DNA separation and fluorescent detection in an optofluidic chip with sub-base-pair resolution

Markus Pollnau, Manfred Hammer, C. Dongre, Hugo Hoekstra

    Research output: Chapter in Book/Report/Conference proceedingConference contributionAcademic

    Abstract

    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.
    Original languageUndefined
    Title of host publicationSolid State Lasers XXIV
    Place of PublicationBellingham, USA
    PublisherSPIE
    PagesPaper 93200J
    Number of pages5
    ISBN (Print)978-1-62841-410-3
    DOIs
    Publication statusPublished - Apr 2015
    EventSolid State Lasers XXIV, San Francisco, USA: Solid State Lasers XXIV - Bellingham, USA
    Duration: 1 Apr 2015 → …

    Publication series

    NameProceedings of the SPIE
    PublisherSPIE
    Volume9320

    Conference

    ConferenceSolid State Lasers XXIV, San Francisco, USA
    CityBellingham, USA
    Period1/04/15 → …

    Keywords

    • single base-pair insertion/deletion
    • EWI-25962
    • IOMS-SNS: SENSORS
    • Capillary electrophoresis
    • IR-95788
    • Fluorescent detection
    • optofluidic chip
    • sub-base-pair sizing accuracy
    • METIS-312571
    • DNA separation

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