CN102516458B - Molecularly imprinted polymer specially combined with specified glycoprotein and preparation method and application of molecularly imprinted polymer - Google Patents
Molecularly imprinted polymer specially combined with specified glycoprotein and preparation method and application of molecularly imprinted polymer Download PDFInfo
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Abstract
The invention discloses a molecularly imprinted polymer specially combined with a specified glycoprotein. Substituted phenylboronic acid containing double bonds is used as a functional monomer. Under the alkali condition, the specified glycoprotein serving as a template (imprinting molecule) and the substituted phenylboronic acid (functional monomer) containing the double bonds form a complex; the complex is mixed with a cross linker, an initiator and a pore forming agent; copolymerization reaction between the functional monomer and the cross linker and in the cross linker is initiated under the irradiation of ultraviolet light to form a polymer; and the template modules (specified glycoprotein) in the polymer are removed by extraction of an acidic solution, and thus the molecularly imprinted polymer containing a phenylboronic acid site reversibly combined with the glycosyl of the glycoprotein and a cavity complementary with the shape of the template modules is obtained. The molecularly imprinted polymer specially combined with the specified glycoprotein has the advantages of high specificity, strong anti-interference capacity, wide practical sample range, good repeatability, simplicity in preparation and the like, and can be directly used for detecting antigens to be detected in a complex biological system of serum, saliva and the like. The invention discloses a preparation method for the molecularly imprinted polymer.
Description
Technical field
The present invention relates to a kind of single-minded molecularly imprinted polymer in conjunction with glycoprotein and method for making and the application in detectable antigens glycoprotein.
Background technology
Glycoprotein is the important protein of a class.In Mammals, the over half of total protein is glycoprotein.Glycoprotein has vital role in physiological process, as molecular recognition, the intra/inter-signal of cell, immunne response, fertilization and developmental regulation etc.Glycoprotein is significant in the early diagnosis of major disease, in the disease marker of food and drug administration (FDA) approval, the overwhelming majority is glycoprotein, as alpha-fetoprotein (AFP), carcinomebryonic antigen (CEA) and prostate specific antigen (PSA) etc.In addition, the glycoprotein such as erythropoietin (EPO) and hCG (hCG) are the materials banned use of in sports, it is that challenging task is (referring to F.Lasne that these anti-depressant rapid sensitives detect, J.de Geaurriz, Nature 2000,405,635; He Jiangang, Liu Zhen, Liu Jing, Dou Peng, Chen Hongyuan, chromatogram 2008,28,402-407).
Antibody is indispensable core element instrument in the fields such as molecular biology research, protein research and medical diagnosis on disease.But there are several unfavorable factors in the use of antibody.At first, the preparation difficulty of antibody, expensive.Secondly, the poor stability of antibody.Moreover the specificity of some antibody is poor.Therefore, single-minded, cheap and replacer good stability of research and development selectivity not only has important scientific meaning, and has considerable market outlook.Molecularly imprinted polymer (MIP) (referring to G.Wulff, Angew.Chem.Int.Ed.Engl.1995,34,1812-1832; G.Vlatakis, L.I.Andersson, P.Muller, K.Mosbach, Nature 1993,361, are 645-647) biomimetic materials of important antibody.
The ultimate principle of molecular imprinting is: at first template molecule and function monomer are formed to title complex by a certain percentage, thereby then add linking agent to form polymkeric substance and title complex is fixed and is wrapped in polymkeric substance inside, finally adopt certain method that template molecule is extracted, thus in polymkeric substance, stay binding site optionally and with template molecule shape complementary cavity mutually.Molecular imprinting has the following advantages: one, precordainment can prepare according to the difference of application target different molecularly imprinted polymers; Two, specificity, single-minded ground of energy recognition template molecule, and form the interaction (therefore molecularly imprinted polymer is called as " plastics antibody " or " artificial antibody ") be similar between antibody and antigen between template molecule; Three, practicality, can prepare by chemosynthesis by molecularly imprinted polymer, cheap on a large scale, and is applicable to that various reaction conditionss, stability are high, long service life.Because these advantages, molecularly imprinted polymer is representing good application prospect (referring to L.X.Chen to fields such as optical isomer fractionation, Solid-Phase Extraction, chemobionics sensing, mimetic enzyme catalysis and pharmaceutical analysis technology, S.F.Xu, J.H.Li, Chem.Soc.Rev.2011,40,2922-2942).
The trace of biomacromolecule (especially protein) has suitable challenge, mainly has the difficulty of two aspects: the first, and even sex change of change of configuration easily occurs in biomacromolecule under common polymerizing condition; Secondly, the mass transfer of biomacromolecule in polymer network is slow, template molecule remove difficulty (referring to Y.Hoshino, K.J.Shea, J.Mater.Chem.2011,21,3517-3521; N.W.Turner, C.W.Jeans, K.R.Brain, C.J.Allender, V.Hlady, D.W.Britt, Biotechnol.Prog.2006,22,1474-1489).In order to solve above difficulty, the trace of several method for biomacromolecule arranged, comprise surface imprinted (referring to M.Kempe, M.Glad, K.Mosbach, J.Mol.Recognit.1995, 8, 35-39), the epitope trace is (referring to N.Nishino, C.S.Huang, K.J.Shea, Angew.Chem.Int.Ed.2006, 45, 2392-2396), the metal companion method is (referring to L.Qin, X.-W.He, W.Zhang, W.-Y.Li, Y.-K.Zhang, Anal.Chem.2009, 81, 7206-7216) and nanotechnology (referring to D.Cai, L.Ren, H.Z.Zhao, et al.Nat.Nanotech.2010, 5, 597-601) etc.With respect to non-glycoprotein, the molecular imprinting bibliographical information of glycoprotein is less.Hjerten etc. adopt the physically trapping method, prepared the low crosslinking degree gel that can trace comprises the multiple proteins of glycoprotein Transferrins,iron complexes (referring to J.-L.Liao, Y.Wang, S.Hjerten, Chromatographia 1996,42,259-262; S.Hjerten, J.L.Liao, K.Nakazato, Y.Wang, G.Zamaratskaia, H.-X.Zhang, Chromatographia 1997,44,227-234; D.Tong, Cs.Hetenyi, Zs.Bikadi, J.-P.Gao, S.Hjerten, Chromatographia 2001,54,7-14).Although the entrapping method engram technology is fairly simple, and may be a kind of blanket method, exist many drawbacks, as low as the microsphere utilization ratio, microsphere be difficult for wash-out, and medium internal divergence resistance is large, and the medium form is irregular etc.Adopt surface imprinted method, on the polysiloxane copolymer on the porous silica gel particle surface such as Mosbach trace the glycoprotein Transferrins,iron complexes, but this imprinted polymer is faint (referring to M.Glad to the specificity of Transferrins,iron complexes, 0.Norrlow, B.Sellergren, N.Siegbahn, K.Mosbach, J.Chromatogr. A 1985,347,11-23).Ratner etc. have developed the new surface imprinted technology of a class: at first protein be adsorbed onto on mica, then two glycan molecules of skim are coated on the protein of absorption, the sugar layer passes through hydrogen bonded with protein, follow the fluorescent polymer thin layer smooth at glycan molecule surface aggregate last layer, finally remove mica and dissolve trace protein, generated there is protein shape hole poly-disaccharides surface imprinted polymer (referring to H.Q.Shi, B.D.Ratner, J.Biomed.Mater.Res., 2000,49,1-11; H.Q.Shi, W.-B.Tsai, S.Ferrari, B.D.Ratner, Nature 1999,398,593-597).Adopt this technology, comprise the trace that the multiple proteins of glycoprotein, immunoglobulin (Ig) is succeeded.This surface imprinted technology has good application prospect preparing aspect the biological diagnosis chip.Make template with protein, oxypolymerization by m-aminophenyl boric acid on the polystyrene material surface, Bossi etc. have successfully prepared the molecular engram film of the multiple proteins that comprises the glycoprotein horseradish peroxidase (referring to A.Bossi, S.A.Piletsky, E.V.Piletska, P.G.Righetti, A.P.F.Turner, Anal.Chem.2001,73,5281-5286), because this molecularly imprinted polymer stability is high, specificity good, be expected at the aspects such as biotechnology, bioanalysis and sensing and be widely used.Ramanavicius etc. on platinum black electrode surface by the electropolymerization legal system standby trace the polypyrrole of bovine leukemia virus glycoprotein gp51, but the method circulation ratio is poor (referring to A.Ramanaviciene, A.Ramanavicius, Biosens.Bioelectron.2004,20,1076-1082).Miyata etc. have reported the molecular imprinting gel that glycoprotein alpha-fetoprotein (AFP) is had to specific recognition and shrinking effect, it is function monomer that the method be take concanavalin A (Con A) and the anti-alpha-fetoprotein antibody (anti-AFP) of having modified two keys, by forming Con A-AFP-anti-AFP mixture, make blot gel shrink when AFP exists, thereby the existence to AFP is responded (referring to T.Miyata, M.Jige, T.Nakaminami, T.Uragami, Proc.Nat.Acad.Sci.USA 2006,103,1190-1193).But these current methods also exist that preparation method's step is many, the cycle is long and the shortcoming such as poor anti jamming capability, and what is more important, also do not have a kind of blanket method to can be used for the molecular imprinting of glycoprotein.
Summary of the invention
The present invention overcomes the deficiency of existing molecular imprinting, invents out a kind of rapid and convenient, is produced on a large scale, the artificial antibody's who prepares specified sugar albumen of universality molecular imprinting.
Technical solution of the present invention is as follows:
A kind of single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, it is to using double bond containing substituted benzene boric acid as function monomer, under alkaline condition, by the glycoprotein of appointment, be that template (microsphere) forms mixture with double bond containing substituted benzene boric acid (function monomer), with the linking agent containing the diene key, the light trigger of uv cure system and pore-creating agent mix, by UV-irradiation, cause between function monomer and linking agent, copolyreaction between linking agent and linking agent forms polymkeric substance, then extract with acidic solution the template molecule (glycoprotein of appointment) of removing in polymkeric substance, obtain containing with the phenylo boric acid site of glycoprotein glycosyl Reversible binding and with the template molecule shape molecularly imprinted polymer of complementary cavity mutually.
The above-mentioned single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, described specified sugar albumen can be horseradish peroxidase, ribonuclease B, alpha-fetoprotein or anti-alpha-fetoprotein antibody.
The above-mentioned single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, described substituted benzene boric acid can be to vinylphenylboronic acid, a vinylphenylboronic acid or an acrylamido phenylo boric acid.
The above-mentioned single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, described linking agent can be polyethyleneglycol diacrylate, ethylene glycol dimethacrylate and methylene diacrylamide.
The above-mentioned single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, the light trigger of described uv cure system can be dimethoxybenzoin, Diisopropyl azodicarboxylate, 1-hydroxycyclohexylphenylketone (Irgacure 184) or Benzoin ethyl ether.
The above-mentioned single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, described pore-creating agent can be polyoxyethylene glycol.
A kind of method for preparing the above-mentioned single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, it comprises the steps:
Step 1. presses 1 by the template molecule of specified sugar albumen and function monomer containing the substituted benzene boric acid of ethylene linkage: 100-1: 10000 mass ratio, under weak basic condition, mix, to form covalent complex, add light trigger, linking agent and pore-creating agent are by 1: 4-1: 8 volume ratio adds, linking agent and function monomer are 1 containing the mass ratio of the substituted benzene boric acid of ethylene linkage: 100-1: 1, mix, the solution obtained becomes pre-polymerization liquid, in finishing the substrate of two keys as dripped pre-polymerization liquid on sheet glass or filter paper, coating is evenly;
Step 2. will cover with the mask of specific pattern on above-mentioned substrate (as the specific pattern of embodiments of the invention is a plurality of round dots, pattern can be also other shape), under UV-light, expose, polymerization is brought out because of UV-irradiation in light transmission part, the not polymerization of part be blocked;
Step 3. removes mask, with solvent (as acetonitrile/water solution), cleans, and removes not polymeric part, obtains with the consistent molecular engram material of mask pattern;
Step 4. is swung and is washed the molecular engram material that step 3 obtains with acidic solution, removes the microsphere in polymkeric substance, obtains the single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, also is the single-minded artificial antibody in conjunction with specified sugar albumen.
The preparation method of the above-mentioned single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, described specified sugar albumen can be horseradish peroxidase, ribonuclease B, alpha-fetoprotein or anti-alpha-fetoprotein antibody; The described substituted benzene boric acid containing ethylene linkage can be to vinylphenylboronic acid, a vinylphenylboronic acid or an acrylamido phenylo boric acid; Described linking agent can be polyethyleneglycol diacrylate, ethylene glycol dimethacrylate and methylene diacrylamide; The light trigger of described uv cure system can be dimethoxybenzoin, Diisopropyl azodicarboxylate, 1-hydroxycyclohexylphenylketone or Benzoin ethyl ether; Described pore-creating agent can be polyoxyethylene glycol.
Above-mentioned preparation process has adopted current generally popular soft lithography, and preparation process is simple and convenient, does not need complex apparatus, is produced on a large scale.Polymerization time is usually in 1 minute, and removing of template molecule is also quite easy, usually only need to swing and wash 2 hours with acidic solution.
The technology of preparing of the above-mentioned single-minded molecularly imprinted polymer in conjunction with specified sugar albumen is a universality method, during the specific glycoprotein of trace, only needs the ratio of fine setting linking agent and pore-creating agent.Utilize this engram technology, successfully prepared ribonuclease B (RNase B, molecular weight 1.5 ten thousand, containing 1 glycosylation site, iso-electric point 8.9), horseradish peroxidase (HRP, molecular weight 4.4 ten thousand, containing 9 glycosylation sites, iso-electric point 3.0-9.0), alpha-fetoprotein (AFP, molecular weight 6.7 ten thousand, containing 11 glycosylation sites, iso-electric point 4.7,5.3) and anti-alpha-fetoprotein antibody (anti-AFP, molecular weight 150,000, containing 2 glycosylation sites, iso-electric point 7.5-7.9).These glycoprotein have good representativeness, and molecular weight distribution is across 1 order of magnitude, and the glycosyl number from 1 to 11 on molecule does not wait, and iso-electric point comprises acid to alkaline range.
The present invention utilizes covalent interaction reversible between the substituted boracic acid be subject to the pH regulation and control and glycosyl, using double bond containing substituted boracic acid as function monomer, under alkaline condition, glycoprotein template (microsphere) and function monomer are formed to mixture, add linking agent, initiator and pore-creating agent, by UV-irradiation, cause between function monomer and linking agent and the copolyreaction between linking agent and linking agent, use acidic solution to extract the template molecule of removing in polymkeric substance, obtain containing can with the phenylo boric acid site of glycoprotein glycosyl Reversible binding and with the template molecule shape molecularly imprinted polymer (principle is shown in Fig. 1) of complementary cavity mutually.This molecularly imprinted polymer divides the period of the day from 11 p.m. to 1 a.m at recognition template, due to the synergy that cavity is arranged, no longer needs common, desired alkaline condition while there is no trace, under neutral and solutions of weak acidity, can carry out single-minded interaction with template molecule; Therefore, under this condition, common sugar, as glucose, fructose and seminose etc., with the substituted boracic acid reactive force extremely a little less than, it is effectively restrained the interference of the recognition reaction of template molecule imprinted material.With other take the affine functional materials of boron that substituted boracic acid is aglucon (referring to L.B.Ren, Z.Liu, Y.C.Liu, P.Dou, H.-Y.Chen, Angew.Chem.Int.Ed.2009,48,6704-6707; L.B.Ren, Y.C.Liu, M.M.Dong, Z.Liu, J.Chromatogr.2009,1216,8421-8425; L.Liang, Z.Liu, Chem.Commun.2011,47,2255-2257; Y.C.Liu, L.B.Ren, Z.Liu, Chem.Commun.2011,47,5067-5069; H.Y.Li, Y.C.Liu, J.Liu, Z.Liu, Chem.Commun.2011,47,8169-8171) to compare, the molecularly imprinted polymer that this invention obtains has two significant advantages.The first, adopt common substituted benzene boric acid as aglucon, can carry out single-minded interaction with template molecule under neutral and slightly acidic pH condition, do not need to regulate pH, can direct applied sample wide ranges.This is very important for practical applications such as biomedical research and clinical diagnosises because the pH of physiologically sample commonly used be slightly acidic to weakly alkaline, for example, the pH of blood is 7.35-7.4, the pH of tears is 6.5-7.6.The second, this molecular engram material has extremely strong immunity from interference, and this analysis for complex sample especially blood sample is highly beneficial.
Adopt the means such as highly sensitive color reaction, chemiluminescence detection and mass spectrometric detection, the antigen glycoprotein that the single-minded molecularly imprinted polymer in conjunction with specified sugar albumen that trace is obtained captures detects, acquired results shows, the molecularly imprinted polymer prepared by the present invention has very outstanding specificity and immunity from interference.In addition, microheterogeneity is the notable feature of glycoprotein, even only have the glycoprotein of a glycosylation site, due to the difference of glycosyl structure, also has different sugared types.Utilize mass spectrum as testing tool, experimental result shows, the antibody material prepared by the present invention can intactly retain the microheterogeneity of microsphere, illustrates that this artificial antibody has good glycosyl difference identification ability.Utilize this character, can there be for the preparation of identification glycosyl structure the glycoprotein of fine difference in the present invention.
The single-minded molecularly imprinted polymer in conjunction with specified sugar albumen of the present invention has that specificity is high, immunity from interference is strong, reproducible, prepare the advantages such as simple, can directly in the complex biological systems such as serum, urine, detect determined antigen.This single-minded molecularly imprinted polymer in conjunction with specified sugar albumen can, with high-sensitive detection method as combinations such as chemoluminescences, can be used for the detection of glycoprotein in actual sample.
The accompanying drawing explanation
Fig. 1. glycoprotein molecule trace principle schematic of the present invention.
Fig. 2. photo (left side) and the Electronic Speculum figure (right side) of the imprinted polymer of the HRP that the glass of take is substrate and non-imprinted polymer (not containing HRP in pre-polymerization liquid).MIP, molecularly imprinted polymer; NIP, non-imprinted polymer.
The imprinted polymer of Fig. 3 .HRP and non-imprinted polymer under condition of different pH to the recognition capability of microsphere.Upper, NIP (non-imprinted polymer); Under, MIP (molecularly imprinted polymer).NIP is not colour developing in pH=6.0, just catches HRP colour developing in pH=8.5.MIP just can identify HRP colour developing in pH=6.0.
Fig. 4 .HRP imprinted polymer in omplicated solution to the single-minded identification of HRP and open hole detection.In figure the 3rd, 4 row manifest blueness, show the HRP imprinted polymer in omplicated solution, can identify HRP and the colour developing.
Fig. 5. imprinted polymer photo and the open hole detection of the HRP that the filter paper after the BABEA of take processes is substrate.Upper, the photo of blank polymkeric substance, under, capture HRP and the photo of polymkeric substance.After colour developing in figure I, II iris out existing yellow, show to take that the imprinted polymer of the HRP that filter paper is substrate can identify equally HRP and develop the color under solutions of weak acidity.
Fig. 6. the ribonuclease B imprinted polymer is to the matrix-assisted laser desorption ionization of the target substance that in the solution of qiagen rnase enzyme B, ribonuclease A and myohaemoglobin, single-minded crawl obtains-time-of-fight mass spectrometry figure.A, the mass spectrum of ribonuclease T. B, ribonuclease A and myohaemoglobin mixing solutions (mass ratio=1: 1: 1) direct analysis; B, the mass spectrum of ribonuclease T. B direct analysis; C, the mass spectrum of ribonuclease T. B, ribonuclease A and myohaemoglobin mixing solutions (mass ratio=1: 1: 1) elutant after molecularly imprinted polymer captures wash-out.
The molecularly imprinted polymer photo of Fig. 7 .AFP (on) and AFP in solution is captured after chemiluminescence signal and the relation of AFP concentration in solution (under).
Fig. 8. crawl and the chemiluminescence detection of the molecularly imprinted polymer of AntiAFP antibody to AntiAFP antibody in human serum.
Fig. 9. the Photomicrograph of the imprinted polymer (B) of the HRP that filter paper (A) and the filter paper of take are substrate.
Embodiment
Embodiment 1: the preparation of horseradish peroxidase (HRP) imprinted polymer
The preparation process of HRP molecularly imprinted polymer is as follows:
1, prepare pre-polymerization liquid, 0.005g is dissolved in to the Macrogol 200 (PEG200 of 400 μ l to the dimethoxybenzoin of vinylphenylboronic acid and 0.001g, molecular-weight average 200), the polyethyleneglycol diacrylate of 100 μ l (PEGMA), the HRP solution 5 μ l that add 10mg/ml after phosphate buffer soln adjustment pH value to 8.5, vortex 2 minutes (specifying glycoprotein h RP and function monomer in the present embodiment is 1: 100 to the mass ratio of vinylphenylboronic acid).
2, drip 100 μ l pre-polymerization liquid on the derivative glass substrate that two keys are arranged, the mask plate that attaching contains ad hoc structure, UV (365nm) 40s that exposes.Remove mask plate, polymkeric substance is soaked in to (the ethanol volume ratio is 40%) vibration 30min in the mixing solutions of ethanol/water, again polymkeric substance is immersed in: in acetonitrile/10M phosphoric acid solution (volume ratio 3: 7) 2 hours, finally with clear water, clean 30 minutes, obtain the HRP molecularly imprinted polymer.
In addition, adopt with formula identical in 1 but do not contain HRP in pre-polymerization liquid, adopting and prepare non-imprinted polymer with step identical in 2.
The photo of HRP molecularly imprinted polymer and non-imprinted polymer and Electronic Speculum figure are shown in Fig. 2.The Electronic Speculum result shows that the molecularly imprinted polymer of gained is vesicular structure.This vesicular structure is conducive to the mass transfer of microsphere in imprinted polymer.
Embodiment 2:HRP imprinted polymer to the identification of HRP and with the comparison of non-imprinted polymer
With HRP imprinted polymer and non-imprinted polymer, HRP is identified respectively, and adopt 3,3 ', 5,5 '-tetramethyl benzidine (TMB) development process carries out open hole detection:
1, preparation TMB nitrite ion, sodium-acetate 0.136g, citric acid 0.016g, 30% hydrogen peroxide 3 μ l, TMB 1.5mg, distilled water adds to 5ml.
2, list at A, B, the C of non-imprinted polymer (A on Fig. 3) water (pH 6.0), the HRP aqueous solution (the 10 μ g/ml that drip 5 μ l respectively, pH 6.0) and HRP phosphate solution (10 μ g/ml, pH 8.5), rinse non-imprinted polymer 1 minute with the mixing solutions of acetonitrile/water (long-pending than be 30: 70) after 2 minutes, dry up polymkeric substance, drip 5 μ l TMB nitrite ions on each point, lucifuge reaction 10 minutes, digital camera record (on Fig. 3) for the polymkeric substance after colour developing.
2, list at A, B, the C of HRP imprinted polymer (under Fig. 3) water (pH6.0), the HRP aqueous solution (the 10 μ g/ml that drip 5 μ l respectively, pH 6.0) and the HRP aqueous solution (5 μ g/ml, pH 6.0), mixing solutions (volume ratio 30: 70) by acetonitrile/water after 2 minutes rinses imprinted polymer 1 minute, dry up polymkeric substance, drip 5 μ l TMB nitrite ions on each point, lucifuge reaction 10 minutes, digital camera record (under Fig. 3) for the polymkeric substance after colour developing.
TMB colour developing photo shows, non-imprinted polymer need to could be identified glycoprotein h RP at alkaline condition (pH=8.5), and the HRP imprinted polymer can be identified HRP in solutions of weak acidity (pH=6.0) time.Recognition capability under this solutions of weak acidity is highly beneficial for the analysis of the authentic samples such as blood.
The immunity from interference of embodiment 3 molecularly imprinted polymers
Adopt preparation process in the same manner as in Example 1, in formula, phosphate buffer soln is adjusted pH value to 8, preparation HRP imprinted polymer.Utilize TMB development process bore hole observation, the HRP imprinted polymer in complex system to the identification of HRP:
1, preparation omplicated solution: 1) 50 μ gHRP, 1mg fructose, 1mg oralbumin, 1mg human hemoglobin, 1mg bovine serum albumin are dissolved in the 1ml human serum; 2) 10 μ gHRP, 1mg fructose, 1mg oralbumin, 1mg human hemoglobin, 1mg bovine serum albumin are dissolved in the 1ml human serum; 3) 5 μ gHRP, 1mg fructose, 1mg oralbumin, 1mg human hemoglobin, 1mg bovine serum albumin are dissolved in the 1ml human serum.
2, list at 1,2,3,4,5 of HRP imprinted polymer (Fig. 4 A) water, serum, the solution 1 that drips 5 μ l respectively), solution 2), solution 3), mixing solutions (volume ratio 30: 70) with ethanol/water after 2 minutes rinses the HRP imprinted polymer 1 minute, dry up polymkeric substance, drip 5 μ l TMB nitrite ions on each point, lucifuge reaction 10 minutes, digital camera record (Fig. 4 A and Fig. 4 B) for the polymkeric substance after colour developing.
TMB colour developing photo shows, the HRP imprinted polymer can be identified HRP in complex system, has good immunity from interference.This immunity from interference is highly beneficial for the analysis of the authentic samples such as blood.
Embodiment 4 be take the imprinted polymer of the HRP that filter paper that BABEA (bisphenol A epoxy acrylate) processed is substrate and to identification and the detection of HRP
The preparation process of the HRP molecularly imprinted polymer that the filter paper of take is substrate is as follows:
1, preparation epoxy acrylate pre-polymerization liquid, bisphenol A epoxy acrylate 100mg, Benzoin ethyl ether 1mg, ultrasonic dissolution is in 200 μ l ethanol.
2, at filter paper surface spin coating one deck epoxy acrylate pre-polymerization liquid, 60 ℃ of thermal treatment 30 minutes, attach the mask plate that contains microstructure, UV (365nm) 20s that exposes.Remove mask plate, filter paper is immersed in to 30s in acetonitrile, process 30 minutes under 90 ℃, clear water soaks 10 minutes post-dryings, obtains the filter paper (Fig. 5 A) that contains microstructure.
3, prepare pre-polymerization liquid, by 0.001g acrylamido phenylo boric acid and 0.005g
diisopropyl azodicarboxylate(AIBN) be dissolved in the Macrogol 200 (PEG200 of 400 μ l, molecular-weight average 200), the ethylene glycol dimethacrylate of 100 μ l (EDMA), the HRP solution 1 μ l that adds 10mg/ml after phosphate buffer soln adjustment pH value to 8.5, vortex 2 minutes (specifying the mass ratio of glycoprotein h RP and function monomer acrylamido phenylo boric acid in the present embodiment is 1: 10000).
4, the pre-polymerization liquid of dropping 1 μ l in each point of filter paper microstructure, UV (365nm) 40 s that expose.Filter paper is soaked in to (the acetonitrile volume ratio is 30%) vibration 30min in the mixing solutions of acetonitrile/water, again polymkeric substance is immersed in to the acetonitrile of 30: 70: in the 10M phosphoric acid solution 2 hours, finally with clear water, clean 30 minutes the HRP molecularly imprinted polymer that to obtain take filter paper be substrate.
The HRP imprinted polymer that the filter paper that the BABEA of take processed is substrate is identified HRP, adopts TMB development process open hole detection:
Drip respectively the HRP aqueous solution (the 100 μ g/ml of 1 μ l on white point in I, II, III, IV circle, pH 6.0), the HRP aqueous solution (10 μ g/ml, pH 6.0), the HRP aqueous solution (1 μ g/ml, pH 6.0), water, the mixing solutions of ethanol/water after 2 minutes (the ethanol volume ratio is 30%) rinses the HRP imprinted polymer 1 minute, dry up polymkeric substance, drip 2 μ l TMB nitrite ions on each point, lucifuge reaction 10 minutes, drip the sulfuric acid color development stopping reaction of 2 μ l 0.1M on each point.Digital camera record (Fig. 5 B) for imprinted polymer after colour developing.
TMB colour developing photo shows, this molecularly imprinted polymer can be identified HRP under solutions of weak acidity.
The preparation of the molecularly imprinted polymer of embodiment 5 ribonuclease Bs reaches the identification to ribonuclease B different sugar type
Adopt preparation process in the same manner as in Example 1, the HRP in formula changes ribonuclease B into, and the initiator dimethoxybenzoin changes the 1-hydroxycyclohexylphenylketone into, prepares the ribonuclease B molecularly imprinted polymer.Adopt matrix-assisted laser desorption ionization (MALDI)-time flight (TOF) mass spectroscopy, the ribonuclease B that this imprinted polymer is grabbed sugar type is detected.
1, prepare solution to be measured: ribonuclease B, ribonuclease A, each 1mg of myohaemoglobin are dissolved in the water of 1ml.
2, drip the solution to be measured of 10 μ l on the ribonuclease B molecularly imprinted polymer, mixing solutions (volume ratio 20: 80) minute by the 1ml acetonitrile/water after 30 minutes rinses imprinted polymer 3 times, dry up polymkeric substance, acetonitrile with 10 μ l: the ribonuclease B that 1M acetic acid mixed solution (volume ratio 30: 70) desorb grabs, the micropipette rifle is drawn and is transferred to Eppendorf tube.Get the above-mentioned stripping liquid point plate of 0.5 μ l, after dry, get 0.5 μ l styracin solution (10mg/ml, 50% acetonitrile, 0.1% trifluoroacetic acid) as matrix point on sample.After it is dry, carry out mass spectroscopy.
Mass spectrometry results is shown in Fig. 6.The mass spectrum result shows, the ribonuclease B molecularly imprinted polymer can be in mixing solutions single-minded crawl ribonuclease B, and intactly retained the microheterogeneity of this glycoprotein.Illustrate that molecular imprinting proposed by the invention has good glycosyl difference identification ability.Utilize this character, can there be for the preparation of identification glycosyl structure the glycoprotein of fine difference in the present invention, as for identifying endogenous and ectogenic glycoprotein.In addition, this result shows, the present invention can be used for the mixture of a plurality of glycoprotein of trace.The preparation of embodiment 6 alpha-fetoprotein molecularly imprinted polymers reaches and the detection of chemiluminescence detection coupling to alpha-fetoprotein
Adopt preparation process in the same manner as in Example 1, HRP in formula changes alpha-fetoprotein into, 400 μ l Macrogol 200s are replaced with 500 μ l Liquid Macrogols, the polyethyleneglycol diacrylate of 100 μ l (PEGMA) is replaced with the methylene diacrylamide of 0.05g, preparation alpha-fetoprotein molecularly imprinted polymer.
Adopt chemiluminescent method to detect the identification of alpha-fetoprotein imprinted polymer to Serum Alpha Fetoprotein:
1, preparation luminol,3-aminophthalic acid cyclic hydrazide mixed solution:
A)
luminol177mg is dissolved in the NaOH of 100ml 0.1M, keeps in Dark Place;
B)
to iodophenol(PIP) 110mg is dissolved in the dimethyl sulfoxide (DMSO) (DMSO) of 5ml, and distilled water diluting, to 50ml, keeps in Dark Place;
C) get respectively 50 μ l solution A and solution B before chemiluminescence detection and mix, with the phosphate buffer soln of pH 8.5, be diluted to 1000 μ l, add 3% hydrogen peroxide 3.4 μ l.
2, prepare solution to be measured: 1) the 10ng alpha-fetoprotein is dissolved in the 1ml human serum; 2) the 20ng alpha-fetoprotein is dissolved in the 1ml human serum; 3) the 30ng alpha-fetoprotein is dissolved in the 1ml human serum.
3, list at 1,2,3,4,5 of alpha-fetoprotein imprinted polymer (Fig. 7 A) water, serum, the solution 1 that drips 2 μ l respectively), solution 2), solution 3), rinse the alpha-fetoprotein imprinted polymer 1 minute with ethanol/water mixing solutions (volume ratio 30: 70) after 2 minutes, drip the anti-alpha-fetoprotein antibody of the 0.1mg/ml HRP mark of 2 μ l on each point, rinse the alpha-fetoprotein imprinted polymer 1 minute with ethanol/water mixing solutions (volume ratio 30: 70) after 2 minutes, dry up polymkeric substance.
4, drip 2 μ l luminol,3-aminophthalic acid cyclic hydrazide mixed solutions on each is selected, detect and light intensity with chemiluminescence detector immediately, 3 repeated experiments, obtain the histogram in Fig. 7 B.
Experimental result shows, the alpha-fetoprotein imprinted polymer can be identified the micro-alpha-fetoprotein in human serum, illustrates that this artificial antibody has very outstanding specific recognition ability and immunity from interference.
The preparation of the molecularly imprinted polymer of embodiment 7 anti-alpha-fetoprotein antibodies reaches and the detection of chemiluminescence detection coupling to anti-alpha-fetoprotein antibody
Adopt preparation process in the same manner as in Example 1, the HRP in formula changes mouse-anti human a-fetoprotein antibody into, and 400 μ l Macrogol 200s are replaced with 800 μ l Macrogol 200s, prepares mouse-anti human a-fetoprotein antibody molecule imprinted polymer.
Adopt chemiluminescent method to detect the identification of mouse-anti human a-fetoprotein antibody imprinted polymer to mouse-anti human a-fetoprotein antibody in serum:
1, prepare solution to be measured: 1) the mouse-anti human a-fetoprotein antibody of 1ng HRP mark is dissolved in the 1ml human serum; 2) the mouse-anti human a-fetoprotein antibody of 5ng HRP mark is dissolved in the 1ml human serum; 3) the mouse-anti human a-fetoprotein antibody of 10ng HRP mark is dissolved in the 1ml human serum; 4) the mouse-anti human a-fetoprotein antibody of 100ngHRP mark is dissolved in the 1ml human serum.
2, list at 1,2,3,4,5 of mouse-anti human a-fetoprotein antibody imprinted polymer water, the solution 1 that drips 2 μ l respectively), solution 2), solution 3), solution 4), rinse mouse-anti human a-fetoprotein antibody imprinted polymer 1 minute with ethanol/water mixing solutions (volume ratio 30: 70) after 2 minutes, dry up polymkeric substance.
3, drip 2 μ l luminol,3-aminophthalic acid cyclic hydrazide mixed solutions on each is selected, detect and light intensity with chemiluminescence detector immediately, obtain result in Fig. 8.
Experimental result shows, the micro-mouse-anti human a-fetoprotein antibody of the imprinted polymer of mouse-anti human a-fetoprotein antibody in can the direct-detection human serum.This detection method has excellent single-minded selectivity and detection sensitivity, detects lower limit and has reached 1ng/ml.
Embodiment 8: the preparation of the imprinted polymer of the HRP that the filter paper of take is substrate
Adopt preparation process in the same manner as in Example 1, in formula, the polyethyleneglycol diacrylate of 100 μ l (PEGMA) is replaced with the methylene diacrylamide of 0.05g, 0.001g vinylphenylboronic acid is replaced with to vinylphenylboronic acid between 0.1g, directly the circular HRP imprinted polymer of preparation on filter paper.The microphotograph of the HRP molecularly imprinted polymer that the filter paper of take is substrate is shown in Fig. 9 B.Fig. 9 illustrates can directly prepare on filter paper by molecularly imprinted polymer, and filter paper can be without pre-treatment.
Claims (5)
1. the single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, it is characterized in that: it is prepared by following method:
Step 1. is pressed the mass ratio of 1:100-1:10000 by the template molecule of specified sugar albumen and function monomer containing the substituted benzene boric acid of ethylene linkage, under weak basic condition, mix, to form covalent complex, add light trigger, linking agent and pore-creating agent add by the volume ratio of 1:4-1:8, linking agent and function monomer are 1:100-1:1 containing the mass ratio of the substituted benzene boric acid of ethylene linkage, mix, the solution obtained becomes pre-polymerization liquid, in finishing drip pre-polymerization liquid on the substrate of two keys, coating is evenly;
Step 2. will cover with the mask of specific pattern on above-mentioned substrate, under UV-light, expose, and polymerization is brought out because of UV-irradiation in light transmission part, the not polymerization of part be blocked;
Step 3. removes mask, uses solvent cleaning, removes not polymeric part, obtains with the consistent molecular engram material of mask pattern;
Step 4. is swung and is washed the molecular engram material that step 3 obtains with acidic solution, removes the microsphere in polymkeric substance, obtains the single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, also is the single-minded artificial antibody in conjunction with specified sugar albumen; The described substituted benzene boric acid containing ethylene linkage is to vinylphenylboronic acid, a vinylphenylboronic acid or an acrylamido phenylo boric acid; Described specified sugar albumen is horseradish peroxidase, ribonuclease B, alpha-fetoprotein or anti-alpha-fetoprotein antibody.
2. the single-minded molecularly imprinted polymer in conjunction with specified sugar albumen according to claim 1 is characterized in that: the described linking agent containing the diene key is polyethyleneglycol diacrylate, ethylene glycol dimethacrylate and methylene diacrylamide.
3. the single-minded molecularly imprinted polymer in conjunction with specified sugar albumen according to claim 1, it is characterized in that: described pore-creating agent is polyoxyethylene glycol.
4. a method for preparing the single-minded molecularly imprinted polymer in conjunction with specified sugar albumen claimed in claim 1, is characterized in that it comprises the steps:
Step 1. is pressed the mass ratio of 1:100-1:10000 by the template molecule of specified sugar albumen and function monomer containing the substituted benzene boric acid of ethylene linkage, under weak basic condition, mix, to form covalent complex, add light trigger, linking agent and pore-creating agent add by the volume ratio of 1:4-1:8, linking agent and function monomer are 1:100-1:1 containing the mass ratio of the substituted benzene boric acid of ethylene linkage, mix, the solution obtained becomes pre-polymerization liquid, in finishing drip pre-polymerization liquid on the substrate of two keys, coating is evenly;
Step 2. will cover with the mask of specific pattern on above-mentioned substrate, under UV-light, expose, and polymerization is brought out because of UV-irradiation in light transmission part, the not polymerization of part be blocked;
Step 3. removes mask, uses solvent cleaning, removes not polymeric part, obtains with the consistent molecular engram material of mask pattern;
Step 4. is swung and is washed the molecular engram material that step 3 obtains with acidic solution, removes the microsphere in polymkeric substance, obtains the single-minded molecularly imprinted polymer in conjunction with specified sugar albumen, also is the single-minded artificial antibody in conjunction with specified sugar albumen.
5. the single-minded molecularly imprinted polymer in conjunction with specified sugar albumen claimed in claim 1 application in specific glycoprotein in detecting sample.
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