Metal ion chelation-based color generation for alkaline phosphatase-linked high-performance visual immunoassays

A novel colorimetric immunoassay strategy is developed based on metal ion chelation-induced color generation and alkaline phosphatase(ALP)-catalyzed signal amplification. The intense purple-colored Cu(I)-bicinchoninic acid (BCA) complex is utilized as a promising chromogenic reporter for the visual ALP-linked immunoassay. In the presence of target, ALP is introduced to catalyze the cascade conversion of L-ascorbic acid 2-phosphate (AAO) to L-ascorbic acid (AA), which results in Cu 2+ reduction to Cu + and subsequently in situ formation of purple-colored Cu(I)-BCA complex. The complex is water-soluble and exhibits a strong absorbance at 562 nm due to the ligand-to-metal charge-transfer (LMCT). The absorbance value is in turn proportional to the level of target analyte. Based on this concept, an ALP-linked colorimetric immunoassay is established with Cu(II)-BCA mixture as a color developing reagent for rabbit IgG detection, achieving a linear range from 0.1 ng mL −1 to 25 ng mL −1 and a LOD of 0.05 ng mL −1 . Its potentiality for practical application is also investigated by detection of a cancer biomarker, prostate specific antigen (PSA), in spiked human serum. A linear range from 0.5 ng mL −1 to 25 ng mL −1 and a LOD at 0.38 ng mL −1 are achieved, which is much lower than the cut-off value of PSA in human blood. The proposed method holds many advantages including low cost, good color stability, reliability and excellent compatibility with the existing ELISA platform, providing a promising colorimetric immunoassay platform for bio-chemical analysis in a variety of fields.