A fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone and a preparation method and application thereof

Abstract

This invention belongs to the field of food analysis, specifically disclosing a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone, its preparation method, and its application. First, a pseudo-template molecule of zearalenone is pre-assembled with a functional monomer. Then, a red-emitting carbon dot ethanol solution is added as a fluorescent indicator. Finally, a crosslinking agent is added to carry out a polymerization reaction. After polymerization, the pseudo-template is eluted to obtain a fluorescent molecularly imprinted polymer capable of specifically recognizing zearalenone. When detecting zearalenone, quantitative identification can be achieved based on the fluorescence quenching rate of the red-emitting carbon dots. This method features simple operation, high specificity, strong stability, and low cost, enabling rapid detection of zearalenone in grains and providing a new fluorescent molecular recognition material for the development of rapid detection products for food safety.

Description

Technical Field

[0001] This invention belongs to the field of food analysis, specifically relating to a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone, its preparation method, and its application. Background Technology

[0002] Zearalenone (ZEN) is a secondary metabolite produced by fungi of the genus *Fusarium*. These fungi easily infect crops in high-humidity, low-temperature storage or field environments, leading to contamination of grains such as corn, wheat, and barley, as well as their products. ZEN possesses strong estrogenic activity and carcinogenicity, posing a significant threat to food safety and has been classified as a Group 3 carcinogen by the World Health Organization. Currently, common methods for ZEN detection include high-performance liquid chromatography (HPLC), HPLC-MS, and enzyme-linked immunosorbent assay (ELISA). Chromatography offers high sensitivity and accurate, reliable results, but it is costly and time-consuming, making it unsuitable for rapid on-site detection. While ELISA has led to the development of portable products such as kits and test strips, it suffers from poor resistance to matrix interference, and antibody development is costly and time-consuming. Therefore, developing a low-cost, simple, high-throughput, and fast-response ZEN detection method is essential.

[0003] Molecular imprinting technology (MIT) originated in immunology. Its mechanism mimics the recognition mechanism between antigens and antibodies; therefore, molecularly imprinted polymers (MIPs) are often referred to as "artificial antibodies." The detection principle of MIPs is to capture target substances by constructing a rigid three-dimensional network structure that is complementary to the target substance in shape, size, and functional groups. It has advantages such as high specificity, good stability, simple preparation, and low cost, and has been widely studied and applied in separation science, sensing analysis, drug delivery, and enzyme catalysis simulation. Carbon dots (CDs), discovered in single-walled carbon nanotubes in 2004, have advantages such as simple preparation, good fluorescence performance, good biocompatibility, low cost, low toxicity, and convenient surface functionalization, making them one of the preferred signal outputs for fluorescent molecularly imprinted polymers. Currently, most carbon dots combined with MIPs in the detection field are blue-green carbon dots, which are easily interfered with by fluorescent substances in the matrix environment, thus adversely affecting the accuracy of the detection results. Summary of the Invention

[0004] Technical problems solved: To address the technical problems of high detection cost, long time consumption, poor anti-interference ability, and inability to meet the requirements of rapid on-site detection in the existing technologies mentioned above, this invention proposes a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone, its preparation method, and its application. Red-emitting carbon dots (RCDs) are obtained through a solvothermal reaction, and then used as fluorescent indicators. Fluorescent carbon dot molecularly imprinted polymers are synthesized using sol-gel technology for the quantitative detection of ZEN in actual samples, thus developing a sensitive and rapid on-site detection method.

[0005] Technical Solution: The first objective of this invention is to provide a method for preparing a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone, comprising the following steps: adding a pseudo template molecule of zearalenone to a water / ethanol co-solvent, mixing with ultrasonic assistance, dispersing evenly, adding a functional monomer, stirring, adding a red light emitting carbon dot ethanol solution and continuing stirring, and finally adding a crosslinking agent to react in the dark; after the reaction is complete, centrifuging to obtain a precipitate, and washing away unreacted substances and the template with ethanol, methanol, and acetic acid elution buffers respectively, and finally collecting the sample in ethanol and storing it in the dark at 4°C to obtain a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone based on red light emitting carbon dots.

[0006] As a preferred embodiment, the preparation method of the red light emitting carbon dot ethanol solution is as follows: Mulberry leaf powder is mixed with anhydrous ethanol and heated in a reaction vessel at 120°C for 6 h. After the reaction is completed, the mixture is cooled to room temperature, centrifuged, and the upper liquid is filtered through a 0.22 μm organic filter membrane. The sample is collected and stored at 4°C in the dark to obtain red light emitting carbon dots. After diluting the solution by 50 times with ethanol, the red light emitting carbon dot ethanol solution is obtained.

[0007] Preferably, the ratio of mulberry leaf powder to anhydrous ethanol is 2 g: 20 mL.

[0008] Preferably, the pseudotemplate molecule of the zearalenone is quercetin, the functional monomer is 3-aminopropyltriethoxysilane (APTES), and the crosslinking agent is tetraethoxysilane (TEOS).

[0009] Preferably, the molar ratio of the pseudo-template molecule, functional monomer, and crosslinking agent is 1:2-6:10, and the ratio of the pseudo-template molecule to the red light emitting carbon dot ethanol solution is 0.1 mmol:3 mL.

[0010] Preferably, the volume ratio of the water / ethanol co-solvent is 4:5; and the volume ratio of methanol to acetic acid in the methanol-acetic acid eluent is 9:1.

[0011] The second objective of this invention is to prepare a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone based on the above method.

[0012] The third objective of this invention is the application of the aforementioned fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone in the quantitative detection of zearalenone.

[0013] Preferably, the quantitative detection steps are as follows: the initial fluorescence value of the fluorescent molecularly imprinted polymer solution is measured using a fluorescence spectrophotometer, the sample to be tested is added, the fluorescence change rate is measured, and a standard curve is established, thereby performing quantitative analysis of zearalenone.

[0014] Preferably, the concentration of the fluorescent molecularly imprinted polymer solution is 0.01-0.5 mg / mL, and the solvent is anhydrous ethanol; after adding the sample to be tested for 1-15 min, the fluorescence change rate is measured and a standard curve is established.

[0015] Zearalenone (ZEN) is a common biotoxin found in food, exhibiting strong reproductive toxicity and potential carcinogenic risks. The fluorescent molecularly imprinted polymer for detecting ZEN based on red-emitting carbon dots described in this invention uses ZEN as a template and red-emitting carbon dots as fluorescent indicators. The polymer is designed and synthesized using molecular imprinting technology, allowing for sensitive and rapid identification of ZEN based on fluorescence variability. It features high specificity, strong stability, simple operation, low cost, and ease of real-time on-site detection.

[0016] Beneficial effects:

[0017] 1. This invention employs sol-gel polymerization with silane reagents. The reaction conditions are mild and carried out at room temperature, which ensures the stability of the molecularly imprinted polymer and facilitates the acquisition of highly specific polymers.

[0018] 2. The precursor for preparing carbon dots in this invention is mulberry leaf, a biomass material, which meets the requirements of green and environmentally friendly practices.

[0019] 3. The red-emitting carbon dots in this invention have a longer emission wavelength, which can avoid fluorescence interference from most impurities. The emission spectrum data shows that the signal of the red-emitting carbon dots prepared in this invention is around 675 nm. Most fluorescent impurities have no fluorescence value at 675 nm, so the interference of most fluorescent impurities can be completely shielded.

[0020] 4. The fluorescent molecularly imprinted polymer for rapid detection of ZEN prepared in this invention can sensitively and rapidly identify ZEN based on fluorescence change rate, and achieve accurate quantification of it.

[0021] 5. The fluorescent molecularly imprinted polymer prepared in this invention has strong specificity for ZEN.

[0022] 6. This detection method is simple to operate, has low production costs, and a short analysis time, which greatly improves the efficiency and sensitivity of the detection and makes it easy to perform on-site sampling and analysis. Attached Figure Description

[0023] Figure 1 A schematic diagram of the synthesis process and detection procedure (c) of red light emitting carbon dots (a) and fluorescent molecularly imprinted polymers (b);

[0024] Figure 2 Transmission electron microscope image of red-emitting carbon dots;

[0025] Figure 3 This is a particle size distribution diagram of red-emitting carbon dots;

[0026] Figure 4 Fluorescence spectra of ethanol extracts from three cereal matrices: (a) rice, (b) wheat, and (c) corn;

[0027] Figure 5 The effects of adding rice matrix extract on the fluorescence of (a) red carbon dots, (b) blue carbon dots, and (c) green carbon dots were investigated.

[0028] Figure 6 Adsorption results of molecularly imprinted polymers with different template-to-functional monomer molar ratios are shown in the figure.

[0029] Figure 7 Scanning electron microscope images of ZEN-RCDS@FMIPs (a), (c), (e) and ZEN-RCDS@FNIPs (b), (d), (f);

[0030] Figure 8 Infrared spectra of ZEN-RCDS@FMIPs and ZEN-RCDS@FNIPs;

[0031] Figure 9 Figure showing the optimized test concentration results for ZEN fluorescently imprinted polymers;

[0032] Figure 10 The figure shows the kinetic test results of the ZEN fluorescently imprinted polymer.

[0033] Figure 11 The figure shows the specificity test results of the ZEN fluorescently imprinted polymer;

[0034] Figure 12 Thermodynamic test results of ZEN fluorescent molecularly imprinted polymers;

[0035] Figure 13 The standard curve represents the linear detection range of ZEN fluorescently imprinted polymers. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0037] Unless otherwise specified, the experimental methods described in the embodiments of this specification are conventional methods. Unless otherwise specified, the reagents and materials mentioned are commercially available.

[0038] Example 1

[0039] This embodiment provides a method for preparing a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone, see [link to documentation]. Figure 1 The steps are as follows:

[0040] Step 1: Preparation of red-emitting carbon dots (hereinafter referred to as red-emitting carbon dots or RCDs), the route is as follows: Figure 1 As shown in (a):

[0041] Weigh 2.0 g of dried mulberry leaf powder that has passed through a 0.3 mm sieve into 20 mL of anhydrous ethanol, stir and mix overnight, place it in a reaction vessel and heat at 120 °C for 6 h. After the reaction is complete, let it cool to room temperature, centrifuge at 5000 rpm for 3 min, take the upper liquid and filter it through a 0.22 μm organic filter membrane, collect the sample and store it at 4 °C in the dark, dilute it 50 times with ethanol to obtain RCDs ethanol solution.

[0042] Figure 2 The image shows a transmission electron microscope (TEM) image of the prepared carbon dots. The carbon dots are dispersed in the field of view, exhibiting good monodispersity and no obvious aggregation. The lattice fringe spacing is 0.21 nm, indicating quasi-spherical carbon dots. Statistical analysis was also performed on hundreds of random particles, such as... Figure 3 As shown, the particle size distribution of the red-emitting carbon dots ranges from 1.5 to 3.7 nm, with an average particle size of 2.45 ± 0.42 nm.

[0043] Step 2: Prepare ZEN fluorescent molecularly imprinted polymers. The specific synthetic route is as follows: Figure 1 As shown in (b):

[0044] First, add 4 mL of water and 5 mL of ethanol to a 25 mL round-bottom flask and sonicate to mix. Then, add 0.1 mmol (0.0302 g) of the ZEN pseudotemplate molecule quercetin, and sonicate to mix. After the mixture is evenly dispersed, add 0.2 mmol (46.8 μL) of the functional monomer APTES. Stir for 60 min, then add 3 mL of the RCDs ethanol solution prepared in step one (adjusted to pH 9 with ammonia beforehand). Stir for 30 min, and finally add 1 mmol (223.29 μL) of TEOS. React at room temperature in the dark for 24 h.

[0045] After the reaction was completed, the sample was collected in a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min to obtain the precipitate. Unreacted substances and ZEN pseudotemplate were removed by elution with ethanol and methanol-acetic acid (v / v, 9 / 1) buffer, respectively. Finally, the ZEN-RCDS@FMIPs sample was collected in ethanol and stored in the dark at 4 °C and its solid content was measured.

[0046] Example 2

[0047] This embodiment investigates the influence of the matrix on red carbon dots by preparing an ethanol extract of grains.

[0048] Figure 4 Ethanol extracts of three grains (rice, wheat, and corn) were prepared. 4 mg of each grain was weighed, 10 mL of ethanol was added, and the mixture was stirred and incubated overnight. After standing, the supernatant was filtered through a 0.22 μm organic filter membrane, and an appropriate amount of water was added to prepare an ethanol / water (4 / 1, v / v) extract for fluorescence testing. The tests revealed that the fluorescence emission spectra of the three grains were very similar. Because rice (… Figure 4 a) showed the strongest fluorescence value, so rice was chosen to investigate the effect of its matrix extract on carbon dot fluorescence.

[0049] By replacing the mulberry leaves in Example 1 with tobacco leaves and sycamore leaves respectively, blue and green light carbon dots can be prepared.

[0050] Figure 5 To test the effect of rice extract on three types of carbon dots (red carbon dots prepared in Example 1 of this invention / existing blue carbon dots / existing green carbon dots), 2 mL of carbon dot solution was added to each group and its fluorescence value F0 was measured. Then, 100 μL of rice matrix extract was added, and after stirring for 2 min, F1 was measured. Simultaneously, another 2 mL of carbon dot solution from each group was added to a cuvette and its fluorescence value KB-F0 was measured. 100 μL of ethanol / water (4 / 1, v / v) co-solvent was added as a blank control, and after stirring for 2 min, KB-F1 was measured. The results showed that when rice matrix was added to the three types of carbon dots, except for the red carbon dots, the other two carbon dots showed varying degrees of quenching after the addition of the rice matrix, while the red carbon dots were least affected by the matrix.

[0051] Comparative Example 1

[0052] This comparative example provides a method for preparing ZEN molecularly imprinted polymers (ZEN@MIPs), the details of which are as follows:

[0053] First, add 4 mL of water and 8 mL of ethanol to a 25 mL round-bottom flask and sonicate to mix. Then, add 0.1 mmol (0.0302 g) of ZEN pseudotemplate quercetin, sonicate to mix, and after even dispersion, add 0.2 mmol (46.8 μL) of functional monomer APTES. After stirring for 1 h, add 1 mmol (223.29 μL) of TEOS and react at room temperature for 24 h.

[0054] After the reaction was completed, the sample was collected in a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min to obtain the precipitate. Unreacted substances and the pseudotemplate of ZEN, quercetin, were removed by elution with ethanol and methanol-acetic acid (v / v, 9 / 1), respectively. Finally, excess elution was washed away with ethanol and the solid powder sample (ZEN@M1) was obtained by vacuum drying and stored in the dark at 4 °C.

[0055] The molar ratio of template to functional monomer affects the selectivity of the synthesized molecularly imprinted polymer to the target. Therefore, two other ZEN@MIPs were prepared with the molar ratio of template to functional monomer set to 1:4 and 1:6, respectively, and named ZEN@M2 and ZEN@M3.

[0056] Comparative Example 2

[0057] Non-imprinted polymers (ZEN@NIPs) were prepared as controls. Except for omitting the template, the preparation was carried out in accordance with the preparation of Comparative Example 1, using the same conditions as the molecularly imprinted polymers. They were named ZEN@N1, ZEN@N2, and ZEN@N3, respectively.

[0058] To investigate the binding ability of the molecularly imprinted polymers prepared in Comparative Example 1 and Comparative Example 2 to the target compound ZEN, their adsorption capacity for ZEN was tested. The test procedure is as follows:

[0059] (1) Preparation of standard curve: First, prepare a series of ZEN solutions with concentrations of 0.5, 1, 2.5, 5 and 10 μg / mL, with ethanol as the solvent. Then, use a UV / Vis spectrophotometer to record the absorbance at a wavelength of 274 nm. Based on the concentration and absorbance, establish a standard curve for ZEN concentration.

[0060] (2) Weigh 1 mg of polymer and dissolve it in 1 mL of ethanol, then add 1 mL of ZEN solution with a concentration of 5 μg / mL and shake overnight (a total of 3 parallel samples and one blank sample were set up). After the process, centrifuge (10000 rpm, 10 min) and take the supernatant. Record its absorbance at a wavelength of 274 nm using a UV / Vis spectrophotometer. Calculate the amount of ZEN adsorbed by the polymer, p, according to the standard curve and calculate the imprinting factor IF.

[0061] The formula for calculating p is as follows:

[0062] ,

[0063] Where C0 is the concentration of the target compound before adsorption by the polymer, in μg / mL; C is the concentration of the target compound after adsorption by the polymer, in μg / mL; C m The value represents the polymer concentration, in μg / mL.

[0064] The formula for calculating the imprinting factor IF, with adsorption amount p as a reference, is as follows:

[0065] ,

[0066] Where P M P represents the adsorption capacity of ZEN@MIPs for the target substance. N This represents the adsorption capacity of ZEN@NIPs on the target substance.

[0067] The results are as follows Figure 6 As shown, ZEN@M1 and ZEN@M3 are selective for the target ZEN, with ZEN@M1 exhibiting the highest adsorption capacity and the highest imprinting factor of 4.3. Therefore, the formulation of ZEN@M1 was ultimately selected for the subsequent synthesis of fluorescent carbon dot molecularly imprinted polymers.

[0068] Comparative Example 3

[0069] A fluorescent non-imprinted polymer was prepared as a control. Except for omitting the template, the preparation was carried out in accordance with the preparation of Example 1, using the same conditions as the molecularly imprinted polymer, and named ZEN-RCDS@FNIPs.

[0070] Figure 7 The images show transmission electron microscopy (TEM) images of ZEN-RCDS@FMIPs and ZEN-RCDS@FNIPs. A comparison reveals that the particle size of ZEN-RCDS@FMIPs is smaller than that of ZEN-RCDS@FNIPs, consistent with the pattern observed in the polymers synthesized in Comparative Examples 1 and 2. Smaller particle sizes generally exhibit better selectivity for the target analyte. However, the dispersibility and uniformity of ZEN-RCDS@FMIPs are slightly worse than those of ZEN-RCDS@FNIPs. Most ZEN-RCDS@FMIPs are cross-linked, indicating that the template addition affects the polymerization process. Furthermore, the surface roughness of ZEN-RCDS@FMIPs is significantly greater than that of ZEN-RCDS@FNIPs, possibly due to imprinted cavities on its surface that specifically recognize the target substance.

[0071] Figure 8Fourier transform infrared (FTIR) spectra of ZEN-RCDs@FMIPs after template removal and ZEN-RCDs@FNIPs are shown. Comparing the peak shapes, there is almost no significant difference between the two, which is consistent with theoretical results. The peak shape at 1631 cm⁻¹ is particularly prominent. -1 The absorption peak at this location represents the bending vibration of the NH group, providing valid proof of the successful synthesis of the fluorescently imprinted polymer.

[0072] Performance tests were performed on the ZEN-RCDS@FMIPs prepared in Example 1 and the ZEN-RCDS@FNIPs prepared in Comparative Example 3:

[0073] (I) Concentration Optimization Experiment

[0074] A certain amount of ZEN-RCDS@FMIPs was diluted with anhydrous ethanol to concentrations of 0.5, 0.1, 0.05, and 0.01 mg / mL, respectively. 2 mL of each concentration was then added to a fluorescence cuvette. After the fluorescence values ​​stabilized, the initial fluorescence signal F0 (excitation wavelength 405 nm, emission wavelength 670 nm) was measured using a fluorescence spectrophotometer. 20 μL of ZEN ethanol solution was added dropwise to each fluorescence cuvette to achieve a system concentration of 0.1 μg / mL. After reacting for 15 min, the fluorescence response rate ΔF was measured, and the change in fluorescence response was calculated. M The ΔF of the corresponding ZEN-RCDS@FNIPs was measured using the same method. N Calculate the imprinting factor (IF) for each polymer concentration.

[0075] The formula for calculating the rate of change of fluorescence response is as follows:

[0076] .

[0077] The formula for calculating the imprinting factor based on the fluorescence change rate is as follows:

[0078] .

[0079] The concentration of the polymer affects the fluorescence response to the target substance. Figure 9 The test results are shown in the figure. Calculations show that the imprinting factors of ZEN-RCDS@FMIPs for ZEN at the four concentrations are 4.8, 2.6, 2.3, and 1.5, respectively. The polymer at 0.1 mg / mL has the highest fluorescence response, which means that the polymer at this concentration shows the most significant fluorescence response after binding the target ZEN. Therefore, the concentration of ZEN-RCDS@FMIPs for subsequent tests was set to 0.1 mg / mL.

[0080] (ii) Dynamic testing

[0081] A certain amount of ZEN-RCDS@FMIPs was diluted with anhydrous ethanol to 0.1 mg / mL. 2 mL of this solution was then added to a fluorescence cuvette. After the fluorescence value stabilized, the initial fluorescence signal F0 (excitation wavelength 405 nm, emission wavelength 670 nm) was measured using a fluorescence spectrophotometer. 20 μL of ZEN ethanol solution was added dropwise to the fluorescence cuvette to make the system concentration 0.1 μg / mL. The reaction time was 1, 3, 5, 10, and 15 min, and F was measured. ΔF was calculated. M The ΔF of ZEN-RCDS@FNIPs was measured using the same method. N .

[0082] Depend on Figure 10 It was observed that after adding ZEN, the fluorescence intensity of ZEN-RCDS@FMIPs gradually decreased over time, stabilizing around 10 minutes. In contrast, ZEN-RCDS@FNIPs reached saturation quickly, with a fluorescence response far lower than that of ZEN-RCDS@FMIPs. Therefore, 10 minutes was ultimately chosen as the incubation time for subsequent ZEN-RCDS@FMIPs testing, as this is significantly faster than most detection methods.

[0083] (iii) Specificity test

[0084] A certain amount of ZEN-RCDS@FMIPs was diluted with anhydrous ethanol to 0.1 mg / mL. 2 mL of each solution was then added to a fluorescence cuvette. After the fluorescence value stabilized, the initial fluorescence signal F0 (excitation wavelength 405 nm, emission wavelength 670 nm) was measured using a fluorescence spectrophotometer. 20 μL of ethanol solutions of quercetin and biotoxins (ZEN, OTA, AFB1, FB1, DON) were added dropwise to the fluorescence cuvettes to a concentration of 0.1 μg / mL. After reacting for 10 min, F was measured, and ΔF was calculated. M The ΔF of ZEN-RCDS@FNIPs was measured using the same method. N And calculate the imprint factor (IF) and cross-reactivity factor for each group.

[0085] The formula for calculating the cross-reactivity factor is as follows:

[0086] ,

[0087] Where ΔF M(ZEN) and ΔF M These represent the fluorescence response changes of zearalenone and other biotoxins under the same ZEN-RCDS@FMIPs conditions.

[0088] Molecularly imprinted polymers, as "artificial antibodies" with excellent selectivity, rely on their specific recognition ability for target analytes as a key indicator of their effectiveness. Fluorescence response tests were performed on ZEN and its pseudotemplate quercetin, as well as common biotoxins. Their respective fluorescence quenching rates are shown below. Figure 11 As shown in the figure, ZEN-RCDS@FMIPs exhibit significant specificity for ZEN and pseudotemplate quercetin. The calculated cross-reactivity factors of ZEN-RCDS@FMIPs with other fungal toxins are 8.37 (AFB1), 4.24 (FB1), and 5.21 (DON), respectively. Since OTA exhibits fluorescence enhancement for polymers, its difference from ZEN is even more significant. Based on the above conclusions, ZEN-RCDS@FMIPs demonstrates a high degree of specificity in recognizing the target zearalenone.

[0089] (iv) Thermodynamic Testing

[0090] A certain amount of ZEN-RCDS@FMIPs was diluted with anhydrous ethanol to 0.1 mg / mL. 2 mL of this solution was then added to a fluorescence cuvette. After the fluorescence value stabilized, the initial fluorescence signal F0 (excitation wavelength 405 nm, emission wavelength 670 nm) was measured using a fluorescence spectrophotometer. Different concentrations of ZEN stock solution were then added dropwise to fluorescence cuvettes, resulting in system concentrations of 1, 5, 10, 50, 100, 500, 1000, 5000, and 10000 ng / mL. After reacting for 10 min, F was measured, and ΔF was calculated. M And the ΔF of ZEN-RCDS@FNIPs was measured using the same method. N .

[0091] Figure 12 The fluorescence of ZEN-RCDS@FMIPs was gradually quenched as the concentration of ZEN added increased, ranging from 1 to 500 ng / mL, with a binding constant Ka of 2.16 × 10⁻⁶. 10 M -2 In contrast, the fluorescence change of ZEN-RCDs@FNIPs reaches saturation at low concentrations and remains essentially unchanged at high concentrations. This is because the surface of ZEN-RCDs@FNIPs lacks imprinted cavities and can only rely on physical adsorption of small amounts of ZEN. Linear detection range and detection limit are important indicators for evaluating a detection method, such as... Figure 13 As shown, the linear detection range of this sensor is between 1 and 500 ng / mL. After logarithmic processing of the data, the fitting equation was obtained as y = 0.05988x + 0.06577 (R²).2 = 0.96); at the same time, its limit of quantitation can reach 1 ng / mL, which has high sensitivity.

[0092] Application Example 1

[0093] For testing of actual samples (corn flour, rice flour, and wheat flour purchased from supermarkets), the route is as follows: Figure 1 As shown in (c):

[0094] Sample pretreatment: Accurately weigh 5 g of rice flour into 50 mL centrifuge tubes. Add 0.025, 0.25, and 2.5 mL of 1 μg / mL zearalenone stock solution to the three weighed rice flour samples, respectively, and dilute to 20 mL with methanol. Vortex the samples to thoroughly mix with the toxin and place them in a fume hood to allow the methanol to evaporate. Add 0.5 g of sodium chloride and 25 mL of acetonitrile / water (v / v, 9 / 1) mixture to the evaporated samples. Homogenize the mixture and then shake it on a shaker for 30 min to extract the toxin. After shaking, filter the sample solution through rapid qualitative filter paper, and then filter it multiple times through a 0.22 μm organic filter before storing it in a refrigerator to obtain the ZEN-added cereal sample extract. The processing method for corn and wheat samples is the same.

[0095] Take 20 mL of the extract of the grain sample with added ZEN, blow the solvent dry with a nitrogen blower and reconstitute with 200 μL of acetonitrile-water (v / v, 7 / 3) solution.

[0096] A certain amount of ZEN-RCDS@FMIPs was diluted with anhydrous ethanol to 0.1 mg / mL. 2 mL of each solution was then added to a fluorescence cuvette. After the fluorescence value stabilized, the initial fluorescence signal F0 (excitation wavelength 405 nm, emission wavelength 670 nm) was measured using a fluorescence spectrophotometer. 40 μL of standard ZEN stock solution was added dropwise to the fluorescence cuvette to make system concentrations of 2, 20, and 200 ng / mL, respectively. After reacting for 10 min, F was measured, and ΔF was calculated. M0 .

[0097] The ΔF of the cereal sample extract with added ZEN was tested using the method described above. M The recovery rates for the three samples at corresponding concentrations were calculated, and the results are shown in Table 1 below. The recovery rate formula is as follows:

[0098] .

[0099] Table 1. Spiked recovery test of ZEN in real samples

[0100]

[0101] As shown in Table 1, the spiked recoveries of ZEN in maize by the fluorescent molecularly imprinted polymer ZEN-RCDS@FMIPs were 83%–114%, in rice 83%–103%, and in wheat 88%–110%, indicating that they have relatively good recoveries.

[0102] The present invention and its embodiments have been described above illustratively. This description is not restrictive, and the figures shown are only one embodiment of the present invention; the actual structure is not limited thereto. Therefore, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method for preparing a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone, characterized in that, The steps are as follows: In a water / ethanol co-solvent, a pseudo-template molecule of zearalenone is added, and the mixture is ultrasonically assisted to achieve uniform dispersion. After dispersion, a functional monomer is added, and the mixture is stirred. Then, a red light emitting carbon dot ethanol solution is added and stirred continuously. Finally, a crosslinking agent is added to react in the dark. After the reaction is complete, the precipitate is obtained by centrifugation, and unreacted substances and the template are washed away with ethanol, methanol, and acetic acid elution buffers, respectively. Finally, the sample is collected in ethanol and stored in the dark at 4°C to obtain a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone based on red light emitting carbon dots.

2. The method for preparing a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone according to claim 1, characterized in that, The preparation method of the red light emitting carbon dot ethanol solution is as follows: Mulberry leaf powder is mixed with anhydrous ethanol and heated in a reaction vessel at 120°C for 6 h. After the reaction is completed, it is cooled to room temperature, centrifuged, and the upper liquid is filtered through a 0.22 μm organic filter membrane. The sample is collected and stored at 4°C in the dark to obtain red light emitting carbon dots. After diluting with ethanol 50 times, the red light emitting carbon dot ethanol solution is obtained.

3. The method for preparing a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone according to claim 2, characterized in that, The ratio of mulberry leaf powder to anhydrous ethanol is 2 g: 20 mL.

4. The method for preparing a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone according to claim 1, characterized in that, The pseudotemplate molecule of the zearalenone is quercetin, the functional monomer is 3-aminopropyltriethoxysilane, and the crosslinking agent is tetraethoxysilane.

5. The method for preparing a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone according to claim 2, characterized in that, The molar ratio of the pseudotemplate molecule, functional monomer and crosslinking agent is 1:2-6:10, and the ratio of the pseudotemplate molecule to the red light emitting carbon dot ethanol solution is 0.1 mmol:3 mL.

6. The method for preparing a fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone according to claim 1, characterized in that, The volume ratio of the water / ethanol co-solvent is 4:5; the volume ratio of methanol to acetic acid in the methanol-acetic acid eluent is 9:

1.

7. A fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone prepared based on the method described in any one of claims 1-6.

8. The application of the fluorescent carbon dot molecularly imprinted polymer for detecting zearalenone as described in claim 7 in the quantitative detection of zearalenone.

9. The application according to claim 8, characterized in that, The quantitative detection steps are as follows: the initial fluorescence value of the fluorescent molecularly imprinted polymer solution is measured using a fluorescence spectrophotometer, the sample to be tested is added, the fluorescence change rate is measured, and a standard curve is established, thereby performing quantitative analysis of zearalenone.

10. The application according to claim 9, characterized in that, The concentration of the fluorescent molecularly imprinted polymer solution is 0.01-0.5 mg / mL, and the solvent is anhydrous ethanol. After adding the sample to be tested for 1-15 min, the fluorescence change rate is measured and a standard curve is established.

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