A sarafloxacin stimuli-responsive hydrogel colorimetric sensor based on aptamer, enzyme and MOF hybrid

Abstract

The present invention discloses a sarafloxacin stimulus-responsive hydrogel colorimetric sensor based on aptamer, enzyme and MOF hybridization. A colorimetric aptamer sensor is designed by sequentially coating natural catalase with multi-shell MOFs and hyaluronic acid-DNA hydrogel, specifically: first, MOF shell is used to coat catalase to form an enzyme-MOF nanozyme complex MOF@cat@MOF, and an aptamer e-apt with higher affinity is obtained through aptamer optimization, and it is designed as a synthetic component of DNA hydrogel, and then the DNA hydrogel is deposited on the surface of multi-shell MOFs through DNA hybridization chain reaction. Due to the integration of specific aptamers in the hydrogel DNA network structure, the introduction of targets will cause the hydrogel structure to disintegrate and expose the encapsulated MOFzyme. The disintegration amplitude of the DNA hydrogel is positively correlated with the content of the target, and the quantification of sarafloxacin can be achieved. The developed biosensor has the potential to become a universal platform in the fields of medical auxiliary diagnosis, food safety and environmental hazards.

Description

Technical Field

[0001] The invention relates to a sarafloxacin stimulus-responsive hydrogel colorimetric sensor based on aptamer, enzyme and MOF hybridization, belonging to the technical field of food safety. Background Art

[0002] Sarafloxacin (SAR) is a broad-spectrum veterinary antibiotic synthesized with a quinoline-4-one ring as its basic structure. It is mainly used to treat bacterial infections caused by a variety of bacteria such as Streptococcus suis, Escherichia coli and Salmonella. After being ingested into the animal's body, sarafloxacin will quickly penetrate into the tissue through the blood, resulting in a large amount of residues in animal-derived foods. In addition, excessive and long-term irrational use will lead to its accumulation in farmed animals, posing a serious threat to the ecological environment and human health. Due to its high residual hazards to human health and the ecological environment, the European Union and China have established strict maximum residue limits for the content of animal-derived sarafloxacin. For example, the maximum content of SAR in muscle tissue cannot exceed 10μg / Kg.

[0003] Capillary electrophoresis, high performance liquid chromatography (HPLC) and LC-MS are the most stable and accurate detection methods for sarafloxacin. However, the cumbersome instrument operation steps and expensive maintenance costs greatly limit the efficiency of these instrument methods. In addition, the structural similarity of quinolone antibiotics also increases the difficulty of screening specific antibodies, which in turn hinders the detection efficiency of antibody-based biosensors. Therefore, the development of a rapid, reliable, specific, and cost-effective method for the quantitative determination of sarafloxacin has great application prospects. Summary of the invention

[0004] To solve the above problems, the present invention provides a sarafloxacin-targeted stimulus-responsive hydrogel colorimetric sensor based on an evolved aptamer, catalase, and a metal-organic framework (MOF) hybrid. The present invention first uses a multi-shell MOF to encapsulate catalase to form an enzyme-MOF nanozyme complex (MOF@cat@MOF), obtains an evolved aptamer (e-apt) with higher affinity through a systematic aptamer optimization process, and carefully designs it into a synthetic component of a DNA hydrogel. Subsequently, the DNA hydrogel is deposited on the surface of the multi-shell MOFs by a DNA hybridization chain reaction (HCR). Due to the integration of specific aptamers in the hydrogel DNA network structure, the introduction of the target causes the hydrogel structure to disintegrate and expose the encapsulated MOFzyme. In addition, the disintegration amplitude of the DNA hydrogel is positively correlated with the content level of the target, so the quantification of sarafloxacin can be achieved effortlessly.

[0005] The first object of the present invention is to provide a method for detecting sarafloxacin, comprising the following steps:

[0006] S1, synthesizing enzyme-MOF nanozyme (enzyme and MOF hybridization) by using MOF material (metal organic framework material) and enzyme in a biomineralization manner, and covalently linking the enzyme-MOF nanozyme to the first nucleic acid chain;

[0007] S2, incubating the product of S1 with the second nucleic acid chain to make the second nucleic acid chain complementary to the first nucleic acid chain, and removing the redundant second nucleic acid chain;

[0008] S3, allowing hyaluronic acid to undergo coupling reaction with the third nucleic acid chain and the fifth nucleic acid chain, respectively, to obtain a hyaluronic acid-third nucleic acid chain polymer and a hyaluronic acid-fifth nucleic acid chain polymer;

[0009] S4, co-incubating the hyaluronic acid-third nucleic acid chain polymer obtained in S3 with the fourth nucleic acid chain to obtain a hyaluronic acid-third nucleic acid chain-fourth nucleic acid chain copolymer;

[0010] S5, mixing and incubating the product of S2, the hyaluronic acid-fifth nucleic acid chain polymer obtained in S3, and the hyaluronic acid-third nucleic acid chain-fourth nucleic acid chain copolymer obtained in S4 to obtain a DNA hydrogel-coated enzyme-MOF nanozyme;

[0011] S6, mixing sarafloxacin solutions of different known concentrations with the DNA hydrogel-coated enzyme-MOF nanozyme and the nanozyme-catalyzed substrate solution to react, measuring the enzyme-catalyzed reaction signal, and establishing a coordinate system of the reaction signal and the sarafloxacin concentration;

[0012] S7, repeat step S6 for the sample to be tested, measure the reaction signal, and substitute it into the coordinate system of S6 to obtain the content of sarafloxacin in the sample to be tested;

[0013] Wherein, the sequences of the first nucleic acid chain, the second nucleic acid chain, the third nucleic acid chain, the fourth nucleic acid chain and the fifth nucleic acid chain are shown as SEQ ID NO.2-6.

[0014] Further, the selection of MOF includes but is not limited to the MOF series.

[0015] Furthermore, the selection of enzymes includes but is not limited to hydrogen peroxide. Accordingly, the nanozyme catalyzed substrate solution includes hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine, and the detected reaction signal is a colorimetric signal.

[0016] Furthermore, in step S1, the preparation method of enzyme-MOF nanozyme is to mix MOF material, catalase, organic ligand, and metal salt, react under solvent thermal conditions, and repeatedly add metal salt to react under solvent thermal conditions to achieve hybridization of catalase and MOF.

[0017] Further, in step S3, the nucleic acid chain is coupled to the hyaluronic acid via the amino group.

[0018] Furthermore, in step S5, the molar ratio of the product of S2, the hyaluronic acid-fifth nucleic acid chain polymer obtained in S3, and the hyaluronic acid-third nucleic acid chain-fourth nucleic acid chain copolymer obtained in S4 is 1.5-2.5:0.5-1.5:1.5-2.5.

[0019] The second object of the present invention is to provide a sarafloxacin stimuli-responsive hydrogel colorimetric sensor based on an aptamer, a catalase and a metal organic framework hybrid, wherein the sensor comprises a DNA hydrogel-coated enzyme-MOF nanozyme, wherein the DNA hydrogel-coated enzyme-MOF nanozyme comprises a hybridized catalase and a metal organic framework formed by biomineralization, and a hydrogel coated on the surface of the hybrid, wherein:

[0020] The hydrogel is composed of hyaluronic acid, a first nucleic acid chain, a second nucleic acid chain, a third nucleic acid chain, a fourth nucleic acid chain and a fifth nucleic acid chain, and the first nucleic acid chain is covalently connected to the metal organic framework, and the sequences of the first nucleic acid chain, the second nucleic acid chain, the third nucleic acid chain, the fourth nucleic acid chain and the fifth nucleic acid chain are shown in SEQ ID NO.2-6.

[0021] Furthermore, the preparation method of the DNA hydrogel-coated enzyme-MOF nanozyme comprises the following steps:

[0022] S1, synthesizing the enzyme-MOF nanozyme by biomineralization of the metal organic framework material and the enzyme, and covalently linking the enzyme-MOF nanozyme to the first nucleic acid chain;

[0023] S2, incubating the product of S1 with the second nucleic acid chain to make the second nucleic acid chain complementary to the first nucleic acid chain, and removing the redundant second nucleic acid chain;

[0024] S3, allowing hyaluronic acid to undergo coupling reaction with the third nucleic acid chain and the fifth nucleic acid chain, respectively, to obtain a hyaluronic acid-third nucleic acid chain polymer and a hyaluronic acid-fifth nucleic acid chain polymer;

[0025] S4, co-incubating the hyaluronic acid-third nucleic acid chain polymer obtained in S3 with the fourth nucleic acid chain to obtain a hyaluronic acid-third nucleic acid chain-fourth nucleic acid chain copolymer;

[0026] S5. Mix and incubate the product of S2, the hyaluronic acid-fifth nucleic acid chain polymer obtained in S3, and the hyaluronic acid-third nucleic acid chain-fourth nucleic acid chain copolymer obtained in S4 to obtain the DNA hydrogel-coated enzyme-MOF nanozyme.

[0027] The third object of the present invention is to provide an aptamer that specifically recognizes sarafloxacin, the sequence of which is shown in SEQ ID NO.1.

[0028] Furthermore, the 3' end or the 5' end of the aptamer is modified with a functional group or molecule.

[0029] Furthermore, the functional group or molecule is an isotope, an electrochemical marker, an enzyme marker, a fluorescent group, biotin, an affinity ligand or a thiol group.

[0030] The fourth object of the present invention is to provide use of the aptamer or sensor in detecting sarafloxacin.

[0031] The fifth object of the present invention is to provide a product for detecting sarafloxacin, wherein the product contains the aptamer.

[0032] Beneficial effects of the present invention:

[0033] (1) In the present invention, a novel colorimetric aptamer sensor for sensitive and accurate quantification of sarafloxacin was developed by encapsulating catalase with porous metal organic frameworks and hyaluronic acid (HA)-DNA hydrogels. First, an evolved aptamer (e-apt) specifically targeting sarafloxacin was initially obtained, which had a higher affinity and was carefully designed as a synthetic component of the downstream DNA hydrogel network structure. At the same time, a hybrid MOFzyme with excellent catalytic activity was obtained by gradually encapsulating natural catalase in multi-shell MOFs. Thereafter, the DNA hydrogel was deposited on the surface of the multi-shell MOFs by DNA hybridization chain reaction (HCR). The integration of e-apt endowed the constructed hydrogel with stimulus-responsive properties. By introducing sarafloxacin, the structural disintegration of the hydrogel caused the exposure of the encapsulated MOFzyme, and the exposure amplitude was positively correlated with the content level of sarafloxacin. Therefore, with the sensitive stimulus responsiveness of DNA hydrogel and the high catalytic efficiency of MOFzymes, accurate and effective trace colorimetric detection of sarafloxacin can be achieved. In brief, a colorimetric aptamer sensor targeting sarafloxacin was constructed by encapsulating catalase with multi-shell MOFs and DNA hydrogel. Thanks to the high affinity of e-apt, the excellent catalytic activity of MOFzyme and the sensitive responsiveness of DNA hydrogel, the developed aptamer sensor has excellent trace detection performance and anti-interference ability. Its successful application in livestock and poultry samples also shows that it has great application prospects.

[0034] (2) The materials used in the present invention are easy to prepare on a large scale, and the operation is simple and convenient, and does not rely on precision instruments and equipment; the present invention outputs a color signal, which has a good linear relationship with the concentration of the target sarafloxacin, and can be semi-quantitatively detected by naked eyes, with a linear range of 0.003 and 200 ng / mL, and the detection limit of accurate determination is lower than 0.6 pg / mL, with high sensitivity, and can achieve trace quantitative detection; and the target is recognized by the nucleic acid aptamer, which has good specificity; the present invention uses an enzyme reader or an ultraviolet detector to effectively distinguish and differentiate trace concentrations of sarafloxacin; the rationality and practicality of the colorimetric sensor are preliminarily verified by actually adding Taihu water, milk and beef samples for sample addition analysis; in addition, the method is simple and portable to operate, which is conducive to the realization of rapid on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a detection flow chart of the present invention.

[0036] Figure 2 Comparison of catalase content determination in the supernatant (10× dilution) before and after coating reaction.

[0037] Figure 3 The standard curve diagram of normalized absorbance and sarafloxacin concentration in Example 1 is shown in FIG.

[0038] Figure 4 This is the detection specificity result of the colorimetric detection of sarafloxacin in Example 1. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0040] The scheme of the present invention is as follows:

[0041] Catalase is precisely encapsulated in layered MOFs through a step-by-step in situ growth reaction to form a MOF@cat@MOF heterostructure, and then the thiol-modified DNA (1) is coupled to the MOFzyme through a typical bridging reaction method to form a DNA (1)-MOF nanozyme. The DNA nucleic acid chain (2) is then combined by the complementary base pairing of the nucleic acid chain, and under the action of the initiator nucleic acid chain component in the nucleic acid (2), an HCR chain reaction is generated between the hybrid double chain (1) / (2), the hyaluronic acid-modified copolymer HA-(3) / (4) and the copolymer HA-(5), that is, the nucleic acid chain (2) and (4) interact to open the hairpin structure of (4) and form a bridge element with the hairpin structure (5), and the nucleic acid chain (5) is linked to the hairpin (4) again, and this process is repeated continuously, so that a layer of hydrogel (HA-DNA) is coated on the outer surface of the MOFzyme. Since the evolved sarafloxacin-specific aptamer sequence is designed to enter the DNA network structure of the hydrogel, in the presence of sarafloxacin, the binding of the sarafloxacin / aptamer complex will trigger the dissociation of the polymer chain, leading to the disintegration of the DNA hydrogel and exposing the MOFzyme. The enzyme exposure level is positively correlated with the target content in the system. Moreover, the absorbance signal of the color product generated by the enzyme-catalyzed substrate H2O2 and TMB is linearly correlated with the target content in the reaction system. Therefore, sarafloxacin quantification can be achieved by measuring the absorbance of the catalytic substrate.

[0042] Example 1 Construction of Sarafloxacin Biosensor

[0043] First, 20.0 mg of 2-aminoterephthalic acid and 30.0 mg of FeCl3·6H2O were accurately weighed and dissolved in 20 mL of deoxygenated ethanol solution under ultrasonic conditions. Subsequently, the mixture was heated at 40 °C for 15 min by oil bath. The MOF was obtained by centrifugation at 8000 rpm for 10 min, washed three times with deoxygenated ethanol, and resuspended in 500 μL of anhydrous ethanol.

[0044] Next, 500 μL of the above product, 500 μL PVP (2.0 mg / mL) solution, FeCl3·6H2O (30.0 mg), catalase (catalase, 20 mg) and NH2-BDC (20.0 mg) were mixed in 15 mL of deionized water and heated in an oil bath at 40 ° C for 2 hours. Subsequently, the product (MOF@cat-MOF) was collected by centrifugation at 8000 rpm for 10 minutes and then resuspended in 10 mL of water. The catalase encapsulation efficiency was determined by the BCA protein kit method.

[0045] In order to synthesize the MOF@cat@MOF nanozyme composite material, another portion of 30.0 mg FeCl3·6H2O and 20.0 mg NH2-BDC was added to the MOF@cat-MOF solution prepared above, sealed and heated in a 40°C oil bath for 30 minutes. Finally, the precipitated product was obtained by centrifugation at 8000 rpm for 10 minutes. The obtained product was washed three times with anhydrous ethanol to obtain the enzyme-MOF nanozyme complex MOFzyme (MOF@cat@MOF). At the same time, the MOF@MOF structure was also synthesized without the participation of catalase.

[0046] To further functionalize MOFzyme, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC) (2 mg) was introduced into MOFzymes (5 mg) in HEPES buffer. After incubation at 25°C for 2 hours, excess Sulfo-SMCC was removed by centrifugation (8000 rpm, 10 min). Then, the obtained activated MOFzyme was incubated with 300 nmol of thiol (SH)-modified nucleic acid (1) solution and kept at 25°C overnight to prepare nucleic acid (1)-functionalized MOFzyme nanoprobes.

[0047] Next, NaCl solution was gradually added to the above product in three times within 8 hours to make the final concentration of NaCl 0.5M, which was used to stabilize the DNA chain and promote the binding between the nucleic acid chain (1) and the nanomaterial. The change in absorbance at a wavelength of 260nm before and after the coupling reaction was used to evaluate the number of nucleic acid chains successfully connected to the MOFzyme. Next, the product was obtained by centrifugation and washed three times with 0.1M PBS (pH 7.0) solution to remove loosely bound nucleic acids (1).

[0048] After conformational unification, nucleic acid chain (2) was added at a ratio of 1.2:1.0 to hybridize with nucleic acid chain (1) attached to MOFzyme through base complementary pairing. After 8 hours of binding reaction, unhybridized downstream reaction initiation element nucleic acid chain (2) was removed by washing several times. Next, the obtained nucleic acid chain (1) / (2)-functionalized MOFzyme was stored at 4°C for further use.

[0049] The nucleic acid chain solution of amino-modified DNA sequence (3) and hairpin sequence (5) was heated at 95°C for 5 minutes and cooled at 0°C for 2 minutes to avoid the formation of DNA aggregates. A 1% hyaluronic acid (HA) solution was activated with 0.2mmol EDC and 0.15mmol NHS for 30 minutes. Then, the final concentration of 100μM DNA chain (3) or hairpin (5) was coupled with HA overnight to obtain polymers HA-(3) and HA-(5). The unreacted HA-(3) and HA-(5) were removed from the mixture by three ultrafiltration purification processes. The amount of nucleic acid chain (3) was quantified by ultraviolet spectroscopy and incubated with an equal concentration of nucleic acid chain (4) at 37°C for 6 hours to produce a copolymer HA-(3) / (4). Among them, e-apt was cleverly designed as a part of nucleic acid chains (4) and (5) with hairpin structures. Thereby giving the DNA network a specific recognition response function in the hydrogel. Among them, e-apt was modified from the sarafloxacin aptamer SAR-Origin. The sequences and affinities before and after the modification are shown in Table 1.

[0050] Nucleic acid (2) immobilized on MOFzyme can trigger the HCR process during the formation of DNA hydrogel, thereby forming a hydrogel layer on MOFzyme. In detail, nucleic acid chain (1) / (2)-functionalized MOFzyme was mixed with HA-(3) / (4), HA-(5) and incubated at 42°C in a ratio of 2:2:1 for 12 hours. Subsequently, the product was purified three times to remove unreacted substances, and the synthesized DNA hydrogel-coated MOFzyme was stored at 4°C for specific detection of sarafloxacin.

[0051] The sequences involved above are shown in Table 2.

[0052] Table 1 Comparison of affinity of aptamer nucleic acid sequences before and after optimization

[0053]

[0054] Table 2 Hydrogel DNA nucleic acid chain sequence

[0055]

[0056]

[0057] Note: The bold part represents the aptamer sequence

[0058] Example 2 Detection of Sarafloxacin

[0059] This embodiment provides a colorimetric detection method for sarafloxacin:

[0060] Adding a gradient diluted sarafloxacin sample to the DNA hydrogel-coated MOFzyme prepared in the embodiment triggers the hydrogel disintegration reaction. In addition, H2O2 and TMB substrate solutions (25mM, 5mM) were added in sequence and incubated at room temperature for 25 minutes. Then, the absorbance of the supernatant at 650nm was detected by a microplate reader. The detection mechanism is that in the presence of sarafloxacin in the reaction system, the binding of the sarafloxacin / aptamer complex will trigger the dissociation of the polymer chain, thereby causing the disintegration of the DNA hydrogel to expose the MOFzyme. The enzyme exposure level is positively correlated with the target content in the system. Moreover, the absorbance signal of the enzyme-catalyzed substrate H2O2 and TMB to generate a color product is linearly correlated with the target content in the reaction system. Therefore, quantification of sarafloxacin can be achieved by measuring the absorbance of the catalytic substrate.

[0061] in:

[0062] (1) The preparation method of the nanozyme catalytic substrate solution is:

[0063] Weigh 0.024 g of 3,3',5,5'-tetramethylbenzidine (TMB) and dissolve it in 5 mL of ethylene glycol to prepare a 20 mM TMB stock solution;

[0064] A chromogenic substrate solution was prepared by mixing 10 μL of TMB solution (5 mM), 10 μL of aqueous hydrogen peroxide solution (25 mM), and 5 μL of sodium acetate buffer (pH = 4, 20 mM);

[0065] (2) A colorimetric method for detecting sarafloxacin, comprising:

[0066] Sensitivity analysis of the detection method:

[0067] 50 μL of DNA hydrogel-coated MOFzyme and 50 μL of sample were thoroughly mixed in a 1.5 mL centrifuge tube. In addition, 10 μL of H2O2 (25 mM) and 10 μL of TMB substrate solution (5 mM) were added in sequence and incubated at room temperature for 30 min. Then, MOFzyme was removed by filtration membrane, and the collected supernatant was detected by microplate reader. Finally, the absorbance of the mixture at 650 nm was measured by UV-1800 spectrophotometer to quantitatively analyze sarafloxacin. Results are shown in Figure 3 .

[0068] Specificity analysis of the detection method:

[0069] Sarafloxacin (SAR), norfloxacin (NOR), pefloxacin (PEF), lomefloxacin (LOM), enrofloxacin (ENR), ofloxacin (OFI), pazufloxacin (PAZ), ciprofloxacin (CIP), calcium ion (Ca 2+ ), potassium ion (K+), zinc ion (Zn3+ ), kanamycin (KAN), neomycin (NOV), netilmicin (NET), gentamicin (GEN), tobramycin (TOB). (The concentration of sarafloxacin is 10 ng / mL, and the concentration of other interfering substances is 20 ng / mL), respectively, according to the above colorimetric sensing method, the absorbance of the supernatant is measured at 650 nm; the specificity results of the colorimetric detection method can be found in Figure 4 .

[0070] Example 3 Detection of actual samples

[0071] The actual spiked Taihu Lake water, milk and beef samples were tested, including:

[0072] According to the sarafloxacin colorimetric detection method established in Example 2, the absorbance of Taihu water, milk and beef samples with different sarafloxacin contents was measured, and the corresponding concentration values ​​were calculated. Then, the values ​​were compared with the LC-MS results, and the recovery rate and relative standard deviation were calculated. The results are shown in Table 3. The recovery rates in the samples were between 90.59 and 99.48, and the relative standard deviations were all lower than 10%, indicating that the method has high accuracy and is feasible for the detection of actual samples.

[0073] Table 3 Results of determination of Taihu water, milk and beef samples by LC-MS method and colorimetric method of the present invention

[0074]

[0075]

[0076] a ND: not detected.

[0077] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for detecting sarafloxacin, characterized in that: The following steps are involved: S1, synthesizing the enzyme-MOF nanozyme by biomineralization of the metal organic framework material and the enzyme, and covalently linking the enzyme-MOF nanozyme to the first nucleic acid chain; S2, incubating the product of S1 with the second nucleic acid chain to make the second nucleic acid chain complementary to the first nucleic acid chain, and removing the redundant second nucleic acid chain; S3, allowing hyaluronic acid to undergo coupling reaction with the third nucleic acid chain and the fifth nucleic acid chain, respectively, to obtain a hyaluronic acid-third nucleic acid chain polymer and a hyaluronic acid-fifth nucleic acid chain polymer; S4, co-incubating the hyaluronic acid-third nucleic acid chain polymer obtained in S3 with the fourth nucleic acid chain to obtain a hyaluronic acid-third nucleic acid chain-fourth nucleic acid chain copolymer; S5, mixing and incubating the product of S2, the hyaluronic acid-fifth nucleic acid chain polymer obtained in S3, and the hyaluronic acid-third nucleic acid chain-fourth nucleic acid chain copolymer obtained in S4 to obtain a DNA hydrogel-coated enzyme-MOF nanozyme; S6, mixing sarafloxacin solutions of different known concentrations with the DNA hydrogel-coated enzyme-MOF nanozyme and the nanozyme-catalyzed substrate solution to react, measuring the enzyme-catalyzed reaction signal, and establishing a coordinate system of the reaction signal and the sarafloxacin concentration; S7, repeat step S6 for the sample to be tested, measure the reaction signal, and substitute it into the coordinate system of S6 to obtain the content of sarafloxacin in the sample to be tested; Wherein, the sequences of the first nucleic acid chain, the second nucleic acid chain, the third nucleic acid chain, the fourth nucleic acid chain and the fifth nucleic acid chain are shown as SEQ ID NO.2-6.

2. The detection method according to claim 1, characterized in that: In step S1, the preparation method of enzyme-MOF nanozyme is to use MOF, catalase, organic ligand required for synthesizing MOF and metal salt as raw materials, react under solvent thermal conditions, and repeatedly add the metal salt to react under solvent thermal conditions to achieve hybridization of catalase and MOF.

3. The detection method according to claim 1, characterized in that: In step S3, the nucleic acid chain is coupled to the hyaluronic acid via the amino group.

4. The detection method according to claim 1, characterized in that: In step S5, the molar ratio of the product of S2, the hyaluronic acid-fifth nucleic acid chain polymer obtained in S3, and the hyaluronic acid-third nucleic acid chain-fourth nucleic acid chain copolymer obtained in S4 is 1.5-2.5:0.5-1.5:1.5-2.

5.

5. A sarafloxacin stimuli-responsive hydrogel colorimetric sensor based on aptamer, catalase and metal organic framework hybrid, characterized in that: The sensor includes a DNA hydrogel-coated enzyme-MOF nanozyme, wherein the DNA hydrogel-coated enzyme-MOF nanozyme includes a hybrid catalase and a metal organic framework formed by biomineralization, and a hydrogel coated on the surface of the hybrid, wherein: The hydrogel is composed of hyaluronic acid, a first nucleic acid chain, a second nucleic acid chain, a third nucleic acid chain, a fourth nucleic acid chain and a fifth nucleic acid chain, and the first nucleic acid chain is covalently connected to the metal organic framework, and the sequences of the first nucleic acid chain, the second nucleic acid chain, the third nucleic acid chain, the fourth nucleic acid chain and the fifth nucleic acid chain are shown in SEQ ID NO.2-6.

6. The sensor according to claim 5, characterized in that The preparation method of the DNA hydrogel-coated enzyme-MOF nanozyme comprises the following steps: S1, synthesizing the enzyme-MOF nanozyme by biomineralization of the metal organic framework material and the enzyme, and covalently linking the enzyme-MOF nanozyme to the first nucleic acid chain; S2, incubating the product of S1 with the second nucleic acid chain to make the second nucleic acid chain complementary to the first nucleic acid chain, and removing the redundant second nucleic acid chain; S3, allowing hyaluronic acid to undergo coupling reaction with the third nucleic acid chain and the fifth nucleic acid chain, respectively, to obtain a hyaluronic acid-third nucleic acid chain polymer and a hyaluronic acid-fifth nucleic acid chain polymer; S4, co-incubating the hyaluronic acid-third nucleic acid chain polymer obtained in S3 with the fourth nucleic acid chain to obtain a hyaluronic acid-third nucleic acid chain-fourth nucleic acid chain copolymer; S5. Mix and incubate the product of S2, the hyaluronic acid-fifth nucleic acid chain polymer obtained in S3, and the hyaluronic acid-third nucleic acid chain-fourth nucleic acid chain copolymer obtained in S4 to obtain the DNA hydrogel-coated enzyme-MOF nanozyme.

7. Use of the sensor according to claim 5 or 6 in detecting sarafloxacin.

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