A fluorescent molecularly imprinted polymer based on biomass red-emitting carbon dots and a preparation method and application thereof

Abstract

This invention discloses a fluorescent molecularly imprinted polymer based on biomass red-emitting carbon dots, its preparation method, and its applications, relating to the field of analytical chemistry. The preparation method of the fluorescent molecularly imprinted polymer based on biomass red-emitting carbon dots includes: preparing biomass red-emitting carbon dots with emission wavelengths above 650 nm using mulberry leaves under optimized conditions; then adding these carbon dots to a pre-assembled solution of template molecules and functional monomers; finally adding a crosslinking agent to carry out a polymerization reaction; after polymerization, removing the template molecules using an eluent to obtain a near-infrared fluorescent molecularly imprinted polymer capable of specifically recognizing the target ochratoxin A molecule. Based on the fluorescence intensity response of the near-infrared fluorescent molecularly imprinted polymer to ochratoxin A, this invention establishes a rapid quantitative analysis method with high sensitivity, strong specificity, resistance to matrix interference, and low cost, providing a new fluorescent molecular recognition material for the development of rapid on-site detection methods for mycotoxins.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to a fluorescent molecularly imprinted polymer based on red-emitting carbon dots from biomass, its preparation method, and its application. Background Technology

[0002] Carbon dots (CDs) were discovered in single-walled carbon nanotubes in 2004 and have attracted widespread attention due to their superior advantages over traditional quantum dots. They possess advantages such as simple preparation, strong fluorescence, good biocompatibility, low cost, low toxicity, and convenient surface functionalization. As optical probes or sensor elements, CDs can be used to detect various target analytes, including environmental pollutants, drugs, and metal ions. However, fluorescent interfering substances in the sample matrix can interfere with the sensitivity and accuracy of CDs in detecting target analytes. Since most carbon dots have relatively short emission wavelengths and are greatly affected by the sample matrix, developing near-infrared fluorescent carbon dots can greatly avoid interference from sample substrate fluorescence and improve the sensitivity of detection methods. Furthermore, to improve the selectivity of CD-based probes or sensors, CDs can be combined with molecular imprinting technology as fluorescent indicators to prepare novel fluorescent molecular recognition materials. These materials can utilize the fluorescence properties of near-infrared CDs to perform highly sensitive and accurate quantitative analysis of target analytes while retaining the high selectivity and specificity of molecularly imprinted polymers.

[0003] Molecular imprinting is a technique for preparing functional polymers based on template molecules. It involves the covalent or non-covalent binding of a target analyte (template molecule) with a functional monomer to form a template-monomer complex. After the addition of a cross-linking agent, the monomer is immobilized around the template molecule under the action of an initiator, forming a highly cross-linked, rigid polymer with an imprinted cavity. Removing the template molecule yields the molecularly imprinted polymer. Compared to antibodies, molecularly imprinted polymers not only possess the specific recognition capabilities of antibodies but also offer advantages such as lower cost, simpler preparation methods, greater stability, and easier transportation. They have already been widely researched and applied in fields such as separation science, sensor analysis, drug delivery, and enzyme catalysis simulation.

[0004] Ochratoxin A (OTA) is a toxic metabolite produced by Penicillium and Aspergillus fungi, widely found in foods such as grains, coffee, grape juice, wine, soybeans, milk, and nuts. OTA has high chemical stability and a long half-life, affects the immune function of organs such as the liver, kidneys, and thymus, and is carcinogenic, posing a serious threat to human health. Currently, the main methods for detecting OTA are enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). ELISA is relatively cumbersome, requires expensive antibodies, and is prone to false positives. While HPLC has high sensitivity, it requires a lengthy sample processing time, relies on expensive and large-scale instruments, and demands highly skilled operators, making rapid on-site detection difficult. Therefore, developing a simple, low-cost, and highly sensitive rapid detection method for OTA is essential. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a fluorescent molecularly imprinted polymer based on biomass red light emitting carbon dots, its preparation method and application.

[0006] This invention focuses on ochratoxin A (OTA) and investigates the preparation and application of near-infrared fluorescent molecularly imprinted polymers based on biomass red-emitting carbon dots. To develop a sensitive and rapid method for on-site detection of OTA, this invention first synthesizes near-infrared emitting biomass red-emitting carbon dots and uses them as fluorescent indicators. These carbon dots are then combined with a molecularly imprinted polymer via sol-gel polymerization to prepare a fluorescent molecularly imprinted polymer for the detection of OTA in real samples.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing a fluorescent molecularly imprinted polymer based on biomass red-emitting carbon dots, the preparation method comprising the following steps: Mulberry leaf powder was dissolved in anhydrous ethanol to obtain a mulberry leaf solution; the mulberry leaf solution was heated at 110~130℃ for 4~8h, and after the reaction was completed, it was cooled, centrifuged, and the upper liquid was filtered through a membrane to obtain a biomass red light emitting carbon dot stock solution; the biomass red light emitting carbon dot stock solution was diluted with anhydrous ethanol to obtain a biomass red light emitting carbon dot ethanol solution. The solvent and the pseudo template molecule of the analyte are mixed evenly, the functional monomer is added and mixed evenly, the biomass red light emitting carbon dot ethanol solution is added and mixed evenly, and finally the crosslinking agent is added to carry out the light-shielded reaction. After the reaction was completed, the precipitate was obtained by centrifugation. The precipitate was washed, centrifuged, and the sample was collected to obtain the fluorescent molecularly imprinted polymer.

[0008] Preferably, the preparation method of the biomass red light emitting carbon dot stock solution specifically includes: Mulberry leaf powder was dissolved in anhydrous ethanol to obtain a mulberry leaf solution; the mulberry leaf solution was heated at 120°C for 6 hours, and after the reaction was completed, it was cooled, centrifuged, and the upper liquid was filtered to obtain biomass red light emitting carbon dots.

[0009] Preferably, the analyte to be detected includes ochratoxin A, and the pseudo-template molecule includes N-fluorenylmethoxycarbonyl-D-phenylalanine.

[0010] Preferably, the functional monomers are 3-aminopropyltriethoxysilane and phenyltrimethoxysilane; The crosslinking agent is tetraethoxysilane; The solvent is a water / ethanol co-solvent, wherein the volume ratio of water to ethanol is 1:1.5~2.5.

[0011] Preferably, the molar ratio of the pseudo-template molecule to 3-aminopropyltriethoxysilane and phenyltrimethoxysilane is 1:1.5~2.5:3.5~4.5; The ratio of the pseudotemplate molecule to the biomass red light emitting carbon dot ethanol solution is 0.1 mmol: 2~4 mL.

[0012] Preferably, the concentration of the biomass red light emitting carbon dot ethanol solution is 1 / 10 to 1 / 100 of the concentration of the biomass red light emitting carbon dot stock solution; more preferably, it is 1 / 40 to 1 / 60.

[0013] Preferably, a method for preparing a fluorescently imprinted polymer for detecting OTA based on biomass red light emission carbon dots includes the following steps: Step 1. Preparation of red-emitting carbon dots from biomass: Weigh out dried mulberry leaf powder and dissolve it in anhydrous ethanol. Stir and mix overnight. Then place it in a reaction vessel and heat at 120℃ for 6 hours. After the reaction is complete, let it cool to room temperature, centrifuge and take the upper liquid. Filter it through a 0.22μm organic filter membrane and collect the sample. Store it at 4℃ in the dark. Step 2. Preparation of fluorescent molecularly imprinted polymers based on biomass red-emitting carbon dots: First, a certain amount of water / ethanol co-solvent was added to a round-bottom flask. Then, OTA pseudo-template molecules were added, and the mixture was ultrasonically assisted to ensure uniform dispersion. After this, the functional monomer was added, and the mixture was stirred for 1 hour. Then, an ethanol solution containing carbon dots was added, and the mixture was stirred for 30 minutes. Finally, a crosslinking agent was added, and the reaction was carried out in the dark for 48 hours. After the reaction was completed, the precipitate was obtained by centrifugation. Unreacted substances and the template were removed by elution with ethanol, methanol, and acetic acid, respectively. Finally, the sample was collected in ethanol and stored in the dark at 4°C.

[0014] As an improvement, the pseudo-template molecule of OTA in step two is N-fluorenylmethoxycarbonyl-D-phenylalanine (Fmoc-D-Phe), and the functional monomers are 3-aminopropyltriethoxysilane (APTES) and phenyltrimethoxysilane (PTMOS).

[0015] As an improvement, the concentration of pseudotemplate molecules in the reaction system of step two is 8 μmol / mL, the concentrations of functional monomers are 17 μmol / mL (APTES) and 33 μmol / mL (PTMOS), and the concentration of biomass red light emitting carbon dots is 1 / 50 of the original solution.

[0016] As an improvement, the molar ratio of pseudotemplate molecule to functional monomer in step two is Fomc-D-Phe : APTES : PTMOS = 1 : 2 : 4.

[0017] As an improvement, the crosslinking agent in the reaction system of step two is tetraethoxysilane (TEOS), and the concentration is 83 μmol / mL.

[0018] Secondly, the present invention provides a fluorescent molecularly imprinted polymer based on biomass red light emitting carbon dots, which is obtained by the preparation method described above.

[0019] Thirdly, the present invention provides the application of the fluorescent molecularly imprinted polymer in the quantitative detection of ochratoxin A in food testing.

[0020] Preferably, the quantitative detection and analysis steps are as follows: first, the initial fluorescence value of the fluorescent molecularly imprinted polymer solution is measured using a fluorescence spectrophotometer; then, the sample to be tested is added to the solution and incubated for a period of time, and the fluorescence signal value is measured; a standard curve is established based on the fluorescence enhancement rate, thereby performing quantitative analysis of OTA.

[0021] The fluorescent molecularly imprinted polymer for detecting OTA based on biomass red light emitting carbon dots described in this invention uses OTA as the research object and biomass red light emitting carbon dots as fluorescent indicators. Fluorescent molecularly imprinted polymers with OTA as templates are designed and synthesized through molecular imprinting technology. It can sensitively and rapidly identify OTA based on changes in fluorescence enhancement rate. It has the characteristics of good specificity, simple operation, low cost, and convenient real-time on-site detection.

[0022] Fourthly, the present invention provides a method for detecting ochratoxin A using the aforementioned fluorescent molecularly imprinted polymer, comprising the following steps: The fluorescently imprinted polymer was added to anhydrous ethanol to obtain a polymer solution; The initial fluorescence value of the polymer solution was determined using a fluorescence spectrophotometer; The sample to be tested is added dropwise to the polymer solution, incubated, and the fluorescence signal value of the sample to be tested is measured. The fluorescence change rate of the sample to be tested is calculated. The concentration of the sample is obtained by substituting the fluorescence change rate of the sample into the standard curve.

[0023] Preferably, the concentration of the polymer solution is 0.05~0.1 mg / mL; The excitation wavelength used in the fluorescence spectrophotometer was 405 nm, and the emission wavelength was 673 nm. The incubation time is 3 to 10 minutes.

[0024] This invention has at least one of the following beneficial effects: This invention provides a fluorescent molecularly imprinted polymer based on biomass red-emitting carbon dots, its preparation method, and its application. This polymer can be used to detect OTA (anti-adrenergic oxidase). Compared with existing technologies, this invention has the following advantages: 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.

[0025] 2. The precursor for the synthesis of carbon dots in this invention is mulberry leaf, a biomass material, which meets the requirements of green and environmentally friendly practices.

[0026] 3. Compared with carbon dots prepared using tobacco leaves and sycamore leaves as precursors, the biomass red light emitting carbon dots prepared using mulberry leaves as precursors in this invention have a higher emission wavelength, which is located in the near-infrared region. This can filter out the influence of most impurities in the matrix and has stronger anti-interference ability.

[0027] 4. The fluorescent molecularly imprinted polymer for rapid detection of OTA prepared in this invention can sensitively and rapidly identify OTA based on changes in fluorescence enhancement rate, and achieve accurate quantification of it.

[0028] 5. The fluorescent molecularly imprinted polymer prepared in this invention has strong specificity for OTA.

[0029] 6. This detection method is simple to operate, requires little reagent, and has 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

[0030] Figure 1 A schematic diagram of the synthesis process and detection principle of biomass red light emitting carbon dots (a), fluorescent molecularly imprinted polymers (b); Figure 2 Fluorescence spectrum (a) and particle size distribution (b) of red-emitting carbon dots in biomass; Figure 3The fluorescence spectrum of rice matrix ethanol extract (a) and the fluorescence response changes after rice matrix ethanol extract was added to (b) carbon dots of sycamore leaves, (c) carbon dots of tobacco leaves and (d) carbon dots of biomass red light emitting light. Figure 4 Scanning electron microscope images of RFM2(a) and RFN2(b), and DLS images of RFM2(c) and RFN2(d); Figure 5 Selectivity results of OTA fluorescently imprinted polymers synthesized with different amounts of carbon dots; Figure 6 The graph shows the selectivity results of polymers with different concentrations of OTA fluorescent molecular imprints. Figure 7 The figure shows the kinetic test results of the OTA fluorescently imprinted polymer. Figure 8 The figure shows the specificity test results of the OTA fluorescently imprinted polymer. Figure 9 Thermodynamic test results of OTA fluorescent molecularly imprinted polymer are shown in the figure. Figure 10 The standard curve represents the linear detection range of OTA fluorescently imprinted polymers. Detailed Implementation

[0031] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0032] Example 1 This embodiment provides a method for preparing biomass red-emitting carbon dots (RCDs). The synthetic route for preparing biomass red-emitting carbon dots is as follows: Figure 1 As shown in (a): Weigh 2.0 g of dried mulberry leaf powder and dissolve it in 20 mL of anhydrous ethanol. Stir and mix overnight. Then 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 and 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 to obtain biomass red light emitting carbon dot stock solution (RCD).

[0033] Figure 2 (a) is the fluorescence spectrum of the synthesized carbon dots, which shows that the maximum excitation wavelength of the synthesized biomass red-emitting carbon dots is 427 nm and the maximum emission wavelength is 675 nm, which is in the red light region. This is a wavelength region that most impurities cannot reach, and their influence can be filtered out. Figure 2(b) The particle size distribution of the carbon dots was statistically analyzed using transmission electron microscopy (TEM) images. The calculated average particle size is approximately 2.45 nm. In addition, the fluorescence quantum yield of the obtained carbon dots is approximately 8.39% (using Rhodamine 6G as the standard), which meets our requirements for fluorescent indicators.

[0034] Comparative Example 1 The types of raw materials for the preparation of biomass red-emitting carbon dots (RCDs) were optimized, and the optimization methods are as follows: 1. In Example 1, “A method for preparing biomass red light emitting carbon dots (RCD),” the precursor is replaced with “tobacco powder”, and everything else is the same as in Example 1.

[0035] 2. In Example 1, “A method for preparing biomass red light emitting carbon dots (RCD),” the precursor is replaced with “mulberry leaf powder”, and everything else is the same as in Example 1.

[0036] The fluorescence spectra of the carbon dots prepared in Example 1 and Comparative Example 1 in the ethanol extract of rice matrix were measured using the following method: The carbon dots prepared from tobacco leaves and sycamore leaves were named tobacco carbon dots and sycamore leaf carbon dots, respectively. In addition, we prepared an ethanol extract of the rice matrix, the specific steps of which are as follows: accurately weigh 4 g of rice sample and mix with 10 mL of ethanol, shake overnight; after natural sedimentation, take the supernatant and filter it through a 0.22 μm filter membrane.

[0037] like Figure 3 As shown, (a) is the fluorescence spectrum of the ethanol extract of the rice matrix from the grain sample, and (b), (c), and (d) are the fluorescence responses of tobacco leaf carbon dots (Comparative Example 1), sycamore leaf carbon dots (Comparative Example 2), and biomass red-emitting carbon dots (Example 1) after the addition of the rice matrix ethanol extract, respectively. First, comparing the emission spectra of the three carbon dots reveals that the biomass red-emitting carbon dots prepared in Example 1 have the largest emission wavelength. Therefore, after adding the fluorescent rice matrix ethanol extract, its fluorescence response changes the least. This is because the fluorescence emission wavelength of the rice matrix ethanol extract is only 409 nm, which is significantly different from the maximum emission wavelength of the biomass red-emitting carbon dots prepared in Example 1. The fluorescence of the sycamore leaf and tobacco leaf carbon dots, with their smaller emission wavelengths, is more significantly affected by this. In conclusion, it can be demonstrated that the biomass red-emitting carbon dots prepared with "mulberry leaf powder" in Example 1 can avoid fluorescence interference from most impurities in the grain matrix, and its anti-interference performance is superior to that of common green and blue carbon dots.

[0038] Comparative Example 2 The reaction temperature and time during carbon dot preparation affect the degree of carbonization of the precursor, which in turn affects its fluorescence spectrum and fluorescence quantum yield. Therefore, we optimized the temperature and time during carbon dot preparation, setting three temperatures of 120℃, 150℃, and 180℃, and three reaction times of 4 h, 6 h, and 8 h. By combining these two conditions one-to-one, nine types of carbon dots were obtained. The optimal preparation conditions were determined based on their fluorescence spectrum and fluorescence quantum yield, and the specific optimization method is as follows: 1. In Example 1, “A method for preparing biomass red light emitting carbon dots (RCD),” “heating at 120°C for 6 hours” is changed to “heating at 120°C for 4 hours”, and the rest is the same as in Example 1.

[0039] 2. In Example 1, “A method for preparing biomass red light emitting carbon dots (RCD),” “heating at 120°C for 6 hours” is changed to “heating at 120°C for 8 hours”, and the rest is the same as in Example 1.

[0040] 3. In Example 1, “A method for preparing biomass red light emitting carbon dots (RCD),” “heating at 120°C for 6 hours” is changed to “heating at 150°C for 4 hours”, and the rest is the same as in Example 1.

[0041] 4. In Example 1, “A method for preparing biomass red light emitting carbon dots (RCD),” “heating at 120°C for 6 hours” is changed to “heating at 150°C for 6 hours”, and the rest is the same as in Example 1.

[0042] 5. In Example 1, “A method for preparing biomass red light emitting carbon dots (RCD),” “heating at 120°C for 6 hours” is changed to “heating at 150°C for 8 hours”, and the rest is the same as in Example 1.

[0043] 6. In Example 1, “A method for preparing biomass red light emitting carbon dots (RCD),” “heating at 120°C for 6 hours” is changed to “heating at 180°C for 4 hours”, and the rest is the same as in Example 1.

[0044] 7. In Example 1, “A method for preparing biomass red light emitting carbon dots (RCD),” “heating at 120°C for 6 hours” is changed to “heating at 180°C for 6 hours”, and the rest is the same as in Example 1.

[0045] 8. In Example 1, “A method for preparing biomass red light emitting carbon dots (RCD),” “heating at 120°C for 6 hours” is changed to “heating at 180°C for 8 hours”, and the rest is the same as in Example 1.

[0046] The maximum emission wavelength, quantum yield, particle size, and PI value of the carbon dots prepared in Example 1 and Comparative Example 1 were determined, and the results are shown in Table 1.

[0047] Table 1 shows the maximum emission wavelengths of mulberry leaf carbon dots prepared at different reaction temperatures and times. A comparison reveals that the nine carbon dots show very little difference in fluorescence spectra, with maximum emission wavelengths all around 675 nm, differing by only a few nanometers. Table 1 also shows the fluorescence quantum yield test results for the nine carbon dots. The group at 120℃ and 6 h had the highest fluorescence quantum yield at 8.39%, while the three groups at 180℃ had the lowest, possibly due to excessive carbonization caused by the high temperature. In addition, we measured the particle size of the synthesized carbon dots using dynamic light scattering and summarized it in Table 1. A lower PI value indicates a more accurate detection result. The table shows that the three groups at 120℃ have a relatively uniform particle size distribution, low PI value, and small particle size; the three groups at 150℃ have less uniform particle size distribution and larger particle size, except for the group at 150℃ and 6 h, which has a smaller particle size; the three groups at 180℃ have uneven particle size distribution and large particle size, presumably due to carbon dot aggregation caused by excessive temperature. After comprehensive consideration, we finally chose 120℃ for 6 h as the reaction temperature and time for the subsequent mulberry leaf carbon dots, and named the carbon dots red light emitting carbon dots.

[0048] Table 1. Maximum emission wavelength, quantum yield, particle size, and PI value of carbon dots from mulberry leaves under different reaction conditions. Example 2 This embodiment provides a method for preparing molecularly imprinted fluorescent nanoparticles (fluorescent molecularly imprinted polymer RFM). The specific synthetic route for preparing molecularly imprinted fluorescent nanoparticles is as follows: Figure 1 As shown in (b): First, a certain amount of water / ethanol co-solvent was added to a 25 mL round-bottom flask (the final volume of water was 4 mL and the volume of ethanol was 8 mL). Then, 0.1 mmol (0.0388 g) of OTA pseudotemplate molecule (FD-Phe) was added, and the mixture was sonicated to aid mixing. After uniform dispersion, 0.2 mmol (46.8 μL) of APTES and 0.4 mmol (97.2 μL) of PTMOS were added. After stirring for 1 h, 3 mL of LRCD ethanol solution was added (i.e., the biomass red light emitting carbon dot stock solution prepared in Example 1 was diluted with anhydrous ethanol at dilution ratios of 1 / 10, 1 / 50, and 1 / 100 of the carbon dot stock solution, pH=9.5). The mixture was stirred for 30 min, and finally, 1 mmol (223.29 μL) of TEOS was added and the reaction was carried out in the dark for 48 h.

[0049] After the reaction was completed, the precipitate was obtained by centrifugation, and unreacted substances and template were removed by elution with ethanol and methanol / acetic acid, respectively, to obtain the fluorescent molecularly imprinted polymers RFM of OTA, which were named RFM1 (RCD ethanol solution concentration was 1 / 10 of the carbon dot stock solution), RFM2 (RCD ethanol solution concentration was 1 / 50 of the carbon dot stock solution), and RFM3 (RCD ethanol solution concentration was 1 / 100 of the carbon dot stock solution), respectively. The samples were collected in ethanol and stored in the dark at 4°C.

[0050] Comparative Example 3 Non-molecularly imprinted fluorescent nanoparticles (RFNs) were prepared as a control. Except for omitting the template (OTA pseudo-template molecule), the preparation was carried out in accordance with Example 2. Non-molecularly imprinted fluorescent nanoparticles (RFNs) were generated under the same conditions as the nanoparticles and named RFN1 (RCD ethanol solution concentration was 1 / 10 of the original solution), RFN2 (RCD ethanol solution concentration was 1 / 50 of the original solution), and RFN3 (RCD ethanol solution concentration was 1 / 100 of the original solution).

[0051] The nanoparticles prepared above were measured, and the results are as follows: Figure 4 (a) and (b) are scanning electron microscope images of RFM2 and RFN2, respectively. Figure 4 (c) and (d) are DLS plots of RFM2 and RFN2, respectively. The results show that the particle size of RFM2 is much smaller than that of RFN2. Although the numerical results differ from those of DLS, the trend is consistent and the two can be mutually verified.

[0052] The amount of carbon dots added affects the selectivity of the synthesized fluorescently imprinted polymers. Therefore, we optimized the amount of RCD added to the synthesized fluorescently imprinted polymers, setting the RCD system concentrations to 1 / 10 (RFM1 & RFN1), 1 / 50 (RFM2 & RFN2), and 1 / 100 (RFM3 & RFN3) of the original solution, and then performed selectivity tests on them. Figure 5 As shown, all RFMs exhibit selectivity, with RFM2 showing the best selectivity and an imprinting factor (IF) of around 4. Furthermore, the non-specific adsorption of RFN in this test system is minimal, which is more conducive to template recognition. Therefore, the amount of carbon dots added in subsequent RFM / RFN synthesis is 1 / 50 of the original solution, i.e., RFM2 and RFN2.

[0053] In the following text, RFM stands for RFM2 and RFN stands for RFN2.

[0054] Example 3 The concentration optimization experiment was carried out on the OTA molecularly imprinted fluorescent nanoparticles prepared in Example 2.

[0055] RFM was dissolved in anhydrous ethanol to obtain polymer solutions at concentrations of 1, 0.5, 0.1, and 0.05 mg / mL. 2 mL of each polymer solution was added to a fluorescence cuvette, and the mixture was stirred until the fluorescence value stabilized. The initial fluorescence signal F0 (excitation wavelength 405 nm, emission wavelength 673 nm) was measured using a fluorescence spectrophotometer. 20 μL of OTA ethanol solution was added to the fluorescence cuvette to achieve a system concentration of 1 μg / mL. After stirring for 10 min, F was measured, and the fluorescence change rate ΔF was calculated. M The fluorescence change rate ΔF of RFN was measured using the same method. N Calculate the imprint factor (IF). Where ΔF = ((F-F0) / F0), IF = ΔF M / ΔF N .

[0056] The concentration of the polymer affects the fluorescence response to the target substance, according to... Figure 6 The results showed that lower RFM concentrations exhibited better selectivity, and the fluorescence change rate of RFN was very small at each concentration, resulting in a high imprinting factor, reaching 29 at a concentration of 0.05 mg / mL. Further reducing the concentration might yield even better results, but the fluorescence value of FMIP was already low at 0.05 mg / mL (around 2000). Further reducing the concentration could easily lead to unstable polymer fluorescence values; therefore, a polymer concentration of 0.05 mg / mL was used for subsequent experiments.

[0057] Example 4 Kinetic tests were performed on the OTA molecularly imprinted fluorescent nanoparticles prepared in Example 2.

[0058] RFM was dissolved in anhydrous ethanol to a concentration of 0.05 mg / mL to obtain a polymer solution. 2 mL of the polymer solution was added to a fluorescence cuvette and stirred until the fluorescence value stabilized. The initial fluorescence signal F0 (excitation wavelength 405 nm, emission wavelength 673 nm) was measured using a fluorescence spectrophotometer. 20 μL of OTA ethanol solution was added to the fluorescence cuvette to a concentration of 1 μg / mL. After stirring for 1, 3, 5, 10, and 15 min, F was measured, and the fluorescence change rate ΔF was calculated. M The fluorescence change rate ΔF of RFN was measured using the same method. N .

[0059] Depend on Figure 7It can be seen that after the addition of OTA, the fluorescence intensity of RFM increases over time, and the adsorption of OTA is almost completed within 3 minutes, reaching its peak at 10 minutes. This is longer than the response time ΔF of most fluorescently imprinted polymers. M To keep it short, considering the time spent on testing, we ultimately chose 5 minutes as the subsequent testing time.

[0060] Example 5 The specificity of the OTA molecularly imprinted fluorescent nanoparticles prepared in Example 2 was tested.

[0061] RFM was dissolved in anhydrous ethanol to a concentration of 0.05 mg / mL to obtain a polymer solution. 2 mL of the polymer solution was added to a fluorescence cuvette and stirred until the fluorescence value stabilized. The initial fluorescence signal F0 (excitation wavelength 405 nm, emission wavelength 673 nm) was measured using a fluorescence spectrophotometer. 20 μL of ethanol solutions of the biotoxins OTA, ochratoxin B (OTB), aflatoxin B1 (AFB1), aflatoxin G2 (AFG2), fumonisin B1 (FB1), zearalenone (ZEN), and vomitoxin (DON) were added to the fluorescence cuvettes to a concentration of 1 μg / mL. After stirring for 5 min, F was measured, and the fluorescence change rate ΔF was calculated. M The fluorescence change rate ΔF of RFN was measured using the same method. N .

[0062] like Figure 8 As shown, we selected six biotoxins—OTB, AFB1, AFG2, FB1, ZEN, and DON—and OTA at the same concentration (1 μg / mL) under the same conditions for fluorescence response testing. It is evident that RFM exhibits selective efficacy only towards OTA, and displays a strong fluorescence response. Notably, this polymer can accurately distinguish between OTA and OTB, a capability not found in previous polymers. Therefore, we conducted repeatability tests on OTB, and the results showed that RFM's response to OTB remained minimal, ensuring its accuracy and repeatability.

[0063] Example 6 Thermodynamic tests were performed on the OTA molecularly imprinted fluorescent nanoparticles prepared in Example 2.

[0064] RFM was dissolved in anhydrous ethanol to a concentration of 0.05 mg / mL to obtain a polymer solution. 2 mL of the polymer solution was added to a fluorescence cuvette and stirred until the fluorescence value stabilized. The initial fluorescence signal F0 (excitation wavelength 405 nm, emission wavelength 673 nm) was measured using a fluorescence spectrophotometer. Different grades of OTA stock solution were added to fluorescence cuvettes to achieve system concentrations of 0.01, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, and 5 μg / mL. After stirring for 5 min, F was measured, and the fluorescence change rate ΔF was calculated. M The fluorescence change rate ΔF of RFN was measured using the same method. N .

[0065] like Figure 9 As shown, the fluorescence intensity of RFM gradually increases with the increase of the added OTA concentration, and the response range can be from 0.01 μg / mL to 5 μg / mL. Judging from its growth trend, RFM can adsorb higher concentrations of OTA and thus produce a stronger fluorescence response. However, considering the low concentration requirement of the target substance for detection, the test concentration of OTA is not increased. On the other hand, the fluorescence change of RFN is relatively stable, and in most cases, the fluorescence is quenched, which shows the good selectivity of the polymer.

[0066] Linear detection range and detection limit are important indicators for evaluating a detection method, such as Figure 10 As shown, the linear detection range of this sensor is between 0.05 and 2.5 μg / mL. We performed simple data processing and obtained the fitting equation: y = 0.359x + 0.016 (R²). 2 =0.98); at the same time, its limit of detection can reach 10 ng / mL, which shows good sensitivity.

[0067] Example 7 The OTA molecularly imprinted fluorescent nanoparticles prepared in Example 2 were used for the detection of actual samples (corn, rice, wheat).

[0068] Sample pretreatment: Weigh 5g of corn sample, rice, and wheat powder into 50mL centrifuge tubes, add a certain amount of OTA methanol solution (OTA spiked concentrations of 0.1, 0.5, and 1μg / g, respectively), mix well, and then place them in a fume hood to air dry naturally for later use. Take the dried sample, add 0.5g of sodium chloride and 25mL of a mixed solution of methanol and water, homogenize, and then sonicate for 30min to extract OTA. Then filter the sample with rapid qualitative filter paper to obtain the extract, and then filter the extract with a 0.22μm organic filter membrane. The obtained filtrate is stored in a refrigerator for RFM recovery testing.

[0069] Recovery rate determination: RFM was placed in anhydrous ethanol to a concentration of 0.05 mg / mL to obtain a polymer solution; 2 mL of the polymer solution was added to a fluorescence cuvette and stirred until the fluorescence value stabilized. The initial fluorescence signal F0 (excitation wavelength 405 nm, emission wavelength 673 nm) was measured using a fluorescence spectrophotometer; 20 μL of OTA ethanol solution of a certain concentration was added to a fluorescence cuvette and stirred for 5 min. F was measured and the fluorescence change rate was calculated; the same method was used to test the OTA-containing sample solution of the same concentration and calculate the fluorescence change rate to obtain the recovery rate of OTA in the sample. The results are shown in Table 2.

[0070] Table 2 Spiked recovery test of OTA in real grain samples As shown in Table 2, the spiked recoveries of this method on real samples were in the range of 80% to 116%, which demonstrates the reliability and accuracy of the method.

[0071] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a fluorescent molecularly imprinted polymer based on biomass red-emitting carbon dots, characterized in that, The preparation method includes the following steps: Mulberry leaf powder was dissolved in anhydrous ethanol to obtain a mulberry leaf solution; the mulberry leaf solution was heated at 110~130℃ for 4~8h, and after the reaction was completed, it was cooled, centrifuged, and the upper liquid was filtered through a membrane to obtain a biomass red light emitting carbon dot stock solution; the biomass red light emitting carbon dot stock solution was diluted with anhydrous ethanol to obtain a biomass red light emitting carbon dot ethanol solution. The solvent and the pseudo template molecule of the analyte are mixed evenly, the functional monomer is added and mixed evenly, the biomass red light emitting carbon dot ethanol solution is added and mixed evenly, and finally the crosslinking agent is added to carry out the light-shielded reaction. After the reaction was completed, the precipitate was obtained by centrifugation. The precipitate was washed, centrifuged, and the sample was collected to obtain the fluorescent molecularly imprinted polymer.

2. The preparation method according to claim 1, characterized in that, The preparation method of the biomass red light emitting carbon dot stock solution specifically includes: Mulberry leaf powder was dissolved in anhydrous ethanol to obtain a mulberry leaf solution; the mulberry leaf solution was heated at 120°C for 6 hours, and after the reaction was completed, it was cooled, centrifuged, and the upper liquid was filtered to obtain biomass red light emitting carbon dots.

3. The preparation method according to claim 1, characterized in that, The analyte to be detected includes ochratoxin A, and the pseudo-template molecule includes N-fluorenylmethoxycarbonyl-D-phenylalanine.

4. The preparation method according to claim 1, characterized in that, The functional monomers are 3-aminopropyltriethoxysilane and phenyltrimethoxysilane; The crosslinking agent is tetraethoxysilane; The solvent is a water / ethanol co-solvent, wherein the volume ratio of water to ethanol is 1:1.5~2.

5.

5. The preparation method according to claim 4, characterized in that, The molar ratio of the pseudo-template molecule to 3-aminopropyltriethoxysilane and phenyltrimethoxysilane is 1:1.5~2.5:3.5~4.5; The ratio of the pseudotemplate molecule to the biomass red light emitting carbon dot ethanol solution is 0.1 mmol: 2~4 mL.

6. The preparation method according to claim 1, characterized in that, The concentration of the biomass red light emitting carbon dot ethanol solution is 1 / 10 to 1 / 100 of the concentration of the biomass red light emitting carbon dot stock solution.

7. A fluorescent molecularly imprinted polymer based on biomass red-emitting carbon dots, characterized in that, It is obtained by the preparation method described in any one of claims 1 to 6.

8. The application of the fluorescent molecularly imprinted polymer of claim 7 in the quantitative detection of ochratoxin A in food testing.

9. A method for detecting ochratoxin A using the fluorescent molecularly imprinted polymer of claim 7, characterized in that, Includes the following steps: The fluorescently imprinted polymer was added to anhydrous ethanol to obtain a polymer solution; The initial fluorescence value of the polymer solution was determined using a fluorescence spectrophotometer; The sample to be tested is added dropwise to the polymer solution, incubated, and the fluorescence signal value of the sample to be tested is measured. The fluorescence change rate of the sample to be tested is calculated. The concentration of the sample is obtained by substituting the fluorescence change rate of the sample into the standard curve.

10. The method according to claim 9, characterized in that, The concentration of the polymer solution is 0.05~0.1 mg / mL; The excitation wavelength used in the fluorescence spectrophotometer was 405 nm, and the emission wavelength was 673 nm. The incubation time is 3 to 10 minutes.

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