title: Enzyme-containing hydrogel micropatterns serving a dual purpose of cell sequestration and metabolite detection.
abstract: 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 (H(2)O(2)) 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 H(2)O(2) and HRP, was either immobilized inside hydrogel elements alongside enzyme molecules or added into the cell culture media during cell activation. The production of H(2)O(2) 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.
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, 451 Health Sciences Drive #2519, Davis, CA 95616, United States.
authors.forename: Jun
authors.initials: J
authors.lastname: Yan
authors.forename: Yinghua
authors.initials: Y
authors.lastname: Sun
authors.forename: He
authors.initials: H
authors.lastname: Zhu
authors.forename: Laura
authors.initials: L
authors.lastname: Marcu
authors.forename: Alexander
authors.initials: A
authors.lastname: Revzin
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods; D015374:Biosensing Techniques / Q000295:instrumentation; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002460:Cell Line; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020100:Hydrogels / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000032:analysis*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008846:Micromanipulation / Q000295:instrumentation / Q000379:methods; D010088:Oxidoreductases / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
chemical_list: D004800:Enzymes, Immobilized; D020100:Hydrogels; D006861:Hydrogen Peroxide; D010088:Oxidoreductases