This study focuses on the development of fluorescence-based biosensor to measure the C-telopeptide fragments of type II collagen (CTX-II) as an osteoarthritis (OA) biomarker. When the OA progresses, joint components such as cartilage collagen are degraded by protease and secreted into urine. Therefore the OA could be monitored by measuring the urinary CTX-II (uCTX-II). Because urinary CTX-II has monomeric or variant monomeric epitope, conventional sandwich assay format is unavailable. Thus, we introduced a competitive immunoassay to accurately measure the uCTX-II. In this study, the PEG4-EKGPDP was used as a competitive molecule for uCTX-II and antibody-conjugated fluoro-microbeads was employed as an optical probe. The optical probes are competitively reacted to the PEG4-EKGPDP-immobilized sensing surface with the target uCTX-II in urine sample. uCTX-II concentration from 200 ng/mmol to 1,400 ng/mmol (corrected value vs. creatinine) was applied. By counting the number of beads from result images, the uCTX-II was quantified. The developed competitive immunoassay for uCTX-II analysis exhibited good correlation with conventional ELISA with fast response, covering the required clinical detection ranges for osteoarthritis diagnosis. Based on the result, we suggest that the developed assay could be a promising tool for OA diagnosis.
