JP2012247432A - Nanotube sensor device for dna detection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect a specific target DNA sequence quickly with high sensitivity.SOLUTION: 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 140. 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 and dried, and then the deposition is 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 provided which employ linker groups to attach ssDNA probes to the nanotube sensor device.

Description

  The present invention relates to a sensor for a specific DNA sequence, comprising a nanotube as an electronic transducer for DNA hybridization.

CROSS REFERENCE OF RELATED APPLICATIONS S. C. 119 (e), based on US Provisional Application No. 60/604293 (Filing Date: August 24, 2004) and US Provisional Application No. 60/629604 (Filing Date: November 19, 2004) The contents disclosed in the specifications of these applications also form part of the present specification.

  In addition, this application is filed with US patent application Ser. No. 10/345833 (filing date: January 2003) which claims priority based on US Provisional Patent Application No. 60/349670 (filing date: January 16, 2002). 16th) [Title of Invention: Electronic detection of biological / chemical causes using functionalized nanostructure; Publication number: 2003-0134433] The contents disclosed in the specifications of these applications also form part of the present specification.

  In addition, this application is filed with US patent application Ser. No. 10 / 704,066 (filing date: November 2003), which claims priority based on US Provisional Patent Application No. 60 / 424,892 (filing date: November 8, 2002). 7th) [Title of Invention: Electronic detection of biomolecules based on the use of nanotubes; Publication number: 2004-0132070 (publication date: July 8, 2004)] The contents disclosed in the specifications of these applications are also part of this specification.

2. Description of the Prior Art Since base sequences in polynucleotides encode genetic information, the decipherability of these sequences has contributed to many advances in biotechnology. This study identifies a number of important sequences related to medical conditions. For example, the BRCA gene is present in the body of women who usually suffer from breast cancer. Many techniques have been developed to scan tissue samples to see if certain important sequences are expressed in order to take advantage of such associations in medical tests. These techniques are disadvantageous in that they are expensive, slow and complex, making them unsuitable for routine medical examinations.

  In general, these techniques rely on the tendency of polynucleotides to form hybrids. The strands of short DNA (ssDNA) in solution readily bind to complementary strand DNA (cDNA) containing opposite bases that pair with each base in ssDNA. As a result of this binding, double-stranded DNA (dsDNA) is formed. dsDNA can be separated from ssDNA by processing. Thus, to scan a specific target sequence, the experimenter provides the appropriate cDNA as a probe sequence. When the target sequence is present in the sample, the target ssDNA hybridizes with the probe ssDNA to generate dsDNA. This hybridization can be detected by several methods.

  The first problem in this case is due to the fact that many methods for detecting this hybridization include a process of denaturing ssDNA in the sample before hybridization. Often, fluorescent molecules are bound to ssDNA. Such molecules, known as labels, allow the detection of ssDNA by optical instruments (eg, microscopes, spectrometers, etc.). Labeling is used to detect the DNA sample after hybridization. When the target sequence is present in the labeled sample, the labeled ssDNA is incorporated into the labeled dsDNA, so that the dsDNA can be detected using an optical instrument. The use of the optical detection method makes the above method simple, but the chemical reaction for labeling DNA is expensive and takes a long time. Detection methods that do not require labeling will significantly increase the utility of DNA scanning methods for routine medical testing.

  The second problem is due to the low sensitivity of traditional detection methods. Some methods of this type are sensitive to low concentrations of DNA, but they require a large number of DNA molecules as absolute numbers. In medical applications, usually only a few cells are available, so there are only a few target sequence DNA molecules present in the sample. This problem is ameliorated by the use of polymerase chain reaction (PCR), which can amplify the amount of target DNA by a factor of one million. However, as in the case of the labeling method, PCR is a complex chemical reaction, so that the cost required for the test is high and it takes time.

  Therefore, in this field, there is a demand for a technique that can detect a specific target DNA sequence quickly with high sensitivity and that does not use labeling or PCR.

  The present invention is an electronic sensor device for detecting a specific target sequence of a polynucleotide, which is a nanostructured element that reacts with the polynucleotide and acts as a detecting element (for example, single-walled and / or multi-walled carbon nanotubes) And / or a connectivity network structure containing the nanotubes). In the specific example detailed below, the nanostructured element contains carbon nanotubes, particularly a network of randomly arranged carbon nanotubes. In these examples, the nanotubes are modified by adsorption of ssDNA probe sequences prior to detection. No labeling of DNA is necessary. The present invention further provides a method of using the electronic sensor device.

  As used herein, the term “nanostructure” means a structure containing at least one sheet of crystalline material having at least one dimension less than 100 nm and having a graphite-like chemical bond. Such a structure is not particularly limited, and examples thereof include single-wall nanotubes, double-wall nanotubes, multi-wall nanotubes, and onion-like structures. The chemical component of such a crystalline substance is not particularly limited, and examples thereof include carbon, boron nitride, molybdenum disulfide, and tungsten disulfide.

  For simplicity, the nanostructures included in the examples detailed below are referred to as “nanotubes”. In the embodiment, an embodiment including one or a plurality of carbon nanotubes is preferable, and an embodiment including one or a plurality of single-walled nanotubes is more preferable. In another embodiment, another nanostructure may be contained in the nanostructure sensor without departing from the technical idea of the present invention.

  As used herein, the term “nanotube network” refers to a film of nanotubes disposed within a specific area on a substrate. The nanotube film contains at least one nanotube disposed on the substrate substantially parallel to the substrate. Such a film may contain a number of nanotubes oriented parallel to each other. Alternatively, the film may contain a number of randomly oriented nanotubes. The film may also contain a small number of nanotubes arranged in selected areas of the substrate, and the film contains a large number of nanotubes arranged in selected areas of the substrate. May be. The number of nanotubes arranged in the region of the substrate is called the density of the network. Preferably, the film contains a number of randomly oriented nanotubes, the density of which passes through the network from one side of a particular region of the network toward the other. High enough to flow (eg via nanotube-to-nanotube contacts).

The substrate is generally a flat object that includes an electrically insulating surface. The chemical composition of the substrate is not particularly limited, and examples include silicon oxide, silicon nitride, aluminum oxide, polyimide, and polycarbonate. In many embodiments described herein, the substrate comprises silicon oxide, SiO _2, and one or more layers present a SiN ₄ or the like on the silicon wafer or silicon chip, a film or coating.

  The nanotube network may be prepared by chemical vapor deposition (CVD) using traditional lithography, solvent suspension deposition, vacuum deposition, or the like. In this regard, reference is made to the following documents: US patent application Ser. No. 10 / 177,929 (corresponding to WO 2004/040671); No. 10/280265; No. 10/846072. Specification; Fu et al., Nano Letters (2004), Vol. 12, No. 12, pp. 2513-2517 ("Percolation in Transparent Conductive Carbon Nanotube Networks"). The disclosures of these documents are also part of this specification.

  The characteristics of the nanostructure element (eg, nanotube network) may be measured using a contact. The contact includes a conductive element arranged to be electrically connected to a nanostructure element such as a nanotube network. For example, the contacts may be placed directly on the surface of the substrate or may be placed on the nanotube network. The current flowing through the nanotube network may be measured using at least two contacts that are electrically connected to the network within a particular region of the nanotube network.

  In some embodiments of the present invention, additional conductive elements called gate electrodes or counter electrodes are disposed. That is, the conductive element is not electrically connected to the nanostructure element (eg, at least one nanotube), and is disposed such that an electrical capacitance exists between the Kate electrode and the nanostructure element. The

  In a preferred embodiment, the gate electrode is a conductive plane in the substrate below the silicon oxide. Examples of this type of nanotube electronic device include those described in the following documents: US Patent Application Nos. 10/656898 (filing date: September 5, 2003) and 10 / 704,066. (Application date: November 7, 2003) (The application was published as US 2004/0132070). The contents of these documents are also part of this specification. Nanotube resistance, impedance, transconductance and other properties may be selected or measured under the influence of variable gate voltage.

  In another preferred embodiment, the gate electrode is a conductive element in contact with a conductive liquid in contact with the nanotube structure. Examples of this embodiment are described in particular in the following document, the disclosure of which is also part of this specification: Bradley et al., Phys. Rev. Lett., 91, 218301. Page (2003).

  In another aspect, the capacitance of the network may be measured as an electric field is induced in the nanotube network relative to the counter electrode or gate electrode by applying a voltage to one or more contacts. As another or additional sensor signal for measuring one or more channel transconductance characteristics, by using appropriate circuit components to measure the channel capacitance relative to the gate electrode, a nanostructured channel (eg, It is convenient to use the power supply (and / or drain) and gate electrode of a transistor having a nanotube network. Other aspects set to optimize the measurement of capacitance or other characteristics are possible without departing from the spirit of the invention.

  The conductive element provides a connector to an electrical circuit for observing the electrical characteristics of the nanotube sensor. Any suitable electrical property may be the basis for the sensitivity of the sensor, such as electrical resistance, conductivity, current, voltage, capacitance, transistor on-current, transistor off-current, and transistor limitations. Examples include voltage. A person skilled in the art can easily observe and measure other electrical characteristics. Accordingly, the above exemplified electrical characteristics are not meant to limit the types of device characteristics that can be measured.

  In a preferred embodiment, the nanotube sensor device includes a transistor. The transistor has the highest conductivity, i.e. the highest conductivity measured using a given range of gate voltages, and the lowest conductivity, i.e. the lowest conductivity measured with a given range of gate voltages. . The transistor has an on-off ratio that is the ratio of the maximum conductivity to the minimum conductivity. In order to produce a highly sensitive chemical sensor, the on-off ratio of the nanotube transistor is preferably greater than 1.2, more preferably greater than 2, and most preferably greater than 10. It is a big value.

  For example, FIG. 1 illustrates a conductivity curve as a function of gate voltage (+10 V to −10 V) in a nanotube electronic device. At gate voltages less than about −5V, a relatively high conductivity occurs in the “on” curve portion 101, and at gate voltages greater than about 0V, the “off” curve portion 102 has a relatively low conductivity. Will occur. The on-off ratio for this device is about 100.

  As used herein, the term “DNA” refers to a polynucleotide. Examples of the polynucleotide include, but are not limited to, deoxyribonucleic acid, ribonucleic acid, messenger ribonucleic acid, transfer ribonucleic acid, and peptide nucleic acid. A distinctive feature of a polynucleotide is the sequence of nucleic acid strands and bases, each base chemically binding to a nucleic acid, and each base paired with an appropriate base on the matching sequence. Form.

  One skilled in the art can prepare other modified derivatives of polynucleotides having these distinct characteristics. Accordingly, the “single-stranded DNA” (hereinafter referred to as “ssDNA”) may be a single strand of deoxyribonucleic acid, ribonucleic acid or other such polynucleotides. The “complementary DNA” (hereinafter referred to as “cDNA”) may be any strand of the above-mentioned polynucleotide that is a single-stranded sequence complementary to the already mentioned single-stranded sequence.

  In certain embodiments, the present invention provides a nanotube sensor device comprising a nanotube network, one or more contacts, and ssDNA in contact with the nanotubes. Several methods are available for preparing ssDNA in contact with nanotyb. In one embodiment, solution ssDNA is mixed with suspension nanotubes according to the methods described in the following literature: Zeng et al., Nature Materials, 2003, No. 2, pages 338-342. The resulting solution contains nanotubes covered with ssDNA strands. By casting this solution on a substrate, nanotubes coated with ssDNA are deposited on the substrate. After placing the nanotubes, contacts are prepared using standard techniques of lithography and metal deposition. In a preferred embodiment, a nanotube network is placed on a substrate and then contacts are prepared. The resulting electronic device is brought into contact with a solution containing ssDNA. By removing the solution, the ssDNA coats the nanotube network without coating the substrate.

  In certain embodiments, an apparatus is provided in which ssDNA contacts the nanotubes directly without using an intervening linker molecule. Furthermore, the ssDNA contacts the nanotubes, but not the regions on the substrate that do not contact the nanotubes.

  The ssDNA in a particular sensor device may be selected to be a cDNA for a particular target sequence. The target sequence is a base sequence to be detected by the sensor device. The cDNA for the target sequence is known as the probe sequence. Once the target sequence has been identified, a certain amount of DNA with the probe sequence must be obtained. Various techniques are known for synthesizing DNA having a specific sequence and synthesizing DNA complementary to a predetermined sequence. Those skilled in the art have knowledge of these techniques. CDNA or other polynucleotides suitable for preparing probes specific for the desired target sequence are generally available from known commercial manufacturers associated with the biotechnology industry.

  The sensor device may be used by contacting the nanotube network with a solution containing the sample ssDNA. The network should be in contact with the solution for a time long enough for hybridization to occur. This contact time depends on the concentration of the sample DNA, the amount of the solution, the room temperature, the pH of the solution, and other variable factors. Those skilled in the art are familiar with the effects of these variables on DNA hybridization and will determine the appropriate contact time, solution composition, temperature and other conditions associated with hybridization without undue experimentation. Can be selected.

Various uses of the sensor device are disclosed.
In one embodiment, the initial measurement of the sensor device is performed by changing the gate voltage applied by a conductive plane that exists below the insulator of the substrate. The reticulate is then contacted with the solution containing the sample ssDNA for the time described above. After removing the solution, it is left for a time sufficient for the substrate to dry substantially. This standing time can be shortened by taking measures to accelerate the drying process. For example, dry air may be blown onto the substrate. After the substrate is dried, the sensor device is measured again by changing the gate voltage. Whether the dsDNA is present is confirmed by comparing the obtained measurement result with the first measurement result.

  In another embodiment, reference data is obtained by contacting the mesh with pure water. The initial measurement of the sensor device is performed by changing the gate voltage applied by a conductive plane that exists below the insulator of the substrate. Next, the network is brought into contact with a solution containing sample DNA in pure water. When the sample DNA contains the target DNA, hybridization occurs over time, and the measurement result of the obtained sensor device changes compared to the initial measurement result.

  In yet another embodiment, reference data is obtained by contacting the mesh with pure water. The first measurement of the sensor device is performed by changing the gate voltage applied by the conductive plane that exists below the insulator of the substrate. The network is then contacted with a solution containing the sample DNA in a buffer adjusted in terms of temperature, pH, dissolved species, etc. to promote hybridization. After hybridization is performed for a predetermined time, DNA that has not been hybridized and other deposits may be removed by washing the mesh. After washing, the network is brought into contact with pure water again and the measurement is performed again. If the sample DNA contains target DNA, the hybridization of this DNA results in a characteristic and measurable change in the sensor device compared to the initial measurement.

  In yet another embodiment, the baseline measurement is performed in the same buffer used for hybridization. The reticulate is then contacted with a solution containing sample DNA in a hybridization buffer. After hybridization, the measurement is repeated. If the sample DNA contains target DNA, the hybridization of this DNA results in a characteristic and measurable change in the sensor device compared to the initial measurement.

  In other embodiments, the mesh is contacted with a conductive liquid. Preferably, the conductive liquid is a buffer suitable for physiological fluids. Most preferably, the conductive liquid is phosphate buffer (PBS). The initial measurement of the sensor device is performed by changing the gate voltage applied by the conductive element in contact with the conductive liquid. The network is then contacted with a solution of sample DNA in the same conductive liquid. The sensor device is measured by changing the gate voltage while the network is in contact with the solution. When the sample DNA contains the target DNA, hybridization is performed over time, and the measurement result obtained for the sensor device changes compared to the initial measurement result.

  In yet another embodiment, an electronic sensor system includes a sensor platform having a substrate, one or more electrodes, a nanostructure element disposed adjacent to the substrate that is electrically connected to the at least one electrode, and And an electronic measurement circuit component connected to the electrode and configured to measure one or more electrical characteristics of the sensor platform. The electronic sensor system includes at least one detection probe that is operatively coupled to a sensor platform. The probe is (a) a linker group disposed in association with a sensor platform, the linker group connected to one or more members selected from nanostructure elements, substrates and electrodes; (b) A detection biomolecule having binding affinity for an analyte (polynucleotide); and (c) a binding connection between the linker group and the detection biomolecule.

  The detection biomolecule may include a species having selective affinity for the polynucleotide. Examples of such species include complementary polynucleotides, transcription factors and / or transcription promoters, synthetic variants and analogs thereof. In a preferred embodiment, the detection biomolecule comprises a detection polynucleotide having at least one nucleotide sequence that is at least partially complementary to the nucleotide target sequence of the analyte (polynucleotide). In the embodiment described in detail, the sensor system measures properties that are affected by the binding of the analyte (polynucleotide) and the probe by at least partial hybridization of the target sequence.

  It should be noted that the occurrence, rate and specificity of polynucleotide hybridization in all the sensor embodiments described above depend on various conditions. In each hybridization process, the binding energy of dsDNA can be measured by rigorous techniques. Such a measurement can be performed by increasing the temperature or changing the buffer to, for example, sodium hydroxide.

  Additional rigorous controls may contain various ionic components of the hybridization medium, such as sodium or magnesium ions. Alternatively, or in addition, the behavior of the polynucleotide may be affected by applying a voltage to the sensor element (eg, nanotube network) before, during and / or after the formation of the hybrid. For example, polynucleotides such as cDNA have a phosphate-based backbone that is generally ionized in the hybridization medium and carries a localized negative charge. A selectively charged sensor element may be used, for example, during incubation or rinsing to destabilize the SNP-mismatched probe hybrid relative to the corresponding hybrid of the corresponding fully matched probe. And may be used to provide strict or repulsive regulators.

  By making strict changes, it is possible to differentiate the binding of complete or incomplete complementary base pairs to strands. The change in the electrical properties of the nanotubes in response to a rigorous process allows in particular differentiation of single base mismatches (SNPs). Those skilled in the art will understand the hybridization conditions to adapt the operation of a particular embodiment of the sensor according to the invention in order to obtain a selectivity of sensitivity to complete and incomplete hybridization of the target sequence. Can be changed.

  For example, in an assay to distinguish between a DNA sample that is homozygous for a particular allele and other comparative samples that are heterologous to that allele, the exact conditions of hybridization, for example, change the temperature By making adjustments, the measurement response of the apparatus clearly different between the same-type sample and the different-type sample may be obtained.

  In each sensor embodiment having features of the present invention, these sensors may be constructed as an array (eg, an array of transistor sensors functionalized to a plurality of different target DNA fragments). . In this regard, reference is made to the following document: US Patent Application No. 10/388701 (Patent Publication US2003-0175161) (Title of Invention: Correction of Detection Sensitivity of Nanostructure Device Array). The contents of this document are also part of this specification.

  A more thorough understanding of the nanotube sensor device by those skilled in the art and the realization of further advantages and objects of the device will be made by reference to the following detailed description of the preferred embodiments.

The accompanying drawings will be briefly described.
FIG. 1 is a schematic diagram illustrating an exemplary conductivity curve for a nanotube transistor device.
FIG. 2 is a schematic diagram showing an exemplary configuration of a nanotube sensor using a random network of nanotubes.
FIG. 3 is a schematic cross-sectional view of the exemplary nanotube sensor shown in FIG.
FIG. 4 is a flowchart illustrating exemplary steps of a method of manufacturing a nanoelectronic sensor according to the present invention described in Example A.
FIG. 5 is a flowchart showing exemplary steps of a polynucleotide detection method according to the present invention.
FIG. 6 is a chart showing conductivity as a function of gate voltage for nanotube electronic devices in three environments, which will be further described in the detailed description of the preferred embodiment below.

FIG. 7A shows the device characteristics of the sensor described in Example B after being subjected to a functionalization process using pyrene-DNA conjugates and a process using cDNA.
FIG. 7B shows the device characteristics of the sensor described in Example B after being subjected to functionalization treatment using pyrene-DNA conjugate, treatment using SNP-DNA, and treatment using cDNA.
FIG. 8A illustrates an embodiment of an exemplary DNA assay that uses a detection probe coupled to a sensor, according to certain aspects of the invention.
8B-8F illustrate an embodiment of another DNA assay using an electronically active incalator, according to certain aspects of the invention.
9A-9D illustrate an embodiment according to a particular aspect of the present invention, illustrating another DNA assay embodiment using an amplification group. FIGS. 9E and 9F are embodiments according to a particular aspect of the present invention. FIG. 5 illustrates another DNA assay embodiment using antibody-antigen binding that binds a detection probe to a sensor.
10A-10D illustrate a sensor having two alternative structures according to certain aspects of the present invention, wherein the detection probe is bound to a nanostructure such as a nanotube.
FIGS. 11A-11C illustrate a sensor having two alternative structures according to certain aspects of the present invention, wherein the detection probe is coupled to a sensor substrate.
12A and 12B illustrate a sensor having two alternative structures according to certain aspects of the present invention, wherein the detection probe is coupled to a sensor electrode.

In the following, preferred embodiments of the invention will be described in more detail.
In certain embodiments, a nanotube sensor device for detecting a target DNA sequence is provided. This device does not require labeling of the target DNA and responds electronically to the presence of the target DNA. In the following detailed description, numerals indicating the same elements are used to indicate the same elements appearing in one or more drawings.

  2 and 3, the nanotube DNA sensor 100 according to the present invention may include a suitable substrate 140, such as a degenerately doped silicon wafer. Other substrates include, for example, other semiconductors or insulating substrates such as ceramic or polymer. The substrate 140 may be passivated with a silicon oxide film 180 as is known in the art.

If desired, the gate electrode 170 may be formed in the lower layer of the substrate and connected to the contact 176 via a suitable conductor 175. Alternatively, the substrate may contain a conductive substrate (eg, doped silicon, etc.) covered by an insulating layer (eg, SiO ₂ layer) (in this case, the conductive substrate is connected to the circuit). And function as a gate electrode or a counter electrode).

  In the illustrated embodiment, a randomly arranged nanotube network 120 is disposed on top of a silicon substrate 140 and the device includes a pair of contacts having interleaves disposed on top of the network 120. 101 and 110 are included. The mesh provides a conductive groove between the contact pairs. The substrate 140 outside the generally rectangular region 130 is substantially free from the nanotube network 120.

  In another embodiment, one or more nanotubes are disposed adjacent to the substrate, and the nanotubes are in electrical connection with the one or more contacts. In some embodiments, most or all nanotubes may extend in a conductive manner between a pair of adjacent contact pairs.

  However, in the illustrated embodiment, in a randomly arranged and interconnected nanotube network 120, not all or the majority of nanotubes need be in electrical contact with one or more electrodes. The contacts between the nanotubes may provide a conductive path that allows transmission of current or charge through the network.

  In the illustrated embodiment, the contacts 101 and 110 are located on top of the mesh 120. Alternatively, the contact point may be disposed on the substrate 140, and the mesh 120 may be formed on the contact point.

  One or more contacts 101 and 110 may be disposed, and the contacts may optionally have a passive coating layer 180, as is known in the art. For example, the contacts 101 and 110 may include one or more metal layers (eg, a titanium layer and a gold layer).

  The contacts 101 and 110 may include a plurality of alternating portions disposed on top of a generally rectangular region 130. This interleaved structure is advantageous in that it increases the surface area of the contacts that can contact the nanotube film between the contacts. Other arrangements of contacts may be used, for example, a suitable parallel labyrinth having any desired form or other arrangement that provides a sensor area between opposing contacts. The rectangular shape of region 130 is merely exemplary, and the region may have any desired shape. The contacts 101 and 110 may have a structure that functions as a power source or drain electrode for a field effect transistor, or a structure that functions as a connection member to a resistance sensor or a capacitance sensor. You may do it. In certain embodiments, a single contact (eg, 101) may be used to provide the sensor signal by inducing the capacitance or electric field of the mesh 120 relative to the gate electrode or other counter electrode. Good.

  The portions other than the contacts (101 and 110) and the contacts of the substrate 140 may be protected by the blocking material 160. For example, an epoxy resin or other suitable polymer material or resin material may be deposited to form a blocking layer, and then the blocking layer in the region between the contacts 101 and 110 facing each other may be removed by etching or the like.

  The plurality of single-stranded DNA molecules 150 may be arranged on the nanotube film by any appropriate method (for example, a method described below). The DNA molecules may be attached directly to the nanotubes in the nanotube film, or may be placed on top of the substrate 140 proximate to the nanotubes in the nanotube film. In another embodiment, the DNA molecule may be placed on the inclusion between the nanotube membrane and the DNA. However, the DNA should be placed sufficiently close to the nanotube membrane so that the reaction between the ssDNA strand and the complementary ssDNA strand affects the measured electrical properties of the sensor 100.

  In one embodiment of the invention (see Example A), the ssDNA is in direct contact with the nanotube without the use of an intervening linker molecule. Furthermore, the ssDNA contacts the nanotubes but not the substrate region that does not contact the nanotubes. The ssDNA molecule 150 may be removed from the substrate (140) region other than the upper part of the nanotube film 120.

  The ssDNA in a particular sensor device is selected to be cDNA for a particular target sequence. The target sequence is a base sequence to be detected by the sensor device. The cDNA for the target sequence is known as the probe sequence. Once the target sequence has been defined, a significant amount of DNA with the probe sequence must be obtained. Various techniques are known for synthesizing DNA having a prescribed sequence and DNA complementary to a predetermined sequence. Those skilled in the art have knowledge of this type of technology. In addition, cDNA is often available as a commercial product.

  It should be noted that after a plurality of nanotube sensors similar to sensor 100 are formed in parallel on a single substrate, each sensor may be separated. The device to be separated allows detection of the target polynucleotide by incorporating it into a chip carrier as known in the art and then integrating with a conventional electronics device. Useful measuring devices may be manufactured. By incorporating multiple sensors in the electronic device that sense different sequences, various polynucleotide sequences may be detected simultaneously. In accordance with the disclosure herein, a suitable electronic device for a detection instrument may be manufactured by using any conventional technique.

  FIG. 4 shows exemplary steps in a method 400 for manufacturing an electronic sensor for a specific DNA sequence. Steps 410 to 490 may be performed in any operation order.

  In step 410, the gate electrode may be formed on top of a substrate (eg, a passivated silicon or other semiconductive substrate) or a semiconductive substrate such as a ceramic or polymer material. The electrode may contain a metal or other conductive material, and the electrode may be obtained by photographic printing and lift-off methods known in the art, or other suitable methods. It may be formed. In a particular embodiment, the gate electrode contains a bulk silicon substrate wafer material connected to a suitable circuit.

  In step 420, the substrate (and the buried gate electrode when a gate electrode is included) may be coated with a passivating layer or insulating layer, for example, a silicon oxide layer as known in the art.

  In step 440, one or more nanotubes are placed in the substrate in electrical connection with each opposing contact. For example, the substrate 140 may be coated with carbon nanotubes that are randomly reticulated as described in previously referenced US patent application Ser. No. 10 / 177,929. Alternatively, other methods known in the art may be used to form nanotubes between the contacts. The resulting nanotubes may be arranged in a specific manner or randomly. In the case of a random arrangement, the nanotubes should provide a network of connected nanotubes that connect opposing contacts via at least one path. The nanotubes should be removed from the substrate by any suitable method (eg, plasma etching) in regions other than between the contacts that face each other.

  In step 430, a pair of opposing contacts (eg, a power source and a drain electrode) may be formed on the substrate. The contacts may be placed above the nanotubes, or may be placed between the nanotubes and the substrate. For example, a counter contact may be formed by forming a titanium contact and coating the contact with a gold layer using a photographic printing method and a lift-off method. Such contacts may include a plurality of interleaved portions disposed on the intermediate region having any desired form.

  In step 450, if desired, a layer of barrier material may be disposed on the contact. Various polymers and resins are known in the art, and these may contain suitable barrier materials. In one embodiment of the invention, an epoxy coating may be used. The blocking material may be applied only to a specific area of the substrate, or after the blocking material is applied to the entire substrate, the blocking material is removed from the area corresponding to the sensor operation area (for example, the area between the contacts) Also good. The barrier may provide electrical insulation, thereby preventing a short circuit when in contact with the conductive fluid, or protecting the sensor from exposure to the environment. The blocking material may help to control the deposition of other materials, including but not limited to nanotubes and DNA molecules. Any number of barrier layers may be used.

In step 460, a solution of oligonucleotide (ssDNA) may be prepared. The desired ssDNA (“probe sequence”) may be obtained from a commercial product, or may be synthesized by methods known in the art. An aqueous solution or an organic solvent solution of the probe array may be prepared at an appropriate concentration. For example, a 10 ⁻⁴ M concentration solution may be prepared by dissolving 100,000 picomoles of oligonucleotide in 1000 μL of pure water (18 MΩ). Other solvents compatible with ssDNA may be used. Prior to the deposition of ssDNA, the electrical characteristics of the sensor device may be recorded as appropriate.

  In step 470, the oligonucleotide solution may be applied onto the active area 130 of the sensor device. For example, a droplet of DNA solution may be placed on a chip on region 130. The solution may then be dried and the carrier evaporated to leave the ssDNA intact. For example, the chip may be left to dry in a humidified chamber at room temperature. The chip is then removed from the chamber, rinsed with pure water (18 MΩ), and air-dried with a stream of dry nitrogen.

  In step 490, excess ssDNA may be removed. This removal process may be performed by the rinsing process and the air drying process as described above. When excess ssDNA is attached to other areas of the substrate, a more powerful method (eg, an etching method) may be used. Alternatively, excess DNA may be left as is if it does not interfere with sensor operation.

  The electrical characteristics of the sensor device may be measured again and compared with the above reference characteristics. If ssDNA is successfully deposited, a change in electrical properties should be observed. Properties that may be measured include, for example, the sensor's gate voltage, conductivity, resistance, or combinations thereof, curves or hysteresis associated with these or other electrical properties, and the like.

  FIG. 5 illustrates exemplary steps of a method 500 of using a sensor device according to the present invention. The use of the sensor essentially consists of measuring the change in the electrical properties of the sensor after exposing the solution containing the sample ssDNA to the nanotube network. In step 510, the sample is prepared by methods known in the art. For example, DNA may be extracted from a patient's cells by lysis. Double stranded DNA should be reduced to ssDNA by methods known in the art. If a sufficiently large amount of DNA sample is available, it is possible to avoid using the PCR method to increase the concentration of DNA. Since the sensor according to the present invention can be operated with a remarkably small volume (for example, less than 100 μL), the use of the PCR method may be omitted in some cases.

  In step 520, the sensor is exposed to the sample solution. The sensor standing time in the solution should be sufficient for hybridization to occur between at least one ssDNA molecule on the nanotube network and a complementary ssDNA molecule in solution. . This standing time depends on the concentration of the sample DNA, the amount of solution, the room temperature, the pH of the solution and other variables. One skilled in the art is familiar with the effects of these variables on DNA hybridization and can select an appropriate standing time.

  In step 530, the electrical response of the sensor is measured. Various different characteristics are available depending on the structure of the sensor. In one embodiment, the initial measurement of the sensor device is performed by changing the gate voltage applied by the conductive plane present under the insulator of the substrate. The network is then exposed to the solution containing the sample ssDNA for the time period described above, after which the solution is removed and left for a time sufficient for the substrate to substantially dry. This standing time may be shortened by taking measures to accelerate the drying process. For example, dry air may be blown onto the substrate. When the substrate is dry, the sensor device is measured again by changing the gate voltage. The presence or absence of dsDNA is confirmed by comparing the obtained measurement result with the first measurement result.

  In another embodiment, the network structure is exposed to pure water. An initial measurement of the sensor device is performed by changing the gate voltage applied by the conductive plane present under the insulator of the substrate. Next, the network structure is exposed to a solution containing sample DNA in pure water. While the network structure is exposed to the solution, the sensor device is measured by changing the gate voltage. When the sample DNA contains the target DNA, hybridization occurs over time, and the result obtained in the sensor device changes compared to the initial measurement result.

  In yet another embodiment, the network is exposed to a conductive liquid. Preferably, the conductive liquid is a buffer suitable for physiological liquids, and the most preferred conductive liquid is phosphate buffer (PBS). An initial measurement of the sensor device is performed by changing the gate voltage applied by the conductive element in contact with the conductive liquid. The network is then exposed to a sample DNA solution in the same conductive liquid. While the network structure is exposed to the solution, the sensor device is measured by changing the gate voltage. When the sample DNA contains the target DNA, hybridization occurs over time, and the measurement result obtained for the sensor device changes compared to the initial measurement result.

  In step 540, a positive or negative result is determined by correlating the measured electrical response with the target species. For example, in a genetic test, the presence or absence of a target sequence is determined. The reaction between the sensor and the target gene should provide consistent and reproducible results with a given type of sensor. Thus, for the same type of sensor, positive or negative results and confidence may be based on a comparison of the response of a particular sensor with statistical control data. Reliability in the results may be increased by multiple measurements using multiple sensors simultaneously.

Example A
As described in previously referenced US patent application Ser. No. 10 / 177,929, a degenerately doped silicon wafer having a silicon dioxide film can be formed into a random network of carbon, generally as described above. Covered with nanotubes. A counter contact was formed by depositing a titanium contact (thickness: 30 nm) coated with a gold contact (thickness: 120 nm) by photographic printing and lift-off to form a pattern. Each of these contacts includes a plurality of interleaves disposed over a generally rectangular region. A network of randomly arranged nanotubes is placed over the silicon substrate. The nanotubes in the network structure are in electrical contact with the alternating contacts. After deposition of the contacts, the nanotubes outside the generally rectangular region are removed by oxygen plasma etching, leaving the remaining nanotube network. In general, the interleaving of a network of nanotubes and metal electrodes interleaved between interleaved contacts results in a large number of nanotubes connected in parallel across the electrodes.

  The die is separated from the wafer and placed in a standard 40-pin chip carrier, with the wires interleaved on the chip and the contacts on the chip carrier connected by wires. The wire and contact pads on the package are coated with epoxy resin and the resin is cured. The chips in the package thus prepared were rinsed with acetone, isopropanol and deionized water, and finally rinsed with pure water (18 MΩ).

A 10 ⁻⁴ M solution of an oligonucleotide (5′-CCT AAT AAC AAT-3 ′) was prepared by dissolving the oligonucleotide 84500 pmol in 845 μL of pure water (18 MΩ) (manufactured by Nanopure Infinity UV Water System). did. The chip prepared as described above was measured by changing the gate voltage applied by the conductive surface under the insulator. The resulting curve is shown at 600 in FIG. A droplet containing 20 μL of DNA solution was placed on the chip. The chip was left in a humid chamber at room temperature for 12 hours. Next, the chip was taken out from the wet chamber, rinsed with pure water (18 MΩ), and dried by blowing dry nitrogen gas. The chip was measured by changing the gate voltage. The resulting curve is shown at 610 in FIG. This curve shows the characteristics of a sensor device prepared for use as a sensor. At this stage, the nanotube network is in contact with ssDNA having a probe sequence. Curve 610 shifts to the left of curve 600 due to the effect of the ssDNA coating on the electronic measurements.

To prove contact between the nanotubes and the probe ssDNA, a chip was prepared using labeled ssDNA. Labeled ssDNA is not necessary in the preferred embodiment, but is described here for demonstration purposes only. A 10 ⁻⁵ M solution (solvent: 18 MΩ pure water) of oligonucleotide (5′-HS- (CH ₂ ) ₆ -CCT AAT AAC AAT-fluorescein-3 ′) was prepared as a receptor DNA sequence. The chip was exposed to this solution overnight, subjected to a rinsing treatment and then dried using nitrogen gas. When an optical fluorescence micrograph of this chip was observed, a green fluorescein label appeared as a bright area only in a specific area where the nanotube network was present, but the label did not appear in other areas of the substrate. It was. This proves that the receptor DNA strand is bound to the sensor nanotube.

Next, a solution of the oligonucleotide 5′-ATT GTT ATT AGG-3 complementary to the target DNA having a concentration of 10 ⁻⁴ M, that is, the receptor DNA strand, was added to the oligonucleotide 132000 p mole with pure water (18 MΩ). Prepared by dissolving in 1320 μL. Using this solution, a diluted solution of target DNA (10 ⁻⁸ M) was prepared. The chip was exposed to a drop of this solution (20 μL) for 1 hour in a humidified chamber at room temperature. The chip was taken out from the chamber, rinsed with pure water (18 MΩ), and dried by blowing dry nitrogen.

  The resulting curve is shown at 620 in FIG. This curve shows the result of hybridization with probe DNA and target DNA. Curve 620 shifted to the right of curve 600 due to the effect of target DNA hybridization in the electronic measurement.

Example B
In this example, non-covalent chemical functionalization of a carbon nanotube device for detecting single base mismatched DNA is described.
B-1: Overview In one embodiment according to the present invention, a nanotube sensor comprises a carbon nanotube network field effect transistor (“NTFET” or “NTNFET”) functionalized by single-stranded DNA (ssDNA). . In certain embodiments, ssDNA may be immobilized to the NTFET device via polymers and polyaromatic molecules non-covalently bound to carbon nanotubes. Significant differences in electronic response to complementary single stranded DNA (cDNA) and single base mismatched single stranded DNA (sbmDNA) may be measured. This exemplary sensor includes the following structures, elements and functions.

a) one or more carbon nanotube FET devices comprising at least a single nanotube and / or a network of nanotubes arranged such that a conductive trench is formed between a power source and a drain electrode The device.
b) The FET structure may have a bottom gate electrode and / or a liquid gate electrode.
c) The polymer and / or aromatic linker molecule is non-covalently bonded to the carbon nanotube.
d) ssDNA chemically binds to the linker molecule to form a probe.
e) In operation, when complementary cDNA is exposed to the sensor, the DNA hybridizes with the probe, which has a measurable effect on the electrical properties of the device.
f) When single base mismatched sbm DNA is exposed to a sensor, the DNA hybridizes with the probe, which results in different instrumental properties in measurement.

  NTNFET devices were prepared according to the procedures described in the prior patent literature, particularly US patent application Ser. Nos. 10 / 177,929, 10/656898, and 10 / 704,066. The contents of these patent documents are also part of this specification. Current is an electrical property that can be measured using a contact. The contacts have conductive elements that may be disposed on the substrate, and the conductive elements are electrically connected to the nanotube network.

  In some embodiments of the present invention, an additional conductive element (gate electrode) is disposed, the electrode not electrically connected to at least one nanotube, and at least one of the gate electrode and the electrode. There is an electrical capacitance between the nanotubes. In one preferred exemplary embodiment, the gate electrode is a conductive surface in the underlying substrate of silicon oxide. Examples of this type of nanotube electronic device are described in the aforementioned US patent application Ser. Nos. 10 / 656,898 and 10 / 704,066.

  The sensor NT device may be formed on a 100 mm wafer, for example, by standard, standard photographic printing methods. The NTFET device may be manufactured using SWNTs grown by chemical vapor deposition (CVD) at 900 ° C. using dispersed iron nanoparticles (growth promoter) and methane / hydrogen mixed gas. The electrical lead was formed in a pattern from the titanium film (thickness: 30 nm) covered with the gold layer (thickness: 120 nm) to the top of the nanotube. After the initial electrical measurements were made to determine the characteristics of the device, the substrates were connected with wires and housed in a 40-pin CERDIP package before performing the DNA experiments. The wires and contact pads on the package were coated with epoxy resin and the resin was cured. DNA experiments were performed by dropping a drop of DNA solution onto the package. The package was housed in a sealed jar containing a beaker containing approximately 100 mL of water to prevent evaporation of the droplets.

  The measurement of the electronic characteristics of the NTFET device, for example the measurement of the current between the S / D electrodes as a function of the applied gate voltage, was performed using a simultaneous measurement system (PMS). When using this system, it is possible to simultaneously measure the device properties of up to 12 sensors based on nanotubes. A set of 32 independent analog switches is digitally adjusted via the PC, whereby the contact to be measured is selected by the user. The applied power supply-drain bias and gate voltage are defined by the user (amplitude, frequency, function). By using this system, the conductivity of the device can be measured as a function of time and gate voltage.

B-2: Preparation Procedure B-2.1 Chip Preparation Before using each chip, the chip was subjected to packaging processing, then the wires and contacts were coated with epoxy resin, and the epoxy was cured. The tip is rinsed with acetone, isopropanol, deionized water from a jar, and subjected to a cleaning process according to the last defined cleaning method (see section B-2.2 below), followed by initial I- A Vg curve was obtained.

B-2.2 Cleaning Procedure The specimen on the surface was removed by simply cleaning the packaged chip by spraying with pure water (18 MW). In the crystallization dish, about 50 ml of salt solution (pH: 7.4 / 25 ° C.) buffered with 0.01 M phosphate was poured onto the chip. The chip was washed for 5 minutes on an orbital shaker (speed setpoint: 6) and then the solution was discarded. Further, the chip was washed four times with 18 MW of water by the same operation.

B-2.3 I-Vg Curve I-Vg curves (curves plotting NTFET current against scan gate voltage) were obtained for all devices on the chip, but are shown here for only one of each chip. . The curves shown in each case should be considered representative of the curves obtained for each chip.

B-3 Pyrene-labeling and DNA
B-3.1 Formation of Pyrene Single Layer After the packaged chip (in this case W517 26:21) was cleaned, initial I-Vg measurements were performed. A 2.5 mg / ml solution of pyrenebutanoic acid succinimidyl ester using N, N-dimethylformamide (DMF) as a solvent was dissolved in 3.032 mg of the pyrene derivative in 1.232 ml of DMF. 50 ml of this solution was present on the surface of the chip, and the chip was sealed for 2 hours at room temperature inside a chamber equipped with an open container containing DMF in order to prevent generation of droplets due to evaporation. The chip was then removed, rinsed with DMF, acetone and isopropanol and subjected to a cleaning process (see section 2.2) to obtain an I-Vg curve.

B-3.2 Covalent Binding of DNA 20 ml of DNA-NH ₂ solution is present on the surface of the chip, and then the chip is moistened and prevented from generating droplets due to evaporation. The inside of the chamber provided with the open container containing water was sealed overnight at room temperature. The chip was removed and washed according to the washing procedure, and then an I-Vg curve was obtained.

B-4 Detection of DNA Hybridization B-4.1 Detection Procedure Dilute a 10 ^-4 M solution of DNA oligonucleotides with a 0.01 M salt solution (pH: 7.4 / 25 ° C) buffered with phosphate. As a result, a 10 ⁻⁶ M solution of the oligonucleotide was prepared. 20 ml of this DNA solution was present on the surface of the chip functionalized with a DNA-pyrene layer according to section B-3. The chip was sealed overnight at room temperature in a chamber with an open container containing water in order to apply moisture and prevent the generation of droplets due to evaporation. After taking out the chip and subjecting it to a cleaning treatment, an I-Vg curve was obtained.

B-4.2 cDNA
The chip (W517 26:21) was functionalized according to section B-3 and then processed with cDNA according to section B-4.1.

  FIG. 7A shows the I-Vg curve, which is shifted to the right, indicating that the device can detect the hybridization of covalently bound DNA and cDNA. The shift to the right of the curve is consistent with the shift observed in previous experiments where double stranded DNA was present.

B-4.3 SNP-DNA
The chip (W 517 26:24) was functionalized according to section B-3 and then treated with SNP-DNA according to section B-4.1.

  FIG. 7B shows an I-Vg curve, and the degree of shift to the right side of the curve is not very large. This may be due to partial and incomplete binding of the SNP-DNA to the DNA bound to the device or due to the SNP-DNA being washed away during the washing process. This is because this degree of shift has been shown to be related to drift (which may be due to a very thin layer of water adsorbed on the nanotubes). In any case, it is clear that the device can distinguish between cDNA and SNP.

B-4.4 SNP-DNA + cDNA
A chip pre-treated with SNP-DNA (W 517 26:24) was treated with cDNA according to section B-4.1.

  FIG. 7B shows the I-Vg curve, which is shifted to the right, which is similar to the shift seen when using cDNA in section B-4.2. This indicates that the device can detect cDNA after exposure to SNP-DNA. If the SNP-DNA is not washed away in section B-4.3, the cDNA will replace the SNP-DNA, thereby obtaining for hybridization in section B-4.2 or other parts of the specification. Results that are consistent with the captured data.

  NTFET, a nanoscale electronic device, may be used for real-time monitoring and detection of small amounts of nucleic acids (RNA and DNA). For DNA oligonucleotide hybridization assays, a small amount of single-stranded DNA (ssDNA) can be detected using the NTNFET device. Such assays can be performed quickly and with a higher sensitivity than conventional methods, and, for example, reduce the number of cycles required for DNA duplication or eliminate the need for PCR.

  Hybridization occurs when a single mismatched base is present between the two DNA strands, but the stability of the hybridization complex with the kink due to mismatch is poor. Such incompatibility is called single nucleotide polymorphism (SNP), which was discovered as a result of the Human Genome project. SNPs are key targets for commercial genetic testing and can potentially be identified by NTNFET devices.

Example C
DNA Assays Using Nanoelectronic Devices A number of different exemplary DNA or other polynucleotide assay embodiments having features of the invention are shown in FIGS. The structures and methods described below are exemplary, and in other embodiments, structures and methods described elsewhere in this specification may be used. In the different embodiments, where substantially similar elements are included, the same reference numerals are used to denote the elements in the description of each embodiment.

C-1 Structure As shown in FIGS. 8-12, the sensor 10 includes a platform having at least one nanostructure. Examples of the nanostructure include a nanotube 12 that is disposed adjacent to the substrate 14 and electrically connected at least between the power supply electrode 16 and the drain electrode 18.

  If desired, the device may comprise at least one additional electrode, for example a gate electrode 20 disposed adjacent to the nanotube 12. The illustrated gate electrode 20 is embedded in the substrate 14, but as described above in connection with other NTFET sensor embodiments, other types of electrodes (eg, bottom gate electrode, top gate electrode, and / or Alternatively, a liquid gate electrode or the like) may be arranged by another installation method.

  In FIGS. 6-9, a single nanotube 12 is schematically shown in a simple end contact manner with electrodes 16 and 18, but as described above without departing from the technical spirit of the present invention. Thus, another nanostructure installation mode may be employed.

  10A and 10B schematically show another structure. FIG. 10A shows a “island” of multiple conductors interconnected by nanotubes. FIG. 10B shows an embodiment of a nanotube network structure, in which a plurality of nanotubes form a network or film of nanotubes that are in communication with each other, thereby conducting between the power electrode and the drain electrode. Sex grooves are provided. In this type of nanotube film, each nanotube does not require a span between the power electrode and the drain electrode, and the conductive groove is one or more via a plurality of nanotubes connected in series with each other. Multiple grooves or paths may be included. Preferably, the density and / or composition of this type of nanotube network is selected by tailored formation and / or post-formation modifications to achieve the desired conductivity and sensor sensitivity. If desired, a plurality of power supplies and / or a plurality of drain electrodes may be included, for example, a series of electrodes interdigitated with such electrodes. The nanotubes 12 may be placed on the bottom and / or side and / or top of the electrode.

  The nanotube film may be directly formed on the substrate by, for example, CVD or solution deposition method mediated by a nano-dispersed catalyst. Alternatively, a nanotube film may be prepared separately and deposited as a separate step directly on the substrate 14 including the film carrier layer. In this regard, reference is made to the aforementioned US patent application Ser. Nos. 10/177929 and 10/846072. The substrate 14 may be a hard structure (for example, a semiconductor wafer, single crystal silicon, or polycrystalline silicon), or may be a flexible body (for example, a polymer sheet or web). Should be noted. The nanotube film may be tuned in the context of electrodes 16 and 18 by selectively removing a portion of the nanotube film from a portion of the substrate.

  Similarly, contacts or electrodes (16 and 18) and / or gate electrodes or additional electrodes may be deposited or formed before or after nanotube 12 is formed. If desired, additional electronic circuitry may be integrally formed with the sensor 10 on the substrate 14 for signal processing and the like. In the manufacture of the sensor 10 and the desired device, known methods for manufacturing devices and layers of integrated electronic circuits (eg, CVD, vacuum deposition, photo printing, masking, chemical etching, spin coating, substrate doping, Substrate oxide formation and substrate nitride formation may be employed.

  Similarly, the illustrated sensor may be included in an integrated array of sensors as described above. If desired, in connection with other sensor and NTFET aspects (eg, contact passivation, dielectric and / or catalyst-carrying layers covering the substrate, and nanostructured hydrophobic coating layers, etc.) It should be noted that the described enhancements and other elements may be included in the embodiments described below.

C-2 Detection Probe As shown in FIGS. 8-10, the sensor comprises a detection probe (e.g., probe 22), which has a linker group, e.g., linker 26, which is a nanotube 12 (Preferably non-covalently linked), thereby binding the probe to the sensor 10. cDNA 24 binds to linker 26 at one site (preferably covalently bound). In this case, the cDNA also has an exposed complementary base sequence extending outward from the linker 26. The linker is a molecule or group having a structure that binds non-covalently to nanotube 12 and covalently to cDNA 24 (eg, an aromatic molecule and / or polymer such as pyrene). ).

The linker group may be attached to more than one cDNA, or conversely, the cDNA may be attached to more than one linker group depending on the properties and conformation of the linker. For example, a liner group comprising a distributed polymer layer may have a plurality of cDNA molecules attached to different points of the polymer layer.

  In this regard, attention should be paid to the definition of dsDNA, ssDNA, cDNA and other nucleotide species described in the summary of the invention in this specification.

  In another specific embodiment, the cDNA need not be a deoxyribose polynucleotide, but other target-specific polynucleotide species (eg, RNA,) that result in at least partial hybridization with a selected target sequence. And denatured or substituted DNA, etc.).

  Similarly, the target “ssDNA” molecule need not necessarily be a discrete, fully denatured deoxyribose polynucleotide chain, but may include RNA, dsDNA, partially denatured dsDNA, species with sticky ends, and the like. . In this case, the target molecule comprises a target nucleotide sequence that results in at least partial hybridization with the probe cDNA.

  In the embodiment shown in FIG. 8A, a probe 22 for detecting a single-stranded fragment of DNA 30 by hybridization with a target base sequence 32 is illustrated. By coupling a suitable sensor circuit (not shown in FIGS. 8-12) to sensor 10, the electrical response of sensor 10 to DNA 30 hybridization is described above in connection with another sensor embodiment. Detected and / or quantified by the determined method. For example, the conductivity between the power supply 16 and the drain 18 changes due to hybridization and this change is measured. Alternatively, in the NTFET DNA sensor embodiment, DNA 30 hybridization results in a shift in device characteristics of the sensor 10 that occurs when the voltage at the gate electrode 20 changes within a selected voltage range. In order to detect hybridization, an additional characteristic or another characteristic of the sensor 10 may be measured.

  In certain applications according to aspects of the present invention, a relatively complete hybrid match between cDNA 24 and a selected target sequence and partial discontinuities and / or loops as opposed to this of the target sequence Sensor 10 may be used to discriminate between the hybridization. The sensor 10 is electrically responsive to this hybridization phenomenon and provides a signal characteristic that reflects the degree and / or characteristics of hybridization of the probe cDNA 24 to the target sequence 32. For example, a signal resulting from partial hybridization of a sequence having a single base mismatch (sbmDNA) to the corresponding probe sequence can identify complete matching sequence hybridization. This function of the sensor 10 provides in particular the properties of single nucleotide polymorphism (SNP).

  In this regard, the ssDNA is an RNA polynucleotide, hetero- or denatured polynucleotide, plasmid, viral fragment, double-stranded DNA fragment (eg, a binding end or other exposed strand target obtained for hybridization with probe 22). Part), partially annealed dsDNA fragments or oligonucleotides and the like.

  In an exemplary method according to the present invention, probe 22 may be prepared to match a selected target sequence 32, and cDNA is obtained by known methods. Commercial materials are available for conventional synthesis of oligonucleotides having a defined sequence, and related sequences are obtained by a number of known methods (eg, PCR, reverse transcription, plasmid amplification, etc.) and denatured. And / or can be amplified. In this regard, the cDNA may comprise a target sequence 32 (eg, a tail or head selected for binding to linker 26, a tail or head selected for purification, amplification and / or processing, It should be noted that nucleotide sequences may be included that are complementary to (and optionally labeling groups, etc.). Probe 22 may then be prepared by conjugating cDNA 24 to linker group 26 (eg, pyrene) by known reactions and methods (eg, the formation of pyrene's DNA-5'-amine).

  A pre-prepared sensor platform 10 is treated, for example, with a solution or suspension of probe 22 to link linker 26 to nanotube 12, for example, π-π deposition of pyrene molecules bound to the graphite lattice of nanotube 12 ( After bonding by stacking), it may be functionalized by subjecting it to a washing and drying process. The functionalized sensor 10 may then be used to detect ssDNA, which is an analyte having a target sequence 32 suspended in the sample medium. Appropriate calibration tests may be performed. This calibration test can be performed, for example, by exposing the sensor 10 to the same sample medium containing a known ssDNA lacking the target sequence 32.

  In another exemplary method of use, a pre-prepared platform of sensor 10 is linked to a linker group material 26 (eg, a polymer selected to react or bind to a portion of cDNA 24) target specific cDNA 24. And then the probe 22 may be prepared in situ by functionalizing the sensor 10 by binding of the cDNA 24.

  In another sensor embodiment (not shown) in accordance with aspects of the present invention, the array sensor system comprises a plurality of spaced apart sensors 10. This array may be pre-prepared as described above, and the sensor 10 may include a plurality of different probes (each probe having a cDNA specific to a particular selected target sequence). One may be functionalized separately. For example, an array having a preset functionalized pattern may be processed by using an ink jet coating method. Such multi-functionalized arrays may be used so that tests on multiple different target DNA sequences can be performed substantially simultaneously using a single analyte sample medium.

  By using a signal processing circuit having a known configuration, signals from an array of sensors 10 arranged in series, in parallel or in any desired pattern may be processed. Ancillary elements (eg, microfluidic reservoirs, channels, needles, valves, pumps and / or injectors, etc.) may be included in the array embodiment, according to which controlled sensor functionalization, sensor Cleaning / reconditioning, and / or controlled sensor calibration, etc. are possible.

C-3 Another Assay Embodiment FIGS. 8B and 9 show another assay embodiment according to the present invention. One or more of these embodiments may be used in place of the above-described embodiments, or may be used in combination with these embodiments.

  FIG. 8B schematically illustrates another embodiment according to aspects of the present invention in which an electrically active intercalator 34 is disposed, the intercalator is introduced into the sample medium, and / or It is introduced separately after hybrid formation. The intercalator 34 communicates with the hybridization part (duplex region) of the complex between the probe 22 and the target sequence 32, thereby amplifying and / or correcting the measured response of the sensor 10, and the hybrid Formation measurement and / or detection is facilitated.

As shown in the structures of FIGS. 8C-8F, these embodiments include electrically active intercalators (eg, daunomycin, methylene blue, Ir (bpy) (phen) ₃ ^+, etc.) and groove binders (eg, Ru (NH ₃ ) ₅ Cl ₂ ⁺ or the like) or a combination thereof is included.

  FIG. 9A schematically illustrates another assay embodiment according to aspects of the present invention. In this case, a secondary or sandwich probe 40 having a form that hybridizes with the second site of ssDNA 30 is used (sandwich array 44). The sandwich probe 40 includes a second cDNA 42 having a portion including a base sequence complementary to the sandwich sequence 44. cDNA 42 preferably includes a moiety covalently linked to amplification group 46. This amplification group 46 is used for amplification or correction of the signal response of the sensor 10 upon hybridization of the target sequence 32 to the detection probe 22. The amplification group 46 may be a group or label that provides the sensor 10 with a signal that can be detected and / or quantified without further reaction. Alternatively, the amplification group 46 may be a group that provides the sensor 10 with a signal that can be detected and / or quantified by further reaction with other promoter substances (eg, chemical or biochemical substrates, etc.). 9A to 9C illustrate examples of the amplification group.

There are many uses for the assay shown in FIG. 9A according to aspects of the present invention. For example, the use aspect including the following process a) -f) is mentioned:
a) binding the probe 22 to the nanotube 12 of the sensor 10;
b) When the sensor 10 is processed using a sample presumed to contain the analyte ssDNA having the target sequence 32, if ssDNA 30 is present, it is bound to the probe 22 and then washed. Do it,
c) When the sensor 10 is treated with a solution containing the sandwich probe 40 having the selected amplification group 46, and the ssDNA 30 is present, the sandwich probe 40 is bound thereto, followed by a washing treatment. Do
d) if necessary for the selected amplification group 46, treatment with a solution containing another promoter substance,
e) Obtain the signal from the sensor 10, and then f) analyze the signal to determine the presence and concentration of the analyte ssDNA 30.

  The above steps a) to f) may be performed in a different order. For example, the process c) may be a pre-processing of the specimen sample performed before the process b). Similarly, additional calibration processes may be performed as many times as desired. The cleaning process is exemplary, and those skilled in the art can easily adapt the method for a particular application without undue experimentation without departing from the technical idea of the present invention. Or it can be optimized, thereby avoiding multiple sources of contamination and other errors.

  In a particular embodiment, the sandwich sequence 44 may be a common sequence that is expected to be present in the sample DNA fragment, and the target sequence 32 may be an analyte that is unknown in the sample. It may be a specific sequence. Alternatively, the probe 46 may have a structural form that exhibits a relatively non-specific binding property to the sample DNA as compared to the relatively high target-specific binding property of the probe 22. In this regard, probe 46 may optionally include additional groups that promote binding to sample DNA and / or prevent unwanted blocking of probe 22.

  In certain embodiments, the amplification group 46 exerts a detectable response to the sensor 10 by effecting oxidation / reduction or other reaction with a chemical or biochemical substrate. Various promoters or catalysts (eg, enzymes, etc.). For example, the amplification group 46 may contain urease. The above step d) may include a step of generating ammonia and carbon dioxide by treatment with a urea solution when a bound probe is present, whereby the pH of the solution is reduced. Modified, resulting in a detectable change in the signal from the sensor 10. Examples of other enzyme systems that may be used include corynesterase, peroxidase (for example, HRP) and glucose oxidase. Examples of the amplification group 46 further include ferrocene, nanometal particles, and labels (for example, nanoparticles and proteins).

  FIG. 9E schematically illustrates another embodiment of an assay according to aspects of the present invention. In this assay embodiment, sandwich probe 40 and ssDNA 30 are generally similar to those described in connection with FIG. 9A.

  However, in the embodiment shown in FIG. 9E, the detection probe 50 includes a tether group 57 and a corresponding detector group 53, which groups are linked or attached to each other. . The constraining group 57 includes a linker 58 connected to a constraining species (in this case, antibody 56). Detection group 53 includes cDNA bound to a constraining group-compatible species. In this case, the antigen 54 is selected to include an epitope having a structural form that binds to the binding site of the receptor or antibody 56.

  For example, as shown in FIG. 9E, the antigen 54 may contain biotin, and the antibody 56 may contain streptavidin. In another embodiment (not shown), the arrangement may be the reverse of that shown in FIG. 9E. For example, the antigen may be linked to a linker and the antibody may be linked to cDNA. Another embodiment of combining a constraining group and a constraining group-binding species may be employed. In this case, the constraining group and the constraining group-binding species are selected to form a self-assembled detection probe 50 by easily linking or binding to each other.

  It should be noted that in general, the constraining group 57 and the detection group 53 may be prepared separately. For example, a partially functionalized sensor platform including the constraining group 57 may be prepared, supplied and stored without the detection group 53. Such subassemblies are not damaging to substances or conditions that specifically degrade polynucleotides (eg, endonucleases and exonucleases, etc.). Thus, this embodiment provides a convenient method (self-testing) for pre-preparing a sensor assembly (ie, a relatively common sensor) that does not contain target-specific cDNA, and then into a constraining group during or immediately prior to measurement of the sample. It is particularly suitable for applications where it is desirable to introduce a detection group of any one of a number of different target-specific detection groups that can be linked or bound by assembly or simple reaction methods). A rapid and powerful selective antigen-antibody binding reaction is a preferred embodiment of the restraining group / constraining group-compatible system.

  The constraining group / constraining group-compatible system shown in FIG. 9E may be used in conjunction with another embodiment according to aspects of the present invention (eg, the embodiment shown in FIGS. 8-9D that provides similar advantages). Similarly, a particular sensor 10 may be functionalized using more than one combination of restraint group / constraint group-compatible system. In this case, each detection group has a selected homologous cDNA. In certain applications where specific cross-reactivity is required, more than one cDNA may be used for a particular sensor. More generally, however, the sensor is set to provide maximum target selectivity.

  Furthermore, the self-assembling constraining group / constraining group-compatible system is a sensor array according to an aspect of the present invention in that a relatively common sensor array can be prepared in advance using constraining groups coupled to a plurality of sensors. In particular embodiments. Then, to complete the patterned target-specific functionalization of the sensor array, the selected pattern of different target-specific detection groups can be obtained in a known manner (eg, using a multiplexed or automated pipette system). Or “inkjet” method).

C-4 Alternative Sensor Assembly Devices FIGS. 10A and 10B show an “island” sensor 70 and a “nanotube network” sensor 72, respectively, both of which are embodiments according to certain aspects of the invention. These aspects generally apply to the assay aspects shown in FIGS. The nanostructure 12 (in this case, a single-walled carbon nanotube; “SWCNT” or “NT” for short) is electrically connected to the power supply electrode 16 and the drain electrode 18. In one embodiment 72, the plurality of nanotubes 12 form a network structure interconnected between the power electrode 16 and the drain electrode 18. The linker group 76 may connect the cDNA chain 74 to the nanotube 12. The ssDNA strand 78 may diffuse to the vicinity of the cDNA strand 74.

  FIGS. 10C and 10D schematically show that the linkage between the cDNA strand 74 and the nanotube 12 is due to the action of the linker group 76. As illustrated in FIG. 10C, the organic group 76 (eg, pyrene) is covalently bonded to cDNA 74 or the like, and as illustrated in FIG. 10D, the reactive polymer group 76 ′ is covalently bonded to cDNA 74 or the like. To join. Both groups may be covalently linked to cDNA 74 or the like.

  FIGS. 11A and 11B show alternative sensor structures 80 and 82. In this case, cDNA 84 binds to the surface of substrate 14 '(for example, a silicon dioxide layer covering a silicon wafer) via a chemical bond (for example, a covalent bond). FIG. 11C shows another binding method that includes a sequence of steps for binding cDNA 84 to substrate 14 by employing known reactants and methods, as shown in the sequence of steps (steps 1-3). Hybridization with ssDNA 88 and cDNA 84 affects the electrical properties of sensor 80 or 82, respectively, thereby providing a detection signal that is generally similar to the various assay embodiments described above.

  12A and 12B show alternative sensor structures 90 and 92. FIG. In this case, cDNA 94 binds to the surface of electrodes or contacts 16 'and 18' via a chemical bond (eg, covalent bond, etc.) by a known reactant and method (eg, DNA at the 5 'end of the cDNA). Binding by formation of -5'-thiol). The electrode may be a bare electrode or an electrode having an oxidized or coated surface. Alternatively, cDNA 94 may be bound to contacts 16 'and 18' by a polymer linker group or the like. Hybridization with ssDNA 98 and cDNA 94 affects the electrical properties of sensor 90 or 92, respectively, thereby providing a detection signal that is generally similar to the various assay embodiments described above.

C-5 Separate Separation and Purification To separate target DNA from genomic DNA in the vicinity of the nanotube device 10, various labeling groups known in the art may be used. For example, a label (eg, nanoparticle, protein, etc.) may be used to separate target DNA from genomic DNA as an additional step. Alternatively, magnetic beads or antibodies may be used for such separation. In certain embodiments according to aspects of the present invention, preliminary measurement sample DNA purification and / or segregation, etc. may be performed as part of an integrated sample processing / measurement system in a sensor or array embodiment. The operation is performed in the vicinity of the sensor array, and such operations may include magnetic control and / or electrostatic control. If desired, the purification and / or separation of sample DNA and the detection and measurement of the sample may be controlled and adjusted by incorporating a microprocessor or computer.

  In the foregoing description, preferred embodiments of nanotube sensor devices have been described, but certain advantages associated with these device systems have been obtained, as will be apparent to those skilled in the art. Further, various modifications, improvements, changes, and the like of the above-described embodiments may be made within the scope of the present invention and the technical idea. The present invention described above is defined by the description of the scope of claims.

2 is a schematic diagram illustrating an exemplary conductivity curve for a nanotube transistor device. FIG. It is a schematic diagram which shows the exemplary form regarding the nanotube sensor using the random network structure of a nanotube. FIG. 3 is a schematic cross-sectional view of the exemplary nanotube sensor shown in FIG. 2 is a flowchart showing exemplary steps of a method for manufacturing a nanoelectronic sensor according to the present invention described in Example A. 2 is a flowchart showing exemplary steps of a polynucleotide detection method according to the present invention. FIG. 6 is a chart showing conductivity as a function of gate voltage for nanotube electronic devices in three environments. The device characteristics of the sensor described in Example B after being subjected to a functionalization treatment using a pyrene-DNA conjugate and a treatment using cDNA are shown. The apparatus characteristics of the sensor described in Example B after being subjected to functionalization treatment using pyrene-DNA conjugate, treatment using SNP-DNA, and treatment using cDNA are shown. FIG. 6 illustrates an exemplary DNA assay embodiment using a detection probe coupled to a sensor, in accordance with certain aspects of the present invention. Fig. 6 illustrates an embodiment of another DNA assay using an electroactive incalator according to certain aspects of the invention. Fig. 3 illustrates an embodiment according to a particular aspect of the present invention, the components of another DNA assay embodiment using an electroactive incalator. Fig. 3 illustrates an embodiment according to a particular aspect of the present invention, the components of another DNA assay embodiment using an electroactive incalator. Fig. 3 illustrates an embodiment according to a particular aspect of the present invention, the components of another DNA assay embodiment using an electroactive incalator. Fig. 3 illustrates an embodiment according to a particular aspect of the present invention, the components of another DNA assay embodiment using an electroactive incalator. Fig. 4 illustrates an embodiment of another DNA assay that uses an amplification group according to certain aspects of the invention. Fig. 4 illustrates an embodiment according to a particular aspect of the present invention, the components of another DNA assay embodiment using an amplification group. Fig. 4 illustrates an embodiment according to a particular aspect of the present invention, the components of another DNA assay embodiment using an amplification group. Fig. 4 illustrates an embodiment according to a particular aspect of the present invention, the components of another DNA assay embodiment using an amplification group. FIG. 4 illustrates another DNA assay embodiment using an antibody-antigen binding that binds a detection probe to a sensor, in accordance with certain aspects of the invention. FIG. 4 illustrates components of another DNA assay embodiment using an antibody-antigen binding that binds a detection probe to a sensor, in accordance with certain aspects of the invention. FIG. 3 illustrates a sensor having two alternative structures according to certain aspects of the present invention, wherein a detection probe is bound to a nanostructure such as a nanotube. FIG. 3 illustrates a sensor having two alternative structures according to certain aspects of the present invention, wherein a detection probe is bound to a nanostructure such as a nanotube. FIG. 3 illustrates a sensor having two alternative structures according to certain aspects of the present invention, wherein a detection probe is bound to a nanostructure such as a nanotube. FIG. 3 illustrates a sensor having two alternative structures according to certain aspects of the present invention, wherein a detection probe is bound to a nanostructure such as a nanotube. Fig. 3 illustrates a sensor having two alternative structures according to certain aspects of the present invention, wherein a detection probe is coupled to a sensor substrate. Fig. 3 illustrates a sensor having two alternative structures according to certain aspects of the present invention, wherein a detection probe is coupled to a sensor substrate. Fig. 3 illustrates a sensor having two alternative structures according to certain aspects of the present invention, wherein a detection probe is coupled to a sensor substrate. Fig. 4 illustrates a sensor having two alternative structures according to certain aspects of the present invention, wherein a detection probe is coupled to a sensor electrode. Fig. 4 illustrates a sensor having two alternative structures according to certain aspects of the present invention, wherein a detection probe is coupled to a sensor electrode.

10 sensor 12 nanotube 14 substrate 16 power supply electrode 18 drain electrode 20 gate electrode 22 probe 24 cDNA
26 linker 30 ssDNA
32 target sequence 34 intercalator 40 sandwich probe 46 amplification group 50 detection probe 53 detection group 56 antibody 57 constraining group 58 linker 70 island sensor 72 nanotube network sensor 74 cDNA chain 76 linker group 78 ssDNA chain 80 sensor structure 82 sensor structure 84 cDNA
88 ssDNA
90 Sensor structure 92 Sensor structure 94 cDNA
98 ssDNA

Claims (50)

A nanotube sensor for polynucleotide detection comprising the following components i) to iv):
i) substrate,
ii) first nanotubes disposed on the substrate;
iii) at least one conductive element electrically connected to the first nanotube, and
iv) at least one single-stranded DNA molecule operatively associated with the first nanotube, configured to interact with a complementary target DNA strand to alter the electrical properties of the nanotube sensor The single-stranded DNA molecule.   The nanotube sensor of claim 1, wherein at least one single-stranded DNA is directly bound to the first nanotube.   The nanotube sensor according to claim 1, wherein the first nanotube is selected from the group consisting of a single-walled nanotube, a double-walled nanotube, a multi-walled nanotube, and an onion-shaped nanotube. The nanotube sensor according to claim 1, wherein the first nanotube contains at least one element selected from the following group:
Carbon, boron, boron nitride, carbon boron nitride, silicon, germanium, gallium nitride, zinc oxide, indium phosphide, molybdenum disulfide, and silver.   The nanotube sensor according to claim 1, wherein the first nanotube comprises a single-walled carbon nanotube.   The nanotube sensor according to claim 1, wherein the at least one conductive element includes an electrode including at least one substance selected from the group consisting of metal, carbon, and a conductive polymer.   The nanotube of claim 1, wherein the electrical property of the nanotube sensor is a capacitance of at least one first nanotube, and the nanotube sensor further comprises a counter electrode configured to allow measurement of the capacitance. sensor.   The nanotube sensor of claim 1, wherein the at least one conductive element contains at least two conductive elements, and the conductive elements contain a metal electrode.   9. The nanotube sensor of claim 8, wherein the conductive element is in direct physical contact with the first nanotube.   The nanotube sensor according to claim 8, further comprising a gate electrode adjacent to the nanotube.   The nanotube sensor according to claim 8, further comprising an inhibitor layer covering a region of the sensor adjacent to the connection portion between the conductive elements.   9. The nanotube sensor according to claim 8, wherein the nanotube further comprises a network of two-dimensional nanotubes disposed on a substrate between two conductive elements.   The nanotube sensor according to claim 12, wherein the network of nanotubes contains a plurality of carbon nanotubes randomly oriented.   The nanotube sensor of claim 12, wherein the two conductive elements comprise a pair of interdigitated electrodes.   13. The nanotube sensor of claim 12, wherein the at least one single-stranded DNA further comprises a plurality of identical DNA receptor strands distributed on a two-dimensional nanotube network.   The nanotube sensor according to claim 15, wherein a plurality of identical DNA receptor chains are directly bonded to the nanotubes of the two-dimensional nanotube network structure. A method for producing a nanoelectronic sensor comprising the following steps i) to iii):
i) orienting a nanotube film onto an electrode placed on a substrate;
ii) A solution of single-stranded DNA set to be a complement to the target DNA sequence is deposited on the substrate. Then
iii) A single-stranded DNA deposit is formed on the substrate by drying the solution.   18. The method of claim 17, further comprising the step of removing the single-stranded DNA deposit from the substrate other than the region between the electrodes.   The method of claim 17, further comprising depositing a conductive deposit as an electrode on the substrate.   20. The method of claim 19, wherein the electrode depositing step comprises setting an electrode having a plurality of interdigitated fingers.   The method of claim 17, further comprising forming a gate electrode configured to operate on a region between the electrodes.   The method according to claim 21, further comprising the step of removing single-stranded DNA deposits from a substrate other than the interelectrode region.   18. The method of claim 17, further comprising the step of coating the electrode with a blocking material prior to the depositing step.   The method according to claim 17, further comprising the step of preparing a single-stranded DNA solution containing the oligonucleotide dissolved in water. A method of detecting a specific target polynucleotide sequence comprising the following steps i) and ii):
i) contacting the test solution with a nanoelectronic sensor containing a polynucleotide complementary to a target polynucleotide sequence attached to at least one nanotube disposed in the region between the conductive electrodes;
ii) measuring at least one electrical property of the nanoelectronic sensor during the contacting step.   26. The method of claim 25, wherein the contacting step further comprises contacting the solution with a nanoelectronic sensor comprising a field effect transistor.   27. The method of claim 26, wherein the measuring step includes measuring at least one electrical characteristic including a gate voltage.   26. The method of claim 25, further comprising comparing electrical characteristics measured during the measurement process with corresponding characteristics measured prior to the measurement process. An electronic sensor system for a target polynucleotide comprising the following components a) to c):
(A) at least one sensor platform having at least one electrical characteristic, the sensor platform comprising the following elements i) to iii):
i) substrate,
ii) at least one electrode disposed adjacent to the substrate; and
iii) at least one nanostructured element disposed adjacent to the substrate, the nanostructured element electrically connected to the electrode;
(B) at least one detection probe operably connected to the sensor platform, the detection probe comprising the following elements i) to iii):
i) a linker group disposed in connection with the sensor platform, the linker group linked to one or more elements of the nanostructured element, the substrate and the electrode;
ii) a detection biomolecule that exhibits binding affinity for the test polynucleotide;
iii) a bond junction between the linker group and the biomolecule;
(C) An electronic measurement circuit connected to the electrode, the electronic measurement circuit configured to measure at least one electrical characteristic of the sensor platform.   30. The electronic sensor system of claim 29, wherein the detection biomolecule is selected from the group consisting of a polynucleotide, a transcription factor and a transcription promoter.   30. The electronic sensor system of claim 29, wherein the detection biomolecule comprises a detection polynucleotide having at least one nucleotide sequence that is at least partially complementary to the nucleotide target sequence of the test polynucleotide.   At least one detection probe operably connected to the sensor platform affects at least one electrical property when the probe binds to the test polynucleotide by at least partial hybridization of the target sequence. 32. The electronic sensor system of claim 31 configured to exert.   (A) the at least one electrode includes at least two electrodes disposed adjacent to the substrate at an interval, each electrode being electrically connected to the nanostructure element, wherein the nanostructure element is between the electrodes; 33. The electronic sensor system of claim 32, wherein the electronic sensor system is configured to form at least one conductive groove and (b) at least two electrodes are connected to an electronic measurement circuit and measure at least one electrical characteristic of the sensor platform. .   33. The electronic sensor system of claim 32, wherein the nanostructured element includes a network structure containing a number of single-walled carbon nanotubes interconnected.   And further comprising a gate electrode disposed adjacent to the nanostructured element and connected to the electronic measurement circuit, the electronic measurement circuit being selected relative to the gate electrode during measurement of at least one electrical property of the sensor platform 33. The electronic sensor system of claim 32, wherein the electronic sensor system is configured to bias at least 1 volt.   33. The electronic sensor system of claim 32, wherein the binding link between the linker group and the detection polynucleotide comprises at least a chemical bond between the linker group and the detection polynucleotide.   37. The electronic sensor system of claim 36, wherein the binding link between the linker group and the detection polynucleotide comprises at least a covalent bond between the linker group and the detection polynucleotide.   38. The electronic sensor system of claim 37, wherein the linker group comprises an aromatic compound configured to interact non-covalently with the nanostructure.   The binding link between the linker group and the detection polynucleotide molecule contains at least one constraining group bound to the linker group and at least one constraining group-compatible group bound to the detection polynucleotide; 35. The constraining group-compatible group (these are collectively referred to as constraining species) has a mutual affinity that increases a self-assembled binding linkage between the constraining group and the constraining group-compatible group. Electronic sensor system.   40. The electronic sensor system of claim 39, wherein the at least one constrained species contains a non-biomolecule.   40. The electronic sensor system of claim 39, wherein the at least one constrained species contains a biopolymer.   40. The electronic sensor system of claim 39, wherein the at least one constrained species comprises a synthetic polymer substantially equivalent to a naturally occurring biopolymer.   At least one constraining group contains an antibody bound to a linker group, and at least one constraining group-compatible group contains a corresponding antigen bound to a detection polynucleotide, the constraining group and constraining group-compatible 40. The electronic sensor system of claim 39, wherein the mutual affinity between the groups comprises the affinity of the antibody binding site for an epitope of the antigen.   At least one constraining group contains an MHC receptor bound to a linker group, and at least one constraining group-compatible group contains a corresponding binding peptide bound to a detection polynucleotide, the constraining group and the constraining group 40. The electronic sensor system of claim 39, wherein the mutual affinity between the compatible groups comprises MHC receptor affinity for the corresponding peptide.   At least one constraining group contains a mammalian cell surface receptor bound to a linker group, and at least one constraining group-compatible group contains a specific binding ligand for the corresponding receptor bound to a detection polynucleotide. 40. The electronic sensor system of claim 39, wherein the mutual affinity between the constraining group and the constraining group-compatible group comprises the affinity of the cell surface receptor for the corresponding ligand.   At least one constraining group contains a bilon host adhesion promoting surface group and / or a bilon endocytosis promoting surface group attached to a linker group, wherein the at least one constraining group-compatible group is a detection polynucleotide A corresponding mammalian cell-surface receptor bound to the molecule, wherein the mutual affinity between the constraining group and the constraining group-compatible group includes the affinity of the mammalian cell-surface receptor for the corresponding virion surface group. Item 40. The electronic sensor system according to Item 39.   The order of attachment of the constraining group binding species and constraining group-compatible group binding species is reversed, at least one constraining group is attached to the detection polynucleotide, and at least one constraining group-compatible group is attached to the linker group. 40. The electronic sensor system according to claim 39.   40. The electronic sensor system of claim 39, wherein the one or more constrained species is a synthetic material.   The system further comprises a plurality of the sensors, and for two or more sensors, of the plurality of sensors to allow self-assembly of selected complex patterns of different target-specific cDNA probes. 40. The electronic sensor system of claim 39, wherein the different sensors contain a plurality of constrained species pairs having distinctly different mutual binding affinities.   40. The electronic sensor system of claim 39, wherein the electrical characteristic of the sensor platform is at least the capacitance of the nanostructured device and further comprises a counter electrode configured to allow measurement of the capacitance.

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