Simultaneous immunoassay of phosphorylated proteins based on apoferritin templated metallic phosphates as voltammetrically distinguishable signal reporters

A novel electrochemical immunosensor has been developed to detect phosphorylated proteins, phospho-p5315 and phospho-p53392, simultaneously. Different apoferritin templated metal phosphates were used as distinguishable signal reporters (apoferritin templated cadmium phosphates (ATCP) and apoferritin templated lead phosphates (ATLP)) to enhance the detection sensitivity. Here, magnetic Fe3O4 nanoparticles functionalized phospho-p5315 capture antibody (MP-p5315c-Ab) and phospho-p53392 capture antibody (MP-p53392c-Ab), respectively, were used to specifically capture phospho-p5315 and phospho-p53392 antigens, followed by immunorecognition with p5315 detection antibody (p5315d-Ab) and p53392 detection antibody (p53392d-Ab) to form sandwich-like immunocomplexes. SiO2@Au nanocomposites served as nanocarriers for co-immobilization of both d-Ab and signal reporters (ATCP/SiO2@Au/p5315d-Ab, ATLP/SiO2@Au/p53392d-Ab), which greatly amplified the detection signal. The distinguished current responses were achieved by electrochemical detection of cadmium ions and lead ions with square wave voltammetry (SWV) after dissolution with acid. The proposed immunoassay exhibited high sensitivity and selectivity for the detection of phospho-p5315 and phospho-p53392 simultaneously. The linear relationships between electrochemical responses and the concentrations of phospho-p5315 and phospho-p53392 were obtained in the range of 0.1-20 ng/mL and 0.05-20 ng/mL, respectively. The detection limits were 0.05 and 0.02 ng/mL (S/N=3), respectively. This strategy provides a new platform for simultaneous immunoassay of multiple protein biomarkers.