3-ethyl-4-methyl-3-pyrroline-2-one false template molecularly imprinted adsorption material for removing bilirubin as well as preparation method and application of 3-ethyl-4-methyl-3-pyrroline-2-one false template molecularly imprinted adsorption material

Abstract

The invention discloses a 3-ethyl-4-methyl-3-pyrrolin-2-one false template molecularly imprinted adsorption material for removing bilirubin as well as a preparation method and application of the 3-ethyl-4-methyl-3-pyrrolin-2-one false template molecularly imprinted adsorption material. 3-ethyl-4-methyl-3-pyrroline-2-ketone with a molecular structure similar to that of bilirubin is used as a false template molecule, 3-aminopropyltriethoxysilane is used as a functional monomer, tetraethoxysilane is used as a cross-linking agent, absolute ethyl alcohol is used as a solvent, and ultrapure water is used for catalytic hydrolysis to obtain the bilirubin fluorescent probe. A molecularly imprinted polymer adsorbent material is prepared and synthesized by using a sol-gel method; after the false template molecules in the adsorbent material are eluted, imprinted holes left in the material can specifically adsorb bilirubin molecules similar to the false template molecules in structure, so that the purpose of highly selectively adsorbing and removing the bilirubin molecules is achieved. The preparation method disclosed by the invention is simple and convenient to operate, low in cost and high in repeatability, and the 3-ethyl-4-methyl-3-pyrroline-2-one false template molecularly imprinted adsorption material has relatively high adsorption capacity, strong specificity and high biological safety.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin, a preparation method and an application thereof. Background Art

[0002] Bilirubin (BR), an endogenous toxin in the human body, is a tetrapyrrole dicarboxylic acid (Mw = 584.67) produced by the metabolism of hemoglobin in red blood cells. BR binds to serum albumin to form a water-soluble complex, allowing for transport in the blood and reducing its toxicity. It is negatively charged, insoluble in water, poorly soluble in alcohol and ether, and readily soluble in alkali. When red blood cells age, they are destroyed by the spleen, producing BR. In the liver, BR combines with glucuronic acid to form a glucuronide ester. This ester then enters the small intestine with bile, where it is deglucuronidated to form bilirubinogen. This ester then travels through the enterohepatic circulation to the liver, where it is mostly excreted in the intestine in feces, while a small portion enters the systemic circulation and is excreted in the urine. Therefore, when liver failure or glucuronic acid deficiency occurs, BR accumulates and deposits in various tissues. Free BR is toxic, and excessive amounts can cause hyperbilirubinemia, jaundice, and irreversible damage to the brain and nervous system. Currently, hemoperfusion has become a promising treatment option for hyperbilirubinemia.

[0003] Hemoperfusion (HP) is a blood purification therapy in which a patient's blood is drawn from the body into an extracorporeal circulation system. Toxins, drugs, and metabolites are removed by adsorption onto adsorbents within the perfusion device, ultimately clearing exogenous or endogenous toxins and other pathogenic substances. The purified blood is then returned to the body. The adsorbent is central to this treatment. Hemoperfusion adsorbents are generally categorized as nonspecific (activated carbon, synthetic resins such as adsorption resins and ion exchange resins) or specific (immunoadsorbents), with activated carbon and adsorption resins being the most commonly used. However, commonly used adsorbents have some unavoidable drawbacks. For example, activated carbon has poor hemocompatibility and can severely damage red blood cells, white blood cells, and particularly platelets, leading to coagulation disorders. Furthermore, particles that shed into the bloodstream can form microemboli, necessitating encapsulation. Ion exchange resins can affect electrolyte balance in the human body and are therefore less commonly used in hemoperfusion.

[0004] Molecularly imprinted polymers (MIPs) are polymers with chemically specific recognition and selective adsorption properties, derived from the principle of antigen-antibody specific binding. They utilize natural or synthetic compounds to mimic biological systems. Their preparation process can be summarized in three steps: First, a template molecule (target molecule) and a functional monomer bind to each other through covalent or non-covalent bonds to form a complex. Second, a crosslinker and solvent (porogen) initiate polymerization under the influence of light, heat, or an initiator, placing the complex within a three-dimensional polymer network. Finally, the template molecule is eluted or dissociated from the polymer by appropriate means, forming recognition pores in the polymer that match the template molecule in shape, size, and spatial arrangement. These pores can effectively and specifically recognize and rebind to the template (or its analogue). Common methods for synthesizing MIPs are categorized by the interaction between the template molecule and the functional monomer: covalent, non-covalent (bulk polymerization, precipitation polymerization, emulsion polymerization, surface imprinting, etc.), semi-covalent, and ionic imprinting techniques. The non-covalent method is the most widely used. The functional monomer and template molecule form a prepolymer through hydrophobic interactions, hydrogen bonds, ionic interactions, and electrostatic interactions. The easily cleavable chemical bonds facilitate elution of the template molecule and prevent structural collapse of the polymer. Furthermore, MIPs offer advantages such as excellent affinity, high selectivity, high stability, and simple preparation methods. Due to the multifunctionality of the template molecule, MIPs are more ideal for recognizing target antigens than traditional antibodies. Therefore, the application of MIP is mainly concentrated in the application of analytical sensors (such as electrochemical, photochemical, and mass sensors), applications in the separation field (such as membrane separation, solid phase extraction, chromatographic separation, chiral separation and analysis of clinical drugs), detection of pesticides and environmental pollutants, identification, separation and optical tracking of biomacromolecules (such as proteins), and applications in the field of medical diagnosis (such as the identification and detection of cancer marker glycoproteins).

[0005] Currently, most hemoperfusion adsorbents used clinically are nonspecific, so while removing toxins, they also remove emergency medications. They can also adsorb amino acids (especially aromatic amino acids), thyroid hormones, growth hormones, and insulin. Furthermore, research on adsorbents for removing bilirubin molecules has drawbacks such as long synthesis times, cumbersome preparation methods, poor biocompatibility due to the frequent use of organic solvents, and a lack of specificity. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin, as well as a preparation method and application.

[0007] The technical solution adopted by the present invention is: a method for preparing a 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material for removing bilirubin, wherein 3-ethyl-4-methyl-3-pyrroline-2-one is used as a pseudo-template molecule to prepare a polymer adsorbent material, and 3-ethyl-4-methyl-3-pyrroline-2-one in the polymer adsorbent material is eluted to obtain the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material for removing bilirubin.

[0008] Preferably, 3-aminopropyltriethoxysilane is used as a functional monomer, tetraethoxysilane is used as a cross-linking agent, and 3-ethyl-4-methyl-3-pyrroline-2-one is used as a pseudo-template molecule, and ultrapure water is used for a catalytic hydrolysis reaction to obtain a polymer adsorbent material; after ultrasonically removing the pseudo-template molecule 3-ethyl-4-methyl-3-pyrroline-2-one, a 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material for removing bilirubin is obtained.

[0009] Preferably, the specific steps are as follows:

[0010] Step 1: dissolving 3-ethyl-4-methyl-3-pyrroline-2-one and 3-aminopropyltriethoxysilane in anhydrous ethanol, performing a prepolymerization reaction under stirring, adding a crosslinking agent tetraethoxysilane and ultrapure water, performing a catalytic hydrolysis reaction, and centrifuging after the reaction to obtain a polymer adsorbent material; the molar ratio of 3-ethyl-4-methyl-3-pyrroline-2-one, 3-aminopropyltriethoxysilane, and tetraethoxysilane is 1:4-12:4-12;

[0011] Step 2: Using 50% (v / v) ethanol-ultrapure water solution, ultrasonically remove the pseudo-template molecule 3-ethyl-4-methyl-3-pyrroline-2-one, and after drying, obtain the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin.

[0012] The 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin prepared by the preparation method is disclosed.

[0013] Application of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted adsorption material for bilirubin removal in the adsorption of bilirubin molecules.

[0014] Preferably, the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin, or a mixture of the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin and a styrene-based anion adsorbent material, is filled into an empty medium-pressure chromatography column for adsorption of bilirubin molecules in plasma samples.

[0015] Preferably, the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material used for removing bilirubin accounts for 0%-100% of the total filler amount.

[0016] Preferably, the flow rate of the plasma sample is controlled to be 20 mL / min-50 mL / min by a constant flow peristaltic pump.

[0017] A plasma bilirubin adsorber filler comprises a 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin.

[0018] The advantages and positive effects of the present invention are as follows: 3-ethyl-4-methyl-3-pyrroline-2-one, which has a similar molecular structure to bilirubin, is used as a pseudo-template molecule for preparing an adsorbent material; after the pseudo-template molecule is eluted and removed, when the bilirubin molecule is adsorbed again, the specific recognition pores remaining in the adsorbent material can be adsorbed and combined with the pseudo-template molecule, thereby selectively adsorbing and removing the bilirubin molecule by the adsorbent material; the synthesis method of the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent material for removing bilirubin by blood perfusion is simple, anhydrous ethanol is used as a solvent, and relevant biosafety experimental evaluation shows that the material has better blood compatibility than a clinically used BS330 bilirubin adsorber, and can be used as a blood perfusion adsorbent material to remove bilirubin molecules.

[0019] The preparation method of 3-ethyl-4-methyl-3-pyrroline-2-one as an adsorbent material is simple to operate, low in cost, highly reproducible, has good adsorption effect, and high biosafety, and can be used as an adsorbent material for blood perfusion to remove bilirubin molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Flow chart for the preparation of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material for bilirubin removal by hemoperfusion.

[0021] Figure 2 Scanning electron micrographs of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent materials (MIPs) and non-imprinted polymer adsorbent materials (NIPs).

[0022] Figure 3 Transmission electron micrographs of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent materials (MIPs) and non-imprinted polymer adsorbent materials (NIPs).

[0023] Figure 4 Fourier transform infrared spectra of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent materials (MIPs) and non-imprinted polymer adsorbent materials (NIPs).

[0024] Figure 5 Thermogravimetric analysis of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent materials (MIPs) and non-imprinted polymer adsorbent materials (NIPs).

[0025] Figure 6 The equilibrium adsorption binding curves of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent materials (MIPs) and non-imprinted polymer adsorbent materials (NIPs).

[0026] Figure 7 The adsorption kinetic curves of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent materials (MIPs) and non-imprinted polymer adsorbent materials (NIPs).

[0027] Figure 8 Schematic diagram of the chemical structures of the pseudo-template molecule 3-ethyl-4-methyl-3-pyrrolidone-2-one, bilirubin molecule, cholesterol molecule and testosterone molecule.

[0028] Figure 9 The selective adsorption evaluation experiment of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent material was conducted.

[0029] Figure 10 This is an experiment to evaluate the reusability performance of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent materials.

[0030] Figure 11 This is an experiment to evaluate the pressure resistance of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent materials.

[0031] Figure 12 This is an evaluation chart for the hemolysis rate determination of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent material and styrene-based anion adsorbent material in BS330 bilirubin adsorber.

[0032] Figure 13 This is the evaluation diagram of the four coagulation tests of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent material and styrene-based anion adsorbent material in BS330 bilirubin adsorber.

[0033] Figure 14 This is a diagram for evaluating the cytotoxicity assay of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent material and styrene-based anion adsorbent material in BS330 bilirubin adsorber.

[0034] Figure 15 This is an evaluation diagram of the adsorption of bilirubin molecules in pig plasma samples after passing through a mixed column of different ratios of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent material and styrene-based anion adsorbent material in a BS330 bilirubin adsorber. DETAILED DESCRIPTION

[0035] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0036] The present invention relates to a 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material for bilirubin removal, as well as its preparation method and application. The molecularly imprinted polymer adsorbent material is synthesized using a sol-gel method, using 3-ethyl-4-methyl-3-pyrroline-2-one, which has a similar molecular structure to bilirubin, as a pseudo-template molecule, 3-aminopropyltriethoxysilane as a functional monomer, tetraethoxysilane as a cross-linking agent, anhydrous ethanol as a solvent, and ultrapure water for catalytic hydrolysis. After eluting the pseudo-template molecules from the adsorbent material, the imprinted pores left within the material can specifically adsorb bilirubin molecules with a similar structure to the pseudo-template molecules, thereby achieving the purpose of highly selective adsorption and removal of bilirubin molecules.

[0037] Because bilirubin molecules are susceptible to oxidation, which can alter their structure and affect the formation of imprinted pores during the preparation of molecularly imprinted polymer adsorbent materials, 3-ethyl-4-methyl-3-pyrrolidone, a molecule with a similar structure to bilirubin, was used as a pseudo-template molecule in the preparation of the adsorbent material to prepare an adsorbent capable of specific adsorption of bilirubin. After the pseudo-template molecule is eluted and removed, the specific recognition pores remaining in the adsorbent material can bind to the bilirubin molecules, resulting in selective adsorption and removal of the bilirubin molecules.

[0038] Preparation method of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material for removing bilirubin Figure 1As shown, 3-aminopropyltriethoxysilane is used as a functional monomer, tetraethoxysilane is used as a cross-linking agent, and 3-ethyl-4-methyl-3-pyrrolin-2-one is used as a pseudo-template molecule. Ultrapure water is used to carry out a catalytic hydrolysis reaction to obtain a polymer adsorbent material. After ultrasonic removal of the pseudo-template molecule 3-ethyl-4-methyl-3-pyrrolin-2-one, a 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-template molecularly imprinted adsorption material for removing bilirubin is obtained. The specific preparation method is as follows:

[0039] Step 1: Dissolve 0.125 g of the pseudo-template molecule 3-ethyl-4-methyl-3-pyrrolidone, 1 mL of the functional monomer 3-aminopropyltriethoxysilane (APTES), and 1 mL of the cross-linking agent tetraethoxysilane (TEOS) in 30 mL of anhydrous ethanol. Stir at room temperature for 2-4 hours for prepolymerization. Then, add 1 mL of ultrapure water to catalyze the hydrolysis reaction, and then stir at room temperature for 24 hours for polymerization.

[0040] 3-Ethyl-4-methyl-3-pyrrolidone is an organic compound with a boiling point of 279°C, a relative density (25°C) of 0.977 g / mL, and a refractive index (20°C) of 1.467. It is a pale yellow or off-white crystalline powder and is an organic and pharmaceutical synthetic intermediate used in the synthesis of glimepiride. APTES is a chemical substance with the molecular formula H2NCH2CH2CH2Si(OC2H5)3, a boiling point of 217°C, a relative density (25°C) of 0.946 g / mL, and a refractive index (20°C) of 1.422. It is a colorless liquid with a distinctive ammoniacal odor. APTES readily hydrolyzes, releasing ethanol to form the corresponding silanol condensate. The amino group within the C-NH2 bond of the molecule reacts with acids, carboxylates, aldehydes, ketones, halogenated hydrocarbons, amides, and nitriles. It is soluble in solvents such as alcohols, chain hydrocarbons, and aromatic hydrocarbons. TEOS is an organic compound with the molecular formula (CH3CH2O)4Si, a boiling point of 168°C, a relative density (20°C) of 0.933 g / mL, and a refractive index (20°C) of 1.382. It is a colorless, transparent liquid. TEOS is stable in anhydrous conditions but decomposes into ethanol and silicic acid upon contact with water. It can become turbid in humid air and is soluble in organic solvents such as alcohol and ether.

[0041] Step 2: Eluting and removing the pseudo-template molecule 3-ethyl-4-methyl-3-pyrroline-2-one; using a 50% (v / v) ethanol-ultrapure water solution, under the assistance of ultrasound, eluting and removing the pseudo-template molecule 3-ethyl-4-methyl-3-pyrroline-2-one until the pseudo-template molecule can no longer be detected using an ultraviolet spectrophotometer, and placing the adsorbent material at 50°C-65°C for vacuum drying.

[0042] Existing adsorbents for removing bilirubin molecules require lengthy synthesis and complex preparation methods. They often use organic solvents, resulting in poor biocompatibility and damage to blood components, such as red blood cells, white blood cells, and platelets. Furthermore, adsorbent particles can shed and enter the bloodstream, potentially forming microemboli, making it difficult to meet the clinical needs of hemoperfusion. However, the 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material for hemoperfusion bilirubin removal, prepared by the aforementioned method, features a simple synthesis method using anhydrous ethanol as a solvent. Biosafety evaluation demonstrated that it exhibits superior hemocompatibility to a clinically used BS330 bilirubin adsorber and can be used as a hemoperfusion adsorbent for bilirubin removal. The 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material combines excellent bilirubin selective removal performance with good biocompatibility, demonstrating its potential as a hemoperfusion adsorbent for the clinical treatment of hyperbilirubinemia.

[0043] In certain embodiments of the present invention, the adsorption performance of the adsorbent material can be determined by adsorption experiments. 10 mg of a 3-ethyl-4-methyl-3-pyrrolidone pseudo-templated molecularly imprinted polymer adsorbent material is placed in 10 mL of a solution containing a bilirubin molecular standard (100-1000 mg / L). After an adsorption reaction of 1.0-4.0 hours, the reacted solution is centrifuged at 10,000 rpm to obtain a supernatant that is diluted and placed in a quartz cuvette. The absorbance of the solution is measured at a wavelength of 441 nm, and the amount of bilirubin adsorbed by the adsorbent material is calculated based on the change in absorbance. The optimal adsorption reaction time for the molecularly imprinted polymer adsorbent material on the bilirubin molecular standard is 4.0 hours.

[0044] In certain embodiments of the present invention, the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material can be mixed with the adsorbent material in a commercially available disposable plasma bilirubin adsorber (BS330 model) to adsorb bilirubin in plasma samples. The adsorber chromatography column can be completely filled with the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material, or the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material can be mixed with the original adsorbent material and filled into the bilirubin adsorber.

[0045] The BS330 bilirubin adsorber uses a polyethylene-based anionic adsorbent. Due to its nonspecific nature, while removing bilirubin molecules during the hemoperfusion process, it may also adsorb some beneficial molecules, such as amino acids (particularly aromatic amino acids). A 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material is mixed with the styrene-based anionic adsorbent material in the BS330 bilirubin adsorber and then filled into an empty medium-pressure chromatography column for bilirubin adsorption from plasma samples. The 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material can comprise 0% to 100% of the total filler material. A constant flow peristaltic pump is used to control the flow rate of the actual plasma sample, passing it through the filled medium-pressure chromatography column at a constant flow rate for a specified period of time to adsorb and remove bilirubin molecules from the sample. The actual plasma sample flow rate, controlled by a constant-flow peristaltic pump, ranged from 20 mL / min to 50 mL / min, and the column flow time was 1 to 3 hours. Experimental results indicate that the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material effectively reduces the adsorption of aromatic amino acids in actual plasma samples during hemoperfusion.

[0046] The present invention is described below with reference to the accompanying drawings. Experimental methods not specifically described were performed according to the corresponding commercial specifications. Unless otherwise specified, the instruments, reagents, and consumables used in the examples are all commercially available. The mass fraction of 3-ethyl-4-methyl-3-pyrrolidone (3-pyrrolidone) is 98%, the mass concentration of 3-aminopropyltriethoxysilane (APTES) is 99%, and the mass concentration of tetraethoxysilane (TEOS) is 99%. Furthermore, porcine plasma, anticoagulated porcine blood, and a BS330 bilirubin adsorber used in the following examples are all commercially available.

[0047] Example 1: Preparation of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material

[0048] Preparation of polymer adsorbent materials:

[0049] 0.125 g of the pseudo-template molecule 3-ethyl-4-methyl-3-pyrroline-2-one, 1 mL of the functional monomer 3-aminopropyltriethoxysilane (APTES), and 1 mL of the cross-linking agent tetraethoxysilane (TEOS) were dissolved in 30 mL of anhydrous ethanol and stirred at room temperature for 2 h for prepolymerization reaction. 1 mL of ultrapure water was added to catalyze the hydrolysis reaction, and then the polymerization reaction was carried out by stirring at room temperature for 24 h.

[0050] Elution removal of pseudo-template molecule 3-ethyl-4-methyl-3-pyrrolidone-2-one:

[0051] Using a 50% (v / v) ethanol-ultrapure water solution, the pseudo-template molecule 3-ethyl-4-methyl-3-pyrroline-2-one was eluted and removed under ultrasound assistance until the pseudo-template molecule could no longer be detected using an ultraviolet spectrophotometer. The adsorbent material was then vacuum dried at 55°C to obtain a molecularly imprinted polymer adsorbent material (MIPs).

[0052] According to the above method, the non-imprinted polymer adsorbent materials (NIPs) of the present invention were prepared without adding the pseudo template molecule 3-ethyl-4-methyl-3-pyrrolidone. The prepared MIPs and NIPs were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. The test results are as follows: Figure 2-3 As shown, the prepared MIPs and NIPs are approximately microspherical, with a size of about 200 nm; the MIPs and NIPs maintain consistent morphology, indicating that the adsorbent material has been successfully prepared.

[0053] The structural characteristics of the prepared MIPs and NIPs were analyzed, such as Figure 4 The following are the Fourier transform infrared spectra of MIPs and NIPs. -1 The absorption peak is the asymmetric stretching of Si-O-Si, 781 cm -1 and 456cm -1 The characteristic peak is the vibration peak of Si-O, which proves the formation of Si-O-Si structure; 1646cm -1 The absorption peak is the vibration peak of C=O in the amide bond, 1554 cm -1 The peaks of the CN stretching vibration of the amide bond and the NH bending vibration peak are shown in Table 1. In addition, the characteristic peaks of MIPs and NIPs are basically consistent, indicating that their structural compositions are similar. Figure 5 The thermogravimetric analysis diagrams of MIPs and NIPs are shown. As can be seen from the figure, two stages of weight loss appear as the temperature increases. The weight loss at around 100°C is due to the dehydration of adsorbed water, while the weight loss at around 450°C is due to the decomposition of the adsorbent material, indicating that the adsorbent material prepared by the present invention has good thermal stability.

[0054] Example 2: Evaluation of the adsorption performance of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material

[0055] 2.1 Equilibrium adsorption binding experiments

[0056] 10 mg of the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material prepared in Example 1 was placed in 10 mL of a solution containing a bilirubin standard (100-1000 mg / L), and the adsorption reaction was carried out at a shaking speed of 250 rpm and a temperature of 37° C. in the dark for 4.0 h. Then, the supernatant obtained by centrifuging the reacted solution at a speed of 10,000 rpm was diluted and placed in a quartz cuvette. The absorbance of the solution was measured at a wavelength of 441 nm. The adsorption amount Q (mg / g) of the adsorbent material on the bilirubin molecule was calculated according to the change in the absorbance value according to the following formula:

[0057]

[0058] Where C0 (mg / L) is the concentration of the bilirubin standard solution before adsorption, C e (mg / L) is the concentration of the bilirubin standard solution after adsorption, V (L) is the volume of the bilirubin standard solution, and m (mg) is the mass of the added 3-ethyl-4-methyl-3-pyrrolidine-2-one pseudo-templated molecularly imprinted polymer adsorbent material.

[0059] The imprinting factor (IF) of the adsorbent material can be calculated by the following formula:

[0060]

[0061] Where Q MIP (mg / g) is the equilibrium adsorption capacity of molecularly imprinted polymer adsorbent materials (MIPs), Q NIP (mg / g) is the equilibrium adsorption capacity of non-imprinted polymer adsorbent materials (NIPs).

[0062] like Figure 6 Shown are the equilibrium adsorption and binding curves for the 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent. The figure shows that as the initial bilirubin concentration increases, the adsorption capacity of the target molecule by both adsorbents gradually increases. The calculated equilibrium adsorption capacity of the imprinted polymer adsorbent was 135.67 mg / L, while that of the non-imprinted polymer adsorbent was 34.05 mg / L. The calculated imprinting factor (IF) for the 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent was 3.98, indicating that the adsorbent material exhibited superior adsorption of the target bilirubin molecule. This result can be attributed to the absence of the pseudo-template molecule 3-ethyl-4-methyl-3-pyrrolin-2-one during the preparation of the non-imprinted polymer adsorbent. This results in the formation of amorphous pores within the adsorbent material, which are unable to attract and bind the target molecule, resulting in lower adsorption capacity.

[0063] 2.2 Adsorption kinetics experiments

[0064] 10 mg of the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material prepared in Example 1 was placed in 10 mL of a solution containing a bilirubin standard (500 mg / L), and the mixture was subjected to adsorption reaction at a shaking speed of 250 rpm and a temperature of 37° C. for 1.0-4.0 h in the dark. Then, the supernatant obtained by centrifuging the reaction solution at a speed of 10,000 rpm was diluted and placed in a quartz cuvette. The absorbance of the solution was measured at a wavelength of 441 nm. The adsorption amount Q (mg / g) of the bilirubin molecule by the adsorbent material was calculated according to the change in the absorbance value according to the following formula:

[0065]

[0066] Where C0 (mg / L) is the concentration of the bilirubin standard solution before adsorption, C e (mg / L) is the concentration of the bilirubin standard solution after adsorption, V (L) is the volume of the bilirubin standard solution, and m (mg) is the mass of the added 3-ethyl-4-methyl-3-pyrrolidine-2-one pseudo-templated molecularly imprinted polymer adsorbent material.

[0067] Figure 7 The adsorption kinetics experimental results of the 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material are shown. The figure shows that the adsorption equilibrium time of bilirubin molecules on the 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material is approximately 4.0 hours.

[0068] 2.3 Selective adsorption evaluation experiment

[0069] Two substances with similar molecular weight and structure to bilirubin and coexisting in the blood (cholesterol and testosterone) were selected as targets. At the same time, for comparison, 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material and styrene-based anion adsorbent material in BS330 bilirubin adsorber were used for adsorption, and the adsorption amount of each target molecule by the two adsorbent materials was calculated to investigate the selectivity of the adsorbent materials.

[0070] The specific experimental operation is as follows: 10 mg of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting polymer adsorbent material or BS330 adsorbent material is dispersed in 10 mL of bilirubin, cholesterol or testosterone solution with a concentration of 500 mg / L, and then the sample is adsorbed at 37°C for 4 hours, centrifuged to obtain each supernatant, and finally the concentration of each substance in the supernatant is measured using a UV-visible spectrophotometer, and the adsorption amount of each target molecule by the two adsorbent materials is calculated to investigate the selectivity of the adsorbent material. Among them, the structures of the pseudo-template molecule 3-ethyl-4-methyl-3-pyrroline-2-one, bilirubin molecule, cholesterol molecule and testosterone molecule are as follows: Figure 8 shown.

[0071] Adsorption selectivity evaluation results are as follows Figure 9 As shown, the adsorption amount of cholesterol and testosterone by the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent material is lower than that of the styrene-based anion adsorbent material in the BS330 bilirubin adsorber, which proves that the adsorbent material obtained by the present invention has good adsorption selectivity for bilirubin molecules.

[0072] Example 3: Evaluation of the Reusability of 3-Ethyl-4-methyl-3-pyrroline-2-one Pseudo-Template Molecularly Imprinted Polymer Adsorbent Materials

[0073] Plasma bilirubin adsorbers commonly used in clinical practice are disposable, whereas the molecularly imprinted polymer material prepared in the present invention is reusable and has the potential to be an ideal adsorbent material. Therefore, a reusability performance test of the adsorbent material was conducted.

[0074] 10 mg of 3-ethyl-4-methyl-3-pyrrolidine-2-one pseudo-templated molecularly imprinted polymer adsorbent material was placed in 10 mL of a solution containing a bilirubin molecular standard (100-1000 mg / L). The adsorption reaction was allowed to proceed for 1.0-4.0 hours. The resulting supernatant was diluted and placed in a quartz cuvette. The absorbance of the solution was measured at 441 nm. The amount of bilirubin adsorbed by the adsorbent material was calculated based on the change in absorbance. The adsorbent material was then eluted and placed again in 10 mL of a solution containing a bilirubin molecular standard (100-1000 mg / L).

[0075] The specific experimental procedure is as follows: 10 mg of 3-ethyl-4-methyl-3-pyrrolidine-2-one pseudo-templated molecularly imprinted polymer adsorbent material was placed in 10 mL of a 500 mg / L bilirubin solution. After adsorption at 37°C for 4 hours, the supernatant was collected by centrifugation. The absorbance of the supernatant was measured using a UV-visible spectrophotometer, and the amount of bilirubin adsorbed by the material was calculated. The resulting precipitate was then washed with 0.1 M NaOH solution until no significant UV absorption peak was detected at 441 nm. The material was then washed once with deionized water to complete the adsorption experiment.

[0076] The adsorbent material was used to repeat the adsorption-elution cycle of bilirubin molecules four times to verify the reusability of the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent material. The adsorption amount of each time was detected and statistically analyzed. The results are as follows: Figure 10 As shown in the adsorption data, it can be seen that after four reuses, the adsorption amount of bilirubin molecules by the adsorbent material can still maintain more than 90% of the adsorption amount after the first cycle, confirming its excellent reusability.

[0077] Example 4: Evaluation of the pressure resistance of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material

[0078] Since clinically used blood perfusion devices need to be sterilized, and most sterilization processes are performed by moist heat sterilization, the pressure resistance of the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material prepared by the present invention was measured and evaluated.

[0079] The experimental procedure was as follows: The prepared 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material was sterilized in an autoclave at 121°C for 15 minutes. After cooling to room temperature, 10 mg of the 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material was placed in 10 mL of bilirubin solutions at concentrations of 100, 200, 300, and 500 mg / L, respectively. After adsorption at 37°C for 4 hours, the supernatants were collected by centrifugation, and the absorbance of each supernatant was measured using a UV-visible spectrophotometer to calculate the bilirubin adsorption capacity of the material. The morphology of the sterilized adsorbent material was observed using a transmission electron microscope (TEM).

[0080] like Figure 11Shown are comparisons of the adsorption capacity of bilirubin molecules by a 3-ethyl-4-methyl-3-pyrrolidone pseudo-templated molecularly imprinted polymer adsorbent material before and after sterilization, as well as transmission electron microscopy characterization of the adsorbent material before and after sterilization. The results show that after sterilization, the adsorbent material's adsorption capacity for bilirubin molecules decreases slightly; however, it still maintains efficient adsorption. Furthermore, pressure also causes the material's surface to become smoother and its morphology to become more regular. Finally, the adsorbent material's pressure resistance is demonstrated, demonstrating that the adsorbent material prepared by this invention maintains its adsorption capacity for bilirubin molecules after sterilization without significant morphological changes.

[0081] Example 5: Evaluation of biocompatibility of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent materials

[0082] 5.1 Hemolytic performance evaluation

[0083] Because of direct contact with blood, the biocompatibility of adsorbent materials in hemoperfusion devices is a primary prerequisite for their application, and hemolytic performance is one aspect of biocompatibility that requires examination. Therefore, to demonstrate the good biocompatibility of the 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material prepared in this invention, the hemolytic performance of the adsorbent material was measured and evaluated. The specific method is as follows:

[0084] The experiment used red blood cells separated from fresh anticoagulated pig blood for hemolysis test. Take 2mL of pig blood, centrifuge at 3000rpm for 10min, wash the sediment layer with PBS solution 5 times to obtain fresh red blood cells; next, take 300μL of red blood cells and mix them with 1200μL of deionized water and PBS solution respectively as positive control samples and negative control samples. Then, the adsorbent material prepared in Example 1 and the styrene-based anion adsorbent material in the BS330 bilirubin adsorber were immersed in the resuspended red blood cell solution (diluted with PBS solution) respectively, so that the ratio of red blood cells to the two adsorbent materials was 1:11-1:15, and the sample was incubated at 37°C for 2h, centrifuged at 8000rpm for 5min, and the absorbance of the obtained supernatant was measured at 541nm. The calculation formula of the hemolysis rate is as follows:

[0085]

[0086] Where A is the absorbance value of each sample obtained by measurement, A N A is the absorbance value of the negative control sample obtained. P is the absorbance value of the positive control sample obtained by measurement.

[0087] The results of the test are as follows Figure 12Figure 2 shows the hemolysis rate evaluation of the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material and the styrene-based anion adsorbent material in the BS330 bilirubin adsorber. Generally speaking, a lower hemolysis rate means a lower hemolysis risk. Among all tested samples, the hemolysis rate of the adsorbent prepared in Example 1 ranged from 0.10% to 1.35%. Even at a high contact dose between the adsorbent material and red blood cells, the hemolysis rate was below the 5% safety threshold required for biomaterials (GB / T 16886.4-2022). The hemolysis rate of the styrene-based anion adsorbent material in the BS330 bilirubin adsorber ranged from 0.84% ​​to 2.80%, higher than that of the adsorbent material prepared in the present invention. This indicates that the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material has good hemocompatibility and can be used in hemoperfusion therapy.

[0088] 5.2 Coagulation performance evaluation

[0089] The second aspect of biocompatibility that needs to be examined is the coagulation performance of the adsorbent material. To this end, the prepared 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material was evaluated by four coagulation tests. The specific methods are as follows:

[0090] The anticoagulant activity of the materials was evaluated by measuring activated partial thromboplastin time (APTT), prothrombin time (PT), prothrombin time (TT), and fibrinogen ionization (FIB). 450 μL of fresh porcine platelet-poor plasma (PPP) was added to 50 μL of 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material and 50 μL of styrene-based anion adsorbent material in a BS330 bilirubin adsorber (both adsorbent concentrations were 500 μg / mL). After incubation at 37°C for 30 minutes, the sample solutions were centrifuged to allow the sample to settle to the bottom. 100 μL of plasma was aspirated and the APTT, PT, TT, and FIB values ​​were measured using a coagulation analyzer. An equal volume of normal saline was added to the blank control group in place of the sample solution.

[0091] The results are as follows Figure 13Figure 2 shows the four coagulation evaluation tests for a 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material and a styrene-based anion adsorbent material in a BS330 bilirubin adsorber. For the four coagulation evaluation tests, APTT and TT are used to assess the sample's in vitro antithrombotic properties, PT is used to assess the sample's exogenous coagulation capacity, and FIB content is associated with proteins involved in coagulation activation. The results show that the APTT, TT, and PT values ​​of the adsorbent material prepared by the present invention are all higher than those measured for the styrene-based anion adsorbent material in a BS330 bilirubin adsorber, effectively prolonging coagulation time and demonstrating that the adsorbent material has certain anticoagulant properties. Furthermore, compared to the platelet-poor plasma control group, the plasma FIB concentration of the adsorbent material prepared by the present invention did not change significantly, indicating that coagulation is less likely to occur. These results demonstrate that the 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material prepared by the present invention has good biocompatibility, ensuring its safety in hemoperfusion applications.

[0092] 5.3 Cytotoxicity studies

[0093] Biocompatibility also requires the examination of the cytotoxicity of the adsorbent material to evaluate its safety. To this end, the cytotoxicity of the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material prepared by the present invention was evaluated. The specific method is as follows:

[0094] Cell culture: Human embryonic kidney cells (HEK293) and human hepatoma cells (HepG2) were revived and grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin). The cells were cultured in a humidified incubator at 37°C with 5% CO2 for 24 h.

[0095] Sample solution preparation: The 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted polymer adsorbent material prepared by the present invention and the styrene-based anion adsorbent material in the BS330 bilirubin adsorber were respectively prepared into sample solutions with concentrations of 10, 50, 100, 200, and 500 μg / mL using PBS solution.

[0096] Cytotoxicity assay: The standard CCK-8 method was used to evaluate the cytotoxicity of the two adsorbent materials. The specific method was as follows: about 1×10 5Cells (approximately 100 μL of cell suspension per well) were cultured for 24 hours. 10 μL of sample solution at concentrations of 10, 50, 100, 200, and 500 μg / mL was added to each well. After another 24 hours of co-incubation, 10 μL of CCK-8 reagent was added. The cells were then incubated in an incubator for another 2 hours. The absorbance of the samples was measured at 450 nm. The cell viability of the two cell types co-cultured with samples of different concentrations was calculated to evaluate the cytotoxicity of the adsorbent material. An equal amount of DMEM medium was added to the control group instead of the sample material for incubation.

[0097] The results are as follows Figure 14 As shown, Figure 14 Figures show cytotoxicity assay evaluations of a 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material and a styrene-based anion adsorbent material in a BS330 bilirubin adsorber. The results show that as the concentration of the adsorbent material co-cultured with cells increases (10, 50, 100, 200, and 500 μg / mL), the viability of both cell types decreases to varying degrees. However, the adsorbent material prepared in the present invention has a higher cell viability than the styrene-based anion adsorbent material in a BS330 bilirubin adsorber, indicating that the 3-ethyl-4-methyl-3-pyrrolin-2-one pseudo-templated molecularly imprinted polymer adsorbent material has better biocompatibility than clinically used adsorbent materials.

[0098] Example 6: Application of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material in the adsorption and removal of bilirubin molecules

[0099] The 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material was mixed with a clinically used BS330 bilirubin adsorber in a certain proportion and then packed together to adsorb and remove bilirubin molecules. The specific method is as follows:

[0100] The 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material was mixed with the styrene-based anion adsorbent material in the BS330 bilirubin adsorber in a ratio of 0% to 100% of the total filler. In this embodiment, experimental groups with molecularly imprinted polymer adsorbent material accounting for 0% (control), 10%, 30%, 50%, and 100% were prepared in sequence. Then, a constant flow peristaltic pump was used to control the flow rate of the porcine plasma sample at 20 mL / min through the filled mixing column. After the porcine plasma sample was adsorbed for 2 hours, the porcine plasma sample after the column was taken, diluted, and placed in a quartz cuvette. The absorbance of the solution was measured at a wavelength of 441 nm using an ultraviolet spectrophotometer, and the adsorption amount of bilirubin molecules by the adsorbent material was calculated based on the change in absorbance value.

[0101] High-performance liquid chromatography (HPLC) was used to determine the tyrosine and phenylalanine content in porcine plasma samples after passage through columns containing various ratios of mixed adsorbent materials (calculated from a calibration curve of amino acid standards obtained simultaneously). Chromatographic conditions were as follows: an AccQ.Tag amino acid analysis column (3.9 × 150 mm), mobile phase A consisting of AccQ.Tag mobile phase diluent (pH 5.02), mobile phase B consisting of acetonitrile, an excitation wavelength of 250 nm, an emission wavelength of 395 nm, a flow rate of 1.0 mL / min, an injection volume of 10 μL, and a column temperature of 37°C.

[0102] The following steps were used to process and derivatize the porcine plasma samples after column chromatography: After centrifugation, the porcine plasma samples were added with 0.1M HCl and 24% perchloric acid to precipitate proteins. The supernatant was filtered through a membrane, and 70 μL of AccQ.Tag Amino Acid Analysis Buffer and 20 μL of AccQ.Tag Amino Acid Analysis Derivatization Reagent were added to 10 μL of the sample solution. The mixture was reacted at 55°C for 10 minutes. After cooling to room temperature, the sample was placed in a desiccator overnight to allow for full derivatization before injection and testing. The derivatization procedures for amino acid standards were the same as above.

[0103] In addition, since tryptophan needs to generate a chromogenic substance under alkaline conditions and cannot be determined using the AccQ.Tag method (derivation under neutral conditions), the spectrophotometric method was used according to the national standard method (GB / T 15400-2018). The specific steps are as follows:

[0104] The processing steps for the pig plasma samples after column are the same as above. Take 100 μL of sample solution and place it in a cold water bath, add 250 μL of p-dimethylaminobenzaldehyde solution-sulfuric acid solution and shake well, take it out and let it stand at room temperature for 30 minutes, then add 100 μL of 0.2% sodium nitrite solution, let it stand at room temperature for 25 minutes, and then use a UV spectrophotometer to measure the absorbance of the sample at a wavelength of 590 nm. The blank calibration solution of the sample is processed as follows: take 100 μL of sample solution and place it in a cold water bath, add 250 μL of sulfuric acid solution (21.2 mol / L) and shake well, take it out and let it stand at room temperature for 30 minutes, then add 100 μL of 0.2% sodium nitrite solution, let it stand at room temperature for 25 minutes, and then measure the absorbance at a wavelength of 590 nm.

[0105] At the same time, the absorbance of tryptophan standards of different concentrations was measured to obtain a standard curve for calculating the tryptophan content in the sample. The specific operation was as follows: 500 μL of tryptophan standard solution of each concentration was taken, and 500 μL of 10% potassium hydroxide solution was added, mixed, and placed in a cold water bath. Then, 2.5 mL of p-dimethylaminobenzaldehyde solution-sulfuric acid solution was added, taken out and shaken, and placed at room temperature for 30 minutes. Then, 100 μL of 0.2% sodium nitrite solution was added, and after placing at room temperature for 25 minutes, the absorbance was measured using an ultraviolet spectrophotometer at a wavelength of 590 nm, where water was used as the blank calibration solution.

[0106] The results are as follows Figure 15 As shown, Figure 15 The following graph shows the adsorption of bilirubin molecules in porcine plasma samples after passage through a column containing mixed adsorbents of varying ratios of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer and styrene-based anion adsorbent in a BS330 bilirubin adsorber. Furthermore, Table 1 shows the changes in the aromatic amino acid content in porcine plasma samples after passage through a column containing mixed adsorbents of varying ratios of 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer and styrene-based anion adsorbent in a BS330 bilirubin adsorber.

[0107] Table 1 Aromatic amino acid content in pig plasma samples after passing through the columns with adsorbent materials mixed in two different ratios

[0108]

[0109] The data in the table show that as the proportion of the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material in the mixed adsorbent material column increases, the content of the three aromatic amino acids in the pig plasma sample after passing through the column increases, indicating that the addition of the adsorbent material prepared by the present invention can effectively avoid the adsorption of aromatic amino acids in the plasma sample, further demonstrating the specific adsorption performance of the adsorbent material; and because the BS330 bilirubin adsorber used clinically is a polyethylene-based anion adsorbent with no specificity, it can adsorb aromatic amino groups to varying degrees while removing bilirubin molecules during the blood perfusion process. In summary, the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-templated molecularly imprinted polymer adsorbent material prepared by the present invention and the styrene-based anion adsorbent material in a clinically used BS330 bilirubin adsorber are mixed in a certain proportion and then co-filled into a column for adsorption and removal of bilirubin molecules.

[0110] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for preparing a 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material for removing bilirubin, characterized in that: A polymer adsorbent material was prepared using 3-ethyl-4-methyl-3-pyrroline-2-one as a pseudo-template molecule. After eluting 3-ethyl-4-methyl-3-pyrroline-2-one from the polymer adsorbent material, a 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material for removing bilirubin was obtained.

2. The method for preparing the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material for removing bilirubin according to claim 1, characterized in that: Using 3-aminopropyltriethoxysilane as the functional monomer, tetraethoxysilane as the cross-linker, and 3-ethyl-4-methyl-3-pyrroline-2-one as the pseudo-template molecule, a polymer adsorbent material was obtained by catalytic hydrolysis reaction using ultrapure water; after ultrasonic removal of the pseudo-template molecule 3-ethyl-4-methyl-3-pyrroline-2-one, a 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin was obtained.

3. The method for preparing the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material for removing bilirubin according to claim 2, characterized in that: The specific steps are as follows: Step 1: dissolving 3-ethyl-4-methyl-3-pyrroline-2-one and 3-aminopropyltriethoxysilane in anhydrous ethanol, performing a prepolymerization reaction under stirring, adding a crosslinking agent tetraethoxysilane and ultrapure water, performing a catalytic hydrolysis reaction, and centrifuging after the reaction to obtain a polymer adsorbent material; the molar ratio of 3-ethyl-4-methyl-3-pyrroline-2-one, 3-aminopropyltriethoxysilane, and tetraethoxysilane is 1:4-12:4-12; Step 2: Using a 50% (v / v) ethanol-ultrapure water solution, ultrasonically remove the pseudo-template molecule 3-ethyl-4-methyl-3-pyrroline-2-one, and after drying, obtain a 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin.

4. A 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material for removing bilirubin prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecularly imprinted adsorption material for removing bilirubin according to claim 4 in the adsorption of bilirubin molecules.

6. The use according to claim 5, characterized in that: The 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin, or a mixture of the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin and a styrene-based anion adsorbent material, is filled into an empty medium-pressure chromatography column for adsorbing bilirubin molecules in a plasma sample.

7. The use according to claim 6, characterized in that: The 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material used to remove bilirubin accounts for 0%-100% of the total filler amount.

8. The use according to claim 6, characterized in that: The flow rate of the plasma sample was controlled by a constant flow peristaltic pump at 20 mL / min-50 mL / min.

9. A plasma bilirubin adsorber filler, characterized in that: The invention comprises the 3-ethyl-4-methyl-3-pyrroline-2-one pseudo-template molecular imprinting adsorption material for removing bilirubin as claimed in claim 4.

Images