A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser.
A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser.
复制标题
一种设计和研究通过流通式微分配器形成的酶微结构的方法。
DOI：
10.1021/ac010214e
复制
发表时间：
2001
影响因子：
7.4
通讯作者：
W. Schuhmann
中科院分区：
文献类型：
--
作者：
S. Gáspár;M. Mosbach;L. Wallman;T. Laurell;E. Csöregi;W. Schuhmann
使用基于流通式微量分配器的新颖方法在金表面上制造微米大小的酶网格。该方法涉及在金底物相对于微分配器喷嘴相对运动的过程中分配非常小的酶溶液液滴（约 100 pL），从而产生线宽约 100 微米的酶图案。已经评估了不同的固定方法，使用功能化自组装硫醇单层进行酶的共价结合来产生酶单层，或者通过交联或将酶捕获在聚合物膜中来产生酶多层。后者的固定技术允许形成偶联的多酶结构。基于这一特征，制备了耦合双酶（葡萄糖氧化酶和过氧化氢酶）或三酶（α-葡萄糖苷酶、变旋酶和葡萄糖氧化酶）微结构，该微结构由一种酶的线图案与其他酶的图案线相交组成。通过在发生器-收集器模式下运行的扫描电化学显微镜 (SECM)，利用酶促产生/消耗的 H2O2 的局部检测来可视化酶的微观结构及其完整性。通过随后对葡萄糖氧化酶微结构进行 SECM 线扫描以增加葡萄糖浓度，可以获得葡萄糖的校准曲线，这证明了从制备的微结构中获得局部定量数据的可能性。强调了这些酶微结构在多分析物检测和干扰消除以及筛选不同生物传感器配置方面的可能应用。
Micrometer-sized enzyme grids were fabricated on gold surfaces using a novel method based on a flow-through microdispenser. The method involves dispensing very small droplets of enzyme solution (approximately 100 pL) during the concomitant relative movement of a gold substrate with respect to the nozzle of a microdispenser, resulting in enzyme patterns with a line width of approximately 100 microm. Different immobilization methods have been evaluated, yielding either enzyme monolayers using functionalized self-assembled thiol monolayers for covalent binding of the enzyme or enzyme multilayers by cross-linking or entrapping the enzymes in a polymer film. The latter immobilization techniques allow the formation of coupled multienzyme structures. On the basis of this feature, coupled bienzyme (glucose oxidase and catalase) or three-enzyme (alpha-glucosidase, mutarotase, and glucose oxidase) microstructures consisting of line patterns of one enzyme intersecting with the patterned lines of the other enzyme(s) were fabricated. By means of scanning electrochemical microscopy (SECM) operated in the generator-collector mode, the enzyme microstructures and their integrity were visualized using the localized detection of enzymatically produced/consumed H2O2. A calibration curve for glucose could be obtained by subsequent SECM line scans over a glucose oxidase microstructure for increasing glucose concentrations, demonstrating the possibility of obtaining localized quantitative data from the prepared microstructures. Possible applications of these enzyme microstructures for multianalyte detection and interference elimination and for screening of different biosensor configurations are highlighted.