Horseradish peroxidase-triggered direct in situ fluorescent immunoassay platform for sensing cardiac troponin I and SARS-CoV-2 nucleocapsid protein in serum.

Direct in situ fluorescent enzyme-linked immunosorbent assay (ELISA) is rarely investigated and reported. Herein, a direct in situ high-performance HRP-labeled fluorescent immunoassay platform was constructed. The platform was developed based on a rapid in situ fluorogenic reaction between Polyethyleneimine (PEI) and p-Phenylenediamine (PPD) analogues to generate fluorescent copolymer nanoparticles (FCNPs). The formation mechanism of FCNPs was found to be the oxidation of •OH radicals, which was further proved by nitrogen protection and scavenger of •OH radicals. Meantime, the fluorescence wavelength of FCNPs could be adjusted from 471 to 512 nm by introducing various substitution groups into the PPD structure. Using cardiac troponin I (cTnI) and SARS-CoV-2 nucleocapsid protein (N-protein) as the model antigens, the proposed fluorescent ELISA exhibited a wide dynamic range of 5-180 ng/mL and a low limit of detection (LOD) of 0.19 ng/mL for cTnI, and dynamic range of 0-120 ng/mL and a LOD of 0.33 ng/mL for SARS-CoV-2 N protein, respectively. Noteworthy, the proposed method was successful applied to evaluate the cTnI and SARS-CoV-2 N protein levels in serum with satisfied results. Therefore, the proposed platform paved ways for developing novel fluorescence-based HRP-labeled ELISA technologies and broadening biomarker related clinical diagnostics.

Fluorescence copolymer-based dual-signal monitoring tyrosinase activity and its inhibitor screening via blue-green emission transformation.

Tyrosinase (TYR) is a crucial enzyme in melanin metabolism and catecholamine production, its abnormal overexpression is closely associated with many human diseases involving melanoma cancer, vitiligo, Parkinson's disease and so on. Herein, a dual-signal fluorescence sensing system for monitoring TYR activity is constructed depending on the transformation of blue-green fluorescence emission of copolymer. The developed sensing system is based on TYR catalyzing the hydroxylation of mono-phenol to o-diphenol and the conversion of fluorescence copolymer (FCP) blue emission (430 nm) and green emission (535 nm) in the presence of PEI. In the system, both blue and green emission exhibit a high selectivity and sensitivity (S/B up to 300 and 30 for blue and green emission, respectively) toward TYR in the range from 0.5 to 2.5 U/mL with the detection limit of 0.002 U/mL and 0.06 U/mL, respectively. Additionally, this assay is used to detect TYR in human serum with excellent recovery even at 30% human serum concentrations. Furthermore, it still has been successfully applied to TYR inhibitor screening by taking kojic acid as a model. We believe that our developed sensor has great potential application in TYR-associated disease diagnosis and treatment and drug discovery.