Analytical Sciences

Single-step Trypsin Inhibitor Assay on a Microchannel Array Device Immobilizing Enzymes and Fluorescent Substrates by Inkjet Printing

Yuko KAWAI,* Kotaro IDEGAMI,** Kenji SUEYOSHI,*,*** Tatsuro ENDO,* and Hideaki HISAMOTO*

*Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka, Sakai, Osaka 599-8531, Japan
**Sysmex Corporation, 1-5-1 Wakinohamakaigandori, Chuo, Kobe, Hyogo 651-0073, Japan
***Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), 5-3 Yonban-cho, Chiyoda, Tokyo 102-8666, Japan

In this paper, we report a single-step trypsin inhibitor assay on a microchannel array device immobilizing enzymes and substrates by inkjet printing. The microdevice is composed of a poly(dimethylsiloxane) (PDMS) microchannel array that immobilizes trypsin and fluorescent substrates as reactive reagents at the two bottom corners of a microchannel. Inkjet printers allow simple, accurate, and position-selective immobilization of reagents as nanoliter spots. Therefore, plural reactive reagents, such as enzymes and substrates, can be separately immobilized at different positions in the same microchannel without mixing, and thus allowing for single-step operation by simply introducing a sample solution through capillary action. Furthermore, reproducible fabrication and mass production of the device could be expected. In this study, the efficiency of an acidic solution as a spotting agent for protease immobilization to prevent decrease in the fluorescence intensity was confirmed. Additionally, single-step trypsin inhibitor screening was performed using three inhibitors. Finally, we investigated the storage stability of the device and confirmed that it remained stable for at least 10 days.

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