C shows a SEM micrograph of an experimental anodic alumina membrane with a hexagonal pore arrangement of about 5 nm diameter (University of Twente, Nederlands). See also, R Schmuhl, et at, Blank, "Si-supported mesoporous and microporous oxide interconnects as electrophoretic gates for application in microfluidic devices," Anal Chem (2005) 77, pp178-84; and S Roy Chowdhury et al, "Pore size and surface chemistry effects on the transport of hydrophobic and hydrophilic solvents through mesoporous g-alumina and silica MCM-48," J Membrane Sci, (2003) 225 pp177- 86, each of which is incorporated by reference.

[00430] FIGS. 33A-33D illustrate alternative exemplary embodiments of nanosensors having aspects of the invention and providing for flow of analyte medium through a porous substrate, and show an exemplary module for fluidic sample analysis.

[00431] FIG. 33A is a is a cross-sectional diagram of a nanosensor embodiment 4Op similar in a number of respects to the capacitive sensor shown in FIG. 16. The reference numerals refer generally to comparable elements as in FIG. 16. In this example, a nanotube network 41 (and optionally any selected functionalization material) is deposited on a microporous membrane 41a, and is shown overlain by an interdigitated pair of contacts d and c2 (44a, 44b). The network is restricted in coverage, so as to leave a nonconducting gap "g" between the network portion "d" and the contact 44a. The microporous membrane 41a is optionally supported by a porous support 41 b, the porous substrate thus comprising 41a and 41b. Optionally a sealant 45 may be deposited on portions of the membrane 41a and support 41 b not covered by sensing elements, so as to guide analyte medium to percolate through the sensor device. [00432] FIG. 33B is a is a cross-sectional diagram of a nanosensor embodiment 100p similar in a number of respects to the NTFET sensor shown in FIG. 1A. In this example, one or more optional gate electrodes 114' (e.g., a porous or perforated conductor) are embedded within microporous membrane 41 a (or alternatively, the gate '114 is disposed above the membrane 41a and covered by a thin porous insulator 46). Nanotube network 106 (and optionally any selected functionalization material) is deposited upon membrane 41a, and is contacted by a pair of contacts 110, 112. The microporous membrane 41 a is optionally supported by a porous support 41 b, the porous substrate thus comprising 41a and 41b. [00433] FIGS. 33C and 33D are two orthogonal cross-sectional diagrams of an exemplary flow-though micro-fluidic sensor module 190 providing for the conduct of a gaseous or liquid analyte medium, and including one or more sensor devices disposed on porous substrates, such as the sensors 4Op and 10Op depicted in FIGS. 33A-33B.

[00434] In the example shown, there are four such sensors, arranged to share a common porous substrate 41 comprising a microporous membrane 41a and a porous support 41 b, so that the combined devices 4Op, 10Op and substrate 41 form a "porous chip" 194. The chip 194 is mounted within a module housing comprising an upper portion 191a defining an analyte media inlet 192, and a lower portion 191 b defining an analyte media outlet 193. Circuit leads 195 connect the devices 4Op and 10Op of chip 194 through a via in body 191 to an external signal connector 196. It is apparent to one of ordinary skill in the art that there are alternative fluidic arrangements and structures that may be employed without departing from the spirit of the invention. For example, body 191 may comprise an assemblage of planar portions such as glass slides, separated by spacers, shaped and formed to provide mountings and conduits (e.g., etched polymer or glass, bonded to planar portions, such as by adhesives, US welding, and the like). Sensor module 190 is preferably integrated in a detector system (not shown) providing for controlled sampling and flow of gaseous or liquid analyte media, and for the analysis and output of measurement date. [00435] Electric Field Stringency Control. Nanotubes can generate high electric field strengths (>106 V/cm) due to their geometry. This permits sensor embodiments having aspects of the invention to employ electrical modulation of binding interactions, so as to provide increased specificity and increased reaction rates, and to avoid mismatched hybridization and nonspecific binding. This also permits adjustable operating modes to favor selected priorities, e.g, higher sensitivity/higher false positives vs lower sensitivity/lower false positives.

[00436] FIGS. 34A-34b illustrate alternative exemplary embodiments of nanosensors having aspects of the invention and providing for electric field stringency to control binding of non-specific polynucleotides. In this diagram, device 10 comprises a nanotube 2 is in contact with at least one contact 3 (substrate not shown) and has attached one or more polynucleotide probes 5. [00437] FIG. 34A shows device 10 with the nanotube 2 in an uncharged state. A highly complementary target 5 is hybridized to probe 4 and a less complementary target 6 is partially hybridized to probe 4'. An excess or non- complementary target 7 is bound to the nanotube 2 but not to a probe. [00438] FIG. 34B shows device 10 with the nanotube 2 in an uncharged state. The negatively charged nanotube 2 tends to repel the target polynucleotides, permiting targets 6 and 7 to be rinsed away, while highly complementary target 5 remains hybridized to probe 4. The voltage or field strength (as well as temperature, pH, buffer concentrations, and the like) may be adjusted to obtain desired stringency. [00439] FIG. 35 illustrates alternative exemplary embodiments of nanosensors having aspects of the invention having capillary delivery of samples. The assay device 20 may be configured as a disposable or partially disposable test strip. Device 20 has a detector 10 which is preferably a flow-through sensor as described herein. Sample 1 is applied to a porous intake portion 2 which wicks the sample to contact sensor 10. Optionally reagents 3 or bioactive species 4 (enzymes, antibodies and the like) may be dissolved in sample 1 as it flows towwards sensor 10. Circuitry 8 receives a signal from sensor 10 by connector 7, so as to output a measurement to a user.

[00440] Surface-deposited networks. FIGS. 36A-36C illustrate alternative exemplary embodiment 30 of a nanosensor having aspects of the invention, and having a network of nanotubes 2 superficially applied from solution to a porous membrane 3 (such as that shown in Fig. 32B). The nanotube net work may be prepared and deposited generally in the manner described in US Patent Applications No. 60/748,834, filed December 9, 2005, entitled "Nanoelectronic Sensors Having Substrates With Pre-Patterned Electrodes, And Environmental Ammonia Control System", and No. 11/274,747, filed November 14, 2005, entitled "Carbon Nanotube based Glucose sensing", each of which is incorporated by this reference. In the methods described below, the use of heating can assist in drying or curing the solution to form a network without substantially penetrating the pores 4 of membrane 3. One or more contacts 5 may be applied in communication with the network 2, for example using a conductive polymer material, or a conductive ink preparation. [00441] A suitable aqueous deposition solution may be made by suspending SWNT-PABS powder in water (preferably at a concentration of about 1 mg/mL), and ultrasonication may be employed to assist in making a homogeneous dispersion. The carbon nanotube dispersion may be sprayed with an air brush to coat the substrate.

[00442] A composition of SWNT-PABS powder (poly (m-aminobenzene sulfonic acid or PABS covalently attached to SWNTs) is commercially available from Carbon Solutions, Inc. of Riverside CA, and may be made as described in B Zhao et al, "Synthesis and Properties of a Water-Soluble Single-Walled Carbon Nanotube-Poly(m-aminobenzene sulfonic acid) Graft Copolymer", Adv Funct Mater (2004) VoI 14, No 1 pp 71-76, which article is incorporated by reference. An aqueous solution of SWNT-PABS may be prepared by ultrasonication (e.g., 1 mg/mL). After brief sonication, a homogeneous dispersion of carbon nanotubes was obtained. [00443] Preferably the deposition is done in several light coating steps with intermediate drying (for example on a hotplate with the temperature of about 55 to 75 degree C). The film resistance may be measured between steps until the selected resistance is obtained (the measurement may be between printed traces, or may be by pin probes on the network coating. For example, the deposition may be continued until resistance with a half-inch pin probe spacing is about 15 K Ohm.

[00444] Note that in the above example, the PABS assists in creating the aqueous dispersion. For hydrophobic nanotube or nanosturctures, alternative solvent methods may be employed, such as are described in US Application No. 10/846,072 filed May 14, 2004, entitled "Flexible nanotube transistors", which is incorporated by reference.

[00445] Functionalization. It should be understood that sensors such as those described above may be functionalized by a variety of methods, so as to provide a suitable probe or recognition group. In addition, the following publications (each of which is incorporated herein by reference as further description, without any implication that these constitute prior art with respect to any aspect of invention herein) describe additional alternative functionalization techniques that may be included in sensors having aspects of the invention without departing from the spirit of the invention:

[00446] R. Bashir, "DNA-mediated artificial nanobiostructures: state of the art and future directions" Invited Review in Superlattices and Microstructures, School of Electrical and Computer Engineering and Department of Biomedical

Engineering, Purdue University, Vol. 29, No. 1 , 2001.

[00447] A. Bianco et al, "Can carbon nanotubes be considered useful tools for biological applications?", Adv. Mater. 2003, 15, No. 20., 1765-68.

[00448] A. Cassell, "Ultrasensitive Carbon Nanoelectrode Biosensor Technology" Center For Nanotechnology, UC-Santa Cruz, Moffett Field CA, April

22, 2004

[00449] R. Chen et al, "Controlled Precipitation of Solubilized Carbon

Nanotubes by Delamination of DNA", American Chemical Society, 2005.

[00450] C. Dwyer et al, "DNA-functionalized single-walled carbon nanotubes", Nanotechnology 13 (2002) 601-604

[00451] C. Hu et al, "DNA Functionalized Single-Walled Carbon Nanotubes for Electrochemical Detection", J. Phys. Chem. B, Vol. 109, No. 43, 2005 20072-

76

[00452] K. Kerman et al, "DNA-Directed Attachment of Carbon Nanotubes for Enhanced Label-Free Electrochemical Detection of DNA Hybridization",

Electroanalysis 2004, 16, No. 20, 1667-72

[00453] J. Li et al, "Carbon Nanotube Nanoelectrode Array for Ultrasensitive

DNA Detection", Nano Lett., Vol. 3, No. 5, 2003, 597-602

[00454] M. J. Moghaddam et al, "Highly Efficient Binding of DNA on the Sidewalls and Tips of Carbon Nanotubes Using Photochemistry", Nano Lett.,

2004 VoI. 4, No. 1 , 89-93

[00455] J. Wang et al, "Ultrasensitive Electrical Biosensing of Proteins and

DNA: Carbon-Nanotube Derived Amplification of the Recognition and

Transduction Events", J. AM. CHEM. SOC. 2004, 126, 3010-3011. [00456] CONCLUSION [00457] One of ordinary skill in the art will understand that a number useful alternative industrial applications and alternative embodiments are possible without departing from the spirit of the invention.

[00458] Alternative embodiments of nanosensor devices having aspects of the invention exploit one or more of a number of device properties, such as capacitance, transconductance, resistance, impedance, inductance, magnetic or electromechanical properties, piezoelectric effects, electro-optical effects, and the like. Additional conducting elements may be include to permit desired properties to be measured electronically and provide the detector signal or signals, such as source and drain electrodes, counter electrodes, gate electrodes, reference electrodes, pseudo-reference electrodes and the like. [00459] The device embodiments may include other elements which enhance nanosensor performance. For example, a micro-hotplate may be include a sensor chip to permit thermal control to enhance sensor speed and/or sensitivity, and to facilitate device recovery or cycling. The device embodiments may be integrated with other components which extend nanosensor system operation, such as a microfluidic sampling device, purification device, PCR or other amplification device, power sources, remote or wireless communication devices, and the like. [00460] The devices may be made by known processes used in the semiconductor industry, preferably as a plurality of devices arranged on a wafer. Typically one or a plurality of devices are disposed on each distinct die of the wafer, the die being separated following fabrication and packaged by conventional methods to facilitate integration into an electronic measurement system. [00461] Known microprocessors, output devices, displays and/or power sources and the like may be included in the sensor system. Alternative sensor devices having aspects of the invention may be configured for systems including wireless sensor and base receiver/transceiver units to permit dispersed placement of sensor units, allowing convenient integration into existing and/or conventional monitoring and alarm systems (e.g., industrial/environmental monitoring systems, wearable patient monitoring units, and the like). [00462] Additional exemplary embodiments having aspects of the invention comprise an electronic sensor device which is biocompatible and configured to be operated with all or a portion of the device emplaced or inserted within a patient's body. Known biocompatible materials may be readily used to construct the sensor device. In certain embodiments, one or more sensor devices are integrated into or coupled to a drug delivery system. The electronic sensor device is configured so as to control the release of one or more drugs in relation to the measured blood concentration of one or more target species. [00463] Sensor devices made according to aspects of the invention may take a number of alternative forms. For example, in certain embodiments, it is advantageous to make each sensor a single-use device, e.g., a disposable sensor unit may be configured to mate to a reusable electronic measurement system, or the measurement system itself may be economically produced so as to be disposable as well. Alternative embodiments may include an array with multiple sensor elements on a chip, wherein the multiple sensors are functionalized for a plurality of different species, so that the device can provide a plurality of distinct measurements.

[00464] Having thus described a preferred embodiment of the nanotube sensor device, it should be apparent to those skilled in the art that certain advantages of the within system have been achieved. It should also be appreciated that various modifications, adaptations, and alternative embodiments thereof may be made within the scope and spirit of the present invention. The invention is further defined by the following claims.

Claims

CLAIMSWhat is Claimed is:

1. A nanotube sensor for sensing an polynucleotide, comprising: a substrate; a first nanotube over the substrate; at least two conducting elements in electrical communication with the first nanotube; and at least one single-strand DNA molecule operatively associated with the first nanotube, the at least one single-strand DNA molecule configured for interacting with a complimentary target DNA strand to alter an electrical property of the nanotube sensor.

2. The nanotube sensor of Claim 1 , wherein the at least one single- strand DNA is directed attached to the first nanotube.

3. The nanotube sensor of Claim 1 , wherein the first nanotube is selected from the group consisting of single-walled nanotubes, double-walled nanotubes, multi-walled nanotubes, and onions.

4. The nanotube sensor of Claim 1, wherein the first nanotube comprises at least one element selected from the group consisting of carbon, boron, boron nitride, and carbon boron nitride, silicon, germanium, gallium nitride, zinc oxide, indium phosphide, molybdenum disulphide, and silver.

5. The nanotube sensor of Claim 1, wherein the first nanotube comprises a single-wall carbon nanotube.

6. The nanotube sensor of Claim 1 , wherein the conducting elements comprise metal electrodes.

7. The nanotube sensor of Claim 1 , wherein the conducting elements are in direct physical contact with the first nanotube.

8. The nanotube sensor of Claim 1 , further comprising a gate electrode in proximity to the nanotube.

9. The nanotube sensor of Claim 1 , further comprising a layer of inhibiting material covering regions of the sensor adjacent to the connections between the conductive elements.

10. The nanotube sensor of Claim 1 , wherein the nanotube further comprises a two-dimensional nanotube network disposed over the substrate between the two conducting elements.

11. The nanotube sensor of Claim 10, wherein the nanotube network comprises a plurality of randomly-oriented carbon nanotubes.

12. The nanotube sensor of Claim 10, wherein the two conducting elements comprise a pair of interdigitated electrodes.

13. The nanotube sensor of Claim 10, wherein the at least one single- strand DNA further comprises a plurality of identical DNA receptor strands distributed over the two-dimensional nanotube network.

14. The nanotube sensor of Claim 13, wherein the plurality of identical DNA receptor strands are directed attached to nanotubes of the two-dimensional nanotube network

15. A method for making a nanoelectronic sensor, comprising: disposing a film of nanotubes over electrodes on a substrate; depositing a solution of single-stranded DNA over the substrate, wherein the single-stranded DNA is configured as a complement to a target DNA sequence; and drying the solution to leave a deposit of the single-stranded DNA over the substrate.

16. The method of Claim 15, further comprising removing the deposit of the single-stranded DNA from the substrate except in a region between the electrodes.

17. The method of Claim 15, further comprising depositing the electrodes as a metallic deposit over the substrate.

18. The method of Claim 17, wherein the depositing the electrodes step further comprises configuring the electrodes to have a plurality of interdigitated fingers.

19. The method of Claim 15, further comprising forming a gate electrode configured to operate on a region between the electrodes.

20. The method of Claim 19, further comprising removing the deposit of the single-stranded DNA from the substrate except in the region between the electrodes.

21. The method of Claim 15, further comprising coating the electrodes with barrier material prior to the depositing step.

22. The method of Claim 15, further comprising preparing the solution of single-stranded DNA comprising an oligonucleotide dissolved in water.

23. A method for sensing a particular target polynucleotide sequence, comprising: exposing a solution to a nanoelectronic sensor, wherein the nanoelectronic sensor comprises a complementary polynucleotide for the target polynucleotide sequence attached to at least one nanotube in a region between conducting electrodes; and [00465] observing at least one electrical property of the nanoelectronic sensor during the exposing step.

24. The method of Claim 23, wherein the exposing step further comprises exposing the solution to the nanoelectronic sensor comprising a field- effect transistor.

25. The method of Claim 24, wherein the observing step comprises observing the at least one electrical property comprising a gate voltage.

26. The method of Claim 23, further comprising comparing an observed electrical property observed during the observing step to a corresponding property observed prior to the observing step.