BACKGROUND: Conventional protein microarrays prepared on hard, dry substrates, such as glass and silicone, have several limitations, as proteins may easily denature and lose their structure. To overcome such problems, the fabrication of wet protein microarrays on non-fouling and hydrated PEG-based hydrogels was investigated. RESULT: Bovine serum albumin (BSA) and glucose oxidase (GOX), chosen as model proteins, were covalently immobilized on PEG hydrogel surfaces via 5-azidonitrobenzoyloxy N-hydroxysuccinimide, a photoreactive bifunctional linker. Successful fixation of the bifunctional linker and subsequent immobilization of the proteins on the PEG hydrogel surfaces were confirmed with X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) studies. GOX immobilized on the hydrogel surface maintained approximately 50% of its initial activity after 24 h when left in dry conditions, but maintained only 20% when immobilized on a dry substrate. Photochemical fixation combined with photolithography produced well-defined protein micropatterns with sizes ranging from 50-500 mu m, and molecular recognition-mediated specific binding between biotin and streptavidin was successfully assayed using microarrays on PEG hydrogels. CONCLUSION: A protein-repel lent PEG hydrogel surface was photochemically modified to covalently immobilize proteins and create protein microarrays. The use of hydrated hydrogels as substrates for protein microarrays could minimize the deactivation of proteins in dry conditions, and the non-fouling property of PEG hydrogels allows the passivation step of protein microarray preparation to be skipped. (C) 2008 Society of Chemical Industry