DNA hydrogel sensor for nucleic acid detection and preparation method thereof

Abstract

The invention discloses a DNA (deoxyribonucleic acid) hydrogel sensor for nucleic acid detection and a preparation method of the DNA hydrogel sensor. The DNA hydrogel sensor comprises a CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats) reaction system and DNA hydrogel, the CRISPR-Cas12a reaction system comprises a Cas12a protein and sgRNA corresponding to the Cas12a protein, and a part of nucleotide sequence of the sgRNA and a part of nucleotide sequence of target nucleic acid to be detected are complementary in base; the DNA hydrogel is prepared from a DNA hydrogel matrix and a color developing agent embedded in the DNA hydrogel matrix; the DNA hydrogel matrix is formed by complementary pairing of a long Y structure and a short Y structure through bases. Due to the structural characteristics of the DNA hydrogel, compared with the existing hydrogel, the DNA hydrogel disclosed by the invention can realize response to activated Cas12a more quickly so as to release a color developing agent, so that the DNA hydrogel sensor comprising the DNA hydrogel can realize quick detection of nucleic acid. The preparation method of the DNA hydrogel sensor is low in cost, safe, free of pollution and suitable for large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a DNA hydrogel sensor for nucleic acid detection and a preparation method thereof. Background Art

[0002] Existing hydrogel sensors for nucleic acid detection are prepared by chemically grafting short DNA chains onto linear polymers, and mixing to prepare a hydrogel with a polymer as the backbone and double-stranded DNA as the cross-linking site. The cross-linking site of this hydrogel contains a single-stranded portion, which will be cut by the CRISPR-Cas12a protein after identifying the target nucleic acid sequence, thereby achieving a phase transition from gel to solution, indicating that the target nucleic acid sequence is contained in the detected solution (MA English, LR Soenksen, RV Gayet, H. de Puig, NM Angenent-Mari, ASMao, PQ Nguyen, JJ Collins, Programmable CRISPR-responsive smart materials, Science 365 (2019) 780-785.).

[0003] However, the existing hydrogel sensors for nucleic acid detection show very slow responsiveness after the addition of target DNA due to the physical entanglement of long polymer chains, and are not suitable for rapid detection of nucleic acids. In addition, their preparation methods are complex, costly, and difficult to control. The preparation process involves chemical reactions that discharge waste liquids and pollute the environment.

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a DNA hydrogel sensor for nucleic acid detection and a preparation method thereof, aiming to solve the problems of slow response speed, complex preparation method and high preparation cost of the existing hydrogel sensor for nucleic acid detection.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect of the present invention, a DNA hydrogel sensor for nucleic acid detection is provided, wherein the DNA hydrogel sensor comprises a CRISPR-Cas12a reaction system and a DNA hydrogel;

[0008] The CRISPR-Cas12a reaction system includes: a Cas12a protein and an sgRNA corresponding to the Cas12a protein, wherein a partial nucleotide sequence of the sgRNA is complementary to a partial nucleotide sequence of the target nucleic acid to be detected;

[0009] The DNA hydrogel comprises a DNA hydrogel matrix and a color developer embedded in the DNA hydrogel matrix;

[0010] The DNA hydrogel matrix is ​​composed of a long Y structure and a short Y structure through base complementary pairing;

[0011] The long Y structure is composed of a first DNA strand, a second DNA strand and a third DNA strand through complementary base pairing;

[0012] The short Y structure is composed of the fourth DNA strand, the fifth DNA strand and the sixth DNA strand through complementary base pairing;

[0013] The nucleotide sequence of the first DNA chain is shown as SEQ ID NO.1, the nucleotide sequence of the second DNA chain is shown as SEQ ID NO.2, the nucleotide sequence of the third DNA chain is shown as SEQ ID NO.3, the nucleotide sequence of the fourth DNA chain is shown as SEQ ID NO.4, the nucleotide sequence of the fifth DNA chain is shown as SEQ ID NO.5, and the nucleotide sequence of the sixth DNA chain is shown as SEQ ID NO.6.

[0014] According to a preferred technical solution, the developer is selected from the group consisting of gold nanoparticles, graphite particles, and ink.

[0015] According to a preferred technical solution, in the DNA hydrogel matrix, the molar ratio of the long Y structure to the short Y structure is 1:1.

[0016] The second aspect of the present invention provides a method for preparing the DNA hydrogel sensor as described in the first aspect, comprising:

[0017] (1) Preparation of CRISPR-Cas12a reaction system:

[0018] The Cas12a protein and the sgRNA corresponding to the Cas12a protein are configured as a solution;

[0019] (2) Preparation of DNA hydrogel:

[0020] Designing and synthesizing a long Y recombinant plasmid comprising a first DNA chain nucleotide sequence, a second DNA chain nucleotide sequence, a third DNA chain nucleotide sequence and an I-R3 deoxyribozyme nucleotide sequence; transforming the long Y recombinant plasmid into competent cells, adding a helper phage to assist amplification, and obtaining long Y phage particles; lysing the long Y phage particles to obtain a long Y precursor single-stranded DNA; adding the long Y precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage, and obtaining a long Y cleavage product comprising a first DNA chain, a second DNA chain, and a third DNA chain;

[0021] Designing and synthesizing a short Y recombinant plasmid comprising a fourth DNA chain nucleotide sequence, a fifth DNA chain nucleotide sequence, a sixth DNA chain nucleotide sequence and an I-R3 deoxyribozyme nucleotide sequence; transforming the short Y recombinant plasmid into competent cells, adding a helper phage to assist amplification, and obtaining short Y phage particles; lysing the short Y phage particles to obtain a short Y precursor single-stranded DNA; adding the short Y precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage, and obtaining a short Y cleavage product comprising a fourth DNA chain, a fifth DNA chain, and a sixth DNA chain;

[0022] The long Y cleavage product, the short Y cleavage product and the color developer are mixed and assembled to obtain a DNA hydrogel;

[0023] Among them, the nucleotide sequence of the long Y recombinant plasmid is shown as SEQ ID NO.7, and the nucleotide sequence of the short Y recombinant plasmid is shown as SEQ ID NO.8.

[0024] In a preferred technical solution, the competent cells are selected from one of JM109 competent cells, DH5α competent cells, Top10 competent cells, and XL1-Blue competent cells.

[0025] In a preferred technical solution, the helper phage is selected from one of M13KO7, VCSM13, and AbMole super helper phage.

[0026] In a preferred technical solution, the first buffer comprises 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride, zinc acetate and water.

[0027] In a preferred technical solution, the second buffer comprises tris(hydroxymethyl)aminomethane, acetic acid, ethylenediaminetetraacetic acid, magnesium chloride and water.

[0028] According to a preferred technical solution, in the first buffer solution, the concentration of zinc ions is 0.4-3.2 mM.

[0029] A preferred technical solution is that in the step of adding the long Y precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage, the added concentration of the long Y precursor single-stranded DNA is 100 to 1200 nM.

[0030] A preferred technical solution is that in the step of adding the short Y precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage, the added concentration of the short Y precursor single-stranded DNA is 100 to 1200 nM.

[0031] According to a preferred technical solution, the reaction temperature of the self-cutting is 25 to 50° C., and the reaction time is 1 to 36 hours.

[0032] Beneficial effects: The present invention provides a DNA hydrogel sensor for nucleic acid detection and a preparation method thereof. The DNA hydrogel matrix in the DNA hydrogel sensor is composed of a long Y structure and a short Y structure through base complementary pairing. Due to the structural characteristics of the DNA hydrogel matrix, it can respond to the activated Cas12a faster than the existing hydrogel sensor to release the developer, thereby realizing rapid detection of nucleic acids. The preparation method of the DNA hydrogel sensor is low-cost, safe and pollution-free, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a diagram of the DNA nucleotide sequence of the long Y structure.

[0034] Figure 2 It is a DNA nucleotide sequence diagram of the short Y structure.

[0035] Figure 3 This is a flow chart of the preparation of DNA hydrogel.

[0036] Figure 4 Schematic diagram of I-R3 DNAzyme self-cleavage.

[0037] Figure 5 It is the agarose gel electrophoresis diagram of the long Y cleavage product and the short Y cleavage product.

[0038] Figure 6 It is the agarose gel electrophoresis diagram after the long Y cleavage product and the short Y cleavage product are assembled respectively.

[0039] Figure 7 This is a scanning electron micrograph of a cross-section of a DNA hydrogel.

[0040] Figure 8 This is a rendering of the effect of DNA hydrogel sensor applied to nucleic acid detection. DETAILED DESCRIPTION

[0041] The present invention provides a DNA hydrogel sensor for nucleic acid detection and a preparation method thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0042] An embodiment of the present invention provides a DNA hydrogel sensor for nucleic acid detection, wherein the DNA hydrogel sensor includes a CRISPR-Cas12a reaction system and a DNA hydrogel;

[0043] The CRISPR-Cas12a reaction system includes: a Cas12a protein and an sgRNA corresponding to the Cas12a protein, wherein a partial nucleotide sequence of the sgRNA is complementary to a partial nucleotide sequence of the target nucleic acid to be detected;

[0044] The DNA hydrogel comprises a DNA hydrogel matrix and a color developer embedded in the DNA hydrogel matrix;

[0045] The DNA hydrogel matrix is ​​composed of a long Y structure and a short Y structure through base complementary pairing;

[0046] The long Y structure is composed of a first DNA strand, a second DNA strand and a third DNA strand through complementary base pairing;

[0047] The short Y structure is composed of the fourth DNA strand, the fifth DNA strand and the sixth DNA strand through complementary base pairing;

[0048] The nucleotide sequence of the first DNA chain is shown as SEQ ID NO.1, the nucleotide sequence of the second DNA chain is shown as SEQ ID NO.2, the nucleotide sequence of the third DNA chain is shown as SEQ ID NO.3, the nucleotide sequence of the fourth DNA chain is shown as SEQ ID NO.4, the nucleotide sequence of the fifth DNA chain is shown as SEQ ID NO.5, and the nucleotide sequence of the sixth DNA chain is shown as SEQ ID NO.6.

[0049] Specifically, if Figure 1 As shown, the present invention uses a first DNA chain (Cas12aL-Y3-1), a second DNA chain (Cas12aL-Y3-2) and a third DNA chain (Cas12aL-Y3-3) to form a long Y structure through base complementary pairing; Figure 2 As shown, the present invention uses the fourth DNA chain (Cas12aS-Y3-1), the fifth DNA chain (Cas12aS-Y3-2) and the sixth DNA chain (Cas12aS-Y3-3) to form a short Y structure through base complementary pairing. The branched ends of the long Y structure and the short Y structure can be complementary paired, and after pairing, there is also a single-stranded nucleotide sequence ((TTATT)3) in the branched chain connected to the long Y structure and the short Y structure.

[0050] When the DNA hydrogel sensor is used for nucleic acid detection, the sample to be tested is first added to the CRISPR-Cas12a reaction system, and then the DNA hydrogel is added. When there is target nucleic acid in the sample to be tested, the Cas12a protein in the CRISPR-Cas12a reaction system is activated, and the single-stranded nucleotide sequence ((TTATT)3) in the DNA hydrogel is cut by the trans-cutting function activated by the Cas12a protein, so that the long Y structure and the short Y structure are dissociated, and then the developer in the DNA hydrogel is released. Therefore, it can be quickly judged whether there is the presence of target nucleic acid by placing it in water.

[0051] In some embodiments, the developer is selected from one of gold nanoparticles, graphite particles, and ink.

[0052] The embodiment of the present invention also provides a method for preparing the DNA hydrogel sensor as described above, comprising:

[0053] (1) Preparation of CRISPR-Cas12a reaction system:

[0054] The Cas12a protein and the sgRNA corresponding to the Cas12a protein are configured as a solution;

[0055] (2) Preparation of DNA hydrogel:

[0056] Design and synthesize a long Y recombinant plasmid comprising a first DNA chain nucleotide sequence, a second DNA chain nucleotide sequence, a third DNA chain nucleotide sequence and an I-R3 deoxyribozyme nucleotide sequence;

[0057] The long Y recombinant plasmid is transformed into competent cells, and a helper phage is added to assist amplification to obtain long Y phage particles;

[0058] Lysing the long Y phage particles to obtain long Y precursor single-stranded DNA;

[0059] The long Y precursor single-stranded DNA is added to a first buffer containing zinc ions for self-cleavage to obtain a long Y cleavage product containing a first DNA chain, a second DNA chain, and a third DNA chain;

[0060] Design and synthesize a short Y recombinant plasmid containing a fourth DNA chain nucleotide sequence, a fifth DNA chain nucleotide sequence, a sixth DNA chain nucleotide sequence and an I-R3 deoxyribozyme nucleotide sequence;

[0061] The short Y recombinant plasmid is transformed into competent cells, and a helper phage is added to assist amplification to obtain short Y phage particles;

[0062] Lysing the short Y phage particles to obtain short Y precursor single-stranded DNA;

[0063] The short Y precursor single-stranded DNA is added to a first buffer containing zinc ions for self-cleavage to obtain a short Y cleavage product containing a fourth DNA chain, a fifth DNA chain, and a sixth DNA chain;

[0064] The long Y cleavage product, the short Y cleavage product and the color developer are mixed and assembled to obtain a DNA hydrogel;

[0065] Among them, the nucleotide sequence of the long Y recombinant plasmid is shown in SEQ ID NO.7, and the nucleotide sequence of the short Y recombinant plasmid is shown in SEQ ID NO.8.

[0066] Specifically, if Figure 3 As shown, in this embodiment, the three DNA chains for assembling the long Y structure are respectively inserted between two I-R3 deoxyribozymes and connected, and then cloned into a plasmid vector to obtain a long Y recombinant plasmid; the long Y recombinant plasmid is amplified by a helper phage in a competent cell to obtain a long Y phage particle containing a long Y precursor single-stranded DNA, and then the long Y phage particle is lysed to obtain a long Y precursor single-stranded DNA; the long Y precursor single-stranded DNA contains the I-R3 deoxyribozyme, which is a zinc ion-dependent self-cleaving deoxyribozyme. When the auxiliary factor zinc ion is present, it can specifically recognize the "GTTGAAAG" sequence and self-cleave ( Figure 4 ), therefore, in a first buffer containing zinc ions, the long Y precursor single-stranded DNA is cut to generate a long Y cleavage product containing a first DNA chain (Cas12aL-Y3-1), a second DNA chain (Cas12aL-Y3-2) and a third DNA chain (Cas12aL-Y3-3); the preparation method of the short Y cleavage product containing a fourth DNA chain (Cas12aS-Y3-1), a fifth DNA chain (Cas12aS-Y3-2) and a sixth DNA chain (Cas12aS-Y3-3) is similar to that of the long Y cleavage product, except that the nucleotide sequences of the DNA chains constituting the Y structure are different.

[0067] In some embodiments, the competent cells are selected from one of JM109 competent cells, DH5α competent cells, Top10 competent cells, and XL1-Blue competent cells.

[0068] In some embodiments, the helper phage is selected from one of M13KO7, VCSM13, and AbMole super helper phage.

[0069] In some embodiments, the first buffer comprises 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride, zinc acetate, and water.

[0070] In some embodiments, the second buffer comprises tris(hydroxymethylaminomethane), acetic acid, ethylenediaminetetraacetic acid, magnesium chloride and water.

[0071] In some embodiments, the concentration of zinc ions in the first buffer is 0.4-3.2 mM.

[0072] In some embodiments, in the step of adding the long Y precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage, the added concentration of the long Y precursor single-stranded DNA is 100 to 1200 nM.

[0073] In some embodiments, in the step of adding the short Y precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage, the added concentration of the short Y precursor single-stranded DNA is 100 to 1200 nM.

[0074] In some embodiments, the specific conditions of the assembly include: incubating at 95° C. for 5 min, and then cooling to 25° C. within 2 hours.

[0075] In some embodiments, the self-cleavage reaction temperature is 25 to 50° C., and the reaction time is 1 to 36 hours.

[0076] The present invention will be further described below by means of specific examples.

[0077] Example 1

[0078] This embodiment provides a DNA hydrogel sensor for nucleic acid detection, and the preparation method thereof is as follows:

[0079] The first DNA chain, the second DNA chain, and the third DNA chain were respectively inserted between the two I-R3 deoxyribozymes and connected, and then cloned into a customized plasmid vector with an ampicillin resistance gene to obtain a long Y recombinant plasmid; the fourth DNA chain, the fifth DNA chain, and the sixth DNA chain were respectively inserted between the two I-R3 deoxyribozymes and connected, and then cloned into a customized plasmid vector with an ampicillin resistance gene to obtain a short Y recombinant plasmid; the long Y recombinant plasmid and the short Y recombinant plasmid were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and sequenced and verified.

[0080] Among them, the nucleotide sequence of the first DNA chain is shown as SEQ ID NO.1, the nucleotide sequence of the second DNA chain is shown as SEQ ID NO.2, the nucleotide sequence of the third DNA chain is shown as SEQ ID NO.3, the nucleotide sequence of the fourth DNA chain is shown as SEQ ID NO.4, the nucleotide sequence of the fifth DNA chain is shown as SEQ ID NO.5, the nucleotide sequence of the sixth DNA chain is shown as SEQ ID NO.6, the nucleotide sequence of the long Y recombinant plasmid is shown as SEQ ID NO.7, and the nucleotide sequence of the short Y recombinant plasmid is shown as SEQ ID NO.8.

[0081] The constructed long Y recombinant plasmid was transformed into JM109 competent cells by heat shock. In a 250 mL shake flask, a fresh clone was selected and added to 50 mL of 2×YT medium containing 5 mM MgCl2 and 50 μg / mL ampicillin. The cells were cultured at 37°C and 200 rpm for 2 hours until slightly turbid (A600 < 0.05, < 10 Klett). 50 μL M13KO7 helper phage (1×10 11 pfu / mL), the final concentration was 1×10 8 pfu / mL, then cultured at 37°C, 200rpm for 90 minutes. Kanamycin was added to the culture medium to a final concentration of 70 μg / mL, and then the bacteria were cultured overnight at 37°C, 250rpm. The bacteria were precipitated by centrifugation at 12000g for 10 minutes, and then 10mL of 2.5M NaCl / 20% PEG-8000 was added to 50mL of the supernatant to precipitate the long Y phage particles in the supernatant, and centrifuged at 12000g for 10 minutes. The long Y phage particles were resuspended in 5mL 1×TE buffer (10mM Tris, 1mM EDTA, pH=8), and then centrifuged at 12000g for 10 minutes again to precipitate any remaining cells. The long Y precursor single-stranded DNA was isolated from the long Y phage particles using alkaline lysis.

[0082] The constructed short Y recombinant plasmid was transformed into JM109 competent cells by heat shock. In a 250 mL shake flask, a fresh clone was selected and added to 50 mL of 2×YT medium containing 5 mM MgCl2 and 50 μg / mL ampicillin. The cells were cultured at 37°C and 200 rpm for 2 hours until slightly turbid (A600 < 0.05, < 10 Klett). 50 μL M13KO7 helper phage (1×10 11 pfu / mL), the final concentration was 1×10 8pfu / mL, then cultured at 37°C, 200rpm for 90 minutes. Kanamycin was added to the culture medium to a final concentration of 70 μg / mL, and then the bacteria were cultured overnight at 37°C, 250rpm. The bacteria were precipitated by centrifugation at 12000g for 10 minutes, and then 10mL of 2.5M NaCl / 20% PEG-8000 was added to 50mL of the supernatant to precipitate the short Y phage particles in the supernatant, and centrifuged at 12000g for 10 minutes. The short Y phage particles were resuspended in 5mL 1×TE buffer (10mM Tris, 1mM EDTA, pH=8), and then centrifuged at 12000g for 10 minutes again to precipitate any remaining cells. The short Y precursor single-stranded DNA was isolated from the short Y phage particles using alkaline lysis.

[0083] 2× buffer was prepared by dissolving 4 mM zinc acetate in a buffer containing 100 mM HEPES and 200 mM NaCl, with a final pH of 7. Long Y precursor single-stranded DNA and short Y precursor single-stranded DNA were mixed with 2× buffer respectively and incubated at 37°C for 36 hours to obtain long Y self-cleavage products and short Y self-cleavage products. Among them, the long Y self-cleavage product contains the first DNA chain, the second DNA chain, and the third DNA chain, and the short Y self-cleavage product contains the fourth DNA chain, the fifth DNA chain, and the sixth DNA chain. The long Y self-cleavage product and the short Y self-cleavage product were precipitated with 75% cold ethanol respectively and quantified on NanoDrop One (Thermo Scientific, USA).

[0084] The long Y self-cleavage product and the short Y self-cleavage product were dissolved in 1×TAE / Mg 2+ Buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA and 12.5 mM Mg 2+ ) and run on a 12% denaturing polyacrylamide gel containing 8 M urea at 250 V for 40 minutes. The gel was removed and stained with GelRed and imaged on a Fujifilm LAS-3000Imager (Fuji Photo Film, Japan). The results are shown in Figure 5 shown.

[0085] Figure 5 In the figure, lane 1 is the self-cleavage product of the short Y; lane 2 is the self-cleavage product of the long Y.

[0086] The chemically synthesized DNA chain, the long Y self-cleavage product produced by biotechnology, and the short Y self-cleavage product produced by biotechnology were dissolved in 1×TAE / Mg 2+ Buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, and 12.5 mM Mg 2+) and incubated at 95°C for 5 minutes, the solution was slowly cooled to room temperature over 2 hours. 2+ The gel was removed and stained with GelRed and imaged on a Fujifilm LAS-3000Imager (Fuji Photo Film, Japan). Figure 6 shown.

[0087] Figure 6 In the figure, lane 1 is the chemically synthesized first DNA chain; lane 2 is the assembly of the chemically synthesized first and second DNA chains; lane 3 is the long Y structure assembled from the chemically synthesized first, second and third DNA chains; lane 4 is the mixture assembled from the long Y self-cleavage products; lane 5 is the chemically synthesized fourth DNA chain; lane 6 is the assembly of the chemically synthesized fourth and fifth DNA chains; lane 7 is the short Y structure assembled from the chemically synthesized fourth, fifth and sixth DNA chains; lane 8 is the mixture assembled from the short Y self-cleavage products.

[0088] The short Y self-cleavage product and the long Y self-cleavage product were equimolar in 1xTAE / Mg 2+ The mixture was mixed in a buffer solution so that the final long Y structure and short Y structure were both 1 mM; polyethylene glycol-modified gold nanoparticles (PEG-AuNP) were added to the mixed solution so that the final concentration of the gold nanoparticles was 20 mg / mL. DNA hydrogel was prepared by annealing from 95°C to 25°C within 2 hours. The cross-sectional scanning electron microscopy image of the DNA hydrogel is shown in Figure 7 shown.

[0089] A solution containing Cas12a protein, sgRNA and target dsDNA was added to the DNA hydrogel, and the final mixture contained 11.6nM Cas12a, 17.4nM sgRNA and 38.4nM target dsDNA. The mixture was incubated at 37°C. A negative control group containing non-target dsDNA and a blank control group containing PBS were also performed. The release of purple-red gold nanoparticles from the DNA hydrogel into the solution was observed and photographed. The results are shown in Figure 2. Figure 8 shown.

[0090] Among them, the nucleotide sequence of the sgRNA is shown in SEQ ID NO.9, the nucleotide sequence of the target dsDNA is shown in SEQ ID NO.10, and the nucleotide sequence of the non-target dsDNA is shown in SEQ ID NO.11, as shown in the following table.

[0091]

[0092]

[0093] In summary, the present invention provides a DNA hydrogel sensor for nucleic acid detection and a preparation method thereof, wherein the DNA hydrogel sensor comprises a CRISPR-Cas12a reaction system and a DNA hydrogel; the CRISPR-Cas12a reaction system comprises: a Cas12a protein and an sgRNA corresponding to the Cas12a protein, wherein a partial nucleotide sequence of the sgRNA is complementary to a partial nucleotide sequence of a target nucleic acid to be detected; the DNA hydrogel comprises a DNA hydrogel matrix and a developer embedded in the DNA hydrogel matrix; the DNA hydrogel matrix is ​​composed of a long Y structure and a short Y structure through base complementary pairing; the long Y structure is composed of a first DNA chain, a second DNA chain and a third DNA chain through base complementary pairing; the short Y structure is composed of a fourth DNA chain, a fifth DNA chain and a sixth DNA chain through base complementary pairing; the nucleotide sequence of the first DNA chain is shown in SEQ ID NO.1, the nucleotide sequence of the second DNA chain is shown in SEQ ID NO.2, the nucleotide sequence of the third DNA chain is shown in SEQ ID NO.3, and the nucleotide sequence of the fourth DNA chain is shown in SEQ ID The nucleotide sequence of the fifth DNA chain is shown in SEQ ID NO.4, the nucleotide sequence of the sixth DNA chain is shown in SEQ ID NO.6.

[0094] The DNA hydrogel of the present invention can respond to activated Cas12a faster than existing hydrogels due to its structural characteristics, thereby releasing the developer, so the DNA hydrogel sensor including the DNA hydrogel can achieve rapid detection of nucleic acids. And the preparation method of the DNA hydrogel sensor is low in cost, safe and pollution-free, and suitable for large-scale production.

[0095] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A DNA hydrogel sensor for nucleic acid detection, characterized in that: The DNA hydrogel sensor includes a CRISPR-Cas12a reaction system and a DNA hydrogel; The CRISPR-Cas12a reaction system includes: a Cas12a protein and an sgRNA corresponding to the Cas12a protein, wherein a partial nucleotide sequence of the sgRNA is complementary to a partial nucleotide sequence of the target nucleic acid to be detected; The DNA hydrogel comprises a DNA hydrogel matrix and a color developer embedded in the DNA hydrogel matrix; The DNA hydrogel matrix is ​​composed of a long Y structure and a short Y structure through base complementary pairing; The long Y structure is composed of a first DNA strand, a second DNA strand and a third DNA strand through complementary base pairing; The short Y structure is composed of the fourth DNA strand, the fifth DNA strand and the sixth DNA strand through complementary base pairing; The nucleotide sequence of the first DNA chain is shown as SEQ ID NO.1, the nucleotide sequence of the second DNA chain is shown as SEQ ID NO.2, the nucleotide sequence of the third DNA chain is shown as SEQ ID NO.3, the nucleotide sequence of the fourth DNA chain is shown as SEQ ID NO.4, the nucleotide sequence of the fifth DNA chain is shown as SEQ ID NO.5, and the nucleotide sequence of the sixth DNA chain is shown as SEQ ID NO.

6.

2. The DNA hydrogel sensor according to claim 1, characterized in that The developer is selected from one of gold nanoparticles, graphite particles and ink.

3. The DNA hydrogel sensor according to claim 1, characterized in that In the DNA hydrogel matrix, the molar ratio of the long Y structure to the short Y structure is 1:

1.

4. A method for preparing a DNA hydrogel sensor according to any one of claims 1 to 3, characterized in that: include: (1) Preparation of CRISPR-Cas12a reaction system: The Cas12a protein and the sgRNA corresponding to the Cas12a protein are configured as a solution; (2) Preparation of DNA hydrogel: Designing and synthesizing a long Y recombinant plasmid comprising a first DNA chain nucleotide sequence, a second DNA chain nucleotide sequence, a third DNA chain nucleotide sequence and an I-R3 deoxyribozyme nucleotide sequence; transforming the long Y recombinant plasmid into competent cells, adding a helper phage to assist amplification, and obtaining long Y phage particles; lysing the long Y phage particles to obtain a long Y precursor single-stranded DNA; adding the long Y precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage, and obtaining a long Y cleavage product comprising a first DNA chain, a second DNA chain, and a third DNA chain; Designing and synthesizing a short Y recombinant plasmid comprising a fourth DNA chain nucleotide sequence, a fifth DNA chain nucleotide sequence, a sixth DNA chain nucleotide sequence and an I-R3 deoxyribozyme nucleotide sequence; transforming the short Y recombinant plasmid into competent cells, adding a helper phage to assist amplification, and obtaining short Y phage particles; lysing the short Y phage particles to obtain a short Y precursor single-stranded DNA; adding the short Y precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage, and obtaining a short Y cleavage product comprising a fourth DNA chain, a fifth DNA chain, and a sixth DNA chain; Adding the long Y cleavage product, the short Y cleavage product and a color developer into a second buffer for assembly to obtain a DNA hydrogel; Among them, the nucleotide sequence of the long Y recombinant plasmid is shown as SEQ ID NO.7, and the nucleotide sequence of the short Y recombinant plasmid is shown as SEQ ID NO.

8.

5. The preparation method according to claim 4, characterized in that: The competent cells are selected from one of JM109 competent cells, DH5α competent cells, Top10 competent cells, and XL1-Blue competent cells.

6. The preparation method according to claim 4, characterized in that: The helper phage is selected from one of M13KO7, VCSM13, and AbMole super helper phage.

7. The preparation method according to claim 4, characterized in that: The first buffer comprises 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride, zinc acetate and water; And / or, the second buffer comprises tris(hydroxymethyl)aminomethane, acetic acid, ethylenediaminetetraacetic acid, magnesium chloride and water.

8. The preparation method according to claim 3, characterized in that: In the first buffer solution, the concentration of zinc ions is 0.4-3.2 mM.

9. The preparation method according to claim 4, characterized in that: In the step of adding the long Y precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage, the concentration of the long Y precursor single-stranded DNA added is 100 to 1200 nM; And / or, in the step of adding the short Y precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage, the added concentration of the short Y precursor single-stranded DNA is 100-1200 nM.

10. The preparation method according to claim 4, characterized in that: The reaction temperature of the self-cutting is 25-50° C., and the reaction time is 1-36 hours.

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