完整糖蛋白为准模板的有机相中聚糖的硼亲和可控定向表面印迹
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摘要
糖蛋白具有十分重要的生理功能和临床价值,对于糖蛋白的特异性识别具有重要的科学意义和应用价值.分子印迹技术是制备具有特异性分子识别的功能聚合物的重要方法,已经用于糖蛋白的识别.但是,在有机相中以蛋白质为模板的印迹方法容易导致模板蛋白质的变性,不易获得良好的分子识别性能,而以糖链为模板的印迹方法需要比较复杂的糖链制备过程.本文提出了直接以完整糖蛋白为准模板,在乙醇相中可控地印迹其糖链,用于制备能识别糖蛋白的分子印迹聚合物的方法.本文以碱性磷酸酶为目标蛋白,以硼亲和磁性纳米颗粒作为基础材料,通过硼亲和作用固定目标糖蛋白,利用原硅酸四乙酯-无水乙醇印迹体系,根据糖蛋白糖链的结构,无须优化,直接控制印迹时间,仅印迹糖链部分,得到磁性分子印迹纳米颗粒.所得磁性分子印迹纳米颗粒对目标糖蛋白具有良好的特异性识别能力,即使在复杂的实际样品中,也可以保持这种特性.本方法直接使用完整糖蛋白作为模板,无须通过酶切等烦琐步骤获得模板,方法效率高.本文方法在一定程度上克服了传统印迹方法中无法在有机相中印迹完整蛋白的局限,拓展了分子印迹技术的应用范围,在亲和分离与疾病诊断等重要领域中具有重要的应用潜力.
Glycoproteins are a large family of proteins which have great biological and clinical importance including molecular recognition, signal transduction, cell adhesion and immune response. Many glycoproteins are important biomarkers for disease diagnosis and as therapeutic targets. However, glycoproteins are present in very low abundance in the body and coexist alongside interfering species present at much higher abundance. So, specific recognition of glycoproteins is of great significance. Although antibodies are commonly used to solve this issue, they are associated with some apparent drawbacks, such as, difficult to prepare, poor storage stability, high cost and requiring harsh conditions for target release. Therefore, novel alternatives are highly desirable. Molecularly imprinted polymers(MIPs), which are synthesized through polymerization in the presence of a template, exhibit specific binding towards the template molecules. A series of molecular imprinting approaches have been developed to produce glycoprotein-specific MIPs. Boronic acids can covalently bind cis-diol groups of cis-diol-containing compounds like glycoproteins and glycopeptides, to form stable fiveor six-membered cyclic esters at relatively high pH values. The esters dissociate reversibly when the pH of the environment is made acidic. In recent years, our group has developed a series of boronate affinity molecular imprinting techniques for specific recognition of glycoproteins, especially boronate affinity controllable-oriented surface imprinting method. Based on silane hydrolysis in alcohol phase, this method permits getting the desired thickness of the imprinting layer according to the size of the template molecules. The method allows for easy and efficient preparation of MIPs specific to glycoproteins, glycans, and monosaccharides, enabling promising applications. Recently, we can even achieve precision imprinting of glycopeptides for the facile preparation of glycan-specific MIPs. However, the molecular imprinting of intact proteins in organic phase often results in the denature of the template proteins while imprinting approaches using glycans as the templates require relatively tedious glycan preparation procedure. Herein, we developed a new approach for imprinting in organic phase using an intact target glycoprotein as a semi-template. This method allowed for the imprinting of only the glycans on the target glycoprotein, without worrying about possible denature of the target protein in organic phase. Alkaline phosphatase(ALP), which is a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was used as the target. ALP-specific molecularly imprinted magnetic nanoparticles(MI-MNPs) were prepared using this method. By using boronic acid-functionalized MNPs as the nanocores, the target molecules were immobilized onto the nanocores via boronate affinity. Then, a thin-layer of silica was controllably formed to cover the templates to an appropriate thickness through polycondensation of tetraethyl orthosilicate(TEOS) in ethanol. After that, the templates are removed facilely by washing with an acidic solution to disrupt the boronate affinity interaction. The prepared MIPs exhibited good specificity towards ALP, and this characteristic was preserved even in a complicated real sample. The developed approach exhibited high imprinting efficiency. It overcame the limitations of previous imprinting approaches. Also, the MI-MIPs synthesized in this work were simple to prepare, stable, cost-efficient and easy to collect. Besides, the target release just needed gentle conditions. It can be a promising means to produce glycoprotein-specific MIPs for important applications such as affinity separation and disease diagnosis.
Glycoproteins are a large family of proteins which have great biological and clinical importance including molecular recognition, signal transduction, cell adhesion and immune response. Many glycoproteins are important biomarkers for disease diagnosis and as therapeutic targets. However, glycoproteins are present in very low abundance in the body and coexist alongside interfering species present at much higher abundance. So, specific recognition of glycoproteins is of great significance. Although antibodies are commonly used to solve this issue, they are associated with some apparent drawbacks, such as, difficult to prepare, poor storage stability, high cost and requiring harsh conditions for target release. Therefore, novel alternatives are highly desirable. Molecularly imprinted polymers(MIPs), which are synthesized through polymerization in the presence of a template, exhibit specific binding towards the template molecules. A series of molecular imprinting approaches have been developed to produce glycoprotein-specific MIPs. Boronic acids can covalently bind cis-diol groups of cis-diol-containing compounds like glycoproteins and glycopeptides, to form stable fiveor six-membered cyclic esters at relatively high pH values. The esters dissociate reversibly when the pH of the environment is made acidic. In recent years, our group has developed a series of boronate affinity molecular imprinting techniques for specific recognition of glycoproteins, especially boronate affinity controllable-oriented surface imprinting method. Based on silane hydrolysis in alcohol phase, this method permits getting the desired thickness of the imprinting layer according to the size of the template molecules. The method allows for easy and efficient preparation of MIPs specific to glycoproteins, glycans, and monosaccharides, enabling promising applications. Recently, we can even achieve precision imprinting of glycopeptides for the facile preparation of glycan-specific MIPs. However, the molecular imprinting of intact proteins in organic phase often results in the denature of the template proteins while imprinting approaches using glycans as the templates require relatively tedious glycan preparation procedure. Herein, we developed a new approach for imprinting in organic phase using an intact target glycoprotein as a semi-template. This method allowed for the imprinting of only the glycans on the target glycoprotein, without worrying about possible denature of the target protein in organic phase. Alkaline phosphatase(ALP), which is a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was used as the target. ALP-specific molecularly imprinted magnetic nanoparticles(MI-MNPs) were prepared using this method. By using boronic acid-functionalized MNPs as the nanocores, the target molecules were immobilized onto the nanocores via boronate affinity. Then, a thin-layer of silica was controllably formed to cover the templates to an appropriate thickness through polycondensation of tetraethyl orthosilicate(TEOS) in ethanol. After that, the templates are removed facilely by washing with an acidic solution to disrupt the boronate affinity interaction. The prepared MIPs exhibited good specificity towards ALP, and this characteristic was preserved even in a complicated real sample. The developed approach exhibited high imprinting efficiency. It overcame the limitations of previous imprinting approaches. Also, the MI-MIPs synthesized in this work were simple to prepare, stable, cost-efficient and easy to collect. Besides, the target release just needed gentle conditions. It can be a promising means to produce glycoprotein-specific MIPs for important applications such as affinity separation and disease diagnosis.
引文
1 Wu L,Qu X G.Cancer biomarker detection:Recent achievements and challenges.Chem Soc Rev,2015,44:2963-2997
2 Dwek R A.Glycobiology:Toward understanding the function of sugars.Chem Rev,1996,96:683-720
3 Ohtsubo K,Marth J D.Glycosylation in cellular mechanisms of health and disease.Cell,2006,126:855-867
4 Chandler K,Goldman R.Glycoprotein disease markers and single protein-omics.Mol Cell Proteomics,2013,12:836-845
5 Rudd P M,Wormald M R,Dwek R A.Glycosylation and the immune system.J Protein Chem,1998,17:519-519
6 Morelle W,Michalski J C.Analysis of protein glycosylation by mass spectrometry.Nat Protoc,2007,2:1585-1602
7 Ludwig J A,Weinstein J N.Biomarkers in cancer staging,prognosis and treatment selection.Nat Rev Cancer,2005,5:845-856
8 Tian Y,Zhang H.Characterization of disease-associated n-linked glycoproteins.Proteomics,2013,13:504-511
9 Bordon Y.Pattern recognition receptors picking lox to find antibodies.Nat Rev Immunol,2014,14:716-716
10 Baker M.Blame it on the antibodies.Nature,2015,521:274-276
11 Baker M.Antibody anarchy:A call to order.Nature,2015,527:545-551
12 Wulff G,Sarhan A.The use of polymers with enzyme-analogous structures for the resolution of racemates.Angew Chem Int Edit,1972,11:341-346
13 Vlatakis G,Andersson L I,Muller R,et al.Drug assay using antibody mimics made by molecular imprinting.Nature,1993,361:645-647
14 Wulff G.Molecular imprinting in cross-linked materials with the aid of molecular templates-A way towards artificial antibodies.Angew Chem Int Edit,1995,34:1812-1832
15 Shi H Q,Tsai W B,Garrison M D,et al.Template-imprinted nanostructured surfaces for protein recognition.Nature,1999,398:593-597
16 Nishino H,Huang C S,Shea K J.Selective protein capture by epitope imprinting.Angew Chem Int Edit,2006,45:2392-2396
17 Haupt K,Mosbach K.Molecularly imprinted polymers and their use in biomimetic sensors.Chem Rev,2000,100:2495-2504
18 Hart B R,And D J R,Shea K J.Discrimination between enantiomers of structurally related molecules:Separation of benzodiazepines by molecularly imprinted polymers.J Am Chem Soc,2000,122:460-465
19 Nematollahzadeh A,Sun W,Aureliano C S A,et al.High-capacity hierarchically imprinted polymer beads for protein recognition and capture.Angew Chem Int Edit,2011,50:495-498
20 Kugimiya A,Takeuchi T.Surface plasmon resonance sensor using molecularly imprinted polymer for detection of sialic acid.Biosens Bioelectron,2001,16:1059-1062
21 Fuchs Y,Soppera O,Mayes A G,et al.Holographic molecularly imprinted polymers for label-free chemical sensing.Adv Mater,2013,25:566-570
22 Kunath S,Panagiotopoulou M,Maximilien J,et al.Cell and tissue imaging with molecularly imprinted polymers as plastic antibody mimics.Adv Healthc Mater,2015,4:1322-1326
23 Yin D Y,Wang S S,He Y J,et al.Surface-enhanced Raman scattering imaging of cancer cells and tissues via sialic acid-imprinted nanotags.Chem Commun,2015,51:17696-17699
24 Wang S S,Wen Y R,Wang Y J,et al.Pattern recognition of cells via multiplexed imaging with monosaccharide-imprinted quantum dots.Anal Chem,2017,89:5646-5652
25 Liu J,Yin D Y,Wang S S,et al.Probing low-copy-number proteins in a single living cell.Angew Chem Int Edit,2016,55:13215-13218
26 Sellergren B,Allender C J.Molecularly imprinted polymers:A bridge to advanced drug delivery.Adv Drug Deliver Rev,2005,57:1733-1741
27 Cecchini A,Raffa V,Canfarotta F,et al.In vivo recognition of human vascular endothelial growth factor by molecularly imprinted polymers.Nano Lett,2017,17:2307-2312
28 Hoshino Y,Koide H,Urakami T,et al.Recognition,neutralization,and clearance of target peptides in the bloodstream of living mice by molecularly imprinted polymer nanoparticles:A plastic antibody.J Am Chem Soc,2010,132:6644-6645
29 Li S W,Yang K G,Deng N,et al.Thermoresponsive epitope surface-imprinted nanoparticles for specific capture and release of target protein from human plasma.ACS Appl Mater Inter,2016,8:5747-5751
30 Bossi A,Piletsky S A,Piletska E V,et al.Surface-grafted molecularly imprinted polymers for protein recognition.Anal Chem,2001,73:5281-5286
31 James T D,Sandanayake K,Shinkai S.Saccharide sensing with molecular receptors based on boronic acid.Angew Chem Int Edit,1996,35:1910-1922
32 Li D J,Chen Y,Liu Z.Boronate affinity materials for separation and molecular recognition:Structure,properties and applications.Chem Soc Rev,2015,44:8097-8123
33 Liu Z,He H.Synthesis and applications of boronate affinity materials:From class selectivity to biomimetic specificity.Accounts Chem Res,2017,50:2185-2193
34 Li L,Lu Y,Bie Z J,et al.Photolithographic boronate affinity molecular imprinting:A general and facile approach for glycoprotein imprinting.Angew Chem Int Edit,2013,52:7451-7454
35 Ye J,Chen Y,Liu Z.A boronate affinity sandwich assay:An appealing alternative to immunoassays for the determination of glycoproteins.Angew Chem Int Edit,2014,53:10386-10389
36 Wang S S,Ye J,Bie Z J,et al.Affinity-tunable specific recognition of glycoproteins via boronate affinity-based controllable oriented surface imprinting.Chem Sci,2014,5:1135-1140
37 Bie Z J,Chen Y,Ye J,et al.Boronate-affinity glycan-oriented surface imprinting:A new strategy to mimic lectins for the recognition of an intact glycoprotein and its characteristic fragments.Angew Chem Int Edit,2015,54:10211-10215
38 Xing R R,Wang S S,Bie Z J,et al.Preparation of molecularly imprinted polymers specific to glycoproteins,glycans and monosaccharides via boronate affinity controllable-oriented surface imprinting.Nat Protoc,2017,12:964-987
39 Bie Z J,Xing R R,He X P,et al.Precision imprinting of glycopeptides for facile preparation of glycan-specific artificial antibodies.Anal Chem,2018,90:9845-9852
40 Forneris F,Mattevi A.Enzymes without borders:Mobilizing substrates,delivering products.Science,2008,321:213-216
41 Wang L Y,Bao J,Wang L,et al.One-pot synthesis and bioapplication of amine-functionalized magnetite nanoparticles and hollow nanospheres.Chem-Eur J,2006,12:6341-6347
42 Bublitz R,Hoppe H,Cumme G A,et al.Structural study on the carbohydrate moiety of calf intestinal alkaline phosphatase.J Mass Spectrom,2001,36:960-972
2 Dwek R A.Glycobiology:Toward understanding the function of sugars.Chem Rev,1996,96:683-720
3 Ohtsubo K,Marth J D.Glycosylation in cellular mechanisms of health and disease.Cell,2006,126:855-867
4 Chandler K,Goldman R.Glycoprotein disease markers and single protein-omics.Mol Cell Proteomics,2013,12:836-845
5 Rudd P M,Wormald M R,Dwek R A.Glycosylation and the immune system.J Protein Chem,1998,17:519-519
6 Morelle W,Michalski J C.Analysis of protein glycosylation by mass spectrometry.Nat Protoc,2007,2:1585-1602
7 Ludwig J A,Weinstein J N.Biomarkers in cancer staging,prognosis and treatment selection.Nat Rev Cancer,2005,5:845-856
8 Tian Y,Zhang H.Characterization of disease-associated n-linked glycoproteins.Proteomics,2013,13:504-511
9 Bordon Y.Pattern recognition receptors picking lox to find antibodies.Nat Rev Immunol,2014,14:716-716
10 Baker M.Blame it on the antibodies.Nature,2015,521:274-276
11 Baker M.Antibody anarchy:A call to order.Nature,2015,527:545-551
12 Wulff G,Sarhan A.The use of polymers with enzyme-analogous structures for the resolution of racemates.Angew Chem Int Edit,1972,11:341-346
13 Vlatakis G,Andersson L I,Muller R,et al.Drug assay using antibody mimics made by molecular imprinting.Nature,1993,361:645-647
14 Wulff G.Molecular imprinting in cross-linked materials with the aid of molecular templates-A way towards artificial antibodies.Angew Chem Int Edit,1995,34:1812-1832
15 Shi H Q,Tsai W B,Garrison M D,et al.Template-imprinted nanostructured surfaces for protein recognition.Nature,1999,398:593-597
16 Nishino H,Huang C S,Shea K J.Selective protein capture by epitope imprinting.Angew Chem Int Edit,2006,45:2392-2396
17 Haupt K,Mosbach K.Molecularly imprinted polymers and their use in biomimetic sensors.Chem Rev,2000,100:2495-2504
18 Hart B R,And D J R,Shea K J.Discrimination between enantiomers of structurally related molecules:Separation of benzodiazepines by molecularly imprinted polymers.J Am Chem Soc,2000,122:460-465
19 Nematollahzadeh A,Sun W,Aureliano C S A,et al.High-capacity hierarchically imprinted polymer beads for protein recognition and capture.Angew Chem Int Edit,2011,50:495-498
20 Kugimiya A,Takeuchi T.Surface plasmon resonance sensor using molecularly imprinted polymer for detection of sialic acid.Biosens Bioelectron,2001,16:1059-1062
21 Fuchs Y,Soppera O,Mayes A G,et al.Holographic molecularly imprinted polymers for label-free chemical sensing.Adv Mater,2013,25:566-570
22 Kunath S,Panagiotopoulou M,Maximilien J,et al.Cell and tissue imaging with molecularly imprinted polymers as plastic antibody mimics.Adv Healthc Mater,2015,4:1322-1326
23 Yin D Y,Wang S S,He Y J,et al.Surface-enhanced Raman scattering imaging of cancer cells and tissues via sialic acid-imprinted nanotags.Chem Commun,2015,51:17696-17699
24 Wang S S,Wen Y R,Wang Y J,et al.Pattern recognition of cells via multiplexed imaging with monosaccharide-imprinted quantum dots.Anal Chem,2017,89:5646-5652
25 Liu J,Yin D Y,Wang S S,et al.Probing low-copy-number proteins in a single living cell.Angew Chem Int Edit,2016,55:13215-13218
26 Sellergren B,Allender C J.Molecularly imprinted polymers:A bridge to advanced drug delivery.Adv Drug Deliver Rev,2005,57:1733-1741
27 Cecchini A,Raffa V,Canfarotta F,et al.In vivo recognition of human vascular endothelial growth factor by molecularly imprinted polymers.Nano Lett,2017,17:2307-2312
28 Hoshino Y,Koide H,Urakami T,et al.Recognition,neutralization,and clearance of target peptides in the bloodstream of living mice by molecularly imprinted polymer nanoparticles:A plastic antibody.J Am Chem Soc,2010,132:6644-6645
29 Li S W,Yang K G,Deng N,et al.Thermoresponsive epitope surface-imprinted nanoparticles for specific capture and release of target protein from human plasma.ACS Appl Mater Inter,2016,8:5747-5751
30 Bossi A,Piletsky S A,Piletska E V,et al.Surface-grafted molecularly imprinted polymers for protein recognition.Anal Chem,2001,73:5281-5286
31 James T D,Sandanayake K,Shinkai S.Saccharide sensing with molecular receptors based on boronic acid.Angew Chem Int Edit,1996,35:1910-1922
32 Li D J,Chen Y,Liu Z.Boronate affinity materials for separation and molecular recognition:Structure,properties and applications.Chem Soc Rev,2015,44:8097-8123
33 Liu Z,He H.Synthesis and applications of boronate affinity materials:From class selectivity to biomimetic specificity.Accounts Chem Res,2017,50:2185-2193
34 Li L,Lu Y,Bie Z J,et al.Photolithographic boronate affinity molecular imprinting:A general and facile approach for glycoprotein imprinting.Angew Chem Int Edit,2013,52:7451-7454
35 Ye J,Chen Y,Liu Z.A boronate affinity sandwich assay:An appealing alternative to immunoassays for the determination of glycoproteins.Angew Chem Int Edit,2014,53:10386-10389
36 Wang S S,Ye J,Bie Z J,et al.Affinity-tunable specific recognition of glycoproteins via boronate affinity-based controllable oriented surface imprinting.Chem Sci,2014,5:1135-1140
37 Bie Z J,Chen Y,Ye J,et al.Boronate-affinity glycan-oriented surface imprinting:A new strategy to mimic lectins for the recognition of an intact glycoprotein and its characteristic fragments.Angew Chem Int Edit,2015,54:10211-10215
38 Xing R R,Wang S S,Bie Z J,et al.Preparation of molecularly imprinted polymers specific to glycoproteins,glycans and monosaccharides via boronate affinity controllable-oriented surface imprinting.Nat Protoc,2017,12:964-987
39 Bie Z J,Xing R R,He X P,et al.Precision imprinting of glycopeptides for facile preparation of glycan-specific artificial antibodies.Anal Chem,2018,90:9845-9852
40 Forneris F,Mattevi A.Enzymes without borders:Mobilizing substrates,delivering products.Science,2008,321:213-216
41 Wang L Y,Bao J,Wang L,et al.One-pot synthesis and bioapplication of amine-functionalized magnetite nanoparticles and hollow nanospheres.Chem-Eur J,2006,12:6341-6347
42 Bublitz R,Hoppe H,Cumme G A,et al.Structural study on the carbohydrate moiety of calf intestinal alkaline phosphatase.J Mass Spectrom,2001,36:960-972