title: A highly sensitive electrochemiluminescence assay for protein kinase based on double-quenching of graphene quantum dots by G-quadruplex-hemin and gold nanoparticles.
abstract: A highly sensitive electrochemiluminescence (ECL) strategy was developed for the protein kinase A (PKA) activity and inhibition assay based on double-quenching of graphene quantum dots (GQDs) ECL by G-quadruplex-hemin DNAzyme and gold nanoparticles (AuNPs). In this strategy, the GQDs were modified onto the indium-tin oxide (ITO) electrode and further assembled with substrate peptide of target protein kinase through covalent coupling, which can exhibit high and stable ECL signal. The AuNPs, functionalized with the phosphorylated DNA and G-quadruplex-hemin DNAzyme via Au-S chemistry, were selected as quenching probes. In the presence of PKA, the peptide on the electrode was phosphorylated and the AuNPs functionalized with the phosphorylated DNA and G-quadruplex-hemin DNAzyme were subsequently integrated onto the phosphorylated peptide by Zr(4+). Owing to the reduction of coreactant H2O2 resulting from G-quadruplex-hemin DNAzyme catalytic reaction and the ECL energy transfer (ECL-RET) between AuNPs and GQDs, the ECL intensity of GQDs was significantly decreased. By taking advantage of the double-quenching effect, this assay can detect PKA with a linear range of 0.05 to 5 U mL(-1) and a detection limit of 0.04 U mL(-1). In addition, the PKA inhibition assay and interferences experiments of CK2 and T4 PNK have been studied respectively. This assay was also successfully applied to PKA assay in serum samples and cell lysates, indicating that the developed method have the potential applications in protein kinase-related biochemical fundamental research and clinical diagnosis.
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors: Jinquan
authors: J
authors: Liu
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China. Electronic address: xiaoxiaohe@hnu.edu.cn.
authors: Xiaoxiao
authors: X
authors: He
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China. Electronic address: kmwang@hnu.edu.cn.
authors: Kemin
authors: K
authors: Wang
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors: Dinggeng
authors: D
authors: He
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors: Yonghong
authors: Y
authors: Wang
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors: Yinfei
authors: Y
authors: Mao
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors: Hui
authors: H
authors: Shi
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors: Li
authors: L
authors: Wen
mesh_terms: D003217:Conductometry / Q000295:instrumentation; D021881:DNA, Catalytic / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D054856:G-Quadruplexes; D006046:Gold / Q000737:chemistry; D006108:Graphite / Q000737:chemistry*; D006427:Hemin / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008163:Luminescent Measurements / Q000295:instrumentation*; D053768:Metal Nanoparticles / Q000737:chemistry*; D011494:Protein Kinases / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D045663:Quantum Dots; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation
chemical_list: D021881:DNA, Catalytic; D006427:Hemin; D006046:Gold; D006108:Graphite; D011494:Protein Kinases
keywords: Electrochemiluminescence; G-quadruplex–hemin DNAzyme; Gold nanoparticles; Graphene quantum dots; Protein kinase A