Colorimetric aptamer sensor and application thereof

Abstract

The invention discloses a colorimetric aptamer sensor and application thereof, and belongs to the technical field of biological detection. The colorimetric aptamer sensor comprises Cas12a protein, HP, crRNA, dsDNA (double-stranded deoxyribonucleic acid) and a colorimetric probe. The colorimetric biosensor is applied to detection of the ochratoxin A, the content of the ochratoxin A is detected through the colorimetric biosensor, and detection and analysis of a target object can be achieved; cas12a aptamer inhibitors are developed as molecular switches for biosensors, which mediate target recognition through engineering modification of HP hairpin sequences. A new thought for improving the mycotoxin detection sensitivity is explored, and good detection potential is expected to be shown in the field of food safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a colorimetric aptamer sensor and application thereof. BACKGROUND

[0002] As toxic compounds produced by fungal metabolic activity, mycotoxins frequently contaminate various agricultural products such as cereals, nuts, fruits, etc. These substances, due to their widespread existence and high tolerance, continuously contaminate food and feed in the whole chain from field production to processing and storage. The damage caused by them not only reflects in the loss of commodity value, but the core risk is to induce complex toxic effects in mammals, including carcinogenicity, teratogenicity and genetic mutation. Among the mycotoxin family, ochratoxin and aflatoxin are both high-risk substances globally regulated. The representative component ochratoxin A (OTA) has been rated as a 2B carcinogen by the International Agency for Research on Cancer (IARC) due to its extremely strong toxicity, widespread contamination and serious health threat. In the face of the outstanding environmental persistence and universal contamination of OTA, China and other major countries and international authoritative organizations have jointly formulated strict food residue limit standards. Under the current regulatory framework in China, the upper limit of OTA residue in products such as cereals, beans, grape wine and coffee is strictly limited to the range of 2 to 10 µg / kg. Therefore, building a precise and reliable OTA detection platform is a key link to ensure food safety and reduce the health risk of the population, and has significant application value.

[0003] Although the traditional detection method of OTA is accurate in quantification, its application promotion is affected by multiple factors: high cost of instrument purchase, complex operation procedure, long detection time, complicated sample pretreatment and high requirement for the professional level of the operator. In view of this, it is of great significance and application prospect to study a new type of mycotoxin detection technology with simple operation, rapid response and low cost. SUMMARY

[0004] In view of the above prior art, the application provides a colorimetric aptamer sensor and application thereof, which solves the problems of high detection cost, long time consumption, complex instrument, and complicated pretreatment process of ochratoxin A in the prior art.

[0005] In order to achieve the above purpose, the technical scheme adopted by the application is to provide a colorimetric aptamer sensor, which comprises Cas12a protein, HP, crRNA, dsDNA and colorimetric probe. The nucleotide sequence of HP is shown in SEQ ID NO. 3, the nucleotide sequence of crRNA is shown in SEQ ID NO. 4, the nucleotide sequence of dsDNA is shown in SEQ ID NO. 5, and the colorimetric probe is MOF@Pt@MOF-DNA-MB.

[0006] On the basis of the above technical scheme, the application can be further improved as follows.

[0007] Further, the preparation method of the colorimetric probe comprises the following steps: (1) synthesis of Fe-MOF: 2-amino terephthalic acid, ferric chloride and glacial acetic acid are dissolved in N,N-dimethylformamide, and Fe-MOF is obtained after heating reaction; (2) preparation of Pt NPs: polyvinylpyrrolidone and chloroplatinic acid hexahydrate are dissolved in deionized water, and sodium borohydride solution is added dropwise under stirring, and the mixture is continuously stirred after turning brown-black, and Pt NPs are obtained; (3) assembly of MOF@Pt@MOF nanoscale enzyme: Fe-MOF@Pt is obtained by mixing and stirring Pt NPs and Fe-MOF; Fe-MOF@Pt is resuspended in N,N-dimethylformamide, and mixed with a solution containing 2-amino terephthalic acid and ferric chloride, and MOF@Pt@MOF nanoscale enzyme is obtained after reaction; (4) preparation of MOF@Pt@MOF-ssDNA: 1-ethyl- (3-dimethylaminopropyl) carbonyldiimidazole and N-hydroxysuccinimide are added in PBS buffer, and Nanozyme-ssDNA is activated; the activated Nanozyme-ssDNA is co-incubated with MOF@Pt@MOF nanoscale enzyme to obtain MOF@Pt@MOF-ssDNA; the nucleotide sequence of Nanozyme-ssDNA is shown in SEQ ID NO. 7; (5) preparation of MB-ssDNA: streptavidin modified magnetic beads are co-incubated with biotinylated MNPs-ssDNA to obtain MB-ssDNA; the nucleotide sequence of MNPs-ssDNA is shown in SEQ ID NO. 8; (6) preparation of colorimetric probe MOF@Pt@MOF-DNA-MB: MOF@Pt@MOF-ssDNA is mixed with MB-ssDNA, and the colorimetric probe MOF@Pt@MOF-DNA-MB is obtained after magnetic separation and purification.

[0008] Further, the amount ratio of 2-amino terephthalic acid, ferric chloride, glacial acetic acid and N,N-dimethylformamide in step (1) is 0.126 g:0.69 mmol:3.45 mmol:15 mL, the temperature of heating reaction is 120 ℃, and the time is 4 h; the amount ratio of deionized water, polyvinylpyrrolidone, chloroplatinic acid hexahydrate and sodium borohydride in step (2) is 45 mL:51.4 mg:0.1 mmol:0.465 mmol, and the solution is stirred for 2 h after it becomes dark brown; the amount ratio of N,N-dimethylformamide, 2-amino terephthalic acid and ferric chloride in step (3) is 12 mL:0.042 g:0.229 mmol, the temperature of heating reaction is 120 ℃, and the time is 8 h; the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in step (4) is 1:1, the amount of Nanozyme-ssDNA added is 0.1 nmol, and the solution is stirred for 24 h at room temperature after the addition of Nanozyme-ssDNA; the incubation time of activated Nanozyme-ssDNA and MOF@Pt@MOF nanoscale enzyme is 24 h; the amount ratio of streptavidin-modified magnetic beads and biotinylated MNPs-ssDNA in step (5) is 100 µL:0.1 nmol, and the co-incubation is carried out at room temperature for 1 h; the volumes of MOF@Pt@MOF-ssDNA and MB-ssDNA are mixed in step (6), the temperature of mixing reaction is 37 ℃, and the time is 60 min.

[0009] Further, the application of the colorimetric aptamer sensor in detecting ochratoxin A.

[0010] Further, the application of the colorimetric aptamer sensor in detecting ochratoxin A.

[0011] Further, the method for detecting ochratoxin A comprises the following steps: (1) mixing 0.6-1.0 µM HP with a sample to be tested, and incubating for 10-40 min; the nucleotide sequence of HP is shown in SEQ ID NO. 3; (2) adding Cas12a protein and crRNA into the reaction system, and the molar ratio of the two is 2:0.5-1, and then incubating for 20-25 min after mixing; the nucleotide sequence of crRNA is shown in SEQ ID NO. 4; (3) adding 400-800 nM dsDNA and 5-10 µL colorimetric probe MOF@Pt@MOF-DNA-MB into the reaction system, and then incubating the mixed solution for 30-60 min; the nucleotide sequence of dsDNA is shown in SEQ ID NO. 5; (4) After the incubation is completed, a magnetic bead separation operation is performed, and the supernatant is collected and washed with PBS buffer three times; (5) The supernatant obtained after the washing in step (4) is mixed with acetic acid-sodium acetate buffer, and H2O2 and a color developing substrate are sequentially added for color developing reaction; after the color developing reaction is terminated, the absorbance value of the solution is measured using an ultraviolet spectrophotometer; Prepare ochratoxin A standard solutions with different concentrations, detect the ochratoxin A standard solutions according to the above steps, record the absorbance generated by each standard solution, and draw a standard curve respectively; detect the absorbance generated by the sample to be tested, and compare with the standard curve to obtain the concentration of ochratoxin A in the sample to be tested.

[0012] Further, the temperature of the mixed incubation in steps (1) and (3) is 37 °C.

[0013] Further, the concentration of HP in step (1) is 0.8 µM, and the incubation time after adding HP and the sample to be tested is 10 min; the molar ratio of Cas12a protein and crRNA in step (2) is 2:0.75, and the incubation time after adding Cas12a protein and crRNA is 20 min; the concentration of dsDNA in step (3) is 600 nM, and the mixed incubation time is 60 min; the molar ratio of H2O2 and color developing substrate in step (5) is 20 µmol:1.005 µmol.

[0014] Further, the color developing substrate is TMB, ABTS or OPD, and the detection wavelength is the absorbance at 650 nm, 420 nm or 450 nm, respectively.

[0015] The present application has the beneficial effects that: the present application constructs a colorimetric sensor for OTA trace detection, the core mechanism of which relies on the efficient cooperation between the CRISPR / Cas12a system and the MOF@Pt@MOF nanometer enzyme, which significantly improves the detection sensitivity. The key design of the sensor is to use aptamer as a high-specificity recognition unit to accurately capture the OTA target molecule, accompanied by significant conformational changes. This conformational conversion eliminates the blocking effect of the aptamer on the crRNA, thereby activating the non-specific nuclease activity of the Cas12a protein, and finally realizing the cleavage of the specific signal reporter molecule. The signal amplification unit innovatively selects MOF@Pt@MOF nanometer enzyme, which is a structure based on MOF-derived carbon templates to fix ultrafine platinum nanocrystals. With the synergistic effect between components, the catalytic ability of the peroxidase simulation is greatly improved, overcoming the performance bottleneck of current nanometer enzyme technology. The functionalized single-stranded DNA is combined with the nanometer enzyme to form the final colorimetric probe. The enzyme cutting of Cas12a on the signal probe releases active nanometer enzyme fragments, which then drive the conversion of the color developing substrate to generate a color response that can be distinguished by the naked eye. This strategy realizes the CRISPR activation and enzyme catalytic detection analysis of OTA, with a detection limit as low as 0.055 ng / mL in the colorimetric mode. This signal mechanism not only guarantees excellent detection sensitivity, but also endows the system with excellent operation stability, result reliability and anti-interference characteristics, maximizing the avoidance of false positive results. This strategy provides an effective solution to break through the bottleneck of mycotoxin analysis, and has important practical value in food safety monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a schematic diagram of the CRISPR-Cas12a colorimetric aptamer sensor using MOF@Pt@MOF nanometer enzyme catalytic signal amplification, and a working mechanism diagram for detecting OTA; wherein A is the preparation process of the colorimetric probe MOF@Pt@MOF-ssDNA-MB, B and C are the working mechanism for detecting OTA; Figure 2 It is an EDS element distribution area scanning diagram of MOF@Pt@MOF; Figure 3 It is a structure characterization of Fe-MOF and MOF@Pt@MOF; wherein A is an XPS full spectrum comparison, B is a C 1s XPS spectrum comparison, C is a Fe 2p XPS spectrum comparison, D is a Pt 4f XPS spectrum comparison, E is a N2 adsorption-desorption isotherm of Fe-MOF, and F is a N2 adsorption-desorption isotherm of Fe-MOF@Pt; Figure 4 It is a structure analysis spectrum of Fe-MOF and MOF@Pt@MOF based on XRD technology; Figure 5MOF@Pt@MOF enzyme-like catalytic characterization; wherein, A-C are the UV-vis spectra of TMB (650 nm), ABTS (420 nm), OPD (450 nm) substrates, and D is the activity comparison of three materials (Fe-MOF, Fe-MOF@Pt, MOF@Pt@MOF) in the TMB / H2O2 system; Figure 6 MOF@Pt@MOF enzyme-like catalytic characterization; wherein, A is the spectral difference between magnetic bead monomers and MOF@Pt@MOF in TMB / H2O2, B and C are the influence curves of MOF@Pt@MOF concentration (0.5-5 mg / L) on TMB color development intensity, and D is the EPR spectrum comparison between MOF@Pt@MOF and the blank group; Figure 7 Steady-state kinetic data of MOF@Pt@MOF catalysis at different concentrations; wherein, A is the reaction rate curve of TMB as substrate, B is the double-reciprocal plot of Figure A, C is the reaction rate curve of H2O2 as substrate, and D is the double-reciprocal plot of Figure C; Figure 8 Steady-state kinetic data of Fe-MOF catalysis at different concentrations; wherein, A is the reaction rate curve of TMB as substrate, B is the double-reciprocal plot of Figure A, C is the reaction rate curve of H2O2 as substrate, and D is the double-reciprocal plot of Figure C; Figure 9 Infrared spectrum characterization (A) and Zeta potential analysis (B) of Fe-MOF and its platinum-modified composite materials; wherein, a is the original MOF@Pt@MOF, b is the Nanozyme-ssDNA modified MOF@Pt@MOF, c is the original MB material, and d is the MB-ssDNA conjugated MB composite; Figure 10 OTA biosensor working mechanism verification; wherein, A is the 10% non-denaturing PAGE characterization of CRISPR / Cas12a system activity, B is the feasibility verification of fluorescence signal output module, C is the catalytic activity change during the hierarchical functionalization of nanozyme composite, and D is the feasibility verification of colorimetric signal output module; Figure 11 Functional analysis of hairpin structure (HP) in CRISPR detection system; wherein, A is the regulation mechanism of HP molecules on system activity, B is the recognition specificity of Cas12a-crRNA-HP complex for activated ssDNA / dsDNA, C is the schematic diagram of HP configuration design variants, and D is the influence law of HP structure difference on fluorescence signal output; Figure 12For the performance optimization path of the biosensor; wherein A is the working concentration of the HP probe (signal conversion unit), B is the binding ratio of Cas12a / crRNA (core enzyme system), C is the dsDNA (activation mechanism) concentration response curve, D is the OTA binding reaction time, and E is the RNP complex maturation time. Figure 13 For the performance verification of the biosensor analysis; wherein A and B are the characteristic absorption spectrum responses of the OTA concentration gradient, C is the dose-signal linear quantitative model at 650 nm, D is the evaluation of biological recognition specificity cross-reaction, and E is the storage stability test of the sensing interface. DETAILED DESCRIPTION

[0017] The specific embodiments of the application will be described in detail below with reference to the examples.

[0018] The nucleotide sequences involved in the examples are shown in Table 1: Table 1 DNA sequence (5'-3')

[0019] Example 1 A colorimetric aptamer sensor coupled with the transcleavage activity of CRISPR / Cas12a and the high-efficiency peroxidase activity of MOF@Pt@MOF nanozyme to realize the detection and analysis of target objects, including Cas12a protein, HP, crRNA, dsDNA, colorimetric probe MOF@Pt@MOF-DNA-MB; the nucleotide sequence of HP is shown as SEQ ID NO. 3, the nucleotide sequence of crRNA is shown as SEQ ID NO. 4, the nucleotide sequence of dsDNA is shown as SEQ ID NO. 5, and the colorimetric probe MOF@Pt@MOF-DNA-MB is prepared by the following steps: (1) Solvothermal synthesis of Fe-MOF: 0.126 g of 2-amino terephthalic acid, 0.69 mmol of iron chloride and 3.45 mmol of glacial acetic acid are mixed and dissolved in 15 mL of N,N-dimethylformamide, and the mixed solution is transferred to a reaction kettle liner, and hydrothermal reaction is carried out at 120°C for 4 hours. After the system returns to room temperature, centrifugal purification is carried out with N,N-dimethylformamide and anhydrous ethanol (8000 rpm x 10 min / time, repeated three times to collect the precipitate), and the precipitate is Fe-MOF material. Then the Fe-MOF material is dispersed in 8 mL of N,N-dimethylformamide to obtain a Fe-MOF solution.

[0020] (2) Preparation of Pt NPs by chemical reduction method: 30 mL of aqueous NaBH4 solution with a concentration of 15.5 mM was added dropwise into the mixed solution under magnetic stirring, during which the solution color gradually changed and finally presented a brown-black color; the mixed solution was uniformly mixed by 45 mL of deionized water, 5 mL of 20 mM chloroplatinic acid hexahydrate solution, and 51.4 mg of polyvinylpyrrolidone. After the dropwise addition was completed, the obtained reaction mixture was then maintained under stirring for 2 hours, and finally a colloidal suspension of Pt NPs was generated and stored at 4°C in the dark for subsequent experiments.

[0021] (3) Layer-by-layer assembly of MOF@Pt@MOF nanoszyme: To construct the MOF@Pt@MOF nanoszyme, first, 8 mL of Fe-MOF solution prepared in step (1) was mixed with 12 mL of Pt NPs colloidal suspension prepared in step (2) under high-speed stirring, and after continuous stirring at room temperature for 2 hours, the obtained Fe-MOF@Pt was separated by centrifugal purification with anhydrous ethanol (8000 rpm x 10 min / time, repeated twice to collect the precipitate). Subsequently, the obtained Fe-MOF@Pt was re-dispersed in 12 mL of N,N-dimethylformamide, and 5 mL of precursor solution containing 0.042 g of 2-amino terephthalic acid and 0.229 mmol of ferric trichloride was added, and reacted at 120°C for 8 hours. Finally, the target nanoszyme MOF@Pt@MOF was collected by washing (5000 rpm x 10 min / time, repeated twice to collect the precipitate), and the obtained MOF@Pt@MOF nanoszyme was dried in an 80°C oven and stored for later use.

[0022] (4) Covalent coupling preparation of MOF@Pt@MOF-ssDNA: The MOF@Pt@MOF-ssDNA complex was resuspended in 1 mL of 0.01 M PBS buffer at pH 7.4 and stored at 4°C for later use.

[0023] The preparation process of the MOF@Pt@MOF-ssDNA complex comprises the following steps: ① Carboxyl activation stage: take 600 μL of PBS buffer solution with pH 7.4, and add 3 mg of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and 3 mg of N-hydroxysuccinimide of equal mass; ② DNA activation: add 100 μL of 1 μM single-stranded DNA solution (i.e. Nanozyme-ssDNA, the nucleotide sequence is shown in SEQ ID No. 7) to the above system, and stir at room temperature for 24 hours to generate activated DNA; ③ Nanoparticle enzyme coupling: prepare a nanoparticle enzyme solution from the MOF@Pt@MOF nanoparticle enzyme obtained in step (3), take 1 mL of 1 mg / mL nanoparticle enzyme solution and mix with the activated DNA, and perform 24 hours of covalent combination under gentle stirring; ④ Purification: remove the free components by centrifugal washing with PBS buffer solution for three times to obtain the final product MOF@Pt@MOF-ssDNA complex.

[0024] (5) Covalent coupling preparation of MB-ssDNA: take 100 μL of streptavidin functionalized magnetic beads (MB) and 1 μM single-stranded DNA solution (i.e. MNPs-ssDNA, the nucleotide sequence is shown in SEQ ID No. 8) respectively, and mix in 1 mL of PBS buffer solution with pH 7.4. After continuous rotation mixing at room temperature for 24 hours, solid-liquid separation is realized by washing with PBS buffer solution for three times, and the obtained product is MB-ssDNA complex. Covalent fixation is realized by incubation at room temperature for 24 hours. Finally, the complex is dispersed in 0.01 M PBS buffer solution with pH 7.4, and is stored at 4°C for standby use.

[0025] (6) Preparation of colorimetric probe MOF@Pt@MOF-ssDNA-MB: mix the MOF@Pt@MOF-ssDNA solution obtained in step (4) and the MB-ssDNA solution prepared in step (5) according to equal volume ratio, and react at 37°C for 60 minutes. After removing the liquid phase of the reaction system by magnetic field separation technology, the precipitate is purified by washing with PBS buffer solution for three times, and finally the colorimetric probe MOF@Pt@MOF-ssDNA-MB is obtained. The colorimetric probe MOF@Pt@MOF-ssDNA-MB is quantitatively characterized by ultraviolet spectrophotometry.

[0026] Example 2 A colorimetric aptamer sensor based on CRISPR / Cas12 coupled MOF@Pt@MOF nanoparticle enzyme for detection of ochratoxin A, comprising the following steps: (1) 6 μL of mixed solution containing 800 nM HP and OTA is added to the reaction system, and incubated at 37°C for 40 minutes; the nucleotide sequence of HP is shown in SEQ ID No. 3; (2) sequentially add 3 µL 10×Reaction Buffer I (containing 500 mM NaCl, 100 mM Tris-Tricine (pH 7.9), 100 mM MgCl2 and 10 mM DTT), 2 µL 1 µM Cas12a protein solution and 0.75 µL 1 µM crRNA solution, and react for 20 minutes; the nucleotide sequence of the crRNA is shown as SEQ ID NO. 4; (3) add 2 µL 600 nM dsDNA and 10 µL colorimetric probe MOF@Pt@MOF-ssDNA-MB, adjust the total reaction volume to 23.75 µL, and continue to react at 37℃ for 1 hour; the nucleotide sequence of the dsDNA is shown as SEQ ID NO. 5; (4) magnetic separation to obtain supernatant and wash with PBS buffer solution for three times; (5) color development stage: take 400 µL 0.1 M acetic acid-sodium acetate solution, add 20 µL 1 M H2O2 solution and 30 µL 33.5 mM TMB solution to start color development, and finally inject 200 µL stop solution to fix the color development signal; the color development system is quantitatively analyzed by ultraviolet spectrophotometry at a wavelength of 650 nm.

[0027] The colorimetric method for OTA quantitative detection based on the transcleavage activity of CRISPR / Cas12a and the excellent peroxidase activity of MOF@Pt@MOF nanoscale enzyme provided by the application has the mechanism as Figure 1The MOF@Pt@DNA-DNA-MB colorimetric probe is constructed based on the principle of base complementary pairing. In the front-end link of recognizing OTA, a composite recognition element (HP) is constructed. The element integrates multiple functional regions: a sequence capable of specifically binding to the target molecule, an aptamer seed region for regulating the activity of Cas12a, a necessary CRISPR-related PAM recognition site, and a support framework for maintaining the overall structural conformation stability. In the absence of the target, the multifunctional probe (HP) can preferentially bind to crRNA due to the PAM module and seed sequence contained therein, thereby hindering the effective pairing of double-stranded DNA and crRNA, resulting in a lower signal response of the system. Conversely, when the target exists, specific binding will induce structural rearrangement of HP, thereby releasing the binding inhibition of crRNA. This allows dsDNA to bind to crRNA again, thereby activating the non-specific transcleavage function of Cas12a protein. After magnetic separation, a large number of free nanoscale enzymes are enriched in the supernatant. At this time, the free nanoscale enzymes can fully exhibit their peroxidase-like activity, and in the coexistence of H2O2 and color developing substrate TMB, they can catalyze the oxidation reaction to induce the solution to show blue color, realizing the visual colorimetric signal output. The application innovatively uses aptamers as sensing and recognition units. After the aptamers bind to specific targets, they undergo conformational changes, which cooperatively trigger the non-specific cleavage function of the CRISPR / Cas12a system and the excellent peroxidase-like catalytic performance of the nanoscale enzyme. The three work together to ultimately realize the visual colorimetric detection of the target mycotoxin. The stability, reliability and operation flexibility of the detection are significantly improved, and the interference of environmental factors is effectively overcome, which opens up an innovative path for significantly improving the detection sensitivity of mycotoxins.

[0028] In order to further analyze the basic composition of the MOF@Pt@MOF composite material and its precursor Fe-MOF, the application systematically uses various characterization techniques. Figure 2 It is clearly shown that the Pt (cyan) element is in a highly uniform dispersion state in the entire MOF@Pt@MOF composite material. At the same time, C (red), N (green), O (blue) and Fe (yellow) elements are also significantly detected in the spectrum, and the coexistence of these key elements strongly supports the successful construction of the target core-shell structure MOF@Pt@MOF. In order to further confirm its chemical composition, Figure 3A comparison shows the XPS full spectra of the precursor Fe-MOF and the composite material MOF@Pt@MOF. The comparison results clearly confirm that MOF@Pt@MOF does indeed contain the five characteristic elements Fe, O, N, C, and Pt. Further fine XPS spectral analysis of the MOF@Pt@MOF composite material reveals its surface chemical state. Its C1s spectrum (… Figure 3 B) can be deconvolved into three characteristic peaks: the peak at 284.8 eV belongs to the C=C bond, the peak at 286.1 eV corresponds to the CO bond, and the peak at 288.5 eV is attributed to the C=O bond. For the Fe 2p spectrum ( Figure 3 C), two distinct broadening peaks can be observed, with binding energies at 710.9 eV (Fe 2p). 3 / 2 ) and 724.4 eV (Fe 2p 1 / 2 This clearly confirms that iron in the material mainly exists in the Fe(III) valence state. Of particular note is the composite material's performance at 71.4 eV (Pt 4f). 7 / 2 ) and 74.7 eV (Pt 4f 5 / 2 Two new characteristic peaks appeared at ( ) Figure 3 D). The appearance of these Pt 4f characteristic peaks not only directly proves that Pt NPs have been successfully loaded onto the Fe-MOF substrate, but also provides key spectroscopic evidence for the final formation of the core-shell structure of MOF@Pt@MOF composite material.

[0029] When evaluating the synthesis effect of MOF@Pt@MOF composites, Figure 4 The comparative analysis of the provided XRD patterns is crucial. The diffraction characteristics of the composite material after synthesis closely match the typical spectra of its core component, Fe-MOF, clearly confirming the preservation of its main crystal structure. The material exhibits five distinct Bragg diffraction responses at specific diffraction angles, precisely corresponding to 2θ = 8.8°, 10.8°, 15.4°, 17.7°, and 19.0°. Indexing analysis clearly identifies these characteristic peaks as originating from coherent scattering of the (002), (101), (103), (200), and (201) crystal plane families. Particularly noteworthy is the high-intensity, sharp morphology of the observed diffraction peaks, an undeniable indication of the highly regular atomic arrangement within the sample, i.e., the presence of a well-developed, long-range ordered crystal structure. The significant features of the XRD patterns—namely, the clear reproduction of the Fe-MOF main structure without impurity phase interference—fully validate the successful preparation of the MOF@Pt@MOF core-shell structure material.

[0030] Results of nitrogen physical adsorption analysis ( Figure 3 E and Figure 3F) The significant evolution of the material's pore structure is clearly revealed. The unmodified Fe-MOF sample exhibits a standard Type I adsorption behavior, which is characterized by a very steep adsorption amount jump step in the low pressure region. This behavior is a hallmark response of microporous materials, and unambiguously confirms that its skeleton is mainly composed of small pores. In sharp contrast, the Fe-MOF@Pt composite material obtained after loading Pt NPs, its adsorption isotherm morphology has undergone a fundamental change, evolving into a Type IV curve with a clear adsorption-desorption branch separation. This isothermal curve transformation from Type I to Type IV is direct experimental evidence for the transformation of the material's internal pore system from a single micropore dominated to a hierarchical pore structure containing significant mesoporous components. This modified hierarchical pore structure has dual functional advantages: first, it significantly improves the transmission efficiency of Pt precursor molecules inside the material and their enrichment at active sites; second, the newly formed mesoporous network physically isolates metal nanoparticles through steric hindrance, effectively inhibiting the sintering phenomenon commonly seen during preparation or use, maintaining the high dispersity and activity of the nanoparticles.

[0031] Example 3 Evaluation of the catalytic performance of nanomaterials: To systematically evaluate the catalytic performance of the prepared MOF@Pt@MOF core-shell composite material, the present application selects three typical color developing substrates: TMB, ABTS and OPD. The detection principle of catalytic activity is based on the color developing reaction of the substrate: in the presence of H2O2, the peroxidase-like nanocatalyst can catalyze the decomposition of H2O2 to produce reactive oxygen species, which in turn efficiently oxidizes these colorless substrate molecules. Their oxidation products respectively present distinct blue (TMB), green (ABTS) and yellow (OPD). By tracking this color change using UV-visible spectrophotometry, the appearance and intensity growth of the characteristic absorption peak can be observed at a specific wavelength, and the corresponding maximum absorption wavelengths are: 650 nm for TMB oxidation products, 420 nm for ABTS oxidation products, and 450 nm for OPD oxidation products. The significant increase in these characteristic absorbance is the direct basis for quantitatively characterizing the catalytic activity of the material's enzyme-like activity.

[0032] UV-Vis spectral analysis results Figure 5A-5C) system reveals the color development mechanism and material activity of the catalytic reaction system. The spectral control experiment clearly shows that when the system only contains isolated hydrogen peroxide or isolated color developing substrate, neither color change of the solution is observed, nor characteristic absorption response beyond the instrument baseline noise is detected at 650 nm, 420 nm and 450 nm. Secondly, when both of them coexist, the signal-to-noise ratio greater than 1 but significantly lower than the effective threshold can be observed at the characteristic wavelength. This sub-stable weak response is derived from the intrinsic instability of H2O2, which undergoes spontaneous homolysis reaction driven by thermodynamics. To accurately evaluate the enzyme-like catalytic performance of MOF@Pt@MOF nanocomposites, three groups of comparative experiments were designed: (1) pure MOF@Pt@MOF dispersion, (2) MOF@Pt@MOF and single substrate (TMB / ABTS / OPD) mixed solution, (3) MOF@Pt@NP@MOF, substrate (TMB / ABTS / OPD) and H2O2 complete reaction system. Key data show that compared with the almost no response baseline of the first two groups, the third group reaction system exhibits sharply enhanced characteristic absorption peaks at the characteristic peaks of TMB, ABTS and OPD, and the absorbance values show an order of magnitude increase. This strong positive catalytic effect confirms that the dual functional active sites in MOF@Pt@MOF can efficiently simulate the catalytic pocket of peroxidase, greatly accelerate the decomposition of H2O2 to produce high concentration of ·OH free radicals, and then efficiently catalyze the substrate oxidation color development reaction.

[0033] By typical color development reaction Figure 5 D): The preliminary Fe-MOF has certain catalytic activity, but only a weak absorbance signal is detected at the characteristic wavelength of 650 nm. However, the catalytic efficiency of Fe-MOF@Pt has been greatly improved due to the loading of Pt NPs on the shell. However, MOF@Pt@MOF realizes a further leap in catalytic activity by virtue of its unique MOF-on-MOF hierarchical structure. This delicate structure not only completely covers the Pt NPs inside, effectively inhibiting their tendency to agglomerate, but also ensures the high stability of the overall activity of the catalyst. The above phenomena and data together confirm the excellent catalytic performance of MOF@Pt@MOF.

[0034] Spectrophotometric analysis data Figure 6 A) clearly confirms that MB does not trigger color development when it is alone, and its absorbance signal at the characteristic wavelength of 650 nm is entirely attributed to the inherent optical properties of its own chromophore. When MB coexists with the TMB+H2O2 reaction system, the detected absorbance change is in the same fluctuation range as the pure TMB+H2O2 system, and this quantitative analysis shows that MB molecules neither promote the oxidation and conversion of TMB nor have detectable peroxidase-like activity.

[0035] To analyze the concentration dependence of the catalytic behavior of the nanoscale enzyme, seven gradient dose sequences (0, 0.5, 1, 2, 3, 4, 5 mg / L, corresponding to Figure 6 B from bottom to top 7 curves) were designed for kinetic response test. The monitoring data ( Figure 6 B-6C) revealed a significant mass effect: as the mass concentration of MOF@Pt@MOF nanoscale enzyme in the reaction system increased, the absorbance response at the characteristic wavelength 650 nm showed a strictly monotonic increasing trend. This linear positive correlation confirmed the stoichiometric law between the number of catalytic active centers and the substrate conversion efficiency, and verified from the experimental level that the nanoscale enzyme followed the classical enzyme kinetics principle - that is, the catalytic efficiency was directly regulated by the concentration of active components. Based on the theoretical assumption of free radical-mediated catalytic pathway, in-situ capture and identification of reaction intermediates were realized by electron paramagnetic resonance spectroscopy. EPR spectrum fingerprint analysis ( Figure 6 D) captured a four-line resonance peak with typical magnetic anisotropy splitting characteristics (intensity ratio 1:2:2:1), which revealed the inherent mechanism of its strong peroxidase-like activity from the molecular level.

[0036] Example 4 Kinetic analysis of nanomaterials: Based on the Michaelis equation, the steady-state kinetic analysis framework systematically revealed the response law of MOF@Pt@MOF and Fe-MOF peroxidase activity to substrate concentration. The experiment observed the characteristic catalytic curve by adjusting the TMB / H2O2 concentration: the initial reaction rate showed super-linear growth with the increase of substrate, and gradually transitioned to zero-order reaction kinetics platform after the critical concentration. To further explore the catalytic performance of MOF@Pt@MOF composite material, we analyzed its reaction kinetics characteristics in the double-substrate system (TMB and H2O2). By determining the corresponding kinetic parameters (Km and Vmax), the experimental data after nonlinear regression analysis was consistent with the classical Michaelis kinetic model. The results showed that the composite material exhibited typical enzyme kinetics behavior in the catalytic process, and its kinetic curve clearly followed the expected law of Michaelis equation, verifying the effectiveness of its simulated enzyme catalytic mechanism. Figure 7 With Figure 8The catalytic performance of MOF@Pt@MOF composite material and single component Fe-MOF was compared. In terms of substrate affinity, MOF@Pt@MOF showed a significant advantage, with a lower Km value, which directly reflected a stronger substrate binding capacity: specifically, for the TMB substrate, the Km value decreased to 0.93 mM; and for the H2O2 substrate, the Km value was only 1.03 mM. At the same time, the catalytic efficiency of the composite material also achieved a significant leap. The determination results of the maximum reaction rate provided strong evidence: in the reaction with TMB as the substrate, the Vmax was 2.47 µM / min; in the H2O2 catalytic reaction, the Vmax value reached 0.73 µM / min. These kinetic parameters together revealed the synergistic optimization of MOF@Pt@MF compared to Fe-MOF in substrate recognition and conversion efficiency, marking a substantial enhancement of its catalytic performance.

[0037] Demonstration of the feasibility of the biosensor: In order to provide key support for the establishment of a stable and reliable biological coupling interface between MOF@Pt@MOF nanoszyme and Nanozyme-ssDNA, the surface chemical properties of Fe-MOF and its derived composite material MOF@Pt@MOF were investigated by FT-IR spectroscopy. Figure 9 A). Fine analysis of the Fourier transform infrared spectra of the two materials found that a set of clear and distinguishable characteristic absorption bands were observed in the key fingerprint region (3000-4000 cm -1 ) representing hydrogen-containing group stretching vibration. These high-frequency regionally distributed absorption characteristics, with peak values accurately falling within the typical identification range of primary / secondary amine functional group N-H bond vibration, provided important spectroscopic evidence for subsequent in-depth analysis of surface chemistry. To further verify the effectiveness of probe assembly, we systematically determined the Zeta potential of the relevant nanomaterials, whose values directly reflect the charge state of the particle surface. Figure 9 B). Basic characterization showed that both unmodified MOF@Pt@MOF nanoparticles and magnetic beads exhibited positive electrification in aqueous dispersion systems, with surface potentials of +3.59 mV and +4.93 mV, respectively. When single-stranded DNA modified with carboxyl groups at the end was successfully assembled onto the surface of MOF@Pt@MOF through covalent bonding to form MOF@Pt@MOF-ssDNA functional complex, its Zeta potential value changed from the original weak positive electrification to strong negative electrification, with a measured value of -37.52 mV. Similarly, after MNPs-ssDNA combined with magnetic beads, the obtained MB-ssDNA also showed excellent negative surface charge characteristics, with a Zeta potential value of -25.71 mV. This result strongly confirmed the effective biological coupling between MNPs-ssDNA and MB, Nanozyme-ssDNA and nanoszyme.

[0038] The running efficiency of the CRISPR-mediated biosensor was intuitively verified by polyacrylamide gel electrophoresis (10% PAGE) analysis. In order to determine the electrophoretic reference position of different nucleic acid components, Figure 10 The PAGE experiment of A: lane 1 contains MNPs-ssDNA, lane 2 contains dsDNA, lane 3 contains hairpin structure HP, which shows the specific migration position and band characteristics of three different nucleic acid configurations in the gel. Lane 4 reveals the activation state of the Cas12a system: when the system contains lane 1 nucleic acid molecules, lane 2 nucleic acid molecules and Cas12a / crRNA complex, the trans-nuclease activity of Cas12a is effectively activated due to the complementary pairing of dsDNA and crRNA, and the start of this enzyme activity triggers the cutting and degradation process of non-specific reporter substrate MNPs-ssDNA in the system. The direct manifestation of this molecular event on the PAGE map is the complete disappearance of the characteristic band of MNPs-ssDNA in lane 4. The response function of the biosensor to the target OTA is directly verified by the comparison of lanes 5 and 6. In lane 5 (without OTA), HP continuously inhibits crRNA, hindering the activation of Cas12a. In lane 6 (with OTA), the presence of OTA removes its blocking effect on crRNA, ultimately leading to the degradation of 73 bp of MNPs-ssDNA reporter probe; fully proving the successful application of CRISPR-Cas12a mechanism in mediating the signal transduction of the biosensor. In order to further comprehensively evaluate the comprehensive performance and practical potential of the constructed biosensor platform, the present application adopts two complementary optical detection strategies for verification: fluorescence analysis method (using fluorescence probe FQ-repoter) and colorimetric analysis method (using colorimetric probe MOF@Pt@MOF-DNA-MB). Figure 10 B shows the results of evaluating the performance of the sensor using fluorescence intensity changes: the fluorescence signal intensity of the experimental group containing the target is much higher than that of the blank control group without OTA. This phenomenon is directly attributed to the activation of Cas12a protease activity induced by OTA, thereby amplifying the fluorescence reporter signal. Based on the fluorescence verification, the present application further switches the signal reporting mechanism to the colorimetric mode. By collecting the ultraviolet-visible absorption spectrum of the reaction solution, Figure 10The C-10D nanobiosensor clearly identifies characteristic absorbance changes triggered by the target analyte. The visualized signal output based on this optical absorption change directly provides experimental evidence that the biosensor possesses colorimetric detection capabilities. The presence of OTA activates Cas12a cleavage, releasing the pre-loaded MOF@Pt@MOF nanozyme probe. After impurity removal via magnetic separation, the nanozyme exhibits strong peroxidase-like catalytic activity in solutions containing H2O2 and TMB, converting H2O2 into highly oxidizing ·OH. The rapid oxidization of TMB by ·OH triggers a color change in the solution, manifested as a sustained increase in absorbance in the UV-Vis absorption spectrum. This observable absorbance enhancement induced by OTA specificity provides solid evidence for the feasibility of detecting OTA using a CRISPR biosensor amplified from MOF@Pt@MOF nanozymes.

[0039] Example 5 Optimization of conditions for biosensors: Figure 11 The data presented in section A strongly supports the central role and indispensability of the HP component in this system. When HP is absent, significant background interference, particularly non-specific signals, is observed during detection. However, the introduction of HP into the experimental group effectively reduces the background signal intensity. This HP-mediated background noise suppression directly contributes to a significant improvement in the detection signal-to-noise ratio and specificity. In this system, as... Figure 11 As shown in Figure B, dsDNA (its nucleotide sequence is shown in SEQ ID No. 5) exhibits superior background control compared to ssDNA (its nucleotide sequence is shown in SEQ ID No. 6): the signal difference induced by dsDNA is significantly increased between the blank control and the experimental treatment groups. This expanded signal difference window directly reflects that dsDNA can more effectively suppress non-specific background interference, thus exhibiting a relatively lower background signal level. Therefore, based on its significant advantages in reducing background noise and enhancing detection specificity, dsDNA was identified as the most suitable activation trigger factor for the system. This invention systematically constructed and verified three HP variants with different functional domain loss characteristics (… Figure 11 C. The nucleotide sequences of HP-Ⅰ, HP-Ⅱ, and HP-Ⅲ are shown in SEQ ID No. 1-SEQ ID No. 3, respectively. HP-Ⅰ was designed to remove the PAM recognition site, HP-Ⅱ lacked the region complementary to the crRNA seed sequence, while HP-Ⅲ, as a control, fully preserved both the PAM region and the seed region—two key elements. Figure 11As shown in Figure D, compared to HP-III, which contains the complete PAM and seed region, both HP-I and HP-II variants lacking key functional domains exhibited significant background fluorescence interference. In both HP-I and HP-II systems, regardless of target activation, the detection systems showed highly consistent fluorescence intensity, indicating excessively high levels of nonspecific signal. HP-III, by simultaneously retaining the PAM recognition site and the crRNA seed sequence binding region, showed a significant decrease in fluorescence response value in its negative control group compared to the HP-I / HP-II system. This phenomenon is attributed to HP-III more effectively promoting the stable assembly of the Cas12a-crRNA-HP ternary complex, thereby significantly inhibiting the nonspecific cleavage activity of Cas12a in the inactive state. These results demonstrate the superior performance of the HP-III conformation in reducing the detection limit and improving analytical specificity.

[0040] The detection sensitivity and signal-to-noise ratio of this biosensor system are significantly affected by multiple reaction conditions. Key regulatory factors include the amount of HP, the molar ratio of Cas12a enzyme to crRNA, the concentration of dsDNA substrate, the incubation time of the target OTA, and the pre-assembly time of the Cas12a-crRNA binary complex. Figure 12 As shown in Figure A, the fluorescence response of the system initially increased and then decreased with increasing HP concentration, reaching a peak at 0.8 µM, indicating that the signal output was most significant at this concentration. Based on this, 0.8 µM HP was established as the optimal concentration for subsequent experiments. Four key reaction variables were systematically investigated: the molar ratio of Cas12a enzyme to crRNA, the concentration of dsDNA substrate, the reaction time of the target OTA, and the pre-assembly time of the Cas12a-crRNA binary complex. Figure 12 (B-12E). The optimization experiments ultimately established the following optimal parameter combinations: a Cas12a:crRNA ratio of 2:0.75; an optimal working concentration of dsDNA of 600 nM; an OTA incubation period of 10 minutes; and a Cas12a-crRNA pre-assembly process requiring 20 minutes. Future research will strictly adhere to these optimized parameters to further explore the performance of the CRISPR / Cas12a coupled nanozyme sensing system.

[0041] Example 6 Analytical performance study of biosensors: Based on the above optimal conditions, the core detection performance of the CRISPR / Cas12a coupled nanozyme biosensing platform was evaluated in depth. For example... Figure 13As shown in A-13C, the increase of the target OTA concentration in the system directly leads to the increase of the overall absorbance value of the UV-Vis absorption spectrum, accompanied by a significant visual color transition of the reaction solution - from the initial colorless state to a clear blue color. The intensity of the characteristic absorption peak at 650 nm was selected as the quantitative analysis index. The sensor system exhibits excellent linear response in the OTA concentration range of 50 ng / mL to 500 ng / mL. Through linear regression analysis, the fitting equation is y=0.5376x-0.8525, and the correlation coefficient R 2 = 0.9910, which proves a strong linear correlation. Based on the signal-to-noise ratio (S / N=3), the detection limit (LOD) of the method is as low as 0.055 ng / mL, indicating that it has ultra-high detection sensitivity. The ultra-low detection limit (0.055 ng / mL) achieved by the sensor is significantly lower than the strict maximum residue limits (MRLs) set by the current Chinese regulations for OTA in major agricultural products and processed products (range between 2 µg / kg and 10 µg / kg). This performance advantage means that the method has sufficient sensitivity margin and can easily meet or even exceed the stringent requirements of regulations for trace OTA pollution monitoring, providing strong technical support for rapid and compliant screening of the above high-risk food substrates.

[0042] To systematically evaluate the anti-interference ability and specificity of the constructed CRISPR / Cas12a-nanozyme cooperative aptamer sensing platform for target OTA recognition, the present application strictly screened seven kinds of widely existing and coexisting mycotoxins in food substrates as potential interferents for analysis, including: DON, ZEN, FB1, PAT, T-2, AFB1, and the target OTA itself. As Figure 13 D clearly discloses that when only OTA exists as an analyte in the reaction system, the sensing platform induces a significant absorbance signal shift that can be captured by optical means. It verifies that the HP has a unique molecular recognition mechanism for the target OTA molecule: not only does it exhibit strict binding selectivity, but it also achieves high-intensity specific interaction. The HP can accurately distinguish from other potential interferents and only drives observable spectral signal changes when interacting with OTA, fully embodying its excellent specificity as a recognition element. To fully evaluate the practicality of the constructed CRISPR / Cas12a-nanozyme hybrid aptamer sensing platform, its storage stability was systematically investigated (A-14C) Figure 13E). The evaluation strategy selected three key time nodes, day 1 (initial state), day 7 (one week later) and day 14 (two weeks later), to analyze the reproducibility of the sensor signal by repeating the colorimetric detection. The experimental data showed that the RSD of the signal response values measured under different storage durations was maintained below 5%, which was a strict quantitative indicator, and effectively confirmed that the sensing system had good time stability and reliable detection reproducibility within the test period, laying a foundation for its practical application.

[0043] Example 7 Real sample detection: To verify the practical value of the developed biosensing platform and its analysis performance in real complex matrices, the present application selected three types of representative cereals and oil crops, namely, sorghum, wheat and peanut, as detection objects to perform quantitative analysis of OTA. As shown in Table 2, excellent accuracy and reliability were exhibited. In the spiked recovery experiment, the determination recovery rate of the sorghum sample was concentrated in a narrow interval of 97.38% to 100.39%; the recovery rate result of the wheat sample was distributed between 97.24% and 101.98%; and the recovery rate of the peanut sample fell within the range of 95.36% to 109.37%. The series of recovery rate data of all three types of representative agricultural products strictly controlled the RSD below 5%. A widely recognized commercial ELISA detection kit was used as a reference standard for parallel comparison analysis. The experimental data showed that the quantitative results obtained based on the ELISA method and the OTA content values measured in the same actual samples by the aptamer sensor constructed by the present application showed a highly consistent trend, and there was significant statistical correlation between the two. Therefore, it was fully confirmed that the method proposed by the present application had high accuracy, good precision and analysis performance meeting the requirements of practical application when performing quantitative detection of OTA in real food samples.

[0044] Table 2 OTA detection results in sorghum, wheat and peanut

[0045] Although the specific embodiments of the present application are described in detail in combination with the embodiments, it should not be understood as limiting the protection scope of the present patent. Various modifications and variations made by those skilled in the art within the scope described in the claims are still within the protection scope of the present patent.

Claims

1. A colorimetric aptamer sensor, characterized in that: The application relates to a colorimetric probe, which comprises a Cas12a protein, an HP, a crRNA, dsDNA and a colorimetric probe; the nucleotide sequence of the HP is shown in SEQ ID NO. 3, the nucleotide sequence of the crRNA is shown in SEQ ID NO. 4, the nucleotide sequence of the dsDNA is shown in SEQ ID NO. 5, and the colorimetric probe is MOF@Pt@MOF-DNA-MB.

2. The colorimetric aptamer sensor of claim 1, wherein: The preparation method of the colorimetric probe comprises the following steps: (1) synthesis of Fe-MOF: 2-amino terephthalic acid, iron trichloride and glacial acetic acid are dissolved in N,N-dimethylformamide, Fe-MOF is obtained after heating reaction; (2) preparation of Pt NPs: polyvinylpyrrolidone and chloroplatinic acid hexahydrate are dissolved in deionized water, a sodium borohydride solution is added dropwise under stirring, the mixed solution is brown-black, and Pt NPs are obtained after continuous stirring; (3) assembly of MOF@Pt@MOF nanoscale enzyme: the Pt NPs are mixed and stirred with Fe-MOF to obtain Fe-MOF@Pt; the Fe-MOF@Pt is resuspended in N,N-dimethylformamide and mixed with a solution containing 2-amino terephthalic acid and iron trichloride, and MOF@Pt@MOF nanoscale enzyme is obtained after reaction; (4) preparation of MOF@Pt@MOF-ssDNA: 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide are added in PBS buffer solution, and Nanozyme-ssDNA is added for activation; the activated Nanozyme-ssDNA is co-incubated with the MOF@Pt@MOF nanoscale enzyme to obtain MOF@Pt@MOF-ssDNA; the nucleotide sequence of the Nanozyme-ssDNA is shown in SEQ ID NO. 7; (5) preparation of MB-ssDNA: streptavidin-modified magnetic beads are co-incubated with biotinylated MNPs-ssDNA to obtain MB-ssDNA; the nucleotide sequence of the MNPs-ssDNA is shown in SEQ ID NO. 8; (6) preparation of the colorimetric probe MOF@Pt@MOF-DNA-MB: the MOF@Pt@MOF-ssDNA is mixed with the MB-ssDNA, magnetic separation and purification are carried out, and the colorimetric probe MOF@Pt@MOF-DNA-MB is obtained.

3. The colorimetric aptamer sensor of claim 2, wherein: The amount ratio of 2-amino terephthalic acid, ferric chloride, glacial acetic acid and N, N-dimethylformamide in the step (1) is 0.126 g:0.69 mmol:3.45 mmol:15 mL, the temperature of heating reaction is 120 ℃, and the time is 4 h; the amount ratio of deionized water, polyvinylpyrrolidone, chloroplatinic acid hexahydrate and sodium borohydride in the step (2) is 45 mL:51.4 mg:0.1 mmol:0.465 mmol, the solution turns into dark brown after stirring for 2 h; the Pt NPs are added into the Fe-MOF in the step (3), and stirring is carried out at room temperature for 2 h; the amount ratio of N, N-dimethylformamide, 2-amino terephthalic acid and ferric chloride in the step (3) is 12 mL:0.042 g:0.229 mmol, the temperature of heating reaction is 120 ℃, and the time is 8 h; the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and N-hydroxysuccinimide in the step (4) is 1:1, the adding amount of Nanozyme-ssDNA is 0.1 nmol, and stirring is carried out at room temperature for 24 h after adding the Nanozyme-ssDNA; the Nanozyme-ssDNA after activation is co-incubated with the MOF@Pt@MOF nanoscale enzyme for 24 h; the amount ratio of streptavidin-modified magnetic beads and biotinylated MNPs-ssDNA in the step (5) is 100 µL:0.1 nmol, and co-incubation is carried out at room temperature for 1 h; the MOF@Pt@MOF-ssDNA and the MB-ssDNA are mixed in equal volume in the step (6), the temperature of mixing reaction is 37 ℃, and the time is 60 min.

4. Use of the colorimetric aptamer sensor of any one of claims 1-3 in detecting ochratoxin A.

5. Use according to claim 4, characterized in that: Detecting the content of ochratoxin A in food.

6. Use according to claim 4, characterized in that, The method for detecting ochratoxin A comprises the following steps: (1) mixing 0.6-1.0 µM HP with the sample to be detected, and incubating for 10-40 min; the nucleotide sequence of the HP is shown in SEQ ID NO. 3; (2) adding Cas12a protein and crRNA into the reaction system, and the molar ratio of the two is 2:0.5-1, and then incubating for 20-25 min after mixing; the nucleotide sequence of the crRNA is shown in SEQ ID NO. 4; (3) adding 400-800 nM dsDNA and 5-10 µL colorimetric probe MOF@Pt@MOF-DNA-MB into the reaction system, and then incubating the mixed solution for 30-60 min; the nucleotide sequence of the dsDNA is shown in SEQ ID NO. 5; (4) after incubation, magnetic bead separation operation is carried out, the supernatant is collected and washed with PBS buffer for 3 times; (5) The supernatant obtained after the final washing in step (4) is mixed with an acetic acid-sodium acetate buffer, and H2O2 and a chromogenic substrate are sequentially added to perform a color development reaction; after the color development reaction is terminated, the absorbance value of the solution is measured using an ultraviolet spectrophotometer; Prepare ochratoxin A standard solutions with different concentrations, detect the ochratoxin A standard solutions according to the above steps, record the absorbance generated by each standard solution, and draw a standard curve; detect the absorbance generated by the sample to be tested, and compare with the standard curve to obtain the concentration of ochratoxin A in the sample to be tested.

7. Use according to claim 6, characterized in that: The temperature of the mixed incubation in steps (1) and (3) is 37 ℃.

8. Use according to claim 6, characterized in that: The concentration of HP in step (1) is 0.8 µM, and the incubation time after adding HP and the sample to be tested is 10 min; the molar ratio of Cas12a protein and crRNA in step (2) is 2:0.75, and the incubation time after adding Cas12a protein and crRNA is 20 min; the concentration of dsDNA in step (3) is 600 nM, and the mixed incubation time is 60 min; the molar ratio of H2O2 and chromogenic substrate in step (5) is 20 µmol:1.005 µmol.

9. Use according to claim 6, characterized in that: The chromogenic substrate is TMB, ABTS or OPD, and the detection wavelength is the absorbance at 650 nm, 420 nm or 450 nm, respectively.

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