Fluorescence immunoassay based on alkaline phosphatase-induced in situ generation of fluorescent non-conjugated polymer dots

Alkaline phosphatase (ALP) activity assay is not only significant to the clinical diagnosis of some related disease, but also momentous to the construction of ALP-based enzyme-linked immunosorbent assay (ELISA). Herein, for the first time, we have discovered that ascorbic acid (AA) can specially react with N-methylethylenediamine (N-MEDA) to generate fluorescent non-conjugated polymer dots (NCPDs) under mild conditions. On the basis of the AA-responsive emission and ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate (AA2P) to AA, we have exploited a fluorometric ALP activity assay with high sensitivity and selectivity. Furthermore, by means of conventional ALP-based ELISA platform, a conceptual fluorescent ELISA has been constructed and applied in the potential clinical diagnosis, during which cardiac troponin I (cTnI), a well-established biomarker of acute myocardial infarction, has been chosen as the model target. We envision that such original fluorescent NCPDs generation-enabled ELISA could become a versatile tool in biochemical sensing and medical diagnosis in the future.