CN108246271A - The preparation method for the molecular blotting polymer microsphere that one kind detects for 2,4,6- trinitrophenols - Google Patents

The preparation method for the molecular blotting polymer microsphere that one kind detects for 2,4,6- trinitrophenols Download PDF

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CN108246271A
CN108246271A CN201810155489.8A CN201810155489A CN108246271A CN 108246271 A CN108246271 A CN 108246271A CN 201810155489 A CN201810155489 A CN 201810155489A CN 108246271 A CN108246271 A CN 108246271A
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microsphere
preparation
molecular
trinitrophenols
detection
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CN108246271B (en
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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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Hefei College
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28016—Particle form
    • B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/56—Acrylamide; Methacrylamide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching

Abstract

The preparation method for the molecular blotting polymer microsphere that one kind detects for 2,4,6 trinitrophenols, it is characterised in that：2,4,6 trinitrophenols and N,More nitro amphyl microspheres of N ' diisopropylcarbodiimides reaction generation have glassy yellow fluorescence,And its fluorescence has closing characteristic under acid-base condition,Elution is located at the microsphere in imprinted layer in the molecular blotting polymer microsphere,The interior formation of imprinted layer has and microsphere structure,Size and the void structure of function base complementation,The polymer microballoon of microsphere is eluted with the specific recognition site to target analyte molecule,Target molecule enters specific recognition site,It realizes to target analyte molecule Selective recognition,It is realized by the opening and closing of target molecule fluorescence to its sensitivity Detection,Its molecular engram microsphere large specific surface area,The space structure for forming recognition site is stablized,Effective site is more,Identification highly selective to target molecule and high sensitive detection.

Description

The system for the molecular blotting polymer microsphere that one kind detects for 2,4,6- trinitrophenols Preparation Method
Technical field
The present invention relates to materials science field, more particularly to the preparation side of 2,4,6- trinitrophenol molecular engrams of detection Method.
Background technology
World today's international relations is complicated and changeable, and the terrorist violences event such as explosion frequently occurs, and it is each to have seriously endangered the world The security of the lives and property and social stability of the state people, and threat is constituted to the safety and stablization of country, area.Therefore, The detection and analysis of explosive cause the great attention of research institution of various countries, it is intended to be prejudged and exploded by analysis and detection technology means Object exists, and reduces injury of the explosive to the mankind.2,4,6- trinitrophenols（2,4,6-trinitrophenol, TNP）Belong to Aldehydes matter, also known as picric acid（Picric acid, PA）.Its raw material is easy to get, and prepares simply, brisance is big, is common One of explosive.Meanwhile picric acid is also used for fungicide and mildew removing agent agriculturally, fungicide and astringent pharmaceutically.It is long Phase contact picric acid can cause the symptoms such as headache, nausea, diarrhea, fever, very big to actual bodily harm.With industrialized development, A large amount of picric acid is discharged into water, causes pollution of waterhead.The extensive purposes of picric acid, improperly use can generate the mankind very big Harm, therefore, there is an urgent need to the technological means of a kind of quick identification and sensitive detection to detect explosive to be effectively reduced Injury of the explosive to the mankind, is of great practical significance.
The method of detection explosive has spectrophotometry, electrochemical process, capillary electrophoresis, high performance liquid chromatography at present Method, gas chromatography etc.（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）, But column pollution easily occurs for these method poor universalities, therefore, it is still necessary to constantly explore new detection method.Ronita L are delivered Scientific paper（Talanta, 2005,66 (2)：581–590）High performance liquid chromatography-photoelectricity assisted electrochemical detection（High- Performance Liquid Chromatography Photoassistedelectro-Chemical Detection, HPLC-PAED）With UV absorption（Ultraviolet Absorbance, UV）Detection is used in combination, for determination of the environment sample In explosive.The system utilizes on-line solid phase extraction technology（Solid-Phase Extract, SPE）Carry out sample pretreatment （It is fractionated and concentrates）, SPE can carry out separation and pre-concentration target analytes according to simple chromatographic principles, suitable for underground water The assessment of middle explosive.Roger T publish thesis（Anal. Chem.1999,71,2739-2744）Propose it is quick continuously into Sample spectrophotometry measures 2,4,6-trinitrotoluene in pedotheque（2,4,6-trinitrotoluene, TNT）Method. This method is based on TNT and derivative reaction of the sodium sulfite in alkaline medium-acetone.And used sample in reacting, the third of use Ketone percentage, reagent volume and the mixing conditions such as coil volume, the time of reaction are optimised.It was found that the reaction is dense to acetone Spend it is especially sensitive, in 88% acetone/water（v/v）It is best in medium.And this method is to the response investigations table of other explosives It is bright, only there is specific selectivity to TNT.Cao Xinhua of Xinyang Normal College et al. discloses patent of invention （CN201610716172.8）" a kind of organogel compound of naphthalimide and preparation method thereof, gel and application ", the hair Bright bromo- 1, the 8- naphthalene anhydrides of 4- and alkylamine in molar ratio 1:1.2 heating reflux reaction generates N- alkyl -4- bromines in toluene solution 1,8- naphthalimide；Then under the conditions of existing for potassium carbonate, the N- alkyl -4- bromines 1 in dimethyl sulphoxide solution, 8- naphthoyls Asia Amine and 4- pyridones in molar ratio 1:1.5,100 DEG C of conditioned responses 12 hours, being introduced using simple alkylamine has fluorescence Property naphthalimide groups, and gel can be formed, the gel is for 2,4,6- trinitrophenols of detection.
Fluorescence analysis method is also a kind of common method for detecting explosive.Arvin Sain Tanwar et al. deliver opinion Text（ACS Sens.2016,1,1,070 1077）A kind of new polyfluorene derivative is described, is closed by Suzuki coupling polymerization method The nitro explosivity picric acid of 22.9 pg levels is identified into the rapid of high yield and specifically, by nuclear magnetic resonance, ultraviolet It can be seen that, fluorescence, time resolution photoluminescence spectra and cyclic voltammetry good characterization has been carried out to polymer.The derivative leads to Crossing powerful inside filter effect realizes the signal response for being exclusively used in picric amplification, which is different from wide coverage Based on nitro-aromatic detection ground state electric charge transfer or based on Resonance energy transfer（Fluorescence Renounce Energy Transfer, FRET）Probe, pass through protonation assist photo induced electron transfer（Photoinduced Electron Transfer, PET）Even if in most common interference nitro explosive and other analyses usually found in natural water In the presence of object, the side group amine groups on polyfluorene derivative side chain can also provide higher sensitivity and outstanding selectivity.Therefore, The trace of PA can be monitored based on the platform of polyfluorene derivative even if in solution and solid competitive environment with extremely low level Mark.Lisa C et al. exist（Anal. Chem.1995,67,2431-2435）In order to detect most widely used explosive TNT, develop Competitive immunoassay method.Trinitrobenzene sulfonic acid（2,4,6-trinitrobenzenesulfonic acid, TNB）It is TNT Analog, marked with fluorogen and be used as analyte competitor.By what is marked containing 7.5 ng/mL sulfo group indigo cyanines 5- ethylenediamines TNB（Cy5-EDA-TNB）Solution be exposed to the optical fiber of antibody cladding, generate and correspond to 100% or with reference to letter higher than background Number signal specific, the inhibition of 100% signal is proportional to the TNT concentration in sample.10 ng/mL in buffer solution can be detected TNT（8 ppb）Sensitivity.Because there are other analytes, interference is easily generated, reduces the accuracy of detection.Nanjing postal TV university stone is the open patent of invention of grace et al.（CN201610219036.8）" a kind of six rib piece of europium Base Metal organic frame and its The n,N-Dimethylformamide of six rib piece of europium Base Metal organic frame is prepared in preparation method and application "（N, N- Dimethylformamide, DMF）Dispersion liquid is filtered through filter paper, obtains the film of six rib sheet materials preparation, is 365 in wavelength Under the ultraviolet excitation light of nm, which sends out red fluorescence, has been added dropwise after the DMF solution of PA, is 365 nm's in wavelength Under ultraviolet light, red fluorescence originally almost quenches, and 2,4- dinitrotoluene (DNT)s are added dropwise（2,4-dinitrotoluene, DNT）DMF solution after, wavelength be 365 nm ultraviolet light under, red fluorescence originally significantly weakens.Quenching degree is not It is both because the six rib piece film of europium Base Metal organic frame has different explosives different detection results.With reference to fluorescence point Principle is analysed, optical sensor becomes detection device all the fashion, Priyanka Dutta et al.（ACS Appl. Mater. Interfaces2015,7,24,778 24790）Report a kind of polyvinyl alcohol graft copolymerized polyaniline of high fluorescent material（PPA）And its With 2- mercapto succinic acids（MSA）The CdTe quantum of sealing end（PPA-Q）With the CdTe/ZnS core shell quantum dots of MSA sealing ends（PPA- CSQ）Nanocomposite single step synthesis Raolical polymerizable.ZnS shells are formed on CdTe cores and increase quantum dot （Quantum Dots, QDs）Stability and quantum yield, and largely reduce its toxicity.This work report The road development of high fluorescent material is showed with the picric acid explosive fluorescent quenching in selectivity and effective detection nanometer range As the mechanism run in quenching phenomenon is considered as strong self-filtering effect and the ground state electrostatic between polymer and picric acid The combination of interaction.And the portable and inexpensive electronic equipment of vision-based detection is carried out to picric acid with sensor-based system It successfully manufactures, which is further used for picric quantitative detection in actual water sample.This method poor selectivity, easily by To the interference of other quenchers.Varun Vij et al. have a learned dissertation published（ACS Appl. Mater. Interfaces2013, 5,5,373 5380）It is middle to design the derivative for having synthesized oneself six benzo nonenes 5 and 6（HBC）, their shapes in aqueous medium is mixed Into fluorescence aggregation, these derivatives have the tertiary butyl of space requirement in periphery, and solubility greatly increases.In addition, introduce two As rotor, this to generate aggregation inducing transmitting enhancing phenomenon in coronene molecule a rotatable phenyl, increases HBC As the effectiveness of fluorescence chemical sensor, these aggregations can detect the PA of steam condition.This is that HBC reports that aggregation lures for the first time Enhancing is put in guide（AIEE）Phenomenon, providing fluorescence aggregation, alternatively property chemical sensor detects PA and the steaming in solution Gaseity.For convenient and inexpensive detection PA, fluorescent test paper is prepared for, this test strips can detect steam and contact mould Ultra trace PA under formula contributes to the trace detection of PA.Akhtar Hussain Malik et al. are in paper（ACS Appl. Mater. Interfaces2015,7,26,968 26976）It is middle to prepare a kind of Optical Properties of Novel Conjugated material PFMI, in polymer Specific recognition site of the cationic imidazole salts group as PA on PFMI side chains, also promotes the probe in polar solvent （DMSO, methanol etc.）In solubility.Good nano particle is generated using spontaneous reprecipitation method, on picomolar Picric detection is realized for the first time.Conjugated polymer nano-particle is used in 100% aqueous medium respectively（CPNs）With solid Under body state picric acid, while both PFMI nano particles are measured using moveable paper tape（PFMI-NPS）The terminal of preparation Sensor device provides a kind of means for detections of the PA under steam condition.The hypersensitivity machine of PFMI-NPS probe detections PA Reason is based on " molecular line effect ", electrostatic interaction, photo induced electron transfer and Resonance energy transfer principle.Chinese science Xinjiang physiochemical techniques research institute of institute Dou Xin deposits et al. open patent of invention（CN201610344253.X）" one kind is based on photoelectric respone Explosive vapors recognition detection method ".The invention is related to a kind of explosive vapors recognition detection method based on photoelectric respone, Device involved in this method is made of sensor, light source, power supply, ammeter, signal processor and alarm, using can week Phase property switchs and changes the light source of light intensity variation, and irradiation has on the single sensor of rapid photoelectric response, measures explosive and steams Gas photocurrent variations caused by sensor sensing material surface adsorbs pass through principal component analysis, linear discriminant analysis, artificial god Data processing is carried out through the recognition methods of network isotype, realizes the standard that sensor array responds variety classes explosive vapors Database by the way that the data processed result of suspected explosive is compared with database, is finally reached recognition detection explosive vapors Purpose.South China Normal University's Wang's southern exposure et al. discloses patent of invention（CN106008358A）" one kind is quick-fried for nitro-aromatic Benzimidazole based chemical sensors of fried object fluorescent quenching detection and preparation method thereof ", with two（Benzimidazole）Naphthalene and halogenated hydrocarbons For raw material, substitution reaction is mainly alkylated by simple and practicable N-, is made to detect based on picric acid, it is highly selective, high The explosive nitroaromatic of sensitivity detection.Benzimidazolyl fluorescence chemical sensor obtained is in the UV lamp using glimmering Visual detection picric acid in optical quenching solution and under solid-state, and can realize the picric detection of gaseous state.The above method obtains Very big progress, also achieves the picric problem of Site Detection, but complex manufacturing process, of high cost, recycling rate of waterused It is low.
In recent years, higher and higher for the requirement of the detection technique of explosive, the detection means of trace materials also appears in greatly In many visuals field.It is tall and big bright et al. to carry out Resonance energy transfer-amplification fluorescent quenching on Nano particles of silicon dioxide surface（Anal. Chem. 2008,80,8545-8553）, fluorescent dye and aminopropyl covalent modification to the surface of Nano particles of silicon dioxide, shape Into dye fluorescence group and the hydridization individual layer of ammonia ligand.Fluorescent silicon dioxide particle can pass through electron rich ammonia ligand and electron deficient virtue Charge-transfer complexation interaction specific binding TNT substances between fragrant ring.Nano-particle assembly array on silicon chip can be with It delicately detects down to about 1 nmolL-1TNT, only only used the TNT solution of 10 μ L（~2 pg TNT）With several ppb TNT steams.This nano-particle sensor based on FRET shows stable fluorescent brightness, to target analytes affinity Therefore strong and good assembling flexibility, can be applied super to trace target analytes detection field.Southern Yangtze University Wei Wei etc. People discloses patent of invention（CN2014000560284.X）" it is a kind of detect 2,4,6- trinitrophenols fluorescence chemical sensor and Preparation method " with hexachlorocyclotriphosphazene and curcumin by the method for an one-step polycondensation, is prepared a kind of with highly cross-linked structure Organic-inorganic hybrid polymer fluorescent optical sensor, realize liquid phase in 2,4,6- trinitrophenols specific recognition and detection, Suitable for trace explosive detection and environmental monitoring, have broad application prospects.What credit of University Of Suzhou et al. open hair Bright patent（CN201710253748.6）" detection method of a kind of silicon substrate SERS chips and preparation method thereof and TNT ", it is open It is a kind of for detecting the enhancing Raman scattering of the silicon substrate surface of TNT（Surface-Enhanced Raman Spectroscopy, SERS）Chip and preparation method thereof, the silver nitrate reduction method which is assisted by hydrofluoric acid, is repaiied on silicon chip in situ Silver nano-grain on decorations, the compound then to interact in the surface modification of chip with TNT, which constructs examines when participating in the cintest Survey the portable stage of TNT, the TNT of trace suitable for qualitative analysis actual environment sample.Lanzhou University is old to make the country prosperous et al. public affairs Patent of invention is opened（CN201610964476.6）" the highly selective water soluble fluorescence nano silicon particles synthesis for measuring trace TNP And application ", it is characterised in that utilize N-（2- aminoethyls）- 3- aminopropyl trimethoxysilanes are as silicon source, and catechol is also Former agent synthesizes the water soluble fluorescence nano silicon particles, and the two molar ratio is 9.3:100, and this method can be used for making test paper or examination Agent is convenient for carrying, and is easy to detect.Wayne H. Griest are in paper（Anal. Chem.1998,70,3015-3020）In carry And cannot most of trace analysises directly be obtained using required selectivity and sensitivity by analyzing complicated matrix, for solution Certainly this problem, before gas-chromatography/ion trap mass spectrometry, using solid phase micro-extraction technique, selectively pre-concentration is analyzed Object.The trace analysis of this method explosive and its metabolin suitable for seawater, but the method equilibration time is long, it is impossible to it realizes Quick detection, the accuracy of measurement result also remain to be discussed.
Molecular imprinting technology is a kind of artificial synthesized means with specific Selective recognition molecule, as comparing in recent years Popular detection technique, molecular engram material stability is good, easily prepares and at low cost, has specific selectivity, extensively should For the various fields such as biology, medicine, chemistry（Eersels K.ACS Appl. Mater. Interfaces2013,5, 7258−7267；Lee J-D.Org. Lett., 2005,7,963-966； Li H.ACS Appl. Mater. Interfaces2013,5,10,502 10509；Schirhagl R.Anal. Chem.2014,86,250 261）.
Molecular engram early stage proposes it is that the service life based on biosensor material is short, poor repeatability, to overcome these Difficulty, artificial synthesized imitative biomaterial come into being（Zhang Z.P.Anal. Chem.2014,86,1,123 1130）. Meng Fanli in 2016 et al. exists（ACS Appl. Mater. Interfaces2016,8,14,133 14141）Pass through what will be catalyzed Ser/His/Asp triplets and two phenylalanine of N- fluorenylmethyloxycarbonyls（Fmoc-FF）Combination, is then assembled into Nanowire by peptide Dimension（CoAHSD）, develop artificial hydrolase.Peptide based nano-fiber provides ideal supermolecule skeleton to support functional group, with Contain only the self assembly catalytic nanometer fiber of catalysis histidine residues（SA-H）It compares, works as histidine, serine and aspartic acid The ratio of residue is 40:1:When 1, the active highest of CoA-HSD, this shows orderly nanofibrous structures and serine and asparagus fern The synergistic effect of histidine residue helps to improve activity.In addition, to further improve peptidyl artificial for first Application molecular imprinting technology Enzyme（CoA-HSD）Activity, using p-NPA as molecular template, with the Ser/His/Asp tri- of appropriate direction arrangement catalysis Body residue.Therefore, the activity of trace CoAHSD nanofibers is 7.86 times of non-trace CoA-HSD, is 13.48 times of SA-H. Davide Carboni et al. exist（ACS Appl. Mater. Interfaces2016,8,34,098 34107）It is printed by molecule Mark technology improves the selective efficiency of graphene mediation enhancing Raman scattering.Xinjiang Academy of Land Reclamation ＆. Cultivation Lu Chunxia et al. is open Patent of invention（CN2017000535808.3）" a kind of preparation method of phthalate molecular imprinted polymer on surface and Using ", which is prepared for phthalic acid two（10- methoxyl group -10- oxo the last of the ten Heavenly stems）Ester compounds, and as virtual template, Mating surface molecular imprinting technology, the PAEs molecularly imprinted polymers prepared by two-step precipitation polymerization, solves in current technology The shortcomings that template embedding is too deep, template is revealed, association rate is low, obtained polymer morphology rule, uniform particle diameter, monodispersity Good, large amount of adsorption, while meet 10 kinds of PAEs multi-residue analysis demands in food.Nanjing Medical University's Chen Li Nas et al. are open to be sent out Bright patent（CN201710474093.5）" a kind of magnetism artificial receptors and its preparation method and application ", mainly include：（1） Fe3O4@SiO2The preparation of composite particles；（2）（1）Fe3O4@SiO2The preparation of@MPS composite particles；（3）The system of magnetic artificial receptors It is standby.Using ZL006 as template, synthesizing ordered, dense molecular trace shell is designed on the surface of magnetic Nano silica gel material, is reduced The phenomenon that imprinted sites are by " embedding ", association rate and separative efficiency are higher.Simultaneously by magnetic material, nanometer silica gel and table Face molecular imprinting technology is combined, and can be realized under external magnetic field and the quick of target substance is directly separated, and is active Chinese drug component The selective fast separating concentration of substance provides a kind of new strong means with detection.Hefei College's paint day precious jade et al. is open Patent of invention（CN201710185685.5）A kind of " Fe for detecting thifensulfuronmethyl3O4@PEG@SiO2The preparation side of artificial antibody Method " is included in Fe3O4The surface modification polyethylene glycol 2000 of magnetic nano-particle, surface coated Si O2Shell, formation core-shell- Shell structure is located at SiO by elution2Microsphere in shell, formation have the function of mutual with microsphere structure, size and base The specific recognition site hole of benefit, is realized to target analyte molecule Selective recognition and detection.Obtain that there is selectivity to know The FeO of other microsphere4@PEG@SiO2Artificial antibody, the maximum saturation binding capacity to thifensulfuronmethyl is 41.28 mg/g, preceding In 30 min, the rate of adsorption is 0.45 mg/gmin, is 5.34 times and 3.46 times of non-immunoblot method respectively.Nanjing medical courses in general The open patent of invention of university's Chen Li Nas et al.（CN201310408025.0）" baicalein molecularly imprinted polymer and preparation method thereof And application ".Precipitate molecules imprinted polymer microballoon prepared by the invention, compared with traditional bulk mole imprinted polymer Compared with having that monodispersity is good, uniform particle diameter, size are controllable, prepare simple, shorter etc. excellent without complicated last handling process and period Point.The open patent of invention of Sichuan University's indigo plant virtue et al.（CN201410432424.5）It is " a kind of super with protein molecule engram Paramagnetism composite Nano ball and preparation method and application " using a kind of improved microemulsion polymerization method, makes superparamagnetism receive Rice grain is arranged closely in polymer nanocomposite ball, remains high saturation magnetization.By in the composite Nano ball Abundant hydrogen bond is formed between the hydroxylating modified layer and polymer shell of the periphery of core, the protein molecular of trace, utilizes ice twice Bath is ultrasonically treated, and the protein molecule for promoting trace uniformly, is firmly distributed in polymer shell, is effectively reduced non-spy Different in nature binding site improves the selective binding ability of superparamagnetism composite Nano ball and target protein molecules, realizes high The separation purpose of selectivity.The open patents of invention of Beijing Agricultural College Jia Minghong et al.（CN201410688522.5）" pyrazoles ether bacterium The preparation method of ester molecule imprinted polymer ", by template molecule pyraclostrobin, function monomer methacrylic acid and crosslinking agent second Diol dimethacrylate, by template molecule:Function monomer:Crosslinking agent molar ratio 1:4:20, mixed dissolution is in perforating agent second In nitrile, two fourth acetonitrile of initiator azo is added in, is protected through nitrogen, water-bath at 60-65 DEG C, the template molecule polymerization of acquisition Object after further vacuum drying is ground, by soxhlet extraction, removes template molecule, and be dried under vacuum to constant weight at 60 DEG C, It is final to obtain pyraclostrobin molecularly imprinted polymer.Molecularly imprinted polymer prepared by the present invention can efficiently separate and be enriched with multiple Pyraclostrobin in miscellaneous matrix.This method can carry out highly selective identification to target analytes, can not realize to entering The target analytes of recognition site are with the output of sensitive optical signalling or electromagnetic signal.
And fluorescence analysis and molecular imprinting technology are combined to the obtained detection material with fluorescence property to be had well Recognition performance（Li H.；Wang L. Y.ACS Appl. Mater. Interfaces2013,5,10,502 10509）, Sensitive detection.The open patent of invention of Jiangsu University's Li Hong Jis et al.（CN201710568072 .X）" one kind is based on surface enhanced 2,6- chlorophenesic acid trace sensors of Raman technology and its preparation method and application ", the invention is by Raman detection technology with dividing Sub- engram technology combines so that product has the selectivity of sensitive detection property and height, in the invention, by Ag/CdTe bases Bottom is combined with molecular imprinting technology, and wherein CdTe is outstanding semiconductor, possesses unique optical characteristics, can be applied to pair The detection of 2,6- chlorophenesic acids.The SERS materials of high-sensitivity metal-semiconductor heterostructure have stronger, more sensitive table Face enhances Raman signal.The open patent of invention of Jiangsu University's poplar civilization et al.（CN201610720836.8）A kind of " fluorescent molecular The preparation method and its usage of trace adsorption and separation material " provides a kind of preparation of fluorescent molecular trace adsorption and separation material Method and application thereof includes the following steps：Step 1 is SiO2The preparation of nano-particle；Step 2 is to adulterate the zinc sulphide quantum of manganese The synthesis of point；Step 3 is the SiO of double bond modification2The preparation of nano-particle；Step 4 is the zinc sulphide amount of double bond modification doping manganese The preparation of son point；Step 5 is the preparation of fluorescent molecular trace adsorption and separation material.This fluorescent molecular imprinted polymer prints molecule The selectivity of mark polymer and the photoluminescent property of quantum dot combine.Can by the change of fluorescence intensity to target molecule into Row quickly detection.The open patent of invention of Nanjing Medical University's Chinese fiddle et al.（CN201210382810.9）“CdTe@SiO2Quantum The preparation method of point surface monoamine neurotransmitter molecularly imprinted polymer " is that CdTe@SiO are synthesized in water phase2Quantum dot, Rotary evaporation to water volatilization finishes, and unreacted substrate is washed away with absolute ethyl alcohol, by gained CdTe SiO2Quantum dot is added to cause In the agent of hole, after ultrasonic disperse is uniform, template molecule, function monomer and crosslinking agent are sequentially added, in N2Under atmosphere, it is stirred to react 20 H centrifuges reaction solution, discards supernatant liquid, after obtained solid washing removes template molecule, obtains CdTe@SiO2Quantum dot surface Monoamine neurotransmitter molecular engram material.Synthesized quantum dot fluorescence probe imprinted polymer material have high sensitivity, The advantages of highly selective, high-affinity and easy detection, it may be directly applied to the survey of monoamine neurotransmitter in biological fluid It is fixed.The open patent of invention of Southwestern University Tan Ke person of outstanding talent et al.（CN201410520007.6）" one kind is printed based on core-shell quanta dots molecule Mark polymer and application thereof " is related to a kind of CdTe/CdS quantum dots-molecularly imprinted polymer（QDs-MIP）, and in this, as glimmering Light probe is applied to the detection and analysis of perfluoro caprylic acid, which provides a kind of quick, easy, sensitive, highly selective detection perfluor The method of octanoic acid.Molecular recognition site is embedded in the coated CdTe/CdS quantum dot surfaces of TEOS by the polymer, with perfluoro caprylic acid The fluorescent molecular imprinted polymer being prepared for template molecule.Molecularly imprinted polymer prepared by the present invention has sensitivity It is high, detection is easy, the advantages that highly selective, high-affinity, can be directly used for the perfluoro caprylic acid in detection environment, can both reduce The cost of detection, and the efficiency of detection can be improved.
Xinyang Normal College's sweetness et al. discloses patent of invention（CN201611031452.1）" a kind of trace quickly detects Molecular imprinting electrochemical sensor of cyromazine and preparation method and application ", it is characterised in that：Working electrode is glass carbon electricity Pole, first using monodispersed SiO2@TiO2Core-shell type nano ball is modified, and then utilizes in-situ electrochemical polymerization method and molten Glue-gel method further prepares specific recognition of the surface molecule print film for cyromazine.Tall and big bright et al. the public affairs of Hefei College Patent of invention is opened（CN201510538714.2）A kind of " Eu of detection residual to agriculture3+Mark molecule trace sensor preparation side Method ", Eu3+It is pre-assembled with amino in APTS and the residual molecule of agriculture, obtain Eu after being condensed with TEOS hydrolytic crosslinkings3+Residual point of the agriculture of label Sub- trace Nano particles of silicon dioxide sensor, after eluting the residual molecule of agriculture, the recognition site for possessing the residual molecule selectivity to agriculture is empty Cave, after the residual molecule of agriculture is again introduced into the recognition site of sensor, by with the Eu on recognition site3+Chelate, the residual molecule of agriculture with Eu3+Fluorescence intensity after chelating increases, and using the change of fluorescence intensity, realizes, Gao Jie highly selective to trace pesticide molecule Resultant and hypersensitivity detection.The open patents of invention of Nanjing Medical University Zhou Xuemin et al.（CN201310023869.3）It is " a kind of The preparation method of molecular imprinting electrochemical sensor mixed for the Jenner's grain of rice for detecting dopamine ", the method is pre- through gold electrode Prepared by processing, Jenner's grain of rice self-assembled modified electrode, molecular engram self assembly solution is prepared, electric polymerization reaction and template molecule are washed It is de- and etc. to prepare molecular engram film electrochemical sensor, the selectivity of traditional electrochemical sensor is improved, and ring Should rapid, stability and tolerance it is good, it can be achieved that efficient, sensitive and real-time detection to dopamine in biological sample.To clinic Diagnosis, pathological research etc. are of great significance.The open patents of invention of Agricultural University Of Hunan Liu Xia et al. （CN201410471090.2）" the collosol and gel molecular imprinting electrochemical sensor detection acryloyl based on nano material compound The method of amine ", this method are after the compound of carbon nanotube, gold nanoparticle and chitosan is overlying on glassy carbon electrode surface, are incited somebody to action It is placed in containing template molecule, function monomer, crosslinking agent sol gel solution in carry out electrochemical deposition, removed after the completion of reaction Template molecule, obtained molecular imprinting electrochemical sensor are directly detected the acrylamide in sample.The method of the present invention Simply, and sample pre-treatments are easy to operate, and detection speed is fast, of low cost.In addition, selectivity that this method has had and reproduction Property, the rate of recovery meets the requirements, directly the acrylamide in the fried foods such as potato chips can be detected, answered with important reality With value.The open patent of invention of Nanjing Medical University's Chen Li Nas et al.（CN 201710592816 .1）A kind of " fluorescence nano point Sub- trace biomimetic sensor and its preparation method and application ".Template molecule, two kinds of different function monomers are added to pore-foaming agent In, after ultrasonic dissolution, prepolymerization is stirred at room temperature, obtains pre-assembled solution A；By the carbon dots ultrasonic disperse of surface double-bond functionalization Solution B is obtained in pore-foaming agent；After solution A and solution B mixing, crosslinking agent and initiator are added in, leads to nitrogen gas stirring, centrifugation is received Collection precipitation, is washed with distilled water；Finally with HAc-SDS eluted template albumen, products therefrom is lyophilized to get fluorescence nano molecule Trace biomimetic sensor.
Although the above method has the characteristics of respective respectively, have no that document report prepares target molecule itself with glimmering There is yellow fluorescence target molecule to enter recognition site and exported by own optical signal sensitivity for light characteristic.Have no document simultaneously Report has the more nitros of glassy yellow fluorescence by the reaction generation of 2,4,6- trinitrophenols and N, N '-diisopropylcarbodiimide Amphyl trace is prepared into polymer microballoon, into the trace derivative of recognition site, is realized by the opening and closing of autofluorescence To the report for identifying and detecting.Therefore, synthesis possesses highly selective and high sensitivity emission band and is visited for yellow autofluorescence The preparation method of the imprinted polymer microballoon of needle, realize has its necessity to ultra trace picric acid molecular recognition and detection.
In the present invention, we report a kind of molecular blotting polymer microsphere for 2,4,6- trinitrophenols of detection Preparation method, realize the identification and detection to 2,4,6- trinitrobenzen amphyl of trace.Imprinted polymer microballoon is especially suitable Identification material is closed, is easily had the function of to microsphere in the interior formation of imprinted layer complementary with microsphere structure, size and base Void structure, eluting the polymer microballoon of microsphere has to the specific recognition site of target analyte molecule, realizes To target analyte molecule Selective recognition.In imprinted polymer function monomer can according to the design feature of target analytes, Select different function monomers.In view of 2,4,6- trinitrophenol derivative molecular structure short of electricity sub-features, electron rich is selected Acrylamide for function monomer, recognition site is entered by the confession in function monomer by non-covalent strong effect target molecule The amino binding of electronics, realizes and 2,4,6- trinitrophenol derivative moleculars is identified.Enter print in view of general objectives molecule It is no signal output problem after mark recognition site, we are devising 2,4,6- trinitrophenols and N, N '-diisopropyl carbon More nitro amphyls of imidodicarbonic diamide reaction generation have glassy yellow fluorescence, and its fluorescence has opening and closing under acid-base condition Therefore characteristic, using 2,4,6- trinitrobenzen amphyl autofluorescence characteristics, has autofluorescence after entering print identification site Optical signalling exports, and realizes identification highly selective to 2,4,6- trinitrobenzen amphyls and high sensitive trace detection.
Invention content
Goal of the invention：For shortcoming existing for currently available technology, the present invention utilizes the bitter taste acid derivative synthesized With soda acid fluorescent switch characteristic, the chemical preparation process of the molecularly imprinted polymer of identification bitter taste acid derivative is prepared for, and Realize the identification of trace bitter taste derivative molecules and trace detection.It is by nucleopilic reagent N, N '-two acyl of diisopropyl carbon Asia first Amine is added to reaction in picric acid and forms bitter taste acid derivative, then carries out separating-purifying, obtains having soda acid fluorescent switch Derivative as target molecule, adds function monomer, crosslinking agent and initiator etc. and prepares bitter taste derivative molecules print Mark polymer microballoon after eluent microsphere, obtains having bitter taste acid derivative Selective recognition and sensitive Property detection molecular blotting polymer microsphere.
The technical scheme is that：One kind is for the molecular blotting polymer microsphere of 2,4,6- trinitrophenols detection Preparation method, it is characterised in that：More nitros of 2,4,6- trinitrophenols and N, N '-diisopropylcarbodiimide reaction generation Amphyl has glassy yellow fluorescence, and its fluorescence has closing characteristic, molecular engram polymerization under acid-base condition Elution is located at the microsphere in imprinted layer in object microballoon, and the interior formation of imprinted layer has and microsphere structure, size and work( The void structure of energy base complementation elutes the polymer microballoon of microsphere with the specific recognition position to target analyte molecule Point realized to target analyte molecule Selective recognition and detection, above-mentioned 2, the molecular engram of 4,6- trinitrophenols detection The preparation process of polymer microballoon includes following four steps：
The first step is the synthesis of 2,4,6- trinitrobenzen amphyls：Weigh 0.4~0.6 g treated 2,4,6- trinitrophenols It is placed in 100 mL three-necked flasks, then adds in 7~8 mL dichloromethane ultrasonic disperses, add the N of 0.2 ~ 0.4 g, N '- Diisopropylcarbodiimide, solution are immediately turned into rufous, 1 ~ 3 h are stirred under nitrogen atmosphere, due to methylene chloride Volatilization, solution becomes thick, and dry 0.5 ~ 1.5 h obtains red brown solid powder in 30 ~ 50 DEG C of vacuum drying chambers End has glassy yellow fluorescent characteristic, the methanol solution of the boiling of 20 mL is poured into the red brown solid powder of above-mentioned preparation, 10 ~ 20 mL distilled water are added, are placed in progress 1 ~ 3 h of water-bath in 0 DEG C of ice water mixed liquor, discovery has red-brown crystals analysis Go out, filtered to get to be prepared 2,4,6- trinitrobenzen amphyls；
Second step is the purification of 2,4,6- trinitrobenzen amphyls：Silicagel column is vertically installed on iron stand, with acetone and stone After oily ether rinse 3 times, 150 ~ 250g silica gel is added in 250 mL beakers, it is uniform with petroleum ether and stirring, then it is filled with To silicagel column, air pump is opened, is pressurizeed to solution downwards, and pillar is beaten with ear washing bulb, silica gel concussion is enriched, treats silicagel column When the height of son reaches the requirement of separation, silicagel column top layer is kept horizontal, after pillar strikes reality, adds petroleum ether, then It is being pressurizeed downwards using air pump, so operation 3 times, until petroleum ether liquid level and silica gel cylinder flush soon, is adding in a small amount of prepare Good eluent flows down naturally, 2,4,6- trinitrobenzen amphyl samples of above-mentioned preparation is drawn with rubber head dropper, along column The slow uniform loading of wall, if sample size is excessive, is pressurizeed downwards with air pump, followed by loading, after sample to be separated all go up, and use Air pump is depressed into be flushed with silica gel cylinder, after end of the sample, the eluent prepared in advance is slowly added into further along post jamb, finally Obtain that 2,4,6- trinitrobenzen amphyls are separated first is Chinese red liquid（PAD1）, secondly yellow liquid （PAD2）, 5 ~ 15 uL of liquid of above-mentioned gained is taken to be added dropwise in silica gel plate respectively with microsyringe, after determining separating-purifying, Obtained PAD1 and PAD2 is evaporated under reduced pressure, 7 ~ 9 h is finally dried in vacuum drying oven at 50 ~ 70 DEG C, finally distinguishes Obtain orange-yellow and yellow-brown solid powder；
Third step is the molecular blotting polymer microsphere synthesis of target analytes：20 are weighed respectively with the electronic balance of a ten thousandth ~ 40 mg orange/yellow solid powder PAD1, the acrylamide of 17 ~ 19 mg, the azodiisobutyronitrile of 15 ~ 25 mg are placed in In the conical flask of 250 mL, it is 9 then to measure 50 mL methanol with acetonitrile volume ratio with the graduated cylinder of 100 mL:1 mixed liquor adds in Into above-mentioned conical flask, ultrasonic disperse 5 minutes or so, finally again with the ethylene glycol two of 190 ~ 210 μ L of microsyringe measurement Methacrylate is added drop-wise in above-mentioned solution, after leading to 20~30 min nitrogen, is uniformly coated with vacuum grease in grinding port plug, is covered cone Shape bottle, then wrapped up with preservative film, then conical flask is positioned in constant temperature oscillator, at 50 ~ 60 DEG C, oscillating reactions 2 ~ 4 h, then temperature is adjusted to 8 ~ 10 h of oscillating reactions at 60 ~ 70 DEG C, at last 70 ~ 85 DEG C, 1 ~ 3 h of oscillating reactions takes Go out to be cooled to room temperature, reaction mixture be transferred in 15 mL centrifuge tubes, with 5000 ~ 7000 rpm rotating speeds centrifugation 5 ~ 10 min remove supernatant, add in detergent and continue to centrifuge, and ultrasonic disperse centrifuges again, so operates 3 times repeatedly, obtains trace The molecular blotting polymer microsphere of target molecule；
4th step is the preparation of the polymer microballoon of Selective recognition and detection target analytes：It is with acetic acid and methanol volume ratio 2：8 200 mL mixed liquors elute the template point in above-mentioned obtained molecular blotting polymer microsphere in Soxhlet extractor Son until not having fluorescence signal in eluent, has been obtained to target analyte molecule Selective recognition and optical signalling inspection The polymer microballoon of survey.
As a further improvement of existing technologies, in the molecular engram microsphere of the preparation target analyte molecule and Microsphere is the PAD1 with fluorescent characteristic in 2,4,6- trinitrobenzen amphyls；The molecular engram of the preparation is micro- Soda acid refers respectively to perchloric acid and potassium tert-butoxide used in ball；Pillar is crossed used in the molecular engram microsphere of the preparation Eluent ratio and to climb the ratio of eluent of silica gel plate be with 1 respectively according to ethyl acetate and petroleum ether:3 and 1:5 Volume ratio mixes；Detergent used in the molecular engram microsphere of the preparation is with 1 according to deionized water and acetonitrile: 1 volume ratio mixes；Function monomer acrylamide in the molecular engram microsphere of the preparation；The molecule of the preparation Crosslinking agent is ethylene glycol dimethacrylate in trace microballoon；Initiator azo two in the molecular engram microsphere of the preparation Isobutyronitrile；Target molecule 2 in the molecular engram microsphere of the preparation, PAD1 is in soda acid item in 4,6- trinitrobenzen amphyls There is closing characteristic under part.
Relative to the advantageous effect of the prior art：The terrorist violences event such as explosion frequently occurs in recent years, seriously endangers The security of the lives and property and social stability of the people of other countries masses, and threat is constituted to the safety and stablization of country, area. Therefore, the detection and analysis of explosive cause the great attention of research institution of various countries.Southern Yangtze University Zhang Jinfang in 2016 et al. public affairs Open patent of invention（CN201610120560.X）" there is luminescent crystal the material { [Cu (DMSO) of detection picric acid energy5] [Cu4I6(DMSO)]}nPreparation method ", a kind of picric luminescent crystal material of detection of the disclosure of the invention.By cuprous iodide It is added in dimethyl sulfoxide in solution, is stirred to react several minutes, hydrochloric acid is then added dropwise dropwise to pH value of solution=2~4, stirring two is small When, the solution containing luminescent crystal material and filtering is made, precipitating reagent is added in most backward luminescent crystal material solution obtained, By crystallizing, being filtered, washed, drying to obtain luminescent crystal material, only preparation process takes longer.Huang Wei et al. （ACS Appl. Mater. Interfaces 2017,9,3,068 3074）In report and be prepared for four kinds of polymer, carry out The test of nitryl aromatic race analyte simultaneously shows different fluorescent quenching responses.Efficiency is quenched depending on target analytes absorbance Spectra overlapping between emitting with fluorescent polymer obtains optical finger print based on analyte to unique response modes of polymer, Using this Miniature Sensor array, nine kinds of nitryl aromatic race analytes can be accurately distinguished with 100%, but prepared by the method It is of high cost, for picric detection there is still a need for finding better method.Kentaro Shiraishi scientific papers（ACS Appl. Mater. Interfaces 2009,1,1379-1382）In mention and being detected by Fluorescence Quenching Principle with phosphorous oxides Explosive, particularly nitryl aromatic race explosive（Such as TNT）.In the image sensing of the explosive sprayed with phosphorescence oxide, permit Perhaps visual fluorescence quenching determines that nitryl aromatic race explosive is limited in the detection of nanogram level lower limit.Nilanjan Dey exist（ACS Appl. Mater. Interfaces2013,5,8,394 8400）It is middle to detect picric acid, and nitric acid aromatic compound using a kind of test-strips Object（NACs）The selective enumeration method of nanomolar concentration is in a variety of media of micella or organogel and to make including water for the first time It is realized in test-strips.The mechanism to styryl interaction of molecules for describing NACs and high fluorescence is used as from electron rich hair Color probe is to the electronics transfer phenomena of electron deficient NACs.The selectivity of sensing is guided by the pKa and the NACs that is considered of probe. The selective gel that TNP is induced in medium tetrahydrofuran-colloidal sol transformation is observed also by the recombination of molecular self-assembling. Only the method does not mention whether test-strips are insoluble materials in use, if whether can dilute fluorescent material in water And lead to the change of fluorescence intensity so as to influence result.Firm et al. the open patent of invention of Qilu University of Technology's lid profit （CN201510902169.0）" a kind of preparation method for being used to detect picric polymer with nitrogen quantum dot ", contains in the invention The preparation process of nitrogen polymer quantum dot is using more bromo pyrroles as raw material, under microwave radiation technology, alkali heat condition, passes through more bromo pyrroles Debrominate cyclisation, hydroxyl nucleophilic substitution are coughed up, can be reacted at a lower temperature.Minoo Bagheri exist（ACS Appl. Mater. Interfaces2016,8,21,472 21479）In report laser dye rhodamine B be introduced into porous framework Dye sensitization metal organic framework TMU-5S has been synthesized on the basis of TMU-5.TMU-5S has been investigated as detecting explosive nitro The ratio fluorescent sensor of aromatic compound, and show than existing any picric acid based on high four times luminous of sensor Selectivity, and in the presence of other nitro-aromatic compounds and volatile organic compounds, it can selectively be distinguished Bitter taste acid concentration.But this method has used fluorescent dye, not friendly to the environment.Beijing University of Chemical Technology Yan winter roc et al. public affairs Open patent of invention（CN201710219563.3）" a kind of hypersensitive Electrochemiluminescsensor sensor is constructed and its in trinitro- first The application of benzene context of detection " discloses a kind of constructing and its being examined in trinitrotoluene for hypersensitive Electrochemiluminescsensor sensor Application in terms of survey.First by negative electricity small molecule luminol mono-sodium salt be inserted into hydrotalcite layers, then using luminol intercalation after Hydrotalcite forms the electrogenerated chemiluminescence of supermolecule laminated film with negative electricity quantum dot by electrostatic attraction layer-by-layer （Electrogenerated Chemiluminescence, ECL）Sensor, so as to form ECL Resonance energy transfers （Electrogenerated Resonance Energy Transfer, ERET）System, for detecting TNT.But this biography Sensor is more fragile, is easily damaged, and practicability is not high.
Based on the shortcomings that above-mentioned invention detects picric acid, complicated, time-consuming, severe reaction conditions, nothing are such as synthesized Recognition site, selectivity is poor, some have recognition site, but enters the output of recognition site no signal, so as to undetectable Shortcoming；It has no that document report prepares target analytes autofluorescence emission spectrum as Yellow luminous band simultaneously, is printed by preparing Mark polymer microballoon realizes identification highly selective to target analytes and high sense optical signal detection.Therefore, synthesis possesses height The emission band of selectivity and high sensitivity is micro- for the bitter taste derivative molecules imprinted polymer of yellow fluorescence optical signalling Ball, realize has its necessity to the identification of trace bitter taste derivative molecules and detection.
The present invention's is the synthesis of 2,4,6- trinitrobenzen amphyls first：Weigh 0.4~0.6 g treated 2,4,6- Trinitrophenol is placed in 100 mL three-necked flasks, then adds in 7~8 mL dichloromethane ultrasonic disperses, add 0.2 ~ The N of 0.4 g, N '-diisopropylcarbodiimide, solution are immediately turned into rufous, 1 ~ 3 h are stirred under nitrogen atmosphere, due to The volatilization of methylene chloride, solution become thick, and dry 0.5 ~ 1.5 h is obtained in 30 ~ 50 DEG C of vacuum drying chambers Red brown solid powder has glassy yellow fluorescent characteristic, and the methanol solution of the boiling of 20 mL is poured into the rufous of above-mentioned preparation In solid powder, 10 ~ 20 mL distilled water are added, are placed in progress 1 ~ 3 h of water-bath in 0 DEG C of ice water mixed liquor, discovery has red Brown crystal is precipitated, and is filtered to get to be prepared 2,4,6- trinitrobenzen amphyl PAD1；
Secondly 2,4,6- trinitrobenzen amphyls purify：Silicagel column is vertically installed on iron stand, with acetone and oil After ether rinse 3 times, in 250 mL beakers add in 150 ~ 250g silica gel, it is uniform with petroleum ether and stirring, be then filled with to In silicagel column, air pump is opened, is pressurizeed to solution downwards, and pillar is beaten with ear washing bulb, silica gel concussion is enriched, treats silicon gel column Height when reaching the requirement of separation, silicagel column top layer is kept horizontal, after pillar strikes reality, adds petroleum ether, Ran Hou It is pressurizeed downwards using air pump, so operation 3 times, until petroleum ether liquid level and silica gel cylinder flush soon, addition prepares on a small quantity Eluent, flow down naturally, 2,4,6- trinitrobenzen amphyl samples of above-mentioned preparation drawn with rubber head dropper, along post jamb Slow uniform loading, if sample size is excessive, is pressurizeed downwards with air pump, followed by loading, after sample to be separated has all been gone up, uses gas Pump is depressed into be flushed with silica gel cylinder, and after end of the sample, the eluent prepared in advance is slowly added into further along post jamb, finally What is be separated first to 2,4,6- trinitrobenzen amphyls is Chinese red liquid（PAD1）, secondly yellow liquid （PAD2）, 5 ~ 15 uL of liquid of above-mentioned gained is taken to be added dropwise in silica gel plate respectively with microsyringe, after determining separating-purifying, Obtained PAD1 and PAD2 is evaporated under reduced pressure, 7 ~ 9 h is finally dried in vacuum drying oven at 50 ~ 70 DEG C, finally distinguishes Obtain orange-yellow and yellow-brown solid powder；
Followed by the molecular blotting polymer microsphere synthesis of target analytes：20 are weighed respectively with the electronic balance of a ten thousandth ~ 40 mg orange/yellow solid powder PAD1, the acrylamide of 17 ~ 19 mg, the azodiisobutyronitrile of 15 ~ 25 mg are placed in 250 In the conical flask of mL, it is 9 then to measure 50 mL methanol with acetonitrile volume ratio with the graduated cylinder of 100 mL:1 mixed liquor is added to It states in conical flask, ultrasonic disperse 5 minutes or so, finally measures the ethylene glycol dimethyl of 190 ~ 210 μ L with microsyringe again Acrylate is added drop-wise in above-mentioned solution, after leading to 20~30 min nitrogen, is uniformly coated with vacuum grease in grinding port plug, is covered taper Bottle, then wrapped up with preservative film, then conical flask is positioned in constant temperature oscillator, at 50 ~ 60 DEG C, oscillating reactions 2 ~ 4 H, then temperature is adjusted to 8 ~ 10 h of oscillating reactions at 60 ~ 70 DEG C, at last 70 ~ 85 DEG C, 1 ~ 3 h of oscillating reactions takes out It is cooled to room temperature, reaction mixture is transferred in 15 mL centrifuge tubes, 5 ~ 10 are centrifuged with 5000 ~ 7000 rpm rotating speeds Min removes supernatant, adds in detergent and continues to centrifuge, and ultrasonic disperse centrifuges again, so operates 3 times repeatedly, obtains trace mesh Mark the molecular blotting polymer microsphere of molecule；
It is finally the preparation of the polymer microballoon of Selective recognition and detection target analytes：It is 2 with acetic acid and methanol volume ratio： 8 200 mL mixed liquors elute the template point in above-mentioned obtained molecular blotting polymer microsphere in Soxhlet extractor Son until not having fluorescence signal in eluent, has been obtained to target analyte molecule Selective recognition and optical signalling inspection The polymer microballoon PAD1 of survey.
In conclusion the molecular blotting polymer microsphere of gained can be used to detect 2,4,6- trinitrobenzen amphyls.
One：2,4,6- trinitrophenols itself do not have fluorescent characteristic, can not be exported into recognition site with optical signalling, Also it can not just detect, however 2,4,6- obtained after the reaction of 2,4,6- trinitrophenols and N, N '-diisopropylcarbodiimide Trinitrobenzen amphyl PAD1 has the fluorescence of yellow spectral band, and there is the fluorescence under acid or alkali environment to open and close characteristic, Therefore, into the 2 of print identification site, 4,6- trinitrobenzen amphyl PAD1, by the output of autofluorescence optical signalling, It realizes to its trace detection.
Secondly：Compared with traditional fluorescent dye or quantum dot modification to recognition site, microsphere enters identification Site does not need to interact with fluorescent dye and quantum dot of the modification in recognition site, and Fluorescence Resonance Energy energy is recycled to turn Telephone-moving reason or photoelectron induction transfer mechanism are detected target analytes.
Thirdly：Compared with traditional imprinted material, polymer microballoon has larger specific surface area, possesses effective identification Site is more, and it is small to be located at skin resistance for recognition site in microballoon, there is fast binding kinetics, larger saturation binding capacity.
It four：In method provided by the present invention, imprinted polymer microspherulite diameter is controllable, can pass through regulatory function list The amount of body, crosslinking agent and solvent is controlled.
It five：2,4,6- obtained after selection 2,4,6- trinitrophenols and the reaction of N, N '-diisopropylcarbodiimide Purposes of the trinitrobenzen amphyl PAD1 as template molecule, because it has the following advantages：（1）Recognition site do not need into Row fluorescent marker;（2）Recognition site has target molecule substantially just to have optical signalling output；（3）Structure fluorescence resonance is not needed to Energy transfer mechanism；（4）It has been discriminated whether by the presence or absence of fluorescence there are target analytes, by the power of fluorescence intensity, Realize the detection quantitative to target analytes.
Description of the drawings
Fig. 1 is the bitter taste acid derivative imprinted polymer microballoon signal of the autofluorescence function of the synthesis prepared by the present invention Figure.
Fig. 2 is the N prepared by the present invention, and the sub- amide of N- diisopropyls carbon two reacts generation bitter taste acid derivative with picric acid PAD1 and PAD2 are schematically illustrate.
Fig. 3 is the bitter taste acid derivative PAD1 nuclear magnetic resonance spectroscopies prepared by the present invention.
Fig. 4 is the picric acid prepared by the present invention（a）With bitter taste acid derivative PAD1（b）Infared spectrum.
Fig. 5 is the picric acid prepared by the present invention（a）And PAD1（b）Solution normalizes uv-vis spectra and PAD1 （c）Solution normalizes fluorescence emission spectrum.
Fig. 6 is bitter taste acid derivative PAD1 prepared by the present invention respectively in tertiary butyl potassium alcoholate and perchloric acid solution molecule knot The transformation of structure.
Fig. 7 is that the diameter of the bitter taste acid derivative PAD1 traces prepared by the present invention is about 2um polymer microballoons SEM。
Fig. 8 is the comparison of the molecular recognition performance of polymer prepared by the present invention.
Fig. 9 is the bitter taste acid derivative PAD1 molecular dynamics curves prepared by the present invention.
Figure 10 be prepared by the present invention in perchloric acid solution with target analytes PAD1 concentration increase fluorescence intensity Increase evolution process.
Specific embodiment is explained further with reference to the accompanying drawings
Fig. 1 is the bitter taste acid derivative imprinted polymer microballoon schematic diagram of the autofluorescence function of the synthesis prepared by the present invention. In Fig. 1：1-2：Bitter taste the acid derivative PAD1 and PAD2 of the sub- amide reaction generation of picric acid and N, N- diisopropyl carbon two first, Using PAD1 as template molecule after separating-purifying；2-3：Bitter taste acid derivative template molecule PAD1 and function monomer acrylamide, Crosslinking agent ethylene glycol dimethacrylate and initiator azo-bis-isobutyl cyanide copolymerization prepare trace bitter taste acid derivative The polymer microballoon of PAD1；3-4：Derive from the recognition site removal of imprinted polymer microballoon and in conjunction with template molecule picric acid Object PAD1 is realized to bitter taste acid derivative PAD1 Selective recognitions and sensitivity Detection.
Fig. 2 is the N prepared by the present invention, and the sub- amide of N- diisopropyls carbon two reacts generation bitter taste acid derivative with picric acid PAD1 and PAD2 are schematically illustrate.With N, N '-diisopropylcarbodiimide is as nucleopilic reagent, 2,4,6- trinitrophenol conducts Substitution occurs on aromatic ring under room temperature under nitrogen for substrate and phenol derivatives PAD1 and PAD2 is made in addition reaction.
Fig. 3 is the bitter taste acid derivative PAD1 nuclear magnetic resonance spectroscopies 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), it is further determined as PAD1.
Fig. 4 is the picric acid prepared by the present invention（a）With bitter taste acid derivative PAD1（b）Infared spectrum.It can by a in figure Know in 3500~3400 cm-1With 3000~2900 cm-1There are absorption peak correspond to phenyl ring in c h bond stretching vibration with And in 2000~1500 cm-1The absorption peak at place should exactly demonstrate,prove the benzene radicals in picric acid.In 1630~1480 cm-1Place There is NO2Stretching vibration peak, and in 1630~1480 cm-1There is the bending vibrations of OH.B in figure it is found that 3200~ 3000 cm-1Between and 1000~600 cm-1Existing absorption peak, corresponding to-NH2Stretching vibration, in 1700~1500 cm-1 Between there are C=O stretching vibration peak, in 1700~1500 cm-1Between there is C=N keys stretching vibration.
Fig. 5 is the picric acid prepared by the present invention（a）And PAD1（b）Solution normalizes uv-vis spectra and PAD1 （c）Solution normalizes fluorescence emission spectrum.Colour plate（a）With（b）Picric acid and bitter taste acid derivative PAD1 respectively from The lower photo for taking solution of right light, and（c）It is then the solution thereon of the PAD1 under 365 nm length ultraviolet lamps.It is bitter under natural light Sour and bitter taste acid derivative PAD1 solution is light green respectively and orange red, and the PAD1 under 365 nm length ultraviolet lamps is molten Liquid is the fluorescence spectral bands for sending out yellow, and maximum emission wavelength is at 567 nm.
Fig. 6 is PAD1 prepared by the present invention respectively in the transformation of tertiary butyl potassium alcoholate and perchloric acid solution molecular structure.It is color Color illustration is that the tertiary butyl potassium alcoholate prepared is added dropwise into solution, it is found that fluorescence is gradually dimmed in solution, adds to glimmering in solution Light disappears；Then perchloric acid solution is added in solution again, it is found that solution colour gradually restores original yellow fluorescence.
Fig. 7 is the SEM that the diameter of the PAD1 traces prepared by the present invention is about 2 um polymer microballoons.As seen from the figure, The polymer glomeration, granular size is consistent and is evenly distributed, and surface is smooth, about 2 um of microsphere diameter.
Fig. 8 is the comparison figure of the molecular recognition performance prepared by the present invention.About 2 um of grain size of the PAD1 traces of 20 mg Polymer microballoon respectively to PAD1 () and PAD2 (zero) in conjunction with amount；About 4 um's of grain size of the PAD1 traces of 20 mg Polymer beads are to PAD1 (Δ) in conjunction with amount；The polymer microballoon pair of about 2 um of grain size of the non-traces of PAD1 of 20 mg PAD1 (▽) is in conjunction with amount；The efficient selective of PAD1 is identified it can be seen that this trace microballoon has, compared to PAD2 high 4 Times.4 um traces particles are about 50 nmol to PAD1 binding capacities, and 2um traces microballoon is about 230 to PAD1 binding capacities Nmol is 4.6 times of 4 um trace particles.Under same particle size, this molecular blotting polymer microsphere has compared with non-trace microballoon There is higher recognition performance, be 6 times of non-trace microballoon.
Fig. 9 is the bitter taste acid derivative PAD1 molecular dynamics curves prepared by the present invention.(a) polymerization of PAD1 traces The binding capacity () that object microballoon develops at any time；(b) conventional print polymer beads at any time and develop binding capacity (zero)； It is 1.39 nmolmin that the polymer microballoon of PAD1 traces, which reaches the kinetic rate before balancing, as seen from the figure-1, PAD1 routines It is 0.18 nmolmin that trace polymerization composition granule, which reaches the kinetic rate before balancing,-1, polymer microballoon kinetic rate is 7.72 times of the kinetic rate of conventional print polymer beads.The polymer microballoon material of trace has faster dynamics, Effect is more preferable.Above-mentioned binding kinetics data are 20mg imprinted polymers 5.0 × 10-5 mol•L-1The acquisition of PAD1 solution 's.
Figure 10 be prepared by the present invention in perchloric acid solution with target analytes PAD1 concentration increase fluorescence intensity Increase evolution process.Various concentration bitter taste acid derivative PAD1 enters in imprinted polymer Selective recognition site, is measured The evolution process of fluorescence intensity, concentration are 1 × 10 successively from the bottom up-9mol•L-1, 1 × 10-8mol•L-1, 1 × 10-7mol•L-1, 1 × 10-6mol•L-1With 1 × 10-5mol•L-5, with the increase of PAD1 concentration, the intensity of fluorescence is also stronger, passes through fluorescence The change of intensity is realized to PAD1 trace detections.
Specific embodiment
The preparation method for the molecular blotting polymer microsphere that one kind detects for 2,4,6- trinitrophenols, feature exist In：More nitro amphyls of 2,4,6- trinitrophenols and N, N '-diisopropylcarbodiimide reaction generation have bright orange Color fluorescence, and its fluorescence has closing characteristic under acid-base condition, elutes and is located in the molecular blotting polymer microsphere Microsphere in imprinted layer, the interior formation of imprinted layer have the function of to tie with the hole of microsphere structure, size and base complementation Structure elutes the polymer microballoon of microsphere with the specific recognition site to target analyte molecule, realizes to target point Object molecule Selective recognition and detection are analysed, above-mentioned preparation process includes following four steps：
The first step is the synthesis of 2,4,6- trinitrobenzen amphyls：Weigh 0.4~0.6 g treated 2,4,6- trinitrophenols It is placed in 100 mL three-necked flasks, then adds in 7~8 mL dichloromethane ultrasonic disperses, add the N of 0.2 ~ 0.4 g, N '- Diisopropylcarbodiimide, solution are immediately turned into rufous, 1 ~ 3 h are stirred under nitrogen atmosphere, due to methylene chloride Volatilization, solution becomes thick, and dry 0.5 ~ 1.5 h obtains red brown solid powder in 30 ~ 50 DEG C of vacuum drying chambers End has glassy yellow fluorescent characteristic, the methanol solution of the boiling of 20 mL is poured into the red brown solid powder of above-mentioned preparation, 10 ~ 20 mL distilled water are added, are placed in progress 1 ~ 3 h of water-bath in 0 DEG C of ice water mixed liquor, discovery has red-brown crystals analysis Go out, filtered to get to be prepared 2,4,6- trinitrobenzen amphyls；
Second step is the purification of 2,4,6- trinitrobenzen amphyls：Silicagel column is vertically installed on iron stand, with acetone and stone After oily ether rinse 3 times, 150 ~ 250g silica gel is added in 250 mL beakers, it is uniform with petroleum ether and stirring, then it is filled with To silicagel column, air pump is opened, is pressurizeed to solution downwards, and pillar is beaten with ear washing bulb, silica gel concussion is enriched, treats silicagel column When the height of son reaches the requirement of separation, silicagel column top layer is kept horizontal, after pillar strikes reality, adds petroleum ether, then It is being pressurizeed downwards using air pump, so operation 3 times, until petroleum ether liquid level and silica gel cylinder flush soon, is adding in a small amount of prepare Good eluent flows down naturally, 2,4,6- trinitrobenzen amphyl samples of above-mentioned preparation is drawn with rubber head dropper, along column The slow uniform loading of wall, if sample size is excessive, is pressurizeed downwards with air pump, followed by loading, after sample to be separated all go up, and use Air pump is depressed into be flushed with silica gel cylinder, after end of the sample, the eluent prepared in advance is slowly added into further along post jamb, finally Obtain that 2,4,6- trinitrobenzen amphyls are separated first is Chinese red liquid（PAD1）, secondly yellow liquid （PAD2）, 5 ~ 15 uL of liquid of above-mentioned gained is taken to be added dropwise in silica gel plate respectively with microsyringe, after determining separating-purifying, Obtained PAD1 and PAD2 is evaporated under reduced pressure, 7 ~ 9 h is finally dried in vacuum drying oven at 50 ~ 70 DEG C, finally distinguishes Obtain orange-yellow and yellow-brown solid powder；
Third step is prepared by the molecular blotting polymer microsphere of target analytes：20 are weighed respectively with the electronic balance of a ten thousandth ~ 40 mg orange/yellow solid powder PAD2, the acrylamide of 17 ~ 19 mg, the azodiisobutyronitrile of 15 ~ 25 mg are placed in In the conical flask of 250 mL, it is 9 then to measure 50 mL methanol with acetonitrile volume ratio with the graduated cylinder of 100 mL:1 mixed liquor adds in Into above-mentioned conical flask, ultrasonic disperse 5 minutes or so, finally again with the ethylene glycol two of 190 ~ 210 μ L of microsyringe measurement Methacrylate is added drop-wise in above-mentioned solution, after leading to 20~30 min nitrogen, is uniformly coated with vacuum grease in grinding port plug, is covered cone Shape bottle, then wrapped up with preservative film, then conical flask is positioned in constant temperature oscillator, at 50 ~ 60 DEG C, oscillating reactions 2 ~ 4 h, then temperature is adjusted to 8 ~ 10 h of oscillating reactions at 60 ~ 70 DEG C, at last 70 ~ 85 DEG C, 1 ~ 3 h of oscillating reactions takes Go out to be cooled to room temperature, reaction mixture be transferred in 15 mL centrifuge tubes, with 5000 ~ 7000 rpm rotating speeds centrifugation 5 ~ 10 min remove supernatant, add in detergent and continue to centrifuge, and ultrasonic disperse centrifuges again, so operates 3 times repeatedly, obtains trace The molecular blotting polymer microsphere of target molecule；
4th step is the preparation of the polymer microballoon of Selective recognition and detection target analytes：It is with acetic acid and methanol volume ratio 2：8 200 mL mixed liquors elute the template point in above-mentioned obtained molecular blotting polymer microsphere in Soxhlet extractor Son until not having fluorescence signal in eluent, has been obtained to target analyte molecule Selective recognition and optical signalling inspection The polymer microballoon of survey.
When target analyte molecule encounters this polymer microballoon, by concentration difference motive force, recognition site is again introduced into, This recognition site has target analytes highly selective identification, while into the target of recognition site point in acidic environment It analyses object to export with high sensitive fluorescence signal, realize to target analytes trace detection.
Specific embodiment
More nitros that 2,4,6- trinitrophenols and N, N '-diisopropylcarbodiimide are generated by nucleophilic addition Amphyl obtains 2,4,6- trinitrobenzen amphyl PAD1 after separating-purifying and is total to function monomer, crosslinking agent and initiator It is poly- that molecular blotting polymer microsphere is prepared, obtain that there is Selective recognition and fluorescence to PAD1 after eluted template molecule PAD1 The polymer microballoon of signal output.
The first step is the synthesis of 2,4,6- trinitrobenzen amphyls：Weigh 0.5 g treated 2,4,6- trinitrophenols It is placed in 100 mL three-necked flasks, then adds in 7.5 mL dichloromethane ultrasonic disperses, add the N, N '-diisopropyl of 0.3 g Base carbon imidodicarbonic diamide, solution is immediately turned into rufous, 2 h is stirred under nitrogen atmosphere, due to the volatilization of methylene chloride, solution Become thick, dry 1 h obtains red brown solid powder in 40 DEG C of vacuum drying chambers, has glassy yellow fluorescent characteristic, will The methanol solution of the boiling of 20 mL is poured into the red brown solid powder of above-mentioned preparation, is added 15 mL distilled water, is placed in 0 DEG C 2 h of water-bath is carried out in ice water mixed liquor, discovery has red-brown crystals precipitation, is filtered to get to be prepared 2,4,6- Trinitrobenzen amphyl；
Second step is the purification of 2,4,6- trinitrobenzen amphyls：Silicagel column is vertically installed on iron stand, with acetone and stone After oily ether rinse 3 times, 200 g silica gel are added in 250 mL beakers, it is uniform with petroleum ether and stirring, it is then filled with to silica gel In column, air pump is opened, is pressurizeed to solution downwards, and pillar is beaten with ear washing bulb, silica gel concussion is enriched, treats the height of silicon gel column When degree reaches the requirement of separation, silicagel column top layer is kept horizontal, after pillar strikes reality, petroleum ether is added, is then utilizing Air pump pressurizes downwards, so operation 3 times, until petroleum ether liquid level and silica gel cylinder flush soon, adds in a small amount of prepared leaching Lotion flows down naturally, and 2,4,6- trinitrobenzen amphyl samples of above-mentioned preparation are drawn with rubber head dropper, slow along post jamb Uniform loading if sample size is excessive, is pressurizeed downwards with air pump,, will with air pump after sample to be separated has all been gone up followed by loading It is depressed into flushes with silica gel cylinder, after end of the sample, the eluent prepared in advance is slowly added into further along post jamb, finally obtains 2, What 4,6- trinitrobenzen amphyls were separated first is Chinese red liquid（PAD1）, secondly yellow liquid（PAD2）, 10 uL of liquid of above-mentioned gained is taken to be added dropwise in silica gel plate respectively with microsyringe, it, will be obtained after determining separating-purifying PAD1 and PAD2 vacuum distillations, finally dry 8 h at 60 DEG C in vacuum drying oven, finally respectively obtain orange-yellow and brown color and consolidate Body powder；
Third step is the molecular blotting polymer microsphere synthesis of target analytes：30 are weighed respectively with the electronic balance of a ten thousandth Mg orange/yellow solid powder PAD1, the acrylamide of 18 mg, the azodiisobutyronitrile of 20 mg are placed in the conical flask of 250 mL In, it is 9 then to measure 50 mL methanol with acetonitrile volume ratio with the graduated cylinder of 100 mL:1 mixed liquor is added to above-mentioned conical flask In, ultrasonic disperse 5 minutes or so, the ethylene glycol dimethacrylate for finally measuring 200 μ L with microsyringe again is added drop-wise to In above-mentioned solution, after leading to 25 min nitrogen, vacuum grease is uniformly coated in grinding port plug, covers conical flask, then wrapped up with preservative film, so Conical flask is positioned in constant temperature oscillator afterwards, at 55 DEG C, 3 h of oscillating reactions, then temperature is adjusted to vibrate instead at 65 DEG C Answer 9 h, at last 80 DEG C, 2 h of oscillating reactions, taking-up is cooled to room temperature, and reaction mixture is transferred to 15 mL centrifuge tubes In, 10 min are centrifuged with 6000 rpm rotating speeds, remove supernatant, detergent is added in and continues to centrifuge, ultrasonic disperse centrifuges again, such as This is operated 3 times repeatedly, obtains the molecular blotting polymer microsphere of trace target molecule；
4th step is the preparation of the polymer microballoon of Selective recognition and detection target analytes：It is with acetic acid and methanol volume ratio 2:8 200 mL mixed liquors elute the template point in above-mentioned obtained molecular blotting polymer microsphere in Soxhlet extractor Son until not having fluorescence signal in eluent, has been obtained to target analyte molecule Selective recognition and optical signalling inspection The polymer microballoon of survey.
When target analyte molecule encounters this polymer microballoon with recognition site again, pushed by concentration difference Power, is again introduced into Selective recognition site, and this recognition site has highly selective identification, while in acidity to target analytes It is exported in environment into the target analytes of recognition site with high sensitive fluorescence signal, realizes and spread out to target analytes picric acid Biological PAD1 trace detections.

Claims (9)

1. one kind is for the preparation method of the molecular blotting polymer microsphere of 2,4,6- trinitrophenols detection, it is characterised in that： More nitro amphyls of 2,4,6- trinitrophenols and N, N '-diisopropylcarbodiimide reaction generation are glimmering with glassy yellow Light, and its fluorescence has closing characteristic under acid-base condition, is eluted in the molecular blotting polymer microsphere and is located at trace Microsphere in layer, the interior formation of imprinted layer have the function of the void structure with microsphere structure, size and base complementation, wash The polymer microballoon of de- microsphere has the specific recognition site to target analyte molecule, realizes to target analytes point Sub- Selective recognition and detection, the preparation process of above-mentioned molecular blotting polymer microsphere include following four steps：
1.1 first steps are the synthesis of 2,4,6- trinitrobenzen amphyls：Weigh 0.4~0.6 g treated 2,4,6- trinitro-s Phenol is placed in 100 mL three-necked flasks, is then added in 7~8 mL dichloromethane ultrasonic disperses, is added 0.2 ~ 0.4 g's N, N '-diisopropylcarbodiimide, solution are immediately turned into rufous, 1 ~ 3 h are stirred under nitrogen atmosphere, due to solvent dichloro The volatilization of methane, solution become thick, and dry 0.5 ~ 1.5 h obtains rufous and consolidates in 30 ~ 50 DEG C of vacuum drying chambers Body powder has glassy yellow fluorescent characteristic, and the methanol solution of the boiling of 20 mL is poured into the red brown solid powder of above-mentioned preparation In, 10 ~ 20 mL distilled water are added, are placed in progress 1 ~ 3 h of water-bath in 0 DEG C of ice water mixed liquor, discovery there are red-brown crystals It is precipitated, is filtered to get to be prepared 2,4,6- trinitrobenzen amphyls；
1.2 second steps are the purifications of 2,4,6- trinitrobenzen amphyls：Silicagel column is vertically installed on iron stand, with acetone and After petroleum ether rinse 3 times, 150 ~ 250g silica gel is added in 250 mL beakers, it is uniform with petroleum ether and stirring, then filled out It is charged in silicagel column, opens air pump, pressurize to solution downwards, and pillar is beaten with ear washing bulb, enrich silica gel concussion, treat silica gel When the height of pillar reaches the requirement of separation, silicagel column top layer is kept horizontal, after pillar strikes reality, adds petroleum ether, so It is being pressurizeed downwards using air pump afterwards, so operation 3 times, until petroleum ether liquid level and silica gel cylinder flush soon, addition is matched on a small quantity The eluent made flows down naturally, and 2,4,6- trinitrobenzen amphyl samples of above-mentioned preparation are drawn with rubber head dropper, along The slow uniform loading of post jamb, if sample size is excessive, is pressurizeed downwards with air pump, followed by loading, after sample to be separated has all been gone up, It is depressed into air pump and is flushed with silica gel cylinder, after end of the sample, the eluent prepared in advance is slowly added into further along post jamb, most After obtain that 2,4,6- trinitrobenzen amphyls are separated first is Chinese red liquid（PAD1）, secondly yellow liquid （PAD2）, 5 ~ 15 uL of liquid of above-mentioned gained is taken to be added dropwise in silica gel plate respectively with microsyringe, after determining separating-purifying, Obtained PAD1 and PAD2 is evaporated under reduced pressure, 7 ~ 9 h is finally dried in vacuum drying oven at 50 ~ 70 DEG C, finally distinguishes Obtain orange-yellow and yellow-brown solid powder；
1.3 thirds step is the molecular blotting polymer microsphere synthesis of target analytes：Claimed respectively with the electronic balance of a ten thousandth Take 20 ~ 40 mg orange/yellow solid powder PAD1, the acrylamide of 17 ~ 19 mg, the azodiisobutyronitrile of 15 ~ 25 mg, It is placed in the conical flask of 250 mL, it is 9 then to measure 50 mL methanol with acetonitrile volume ratio with the graduated cylinder of 100 mL:1 mixed liquor It is added in above-mentioned conical flask, ultrasonic disperse 5 minutes or so, finally measures the second two of 190 ~ 210 μ L with microsyringe again Alcohol dimethylacrylate is added drop-wise in above-mentioned solution, and after leading to 20~30 min nitrogen, vacuum grease is uniformly coated in grinding port plug, is covered Upper conical bottle, then wrapped up with preservative film, then conical flask is positioned in constant temperature oscillator, at 50 ~ 60 DEG C, oscillation is anti- It answers 2 ~ 4 h, then temperature is adjusted to 8 ~ 10 h of oscillating reactions at 60 ~ 70 DEG C, at last 70 ~ 85 DEG C, oscillating reactions 1 ~ 3 h, taking-up are cooled to room temperature, and reaction mixture is transferred in 15 mL centrifuge tubes, with 5000 ~ 7000 rpm rotating speeds from 5 ~ 10 min of the heart removes supernatant, adds in detergent and continues to centrifuge, and ultrasonic disperse centrifuges again, so operates 3 times, obtains repeatedly To the molecular blotting polymer microsphere of trace target molecule；
1.4 the 4th steps are the preparations of the polymer microballoon of Selective recognition and detection target analytes：With acetic acid and methanol volume Than being 2:8 200 mL mixed liquors elute the mould in above-mentioned obtained molecular blotting polymer microsphere in Soxhlet extractor Plate molecule until not having fluorescence signal in eluent, has been obtained to target analyte molecule Selective recognition and optics letter Number detection polymer microballoon.
2. a kind of system of molecular blotting polymer microsphere for 2,4,6- trinitrophenols of detection according to claim 1 Preparation Method, it is characterized in that：Target analyte molecule and microsphere are 2,4,6- trinitro-s in prepared molecular engram microsphere The PAD1 with fluorescent characteristic in phenol derivatives.
3. a kind of system of molecular blotting polymer microsphere for 2,4,6- trinitrophenols of detection according to claim 1 Preparation Method, it is characterized in that：Soda acid used in prepared molecular engram microsphere refers respectively to perchloric acid and potassium tert-butoxide.
4. a kind of system of molecular blotting polymer microsphere for 2,4,6- trinitrophenols of detection according to claim 1 Preparation Method, it is characterized in that：The ratio of the eluent of pillar is crossed used in prepared molecular engram microsphere and climbs silica gel plate The ratio of eluent is with 1 respectively according to ethyl acetate and petroleum ether:3 and 1:5 volume ratio mixes.
5. a kind of system of molecular blotting polymer microsphere for 2,4,6- trinitrophenols of detection according to claim 1 Preparation Method, it is characterized in that：Detergent used in prepared molecular engram microsphere is with 1 according to deionized water and acetonitrile:1 Volume ratio mixes.
6. a kind of system of molecular blotting polymer microsphere for 2,4,6- trinitrophenols of detection according to claim 1 Preparation Method, it is characterized in that：Function monomer acrylamide in prepared molecular engram microsphere.
7. a kind of system of molecular blotting polymer microsphere for 2,4,6- trinitrophenols of detection according to claim 1 Preparation Method, it is characterized in that：Crosslinking agent is ethylene glycol dimethacrylate in prepared molecular engram microsphere.
8. a kind of system of molecular blotting polymer microsphere for 2,4,6- trinitrophenols of detection according to claim 1 Preparation Method, it is characterized in that：Initiator azodiisobutyronitrile in prepared molecular engram microsphere.
9. a kind of system of molecular blotting polymer microsphere for 2,4,6- trinitrophenols of detection according to claim 1 Preparation Method, it is characterized in that：Target molecule 2 in prepared molecular engram microsphere, PAD1 exists in 4,6- trinitrobenzen amphyls There is closing characteristic under acid-base condition.
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