Nowall W B, Dontha N, Kuhr W G
Department of Chemistry, University of California, Riverside 92521, USA.
Biosens Bioelectron. 1998 Nov 15;13(11):1237-44. doi: 10.1016/s0956-5663(98)00030-x.
Photolithographic techniques using a laser interference pattern were used to attach photobiotin to micron-sized stripes on the surface of a carbon electrode. Fluorophore-tagged avidin was attached to this spatially-patterned biotin with essentially no loss in spatial resolution. The kinetics of the glassy carbon surface were examined to see if electron transfer sites could indeed be segregated from the attachment sites of photobiotin-immobilized avidin. The ECL of luminol and SECM were used to verify the segregation between underivatized sites (which exhibit normal electron transfer kinetics) and extensively derivatized biotin/avidin surfaces (which presumably exhibit slow electron transfer kinetics). Both techniques were found to be capable of differentiating the protein-covered surface from bare carbon with sufficient resolution to tell whether a significant portion of the carbon surface is still active and available to detect the product of an enzyme generated analyte. These results indicate that extensive biotin/avidin derivatization of the surface does decrease the electron transfer rate of a carbon electrode, and that the photolithographic approach was able to modify specific sections of the electrode surface, while leaving other regions untouched and available for facile electron transfer. This leads to a more general protocol for the construction of enzyme-based biosensors which utilize diffusable mediators.
利用激光干涉图案的光刻技术被用于将光生物素附着到碳电极表面的微米级条纹上。带有荧光团的抗生物素蛋白附着到这种空间图案化的生物素上，空间分辨率基本没有损失。研究了玻碳表面的动力学，以确定电子转移位点是否确实可以与光生物素固定化抗生物素蛋白的附着位点分离。使用鲁米诺的电化学发光和扫描电化学显微镜来验证未衍生化位点（表现出正常电子转移动力学）和大量衍生化的生物素/抗生物素蛋白表面（推测表现出缓慢电子转移动力学）之间的分离。发现这两种技术都能够以足够的分辨率区分蛋白质覆盖的表面和裸露的碳，从而判断碳表面的很大一部分是否仍然活跃并可用于检测酶产生的分析物的产物。这些结果表明，表面的大量生物素/抗生物素蛋白衍生化确实会降低碳电极的电子转移速率，并且光刻方法能够修饰电极表面的特定部分，同时使其他区域保持不变并可用于便捷的电子转移。这导致了一种更通用的构建基于酶的生物传感器的方案，该方案利用可扩散的介质。
