A method for detecting carbendazim in food based on a bimetallic nanocluster self-assembly compound

Abstract

The application discloses a method for detecting carbendazim in food based on a bimetallic nanocluster self-assembly compound, gold nanoclusters and silver nanoclusters are prepared respectively by taking 6-azido-2-thiothymine and arginine as templates, nanocomposites are self-assembled through a one-step method, and gold-silver nanoclusters AuAgNCs-CD of carboxymethyl-beta-cyclodextrin which is modified and has fluorescence enhancement are obtained, FRET between the AuAgNCs-CD and RhB can occur, and the carbendazim can complete the indicator substitution analysis through host-guest recognition and competition with the RhB, at this time, anti-FRET effect occurs, and a ratio fluorescence sensing method of the carbendazim is constructed accordingly; meanwhile, a ratio fluorescence lateral flow chromatography test paper is developed, the test paper has high sensitivity, rapidness, excellent selectivity and anti-interference capacity, and has high potential for application in carbendazim detection.

Description

Technical Field

[0001] This invention belongs to the field of optical sensors, specifically relating to a method for detecting carbendazim in food based on a bimetallic nanocluster self-assembled complex. Background Technology

[0002] Carbendazim (CBZ), as a fungicide, can effectively control diseases of crops, fruits, and vegetables caused by fungi. It has many advantages, including a broad fungicidal spectrum, low cost, and good antifungal effect. However, its good chemical stability allows it to persist in soil for a long time. Ingestion of CBZ residues by humans or animals through food can pose various health risks. Therefore, developing rapid and portable sensing methods for carbendazim residues is of great significance.

[0003] Currently, many detection methods have been established for carbendazim residues in food. These include high-performance liquid chromatography (HPLC), surface-enhanced Raman scattering, capillary electrophoresis, electrochemical methods, and immunoassay. However, most of these methods suffer from drawbacks such as expensive equipment, time-consuming sample processing, difficulty in preparing biological reagents, or high skill requirements for operators.

[0004] Fluorescence resonance energy transfer (FRET)-based sensing methods offer advantages such as high sensitivity, strong anti-interference capabilities, and visualization. Therefore, sensing methods based on metal nanoclusters combined with FRET have attracted considerable attention from researchers. Based on supramolecular recognition and guest competition principles, indicator substitution analysis has led to the development of numerous fluorescence detection methods for pesticides; however, their single signal output mode is susceptible to interference. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting carbendazim in food based on bimetallic nanocluster self-assembled complexes.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for detecting carbendazim in food based on a bimetallic nanocluster self-assembled complex, comprising,

[0009] Preparation of ATT-AuNCs nanocluster solution;

[0010] Preparation of Arg-AuNCs nanocluster solution;

[0011] Preparation of AuAgNCs nanoclusters: Arg-AgNCs nanocluster solution was mixed into ATT-AuNCs nanocluster solution and reacted at 30-37℃ for 12-24h. The synthesized AuAgNCs were precipitated with isopropanol, centrifuged, and the centrifuged product was redispersed in deionized water to obtain AuAgNCs nanocluster solution.

[0012] Preparation of AuAgNCs-CD nanoclusters: Take AuAgNCs nanocluster solution, add CM-β-CD solution, incubate at 40-50℃ for 2-3 h, and then filter with an ultrafiltration tube to remove excess reactants to obtain AuAgNCs-CD nanocluster solution;

[0013] Detection of carbendazim in food: After processing the food, carbendazim solution is prepared by dissolving it in methanol;

[0014] Rhodamine B solution and carbendazim solution were added sequentially to AuAgNCs-CD nanocluster solution. Simultaneously, under 410 nm excitation, the fluorescence emission spectra of the solution at dual wavelengths of 530 nm and 580 nm were recorded. Based on the ratio of fluorescence intensity, a standard curve for carbendazim concentration was constructed to determine the carbendazim content in food.

[0015] As a preferred embodiment of the method described in this invention, the preparation of the ATT-AuNCs nanocluster solution includes,

[0016] Dissolve 6-aza-2-thiothymine (ATT) in 0.2 mol / L NaOH solution to form an 80 mmol / L ATT solution, and mix it with a 10 mg / mL HAuCl4 solution.

[0017] After continuous stirring for 1–2 hours under light-protected conditions, the obtained ATT-AuNCs were washed twice with isopropanol precipitation, dissolved in deionized water to form a stock solution, and stored at 4°C in the dark to obtain ATT-AuNCs nanoclusters; among which,

[0018] The ratio of NaOH solution to HAuCl4 solution is 3 mL: 3 mL;

[0019] The stirring speed was 800 r / min and the stirring temperature was 25℃.

[0020] The concentration of the stock solution is 15 mg / mL.

[0021] As a preferred embodiment of the method described in this invention, the preparation of the Arg-AuNCs nanocluster solution includes,

[0022] Add 0.5M Arg solution dropwise to AgNO3 solution, adjust the pH of the solution to 10 with 1M NaOH, and incubate at 37℃ for 6 hours.

[0023] Arg-AgNCs were extracted by ultrafiltration and stored at 4°C in the dark to obtain an Arg-AuNCs nanocluster solution; wherein,

[0024] The ratio of Arg solution to AgNO3 solution is 1 mL: 1 mL, and the concentration of AgNO3 solution is 10 mmol / L.

[0025] In a preferred embodiment of the method described in this invention, the preparation of AuAgNCs nanoclusters involves a ratio of 6 mL to 18 mL between Arg-AgNCs nanocluster solution and ATT-AuNCs nanocluster solution, a pH of 10 for the Arg-AgNCs nanocluster solution, a centrifugation speed of 10000 rpm, and a centrifugation time of 5 min.

[0026] In a preferred embodiment of the method described in this invention, the food is processed, including...

[0027] After drying the food, cut it into pieces and then crush it using a high-speed shearing machine.

[0028] Take 5g of sample, add 10mL of dichloromethane, sonicate for 5min, and centrifuge at 8000r / min to remove insoluble precipitate;

[0029] The sample solution was prepared by water bath at 50℃ for 10 min, 2 mL of methanol was added and diluted with ultrapure water to 20 mL.

[0030] The food products mentioned include apples.

[0031] In a preferred embodiment of the method described in this invention, the step of constructing a standard curve for carbendazim concentration based on the ratio of fluorescence intensities includes:

[0032] Based on the ratio of fluorescence intensity at different wavelengths in the solution, respectively using F 580 / F 530 Regarding the concentration of Rhodamine B and F 530 / F 580 A standard curve was constructed for carbendazim concentration to determine the carbendazim content in food; among which,

[0033] F 530 and F 580 These represent the highest fluorescence intensities at the characteristic emission peaks of CD-AuAgNCs and Rhodamine B, respectively.

[0034] As a preferred embodiment of the method described in this invention, it further includes:

[0035] A fluorescent lateral flow chromatography test strip (RFLFS) for carbendazim determination is prepared, wherein the fluorescent lateral flow chromatography test strip (RFLFS) consists of a sample pad, a nitrocellulose membrane (NC membrane), an absorbent pad, a PVC board, and a T-line;

[0036] The fluorescence value on the T line was measured using a fluorescence reader, and the fluorescence change rate F was compared with the concentration of carbendazim. 510 / F 610 A standard curve for carbendazim on a paper substrate was constructed using graphing, enabling the detection of carbendazim.

[0037] As a preferred embodiment of the method described in this invention, the linear range of carbendazim detection is 0.2 μmol / L to 1.4 μmol / L, and the detection limit is calculated to be 60 nmol / L using the 3σ / s formula.

[0038] In a preferred embodiment of the method described in this invention, the preparation method of the fluorescence side-flow chromatography strip RFLFS includes:

[0039] The sample pad was soaked in 0.01 mol / L phosphate buffer at pH 7.4 for 30 min and dried at 37 °C for 8 h. The phosphate buffer contained 0.01 mol / L Na2HPO4 and 0.01 mol / L NaH2PO4.

[0040] 30 μL of protamine sulfate was sprayed onto the NC membrane as a T-line using a three-dimensional spray nozzle and dried for 2 h. The distance between the T-line and the absorbent pad was 24 mm. Then, 30 μL of CD-AuAgNCs-RhB was sprayed onto the same position and fixed, and dried at 37 °C for 2 h.

[0041] Attach all components to the PVC board, and place the sample pad and absorbent pad on the NC film, overlapping it by 2mm respectively.

[0042] Cut the assembled PVC board into strips 4mm long and 1mm wide, and store them in a dark, sealed container.

[0043] In a preferred embodiment of the method described in this invention, the CD-AuAgNCs are diluted by a factor of 200, and the protamine concentration is 1.25 mg / mL.

[0044] Beneficial effects of this invention:

[0045] This invention uses 6-aza-2-thiothymidine (ATT) and arginine (Arg) as templates to prepare gold nanoclusters and silver nanoclusters, respectively. Through a one-step self-assembly of nanocomposites, gold and silver nanoclusters (AuAgNCs-CD) modified with carboxymethyl-β-cyclodextrin (CM-β-CD) and enhanced fluorescence were obtained. This AuAgNCs-CD can undergo FRET with RhB. Carbendazim can complete the indicated substitution analysis through host-guest recognition and competition with RhB, accompanied by an anti-FRET effect. Based on these effects, a ratiometric fluorescence sensing method for carbendazim was constructed. Furthermore, based on the above work, a ratiometric fluorescence side-flow chromatography strip (RFLFS) was developed. This RFLFS strip exhibits high sensitivity, speed, and excellent selectivity and anti-interference ability, thus possessing great potential for application in carbendazim detection. Attached Figure Description

[0046] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0047] Figure 1 This is a schematic diagram of the detection of CBZ in food based on the self-assembled bimetallic nanocluster complex in an embodiment of the present invention. In this diagram, A is a schematic diagram of the construction of the homogeneous fluorescence detection method for carbendazim, and B is a schematic diagram of the construction of the ratiometric fluorescence side-flow chromatography test strip for carbendazim.

[0048] Figure 2 The images shown are TEM images and particle size distribution maps in this embodiment of the invention. (a) and (b) are TEM images of AuAgNCs and AuAgNCs-CD, respectively, and (c) and (d) are particle size distribution maps of AuAgNCs and AuAgNCs-CD, respectively.

[0049] Figure 3 The images show the fluorescence spectra and standard curves of AuAgNCs-CD at different RhB concentrations in this embodiment of the invention; where a is the fluorescence spectrum of AuAgNCs-CD at different RhB concentrations, and b is the standard curve of RhB.

[0050] Figure 4 The diagram shows the feasibility analysis verification results and the response of CBZ in the embodiments of the present invention; where a is the feasibility analysis verification results and b is the response of different nanoclusters as probes to CBZ.

[0051] Figure 5This is a diagram showing the selective experimental results of potential interfering substances in an embodiment of the present invention.

[0052] Figure 6 The figures show the fluorescence spectra of the probe at different CBZ concentrations and the standard curve for CBZ detection in this embodiment of the invention; where a is the fluorescence spectrum of the AuAgNCs-CD-RhB probe at different CBZ concentrations, and b is the standard curve for CBZ detection.

[0053] Figure 7 This is a diagram showing the optimized conditions of the Anti-FRET-LFA test strip in an embodiment of the present invention; where a is the optimized result of the PROT polyelectrolyte concentration, and b is the optimized structure diagram of the AuAgNCs-CD concentration.

[0054] Figure 8 This is a schematic diagram of the RFLFS structure in an embodiment of the present invention.

[0055] Figure 9 The above is a graph showing the RFLFS detection results in an embodiment of the present invention; where a is the fluorescence spectrum of CBZ detected by RFLFS at different concentrations, and b is the standard curve of CBZ detection on RFLFS. Detailed Implementation

[0056] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0057] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0058] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0059] Example 1:

[0060] This embodiment provides a synthesis method based on dual gold nanoclusters, the main steps of which are as follows:

[0061] (1) Preparation of ATT-AuNCs nanoclusters:

[0062] Dissolve 6-aza-2-thiothymidine (ATT) (80 mmol / L) in 3 mL of NaOH solution (0.2 mol / L) and mix it with 3 mL of HAuCl4 solution (10 mg / mL);

[0063] The ATT-AuNCs were stirred continuously for 1 hour (25℃, 800 r / min) under light-protected conditions. The resulting ATT-AuNCs were then washed twice with isopropanol precipitation and dissolved in deionized water to form a 15 mg / mL stock solution. The solution was stored at 4℃ in the dark to obtain ATT-AuNCs nanoclusters.

[0064] (2) Preparation of Arg-AuNCs nanoclusters:

[0065] 1 mL of Arg solution (0.5 M) was added dropwise to 1 mL of AgNO3 solution (10 mmol / L);

[0066] After adjusting the pH of the solution to 10 with NaOH (1M), it was incubated at 37℃ for 6 hours.

[0067] Arg-AgNCs were extracted by ultrafiltration (Millipore, 50 kDa) and stored at 4°C in the dark.

[0068] (3) Preparation of AuAgNCs nanoclusters:

[0069] 6 mL of the prepared Arg-AgNCs (pH 10) was mixed into 18 mL of ATT-AuNCs and reacted at 37 °C for 24 h.

[0070] The synthesized AuAgNCs were precipitated with isopropanol, centrifuged at 10,000 rpm for 5 min, and then redispersed in 10 mL of deionized water.

[0071] (4) Preparation of AuAgNCs-CD nanoclusters:

[0072] Take 970 μL of AuAgNCs-CD nanoclusters, add 30 μL of CM-β-CD solution (10 mmol / L), and incubate at 50 °C for 3 h;

[0073] The solution was then filtered using an ultrafiltration tube (Millipore, 3kDa) to remove excess reactants, yielding an AuAgNCs-CD nanocluster solution, which was stored at 4°C in the dark.

[0074] TEM images of AuAgNCs and AuAgNCs-CD are shown below. Figure 2 (a) and 2(b), particle size distribution diagrams are shown below. Figure 2 (c) 2(d).

[0075] Example 2:

[0076] Method feasibility verification:

[0077] After introducing RhB into the system, the fluorescence emission intensity of CD-AuAgNCs under 410 nm excitation gradually decreased with increasing RhB concentration in the range of 10 nmol / L–90 nmol / L. This is because the increase in the RhB acceptor concentration induced energy transfer in the donor CD-AuAgNCs, and the linear relationship is F. 580 / F 530 =0.0279C + 0.044(R) 2 =0.9974)( Figure 3 b) This also verifies the feasibility of CD-AuAgNCs-RhB as an indicator to replace the analytical sensor.

[0078] Ultimately, 90 nmol / L RhB was chosen as the starting condition for subsequent testing of carbendazim.

[0079] The changes in the fluorescence spectra of CD-AuAgNCs before and after the addition of RhB were compared. It was found that... Figure 4 (a) shows two typical emission bands. The fluorescence emission peak of CD-AuAgNCs at 530 nm is quenched, while the characteristic peak of RhB appears at 580 nm, indicating that RhB enters the cavity and forms an inclusion complex with cyclodextrin, resulting in energy transfer from the donor (CD-AuAgNCs) to the acceptor (RhB).

[0080] The addition of carbendazim resulted in an Anti-FRET effect, causing the bimodal peaks to show opposite trends, thus restoring the fluorescence of the nanoclusters.

[0081] Using the synthesized ATT-AuNCs as a precursor, 1 mL of Arg solution (0.5 M) was added dropwise. After adjusting the pH of the solution to 10 with NaOH (1 M), the solution was incubated at 37 °C for 6 h and then filtered through an ultrafiltration tube (Millipore, 3 kDa) to synthesize Arg / ATT-AuNCs.

[0082] 80 μL of RhB was added to Arg / ATT-AuNCs, AuAgNCs, and CD-AuAgNCs, respectively.

[0083] A fluorescent probe was constructed using a (225 nmol / L) solution, and CBZ was added. Under excitation at 410 nm, the ratio of fluorescence emission intensity (Fi) at dual wavelengths of 530 nm and 580 nm was recorded. 530 / F 580 ), compare the response to CBZ.

[0084] Figure 4(b) The results show that the Arg / ATT-AuNCs and AuAgNCs complex without cyclodextrin are not sensitive to the target, which may be due to the excellent optical properties and special recognition effect of the self-assembled nanoclusters, which significantly enhances the FRET effect and improves the sensitivity.

[0085] Example 3:

[0086] Selectivity analysis:

[0087] 80 μL of Rhodamine B (225 nmol / L) solution and 20 μL of different pesticide interferons were added sequentially to 100 μL of prepared CD-AuAgNCs.

[0088] The concentrations of interfering substances were as follows: carbendazim final concentration was 0.6 μmol / L, Ca... 2+ Na + K + Al 3+ The final concentration of other interfering agents, such as diuron, flusulfanilamide, bensulfuron-methyl, carbofuran, dimethoate, fenpropathrin, atrazine, imidacloprid, chlorpyrifos, and chlorothalonil, was 6 μmol / L. The effects of different interfering agents on the selectivity of the system were recorded under optimal excitation conditions (410 nm).

[0089] The measurement results are shown below. Figure 5 ,from Figure 5 It can be seen that the nanosensor constructed in this invention has good anti-interference performance and good specific response to carbendazim.

[0090] Example 4:

[0091] Establishment of the standard analytical curve for carbendazim:

[0092] Dissolve carbendazim in methanol and dilute it with PB buffer (pH 7.0) to prepare a series of carbendazim solutions of different concentrations.

[0093] 80 μL of Rhodamine B (225 nmol / L) solution and 20 μL of carbendazim solutions of different concentrations (1.5-40 μmol / L) were added sequentially to 100 μL of prepared CD-AuAgNCs.

[0094] Under 410 nm excitation, the fluorescence emission spectra of the above solution at dual wavelengths of 530 nm and 580 nm were recorded.

[0095] Based on the ratio of fluorescence intensity at different wavelengths in the solution, respectively using F 580 / F 530 Regarding the concentration of Rhodamine B and F 530 / F 580 A standard curve was constructed for the concentration of carbendazim.

[0096] F 530 and F 580 These represent the highest fluorescence intensities at the characteristic emission peaks of CD-AuAgNCs and Rhodamine B, respectively.

[0097] like Figure 6 As shown, different concentrations of carbendazim exhibited effects on the ratio (F) of CD-AuAgNCs and RhB characteristic peaks in the sensing system. 530 / F 580 The two substances exhibit high sensitivity, showing a linear relationship in the range of 0.15 μmol / L–4 μmol / L, with a correlation index (R0). 2 The limit of detection (LOD) was 0.9974, and the limit of detection (LOD) was 18 nmol / L (3σ / s).

[0098] The green fluorescence of CD-AuAgNCs quenched by RhB gradually recovered, accompanied by a clear color change from orange to green under a 365nm UV lamp.

[0099] Example 5:

[0100] Construction and optimization of anti-FRET-LFA test strips:

[0101] Since CD-AuAgNCs-RhB cannot form a strong interaction with the nitrocellulose membrane and thus cannot be fixed onto it, it will migrate along with the solution during testing. Therefore, it is necessary to study the fixation conditions when CD-AuAgNCs-RhB is sprayed onto the nitrocellulose membrane.

[0102] PROT polyelectrolyte was mixed with CD-AuAgNCs-RhB to achieve its fixation on the T-line. For example... Figure 7 When the protamine concentration was 1.25 mg / mL, the detection sensitivity was highest and the detection effect was best when the volume ratio of PROT polyelectrolyte:RhB:CD-AuAgNCs was 100:99:1 (CD-AuAgNCs was diluted by 200). Based on this condition, LFA test strips were constructed.

[0103] The structure of Anti-FRET-LFA is shown in the diagram. Figure 8 As shown. PROT polyelectrolyte, RhB, and CD-AuAgNCs were mixed in a volume ratio of 100:99:1 and sprayed onto the T line. The concentration of protamine sulfate sprayed was 1.25 mg / mL, the dilution factor of CD-AuAgNCs was 200, and the concentration of rhodamine B was 90 nmol / L.

[0104] LFA test strips consist of four parts: a sample pad, a nitrocellulose membrane (NC membrane), an absorbent pad, and a PVC board.

[0105] The sample pad was soaked in 0.01 mol / L phosphate buffer (PBS, 0.01 mol / L Na2HPO4, 0.01 mol / L NaH2PO4) at pH 7.4 for 30 min and then dried at 37 °C for 8 h.

[0106] 30 μL of protamine sulfate was sprayed onto the NC membrane as a T-line using a three-dimensional spray nozzle and dried for 2 h. The distance between the T-line and the absorbent pad was 24 mm. Then, 30 μL of CD-AuAgNCs-RhB was sprayed onto the same position and fixed, and dried at 37 °C for 2 h.

[0107] Finally, all components were glued onto the PVC board, with the sample pad and absorbent pad placed on the NC film, overlapping it by 2mm.

[0108] Cut the assembled PVC board into strips 4mm long and 1mm wide, and store them in a dark, sealed container.

[0109] Example 6:

[0110] Establishment of the standard detection curve for Anti-FRET-LFA test strips:

[0111] The prepared Anti-FRET-LFA was used to detect different concentrations of carbendazim solutions, and the fluorescence value on the T line was measured using a fluorescence reader. Figure 9 a). The effect of carbendazim concentration on fluorescence change rate F 510 / F 610 The standard curve for carbendazim on paper was successfully constructed by plotting the graph.

[0112] like Figure 9 (b) The fluorescence enhancement efficiency was directly proportional to the concentration of carbendazim, with a linear range of 0.2 μmol / L–1.4 μmol / L (0.038 mg / kg–0.268 mg / kg, R0). 2 =0.997), and the detection limit was calculated to be 60 nmol / L (0.011 mg / kg) using the 3σ / s formula, which is consistent with the sensitivity of existing immunochromatographic assays and commercial carbendazim test strips from Shanghai Feice Biotechnology Co., Ltd.

[0113] Example 7:

[0114] Detection of carbendazim in actual samples:

[0115] Add the standard solution to the apple sample and let it stand overnight at room temperature.

[0116] After drying the apples, cut them into small pieces and crush them using a high-speed shearing machine. Take 5g of sample, add 10mL of dichloromethane, sonicate for 5min, and centrifuge at 8000r / min to remove insoluble precipitate. Incubate in a water bath at 50℃ for 10min, add 2mL of methanol, and dilute to 20mL with ultrapure water. Use the obtained sample solution for subsequent detection.

[0117] The homogeneous solution detection and the test strip detection of Example 6 were performed according to the steps in Example 4 (see schematic diagram). Figure 1 (A is a schematic diagram of the construction of the ratio fluorescence nanosensing method for carbendazim, and B is a schematic diagram of the construction of the ratio fluorescence side-flow chromatography test paper for carbendazim.) The detection results are shown in Table 1 and Table 2.

[0118] Table 1. Detection of carbendazim in actual samples using the homogeneous fluorescence method developed in this invention (n=3)

[0119]

[0120] Note: RSD = (SD / mean) × 100%.

[0121] Table 2 shows the detection of carbendazim in actual samples using the fluorescent strategy test strips developed in this invention (n=3).

[0122]

[0123]

[0124] Note: RSD = (SD / mean) × 100%.

[0125] It can be seen that the nanosensor constructed in this invention has good anti-interference performance and good specific response to carbendazim.

[0126] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for detecting carbendazim in food based on a bimetallic nanocluster self-assembled complex, characterized in that: include, Preparation of ATT-AuNCs nanocluster solution; Preparation of Arg-AuNCs nanocluster solution; Preparation of AuAgNCs nanoclusters: Arg-AgNCs nanocluster solution was mixed into ATT-AuNCs nanocluster solution and reacted at 30-37℃ for 12-24h. The synthesized AuAgNCs were precipitated with isopropanol, centrifuged, and the centrifuged product was redispersed in deionized water to obtain AuAgNCs nanocluster solution. Preparation of AuAgNCs-CD nanoclusters: Take AuAgNCs nanocluster solution, add CM-β-CD solution, incubate at 40-50℃ for 2-3 h, and then filter with an ultrafiltration tube to remove excess reactants to obtain AuAgNCs-CD nanocluster solution; Detection of carbendazim in food: After processing the food, carbendazim solution is prepared by dissolving it in methanol; Rhodamine B solution and carbendazim solution were added sequentially to AuAgNCs-CD nanocluster solution. Simultaneously, under 410 nm excitation, the fluorescence emission spectra of the solution at dual wavelengths of 530 nm and 580 nm were recorded. Based on the ratio of fluorescence intensity, a standard curve for carbendazim concentration was constructed to determine the carbendazim content in food.

2. The method as described in claim 1, characterized in that: The preparation of the ATT-AuNCs nanocluster solution includes, Dissolve 6-aza-2-thiothymine (ATT) in 0.2 mol / L NaOH solution to form an 80 mmol / L ATT solution, and mix it with a 10 mg / mL HAuCl4 solution. After continuous stirring for 1–2 hours under light-protected conditions, the obtained ATT-AuNCs were washed twice with isopropanol precipitation, dissolved in deionized water to form a stock solution, and stored at 4°C in the dark to obtain ATT-AuNCs nanoclusters; among which, The ratio of NaOH solution to HAuCl4 solution is 3 mL: 3 mL; The stirring speed was 800 r / min and the stirring temperature was 25℃. The concentration of the stock solution is 15 mg / mL.

3. The method as described in claim 1, characterized in that: The preparation of the Arg-AuNCs nanocluster solution includes, Add 0.5M Arg solution dropwise to AgNO3 solution, adjust the pH of the solution to 10 with 1M NaOH, and incubate at 37℃ for 6 hours. Arg-AgNCs were extracted by ultrafiltration and stored at 4°C in the dark to obtain an Arg-AuNCs nanocluster solution; wherein, The ratio of Arg solution to AgNO3 solution is 1 mL: 1 mL, and the concentration of AgNO3 solution is 10 mmol / L.

4. The method according to any one of claims 1 to 3, characterized in that: The preparation of AuAgNCs nanoclusters involves a ratio of 6 mL to 18 mL of Arg-AgNCs nanocluster solution to ATT-AuNCs nanocluster solution, a pH of 10 for the Arg-AgNCs nanocluster solution, a centrifugation speed of 10000 rpm, and a centrifugation time of 5 min.

5. The method as described in claim 4, characterized in that: The food has been processed, including, After drying the food, cut it into pieces and then crush it using a high-speed shearing machine. Take 5g of sample, add 10mL of dichloromethane, sonicate for 5min, and centrifuge at 8000r / min to remove insoluble precipitate; The sample solution was prepared by water bath at 50℃ for 10 min, 2 mL of methanol was added and diluted with ultrapure water to 20 mL. The food products mentioned include apples.

6. The method as described in claim 1, characterized in that: The construction of a standard curve for carbendazim concentration based on the ratio of fluorescence intensities includes, According to the ratio of fluorescence intensity at different wavelengths in the solution, F 580 / F 530 To the concentration of rhodamine B, F 530 / F 580 To the concentration of carbendazim, a standard curve is constructed to determine the content of carbendazim in food; wherein, F 530 and F 580 These represent the highest fluorescence intensities at the characteristic emission peaks of CD-AuAgNCs and Rhodamine B, respectively.

7. The method according to any one of claims 1 to 3, 5 or 6, characterized in that: It also includes, A fluorescent lateral flow chromatography test strip (RFLFS) for carbendazim determination is prepared, wherein the fluorescent lateral flow chromatography test strip (RFLFS) consists of a sample pad, a nitrocellulose membrane (NC membrane), an absorbent pad, a PVC board, and a T-line; The fluorescence value on the T line was measured using a fluorescence reader, and the fluorescence change rate F was compared with the concentration of carbendazim. 510 / F 610 A standard curve for carbendazim on a paper substrate was constructed using graphing, enabling the detection of carbendazim.

8. The method as described in claim 7, characterized in that: The linear range for carbendazim detection is 0.2 μmol / L to 1.4 μmol / L, and the detection limit is calculated to be 60 nmol / L using the 3σ / s formula.

9. The method as described in claim 8, characterized in that: The preparation method of the fluorescence side-flow chromatography test strip RFLFS includes, The sample pad was soaked in 0.01 mol / L phosphate buffer at pH 7.4 for 30 min and dried at 37 °C for 8 h. The phosphate buffer contained 0.01 mol / L Na2HPO4 and 0.01 mol / L NaH2PO4. 30 μL of protamine sulfate was sprayed onto the NC membrane as a T-line using a three-dimensional spray nozzle and dried for 2 h. The distance between the T-line and the absorbent pad was 24 mm. Then, 30 μL of CD-AuAgNCs-RhB was sprayed onto the same position and fixed, and dried at 37 °C for 2 h. Attach all components to the PVC board, and place the sample pad and absorbent pad on the NC film, overlapping it by 2mm respectively. Cut the assembled PVC board into strips 4mm long and 1mm wide, and store them in a dark, sealed container.

10. The method as described in claim 9, characterized in that: The CD-AuAgNCs were diluted 200 times, and the protamine concentration was 1.25 mg / mL.

Images