Ratio-dependent fluorescence sensor based on FRET effect for detecting patulin

Abstract

The invention relates to the technical field of analytical chemistry, and discloses a ratio-dependent fluorescence sensor based on FRET effect for detecting patulin, the ratio-dependent fluorescence sensor comprises: an N, S-GQDs (at) apt fluorescence probe; a gold nanocluster solution with a concentration range of 20-70 [mu] g / mL; a metal organic framework solution with a concentration range of 5-30 [mu] g / mL; wherein the N, S-GQDs (at) apt fluorescent probe is prepared by mixing a patulin aptamer solution and a nitrogen and sulfur doped graphene quantum dot solution with the concentration range of 25-100 [mu] g / mL according to the volume ratio of 1: 2. The nitrogen and sulfur doped graphene quantum dots and a metal organic framework are creatively constructed into a fluorescence resonance energy transfer pair, the surface of the energy donor nitrogen and sulfur doped graphene quantum dots is modified with an aptamer, the detection limit of 3.29 pg / mL for patulin is obtained, and extremely high detection sensitivity is achieved.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to a ratiometric fluorescence sensor based on the FRET effect for the detection of patulin. Background Technology

[0002] Patulin is a toxin produced by specific molds that commonly contaminates fruits such as apples and hawthorns, as well as their processed products, such as juices and jams. This toxin poses a potential threat to food safety and human health. Therefore, establishing a rapid and accurate method for detecting patulin content in food is of great significance for effective quality control and risk assessment.

[0003] Currently, various technologies are applied to the detection of patulin. High-performance liquid chromatography (HPLC) is the recognized standard analytical technique for the confirmation and quantification of patulin, providing accurate quantitative results. Immunoassay techniques such as enzyme-linked immunosorbent assay (ELISA) enable high-throughput screening of large batches of samples. Fluorescence sensing technology based on molecular recognition has also provided new ideas for the detection of patulin, utilizing recognition elements such as aptamers or antibodies to bind to the target analyte, triggering a change in fluorescence signal.

[0004] However, in practical applications, existing technologies still face challenges. First, in the pursuit of higher detection sensitivity, existing technologies have encountered bottlenecks. The energy transfer pairs used in traditional fluorescence sensing systems have inherent limitations in quenching efficiency or fluorescence signal intensity, which directly restricts further reduction of the detection limit and makes it difficult to meet the detection requirements for trace pollutants in samples. Second, most fluorescence sensing methods rely on changes in a single fluorescence signal for judgment. This single-signal output mode is highly susceptible to interference from instrument background, excitation light fluctuations, and environmental factors, challenging the accuracy and reproducibility of the detection results. In addition, existing analytical methods also have shortcomings in terms of ease of operation and timeliness. Chromatography usually involves cumbersome sample pretreatment and long instrument analysis time, limiting its application in rapid on-site screening. To address these issues, those skilled in the art propose a ratiometric fluorescence sensor based on the FRET effect for the detection of patulin. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a ratiometric fluorescence sensor based on the FRET effect for detecting patulin, which solves the technical problems of insufficient detection sensitivity, poor accuracy due to interference in single-signal sensing, and complex operation procedures in existing technologies.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention provides a ratiometric fluorescence sensor for detecting patulin based on the fluorescence resonance energy transfer (FRET) effect, which is a mixture comprising the following components: N,S-GQDs@apt fluorescent probe; A solution of gold nanoclusters (AuNCs) with a concentration range of 20-70 μg / mL was used as an internal reference signal; A metal-organic framework solution of MIL-101(Cr) with a concentration range of 5-30 μg / mL was used as an energy acceptor.

[0007] The N,S-GQDs@apt fluorescent probe was prepared by mixing a patulin aptamer solution with a nitrogen- and sulfur-doped graphene quantum dot (N,S-GQDs) solution. The concentration of the patulin aptamer solution ranged from 10 to 30 μM, and the concentration of the nitrogen- and sulfur-doped graphene quantum dot (N,S-GQDs) solution ranged from 25 to 100 μg / mL, with a mixing volume ratio of 1:2.

[0008] In one embodiment, the mixing volume ratio of the component N,S-GQDs@apt fluorescent probe, the component gold nanoclusters (AuNCs) solution, and the metal-organic framework MIL-101(Cr) solution is 1:1:1.

[0009] In one embodiment, the patulin aptamer is an oligonucleotide. The nitrogen- and sulfur-doped graphene quantum dots (N,S-GQDs) are energy donors, synthesized from citric acid and L-cysteine ​​via a hydrothermal method.

[0010] In one embodiment, the gold nanoclusters (AuNCs) are bovine serum albumin (BSA) stabilized gold nanoclusters. The metal-organic framework MIL-101(Cr) is a product prepared by reacting chromium(III) nitrate trihydrate with terephthalic acid.

[0011] In the sensor system: In the absence of patulin, the nitrogen- and sulfur-doped graphene quantum dot (N,S-GQDs) solution on the N,S-GQDs@apt fluorescent probe is spatially close to the metal-organic framework (MIL-101(Cr)) solution, satisfying the conditions for FRET. Under specific wavelength excitation, the energy of the N,S-GQDs solution is transferred to the MIL-101(Cr) solution non-radiatively, resulting in the quenching of the fluorescence of the N,S-GQDs solution at 453 nm. Simultaneously, the fluorescence of the gold nanoclusters (AuNCs) solution at 678 nm is unaffected by this process.

[0012] In the presence of patulin, the patulin molecule specifically binds to the aptamer on the N,S-GQDs@apt fluorescent probe. This binding event causes a conformational change in the aptamer, leading to an increase in the spatial distance between the N,S-GQDs@apt fluorescent probe and the metal-organic framework MIL-101(Cr) solution, thus blocking the FRET process. Consequently, the fluorescence of the nitrogen- and sulfur-doped graphene quantum dot (N,S-GQDs) solution at 453 nm is recovered, and its recovery intensity is correlated with the concentration of patulin. During this process, the fluorescence intensity of the gold nanoclusters (AuNCs) solution remains constant at 678 nm.

[0013] A second aspect of the present invention provides a method for detecting patulin using the ratiometric fluorescence sensor described in any of the foregoing embodiments, the method comprising the following steps: S1. Mix the ratiometric fluorescence sensor with the sample to be tested and incubate it. S2. At an excitation wavelength of 400 nm, detect the fluorescence emission spectrum of the mixed solution obtained after incubation in step S1 in the range of 400-800 nm, and record the fluorescence intensity value F at 453 nm. 453 and the fluorescence intensity value F at 678 nm 678 ; S3, Calculate the fluorescence intensity ratio F 453 / F 678 The presence or concentration of patulin in the sample to be tested is determined based on this ratio.

[0014] In one embodiment, the incubation time in step S1 is 1-10 minutes.

[0015] In one embodiment, in step S3, the calculated fluorescence intensity ratio F is used... 453 / F 678 The concentration of patulin was determined by comparing it with a pre-established standard curve.

[0016] In one embodiment, the standard curve is established as follows: using a series of patulin standard solutions of known concentrations as the detection target, steps S1 to S3 are performed to obtain the corresponding fluorescence intensity ratios; data fitting is performed with patulin concentration as the independent variable and fluorescence intensity ratio as the dependent variable. The standard curve is obtained within a concentration range of 0.01-300 ng / mL, where the patulin concentration to fluorescence intensity ratio F... 453 / F 678 The relationship is linear.

[0017] In one embodiment, the method further includes a pretreatment step for the sample to be tested before step S1, wherein the pretreatment includes extraction of the sample to be tested using an organic solvent.

[0018] In one embodiment, the organic solvent used for extraction is ethyl acetate.

[0019] This invention provides a ratiometric fluorescence sensor based on the FRET effect for detecting patulin. It has the following advantages: 1. This invention creatively constructs a fluorescence resonance energy transfer pair by combining nitrogen- and sulfur-doped graphene quantum dots (N,S-GQDs) with a metal-organic framework (MIL-101(Cr)). The surface of the energy donor nitrogen- and sulfur-doped graphene quantum dots (N,S-GQDs) is modified with aptamers, achieving a detection limit of 3.29 pg / mL for patulin and realizing extremely high detection sensitivity. Compared with existing technologies that rely on complex instruments and cumbersome pretreatment chromatographic methods or conventional fluorescence analysis methods with limited sensitivity, this method solves the problem of insufficient detection limit and inability to meet the needs of trace analysis.

[0020] 2. This invention introduces gold nanoclusters (AuNCs) with stable red fluorescence as an internal reference calibration signal into the sensing system. The detection output uses the intensity ratio of blue fluorescence to red fluorescence, which can effectively calibrate and eliminate signal errors caused by instrument fluctuations or environmental changes, significantly improving the accuracy and reliability of the detection results. It overcomes the inherent defects of existing single-signal fluorescence sensors, which are susceptible to background interference and have poor stability, resulting in poor reproducibility of the results.

[0021] 3. This invention provides a rapid detection method with simple preparation steps and an incubation time of only 1-10 minutes by directly mixing aptamer-functionalized sulfur-doped graphene quantum dots (N,S-GQDs), gold nanoclusters (AuNCs) internal references, and metal-organic framework MIL-101(Cr) quencher. As the concentration of patulin increases, the fluorescence color of the system clearly changes from red to blue, realizing a visual semi-quantitative judgment in addition to quantitative detection. This solves the technical problems of complex operation procedures, long time consumption, and inability to perform rapid on-site screening that are common in existing chromatographic or immunoassay methods. Attached Figure Description

[0022] Figure 1 Figures showing experimental data related to the principle verification of the ratiometric aptamer sensor based on the FRET effect for detecting patulin prepared in this embodiment of the invention; Figure 2 Figures showing experimental data related to the response mechanism verification of patulin detection by the ratiometric aptamer sensor based on the FRET effect prepared in this embodiment of the invention; Figure 3 The figure shows the relevant experimental data related to parameter optimization for the ratiometric aptamer sensor based on the FRET effect prepared in the embodiments of the present invention for detecting patulin; Figure 4 This is a fluorescence curve showing the change in fluorescence intensity of the ratiometric aptamer sensor based on the FRET effect prepared in this embodiment of the invention as a function of patulin concentration. Figure 5 The linear fitting curve and equation of the fluorescence ratio F453 / F678 of the ratiomated aptamer sensor based on the FRET effect prepared in the embodiments of the present invention as a function of patulin concentration. Figure 6 This is a bar chart showing the specificity of the ratiometric aptamer sensor based on the FRET effect prepared in this embodiment of the invention for detecting patulin. Figure 7 This is a schematic diagram illustrating the detection principle of the AuNCs / GQDs@apt-MIL ratiometric aptamer sensor prepared in this embodiment of the invention for detecting patulin. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0025] Citric acid, anhydrous, CAS: 77-92-9; L-cysteine, CAS: 52-90-4; Chromium nitrate trihydrate (III), CAS: 7789-02-8; Terephthalic acid, CAS: 100-21-0; Hydrofluoric acid, 40% aqueous solution, CAS: 7664-39-3; Tetrachloroauric acid trihydrate, HAuCl4·3H2O, CAS: 16961-25-4; Bovine serum albumin, CAS: 9048-46-8; Sodium hydroxide, CAS: 1310-73-2; N,N-Dimethylformamide, CAS: 68-12-2; Ethyl acetate, CAS: 141-78-6; Anhydrous ethanol, CAS: 64-17-5; Patulin aptamer, 5'-GGC CCG CCA ACC CGC ATC ATC TAC ACT GAT ATT TTA CCTT-NH2C6-3', was custom-synthesized by a commercial company; The water used in the experiment was ultrapure water with a resistivity greater than 18.2 MΩ·cm.

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. (Appendix) Figure 7 This is a schematic diagram illustrating the working principle of the ratiometric fluorescence sensor described in this invention.

[0027] Example 1: Preparation of nitrogen- and sulfur-doped graphene quantum dots (N,S-GQDs): 1.0 g of citric acid and 0.3 g of L-cysteine ​​were added to 25 mL of ultrapure water and stirred magnetically until completely dissolved and homogeneous. The resulting mixture was transferred to a 100 mL high-pressure hydrothermal synthesis reactor lined with polytetrafluoroethylene. The reactor was sealed and placed in an oven at 180 °C for 2 hours. After the reaction, the reactor was allowed to cool naturally to room temperature. The resulting orange solution was freeze-dried, and the collected orange powder was identified as nitrogen- and sulfur-doped graphene quantum dots (N,S-GQDs), which was then sealed and stored in a dry environment for later use.

[0028] Example 2: Preparation of metal-organic framework (MIL-101(Cr)): 0.8 g of chromium(III) nitrate trihydrate and 332 mg of terephthalic acid were added to 50 mL of ultrapure water and stirred vigorously at room temperature for 30 minutes to form a suspension. Then, 100 μL of 40 wt% hydrofluoric acid was added to the suspension, and the mixture was sonicated for another 10 minutes. The resulting mixture was transferred to a 100 mL high-pressure hydrothermal synthesis reactor lined with polytetrafluoroethylene. The reactor was placed in an oven at 220 °C and heated for 8 hours. After the reaction was completed, the mixture was allowed to cool naturally to room temperature. The product was centrifuged at 6000 rpm for 10 minutes, and the supernatant was discarded to collect the precipitate. The precipitate was washed three times each with N,N-dimethylformamide (DMF) and anhydrous ethanol, with centrifugation performed after each wash. Finally, the washed product was vacuum dried at 40 °C to obtain a metal-organic framework (MIL-101(Cr)) powder.

[0029] Example 3: Preparation of bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs): Under light-protected conditions, 5 mL of a 10 mM tetrachloroauric acid trihydrate aqueous solution and 5 mL of a 50 mg / mL bovine serum albumin (BSA) aqueous solution were mixed. The mixture was stirred continuously for 30 minutes under light-protected conditions. Subsequently, 0.5 mL of a 1 M sodium hydroxide (NaOH) solution was added to adjust the pH of the system. The system was transferred to a 50°C constant temperature water bath and stirred continuously for 7 hours. After the reaction was completed, the resulting brownish-red solution was centrifuged at 6000 rpm for 5 minutes, and the supernatant was carefully collected. This supernatant is the bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) solution and was stored at 4°C under light-protected conditions for later use.

[0030] Example 4: Construction of a ratiometric fluorescence sensor: First, prepare the working solutions for each component: (1) The N,S-GQDs powder prepared in Example 1 was prepared into an N,S-GQDs solution with a concentration of 75 μg / mL using ultrapure water. (2) The MIL-101(Cr) powder prepared in Example 2 was prepared into a MIL-101(Cr) solution with a concentration of 20 μg / mL using ultrapure water; (3) The BSA-AuNCs solution prepared in Example 3 was diluted with ultrapure water to a concentration of 50 μg / mL. (4) Prepare an aptamer solution with a concentration of 25 μM by using commercially synthesized patulin aptamer dry powder with ultrapure water.

[0031] Secondly, the N,S-GQDs@apt fluorescent probe was constructed: the above aptamer solution and N,S-GQDs solution were mixed at a volume ratio of 1:2 and thoroughly mixed to obtain the N,S-GQDs@apt fluorescent probe.

[0032] Finally, assemble the final sensor: Mix the prepared N,S-GQDs@apt fluorescent probe, BSA-AuNCs solution, and MIL-101(Cr) solution at a volume ratio of 1:1:1 at room temperature. The resulting solution is the ratiometric fluorescent sensor of this invention, which can be directly used for subsequent detection.

[0033] This comparative example aims to evaluate the response specificity of the ratiometric fluorescence sensor prepared in Example 4 to the target analyte patulin (PAT).

[0034] The experimental method is as follows: A solution of patulin (PAT) at a concentration of 300 ng / mL was prepared, along with solutions of various potential interfering agents, including aflatoxin B1 (AFB1), aflatoxin M1 (AFM1), deoxynivalenol (DON), zearalenone (ZEN), and fumonisin B1 (FB1). Simultaneously, a mixed solution (MIX) containing all of the above interfering agents at a concentration of 300 ng / mL was prepared. A blank control group was established using ultrapure water instead of the sample solution.

[0035] Take equal volumes of the ratiometric fluorescence sensor prepared in Example 4 and mix them with equal volumes of the above-mentioned test solutions (PAT, AFB1, AFM1, DON, ZEN, FB1, MIX and blank control), and incubate at room temperature for 5 minutes.

[0036] After incubation, a fluorescence spectrophotometer was used to collect the fluorescence emission spectra of each mixed solution at an excitation wavelength of 400 nm, and the fluorescence intensity value F at 453 nm was recorded. 453 and the fluorescence intensity value F at 678 nm 678 And calculate the fluorescence intensity ratio F. 453 / F 678 .

[0037] The experimental results are attached. Figure 6 As shown. (Attached) Figure 6 This is a bar chart showing the sensor's response to different fungal toxins. The data in the chart shows that, compared to the blank control group, only the experimental group with added patulin (PAT) showed a significantly higher fluorescence ratio (F). 453 / F 678 A significant increase was observed. However, in the experimental groups with single interfering agents such as AFB1, AFM1, DON, ZEN, and FB1, as well as mixtures thereof (MIX), the fluorescence ratios did not change significantly and remained largely consistent with the values ​​in the blank control group. This result indicates that the ratiometric fluorescence sensor described in this invention exhibits high selectivity for patulin.

[0038] Test Example 1: This test case aims to verify the working principle of the ratiometric fluorescence sensor described in this invention.

[0039] First, the "off-on" behavior of the sensor fluorescence was verified. The following three solutions were prepared: (a) The N,S-GQDs@apt fluorescent probe prepared in Example 4 was mixed with BSA-AuNCs solution at a volume ratio of 1:1; (b) Add an equal volume of MIL-101(Cr) solution to solution (a) to the solution; (c) Add patulin (PAT) to solution (b) to achieve a final concentration of 300 ng / mL.

[0040] Fluorescence spectra of solutions (a), (b), and (c) were performed at an excitation wavelength of 400 nm. The experimental results are attached. Figure 1 As shown. (Attached) Figure 1 The fluorescence emission spectra are used to verify the working principle of the sensor. Curve a is the spectrum of solution (a), which shows two fluorescence emission peaks at 453 nm and 678 nm, corresponding to N,S-GQDs and AuNCs, respectively. Curve b is the spectrum of solution (b), showing that after the addition of MIL-101(Cr), the intensity of the fluorescence emission peak at 453 nm is significantly reduced, while the intensity of the fluorescence peak at 678 nm remains basically unchanged. Curve c is the spectrum of solution (c), showing that after the addition of patulin, the intensity of the fluorescence emission peak at 453 nm is restored.

[0041] Secondly, the mechanism of fluorescence quenching needs to be confirmed.

[0042] The absorption spectrum of MIL-101(Cr) solution was measured using a UV-Vis spectrophotometer, and the fluorescence emission spectrum of N,S-GQDs solution under 400 nm excitation was measured using a fluorescence spectrophotometer. The experimental results are attached. Figure 2 As shown in Figure A. (Attachment) Figure 2 A is an overlay diagram of the emission spectrum of N,S-GQDs and the absorption spectrum of MIL-101(Cr). The figure shows that the fluorescence emission spectrum of N,S-GQDs and the absorption spectrum of MIL-101(Cr) have significant overlap in the wavelength range of 420-550 nm, which meets the spectral conditions for fluorescence resonance energy transfer (FRET).

[0043] Furthermore, using a fluorescence lifetime spectrometer, the fluorescence lifetime of the N,S-GQDs@apt probe was measured before and after the addition of MIL-101(Cr) under 400 nm excitation. The experimental results are attached. Figure 3 As shown in B. (Attachment) Figure 3 B shows the fluorescence decay curves of the N,S-GQDs@apt probe before and after the addition of MIL-101(Cr). The data in the figure show that the fluorescence lifetime of the N,S-GQDs@apt probe itself is 10.11 ns. After the addition of MIL-101(Cr), its fluorescence lifetime decreased to 4.83 ns. This significant decrease in fluorescence lifetime is direct evidence of fluorescence resonance energy transfer (FRET), ruling out other static quenching mechanisms such as the inner filter effect (IFE).

[0044] Test Example 2: This test case aims to determine the optimal operating parameters of the ratiometric fluorescence sensor constructed in Example 4, including the concentration of the quencher MIL-101(Cr) and the incubation time between the sensor and the target.

[0045] (1) Optimization of the concentration of quencher MIL-101(Cr): To determine the optimal working concentration of MIL-101(Cr), a series of mixed systems containing fixed concentrations of N,S-GQDs@apt probes and BSA-AuNCs solutions were prepared, and MIL-101(Cr) solutions of different final concentrations were added to each system, so that the final concentration range covered 0 to 30 μg / mL.

[0046] At an excitation wavelength of 400 nm, the fluorescence emission spectra of each system were measured, and the fluorescence intensity ratio F was recorded and calculated. 453 / F 678 The experimental results are attached. Figure 3 As shown in Figure A. (Attachment) Figure 3 A is the fluorescence ratio F 453 / F 678 A graph showing the effect of MIL-101(Cr) concentration on fluorescence ratio. The data in the graph show that as the MIL-101(Cr) concentration increases, the fluorescence ratio F... 453 / F 678 The gradually decreasing concentration indicates an increase in quenching efficiency. When the concentration of MIL-101(Cr) reaches 20 μg / mL, the fluorescence ratio reaches its lowest point and tends to stabilize. Therefore, 20 μg / mL was chosen as the optimal working concentration of MIL-101(Cr) for subsequent experiments.

[0047] (2) Optimization of incubation time: To determine the optimal incubation time required for the sensor to react with the target analyte, the ratiometric fluorescence sensor prepared in Example 4 (with a MIL-101(Cr) concentration of 20 μg / mL) was mixed with patulin solution (final concentration of 300 ng / mL). The fluorescence ratio F of the system was measured at different time points after mixing (0 to 15 minutes). 453 / F 678 .

[0048] The experimental results are attached. Figure 3 As shown in B. (Attachment) Figure 3 B is the fluorescence ratio F. 453 / F 678 A graph showing the change in fluorescence ratio over incubation time. The data in the graph shows that after the addition of patulin, the fluorescence ratio F... 453 / F 678The incubation time increased rapidly over time, reaching a maximum at 5 minutes, and then remained stable. This result indicates that the binding reaction between the sensor and patulin reached equilibrium within 5 minutes. Therefore, 5 minutes was chosen as the optimal incubation time for subsequent detection experiments.

[0049] Test Example 3: This test case aims to evaluate the analytical performance of the ratiometric fluorescence sensor constructed in Example 4 and optimized in Test Case 2 for the quantitative detection of patulin, including its linear response range and limit of detection (LOD).

[0050] The experimental method is as follows: A series of patulin (PAT) standard solutions with gradient concentrations were prepared, resulting in final concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 10, 50, 100, 200, and 300 ng / mL. Each of these standard solutions was mixed with an equal volume of the ratiometric fluorescence sensor prepared in Example 4 and incubated at room temperature for 5 minutes.

[0051] After incubation, fluorescence emission spectrophotometers were used with an excitation wavelength of 400 nm to collect the fluorescence emission spectra of each mixed solution. The experimental results are attached. Figure 4 As shown. (Attached) Figure 4 The figure shows the fluorescence emission spectra of the sensor in response to different concentrations of patulin. The data in the figure show that as the concentration of patulin increases, the intensity of the fluorescence emission peak at 453 nm gradually increases, while the intensity of the fluorescence emission peak at 678 nm remains constant.

[0052] The above spectral data were processed, and the fluorescence intensity value F at 453 nm was recorded for each concentration. 453 and the fluorescence intensity value F at 678nm 678 And calculate its ratio F. 453 / F 678 Plotting patulin concentration (x-axis) on the x-axis, the corresponding fluorescence ratio F... 453 / F 678 Use the y-axis as the vertical axis to plot the standard curve.

[0053] The results are attached. Figure 5 As shown. (Attached) Figure 5 This is a standard curve for patulin detection using this sensor. The data in the figure shows that within the concentration range of 0.01 ng / mL to 300 ng / mL, the fluorescence ratio F... 453 / F 678 It showed a good linear relationship with the concentration of patulin. The linear regression equation is as follows: y = 0.3849x + 0.889; Where y is the fluorescence intensity ratio F 453 / F 678 , where x is the concentration of patulin (ng / mL). The coefficient of determination R for this linear relationship is... 2 It is 0.9942.

[0054] Based on the calculation rule of 3σ / k (where σ is the standard deviation of the blank signal and k is the slope of the standard curve), the detection limit (LOD) of the method of the present invention is calculated to be 3.29 pg / mL.

[0055] Test Example 4: This test case aims to evaluate the accuracy and reliability of the ratiometric fluorescence sensor described in this invention in detecting patulin in a real sample matrix. Commercially available apple juice and grape juice were selected as the actual samples for the experiment.

[0056] The sample pretreatment steps are as follows: Take 1 mL of fruit juice sample, add 1 mL of ethyl acetate, and vortex for 2 minutes to ensure complete extraction. Then, centrifuge the mixture at 5000 rpm for 5 minutes. Take the upper organic phase and filter it through a 0.22 μm filter membrane; collect the filtrate as the sample solution to be tested.

[0057] The standard addition-recovery method was used in the experiments. Patulin standard at three different concentration levels (10, 50, 100 ng / mL) was added to pretreated apple juice and grape juice samples. These spiked samples were used as test solutions, and the methods established in Example 4 and Test Examples 2 and 3 were used for detection. Each concentration level was measured three times (n=3), and the recovery rate and relative standard deviation (RSD) were calculated. The experimental results are summarized in Table 1 below.

[0058] Table 1: Spiked recovery results of patulin in actual samples Table 1 shows that the recoveries of patulin in apple juice and grape juice were between 96.41% and 100.30% and 97.54% and 102.13%, respectively, indicating that this method can be applied to the detection of patulin in actual samples.

[0059] This method for detecting patulin can also be applied to the development of other mycotoxins. The detection of other mycotoxins can be achieved by changing the aptamer for patulin.

[0060] This invention uses N,S-GQDs as the energy donor and selects MIL-101(Cr), whose absorption spectrum overlaps well with that of N,S-GQDs, as the energy acceptor, ensuring efficient energy transfer. AuNCs serve as an internal reference signal, solving the problem of high background signal and low sensitivity in FRET sensors. Furthermore, the detection result shows a visible change in fluorescence color from red to blue, offering advantages in visual detection. The detection limit is 3.29 pg / mL, with excellent selectivity for PAT. The AuNCs / GQDs@apt-MIL sensor exhibits high sensitivity, excellent selectivity, and good stability.

[0061] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A ratiometric fluorescence sensor based on the FRET effect, characterized in that, Includes the following components: N,S-GQDs@apt fluorescent probe; Gold nanocluster solutions with concentrations ranging from 20 to 70 μg / mL; Metal-organic framework solutions with concentrations ranging from 5 to 30 μg / mL; The N,S-GQDs@apt fluorescent probe is prepared by mixing a solution of patulin aptamer with a nitrogen- and sulfur-doped graphene quantum dot solution with a concentration range of 25-100 μg / mL at a volume ratio of 1:

2. The concentration range of the patulin aptamer solution is 10-30 μM.

2. The ratiometric fluorescence sensor based on the FRET effect according to claim 1, characterized in that, The volume ratio of the N,S-GQDs@apt fluorescent probe, gold nanocluster solution, and metal-organic framework solution is 1:1:

1.

3. A ratiometric fluorescence sensor based on the FRET effect according to claim 1, characterized in that, The nitrogen and sulfur-doped graphene quantum dot solution is synthesized from citric acid and L-cysteine ​​via a hydrothermal method, and the patulin aptamer is an oligonucleotide.

4. A ratiometric fluorescence sensor based on the FRET effect according to claim 1, characterized in that, The gold nanoclusters are gold nanoclusters stabilized by bovine serum albumin, and the metal-organic framework is prepared by reacting chromium nitrate trihydrate with terephthalic acid.

5. A method for detecting patulin using a ratiometric fluorescence sensor based on the FRET effect as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Incubate the ratiometric fluorescence sensor with the sample to be tested; S2. At an excitation wavelength of 400 nm, the fluorescence emission spectrum of the incubated mixed solution was detected, and the fluorescence intensity value F at 453 nm was recorded. 453 and the fluorescence intensity value F at 678 nm 678 ; S3, Calculate the fluorescence intensity ratio F 453 / F 678 The presence or concentration of patulin in the sample to be tested is determined based on this ratio.

6. The method for detecting patulin using a ratiometric fluorescence sensor based on the FRET effect according to claim 5, characterized in that, In step S1, the incubation time is 1-10 minutes.

7. The method for detecting patulin using a ratiometric fluorescence sensor based on the FRET effect according to claim 5, characterized in that, In S3, the fluorescence intensity ratio F 453 / F 678 The concentration of patulin was determined by comparing it with a pre-established standard curve.

8. The method for detecting patulin using a ratiometric fluorescence sensor based on the FRET effect according to claim 7, characterized in that, The standard curve is defined as the ratio of patulin concentration to fluorescence intensity F within a concentration range of 0.01-300 ng / mL. 453 / F 678 The relationship is linear.

9. The method for detecting patulin using a ratiometric fluorescence sensor based on the FRET effect according to claim 5, characterized in that, Before step S1, there is a step of pretreatment of the sample to be tested, which includes extraction using an organic solvent.

10. The method for detecting patulin using a ratiometric fluorescence sensor based on the FRET effect according to claim 9, characterized in that, The organic solvent is ethyl acetate.

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