A gene chip sensor for single nucleotide polymorphism detection and its application

Abstract

A gene chip sensor for single nucleotide polymorphism detection and its application belong to the field of biosensor detection technology. In order to solve the technical problems of low specificity and accuracy, long processing time and high cost in the existing single nucleotide polymorphism detection methods, the present invention provides a gene chip sensor for single nucleotide polymorphism detection, wherein the gene chip sensor comprises WT wild-type and SNP mutant nucleotide sequences, LSL probe, and CRISPR / Cas 12a system. LSL probe is used as a recognition intermediate to activate the CRISPR / Cas12a system, iron-cobalt nanomaterials are used to provide a stable signal source for sample detection, and screen-printed electrodes are combined with electrochemical sensors; the application of CRISPR / Cas12a and nanomaterial technology in molecular breeding is expanded, and technical support is provided for economical and efficient SNP detection.

Description

Technical Field

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

[0002] Soybean ( Glycine max ) is one of the world's most important oilseed crops, not only providing high-quality vegetable oil for the human diet but also serving as a key renewable feedstock for biodiesel. Driven by shifting dietary patterns driven by population growth, bioenergy policies, and innovation-driven transformation and upgrading of the soybean deep processing industry, global soybean consumption continues to grow steadily annually. However, in the face of this rigid and growing demand, traditional domestication and selective breeding have shown diminishing marginal returns in increasing yield potential. Therefore, the industry urgently needs advances in molecular design breeding technologies. With the publication of the first whole-genome sequence of the American soybean variety "Williams 82," an increasing number of single nucleotide polymorphisms (SNPs) have been found to play a key role in gene functional differentiation; SNP analysis enables breeders to accurately assess the genetic diversity of soybean germplasm resources, thereby facilitating the selection of superior materials and optimal parent combinations.

[0003] In recent years, with the continuous advancement of molecular marker technology, soybean single nucleotide polymorphism (SNP) detection systems have developed into a multi-technology framework. DNA sequencing is considered the "gold standard" for SNP detection due to its high specificity, but its use in soybean breeding is limited by 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 associated with specific traits. Furthermore, the accuracy of chip-based hybridization is affected when analyzing complex genomes rich in repetitive sequences. New SNP genotyping strategies based on various isothermal nucleic acid amplification technologies (such as rolling circle amplification, hybridization chain reaction, and catalytic hairpin assembly) have improved detection sensitivity but are susceptible to interference from repetitive genomic sequences, and sequence complexity may lead to nonspecific amplification or genotyping failure.

[0004] As a third-generation gene editing tool, the CRISPR / Cas12a system uses the Cas12a protein to form a complex with crRNA to directly recognize double-stranded or single-stranded DNA targets, activating cis-cleavage of the target DNA and trans-cleavage of any non-complementary single-stranded DNA. In addition, CRISPR / Cas12a only requires a complementary sequence of 24-26 bases to crRNA, and has a high degree of design flexibility and excellent nucleic acid cleavage ability, making it highly applicable in SNP genotyping. However, the high sequence similarity of the allopolyploid soybean genome increases the risk of Cas12a being erroneously activated by non-target wild-type (WT) sequences, limiting single-base resolution; how to effectively transmit single-point mutation signals in SNP sequences to the Cas enzyme to activate its function remains a key challenge that those skilled in the art 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 aims to solve the technical problems of low specificity and accuracy, long processing time and high cost in the existing single nucleotide polymorphism detection methods, and provides a gene chip sensor for single nucleotide polymorphism detection and its application.

[0007] One of the objects 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;

[0008] The WT wild-type and SNP mutant nucleotide sequences are designed based on the target gene sequence fragment;

[0009] The dumbbell-shaped LSL probe is designed for the mutation 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 of nucleotides of the same sequence, and are both completely complementary to the SNP mutation 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 of 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 the part c is complementary to the crRNA part in the CRISPR system;

[0010] The CRISPR / Cas 12a system is composed of crRNA and Cas 12a protein, and the crRNA is a nucleotide sequence that is complementary to the c part of the dumbbell-shaped LSL probe stem.

[0011] In a preferred embodiment of the present invention, the gene chip sensor further comprises a modified gold-plated screen-printed electrode, wherein the modified gold-plated screen-printed electrode comprises: gold nanoparticles modified on the surface of the screen-printed electrode, a gold-plated screen-printed electrode modified with a P0 probe, and a FeCo nanomaterial modified with a CP probe;

[0012] The P0 probe is a nucleotide sequence containing a CRISPR system cleavage site, which is divided into an upstream sequence A and a downstream sequence B from the P0 probe cleavage site; the CP probe is a nucleotide sequence that is completely complementary to the P0 probe cleavage site and the downstream sequence B, and is not completely complementary to the upstream sequence A.

[0013] In a preferred embodiment of the present invention, the preparation method of the gold-plated screen-printed electrode modified with the P0 probe is as follows: using TCEP to treat the P0 probe modified with a thiol group at the 5' end, incubating the treated P0 probe with the gold-plated screen-printed electrode at 4°C overnight to obtain a modified screen-printed electrode; adding MCH solution to 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.

[0014] In a preferred embodiment of the present invention, the preparation method of the FeCo nanomaterial modified with the CP probe is as follows: 0.3 g Co(Ac)2·4H2O, 0.33 g Fc(COOH)2 and 3.5 g PVP are dissolved in 24 mL dimethylformamide solution, ultrasonically mixed for 30 minutes, the mixture is transferred to a reaction vessel, heated at 125°C for 5 hours, centrifuged, and washed to obtain a CoFe MOF solution; 6 μL of capture agent and 200 μL of EDC are added to 200 μL of the above CoFe MOF solution, incubated in a metal bath at 25°C at an oscillation speed of 1000 rpm, centrifuged, and washed to obtain the FeCo nanomaterial modified with the CP probe.

[0015] A second object of the present invention is to provide an application of the above-mentioned gene chip sensor in detecting single nucleotide polymorphisms.

[0016] A third object of the present invention is to provide a method for detecting single nucleotide polymorphisms using the above-mentioned gene chip sensor, the method comprising the following steps:

[0017] S1: Mix the sample to be tested with the dumbbell-shaped LSL probe and perform annealing treatment to obtain SNP recognition solution;

[0018] S2: Dilute Cas12a and crRNA separately with 1×NE buffer, then mix the diluted Cas12a and crRNA in proportion, and incubate in a 25°C metal bath for 30 minutes to obtain a Cas12a / crRNA solution;

[0019] 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;

[0020] S4: The test solution a obtained in S3 is added dropwise to the working area of ​​the gold-plated screen-printed electrode modified with the P0 probe to perform the Cas12a / crRNA system reaction; the FeCo nanomaterial modified with the CP probe is added dropwise to the surface of the above-mentioned electrode, and after the reaction is completed, the electrochemical redox signal of the dipentyl iron active site in the FeCo nanomaterial is electrochemically detected, and the detection results are substituted into the linear regression equation of the standard curve for calculation, and judgment is made based on the calculation results.

[0021] In a preferred embodiment of the present invention, the mixing volume ratio of the test sample and the dumbbell-shaped LSL probe in S1 is 1:1, the concentration of the dumbbell-shaped LSL probe is 40 nM, and the annealing conditions are: 95°C for 5 minutes and 25°C for 30 minutes.

[0022] In a preferred embodiment of the present invention, the concentration of the diluted Cas12a in S2 is 12 nM, the concentration of the diluted crRNA is 24 nM, and the mixed volume ratio of the diluted Cas12a to crRNA is 1:1.

[0023] In a preferred embodiment of the present invention, the mixed volume of the SNP recognition solution and the Cas12a / crRNA solution in S3 is 1:1.

[0024] In a preferred embodiment of the present invention, the electrolyte for electrochemical detection in S4 is 1×PBS solution and the voltage is 0-0.6 V; the judgment standard is: minimum linear concentration ≤ calculated result ≤ maximum linear concentration, and it is judged that the sample to be tested contains a SNP mutation site.

[0025] Beneficial effects of the present invention: The present invention provides 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 / Cas12a system; a CRISPR / Cas12a single-base precise recognition system utilizing an LSL (self-folding dumbbell-shaped functional nucleic acid) probe as a signal transduction medium, wherein both ends of the dumbbell-shaped LSL probe are a closed hairpin loop for target identification, and the stem segment thereof complements the crRNA for activating the CRISPR / Cas12a system.

[0026] In the gene chip sensor provided by the present invention, in the absence of a SNP target, the dumbbell-shaped LSL probe will spontaneously fold into a closed structure, locking its stem segment to prevent it from interacting with crRNA; in the presence of a 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 cutting function of the CRISPR / Cas12a system. On the contrary, the WT wild-type nucleotide sequence cannot unlock the Loop segment of the dumbbell-shaped LSL probe due to a single base mismatch, and therefore cannot activate the cutting function of the CRISPR / Cas12a system; the design of the Loop segments at both ends of the dumbbell-shaped LSL probe gives the gene chip sensor structure stability, and the three-dimensional space hindering effect of the annular structure effectively suppresses nonspecific interactions.

[0027] The present invention combines the CRISPR / Cas system with electrochemical analysis to construct an electrochemical sensor for single nucleotide polymorphism detection based on the coordinated 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; wherein 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); thereby, in the electrochemical system, the dipentyl iron active center of the FeCo nanomaterial generates a strong redox signal; further, after Cas12a is activated by the target, the P0 probe is cut and detached from the electrode surface, resulting in a sharp drop in the electrochemical signal; by comparing the current before and after Cas12a treatment, specific detection of the SNP target is achieved.

[0028] The gene chip sensor provided by the present invention features a highly sensitive, easy-to-use, and highly specific electrochemical biosensing platform capable of accurately identifying and distinguishing wild-type (WT) and mutant (SNP) nucleotide sequences. Utilizing CP@FeCo nanomaterials as a signal source, it provides reliable analytical performance, reduces false-positive results, and can be used to detect single nucleotide polymorphisms (SNPs). Furthermore, the present invention has demonstrated its ability to detect SNPs in soybean genes, demonstrating excellent analytical performance, including low detection limits, fast response times, and high single-base discrimination capabilities. The gene chip sensor provided by the present invention utilizes iron-cobalt nanomaterials to provide a stable signal source for sample detection. The combination of screen-printed electrodes and electrochemical sensing further enhances sensitivity, enabling accurate analysis of the dosage effect of SNPs in soybeans. By integrating synthetic biology, nanotechnology, and precision agriculture, the sensor expands the application of CRISPR / Cas12a and nanomaterial technologies in molecular breeding, providing new strategies and technical support for cost-effective SNP detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Flowchart for detecting soybean gene single nucleotide polymorphisms using gene chip sensors;

[0030] Figure 2 This is a polyacrylamide gel electrophoresis verification diagram during the gene chip sensor detection process;

[0031] Figure 3 This is a step-by-step characterization diagram of EIS during the detection process of the gene chip sensor;

[0032] Figure 4 This is the result of detecting soybean gene single nucleotide polymorphism using gene chip sensor;

[0033] Figure 5 The figure shows the linear calibration curve between the gene chip sensor and the concentration of the SNP mutant nucleotide sequence. DETAILED DESCRIPTION

[0034] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine 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.

[0035] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0036] The following examples are based on the detection of soybean Glyma.20G116200 The technical solution of the present invention is specifically described by taking the SNP of the gene as an example. The WT wild-type soybean Glyma.20G116200 The nucleotide sequence of the gene is shown in SEQ ID NO.7, and the SNP mutant soybean Glyma.20G116200 The nucleotide sequence of the gene is shown in SEQ ID NO.8;

[0037] The oligonucleotide chains involved in the examples were synthesized by Shanghai Sangon Biotechnology Service Co., Ltd.

[0038] Example 1: Application of a gene chip sensor in detecting soybean single nucleotide polymorphisms

[0039] The gene chip sensor provided in this embodiment includes soybean Glyma.20G116200 The WT wild-type and SNP mutant nucleotide sequences of the gene, the dumbbell-shaped LSL probe, and the CRISPR / Cas 12a system; the SNP mutant nucleotide sequence is shown in SEQ ID NO.1; the WT wild-type nucleotide sequence is shown in SEQ ID NO.2; the dumbbell-shaped LSL probe nucleotide sequence 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 dumbbell-shaped LSL probe stem segment; the crRNA nucleotide sequence is shown in SEQ ID NO.6.

[0040] The gene chip sensor also includes a gold-plated 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 a 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.

[0041] Method for detecting soybean single nucleotide polymorphism using gene chip sensor:

[0042] S1: Dissolve the nucleic acid sample powder in DEPC water to prepare a nucleic acid stock solution with a final concentration of 100 μM and store at -20°C for subsequent use. Dilute the nucleic acid stock solution to 10 μM using 50 mM Tris-HCl buffer (pH 7.4, 10 mM MgCl2, dissolved in DEPC water).

[0043] The nucleic acid stock solution was mixed with a dumbbell-shaped LSL probe (shown in SEQ ID NO. 3) and annealed to form a stable secondary structure, thereby obtaining a SNP identification solution. The volume ratio of the test sample to the dumbbell-shaped LSL probe was 1:1, the concentration of the dumbbell-shaped LSL probe was 40 nM, and the annealing conditions were: 95°C for 5 minutes and 25°C for 30 minutes.

[0044] 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 diluted Cas12a and crRNA at a volume ratio of 1:1, and incubate in a metal bath at 25°C for 30 minutes to obtain a Cas12a / crRNA solution;

[0045] S3: Add the Cas12a / crRNA solution obtained in S2 to the SNP recognition solution obtained in S1 and mix evenly to obtain a test solution a; the mixed volume of the SNP recognition solution and the Cas12a / crRNA solution is 1:1;

[0046] S4: Preparation of gold-coated screen-printed electrodes modified with P0 probes:

[0047] The disposable SPCE was cleaned and dried with nitrogen to remove surface contaminants; 200 μL of 0.1% HAuCl4 solution was dropped on the SPCE working area and deposited at a deposition potential of -0.2 V for 100 seconds. The SPCE / Au was rinsed with deionized water and PBS to obtain a gold-plated screen-printed electrode SPCE / Au; the thiolated P0 probe (1 μM) with the nucleotide sequence shown in SEQ ID NO.4 was mixed with TCEP (10 mM) in a volume ratio of 1:1 and incubated at room temperature for 1 hour to reduce disulfide bonds and obtain a P0 probe solution; then 14 μL of the TCEP-treated P0 probe solution was dropped on the SPCE / Au surface and incubated overnight at 4°C to fix the P0 probe on the electrode through the Au-S bond (SPCE / Au / P0); 14 μL of 1 was further dropped on the electrode. mMMCH, reacted at 25°C for 30 minutes to block the remaining sites on the electrode (forming a GCE / Au / P0 / MCH structure), and obtained a gold-plated screen-printed electrode modified with P0 probe (SPCE electrode b);

[0048] S5: Preparation of FeCo nanomaterials modified with CP probes:

[0049] 0.3 g Co(Ac)2·4H2O, 0.33 g Fc(COOH)2, and 3.5 g PVP were dissolved in 24 mL dimethylformamide solution and ultrasonically mixed for 30 minutes. The mixture was then transferred to a reaction vessel and heated at 125°C for 5 hours. After the reaction was completed, the reaction product was centrifuged (at 10,000 rpm for 10 minutes) and washed three times to obtain a CoFeMOF solution. 6 μL of the capture agent (40 mM) and 200 μL of EDC (40 mM) were added to 200 μL of the above CoFeMOF solution (0.1 mg / ml). The mixture was incubated in a metal bath at 25°C at an oscillation speed of 1000 rpm, centrifuged, and washed three times to obtain FeCo nanomaterials modified with CP probes (diluted with 50 mM Tris-HCl buffer).

[0050] S6: The test solution a obtained in S3 was added dropwise to the working area of ​​the SPCE electrode b obtained in S4, and incubated at 37°C for 40 minutes to cleave the P0 probe fixed on the electrode and perform the Cas12a / crRNA system reaction; 14 μL of the FeCo nanomaterial modified with the CP probe (shown in SEQ ID NO.5) obtained in S5 was added dropwise to the surface of the above electrode, incubated at room temperature for 60 minutes, and washed three times with 40 μL of 1×PBS. The electrochemical redox signal of the dipentyl iron active site in the FeCo nanomaterial was electrochemically detected, and the detection results were substituted into the linear regression equation of the standard curve for calculation, and judgment was made based on the calculation results;

[0051] The electrochemical detection steps were as follows: electrochemical measurements were performed at 25°C using a CHI 660E instrument (Shanghai, China) by scanning the potential from -1.0 V to 0.8 V for differential pulse voltammetry (DPV) measurements. The electrolyte used was 1× PBS. To further evaluate the prepared gene chip sensor and its function, 5 mM [Fe(CN)6] 3- / 4- Electrochemical impedance spectroscopy (EIS) measurements were performed in the range of −0.2 V to 0.6 V (0.1 M KCl) with an amplitude potential of 5 mV and a sweep frequency range of 0.1 to 10 5 Hz;

[0052] The judgment standard is: minimum linear concentration ≤ calculated result ≤ maximum linear concentration, and it is judged that the sample to be tested contains a SNP mutation site.

[0053] Effect experiment:

[0054] (1) The flow chart of the gene chip sensor provided in this embodiment for detecting soybean gene single nucleotide polymorphisms is as follows: Figure 1 As shown, in the absence of a SNP target, 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 to prevent it from interacting with crRNA; in the presence of a 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 cutting function of the CRISPR / Cas12a system. On the contrary, the WT wild-type nucleotide sequence cannot unlock the Loop segment of the dumbbell-shaped LSL probe due to a single base mismatch, and therefore cannot activate the cutting function of the CRISPR / Cas12a system; the design of the Loop segments at both ends of the dumbbell-shaped LSL probe gives the gene chip sensor structure stability, and the three-dimensional space hindering effect of the annular structure effectively suppresses nonspecific interactions.

[0055] (2) In this example, the gene chip sensor used in the detection of soybean single nucleotide polymorphism was verified by polyacrylamide gel electrophoresis. The results are as follows: Figure 2 As shown, it is shown that the design of the DNA chain used in the gene chip sensor provided by the present invention is feasible.

[0056] (3) EIS step-by-step characterization:

[0057] like Figure 3 As shown in curve a, the unmodified SPCE electrode exhibits high impedance; after the deposition of the gold nanoparticle layer, the impedance of the SPCE electrode decreases (as shown in Figure 3 The reason for this is 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 via the Au-S bond, and an increase in the impedance of the SPCE electrode can be observed (as shown in curve b). Figure 3 This is because the negatively charged phosphate chain backbone of DNA; when MCH, which is used to block nonspecific sites, is assembled on the electrode, the impedance of the SPCE electrode further increases (as shown by curve c). Figure 3 As shown by the middle curve d), this indicates that the MCH modification is successful; when the dumbbell-shaped LSL probe recognizes the SNP target, Cas12a / crRNA is activated and introduced into the electrode surface. The activated complex cuts the single-stranded DNA, reducing the coverage of the P0 probe on the electrode surface, thereby further reducing the impedance of the SPCE electrode (as shown in Fig. Figure 3 Finally, the FeCo nanomaterial modified with the CP probe was hybridized with the SPCE electrode through the complementary capture chain CP probe, resulting in a significant increase in the impedance of the SPCE electrode (as shown in the curve e). Figure 3 As shown by the middle curve f). It can be seen that the above results all prove that the step-by-step assembly of the gene chip sensor provided by the present invention is successfully completed.

[0058] (4) This embodiment is based on Glyma.20G116200 The wild-type and SNP mutant nucleotide sequences of the gene and Tris-HCl buffer (Blank) were used as the test samples, respectively. The soybean single nucleotide polymorphism was detected using the gene chip sensor. The results are shown in Figure 2. Figure 4 As shown in the figure, the blank group exhibited a peak at +0.36 V. However, the WT and SNP groups showed a decrease in current at this peak, with the SNP group experiencing a more significant decrease. This is due to SNP-mediated activation of CRISPR / Cas12a, which cleaves the P0 probe attached to the electrode surface, thereby reducing the capture efficiency of the FeCo nanomaterial modified with the CP probe.

[0059] In summary, the gene chip sensor provided by the present invention can effectively distinguish Glyma.20G116200 Gene WT wild type and SNP mutant nucleotide sequences.

[0060] (5) Drawing of standard curve

[0061] According to the optimized experimental conditions, different concentrations of 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, and 1000 pM were prepared. Glyma.20G116200 The SNP mutant nucleotide sequence solution was then annealed with the dumbbell-shaped LSL probe solution. Differential pulse voltammetry (DPV) was performed using a gene chip sensor to draw a standard curve. The potential was from 0 to 0.6 V, and the electrolyte used was 10 mM PBS. The electrochemical signal difference ΔI = I was recorded when different concentrations of standard solution were added. blank -I, where I blank is the blank current, and I is the test current.

[0062] The results are as follows Figure 5 As shown in the figure, within the wide detection range of 100 aM to 10 pM, as the concentration of the SNP mutant nucleotide sequence solution increases, the peak current gradually decreases, and the signal difference ΔI is significantly linearly correlated with the negative logarithm of the SNP mutant nucleotide sequence solution concentration, and the linear regression equation is: y = 117.16 × log C MT +663.55 (R 2 =0.9960).

[0063] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A gene chip sensor for single nucleotide polymorphism detection, characterized in that: The gene chip sensor comprises WT wild-type and SNP mutant nucleotide sequences, dumbbell-shaped LSL probes, and CRISPR / Cas 12a system; The WT wild-type and SNP mutant nucleotide sequences are designed based on the target gene sequence fragment; The dumbbell-shaped LSL probe is designed for the mutation 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 of nucleotides of the same sequence, and are both completely complementary to the SNP mutation 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 of 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 the 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, wherein the crRNA is a nucleotide sequence complementary to the c portion of the dumbbell-shaped LSL probe stem; The gene chip sensor further comprises a modified gold-plated screen-printed electrode, wherein the modified gold-plated screen-printed electrode comprises: gold nanoparticles modified on the surface of the screen-printed electrode, a gold-plated screen-printed electrode modified with a P0 probe, and a FeCo nanomaterial modified with a CP probe; The P0 probe is a nucleotide sequence containing a CRISPR system cleavage site, which is divided into an upstream sequence A and a downstream sequence B from the P0 probe cleavage site; the CP probe is a nucleotide sequence that is completely complementary to the P0 probe cleavage site and the downstream sequence B, and is not completely complementary to the upstream sequence A.

2. The gene chip sensor according to claim 1, characterized in that: The preparation method of the gold-plated screen-printed electrode modified with the P0 probe is as follows: The P0 probe with a thiol group modified at the 5' end was treated with TCEP, and the treated P0 probe was incubated with a gold-plated screen-printed electrode at 4°C overnight to obtain a modified screen-printed electrode; MCH solution was added dropwise to the surface of the modified screen-printed electrode, and the mixture was allowed to react at 25° C. for 30 minutes to obtain a screen-printed electrode modified with a P0 probe.

3. The gene chip sensor according to claim 1, characterized in that: The preparation method of the FeCo nanomaterial modified with the CP probe is as follows: 0.3 g Co(Ac)2·4H2O, 0.33 g Fc(COOH)2 and 3.5 g PVP were dissolved in 24 mL dimethylformamide solution and ultrasonically mixed for 30 minutes. The mixture was transferred to a reaction vessel, heated at 125°C for 5 hours, centrifuged and washed to obtain a CoFeMOF solution. To 200 μL of the above CoFeMOF solution, 6 μL of capture agent and 200 μL of EDC were added, and the mixture was incubated in a metal bath at 25°C at an oscillation speed of 1000 rpm, centrifuged and washed to obtain FeCo nanomaterials modified with CP probes.

4. Use of the gene chip sensor according to any one of claims 1 to 3 in detecting single nucleotide polymorphisms.

5. The method for detecting single nucleotide polymorphism using a gene chip sensor according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: S1: Mix the sample to be tested with the dumbbell-shaped LSL probe and perform annealing treatment to obtain SNP recognition solution; S2: Dilute Cas12a and crRNA separately with 1×NE buffer, then mix the diluted Cas12a and crRNA in proportion, and incubate 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: The test solution a obtained in S3 is added dropwise to the working area of ​​the gold-plated screen-printed electrode modified with the P0 probe to perform the Cas12a / crRNA system reaction; the FeCo nanomaterial modified with the CP probe is added dropwise to the surface of the above-mentioned electrode, and after the reaction is completed, the electrochemical redox signal of the dipentyl iron active site in the FeCo nanomaterial is electrochemically detected, and the detection results are substituted into the linear regression equation of the standard curve for calculation, and judgment is made based on the calculation results.

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

7. The method according to claim 5, wherein The concentration of the diluted Cas12a in S2 is 12 nM, the concentration of the diluted crRNA is 24 nM, and the mixed volume ratio of the diluted Cas12a and crRNA is 1:

1.

8. The method according to claim 5, characterized in that The mixed volume of the SNP recognition solution and Cas12a / crRNA solution described in S3 is 1:

1.

9. The method according to claim 5, wherein The electrolyte for the electrochemical detection described in S4 was 1× PBS solution and the voltage was 0-0.6 V; The judgment standard is: minimum linear concentration ≤ calculated result ≤ maximum linear concentration, and it is judged that the sample to be tested contains a SNP mutation site.

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