|نسخه تمام متن
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• This research developed a simple and effective method to analyze nucleic acid by CGE-UV sensitively.
• We combined matrix field-amplified with head-column field-amplified stacking injection (C-FASI) to employ the advantages of two methods, which obtained a limit of detection of 0.13 ng/ml (signal/noise = 3) in a 300,000-fold diluted sample.
• No complicated device modification or special mechanism was needed.
• RSD of migration time and peak area were (0.03–1.15) and (0.72–6.42) respectively demonstrated the methods had good qualitative abilities and quantitative abilities.
• A φX174-Hae III digest DNA product without purification and a 400 bp PCR product without purification were tested to prove C-FASI was effective for the real sample. Our present study may be the first to demonstrate enhanced detection sensitivity for DNA using CGE-UV without special complicated mechanisms or device reconfigurations while still retaining superior analytical sensitivity.
To develop a highly sensitive method for analyzing nucleic acids using capillary gel electrophoresis with ultraviolet detection (CGE-UV), we combined matrix field-amplified with head-column field-amplified stacking injection (C-FASI) to employ the advantages of two methods. Without diminishing the resolution, a limit of detection of 0.13 ng/ml (signal/noise = 3) in a 300,000-fold diluted sample was obtained, the sensitivity is 102,308 times higher than that achieved with normal pressure injection, 3077 times that with normal electrokinetic injection, 154 times that with pressure field-amplified sample stacking injection, and 31 times that with matrix field-amplified stacking injection. After establishing the method, we tested the detection of a φX174-Hae III digest DNA product without purification and with a high ionic strength. At the lowest dilution of 5000-fold, sample at a concentration of 10 ng/ml was enriched and detected. The relative standard deviations for migration time and peak area (n = 3) were 0.03–1.15 and 0.72–6.42, respectively. To further validate C-FASI was applicable for real sample, a 400 bp PCR product without purification was directly detected with a limit of detection at the concentration of 6000-fold dilution (signal/noise = 3), The relative standard deviations for migration time and peak area (n = 6) were 0.44 and 4.8, respectively. These results indicated that C-FASI had good qualitative and quantitative detection abilities and CGE-UV based on C-FASI is easy to perform, practical, highly-sensitive and robust for nucleic acid detection, which makes it a highly valuable tool for genetic diagnostics based on nucleic acid analysis.
Journal: Journal of Chromatography B - Volumes 978–979, 26 January 2015, Pages 29–42