We have developed a new strategy to construct a colorimetric biosensor on a miniaturized platform. Polydiacetylene (PDA), which is widely known to change color by external stimuli, was employed as a sensing material. To construct a gel-based portable colorimetric biosensor using PDA, PDA was incorporated into poly(ethylene glycol) (PEG)-based hydrogel upon photopolymerization and PDA-embedded PEG hydrogel matrix was patterned within PDMS-based microfluidic channels. The resulting intrachannel hydrogel matrix containing PDA was reacted with alpha-cyclodextrin as a model target sample, which causes a color change of PDA from blue to red. Parallel analyses were performed inside microchannels for multiple solutions containing different amounts of alpha-cyclodextrin and color change gradients were successfully demonstrated depending on the contacting time and amount of alpha-cyclodextrin. (c) 2005 Elsevier B.V. All rights reserved.