Dontha N, Nowall W B, Kuhr W G
Department of Chemistry, University of California, Riverside 92521, USA.
J Pharm Biomed Anal. 1999 Feb;19(1-2):83-91. doi: 10.1016/s0731-7085(98)00292-1.
Sub-micron sized domains of a carbon surface are derivatized with antibodies using biotin/avidin technology. These sites are spatially-segregated from, and directly adjacent to, electron transfer sites on the same electrode surface. The distance between these electron transfer sites and enzyme-loaded domains are kept to a minimum (e.g. less than a micron) to maintain the high sensitivity required for the measurement of enzyme-linked cofactors in an enzyme-linked immunoassay (ELISA). This is accomplished through the use of photolithographic attachment of photobiotin using an interference pattern from a UV laser generated at the electrode surface. This allows the construction of microscopic arrays of active ELISA sites on a carbon substrate while leaving other sites underivatized to facilitate electron transfer reactions of redox mediators; thus maximizing sensitivity and detection of the enzyme mediator. The carbon electrode surface is characterized with respect to its chemical structure and electron transfer properties following each step of the antibody immobilization process. The characterization of specific modifications of micron regions of the carbon surface requires analytical methodology that has both high spatial resolution and sensitivity. We have used fluorescence microscopy with a cooled CCD imaging system to visualize the spatial distribution of enzyme immobilization sites (indicated by fluorescence from Texas-Red labeled antibody) across the carbon surface. The viability of the enzyme attached to the surface in this manner was demonstrated by imaging the distribution of an insoluble, fluorescent product.
利用生物素/抗生物素蛋白技术，用抗体对碳表面的亚微米级区域进行衍生化处理。这些位点在空间上与同一电极表面的电子转移位点分隔开且直接相邻。这些电子转移位点与酶负载区域之间的距离保持在最小值（例如小于一微米），以维持酶联免疫吸附测定（ELISA）中测量酶联辅因子所需的高灵敏度。这是通过利用来自电极表面产生的紫外激光的干涉图案，对光生物素进行光刻附着来实现的。这使得能够在碳基底上构建活性ELISA位点的微观阵列，同时使其他位点未衍生化，以促进氧化还原介质的电子转移反应；从而最大限度地提高酶介质的灵敏度和检测能力。在抗体固定化过程的每一步之后，对碳电极表面的化学结构和电子转移特性进行表征。对碳表面微米区域的特定修饰进行表征需要具有高空间分辨率和灵敏度的分析方法。我们使用配备冷却电荷耦合器件（CCD）成像系统的荧光显微镜来观察酶固定位点（由德克萨斯红标记抗体的荧光指示）在整个碳表面的空间分布。通过对不溶性荧光产物的分布进行成像，证明了以这种方式附着在表面的酶的活性。
