Electrochemical biosensor for protein kinase A activity assay based on gold nanoparticles-carbon nanospheres, phos-tag-biotin and β-galactosidase

A sensitive and selective electrochemical biosensor was fabricated for protein kinase A (PICA) activity assay. Multiple signal amplification techniques were employed including the nanocomposite of gold nanoparticles and carbon nanospheres (Au@C), the biocomposite of SiO2 and streptavidin (SiO2-SA), the composite of AuNPs and biotinylated beta-galactosidase (AuNPs-B-Gal) and in situ enzymatic generation of electrochemical activity molecule of p-aminophenol. After peptides were assembled on Au@C modified electrode surface, they were phosphorylated by PICA in the presence of ATP. Then, biotinylated Phos-tag was modified on electrode surface through the specific interaction between Phos-tag and phosphate group. Finally, SiO2-SA and AuNPs-B-Gal were captured through the specific interaction between biotin and streptavidin. Because the electrochemical response of p-aminophenol was directly related to PICA concentration, an innovative electrochemical assay could be realized for PICA detection. The detection limit was 0.014 unit/mL. The developed method showed high detection sensitivity and selectivity. In addition, the fabricated biosensor can be also applied to detect PICA in human normal gastricepithelial cell line and human gastric carcinoma cell line with satisfactory results. (C) 2016 Elsevier B.V. All rights reserved.