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光化学活化电极:在可逆免疫传感器和抗体图案化界面设计中的应用。

Photochemically-activated electrodes: application in design of reversible immunosensors and antibody patterned interfaces.

作者信息

Blonder R, Ben-Dov I, Dagan A, Willner I, Zisman E

机构信息

Institute of Chemistry, Hebrew University of Jerusalem, Israel.

出版信息

Biosens Bioelectron. 1997;12(7):627-44. doi: 10.1016/s0956-5663(97)00022-5.

Abstract

Antigen monolayers assembled onto Au electrodes associated with a quartz crystal act as electrochemical or microgravimetric quartz-crystal-microbalance (QCM) sensing interfaces for the complementary antibody. Electrochemical analysis of the antibody (Ab) is based on the insulation of the antigen monolayer electrode by the associated Ab towards a redox probe in the electrolyte solution. Ferrocene-modified glucose oxidase (Fc-GOx) and glucose are employed as redox probes for the amperometric transduction of the Ab association to the electrode. Bioelectrocatalyzed oxidation of glucose provides an electrochemical route to amplify the antigen-Ab complex formation. Electrochemical analysis of the dinitrophenyl antibody, DNP-Ab, by a dinitrophenyl-lysine monolayer electrode is presented. QCM analysis of the Ab is based on the frequency changes of the quartz crystal resulting from the association of the Ab to the crystal assembly. This method is discussed with the analysis of the fluorescein antibody, Flc-Ab, using a fluorescein monolayer-modified quartz crystal. A novel method to tailor reversible immunosensor devices by the application of photoisomerizable antigen monolayers on electrodes is presented. The antigen is modified by photoactive units exhibiting reversible photoisomerizable properties. In one photoisomer state, the antigen exhibits affinity for the Ab and enables its electrochemical or QCM analysis. Photoisomerization to the complementary state perturbs the antigen structure and the monolayer lacks affinity for the Ab. This enables the washing-off of the Ab and the regeneration of the actively sensing interface by a second illumination process that restores the antigen monolayer-modified surface. This method is exemplified by the development of a reversible DNP-Ab sensing electrode. N-Mercaptobutyl dinitrospiropyran was assembled as a photoisomerizable monolayer on a Au electrode. The dinitrospiropyran monolayer, SP-state, exhibits affinity for the DNP-Ab and enables the amperometric detection of the Ab using Fc-GOx and glucose as redox probe. The complementary photoisomerized protonated dinitromerocyanine monolayer, MRH(+)-state, lacks affinity for the DNP-Ab. By photoisomerization of the DNP-Ab associated with the SP-monolayer electrode to the MRH(+)-monolayer state, the DNP-Ab is washed-off, and by a second illumination process, the MRH(+)-monolayer is re-isomerized to the SP-monolayer assembly, which is the active interface for further analysis of the DNP-Ab. Cyclic amperometric detection of the DNP-Ab by the photoisomerizable dinitrospiropyran monolayer is demonstrated. The association of the DNP-Ab to the SP-monolayer electrode and the dissociation of the Ab from the MRH(+)-monolayer electrode are confirmed by QCM experiments using a dinitrospiropyran monolayer-modified quartz crystal. The insulating features of an antigen-Ab complex on a conductive surface and the photochemically controlled association of an antibody to a photoisomerizable monolayer assembled onto the surface were used to develop means for micropatterning of surfaces by the antibody. A dinitrospiropyran antigen monolayer was assembled onto conductive ITO glass. A DNP-Ab solution was used as 'ink solution' to pattern the surface. The Ab-pattern was imaged by electrochemical copper deposition onto the Ab-lacking surface domains. The dinitrospiropyran monolayer assembled onto ITO or Pyrex glass surfaces was employed as an active interface for the photolithographic patterning of the surface with the DNP-Ab. (ABSTRACT TRUNCATED)

摘要

组装在与石英晶体相连的金电极上的抗原单层,可作为互补抗体的电化学或微重力石英晶体微天平(QCM)传感界面。抗体(Ab)的电化学分析基于相关抗体使抗原单层电极对电解质溶液中的氧化还原探针绝缘。二茂铁修饰的葡萄糖氧化酶(Fc-GOx)和葡萄糖用作氧化还原探针,用于将抗体与电极的结合进行安培检测。葡萄糖的生物电催化氧化提供了一条电化学途径来放大抗原-抗体复合物的形成。本文介绍了通过二硝基苯基赖氨酸单层电极对二硝基苯基抗体(DNP-Ab)进行电化学分析。抗体的QCM分析基于抗体与晶体组件结合导致石英晶体的频率变化。使用荧光素单层修饰的石英晶体对荧光素抗体(Flc-Ab)进行分析时讨论了该方法。提出了一种通过在电极上应用光致异构化抗原单层来定制可逆免疫传感器装置的新方法。抗原通过具有可逆光致异构化特性的光活性单元进行修饰。在一种光异构体状态下,抗原对抗体表现出亲和力,并能够对其进行电化学或QCM分析。光异构化为互补状态会扰乱抗原结构,单层对抗体缺乏亲和力。这使得抗体能够被洗脱,并通过恢复抗原单层修饰表面的第二次光照过程使活性传感界面再生。通过开发可逆的DNP-Ab传感电极来举例说明该方法。N-巯基丁基二硝基螺吡喃作为光致异构化单层组装在金电极上。二硝基螺吡喃单层(SP态)对DNP-Ab表现出亲和力,并能够使用Fc-GOx和葡萄糖作为氧化还原探针进行抗体的安培检测。互补的光异构化质子化二硝基部花青单层(MRH(+)态)对DNP-Ab缺乏亲和力。通过将与SP单层电极结合的DNP-Ab光异构化为MRH(+)单层状态,DNP-Ab被洗脱,并且通过第二次光照过程,MRH(+)单层重新异构化为SP单层组件,这是用于进一步分析DNP-Ab的活性界面。展示了通过光致异构化二硝基螺吡喃单层对DNP-Ab进行循环安培检测。使用二硝基螺吡喃单层修饰的石英晶体通过QCM实验证实了DNP-Ab与SP单层电极的结合以及抗体从MRH(+)单层电极的解离。导电表面上抗原-抗体复合物的绝缘特性以及抗体与组装在表面上的光致异构化单层的光化学控制结合被用于开发通过抗体对表面进行微图案化的方法。将二硝基螺吡喃抗原单层组装在导电的ITO玻璃上。使用DNP-Ab溶液作为“墨水溶液”对表面进行图案化。通过将电化学铜沉积到缺乏抗体的表面区域来对抗体图案进行成像。组装在ITO或派热克斯玻璃表面上的二硝基螺吡喃单层用作与DNP-Ab进行表面光刻图案化的活性界面。(摘要截断)

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