Molecularly imprinted polymer membrane based on microfluidic confinement as well as preparation method and application of molecularly imprinted polymer membrane

Abstract

The invention provides a molecularly imprinted polymer membrane based on microfluidic confinement and a preparation method and application thereof, the preparation method comprises the following steps: taking a base material, and arranging a microchannel on the base material; adding the prepolymerization solution into the microchannel; the prepolymerization solution comprises a silane coupling agent, template molecules, a cross-linking agent and a catalyst; and carrying out polymerization reaction on the prepolymerization solution under a confinement condition to prepare the molecularly imprinted polymer membrane. The microchannel is used for providing a confinement environment for in-situ growth of the molecularly imprinted polymer film, simultaneous imprinting of multiple template molecules is achieved, the obtained molecularly imprinted polymer film is uniform and controllable in thickness and matched with a detection probe, simultaneous detection of multiple substances can be achieved, the preparation method is simple, and preparation conditions are mild. According to the invention, high-integration-level detection of MIPs and NIPs is realized on the same chip, and the chip is simultaneously applied to simultaneous identification, separation, enrichment and detection of simultaneous detection of various substances.

Description

A molecularly imprinted polymer film based on microfluidic confinement, its preparation method and application Technical Field

[0001] This invention belongs to the field of molecular detection technology, specifically relating to a molecularly imprinted polymer membrane based on microfluidic confinement, its preparation method, and its application. Background Technology

[0002] Molecular imprinting is a highly selective and sensitive antigen-antibody recognition system. Synthesized molecularly imprinted polymers (MIPs) exhibit high specificity and selectivity for target molecules in terms of shape, size, and structure, while also possessing low cost, physical and chemical stability, and excellent reusability in detecting, extracting, quantifying, recovering, and removing specific substances. Microfluidics provides a powerful tool for detection applications due to its advantages such as portability, miniaturization, automation, multi-channel sample detection, reduced handling of hazardous substances, and cost savings. Compared to traditional methods, the greatest advantage of microfluidics lies in its ability to create controlled microenvironments, enabling precise actuation and control of fluids within microfluidic channels, thereby improving detection sensitivity. However, integrating effective biorecognition molecules onto microfluidic chips remains a challenge. Therefore, combining both approaches holds promise for developing highly sensitive, low-sample-volume, and rapidly portable sensors. However, the preparation method of microfluidic molecularly imprinted polymers and the effective control of their thickness are crucial to their selectivity, reproducibility, and sensitivity. On the other hand, most existing MIPs are synthesized for the detection of a single analyte, which hinders their applicability in more complex scenarios where accurate decisions require the determination of multiple substances. Therefore, it is of great significance to design microfluidic molecularly imprinted polymers that allow for effective control of MIP thickness and the preparation of polymers with high selectivity and sensitivity for the simultaneous detection of multiple templates. Summary of the Invention

[0003] To overcome the problems existing in the prior art, one objective of this invention is to provide a method for preparing a molecularly imprinted polymer membrane based on microfluidic confinement. A second objective of this invention is to provide a molecularly imprinted polymer membrane prepared by the above method. A third objective of this invention is to provide a detection chip. A fourth objective of this invention is to provide applications of the molecularly imprinted polymer membrane and the detection chip. A fifth objective of this invention is to provide a method for molecularly imprinted detection.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a method for preparing a molecularly imprinted polymer membrane based on a microfluidic confinement, comprising the following steps: taking a first substrate and a second substrate, wherein a microfluidic channel is provided on one side of the first substrate, and the side of the first substrate with the microfluidic channel is sealed and connected to the second substrate to form a microfluidic confinement; adding a prepolymerization solution into the microfluidic confinement; wherein the prepolymerization solution includes a silane coupling agent, a template molecule, a crosslinking agent, and a catalyst; and undergoing a polymerization reaction under confinement conditions to obtain the molecularly imprinted polymer membrane.

[0005] Preferably, the method further includes the following step: the first substrate is first subjected to a hydrophilic treatment.

[0006] More preferably, the hydrophilic treatment includes the following steps: adding a surfactant containing a polyoxyethylene chain to the microfluidic channel of the first substrate for hydrophilic treatment.

[0007] More preferably, the surfactant containing the polyoxyethylene chain includes polyethylene glycol isooctylphenol ether.

[0008] More preferably, the surfactant containing the polyoxyethylene chain is Triton X-100.

[0009] More preferably, the substrate is first cleaned by immersing it in an alcohol solvent before undergoing hydrophilic treatment.

[0010] Preferably, the silane coupling agent comprises at least one of benzyltriethoxysilane (BnTES), isobutyltriethoxysilane (IBTES), 3-triethoxysilyl-1-propylamine (APTES), 3-ureapropyltriethoxysilane (UPTES), phenyltriethoxysilane (PTES), and ureapropyltrimethoxysilane (UPTMS).

[0011] Preferably, the crosslinking agent includes at least one of tetraethyl orthosilicate, methyl orthosilicate, and propyl orthosilicate.

[0012] Preferably, the catalyst comprises ammonia.

[0013] Preferably, the template molecule includes at least one of okadaic acid (OA), chlorogenic acid, vanillin, ferulic acid, benzoic acid, and pyrophylloidin-2 (PbTx-2).

[0014] More preferably, the template molecules are of no less than two types.

[0015] Preferably, the solvent of the prepolymer solution is ethanol.

[0016] Preferably, the microfluidic channel includes an inlet, a detection channel, and an outlet; the detection channel includes a central groove and side grooves located on both sides of the central groove; the width of the central groove is greater than that of the side grooves; the inlet and outlet are respectively connected to the side grooves on both sides.

[0017] The central groove and side groove are blind grooves that do not penetrate the substrate; the inlet and outlet are through holes that penetrate the substrate.

[0018] There are no special restrictions on the substrate material and height; some commonly used materials include silicon wafers, glass, and polycarbonate. Polystyrene alloy (PCPS), polymethyl methacrylate (PMMA), etc., can all be used as substrates. The height of the substrate only needs to be suitable for forming the microfluidic channels. The provided microfluidic chip is small in size and easy to carry.

[0019] Preferably, the materials of the first substrate and the second substrate are independently selected from at least one of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polyethylene terephthalate (PET), and polyvinyl chloride (PVC).

[0020] More preferably, the method for preparing the microfluidic confinement includes the following steps: bonding a first substrate containing microfluidic channels to another complete second substrate to obtain a composite substrate with microfluidic confinement.

[0021] More preferably, the parameters of the microfluidic channel are selected from at least one of the following: a) the width of the side groove is 0.1-1 mm; b) the width of the central groove is 0.5-2 mm; c) the depth of the microchannel is 0.1-1 mm; d) the length of the central groove accounts for 5%-30% of the microchannel.

[0022] More preferably, the projected shape of the intermediate groove is circular or polygonal.

[0023] More preferably, the side groove is a straight groove.

[0024] The side grooves are used to deliver fluid to the entire microfluidic channel, while the middle grooves are used for in-situ synthesis and detection.

[0025] Preferably, the polymerization reaction takes 1-12 hours.

[0026] More preferably, the reaction time is 4-8 h.

[0027] A third aspect of the present invention provides a detection chip, comprising the molecularly imprinted polymer film and the detection substrate described in the second aspect; the detection substrate has a microfluidic channel disposed therein; and the molecularly imprinted polymer film is loaded on the surface of the microfluidic channel.

[0028] Preferably, the detection chip is prepared by referring to the method for preparing the molecularly imprinted polymer film described in the first aspect, and the prepared molecularly imprinted polymer film and the first and second substrates constitute the detection chip.

[0029] The fourth aspect of the present invention provides the application of the molecularly imprinted polymer membrane described in the second aspect, or the detection chip described in the third aspect, in molecularly imprinted detection.

[0030] The fifth aspect of the present invention provides a method for molecular imprint detection, comprising the following steps: injecting a solution to be detected into the microfluidic channel of the detection chip described in the third aspect for adsorption of the analyte, and washing away the solution to be detected; adding a detection probe into the microfluidic channel of the detection chip, washing away the detection probe, and monitoring the fluorescence signal in the detection chip using a fluorescence microscope.

[0031] Preferably, the method further includes the following steps: (1) injecting standard sample solutions of different concentrations into the microfluidic confined channel of the detection chip for adsorption, cleaning, then adding a detection probe for binding, cleaning again, and finally measuring, using a fluorescence inverted microscope to read the fluorescence signal in the detection chip, plotting the linear relationship between concentration and signal intensity, and obtaining a standard curve; (2) injecting the solution to be detected into the microfluidic confined channel of the detection chip for adsorption, cleaning, then adding a detection probe, cleaning again, and finally measuring, and then comparing with the standard curve to obtain the concentration of the solution to be detected.

[0032] Preferably, the adsorption process of the analyte is carried out under ultrasonic conditions.

[0033] Preferably, the procedure includes the following steps: adding the detection probe into the microfluidic channel of the detection chip, letting it stand for 10-60 minutes, and then washing away the detection probe.

[0034] Preferably, the detection probe includes ZIF-8-NH2@DEAC~OA Apt and / or ZIF-8-NH2@RhB~PbTx-2Apt.

[0035] Preferably, an inverted fluorescence microscope is used to capture images of the microchannels of the chip, and then image analysis software is used to read the fluorescence.

[0036] Preferably, the fluorescence signal of the central groove of the detection chip is monitored using a fluorescence inverted microscope.

[0037] Preferably, the washing solution used to remove the solution to be tested is water.

[0038] Preferably, the washing solution used to remove the detection probe is water.

[0039] Preferably, the method further includes a method for regenerating the detection chip, wherein the regeneration includes the following steps: after detection, the template molecules are eluted with an eluent.

[0040] More preferably, the eluent is a 5-20% sodium dodecyl sulfonate solution containing acetic acid.

[0041] The beneficial effects of this invention are: This invention utilizes microchannels to provide a confined environment for the in-situ growth of molecularly imprinted polymer films, enabling the simultaneous imprinting of multiple template molecules. This can solve the problem of uneven surface or size of molecularly imprinted polymers prepared by traditional methods. The resulting molecularly imprinted polymer films have uniform and controllable thickness, are matched with detection probes, and can achieve simultaneous detection of multiple substances. The preparation method is simple and the preparation conditions are mild.

[0042] This invention achieves highly integrated detection of MIPs and NIPs on a single chip, and is simultaneously applied to the detection of multiple substances such as OA and PbTx-2, enabling simultaneous identification, separation, enrichment, and detection. The detection offers advantages such as high selectivity, high sensitivity, good repeatability, reproducibility, and stability, fast analysis speed, low reagent consumption, and the small, portable nature of the microfluidic chip. Specifically, the limit of detection (LOD) for okadaic acid (OA) can be as low as 0.58 µg / L, and the LOD for PbTx-2 can be as low as 6.45 ng / L. The multi-template molecularly imprinted polymer membrane prepared based on microfluidic confinement in this invention can be reused nine times without a significant decrease in signal strength during reuse. Attached Figure Description

[0043] Figure 1 is a flowchart of the preparation method of multi-template molecularly imprinted polymer films based on microfluidic confinement.

[0044] Figure 2 shows the SEM characterization of the fluorescent probes ZIF-8-NH2@DEAC and ZIF-8-NH2@RhB.

[0045] Figure 3 shows the XRD patterns of the fluorescent probes ZIF-8-NH2, ZIF-8-NH2@DEAC, and ZIF-8-NH2@RhB.

[0046] Figure 4 shows the Fourier transform infrared (FTIR) characterization of the fluorescent probes ZIF-8-NH2, ZIF-8-NH2@DEAC, and ZIF-8-NH2@RhB.

[0047] Figure 5 shows the thermogravimetric analysis of ZIF-8-NH2, ZIF-8-NH2@DEAC and ZIF-8-NH2@RhB in the fluorescent probes.

[0048] Figure 6 shows the zeta potential characterization of the fluorescent probes ZIF-8-NH2, ZIF-8-NH2@DEAC, ZIF-8-NH2@RhB, ZIF-8-NH2@DEAC~OA Apt, and ZIF-8-NH2@RhB~PbTx-2 Apt.

[0049] Figure 7 shows the CAD schematic diagram and physical image of the microfluidic chip in Example 1.

[0050] Figure 8 shows microscopic images of the MIPs and NIPs channels of the PMMA chip, the Triton X-100 modified PMMA chip, and the microfluidic confined multitemplate molecularly imprinted polymer film in Example 1.

[0051] Figure 9 shows the effect of different proportions of OA and PbTx-2 on the detection of microfluidic confined multitemplate molecularly imprinted polymer films prepared in Example 1.

[0052] Figure 10 shows the effect of different fluorescent probe ratios on the detection of microfluidically confined multitemplate molecularly imprinted polymer films in Example 1.

[0053] Figure 11 shows fluorescence microscopy images of the microfluidic confined single-template molecularly imprinted polymer membrane and the microfluidic confined single-template non-molecularly imprinted polymer membrane prepared with OA as a template in Example 2 for OA detection.

[0054] Figure 12 shows fluorescence microscopy images of PbTx-2 detection in microfluidically confined single-template molecularly imprinted polymer membranes and microfluidically confined single-template non-molecularly imprinted polymer membranes prepared with PbTx-2 as a template in Example 3.

[0055] Figure 13 shows the effect of the dosage of different functional monomers APTES, BnTES, UPTES, UPTMS and crosslinking agent TEOS on the preparation of microfluidic confined multitemplate molecularly imprinted polymer films in Example 1.

[0056] Figure 14 shows the effect of different synthesis times on the detection of microfluidic confined multitemplate molecularly imprinted polymer films prepared in Example 1.

[0057] Figure 15 shows the effect of different elution times of template molecules on the detection of microfluidic confined multitemplate molecularly imprinted polymer films prepared in Example 1.

[0058] Figure 16 shows fluorescence microscopy images of the microfluidic confined multitemplate molecularly imprinted polymer membrane and the microfluidic confined multitemplate non-molecularly imprinted polymer membrane prepared using OA and PbTx-2 as templates in Example 1 for the detection of OA and PbTx-2.

[0059] Figure 17 shows the fluorescence response and standard curves of different concentrations of OA and PbTx-2 detected by the microfluidic confined multitemplate molecularly imprinted polymer membrane in Example 1.

[0060] Figure 18 is a flowchart of the preparation method of the microfluidic unconfined multitemplate molecularly imprinted polymer film of Comparative Example 1.

[0061] Figure 19 is a microscopic image of the microfluidic confined multitemplate molecularly imprinted polymer film of Example 1 and the microfluidic unconfined dual-template molecularly imprinted polymer film of Comparative Example 1.

[0062] Figure 20 shows the detection fluorescence microscopy images of the microfluidic confined multitemplate molecularly imprinted polymer film of Example 1 and the microfluidic unconfined dual-template molecularly imprinted polymer film of Comparative Example 1.

[0063] Figure 21 shows the ion interference test results of the microfluidic confined multi-template molecularly imprinted polymer film of Example 1.

[0064] Figure 22 shows the selectivity test results for OA and PbTx-2 in the microfluidic confined multitemplate molecularly imprinted polymer film of Example 1.

[0065] Figure 23 is a schematic diagram illustrating the mechanism of the detection of OA and PbTx-2 selectively in the microfluidic confined multitemplate molecularly imprinted polymer film of Example 1.

[0066] Figure 24 shows the stability test results of the microfluidic confined multitemplate molecularly imprinted polymer film in Application Example 1.

[0067] Figure 25 shows the repeatability test results of the microfluidic confined multitemplate molecularly imprinted polymer film in Application Example 1. Detailed Implementation

[0068] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0069] In one aspect of the present invention, a detection chip is provided, comprising a first substrate and a second substrate bonded and sealed together, the two polymer substrates being arranged in a stacked structure; wherein a microfluidic channel is provided on one side of the first substrate, and the side of the first substrate with the microfluidic channel and the second substrate together form a microfluidic confined channel; the surface of the microfluidic channel is covered with a single-template / multi-template molecularly imprinted polymer film substrate; in this example, the microfluidic confined channel consists of an inlet, a straight channel 1, a hexagonal channel, a straight channel 2, and an outlet; wherein the straight channels 1, 2, and the hexagonal channel are blind grooves that do not penetrate the substrate, and the inlet and outlet are through holes that penetrate the substrate; the inlet is connected to the straight channel 1, the straight channels 1 and 2 are respectively located on both sides of the hexagonal channel, and the straight channel 2 is connected to the outlet; the straight channels on both sides are used to transport fluid to the entire microfluidic channel, and the hexagonal channel is used for detection; wherein the total length of the microfluidic confined channel is 21 mm, the depth is 0.4 mm, the width of the straight channel is 0.4 mm, and the length of the hexagonal channel is 3 mm. The diameter of the inlet and outlet holes is 1 mm, the width is 1.3 mm, the angle between the hexagonal channel and the straight channel is 30°, and the diameter of the through holes is 1 mm.

[0070] The first and second substrates are made of PMMA board, with a thickness of 1.5 mm, a length of 34 mm, and a width of 20 mm.

[0071] In one aspect of the present invention, the present invention provides a method for preparing the above-mentioned detection chip and molecularly imprinted polymer film. The preparation process is shown in Figure 1. The specific steps are as follows: (1) Preparation of PMMA chip and hydrophilic modification: The first PMMA substrate is engraved using a chip engraving machine. Its size is 34 mm × 20 mm. It has two straight channels and a hexagonal channel in the middle. The angle between the hexagonal channel and the straight channel is 30°. The total length is 21 mm, the width is 0.4 mm, and the depth is 0.4 mm. The engraved PMMA substrate is placed in 95% ethanol solution for ultrasonic cleaning for 15 min, and then placed in distilled water for ultrasonic cleaning for 15 min. After cleaning, it is naturally air-dried. After cleaning, it is naturally air-dried. 2.0% Triton X-100 is added to 5 µL into the microfluidic channel and evaporated in the air for 15 min. The blank PMMA of the second substrate and the first substrate containing the microfluidic channel are bonded at 108°C for 5 min, and then naturally cooled to room temperature to obtain the PMMA confined chip.

[0072] (2) Preparation of microfluidically confined multi-template molecularly imprinted polymer film: Template molecules (single template or multiple templates, such as OA and / or PbTx-2) and silane coupling agents APTES, BnTES, UPTES and UPTMS were added to 3 mL of ethanol, wherein the template molecules OA (30 µL, 1 µmol / L), PbTx-2 (5 µL, 1 µmol / L), and APTES, BnTES, UPTES and UPTMS were all 80 µL. 25 µL of NH3·H2O and 100 µL of TEOS were added under stirring. After stirring for 30 minutes, a prepolymer solution was obtained.

[0073] 5 µL of the prepolymer solution was added to the microfluidic confinement channel of a 0.4 mm PMMA chip and allowed to stand for 6 hours. The prepolymer was then added to the 0.4 mm chip. The preparation method for non-imprinted polymers (NIPs) differed from the above method in that template molecules OA and PbTx-2 were not added; all other steps were the same. After the reaction, the microfluidic confinement multi-template molecularly imprinted polymer membrane was washed with ultrapure water at a flow rate of 48 µL / min for 15 min. Then, the template molecules were eluted with an eluent containing 10% sodium dodecyl sulfate solution of 0.1 mol / L acetic acid for 3 h. Finally, the membrane was washed with ultrapure water for 15 min to remove residual eluent from the channel, yielding the microfluidic confinement multi-template molecularly imprinted polymer membrane.

[0074] Preparation of fluorescent probes: Two fluorescent probes were prepared as follows: Triethylamine (TEA) (0.1 mL, 0.7 mmol) was added to 0.8 mL of aqueous solution containing Zn(NO3)2·6H2O (199.3 mg, 0.67 mmol). DEAC or RhB (4 mL, 8 μmol) was added while stirring. Then, a mixture of 2-aminobenzimidazole (275.6 mg, 2.07 mmol) and 2-methylimidazole (396.5 mg, 4.83 mmol) dissolved in 4 mL of water and 2 mL of methanol was added to the above mixture. Methanol was then added to make a final volume of 24 mL. The mixture was stirred at room temperature for 30 min, centrifuged, and washed several times with methanol until no rhodamine fluorescence was observed in the supernatant. Next, 10 μL of OA Apt or PbTx-2 Apt (10 μmol / L) was added to 240 μL of PBS (pH=7.4, 10 mmol / L) buffer, followed by 125 μL of EDC (1 mol / L) and 125 μL of NHS (0.25 mol / L). The mixture was then shaken at 37 °C for 1 h to activate the OA Apt or PbTx-2 Apt. ZIF-8-NH2@RhB (10 mg) was dispersed in PBS (4.5 mL, pH 7.4) buffer, and the activated OA Apt or PbTx-2 was added. The mixture was then shaken overnight at 37 °C. After centrifugation and washing with PBS to remove unbound aptamers, ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt were obtained and prepared at 2 mg / mL and stored at 4 °C.

[0075] Figure 2 shows the SEM characterization of ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt during the preparation of fluorescent probes ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt. In Figure 2, A and B are the morphologies of ZIF-8-NH2@DEAC and ZIF-8-NH2@RhB, respectively. As can be seen from the figure, the original dodecahedral structure was not changed after ZIF-8-NH2 encapsulated DEAC and RhB.

[0076] Figure 3 shows the XRD patterns of ZIF-8-NH2, ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt during the preparation of the fluorescent probes ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt in Example 2. As shown in Figure 3, the XRD patterns are consistent with those reported in the literature. The XRD patterns of ZIF-8-NH2@RhB coated with ZIF-8-NH2 and RhB are also consistent with those of ZIF-8-NH2, further indicating that the modification in each step did not change the original crystal structure of ZIF-8-NH2.

[0077] Figure 4 shows the FTIR characterization of ZIF-8-NH2, ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt during their preparation. The insets show the physical images of the corresponding materials, with the FTIR spectra at 3470 / 3385 cm⁻¹. -1 and 916 / 848 cm -1 The peak at the specified position corresponds to the -NH2 and -NH groups in 2-aminobenzimidazole. The FTIR spectrum of ZIF-8-NH2@RhB is consistent with that of ZIF-8-NH2@DEAC and ZIF-8-NH2@RhB, indicating that the encapsulation of DEAC and RhB did not cause structural changes in ZIF-8-NH2. As shown in the inset of Figure 4, the color changed from white to purplish-red after encapsulation of RhB, further confirming that RhB was successfully encapsulated by ZIF-8-NH2, demonstrating the successful preparation of the above-mentioned material in this invention.

[0078] Figure 5 shows the thermogravimetric analysis of ZIF-8-NH2, ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt during the preparation of fluorescent probes ZIF-8-NH2@DEAC and ZIF-8-NH2@RhB in Example 2. As shown in Figure 5, the weights of ZIF-8-NH2, ZIF-8-NH2@DEAC, and ZIF-8-NH2@RhB did not decrease significantly before 400℃. This indicates that ZIF-8-NH2@DEAC and ZIF-8-NH2@RhB possess excellent thermal stability, and introducing DEAC and RhB into the channels of ZIF-8-NH2 did not alter the material's thermal stability.

[0079] Figure 6 shows the zeta potential characterization of ZIF-8-NH2, ZIF-8-NH2@DEAC~OA Apt, ZIF-8-NH2@RhB~PbTx-2 Apt during the preparation of fluorescent probes ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt in Example 2. The potential of ZIF-8-NH2 is approximately +27. After encapsulating DEAC and RhB, the potentials of ZIF-8-NH2@DEAC and ZIF-8-NH2@RhB are approximately +7 and +22, respectively. The potentials of grafted OA Apt and PbTx-2 Apt are... After Apt, the potentials were approximately -19 and -25, respectively. This is due to the potential changes caused by the binding of -COOH on the Apt surface to -NH2 on the ZIF-8-NH2@DEAC and ZIF-8-NH2@RhB surfaces, as well as the phosphate groups on the Apt backbone. These results further confirm the successful preparation of the fluorescent probes ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt in this invention.

[0080] Example 1 The preparation method of the microfluidic confined multitemplate molecularly imprinted polymer membrane and the detection chip in this example refers to the preparation method of the detection chip and molecularly imprinted polymer membrane described above. In this example, the template molecules in step (2) are OA and PbTx-2 dual templates.

[0081] Figure 7 shows a schematic diagram and a physical image of the microfluidically confined multi-template molecularly imprinted polymer film prepared above. Figure 7A shows a schematic diagram of the microfluidically confined channels drawn using CAD. Figure 7B shows a physical image of the microfluidically confined multi-template molecularly imprinted polymer film prepared using a PMMA substrate according to the method in Example 1. As can be seen from Figure 7, this substrate is miniaturized, highly integrated, requires less reagent, and can save costs.

[0082] The morphology of the chip fabrication process was analyzed using a microscope: Figure 8 shows microscopic images of the PMMA chip, the Triton X-100 modified PMMA chip, and the MIPs and NIPs microchannels of the microfluidic confined multi-template molecularly imprinted polymer film in Example 1. In Figure 8, A is the PMMA chip, and the blank PMMA chip etched by the chip etching machine shows a striped pattern. After Triton X-100 modification, the chip surface becomes rough, as shown in Figure 8, B. Figures 8, C and D are microscopic images of the MIPs and NIPs microchannels of the microfluidic confined multi-template molecularly imprinted polymer film. As can be seen from the figures, the MIPs microchannels exhibit a rougher and more porous morphology and no longer show a striped pattern. The NIPs microchannels also exhibit a rough morphology, but compared to the MIPs microchannels, they do not have obvious porosity. This proves that the MIPs have obvious imprinting sites that can be used for the adsorption of target substances. The above results indicate that the microfluidic confined multi-template molecularly imprinted polymer film of this invention was successfully prepared.

[0083] The microfluidic confined dual-template molecularly imprinted polymer membrane used in this example was applied to the detection of OA and PbTx-2. The specific detection process was as follows: 5 μL of a mixed solution of OA and PbTx-2 was added to the MIPs and NIPs microchannels of the PMMA chip. After sonication for 20 minutes, the solution was removed, and the MIPs / NIPs were washed with ultrapure water. Then, 5 µL of a 3:1 volume ratio mixture of ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt was added to the above MIPs / NIPs. The mixture was allowed to stand for 20 minutes, and then rinsed with a syringe pump at a flow rate of 48 µL / min for 20 minutes. Fluorescence detection was performed using a fluorescence inverted microscope.

[0084] Test conditions: OA detection was performed using a 361-389 nm filter with an exposure time of 1 second; PbTx-2 detection was performed using a 537-552 nm filter with an exposure time of 200 milliseconds.

[0085] Figure 9A shows the fluorescence response of microfluidically confined multi-template molecularly imprinted polymer films with different template ratios of OA and PbTx-2 in Example 1 for detecting OA; Figure 9B shows the fluorescence response of microfluidically confined multi-template molecularly imprinted polymer films with different template ratios of OA and PbTx-2 for detecting PbTx-2. As shown in the figures, the ratio of template OA to PbTx-2 is a crucial factor in microfluidically confined synthesis. By controlling the ratio of template OA to PbTx-2, dual-template molecularly imprinted polymer films with more sites can be prepared more quickly and efficiently within the confined space, suitable for the simultaneous fluorescence detection of OA and PbTx-2. The figures also show that a 6:1 ratio of template OA to PbTx-2 is more favorable for fluorescence detection, considering the combined fluorescence signal response values ​​of both OA and PbTx-2.

[0086] Figure 10 shows the effect of different ratios of the fluorescent probes ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt on the fluorescence intensity during detection. The ratios of ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt were 1:1, 2:1, 3:1, 4:1, and 5:1. The relationship between the fluorescence intensity and the ratio of the fluorescent probes in the microfluidic confined multi-template molecularly imprinted polymer films prepared with different fluorescent probe ratios was then tested. The specific test results are shown in the figures. In Figure 10, A represents the fluorescence response of the microfluidic confined multi-template molecularly imprinted polymer films prepared with different fluorescent probe ratios for detecting OA; B represents the fluorescence response of the microfluidic confined multi-template molecularly imprinted polymer films prepared with different fluorescent probe ratios for detecting PbTx-2. As can be seen from the figures, when the ratio of the fluorescent probes increases sequentially, the fluorescence intensity of the microfluidic confined multi-template molecularly imprinted polymer films prepared with different fluorescent probe ratios increases. The calculated OA-M... The ratio of fluorescence intensity of IPs to OA-NIPs showed that the ratio increased sequentially with increasing proportion, stabilizing at approximately 3:1. However, for PbTx-2 detection, the ratio of fluorescence intensity of PbTx-2-MIPs to PbTx-2-NIPs decreased sequentially with increasing proportion of fluorescent probes. Considering the combined effects of simultaneous detection, a ratio of 3:1 between fluorescent probes ZIF-8-NH2@DEAC~OAApt and ZIF-8-NH2@RhB~PbTx-2 Apt is more favorable for the fluorescence detection of both.

[0087] Example 2 The preparation method of the microfluidic confined multitemplate molecularly imprinted polymer membrane and the detection chip in this example refers to the preparation method of the detection chip and molecularly imprinted polymer membrane described above. The template molecule in step (2) of this example is OA.

[0088] The microfluidic confined single-template molecularly imprinted polymer membrane used in this example was applied to OA detection. The specific detection process was as follows: 5 μL of OA solution was added to the MIPs and NIPs microchannels of the PMMA chip. After sonication for 20 minutes, the solution was removed, and the MIPs / NIPs were washed with ultrapure water. Then, 5 µL of a 3:1 volume ratio mixture of ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2Apt was added to the above MIPs / NIPs. The mixture was allowed to stand for 20 minutes, and then rinsed with a syringe pump at a flow rate of 48 µL / min for 20 minutes. Fluorescence detection was performed using a fluorescence inverted microscope.

[0089] Test conditions: OA detection was performed using a 361-389 nm filter with an exposure time of 3 seconds.

[0090] The fluorescence response of the microfluidically confined single-template molecularly imprinted polymer membrane prepared in this example for detecting OA is shown in the figures. Figure 11A shows the fluorescence response of the microfluidically confined single-template molecularly imprinted polymer membrane (MIPs) prepared with OA as the template molecule for detecting RhB; Figure 11B shows the fluorescence response of the microfluidically confined single-template non-molecularly imprinted polymer membrane (NIPs) prepared with OA as the template molecule for detecting OA. As can be seen from the figures, the MIPs prepared with OA as the template molecule have a good fluorescence response for detecting OA. At the same time, compared with the NIPs for detecting the same concentration of OA, the fluorescence response value of MIPs (203.0) is higher than that of NIPs (165.5). Therefore, it is proved that a single-template molecularly imprinted polymer membrane for OA can be prepared in a microfluidically confined space.

[0091] Example 3 The preparation method of the microfluidic confined multitemplate molecularly imprinted polymer film and the detection chip in this example refers to the preparation method of the detection chip and molecularly imprinted polymer film described above. In this example, the template molecule in step (2) is PbTx-2.

[0092] The microfluidic confined single-template molecularly imprinted polymer membrane used in this example was applied to PbTx-2 detection. The specific detection process was as follows: 5 μL of PbTx-2 solution was added to the MIPs and NIPs microchannels of the PMMA chip. After sonication for 20 minutes, the solution was removed, and the MIPs / NIPs were washed with ultrapure water. Then, 5 µL of a 3:1 volume ratio mixture of ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt was added to the above MIPs / NIPs. The mixture was allowed to stand for 20 minutes, and then rinsed with a syringe pump at a flow rate of 48 µL / min for 20 minutes. Fluorescence detection was performed using a fluorescence inverted microscope.

[0093] Test conditions: PbTx-2 detection was performed using a 537-552 nm filter with an exposure time of 200 milliseconds.

[0094] The fluorescence response of the microfluidically confined single-template molecularly imprinted polymer membrane prepared in this example to detect PbTx-2 is shown in Figure 12. Figure 12A shows the fluorescence response of the microfluidically confined single-template molecularly imprinted polymer membrane (MIPs) prepared with PbTx-2 as the template molecule to detect RhB; Figure 12B shows the fluorescence response of the microfluidically confined single-template non-molecularly imprinted polymer membrane (NIPs) prepared with PbTx-2 as the template molecule to detect PbTx-2. As can be seen from the figures, the MIPs prepared with PbTx-2 as the template molecule have a good fluorescence response to detect PbTx-2. At the same time, compared with the NIPs to detect the same concentration of PbTx-2, the fluorescence response value of MIPs (169.3) is higher than that of NIPs (61.34). Therefore, it is proved that a single-template OA molecularly imprinted polymer membrane can be prepared in a microfluidically confined space.

[0095] Multi-factor optimization investigation 1 Table 1, Figure 13 and Figure 14 show the dosage of functional monomers APTES, BnTES, UPTES, UPTMS and crosslinking agent TEOS in Example 1 and the synthesis time. Specifically, the dosage of functional monomer APTES was 20 µL, 40 µL, 60 µL, 80 µL and 100 µL; the dosage of BnTES was 20 µL, 40 µL, 60 µL and 80 µL; the dosage of UPTES was 20 µL, 40 µL, 60 µL and 80 µL; the dosage of UPTMS was 20 µL, 40 µL, 60 µL and 80 µL; the dosage of crosslinking agent TEOS was 40 µL, 60 µL, 80 µL and 100 µL; and the synthesis time was 2 h, 4 h, 6 h, 8 h and 10 h. The fluorescence intensity of microfluidically confined multi-template molecularly imprinted polymer films prepared under different conditions was further tested in relation to the amounts of template, functional monomers, crosslinking agent, and synthesis time. Specific test results are shown in Table 1, Figure 13, and Figure 14. Table 1 shows the optimized amounts of different functional monomers APTES, BnTES, UPTES, UPTMS, and crosslinking agent TEOS. Figure 13 shows A representing the microfluidically confined multi-template molecularly imprinted polymer films prepared under different amounts of APTES, BnTES, UPTES, UPTMS, and crosslinking agent TEOS. The figures show the fluorescence response of the microfluidic confined multitemplate molecularly imprinted polymer membrane for detecting OA; Figure 13B shows the fluorescence response of the microfluidic confined multitemplate molecularly imprinted polymer membrane for detecting PbTx-2 prepared with different amounts of functional monomers APTES, BnTES, UPTES, UPTMS, and crosslinking agent TEOS; Figure 14A shows the fluorescence response of the microfluidic confined multitemplate molecularly imprinted polymer membrane for detecting OA prepared with different synthesis times; Figure 14B shows the fluorescence response of the microfluidic confined multitemplate molecularly imprinted polymer membrane for detecting PbTx-2 prepared with different synthesis times. As can be seen from the figures, the amounts of functional monomers APTES, BnTES, UPTES, UPTMS, and crosslinking agent TEOS, as well as the synthesis time, are important factors in microfluidic confined synthesis. By controlling these conditions, dual-template molecularly imprinted polymer membranes can be prepared more quickly and efficiently within the confined space, suitable for the simultaneous fluorescence detection of OA and PbTx-2. As shown in the figure, when the amounts of functional monomers APTES, BnTES, UPTES, UPTMS and crosslinking agent TEOS are 80 µL, 80 µL, 80 µL, 80 µL and 100 µL respectively, and the reaction time is 6 h, the fluorescence signal response values ​​of simultaneous detection of OA and PbTx-2 are more favorable for fluorescence detection.

[0096] Table 1 Optimization of Functional Monomer and Crosslinking Agent Dosage

[0097] Figure 15 shows the elution time of template molecules in Example 1, specifically: the elution time of template molecules was 1 h, 2 h, 3 h and 4 h. The fluorescence intensity of microfluidically confined multi-template molecularly imprinted polymer films prepared under different template elution time conditions was further tested in relation to the template elution time. The specific test results are shown in Figure 15. Figure 15A shows the fluorescence response of microfluidically confined multi-template molecularly imprinted polymer films prepared under different template elution times for detecting OA; Figure 15B shows the fluorescence response of microfluidically confined multi-template molecularly imprinted polymer films prepared under different template elution times for detecting PbTx-2. As can be seen from the figures, when the elution time is 4 h, the fluorescence intensity of OA-MIPs and OA-NIPs is similar after adding ZIF-8-NH2@DEAC~OA Apt. Similarly, when the elution time is 4 h, the fluorescence intensity of PbTx-2-MIPs and PbTx-2-NIPs is similar after adding ZIF-8-NH2@RhB~PbTx-2 Apt. Therefore, when the elution time is 4 h... At h, it was shown that OA and PbTx-2 were basically completely eluted, which is more conducive to fluorescence detection.

[0098] The fluorescence response of the microfluidically confined dual-template molecularly imprinted polymer membrane prepared in this example for the simultaneous detection of OA and PbTx-2 is shown in Figure 16. Figure 16A shows the fluorescence response of the prepared microfluidically confined dual-template molecularly imprinted polymer membrane (MIPs) for the simultaneous detection of OA; Figure 16B shows the fluorescence response of the prepared microfluidically confined dual-template non-molecularly imprinted polymer membrane (NIPs) for the detection of OA. As shown in the figures, the prepared MIPs exhibit good fluorescence response for the simultaneous detection of OA. Furthermore, compared with NIPs for the detection of the same concentration of OA, the fluorescence response value of MIPs (187.4) is higher than that of NIPs (48.43). Similarly, Figure 16C shows the fluorescence response of the prepared microfluidic confined dual-template molecularly imprinted polymer membrane (MIPs) for simultaneous detection of PbTx-2; Figure 16D shows the fluorescence response of the prepared microfluidic confined dual-template non-molecularly imprinted polymer membrane (NIPs) for detecting PbTx-2. As shown in the figures, the prepared MIPs have a good fluorescence response for detecting PbTx-2 in simultaneous detection. Compared with NIPs for detecting the same concentration of PbTx-2, the fluorescence response value of MIPs (231.1) is higher than that of NIPs (28.86). Therefore, it is proven that molecularly imprinted polymer membranes with dual templates OA and PbTx-2 can be prepared in a microfluidic confined space.

[0099] The method for preparing the microfluidic confined multitemplate molecularly imprinted polymer membrane in this example differs from that in Example 1 in that: the ratio of template OA and PbTx-2 in step (2), the amount of functional monomers APTES, BnTES, UPTES, UPTMS and crosslinking agent TEOS, the synthesis time in step (2), and the elution time of template molecules in step (3) are changed as follows: the ratio of template OA and PbTx-2 is 6:1, the amounts of functional monomers APTES, BnTES, UPTES, UPTMS and crosslinking agent TEOS are 80 µL, 80 µL, 80 µL, 80 µL and 100 µL respectively, the reaction time is 6 h, and the elution time of template molecules is 3 h.

[0100] The microfluidic confined multitemplate molecularly imprinted polymer membrane used in this example was employed for the simultaneous detection of OA and PbTx-2. The specific detection process was as follows: OA solutions of different concentrations (0.8 µg / L, 4.0 µg / L, 8.0 µg / L, 12.0 µg / L, 16.0 µg / L, 20.0 µg / L) and PbTx-2 (10.0 µg / L, 45.0 µg / L, 250.0 µg / L, 450.0 µg / L, 600 µg / L, 900.0 µg / L) were added to MIPs and NIPs chips. After sonication for 20 minutes, the solution was removed, and the MIPs / NIPs were washed with ultrapure water. Subsequently, 5 µL of a 3:1 volume ratio mixture of ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt was taken. µL was added to the above MIPs / NIPs and allowed to stand for 20 minutes. Then, the mixture was flushed with a syringe pump at a flow rate of 48 µL / min for 20 minutes. Fluorescence was detected using an inverted fluorescence microscope. An OA detection was performed using a 361-389 nm filter with an exposure time of 1 second. A PbTx-2 detection was performed using a 537-552 nm filter with an exposure time of 200 milliseconds.

[0101] The test results are shown in Figure 17. Image A in Figure 17 corresponds to the test images of OA solutions with concentrations of 0.8 µg / L, 4.0 µg / L, 8.0 µg / L, 12.0 µg / L, 16.0 µg / L, and 20.0 µg / L. ImageJ was then used to read the values, and a standard curve for OA was plotted based on these readings, as shown in Figure 17 (B). The linear equation of the OA standard curve is FL = 6.835C. OA +72.32, where FL represents the fluorescence value of different concentrations of OA, and C OAThe concentration of OA (unit: µg / L) has a linear range of 0.8 µg / L to 20.0 µg / L, a correlation coefficient r = 0.9940, and a limit of detection (LOD) of 0.58 µg / L (S / N = 3). The established analytical method for OA meets the requirements of actual sample analysis in terms of both linear range and detection limit. Similarly, the images in C of Figure 17 correspond to the test images of PbTx-2 solutions with concentrations of 10.0 µg / L, 45.0 µg / L, 250.0 µg / L, 450.0 µg / L, 600 µg / L, and 900.0 µg / L. ImageJ was then used to read the values, and a standard curve for PbTx-2 was plotted based on these readings, as shown in D of Figure 17. The linear equation of the PbTx-2 standard curve is FL = 0.1107C. PbTx-2 +139.7, where FL represents the fluorescence value of PbTx-2 at different concentrations, and C PbTx-2 The linear range for PbTx-2 concentration (µg / L) was 10 ng / L to 900.0 ng / L, with a correlation coefficient r = 0.9924 and a limit of detection (LOD) of 6.45 ng / L (S / N = 3). The established analytical method for PbTx-2 meets the requirements for analysis of practical samples in terms of both linear range and detection limit.

[0102] Comparative Example 1 The preparation method of the microfluidic unconfined multitemplate molecularly imprinted polymer film in the comparative example is the same as that in Example 1. The preparation method is shown in Figure 18. The difference is that the microfluidic confined synthesis step in step (1) of the comparative example is changed. Specifically, 2.0% Triton X-100 is added to 5 µL into the chip channel and evaporated in air for 15 minutes without bonding with another PMMA plate to obtain a PMMA unconfined chip. The PMMA unconfined chip is then immersed in the prepolymer solution for 6 h.

[0103] Figure 19 shows the microscopic morphology of the microfluidically confined multitemplate molecularly imprinted polymer film and the microfluidically unconfined multitemplate molecularly imprinted polymer film in Comparative Example 1. In Figure 19, A is the multitemplate molecularly imprinted polymer film prepared in a microfluidically confined environment, and B is the multitemplate molecularly imprinted polymer film prepared in a microfluidically unconfined environment. As can be seen from the figure, the multitemplate molecularly imprinted polymer film prepared in the microfluidically confined environment exhibits a uniform and dense morphology, while the multitemplate molecularly imprinted polymer film prepared in the microfluidically unconfined environment exhibits a non-uniform, multilayered structure.

[0104] The detection of OA and PbTx-2 using the microfluidically confined multi-template molecularly imprinted polymer membrane and the microfluidically unconfined multi-template molecularly imprinted polymer membrane in this example is shown in Figure 20. Figure 20A shows the fluorescence response of the microfluidically confined multi-template molecularly imprinted polymer membrane for detecting OA; Figure 20B shows the fluorescence response of the microfluidically confined multi-template molecularly imprinted polymer membrane for detecting PbTx-2. As shown in the figures, the multi-template molecularly imprinted polymer membrane prepared in the microfluidically confined space exhibits a uniform fluorescence distribution for both OA and PbTx-2. Figure 20C shows the fluorescence response of the microfluidically unconfined multi-template molecularly imprinted polymer membrane for detecting OA; Figure 20D shows the fluorescence response of the microfluidically unconfined multi-template molecularly imprinted polymer membrane for detecting PbTx-2. As shown in the figure, the multi-template molecularly imprinted polymer film prepared in the microfluidic unconfined space exhibits a very uneven fluorescence distribution for both OA and PbTx-2. Therefore, the microfluidic confined multi-template molecularly imprinted polymer film prepared by the method of this invention can produce a uniform and stable substrate, which is more conducive to fluorescence detection.

[0105] The ion interference test of the microfluidic confined multi-template molecularly imprinted polymer membrane in Example 1 was conducted to detect the ion interference of OA and PbTx-2 simultaneously. The specific detection process was as follows: (1) A mixed solution of 20 µg / L OA and 900.0 µg / L PbTx-2 was prepared and different interfering ions, including Na+, were added to it. + K + Mg 2+ Ca 2+ HCO3 2- A mixed solution of phenylalanine, L-alanine, L-proline, glucose, and the aforementioned interfering ions.

[0106] (2) Use a pipette to take 5 µL of the mixed solution of OA and PbTx-2 and the solution containing interfering ions and add them to the MIPs and NIPs microchannels of the microfluidic confined multitemplate molecularly imprinted polymer membrane prepared in this example. After sonication for 20 min, remove the solution and wash the MIPs / NIPs with ultrapure water.

[0107] (3) Take another 5 µL of a 3:1 mixture of ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt and add it to the MIPs and NIPs microchannels of the microfluidic confined multitemplate molecularly imprinted polymer film. Let it stand for 20 minutes, and then rinse it with a syringe pump at a flow rate of 48 µL / min for 20 minutes. Take pictures of the microfluidic confined chip using a fluorescence inverted microscope. (4) Use ImageJ to perform fluorescence readings on the pictures of the microfluidic confined chip.

[0108] The results obtained according to the above testing method are shown in Figure 21. Figure A in Figure 21 is a fluorescence spectrum of the detection of OA and the interference of different ions by the microfluidic confined multi-template molecularly imprinted polymer membrane. As can be seen from the figure, different interfering ions have little effect on the detection of OA by the microfluidic confined multi-template molecularly imprinted polymer membrane, with an RSD of 2.6%. Similarly, Figure B in Figure 21 is a fluorescence spectrum of the detection of PbTx-2 and the interference of different ions by the microfluidic confined multi-template molecularly imprinted polymer membrane. As can be seen from the figure, different interfering ions did not have a significant effect on the detection of PbTx-2 by the microfluidic confined multi-template molecularly imprinted polymer membrane, with an RSD of 3.0%. This indicates that the microfluidic confined multi-template molecularly imprinted polymer membrane of the present invention has good anti-interference ability and can be used for the analysis of actual samples.

[0109] Selectivity test was performed to test the selectivity of the microfluidic confined multitemplate molecularly imprinted polymer membrane in the simultaneous detection of OA and PbTx-2. The specific detection process was as follows: (1) Prepare a mixed solution of 20 µg / L OA and 900.0 µg / L PbTx-2, 20 µg / L and 900.0 µg / L DA, TTX, MC-LA, MC-LR and a mixed solution of 20 µg / L and 900.0 µg / L OA, PbTx-2, DA, TTX, MC-LA, MC-LR.

[0110] (2) Use a pipette to take 5 µL of a mixed solution of OA and PbTx-2 or DA, TTX, MC-LA, MC-LR or a mixed solution of OA, PbTx-2, DA, TTX, MC-LA, MC-LR and add it to the MIPs and NIPs microchannels of the microfluidic confined multitemplate molecularly imprinted polymer membrane prepared in Example 7. After sonication for 20 min, remove the solution and wash the MIPs / NIPs with ultrapure water.

[0111] (3) Take another 5 µL of a 3:1 mixture of ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt and add it to the MIPs and NIPs microchannels of the microfluidic confined multitemplate molecularly imprinted polymer film. Let it stand for 20 minutes, and then rinse it with a syringe pump at a flow rate of 48 µL / min for 20 minutes. Take pictures of the microfluidic confined chip using a fluorescence inverted microscope. (4) Use ImageJ to perform fluorescence readings on the pictures of the microfluidic confined chip.

[0112] The results obtained according to the above testing method are shown in Figure 22. As can be seen from A in Figure 22, the fluorescence signal for detecting OA is significantly stronger than that for other toxins, and the mixture of OA with DA, TTX, MC-LA, and MC-LR does not significantly affect the detection results of OA. This indicates that the microfluidic confined multi-template molecularly imprinted polymer membrane of the present invention has higher selectivity for OA than for other toxins. Similarly, as can be seen from B in Figure 22, the fluorescence signal for detecting PbTx-2 is significantly stronger than that for other toxins, and the mixture of PbTx-2 with DA, TTX, MC-LA, and MC-LR does not significantly affect the detection results of PbTx-2. This indicates that the microfluidic confined multi-template molecularly imprinted polymer membrane of the present invention has higher selectivity for PbTx-2 than for other toxins. Further analysis of the size of OA, PbTx-2, DA, TTX, MC-LA, and MC-LR, as well as the hydrogen bonding and binding energy between the polymer material and OA, PbTx-2, DA, TTX, MC-LA, and MC-LR, was conducted. The size analysis results are shown in Figure 23(A). The sizes of OA and PbTx-2 differ significantly from those of DA, TTX, MC-LA, and MC-LR, leading to a mismatch in the size and shape of the imprinted cavity, making it difficult for the polymer to partially enter the cavity. The hydrogen bonding analysis results between the polymer material and OA, PbTx-2, DA, TTX, MC-LA, and MC-LR are shown in Figure 23(B). The polymer material is mainly bonded to OA and PbTx-2 via hydrogen bonds, with binding energies (ΔE) of -32.53 Kcal / mol and -17.48 Kcal / mol, respectively. The Kcal / mol polymer material has a higher binding energy than polymer materials and other toxins. Comprehensive size analysis, hydrogen bonding analysis and binding energy analysis show that the microfluidic confined multi-template molecularly imprinted polymer film of this invention has high selectivity for OA and PbTx-2.

[0113] Application Example 11: Actual Sample Detection. The microfluidic confined multitemplate molecularly imprinted polymer membrane from Example 1 was used to detect OA and PbTx-2 in four types of shellfish (mussels, white clams, clams, and oysters). The specific detection steps were as follows: After sample pretreatment of the test solution, OA or PbTx-2 was added to the microchannels of the microfluidic confined chips MIPs and NIPs. The membrane was loaded for 20 min, the solution was removed, and the MIPs / NIPs were washed with ultrapure water. Then, 5 µL of a 3:1 mixture of ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt was added to the above MIPs / NIPs and allowed to stand for 20 min. Subsequently, the membrane was rinsed for 20 min at a flow rate of 48 µL / min using a syringe pump. Fluorescence detection was performed using a fluorescence inverted microscope. The specific testing process is as follows: (1) Preparation of the test solution: Remove the shells of the shellfish (mussels, clams, oysters, and mussels), wash the shellfish meat to remove the mud and sand, wipe the surface of the shellfish meat dry with absorbent paper, and cut the shellfish meat into a paste. Then add 1 gram of shellfish meat to 5 ml of methanol (90%) and shake at 300 rpm for 60 minutes. Then centrifuge the mixture at 6000 rpm for 10 minutes. Mix the supernatant with three times its volume of PBS (pH 7.4, 10 mmol / L) to obtain the test solution.

[0114] (2) Take 5 µL of the test solution with a pipette and add it to the MIPs and NIPs microchannels of the microfluidic confined multitemplate molecularly imprinted polymer membrane prepared in Example 1. After sonication for 20 min, remove the solution and wash the MIPs / NIPs with ultrapure water.

[0115] (3) Take 5 µL of a 3:1 mixture of ZIF-8-NH2@DEAC~OA aptamer and ZIF-8-NH2@RhB~PbTx-2 aptamer and add it to the MIPs and NIPs microchannels of the microfluidic confined multitemplate molecularly imprinted polymer film. Let it stand for 20 minutes, and then rinse it with a syringe pump at a flow rate of 48 µL / min for 20 minutes. Take pictures of the microfluidic confined chip using a fluorescence inverted microscope. Select a 361-389 nm filter for OA detection with an exposure time of 1 second; select a 537-552 nm filter for PbTx-2 detection with an exposure time of 200 milliseconds.

[0116] (4) Image J was used to take readings of the images of the microfluidic confined chip.

[0117] The test results are shown in Tables 2 and 3 below: Table 2: Detection of OA in aquatic products

[0118] Table 3. Detection of PbTx-2 in aquatic products

[0119] As shown in the table, the results obtained using the microfluidic-confined multi-template molecularly imprinted polymer membrane of this invention are consistent with the results obtained using the standard ELISA method. The spiked recoveries of OA measured using the microfluidic-confined multi-template molecularly imprinted polymer membrane of this invention were 89.7–108.9%, with RSDs of 3.0–6.8% (n=3), and the spiked recoveries of PbTx-2 were 91.0–109.0%, with RSDs of 3.1–7.3% (n=3).

[0120] 2. Stability test The stability of the microfluidic confined multi-template molecularly imprinted polymer membrane in detecting OA and PbTx-2 was tested. The specific test method is as follows: (1) Prepare a mixed solution of 20 µg / L OA and 900.0 µg / L PbTx-2.

[0121] (2) Use a pipette to take 5 µL of OA and PbTx-2 mixed solution and add it to the MIPs and NIPs microchannels of the microfluidic confined multitemplate molecularly imprinted polymer membrane prepared in Example 1. After loading and treating the same batch and different batches of microfluidic confined multitemplate molecularly imprinted polymer membranes for 20 min, remove the solution and wash the MIPs / NIPs with ultrapure water.

[0122] (3) Take another 5 µL of a 3:1 mixture of ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt and add it to the MIPs and NIPs microchannels of the microfluidic confined multitemplate molecularly imprinted polymer film. Let it stand for 20 minutes, and then rinse it with a syringe pump at a flow rate of 48 µL / min for 20 minutes. Take pictures of the microfluidic confined chip using a fluorescence inverted microscope. (4) Use ImageJ to perform fluorescence readings on the pictures of the microfluidic confined chip.

[0123] The results obtained according to the above testing method are shown in Figure 24. In Figure 24, A and B represent the test results of the same batch and different batches of microfluidic-confined multi-template molecularly imprinted polymer membranes for detecting OA, respectively. As shown in the figure, the RSD for OA detection by the microfluidic-confined multi-template molecularly imprinted polymer membrane within the same batch is 3.4%, while the RSD for OA detection by the microfluidic-confined multi-template molecularly imprinted polymer membrane within different batches is 6.7%. Similarly, C and D in Figure 24 represent the test results of the same batch and different batches of microfluidic-confined multi-template molecularly imprinted polymer membranes for detecting PbTx-2, respectively. As shown in the figure, the RSD for PbTx-2 detection by the microfluidic-confined multi-template molecularly imprinted polymer membrane within the same batch is 3.0%, while the RSD for PbTx-2 detection by the microfluidic-confined multi-template molecularly imprinted polymer membrane within different batches is 6.1%. This indicates that the microfluidic-confined multi-template molecularly imprinted polymer membrane prepared using the method described in this invention exhibits excellent stability in detecting OA and PbTx-2. By preparing a dual-template molecularly imprinted polymer film in a microfluidic confined channel with a smooth and uniform surface morphology, the problem of uneven surface or size of molecularly imprinted polymers prepared by traditional methods can be reduced. Therefore, the microfluidic confined multi-template molecularly imprinted polymer film prepared by the method of the present invention helps to reduce errors.

[0124] 3. Repeatability test The repeatability of the microfluidic confined multi-template molecularly imprinted polymer membrane in detecting OA and PbTx-2 is tested. The specific test method is as follows: (1) Prepare a mixed solution of 20 µg / L OA and 900.0 µg / L PbTx-2.

[0125] (2) Use a pipette to take 5 µL of OA and PbTx-2 mixed solution and add it to the MIPs and NIPs microchannels of the microfluidic confined multitemplate molecularly imprinted polymer membrane prepared in Example 7. After sonication for 20 min, remove the solution and wash the MIPs / NIPs with ultrapure water.

[0126] (3) Take another 5 µL of a 3:1 mixture of ZIF-8-NH2@DEAC~OA Apt and ZIF-8-NH2@RhB~PbTx-2 Apt and add it to the MIPs and NIPs microchannels of the microfluidic confined multitemplate molecularly imprinted polymer film. Let it stand for 20 minutes, and then rinse it with a syringe pump at a flow rate of 48 µL / min for 20 minutes. Take pictures of the microfluidic confined chip using a fluorescence inverted microscope. (4) Use ImageJ to perform fluorescence readings on the pictures of the microfluidic confined chip.

[0127] (5) The template molecules were then eluted for 3 h with an elution buffer containing 10% sodium dodecyl sulfonate solution of 0.1 mol / L acetic acid, and then washed with ultrapure water for 15 min to remove the residual elution buffer in the channel.

[0128] (6) Repeat steps (1) to (5) until the fluorescence signal shows a significant decrease. The results obtained according to the above test method are shown in Figure 25. As can be seen from A in Figure 25, the fluorescence signal of OA showed no significant change within 9 tests, with an RSD of 5.4%. Similarly, as can be seen from B in Figure 25, the fluorescence signal of PbTx-2 showed no significant change within 9 tests, with an RSD of 5.9%. This indicates that the microfluidic confined multi-template molecularly imprinted polymer membrane in this invention can be reused at least 9 times, exhibiting good reusability and significantly reducing detection costs.

[0129] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a molecularly imprinted polymer film based on microfluidic confinement, characterized in that, The process includes the following steps: taking a first substrate and a second substrate, wherein a microfluidic channel is provided on one side of the substrate, and sealing the side of the first substrate with the microfluidic channel and the second substrate together to form a microfluidic confinement area; A prepolymerization solution is added to a microfluidic confinement space; the prepolymerization solution comprises a silane coupling agent, a template molecule, a crosslinking agent, and a catalyst. The prepolymer solution undergoes a polymerization reaction under confined conditions to obtain the molecularly imprinted polymer film.

2. The method for preparing a molecularly imprinted polymer film based on microfluidic confinement according to claim 1, characterized in that, It also includes the following steps: the first substrate is first subjected to hydrophilic treatment.

3. The method for preparing a molecularly imprinted polymer film based on microfluidic confinement according to claim 1, characterized in that, The silane coupling agent includes at least one of benzyltriethoxysilane, isobutyltriethoxysilane, 3-triethoxysilyl-1-propane, 3-ureapropyltriethoxysilane, phenyltriethoxysilane, and ureapropyltrimethoxysilane.

4. The method for preparing a molecularly imprinted polymer film based on microfluidic confinement according to claim 1, characterized in that, The crosslinking agent includes at least one of tetraethyl orthosilicate, methyl orthosilicate, and propyl orthosilicate; and / or, the catalyst includes ammonia.

5. The method for preparing a molecularly imprinted polymer film based on microfluidic confinement according to claim 1, characterized in that, The microfluidic channel includes an inlet, a detection channel, and an outlet; the detection channel includes a central groove and side grooves located on both sides of the central groove; the width of the central groove is greater than that of the side grooves; the inlet and outlet are respectively connected to the side grooves on both sides.

6. The method for preparing a molecularly imprinted polymer film based on microfluidic confinement according to claim 1, characterized in that, The reaction time for the polymerization reaction is 1-12 h.

7. The molecularly imprinted polymer film prepared by the preparation method according to any one of claims 1-6.

8. A detection chip, characterized in that, The invention includes the molecularly imprinted polymer membrane and the detection substrate as described in claim 7; the detection substrate is provided with microfluidic channels; and the molecularly imprinted polymer membrane is loaded on the surface of the microchannels.

9. The application of the molecularly imprinted polymer membrane of claim 7, or the detection chip of claim 8, in molecularly imprinted detection.

10. A method for detecting molecular imprinting, characterized in that, The method includes the following steps: injecting the solution to be tested into the microchannel of the detection chip according to claim 8 for adsorption of the analyte, and washing away the solution to be tested; adding the detection probe into the microchannel of the detection chip, washing away the detection probe, and monitoring the fluorescence signal in the detection chip using a fluorescence inverted microscope.

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