Immobilization of multi-enzyme microreactors inside microfluidic devices

A simple method to fabricate enzyme-containing microscopic hydrogel structures in microfluidic devices for the potential use in micro total analysis systems (μ-TAS) is described. Poly(ethylene glycol)-based hydrogel microstructures were prepared inside microchannels by photolithography and enzymes conjugated to a pH sensitive fluorophore (SNAFL-1) were incorporated into these hydrogel microstructures. Because of the ratiometric pH-dependent nature of SNAFL fluorescence, hydrogel microstructures exhibited a different emission intensity ratio with pH and this intensity ratio changed almost linearly between pH 7 and 12. When alkaline phosphatase-containing microreactors were exposed to p-nitrophenylphosphate (pNPP) as a substrate, phosphoric acid was produced inside the microstructure by enzymatic-catalyzed hydrolysis of the substrate and subsequently decreased the microenvironment pH. Because of the relatively rapid mass transport of analyte through the hydrogel, enzyme-catalyzed reaction was easily detected by change in emission intensity ratio before and after exposure to substrates. Enzyme-catalyzed reactions were quite fast and reached 90% of maximum value within 10 min. Data were analyzed using a modified Michaelis–Menten equation and apparent Michaelis constants could be obtained. This system was also successfully applied to urea hydrolysis by urease.