A one-step method for detecting multiple mycotoxins based on Y-shaped multiple DNA tweezers

Abstract

The present invention provides a one-step method for detecting multiple mycotoxins based on Y-shaped multiple DNA tweezers. The Y-shaped multiple DNA tweezers forms a Y-shaped double DNA arm with two DNA tweezers at the end, and the aptamer sequence at the third end can bind to the target mycotoxin with strong affinity, and then release two DNA fragments. The released DNA fragments can open the DNA tweezers at the end of the Y-shaped DNA arm. The amount of aflatoxin B1 and ochratoxin A can be quantitatively detected by restoring the fluorescence intensity. The method controls the "opening" of the Y-shaped DNA tweezers through an automatic dynamic DNA hybridization reaction, which can be completed within 60 minutes and only requires one step. It has also been successfully applied to the detection of multiple mycotoxins in real food samples. Importantly, the application can be extended to the detection of other mycotoxins by simply replacing the corresponding aptamer sequences.

Description

Technical Field

[0001] The invention relates to a method for detecting mycotoxins, in particular to the field of aflatoxin B1 and ochratoxin A detection methods. Background Art

[0002] Mycotoxins are different extracellular metabolites mainly produced by certain strains of molds. Feed or food is often contaminated by various molds during growth, harvesting, storage and processing. Aflatoxin B1 (AFB1), ochratoxin A (OTA), fumonisin B1 (FB1) and deoxynivalenol (DON) are the most toxic mold metabolites widely present in the environment. Global incidence data show that the incidence of mycotoxin contamination in raw cereals is as follows: 55% for aflatoxins, 29% for ochratoxin A, 61% for fumonisins, 58% for deoxynivalenol and 46% for zearalenone. Mycotoxins remain a major public health concern. Due to their carcinogenic, immunosuppressive and neurotoxic effects in humans and animals, aflatoxin B1 and ochratoxin A have been listed as one of the most potent renal carcinogens by the International Agency for Research on Cancer (IARC). Aflatoxin B1 and ochratoxin A are regulated in a variety of foods in many countries around the world, including the European Union and the United States.

[0003] Mycotoxins can enter the human food chain directly or indirectly. Due to good chemical stability and long half-life in mammalian tissues, it can accumulate in plants and animals. Even small doses of food can lead to accumulation of mycotoxins in humans and animals. In the past decades, many studies have examined the interactions of mycotoxins in biological models, showing additive and synergistic effects. This means that the combined intake of mycotoxins may lead to a higher risk of adverse health effects than the intake of one of the mycotoxins alone. Therefore, the occurrence of mycotoxins in food and the environment should be monitored to assess the risks associated with mycotoxins and to understand the interactions between mycotoxins in vitro.

[0004] In the past few years, significant progress has been made in the detection of mycotoxins, including ultraviolet detection, fluorescence, electrochemical detection, and mass spectrometry. The commonly used chromatography method is accurate and sensitive for the determination of mycotoxins. However, chromatography requires complex pretreatment, experienced operators, and expensive instruments. Aptamers are single-stranded DNA or RNA oligonucleotides. It is a new type of chemical antibody with unique advantages such as simple chemical synthesis and modification, friendly storage conditions, low immunogenicity, and low cost. It also has considerable affinity and specificity with the target of the antibody. Compared with traditional instrumental methods and immunological methods, it has been used to detect a variety of mycotoxins, including ochratoxin A, aflatoxin B1, aflatoxin M1 (AFM1), and deoxynivalenol (DON) and zeacrylonitrile (ZEN). Therefore, this aptamer has better application prospects than antibodies in the fields of environmental monitoring, food analysis, toxicological research, drug screening, and clinical diagnosis. Summary of the invention

[0005] The present invention provides a one-step method for detecting multiple mycotoxins based on Y-shaped multiple DNA tweezers.

[0006] The method comprises the following steps:

[0007] 1) Synthesis of Y-shaped multiple DNA tweezers: Y-shaped multiple DNA tweezers are formed in two steps. In the first step, sequence Y1 containing aflatoxin B1 aptamer, sequence Y3 containing ochratoxin A aptamer, sequence Y2 that can partially pair with the DNA tweezer sequence, and DNA fragments of ochratoxin A and aflatoxin B1 are mixed to form a Y-shaped arm structure; in the second step, formation of DNA tweezers: the Y-shaped arm structure formed in the first step is mixed with DNA tweezers sequences 1, 1', 2, 2' and ochratoxin A chain sequence and aflatoxin B1 chain sequence to form a Y-shaped multiple DNA tweezers;

[0008] 2) Simultaneous detection of ochratoxin A and aflatoxin B1: adding multiple mycotoxin samples to the Y-shaped multiple DNA tweezers obtained in step 1), incubating and performing fluorescence spectrum detection, and calculating the concentrations of ochratoxin A and aflatoxin B1 using the standard curve method.

[0009] Preferably, in step 1), the sequence Y1 (5' to 3') is GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACTTTGTA-CCGG-CCATTCGG-AACCGGTA-CGCGGAGGAAGC, the sequence Y2 (5'to3') is CTCTGTGTTATG-CTGGGTGT-CCGAATGG-AAAA-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCG GACAAAGT GG , sequence Y3 (5' to 3') is CAGGATAGATAC-TACCGGTT-ACACCCAG-ATCTGAGTCCTA.

[0010] Preferably, in step 1), the DNA fragment (5' to 3') of ochratoxin A is CC-ACTTTG-TC -ATTAAT-AT, the DNA fragment of aflatoxin B1 (5' to 3') is CG-CGCGGG- CG-TGCCCA-AC.

[0011] Preferably, in the step 1), the DNA tweezers sequence 1 (5'to3') is Cy5-CGCGGG-GCTTCCTCCGCG, the DNA tweezers sequence 1' (5'to3') is GTATCTATCCTG-TGCCCA-DABCYL, the DNA tweezers sequence 2 (5'to3') is BHQ3-ACTTTG-TAGGACTCAGAT, and the DNA tweezers sequence 2' (5'to3') is CATAACACAGAG-ATTAAT-FAM.

[0012] Preferably, in step 1), the ochratoxin A chain sequence (5' to 3') is AT-ATTAAT-GA-CAAAGT-GG, and the aflatoxin B1 chain sequence (5' to 3') is GT-TGGGCA-CG-CCCGCG-CG.

[0013] Preferably, the first step in step 1) is as follows: Sequences Y1, Y2, and Y3 are mixed with the DNA fragment of ochratoxin A and the DNA fragment of aflatoxin B1 and heated to 90° C. After being kept for 5 minutes, the mixture is slowly cooled to form a Y-shaped arm structure.

[0014] Preferably, the second step in step 1) is specifically that the Y-shaped arm structure formed in the first step is mixed with the DNA tweezers sequences 1, 1', 2, 2' and the ochratoxin A chain sequence and the aflatoxin B1 chain sequence, and hybridized at room temperature for 30 minutes to form Y-shaped multiple DNA tweezers.

[0015] Preferably, in step 2), the specific incubation time is 40 minutes.

[0016] In this paper, a versatile Y-shaped multiple DNA tweezers was developed for the simple, rapid, and one-step simultaneous detection of aflatoxin B1 and ochratoxin A. Two complete DNA tweezers were used at both ends to form a Y-shaped double DNA arm. The third end tail contained two aptamers (aflatoxin B1 and ochratoxin A). These two aptamers can bind to specific mycotoxins, release the originally bound DNA fragments of ochratoxin A and aflatoxin B1, and open the DNA tweezers, respectively. The opening of the DNA tweezers can lead to the separation of the fluorophore and the quencher, resulting in a significant recovery of the fluorescence intensity. Finally, the fluorescence intensity of FAM and Cy5 increased significantly. The content of aflatoxin B1 and ochratoxin A can be quantitatively analyzed by the corresponding fluorescence intensity. This method is simple and rapid, and the "opening" of the Y-shaped DNA tweezers is controlled by the automated DNA hybridization technology. In addition, all the detection processes can be completed within 60 min with only one-step operation. Importantly, it can be extended to other mycotoxins by replacing the corresponding aptamer sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the detection process of the present invention.

[0018] Figure 2 The fluorescence signal diagrams obtained from testing different samples.

[0019] Figure 3 Figure 1 shows the effects of (A) the number of base pairs of DNA tweezers arms, (B) the number of base pairs between the aptamer and the short DNA fragment, (C) the incubation temperature, and (D) the reaction time on the fluorescence intensity of the Y-shaped multiple DNA tweezers to optimize the experimental conditions.

[0020] Figure 4 The analytical performance diagram for the simultaneous detection of aflatoxin B1 and ochratoxin A, including (A) fluorescence spectra of different concentrations of aflatoxin B1 (ae: 0, 5, 50, 100 and 200 ng / mL) and ochratoxin A (ae: 4, 50, 100, 200 and 300 ng / mL). (B) Linearity plot between aflatoxin B1 concentration and fluorescence intensity in the range of 0.5-200 ng / mL. (C) Linearity plot between ochratoxin A concentration and fluorescence response in the range of 4-300 ng / mL. (D) Anti-interference effect and selectivity of the method for some other mycotoxins (blank: buffer, mixed with other mycotoxins: aflatoxin M1 (AFM1) and zeacrylonitrile (ZEN) and deoxynivalenol (DON)) Specific embodiments

[0021] The present invention will be further described in detail below in conjunction with the embodiments.

[0022] The detection principle of this method is as follows Figure 1 As shown, sequences Y1, Y2, and Y3 are mixed with DNA fragments of ochratoxin A and DNA fragments of aflatoxin B1 to form a Y-shaped arm structure; the Y-shaped arm structure is then mixed with DNA tweezers sequences 1, 1', 2, and 2' and ochratoxin A chain sequences and aflatoxin B1 chain sequences to form Y-shaped multiple DNA tweezers. At this time, the DNA tweezers are closed and the fluorescence intensity is quenched. In the presence of target mycotoxins, aflatoxin B1 and ochratoxin A compete with the DNA fragments of ochratoxin A and aflatoxin B1 on the Y-shaped multiple DNA tweezers, resulting in the release of the DNA fragments of ochratoxin A and aflatoxin B1 on the Y-shaped multiple DNA tweezers. These DNA fragments are complementary to the chain sequences of the DNA tweezers, respectively. They can open the DNA tweezers, resulting in the separation of the fluorophore and the quencher, and ultimately leading to the recovery of the fluorescence signal. The concentrations of aflatoxin B1 and ochratoxin A can be determined by the corresponding fluorescence intensity. By changing the aptamer sequence, it can be expanded to detect other target mycotoxins.

[0023] Example 1

[0024] Synthesis of Y-shaped multiple DNA tweezers: The Y-shaped multiple DNA tweezers were formed in two steps: In the first step, the sequences Y1, Y2, and Y3 (sequence Y1 (5' to 3') are GTTGGGCACG TGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACTTTGTA-CCGG-CCATTCGG-AACCGGTA-CGCGGAGGAAGC, the sequence Y2 (5'to3') is CTCTGTGTTATG-CTGGGTGT-CCGAATGG-AAAA-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCG GACAAAGTGG , sequence Y3 (5' to 3') is CAGGATAGATAC-TACCGGTT-ACACCCAG-ATCTGAGTCCTA) and the DNA fragment of ochratoxin A and the DNA fragment of aflatoxin B1 (the DNA fragment of ochratoxin A (5' to 3') is CC-ACTTTG-TC -ATTAAT-AT, the DNA fragment of aflatoxin B1 (5' to 3') is CG-CGCGGG- CG-TGCCCA-AC) was mixed and heated to 90°C, kept for 5 minutes, and then slowly cooled to form a Y-shaped arm structure; the second step was the formation of DNA tweezers: the Y-shaped arm structure formed in the first step was combined with DNA tweezers sequences 1, 1', 2, 2' (DNA tweezers sequence 1 (5' to 3') was Cy5-CGCGGG-GCTTCCTCCGCG, DNA tweezers sequence 1' (5' to 3') was GTATCTATCCTG-TGCCCA-DABCYL, DNA tweezers sequence 2 (5' to 3') was BHQ3-ACTTTG- TAGGACTCAGAT, DNA tweezers sequence 2' (5' to 3') is CATAACACAGAG-ATTAAT-FAM) and ochratoxin A chain sequence, aflatoxin B1 chain sequence (ochratoxin A chain sequence (5' to 3') is AT-ATTAAT-GA-CAAAGT-GG, aflatoxin B1 chain sequence (5' to 3') is GT-TGGGCA-CG-CCCGCG-CG) were mixed and hybridized at room temperature for 30 min to form Y-shaped multiple DNA tweezers;

[0025] Simultaneous detection of ochratoxin A and aflatoxin B1: multiple mycotoxin samples were added to the Y-shaped multiple DNA tweezers obtained in step 1), incubated for 40 minutes, and then fluorescence spectrum detection was performed, and the concentrations of ochratoxin A and aflatoxin B1 were calculated using the standard curve method.

[0026] Example 2

[0027] Feasibility study of the present invention

[0028] We designed some samples under different conditions and studied the mechanism of action of this method. The test results are as follows: Figure 2 As shown. The blank sample without aflatoxin B1 and ochratoxin A showed background fluorescence intensity, indicating that the DNA tweezers could not be opened without the presence of aflatoxin B1 and ochratoxin A (sample 1). The sequence of the aptamer (Y1) of aflatoxin B1 in sample 2 was changed, resulting in a lower fluorescence intensity of FAM (sample 2). A similar situation occurred in sample 3, in which the sequence of the aptamer (Y2) of ochratoxin A was changed, resulting in a lower fluorescence intensity of Cy5. Sample 4 (without aflatoxin B1 fragment) and sample 5 (without ochratoxin A fragment) both showed similar background fluorescence intensity on the corresponding signal channels. This can be explained by the fact that the Y-shaped multiple DNA tweezers do not have their specific complementary DNA fragments. The samples under standard conditions showed higher fluorescence intensity on both signal channels (Example 1, sample 6), and all the results indirectly proved the working mechanism and feasibility of the method.

[0029] Example 3

[0030] Optimize relevant experimental conditions

[0031] Several experimental parameters have a significant impact on the performance of this method and therefore need to be optimized to achieve the best efficiency, such as the number of base pairs of the DNA tweezers arms, the number of base pairs between the aptamer and the short DNA fragment, the incubation temperature, and the reaction time.

[0032] The number of base pairs in the DNA tweezers arm significantly affects the stability of the DNA tweezers. The results show that when the number of base pairs in the arm is less than 6, the fluorescence signal is stable. However, when the number of base pairs is 10, the fluorescence signal decreases significantly. This can be explained by the strong stability of the DNA tweezers and the low hybridization efficiency between the short DNA fragment and the lock chain sequence ( Figure 3 A). In addition, when the arm base pair number is greater than 6, the background signal (no target) decreases significantly. Therefore, the arm base pair number of the DNA tweezers is selected to be 6.

[0033] The binding efficiency of mycotoxins to aptamers is affected by the number of base pairs between the aptamer and the short DNA fragment. Figure 3 B. When the number of base pairs is low, the fluorescence signal is high due to the high binding affinity between the aptamer and the mycotoxin. When the number of base pairs increases to 20, the fluorescence signal is greatly reduced. This is due to the high stability between the aptamer and the mycotoxin short DNA fragment, which greatly increases the difficulty of replacing the DNA fragment with the mycotoxin. However, when the number of base pairs is lower than 10, the melting temperature of the aptamer and the short DNA fragment is lower than room temperature, which is calculated by the oligomerization analyzer. Therefore, the number of base pairs between the aptamer and the short DNA fragment was selected to be 10.

[0034] The incubation temperature affects the stability and opening efficiency of the Y-shaped multiple DNA tweezers. Figure 3 As shown in C, the fluorescence signal increases with temperature and stabilizes at around 25°C, but due to the weak thermal stability of the Y-shaped multi-DNA tweezers, the background increases significantly with temperature. Therefore, the optimal incubation temperature of this method is selected at 25°C.

[0035] The incubation time is related to the degree of completion of the opening of the Y-shaped multiple DNA tweezers. Therefore, the incubation time was optimized here ( Figure 3 D). The results show that the fluorescence intensity becomes positive with the extension of incubation time. The signal increases significantly with time and remains stable at around 40 min. This result indicates that aptamer binding and DNA tweezers opening are almost complete. Therefore, the incubation time is selected as 40 min.

[0036] Example 4

[0037] Analytical performance for simultaneous detection of aflatoxin B1 and ochratoxin A

[0038] Under the conditions of Specific Example 1, the analytical performance including linear range and sensitivity was studied ( Figure 4 A). The fluorescence intensity of FAM increased significantly with the increase of aflatoxin B1 concentration, and showed a linear relationship in the range of 0.5-200 ng / mL (R2=0.995), with a detection limit (LOD) of 0.1 ng / mL ( Figure 4 B). In addition, the fluorescence intensity of Cy5 also increased significantly with the increase of ochratoxin A concentration, showing a good linear range between 4-300 ng / mL (R2=0.990), and the LOD was 2 ng / mL ( Figure 4 C). The LOD was calculated as three times the standard deviation of the procedural blank sample. The LOD of this method is comparable to those of aptamer-based methods that detect only one mycotoxin (No: 1-5). In addition, the LOD is as good as those of multiple mycotoxin detection methods (No: 6-7). In addition, our method is very simple, requires only one step, and can be completed within 60 min.

[0039] In mycotoxin analysis, joint mycotoxin separation is a key challenge for practical application. Under the same experimental procedure, the specificity of some other common mycotoxins, including aflatoxin M1 (AFM1) and zearalenone and deoxynivalenol, was explored by comparison. Figure 4 As shown in Figure D, the signals of aflatoxin M1 (AFM1) and zearalenone, deoxynivalenol and their mixtures are almost negligible. Only aflatoxin B1 or ochratoxin A and their mixtures show strong fluorescence signals in the corresponding signal channels. These results can be attributed to the weak affinity of these related mycotoxins with specific aptamers, indicating that the proposed method has good specificity for the detection of multiple mycotoxins.

[0040] Example 5

[0041] Analysis of real food samples

[0042] Its practical application ability was evaluated using food samples obtained directly from local supermarkets. All samples were purchased randomly, with at least 1 kg for solid samples and at least 1 liter for liquid samples. Some simple pretreatment is required before testing. Solid samples need to be crushed and then extracted with a methanol-water (70:30 v / v) mixed solution. Liquid samples are directly extracted with a methanol-water (70:30 v / v) mixed solution. After shaking for 30 minutes, the precipitate is removed by the following centrifugation. The supernatant is filtered with a 0.22 μm membrane and then the mycotoxins are detected according to the steps in Example 1.

[0043] The results in Table 1 show that the concentrations of aflatoxin B1 and ochratoxin A in the food samples were lower than the specified maximum residual amounts. In addition, two different concentrations of aflatoxin B1 and ochratoxin A were added to the food samples for recovery tests. The recovery of this method was between 92.0% and 107.0%, and the RSD values ​​were between 4.6% and 8.5%, which met the requirements of practical application. These results were also verified by the standard HPLC method.

[0044] Table 1 Determination of aflatoxin B1 and ochratoxin A in real food samples

[0045]

[0046] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. Sequence Listing <110> Wenzhou Medical University <120> A one-step method for detecting multiple mycotoxins based on Y-shaped multiple DNA tweezers <140> 2022102429656 <141> 2022-03-11 <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> 87 <212> DNA <213> Artificial Sequence <400> 1 gttgggcacg tgttgtctct ctgtgtctcg tgcccttcgc taggcccact ttgtaccggc 60 cattcggaac cggtacgcgg aggaagc 87 <210> 2 <211> 74 <212> DNA <213> Artificial Sequence <400> 2 ctctgtgtta tgctgggtgt ccgaatggaa aagatcgggt gtgggtggcg taaagggagc 60 atcggacaaa gtgg 74 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <400> 3 caggatagat actaccggtt acacccagat ctgagtccta 40 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <400> 4 ccactttgtc attaatat 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <400> 5 cgcgcgggcg tgcccaac 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <400> 6 cgcggggctt cctccgcg 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <400> 7 gtatctatcc tgtgccca 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <400> 8 actttgtagg actcagat 18 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <400> 9 cataacacag agattaat 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <400> 10 atattaatga caaagtgg 18 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <400> 11 gttgggcacg cccgcgcg 18

Claims

1. A one-step method for detecting multiple mycotoxins based on Y-shaped multiple DNA tweezers, comprising the following steps: 1) Synthesis of Y-shaped multiple DNA tweezers: Y-shaped multiple DNA tweezers are formed in two steps. In the first step, sequence Y1 containing aflatoxin B1 aptamer, sequence Y3 containing ochratoxin A aptamer, sequence Y2 that can partially pair with the DNA tweezer sequence, and DNA fragments of ochratoxin A and aflatoxin B1 are mixed to form a Y-shaped arm structure; the second step is the formation of DNA tweezers: the Y-shaped arm structure formed in the first step is mixed with DNA tweezers sequences 1, 1', 2, 2' and ochratoxin A chain sequence and aflatoxin B1 chain sequence to form a Y-shaped multiple DNA tweezers; 2) Simultaneous detection of ochratoxin A and aflatoxin B1: adding multiple mycotoxin samples to the Y-shaped multiple DNA tweezers obtained in step 1), incubating and performing fluorescence spectrum detection, and calculating the concentrations of ochratoxin A and aflatoxin B1 using a standard curve method; in, The DNA fragment (5' to 3') of ochratoxin A is CC-ACTTTG-TC -ATTAAT-AT, the DNA fragment of aflatoxin B1 (5' to 3') is CG-CGCGGG- CG-TGCCCA-AC; The chain sequence (5' to 3') of ochratoxin A is AT-ATTAAT-GA-CAAAGT-GG, and the chain sequence (5' to 3') of aflatoxin B1 is GT-TGGGCA-CG-CCCGCG-CG.

2. The method according to claim 1, characterized in that: In step 1), the sequence Y1 (5' to 3') is GTTGGGCA CG TGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACTTTGTA-CCGG-CCATTCGG-AACCGGTA-CGCGGAGGAAGC, the sequence Y2 (5'to3') is CTCTGTGTTATG-CTGGGTGT-CCGAATGG-AAAA-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCG GACAAAGTGG , sequence Y3 (5' to 3') is CAGGATAGATAC-TACCGGTT-ACACCCAG-ATCTGAGTCCTA.

3. The method according to claim 1, characterized in that: In the step 1), the DNA tweezers sequence 1 (5' to 3') is Cy5-CGCGGG-GCTTCCTCCGCG, the DNA tweezers sequence 1' (5' to 3') is GTATCTATCCTG-TGCCCA-DABCYL, the DNA tweezers sequence 2 (5' to 3') is BHQ3-ACTTTG-TAGGACTCAGAT, and the DNA tweezers sequence 2' (5' to 3') is CATAACACAGAG-ATTAAT-FAM.

4. The method according to claim 1, characterized in that: In the step 1), the first step is specifically: the sequences Y1, Y2, Y3 and the DNA fragments of ochratoxin A and aflatoxin B1 are mixed and heated to 90° C., maintained for 5 minutes, and then slowly cooled to form a Y-shaped arm structure.

5. The method according to claim 1, characterized in that: In the step 1), the second step is specifically to mix the Y-shaped arm structure formed in the first step with the DNA tweezers sequences 1, 1', 2, 2' and the ochratoxin A chain sequence and the aflatoxin B1 chain sequence, and hybridize at room temperature for 30 minutes to form Y-shaped multiple DNA tweezers.

6. The method according to claim 1, characterized in that: In the step 2), the specific incubation time is 40 minutes.

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