Preparation method of fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material

Abstract

The invention belongs to the technical field of biosensing detection, and discloses a preparation method and application of a fluorescent colorimetric dual-mode sensor based on a CRISPR / Cas12a integrated MXene composite material. CeO2 NPs is loaded on the surface of an MXene-Nb2C material to prepare the difunctional composite material with fluorescence quenching capability and peroxidase-like activity. The activation of a CRISPR / Cas12a system is realized through HCR cyclic amplification, and a fluorescent and colorimetric dual-mode channel is ingeniously constructed. A CRISPR / Cas12a system is used for shearing a DNA single chain marked by a fluorophore FAM, so that the DNA single chain is not adsorbed by the Nb2C / CeO2 NPs material, and the recovery of fluorescence in a fluorescence system and the chromogenic reaction in a colorimetric system are realized. Quantitative analysis of AFB1 is realized according to enhancement of fluorescence intensity and absorbance in a dual-mode system. The dual-mode sensor constructed by the invention is simple and convenient to operate and sensitive and reliable in detection result, the detection limits of a fluorescence mode and a colorimetric mode are 3.61 pg / mL and 0.064 ng / mL respectively, the dual-mode sensor has good anti-interference capability and practical application capability, and a novel biosensing platform is provided for practical sample analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a method for preparing a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material and an application thereof in detecting aflatoxin B1. Background Art

[0002] Mycotoxins are a type of secondary metabolites produced by molds. They are widely distributed in nature and are very common in food contamination. Aflatoxin (AF) is a highly toxic secondary metabolite produced by the fungi Aspergillus flavus and Aspergillus parasiticus. It is reported that aflatoxin is a highly stable natural mycotoxin. Among them, aflatoxin B1 (AFB1) is the most toxic of the aflatoxins and has been classified as a Class I carcinogen by the International Agency for Research on Cancer. AFB1 has varying degrees of harm to the liver, immune system, and reproductive system of humans and some animals. In order to protect public health and food safety, it is of great significance to strengthen the detection of AFB1 in food.

[0003] Existing AFB1 detection methods mainly include chromatographic analysis methods, such as liquid chromatography, liquid chromatography tandem mass spectrometry and gas chromatography tandem mass spectrometry. Although these methods can achieve high-sensitivity detection of AFB1, most of these methods have high requirements for equipment and technical operation, complex sample processing, time-consuming, high cost, etc., and are not suitable for large-scale food safety testing. Therefore, it is extremely important to construct a simple, efficient and rapid detection method to detect AFB1 in food.

[0004] Fluorescent aptamer sensors are based on aptamers for molecular recognition, using fluorescent groups as signal converters to achieve high sensitivity and high selectivity for rapid detection of the target to be detected. Aptamers are molecular probes that can specifically bind to targets. Compared with antibodies, aptamers have the advantages of simple structure, easy synthesis and modification, wide adaptability, easy preparation and storage. The CRISPR / Cas system makes it possible to establish a direct linear positive correlation between fungal toxins and fluorescent signals. The CRISPR-Cas system can achieve binding with matching RNA or DNA by designing specific guide RNA chains, making it an ideal molecular detection tool based on this feature. The CRISPR / Cas12a system is based on the Cas12a enzyme. When the functional DNA encounters the target, the Cas12a enzyme indiscriminately cuts the surrounding single-stranded DNA (ssDNA) probes. In addition, Cas12a has a low intrinsic tolerance to crRNA target DNA mismatches and requires a high degree of complementarity. The sensor is not restricted by the interface space when capturing the target molecule, which significantly improves the recognition efficiency.

[0005] As a new type of two-dimensional transition metal carbides and nitrides, MXene has attracted much attention due to its excellent physical and chemical properties. Due to its rich oxygen or hydroxyl terminal surface and complete metal atomic layer, MXene can interact with most biomolecules through hydrogen bonds, van der Waals forces, electrostatic interactions, coordination bonds, etc., which makes it a unique nanobiological unit in the structure of biosensors. Compared with graphene oxide, MXene has obvious advantages in light absorption from visible light to near-infrared region. MXene can adsorb single-stranded DNA and effectively quench the fluorescence of fluorescent groups.

[0006] Although the dual-mode sensors currently constructed have the advantages of accurate and reliable detection results, most of them rely on certain properties of two or even more complex nanomaterials, which leads to a complex and time-consuming preparation process of the sensor. The combination of multiple nanomaterials with special properties can obtain a new multifunctional material with all their properties, which can simplify the construction process of dual-mode sensors and improve the stability of sensors. CeO2 NPs are excellent candidates for peroxidase mimics. MXene has attracted widespread attention as a new type of two-dimensional nanomaterial with good reducing and fluorescence quenching abilities. Therefore, it can also be used to load CeO2 NPs to prepare MXene / CeO2 NPs composites, which have peroxidase-like activity, adsorption of single-stranded DNA and fluorescence quenching properties.

[0007] The present invention aims at the problems existing in the existing detection technology, such as low sensitivity, poor detection accuracy, unstable detection results, etc., and constructs a new type of fluorescent colorimetric dual-mode sensor. First, THMS is used to specifically identify the target AFB1 to enhance the detection specificity; secondly, the STP-mediated HCR cycle strategy is used to achieve signal amplification and improve the detection sensitivity; then, the magnetic separation technology is used to cleverly separate the reaction system to construct the CRISPR / Cas12a trans-cutting system. Greatly reduce environmental interference, avoid false positive signals, and improve the reliability of the sensor; finally, introduce a bifunctional MXene / CeO2 NPs composite material into the reaction system, which not only realizes the signal response caused by the target, but also simplifies the experimental operation. Utilizing the strong adsorption capacity of MXene for single-stranded DNA and its advantages in visible light absorption, MXene can effectively quench the fluorescence of single-stranded DNA labeled with fluorophores, thereby significantly reducing the background signal. The dual-mode sensing strategy based on the present invention not only overcomes the problems existing in the above prior art, but also improves the sensitivity of the biosensor and reduces the fluorescence background signal of the establishment method. Therefore, the fluorescent biosensor based on CRISPR / Cas12a integrated with MXenes can quickly, sensitively and accurately detect AFB1 in food, and has good application prospects. Summary of the invention

[0008] The main purpose of the present invention is to provide a novel biosensor for detecting the content of AFB1, which can be combined with the HCR amplification strategy and CRISPR / Cas12a trans-cleavage to achieve amplification of the fluorescence signal when detecting AFB1, which is beneficial to reduce the background signal and improve the sensitivity of the sensor.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions: A method for preparing a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material is carried out according to the following steps: (1) Preparation of bifunctional MXene materials: Ce(NO3)3⋅6H2O was dissolved in 100 mL of water-ethylene glycol mixture, the solution was heated to 60°C, and then 16 mL of 25% ammonium hydroxide solution was quickly added. The mixture was stirred vigorously at 60°C, and the mixture first turned black purple and finally yellow. The obtained solid was collected by centrifugation and washed three times with ethanol to remove excess reactants to obtain CeO2 NPs. The MAX phase material was etched with an etching solution to remove the Al element. The material was centrifuged and washed with ultrapure water, the pH was adjusted to neutral, the precipitate was collected, and the two-dimensional layered MXene material was obtained after vacuum freeze drying. The synthesized CeO2 NPs were added, and nanoparticles with peroxidase-like activity were loaded on the surface of the MXene to prepare a bifunctional MXene / CeO2 NPs composite material, which was also used for dual-mode channels.

[0010] (2) Preparation of triplex-lock structure: Take 100 μM aptamer chain and 100 μM primer chain, add 50 mM Tric-HCl buffer solution into a sterile centrifuge tube, anneal, and assemble into a triplex-lock structure. After preparation, store it in a low temperature environment.

[0011] (3) Pretreatment of DNA chains H1 and H2: After H1 and H2 are activated and configured into a 100 μM stock solution, they should be annealed before use to form a hairpin structure. The specific steps are as follows: Take 100 μM H1 and H2 and add them to two sterilized centrifuge tubes, then add 50 mM Tric-HCl buffer solution to each centrifuge tube, anneal the centrifuge tubes at high temperature, and then let them stand and slowly cool to room temperature to obtain the reaction product, and store the reaction product in a low temperature environment.

[0012] (4) Construction of CRISPR / Cas12a trans-cleavage system: crRNA, LbCas12a, and Cas12a buffer (10×) were added to a sterilized centrifuge tube, and the volume was supplemented with buffer solution to make the final concentration of the above substances reach the concentration required for the experiment.

[0013] (5) Construction of dual-mode aptamer sensor: First, a triplex-lock structure must be prepared in advance. The STP is fixed to the surface of the magnetic beads (MBs) through the interaction between the biotin on the primer chain STP and the streptavidin on the surface of the magnetic beads. When the sample contains AFB1, the aptamer chain can specifically bind to AFB1, thereby opening the "lock" structure and exposing the STP in the triplex-lock structure. After magnetic separation, the magnetic beads are washed with magnetic bead washing solution and the supernatant is discarded. H1 and H2 that were annealed in advance in step (3) are added to perform HCR reaction. After the HCR reaction is completed, magnetic separation is performed, the supernatant is discarded, and the magnetic beads are washed with magnetic bead washing solution. Then, the CRISPR / Cas12a trans-cleavage system constructed in step (4) is added, and single-stranded DNA labeled with a fluorescent group is added. Then, the dual-functional material MXene / CeO2 NPs is added to the reaction system to quench the single-stranded DNA that has not been cut. Fluorescence channel: The shredded single-stranded DNA will not be adsorbed to the surface of the material, which will enhance the fluorescence of the reaction system. Colorimetric channel: Hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB) are continued to be added to the reaction system, and the system changes color after a short incubation. According to the increase in fluorescence intensity in the fluorescence channel and the decrease in absorbance in the colorimetric channel after the addition of AFB1, the relationship curve between the fluorescence signal and the colorimetric signal response value and the target AFB1 concentration is established to achieve dual-mode quantitative analysis.

[0014] (6) Application to the detection of food samples: The fluorescence signal of the food sample with unknown concentration is measured in step (5), and the detection result is substituted into the standard curve to obtain the concentration of AFB1 in the sample.

[0015] It is further defined that in step (1), the ratio of the water-ethylene glycol mixture is one of 2:1, 1:1, and 1:2; the stirring time at 60°C is 1 to 4 hours; the etching solution is one or more of hydrofluoric acid, ammonium bifluoride, or a mixture of hydrochloric acid and lithium fluoride; the MAX phase is one of niobium aluminum carbide, titanium aluminum carbide, or titanium silicon carbide; the vacuum freeze-drying time is between 12 and 48 hours, and the temperature is between -80 and -20°C.

[0016] It is further defined that in steps (2) and (3), the annealing temperature is 95°C, the duration is 10 min, and the low-temperature storage environment of the reaction product is 4°C.

[0017] It is further defined that in step (4), the final concentrations of the crRNA and LbCas12a are 100 nM and 200 nM respectively, the Cas12a buffer is 1×, and the volume of the reaction system is 10-20 μL.

[0018] It is further defined that in step (5), the volume of MBs is 1 to 10 μL, and the concentration is 5 to 10 mg / mL; the fluorescent group is one of FAM, ROX, Cy3 or Cy5; the volume of MXene / CeO2 NPs composite material is 1 to 10 μL, and the concentration is 1 to 20 mg / mL; the volume of H2O2 is 1 to 10 μL, and the concentration is 1 to 20 mM / mL; the volume of TMB is 10 to 20 μL, and the concentration is 1 to 20 mM / mL.

[0019] It is further defined that the sequence of the adaptor chain in steps (2), (3), (4) and (5) is 5'-CTC TCT TGC ACGTGT TGT CTC TCT GTG TCT CGT GCT CTC TC -3'; the sequence of the primer chain is 5'-AGA AGA AGGTGT TTA AGT AGA GAG AGA GAG AGA TTT TTT TT-Biotin-3'; the sequence of the crRNA chain is 5'-UAA UUU CUA CUA AGU GUA GAU AAA AAA AAA AAA AAA AAA AA-3'; the sequence of the DNA chain H1 is 5'-AGG GCG GGT GGG TGT TTA AGT TGG AGA ATT GTA CTT AAA CAC CTT CTT CTT GGGTCT TTT TTT TTT TTT TTT TTT TTT TTT-3'; the sequence of the DNA chain H2 is 5'-TTT TTT TTT TTTTTT TTT TTT TTT TGG GTC AAT TCT CCA ACT TAA ACT AGA AGA AGG TGT TTA AGT TGGGTA GGG CGG G -3'; the single-stranded DNA sequence labeled with the fluorescent group is 5'-FAM-ACC AAC CCA ACC TTATTA TT-3' Compared with the prior art, the present invention has the following significant advantages: The present invention utilizes a precisely designed triple-helix molecular switch to skillfully pair the aptamer chain of AFB1 with the primer chain that can initiate the hybridization chain reaction HCR through two pairing methods: base complementary pairing and Hoostein base pairing to form a triplex-lock structure. Compared with the double-helix DNA molecular switch, the triplex-lock structure has the advantages of high sensitivity, good stability, selective retention and original aptamer affinity.

[0020] The present invention combines magnetic separation technology to not only cleverly construct a fluorescence / colorimetric dual-mode channel, but also reduce the interference of background signals and improve detection accuracy and reliability.

[0021] The present invention adopts a hybridization chain reaction (HCR) cycle strategy to achieve signal amplification, improves the activation efficiency of Cas12a, and enhances the sensitivity of the dual-mode sensor.

[0022] The present invention successfully prepares a new bifunctional MXene / CeO2 NPs composite material, and utilizes its fluorescence quenching ability and peroxidase-like activity to realize fluorescence and colorimetric dual-mode detection, which can reduce experimental operations and detection environment interference while improving the reliability of the sensor.

[0023] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of a method for preparing a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material.

[0025] Figure 2 The fluorescence / colorimetric intensity of the sensor constructed in Example 1 of the present invention in the absence of AFB1 (dotted line), the presence of 5 ng / mL AFB1 (dashed line), and the presence of 50 ng / mL AFB1 (solid line), (A) is the fluorescence channel, and (B) is the colorimetric channel.

[0026] Figure 3 The fluorescence (A) and colorimetric (B) standard curves of AFB1 detected by the dual-mode sensor constructed in Example 1 of the present invention.

[0027] Figure 4 This is a comparison chart of the selectivity of the dual-mode aptasensor constructed in Example 1 of the present invention for AFB1 in the presence of other interfering toxins, (A) is the fluorescence channel, and (B) is the colorimetric channel. DETAILED DESCRIPTION

[0028] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Example 1

[0029] A preparation method and application of a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material, the implementation method thereof is as follows Figure 1 shown.

[0030] A method for preparing a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material, comprising the following steps: (1) Preparation of MXene / CeO2 NPs composites: Synthesis of CeO2NPs: Ce(NO3)3⋅6H2O (5.8mmol) was dissolved in 100 mL of water-ethylene glycol mixture (1:1). The solution was heated to 60°C, and then 16 mL of 25% ammonium hydroxide was quickly added and stirred vigorously. After stirring at 60°C for 3 h, the mixture first turned black purple and finally yellow. The obtained solid was collected by centrifugation and washed three times with ethanol to remove excess reactants to obtain CeO2NPs. Synthesis of two-dimensional layered MXene materials: The aluminum (Al) layer of MAX phase Nb2AlC was removed by acid etching in 40% hydrofluoric acid (HF). Briefly, 2 g of Nb2AlC powder was slowly added to 80 mL of HF (40%) solution in batches within 10 min, and then stirred for 24 h in a 60°C water bath and sealed conditions to remove the Al layer. Secondly, the above suspension was centrifuged at 10000 r / min for 10 min, then the supernatant was removed and the precipitate was collected. Again, the precipitate was mixed with 10 mL HF (40%) and ultrasonically treated for 3 h. After ultrasonic treatment, the mixture was centrifuged at 10000 r / min for 10 min, the precipitate was collected, and washed with deionized water until neutral. Finally, the precipitate was frozen in an ultra-low temperature freezer at -80 ° C for 12 h and freeze-dried for 24 h to obtain a two-dimensional layered MXene material. 40 mg Nb2C was dispersed in 40 mL water, ultrasonically treated for 1 h, and 40 mg CeO2 NPs were dispersed in 10 mL water. The two solutions were slowly mixed, and then the mixture was shaken at 600 r / min at room temperature for 24 h. Finally, it was washed three times by centrifugation with deionized water.

[0031] (2) Preparation of triplex-lock structure: Take an aptamer chain with a concentration of 100 μM and a primer chain with a concentration of 100 μM, and add 50 mM Tric-HCl buffer solution to a sterile centrifuge tube for annealing at 95°C for 10 min; then let it stand and slowly cool to room temperature to assemble into a triplex-lock structure, and then store it in a low-temperature environment at 4°C; the volumes of the selected aptamer chain, primer chain, and 50 mM Tric-HCl are 10 μL, 10 μL, and 480 μL, respectively.

[0032] (3) Pretreatment of DNA chains H1 and H2: Centrifuge H1 and H2 at 4000 r / min for 5 min to prevent them from being dispersed. Then open the can lid and add different volumes of sterilized 50 mM Tris-HCl to the tube according to the instructions to prepare a 100 μM mother solution, and vortex to mix thoroughly. The above DNA chains should be annealed before use to form a hairpin structure. The specific steps are as follows: add 5 μL of H1 chain with an initial concentration of 100 μM and 5 μL of H2 chain with an initial concentration of 100 μM to two sterilized centrifuge tubes, then add 495 μL of 50 mM Tris-HCl buffer solution to each centrifuge tube, and vortex to mix thoroughly. Then, anneal the centrifuge tube at high temperature at 95°C for 10 min, then let it stand and slowly cool to room temperature to obtain the reaction product, and store the reaction product in a low temperature environment at 4°C.

[0033] (4) Construction of CRISPR / Cas12a trans-cleavage system: Add 2 μL 1 μM crRNA, 0.4 μL 10 μM LbCas12a, and 2 μL Cas12a buffer (10×) into a sterilized centrifuge tube, and add buffer solution to 20 μL to make the final concentration of the above substances reach 100 nM crRNA, 200 nM LbCas12a, and Cas12a buffer (1×).

[0034] (5) Construction of dual-mode aptamer sensor: 10 μL of 1 mg / mL streptavidin MBs was placed in a 200 μL centrifuge tube and washed three times with 50 μL magnetic bead washing solution. 20 μL of the triplex-lock structure prepared in step (2) was placed in a centrifuge tube containing magnetic beads and incubated at 37°C for 1 h. The triplex-lock structure was fixed to the surface of the magnetic bead MBs by the interaction between the biotin on the primer chain STP and the streptavidin on the surface of the magnetic beads. After the reaction was completed, magnetic separation was performed and the mixture was washed three times with magnetic bead washing solution, and the supernatant was removed. 10 μL of different concentrations of AFB1 was added to the reaction system. The aptamer chain can specifically bind to AFB1, thereby opening the "lock" structure and exposing the primer chain STP in the triplex-lock structure. After magnetic separation, the magnetic beads were washed with magnetic bead washing solution and the supernatant was removed. Add 10 μL of 1 μM H1 and H2 after pre-annealing treatment in step (3) and incubate at 37°C for 1 h. After the HCR reaction is completed, perform magnetic separation, remove the supernatant, and wash the magnetic beads 3 times with magnetic bead washing solution. Then add the CRISPR / Cas12 trans-cutting system constructed in step (4), and mix 10 μL of 1 μmol / L fluorescent group-modified single-stranded DNA with 20 μL, and incubate at 37°C for 30 min. Take the supernatant after magnetic separation and place it in a sterilized centrifuge tube. Add 10 μL of 10mg / mL MXene / CeO2 NPs, a dual-functional material, to the reaction system to quench the single-stranded DNA that has not been sheared. Incubate at 37°C for 30 min, add buffer to make up the volume to 200 μL, and perform fluorescence measurement. Then, 10 μL 15 mmol / L H2O2 and 10 μL 15 mmol / L TMB were added to the reaction system, incubated at 37°C for 10 min to develop color, and the absorbance was measured.

[0035] (6) Establishment of standard curve: 10 μL of AFB1 standard solution of different concentrations was added to step (5) to obtain sample test solutions of different gradients, which were detected by fluorescence spectrophotometer and UV spectrophotometer, respectively. The logarithmic value of AFB1 concentration was used as the horizontal axis and the difference of FAM fluorescence intensity before and after the addition of the standard solution was used as the vertical axis to establish the standard curve of the fluorescence channel. The logarithmic value of AFB1 concentration was used as the horizontal axis and the difference of absorbance before and after the addition of the standard solution was used as the vertical axis to establish the standard curve of the colorimetric channel.

[0036] (7) Application to food sample detection: The fluorescence signal of the food sample with unknown concentration is measured in step (5), and the detection result is substituted into the standard curve to obtain the AFB1 concentration in the sample.

[0037] like Figure 2Shown are the fluorescence (A) and colorimetric (B) intensities of the dual-mode sensor constructed in Example 1 of the present invention in the absence of AFB1 (dotted line), the presence of 5 ng / mL AFB1 (dashed line) and 50 ng / mL AFB1 (solid line).

[0038] like Figure 3 As shown, the fluorescence (A) and colorimetric (B) standard curves of the dual-mode sensor constructed in Example 1 of the present invention for detecting AFB1.

[0039] To further illustrate the present invention, the biosensor prepared by the present invention is used to detect AFB1. Example 2

[0040] A preparation method and application of a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material, comprising the following steps: In order to verify that the prepared fluorescence colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material has specific recognition of AFB1, AFB1 standard was added to 50 mM Tric-HCl buffer to make the concentration of AFB1 in the sample 10 ng / mL; 50 mM Tric-HCl buffer was used to prepare other interfering toxins (AFG1, AFM1, OTA, AFB2, FB1) standard solutions, all with a concentration of 100 ng / mL. The fluorescence signal intensity corresponding to the ordinate is the difference between different targets and blank samples at 520 nm ( Figure 4 A), Figure 4 The absorbance corresponding to the vertical axis B is the difference between the blank sample and the different target substances at 652 nm. The above-mentioned different interfering toxin standards were tested according to the detection system constructed in Example 1. The test results are shown in FIG. Figure 4 As shown, it is shown that the method of the present invention has a higher selectivity for AFB1. Example 3

[0041] A preparation method and application of a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material, comprising the following steps: (1) Actual sample processing: Take 0.5 g of spiked food sample, add it to 10 mL of methanol / water solution (ratio 7:3), place it on a shaker for 20 minutes, then take it out and centrifuge it for 10 minutes at a speed of 10000 r / min. After the centrifugation is completed, take out the supernatant to obtain the food sample extract.

[0042] (2) Sample detection: The fluorescence and colorimetric intensity signals were measured according to the steps in Example 1, and the concentration of AFB1 in the sample was obtained by substituting the standard curve into the standard curve.

[0043] The calculated reproducibility RSD values ​​of the sensor were 2.29% (fluorescence) and 1.37% (colorimetric), and the stability RSD values ​​were 1.16% (fluorescence) and 1.3% (colorimetric), respectively, indicating that the dual-mode sensor has excellent repeatability and reproducibility. It was verified that the prepared dual-mode aptamer sensor has the characteristics of high accuracy, good sensitivity, good reproducibility and stability for the detection of AFB1.

[0044] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0045] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material, characterized in that: The following steps are involved: (1) Preparation of bifunctional MXene materials: Ce(NO3)3⋅6H2O was dissolved in 100 mL of water-ethylene glycol mixture, and the solution was heated to 60°C. Then, 16 mL of 25% ammonium hydroxide solution was quickly added and stirred vigorously at 60°C. The mixture first turned dark purple and finally yellow. The obtained solid was collected by centrifugation and washed three times with ethanol to remove excess reactants to obtain CeO2 NPs. The MAX phase material was etched with an etching solution and vacuum freeze-dried to obtain a two-dimensional layered material MXene with single-stranded DNA adsorption and fluorescence quenching properties. The synthesized CeO2 NPs were added, and nanoparticles with peroxidase-like activity were loaded on the surface of the MXene to prepare a bifunctional MXene / CeO2 NPs composite material, which was also used for dual-mode channels. (2) Fixation of triplex-lock structure: The triplex-lock structure is fixed to the surface of magnetic bead MBs through the interaction between the biotin labeled by the primer chain STP and the streptavidin on the surface of the magnetic beads, and then incubated at a constant temperature; when the sample contains AFB1, the aptamer chain APT can specifically bind to AFB1, thereby opening the "lock" structure and exposing the STP in the triplex-lock structure. The magnetic separation technology is further used to construct a target-induced fluorescence and colorimetric dual-mode channel; (3) Construction of fluorescence / colorimetric dual-mode sensor: After the above mixture is magnetically separated, the pre-annealed DNA hairpins H1 and H2 are added to carry out HCR reaction, and then the CRISPR / Cas12a trans-cleavage system and single-stranded DNA labeled with fluorescent groups are added; MXene / CeO2NPs composite materials are added to the reaction system to quench the single-stranded DNA that has not been sheared; fluorescence channel: the sheared single-stranded DNA will not be adsorbed to the surface of the material, so that the fluorescence of the reaction system is enhanced; colorimetric channel: hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine are continued to be added to the reaction system, and the color of the system changes after a short incubation; based on the increase in fluorescence intensity in the fluorescence channel and the decrease in absorbance in the colorimetric channel after the addition of AFB1, a relationship curve between the fluorescence signal response value and the concentration of the target AFB1 is established to achieve dual-mode quantitative analysis.

2. The method for preparing a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material according to claim 1, characterized in that: In step (1), the ratio of the water-ethylene glycol mixture is one of 2:1, 1:1, and 1:2; the time of vigorous stirring at 60° C. is 1 to 4 h; the etching solution is one or more of hydrofluoric acid, ammonium bifluoride, or a mixture of hydrochloric acid and lithium fluoride; and the MAX phase is one of niobium aluminum carbide, titanium aluminum carbide, or titanium silicon carbide.

3. The method for preparing a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material according to claim 1, characterized in that: In step (2), the volume of the MBs is 1 to 10 μL, and the concentration is 5 to 10 mg / mL; the concentration of the aptamer chain and the primer chain is 0.5 to 2 μmol / L, the volume is 5 to 10 μL, and the constant temperature incubation time is 0.5 to 2 h.

4. The method for preparing a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material according to claim 1, characterized in that: It is further defined that in step (3), the CRISPR / Cas12a trans-cleavage system includes crRNA, LbCas12a, and Cas12a buffer (10×); wherein the final concentrations of crRNA and LbCas12a are 100 nM and 200 nM, respectively, the Cas12a buffer is 1×, the volume of the reaction system is 10 to 20 μL, and the reaction time is 10 to 40 min.

5. The method for preparing a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material according to claim 1, characterized in that: In step (3), the fluorescent group is one of FAM, ROX, Cy3 or Cy5; the volume of the MXene / CeO2 NPs composite material is 1 to 10 μL, and the concentration is 1 to 20 mg / mL; the volume of the hydrogen peroxide is 1 to 10 μL, and the concentration is 1 to 20 mM / mL; the volume of the 3,3',5,5'-tetramethylbenzidine is 10 to 20 μL, and the concentration is 1 to 20 mM / mL.

6. The method for preparing a fluorescent colorimetric dual-mode sensor based on CRISPR / Cas12a integrated MXene composite material according to claim 1, characterized in that: The sequence of the aptamer chain is shown in SEQ ID NO.1: 5'-CTC TCT TGCACG TGT TGT CTC TCT GTG TCT CGT GCT CTC TC -3'; the sequence of the primer chain is shown in SEQ ID NO.2: specifically 5'-AGA AGA AGG TGT TTA AGT AGA GAG AGA GAG AGA TTT TTT TT-Biotin-3'; the sequence of the crRNA chain is shown in SEQ ID NO.3: specifically 5'-UAA UUU CUA CUA AGU GUA GAUAAA AAA AAA AAA AAA AAA AA-3'; the sequence of the DNA hairpin H1 is shown in SEQ ID NO.4: specifically 5'-AGG GCG GGT GGG TGT TTA AGT TGG AGA ATT GTA CTT AAA CAC CTT CTT CTT GGG TCTTTT TTT TTT TTT TTT TTT TTT TTT-3'; the sequence of the DNA hairpin H2 is shown in SEQ ID NO. ID NO.5: specifically 5'-TTT TTT TTT TTT TTT TTT TTT TTT TGG GTC AAT TCT CCA ACT TAA ACT AGA AGAAGG TGT TTA AGT TGG GTA GGG CGG G -3'; the single-stranded DNA sequence labeled with the fluorescent group is shown in SEQ ID NO.6: specifically 5'-FAM-ACC AAC CCA ACC TTA TTA TT-3'.

Images