Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip.
A rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection (ED) based on microchip was described. We employed a biotin-modified DNA, which reacted with avidin-conjugated horseradish peroxidase (avidin-HRP) to obtain the HRP-labeled DNA probe and hybridized with its complementary target. After hybridization, the mixture containing dsDNA-HRP, excess ssDNA-HRP, and remaining avidin-HRP was separated by MCE. The separations were performed at a separation voltage of +1.6 kV and were completed in less than 100 s. The HRP was used as catalytic labels to catalyze H(2)O(2)/o-aminophenol reaction. Target DNA could be detected by the HRP-catalyzed reduction with ED. With this protocol, the limits of quantification for the hybridization assay of 21- and 39-mer DNA fragments were of 8 x 10(-12) M and 1.2 x 10(-11) M, respectively. The proposed method has been applied satisfactorily in the analysis of Escherichia coli genomic DNA. We selected the detection of PCR amplifications from the gene of E. coli to test the real applicability of our method. By using an asymmetric PCR protocol, we obtained ssDNA targets of 148 bp that could be directly hybridized by the single-stranded probe and detected with ED.
Cao
Wei
W
College of Life Science and Pharmacy, Nanjing University of Technology, Nanjing, P R China.
Su
Mingming
M
Zhang
Shusheng
S
D000627:Aminophenols / Q000737:chemistry; D001360:Avidin / Q000737:chemistry*; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D046209:Electrophoresis, Microchip / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D011136:Polysorbates / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000627:Aminophenols; D015342:DNA Probes; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D011136:Polysorbates; C083986:avidin-horseradish peroxidase complex; D001360:Avidin; C027667:2-aminophenol; D004247:DNA; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase