Serotonin molecularly imprinted polymer, its preparation method and application

Abstract

The present application relates to the field of organic synthesis, and discloses a serotonin molecularly imprinted polymer, a preparation method and application thereof.The molecularly imprinted polymer is prepared by using a bulk polymerization method; in the preparation, a dummy template 5-methoxytryptamine is preassembled with a functional monomer in a solution, then a crosslinking agent and an initiator are added, and the polymerization product is obtained under the protection of inert gas; and after the template molecule is eluted, the serotonin molecularly imprinted polymer is obtained.The present application reduces the preparation cost through the dummy template strategy, and avoids the occurrence of template leakage in application, thereby avoiding the problem of false positive results; meanwhile, the synergistic effect of the bifunctional monomer improves the selectivity.The imprinted factor of the polymer provided by the present application for serotonin can reach 3.54, and the selectivity is excellent and the stability is good; the polymer can be used as a solid-phase extraction adsorbent, and is used for efficient separation, enrichment and detection of serotonin in biological samples (especially urine), and has a good application prospect in the field of biomarker detection related to disease diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, and relates to a serotonin molecularly imprinted polymer, its preparation method, and its application. Background Technology

[0002] Serotonin, also known as 5-hydroxytryptamine, is an important biogenic amine neurotransmitter. Serotonin plays a positive role in the cardiovascular, central nervous, and gastrointestinal systems, participating in the regulation of mood, emotion, sleep, and appetite. Abnormal serotonin levels are closely related to mental illnesses such as depression, anxiety, and Parkinson's disease. Accurate detection of serotonin levels in biological samples is crucial for disease diagnosis and treatment. However, serotonin is present in low concentrations in biological samples, is chemically unstable, and the biological matrix is ​​complex, making direct detection challenging and often requiring pretreatment. Traditional pretreatment methods such as liquid-liquid extraction and solid-phase extraction are cumbersome, time-consuming, and have poor selectivity. Therefore, there is a need to develop efficient and selective sample pretreatment methods.

[0003] Molecularly imprinted polymers (MIPs) possess recognition cavities that are highly matched to target molecules in terms of shape, size, and functional group distribution. They exhibit excellent selectivity and stability, making them ideal solid-phase extraction adsorption materials. They have been widely used for the separation and enrichment of target molecules in complex matrices such as those in the environment, food, and biological fields.

[0004] However, when traditional molecularly imprinted polymers are used as adsorbents to treat samples, they face the problem of template molecule leakage interfering with the detection results. The virtual template strategy is a feasible solution. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a serotonin molecularly imprinted polymer, its preparation method, and its applications, which can solve the problems of small capacity, long processing time, and poor selectivity in existing pretreatment methods. Using a virtual template can effectively prevent template molecule leakage, thereby avoiding interference with serotonin content analysis results in practical applications.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing a serotonin molecularly imprinted polymer includes the following steps:

[0008] Step 1: Dissolve the virtual template molecule 5-methoxytryptamine and the functional monomer in a mixed solvent of chloroform and methanol, and pre-assemble them by stirring in a closed container at room temperature to form a template-functional monomer complex.

[0009] Step 2: Add crosslinking agent and initiator to the solution obtained in Step 1, and carry out polymerization reaction under argon protection and heating conditions to obtain solid polymer;

[0010] Step 3: After crushing, grinding and sieving the polymer obtained in Step 2, the template molecules are removed by elution with a mixed solution of methanol and acetic acid, and the polymer is dried to obtain the serotonin molecularly imprinted polymer.

[0011] In step one, the functional monomer is a mixture of methacrylic acid and acrylamide; the molar ratio of 5-methoxytryptamine, methacrylic acid, and acrylamide is 1:0.5–4:0.5–4. More preferably, the molar ratio of 5-methoxytryptamine, methacrylic acid, and acrylamide is 1:1:1.

[0012] Preferably, the volume ratio of chloroform to methanol in the mixed solvent in step one is 5 to 20:1. More preferably, the volume ratio of chloroform to methanol is 10:1.

[0013] Preferably, the crosslinking agent in step two is ethylene glycol dimethacrylate; the molar ratio of 5-methoxytryptamine to ethylene glycol dimethacrylate is 1:10 to 35. More preferably, the molar ratio of 5-methoxytryptamine to ethylene glycol dimethacrylate is 1:25.

[0014] Preferably, the initiator in step two is azobisisobutyronitrile, and the molar ratio of azobisisobutyronitrile to 5-methoxytryptamine is 0.15~0.6:1.

[0015] Preferably, the pre-assembly time in step one is 10 to 35 minutes.

[0016] Preferably, the polymerization reaction temperature in step two is 50–70°C, and the reaction time is 12–24 h.

[0017] Preferably, the volume ratio of methanol to acetic acid in step three is 5 to 15:1. More preferably, the volume ratio of methanol to acetic acid is 9:1.

[0018] This invention also claims protection for a serotonin molecularly imprinted polymer prepared by the aforementioned method.

[0019] This invention also claims protection for the application of the serotonin molecularly imprinted polymer, which can be used as a solid-phase extraction adsorbent for the selective adsorption, separation, and enrichment of serotonin in biological samples. Preferably, the biological sample is a urine sample.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. A virtual template, 5-methoxytryptamine, was used to replace serotonin, thus avoiding leakage of serotonin as a template molecule that could interfere with the analytical results.

[0022] 2. This invention uses 5-methoxytryptamine as a template molecule, with functional monomers methacrylic acid and acrylamide working synergistically to recognize target molecules through ionic interactions, electrostatic interactions, and hydrogen bonding. After polymerization with a crosslinking agent, a stable three-dimensional cavity structure is generated, thereby achieving the selective adsorption of serotonin molecules.

[0023] 3. The preparation method of the present invention is simple, low-cost and highly selective. The serotonin molecular imprinted polymer prepared by the present invention has an imprinting factor of 3.54 for serotonin molecules, which can be used for the separation and enrichment of serotonin molecules in biological samples.

[0024] This invention effectively avoids template leakage interference through a virtual template strategy, and the synergistic effect of bifunctional monomers enhances selectivity. The prepared polymer exhibits an imprinting factor of 3.54 for serotonin, demonstrating excellent selectivity and good stability. This polymer can be used as a solid-phase extraction adsorbent for the efficient separation, enrichment, and detection of serotonin in biological samples (especially urine). It features simple pretreatment, low cost, and high recovery rate, making it suitable for the precise analysis of serotonin in complex biological matrices. It shows promising application prospects in the field of biomarker detection related to disease diagnosis. Attached Figure Description

[0025] Figure 1 Infrared spectra of MIPs-21 and NIPs-21 prepared in Example 21 and Comparative Example 22.

[0026] Figure 2 Scanning electron microscope image of the serotonin molecularly imprinted polymer MIPs-21 prepared in Example 21.

[0027] Figure 3 The nitrogen adsorption-desorption isotherm of the serotonin molecularly imprinted polymer prepared in Example 21 is shown.

[0028] Figure 4 The pore size distribution diagram is shown for the serotonin molecularly imprinted polymer prepared in Example 21. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available reagents and materials.

[0030] I. The following examples provide a method for preparing a serotonin molecularly imprinted polymer, comprising the following steps:

[0031] Step 1: Accurately weigh 0.4 mmol of the virtual template molecule 5-methoxytryptamine, transfer it to a 15 mL Schlank flask, add 2 mL of chloroform and 0.2 mL of methanol to dissolve it, then add 0.4 mmol of functional monomer methacrylic acid and 0.4 mmol of acrylamide, seal the flask, and stir for 30 min at room temperature under sealed conditions to pre-assemble the template-functional monomer complex.

[0032] Step 2: Add 10 mmol of crosslinking agent ethylene glycol dimethacrylate and 30 mg of initiator azobisisobutyronitrile to the pre-assembled solution in Step 1, and react at 60 °C for 20 h to obtain the polymer. The feeding and reaction were carried out in an argon atmosphere.

[0033] Step 3: The polymer obtained in Step 2 is crushed and ground, passed through a 300-mesh sieve, and repeatedly eluted with a methanol / acetic acid (v / v, 9 / 1) solution to remove the template molecules from the polymer. Then, the acetic acid in the polymer is washed away with methanol, and the polymer is dried under vacuum to obtain the serotonin molecularly imprinted polymer.

[0034] II. The present invention provides an application of the above-mentioned serotonin molecularly imprinted polymer, wherein the serotonin molecularly imprinted polymer of the present invention can adsorb serotonin molecules in urine samples.

[0035] The specific embodiments and comparative examples of the present invention will be further described below.

[0036] 1. Selection of template molecules

[0037] Example 1

[0038] Step 1: Accurately weigh 0.4 mmol of the virtual template molecule 5-methoxytryptamine, transfer it to a 15 mL Schlank flask, add 2 mL of chloroform and 0.2 mL of methanol to dissolve it, then add 0.8 mmol of functional monomers methacrylic acid and 0.8 mmol of acrylamide, seal the flask, and stir for 30 min at room temperature under sealed conditions to pre-assemble the template-functional monomer complex.

[0039] Step 2: Add 8 mmol of crosslinking agent ethylene glycol dimethacrylate and 30 mg of initiator azobisisobutyronitrile to the pre-assembled solution in Step 1, and react at 60 °C for 20 h to obtain the polymer. The feeding and reaction were carried out in an argon atmosphere throughout.

[0040] Step 3: The polymer obtained in Step 2 is crushed and ground, passed through a 300-mesh sieve, and the template molecules in the polymer are repeatedly eluted with a methanol / acetic acid (v / v, 9 / 1) solution. The acetic acid in the polymer is then washed away with methanol, and the polymer is dried under vacuum to obtain serotonin molecularly imprinted polymer 5-MT-MIPs-1.

[0041] Comparative Example 1

[0042] Based on Example 1, the template molecule was replaced with serotonin, while the other preparation methods remained the same as in Example 1, to obtain serotonin molecularly imprinted polymers 5-HT-MIPs.

[0043] Test Example 1

[0044] Weigh 5 mg of the molecularly imprinted polymers from Example 1 and Comparative Example 1 into a centrifuge tube, add 1 mL of 100 µg / mL chloroform standard solution of 5-HT, stir and adsorb at room temperature for 20 min. After adsorption is complete, centrifuge and collect the supernatant, then filter through a 0.22 µm filter membrane. Analyze the filtrate using HPLC. The adsorption capacity Q is used to measure the polymer's adsorption capacity, and the imprinting factor IF is used to measure the selectivity of the molecularly imprinted polymer. The formulas for calculating Q and IF are as follows: ;

[0045] In the formula, Q (mg / g) represents the adsorption capacity (MIPs or NIPs); C0 (µg / mL) represents the initial concentration of the target analyte; C e (µg / mL) represents the equilibrium concentration of the target analyte; V (mL) represents the solution volume; m (mg) represents the polymer mass (MIPs or NIPs). The test results are shown in Table 1:

[0046] Table 1. Adsorption capacity of polymers in Example 1 and Comparative Example 1

[0047] As shown in Table 1, when comparing the adsorption capacity of serotonin and 5-methoxytryptamine as template molecules, the adsorption capacity of the polymer is comparable and there is no significant decrease. Therefore, choosing 5-methoxytryptamine as a virtual template to prepare serotonin molecularly imprinted polymers is a feasible approach.

[0048] 2. Selection of different preparation solvents

[0049] Example 2

[0050] Based on Example 1, adjustments were made, the difference being that the solvent was replaced with 2 mL of tetrahydrofuran, the amount of methanol was replaced with 0.1 mL, and the other preparation methods remained the same as in Example 1, to obtain serotonin molecularly imprinted polymers MIPs-2.

[0051] Example 3

[0052] Based on Example 1, adjustments were made, the difference being that the solvent was replaced with 2 mL of isopropanol and the amount of methanol was replaced with 0.4 mL, while the other preparation methods remained the same as in Example 1, to obtain serotonin molecularly imprinted polymers MIPs-3.

[0053] Example 4

[0054] Based on Example 1, adjustments were made, the difference being that the solvent was replaced with 2 mL of toluene and the amount of methanol was replaced with 0.4 mL, while the other preparation methods remained the same as in Example 1, to obtain serotonin molecularly imprinted polymers MIPs-4.

[0055] Example 5

[0056] Based on Example 1, the following adjustments were made: the solvent was replaced with 2 mL of N,N-dimethylformamide, and methanol was not added. The other preparation methods remained the same as in Example 1, and serotonin molecularly imprinted polymers MIPs-5 were obtained.

[0057] Example 6

[0058] Based on Example 1, adjustments were made, the difference being that the solvent was replaced with 2 mL of dimethyl sulfoxide, and methanol was not added. The other preparation methods remained the same as in Example 1, and serotonin molecularly imprinted polymers MIPs-6 were obtained.

[0059] Example 7

[0060] Based on Example 1, adjustments were made, the difference being that the solvent was replaced with 2 mL of hexafluoroisopropanol, and methanol was not added. The other preparation methods remained the same as in Example 1, and serotonin molecularly imprinted polymers MIPs-7 were obtained.

[0061] Example 8

[0062] Based on Example 1, the following adjustments were made: the solvent was replaced with 2 mL of methanol, while the other preparation methods remained the same as in Example 1, to obtain serotonin molecularly imprinted polymers MIPs-8.

[0063] Comparative Example 2

[0064] Based on Example 1, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 1, to obtain the non-molecularly imprinted polymer NIPs-1.

[0065] Comparative Example 3

[0066] Based on Example 2, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 2, to obtain the non-molecularly imprinted polymer NIPs-2.

[0067] Comparative Example 4

[0068] Based on Example 3, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 3, to obtain the non-molecularly imprinted polymer NIPs-3.

[0069] Comparative Example 5

[0070] Based on Example 4, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 4, to obtain the non-molecularly imprinted polymer NIPs-4.

[0071] Comparative Example 6

[0072] Based on Example 5, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 5, to obtain the non-molecularly imprinted polymer NIPs-5.

[0073] Comparative Example 7

[0074] Based on Example 6, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 6, to obtain the non-molecularly imprinted polymer NIPs-6.

[0075] Comparative Example 8

[0076] Based on Example 7, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 7, to obtain the non-molecularly imprinted polymer NIPs-7.

[0077] Comparative Example 9

[0078] Based on Example 8, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 8, to obtain the non-molecularly imprinted polymer NIPs-8.

[0079] Test Example 2

[0080] Weigh 5 mg of the MIPs and NIPs prepared in Examples 1-8 and Comparative Examples 2-9 and add them to a centrifuge tube. Add 1 mL of chloroform standard solution of 100 µg / mL 5-MT, stir and adsorb for 20 min at room temperature. After adsorption is complete, centrifuge and collect the supernatant. Filter the supernatant through a 0.22 µm filter membrane, and analyze the filtrate using HPLC. The adsorption capacity Q is used to measure the polymer's adsorption capacity, and the imprinting factor IF is used to measure the selectivity of the molecularly imprinted polymer. The formulas for calculating Q and IF are the same as in Test Example 1. The test results are shown in Table 2.

[0081] Table 2. Adsorption capacity and imprinting factor of polymers in Examples 1-8 and Comparative Examples 2-9

[0082] As shown in Table 2, the adsorption capacity and imprinting factor of the polymer both reach their maximum values ​​when the solvent is 2 mL of chloroform and 0.2 mL of methanol. Therefore, the optimal preparation solvent is chloroform / methanol (v / v, 10 / 1).

[0083] 3. Selection of different functional monomers in the preparation process

[0084] Example 9

[0085] Based on Example 1, adjustments were made, the difference being that the functional monomer was replaced with 1.6 mmol of methacrylic acid, while the other preparation methods remained the same as in Example 1, to obtain serotonin molecularly imprinted polymers MIPs-9.

[0086] Example 10

[0087] Based on Example 1, adjustments were made, the difference being that the functional monomers were replaced with 1.2 mmol of methacrylic acid and 0.4 mmol of acrylamide, while the other preparation methods remained the same as in Example 1, to obtain serotonin molecularly imprinted polymers MIPs-10.

[0088] Example 11

[0089] Based on Example 1, adjustments were made, the difference being that the functional monomers were replaced with 0.4 mmol of methacrylic acid and 1.2 mmol of acrylamide, while the other preparation methods remained the same as in Example 1, to obtain serotonin molecularly imprinted polymers MIPs-11.

[0090] Example 12

[0091] Based on Example 1, adjustments were made, the difference being that the functional monomer was replaced with 1.6 mmol of acrylamide, while the other preparation methods remained the same as in Example 1, to obtain serotonin molecularly imprinted polymer MIPs-12.

[0092] Example 13

[0093] Based on Example 1, adjustments were made, the difference being that the functional monomer was replaced with 0.4 mmol of methacrylic acid, while the other preparation methods remained the same as in Example 1, to obtain serotonin molecularly imprinted polymer MIPs-13.

[0094] Example 14

[0095] Based on Example 1, adjustments were made, the difference being that the functional monomers were replaced with 0.4 mmol of methacrylic acid and 0.4 mmol of acrylamide, while the other preparation methods remained the same as in Example 1, to obtain serotonin molecularly imprinted polymers MIPs-14.

[0096] Comparative Example 10

[0097] Based on Example 9, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 9, to obtain the non-molecularly imprinted polymer NIPs-9.

[0098] Comparative Example 11

[0099] Based on Example 10, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 10, to obtain the non-molecularly imprinted polymer NIPs-10.

[0100] Comparative Example 12

[0101] Based on Example 11, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 11, to obtain the non-molecularly imprinted polymer NIPs-11.

[0102] Comparative Example 13

[0103] Based on Example 12, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 12, to obtain the non-molecularly imprinted polymer NIPs-12.

[0104] Comparative Example 14

[0105] Based on Example 13, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 13, to obtain the non-molecularly imprinted polymer NIPs-13.

[0106] Comparative Example 15

[0107] Based on Example 14, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 14, to obtain the non-molecularly imprinted polymer NIPs-14.

[0108] Test Example 3

[0109] The test method was the same as in Test Example 2, except that the adsorbent material was changed to that used in Examples 1 and 9-14, and Comparative Examples 2 and 10-15. The test results are shown in Table 3.

[0110] Table 3 shows the adsorption capacity and imprinting factor of the polymers in Examples 1 and 9-14 and Comparative Examples 2 and 10-15.

[0111] Table 3 shows that increasing the amount of methacrylic acid significantly improves the polymer's adsorption capacity, but decreases the imprinting factor. The imprinting factor reaches its maximum value when the molar ratio of methacrylic acid to acrylamide is 1:3, indicating the optimal imprinting effect. Furthermore, the data reveals that using only acrylamide as the functional monomer results in low polymer adsorption capacity and a low imprinting factor. Therefore, optimizing the acrylamide addition amount under the condition of a 1:3 molar ratio of methacrylic acid to acrylamide, a 1:1 molar ratio of methacrylic acid to acrylamide yields good adsorption capacity and imprinting effect. Thus, the optimal preparation conditions are a molar ratio of template molecule 5-methoxytryptamine:methacrylic acid:acrylamide of 1:1:1.

[0112] 4. Selection of different crosslinking agents in the preparation process

[0113] Example 15

[0114] Based on Example 14, adjustments were made, the difference being that the crosslinking agent was replaced with 1,4-butanediol diacrylate, while the other preparation methods remained the same as in Example 14, to obtain serotonin molecularly imprinted polymers MIPs-15.

[0115] Example 16

[0116] Based on Example 14, the crosslinking agent was replaced with trimethylolpropane trimethacrylate, while the other preparation methods remained the same as in Example 14, to obtain serotonin molecularly imprinted polymers MIPs-16.

[0117] Example 17

[0118] Based on Example 14, the following adjustments were made: the crosslinking agent was replaced with pentaerythritol triacrylate, while the other preparation methods remained the same as in Example 14, to obtain serotonin molecularly imprinted polymers MIPs-17.

[0119] Example 18

[0120] Based on Example 14, the crosslinking agent was replaced with divinylbenzene, while the other preparation methods remained the same as in Example 14, to obtain serotonin molecularly imprinted polymers MIPs-18.

[0121] Comparative Example 16

[0122] Based on Example 15, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 15, to obtain the non-molecularly imprinted polymer NIPs-15.

[0123] Comparative Example 17

[0124] Based on Example 16, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 16, to obtain the non-molecularly imprinted polymer NIPs-16.

[0125] Comparative Example 18

[0126] Based on Example 17, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 17, to obtain the non-molecularly imprinted polymer NIPs-17.

[0127] Comparative Example 19

[0128] Based on Example 18, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 18, to obtain the non-molecularly imprinted polymer NIPs-18.

[0129] Test Example 4

[0130] The test method was the same as in Test Example 2, except that the adsorbent material was replaced with that used in Examples 14-18 and Comparative Examples 15-19. The test results are shown in Table 4.

[0131] Table 4. Adsorption capacity and imprinting factor of polymers in Examples 14-18 and Comparative Examples 15-19

[0132] Table 4 shows that when ethylene glycol dimethacrylate is used as the crosslinking agent, the adsorption capacity and imprinting factor of the polymer both reach their maximum values. Ethylene glycol dimethacrylate is a bifunctional dimethacrylate, and its ester groups can also play a certain role in recognizing the template molecules. Furthermore, the synthesized polymer has a stable structure. Therefore, ethylene glycol dimethacrylate is the optimal crosslinking agent for preparation.

[0133] 5. Selection of different crosslinking agent dosages during preparation

[0134] Example 19

[0135] Based on Example 14, adjustments were made, the difference being that the molar amount of the crosslinking agent ethylene glycol dimethacrylate was replaced with 4 mmol, while the other preparation methods remained the same as in Example 14, to obtain serotonin molecularly imprinted polymers MIPs-19.

[0136] Example 20

[0137] Based on Example 14, adjustments were made, the difference being that the molar amount of the crosslinking agent ethylene glycol dimethacrylate was replaced with 6 mmol, while the other preparation methods remained the same as in Example 14, to obtain serotonin molecularly imprinted polymers MIPs-20.

[0138] Example 21

[0139] Based on Example 14, the following adjustments were made: the molar amount of the crosslinking agent ethylene glycol dimethacrylate was replaced with 10 mmol, while the other preparation methods remained the same as in Example 14, to obtain serotonin molecularly imprinted polymer MIPs-21.

[0140] Example 22

[0141] Based on Example 14, the following adjustments were made: the molar amount of the crosslinking agent ethylene glycol dimethacrylate was replaced with 12 mmol, while the other preparation methods remained the same as in Example 14, to obtain serotonin molecularly imprinted polymer MIPs-22.

[0142] Example 23

[0143] Based on Example 14, the following adjustments were made: the molar amount of the crosslinking agent ethylene glycol dimethacrylate was replaced with 14 mmol, while the other preparation methods remained the same as in Example 14, to obtain serotonin molecularly imprinted polymer MIPs-23.

[0144] Comparative Example 20

[0145] Based on Example 19, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 19, to obtain the non-molecularly imprinted polymer NIPs-19.

[0146] Comparative Example 21

[0147] Based on Example 20, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 20, to obtain the non-molecularly imprinted polymer NIPs-20.

[0148] Comparative Example 22

[0149] Based on Example 21, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 21, to obtain the non-molecularly imprinted polymer NIPs-21.

[0150] Comparative Example 23

[0151] Based on Example 22, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 22, to obtain the non-molecularly imprinted polymer NIPs-22.

[0152] Comparative Example 24

[0153] Based on Example 23, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 23, to obtain the non-molecularly imprinted polymer NIPs-23.

[0154] Test Example 5

[0155] The test method was the same as in Test Example 2, except that the adsorbent material was replaced with that used in Examples 14 and 19-23 and Comparative Examples 15 and 20-24. The test results are shown in Table 5.

[0156] Table 5 shows the adsorption capacity and imprinting factor of the polymers in Examples 14 and 19-23 and Comparative Examples 15 and 20-24.

[0157] Table 5 shows that as the molar amount of the crosslinking agent ethylene glycol dimethacrylate increases, the polymer adsorption capacity increases, but non-specific adsorption also increases, leading to a decrease in the imprinting factor. A good adsorption capacity and the highest imprinting factor are obtained when the molar amount is 10 mmol. Therefore, 10 mmol of the crosslinking agent ethylene glycol dimethacrylate is selected as the optimal preparation condition.

[0158] 6. Selection of initiator dosage during preparation

[0159] Example 24

[0160] Based on Example 21, adjustments were made, the difference being that the amount of initiator azobisisobutyronitrile was changed to 10 mg, while the other preparation methods remained the same as in Example 21, to obtain serotonin molecularly imprinted polymers MIPs-24.

[0161] Example 25

[0162] Based on Example 21, adjustments were made, the difference being that the amount of initiator azobisisobutyronitrile was changed to 20 mg, while the other preparation methods remained the same as in Example 21, to obtain serotonin molecularly imprinted polymers MIPs-25.

[0163] Example 26

[0164] Based on Example 21, adjustments were made, the difference being that the amount of initiator azobisisobutyronitrile was changed to 40 mg, while the other preparation methods remained the same as in Example 21, to obtain serotonin molecularly imprinted polymers MIPs-26.

[0165] Comparative Example 25

[0166] Based on Example 24, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 24, to obtain the non-molecularly imprinted polymer NIPs-24.

[0167] Comparative Example 26

[0168] Based on Example 25, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 25, to obtain the non-molecularly imprinted polymer NIPs-25.

[0169] Comparative Example 27

[0170] Based on Example 26, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 26, to obtain the non-molecularly imprinted polymer NIPs-26.

[0171] Test Example 6

[0172] The test method was the same as in Test Example 2, except that the adsorbent material was replaced with that used in Examples 21 and 24-26 and Comparative Examples 22 and 25-27. The test results are shown in Table 6.

[0173] Table 6 shows the adsorption capacity and imprinting factor of the polymers in Examples 21 and 24-26 and Comparative Examples 22 and 25-27.

[0174] Table 6 shows that when the amount of azobisisobutyronitrile (AIBN) initiator is 30 mg, the adsorption capacity and imprinting factor of the polymer both reach their maximum values. Under these conditions, the polymerization rate is moderate, and excessively fast reaction rates will not lead to excessively high local temperatures, thus preventing damage to the pre-assembly effect of the system. The synthesized polymer structure is stable. Therefore, the optimal amount of azobisisobutyronitrile (AIBN) initiator is 30 mg.

[0175] 7. Selection of reaction time during preparation

[0176] Example 27

[0177] Based on Example 21, adjustments were made, the difference being that the reaction time was changed to 2 h, while the other preparation methods remained the same as in Example 21, to obtain serotonin molecularly imprinted polymers MIPs-27.

[0178] Example 28

[0179] Based on Example 21, adjustments were made, the difference being that the reaction time was changed to 8 h, while the other preparation methods remained the same as in Example 21, to obtain serotonin molecularly imprinted polymers MIPs-28.

[0180] Example 29

[0181] Based on Example 21, adjustments were made, the difference being that the reaction time was changed to 14 h, while the other preparation methods remained the same as in Example 21, to obtain serotonin molecularly imprinted polymers MIPs-29.

[0182] Comparative Example 28

[0183] Based on Example 27, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 27, to obtain the non-molecularly imprinted polymer NIPs-27.

[0184] Comparative Example 29

[0185] Based on Example 28, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 28, to obtain the non-molecularly imprinted polymer NIPs-28.

[0186] Comparative Example 30

[0187] Based on Example 29, adjustments were made, the difference being that no template molecules were added, while the other preparation methods remained the same as in Example 29, to obtain the non-molecularly imprinted polymer NIPs-29.

[0188] Test Example 7

[0189] The test method was the same as in Test Example 2, except that the adsorbent material was replaced with that used in Examples 21 and 27-29 and Comparative Examples 22 and 28-30. The test results are shown in Table 7.

[0190] Table 7 shows the adsorption capacity and imprinting factor of the polymers in Examples 21 and 27-29 and Comparative Examples 22 and 28-30.

[0191] As shown in Table 7, the polymerization time directly affects the degree of completion of the polymerization reaction. Extending the polymerization time increases both the adsorption capacity and imprinting factor of the polymer. Therefore, the optimal polymerization time is 20 h.

[0192] 8. Selectivity test of serotonin molecularly imprinted polymers

[0193] To investigate the selectivity of the prepared serotonin molecularly imprinted polymer for 5-HT, several substances that may interfere with the detection of 5-HT content in body fluids were selected for selective adsorption experiments, including dopamine (DA), L-adrenaline (LE), L-norepinephrine (L-NE), histamine (HA), and ascorbic acid (AA).

[0194] Test Example 8

[0195] Weigh 5 mg of MIPs-21 and NIPs-21 prepared in Examples 21 and 22 into centrifuge tubes, add 1 mL of a 100 µg / mL solution of 5-HT, DA, LE, L-NE, HA, and AA, and stir for adsorption at room temperature for 20 min. After adsorption is complete, centrifuge and collect the supernatant, then filter through a 0.22 µm filter membrane. Analyze the filtrate using HPLC. Calculate the adsorption capacity Q and the imprinting factor IF using the same method as in Test Example 1. Calculate the selectivity coefficient β, which is used to evaluate the recognition ability of target molecules in complex systems. The calculation formula is as follows:

[0196] IF in the formula template The imprinting factor of the molecularly imprinted polymer on the target analyte; IF other This represents the imprinting factor of the molecularly imprinted polymer on other substances. The test results are shown in Table 8:

[0197] Table 8. Selective adsorption results of serin molecularly imprinted polymers

[0198] As shown in Table 8, the serotonin molecularly imprinted polymer prepared in this invention has the best recognition effect on serotonin molecules, and its adsorption capacity and selectivity are significantly better than other interfering substances. This indicates that an imprinted cavity matching the shape, size and functional group position of the target molecule has been successfully created inside the polymer, and the imprinting factor IF value reaches 3.54.

[0199] 9. Applications of serotonin molecularly imprinted polymers

[0200] To investigate the applicability of serotonin molecularly imprinted polymers in real samples, artificial urine was used to simulate a biological sample environment for serotonin spiking and recovery experiments.

[0201] The pretreatment process for molecularly imprinted solid-phase extraction is as follows:

[0202] An empty SPE column was prepared by inserting a ethylene gasket into the column, accurately weighing 100 mg of serotonin-imprinted polymer (SPE), and then placing another ethylene gasket on top of the SPE. The column was then compressed to obtain the SPE column. 5 mL of methanol and 5 mL of ultrapure water were added to activate the SPE column. 1 mL of spiked urine sample was loaded into the solid-phase extraction column, washed with 1 mL of ultrapure water, and eluted with 1 mL of methanol / acetic acid (v / v, 8 / 2). The eluent was collected, dried under nitrogen, reconstituted with ultrapure water, and then filtered through a 0.22 µm filter membrane. The filtrate was analyzed by HPLC, and the recovery rate was calculated.

[0203] Test Example 9

[0204] Using the molecularly imprinted solid-phase extraction (SPE) process described above, the serotonin molecularly imprinted polymer MIPs-21 prepared in Example 21 was used as the adsorbent for SPE. Urine samples spiked with concentrations of 1, 10, and 40 µg / mL were treated, and the recoveries were calculated using HPLC. The test results are shown in Table 9.

[0205] Table 9. Results of serotonin spike recovery in urine samples (n=5)

[0206] As shown in Table 9, when serotonin molecularly imprinted polymer was used as a solid-phase extraction adsorbent to treat urine samples, the spiked recovery rate remained at a high level, with relative standard deviations all below 5%. This demonstrates that the method has good accuracy and practicality in treating urine samples.

[0207] 10. Characterization of serotonin molecularly imprinted polymers

[0208] Test Case 10

[0209] The MIPs-21 and NIPs-21 prepared in Example 21 and Comparative Example 22 were measured using a Fourier transform infrared spectroscopy (FTIR) spectra. The FTIR spectra of MIPs-21 and NIPs-21 are shown below. Figure 1 As shown in the figure, curve a represents MIPs-21, and curve b represents NIPs-21. Based on the information in the figure, at 2986 cm... -1 and 2950 cm -1 The characteristic peak at 1718 cm⁻¹ is generated by the CH stretching vibration, and is present in all monomers involved in the polymerization, with EGMDA providing the most -CH₂-. -1 and 1633 cm -1 The absorption peak at 1144 cm⁻¹ is attributed to the stretching vibration of C=O, and this functional group originates from the crosslinking agent EGDMA and the functional monomers MAA and AAm. -1The vicinity contains the characteristic strong absorption regions of the COC stretching vibrations in the EGDMA ester group and the CO in the MAA. These characteristic peaks collectively indicate the completion of the polymerization reaction. The highly similar spectral data of MIPs-21 and NIPs-21 suggest that the addition of template molecules does not hinder the polymerization reaction, and that MIPs-21 successfully removed the template molecules during the elution step, providing space for subsequent adsorption.

[0210] Test Example 11

[0211] The MIPs-21 prepared in Example 21 were characterized using scanning electron microscopy to observe their microstructure. The scanning electron microscopy results are as follows: Figure 2 As shown in the figure, MIPs-21 exhibits an irregular blocky structure with a rough surface and numerous pores, forming a loose network structure. The pores on the surface of MIPs-21 originate from the imprinted cavities left on the surface after the template molecules are eluted. This is the basis for the specific recognition and adsorption of serotonin molecules by MIPs-21, indicating that MIPs-21 can be used as an adsorbent material for solid-phase extraction to separate and enrich serotonin molecules in complex samples.

[0212] Test Example 12

[0213] N2 adsorption-desorption experiments were conducted on the MIPs-21 prepared in Example 21, and the specific surface area and pore size of the polymer were analyzed. The N2 adsorption-desorption isotherms and pore size distribution curves are shown below. Figure 3 , Figure 4 As shown in the figure. The results indicate that the specific surface area of ​​MIPs-21 is 68 m². 2 / g, total pore volume is 0.075 cm³ 3 The average pore size is 2.19 nm, the isotherm is type II without hysteresis loops, and the pore volume is small, with only a few micropores left by the elution of template molecules. These results are consistent with the typical characteristics of molecularly imprinted polymers synthesized by bulk polymerization, and their dense polymer network structure endows the material with good mechanical strength and stability.

[0214] Obviously, the specific implementation schemes described above are merely a further detailed explanation of the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a serotonin molecularly imprinted polymer, characterized in that, Includes the following steps: Step 1: Dissolve the virtual template molecule 5-methoxytryptamine and the functional monomer in a mixed solvent of chloroform and methanol, and pre-assemble them by stirring in a closed container at room temperature to form a template-functional monomer complex. Step 2: Add crosslinking agent and initiator to the template-functional monomer complex obtained in Step 1, and carry out polymerization reaction under argon protection and heating conditions to obtain solid polymer; Step 3: After crushing, grinding and sieving the polymer obtained in Step 2, the template molecules are removed by elution with a mixed solution of methanol and acetic acid, and the polymer is dried to obtain the serotonin molecularly imprinted polymer. In step one, the functional monomer is a mixture of methacrylic acid and acrylamide; the molar ratio of 5-methoxytryptamine, methacrylic acid, and acrylamide is 1:0.5-4:0.5-4.

2. The method for preparing the serotonin molecularly imprinted polymer according to claim 1, characterized in that, In step one, the volume ratio of chloroform to methanol in the mixed solvent is 5 to 20:

1.

3. The method for preparing the serotonin molecularly imprinted polymer according to claim 1, characterized in that, The crosslinking agent mentioned in step two is ethylene glycol dimethacrylate; the molar ratio of 5-methoxytryptamine to ethylene glycol dimethacrylate is 1:10 to 35.

4. The method for preparing the serotonin molecularly imprinted polymer according to claim 1, characterized in that, In step two, the initiator is azobisisobutyronitrile (AIBN), and the molar ratio of AIBN to 5-methoxytryptamine is 0.15~0.6:

1.

5. The method for preparing the serotonin molecularly imprinted polymer according to claim 1, characterized in that, The pre-assembly time in step one is 10 to 35 minutes.

6. The method for preparing the serotonin molecularly imprinted polymer according to claim 1, characterized in that, In step two, the polymerization reaction temperature is 50–70℃ and the reaction time is 12–24 h.

7. The method for preparing the serotonin molecularly imprinted polymer according to claim 1, characterized in that, In step three, the volume ratio of methanol to acetic acid is 5 to 15:

1.

8. A serotonin molecularly imprinted polymer, characterized in that, It is prepared by the preparation method according to any one of claims 1-7.

9. The application of the serotonin molecularly imprinted polymer according to claim 8, characterized in that, It is used as a solid-phase extraction adsorbent for the selective adsorption, separation, and enrichment of serotonin in biological samples.

10. The application according to claim 9, characterized in that, The biological sample is a urine sample.

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