Electrochemical DNA biosensor based on proximity-dependent DNA ligation assays with DNAzyme amplification of hairpin substrate signal.
This paper describes a novel electrochemical DNA sensor for the simple, sensitive and specific detection of nucleic acids based on proximity-dependent DNA ligation assays with the DNAzyme amplification of hairpin substrate signal. A long DNA strand contains the catalytic motif of Mg(2+)-dependent 10-23 DNAzyme, acting as the recognition probe. When the target DNA was introduced into the system, part of it was complementary to 5'-end of the recognition probe, resulting in the ligation of a stable duplex, the unbinded part of the target DNA was acted as one binding arm for the DNAzyme. This duplex containing a complete 10-23 DNAzyme structure could cleave the purine-pyrimidine cleavage site of the hairpin substrate, which resulted in the fragmentation of the hairpin structure and the release of two single-stranded nucleic acids, one of which was biotinylated and acted as the signal probe. An immobilized thiolated capture probe could bind with the signal probe, using biotin as a tracer in the signal probe, and streptavidin-alkaline phosphatase (SA-ALP) as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 5 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The results revealed that the sensor showed a sensitive response to complementary target sequences of H. pylori in a concentration range from 100 fM to 1 nM, with a detection limit of 50 fM. In addition, the sensing system could discriminate the complementary sequence from mismatched sequences, with high sensitivity and reusability.
Sun
Chenhu
C
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Zhang
Liangliang
L
Jiang
Jianhui
J
Shen
Guoli
G
Yu
Ruqin
R
D015374:Biosensing Techniques / Q000295:instrumentation*; D003217:Conductometry / Q000295:instrumentation*; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D021881:DNA, Catalytic / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D022521:Ligase Chain Reaction / Q000295:instrumentation*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D021881:DNA, Catalytic; D004247:DNA