Preparation method of a dual-mode sensor based on ZnNi nanoflowers and bifunctional NiFe Prussian blue analogues

Abstract

The present invention relates to a preparation method and application of an electrochemical-colorimetric dual-mode sensor based on nanoflowers and bifunctional NiFe-PBA nanozymes. NiFe-PBA-loaded gold-platinum bimetallic nanoparticles have excellent redox activity and peroxidase activity, and are used as bifunctional probes to construct an electrochemical colorimetric dual-mode sensor. The branched DNA structure introduces more signal probes to participate in the reaction, and the sensor specificity is enhanced by the specific recognition of OTA by the aptamer. Finally, the magnetic separation technology is used to cleverly construct an electrochemical and colorimetric dual-mode channel, effectively reducing environmental interference and avoiding false positive signals. NiFe-PBA-loaded gold-platinum bimetallic nanoparticles are involved in both dual-mode channels, realizing the signal response caused by the target while simplifying the experimental operation. The dual-mode sensor constructed by the present invention is sensitive, reliable, fast and stable, which is conducive to the promotion and application of the invention. It also has good anti-interference ability and practical application ability, providing a new type of biosensor platform for actual sample analysis.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of a dual-mode sensor based on ZnNi nanoflowers and dual-functional NiFe Prussian blue analogues. 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 a key group of mycotoxins, primarily produced by the genera Aspergillus and Penicillium. It is a typical biotoxin. Studies have shown that of the six known ochratoxins, OTA is the most toxic, a highly toxic chemical that can damage the liver and kidneys, and long-term exposure may increase the risk of cancer. OTA also has immunosuppressive effects, potentially weakening the host immune system and making it more susceptible to other pathogens. For this reason, the International Agency for Research on Cancer classified OTA as a possible human carcinogen, Group 2B, in 1993 and has implemented strict regulations to limit OTA levels in food. For example, the European Union sets a maximum residue limit (MRL) of 0.5 μg / kg for cereals, 0.2 μg / kg for wine (or grape juice), and 0.5 μg / kg for coffee products. 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 alcoholic beverages is 2.0 μg / kg.

[0003] Currently, traditional analytical methods for OTA detection include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), high-performance liquid chromatography-mass spectrometry (HPLC-MS), and high-performance liquid chromatography-fluorescence detection (HPLC-FD). While these traditional methods offer excellent sensitivity and accuracy, they also have limitations, particularly in rapid on-site testing. Therefore, the development of cost-effective, simple, and stable detection methods is crucial for rapid on-site testing.

[0004] Electrochemical, colorimetric, and fluorescent sensors, as emerging detection technologies, are gaining widespread attention both within and beyond the industry. Compared to traditional detection methods, these sensing technologies offer significant advantages, such as ease of operation, low cost, and rapid response. However, most of these sensing methods rely on a single, target-induced signal change, making them susceptible to interference from operating methods and environmental factors, leading to signal fluctuations. To improve detection resistance, reliability, and accuracy, an innovative strategy has emerged in recent years: colorimetric-electrochemical dual-mode sensors. These sensors combine the high sensitivity of electrochemical signals with the intuitive color changes of colorimetry, achieving more precise detection through dual signal channels. Dual-mode detection technology not only leverages the strengths of each channel but also enables self-verification by comparing the detection results of the two channels, thereby improving the accuracy and reliability of detection results.

[0005] Prussian blue analog (PBA) is a typical hexacyanoferrate coordination compound. The redox activity of the various metal ions it contains gives it excellent redox activity, enabling it to provide a clear redox current for electrochemical analysis. Interestingly, as an iron-containing nanomaterial, PBA exhibits excellent peroxidase-like activity, catalyzing the decomposition of hydrogen peroxide and the oxidation of a chromogenic substrate, facilitating visual observation. Based on these considerations, PBA has become an ideal bifunctional probe, enabling the simple fabrication of electrochemical-colorimetric dual-signal biosensors for accurate and reliable analysis.

[0006] The present invention addresses the problems existing in existing detection technologies, 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 was prepared, and applied to the construction of an electrochemical colorimetric dual-signal readout sensor. The branched DNA structure was designed to allow more signal probes to participate in the reaction, and the sensor specificity was enhanced by combining the specific recognition of OTA by the aptamer. Finally, the magnetic separation technology was used to cleverly construct an electrochemical and colorimetric dual-mode channel, which greatly reduced environmental interference, avoided false positive signals, and improved the reliability of the sensor. NiFe-PBA-loaded gold-platinum bimetallic nanoparticles are involved in both dual-mode channels, 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-mentioned 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 bifunctional NiFe Prussian blue analogue is carried out according to the following steps:

[0008] (1) Preparation of ZnNi nanoflowers: Zinc nitrate, nickel acetylacetonate, terephthalic acid, and polyvinylpyrrolidone were dissolved in a mixed solution of N, N-dimethylformamide, N, N-dimethylacetamide, and ethanol and dispersed evenly by magnetic stirring. The resulting mixed solution was transferred to an autoclave for incubation, and the product was collected by high-speed centrifugation and vacuum dried to constant weight to prepare ZnNi nanoflowers.

[0009] (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. After ultrasonication, chloroauric acid solution was added 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 the ZnNi nanoflower-loaded gold nanoparticle composite material.

[0010] (3) Preparation of NiFe-PBA nanozyme: Nitrate and trisodium citrate were dissolved in ultrapure water to obtain solution A, and potassium ferrocyanide was dissolved in ultrapure water to obtain solution B. Solution A and solution 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.

[0011] (4) Preparation of nanozyme-loaded bimetallic nanoparticles: First, the above-mentioned NiFe-PBA was dispersed in water, and precious metal salt solution A and precious metal salt solution B were added and stirred thoroughly. Then, a reducing agent was added and stirred thoroughly. The product was collected by high-speed centrifugation and vacuum dried to constant weight to obtain a NiFe-PBA-loaded bimetallic nanoparticle composite material.

[0012] (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, and then mix the products in proportion to make the C1 chain and C2 chain hybridize to form a double chain. The products are collected by centrifugation and dispersed with buffer solution to obtain a branched bifunctional nanoprobe, which is placed at 4°C for use.

[0013] (6) Preparation of MBs@OTA apt system and identification of target: The aptamer is connected to the MBs through the strong binding interaction between the amino groups on the aptamer chain and the carboxyl groups 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 be complementary to the OTA apt base to form a double-stranded structure. When OTA is present, OTA apt binds preferentially to OTA, freeing the C1-C2 system. Detection is performed using the electrochemical and colorimetric dual-mode channels using magnetic separation technology.

[0014] (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 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.

[0015] It is further defined that in step (3), the nitrate is one of nickel nitrate, copper nitrate and manganese nitrate.

[0016] 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.

[0017] 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'; and the sequence of the hairpin chain H1 is: 5'-GTG CCC GTG AGC ATC GGA CAT TC-3'.

[0018] 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.

[0019] 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.

[0020] Compared with the prior art, the present invention has the following significant advantages:

[0021] 1. The present invention prepares bifunctional NiFe-PBA loaded bimetallic nanoparticles, which simultaneously have electrochemical signals and peroxidase activity, realize electrochemical and colorimetric dual-mode detection, and improve the anti-interference ability, reliability and accuracy of sensor detection.

[0022] 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.

[0023] 3. The present invention successfully prepared ZnNi bimetallic nanoflowers with high specific surface area and loaded with a large number of metal nanoparticles, providing a large number of active sites and high conductivity for the sensor, achieving signal amplification.

[0024] 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

[0025] Figure 1 Schematic diagram of the preparation method of a dual-mode sensor based on ZnNi nanoflowers and bifunctional NiFe Prussian blue analogs.

[0026] 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.

[0027] Figure 3 These are the electrochemical (A) and colorimetric (B) standard curves of the dual-mode sensor constructed in Example 1 of the present invention.

[0028] Figure 4 The electrochemical (A) and colorimetric (B) selectivity of the dual-mode sensor constructed in Example 1 of the present invention for OTA in the presence of other interfering toxins. DETAILED DESCRIPTION

[0029] The following embodiments of the present invention are described in further detail with reference to 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

[0030] A preparation method and application of a dual-mode sensor based on ZnNi nanoflowers and dual-functional NiFe Prussian blue analogs, the implementation method of which is as follows Figure 1 shown.

[0031] The specific steps are as follows:

[0032] (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 solutions A and 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 dried in vacuum at 60°C.

[0033] (2) Preparation of AuPt NPs / NiFe-PBA: 4 mg of Ni-Fe PBA was weighed and 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 and magnetically stirred for 30 min. Then, 700 μL of 10 mM ascorbic acid solution was added dropwise to the mixed solution and 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.

[0034] (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 of zinc nitrate, 37 mg of nickel acetylacetonate, 12 mg of terephthalic acid, and 1.3658 g of polyvinylpyrrolidone 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 vacuo at 60 °C to obtain flower-shaped ZnNi-MOF.

[0035] (4) Preparation of Au NPs / ZnNi-MOF: 3 mg of ZnNi-MOF was ultrasonically dispersed in 1 mL of water. 225 μL of 25 mM tetrachloroauric acid solution was then added and magnetically stirred for 20 min. 2 mL of 0.1 M sodium borohydride solution was added dropwise to the above solution and magnetically stirred for 30 min. The precipitate was collected by centrifugation, washed with ultrapure water, and dried in vacuo at 60°C.

[0036] (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 with shaking for 2 h. After centrifugation, the mixture was dispersed with Tris-HCl and stored at 4°C 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 with shaking for 1 h. After centrifugation, the mixture was dispersed with Tris-HCl and stored at 4°C to obtain AuPt NPs / NiFe-PBA-C1-C2.

[0037] (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 on a shaker overnight. After magnetic separation, the mixture was washed and dispersed with PBS to obtain the MBs@OTA apt system. 15 μL OTA standard solution of different concentrations and 15 μL of the above-mentioned AuPt NPs / NiFe-PBA-C1-C2 were added to the MBs@OTA apt system and incubated on a shaker for 40 min. As the OTA concentration changed, the amount of AuPt NPs / NiFe-PBA-C1-C2 attached to the MBs changed. The detection was performed by electrochemical and colorimetric dual-mode channels using magnetic separation technology.

[0038] (7) Construction of dual-mode channel: After magnetic separation of the above mixed solution, the supernatant was used for electrochemical detection and the precipitate was used for colorimetric 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 hour. Then 5 μL of 1.5 μM hairpin chain H1 was added and incubated at 37°C for 1 hour, 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 hour. After each step, the electrode surface was washed with Tris-HCl buffer solution, and the electrode was placed in PBS buffer solution. The changes in electrochemical signals 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 minutes, a color change occurred and the absorbance change at 652 nm was detected by UV spectrophotometer.

[0039] (8) Establishment of standard curve: OTA standard solutions of different concentrations were added to step (6) to obtain sample test solutions of different gradients. These solutions were tested using an electrochemical workstation and a UV spectrophotometer, respectively. A linear fit was performed using the logarithmic value of the OTA concentration as the horizontal axis and the current signal as the vertical axis to establish a standard curve for the electrochemical mode. A linear fit was performed using the logarithmic value of the OTA concentration as the horizontal axis and the absorbance as the vertical axis to establish a standard curve for the colorimetric mode.

[0040] 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.

[0041] 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

[0042] A method for preparing a dual-mode sensor based on ZnNi nanoflowers and a dual-functional NiFe Prussian blue analogue, and its practical application, comprises the following steps:

[0043] 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 a concentration of 50 ng / mL; other interfering toxins (FB1, T-2, AFB1, OTB, DON, ZEN) standard solutions were prepared in the buffer solution at a concentration of 500 ng / mL. The above-mentioned different interfering toxin standards were detected according to the detection system constructed in Example 1. The detection results are shown in Figure 2. Figure 4 Electrochemical (A) and colorimetric (B) results show that the method of the present invention has high selectivity for OTA. Example 3

[0044] A method for preparing a dual-mode sensor based on ZnNi nanoflowers and a dual-functional NiFe Prussian blue analogue, and its practical application, comprises the following steps:

[0045] Actual sample preparation: For edible oil samples, 5.0 g of spiked edible oil sample was weighed into a 50 mL stoppered graduated cylinder. 20 mL of petroleum ether was added and shaken to dissolve the oil sample. 10 mL of a methanol / water solution (ratio 7:3) was added and extracted by high-speed vortex mixing for 5 minutes. After standing for stratification, 5 mL of the lower layer extract was aspirated and placed in a refrigerator. It was quickly frozen at -15°C for 20 minutes. The extract was filtered through a 0.45 μm filter while still cold and mixed with OTA standard solutions of varying concentrations to obtain spiked edible oil extracts. For red wine samples, 1 mL of wine sample was mixed with OTA standard solutions of varying concentrations and filtered through a 0.45 μm organic filter to obtain spiked red wine extracts.

[0046] Sample detection: Take 2.5 μL of sample solution, measure the electrical signal and absorbance according to the steps in Example 1, and insert them into the standard curve to obtain the concentration of OTA in the sample.

[0047] When measuring edible oil as the actual sample, 0.1 times and 10 times the standard amount of OTA standard were added to corn flour, respectively, with a 10 ng / mL addition amount as the reference. 2.5 μL of sample solution was taken, and the electrical signal and absorbance were measured according to steps (1) to (8) of Example 1. The OTA concentration in the sample was obtained by substituting the standard curve detected in Example 1 into the standard curve. Each sample was measured three times and the average value was taken. The average recovery rate in the electrochemical mode was calculated to be 95.7%-106.3%, and the average recovery rate in the colorimetric mode was 94.9%-102.9%.

[0048] The electrochemical-colorimetric dual-mode sensor has been demonstrated to detect OTA with high accuracy, good sensitivity, and excellent stability. Furthermore, test results on real samples (such as cooking oil and red wine) demonstrate the sensor's excellent practical application value.

[0049] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0050] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method 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 can also be appropriately combined to form other implementation methods 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 a dual-functional NiFe Prussian blue analogue, 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 ferricyanide 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. The C1 chain and the 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 groups on the aptamer chain and the carboxyl groups 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 forms a double-stranded structure by base complementary pairing with the OTAapt. When OTA is present, OTA apt binds preferentially to OTA, freeing the C1-C2 system. Electrochemical and colorimetric dual-mode channel detection is performed using magnetic separation technology; (4) Construction of dual-mode channel: OTA standard solution of different concentrations and the above-mentioned branched bifunctional nanoprobe were added to the MBs@OTA apt system. The supernatant and precipitate of the above-mentioned mixed solution after magnetic separation were used for electrochemical channel and colorimetric channel detection, respectively. In the electrochemical mode, the ZnNi nanoflower-loaded gold nanoparticle composite material was first modified on the electrode surface, and then the hairpin chain H1 was added to fix it on the electrode surface, and MCH was used to block the binding site. Finally, the supernatant obtained by magnetic separation was added to the electrode. The C1 chain in the supernatant was complementary to H1, so that the branched bifunctional nanoprobe was fixed on the electrode to generate an electrochemical signal response. In the colorimetric mode, the precipitate obtained by magnetic separation was washed with buffer solution, and only a small amount of C1 chain was connected to MBs through base complementary pairing with OTA apt. After incubation with hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine, a color change occurred, 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 a dual-functional NiFe Prussian blue analogue according to claim 1, characterized in that: In steps (2)(3)(4), the DNA chain is 0.5 ~ 4 μM, the volume used is 1 ~ 20 μL, and the incubation time is 0.5 ~ 2.5 h; the sequence of the C1 chain is: 5'-TTT TTT CTA TCT AAT TGA GGGAAT GTC CGA TGC TCC CTT TA-3'; the sequence of the C2 chain 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 a dual-functional NiFe Prussian blue analogue 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.

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