A detection system and method for NbY24 coupled with carbon quantum dots.

Abstract

This invention discloses a detection system and method for NbY24 conjugated with carbon quantum dots, belonging to the field of immunoassay technology. The detection system for NbY24 conjugated with carbon quantum dots provided by this invention includes the following components: a CQDs-NbY24 solution prepared by bioconjugating NbY24 with carbon quantum dots; and a standard curve established with the fluorescence quenching efficiency calculated from the fluorescence intensity of the CQDs-NbY24 solution and the fluorescence intensity of the CQDs-NbY24 solution mixed with a gradient concentration of AFM1 solution as the ordinate and the AFM1 solution concentration as the abscissa. This invention employs a fluorescence signal amplification strategy, conjugating NbY24 with CQDs, to establish a highly sensitive, selective, simple, and rapid immunoassay method based on FIA for the detection of AFM1 in dairy products, providing a reference for future monitoring of AFM1 contaminants.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, and particularly relates to a detection system and method for NbY24 coupled with carbon quantum dots. Background Technology

[0002] Aflatoxin M1 (AFM1) is a fungal toxin found in dairy products. It is carcinogenic, hepatotoxic, and immunosuppressive. However, AFM1 is highly heat-resistant and stable, and its presence cannot be eliminated even after high-temperature processing such as sterilization of dairy products, thus severely impacting the quality of the finished product. Dairy products, as nutrient-rich foods, play a vital role in the diets of infants and the elderly. They not only provide essential nutrients such as protein and calcium but also have a profound impact on the health and growth of these vulnerable groups. Therefore, monitoring aflatoxin M1 (AFM1) in dairy products is crucial. Establishing rapid and low-cost detection methods with high sensitivity and specificity for AFM1 detection can not only ensure the quality and safety of dairy products but also effectively prevent potential threats to consumer health posed by harmful substances.

[0003] Currently, AFM1 detection technologies mainly include instrumental analytical methods based on chromatography and immunoassay methods based on the specific binding of antigens and antibodies. Rapid detection methods can be used to determine the presence of the toxin in a sample, while quantitative methods are more suitable for accurately detecting the content of analytes. Each method has its own advantages and limitations, and each method has its own characteristics. For example, immunoassays utilize the principle of antigen-antibody interactions to detect and separate specific antigens or antibodies in biological samples. They have advantages such as simple operation, rapid results, and no need for complex instruments or special culture conditions, and are therefore widely used in clinical diagnostics, biological research, and biopharmaceuticals. Currently, enzyme-linked immunosorbent assay (ELISA) is used for the detection of AFM1.

[0004] Carbon quantum dots (CQDs) are an emerging nanomaterial composed of carbon atoms. They possess extremely small size and unique photoelectric properties, exhibiting a quasi-spherical structure. Compared to traditional quantum dots and molecular dyes, they have attracted significant attention due to their superior photoluminescence properties, high water solubility, high biocompatibility, ease of chemical synthesis and modification, environmental friendliness, abundant raw materials, and low cost. In 2004, these highly fluorescent nanoparticles with multicolor emission were accidentally discovered. Using graphite powder as a precursor, multicolor fluorescent carbon nanoparticles were obtained and named "carbon dots." Carbon quantum dots have developed rapidly and have found wide applications in biomedical imaging, optoelectronic devices, catalysts, and sensors, providing new solutions for problems in medical diagnosis, renewable energy, and environmental monitoring, demonstrating broad application prospects.

[0005] Previous studies have developed a novel fluorescent immunoassay probe by combining boron, nitrogen, and sulfur-doped carbon quantum dots (CQDs) with dopamine. The addition of fluoride induces electron transfer between CQDs and dopamine, leading to fluorescence quenching and achieving highly efficient sensing of fluoride. This sensor has been successfully applied to detect fluoride in human serum samples, exhibiting an extremely low detection limit, and has been effectively used to monitor fluoride contamination in living cells. The excellent fluorescence properties of CQDs make them ideal choices for fluorescent labeling; upon binding with biomolecules, carbon quantum dots can generate bright and stable fluorescent signals for the detection and quantification of biomolecule concentrations. However, a method for detecting aflatoxin M1 using carbon quantum dots has not yet been developed. Summary of the Invention

[0006] To address the aforementioned technical issues, this invention employs a fluorescence signal amplification strategy, conjugating Nb-Y24 prepared in the laboratory with CQDs to establish a high-sensitivity, highly selective, simple, and rapid immunoassay based on FIA for the detection of AFM1 in dairy products, providing a reference for future monitoring of AFM1 contaminants.

[0007] To achieve the above objectives, this invention provides a detection system for NbY24 coupled with carbon quantum dots, comprising the following components: a CQDs-NbY24 solution prepared by bio-coupling NbY24 with carbon quantum dots; and a standard curve established with the fluorescence quenching efficiency calculated from the fluorescence intensity of the CQDs-NbY24 solution and the fluorescence intensity of the CQDs-NbY24 solution mixed with a gradient concentration of AFM1 solution as the ordinate and the concentration of AFM1 solution as the abscissa.

[0008] Preferably, the preparation method of the CQDs-NbY24 solution includes the following steps:

[0009] (1) Citric acid and glycine were mixed with deionized water and reacted at 220℃ for 14h to obtain a reaction solution. The supernatant was collected by centrifugation and the centrifugation was repeated 3 times. The solution was filtered, dialyzed once for 24h, and then freeze-dried to obtain carbon quantum dot powder.

[0010] (2) The carbon quantum dot powder described in step (1) is mixed with PBST buffer to obtain CQDs solution. The CQDs solution is then mixed with EDC-NHS solution and NbY24 solution, and incubated with shaking in the dark for 1 hour. After a second dialysis for 12-16 hours, CQDs-NbY24 solution is obtained.

[0011] Preferably, the mixing ratio of citric acid, glycine and deionized water in step (1) is 1g:1.2g:10mL.

[0012] Preferably, the centrifugation speed in step (1) is 6000 rpm, and the centrifugation time for each step is 10 min; the filtration in step (1) uses a 0.22 μm filter membrane, and the dialysis in step (1) uses a dialysis bag with a capacity of 30-40 kDa; the freeze-drying temperature in step (1) is -80℃, and the freeze-drying time is 48 h.

[0013] Preferably, in step (2), the mixing ratio of carbon quantum dot powder to PBST buffer is 1 mg: 1 mL; in step (2), the EDC concentration in the EDC-NHS solution is 0.08 mg / mL, and the NHS concentration in the EDC-NHS solution is 0.22 mg / mL; in step (2), the NbY24 concentration in the NbY24 solution is 1 mg / mL; in step (2), the mixing ratio of CQDs solution, EDC-NHS solution and NbY24 solution is 1 mL: 100 μL: 100 μL; in step (2), the temperature for light-protected shaking incubation is 37°C; in step (2), the temperature for secondary dialysis is 4°C, and the secondary dialysis uses a dialysis bag with a capacity of 30–40 kDa.

[0014] Preferably, the formula for calculating the fluorescence quenching efficiency based on the fluorescence intensity of the CQDs-NbY24 solution and the fluorescence intensity of the CQDs-NbY24 solution mixed with a gradient concentration of AFM1 solution is as follows:

[0015] E = (FI0 - FI) / FI0, where E is the fluorescence quenching efficiency, FI0 is the fluorescence intensity of the CQDs-NbY24 solution, and FI is the fluorescence intensity of the CQDs-NbY24 solution mixed with gradient concentration AFM1 solution.

[0016] The present invention also provides a method for detecting aflatoxin M1 in dairy products using the aforementioned detection system, comprising the following steps:

[0017] A. Sample pretreatment: Mix 4g of the emulsion sample to be tested with 10mL of methanol, centrifuge, take the supernatant and mix it with 40mL of PBS buffer to obtain the sample matrix extract;

[0018] B. Establishing a standard curve: CQDs-NbY24 solution was mixed with AFM1 solution of gradient concentration to obtain a mixed solution; the mixture was shaken at 37℃ in the dark for 1 h and incubated for 30 min. The fluorescence intensity of the CQDs-NbY24 solution was recorded as FI0, and the fluorescence intensity of the CQDs-NbY24 solution mixed with AFM1 solution of gradient concentration was recorded as FI. The fluorescence quenching efficiency E=(FI0-FI) / FI0 was calculated. A standard curve was established with the concentration of AFM1 solution as the abscissa and the fluorescence quenching efficiency as the ordinate.

[0019] C. Sample detection: The CQDs-NbY24 solution was mixed with the sample matrix extract to obtain the test mixture. The mixture was shaken at 37°C in the dark for 1 hour and incubated for 30 minutes. The fluorescence intensity was detected and the result was substituted into the standard curve to calculate the concentration of AFM1 in the sample matrix extract.

[0020] Preferably, the centrifugation temperature in step A is 4°C, the centrifugation speed is 12000 r / min, and the centrifugation time is 10 min.

[0021] Preferably, the mixing volume ratio of the CQDs-NbY24 solution to the gradient concentration AFM1 solution in step B is 9:1, and the total volume of the mixture in step B is 1 mL.

[0022] Preferably, the volume ratio of the CQDs-NbY24 solution to the sample matrix extract in step C is 9:1, and the total volume of the mixture to be tested in step C is 1 mL.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] This invention synthesizes water-soluble CQDs using a hydrothermal method and characterizes them. Subsequently, Nb-Y24 is coupled to the surface of the CQDs using EDC-NHS, establishing a CQDs-NbY24-FIA analytical method. The sensitivity of the CQDs-NbY24 FIA analytical method is evaluated by testing AFM1 solutions of different concentrations, and it is used to detect AFM1 residues in dairy products.

[0025] A fluorescence immunoassay (FIA) for the detection of AFM1 in dairy products was established based on CQDs-NbY24. The CQDs synthesized via a one-step hydrothermal method were characterized by high-resolution transmission electron microscopy (HRTEM) and energy-dispersive X-ray photoelectron spectroscopy (EDX). Previously prepared AFM1-resistant NbY24 was covalently linked to the CQDs via EDC / NHS, and parameters such as reaction time, pH, methanol concentration, ionic strength, and Tween 20 concentration were optimized. Under optimal conditions, the IC50 of CQDs-NbY24-FIA was [value missing]. 50 The concentration was 0.365 ng / mL, the LOD was 0.010 ng / mL, and the linear range was 0.039–3.439 ng / mL, meeting the Chinese standard for AFM1 content in dairy products of less than 0.5 ng / mL. It showed no fluorescent response to other mycotoxins, and the recoveries ranged from 96.72% to 103.64%, with coefficients of variation ranging from 1.969% to 8.191%. Furthermore, it showed high consistency with HPLC, with R... 2=0.999. Furthermore, it retains strong fluorescence characteristics even after storage at -80°C for 180 days. This indicates that the detection system described in this invention possesses good accuracy, stability, and reliability. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in 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.

[0027] Figure 1 The images show the TEM observations, EDX spectra, and particle size distributions of CQDs. In the images, A is the TEM observation with a scale bar of 100 nm, B is the EDX spectrum, and C is the particle size distribution.

[0028] Figure 2 The figure shows the fluorescence spectrum of CQDs, with the inset showing a photograph of CQDs under ultraviolet light irradiation.

[0029] Figure 3 The FTIR spectra of CQDs;

[0030] Figure 4 The images show the FTIR and fluorescence spectra of CQDs and CQDs-NbY24, the CD spectra of different NbY24 concentrations, and the calibration curves. In the images, A represents the FTIR spectra of CQDs and CQDs-NbY24, B represents the fluorescence spectra of CQDs and CQDs-NbY24, C represents the CD spectra of different NbY24 concentrations, and D represents the CD spectrum calibration curves of NbY24.

[0031] Figure 5 The effect of reaction time on the fluorescence intensity of CQDs-NbY24-FIA;

[0032] Figure 6 The effect of pH on the fluorescence intensity of CQDs-NbY24-FIA solution;

[0033] Figure 7 The effect of methanol concentration on the fluorescence intensity of CQDs-NbY24-FIA;

[0034] Figure 8 The effect of ionic strength on the fluorescence intensity of CQDs-NbY24-FIA;

[0035] Figure 9 The effect of Tween-20 concentration on the fluorescence intensity of CQDs-NbY24-FIA;

[0036] Figure 10The fluorescence response of CQDs-NbY24-FIA to AFM1, AFM2, AFB1, AFB2, AFG1, AFG2, DON and ZEN;

[0037] Figure 11 The fluorescence response of CQDs-NbY24-FIA to different concentrations of AFM1;

[0038] Figure 12 The figure shows the fluorescence quenching curves of CQDs-NbY24-FIA with different concentrations of AFM1 added. The inset is the linear calibration curve.

[0039] Figure 13 Fluorescence changes of CQDs-NbY24-FIA stored at different temperatures for 120 days;

[0040] Figure 14 The matrix effect of CQDs-NbY24-FIA on different samples;

[0041] Figure 15 The matrix effect of CQDs-NbY24-FIA on diluted yogurt;

[0042] Figure 16 Correlation analysis of CQDs-NbY24-FIA and HPLC methods. Detailed Implementation

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0046] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0047] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0048] NbY24 is derived from Liu Haiyuan. Construction of icELISA method for AFM1 detection based on nanobody and research on antibody site-directed mutagenesis [D]. Inner Mongolia Agricultural University, 2023. DOI:10.27229 / d.cnki.gnmnu.2023.001452.

[0049] Example 1

[0050] AFM1 standard solution: 50 μg of AFM1 standard was fully dissolved in 5 mL of methanol (chromatographic grade), and the concentration of the stock solution was 10 μg / mL.

[0051] 0.01M PBS buffer (pH 7.4): Weigh 8g NaCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O and 0.2g KCl, dissolve in 1L deionized water, adjust pH to 7.4, and store at room temperature.

[0052] PBST: Measure 1000 mL of 0.01 M PBS buffer, add 0.5 mL of L-20, and store at room temperature.

[0053] 1% D-gluconic acid solution: Take 1 mL of D-gluconic acid (Macklin Reagent Company), add deionized water to make up to 100 mL, and store at room temperature.

[0054] CQDs-NbY24 solution: (1) Mix 1g citric acid and 1.2g glycine with 10mL deionized water, react at 220℃ for 14h to obtain the reaction solution, centrifuge at 6000rpm for 10min, take the supernatant, repeat centrifugation 3 times, filter with a 0.22μm filter membrane, dialyze once with a 30~40kDa dialysis bag for 24h, freeze dry at -80℃ for 48h to obtain carbon quantum dot powder;

[0055] (2) Mix 1 mg of carbon quantum dot powder with 1 mL of PBS buffer to obtain CQDs solution. Mix the CQDs solution with 100 μL of LEDC-NHS solution (EDC concentration is 0.08 mg / mL, NHS concentration is 0.22 mg / mL, PBS buffer is used as solvent) and 100 μL of 1 mg / mL NbY24 solution (PBS buffer is used as solvent). Incubate at 37°C in the dark with shaking for 1 h. Use a 30-40 kDa dialysis bag and dialyze twice at 4°C for 14 h to obtain CQDs-NbY24 solution.

[0056] CQDs-NbY24 solution was mixed with AFM1 solutions of gradient concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 20 ng / mL to obtain a mixed solution. The mixture was then incubated at 37°C in the dark for 1 hour with shaking, followed by 30 minutes of incubation. The fluorescence intensity of the CQDs-NbY24 solution was recorded as FI0, and the fluorescence intensity of the CQDs-NbY24 solution mixed with the gradient concentrations of AFM1 solutions was recorded as FI. The fluorescence quenching efficiency E = (FI0 - FI) / FI0 was calculated. A standard curve was established with the AFM1 solution concentration as the x-axis and the fluorescence quenching efficiency as the y-axis.

[0057] In the formula, E is the fluorescence quenching efficiency, FI0 is the fluorescence intensity of the CQDs-NbY24 solution, and FI is the fluorescence intensity of the CQDs-NbY24 solution mixed with gradient concentration AFM1 solutions.

[0058] Experimental Example 1

[0059] 1. Materials and Methods

[0060] 1.1 Experimental Materials and Equipment

[0061] 1.1.1 Test Instruments and Equipment

[0062] The main instruments and equipment used in the experiment are shown in Table 1.

[0063] Table 1. List of Main Instruments and Equipment

[0064] Instruments and equipment Manufacturer SpectraMax M2 fluorescent microplate reader ThermoFisherScientific, USA High-speed refrigerated centrifuge HC-3018R Eppendorf, Germany ZWYR-200D Constant Temperature Shaking Incubator American Precision Company SYESTEMS-20A High Performance Liquid Chromatograph Shimadzu Corporation of Japan Nitrogen purging device ND100-2 Hangzhou Ruicheng Instrument Co., Ltd. Muffle furnace BSX2-2.5-12P Tianjin Tester Instruments Co., Ltd. KQ2200E Ultrasonic Cleaner Kunshan Ultrasonic Instruments Co., Ltd. Fourier transform infrared spectrometer IRAffinty-1 Shimadzu Corporation of Japan Rotary Evaporator RV10 German company ECAR

[0065] 1.1.2 Experimental Materials and Reagents

[0066] The main materials and reagents are shown in Table 2.

[0067] Table 2 Main Materials and Reagents List

[0068]

[0069]

[0070] 1.1.3 Preparation of test solutions

[0071] (1) AFM1 standard solution: 50 μg of AFM1 standard was fully dissolved in 5 mL of methanol (chromatographic grade), and the concentration of the stock solution was 10 μg / mL.

[0072] (2) 0.01M phosphate buffer (PBS, pH 7.4): Weigh 8g NaCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O and 0.2g KCl, dissolve in 1L deionized water, adjust pH to 7.4, and store at room temperature.

[0073] (3) 0.01% / 0.05% / 0.1% / 0.3% / 0.5% PBST buffer: Measure 1000 mL of 0.01 M PBS buffer, add 0.1 / 0.5 / 1 / 3 / 5 mL of PBST buffer, and store at room temperature.

[0074] (4) 2.5 / 5 / 10 / 20 / 40% methanol concentration PBS buffer: Measure 12.5 / 25 / 50 / 100 / 200 mL of methanol (chromatographic grade), add 0.01 M PBS solution to make up to 500 mL, and store at room temperature.

[0075] (5) 10 / 20 / 50 / 100 / 200mM ionic strength PBS buffer: Measure 500mL of 0.01M PBS solution, add 0.58 / 1.16 / 2.90 / 5.8 / 11.6g NaCl, and store at room temperature.

[0076] (6) 1% D-gluconic acid solution: Take 1 mL of D-gluconic acid, add deionized water to make up to 100 mL, and store at room temperature.

[0077] 1.2 Detection of AFM1 in dairy products using a CQDs-NbY24-based fluorescence immunoassay

[0078] 1.2.1 Synthesis of CQDs

[0079] 1 g citric acid (CA) and 1.2 g glycine (Gly) were mixed with 10 mL deionized water and reacted at 220 °C for 14 h to obtain a reaction solution. The solution was centrifuged at 6000 rpm for 10 min, and the supernatant was collected. The centrifugation was repeated 3 times. The solution was filtered through a 0.22 μm filter membrane and dialyzed once for 24 h using a 30–40 kDa dialysis bag. The solution was then freeze-dried at -80 °C for 48 h to obtain carbon quantum dot (CQD) powder.

[0080] 1.2.2 Characterization of CQDs

[0081] (1) The general structure and surface morphology of CQDs were characterized using HRTEM equipment, and the structure of CQDs was observed.

[0082] (2) The elemental composition of CQDs was characterized using an EDX device;

[0083] (3) The particle size distribution of CQDs was analyzed using a nanoparticle size and zeta potential analyzer (DLS);

[0084] (4) Its fluorescence properties were characterized using a fluorescence microplate reader (FEIA);

[0085] (5) The structure and surface functional groups of CQD were analyzed using Fourier transform infrared spectroscopy (FTIR).

[0086] 1.2.3 Bioconjugation between NbY24 and CQDs

[0087] 1 mg of CQDs powder was mixed with 1 mL of PBS buffer to obtain a CQDs solution. The CQDs solution was then mixed with 100 μL of EDC-NHS solution (EDC concentration of 0.08 mg / mL, NHS concentration of 0.22 mg / mL, PBS buffer as solvent) and 100 μL of 1 mg / mL NbY24 solution (PBS buffer as solvent). The mixture was incubated at 37°C in the dark with shaking for 1 h. The mixture was then dialyzed twice at 4°C for 14 h using a 30–40 kDa dialysis bag to obtain a CQDs-NbY24 solution.

[0088] 1.2.4 Characterization of CQDs-NbY24 solution

[0089] (1) Different concentrations of NbY24 solution and purified CQDs-NbY24 solution were detected using a dichroic spectrometer (CD).

[0090] (2) CQD and CQD-Y24 were characterized using an FTIR device.

[0091] (3) Use the FEIA device to characterize CQD and CQD-Y24 respectively, observe the differences between the two, and determine whether the coupling was successful.

[0092] 1.2.5 Conditional Optimization of CQDs-NbY24-FIA

[0093] 1.2.5.1 Effect of different reaction times on the fluorescence intensity of CQDs-NbY24-FIA

[0094] Under light-protected conditions, 900 μL of CQDs-NbY24 solution was mixed with 100 μL of 0.1 mg / mL AFM1 and shaken for 1 h to achieve a final volume of 1 mL. The mixture was incubated for 5, 10, 20, 30, 60, 90, and 120 min, and the fluorescence intensity after quenching was measured. The fluorescence intensity of the CQDs-NbY24 solution was denoted as FI0, and the fluorescence intensity of the CQDs-NbY24 solution mixed with AFM1 was denoted as FI. The fluorescence quenching efficiency E was calculated as E = (FI0 - FI) / FI0.

[0095] 1.2.5.2 Effect of different pH values ​​on the fluorescence intensity of CQDs-NbY24-FIA

[0096] Based on the optimized conditions in 1.2.5.1, CQDs-NbY24 solutions dispersed in double-distilled water were prepared to pH values ​​of 4.4, 5.4, 6.4, 7.4, 8.4, 9.4, and 10.4, respectively, using 0.1M HCl and 0.1M NaOH. The effect of different pH values ​​on the fluorescence intensity of CQDs-NbY24-FIA was then detected.

[0097] 1.2.5.3 Effect of different methanol concentrations on the fluorescence intensity of CQDs-NbY24-FIA

[0098] Based on the optimized conditions in 1.2.5.2, CQDs-NbY24 solutions were prepared using PBS with different methanol concentrations (0, 2.5, 5, 10, 20, 30, and 50%), and the effect of different methanol concentrations on the fluorescence intensity of CQDs-NbY24-FIA was detected.

[0099] 1.2.5.4 Effect of different ionic strengths on the fluorescence intensity of CQDs-NbY24-FIA

[0100] Based on the optimized conditions in 1.2.5.3, CQDs-NbY24 solutions were prepared using PBS solutions with different ionic strengths (NaCl concentrations: 0, 10, 20, 50, 100, 150, 200 mM) to detect the effect of different ionic strengths on the fluorescence intensity of CQDs-NbY24-FIA.

[0101] 1.2.5.5 Effect of different Tween-20 concentrations on the fluorescence intensity of CQDs-NbY24-FIA

[0102] Based on the optimized conditions in 1.2.5.4, CQDs-NbY24 solutions were prepared with different Tween-20 concentrations (0%, 0.01%, 0.05%, 0.1%, 0.3%, 0.5%, 1%) to detect the effect of different Tween-20 concentrations on the fluorescence intensity of CQDs-NbY24-FIA.

[0103] Stability of 1.2.5.6CQDs-NbY24-FIA

[0104] Based on the above optimal experimental conditions, CQDs-NbY24 solution was prepared, and the fluorescence intensity of CQDs-NbY24 solution was detected at different time periods from day 1 to day 120 at -80℃, -20℃, 4℃ and 25℃.

[0105] 1.2.6 Specificity of CQDs-NbY24-FIA

[0106] Under optimal experimental conditions, the fluorescence responses of CQDs-NbY24-FIA to AFM1, AFM2, AFB1, AFB2, AFG1, AFG2, DON, and ZEN were recorded to determine their specificity.

[0107] 1.2.7 Establishment of the CQDs-NbY24-FIA Standard Curve

[0108] CQDs-NbY24 solution was mixed with AFM1 solutions of gradient concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 20 ng / mL to obtain a mixed solution. The mixture was then incubated at 37°C in the dark for 1 hour with shaking, followed by 30 minutes of incubation. The fluorescence intensity of the CQDs-NbY24 solution was recorded as FI0, and the fluorescence intensity of the CQDs-NbY24 solution mixed with the gradient concentrations of AFM1 solutions was recorded as FI. The fluorescence quenching efficiency E = (FI0 - FI) / FI0 was calculated. A standard curve was established with the AFM1 solution concentration as the x-axis and the fluorescence quenching efficiency as the y-axis.

[0109] 1.2.8 Establishment of a homogeneous immunoassay method for CQDs-NbY24-FIA

[0110] 1.2.8.1 Sample Pretreatment

[0111] Three samples—milk, milk powder, and yogurt—that were free of AFM1 (as determined by HPLC) were selected for pretreatment. 4g of milk or yogurt was placed in a 50mL centrifuge tube, 10mL of methanol was added, and the tube was vortexed for 3 minutes. The tubes were then centrifuged at 12000 rpm for 10 minutes at 4℃. The supernatant was transferred to a beaker, diluted with 40mL of PBS buffer, and stored at 4℃ for later use.

[0112] 1.2.8.2 Matrix effect

[0113] Add AFM1 standard at concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 ng / mL to the pretreated milk, milk powder, and yogurt as described above, with three replicates for each concentration. Based on the optimized experimental conditions, mix 900 μL of CQDs-NbY24 solution with 100 μL of samples containing different concentrations of AFM1, and shake in the dark for 1 h to achieve a final volume of 1 mL. Incubate the mixture for 30 min and detect the fluorescence intensity after quenching. The fluorescence intensity of the CQDs-NbY24 solution is denoted as FI0, and the fluorescence intensity of CQDs-NbY24 mixed with AFM1 solution is denoted as FI. Calculate the fluorescence quenching efficiency E = (FI0 - FI) / FI0.

[0114] 1.2.8.3 Spiked Recycling

[0115] AFM1 standard was added to pretreated milk, milk powder, and yogurt to final concentrations of 0.05, 0.25, and 0.5 ng / mL, respectively. The AFM1 content was determined using the established CQDs-NbY24-FIA homogeneous quenching curve, and the spiked recovery rate and coefficient of variation were calculated. Three replicates were performed for each concentration.

[0116] 1.2.8.4 Actual Sample Testing

[0117] (1) The AFM1 of the sample obtained by pretreatment according to 1.2.8.1 was determined by HPLC according to GB 5009.24-2016;

[0118] (2) In the same method determined by matrix effect, the pretreated dairy samples were measured in CQDs-NbY24-FIA. AFM1 standard was added to four AFM1 negative pretreated samples at final concentrations of 0.25, 0.5, 0.75 and 1 ng / mL, respectively. Based on the established CQDs-NbY24-FIA quenching curve, the measured concentration and spiked recovery rate were calculated. The results were compared with HPLC and commercially available aflatoxin M1 enzyme-linked immunosorbent assay kit.

[0119] 1.2.9 Experimental Data Analysis

[0120] Experimental data are presented as mean ± standard deviation (mean ± SD). Each experiment was repeated three times. Statistical analysis was performed using SPSS 26.0 and Microsoft Office Excel 2020 software. Fluorescence spectra were plotted using OriginLab Corporation software, and standard curves were plotted using GraphPad Prism 8 software.

[0121] 2 Results and Analysis

[0122] 2.1 Establishment of CQDs-NbY24-FIA method for detecting AFM1 in dairy products based on immunological principles

[0123] 2.1.1 Synthesis and Characterization of CQDs

[0124] CQDs were prepared using CA and Gly as raw materials via a one-step hydrothermal method. Both CA and Gly consist of multiple functional groups to achieve covalent bonding between amino (Gly) and carboxyl (CA) groups. The overall structure and surface morphology of the synthesized CQDs were characterized using HRTEM. Figure 1 As shown in Figure A, the structure of the CQDs was observed. They exhibited a quasi-spherical structure with uniform distribution and a size of approximately 10 nm. EDX analysis of the elemental composition of the CQDs revealed that the synthesized CQDs were composed of C (38.75%), N (28.56%), and O (32.69%), with no other impurities (such as...). Figure 1 As shown in Figure B), the particle size distribution is uniform, with a size of approximately 10 nm (as shown in Figure B). Figure 1 (As shown in C).

[0125] CQDs were spectroscopically characterized using FEIA (e.g., Figure 2 As shown in the figure, the results show that CQDs exhibit fluorescence behavior related to the excitation wavelength. At the initial excitation wavelength of 400 nm, the emission peak intensity is low, gradually increasing with increasing wavelength until reaching 430 nm. Beyond 430 nm, the excitation fluorescence intensity decreases. Therefore, the optimal excitation wavelength for CQDs is 430 nm. According to the FTIR spectrum, CQDs exhibit fluorescence intensity at 3388 cm⁻¹. -1 and 1682cm -1 The presence of absorption bands at the point indicates the presence of surface hydroxyl and amine functional groups (NH2), which are associated with stretching and bending vibrations of OH and NH bonds. Figure 3 (As shown).

[0126] 2.1.2 Synthesis and Characterization of CQDs-NbY24

[0127] After NbY24 is attached to CQDs, at 3388cm -1 The wide absorption band at that point decreased to 3309 cm⁻¹ -1 With 2917cm -1 Narrow absorption bands at such locations (e.g.) Figure 4 (As shown in Figure A). This result indicates that an amide bond (-CO-NH-) is formed between the carboxyl group of NbY24 (activated by EDC-NHS coupling) and the amino group of CQDs, located at 1196 cm⁻¹. -1 The band at this point represents an amide bond, confirming the amidation reaction between CQDs and the AFM1 antibody. Furthermore, at 1789 cm⁻¹... -1 and 1052cm-1 The wavelength range at this point can be attributed to the stretching vibrations of C=O and C=N in the amide bond. Therefore, the FTIR results indicate that NbY24 is successfully coupled with CQDs. The fluorescence emission properties of CQDs and CQDs-Nb show (e.g.) Figure 4 As shown in Figure B), at an excitation wavelength of 430 nm, the fluorescence intensity of the CQDs-Nb solution is slightly lower than that of CQDs alone, and the emission peak exhibits a blue shift. This decrease in fluorescence intensity may be related to the absorption of some excitation energy by antibody molecules attached to the surface of CQDs. The CD spectra of different concentrations of NbY24 (0.01–0.2 μg / mL) and CQDs-NbY24 were measured (e.g., [Figure B is missing]). Figure 4 As shown in Figure C), the calibration curve of NbY24 was obtained (as shown in Figure C). Figure 4 As shown in Figure D, the amount of NbY24 bound to CQDs in CQDs-NbY24 was measured to be approximately 0.025 mg / mL.

[0128] 2.1.3 Conditional Optimization of CQDs-NbY24-FIA

[0129] 2.1.3.1 Effect of different reaction times on the fluorescence intensity of CQDs-NbY24-FIA

[0130] An immune response is a dynamic process of "binding-dissociation" in which antibodies recognize antigens. Sufficient reaction time is beneficial for the full binding of antigens and antibodies, and the reaction tends to stabilize after sufficient reaction. Therefore, this invention incubated 900 μL of CQDs-NbY24 solution with 100 μL of AFM1 at room temperature and measured the effect of different reaction times (5–120 min) on its fluorescence intensity. Figure 5 It can be seen that the fluorescence intensity reaches a certain level at approximately 30 minutes, after which it tends to stabilize without significant change. Therefore, the optimal response time of this sensor is 30 minutes.

[0131] 2.1.3.2 Effect of different pH values ​​on the fluorescence intensity of CQDs-NbY24-FIA

[0132] Under different pH conditions, the charge state of proteins changes, thereby affecting the charge complementarity between antigens and antibodies and reducing their binding ability. This invention explored the effect of different pH values ​​on fluorescence intensity at the optimal reaction time, using fluorescence quenching efficiency E = (FI0 - FI) / FI0 as the indicator. The results showed that CQDs-NbY24-FIA exhibited the best fluorescence performance at a neutral pH of 7.4, with a fluorescence quenching efficiency of 47.67% (e.g., FI0 - FI0) / FI0. Figure 6(As shown in the figure). This indicates that a neutral environment is beneficial for maintaining the fluorescence signal intensity and stability of CQDs-Nb-FIA. Therefore, pH 7.4 was chosen as the optimal pH value for CQDs-NbY24-FIA.

[0133] 2.1.3.3 Effect of different methanol concentrations on the fluorescence intensity of CQDs-NbY24-FIA

[0134] Methanol can alter the conformation and properties of proteins, thereby affecting the binding efficiency of antigens and antibodies. Therefore, this invention investigated the effect of different methanol concentrations on the fluorescence quenching efficiency of CQDs-NbY24-FIA under optimal reaction time and pH conditions. The results are as follows: Figure 7 As shown, the fluorescence quenching efficiency decreases with increasing methanol concentration. This may be because excessively high methanol concentrations disrupt the antigen-antibody reactivity, thus affecting the fluorescence quenching efficiency. Therefore, methanol is not suitable for the CQDs-NbY24-FIA method.

[0135] 2.1.3.4 Effect of different ionic strengths on the fluorescence intensity of CQDs-NbY24-FIA

[0136] Appropriate ionic strength can maintain the native conformation and stability of antibodies and promote antigen-antibody binding. However, excessively high or low ionic strength may affect this interaction, leading to a decrease in binding efficiency. Under the optimized conditions described above, the effect of ionic strength on the fluorescence quenching rate of CQDs-NbY24-FIA was further investigated, and the results are as follows: Figure 8 As shown, the fluorescence quenching efficiency decreases with increasing ionic strength. This may be because excessively high NaCl concentrations lead to protein aggregation and precipitation, affecting their solubility and binding properties, thus reducing the fluorescence quenching efficiency. Therefore, the established CQDs-NbY24-FIA method is not suitable for adding NaCl.

[0137] 2.1.3.5 Effect of different Tween-20 concentrations on the fluorescence intensity of CQDs-NbY24-FIA

[0138] Tween-20, a commonly used nonionic surfactant, promotes protein dissolution and dispersion, facilitates the uniform distribution of antigens and antibodies in solution, reduces adsorption on reagent tube walls, minimizes nonspecific adsorption, and enhances antigen-antibody binding, thereby increasing the specific binding signal. Under the optimized conditions described above, the effect of different Tween-20 concentrations on the binding of NbY24 to AFM1 was investigated. The results are as follows: Figure 9As shown, the fluorescence quenching efficiency initially increased and then decreased with increasing Tween-20 concentration. This may be because high concentrations of Tween-20 cause protein degradation, reducing binding efficiency and resulting in a weak fluorescence signal. Therefore, 0.05% was selected as the optimal addition amount of Tween-20.

[0139] 2.1.4 Specificity of CQDs-NbY24-FIA

[0140] To evaluate the specificity of the CQDs-NbY24-FIA detection system for other mycotoxins, common mycotoxins such as AFM1, AFM2, AFB1, AFB2, AFG1, AFG2, DON, and ZEN were introduced into the detection system, and the fluorescence quenching efficiency was measured. Figure 10 The results showed that CQDs-NbY24-FIA was specific to AFM1, and the cross-reactivity rates with other fungal toxins, from high to low, were AFM1 > AFG1 > AFB2 > ZEN > AFB1 > AFG2 > DON, indicating that the method had good specificity.

[0141] 2.1.5 Establishment of the CQDs-NbY24-FIA Standard Curve

[0142] Under the aforementioned optimal conditions, the effect of different concentrations of AFM1 (0–20 ng / mL) on the fluorescence quenching rate of CQDs-NbY24-FIA was investigated. Figure 11 It was observed that with increasing AFM1 concentration, the fluorescence intensity of CQDs-NbY24-FIA gradually decreased, while the quenching rate increased. This concentration-dependent fluorescence quenching may be due to the reduction in the effective excitation energy of CQDs-NbY24-FIA after AFM1 binds to the loaded CQDs-NbY24. Furthermore, a slight blue shift in the fluorescence emission peak was observed. Based on the fluorescence quenching, fluorescence quenching rate spectra and calibration curves (e.g., for analyte concentrations ranging from 0.01 to 20 ng / mL) of standards were obtained. Figure 12 As shown), IC 50 The effective concentration was 0.365 ng / mL, the LOD was 0.010 ng / mL, the LOQ was 0.035 ng / mL, and the linear range was 0.039–3.439 ng / mL, meeting the detection standards in GB5009.24-2016.

[0143] 2.1.6 Stability of CQDs-NbY24-FIA

[0144] To investigate the stability of CQDs-NbY24-FIA, this invention investigated the changes in fluorescence intensity of CDDs-NbY24 during 120 days of storage at different temperatures (-80℃, -20℃, 4℃ and room temperature). Figure 13(As shown in the figure). The results showed that the fluorescence intensity decreased slightly at room temperature, while the fluorescence intensity did not change significantly at low temperature. This result demonstrates the fluorescence stability of CQDs-NbY24 solution and the wide applicability of CQDs-NbY24-FIA.

[0145] 2.1.7 Matrix effect

[0146] Dairy products have a complex matrix, including fat, protein, and carbohydrates. Fat and protein can bind to or encapsulate AFM1, making some AFM1 difficult to detect and leading to inaccurate results. Appropriate sample dilution can effectively reduce matrix effects and lower the concentration of interfering substances in the sample; however, excessive dilution may reduce AFM1 levels to undetectable levels. Therefore, choosing an appropriate dilution ratio can strike a balance between reducing matrix effects and maintaining sufficient detection sensitivity. After pretreatment of milk, milk powder, and yogurt samples, they were diluted 10-fold and different concentrations of AFM1 standards (0–10 ng / mL) were added before detection in the CQDs-NbY24-FIA system. The results showed (e.g.) Figure 14 As shown in the figure, when milk and milk powder were diluted 10 times, the detection curves showed a consistent trend with the standard curve unaffected by the matrix. However, under the same dilution conditions, the detection rate of yogurt was lower, which is related to the increased viscosity of milk after fermentation. After re-diluting yogurt samples to 15, 20, and 30 times, the results showed that when the yogurt samples were diluted 20 times, the matrix effect was minimized, and its curve was close to the standard curve unaffected by the matrix (as shown in the figure). Figure 15 (As shown).

[0147] 2.1.8 Spiked Recycling

[0148] AFM1 standard was added to milk, yogurt, and milk powder samples to final concentrations of 0.05, 0.25, and 0.5 ng / mL, respectively. The samples were then analyzed using CQDs-NbY24-FIA and HPLC methods. Intergroup comparisons were performed to evaluate the accuracy and reliability of CQDs-NbY24-FIA. The results are shown in Table 3. The average recovery rate of CQDs-NbY24-FIA was 96.72%–103.64%, with a coefficient of variation (CV) of 1.969%–8.191%; the average recovery rate of HPLC was 94.55%–101.40%, with a CV of 2.111%–8.831%. The two methods showed good correlation, with a correlation coefficient R0. 2 =0.999 (e.g.) Figure 16 As shown in the figure, this indicates that the established CQDs-NbY24-FIA is suitable for actual sample analysis.

[0149] Table 3. CQDs-NbY24-FIA and HPLC Spike Recovery Analysis

[0150]

[0151] 2.1.9 Actual Sample Testing

[0152] Twenty samples of cow's milk, camel's milk, goat's milk, yogurt, and milk powder were purchased from local supermarkets in Hohhot, Inner Mongolia, and from herders in Alxa League. The samples were analyzed using three methods: CQDs-NbY24-FIA, HPLC, and ELISA kits. The results were compared and analyzed. Table 4 shows that only three of the 20 samples, raw milk collected from pastoral areas, contained trace amounts of AFM1. The AFM1 content detection results from CQDs-NbY24-FIA and HPLC were consistent, and the method showed better sensitivity compared to the ELISA kit. This is attributed to the superior sensitivity of Nb compared to mAb and the superiority of fluorescence detection over color reactions, indicating that the established method can be used for rapid screening of AFM1 in real samples.

[0153] Table 4. Detection results of AFM1 content in actual samples

[0154]

[0155]

[0156] Compared with conventional antibodies, nanobodies are small in size, highly stable, highly soluble, and have high affinity. They are also readily produced and easily modified, making them promising antibody elements for immunoassays. Carbon quantum dots (CQDs) offer significant advantages in the quantitative / qualitative analysis of small molecules due to their broad-wavelength excitation and narrow-wavelength emission, high brightness and photostability, tunable size, and ease of modification. Fluorescence immunoassay (FIA), a promising and reliable method, has attracted considerable attention due to its rapid and efficient analysis. Therefore, this invention employs a fluorescence signal amplification strategy, conjugating previously prepared Nb-Y24 with CQDs to establish a high-sensitivity, highly selective, simple, and rapid FIA-based immunoassay for the detection of AFM1 in dairy products, providing a reference for future monitoring of AFM1 contaminants.

[0157] The FIA ​​method is an immunoassay method established based on the fluorescence properties of fluorescent substances. Compared with the color reaction of the ELISA method, it is more sensitive, has a lower detection limit, and a wider detection range. Therefore, this invention utilizes the prepared CQDs coupled with NbY24 to establish a CQDs-NbY24-based FIA method for the detection of AFM1 in dairy products, and optimizes the key parameters. Under optimal conditions, the LOD of CQDs-NbY24-FIA is 0.010 ng / mL, and the IC50 is [missing value]. 50The effective concentration was 0.365 ng / mL, with a linear range of 0.039–3.439 ng / mL. The elimination of coating and blocking reduced detection time by more than 40 times and simplified operation. It also improved the problem of potentially large errors caused by weak color reaction signals. Compared with similar studies using mAbs, the LOD was improved by approximately 7 times, and the linear range was expanded by more than 5 times. However, the non-directional coupling method used in this invention, such as EDC, has a certain degree of unpredictability. This invention maximizes the coupling rate by using an excess of Nb. Further targeted coupling methods mediated by glutaric acid succinamide-PEG-maleimide (SMPEG) can be used to further improve antibody coupling efficiency, thereby increasing detection sensitivity.

[0158] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for detecting aflatoxin M1 in dairy products using carbon quantum dot-coupled NbY24, characterized in that, Includes the following steps: A. Sample pretreatment: Mix 4g of the emulsion sample to be tested with 10mL of methanol, centrifuge, take the supernatant and mix it with 40mL of PBS buffer to obtain the sample matrix extract; B. Establishing a standard curve: CQDs-NbY24 solution was mixed with AFM1 solution of gradient concentration to obtain a mixed solution; the mixture was shaken at 37℃ in the dark for 1 h and incubated for 30 min. The fluorescence intensity of the CQDs-NbY24 solution was recorded as FI0, and the fluorescence intensity of the CQDs-NbY24 solution mixed with AFM1 solution of gradient concentration was recorded as FI. The fluorescence quenching efficiency was calculated as E = (FI0 - FI) / FI0. A standard curve was established with the concentration of AFM1 solution as the x-axis and the fluorescence quenching efficiency as the y-axis. C. Sample detection: The CQDs-NbY24 solution was mixed with the sample matrix extract to obtain the test mixture. The mixture was shaken at 37°C in the dark for 1 hour and incubated for 30 minutes. The fluorescence intensity was detected and substituted into the standard curve to calculate the concentration of AFM1 in the sample matrix extract. The preparation method of the CQDs-NbY24 solution includes the following steps: (1) Citric acid and glycine were mixed with deionized water and reacted at 220℃ for 14h to obtain a reaction solution. The supernatant was collected by centrifugation and the centrifugation was repeated 3 times. The solution was filtered, dialyzed once for 24h, and then freeze-dried to obtain carbon quantum dot powder. (2) The carbon quantum dot powder described in step (1) is mixed with PBS buffer to obtain CQDs solution. The CQDs solution is then mixed with EDC-NHS solution and NbY24 solution, and incubated with shaking in the dark for 1 hour. After a second dialysis for 12-16 hours, CQDs-NbY24 solution is obtained. The mixing ratio of carbon quantum dot powder to PBS buffer in step (2) is 1 mg: 1 mL; the EDC concentration in the EDC-NHS solution in step (2) is 0.08 mg / mL, and the NHS concentration in the EDC-NHS solution is 0.22 mg / mL; the NbY24 concentration in the NbY24 solution in step (2) is 1 mg / mL; the mixing ratio of CQDs solution, EDC-NHS solution and NbY24 solution in step (2) is 1 mL: 100 μL: 100 μL; the temperature for light-protected shaking incubation in step (2) is 37 °C; the temperature for secondary dialysis in step (2) is 4 °C, and the secondary dialysis uses a dialysis bag with a capacity of 30~40 kDa.

2. The method according to claim 1, characterized in that, The mixing ratio of citric acid, glycine and deionized water in step (1) is 1g:1.2g:10mL.

3. The method according to claim 1, characterized in that, The centrifugation speed in step (1) is 6000 rpm, and the centrifugation time is 10 min each time; the filtration in step (1) uses a 0.22 μm filter membrane, and the dialysis in step (1) uses a dialysis bag with a capacity of 30~40 kDa; the freeze-drying temperature in step (1) is -80℃, and the freeze-drying time is 48 h.

4. The method according to claim 1, characterized in that, In step A, the centrifugation temperature is 4°C, the centrifugation speed is 12000 r / min, and the centrifugation time is 10 min.

5. The method according to claim 1, characterized in that, The mixing volume ratio of the CQDs-NbY24 solution and the gradient concentration AFM1 solution in step B is 9:1, and the total volume of the mixture in step B is 1 mL.

6. The method according to claim 1, characterized in that, The volume ratio of the CQDs-NbY24 solution to the sample matrix extract in step C is 9:1, and the total volume of the mixture to be tested in step C is 1 mL.

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