Gene chip sensor for single nucleotide polymorphism detection and application thereof

Abstract

The invention discloses a gene chip sensor for single nucleotide polymorphism detection and application thereof, and belongs to the technical field of biosensing detection. In order to solve the technical problems of low specificity and accuracy, long processing time and high cost of a single nucleotide polymorphism detection method in the prior art, the invention provides a gene chip sensor for single nucleotide polymorphism detection, and the gene chip sensor comprises WT wild type and SNP mutant nucleotide sequences, an LSL probe and a CRISPR / Cas 12a system. An LSL probe is used as a recognition intermediate to activate a CRISPR / Cas12a system, an iron-cobalt nano material is used to provide a stable signal source for sample detection, and a screen-printed electrode is combined with electrochemical sensing; the application of CRISPR / Cas12a and nanomaterial technology in molecular breeding is expanded, and technical support is provided for economic and effective SNP detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensing detection, and particularly relates to a gene chip sensor for single nucleotide polymorphism detection and its application. Background Art

[0002] Soybean ( Glycine max ) is one of the most important oil crops in the world, providing high-quality vegetable oil for human diet and serving as a key renewable raw material for biodiesel. Due to the dietary pattern transformation brought about by population growth, bioenergy policies, and innovation-driven transformation and upgrading of the soybean deep-processing industry, the annual global soybean consumption has been steadily increasing. However, in the face of this rigid and growing demand, traditional domestication and selective breeding have shown a trend of diminishing marginal benefits in improving yield potential. Therefore, the industry urgently needs the progress of molecular design breeding technology. With the publication of the first whole-genome sequence of the soybean variety "Williams 82" in the United States, more and more single nucleotide polymorphisms (SNPs) have been found to play a key role in gene function differentiation; SNP analysis enables breeders to accurately evaluate the genetic diversity of soybean germplasm resources, thus helping to select excellent materials and the optimal parental combination.

[0003] In recent years, with the continuous progress of molecular marker technology, the soybean single nucleotide polymorphism (SNP) detection system has developed into a multi-technical framework. DNA sequencing is regarded as the "gold standard" for SNP detection due to its high specificity, but it is limited in soybean breeding due to the high cost of whole-genome sequencing. Microarray chips are suitable for high-throughput detection, but their complex data analysis and long processing time make them unsuitable for rapid SNP screening related to specific traits, and when analyzing complex genomes rich in repetitive sequences, the hybridization accuracy based on the chips will be affected. New SNP genotyping strategies based on various isothermal nucleic acid amplification technologies (such as rolling circle amplification, hybridization chain reaction, and catalytic hairpin assembly), although improving the detection sensitivity, are easily interfered by repetitive genomic sequences, and sequence complexity may lead to non-specific amplification or genotyping failure.

[0004] As a third-generation gene editing tool, the CRISPR / Cas12a system uses Cas12a protein to form a complex with crRNA to directly recognize double-stranded or single-stranded DNA targets, activate cis-cutting of target DNA and trans-cutting of any non-complementary single-stranded DNA. In addition, CRISPR / Cas12a only needs to have a complementary sequence of 24-26 bases with crRNA, and has a high degree of design flexibility and excellent nucleic acid cutting ability, making it highly applicable in SNP genotyping. However, the high sequence similarity of the allopolyploid soybean genome increases the risk of Cas12a being mistakenly activated by non-target wild-type (WT) sequences, limiting the single-base resolution; how to effectively transmit single-point mutation signals in SNP sequences to Cas enzymes to activate their functions is still a key challenge that technicians in this field need to solve.

[0005] Therefore, those skilled in the art are eager to develop a rapid SNP genotyping technology with high sensitivity, high specificity and simple operation. Summary of the invention

[0006] The present invention provides a gene chip sensor for single nucleotide polymorphism detection and its application in order to solve the technical problems of low specificity and accuracy, long processing time and high cost in the prior art single nucleotide polymorphism detection method.

[0007] One of the purposes of the present invention is to provide a gene chip sensor for single nucleotide polymorphism detection, wherein the gene chip sensor comprises WT wild-type and SNP mutant nucleotide sequences, a dumbbell-shaped LSL probe, and a CRISPR / Cas 12a system; The WT wild-type and SNP mutant nucleotide sequences are designed and obtained based on the target gene sequence fragment; The dumbbell-shaped LSL probe is designed for the mutant fragment of the target gene sequence. The dumbbell-shaped LSL probe is a single-stranded molecule composed of five parts: a, b, c, d, and e, and can self-assemble into a dumbbell structure; wherein parts b and d are the loop parts of the dumbbell-shaped LSL probe, and the two loops contain 18 nt nucleotides of the same sequence, and are completely complementary to the SNP mutant nucleotide sequence; parts a, e, and c are the stem parts of the dumbbell-shaped LSL probe, and parts a and e both contain 11 nt nucleotides, and parts a and e are completely complementary to the sequence of part c containing 22 nt to form a closed stem segment, and part c is complementary to the crRNA part in the CRISPR system; The CRISPR / Cas 12a system is composed of crRNA and Cas 12a protein, and the crRNA is a nucleotide sequence complementary to the c part of the dumbbell-shaped LSL probe stem.

[0008] In a preferred embodiment of the present invention, the gene chip sensor further comprises a modified gold-plated wire screen-printed electrode, and the modified gold-plated wire screen-printed electrode includes: gold nanoparticles modified on the surface of the screen-printed electrode, a gold-plated wire screen-printed electrode modified with a P0 probe, and an FeCo nanomaterial modified with a CP probe; The P0 probe is a nucleotide sequence containing a cleavage site of the CRISPR system, and is divided into an upstream sequence A and a downstream sequence B from the cleavage site of the P0 probe; the CP probe is a nucleotide sequence that is completely complementary to the cleavage site of the P0 probe and the downstream sequence B and is incompletely complementary to the upstream sequence A.

[0009] In a preferred embodiment of the present invention, the preparation method of the gold-plated wire screen-printed electrode modified with the P0 probe is as follows: treating the P0 probe modified with a thiol group at the 5' end with TCEP, incubating the treated P0 probe with the gold-plated wire screen-printed electrode at 4 °C overnight to obtain a modified screen-printed electrode; dropping an MCH solution on the surface of the modified screen-printed electrode and reacting at 25 °C for 30 minutes to obtain a screen-printed electrode modified with the P0 probe.

[0010] In a preferred embodiment of the present invention, the preparation method of the FeCo nanomaterial modified with the CP probe is as follows: dissolving 0.3 g of Co(Ac)2·4H2O, 0.33 g of Fc(COOH)2 and 3.5 g of PVP in 24 mL of dimethylformamide solution, ultrasonically mixing for 30 minutes, transferring the mixture to a reaction vessel, heating at 125 °C for 5 h, centrifuging and washing to obtain a CoFe MOF solution; adding 6 μL of a capture agent and 200 μL of EDC to 200 μL of the above CoFe MOF solution, incubating in a 25 °C metal bath at an oscillation speed of 1000 revolutions per minute, centrifuging and washing to obtain an FeCo nanomaterial modified with the CP probe.

[0011] The second object of the present invention is to provide the application of the above gene chip sensor in the detection of single nucleotide polymorphisms.

[0012] The third object of the present invention is to provide a method for detecting single nucleotide polymorphisms by the above gene chip sensor, and the method comprises the following steps: S1: annealing the sample to be detected with a dumbbell-shaped LSL probe to obtain an SNP recognition solution; S2: diluting Cas12a and crRNA respectively with 1×NE buffer, then mixing the diluted Cas12a and crRNA evenly according to a ratio, and incubating in a 25 °C metal bath for 30 minutes to obtain a Cas12a / crRNA solution; S3: Add the Cas12a / crRNA solution obtained in S2 to the SNP recognition solution obtained in S1 and mix evenly to obtain the test solution a. S4: Drop the test solution a obtained in S3 onto the working area of the gold wire mesh printed electrode modified with the P0 probe to carry out the Cas12a / crRNA system reaction; drop the FeCo nanomaterial modified with the CP probe onto the surface of the above electrode. After the reaction is completed, perform electrochemical detection on the electrochemical oxidation-reduction signal of the ferrocene active site in the FeCo nanomaterial, substitute the detection result into the linear regression equation of the standard curve for calculation, and make a judgment based on the calculation result.

[0013] In a preferred embodiment of the present invention, the mixing volume ratio of the test sample to the dumbbell-shaped LSL probe in S1 is 1:1, the concentration of the dumbbell-shaped LSL probe is 40 nM, and the conditions for the annealing treatment are: treatment at 95 °C for 5 minutes and treatment at 25 °C for 30 minutes.

[0014] In a preferred embodiment of the present invention, the diluted concentration of Cas12a in S2 is 12 nM, the diluted concentration of crRNA is 24 nM, and the mixing volume ratio of the diluted Cas12a to crRNA is 1:1.

[0015] In a preferred embodiment of the present invention, the mixing volume of the SNP recognition solution to the Cas12a / crRNA solution in S3 is 1:1.

[0016] In a preferred embodiment of the present invention, the electrolyte for the electrochemical detection in S4 is 1×PBS solution and the voltage is 0 - 0.6 V; the judgment criterion is: the minimum linear concentration ≤ calculation result ≤ the maximum linear concentration, and it is judged that the test sample contains SNP mutation sites.

[0017] Advantages of the present invention: The present invention provides a gene chip sensor for single nucleotide polymorphism detection, and the gene chip sensor includes WT wild-type and SNP mutant nucleotide sequences, dumbbell-shaped LSL probes, and a CRISPR / Cas12a system; a CRISPR / Cas12a single-base precise recognition system using the LSL (self-folding dumbbell-shaped functional nucleic acid) probe as a signal transduction medium, and both ends of the dumbbell-shaped LSL probe are a closed hairpin loop (Loop) for target recognition, and its stem segment is complementary to crRNA for activating the CRISPR / Cas12a system.

[0018] In the absence of SNP targets, the dumbbell-shaped LSL probe of the gene chip sensor provided by the present invention will spontaneously fold into a closed structure, locking its stem segment and preventing it from interacting with crRNA. In the presence of SNP targets, specific recognition opens the Loop segments at both ends of the dumbbell-shaped LSL probe, releasing the stem segment sequence and activating the cleavage function of the CRISPR / Cas12a system. On the contrary, for the WT wild-type nucleotide sequence, due to a single-base mismatch, the Loop segment of the dumbbell-shaped LSL probe cannot be unlocked, and therefore, the cleavage function of the CRISPR / Cas12a system cannot be activated. The design of the Loop segments at both ends of the dumbbell-shaped LSL probe endows the gene chip sensor with structural stability, and the steric hindrance effect of the ring structure effectively inhibits non-specific interactions.

[0019] The present invention combines the CRISPR / Cas system with electrochemical analysis to construct an electrochemical sensor for single nucleotide polymorphism detection based on the synergistic regulation of CRISPR / Cas12a and nanomaterials, and selects nanomaterials as signal amplification materials. The gene chip sensor also includes a screen-printed electrode, which includes: gold nanoparticles modified on the surface of the screen-printed electrode, a gold-plated screen-printed electrode modified with a P0 probe, and an FeCo nanomaterial modified with a CP probe. Among them, the P0 probe modified on the electrode is used to capture the FeCo nanomaterial connected to the CP probe through a chemical bond (EDC / NHS). Thus, in the electrochemical system, the ferrocene active center of the FeCo nanomaterial generates a strong redox signal. Further, after Cas12a is activated by the target, the P0 probe is cleaved and detached from the electrode surface, resulting in a sharp decrease in the electrochemical signal. By comparing the currents before and after Cas12a treatment, specific detection of SNP targets is achieved.

[0020] The gene chip sensor provided by the present invention has a highly sensitive, easy-to-operate and highly specific electrochemical biosensing platform, which can accurately identify and distinguish WT wild-type and SNP mutant nucleotide sequences, and uses CP@FeCo nanomaterials as signal sources to provide reliable analytical performance, reduce false positive results, and can be used to detect single nucleotide polymorphisms (SNPs). Moreover, the present invention has verified the detection ability of detecting single nucleotide polymorphisms in soybean genes, and the results show excellent analytical performance, including: low detection limit, fast response time and high single-base discrimination ability. The gene chip sensor provided by the present invention uses iron-cobalt nanomaterials to provide a stable signal source for sample detection, combines screen-printed electrodes with electrochemical sensing, further improves the sensitivity, and can accurately analyze the dose effect of single nucleotide polymorphisms in soybeans; by combining synthetic biology, nanotechnology and precision agriculture, it expands the application of CRISPR / Cas12a and nanomaterial technology in molecular breeding, and provides new strategies and technical support for cost-effective SNP detection. Brief Description of the Drawings

[0021] Figure 1 It is a flow chart for the gene chip sensor to detect single nucleotide polymorphisms in soybean genes; Figure 2 It is a verification diagram of polyacrylamide gel electrophoresis during the detection process of the gene chip sensor; Figure 3 It is a step-by-step characterization diagram of EIS during the detection process of the gene chip sensor; Figure 4 It is a result diagram of the gene chip sensor detecting single nucleotide polymorphisms in soybean genes; Figure 5 It is a linear calibration curve diagram between the gene chip sensor and the concentration of SNP mutant nucleotide sequences. Detailed Embodiments

[0022] Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0023] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in this field, and those skilled in the art can obtain them through commercial channels without special instructions.

[0024] The following examples take the detection of soybeans Glyma.20G116200 gene SNPs as an example to specifically illustrate the technical solution of the present invention. The nucleotide sequence of the WT wild-type soybean Glyma.20G116200 gene is shown in SEQ ID NO.7, and the nucleotide sequence of the SNP mutant soybean Glyma.20G116200 gene is shown in SEQ ID NO.8; The oligonucleotide chains involved in the examples were all synthesized by Shanghai Sangon Biotech Co., Ltd.

[0025] Example 1: Application of a gene chip sensor in the detection of soybean single nucleotide polymorphisms The gene chip sensor provided in this example includes the WT wild-type and SNP mutant nucleotide sequences of the soybean Glyma.20G116200 gene, dumbbell-shaped LSL probe, and CRISPR / Cas 12a system; the nucleotide sequence of the SNP mutant is shown in SEQ ID NO.1; the nucleotide sequence of the WT wild-type is shown in SEQ ID NO.2; the nucleotide sequence of the dumbbell-shaped LSL probe is shown in SEQ ID NO.3; the CRISPR / Cas 12a system is composed of crRNA and Cas 12a protein, and the crRNA is a nucleotide sequence complementary to the stem segment of the dumbbell-shaped LSL probe; the nucleotide sequence of the crRNA is shown in SEQ ID NO.6.

[0026] The gene chip sensor also includes a silk-gold screen-printed electrode, including: gold nanoparticles modified on the surface of the screen-printed electrode, a gold-plated screen-printed electrode modified with a P0 probe, and an FeCo nanomaterial modified with a CP probe; the nucleotide sequence of the P0 probe is shown in SEQ ID NO.4; the nucleotide sequence of the CP probe is shown in SEQ ID NO.5.

[0027] Method for detecting soybean single nucleotide polymorphisms by a gene chip sensor: S1: Dissolve the nucleic acid sample powder to be tested in DEPC water to prepare a nucleic acid stock solution with a final concentration of 100 μM, and store it at -20 °C for subsequent use; dilute the nucleic acid stock solution to 10 μM with 50 mM Tris-HCl buffer (pH 7.4, 10 mM MgCl2, dissolved in DEPC water); Mix the nucleic acid stock solution with the dumbbell-shaped LSL probe (shown as SEQ ID NO.3) for annealing treatment to form a stable secondary structure and obtain the SNP recognition solution; the mixing volume ratio of the sample to be tested and the dumbbell-shaped LSL probe is 1:1, the concentration of the dumbbell-shaped LSL probe is 40 nM, and the conditions for the annealing treatment are: treat at 95 °C for 5 minutes and at 25 °C for 30 minutes; S2: Add 2.4 μL of Cas12a (Cpf1, 1 μM) to 1×NE buffer to obtain a 12 nM Cas12a solution; dilute the crRNA with 1×NE buffer to obtain a 24 nM crRNA (shown as SEQ ID NO.6) solution; mix the above-diluted Cas12a and crRNA evenly according to a mixing volume ratio of 1:1, and incubate in a 25 °C metal bath for 30 minutes to obtain the Cas12a / crRNA solution; S3: Add the Cas12a / crRNA solution obtained in S2 to the SNP recognition solution obtained in S1 and mix evenly to obtain the test solution a; the mixing volume of the SNP recognition solution and the Cas12a / crRNA solution is 1:1; S4: Preparation of a gold wire screen-printed electrode modified with the P0 probe: Wash the disposable SPCE, and blow it dry with nitrogen to remove surface contaminants; take 200 μL of 0.1% HAuCl4 solution and drop it on the working area of the SPCE, and deposit it at a deposition potential of -0.2 V for 100 seconds, and rinse the SPCE / Au with deionized water and PBS to obtain the gold wire screen-printed electrode SPCE / Au; mix the thiolated P0 probe (1 μM) with the nucleotide sequence shown as SEQ ID NO.4 and TCEP (10 mM) in a volume ratio of 1:1, and incubate at room temperature for 1 hour to reduce disulfide bonds to obtain the P0 probe solution; then take 14 μL of the P0 probe solution treated with TCEP and drop-coat it on the surface of the SPCE / Au, and incubate overnight at 4 °C to fix the P0 probe on the electrode through the Au-S bond (SPCE / Au / P0); continue to drop 14 μL of 1 mM MCH on the electrode and react at 25 °C for 30 minutes to block the remaining sites on the electrode (forming the GCE / Au / P0 / MCH structure) to obtain the gold wire screen-printed electrode modified with the P0 probe (SPCE electrode b); S5: Preparation of FeCo nanomaterials modified with CP probe: Dissolve 0.3 g of Co(Ac)₂·4H₂O, 0.33 g of Fc(COOH)₂ and 3.5 g of PVP in 24 mL of dimethylformamide solution, perform ultrasonic mixing for 30 minutes, then transfer the mixture to a reaction vessel, heat at 125 °C for 5 h. After the reaction is completed, centrifuge the reaction product (at a speed of 10,000 revolutions per minute for 10 minutes) and wash it 3 times to obtain a CoFe MOF solution; add 6 μL of a capture agent (40 mM) and 200 μL of EDC (40 mM) to 200 μL of the above CoFe MOF solution (0.1 mg / ml), incubate in a metal bath at 25 °C with an oscillation speed of 1,000 revolutions per minute, centrifuge and wash 3 times to obtain FeCo nanomaterials modified with CP probe (diluted with 50 mM Tris-HCl buffer); S6: Drop the test solution a obtained in S3 onto the working area of the SPCE electrode b obtained in S4, incubate at 37 °C for 40 minutes to lyse the P0 probe immobilized on the electrode and carry out the Cas12a / crRNA system reaction; drop 14 μL of the FeCo nanomaterials modified with the CP probe (shown as SEQ ID NO.5) obtained in S5 onto the surface of the above electrode, incubate at room temperature for 60 minutes, wash 3 times with 40 μL of 1×PBS, perform electrochemical detection on the electrochemical oxidation-reduction signal of the ferrocene active site in the FeCo nanomaterials, substitute the detection results into the linear regression equation of the standard curve for calculation, and make a judgment according to the calculation results; The steps of the electrochemical detection are as follows: At 25 °C, electrochemical measurements were carried out using a CHI 660E instrument (Shanghai, China). Differential pulse voltammetry (DPV) measurements were carried out by scanning the potential from -1.0 V to 0.8 V, and the electrolyte used was 1×PBS; To further evaluate the prepared gene chip sensor and its function, 5 mM [Fe(CN)₆] 3- / 4- (0.1 M KCl) was used to carry out electrochemical impedance spectroscopy (EIS) measurements in the range of -0.2 V to 0.6 V, the amplitude potential was 5 mV, and the scanning frequency range was 0.1 to 10 5 Hz; The judgment criterion is: minimum linear concentration ≤ calculation result ≤ maximum linear concentration, and it is judged that the test sample contains SNP mutation sites.

[0028] Effect experiment: (1) The flow chart of the gene chip sensor provided in this example for detecting single nucleotide polymorphisms of soybean genes is as Figure 1As shown, in the absence of the SNP target, the dumbbell-shaped LSL probe will spontaneously fold into a closed structure, locking its stem segment and preventing it from interacting with crRNA; in the presence of the SNP target, specific recognition opens the Loop segments at both ends of the dumbbell-shaped LSL probe, releasing the stem segment sequence and activating the cleavage function of the CRISPR / Cas12a system. Conversely, for the WT wild-type nucleotide sequence, due to a single-base mismatch, the Loop segment of the dumbbell-shaped LSL probe cannot be unlocked, and thus, the cleavage function of the CRISPR / Cas12a system cannot be activated; the design of the Loop segments at both ends of the dumbbell-shaped LSL probe endows the gene chip sensor with structural stability, and the steric hindrance of the ring structure effectively inhibits non-specific interactions.

[0029] (2) In this example, the polyacrylamide gel electrophoresis was used to verify the gene chip sensor during the detection of soybean single nucleotide polymorphisms. The results are as Figure 2 shown, indicating that the design of the DNA strand used in the gene chip sensor provided by the present invention is feasible.

[0030] (3) EIS step-by-step characterization: As Figure 3 shown by curve a in, the unmodified SPCE electrode exhibits high impedance; after depositing the gold nanoparticle layer, the impedance of the SPCE electrode decreases (as Figure 3 shown by curve b in), the reason being that the gold nanoparticles modified on the surface of the screen-printed electrode provide enhanced electron transfer; subsequently, the P0 probe is fixed on the electrode through the Au-S bond, and it can be observed that the impedance of the SPCE electrode increases (as Figure 3 shown by curve c in), which is because of the negatively charged phosphate backbone of DNA; when MCH used to block non-specific sites is assembled onto the electrode, the impedance of the SPCE electrode further increases (as Figure 3 shown by curve d in), indicating the successful modification of MCH; when the dumbbell-shaped LSL probe recognizes the SNP target, Cas12a / crRNA is activated and introduced onto the electrode surface, and the activated complex cleaves the single-stranded DNA, reducing the coverage of the P0 probe on the electrode surface, thereby resulting in a further decrease in the impedance of the SPCE electrode (as Figure 3 shown by curve e in); finally, the FeCo nanomaterial modified with the CP probe is hybridized with the SPCE electrode through the complementary capture strand CP probe, resulting in a significant increase in the impedance of the SPCE electrode (as Figure 3 shown by curve f in). It can be seen that the above results all prove the successful completion of the step-by-step assembly of the gene chip sensor provided by the present invention.

[0031] (4) In this example, Glyma.20G116200The nucleotide sequences of the wild-type gene WT and the SNP mutant, and the Tris-HCl buffer (Blank) were used as samples to be tested respectively. The single nucleotide polymorphism of soybeans was detected using a gene chip sensor, and the results are as Figure 4 shown. A peak appeared at +0.36 V in the Blank experimental group. When the WT and SNP experimental groups were tested, the current at the peak at +0.36 V decreased, and the current decrease in the SNP experimental group was more significant. The reason is that the SNP-mediated CRISPR / Cas12a was activated and cleaved the P0 probe attached to the electrode surface, thereby reducing the capture efficiency of the FeCo nanomaterial for the modified CP probe.

[0032] In summary, the gene chip sensor provided by the present invention can effectively distinguish Glyma.20G116200 the nucleotide sequences of the wild-type gene WT and the SNP mutant.

[0033] (5) Plotting of the standard curve According to the optimized experimental conditions, nucleotide sequence solutions of the gene SNP mutant with different concentrations of 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000 pM were prepared, and then annealed with the dumbbell-shaped LSL probe solution respectively. Differential pulse voltammetry (DPV) measurements were carried out using the gene chip sensor to plot the standard curve; the potential ranged from 0 to 0.6 V, and the electrolyte used was 10 mM PBS. The electrochemical signal difference ΔI = I Glyma.20G116200 -I was recorded when different concentrations of standard solutions were added, where I blank is the blank current and I is the test current. blank

[0034] The results are as Figure 5 shown. In the wide detection range from 100 aM to 10 pM, as the concentration of the SNP mutant nucleotide sequence solution increased, the peak current gradually decreased, and the signal difference ΔI had a significant linear correlation with the negative logarithm of the concentration of the SNP mutant nucleotide sequence solution. The linear regression equation was: y = 117.16×logC MT + 663.55 (R 2 = 0.9960).

[0035] The content not described in detail in the specification of the present invention is well-known technology to those skilled in the art. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A gene chip sensor for single nucleotide polymorphism detection, characterized in that, The gene chip sensor includes WT wild-type and SNP mutant nucleotide sequences, dumbbell-shaped LSL probes, and a CRISPR / Cas 12a system; The WT wild-type and SNP mutant nucleotide sequences are designed for the target gene sequence fragment; The dumbbell-shaped LSL probe is designed for the mutant fragment of the target gene sequence. The dumbbell-shaped LSL probe is a single-stranded molecule composed of five parts, a, b, c, d, and e, which can self-assemble into a dumbbell structure. Among them, parts b and d are the loop parts of the dumbbell-shaped LSL probe. The two loops contain 18 nt of the same sequence of nucleotides and are both completely complementary to the SNP mutant nucleotide sequence. Parts a, e, and c are the stem parts of the dumbbell-shaped LSL probe. Parts a and e both contain 11 nt of nucleotides, and parts a and e are completely complementary to the 22-nt c part sequence to form a closed stem segment. The c part is complementary to the crRNA part in the CRISPR system; The CRISPR / Cas 12a system is composed of crRNA and Cas 12a protein. The crRNA is a nucleotide sequence complementary to the c part of the stem of the dumbbell-shaped LSL probe.

2. The gene chip sensor according to claim 1, characterized in that, The gene chip sensor also includes a modified gold-plated wire screen-printed electrode, and the modified gold-plated wire screen-printed electrode includes: gold nanoparticles modified on the surface of the screen-printed electrode, a gold-plated wire screen-printed electrode modified with a P0 probe, and an FeCo nanomaterial modified with a CP probe; The P0 probe is a nucleotide sequence containing the cleavage site of the CRISPR system, and is divided into an upstream sequence A and a downstream sequence B from the cleavage site of the P0 probe; the CP probe is a nucleotide sequence that is completely complementary to the cleavage site of the P0 probe and the downstream sequence B and is not completely complementary to the upstream sequence A.

3. The gene chip sensor according to claim 2, wherein The preparation method of the gold-plated wire screen-printed electrode modified with the P0 probe is as follows: Treat the P0 probe modified with a thiol group at the 5' end with TCEP, and incubate the treated P0 probe with the gold-plated wire screen-printed electrode at 4 °C overnight to obtain a modified screen-printed electrode; Drop MCH solution on the surface of the modified screen-printed electrode obtained above, and react at 25 °C for 30 minutes to obtain a screen-printed electrode modified with the P0 probe.

4. The gene chip sensor according to claim 2, wherein The preparation method of the FeCo nanomaterial modified with the CP probe is as follows: Dissolve 0.3 g of Co(Ac)2·4H2O, 0.33 g of Fc(COOH)2, and 3.5 g of PVP in 24 mL of dimethylformamide solution, ultrasonically mix for 30 minutes, transfer the mixture to a reaction vessel, heat at 125 °C for 5 h, centrifuge and wash to obtain a CoFe MOF solution; add 6 μL of a capture agent and 200 μL of EDC to 200 μL of the above CoFe MOF solution, incubate in a 25 °C metal bath at an oscillation speed of 1000 revolutions per minute, centrifuge and wash to obtain an FeCo nanomaterial modified with the CP probe.

5. Use of the gene chip sensor according to any one of claims 1 to 4 in the detection of single nucleotide polymorphisms.

6. The method for detecting single nucleotide polymorphisms by the gene chip sensor according to any one of claims 1 to 4, characterized in that The method includes the following steps: S1: Mix the sample to be tested with dumbbell-shaped LSL probes and perform annealing treatment to obtain SNP recognition solution; S2: Dilute Cas12a and crRNA with 1×NE buffer respectively, then mix the diluted Cas12a and crRNA evenly according to a ratio, and incubate in a 25°C metal bath for 30 minutes to obtain Cas12a / crRNA solution; S3: Add the Cas12a / crRNA solution obtained in S2 to the SNP recognition solution obtained in S1 and mix evenly to obtain test solution a; S4: Drop the test solution a obtained in S3 onto the working area of the gold wire screen-printed electrode modified with P0 probe to carry out the Cas12a / crRNA system reaction; drop the FeCo nanomaterial modified with CP probe onto the surface of the above electrode. After the reaction ends, perform electrochemical detection on the electrochemical redox signal of the ferrocene active site in the FeCo nanomaterial, substitute the detection result into the linear regression equation of the standard curve for calculation, and make a judgment according to the calculation result.

7. The method according to claim 6, characterized in that, In S1, the mixing volume ratio of the sample to be tested and the dumbbell-shaped LSL probe is 1:1, the concentration of the dumbbell-shaped LSL probe is 40 nM, and the conditions of the annealing treatment are: treatment at 95°C for 5 minutes and treatment at 25°C for 30 minutes.

8. The method according to claim 6, characterized in that In S2, the diluted concentration of Cas12a is 12 nM, the diluted concentration of crRNA is 24 nM, and the mixing volume ratio of the diluted Cas12a and crRNA is 1:

1.

9. The method according to claim 6, wherein In S3, the mixing volume of the SNP recognition solution and the Cas12a / crRNA solution is 1:

1.

10. The method according to claim 6, wherein In S4, the electrolyte for the electrochemical detection is 1×PBS solution and the voltage is 0 - 0.6 V; The judgment criterion is: minimum linear concentration ≤ calculation result ≤ maximum linear concentration, and it is judged that the sample to be tested contains SNP mutation sites.

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