We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location.First,a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsi-loxane)/glass microchannel.The channel was then filled with an aqueous biotin-linked dye solution.Using an Ar~+/ Kr~+ laser,the fluorophore moieties were bleached to create highly reactive species.These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism.Enzymes linked to strepta-vidin or avidin could then be immobilized via (strept)-avidin/biotin binding.Using this process,we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions.The density of alkaline phosphatase in the patches was calculated to be ~5% of the maximum possible density by comparison with known standards.Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy.A more complex two-step enzyme reaction was also designed.In this case,avidin-linked glucose oxidase and streptavidin-linked horseradish per-oxidase were sequentially patterned in separate patches inside straight microfluidic channels.Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase,patterned downstream,where fluorogenic substrate turnover was recorded.
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