DNAzyme-based gene chip biosensor and application thereof

Abstract

The application relates to a DNAzyme-based gene chip biosensor and application thereof, and belongs to the technical field of electrochemical sensors. In order to solve the technical problems of low specificity and accuracy, long processing time and high cost in the single nucleotide polymorphism detection method in the prior art, a DNAzyme-based gene chip biosensor is provided, which comprises a DNAzyme molecular probe SNP recognition system and a solid-liquid phase mixed catalytic network system. The application provides a proteinase-free and space-oriented biosensing strategy, utilizes a molecular switch based on a DNAzyme probe to realize accurate SNP recognition, utilizes a space steric hindrance parallel amplification system to enhance signal amplification and simultaneously inhibit background noise, so that the detection performance is remarkably improved; and the gene chip biosensor can be applied to genotyping and trait selection in crop improvement and other applications.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensor technology, specifically relating to a DNAzyme-based gene chip biosensor and its applications. Background Technology

[0002] Soybeans Glycine max Soybean is a crucial strategic crop at the core of the global food security system. However, traditional domestication and selection breeding methods are inefficient, and the urgent need for industrial upgrading has spurred the development of molecular design breeding technology to overcome yield potential bottlenecks. Single nucleotide polymorphisms (SNPs) are the most common forms of genetic variation in the soybean genome, typically associated with phenotypic traits, disease susceptibility, and agronomic traits. In the context of crop science, rapid and accurate detection of SNPs is essential for genotype-phenotype association studies, marker-assisted selection, and trait-specific breeding.

[0003] Traditional DNA sequencing technology is considered the "gold standard" for SNP detection due to its high specificity, but the high cost associated with full-length DNA analysis limits its widespread application in soybean breeding. Currently, commonly used SNP detection strategies, such as allele-specific PCR, Sanger sequencing, and high-throughput genotyping platforms, all require complex instruments with limited portability. These factors hinder their application in field diagnostics or resource-scarce environments. Therefore, those skilled in the art are eager to develop a cost-effective, scalable technique capable of detecting single nucleotide polymorphisms (SNPs) in complex biological samples with minimal sample processing.

[0004] Electrochemical biosensors, with their advantages of high sensitivity, rapid response, low cost, great potential for miniaturization, and suitability for field applications, are gradually becoming promising tools for single nucleotide polymorphism (SNP) detection. However, traditional enzyme-linked immunosorbent assay (ELISA) strategies for SNP detection often rely on multi-component strand substitution reactions between the target sequence and double-stranded DNA with single-stranded ends; but they suffer from limited single-base resolution (1→1 distinction) and require precise stoichiometric adjustments to avoid interference from unbound single strands, severely limiting their application in complex samples.

[0005] In recent years, DNAzyme-based catalytic systems, especially those employing 10⁻²³ DNAzyme systems capable of cleaving RNA, have attracted widespread attention. Unlike traditional proteases, DNAzymes are composed entirely of DNA, typically consisting of a catalytic core and two substrate-binding arms. The catalytic core is responsible for cleaving the RNA substrate, while the binding arms can be designed to exhibit high selectivity for specific target sequences. This modular design allows DNAzymes to bind to target molecules in a sequence-specific manner and catalyze their activity. However, existing DNAzymes exhibit poor sequence selectivity when dealing with single nucleotide polymorphisms (SNPs), and their full potential in SNP detection has not yet been fully realized. Summary of the Invention

[0006] To address the technical problems of low specificity and accuracy, long processing time, and high cost in existing single nucleotide polymorphism detection methods, this invention provides a DNAzyme-based gene chip biosensor and its application.

[0007] One objective of this invention is to provide a DNAzyme-based gene chip biosensor, which includes a DNAzyme probe SNP recognition system and a fuel chain-guided solid-liquid hybrid catalytic network; the DNAzyme probe SNP recognition system includes MTzy hairpin probes and WTzy hairpin probes.

[0008] The MTzy hairpin probe is designed based on the SNP mutant nucleotide sequence. The specific steps are as follows: the SNP mutant nucleotide sequence is divided into an upstream b' sequence and a downstream a' sequence at the SNP mutation site. The MTzy hairpin probe comprises four parts: a, b, c, and d. Parts a and c are the stem segments of the MTzy hairpin probe. Part a also includes a 15nt raised loop, with 10nt nucleotides on each side of the raised loop. This raised loop serves as the catalytic core of the 10⁻²³ DNAzyme probe. Part b is a loop containing the "TTTT" sequence. Part c is complementary to the sequences on both sides of the raised loop in part a, and these sequences are identical to the downstream a' sequence. Part d contains an 8-base toehold sequence. Part d is complementary to the SNP mutation site and the upstream b' sequence, and the first base of part d is complementary to the SNP mutation site.

[0009] The WTzy hairpin probe is designed based on the WT wild-type nucleotide sequence. The specific steps are as follows: the WT wild-type nucleotide sequence is divided into an upstream b' sequence and a downstream a' sequence at the unmutated SNP site. The WTzy hairpin probe contains four parts: a, b, c, and d. Parts a and c are the stem segments of the WTzy hairpin probe. Part a also contains a 15nt raised loop, and nucleotides on both sides of the raised loop are 10nt each. This raised loop does not have the catalytic activity of 10⁻²³ DNAzyme. Part b is a loop containing the "TTTT" sequence. Part c is complementary to the sequences on both sides of the raised loop in part a. The sequences on both sides of the raised loop in part a are the same as the downstream a' sequence. Part d contains a toehold composed of an 8-base fulcrum sequence. Part d is complementary to the unmutated SNP site and the upstream b' sequence, and the first base of part d is complementary to the unmutated SNP site.

[0010] In a preferred embodiment of the present invention, the fuel chain Fuel-guided solid-liquid phase hybrid catalytic network consists of a fuel chain Fuel and an electrochemical three-electrode system. The fuel chain Fuel is designed based on SNP mutant nucleotide sequences and consists of two RNA base-modified nucleotide sequences. The nucleotide sequences are complementary to the sequences on both sides of the protruding loop of the MTzy hairpin probe a portion, respectively, and serve as substrates for DNAzyme digestion reactions.

[0011] In a preferred embodiment of the present invention, the electrochemical three-electrode system comprises a working electrode, a counter electrode, and a reference electrode; the counter electrode is a platinum wire electrode, and the reference electrode is a silver chloride electrode.

[0012] In a preferred embodiment of the present invention, the working electrode is composed of a modification electrode and a signal molecule hairpin;

[0013] The preparation steps of the signal molecule hairpin are as follows: Heme and pretreated SWCNT solution are mixed at a volume ratio of 1:3, ultrasonicated until uniformly dispersed, incubated at room temperature for 1 h, centrifuged, washed, and Hemin@SWCNT stock solution is obtained; The above Hemin@SWCNT, 40 mM EDC coupling agent, and 15 μM amino-modified hairpin DNA are mixed at a volume ratio of 200:200:6 and ultrasonicated, wherein the amino-modified hairpin DNA is H2 hairpin or H4 hairpin, shaken at 25℃ for 1 h, centrifuged, and Hemin@SWCNT-H2 and Hemin@SWCNT-H4 signal molecule hairpins are obtained respectively;

[0014] The preparation steps of the modified electrode are as follows: hairpins H1, H3, and H5 with thiol-modified 5' ends are treated with 50 mM TCEP for 1 h at room temperature; the hairpins H1, H3, and H5 are mixed in equal proportions to a final concentration of 0.8 μM to obtain a hairpin mixture; the hairpin mixture is dropped onto the surface of a polished and gold-plated glassy carbon electrode GCE / Au, and incubated overnight at 4°C to obtain a GCE / Au / H135 electrode; 8 μL of 1 mM MCH is added dropwise to the GCE / Au / H135 electrode, and the reaction is carried out at 25°C for 30 minutes, followed by washing to obtain a GCE / Au / H135 / MCH modified electrode.

[0015] In a preferred embodiment of the present invention, the hairpins H1, H2, H3, H4 and H5 are obtained according to the fuel chain design;

[0016] The fuel chain Fuel complements the stem sequence of the H1 hairpin, opening the H1 hairpin and releasing the hidden toe chain 1;

[0017] The toe chain 1 complements the stem sequence of the secondary H2 hairpin, opening the H2 hairpin and releasing the hidden toe chain 2;

[0018] The toe chain 2 complements the stem sequence of the secondary H3 hairpin, opening the H3 hairpin and releasing the hidden toe chain 3;

[0019] The toe chain 3 complements the stem sequence of the secondary H4 hairpin, opening the H4 hairpin and releasing the hidden toe chain 4.

[0020] The toe chain 4 complements the stem sequence of the secondary H5 hair clip, opening the H5 hair clip and releasing the hidden toe chain 5.

[0021] The toe chain is a nucleotide from the loop portion of the hairpin to the toehold portion, including the loop portion and the toehold.

[0022] The second objective of this invention is to provide the application of the above-mentioned gene chip biosensor in the detection of single nucleotide polymorphisms.

[0023] A third objective of this invention is to provide a method for detecting single nucleotide polymorphisms using the aforementioned gene chip biosensor, the method comprising the following steps:

[0024] S1: Perform SNP recognition on the sample to be tested with MTzy probe and WTzy probe respectively to obtain double-stranded complexes MTzy / SNP and WTzy / WT;

[0025] S2: The MTzy / SNP and WTzy / WT double-stranded complexes obtained in S1 are subjected to DNAzyme digestion with the fuel chain Fuel to obtain DNAzyme reaction solution;

[0026] S3: The DNAzyme reaction solution obtained in S2 was mixed with the hairpins of Hemin@SWCNT-H2 and Hemin@SWCNT-H4 signal molecules in a volume ratio of 2:3:3. The mixture was then dropped onto the surface of the modified electrode GCE / Au / H135 / MCH and reacted for 30 minutes for isothermal amplification to obtain the working electrode GCE / Au / H135 / MCH / H24. Electrochemical detection was performed on the working electrode, and the detection results were substituted into the linear regression equation of the standard curve for calculation. The results were then used to make a judgment.

[0027] In a preferred embodiment of the present invention, the conditions for SNP recognition in S1 are: reaction at 95°C for 5 minutes and reaction at 4°C for 20 minutes; the concentrations of the MTzy probe and the WTzy probe are both 150 nM; the conditions for the enzyme digestion reaction in S2 are: reaction at 25°C for 1 h.

[0028] In a preferred embodiment of the present invention, the isothermal amplification reaction conditions in S3 are 1 hour at 5°C; the electrolyte for electrochemical detection is 10 mM PBS buffer, the voltage is -0.8 to 0 V, and the scan rate is 0.05 V / s.

[0029] In a preferred embodiment of the present invention, the method for establishing the standard curve in S3 is as follows:

[0030] Target sequences containing SNP mutation sites were synthesized, and standard solutions with concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, and 1000 pM were prepared. Electrochemical detection was performed using a gene chip biosensor, and a standard curve between concentration and current difference was established to obtain a linear regression equation.

[0031] The judgment criteria are: minimum linear concentration ≤ calculation result ≤ maximum linear concentration, indicating that the sample to be tested contains SNP mutation sites.

[0032] The beneficial effects of this invention are as follows: This invention provides a DNAzyme-based gene chip biosensor, which comprises a DNAzyme probe SNP recognition system and a fuel chain fuel-guided solid-liquid hybrid catalytic network. The DNAzyme probe SNP recognition system includes MTzy hairpin probes and WTzy hairpin probes. The fuel chain fuel-guided solid-liquid hybrid catalytic network consists of a fuel chain fuel and an electrochemical three-electrode system, which comprises a working electrode, a counter electrode, and a reference electrode. The counter electrode is a platinum wire electrode, and the reference electrode is a silver chloride electrode. The MTzy probe contains a 15nt raised loop, with 10nt nucleotides on each side of the raised loop. This raised loop serves as the catalytic core of the 10⁻²³ DNAzyme probe, ensuring that the DNAzyme hairpin successfully opens the fuel chain fuel, exhibiting high catalytic efficiency and specificity. Under normal conditions, the stem-binding arms located on both sides of the 10⁻²³ DNAzyme catalytic core are completely closed, causing DNAzyme inactivation. This thermodynamically stable intramolecular interaction contributes to better assembly and locking efficiency.

[0033] To avoid interference from a large number of wild-type WT sequences, this invention designs a WTzy probe with the same structure, but whose raised loop lacks catalytic activity. During detection, SNP targets containing SNP mutation sites tend to bind to the adapter of the WTzy hairpin probe, thereby initiating a strand displacement reaction, exposing the catalytic core, activating the cleavage reaction, and continuously cleaving the fuel chain (Fuel) containing RNA bases. Meanwhile, wild-type WT sequences without SNP mutation sites tend to bind to the adapter of the WTzy hairpin probe, but due to the lack of a catalytic core, they cannot cleave the fuel chain (Fuel), thus enabling the detection of single nucleotide polymorphisms.

[0034] This invention constructs a gene chip biosensor based on competitive DNAzyme circuits and parallel amplification for SNP mutation sites. It achieves SNP-specific recognition through competitive strand substitution and an "activation-inhibition" switch in the DNAzyme catalytic core, and promotes signal transduction through solid-liquid phase cross-amplification. This enables sensitive and specific detection of single nucleotide polymorphisms. Furthermore, the detection process requires no thermal cycling or the addition of protease additives, making it simple to operate and low in cost. It can be applied to genotyping and phenotypic selection in crop improvement and other applications, providing a novel method for SNP detection and phenotypic association studies in complex biological samples. Attached Figure Description

[0035] Figure 1 A flowchart for detecting single nucleotide polymorphisms using a gene chip biosensor;

[0036] Figure 2This is a polyacrylamide gel electrophoresis verification image during the gene chip biosensor detection process.

[0037] Figure 3 EIS stepwise characterization diagram during the gene chip biosensor detection process;

[0038] Figure 4 The image shows the results of detecting single nucleotide polymorphisms in soybean genes using a gene chip biosensor.

[0039] Figure 5 This is a graph showing the difference in electrochemical signals of gene chip biosensors under parallel and perpendicular conformation conditions.

[0040] Figure 6 Linear calibration curve between gene chip biosensors and SNP mutant nucleotide sequence concentrations;

[0041] Figure 7 This is a graph showing the results of single nucleotide polymorphism detection of dsDNA and ssDNA using a gene chip biosensor. Detailed Implementation

[0042] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0044] The following examples are used to detect soybeans Glyma.20G116200 The technical solution of the present invention will be specifically described using SNPs of genes as an example, wherein the WT wild-type soybean Glyma.20G116200 The gene nucleotide sequence is shown in SEQ ID NO.12, describing the SNP mutant soybean. Glyma.20G116200 The gene nucleotide sequence is shown in SEQ ID NO.13;

[0045] All oligonucleotide chains involved in the examples were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0046] Example 1: Application of gene chip biosensors in detecting single nucleotide polymorphisms

[0047] This embodiment uses soybeans. Glyma.20G116200 The MTzy hairpin probe and fuel chain Fuel were designed using the gene SNP mutant nucleotide sequence as a template. The nucleotide sequence of the MTzy hairpin probe is shown in SEQ ID NO.1, and the nucleotide sequence of the fuel chain Fuel is shown in Table 1.

[0048] soybeans Glyma.20G116200 Genetically modified WT wild-type soybean Glyma.20G116200 The WTzy hairpin probe was designed using the gene nucleotide sequence as a template. The nucleotide sequence of the WTzy hairpin probe is shown in SEQ ID NO.2.

[0049] S1: Competitive DNAzyme-assisted recovery reaction:

[0050] The lyophilized MTzy probe (nucleotide sequence shown in SEQ ID NO.1) and WTzy probe (nucleotide sequence shown in SEQ ID NO.2) were dissolved in ultrapure water to obtain 100 μM MTzy probe and WTzy probe stock solutions; the MTzy probe and WTzy probe stock solutions were diluted to 10 μM using 50 mM Tris-HCl buffer (containing 50 mM MgCl2, 150 mM NaCl, pH 7.4);

[0051] The diluted MTzy and WTzy probe stock solutions were reacted at 95°C for 5 minutes, then rapidly cooled to 4°C and reacted for 20 minutes to complete the annealing process. The reacted MTzy and WTzy probe stock solutions were mixed to a final concentration of 150 nM. 100 nM of the test sample was added to the mixture, and the mixture was reacted at 95°C for 5 minutes, then rapidly cooled to 4°C and reacted for 20 minutes to perform SNP recognition, thus obtaining the MTzy / SNP and WTzy / WT double-stranded complexes.

[0052] The MTzy / SNP and WTzy / WT double-stranded complexes obtained above were mixed with fuel chain Fuel to a final concentration of 10 nM. The mixture was placed at 25°C for 1 hour for enzymatic digestion to carry out DNAzyme reaction, and the DNAzyme reaction solution was then incubated at 25°C for 2 hours to promote DNAzyme-assisted cyclic amplification.

[0053] S2: Preparation of signal molecule hairpins:

[0054] Pretreatment of single-walled carbon nanotubes: Single-walled carbon nanotubes (SWCNTs) with carboxyl-rich sidewalls were placed in a mixture (98% sulfuric acid: 70% nitric acid = 3:1), and sonicated for 5 minutes to disperse them evenly. They were then incubated overnight to fully dissolve them and obtain a mixture. The mixture was centrifuged at 12,000 rpm and washed until the pH reached neutral to obtain a 0.2 mg / mL SWCNT solution. Before each use, the mixture needs to be sonicated for 2 hours to obtain the SWCNT solution.

[0055] Mix 500 μL of 16 μM hemin with 1500 μL of pretreated SWCNT solution, sonicate until uniformly dispersed, incubate at room temperature for 1 hour, centrifuge at 10000 rpm for 10 minutes, wash 3 times to remove unbound Hemin, and resuspend the precipitate in water to obtain Hemin@SWCNT stock solution.

[0056] 200 μL of Hemin@SWCNT, 200 μL of 40 mM EDC coupling agent (dissolved in 20 mM MES buffer at pH 5.5), and 6 μL of 15 μM amino-modified hairpin DNA (H2 hairpin or H4 hairpin) were mixed and sonicated. The mixture was shaken at 25°C for 1 hour. The hairpin DNA (H2 hairpin or H4 hairpin) was covalently attached to Hemin@SWCNT. After centrifugation to remove unreacted DNA, Hemin@SWCNT-H2 and Hemin@SWCNT-H4 were obtained, respectively. The nucleotide sequence of the H2 hairpin is shown in SEQ ID NO.6, and the nucleotide sequence of the H4 hairpin is shown in SEQ ID NO.8.

[0057] S3: Preparation of modified electrodes:

[0058] Hair clips H1 (nucleotide sequence shown in SEQ ID NO.5), H3 (nucleotide sequence shown in SEQ ID NO.7), and H5 (nucleotide sequence shown in SEQ ID NO.9) with 5' end modified with thiol groups were pretreated with 50 mM TCEP for 1 hour at room temperature to cleave disulfide bonds. The hair clips H1, H3, and H5 were mixed in equal proportions to a final concentration of 0.8 μM to obtain a hair clip mixture.

[0059] Take 8 μL of the above hairpin mixture and add it dropwise to the surface of the polished and gold-plated glassy carbon electrode (GCE / Au). Incubate overnight at 4°C to anchor hairpins H1, H3, and H5 onto the electrode to obtain GCE / Au / H135. Continue to add 8 μL of 1 mM MCH to the GCE / Au / H135 electrode and react at 25°C for 30 minutes to block the remaining sites on the electrode, thus obtaining the modified electrode GCE / Au / H135 / MCH.

[0060] S4: Mix the DNAzyme reaction solution obtained in S1 with Hemin@SWCNT-H2 and Hemin@SWCNT-H4 obtained in S2 at a volume ratio of 2:3:3. Add the mixture to the surface of the modified electrode GCE / Au / H135 / MCH obtained in S3. React for 30 minutes until the solid-phase reaction is completed on the electrode surface. Perform isothermal amplification reaction at 25℃ for 1 hour to obtain the working electrode GCE / Au / H135 / MCH / H24. After each modification step, wash the modified electrode 2-3 times with 24 μL of Tris buffer containing 0.1% Tweent 20 to prepare for the subsequent differential pulse voltammetry (DPV) test.

[0061] S5: Electrochemical measurements were performed at 25°C using a CHI 660E instrument (Shanghai, China) employing a three-electrode system, including a working electrode, a counter electrode, and a reference electrode. The working electrode was the GCE / Au / H135 / MCH / H24 electrode obtained in S4; the counter electrode was a platinum wire electrode (Pt); and the reference electrode was a silver chloride electrode (Ag / AgCl). Differential pulse voltammetry (DPV) measurements were performed by scanning the potential from -0.8 V to 0 V at a scan rate of 0.05 V / s, using 10 mM PBS as the electrolyte; 5 mM [Fe(CN)6] was used. 3- / 4- Electrochemical impedance spectroscopy (EIS) measurements were performed on (0.1 M KCl) in the range of -0.2 V to 0.6 V, with an amplitude potential of 5 mV and a scan frequency range of 0.1 to 10 mV. 5 Hz; The detection results are substituted into the linear regression equation of the standard curve for calculation, and the judgment is made based on the results; The method for establishing the standard curve is as follows: Target sequences containing SNP mutation sites are synthesized, and standard solutions with concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, and 1000 pM are prepared respectively. Electrochemical detection is performed on each solution, and a standard curve between concentration and current difference is established to obtain the linear regression equation; The judgment criterion is: minimum linear concentration ≤ calculation result ≤ maximum linear concentration, indicating that the sample to be tested contains SNP mutation sites.

[0062] Table 1

[0063]

[0064] Note: In the table, rU represents a nucleotide sequence modified by the RNA base U.

[0065] Effect Experiment:

[0066] (1) The flowchart of the gene chip biosensor for detecting soybean gene single nucleotide polymorphisms provided in this embodiment is as follows: Figure 1 As shown, during the detection process, SNP targets containing SNP mutation sites tend to bind to the adapter of the MTzy hairpin probe, thereby initiating a strand displacement reaction, exposing the catalytic core, activating the cleavage reaction, and continuously cleaving the fuel chain containing RNA bases (Fuel). This prevents the liquid-solid phase cross-reaction on the working electrode surface from occurring, thus preventing amplification and signal amplification. Conversely, WT wild-type sequences without SNP mutation sites tend to bind to the adapter of the WTzy hairpin probe, but due to the lack of a catalytic core, they cannot cleave the fuel chain (Fuel). Therefore, the fuel chain (Fuel) can open the hairpin structure on the working electrode surface, thereby initiating a "parallel amplification" reaction, resulting in a significant electrochemical signal. Enhancement is used to identify and amplify SNPs; the "parallel amplification" reaction refers to the following: the fuel chain Fuel is complementary to the stem sequence of the H1 hairpin, opening the H1 hairpin and releasing the hidden toe chain 1; the toe chain 1 is complementary to the stem sequence of the secondary H2 hairpin, opening the H2 hairpin and releasing the hidden toe chain 2; the toe chain 2 is complementary to the stem sequence of the secondary H3 hairpin, opening the H3 hairpin and releasing the hidden toe chain 3; the toe chain 3 is complementary to the stem sequence of the secondary H4 hairpin, opening the H4 hairpin and releasing the hidden toe chain 4; the toe chain 4 is complementary to the stem sequence of the secondary H5 hairpin, opening the H5 hairpin and releasing the hidden toe chain 5; finally, they are joined to form a longer double-stranded DNA structure.

[0067] (2) In this embodiment, the use of the gene chip biosensor in detecting soybean single nucleotide polymorphisms was verified by polyacrylamide gel electrophoresis, and the results are as follows: Figure 2 As shown, the design of the DNA strand used in the gene chip sensor provided by the present invention is feasible.

[0068] (3) Stepwise characterization of EIS:

[0069] like Figure 3 As shown in curve a, the exposed glassy carbon electrode (GCE) has a low resistance; after gold deposition, the impedance of the GCE / Au electrode decreases significantly (e.g., ...). Figure 3 (As shown in curve b); when equimolar H1, H3, and H5 hairpins are fixed to the electrode surface, the impedance of the GCE / Au / H135 electrode increases (e.g., as shown in curve b). Figure 3 (As shown in curve c), this is because the DNA backbone carries a negative charge; when the blocking agent MCH is assembled onto the electrode, the impedance of the GCE / Au / H135 / MCH electrode further increases (as shown in curve c). Figure 3As shown in curve d), this indicates that the MCH modification was successful; after the Hemin@SWCNT-H2 and Hemin@SWCNT-H4 mixture was drop-coated onto the electrode and incubated, the impedance of the GCE / Au / H135 / MCH / H24 electrode was significantly reduced (as shown in curve d). Figure 3 (As shown in curve e), this is because the modification of the electrode with SWCNT promotes electron transfer.

[0070] In summary, this invention demonstrates that the electrochemical signaling molecules Hemin@SWCNT-H2 and Hemin@SWCNT-H4 were successfully assembled onto the electrode via DNA hybridization, indicating the successful preparation of the sensing interface.

[0071] (4) In this embodiment, Tris-HCl buffer was selected to replace the test sample added in S1 as a control group; soybean samples were tested with 100 nM SNP mutant test sample and 100 nM WT wild-type test sample added, and the results were analyzed using a gene chip biosensor. Glyma.20G116200 Detection of single nucleotide polymorphisms in genes.

[0072] The results are as follows Figure 4 As shown, compared with the control group and the WT wild-type treatment group, the SNP mutant treatment group exhibited a significantly reduced electrochemical signal obtained by the gene chip biosensor JinCe. This is because, in the presence of the SNP target sequence, the SNP mutation site activates the cleavage activity of the MTzy probe, cleaving the fuel chain (Fuel) and preventing it from opening the hairpin structure, thus reducing the Hemin@SWCNT load on the electrode surface and significantly decreasing the electrochemical signal of heme. In contrast, the control group and the WT wild-type treatment group did not activate the cleavage activity of the WTzy probe, nor did they cleave the fuel chain (Fuel).

[0073] (5) This invention uses solid-liquid phase cross-linking to reconfigure the DNA strands into a parallel arrangement relative to the electrode surface, compared with the traditional method of arranging reaction products perpendicularly to the electrode surface. The results are as follows: Figure 5As shown, when using the same concentration of fuel chain to construct vertical and parallel conformations, the DPV detection results showed that the parallel conformation had a higher current compared to the vertical conformation. This is because, for the vertical conformation arrangement on the electrode surface, the nucleotide distances between the Hemin@SWCNTs introduced by H2 and H4 hairpins and the electrode surface were 21, 55, and 89 nucleotides, respectively; while for the parallel conformation arrangement, the nucleotide distances between the electrode surface and the Hemin@SWCNTs decreased to 5 and 21 nucleotides, respectively. This parallel conformation arrangement shortens the electron transfer path, thus significantly enhancing signal transduction. It is evident that the DNA double helix structure is not strictly vertical, and single-stranded DNA exhibits a certain degree of conformational flexibility. Therefore, the current difference observed in DVP detection is not directly proportional to the number of nucleotides.

[0074] (6) Drawing the standard curve

[0075] Based on optimized experimental conditions, different concentrations of [agent name] were prepared at pM: 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, and 1000. Glyma.20G116200 The SNP mutant nucleotide sequence solution was then subjected to a competitive DNAzyme-assisted recovery reaction with a DNAzyme probe. Differential pulse voltammetry (DPV) measurements were performed using a gene chip biosensor to plot a standard curve. The potential was scanned from -0.8 V to 0 V, using 10 mM PBS as the electrolyte. The electrochemical signal difference ΔI=I was recorded when different concentrations of standard solution were added. blank -I, where I blank I represents the blank current, and I represents the test current.

[0076] like Figure 6 As shown, within a wide detection range of 100 aM to 100 pM, the peak current gradually decreases with increasing concentration of the SNP mutant nucleotide sequence solution. The signal difference ΔI shows a significant linear correlation with the negative logarithm of the SNP mutant nucleotide sequence solution concentration, and its linear regression equation is: y = 46.22 × logC MT + 1101.94 (R) 2 =0.9964).

[0077] (7) Determination of actual samples

[0078] To evaluate the ability of the single nucleotide polymorphism (SNP) detection of this invention to distinguish phenotypic variations, a comparative analysis was conducted on two representative soybean genotypes, in which the MT sequence (nucleotide sequence shown in SEQ ID NO.3) corresponds to the pointed leaf phenotype and the WT sequence (nucleotide sequence shown in SEQ ID NO.4) corresponds to the round leaf phenotype.

[0079] Fresh leaf tissue was used as a sample. The sample underwent a standardized pretreatment process, including rapid freezing in liquid nitrogen, low-temperature grinding, and DNA extraction and purification. Using the genomic DNA obtained above as a template, a 500 bp DNA fragment was obtained by PCR amplification using upstream primer F (nucleotide sequence shown in SEQ ID NO.10) and downstream primer R (nucleotide sequence shown in SEQ ID NO.11). Sanger sequencing confirmed the existence of a single nucleotide variation (G→C) between the WT and MT sequences.

[0080] This invention fragments the extracted genomic DNA to obtain double-stranded DNA (dsDNA), and then digests it with an exonuclease to produce single-stranded DNA (ssDNA). To evaluate the detection capability of the gene chip biosensor provided by this invention under different DNA forms, single nucleotide polymorphism (SNP) detection was performed on the above-mentioned double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) samples.

[0081] The results are as follows Figure 7 As shown, the signal of the pointed leaf genotype (MT sequence) was significantly reduced compared to the round leaf genotype (WT sequence). p <0.001 indicates the presence of SNP mutations in the pointed leaf genotype (MT sequence), a result consistent with Sanger sequencing results; further evidence shows that single-base variations exist between the pointed leaf genotype (MT sequence) and the round leaf genotype (WT sequence) samples; and, the target detection signals of dsDNA and ssDNA derived from the pointed leaf and round leaf samples show significant differences. This stable and obvious signal difference indicates that the gene chip biosensor provided by this invention is not only suitable for ssDNA detection, but also has the ability to significantly distinguish DNA forms.

[0082] The contents 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, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A DNAzyme-based gene chip biosensor, characterized by, The gene chip biosensor comprises a DNAzyme probe SNP recognition system and a fuel chain Fuel guided solid-liquid phase mixed catalytic network; the DNAzyme probe SNP recognition system comprises an MTzy hairpin probe and a WTzy hairpin probe; The MTzy hairpin probe is obtained according to a SNP mutant nucleotide sequence, and the specific steps are as follows: the SNP mutant nucleotide sequence is divided into an upstream b' sequence and a downstream a' sequence from a SNP mutation site; the MTzy hairpin probe comprises four parts a, b, c and d; the parts a and c are stem segments of the MTzy hairpin probe, wherein the part a further comprises a 15 nt bulge loop, and each of the two sides of the bulge loop comprises 10 nt nucleotides, and the bulge loop serves as a catalytic core of a 10-23 DNAzyme probe; the part b is a loop part comprising a "TTTT" sequence; the part c is complementary to the two sides of the bulge loop of the part a, and the two sides of the bulge loop of the part a are identical to the downstream a' sequence; and the part d comprises a toehold composed of eight base branch point sequences, the part d is complementary to the SNP mutation site and the upstream b' sequence, and the first base of the part d is complementary to the SNP mutation site; The WTzy hairpin probe is obtained according to a WT wild type nucleotide sequence, and the specific steps are as follows: the WT wild type nucleotide sequence is divided into an upstream b' sequence and a downstream a' sequence from a non-mutated SNP site; the WTzy hairpin probe comprises four parts a, b, c and d; the parts a and c are stem segments of the WTzy hairpin probe, wherein the part a further comprises a 15 nt bulge loop, and each of the two sides of the bulge loop comprises 10 nt nucleotides, and the bulge loop does not have catalytic activity of a 10-23 DNAzyme; the part b is a loop part comprising a "TTTT" sequence; the part c is complementary to the two sides of the bulge loop of the part a, and the two sides of the bulge loop of the part a are identical to the downstream a' sequence; and the part d comprises a toehold composed of eight base branch point sequences, the part d is complementary to the non-mutated SNP site and the upstream b' sequence, and the first base of the part d is complementary to the non-mutated SNP site; The fuel chain Fuel guided solid-liquid phase mixed catalytic network is composed of a fuel chain Fuel and an electrochemical three-electrode system; the fuel chain Fuel is obtained according to a SNP mutant nucleotide sequence, and is composed of two RNA base modified nucleotide sequences, and the nucleotide sequences are respectively complementary to the two sides of the bulge loop of the part a of the MTzy hairpin probe, and serve as substrates of a DNAzyme enzyme cutting reaction; The electrochemical three-electrode system is a working electrode, a counter electrode and a reference electrode; the counter electrode is a platinum wire electrode, and the reference electrode is a silver-silver chloride electrode; The working electrode is composed of a modified electrode and a signal molecule hairpin. The preparation steps of the signal molecule hairpin are as follows: mixing hemin and the pretreated SWCNT solution according to a mixing volume ratio of 1:3, ultrasonic treatment until dispersed uniformly, incubation at room temperature for 1 h, centrifugation, washing, and obtaining a Hemin@SWCNT stock solution; mixing the above Hemin@SWCNT, 40 mM EDC coupling agent, and 15 μM amino-modified hairpin DNA according to a volume ratio of 200:200:6, the amino-modified hairpin DNA being H2 hairpin or H4 hairpin, ultrasonic treatment, oscillation at 25℃ for 1 h, centrifugation, and obtaining Hemin@SWCNT-H2 and Hemin@SWCNT-H4 signal molecule hairpins respectively; The preparation steps of the modified electrode are as follows: treating 5'-end thiol-modified H1, H3, and H5 hairpins with 50 mM TCEP at room temperature for 1 h, mixing the above H1, H3, and H5 hairpins in equal proportions to a final concentration of 0.8 μM to obtain a hairpin mixture; dropping the above hairpin mixture on a polished gold-plated glassy carbon electrode GCE / Au surface, incubating overnight at 4℃, and obtaining a GCE / Au / H135 electrode; continuously dropping 8 μL of 1 mM MCH on the GCE / Au / H135 electrode, reacting at 25℃ for 30 minutes, washing, and obtaining a GCE / Au / H135 / MCH modified electrode.

2. The gene chip biosensor according to claim 1, wherein, The H1, H2, H3, H4, and H5 hairpins are obtained according to a fuel chain Fuel design; The fuel chain Fuel is complementary to the stem sequence of the H1 hairpin, opens the H1 hairpin, and releases a hidden toehold strand 1; The toehold strand 1 is complementary to the stem sequence of the secondary H2 hairpin, opens the H2 hairpin, and releases a hidden toehold strand 2; The toehold strand 2 is complementary to the stem sequence of the secondary H3 hairpin, opens the H3 hairpin, and releases a hidden toehold strand 3; The toehold strand 3 is complementary to the stem sequence of the secondary H4 hairpin, opens the H4 hairpin, and releases a hidden toehold strand 4; The toehold strand 4 is complementary to the stem sequence of the secondary H5 hairpin, opens the H5 hairpin, and releases a hidden toehold strand 5; The toehold strand is a nucleotide from the loop part to the toehold part of the hairpin, comprising the loop part and the toehold.

3. Use of the gene chip biosensor of any one of claims 1 to 2 in detection of single nucleotide polymorphism.

4. The method of detecting single nucleotide polymorphisms using a gene chip biosensor according to any one of claims 1 to 2, wherein, The method comprises the following steps: S1: performing SNP identification on the sample to be tested with MTzy probes and WTzy probes respectively to obtain double-stranded complexes MTzy / SNP and WTzy / WT; S2: performing DNAzyme enzyme cutting reaction on the double-stranded complexes MTzy / SNP and WTzy / WT obtained in S1 with a fuel chain Fuel to obtain a DNAzyme reaction solution; S3: The DNAzyme reaction solution obtained in S2 is mixed with Hemin@SWCNT-H2 and Hemin@SWCNT-H4 signal molecule hairpins in a volume ratio of 2:3:3, and the mixture is added dropwise to the modified electrode GCE / Au / H135 / MCH surface to react for 30 minutes, isothermal amplification is performed, and a working electrode GCE / Au / H135 / MCH / H24 is obtained; the working electrode is subjected to electrochemical detection, the detection result is substituted into the linear regression equation of the standard curve for calculation, and the calculation result is used for judgment.

5. The method of claim 4, wherein, The SNP recognition condition in S1 is 5 minutes of reaction at 95°C and 20 minutes of reaction at 4°C; the concentrations of the MTzy probe and the WTzy probe are both 150 nM; and the enzyme digestion reaction condition in S2 is 1 hour of reaction at 25°C.

6. The method of claim 4, wherein, The isothermal amplification reaction condition in S3 is 1 hour of reaction at 5°C; and the electrolyte for electrochemical detection is 10 mM PBS buffer, the voltage is -0.8-0 V, and the scanning rate is 0.05 V / s.

7. The method of claim 4, wherein, The standard curve in S3 is established by: A target sequence containing a SNP mutation site is synthesized, standard solutions with concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, and 1000 pM are prepared respectively, electrochemical detection is performed by a gene chip biosensor, a standard curve of concentration versus current difference is established, and a linear regression equation is obtained; The judgment standard is that the minimum linear concentration ≤ the calculation result ≤ the maximum linear concentration, and it is judged that the SNP mutation site is contained in the sample to be tested.

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