Zr4+-mediated hybrid chain reaction and its application for highly sensitive electrochemical detection of protein kinase A

Abstract

An electrochemical platform has been developed to detect protein kinase activity through the combined actions of Zr4+ mediated signal transition and hybridization chain reaction (HCR)-stimulated DNAzymes nanowires. First of all, protein kinase A (PKA) phosphorylates substrate peptides immobilized on gold electrode surface. Thereafter, the DNA1 containing 5′-phosphoryl ends is linked to the phosphorylated substrate peptide via the robust phosphate-Zr4+-phosphate linkages. By the introduction of molecular beacons (MBs), the DNA1 can open the hairpin structures of MBs through toehold mediated strand displacement (TMSDR), leading to an autonomous stem-opening process and subsequent assembly of G-quadruplex-containing DNA chains by HCR. After the addition of hemin, the formed HRP-mimicking DNAzymes can catalyze the hydroquinone-H2O2 system to generate amplified electrochemical signals. As expected, this method can achieve ultrahigh analytical performance with a low detection limit of 0.02U/mL and exhibit high cost-savings potential without the need for antibody, protease and labeling. Therefore, this method can serve as a new tool for the assay of protein kinase A and its inhibitor screening in the future.