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| We firstly transformed the traditional Michaelis–Menten equation into an off-line form which can be used for evaluating the Michaelis–Menten constant after the enzymatic reaction. For experimental estimation of the kinetics of enzymatic reactions, we developed a facile and effective method integrating an enzyme microreactor into a direct-printing polymer microchip. Strong nonspecific adsorption of proteins was utilized to immobilize effectively enzyme onto microchannel wall, forming the integrated on-column enzyme microreactor in a microchip. The property of the integrated enzyme microreactor was evaluated by using the enzymatic reaction of glucose oxidase (GOx) with its substrate glucose. Reaction product-hydrogen peroxide was electrochemically (EC) detected using a Pt microelectrode. Rapid determination of the enzyme kinetics using our off-line form of the Michaelis–Menten equation was achieved (Km= 2.64 mM), which is much smaller than that reported in solution (Km= 6.0 mM). Due to the hydrophobic property and the native mesoscopic structure of the poly(ethylene terephthalate) film as indicated by the atomic force microscopic measurement, the immobilized enzyme in the microreactor shows good stability and bioactivity under the flowing conditions. |
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| Keywords:kinetics of enzymatic reactions, microreactor, enzyme, polymer microchip, glucose oxidase |
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