Cascade nano bioreactor based on mesoporous silica and application

Abstract

The invention belongs to the technical field of new functional materials and biosensing detection, and particularly relates to a cascaded nano-bioreactor based on mesoporous silica and application, the cascaded nano-bioreactor based on mesoporous silica takes mesoporous silica as a carrier, and AChE, CHO and HRP are encapsulated in the mesoporous silica. AChE, CHO and HRP are fixed in mesoporous silica at the same time to construct a cascade catalysis platform, the cascade catalysis platform is used for colorimetric fluorescence dual-mode sensing detection of organophosphorus pesticides, and the cascade catalysis platform has the advantages of being easy to operate, free of precise equipment, good in detection performance, good in long-term stability and the like. The invention not only provides a new tool for high-sensitivity and convenient monitoring of pesticide residues, but also inspires the development of a high-efficiency multienzyme sensing platform, and has a wide application prospect in field analysis of the pesticide residues.

Description

Technical Field

[0001] The invention belongs to the technical field of new functional materials and biosensor detection, and specifically relates to a cascade nano bioreactor based on mesoporous silica and its application. Background Art

[0002] The monitoring of pesticide residues in food and environmental matrices is undoubtedly the key to ensuring food safety and ecological health. At present, many laboratory-based technologies have been developed to detect organophosphorus pesticides (OPs), including gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, etc. Although these methods are quite mature at the technical level and have high accuracy and sensitivity, there are still certain limitations for the analysis of biological samples of organophosphorus pesticides, such as expensive instruments and equipment, complex and time-consuming sample pretreatment, and the need for professional laboratory personnel to complete the operation, which greatly limits their on-site detection and use. Therefore, in order to further simplify the sample analysis steps, improve the selectivity and sensitivity of the method, and realize the visualization and intelligent detection of biological sample detection, it is urgent to develop a simple, fast and instant new method to detect trace organophosphorus pesticide residues in fruits, vegetables and environmental samples. Summary of the invention

[0003] In order to solve the problems in the prior art, the present invention provides a cascade nanobioreactor based on mesoporous silica and its application, which is simple to operate, does not require precise equipment, has good detection performance, and has good long-term stability, thereby achieving the purpose of colorimetric and fluorescent dual-mode detection of organophosphorus pesticides.

[0004] Enzyme inhibition method has become the most promising alternative to traditional methods because it does not require large instruments, is low-cost, and is simple and rapid for target detection. Natural enzymes such as acetylcholinesterase (AChE) have been widely used to determine OPs by inhibiting enzyme activity. It is worth noting that natural enzymes are easily inactivated by extreme pH and temperature changes when exposed to external conditions. The introduction of immobilized enzymes is a major breakthrough in improving enzyme stability.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] The present invention aims to provide a cascade nano bioreactor based on mesoporous silica, in which the mesoporous silica is used as a carrier and AChE, CHO and HRP are encapsulated in the mesoporous silica.

[0007] Mesoporous silica is a typical representative of mesoporous nanomaterials. The adjustable pores and high loading capacity of mesoporous silica ensure the space required for the reaction and promote the internal material transport. In addition, the high stability of mesoporous silica can provide effective protection for internal reactions. Moreover, it has good biocompatibility and is suitable as a carrier for various biomolecules. Compared with other materials used in nanoreactors, these unique physical and chemical properties give mesoporous silica obvious advantages and make it an ideal material for building nanoreactors.

[0008] Furthermore, the preparation method of mesoporous silica comprises the following steps:

[0009] 1) Mix cetyltrimethylammonium bromide (CTAB) and triethanolamine (TEA) in a solution system and react at 60° C. for 1 hour;

[0010] 2) adding a mixed solution of tetraethyl silicate (TEOS) and cyclohexane into the solution system of 1) and mixing, and continuing the reaction at 60° C. for 24 hours, and washing by centrifugation to obtain a white precipitate;

[0011] 3) Dissolve the white precipitate obtained in 2) in acetone solution, react at 70° C. for 12 hours, and centrifuge and wash to obtain a white precipitate;

[0012] 4) Dissolve the white precipitate obtained in 3) in an ethanol solution, add 3-aminopropyltriethoxysilane (APTES) solution into the solution system, mix, react at 70°C for 8 hours, centrifuge and wash to obtain a white precipitate, and dry at 50°C to obtain a white powder, which is mesoporous silica.

[0013] Furthermore, the preparation method of the cascade nanobioreactor based on mesoporous silica comprises the following steps:

[0014] 1) Mix mesoporous silica, AChE, CHO, HRP, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a solution system and react;

[0015] 2) Adding bovine serum albumin (BSA) solution into the solution system of step 1, mixing, and continuing the reaction, collecting the precipitate by centrifugation, and obtaining a mesoporous silica-based cascade nanobioreactor (AChE / CHO / HRP@SiO2).

[0016] The integrated AChE / CHO / HRP@SiO2 has the advantages of simple operation, no need for precision equipment, good detection performance, and good long-term stability. This study not only provides a new tool for high-sensitivity and convenient monitoring of pesticide residues, but also will inspire the development of efficient multi-enzyme sensing platforms, which has broad application prospects in on-site analysis of pesticide residues.

[0017] Furthermore, after the solution system of 1) is mixed, it is stirred and reacted at room temperature for 2 hours, and the BSA solution of 2) is added to the solution system and mixed, and the stirring reaction is continued at room temperature for 2 hours.

[0018] A mesoporous silica-based cascade nanobioreactor for the detection of organophosphorus pesticides.

[0019] Although strategies based on enzyme inhibition have been considered as a potential approach and have been intensively explored in pesticide analysis, fragile enzyme activity and cumbersome operations have greatly hindered their practical applications. The development of portable and efficient pesticide sensing platforms remains challenging. Herein, we developed a mesoporous silica nanomaterial. On this basis, a cascade catalytic platform was constructed by simultaneously immobilizing acetylcholinesterase (AChE), choline oxidase (CHO), and horseradish peroxidase (HRP) in mesoporous silica for colorimetric and fluorescence dual-mode sensing of organophosphorus pesticides (OPs).

[0020] Furthermore, the organophosphorus pesticide detection method includes: dispersing the organophosphorus and the mesoporous silica-based cascade nanobioreactor in PBS buffer for incubation, adding ACh solution for continued incubation, and then adding HAc-NaAc buffer and a colorimetric substrate for incubation, and recording the changes in absorbance and fluorescence intensity.

[0021] Furthermore, the organophosphorus pesticide is paraoxon.

[0022] Furthermore, the incubation temperature is 15-60°C, preferably 37°C, the incubation time of the organophosphorus in PBS buffer is 1-10 min, preferably 1 min, the ACh concentration is 0.08-2.00 mM, preferably 0.80 mM, and the incubation time after adding ACh is 10-70 min, preferably 40 min.

[0023] Furthermore, the pH of PBS buffer is 3-10, preferably 7, and the pH of HAc-NaAc buffer is 3-9, preferably 6.5.

[0024] Furthermore, the chromogenic substrates include TMB (3,3',5,5'-tetramethylbenzidine) and OPD (o-phenylenediamine), with a TMB concentration of 0.08-1.20 mM, preferably 0.20 mM, a TMB reaction time of 5-35 min, preferably 20 min, an OPD concentration of 1-20 mM, preferably 4 mM, and an OPD reaction time of 10-70 min, preferably 40 min.

[0025] Compared with the prior art, the beneficial technical effects of the present invention are:

[0026] 1. The present invention uses mesoporous silica as an immobilized carrier, and encapsulates AChE, CHO and HRP in the mesoporous silica. Based on the multi-enzyme cascade reaction and the Fenton reaction, a new type of nanobioreactor is constructed, which not only effectively improves the various properties of the material, but also promotes the efficient mass transfer of the substrate, and can achieve highly sensitive detection of the target paraxenphos. The detection of organophosphorus pesticides based on the nanobioreactor of the present invention has the characteristics of simple operation, high sensitivity, good stability and strong anti-interference ability.

[0027] 2. The present invention is based on the AChE / CHO / HRP@SiO2-mediated cascade catalytic reaction combined with the Fenton reaction, and successfully realizes the amplification of colorimetric and fluorescent dual detection signals. This nanobioreactor integrating multiple sensing elements provides a new way to detect pesticide residues in food.

[0028] 3. The cascade nanobioreactor prepared by the present invention has the remarkable characteristics of simple operation, high sensitivity and high accuracy for the detection of organophosphorus pesticides, has good application prospects, and provides favorable protection for food safety.

[0029] 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, it can be implemented according to the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is an experimental test diagram in Examples 1-5 of the present invention.

[0031] Figure 2 These are experimental test diagrams in Examples 5 and 6 of the present invention.

[0032] Figure 3 This is a schematic diagram of the preparation, detection and working principle of the mesoporous silica-based cascade nanobioreactor of the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0034] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0035] Example 1

[0036] Preparation of mesoporous silica-based cascade nanobioreactor:

[0037] S1. Synthesis of mesoporous silica: Add CTAB aqueous solution (20 mL, 1 mg / mL) and TEA (50 μL) together and react at 60°C for 1 hour. Add a mixed solution of TEOS (1.5 mL) and cyclohexane (6 mL) to the above solution system and mix, and continue to react at 60°C for 24 hours, and centrifuge and wash to obtain a white precipitate. Dissolve the white precipitate in acetone solution (30 mL), react at 70°C for 12 hours, and centrifuge and wash to obtain a white precipitate. Dissolve the white precipitate in ethanol solution (30 mL), add APTES solution (2 mL) to the above solution system and mix, react at 70°C for 8 hours, centrifuge and wash to obtain a white precipitate, and dry at 50°C to obtain white powder SiO2, which is the mesoporous silica. Collect the powder and store the obtained SiO2 in a refrigerator at 4°C for further use.

[0038] S2. Synthesis of AChE / CHO / HRP@SiO2: AChE aqueous solution (100 μL, 0.5 mg / mL), CHO aqueous solution (100 μL, 0.5 mg / mL), HRP aqueous solution (100 μL, 0.5 mg / mL), EDC aqueous solution (50 μL, 2 mg / mL), and NHS aqueous solution (50 μL, 1 mg / mL) were added to SiO2 aqueous solution (0.3 mL, 0.17 mg / mL) and stirred for 2 h. Then, it was mixed with BSA (50 μL, 10%) and stirred at room temperature for 2 h. The pale yellow precipitate was collected by centrifugation, dissolved in 100 μL BBS buffer, and the obtained AChE / CHO / HRP@SiO2 was stored in a refrigerator at 4°C for further use.

[0039] like Figure 1 As shown, Figure A is a SEM image of mesoporous silica, and Figure B is a TEM image of mesoporous silica.

[0040] Example 2

[0041] Feasibility analysis of AChE / CHO / HRP@SiO2 detection of organophosphorus:

[0042] In this study, paraoxon was selected as a model of organophosphorus pesticides to verify the feasibility of AChE / CHO / HRP@SiO2 for the detection of organophosphorus pesticides. Specifically, paraoxon (10μL, 1mg / mL) and AChE / CHO / HRP@Si O2 (10μL, 1mg / mL) were first dispersed in 80μL PBS buffer and incubated at 37°C for 10min. ACh solution (50μL, 10mmol / L) was added and incubated at 37°C for 10min, and then HAc-NaAc buffer (100μL, 0.1mol / L) and TMB colorimetric solution (50μL) were added and incubated at 37°C for 10min. The absorbance changes at 652nm were recorded. Figure 1 As shown in C, the feasibility analysis of the cascade encapsulation system for detecting organophosphorus.

[0043] Example 3

[0044] Determination of enzyme loading and immobilization efficiency of AChE, CHO and HRP:

[0045] AChE, CHO, and HRP were labeled with FITC. After encapsulation, the fluorescence spectra of the encapsulated enzymes were recorded, and the loading amount of the individual enzymes was calculated based on the corresponding calibration curves. The loading amount was defined as the ratio of the amount of enzyme encapsulated in SiO2 to the weight of AChE / CHO / HRP@SiO2. The immobilization efficiency was defined as the ratio of the amount of enzyme encapsulated in SiO2 to the mass of the enzyme initially added to the solution. Figure 1 D, E, and F are fluorescence curves and calibration curves for determining enzyme loading and enzyme immobilization efficiency.

[0046] Example 4

[0047] Comparison of catalytic kinetics between free enzyme and encapsulated system:

[0048] The catalytic activities of AChE, CHO and HRP were measured in AChE / CHO / HRP@SiO2. According to the calculated loading amount, the concentration of free enzyme was kept consistent with the concentration of the corresponding enzyme in AChE / CHO / HRP@SiO2, and then the activities of free enzyme and immobilized enzyme were measured. The catalytic activity of AChE was evaluated using formula (1) (2). The catalytic activity of CHO was evaluated using formula (3) (4). The catalytic activity of HRP was evaluated using formula (4).

[0049] (1):

[0050] (2): Thiocholine+DTNB→TNBA+Mixed disulfide

[0051] (3):

[0052] (4):

[0053] Example 5

[0054] Comparison of stability between free enzyme and encapsulated system:

[0055] S1. For the thermal stability test, AChE / CHO / HRP@SiO2 was placed at 15°C, 25°C, 37°C, 40°C, 50°C and 60°C for 30 minutes. The free enzyme was also treated as above. After the above treatment, 30 μL of 1 mg / mL AChE / CHO / HRP@SiO2 was added to a 96-well plate, 50 μL of ACh (5 mM) was added, and incubated at 37°C for 10 minutes. 50 μL of TMB colorimetric solution and 100 μL of HAc-NaAc buffer (pH = 6.5, 0.1 M) were added and incubated at 37°C for 10 minutes. The absorbance was quantified at a wavelength of 652 nm by an enzyme reader. The relative activity of AChE / CHO / HRP@SiO2 was calculated by comparing the activity with the initial activity. Untreated free enzyme and AChE / CHO / HRP@SiO2 were used as controls, and their activities were defined as 100%. As Figure 1 As shown, G and H in the figure are the anti-interference performance of the encapsulation system. I in the figure is the comparison of the thermal stability of the free enzyme and the encapsulation system.

[0056] S2. For pH stability test, AChE / CHO / HRP@SiO2 was placed in HAc-NaAc buffer (pH=3, 4, 5, 6, 7, 8, 9) and PBS buffer (pH=3, 4, 5, 6, 7, 8, 9, 10) for 30 minutes. The free enzyme was also treated as above. After the above treatment, 30μL 1mg / mL AChE / CHO / HRP@SiO2 was added to a 96-well plate, 50μL ACh (5mM) was added, and incubated at 37°C for 10 minutes. 50μL TMB colorimetric solution and 100μL HAc-NaAc buffer (pH=6.5, 0.1M) were added and incubated at 37°C for 10 minutes. The absorbance was quantified at a wavelength of 652nm by an ELISA reader. The relative activity of AChE / CHO / HRP@SiO2 was calculated by comparing the activity with the initial activity. Untreated free enzyme and AChE / CHO / HRP@SiO2 were used as controls, and their activities were defined as 100%. Figure 2 As shown, J in the figure is a comparison of the pH stability of the free enzyme and the encapsulated system: HAc-NaAc buffer; K in the figure is a comparison of the pH stability of the free enzyme and the encapsulated system: PBS buffer.

[0057] S3. For the chemical stability test, AChE / CHO / HRP@SiO2 was exposed to organic solvents (ethanol, ether, acetone, isopropanol, cyclohexane, dichloromethane, chloroform) for 30 minutes. The free enzyme was also treated as above. After the above treatment, 30 μL of 1 mg / mL AChE / CHO / HRP@SiO2 was added to a 96-well plate, 50 μL of ACh (5 mM) was added, and incubated at 37°C for 10 minutes. 50 μL of TMB colorimetric solution and 100 μL of HAc-NaAc buffer (pH = 6.5, 0.1 M) were added and incubated at 37°C for 10 minutes. The absorbance was quantified at a wavelength of 652 nm by an enzyme reader. The relative activity of AChE / CHO / HRP@SiO2 was calculated by comparing the activity with the initial activity. Untreated free enzyme and AChE / CHO / HRP@SiO2 were used as controls, and their activities were defined as 100%. Figure 2 As shown, L in the figure is the comparison of the chemical stability of the free enzyme and the encapsulated system.

[0058] S4. For the storage stability test, AChE / CHO / HRP@SiO2 was placed at 4°C for 1, 2, 3, 4, 5, 6 and 7 days, respectively. The free enzyme was also treated as above. After the above treatment, 30 μL of 1 mg / mL AChE / CHO / HRP@SiO2 was added to a 96-well plate, 50 μL of ACh (5 mM) was added, and incubated at 37°C for 10 minutes. 50 μL of TMB colorimetric solution and 100 μL of HAc-NaAc buffer (pH = 6.5, 0.1 M) were added and incubated at 37°C for 10 minutes. The absorbance was quantified at a wavelength of 652 nm by an enzyme reader. The relative activity of AChE / CHO / HRP@SiO2 was calculated by comparing the activity with the initial activity. Untreated free enzyme and AChE / CHO / HRP@SiO2 were used as controls, and their activities were defined as 100%. The results are shown in Figure 2. Figure 2 As shown, M in the figure is the time stability comparison between the free enzyme and the encapsulated system.

[0059] Example 6

[0060] Mesoporous silica-based cascade nanobioreactor for paraoxon detection:

[0061] S1. Add 10μL 1mg / mL AChE / CHO / HRP@SiO2 to a 96-well plate. Then add 10μL of different concentrations of paraoxon. At the same time, replace OPs with the same volume of ultrapure water as a blank control. After 10 minutes, add ACh solution (10μL, 5mmol / L) and incubate at 37℃ for 40 minutes. Add 50μL TMB (1mM) and 100μL HAc-NaAc buffer (pH=6.5,0.1M) and incubate at 37℃ for 20 minutes. The color change can be observed by naked eyes, and the absorbance at a wavelength of 652nm is recorded with a UV spectrophotometer.

[0062] S2, 10μL 1mg / mL AChE / CHO / HRP@SiO2 was added to the 96-well plate. Then 10μL of different concentrations of paraoxon were added. At the same time, the same volume of ultrapure water was used instead of OPs as a blank control. After 10 minutes, ACh solution (10μL, 5mmol / L) was added and incubated at 37℃ for 40 minutes, and 50μL OPD (20mM) and 100μL HAc-NaAc buffer (pH=6.5,0.1M) were added and incubated at 37℃ for 40 minutes. The fluorescence intensity change at 564nm was recorded by fluorescence spectrophotometer under 412nm wavelength excitation.

[0063] The results are as follows Figure 2 As shown, N, O, P, and Q in the figure are colorimetric detection of paraoxon by the encapsulation system; R, S, T, and U in the figure are fluorescent detection of paraoxon by the encapsulation system.

[0064] Example 7

[0065] Mesoporous silica-based cascade nanobioreactor for real sample detection of paraoxon:

[0066] Apples and cabbages were pretreated according to the LS / T 6139-2020 protocol. In brief, a certain amount of apples and cabbages were crushed and extracted with a mixed solvent (composed of 0.2 g / mL Na2SO4, ethyl acetate and methanol) under ultrasound. Afterwards, the supernatant was taken out and dried at 45°C. Subsequently, the samples were redissolved in pure water and measured according to the above steps. OPs in tap water were directly determined after filtration. In addition, different concentrations of paraoxon were added to these real samples to evaluate the detection performance of AChE / CHO / HRP@SiO2.

[0067] Example 8

[0068] Mesoporous silica-based cascade nanobioreactor for colorimetric and fluorescence dual-mode detection of paraoxon with the aid of an electrophoretic device:

[0069] S1. First, prepare AChE / CHO / HRP@SiO2 hydrogel, then place the hydrogel in the electrophoresis model, and achieve the enrichment of paraoxon under the action of electrophoresis. Take out the hydrogel, add ACh solution (10μL, 5mmol / L) and incubate at 37℃ for 40min, add 50μL TMB (1mM) and 100μL HAc-NaAc buffer (pH=6.5,0.1M), and incubate at 37℃ for 20min. The color change can be observed by naked eyes, and the absorbance at a wavelength of 652nm is recorded by UV spectrophotometer.

[0070] S2. First, prepare AChE / CHO / HRP@SiO2 hydrogel, then place the hydrogel in the electrophoresis model, and achieve the enrichment of paraoxon under the action of electrophoresis, take out the hydrogel, add ACh solution (10μL, 5mmol / L) and incubate at 37℃ for 40min, add 50μL OPD (20mM) and 100μL HAc-NaAc buffer (pH=6.5,0.1M), and incubate at 37℃ for 40min. Use a fluorescence spectrophotometer to record the change of fluorescence intensity at 564nm under 412nm wavelength excitation.

[0071] The present invention uses paraoxon as a representative of organophosphorus pesticides and provides a cascade nanobioreactor based on mesoporous silica, such as Figure 3 As shown, the reactor uses an in-situ encapsulation method to simultaneously encapsulate acetylcholinesterase (AChE), choline oxidase (CHO) and horseradish peroxidase (HRP) into the mesopores of mesoporous silica. The constructed nanoreactor improves the detection performance while maintaining the enzyme activity. Based on the excellent performance of the nanoreactor of the present invention, highly sensitive detection of paraoxon is successfully achieved. When there is no target, the substrate acetylcholine (ACh) is catalyzed by the cascade of AChE / CHO / HRP@SiO2 to generate a hydroxyl radical (·OH) with strong oxidizing properties, which can oxidize TMB into TMBox, with an increase in absorbance at 652nm; oxidize OPD into DAP, with an increase in fluorescence intensity at 564nm; and paraoxon, as an inhibitor of AChE, can hinder the occurrence of the cascade reaction, reduce the production of the final product ·OH, and then cause changes in absorbance and fluorescence intensity. By monitoring the changes in absorbance and fluorescence intensity, quantitative detection of paraoxon can be achieved.

[0072] The present invention uses mesoporous silica as an immobilized carrier, and encapsulates AChE, CHO and HRP in the mesoporous silica, respectively, and constructs a novel nanobioreactor based on multi-enzyme cascade reaction and Fenton reaction. Mesoporous silica can protect the internal enzymes and avoid the decline of enzyme activity. In addition, under the action of the constraint effect, the internal reaction can be carried out efficiently, and the amplification of the colorimetric and fluorescent signals is achieved, and the colorimetric and fluorescent dual signals can be free from background interference. The method is simple to operate, has high sensitivity, good stability and strong anti-interference ability.

[0073] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0074] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A cascade nanobioreactor based on mesoporous silica, characterized in that: Using mesoporous silica as a carrier, AChE, CHO and HRP are encapsulated in the mesoporous silica.

2. A cascade nanobioreactor based on mesoporous silica as claimed in claim 1, characterized in that: The preparation method of mesoporous silica comprises the following steps: 1) Mix CTAB and TEA in a solution system and react at 60°C for 1 hour; 2) Add the mixed solution of TEOS and cyclohexane into the solution system of 1) and mix, and continue to react at 60° C. for 24 hours, and centrifuge and wash to obtain a white precipitate; 3) Dissolve the white precipitate obtained in 2) in acetone solution, react at 70° C. for 12 hours, and centrifuge and wash to obtain a white precipitate; 4) Dissolve the white precipitate obtained in 3) in an ethanol solution, add the APTES solution into the solution system, mix, react at 70° C. for 8 hours, centrifuge and wash to obtain a white precipitate, and dry at 50° C. to obtain white powder mesoporous silica.

3. A cascade nanobioreactor based on mesoporous silica as claimed in claim 1 or 2, characterized in that: The preparation method of the cascade nanobioreactor based on mesoporous silica comprises the following steps: 1) Mix mesoporous silica, AChE, CHO, HRP, EDC and NHS in a solution system and react; 2) Add the BSA solution into the solution system of 1) above, mix, and continue the reaction. Collect the precipitate by centrifugation to obtain a cascade nanobioreactor based on mesoporous silica.

4. A cascade nanobioreactor based on mesoporous silica as claimed in claim 3, characterized in that: After the solution system in 1) is mixed, the mixture is stirred and reacted at room temperature for 2 hours. The BSA solution in 2) is added to the solution system and mixed. The mixture is stirred and reacted at room temperature for another 2 hours.

5. Use of a mesoporous silica-based cascade nanobioreactor as claimed in any one of claims 1 to 4 in the detection of organophosphorus pesticides.

6. The use according to claim 5, characterized in that: The organophosphorus pesticide detection method includes: dispersing the organophosphorus pesticide and the mesoporous silica-based cascade nanobioreactor in PBS buffer for incubation, adding ACh solution for continued incubation, then adding HAc-NaAc buffer and a colorimetric substrate for incubation, and recording the changes in absorbance and fluorescence intensity.

7. The use according to claim 5 or 6, characterized in that: The organophosphorus pesticide is paraoxon.

8. The use according to claim 6, characterized in that: The incubation temperature is 15-60°C, the incubation time of the organophosphate in PBS buffer is 1-10 minutes, the ACh concentration is 0.08-2.00 mM, and the incubation time after adding ACh is 10-70 minutes.

9. The use according to claim 6, characterized in that: The pH of PBS buffer is 3-10, and the pH of HAc-NaAc buffer is 3-9.

10. The use according to claim 6, characterized in that: The color developing substrates are TMB and OPD, the TMB concentration is 0.08-1.20 mM, the TMB reaction time is 5-35 min, the OPD concentration is 1-20 mM, and the OPD reaction time is 10-70 min.

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