A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser

Micrometer-sized enzyme grids were fabricated on gold surfaces using a nove l method based on a flow-through microdispenser. The method involves dispen sing very small droplets of enzyme solution (similar to 100 pL) during the concomitant relative movement of a gold substrate with respect to the nozzl e of a microdispenser, resulting in enzyme patterns with a line width of si milar to 100 mum. Different immobilization methods have been evaluated, yie lding either enzyme monolayers using functionalized self-assembled thiol mo nolayers for covalent binding of the enzyme or enzyme multilayers by cross- linking or entrapping the enzymes in a polymer film. The latter immobilizat ion techniques allow the formation of coupled multienzyme structures. On th e basis of this feature, coupled bienzyme (glucose oxidase and catalase) or three-enzyme (alpha -glucosidase, mutarotase, and glucose oxidase) microst ructures consisting of line patterns of one enzyme intersecting with the pa tterned 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 l ocalized detection of enzymatically produced/consumed H2O2. A calibration c urve for glucose could be obtained by subsequent SECM line scans over a glu cose oxidase microstructure for increasing glucose concentrations, demonstr ating the possibility of obtaining localized quantitative data from the pre pared microstructures. Possible applications of these enzyme microstructure s for multianalyte detection and interference elimination and for screening of different biosensor configurations are highlighted.