Parallel assay of inkjet-printed cytochrome P450

Assaying the activities of cytochrome P450 (P450) is important for evaluating the toxicity of chemicals in drugs, food, and the environment. We developed a methodology to immobilize multiple P450 isoforms with a heightened density and assay their activities in parallel. Inkjet printing technology was applied for the deposition of P450 onto a plastic substrate (cycloolefin polymer: COP) having arrayed microwells (diameter: 50 μm, depth: 60 μm). P450 was printed into each microwell and immobilized by gelation of co-printed agarose. Activities of printed P450 isoforms were measured by introducing a substrate solution into the microwells and sealing it with an elastomer sheet, isolating individual wells. In order to avoid uncontrolled initiation of the reaction, we photo-regulated the P450 catalysis by using caged glucose-6-phosphate. The combination of inkjet printing, immobilization in microwells, and photo-regulated initiation of the enzymatic reaction enabled us to assay multiple P450 isoforms on a chip with an unprecedented density. We demonstrate the parallel assay of single nucleotide polymorphs of P450, which play important roles in different drug efficacies in individuals.