Preparation method and application of metalloproteinase 9 antigenic determinant imprinted nanoparticles

Abstract

The present invention discloses a preparation method and application of metalloproteinase-9 antigenic determinant-imprinted nanoparticles. The preparation method adopts the epitope imprinting method to design and synthesize the MMP-9 carboxyl-terminal nonapeptide YDILQCPED as the antigenic determinant template. Zinc acrylate and the green low-melting reagent choline chloride-MAA are used as functional monomers to prepare the imprinted nanoparticles. The polymerization factors that affect the adsorption performance of the imprinted nanoparticles are investigated, and MMP-9 imprinted nanoparticles with excellent adsorption effect, strong specificity, and high reproducibility are obtained. The prepared MIPs can be used as a new highly selective adsorption material for MMP-9 protein and have good application prospects for the enrichment, separation, and detection of trace cancer markers. They are of great significance in the early screening, early diagnosis, treatment guidance, prognosis assessment, and screening of high-risk populations of cancer patients.

Description

Technical Field

[0001] The present invention relates to the field of preparation of molecularly imprinted nanoparticles, and in particular to a method for preparing metalloproteinase 9 antigenic determinant imprinted nanoparticles. Background Art

[0002] Matrix metalloproteinase-9 (MMP-9), also known as gelatinase B, is a highly conserved zinc-dependent proteolytic enzyme whose primary function is to degrade and remodel the dynamic balance of the ECM, playing a key role in cancer cell invasion, metastasis, and tumor growth. The catalytic domain of the MMP-9 protein contains a zinc ion binding site, which is essential for the enzyme's catalytic action. Numerous studies have found that MMP-9 can serve as a potential biomarker for patients with cancers such as pancreatic cancer, non-small cell carcinoma, thyroid tumors, cervical cancer, oral squamous cell carcinoma, bladder cancer, and breast cancer. It plays an important role in cancer screening, early diagnosis, disease monitoring, and predicting therapeutic efficacy. Therefore, the rapid and accurate detection of MMP-9 levels, a cancer biomarker, has become a key area of ​​cancer research.

[0003] In recent years, with the continuous deepening of research, methods such as zymography, enzyme-linked immunosorbent assay (ELISA), fluorescence resonance energy transfer (FRET), chemiluminescence, and electrochemistry have been developed to detect MMP-9 levels. The results have shown good reproducibility and accuracy. However, these methods are time-consuming, expensive, and require high equipment operation, making them difficult to achieve widespread clinical application. Therefore, the development of a time-saving, convenient, and low-cost method for MMP-9 protein detection is particularly important.

[0004] Molecular imprinting technology is rapidly developing and has become a research hotspot due to its high specificity, stability, and reproducibility. Based on the principle of antigen-antibody specific recognition, it uses molecularly imprinted polymers (MIPs) to achieve precise molecular recognition. As synthetic receptors, MIPs possess high chemical stability, strong recognition capabilities, and high selectivity, as well as ease of synthesis, low cost, and low consumption. Consequently, MIPs are widely used in chromatographic separations, biochemical sensors, drug delivery, and catalysis, particularly in biology, analytical chemistry, and medical diagnostics, attracting extensive research and application.

[0005] However, due to the large molecular weight and complex structure of proteins, protein molecular imprinting still faces many difficulties, such as difficult template removal, poor imprinting effect, and laborious and time-consuming shortcomings. Rachkov and Minoura proposed the epitope imprinting technology in 2000, which made a great contribution to protein imprinting. He Jiayuan et al. successfully prepared a new type of magnetic amphiphilic epitope molecular imprinting polymer coated with ferroferric oxide nanoparticles (Fe3O4@DEMIP) for the specific recognition of (transferrin) Trf. The results showed that Fe3O4@DEMIP has good recognition ability for Trf, with an imprinting factor of up to 6.60 and an adsorption capacity of 43.96 mg / g. Zhang Xuemei et al. successfully prepared silicon nanoparticles coated with SiO2 epitope imprinting polymer (SiNP@SiO2@MIP) for the fluorescent determination of cytochrome c. The results showed that SiNP@SiO2@MIP had good recognition ability for Cyt c, with an imprinting factor of 2.43, and the recovery rate of Cyt c in spiked and diluted human serum was between 94.0% and 107.5%.

[0006] Since the MMPs family of proteins has similar structures and functions, the high-sensitivity and selective detection of MMP-9 remains a challenge. So far, there have been no reports on the preparation of MMP-9 imprinted polymers at home and abroad. Summary of the Invention

[0007] The purpose of the present invention is to provide a preparation method and application of MMP-9 imprinted nanoparticles with good adsorption effect, strong specificity and high repeatability.

[0008] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing metalloproteinase 9 antigenic determinant imprinted nanoparticles, comprising the following steps:

[0009] Step (1): synthesizing the functional monomer choline chloride-MAA eutectic reagent;

[0010] Step (2): mixing a preset functional monomer, a preset template molecule, a preset initiator, a preset porogen and a preset cross-linking agent, mixing them evenly and then heating them for reaction;

[0011] Step (3): After the heating reaction is completed, washing is performed with a preset solvent, and the content of the template molecules in the eluate is detected by HPLC until no template molecules are detected, thereby obtaining the target polymer;

[0012] Step (4): vacuum drying the target polymer at a preset temperature to obtain MMP-9 imprinted nanoparticles.

[0013] In a preferred embodiment, the step (1) comprises:

[0014] Choline chloride and MAA with a molar ratio of 1:2 were reacted in a water bath at 85°C for 3 h to obtain a choline chloride-MAA eutectic reagent.

[0015] In a preferred embodiment, in step (2), the preset functional monomers are choline chloride-MAA, zinc acrylate, methacrylic acid, and acrylamide, and the molar numbers of choline chloride-MAA, zinc acrylate, methacrylic acid, and acrylamide are (0-0.048 mmol), (0-0.048 mmol), 0.024 mmol, and 0.024 mmol, respectively.

[0016] In a preferred embodiment, in step (2), the preset template molecule is MMP-9 carboxyl-terminal nonapeptide.

[0017] In a preferred embodiment, in step (2), the preset porogen is a mixture of acetonitrile and water, wherein the volume ratio of acetonitrile to water is 2:8 to 24:6.

[0018] In a preferred embodiment, in step (2), the preset initiator is azobisisobutyronitrile, and the amount of azobisisobutyl is 0.04 g.

[0019] In a preferred embodiment, in step (2), the preset cross-linking agent is N,N-methylenebisacrylamide, and the molar number of the N,N-methylenebisacrylamide is 0.144 mmol to 0.240 mmol.

[0020] In a preferred embodiment, in step (2), the heating reaction after mixing uniformly comprises:

[0021] After ultrasonic mixing for 5 to 10 minutes, the mixture is heated in a water bath at 50 to 70° C. for a reaction time of 12 to 30 hours.

[0022] In a preferred embodiment, in step (3), the cleaning with a preset solvent includes:

[0023] Acetonitrile was used for elution three times, each elution time was 10 min;

[0024] The mixture was eluted three times with a mixed solution of acetonitrile, water and acetic acid, each elution being 12 h, wherein the volume ratio of acetonitrile, water and acetic acid in the mixed solution of acetonitrile, water and acetic acid was 45:45:10.

[0025] In a second aspect, the present invention further provides a use of molecularly imprinted polymer nanoparticles prepared by the method for preparing metalloproteinase 9 antigen determinant imprinted nanoparticles as described in any one of the first aspects in the enrichment, separation and detection of trace cancer markers.

[0026] The advantages of the present invention are as follows: the present invention provides a preparation method and application of metalloproteinase 9 antigenic determinant imprinted nanoparticles. In the preparation method, an epitope imprinting method is adopted to design and synthesize MMP-9 carboxyl-terminal nonapeptide YDILQCPED as an antigenic determinant template, zinc acrylate and a green low eutectic reagent choline chloride-MAA are used as functional monomers to prepare the imprinted nanoparticles, polymerization factors affecting the adsorption performance of the imprinted nanoparticles are investigated, and MMP-9 imprinted nanoparticles with good adsorption effect, strong specificity and high repeatability are obtained; the prepared MIPs can be used as a new type of highly selective adsorption material for MMP-9 protein, have good application prospects for the enrichment, separation and detection of trace cancer markers, and are of great significance in the early screening, early diagnosis, treatment guidance, prognosis evaluation and high-risk population screening of cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0028] Figure 1 Scanning electron microscopy and infrared images of the imprinted polymer;

[0029] Figure 2 Schematic diagram of dynamic adsorption fitting curve;

[0030] Figure 3 Schematic diagram of static adsorption fitting curve;

[0031] Figure 4 Schematic diagram of the research results on the repeatability of imprinted polymers;

[0032] Figure 5 Schematic diagram of the solid phase extraction analysis results of three proteins by MIPs / NIPs;

[0033] Figure 6 The figure shows the analysis results of MIPs / NIPs on spiked human serum samples. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but this should not be construed as limiting the present invention. Unless otherwise specified, the techniques used in the following examples, such as centrifugation and elution, are conventional methods well known to those skilled in the art. The materials and reagents used in the following examples, unless otherwise specified, are commercially available.

[0035] As described in the background, MIPs, as synthetic receptors, possess high chemical stability, strong recognition and selectivity, and are easily synthesized, cost-effective, and low-consumption. They have attracted extensive research and application in fields such as biology, analytical chemistry, and medical diagnostics. However, due to the large molecular weight and complex structure of proteins, protein molecular imprinting still faces numerous challenges, such as difficulty in template removal, poor imprinting results, and laborious and time-consuming processes. Because the MMP family of proteins shares similar structures and functions, highly sensitive and selective detection of MMP-9 remains a challenge. To address the above problems, the present invention creatively proposes a method for preparing and applying tumor marker matrix metalloproteinase-9 antigenic determinant-imprinted nanoparticles. Based on the characteristics of the catalytic domain of the MMP-9 protein, the epitope imprinting method is used to design and synthesize the MMP-9 carboxyl-terminal nonapeptide YDILQCPED as an antigenic determinant template. Zinc acrylate and the green low-melting agent choline chloride (MAA) are used as functional monomers to prepare the imprinted nanoparticles. The polymerization factors that affect the adsorption performance of the imprinted nanoparticles are investigated, and MMP-9 imprinted nanoparticles with excellent adsorption effect, strong specificity, and high reproducibility are obtained.

[0036] Example 1: This example provides a method for preparing metalloproteinase 9 antigenic determinant imprinted nanoparticles, comprising the following steps:

[0037] Step (1): synthesizing the functional monomer choline chloride-MAA eutectic reagent.

[0038] In this embodiment, this step includes: the ratio of choline chloride to MAA is 1:2 (mol / mol), accurately weighing 13.962 g of choline chloride, adding 16.96 mL of MAA, and placing in an 85° C. water bath to react for 3 hours to obtain the product.

[0039] Step (2): mixing a preset functional monomer, a preset template molecule, a preset initiator, a preset porogen and a preset cross-linking agent, and heating the mixture to react after uniform mixing.

[0040] In this embodiment, this step includes: accurately weighing 0.012 mmol of the preset template molecule YDILQCPED, 0.012 mmol of zinc acrylate, and 0.192 mmol of N,N-methylenebisacrylamide into a 30 mL vial, then adding 8 mL of acetonitrile, sonicating for 5 minutes, then adding 0.012 mmol of choline chloride-MAA, sonicating for 5 minutes, adding 8 mL of acetonitrile and 4 mL of water, sonicating for 5 minutes, adding 0.04 g of azobisisobutyronitrile, sonicating for 5 minutes, and reacting in a 55°C water bath for 18 hours.

[0041] Step (3): After the heating reaction is completed, washing is performed using a preset solvent until no template molecules are detected in the eluate by HPLC, thereby obtaining the target polymer.

[0042] In this embodiment, this step includes: after the heating reaction is completed, the reaction product is transferred to a 50 mL centrifuge tube, 20 mL of acetonitrile is added and shaken for 10 minutes, centrifuged for 10 minutes (5000 r / min), and the supernatant is removed (to remove residual reagents and monomers), and the above steps are repeated 3 times. Then, 20 mL of acetonitrile / water / acetic acid (45:45:10, v / v / v) eluent is added and shaken for 12 hours, centrifuged for 10 minutes (5000 r / min), and the supernatant is removed (to remove the preset template molecules), and the above steps are repeated 3 times to obtain eluted MIPs.

[0043] Step (4): vacuum drying the target polymer at a preset temperature to obtain MMP-9 imprinted nanoparticles.

[0044] In this embodiment, this step includes: drying the eluted MIPs at 60° C. for 6 h to obtain MIPs solid particles, namely, MMP-9 imprinted nanoparticles.

[0045] Example 2: This example provides a method for preparing tumor marker matrix metalloproteinase-9 antigenic determinant-imprinted nanoparticles, which differs from Example 1 in that:

[0046] Step (2) of this embodiment includes: accurately weighing 0.012 mmol of the preset template molecule YDILQCPED, 0.024 mmol of zinc acrylate, and 0.192 mmol of N,N-methylenebisacrylamide into a 30 mL vial, then adding 8 mL of acetonitrile, sonicating for 5 min, then adding 0.024 mmol of choline chloride-MAA, sonicating for 5 min, adding 8 mL of acetonitrile and 4 mL of water, sonicating for 5 min, adding 0.04 g of azobisisobutyronitrile, sonicating for 5 min, and reacting in a 60°C water bath for 18 h.

[0047] Example 3: This example provides a method for preparing tumor marker matrix metalloproteinase-9 antigenic determinant-imprinted nanoparticles, which differs from Example 1 in that:

[0048] Step (2) of this embodiment includes: accurately weighing 0.012 mmol of the template molecule YDILQCPED, 0.024 mmol of zinc acrylate, and 0.192 mmol of N,N-methylenebisacrylamide into a 30 mL vial, then adding 8 mL of acetonitrile, sonicating for 5 min, then adding 0.024 mmol of choline chloride-MAA, sonicating for 5 min, adding 8 mL of acetonitrile and 4 mL of water, sonicating for 5 min, adding 0.04 g of azobisisobutyronitrile, sonicating for 5 min, and reacting in a 60°C water bath for 24 h.

[0049] Comparative Example 1: NIPs, a non-imprinted polymer, was provided. The preparation process thereof was consistent with the method steps and conditions for preparing MIPs solid particles in Example 1 except that the template molecule MMP-9 carboxyl-terminal nonapeptide was not added.

[0050] Performance test of molecularly imprinted polymers:

[0051] The adsorption properties of MIPs / NIPs to template nonapeptides and proteins were determined by HPLC and visible spectrophotometry, respectively, as follows:

[0052] The HPLC method used a C18 reversed-phase column (4.6×250 mm, 5 μm) to detect the concentration of the template nonapeptide. The mobile phase was 0.1% TFA water and 0.1% TFA acetonitrile (73:27, v / v). The column temperature was 28°C, the flow rate was 1.0 mL / min, the injection volume was 20 μL, and the detection wavelength was 220 nm. The peak areas of the template nonapeptide at different concentrations, the supernatant after adsorption, and the eluate were measured by HPLC. The concentrations of the template nonapeptide in the adsorption solution and the eluate were calculated using the standard curve method.

[0053] The visible spectrophotometric assay was performed according to the Bradbord protein quantification kit: 80 μL of the protein sample solution was accurately pipetted, 240 μL of Coomassie Brilliant Blue G250 solution was added, and the mixture was thoroughly mixed. After standing at room temperature for 3-5 minutes, 0.3 mL of each solution was aspirated and placed in a cuvette for detection. A blank solution without BSA standard was used as a reference, i.e., a mixture of 0.01 mol / L PBS buffer (pH 7.4) and Coomassie Brilliant Blue G250 (1:3, v / v), and colorimetric determination was performed at a wavelength of 595 nm. The absorbance of BSA standard solutions of varying concentrations and the supernatant after protein adsorption was measured by visible spectrophotometry, and the protein concentration was calculated using the standard curve method.

[0054] 2-10 mg of the MIPs / NIPs prepared in Example 1 were weighed and placed in a 5 mL centrifuge tube. 1 mL of a 0.005 mg / mL-0.05 mg / mL MMP-9 nonapeptide-Tris-HCl solution was added. The tube was placed in a thermostatic oscillator for 12 h of adsorption at room temperature, followed by centrifugation at 10,000 rpm for 10 min, and the supernatant was collected. The adsorbed MIPs / NIPs particles were placed in a 5 mL-50 mL centrifuge tube, 1-10 mL of a 0.1 mol / L Tris-HCl eluent (pH 7.0) was added, and the tube was placed in a thermostatic oscillator for 10 min of elution at room temperature, followed by centrifugation at 10,000 rpm for 10 min. The supernatant was collected, and the above elution steps were repeated twice. The supernatant was then eluted with 1-10 mL of methanol / acetic acid (9:1, v / v) in a thermostatic oscillator for 20 h. The supernatant was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. The supernatant was then eluted with 1-10 mL of methanol / acetic acid (9:1, v / v) in a thermostatic oscillator for 12 h. The supernatant was collected and the above steps were repeated for another 12 h. HPLC methods were used to determine the adsorption properties of the template nonapeptide on MIPs / NIPs.

[0055] The adsorption capacity Qe and IF of MIPs / NIPs were calculated according to the following formula:

[0056] The adsorption capacity Qe (mg / g) of MIPs / NIPs for template molecules is calculated as follows:

[0057]

[0058] In formula (1), C0 (mg / L) is the initial concentration of the sample solution, C e (mg / L) is the sample concentration in the supernatant, m (g) is the weight of MIPs / NIPs, and V (L) is the volume of the adsorption solution.

[0059] IF is used to evaluate the specific adsorption performance of MIPs to template molecules, and its calculation formula is:

[0060]

[0061] In formula (2), Q MIPs and Q NIPs are the adsorption capacities of MIPs / NIPs adsorption templates, respectively. Each adsorption capacity is calculated from the results of three parallel experiments.

[0062] Figure 1 The morphological characteristics of MIPs / NIPs are shown. Figure 1(a, b, c, d) are scanning electron micrographs of MIPs and NIPs measured at the same magnification. Figure 1 As can be seen from (a, b), MIPs are composed of nano-scale particles that are nearly spherical, with a rough surface and regular particles. The addition of template molecules during the preparation of MIPs can change the morphology and size of the polymer, increase the specific surface area, provide more binding sites, and increase the mass transfer rate of MIPs adsorption and desorption of template molecules. Figure 1 As can be seen in (c, d), NIPs are blocky, compact, irregular nanoparticles with slightly larger size, smaller specific surface area, and fewer binding sites.

[0063] The MIPs, NIPs and zinc acrylate were analyzed by Fourier transform infrared spectrometer (FTIR). Figure 1 As shown in Figure (f), there is no significant difference in the number of characteristic absorption peaks between MIPs / NIPs, indicating that the same functional monomers and crosslinkers were used in their preparation, resulting in polymers with similar functional groups. The higher wavenumber of characteristic peaks in MIPs compared to NIPs is likely due to the addition of a template nonapeptide during the preparation of MIPs. The presence of the template molecule leads to a different distribution of functional monomers on the polymer surface, causing a deviation in the characteristic absorption peaks. Therefore, the difference in infrared spectral characteristics between MIPs / NIPs and zinc acrylate indicates that the presence of the template molecule affects the distribution of functional monomers on the polymer surface and the successful preparation of imprinted polymers.

[0064] Figure 2 The adsorption kinetics curve of MIPs / NIPs is shown. It can be seen from the figure that within the same adsorption time period, the adsorption capacity of MIPs is significantly higher than that of NIPs. After 8 hours, the adsorption capacity of MIPs tends to be balanced, and the maximum adsorption capacity reaches 0.3804 mg / g. By comparing the linear correlation coefficient R 2 It can be seen that the recognition process of MIPs on the MMP-9 carboxyl-terminal nonapeptide is more consistent with the pseudo-second-order adsorption kinetic model.

[0065] Figure 3 The static adsorption curve of MIPs / NIPs is shown. It can be seen from the figure that at the same concentration, the adsorption capacity of MIPs is higher than that of NIPs, which indicates that MIPs have more specific imprinting sites that bind to the template nonapeptide than NIPs. When the concentration is between 0.001 and 0.004 mg / mL, the adsorption amount of MIPs on the template increases rapidly with the increase of concentration. When the concentration increases to 0.005 mg / mL, the adsorption amount reaches a maximum of 0.318 mg / g. When the concentration is greater than 0.005 mg / mL, the adsorption amount of MIPs on the template gradually tends to equilibrium.

[0066] Figure 4The figure below shows the MIPs' reusability. As the number of adsorption-desorption cycles increases, the MIPs maintain good adsorption of the template molecule. The initial adsorption capacity is 3.59 mg / g, while the fifth adsorption capacity is 2.59 mg / g. Relative to the initial adsorption, the adsorption capacity for the fifth adsorption cycle drops to 72.15%. Overall, the MIPs maintain good adsorption of the MMP-9 carboxyl-terminal nonapeptide after repeated use, demonstrating their excellent regeneration capacity and strong potential for practical application.

[0067] The selectivity of MIPs for MMP-9 and other proteins (BSA, Trypsin) was evaluated by selectivity experiments. We selected BSA (66.43 kDa) and trypsin (18 kDa) as reference proteins. Figure 5 As shown in (a), MIPs have the highest adsorption capacity for MMP-9 (2.49 mg / g), while BSA (0.84 mg / g) and Trypsin (1.71 mg / g) have lower adsorption capacities. Relative to MMP-9, the selectivity factors of MIPs for BSA and Trypsin are 3.0 and 1.46, respectively, both greater than 1, indicating that MIPs have high selectivity and specificity for MMP-9.

[0068] Finally, the MIPs that adsorbed MMP-9 protein three times were eluted, and the proteins in the eluate were qualitatively and quantitatively analyzed by SDS-PAGE. Figure 5 As shown in Figure b, the molecular weight of the protein band isolated from the template eluate is approximately 90 kDa, consistent with the molecular weight of the added template MMP-9 protein (15 kDa-200 kDa). This indicates that the eluted template is MMP-9 protein. Compared with the protein bands from 0.01 mg / mL BSA1 and 0.05 mg / mL BSA2, the width and color depth of the protein bands roughly estimate the MMP-9 protein content in the three elutions to be between 0.01 and 0.05 mg / mL, which is consistent with the total amount of MMP-9 protein adsorbed by the MIPs over the three elutions (approximately 0.015 mg / mL).

[0069] To evaluate the sensitivity and accuracy of the method, 0.05 mg / mL MMP-9 nonapeptide was added to normal human serum to prepare spiked samples. After pretreatment, the samples were subjected to spike recovery experiments and the experiments were repeated five times. Figure 6As shown, the spiked recoveries of MIPs ranged from 87.10% to 92.90%, with an average recovery of 90.04% and a relative standard deviation of 0.04% to 0.24%. The spiked recoveries of NIPs ranged from 53.30% to 63.70%, with an average recovery of 57.13% and a relative standard deviation of 0.0008% to 0.1839%. This blotting method is stable and accurate, and the results are satisfactory.

[0070] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the present invention and implement it accordingly. They are not intended to limit the scope of protection of the present invention. Any modifications made within the spirit of the main technical solution of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A method for preparing metalloproteinase 9 antigenic determinant imprinted nanoparticles, characterized in that: The following steps are involved: Step (1): using choline chloride and MAA in a molar ratio of 1:2 to synthesize a functional monomer choline chloride-MAA eutectic reagent; Step (2): mixing a preset functional monomer, a preset template molecule, a preset initiator, a preset porogen and a preset cross-linking agent, mixing them evenly and then heating them for reaction; The preset functional monomers are choline chloride-MAA and zinc acrylate, and the molar numbers of choline chloride-MAA and zinc acrylate are (0.012-0.024 mmol) and (0.012-0.024 mmol), respectively; The preset template molecule is MMP-9 carboxyl-terminal nonapeptide; The preset initiator is azobisisobutyronitrile, and the amount of the azobisisobutyronitrile is 0.04 g; The preset porogen is a mixture of acetonitrile and water, wherein the volume ratio of acetonitrile to water is 2:8 to 24:6; The preset cross-linking agent is N,N-methylenebisacrylamide, and the molar number of the N,N-methylenebisacrylamide is 0.144mmol~0.240mmol; Step (3): After the heating reaction is completed, washing is performed using a preset solvent, and the content of the template molecules in the eluate is detected by HPLC until no template molecules are detected, thereby obtaining the target polymer; Step (4): vacuum drying the target polymer at a preset temperature to obtain MMP-9 imprinted nanoparticles.

2. The method for preparing metalloproteinase 9 antigenic determinant imprinted nanoparticles according to claim 1, characterized in that: The step (1) comprises: Choline chloride and MAA with a molar ratio of 1:2 were reacted in a water bath at 85°C for 3 h to obtain a choline chloride-MAA eutectic reagent.

3. The method for preparing metalloproteinase 9 antigenic determinant imprinted nanoparticles according to claim 1, characterized in that: In the step (2), the heating reaction after the mixing is uniformly carried out comprises: After ultrasonic mixing for 5 to 10 minutes, the mixture is heated in a water bath at 50 to 70° C. for a reaction time of 12 to 30 hours.

4. The method for preparing metalloproteinase 9 antigenic determinant imprinted nanoparticles according to claim 1, characterized in that: In the step (3), the cleaning with a preset solvent includes: Acetonitrile was used for elution three times, each elution time was 10 min; The mixture was eluted three times with a mixed solution of acetonitrile, water and acetic acid, each elution being 12 h, wherein the volume ratio of acetonitrile, water and acetic acid in the mixed solution of acetonitrile, water and acetic acid was 45:45:

10.

5. Use of MMP-9 imprinted nanoparticles prepared by the method for preparing metalloproteinase 9 antigen determinant imprinted nanoparticles according to any one of claims 1 to 4 in preparing products for enrichment, separation and detection of trace cancer markers.

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