Conductivity sensor for detecting nucleic acid and detection method thereof

Abstract

The present invention provides a sensor for detecting nucleic acid, comprising a substrate, a pair of terminal electrodes arranged on the substrate in a spaced-apart opposing relationship, and a sensor element located between the pair of terminal electrodes and in electrical contact with the pair of terminal electrodes, wherein the sensor element includes: (i) a semiconductor portion of the substrate, wherein a conductive path between the terminal electrodes passes through the semiconductor portion; and (ii) an oligonucleotide on a surface of the semiconductor portion, the oligonucleotide being complementary to a nucleic acid to be detected; and hybridization of the nucleic acid with the oligonucleotide results in a change in the resistance of the sensor.

Description

[Technical field]

[0001] The present invention relates to sensors, and in particular to a conductivity sensor for detecting nucleic acid sequences in fluids and a method for detecting nucleic acids using such a sensor. The present invention has been developed primarily for use in the detection of nucleic acids in and from samples such as body fluids or tissues and will be described below with reference to this exemplary application.

[0002] The following discussion of the background of the invention is intended to facilitate an understanding of the present invention, but it is understood that the discussion is not an affirmation or admission that any of the subject matter referred to was published or publicly known or part of the common general knowledge in Australia or any other country at the priority date of any one of the claims herein. [Background technology]

[0003] The ability to accurately detect different nucleic acids of interest has applications in a wide range of technical fields, but is particularly important in sensors used in medical diagnostics and treatment. For example, this finds application where the presence of a particular nucleic acid sequence in a sample obtained from an individual indicates a genetic mutation in the individual that leads to the individual suffering from a disease, such as cancer. This is particularly important when the difference between the natural nucleic acid (e.g., RNA and subtypes, DNA, methylation type) and the nucleic acid of an individual suffering from a disease is a single point mutation in the nucleic acid and / or when appropriate treatment is determined based on the change in the nucleic acid in the individual.

[0004] An example of this type of disease is melanoma with a BRAF V600E single base mutation. Melanoma is a common skin cancer and a global health concern with a high mortality rate when identified at late stage (approximately 1700 deaths per year in Australia). BRAF V600E is a common oncogenic point mutation in melanoma (approximately 40%) and identification is required for anti-BRAF targeted therapy. While certain treatments target the mutation, the ability to identify such subtle changes in nucleic acid is essential when other methods are ineffective. Given the poor prognosis of patients with these medical diseases, access to the correct treatment is essential to improve mortality. There are multiple other examples of nucleic acid changes / mutations of diagnostic, prognostic and therapeutic importance.

[0005] Given the benefits of detecting specific nucleic acids, such as the ability to detect mutations, significant research has been conducted in these fields. Thus, a wide range of conventional PCR-based techniques, allele-specific PCR, droplet digital PCR, high-resolution melting PCR, and PCR clamping and sequencing, such as Sanger and next-generation sequencing, are widely used to detect mutations in DNA, such as BRAF V600E in melanoma. Unfortunately, conventional mutant DNA detection methods, when used as cancer identification techniques, are greatly plagued by the complexity of sample preparation, sample handling, manipulation, and data analysis. Moreover, the tests are very time-consuming, laborious, and expensive.

[0006] For example, conventional BRAF V600E mutant DNA detection techniques include polymerase chain reaction (PCR)-based techniques, as well as sequencing-based approaches such as traditional Sanger and more recent next-generation sequencing.

[0007] These conventional methods suffer from at least the following general drawbacks: (i) the need for specific technical skills and knowledge; (ii) expensive and bulky equipment and test costs; (iii) undue susceptibility to sample contamination resulting in false positive / negative results; (iv) complex and time-consuming sample preparation methods (varies from hours to days); and (v) the need for careful control of experimental conditions in sample handling, equipment preparation and operation.

[0008] Alternatively, optical sensors have been proposed for detecting mutations, such as point mutations, in nucleic acids, utilizing detection methods such as fluorescence spectroscopy, surface-enhanced Raman spectroscopy, luminescence spectroscopy, and surface plasmon resonance spectroscopy. These optical sensors for detecting point mutations in nucleic acid sequences are limited by the following limitations: (i) the need for bulky measurement equipment, (ii) the need for appropriate optically active tags / labels to detect the biomarkers of interest (e.g., fluorescence spectroscopy), (iii) the close overlap of the optical bands of the target biomarker and background components, which leads to a lack of sensitivity, (iv) the need for nanoparticles for detection (e.g., surface-enhanced Raman spectroscopy and surface plasmon spectroscopy), which may result in nanoparticle cytotoxicity, (v) limited photostability and loss of recognition ability, and (vi) susceptibility to interference from factors such as pH, temperature, and oxygen levels.

[0009] Electrochemical sensors for the detection of mutations, such as point mutations, in nucleic acid sequences have also been proposed, utilizing detection methods such as amperometry, voltammetry (cyclic, square wave, differential pulse), field effect transistors, and electrochemical impedance spectroscopy.

[0010] These electrochemical sensors for detecting mutations, such as point mutation detection in nucleic acid sequences, have the following disadvantages: (i) Conventional electrochemical methods require complex detection techniques (e.g., electrochemical impedance spectroscopy and field effect transistors), whereas the present invention uses a simple conductometric detection method; (ii) Electrochemical methods such as field effect transistors and cyclic voltammetry require multiple electrodes leading to high power consumption, whereas the present invention uses two terminal electrodes for low power consumption; (iii) Some electrochemical methods utilize specific materials and complex sensor fabrication methods (e.g., nanomaterial-related sensor platforms); (iv) Some electrochemical techniques require specific enzymes (e.g., endonucleases) for signal amplification and specific redox couples (e.g., amperometry) for signal generation, whereas the present invention does not require these materials for detection.

[0011] Thus, although it is desirable to develop methods that allow for the detection of nucleic acids, such as DNA sequences, that contain mutations, there remains a need for additional methods to achieve this result.

[0012] The present invention seeks to provide sensors for use in detecting and / or quantifying levels of nucleic acids in a sample, and methods for detecting and / or quantifying levels of altered nucleic acids, which overcome or substantially ameliorate at least some of the shortcomings of the prior art, or at least provide a useful alternative. Summary of the Invention [Means for solving the problem]

[0013] According to a first aspect of the present invention, there is provided a sensor for detecting a nucleic acid, comprising a substrate, a pair of terminal electrodes arranged on the substrate in a spaced-apart opposing relationship, and a sensor element located between the pair of terminal electrodes and in electrical contact with the pair of terminal electrodes, wherein the sensor element comprises: (i) a semiconductor portion of the substrate, wherein a conductive path between the terminal electrodes passes through the semiconductor portion; and (ii) an oligonucleotide on a surface of the semiconductor portion, the oligonucleotide being complementary to a nucleic acid to be detected, wherein hybridization of the nucleic acid with the oligonucleotide results in a change in resistance of the sensor.

[0014] The semiconductor portion of the substrate that forms part of the sensor element can take many forms. In one embodiment, the semiconductor portion comprises a high resistivity non-oxide semiconductor. In one embodiment, the semiconductor portion comprises an oxygen deficient metal oxide.

[0015] In some embodiments, the semiconductor portion has a resistivity greater than 100 ohm-cm. In some embodiments, the semiconductor portion has a resistivity greater than 200 ohm-cm. In some embodiments, the semiconductor portion has a resistivity greater than 500 ohm-cm. In some embodiments, the semiconductor portion has a resistivity greater than 1000 ohm-cm. In some embodiments, the semiconductor portion has a resistivity greater than 2000 ohm-cm. In some embodiments, the semiconductor portion has a resistivity greater than 50 ...

[0016] In some embodiments, the semiconductor portion has a resistivity in the range of about 500 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the semiconductor portion has a resistivity in the range of about 1,000 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the semiconductor portion has a resistivity in the range of about 5,000 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the semiconductor portion has a resistivity in the range of about 1,000 ohm·cm to about 10,000 ohm·cm.

[0017] In some embodiments, the semiconductor portion comprises a high resistivity non-oxide semiconductor. In some embodiments, the non-oxide semiconductor has a resistivity greater than 100 ohm-cm. In some embodiments, the non-oxide semiconductor has a resistivity in the range of about 500 ohm-cm to about 50,000 ohm-cm, or in the range of about 1000 ohm-cm to about 10000 ohm-cm.

[0018] In some embodiments, the sensor has an electrical resistance in the range of about 10 kilohms to about 10,000 kilohms.

[0019] In some embodiments, the non-oxide semiconductor is selected from the group consisting of elemental semiconductors and compound semiconductors, hi some embodiments, the non-oxide semiconductor is an elemental semiconductor.

[0020] In some embodiments, the non-oxide semiconductor is a silicon semiconductor. The silicon semiconductor may be an intrinsic silicon semiconductor. The silicon semiconductor may be a float-zone type silicon semiconductor.

[0021] In some embodiments, the substrate includes a semiconductor portion as an integral part thereof. The substrate may be a wafer of a non-oxide semiconductor.

[0022] In another embodiment, the semiconductor portion comprises an oxygen deficient metal oxide. In some embodiments, the oxygen deficient metal oxide has a resistivity in the range of about 500 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the oxygen deficient metal oxide has a resistivity in the range of about 1,000 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the oxygen deficient metal oxide has a resistivity in the range of about 5,000 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the oxygen deficient metal oxide has a resistivity in the range of about 1,000 ohm·cm to about 10,000 ohm·cm.

[0023] In some embodiments, the oxygen-deficient metal oxide is selected from the group consisting of zinc oxide (ZnO), strontium titanium oxide (STO), tin oxide (SnO2), and titanium dioxide (TiO2). In some embodiments, the oxygen-deficient metal oxide layer is oxygen-deficient zinc oxide.

[0024] In some embodiments, the oligonucleotide is chemically bonded to the semiconductor portion by an organic linker, which may be, for example, a residue of a silanizing agent. The oligonucleotide may be chemically bonded to the semiconductor layer by a process comprising: (i) silanizing a non-oxide semiconductor with a silanizing agent having a terminal functional group selected from the group consisting of an epoxy group, a thiol group, an amino group, a carboxy group, and a hydroxy group; and (ii) reacting a precursor comprising an oligonucleotide with the terminal functional group. The silanizing agent is selected from the group consisting of (3-glycidyloxypropyl)trimethoxysilane (GPS), (3-mercaptopropyl)trimethoxysilane (MTS), (3-aminopropyl)triethoxysilane (APTES), and N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (AEAPTS).

[0025] In some embodiments, the oligonucleotide is complementary to a nucleic acid having a single point mutation relative to a native DNA sequence. In some embodiments, the oligonucleotide is complementary to a nucleic acid having an insertion or deletion mutation relative to a native DNA sequence. In some embodiments, the oligonucleotide is attached to the surface of the semiconductor portion via the 3' end of the oligonucleotide. In some embodiments, the oligonucleotide is attached to the surface of the semiconductor portion via the 5' end of the oligonucleotide. In some embodiments, the oligonucleotide comprises at least one 2'-O,4'-C-methylene-β-D-ribofuranosyl nucleotide monomer unit, i.e., a locked nucleic acid (LNA) (Vester, B., Wengel, J., LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43, 13233-13241 (2004)).

[0026] In some embodiments, the oligonucleotide is complementary to a sequence of the human BRAF gene. In some embodiments, the oligonucleotide is complementary to a microRNA sequence. In some embodiments, the oligonucleotide is complementary to a pharmacogenetic gene variant.

[0027] The sensor is preferably a conductivity sensor. The sensor may therefore comprise a device for applying a voltage between the terminal electrodes and measuring the current flow through the conductive path of the sensor. This device may preferably be a potentiostat. In an embodiment, the sensor is therefore not a field effect transistor.

[0028] According to a second aspect of the present invention, there is provided a method for detecting a nucleic acid, comprising the steps of: (a) contacting a sensor element of a sensor of the present invention with a substance that may contain a nucleic acid; (b) measuring an electrochemical parameter of the sensor corresponding to the resistance of the sensor; and (c) detecting the presence or absence of a nucleic acid on the sensor element based on the electrochemical parameter measured in step (b).

[0029] The applicants have found that hybridization of the nucleic acid with the oligonucleotide results in a change in the resistance of the sensor. Without wishing to be bound by theory, it is believed that this is due to a change in the electrical environment caused by hybridization of the target nucleic acid to the oligonucleotide. There are several different parameters that can be measured. In one embodiment, measuring the electrochemical parameters of the sensor includes (i) applying a voltage across the sensor and (ii) measuring the current flow through the sensor.

[0030] In some embodiments, measuring an electrochemical parameter of the sensor includes (i) applying a voltage across the sensor, and (ii) measuring a current flow through the sensor.

[0031] In some embodiments, detecting the presence or absence of nucleic acid comprises comparing the electrochemical parameter measured in step b) to a reference value for that parameter for the sensor, in some embodiments, an increase in the resistance of the sensor relative to the reference resistance of the sensor indicates the presence of nucleic acid on the sensor and thus in the sample.

[0032] In some embodiments, the material is a sample solution from nucleic acids extracted from tissues and / or bodily fluids.

[0033] According to a third aspect of the present invention, there is provided a method for manufacturing a sensor for detecting nucleic acids, the method comprising the steps of: preparing a substrate including a semiconductor portion; producing a pair of terminal electrodes on the substrate that are spaced apart and facing each other, wherein the semiconductor portion of the substrate is disposed between and in electrical contact with the terminal electrodes, and a conductive path between the terminal electrodes passes through the semiconductor portion; and immobilizing oligonucleotides complementary to the nucleic acid to be detected on a surface of the semiconductor portion, thereby producing a sensor element comprising (i) the semiconductor portion and (ii) the oligonucleotide.

[0034] Other aspects of the invention are also disclosed.

[0035] Notwithstanding other forms which may fall within the scope of the invention, preferred embodiments of the invention will now be described by way of example only with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 shows a schematic diagram of one embodiment of a conductivity sensor for detecting nucleic acids according to certain embodiments of the present invention, the sensor having a sensor element including a semiconductor portion of a sensor substrate having oligonucleotides immobilized on a surface of the semiconductor portion, the oligonucleotides being complementary to the nucleic acid sequence to be detected. [Diagram 2] FIG. 2 shows a schematic diagram of a method for manufacturing the conductivity sensor shown in FIG. [Diagram 3] FIG. 3 shows a schematic diagram of another embodiment of a conductivity sensor for detecting nucleic acids according to certain embodiments of the present invention, the sensor having a sensor element including a semiconductor portion of a sensor substrate having oligonucleotides immobilized on a surface of the semiconductor portion, the oligonucleotides being complementary to the nucleic acid sequence to be detected. [Figure 4] FIG. 4 shows a schematic diagram of a method for manufacturing the conductivity sensor shown in FIG. [Diagram 5] FIG. 5 shows a schematic diagram of sensor formation and nucleic acid detection on a high resistivity non-oxide semiconductor. [Figure 6] FIG. 6 shows a schematic diagram of sensor formation and nucleic acid sequence detection on oxygen-deficient metal oxides. [Figure 7] FIG. 7 shows a graphical representation of the detection of single-point mutated DNA on (a) 3′ and (b) 5′ amine immobilized oligonucleotides on metal oxide (ZnO(1-x) sensors). [Figure 8] FIG. 8 shows a graphical representation of the selectivity experiment of mutant DNA on (a) 3′ and (b) 5′ amine immobilized oligonucleotides on metal oxide (ZnO(1-x) sensors). [Figure 9] FIG. 9 shows a graphical representation of the detection of single point mutant DNA on a 5′ amine immobilized oligonucleotide on a silicon sensor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Summary of sequence identifiers [Table 1]

[0038] BRAF 3'-amine DNA oligonucleotide (SEQ ID NO:1) (5'-GGTCTAGCTACAGAGAAATCTCGAT / 3AmMO / -3')

[0039] BRAF 5'-amine DNA oligonucleotide (SEQ ID NO:2) (5'- / AmMC6 / GGTCTAGCTACAGAGAAATCTCGAT-3')

[0040] The present invention relates to a conductivity sensor for detecting nucleic acids. The sensor comprises a substrate, a pair of terminal electrodes arranged on the substrate in a spaced apart and opposing relationship, and a sensor element arranged between and in electrical contact with the pair of terminal electrodes. The sensor element includes (i) a semiconductor portion of the substrate, and (ii) one or more oligonucleotides on a surface of the semiconductor portion, the oligonucleotides being complementary to a nucleic acid to be detected. An electrical conductive path between the terminal electrodes passes through the semiconductor portion. In use, binding of a nucleic acid sequence to the oligonucleotide causes a change in the electrical resistance of the sensor. Without wishing to be bound by theory, it is believed that binding of a nucleic acid sequence to the oligonucleotide causes a change in the electron density on the sensor, which in turn causes a change in the resistance of the sensor. Thus, the increase in resistance can be determined by measuring an electrochemical parameter of the sensor that corresponds to the resistance of the sensor. For example, the resistance can be measured by measuring a current response when a voltage is applied across the sensor, thus detecting the presence and / or concentration of a nucleic acid. In one embodiment, the presence of a nucleic acid of interest in a sample is indicated by an increase in the resistance of the sensor after the sensor is incubated with the sample. In yet another embodiment, the presence of a nucleic acid of interest in a sample is indicated by an increase in the resistance of the sensor after incubation of the sensor with the sample, while incubation with the native sequence results in a decrease in resistance.

[0041] Thus, the sensor of the present invention uses conductivity detection technology to detect a range of potential nucleic acids in a sample, e.g., in a fluid. Exemplary fluids include bodily fluids such as human saliva, blood, plasma, interstitial fluid, cerebrospinal fluid, tears and / or sweat, or nucleic acids extracted from tissues for prognosis / treatment of medical diseases or other characteristics of an individual, and for pharmacogenomics. The sensor can also be used to detect nucleic acids in gas samples, such as respiratory aerosol droplets or ventilation systems. As described in more detail below, the conductivity sensor has a simple and relatively easy to manufacture device structure, which provides a cost-effective alternative to conventional non-invasive sensors that either require specialized substrates or employ detection technologies that limit their accuracy.

[0042] Below is a detailed description of a non-invasive conductivity sensor and its application method for detecting a range of nucleic acid levels in a sample, e.g., in a fluid. It should be noted that in the following description, similar or identical reference numbers in different embodiments indicate the same or similar features.

[0043] Sensors 1, a sensor 100 includes a substrate 102, a pair of terminal electrodes 104, 106 disposed in spaced apart facing relationship on the substrate, and a sensor element 108 between and in electrical contact with the terminal electrodes 104, 106. The sensor element 108 includes a semiconductor portion 110 including a semiconductor material 112 and oligonucleotides 114 on a surface 116 of the semiconductor portion 110. A conductive path 120 between the terminal electrodes 104 and 106 passes through the semiconductor portion 110 and thus through the semiconductor material 112.

[0044] In the embodiment shown in Figure 1, the substrate 102 includes the semiconductor portion 110 as an integral part of the substrate, and therefore the remainder of the substrate is composed of the same semiconductor material 112. The conductive path 120 between the terminals 104 and 106 is substantially confined to a surface layer of the substrate (the surface layer corresponding to the semiconductor portion 110) by the electric field lines that are established when a voltage is applied across the sensor in use. Advantageously, therefore, there is no need to fabricate a separate thin-film semiconductor layer on the sensor substrate. The substrate 102 may therefore be of any convenient thickness, for example as provided when using a wafer of the semiconductor material 112.

[0045] Alternatively, the sensor element 108 may include a semiconductor portion 110 formed as a separate surface layer on the substrate 102, at least between the terminal electrodes 104 and 106, but optionally extending across the entire substrate surface. In such embodiments, the substrate 102 may be constructed from any suitable material capable of receiving and supporting the semiconductor layer 110.

[0046] In use, the sensor 100 is contacted with a substance, such as a sample solution 122, which contains (or may contain) a nucleic acid 124. The nucleic acid, if present, hybridizes with the oligonucleotide 114, thereby causing a change in the electrical resistance of the sensor. The change in electrical resistance occurs due to a charge transfer by donating an electron to the semiconductor or accepting an electron from the semiconductor when an incoming complementary DNA strand hybridizes with the oligonucleotide. When a voltage is applied across the sensor, i.e. between the terminal electrodes 104 and 106, the resulting current flowing between the terminal electrodes along the conductive path 120 can be measured, and thus the electrical resistance of the sensor can be determined. By comparing this resistance with a predefined reference resistance of the sensor, the presence or absence of a nucleic acid in the sample solution 122 can be detected.

[0047] As will be appreciated by those skilled in the art, the sensor element 108 typically includes a plurality of oligonucleotides 114, and the percentage of those oligonucleotides that hybridize to 124 may depend on the concentration of nucleic acid in the sample solution 122. Because the resistance of the conductive pathway 120 is proportional to the percentage of hybridized oligonucleotide sites 114, the concentration of nucleic acid 124 in the fluid 122 can be determined, for example, by comparing the determined resistance with a calibration curve.

[0048] In another form, as shown in the schematic diagram of Figure 3, a sensor 100 comprises a substrate 102, a pair of terminal electrodes 104, 106 disposed in spaced apart facing relationship on the substrate, and a sensor element 108 between and in electrical contact with the terminal electrodes 104, 106. The sensor element 108 comprises a semiconductor portion 110 including a semiconductor layer 112 on an underlying substrate, and oligonucleotides 114 on a surface 116 of the semiconductor portion 110. A conductive path 120 between the terminal electrodes 104 and 106 passes through the semiconductor portion 110 and thus through the semiconductor material 112.

[0049] 3, the substrate 102 includes the semiconductor portion 110 as a layer portion of the substrate, with the remainder of the substrate being composed of the support material. The conductive path 120 between the terminals 104 and 106 is substantially confined to the layer corresponding to the semiconductor portion 110.

[0050] Thus, in this embodiment, the sensor element 108 includes a semiconductor portion 110 formed as a separate surface layer on the substrate 102, at least between the terminal electrodes 104 and 106, but optionally extending across the entire substrate surface. In such an embodiment, the substrate 102 may be constructed from any suitable material capable of receiving and supporting the semiconductor layer 110.

[0051] In use, the sensor 100 is contacted with a substance, such as a sample solution 122, which contains (or may contain) a nucleic acid 124. The nucleic acid, if present, binds to the oligonucleotide 114, thereby causing a change in the electrical resistance of the sensor. The change in electrical resistance occurs due to a charge transfer by donating an electron to or accepting an electron from the semiconductor when an incoming complementary DNA strand hybridizes with the oligonucleotide. When a voltage is applied across the sensor, i.e., between the terminal electrodes 104 and 106, the resulting current flowing between the terminal electrodes along the conductive path 120 can be measured, and thus the electrical resistance of the sensor can be determined. By comparing this resistance with a predefined reference resistance of the sensor, the presence or absence of the oligonucleotide in the sample solution 122 can be detected.

[0052] As mentioned above, those skilled in the art will appreciate that the sensor element 108 typically includes a plurality of oligonucleotide sites 114, and the proportion of those binding sites that hybridize to the nucleic acid 124 may depend on the concentration of the nucleic acid in the sample solution 122. Because the resistance of the conductive path 120 is proportional to the proportion of hybridized oligonucleotides 114, the concentration of the nucleic acid 124 in the fluid 122 can be determined, for example, by comparing the determined resistance with a calibration curve.

[0053] Each component of the conductivity sensor will now be described.

[0054] substrate In the broadest form of the invention, the substrate is generally not particularly limited and may be made of a material selected from the group consisting of, for example, a semiconductor, a polymer, a glass, or a ceramic.

[0055] For example, a suitable polymer for use as the substrate may be selected from the group consisting of polydimethylsiloxane (PDMS), polyimide (PI) and polyethylene naphthalate (PEN). A suitable ceramic may be selected from the group consisting of aluminum oxide (Al2O3), sapphire and silicon nitride (Si3N4).

[0056] As mentioned above, at least a portion of the substrate includes a semiconductor portion that forms part of the sensor element. In certain embodiments, the semiconductor portion corresponds to the entire substrate. In certain embodiments, the semiconductor portion corresponds to only a portion of the substrate as a whole. In such embodiments, the semiconductor portion of the sensor element may be supported on a support layer of the substrate, optionally only in the substrate area covered by the sensor element. In certain embodiments, the semiconductor portion of the substrate that forms the sensor element is in the form of a layer on one side of the support layer of the substrate. In certain embodiments, the semiconductor portion of the substrate that forms the sensor element is in the form of a strip recessed on or in the support layer of the substrate between the electrodes. However, in some preferred embodiments, the substrate comprises or consists of a semiconductor portion. In this embodiment, the semiconductor portion of the substrate corresponds to the entire substrate. Thus, as can be seen in FIG. 1, the semiconductor portion of the sensor element may be an integral part of the substrate, simplifying the device structure. In some embodiments, the substrate is a wafer of semiconductor material.

[0057] electrode The sensor includes a pair of terminal electrodes disposed on a substrate in spaced-apart, opposing relationship such that a sensor element of the sensor is disposed in a detection region between the spaced-apart terminal electrodes, and as will be appreciated by those skilled in the art, the terminal electrodes are conductive and configured for electrical connection to a device, such as a potentiostat, for applying a voltage across the sensor.

[0058] As shown in Figure 1, the terminal electrodes are formed as separate structures above the substrate surface and are in electrical contact with the semiconductor portion of the substrate below, although other configurations are contemplated. For example, the terminal electrodes may be recessed into the substrate, and the semiconductor portion of the sensor element may lie horizontally between the terminal electrodes along the substrate surface.

[0059] The terminal electrodes may comprise a conductive metal or alloy, preferably a chemically inert metal or alloy. Gold is one example of a suitable metal.

[0060] In some embodiments, terminal electrodes are formed on the substrate by microfabrication techniques. Gold terminal electrodes can be formed by evaporating a thin gold film (250 nm with a 100 nm chromium adhesion layer) onto the semiconductor layer using electron beam lithography. The as-deposited gold film is then patterned using standard photolithography and wet etching techniques to define a pair of terminal electrodes.

[0061] The terminal electrodes may generally be sized and positioned relative to one another in any configuration suitable for the conductivity sensor. In some embodiments, the terminal electrodes are spaced apart by a distance ranging from 1 micrometer to 100 micrometers. In some embodiments, the terminal electrodes have a length (i.e., the length in the direction perpendicular to the inter-electrode gap distance) ranging from 200 to 4000 micrometers. The inventors have obtained good results using two parallel electrodes spaced 40 micrometers apart and 4000 micrometers long, achieving a 16×10 -8 m 2 A detection area with an area of

[0062] In principle, the sensor can function with a wide variety of sensor geometries and dimensions for the electrodes. Nevertheless, the electrodes are typically spaced 1 μm to 200 μm apart. In one embodiment, the electrodes are spaced 1 μm to 100 μm apart. In one embodiment, the electrodes are spaced 10 μm to 80 μm apart. In one embodiment, the electrodes are spaced 20 μm to 60 μm apart. In one embodiment, the electrodes are spaced 30 μm to 50 μm apart. In one embodiment, the electrodes are spaced 35 μm to 45 μm apart. In one embodiment, the electrodes are spaced about 40 μm apart.

[0063] The detection electrodes may also vary in width, typically between 200 and 4000 μm wide. In one embodiment, the detection electrodes are between 400 and 3000 μm wide. In one embodiment, the detection electrodes are between 800 and 2000 μm wide. In one embodiment, the detection electrodes are between 1000 and 1500 μm wide.

[0064] Sensor element The sensor comprises a sensor element comprising (i) a semiconductor portion of a substrate, and (ii) an oligonucleotide on the surface of the semiconductor portion, the oligonucleotide being complementary to the nucleic acid to be detected. As will be appreciated by those skilled in the art, the sensor may be manufactured with one type of oligonucleotide on the surface, or with multiple different oligonucleotides depending on the end use. In one embodiment, the sensor is manufactured with a single oligonucleotide. In one embodiment, the sensor is manufactured with multiple different oligonucleotides. As will be further appreciated, the sensor is more sensitive when only one type of oligonucleotide is present on the surface, since the possibility of interference between different oligonucleotides is reduced. Nevertheless, in principle, the sensor may comprise several different oligonucleotides, such that multiple different nucleic acids may be detected in one test. The nucleic acids may be of any class and type.

[0065] The sensor element is located between and in electrical contact with both of the terminal electrodes such that the device is configured such that an electrical conductive path between the terminal electrodes passes through the semiconductor portion and thus through the semiconductor portion.

[0066] Semiconductor part In some embodiments, the semiconductor portion is an integral part of the substrate, specifically a region or surface portion of the substrate that extends across the sensing region between the terminal electrodes.

[0067] In other embodiments, the semiconductor portion is a separate surface layer of the substrate supported on a support layer of the underlying substrate. The semiconductor layer is located at least in the detection region between the terminal electrodes, but may optionally extend across the entire substrate surface. In such embodiments, the terminal electrodes may be formed, for example by metal deposition, on the surface of the separate semiconductor layer of the substrate. Alternatively, the terminal electrodes may be formed on the support layer, and the semiconductor portion of the substrate is subsequently formed on the support layer of the substrate at least in the detection region between the terminal electrodes.

[0068] The semiconductor portion of the substrate can take several different forms. In one embodiment, the semiconductor portion comprises, and typically consists of, a high resistivity non-oxide semiconductor. As used herein, non-oxide semiconductor includes both elemental and compound semiconductor materials, but does not include metal oxide semiconductors. In another embodiment, the semiconductor portion comprises an oxygen-deficient metal oxide.

[0069] Common semiconductors used in electrochemical devices, including many non-oxide semiconductors such as doped silicon, are too conductive for use in conductivity sensor elements. Any effect on the electronic properties of such semiconductors caused by the binding of oligonucleotides to the surface is too small to provide sufficient sensitivity. For this reason, conventional conductivity sensors are typically constructed with a separate conductivity sensing layer of high resistivity polymer or metal oxide material.

[0070] Applicants have surprisingly found that in order to be able to achieve a suitably sensitive sensor capable of detecting the sometimes very small changes in the electrical environment caused by hybridization of nucleic acid sequences to pendant oligonucleotides bound to the sensor surface, the semiconductor material used must have a very high resistivity. In some embodiments, the semiconductor material has a resistivity of greater than 100 ohm-cm, or greater than 200 ohm-cm. Resistivities of this type or greater have been found to provide the sensor with suitable sensitivity in detecting bound oligonucleotides.

[0071] In some embodiments, the semiconductor portion has a resistivity greater than 100 ohm-cm. In some embodiments, the semiconductor portion has a resistivity greater than 200 ohm-cm. In some embodiments, the semiconductor portion has a resistivity greater than 500 ohm-cm. In some embodiments, the semiconductor portion has a resistivity greater than 1000 ohm-cm. In some embodiments, the semiconductor portion has a resistivity greater than 2000 ohm-cm. In some embodiments, the semiconductor portion has a resistivity greater than 50 ...

[0072] In some embodiments, the semiconductor portion has a resistivity in the range of about 500 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the semiconductor portion has a resistivity in the range of about 1,000 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the semiconductor portion has a resistivity in the range of about 5,000 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the semiconductor portion has a resistivity in the range of about 1,000 ohm·cm to about 10,000 ohm·cm.

[0073] Thus, it has now surprisingly been found that the use of high resistivity non-oxide semiconductors in the conductivity sensor element can result in good conductivity sensor performance. By selecting a non-oxide semiconductor with high resistivity, the sensor has an overall resistance that falls within a range suitable for detecting changes in the electrical environment caused by hybridization of nucleic acid sequences to pendant oligonucleotides attached to the sensor element surface.

[0074] In some embodiments, high resistivity non-oxide semiconductors have a resistivity of greater than 100 ohm-cm, or greater than 200 ohm-cm In contrast, doped silicon semiconductors commonly used in electrochemical sensing devices generally have a resistivity of about 1 to 10 ohm-cm.

[0075] In some embodiments, the high resistivity non-oxide semiconductor has a resistivity in the range of 500 ohm-cm to about 50,000 ohm-cm, for example, in the range of about 1000 ohm-cm to about 10000 ohm-cm. The inventors have had good results with non-oxide semiconductors having resistivities of 1000 ohm-cm and 5000-10000 ohm-cm.

[0076] The high resistivity non-oxide semiconductor may be selected so that the sensor has an appropriate electrical resistance when measured between the terminal electrodes (and along the conductive path). In some embodiments, the sensor has an electrical resistance in the range of about 10 kilohms to about 10,000 kilohms, for example, in the absence of nucleic acid sequences hybridized to the oligonucleotides. The inventors have found that the use of low resistance sensors results in very low sensitivity to bioanalytes.

[0077] In some embodiments, the non-oxide semiconductor is selected from the group consisting of elemental semiconductors and compound semiconductors.

[0078] Suitable elemental semiconductors include silicon and germanium semiconductors, preferably silicon semiconductors. High purity intrinsic (undoped) silicon semiconductors have been found to be particularly suitable due to their resistive properties. The intrinsic silicon semiconductor may be float zone silicon, which is high purity silicon prepared by a float zone purification technique, in which a molten zone passes slowly along a rod of silicon and impurities preferentially remain in the molten zone rather than being reincorporated into the recrystallized silicon. In contrast, most silicon semiconductors are produced by the Czochralski process and therefore incorporate a higher degree of impurities. Suitable float zone silicon is: <100> These are 3" and 4" wafers with different orientations.

[0079] Intrinsic silicon semiconductors have been found to be particularly suitable, but this is not to be excluded as the non-oxide semiconductor may be a doped elemental semiconductor, provided that the doping level is sufficiently low to maintain the semiconductor highly resistive.

[0080] Suitable compound semiconductors include binary semiconductors such as gallium arsenide (GaAs), indium phosphide (InP) and indium antimony (InSb), and ternary semiconductors such as gallium aluminum arsenide (GaAlAs).

[0081] As previously mentioned, in certain embodiments, the semiconductor portion comprising the high resistivity non-oxide semiconductor may be an integral part of the substrate, i.e., the substrate may comprise or consist of a non-oxide semiconductor. For example, the substrate may be a wafer of a non-oxide semiconductor, such as a wafer of a high resistivity intrinsic silicon semiconductor.

[0082] In still further embodiments, the semiconductor portion comprises an oxygen deficient metal oxide. In some embodiments, the oxygen deficient metal oxide has a resistivity in the range of about 500 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the oxygen deficient metal oxide has a resistivity in the range of about 1,000 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the oxygen deficient metal oxide has a resistivity in the range of about 5,000 ohm·cm to about 5,000,000 ohm·cm. In some embodiments, the oxygen deficient metal oxide has a resistivity in the range of about 1,000 ohm·cm to about 10,000 ohm·cm.

[0083] A number of suitable oxygen-deficient metal oxides can be used for the semiconductor portion. In one embodiment, the oxygen-deficient metal oxide semiconductor portion can be formed using any suitable metal oxide selected from the group consisting of zinc oxide (ZnO), strontium titanium oxide (STO), tin oxide (SnO2), and titanium dioxide (TiO2).

[0084] In a preferred embodiment, the semiconductor portion is an oxygen-deficient metal oxide layer formed using zinc oxide (ZnO) or strontium titanium oxide (STO). As described below, the inventors have found that good results are obtained when the metal oxide layer is a thin oxygen-deficient zinc oxide (ZnO) layer.

[0085] The oxygen deficient metal oxide semiconductor portion may be deposited on the substrate surface by a technique selected from the group consisting of reactive sputtering, physical vapor deposition (PVD), chemical vapor deposition (CVD), metalorganic chemical vapor deposition (MOCVD), pulsed laser deposition (PLD) and molecular beam epitaxy (MBE).

[0086] In one embodiment, an oxygen-deficient metal oxide layer is deposited on the surface of a rigid (SiO2 / Si) wafer or a flexible polyimide foil by reactive sputtering to obtain a thin metal oxide film having a thickness ranging from about 50 nm to about 200 μm.

[0087] In a particular embodiment, the semiconductor portion is formed by zinc oxide sputtered onto the surface of a rigid (SiO2 / Si) wafer to obtain an oxygen-deficient zinc oxide layer (ZnO1-x) presenting a plurality of hydroxyl (OH) groups on the surface. The as-deposited oxygen-deficient ZnO layer may be of any suitable thickness to suit the desired application. Applicants have found that good results are obtained when the oxygen-deficient ZnO layer has a thickness falling within the range of about 10 nm to about 1 μm.

[0088] Zinc oxide (ZnO) Two different types of ZnO thin films with different oxygen content ratios are prepared by magnetron sputtering, which results in different stoichiometries of the sputtered thin films. The sputtering parameters and associated electrical conductivity are listed in Table 1.

[0089] [Table 2]

[0090] The sputtering parameters are selected to design a thin film with a conductivity in the range of 0.08-0.6 S / m, which gives good sensitivity of the sensor.

[0091] Strontium Titanium Oxide (STO) Two different types of strontium titanium oxide (SrTiO3:STO) thin films are prepared by magnetron sputtering with different oxygen contents. The sputtering parameters are summarized in Table 3.

[0092] [Table 3]

[0093] Oligonucleotides The sensor element of the sensor comprises at least one oligonucleotide, typically a plurality of oligonucleotides, on the surface of the semiconductor portion. The oligonucleotide is selected to be complementary to the nucleic acid sequence or sequences to be detected. As used herein, the term "complementary" means that the nucleic acid sequence or sequences to be detected bind to the oligonucleotide as a result of base pairing over substantially the entire length of the nucleic acid sequence. In some embodiments, the sensor element comprises a plurality of oligonucleotides on the surface of the semiconductor portion.

[0094] The oligonucleotides may be immobilized on the semiconductor portion of the substrate by either physical absorption or chemical bonding. In a preferred form, the oligonucleotides are chemically bonded to the surface of the semiconductor portion.

[0095] Semiconductors such as oxygen-deficient metal oxides or non-oxide semiconductors, including silicon semiconductors, typically contain surface functional groups, such as hydroxyl groups, that are susceptible to covalent bond-forming reactions with surface modifiers, such as silanizing agents (surface modifiers that contain silanizing groups, such as alkoxysilanes). Thus, oligonucleotides can be chemically bonded to the semiconductor moiety by a process that includes: (i) silanizing the non-oxide semiconductor with a silanizing agent having a terminal functional group selected from the group consisting of epoxy groups, thiol groups, amino groups, carboxy groups, and hydroxy groups; and (ii) reacting an appropriately functionalized oligonucleotide with the terminal functional group. As a result of this process, the binding site is fixed to the surface of the semiconductor moiety by an organic linker that is the residue of the silanizing agent.

[0096] Suitable silanizing agents include (3-glycidyloxypropyl)trimethoxysilane (GPS), (3-mercaptopropyl)trimethoxysilane (MTS), (3-aminopropyl)triethoxysilane (APTES), and N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (AEAPTS). For example, when an epoxy-functionalized silanizing agent such as (3-glycidyloxypropyl)trimethoxysilane (GPS) is used, the silanization of the non-oxide semiconductor functionalizes the surface with pendant epoxy groups. Thus, appropriately functionalized oligonucleotides, such as amino-functionalized oligonucleotides, can be immobilized on this surface by conjugation reaction of the epoxy-reactive functional group with an amine.

[0097] In another set of embodiments, the oligonucleotides are initially present on molecules that are pre-functionalized with surface-reactive functional groups, such as silanized groups. Thus, the oligonucleotides can be chemically bound to the semiconductor moiety by contacting the pre-functionalized biomolecule (or other entity) with the semiconductor moiety under conditions suitable to allow covalent bond formation and thus surface immobilization.

[0098] In certain embodiments, the oligonucleotide is attached to the surface of the semiconductor portion via the 3' end of the oligonucleotide.In certain embodiments, the oligonucleotide is attached to the surface of the semiconductor portion via the 5' end of the oligonucleotide.

[0099] The sensor of the present invention can be used to detect a wide variety of nucleic acids, the only practical limitation being that in order to detect a nucleic acid, it is necessary to attach an oligonucleotide (as described above) complementary to the nucleic acid to be detected to the semiconductor moiety. In general, this does not pose a significant difficulty, since with advances in nucleic acid sequencing and oligonucleotide synthesis, the target nucleic acid to be detected is usually well characterized as it relates to the medical disease being diagnosed.

[0100] In one embodiment, the oligonucleotide is complementary to a nucleic acid sequence that has a single point mutation compared to a native DNA sequence. In one embodiment, the oligonucleotide is derived from human BRAF. In one embodiment, the oligonucleotide is derived from the human KRAS gene. In one embodiment, the oligonucleotide is derived from the human PIK3CA gene. In one embodiment, the oligonucleotide is complementary to a nucleic acid that has an insertion or deletion mutation relative to a native DNA sequence. In one embodiment, the oligonucleotide is derived from the human EGFR gene. In one embodiment, the oligonucleotide is complementary to a human microRNA nucleic acid. In one embodiment, the oligonucleotide is derived from the human microRNA miR-371a. In one embodiment, the oligonucleotide is complementary to a nucleic acid that has a common mutation in a human native DNA sequence. In one embodiment, the oligonucleotide is derived from the human DPYD gene. In one embodiment, the oligonucleotide is SEQ ID NO:1 or SEQ ID NO:2.

[0101] The semiconductor portion of the substrate may include an oxide surface layer on the semiconductor portion, and the oxide layer may include surface functional groups that are susceptible to covalent bond formation reactions with the surface modifier. Such passivation layers are generally very thin, so that the binding of oligonucleotides causes a change in the resistance of the underlying high resistivity semiconductor during use. Thus, any oxide surface layer on the surface of the semiconductor portion may be less than 10 nm in thickness.

[0102] Detection Method The present invention also relates to a method for detecting a nucleic acid, comprising the steps of: (a) contacting a sensor element of a sensor described herein with a substance that may contain a nucleic acid, (b) measuring an electrochemical parameter of the sensor that corresponds to the resistance of the sensor, and (c) detecting the presence or absence of a nucleic acid on the sensor element based on the electrochemical parameter measured in step (b).

[0103] The nucleic acid to be detected is typically located in a substance, which may be any substance that contains or may contain a nucleic acid sequence of interest, in some embodiments the substance is a sample solution, for example a bodily fluid such as saliva, sweat, blood or urine, or a liquid sample that is or contains nucleic acid extracted from a tissue or tumor.

[0104] As mentioned above, this method relies on the nucleic acid to be detected being located on the sensor and hybridizing with the oligonucleotide that is complementary to the nucleic acid to be detected.Therefore, the sensor element of the present invention needs to be contacted with the substance for a sufficient period of time for hybridization to occur.For example, if the substance is a fluid, the nucleic acid needs to be given sufficient time to diffuse through the liquid to the oligonucleotide and hybridize with the oligonucleotide.

[0105] In principle, the contact time between the sensor element and the substance sample can be any length of time, so long as the nucleic acid to be detected is in contact with the substance for a sufficient time to hybridize with the oligonucleotide. In certain embodiments, the sensor element is in contact with the substance for a period of 1 minute to 60 minutes. In certain embodiments, the sensor element is in contact with the substance for a period of 1 minute to 30 minutes. In certain embodiments, the sensor element is in contact with the substance for a period of 1 minute to 20 minutes. In certain embodiments, the sensor element is in contact with the substance for a period of 1 minute to 10 minutes. In certain embodiments, the sensor element is in contact with the substance for a period of 5 minutes to 10 minutes.

[0106] After contacting the sensor element with the sample, the sensor element is typically washed with phosphate buffered saline (PBS, pH 7.4) to remove any material from the sensor surface.

[0107] As mentioned above, hybridization of the nucleic acid to the oligonucleotide results in a change in the resistance of the sensor, and therefore, after contacting the sensor element with a substance, an electrochemical parameter of the sensor corresponding to the resistance of the sensor is measured.

[0108] As will be appreciated by those skilled in the art, there are several different electrochemical parameters of a sensor that can be measured that correspond to the resistance of the sensor. For example, a fixed current can be applied across the sensor and the voltage across the sensor can be measured. Alternatively, a fixed voltage can be applied across the sensor and the current flow can be measured.

[0109] In a typical operation of a conductivity sensor, the parameter that is directly measured is the current response when a known voltage (or voltage profile) is applied across the sensor. Thus, in some embodiments, a method for measuring an electrochemical parameter of a sensor includes (i) applying a voltage across the sensor and measuring the current flow through the sensor. Conventional equipment for conductivity sensors, such as a potentiostat, can be used to apply the voltage and measure the current flow.

[0110] However, it is not excluded that a different electrochemical parameter corresponding to the sensor resistance may be measured. For example, it is in principle possible to pass a given current through the sensor and measure the voltage required to achieve this current. The measured voltage then corresponds to the sensor resistance. In one embodiment, an increase in the resistance of the sensor compared to a reference resistance indicates the presence of nucleic acid in the sample.

[0111] The presence or absence of nucleic acid on the sensor element can be detected by comparing the measured electrochemical parameter to a reference value of that parameter for the sensor. If the measured parameter is a current response, the current flow or electrical resistance of the sensor determined from the current flow is relative to a predefined reference current flow or resistance of the sensor corresponding to the presence or absence of nucleic acid on the sensor element. For example, the current flow (or resistance) of the sensor after contact with a substance can be compared to the current flow (or resistance) of the sensor after contact with a reference solution that does not contain nucleic acid. In its simplest form, such a comparison can be used to determine the presence or absence of nucleic acid in a substance. Alternatively, the current flow (or resistance) of the sensor after contact with a sample solution containing nucleic acid can be compared to a calibration curve that plots the current flow (or resistance) of the sensor after contact with a series of reference solutions with known concentrations of nucleic acid. In this way, the concentration of nucleic acid in the sample solution can be calculated.

[0112] The method can optionally include one or more preparation steps between contacting the sensor element with the substance and applying the voltage. For example, if the substance is a sample solution, the sensor element can be incubated at a defined condition (e.g., temperature) for a defined time to allow binding of nucleic acid (if present in the sample solution) to the oligonucleotide site. The sample solution can then be removed from the sensor, and the sensor element can be washed and / or dried before performing a conductivity measurement.

[0113] Alternatively, the sensor can be used as an invasive sensor inserted into the human body for in situ detection of nucleic acids, for example when incorporated into a microneedle, hi another embodiment, the sensor is incorporated into a wearable device for monitoring nucleic acids in human sweat.

[0114] In principle, the sensor of the present invention can be used to detect the presence of any nucleic acid of interest. Indeed, although the sensor is illustrated with respect to BRAF and cancer-associated sequences, in principle, the sensor can be used to detect any nucleic acid. The only limitation to the sensor's ability to detect nucleic acid sequences.

[0115] How the sensor is manufactured The present invention also relates to a method for producing a sensor for detecting nucleic acids, the method comprising the steps of: preparing a substrate including a semiconductor portion; a pair of terminal electrodes are fabricated on the substrate in a spaced apart and opposing relationship such that the semiconductor portion of the substrate is disposed between and in electrical contact with the terminal electrodes, and a conductive path between the terminal electrodes passes through the semiconductor portion; oligonucleotides are then immobilized on the surface of the semiconductor portion, thereby fabricating a sensor element including (i) the semiconductor portion and (ii) the oligonucleotides on the surface of the semiconductor portion.

[0116] In one form of the invention, a substrate 102 is provided in step A, as shown in the schematic diagram of Figure 2. The substrate 102 includes a semiconductor portion 110 that includes a semiconductor material 112 as described herein. In the embodiment shown in Figure 2, the substrate 102 includes the semiconductor portion 110 as an integral part of the substrate, such that the remainder of the substrate is composed of the same semiconductor material 112. Alternatively, the substrate 102 may include the semiconductor portion 110 formed as a separate thin surface layer on an underlying support layer, which may be composed of any suitable material capable of accepting and supporting the semiconductor layer 110.

[0117] In step B, a pair of terminal electrodes 104, 106 are fabricated on the substrate 102 in a spaced apart, opposing relationship. The electrodes are fabricated such that the semiconductor portion 110 of the substrate is disposed between and in electrical contact with the terminal electrodes 104, 106. Thus, the conductive path 120 between the terminal electrodes 104, 106 passes through the semiconductor portion 110 and thus through the semiconductor material 112.

[0118] In step C, oligonucleotides 114 are immobilized to surface 116 of the semiconductor portion, thereby creating sensor element 108. Although Figure 1 shows a single binding site, it will be appreciated that multiple oligonucleotides 114 may be immobilized to surface 116. Sensor element 108 includes semiconductor portion 110 and oligonucleotides 114. Thus, sensor 100 is fabricated after performing steps A, B, and C, as previously described herein with reference to Figure 1.

[0119] In another form of the manufacturing method of the present invention, a substrate 102 is provided in step A, as shown in the schematic diagram of Figure 4. The substrate 102 includes a semiconductor portion 110 present as a layer 112 of a semiconductor material as described herein. In the embodiment shown in Figure 4, the substrate 102 includes the semiconductor portion 110 formed as a separate thin surface layer 112 on an underlying support layer, which may be composed of any suitable material capable of accepting and supporting the semiconductor layer 110.

[0120] In step B, a pair of terminal electrodes 104, 106 are fabricated on the substrate 102 in a spaced apart, opposing relationship. The electrodes are fabricated such that the semiconductor portion 110 of the substrate is disposed between and in electrical contact with the terminal electrodes 104, 106. Thus, the conductive path 120 between the terminal electrodes 104, 106 passes through the semiconductor portion 110 and thus through the semiconductor material 112.

[0121] In step C, oligonucleotides 114 are immobilized to surface 116 of the semiconductor portion, thereby creating sensor element 108. Although Figure 4 shows a single oligonucleotide, it will be appreciated that multiple oligonucleotide moieties 114 may be immobilized to surface 116. Sensor element 108 includes semiconductor portion 110 and oligonucleotide 114. Thus, sensor 100 is fabricated after performing steps A, B, and C, as previously described herein with reference to Figure 3.

[0122] The substrate including the semiconductor portion may be according to any of the embodiments described herein in relation to the sensor of the present invention.

[0123] The terminal electrodes can be fabricated on the substrate by any suitable method. In some embodiments, the terminal electrodes are formed by microfabrication techniques. Gold terminal electrodes can be formed by evaporating a thin gold film (250 nm with a 100 nm chromium adhesion layer) onto the semiconductor layer using electron beam lithography. The as-deposited thin gold film is then patterned using standard photolithography and wet etching techniques to define a pair of terminal electrodes.

[0124] The oligonucleotide binding sites may be immobilized on the surface of the semiconductor moiety by either physical absorption or chemical bonding. In a preferred form, the oligonucleotides are chemically bonded to the surface of the semiconductor moiety.

[0125] Materials used for the semiconductor portion of the substrate of the present invention, such as silicon semiconductors or non-oxide semiconductors including oxygen-deficient metal oxides, typically contain surface functional groups, such as hydroxy groups, that are susceptible to covalent bond formation reactions with surface modifiers, such as silanizing agents (surface modifiers that contain silanizing groups, such as alkoxysilanes). Thus, oligonucleotides can be chemically bonded to the semiconductor portion by a process that includes: (i) silanizing the semiconductor portion with a silanizing agent having a terminal functional group selected from the group consisting of epoxy groups, thiol groups, amino groups, carboxy groups, and hydroxy groups; and (ii) reacting a precursor that includes an oligonucleotide with the terminal functional group. As a result of this process, the oligonucleotide is immobilized on the surface of the semiconductor portion by an organic linker that is the residue of the silanizing agent.

[0126] Suitable silanizing agents include (3-glycidyloxypropyl)trimethoxysilane (GPS), (3-mercaptopropyl)trimethoxysilane (MTS), (3-aminopropyl)triethoxysilane (APTES), and N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (AEAPTS).

[0127] As shown in the schematic diagram of FIG. 5(1), the semiconductor portion of the substrate beneath and between the gold (Au) terminal electrodes comprises a semiconductor material, in this case a high resistivity intrinsic silicon wafer. In step (2), the surface of the semiconductor portion is contacted with a silanizing agent, which may optionally be an epoxy-functionalized silanizing agent such as (3-glycidyloxypropyl)trimethoxysilane (GPS). The silanizing agent reacts with the surface hydroxy (-OH) functional groups of the semiconductor portion, thereby immobilizing the silanizing agent to the surface via a covalent bond and functionalizing the surface with a pendant conjugated group, in this case an epoxy group. Then, in step (3), an oligonucleotide is immobilized to the surface by a conjugation reaction of an epoxy-reactive functional group present in the oligonucleotide, in this case an amine (-NH2). That is, the oligonucleotide is immobilized to the surface of the semiconductor portion by an organic linking group that is a residue of the silanizing agent. In use, as shown in step 4, a nucleic acid hybridizes to the oligonucleotide to form an immobilized nucleic acid molecule on the sensor.

[0128] The same process steps for another embodiment are shown in the schematic diagram of FIG. 6. In FIG. 6(1), a sensor is provided with the semiconductor portion of the substrate provided as a layer on the support of the substrate, below and between the gold (Au) terminal electrodes. The layer shown is an oxygen-deficient zinc oxide layer. In step (2), the surface of the semiconductor portion is contacted with a silanizing agent, which may optionally be an epoxy-functionalized silanizing agent such as (3-glycidyloxypropyl)trimethoxysilane (GPS). The silanizing agent reacts with the surface hydroxy (-OH) functional groups of the semiconductor portion, thereby immobilizing the silanizing agent to the surface via a covalent bond and functionalizing the surface with a pendant conjugated group, in this case an epoxy group. Then, in step (3), an oligonucleotide is immobilized to the surface by a conjugation reaction of an epoxy-reactive functional group present in the oligonucleotide, in this case an amine (-NH2). That is, the oligonucleotide is immobilized to the surface of the semiconductor portion by an organic linking group that is the residue of the silanizing agent. In use, as shown in step 4, the nucleic acid hybridizes with the oligonucleotide to form an immobilized nucleic acid molecule on the sensor. EXAMPLES

[0129] Materials and Methods High resistivity silicon wafers (diameter 100 mm) with resistivity of 1000-2000 ohm·cm were purchased from D&X Co., Ltd., Japan, and were single-sided polished silicon wafers. The orientation of the 1000-2000 ohm·cm wafers was <100> and the thickness was 500±10 μm.

[0130] Silicon wafer sensors were fabricated by patterning two terminal in-plane electrodes on a high resistivity silicon wafer using standard photolithography processes. The electrode gap can range from 1-2 μm to 100 μm. However, this electrode gap was optimized to be 40 μm for best sensor performance. The electrode length ranged from 200 μm to 4000 μm. The optimal electrode length was 4000 μm. The sensor element area (silicon substrate area between the electrodes) was 16 × 10 -8 m 2 It was confirmed that the metal oxide layer had the same specifications.

[0131] The conductance of the sensor was measured using a commercial current source meter (B2901A precision source / measurement unit from Keysight Technologies). The sensor was placed on an LTS120 Linkam stage as a sensor holder for all measurements. Keysight Quick IV Measurement software was used for data acquisition. The bias between the electrodes was maintained at 1.8 V. Resistance measurements of the sensor were taken after immobilization of the oligonucleotides and hybridization of the complementary DNA. The data acquisition time for a given sensor was 1 min.

[0132] Wild type DNA (NA 12878) in PBS and melanoma A375 DNA carrying the point mutation V600E in PBS were provided by the Peter MacCallum Cancer Centre and were used as received.

[0133] Example 1. Preparation of GPS silanized silicon wafer sensor: Silicon wafer sensors were fabricated by patterning two terminal in-plane electrodes on a high resistivity silicon wafer using standard photolithography processes. The electrode gap can range from 1-2 μm to 100 μm. However, this electrode gap was optimized to be 40 μm for best sensor performance. The electrode length ranged from 200 μm to 4000 μm. The optimal electrode length was 4000 μm. The sensor element area (silicon substrate area between the electrodes) was 16 × 10 -68 m 2 It was.

[0134] Silanization of silicon wafer sensor surfaces with (3-glycidyloxypropyl)trimethoxysilane (GPS) was performed after exposing freshly prepared sensor devices to O2 plasma (Plasma Cleaner PDC-002, Harrick Plasma) for 10 min to activate the hydroxyl groups on the silicon surface. Then, 20 μL of the freshly prepared GPS solution was drop-cast onto an Al foil, which was placed in a vacuum desiccator, resulting in GPS vapor within the desiccator. The O2 plasma-cleaned silicon sensors were then exposed to this GPS vapor for 30–45 min in an LC200 glove box system. The silanized silicon wafer sensors were then rinsed thoroughly with Milli-Q water for 2 min to remove unbound silane groups from the surface. The cleaned sensors were then heated at 150 °C for 10 min to enhance the binding of the silane groups to the silicon wafer surface. These GPS-silanized silicon wafer sensors, functionalized with surface epoxide functional groups chemically bonded to the substrate surface, were then used to immobilize oligonucleotides.

[0135] Example 2. Preparation of GPS silanized zinc oxide wafer sensor: The sensors were fabricated by depositing 80-100 nm thick thin films of metal oxides, such as oxygen-deficient zinc oxide (ZnO), which act as the sensing layer in the biosensor, on rigid substrates (50-300 nm SiO2 / 500 μm Si) and flexible plastic substrates (polyimide foil, 75-125 μm thick) according to standard micro-nano fabrication techniques. The sensing layer composition was fabricated by reactive sputtering, resulting in oxygen-deficient metal oxide films with conductances in the range of 1-2 Siemens / m. For conductance measurements, a 16×10 -8 m 2 Two terminal in-plane electrodes with a detection area of ​​100 nm are patterned and fabricated.

[0136] Freshly prepared ZnO devices were exposed to O2 plasma (commercially available Plasma Cleaner PDC-002, Harrick Plasma) for 10 min to remove organic contaminants from the device surface and to activate the hydroxyl groups on the ZnO surface. Then, 20 μL of the freshly prepared GPS solution was drop-cast onto a commercial Al foil placed in a vacuum desiccator, resulting in GPS vapor within the desiccator. The O2 plasma-cleaned ZnO sensors were then exposed to this GPS vapor for 1–2 h. The exposure of the ZnO sensors to GPS vapor was performed within an LC200 glove box system. Once the exposure to GPS vapor was complete, the ZnO sensors were thoroughly rinsed with Mili-Q water for 2 min to remove unbound silane groups from the ZnO devices. The cleaned ZnO sensors were then heated at 150 °C for 10 min to enhance the binding of the silane groups to the ZnO surface. These GPS-silanized sensors were used for the immobilization of oligonucleotides.

[0137] Example 3. Immobilization of oligonucleotides This example illustrates the immobilization of oligonucleotides to the ZnO sensor of Example 2. Similar methods can be used to immobilize oligonucleotides to other sensors.

[0138] BRAF 3'-amine DNA oligonucleotide (5'-GGTCTAGCTACAGAGAAATCTCGAT / 3AmMO / -3') and BRAF 5'-amine DNA oligonucleotide (5'- / AmMC6 / GGTCTAGCTACAGAGAAATCTCGAT-3') were purchased from Integrated DNA Technologies, Inc., USA and used as received. A 15 μL volume of freshly prepared 35 pM oligonucleotide solution (prepared in pH 7.4 phosphate-buffered saline (PBS)) was drop-cast onto each freshly GPS-silanized ZnO sensor and incubated for 1 h to allow the oligonucleotides to be immobilized onto the ZnO sensor. The sensors were then rinsed extensively with pH 7.4 PBS solution to remove unbound oligonucleotides. The PBS-washed ZnO sensors were then dried under N2 gas for 2 min prior to conductivity measurements.

[0139] Example 4. Conductivity measurement of DNA-ZnO sensor Wild-type DNA (NA 12878) in PBS and melanoma A375 DNA with point mutant V600E in PBS were provided by Peter MacCallum Cancer Centre and used as received. Prior to the addition of DNA samples, the baseline conductance of the oligonucleotide-immobilized ZnO sensor was measured. A volume of 15 μL of DNA solution (wild-type DNA or point mutant DNA) was drop-cast onto the oligonucleotide-immobilized ZnO sensor and incubated for 10 min. After 10 min, the remaining DNA solution on the sensor was removed and the surface was dried under N2 gas before conductance measurements were performed. For cross-selection measurements, a series of DNA mixtures were prepared by premixing the two types of DNA samples at volume ratios of 1:200, 1:100, 1:1, 100:1, and 200:1 (point mutant DNA:wild-type DNA). As the molar concentrations of the stock DNA samples were the same, the volume ratio of the two DNA types was the same as the molar ratio of the two DNA types.

[0140] Both 3'-amine and 5'-amine oligonucleotides showed a reversal of polarity in the resistance change upon hybridization with the point mutant DNA strand compared to the resistance change obtained for hybridization with the wild-type DNA strand (Figure 7). The change in resistance is the percentage change in the resistance of the device after DNA hybridization relative to the baseline resistance. The baseline resistance is the resistance of the oligonucleotide-immobilized device before DNA addition. The resistance of the device increased upon hybridization with the point mutant DNA, whereas the resistance of the device decreased after hybridization with the wild-type DNA. This result suggests that the point mutant DNA acts as an electron acceptor and the wild-type DNA acts as an electron donor upon hybridization with both types of oligonucleotides. The reverse polarity of the resistance change for the point mutant DNA highlights the high sensitivity of these conductometric devices for detecting cancer DNA such as melanoma that has a single base pair difference (V600E mutant) compared to healthy DNA. The absolute change in resistance obtained for all samples tested is higher in the 3'-amine oligonucleotide-immobilized device than in the 5'-amine oligonucleotide-immobilized device. This indicates that the charge transfer process is much more likely to occur in 3'-amine oligonucleotides than in 5'-amine oligonucleotides. The resistance change in PBS solvent is of the same polarity as that of wild-type DNA, but not the same polarity as that of the point mutant DNA. Therefore, the contribution from the matrix of the point mutant DNA solution to the resistance change can be neglected.

[0141] Example 5. Cross-selectivity experiments Cross-selectivity measurements were also performed to investigate the feasibility of the conductance measurement devices for detecting V600E point mutant DNA in the presence of other DNA. Both types of oligonucleotide immobilized devices selectively detected V600E point mutant DNA in the presence of wild-type DNA (Figure 8). The resistance change increased with increasing V600E point mutant DNA fraction and reached saturation values ​​when the molar fraction of point mutant DNA:wild-type DNA exceeded 100:1. This trend was observed for both types of oligonucleotides. Both oligonucleotides successfully detected V600E point mutant DNA at a point mutant DNA:wild-type DNA molar ratio of 1:200, which corresponds to a clinically significant allele fraction ratio (i.e., 0.5%). This result highlights the high sensitivity and high selectivity of these conductance sensors in detecting V600E point mutant DNA.

[0142] Example 6. Conductivity measurement of DNA on a Si sensor The experiments were carried out using the method of Example 4, but using the silicon sensor with immobilized 5'-amine oligonucleotides of Example 1. The results of the conductance measurement experiments are shown in FIG.

[0143] definition Whenever a range is given herein, e.g., a temperature range, a time range, or a concentration range, all intermediate ranges and subranges, as well as all individual values ​​that fall within the given range, are intended to be included in the disclosure. Of course, any subrange contained in the description herein, or any individual value within a range or subrange, can be excluded from the claims herein.

[0144] All definitions and those used herein should, of course, be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0145] As used herein, the indefinite articles "a" and "an" should be understood to mean "at least one," unless expressly indicated otherwise.

[0146] The phrase "and / or" as used herein should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related to the elements specifically identified or not. Thus, as a non-limiting example, a reference to "A and / or B", when used with an open-ended expression such as "comprising", may in one embodiment refer to only A (optionally including elements other than B), in another embodiment may refer to only B (optionally including elements other than A), and in yet another embodiment may refer to both A and B (optionally including other elements), and so forth.

[0147] While the invention has been described in conjunction with a limited number of embodiments, it will be apparent to those skilled in the art that many alternatives, modifications, and variations are possible in light of the foregoing description, and therefore, the present invention is intended to embrace all such alternatives, modifications, and variations that may fall within the spirit and scope of the invention disclosed.

[0148] When the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims), they should be interpreted as specifying the presence of stated features, integers, steps or components, but not excluding the presence of one or more other features, integers, steps or components, or groups thereof.

Claims

1. A sensor for detecting nucleic acid, comprising: a substrate; a pair of terminal electrodes disposed on the substrate in a spaced-apart and opposing relationship; a sensor element between the pair of terminal electrodes and in electrical contact with the pair of terminal electrodes, the sensor element comprising: (i) a semiconductor portion of the substrate, wherein a conductive path between the terminal electrodes penetrates the semiconductor portion; and (ii) an oligonucleotide on a surface of the semiconductor portion, the oligonucleotide being complementary to the nucleic acid to be detected; wherein hybridization between the nucleic acid and the oligonucleotide causes a change in the resistance of the sensor. A sensor for detecting nucleic acid, wherein hybridization between the nucleic acid and the oligonucleotide causes a change in the resistance of the sensor.

2. The sensor according to claim 1, wherein the semiconductor portion has a resistivity greater than 100 ohm·cm.

3. The sensor according to claim 1, wherein the semiconductor portion has a resistivity in the range of about 1000 ohm·cm to about 10000 ohm·cm.

4. The sensor according to claim 1, wherein the semiconductor portion comprises a high-resistivity non-oxide semiconductor having a resistivity greater than 100 ohm·cm.

5. The sensor according to claim 4, wherein the non-oxide semiconductor is an intrinsic silicon semiconductor.

6. The sensor according to claim 5, wherein the silicon semiconductor is a float-zone silicon semiconductor.

7. The sensor according to claim 1, wherein the semiconductor portion comprises an oxygen-deficient metal oxide selected from the group consisting of zinc oxide (ZnO), strontium titanate (STO), tin oxide (SnO₂), and titanium dioxide (TiO₂).

8. The sensor according to claim 1, wherein the oligonucleotide is chemically bonded to the semiconductor portion by a process comprising: (i) silanizing the semiconductor portion with a silanizing agent having a terminal functional group selected from the group consisting of an epoxy group, a thiol group, an amino group, a carboxy group, and a hydroxy group; and (ii) reacting the oligonucleotide with the terminal functional group.

9. The sensor according to claim 1, wherein the oligonucleotide is complementary to a nucleic acid having a single-point mutation with respect to a natural DNA sequence.

10. The sensor according to claim 1, wherein the oligonucleotide is a BRAF oligonucleotide.

11. A method for detecting nucleic acid, comprising: Step (a) of contacting the sensor element of the sensor according to claim 1 with a substance that may contain nucleic acid; Step (b) of measuring the electrochemical parameter of the sensor corresponding to the resistance of the sensor; Step (c) of detecting the presence or absence of nucleic acid on the sensor element based on the electrochemical parameter measured in step (b); A method comprising the above steps.

12. A method for manufacturing a sensor for detecting nucleic acid, comprising: Step a. of preparing a substrate including a semiconductor portion; Step b. of fabricating a pair of terminal electrodes on the substrate in a spaced-apart and facing relationship, wherein the semiconductor portion of the substrate is disposed between the terminal electrodes and in electrical contact with the terminal electrodes, and the conductive path between the terminal electrodes penetrates the semiconductor portion; Step c. of immobilizing an oligonucleotide complementary to the nucleic acid to be detected on the surface of the semiconductor portion, thereby fabricating a sensor element including (i) the semiconductor portion and (ii) the oligonucleotide; A method comprising the above steps.

13. The method according to claim 12, wherein the semiconductor portion has a resistivity greater than 100 ohm·cm.

14. The method according to claim 13, wherein the oligonucleotide is chemically bonded to the semiconductor portion.

15. The method according to claim 14, wherein the oligonucleotide is chemically bonded to the semiconductor portion by a process including (i) silanizing the semiconductor portion with a silanizing agent having a terminal functional group selected from the group consisting of an epoxy group, a thiol group, an amino group, a carboxy group, and a hydroxy group, and (ii) reacting the oligonucleotide with the terminal functional group.

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