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5 March 2015 DNA separation and fluorescent detection in an optofluidic chip with sub-base-pair resolution
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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 = 4×10-4. 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 = 4×10-4, enabling detection of single base-pair insertion/deletion in a lab-on-a-chip.
© (2015) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Markus Pollnau, Manfred Hammer, Chaitanya Dongre, and Hugo J.W.M. Hoekstra "DNA separation and fluorescent detection in an optofluidic chip with sub-base-pair resolution", Proc. SPIE 9320, Microfluidics, BioMEMS, and Medical Microsystems XIII, 93200J (5 March 2015); https://doi.org/10.1117/12.2077515
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