Cu²⁺-triggered wavelength-tunable real-time in situ fluorogenic immunoassay for detection of cTnI in human serum

Using a single emission-wavelength to accurately/sensitively quantify disease markers in complex environments still faces enormous challenges. In this study, we construct a Cu²⁺-triggered wavelength-tunable fluorescent immunoassay for real-time detection of cTnI in human serum. The developed platform is based on an “on-off-on” strategy, which involves the rapid in-situ fluorogenic reaction of dopamine (DA) and phenol analogue by Cu²⁺, strong coupling between PPi and Cu²⁺ as well as the specific hydrolysis of PPi into orthophosphate by alkaline phosphatase. By using cTnI as a model antigen, the assay shows good linearity relationships between the fluorescence response and cTnI concentration. Furthermore, we used different phenol analogues (1,3-dihydroxynaphthalene, 8-hydroxyjulolidine, 1,5-dihydroxynaphthalene) as fluorescence reaction substrates to achieve multi-channel (blue, green and yellow) real-time monitoring of cTnI with detection limits of 0.17 ng/mL, 0.17 ng/mL and 0.33 ng/mL, respectively. We successfully applied the wavelength-tunable ELISA to detect cTnI levels in real human serum, and the results were compared with a commercial ELISA with satisfactory outcomes. Therefore, our proposed platform demonstrates great potential in the early diagnosis of acute myocardial infarction.