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


Dongre, Chaitanya (2010) Multi-color fluorescent DNA analysis in an integrated optofluidic lab on a chip. thesis.

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.
Item Type:Thesis
Electrical Engineering, Mathematics and Computer Science (EEMCS)
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