A novel and convenient colorimetric bio-detection method for acetamiprid based on fe-n-c sacs and aptamer

Abstract

The application discloses a novel convenient colorimetric biological detection method based on iron monatomic nanoscale enzyme (Fe-N-C SACs) and aptamer. And taking acetamiprid (ACE) as a detection object, a simple and rapid method for quantitatively detecting ACE is established. Fe-N-C SACs are synthesized by double-constrained path synthesis, and the material has high efficient oxidase-like activity. Fe-N-C SACs-TMB is used as a color developing sensing platform, and the ACE aptamer modified with a mercapto group is used as a specific recognition element, so that a novel convenient colorimetric biological detection technology for ACE is constructed by virtue of the SH-APT aptamer, which can double inhibit the interaction of Fe-N-C SACs-TMB on one hand and highly specifically recognize ACE on the other hand. Qualitative and quantitative detection of ACE is realized by directly observing the color of a reaction system and detecting the absorbance.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological detection, and particularly relates to a novel convenient pyridaben biological detection method based on Fe-N-C SACs and aptamers. BACKGROUND

[0002] Aptamers are molecular recognition elements with high affinity and specificity, and various detection methods based on aptamers have been developed, mainly including colorimetric, fluorescent and electrochemical methods.

[0003] The invention patent with publication number CN 113203781 A relates to an electrochemical detection method based on aptamers, but the method is relatively complex, and the result cannot be directly observed, and needs to be assisted by large instruments and professional operators. The invention patent with publication number CN111190002 A relates to a nano-gold-nucleic acid aptamer colorimetric detection method, but the colorimetric indicator-nano-gold used in the method has the disadvantages of instability, difficulty in long-term preservation, and large batch difference.

[0004] Therefore, it is necessary to establish a direct, convenient and sensitive colorimetric detection method. SUMMARY

[0005] The purpose of the present application is to provide a novel convenient pyridaben biological detection method based on Fe-N-C SACs and aptamers, which is a Fe-N-C SACs nanomaterial with good oxido-mimetic enzyme activity, and constructs an aptamer colorimetric biosensor which is intuitive, convenient and sensitive. In addition, we realize the detection of the pesticide pyridaben based on the detection material and principle, and the minimum detection limit is 16.9nM, and the detection range is 200-1200nM.

[0006] To solve the technical problem, a Fe-N-C SACs nanomaterial with excellent oxido-mimetic enzyme activity is synthesized by using a double-constraint approach. The aptamer modified with a thiol group is used as a recognition element, and a single atom and a TMB system are used as a signal transduction, and an aptamer biosensor based on single-atom catalysis is developed. The single-atom catalyst can efficiently and quickly catalyze O2 in water to produce free radicals to make TMB change color, and the thiol-modified aptamer can double inhibit the oxido-mimetic enzyme activity of Fe-N-C SACs, and the pyridaben aptamer has a specific recognition effect on pyridaben, and the A450 absorbance value of the system after the addition of the target pyridaben is recorded. The reaction pH, reaction temperature, reaction content and aptamer concentration of Fe-N-C SACs are optimized, a standard curve is drawn, and the accurate pyridaben concentration is obtained by comparison with the standard working curve. Compared with the existing method, the operation is simple, the specificity is high, and the time and cost consumption are less.

[0007] The application provides a novel convenient colorimetric acetamiprid biological detection method based on Fe-N-C SACs and aptamers, and comprises the following steps:

[0008] S1, synthesis of iron monatomic nanoscale enzyme Fe-N-C SACs:

[0009] Ferrous chloride and 1,10-phenanthroline are dissolved in an organic solvent, and then porous carbon (PC) is added and ultrasonically shaken, high-temperature stirring is carried out until the organic solvent volatilizes, and drying is carried out; melamine is added and ground, and iron monatomic nanoscale enzyme Fe-N-C SACs is obtained after calcination;

[0010] S2, construction of an acetamiprid aptamer (ACE aptamer) / iron monatomic nanoscale enzyme (Fe-N-C SACs) reaction system

[0011] First, different concentrations of standard acetamiprid (ACE) solution are mixed with thiol-modified aptamer (SH-APT) mother liquor for incubation, and then iron monatomic nanoscale enzyme Fe-N-C SACs solution is added for reaction, so as to prepare an ACE aptamer (acetamiprid aptamer) / Fe-N-C SACs reaction system;

[0012] Or, thiol-modified aptamer (SH-APT) mother liquor is mixed with iron monatomic nanoscale enzyme Fe-N-C SACs solution for reaction, and then different concentrations of standard acetamiprid (ACE) solution are added for incubation, so as to prepare an ACE aptamer (acetamiprid aptamer) / Fe-N-C SACs reaction system;

[0013] S3, drawing of a standard curve of acetamiprid

[0014] In the reaction system obtained in step S2, TMB solution is added, and then pH is adjusted, and after reaction, UV-Vis absorbance is recorded, and a standard curve is linearly fitted according to the absorbance and different concentrations of standard acetamiprid (ACE) solution;

[0015] S4, determination of the concentration of acetamiprid

[0016] S4-1, construction of a reaction system according to the method of step S2

[0017] The acetamiprid (ACE) solution with a to-be-detected concentration is mixed with thiol-modified aptamer (SH-APT) for incubation, and then iron monatomic nanoscale enzyme Fe-N-C SACs solution is added for reaction;

[0018] Or, thiol-modified aptamer (SH-APT) is mixed with iron monatomic nanoscale enzyme Fe-N-C SACs solution for reaction, and then the standard acetamiprid (ACE) solution with a to-be-detected concentration is added for incubation;

[0019] S4-2, test the concentration according to the method of step S3

[0020] Add TMB solution to the resulting reaction system, adjust the pH, and after the reaction, terminate the reaction with TMB termination color developing solution, and record the UV-Vis absorbance, and substitute it into the standard curve obtained in step S3 to measure the concentration of acetamiprid (ACE) solution.

[0021] Preferably, in step S1, the porous carbon (PC) is prepared by heating potassium citrate, soaking the resulting black powder in a sulfuric acid solution, stirring, filtering, and collecting the dried porous carbon powder to obtain the porous carbon (PC). If there are too many impurities, the acid washing can be performed several times.

[0022] Preferably, the heating temperature is 700-800°C, the heating time is 1-2h, and the heating rate is 2-10°C / min; heating is performed in a nitrogen environment. The sulfuric acid concentration is 0.5-1M, and the stirring time is 0.2-0.5h. After acid washing, the filtration is performed by water suction filtration. The acid washing and filtration can remove residual metal ions and acid. The drying temperature is 70-100°C, and the drying time is 10-12h.

[0023] Preferably, in step S1, the amount ratio of ferrous chloride, 1,10-phenanthroline, ethanol solution, porous carbon (PC), and melamine is 0.5-07mmoL:2.5-3.5mmoL:15-30mL:250-350mg:2-3g, and preferably 0.6mmoL:3mmoL:20mL:300mg:2g.

[0024] Preferably, in step S1, the high-temperature stirring temperature is 60-70°C, and the stirring is performed until the ethanol evaporates, and then drying.

[0025] Preferably, in step S1, the calcination temperature is 650-750°C, the calcination time is 0.5-1.5h, and the heating rate is 4-6°C / min, and preferably the calcination temperature is 700°C, the calcination time is 1h, and the heating rate is 5°C / min.

[0026] Preferably, in step S1, the obtained Fe-N-C SACs are further treated with a sulfuric acid solution at 75-85°C for 1-3h, and preferably at 80°C for 2h, for removing unstable iron species; the sulfuric acid solution has a concentration of 0.4-0.6M, and preferably 0.5M.

[0027] Preferably, in step S2, the concentration of acetamiprid is 200-1200 nM. The volume ratio of the thiol-modified aptamer (SH-APT) mother liquor to the iron monatomic nanoscale enzyme Fe-N-C SACs is 140-196:40-60. The concentration of the thiol-modified aptamer mother liquor is 8-12 μM, preferably 10 μM, and more preferably the final concentration of the aptamer in the reaction system is 2-2.8 uM; the concentration of the iron monatomic nanoscale enzyme Fe-N-C SACs solution is 0.4-0.9 mg / ml.

[0028] Preferably, in step S2, the thiol-modified aptamer (SH-APT) is obtained by chemically modifying a thiol group at the 5' end of the aptamer, and the sequence of the aptamer is SEQ ID NO. 1 (5'-CTG ACA CCA TAT TAT GAA GA-3').

[0029] Preferably, in step S2, the incubation temperature is 15-60°C, and the time is 15-25 min; the reaction time is 10-15 min.

[0030] Preferably, in step S3, the concentration of the TMB solution is 0.05-0.15 mM, preferably 0.1 mM, and the volume ratio of the TMB solution to the reaction system is 20-30:65-75, preferably 25:70.

[0031] Preferably, in step S3, the pH is adjusted to 3-7 using acetic acid buffer.

[0032] Preferably, in step S3, the reaction time is 3-4 minutes, preferably 3 minutes, and the UV-Vis absorbance is recorded at a wavelength of 300-600 nm, preferably 450 nm. After the reaction, the reaction is terminated with TMB termination color developing solution.

[0033] A SH-APT inhibited Fe-N-C SACs-TMB colorimetric nanobiosensor is prepared by the following method: ferrous chloride and 1,10-phenanthroline are dissolved in an organic solvent, and then porous carbon is added and ultrasonically shaken. After high-temperature stirring until the organic solvent volatilizes, drying is performed. Melamine is added and ground, and after calcination, iron monatomic nanoscale enzyme Fe-N-C SACs are obtained. The thiol-modified aptamer mother liquor and the iron monatomic nanoscale enzyme Fe-N-C SACs solution are mixed and reacted to obtain the SH-APT inhibited Fe-N-CSACs-TMB colorimetric nanobiosensor.

[0034] The step 1 of the present application prepares a unique Fe-N-C SACs nanomaterial with excellent oxidase-like activity. The step 2 constitutes a biosensing interface for specifically recognizing ACE, and the specific binding of ACE aptamer and ACE and the peroxidase-like property of the Fe-N-CSACs nanomaterial are utilized; when there is no target ACE in the detection system, SH-APT binds with the Fe-N-C SACs, and the oxidase-like activity is reduced, and TMB does not change color. When the detection system contains the target ACE, SH-APT preferentially binds with ACE, and the active site of the Fe-N-C SACs is released to catalyze the color change of TMB, and the synergistic effect and catalysis of TMB as an indicator are realized. The construction of the biosensing interface in the step 2 is an indispensable key step in the colorimetric detection of ACE in the step 3. It can be seen that the steps 1-3 support each other and work together to realize the detection of ACE by using the Fe-N-C SACs-SH-APT-TMB nanosignal probe.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] (1) At present, most of the colorimetric detection methods based on aptamers are based on nanogold colorimetric method, and the nanogold is unstable, has large batch difference, and has relatively high preparation cost, which hinders the practical application. However, the main material Fe-N-C SACs used in the present colorimetric method is simple to prepare and low in cost, and there is no need to worry about the problems of preservation and transportation in the use process, which is beneficial to the later commercial development.

[0037] (2) The fluorescence and electrochemical detection methods based on aptamers are complex to operate and high in cost. The present application is simple to operate, and the color change visible to the naked eye can be used for rapid, efficient and sensitive qualitative detection of pesticides. BRIEF DESCRIPTION OF DRAWINGS

[0038] Other features, objects and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments with reference to the following drawings:

[0039] Figure 1 A schematic diagram of the present application based on the colorimetric aptamer sensor for detecting ACE by using Fe-N-C SACs nanoscale enzyme and aptamer;

[0040] Figure 2 The electron microscope image of the Fe-N-C SACs nanoscale enzyme prepared in the present application example 1;

[0041] Figure 3 The curve graph of the Fe-N-C SACs nanoscale enzyme reaction condition exploration in the present application example 1; A is the curve graph for different pH values, B is the curve graph for different temperatures, and C is the curve graph for different Fe-N-C SACs concentrations;

[0042] Figure 4 Concentration optimization curve for SH-APT in Example 1 of the present application;

[0043] Figure 5 Working standard curve of ACE obtained in Example 1 of the present application. DETAILED DESCRIPTION

[0044] The present application will be described in detail below with reference to the accompanying drawings and specific examples. The following examples are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are provided, which will help those skilled in the art to further understand the present application. It should be pointed out that the protection scope of the present application is not limited to the following examples, and several adjustments and improvements made on the premise of the concept of the present application all belong to the protection scope of the present application.

[0045] The principle diagram of a catalytic TMB colorimetric aptamer sensor for detecting acetamiprid based on Fe-N-C SACs mimetic oxide nanoszyme and aptamer is shown in Figure 1 First, Fe-N-C SACs mimetic oxide nanoszyme is prepared. The prepared Fe-N-C SACs mimetic oxide nanoszyme has good oxidase effect and can catalytically decompose oxygen in the supporting solution to produce active oxygen, and the active oxygen further catalyzes the color change of TMB. Since the thiol-modified aptamer is covered on the active site of the Fe-N-C SACs mimetic oxide nanoszyme by physical adsorption and other intermolecular forces, the thiol-modified aptamer can double inhibit the activity of the Fe-N-C SACs mimetic oxide nanoszyme, and a SH-APT inhibits Fe-N-C SACs-TMB colorimetric nanobiosensor is constructed. After adding the pesticide acetamiprid ACE to the biosensor interface, the ACE can specifically bind with the ACE aptamer, thereby releasing the active site of the Fe-N-C SACs, so as to catalytically decompose oxygen to produce active substances such as superoxide free radicals to catalyze the color change of TMB. By recording the A450 absorbance signal after detecting the pesticide acetamiprid, the relationship curve between the absorbance and the concentration of acetamiprid is drawn, so as to realize the detection of acetamiprid.

[0046] Example 1

[0047] The implementation steps are as follows:

[0048] 1. Preparation of Fe-N-C SACs

[0049] (1) Heat 10.0 g of potassium citrate at 800℃ for 1 h under a nitrogen environment (heating rate: 3℃ / min). Soak the obtained black powder in a 0.5M sulfuric acid solution and stir for 2 h. Use distilled water to extract and remove residual metal ions and acid. Collect the obtained porous carbon powder after drying in an oven at 70℃ for 10 h.

[0050] (2) 0.6 mmoL FeSO4.7H2O and 3 mmoL 1,10-phenanthroline hydrate were dissolved in 20 mL ethanol solution, and ultrasonic vibration was performed for 20 min, and was ready for use.

[0051] (3) 300 mg of porous carbon was added to the prepared ethanol solution, and ultrasonic vibration was continued for 20 min, and was stirred at 65°C until the ethanol was volatilized, and was dried. 2 g of melamine was added and ground, and then was transferred to a stone boat, and was calcined at 700°C in a tube furnace under nitrogen atmosphere for 1 h (heating rate: 5°C / min). After cooling, 1.0 M sulfuric acid solution was used to treat at 80°C for 2 h to remove unstable iron species. Figure 2 The electron microscope image of the prepared Fe-N-C SACs was in a sheet shape, and there was no iron cluster.

[0052] 2, Study on the peroxide mimetic enzyme activity of Fe-N-C SACs

[0053] (1) Exploration of the optimum pH of Fe-N-C single atom catalysis

[0054] In order to explore the optimum pH, 50 μL of 0.1 mM TMB and 50 μL of 1 mg / mL Fe-N-C SACs were added to acetic acid buffer with pH of 3, 4, 5, 6, 7, 8, and 9, respectively, and the total volume of the reaction system was kept at 2 mL. The mixed solution was incubated at 25°C, the reaction was terminated by using TMB color developing termination liquid, and the UV-Vis absorbance was recorded at 450 nm. The detection results are as follows Figure 3 A, with the increase of pH, the catalyst activity decreased obviously, which indicated that the catalyst activity was good in acidic system.

[0055] (2) Exploration of the influence of temperature on Fe-N-C single atom catalysis

[0056] In order to explore the optimum temperature, 50 μL of 0.1 mM TMB and 50 μL of 1 mg / mL Fe-N-C SACs were added to the buffer, and the total volume of the reaction system was kept at 2 mL. The mixed solution was incubated at 15°C, 20°C, 25°C, 30°C, 35°C, 45°C, and 60°C, respectively, the reaction was terminated by using TMB color developing termination liquid, and the UV-Vis absorbance was recorded at 450 nm. The detection results are as follows Figure 3 B, with the increase of temperature, the catalytic activity gradually increased, and the best reaction temperature was 45°C. 45°C was selected as the reaction temperature in the subsequent study.

[0057] (3) Optimization of catalyst concentration

[0058] To explore the most suitable catalyst concentration, 50 μΐ, 0.1 mM TMB and 50 μΐ, of different concentrations of Fe-N-C SACs were added to acetic acid buffer with pH 3 in 700 μΐ, reaction system. The mixed solution was incubated at 45 °C, and the reaction was terminated by TMB color development termination solution, and the UV-Vis absorbance was recorded at 450 nm. We believe that the oxygen content in the 700 μΐ, system is certain. If the monatomic material is too much, it will decompose O2 to produce a large amount of active substances, which is not conducive to the subsequent masking of aptamer, and if the monatomic material is too little, it will not produce enough active substances to make TMB color. The detection results are as follows Figure 3 C, with the increase of monatomic material in the system, the absorbance is continuously improved, and when the concentration is 0.7 mg / ml, the absorbance value tends to be stable. Therefore, we choose the concentration of 0.7 mg / ml as the best catalyst concentration.

[0059] 3, concentration optimization of SH-aptamer

[0060] The concentration of aptamer is a key factor for the successful quantification of aptamer nanozyme biosensor. Therefore, we studied the color reaction of different final concentrations of thiol-modified aptamer SH-APT (2, 2.2, 2.4, 2.6, 2.8 μΜ) and Fe-N-C SACs-TMB after adding the aptamer to the reaction system, and determined the best concentration of SH-APT. Thiol-modified aptamer (SH-APT) is obtained by chemically modifying thiol on the 5' terminal nucleotide of the aptamer, and the sequence of the aptamer is SEQ ID NO. 1 (5'-CTG ACA CCA TAT TAT GAA GA-3'). If the aptamer is too much, the excess aptamer will interfere with the detection system, which is not conducive to the minimum detection limit of the target; and if the aptamer is too little, it cannot bind more targets, and the threshold of this detection method will be greatly reduced. The detection results are as follows Figure 4 , with the increase of the concentration of aptamer, the absorbance is continuously reduced. When the concentration of aptamer is more than 2.6 uM, the trend of absorbance decrease of the system is significantly slowed down, indicating that the aptamer is excessive. Therefore, we choose 2.6 uM as the best aptamer concentration.

[0061] 4, drawing of ACE working curve of pesticide acetamiprid

[0062] Under the optimal conditions, the ACE working curve was drawn.

[0063] The specific method and conditions are as follows: First, ACE (20 μL, 200, 400, 600, 800, 1000, 1200 nM) and SH-APT stock solution (10 μM, 182 μL) were incubated at room temperature for 20 min. Then, 50 μL of 0.7 mg / mL Fe-N-CSACs was added, and the reaction time was 10 min. Afterward, TMB (25 μL, 0.1 mM) and acetate buffer (0.1 M, pH = 4) were added, maintaining a total volume of 700 μL. Finally, after three minutes of reaction, the reaction was terminated with 5 μL of TMB stop solution, and the UV-Vis absorbance was recorded at 450 nm. Figure 5 Linear fitting calculations show that when the ACE concentration is in the range of 200-1200 nM, the relationship between absorbance (Y) and ACE concentration (X) is linear, with the linear regression equation being Y = 0.0001772*X + 0.1190 and a correlation coefficient of 0.91. The linear range of the working curve is 200-1200 nM. Using three standard deviations of the blank control as the limit of detection, the lowest detection limit for ACE is calculated to be 16.9 nM.

[0064] We summarized other ACE detection methods based on aptamer colorimetry, as shown in Table 1. Most ACE detection methods have good linear operating ranges, but their LODs are all above 40 nM. Furthermore, for colorimetric detection methods using gold nanoparticles, the preparation cost of gold nanoparticles is high, and they are difficult to preserve. Compared to these methods, our developed single-atom-aptamer detection method has a wider linear range and good LOD. Moreover, the preparation method of single atoms is simple, inexpensive, easy to preserve and transport, and more practical.

[0065] Table 1. Comparison of colorimetric detection methods for different aptamers of ACE

[0066]

[0067] 5. Spiked recycling

[0068] To evaluate whether this detection method can be used for environmental samples, we conducted a spiked recovery experiment with river water as an example. First, the collected water sample was added to a 1.5 mL centrifuge tube and centrifuged at 6000 rpm for 10 minutes. 1 mL of supernatant was taken and filtered with a 0.22 μM filter to remove impurities. ACE standard solution was added to the pretreated river water to make the ACE concentration in the sample 300 nM, 800 nM and 1200 nM. Using the optimized experimental conditions, the ACE concentration in the water sample was determined by the detection method. Each group of experiments was repeated 3 times, and the recovery rate and relative standard deviation (RSD) were calculated. According to the experimental results, as shown in Table 2, the recovery rate was 97.58-105.75%, and the RSD was 4.33-10.13%. These data indicate that our current detection method is suitable for ACE detection in actual water samples.

[0069] Table 2 Spiked recovery

[0070]

[0071] Comparative Example 1

[0072] This comparative example 1 provides a new convenient colorimetric acetamiprid bio-detection method based on Fe-N-C SACs and aptamer, the steps are basically the same as example 1, the only difference is that the aptamer is not modified with thiol. The thiol used in this invention can easily coordinate with Fe, masking its activity, effectively reducing the catalytic activity of Fe-N-C SACs. In this comparative example, the aptamer alone cannot effectively reduce the catalytic activity of Fe-N-C SACs, and cannot be used for subsequent detection development.

[0073] The above only describes the preferred embodiments of the present application, and does not limit the present application in any form. Any simple modification, equivalent change and modification made according to the technical essence of the present application to the above embodiments are within the scope of the technical solutions of the present application.

Claims

1. A convenient colorimetric bio-detection method of acetamiprid based on Fe-N-C SACs and aptamer, characterized in that, Comprising the following steps: S1, synthesis of iron monatomic nanoscale enzyme Fe-N-C SACs: Dissolve ferrous chloride and 1,10-phenanthroline in an organic solvent, then add porous carbon ultrasonic oscillation, high temperature stirring until the organic solvent volatilizes, and dry; add melamine and grind, and obtain iron monatomic nanoscale enzyme Fe-N-C SACs after calcination; S2, construction of the reaction system of acetamiprid aptamer / iron monatomic nanoscale enzyme: First, mix different concentrations of standard acetamiprid solution with the thiol-modified aptamer mother liquor and incubate, then add the iron monatomic nanoscale enzyme Fe-N-C SACs solution and react, to obtain the ACE aptamer / Fe-N-C SACs reaction system; Or, mix the thiol-modified aptamer mother liquor with the iron monatomic nanoscale enzyme Fe-N-C SACs solution and react, then add different concentrations of standard acetamiprid solution and incubate, to obtain the ACE aptamer / Fe-N-C SACs reaction system; S3, drawing of the standard curve of acetamiprid: Add TMB solution to the reaction system obtained in step S2, adjust the pH, record the UV-Vis absorbance after reaction, and linearly fit the standard curve according to the absorbance and different concentrations of standard acetamiprid solution; S4, determination of the concentration of acetamiprid: S4-1, construct the reaction system according to the method of step S2 Take the acetamiprid solution of the concentration to be determined and mix with the thiol-modified aptamer, then add the iron monatomic nanoscale enzyme Fe-N-C SACs solution and react; Or, mix the thiol-modified aptamer with the iron monatomic nanoscale enzyme Fe-N-C SACs solution and react, then add the standard acetamiprid solution of the concentration to be determined and incubate; S4-2, test the concentration according to the method of step S3 Add TMB solution to the reaction system obtained, adjust the pH, terminate the reaction with TMB termination color developing solution after reaction, and record the UV-Vis absorbance; Substitute the measured absorbance into the standard curve obtained in step S3 to obtain the concentration of the acetamiprid solution; In step S2, the thiol-modified aptamer is obtained by chemically modifying thiol on the 5' terminal nucleotide of the aptamer, and the sequence of the aptamer is: 5'-CTGACACCATATTATGAAGA-3'; The prepared Fe-N-C SACs oxide nanoscale enzyme has good oxidase effect and can catalyze the decomposition of oxygen in the supporting liquid to produce active oxygen, and the active oxygen further catalyzes the color change of TMB. Since the thiol-modified aptamer covers the active site of the Fe-N-C SACs oxide nanoscale enzyme through intermolecular forces, the thiol-modified aptamer can double inhibit the activity of the Fe-N-C SACs oxide nanoscale enzyme. After adding the pesticide acetamiprid ACE in the biosensing interface, ACE can specifically bind with ACE aptamer, thereby releasing the active site of Fe-N-C SACs, thereby catalyzing the decomposition of oxygen to produce active substances to catalyze the color change of TMB.

2. The bioassay method according to claim 1, wherein, In step S1, the porous carbon is prepared by heating potassium citrate, soaking the obtained black powder in a sulfuric acid solution, stirring, filtering, drying the obtained porous carbon powder, and collecting the porous carbon.

3. The bioassay method according to claim 1, wherein, In step S1, the amount ratio of ferrous chloride, 1,10-phenanthroline, and porous carbon is 0.5-0.6 mmol: 2.5-3.5 mmol: 250-350 mg.

4. The bioassay method according to claim 1, wherein, In step S1, the calcination temperature is 650-750 ℃, and the calcination time is 0.5-1.5 h.

5. The bioassay method according to claim 1, wherein, In step S2, the different concentrations of acetamiprid are 200-1200 nM.

6. The bioassay method according to claim 1, wherein, The volume ratio of the mother liquor of thiol-modified aptamer to the amount of iron monatomic nanoscale enzyme Fe-N-C SACs is 140-196:40-60; the concentration of the mother liquor of thiol-modified aptamer is 8-12 μM; and the concentration of the iron monatomic nanoscale enzyme Fe-N-C SACs solution is 0.4-0.9 mg / ml.

7. The bioassay method according to claim 1, wherein, In step S2, the incubation temperature is 15-60 ℃, the time is 15-25 min, and the reaction time is 10-15 min.

8. The bioassay method according to claim 1, wherein, In step S3, the concentration of the TMB solution is 0.05-0.15 mM, and the volume ratio of the TMB solution to the reaction system is 20-30:65-75.

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