分子印迹电化学传感器制备及在蛋白质检测上的应用
2017-07-18刘艳丽李小军贺晓荣李延斌李红朝
刘艳丽,李小军,贺晓荣,李延斌,李红朝
分子印迹电化学传感器制备及在蛋白质检测上的应用
刘艳丽1,李小军2,贺晓荣1,李延斌1,李红朝1
(1中北大学化学系,山西太原 030051;2中国五环工程有限公司,湖北武汉430223)
分子印迹电化学传感器是分子印迹技术与分析传感器技术相结合的一种先进技术,它结合分子印迹的优点,避免了传统传感器的缺点,提高了电化学传感器的选择性和灵敏度,并且缩短了响应时间,更因其设计简单、经济实用等优点受到越来越多领域的欢迎。本文介绍了分子印迹传感器的5种常用的制备方法,包括涂层法、原位聚合法、电聚合法、溶胶-凝胶法和自组装法以及这5种方法在实际中的应用,重点介绍了4种分子印迹传感器(MIPs电容/阻抗型、MIPs电导型、MIPs电位型、MIPs电流型)在蛋白质检测上的应用,并且其检测方式以及时间都达到了预期的效果,相信随着技术的更新发明与创造,分子印迹电化学传感器的检测领域会拓展到更多的领域。
分子印迹;电化学传感器;蛋白质
分子印迹技术作为近年迅速发展起来的一门新的化学分析技术,主要过程就是形成与目标分子的化学功能互补的人工识别元素或者腔体[1]。如图1所示,一个分子印迹的基本过程主要包括3个步骤:①形成功能单体——模板复合物;②功能单体间的聚合及交联;③将模板分子从聚合物中脱去。多次实验结果表明,分子印迹技术在不同的条件下都表现出其易制备、选择性及稳定性良好的特点,并且可以建立特异的结合主位点[2],从而在传感器[3]、色谱[4]、药物的释放[5]以及固相萃取[6]等方面有广泛的应用,同时在化学和生物交叉的学术领域中得到了快速的发展和应用。
分子印迹电化学传感器是将分子印迹技术应用在传感器上,该技术综合二者的显著优点,在对生物大分子的分析检测上,已经表现出拥有简单、低成本的设计以及合理的准确度和精密度等优点[7-13]。而且与其他技术相比,电化学传感器低敏感性的特征使其在检测过程中减少了由于衍生化和提取步骤的时间,更加缩短了响应时间[14]。1990年电化学传感器的出现,更加引起人们对将分子印迹技术应用于蛋白质[15-17]、DNA[1,18-21]等关于生命的生物大分子研究的关注。而蛋白质是人身体组织、器官的重要构成,直接影响并参与大部分生命活动[22]。随着社会进步、人类生命质量的提高以及科学技术的进步,各类有关蛋白质的检测技术包括电泳、离子交换色谱法、高效液相离子交换层析(HPLC)、硼酸亲和层析、免疫分析法等都快速推进,但这些方法都存在特异性差、稳定性不足、测定时间长等劣势。因此建立一种具有高效、专一、特异性强、方便快速的检测方法也成为重点研究的对象。而分子印迹传感器的出现,不仅解决了传统方法的缺陷,更是对医学上蛋白质检测的发展提供了更适宜的技术[23]。近几年来,分子印迹技术与传感器的结合已经开始逐渐成为蛋白质检测的可行的替代方案[24-28],且该新技术在保质期、稳定性、稳健性、成本以及制备工艺方面有着更为突出的优点[29]。然而,蛋白质 这种大型的生物大分子因其本身易受各类因素 的影响,形成的诸多不稳定的可变构象对印迹过程提出了各种挑战[28,30-32]。这是由于在分子印迹合成的过程中,大尺寸的蛋白质和分子印迹网的密度一起影响着在本体和表面上的形成的分子印迹聚 合物[33]。
1 分子印迹电化学传感器的制备方法
分子印迹电化学传感器的制备方法主要有涂附分子印迹聚合物法、原位引发聚合法、电聚合法、溶胶-凝胶法以及自组装5种方法。
1.1 涂附分子印迹聚合法
涂层法是指通过蘸涂、滴涂、旋涂的方法将制备好的分子印迹聚合物修饰到电极表面上,待其表面溶剂蒸发掉后就在电极表面形成了分子印迹聚合物敏感膜。这种方法作为最简单的制备方法不需要额外加入交联剂和引发剂。FANG等[34]利用该方法在金电极的表面均匀涂附上光敏聚合物,待金电极表面溶剂蒸发以后就形成可以检测葡萄糖的分子印迹电化学传感器,但是该传感器的选择性略差,所以在实际制备中应用的较少。
1.2 原位引发聚合法
原位引发聚合法是指利用光或热的作用下,将含有单体、模板分子、引发剂的混合溶液涂附到传导装置,并且在传导装置上引发聚合,然后在其表面形成分子印迹膜。例如,BAI等[35]利用原位聚合法以丙烯酰胺(AM)为功能单体,乙二醇二甲基丙烯酸酯(EGDMA)为交联剂,在石墨烯表面修饰玻碳电极制作出青蒿素分子印迹膜(ART-MIMS),在最优条件下优化制作出的分子印迹传感器ART-MIM/G/GCE在测定青蒿素以及青蒿素的类似物例时有较高的选择性、灵敏性和抵抗性。RIBEIRO等[36]利用原位热聚合法,以绿原酸为模板,将多壁碳纳米管(MWCNT)与改性过后玻碳极与分子印迹硅氧烷结合起来,所制备的传感器可以从一些高等植物如水果、蔬菜、红茶或者某些中药中检测出绿原酸,其检出限高达0.035µmol/L。
1.3 电化学聚合法
电化学聚合法是直接在传感器的表面上制作分子印迹膜的一个过程,起初传感器是充当一个模板的作用[37],随后模板去除后就形成了与模板分子互补的分子印迹结合位点[38-39]。其中印迹膜的厚度控制着电荷的转移,溶剂和电解质的选择影响着表面形态,而膜的刚性和孔隙度是由溶剂的溶胀度和电解质中离子含量的多少决定的[40]。例如YANG 等[41]以速灭威为模板,用电化学聚合法氧化还原氨基苯甲酸(P-ABA),通过结合有序介孔材料(CMK-3)提高修饰电极的结构促进电荷的转移,并以普鲁士蓝(PB)作为固有的电化学活性探针,制备出具有三维结构的分子印迹电化学传感器,见图2。该传感器具有高表面多孔结构,对目标分子——速灭威具有稳定良好的特异性选择吸附,其快速响应和可重复性利用的特点也使其在环境和安全检测方面有很好的应用。龙芳等[42]在磁性石墨烯修饰的碳电极表面以辛基酚为模板分子,多巴胺为功能单体,利用电化学聚合法制备出对辛基酚有高灵敏度和高选择性的分子印迹电化学传感器,与传统传感器相比,该传感器有较宽的线性范围和检出限,同时其磁性石墨烯的利用提高了稳定性和重现性,为以后的制备技术提供了更好的思路。
1.4 溶胶-凝胶法
分子印迹溶胶-凝胶法制备出来的分子印迹聚合膜最显著的特点是该膜除了具有特异性识别的膜之外,其孔大的特点允许无机或有机分子在里面自由穿透,并且刚性的结构在面对强酸、强碱、高温、高压时不易被破坏。该方法结合了分子印记技术和溶液-溶胶技术的基本上所有的优点,使得该传感器技术有更高的选择特异性,并且大大消除了分子印迹技术在稳定性、惰性和刚性方面的缺点。SANTOS等[43]用咖啡因和其他类似分子为模板,利用溶胶-凝胶技术将用多层碳纳米管和乙烯基三甲氧基硅烷修饰的玻碳电极与分子印迹硅氧烷结合起来,其制备的传感器对咖啡因及其他类似分子的检出限达0.22µmol/L。DING等[44]通过该技术在Ru2+修饰后的玻碳电极表面沉积聚合成ECL-MIP(电化学发光分子印迹)传感器,该传感器可成功快速检测到L-苯基丙氨酸,检出限高达3.1×10–12mol/L。
1.5 自组装法
自组装法主要特点是借助单体和印迹分子之间以非共价键、氢键、范德华力、螯合力等弱作用力以及模板分子与功能单体之间的自组织排列,自发形成具有多重作用位点的单体模板分子复合物,经交联聚合后这种作用保存下来。该方法形成的传感器具有稳定性强、构造简单且不受基地材料影响的特点[45]。例如PAN等[46]研究出以甲氟磷酸异丙基酯为模板,用环糊精自组装的方法制作出具有高度选择性和灵敏性的分子印传感器,该传感器在不同的温度下都能够检测到低浓度的沙林。此外,该技术还可以与其他制备方法相结合,如张燕等[47]将自组装法和电化学聚合法结合起来,在金电极的表面制备了对一种选择性检测盐酸阿霉素的分子印迹电化学传感器。LUO等[48]将分子自组装法和分子印迹技术结合起来,以扑息通为模板分子,将纳米粒子嵌入模板形成分子印迹的电活性纳米颗粒后,在其表面形成一层稳定的分子印迹膜的同时聚合一个咔唑单元,在移除模板扑息通模板分子以后,就形成一个对扑息通有选择性的电化学传感器,同时该传感器优越的稳定性和重复性已经能够应用于药物和人类尿液的检测上,检出限高达0.3µmol。
GCE—玻碳电极;CMK-3/GCE—介孔有序材料玻碳电极;PB-CMK—3/GCE-普鲁士蓝-介孔有序材料-玻碳电极
1.6 5种制备方法的优缺点
通过对5种制备方法的内容及应用的分析,其优缺点的归纳如表1所示。
表1 5种方法的优缺点
2 分子印迹电化学传感器的应用
分子印迹电化学传感器有MIPs电容/阻抗型、MIPs电导型、MIPs电位型、MIPs电流型4种电化学传感器。其中MIPs电流型传感器的应用最为 广泛。
2.1 MIPs电容/阻抗型传感器在蛋白质检测上的应用
该类型的传感器不需要加入试剂或探针,以印迹膜识别前后电容或者阻抗的与慕白哦分析物的变化关系作为识别信号,操作简单并且灵敏度高。在实际生产中制造超薄和超高的绝缘性能分子印迹薄膜是该类传感器制造过程中的关键技术。CAI等[49]以苯酚为基体,在玻璃基板上生长碳纳米管阵列,嵌入SU8-2002聚合材料,用以检测人体铁蛋白阻抗型传感器。KHAN等[50]发明了一种检测蛋白的阻抗型电化学传感器,在制备过程中,先将单壁碳纳米管放置在丝网印刷电极上用于制备分子印迹聚合物,然后再用循环伏安法以蛋白为模板,聚合3-对氨基苯酚里制备具有高选择和高精确度的阻抗型传感器,其中电化学阻抗谱会记录随着蛋白的浓度所引起的工作电极点特性的变化,检出限高达16.3nmol/L。
2.2 MIPs电导型传感器在蛋白质检测上的应用
该类型的传导器只需简单地将分子印迹膜固化在探头表面,以被测分子与印迹膜键和引起的导电性能的变化作为识别信号。在实际应用中,尽量减少微量杂质的含量,以减少在膜的制备和冲洗过程中引起的电导性能的变化。LI等[51]采用光聚合法在丝网印刷电极表面原位聚合制备出含有琥珀酸氯霉素(ns—CAP)分子印迹位点的分子印迹膜。将修饰有分子印迹膜的丝网印刷电板与电导分析仪相连接,组装可检测牛奶蛋白样品中氯霉素的电导型传感器,结果表明,传感器对该分子具有良好的特异性识别能力。检出限高达0.05mg/L。
2.3 MIPs电位型传感器在蛋白质检测上的应用
该类型的传导器对印迹分子的大小没有限制,探针无需经过印迹膜直接利用分子在短暂的识别过程前后电极电位的变化作为识别信号,其化学修饰后用于导入的电极直接影响着对目标物种的选择性和灵敏度[52]。MOREIRA等[53]通过在硅珠表面自组装有机硅烷单分子膜制备了对肌红蛋白特异性选择的电位型分子印迹传感器。WANG等[54]用类似方法,在Au涂层表面自组装多羟基硫醇单分子膜,建立起的电位型分子印迹传感器对肌红蛋白和血红蛋白都有优异的选择性。
2.4 MIPs电流型传感器在蛋白质检测上的应用
该类型的传感器是目前应用最多的一类传感器,可以直接测定含有电活性的物质,通过底物分子特异性识别前后,印迹分子的浓度和固定电位下的响应电流的变化作为识别信号,也可以将没有活性的物质在加入电活性物质如铁氰化钾[55]作为探针后再进行特异性识别后间接测定。目前该类传感器经常与纳米材料相结合来提高对蛋白质的识别与测定[56]。LI等[57]就将二者结合起来,在玻璃电极表面加入了金纳米颗粒和普鲁士蓝离子用来提高电化学传感器的敏感性,在模板蛋白存在的基础下通过电化学聚合的方法氧化还原丙烯酰胺。模板提取蛋白质后,印迹腔可以选择性地重新绑定血红蛋白,并且作为电极通道影响着普鲁士蓝的电流峰值,检出限高达0.05mg/L。VICTORIA等[58]制备了一种可检测人类血清中肌红蛋白的分子印迹电化学传感器,传感器的制备方法如图3所示,其血红蛋白的浓度直接影响着电流的大小,检出限高达9ng/mL,该检测也可直接用于检测心肌梗塞患者和健康的捐赠者血清中含有的血红蛋白的多少,在医学方面将会有更广泛的应用领域。
3 结语
分子印迹电化学传感器的高度特异性和对蛋白质的高亲和性以及良好的稳定性为其在其他生物大分子测定中的应用奠定了扎实的基础。但是分子印迹电化学传感器还存在很大的进步空间,比如将来更多的信号不单单只局限在二维上,应该更多地产生三维图像,随着该类技术的日益成熟,分子印迹电化学传感器将不再只局限在蛋白质一类物质的测定上,还会应用在其他的一些大分子如核酸、DNA、多糖等分子上的测定。而且新型材料的应用,例如石墨烯、荧光技术、MOF等材料与磁性材料、纳米材料的结合应用也会对该传感器的性能产生更大的提升空间。
-PD—邻苯二胺;Mb—肌红蛋白;Poly--PD—聚邻苯二胺
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The fabrication of molecularly imprinted electochemical sensorand its application in protein detection
LIU Yanli1,LI Xiaojun2,HE Xiaorong1,LI Yanbin1,LI Hongchao1
(1Department of Chemistry,North University of China,Taiyuan 030051,Shanxi,China;2China Rings Engineering Co.,Ltd.,Wuhan 430223,Hubei,China)
Molecular imprinting electrochemical sensor is a combination of molecular imprinting technology and analytical sensor technology,which possess the advantages of molecular imprinting technique,avoids the disadvantages of traditional sensors,improves the electrochemical sensor sensitivity and selectivity,and shortens the response time. Because of its simple design,economical and practical advantages,this technology becomes popular in more and more areas. In this paper,five commonly used preparation methods for molecular imprinting sensors include coating method,polymerization method,electric polymerization method,sol-gel method and self-assembly method are introduced,and the application of these five methods are discussed. The application of four molecular imprinted sensors(MIPs capacitance/impedance type,MIPs conductivity type,MIPs potential type,MIPs current type)in protein detection was introduced,and its detection methods and time to achieve the desired results. Believe that with the technical update of the invention and creation,molecular imprinting electrochemical sensor detection field will be expanded to more areas.
molecular imprinting;electrochemical sensors;protein
TQ317
A
1000–6613(2017)07–2533–07
10.16085/j.issn.1000-6613.2016-2080
2016-11-14;
2017-03-17。
国家自然科学基金青年基金项目(21404093)。
刘艳丽(1990—),女,硕士研究生。E-mail:707933269@qq.com。
联系人:李延斌,博士,副教授。E-mail:lyb2010@nuc.edu.cn。