A mineralized bacterial nanozyme probe, its preparation method, and a colorimetric / fluorescence dual-mode paper-based sensor made therefrom.

Abstract

This invention discloses a mineralized bacterial nanozyme probe, its preparation method, and a colorimetric / fluorescence dual-mode paper-based sensor made therefrom. Based on the mineralized bacterial nanozyme probe, a colorimetric and fluorescence dual-mode visualization paper-based sensor for mycotoxins is constructed. The two modes mutually verify and complement each other, resulting in high accuracy. Furthermore, by changing the aptamer type, it can detect a variety of mycotoxins, demonstrating strong versatility.

Description

Technical Field

[0001] This invention belongs to the field of fungal toxin detection technology, specifically relating to a mineralized bacterial nanozyme probe and its preparation method, and a colorimetric / fluorescence dual-mode paper-based sensor and its detection method made using the probe. Background Technology

[0002] Mycotoxins are a class of secondary metabolites produced by fungi, exhibiting significant toxicity and biological activity. Currently, common mycotoxin contamination in food mainly includes aflatoxin B1 (AFB1), ochratoxins (OTs), fumonisins (FB), aflatoxins (AFTs), deoxynivalenol (DON), T-2 toxin, zearalenone (ZEN), and patulin (PAT). These toxins not only pose a serious threat to food safety but may also cause long-term harm to human health.

[0003] In recent years, nanozymes have been widely used to construct colorimetric biosensors to amplify detection signals due to their advantages such as high catalytic efficiency, good stability, and low cost. However, most nanozymes themselves lack specific recognition functions and need to be used in combination with recognition elements such as antibodies or aptamers. Meanwhile, nucleic acid aptamers, as single-stranded DNA or RNA molecules obtained through SELEX screening technology, have advantages such as simple synthesis, high stability, easy modification, and a wide target range, and have become an ideal alternative to antibodies.

[0004] Although existing technologies have combined aptamers with nanozymes for the detection of specific mycotoxins, most current schemes are single-signal output modes and are usually limited to solution systems, resulting in shortcomings in portability and interference resistance. Furthermore, the potential to achieve universal detection of multiple mycotoxins using a single detection platform by changing the aptamer has not yet been fully explored and applied. Summary of the Invention

[0005] To address the shortcomings of the prior art, this invention provides a mineralized bacterial nanozyme probe, its preparation method, and a colorimetric / fluorescence dual-mode paper-based sensor made using the probe. Based on the mineralized bacterial nanozyme probe, a colorimetric and fluorescence dual-mode visualization paper-based sensor for mycotoxins is constructed. The two modes mutually verify and complement each other, resulting in high accuracy. Furthermore, by changing the aptamer type, it can detect a variety of mycotoxins, demonstrating strong versatility.

[0006] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows: A method for preparing a mineralizing bacterial nanozyme probe includes the following steps: (1) The bacteria were sterilized, centrifuged and the supernatant was discarded. Tris-HCl buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added first, followed by phosphomolybdic acid and soluble metal M salt. After shaking the reaction, the supernatant was discarded by centrifugation. Finally, the mixture was washed with Tris-HCl and resuspended to obtain B-MoM. (2) After freezing and centrifuging the Aptamer dry powder, prepare a stock solution with PBS, mix it thoroughly with B-MoM, centrifuge and discard the supernatant, block the non-specific binding sites with BSA, and then wash with Tris-HCl to obtain the mineralized bacterial nanozyme probe, denoted as B-MoM@Apt.

[0007] This invention first synthesizes B-MoM in situ on the surface of bacterial cells using a biomineralization method. Then, a nucleic acid aptamer with high affinity and specificity to bind to specific fungal toxins is covalently modified onto the surface of B-MoM via an EDC / NHS cross-linking method to construct a mineralized bacterial nanozyme probe, B-MoM@Apt. This mineralized bacterial nanozyme probe exhibits excellent peroxidase-like activity, catalyzing the oxidation of the colorless substrate TMB by H2O2 to produce blue oxTMB. Simultaneously, the absorption spectrum of the B-MoM nanozyme overlaps with the excitation spectrum of RhB, resulting in fluorescence resonance energy transfer (FRET) between the two, which quenches the fluorescence of RhB, achieving colorimetric / fluorescence dual-mode signal detection.

[0008] Furthermore, in step (1), the bacteria are Staphylococcus aureus or Listeria.

[0009] Further, in step (1), the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide added is: 100 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride with a concentration of 5 mg / mL and 100 μL of N-hydroxysuccinimide with a concentration of 5 mg / mL are added to each 1 mL of bacteria.

[0010] Further, in step (1), the soluble metal M salt is a soluble salt of iron, nickel, or copper, or a hydrate thereof.

[0011] Furthermore, in step (1), the concentrations of both the phosphomolybdic acid and the soluble metal M salt are 1-5%. w / V.

[0012] Further, in step (2), 10 μL of Aptamer stock solution with a concentration of 100 μmol / L is added to each 1 mg B-MoM.

[0013] In this invention, there are no special requirements for the Aptamer type, which can be changed according to actual detection needs, making the paper-based sensor applicable to the detection of various mycotoxins such as AFB1, OTA, and ZEN, with strong versatility.

[0014] The present invention also provides a mineralized bacterial nanozyme probe prepared by the above preparation method.

[0015] A colorimetric / fluorescence dual-mode paper-based sensor includes a polydopamine (PDA) modified test paper and the aforementioned mineralized bacterial nanozyme probe.

[0016] A detection method for a colorimetric / fluorescence dual-mode paper-based sensor involves first adding the sample solution to be tested onto a polydopamine-modified test paper, then adding a mineralized bacterial nanozyme probe to specifically bind to mycotoxins, washing away any non-specifically bound mineralized bacterial nanozyme probes, and finally adding a chromogenic substrate and a fluorescent molecule. The detection of mycotoxins is achieved by detecting changes in the color and fluorescence signal of the test paper. The chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), and the fluorescent molecule is rhodamine B (RhB).

[0017] In this invention, polydopamine, through its abundant conjugated structure, undergoes π-π stacking with mycotoxins, thereby efficiently enriching trace amounts of mycotoxins in the sample solution onto the surface of the test strip, serving as a pre-concentration and immobilization function. Meanwhile, the mineralized bacterial nanozyme probe can specifically recognize and bind to the enriched mycotoxins, and based on its peroxidase-like activity, catalyze the substrate to undergo a colorimetric reaction. At the same time, it can quench the fluorescence of fluorescent molecules, thereby achieving dual-mode detection of mycotoxins using both colorimetric and fluorescence methods.

[0018] The dual-mode signal generation mechanism is as follows: Colorimetric channel: When the target mycotoxin is present in the sample to be tested, the aptamer on the B-MoM@Apt probe specifically binds to the mycotoxin, thereby fixing the entire probe onto the test strip. After the TMB / H2O2 substrate is added, the immobilized B-MoM nanozyme catalyzes the colorimetric reaction, making the test strip turn blue, and the color depth is positively correlated with the toxin concentration. Fluorescent channel: When RhB is introduced into the reaction system, the absorption spectrum of B-MoM nanozyme overlaps with the excitation spectrum of RhB, resulting in fluorescence resonance energy transfer (FRET) between them, which quenches the fluorescence of RhB. When the probe is fixed on the test strip, its local concentration increases, which enhances the fluorescence quenching effect of RhB, resulting in a decrease in the fluorescence intensity of the system. The degree of decrease is positively correlated with the toxin concentration.

[0019] Furthermore, the color change is analyzed by capturing images with a smartphone and analyzing RGB values ​​for colorimetric analysis; the fluorescence signal change is analyzed by detecting the fluorescence intensity of the solution with a fluorescence spectrophotometer for fluorescence analysis.

[0020] Compared with the prior art, the present invention has the following advantages: (1) High accuracy and reliability: A dual signal output channel for colorimetry and fluorescence was constructed. The signals of the two channels verify each other and complement each other, effectively avoiding single signal errors caused by sample matrix or operating environment, and greatly improving the accuracy and reliability of detection results.

[0021] (2) High sensitivity: Based on the signal amplification effect of the efficient enzyme-like catalytic activity of B-MoM nanozymes and the enrichment effect of PDA on the target, this method has high sensitivity and low detection limit for the detection of fungal toxins, which can meet the requirements of trace analysis.

[0022] (3) High specificity: Nucleic acid aptamers are used as recognition elements, which have high affinity and specificity for the target fungal toxins similar to antibodies, effectively avoiding interference from other structural analogs or coexisting components.

[0023] (4) Portability and speed: Based on the paper-based platform and combined with smartphone readings, the detection is portable and on-site. The entire detection process can be completed in 30-40 minutes, which is much faster than traditional chromatography methods.

[0024] (5) Versatility and low cost: The core technology of this invention is a universal architecture of "PDA paper-based platform + nanozyme signal amplification + dual-mode readout". By changing the type of aptamer modified on B-MoM, the sensor platform can be used to detect a variety of fungal toxins such as AFB1, OTA, and ZEN, realizing the universal function of "one platform, multiple detections". Attached Figure Description

[0025] Figure 1 The full UV-Vis absorption spectra of B-MoM materials in the wavelength range of 500~800 nm are shown, where A and B are the absorption spectra of the B-MoCu system; C and D are the absorption spectra of the B-MoFe system; and E and F are the absorption spectra of the B-MoNi system.

[0026] Figure 2 This is a TEM image of B-MoFe.

[0027] Figure 3 The elemental mapping diagram (A) and EDS diagram (B) of B-MoFe material are shown.

[0028] Figure 4 The image shows the XRD pattern of B-MoFe material.

[0029] Figure 5 XPS image of B-MoFe material.

[0030] Figure 6 SEM images of the test strips (before modification (A), after PDA modification (B), after AFB1+PDA modification (C), after B-MoFe@Apt + AFB1 + PDA modification (D)).

[0031] Figure 7 Enzyme kinetic radar diagram PMA(A), Fe 3+ (B) PMA + Fe 3+ (C), B-PMA (D), B-Fe 3+ (E) and B-MoFe(F).

[0032] Figure 8 The effect of B-MoFe on the viability of A549 cells.

[0033] Figure 9 The UV-Vis absorption spectra of B-MoFe with different chromogenic substrates (A), UV-Vis absorption spectra in different reaction systems (B), and UV-Vis absorption spectra of different enzyme activities (C).

[0034] Figure 10 To detect the linear relationship between the colorimetric test strip R0-R and the logarithm of AFB1 concentration (A), and the linear relationship between the fluorescent test strip R-R0 and the logarithm of AFB1 concentration (B). Detailed Implementation

[0035] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the protection scope of the present invention.

[0036] The following are examples of the preparation method and application of the dual-mode paper-based sensor for detecting fungal toxins (with aflatoxin B1, i.e., AFB1 as the main research object), but the content of the present invention is not limited thereto.

[0037] Example 1

[0038] 1. Preparation of B-MoM (where M is Cu) 2+ Fe 3+ Ni 2+ )Material Take 1 mL (approximately 10) 9Add CFU / mL Listeria monocytogenes to a centrifuge tube, sterilize at 70°C for 1 h, centrifuge, discard the supernatant, and add 600 μL (10 mmol / L, pH 8.5) Tris-HCl, 100 μL 5 mg / mL EDC, 100 μL 5 mg / mL NHS, and 100 μL 2% HCl. w / V phosphomolybdic acid solution and 100 μL 2% w / V Copper sulfate pentahydrate solution (or ferric chloride hexahydrate solution; or nickel chloride hexahydrate solution) was shaken thoroughly in a shaker for 2 h. After shaking, the mixture was stirred at 7000 r / 12 min, the supernatant was removed, and 1 mL of Tris-HCl was added to mix and precipitate was formed. The mixture was stirred at 7000 r / 6 min, the supernatant was removed, and 1 mL of Tris-HCl reagent was added to mix and set aside.

[0039] 2. Verification of peroxidase-like activity of B-MoM materials To verify the peroxidase-like catalytic activity of B-MoM material, TMB was selected as the chromogenic substrate. After adding B-MoM to a NaAc-HAc buffer (pH 4.0) composed of 5 mmol / L TMB and 10 mmol / L H2O2, the oxidation product exhibited maximum absorbance at l = 650-670 nm. Figure 1 As shown, Listeria monocytogenes mineralized with phosphomolybdic acid (B-Mo) reacts with metal ions to produce a colored reaction, indicating that B-MoM possesses peroxidase activity. Therefore, nanozymes synthesized using bacterial-based materials can enhance peroxidase-like activity. Furthermore, the reaction with Cu... 2+ Fe 3+ and Ni 2+ The doped B-Mo showed a significant increase in peroxidase-like activity, indicating that B-MoM not only possesses oxidase activity but also the catalytic ability of peroxidase, and can effectively catalyze the oxidation reaction of TMB by H2O2 to generate the blue product oxTMB.

[0040] Example 2

[0041] 1. Preparation of molybdenum and iron mineralization bacterial nanozyme (B-MoFe) probe 1.1 Preparation of B-MoFe materials Take 1 mL (approximately 10) 9 Staphylococcus aureus at cfu / mL was sterilized at 70℃ for 1 h. After centrifugation and discarding the supernatant, 500 μL (10 mmol / L, pH 8.5) Tris-HCl buffer, 100 μL 5 mg / mL EDC, 100 μL 5 mg / mL NHS, and 100 μL 2% HCl were added. w / V phosphomolybdic acid solution and 100 μL 2% w / VThe ferric chloride hexahydrate solution was shaken thoroughly in a shaker for 4 h, centrifuged at 5400 rpm for 5 min and the supernatant was discarded. The solution was washed three times with Tris-HCl and resuspended for later use. 1.2 Preparation of B-MoFe@Apt Aptamer dry powder was centrifuged at 13000 rpm for 5 min, and a 100 μL stock solution of 100 μmol / L PBS was prepared. The solution was then incubated at 95°C for 5 min, cooled in an ice bath for 5 min, and slowly cooled to room temperature. 10 μL of the Aptamer stock solution was mixed with 1 mg of β-MoFe activated for 30 min and stirred for 4 h. The mixture was centrifuged at 5400 rpm for 5 min, and the supernatant was discarded. The solution was then diluted with 0.5% PBS. w / V BSA was used to block non-specific binding sites, followed by washing three times with Tris-HCl, and finally storage at 4°C.

[0042] Characterization of B-MoFe materials: Figure 2 This indicates that the B-MoFe material has a spherical structure.

[0043] Figure 3 The presence of Mo, Fe, and P elements on the surface of the Staphylococcus aureus cell wall further demonstrates the consistency between the elements and the spherical structure.

[0044] Figure 4 This indicates that the crystal structure of B-MoFe material is amorphous.

[0045] Figure 5 This indicates that the valence states of Mo(IV) / Mo(VI) and Fe(II) / Fe(III) coexist, and the catalytic active sites are clearly defined.

[0046] Figure 6 The image shows a SEM image of the test strip, confirming that the PDA successfully modified the test strip and that B-MoFe@Apt can be fixed to the test strip surface via AFB1.

[0047] Figure 7 The kinetic parameters for the oxidation of TMB by H2O2 catalyzed by B-MoFe were shown to be 0.51 mmol / L for H2O2 and Vmax for H2O2 to be 99.40 × 10⁻⁶. -8 mol / L·s -1 The Vmax for TMB reaches 33.47 × 10⁻⁶. -8 mol / L·s -1 It has a much lower concentration than natural horseradish peroxidase, resulting in superior catalytic activity.

[0048] 2. Cell viability experiment 2.1 Culture of A549 lung cancer epithelial cells Set the water bath to 37°C, remove the A549 cell cryovials, and thaw them rapidly in the water bath. After thawing, transfer the cell culture to a culture flask, add approximately 5 mL of DMEM containing 10% FBS, and place in an incubator at 37°C and 5% CO2. The next day, change the culture medium, remove the cryovial solution, and replace it with fresh DMEM containing 10% FBS. Check the color of the culture medium daily; if it turns yellow, change the medium and wash the cells. If the cell density exceeds 80%, passage the cells.

[0049] 2.2 Cytotoxicity assay A549 cells in good growth condition were harvested, digested with trypsin containing EDTA, and then the cell concentration was adjusted to 1×10⁻⁶. 5 Cells were seeded at 100 μL / mL in 96-well plates, divided into three groups: cell control, material control, and material and cell treatment group. Each well contained 100 μL of B-MoFe solution, and the cells were incubated at 37°C with 5% CO2 for 24 h to allow cell adhesion. 600 μL of B-MoFe solution was diluted 5-fold with culture medium. The supernatant from each well was discarded. 100 μL of DMEM medium containing 10% FBS was added to the cell control group, while 100 μL of B-MoFe material dilution was added to both the material control and material and cell treatment groups. The cells were incubated overnight. CCK-8 solution was diluted 10-fold with DMEM medium containing 10% FBS. The supernatant from each well was discarded, and 100 μL of working solution was added to each well. The cells were incubated at 37°C for 30–40 min, and the absorbance was measured at 450 nm to calculate cell viability and assess the cytotoxicity of the B-MoFe material.

[0050] The effect of B-MoFe on the viability of A549 cells was detected using a CCK-8 assay kit. Based on absorbance values, the survival rate of A549 cells treated with B-MoFe was calculated to be 99.2%. Figure 8 As shown, B-MoFe did not cause any significant difference in the activity of A549 cells, indicating that the nanomaterial has good biocompatibility and no cytotoxicity.

[0051] 3. Preparation of agarose gel electrophoresis Weigh 2.4 g of agarose into an Erlenmeyer flask, add 60 mL of 1× TAE electrophoresis buffer, and heat the solution in the flask using a microwave oven for about 2 minutes until a clear, bubble-free gel forms. When the gel temperature drops to about 70°C, quickly pour it into a plastic plate mold with a comb inserted. After the gel plate cools and solidifies, remove the comb and the mold to complete the gel plate preparation. Place the gel plate in an electrophoresis tank containing 1× TAE electrophoresis buffer, ensuring the buffer completely covers the plate. Take 10 μL of sample solution, 2 μL of 6× DNA loading buffer, and 1 μL of 50× GelRed nucleic acid dye, and vortex to mix. Then, add the mixture to the sample wells, set the voltage to 220 V, and the electrophoresis time to 40 minutes. After electrophoresis, observe the bands using a gel imaging system and determine the DNA molecule ligation based on the band migration positions. Multiple samples were prepared, including: (1) Sample 1 DNA Marker; (2) Sample 2 B-MoFe; (3) Sample 3 Aptamer; (4) Sample 4 B-MoFe@Apt; (5) Sample 5 B-MoFe@Apt + AFB1 4. Peroxidase-like properties of B-MoFe materials To verify the peroxidase-like catalytic activity of B-MoFe material, TMB was selected as the chromogenic substrate. After adding B-MoFe to a NaAc-HAc buffer (pH 4.0) consisting of 5 mmol / L TMB and 10 mmol / L H₂O₂, its oxidation product exhibited a characteristic absorption peak at 652 nm. Figure 9 As shown, different combinations of TMB and B-MoFe exhibit a weak absorption peak at 652 nm. However, when TMB, H2O2, and B-MoFe are mixed, a significantly enhanced absorption peak is observed at 652 nm, and the solution turns blue. This indicates that B-MoFe not only possesses oxidase activity but also peroxidase catalytic ability, effectively catalyzing the oxidation of TMB by H2O2 to generate the blue product oxTMB.

[0052] 5. Fabrication and Detection of Colorimetric / Fluorescence Paper-Based Dual-Mode Paper-Based Sensor 5.1 Fabrication of Paper-Based Sensors Circular test strips with a diameter of 6 mm were prepared on a glass fiber membrane using a punch. These strips were then incubated in a 0.5 mg / mL pH 8.5 dopamine (DA) solution for 12 h to form a polydopamine (PDA) film on the surface. The films were then dried at 37°C for later use. 30 μL of AFB1 at different concentrations was added to each test strip, and incubation was performed for 10 min to immobilize the toxin. Subsequently, a B-MoFe@Apt probe was added, which specifically bound to AFB1 for 10 min. Excess probe was then washed away with a pH 8.5 Tris-HCl buffer. Finally, 10 μL of 0.5 mmol / L Rhodamine B (RhB), along with a TMB and H2O2 catalytic system, was added to initiate a colorimetric reaction, completing the sensor preparation. The sensor was then integrated with the "Color Picker" app, an RGB recognition software for smartphones, for quantitative analysis.

[0053] 5.2 Plotting the Standard Curve This experiment used aflatoxin AFB1 as an example. Under optimal conditions (PDA synthesis time 12 h, TMB 0.5 mmol / L, H2O2 0.3 mmol / L, pH 4.0), as the concentration of AFB1 increased, the R channel value of the colorimetric test strip gradually increased, while the intensity value of the fluorescence system at 570 nm gradually decreased. The linear range for both was 1–1000 ng / mL, and the LOD was 0.82 ng / mL (R0.05). 2 =0.9942) and 0.35 ng / mL (R 2 =0.9914)( Figure 10 ).

[0054] 5.3 Analysis of actual samples To verify the applicability of the method, the analytical method established in this study was applied to the detection of aflatoxin B1 in tap water and peanuts.

[0055] Water samples were taken from laboratory tap water and only required filtration. Food sample pretreatment followed the national food safety standard GB-5009.22-2016. The pretreatment method was as follows: For peanuts, they were first ground into powder using a grinder. 5 g of powder was accurately weighed and placed in a 50 mL centrifuge tube. 20.0 mL of methanol-water solution (v / v 7:3) was added, and the mixture was vortexed and shaken for 20 minutes. The mixture was then centrifuged at 6000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter membrane and used for further processing. Wheat flour quality control samples were purchased from Guangzhou Shuopu Biotechnology Co., Ltd., and were wheat with a standard value of 49.1 μg / kg. The pretreatment method was the same as for peanuts. The established method was then used to verify its practicality. For the sample spike recovery experiment, the samples were replaced with spiked samples (AFB1 concentrations of 10, 100, and 500 ng / mL).

[0056] The method established in this study has been applied to the detection of aflatoxin B1 (AFB1) in tap water, peanuts, and wheat flour. After sample pretreatment, the detection results were compared with those of the national standard method. The analysis showed that AFB1 was not detected in tap water and peanut samples, and the concentration of the spiked samples was consistent with the actual values. The recovery rate of the colorimetric method was 92.5% to 101%, and the relative standard deviation (RSD) was ( ). RSD The recovery rate was 1.2% to 5.7%; the recovery rate of the fluorescence method was 95.0% to 103%. RSD The percentages ranged from 2.1% to 5.6%. The test results for wheat flour samples were consistent with the national standard method, thus verifying the reliability and application prospects of this method.

[0057] Table 1. Determination of AFB1 in wheat flour quality control samples ( n =3) Wheat (QC) 2.352 2.281 2.302 0.5180>0.05

[0058] Table 2. Determination of AFB1 in actual samples

Claims

1. A method for preparing a mineralizing bacterial nanozyme probe, characterized in that, Includes the following steps: (1) The bacteria were sterilized, centrifuged and the supernatant was discarded. Tris-HCl buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added first, followed by phosphomolybdic acid and soluble metal M salt. After shaking the reaction, the supernatant was discarded by centrifugation. Finally, the supernatant was washed with Tris-HCl and resuspended to obtain B-MoM. (2) After freezing and centrifuging the Aptamer dry powder, prepare a stock solution with PBS, mix it thoroughly with B-MoM, centrifuge and discard the supernatant, block the non-specific binding sites with BSA, and then wash with Tris-HCl to obtain the mineralized bacterial nanozyme probe, denoted as B-MoM@Apt.

2. The preparation method according to claim 1, characterized in that, In step (1), the bacteria are Staphylococcus aureus or Listeria.

3. The preparation method according to claim 1, characterized in that, In step (1), the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide added is: 100 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride with a concentration of 5 mg / mL and 100 μL of N-hydroxysuccinimide with a concentration of 5 mg / mL are added to each 1 mL of bacteria.

4. The preparation method according to claim 1, characterized in that, In step (1), the soluble metal M salt is a soluble salt of iron, nickel, or copper, or a hydrate thereof.

5. The preparation method according to claim 1, characterized in that, In step (1), the concentrations of both the phosphomolybdic acid and the soluble metal M salt are 1-5%. w / V.

6. The preparation method according to claim 1, characterized in that, In step (2), 10 μL of Aptamer stock solution with a concentration of 100 μmol / L is added to each 1 mg B-MoM.

7. The mineralized bacterial nanozyme probe prepared by the preparation method according to any one of claims 1-6.

8. A colorimetric / fluorescence dual-mode paper-based sensor, characterized in that, This includes polydopamine-modified test strips and the mineralized bacterial nanozyme probe as described in claim 7.

9. A detection method for a colorimetric / fluorescence dual-mode paper-based sensor, characterized in that, First, the sample solution to be tested is dropped onto a polydopamine-modified test strip. Then, a mineralized bacterial nanozyme probe is added to specifically bind to the mycotoxin. The non-specifically bound mineralized bacterial nanozyme probe is washed away. Finally, a chromogenic substrate and a fluorescent molecule are added. The mycotoxin is detected by detecting the color change and fluorescence signal change of the test strip. The chromogenic substrate is 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide, and the fluorescent molecule is rhodamine B.

10. The detection method according to claim 9, characterized in that, The color change is analyzed by capturing images with a smartphone and analyzing RGB values ​​for colorimetric analysis; the fluorescence signal change is analyzed by detecting the fluorescence intensity of the solution with a fluorescence spectrophotometer for fluorescence analysis.

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