Enzyme-containing hydrogel micropatterns serving a dual purpose of cell sequestration and metabolite detection

The integration of sensing elements with small groups of cells is a critical step towards miniaturization of cell cultivation and analysis. This paper describes the development of an optical, enzyme-based biosensor for local detection of hydrogen peroxide (H₂O₂) secreted by stimulated macrophages. Photolithographic patterning of horseradish peroxidase (HRP)-containing poly (ethylene glycol) (PEG) hydrogel microstructures was used to create sensing structures on the glass surface. Importantly, enzyme-entrapping hydrogel micropatterns did not support protein or cell deposition and allowed to guide attachment of macrophages next to the sensing elements. Amplex Red, an organic molecule that becomes fluorescent in the presence of H₂O₂ and HRP_, was either immobilized inside hydrogel elements alongside enzyme molecules or added into the cell culture media during cell activation. The production of H₂O₂ after mitogenic stimulation of macrophages resulted in appearance of fluorescence in the HRP-containing hydrogel microstructures, with fluorescence intensity being a strong function of analyte concentration. The novel cell culture system with integrated sensing elements described here may be enhanced in the future by incorporating additional biorecognition elements to enable multi-metabolite detection at the site of a cell.