(A) study on the enzyme assay in microfluidic channels

Abstract

A novel platform technology of enzyme assay using microfluidic channels was developed. The microfluidic device for alkaline phosphatase assay was fabricated by micromolding processes with PDMS (polydimethylysiloxane) polymer. This microfluidic device can be provided a novel assay platform which can overcome limitation of the conventional assay platform, which contains the requirement of special detection instrument, manual dilution of the substrate solution and individual measurement of enzyme reactions. In this study, ELISA reader instrument was used as a detection system and the substrate solution of different concentration was generated by microfluidic channel network. Enzyme reactions were measured in ELISA reader instrument, which occurred in six reaction chambers containing each different concentration of substrate solution. Reaction chambers also functioning detection chambers has been arranged in 96-well formats to be operated by ELISA reader. Overall dimension of the microfluidic device was the same size as a 96-well microplate. Enzyme activity was determined with the continuous methods that are generally an instantaneous observation, which was measured by ELISA reader instrument. Also the enzyme activity is possible to be estimated by stop method. In this thesis, stop method of enzyme assay was used as a standard assay, which showed error bars of less than ±1.7% enzyme activity. Employing the patterns of microfluidic channels, the substrate solution was diluted in serial and the enzyme solution was injected into the detection wells simultaneously. Dilution effect was examined by red food dye with error bars of less than ±9.43% absorbance and the experimental results have difference from the predicted value less than 1.82 % relative absorbance. The predicted values are calculated by the concentration equation derived in this paper. Initial velocity of enzyme reaction is measurable soon after enzyme and substrate solution is mixed together at six detect...