CN111850168A - Field effect transistor sensor for detecting virus SARS-CoV-2 nucleic acid and its preparing method and use - Google Patents
Field effect transistor sensor for detecting virus SARS-CoV-2 nucleic acid and its preparing method and use Download PDFInfo
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- G01N27/4146—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS involving nanosized elements, e.g. nanotubes, nanowires
Abstract
The invention relates to a field effect transistor sensor for detecting virus SARS-CoV-2 nucleic acid and its preparation method and application, the field effect transistor sensor includes insulating substrate, semiconductor layer and electrode arranged on the insulating substrate, the semiconductor layer is provided with exposed semiconductor channel, the semiconductor channel is internally decorated with fixed DNA probe; the DNA probe can be directly combined with a novel coronavirus SARS-CoV-2 nucleic acid sequence to be detected or combined with the DNA subjected to reverse transcription through base complementary matching, so that the novel coronavirus SARS-CoV-2 nucleic acid sequence to be detected or the DNA subjected to reverse transcription of the novel coronavirus nucleic acid sequence to be detected contacts the surface of the field effect transistor sensor. The invention realizes the purpose of accurately detecting the novel coronavirus nucleic acid, and compared with the common polymerase chain reaction and fluorescent quantitative polymerase chain reaction methods used for detecting the virus nucleic acid at present, the detection time is greatly shortened, the sensitivity is high, the specificity is good, and the application prospect is good.
Description
Technical Field
The invention belongs to the technical field of sensors, and particularly relates to a field effect transistor sensor for detecting virus SARS-CoV-2 nucleic acid, and a preparation method and application thereof.
Background
The new type coronavirus pneumonia (SARS-CoV-2) is more contagious than SARS virus, so that it is very important to establish a quick detection and diagnosis method for preventing and controlling disease condition. The new type of coronavirus belongs to the genus beta coronavirus, its genetic material is single RNA chain, and its nucleic acid (DNA or RNA) is the genetic material of the virus, and its unique characteristic nucleic acid sequence is screened and tested so as to determine the pathogen.
At present, the detection of novel coronavirus nucleic acid mainly depends on common polymerase chain reaction and fluorescent quantitative polymerase chain reaction methods, and the detection methods have high requirements on instruments and laboratory environments, are complex in operation process and long in detection time, and inevitably have false negative results, so that the development of a simple, high-efficiency and high-specificity novel coronavirus nucleic acid detection method is urgently needed.
Disclosure of Invention
The invention aims to overcome the defects of the common polymerase chain reaction and fluorescence quantitative polymerase chain reaction method in the aspect of novel coronavirus nucleic acid detection, and provides a field effect transistor sensor for detecting virus SARS-CoV-2 nucleic acid, a preparation method and application thereof.
The invention realizes the purpose of accurately detecting the novel coronavirus nucleic acid, and compared with the common polymerase chain reaction and fluorescent quantitative polymerase chain reaction methods used for detecting the virus nucleic acid at present, the detection time is greatly shortened, the sensitivity is high, the specificity is good, and the application prospect is good.
The purpose of the invention is realized by the following technical scheme:
a field effect transistor sensor for detecting novel coronavirus SARS-CoV-2 nucleic acid comprises an insulating substrate, a semiconductor layer and an electrode, wherein the semiconductor layer and the electrode are arranged on the insulating substrate, an exposed semiconductor channel is arranged on the semiconductor layer, and a fixed DNA probe is modified in the semiconductor channel;
the DNA probe can be directly combined with a novel coronavirus SARS-CoV-2 nucleic acid sequence to be detected or can be combined with DNA subjected to reverse transcription of a novel coronavirus SARS-CoV-2 nucleic acid characteristic sequence to be detected through base complementary matching, so that the novel coronavirus SARS-CoV-2 nucleic acid sequence to be detected or the DNA subjected to reverse transcription of the novel coronavirus SARS-CoV-2 nucleic acid sequence to be detected contacts the surface of the field effect transistor sensor, and whether the novel coronavirus SARS-CoV-2 nucleic acid exists is detected through detecting the current change of the field effect transistor sensor in real time.
Preferably, the DNA probe is a single DNA strand or a two-dimensional, three-dimensional nucleic acid nanostructure self-assembled by oligonucleotide strand complementary pairing, wherein various nano-two-dimensional and three-dimensional nucleic acid structures are self-assembled by oligonucleotide strand complementary pairing;
wherein, the nucleic acid characteristic sequence of the novel coronavirus SARS-CoV-2 is one or more of a coding non-structural protein gene (ORF1ab), a coding spike protein gene (S), a coding nucleocapsid protein gene (N) or a coding envelope protein gene (E).
Preferably, the DNA probe is modified and fixed to the surface of the semiconductor channel by an adsorption method, a cross-linking method, a covalent bonding method, an entrapment method, or a biological tissue fixation method.
Preferably, the insulating substrate is silicon dioxide, quartz, insulating glass, mica, a polyethylene terephthalate film, a polyimide film or a polydimethylsiloxane film.
Preferably, the semiconductor layer is graphene, an oxide semiconductor, a transition metal chalcogenide, silicon, or germanium.
Preferably, the electrode is a patterned electrode, the thickness of the electrode material is 20-2000 nm, and the electrode can adopt metal simple substances such as gold, silver, copper, titanium, chromium and the like, conductive silicide, carbide, conductive polymer and the like.
A method for preparing a field effect transistor sensor for detecting novel coronavirus SARS-CoV-2 nucleic acid comprises the following steps:
(1) preparing a field effect transistor device with a semiconductor channel exposed;
(2) modifying and fixing a DNA probe on the surface of the semiconductor channel;
(3) when the liquid groove or the micro-fluidic channel is manufactured on the field effect transistor, an object to be detected is added into the liquid groove or the micro-fluidic channel to enable the object to be detected to contact with a semiconductor channel, and high-sensitivity detection of the novel coronavirus SARS-CoV-2 nucleic acid is realized through real-time current change.
Preferably, the specific method in the step (2) is that a DNA probe is designed, a liquid tank is manufactured on a semiconductor channel, the DNA probe solution is placed in the liquid tank, the DNA probe is modified and fixed on the surface of the semiconductor channel through an adsorption method, a cross-linking method, a covalent bonding method, an embedding method or a biological tissue fixing method, the DNA probe is formed through self-assembly, the self-assembly process is realized through a heating annealing method, the heating temperature is kept at 85-95 ℃ for 2-20 minutes, and then the temperature is reduced to 10-20 ℃ at a constant speed within 1-10 minutes.
The field effect transistor sensor is used for preparing a novel nucleic acid detection reagent for coronavirus SARS-CoV-2.
The novel coronavirus SARS-CoV-2 nucleic acid is extracted from a sampled virus specimen after treatment, and the specific method comprises the following steps: extracting a proper amount of virus specimen in a collection tube of a virus sample, putting the virus specimen into a nucleic acid extraction kit, then putting the kit into an automatic or semi-automatic nucleic acid extractor, and extracting novel coronavirus SARS-CoV-2 nucleic acid from the kit, wherein the virus specimen can be a blood specimen or an upper respiratory tract specimen, such as a throat swab and a nose swab.
The DNA sequence obtained by reverse transcription of the nucleic acid sequence of the novel coronavirus SARS-CoV-2 to be detected comprises the following specific steps: and carrying out reverse transcription treatment on the extracted novel coronavirus SARS-CoV-2 nucleic acid by using a reverse transcription kit, wherein the treatment method comprises the steps of carrying out heating annealing after the treatment by using the reverse transcription kit, keeping the heating temperature at 20-30 ℃ for 5-20 minutes, keeping the heating temperature at 35-45 ℃ for 1-2.5 hours, and keeping the temperature at 80-90 ℃ for 2-10 minutes.
The field effect transistor sensor is a sensing device which can change the performance of a semiconductor in the adsorption and desorption processes of conducting and monitoring charged molecules, ions and the like and then respond to a detection object in the form of an electric signal, and has the advantages of no mark, high sensitivity, high selectivity, real-time monitoring and the like.
In the invention, during detection, a liquid groove or a micro-fluidic channel is manufactured on a field effect transistor, and a solution to be detected is added in the liquid groove or the micro-fluidic channel, so that a DNA probe modified on the field effect transistor is complementarily combined with a novel coronavirus SARS-CoV-2 nucleic acid sequence in the solution to be detected or DNA obtained after reverse transcription of the novel coronavirus nucleic acid, the DNA probe is contacted with the surface of a channel of the field effect transistor, current change is detected in real time, and the minimum detection concentration is 0.1 copy number/microliter.
Compared with the common polymerase chain reaction and the fluorescent quantitative polymerase chain reaction, the method has the advantages that: a field effect transistor sensor for detecting SARS-CoV-2 nucleic acid is constructed as a new method for detecting novel coronavirus nucleic acid, and its principle is that the change of device conductivity is caused by designing and synthesizing DNA probes with various different structures and novel coronavirus SARS-CoV-2 nucleic acid sequence or reverse transcription DNA sequence complementary hybridization of novel coronavirus nucleic acid gene to implement real-time detection.
Drawings
FIG. 1 is a schematic view of the surface of a field effect transistor in embodiment 1;
FIG. 2 is a scanning electron micrograph obtained in example 1;
FIG. 3 is a current response curve for detecting the novel coronavirus SARS-CoV-2 nucleic acid extracted from a blood sample in example 1;
FIG. 4 is a graph showing the current response curve for detecting the novel coronavirus SARS-CoV-2 nucleic acid extracted from the upper respiratory tract pharyngeal swab specimen in example 2.
Detailed Description
For the purpose of enhancing the understanding of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
Example 1
Firstly, preparing single-layer graphene on a copper foil with the thickness of 25 microns by using a chemical vapor deposition method, and transferring the prepared graphene to a clean silicon dioxide/silicon substrate by using an electrochemical stripping method. And preparing a patterned electrode by adopting an ultraviolet lithography method and an oxygen plasma etching technology, and preparing a chromium/gold (5/40 nm) source/drain electrode by adopting a thermal evaporation technology to obtain the graphene field effect transistor.
And then, soaking the graphene field effect transistor in an N-hydroxysuccinimide benzoate solution for 1-2 hours, then washing the graphene field effect transistor with ultrapure water, and adsorbing the pyrenyl group at one end of the molecule to the surface of the field effect transistor through pi-pi stacking effect. A liquid tank is made to be placed on the graphene channel, and the capacity of the liquid tank is about 80-100 microliters.
Designing a DNA three-dimensional nanostructure probe which can be combined with a reverse transcription DNA sequence of a novel coronavirus SARS-CoV-2 nucleic acid gene. The probe is formed by self-assembling four DNA single strands through base complementary pairing, and the method for synthesizing the DNA probe is to heat DNA mixed liquor to 95 ℃ for 5 minutes, then uniformly cool the mixed liquor to 15 ℃ within 1 minute, and finally store the mixed liquor at 4 ℃. And then, placing the DNA probe solution in a liquid tank, wherein an amino group connected to a base of the DNA probe can be combined with an N-hydroxysuccinimide group at the other end of the N-hydroxysuccinimide benzoate molecule to fix the probe on an interface of a graphene transistor, wherein the required time is about 8-12 hours, and then, washing the graphene transistor with ultra-pure water.
Fig. 1 is a schematic view of the surface of a field effect transistor in example 1, and fig. 2 is a scanning electron micrograph obtained in example 1.
The blood sample of the novel coronavirus SARS-CoV-2 is treated to extract virus nucleic acid, the extraction method is that 200 microliter of sample is extracted from the collecting tube of the virus blood sample, and then the sample is put into a nucleic acid extraction kit, and then the kit is put into an automatic or semi-automatic nucleic acid extractor, and the extracted novel coronavirus SARS-CoV-2 nucleic acid is stored at 4 ℃.
Then the extracted novel coronavirus SARS-CoV-2 nucleic acid is treated by reverse transcription by a reverse transcription kit, wherein the treatment method comprises the steps of heating to 25 ℃ for 10 minutes, then heating to 37 ℃ for 2 hours, and finally heating to 85 ℃ for 5 minutes. The DNA sequence obtained after reverse transcription was stored at 4 ℃.
Finally, the solution containing the reverse transcription DNA sequence of the novel coronavirus nucleic acid prepared as described above was added to the liquid tank, and the minimum detection concentration was 0.1 copy number/microliter, and FIG. 3 is a current response curve for detecting the novel coronavirus SARS-CoV-2 nucleic acid extracted from the blood specimen in example 1, whereby it was found that the sensor constructed in this manner had a highly sensitive response to the novel coronavirus SARS-CoV-2 nucleic acid extracted from the blood specimen, and the detection limit was 0.3 copy number/microliter.
Example 2
Firstly, preparing single-layer graphene on a copper foil with the thickness of 25 microns by using a chemical vapor deposition method, and transferring the prepared graphene to a clean silicon dioxide/silicon substrate by using an electrochemical stripping method. And preparing a patterned electrode by adopting an ultraviolet lithography method and an oxygen plasma etching technology, and preparing a chromium/gold (5/40 nm) source/drain electrode by adopting a thermal evaporation technology to obtain the graphene field effect transistor.
And then, soaking the graphene field effect transistor in an N-hydroxysuccinimide benzoate solution for 1-2 hours, then washing the graphene field effect transistor with ultrapure water, and adsorbing the pyrenyl group at one end of the molecule to the surface of the field effect transistor through pi-pi stacking effect. A liquid tank is made to be placed on the graphene channel, and the capacity of the liquid tank is about 80-100 microliters.
Designing a DNA three-dimensional nanostructure probe which can be combined with a reverse transcription DNA sequence of a novel coronavirus SARS-CoV-2 nucleic acid gene. The probe is formed by self-assembling four DNA single strands through base complementary pairing, and the method for synthesizing the DNA probe is to heat DNA mixed liquor to 95 ℃ for 5 minutes, then uniformly cool the mixed liquor to 15 ℃ within 1 minute, and finally store the mixed liquor at 4 ℃. And then, placing the DNA probe solution in a liquid tank, wherein an amino group connected to a base of the DNA probe can be combined with an N-hydroxysuccinimide group at the other end of the N-hydroxysuccinimide benzoate molecule to fix the probe on an interface of a graphene transistor, wherein the required time is about 8-12 hours, and then, washing the graphene transistor with ultra-pure water.
The upper respiratory tract pharynx swab sample of the novel coronavirus SARS-CoV-2 is processed to extract virus nucleic acid, the extraction method comprises the steps of extracting 200 microliter of sample from a collection tube of the virus upper respiratory tract pharynx swab sample, putting the sample into a nucleic acid extraction kit, then putting the kit into an automatic or semi-automatic nucleic acid extractor, and extracting to obtain the novel coronavirus SARS-CoV-2 nucleic acid and storing at 4 ℃.
Then the extracted novel coronavirus SARS-CoV-2 nucleic acid is treated by reverse transcription by a reverse transcription kit, wherein the treatment method comprises the steps of heating to 25 ℃ for 10 minutes, then heating to 37 ℃ for 2 hours, and finally heating to 85 ℃ for 5 minutes. The DNA sequence obtained after reverse transcription was stored at 4 ℃. Finally, the solution containing the reverse transcription DNA sequence of the novel coronavirus nucleic acid prepared as described above was added to the liquid tank, and the minimum detection concentration was 0.1 copy number/microliter, and FIG. 4 is a current response curve for detecting the novel coronavirus SARS-CoV-2 nucleic acid extracted from the upper respiratory tract pharyngeal swab specimen in example 2, whereby it was found that the sensor constructed in this manner had a highly sensitive response to the novel coronavirus SARS-CoV-2 nucleic acid extracted from the upper respiratory tract pharyngeal swab specimen, and the detection limit was 0.1 copy number/microliter.
Example 3
Firstly, preparing single-layer graphene on a copper foil with the thickness of 25 microns by using a chemical vapor deposition method, and transferring the prepared graphene to a clean silicon dioxide/silicon substrate by using an electrochemical stripping method. And preparing a patterned electrode by adopting an ultraviolet lithography method and an oxygen plasma etching technology, and preparing a chromium/gold (5/40 nm) source/drain electrode by adopting a thermal evaporation technology to obtain the graphene field effect transistor.
And then, soaking the graphene field effect transistor in an N-hydroxysuccinimide benzoate solution for 1-2 hours, then washing the graphene field effect transistor with ultrapure water, and adsorbing the pyrenyl group at one end of the molecule to the surface of the field effect transistor through pi-pi stacking effect. A liquid tank is made to be placed on the graphene channel, and the capacity of the liquid tank is about 80-100 microliters.
Designing a DNA three-dimensional nano-structure probe which can be directly combined with a nucleic acid sequence of a novel coronavirus SARS-CoV-2 to be detected. The probe is formed by self-assembling four DNA single strands through base complementary pairing, and the method for synthesizing the DNA probe is to heat DNA mixed liquor to 95 ℃ for 5 minutes, then uniformly cool the mixed liquor to 15 ℃ within 1 minute, and finally store the mixed liquor at 4 ℃. And then, placing the DNA probe solution in a liquid tank, wherein an amino group connected to a base of the DNA probe can be combined with an N-hydroxysuccinimide group at the other end of the N-hydroxysuccinimide benzoate molecule to fix the probe on an interface of a graphene transistor, wherein the required time is about 8-12 hours, and then, washing the graphene transistor with ultra-pure water.
The upper respiratory tract pharynx swab sample of the novel coronavirus SARS-CoV-2 is processed to extract virus nucleic acid, the extraction method comprises the steps of extracting 200 microliter of sample from a collecting tube of a virus blood sample, putting the sample into a nucleic acid extraction kit, then putting the kit into an automatic or semi-automatic nucleic acid extractor, and storing the extracted novel coronavirus SARS-CoV-2 nucleic acid at 4 ℃.
Finally, the solution containing the novel coronavirus nucleic acid sequence prepared above was added to the liquid tank at a minimum concentration of 0.1 copy number/μ l for detection.
Example 4
Firstly, preparing single-layer graphene on a copper foil with the thickness of 25 microns by using a chemical vapor deposition method, and transferring the prepared graphene to a clean silicon dioxide/silicon substrate by using an electrochemical stripping method. And preparing a patterned electrode by adopting an ultraviolet lithography method and an oxygen plasma etching technology, and preparing a chromium/gold (5/40 nm) source/drain electrode by adopting a thermal evaporation technology to obtain the graphene field effect transistor.
And then, soaking the graphene field effect transistor in an N-hydroxysuccinimide benzoate solution for 1-2 hours, then washing the graphene field effect transistor with ultrapure water, and adsorbing the pyrenyl group at one end of the molecule to the surface of the field effect transistor through pi-pi stacking effect. A liquid tank is made to be placed on the graphene channel, and the capacity of the liquid tank is about 80-100 microliters.
The designed DNA probe is single-stranded DNA, and the single-stranded DNA probe can be combined with the reverse transcription DNA sequence of the novel coronavirus SARS-CoV-2 nucleic acid gene. Placing the single-stranded DNA probe solution in a liquid tank, enabling an amino group at the tail end of the DNA probe to be combined with an N-hydroxysuccinimide group at the other end of a benzoic acid N-hydroxysuccinimide ester molecule to fix the probe on an interface of a graphene transistor, wherein the required time is about 8-12 hours, and then washing the graphene transistor by using ultrapure water.
The upper respiratory tract pharynx swab sample of the novel coronavirus SARS-CoV-2 is processed to extract virus nucleic acid, the extraction method comprises the steps of extracting 200 microliter of sample from a collection tube of the virus upper respiratory tract pharynx swab sample, putting the sample into a nucleic acid extraction kit, then putting the kit into an automatic or semi-automatic nucleic acid extractor, and extracting to obtain the novel coronavirus SARS-CoV-2 nucleic acid and storing at 4 ℃.
Then the extracted novel coronavirus SARS-CoV-2 nucleic acid is treated by reverse transcription by a reverse transcription kit, wherein the treatment method comprises the steps of heating to 25 ℃ for 10 minutes, then heating to 37 ℃ for 2 hours, and finally heating to 85 ℃ for 5 minutes. The DNA sequence obtained after reverse transcription was stored at 4 ℃. Finally, the solution containing the reverse transcribed DNA sequence of the novel coronavirus nucleic acid prepared above was added to the liquid tank at the lowest detection concentration of 0.1 copy number/microliter.
Example 5
Firstly, stripping a molybdenum disulfide thin layer with the size larger than 10 nanometers by using a mechanical stripping method, transferring the molybdenum disulfide thin layer onto a quartz substrate, preparing a patterned electrode by using an ultraviolet lithography method and an oxygen plasma etching technology, and preparing a titanium/gold (5/40 nanometers) source/drain electrode by using a thermal evaporation technology to obtain the molybdenum disulfide field effect transistor.
And then soaking the molybdenum disulfide field effect transistor in 1-pyrenebutyric acid N-hydroxysuccinimide ester solution for 1-2 hours, then washing the transistor with ultrapure water, and adsorbing the pyrenyl group at one end of the molecule to the surface of the field effect transistor through pi-pi stacking effect. A liquid tank is made to rest on the device channel, the liquid tank having a capacity of about 80-100 microliters.
Designing a DNA three-dimensional nanostructure probe which can be combined with a reverse transcription DNA sequence of a novel coronavirus SARS-CoV-2 nucleic acid gene. The probe is formed by self-assembling four DNA single strands through base complementary pairing, and the method for synthesizing the DNA probe is to heat DNA mixed liquor to 95 ℃ for 5 minutes, then uniformly cool the mixed liquor to 15 ℃ within 1 minute, and finally store the mixed liquor at 4 ℃. And then, placing the DNA probe solution in a liquid tank, wherein amino groups connected to a DNA probe base can be combined with an N-hydroxysuccinimide group at the other end of a 1-pyrenebutyric acid N-hydroxysuccinimide ester molecule to fix the probe on an interface of a molybdenum disulfide transistor, the required time is about 8-12 hours, and then, washing the probe with ultra-pure water.
The upper respiratory tract pharynx swab sample of the novel coronavirus SARS-CoV-2 is processed to extract virus nucleic acid, the extraction method comprises the steps of extracting 200 microliter of sample from a collection tube of the virus upper respiratory tract pharynx swab sample, putting the sample into a nucleic acid extraction kit, then putting the kit into an automatic or semi-automatic nucleic acid extractor, and extracting to obtain the novel coronavirus SARS-CoV-2 nucleic acid and storing at 4 ℃.
Then the extracted novel coronavirus SARS-CoV-2 nucleic acid is treated by reverse transcription by a reverse transcription kit, wherein the treatment method comprises the steps of heating to 25 ℃ for 10 minutes, then heating to 37 ℃ for 2 hours, and finally heating to 85 ℃ for 5 minutes. The DNA sequence obtained after reverse transcription was stored at 4 ℃. Finally, the solution containing the reverse transcribed DNA sequence of the novel coronavirus nucleic acid prepared above was added to the liquid tank at the lowest detection concentration of 0.1 copy number/microliter.
Example 6
Firstly, depositing a silicon dioxide thin sheet with the thickness of 20 nanometers on an insulating silicon sheet with the thickness of 50 nanometers by using a plasma enhanced chemical vapor deposition method, processing a silicon dioxide layer by using a stepping photoetching instrument to obtain a required pattern, putting the silicon sheet into 100 milliliters of 25 percent tetramethylammonium hydroxide solution for wet etching (70 ℃ for 23.5 minutes), and then depositing a nickel/gold (20 nanometers/100 nanometers) double-layer structure on the surface of the silicon sheet by using a metal deposition method. Forming a passivation layer structure of silicon dioxide/silicon nitride (200 nm/200 nm) on the surface of a silicon wafer by adopting a plasma enhanced chemical vapor deposition method, preparing a silicon nanowire by adopting a photoetching method, removing a silicon nitride layer on the surface of a device by adopting a reactive ion etching method, and finally removing a silicon dioxide layer by adopting a buffer oxide corrosive method to obtain the silicon nanowire field effect transistor.
Then, cleaning the surface of the silicon nanowire for 5 minutes by using an oxygen plasma method to generate hydroxyl; and then soaking the silicon nanowire field effect transistor in 3-aminopropyltriethoxysilane anhydrous ethanol solution for 1-2 hours, and then washing the silicon nanowire field effect transistor with ultrapure water to clean the silicon nanowire field effect transistor so that amino and hydroxyl at one end of a 3-aminopropyltriethoxysilane molecule are combined and adsorbed on the surface of the field effect transistor. And then soaking the device in 2.5% glutaraldehyde water solution for 1 hour, and then washing the device to be clean, so that glutaraldehyde molecules and hydroxyl at the tail end of 3-aminopropyltriethoxysilane molecules are combined to form aldehyde groups. A liquid tank is made to rest on the device channel, the liquid tank having a capacity of about 80-100 microliters.
Designing a DNA three-dimensional nanostructure probe which can be combined with a reverse transcription DNA sequence of a novel coronavirus SARS-CoV-2 nucleic acid gene. The probe is formed by self-assembling four DNA single strands through base complementary pairing, and the method for synthesizing the DNA probe is to heat DNA mixed liquor to 95 ℃ for 5 minutes, then uniformly cool the mixed liquor to 15 ℃ within 1 minute, and finally store the mixed liquor at 4 ℃. And then, placing the DNA probe solution in a liquid tank, wherein amino connected to a DNA probe base can be combined with aldehyde groups formed on the surface of the device through covalent bonding, so that the probe is fixed on an interface of a graphene transistor for about 8-12 hours, and then washing the graphene transistor with ultrapure water.
The upper respiratory tract pharynx swab sample of the novel coronavirus SARS-CoV-2 is processed to extract virus nucleic acid, the extraction method comprises the steps of extracting 200 microliter of sample from a collection tube of the virus upper respiratory tract pharynx swab sample, putting the sample into a nucleic acid extraction kit, then putting the kit into an automatic or semi-automatic nucleic acid extractor, and extracting to obtain the novel coronavirus SARS-CoV-2 nucleic acid and storing at 4 ℃.
Then the extracted novel coronavirus SARS-CoV-2 nucleic acid is treated by reverse transcription by a reverse transcription kit, wherein the treatment method comprises the steps of heating to 25 ℃ for 10 minutes, then heating to 37 ℃ for 2 hours, and finally heating to 85 ℃ for 5 minutes. The DNA sequence obtained after reverse transcription was stored at 4 ℃. Finally, the solution containing the reverse transcribed DNA sequence of the novel coronavirus nucleic acid prepared above was added to the liquid tank at the lowest detection concentration of 0.1 copy number/microliter.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (9)
1. A field effect transistor sensor for detecting novel coronavirus SARS-CoV-2 nucleic acid is characterized by comprising an insulating substrate, a semiconductor layer and an electrode, wherein the semiconductor layer and the electrode are arranged on the insulating substrate, an exposed semiconductor channel is arranged on the semiconductor layer, and a DNA probe is modified and fixed in the semiconductor channel;
the DNA probe can be directly combined with a novel coronavirus SARS-CoV-2 nucleic acid sequence to be detected or combined with DNA subjected to reverse transcription of a novel coronavirus SARS-CoV-2 nucleic acid characteristic sequence to be detected through base complementary matching, so that the novel coronavirus SARS-CoV-2 nucleic acid sequence to be detected or the DNA subjected to reverse transcription of the novel coronavirus nucleic acid sequence to be detected contacts the surface of the field effect transistor sensor.
2. The FET sensor for detecting SARS-CoV-2 nucleic acid of claim 1, wherein the DNA probe is a single DNA strand or a two-dimensional or three-dimensional nucleic acid nanostructure self-assembled by oligonucleotide strand complementary pairing, wherein the various nano two-dimensional and three-dimensional nucleic acid structures are self-assembled by oligonucleotide strand complementary pairing;
wherein, the characteristic sequence of the novel coronavirus SARS-CoV-2 nucleic acid is one or more of a coding non-structural protein gene, a coding thorn protein gene, a coding nucleocapsid protein gene or a coding envelope protein gene.
3. The FET sensor for detecting SARS-CoV-2 nucleic acid of claim 1, wherein the DNA probe is modified and immobilized on the surface of the semiconductor channel by adsorption, crosslinking, covalent bonding, entrapment or biological tissue immobilization.
4. The FET sensor for detecting SARS-CoV-2 nucleic acid of claim 1, wherein the insulating substrate is silica, quartz, insulating glass, mica, polyethylene terephthalate film, polyimide film or polydimethylsiloxane film.
5. The FET sensor for detecting SARS-CoV-2 nucleic acid, according to claim 1, wherein the semiconductor layer is graphene, oxide semiconductor, chalcogenide of transition metal, silicon or germanium.
6. The FET sensor for detecting SARS-CoV-2 nucleic acid of claim 1, wherein the electrode is a patterned electrode and the thickness of the electrode material is 20-2000 nm.
7. The method of claim 1, wherein the method comprises the steps of:
(1) Preparing a field effect transistor device with a semiconductor channel exposed;
(2) modifying and fixing a DNA probe on the surface of the semiconductor channel;
(3) liquid channels or microfluidic channels are fabricated on the field effect transistors.
8. The method of claim 7, wherein the nucleic acid sequence of the FET sensor for detecting the novel coronavirus SARS-CoV-2 is selected from the group consisting of,
designing a DNA probe, manufacturing a liquid groove on the semiconductor channel, placing the DNA probe solution in the liquid groove, and modifying and fixing the DNA probe solution on the surface of the semiconductor channel by an adsorption method, a crosslinking method, a covalent bonding method, an embedding method or a biological tissue fixing method.
9. The use of the field effect transistor sensor for detecting the novel coronavirus SARS-CoV-2 nucleic acid according to claim 1, wherein the field effect transistor sensor is used for preparing a novel coronavirus SARS-CoV-2 nucleic acid detection reagent.
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