DNA hybridization detection in a microfluidic channel using two fluorescently labelled nucleic acid probes |
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Authors: | Chen Lingxin Lee Sangyeop Lee Moonkwon Lim Chaesung Choo Jaebum Park Joong Yull Lee Sanghoon Joo Sang-Woo Lee Kyeong-Hee Choi Young-Wook |
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Institution: | Department of Applied Chemistry, Hanyang University, Sa-1-dong 127, Ansan, Kyunggi-do 426-791, Republic of Korea. |
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Abstract: | A conceptually new technique for fast DNA detection has been developed. Here, we report a fast and sensitive online fluorescence resonance energy transfer (FRET) detection technique for label-free target DNA. This method is based on changes in the FRET signal resulting from the sequence-specific hybridization between two fluorescently labelled nucleic acid probes and target DNA in a PDMS microfluidic channel. Confocal laser-induced microscopy has been used for the detection of fluorescence signal changes. In the present study, DNA hybridizations could be detected without PCR amplification because the sensitivity of confocal laser-induced fluorescence detection is very high. Two probe DNA oligomers (5'-CTGAT TAGAG AGAGAA-TAMRA-3' and 5'-TET-ATGTC TGAGC TGCAGG-3') and target DNA (3'-GACTA ATCTC TCTCT TACAG GCACT ACAGA CTCGA CGTCC-5') were introduced into the channel by a microsyringe pump, and they were efficiently mixed by passing through the alligator teeth-shaped PDMS microfluidic channel. Here, the nucleic acid probes were terminally labelled with the fluorescent dyes, tetrafluororescein (TET) and tetramethyl-6-carboxyrhodamine (TAMRA), respectively. According to our confocal fluorescence measurements, the limit of detection of the target DNA is estimated to be 1.0 x 10(-6) to 1.0 x 10(-7)M. Our result demonstrates that this analytical technique is a promising diagnostic tool that can be applied to the real-time analysis of DNA targets in the solution phase. |
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Keywords: | FRET Microfluidic sensor DNA hybridization Confocal laser-scanning microscopy Real-time analysis |
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