Preparation of bifunctional imprinting fluorescent probe and sensitive detection of marine toxins and antiviral drugs

Abstract

The application belongs to the field of analysis and detection, and particularly relates to preparation of a bifunctional imprinting fluorescent probe and sensitive detection of marine biological toxins and antiviral drugs. The application adopts valganciclovir hydrochloride (VAL) similar in structure to saxitoxin (STX) as a pseudo-template, avoids the high toxicity of saxitoxin (STX) and the risk of template leakage, adopts a primary-secondary functional monomer synergistic strategy to construct a high-specificity recognition site, combines a fluorescent dye occupation-competition release mechanism to realize fluorescence signal transduction, and prepares an imprinting polymer fluorescent probe which has high selectivity, high sensitivity and fast response speed for STX and VAL, and can simultaneously adapt to the detection requirements of marine environmental samples and clinical biological samples, and still maintains excellent recovery rate and stability in a complex matrix.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection, specifically relating to the preparation of a bifunctional blotted fluorescent probe and its application in the sensitive detection of marine biological toxins and antiviral drugs. Background Technology

[0002] Stomatoxin (STX) is the most potent analogue of paralytic shellfish toxins. Against the backdrop of global climate change and increasing coastal eutrophication, frequent harmful algal blooms lead to the bioaccumulation of STX in shellfish and other seafood. Ingestion can block voltage-gated sodium channels in human nerve cells, causing respiratory paralysis and even death, seriously threatening seafood safety and public health. Valganciclovir hydrochloride (VAL) is a commonly used anti-cytomegalovirus drug in clinical practice. Its efficacy and toxicity are strongly dose-dependent; therapeutic drug monitoring (TDM) is crucial for ensuring medication safety in transplant recipients and immunocompromised individuals.

[0003] Currently, the mainstream detection methods for STX are mouse bioassay and liquid chromatography-tandem mass spectrometry (LC-MS / MS). The former has ethical concerns and insufficient sensitivity, while the latter relies on expensive instruments and requires complex pre-column derivatization, failing to meet the needs of rapid on-site screening. Alternative solutions such as immunoassay and aptamer sensing excel in rapid detection, but their lack of cross-reactivity, matrix effects, and standardization leads to insufficient stability and reproducibility. Clinical quantification of VAL largely relies on chromatographic mass spectrometry platforms, which involve cumbersome pretreatment and long detection cycles, making rapid clinical monitoring difficult. Rapid sensing technologies that can directly achieve fluorescence readout have significant limitations in clinically deployable detection areas.

[0004] Molecularly imprinted polymers (MIPs), hailed as "artificial antibodies," possess advantages such as strong specificity, high stability, and low preparation cost, and have been widely used for the enrichment and detection of targets in complex matrices. However, using highly toxic STX as a direct template for imprinting presents challenges such as expensive raw materials, high operational risks, and the potential for false positives due to template leakage. Therefore, this application employs valganciclovir hydrochloride (VAL), structurally similar to STX, as a pseudo-template to mitigate these risks. Furthermore, existing imprinting materials are mostly designed for single-target detection, with few dual-functional systems suitable for both marine environmental toxin monitoring and clinical drug detection. Moreover, for STX, which lacks natural fluorescence, achieving integrated rapid detection of both recognition and signal transduction is difficult. Therefore, developing a low-toxicity, safe, dual-functional, highly sensitive, and interference-resistant molecularly imprinted fluorescent probe has significant application value. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing a bifunctional blotted fluorescent probe and its application in the sensitive detection of marine biotoxins and antiviral drugs. It possesses specific recognition capabilities for the marine toxin STX and the antiviral drug VAL, enabling sensitive detection.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides a method for preparing a bifunctional blotted fluorescent probe, the method comprising:

[0008] S1. A prepolymer is obtained by reacting a mixture of pseudo-template molecules, main functional monomers, auxiliary functional monomers, initiators and porogens.

[0009] S2. The prepolymer is reacted with a crosslinking agent to obtain a polymer product;

[0010] S3. Purify the polymerization product until pseudo-template molecules are undetectable, dry it, and obtain molecularly imprinted polymer (MIP) solid particles.

[0011] S4. The molecularly imprinted polymer (MIP) solid particles are dispersed and then mixed with a fluorescent dye solution to obtain a bifunctional imprinted fluorescent probe.

[0012] In some embodiments, in S1, the pseudo-template molecule includes valganciclovir hydrochloride.

[0013]

[0014] In some embodiments, in S1, the primary functional monomer includes 2-acrylamide-2-methylpropanesulfonic acid, and the secondary functional monomer includes sodium methpropanesulfonate;

[0015] In some embodiments, in S1, the porogen is selected from one or more of methanol, acetonitrile, and a methanol-acetonitrile mixture;

[0016] In some embodiments, in S1, the initiator is selected from one or more of dimethyl azobisisobutyrate or azobisisobutyronitrile;

[0017] In some embodiments, in S1, the molar amount of the pseudo-template molecule is 1 part, the molar amount of the main functional monomer is 4 to 8 parts, the molar amount of the auxiliary functional monomer is 1 to 4 parts, and the molar amount of the initiator is 5 to 9 parts.

[0018] In some embodiments, the method of obtaining a prepolymer by reacting the mixed pseudo-template molecules, main and auxiliary functional monomers, initiator and porogen in S1 includes: mixing the pseudo-template molecules, main and auxiliary functional monomers, initiator and porogen and then sonicating for 5-10 min; and letting stand for 30-60 min.

[0019] In some embodiments, in S2, the crosslinking agent is selected from one or more of trimethylolpropane trimethacrylate or ethylene glycol dimethacrylate;

[0020] In some embodiments, in S2, the molar ratio of the pseudo-template molecule to the crosslinking agent is 1:(16~20).

[0021] In some embodiments, in S2, the method for reacting the prepolymer with the crosslinking agent to obtain the polymer product includes: reacting the prepolymer with the crosslinking agent in a closed, nitrogen-protected environment with water bath heating; the water bath heating temperature is 50-70°C, and the reaction time is 8-24 hours.

[0022] In some embodiments, in step S3, purification includes adding methanol to the product, shaking, centrifuging, removing the supernatant, adding methanol-acetic acid (9:1, v / v) elution buffer, shaking, centrifuging, until pseudo-template molecules are undetectable.

[0023] In some embodiments, in step S4, the dispersion medium is selected from one or more of pure water, hydrochloric acid aqueous solution, and acetic acid aqueous solution; preferably, it is a 1.5% hydrochloric acid aqueous solution.

[0024] In some embodiments, in S4, the fluorescent dye is selected from one or more of acridine yellow G, rhodamine B, rhodamine 6G, fluorescein isothiocyanate (FITC), sodium fluorescein, and 2-aminopurine;

[0025] In some embodiments, in step S4, the concentration of the fluorescent dye solution is 0.0025-0.025 mg / mL;

[0026] In some embodiments, in step S4, the solid-liquid ratio when the molecularly imprinted polymer is mixed with the fluorescent dye solution is 1~5 mg / mL.

[0027] In some embodiments, in step S4, the reaction conditions for the mixed load are: stirring for 5-20 minutes at room temperature and in the dark until the fluorescent dye is saturated on the molecularly imprinted polymer, to obtain a bifunctional imprinted fluorescent probe suspension working solution.

[0028] This invention provides the application of the bifunctional blotted fluorescent probe prepared by the aforementioned method in the sensitive detection of marine biotoxins and antiviral drugs, wherein the marine biotoxin is STX, and its structural formula is [insert structural formula here].

[0029]

[0030] The antiviral drug is VAL, with the following structural formula:

[0031] .

[0032] The beneficial effects of this invention are:

[0033] 1. Using valganciclovir hydrochloride, which is structurally highly similar to STX, as a pseudo-template completely avoids the high toxicity, high cost, and false positive problems caused by template leakage of direct STX imprinting. At the same time, it realizes the simultaneous detection of VAL of clinical drugs, making one material dual-purpose and covering two major scenarios: seafood safety monitoring and clinical therapeutic drug monitoring.

[0034] 2. By adopting a master-auxiliary functional monomer synergistic strategy, with AMPS as the master monomer and SMAS as the auxiliary monomer, a specific recognition site with multi-point hydrogen bonding and electrostatic interaction is constructed, which significantly improves the imprinting factor and adsorption selectivity and effectively reduces non-specific interference from complex matrices.

[0035] 3. A fluorescent signal transduction system was constructed based on the fluorescent dye site occupancy-target competitive release mechanism, which transforms the recognition event of STX without natural fluorescence into a quantifiable fluorescent signal, realizing the integration of recognition and signal transduction. The detection process is simple, the response is fast, and the sensitivity is high. Quantitative analysis can be completed without large instruments. 4. The prepared bifunctional blotted fluorescent probe can be adapted to marine environmental samples such as high-salt seawater and shellfish extracts, as well as clinical biological samples such as serum. It maintains excellent recovery rate, repeatability and stability in complex matrices, providing a general and low-cost technical platform for rapid screening of targets in both domains. Attached Figure Description

[0036] Figure 1 Scanning electron microscope image of molecularly imprinted polymer;

[0037] Figure 2 A comparison of the adsorption effects of pseudo-template molecularly imprinted polymers obtained by using different proportions of main and auxiliary monomers in the preparation method of the bifunctional imprinted fluorescent probe provided in the embodiments of this application.

[0038] Figure 3 This is a comparison diagram of the response signals of fluorescent probes made from different fluorophores in the preparation method of the bifunctional imprinted fluorescent probe provided in the embodiments of this application;

[0039] Figure 4 Comparison of adsorption response signals of fluorescent probes made by adsorbing fluorescent dyes with MIP / NIP at different concentrations;

[0040] Figure 5 For the appendix Figure 5 This is a schematic diagram of the fluorescent probe recognition mechanism;

[0041] Figure 6The recovery rates of STX-spiked seawater samples and VAL-spiked samples from 10% mouse serum are shown in the figure.

[0042] Figure 7 A comparison chart of imprinting factors for different toxins at a certain concentration. Detailed Implementation

[0043] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the invention to the scope of the embodiments described.

[0044] As described in the background section, existing STX detection methods suffer from high instrument dependence, complex operation, and high template toxicity, leaving a technological gap in rapid clinical VAL detection. Molecularly imprinted polymers (MIPs) are a class of functional materials hailed as "artificial antibodies," possessing specific recognition capabilities. They are highly stable, easy to prepare, and inexpensive. MIPs are prepared by polymerizing functional monomers in the presence of template molecules, forming specific cavities highly complementary to the size, shape, and functional group orientation of the template molecules, thereby achieving specific recognition and detection of target molecules. Currently, MIPs are widely used for the selective enrichment and sample purification of various toxins. While traditional MIPs exhibit selectivity, they are mostly single-target designs, making it difficult to simultaneously adapt to complex marine and clinical matrices. Furthermore, they typically lack signal transduction capabilities, requiring separate quantitative steps and failing to achieve rapid signal readout for non-fluorescent toxins such as STX.

[0045] To address the aforementioned issues, this application creatively proposes a method for preparing and applying a bifunctional imprinted fluorescent probe. Using valganciclovir hydrochloride (VAL), structurally similar to salicornin (STX), as a pseudo-template, the high toxicity and template leakage risk of STX are avoided. A synergistic strategy of primary and secondary functional monomers is employed to construct highly specific recognition sites. Combined with a fluorescent dye occupancy-competitive release mechanism, fluorescent signal transduction is achieved. The prepared imprinted polymer fluorescent probe exhibits high selectivity and sensitivity for both STX and VAL, with a fast response speed. It can simultaneously meet the detection needs of marine environmental samples and clinical biological samples, maintaining excellent recovery and stability even in complex matrices.

[0046] The method for preparing the bifunctional imprinted fluorescent probe provided in this application, based on the common molecular structures of valganciclovir hydrochloride (VAL) and scutellarin (STX), and the functional group characteristics of 2-acrylamide-2-methylpropanesulfonic acid (AMPS) and sodium methacrylate sulfonate (SMAS), allows the sulfonic acid groups in the master and co-functional monomers to form stable ion-pair complexes with the guanidinyl and amino groups in the pseudo-template molecule VAL. Simultaneously, the amide groups form multi-point hydrogen bonds with the hydroxyl and amino groups of the template molecule. The synergistic combination of master and co-functional monomers can precisely match the spatial size and functional group orientation of the template molecule, playing a supporting and stabilizing role in the cross-linking polymerization process, thus providing a structural basis for the specific occupancy of fluorescent dyes and competitive recognition of target analytes. The prepared molecularly imprinted polymer, after being loaded with fluorescent dye, forms a bifunctional imprinted fluorescent probe. During the detection of the target analyte, the imprinted system constructed by the master-auxiliary monomers not only endows the imprinted cavity with the specific recognition ability of VAL characteristic functional groups, but also achieves cross-specific synergistic recognition of STX, which is highly similar to VAL structure, through spatial structural complementarity and functional group interaction matching. The target analyte can competitively replace the fluorescent dye in the imprinted cavity, causing the dye to be released into the system and generate a quantitative change in fluorescence signal, transforming the molecular recognition event into an intuitive fluorescence response, significantly improving the sensitivity and selectivity of dual-target analyte detection, and realizing rapid fluorescence quantitative detection of VAL and STX.

[0047] The solution of this application will now be described in detail with reference to the accompanying drawings and various embodiments.

[0048] Example 1

[0049] This embodiment provides a method for preparing a bifunctional blotted fluorescent probe, including:

[0050] 0.03 mmol of valganciclovir hydrochloride, 0.12 mmol of 2-acrylamide-2-methylpropanesulfonic acid, 0.03 mmol of sodium methacrylamide sulfonate, and 0.04 g of dimethyl azobisisobutyrate were added to a polymerization flask. 3.0 mL of methanol and 1.0 mL of acetonitrile were added, and the mixture was sonicated for 8 min to ensure homogeneity. The mixture was then allowed to stand at room temperature for 60 min to allow prepolymerization. Next, 0.60 mmol of trimethylolpropane trimethacrylate was added, and the mixture was thoroughly mixed. The mixture was then purged with nitrogen for 5 min, and the cap was quickly tightened to seal the flask. The joint was sealed with sealing film. The polymerization flask was placed in a water bath and reacted at 60 °C for 20 h. After the reaction, the polymerization product was transferred to a centrifuge tube, methanol was added and shaken for 10 min, centrifuged at 10000 rpm for 10 min, and the supernatant was removed. Then, methanol-acetic acid (9:1, v / v) elution buffer was added and shaken for 3 h. After centrifugation under the same conditions, the supernatant was removed. The above steps were repeated until the characteristic absorption of valganciclovir hydrochloride was no longer detectable by UV spectrophotometer in the supernatant. The eluted polymer was washed once more with pure methanol and dried at 60℃ for 6 h to obtain molecularly imprinted polymer (MIP) solid particles. A 0.025 mg / mL solution of rhodamine 6G was prepared using a 1.5% (v / v) hydrochloric acid aqueous solution as the dispersion medium. The MIP particles were added at a solid-liquid ratio of 1 mg / mL and stirred at room temperature in the dark for 15 min until adsorption saturation, yielding a bifunctional imprinted fluorescent probe suspension working solution, which was stored at 4℃ in the dark.

[0051] Example 2

[0052] This embodiment provides a method for preparing a bifunctional blotted fluorescent probe, including:

[0053] 0.03 mmol of valganciclovir hydrochloride, 0.20 mmol of 2-acrylamide-2-methylpropanesulfonic acid, 0.10 mmol of sodium methacrylate, and 0.04 g of dimethyl azobisisobutyrate were added to a polymerization flask. 4.0 mL of methanol was added, and the mixture was sonicated for 5 min to ensure homogeneity. The mixture was then allowed to stand at room temperature for 30 min for prepolymerization. Next, 0.48 mmol of trimethylolpropane trimethacrylate was added, and the mixture was thoroughly mixed. The mixture was then purged with nitrogen for 5 min, and the cap was quickly tightened to seal the flask. The joint was sealed with sealing film. The polymerization flask was placed in a water bath and reacted at 65 °C for 20 h. After the reaction, the polymerization product was transferred to a centrifuge tube, methanol was added and shaken for 10 min, centrifuged at 10000 rpm for 10 min, and the supernatant was removed. Then, methanol-acetic acid (9:1, v / v) elution buffer was added and shaken for 3 h. After centrifugation under the same conditions, the supernatant was removed. This process was repeated until the characteristic absorption of valganciclovir hydrochloride was no longer detectable by a UV spectrophotometer in the supernatant. The eluted polymer was washed once more with pure methanol and dried at 60 °C for 6 h to obtain molecularly imprinted polymer (MIP) solid particles. A 0.025 mg / mL Rhodamine B solution was prepared using a 2.0% (v / v) acetic acid aqueous solution as the dispersion medium. The MIP particles were added at a solid-liquid ratio of 5 mg / mL and stirred at room temperature in the dark for 10 min until adsorption saturation, yielding a bifunctional imprinted fluorescent probe suspension working solution, which was stored at 4 °C in the dark.

[0054] Example 3

[0055] This embodiment provides a method for preparing a bifunctional blotted fluorescent probe, including:

[0056] Add 0.03 mmol of valganciclovir hydrochloride, 0.20 mmol of 2-acrylamide-2-methylpropanesulfonic acid, 0.10 mmol of sodium methacrylate, and 0.04 g of azobisisobutyronitrile to a polymerization flask, add 4.0 mL of methanol, sonicate for 10 min to mix thoroughly, and let stand at room temperature for 30 min to allow prepolymerization. Then add 0.48 mmol of trimethylolpropane trimethacrylate, mix thoroughly, purge with nitrogen for 5 min, quickly tighten the cap to seal, and seal the joint with sealing film. Place the polymerization flask in a water bath and react at 65 °C for 20 h. After the reaction, the polymerization product was transferred to a centrifuge tube, methanol was added and shaken for 10 min, centrifuged at 10000 rpm for 10 min, and the supernatant was removed. Then, methanol-acetic acid (9:1, v / v) elution buffer was added and shaken for 3 h. After centrifugation under the same conditions, the supernatant was removed. The above steps were repeated until the characteristic absorption of valganciclovir hydrochloride was no longer detectable by UV spectrophotometer in the supernatant. The eluted polymer was washed once more with pure methanol and dried at 60 °C for 6 h to obtain molecularly imprinted polymer (MIP) solid particles. Using a pre-determined aqueous acetic acid solution suitable for the target analyte as the dispersion medium, a 0.025 mg / mL solution of rhodamine 6G was prepared. The MIP particles were added at a solid-liquid ratio of 5 mg / mL and stirred at room temperature in the dark for 10 min until adsorption saturation, yielding a bifunctional imprinted fluorescent probe suspension working solution, which was stored at 4 °C in the dark.

[0057] Comparative Example 1

[0058] In this comparative example, a monofunctional monomer-imprinted polymer fluorescent probe was prepared. The preparation process was the same as in Example 1, except that the auxiliary functional monomer sodium methacrylate was not added, and only 0.12 mmol of 2-acrylamide-2-methylpropanesulfonic acid was used as the single functional monomer.

[0059] Comparative Example 2

[0060] In this comparative example, a monofunctional monomer-imprinted polymer fluorescent probe was prepared. The preparation process was the same as in Example 1, except that 0.03 mmol of the auxiliary functional monomer sodium methylpropenesulfonate was not added, but 0.06 mmol of the auxiliary functional monomer sodium methylpropenesulfonate was added instead.

[0061] Comparative Example 3

[0062] In this comparative example, a monofunctional monomer-imprinted polymer fluorescent probe was prepared. The preparation process was the same as in Example 1, except that 0.03 mmol of the auxiliary functional monomer sodium methylpropenesulfonate was not added, but 0.09 mmol of the auxiliary functional monomer sodium methylpropenesulfonate was added instead.

[0063] Comparative Example 4

[0064] In this comparative example, a monofunctional monomer-imprinted polymer fluorescent probe was prepared. The preparation process was the same as in Example 1, except that 0.03 mmol of the auxiliary functional monomer sodium methylpropenesulfonate was not added, but 0.12 mmol of the auxiliary functional monomer sodium methylpropenesulfonate was added instead.

[0065] Comparative Example 5

[0066] In this comparative example, Rhodamine 6G was used to load MIP to prepare a fluorescent probe. The preparation process was the same as in Example 2, except that Rhodamine B was not used and Rhodamine 6G was used instead.

[0067] Comparative Example 6

[0068] In this comparative example, 2-aminopurine was used to load MIP to prepare a fluorescent probe. The preparation process was the same as in Example 2, except that Rhodamine B was not used and 2-aminopurine was used instead.

[0069] Comparative Example 7

[0070] In this comparative example, a fluorescent probe was prepared using FITC-loaded MIP. The preparation process was the same as in Example 2, except that Rhodamine B was not used, and FITC was used instead.

[0071] Comparative Example 8

[0072] In this comparative example, a fluorescent probe was prepared by loading MIP with sodium fluorescein. The preparation process was the same as in Example 2, except that Rhodamine B was not used and sodium fluorescein was used instead.

[0073] Comparative Example 9

[0074] In this comparative example, acridine yellow G was used to load MIP to prepare a fluorescent probe. The preparation process was the same as in Example 2, except that rhodamine B was not used and acridine yellow G was used instead.

[0075] Comparative Example 10

[0076] In this comparative example, a non-imprinted polymer NIP loaded with fluorescein was prepared to form a fluorescent probe. The preparation process of NIP was the same as in Example 2, except that the template molecule VAL was not added.

[0077] Performance tests were conducted on the molecularly imprinted polymers prepared in the above examples and comparative examples:

[0078] The imprinting ability of MIPs and NIPs was investigated by adsorption equilibrium experiments. 12 mg of dried MIPs / NIPs were weighed and dispersed in 6 mL of valganciclovir hydrochloride solution (concentration 0.1 mg / mL), serving as either the MIPs or NIPs experimental group. Adsorption was carried out at room temperature with shaking for 5 h. After centrifugation, the supernatant was collected and filtered through a 0.45 μm filter to remove remaining particles. UV detection (λ: 255 nm) was used, and the concentration of the supernatant was calculated using the standard curve method. The adsorption capacity (Qe) of the imprinted microspheres was calculated using formula (1):

[0079] (1).

[0080] Where C0 and Ce represent the initial and equilibrium concentrations of the template in the solution (g / mL), respectively, V represents the volume of the solution (mL), and M is the mass of the polymer (mg).

[0081] The adsorption experiment data were fitted using the Langmuir-Freundlich (LF) model, and the formula for the Langmuir-Freundlich (LF) model is formula (2):

[0082] (2).

[0083] Where Qmax is the apparent maximum number of template binding sites, and K is a constant (mmol / L) related to adsorption energy or net enthalpy.

[0084] The imprinting effect of MIPs is evaluated using the imprinting factor IF:

[0085] .

[0086] QMIP represents the amount of target molecules bound to MIP, and QNIP represents the amount of target molecules bound to NIP.

[0087] Performance testing of the bifunctional imprinted fluorescent probes prepared in the above embodiments and comparative examples:

[0088] Take 4.0 mL of the test solution containing the target analyte (drug / marine biotoxin) and place it in a vial. Prepare the test system using a pre-determined concentration of aqueous acetic acid as the dispersion medium. Add 1.0 mL of MIP@Rh6G working suspension, mix well, and react at 35 ℃ with magnetic stirring in the dark (15 min for the drug system, 20 min for the marine biotoxin system). After the reaction is complete, the fluorescent dye is competitively released. Filter the solution through a 0.45 μm aqueous filter membrane to remove particles, and measure the fluorescence intensity of the filtrate, denoted as F.

[0089] The blank control is prepared by replacing the test solution with an equal volume of dispersion medium without the target analyte, while the remaining steps are the same. The fluorescence intensity is measured and recorded as F0. ΔF = F − F0 is used as the quantitative signal.

[0090] The adsorption effects of pseudo-template molecularly imprinted polymers obtained by using different proportions of master and auxiliary monomers in Examples 1 and 2, 3, and 4 are shown in the appendix. Figure 2 As shown, when AMPS:SMAS = 4:2, the spatial matching between the imprinted sites and the template molecules is optimal, and non-specific adsorption is minimized. An appropriate amount of SMAS can synergistically interact with the host monomer AMPS to form more oriented ionic interaction sites and synergistic hydrogen bonding sites. However, excessive SMAS can lead to an overly dense ionic group concentration within the system, resulting in disordered pre-assembly configurations of the template and monomer, and even stronger non-specific electrostatic adsorption and mass transfer resistance after polymerization. Ultimately, this significantly reduces the adsorption capacity Q and imprinting factor IF of the MIP.

[0091] Example 2 and Comparative Examples 5, 6, 7, 8, and 9 used different fluorescent dyes to construct fluorescent probes, and the results are shown in the appendix. Figure 3 As shown, only Rhodamine B and Rhodamine 6G can produce a clear competitive release response, and Rhodamine 6G has the best response amplitude; although 2-aminopurine has a similar structure to the target substance, it has no significant response, indicating that the competitive release efficiency depends not only on structural similarity, but also on factors such as charge matching between the dye and the imprinted microenvironment, hydrophobic / hydrophilic balance and adsorption strength.

[0092] Example 2 and Comparative Example 10 used MIP and NIP-loaded fluorophore probes, and the responses at different concentrations are shown in the attached figures. Figure 4 As shown, a reaction system of 4.0 mL of the test solution and 1.0 mL of the fluorescent probe working solution was used, and quantification was performed using ΔF. A series of STX standard solutions ranging from 2 to 400 ng / mL were prepared, and scatter plot analysis of ΔF versus concentration was performed. NIP control experiments were conducted at concentrations ranging from 10 to 200 ng / mL (see attached diagram). Figure 4 A). Prepare a series of VAL standard solutions ranging from 12.5 to 1000 ng / mL, and perform scatter plot analysis of ΔF versus concentration; perform NIP control experiments under the same conditions at concentrations ranging from 50 to 600 ng / mL (see attached). Figure 4 B). The scatter plot shows that the signal response of the fluorescent probe constructed by MIP is significantly better than that of the fluorescent probe constructed by NIP, both in terms of STX and VAL. Furthermore, the highest IF at the 100 ng / mL concentration point reaches 4.07, and the highest VAL reaches 3.01, further demonstrating the good specificity of this fluorescent probe.

[0093] Appendix Figure 5 This is a schematic diagram of the fluorescent probe recognition mechanism.

[0094] Appendix Figure 6 Figure A shows the recovery rate of STX-spiked seawater samples. Figure 6 Figure B shows the recovery rate of VAL samples spiked with 10% mouse serum. The results show that after desalination and demineralization, the highest recovery rate (91.77%) was achieved at different spiking levels, with an RSD < 4.83%. The relatively low recovery rate is likely due to potential loss of spiked STX during the desalination and demineralization process, leading to a decrease in recovery. However, the overall repeatability remains acceptable and does not affect the basic applicability of the method. With different spiking levels, the overall recovery rate of mouse serum simulated samples (VAL) reached 98.76%, corresponding to an RSD < 3.13%. Diluting the serum to 10% and adjusting the volume with an acetic acid-water system effectively weakened the matrix effect, reducing interference from the serum matrix on the detection system. This indicates that the method has good accuracy and precision in a serum matrix and can meet the requirements for in vivo drug detection.

[0095] To evaluate the specificity and anti-interference performance of the MIP fluorescent probe, selectotoxin GTX1 / 4, domoic acid (DA), and scallop toxin 2 (PTX2) for selectivity verification. The experiment used a detection system of 4.0 mL test solution + 1.0 mL MIP fluorescent probe working solution, with ΔF as the response signal. Detection was carried out under the conditions of dispersion in 1.5% (v / v) acetic acid aqueous solution and reaction at 35 ℃ for 20 min. Response signals at different concentrations were analyzed using 5–300 ng / mL GTX1 / 4 standard solution, 25–200 ng / mL DA standard solution, and 25–200 ng / mL PTX2 standard solution. Corresponding NIP control experiments were also set up. The concentration range for the GTX1 / 4 NIP control was 10–250 ng / mL, for the DA NIP control was 25–200 ng / mL, and for the PTX2 NIP control was 50–200 ng / mL. The concentration was set at 100 ng / mL to examine the cross-recognition of different marine biological toxins by the sensing system. At the same time, the selectivity factor (SF) of scutellarin relative to each toxin was calculated based on a concentration of 100 ng / mL, so as to achieve a quantitative evaluation of the specificity and anti-interference ability of MIP.

[0096] This invention selected three other toxins for specificity testing: GTX1 / 4 (Gymnotoxin 1 / 4), DA (domolybdic acid), and PTX2 (scallop toxin 2). The selectivity factors of MIP for these three toxins were 1.30, 3.37, and 2.97, respectively.

[0097] The bifunctional blotted fluorescent probe provided by this invention uses valganciclovir hydrochloride, which has a structure similar to that of salicornin, as a low-risk pseudo-template. It is prepared using a master-slave functional monomer synergistic strategy, combined with dye occupancy and target analyte competitive release mechanism to achieve fluorescence quantification. It has high selectivity and high sensitivity for both STX and VAL, and can be adapted to both marine environments and complex clinical biological matrices. It has good detection accuracy and spike recovery rate. The preparation method is simple, low-cost, and highly stable, and has broad industrialization potential and large-scale application prospects.

Claims

1. A method for preparing a bifunctional blotted fluorescent probe, the method comprising: S1. A prepolymer is obtained by reacting a mixture of pseudo-template molecules, main functional monomers, auxiliary functional monomers, initiators and porogens. S2. The prepolymer is reacted with a crosslinking agent to obtain a polymer product; S3. Purify the polymerization product until pseudo-template molecules are undetectable, dry it, and obtain molecularly imprinted polymer (MIP) solid particles. S4. The molecularly imprinted polymer (MIP) solid particles are dispersed and then mixed with a fluorescent dye solution for loading to obtain a bifunctional imprinted fluorescent probe. In S1, the pseudo-template molecule includes valganciclovir hydrochloride. ； In S1, the main functional monomer includes 2-acrylamide-2-methylpropanesulfonic acid, and the auxiliary functional monomer includes sodium methpropanesulfonate; In S1, the pore-forming agent is selected from one or more of methanol, acetonitrile, and a methanol-acetonitrile mixture; In S1, the initiator is selected from one or more of dimethyl azobisisobutyrate or azobisisobutyronitrile; In step S2, the crosslinking agent is selected from one or more of trimethylolpropane trimethacrylate or ethylene glycol dimethacrylate. In step S4, the dispersion medium is selected from one or more of pure water, hydrochloric acid aqueous solution, and acetic acid aqueous solution; preferably, it is a 1.5% hydrochloric acid aqueous solution. In S4, the fluorescent dye is selected from one or more of the following: Acridine Yellow G, Rhodamine B, Rhodamine 6G, FITC (fluorescein isothiocyanate), sodium fluorescein, and 2-aminopurine.

2. The preparation method according to claim 1, characterized in that, In S1, the molar amount of the pseudo-template molecule is 1 part, the molar amount of the main functional monomer is 4 to 8 parts, the molar amount of the auxiliary functional monomer is 1 to 4 parts, and the molar amount of the initiator is 5 to 9 parts.

3. The preparation method according to claim 1, characterized in that, In S1, the method for obtaining the prepolymer by reacting the mixed pseudo-template molecules, main and auxiliary functional monomers, initiator and porogen includes: mixing the pseudo-template molecules, main and auxiliary functional monomers, initiator and porogen, followed by sonication for 5-10 min; and standing for 30-60 min.

4. The preparation method according to claim 1, characterized in that, In S2, the molar ratio of pseudo-template molecules to crosslinking agents is 1:(16~20).

5. The preparation method according to claim 1, characterized in that, In step S2, the method for obtaining the polymer product by reacting the prepolymer with the crosslinking agent includes: reacting the prepolymer with the crosslinking agent in a closed, nitrogen-protected environment with water bath heating; the water bath heating temperature is 50-70°C, and the reaction time is 8-24 hours.

6. The preparation method according to claim 1, characterized in that, In step S3, purification includes adding methanol to the product, shaking, centrifuging, removing the supernatant, adding a methanol-acetic acid eluent with a volume ratio of 9:1, shaking, centrifuging, until pseudo-template molecules are undetectable.

7. The preparation method according to claim 1, characterized in that, In step S4, the concentration of the fluorescent dye solution is 0.0025-0.025 mg / mL.

8. The preparation method according to claim 1, characterized in that, In step S4, the solid-liquid ratio when the molecularly imprinted polymer is mixed with the fluorescent dye solution is 1~5 mg / mL.

9. The preparation method according to claim 1, characterized in that, In step S4, the reaction conditions for the mixed loading are: stirring for 5-20 minutes at room temperature and in the dark until the fluorescent dye is saturated on the molecularly imprinted polymer, thus obtaining a bifunctional imprinted fluorescent probe suspension working solution.

10. The application of the bifunctional blotted fluorescent probe prepared by the method of claim 1 in the sensitive detection of marine biotoxins and antiviral drugs, wherein the marine biotoxin is STX, and its structural formula is [insert structural formula here]. The antiviral drug is VAL, with the following structural formula: 。

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