Preparation method of dual-mode sensor based on ZnNi nanoflower and difunctional NiFe Prussian blue analogue

Abstract

The invention relates to a preparation method and application of an electrochemical-colorimetric dual-mode sensor based on nanoflowers and a bifunctional NiFe-PBA nano enzyme. The NiFe-PBA loaded gold-platinum bimetallic nanoparticles have excellent oxidation-reduction activity and peroxidase activity, and are used as a bifunctional probe to construct an electrochemical colorimetric dual-mode sensor. More signal probes are introduced into the branched DNA structure to participate in the reaction, the specificity of the sensor is enhanced through specific recognition of the aptamer on OTA, and finally, an electrochemical and colorimetric dual-mode channel is ingeniously constructed by utilizing a magnetic separation technology, so that environmental interference is effectively reduced, and false positive signals are avoided. NiFe-PBA loaded gold-platinum bimetallic nanoparticles participate in the dual-mode channels, so that the experimental operation is simplified while the signal response caused by the target is realized. The dual-mode sensor constructed by the invention has the characteristics of sensitivity, reliability, rapidness and stability, and is beneficial to popularization and application. And the biosensor has good anti-interference capability and practical application capability, and provides a novel biosensing platform for practical sample analysis.

Description

Technical Field

[0001] The invention relates to a preparation method and application of a dual-mode sensor based on ZnNi nanoflowers and dual-functional NiFe Prussian blue analogs. Background Art

[0002] Mycotoxins are a class of highly toxic secondary metabolites produced by specific fungi. Their presence in food and feed poses a serious threat to human and animal health. Ochratoxin A (OTA) is an important group of mycotoxins. It is mainly produced by two genera, Aspergillus and Penicillium, and is a typical biotoxin. Studies have shown that among the six known ochratoxins, OTA is the most toxic. It is a highly toxic chemical that can cause damage to the liver and kidneys. Long-term exposure may also increase the risk of cancer. In addition, OTA has an immunosuppressive effect and may weaken the host's immune system, making it more susceptible to invasion by other pathogens. For this reason, the International Agency for Research on Cancer classified OTA as a possible human carcinogen Group 2B in 1993 and strictly regulated the level of OTA in food. Taking the European Union as an example, the maximum residue limit of OTA in cereals is 0.5 μg / kg, and the maximum residue limits of wine (or grape juice) and coffee products are 0.2 μg / kg and 0.5 μg / kg, respectively. GB 2761-2017 "National Food Safety Standard Limits of Mycotoxins in Food" stipulates that the limit for cereals and their products, beans and their products, nuts and seeds, and ground coffee is 5.0 μg / kg, and the limit for alcohol is 2.0 μg / kg.

[0003] At present, the traditional analytical methods for OTA detection include thin layer chromatography (TLC), high performance liquid chromatography (HPLC), high performance liquid chromatography-mass spectrometry (HPLC-MS), high performance liquid chromatography-fluorescence detection (HPLC-FD), etc. Although traditional detection methods have excellent performance in sensitivity and accuracy, they also have some limitations, especially in the application of rapid on-site detection. Therefore, it is of great significance to seek cost-effective, easy-to-operate and stable detection methods for rapid on-site detection.

[0004] As new detection technologies, electrochemical sensors, colorimetric sensors and fluorescent sensors are gradually receiving widespread attention from both inside and outside the industry. Compared with traditional detection methods, these sensing technologies have significant advantages such as simple operation, low cost and fast response speed. However, most of these sensing methods are based on single signal changes induced by the target, which are easily interfered by the operation method and environmental factors, resulting in signal fluctuations. In order to improve the anti-interference ability, reliability and accuracy of detection, an innovative strategy has emerged in recent years-a dual-mode sensor based on colorimetry-electrochemistry. This sensor combines the high sensitivity of electrochemical signals with the intuitive color changes of colorimetry to achieve more accurate detection through dual signal channels. Dual-mode detection technology can not only give full play to the advantages of each channel, but also self-verify by comparing the detection results of the two channels, thereby improving the accuracy and reliability of the detection results.

[0005] Prussian blue analog (PBA) is a typical hexacyanoferrate coordination compound. The redox of the various metal ions it contains makes it have excellent redox activity and can provide a clear redox current for electrochemical analysis. Interestingly, as an iron-containing nanomaterial, PBA has excellent peroxidase-like activity and can catalyze the decomposition of hydrogen peroxide and the oxidation of the chromogenic substrate, which is conducive to naked eye observation. Based on the above considerations, PBA has become an ideal bifunctional probe that can simply make an electrochemical-colorimetric dual-signal biosensor for accurate and reliable analysis.

[0006] The present invention aims at the problems existing in the existing detection technology, such as poor detection accuracy and unstable detection results, and constructs a novel electrochemical colorimetric dual-mode sensor. First, a bifunctional NiFe-PBA-loaded gold-platinum bimetallic nanoparticle probe with excellent redox activity and peroxidase-like activity is prepared, and it is applied to construct an electrochemical colorimetric dual-signal readout sensor. The branched DNA structure is designed to allow more signal probes to participate in the reaction, and the specific recognition of OTA by the aptamer is combined to enhance the sensor specificity. Finally, the electrochemical and colorimetric dual-mode channels are cleverly constructed using magnetic separation technology, which greatly reduces environmental interference, avoids false positive signals, and improves the reliability of the sensor. NiFe-PBA-loaded gold-platinum bimetallic nanoparticles are involved in the dual-mode channel, which not only realizes the signal response caused by the target, but also simplifies the experimental operation. The dual-mode sensing strategy based on the present invention has the characteristics of being more sensitive, reliable, fast and stable while overcoming the problems existing in the above prior art, which is conducive to the promotion and application of the invention. Summary of the invention

[0007] A method for preparing a dual-mode sensor based on ZnNi nanoflowers and a dual-functional NiFe Prussian blue analogue is carried out according to the following steps: (1) Preparation of ZnNi nanoflowers: Dissolve zinc nitrate, nickel acetylacetonate, terephthalic acid and polyvinyl pyrrolidone in a mixed solution of N, N-dimethylformamide, N, N-dimethylacetamide and ethanol and disperse them evenly by magnetic stirring. Transfer the obtained mixed solution to a high-pressure reactor for incubation, collect the product by high-speed centrifugation, and vacuum dry to constant weight to prepare ZnNi nanoflowers.

[0008] (2) Preparation of ZnNi nanoflowers loaded with gold nanoparticles: Metal nanoparticles were generated in situ on the nanoflowers prepared above by an in situ growth method. First, the ZnNi nanoflowers were dispersed in water, and chloroauric acid solution was added after ultrasonication and stirred thoroughly. Then, sodium borohydride was added and stirred thoroughly for 30 minutes. The product was collected by high-speed centrifugation and vacuum dried to constant weight to obtain a ZnNi nanoflower-loaded gold nanoparticle composite material.

[0009] (3) Preparation of NiFe-PBA nanozyme: Nitrate and trisodium citrate were dissolved in ultrapure water to obtain liquid A, and potassium ferrocyanide was dissolved in ultrapure water to obtain liquid B. Liquid A and liquid B were mixed and stirred overnight, the product was collected by high-speed centrifugation, and vacuum dried to constant weight to prepare NiFe-PBA nanozyme.

[0010] (4) Preparation of nanozyme-loaded bimetallic nanoparticles: First, the above-mentioned NiFe-PBA is dispersed in water, and precious metal salt solution A and precious metal salt solution B are added and stirred thoroughly. Then, a reducing agent is added and stirred thoroughly. The product is collected by high-speed centrifugation and vacuum dried to constant weight to obtain a NiFe-PBA-loaded bimetallic nanoparticle composite material.

[0011] (5) Preparation of bifunctional signal probe: Take a certain amount of NiFe-PBA loaded bimetallic nanoparticles and dissolve them in water, connect them to the C1 chain and C2 chain respectively, then mix the products in proportion to make the C1 chain and C2 chain hybridize to form a double chain, collect the products by centrifugation, and disperse them with buffer solution to obtain a branched bifunctional nanoprobe, which is placed at 4 °C for use.

[0012] (6) Preparation of MBs@OTA apt system and identification of target: The aptamer is connected to the MBs through the strong binding between the amino group on the aptamer chain and the carboxyl group coated on the surface of the magnetic beads MBs. The C1 chain and the C2 chain form a branched structure through base complementary pairing. The other end of the C1 chain can form a double-stranded structure by base complementary pairing with the OTA apt. When OTA is present, OTA apt binds preferentially to OTA, freeing the C1-C2 system, and the electrochemical and colorimetric dual-mode channel detection is performed using magnetic separation technology.

[0013] (7) Construction of dual-mode channel: After magnetic separation of the above mixed solution, the supernatant and precipitate are used for detection of electrochemical channel and colorimetric channel respectively. In the electrochemical mode, the nanoflower loaded with metal nanoparticles is first modified on the electrode surface, and then the hairpin chain H1 is added to fix it on the electrode surface, and the binding site is blocked with MCH. Finally, the supernatant obtained by magnetic separation is added to the electrode. The C1 chain in the supernatant is complementary to H1, so that the branched bifunctional nanoprobe is fixed on the electrode to generate an electrochemical signal response. In the colorimetric mode, the precipitate obtained by magnetic separation is washed with a buffer solution, and only a small amount of C1 chain is connected to MBs by base complementary pairing with OTA apt. After incubation with hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine, a color change occurs, resulting in a change in absorbance.

[0014] It is further defined that in step (3), the nitrate is one of nickel nitrate, copper nitrate and manganese nitrate.

[0015] It is further defined that in step (4), the noble metal salt solution is one or more of tetrachloroauric acid, chloroplatinic acid, and chloropalladic acid, and the reducing agent is one or more of sodium borohydride, ascorbic acid, citrate, and hydrazine hydrate.

[0016] It is further defined that in steps (5) and (6), the concentration of the DNA chain is 0.5 to 4 μM, the volume used is 1 to 20 μL, and the incubation time is 0.5 to 2.5 h. The sequence of the single-chain C1 is: 5'-TTT TTT CTA TCT AAT TGAGGG AAT GTC CGA TGC TCC CTT TA-3'; the sequence of the single-chain C2 is: 5'-CCC TCA ATT AGA TAG TTTTTT-3'; the sequence of the hairpin chain H1 is: 5'-GTG CCC GTG AGC ATC GGA CAT TC-3'.

[0017] It is further defined that in step (7), the volume of the hydrogen peroxide is 10 ~ 20 μL, and the concentration is 1 ~ 30 mM; the volume of the 3,3',5,5'-tetramethylbenzidine is 1 ~ 10 μL, and the concentration is 1 ~ 10 mM, and the incubation time is 5 ~ 10 min.

[0018] It is further defined that in steps (5), (6) and (7), the buffer solution is one or more of Tris-HCl buffer, PBS buffer, acetate buffer solution and PB buffer.

[0019] Compared with the prior art, the present invention has the following significant advantages: 1. The present invention prepares bifunctional NiFe-PBA loaded bimetallic nanoparticles, which have both electrochemical signals and peroxidase activity, realize electrochemical and colorimetric dual-mode detection, and improve the anti-interference ability, reliability and accuracy of sensor detection.

[0020] The present invention designs a branched DNA structure to allow more signals to participate in the reaction, and combines target-specific recognition and magnetic separation technology to achieve sensitive detection of the target.

[0021] 3. The present invention successfully prepared ZnNi bimetallic nanoflowers with high specific surface area loaded with a large number of metal nanoparticles, which provided a large number of active sites and high conductivity for the sensor to achieve signal amplification.

[0022] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the preparation method of a dual-mode sensor based on ZnNi nanoflowers and bifunctional NiFe Prussian blue analogs.

[0024] Figure 2 Electrochemical (A) and colorimetric (B) detection results of the dual-mode sensor constructed in Example 1 of the present invention before (dashed line) and after (solid line) the addition of 50 ng / mL OTA.

[0025] Figure 3 Electrochemical (A) and colorimetric (B) standard curves of the dual-mode sensor constructed in Example 1 of the present invention.

[0026] Figure 4 The electrochemical (A) and colorimetric (B) selectivity of the dual-mode sensor constructed according to Example 1 of the present invention for OTA in the presence of other interfering toxins. DETAILED DESCRIPTION

[0027] 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

[0028] A preparation method and application of a dual-mode sensor based on ZnNi nanoflowers and dual-functional NiFe Prussian blue analogs, the implementation method thereof is as follows Figure 1 shown.

[0029] The specific steps are as follows: (1) Preparation of NiFe-PBA nanozyme: 0.1745 g of nickel nitrate hexahydrate and 0.2323 g of trisodium citrate were dissolved in 20 mL of ultrapure water to prepare solution A. Then, 0.1317 g of potassium ferrocyanide was dissolved in another 20 mL of ultrapure water to prepare solution B. The above solution A and solution B were mixed and vigorously stirred at room temperature for 12 h to finally obtain a bright yellow Ni-Fe PBA product. The precipitate was collected by centrifugation, washed with ultrapure water, and vacuum dried at 60°C.

[0030] (2) Preparation of AuPt NPs / NiFe-PBA: 4 mg of Ni-Fe PBA was ultrasonically dispersed in 2 mL of ultrapure water. 250 μL of 10 mM tetrachloroauric acid solution and 250 μL of 10 mM chloroplatinic acid solution were added respectively. The mixture was magnetically stirred for 30 min. Then, 700 μL of 10 mM ascorbic acid solution was added dropwise to the mixed solution. The mixture was magnetically stirred for 1 h. The precipitate was collected by centrifugation and washed with ultrapure water, and then dried in a vacuum at 60 °C.

[0031] (3) Preparation of ZnNi-MOF: N, N-dimethylacetamide, N, N-dimethylformamide and ethanol were prepared into a 25.6 mL mixed solution in a volume ratio of 5:5:6. 25 mg zinc nitrate, 37 mg nickel acetylacetonate, 12 mg terephthalic acid and 1.3658 g polyvinyl pyrrolidone were added to the mixed solution. After thorough dissolution, the resulting mixture was transferred to a 50 ml Teflon-lined autoclave and reacted at 150 °C for 4 hours. The precipitate was collected by centrifugation, washed alternately with N, N-dimethylacetamide and ethanol, and dried in vacuum at 60 °C to obtain a flower-like ZnNi-MOF.

[0032] (4) Preparation of Au NPs / ZnNi-MOF: Weigh 3 mg ZnNi-MOF and ultrasonically disperse it in 1 mL water. Then add 225 μL 25 mM tetrachloroauric acid solution and stir magnetically for 20 min. Add 2 mL 0.1 M sodium borohydride solution to the above solution and stir magnetically for 30 min. Collect the precipitate by centrifugation, wash it with ultrapure water, and dry it in vacuum at 60 °C.

[0033] (5) Preparation of AuPt NPs / NiFe-PBA-C1-C2: 2 μL of 100 μM C1 chain was added to 10 μL TCEP and incubated at 37°C for 1 h to reduce disulfide bonds. Then, 25 μL 8 mg / mL and 11 μL Tris-HCl were added and incubated at 37°C for 2 h with shaking. After centrifugation, the mixture was dispersed with Tris-HCl for later use to obtain AuPt NPs / NiFe-PBA-C1. The same method was used to obtain AuPt NPs / NiFe-PBA-C2. AuPt NPs / NiFe-PBA-C1 and AuPt NPs / NiFe-PBA-C2 were mixed in equal proportions and incubated at 37°C for 1 h with shaking. After centrifugation, the mixture was dispersed with Tris-HCl and stored at 4°C to obtain AuPt NPs / NiFe-PBA-C1-C2.

[0034] (6) Preparation of magnetic bead OTA apt system and identification of target: 3 μL 10 μM OTA apt chain, 3 μL 30 mg / mL MBs and 24 μL Tris-HCl were mixed and incubated overnight on a shaker. After that, the mixture was washed and dispersed with PBS by magnetic separation to obtain the MBs@ OTA apt system. 15 μL OTA standard solution of different concentrations and 15 μL of the above AuPt NPs / NiFe-PBA-C1-C2 were added to the MBs@ OTA apt system and incubated on a shaker for 40 min. With the change of OTA concentration, the amount of AuPt NPs / NiFe-PBA-C1-C2 connected to MBs changed, and the electrochemical and colorimetric dual-mode channel detection was performed by magnetic separation technology.

[0035] (7) Construction of dual-mode channel: After magnetic separation of the above mixed solution, the supernatant was used for electrochemical mode detection and the precipitate was used for colorimetric mode detection. In the electrochemical mode, 5 μL of 0.25 mg / mL Au NPs / ZnNi-MOF was first added to the electrode and incubated at 37°C for 1 h. Then 5 μL of 1.5 μM hairpin chain H1 was added and incubated at 37°C for 1 h, and then MCH was used to block the unbound sites. Finally, 5 μL of the supernatant obtained by magnetic separation was added to the electrode surface and incubated at 37°C for 1 h. After each step, the electrode surface was cleaned with Tris-HCl buffer solution, and the electrode was placed in PBS buffer solution, and the electrochemical signal changes were detected by square wave voltammetry. In the colorimetric mode, the precipitate obtained by magnetic separation was washed with Tris-HCl buffer solution, and then 10 μL of 5mM 3,3',5,5'-tetramethylbenzidine, 15 μL of 30mM hydrogen peroxide and 180 μL of acetate buffer solution were added. After incubation at room temperature for 5 min, a color change occurred and the absorbance change at 652 nm was detected by a UV spectrophotometer.

[0036] (8) Establishment of standard curve: Add OTA standard solutions of different concentrations to step (6) to obtain sample test solutions of different gradients, and detect them using an electrochemical workstation and a UV spectrophotometer, respectively. Use the logarithmic value of OTA concentration as the horizontal axis and the current signal as the vertical axis to perform linear fitting to establish a standard curve for the electrochemical mode. Use the logarithmic value of OTA concentration as the horizontal axis and the absorbance as the vertical axis to perform linear fitting to establish a standard curve for the colorimetric mode.

[0037] like Figure 2 Shown are the chemical (A) and colorimetric (B) detection results of the dual-mode sensor constructed in Example 1 of the present invention before (solid line) and after (dashed line) the addition of 50 ng / mL OTA.

[0038] like Figure 3 As shown, the electrochemical (A) and colorimetric (B) standard curves of the dual-mode sensor constructed in Example 1 of the present invention for detecting AFB1. Example 2

[0039] A method for preparing a dual-mode sensor based on ZnNi nanoflowers and a dual-functional NiFe Prussian blue analog, and its practical application, comprises the following steps: In order to verify that the prepared electrochemical-colorimetric dual-mode sensor based on nanoflowers and bifunctional NiFe-PBA nanozymes has specific recognition of OTA, OTA standard was added to the buffer solution to make its concentration 50 ng / mL; other interfering toxins (FB1, T-2, AFB1, OTB, DON, ZEN) standard solutions with a concentration of 500 ng / mL were prepared with the buffer solution. The above-mentioned different interfering toxin standards were detected according to the detection system constructed in Example 1, and the detection results are shown as follows: Figure 4 As shown in electrochemistry (A) and colorimetry (B), it is shown that the method of the present invention has high selectivity for OTA. Example 3

[0040] A method for preparing a dual-mode sensor based on ZnNi nanoflowers and a dual-functional NiFe Prussian blue analog, and its practical application, comprises the following steps: Actual sample processing: For edible oil samples, weigh 5.0 g of edible oil spiked sample into a 50 mL stoppered measuring cylinder, add 20 mL of petroleum ether, shake to dissolve the oil sample, add 10 mL of methanol / water solution (ratio 7:3), high-speed vortex mixing and extraction for 5 min, let stand for stratification, take 5 mL of the lower layer extract and put it into the refrigerator, quickly freeze it at -15 °C for 20 min, filter the extract with a 0.45 μm filter membrane while it is cold, and mix it with OTA standard solutions of different concentrations to obtain spiked edible oil extract. For red wine samples, take 1 mL of wine sample, mix it with OTA standard solutions of different concentrations, and collect the mixed sample by filtering with a 0.45 μm organic filter membrane to obtain spiked red wine extract.

[0041] Sample detection: Take 2.5 μL of sample solution, measure the electrical signal and absorbance according to the steps of Example 1, and insert them into the standard curve to obtain the concentration of OTA in the sample.

[0042] When edible oil is used as the actual sample for determination, 10 ng / mL is used as the reference, and 0.1 times and 10 times the reference amount of OTA standard are added to corn flour, respectively. 2.5 μL of sample solution is taken, and the electrical signal and absorbance are measured according to steps (1) to (8) of Example 1, and the standard curve detected in Example 1 is substituted to obtain the OTA concentration in the sample. Each sample is measured three times and the average value is taken. The average recovery rate in the electrochemical mode is calculated to be 95.7%-106.3%, and the average recovery rate in the colorimetric mode is 94.9%-102.9%.

[0043] It has been verified that the prepared electrochemical-colorimetric dual-mode sensor has the characteristics of high accuracy, good sensitivity and good stability in the detection of OTA. At the same time, the test results of actual samples (such as edible oil and red wine) show that the prepared dual-mode sensor has very good practical application value.

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

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

Claims

1. A method for preparing a dual-mode sensor based on ZnNi nanoflowers and dual-functional NiFe Prussian blue analogs, characterized in that: The following steps are involved: (1) Preparation of ZnNi nanoflowers and gold nanoparticles loaded thereon: zinc nitrate, nickel acetylacetonate, terephthalic acid and polyvinyl pyrrolidone were dissolved in a mixed solution of N, N-dimethylformamide, N, N-dimethylacetamide and ethanol, and incubated in a high-pressure reactor to obtain ZnNi nanoflowers, the ZnNi nanoflowers were ultrasonically dispersed in water, chloroauric acid solution was added and stirred thoroughly, and then sodium borohydride was added and stirred thoroughly to obtain a ZnNi nanoflower-loaded gold nanoparticle composite material; (2) Preparation of bifunctional nanoprobes: A mixed solution of nitrate and trisodium citrate was mixed with a potassium ferrocyanide solution to obtain a NiFe Prussian blue analog nanozyme NiFe-PBA; NiFe-PBA was dispersed in water, and noble metal salt solution A and noble metal salt solution B were added and stirred thoroughly; then a reducing agent was added and stirred thoroughly to obtain a NiFe-PBA-loaded bimetallic nanoparticle composite material; C1 chain and C2 chain were respectively connected to the NiFe-PBA-loaded bimetallic nanoparticle composite material, and then mixed in equal proportions to obtain a branched bifunctional nanoprobe; the probe was dispersed in a buffer solution and placed at 4°C for use; (3) Preparation of magnetic beads and OTA apt system and identification of targets: The aptamer is connected to the MBs through the strong binding interaction between the amino group on the aptamer chain and the carboxyl group coated on the surface of the magnetic beads MBs. The MBs@OTA apt system is obtained by magnetic separation and washing with buffer solution. The C1 chain and the C2 chain form a branched structure through base complementary pairing. The other end of the C1 chain and the OTAapt base complementary pairing form a double-stranded structure. When OTA is present, OTA apt binds preferentially to OTA, freeing the C1-C2 system. Magnetic separation technology is used for electrochemical and colorimetric dual-mode channel detection. (4) Construction of dual-mode channel: After magnetic separation of the above mixed solution, the supernatant and precipitate are used for detection of electrochemical channel and colorimetric channel, respectively. In the electrochemical mode, the ZnNi nanoflower-loaded gold nanoparticle composite material is first modified on the electrode surface, and then the hairpin chain H1 is added to fix it on the electrode surface, and the binding site is blocked with MCH. Finally, the supernatant obtained by magnetic separation is added to the electrode. The C1 chain in the supernatant is complementary to H1, so that the branched bifunctional nanoprobe is fixed on the electrode to generate an electrochemical signal response. In the colorimetric mode, the precipitate obtained by magnetic separation is washed with buffer solution, and only a small amount of C1 chain is complementary to OTA apt and connected to MBs. After incubation with hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine, a color change occurs, resulting in a change in absorbance.

2. The method for preparing a dual-mode sensor based on ZnNi nanoflowers and bifunctional NiFe Prussian blue analogues according to claim 1, characterized in that: In step (2), the nitrate is one of nickel nitrate, copper nitrate and manganese nitrate; the noble metal salt solution A and the noble metal salt solution B are two of tetrachloroauric acid, chloroplatinic acid and chloropalladic acid; the reducing agent is one or more of sodium borohydride, ascorbic acid, citrate and hydrazine hydrate; and the stirring time is between 0.5 and 12 h.

3. The method for preparing a dual-mode sensor based on ZnNi nanoflowers and bifunctional NiFe Prussian blue analogues according to claim 1, characterized in that: In step (3), the volume of the MBs is 1 to 10 μL.

4. The method for preparing a dual-mode sensor based on ZnNi nanoflowers and bifunctional NiFe Prussian blue analogues according to claim 1, characterized in that: In steps (2), (3) and (4), the DNA chain is 0.5 to 4 μM, the volume used is 1 to 20 μL, and the incubation time is 0.5 to 2.5 h; the sequence of the single-stranded C1 is: 5'-TTT TTT CTA TCT AAT TGAGGG AAT GTC CGA TGC TCC CTT TA-3'; the sequence of the single-stranded C2 is: 5'-CCC TCA ATT AGA TAG TTTTTT-3'; the sequence of the hairpin chain H1 is: 5'-GTG CCC GTG AGC ATC GGA CAT TC-3'.

5. The method for preparing a dual-mode sensor based on ZnNi nanoflowers and bifunctional NiFe Prussian blue analogues according to claim 1, characterized in that: In step (4), the volume of hydrogen peroxide is 10 to 20 μL, and the concentration is 1 to 30 mM; the volume of 3,3',5,5'-tetramethylbenzidine is 1 to 10 μL, and the concentration is 1 to 10 mM, and the incubation time is 5 to 10 min.

6. The method for preparing a dual-mode sensor based on ZnNi nanoflowers and bifunctional NiFe Prussian blue analogues according to claim 1, characterized in that: In steps (2), (3) and (4), the buffer solution is one or more of Tris-HCl buffer, PBS buffer, acetate buffer solution and PB buffer.

Images