Multi-mycotoxin target detection method based on CRISPR-Cas12a

Abstract

According to the multiple mycotoxin target detection method based on CRISPR-Cas12a provided by the invention, multiple detection of OTA and AFB1 is successfully realized by utilizing a transverse flow analysis platform assisted by CRISPR / Cas12a under the condition that Cas12a and crRNA are not replaced, and the experiment cost is reduced. Compared with a commercial colloidal gold test strip, the detection sensitivity is improved by 1-2 orders of magnitude. Under the condition that a complex magnetic separation step is not needed, a Signal-on signal mode can be directly achieved, and complex experiment operation is reduced. The research shows the potential of the MC-LFA strategy for food safety detection in a resource limited environment, and the detection target range can be effectively expanded only by replacing the aptamer sequence, so that the MC-LFA strategy becomes a general technology suitable for various analysis schemes, and has potential application prospects in multiple fields such as public health, agriculture and environment monitoring.

Description

Technical Field

[0001] The present invention relates to the technical field of target detection, and in particular to a multiple mycotoxin target detection method based on CRISPR-Cas12a. Background Art

[0002] Mycotoxins, secondary metabolites produced by fungi, are easily spread in contaminated crops such as wheat, corn and rice. Most mycotoxins are carcinogenic, mutagenic and hepatotoxic. Ochratoxin A (OTA) and aflatoxin B1 (AFB1) are the two most common toxins in cereals. Due to their heat stability, they can easily enter the human body through the food chain and respiratory system, causing serious harm to human and animal health. Many organizations have established regulatory thresholds for the levels of these contaminants in food. For example, the European Commission stipulates that the maximum acceptable content range of AFB1 in different foods is 2-12μg / kg (depending on the specific food type), while the maximum content of OTA in unprocessed cereals should not exceed 5μg / kg. The European Food Safety Authority stipulates that the acceptable weekly intake of OTA is 120ng / kg. Due to the genotoxicity of AFB1, an acceptable weekly intake cannot be established for it. Despite these limits, mycotoxin contamination continues worldwide. Therefore, it is crucial to establish an accurate and effective detection method that can simultaneously identify and distinguish the two toxins to ensure food safety.

[0003] Traditional methods for detecting mycotoxins include liquid chromatography, gas chromatography-tandem mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). However, these technologies usually require expensive large-scale instruments and equipment, lengthy sample preparation steps, and professional technicians. Considering the actual application needs, colloidal gold test strips have emerged as a point-of-care testing (POCT) solution. Its advantages include easy and rapid operation, simple and intuitive results, and no need for other complex equipment. However, most commercial mycotoxin detection test strips on the market cannot meet the needs of multiple detection, and most of them use gold nanoparticles as signal markers. Gold nanoparticles are unstable in complex matrices, so the sensitivity still needs to be improved.

[0004] Recently, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems, known as the adaptive defense mechanism of prokaryotes against exogenous nucleic acids, have attracted attention not only as key gene editing tools, but also in the field of biosensing due to their diverse designs, precise target recognition, and efficient cleavage capabilities. Studies have reported that Cas12a can be activated by DNA and exhibits efficient and nonspecific single-stranded DNA cleavage activity. Summary of the invention

[0005] The present invention proposes a CRISPR-Cas12a-based multiple mycotoxin target detection method (MC-LFA), which uses the activation and cutting preference of Cas12a to achieve simultaneous detection of OTA and AFB1. This goal can be achieved by simply replacing the aptamer without changing the Cas12a or crRNA sequence, thereby reducing the experimental cost. In addition, a separate streptavidin (SA) line is introduced into the test strip to replace the conventional magnetic separation step, and the experimental operation process is simplified by the "Signal-on" mode. The introduction of functionalized quantum dots helps to detect target signals with ultra-high sensitivity and improve the signal amplification effect. Lateral flow analysis is considered to be an economical, efficient and environmentally friendly downstream detection method, so the method proposed in the present invention is expected to become an on-site solution that can be used to quantitatively and sensitively identify OTA and AFB1.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A method for detecting multiple mycotoxin targets based on CRISPR-Cas12a, comprising the following steps:

[0008] Step 1: Design DNA double-strand switches for each target;

[0009] Step 2: Add the DNA double-strand switch to the target analyte for incubation reaction;

[0010] Step 3: Add Cas12a, crRNA, and HBR to the solution after the reaction in step 2 for reaction;

[0011] Step 4: Add Anti-FAM-QD to the solution after the reaction in step 3 to react;

[0012] Step 5: Perform lateral flow analysis on the solution after the reaction in step 4 to detect the T-line fluorescence signal to determine whether it contains the target

[0013] Preferably, the target is OTA or AFB1.

[0014] Preferably, in the DNA double-strand switch designed for the target OTA in step 1, the nucleic acid sequences of the two DNA single-strands Probe1 and Probe2 are respectively:

[0015]

[0016] Preferably, in the DNA double-strand switch designed for the target AFB1 in step 1, the nucleic acid sequences of the two DNA single-strands Probe1 and Probe 2 are respectively:

[0017]

[0018]

[0019] Preferably, the steps of preparing Anti-FAM-QDs include: first mixing a carboxyl water-soluble quantum dot stock solution with ultrapure water, then adding HEPES, polyethylene glycol 3350, EDC and Anti-FAM, and mixing evenly; then subjecting the mixture to ultracentrifugation to purify Anti-FAM-QDs; and finally adding Triton X-100 to prevent aggregation of Anti-FAM-QDs.

[0020] Preferably, the HBR corresponding to the detection of OTA is Dig / FAM-Biotin; the HBR corresponding to the detection of AFB1 is TAMRA / FAM-Biotin.

[0021] Preferably, in step 5, fluorescence detection is performed using a lateral flow analysis test strip; the lateral flow analysis test strip comprises a nitrocellulose membrane, a sample pad, a water-absorbing pad and a polyvinyl chloride bottom plate; streptavidin, Anti-Dig, Anti-TAMRA and Anti-rabbit-antibody are used as SA, T1, T2 and C lines on the nitrocellulose membrane, the unconsumed complete HBR and the single-stranded sequence cut from the HBR are fixed on the SA line, T1 is used to identify and detect OTA, T2 is used to identify and detect AFB1, and the secondary antibody on the C line is always combined with Anti-FAM-QDs.

[0022] Preferably, Image J is used to calculate the fluorescence intensity ratio between the T1 or T2 line and the C line to quantitatively detect OTA or AFB1.

[0023] Beneficial effects: The present invention successfully achieved multiple detections of OTA and AFB1 using a CRISPR / Cas12a-assisted lateral flow analysis platform without replacing Cas12a and crRNA, thereby reducing experimental costs. Compared with commercial colloidal gold test strips, the detection sensitivity is improved by 1 to 2 orders of magnitude. The Signal-on signal mode can be directly achieved without the need for complex magnetic separation steps, reducing complex experimental operations. This study demonstrates the potential of the MC-LFA strategy for food safety testing in resource-limited environments. Replacing only the aptamer sequence can effectively expand the range of detection targets, making it a general technology suitable for various analysis schemes, and has potential application prospects in many fields such as public health, agriculture, and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Schematic diagram of mycotoxin detection based on MC-LFA.

[0025] Figure 2 :Verification of OTA / AFB1 and its corresponding aptamer binding.

[0026] Figure 3 :Feasibility verification of HBR trans-cleavage.

[0027] Figure 4 :Verification of downstream detection feasibility.

[0028] Figure 5 : The ratio of Probe 1 and Probe 2 is optimized.

[0029] Figure 6 : Optimization of key experimental parameters.

[0030] Figure 7 : Sensitivity analysis of MC-LFA. DETAILED DESCRIPTION

[0031] The present invention will be further explained below in conjunction with the embodiments.

[0032] Nucleic acid pretreatment: All oligonucleotide sequences were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. and dissolved in enzyme-free water. Probe 1 was heated at 95°C for 5 min and immediately cooled on ice to maintain its single-stranded conformation. Probe 2 was heated at 95°C for 5 min and then gradually cooled to room temperature to maintain its double-stranded hairpin structure. Probe 1 and Probe 2 were mixed at a concentration ratio of 3:1 in a high salt buffer (20 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 10 mM MgCl 2 , pH = 7.3) to form a double-stranded switch, and stored at 4°C for subsequent experiments. The nucleic acid sequences used in the experiment are shown in Table 1.

[0033] Table 1. Nucleotide sequences used in this experiment

[0034]

[0035] Preparation of 12% polyacrylamide gel: First, 10mL 30% acrylamide / methylenebisacrylamide solution (29:1), 8.25mL distilled water, 6.25mL 5x TBE buffer (89.2mM Tris, 89.2mM boric acid, 0.2mM EDTA), 250μL 10% ammonium persulfate solution and 25μL N,N,N',N'-tetramethylenediamine (TEMED) were mixed and placed at room temperature for 2h. 5μL of DNA sample was mixed with 1μL 6x loading buffer, added to the prepared gel wells, and run at 180V for 6min to stabilize nucleic acid migration, then the voltage was adjusted to 135V and run for another 30min. Then the gel was stained with fluorescent dye (Gel-Red) and finally imaged on a gel imager. DNA is subjected to gel electrophoresis to check the position of bands, analyze the size of nucleic acid molecules and verify the mechanism of nucleic acid reaction.

[0036] Preparation of Anti-FAM-QDs: Anti-FAM was provided by Sangon Biotech (Shanghai) Co., Ltd., and carboxyl water-soluble quantum dots (QDs) were provided by Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd., China. First, 8 μL of carboxyl water-soluble quantum dot stock solution was mixed with 1 mL of ultrapure water in a glass bottle, followed by the addition of HEPES (20 μL, 1 mol / L, pH 7.3), polyethylene glycol 3350 (20 μL, 5 wt%), EDC (4 μL, 1 mg / mL) and Anti-FAM (60 μL, 0.55 mg / mL), and mixed for 3 h in the dark. The mixture was then transferred to an ultracentrifuge tube for ultracentrifugation (3000 rpm, 2 min) to purify Anti-FAM-QDs. Finally, Triton X-100 (80 μL, 2.5%) was added to prevent aggregation of Anti-FAM-QDs.

[0037] Construction of lateral flow analysis test strip: The test strip consists of four parts: nitrocellulose (NC) membrane, sample pad, absorbent pad and polyvinyl chloride (PVC) base. First, the sample pad was soaked in pretreatment buffer (0.05% polyvinyl pyrrolidone, 0.1% Triton X-100, 0.01% bovine serum albumin, 1x PBS) for 30 min and then dried at 37 ° C for 12 h. Subsequently, the sample pad, NC membrane and absorbent pad were overlapped on the PVC base in sequence, overlapping by about 2 mm to form a complete test strip assembly. SA (1 mg / mL), Anti-Dig (0.5 mg / mL), Anti-TAMRA (0.05 mg / mL) and Anti-rabbit-antibody (1 mg / mL) were streaked on the NC membrane at a rate of 1 μL / cm using a film spray gold instrument. SA, T1, T2 and C lines were formed respectively. Finally, the test strips were precisely cut into a size of 6 cm × 2 mm using a programmable strip cutter and stored at room temperature after being sealed with a light-proof bag and desiccant for future use.

[0038] MC-LFA detection process: In tube A, taking OTA as an example, add the double-strand switch (10 μL) to different concentrations of target analytes and incubate at 37°C for 45 min to promote the binding of the target and the aptamer and release Probe 2. Take the above 3 μL mixed solution, add 2 μL 10× HOLMES buffer, 0.3 μL Cas12a, 0.3 μL crRNA, 8 μL HBR and 6.4 μL ddH 2 O, the total reaction volume was 20 μL, and then the mixed solution was reacted at 37°C for 15 min. The target of tube B was AFB1. The reaction conditions of tube A and tube B were the same. After the reaction, tube A and tube B were mixed and 54 μL ddH 2 O and 6 μL Anti-FAM-QDs to make the total volume reach 100 μL. Finally, immerse the end of the test strip with the sample pad in the mixed solution, making sure it does not submerge the sample pad, and read the fluorescence results after 5 minutes.

[0039] Spiked sample testing process: 100 mg corn flour or wheat flour was dissolved in 1 mL methanol / water (60 / 40, v / v), mixed at room temperature for 30 min, and then the extract was subjected to ultracentrifugation (10000 rpm, 12 min), the supernatant was diluted 10 times with 1x PBS, and three different concentrations of target analytes were prepared (OTA: 0.5 ng / mL, 50 ng / mL and 2 μg / mL; AFB1: 0.5 ng / mL, 50 ng / mL and 4 μg / mL). Finally, under the optimized conditions, MC-LFA was used to perform the target analyte recovery experiment.

[0040] MC-LFA detection principle: The detection principle of MC-LFA is as follows Figure 1 As shown in A. Here, the present invention designs a "double-strand switch" composed of two different DNA molecules, respectively referred to as "Probe 1" and "Probe 2". Probe 1 contains an aptamer sequence and a non-aptamer sequence. The non-aptamer sequence and part of the aptamer sequence are designed to be complementary to part of the sequence in Probe 2, thereby forming a strong double-stranded structure, which can effectively prevent crRNA from approaching the red sequence in Probe 2, and therefore cannot activate the trans-cutting activity of Cas12a. The target molecule binds to the aptamer sequence in Probe 1 through the stacking of aromatic rings, electrostatic interactions, van der Waals forces and hydrogen bonds, thereby releasing Probe 2 and activating the trans-cutting activity of Cas12a. In order to be combined with the lateral flow analysis platform, a half-blocking reporter (half-blocking-reporter, HBR) was designed, and Dig / TAMRA, Biotin and FAM were used at the end of the chain for modification. When the released Probe 2 binds to crRNA, Cas12a will trans-cut the single-stranded sequence on the HBR to generate Dig / FAM and TAMRA / FAM double-stranded products. These double-stranded products can effectively bind to Anti-FAM-QDs. Tube A is used to detect OTA, and its corresponding HBR is Dig / FAM-Biotin. Tube B is used to detect AFB1, and its corresponding HBR is TAMRA / FAM-Biotin.

[0041] according to Figure 1 As shown in B, the present invention designs four lines for signal output of MC-LFA. Among them, SA, Anti-Dig, Anti-TAMRA and Anti-rabbit-antibody are used as SA, T1, T2 and C lines respectively. Through the interaction of biotin-streptavidin, the unconsumed complete HBR and the single-stranded sequence under cleavage will be fixed on the SA line to replace the conventional magnetic separation step. Since Anti-digoxin can recognize the double-stranded product labeled with Dig / FAM, T1 is used to identify and detect OTA. At the same time, since Anti-TAMRA can recognize the double-stranded product labeled with TAMRA / FAM, T2 is used to identify and detect AFB1. The secondary antibody on the C line is always combined with Anti-FAM-QDs to ensure that the C line always emits a fluorescent signal to accurately reflect the flow validity of the test strip. With the help of a handheld ultraviolet lamp (UV, 365nm) and a smartphone (for taking images of the strips), multiple qualitative detections of OTA and AFB1 can be achieved. The fluorescence intensity ratio (FI) between T line and C line was calculated by Image J.T / FI C ), which can be further used for multiplex quantitative detection of OTA and AFB1.

[0042] The non-aptamer sequence and part of the aptamer sequence of Probe 1 prevent the contact between the Cas12a-crRNA complex and the red sequence, while the aptamer-target binding can eliminate this obstacle. The biggest advantage of this double-stranded switch is that its aptamer sequence is independent of the activation sequence (red sequence). Therefore, without changing the Cas12a, crRNA and HBR sequences, the double-stranded switch can be applied to any target by simply changing the aptamer sequence, thereby reducing the experimental cost and broadening the detection range of the target.

[0043] Feasibility verification of MC-LFA: The absorption peaks of the target analyte, Probe1 and their complex were determined by UV absorption spectroscopy to evaluate the binding ability between the target analyte and the aptamer. Figure 2 As shown in A and B, after incubating OTA / AFB1 and its corresponding aptamer at 37°C for 45 min, a decrease in the peak intensity at a wavelength of 260 nm (typical nucleic acid absorption peak) was observed, indicating that OTA / AFB1 successfully bound to the aptamer and released Probe 2.

[0044] In the absence of a target, Figure 3 Middle A shows that Cas12a does not mediate the cutting process of ssDNA and HBR (lane 2, lane 4). Figure 3 B shows that when Probe 2 or S (red sequence) is present, the ssDNA substrate is degraded (lane 3, lane 5). In addition, the present invention conducted a fluorescence cleavage experiment to verify the response of the system to ssDNA ( Figure 3 In the middle C), when the target is present, the FAM signal (520 nm) is observed to be enhanced, so Cas12a mediates the ssDNA cutting process.

[0045] According to the hypothesis of the present invention, the single-stranded portion will be cut, while the double-stranded region will remain intact. Therefore, the present invention designs a semi-blocking reporter, and the gel electrophoresis results are Figure 3 The retention of the double-stranded region was verified in D. In addition, to verify the feasibility of HBR, the present invention conducted a fluorescent cleavage experiment. The two chains were modified with HEX and FAM / BHQ1, respectively. It can be found that when there is no target, neither 520nm (FAM) nor 556nm (HEX) fluorescence signals can be detected. In the presence of the target, only the FAM signal enhancement phenomenon was observed through the trans-cleavage mediated by Cas12a ( Figure 3 E), thus verifying that only single-stranded nucleic acid fragments modified with FAM will be cut. Figure 3 The observed decrease in HEX signal in F may be due to the increased quenching effect of BHQ1 after the FAM-modified single-stranded nucleic acid is cleaved. The verification of Cas12a's preference for specific cleavage sites for HBR further indicates that the double-stranded region will remain intact and can be used for subsequent lateral flow analysis.

[0046] To achieve the goal of multiple visual readouts, the present invention designed a four-line lateral flow strip to verify the MC-LFA system. The intact HBR substrate and the cleaved ssDNA accumulated at the SA. The cleavage of HBR resulted in the visualization of T1 and T2. Figure 4 As shown in A, when the antibody streaking concentration ratio of T1 and T2 is 1:1, OTA will cause T2 to produce nonspecific fluorescence signals. In order to reduce nonspecific phenomena, the present invention optimizes the antibody streaking concentration ratio of T1 and T2, and finds that when T1:T2 is 10:1, the test strip can distinguish OTA from AFB1. When T1:T2 is 15:1, T2 has an empty band phenomenon, which may be due to the fact that the T2 antibody streaking concentration is too low and unevenly distributed on the NC membrane. Therefore, 10:1 was selected for subsequent experiments, and the effectiveness of MC-LFA in downstream detection was verified.

[0047] Optimization of experimental conditions: In order to optimize the analytical performance of MC-LFA, the present invention studied several key experimental parameters. By adjusting the concentration ratio of Probe 1 and Probe 2, it was found that the background signal generated was the lowest when the ratio was 3:1 (see Figure 5 According to A and B Figure 6 The results of AC suggest using 150nM Cas12a, 150nM crRNA and 8μL HBR to improve the cutting efficiency of Cas12a, and the optimal cutting time is 15min ( Figure 6 The optimal reaction temperature is 37°C ( Figure 6 Middle E), 6 μL of Anti-FAM-QDs showed the strongest fluorescence signal and signal-to-noise ratio (S / N) ( Figure 6 F). When the streaking concentration of SA was 1 mg / ml, the complete HBR could be captured in the absence of target ( Figure 6 The signal reading time is 5min ( Figure 6 Middle H).

[0048] Sensitivity analysis: According to Figure 7The results in Figures A and B show that there is a significant linear relationship between the concentration range of OTA and AFB1 and the fluorescence intensity. The linear detection ranges of OTA and AFB1 are 0.05ng / mL to 2.5μg / mL and 0.5ng / mL to 5μg / mL, respectively. Compared with commercial colloidal gold test strips, the visual sensitivity of MC-LFA to OTA (0.05ng / mL vs 5ng / mL) at 37°C is increased by two orders of magnitude ( Figure 7 C), visual sensitivity to AFB1 (0.25 ng / mL vs 5 ng / mL) was increased by an order of magnitude ( Figure 7 Considering that the 0.2 ng / mL spike concentration is equivalent to 2.2 ng / g sample, the visual detection limits of OTA and AFB1 are approximately 0.55 ng / g and 2.75 ng / g, respectively. Therefore, the detection sensitivity of this platform fully meets the highest detection standards set by the European Union (10 ng / g for OTA and 5 ng / g for AFB1).

[0049] Analysis of actual spiked samples: This example uses commercially available corn flour and wheat flour as matrices, and tests were carried out under different concentrations of target doping to further verify the feasibility of the detection platform. As can be seen from Table 2, when the target OTA is added to wheat flour, its recovery rate is between 92% and 108.66%. When OTA is added to corn flour, its recovery rate is 101.62% to 112%. For AFB1, the recovery rate in wheat flour is 94% to 106.65%. When AFB1 is added to corn flour, its recovery rate ranges from 97.74% to 106%. These recovery results obtained within the linear range show that the test strip is suitable for multiple rapid detection of mycotoxins in complex matrices and can be used as an on-site auxiliary means to monitor potential safety issues of crops.

[0050] Table 2. Recovery of OTA and AFB1 at different concentrations in corn flour and wheat flour (n=3)

[0051]

Claims

1. A method for detecting multiple mycotoxin targets based on CRISPR-Cas12a, characterized in that: The steps include: Step 1: Design DNA double-strand switches for each target; Step 2: Add the DNA double-strand switch to the target analyte for incubation reaction; Step 3: Add Cas12a, crRNA, and HBR to the solution after the reaction in step 2 for reaction; Step 4: Add Anti-FAM-QDs to the solution after the reaction in step 3 for reaction; Step 5: Perform lateral flow analysis on the solution after the reaction in step 4, detect the T-line fluorescence signal, and determine whether it contains the target.

2. A method for detecting multiple mycotoxin targets based on CRISPR-Cas12a according to claim 1, characterized in that: The target is OTA or AFB1.

3. A CRISPR-Cas12a-based multiple mycotoxin target detection method according to claim 2, characterized in that: In the DNA double-strand switch designed for the target OTA in step 1, the nucleic acid sequences of the two DNA single-strands Probe 1 and Probe 2 are:

4. A CRISPR-Cas12a-based multiple mycotoxin target detection method according to claim 2, characterized in that: In the DNA double-strand switch designed for the target AFB1 in step 1, the nucleic acid sequences of the two DNA single-strands Probe1 and Probe 2 are:

5. A method for detecting multiple mycotoxin targets based on CRISPR-Cas12a according to claim 1, characterized in that: The steps of preparing Anti-FAM-QDs include: firstly mixing the carboxyl water-soluble quantum dot stock solution with ultrapure water, then adding HEPES, polyethylene glycol 3350, EDC and Anti-FAM, and mixing them evenly; then subjecting the mixture to ultracentrifugation to purify Anti-FAM-QDs; and finally adding Triton X-100 to prevent the aggregation of Anti-FAM-QDs.

6. A CRISPR-Cas12a-based multiple mycotoxin target detection method according to claim 2, characterized in that: The HBR corresponding to the detection of OTA is Dig / FAM-Biotin; the HBR corresponding to the detection of AFB1 is TAMRA / FAM-Biotin.

7. A CRISPR-Cas12a-based multiple mycotoxin target detection method according to claim 6, characterized in that: In step 5, fluorescence detection is performed using a lateral flow analysis test strip; the lateral flow analysis test strip includes a nitrocellulose membrane, a sample pad, a water-absorbing pad and a polyvinyl chloride bottom plate; streptavidin, Anti-Dig, Anti-TAMRA and Anti-rabbit-antibody are used as SA, T1, T2 and C lines on the nitrocellulose membrane, unconsumed complete HBR and the single-stranded sequence cut from the HBR are fixed on the SA line, T1 is used to identify and detect OTA, T2 is used to identify and detect AFB1, and the secondary antibody on the C line is always combined with Anti-FAM-QDs.

8. A method for detecting multiple mycotoxin targets based on CRISPR-Cas12a according to claim 7, characterized in that: Image J was used to calculate the fluorescence intensity ratio between the T1 or T2 line and the C line to quantitatively detect OTA or AFB1.

9. A method for detecting multiple mycotoxin targets based on CRISPR-Cas12a according to claim 7, characterized in that: The antibody streak concentration ratio of T1 and T2 was 10:

1.

10. A method for detecting multiple mycotoxin targets based on CRISPR-Cas12a according to claim 1, characterized in that: The concentration ratio of the two DNA single strands Probe1 and Probe 2 in the DNA double-strand switch is 3:1.

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