title: Enhanced Photoelectrochemical Method for Sensitive Detection of Protein Kinase A Activity Using TiO2/g-C3N4, PAMAM Dendrimer, and Alkaline Phosphatase.
abstract: A novel photoelectrochemical (PEC) assay is developed for sensitive detection of protein kinase A (PKA) activity based on PKA-catalyzed phosphorylation reaction in solution and signal amplification strategy triggered by PAMAM dendrimer and alkaline phosphatase (ALP). In this strategy, it is noteworthy at this point that PKA phosphorylation was achieved in solution instead of on the surface of the electrode, which has advantages of the good contact in reactants and simple experimental procedure. For immobilizing the phosphorylated peptide (P-peptide) on electrode surface, graphite-like carbon nitride (g-C3N4) and titanium dioxide (TiO2) complex is synthesized and characterized, which plays a significant role for TiO2conjugating phosphate groups and g-C3N4providing PEC signal. Subsequently, PAMAM dendrimer and ALP can be captured on P-peptide and TiO2/g-C3N4modified ITO electrode via interaction between the -COOH groups on the surface of PAMAM dendrimer and the -NH2groups of peptide and ALP, which can lead to the increase of ALP amount on the modified electrode surface assisted with the PAMAM dendrimer. As a result, the amount of ALP catalyzes of L-ascorbic acid 2-phosphate trisodium salt (AAP) to produce electron donor of ascorbic acid (AA), resulting in an increased photocurrent. The proposed detection assay displays high selectivity and low detection limit of 0.048 U/mL (S/N = 3) for PKA activity. This biosensor can also be applied for the evaluation of PKA inhibition and PKA activity assay in cell samples. Therefore, the fabricated PEC biosensor is potentionally well in PKA activity detection and inhibitor screening.
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University , Taian, 271018, P. R. China.
authors.forename: Xue
authors.initials: X
authors.lastname: Li
authors.affiliation: College of Resources and Environment, Shandong Agricultural University , Taian, 271018, P. R. China.
authors.forename: Lusheng
authors.initials: L
authors.lastname: Zhu
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University , Taian, 271018, P. R. China.
authors.forename: Yunlei
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University , Taian, 271018, P. R. China.
authors.forename: Huanshun
authors.identifier: 0000-0002-5022-0314
authors.initials: H
authors.lastname: Yin
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University , Taian, 271018, P. R. China.
authors.forename: Shiyun
authors.initials: S
authors.lastname: Ai
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D001205:Ascorbic Acid / Q000737:chemistry; D055162:Biocatalysis; D015374:Biosensing Techniques; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000737:chemistry; D050091:Dendrimers / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000379:methods*; D004566:Electrodes; D006108:Graphite / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D053758:Nanoparticles / Q000737:chemistry; D009570:Nitriles / Q000737:chemistry*; D010766:Phosphorylation; D017971:Tin Compounds / Q000737:chemistry; D014025:Titanium / Q000737:chemistry*
chemical_list: D050091:Dendrimers; D009570:Nitriles; C104700:PAMAM Starburst; D017971:Tin Compounds; C009495:titanium dioxide; C011206:cyanogen; C109984:indium tin oxide; D006108:Graphite; D014025:Titanium; D017868:Cyclic AMP-Dependent Protein Kinases; D000469:Alkaline Phosphatase; D001205:Ascorbic Acid