Miniaturization of a colorimetric cellulase activity assay on a microfluidic sensor platform

Enzymes are important commercial products with widespread uses as industrial catalysts, processing aids and analytical reagents. Biological activity is the major critical quality attribute monitored during the enzyme production. Assaying the enzyme activity often requires laborious procedures that are challenging to implement on microfluidic chips for facile on-site use in hand-held mobile devices. Here, we show the integration of a colorimetric assay of cellulase activity into a prototype microfluidic sensor platform on cyclic olefin copolymer foil obtained by roll-to-roll processing. The activity assay was based on resorufin dye release from the corresponding β-cellobioside substrate. Low water solubility of the substrate necessitated a concentration co-optimization for substrate and dimethylsulfoxide co-solvent used in the assay, considering that the co-solvent caused substantial activity loss of the enzyme. A flexible, chip-amenable read-out method was developed based on CIELAB colour space image analysis to quantify both dye release for enzyme activity determination and substrate solubilization at the same time. Information on the substrate solubilization kinetics on chip was crucial to establish the activity assay on the microfluidic platform designed to contain the substrate as a spotted array of resorufin β-cellobioside droplets. We compared the assay conducted in the microfluidic chip with that in conventional microwell plate and found that both formats of analytical measurement could be used with the colour-based read-out method. Overall, we demonstrate cellulase activity monitoring on chip based on identification and optimization of critical parameters of microfluidic assay integration. The analytical approach shown for cellulase may be broadly applicable to the assay of enzyme activity.