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 (PKA) activity assay. Multiple signal amplification techniques were employed including the nanocomposite of gold nanoparticles and carbon nanospheres ( [email protected] ), the biocomposite of SiO 2 and streptavidin (SiO 2 -SA), the composite of AuNPs and biotinylated β -galactosidase (AuNPs-B-Gal) and in situ enzymatic generation of electrochemical activity molecule of p -aminophenol. After peptides were assembled on [email protected] modified electrode surface , they were phosphorylated by PKA 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, SiO 2 -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 PKA concentration, an innovative electrochemical assay could be realized for PKA 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 PKA in human normal gastricepithelial cell line and human gastric carcinoma cell line with satisfactory results.