Dual-point dual-wavelength fluorescence monitoring of DNA separation in a lab on a chip

Abstract

We present a simple approach in electrophoretic DNA separation and fluorescent monitoring that allows to identify the insertion or deletion of base-pairs in DNA probe molecules from genetic samples, and to perform intrinsic calibration/referencing for highly accurate DNA analysis. The principle is based on dual-point, dual-wavelength laser-induced fluorescence excitation using one or two excitation windows at the intersection of integrated waveguides and microfluidic channels in an optofluidic chip and a single, color-blind photodetector, resulting in a limit of detection of ~200 pM for single-end-labeled DNA molecules. The approach using a single excitation window is demonstrated experimentally, while the option exploiting two excitation windows is proposed theoretically.

Fig. 1

Layout of the optofluidic chip indicating the MF reservoirs, MF channels, and the two DWs, each comprising two WGs crossing the MCE separation channel perpendicularly in plane

Fig. 2

Electropherograms depicting the MCE separation of two fluorescently labeled DNA molecules: (a) cumulative signal during simultaneous dual-wavelength excitation of migrating 19-nt-AF647 and 19-nt-Cy3 molecules; (b) individual signals detected during different flow experiments applying single-wavelength excitation with only one of the two lasers switched on, temporally superimposed on each other; (c) and (d) the same for 19-nt-AF647 and 20-nt-Cy3 molecules.

Fig. 3

Simulated electropherograms as would be detected from the two DWs with swapped excitation wavelengths during an experiment with internal calibration using a green-labeled DNA sample (“S”) consisting of four different molecule sizes (250 bp, 300 bp, 450 bp, and 700 bp) and a red-labeled DNA reference (“R”) consisting of three different molecule sizes (150 bp, 355 bp, and 1000 bp). Fluorescence signals S were excited through WG1, R through WG2, R' through WG3 and S' through WG4.