Application of copper-gold nanoclusters / carbon quantum dots ratio fluorescent probe in detection of aflatoxin

Abstract

The application discloses application of a copper-gold nanocluster / carbon quantum dot ratio fluorescence probe in detection of aflatoxin, and belongs to the technical field of ratio fluorescence probes.The application constructs a ratio fluorescence probe by using copper-gold nanoclusters / carbon quantum dots, establishes an aflatoxin fluorescence detection system based on nanocluster aggregation fluorescence quenching according to the structure and property characteristics of the target aflatoxin, and realizes convenient and accurate detection of the aflatoxin.The application adopts a chemical reduction method to prepare the copper-gold nanoclusters, and the method is simple, easy to operate, simple in process and controllable;the ratio fluorescence probe replaces a traditional on-off fluorescence detection probe, is not dependent on single signal change, and greatly reduces background and environmental errors.The copper-gold nanocluster / carbon quantum dot ratio fluorescence probe lays a theoretical and experimental foundation for accurate and rapid detection of aflatoxin.

Description

Technical Field

[0001] This invention relates to the field of ratiometric fluorescent probe technology, specifically to the application of a copper-gold nanocluster / carbon quantum dot ratiometric fluorescent probe in the detection of aflatoxin. Background Technology

[0002] Aflatoxins (AFs) are a class of heterocyclic aromatic hydrocarbon secondary metabolites, mainly produced by Aspergillus flavus and Aspergillus parasiticus under humid and hot conditions. The World Health Organization classifies them as Group 1 carcinogens. Aflatoxins have derivatives such as B1, B2, G1, G2, M1, M2, GM, P1, and Q1. Among them, aflatoxin B1 (AFB1) is the strongest known natural carcinogen and has the highest toxicity. AFB1 is highly carcinogenic, mutagenic, and teratogenic, and can induce primary liver cancer, gastric cancer, lung cancer, etc. It is also a major risk factor in the multifactorial etiology of human hepatocellular carcinoma (HCC).

[0003] Currently, the main methods used for aflatoxin detection include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), high-performance liquid chromatography-mass spectrometry (HPLC-MS), and enzyme-linked immunosorbent assay (ELISA). These methods suffer from drawbacks such as cumbersome sample pretreatment, lengthy detection times, high testing costs, and demanding testing equipment requirements, failing to meet the needs for rapid on-site detection of aflatoxins (AFs). Fluorescence detection has attracted researchers' attention due to its high specificity, high sensitivity, and simple operation; however, current fluorescence detection methods rely on changes in a single signal output, making it impossible to eliminate background and environmental errors. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide an application of a copper-gold nanocluster / carbon quantum dot ratio fluorescent probe in the detection of aflatoxin, thereby achieving accurate and rapid detection of aflatoxin.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: an application of a copper-gold nanocluster / carbon quantum dot ratio fluorescent probe in the detection of aflatoxin is provided. The copper-gold nanocluster / carbon quantum dot ratio fluorescent probe is prepared by the following method: mixing copper-gold nanoclusters and silica-coated carbon quantum dots.

[0006] Furthermore, the volume ratio of copper-gold nanoclusters to silica-coated carbon quantum dots is 1:3-6.

[0007] Furthermore, copper-gold nanoclusters were prepared by the following method: using chloroauric acid and copper salt as raw materials and glutathione as ligand, copper-gold nanoclusters were prepared by chemical reduction.

[0008] Furthermore, copper-gold nanoclusters were prepared by the following method: CuCl2·2H2O solution, HAuCl4·4H2O solution and glutathione solution were mixed evenly and reacted for 20-40 min. Then the reaction system was adjusted to acidity and the reaction was continued for 60-90 min to obtain the nanoclusters.

[0009] Furthermore, the molar ratio of CuCl2·2H2O, HAuCl4·4H2O and glutathione is 45-55:1:85-95.

[0010] Furthermore, adjusting the reaction system to acidic conditions involves adjusting the pH of the reaction system to 3-5.

[0011] Furthermore, silica-coated carbon quantum dots are prepared by the following methods: (1) a carbon quantum dot solution is prepared by hydrothermal method using citric acid and ethylenediamine as raw materials; (2) a carbon quantum dot solution is prepared by hydrothermal method using ammonia, tetraethyl orthosilicate and the carbon quantum dot solution prepared in step (1) as raw materials. Carbon quantum dots coated with silica were prepared by a method.

[0012] Furthermore, carbon quantum dots coated with silica are prepared by the following methods: (1) Citric acid and ethylenediamine are dissolved in deionized water, then heated at 180-220℃ for 4-6 hours, and then transferred to a dialysis bag for dialysis for 20-30 hours to obtain a carbon quantum dot solution; (2) Ammonia and tetraethyl orthosilicate are added to anhydrous ethanol, reacted for 20-40 minutes, and then the carbon quantum dot solution prepared in step (1) is added, reacted for 2-3 hours, and then transferred to a dialysis bag for dialysis for 20-30 hours to obtain the solution.

[0013] Furthermore, in step (1), the mass ratio of citric acid to ethylenediamine is 1:1-1.5.

[0014] Furthermore, in step (2), the mass ratio of ammonia, tetraethyl orthosilicate and carbon quantum dot solution is 1:2-3:1-1.5.

[0015] Furthermore, the molecular weight cutoff of the dialysis bag in steps (1) and (2) is 1000-5000 Da.

[0016] The present invention has the following beneficial effects:

[0017] (1) Copper nanoclusters possess similar fluorescence responsiveness and spectral tunability to noble metal nanoclusters, and also have broad application prospects due to their abundant sources and low cost. However, nanoclusters formed from single metallic copper also suffer from low fluorescence intensity and poor stability, making their sensitivity and reproducibility in biofluorescence detection unsatisfactory. Compared with single-metal nanoclusters, the electronic structures and properties of the two metals work synergistically, concentrating the performance advantages of the two metals into a single nanocluster. This not only improves the stability of single-metal nanoclusters but also avoids their exposure. Compared with single copper or gold nanoclusters, copper-gold nanoclusters can form both long straight chains and dense curved chain structures. This structure can improve the fluorescence intensity of copper-gold nanoclusters, thereby improving the sensitivity of fluorescent probes.

[0018] (2) This invention constructs a ratiometric fluorescent probe using copper-gold nanoclusters / carbon quantum dots (CuAuNCs / CDs@SiO2). In the CuAuNCs / CDs@SiO2 ratiometric fluorescent probe, both carbon quantum dots and copper-gold nanoclusters can emit fluorescence. CDs@SiO2, due to the protection of the inert silica coating, hardly reacts with the medium, thus providing a stable reference signal. Meanwhile, the thiol groups of glutathione on the copper-gold nanoclusters have strong reactivity and are greatly affected by the surrounding medium, thus providing a response signal.

[0019] (3) Based on the structural and property characteristics of the target analyte aflatoxin, an aflatoxin fluorescence detection system based on nanocluster aggregation fluorescence quenching was established. First, Hg was used... 2+ Ions bind to thiol groups, mediating the aggregation of copper-gold nanoclusters, which then undergo fluorescence quenching. Upon addition of aflatoxin, aflatoxin reacts with Hg... 2+ A strong complexing reaction occurs, detaching Hg from glutathione. 2+ The ions disaggregate the aggregated copper-gold nanoclusters, allowing the fluorescence to be restored. Finally, the degree of fluorescence restoration of the copper-gold nanoclusters can be used to detect aflatoxin.

[0020] (4) This invention does not require expensive and complex chromatographs. The limit of detection for fluorescence detection is 0.56 ng / mL, the linear range is 0–55 ng / mL, the sensitivity is 33.15 mL / ng, the spiked recovery rate is between 99.2% and 105%, and the RSD is less than 5%, demonstrating accuracy. Meanwhile, the RSDs for selectivity and interference resistance are 2.2% and 1.1%, respectively, thus exhibiting certain selectivity and interference resistance.

[0021] (5) The present invention uses a ratiometric fluorescent probe instead of a traditional switch-type fluorescent detection probe, which does not rely on a single signal change and greatly reduces background and environmental errors. Attached Figure Description

[0022] Figure 1 Transmission electron microscopy and hydration particle size distribution of copper-gold nanoclusters;

[0023] Figure 2 X-ray diffraction pattern and infrared spectrum of copper-gold nanoclusters;

[0024] Figure 3 X-ray photoelectron spectrum of copper-gold nanoclusters;

[0025] Figure 4 Ultraviolet spectrum and matrix-assisted laser desorption / ionization time-of-flight mass spectrum of copper-gold nanoclusters;

[0026] Figure 5 A schematic diagram illustrating the principle of fluorescence quenching in nanocluster aggregation;

[0027] Figure 6 Transmission electron microscopy (TEM) image and hydration particle size distribution diagram of copper-gold nanoclusters after the addition of aflatoxin;

[0028] Figure 7 The response of the CuAuNCs / CDs@SiO2 ratio probe to different concentrations of aflatoxin;

[0029] Figure 8 To add Hg 2+ Scanning electron microscope images of copper-gold nanoclusters before and after ionization;

[0030] Figure 9 For different concentrations of Hg 2+ Effect of this effect on the fluorescence intensity of the CuAuNCs / CDs@SiO2 ratio fluorescent probe;

[0031] Figure 10 To assess the selectivity and anti-interference properties of the CuAuNCs / CDs@SiO2 ratiometric fluorescent probe for aflatoxin detection. Detailed Implementation

[0032] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0033] Example 1:

[0034] A method for preparing a copper-gold nanocluster / carbon quantum dot (CuAuNCs / CDs@SiO2) ratiometric fluorescent probe includes the following steps:

[0035] (1) Copper-gold nanoclusters (CuAuNCs) were prepared by chemical reduction using gold chlorate tetrahydrate and copper chloride dihydrate as the main raw materials and glutathione as the ligand. The specific operation was as follows: 0.05 mmol CuCl2·2H2O, 0.001 mmol HAuCl4·4H2O and 0.92 mmol glutathione were dissolved in 5 mL of ultrapure water. The CuCl2·2H2O solution and the HAuCl4·4H2O solution were mixed evenly and then quickly poured into the glutathione solution. The mixture was stirred for 20 min, and then 1 M NaOH solution was added to adjust the pH of the system to 4. The mixture was stirred for another 1 h. After the reaction was completed, the reaction product was transferred to a centrifuge tube, anhydrous ethanol was added, and the tube was centrifuged at high speed (10000 rpm, 5 min). After centrifugation, the supernatant was discarded, and the precipitate was washed three times with ultrapure water and anhydrous ethanol to obtain CuAuNCs. Anhydrous ethanol was added and the tube was stored at 4 °C for later use.

[0036] (2) Dissolve 0.42 g citric acid and 536 μL ethylenediamine in 10 mL deionized water, then transfer the solution to a high-pressure reactor with a polytetrafluoroethylene (Teflon) liner, heat at 200 °C for 5 h, cool to room temperature after the reaction is complete, and then dialyze with a cellulose ester membrane bag (mw = 1000 Da) for 24 h to obtain a carbon quantum dot solution, and then put it in a 4 °C refrigerator for later use;

[0037] (3) Add 0.5 mL of ammonia and 0.2 mL of tetraethyl orthosilicate to 50 mL of ethanol. After reacting for 30 min, add 1 mL of the carbon quantum dot solution obtained in step (2). Stir and react for 2 h. Then dialyze with a cellulose ester membrane bag (mw = 3000 Da) for 24 h to obtain silica-coated carbon quantum dots (CDs@SiO2). Store in a 4℃ refrigerator for later use.

[0038] (4) The CuAuNCs and CDs@SiO2 prepared in steps (1) and (3) are mixed at a volume ratio of 1:4 to obtain the final product.

[0039] Example 2: Characterization of copper-gold nanoclusters

[0040] (1) The morphology and particle size of the copper-gold nanoclusters prepared in Example 1 were scanned using transmission electron microscopy, and the results are as follows: Figure 1 As shown. By Figure 1 It can be seen that the copper-gold nanoclusters have a spherical morphology and no obvious lattice arrangement. Their average hydrated particle size is 94.34 nm (PDI = 0.242).

[0041] (2) The copper-gold nanocluster structure prepared in Example 1 was characterized using X-ray diffraction and infrared spectroscopy, and the results are as follows: Figure 2 As shown. By Figure 2It can be seen that the XRD peaks of the copper-gold nanoclusters are very broad, and the non-sharp diffraction narrow peaks indicate that they are amorphous structures (see...). Figure 2 a). In the infrared absorption spectrum of copper-gold nanoclusters (see...) Figure 2 b) SH characteristic tensile vibration peak at 2515cm -1 The disappearance of glutathione means that a complex was formed between the thiol group of glutathione and copper or gold, and glutathione was successfully bound to copper-gold nanoclusters, forming glutathione-bound copper-gold nanoclusters.

[0042] (3) The elemental composition of the copper-gold nanoclusters prepared in Example 1 was analyzed using X-ray photoelectron spectroscopy. Figure 3 As can be seen from a, the copper-gold nanoclusters prepared in Example 1 are composed of C, N, O, S, Cu, and Au elements. The high-resolution spectrum of Cu 2p (see...) Figure 3 b) shows the Cu 2p of Cu(0). 1 / 2 Cu 2p and Cu(Ⅰ) 3 / 2 The electron peaks at 951.88 eV and 932.28 eV, and the absence of the characteristic peak for Cu(II) at 942 eV, indicate the absence of Cu(II) in the copper-gold nanoclusters. (See the high-resolution spectrum of Au 4f (see...)) Figure 3 In c), the characteristic peaks at 84.38 eV and 87.98 eV are considered to be Au4f of Au(0). 7 / 2 Au(Ⅰ) Au 4f 5 / 2 The peak value of the orbital electron. Furthermore, the S2p high-resolution spectrum (see...) Figure 3 d) shows two asymmetric peaks at 162.98 eV and 161.98 eV, representing S coordination with Cu and Au, respectively, further confirming the formation of glutathione-bound copper-gold nanoclusters.

[0043] (4) The copper-gold nanoclusters prepared in Example 1 were analyzed using ultraviolet spectroscopy and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and the results are as follows: Figure 4 As shown. By Figure 4 As can be seen from a, the copper-gold nanoclusters exhibit a UV absorption peak at 220 nm, which is attributed to the n-π* electronic transition of Au, while the weak shoulder peak at 270 nm is attributed to the LMCT transition; the absence of localized surface plasmon resonance absorption peaks in the visible light region indicates the absence of large-sized particles. Figure 4 As shown in b, the CIE chromaticity coordinates of the copper-gold nanoclusters are (0.4620, 0.4923), and they emit orange fluorescence. From... Figure 4 c indicates that a peak was observed at m / z 685.4 in the MALDI-TOF-MS spectrum, which may be the [Cu3Au1GSH] molecule.

[0044] Example 3: Detection of aflatoxin using a CuAuNCs / CDs@SiO2 ratiometric fluorescent probe

[0045] The core principle of CuAuNCs / CDs@SiO2 ratiometric fluorescent probe for aflatoxin detection is the fluorescence quenching of nanoclusters, as illustrated in the schematic diagram below. Figure 5 As shown. Hg 2+ The ion chelates with the two adjacent carbonyl oxygen groups (C=O) in the aflatoxin molecule, Hg 2+ It acts as a bridge, forming something like "AFs-Hg" 2+ The structure of "-AFs". Hg 2+ The chelation between the ion and the carbonyl C=O group is very stable, and its binding force is higher than that of Hg. 2+ Complexation with glutathione and Hg 2+ With Au + The affinity of aflatoxin for Hg. Therefore, aflatoxin has an affinity for Hg. 2+ The formation of ion chelates involves aflatoxin competing with copper-gold nanoclusters for Hg. 2+ The subsequent result, Hg 2+ The Hg was extracted from the copper-gold nanoclusters, causing the aggregated nanoclusters to be deprived of Hg. 2+ The bridging effect leads to depolymerization. Different amounts of aflatoxin result in different amounts of Hg. 2+ Ions are displaced from copper-gold nanoclusters, causing varying degrees of deaggregation and resulting in different levels of fluorescence recovery. Therefore, aflatoxin can be detected based on changes in fluorescence intensity. AFB1, AFB2, AFG1, and AFG2 are some of the most common derivatives of aflatoxin, and in most cases, the mass ratio of AFB1, AFB2, AFG1, and AFG2 is 1.0:0.1:0.3:0.03. To simulate naturally occurring aflatoxin, a solution with a total concentration of 5 μg / mL was prepared according to a ratio of 1.0:0.1:0.3:0.03. Figure 6 Transmission electron microscopy (TEM) images and hydration particle size distribution diagrams of copper-gold nanoclusters after the addition of aflatoxin. Figure 6 It can be seen that, compared with the addition of aflatoxin, the average hydrated particle size of copper-gold nanoclusters decreased from 279.3 nm under aggregation to 97.62 nm, indicating that the copper-gold nanoclusters underwent deagglomeration rather than further aggregation.

[0046] To investigate the response of GSH-CuAuNCs / CDs@SiO2 to aflatoxin, the fluorescence of the copper-gold nanoclusters was quenched after adding mercuric nitrate solution (0.75 μM, 500 μL) to 1 mL of CuAuNCs / CDs@SiO2 dispersion. The lowest and most stable fluorescence intensity was detected at 560 nm. Then, different volumes of aflatoxin solution (5 μg / mL) were added and diluted to 10 mL with disodium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 7.0) to achieve equivalent aflatoxin concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65 ng / mL. After mixing and shaking, and standing for 30 min, the fluorescence intensity at the maximum emission wavelength was recorded at different aflatoxin concentrations using excitation at 360 nm. The results are as follows: Figure 7 As shown, as the aflatoxin concentration increased from 0 to 65 ng / mL, the reference signal F1 remained essentially constant, while the response signal F2 gradually increased, and the F1 / F2 ratio gradually decreased. Within the aflatoxin concentration range of 0–55 ng / mL, F1 / F2 exhibited a strong linear relationship with the aflatoxin concentration, with the linear equation being F1 / F2 = -0.0612 × C. AFs +4.6000(R 2 =0.9986, n=3). According to the formula for calculating the limit of detection (LOD): LOD = 3SD / k (where SD is the standard deviation of 11 blanks, and k is the slope of the standard curve), the limit of detection for aflatoxin in this invention is calculated to be 0.56 ng / mL. Similarly, since the reference signal F1 remains essentially constant, the response signal F2 is compared with the aflatoxin concentration C. AFs A linear fit is performed, and the absolute value of the slope of the fitted curve is used as the detection sensitivity. The fitted linear equation is: F2 = 33.1468 × C AFs +906.3(R 2 =0.9480, n=3). Therefore, the sensitivity of the CuAuNCs / CDs@SiO2 ratiometric fluorescent probe for fluorescence detection of aflatoxin is 33.15 mL / ng.

[0047] The above results indicate that the CuAuNCs / CDs@SiO2 ratiometric fluorescent probe of the present invention has the advantages of high sensitivity and low detection limit for the detection of aflatoxin.

[0048] Example 4: Hg 2+ Mediated aggregation and fluorescence quenching of copper-gold nanoclusters

[0049] Hg 2+ The outer electron configuration is 5s 2 5p 6 5d 10 6s0 Its empty electron orbitals can coordinate with S, which has lone pairs of electrons, Hg 2+ -S complexes, due to the symmetry of S, sp 3 Hybridization results in a tetrahedral spatial configuration. Furthermore, Hg 2+ With Au + The outer d orbitals are all filled with 10 electrons and have strong d-coefficients with each other. 10 -d 10 Metalophilic interaction. Therefore, Hg 2+ The complexation reaction proceeds after addition and Hg 2+ Au + The affinity between them can mediate the aggregation of copper-gold nanoclusters. Figure 8 To add Hg 2+ Scanning electron microscopy images of copper-gold nanoclusters before and after ionization, by Figure 8 It can be known that Hg 2+ This causes copper-gold nanoclusters to aggregate (see...) Figure 8 (a and 8b), and the tested hydrated particle size increased from 59.37 nm to 279.3 nm (see a and b). Figure 8 c and 8d).

[0050] Figure 9 For different concentrations of Hg 2+ The effect of (0, 0.15, 0.30, 0.45, 0.60, 0.75, 0.90 μM) on the fluorescence intensity of the CuAuNCs / CDs@SiO2 ratiometric fluorescent probe. The test results show that as Hg... 2+ The concentration increases because the reference signal F1 remains stable, while the ratio F1 / F2 increases due to the weakening of the response signal F2. Therefore, Hg 2+ The aggregation of copper-gold nanoclusters caused by the addition of Hg leads to fluorescence quenching, but when Hg 2+ After increasing the concentration to 0.75 μM, the F1 / F2 ratio essentially remained unchanged. Therefore, 0.75 μM was chosen as the concentration for Hg in subsequent experiments. 2+ The working concentration.

[0051] Example 5: Selectivity and anti-interference ability of CuAuNCs / CDs@SiO2 ratiometric fluorescent probe for aflatoxin detection

[0052] The selectivity and anti-interference ability of the CuAuNCs / CDs@SiO2 ratiometric fluorescent probe were investigated by studying its response to other toxins. Ochratoxin (OTA), patulin (PAT), deoxynivalenol (DON), nivalenol (NIV), and zeinone (ZEN) were used as interfering agents, all at a concentration of 50 ng / mL. Figure 10 It can be seen that after adding other substances, the fluorescence intensity ratio F1 / F2 is basically the same as that of the blank sample (see...). Figure 10 The pink column on the left shows a relative standard deviation of 2.2%, indicating that these toxins do not elicit a response from the fluorescent probe, demonstrating the probe's excellent selectivity. Adding AFs to the aforementioned interfering standard solutions resulted in a significant decrease in fluorescence intensity compared to F1 / F2, but the measured F1 / F2 ratios for each group were very similar (see [link to F1 / F2]). Figure 10 The blue bar on the right shows a relative standard deviation of 1.1%, indicating that this ratio fluorescent probe test system has a good ability to resist interference from other toxins in the detection of AFs.

[0053] Example 6: Accuracy of CuAuNCs / CDs@SiO2 ratiometric fluorescent probe for aflatoxin detection

[0054] The accuracy of the CuAuNCs / CDs@SiO2 ratiometric fluorescent probe for aflatoxin detection was evaluated using a spiked recovery experiment. The results of the standard addition method are shown in Table 1. Table 1 shows that the average spiked recovery rate of aflatoxin ranged from 99.2% to 102.5%, with an RSD of less than 5%. This indicates that the CuAuNCs / CDs@SiO2 ratiometric fluorescent probe of this invention has high accuracy and potential application value for aflatoxin detection.

[0055] Table 1. Determination of aflatoxin using the standard addition method.

[0056]

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a copper-gold nanocluster / carbon quantum dot ratio fluorescent probe in the detection of aflatoxin, characterized in that, The copper-gold nanocluster / carbon quantum dot ratio fluorescent probe was prepared by mixing copper-gold nanoclusters and silica-coated carbon quantum dots. The copper-gold nanoclusters were prepared by the following method: using chloroauric acid and copper salt as raw materials and glutathione as ligand, copper-gold nanoclusters were prepared by chemical reduction. The silica-coated carbon quantum dots are prepared by the following methods: (1) using citric acid and ethylenediamine as raw materials, a carbon quantum dot solution is prepared by hydrothermal method; (2) using ammonia, tetraethyl orthosilicate and the carbon quantum dot solution prepared in step (1) as raw materials, silica-coated carbon quantum dots are prepared by Stöber method. The detection method is as follows: First, use Hg 2+ Ions bind to thiol groups, mediating the aggregation of copper-gold nanoclusters, which then undergo fluorescence quenching. Upon addition of aflatoxin, aflatoxin reacts with Hg... 2+ A strong complexation reaction occurs, which removes Hg bound to glutathione. 2+ Ions are used to depolymerize the aggregated copper-gold nanoclusters, thus restoring their fluorescence. Finally, the degree of fluorescence restoration of the copper-gold nanoclusters is used to detect aflatoxin.

2. The application of the copper-gold nanocluster / carbon quantum dot ratio fluorescent probe according to claim 1 in the detection of aflatoxin, characterized in that, The volume ratio of the copper-gold nanoclusters to the silica-coated carbon quantum dots is 1:3-6.

3. The application of the copper-gold nanocluster / carbon quantum dot ratio fluorescent probe according to claim 1 in the detection of aflatoxin, characterized in that, The copper-gold nanoclusters were prepared by the following method: CuCl2·2H2O solution, HAuCl4·4H2O solution and glutathione solution were mixed evenly and reacted for 20-40 min. Then the reaction system was adjusted to acidity and the reaction was continued for 60-90 min to obtain the nanoclusters.

4. The application of the copper-gold nanocluster / carbon quantum dot ratio fluorescent probe according to claim 3 in the detection of aflatoxin, characterized in that, The molar ratio of CuCl2·2H2O, HAuCl4·4H2O, and glutathione is 45-55:1:85-95.

5. The application of the copper-gold nanocluster / carbon quantum dot ratio fluorescent probe according to claim 1 in the detection of aflatoxin, characterized in that, The silica-coated carbon quantum dots are prepared by the following method: (1) Citric acid and ethylenediamine are dissolved in deionized water, heated at 180-220 °C for 4-6 h, and then transferred to a dialysis bag for dialysis for 20-30 h to obtain a carbon quantum dot solution; (2) Ammonia and tetraethyl orthosilicate are added to anhydrous ethanol, reacted for 20-40 min, and then the carbon quantum dot solution prepared in step (1) is added, reacted for 2-3 h, and then transferred to a dialysis bag for dialysis for 20-30 h to obtain the solution.

6. The application of the copper-gold nanocluster / carbon quantum dot ratio fluorescent probe according to claim 5 in the detection of aflatoxin, characterized in that, The mass ratio of citric acid and ethylenediamine in step (1) is 1:1-1.

5.

7. The application of the copper-gold nanocluster / carbon quantum dot ratio fluorescent probe according to claim 5 in the detection of aflatoxin, characterized in that, The mass ratio of the ammonia, the tetraethyl orthosilicate and the carbon quantum dot solution in step (2) is 1:2-3:1-1.

5.

8. The application of the copper-gold nanocluster / carbon quantum dot ratio fluorescent probe according to claim 5 in the detection of aflatoxin, characterized in that, The molecular weight cutoff of the dialysis bags mentioned in steps (1) and (2) is 1000-5000 Da.

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