分子印迹技术及其在药物分离与分析中的应用

傅 强，罗智敏，郭鹏琦

(西安交通大学医学部药学院药物分析学系，陕西西安 710061)



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分子印迹技术及其在药物分离与分析中的应用

傅 强，罗智敏，郭鹏琦

(西安交通大学医学部药学院药物分析学系，陕西西安 710061)

复杂体系微量药物和药物相关物质的分离与分析是药学研究领域的共性问题。建立高灵敏度、高选择性、高速度、自动化、连续化、智能化的分析技术是药物分析学科发展的重要方向。新方法的建立对于进一步了解生命过程和药物的作用、保障药品质量、提高药品疗效发挥了积极的推动作用。近年来发展起来的分子印迹技术具有构效预定性、识别特异性、长期稳定性和广泛适用性等特点，得到研究者的广泛关注。本文简述分子印迹技术的原理和制备方法，综述了分子印迹技术在手性药物分析、体内药物分析、中药活性成分和生物大分子的分离与分析等中的应用，并对其面临的问题及应用前景进行了展望。

分子印迹技术；药物分离；药物分析

药物与生命体的相互作用，本质上是物质间的相互作用。灵敏度高、特异性强的分析技术对于进一步了解生命过程和药物的作用、保障药品质量、提高药品疗效发挥了积极的推动作用。分子印迹技术(molecular imprinting technology, MIT)是一种高选择性、特异性的分离和分析技术，这种技术的思想源于抗原-抗体专一性识别。基于分子识别的聚合物材料——分子印迹聚合物具有构效预定性、识别特异性、长期稳定性和广泛适用性等特点，且具有抗恶劣环境能力强、稳定性好、使用寿命长等优点，使得MIT在诸多领域均展现了良好的应用前景。

1 原理与聚合物制备

1.1 基本原理 分子印迹的概念是由POLYAKOV 1931年提出，MOSBACH研究组在Nature上发表了茶碱和安定分子印迹聚合物的报道，引起了人们对MIT的极大关注，使得MIT得到了迅速的发展[1-2]。

MIT是一个为目标分子合成人工抗体的过程。分子印迹聚合物(molecularly imprinted polymers, MIPs)的合成过程是以目标分子为模板(template)，将具有结构互补的功能单体(functional monomers)与模板分子结合后，使用交联剂(cross linker)聚合在一起，然后将目标分子去除，便会留下一系列大小、形状与目标分子相匹配的结合位点。MIPs不仅具有类似天然抗体识别的特异性、高选择性和高结合能力等特点，还具有更好的稳定性、制备过程简单并可重复使用等优点[3]。

1.2 MIPs的制备 MIPs的制备过程如图1所示。根据模板分子与功能单体相互作用的原理不同，传统MIPs的制备方法主要包括预组装法、自组装法、结合法和金属螯合法[1]。

图1 MIPs制备过程示意图Fig.1 The preparation process of molecularly imprinted polymers

然而，传统的MIPs制备方法均为不可控的自由基聚合法，这就导致MIPs存在非特异性的结合位点、结合位点分布不均一以及聚合层厚度不均一等问题。为了克服这些缺点，近几年研究人员将可控活性自由基聚合法引入到MIT中，该法通过控制溶液中参与反应的自由基的钝化，从而控制整个反应的速度和进程[4]。可控活性自由基聚合法主要包括以下几种：

1.2.1 可逆加成-断裂链转移聚合法(reversible addition fragmentation chain transfer polymerization, RAFT) 该法制备过程中需要有可逆加成序列的存在，通过控制该序列中活跃链和休眠链间双硫酯键的转移来控制聚合的进程。该法所得聚合物的分子量分布、分子大小和分子结构都可以实现有效控制[5]。

1.2.2 引发-转移-终止聚合法(initiation and transfer termination polymerization, ITTP) 该法是利用二硫代氨基甲酸盐分裂成初始化的烷基以及稳定的第二种自由基过程来制备。由于第二种自由基无法引发新的聚合链，当向该反应供给热量或者减少紫外辐射时两种自由基会重组而停止链增长，之后再进行下一次引发聚合，因此会在一定程度上实现了在线控制[6]。

1.2.3 原子转移自由基聚合法(atom transfer radical polymerization, ATRP) 该法是通过可逆的氧化还原反应产生多种活性基团，经金属离子/配体复合物所催化，该催化剂则可以被从休眠态到活化态所转移的卤素原子所氧化，从而使复合物到达一个高氧化状态[7]。

1.2.4 氮氧自由基聚合法(nitroxide mediated polymerization, NMP) 该法是一个热可逆终止反应，烷氧基胺C-ON键的均裂会促使该反应的发生，从而烷基和氮氧自由基被激活，可以通过控制自由基产生的量来控制整个反应的进程[8]。

2 MIPs在药物分离与分析中的应用

与常规的分离或分析用的色谱固定相比较，MIPs的突出特点是对目标分子具有高度的选择性，同时还具有良好的物理化学稳定性，能够耐受高温、高压、酸碱、有机溶剂等，容易保存、制备简单、易于实现规模化制备，因而得到比较广泛的应用。MIPs在药物分离与分析中主要被应用于手性药物分析、体内药物分析、中药活性成分分离与分析以及药物杂质分离与分析等几个方面。

2.1 手性药物分析 手性药物(chiral drug)的拆分一直是制药工业、临床药物分析和环境检测领域的研究热点。目前世界临床使用的合成药物中，手性药物占40%，且87%以上的手性药物是以外消旋体的形式出售[9]。随着对手性药物研究的不断深入，人们已经发现手性药物的药效学、药动学和毒理学可表现出质和量的区别，这就对分离手性药物的技术提出了新的要求。尽管目前已有手性合成、酶拆分以及其它的分离技术，但MIPs在分离对映体方面有独到之处，而且MIPs作为色谱固定相进行手性拆分也是MIPs早期的研究重点。目前已成功分离的手性药物见表1。

表1 MIPs用于对手性药物的分析Tab.1 The application of MIPs for the analysis of chiral drugs

尽管MIPs作为手性固定相已经研究二十余年，但其仍未投入商业使用，原因可能是由于MIPs手性固定相仅能针对特定手性药物进行分离，广谱适用性较差；另外，缺乏高纯度的光学异构体作为模板，也限制了其应用。

2.2 体内药物分析 体液成份复杂，干扰物质多，而待测药物浓度一般都很低。如何方便、快捷地对样品进行预处理，将少量的目标药物从大量复杂的生物基质中分离出来，以便准确定性、定量，是体内药物分析面临的首要问题[28]。近年来，随着药物分析技术的不断提高，生物样品预处理技术得到迅速发展，并克服了传统萃取方式回收率低、繁琐费时、溶剂毒性大、易乳化等不足。其中，固相萃取技术(solid phase extraction, SPE)因其对目标物具有回收率高、有机溶剂用量少、无相分离操作、能处理微量样品以及易于自动化等优点而被广泛和普遍应用的样品前处理方法[29]。然而，由于常规固相萃取吸附材料(如硅胶或C18材料等)与目标分析物之间的作用是非特异性的，常常因萃取介质对目标物的选择性较差的原因，在吸附目标物的同时也引入了部分基质和干扰物，使得对目标物的分析受到干扰[30]。而分子印迹固相萃取是以MIPs为萃取介质的固相萃取技术，MIPs具有从复杂样品中特异地吸附目标分子的能力，这一性质使得MIPs非常适合作为SPE萃取介质实现对复杂样品中的目标物选择性的分离和富集[31]。

自1994年SELLERGREN等[32]以戊咪为模板分子制备了MIPs并将其作为固相萃取吸附剂用于对尿液中戊咪的分离后，将MIPs与SPE结合用于对体内药物分析的研究呈现出迅速发展。分子印迹-固相萃取(molecularly imprinted-solid phase extraction, MISPE)可用于对各种复杂样品中目标物的分离、纯化和浓缩，涉及到的生物样品包括各种体液、组织和排泄物等[33]，被检测过化合物多为药物及其代谢物、毒物或其他有害物质等(表2)。

2.3 环境样品中药物的分离与分析 鉴于环境样品中目标物的浓度低、组分复杂且易变化，因而其检测对技术的要求越来越高。MIPs用作固相萃取剂可克服环境样品体系复杂的预处理手续，为这些样品的采集、富集和分析提供极大的方便，尤其在痕量分析中发挥重要作用，毫摩尔水平下的痕量物质经这一方法预富集处理后，在色谱中则易于检出(表3)。随着MIT的不断深入和应用领域的不断拓展，MIT在环境领域定会有更广阔的应用前景。

2.4 中药活性成分的分离与分析 中药是一个复杂的、结构多样性的天然组合化学库，其所含的化合物结构类型多样、量悬殊且许多成分未知，导致对中药活性成分分离和分析的难度较大[76]。目前分离和分析中药活性成分主要依赖于硅胶柱色谱(silica gel column chromatography)、大孔吸附树脂柱色谱(macroporous adsorption resin column chromatography)、聚酰胺柱色谱(polyamide column chromatography)、凝胶柱色谱(gel column choromatography)、高速逆流色谱(high-speed countercurrent chromatography)、制备型高效液相色谱(preparative high performance liquid chromatography)等技术，为了更好地对活性成分进行分离和分析，常需经多种溶剂萃取和反复柱色谱，不仅溶剂消耗量大、环境污染严重，而且效率和收率也低[77]。基于MIT制备的MIPs具有亲和性强和选择性高、抗恶劣环境能力强、稳定性好、使用寿命长、应用范围广等特点[78]，而且MIPs具有其他分离材料所不具备的特异性和选择性，MIT将在中药目标成分的分离和分析中具有广阔的应用前景。目前用MIT研究的中药成分有黄酮类、多元酚类、生物碱类、甾体类、香豆素类和木脂素类等(表4)，均取得了良好的效果。

表2 MIPs用于对体内样品中目标物的分析Tab.2 The application of MIPs for the analysis of targets in biological samples

表3 MIPs用于对环境样品中药物的分离与分析Tab.3 The application of MIPs for the analysis of different types of drugs

表4 MIPs用于中药活性成分的分离与分析Tab.4 The application of MIPs for the separation and analysis of the active ingredients in traditional Chinese medicines

然而，MIPs在中药活性成分分离与分析中的应用也有一定的局限性：①有些模板十分昂贵或难于获取，且模板需用量较大，导致成本过高；②目前制备相对分子质量较大的目标物的MIPs还有一定的困难，需进一步开发中药活性成分的模板；③MIPs的吸附容量受外界条件影响较大、较不稳定，因此对目标物的回收率常出现波动；④MIPs对模板的结构类似物也会有一定的交叉识别作用，因此吸附物很难保证是单一模板化合物，吸附后还需进一步纯化等。

2.5 药物杂质的分离与分析 药物杂质控制是药品质量研究中的一项重要内容。药物中的杂质含量低、来源广泛、结构类型多且与主成分类似，必须选择合适的分析技术进行研究[110]。MIPs的高选择性、高强度、高耐用性及可重复使用等诸多优点使得MIT在药物杂质分离和分析中也显示出良好的应用前景。

NAJAFI等[111]采用MIPs-电位传感器分析吡罗昔康中的杂质2-氨基吡啶(2-AP)，结果显示，该传感器对2-AP有高度识别性；HASHEMI等[112-113]制备的MIPs分别用于从盐酸苯海拉明原料药和氟伏沙明原料药中特异识别具有潜在遗传毒性的杂质；BALAMURUGAN等[114]合成了分子印迹液相色谱柱用于检测伪麻黄碱硫酸盐对映体杂质，结果显示，两个对映体的分离度不低于2，拖尾因子小于3.5，分离效果良好；LUO等[115-116]采用表面分子印迹法制备了MIPs，该MIPs可以从青霉素原料药中特异识别具潜在致敏性的青霉噻唑酸；DU等[117]采用溶胶凝胶法制备的MIPs可从氯唑西林钠原料药中特异识别氯唑西林噻唑酸杂质；SZÉKELY等[118]制备的MIPs结合有机溶剂纳滤，可实现从药物活性组分中对具有遗传毒性的1,3-diisopropylurea的特异识别和分离。因此，MIT的不断发展为药物杂质的分离和分析提供了新的方法。

2.6 生物大分子的分离与分析 以蛋白质、DNA、多糖等生物大分子为模板的MIT是一个崭新而且富有挑战性的研究。MIT利用仿生原理进行分子识别，它具有的高特异性、高亲和性以及良好的稳定性都为其在生物大分子的分析应用奠定了基础，已经成功被印迹的生物大分子具体见表5。

表5 MIPs用于生物大分子的分离与分析Tab.5 The application of MIPs for the separation and analysis of biomacromolecules

目前，分子印迹作为一种新型的技术用于生物大分子印迹也有许多局限性[118,142]：①生物大分子和聚合物单体官能团间的相互作用缺乏系统研究，在识别过程中是孔穴的三维空间结构还是功能基团或其他方面起主导作用还无共识；②目前还没有令人满意的聚合方法以得到高吸附量的MIPs，这就使得MIT远远不能满足实际应用的需要；③尚未找到合适的方法洗脱蛋白质印迹分子，活性蛋白质很难得到回收利用。

3 展 望

近年来，随着MIT的不断发展，其作为一种方便、有效、操作简单的分离技术，已经被广泛应用于生物样品预处理、体内微量药物和代谢产物的检测、毒物和药物滥用分析、食品中药物的非法添加和残留分析、原料药及制剂中有关物质的富集和检查等方面；更由于MIT具有易于产业化等优点，越来越受到广大研究者的重视，在药学领域中也发挥了越来越大的作用，并显示出独特的优势和良好的应用前景。

然而，作为一种新型的分离、富集手段，MIT自身还存在一定的问题：①MIPs与目标物识别的作用机制研究相对肤浅；②功能单体、交联剂和聚合方法的选择有很大局限性；③该技术在诸多的应用研究较多，但目前商业化的步伐缓慢，如何加速MIT的商业化是研究者需要关注的问题；④MIPs分离和富集目标物的过程易于受到外界诸多因素的影响，如何提高其重复性的问题亟待解决；⑤尽管对生物大分子的印迹研究已取得了一定的进展，但所采用的方法多数普适性较差，方法本身也不够成熟，实验结果的重现性不好，制备过程较烦琐，迄今研究过的对象也比较少；⑥由于聚合条件的限制，MIPs在水相条件下聚合以及在水相条件下应用的研究相对滞后，而且其在水环境下的识别机制也不甚清楚。因此，MIT还需要随着新型材料的应用和新技术的推进从而进一步发展和完善。

尽管MIT具有上述种种局限性，但由于其具备很高的选择性以及优良的理化特性，其必然会随着技术本身的不断发展和应用的逐渐增多，将在药物的分离与分析领域发挥越来越大的作用。

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(编辑 卓选鹏)

Molecular imprinting technology and its application in separation and analysis of drugs

FU Qiang, LUO Zhi-min, GUO Peng-qi

(Department of Pharmaceutical Analysis, School of Pharmacy of Xi’an Jiaotong University Health Science Center, Xi’an 710061, China)

The separation and analysis of trace drugs and drug-related substances in complex systems is a common issue in the field of pharmaceutical research. Establishment of high-sensitivity, high-selectivity, high-speed, automatic, continuous, and intelligent analysis technology is an important direction for the development of pharmaceutical analysis. The establishment of the new method has played a positive role in promoting further understanding of life process and the role of drug effect, guaranteeing drug quality and improving drug efficacy. Molecular imprinting technology (MIT) has attracted wide concern among researchers in recent years because of its characteristics of predetermined structure-activity, recognition specificity, long-term stability and wide applicability. This articles briefly introduces the principles and preparation methods; and reviews the application of MIT in the separation and analysis of chiral drugs, drugsinvivo, the active ingredients of traditional Chinese medicines, and the biomacromolecules. Besides, the problems and future development are also discussed.

molecular imprinting technology; drug separation; pharmaceutical analysis

傅强，西安交通大学医学部药学院药物分析学系教授，医学博士，博士生导师，享受国务院政府特殊津贴专家。主要从事药物色谱分析、毒物分析及药品质量标准研究工作。近年来主持国家自然科学基金面上项目4项，发表研究论文156篇，授权发明专利7项，主编教材和著作16部。已指导毕业硕士研究生45名，博士研究生8名。主持的项目曾获陕西省高等学校科学技术一等奖、陕西省科学技术一等奖、吴阶平医学研究奖——保罗·杨森药学研究奖、西安交通大学教学名师奖、王宽诚育才奖。兼任教育部高等学校药学类专业教学指导委员会委员(2013～2017)，中国高等医学教育药学教育研究会常务理事，中国药学会药物分析专业委员会委员(第7届、第8届)，中国药学会药学教育专业委员会委员(第1届)，J PharmANal、药物分析杂志、西安交通大学学报(医学版)等杂志编委。

2016-09-06

2016-09-28

国家自然科学基金资助项目(No.81573391, 81603073)

Supported by the National Natural Science Foundation of China (No.81573391 and 81603073)

傅强. E-mail: fuqiang@xjtu.edu.cn

R914.1

A

10.7652/jdyxb201606001

专家介绍

优先出版：http://www.cnki.net/kcms/detail/61.1399.R.20161010.1506.002.html(2016-10-10)