Enzyme enhanced quantitative determination of multiple DNA targets based on capillary electrophoresis

• Published in: Journal of Chromatography A Vol. 1216; no. 12; pp. 2567 - 2573
• Main Authors: Li, Xuemei, Zhan, Zhiming, Zhang, Shusheng, Chen, Hongyuan
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 20.03.2009; Amsterdam; New York: Elsevier; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Avidin - metabolism; Biological and medical sciences; Biotin - metabolism; Capillary electrophoresis; DNA; DNA - analysis; DNA - metabolism; Dna, deoxyribonucleoproteins; Electrochemical detection; Electrochemical Techniques - methods; Electrophoresis, Capillary - methods; Enzyme enhancement; Fundamental and applied biological sciences. Psychology; Horseradish Peroxidase - metabolism; Hydrogen-Ion Concentration; Linear Models; Multianalyte determination; Nucleic acids; Reproducibility of Results; Sensitivity and Specificity; Multianalyte determination; Enzyme enhancement; Electrochemical detection; Capillary electrophoresis; DNA; Chemical analysis; Enzyme; Avidin; Electrochemical detector; Analysis method; Peroxidases; Amperometry; Peroxidase; Oxidoreductases; Conjugated compound; Quantitative analysis
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2009.01.019

In the current paper, enzyme enhanced simultaneous quantitative determination of multiple DNA targets based on capillary electrophoresis (CE) was described. We used three biotin-modified DNA probes, which reacted with avidin-conjugated horseradish peroxidase (avidin-HRP) conjugate to obtain the HRP labeled probes, to hybridize with three corresponding targets. The resulting mixture containing double-strand DNA (dsDNA)-HRP, excess single-strand DNA (ssDNA)-HRP and remaining avidin-HRP was separated by capillary electrophoresis, and then the system of HRP catalyzing H 2O 2/ o-aminophenol (OAP) reaction was adopted. The catalytic product was detected with electrochemical detection. With this protocol, the limits of quantification for the hybridization assay of 21-, 39- and 80-mer DNA fragments were of 1.2 × 10 −11, 2.4 × 10 −11 and 3.0 × 10 −11 M, respectively. The multiplex assay also provided good specificity without any cross-reaction.