Dontha N, Nowall W B, Kuhr W G
Department of Chemistry, University of California, Riverside 92521, USA.
Anal Chem. 1997 Jul 15;69(14):2619-25. doi: 10.1021/ac9702094.
Micrometer-sized domains of a carbon surface are modified to allow derivatization to attach redox enzymes with 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 must be kept to a minimum (e.g., less than 5 microns) to maintain the fast response time and high sensitivity required for the measurement of neurotransmitter dynamics. 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 will allow the construction of microscopic arrays of active enzyme sites on a carbon fiber substrate while leaving other sites underivatized to facilitate electron transfer reactions of redox mediators, thus maximizing enzyme activity and detection of the enzyme mediator. The ultimate sensitivity of these sensors will be realized only through careful characterization of the carbon electrode surface with respect to its chemical structure and electron transfer properties following each step of the enzyme immobilization process. The characterization of specific modifications of micrometer 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 avidin) 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. An atomic force microscope was used to obtain high-resolution images that probe the heterogeneity of the enzyme sites.
对碳表面的微米级区域进行修饰，以便利用生物素/抗生物素蛋白技术进行衍生化，从而附着氧化还原酶。这些位点在空间上与同一电极表面上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必须保持在最小限度（例如，小于5微米），以维持测量神经递质动力学所需的快速响应时间和高灵敏度。这是通过利用从电极表面产生的紫外激光的干涉图案，通过光刻法附着光生物素来实现的。这将允许在碳纤维基板上构建活性酶位点的微观阵列，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活性和酶介质的检测。只有在酶固定化过程的每一步之后，通过仔细表征碳电极表面的化学结构和电子转移特性，才能实现这些传感器的最终灵敏度。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成像系统的荧光显微镜来可视化酶固定位点（由德克萨斯红标记的抗生物素蛋白的荧光指示）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。使用原子力显微镜获得高分辨率图像，以探测酶位点的异质性。
