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DNA separation and fluorescent detection in an optofluidic chip with sub-base-pair resolution
KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics. Univ Twente, Netherlands.
2015 (English)In: Microfluidics, BioMEMS, and Medical Microsystems XIII, SPIE - International Society for Optical Engineering, 2015, Vol. 9320, 93200JConference paper (Refereed)
Abstract [en]

DNA sequencing in a lab-on-a-chip aims at providing cheap, high-speed analysis of low reagent volumes to, e.g., identify genomic deletions or insertions associated with genetic illnesses. Detecting single base-pair insertions/deletions from DNA fragments in the diagnostically relevant range of 150-1000 base-pairs requires a sizing accuracy of S < 10(-3). Here we demonstrate S = 4x10(-4). A microfluidic chip was post-processed by femtosecond-laser writing of an optical waveguide. 12 blue-labeled and 23 red-labeled DNA fragments were separated in size by capillary electrophoresis, each set excited by either of two lasers power-modulated at different frequencies, their fluorescence detected by a photomultiplier, and blue/red signals distinguished by Fourier analysis. Different calibration strategies were tested: a) use either set of DNA molecules as reference to calibrate the set-up and identify the base-pair sizes of the other set in the same flow experiment, thereby eliminating variations in temperature, wall-coating and sieving-gel conditions, and actuation voltages; b) use the same molecular set as reference and sample with the same fluorescence label, flown in consecutive experiments; c) perform cross-experiments based on different molecular sets with different labels, flown in consecutive experiments. From the results we conclude: Applying quadratic instead of linear fit functions improves the calibration accuracy. Blue-labeled molecules are separated with higher accuracy. The influence of dye label is higher than fluctuations between two experiments. Choosing a single, suitable dye label combined with reference calibration and sample investigation in consecutive experiments results in S = 4x10(-4), enabling detection of single base-pair insertion/deletion in a lab-on-a-chip.

Place, publisher, year, edition, pages
SPIE - International Society for Optical Engineering, 2015. Vol. 9320, 93200J
, Proceedings of SPIE, ISSN 0277-786X ; 9320
Keyword [en]
DNA separation, optofluidic chip, capillary electrophoresis, fluorescent detection, single base-pair insertion/deletion, sub-base-pair sizing accuracy
National Category
Other Engineering and Technologies
URN: urn:nbn:se:kth:diva-169304DOI: 10.1117/12.2077515ISI: 000354516300014ISBN: 978-1-62841-410-3OAI: diva2:822679
Conference on Microfluidics, BioMEMS, and Medical Microsystems XIII, FEB 07-09, 2015, San Francisco, CA, United States

QC 20150617

Available from: 2015-06-17 Created: 2015-06-12 Last updated: 2015-06-17Bibliographically approved

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Pollnau, Markus
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