Multi-color fluorescent DNA analysis in an integrated optofluidic lab on a chip

Abstract: Sorting and sizing of DNA molecules within the human genome project has enabled the genetic mapping of various illnesses. Furthermore by employing tiny lab-on-a-chip device, integrated DNA sequencing and genetic diagnostics have become feasible. We present the combination of capillary electrophoresis with laser-induced fluorescence for optofluidic integration toward an on-chip bio-analysis tool. Integrated optical fluorescence excitation allows for a high spatial resolution (12 μm) in the electrophoretic separation channel, and can lead to a further 20-fold enhancement as soon as improved microfluidic protocols become available. We demonstrate accurate sizing (with > 99% sizing accuracy) and highly sensitive (LOD = 220 femtomolar, corresponding to merely 6 molecules in the excitation volume) fluorescence detection of double-stranded DNA molecules by integrated waveguide laser excitation. Subsequently, we introduced a principle of parallel optical processing to this optofluidic lab on a chip. In this approach, different sets of exclusively color-labeled DNA fragments - otherwise rendered indistinguishable by their spatial (in the microchip CE separation channel) and temporal (in the consequent electropherogram) coincidence - are traced back to their origin by modulation-frequency-encoded multi-wavelength laser excitation, fluorescence detection with a color-blind photomultiplier, and Fourier-analytical decoding. As a proof of principle, fragments obtained by multiplex ligation-dependent probe amplification from independent human genomic segments, associated with genetic predispositions to breast cancer and anemia, are simultaneously analyzed. The techniques described in this thesis for multiple, yet unambiguous optical identification of biomolecules will potentially open new horizons for “enlightened‿ lab-on-a-chip devices in the future