Modulation-frequency encoded multi-color fluorescent DNA analysis in an optofluidic chip

By capillary electrophoresis (CE) in miniaturized lab-on-a-chip devices, integrated DNA sequencing and genetic diagnostics have become feasible. We introduce a principle of parallel optical processing to significantly enhance analysis capabilities. In a commercial microfluidic chip, a plug of DNA molecules was injected and the DNA molecules were CE-separated with a high relative sizing accuracy of >99%. Through an optical waveguide inscribed by femtosecond-laser writing a laser was launched perpendicularly into the microfluidic channel. A photomultiplier collected the fluorescence signals from a small detection window with a limit of detection of ~8 DNA molecules. In our approach, different sets of exclusively end-labeled DNA fragments are unambiguously identified by simultaneously launching several continuous-wave lasers, each modulated with a different frequency, detection of the frequency-encoded signals at different fluorescence wavelengths by a single ultrasensitive, albeit color-blind photomultiplier, and Fourier-domain frequency decoding. As a proof of principle, fragments from independent human genomic segments, associated with genetic predispositions to breast cancer and anemia, are simultaneously analyzed in a single flow experiment. This novel method of modulation-frequency-encoded fluorescence excitation opens new opportunities in genetic diagnostics. It enables the identification of end-labeled DNA samples of different genetic origin during their electrophoretic separation, opening perspectives for intrinsic size calibration, malign / healthy sample comparison, and exploitation of multiplex ligation-dependent probe amplification.