Sensitive electrochemical assay of alkaline phosphatase activity based on TdT-mediated hemin/G-quadruplex DNAzyme nanowires for signal amplification.
Taking TdT-mediated hemin/G-quadruplex DNAzyme nanowires as NADH oxidase and HRP-mimicking DNAzyme, a novel DNA-based electrochemical method has been developed for sensitive and selective assay of alkaline phosphatase (AP) activity. The double-stranded DNA (dsDNA) probe consisted of thiol-functionalized DNA1 and 3'-phosphorylated DNA2, was immobilized on a gold nanoparticles (AuNPs) modified glassy carbon (GC) electrode. In the presence of AP, 3'-phosphoryl end of DNA2 was dephosphorylated. Terminal deoxynucletidyl transferase (TdT) catalyzed the sequential addition of deoxynucleotides (dTTPs) at 3'-OH end of DNA2 to extend DNA2 with a poly-T sequence. Then, G-rich DNA3 strand hybridized with the poly-T sequence of DNA2. Upon addition of hemin, the hemin/G-quadruplex DNAzyme was formed. In the presence of NADH, the hemin/G-quadruplex DNAzyme oxidased NADH to NAD+, accompanied by the formation of H2O2which was further catalyzed by hemin/G-quadruplex DNAzyme (served as a HRP-mimicking DNAzyme) with the thionine (Thi) as electron transfer mediator, leading to the amplified electrochemical signal. Under optimized conditions, the response peak current was linear with the concentration of AP in the range from 0.1UL-1to 5UL-1with the detection limit of 0.03UL-1. Also, the developed biosensor possessed good selectivity, reproducibility and stability, and simple sensing structure, showing promising practical applications in AP activity assay.
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Yunqing
Y
Liu
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Erhu
E
Xiong
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Xiaoyu
X
Li
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Junjing
J
Li
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China. Electronic address: mickyxie@hnu.edu.cn.
Xiaohua
X
Zhang
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China. Electronic address: chenjinhua@hnu.edu.cn.
Jinhua
J
Chen
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000097:blood; D000818:Animals; D001156:Artemia / Q000201:enzymology; D004253:DNA Nucleotidylexotransferase / Q000737:chemistry; D021881:DNA, Catalytic / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods; D054856:G-Quadruplexes; D006427:Hemin / Q000737:chemistry*; D006801:Humans; D057230:Limit of Detection; D053770:Nanowires / Q000737:chemistry* / Q000648:ultrastructure
D021881:DNA, Catalytic; D006427:Hemin; D004253:DNA Nucleotidylexotransferase; D000469:Alkaline Phosphatase
Alkaline phosphatase; Electrochemical biosensor; Hemin/G-quadruplex DNAzyme; Terminal deoxynucletidyl transferase