CN101555289A - Glycosyl-functionalization bacterial toxin molecularly imprinted polymer, preparation method and application - Google Patents
Glycosyl-functionalization bacterial toxin molecularly imprinted polymer, preparation method and application Download PDFInfo
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Abstract
The invention relates to a glycosyl-functionalization bacterial toxin molecularly imprinted polymer and a preparation method thereof. The glycosyl-functionalization bacterial toxin molecularly imprinted polymer is prepared by mixing 0.01-10molof bacterial toxin template molecule, 1mol of functional monomer, 0.5-8mol of cross linker, 30-65molof pore-foaming agent and 0.05-0.20mol of evocating agent and carrying out in-situ polymerization. The invention also discloses the application of a glucide compound as a functional monomer in preparing the bacterial toxin molecularly imprinted polymer. The glycosyl-functionalization bacterial toxin molecularly imprinted polymer provided by the invention combines the special distinguishing mechanism of glucid and bacterial toxin and the special distinguishing mechanism of molecularly imprinted polymer to the bacterial toxin, thereby having favorable molecular distinguishing performance and greatly improving the sensitivity and selectivity for the detecting bacterial toxin.
Description
Technical field
The present invention relates to a kind of glycosyl-functionalization bacterial toxin molecularly imprinted polymer and preparation method thereof, be specifically related to a kind of glycosyl-functionalization molecularly imprinted polymer that the template molecule bacteriotoxin is had single-minded recognition capability and preparation method thereof; The invention still further relates to saccharide compound as the application of functionalization monomer in the preparation bacterial toxin molecularly imprinted polymer.
Background technology
Many bacterial pathogens synthetic toxin are morbific primary factor.This toxigenic ability is the morbific potential mechanisms of many bacterial pathogens.Bacteriotoxin mainly contains three kinds: extracellular toxin, intracellular toxin and non-proteotoxin.Extracellular toxin is a kind of typical water-soluble protein, be bacterium at the known the strongest bacteriotoxin of human toxicity that infects of excretory exponential phase of growth, when lower concentration very, just have very high toxicity.Therefore, develop a kind of accuracy height, high specificity, the bacteriotoxic method of rapid detection, burst accidents such as food poisoning, water pollution being made quick diagnosis, and select suitable counter-measure, all is crucial for control of food and environment and national defense safety.
The method of bacterial detection toxin mainly contains biological method at present, as: animal surveys malicious method and cell is surveyed malicious method; Immunological method, as: reverse passive hemagglutination experiment (RPHA), passive immunohemolysis test (PIH), toxin and toxinicide agar diffusion test, radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) are arranged; Molecular biology method, as: the PCR method.Though above-mentioned these method sensitivity are higher, stability better, shortcoming be the operating time long, step is complicated, accuracy is not enough as a result.
Summary of the invention
For solving the problem of above-mentioned existence, the invention provides a kind of glycosyl-functionalization bacterial toxin molecularly imprinted polymer and preparation method thereof, to realize to bacteriotoxic high specific, highly sensitive identification.
The present invention also provides saccharide compound as the application of functionalization monomer in the preparation bacterial toxin molecularly imprinted polymer.
The present invention is achieved by the following technical solutions:
Described glycosyl-functionalization bacterial toxin molecularly imprinted polymer, press 0.01-10 by bacteriotoxin template molecule, function monomer, linking agent, pore-creating agent, initiator: 1: 0.5-8: 30-65: the mixed in molar ratio of 0.05-0.20 obtains through in-situ polymerization again.Described function monomer is a saccharide compound, and described saccharide compound is A-D-pyrans glycosyl seminose, N-acetyl osamine, N-acetyl lactosamine, ganglioside sialic acid or ceramide three hexosides.
Described bacteriotoxin is diphtheria toxin, tetanus toxin, Toxins, botulin, Pseudomonas aeruginosa exotoxin A, toxic shock toxin 1, SPE, Toxins,exo-, cholera, Pseudomonas aeruginosa extracellular toxin a or shiga toxin.
Described linking agent is trimethylolpropane trimethacrylate, N, N-methylene diacrylamine, N, N-1,4-phenylene diacrylamine, 3,5-two (acrylamide) phenylformic acid, ethylene glycol dimethacrylate, Vinylstyrene, N, O-two acryloyls-L-amphetamine alcohol, pentaerythritol triacrylate or tetramethylol methane tetraacrylate; Be preferably ethylene glycol dimethacrylate or Vinylstyrene.
Described pore-creating agent is methylene dichloride, chloroform, acetonitrile, methyl alcohol, Virahol, tetracol phenixin, heterogeneous ring compound acid amides or sulfone compound, is preferably methylene dichloride or chloroform.
Described initiator is organo-peroxide or azo compound, is preferably Diisopropyl azodicarboxylate.
The preparation method of above-mentioned glycosyl-functionalization bacterial toxin molecularly imprinted polymer may further comprise the steps:
(1) bacteriotoxin template molecule, function monomer, linking agent, pore-creating agent, initiator are mixed by certain mol proportion, add in the reactor, feed nitrogen-sealed, under thermal initiation or light-initiated condition, carry out in-situ polymerization, obtain containing bacteriotoxic polymkeric substance;
(2) the above-mentioned polymkeric substance that obtains is taken out from reactor, grind, sieve, be eluted to elutriant and do not contain bacteriotoxin, clean to neutral with organic solvent then, dry under the vacuum, promptly make molecularly imprinted polymer of the present invention.
Described thermal initiation condition is: polymerization temperature is controlled at 60 ℃～90 ℃, isothermal reaction 12～48 hours; The Photoinitiated reactions condition is: polymerization is 12～48 hours under 120w or 150w high voltage mercury lamp.
Described elutriant is the mixed solution of methyl alcohol and acetate or the mixed solution of acetonitrile and acetate, and the volume ratio of methyl alcohol or acetonitrile and acetate is 30～10: 1, and described organic solvent is a methyl alcohol.
The invention provides saccharide compound as the application of function monomer in the preparation bacterial toxin molecularly imprinted polymer.
Because most of toxin are multivalence attitudes, it can combine with special glycoprotein receptor on the host cell membrane, as initial identification and binding site.By studying the mechanism of bacteriotoxic character and intrusion host cell thereof, we find that saccharide compound acts on this performance on mainly, it is not that simple monomer combines with bacteriotoxic interaction, but widow or polysaccharide mechanism of action, therefore to come the bacterial detection toxin with the acceptor molecule of bacteriotoxin specificity bonded carbohydrate structure be very favorable if can utilize.Based on above-mentioned thinking, the present invention has broken traditional with acrylic acid or the like, pyridines, amides prepares the method for molecularly imprinted polymer as function monomer, and chosen the various saccharides compound as function monomer, dexterously saccharide compound can be carried out the mechanism of specific recognition and molecularly imprinted polymer to bacteriotoxin combines to the mechanism of microsphere-bacteriotoxin specific recognition, the glycosyl-functionalization bacterial toxin molecularly imprinted polymer of preparing thus has higher selectivity to target molecule, thereby has improved the specificity and the accuracy of bacterial detection toxin.
Beneficial effect of the present invention:
The glycosyl-functionalization bacterial toxin molecularly imprinted polymer that the present invention developed, carbohydrate is combined to bacteriotoxic specific recognition mechanism with bacteriotoxic specific recognition mechanism and molecularly imprinted polymer, have good molecular recognition performance, improved the sensitivity and the selectivity of bacterial detection toxin greatly.The preparation method of glycosyl-functionalization bacterial toxin molecularly imprinted polymer provided by the invention, simple, efficient, for the preparation of the bacteriotoxic molecularly imprinted polymer of glycosyl-functionalization provides a kind of feasible preparation scheme.
The present invention has proposed saccharide compound first as the application of function monomer in the preparation bacterial toxin molecularly imprinted polymer.
Embodiment
The invention will be further described below in conjunction with specific embodiment.
Embodiment 1
Take by weighing 0.5220g reaction monomers A-D-pyrans glycosyl seminose, 0.0235g template molecule diphtheria toxin, 4.6500g linking agent trimethylolpropane trimethacrylate, 0.0745g initiator Diisopropyl azodicarboxylate respectively, 15ml pore-creating agent methylene dichloride, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, reaction is 48 hours in 60 ℃ of baking ovens.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt methyl alcohol: the mixing solutions of acetate (volume ratio 10: 1) is the polymkeric substance that makes of ultrasonic cleaning repeatedly, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the diphtheria toxin molecularly imprinted polymer of A-D-pyrans glycosyl seminose glycosyl-functionalization.
Take by weighing 0.4365g reaction monomers vinylformic acid, 0.0235g template molecule diphtheria toxin, 4.6500g linking agent trimethylolpropane trimethacrylate, 0.0745g initiator Diisopropyl azodicarboxylate respectively, 15ml pore-creating agent methylene dichloride, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, reaction is 48 hours in 60 ℃ of baking ovens.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt methyl alcohol: the mixing solutions of acetate (volume ratio 10: 1) is the polymkeric substance that makes of ultrasonic cleaning repeatedly, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the diphtheria toxin molecularly imprinted polymer.
Respectively above-mentioned two kinds of molecularly imprinted polymers are filled in the glass syringe from preparing solid-phase extraction column (SPE post), the successful SPE post of preparation are connected to chemiluminescent analyzer, concentration 2.5 * 10
-4Mol/L luminol solution and sample solution pump into chemiluminescent analyzer respectively, and the diphtheria toxin in the sample is detected, and investigate molecular recognition and the recovering effect of this SPE post to diphtheria toxin in the water sample, the results are shown in Table 1.
The result as can be seen from table 1: utilize glycan molecule (A-D-pyrans glycosyl seminose) than the diphtheria toxin molecularly imprinted polymer with common monomer (vinylformic acid) preparation diphtheria toxin to be had better recognition specificity and recovering effect as the diphtheria toxin molecularly imprinted polymer of monomer preparation.
Embodiment 2
Take by weighing 0.3820g reaction monomers ganglioside sialic acid, 0.03220g template molecule tetanus toxin, 3.8820g linking agent Vinylstyrene, 0.0750g initiator Diisopropyl azodicarboxylate respectively, 15ml pore-creating agent chloroform, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, polymerization is 48 hours under the 120w high voltage mercury lamp.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt acetonitrile: the mixing solutions of acetate (volume ratio 15: 1) is the polymkeric substance that makes of ultrasonic cleaning repeatedly, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the tetanus toxin molecule imprinted polymer of ganglioside sialic acid glycosyl-functionalization.
Take by weighing 0.4025g reaction monomers methacrylic acid, 0.03220g template molecule tetanus toxin, 3.8820g linking agent Vinylstyrene, 0.0750g initiator Diisopropyl azodicarboxylate respectively, 15ml pore-creating agent chloroform, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, polymerization is 48 hours under the 120w high voltage mercury lamp.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt acetonitrile: (15: 1 mixing solutions of volume ratio is the polymkeric substance that makes of ultrasonic cleaning repeatedly for acetate, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the tetanus toxin molecule imprinted polymer.
Respectively above-mentioned two kinds of molecularly imprinted polymers are filled in the glass syringe from preparing solid-phase extraction column (SPE post), and investigate molecular recognition and the recovering effect of this SPE post, the results are shown in Table 1 tetanus toxin in the water sample.
The result as can be seen from table 1: utilize glycan molecule (ganglioside sialic acid) than the tetanus toxin molecule imprinted polymer with common monomer (methacrylic acid) preparation tetanus toxin to be had better recognition specificity and recovering effect as the tetanus toxin molecule imprinted polymer of monomer preparation.
Embodiment 3
Take by weighing 0.4100g reaction monomers N-acetyl osamine, 0.03560g template molecule Toxins, botulin, 3.9600g linking agent pentaerythritol triacrylate, 0.0810g initiator Diisopropyl azodicarboxylate respectively, 15ml pore-creating agent acetonitrile, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, reaction is 36 hours in 70 ℃ of baking ovens.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt methyl alcohol: the mixing solutions of acetate (volume ratio 20: 1) is the polymkeric substance that makes of ultrasonic cleaning repeatedly, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the botulinum toxin molecules imprinted polymer of N-acetyl osamine glycosyl-functionalization.
Take by weighing 0.4365g reaction monomers 4-vinylpridine, 0.03560g template molecule Toxins, botulin, 3.9600g linking agent pentaerythritol triacrylate, 0.0810g initiator Diisopropyl azodicarboxylate respectively, 15ml pore-creating agent acetonitrile, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, reaction is 36 hours in 70 ℃ of baking ovens.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt methyl alcohol: the mixing solutions of acetate (volume ratio 20: 1) is the polymkeric substance that makes of ultrasonic cleaning repeatedly, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the botulinum toxin molecules imprinted polymer.
Respectively above-mentioned two kinds of molecularly imprinted polymers are filled in the glass syringe from preparing solid-phase extraction column (SPE post), and investigate this SPE post, the results are shown in Table 1 botulinal molecular recognition and recovering effect in the water sample.
The result as can be seen from table 1: utilize glycan molecule (N-acetyl osamine) than the botulinum toxin molecules imprinted polymer with common monomer (4-vinylpridine) preparation Toxins, botulin to be had better recognition specificity and recovering effect as the botulinum toxin molecules imprinted polymer of monomer preparation.
Embodiment 4
Take by weighing 0.4150g reaction monomers ceramide three hexosides, 0.0330g template molecule shiga toxin, 3.8995g linking agent ethylene glycol dimethacrylate, 0.0780g initiator Diisopropyl azodicarboxylate respectively, 15ml pore-creating agent methyl alcohol, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, polyase 13 is 6 hours under the 130w high voltage mercury lamp.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt acetonitrile: (25: 1 mixing solutions of volume ratio is the polymkeric substance that makes of ultrasonic cleaning repeatedly for acetate, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the shiga toxin molecularly imprinted polymer of ceramide three hexoside glycosyl-functionalizations.
Take by weighing 0.4090g reaction monomers 4-vinylpridine, 0.0330g template molecule shiga toxin, 3.8995g linking agent ethylene glycol dimethacrylate, 0.0780g initiator Diisopropyl azodicarboxylate respectively, 15ml pore-creating agent methyl alcohol, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, polyase 13 is 6 hours under the 130w high voltage mercury lamp.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt acetonitrile: (25: 1 mixing solutions of volume ratio is the polymkeric substance that makes of ultrasonic cleaning repeatedly for acetate, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the shiga toxin molecularly imprinted polymer.
Respectively above-mentioned two kinds of molecularly imprinted polymers are filled in the glass syringe from preparing solid-phase extraction column (SPE post), and investigate molecular recognition and the recovering effect of this SPE post, the results are shown in Table 1 shiga toxin in the water sample.
The result as can be seen from table 1: utilize glycan molecule (ceramide three hexosides) than the shiga toxin molecularly imprinted polymer with common monomer (4-vinylpridine) preparation shiga toxin to be had better recognition specificity and recovering effect as the shiga toxin molecularly imprinted polymer of monomer preparation.
Embodiment 5
Take by weighing 0.4020g reaction monomers N-acetyl lactosamine, 0.0345g template molecule toxic shock toxin 1,3.9880g linking agent N respectively, O-two acryloyls-L-amphetamine alcohol, 0.0865 initiator Diisopropyl azodicarboxylate, 15ml pore-creating agent Virahol, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, reaction is 24 hours in 80 ℃ of baking ovens.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt methyl alcohol: the mixing solutions of acetate (volume ratio 30: 1) is the polymkeric substance that makes of ultrasonic cleaning repeatedly, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the toxic shock toxin molecularly imprinted polymer of N-acetyl lactosamine glycosyl-functionalization.
Take by weighing 0.3990g reaction monomers methacrylic acid, 0.0345g template molecule toxic shock toxin 1,3.9880g linking agent N respectively, O-two acryloyls-L-amphetamine alcohol, 0.0865 initiator Diisopropyl azodicarboxylate, 15ml pore-creating agent Virahol, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, reaction is 24 hours in 80 ℃ of baking ovens.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt methyl alcohol: the mixing solutions of acetate (volume ratio 30: 1) is the polymkeric substance that makes of ultrasonic cleaning repeatedly, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the toxic shock toxin molecularly imprinted polymer.
Respectively above-mentioned two kinds of molecularly imprinted polymers are filled in the glass syringe from preparing solid-phase extraction column (SPE post), and investigate molecular recognition and the recovering effect of this SPE post, the results are shown in Table 1 target toxic shock toxin in the water sample.
The result as can be seen from table 1: utilize glycan molecule (N-acetyl lactosamine) than toxic shock toxin 1 molecularly imprinted polymer with common monomer (methacrylic acid) preparation toxic shock toxin 1 to be had better recognition specificity and recovering effect as toxic shock toxin 1 molecularly imprinted polymer of monomer preparation.
Embodiment 6
Take by weighing 0.4120g reaction monomers N-acetyl osamine, 0.0400g template molecule SPE, 4.1010g linking agent N respectively, N-1,4-phenylene diacrylamine, 0.0800g initiator Diisopropyl azodicarboxylate, 15ml pore-creating agent tetracol phenixin, mix, in the 25ml ampere bottle of packing into, fed nitrogen 10 minutes, sealing, polymerization is 24 hours under the 140w high voltage mercury lamp.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt acetonitrile: (30: 1 mixing solutions of volume ratio is the polymkeric substance that makes of ultrasonic cleaning repeatedly for acetate, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the SPE molecularly imprinted polymer of N-acetyl osamine glycosyl-functionalization.
Take by weighing 0.4025g reaction monomers vinylformic acid, 0.0400g template molecule SPE, 4.1010g linking agent N respectively, N-1,4-phenylene diacrylamine, 0.0800g initiator Diisopropyl azodicarboxylate, 15ml pore-creating agent tetracol phenixin, mix, in the 25ml ampere bottle of packing into, fed nitrogen 10 minutes, sealing, polymerization is 24 hours under the 140w high voltage mercury lamp.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt acetonitrile: (30: 1 mixing solutions of volume ratio is the polymkeric substance that makes of ultrasonic cleaning repeatedly for acetate, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the SPE molecularly imprinted polymer.
Respectively above-mentioned two kinds of molecularly imprinted polymers are filled in the glass syringe from preparing solid-phase extraction column (SPE post), and investigate molecular recognition and the recovering effect of this SPE post, the results are shown in Table 1 water sample streptococcus intermedius pyrogenic exotoxin.
The result as can be seen from table 1: utilize glycan molecule (N-acetyl osamine) than the SPE molecularly imprinted polymer with common monomer (vinylformic acid) preparation SPE to be had better recognition specificity and recovering effect as the SPE molecularly imprinted polymer of monomer preparation.
Embodiment 7
Take by weighing 0.4125g reaction monomers ganglioside sialic acid respectively, 0.0355g template molecule Toxins,exo-, cholera, 3.9555g linking agent ethylene glycol dimethacrylate, 0.0805g initiator Diisopropyl azodicarboxylate, 15ml pore-creating agent acrylamide, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, reaction is 12 hours in 90 ℃ of baking ovens.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt methyl alcohol: the mixing solutions of acetate (volume ratio 20: 1) is the polymkeric substance that makes of ultrasonic cleaning repeatedly, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the Toxins,exo-, cholera molecularly imprinted polymer of ganglioside sialic acid glycosyl-functionalization.
Take by weighing 0.4365g reaction monomers 4-vinylpridine, 0.0355g template molecule Toxins,exo-, cholera, 3.9555g linking agent ethylene glycol dimethacrylate, 0.0805g initiator Diisopropyl azodicarboxylate respectively, 15ml pore-creating agent acrylamide, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, reaction is 12 hours in 90 ℃ of baking ovens.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt methyl alcohol: the mixing solutions of acetate (volume ratio 20: 1) is the polymkeric substance that makes of ultrasonic cleaning repeatedly, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the Toxins,exo-, cholera molecularly imprinted polymer.
Respectively above-mentioned two kinds of molecularly imprinted polymers are filled in the glass syringe from preparing solid-phase extraction column (SPE post), and investigate molecular recognition and the recovering effect of this SPE post, the results are shown in Table 1 Toxins,exo-, cholera in the water sample.
The result as can be seen from table 1: utilize glycan molecule (ganglioside sialic acid) than the Toxins,exo-, cholera molecularly imprinted polymer with common monomer (4-vinylpridine) preparation Toxins,exo-, cholera to be had better recognition specificity and recovering effect as the Toxins,exo-, cholera molecularly imprinted polymer of monomer preparation.
Embodiment 8
Take by weighing 0.4025g reaction monomers ceramide three hexosides, 0.0340g template molecule Pseudomonas aeruginosa extracellular toxin a, 4.0110g linking agent tetramethylol methane tetraacrylate, 0.0885g initiator Diisopropyl azodicarboxylate respectively, 15ml pore-creating agent dimethyl sulfone, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, polymerization is 12 hours under the 150w high voltage mercury lamp.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt acetonitrile: (15: 1 mixing solutions of volume ratio is the polymkeric substance that makes of ultrasonic cleaning repeatedly for acetate, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains the molecularly imprinted polymer of the Pseudomonas aeruginosa extracellular toxin a of ceramide three hexoside glycosyl-functionalizations.
Take by weighing 0.4120g reaction monomers vinylformic acid, 0.0340g template molecule Pseudomonas aeruginosa extracellular toxin a, 4.0110g linking agent tetramethylol methane tetraacrylate, 0.0885g initiator Diisopropyl azodicarboxylate respectively, 15ml pore-creating agent dimethyl sulfone, mix, pack in the 25ml ampere bottle, fed nitrogen 10 minutes, sealing, polymerization is 12 hours under the 150w high voltage mercury lamp.Polymer monolith is taken out, grind, extracting, drying is sieved.Adopt acetonitrile: (15: 1 mixing solutions of volume ratio is the polymkeric substance that makes of ultrasonic cleaning repeatedly for acetate, until detecting less than template molecule in the maximum absorption wave strong point, remove excessive acetate with washed with methanol again, drying obtains Pseudomonas aeruginosa extracellular toxin a molecularly imprinted polymer.
Respectively above-mentioned two kinds of molecularly imprinted polymers are filled in the glass syringe from preparing solid-phase extraction column (SPE post), and investigate molecular recognition and the recovering effect of this SPE post, the results are shown in Table 1 Pseudomonas aeruginosa extracellular toxin a in the water sample.
The result as can be seen from table 1: utilize glycan molecule (ceramide three hexosides) than the Pseudomonas aeruginosa extracellular toxin a molecularly imprinted polymer with common monomer (vinylformic acid) preparation Pseudomonas aeruginosa extracellular toxin a to be had better recognition specificity and recovering effect as the Pseudomonas aeruginosa extracellular toxin a molecularly imprinted polymer of monomer preparation.
The solid-phase extraction column rate of recovery result of table 1 differing molecular imprinted polymer preparation
Claims (8)
1. glycosyl-functionalization bacterial toxin molecularly imprinted polymer, it is characterized in that: press 0.01-10 by bacteriotoxin template molecule, function monomer, linking agent, pore-creating agent, initiator: 1: 0.5-8: 30-65: the mixed in molar ratio of 0.05-0.20 obtains through in-situ polymerization again; Described function monomer is a saccharide compound.
2. glycosyl-functionalization bacterial toxin molecularly imprinted polymer according to claim 1 is characterized in that: described saccharide compound is A-D-pyrans glycosyl seminose, N-acetyl osamine, N-acetyl lactosamine, ganglioside sialic acid or ceramide three hexosides.
3. glycosyl-functionalization bacterial toxin molecularly imprinted polymer according to claim 1 and 2 is characterized in that: described bacteriotoxin is diphtheria toxin, tetanus toxin, Toxins, botulin, Pseudomonas aeruginosa exotoxin A, toxic shock toxin 1, SPE, Toxins,exo-, cholera, Pseudomonas aeruginosa extracellular toxin a or shiga toxin; Described linking agent is trimethylolpropane trimethacrylate, N, N-methylene diacrylamine, N, N-1,4-phenylene diacrylamine, 3,5-two (acrylamide) phenylformic acid, ethylene glycol dimethacrylate, Vinylstyrene, N, O-two acryloyls-L-amphetamine alcohol, pentaerythritol triacrylate or tetramethylol methane tetraacrylate;
Described pore-creating agent is methylene dichloride, chloroform, acetonitrile, methyl alcohol, Virahol, tetracol phenixin, heterogeneous ring compound acid amides or sulfone compound;
Described initiator is organo-peroxide or azo compound.
4. glycosyl-functionalization bacterial toxin molecularly imprinted polymer according to claim 3 is characterized in that: described linking agent is ethylene glycol dimethacrylate or Vinylstyrene; Described pore-creating agent is methylene dichloride or chloroform; Described initiator is a Diisopropyl azodicarboxylate.
5. the preparation method of claim 1 or 2 described glycosyl-functionalization bacterial toxin molecularly imprinted polymers is characterized in that may further comprise the steps:
(1) bacteriotoxin template molecule, function monomer, linking agent, pore-creating agent, initiator are mixed by certain mol proportion, add in the reactor, feed nitrogen-sealed, under thermal initiation or light-initiated condition, carry out in-situ polymerization, obtain containing bacteriotoxic polymkeric substance; (2) the above-mentioned polymkeric substance that obtains is taken out from reactor, grind, sieve, be eluted to elutriant and do not contain bacteriotoxin, clean to neutral with organic solvent then, dry under the vacuum, promptly make molecularly imprinted polymer of the present invention.
6. preparation method according to claim 5 is characterized in that: described thermal initiation condition is: polymerization temperature is controlled at 60 ℃～90 ℃, isothermal reaction 12～48 hours; The Photoinitiated reactions condition is: polymerization is 12～48 hours under 120w or 150w high voltage mercury lamp.
7. preparation method according to claim 5 is characterized in that: described elutriant is the mixed solution of methyl alcohol and acetate or the mixed solution of acetonitrile and acetate, and the volume ratio of methyl alcohol or acetonitrile and acetate is 30～10: 1, and described organic solvent is a methyl alcohol.
8. saccharide compound is as the application of function monomer in the preparation bacterial toxin molecularly imprinted polymer.
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| CN102590307A (en) * | 2012-01-19 | 2012-07-18 | 济南大学 | Molecularly imprinted membrane electrode based on nucleic acid adapters for detecting organic arsenide in marine product and preparation method of electrode |
| CN103071471A (en) * | 2013-02-01 | 2013-05-01 | 中国科学院新疆理化技术研究所 | Preparation method of epiglloctechin gallate imprinted polymer monolithic column |
| CN105315397A (en) * | 2015-11-30 | 2016-02-10 | 山东省分析测试中心 | Preparation method and application of hydrophilic fake template molecularly imprinted polymer for detecting geniposide |
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| CA2685497A1 (en) * | 2007-03-27 | 2008-10-02 | University Of Maryland, College Park | Imprinted polymeric materials for binding various targets such as viruses |
| CN101308066B (en) * | 2008-06-12 | 2010-07-28 | 济南大学 | Trace amount mycotoxins molecular blotting column preparation method and application |
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| CN101858898A (en) * | 2010-05-18 | 2010-10-13 | 华中师范大学 | Monolithic column solid phase extraction sample pretreatment method |
| CN101858898B (en) * | 2010-05-18 | 2012-08-29 | 华中师范大学 | Monolithic column solid phase extraction sample pretreatment method |
| CN102590307A (en) * | 2012-01-19 | 2012-07-18 | 济南大学 | Molecularly imprinted membrane electrode based on nucleic acid adapters for detecting organic arsenide in marine product and preparation method of electrode |
| CN103071471A (en) * | 2013-02-01 | 2013-05-01 | 中国科学院新疆理化技术研究所 | Preparation method of epiglloctechin gallate imprinted polymer monolithic column |
| CN105315397A (en) * | 2015-11-30 | 2016-02-10 | 山东省分析测试中心 | Preparation method and application of hydrophilic fake template molecularly imprinted polymer for detecting geniposide |
| CN111693586A (en) * | 2020-06-02 | 2020-09-22 | 上海海洋大学 | Bacterial molecularly imprinted polymer, preparation method thereof and bacterial detection method |
| CN111693586B (en) * | 2020-06-02 | 2022-09-09 | 上海海洋大学 | Bacterial molecularly imprinted polymer, preparation method thereof and bacterial detection method |
| CN111521664A (en) * | 2020-07-06 | 2020-08-11 | 南京市食品药品监督检验院 | Listeria monocytogenes imprinted electrochemical sensor and preparation method thereof |
| CN111521664B (en) * | 2020-07-06 | 2020-10-16 | 南京市食品药品监督检验院 | Listeria monocytogenes imprinted electrochemical sensor and preparation method thereof |
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