A crisper-cas12a sensor and a method for detecting mc-lr in water bodies by combining the same with a pregnancy test paper

Abstract

The present application belongs to the technical field of microcystin detection in water body, and particularly relates to a CRISPR-Cas12a sensor and a method for detecting MC-LR in water body by combining the CRISPR-Cas12a sensor and a pregnancy test paper. The present application develops a method for detecting MC-LR based on a CRISPR-Cas12a sensor. The detection method is developed based on the trans-cleavage ability of the CRISPR-Cas12a sensor, the binding ability of cationic surfactants and long chains containing part of hybrid double strands, the ability of cationic surfactants to trigger HCG release, and the binding ability of HCG and pregnancy reagents. The detection method can quickly and accurately realize the detection of MC-LR, does not need special detection test paper, can use commercial pregnancy test paper, and has low cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microcystin detection in water bodies, and particularly relates to a CRISPR-Cas12a sensor and a method for detecting MC-LR in water bodies by combining the sensor with a pregnancy test paper. BACKGROUND

[0002] Microcystin LR (MC-LR) is one of the strongest freshwater cyanotoxins known for its strong hepatotoxicity. It can strongly inhibit the activity of protein phosphatase, causing damage to liver cells, and may lead to pathological changes such as liver enlargement, bleeding, and necrosis. In addition, MC-LR is a strong liver tumor promoter, and long-term drinking may increase the risk of liver cancer. In addition to hepatotoxicity, MC-LR also has nephrotoxicity, reproductive toxicity, neurotoxicity, and genetic toxicity, causing damage to multiple organs and systems. MC-LR is widely distributed in eutrophic freshwater lakes, reservoirs, and rivers, posing a great threat to human health and the environment. Therefore, it is particularly important to detect MC-LR.

[0003] The prior art can detect microcystin LR by real-time assembly of aptamer sensor through plasma-oxidized graphene (p-GO); it mainly consists of two parts: one is the assembly of MC-LR aptamer assembled by biotin / avidin, which is used to recognize the target; the other is the composite of hollow nanometer plate AgClAu and p-GO with aptamer complementary chain, which can enhance the Raman signal. In addition, Raman signal molecules are added to the detection solution to produce SERS signals; this scheme adjusts the competition degree of MC-LR and the complementary chain of aptamer for aptamer, and controls the assembly mode and result of the two parts in real time, thereby producing different SERS signals; the defect is that the material preparation conditions are harsh, the process is complex, the cost is high, and it cannot be separated from large equipment, which is not suitable for on-site analysis.

[0004] The prior art can also analyze based on indirect competitive immunity, and use the total internal reflection property of divergent wave optical fiber to realize ultra-micro toxin analysis; MC-LR and monoclonal MC-LR antibody are incubated together to occupy the recognition site; when the reaction solution is added to the detection pool, the antigen fixed on the probe combines with the free antibody that has not been occupied, producing an optical signal; although this method optimizes the optical technology and greatly improves the detection sensitivity; but the fiber probe, monoclonal antibody, and antigen fixation all require high cost, and the immune analysis has problems such as non-specific reaction and excessive recognition.

[0005] In addition, the prior art can also be based on the sensitive sensor of CRISPR-Cas12a for quantitative detection of MC-LR in real water samples; specifically, the MC-LR aptamer is attached to the magnetic beads (MBs) and hybridized with the blocking DNA strand; after recognizing the MC-LR molecules in the water sample, the blocking DNA is released, combined with Cas12a / crRNA to activate the enzyme cutting activity; the activated Cas12a takes the fluorescently labeled ssDNA substrate as the reporter to obtain the fluorescence signal. In addition, by replacing the reporter molecule, the MC-LR content can be directly observed with a special test strip. Although this method overcomes the defects of immunoassay and improves the detection accuracy. However, a small fluorescence meter is still used in the detection process, and the special test strip used has poor quantitative effect, high cost and poor universality.

[0006] Therefore, it is necessary to develop a MC-LR detection method with high accuracy, suitable for on-site detection, and low cost. SUMMARY

[0007] The present application develops a MC-LR detection method based on CRISPR-Cas12a sensor, the detection method of the present application is developed based on the trans-cleavage ability of CRISPR-Cas12a sensor, the binding ability of cationic surfactant and long chain containing part of hybrid double-stranded, the ability of CTAB triggering HCG release, and the binding ability of HCG and pregnancy reagent, which can quickly and accurately realize the detection of MC-LR, and does not need special detection test paper, and can use commercial pregnancy test paper, which is low in cost.

[0008] In order to achieve the above purpose, the present application can adopt the following technical scheme:

[0009] The present application provides a CRISPR-Cas12a sensor, which comprises Cas12a, crRNA, Cas12a activation chain and MC-LR aptamer; wherein the sequence of crRNA is shown as SEQ ID NO: 1; and / or the Cas12a activation chain is shown as SEQ ID NO: 2; and / or the sequence of MC-LR aptamer is shown as SEQ ID NO: 3.

[0010] The present application provides a water body MC-LR detection reagent or detection kit, which comprises independently packaged CRISPR-Cas12a sensor, long chain containing part of hybrid double-stranded, cationic surfactant or solution thereof, HCG reagent and HCG detection reagent in the present application; wherein the CRISPR-Cas12a sensor comprises Cas12a, crRNA, Cas12a activation chain and MC-LR aptamer.

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

[0012] (a) the sequence of the crRNA is as shown in SEQ ID NO: 1; and / or the sequence of the Cas12a activation chain is as shown in SEQ ID NO: 2; and / or the sequence of the MC-LR aptamer is as 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 as shown in SEQ ID NO: 4, and the sequence of the short single strand is as shown in SEQ ID NO: 5;

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

[0015] (e) the detection reagent or detection kit further comprises a water sample pretreatment reagent;

[0016] (f) the HCG-loaded reagent is HCG-loaded calcium carbonate ball 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 ball vaterite comprises: mixing a CaCl2 solution with an HCG solution, ultrasonicating; then quickly adding a Na2CO3 solution to react to obtain a reaction product; washing the reaction product, dissolving in ethanol, and drying to obtain the HCG-loaded calcium carbonate ball vaterite; wherein the HCG solution is prepared by Tris-HCl.

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

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

[0022] More preferably, the HCG detection reagent is a pregnancy test paper, and in step (4), the pregnancy test paper is used to detect the supernatant 2, and 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 a photo and analyzing the test strip after the detection line 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 strip provided by the present invention avoids the use of large-scale instruments and equipment, does not require the participation of professional personnel, and is more suitable for on-site detection; it solves the problem that the existing test strip detection method has insufficient sensitivity and is not suitable for quantitative analysis; in addition, the present invention can be used for detection using existing commercial pregnancy test strips, shortening the R&D and market conversion cycle and reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 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 in 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 and unloaded with HCG;

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

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

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

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

[0035] Figure 10 Standard working curve of test strip detection line area gray value and standard MC-LR concentration;

[0036] Figure 11 Fixing condition of partial hybridization long chain on magnetic beads with different DNA long and short chain ratios (ultraviolet of supernatant);

[0037] Figure 12 Stability of partial hybridization long chain on magnetic beads after multiple water washing with different DNA long and short chain ratios (ultraviolet of supernatant);

[0038] Figure 13 Detection condition of CTAB with different concentrations combined with MB@dsDNA;

[0039] Figure 14 Detection condition of high concentration CTAB combined with double dosage MB@dsDNA;

[0040] Figure 15 Optimization of CRISPR-Cas12a system reaction condition; wherein, a is the volume condition of the optimized CRISPR / Cas12a system; b is the optimization of Cas12a concentration (Cas12a:crRNA = 1:1, MC-LR:10nM); c is the optimization of Cas12a:crRNA ratio; d is the optimization of cutting time after adding MC-LR;

[0041] Figure 16 Selectivity test condition of the detection method constructed by the application on MC-LR with different concentrations (low, medium and high);

[0042] Figure 17 Selectivity test condition of the detection method constructed by the application on MC-LR (interfering substance is 10 times or 100 times);

[0043] Figure 18 Detection condition of the MC-LR detection method constructed by the application on different environmental water samples. DETAILED DESCRIPTION

[0044] The examples are used to better illustrate the application, but are not the only embodiments of the application. Therefore, those skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above description, which still belong to the protection scope of the application.

[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", "comprise" and the like 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 an MC-LR aptamer; wherein the sequence of the 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 separately packaged CRISPR-Cas12a sensors of the present invention, long chains containing partially hybridized double strands, cationic surfactants or their solutions, HCG-loaded reagents, and HCG detection reagents; wherein the CRISPR-Cas12a sensor comprises Cas12a, crRNA, a Cas12a activation chain, and an MC-LR adaptor.

[0049] It should be noted that the independent packaging in the present invention means that the reagents are 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. Of course, the reagents here include test strips.

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

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

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

[0053] (c) the long chain containing partially hybridized double strands is fixedly connected with magnetic beads; specifically, the long chain containing partially hybridized double strands can also be fixedly connected with magnetic beads, which can better separate the part combined with the cationic surfactant when detecting the signal molecule HCG later;

[0054] (d) the detection reagent or detection kit further comprises a water sample pretreatment reagent; specifically, the water sample can be pretreated before detection, such as filtering the solid impurities in the water sample, and then enriching MC-LR;

[0055] (e) the HCG-loaded reagent is HCG-loaded calcite aragonite; specifically, the HCG-loaded reagent in the present application can preferably be HCG-loaded calcite aragonite, because the cationic surfactant (such as CTAB) is positively charged, and has a strong electrostatic interaction with HCG (isoelectric point is 2.95, and has a net negative charge at pH 8.0) in the calcite microspheres (referring to the literature “Wang Jing, Guo Chen, Liang Xiangfeng, et al. Influence of Cationic Surfactant CTAB on Protein Secondary Structure [C] / / The Fourteenth National Molecular Spectroscopy Conference” or “Zhou Juan, Jin Guiyun, Zhu Li, et al. Spectroscopic Study on the Interaction between Anionic / Cationic Surfactants and Bovine Serum Albumin [J]. Spectroscopy and Laboratory, 2014, 31(2): 6.”), which releases the HCG molecules from the surface or micropores of the calcite after attracting them; in addition, after adding CTAB, part of the Ca 2+Release, that is, a small amount of CaCO3 dissolves under the action of CTAB, will also cause the release of HCG (for reference literature Huang Funing, Wang Caixia, Gao Jianqiang, Liang Yaodong, He Yongjun. Morphology controllable preparation and performance research of calcium carbonate [J]. Chemical Engineering, 2020, 48(8): 5. Or Tang Yanjun, Li Youming, Li Bin. Interface property changes caused by CTAB adsorption on the surface of nano-CaCO3 [J]. Journal of South China University of Technology: Natural Science Edition, 2007, 35(11): 5.);

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

[0057] In some specific examples, the long chain and the short chain in the long chain containing a part of hybrid double-stranded chain described above have a feed molar ratio of 1:4.

[0058] In some specific examples, the method for preparing the HCG-loaded calcium carbonate ball aragonite described above comprises: mixing a CaCl2 solution with an HCG solution, ultrasonicating, and then quickly adding a Na2CO3 solution to react (such as reacting at 30℃ for 30min) to obtain a reaction product; washing the reaction product, dissolving in ethanol, and drying to obtain the HCG-loaded calcium carbonate ball aragonite; wherein the HCG solution is prepared by Tris-HCl.

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

[0060] In some specific examples, the method described above comprises: (1) mixing the CRISPR-Cas12a sensor with the water sample to be detected to obtain a mixed solution 1; mixing the long chain containing a part of hybrid double-stranded chain and the cationic surfactant or the solution thereof to obtain a mixed solution 2; (2) mixing the mixed solution 1 and the mixed solution 2 to react to obtain a reaction product; (3) separating the reaction product to obtain a supernatant 1; mixing the supernatant 1 with the HCG-loaded reagent, centrifuging to obtain a supernatant 2; (4) testing the HCG content in the supernatant 2 by the HCG detection reagent, so as to qualitatively or quantitatively detect the MC-LR in the water.

[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, and then the Cas12a activation chain complementary to the aptamer is released; the activation chain binds to the existing Cas12a / crRNA binary complex in the solution to assemble into a ternary complex Cas12a / crRNA / Activator with enzyme cutting activity (this ternary complex has high and indiscriminate trans-cleavage ability for DNA single strand); in addition, the long chain containing partially hybridized double strands and the 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, if the water sample to be detected contains MC-LR, the ternary complex Cas12a / crRNA / Activator will accurately and rapidly cut the single strand part of the hybrid long chain, and when the length of the long chain DNA containing partially hybridized double strands is less than 20 bases, it cannot be effectively combined with the cationic surfactant, resulting in a large amount of free cationic surfactant remaining in the supernatant; if the water sample to be detected 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 detected contains MC-LR, the free cationic surfactant obtained after mixing with the HCG-loaded reagent releases a large amount of HCG; if the water sample to be detected does not contain MC-LR, no reaction occurs; in step (4), by detecting the content of HCG, the content of cationic surfactant can be obtained, and then the content of aptamer can be obtained, and finally the content of MC-LR can be obtained.

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

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

[0064] In some specific examples, quantitative analysis is performed according to the detection line, including: (i) taking a photo of the test paper strip after the detection line appears for analysis, obtaining the gray value of the test paper strip detection line region; (ii) obtaining the MC-LR concentration in the water sample to be detected according to the working curve of the paper strip detection line region gray value and the standard MC-LR concentration.

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

[0066] In some specific examples, in the above method, the 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 application, the content of the present application will be further illustrated below in combination with specific examples, but the content of the present application is not limited to the following examples.

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

[0069] Table 1: Material sequence involved

[0070]

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

[0072] In the present application, the construction idea of the MC-LR detection method in the water body refers to Figure 1 .

[0073] (I) Competitive recognition of target

[0074] (1) Sample collection and pretreatment: The collected sample (tap water, lake water or river water sample) is pretreated according to the water sample collection, preservation and treatment method recorded in GB / T20466-2006 "Determination of Microcystins in Water".

[0075] (2) Preparation of aptamer-activating chain "lock" structure: The aptamer of MC-LR and the Cas 12a activating chain are annealed at 95℃ for 5min respectively, and cooled in ice bath for 3min; then 10μL of 10μM activating chain and 15μL of 10μM aptamer chain are added to 500μL centrifuge tube ①, denatured at 95℃ for 5min, and then taken out and placed at room temperature for 15min to form "lock" double-stranded.

[0076] (3) Take 10μL of the water sample treated in (1), add to the reaction completed centrifuge tube ①, and add 5μL of RNase scavenger (or RNase-free water), mix well, and then stand at room temperature for 10min to release the activating chain of Cas enzyme.

[0077] (ii) Activation of Cas12a and its cleavage of long hybridized DNA strands

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

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

[0080] Place the DNA immobilized magnetic beads in centrifuge tube ② after reaction in 1 and the complementary short DNA (short single-stranded ssDNA) in a metal bath, anneal at 95°C for 5 min, cool in an ice bath for 3 min, denature at 95°C for 5 min, and take out and cool at room temperature for 15 min. Cool, immobilize the long DNA strands containing partially hybridized double strands on the surface of the magnetic beads to obtain MB@dsDNA.

[0081] Test the particle size and Zeta potential of MB, MB@lsDNA (modified with long single-stranded DNA), and MB@dsDNA (modified with double-stranded DNA) respectively, and the results are shown in Figure 2 After lsDNA and dsDNA modification, the size of the magnetic beads gradually increases; in addition, the Zeta potential shows that after lsDNA and dsDNA modification, the Zeta potential gradually decreases, and when lsDNA and dsDNA are attached to MB, the absorbance at 260 nm increases significantly, which is direct evidence of successful immobilization of DNA on the surface of the magnetic beads.

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

[0083] Table 2 Add substances to centrifuge tube ①

[0084] Substance Amount 1 μM crRNA 50 μL 1 μM Cas12a 25 μL 10× NEBuffer 2.1 20 μL RNase Inhibitor (Beijing Huayueyang Foreign, same below) 10 μL RNase-free Water 45 μL

[0085] 3. Transfer the solution (total volume 200 μL) from the original tube (1) containing 25 μL of the aptamer-activated chain hybridized double-stranded solution, 5 μL of RNase scavenger, and 10 μL of water sample into the prepared magnetic bead tube (2) containing the immobilized double-stranded DNA, and place it in a 37°C water bath for 60 min. Then quickly raise the temperature to 65°C (metal bath) to inactivate Cas12a.

[0086] (Three) Interaction of partially double-stranded structure (hybridized DNA long chain) with CTAB

[0087] After inactivating Cas12a in the above-mentioned tube (2), perform magnetic separation on tube (2) and discard the supernatant. Wash the cleaved magnetic beads with ultrapure water for 3-5 times. Add 200 μL of 300 μM cetyltrimethylammonium bromide (CTAB) aqueous solution, shake and mix, and react at 37°C for 30 min.

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

[0089] 1. Preparation and characterization of HCG-loaded calcium carbonate aragonite

[0090] (1) Preparation of HCG-loaded calcium carbonate aragonite: Take a 10 mL centrifuge tube, first add 1 mL of 1M CaCl2 freshly prepared solution, then 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, constant volume to 10 mL) and ultrasonic for 1 min. Then quickly add 1 mL of 1M Na2CO3 freshly prepared solution to the above centrifuge tube, vortex for 1 min, and then place it in a 30°C water bath for 40 min.

[0091] (2) Removal of residual HCG and drying of particles: centrifuge the reaction system in (1) (speed 7500 r / min, time 10 min), wash with ultrapure water and anhydrous ethanol, each time 5 mL, 3 times each, and centrifuge (conditions remain the same). Then dissolve with 2 mL of anhydrous ethanol and evenly distribute into 10 200 μL centrifuge tubes (3), and dry in a 60°C constant temperature dryer for 2 hours to obtain HCG-loaded calcium carbonate aragonite.

[0092] The prepared HCG-loaded calcium carbonate aragonite was characterized by SEM and EDS, and the results are shown in Figure 4 SEM shows that most of the aragonite is oval-shaped, with a size of about 3-5 μm and monodisperse. In the enlarged image, the rough surface shows a clear porous structure, and the nanoscale particles that make up the aragonite are arranged regularly. In the EDS mapping results, purple, yellow and dark blue represent N, P and S elements in the particles, indicating that HCG has been successfully loaded.

[0093] In addition, the Zeta potentials of the loaded and unloaded HCG vaterite were measured as Figure 5 The results showed that the Zeta potentials of the prepared vaterite were 0.294 mV and -7.0 mV before and after loading HCG, respectively. Since the isoelectric point PI of HCG was 2.95, it was negatively charged in neutral environment, and the electrostatic attraction was the main reason for its loading into CaCO3.

[0094] 2. CTAB triggered HCG release

[0095] (1) CTAB triggered HCG release: The supernatant of the centrifuge tube ② after magnetic separation was added to the particle dispensing centrifuge tube ③, and the supernatant was mixed with the particles by oscillation and vortexing. The reaction was carried out for 10 min.

[0096] (2) Mechanism of CTAB triggered HCG release

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

[0098] Firstly, the loaded HCG vaterite was placed in pure water and aqueous solutions of several surfactants. When the loaded HCG particles were resuspended in pure water, no signal was observed on the detection line, indicating that the loaded HCG vaterite was relatively stable in water. However, the HCG loaded in the vaterite was immediately released after the introduction of CTAB. The results of the pregnancy test showed that the loaded HCG 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 had selective response release characteristics (see Figure 6 ).

[0099] Then, the interaction between CTAB and HCG was characterized by circular dichroism (CD). In the CD spectrum, CTAB had an absorption at 192 nm, consistent with its UV-Vis absorption. The CD peaks of pure HCG at 191 nm and 198 nm represented its secondary conformation (alpha helix and beta fold) and two subunits. After interaction with CTAB (15 min), the peak value of HCG at 220 nm decreased, and the peak values at other wavelengths reversed, which might 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 ), and the results showed that the concentration of HCG in the supernatant gradually increased with the increase of the concentration of CTAB in the range of 50 μM to 400 μM, and the curve followed the 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 CaCO3 and its role in HCG-responsive release was investigated, and the Ca content of vaterite was determined using calcein. 2+ Dissolution of Ca-free 2+ The calcein fluorescence is marked 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 (not loaded with HCG) hardly decomposes in water; HCG-loaded calcium carbonate also does not dissolve Ca in pure water. 2+ , Ca 2+ The release of HCG was even less than that of calcium carbonate without HCG. HCG-loaded calcium carbonate released a considerable amount of Ca when CTAB (300 μM) was introduced. 2+ , indicating that CTAB has a significant effect on the surface properties of CaCO3 and promotes the release of HCG (see Figure 9 ).

[0102] Based on the above facts, it can be concluded that the interaction between CTAB and CaCO3 and the interaction between CTAB and HCG jointly trigger the release of HCG from calcium carbonate particles.

[0103] (5) Signal reading and numerical analysis

[0104] (1) Use a pocket centrifuge (6000 r / 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 Biotech Co., Ltd.), immerse the end of the test strip 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 different concentrations of MC-LR samples, according to the above method to detect test strip detection line gray value, then according to the test strip detection line gray value and standard MC-LR concentration corresponding relationship, obtain the standard working curve, results as shown in Figure 10 The results showed that the gray value of the final pregnancy test strip detection line was positively correlated with the concentration of MC-LR. The linear regression equation obtained was Y = 28882.34 lgC MC-LR +45976.79, the correlation coefficient was 0.9976, the range was 0.1 μg / L to 100 μg / L, and the lowest detection limit (LOD) was calculated as 0.028 μg / L (3σ / slope method).

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

[0109] (I) Optimization of aptamer / activating chain hybrid synthesis of different activating chains

[0110] Aptamer-activating chain hybridization of aptamer ratios (1:1, 1:1.2 and 1:1.5) and "lock" structure were reacted with 10 μg / L MC-LR to select the best ratio. Through comparison of the final detection results of the test strip, it was determined that the best ratio was 1:1.5.

[0111] (II) Optimization of double-stranded structure (hybrid DNA long chain) and CTAB binding in the region

[0112] Different types of DNA (long single-stranded DNA and long chain containing partial double-stranded region formed by DNA hybridization in different ratios (1:3, 1:4, 1:5)) were fixed on magnetic beads, then incubated with CTAB, and the residual CTAB concentration in the supernatant was detected by ultraviolet 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 to MB@lsDNA was limited, and most of the CTAB was still in free state. In order to enhance the interaction between DNA and CTAB, the lsDNA part of MB@DNA was hybridized into rigid double-stranded to expose the phosphate group to interact with the quaternary ammonium group of CTAB; the binding ratio of long chain and short chain (short chain modified with carboxyl to further enhance its binding ability with CTAB) in the designed MB@dsDNA was 1:3.

[0113] In addition, different long-short chain ratios (1:3, 1:4 and 1:5) were used to prepare MB@dsDNA, which was then mixed with the same concentration (200 μM) of CTAB, and the residual CTAB concentration in the supernatant was determined; the absorption curve of MB+ds1:5+CTAB showed that all the CTAB molecules had been bound, and there was no free form of CTAB remaining (see Figure 11). And both 1:4 and 1:5 groups showed no significant CTAB loss after multiple water washes Figure 12 ), proving its stability and durability. Considering the cost factor of synthesis, combined with the MB+ds1:4+CTAB curve (CTAB was basically bound and did not trigger HCG release to produce signals), the final ratio of long chain to single chain was 1:4 to prepare MB@dsDNA.

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

[0115] MB@dsDNA prepared at a ratio of 1:4 (magnetic bead final concentration 5 mg / mL) was combined with 200 μΜ CTAB, triggering HCG release and testing with test strips; the results were consistent with Figure 11 , indicating that the double strands on the MB effectively occupied all CTAB molecules; increasing the concentration of CTAB can enhance the T-line color development intensity, thereby reducing the detection limit; therefore, 300 μΜ CTAB was also tested. However, the obvious color on the T-line indicated that there were too many CTAB molecules that could not be bound by the double strands on the MB (see Figure 13 ).

[0116] To improve the sensitivity of the method, the amount of MB@dsDNA was increased (10 mg / mL), which expanded the range of magnetic bead DNA system to regulate CTAB; even with the addition of 300 μΜ CTAB, the test strip showed no obvious signal; the addition of lower concentrations of MC-LR still produced a visible band on the T-line; the addition of 400 μΜ and 500 μΜ CTAB without any target molecules showed that the MB@dsDNA used could not fully bind CTAB. After considering the signal intensity and detection sensitivity, 300 μΜ was finally selected as the actual CTAB concentration used in the determination (see Figure 14 ).

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

[0118] The magnetic beads (MB@dsDNA) fixed with long-chain DNA containing double-stranded regions are cut for 60 minutes using different final concentrations of 100 nM, 150 nM, 200 nM, 250 nM and 300 nM of Cas12a protein (the mass ratio of Cas12a and crRNA is kept at 1:1); then the ratio of Cas12a to crRNA is adjusted according to the optimized Cas12a concentration (1:1, 1:1.5 and 1:2); the reaction time should be controlled within 60 minutes; the cutting time of CRISPR-Cas12a is also studied; after adding the MC-LR solution, the time is controlled at 30 minutes, 45 minutes, 60 minutes, 90 minutes and 120 minutes, respectively; for the optimization of the above-mentioned CRISPR-Cas12a cutting, the temperature should be kept at 37℃, and the total volume of the reaction solution should be kept at 200 μL; the gray value of the detection line of the pregnancy test paper is 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 parameter is changed, the other parameters remain unchanged, and the test is carried out according to the above-mentioned MC-LR detection method).

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

[0120] Third, the MC-LR detection method constructed by the application is selectively tested

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

[0122] Secondly, 10 μL of each concentration of each substance is taken, and the steps are operated according to the steps described in the section “I, construction of MC-LR detection method in water body” above, and finally the gray value of the detection line region of the test paper strip is read; each substance and each concentration sample needs to be determined in triplicate;

[0123] Finally, the gray value is plotted against different concentrations and different types of substance samples to obtain a three-dimensional statistical chart, as shown in Figure 16 .

[0124] In addition, 1 μg / L MC-LR (target), 10 μg / L MC-YR and MC-RR (both are MC-LR analogues) and 100 μg / L kanamycin, 100 μg / L tetracycline hydrochloride and 100 μg / L lipopolysaccharide (common water pollutants) were selected for plotting, and the results are shown in Figure 17 Fig. 1, which intuitively proves the high selectivity of the present application to MC-LR.

[0125] IV. Real environment sample test

[0126] Water samples were taken from different environments, and all the water samples were tested by the high performance liquid chromatography method recorded in GB / T 20466-2006 "Determination of Microcystins in Water" before being tested by the method, and no MC-LR was found. Then, MC-LR standard was added to prepare standard samples with different concentrations of 1.00 μg / L, 10 μg / L and 100 μg / L, and the MC-LR detection method constructed above was used for testing, and the results are shown in Table 3, and the recovery rate data are plotted as shown in Figure 18 Fig. 2.

[0127] Table 3 Test results of different samples

[0128]

[0129] From the above table, it can be seen that the concentration obtained by detecting different samples by the method of the present application is not much different from the actual sample concentration, which indicates that the MC-LR detection method constructed by the present application has high accuracy.

[0130] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A test reagent or test kit for MC-LR in a water body, characterized in that, The kit comprises a CRISPR-Cas12a sensor, a long chain containing partial hybrid double strands, a cationic surfactant or a solution thereof, a HCG loading reagent, and a HCG detection reagent, respectively independently packaged; the CRISPR-Cas12a sensor comprises Cas12a, crRNA, Cas12a activation chain, and MC-LR aptamer; wherein the sequence of the crRNA is shown as SEQ ID NO: 1; the sequence of the Cas12a activation chain is shown as SEQ ID NO: 2; the sequence of the MC-LR aptamer is shown as SEQ ID NO: 3; in the long chain containing partial hybrid double strands, the sequence of the long single strand is shown as SEQ ID NO: 4, and the sequence of the short single strand is shown as SEQ ID NO: 5; in the long chain containing partial hybrid double strands, a magnetic bead is fixedly connected; the HCG loading reagent is HCG-loaded calcite aragonite; and the cationic surfactant is CTAB.

2. The detection reagent or detection kit according to claim 1, characterized in that The detection reagent or the kit satisfies one or more of the following conditions: (d) The detection reagent or the kit further comprises a water sample pretreatment reagent; (f) The HCG detection reagent is a pregnancy test paper.

3. The detection reagent or detection kit according to claim 1 or 2, characterized in that, In the long chain containing partial hybrid double strands, the feeding molar ratio of the long chain to the short chain is 1:

4.

4. The detection reagent or detection kit according to claim 1 or 2, characterized in that The preparation method of the HCG-loaded calcite aragonite comprises the following steps: mixing a CaCl2 solution with a HCG solution, ultrasonicating, then quickly adding a Na2CO3 solution to react to obtain a reaction product; washing the reaction product, dissolving in ethanol, and drying to obtain the HCG-loaded calcite aragonite; wherein the HCG solution is prepared by Tris-HCl.

5. A method of detecting MC-LR in a water body, characterized in that, The kit comprises the detection reagent or the kit according to any one of claims 1 to 4.

6. The method of claim 5, wherein The kit comprises: (1) mixing the CRISPR-Cas12a sensor with a water sample to be detected to obtain a mixed solution 1; mixing the long chain containing partial hybrid double strands and the cationic surfactant or the solution thereof to obtain a mixed solution 2; (2) mixing and reacting the mixed solution 1 and the mixed solution 2 to obtain a reaction product; (3) separating the reaction product to obtain a supernatant 1; mixing the supernatant 1 with the HCG loading reagent, and centrifuging to obtain a supernatant 2; (4) testing the HCG content in the supernatant 2 by the HCG detection reagent, so as to qualitatively or quantitatively detect MC-LR in the water body.

7. The method of claim 6, wherein, The HCG detection reagent is a pregnancy test paper, and in step (4), the pregnancy test paper is used to detect the supernatant 2, and qualitative or quantitative analysis is performed according to the condition of the detection line.

8. The method of claim 7, wherein, The quantitative analysis according to the condition of the detection line comprises: (i) taking a photo of the detection line of the test paper strip after the detection line appears, and obtaining the gray value of the detection line region of the test paper strip; (ii) obtaining the MC-LR concentration in the water sample to be detected according to the working curve of the gray value of the detection line region of the test paper strip and the standard MC-LR concentration.

9. The method according to any one of claims 6 to 8, characterized in that, The MC-LR in the water sample to be detected is enriched before being mixed with the CRISPR-Cas12a sensor.

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