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Article(id=1199703584867713415, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1199703581889753882, articleNumber=1001-2494(2025)01-0001-08, orderNo=null, doi=10.11669/cpj.2025.01.001, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1715184000000, receivedDateStr=2024-05-09, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1763961224047, onlineDateStr=2025-11-24, pubDate=1736265600000, pubDateStr=2025-01-08, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763961224047, onlineIssueDateStr=2025-11-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763961224047, creator=13701087609, updateTime=1763961224047, updator=13701087609, issue=Issue{id=1199703581889753882, tenantId=1146029695717560320, journalId=1190317699101192196, year='2025', volume='60', issue='1', pageStart='1', pageEnd='104', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=0, articleOrder=1, issueType=-1, specialIssue=null, createTime=1763961223337, creator=13701087609, updateTime=1763967062652, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1199728073798157161, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1199703581889753882, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1199728073798157162, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1199703581889753882, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1, endPage=8, ext={EN=ArticleExt(id=1199703585241006473, articleId=1199703584867713415, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Progress in Preparation Technology of Bioaffinity Monolithic Column and Its Application in Pharmaceutical Analysis, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

Bioaffinity monolithic chromatography is a chromatographic technology developed by combining the latest advances in biology, hylology and chemical analysis. Based on the basic principle of biological affinity, monolithic materials were used to prepare stationary phase, which can simulate intermolecular interactions in vivo to screen and purify active substances. This technology can specifically customize analysis methods for different target active substances and has been widely used in multiple research fields in pharmacy, chemistry and biology. This article focuses on the preparation methods of monolithic columns, systematically reviews the selection for chromatographic columns, as well as the strategies for affinity ligand immobilization such as covalent binding of biomolecules and biological labels. It also summarizes the relevant applications and development directions of bioaffinity monolithic columns in the preparation of active proteins and drug/biological analysis.

, correspAuthors=Yifeng CHAI, Xiaofei CHEN, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Zhuoran SUN, Yanqiu GU, Dan JIA, Yifeng CHAI, Xiaofei CHEN), CN=ArticleExt(id=1199703587958915504, articleId=1199703584867713415, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=生物亲和整体色谱柱制备技术与药物分析应用进展, columnId=1190352408384471863, journalTitle=中国药学杂志, columnName=综述, runingTitle=null, highlight=null, articleAbstract=

生物亲和整体色谱是结合生物学、材料学及分析化学最新进展发展而来的色谱技术。该技术以生物亲和为基本原理,应用整体材料制备固定相,能够模拟生物体内微观分子间相互作用以筛选纯化活性物质。这项技术可以特异性地为不同目标活性物质定制分析方法,目前已经广泛应用于药物、化学和生物学相关的多个研究领域。本研究聚焦整体色谱柱的制备方法,系统综述了色谱柱基质选择以及生物大分子共价结合、生物标签等靶标固定方法策略,并总结了生物亲和整体色谱柱在活性蛋白制备、药物/生物分析中的相关应用和发展方向。

, correspAuthors=柴逸峰, 陈啸飞, authorNote=null, correspAuthorsNote=
*柴逸峰,男,博士,教授,博士生导师 研究方向:药物活性分析 Tel: (021) 81871393;
陈啸飞,男,博士,副教授,博士生导师 研究方向:药物靶标相互作用分析 Tel: (021) 81871335
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孙卓然,男,硕士研究生 研究方向:生物色谱分析

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孙卓然,男,硕士研究生 研究方向:生物色谱分析

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孙卓然,男,硕士研究生 研究方向:生物色谱分析

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DOI:10.1002/biot.201900028., articleTitle=Reversible two-enzyme coimmobilization on ph-responsive imprinted monolith for glucose detection, refAbstract=null), Reference(id=1199727466504880894, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, doi=null, pmid=null, pmcid=null, year=2016, volume=1456, issue=null, pageStart=249, pageEnd=256, url=null, language=null, rfNumber=[35], rfOrder=34, authorNames=HONG T, CHEN X, XU Y, journalName=J Chromatogr A, refType=null, unstructuredReference=HONG T, CHEN X, XU Y, et al. Preparation of graphene oxide-modified affinity capillary monoliths based on three types of amino donor for chiral separation and proteolysis[J]. J Chromatogr A, 2016, 1456: 249-256. DOI:10.1016/j.chroma.2016.06.025., articleTitle=Preparation of graphene oxide-modified affinity capillary monoliths based on three types of amino donor for chiral separation and proteolysis, refAbstract=null), Reference(id=1199727466655875839, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, doi=null, pmid=null, pmcid=null, year=2021, volume=1657, issue=null, pageStart=462567, pageEnd=null, url=null, language=null, rfNumber=[36], rfOrder=35, authorNames=GOTTARDINI A, DUGAS V, DEMESMAY C, journalName=J Chromatogr A, refType=null, unstructuredReference=GOTTARDINI A, DUGAS V, DEMESMAY C. Development of a multi-layering protein grafting process on miniaturized monolithic columns for weak affinity nano liquid chromatography application purposes[J]. J Chromatogr A, 2021, 1657: 462567. 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DOI:10.1007/s00216-015-8775-2., articleTitle=Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum, refAbstract=null), Reference(id=1199727466894951172, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, doi=null, pmid=null, pmcid=null, year=2016, volume=45, issue=8, pageStart=2137, pageEnd=2211, url=null, language=null, rfNumber=[38], rfOrder=37, authorNames=CHEN L, WANG X, LU W, journalName=Chem Soc Rev, refType=null, unstructuredReference=CHEN L, WANG X, LU W, et al. Molecular imprinting: perspectives and applications[J]. Chem Soc Rev, 2016, 45(8): 2137-2211. DOI:10.1039/c6cs00061d., articleTitle=Molecular imprinting: perspectives and applications, refAbstract=null), Reference(id=1199727468031607561, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, doi=null, pmid=null, pmcid=null, year=2019, volume=119, issue=1, pageStart=94, pageEnd=119, url=null, language=null, rfNumber=[39], rfOrder=38, authorNames=BELBRUNO J J, journalName=Chem Rev, refType=null, unstructuredReference=BELBRUNO J J. Molecularly imprinted polymers[J]. Chem Rev, 2019, 119(1): 94-119. 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DOI:10.1016/j.chroma.2020.460890., articleTitle=Purification of supercoiled p53-encoding plasmid using an arginine-modified macroporous support, refAbstract=null), Reference(id=1199727468379734800, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, doi=null, pmid=null, pmcid=null, year=2013, volume=27, issue=11, pageStart=1574, pageEnd=1579, url=null, language=null, rfNumber=[42], rfOrder=41, authorNames=YU X, ZHAO P, ZHANG L, journalName=Biomed Chromatogr, refType=null, unstructuredReference=YU X, ZHAO P, ZHANG L, et al. Screening of phage-displayed human liver cDNA library against doxorubicin with drug-immobilized monolithic polyacrylamide cryogel[J]. Biomed Chromatogr, 2013, 27(11): 1574-1579. DOI:10.1002/bmc.2962., articleTitle=Screening of phage-displayed human liver cDNA library against doxorubicin with drug-immobilized monolithic polyacrylamide cryogel, refAbstract=null), Reference(id=1199727468492981010, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, doi=null, pmid=null, pmcid=null, year=2019, volume=42, issue=11, pageStart=1980, pageEnd=1989, url=null, language=null, rfNumber=[43], rfOrder=42, authorNames=LIN H, ZHANG C, LIN Y, journalName=J Separat Sci, refType=null, unstructuredReference=LIN H, ZHANG C, LIN Y, et al. A strategy for screening trypsin inhibitors from traditional Chinese medicine based on a monolithic capillary immobilized enzyme reactor coupled with offline liquid chromatography and mass spectrometry[J]. J Separat Sci, 2019, 42(11): 1980-1989. DOI:10.1002/jssc.201900169., articleTitle=A strategy for screening trypsin inhibitors from traditional Chinese medicine based on a monolithic capillary immobilized enzyme reactor coupled with offline liquid chromatography and mass spectrometry, refAbstract=null), Reference(id=1199727468585255702, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, doi=null, pmid=null, pmcid=null, year=2021, volume=1656, issue=null, pageStart=462544, pageEnd=null, url=null, language=null, rfNumber=[44], rfOrder=43, authorNames=ZHANG F, JIANG Y, JIAO P, journalName=J Chromatogr A, refType=null, unstructuredReference=ZHANG F, JIANG Y, JIAO P, et al. Ligand fishing via a monolithic column coated with white blood cell membranes: A useful technique for screening active compounds in Astractylodes lancea[J]. J Chromatogr A, 2021, 1656: 462544. DOI:10.1016/j.chroma.2021.462544., articleTitle=Ligand fishing via a monolithic column coated with white blood cell membranes: A useful technique for screening active compounds in Astractylodes lancea, refAbstract=null), Reference(id=1199727468706890521, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, doi=null, pmid=null, pmcid=null, year=2018, volume=1563, issue=null, pageStart=135, pageEnd=143, url=null, language=null, rfNumber=[45], rfOrder=44, authorNames=WANG L, ZHAO Y, ZHANG Y, journalName=J Chromatogr A, refType=null, unstructuredReference=WANG L, ZHAO Y, ZHANG Y, et al. Online screening of acetylcholinesterase inhibitors in natural products using monolith-based immobilized capillary enzyme reactors combined with liquid chromatography-mass spectrometry[J]. J Chromatogr A, 2018, 1563: 135-143. DOI:10.1016/j.chroma.2018.05.069., articleTitle=Online screening of acetylcholinesterase inhibitors in natural products using monolith-based immobilized capillary enzyme reactors combined with liquid chromatography-mass spectrometry, refAbstract=null), Reference(id=1199727468811748124, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, doi=null, pmid=null, pmcid=null, year=2018, volume=119, issue=null, pageStart=150, pageEnd=160, url=null, language=null, rfNumber=[46], rfOrder=45, authorNames=LI C G, YUAN B, DONG M M, journalName=Food Chem Toxicol, refType=null, unstructuredReference=LI C G, YUAN B, DONG M M, et al. Purification and identification of an actinomycin D analogue from actinomycetes associated with Ganoderma applanatum via magnetic molecularly imprinted polymers and tandem mass spectrometry[J]. Food Chem Toxicol, 2018, 119: 150-160. DOI:10.1016/j.fct.2018.05.015., articleTitle=Purification and identification of an actinomycin D analogue from actinomycetes associated with Ganoderma applanatum via magnetic molecularly imprinted polymers and tandem mass spectrometry, refAbstract=null), Reference(id=1199727469000491808, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, doi=null, pmid=null, pmcid=null, year=2022, volume=12, issue=9, pageStart=3682, pageEnd=3693, url=null, language=null, rfNumber=[47], rfOrder=46, authorNames=GU Y, WANG R, CHEN P, journalName=Acta Pharm Sin B(药学学报英文版), refType=null, unstructuredReference=GU Y, WANG R, CHEN P, et al. In situ synthesis and unidirectional insertion of membrane proteins in liposome-immobilized silica stationary phase for rapid preparation of microaffinity chromatography[J]. Acta Pharm Sin B(药学学报英文版), 2022, 12(9): 3682-3693. DOI:10.1016/j.apsb.2022.04.010., articleTitle=In situ synthesis and unidirectional insertion of membrane proteins in liposome-immobilized silica stationary phase for rapid preparation of microaffinity chromatography, refAbstract=null), Reference(id=1199727469201818403, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, doi=null, pmid=null, pmcid=null, year=2023, volume=161, issue=null, pageStart=117015, pageEnd=null, url=null, language=null, rfNumber=[48], rfOrder=47, authorNames=GU Y Q, FAN F, LIU Y, journalName=TrAC Trend Anal Chem, refType=null, unstructuredReference=GU Y Q, FAN F, LIU Y, et al. Cell-free protein synthesis system for bioanalysis: advances in methods and applications[J]. TrAC Trend Anal Chem, 2023, 161: 117015. 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A-环氧胺法;B-席夫碱法;C-乙二胺-戊二醛法;D-溴化氰法;E-DSC/CDI法;F-酰肼法;G-EDC/NHS法;EDA-乙二胺;DCS-N,N二琥珀酰亚胺基碳酸酯;CDI-羰基二咪唑;AADH-己二酸二酰肼;PDA-聚多巴胺;PEI-聚乙烯亚胺;EDC-1-(3-二甲基氨基丙基)-3-乙基碳二亚胺;NHS-N-羟基琥珀酰亚胺基团。

, figureFileSmall=O64aZSD/wmz+2hkuHWsmpA==, figureFileBig=sHfj3CXOq3e9+LeIB215GQ==, tableContent=null), ArticleFig(id=1199727460192453196, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, language=EN, label=null, caption=null, figureFileSmall=/9nPrkxhCS/wyhSHAfm37Q==, figureFileBig=mwcgzirUGAFX8u91AsnqEA==, tableContent=null), ArticleFig(id=1199727460385391187, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, language=CN, label=图3, caption=生物亲和整体色谱柱基质制备中使用亲和标签法固定靶标的策略与机制示意图

A-金属离子亲和法;B-生物标签法。

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应用方向 固定相基质 固定靶标 固定方法 文献
毒素检测 有机聚合物 抗体 环氧基-氨基法 [3]
手性分离 有机聚合物 氨基酸 巯基-烯点击反应 [4]
毒素检测 有机聚合物 寡核苷酸 巯基-烯点击反应 [5]
蛋白质鉴定 有机聚合物 蛋白质 亲和标签法 [6]
蛋白质耗竭 有机聚合物 抗体 酰肼法 [7]
蛋白质纯化 有机聚合物 蛋白质 CDI法 [8]
抗体纯化 有机聚合物 肽 亲和标签法 [9,14]
抗体纯化 有机聚合物 氨基酸 CDI法 [10]
抗体纯化 混合型基质 蛋白质 NHS法 [11]
药物筛选 二氧化硅、有机聚合物 蛋白质 环氧基-氨基法、席夫碱法、生物素标签法 [12]
药物-靶标结合作用研究 二氧化硅 蛋白质 溶胶-凝胶法 [13]
免疫亲和实验 有机聚合物 蛋白质、抗体 CDI法 [15]
蛋白质检测 有机聚合物 抗体 酰肼法 [16]
蛋白质纯化 冷冻凝胶 碳水化合物 乙二胺-戊二醛法 [25]
毒素检测 冷冻凝胶 蛋白质 溴化氰法 [26]
病毒纯化 凝胶 肝素 EDC/NHS法 [29]
蛋白质检测 凝胶 蛋白质 亲和标签法 [30-31]
蛋白质消化 有机聚合物 蛋白质 NHS法 [32]
核酸纯化 有机聚合物 氨基酸 环氧基-氨基法 [33]
生物传感器 有机聚合物 蛋白质 分子印迹 [34]
手性分离 二氧化硅 蛋白质 EDC/NHS法 [35]
药物筛选 有机聚合物 蛋白质 亲和标签法 [36,40]
核酸纯化 大孔树脂 氨基酸 环氧基-氨基法 [41]
蛋白质筛选 冷冻凝胶 药物 戊二醛法 [42]
药物筛选 有机聚合物 蛋白质 环氧基-氨基法 [43,45]
药物筛选 有机聚合物 细胞膜 席夫碱法 [44]
药物筛选 有机聚合物 药物 分子印迹 [46]
), ArticleFig(id=1199727460691575391, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703584867713415, language=CN, label=表1, caption=

生物亲和整体色谱柱代表性应用

, figureFileSmall=null, figureFileBig=null, tableContent=
应用方向 固定相基质 固定靶标 固定方法 文献
毒素检测 有机聚合物 抗体 环氧基-氨基法 [3]
手性分离 有机聚合物 氨基酸 巯基-烯点击反应 [4]
毒素检测 有机聚合物 寡核苷酸 巯基-烯点击反应 [5]
蛋白质鉴定 有机聚合物 蛋白质 亲和标签法 [6]
蛋白质耗竭 有机聚合物 抗体 酰肼法 [7]
蛋白质纯化 有机聚合物 蛋白质 CDI法 [8]
抗体纯化 有机聚合物 肽 亲和标签法 [9,14]
抗体纯化 有机聚合物 氨基酸 CDI法 [10]
抗体纯化 混合型基质 蛋白质 NHS法 [11]
药物筛选 二氧化硅、有机聚合物 蛋白质 环氧基-氨基法、席夫碱法、生物素标签法 [12]
药物-靶标结合作用研究 二氧化硅 蛋白质 溶胶-凝胶法 [13]
免疫亲和实验 有机聚合物 蛋白质、抗体 CDI法 [15]
蛋白质检测 有机聚合物 抗体 酰肼法 [16]
蛋白质纯化 冷冻凝胶 碳水化合物 乙二胺-戊二醛法 [25]
毒素检测 冷冻凝胶 蛋白质 溴化氰法 [26]
病毒纯化 凝胶 肝素 EDC/NHS法 [29]
蛋白质检测 凝胶 蛋白质 亲和标签法 [30-31]
蛋白质消化 有机聚合物 蛋白质 NHS法 [32]
核酸纯化 有机聚合物 氨基酸 环氧基-氨基法 [33]
生物传感器 有机聚合物 蛋白质 分子印迹 [34]
手性分离 二氧化硅 蛋白质 EDC/NHS法 [35]
药物筛选 有机聚合物 蛋白质 亲和标签法 [36,40]
核酸纯化 大孔树脂 氨基酸 环氧基-氨基法 [41]
蛋白质筛选 冷冻凝胶 药物 戊二醛法 [42]
药物筛选 有机聚合物 蛋白质 环氧基-氨基法 [43,45]
药物筛选 有机聚合物 细胞膜 席夫碱法 [44]
药物筛选 有机聚合物 药物 分子印迹 [46]
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生物亲和整体色谱柱制备技术与药物分析应用进展
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孙卓然 1, 2 , 顾妍秋 3 , 贾丹 1 , 柴逸峰 1, * , 陈啸飞 1, *
中国药学杂志 | 综述 2025,60(1): 1-8
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中国药学杂志 | 综述 2025, 60(1): 1-8
生物亲和整体色谱柱制备技术与药物分析应用进展
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孙卓然1, 2, 顾妍秋3, 贾丹1, 柴逸峰1, *, 陈啸飞1, *
作者信息
  • 1 海军军医大学药学院, 上海 200433
  • 2 中国人民解放军联勤保障部队第九〇六医院药剂科, 浙江 宁波 315100
  • 3 上海交通大学医学院附属第九人民医院药剂科, 上海 201999

    • 孙卓然,男,硕士研究生 研究方向:生物色谱分析

通讯作者:

*柴逸峰,男,博士,教授,博士生导师 研究方向:药物活性分析 Tel: (021) 81871393;
陈啸飞,男,博士,副教授,博士生导师 研究方向:药物靶标相互作用分析 Tel: (021) 81871335
Progress in Preparation Technology of Bioaffinity Monolithic Column and Its Application in Pharmaceutical Analysis
Zhuoran SUN1, 2, Yanqiu GU3, Dan JIA1, Yifeng CHAI1, *, Xiaofei CHEN1, *
Affiliations
  • 1 School of Pharmacy, Naval Medical University, Shanghai 200433, China
  • 2 Department of Pharmacy, No.906 Hospital of Joint Logistics Support Force of PLA, Ningbo 315100, China
  • 3 Department of Pharmacy, Shanghai 9th People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201999, China
出版时间: 2025-01-08 doi: 10.11669/cpj.2025.01.001
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摘要
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生物亲和整体色谱是结合生物学、材料学及分析化学最新进展发展而来的色谱技术。该技术以生物亲和为基本原理,应用整体材料制备固定相,能够模拟生物体内微观分子间相互作用以筛选纯化活性物质。这项技术可以特异性地为不同目标活性物质定制分析方法,目前已经广泛应用于药物、化学和生物学相关的多个研究领域。本研究聚焦整体色谱柱的制备方法,系统综述了色谱柱基质选择以及生物大分子共价结合、生物标签等靶标固定方法策略,并总结了生物亲和整体色谱柱在活性蛋白制备、药物/生物分析中的相关应用和发展方向。

生物亲和色谱  /  整体柱  /  固定相制备  /  分离纯化  /  药物分析

Bioaffinity monolithic chromatography is a chromatographic technology developed by combining the latest advances in biology, hylology and chemical analysis. Based on the basic principle of biological affinity, monolithic materials were used to prepare stationary phase, which can simulate intermolecular interactions in vivo to screen and purify active substances. This technology can specifically customize analysis methods for different target active substances and has been widely used in multiple research fields in pharmacy, chemistry and biology. This article focuses on the preparation methods of monolithic columns, systematically reviews the selection for chromatographic columns, as well as the strategies for affinity ligand immobilization such as covalent binding of biomolecules and biological labels. It also summarizes the relevant applications and development directions of bioaffinity monolithic columns in the preparation of active proteins and drug/biological analysis.

bioaffinity chromatography  /  monolithic column  /  stationary phase preparation  /  separation purification  /  pharmaceutical analysis
孙卓然, 顾妍秋, 贾丹, 柴逸峰, 陈啸飞. 生物亲和整体色谱柱制备技术与药物分析应用进展. 中国药学杂志, 2025 , 60 (1) : 1 -8 . DOI: 10.11669/cpj.2025.01.001
Zhuoran SUN, Yanqiu GU, Dan JIA, Yifeng CHAI, Xiaofei CHEN. Progress in Preparation Technology of Bioaffinity Monolithic Column and Its Application in Pharmaceutical Analysis[J]. Chinese Pharmaceutical Journal, 2025 , 60 (1) : 1 -8 . DOI: 10.11669/cpj.2025.01.001
正文
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亲和色谱法是分离分析复杂样品中特定活性化合物或生物分子相互作用的常用方法[1-2],通过在体外实现受体与配体的相互作用,亲和色谱具有特异性和高选择性识别活性分子的能力,常用于分离小分子化合物[3-5]、蛋白质[6-8]和抗体[9-11],成为分析化学、药物分析等多个领域的重要研究策略。
与其他体外蛋白互作实验相比,将靶蛋白键合在固定相上可以显著减少蛋白质需求量。但是,传统亲和色谱柱的制备由于其体积和孔径大,可能需要多达几十毫克的靶蛋白,且获得毫克级别的正确折叠并有稳定活性的蛋白质难度较大。研究者们为减少靶蛋白的使用量尝试缩小色谱柱直径,但内径小的毛细管柱填充对技术要求高。整体柱可将功能单体原位聚合,得到的连续多孔高聚物固体材料,从而产生具有高渗透性的毛细管色谱柱。在整体基质合成后,再通过流动蛋白质溶液进行接枝固定[9-12]。对用于蛋白质纯化的制备性亲和色谱而言,琼脂糖树脂和磁珠是蛋白质体外结合实验中常用的基质。但琼脂糖树脂纯化蛋白质的过程繁琐且耗时,需要手动重复离心和洗涤;磁珠的金属氧化物材料的固有性质导致非特异性吸附,降低了纯化效率。整体色谱基质具有克服以上两种基质缺点的能力,其刚性多孔表面积较大,可以提供更高的结合能力,允许配体轻松地接近靶蛋白[6]。
本研究系统综述了生物亲和整体柱的制备方法,包括基质的选择和靶标固定的策略,生物亲和整体色谱柱在分离纯化和药物分析领域的应用,并对生物亲和整体柱未来的发展进行了展望。
1 生物亲和整体柱的制备方法
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1.1 整体基质选择
整体柱的基质作为液相色谱的固定相,需提供良好的传质基础和活性分子负载能力,能够实现对生物活性大分子的亲和固定能力,目前常用的基质有二氧化硅、有机聚合物、凝胶等。
1.1.1 二氧化硅整体柱
二氧化硅是一种传统的色谱基质材料,也被用作整体柱的基质。这种整体柱将颗粒二氧化硅载体的表面特性与整体柱的传质特性结合起来,表现出很高的柱效率,创造了生物分子可接近表面积,实现了高液体渗透性和高靶标负载能力。但是二氧化硅整体材料在形成过程中很容易收缩,导致其难以直接在柱内原位合成,且其发挥功能需要用缩水甘油氧基丙基三甲氧基硅烷(GPTMS)进行硅烷化,该步骤使得二氧化硅整体柱在与受体相互作用时表现出高水平的非特异性,这可能与新引入的缩水甘油基丙基亲水性较差有关[12]。溶胶-凝胶法是制备二氧化硅整体柱的常用方法,例如将甘油硅烷溶液与含有生物分子、聚乙二醇600(PEG 600)、3-氨丙基三乙氧基硅烷(APTES)的磷酸盐缓冲溶液等体积混合并注入活化的熔融玻璃毛细管中反应,可制得具有良好的生物相容性的整体柱[13]。
1.1.2 有机整体柱
有机整体柱通过功能单体聚合形成,是常见的生物相容性固定相方案。在其制备过程中一般会加入交联剂和致孔剂,得到有较好色谱效率的疏松多孔结构,见图1。在有机整体柱中发挥亲和活性的关键因素是功能单体,甲基丙烯酸缩水甘油酯(GMA)是最常见的有机功能单体之一,它可以通过光化学诱导聚合,过程简单快速,并在表面留下可直接功能化的环氧基。因为其他传统的基质过高的受体密度可能导致空间位阻效应增加,GMA整体柱受体密度的减少并没有导致其结合能力的降低[9]。相比于二氧化硅材料,GMA虽然可加载的活性蛋白较少,但是提高了在与配体相互作用时的特异性。以GMA为功能单体的生物相容性聚合物整料易于制造,具有优异的形态、良好的机械性能、丰富的化学性质以及可忽略的蛋白质非特异性吸附[12,14]。甲基丙烯酸酯等材料也可以作为整体柱的功能单体,已成为成熟商品在市场上出售。这种整体柱被称为“对流相互作用介质”(CIM),用于分离、纯化或清除样品中的生物大分子[7,15-16]。CIM柱具有开放通道的紧凑载体,使流动相能够不间断地流过色谱单元,受体和配体之间的相互作用仅发生在整料内空间的表面,从而排除了扩散过程,与填充色谱柱相比获得了更窄的峰宽且缩短了几个数量级的相互作用时间[8],因其与流速无关的结合能力,更加适合用于高通量筛选与纯化[10]。根据实现功能的不同或分析效果的要求,还有大量其他单体可供选择。例如为了使整体柱表面聚合反应性N-羟基琥珀酰亚胺基团,可以使用4-戊烯酸琥珀酰亚胺酯(PAS)为功能单体[6];或为提高基质亲水性而使用甲基丙烯酸2-羟乙酯(HEMA)[6]及N,N-二甲基-N-丙烯酰氧基-N-(3-磺丙基)甜菜碱[17];或模拟生物膜的各种磷脂环境而采用MDPC80PS20等[18]。使用不同交联剂会改变色谱整体柱固定相的亲水性和选择性[19],改变其与功能单体的使用比例,也会造成色谱填料渗透性和特异性的变化[20]。乙二醇二甲基丙烯酸酯(EGDMA或EDMA)是最常见的交联剂之一,可以与不同的功能单体聚合为整体柱[21]。Zhu等[22]以1-十二烷基-2-(11-甲基丙烯酰胺十二烷基)-sn-甘油-3-磷酸乙醇胺为功能单体,EDMA为交联剂,制备了新型磷脂功能化整体柱,用以模仿细胞膜环境并探究药物与细胞膜间的相互作用。常见的整体柱扫描电子显微镜成像见图1。
1.1.3 冷冻凝胶和凝胶整体柱
在使用有机功能单体的情况下,多孔结构也可以通过冷冻凝胶或凝胶的方法获得。冷冻凝胶是低温凝胶化(cryogelation)的技术产物,是一种孔隙率可控的大孔聚合物网络。其制备过程是以水为溶剂,冷冻后形成冰晶以产生孔隙,最终形成高度连续的多孔结构。由于大孔的存在,冷冻凝胶柱有良好的流通性和传质效率[24-25]。目前已有不少研究报道,使用可以聚合的单体物质,如乙烯醇、HEMA、N,N’-亚甲基双丙烯酰胺(MBA)、丙烯酰胺(AAm)、烯丙基缩水甘油醚(AGE)、聚乙二醇二丙烯酸酯(PEGDA)等,合成了以聚乙烯醇(PVA)、AAM-AGE、HEMA-MBA或HEMA-PEGDA等为基质的冷冻凝胶[24,26]。同时,由高内相乳液制备的材料也受到越来越多的关注。其制备原理是利用高内相乳液这种高度黏稠的糊状乳液,通过聚合其连续相并去除占体积比超过74%的内部分散相液体而产生的称为polyHIPE的高度多孔材料[27]。由水包油(O/W)HIPEs的亲水单体直接制备的水凝胶适用于生物工程,特别是酶固定,组织工程和生物大分子纯化。这归因于它们高度互连的多孔结构和诸如羟基,羧基和氨基等的亲水基团。水凝胶固有的亲水性赋予了它们良好的生物相容性,高度渗透性的结构特点使得其在高流速下也能保持较低的柱压,适合于高通量亲和反应[28-29]。应用此种技术,Gu等[29]以HEMA、MBA、丙烯酸为功能单体,制备了一种表面带有亚氨基的大孔水凝胶,在功能化修饰后用于肝素的固定。Stanti c ˇ等[30]以GMA为功能单体,乙二醇二甲基丙烯酸酯为交联剂制备了polyHIPE凝胶整体柱并成功在整料表面固定了凝集素。Du等[31]也用此法制作了固定凝集素的大孔整体柱,他们选择纤维素作为整体基质并用乙二醇二缩水甘油醚交联以提高力学稳定性。
1.1.4 混合型整体柱
为了结合二氧化硅优秀的柱效率和功能单体亲和的特异性,可以将前述方法混合使用。有报道在旋转柱中原位聚合整体材料,如Ota等[11]在用3-甲基丙烯酰氧基丙基三甲氧基硅烷活化的二氧化硅整体材料表面上聚合HEMA和GMA,制备了Protein A包被的纯化柱。这里把GMA用作固定Protein A的功能单体,用HEMA来减少非特异性吸附。
1.2 靶标固定策略
为了使整体基质获得生物学亲和力并发挥其分析或制备生物活性物质的功能,选择合适的基质十分重要,因其表面可以直接带有方便活性受体锚定的结合基团。不过有时还需要对整体基质表面进行聚合后修饰[32],而后通过新修饰的功能基团来实现生物目标的固定。
1.2.1 官能团共价固定
通过在整体基质的表面修饰出能够与目标活性物质上特定基团共价结合的官能团来实现整料的功能化是一种常见的策略,见图2。
最常用的策略是通过蛋白质赖氨酸侧链的伯胺基的反应将蛋白质直接共价偶联到环氧基上。通过蛋白质在表面上的快速吸附,然后在环氧基质和生物分子的胺基残基之间缓慢形成共价键[10,12],这种方法也能在疏水的条件下固定核酸[33]。表面有环氧基的整体基质也可以直接使用H2SO4进行开环水解生成二醇官能团,再用NaIO4将其氧化获得醛基,可与受体上的胺基反应生成共价固定的肽键[12,30]。此种方法被称为席夫碱法,在与环氧法相同的反应时间下,嫁接密度提高了1.5~4倍。但是这种接枝方法不太适用于脆性蛋白,因在耗时的接枝步骤中蛋白质可能会变性[12]。同样是生成醛基,为了减小空间位阻对于大型靶标分子固定过程的影响,通常需要考虑引入间隔臂,使靶标与固定相表面产生一定距离。例如先将固定相表面的环氧基用乙二胺(EDA)进行处理得到胺基改性的整料[10,25-26,34],然后再用戊二醛将其表面醛基功能化[10,25-26]。除了EDA外,聚乙烯亚胺(PEI)也可作为间隔臂和氨基供体[35]。用EDA将环氧基开环后,也可使用N,N'-二琥珀酰亚胺基碳酸酯(DSC)将基质表面修饰为N-羟基琥珀酰亚胺基团(NHS)。NHS本身是一种交联剂,通过与生物分子表面的胺基形成酰胺键而固定后者[11]。羰基二咪唑(CDI)也可通过NHS固定,市售的CIM整体柱表面即带有这种基团[8,10,15]。CDI基团可以与生物分子的伯胺基共价结合,反应需要进行28 h,但反应完成后不需要特别处理剩余未反应的咪唑基团,因为其会快速自灭[10]。溴化氰(CNBr)也是功能化整体基质的试剂之一。可以用CNBr处理表面有羟基的整体基质,生成的异脲键来固定带有胺基的亲和受体,如Protein A等[26]。酰肼法也是常见的固定方法[7,16]:使用己二酸二酰肼开环环氧基并将酰肼基团修饰在基质表面,之后可与含有醛基的亲和受体(如被氧化成醛基的抗体Fc区上的碳水化合物区域)反应,形成腙键。由于这种固定方式锚定了糖蛋白(抗体)位于抗体Fc区的碳水化合物链,故确保了抗体活性位点(Fab)朝向流动相的方向,使抗原结合位点获得了良好的空间可及性[16]。对于含有羧基的生物大分子,可以使用1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC)和NHS两种物质配合固定。EDC/NHS需要作用于表面带有胺基的整体柱材料,活化受体的羧基并与整体柱表面的胺基形成酰胺键。Gu等[29]已使用此方法成功将肝素结合在固定相上。
1.2.2 亲和标签固定
金属离子亲和色谱(IMAC)是一种成熟的色谱方法。该方法将金属离子用螯合剂修饰在整料表面,这些金属离子会与蛋白质或肽中特定的氨基酸序列发生配位结合,原理见图3A。这种方法为了固定的特异性和方向性,常常需要在受体上的特定位置表达出专门用于固定的标签氨基酸序列。一般使用螯合剂如氨基丁基硝基三乙酸等以及金属离子供体如NiSO4·6H2O等将Ni2+修饰在整体柱表面,并用其固定带有组氨酸(His)标签的目标[6,9,14]。也有通过亚氨基二乙酸(IDA)和CuSO4溶液将Cu2+固定在整体基质上的,使其具有与刀豆蛋白A可逆螯合的能力[31]。
生物特异性固定是一种间接的亲和标签固定方法,它需要整体基质先固定一种生物活性物质作为黏合剂,让亲和目标通过生物特异性识别与固定相表面结合,原理见图3B。链霉亲和素是一种常见的间接黏合剂。它可利用高度特异性和极强的亲和力将带有生物素标签的生物分子固定在色谱基质上。它比共价结合法提供了更多的活性位点,并且允许靶向较低亲和力的目标;可以在使用前几分钟内加载目标蛋白,反应迅速,从而解决了稳定性的问题,蛋白质的活性率超过80%;接枝步骤可以通过紫外线实时监测固定过程,从而减少了蛋白质的浪费。链霉亲和素-生物素接枝方法是普适的,可以应用于任何带有生物素标签的目标[12,36]。蛋白质A也可作为间接黏合剂,常用于捕获各种抗体[26,37]。如Ingavle等[26]在4种不同的冷冻凝胶表面修饰上Protein A,辅以二甲基吡咪酯为交联剂固定非糖基化植物源性人单克隆抗体(PANG)和糖基化人单克隆抗体,使亲和整体柱获得捕获炭疽毒素保护性抗原(PA)的能力。
1.2.3 其他制备策略
分子印迹技术是一种利用分子模板的存在聚合功能单体和交联剂,使得移除模板后的聚合物留下了在形状、大小和化学功能上与模板分子互补印迹腔,可以选择性地固定目标受体[38-39]。通过pH响应等设计修饰,可实现定向可逆的固定。通过表面引发的原子转移自由基聚合技术将带有辣根过氧化物酶(HRP)印迹腔的4-乙烯基苯基硼酸修饰在GMA-co-EDMA的整体基质上,Luo等[34]以此制备了可以通过pH改变以吸附或解吸附而获得再生能力的生物亲和整体柱。
为了提高色谱柱的生物利用效率等方面的考虑,在选择固定策略的时候会额外进行一些优化设计。为了增加官能团密度,可以将聚多巴胺(PDA)和PEI涂布在基质表面。Gu等[29]应用了这种方法修饰的水凝胶整料实现了更高的肝素化效率。为了保证跨膜蛋白质在固定之中及之后仍能保持正确折叠、构象稳定,需要为其提供类似天然的环境。纳米盘就是一种被膜支架蛋白(MSP)包围的自组装可溶性盘状磷脂双层,它可以为跨膜蛋白提供模拟的细胞膜环境。Lecas等将G蛋白偶联受体(GPCRs)家族的AA2AR蛋白嵌入带有生物素标签的纳米盘,并固定在链霉亲和素包被的整体柱上。纳米盘的存在使得AA2AR本身并没有被束缚在固定相上,而是通过生物素化的MSP实现了均匀定向的固定,由于有纳米盘充当间隔臂,确保了蛋白质的结合口袋对小分子完全可及[40]。为了保证生物大分子的结合部位在基质表面正确暴露,需要考虑结合的方向性。可以通过基因编辑技术对靶标蛋白进行重组修饰,以添加充当间隔物和允许定向固定的接头。Stanti c ˇ等[30]通过在固定位置引入间隔臂重组了凝集素,并证明了此方法对于凝集素捕获糖蛋白的效率有促进作用。为了一些特定的功能,可以对整体柱更有针对性地修饰。例如进行活性物质的手性分离,可以用氧化石墨烯(GO)与其他间隔臂一起对整体柱进行修饰。用这种方法成功使固定了人血白蛋白(HSA)的整体柱获得了手性分离的能力。同时,实验证明这种修饰方法也可以用来增加固定了胃蛋白酶的整体柱的酶活性[35]。
2 亲和整体色谱在生物和药学领域的相关应用
收起
亲和整体色谱技术近年来发展迅速,不但有实验室自制整体柱的成熟方案,也有商品化的成熟产品。如BIA Separations公司开发的制备用层析整体柱-CIMmultus®系列和分析用层析整体柱-CIMacTM系列等,在大生物分子纯化、生物分子的HPLC法检测和定量等有很多应用。本文总结亲和整体色谱技术代表性的应用见表1。
2.1 分离纯化应用
生物亲和色谱法可以应用于生物工程的多个领域,可以用来分离纯化蛋白质、病毒、抗体、核酸等生物活性物质。
凝集素是一类对碳水化合物结合具有高度特异性的蛋白质,可用于选择性分离和分析具有聚糖结构的糖蛋白。通过在polyHIPE整体柱上固定凝集素,成功捕获了Fc融合糖蛋白,并完成了结合、解离循环。这项技术可以用于生物过程的在线监测,可以在生产过程中实时检查和控制蛋白质的糖基化水平[30]。凝集素也可被固定在冷冻凝胶等其他整料表面,用以分离纯化HRP或血清中的糖蛋白等[24,31-32]。另一方面,也可以利用这种特异性的生物反应在冷冻凝胶整料表面固定碳水化合物来分离纯化凝集素[25]。肝素可以选择性识别肠道病毒71(EV71),通过将使用PDA和PEI改性的大孔水凝胶整体柱高密度肝素化,高效吸附了EV71。该方法被证明适用于纯化EV71用以生产蛋白质和疫苗以及进行基因治疗研究[29]。在单克隆抗体的生产中,需要快速定量,分离和纯化构建的细胞系中抗体的方法,以及在短时间内分析大量样品的系统。基于Protein A包被的有机/无机混合型整料而开发的一种整体旋转纯化柱,因其高回收率,适用于小溶液体积和低浓度IgG的分析,为中国仓鼠卵巢细胞培养基中IgG的提取和测定提供了快速有效的工具[11]。将蛋白A、G和L固定在CDI修饰的CIM柱上,可以快速同时亲和纯化α(A),γ(G)和μ(M)免疫球蛋白,用于人血清的高通量筛选[8]。使用同样的方式固定L-组氨酸也可以达到类似甚至更优的高通量纯化抗体效果[10]。仿生小肽3,5-二叔丁基-4-羟基苯甲酸-Arg-Arg-Gly固定化的金属亲和整体色谱能够特异性地识别单克隆抗体,并表现出了很好的稳定性,具有在抗体纯化中长期使用的潜力[9]。环肽功能化的整体材料可以抵抗蛋白水解酶的影响,是下游生物加工中抗体纯化的潜在候选材料[14]。在基因工程方面,将赖氨酸或其脱羧产物尸胺直接固定在整体柱表面,可以用来纯化超螺旋微环状DNA[33];用固定化精氨酸的大孔树脂整体柱纯化超螺旋p53编码质粒,其动态结合能力超过同类商品化树脂50%[41]。
2.2 药物分析应用
色谱法是经典的化学分析方法,在化学领域广泛应用。采用新开发的色谱填料,使得色谱柱获得了更高的柱效和生物亲和特性,这类新型色谱方法能够在最前沿的学科交叉的领域有着更加广阔的应用前景,用以分析诸如以下几种生物医药分析领域的研究对象。
Hong等[35]开发了一种GO修饰的整体柱,在固定了HSA后对 (±)-色氨酸等9对外消旋体进行了对映体分离,与之前的技术相比缩短了分析时间并减少了试剂消耗。亲和纯化质谱方法可以用来研究药物及蛋白质之间瞬时、动态的相互作用。Hu等[13]使用固定化胰蛋白酶甘油硅烷整体柱与FAC,结合探究了苦参碱、氧化苦参碱、大豆苷和染料木苷与胰蛋白酶的天然结合特性。Liu等[6]开发了一种固定了FYCO1蛋白的旋转纯化整体柱,在离心机中快速地分离出与靶标相互作物的物质,洗脱液用于无标记定量蛋白质组学分析,鉴定了96种蛋白质并探究了与FYCO1蛋白的GOLD结构域的相互作用。Yu等[42]采用冷冻凝胶法制备整体柱并通过戊二醛法固定抗癌药物阿霉素,从噬菌体展示人肝脏cDNA文库中筛选该药的靶蛋白,选择性地富集了甲基CpG结合蛋白2 (MeCP2),并证明了两者间的相互作用。
装载了链霉亲和素的生物亲和整体色谱可以固定生物素化的纳米盘,并在其中重构膜蛋白,从而为膜蛋白上基于片段的药物设计提供支持。使用区带亲和色谱分析法与质谱联用,可以在每次约30 min的分析中筛选约10~20种化合物,每天从片段文库中筛选约500~1 000种化合物[12]。在基于结构的片段筛选中,可以用竞争实验来区分特异性和非特异性配体/受体结合、评估亲和力以及确定结合位点。Lecas等[40]运用了他们开发的AA2AR纳米盘亲和整体色谱,通过前沿亲和色谱法(FAC)观察到了茶碱和AA2AR纳米盘之间对应于蛋白质结构口袋中的特定相互作用。生物亲和整体色谱同样也是药物评价和筛选的有效方法。Lin等[43]应用整体柱材料制作酶反应器,联用离线液质系统从中药黄芩提取物中筛选出3种胰蛋白酶抑制剂。Zhang等[44]将白细胞的细胞膜包被到整体柱上,对黄芪中的成分进行亲和垂钓,发现了3种潜在的抗炎活性物质。Wang等[45]将含有环氧基的整体柱基质作为载体固定乙酰胆碱酯酶(AChE),建立了一种在线比较配体垂钓平台,用以从延胡索提取物中筛选AChE抑制剂,鉴定出8个具有AChE亲和力的化合物。Li等[46]采用分子印迹技术制备整体柱吸附放线菌素D(Act D)及其类似物,从内生放线菌KLBMP 2541中新鉴定出一种具有比Act D更好的抗肿瘤活性的类似物。
2.3 发展前景
亲和色谱整体柱是一种高效的药物/生物分析技术,整合了整体柱和生物色谱的各自优势,目前已经形成了多种成熟的技术体系。然而,目前该技术仍有改进的需求。如被固定的亲和受体特异性的问题可能导致非特异性的化合物被捕获,广泛存在的非特异性相互作用也是假阳性产生的主要原因,需要对照试验加以排除。例如链霉亲和素会结合腺苷,对结合GPCRs的FAC实验产生影响,需要做额外的实验进行区分[40]。由于细胞裂解物等样品成分复杂,很难避免假阳性[6,40]。近年来基于片段的药物设计兴起,要求色谱扩大亲和力检测范围以鉴定亲和力更低的配体。目前已有通过增加介质密度来提高保留性能的尝试。如利用链霉亲和素具有4个活性位点的特点,反复进行固定操作,得到了3层固定生物素化配体的整体柱,增加了柱上固定的活性蛋白质的量,接枝密度增加1倍的同时保留性能加倍并扩大亲和力范围[36]。目前亲和受体一般通过共价结合的方式固定在整体柱表面,这种方式赋予其优异的结构稳定性。但这种固定是不可逆的,在亲和受体因重复使用后失活后,昂贵的色谱载体就只能被废弃。Du等[31]探索了可逆的固定化方式,对于降低生物亲和整体柱的制备使用成本有积极意义。此外,合成生物学技术在生物色谱中也得到了拓展性应用,本课题组在二氧化硅固定相上原位合成了膜蛋白PDGFRβ用于靶向药物筛选[47]。通过将人工合成的靶标定向地结合在固定相上,这种技术可以成为新的筛选平台以研发小分子、多肽或蛋白质靶向药物[48]。
综上所述,亲和整体色谱的固定相制备策略向特异性更高、亲和力更强、制备更简便的方向发展。靶蛋白等生物大分子的固定策略将与合成生物学等学科紧密交叉,实现原位高活性合成、朝向精准控制、活性位点特异性暴露以及固定蛋白的回收与再生。简便、快速、高效、精准是生物亲和整体柱色谱技术的发展方向,将成为生物大分子的分离纯化、靶向药物筛选和亲和力检测的有力工具。
基金
收起
  • 国家自然科学基金项目资助(82073814)
  • 国家自然科学基金项目资助(82122066)
  • 国家自然科学基金青年科学基金项目资助(82003909)
  • 国家自然科学基金青年科学基金项目资助(82004002)
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2025年第60卷第1期
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doi: 10.11669/cpj.2025.01.001
  • 接收时间：2024-05-09
  • 首发时间：2025-11-24
  • 出版时间：2025-01-08
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  • 收稿日期：2024-05-09
基金
国家自然科学基金项目资助(82073814)
国家自然科学基金项目资助(82122066)
国家自然科学基金青年科学基金项目资助(82003909)
国家自然科学基金青年科学基金项目资助(82004002)
作者信息
    1 海军军医大学药学院, 上海 200433
    2 中国人民解放军联勤保障部队第九〇六医院药剂科, 浙江 宁波 315100
    3 上海交通大学医学院附属第九人民医院药剂科, 上海 201999

通讯作者:

*柴逸峰,男,博士,教授,博士生导师 研究方向:药物活性分析 Tel: (021) 81871393;
陈啸飞,男,博士,副教授,博士生导师 研究方向:药物靶标相互作用分析 Tel: (021) 81871335
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2种不同金属材料的力学参数
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species		 占总种数比例
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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