Microfluidic chip immunoassay based on rolling circle amplification and G-quadruplex/Thioflavin T for multiplex detection of CTX I.

A label-free immunoassay based on rolling circle amplification (RCA) and G-quadruplex/Thioflavin T (G4/ThT) is proposed to realize the sensitive detection of carboxy-terminal cross-linked fragment of type I collagen (CTX I) for bone loss. Under the optimal conditions, as low as 38.02 pg/mL of CTX I can be detected. To improve the detecting throughput and simplify the operation, a microfluidic chip was designed, fabricated, and used for CTX I detection based on the proposed assay. The detection can be completed with only a single on-chip magnetic separation step, which was easy to operate, less time-consuming, and has only low reagent consumption. The limit of detection was 131.83 pg/mL by observing with fluorescence microscope. With further improvement of detection equipment, the sensitivity of on-chip detection can be improved. It can be expected that the proposed RCA/G4/ThT immunoassay for sensitive and high-throughput automated detection of CTX I might be chosen as a potential analytical tool for clinical osteoporosis diagnosis and in-orbit bone loss detection.