Rapid Homogeneous Fluorescent Enzyme-Linked Immunosorbent Assay (ELISA) for N-Terminal Brain Natriuretic Peptide (NT-proBNP) Combining Magnetic Probes and Ampliflu Red

Abstract

Heart failure (HF) is the leading cause of death from cardiovascular disease, and early diagnosis using serum markers holds extraordinary significance for the treatment of patients. N-terminal brain natriuretic peptide (NT-proBNP) is the gold standard for HF diagnosis. Herein, a novel, rapid, sensitive, and immune-sensing strategy for NT-proBNP is reported employing an enzyme-linked immunosorbent assay (ELISA) that combines the use of magnetic biological probes and the fluorogenic substrate Ampliflu Red. Functionalized immunomagnetic nanoparticles with large surface areas provide an abundant reaction platform and reduce the analysis time due to their superparamagnetic properties. Ampliflu Red is catalyzed by horseradish peroxidase (HRP) using hydrogen peroxide (H2O2) to produce strong fluorescence and a low background. The reported method achieved a good detection limit of 2.73 ng mL−1 and a wide linear range from 3.13 to 100 ng mL−1. The analysis time was less than 1 h, which is superior to that of traditional ELISA (3–4 h). In addition, the recoveries of spiked human serum were satisfactory, indicating that the proposed strategy has potential applications for the rapid determination of NT-proBNP and other biomarkers in clinical settings.