Nucleic acid detection sensor based on inse field effect transistor and its application in novel coronavirus detection

Abstract

This invention discloses a nucleic acid detection sensor based on InSe FET and its application in the detection of COVID-19. The sensor is constructed by: (1) using an ultrathin InSe material obtained by mechanical exfoliation as the channel material of the FET sensor; (2) depositing Al2O3 on the ultrathin InSe on a SiO2 / Si substrate using ALD; (3) spin-coating PMMA adhesive and constructing an electrode pattern on the PMMA layer using EBL technology; (4) obtaining high-quality source and drain electrodes through EBE; (5) depositing an Al2O3 passivation layer again; (6) depositing uniformly distributed Au nanoparticles as probe sites through low-rate EBE; (7) creating windows at the channel using EBL for DNA probe modification; and (8) incubating the device in a phosphate buffer containing DNA probes. The InSe FET sensor of this invention has good electrical performance, high detection sensitivity, and can be applied to the detection of ultra-low concentration nucleic acid of COVID-19, while also having the ability to determine nucleic acid mutations.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology and relates to a nucleic acid detection sensor based on InSe field-effect transistors and its application in the detection of the novel coronavirus. Background Technology

[0002] COVID-19 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) and has been declared a pandemic by the World Health Organization.

[0003] Among the many diagnostic methods currently available, field-effect transistor (FET)-based biosensing devices offer advantages, including the ability to perform high sensitivity and instantaneous measurements using small amounts of analyte. This makes this technology more time-efficient and accurate compared to traditional virus detection methods such as PCR.

[0004] Two-dimensional semiconductor materials have been a hot research topic in microelectronic device materials for many years due to their unique optoelectronic properties. Furthermore, due to their large specific surface area, many emerging two-dimensional materials are highly sensitive to various analytes (ions, glucose, nucleic acids, proteins, etc.), and have found wide application in the sensing field. In summary, FET sensors based on two-dimensional materials are well-suited for applications in wearable electronic devices, electronic skin, or biomonitoring devices.

[0005] Indium selenide (InSe) is a typical group III-VI layered material, with each layer consisting of Se-In-In-Se atomic arrangements and van der Waals bonds between the layers. Currently, the mainstream preparation method for ultrathin InSe materials is mechanical exfoliation (Nat. Nanotechnol. 2017, 12, 223-227), and there are also reports of successful synthesis by chemical vapor deposition (CVD) (Small 2018, 14(39):1802351). The band gap of InSe from bulk to monolayer is 1.26-2.11 eV. Due to its small effective electron mass (0.143 m0), excellent carrier transport properties, and intrinsic mobility as high as 10 under ideal conditions, InSe is widely used in chemical vapor deposition. 3 cm 2 v -1 s -1(At room temperature), ultrathin InSe materials have attracted increasing attention in optoelectronics, microelectronics, logic circuits, and flexible electronics (ACS Nano 2014, 8(2):1263-1272; Nano Lett. 2015, 15(6):3815-3819; Adv. Mater. 2018, 30:1803690; ACS Nano 2019, 13(6):7291-7299). Meanwhile, few-layer InSe exhibits excellent gate control capabilities, and its carriers are highly sensitive to various charge scattering effects generated at the contact interface (Adv. Mater. 2014, 26(38):6587-6593), making it an ideal channel material for high-sensitivity FET sensors. However, InSe is prone to oxidation, leading to performance degradation. For example, Po-Hsun Ho et al. performed in-situ characterization of the oxidation process of ultrathin InSe and found that in an air environment, water molecules promote the distortion and disintegration of the InSe crystal structure during oxidation, resulting in severe performance degradation after oxidation (ACS Nano 2017, 11:7362-7370). Meanwhile, Tsung-Han Tsai et al. found that oxidized ultrathin InSe contains InO. x The / InSe interface, where trapped states negatively impact electron transport, is another concern. Additionally, wet oxidation of InO... x The InSe interface quality is far inferior to that of dry oxidation, exhibiting a higher defect state density and more severe performance degradation (npj 2D Mater. Appl. 2019, 3:29). Therefore, improving the stability of InSe is a bottleneck problem for realizing InSe-based biosensors. This is not only because physiological solutions exhibit stronger oxidizing and corrosive effects on the material, but also because biosensors require ultra-thin encapsulation layers (thickness < Debye length). Summary of the Invention

[0006] The purpose of this invention is to provide a nucleic acid detection sensor based on InSe field-effect transistors and its application in the detection of the novel coronavirus. The sensor provided by this invention has good stability, high sensitivity, and significantly reduces the time required compared to traditional PCR detection.

[0007] The technical solution for achieving the objective of this invention is as follows:

[0008] The fabrication method of a nucleic acid detection sensor based on InSe field-effect transistors includes the following steps:

[0009] (1) Mechanically peel off the bulk InSe material to obtain ultrathin InSe material;

[0010] (2) The ultrathin InSe material was soaked in acetone to remove the glue, and then screened to obtain clean ultrathin InSe material;

[0011] (3) Deposit clean, ultrathin InSe material into alumina for 6-8 cycles using atomic layer deposition (ALD) technology;

[0012] (4) Spin-coat polymethyl methacrylate (PMMA) adhesive onto the sample surface obtained in step (3) and dry it. Then, use electron beam lithography (EBL) technology to construct an electrode pattern on the PMMA layer.

[0013] (5) Cr and Au are deposited sequentially on the sample surface obtained in step (4) by electron beam evaporation (EBE). After removing the adhesive with acetone, high-quality Cr / Au source and drain electrodes are obtained.

[0014] (6) The sample surface obtained in step (5) is subjected to ALD deposition of an alumina passivation layer for 20-30 cycles;

[0015] (7) Use the uniformly distributed Au nanoparticles deposited on the sample surface obtained in step (6) by low-coating-rate EBE as probe binding sites.

[0016] (8) Spin-coat PMMA glue to cover the sample obtained in step (7), remove the PMMA glue at the channel using EBL, and finally place the entire device in phosphate buffer (PBS) containing DNA probes for incubation to obtain the InSeFET-based nucleic acid detection sensor.

[0017] Preferably, in step (1), the specific method is as follows: the bulk InSe material is evenly pasted onto the mechanically peeled master tape, the master tape is folded three times to thin it, the sub-tape is covered on the master tape, and the InSe is thinned and transferred to the sub-tape after ten minutes of bonding. After ultrasonic cleaning, the 300nm SiO2 thick silicon substrate is tightly attached to the sub-tape and removed after standing for 24 hours.

[0018] Preferably, in step (2), the soaking time is 1 to 2 hours.

[0019] In step (2) of this invention, the thickness of the screened InSe material is adaptively adjusted according to the nucleic acid sequence to be detected and the cation concentration of the phosphate buffer. In a specific embodiment of this invention, the thickness of the screened InSe material is 6-9 nm, which is related to the Debye length of the sensing element.

[0020] Preferably, in steps (3) and (6), the parameters for ALD deposition of alumina are: process pressure 0.2 Torr, base temperature 200℃; single cycle process flow: Dose trimethylaluminum (TMA) 0.025s; clean 30s; Dose deionized water 0.015s; clean 30s.

[0021] In step (3) of this invention, alumina is deposited by ALD for 6-8 cycles. As an empirical parameter, the alumina thickness is controlled to be approximately 0.8-1.0 nm in this process. If it is too thin (too few cycles), the protective effect during microfabrication will be weak, and if it is too thick (>1 nm), it will increase the contact resistance of the electrode.

[0022] Preferably, in steps (4) and (8), the PMMA spin coating parameters are: first spin coating at 600 rpm for 6 seconds, then spin coating at 2000 rpm for 50 seconds; the drying temperature is 150°C, and the drying time is 5 minutes.

[0023] Preferably, in step (5), the Cr evaporation rate of EBE is... Au for The thickness of the Cr electrode is 3 nm, and the thickness of the Au electrode is 60 nm.

[0024] Preferably, in step (7), the Au deposition rate of EBE is... The thickness is set to 0.4nm.

[0025] Preferably, in step (8), the K of PBS + The ion concentration was 0.01 mol / L, and the incubation time was more than 36 hours.

[0026] This invention also provides an InSe FET sensor fabricated by the above method, wherein the InSe thickness is 6-9 nm, the alumina thickness is approximately 3 nm, and the surface gold particles are approximately 1 nm high and uniformly but discontinuously distributed. The FET on / off ratio can reach 10. 6 The limit of nucleic acid detection by the sensor is less than 10. -15 mol / L.

[0027] Furthermore, this invention provides the application of the above-mentioned nucleic acid detection sensor in the detection of the novel coronavirus. The specific application method is as follows: after heating the above-mentioned nucleic acid detection sensor to 40°C, a target solution is added, and the change in source-leakage current (ΔI) is measured. ds According to the conversion formula The calibration response (ΔV) is calculated. cal Finally, based on the linear relationship between calibration response and concentration, the concentration of the novel coronavirus in the target solution was calculated.

[0028] Preferably, the test range is the gate voltage (V) gs ): 0V~0.5V, and ΔI ds The value of is when V gs =0.5V.

[0029] This invention obtains ultrathin two-dimensional InSe material through mechanical exfoliation, which is used as the channel material for a high-sensitivity FET sensor. The sensor construction includes the following steps: depositing Al2O3 for 6-8 cycles on the mechanically exfoliated InSe along with a Si / SiO2 substrate using atomic layer deposition (ALD); spin-coating PMMA adhesive and constructing electrode patterns on the PMMA layer using electron beam lithography (EBL); sequentially depositing Cr and Au onto the sample using electron beam evaporation (EBE), and obtaining high-quality Cr / Au source / drain electrodes after acetone removal; depositing an Al2O3 passivation layer again using ALD for 20-30 cycles; obtaining uniformly distributed Au nanoparticles above the FET channel using EBE with a low deposition rate method; spin-coating PMMA adhesive to cover the electrodes, and removing the PMMA adhesive at the channel using EBL; and placing the entire device in phosphate buffered saline (PBS) containing probes for incubation. The construction method provided by this invention can obtain a highly sensitive InSe FET sensor with good electrical performance and ideal stability, which can be applied to the detection of ultra-low concentration nucleic acid (sub-fM level) of the novel coronavirus, and also has the ability to judge nucleic acid mutation.

[0030] This invention provides a highly sensitive and stable InSe-based sensor through a two-step ALD passivation method, and validates it using simulated samples of partial nucleic acid fragments (DNA / RNA) from the novel coronavirus. Experimental results show that the nucleic acid detection limit of the sensor provided by this invention is less than 10. -15 At mol / L (without sequence amplification), the sensor can effectively detect both DNA and RNA samples. Furthermore, it can identify the type and location of single-base pair mutations in nucleic acid sequences, enabling multi-dimensional information acquisition for viral nucleic acid detection. This is of great significance for early screening and mutation prediction of the novel coronavirus. Attached Figure Description

[0031] Figure 1 The AFM image of InSe prepared in Example 1;

[0032] Figure 2 Raman spectrum of InSe prepared in Example 1

[0033] Figure 3 The XRD pattern of the InSe block selected in Example 1;

[0034] Figure 4 Output curve of the InSe FET constructed for Example 2;

[0035] Figure 5 The statistical graph of the electrical performance stability of InSe FET constructed for Example 2;

[0036] Figure 6This is a flowchart illustrating the construction process of the InSe FET biosensor used in Example 3;

[0037] Figure 7 Optical mirror image of the InSe FET sensor constructed in Example 3;

[0038] Figure 8 The performance graph of the InSe FET constructed in Example 3 before and after DNA probe modification;

[0039] Figure 9 The energy band diagram of the InSe FET sensing mechanism constructed in Example 3;

[0040] Figure 10 A schematic diagram of the InSe FET nucleic acid sensor constructed in Example 4;

[0041] Figure 11 This is a data graph showing the detection of different concentrations of DNA (ORF1ab) using the InSe sensor constructed in Example 4;

[0042] Figure 12 This is a graph showing the data of different concentrations of RNA (ORF1ab) detected by the InSe sensor constructed in Example 5;

[0043] Figure 13 Linear fitting data for detecting DNA / RNA (ORF1ab) concentration using the InSe sensor constructed in Examples 4 and 5;

[0044] Figure 14 This is a data graph showing the detection of DNA (ORF1ab) single-base mutation types using the InSe sensor constructed in Example 6;

[0045] Figure 15 This is a data graph showing the detection of single-base mutation sites in DNA (ORF1ab) using the InSe sensor constructed in Example 7.

[0046] Figure 16 A graph showing data from the InSe sensor constructed in Example 8 for detecting RNA (ORF8) complementarity, single base mutations, and non-complementarity sequences.

[0047] Table 1 shows the DNA ORF1ab probe, DNA ORF8 probe, and their respective target sequences used in this invention. Detailed Implementation

[0048] The phosphate-buffered saline (PBS) of this invention is prepared from potassium dihydrogen phosphate and dipotassium hydrogen phosphate, wherein K + The concentration was 0.01 mol / L, and the pH was 7.4.

[0049] The method for fabricating a nucleic acid detection sensor based on an InSe field-effect transistor of the present invention includes the following steps:

[0050] (1) Mechanically peel off the bulk InSe material to obtain ultrathin InSe material;

[0051] (2) The ultrathin InSe material was soaked in acetone to remove the glue, and then screened to obtain clean ultrathin InSe material;

[0052] (3) Deposit clean, ultrathin InSe material into alumina for 6-8 cycles using atomic layer deposition (ALD) technology;

[0053] (4) Spin-coat polymethyl methacrylate (PMMA) adhesive onto the sample surface obtained in step (3) and dry it. Then, use electron beam lithography (EBL) technology to construct an electrode pattern on the PMMA layer.

[0054] (5) Cr and Au are deposited sequentially on the sample surface obtained in step (4) by electron beam evaporation (EBE). After removing the adhesive with acetone, high-quality Cr / Au source and drain electrodes are obtained.

[0055] (6) The sample surface obtained in step (5) is subjected to ALD deposition of an alumina passivation layer for 20-30 cycles;

[0056] (7) Use the uniformly distributed Au nanoparticles deposited on the sample surface obtained in step (6) by low-coating-rate EBE as probe binding sites.

[0057] (8) Spin-coat PMMA glue to cover the sample obtained in step (7), remove the PMMA glue at the channel using EBL, and finally place the entire device in phosphate buffer (PBS) containing DNA probes for incubation to obtain the InSeFET-based nucleic acid detection sensor.

[0058] The PBS in this invention contains K + The ion concentration is 0.01 mol / L, and the Debye length at this concentration is λ. D = 7.53nm. Taking into account the yield of mechanical exfoliation and the limitations of Debye length, the InSe material screened in this invention has a thickness of 6-9nm.

[0059] The parameters for ALD deposition of alumina in this invention are: process pressure 0.2 Torr, base temperature 200℃, pipeline temperature 100℃, and N2 gas flow rate 8 sccm. The single-cycle process flow is: Dose trimethylaluminum (TMA) 0.025s; purge 30s; Dose deionized water 0.015s; purge 30s. The average alumina thickness is... Step (3) involves ALD deposition of alumina for 6-8 cycles. As an empirical parameter, the alumina thickness in this process is controlled to be approximately 0.8-1.0 nm. If it is too thin (too few cycles), the protective effect during microfabrication will be weak, and if it is too thick (>1 nm), it will increase the contact resistance of the electrode.

[0060] In this invention, the PMMA spin coating is performed at a low speed of 600 rpm (6 s) and a high speed of 2000 rpm (50 s). It is then baked at 150°C for 5 minutes. This yields a PMMA coating with a thickness of approximately 200 nm. This thickness of PMMA ensures a good electron beam exposure pattern and facilitates the removal of excess Au. Typically, the device channel length L is 2-3 nm, with an aspect ratio of approximately 1:3.

[0061] In this invention, the Cr deposition rate of electrode EBE is: Au for The Cr / Au electrode thicknesses are 3 / 60 nm. A faster Au deposition rate ensures electrode density while reducing the time the material spends in the high-temperature chamber, thus improving electrode contact quality.

[0062] In this invention, the Au deposition rate of gold nanoparticles EBE is: The thickness was set at 0.4 nm. The extremely slow Au evaporation rate, combined with the ultra-thin set thickness, yielded gold nanoparticles with a height of approximately 1 nm and a uniformly distributed, discontinuous structure.

[0063] In this invention, the potassium ion concentration of PBS is 0.01 mol / L, and the incubation time is 36 hours.

[0064] In this invention, all sensor tests were performed on a three-probe semiconductor parameter analyzer platform, with a heating plate configured to enable sensing tests at different temperatures. The target analyte solutions were prepared in phosphate buffer (0.01×) and stored at 4°C to prevent unstable decomposition of nucleic acid single strands. The devices were stored in vacuum-sealed plastic bags.

[0065] In this invention, the stability test of the sensor involves immersing the device in phosphate buffer at room temperature (27°C) every 12 hours, for a total test duration of 48 hours, which meets the application requirements of the sensor. The source-drain voltage V... ds =0.1V, gate voltage (V gs Range: -10V to 10V.

[0066] In the concentration detection experiment of this invention, the gene fragment selected is ORF1ab (SEQ ID No. 1). The target solution containing complementary DNA (SEQ ID No. 2) and RNA (SEQ ID No. 3) was prepared at a concentration of 10. -14 mol / L, 10 -13mol / L, 2×10 - 12 mol / L, 10 -10 mol / L, 10 -8 mol / L, the concentration used for all single base pair mismatch experiments was 10 mol / L. -8 mol / L.

[0067] In the concentration detection experiment of this invention, after heating the device to 40°C using a hot plate, 20 μL of solution is added dropwise using a pipette. Tests are performed every 5 minutes, and the process is stopped after 30 minutes. Appropriate solutions are added during the process to maintain a constant droplet volume.

[0068] In this invention, the directly measured electrical data is the source-drain current change (ΔI). ds The calibration response can be obtained after conversion. This can reduce the error introduced by the performance of different devices on the responsivity.

[0069] In this invention, the standard deviation (SD) of the sensor system represents the system error under blank conditions and test conditions of 40°C in phosphate buffer. After linearly fitting the "response-concentration" data points to y = a + bx, the detection limit (LOD) is obtained as ΔV. cal =3×SD corresponding to the concentration of the detectable.

[0070] Table 1

[0071]

[0072] In this invention, the gene fragment ORF1ab was selected for DNA base variation experiments. One set of experiments was set with a mismatch location at the 6th base pair, and the mismatch types were CA (SEQ ID No. 4), CT (SEQ ID No. 5), and CC (SEQ ID No. 6). Another set of experiments was set with a mismatch type of CT, and the mismatch locations were the 6th (SEQ ID No. 5), 13th (SEQ ID No. 7), and 21st (SEQ ID No. 8) base pairs. Test conditions: V ds =0.1V, temperature 40℃ or 60℃.

[0073] In addition, sensing experiments were conducted on the fully complementary strand (SEQ ID No. 10) and the single-base variant strand (SEQ ID No. 11) of the SARS-CoV-2 N protein encoding the variable gene fragment ORF8 (SEQ ID No. 9). Because the instability of RNA mismatch structures is significant at high temperatures, the test conditions were 60°C.

[0074] In the above-mentioned concentration and variant sequence experiments, the test range of this invention is the gate voltage (V). gs ): 0V~0.5V, and ΔIds The value of is when V gs =0.5V. At this point, the channel is open, the transfer current enters the linear region, and the gate voltage is not much greater than the potential difference caused by the nucleic acid, so the responsivity can be accurately measured.

[0075] To further illustrate the present invention, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0076] Example 1

[0077] Step 1: Evenly adhere the block InSe material onto the mechanically peelable tape (master tape), and fold the master tape three times to thin it.

[0078] Step 2: Cover the mother tape with blue tape (sub-tape) and let it adhere for ten minutes to thin out the InSe and transfer it onto the sub-tape.

[0079] Step 3: After ultrasonically cleaning the 300nm SiO2 thick silicon substrate (10 min each of acetone, ethanol, and deionized water), place it firmly on the sub-strip and let it stand for 24 hours before removing it.

[0080] Step 4: Soak the sample in acetone for 1 hour to remove the sizing agent, and then screen to obtain clean two-dimensional InSe material.

[0081] The AFM pattern of InSe prepared in this embodiment is shown below. Figure 1 As shown in the figure, InSe exhibits typical layered steps, and the sample thickness can be accurately measured in the range of 6-9 nm using AFM.

[0082] The obtained InSe was subjected to Raman spectroscopy. Figure 2 ) and XRD characterization ( Figure 3 The peak positions were calibrated, which verifies that the mechanically exfoliated InSe samples are of good quality.

[0083] Example 2

[0084] (1) The ultrathin InSe material obtained in Example 1 was deposited with alumina for 6 cycles using ALD technology;

[0085] (2) Spin-coating PMMA adhesive onto the sample surface obtained in step (1) and drying it, and then using EBL technology to construct an electrode pattern on the PMMA layer;

[0086] (3) The sample surface obtained in step (2) is successively deposited with Cr and Au by EBE, and after removing the adhesive with acetone, high-quality Cr / Au source electrode and drain electrode are obtained.

[0087] (4) An alumina passivation layer was deposited on the sample surface obtained in step (3) for 20 cycles using ALD to obtain an InSe FET containing an Al2O3 layer. Its output curve is shown in Figure 1. Figure 4 As can be seen, within 48 hours, the on-state current intensity of the FET prepared in this embodiment remained above 74%, and the mobility also remained at 10 cm⁻¹. 2 / (Vs), switching ratio exceeding 10 5 The electrical properties showed no significant degradation, meeting the stability requirements of biosensors. Figure 5 ).

[0088] Example 3

[0089] (1) Au nanoparticles uniformly distributed on the surface of the sample obtained in Example 2 were deposited using low-rate EBE as probe binding sites.

[0090] (2) Spin-coating PMMA glue to cover the sample obtained in step (1), removing the PMMA glue at the channel using EBL, and finally placing the entire device in PBS containing DNA probes for incubation to obtain the InSeFET sensing platform. The complete process is as follows: Figure 6 Actual product image as shown Figure 7 .

[0091] The electrical performance of the InSe FET sensing platform before and after modification is shown in the figure. Figure 8 This verifies that the presence of the Al2O3 layer not only serves as passivation but also acts as an encapsulation layer, separating the gold nanoparticles from the n-type InSe. This prevents the gold particles from causing p-doping effects on the InSe, thereby improving the sensing sensitivity of the n-type InSe. The threshold voltage V after DNA probe modification... th The change from -1.6V to -0.5V indicates that the Al2O3 layer does not modify the p-doping effect brought about by DNA.

[0092] The energy band diagram of the InSe FET sensing mechanism is as follows: Figure 9 Because nucleic acids carry a negative charge, they form a negative potential on the channel surface after binding with the probe. This causes the Fermi level of n-type InSe to shift downward, reducing carrier concentration and mobility, and resulting in a decrease in source-drain current.

[0093] Example 4

[0094] After modifying the DNA probe with the InSe FET sensor obtained in Example 3, it was used to detect PBS solutions with different nucleic acid concentrations. The selected gene fragment was DNA ORF1ab ( Figure 10 The target solution concentration is 10. -14 mol / L, 10 -13 mol / L, 2×10 -12 mol / L, 10-10 mol / L, 10 -8 mol / L. After heating the hot plate to 40°C, add 20 μL of solution dropwise using a pipette. Test every 5 minutes, then stop after 30 minutes. Replace the solution every 5 minutes to maintain a constant concentration of the target analyte.

[0095] The data in this embodiment is as follows: Figure 11 As shown, after linear fitting, from Figure 13 It can be concluded that LOD DNA =0.71fM, sensitivity is b1 = 11.2mV / dec. Effective operating range covers 10 -14 -10 -8 mol / L.

[0096] Example 5

[0097] Similar to Example 4, except that the target substance is changed to RNA ORF1ab.

[0098] The data in this embodiment is as follows: Figure 12 As shown, after linear fitting, from Figure 13 It can be concluded that LOD RNA =0.79fM, sensitivity is b2 = 8.0mV / dec. The effective operating range covers 10... -14 -10 -8 mol / L. The device is slightly less stable for RNA than for the same DNA fragment, because the stability of double-stranded DNA structures is greater than that of double-stranded DNA / RNA structures.

[0099] Example 6

[0100] Different base mutation types of target-PBS solutions were added to the InSe FET sensor obtained in Example 4. The selected gene fragment was DNA ORF1ab. The target mismatch position was the 6th base pair, and the mismatch types were CA, CT, and CC. Test conditions: V ds =0.1V, V gs 0-0.5V. The concentration used for all single base pair mismatch experiments was 10... -8 mol / L.

[0101] The data in this embodiment is as follows: Figure 14 As shown, DNA single base mismatches can be distinguished at 40°C, and the response is consistent with the T value defined under the high-resolution melting curve. m The trends are consistent. The responses to all three types of mismatches were significantly different from those to complementary and non-complementary sequences (P<0.01). Among the single-base mismatch types, except for CA and CT, which showed no statistically significant difference, the results of the other types were all significantly different (P<0.05).

[0102] Example 7

[0103] Similar to Example 6, the difference is that the variation is set to mismatch type CT, and the mismatch positions are the 6th, 13th, and 21st base pairs (starting from the gold particle modification end).

[0104] The data in this embodiment is as follows: Figure 15 As shown, single-base mismatch positions in DNA are resolvable at 60°C, and the response is consistent with the T value defined under the high-resolution melting curve. d The trends are consistent. The responses to all three types of mismatches were significantly different from those to complementary and non-complementary sequences (P<0.05). Among the single-base mismatch types, except for the variant position 13… th With 21 st 6 th Apart from no statistically significant differences between the results, all other results showed significant differences (P<0.05), with a resolution of approximately 15 meters for the position of base variation.

[0105] Example 8

[0106] Similar to Example 6, the difference is that the gene fragment used is DNA ORF8. The mutation scenarios were set to three types: perfect match, CT mismatch at the 12th pair, and complete mismatch.

[0107] The data in this embodiment is as follows: Figure 16 As shown, the complete match and the mutated sequence can be distinguished at 60℃. This is mainly because the stability of the DNA / RNA double strand at room temperature is not significantly stronger than that of the mutated sequence, but the mutated double strand unwinds faster at high temperatures, thus allowing for differentiation. The mutated sequence response differed significantly from that of complementary and non-complementary sequences (P<0.01).

[0108] As can be seen from the above comparative examples and embodiments, the detection method provided by this invention is simple to operate, operates under mild conditions, and has a short detection time. It is also applicable to multiple targets, including DNA and RNA. Furthermore, it possesses the ability to acquire multi-dimensional viral nucleic acid information, making it an effective tool for detecting infectious viruses.

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Nanjing University of Science and Technology <120> Nucleic acid detection sensor based on InSe field-effect transistor and its application in COVID-19 detection <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <400> 1 gcagacgcca tacacctttc ca 22 <210> 2 <211> twenty two <212> DNA <213> Artificial Sequence <400> 2 cgtctgcggt atgtggaaag gt 22 <210> 3 <211> twenty two <212> RNA <213> Artificial Sequence <400> 3 cgucugcugu auguggaaag gu 22 <210> 4 <211> twenty two <212> DNA <213> Artificial Sequence <400> 4 cgtctacggt atgtggaaag gt 22 <210> 5 <211> twenty two <212> DNA <213> Artificial Sequence <400> 5 cgtcttcggt atgtggaaag gt 22 <210> 6 <211> twenty two <212> DNA <213> Artificial Sequence <400> 6 cgtctccggt atgtggaaag gt 22 <210> 7 <211> twenty two <212> DNA <213> Artificial Sequence <400> 7 cgtctgcggt atttggaaag gt 22 <210> 8 <211> twenty two <212> DNA <213> Artificial Sequence <400> 8 cgtctgcggt atgtggaaag tt 22 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <400> 9 gagaagttcc cctactgctg cc 22 <210> 10 <211> twenty two <212> RNA <213> Artificial Sequence <400> 10 cucuucaagg ggaugacgac gg 22 <210> 11 <211> twenty two <212> RNA <213> Artificial Sequence <400> 11 cucuucaagg gaaugacgac gg 22

Claims

1. A method for fabricating a nucleic acid detection sensor based on an InSe field-effect transistor, characterized in that, Includes the following steps: (1) Mechanically peel off the bulk InSe material to obtain ultrathin InSe material; (2) The ultrathin InSe material was soaked in acetone to remove the glue and screened to obtain clean ultrathin InSe material. The soaking time was 1-2 h and the thickness of the screened InSe material was 6-9 nm. (3) Alumina was deposited on the clean ultrathin InSe material for 6-8 cycles using atomic layer deposition technology. The parameters for ALD deposition of alumina were: process pressure 0.2 Torr, base temperature 200℃; single cycle process flow: Dose trimethylaluminum 0.025 s; clean 30 s; Dose deionized water 0.015 s; clean 30 s; (4) Spin-coat polymethyl methacrylate adhesive onto the sample surface obtained in step (3) and dry it. Then, use electron beam lithography to construct an electrode pattern on the PMMA layer. (5) Cr and Au are deposited sequentially on the sample surface obtained in step (4) by electron beam evaporation. After removing the adhesive with acetone, high-quality Cr / Au source and drain electrodes are obtained. (6) The sample surface obtained in step (5) is subjected to ALD deposition of an alumina passivation layer for 20-30 cycles. The parameters for ALD deposition of alumina are: process pressure 0.2 Torr, base temperature 200℃; single cycle process flow: Dose trimethylaluminum 0.025 s; clean 30 s; Dose deionized water 0.015 s; clean 30 s; (7) Use low-coating-rate electron beam evaporation to deposit uniformly distributed Au nanoparticles on the sample surface obtained in step (6) as probe binding sites. (8) Spin-coat PMMA glue to cover the sample obtained in step (7), remove the PMMA glue at the channel using EBL, and finally place the entire device in phosphate buffer containing DNA probes for incubation to obtain the InSeFET-based nucleic acid detection sensor.

2. The preparation method according to claim 1, characterized in that, In step (1), the specific method is as follows: the bulk InSe material is evenly pasted onto the mechanically peeled master tape, the master tape is folded three times to thin it, the sub-tape is covered on the master tape, and the InSe is thinned and transferred to the sub-tape after ten minutes of bonding. After ultrasonic cleaning, the 300 nm SiO2 thick silicon substrate is tightly attached to the sub-tape and removed after standing for 24 hours.

3. The preparation method according to claim 1, characterized in that, In steps (4) and (8), the PMMA spin coating parameters are: first spin coating at 600 rpm for 6 s, then spin coating at 2000 rpm for 50 s; the drying temperature is 150℃ and the drying time is 5 min.

4. The preparation method according to claim 1, characterized in that, In step (5), the Cr deposition rate of EBE is 0.5 Å / s, the Au deposition rate is 2 Å / s, the Cr electrode thickness is 3 nm, and the Au electrode thickness is 60 nm; in step (7), the Au deposition rate of EBE is 0.1 Å / s, and the thickness is set to 0.4 nm; in step (8), the K of PBS... + The ion concentration was 0.01 mol / L, and the incubation time was more than 36 hours.

5. A nucleic acid detection sensor prepared by any one of the preparation methods according to claims 1-4.

6. The application of the nucleic acid detection sensor according to claim 5 in the detection of the novel coronavirus.

7. The application according to claim 6, characterized in that, The specific application method is as follows: After heating the nucleic acid detection sensor to 40°C, add the target solution and measure the change in source-leakage current ∆. I ds According to the conversion formula The calibration response ∆ is calculated. V cal Finally, based on the linear relationship between calibration response and concentration, the concentration of the SARS-CoV-2 virus in the target solution was calculated.

8. The application according to claim 7, characterized in that, The test range is the gate voltage. V gs : 0 V ~ 0.5 V, and ∆ I ds The value of is when V gs = 0.5 V.

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