Abstract

The study developed a novel quantitative detection method of homocysteine (Hcy), an independent risk factor for cardiovascular disease, based on an immunofluorescent test strip. The fluorescent nanospheres (F-NSs) were prepared by embedding fluorophores (Cy5) into poly(styrene-acrylate) copolymer nanospheres, then conjugated with streptavidin (SA) to obtain SA/F-NSs as a signal amplification label stored in sample diluents. Biotin–antibody was fixed on the conjugate pad to specifically capture S -adenosyl Hcy (SAH) in the sample, where SAH was transformed from S -adenosyl methionine (SAM) by Hcy S -methyltransferase catalysis using Hcy as a substrate, the formed SAH could be trapped by SA/F-NSs and biotin–antibody conjugates in a competitive way with SAH–bovine serum albumin fixed on the test line, a detection limit of 0.27 μM Hcy was achieved. The fluorescence intensity of F-NSs remained stable during 273 days of storage, the bioactivity of the test strip was stable during 12 months of storage, and the strip possessed good reproducibility (intra-assay variability of 5.8%). Furthermore, other structural analogues SAM and cysteine showed negative results, validating the excellent specificity of the strips.