Preparation method and application of stationary phase material for screening acetylcholine interferent
The acetylcholine-specific recognition material was prepared through molecular imprinting technology, which solved the problem of efficient screening and separation of acetylcholine interferors and achieved a highly selective and stable detection effect, which is particularly suitable for the rapid screening of complex environmental samples.
Patent Information
- Application Number
- CN202510541419.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2025-10-10
Smart Images
Figure CN120754828A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of new materials, and in particular to a preparation method and application of a fixed phase material for screening acetylcholine interferents BACKGROUND
[0002] As an important neurotransmitter of cholinergic nervous system, the dynamic balance of acetylcholine synthesis, release, receptor binding and hydrolysis metabolism is crucial for maintaining neural signal transmission. Studies have shown that exogenous chemicals (such as carbamate pesticides, neonicotinoid insecticides, etc.) can interfere with cholinergic nerve function through various ways, mainly including: inhibiting acetylcholinesterase, leading to excessive accumulation of synaptic acetylcholine; competitively antagonizing acetylcholine receptors (such as nicotinic or muscarinic receptors), blocking normal neurotransmitter binding; disrupting choline transporter function or inhibiting choline acetyltransferase activity, interfering with acetylcholine synthesis and metabolism balance. However, the heterogeneity of the action target and molecular mechanism of such substances makes it difficult for traditional screening techniques to achieve a balance between broad spectrum coverage and high specificity recognition, limiting the efficient monitoring and risk assessment of neurotoxic interferents.
[0003] Currently, the detection of acetylcholine interferents mainly relies on methods such as radioligand binding experiments, choline transporter inhibition experiments, and acetylcholinesterase activity detection. Although these techniques have biological specificity, there are still the following key bottlenecks: poor stability: enzymes and receptors are prone to inactivation, making long-term preservation difficult; complex operation: multi-step pretreatment, long cycle, high cost; insufficient selectivity: weak ability to distinguish structural analogs; strong matrix interference: high false positive / negative rate in complex samples; inefficient combination technology: chromatography-activity detection tandem process is cumbersome and has low throughput. Affinity chromatography based on biological recognition elements (such as immobilized acetylcholinesterase and acetylcholine receptor) can achieve "recognition-separation" integration, but is still limited by: low immobilization efficiency and rapid activity decay; poor tolerance to organic solvents; difficult regeneration, short column life. These technical limitations have severely restricted the high-throughput screening and environmental risk assessment efficiency of acetylcholine interferents, and there is an urgent need to develop new biomimetic recognition materials and analysis strategies. Molecular imprinting technology, by constructing synthetic receptors with "artificial antibody" characteristics, is expected to break through the stability bottleneck of existing biological recognition elements, providing a new technical path for the screening of acetylcholine interferents in complex environmental matrices.
[0004] The basis for most acetylcholine interferents to produce neurotoxic effects lies in their structural similarity to acetylcholine molecules. Such a class of compounds can mimic the interaction of acetylcholine with cholinergic receptors, or competitively bind the active site of acetylcholinesterase. Based on this property, the development of artificial receptor materials with acetylcholine specific recognition ability can provide a new idea for the identification of acetylcholine interferents in environmental and biological samples. Molecular imprinting technology forms a recognition cavity complementary to the three-dimensional structure of the template molecule (such as acetylcholine) in the polymer network through the directional assembly and cross-linking polymerization of functional monomers and template molecules. This "molecular memory" effect endows the material with specific recognition ability similar to antibodies, while also having high stability (resistant to high temperature, acid and base, and organic solvents, excellent mechanical strength) and controllable selectivity (precise recognition of acetylcholine and its structural analogues through spatial structure memory of the template molecule). By packing acetylcholine molecularly imprinted polymers as a stationary phase in a separation column, specific capture, high-efficiency separation and rapid screening can be achieved. By combining with detection techniques such as mass spectrometry, a screening platform for acetylcholine interferents can be established. Compared with traditional biological affinity chromatography, this separation strategy based on molecular recognition shows stronger applicability in environmental sample analysis, especially for handling actual samples containing organic solvents or complex matrices. SUMMARY
[0005] The present application aims to use molecular imprinting biomimetic recognition strategy to construct a molecularly imprinted material with acetylcholine specific recognition ability, to realize the screening, separation and detection of acetylcholine interferents in environmental and biological samples.
[0006] To achieve the purpose of the present application, the following technical solutions are adopted:
[0007] A preparation method and application of a fixed phase material for screening acetylcholine interferents, comprising the preparation of an acetylcholine interferent fixed phase material and the screening of acetylcholine interferents. The specific steps are as follows:
[0008] 1. Preparation of acetylcholine interferent fixed phase material
[0009] (1) Dissolve a certain mass of acetylcholine in methanol, add functional monomers and cross-linking agents, and add metal catalysts, ligand and initiator modified silica gel microspheres, stir at a temperature of 60-80°C and under a nitrogen atmosphere for 12-24 hours; after polymerization, the silica gel microspheres are washed with organic reagents for 3-6 times, and then Soxhlet extracted in eluent for 24-48 hours;
[0010] According to the above scheme, the mass / volume concentration of acetylcholine in step (1) is 1-10 g / L.
[0011] According to the above scheme, the molar ratio of acetylcholine to the functional monomer in step (1) is 1:2-1:10, and the functional monomer is one of methyl methacrylate, 2-vinylpyridine and histamine acrylamide.
[0012] According to the above scheme, the molar ratio of acetylcholine to the crosslinking agent in step (1) is 1:10-1:60, and the crosslinking agent is one of ethylene glycol dimethacrylate, N,N'-methylene bisacrylamide, trimethylolpropane trimethacrylate, divinylbenzene and pentaerythritol triacrylate.
[0013] According to the above scheme, the metal catalyst in step (1) is cuprous bromide or cuprous chloride, the ligand is one of N,N,N',N",N"-pentamethyldiethylenetriamine, 2,2-bipyridine or tris(2-pyridylmethyl)amine, and the molar ratio of the metal catalyst to the ligand is 1:1-1:3.
[0014] According to the above scheme, the initiator-modified silica gel microspheres in step (1) are 2-bromoisobutyryl or 2-bromopropionyl-modified silica or polystyrene silica microspheres, and the mass-volume concentration is 10-50 g / L.
[0015] According to the above scheme, the preparation method in step (1) is characterized in that the organic solvent is methanol or ethanol.
[0016] According to the above scheme, the eluent in step (1) is a methanol or ethanol solution of acetic acid or ammonia water with a volume ratio of 1:5-10.
[0017] 2. Screening of acetylcholine interferents
[0018] The acetylcholine imprinted material prepared by the method provided in the present application is filled in a chromatographic column, and the size of the chromatographic column is 4.6*100-200 mm. The mobile phase is hexane / isopropanol=80 / 20 (v / v), the flow rate is 0.5-1 mL / min, the column temperature is 25-35°C, and the detection wavelength is 230-290 nm. The evaluated pollutants are phthalate esters, ultraviolet absorbers, polycyclic aromatic hydrocarbons, bisphenols and carbamates, etc.
[0019] The present application has the following advantages:
[0020] (1) The present application is based on the preparation of a specific recognition material for screening acetylcholine interferents by molecular imprinting technology, which can realize high selective recognition and separation of acetylcholine interferents. Compared with traditional biological recognition elements (such as acetylcholine receptor and acetylcholinesterase), the material has the advantages of low preparation cost, high stability and strong organic solvent resistance, and realizes efficient integration of recognition and detection.
[0021] (2) The screening acetylcholine interferent stationary phase material of the application simplifies the complex sample pretreatment process and significantly improves the detection throughput. Actual application advantages: this technology is particularly suitable for rapid screening of acetylcholine interferents in complex matrices such as environmental samples, solving the technical bottlenecks of traditional methods such as complicated operation and poor stability. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 is the infrared spectrum of the bare silica gel and acetylcholine imprinted material of the application.
[0023] Figure 2 is the chromatogram of various pollutants on the acetylcholine imprinted column. DETAILED DESCRIPTION
[0024] The application will be further explained in conjunction with the accompanying drawings and examples, which are only limited to illustrate the application and not limit the scope of the application.
[0025] EXAMPLE
[0026] Example 1 Preparation of acetylcholine imprinted material
[0027] (1) Synthesis of histamine acrylamide functional monomer
[0028] 4g of histamine dihydrochloride was dissolved in 20mL of distilled water, and the solution was cooled to 0℃ in an ice bath. Under nitrogen and magnetic stirring, 1.967g of acryloyl chloride in 20mL of anhydrous dichloromethane solution and NaOH aqueous solution (2.62g of NaOH, 20mL of pure water) were added simultaneously dropwise within 1 hour. Then, the reaction mixture was stirred at room temperature for 4 hours, transferred to a separatory funnel, allowed to stand and separate into layers, the aqueous layer was taken and freeze-dried for 72 hours. The residue was re-suspended in 200mL of ethanol and stirred uniformly, then the insoluble matter was removed by suction filtration, and the filtrate was rotary evaporated. Purification was performed by silica gel column chromatography (volume ratio of dichloromethane:methanol = 10:1), and white solid was obtained after evaporation.
[0029] (2) Preparation of acetylcholine imprinted material
[0030] 182mg of acetylcholine, 990mg of histamine acrylamide were dissolved in 100mL of methanol, ultrasonicated for 15min, pre-polymerized for 6 hours, 4250μL of divinylbenzene, 28.7mg of cuprous bromide, 66μL of N,N,N',N",N"-pentamethyldivinyltriamine, 2g of 2-bromoisobutyryl modified silica sphere initiator were added, vacuumed to remove oxygen, and nitrogen was passed. Stir the reaction at 65℃ for 24h. The polymerized silica gel microspheres were washed with methanol for 5 times, and then Soxhlet extracted in eluent acetic acid / methanol = 1 / 9 (v / v) for 36 hours;
[0031] Preparation of non-imprinted material: 990 mg of histamine acrylamide was dissolved in 100 mL of methanol, ultrasonic for 15 min, pre-polymerization for 6 hours, 4250 μL of divinyl benzene, 28.7 mg of cuprous bromide, 66 μL of N, N, N', N", N"-pentamethyl divinyl triamine, 2 g of 2-bromoisobutyryl modified silica gel initiator were added, vacuumed to remove oxygen, and nitrogen was introduced. The reaction was stirred at 65°C for 24 h. The polymerized silica gel microspheres were washed with methanol for 5 times, and then Soxhlet extracted for 36 h in eluent acetic acid / methanol = 1 / 9 (v / v). Since the non-imprinted material does not add acetylcholine template molecules, specific recognition sites will not be formed.
[0032] Figure 1 The infrared spectrum of the acetylcholine imprinted material. Compared with the unmodified silica gel, the acetylcholine imprinted material has the stretching vibration peaks of amide at 1651 and 1543 cm -1 , which indicates the successful coupling of molecular imprinting.
[0033] Example 2 Screening of acetylcholine interferents
[0034] The acetylcholine imprinted silica gel microspheres were packed in a 4.6*150 mm stainless steel chromatographic column to make an acetylcholine imprinted column. A mixed standard solution of pollutants with a concentration of 10 μg / mL was prepared, and hexane / isopropanol = 80 / 20 (v / v) was used as the mobile phase to analyze the retention time of different pollutants. Figure 2 The chromatogram of the acetylcholine imprinted column separating a variety of pollutants; the results show that the stationary phase can realize the complete separation of different types of a variety of pollutants within 15 min, and the chromatographic peak shape is symmetrical. The retention time of carbamate is higher than that of other pollutants, which proves that the acetylcholine imprinted column has the ability to screen acetylcholine interferents.
Claims
1. A method for preparing a stationary phase material for screening acetylcholine interferors, characterized in that: The specific steps are as follows: dissolving acetylcholine in methanol, adding a functional monomer and a cross-linking agent, adding a metal catalyst, a ligand and silica microspheres modified with an initiator, stirring and reacting at a temperature of 60 to 80°C in a nitrogen atmosphere for 12 to 24 hours; washing the polymerized silica microspheres with an organic solvent for 3 to 6 times, and then performing Soxhlet extraction in an eluent for 24 to 48 hours; the molar ratio of acetylcholine to the functional monomer is 1:2 to 1:10, and the molar ratio of acetylcholine to the cross-linking agent is 1:10 to 1:
60.
2. The preparation method according to claim 1, characterized in that The acetylcholine is dissolved in methanol at a mass volume concentration of 1 to 10 g / L.
3. The preparation method according to claim 1, wherein: The functional monomer is selected from one of methyl methacrylate, 2-vinyl pyridine and histamine acrylamide; the crosslinking agent is selected from one of ethylene glycol dimethacrylate, N,N'-methylenebisacrylamide, trimethylolpropane trimethacrylate, divinylbenzene and pentaerythritol triacrylate.
4. The preparation method according to claim 1, characterized in that The metal catalyst is cuprous bromide or cuprous chloride, the ligand is one of N,N,N',N",N"-pentamethyldiethylenetriamine, 2,2-bipyridine or tris(2-pyridylmethyl)amine, and the molar ratio of the metal catalyst to the ligand is 1:1 to 1:
3.
5. The preparation method according to claim 1, wherein: The initiator-modified silica microspheres are 2-bromoisobutyryl or 2-bromopropionyl-modified silica or polystyrene silica microspheres, and the mass volume concentration thereof is 10-50 g / L.
6. The preparation method according to claim 1, characterized in that The organic solvent is methanol or ethanol.
7. The preparation method according to claim 1, characterized in that The eluent is a solution in which the volume ratio of acetic acid or ammonia water to methanol or ethanol is 1:5-10.
8. A chromatographic column for screening acetylcholine interferors, characterized in that: The stationary phase is filled with the materials described in claims 1-7.
9. A method for screening acetylcholine interferors, characterized in that: The sample to be tested is passed through the chromatographic column according to claim 8, with hexane / isopropanol = 80 / 20 (v / v) as the mobile phase, and screening is achieved by analyzing the retention behavior of each component on the chromatographic column.