A fluorescent immunoassay method for pesticide based on gold cluster-manganese dioxide nanosheet

Abstract

The application discloses a kind of gold cluster-manganese dioxide nanosheet-based pesticide fluorescence immunoassay method.Currently, it is still challenging to develop a high-sensitivity, strong anti-interference ability analysis method to detect pesticides in complex food matrix.In the present application, coated antigen competes with pesticide to recognize enzyme-labeled antibody.After competitive immune reaction, ALP catalyzes substrate to produce ascorbic acid, which triggers the decomposition of AuNCs-MnO2NFs and regulates fluorescence reaction.Methidathion is selected as the analyte to be detected by the method.The results show that the fluorescence immunoassay method has high detection sensitivity (IC 10 = 5.78 pg mL ‑1 ), which is about 56 times higher than the traditional enzyme-linked immunoassay.The present application is successfully used for precise monitoring of pesticide residues and degradation dynamics in pakchoi, and has good anti-interference ability and reproducibility, paving the way for pesticide residue determination in actual samples.

Description

Technical Field

[0001] This invention belongs to the fields of biosensor technology and food and environmental sample quality and safety monitoring. Specifically, it relates to the establishment of a fluorescence immunoassay system. This technology combines the high specificity of enzyme-labeled nanobodies with the good fluorescence properties of gold nanoclusters-manganese dioxide nanosheet composite materials, providing a new method for highly sensitive on-site detection of pesticides and monitoring the residue and degradation dynamics of pesticides in plant tissues. Background Technology

[0002] Pesticides are chemical substances commonly used to protect agricultural products from weeds, pests, and diseases. Organophosphorus pesticides are among the most effective insecticides. Feritrothion (FNT) is a commonly used organophosphorus pesticide, widely used for controlling various pests on vegetables, fruits, and other crops due to its low cost, easy availability, and high efficiency. This widespread use means it is frequently detected in aquatic and terrestrial environments, as well as in agricultural products. Feritrothion is not only toxic to plants, but epidemiological studies have also shown that long-term exposure to FNT may lead to DNA damage, delayed muscle weakness, and reduced sperm concentration in humans, even affecting future generations. Furthermore, fenitrothion accumulates in the environment and can enter the food chain, posing a threat to ecosystems. Therefore, monitoring the degradation and residue levels of FNT in crops is essential. However, the presence of numerous interfering substances in the food matrix makes FNT detection very complex. Traditional enzyme-linked immunosorbent assay (ELISA) is a relatively simple and selective method for pesticide detection; however, its sensitivity is relatively low and cannot meet the increasingly stringent requirements for precision analysis. Therefore, there is an urgent need to develop a detection method with high sensitivity and strong anti-interference capabilities.

[0003] In immunoassays, analytical performance is primarily influenced by two factors: the affinity antibody (recognition unit) and the chromogenic substrate system (signal unit). Compared to traditional monoclonal and polyclonal antibodies, nanobodies exhibit better solubility and thermal stability, higher binding specificity and affinity, and can directly fuse with enzyme gene fragments to form immunoassay reagents, significantly shortening detection time. While traditional chromogenic systems are easy to observe and use, their sensitivity is low when used to detect targets in complex food matrices, and they are limited by toxic chromogenic substrates, such as o-phenylenediamine (OPD). Therefore, fluorescence immunoassay (FIA) has gradually become a superior alternative to traditional colorimetric immunosensing due to its excellent detection sensitivity and signal stability. Replacing traditional colorimetric substrates with fluorescent substances can improve detection sensitivity by leveraging the excellent fluorescence properties of the fluorescent substances. Gold nanoclusters (AuNCs) have attracted widespread attention due to their good biocompatibility, photostability, and unique optical and electrical properties. In addition to the advantages mentioned above, AuNCs are easily synthesized under protein-stable conditions, making them suitable for biosensor applications. Currently, the combination of fluorescent probes and quenching-capable nanomaterials is widely used in the design of biosensors. Manganese dioxide nanoflakes (MnO2 NFs) possess excellent light absorption, a large surface area, and can be decomposed by antioxidants, making them an excellent fluorescent quencher for developing sensing platforms. Therefore, we believe that the combination of AuNCs (fluorophores) and MnO2 NFs (quenchers) will exhibit excellent sensing performance in fluorescent immunoassay systems.

[0004] Based on the above research background, we propose combining a fluorescence sensing platform with an immune system. By using alkaline phosphatase-fused nanobody against FNT-linked-alkaline phosphatase (Nb-FNT-ALP), we can enhance the affinity of the recognition unit in the immune system, thereby improving the selectivity, detection sensitivity, and anti-interference ability of the antibody. Combined with the excellent fluorescence sensing properties of fluorescent substances, this improves the ability of fluorescence immunosensing methods to detect trace pesticides in complex food matrices. Summary of the Invention

[0005] This invention develops a highly sensitive competitive fluorescent immunosensor based on Nb-FNT-ALP (enzyme-labeled nanobody) and AuNCs-MnO2NFs (fluorescent substrate). The coated antigen immobilized on a 96-well plate competitively captures Nb-FNT-ALP with FNT, forming an antigen-antibody complex. The AuNCs-MnO2 composite material is used to identify ALP activity, thereby evaluating FNT residual levels and further improving detection performance. In addition, this method has been successfully applied to the determination of FNT residues in real samples (tap water, Songhua River water, apples, cabbage, lettuce, rice, and tomatoes). Simultaneously, this method was successfully used to monitor the degradation dynamics of FNT in pak choi. This invention integrates the high selectivity of Nb-FNT-ALP with the excellent optical properties of the fluorescent matrix AuNCs-MnO2 composite material to construct a universal immunosensor applicable to a wide range of target analyses. In addition to significantly improving sensitivity, this invention changes the detection process from a two-step method (antigen-primary antibody-secondary antibody) to a one-step method (antigen-enzyme-labeled nanobody), shortening the detection time and effectively improving anti-interference capabilities, providing a powerful tool for monitoring harmful substances in agricultural and environmental samples.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] The development of a pesticide fluorescence immunoassay based on gold clusters and manganese dioxide nanosheets involves the following steps:

[0008] Preparation of AuNCs-MnO2 composite materials:

[0009] Take tetrachloroauric acid (HAuCl4·4H2O, 10 mmol L) -1 ) and bovine serum albumin (BSA, 50 mg / mL) -1 The mixture was incubated at 37°C for 10 minutes, then the two were mixed at a volume ratio of 1:1. After vigorous stirring for 5 minutes, NaOH (1 mol L) was added. -1 Compared to the above mixed solution, a volume ratio of 1:20 was added to the above solution, and the mixture was stirred vigorously at 37°C for 12 h. The resulting yellow AuNCs solution was purified in a dialysis bag (1 kDa). The purified AuNCs solution was mixed with ultrapure water at a volume ratio of 1:3.9 and gently stirred at room temperature for 5 min. Subsequently, manganese chloride (MnCl2·4H2O, 50 mmol / L) was added. -1 A 1:98 ratio of the above mixed solution was added to the above solution, and the mixture was gently stirred for 1 hour. Afterwards, NaOH (1 mol L) was added... -1The above mixed solution was added to the above solution at a volume ratio of 1:99, and the mixture was gently stirred for 4 hours to obtain a brown AuNCs-MnO2 solution. Finally, the AuNCs-MnO2 solution was purified using a dialysis bag (1 kDa). The purified AuNCs solution and the brown AuNCs-MnO2 composite solution need to be stored at 4°C.

[0010] B. Study on the catalytic activity of alkaline phosphatase:

[0011] Ascorbic acid (AA) is produced through a catalytic reaction between alkaline phosphatase (ALP) and L-ascorbic acid-2-phosphate (AAP). AA can then reduce MnO2NFs to Mn. 2+ The fluorescence of AuNCs quenched was restored, and the resulting fluorescence signal change was used to study the enzyme's catalytic activity. AAP (100 μmol L) was added... -1 The solution was mixed with different concentrations of ALP at a volume ratio of 2:1 and incubated at 37°C for 30 min. Then, the above solution was added to Tris-HCl buffer (pH = 9.0, 10 mmol / L). -1 The solution was mixed with AuNCs-MnO2 in a ratio of 3:1:2 and incubated at 37°C for 10 min. Then, the mixture was mixed with ultrapure water in a ratio of 3:7. After mixing, fluorescence was measured using a fluorescence spectrophotometer.

[0012] C. Fluorescent immunoassay for detecting fenitrothion:

[0013] Use carbonate buffer (CBS, 50 mmol / L) -1 (pH=9.6) Dilute the coated antigen to 1 μg / mL -1 100 μL of antigen was coated onto each well of a 96-well plate and incubated overnight at 4°C. The solution was then diluted with PBST (containing 10 mmol / L of 0.05% Tween-20). -1 After washing three times with PBS, 300 μL of bovine serum albumin (BSA, 3 wt%) was added to each well, and the 96-well plate was blocked at 37°C for 60 min. After washing three times with PBST, Nb-FNT-ALP (663.5 ng / mL) was added. -1 FNT and different concentrations were added at a 1:1 ratio to 96-well plates containing the coated antigen. After incubation at 37°C for 90 min, the plates were washed three times with PBST. Then, ultrapure water and AAP (100 μmol / L) were added at 37°C. -1The solution was added to a 96-well plate at a 1:2 ratio and reacted for 30 min. Subsequently, the above solution was mixed with Tris-HCl buffer (pH = 9.0, 10 mmol / L). -1 The solution was mixed with AuNCs-MnO2 in a ratio of 3:1:2 and allowed to stand at 37°C for 10 min. Then, the resulting solution was transferred to a 1.5 mL centrifuge tube and mixed with ultrapure water in a ratio of 3:7, and the fluorescence value was measured.

[0014] The inhibition efficiency (IE%) was used as the response signal to analyze the presence level of FNTs. The formula for calculating IE% is as follows: IE% = (FNTs / FNTs) Max -F FNT ) / (F Max -F Min )×100%. Where F Max The fluorescence intensity represents the fluorescence intensity when the system does not contain FNT. FNT The fluorescence intensity represents the fluorescence intensity in the presence of FNTs in the system. Min The fluorescence intensity is denoted as the fluorescence intensity of the system without Nb-FNT-ALP and FNT.

[0015] D. Monitoring and analysis of pesticide levels in actual food samples:

[0016] Seven samples (tap water, Songhua River water, apple, Chinese cabbage, lettuce, rice, and tomato) were prepared to evaluate the ability of this invention to detect pesticides in actual food samples. Except for the water sample, homogenized samples (0.5 g) were mixed with FNT standard solutions (0.01, 1, 10, and 100 ng / mL). -1 The above samples were added to an extraction buffer solution of acetonitrile and water in a 5:1 ratio. After shaking for 60 min, NaCl (1.5 g) was added to a centrifuge tube. The mixture was vortexed for 5 min to allow precipitation and separation. After standing for 30 min, the supernatant was extracted from the samples and detected using the established fluorescence immunoassay method. Following this, two pots of bok choy were planted; one served as a blank control, and the other was sprayed daily with 10 mL of FNT standard (5.0 μg / mL) starting from day 8 after planting. -1 The mixture was sprayed for two consecutive days. After the final spray, once the surface of the bok choy was dry, FNT was extracted from the roots and leaves of the bok choy using the method described above. Then, the degradation level of FNT in the bok choy was monitored using this invention.

[0017] The mechanism of this invention is as follows:

[0018] MnO2 NFs can effectively quench the fluorescence of AuNCs, resulting in a low fluorescence signal in the AuNCs-MnO2 composite material. ALP can catalyze the formation of AA from AAP, and AA will reduce MnO2 NFs to Mn. 2+This causes the FNT to lose its quenching ability against AuNCs, thereby restoring the fluorescence of AuNCs and resulting in an increase in the fluorescence signal of the system. Using a competitive ELISA, Nb-FNT-ALP and FNT are simultaneously added to a 96-well plate coated with antigen. The coated antigen and FNT compete for Nb-FNT-ALP. After washing, antigen-antibody complexes remain in the 96-well plate, while the FNT-antibody conjugates are washed away. Therefore, the more FNT present, the less Nb-FNT-ALP is present in the system, leading to a decrease in AA production and a weakened fluorescence signal recovery ability. This invention combines the high selectivity of Nb-FNT-ALP with the excellent optical properties of the AuNCs-MnO2 composite material. Nb-FNT-ALP serves as the recognition unit for direct competitive fluorescence immunoassay, saving detection steps and culture time. This method has been successfully applied to the precise monitoring of FNT degradation in Chinese cabbage. This scheme provides a multifunctional sensing tool by changing the recognition unit and points to the development direction of high-performance fluorescence immunosensors. In addition, it provides a powerful tool for the rapid and sensitive monitoring of harmful substances in agricultural and environmental samples.

[0019] The high sensitivity of this invention stems from the following characteristics:

[0020] (1) The fusion of enzyme and nanobody forms an immunoassay reagent, which improves the binding affinity and does not require covalent coupling, thus improving the detection sensitivity.

[0021] (2) The combination of low background signal MnO2 nanosheets and AuNCs is beneficial to improving the sensitivity of quantitative detection of targets.

[0022] (3) AuNCs-MnO2 composite material provides a highly sensitive switchable fluorescence signal. Attached Figure Description

[0023] Figure 1Example 1 illustrates the preparation of the AuNCs-MnO2 composite material. (A) is a TEM image of the AuNCs-MnO2 composite material; (B) is the UV-Vis absorption spectrum of AuNCs, MnO2 nanosheets, and the AuNCs-MnO2 composite material; (C) is the infrared spectrum of MnO2 nanosheets and the AuNCs-MnO2 composite material; (D) is the photostability of the AuNCs-MnO2 composite material; and (E) is the fluorescence intensity of AuNCs immobilized on MnO2 nanosheets at different concentrations (0, 0.125, 0.25, 0.375, 0.5, 0.75, 1, 1.25, 1.5 mM). The inset is a linear graph of (F-F1) / F versus the logarithm of the AuNCs-MnO2NFs concentration. F and F1 represent the fluorescence intensity of AuNCs and the fluorescence intensity of AuNCs-MnO2 composite materials at different concentrations, respectively; (F) is a photograph of AuNCs-MnO2NFs powder under sunlight and handheld UV lamp irradiation and AuNCs-MnO2 solution under handheld UV lamp irradiation.

[0024] Figure 2 Example 2 describes the study of alkaline phosphatase catalytic activity. (A) is a schematic diagram of the principle of ascorbic acid reduction of AuNCs-MnO2 composite material; (B) shows different concentrations of AAP (5, 10, 50, 100, 150 μmol / L). -1 ) and ALP (0, 0.1, 1, 10, 50, 100 U L) -1 (A) Cross-reaction occurs; (F2 and F0 are the fluorescence intensities of the cross-reaction and the fluorescence intensity of the system without ALP, respectively); (C) Feasibility of constructing a fluorescence sensing platform. Fluorescence spectra of AuNCs-MnO2, AuNCs-MnO2+AA, AuNCs-MnO2+AAP+ALP, and AuNCs; (D) Fluorescence spectra of AuNCs at different concentrations of ALP (0, 0.001, 0.004, 0.008, 0.1, 0.4, 0.8, 1 UL). -1 Fluorescence spectra of the AuNCs-MnO2+AAP+ALP system in the presence of ALP. The inset shows the fluorescence intensity trend of the system in the presence of ALP; (E) shows the fluorescence intensity trend of the system in the presence of ALP. ALP A linear plot of -F0) / (F-F0) versus the logarithm of ALP concentration (F ALP (F) represents the fluorescence intensity in the presence of different concentrations of ALP; (F) represents the fluorescence intensity of the AuNCs-MnO2+AAP system and the AuNCs-MnO2+AAP+ALP system in the presence of interfering substances (0.1 μg / mL).

[0025] Figure 3Example 3 describes a fluorescence immunoassay method for detecting fenitrothion. (A) is a schematic diagram of the competitive immunoassay principle; (B) compares the sensitivity of FIA and ELISA for detecting fenitrothion; (C) demonstrates the specificity of this method for detecting fenitrothion (the concentration of fenitrothion and other pesticides is 1.0 ng / mL). -1 (D) shows the anti-interference ability of this method for detecting fenitrothion (the concentration of fenitrothion and other substances is 1.0 ng / mL). -1 ).

[0026] Figure 4 Example 4 describes the monitoring and analysis of pesticide levels in actual food samples. (A) shows images of bok choy, leaves, and roots; (B) shows the degradation of FNT in the leaves and roots. Detailed Implementation

[0027] The invention will now be further described with reference to the accompanying drawings.

[0028] Example 1: Synthesis and Characterization of AuNCs-MnO2

[0029] Take tetrachloroauric acid (HAuCl4·4H2O, 10 mmol L) -1 ) and bovine serum albumin (BSA, 50 mg / mL) -1 The mixture was incubated at 37°C for 10 minutes, then the two were mixed at a volume ratio of 1:1. After vigorous stirring for 5 minutes, NaOH (1 mol L) was added. -1 Compared to the above mixed solution, a volume ratio of 1:20 was added to the above solution, and the mixture was stirred vigorously at 37°C for 12 h. The resulting yellow AuNCs solution was purified in a dialysis bag (1 kDa). The purified AuNCs solution was mixed with ultrapure water at a volume ratio of 1:3.9 and gently stirred at room temperature for 5 min. Subsequently, manganese chloride (MnCl2·4H2O, 50 mmol / L) was added. -1 A 1:98 ratio of the above mixed solution was added to the above solution, and the mixture was gently stirred for 1 hour. Afterwards, NaOH (1 mol L) was added... -1A mixture of AuNCs and MnO2 was added to the above solution at a volume ratio of 1:99 and gently stirred for 4 hours to obtain a brown AuNCs-MnO2 solution. Finally, the AuNCs-MnO2 solution was purified using a dialysis bag (1 kDa). The purified AuNCs solution and the brown AuNCs-MnO2 composite solution needed to be stored at 4°C. Morphological images of AuNCs and AuNCs-MnO2NFs were recorded using a Hitachi transmission electron microscope (TEM); UV-Vis absorption spectra were recorded using a Shimadzu UV-2550 UV-Vis spectrophotometer; Fourier transform infrared (FTIR) spectra were collected using an IRPRESTIGE-21 spectrometer; and fluorescence spectra of the AuNCs-MnO2 composite material were acquired using a Shimadzu RF-5301 fluorescence spectrometer, with an excitation wavelength of 585 nm and an emission wavelength of 615–800 nm. The emission and excitation slits were fixed at 15 nm and 10 nm, respectively.

[0030] The results are as follows Figure 1 As shown, the AuNCs-MnO2 composite material was characterized by transmission electron microscopy (TEM), revealing a typical wrinkled lamellar structure. Figure 1 A). The surface exhibits distinct lattice fringes of 0.21 nm, confirming that AuNCs were successfully anchored on the surface of MnO2 nanosheets. Figure 1 (Inset A). The UV-Vis absorption spectrum shows a broad absorption band in the 300–500 nm range, with the maximum absorption peak at 375 nm. Figure 1 B), which makes MnO2 nanosheets an effective quencher for various energy donors. Subsequently, Fourier transform infrared spectroscopy (FTIR) was performed at 3300 cm⁻¹. -1 1656cm -1 and 514cm -1 The characteristic peaks of OH bond stretching vibration, Mn-OH bending vibration, and Mn-O stretching vibration are shown at the respective locations. Figure 1 C). Under 150W xenon lamp irradiation, the fluorescence emission intensity of the AuNCs-MnO2 composite material exhibits high stability ( Figure 1 D). In the AuNCs-MnO2 composite material, as the concentration of MnO2 nanosheets increases from 0 to 1.5 mM, the fluorescence intensity of AuNCs gradually decreases. Figure 1 E), the fluorescent color changes significantly from bright red to dark red. Figure 1 F). The above conclusions fully demonstrate the successful synthesis of AuNCs-MnO2 composite materials.

[0031] Example 2: Study on the catalytic activity of alkaline phosphatase

[0032] Take Tris-HCl buffer (pH = 9.0, 10 mmol / L) -125 μL, 50 μL of the AuNCs-MnO2 solution synthesized in Example 1, and ascorbic acid (50 μmol / L) -1 50 μL of AAP was added to the same centrifuge tube and reacted at 37 °C for 10 min to determine the reducing power of ascorbic acid. Subsequently, the optimal working concentration of AAP was screened using a checkerboard method. Different concentrations of AAP were mixed with different concentrations of ALP at a ratio of 2:1 and reacted at 37 °C for 30 min. The above solution was then mixed with Tris-HCl buffer (pH = 9.0, 10 mmol / L). -1 ALP and AuNCs-MnO2 solution were mixed in a 3:1:2 ratio and reacted at 37 °C for 10 min. Afterward, the enzyme's catalytic activity was studied by the fluorescence reaction of ALP with L-ascorbic acid-2-phosphate trisodium salt (AAP). AAP (100 μmol L... -1 The solution was mixed with different concentrations of ALP at a volume ratio of 2:1 and incubated at 37°C for 30 min. Then, the above solution was added to Tris-HCl buffer (pH = 9.0, 10 mmol / L). -1 The mixture of AuNCs and AuNCs-MnO2 was prepared in a ratio of 3:1:2 and incubated at 37°C for 10 min. The resulting mixture was then mixed with ultrapure water in a ratio of 3:7. After thorough mixing, fluorescence was measured using a fluorescence spectrophotometer.

[0033] The results are as follows Figure 2 As shown, Figure 2 A is a schematic diagram illustrating the principle of AA reduction of AuNCs-MnO2 composite material; Figure 2 B screened out that the optimal working concentration of AAP was 100 μmol / L. -1 ; Figure 2 C demonstrates that this principle is feasible; Figure 2 D indicates that as the ALP concentration increases, the fluorescence recovery of the system improves, and from... Figure 2 E shows the fluorescence recovery rate (F) ALP -F0) / (F-F0) in the range of 0.001 to 1.0 UL -1 Within the range, it showed a good linear relationship with the logarithmic concentration of ALP (R0). 2 =0.9942). Where, F ALP F0 and F1 represent the fluorescence intensity of the system in the presence and absence of ALP, respectively. Figure 2 F indicates that ALP activity is not affected by proteins (bovine serum albumin), amino acids (histidine, glutamic acid, tryptophan, and tyrosine), lactose, and ions (Na+) in biological samples. + and Mg 2+ Interference from common substances such as )

[0034] Example 3: Fluorescent Immunoassay for Detection of Fenitrothion

[0035] Use carbonate buffer (CBS, 50 mmol / L) -1 (pH=9.6) Dilute the coated antigen to 1 μg / mL -1 100 μL of antigen was coated onto each well of a 96-well plate and incubated overnight at 4°C. The solution was then diluted with PBST (containing 10 mmol / L of 0.05% Tween-20). -1 After washing three times with PBS, 300 μL of bovine serum albumin (BSA, 3 wt%) was added to each well, and the 96-well plate was blocked at 37°C for 60 min. After washing three times with PBST, Nb-FNT-ALP (663.5 ng / mL) was added. -1 FNT and different concentrations were added at a 1:1 ratio to 96-well plates containing the coated antigen. After incubation at 37°C for 90 min, the plates were washed three times with PBST. Then, ultrapure water and L-ascorbic acid-2-phosphate trisodium salt (AAP, 100 μmol / L) were added at 37°C. -1 The solution was added to a 96-well plate at a 1:2 ratio and reacted for 30 min. Subsequently, the above solution was mixed with Tris-HCl buffer (pH = 9.0, 10 mmol / L). -1 The solution was mixed with AuNCs-MnO2 in a 3:1:2 ratio and incubated at 37°C for 10 min. Then, the resulting solution was transferred to a 1.5 mL centrifuge tube and mixed with ultrapure water in a 3:7 ratio, and the fluorescence value was measured. The difference between traditional ELISA and fluorescence immunoassay is that in the traditional ELISA protocol, after incubation at 37°C for 90 min, followed by three washes, 200 μL of the chromogenic substrate 4-nitrophenyl phosphate disodium salt hexahydrate (4-NPP) was directly added and reacted at 37°C for 30 min. Finally, the OD value was measured and calculated according to the analytical formula.

[0036] The inhibition efficiency (IE%) was used as the response signal to analyze the presence level of FNTs. The formula for calculating IE% is as follows: IE% = (FNTs / FNTs) Max -F FNT ) / (F Max -F Min )×100%. Where F Max The fluorescence intensity represents the fluorescence intensity when the system does not contain FNT. FNT The fluorescence intensity represents the fluorescence intensity in the presence of FNTs in the system. Min The fluorescence intensity is the value of the system without Nb-FNT-ALP and FNT. The above formula also applies if the fluorescence intensity is replaced with OD values.

[0037] The results are as follows Figure 3 As shown, Figure 3 A demonstrates the principle of the fluorescence immunoassay method for detecting FNT. Figure 3 B indicates a range of 0.00001–100 ng / mL.-1 Within the concentration range, the inhibition rate (IE%) increased with the FNT concentration (R). 2 The detection limit of FNT (LOD, IC10) was increased by 0.9940 pg / mL, and the detection limit was calculated to be 5.78 pg / mL. -1 The sensitivity of this invention is nearly 56 times higher than that of traditional ELISA. The selectivity of the construction method was investigated using methamidophos, methamidophos, lambda-cyhalothrin, malathion, carbaryl, chlorpyrifos, acetamiprid, paraoxon, methyl paraoxon, and imidacloprid as controls. Figure 3 The results in C clearly show that only FNT (1 ng / mL) -1 This leads to significant changes, demonstrating the high selectivity of the present invention for FNT. In addition, as... Figure 3 As shown in D, common substances in the sample, such as ovalbumin, histidine, glutamic acid, bovine serum albumin, lactose, sodium ions, magnesium ions, tryptophan, and tyrosine, do not significantly affect the fluorescence changes of the system constructed by this method, indicating that the method of this invention has good anti-interference ability.

[0038] Example 4: Monitoring and Analysis of Pesticide Levels in Actual Food Samples

[0039] Seven samples (tap water, Songhua River water, apple, Chinese cabbage, lettuce, rice, and tomato) were prepared to evaluate the ability of this invention to detect pesticides in actual food samples. Except for the water sample, homogenized samples (0.5 g) were mixed with FNT standard solutions (0.01, 1, 10, and 100 ng / mL). -1 The above samples were added to an extraction buffer solution of acetonitrile and water in a 5:1 ratio. After shaking for 60 min, NaCl (1.5 g) was added to a centrifuge tube. The mixture was vortexed for 5 min to allow precipitation and separation. After standing for 30 min, the supernatant was extracted from the samples and detected using the established fluorescence immunoassay method. Subsequently, two pots of bok choy were planted; one served as a blank control, and the other was sprayed daily with 10 mL of FNT standard (5.0 μg / mL) once a day starting from day 8 after planting. -1 The mixture was sprayed for two consecutive days. After the final spray, once the surface of the bok choy was dry, FNT was extracted from the roots and leaves of the bok choy using the method described above. Then, the degradation level of FNT in the bok choy was monitored using this invention.

[0040] The results are shown in Table 1 and , respectively. Figure 4 As shown in Table 1, the recovery rate of FNT in actual samples ranged from 90.49% to 107.21%, with a relative standard deviation (RSD) of less than 7.45%, indicating the potential applicability of this invention for the detection of FNT in complex food and environmental samples. Figure 4As shown in Figure B, after 3 days following the last FNT spray, the residual amount decreased by half, indicating that the residual amount of FNT in the leaves and roots gradually decreased with the extension of the growth period. After 10 days of growth, the residual amount of FNT was less than 0.5 μg / g. -1 This demonstrates the effective metabolism of FNT in bok choy. Figure 4 The FNT degradation process shown in D follows pseudo-first-order kinetics, with leaf Y = 4.3826e (-0.2332x) (R 2 =0.9895), root system Y = 3.672e (-0.3061x) (R 2 =0.9861).

[0041] Table 1

[0042]

Claims

1. A fluorescent immunoassay method for pesticides based on gold cluster-manganese dioxide nanosheet, characterized in that, The steps are as follows: A. Preparation of AuNCs-MnO2 composite material: Take HAuCl4·4H2O and BSA at 37℃ for 10 min, then mix them in a volume ratio of 1:1, stir vigorously for 5 min, then add NaOH to the above solution in a volume ratio of 1:20, continue to stir vigorously at 37℃ for 12 h, put the obtained yellow AuNCs solution into a 1kDa dialysis bag for purification, mix the purified AuNCs solution with ultrapure water in a volume ratio of 1:3.9, stir gently at room temperature for 5 min, then add MnCl2·4H2O to the solution in a ratio of 1:98, stir gently for 1 h, then add NaOH to the solution in a volume ratio of 1:99, stir gently for 4 h, obtain a brown AuNCs-MnO2 solution, finally, purify the AuNCs-MnO2 solution with a 1kDa dialysis bag, and the purified AuNCs solution and the brown AuNCs-MnO2 composite solution need to be stored at 4℃, B. Study on catalytic activity of alkaline phosphatase: Through the catalytic reaction of alkaline phosphatase and L-ascorbic acid-2-phosphate trisodium salt to generate ascorbic acid, ascorbic acid reduces MnO2 nanosheet to Mn 2+ , restore AuNCs fluorescence, produce fluorescence signal change, study enzyme catalytic activity, mix L-ascorbic acid-2-phosphate trisodium salt with different concentrations of ALP according to volume ratio 2:1, and incubate at 37℃ for 30min, then mix solution, Tris-HCl buffer and AuNCs-MnO2 solution in the ratio of 3:1:2, incubate at 37℃ for 10min, then mix the mixture with ultrapure water in the ratio of 3:7, after mixing, use fluorescence spectrophotometer to measure fluorescence, C. Fluorescence immunoassay for detecting fenitrothion: Coat antigen with carbonate buffer to 1 μg / mL -1 , 100 μL / well of antigen was coated on 96-well plates, incubated at 4°C overnight, washed 3 times with PBST, then 300 μL of bovine serum albumin was added to each well, the 96-well plate was blocked at 37°C for 60 min, washed 3 times with PBST, then Nb-FNT-ALP and different concentrations of FNT were added to the 96-well plate with coated antigen at a ratio of 1:1, incubated at 37°C for 90 min, then washed 3 times with PBST, then ultrapure water and L-ascorbic acid-2-phosphate trisodium salt were added to the 96-well plate at a ratio of 1:2 at 37°C and reacted for 30 min, then the solution, Tris-HCl buffer and AuNCs-MnO2 solution were mixed at a ratio of 3:1:2, and left to stand at 37°C for 10 min, then the obtained solution was moved to a 1.5 mL centrifuge tube and mixed with ultrapure water at a ratio of 3:7, and the fluorescence value was measured, D. Monitoring and analysis of pesticide levels in actual food samples: Seven samples were prepared: tap water, Songhua River water, apples, Chinese cabbage, lettuce, rice and tomatoes to evaluate the ability to detect pesticides in actual food samples. Except for water samples, homogeneous samples were added with FNT standard solution. The samples were added with extraction buffer mixed with acetonitrile and water at a ratio of 5:

1. After shaking for 60 min, NaCl was added to the centrifuge tube, and the mixture was vortexed for 5 min to allow the precipitate to separate. After standing for 30 min, the supernatant was extracted from the sample and detected using the established fluorescence immunoassay method. After that, two pots of Chinese cabbage were planted, one as a blank control and the other sprayed with 10 mL of FNT standard every day for 2 consecutive days starting from the 8th day after planting. After the last spraying, the FNT was extracted from the roots and leaves of the Chinese cabbage using the method described above, and then the degradation level of FNT in the Chinese cabbage was monitored.

2. The pesticide fluorescent immunoassay method based on gold cluster-manganese dioxide nanosheet according to claim 1, characterized in that, The entire synthesis process of AuNCs described in step A was carried out at 37℃, and the entire synthesis process of AuNCs-MnO2 was carried out at room temperature.

3. The pesticide fluorescent immunoassay method based on gold cluster-manganese dioxide nanosheet according to claim 1, characterized in that, HAuCl4.4H2O concentration was 10 mmol L -1 BSA concentration was 50 mg mL -1 NaOH concentration was 1 mol L -1 MnCl2.4H2O concentration was 50 mmol L -1 .

4. The pesticide fluorescent immunoassay method based on gold cluster-manganese dioxide nanosheet according to claim 1, characterized in that, The concentration of L-ascorbic acid-2-phosphoric acid trisodium salt described in step B is 100 μmol L -1 Tris-HCl buffer solution pH = 9.0, concentration 10 mmol L -1 The reaction process is carried out at 37 °C.

5. The pesticide fluorescence immunoassay method based on gold cluster-manganese dioxide nanosheets as described in claim 1, characterized in that, The excitation wavelength for fluorescence measurement in step B is 585 nm, and the emission wavelength is 615-800 nm. The emission and excitation slits are fixed at 15 nm and 10 nm, respectively.

6. The method according to claim 1, wherein the method is a gold cluster-manganese dioxide nanosheet-based fluorescent immunoassay for pesticide. The optimal emission wavelength of AuNCs measured in step B is located at 655 nm, and the maximum absorption peak of MnO2 NFs is located at 375 nm.

7. The method according to claim 1, wherein the method is a gold cluster-manganese dioxide nanosheet-based fluorescent immunoassay for pesticide. The carbonate buffer concentration was 50 mmol L -1 pH = 9.6; the coating antigen concentration was 1 μg mL -1 PBST was 10 mmol L -1 PBS, the Nb-FNT-ALP concentration was 663.5 ng mL -1 L-ascorbic acid-2-phosphoric acid trisodium salt concentration was 100 μmol L -1 Tris-HCl buffer pH = 9.0, concentration was 10 mmol L -1 The reaction was carried out at 37 °C.

8. The pesticide fluorescence immunoassay method based on gold cluster-manganese dioxide nanosheets as described in claim 1, characterized in that, The acetonitrile and water described in step D are both analytical pure solutions.

9. The method according to claim 1, wherein the method is a gold cluster-manganese dioxide nanosheet-based fluorescent immunoassay for pesticide. The 10 mL of FNT standard described in step D has a concentration of 5.0 μg mL-1.

10. The method according to claim 1, wherein the method is a gold cluster-manganese dioxide nanosheet-based fluorescent immunoassay for pesticides. This method can construct a general method for detecting multiple pesticides by changing the recognition unit.

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