We develop a micro analysis and detection system for replicase of the hepatitis C virus. The fabricated analysis system consists of a bead based microchip and a computer based controller. Four pneumatic microvalves, one microchannel, two microheaters, two micro temperature sensors and two microreators for bead packing are fully integrated in one microchip to realize several sequences for the detection and the digestion of replicase. Four pneumatic valves are used that two reaction chambers can be connected or disconnected by the valve control. Controllers for the temperature and the PDMS pneumatic valve actuation are realized with the LabVIEW, a commercialized computer program. Two reaction chambers are thermally isolated because the enzymes immobilized on the beads are highly sensitive to the temperature. We use two kinds of beads for capturing and digesting the target protein. The first reaction chamber is packed with the PEG-grafted PS bead on which RNA aptamer of HCV replicase is immobilized. The second reaction chamber is packed with the PEG-grafted bead on which trypsin is immobilized covalently. Heat is applied to release, denaturation and digestion of the captured protein. First of all, 20 μl mixture of proteins (HCV replicase, HCV helicase, BSA, ovalbumin) was injected into the first reaction chamber and then incubated at 25°C. After incubation, the first reaction chamber was washed with buffer intensively to remove non-specifically adsorbed protein. The captured HCV replicase was released from beads through protein denaturation by heating the first reaction chamber. 20μl HCV replicase was transferred to the second reaction chamber packed with trypsin-immobilized PS beads and incubated to digest HCV replicase into peptides. 20μl solution was eluted from microchip finally and analyzed with MALDI-TOF. As a result, we could acquire six matched peaks (12 % sequence coverage) of HCV replicase within about 20 min using a fabricated microchip and a control system.

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