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-p53(15) and phospho-p53(392), 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-p53(15) capture antibody (MP-p53(15)c-Ab) and phospho-p53(392) capture antibody (MP-p53(392)c-Ab), respectively, were used to specifically capture phospho-p53(15) and phospho-p53(392) antigens, followed by immunorecognition with p53(15) detection antibody (p53(15)d-Ab) and p53(392) detection antibody (p53(392)d-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/p53(15)d-Ab, ATLP/SiO2@Au/p53(392)d-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-p53(15) and phospho-p53(392) simultaneously. The linear relationships between electrochemical responses and the concentrations of phospho-p53(15) and phospho-p53(392) 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.