CRISPR / Cas12a-responsive AIE MOF nano-enzyme hydrogel dual-mode biosensor as well as construction method and application of CRISPR / Cas12a-responsive AIE MOF nano-enzyme hydrogel dual-mode biosensor

Abstract

The invention discloses a CRISPR / Cas12a responsive AIE MOF nano-enzyme hydrogel dual-mode biosensor as well as a construction method and application thereof, and belongs to the technical field of food safety rapid detection. In order to solve the technical problems that an existing salmonella detection method is tedious in operation, low in detection speed, dependent on special equipment and professional operators, prone to being polluted by aerosol, high in price and the like, a difunctional AIE MOF nano material with AIE fluorescence performance and POD activity is packaged into DNA hydrogel and is further combined with a CRISPR / Cas12a system, and the detection sensitivity of salmonella is improved. A biosensor for detecting salmonella is constructed, and a high-sensitivity, accurate and rapid method is provided for detecting salmonella in food safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food safety rapid detection, and particularly relates to a CRISPR / Cas12a responsive AIE MOF nanozyme hydrogel dual-mode biosensor and a construction method and application thereof. BACKGROUND

[0002] Salmonella is a zoonotic pathogen existing in the environment and food, and is one of the main causes of food poisoning. According to the report of the World Health Organization, about 25% of foodborne diseases are caused by Salmonella each year. Salmonella infection can cause various diseases such as diarrhea, gastroenteritis and septicemia, which seriously threatens human life and health. Moreover, Salmonella can be rapidly transmitted through river drinking water and food. Even a small amount of Salmonella can rapidly multiply in contaminated milk, vegetables, chicken and other foods, causing humans or animals to become ill. As one of the most contagious and dangerous foodborne pathogens, Salmonella seriously threatens dairy product safety and meat product safety. International and Chinese food standards clearly stipulate that Salmonella is not allowed to be detected in food. Therefore, developing an efficient and sensitive Salmonella detection technology is of great significance to ensure food safety and prevent outbreaks of foodborne diseases caused by Salmonella.

[0003] Although plate counting based on culture is still the "gold standard" for Salmonella detection, its cumbersome operation and long detection period (usually 2 to 7 days) limit its application in rapid detection. Nucleic acid-based methods (such as polymerase chain reaction (PCR) and real-time fluorescent quantitative PCR (qPCR)) can detect Salmonella within a few hours. However, they usually rely on specialized equipment and operators, and are susceptible to aerosol contamination, leading to false positive results. In addition, enzyme-linked immunosorbent assay (ELISA) provides specific and rapid detection results, but the preparation of antibodies is time-consuming and expensive, limiting its further application. Therefore, how to simply, quickly and sensitively detect Salmonella in food is still a challenge.

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein (Cas) (CRISPR / Cas) system-based biosensing technology shows great potential in the field of analytical science due to its precise targeting of nucleic acids. Among them, based on the significant trans-cleavage activity of Cas12a protein, many CRISPR / Cas12a-based biosensors have been developed and used for rapid and accurate detection of pathogenic bacteria. As a programmable molecular component, the CRISPR / Cas12a system not only can accurately recognize a variety of nucleic acid targets, but also can complete the amplification and transduction of sensing signals. Compared with other analytical methods, CRISPR / Cas12a system-mediated biosensors have the advantages of accuracy, flexibility, simplicity, low cost, etc.

[0005] Enhanced luminescent materials have considerable potential in the design and construction of CRISPR / Cas12a-based biosensors. Notably, aggregation-induced emission (AIEgens) exhibits high aggregation emission, low background noise, and excellent photostability in the aggregated state, making it a particularly attractive luminescent nanoprobe for biosensors. In addition, tetraphenylacetylene (TPE) and its derivatives have been widely used as ideal ligands for constructing fluorescent metal-organic framework (MOF) and covalent organic framework (COF) materials. In contrast to aggregation-caused quenching (ACQ) fluorophores, AIEgens exhibit enhanced emission in the aggregated state. In addition, MOFs have a rigid state anchored by metal ions, which highly corresponds to the luminescence mechanism of AIEgens. In the framework materials (such as MOF and COF), AIEgens can be restricted by covalent / coordination bonds, which means that intramolecular vibrations or rotations are restricted, thereby forming a strongly fluorescent metal-AIEgens framework. Importantly, AIE MOFs designed using AIEgens as ligands can overcome the limitations of aggregation-caused quenching (ACQ) effects and low quantum yield (QY) of traditional luminescent materials. The above advantages make AIE MOFs promising candidates for multifunctional fluorescent probes. Therefore, the integration of AIE MOFs into the CRISPR / Cas12a biosensing system can effectively enhance the performance of CRISPR / Cas12a biosensors.

[0006] DNA hydrogel is a three-dimensional (3D) network structure formed by DNA as a structural skeleton or crosslinking agent, with programmable, recognition, stimulus response, biocompatibility, and adjustable functional characteristics. In addition, the porous network structure of DNA hydrogel can effectively encapsulate various signal molecules (such as enzymes, nanomaterials and drugs) to provide additional target recognition, catalytic activity and therapeutic potential, thereby endowing DNA hydrogel with great potential in the field of biosensors and biomedical analysis. With the efficient trans -cleavage activity of Cas12a protein, DNA hydrogel can be quickly cleaved during the detection process, leading to rapid release of signals. Therefore, the integration of DNA hydrogel encapsulating AIE MOF with the CRISPR / Cas12a system is a promising strategy to improve the performance of biosensors, which can achieve rapid and sensitive detection of Salmonella. SUMMARY

[0007] To solve the technical problems of existing Salmonella detection methods, such as complicated operation, slow detection speed, dependence on special equipment and professional operators, susceptibility to aerosol pollution, and high price, the present application provides a CRISPR / Cas12a-responsive AIE MOF bifunctional nanoenzyme hydrogel biosensor, a construction method thereof, and an application thereof in rapid detection of Salmonella. Specifically, an AIE MOF nanoenzyme with fluorescence and peroxidase activity is used as a signal probe, and the AIE MOF nanoenzyme is further encapsulated in a DNA hydrogel, combined with the high-efficiency non-specific ssDNA trans-cleavage activity of CRISPR / Cas12a, to construct a CRISPR / Cas12a-responsive DNA biosensor. In this biosensor, the ssDNA cleavage activity of activated CRISPR / Cas12a causes changes in the physical and chemical properties of the DNA hydrogel, resulting in the release of the loaded AIE MOF nanoenzyme. As a proof-of-concept application, the biosensor is used for detection of the invA gene of Salmonella. In the presence of the target, the trans-cleavage activity of CRISPR / Cas12a can be activated. Subsequently, the activated CRISPR / Cas12a can cleave the ssDNA network of the DNA hydrogel, resulting in the release of the loaded AIE MOF nanoenzyme into the reaction medium. The fluorescence properties and POD activity of the AIE MOF nanoenzyme contribute to the output of fluorescence and colorimetric signals, respectively. The degree of change in the fluorescence / colorimetric signal is proportional to the target concentration. Conversely, the absence of the target prevents the activation of CRISPR / Cas12a, and the DNA hydrogel remains intact, preventing the release of the AIE MOF nanoenzyme into the reaction medium, so the resulting signal response is negligible. The DNA hydrogel has high porosity and high transparency, and the AIE MOF nanoenzyme is loaded as a biosensing module in the hydrogel, enhancing the operability and stability. Due to the enhanced fluorescence properties of the AIE MOF nanoenzyme, the formation and collapse of the DNA hydrogel structure are characterized in real time. Importantly, the precise recognition and strong ssDNA cleavage activity of the CRISPR / Cas12a system, as well as the excellent fluorescence and POD activity of the AIE MOF nanoenzyme, improve the detection sensitivity and specificity of the CRISPR / Cas12a biosensor.

[0008] To solve the above technical problems and achieve the corresponding technical effects, the present application provides the following technical solutions: The first object of the present application is to provide a CRISPR / Cas12a-responsive AIE MOF nanoenzyme hydrogel dual-mode biosensor, which comprises a DNA hydrogel loaded with AIE MOF nanoenzymes and a CRISPR / Cas12a detection system for Salmonella. The preparation method of the DNA hydrogel loaded with the AIE MOF nanoscale enzyme is as follows: 23 mg of anhydrous ZrCl4, 23 mg of TCPE and 2-5 mg of hematin are dissolved in 6 mL of DMF, 230 mg of PVP and 100 muL of H2O are added, heated at 120 DEG C for 6-12 h, centrifuged to collect the precipitate after cooling to room temperature, washed with DMF and anhydrous ethanol for three times, dried to obtain the AIE MOF nanoscale enzyme; 10 muL of 100 muM primer DNA is mixed with 5 muL of 100 muM linear DNA, heated at 95 DEG C for 5 min, cooled to room temperature at a speed of 1 DEG C / min, 3 muL of 10x T4 DNA ligase reaction buffer and 2 muL of 400 U / muL T4 DNA ligase are added, incubated at 25 DEG C for 30 min, and then inactivated at 65 DEG C for 10 min to form a circular DNA; 1.25 muL of the circular DNA is mixed with 10.6 muL of TE buffer, 1.5 muL of 10x phi29 reaction buffer, 1.5 muL of 10 mM dNTPs, 0.15 muL of 10 U / muL phi29 DNA polymerase and AIE MOF nanoscale enzyme to form a mixed solution, and reacted at 37 DEG C for 3 h to form a DNA hydrogel loaded with the AIE MOF nanoscale enzyme; the concentration of the AIE MOF nanoscale enzyme in the mixed solution is 1-10 mg / mL; The CRISPR / Cas12a detection system is composed of Cas12a protein, cleavage buffer, crRNA, Salmonella RPA amplification product and enzyme-free water; the nucleotide sequence of the crRNA is shown in SEQ ID NO. 5.

[0009] In an embodiment of the present application, the biosensor further comprises an RPA amplification reagent for Salmonella detection; the RPA amplification reagent comprises RPA primers with nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0010] A second object of the present application is to provide a construction method of the above-mentioned CRISPR / Cas12a responsive AIE MOF nanoscale enzyme hydrogel dual-mode biosensor, which comprises the following steps: S1, preparing AIE MOF nanoscale enzyme: 23 mg of anhydrous ZrCl4, 23 mg of TCPE and 2-5 mg of hematin are dissolved in 6 mL of DMF, 230 mg of PVP and 100 muL of H2O are added, heated at 120 DEG C for 6-12 h, centrifuged to collect the precipitate after cooling to room temperature, washed with DMF and anhydrous ethanol for three times, dried to obtain the AIE MOF nanoscale enzyme; S2, preparing the DNA hydrogel loaded with AIE MOF nanosomes: 10 μL of 100 μM primer DNA was mixed with 5 μL of 100 μM linear DNA, heated at 95°C for 5 min, cooled to room temperature at a rate of 1°C / min, 3 μL of 10×T4 DNA ligase reaction buffer and 2 μL of 400 U / μL T4 DNA ligase were added, incubated at 25°C for 30 min, and then inactivated at 65°C for 10 min to form circular DNA; 1.25 μL of the circular DNA was mixed with 10.6 μL of TE buffer, 1.5 μL of 10×phi29 reaction buffer, 1.5 μL of 10 mM dNTPs, 0.15 μL of 10 U / μL phi29 DNA polymerase and AIE MOF nanosomes to form a mixed solution, which was reacted at 37°C for 3 h to form a DNA hydrogel loaded with AIE MOF nanosomes; the concentration of AIE MOF nanosomes in the mixed solution was 1-10 mg / mL; S3, 2 μL of Cas12a protein, 2 μL of cleavage buffer, 2 μL of crRNA, 2 μL of Salmonella RPA amplification product and 2 μL of enzyme-free water were mixed uniformly and incubated for 10 min to obtain a CRISPR / Cas12a complex, the CRISPR / Cas12a complex was mixed with the DNA hydrogel loaded with AIE MOF nanosomes obtained in S2 according to a volume ratio of 1:2, and incubated at 30-39°C for 10-90 min.

[0011] In an embodiment of the present application, the amount of hematin added in S1 is 4 mg.

[0012] In an embodiment of the present application, the concentration of AIE MOF nanosomes in the mixed solution in S2 is 4 mg / mL.

[0013] In an embodiment of the present application, the concentration of Cas12a protein in S3 is 1-7 μM, and the final concentration of crRNA is 100-500 nM.

[0014] Preferably, the concentration of Cas12a protein in S3 is 4 μM.

[0015] Preferably, the incubation time in S3 is 70 min.

[0016] In an embodiment of the present application, the concentration of Cas12a protein in S3 is 4 μM.

[0017] A third object of the present application is to provide a method for detecting Salmonella using the above-mentioned CRISPR / Cas12a-responsive AIE MOF nanoszyme hydrogel dual-mode biosensor, which comprises the following steps: 1) Extracting the genomic DNA of the sample to be detected, and obtaining the Salmonella RPA amplification product using the RPA amplification kit; 2) Mixing the Cas12a protein, the cutting buffer, the crRNA, the Salmonella RPA amplification product and the enzyme-free water, and incubating to obtain the crRNA / Cas12a complex; 3) Mixing the crRNA / Cas12a complex and the DNA hydrogel loaded with AIE MOF nanoszyme according to a volume ratio of 1:2, incubating for 10-90 min, and then performing fluorescence detection or colorimetric detection on the reaction system; for fluorescence detection, the supernatant of the reaction system is diluted by 10 times, and then the fluorescence intensity is measured under excitation light of 280-340 nm, and the Molecular Devices SpectraMax i3x is used for quantification; for colorimetric detection, the supernatant of the reaction system is mixed with equal volume of TMB and H2O2 to perform colorimetric reaction, and the absorbance of the reaction system is measured at 652 nm using the ultraviolet spectrophotometer for quantitative detection.

[0018] A fourth object of the present application is to provide the application of the above-mentioned CRISPR / Cas12a-responsive AIE MOF nanoszyme hydrogel dual-mode biosensor in detecting Salmonella contamination in food or food processing.

[0019] A fifth object of the present application is to provide the application of the above-mentioned method in detecting Salmonella contamination in food or food processing.

[0020] The beneficial effects of the present application are: The present application selects TCPE as the AIEgens linker to prepare AIE MOF nanoszyme. At the same time, further compounding hemin gives the AIE MOF nanoszyme activity. The AIE MOF nanoszyme prepared by the method not only has enhanced fluorescence performance, but also has peroxidase-like activity, giving the nanomaterial multifunctional effect. Using the nanomaterial as a detection nanoprobe, different properties can promote different signal outputs, and multiple signals can verify each other, further improving the accuracy of detection.

[0021] The application loads AIE MOF nanoszyme through DNA hydrogel. The DNA hydrogel provides a platform for the application of AIE MOF nanoszyme, and provides a simpler method for preparing probes. During detection, the phase change of the hydrogel can be converted into the concentration of AIE MOF nanoszyme in the solution, and real-time observation of the structure collapse of the hydrogel makes it possible for naked-eye detection. In addition, the fluorescence performance of AIE MOF nanoszyme also provides a method for real-time monitoring of the detection process.

[0022] The application develops a CRISPR / Cas12a responsive DNA hydrogel dual-mode biosensor for the detection of Salmonella, and optimizes the preparation of AIE MOF nanoszyme, the preparation of DNA hydrogel loaded with AIE MOF nanoszyme and the CRISPR / Cas12a detection process, and adopts fluorescence and colorimetric signals as signal output methods. The detection limit of the detection method for S. typhimurium liquid and commercially available artificially contaminated food samples under pure culture conditions can reach 1 CFU / mL. Compared with other traditional fluorescence and colorimetric detection methods, the detection limit is lower, and there is no cross reaction with 6 strains of non-S. typhimurium strains such as Staphylococcus aureus, Enterobacter cloacae, Escherichia coli, Listeria monocytogenes, Cronobacter sakazakii and Shigella flexneri, which can effectively avoid false positive results.

[0023] The application provides a rapid, simple, high-sensitivity and high-specificity method for detecting S. typhimurium in food samples. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a schematic diagram of the technical route and detection principle of the application; Figure 2 It is a result graph of the influence of different amounts of hematin added in the synthesis of AIE MOF nanoszyme on peroxidase-like activity; Figure 3 It is a result graph of the influence of different amounts of hematin added in the synthesis of AIE MOF nanoszyme on fluorescence intensity; Figure 4 It is a result graph of the optimization of the excitation wavelength of AIE MOF nanoszyme; Figure 5 It is a result graph of the peroxidase-like activity evaluation of AIE MOF nanoszyme; wherein, Figure 5 a in the above formula is the Michaelis-Menten curve graph of AIE MOF nanoszyme to TMB, Figure 5 b in the above formula is the Lineweaver-Burk graph of AIE MOF nanoszyme to TMB; Figure 6 It is a result graph of the thermal stability evaluation of AIE MOF nanoszyme; wherein, Figure 6Fig. 1a is a graph of the thermal stability evaluation results of the peroxidase-like activity of the AIE MOF nanoscale enzyme, Figure 6 Fig. 1b is a graph of the thermal stability evaluation results of the fluorescence performance of the AIE MOF nanoscale enzyme; Figure 7 Fig. 2 is a graph of the storage stability evaluation results of the AIE MOD nanoscale enzyme; wherein, Figure 7 Fig. 2a is a graph of the results of the peroxidase-like activity of the AIE MOF nanoscale enzyme changing with storage time, Figure 7 Fig. 2b is a graph of the results of the fluorescence performance of the AIE MOF nanoscale enzyme changing with storage time; Figure 8 Fig. 3 is a graph of the optimization results of the concentration of the AIE MOF nanoscale enzyme in the DNA hydrogel; the inset is a photograph of the DNA hydrogel loaded with different concentrations of the AIE MOF nanoscale enzyme; Figure 9 Fig. 4 is a graph of the optimization results of the Cas12a concentration; Figure 9 Fig. 4a is a graph of the results of the influence of the Cas12a concentration on the volume of the AIE MOF enzyme hydrogel, Figure 9 Fig. 4b is a graph of the results of the influence of the Cas12a concentration on the fluorescence intensity of the supernatant of the reaction system, Figure 9 Fig. 4c is a graph of the results of the influence of the Cas12a concentration on the colorimetric signal intensity of the supernatant of the reaction system; Figure 10 Fig. 5 is a graph of the optimization results of the Cas12a cleavage time; Figure 10 Fig. 5a is a graph of the results of the influence of the Cas12a cleavage time on the volume of the AIE MOF enzyme hydrogel, Figure 10 Fig. 5b is a graph of the results of the influence of the Cas12a cleavage time on the fluorescence intensity of the supernatant of the reaction system, Figure 10 Fig. 5c is a graph of the results of the influence of the Cas12a cleavage time on the colorimetric signal intensity of the supernatant of the reaction system; Figure 11 Fig. 6 is a graph of the specificity verification results of the DNA hydrogel biosensor responding to CRISPR / Cas12a for detecting Salmonella; wherein, Figure 11 Fig. 6a is a graph of the fluorescence intensity obtained by detecting different strains and negative controls using the DNA hydrogel biosensor responding to CRISPR / Cas12a, Figure 11 Fig. 6b is a graph of the colorimetric signal intensity obtained by detecting different strains and negative controls using the DNA hydrogel biosensor responding to CRISPR / Cas12a; Figure 12 Fig. 7 is a graph of the anti-interference performance evaluation results of the DNA hydrogel biosensor responding to CRISPR / Cas12a for detecting Salmonella; wherein, Figure 12a is a fluorescence intensity chart obtained by using a CRISPR / Cas12a response DNA hydrogel biosensor to detect different mixed bacterial liquids, positive controls and negative controls, Figure 12 b is a colorimetric signal intensity chart obtained by using a CRISPR / Cas12a response DNA hydrogel biosensor to detect different mixed bacterial liquids, positive controls and negative controls; Figure 13 a is a fluorescence signal mode sensitivity evaluation result chart of a CRISPR / Cas12a response DNA hydrogel biosensor for detecting Salmonella, Figure 13 a is a detection limit result chart, Figure 13 b is a linear range result chart; Figure 14 a is a fluorescence signal mode sensitivity evaluation result chart of a CRISPR / Cas12a response DNA hydrogel biosensor for detecting Salmonella, Figure 14 a is a detection limit result chart, Figure 14 b is a linear range result chart; Figure 15 a is a recovery rate and accuracy evaluation result chart of a CRISPR / Cas12a response DNA hydrogel biosensor in detecting artificially contaminated food samples. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments and the drawings of the specification. It should be noted that the following examples are only used to explain the present application, but not to limit the scope of the present application. The following examples are only a part of the embodiments of the present application, not all the embodiments. Those skilled in the art can refer to the content herein to appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present application, to achieve and apply the present application technology. In the art, other technicians will not make creative efforts, and the examples they obtain are protected by the present application.

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and instruments used are conventional materials, reagents and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0027] The reagents used in the present application are as follows: LbCas12a nuclease was purchased from Suzhou Coastal Protein Technology Co., Ltd. phi29 DNA polymerase (10 U / μL), T4 DNA ligase (400 U / μL) and deoxynucleotide triphosphates (dNTPs, 10 mM) were purchased from New England Biolabs. ZrCl4 and polyvinylpyrrolidone (PVP) were purchased from Shanghai Aldrin Biochemical Technology Co., Ltd. in China. 1,1,2,2-tetrakis (4-carboxyphenyl) ethylene (TCPE) and hemin were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. in China. N,N-dimethylformamide (DMF) was purchased from Shanghai Reagent Co., Ltd. in China. Acetic acid (HAc) and H2O2 (30%) were purchased from Tianjin Tianli Chemical Reagent Co., Ltd. Dimethyl sulfoxide (DMSO) was purchased from Beijing Bioda Technology Co., Ltd. 3,3',5,5'-tetramethylbenzidine (TMB) was purchased from Solarbio Technology Co., Ltd. All chemicals used in this study were of analytical grade and did not require further purification unless otherwise stated.

[0028] The strain information used in the present application is shown in Table 1.

[0029] Table 1 Strain information

[0030] The technical principle involved in the present application is as follows: An AIE MOF nanoscale enzyme with fluorescent properties and peroxidase-like activity was used as a signal probe, and the AIE MOF nanoscale enzyme was further encapsulated into a DNA hydrogel, combined with the high-efficiency non-specific ssDNA transcleavage activity of CRISPR / Cas12a, to construct a CRISPR / Cas12a-responsive DNA hydrogel dual-mode biosensor, and the detection principle is as follows: Figure 1The ssDNA cleavage activity of activated CRISPR / Cas12a can cause the physical and chemical properties of the DNA hydrogel to change, resulting in the release of the AIE MOF nanoszyme-loaded. The biosensor is used for the detection of Salmonella. In the presence of Salmonella, the trans-cleavage activity of CRISPR / Cas12a can be activated. Subsequently, the activated CRISPR / Cas12a can cleave the ssDNA network of the DNA hydrogel, resulting in the release of the AIE MOF nanoszyme-loaded into the reaction medium. The maximum excitation wavelength (Ex) of the AIE MOF nanoszyme is 310 nm, and the maximum emission wavelength (Em) of FAM is 470 nm. When the AIE MOF is excited at 310 nm, its fluorescence emission at 470 nm is observed, thereby outputting a fluorescence signal. Here, using TMB and hydrogen peroxide as reaction substrates, the AIE MOF nanoszyme can catalyze the reaction system to produce a color signal, and by detecting its absorbance at 602 nm, a colorimetric signal can be output. The degree of change in fluorescence and colorimetric signals is proportional to the target concentration.

[0031] On the contrary, in the absence of Salmonella, the activation of CRISPR / Cas12a is prevented, and the DNA hydrogel remains intact, resulting in the inability of the AIE MOF nanoszyme to be released into the reaction medium, and thus the signal response produced is negligible. The DNA hydrogel has high porosity and high transparency, and the AIE MOF nanoszyme is loaded in the hydrogel as a biosensing module, enhancing the operability and stability. Due to the enhanced fluorescence properties of the AIE MOF nanoszyme, the formation and collapse of the DNA hydrogel structure are characterized in real time. Importantly, the precise recognition and strong ssDNA cleavage activity of the CRISPR / Cas12a system, as well as the excellent fluorescence and POD activity of the AIE MOF nanoszyme, improve the detection sensitivity and specificity of the CRISPR / Cas12a biosensor.

[0032] Example 1: Construction of CRISPR / Cas12a-responsive AIE MOF nanoszyme hydrogel dual-mode biosensor 1. Extraction and RPA amplification of Salmonella genome The Salmonella in the cryovial was transferred to 2 mL of LB liquid medium at an inoculation amount of 2%, and cultured for 10-12 h to obtain a bacterial solution. The bacterial solution was streaked on LB agar medium plates in three zones, and cultured for 12 h. Single colonies were picked and inoculated in LB liquid medium, and cultured for 10-12 h to obtain bacterial solutions in the logarithmic growth phase, respectively. 1 mL of the bacterial solution was taken, and then the concentration of Salmonella was adjusted to a uniform concentration of 10 0 to 10 8CFU / mL for subsequent experiments. The genomic DNA of Salmonella at different concentrations was extracted by a DNA extraction kit (Tiangen Biotech Co., Ltd.).

[0033] The extracted Salmonella genomic DNA was subjected to RPA amplification by an RPA amplification kit (TwistDx Co., Ltd.). The primers used for RPA amplification were RPA-F and RPA-R, and the nucleotide sequences were shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0034] SEQ ID NO. 1: 5'-ACCCATTTGTATTGGTTGTTACGGCTATTTTG-3'; SEQ ID NO. 2: 5'-GCGGCTGCTCGCCTTTGCTGGTTTTAGGTTTG-3'.

[0035] 2. Preparation and characterization of AIE MOF nanozymes 23 mg of anhydrous ZrCl4, 23 mg of TCPE and hemin were dissolved in 6 mL of DMF, and ultrasonic was used to ensure complete dissolution. Then, 230 mg of PVP (Mw 30000) and 100 μL of H2O were added to the mixed solution. The above mixture was transferred to a 50 mL reaction kettle, and the reaction was heated at 120°C for 10 h. After the reaction was completed, it was cooled to room temperature, and the precipitate produced was collected by centrifugation (8000 rpm, 10 min) and washed with DMF and anhydrous ethanol three times. Then, the obtained yellow-white AIE MOF nanozyme was dried at 60°C, and stored at room temperature.

[0036] To evaluate the fluorescence performance of AIE MOF nanozyme, AIE MOF was dispersed in deionized water, and the concentration was set to 1 mg / mL. The fluorescence emission intensity at 470 nm was measured by a multifunctional enzyme marker.

[0037] To evaluate the peroxidase-like activity of AIE MOF nanozyme, 30 μL of TMB solution (5 mM) and 30 μL of AIE MOF nanozyme (1 mg / mL) were mixed with 30 μL of H2O2 solution (10 mM, 200 mM NaAc-HAc buffer, pH 4.0). The absorbance change of the above reaction system at 602 nm was recorded by a multifunctional enzyme marker to evaluate the peroxidase-like activity of AIE MOF nanozyme.

[0038] 3. Preparation of DNA hydrogel loaded with AIE MOF nanozyme Ten microliters of primer DNA (100 mM) was mixed with 5 microliters of linear DNA (100 mM) and then heated at 95 °C for 5 min. Next, the mixture was cooled to room temperature at a rate of 1 °C per minute. Subsequently, 3 microliters of 10x T4 DNA ligase reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM dithiothreitol, pH 7.5) and 2 microliters of T4 DNA ligase (400 U / microliter) were added to the mixture, and incubated at 25 °C for 30 min to form a circular DNA. Finally, the circular DNA was inactivated at 65 °C for 10 min and stored at -20 °C for later use. The nucleotide sequences of the primer DNA and linear DNA are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0039] SEQ ID NO. 3: 5'-TCGGCTTAGCCTCAACCCCCAPO4-3'; SEQ ID NO. 4: 5'-GCTAAGCCGACATTCTTGCATGGTCACACGTCGTTCTAGTACGCTTCTTTGGGGGTTGAG-3'.

[0040] Preparation of AIE MOF nanozyme-loaded DNA hydrogel: 1.25 microliters of circular DNA was mixed with 10.6 microliters of TE buffer, 1.5 microliters of 10x phi29 reaction buffer, 1.5 microliters of dNTPs (10 mM), 0.15 microliters of phi29 DNA polymerase (10 U / microliter), and 5 microliters of AIE MOF nanozyme solution (1-10 mg / mL) to form a mixture. Then, the mixture was reacted at 37 °C for 3 h to form an AIE MOF nanozyme-loaded DNA hydrogel. The obtained DNA hydrogel was washed three times in ultrapure water and stored at 4 °C for later use.

[0041] 4. Detection procedure of CRISPR / Cas12a-responsive AIE MOF nanozyme hydrogel dual-mode biosensor Cutting reaction: 2 μL Cas12a protein, 2 μL cutting buffer, 2 μL crRNA (350 nM), 2 μL Salmonella RPA amplification product and 2 μL enzyme-free water were mixed uniformly and incubated for 10 min. Subsequently, the mixture was mixed with the pre-prepared AIE MOF nanoscale enzyme DNA hydrogel according to a volume ratio of 1:2, and incubated at 37°C to allow the activated Cas12a protein to cut the single-stranded DNA network in the DNA hydrogel, thereby releasing the loaded AIE MOF nanoscale enzyme. The nucleotide sequence of the crRNA is shown in SEQ ID NO. 5.

[0042] SEQ ID NO. 5: 5'-UAAUUUCUACUAAGUGUAGAUAAAUAGAAGAGUACGCUUAAAAC-3'.

[0043] Fluorescence detection procedure: 10 μL of the supernatant of the cutting reaction system was diluted to 200 μL, and then the fluorescence intensity was measured under excitation light at 310 nm, using Molecular Devices SpectraMax i3x for determination.

[0044] Colorimetric detection procedure: 10 μL of the supernatant of the cutting reaction system was mixed with 10 μL TMB and 10 μL H2O2 for colorimetric reaction. The absorbance of the reaction system was determined at 652 nm using a UV spectrophotometer for quantitative detection.

[0045] Example 2: Optimization of AIE MOF nanoscale enzyme hydrogel dual-mode biosensor responsive to CRISPR / Cas12a 1. Optimization of the amount of hemin added in the synthesis of AIE MOF nanoscale enzyme On the basis of other unchanged conditions, hemin was set as 0 mg, 1 mg, 2 mg, 3 mg, 4 mg and 5 mg, respectively. After the synthesis of AIE MOF nanoscale enzyme, its fluorescence intensity at 470 nm and absorbance at 602 nm after catalyzing TMB color development were determined by a multifunctional enzyme label instrument. The optimal amount of hemin was selected by the size of fluorescence intensity and absorbance. The results are shown in Figure 2 and Figure 3 As the amount of hemin added increased, the effect of AIE MOF nanoscale enzyme on catalyzing TMB color development was better. However, the fluorescence intensity of AIE MOF nanoscale enzyme gradually decreased with the increase of the amount of hemin added. Considering its fluorescence and peroxidase-like activity, 4 mg of hemin was selected for the synthesis of AIE MOF nanoscale enzyme.

[0046] 2. Optimization of the best excitation wavelength of AIE MOF nanoscale enzyme With other conditions remaining constant, the excitation wavelengths of the AIE MOF nanozyme were set to 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, and 380 nm. After triggering the overall reaction, the fluorescence intensity at 470 nm was measured using a multi-functional microplate reader. The optimal excitation wavelength of the AIE MOF nanozyme was selected based on the fluorescence intensity. The results are as follows: Figure 4 As shown, the AIE MOF nanozyme exhibits optimal emission in the range of 280 nm to 340 nm, independent of the excitation wavelength. When the excitation wavelength is further increased to 380 nm, its fluorescence intensity decreases. Therefore, any wavelength within the range of 280 nm to 340 nm can be selected as the excitation wavelength; this invention selects 310 nm as the excitation wavelength for subsequent experiments.

[0047] 3. Evaluation of peroxidase-like activity of AIE MOF nanozymes With other conditions remaining constant, the concentration of TMB was set to 0–2.4 mM. Further, 30 μL of TMB solutions of different concentrations and 30 μL of AIE MOF nanozyme (4 mg / mL) were mixed with 30 μL of H2O2 solution (100 mM, 200 mM NaAc-HAc buffer, pH 4.0). The absorbance changes of the above reaction system at 602 nm were recorded using a multi-mode microplate reader to evaluate the peroxidase-like activity of the AIE MOF nanozyme. The Michaelis-Menten curve of the AIE MOF nanozyme was fitted based on the TMB concentration (the molar absorptivity (ε) of TMB at 652 nm was 39000 M). -1 cm -1 The kinetic constants Km and Vmax of the AIE MOF nanozyme were calculated using the Lineweaver-Burk equation.

[0048]

[0049] In the formula, V is the initial reaction rate, V max C represents the maximum reaction rate, and C is the TMB concentration.

[0050] The results are as follows Figure 5 As shown, typical Michaelis-Menten curves were obtained within the TMB concentration range of 0–2.4 mM. The Lineweaver-Burk fitting curve for the AIE MOF nanozyme was y = 0.0145 × x + 0.0221 (R²). 2 =0.972). The Michaelis constant (Km) and maximum initial velocity (V) of the AIE MOF nanozyme were calculated.max ) were 0.65 mM and 45.49 x 10 -7 M s -1 . Lower Km value and higher V max indicated that AIE MOF nanoszyme had higher substrate affinity and catalytic efficiency.

[0051] 4. Thermal stability evaluation of AIE MOF nanoszyme On the basis of other conditions unchanged, 1 mg / mL of AIE MOF nanoszyme was treated at 25℃, 37℃, 50℃, 60℃, 70℃, 80℃ for 2 h, and the thermal stability was evaluated by measuring the absorbance at 602 nm when AIE MOF catalyzed TMB color development and the fluorescence intensity change of AIE MOF nanoszyme itself. The results are shown in Figure 6 , compared with the natural HRP enzyme, the peroxidase-like activity of AIE MOF nanoszyme decreased slightly, and still had high catalytic activity after 2 h treatment at 80℃. In addition, as the treatment temperature increased, the fluorescence intensity of AIE MOF nanoszyme gradually decreased with the increase of temperature, but still had very high fluorescence intensity when the temperature was 80℃. This showed that the prepared AIE MOF nanoszyme had high thermal stability.

[0052] 5. Storage stability evaluation of AIE MOF nanoszyme On the basis of other conditions unchanged, 1 mg / mL of AIE MOF nanoszyme was stored at 25℃ for 30 days, and the storage stability was evaluated by measuring the absorbance at 602 nm when AIE MOF catalyzed TMB color development and the fluorescence intensity change of AIE MOF nanoszyme itself every 5 days. The results are shown in Figure 7 , compared with the natural HRP enzyme, the peroxidase-like activity of AIE MOF nanoszyme decreased slightly, but still maintained 80% of the initial activity after 30 days. In addition, as the treatment temperature increased, the fluorescence intensity of AIE MOF nanoszyme hardly changed. This showed that the prepared AIE MOF nanoszyme had high storage stability.

[0053] 6. Optimization of AIE MOF nanoszyme concentration in DNA hydrogel On the basis of other conditions unchanged, the concentration of AIE MOF nanoszyme was set to 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL and 10 mg / mL. The optimal AIE MOF nanoszyme loading concentration was selected by detecting the residual fluorescence intensity in the supernatant and observing the fluorescence intensity of the hydrogel. The results are shown in Figure 8As shown in Fig. 8a, with the increase of the concentration of AIE MOF nanoszyme, the volume of the DNA hydrogel decreased. When the concentration of AIE MOF nanoszyme was more than 4 mg / mL, the residual fluorescence intensity in the supernatant was significantly enhanced. Therefore, 4 mg / mL of AIE MOF nanoszyme was selected to prepare the AIE MOF nanoszyme-loaded DNA hydrogel.

[0054] 7. Concentration optimization of Cas12a On the basis of keeping other conditions unchanged, the concentration of Cas12a was set as 0 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, and 7 μM. The optimal concentration of Cas12a was selected by detecting the fluorescence intensity in the supernatant of the reaction system and observing the volume change of the hydrogel. The results are shown in Fig. 6a, 6b and 6c. Figure 9 As shown in Fig. 6a, with the increase of the concentration of Cas12a, the volume of the AIE MOF nanoszyme hydrogel gradually decreased. Moreover, the fluorescence and colorimetric signal intensity gradually increased with the extension of the cutting time, and reached the peak value at 4 μM (Fig. 6b and 6c). Therefore, 4 μM was selected as the optimal concentration of Cas12a. Figure 9 Figure 9 As shown in Fig. 6a, with the increase of the concentration of Cas12a, the volume of the AIE MOF nanoszyme hydrogel gradually decreased. Moreover, the fluorescence and colorimetric signal intensity gradually increased with the extension of the cutting time, and reached the peak value at 4 μM (Fig. 6b and 6c). Therefore, 4 μM was selected as the optimal concentration of Cas12a.

[0055] 8. Optimization of the cutting time of Cas12a On the basis of keeping other conditions unchanged, the cutting time of Cas12a was set as 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, and 90 min. The optimal cutting time of Cas12a was selected by detecting the fluorescence intensity in the supernatant of the reaction system and observing the volume change of the hydrogel. The results are shown in Fig. 7a, 7b and 7c. Figure 10 As shown in Fig. 7a, with the extension of the cutting time, the volume of the AIE MOF nanoszyme hydrogel gradually decreased. In addition, the fluorescence and colorimetric signal intensity gradually increased with the extension of the cutting time, and reached the peak value at 70 min (Fig. 7b and 7c). Therefore, 70 min was the optimal cutting time of Cas12a. Figure 10

[0056] Example 3: Specificity verification of the CRISPR / Cas12a-responsive AIE MOF nanoszyme hydrogel dual-mode biosensor for detecting Salmonella In order to evaluate the specificity of the biosensor and the detection method provided by the present application, 7 strains of common pathogenic microorganisms of different species (see Table 1) were selected for specificity test. Among them, Salmonella typhimurium ATCC 14028 was set as the positive control and the blank sample as the negative control.

[0057] ​​The 7 strains in the cryopreservation tube were transferred to 2 mL of LB liquid medium at an inoculation amount of 2%, and cultured for 10-12 h to obtain bacterial liquid. The bacterial liquid was streaked on LB agar medium plates in three zones, and cultured for 12 h. Single colonies were picked and inoculated into LB liquid medium and cultured for 10-12 h to obtain bacterial liquid in the logarithmic growth phase. 1 mL of bacterial liquid was taken, and the concentration of the strain was adjusted to 10 7 CFU / mL using phosphate buffer according to the results of plate colony counting for subsequent experiments. The genomes of different strains were extracted by a DNA extraction kit (Tiangen Biotech Co., Ltd.). The extracted genomes were subjected to RPA amplification by an RPA amplification kit (TwistDx Co.).

[0058] The above 7 strains were detected according to the method of Example 1 combined with the optimal parameters obtained in Example 2, and the results are shown in Table 2. Figure 11 Among them, the strongest fluorescence and colorimetric signals were detected for Salmonella, which indicates that Salmonella can trigger the detection procedure provided by the application and can be judged as a positive reaction. However, the fluorescence and colorimetric signal intensities obtained by detecting the other 6 strains of non-typhoid Salmonella are almost negligible, which indicates that non-Salmonella cannot start the reaction procedure, and thus presents a negative reaction, which indicates that the constructed sensor can only detect Salmonella and has good specificity.

[0059] Example 4: Anti-interference ability verification of CRISPR / Cas12a-responsive AIE MOF nano-enzyme hydrogel dual-mode biosensor for detecting Salmonella In order to evaluate the anti-interference performance of the biosensor and its detection method provided by the application, 7 common pathogenic microorganisms of different species (see Table 1) were selected for anti-interference test. Among them, Salmonella ATCC 14028 was set as a positive control and a blank sample as a negative control, and a mixed group of 10 6 CFU / mL Salmonella and 10 6 CFU / mL Enterobacter cloacae, 10 6 CFU / mL Escherichia coli, 10 6 CFU / mL Listeria monocytogenes, 10 6 CFU / mL Salmonella and 10 6 CFU / mL Staphylococcus aureus, 10 6 CFU / mL Cronobacter sakazakii, and 10 6 CFU / mL Shigella flexneri mixed group, and 10 6 CFU / mL Salmonella and 10 6 CFU / mL Staphylococcus aureus, 10 6 CFU / mL Enterobacter cloacae, 10 6CFU / mL E. coli, 10 6 CFU / mL L. monocytogenes, 10 6 CFU / mL C. rodentium, and 10 6 CFU / mL S. flexneri mixed group to evaluate the anti-interference performance of the biosensor and detection method provided by the present application.

[0060] The seven strains in the cryopreservation tube were transferred to 2 mL of LB liquid medium at an inoculation amount of 2%, and cultured for 10-12 h to obtain bacterial liquid. The bacterial liquid was streaked on LB agar medium plates in three zones, and cultured for 12 h. Single colonies were picked and inoculated in LB liquid medium and cultured for 10-12 h to obtain bacterial liquid in the logarithmic growth phase. 1 mL of bacterial liquid was taken, and the concentration of the strain was adjusted to a uniform concentration of 10 6 CFU / mL using phosphate buffer according to the results of plate colony counting. Salmonella was mixed with Enterobacter cloacae and E. coli, mixed with Staphylococcus aureus, C. rodentium and S. flexneri, and mixed with Staphylococcus aureus, Enterobacter cloacae, E. coli, L. monocytogenes, C. rodentium and S. flexneri. The concentration of different strains in the mixed system was 10 6 CFU / mL. The genomes of different combination strains were extracted by a DNA extraction kit (Tiangen Biotech Co., Ltd.). The extracted genomes were subjected to RPA amplification by an RPA amplification kit (TwistDx Co., Ltd.).

[0061] The above different mixed bacterial liquids were detected according to the method of Example 1 combined with the optimal parameters obtained in Example 2, and the results are shown in Figure 12 , wherein both the single Salmonella and the mixed system of Salmonella and interfering strains are detected to have strong fluorescence and colorimetric signals, which indicates that the interference of other strains on the detection of Salmonella is very small. The detection of Salmonella using the biosensor provided by the present application will not be interfered by non-Salmonella typhi, which proves that the biosensor and detection method provided by the present application have good anti-interference performance.

[0062] Example 4: Sensitivity evaluation of rapid detection of Salmonella typhi using CRISPR / Cas12a-responsive AIE MOF nanozyme hydrogel dual-mode biosensor Sensitivity determination of Salmonella ATCC 14028 under pure culture conditions: 10 8 CFU / mL of Salmonella ATCC 14028 was used, and the Salmonella ATCC 14028 liquid was diluted by 10 times in gradient, and the bacterial liquid concentration ranged from 10 0 -10 8CFU / mL, the fluorescence and colorimetric signal values of different concentrations of bacteria solution were measured according to the detection method in Example 1 above combined with the optimal parameters obtained in Example 2 to evaluate the sensitivity of the DNA hydrogel dual-mode biosensor responding to CRISPR / Cas12a for detecting Salmonella typhimurium.

[0063] The results are shown in Figure 13 The detection limit of the fluorescence signal mode of the method is 1 CFU / mL, and the linear range is 10 1 ~10 8 CFU / mL. As shown in Figure 14 The detection limit of the colorimetric signal mode of the method is 15.9 CFU / mL, and the linear range is 10 2 ~10 8 CFU / mL.

[0064] Example 5: Accuracy evaluation of the AIE MOF nanoszyme hydrogel dual-mode biosensor responding to CRISPR / Cas12a for rapid detection of Salmonella typhimurium In order to evaluate the applicability of the biosensor and its detection method provided by the present application in complex samples, the biosensor was used to detect Salmonella in artificially contaminated milk, egg and meat samples. For meat samples, 25 g of meat was chopped and mixed with sterile water until the solution became transparent. For egg samples, the yolk was mixed with the albumen and diluted under sterile conditions until the mixture became transparent. The milk sample was directly diluted with sterile technique until the mixture became transparent. Subsequently, the milk, egg and meat sample solutions were mixed with Salmonella to prepare artificially contaminated samples with concentrations of 10 4 , 10 5 and 10 6 CFU / mL, respectively. In addition, samples without the addition of Salmonella were used as negative controls. According to the detection method in Example 1 above and combined with the optimal parameters in Example 2, the fluorescence and colorimetric signal values of different concentrations of artificially contaminated samples were measured, and the recovery rate of the method was calculated to evaluate the accuracy of the method of the DNA hydrogel dual-mode biosensor responding to CRISPR / Cas12a for detecting Salmonella typhimurium.

[0065] As shown in Figure 15As shown, the average recovery rates of the Salmonella-added milk, egg and meat samples were 102%-118%, 89%-93% and 102%-112%, respectively. In addition, the coefficients of variation of the Salmonella-added milk, egg and meat samples were 6%-7% (n=3), 5%-9% (n=3) and 5%-9% (n=3), respectively. Importantly, the number of Salmonella detected by the biosensor in the Salmonella-containing milk, egg and meat samples was basically consistent with the result of qPCR, indicating that the biosensor and detection method provided by the application have high accuracy.

[0066] The content not described in detail in the specification of the present application is the technology known to those skilled in the art. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A CRISPR / Cas12a-responsive AIE MOF nanozyme hydrogel dual-mode biosensor, characterized in that, This includes DNA hydrogels loaded with AIE MOF nanozymes and a CRISPR / Cas12a detection system for Salmonella; The method for preparing the DNA hydrogel loaded with AIE MOF nanozymes is as follows: 23 mg anhydrous ZrCl4, 23 mg TCPE, and 2-5 mg heme were dissolved in 6 mL DMF. 230 mg PVP and 100 μL H2O were added, and the mixture was heated at 120 °C for 6-12 h. After cooling to room temperature, the precipitate was collected by centrifugation, washed three times with DMF and anhydrous ethanol, and dried to obtain AIE MOF nanozyme. 10 μL of 100 μM primer DNA was mixed with 5 μL of 100 μM linear DNA, heated at 95 °C for 5 min, and cooled to room temperature at a rate of 1 °C / min. 3 μL of 10×T4 DNA ligase reaction buffer and 2 μL of 400 U / μL T4 DNA ligase were added, and the mixture was incubated at 25 °C for 30 min, followed by inactivation at 65 °C for 10 min to form circular DNA. 1.25 μL of circular DNA was mixed with 10.6 μL TE buffer, 1.5 μL of 10×Phi29 reaction buffer, and 1.5 μL of... 10 mM dNTPs, 0.15 μL 10 U / μL phi29 DNA polymerase and AIE MOF nanozyme were mixed to form a mixture, and the mixture was reacted at 37°C for 3 h to form a DNA hydrogel loaded with AIE MOF nanozyme; the concentration of AIE MOF nanozyme in the mixture was 1-10 mg / mL. The CRISPR / Cas12a detection system consists of Cas12a protein, cleavage buffer, crRNA, Salmonella RPA amplification product, and enzyme-free water; the nucleotide sequence of crRNA is shown in SEQ ID NO.

5.

2. The CRISPR / Cas12a-responsive AIE MOF nanozyme hydrogel dual-mode biosensor according to claim 1, characterized in that, The biosensor also includes an RPA amplification reagent for Salmonella detection; the RPA amplification reagent includes RPA primers with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. The method for constructing the CRISPR / Cas12a-responsive AIE MOF nanozyme hydrogel dual-mode biosensor according to claim 1, characterized in that, Includes the following steps: S1. Preparation of AIE MOF nanozyme: Dissolve 23 mg of anhydrous ZrCl4, 23 mg of TCPE and 2-5 mg of heme in 6 mL of DMF, add 230 mg of PVP and 100 μL of H2O, heat at 120℃ for 6-12 h, cool to room temperature, centrifuge to collect the precipitate, wash three times with DMF and anhydrous ethanol, and dry to obtain AIE MOF nanozyme; S2. Preparation of DNA hydrogel loaded with AIE MOF nanozyme: Mix 10 μL of 100 μM primer DNA with 5 μL of 100 μM linear DNA, heat at 95°C for 5 min, cool to room temperature at a rate of 1°C / min, add 3 μL of 10×T4 DNA ligase reaction buffer and 2 μL of 400 U / μL T4 DNA ligase, incubate at 25°C for 30 min, and then inactivate at 65°C for 10 min to form circular DNA; mix 1.25 μL of circular DNA with 10.6 μL of TE buffer, 1.5 μL of 10×phi29 reaction buffer, 1.5 μL of 10 mM dNTPs, 0.15 μL of 10 U / μL phi29 DNA polymerase and AIE MOF nanozyme to form a mixture, and react at 37°C for 3 h to form a DNA hydrogel loaded with AIE MOF nanozyme; the AIE in the mixture... The concentration of MOF nanozymes is 1-10 mg / mL; S3. Mix 2 μL Cas12a protein, 2 μL cleavage buffer, 2 μL crRNA, 2 μL Salmonella RPA amplification product and 2 μL enzyme-free water thoroughly and incubate for 10 min to obtain a CRISPR / Cas12a complex. Mix the CRISPR / Cas12a complex with the DNA hydrogel loaded with AIE MOF nanozyme obtained in S2 at a volume ratio of 1:2 and incubate at 30-39°C for 10-90 min.

4. The construction method according to claim 3, characterized in that, The amount of heme added as described in S1 is 4 mg.

5. The construction method according to claim 3, characterized in that, The concentration of AIE MOF nanozyme in the mixture described in S2 is 4 mg / mL.

6. The construction method according to claim 3, characterized in that, The concentration of Cas12a protein in S3 is 1-7 μM, and the final concentration of crRNA is 100-500 nM.

7. The construction method according to claim 6, characterized in that, The concentration of the Cas12a protein in S3 is 4 μM.

8. A method for detecting Salmonella using the CRISPR / Cas12a-responsive AIE MOF nanozyme hydrogel dual-mode biosensor as described in claim 1, characterized in that, Includes the following steps: 1) Extract genomic DNA from the sample to be tested and obtain Salmonella RPA amplification products using an RPA amplification kit; 2) Mix Cas12a protein, cleavage buffer, crRNA, Salmonella RPA amplification product and enzyme-free water, and incubate to obtain crRNA / Cas12a complex; 3) The crRNA / Cas12a complex was mixed with DNA hydrogel loaded with AIE MOF nanozyme at a volume ratio of 1:2 and incubated for 10-90 min. The reaction system was then subjected to fluorescence detection or colorimetric detection. For fluorescence detection, the supernatant of the reaction system was diluted 10 times and the fluorescence intensity was measured under excitation light at 280-340 nm. Quantification was performed using a Molecular Devices SpectraMax i3x. For colorimetric detection, the supernatant of the reaction system was mixed with an equal volume of TMB and H2O2 for colorimetric reaction. The absorbance of the reaction system was measured at 652 nm using a UV spectrophotometer for quantitative detection.

9. The application of the CRISPR / Cas12a-responsive AIE MOF nanozyme hydrogel dual-mode biosensor of claim 1 in the detection of Salmonella contamination in food or food processing.

10. The application of the method of claim 8 in detecting Salmonella contamination in food or food processing.

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