CRISPR-Cas12a sensor and method for detecting MC-LR in water body by combining CRISPR-Cas12a sensor with pregnancy test paper

Abstract

The invention belongs to the technical field of detection of microcystic toxins in a water body, and particularly relates to a CRISPR-Cas12a sensor and a method for detecting MC-LR in the water body by combining the CRISPR-Cas12a sensor with pregnancy test paper. According to the present invention, the MC-LR detection method is developed based on the CRISPR-Cas12a sensor, and the MC-LR detection method is developed based on the trans-cleavage capacity of the CRISPR-Cas12a sensor, the binding capacity of the cationic surfactant and the long chain containing the partially hybridized double chains, the HCG release triggering capacity of the cationic surfactant, and the binding capacity of the HCG and the pregnancy reagent; according to the present invention, the MC-LR detection can be rapidly and accurately achieved, the special test paper is not required, the commercial pregnancy test paper is used, and the cost is low.

Description

Technical Field

[0001] The present invention belongs to the technical field of microcystin detection in water bodies, and specifically relates to a CRISPR-Cas12a sensor and a method for detecting MC-LR in water bodies in combination with a pregnancy test paper. Background Art

[0002] Microcystin LR (MC-LR) is one of the most toxic freshwater cyanobacterial toxins known, with strong liver toxicity. It can strongly inhibit the activity of protein phosphatases, causing damage to liver cells, which may lead to pathological changes such as liver enlargement, hemorrhage and necrosis; in addition, MC-LR is also a strong liver tumor promoter, and long-term drinking may increase the risk of liver cancer. In addition to liver toxicity, MC-LR also has kidney toxicity, reproductive toxicity, neurotoxicity and genotoxicity, causing damage to multiple organs and systems. MC-LR is widely distributed in eutrophic freshwater lakes, reservoirs, rivers and other water bodies, posing a great threat to human health and the environment. Therefore, the detection of MC-LR is particularly important.

[0003] The existing technology can detect microcystin LR ultra-sensitively by assembling aptamer sensors in real time with plasma graphene oxide (p-GO); it mainly consists of two parts, one is the collection of MC-LR aptamers assembled with biotin / avidin, which is used to identify the target; the other is the complex of hollow nanoplate AgClAu and p-GO connected with the complementary chain of the aptamer, which can enhance the Raman signal. In addition, Raman signal molecules are added to the detection solution to generate SERS signals; this scheme adjusts the degree of competition between MC-LR and the complementary chain of the aptamer for the aptamer, and regulates the assembly mode and results of the two parts in real time, thereby generating different SERS signals; its disadvantages are that the material preparation conditions are harsh, the process is complicated, the cost is high, it cannot be separated from large equipment, and it is not suitable for on-site instant analysis.

[0004] Existing technology can also perform analysis based on indirect competitive immunity, using the total internal reflection properties of divergent wave optical fiber to achieve ultra-trace toxin analysis; MC-LR and monoclonal MC-LR antibodies are incubated together to occupy recognition sites; when the reaction solution is added to the detection pool, the antigen fixed on the probe combines with the unoccupied free antibody to generate an optical signal; although this method is optimized through optical technology to greatly improve the detection sensitivity; however, the fixation of fiber probes, monoclonal antibodies and antigens all require relatively high costs, and immunoassays have problems such as nonspecific reactions and over-recognition.

[0005] In addition, the existing technology can also be used to quantitatively detect MC-LR in real water samples based on sensitive sensors of CRISPR-Cas12a; specifically, the MC-LR aptamer is attached to magnetic beads (MBs) and hybridized with the blocking DNA chain; after identifying the MC-LR molecules in the water sample, the blocking DNA is released and binds to Cas12a / crRNA to activate the enzyme activity; the activated Cas12a uses the fluorescently labeled ssDNA substrate as a reporter to obtain a fluorescent signal. In addition, by replacing the reporter molecule, the MC-LR content is directly observed with a special test strip. Although this method overcomes the defects of immunoassay and improves the detection accuracy. However, a small fluorometer is still used in the detection process, and the special test strips used have poor quantitative effect, high cost, and poor versatility.

[0006] Therefore, it is necessary to develop a MC-LR detection method that is highly accurate, suitable for on-site detection, and low-cost. Summary of the invention

[0007] The present invention develops an MC-LR detection method based on the CRISPR-Cas12a sensor. The detection method of the present invention is developed based on the trans-cleavage ability of the CRISPR-Cas12a sensor, the binding ability of a cationic surfactant and a long chain containing a partially hybridized double strand, the ability of CTAB to trigger HCG release, and the binding ability of HCG and a pregnancy reagent. The detection of MC-LR can be achieved quickly and accurately, and no special test strip is required. Commercial pregnancy test strips can be used, and the cost is low.

[0008] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0009] On the one hand, the present invention provides a CRISPR-Cas12a sensor, comprising Cas12a, crRNA, a Cas12a activation chain and a MC-LR aptamer; wherein the sequence of crRNA is shown in SEQ ID NO: 1; and / or the Cas12a activation chain is shown in SEQ ID NO: 2; and / or the sequence of the MC-LR aptamer is shown in SEQ ID NO: 3.

[0010] On the other hand, the present invention provides a detection reagent or detection kit for MC-LR in water, comprising the CRISPR-Cas12a sensor of the present invention, a long chain containing a partially hybridized double strand, a cationic surfactant or a solution thereof, a loaded HCG reagent and an HCG detection reagent, which are separately packaged; wherein the CRISPR-Cas12a sensor comprises Cas12a, crRNA, a Cas12a activation chain and a MC-LR aptamer.

[0011] Preferably, the above detection reagent or detection kit meets one or more of the following conditions:

[0012] (a) The sequence of crRNA is shown in SEQ ID NO: 1; and / or the Cas12a activation chain is shown in SEQ ID NO: 2; and / or the sequence of MC-LR aptamer is shown in SEQ ID NO: 3;

[0013] (b) In the long chain containing partially hybridized double strands, the sequence of the long single strand is shown in SEQ ID NO:4, and the sequence of the short single strand is shown in SEQ ID NO:5;

[0014] (c) The magnetic beads are fixedly connected to the long chain containing partially hybridized double strands;

[0015] (e) The detection reagent or detection kit also includes a water sample pretreatment reagent;

[0016] (f) the HCG-loaded reagent is HCG-loaded calcium carbonate vaterite;

[0017] (g) The cationic surfactant is CTAB.

[0018] More preferably, in the above-mentioned long chain containing partially hybridized double strands, the molar ratio of the long chain to the short chain is 1:4.

[0019] More preferably, the preparation method of the above-mentioned HCG-loaded calcium carbonate vaterite comprises: 2 The solution was mixed with HCG solution and ultrasonicated; then Na 2 CO 3 The solution is reacted to obtain a reactant; the reactant is washed, dissolved in ethanol, and dried to obtain HCG-loaded calcium carbonate vaterite; wherein the HCG solution is prepared from Tris-HCl.

[0020] The present invention further provides a method for detecting MC-LR in water, which comprises using the detection reagent or detection kit of the present invention for detection.

[0021] Preferably, the above method comprises: (1) mixing the CRISPR-Cas12a sensor with a water sample to be detected to obtain a mixed solution 1; mixing a long chain containing a partially hybridized double chain and a cationic surfactant or a solution thereof to obtain a mixed solution 2; (2) mixing the mixed solution 1 and the mixed solution 2 to obtain a reactant; (3) separating the reactant to obtain a supernatant 1; mixing the supernatant 1 with a loaded HCG reagent and centrifuging to obtain a supernatant 2; (4) testing the HCG content in the supernatant 2 by using an HCG detection reagent, thereby qualitatively or quantitatively detecting MC-LR in the water body.

[0022] More preferably, the HCG detection reagent is a pregnancy test paper. In step (4), the supernatant 2 is detected using the pregnancy test paper, and a qualitative or quantitative analysis is performed according to the situation of the detection line.

[0023] More preferably, quantitative analysis is performed based on the situation of the detection line, including: (i) taking photos and analyzing the test strip detection line after it appears to obtain the grayscale value of the test strip detection line area; (ii) obtaining the MC-LR concentration in the water sample to be tested based on the working curve of the grayscale value of the test strip detection line area and the standard MC-LR concentration.

[0024] Preferably, in the above method, MC-LR in the water sample to be detected is enriched and then mixed with the CRISPR-Cas12a sensor.

[0025] The beneficial effects of the present invention include: the method for detecting MC-LR in water based on the CRISPR-Cas12a sensor and the combined pregnancy test paper provided by the present invention avoids the use of large-scale instruments and equipment, does not require the participation of professionals, and is more suitable for on-site detection; it solves the problem that the existing test strip detection method is insufficiently sensitive and unsuitable for quantitative analysis; in addition, the present invention can be detected through existing commercial pregnancy test strips, shortening the research and development and market conversion cycle and reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the principle of the method for detecting MC-LR in water based on CRISPR-Cas12a sensor and its combined pregnancy test paper of the present invention;

[0027] Figure 2 The particle size and Zeta potential of MB, MB@lsDNA and MB@dsDNA;

[0028] Figure 3 The UV absorption spectra of MB, MB@lsDNA and MB@dsDNA;

[0029] Figure 4 The SEM and EDS characterization of HCG-loaded calcium carbonate vaterite;

[0030] Figure 5 Zeta potential of vaterite loaded with and without HCG;

[0031] Figure 6 The selective response of HCG-loaded particles to cationic surfactants;

[0032] Figure 7 The circular dichroism characterization of the interaction between CTAB and HCG;

[0033] Figure 8The release curve of HCG release triggered by CTAB;

[0034] Fig. 9 is the Ca content of HCG-loaded calcium carbonate after adding CTAB. 2+ Release conditions;

[0035] Fig.10 It is the standard working curve of the gray value of the test line area of ​​the test strip and the standard MC-LR concentration;

[0036] Fig.11 The fixation of some hybridized long chains with different DNA long-short chain ratios on magnetic beads (supernatant UV);

[0037] Fig.12 The stability of different DNA length ratios after multiple washings after hybridization of long chains in the fixed part of magnetic beads (supernatant UV);

[0038] Fig.13 The detection results after different concentrations of CTAB combined with MB@dsDNA;

[0039] Fig.14 This is the detection result after high concentration CTAB is combined with double dosage MB@dsDNA;

[0040] Fig.15 To optimize the reaction conditions of the CRISPR-Cas12a system; where a is the volume of the optimized CRISPR / Cas12a system; b is the optimization of the Cas12a concentration (Cas12a:crRNA=1:1, MC-LR:10nM); c is the optimization of the Cas12a:crRNA ratio; d is the optimization of the cutting time after adding MC-LR;

[0041] Fig.16 The selectivity test of the detection method constructed by the present invention on MC-LR at different concentrations (low, medium and high);

[0042] Fig.17 The selectivity test results of the detection method constructed by the present invention on MC-LR (the interference is 10 times or 100 times);

[0043] Fig.18 The MC-LR detection method constructed by the present invention detects different environmental water samples. DETAILED DESCRIPTION

[0044] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0045] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0046] An embodiment of the present invention provides a CRISPR-Cas12a sensor, comprising Cas12a, crRNA, a Cas12a activation chain and a MC-LR aptamer; wherein the sequence of crRNA is shown in SEQ ID NO: 1; and / or the Cas12a activation chain is shown in SEQ ID NO: 2; and / or the sequence of the MC-LR aptamer is shown in SEQ ID NO: 3.

[0047] It should be noted that in the present invention, in order to better detect MC-LR through CTAB, HCG and pregnancy reagents, the above-mentioned CRISPR-Cas12a sensor is designed, which has better detection effect than other CRISPR-Cas12a sensors.

[0048] An embodiment of the present invention also provides a detection reagent or detection kit for MC-LR in water, comprising the CRISPR-Cas12a sensor of the present invention, a long chain containing a partially hybridized double strand, a cationic surfactant or its solution, a loaded HCG reagent and an HCG detection reagent, which are separately packaged; wherein the CRISPR-Cas12a sensor comprises Cas12a, crRNA, a Cas12a activation chain and a MC-LR adaptor.

[0049] It should be noted that the independent packaging in the present invention means that each reagent is separated before use; in addition, the HCG-loaded reagent refers to calcium carbonate vaterite loaded with HCG; in addition, the HCG detection reagent refers to a reagent that can qualitatively or quantitatively detect HCG, and of course, the reagent here includes test strips.

[0050] In some specific examples, the above detection reagent or detection kit meets one or more of the following conditions:

[0051] (a) The sequence of crRNA is shown in SEQ ID NO: 1; and / or the Cas12a activation chain is shown in SEQ ID NO: 2; and / or the sequence of the MC-LR aptamer is shown in SEQ ID NO: 3; Specifically, the CRISPR-Cas12a sensor as described above is more suitable for the detection of the present invention;

[0052] (b) In the long chain containing partially hybridized double strands, the sequence of the long single strand is as shown in SEQ ID NO:4, and the sequence of the short single strand is as shown in SEQ ID NO:5; specifically, the long chain containing partially hybridized double strands in the present invention can be obtained by hybridizing any long single strand and short single strand, preferably the sequence of the long single strand is as shown in SEQ ID NO:4, and the sequence of the short single strand is as shown in SEQ ID NO:5, which can better realize the detection in the present invention;

[0053] (c) The long chain containing a partially hybridized double strand is fixedly connected to the magnetic beads; specifically, the long chain containing a partially hybridized double strand can also be fixedly connected to the magnetic beads, so that the part bound to the cationic surfactant can be better separated when the signal molecule HCG is detected later;

[0054] (d) The detection reagent or detection kit also includes a water sample pretreatment reagent; specifically, when the water sample is tested, the water sample can be pretreated, such as filtering solid impurities in the water sample first, and then enriching MC-LR;

[0055] (e) The HCG-loaded reagent is HCG-loaded calcium carbonate vaterite; specifically, the HCG-loaded reagent in the present invention can preferably be HCG-loaded calcium carbonate vaterite. Since the cationic surfactant (such as CTAB) is positively charged, it produces a strong electrostatic interaction with HCG (isoelectric point is 2.95, with a net negative charge at pH 8.0) in the calcium carbonate microspheres (refer to the literature "Wang Jing, Guo Chen, Liang Xiangfeng, et al. The effect of cationic surfactant CTAB on protein secondary structure [C] / / The 14th National Molecular Spectroscopy Academic Conference" or "Zhou Juan, Jin Guiyun, Zhu Li, et al. Spectroscopic study of the interaction between anionic / cationic surfactants and bovine serum albumin [J]. Spectroscopy Laboratory, 2014, 31(2): 6."), attracting HCG molecules and releasing them from the surface or micropores of calcium carbonate; in addition, after adding CTAB, the calcium carbonate loaded with HCG has some Ca 2+ Release, that is, a small amount of CaCO 3 Dissolution will also lead to the release of HCG (refer to the literature "Huang Funing, Wang Caixia, Gao Jianqiang, Liang Yaodong, He Yongjun. Study on the controllable morphology and properties of calcium carbonate [J]. Chemical Engineering, 2020, 48(8): 5. " or "Tang Yanjun, Li Youming, Li Bin. CTAB in nano-CaCO3 Changes in interfacial properties caused by surface adsorption[J]. Journal of South China University of Technology: Natural Science Edition, 2007, 35(11):5.》);

[0056] (g) The cationic surfactant may preferably be CTAB.

[0057] In some specific examples, in the long chain containing partially hybridized double strands, the molar ratio of the long chain to the short chain is 1:4.

[0058] In some specific examples, the preparation method of the above-mentioned HCG-loaded calcium carbonate vaterite includes: 2 The solution was mixed with HCG solution and ultrasonicated; then Na 2 CO 3 The solution is reacted (for example, at 30° C. for 30 minutes) to obtain a reactant; the reactant is washed, dissolved in ethanol, and dried to obtain HCG-loaded calcium carbonate vaterite; wherein the HCG solution is prepared from Tris-HCl.

[0059] The embodiment of the present invention further provides a method for detecting MC-LR in water, which comprises using the detection reagent or detection kit of the present invention for detection.

[0060] In some specific examples, the above method includes: (1) mixing the CRISPR-Cas12a sensor with a water sample to be detected to obtain a mixed solution 1; mixing a long chain containing a partially hybridized double chain and a cationic surfactant or its solution to obtain a mixed solution 2; (2) mixing the mixed solution 1 and the mixed solution 2 to obtain a reactant; (3) separating the reactant to obtain a supernatant 1; mixing the supernatant 1 with a loaded HCG reagent and centrifuging to obtain a supernatant 2; (4) testing the HCG content in the supernatant 2 by an HCG detection reagent, thereby qualitatively or quantitatively detecting MC-LR in the water body.

[0061] It should be noted that in the above step (1), after the CRISPR-Cas12a sensor is mixed with the water sample to be detected to obtain a mixed solution, the MC-LR in the water sample binds to the aptamer, which releases the Cas12a activation chain that is partially complementary to the aptamer; the activation chain combines with the existing Cas12a / crRNA binary complex in the solution to assemble into a ternary complex Cas12a / crRNA / Activator with enzyme cleavage activity (this ternary complex has efficient and indiscriminate trans-cleavage ability for DNA single strands); in addition, the long chain containing partially hybridized double strands and a cationic surfactant or its solution (such as CTAB or CTAB solution) are mixed to obtain a mixed solution 2, and the long chain containing partially hybridized double strands can completely bind to the cationic surfactant; in step (2), when the mixed solution 1 and the mixed solution 2 are mixed Afterwards, if the water sample to be tested contains MC-LR, the ternary complex Cas12a / crRNA / Activator will accurately and quickly cut the single-stranded portion of the hybrid long chain. When the length of the long-chain DNA containing the partially hybridized double-stranded DNA is less than 20 bases, it cannot effectively combine with the cationic surfactant, resulting in a large amount of free cationic surfactant remaining in the supernatant; if the water sample to be tested does not contain MC-LR, no reaction occurs, and the supernatant does not have free CTAB; in step (3), if the water sample to be tested contains MC-LR, the obtained free cationic surfactant is mixed with the loaded HCG reagent to release a large amount of HCG; if the water sample to be tested does not contain MC-LR, no reaction occurs; in step (4), the cationic surfactant content can be obtained by detecting the HCG content, thereby knowing the aptamer content, and then obtaining the MC-LR content.

[0062] In some specific examples, the HCG detection reagent is a pregnancy test paper. In step (4), the pregnancy test paper is used to detect the supernatant 2, and a qualitative or quantitative analysis is performed according to the situation of the detection line.

[0063] It should be noted that in the above step (4), HCG detection can be achieved using commercial pregnancy test strips, and the detection method can refer to the instructions of the commercial pregnancy test strips.

[0064] In some specific examples, quantitative analysis is performed based on the situation of the test line, including: (i) taking a photo and analyzing the test strip after the test line appears to obtain the grayscale value of the test strip test line area; (ii) obtaining the MC-LR concentration in the water sample to be tested based on the working curve of the grayscale value of the test line area and the standard MC-LR concentration.

[0065] It should be noted that the analysis in the present invention can be analyzed and processed using existing software, such as using ImageJ to open the test strip photo, selecting Image-Type-8bit, and then clicking Process-Substract to process the image; clicking Edit-Invert to obtain a grayscale analysis image; using the "box selection" tool to select the test line area, and pressing Ctrl+M to read the grayscale value of the selected area.

[0066] In some specific examples, in the above method, MC-LR in the water sample to be detected is enriched and then mixed with the CRISPR-Cas12a sensor.

[0067] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0068] In the following example, the material sequence involved is shown in Table 1.

[0069] Table 1 Material sequence involved

[0070]

[0071] 1. Construction of MC-LR detection method in water

[0072] In the present invention, the construction idea of ​​the MC-LR detection method in water is referred to Figure 1 .

[0073] (I) Competitive recognition of targets

[0074] (1) Sample pretreatment: The collected samples (such as tap water, lake water or river water samples) shall be pretreated according to the water sample collection, storage and treatment methods described in GB / T20466-2006 "Determination of Microcystins in Water".

[0075] (2) Preparation of the aptamer-activation chain "locked" structure: First, the MC-LR aptamer and Cas 12a activation chain were annealed at 95°C for 5 min, respectively, and cooled in an ice bath for 3 min; then, 10 μL of 10 μM activation chain and 15 μL of 10 μM aptamer chain were added to a 500 μL centrifuge tube ①, and placed in a metal bath at 95°C for denaturation for 5 min. After taking out, it was placed at room temperature for 15 min to form a "locked" double chain.

[0076] (3) Take 10 μL of the water sample treated in step (1) and add it to the centrifuge tube ① where the reaction is completed. Then add 5 μL of RNase scavenger (or RNase-free water), mix well and let it stand at room temperature for 10 minutes to release the activation chain of the Cas enzyme.

[0077] (II) Activation of Cas12a and its cleavage of long hybrid DNA chains

[0078] 1. Preparation and characterization of magnetic beads for immobilizing long DNA containing partially hybridized double strands

[0079] Take 10 μL of 50 μM well-dispersed carboxyl magnetic beads (MB-COOH, also known as MB, Shanghai Titan Technology Co., Ltd., 1 μm, low non-specificity) in a 500 μL centrifuge tube ②, wash with ultrapure water for 3 times, 200 μL each time; then wash the magnetic beads twice with 200 μL MES buffer (0.5 M, pH 8.5, Shanghai MacLean Biochemical Technology Co., Ltd.), add coupling agent EDC and NHS (dissolved in MES buffer) 100 μL each; after reacting at 37 ° C for 30 minutes, wash the magnetic beads (activated magnetic beads) twice with 1× PBS buffer; finally, add 5'-amino-modified long single-stranded DNA and place it on a shaker for overnight reaction to obtain MB@lsDNA.

[0080] The DNA-fixed magnetic beads and complementary short-chain DNA (short single-stranded ssDNA) in the centrifuge tube ② after the reaction in 1 were placed in a metal bath respectively, annealed at 95°C for 5 minutes, cooled in an ice bath for 3 minutes, and then denatured at 95°C for 5 minutes. After taking out, place it at room temperature for 15 minutes to cool, and fix the long-chain DNA containing partially hybridized double strands on the surface of the magnetic beads to obtain MB@dsDNA.

[0081] The particle size and Zeta potential of MB, MB@lsDNA (modified with long single strands) and MB@dsDNA (modified with double strands) were tested respectively. Figure 2 As shown in the figure. After modification with lsDNA and dsDNA, the size of the magnetic beads gradually increased; in addition, the Zeta potential showed that after modification with lsDNA and dsDNA, the Zeta potential gradually decreased. When lsDNA and dsDNA were attached to MB, the absorbance at 260nm increased significantly, which was direct evidence of the successful fixation of DNA to the surface of the magnetic beads.

[0082] 2. After the reaction in (a) above is completed, add the substances shown in Table 2 to the centrifuge tube ① respectively.

[0083] Table 2 Adding substances to centrifuge tube ①

[0084] substance Dosage 1μM crRNA 50μL 1μM Cas12a 25μL 10×NEBuffer 2.1 20μL RNase scavenger (Beijing Huayueyang Waiyuan, the same below) 10μL RNase-free Water 45μL

[0085] 3. Transfer a total volume of 200 μL of solution (centrifuge tube ① originally contained 25 μL of aptamer-activation chain hybrid double-stranded solution, 5 μL of RNase scavenger and 10 μL of water sample) to the magnetic bead tube with fixed double-stranded DNA prepared in (ii). 1 (i.e., centrifuge tube ②), place it in a 37°C water bath for cutting for 60 min; then quickly heat it to 65°C (metal bath) to inactivate Cas12a.

[0086] (III) Interaction between partial double-stranded structure (hybridized DNA long chain) and CTAB

[0087] After inactivating Cas12a in the above centrifuge tube ②, perform magnetic separation on centrifuge tube ② and discard the supernatant; wash the cut magnetic beads 3-5 times with ultrapure water, add 200 μL, 300 μM hexadecyl ammonium bromide (CTAB) aqueous solution, shake and mix, and react at 37 ° C for 30 min.

[0088] (IV) Loading and stimulating release of signal molecules (HCG)

[0089] 1. Preparation and Characterization of HCG-loaded Calcium Carbonate Vaterite

[0090] (1) Preparation of HCG-loaded calcium carbonate vaterite: Take a 10 mL centrifuge tube and add 1 mL of 1 M CaCl 2 Prepare the solution, add 3 mL of 8 mg / mL HCG solution prepared with Tris-HCl (pH 8.5, 100 mM Tris-base + 100 μL concentrated hydrochloric acid, dilute to 10 mL), sonicate for 1 min; then quickly add 1 mL of 1M Na 2 CO 3 Prepare the solution in the above centrifuge tube, vortex for 1 min, and place it in a 30℃ water bath to react for 40 min.

[0091] (2) Removal of residual HCG and drying of particles: The reaction system in (1) was centrifuged (speed 7500 r / min, time 10 min), washed with ultrapure water and anhydrous ethanol, 5 mL each time, washed 3 times, and centrifuged (conditions remain unchanged); then dissolved with 2 mL of anhydrous ethanol, evenly distributed into 10 200 μL centrifuge tubes ③, and dried in a constant temperature dryer at 60°C for 2 hours to prepare HCG-loaded calcium carbonate vaterite.

[0092] The prepared HCG-loaded calcium carbonate vaterite was characterized by SEM and EDS. Figure 4As shown, SEM shows that most of the vaterite is oval, about 3-5μm in size, and monodisperse; in the magnified image, the rough surface shows an obvious porous structure, and the small nanoparticles that make up the vaterite are regularly arranged; in the EDS mapping results, purple, yellow, and dark blue represent the N, P, and S elements in the particles, respectively, indicating that HCG is successfully loaded.

[0093] In addition, the Zeta potential of vaterite loaded and unloaded with HCG is shown in Figure 5 As shown in the figure, the Zeta potential of the prepared vaterite before and after HCG loading was 0.294 mV and -7.0 mV, respectively; since the isoelectric point PI of HCG is 2.95, it is negatively charged in a neutral environment, and electrostatic attraction is the main factor for its loading onto CaCO 3 The main reason.

[0094] 2. CTAB triggers HCG release

[0095] (1) CTAB triggers HCG release: Perform magnetic separation on the centrifuge tube ② that has completed step (iii), take the supernatant and add it to the centrifuge tube ③ containing the particles, and make the supernatant fully contact and mix with the particles by oscillation and vortexing, and react for 10 minutes.

[0096] (2) Study on the mechanism of CTAB-triggered HCG release

[0097] Since HCG release has a decisive influence on the detection results of MC-LR, the response release characteristics of HCG-loaded particles were studied in depth, as follows:

[0098] First, HCG-loaded vaterite was placed in an aqueous solution of pure water and several surfactants. When the HCG-loaded particles were resuspended in pure water, no signal was observed on the detection line, indicating that the HCG-loaded vaterite was relatively stable in water; however, the HCG loaded in the vaterite was immediately released after the introduction of CTAB; the pregnancy test results showed that the HCG-loaded particles responded to cationic surfactants (such as CTAB, DTAB and OTAB), but did not respond to anionic SDS and neutral PVA, indicating that the prepared vaterite carrier has a selective response release characteristic (see Figure 6 ).

[0099] Then, the interaction between CTAB and HCG was characterized by circular dichroism (CD). In the CD spectrum, CTAB absorbs at 192 nm, which is consistent with its UV-Vis absorption. The CD peaks of pure HCG at 191 nm and 198 nm represent its secondary conformation (α helix and β fold) and two subunits. After interacting with CTAB (15 minutes), the peak of HCG at 220 nm decreased, and the peaks of other wavelengths reversed, which may be due to the electrostatic attraction and hydrophobic interaction between CTAB and HCG (see Figure 7 ).

[0100] Secondly, the release curve of CTAB-triggered HCG release was plotted (see Figure 8 ), the results showed that in the range of 50 μM to 400 μM, the HCG concentration in the supernatant gradually increased with the increase of CTAB concentration, and the curve followed equation C HCG =95.82*exp(C CTAB / 396.38)+120.98, indicating that the prepared HCG-loaded calcium carbonate is highly responsive and the release of HCG in the carrier can be well quantitatively regulated by controlling the CTAB concentration.

[0101] Finally, the effect of CTAB on CaCO 3 The effects of calcein on the release of vaterite Ca and its role in HCG response were tested using calcein 2+ Dissolution of MgCl2O3; No Ca added 2+ The calcein fluorescence is shown as a green curve. If any Ca is released from calcium carbonate 2+ , fluorescence enhancement should be observed; the results show that pure calcium carbonate (without HCG loading) hardly decomposes in water; HCG-loaded calcium carbonate also does not dissolve Ca in pure water. 2+ , Ca 2+ The release of HCG-loaded calcium carbonate was even less than that of calcium carbonate without HCG. HCG-loaded calcium carbonate released considerable Ca when CTAB (300 μM) was introduced. 2+ , indicating that CTAB has a strong affinity for CaCO 3 Surface properties have a great influence on promoting HCG release (see Fig. 9 ).

[0102] Based on the above facts, it can be concluded that CTAB and CaCO 3 The interaction between them and the interaction between CTAB and HCG will jointly trigger the release of HCG from calcium carbonate particles.

[0103] (V) Signal reading and numerical analysis

[0104] (1) Use a pocket centrifuge (6000r / min) to centrifuge tube ③ after treatment in (IV).2 to separate the particles from the solution until the upper liquid is clear.

[0105] (2) Use Jinxiuer Early Pregnancy Test Reagent (Colloidal Gold Method) (Guangzhou Wondfo Biotechnology Co., Ltd.), immerse the end into the upper clear liquid of centrifuge tube ③, wait for the liquid to flow through the binding pad, take it out, and place it flat on the table; when the control line of the test strip appears, wait for 5 minutes and read the result directly or take a photo for quantitative analysis.

[0106] (3) Quantitative analysis: Use Image J to open the test strip photo, select Image-Type-8bit, and then click Process-Substract to process the image; click Edit-Invert to obtain the grayscale analysis image; use the "Box Selection" tool to select the test line area, and press Ctrl+M to read the grayscale value of the selected area.

[0107] Set MC-LR samples of different concentrations, detect the grayscale value of the test strip detection line area according to the above method, and then obtain the standard working curve based on the corresponding relationship between the grayscale value of the test strip detection line area and the standard MC-LR concentration. The results are as follows: Fig.10 As shown, the results show that the gray value of the final pregnancy test strip detection line is positively correlated with the MC-LR concentration. The linear regression equation obtained is Y = 28882.34lgC MC-LR +45976.79, correlation coefficient was 0.9976, range was 0.1μg / L to 100μg / L, and the lowest limit of detection (LOD) was calculated to be 0.028μg / L (3σ / slope method).

[0108] 2. Optimization of MC-LR detection method in water

[0109] (I) Optimizing aptamer / activator chain hybrids to synthesize different activation chains

[0110] The hybrids of aptamer-activator chains with different aptamer ratios (1:1, 1:1.2 and 1:1.5) were prepared, and the "locked" structure was reacted with 10 μg / LMC-LR to select the best ratio. After comparing the final test results of the test strips, the best ratio was determined to be 1:1.5.

[0111] (II) Optimization of regional double-stranded structure (hybridized DNA long chain) and CTAB binding

[0112] Different types of DNA (long single-stranded DNA and long chains containing partial double-stranded regions formed by DNA hybridization at different ratios (1:3, 1:4, 1:5)) were fixed on magnetic beads and then incubated with CTAB. The residual CTAB concentration in the supernatant was detected by UV spectrometer; the absorbance of MB@lsDNA was lower than that of pure CTAB, but it was the highest among all MB@DNA incubated with CTAB, indicating that the amount of CTAB bound by MB@lsDNA was limited and most of the CTAB was still in a free state. In order to enhance the interaction between DNA and CTAB, some regions of lsDNA on MB@DNA were hybridized into rigid double strands, exposing the phosphate groups to interact with the quaternary ammonium groups of CTAB; the binding ratio of long chains to short chains (the short chains were modified with carboxyl groups to further enhance their binding ability with CTAB) in the designed MB@dsDNA was 1:3.

[0113] In addition, MB@dsDNA was prepared with different ratios of long and short chains (1:3, 1:4 and 1:5), and then mixed with CTAB of the same concentration (200 μM), and the residual CTAB concentration in the supernatant was measured; the absorption curve of MB+ds1:5+CTAB showed that all CTAB molecules were bound and no free CTAB remained (see Fig.11 ). Moreover, there was no obvious CTAB shedding in the 1:4 and 1:5 groups after multiple washings ( Fig.12 ), proving its stability and durability. Considering the cost of synthesis, combined with the MB+ds1:4+CTAB curve (CTAB is basically bound and will not trigger HCG release to produce signals), MB@dsDNA was finally prepared with a ratio of long chain to single chain of 1:4.

[0114] (III) Optimization of MB@dsDNA binding CTAB concentration

[0115] MB@dsDNA (magnetic beads final concentration 5 mg / mL) prepared at a 1:4 ratio was combined with 200 μM CTAB to trigger HCG release and detect it with a test strip; the results were similar to Fig.11 The results are consistent, indicating that the double strands on the MB effectively occupy all CTAB molecules; increasing the CTAB concentration can enhance the color intensity of the T line, thereby reducing the detection limit; therefore, 300μM CTAB was tested. However, the obvious color on the T line indicates that there are too many CTAB molecules to be bound by the double strands on the MB (see Fig.13 ).

[0116] To improve the sensitivity of the method, the amount of MB@dsDNA was increased (10 mg / mL), which broadened the range of regulation of the magnetic bead DNA system for CTAB. Even when 300 μM CTAB was added, the test strip did not show obvious signals. Adding a lower concentration of MC-LR still produced visible bands on the T line. When 400 μM and 500 μM CTAB were added without any target molecules, the test strip results showed that the MB@dsDNA used could not fully bind to CTAB. After comprehensively considering the signal intensity and detection sensitivity, 300 μM was finally selected as the CTAB concentration actually used in the determination (see Fig.14 ).

[0117] (IV) Optimizing the reaction conditions of the CRISPR-Cas12a system

[0118] The Cas12a protein with different final concentrations of 100nM, 150nM, 200nM, 250nM and 300nM (the mass ratio of Cas12a to crRNA was kept at 1:1) was used to cut the magnetic beads (MB@dsDNA) fixed with long-chain DNA containing double-stranded regions for 60 minutes; then the ratio of Cas12a to crRNA was adjusted according to the optimized Cas12a concentration (1:1, 1:1.5 and 1:2); the reaction time should be controlled within 60 minutes; CRISPR-Cas1 2a cutting time; after adding MC-LR solution, the time was controlled to 30 minutes, 45 minutes, 60 minutes, 90 minutes and 120 minutes respectively; for the optimization of the above CRISPR-Cas12a cutting, the temperature should be maintained at 37°C and the total volume of the reaction solution was maintained at 200 μL; the grayscale value of the pregnancy test strip detection line was used as an analysis parameter to determine the optimal value of all the mentioned conditions to be optimized (it should be noted that when one of the parameters was changed, the other parameters remained unchanged, and the test was performed according to the MC-LR detection method constructed as above).

[0119] After a series of condition optimization, it was finally determined that when the final concentration of MB@dsDNA magnetic beads was 10 mg / mL, the CRISPR system reaction volume was 200 μL, the final concentration of Cas12a protein was 250 nM, the ratio of Cas enzyme to crRNA was 1:2, and the cleavage time of Cas12a on the single-stranded part of MB@dsDNA was 60 min (see Fig.15 ).

[0120] 3. Selectivity test of the MC-LR detection method constructed by the present invention

[0121] First, using deionized water as solvent, 100 μL of standard solutions of 1 μg / L, 10 μg / L and 100 μg / L were prepared with the six substances (MC-LR, MC-RR, MC-YR, kanamycin, tetracycline hydrochloride and lipopolysaccharide) standards respectively for use;

[0122] Secondly, take 10 μL of each substance and each concentration, follow the steps described in the above section "I. Construction of MC-LR detection method in water", and finally read the grayscale value of the test line area of ​​the test strip; each substance and each concentration sample needs to be measured in parallel 3 times;

[0123] Finally, the grayscale values ​​are used to plot samples of different concentrations and types of substances to obtain a three-dimensional statistical graph, such as Fig.16 shown.

[0124] In addition, the results of 1 μg / L MC-LR (target), 10 μg / L MC-YR and MC-RR (both are MC-LR analogs), 100 μg / L kanamycin, 100 μg / L tetracycline hydrochloride and 100 μg / L lipopolysaccharide (common water pollutant) were selected for plotting. The results are shown in Figure 2. Fig.17 As shown, it is to intuitively prove the high selectivity of the present invention for MC-LR.

[0125] 4. Real environment sample test

[0126] Water was collected from different environments. Before using this method for detection, all water samples were tested in advance according to the high performance liquid chromatography method described in GB / T20466-2006 "Determination of Microcystins in Water", and no MC-LR was found. Then, MC-LR standards were added to prepare spiked samples of different concentrations of 1.00μg / L, 10μg / L and 100μg / L, and the samples were tested according to the MC-LR detection method constructed above. The results are shown in Table 3 below, and the recovery rate data are plotted as shown in Fig.18 shown.

[0127] Table 3 Test conditions of different samples

[0128]

[0129] It can be seen from the above table that the concentrations obtained by detecting different samples using the method of the present invention are not much different from the actual concentrations added to the samples, indicating that the MC-LR detection method constructed by the present invention has high accuracy.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered within the scope of the claims of the present invention.

Claims

1. A CRISPR-Cas12a sensor, characterized in that It includes Cas12a, crRNA, Cas12a activation chain and MC-LR adapter; wherein the sequence of crRNA is shown in SEQ ID NO: 1; and / or the Cas12a activation chain is shown in SEQ ID NO: 2; and / or the sequence of MC-LR adapter is shown in SEQ ID NO:

3.

2. A detection reagent or a detection kit for MC-LR in water, characterized in that: It includes independently packaged CRISPR-Cas12a sensors, long chains containing partially hybridized double chains, cationic surfactants or their solutions, loaded HCG reagents and HCG detection reagents; the CRISPR-Cas12a sensor includes Cas12a, crRNA, Cas12a activation chains and MC-LR adapters.

3. The detection reagent or detection kit according to claim 2, characterized in that: The detection reagent or detection kit meets one or more of the following conditions: (a) The sequence of crRNA is shown in SEQ ID NO: 1; and / or the Cas12a activation chain is shown in SEQ ID NO: 2; and / or the sequence of MC-LR aptamer is shown in SEQ ID NO: 3; (b) In the long chain containing partially hybridized double strands, the sequence of the long single strand is shown in SEQ ID NO:4, and the sequence of the short single strand is shown in SEQ ID NO:5; (c) The magnetic beads are fixedly connected to the long chain containing partially hybridized double strands; (d) the detection reagent or detection kit also includes a water sample pretreatment reagent; (e) the HCG-loaded reagent is HCG-loaded calcium carbonate vaterite; (f) HCG test reagent is a pregnancy test strip; (g) The cationic surfactant is CTAB.

4. The detection reagent or detection kit according to claim 3, characterized in that: In the long chain containing partially hybridized double chains, the molar ratio of the long chain to the short chain is 1:

4.

5. The detection reagent or detection kit according to claim 3 or 4, characterized in that: The preparation method of HCG-loaded calcium carbonate vaterite comprises: mixing CaCl2 solution and HCG solution, ultrasonicating; then rapidly adding Na2CO3 solution to react and obtain reactants; washing the reactants, dissolving them in ethanol, and drying them to obtain HCG-loaded calcium carbonate vaterite; wherein the HCG solution is prepared by Tris-HCl.

6. A method for detecting MC-LR in water, characterized in that: The method comprises using the detection reagent or detection kit described in any one of claims 3 to 5 for detection.

7. The method according to claim 6, characterized in that include: (1) Mixing the CRISPR-Cas12a sensor with a water sample to be detected to obtain a mixed solution 1; mixing a long chain containing a partially hybridized double chain with a cationic surfactant or its solution to obtain a mixed solution 2; (2) mixing the mixed solution 1 and the mixed solution 2 to obtain a reactant; (3) separating the reactant to obtain a supernatant 1; mixing the supernatant 1 with a loaded HCG reagent and centrifuging to obtain a supernatant 2; (4) testing the HCG content in the supernatant 2 with an HCG detection reagent, thereby qualitatively or quantitatively detecting MC-LR in the water body.

8. The method according to claim 7, characterized in that The HCG detection reagent is a pregnancy test paper. In step (4), the supernatant 2 is detected using the pregnancy test paper, and a qualitative or quantitative analysis is performed according to the situation of the detection line.

9. The method according to claim 8, characterized in that Quantitative analysis is performed based on the situation of the detection line, including: (i) taking photos and analyzing the test strip detection line after it appears to obtain the grayscale value of the test strip detection line area; (ii) obtaining the MC-LR concentration in the water sample to be tested based on the working curve of the grayscale value of the test strip detection line area and the standard MC-LR concentration.

10. The method according to any one of claims 7 to 9, characterized in that The MC-LR in the water sample to be tested is enriched and then mixed with the CRISPR-Cas12a sensor.

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