Immobilized enzymes in microfluidic systems

Advances in high-throughput chemical and biological analysis have involved the incorporation of biomolecules onto the functional surfaces of microfabricated devices. These devices require biomolecules to be spatially localized and can roughly be divided into two categories: microarrays and microfluidics. While microfluidic systems have the potential to perform more complex biochemical analysis, the development of these fluidic systems has been comparatively slow due in part to the complexities of immobilizing biological molecules within a microfluidic format.; Porous silica sol-gels are developed as an enzyme immobilization matrix suitable for microfluidics. Mercury porosimetry and nitrogen adsorption are used to investigate the pore structures of different gels as a function of the initial sol precursors. It is observed that the appropriate combination of alkoxysilanes produces a favorable bimodal pore structure with macropores to allow fluid transport and micropores to entrap enzyme. The gel utility is demonstrated by observing fluorescent product formation in situ of trypsin entrapped within a microfluidic channel. Laminar flow is used to pattern enzyme-containing gel. This patterned gel is integrated with electrochemical detection (ECD), creating a device capable of measuring the hydrogen peroxide produced from entrapped glucose oxidase.; Porous methacrylate polymers are also developed as a microfluidic enzyme immobilization matrix. Proteins are photo-patterned to the polymer surface and nonspecific protein adsorption is minimized through the use of non-ionic surfactants and grafted poly(ethylene glycol). The fluorescent product of a horseradish peroxidase reaction is measured in situ and the kinetics of the immobilized enzyme are analyzed with various chemical engineering techniques. Finally a sequential reaction involving invertase, glucose oxidase, and horseradish peroxidase illustrates how the direction of fluid flow may be used to determine the sequence of a multi-step reaction.; The immobilization methods presented demonstrate two simple techniques for patterning enzymes in microfluidic channels. The advantages of the microfluidic approach are demonstrated by the use of the fluid flow rate and direction to control the residence time and reaction sequence of multi-enzymatic reactions. These techniques are expected to enable applications such as combinatorial reactions and synthetic metabolic pathways to be performed in a high-throughput microfluidic format.