An ELISA-Inspired Self-Propagating Click-Fluoresce-and-Release Assay for the Ultrasensitive Detection of CD13 in Lung Cancer Patient Plasma.

Reaction-based fluorescent probes often suffer from limited sensitivity in biomarker detection due to slow reaction kinetics and inherent stoichiometric constraints. Inspired by the decoupling of capture and detection in the enzyme-linked immunosorbent assay (ELISA), we developed a click fluoresce and release (CFR) system, which is a two-step detection platform comprising a capture probe and a detection probe. The capture probe converts the detection of an enzyme biomarker into the detection of tetrazine, which is then specifically recognized by the detection probe through a fluorogenic click reaction. Importantly, this click reaction simultaneously regenerates a molecule of tetrazine, establishing a self-propagating cycle for signal amplification. CFR demonstrates superior detection limits and enhanced ability to discriminate low biomarker levels compared to conventional reaction-based fluorescent probes, as validated by ultrasensitive detection of CD13 in complex biofluids. Notably, with this strategy, we found that plasma CD13 levels are significantly elevated in advanced lung cancer patients compared with early-stage patients and healthy participants. Given its modular design, CFR can be readily adapted for the sensitive detection of various biomarkers by simply replacing the capture probe.