CN109467659B - Molecularly imprinted microspheres and application method thereof - Google Patents

Molecularly imprinted microspheres and application method thereof Download PDF

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CN109467659B
CN109467659B CN201811367979.0A CN201811367979A CN109467659B CN 109467659 B CN109467659 B CN 109467659B CN 201811367979 A CN201811367979 A CN 201811367979A CN 109467659 B CN109467659 B CN 109467659B
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谢练武
吴茂
邓慧芸
范亚军
郭亚平
卢丹青
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Central South University of Forestry and Technology
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Abstract

The invention relates to a molecularly imprinted microsphere and an application method thereof. The invention takes organophosphorus pesticide as template molecules, takes ethylene phosphate and 1-octadecene as difunctional monomers to prepare magnetic molecularly imprinted microspheres, and realizes the rapid qualitative and quantitative analysis of organophosphorus through the color development of sodium molybdate. According to the method, sodium molybdate reacts with the phosphate skeleton of the ethylene phosphate on the surface of the microsphere to generate phosphorus-molybdenum blue, organic phosphorus is not required to be digested into inorganic phosphorus, and the visual color development qualitative analysis and the spectrophotometry quantitative detection of the organic phosphorus pesticide residue can be realized by observing the color change (light color) or absorbance before and after the microsphere specifically adsorbs the organic phosphorus pesticide residue. The invention can realize the color-sensitive identification and on-site rapid screening of the molecular imprinting microspheres on organic phosphorus. The method has the advantages of large microsphere adsorption capacity, high sensitivity, high accuracy, no need of digestion of organic phosphorus, high detection speed (less than or equal to 10min) and low detection limit (1.0 multiplied by 10)‑4mmol/L) and the like.

Description

Molecularly imprinted microspheres and application method thereof
Technical Field
The invention belongs to the technical field of organophosphorus pesticide residue detection, and relates to a molecularly imprinted microsphere and a detection method thereof applied to organophosphorus pesticide residue; in particular to an organophosphorus pesticide residue magnetic molecularly imprinted microsphere and application thereof in rapid chromogenic analysis and detection of organophosphorus pesticide residue.
Background
In agricultural production, organophosphorus pesticides are widely used for killing pests and improving yield, and the usage amount of the organophosphorus pesticides accounts for more than 70% of all pesticides, so that non-point source environmental pollution is caused, and the food safety is seriously affected. In addition to acute toxicity caused by exposure to high concentrations, most organophosphorus pesticide residues exhibit high toxicity after absorption by humans, due to the excessive accumulation of acetylcholine in the body as a result of organophosphorus inhibitors of acetylcholinesterase. In order to comprehensively guarantee food safety, an effective method for rapidly detecting organophosphorus pesticide residues is urgently needed to be developed. In recent years, many methods for detecting and analyzing organophosphorus pesticide residues have been used, including: spectroscopy, electrochemical analysis methods, gas chromatography, ultraviolet-visible spectrophotometry and voltammetry, gas chromatography-mass spectrometry, and high performance liquid chromatography-mass spectrometry, among others, with detection limits as low as nmol/L levels. Most of the analysis techniques rely on expensive precise instruments, have the defects of consuming time and organic solvents, and are very complicated in general pretreatment operation and inconvenient for real-time on-site rapid detection.
In the organophosphorus pesticide residue detection, in order to improve the selectivity, the pretreatment of a sample needs to be continuously enhanced. Currently, the more pretreatment technologies are as follows: dispersion liquid-liquid micro-extraction, solid-phase extraction, supercritical fluid extraction, solid-phase micro-extraction, matrix solid-phase dispersion extraction, microwave-assisted extraction, supercritical fluid extraction, stirring rod adsorption extraction and the like. Among them, the solid phase extraction is most widely used and has the highest degree of commercialization because of its simple operation, stable performance and automatic operation. C is often used in solid phase extraction18Or C8Bonded silica gel is a solid phase adsorbent, but the interaction between the adsorbent and the target analyte is non-specific, resulting in less than ideal purification effect, difficulty in completely eliminating matrix interference, and thus, insufficient selectivity. In contrast, the molecules were preparedThe imprinted polymer is used as an adsorbent, and the strong affinity and high specificity of the imprinted polymer are utilized to perform molecular imprinting solid phase extraction, so that the target substance can be separated and enriched with high selectivity, and the imprinted polymer becomes one of the popular sample pretreatment methods in analytical detection. The molecular imprinting technology has the advantages of selective recognition, strong practicability, reusability, long service life and the like.
In recent years, the combination of bifunctional monomers and molecular imprinting technology has led to the rapid development of molecular imprinting technology. The gallic acid imprinted polymer prepared by the inventor by adopting a single-functional monomer has good selective recognition performance (Duhui Yun, Wang bin, Wu Mao, Wenzhi, Maqiang, Guo Yao Ping, Dun, Shi Wu & Shi & lt & gt. Although there are patents (such as Yangxin, Huang Wei, Wang Jing. A method for preparing high-efficiency adsorbed bifunctional monomer polysaccharide molecularly imprinted nanoparticles [ P ]. national intellectual Property office, patent No. 201510955170X, publication No. CN 105542083B,2017.11.03.) the molecularly imprinted material is prepared by using bifunctional monomers, but the bifunctional monomers are m-aminobenzoic acid and 2-propyl amido-2-methyl propanesulfonic acid, and the reversible covalent interaction and hydrogen bonding interaction are generated between the bifunctional monomers and starch, the invention uses the principle that the functional monomers such as ethylene phosphate and the like and organic phosphorus molecules form hydrogen bonds and similar polar attraction, and the nonpolar carbon chain of the auxiliary functional monomer 1-octadecene and the like can enhance the specific adsorption of the molecularly imprinted material on the organic phosphorus through hydrophobic interaction with the weak polar or nonpolar functional group in the organic phosphorus molecules, the imprinting cavity is more stable and the selectivity is stronger.
In addition, in the conventional phosphomolybdic blue spectrophotometry for measuring phosphorus, a sample is firstly digested into phosphate radicals, the phosphate radicals and sodium molybdate quantitatively generate phosphomolybdic blue under a reducing condition, and then the absorbance is measured at the maximum absorption wavelength of the phosphomolybdic blue. In contrast, the method utilizes the reaction of sodium molybdate and a phosphoric acid skeleton (derived from functional monomer ethylene phosphate and the like) on the surface of the microsphere to generate the phosphomolybdic blue, eliminates the organic phosphorus pesticide residue, and realizes the visual color development qualitative and spectrophotometric quantitative detection of the organic phosphorus pesticide residue by observing the color change or absorbance before and after the microsphere absorbs the organic phosphorus molecules. The invention avoids long-time digestion steps, and simultaneously introduces the magnetic rapid separation function of the molecularly imprinted microspheres, thereby greatly shortening the analysis time, and if the microspheres are prepared in advance, the analysis time of a single sample is not more than 10 min. Therefore, the organic phosphorus magnetic molecularly imprinted microsphere of the bifunctional monomer is developed, the specific recognition and high-power enrichment of organic phosphorus molecules are realized, and the phosphoric acid skeleton formed on the surface of the microsphere by the functional monomer and sodium molybdate are utilized to generate phosphorus-molybdenum blue, so that a novel method for rapidly screening and developing color analysis of organic phosphorus pesticide residues is obtained, all organic phosphorus pesticides can be detected, and the application prospect is wide.
Disclosure of Invention
The invention aims to provide a method for realizing rapid detection and analysis of organic phosphorus by combining a molecular imprinting technology with a visual rapid color development analysis method and a corresponding molecular imprinting microsphere product. The problems of complicated analysis steps and low detection speed of organophosphorus pesticide residues in the current fields of environment, food and Chinese medicine can be solved; can be used for qualitative analysis and quantitative detection.
A molecularly imprinted microsphere is prepared by loading imprinted polymer with eluted template molecules on a carrier microsphere, adding pre-assembly liquid formed by organophosphorus template molecules and functional monomers into the carrier microsphere, a cross-linking agent, an initiator and a pore-forming agent, polymerizing and eluting the template molecules; the functional monomer meets the following conditions:
(1) the functional monomer can be combined with the organophosphorus template molecule by utilizing the principle that the functional monomer and the organophosphorus template molecule form hydrogen bonds and the molecules have similar polarities and attract each other;
(2) the functional monomer can react with Na2MoO4Solution or (NH)4)2MoO4The solution is subjected to color reaction under the action of a reducing agent.
Further, the structural formula of the functional monomer is one or more than one of the following formulas 1:
Figure BDA0001869096660000031
the functional monomer comprises: vinyl phosphate, propylene phosphate, butylene phosphate, and pentene phosphate.
Further preferably, the pre-assembly liquid further comprises an auxiliary functional monomer: is a monomer molecule which can be combined with a weak polar or nonpolar functional group in an organophosphorus template molecule through a hydrophobic acting force.
Further preferably, the auxiliary functional monomers include: 1-dodecene, 1-tridecene, 1-tetradecene, 1-pentadecene, 1-hexadecene, 1-heptadecene, 1-octadecene, 1-nonadecene, and 1-eicosene.
Further, the crosslinking agent includes: one or more of ethylene glycol dimethacrylate, pentaerythritol triacrylate, trimethoxypropane trimethacrylate and pentaerythritol tetramethacrylate.
Further, the initiator comprises: one or more of azodiisobutyronitrile and azodiisoheptonitrile.
Further, the pore-forming agent comprises: solvents with equivalent polarity such as acetonitrile, methanol, acetic acid, acetone and the like can be used as long as organic phosphorus and functional monomers can be dissolved into oil-free liquid drops.
Further, the reducing agent includes: ascorbic acid, SnCl2One or more of them.
The microsphere carrier can be prepared from various raw materials or by various methods as long as a reaction site is provided for the molecularly imprinted material.
The carrier microsphere includes: double bond modified Fe3O4@mSiO2Nanoparticles, but not limited to such particles, are preferably prepared by a process comprising:
(1) preparation of magnetic Fe3O4A nanoparticle;
(2) preparation of Fe by sol suspension method3O4@CTAB@mSiO2Nano-particles: magnetic Fe3O4Reacting the nano particles with hexadecyl trimethyl ammonium bromide and tetraethyl orthosilicate to obtain Fe3O4@CTAB@mSiO2A nanoparticle;
(3) preparation of Fe3O4@mSiO2Nano-particles:
mixing Fe3O4@CTAB@mSiO2The nano particles are eluted in acetone to remove CTAB, and Fe is prepared3O4@mSiO2A nanoparticle;
(4) double bond modified Fe3O4@mSiO2Nano-particles:
mixing Fe3O4@mSiO2Suspending the nano particles in MPS solution to obtain double-bond modified Fe3O4@mSiO2And (3) nanoparticles.
The preparation of the molecularly imprinted microsphere comprises the following steps: dissolving organophosphorus template molecules and functional monomers in a pore-foaming agent for preassembly, and using N2Blowing for protection for 1-2 min, then placing the mixture into a refrigerator at 4 ℃ for 10-15 h for later use, and then modifying double-bond modified Fe3O4@mSiO2Nanoparticles, cross-linking agent and initiator are dissolved in a porogen (preferably the same porogen used for pre-assembly, otherwise the polymer is easily swollen, resulting in a change in the three-dimensional structure of the binding site, causing weak binding and poor adsorption effect), ice-working, N2Slowly adding the pre-assembled solution under purging, and then stirring and reacting at the constant temperature of 55-65 ℃ for 20-26 h; after polymerization, Fe was collected by magnetic control3O4@mSiO2@ MIPs, washing the template molecules with organic solvents (including solvents with equivalent polarity such as acetonitrile, methanol, acetic acid, acetone and the like, preferably the same as a pore-forming agent) until the supernatant is transparent, and then eluting (the eluent includes mixed solvents such as methanol-formic acid, ethanol-formic acid, acetonitrile-formic acid, methanol-acetic acid, ethanol-acetic acid, acetonitrile-acetic acid and the like); finally, washing the mixture to be neutral by using methanol or acetone (or other solvents with equivalent polarity), and carrying out vacuum drying at 50-60 ℃ overnight to obtain Fe with the particle size of 300-500 nm3O4@mSiO2@ MIPs microspheres.
Auxiliary functional monomers can also be added during pre-assembly.
The mass ratio of the organophosphorus template molecules to the functional monomer and the auxiliary functional monomer is 0.25: 0.75-1.25; when the auxiliary functional monomer is not added, the mass ratio of the organophosphorus template molecules to the functional monomer is 0.25: 0.75-1.25.
The ratio of the amount of the organophosphorus template molecules to the volume of the pore-forming agent is 0.25mmol: 10-18 mL.
The double bond modified Fe3O4@mSiO2The ratio of the mass of the nanoparticles, the amount of the crosslinking agent and the mass of the initiator is 50mg: 3-6 mmol: 18-22 mg.
The double bond modified Fe3O4@mSiO2The ratio of the mass of the nanoparticles to the volume of the pore-foaming agent is 50mg: 15-20 mL.
The molecular imprinting microsphere is used for qualitatively or quantitatively determining organic phosphorus. The method specifically comprises the following steps:
(1) and (3) qualitative analysis:
A) color development reaction before organic phosphorus is adsorbed by the microspheres:
taking a certain amount of microspheres, adding excessive Na2MoO4Solution or (NH)4)2MoO4The solution and the reducing agent, and the microsphere system is dark blue;
B) and (3) color development reaction after the microspheres absorb organic phosphorus:
adding microspheres with the same quantity as the microspheres obtained in the step A) into a sample solution to be detected, carrying out chemical adsorption on organic phosphorus molecules in the sample and partial phosphoric acid frameworks on the surfaces of the microspheres at the same molar ratio, and adding excessive Na into the system2MoO4Solution or (NH)4)2MoO4Comparing the solution and the reducing agent solution with the step A), observing the color change, wherein the color change is light, which indicates that the sample contains organic phosphorus;
(2) quantitative analysis:
adding organophosphorus standard solutions with the same volume and different concentrations into the microspheres by adopting an external standard method, wherein the phosphoric acid skeleton on the surface part of the microspheres is enough to generate chemical adsorption with organophosphorus molecules and excessive Na2MoO4Solution or (NH)4)2MoO4After the solution reacts, generating phosphomolybdic blue under the condition of a reducing agent, measuring the absorbance at the maximum absorption wavelength of the phosphomolybdic blue by adopting an ultraviolet visible spectrophotometer, and drawing a standard curve by taking the absorbance (A) as a vertical coordinate and the quantity concentration (c) of a substance as a horizontal coordinate; and adsorbing organic phosphorus in the sample solution to be detected by using the same amount of microspheres, reacting according to the same steps, measuring absorbance, and calculating the quantitative concentration of organic phosphorus in the sample according to a standard curve.
The organophosphorus detected by the method contains N, O, F, S one or more elements, namely, the organophosphorus can form hydrogen bonds with functional monomer phosphate, and the nonpolar group and the auxiliary functional monomer octadecene have hydrophobic effect, so that all organophosphorus pesticides can be detected.
The preparation method of the organophosphorus pesticide residue magnetic molecularly imprinted microsphere disclosed by the invention is preferably carried out according to the following steps:
(1) preparation of superparamagnetic Fe3O4Nano-particles:
FeCl is added3·6H2O, sodium acetate and polyethylene glycol were dissolved in ethylene glycol. And then, carrying out ultrasonic treatment on the mixture for 30-40 min, putting the mixture into a stainless steel autoclave with a polytetrafluoroethylene lining, sealing, and heating to 200-250 ℃ for reaction for 8-10 h. After magnetic control separation, washing with deionized water for 3-6 times, and vacuum drying at 50-60 ℃ for 8-10 h to obtain superparamagnetic Fe3O4Nanoparticles (fig. 1);
FeCl described in step (1)3·6H2The mass ratio of O, sodium acetate and polyethylene glycol is 1.35:3.60: 1.00; FeCl3·6H2The ratio of the mass of O to the volume of ethylene glycol is 1.35g: 40-60 mL.
(2) Preparation of Fe by sol suspension method3O4@CTAB@mSiO2Nano-particles:
mixing the above superparamagnetic Fe3O4Suspending the nanoparticles and CTAB in deionized water, and ultrasonically mixing for 25-35 min. Slowly diluting with NaOH solution, performing ultrasonic treatment for 5-10 min, mechanically stirring at 60-65 ℃ for 30-40 min, adding TEOS/ethanol solution (1/4, v/v), and continuously stirringStirring for 5-8 min, and standing for 12-15 h. Magnetic control separation and collection of Fe3O4@CTAB@mSiO2A nanoparticle;
superparamagnetic Fe as set forth in step (2)3O4The mass ratio of the nanoparticles to CTAB is 50: 500; superparamagnetic Fe3O4The ratio of the mass of the nano particles to the volume of the NaOH solution is 50mg: 450-480 mL, wherein the concentration of NaOH is 1 mmol/L; superparamagnetic Fe3O4The ratio of the mass of the nanoparticles to the volume of the TEOS/ethanol solution (1/4, v/v) is 50mg: 2.5-3.5 mL.
(3) Preparation of Fe3O4@mSiO2Nano-particles:
collecting Fe3O4@CTAB@mSiO2Dispersing the nano particles in acetone, carrying out reflux reaction for 2 times at 80-90 ℃, carrying out 18-24 h each time, eluting to remove CTAB, washing with deionized water for 3-6 times, and carrying out vacuum drying for 8-10 h at 50-60 ℃ to obtain Fe3O4@mSiO2And (3) nanoparticles.
(4) Double bond modified Fe3O4@mSiO2Nano-particles:
mixing Fe3O4@mSiO2Suspending the nano particles in MPS solution, stirring for 4-6 h at 50-60 ℃, washing for 3-6 times by deionized water after magnetic control separation and collection, and vacuum drying for 8-10 h at 50-60 ℃ to finish Fe3O4@mSiO2Modifying double bonds of the nanoparticles;
fe described in step (4)3O4@mSiO2The ratio of the mass of the nanoparticles to the volume of the MPS solution is 250mg: 40-50 mL.
(5) Synthesizing the bifunctional monomer organophosphorus magnetic molecularly imprinted microspheres by a surface imprinting polymerization method:
dissolving template molecule organophosphorus and vinyl phosphate (functional monomer) in acetonitrile for first preassembly, adding 1-octadecene (auxiliary functional monomer) for second preassembly to obtain bifunctional monomer preassembly solution, and using N2Blowing and protecting for 1-2 min, and then placing the mixture into a refrigerator with the temperature of 4 ℃ for 10-15 h for later use. Then, double bond modified Fe3O4@mSiO2Nanoparticles, cross-linker EGDMA andinitiator AIBN dissolved in porogen acetonitrile, working on ice, N2And adding the preassembly solution under purging, and then stirring and reacting at the constant temperature of 55-65 ℃ for 20-26 h. After polymerization, Fe was collected by magnetic control3O4@mSiO2@ MIPs, washed with acetonitrile until the supernatant is clear. Then eluted with methanol-acetic acid (9/1, v/v) until the template molecule is completely removed. Finally, washing the mixture to be neutral by methanol, and carrying out vacuum drying overnight at 50-60 ℃ to obtain Fe with the particle size of about 300nm3O4@mSiO2@ MIPs microspheres (FIG. 1);
the quantity ratio of the organic phosphorus of the template molecule to the vinyl phosphate and the 1-octadecene which are functional monomers in the step (5) is 0.25:1.00: 1.00; the ratio of the amount of the organic phosphorus substances in the template molecules to the volume of the acetonitrile is 0.25mmol: 10-18 mL; double bond modified Fe3O4@mSiO2The ratio of the mass of the nanoparticles, the amount of the substance of the crosslinking agent EGDMA and the mass of the initiator AIBN is 50mg:5mmol:20 mg; double bond modified Fe3O4@mSiO2The ratio of the mass of the nano particles to the volume of the pore-foaming agent acetonitrile is 50mg: 15-20 mL.
The organic phosphorus rapid color development analysis method based on the molecular imprinting microsphere is preferably carried out according to the following steps:
(1) and (3) qualitative analysis:
A) color development reaction before organic phosphorus is adsorbed by the microspheres:
taking a certain amount of microspheres, adding excessive Na2MoO4Solution or (NH)4)2MoO4The solution and the reducing agent, and the microsphere system is dark blue;
B) and (3) color development reaction after the microspheres absorb organic phosphorus:
adding microspheres with the same quantity as the microspheres obtained in the step A) into a sample solution to be detected, carrying out chemical adsorption on organic phosphorus molecules in the sample and partial phosphoric acid frameworks on the surfaces of the microspheres at the same molar ratio, and adding excessive Na into the system2MoO4Solution or (NH)4)2MoO4Comparing the solution and the reducing agent solution with the step A), observing the color change, wherein the color change is light, which indicates that the sample contains organic phosphorus;
(2) quantitative analysis:
adding organic phosphorus series standard solution with same volume and different concentrations into a certain amount of microspheres (excess) by adopting an external standard method, and adding excess Na2MoO4Solution or (NH)4)2MoO4After the solution reacts, generating phosphomolybdic blue under the condition of a reducing agent, measuring the absorbance at the maximum absorption wavelength of the phosphomolybdic blue by adopting an ultraviolet visible spectrophotometer, and drawing a standard curve by taking the absorbance (A) as a vertical coordinate and the quantity concentration (c) of a substance as a horizontal coordinate; and adsorbing organic phosphorus in the sample solution to be detected by using the same amount of microspheres, reacting according to the same steps, measuring absorbance, and calculating the quantitative concentration of organic phosphorus in the sample according to a standard curve.
The quantity concentration ratio of substances for preparing a series of organic phosphorus standard solutions with known concentrations in the step (2) is 0.2:0.4:0.8:1.6:2.0: 2.4.
The invention discloses a preparation method of organophosphorus pesticide residue magnetic molecularly imprinted microspheres and a rapid color development analysis method based on phosphorus-molybdenum blue. The invention aims to solve the problems of complicated analysis steps and low detection speed of organophosphorus pesticide residues in the current environment, food and Chinese medicine fields; the magnetic molecularly imprinted microspheres prepared by taking organic phosphorus as template molecules can carry out rapid qualitative analysis on the enriched organic phosphorus based on a phosphomolybdic blue method, and can carry out rapid quantitative analysis by means of an ultraviolet-visible spectrophotometry. Firstly, adopting a hydrothermal method to synthesize superparamagnetic Fe3O4Nano particles, then using CTAB to superparamagnetism Fe by sol suspension method3O4Preparing Fe by carrying out pore-forming on nano particles and then carrying out reflux elution on CTAB in acetone3O4@mSiO2Mesoporous nanoparticles, and then MPS solution to Fe3O4@mSiO2Double bond modification is carried out on nano particles, functional monomers and auxiliary functional monomers are introduced through a surface imprinting polymerization method to prepare organic phosphorus magnetic molecularly imprinted microspheres, and finally Na is used2MoO4Or (NH)4)2MoO4And (3) carrying out rapid color development qualitative and quantitative analysis after the solution and the reducing agent form the phosphomolybdic blue.
The color development principle of the invention is through Na2MoO4Or (NH)4)2MoO4The solution reacts with the phosphoric acid skeleton on the surface of the microsphere to generate colorless PMo with Mo (VI) alpha-Keggin structure12O40 3-In ascorbic acid or SnCl2Mo (VI) with 1/3 is reduced to Mo (V) under the action of the catalyst to generate blue beta-Keggin ion PMo12O40 7-(FIG. 3), blue PMo is produced12O40 7-The amount of (a) is directly proportional to the amount of phosphate backbone present.
PMo12 ⅥO40 3-+4e-→PMo4 ⅤMo8 ⅥO40 7-
Compared with the prior art, the invention has the beneficial effects that: (1) compared with monofunctional or other bifunctional monomer molecularly imprinted polymers, functional monomers such as: the principle that ethylene phosphate and organic phosphorus molecules form hydrogen bonds and have similar polarities and attract each other is as follows: the nonpolar long carbon chain of the 1-octadecene and the weak polar or nonpolar functional group in the organophosphorus molecules can strengthen the specific adsorption of the molecularly imprinted material on the organophosphorus through hydrophobic acting force, so that the imprinted cavity is more stable, the bonding is firmer and the selectivity is stronger. (2) According to the invention, sodium molybdate or ammonium molybdate reacts with the phosphoric acid skeleton on the surface of the microsphere to generate phosphorus-molybdenum blue, so that organic phosphorus pesticide residues do not need to be digested, and visual color development qualitative analysis of the organic phosphorus pesticide residues is realized by observing color change before and after the microsphere adsorbs the organic phosphorus. (3) The method comprises the steps of adsorbing organophosphorus with different concentrations to microspheres containing phosphoric acid frameworks to form phosphorus-molybdenum blue, and measuring the absorbance of a solution after the microspheres process a sample by adopting an external standard method to complete quantitative analysis of the organophosphorus in the sample; (4) the functional monomer and the template molecule organophosphorus are pre-assembled one by one before surface polymerization, so that the functional monomer and the organophosphorus can effectively form hydrogen bonds, the order of intermolecular combination is enhanced, and the imprinted polymer with an ordered structure is generated. (5) The analysis method has the advantages of large microsphere adsorption capacity, high sensitivity, high accuracy, no need of digestion of organic phosphorus, high detection speed (less than or equal to 10min) and the like. (6) The analysis method can be used for spectrophotometry rapid quantitative analysis (figure 3) of organic phosphorusThe limit can be as low as 1.0 x 10-4mmol/L。
Description of the drawings:
FIG. 1 is Fe3O4And Fe3O4@mSiO2A transmission electron microscope image of @ MIPs microspheres;
left: superparamagnetic Fe3O4A nanoparticle; and (3) right: fe3O4@mSiO2@ MIPs microspheres;
FIG. 2 shows the skeleton structure and crystal structure of phosphomolybdic blue;
FIG. 3 is a schematic diagram of the principle of organophosphorus pesticide residue rapid color development analysis based on magnetic molecularly imprinted microspheres.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments, which are not intended to limit the scope of the present invention.
Example 1:
a preparation method of organophosphorus pesticide residue magnetic molecularly imprinted microspheres respectively takes triazophos, thiophenium and quinalphos as template molecules and ethylene phosphate as a monofunctional monomer, and comprises the following steps:
(1) preparation of superparamagnetism Fe by hydrothermal method3O4Nano-particles:
1.35g FeCl3·6H2O, 3.60g sodium acetate and 1.00g polyethylene glycol were dissolved in 40mL ethylene glycol. Then the mixture is put into a stainless steel autoclave with a polytetrafluoroethylene lining after being subjected to ultrasonic treatment for 30min, sealed and heated to 200 ℃ for reaction for 8 h. After magnetic control separation, washing for 3-6 times by deionized water, and vacuum drying for 8h at 50 ℃ to obtain superparamagnetic Fe3O4And (3) nanoparticles.
(2) Preparation of Fe by sol suspension method3O4@CTAB@mSiO2Nano-particles:
the superparamagnetic Fe is added into 100mL deionized water3O450mg of nano particles and 500mg of CTAB are ultrasonically mixed for 30 min. Slowly diluting with 1mmol/L NaOH solution 450mL, ultrasonically treating for 5min, mechanically stirring at 60 deg.C for 30min, adding 2.5mL TEOS/ethanol solution (1/4, v/v), stirring for 5min, standing for 12h. Magnetic control separation and collection of Fe3O4@CTAB@mSiO2And (3) nanoparticles.
(3) Preparation of Fe3O4@mSiO2Nanoparticles
Collecting Fe3O4@CTAB@mSiO2Dispersing the nano particles in acetone, carrying out reflux reaction for 2 times at 80 ℃ for 24 hours each time, eluting to remove CTAB, washing with deionized water for 3-6 times, and vacuum drying at 50 ℃ for 8 hours to obtain Fe3O4@mSiO2And (3) nanoparticles.
(4) Double bond modified Fe3O4@mSiO2Nano-particles:
mixing 250mg of Fe3O4@mSiO2Suspending the nano particles in 40mL of MPS solution, stirring for 5h at 50 ℃, washing for 3-6 times by using deionized water after magnetic control separation, and drying for 8h in vacuum at 50 ℃ to finish Fe3O4@mSiO2And modifying double bonds of the nanoparticles.
(5) Synthesizing organic phosphorus monofunctional monomer magnetic molecularly imprinted microspheres by a surface imprinting polymerization method:
0.25mmol template molecule and 1.00mmol vinyl phosphate are dissolved in 6mL anhydrous acetonitrile for preassembly, and N is used2Blowing and protecting for 1-2 min, and then placing the mixture into a refrigerator at 4 ℃ for storage for 12h for later use. Then, double bond modified Fe3O4@mSiO2Nanoparticles, 5mmol of crosslinking agent EGDMA and 20mg of initiator AIBN were dissolved in 15mL of porogen acetonitrile and worked up on ice, N2The pre-assembled solution is added under purging, and then the reaction is carried out for 24 hours under constant temperature stirring at 60 ℃. After polymerization, Fe was collected by magnetic control3O4@mSiO2@ MIPs, washed clear supernatant with acetonitrile and then eluted with methanol-acetic acid (9/1, v/v) to complete removal of the template molecule. Finally, washing with methanol to neutrality, vacuum drying at 50 deg.C overnight to obtain Fe with particle size of about 300nm3O4@mSiO2@ MIPs Microspheres (MMIPs).
Simultaneous preparation of Fe without template molecules3O4@mSiO2@ NIPs Microspheres (MNIPs) for use in comparative experiments on adsorption performance.
Example 2:
a preparation method of organophosphorus pesticide residue magnetic molecularly imprinted microspheres respectively takes triazophos, thiophenium and quinalphos as template molecules and takes propylene phosphate as a monofunctional monomer, and comprises the following steps:
the steps (1) to (4) may be carried out in the same manner as in example 1, in accordance with the procedure.
(5) Synthesizing organic phosphorus monofunctional monomer magnetic molecularly imprinted microspheres by a surface imprinting polymerization method:
this procedure was the same as in step (5) of example 1, except that the functional monomer was changed to a phosphate acrylate.
Simultaneous preparation of Fe without template molecules3O4@mSiO2@ NIPs Microspheres (MNIPs) for use in comparative experiments on adsorption performance.
Example 3:
a preparation method of organophosphorus pesticide residue magnetic molecularly imprinted microspheres respectively takes triazophos, thiophenon and quinalphos as template molecules and vinyl phosphate and 1-octadecene as bifunctional monomers and comprises the following steps:
the steps (1) to (4) may be carried out in the same manner as in example 1, in accordance with the procedure.
(5) Synthesizing organic phosphorus bifunctional monomer magnetic molecularly imprinted microspheres by a surface imprinting polymerization method:
this procedure is the same as in step (5) of example 1, except that one functional monomer is replaced with two functional monomers: vinyl phosphate, 1-octadecene.
Simultaneous preparation of Fe without template molecules3O4@mSiO2@ NIPs Microspheres (MNIPs) for use in comparative experiments on adsorption performance.
Example 4:
a preparation method of organophosphorus pesticide residue magnetic molecularly imprinted microspheres respectively takes triazophos, thiophenon and quinalphos as template molecules and takes propene phosphate and 1-heptadecene as bifunctional monomers and comprises the following steps:
the steps (1) to (4) may be carried out in the same manner as in example 1, in accordance with the procedure.
(5) Synthesizing organic phosphorus bifunctional monomer magnetic molecularly imprinted microspheres by a surface imprinting polymerization method:
this procedure is the same as in step (5) of example 1, except that one functional monomer is replaced with two functional monomers: propene phosphate and 1-heptadecene.
Simultaneous preparation of Fe without template molecules3O4@mSiO2@ NIPs Microspheres (MNIPs) for use in comparative experiments on adsorption performance.
The adsorption performance of the prepared organophosphorus monofunctional monomer and bifunctional monomer MMIPs, monofunctional monomer and bifunctional monomer MNIPs is evaluated, 10mg of the 24 microspheres are respectively added into 1mmol/L of triazophos, thiophosphoryl and quinalphos standard solutions, and the saturated adsorption quantity Q of different microspheres to respective template molecules is measured after complete adsorption, which is shown in Table 1.
TABLE 1 comparison of adsorption effect of single-and dual-functional monomer organophosphorus molecularly imprinted microspheres
Figure BDA0001869096660000111
Although the preparation method similar to that of the invention is adopted in the patent (such as Huanfu, Zhang jin, Yi Wei, Hou Chang Jun, Hodan group, Luo Xiao gang, Yang eyebrow, Mao Yao Li, Zhang Hai Feng. the molecular imprinting composite membrane for detecting organophosphorus pesticide and the application thereof [ P ]. the national intellectual property office, the patent application No. CN201210563254.5, the grant publication No. CN103172899B,2012.07.25.) the preparation method is similar to that of the invention, but the adsorption capacity of the prepared molecular imprinting microsphere is larger and has larger advantages from the saturated adsorption data.
Example 5:
the fast chromogenic triazophos analysis method based on the molecular engram microsphere (figure 3) is carried out according to the following steps:
(1) and (3) qualitative analysis:
A) and (3) color development reaction before adsorbing triazophos by the microspheres:
20mg of triazophos molecularly imprinted microspheres prepared in examples 1 to 4 was weighed, and 3mL (excess) of Na was added2MoO4Solution or (NH)4)2MoO4Solution (50mmol/L) and 1.5mL of reducing agent SnCl2And (3) reacting the solution (30mmol/L) for developing for 8min, wherein the microsphere system is dark blue.
B) And (3) carrying out color reaction after adsorbing triazophos by the microspheres:
similarly, 20mg of the triazophos molecularly imprinted microspheres prepared in examples 1-4 are weighed, added into a sample solution, and after the triazophos adsorbed by the microspheres is saturated (the triazophos molecules and part of the phosphate skeleton on the surfaces of the microspheres are subjected to chemical adsorption with an equimolar ratio), 3mL (excessive) of Na is added into the system2MoO4Solution or (NH)4)2MoO4Solution (50mmol/L) and 1.5mL SnCl2After the reaction of the solution (30mmol/L) for 8min, the color change was observed in comparison with step A), and if the color becomes lighter, it indicates that the sample contains triazophos.
(2) Quantitative analysis:
respectively weighing 20mg of the triazophos molecularly imprinted microspheres prepared in examples 1-4 by adopting an external standard method, respectively adding 5mL of 0.2, 0.4, 0.8, 1.6, 2.0 and 2.4mmol/L triazophos standard solution and 3mL of excessive Na2MoO4Solution or (NH)4)2MoO4Solution (50mmol/L) and 1.5mL SnCl2Reacting the solution (30mmol/L) to generate phosphomolybdic blue, measuring absorbance at the maximum absorption wavelength of the phosphomolybdic blue through UV-vis, drawing a standard curve by taking the absorbance (A) as an ordinate and the quantity concentration (c) of the substance as an abscissa to obtain a linear equation of-0.6253 c +2.0467 and a correlation coefficient R20.9991, linear range of 0.001-4 mmol/L, limit of detection LOD of 1.0 × 10-4mmol/L, limit of quantitation LOQ of 1.0X 10-3mmol/L; after 20mg of the triazophos molecularly imprinted microspheres prepared in examples 1 to 4 was additionally weighed and adsorbed in 5mL of sample solution, absorbance of the generated phosphomolybdic blue was measured by UV-vis, and the concentration of triazophos in the sample was calculated according to a linear equation, which is shown in Table 2.
Other rapid color development analysis methods of organic phosphorus can also complete qualitative and quantitative analysis according to the method.
Example 6:
the reliability of the analytical method of the invention was verified by HPLC:
(1) standard curve for triazophos by HPLC:
preparing triazophos standard stock solution with concentration of 320mmol/L, diluting the standard stock solution by a certain multiple to obtain standard solutions with concentrations of 3.2, 16, 32, 64, 160 and 320mmol/L, respectively, and performing HPLC (liquid phase condition: chromatographic column is Pgrandil-STC-C)18The mobile phase is acetonitrile and water which are 55:45, the column temperature is 25 ℃, the flow rate is 1mL/min, the detection wavelength is 247nm, and the sample injection amount is 20 mu L). Taking the concentration c (mmol/L) as an abscissa and the peak area S (mu Au S) as an ordinate, a standard curve is drawn, the linear regression equation of the standard curve is S748932.57719 +114123.77828c, and the correlation coefficient R is20.9993, linear relation in the concentration range of 0.001-1000 mmol/L, and a detection limit LOD of 1.0 multiplied by 10-5mmol/L, limit of quantitation LOQ of 1.0X 10-4mmol/L。
(2) The same sample solution as determined in example 5 by the method of the invention was also determined and compared by HPLC: the peak area of the same sample solution was measured by HPLC, the sample introduction was repeated 3 times to obtain the average peak area S, and the concentration of triazophos in the sample was calculated according to the linear regression equation, as shown in Table 2.
TABLE 2 results of determination of triazophos in samples by the method of the present invention and HPLC method
Figure BDA0001869096660000121
Figure BDA0001869096660000131
As can be seen from Table 2, the concentration of triazophos in the sample solution measured by the method of the present invention is basically consistent with that measured by HPLC method, the relative error is less than or equal to 0.6%, and the method meets the analysis requirements. Therefore, the rapid color development analysis method of triazophos based on the molecularly imprinted microspheres has good reliability and great application value.
Example 7:
superparamagnetic Fe prepared by Transmission Electron Microscope (TEM)3O4Nanoparticles with Fe3O4@mSiO2@MThe morphology analysis and the particle size measurement of the IPs microsphere product are carried out, and the product is shown in the attached figure 1.
Example 8:
the rapid organic phosphorus residue chromogenic analysis (shown in figure 3) based on the molecular imprinting microsphere can be carried out according to the following steps:
(1) qualitative analysis of organophosphorus residues:
the molecular imprinting microspheres which respectively take organophosphorus such as triazophos, thiophenon, quinalphos and the like as templates are mixed in quality, and the visual qualitative analysis of the organophosphorus residues can be realized according to the step (1) in the embodiment 5.
(2) Quantitative analysis of organophosphorus residues:
firstly, mixing the molecular imprinting microspheres which respectively take organophosphorus such as triazophos, thiophenon, quinalphos and the like as templates in quality, operating according to the step (2) in the embodiment 5, and establishing a standard curve between the absorbance and the quantity concentration of the total organophosphorus substances; the absorbance of the sample solution was measured to calculate the residual concentration of organophosphorus, as shown in Table 3.
Example 9:
the reliability of the organophosphorus multi-residue rapid chromogenic analytical method is verified by an HPLC method:
(1) standard curve for determination of organophosphorus residues by HPLC method:
organophosphorus pesticides (triazophos, fenthion, quinalphos, etc.) were first mixed in equal amounts, and a standard curve of the total concentration of organophosphorus residues and the sum of peak areas was plotted according to step (1) in example 6. Taking the concentration c (mmol/L) as an abscissa and the sum S (mu Au S) of the peak areas of the organic phosphorus as an ordinate, drawing a standard curve, wherein a linear regression equation is that S is 851107.8+107725.4c, and a correlation coefficient R2Is 0.9993. The linear range is 0.001-1000 mmol/L, the detection limit LOD is 1.0 x 10- 5mmol/L, limit of quantitation LOQ of 1.0X 10-3mmol/L。
(2) The same sample solution in example 8, which was subjected to the chromogenic assay of the invention, was also subjected to HPLC assay and compared: and (3) measuring the peak area of the solution of the same sample by using HPLC, repeatedly injecting the sample for 3 times to obtain the sum S of the average peak areas, and calculating the total concentration of the organophosphorus residues in the sample according to a linear regression equation, wherein the total concentration is shown in Table 3.
TABLE 3 results of determination of organophosphorus residues in samples by the method of the present invention and HPLC method
Figure BDA0001869096660000141
As can be seen from the data in Table 3, the total concentration of organophosphorus residues in the sample solution measured by the method of the present invention is substantially consistent with that measured by HPLC. Therefore, the molecular imprinting microsphere-based organophosphorus multi-residue rapid color development analysis method has a reliability number and a high popularization value.

Claims (10)

1. A molecularly imprinted microsphere is an imprinted polymer with eluted template molecules loaded on a carrier microsphere, and is characterized in that a pre-assembly liquid formed by organophosphorus template molecules and functional monomers is added into the carrier microsphere, a cross-linking agent, an initiator and a pore-forming agent, and the template molecules are eluted after complete polymerization reaction to form the molecularly imprinted microsphere; the structural formula of the functional monomer is shown as formula 1, and the functional monomer is one or more than one of the following:
Figure FDA0002718789040000011
the method comprises the following steps: vinyl phosphate, propylene phosphate, butylene phosphate, and pentene phosphate.
2. The molecularly imprinted microsphere of claim 1, further comprising an auxiliary functional monomer in the pre-assembly liquid: is a monomer molecule which can be combined with a weak polar or nonpolar functional group in an organophosphorus template molecule through a hydrophobic acting force.
3. The molecularly imprinted microsphere of claim 2, wherein the auxiliary functional monomer comprises: 1-dodecene, 1-tridecene, 1-tetradecene, 1-pentadecene, 1-hexadecene, 1-heptadecene, 1-octadecene, 1-nonadecene, and 1-eicosene.
4. The molecularly imprinted microsphere of claim 1, wherein the carrier microsphere comprises: double bond modified Fe3O4@mSiO2And (3) nanoparticles.
5. The molecularly imprinted microsphere of claim 4, wherein the carrier microsphere is prepared by a process comprising:
(1) preparation of magnetic Fe3O4A nanoparticle;
(2) preparation of Fe by sol suspension method3O4@CTAB@mSiO2Nano-particles: magnetic Fe3O4Reacting the nano particles with hexadecyl trimethyl ammonium bromide and tetraethyl orthosilicate to obtain Fe3O4@CTAB@mSiO2A nanoparticle;
(3) preparation of Fe3O4@mSiO2Nano-particles:
mixing Fe3O4@CTAB@mSiO2The nanometer particles are eluted in acetone to remove CTAB, and Fe is obtained3O4@mSiO2A nanoparticle;
(4) double bond modified Fe3O4@mSiO2Nano-particles:
mixing Fe3O4@mSiO2Suspending the nano particles in MPS solution to obtain double-bond modified Fe3O4@mSiO2And (3) nanoparticles.
6. The molecularly imprinted microsphere of claim 5, wherein the molecularly imprinted microsphere preparation comprises: dissolving organophosphorus template molecules and functional monomers in a pore-foaming agent for preassembly, and using N2Purging and protecting for 1-2 min, and then placing the mixture into a refrigerator at 4 ℃ for 10-15 h for later use; then, double bond modified Fe3O4@mSiO2Dissolving nanoparticles, cross-linking agent and initiator in a pore-forming agent, working on ice, N2Blowing, slowly adding the pre-assembled solution, and then keeping the temperature of 55-65 DEG CStirring and reacting for 20-26 h at a warm temperature; after polymerization, Fe was collected by magnetic control3O4@mSiO2@ MIPs, washing with an organic solvent until the supernatant is transparent, and then eluting the template molecules with an eluant; finally, washing the mixture to be neutral by using methanol or acetone, and carrying out vacuum drying overnight at 50-60 ℃ to obtain Fe with the particle size of 300-500 nm3O4@mSiO2@ MIPs microspheres.
7. The molecularly imprinted microsphere of claim 6, wherein an auxiliary functional monomer is further added during pre-assembly.
8. Molecularly imprinted microsphere according to claim 5 or 6,
the mass ratio of the organophosphorus template molecules to the functional monomers is 0.25 (0.75-1.25);
the mass ratio of the organophosphorus template molecules to the functional monomers and the auxiliary functional monomers is 0.25 (0.75-1.25) to 0.75-1.25;
the ratio of the amount of the organophosphorus template molecules to the volume of the pore-foaming agent is 0.25mmol: 10-18 mL;
the double bond modified Fe3O4@mSiO2The mass ratio of the nanoparticles, the amount of the cross-linking agent and the initiator is 50mg: 3-6 mmol: 18-22 mg;
the double bond modified Fe3O4@mSiO2The volume ratio of the mass of the nano particles to the pore-foaming agent is 50mg: 15-20 mL.
9. Molecularly imprinted microspheres as claimed in any one of claims 1 to 8 for the qualitative or quantitative determination of organophosphorus.
10. The application method according to claim 9, comprising the following steps:
(1) and (3) qualitative analysis:
A) color development reaction before organic phosphorus is adsorbed by the microspheres:
taking a certain amount of microspheres, adding excessive Na2MoO4Solution or (NH)4)2MoO4The solution and the reducing agent, and the microsphere system is dark blue;
B) and (3) color development reaction after the microspheres absorb organic phosphorus:
adding microspheres with the same quantity as the microspheres obtained in the step A) into a sample solution to be detected, carrying out chemical adsorption on organic phosphorus molecules in the sample and partial phosphoric acid frameworks on the surfaces of the microspheres at the same molar ratio, and adding excessive Na into the system2MoO4Solution or (NH)4)2MoO4Comparing the solution and the reducing agent solution with the step A), observing the color change, wherein the color change is light, which indicates that the sample contains organic phosphorus;
(2) quantitative analysis:
adding organophosphorus standard solutions with the same volume and different concentrations into the microspheres by adopting an external standard method, wherein the phosphoric acid skeleton on the surface part of the microspheres is enough to generate chemical adsorption with organophosphorus molecules and excessive Na2MoO4Solution or (NH)4)2MoO4After the solution reacts, generating phosphomolybdic blue under the condition of a reducing agent, measuring the absorbance at the maximum absorption wavelength of the phosphomolybdic blue by adopting an ultraviolet visible spectrophotometer, and drawing a standard curve by taking the absorbance (A) as a vertical coordinate and the quantity concentration (c) of a substance as a horizontal coordinate; and adsorbing organic phosphorus in the sample solution to be detected by using the same amount of microspheres, reacting according to the same steps, measuring absorbance, and calculating the quantitative concentration of organic phosphorus in the sample according to a standard curve.
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