Preparation method for detecting ZEN by using chain displacement amplification dual-mode sensor based on AuNCs and ZIF-67 (at) Ag

Abstract

The invention relates to a preparation method for detecting ZEN by using a dual-mode sensor for chain displacement amplification of AuNCs and ZIF-67 (at) Ag, which comprises the following steps: incubating aminated Apt and magnetic beads MBs under certain conditions to obtain an MBs (at) Apt structure; in the presence of ZEN, the cDNA and a hairpin H1 are hybridized to form a double-strand structure, and a DNA polymerase Klenow Fragment and a restriction enzyme Nt.BbvCI are added for a strand displacement amplification reaction, so that a large number of single strands are obtained; a single chain and the fluorescent probe form a complex structure, a fluorescent signal is enhanced, meanwhile, the enzyme-like activity of ZIF-67 (at) Ag is recovered, TMB is catalyzed to generate a strong color development reaction, and the absorbance of a system is enhanced. The chain displacement amplification dual-mode sensor utilizing the AuNCs and the ZIF-67 is obtained. Then, the sensor is used for detecting zearalenone, the food labeled sample is detected according to the steps, and changes of a fluorescence intensity signal and an absorbance intensity signal of the food labeled sample are analyzed. Compared with other sensors for zearalenone detection, the prepared fluorescent colorimetric dual-mode sensor has the advantages of high sensitivity, good repeatability and high accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a preparation method and application of a chain displacement amplification dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag. Background Art

[0002] Zearalenone is a non-steroidal estrogenic mycotoxin, mainly produced by Fusarium and Fusarium graminearum, and widely found in corn, sorghum, barley and wheat. The International Agency for Research on Cancer (IARC) classifies zearalenone as a Class III carcinogen because of its potential neurotoxicity, reproductive toxicity and carcinogenicity. In order to prevent the toxicity of zearalenone, many countries have set maximum residue limits (MRLs) for zearalenone in various crops and their products, ranging from 20 μg / kg to 300 μg / kg. Therefore, in order to effectively control the pollution and hazards of ZEN, it is necessary to establish a rapid, accurate and sensitive ZEN detection technology.

[0003] At present, ZEN detection technologies are mainly divided into two categories: instrumental analysis and immunoassay. Instrumental analysis mainly includes thin layer chromatography, high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), etc. These methods have the advantages of high resolution, high accuracy, and high sensitivity, but they also have disadvantages such as expensive instruments, complex operations, and cumbersome sample pretreatment. Immunoassay mainly includes enzyme-linked immunosorbent assay (ELISA), immunosensor method, etc. These methods have the advantages of being fast, simple, and cheap, but they also have disadvantages such as cross-reaction, poor stability, and low sensitivity. Therefore, the current research direction is to develop new ZEN detection technologies, such as detection technologies based on fluorescent probes, nanomaterials, nucleic acid aptamers, etc., to improve the efficiency and performance of ZEN detection.

[0004] Compared with traditional antibodies, the synthesis method of nucleic acid aptamers is simple, easy to synthesize and modify in vitro, low cost, wide target molecules, and has great development potential. In view of the problems existing in the existing traditional methods, the present invention combines the fluorescence analysis method with aptamers, which has good reproducibility, simple operation and fast response, to construct a fluorescent aptamer sensor. The introduction of aptamers instead of antibodies as recognition molecules reduces the interference of false positive signals and improves the detection sensitivity; at the same time, the present invention introduces restriction endonucleases to specifically shear DNA and induce chain displacement amplification for cyclic amplification reactions. Chain displacement amplification is a typical isothermal polymerization reaction that produces single-stranded DNA products on templates activated by specific primers. The isothermal amplification reaction is carried out at a constant temperature and has the obvious advantages of high amplification efficiency and fast amplification kinetics. Short oligonucleotides are amplified 106~109 times within a few minutes. ZIF-67 is a type of metal organic framework (MOFs) composed of metal cations and imidazoles as ligands. This porous material has the characteristics of adjustable pore size, large active sites, and large specific surface area, and is widely used in catalysis and adsorbents. ZIF-67 can quench single-stranded DNA-modified fluorescent probes through energy resonance transfer, but cannot quench double-stranded DNA-modified fluorescent probes. Based on this, a series of sensors were prepared to detect targets. However, simple ZIF-67 is sometimes not sufficient in terms of adsorption, photocatalysis, and electrocatalysis due to its limited surface functional groups. In this study, we took advantage of the structural characteristics of ZIF-67 and developed a new method to improve the adsorption capacity of ZIF-67 through a mixed metal ion strategy by incorporating two metal ions into one framework. Among various metals, we chose silver because of its biocompatibility. A green and simple strategy was proposed to prepare highly regular ZIF-67@Ag with significantly enhanced physical properties. This synthetic method not only allows Ag to be distributed in the internal structure of ZIF-67, but also on the surface of ZIF-67, thereby expanding the active specific surface area and pore size of ZIF-67. The larger pore parameters play an important role in the adsorption process, ultimately improving the adsorption capacity. The fluorescence-colorimetric sensing strategy based on the present invention not only overcomes the problems existing in the prior art, but also has the characteristics of simple operation steps, no need for complicated processing procedures, and low use cost, which is conducive to the promotion and application of the invention. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of currently commonly used detection methods, such as low sensitivity, high cost, easy occurrence of false positive signals, and complex sensor preparation. A detection method for detecting ZEN using a chain displacement amplification dual-mode sensor of AuNCs and ZIF-67 is provided, which can detect ZEN in food samples with high sensitivity, simple operation, and low cost.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: A strand displacement amplification dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67 comprises the following steps: (1) Loading amino-modified Apt on the surface of magnetic beads MBs: Carboxyl-modified magnetic beads MBs were washed with a certain amount of PBS, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide were added, and the supernatant was removed by magnetic separation after ultrasound, and the beads were washed with buffer solution. Finally, a certain amount of buffer solution was added to the fixed volume, and the pretreatment of magnetic beads MBs was completed; the above-mentioned magnetic beads were mixed with the diluted amino-modified Apt, and incubated in a constant temperature oscillator at 37°C for a period of time to obtain the MBs@Apt complex through carboxylation reaction; (2) Hairpin H1-induced strand displacement amplification mode: Add different concentrations of ZEN and cDNA to MBs@Apt and incubate in a constant temperature oscillator at 37°C for a certain period of time. After magnetic separation, take the supernatant, add hairpin H1 and incubate in a constant temperature oscillator at 37°C for a period of time, add DNA polymerase Klenow Fragment and restriction endonuclease Nt.BbvCI and incubate in a 37°C incubator for a period of time to perform strand displacement amplification reaction; (3) Preparation of ZIF-67@Ag and AuNCs: 2-Methylimidazole and cobalt nitrate hexahydrate were dissolved in methanol respectively, stirred thoroughly, aged for a period of time, and washed with methanol to obtain ZIF-67. ZIF-67 was dissolved in anhydrous ethanol, stirred thoroughly, and silver nitrate was slowly added to ZIF-67. After stirring, ultrasonication, and standing, it was washed with ethanol and dried in a vacuum at 60°C. Sodium borohydride dissolved in methanol was added to the dried product, and it was washed with methanol after continuous stirring. After centrifugal drying, a dark purple powder was obtained as ZIF-67@Ag. AuNCs were obtained by reacting A-rich DNA single strands with chloroauric acid in a 90°C water bath for a period of time. (4) Preparation of fluorescence channel sensor: Add AuNCs to ZIF-67@Ag and incubate at room temperature for a period of time. AuNCs are adsorbed by ZIF-67@Ag and the fluorescence signal decreases. When ZEN is present, add AuNCs and ZIF-67@Ag to the system after the chain displacement amplification mode is completed and incubate for a period of time to produce a large amount of complexes, which promote the desorption of ZIF-67@Ag and restore the fluorescence signal. Set a fixed excitation wavelength, scanning range and measurement voltage, and record the fluorescence intensity of the emission wavelength under a fluorescence spectrophotometer. Preparation of colorimetric channel sensor: After the fluorescence channel reaction is completed, add TMB and H2O2 and incubate at room temperature for a period of time. At this time, the enzyme-like activity of ZIF-67@Ag is restored, catalyzing TMB to produce a strong color reaction, the absorbance of the system is enhanced, and the absorbance is measured using a UV spectrophotometer. At this point, a chain displacement amplification dual-mode sensor using AuNCs and ZIF-67 to detect ZEN is prepared.

[0007] (5) For food sample testing: According to steps (1) to (5), the fluorescence signal of the food sample of unknown concentration to be tested is measured, wherein the different concentrations of ZEN added in step (2) are food sample extracts in the actual sample testing, and the test results are substituted into the standard curve to obtain the ZEN concentration in the sample.

[0008] The detection mechanism of the present invention is as follows: due to the strong adsorption capacity of ZIF-67@Ag, the AuNCs synthesized on the single-stranded DNA are quenched through the fluorescence resonance energy transfer (FRET) effect, and the fluorescence signal is reduced. After magnetic separation, the supernatant contains a lot of cDNA that can hybridize with the sequence of H1 and start the chain displacement amplification cycle. That is, under the action of DNA polymerase, a completely complementary double-stranded structure cDNA-H1 is formed. The restriction endonuclease Nt.BbvCI is sheared at a specific site, and the single-stranded fragments obtained by shearing are double-stranded structures of cDNA-H1. ZIF-67@Ag adsorbed with the single-stranded DNA with AuNCs is added to the reaction system, and the single-stranded fragments hybridize with the single-stranded DNA with AuNCs to form a double-stranded structure, thereby moving away from ZIF-67@Ag, and the fluorescence signal is restored. Then TMB and H2O2 were added. As the DNA single strand with AuNCs detached from the material surface, its enzyme-like activity was restored, catalyzing TMB to produce a strong color reaction, resulting in an increase in the absorbance of the system. Based on the specific recognition of ZEN by the aptamer, the sensor achieved signal amplification through chain displacement amplification, thereby improving the sensitivity of the sensor. The detection results can be observed by the naked eye in a short time, which enhances the practical application and on-site detection capabilities of the sensor, thereby achieving rapid and highly sensitive detection of ZEN.

[0009] It is further defined that the cDNA nucleotide sequence in step (2) is 5'-ACCATAGATAGATGAAAA-3', and the H1 nucleotide sequence is 5'-ACCATAGATAGAGGGGCTGAGGTTTTCATCTATCTATGGT-3'; It is further defined that the A-rich single-stranded DNA nucleotide sequence in step (3) is 5'-AAAAAAAAAAAAAAAAAAAAAAAAAAAACCATAGATAGAGGGGCTGA-3'; It is further defined that the amount of the aptamer chain Apt in the magnetic beads MBs described in step (1) is in the range of 10 to 120 μL, and the concentration range is 1 to 3 μM, 20 to 30 mg / mL; It is further defined that the concentration range of cDNA and H1 in step (2) is 1 ~ 3 μM, and the dosage range is 10 ~ 20 μL; the incubation time in the constant temperature oscillator is 0.5 ~ 2h; the incubation time in the incubator is 1 ~ 3h; the dosage range of DNA polymerase Klenow Fragment and restriction endonuclease Nt.BbvCI is 1 ~ 5μL, and the concentration range is 1 ~ 10U; It is further defined that the concentration of the A-rich DNA single strand in step (3) is 1 to 3 μM; the dosage is 20 to 50 μL; the concentration of chloroauric acid is 1 to 10 μM; It is further defined that the aging time in step (3) is 12 to 36 hours; the stirring time is 0.5 to 1 hour; the ultrasonic time is 0.5 to 1 hour; the standing time is 1 to 3 hours; the vacuum drying is performed at 60°C for 8 to 15 hours; the stirring time is 0.5 to 1 hour; the reaction time in a water bath at 90°C is 0.5 to 1 hour; It is further defined that the incubation time at room temperature in step (4) is 10 to 30 minutes; It is further defined that the incubation time at room temperature in step (5) is 10 to 30 minutes.

[0010] Compared with the prior art, the present invention has the following significant advantages: 1. The present invention successfully prepared a dual-functional nanocomposite material ZIF-67@Ag, which has more excellent optical properties and peroxidase-like activity and is very suitable for the construction of dual-mode sensors.

[0011] 2. Based on the fluorescence colorimetric dual-mode analysis strategy of the present invention, self-verification is performed through two independently acquired signals, so that the detection results have higher accuracy and reliability, and the detection process is convenient and fast, which is conducive to the promotion and application of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the strand displacement amplification dual-mode sensor based on AuNCs and ZIF-67@Ag for detecting ZEN.

[0013] Figure 2 The fluorescence intensity of AuNCs of the sensor constructed in Example 1 of the present invention in the absence of ZEN (solid line) and the presence of 100 ng / mL ZEN (dotted line).

[0014] Figure 3 The absorbance of ZIF-67@Ag of the sensor constructed in Example 1 of the present invention in the absence of ZEN (solid line) and the presence of 100 ng / mL ZEN (dotted line).

[0015] Figure 4Comparative graph of the selectivity of the sensor constructed in Example 1 of the present invention to ZEN in the presence of other interfering toxins (A: fluorescence; B: colorimetry). DETAILED DESCRIPTION

[0016] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Example 1

[0017] A method for preparing and applying a dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag, wherein the method for implementing the method is as follows: Figure 1 shown.

[0018] A method for preparing a dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag chain displacement amplification comprises the following steps: (1) Loading amino-Apt on the surface of magnetic bead MBs: Take 60 μL of carboxylated MBs and wash them three times with PBS, add 100 μL of 10 mg / mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 100 μL of 10 mg / mL N-hydroxysuccinimide, sonicate for 0.5 h, remove the supernatant, wash three times with PBS, and finally add 100 μL of PBS to make up to 30 mg / mL MBs. Take the above 20 μL MBs and combine it with 100 μL Apt (1 μM) chain, shake overnight, wash once with PBS, and make up to 100 μL to obtain MBs@Apt.

[0019] (2) Preparation of hairpin H1-induced strand displacement amplification mode: Take 10 μL MBs@Apt, add 10 μL ZEN of different concentrations and 10 μL cDNA (1 μM), incubate at 37°C for 1 h, take the supernatant after magnetic separation, add 10 μL 1 μM H1, incubate at 37°C for 1 h, add 1 μL 10mM dNTP, 1 μL 5U Klenow Fragment, 1 μL 10U Nt.BbvCI, 4 μL 1×Klenow Buffer, 4 μL 1×Cut Smart Buffer, incubate at 37°C for 2 h to produce a large amount of single-stranded DNA.

[0020] (3) Binding of single-stranded DNA with AuNCs fluorescent probe: 10 μL of 1 mg / mL ZIF-67@Ag was added to the system after the chain displacement amplification reaction and reacted for 10 min, and the fluorescence was enhanced.

[0021] (4) ZIF-67@Ag catalyzed TMB color development reaction: After the fluorescence channel reaction in (3) was completed, 10 μL of 60 mM TMB and 10 μL of 60 mM H2O2 were added and reacted for 10 min. The absorbance increased. Thus, a dual-mode sensor for detecting ZEN using chain displacement amplification of AuNCs and ZIF-67@Ag was prepared.

[0022] (5) Establishment of standard curve: Add 10 μL of ZEN standard solution of different concentrations into step (2), and detect the fluorescence intensity and absorbance after incubation in steps (2), (3), and (4). Perform linear fitting with the logarithm of ZEN concentration as the horizontal axis and the fluorescence intensity (absorbance) as the vertical axis to establish the standard curve of the sensor for ZEN.

[0023] The specific steps for detecting the fluorescence intensity of the sample are as follows: use a micro-cuvette to hold the sample to be tested, set the excitation wavelength of the fluorescence spectrophotometer to 280 nm, the emission wavelength measurement range to 350-650 nm, select the slit to be 10 nm, measure the voltage to be 400 V, and record the fluorescence intensity at the emission wavelength of 490. The specific steps for detecting the absorbance of the sample are as follows: use a micro-cuvette to hold the sample to be tested, scan the wavelength in the range of 500-800 nm, and use an ultraviolet spectrophotometer to measure the absorbance at 652 nm.

[0024] In order to verify that the prepared dual-mode sensor using chain displacement amplification of AuNCs and ZIF-67@Ag has specific recognition of ZEN, ZEN standard was added to 50 mM Tris-HCl to make the concentration of ZEN in the sample 50 ng / mL; 50 mM Tris-HCl was used to prepare other interfering toxins (AFB1, FB1, OTA, OTB, DON) standard solutions, all with a concentration of 500 ng / mL. The above-mentioned different interfering toxin standards were detected according to the detection system constructed in Example 1, and the detection results are shown in the figure. Figure 3 As shown, it is explained that the method of the present invention has a higher selectivity for ZEN. Example 2

[0025] The preparation method and application of the dual-mode sensor of chain displacement amplification of AuNCs and ZIF-67@Ag, and its practical application, include the following steps: (1) Actual sample treatment: Take 1g of food spiked sample (corn flour), add it to 5mL methanol / water solution (ratio 6:4), place it on a shaker for 30 minutes, then take it out and centrifuge it for 10 minutes at 8000 rpm / min. After filtering the supernatant with a 0.45μM organic filter membrane, dilute the filtrate 100 times with 50mM Tris-HCl buffer. Prepare ZEN spiked corn flour sample extracts of different concentrations according to the above pretreatment method. Take an appropriate amount of beer in a 100mL beaker and thoroughly ultrasonicate it to remove bubbles such as carbon dioxide. Measure 1mL of beer sample and mix it with different concentrations of ZEN solution, filter it with a 0.45μM water filter membrane, and collect the sample treatment filtrate. Dilute the filtrate 100 times with 50mM Tris-HCl buffer to prepare spiked beer sample extracts of different concentrations.

[0026] (2) When corn flour is used as the actual sample for determination, 10 times and 40 times the standard amount of OTA standard is added to the corn flour, respectively, with an addition amount of 5 ng / mL as the reference. Take 10 μL of the sample solution, and measure the fluorescence intensity at the emission wavelengths of 350 nm and 650 nm according to steps (1)-(4) of Example 1; and measure the absorbance at 652 nm using an ultraviolet spectrophotometer. Substitute the standard curve detected in Example 1 to obtain the ZEN concentration in the sample, repeat the measurement 3 times for each sample and take the average value. The average recovery rate of the fluorescence channel of the dual-mode sensor is calculated to be 91.3%-119.8%; the average recovery rate of the colorimetric channel of the dual-mode sensor is calculated to be 97.2%-106.7%. When beer is used as the actual sample for determination, 10 times and 40 times the standard amount of ZEN standard is added to the beer, respectively, with an addition amount of 5 ng / mL as the reference. Take 10 μL of sample solution and measure the fluorescence intensity at emission wavelengths of 350 nm and 650 nm according to steps (1) to (4) of Example 1; use a UV spectrophotometer to measure the absorbance at 652 nm. Substitute the standard curve detected in Example 1 to obtain the ZEN concentration in the sample. Repeat the measurement three times for each sample and take the average value. The average recovery rate of the fluorescence channel of the dual-mode sensor is calculated to be 86.8%-100.3%; the average recovery rate of the colorimetric channel of the dual-mode sensor is 100.9%-102.3%.

[0027] It has been verified that the prepared fluorescent aptamer sensor has the characteristics of high accuracy, wide linear range and low detection limit for the detection of zearalenone. At the same time, the detection results of actual samples (such as corn flour and beer) show that the prepared sensor has very good practical application value.

[0028] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0029] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing a dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag, characterized in that: The following steps are involved: (1) Loading amino-modified Apt on the surface of magnetic beads MBs: Carboxyl-modified magnetic beads MBs were washed with a certain amount of buffer, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide were added, the supernatant was removed by magnetic separation after ultrasound, and the beads were washed with a buffer solution. Finally, a certain amount of buffer was added to the fixed volume, and the pretreatment of MBs was completed; the above-mentioned magnetic beads were mixed with the diluted amino-modified Apt, and placed in a constant temperature oscillator at 37°C for a period of time to incubate MBs@Apt through carboxylation reaction; (2) Hairpin H1-induced strand displacement amplification mode: Add different concentrations of ZEN and cDNA to MBs@Apt and incubate in a constant temperature oscillator at 37°C for a certain period of time. After magnetic separation, take the supernatant, add hairpin H1 and incubate in a constant temperature oscillator at 37°C for a period of time, add DNA polymerase Klenow Fragment and restriction endonuclease Nt.BbvCI and incubate in a 37°C incubator for a period of time to perform strand displacement amplification reaction; (3) Preparation of ZIF-67@Ag and AuNCs: 2-Methylimidazole and cobalt nitrate hexahydrate were dissolved in methanol, stirred and aged for a period of time, and washed with methanol to obtain ZIF-67; ZIF-67 was dissolved in anhydrous ethanol, stirred and aged, and then silver nitrate was slowly added to ZIF-67. After stirring, ultrasonication, and standing, the product was washed with ethanol and dried in a vacuum at 60°C; sodium borohydride dissolved in methanol was added to the dried product, stirred continuously, washed with methanol, and centrifuged to obtain a dark purple powder, namely ZIF-67@Ag; A-rich DNA single strands were reacted with chloroauric acid in a 90°C water bath for a period of time to obtain AuNCs as fluorescent probes; (4) Preparation of fluorescence channel sensor: Add AuNCs to ZIF-67@Ag and incubate at room temperature for a period of time. AuNCs are adsorbed by ZIF-67@Ag and the fluorescence signal decreases. When ZEN is present, add AuNCs and ZIF-67@Ag to the system after the chain displacement amplification mode is completed and incubate for a period of time to produce a large number of complexes, promote the desorption of ZIF-67@Ag, and restore the fluorescence signal. Set a fixed excitation wavelength, scanning range and measurement voltage, and record the fluorescence intensity of the emission wavelength under a fluorescence spectrophotometer. Preparation of colorimetric channel sensor: After the fluorescence channel reaction is completed, add TMB and H2O2 and incubate at room temperature for a period of time. At this time, the enzyme-like activity of ZIF-67@Ag is restored, catalyzing TMB to produce a strong color reaction, the absorbance of the system is enhanced, and the absorbance is measured using an ultraviolet spectrophotometer. At this point, a chain displacement amplification dual-mode sensor for detecting ZEN using AuNCs and ZIF-67 was prepared.

2. The method for preparing a dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag according to claim 1, characterized in that: In step (1), the buffer solution is one or more of Tris-HCl, PBS, and HEPES.

3. The method for preparing a dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag according to claim 1, characterized in that: In step (1), the dosage of magnetic beads MBs ranges from 10 to 120 μL, the concentration ranges from 20 to 30 mg / mL, the dosage of amino-Apt ranges from 10 to 120 μL, the concentration ranges from 1 to 3 μM, and the MBs and amino-Apt are incubated at a constant temperature with shaking for 8 to 12 hours.

4. The method for preparing a dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag according to claim 1, characterized in that: In step (2), the cDNA nucleotide sequence is 5'-ACCATAGATAGATGAAAA-3', and the H1 nucleotide sequence is 5'-ACCATAGATAGAGGGGCTGAGGTTTTCATCTATCTATGGT-3'.

5. The method for preparing a dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag according to claim 1, characterized in that: In step (2), the concentration range of cDNA and H1 is 1-3 μM, and the dosage range is 10-20 μL; the incubation time in the constant temperature oscillator is 0.5-2 h; the incubation time in the incubator is 1-3 h; the dosage range of DNA polymerase KlenowFragment and restriction endonuclease Nt.BbvCI is 1-5 μL, and the concentration range is 1-10 U.

6. The method for preparing a dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag according to claim 1, characterized in that: In step (3), the A-rich single-stranded DNA nucleotide sequence is 5'-AAAAAAAAAAAAAAAAAAAAAAAAAAAACCATAGATAGAGGGGCTGA-3'.

7. The method for preparing a dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag according to claim 1, characterized in that: In step (3), the concentration of the A-rich DNA single strand is 1 to 3 μM; the dosage is 20 to 50 μL; the concentration of chloroauric acid is 1 to 10 μM; the dosage is 5 to 20 μL.

8. The method for preparing a dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag according to claim 1, characterized in that: In step (3), the aging time is 12 to 36 hours; the stirring time is 0.5 to 1 hour; the ultrasonic time is 0.5 to 1 hour; the standing time is 1 to 3 hours; the vacuum drying is performed at 60°C for 8 to 15 hours; the stirring time is 0.5 to 1 hour; and the reaction time in a 90°C water bath is 0.5 to 1 hour.

9. The method for preparing a dual-mode sensor for detecting ZEN based on AuNCs and ZIF-67@Ag according to claim 1, characterized in that: In step (4), the room temperature incubation time is 10 to 30 minutes.

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