A signal "on" photoelectrochemical biosensor for assay of protein kinase activity and its inhibitor based on graphite-like carbon nitride, Phos-tag and alkaline phosphatase.

A highly sensitive and selective photoelectrochemical (PEC) biosensor is fabricated for the detection of protein kinase activity based on visible-light active graphite-like carbon nitride (g-C3N4) and the specific recognition utility of Phos-tag for protein kinase A (PKA)-induced phosphopeptides. For assembling the substrate peptides, g-C3N4 and gold nanoparticles (g-C3N4-AuNPs) complex is synthesized and characterized. When the immobilized peptides on g-C3N4-AuNPs modified ITO electrode are phosphorylated under PKA catalysis, they can be specifically identified and binded with biotin functionalized Phos-tag (Phos-tag-biotin) in the presence of Zn(2+). Then, through the specific interaction between biotin and avidin, avidin functionalized alkaline phosphatase (avidin-ALP) is further assembled to catalyze its substrate of l-ascorbic acid-2-phosphate trisodium salt (AAP) to produce electron donor of ascorbic acid (AA), resulting an increased photocurrent compared with the absence of phosphorylation event. Based on the specific identification effect of Phos-tag, the fabricated biosensor presents excellent selectivity for capturing the phosphorylated serine residues in the substrate peptides. With the good photoactivity of g-C3N4 and ALP-catalyzed signal amplification, the fabricated biosensor presents high sensitivity and low detection limit (0.015 unit/mL, S/N = 3) for PKA. The applicability of this PEC biosensor is further testified by the evaluation of PKA inhibition by HA-1077 with the IC50 value of 1.18μM. This new strategy is also successfully applied to detect the change of PKA activity in cancer cell lysate with and without drug stimulation. Therefore, the developed PEC method has great potential in screening of kinase inhibitors and highly sensitive detection of kinase activity.