CN108246271B - Preparation method of molecularly imprinted polymer microspheres for detecting 2, 4, 6-trinitrophenol - Google Patents

Preparation method of molecularly imprinted polymer microspheres for detecting 2, 4, 6-trinitrophenol Download PDF

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CN108246271B
CN108246271B CN201810155489.8A CN201810155489A CN108246271B CN 108246271 B CN108246271 B CN 108246271B CN 201810155489 A CN201810155489 A CN 201810155489A CN 108246271 B CN108246271 B CN 108246271B
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高大明
陈倩云
陈红
刘辰辰
张宇钢
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Holmes Beijing Biotechnology Co ltd
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Abstract

A preparation method of molecularly imprinted polymer microspheres for detecting 2, 4, 6-trinitrophenol is characterized by comprising the following steps: the poly-nitrophenol derivative engram molecule generated by the reaction of 2, 4, 6-trinitrophenol and N, N' -diisopropyl carbodiimide has bright yellow fluorescence, the fluorescence of the molecular engram polymer microsphere has the opening and closing characteristic under the acid-base condition, the engram molecules positioned in the engram layer are eluted from the molecular engram polymer microsphere, a cavity structure which is complementary with the structure, the size and the functional group of the engram molecules is formed in the engram layer, the polymer microsphere for eluting the engram molecules has a specific recognition site for target analyte molecules, the target molecules enter the specific recognition site to realize the selective recognition of the target analyte molecules, the sensitivity detection of the target molecules is realized through the opening and closing of the fluorescence of the target molecules, the molecularly imprinted microspheres have large specific surface area, stable spatial structure of formed recognition sites, more effective sites, high selective recognition and high sensitive detection on target molecules.

Description

Preparation method of molecularly imprinted polymer microspheres for detecting 2, 4, 6-trinitrophenol
Technical Field
The invention relates to the field of material science, in particular to a preparation method for detecting 2, 4, 6-trinitrophenol molecular imprinting.
Background
The international relations in the world are complex and changeable, terrorist violent incidents such as explosion and the like frequently occur, the safety of lives and properties and the social stability of people in all countries in the world are seriously harmed, and the safety and the stability of the countries and the regions are threatened. Therefore, the detection and analysis of explosives draws great attention from research institutions of various countries, and attempts are made to predict the existence of explosives by analyzing and detecting technical means so as to reduce the harm of explosives to human beings. 2, 4, 6-trinitrophenol (2, 4, 6-trinitrophenol, TNP) belongs to a phenolic substance, also known as Picric Acid (PA). The raw materials are easy to obtain, the preparation is simple, the explosive power is large, and the explosive is one of common explosives. Meanwhile, picric acid is also used as a bactericide and mildew remover in agriculture and a bactericide and astringent in medicine. The long-term contact of picric acid can cause symptoms such as headache, nausea, diarrhea, fever and the like, and has great harm to the body. With the development of industrialization, a large amount of picric acid is discharged into water, which causes water source pollution. Picric acid has wide application, and is harmful to human beings due to improper use, so that a technical means for quickly identifying and sensitively detecting explosives is urgently needed to effectively reduce the harm of the explosives to the human beings, and the practical significance is very important.
The existing methods for detecting explosives include spectrophotometry, electrochemistry, capillary electrophoresis, high performance liquid chromatography, gas chromatography and the like (Bowerbank C R; Smith P A; Fetterolfd D.JChromatogr. A，2000，902(2)：413-419；Wayne H Griest；Stacy-Ann Barshick. Anal. Chem.1998, 70, 3015-3020), these methods are less versatile and are prone to column contamination, and therefore new detection methods are continually sought. Ronitia L published academic paper (Talanta,2005, 66(2): 581-590) High Performance Liquid Chromatography-photoelectric assisted electrochemical Detection (High-Performance Liquid Chromatography-assisted electrochemical Detection-Chemical Detection, HPLC-PAED) in combination with Ultraviolet absorption (UV) Detection for the determination of explosives in environmental samples. The system utilizes an online Solid Phase Extraction (SPE) technology to carry out sample pretreatment (namely fractionation and concentration), and the SPE can separate and pre-concentrate target analytes according to a simple chromatographic principle and is suitable for evaluating explosives in underground water. Roger T published paper (Anal. Chem.1999, 71, 2739-2744) proposed a method for rapid serial injection spectrophotometry for the determination of 2, 4, 6-trinitrotoluene (2, 4, 6-trinitrotoluene, TNT) in soil samples. The method is based on the derivatization of TNT and sodium sulfite in an alkaline acetone mediumAnd (4) reacting. And the sample used in the reaction, the percentage of acetone used, the volume of the reagent, the volume of the mixing coil, the reaction time and other conditions are optimized. The reaction was found to be particularly sensitive to acetone concentration, being optimal in an 88% acetone/water (v/v) medium. And response research of the method to other explosives shows that the method only has specific selectivity to TNT. Xinyang academy of teachni Cao Xinhua et al discloses an invention patent (CN 201610716172.8) "an organic gel compound of naphthalimide, a preparation method, gel and application thereof", wherein the invention uses 4-bromine-1, 8-naphthalic anhydride and alkylamine to generate N-alkyl-4-bromine 1, 8-naphthalimide through heating reflux reaction in toluene solution according to the molar ratio of 1: 1.2; then in the presence of potassium carbonate, N-alkyl-4-bromo-1, 8-naphthalimide and 4-hydroxypyridine react in a dimethyl sulfoxide solution at a molar ratio of 1:1.5 at 100 ℃ for 12 hours, naphthalimide groups with fluorescent properties are introduced by using simple alkylamine, and a gel can be formed, and the gel is used for detecting 2, 4, 6-trinitrophenol.
Fluorescence analysis is also a common method for detecting explosives. Arvin Sain Tanwar et al, (supra) published a paperACS Sens.2016, 1, 1070, 1077) describes a novel polyfluorene derivative, a high-yield nitro-explosive picric acid with a level of 22.9 pg is synthesized by a suzuki coupling polymerization method, and the polymer is well characterized by nuclear magnetic resonance, ultraviolet visible, fluorescence, time-resolved photoluminescence spectrum and cyclic voltammetry. The derivative realizes amplified signal response specially used for picric acid through strong internal filtering effect, which is different from widely reported probes based on ground state charge Transfer of nitroarene detection or based on resonance Energy Transfer (FRET), and the pendant amine group on the side chain of the polyfluorene derivative can provide higher sensitivity and excellent selectivity even in the presence of most common interfering nitro explosives and other analytes usually found in natural water through protonation assisted Photoinduced Electron Transfer (PET). Therefore, the platform based on the polyfluorene derivative can be very high even in the competition environment of solution and solidLow levels monitor traces of PA. Lisa C et al inAnal. Chem.1995, 67, 2431-2435) to detect the most widely used explosive TNT, competitive immunoassays were developed. Trinitrobenzenesulfonic acid (2, 4, 6-trinitrobenzenesulfonic acid, TNB) is an analog of TNT, labeled with a fluorophore and used as an analyte competitor. Exposing a solution containing 7.5 ng/mL of sulfoindocyanine 5-ethylenediamine-labeled TNB (Cy 5-EDA-TNB) to an antibody-coated fiber produced a specific signal above background corresponding to 100% or the reference signal, the inhibition of the 100% signal being proportional to the TNT concentration in the sample. The sensitivity of TNT (8 ppb) in the buffer at 10 ng/mL can be measured. Interference is easily generated due to other analytes, and the detection accuracy is reduced. The invention discloses a europium-based metal organic frame hexagonal plate and a preparation method and application thereof in patent CN201610219036.8 published by Nanjing post and electronics university Stone Meter et al, wherein N, N-Dimethylformamide (DMF) dispersion liquid of the europium-based metal organic frame hexagonal plate is prepared, the dispersion liquid is filtered by filter paper to obtain a film prepared from a hexagonal plate material, the film emits red fluorescence under the excitation light of ultraviolet light with the wavelength of 365 nm, the original red fluorescence is almost completely quenched under the ultraviolet light with the wavelength of 365 nm after the DMF solution of PA is dripped, and the original red fluorescence is obviously weakened under the ultraviolet light with the wavelength of 365 nm after the DMF solution of 2, 4-dinitrotoluene (2, 4-dinitrotoluene, DNT) is dripped. The difference in quenching degree is due to the different detection effect of the europium-based metal organic framework hexagonal plate film on different explosives. In combination with the principle of fluorescence analysis, optical sensors have become a very popular detection device, Priyanka Dutta et al (ACS Appl. Mater. Interfaces2015, 7, 24778-24790) reports a high fluorescence material polyvinyl alcohol grafted polyaniline (PPA) and a nanocomposite single-step synthesis free radical polymerization reaction thereof with 2-mercaptosuccinic acid (MSA) terminated CdTe quantum dots (PPA-Q) and MSA terminated CdTe/ZnS core/shell quantum dots (PPA-CSQ). The formation of ZnS shells on CdTe cores increases the stability and Quantum yield of Quantum Dots (QDs) and reduces their toxicity to a large extent. This work reported highly fluorescent materialsThe development of materials to selectively and efficiently detect picric acid explosives fluorescence quenching phenomena in the nanometer range, the mechanism that operates in the quenching phenomena is believed to be a combination of strong internal filtering effects and ground state electrostatic interactions between the polymer and picric acid. And portable and low-cost electronic devices have been successfully manufactured that use sensing systems to visually detect picric acid, which devices are further used for quantitative detection of picric acid in actual water samples. The method has poor selectivity and is easily interfered by other quenchers. Varun Vij et al issue academic papers (ACS Appl. Mater. Interfaces2013, 5, 5373-5380), hexabenzononene 5 and 6 derivatives (HBC) are designed and synthesized, and form fluorescent aggregates in a mixed aqueous medium, and the derivatives have space-required tertiary butyl groups at the periphery, so that the solubility is greatly increased. In addition, the introduction of two rotatable phenyl groups as rotors, which causes an aggregation-induced emission enhancement phenomenon in coronene molecules, increases the utility of HBCs as fluorescent chemical sensors, which are capable of detecting PA in a vapor state. This is the first report by HBC of the aggregation-induced emission enhancement (AIEE) phenomenon, which gives fluorescent aggregates as selective chemical sensors to detect PA in solution as well as vapor states. In order to conveniently detect PA at low cost, a fluorescent test paper is prepared, and the test paper can detect ultra-trace PA in a steam and contact mode and is beneficial to trace detection of PA. Akhtar Hussain Malik et al, (paper ofACS Appl. Mater. Interfaces2015, 7, 26968-26976), a novel conjugated polymer material PFMI is prepared, and a cationic imidazolium salt group on the side chain of the polymer PFMI is used as a specific recognition site of PA, so that the solubility of the probe in polar solvents (DMSO, methanol and the like) is promoted. Good nano-particles are generated by adopting a spontaneous reprecipitation method, and the picric acid detection is realized for the first time on the picomolar level. The picric acid is measured by using Conjugated Polymer Nanoparticles (CPNs) in a 100% aqueous medium and a movable paper tape in a solid state respectively, and an end sensor device prepared by the two PFMI nanoparticles (PFMI-NPS) provides a means for detecting PA in a steam state. Detection of PA (polyamide) superelevation by PFMI-NPS (fiber optic transport-neutral Signal) probeThe sensitivity mechanism may be based on the principles of "molecular line effect", electrostatic interaction, photo-induced electron transfer and resonance energy transfer. The invention patent (CN201610344253. X) discloses a photoelectric response-based explosive vapor identification and detection method. The invention relates to an explosive vapor identification detection method based on photoelectric response, wherein a related device in the method consists of a sensor, a light source, a power supply, an ammeter, a signal processor and an alarm, the light source capable of periodically switching and changing light intensity change is used for irradiating a single sensor with rapid photoelectric response to measure photocurrent change caused by adsorption of explosive vapor on the surface of a sensitive material of the sensor, and data processing is carried out by mode identification methods such as principal component analysis, linear discriminant analysis and artificial neural network, so that a standard database of response of a sensor array to different types of explosive vapor is realized, and the purpose of identifying and detecting the explosive vapor is finally achieved by comparing the data processing result of suspected explosives with the database. The patent of CN 106008358A' Wang dynasty, et al, in south China university discloses a benzimidazolyl chemical sensor for fluorescence quenching detection of nitroarene explosives and a preparation method thereof, wherein di (benzimidazole) naphthalene and halohydrocarbon are used as raw materials, and the explosive nitroaromatic compounds mainly used for detecting picric acid and having high selectivity and high sensitivity are prepared by simple and easy N-alkylation substitution reaction. The prepared benzimidazolyl fluorescent chemical sensor can be used for visually detecting picric acid under the ultraviolet lamp by utilizing a fluorescence quenching solution to neutralize the solid state, and the detection of gaseous picric acid can be realized. The method has the advantages of great progress, realization of the difficult problem of on-site detection of picric acid, complex manufacturing process, high cost and low repeated utilization rate.
In recent years, the detection technology for explosives is more and more demanding, and the detection means for trace substances is also present in the public field of vision. (Kyodaming et al resonance energy transfer-amplified fluorescence quenching on the surface of silica nanoparticles: (Anal. Chem. 2008, 80, 8545-8553)), a fluorescent dye and aminopropyl are covalently modified to the surface of the silica nanoparticleForming a hybrid monolayer of dye fluorophore and ammonia ligand. The fluorescent silica particles can specifically bind to TNT species through charge transfer complexation interactions between the electron-rich ammonia ligand and the electron-deficient aromatic ring. The assembled array of nanoparticles on a silicon wafer can sensitively detect as low as about 1 nmol.L-1Only 10 μ L of TNT solution (-2 pg TNT) and a few ppb of TNT vapor was used. The FRET-based nanoparticle sensor shows stable fluorescence brightness, strong affinity to target analytes and good assembly flexibility, and therefore, can be applied to the field of detection of ultra-trace target analytes. The invention patent (CN2014000560284. X) discloses a fluorescent chemical sensor for detecting 2, 4, 6-trinitrophenol and a preparation method thereof, and an organic-inorganic hybrid polymer fluorescent sensor with a highly cross-linked structure is prepared by a one-step polycondensation method of hexachlorocyclotriphosphazene and curcumin, so that the specific identification and detection of the 2, 4, 6-trinitrophenol in a liquid phase are realized, and the fluorescent chemical sensor is suitable for the fields of trace explosive detection and environmental monitoring and has a wide application prospect. The invention discloses a patent (CN 201710253748.6) of 'silicon-based SERS (SERS) chip and a preparation method thereof and a TNT detection method' of Suzhou university, and discloses a silicon-based Surface-Enhanced Raman scattering (SERS) chip for detecting TNT and a preparation method thereof. The invention patent (CN 201610964476.6) discloses synthesis and application of water-soluble fluorescent silicon nanoparticles for high-selectivity determination of trace TNP, which is characterized in that N- (2-aminoethyl) -3-aminopropyltrimethoxysilane is used as a silicon source, catechol is used as a reducing agent to synthesize the water-soluble fluorescent silicon nanoparticles, the molar ratio of the N- (2-aminoethyl) -3-aminopropyltrimethoxysilane to the catechol is 9.3:100, and the method can be used for manufacturing test paper or reagents, is convenient to carry and is easy to detect. Wayne H. Griest, paper (Anal. Chem.1998，70，3015-3020) cannot be directly analyzed to obtain the selectivity and sensitivity required for most trace analysis applications, and to solve this problem, the analytes are selectively pre-concentrated using solid phase microextraction techniques prior to gas chromatography/ion trap mass spectrometry. The method is suitable for trace analysis of explosives and metabolites thereof in seawater, but the method has long balance time, cannot realize quick detection and has questionable accuracy of measurement results.
The molecular imprinting technology is a means of artificially synthesizing and recognizing molecules with specific selectivity, is a popular detection technology in recent years, has good stability, easy preparation, low cost and specific selectivity, and is widely applied to various fields (Eersels K) such as biology, medicine, chemistry and the like.ACS Appl. Mater. Interfaces2013，5，7258−7267；Lee J-D. Org. Lett.，2005, 7，963-966； Li H. ACS Appl. Mater. Interfaces2013，5，10502−10509；Schirhagl R. Anal. Chem.2014，86，250−261）。
Early proposals for molecular imprinting were based on the short lifetime and poor reproducibility of biosensor materials, and to overcome these difficulties, artificially synthesized biomimetic materials were produced (Zhang Z.P).Anal. Chem.2014, 86, 1123-1130). In 2016 Monsantril et al (ACS Appl. Mater. Interfaces2016, 8, 14133-14141) an artificial hydrolase was developed by combining the catalytic Ser/His/Asp triad with N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) and then assembling the peptides into nanofibers (CoAHSD). Peptidyl nanofibers provide an ideal supramolecular backbone to support functional groups, and the activity of CoA-HSD is highest when the ratio of histidine, serine and aspartate residues is 40:1:1 compared to self-assembling catalytic nanofibers (SA-H) containing only catalytic histidine residues, indicating that the ordered nanofiber structure and the synergistic effect of serine and aspartate residues contribute to increased activity. In addition, the activity of peptidyl-artificial enzyme (CoA-HSD) was further improved for the first time using molecular imprinting technique, using p-NPA as molecular template, arranging catalytic Ser/His/Asp triad residues in the appropriate orientation.Therefore, the activity of the imprinted CoAHSD nanofiber is 7.86 times that of non-imprinted CoA-HSD and 13.48 times that of SA-H. Davide Carboni et al in (ACS Appl. Mater. Interfaces2016, 8, 34098) -34107) through a molecular imprinting technology, the selectivity efficiency of graphene mediated enhanced Raman scattering is improved. The invention discloses a preparation method and application of a phthalate ester surface molecularly imprinted polymer (CN 2017000535808.3) in patent publication (CN 2017000535808.3) of Luchuncui et al, Xinjiang academy of agricultural sciences, wherein a bis (10-methoxy-10-oxodecyl) phthalate compound is prepared by the invention, and the compound is used as a virtual template, and a surface molecularly imprinted technology is combined, so that the PAEs molecularly imprinted polymer prepared by two-step precipitation polymerization is used for solving the defects of too deep template embedding, template leakage and low combination rate in the prior art, and the obtained polymer has regular appearance, uniform particle size, good monodispersity and large adsorption quantity, and simultaneously meets the requirement of analysis on multiple residues of 10 PAEs in food. The invention patent (CN 201710474093.5) published by Chenlina et al of Nanjing medical university discloses a magnetic artificial receptor and a preparation method and application thereof, which mainly comprises the following steps: (1) fe3O4@SiO2Preparing composite particles; (2) (1) Fe3O4@SiO2Preparation of @ MPS composite particles; (3) and (3) preparing the magnetic artificial receptor. The ZL006 is used as a template, and an ordered and compact molecular imprinting shell layer is designed and synthesized on the surface of the magnetic nano silica gel material, so that the phenomenon that imprinting sites are embedded is reduced, and the binding rate and the separation efficiency are high. Meanwhile, the magnetic material, the nano silica gel and the surface molecular imprinting technology are combined, rapid and direct separation of target substances can be realized under the action of an external magnetic field, and a new powerful means is provided for selective and rapid separation, enrichment and detection of active substances of traditional Chinese medicines. Patent of invention (CN 201710185685.5) of Yao et al, a method for detecting Fe in thifensulfuron methyl3O4@PEG@SiO2A method for preparing artificial antibody, which comprises the step of adding Fe3O4The surface of the magnetic nano particle is modified with polyethylene glycol 2000, and the surface is coated with SiO2Shell layer, forming core-shell structure, is located on SiO by elution2The imprinting molecules in the shell form a complex with the imprinting moleculesThe specific recognition site hole with the complementary substructure, size and functional group realizes the selective recognition and detection of target analyte molecules. Obtaining FeO with selective recognition imprinted molecules4@PEG@SiO2The maximum saturation binding capacity of the artificial antibody to thifensulfuron methyl is 41.28 mg/g, and the adsorption rate is 0.45 mg/g.min within the first 30min, which is 5.34 times and 3.46 times of that of the non-blotting method. The invention patent (CN 201310408025.0) discloses baicalein molecularly imprinted polymer, a preparation method and application thereof, of Chenlina et al, Nanjing medical university. Compared with the traditional bulk molecularly imprinted polymer, the prepared precipitated molecularly imprinted polymer microsphere has the advantages of good monodispersity, uniform particle size, controllable size, simple preparation, no complex post-treatment process, short period and the like. The patent of invention (CN 201410432424.5) of blue-fragrant et al of Sichuan university discloses a superparamagnetic composite nanosphere with protein molecular imprinting and a preparation method and application thereof, and an improved microemulsion polymerization method is utilized to ensure that superparamagnetic nanoparticles are closely arranged in a polymer nanosphere, thereby keeping high saturation magnetization. Abundant hydrogen bonds are formed among the hydroxylation modification layer on the periphery of the inner core of the composite nanosphere, the polymer shell and the imprinted protein molecules, and by utilizing ice bath ultrasonic treatment twice, the uniform and firm distribution of the imprinted protein molecules in the polymer shell is promoted, the non-specific binding sites are effectively reduced, the selective binding capacity of the superparamagnetic composite nanosphere and the target protein molecules is improved, and the purpose of high-selectivity separation is realized. A preparation method of pyraclostrobin molecularly imprinted polymer disclosed in the invention patent (CN 201410688522.5) of Beijing college of agriculture and university, i.e., pyraclostrobin, functional monomer methacrylic acid and cross-linking agent ethylene glycol dimethacrylate are mixed and dissolved in pore-forming agent acetonitrile according to the molar ratio of template molecule to functional monomer to cross-linking agent of 1:4:20, initiator azobisbutyronitrile is added, water bath reaction is carried out at 60-65 ℃ under the protection of nitrogen, the obtained template molecular polymer is further dried and ground in vacuum, the template molecule is removed through Soxhlet extraction, and vacuum drying is carried out at 60 ℃ to constant weight, and pyraclostrobin is finally obtainedA molecularly imprinted polymer. The molecularly imprinted polymer prepared by the invention can effectively separate and enrich pyraclostrobin in a complex matrix. The method can perform high-selectivity recognition on the target analyte, and cannot realize the output of sensitive optical signals or electromagnetic signals of the target analyte entering the recognition site.
The detection material with fluorescence property obtained by combining the fluorescence analysis method and the molecular imprinting technology has good identification property (Li H.; Wang L. Y).ACS Appl. Mater. Interfaces2013, 5, 10502-. The invention discloses a patent (CN 201710568072. X) of a 2, 6-dichlorophenol imprinted sensor based on a surface enhanced Raman technology, a preparation method and application thereof, namely Lihongji et al, Jiangsu university, wherein the invention combines the Raman detection technology with the molecular imprinting technology to ensure that a product has sensitive detectability and high selectivity. The SERS material of the high-sensitivity metal-semiconductor heterostructure has stronger and more sensitive surface enhanced Raman signals. The patent of invention (CN 201610720836.8) of published patent of Yangshu university Yangming et al discloses a preparation method and application of a fluorescent molecular imprinting adsorption separation material, and provides a preparation method and application of the fluorescent molecular imprinting adsorption separation material, which comprises the following steps: step 1 is SiO2Preparing nano particles; step 2, synthesizing manganese-doped zinc sulfide quantum dots; step 3 is double bond modified SiO2Preparing nano particles; step 4, preparing double-bond modified manganese-doped zinc sulfide quantum dots; and step 5, preparing the fluorescent molecular imprinting adsorption separation material. The fluorescent molecularly imprinted polymer combines the selectivity of the molecularly imprinted polymer and the fluorescence property of the quantum dot. The target molecule can be rapidly detected through the change of the fluorescence intensity. Patent of invention (CN 201210382810.9) "CdTe @ SiO" of Huqin, university of Nanjing medical science2The preparation method of the molecular imprinting polymer of the monoamine neurotransmitter on the surface of the quantum dot is to synthesize CdTe @ SiO in the water phase2The quantum dots are rotated and evaporated until the water is volatilized, the unreacted substrate is washed away by absolute ethyl alcohol, and the obtained CdTe @ SiO2Adding quantum dots into a pore-foaming agent, ultrasonically dispersing uniformly, then sequentially adding template molecules, functional monomers and a cross-linking agent, and adding N2Stirring and reacting for 20 h under the atmosphere, centrifuging the reaction solution, discarding the supernatant, washing the obtained solid to remove template molecules to obtain CdTe @ SiO2Monoamine neurotransmitter molecularly imprinted material on the surface of quantum dots. The synthesized quantum dot fluorescent probe imprinted polymer material has the advantages of high sensitivity, high selectivity, high affinity and simple and convenient detection, and can be directly applied to the determination of monoamine neurotransmitters in biological body fluid. The invention discloses a patent (CN 201410520007.6) of Tankjun et al, university in southwest, discloses a core-shell quantum dot-based molecularly imprinted polymer (QDs-MIP) and application thereof, relates to a CdTe/CdS quantum dot-molecularly imprinted polymer (QDs-MIP), and the QDs-MIP is used as a fluorescent probe to be applied to detection and analysis of perfluorooctanoic acid. The polymer is a fluorescent molecularly imprinted polymer prepared by embedding molecular recognition sites into the surface of CdTe/CdS quantum dots coated by TEOS and taking perfluorooctanoic acid as template molecules. The molecularly imprinted polymer prepared by the invention has the advantages of high sensitivity, simple and convenient detection, high selectivity, high affinity and the like, can be directly used for detecting the perfluorooctanoic acid in the environment, can reduce the detection cost and can improve the detection efficiency.
Xinyang institute of academy of academic sweets et al discloses an invention patent (CN 201611031452.1) "a molecular imprinting electrochemical sensor for rapidly detecting cyromazine with trace amount, a preparation method and application thereof", which is characterized in that: the working electrode is a glassy carbon electrode, and monodisperse SiO is adopted2@TiO2Modifying the core-shell nanospheres, and then further preparing a surface molecularly imprinted membrane for specific recognition of cyromazine by using an in-situ electrochemical polymerization method and a sol-gel method. "A Eu for detecting pesticide residue" is disclosed in invention patent (CN 201510538714.2) by Gao Da Ming, the institute of compost, and the like3+Preparation method of labeled molecularly imprinted sensor, Eu3+With ammonia in APTSBase and pesticide residue molecules are preassembled and hydrolyzed, crosslinked and condensed with TEOS to obtain Eu3+The marked pesticide residue molecularly imprinted silica nanoparticle sensor has a recognition site hole selective for pesticide residue molecules after the pesticide residue molecules are eluted, and the pesticide residue molecules enter the recognition site of the sensor again and then react with Eu on the recognition site3+Chelation occurs, pesticide residue molecules and Eu3+The chelated fluorescence intensity is increased, and the detection of high selectivity, high binding capacity and high sensitivity to trace pesticide molecules is realized by utilizing the change of the fluorescence intensity. The invention discloses a preparation method of a gold nanoparticle-doped molecularly imprinted electrochemical sensor for detecting dopamine (CN 201310023869.3), which comprises the steps of gold electrode pretreatment, gold nanoparticle self-assembly modified electrode preparation, molecularly imprinted self-assembly solution preparation, electropolymerization reaction, template molecule elution and the like, so that the selectivity of the traditional electrochemical sensor is improved, the response is rapid, the stability and the tolerance are good, and the efficient, sensitive and real-time detection of dopamine in a biological sample can be realized. Has important significance for clinical diagnosis, pathological research and the like. The invention discloses a patent (CN 201410471090.2) of Liuxia of Hunan agriculture university and the like, which discloses a method for detecting acrylamide by a sol-gel molecular imprinting electrochemical sensor based on a nano-material compound, and the method comprises the steps of covering the surface of a glassy carbon electrode with a compound of a carbon nano-tube, a gold nano-particle and chitosan, placing the glassy carbon electrode in a sol-gel solution containing a template molecule, a functional monomer and a cross-linking agent for electrochemical deposition, removing the template molecule after the reaction is finished, and directly detecting the acrylamide in a sample by the obtained molecular imprinting electrochemical sensor. The method is simple, the sample pretreatment operation is simple, the detection speed is high, and the cost is low. In addition, the method has good selectivity and reproducibility, the recovery rate meets the requirement, acrylamide in fried foods such as potato chips and the like can be directly detected, and the method has important practical application value. The patent of invention (CN 201710592816.1) discloses a fluorescent nano molecular imprinting bionic sensor and a preparation method and application thereof. Divide the template intoAdding two different functional monomers into a pore-foaming agent, ultrasonically dissolving, and stirring at room temperature for prepolymerization to obtain a pre-assembled solution A; ultrasonically dispersing carbon points with surface double bond functionalized in a pore-foaming agent to obtain a solution B; uniformly mixing the solution A and the solution B, adding a cross-linking agent and an initiator, introducing nitrogen, stirring, centrifuging, collecting precipitate, and washing with distilled water; and finally, eluting the template protein by HAc-SDS, and freeze-drying the obtained product to obtain the fluorescent nano molecular imprinting bionic sensor.
Although the methods have respective characteristics, no document reports that the prepared target molecules have fluorescence characteristics, and have yellow fluorescence, enter the recognition sites and are sensitively output through self optical signals. Meanwhile, no literature report is found, the poly nitrophenol derivative with bright yellow fluorescence generated by the reaction of 2, 4, 6-trinitrophenol and N, N' -diisopropylcarbodiimide is imprinted to prepare the polymer microsphere, the imprinted derivative enters the recognition site, and the recognition and detection are realized by the opening and closing of autofluorescence. Therefore, the preparation method for synthesizing the imprinted polymer microsphere with the yellow autofluorescence probe in the emission band with high selectivity and high sensitivity has the necessity of realizing the identification and detection of the ultra-trace picric acid molecules.
In the invention, a preparation method of a molecularly imprinted polymer microsphere for detecting 2, 4, 6-trinitrophenol is reported, and the identification and detection of trace 2, 4, 6-trinitrophenol derivatives are realized. The imprinted polymer microspheres are particularly suitable for recognition materials, a cavity structure which is complementary with the structure, the size and the functional group of the imprinted molecules can be easily formed in the imprinted layer for the imprinted molecules, and the polymer microspheres for eluting the imprinted molecules have specific recognition sites for target analyte molecules, so that the target analyte molecules can be selectively recognized. The functional monomers in the imprinted polymer can be different functional monomers according to the structural characteristics of the target analyte. Considering the electron-deficient characteristic of the molecular structure of the 2, 4, 6-trinitrophenol derivative, electron-rich acrylamide is selected as a functional monomer, and a target molecule enters a recognition site and is bound by an electron-donating amino group in the functional monomer through the non-covalent bond effect, so that the 2, 4, 6-trinitrophenol derivative is recognized. Considering that no signal output problem exists after a general target molecule enters an imprinting recognition site, a polynitrophenol derivative generated by the reaction of 2, 4, 6-trinitrophenol and N, N' -diisopropylcarbodiimide has bright yellow fluorescence, and the fluorescence has an opening and closing characteristic under an acid-base condition, so that the 2, 4, 6-trinitrophenol derivative has an autofluorescence characteristic and autofluorescence optical signal output after entering the imprinting recognition site, and high-selectivity recognition and high-sensitivity trace detection of the 2, 4, 6-trinitrophenol derivative are realized.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects existing in the prior art, the invention utilizes the acid-base fluorescence switch characteristic of the synthesized picric acid derivative to prepare the chemical preparation method of the molecularly imprinted polymer for identifying the picric acid derivative, and realizes the molecular identification and trace detection of the trace picric acid derivative. Firstly, adding a nucleophilic reagent N, N' -diisopropylcarbodiimide into picric acid to react to form a picric acid derivative, then separating and purifying to obtain a derivative with an acid-base fluorescence switch, taking the derivative as a target molecule, adding a functional monomer, a cross-linking agent, an initiator and the like to prepare the picric acid derivative molecularly imprinted polymer microsphere, and eluting the imprinted molecule by using an eluent to obtain the molecularly imprinted polymer microsphere with selective recognition and sensitivity detection on the picric acid derivative.
The technical scheme of the invention is as follows: a preparation method of molecularly imprinted polymer microspheres for detecting 2, 4, 6-trinitrophenol is characterized by comprising the following steps: the polynitrophenol derivative generated by the reaction of 2, 4, 6-trinitrophenol and N, N' -diisopropyl carbodiimide has bright yellow fluorescence, the fluorescence has an opening and closing characteristic under the acid-base condition, the molecularly imprinted polymer microsphere elutes imprinted molecules positioned in an imprinting layer, a cavity structure complementary with the imprinting molecular structure, the size and the functional group is formed in the imprinting layer, the polymer microsphere for eluting the imprinted molecules has a specific recognition site for target analyte molecules, and the selective recognition and detection of the target analyte molecules are realized, and the preparation process of the molecularly imprinted polymer microsphere for detecting 2, 4, 6-trinitrophenol comprises the following four steps:
the first step is the synthesis of 2, 4, 6-trinitrophenol derivatives: weighing 0.4-0.6 g of treated 2, 4, 6-trinitrophenol, placing the 2, 4, 6-trinitrophenol in a 100mL three-neck flask, adding 7-8 mL of dichloromethane for ultrasonic dispersion, adding 0.2-0.4 g of N, N' -diisopropyl carbodiimide, immediately turning the solution into reddish brown, stirring for 1-3 h under nitrogen atmosphere, drying for 0.5-1.5 h in a vacuum drying oven at 30-50 ℃ to obtain reddish brown solid powder with bright yellow fluorescence characteristics, pouring 20mL of boiling methanol solution into the prepared reddish brown solid powder, adding 10-20 mL of distilled water, placing in an ice water mixed solution at 0 ℃ for water bath for 1-3 h, finding that reddish brown crystals are separated out, filtering to obtain 2 to be prepared, 4, 6-trinitrophenol derivatives;
the second step is the purification of the 2, 4, 6-trinitrophenol derivative: vertically installing a silica gel column on an iron support, rinsing with acetone and petroleum ether for 3 times, adding 150-250 g of silica gel into a 250mL beaker, uniformly stirring with petroleum ether, filling the silica gel column, starting an air pump, pressurizing the solution downwards, beating the column with an ear washing ball to vibrate and fill the silica gel tightly, keeping the uppermost layer of the silica gel column horizontal when the height of the silica gel column meets the separation requirement, adding petroleum ether after the column is tamped, pressurizing downwards by using the air pump, performing the operation for 3 times, adding a small amount of prepared eluent when the liquid level of the petroleum ether and the column surface of the silica gel are level, naturally flowing downwards, sucking the prepared 2, 4, 6-trinitrophenol derivative sample by using a rubber head dropper, slowly and uniformly loading along the column wall, pressurizing downwards by using the air pump if the sample amount is excessive, then loading, and completely loading the separated sample, pressing the mixture to be flush with the cylindrical surface of the silica gel by using an air pump, after the sample loading is finished, slowly adding a prepared eluent along the wall of the column, and finally obtaining 2, 4, 6-trinitrophenol derivatives, wherein the separated liquid is orange red liquid (PAD 1) firstly, yellow liquid (PAD 2) secondly, 5-15 uL of the obtained liquid is respectively dripped on a silica gel plate by using a trace sample injector, after the separation and purification are confirmed, the obtained PAD1 and PAD2 are distilled under reduced pressure, and finally, the obtained product is dried in a vacuum oven at 50-70 ℃ for 7-9 h, and finally, orange yellow and brown yellow solid powder is respectively obtained;
the third step is the synthesis of the molecular engram polymer microsphere of the target analyte: respectively weighing 20-40 mg of orange-yellow solid powder PAD1, 17-19 mg of acrylamide and 15-25 mg of azodiisobutyronitrile by using a ten-thousandth electronic balance, placing the weighed materials in a 250mL conical flask, then weighing 50mL of mixed solution of methanol and acetonitrile in a volume ratio of 9:1 by using a 100mL measuring cylinder, adding the mixed solution into the conical flask, ultrasonically dispersing for about 5 minutes, finally weighing 190-210 mu L of ethylene glycol dimethacrylate by using a microsyringe, dripping the ethylene glycol dimethacrylate into the solution, introducing nitrogen for 20-30 minutes, uniformly coating vacuum grease on a ground stopper, covering the conical flask, wrapping the conical flask with a preservative film, then placing the conical flask in a constant temperature oscillator, carrying out oscillation reaction for 2-4 hours at 50-60 ℃, regulating the temperature to 60 ℃ and carrying out oscillation reaction for 8-10 hours, finally, carrying out oscillation reaction for 1-3 h at 70-85 ℃, taking out and cooling to room temperature, transferring the reaction mixed solution into a 15mL centrifuge tube, centrifuging at the rotating speed of 5000-7000 rpm for 5-10 min, removing supernatant, adding a detergent, continuing centrifugation, carrying out ultrasonic dispersion, re-centrifuging, and repeating the operation for 3 times to obtain the molecular imprinting polymer microspheres of the imprinting target molecules;
the fourth step is the preparation of polymer microspheres for selectively identifying and detecting target analytes: using acetic acid and methanol in a volume ratio of 2: eluting the template molecules in the obtained molecularly imprinted polymer microspheres in 200mL of mixed solution in a Soxhlet extractor until no fluorescence signal exists in the eluent, thereby obtaining the polymer microspheres for selectively identifying the target analyte molecules and detecting optical signals.
As a further improvement to the prior art, the target analyte molecules and the engram molecules in the prepared molecularly imprinted microspheres are PAD1 with fluorescence characteristics in 2, 4, 6-trinitrophenol derivatives; the acid and the alkali used in the prepared molecular imprinting microsphere respectively refer to perchloric acid and potassium tert-butoxide; the proportion of the eluent passing through the column and the proportion of the eluent climbing the silica gel plate in the prepared molecularly imprinted microspheres are respectively mixed according to the volume ratio of ethyl acetate to petroleum ether of 1:3 and 1: 5; the detergent used in the prepared molecularly imprinted microsphere is prepared by mixing deionized water and acetonitrile in a volume ratio of 1: 1; functional monomer acrylamide in the prepared molecularly imprinted microsphere; the cross-linking agent in the prepared molecularly imprinted microsphere is ethylene glycol dimethacrylate; the initiator azobisisobutyronitrile in the prepared molecularly imprinted microsphere; PAD1 in the target molecule 2, 4, 6-trinitrophenol derivative in the prepared molecularly imprinted microsphere has opening and closing characteristics under acid-base conditions.
Compared with the prior art, the method has the beneficial effects that: in recent years, terrorist violence events such as explosion and the like frequently occur, which seriously jeopardize the life and property safety and social stability of people of all countries in the world and threaten the safety and stability of the countries and regions. Therefore, the detection and analysis of explosives are highly regarded by research institutions of various countries. Zhang jin Fang et al, Jiangnan university, 2016 (CN201610120560. X) "luminescent crystalline material with property of detecting bitter acidity { [ Cu (DMSO)5][Cu4I6(DMSO)]}nThe invention discloses a luminescent crystal material for detecting picric acid. Adding cuprous iodide into a solution in dimethyl sulfoxide, stirring for reacting for several minutes, then dropwise adding hydrochloric acid until the pH value of the solution is 2-4, stirring for two hours to prepare a solution containing the luminescent crystal material, filtering, finally adding a precipitator into the prepared luminescent crystal material solution, crystallizing, filtering, washing and drying to obtain the luminescent crystal material, wherein the preparation process only needs a long time. Huang Wei et al inACS Appl. Mater. Interfaces 2017, 9, 3068-. Quenching efficiency depends on spectral overlap between target analyte absorbance and fluorescent polymer emission, optical fingerprints are obtained based on unique response patterns of analytes to polymers, nine nitroaromatic analytes can be distinguished 100% accurately using such small sensor arrays, but the method producesThe method is high, and a better method still needs to be found for detecting picric acid. Kentaro Shiraishi academic paper (ACS Appl. Mater. Interfaces 2009, 1, 1379-1382) mention the detection of explosives, in particular nitroaromatic explosives (such as TNT), with phosphorus oxides by the principle of fluorescence quenching. Visual fluorescence quenching is allowed to determine the detection limit of nitroaromatic explosives at the lower limit of the nanogram scale in image sensing of explosives projected with phosphorescent oxides. Nilanjan Dey at (ACS Appl. Mater. Interfaces2013, 5, 8394-8400) using a test strip, and selective detection of nanomolar concentrations of aromatic Nitrate (NACs) was first achieved in a variety of media including water, micelles, or organogels and using test strips. The mechanism by which NACs interact with highly fluorescent p-styryl molecules is described as the phenomenon of electron transfer from electron-rich chromogenic probes to electron-deficient NACs. The selectivity of the sensing is guided by the pKa of the probe and the NACs under consideration. The selective gel-sol transition induced by TNP in the medium tetrahydrofuran was also observed by recombination of molecular self-assembly. This method is only silent about whether the test strip is insoluble material or not, and if it is diluted in water, the fluorescence intensity changes and the result is affected. The invention discloses a preparation method of a nitrogen-containing polymer quantum dot for detecting picric acid in patent publication (CN 201510902169.0) of Gelidjust et al, Qilu Industrial university, wherein polybrominated pyrrole is used as a raw material in the preparation process of the nitrogen-containing polymer quantum dot, and the polybrominated pyrrole is subjected to debromination cyclization and hydroxyl nucleophilic substitution under the conditions of microwave assistance and alkali heat, so that the reaction can be carried out at a lower temperature. Minoo Bagheri at (ACS Appl. Mater. Interfaces2016, 8, 21472-21479) reported that a dye-sensitized metal organic framework TMU-5S is synthesized on the basis of introducing a laser dye rhodamine B into a porous framework TMU-5. TMU-5S was studied as a ratiometric fluorescent sensor for the detection of explosive nitroaromatics and showed four times higher picric acid selectivity than any existing luminescence-based sensor and it could selectively react in the presence of other nitroaromatics and volatile organic compoundsThe picric acid concentration is distinguished. However, this method uses a fluorescent dye and is not environmentally friendly. The invention discloses the construction of an ultrasensitive electrochemiluminescence sensor and the application thereof in the detection of trinitrotoluene in patent CN201710219563.3 'Yan Donpeng Peng of Beijing university of chemical industry and the like', and discloses the construction of an ultrasensitive electrochemiluminescence sensor and the application thereof in the detection of trinitrotoluene. The method comprises the steps of firstly inserting negative small molecule luminol monosodium salt into hydrotalcite layers, and then forming an Electrochemiluminescence (ECL) sensor of the supermolecule composite film by using hydrotalcite after luminol intercalation and negative quantum dots through an electrostatic attraction layer-by-layer self-assembly technology, so as to form an ECL Resonance Energy Transfer (ERET) system for detecting TNT. However, such a sensor is fragile, easily damaged, and not highly practical.
The picric acid detection method based on the invention has the defects of complex synthesis, time consumption, harsh reaction conditions, no identification sites, poor selectivity and incapability of detecting the picric acid due to the fact that some identification sites exist and no signal is output when the picric acid enters the identification sites; meanwhile, the target analyte autofluorescence emission spectrum prepared by the method is a yellow luminous band, and the high-selectivity recognition and high-sensitivity optical signal detection of the target analyte are realized by preparing the imprinted polymer microspheres. Therefore, the picric acid derivative molecularly imprinted polymer microsphere which has high selectivity and high sensitivity and emits light with a yellow fluorescent optical signal is synthesized, and the necessity of recognizing and detecting the trace picric acid derivative molecules is realized.
The invention firstly synthesizes the 2, 4, 6-trinitrophenol derivative: weighing 0.4-0.6 g of treated 2, 4, 6-trinitrophenol, placing the 2, 4, 6-trinitrophenol in a 100mL three-neck flask, adding 7-8 mL of dichloromethane for ultrasonic dispersion, adding 0.2-0.4 g of N, N' -diisopropyl carbodiimide, immediately turning the solution into reddish brown, stirring for 1-3 h under nitrogen atmosphere, drying for 0.5-1.5 h in a vacuum drying oven at 30-50 ℃ to obtain reddish brown solid powder with bright yellow fluorescence characteristics, pouring 20mL of boiling methanol solution into the prepared reddish brown solid powder, adding 10-20 mL of distilled water, placing in an ice water mixed solution at 0 ℃ for water bath for 1-3 h, finding that reddish brown crystals are separated out, filtering to obtain 2 to be prepared, 4, 6-trinitrophenol derivative PAD 1;
secondly, purifying the 2, 4, 6-trinitrophenol derivative: vertically installing a silica gel column on an iron support, rinsing with acetone and petroleum ether for 3 times, adding 150-250 g of silica gel into a 250mL beaker, uniformly stirring with petroleum ether, filling the silica gel column, starting an air pump, pressurizing the solution downwards, beating the column with an ear washing ball to vibrate and fill the silica gel tightly, keeping the uppermost layer of the silica gel column horizontal when the height of the silica gel column meets the separation requirement, adding petroleum ether after the column is tamped, pressurizing downwards by using the air pump, performing the operation for 3 times, adding a small amount of prepared eluent when the liquid level of the petroleum ether and the column surface of the silica gel are level, naturally flowing downwards, sucking the prepared 2, 4, 6-trinitrophenol derivative sample by using a rubber head dropper, slowly and uniformly loading along the column wall, pressurizing downwards by using the air pump if the sample amount is excessive, then loading, and completely loading the separated sample, pressing the mixture to be flush with the cylindrical surface of the silica gel by using an air pump, after the sample loading is finished, slowly adding a prepared eluent along the wall of the column, and finally obtaining 2, 4, 6-trinitrophenol derivatives, wherein the separated liquid is orange red liquid (PAD 1) firstly, yellow liquid (PAD 2) secondly, 5-15 uL of the obtained liquid is respectively dripped on a silica gel plate by using a trace sample injector, after the separation and purification are confirmed, the obtained PAD1 and PAD2 are distilled under reduced pressure, and finally, the obtained product is dried in a vacuum oven at 50-70 ℃ for 7-9 h, and finally, orange yellow and brown yellow solid powder is respectively obtained;
then synthesizing the molecular imprinting polymer microspheres of the target analyte: respectively weighing 20-40 mg of orange-yellow solid powder PAD1, 17-19 mg of acrylamide and 15-25 mg of azodiisobutyronitrile by using a ten-thousandth electronic balance, placing the weighed materials in a 250mL conical flask, then weighing 50mL of mixed solution of methanol and acetonitrile in a volume ratio of 9:1 by using a 100mL measuring cylinder, adding the mixed solution into the conical flask, ultrasonically dispersing for about 5 minutes, finally weighing 190-210 mu L of ethylene glycol dimethacrylate by using a microsyringe, dripping the ethylene glycol dimethacrylate into the solution, introducing nitrogen for 20-30 minutes, uniformly coating vacuum grease on a ground stopper, covering the conical flask, wrapping the conical flask with a preservative film, then placing the conical flask in a constant temperature oscillator, carrying out oscillation reaction for 2-4 hours at 50-60 ℃, regulating the temperature to 60 ℃ and carrying out oscillation reaction for 8-10 hours, finally, carrying out oscillation reaction for 1-3 h at 70-85 ℃, taking out and cooling to room temperature, transferring the reaction mixed solution into a 15mL centrifuge tube, centrifuging at the rotating speed of 5000-7000 rpm for 5-10 min, removing supernatant, adding a detergent, continuing centrifugation, carrying out ultrasonic dispersion, re-centrifuging, and repeating the operation for 3 times to obtain the molecular imprinting polymer microspheres of the imprinting target molecules;
finally, preparing the polymer microspheres for selectively identifying and detecting the target analyte: using acetic acid and methanol in a volume ratio of 2: and eluting the template molecules in the obtained molecularly imprinted polymer microspheres in a Soxhlet extractor by using 8mL of mixed solution until no fluorescence signal exists in the eluent, thereby obtaining the polymer microspheres PAD1 for selectively identifying the target analyte molecules and detecting optical signals.
In conclusion, the obtained molecularly imprinted polymer microspheres can be used for detecting 2, 4, 6-trinitrophenol derivatives.
One is as follows: the 2, 4, 6-trinitrophenol has no fluorescence characteristic and cannot be output as an optical signal when entering the recognition site, so that the 2, 4, 6-trinitrophenol cannot be detected, however, the 2, 4, 6-trinitrophenol derivative PAD1 obtained after the 2, 4, 6-trinitrophenol reacts with N, N' -diisopropyl carbodiimide has fluorescence with a yellow spectral band and has the characteristics of fluorescence opening and closing under an acid-base environment, so that the 2, 4, 6-trinitrophenol derivative PAD1 entering the blot recognition site realizes trace detection of the 2, 4, 6-trinitrophenol derivative PAD1 through the output of an autofluorescence optical signal.
The second step is as follows: compared with the traditional method that the fluorescent dye or the quantum dot is modified to the recognition site, the imprinting molecule enters the recognition site without interacting with the fluorescent dye and the quantum dot modified at the recognition site, and then the target analyte is detected by utilizing a fluorescence energy resonance energy transfer mechanism or a photoelectron induced transfer mechanism.
And thirdly: compared with the traditional imprinted material, the polymer microsphere has larger specific surface area, more effective recognition sites, small surface resistance of the recognition sites in the microsphere, fast binding kinetics and larger saturated binding capacity.
Fourthly, the method comprises the following steps: in the method provided by the invention, the particle size of the imprinted polymer microspheres is controllable, and can be controlled by adjusting the amounts of the functional monomer, the crosslinking agent and the solvent.
And fifthly: the 2, 4, 6-trinitrophenol derivative PAD1 obtained after the reaction of 2, 4, 6-trinitrophenol with N, N' -diisopropylcarbodiimide was chosen for the purpose of being a template molecule because it has the following advantages: (1) the recognition site does not need to be fluorescently labeled, (2) the recognition site basically has optical signal output when target molecules exist; (3) a fluorescence resonance energy transfer mechanism does not need to be constructed; (4) whether the target analyte exists or not is judged through the existence of fluorescence, and the quantitative detection of the target analyte is realized through the strength of fluorescence intensity.
Drawings
FIG. 1 is a schematic diagram of a synthesized self-fluorescent functional picric acid derivative imprinted polymer microsphere prepared by the present invention.
FIG. 2 is a schematic representation of the reaction of N, N-diisopropylcarbodiimide prepared according to the invention with picric acid to form the picric acid derivatives PAD1 and PAD 2.
FIG. 3 shows the hydrogen nuclear magnetic resonance spectrum of picric acid derivative PAD1 prepared by the present invention.
FIG. 4 is an infrared spectrum of picric acid (a) and picric acid derivative PAD1 (b) prepared according to the present invention.
FIG. 5 is a normalized ultraviolet-visible spectrum of picric acid (a) and PAD1 (b) solutions and a normalized fluorescence emission spectrum of PAD1 (c) solutions prepared in accordance with the present invention.
FIG. 6 shows the molecular structure transformation of picric acid derivative PAD1 prepared by the present invention in potassium tert-butoxide and perchloric acid solution, respectively.
FIG. 7 is an SEM of approximately 2um diameter polymeric microspheres imprinted with picric acid derivative PAD1 prepared according to the present invention.
FIG. 8 is a comparison of the molecular recognition performance of the polymers prepared according to the present invention.
FIG. 9 shows the molecular dynamics curve of the picric acid derivative PAD1 prepared according to the present invention.
FIG. 10 is a progression of the increase in fluorescence intensity with increasing concentration of target analyte PAD1 in a perchloric acid solution prepared in accordance with the present invention.
The embodiments are further explained with reference to the drawings
FIG. 1 is a schematic diagram of a synthesized self-fluorescent functional picric acid derivative imprinted polymer microsphere prepared by the present invention. In fig. 1: 1-2: firstly, picric acid and N, N-diisopropyl carbodiimide react to generate picric acid derivatives PAD1 and PAD2, and PAD1 is used as a template molecule after separation and purification; 2-3: the picric acid derivative template molecule PAD1 is copolymerized with functional monomer acrylamide, cross-linking agent ethylene glycol dimethacrylate and initiator azobisisobutyronitrile to prepare the polymer microsphere of the imprinted picric acid derivative PAD 1; 3-4: the picric acid derivative PAD1 is removed from the recognition site of the imprinted polymer microsphere and combined with the template molecule, so that the selective recognition and the sensitivity detection of the picric acid derivative PAD1 are realized.
FIG. 2 is a schematic representation of the reaction of N, N-diisopropylcarbodiimide prepared according to the invention with picric acid to form the picric acid derivatives PAD1 and PAD 2. N, N' -diisopropyl carbodiimide is used as a nucleophilic reagent, 2, 4, 6-trinitrophenol is used as a substrate, and substitution and addition reactions are carried out on aromatic rings under the nitrogen atmosphere at room temperature to prepare phenolic derivatives PAD1 and PAD 2.
FIG. 3 shows the hydrogen nuclear magnetic resonance spectrum of picric acid derivative PAD1 prepared by the present invention. 1H NMR (400 MHz, CDCl3) δ 9.01 (s, 2H), 4.23-4.16 (m, 1H), 4.08-4.00 (m, 1H), 3.89-3.84 (m, 1H), 3.18-3.10 (m, 1H), 1.73 (d, J = 6.6 Hz, 6H), 1.46 (d, J = 6.4 Hz, 6H), 1.34 (d, J = 7.3 Hz, 6H), 1.23 (d, J = 6.7 Hz, 6H), further identified as PAD 1.
FIG. 4 is an infrared spectrum of picric acid (a) and picric acid derivative PAD1 (b) prepared according to the present invention. As can be seen from a in the figure, the thickness of the film is 3500 to 3400 cm-1And 3000E2900 cm-1The absorption peak is corresponding to the stretching vibration of C-H bond in benzene ring and is 2000-1500 cm-1The absorption peak is just the benzene ring group in picric acid. At 1630-1480 cm-1In the presence of NO2The peak of the stretching vibration is 1630-1480 cm-1There is bending vibration of the OH. In the figure, b is 3200-3000 cm-1And 1000-600 cm-1The absorption peak present corresponds to-NH2Stretching vibration at 1700-1500 cm-1A C = O expansion vibration peak is arranged between the two vibration peaks and is 1700-1500 cm-1There is a stretching vibration of the C = N key in between.
FIG. 5 is a normalized ultraviolet-visible spectrum of picric acid (a) and PAD1 (b) solutions and a normalized fluorescence emission spectrum of PAD1 (c) solutions prepared in accordance with the present invention. Color insets (a) and (b) are photographs of solutions of picric acid and picric acid derivative PAD1 taken under natural light, respectively, and (c) is a photograph of a solution of PAD1 taken under a 365 nm wavelength ultraviolet lamp. Solutions of picric acid and its derivative PAD1 were greenish and reddish-orange under natural light, respectively, while solutions of PAD1 under a 365 nm wavelength ultraviolet lamp emitted yellow fluorescence bands with a maximum emission wavelength of 567 nm.
FIG. 6 shows the molecular structure transformation of PAD1 prepared according to the present invention in potassium tert-butoxide and perchloric acid solutions, respectively. The color picture is that the prepared potassium tert-butyl alcoholate is added into the solution drop by drop, the fluorescence in the solution is gradually darkened, and the fluorescence disappears when the solution is added; then adding perchloric acid solution into the solution, and finding that the color of the solution gradually recovers the original yellow fluorescence.
FIG. 7 is an SEM of a PAD1 blot prepared according to the present invention of polymer microspheres with a diameter of about 2 um. As can be seen, the polymer is spherical, the particle size is uniform and the distribution is uniform, the surface is smooth, and the diameter of the microsphere is about 2 um.
FIG. 8 is a graph comparing the recognition performance of molecules prepared according to the present invention. 20mg PAD1 imprinted polymer microspheres with a particle size of about 2um were rebinding PAD1(□) and PAD2 (. smallcircle.) respectively; PAD1(Δ) rebinding amount of 20mg PAD1 imprinted polymer particles with a particle size of about 4 um; 20mg of PAD1 non-imprinted polymer microspheres having a particle size of about 2um to PAD1 (. v.) rebinding amount; it can be seen that the imprinted microsphere has high-efficiency selective recognition on PAD1, which is 4 times higher than PAD 2. The 4 um imprinted particles bound approximately 50 nmol to PAD1, while the 2um imprinted microspheres bound approximately 230 nmol to PAD1, which is 4.6 times that of the 4 um imprinted particles. Compared with non-imprinted microspheres, the molecularly imprinted polymer microspheres have higher recognition performance under the same particle size, which is 6 times that of the non-imprinted microspheres.
FIG. 9 shows the molecular dynamics curve of the picric acid derivative PAD1 prepared according to the present invention. (a) PAD1 imprinted polymeric microspheres' binding capacity (□) over time; (b) the amount of binding (°) of the conventional imprinted polymer particles evolving over time; the kinetic rate of PAD1 imprinted polymer microspheres before reaching equilibrium is 1.39 nmol min-1PAD1 kinetic rate of 0.18 nmol min before equilibrium of conventional imprinted polymer particles was reached-1The kinetic rate of the polymer microspheres was 7.72 times that of the conventional imprinted polymer particles. The imprinted polymer microsphere material has faster kinetics and better effect. The binding kinetics data above are 20mg of imprinted polymer at 5.0X 10-5 mol•L-1PAD1 solution acquisition.
FIG. 10 is a progression of the increase in fluorescence intensity with increasing concentration of target analyte PAD1 in a perchloric acid solution prepared in accordance with the present invention. The picric acid derivative PAD1 with different concentrations enters the selective recognition site of the imprinted polymer, and the evolution process of the measured fluorescence intensity is 1 multiplied by 10 from bottom to top-9mol•L-1，1×10-8mol•L-1，1×10-7mol•L-1，1×10-6mol•L-1And 1X 10-5mol•L-5The intensity of fluorescence is stronger as the concentration of PAD1 is increased, and trace detection of PAD1 is realized through the change of the fluorescence intensity.
Detailed Description
A preparation method of molecularly imprinted polymer microspheres for detecting 2, 4, 6-trinitrophenol is characterized by comprising the following steps: the polynitrophenol derivative generated by the reaction of 2, 4, 6-trinitrophenol and N, N' -diisopropyl carbodiimide has bright yellow fluorescence, the fluorescence has the opening and closing characteristic under the acid-base condition, the molecularly imprinted polymer microsphere elutes imprinted molecules positioned in an imprinted layer, a hole structure complementary with the imprinted molecular structure, the size and the functional group is formed in the imprinted layer, the polymer microsphere for eluting the imprinted molecules has specific recognition sites for target analyte molecules, and the selective recognition and detection of the target analyte molecules are realized, and the preparation process comprises the following four steps:
the first step is the synthesis of 2, 4, 6-trinitrophenol derivatives: weighing 0.4-0.6 g of treated 2, 4, 6-trinitrophenol, placing the 2, 4, 6-trinitrophenol in a 100mL three-neck flask, adding 7-8 mL of dichloromethane for ultrasonic dispersion, adding 0.2-0.4 g of N, N' -diisopropyl carbodiimide, immediately turning the solution into reddish brown, stirring for 1-3 h under nitrogen atmosphere, drying for 0.5-1.5 h in a vacuum drying oven at 30-50 ℃ to obtain reddish brown solid powder with bright yellow fluorescence characteristics, pouring 20mL of boiling methanol solution into the prepared reddish brown solid powder, adding 10-20 mL of distilled water, placing in an ice water mixed solution at 0 ℃ for water bath for 1-3 h, finding that reddish brown crystals are separated out, filtering to obtain 2 to be prepared, 4, 6-trinitrophenol derivatives;
the second step is the purification of the 2, 4, 6-trinitrophenol derivative: vertically installing a silica gel column on an iron support, rinsing with acetone and petroleum ether for 3 times, adding 150-250 g of silica gel into a 250mL beaker, uniformly stirring with petroleum ether, filling the silica gel column, starting an air pump, pressurizing the solution downwards, beating the column with an ear washing ball to vibrate and fill the silica gel tightly, keeping the uppermost layer of the silica gel column horizontal when the height of the silica gel column meets the separation requirement, adding petroleum ether after the column is tamped, pressurizing downwards by using the air pump, performing the operation for 3 times, adding a small amount of prepared eluent when the liquid level of the petroleum ether and the column surface of the silica gel are level, naturally flowing downwards, sucking the prepared 2, 4, 6-trinitrophenol derivative sample by using a rubber head dropper, slowly and uniformly loading along the column wall, pressurizing downwards by using the air pump if the sample amount is excessive, then loading, and completely loading the separated sample, pressing the mixture to be flush with the cylindrical surface of the silica gel by using an air pump, after the sample loading is finished, slowly adding a prepared eluent along the wall of the column, and finally obtaining 2, 4, 6-trinitrophenol derivatives, wherein the separated liquid is orange red liquid (PAD 1) firstly, yellow liquid (PAD 2) secondly, 5-15 uL of the obtained liquid is respectively dripped on a silica gel plate by using a trace sample injector, after the separation and purification are confirmed, the obtained PAD1 and PAD2 are distilled under reduced pressure, and finally, the obtained product is dried in a vacuum oven at 50-70 ℃ for 7-9 h, and finally, orange yellow and brown yellow solid powder is respectively obtained;
the third step is the preparation of the molecular engram polymer microsphere of the target analyte: respectively weighing 20-40 mg of orange-yellow solid powder PAD2, 17-19 mg of acrylamide and 15-25 mg of azodiisobutyronitrile by using a ten-thousandth electronic balance, placing the weighed materials in a 250mL conical flask, then weighing 50mL of mixed solution of methanol and acetonitrile in a volume ratio of 9:1 by using a 100mL measuring cylinder, adding the mixed solution into the conical flask, ultrasonically dispersing for about 5 minutes, finally weighing 190-210 mu L of ethylene glycol dimethacrylate by using a microsyringe, dripping the ethylene glycol dimethacrylate into the solution, introducing nitrogen for 20-30 minutes, uniformly coating vacuum grease on a ground stopper, covering the conical flask, wrapping the conical flask with a preservative film, then placing the conical flask in a constant temperature oscillator, carrying out oscillation reaction for 2-4 hours at 50-60 ℃, regulating the temperature to 60 ℃ and carrying out oscillation reaction for 8-10 hours, finally, carrying out oscillation reaction for 1-3 h at 70-85 ℃, taking out and cooling to room temperature, transferring the reaction mixed solution into a 15mL centrifuge tube, centrifuging at the rotating speed of 5000-7000 rpm for 5-10 min, removing supernatant, adding a detergent, continuing centrifugation, carrying out ultrasonic dispersion, re-centrifuging, and repeating the operation for 3 times to obtain the molecular imprinting polymer microspheres of the imprinting target molecules;
the fourth step is the preparation of polymer microspheres for selectively identifying and detecting target analytes: using acetic acid and methanol in a volume ratio of 2: eluting the template molecules in the obtained molecularly imprinted polymer microspheres in 200mL of mixed solution in a Soxhlet extractor until no fluorescence signal exists in the eluent, thereby obtaining the polymer microspheres for selectively identifying the target analyte molecules and detecting optical signals.
When target analyte molecules encounter the polymer microspheres, the target analyte molecules enter the recognition sites again through concentration difference driving force, the recognition sites have high selective recognition on the target analytes, and meanwhile, the target analytes entering the recognition sites in an acidic environment have high sensitive fluorescent signal output, so that the trace detection on the target analytes is realized.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The poly-nitrophenol derivative is generated by nucleophilic addition reaction of 2, 4, 6-trinitrophenol and N, N' -diisopropyl carbodiimide, the poly-nitrophenol derivative is separated and purified to obtain 2, 4, 6-trinitrophenol derivative PAD1, and the poly-nitrophenol derivative PAD1 is copolymerized with a functional monomer, a cross-linking agent and an initiator to prepare the molecularly imprinted polymer microsphere, and the polymer microsphere with selective recognition and fluorescent signal output on PAD1 is obtained after eluting a template molecule PAD 1.
The first step is the synthesis of 2, 4, 6-trinitrophenol derivatives: weighing 0.5 g of treated 2, 4, 6-trinitrophenol, placing the 2, 4, 6-trinitrophenol in a 100mL three-neck flask, adding 7.5 mL of dichloromethane for ultrasonic dispersion, adding 0.3 g of N, N' -diisopropylcarbodiimide, immediately turning the solution into reddish brown, stirring for 2h under nitrogen atmosphere, drying for 1h in a vacuum drying oven at 40 ℃ due to volatilization of the dichloromethane solvent to obtain reddish brown solid powder with bright yellow fluorescence characteristics, pouring 20mL of boiling methanol solution into the prepared reddish brown solid powder, adding 15mL of distilled water, placing in an ice water mixed solution at 0 ℃ for water bath for 2h, finding that reddish brown crystals are separated out, and filtering to obtain the 2, 4, 6-trinitrophenol derivative to be prepared;
the second step is the purification of the 2, 4, 6-trinitrophenol derivative: vertically installing a silica gel column on an iron support, moistening and washing the silica gel column for 3 times by acetone and petroleum ether, adding 200 g of silica gel into a 250mL beaker, uniformly stirring the silica gel with the petroleum ether, filling the silica gel column, starting an air pump, pressurizing the solution downwards, beating the column by an ear washing ball to vibrate and fill the silica gel in a shaking way, keeping the uppermost layer of the silica gel column horizontal when the height of the silica gel column meets the separation requirement, adding the petroleum ether after the column is tamped, then pressurizing downwards by the air pump, performing the operation for 3 times, adding a small amount of prepared eluent when the liquid level of the petroleum ether is quickly aligned with the column surface of the silica gel, naturally flowing downwards, sucking the prepared 2, 4, 6-trinitrophenol derivative sample by a rubber head dropper, slowly and uniformly loading the sample along the column wall, if the sample amount is excessive, pressurizing downwards by the air pump, then loading the sample, and finishing the loading after the separated sample is completely loaded, pressing the mixture to be flush with the cylindrical surface of the silica gel by using an air pump, after the sample loading is finished, slowly adding a prepared eluent along the wall of the column, and finally obtaining the 2, 4, 6-trinitrophenol derivative, wherein the separated liquid is orange red liquid (PAD 1) firstly, and yellow liquid (PAD 2) secondly, 10 uL of the obtained liquid is respectively dripped on a silica gel plate by using a micro sample injector, after the separation and purification are confirmed, the obtained PAD1 and PAD2 are distilled under reduced pressure, and finally, the obtained product is dried in a vacuum oven at 60 ℃ for 8 hours, and finally, orange yellow and brown yellow solid powder is respectively obtained;
the third step is the synthesis of the molecular engram polymer microsphere of the target analyte: respectively weighing 30 mg of orange-yellow solid powder PAD1, 18 mg of acrylamide and 20mg of azodiisobutyronitrile by using a ten-thousandth electronic balance, placing the powder PAD1, the azodiisobutyronitrile into a 250mL conical flask, then weighing 50mL of mixed solution of methanol and acetonitrile with the volume ratio of 9:1 by using a 100mL measuring cylinder, adding the mixed solution into the conical flask, ultrasonically dispersing the mixed solution for about 5 minutes, finally weighing 200 mu L of ethylene glycol dimethacrylate by using a microsyringe, dropwise adding the ethylene glycol dimethacrylate into the solution, introducing 25 min of nitrogen, uniformly coating a ground stopper with vacuum grease, covering the conical flask, wrapping the conical flask with a preservative film, then placing the conical flask into a constant-temperature oscillator, oscillating the conical flask for 3 hours at 55 ℃, then adjusting the temperature to 65 ℃ for 9 hours, finally oscillating the conical flask for 2 hours at 80 ℃, taking out and cooling the conical flask to room temperature, transferring the reaction mixed solution into a 15mL centrifugal tube, centrifuging at 6000 rpm for 10min, removing supernatant, adding detergent, continuing centrifuging, ultrasonically dispersing, centrifuging again, and repeating the operation for 3 times to obtain molecularly imprinted polymer microspheres imprinted with target molecules;
the fourth step is the preparation of polymer microspheres for selectively identifying and detecting target analytes: eluting the template molecules in the obtained molecularly imprinted polymer microspheres in a Soxhlet extractor by using 200mL of mixed solution with the volume ratio of acetic acid to methanol being 2:8 until no fluorescence signal exists in the eluent, thus obtaining the polymer microspheres for selectively identifying the target analyte molecules and detecting optical signals.
When target analyte molecules meet the polymer microspheres with the recognition sites again, the target analyte molecules enter the selective recognition sites again through concentration difference driving force, the recognition sites have high selective recognition on the target analytes, and meanwhile, the target analytes entering the recognition sites in an acidic environment have high sensitive fluorescent signal output, so that trace detection on the target analyte picric acid derivative PAD1 is realized.

Claims (4)

1. A preparation method of molecularly imprinted polymer microspheres for detecting 2, 4, 6-trinitrophenol is characterized by comprising the following steps: the polynitrophenol derivative generated by the reaction of 2, 4, 6-trinitrophenol and N, N' -diisopropyl carbodiimide has bright yellow fluorescence, the fluorescence of the polynitrophenol derivative has an opening and closing characteristic under the acid-base condition, the molecularly imprinted polymer microspheres elute imprinted molecules positioned in an imprinted layer, a cavity structure complementary with the imprinted molecular structure, the size and the functional group is formed in the imprinted layer, the polymer microspheres eluting the imprinted molecules have specific recognition sites for target analyte molecules, the selective recognition and detection of the target analyte molecules are realized, and the preparation process of the molecularly imprinted polymer microspheres comprises the following four steps:
1.1 the first step is the synthesis of 2, 4, 6-trinitrophenol derivatives: weighing 0.4-0.6 g of treated 2, 4, 6-trinitrophenol, placing the 2, 4, 6-trinitrophenol into a 100mL three-neck flask, adding 7-8 mL of dichloromethane for ultrasonic dispersion, then adding 0.2-0.4 g of N, N' -diisopropyl carbodiimide, immediately turning the solution into reddish brown, stirring for 1-3 h under nitrogen atmosphere, drying for 0.5-1.5 h in a vacuum drying oven at 30-50 ℃ to obtain reddish brown solid powder with bright yellow fluorescence characteristics, pouring 20mL of boiling methanol solution into the reddish brown solid powder, then adding 10-20 mL of distilled water, placing the mixture into an ice water mixed solution at 0 ℃ for water bath for 1-3 h, finding out reddish brown crystals, and filtering to obtain the 2, 4, 6-trinitrophenol derivative to be prepared;
1.2 second step purification of 2, 4, 6-trinitrophenol derivatives: vertically installing a silica gel column on an iron support, rinsing with acetone and petroleum ether for 3 times, adding 150-250 g of silica gel into a 250mL beaker, stirring uniformly with petroleum ether, filling the silica gel column, starting an air pump, pressurizing the solution downwards, beating the column with an ear washing ball, vibrating and filling the silica gel, keeping the uppermost layer of the silica gel column horizontal when the height of the silica gel column meets the separation requirement, adding petroleum ether after the column is tamped, then pressurizing downwards by using the air pump, performing the operation for 3 times, adding a small amount of eluent which is prepared and passes through the column when the liquid level of the petroleum ether is flush with the surface of the silica gel column, naturally flowing downwards, sucking the prepared 2, 4, 6-trinitrophenol derivative sample by using a rubber head dropper, uniformly loading the sample along the column wall, pressurizing downwards by using the air pump if the sample amount is excessive, then loading the sample, and finishing the separation of all the samples, pressing the mixture to be flush with the silica gel column surface by using an air pump, adding an eluent prepared in advance for climbing a silica gel plate along the column wall after sample loading is finished, finally obtaining 2, 4, 6-trinitrophenol derivatives, namely, an orange red liquid PAD1 separated out firstly, and a yellow liquid PAD2, respectively taking 5-15 mu L of the orange red liquid and the yellow liquid by using a micro sample injector, dropwise adding the orange red liquid and the yellow liquid on the silica gel plate, after separation and purification are confirmed, carrying out reduced pressure distillation on the obtained PAD1 and PAD2, finally drying in a vacuum oven at 50-70 ℃ for 7-9 h, and finally respectively obtaining orange yellow and brown yellow solid powder;
1.3 the third step is the synthesis of the molecular engram polymer microsphere of the target analyte: respectively weighing 20-40 mg of orange-yellow solid powder, 17-19 mg of acrylamide and 15-25 mg of azodiisobutyronitrile by using a ten-thousandth electronic balance, placing the orange-yellow solid powder, the azodiisobutyronitrile into a 250mL conical flask, then weighing 50mL of mixed solution of methanol and acetonitrile with the volume ratio of 9:1 by using a 100mL measuring cylinder, adding the mixed solution into the conical flask, ultrasonically dispersing for 5 minutes, finally weighing 190-210 mu L of ethylene glycol dimethacrylate by using a microsyringe, dripping the ethylene glycol dimethacrylate into the solution of the conical flask, introducing nitrogen for 20-30 minutes, uniformly coating vacuum grease on a ground stopper, covering the conical flask, wrapping the conical flask with a preservative film, then placing the conical flask into a constant-temperature oscillator, carrying out oscillation reaction for 2-4 hours at 50-60 ℃, then carrying out oscillation reaction for 8-10 hours at the temperature of 60-70 ℃, finally carrying out oscillation reaction for 1-3 hours at the temperature of 70-85 ℃, taking out cooling to room temperature, transferring the reaction mixed solution into a 15mL centrifugal tube, centrifuging at the rotating speed of 5000-7000 rpm for 5-10 min, removing supernatant, adding a detergent, continuing centrifuging, performing ultrasonic dispersion, centrifuging again, and repeating the operation for 3 times to obtain the molecularly imprinted polymer microspheres imprinted with target molecules;
1.4 the fourth step is the preparation of polymeric microspheres for selective recognition and detection of target analytes: eluting the template molecules in the obtained molecularly imprinted polymer microspheres in a Soxhlet extractor by using 200mL of mixed solution with the volume ratio of acetic acid to methanol being 2:8 until no fluorescence signal exists in the eluent, and obtaining the polymer microspheres for selectively identifying the target analyte molecules and detecting optical signals.
2. The method for preparing the molecularly imprinted polymer microsphere for detecting 2, 4, 6-trinitrophenol according to claim 1, wherein the method comprises the following steps: the acid and the base respectively refer to perchloric acid and potassium tert-butoxide.
3. The method for preparing the molecularly imprinted polymer microsphere for detecting 2, 4, 6-trinitrophenol according to claim 1, wherein the method comprises the following steps: the eluent used for passing through the column and the eluent used for climbing the silica gel plate in the prepared molecularly imprinted polymer microspheres are respectively prepared by mixing ethyl acetate and petroleum ether in a volume ratio of 1:3 and 1: 5.
4. The method for preparing the molecularly imprinted polymer microsphere for detecting 2, 4, 6-trinitrophenol according to claim 1, wherein the method comprises the following steps: the detergent used in the prepared molecularly imprinted polymer microsphere is prepared by mixing deionized water and acetonitrile in a volume ratio of 1:1.
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