New Principles of Detecting Specific DNA Targets With Liquid Crystals

Abstract

Detecting DNA targets with specific sequence is important in the identification and detection of single nucleotide polymorphisms and gene expression profile analysis. Traditionally, fluorescence is used to report the presence of DNA targets hybridized to surface-immobilized DNA probes. However, this method requires fluorescent labeling of DNA targets, and the sensitivity remains a challenge. In the first part of this thesis, we used cetyl trimethylammonium bromide (CTAB) for enhancing the fluorescence intensity of 6-carboxy-fluorescene (FAM)-labeled DNA targets hybridized to the immobilized DNA probes. The fluorescence intensity shows a 26-fold increase for perfect-match DNA targets. The contrast ratio between perfect-match and 1-mismatch DNA is increased from 1.3-fold to 15-fold. This method offers a simple and efficient technique to enhance the fluorescence detection limit on solid surface.

In the second part, we used liquid crystal (LCs) as an imaging tool to detect DNA targets. The imaging principle is based on the disruption of the orientations of LCs by single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). Because LCs are birefringent materials, disruption of their orientations can manifest as optical signals visible to the naked eye. Firstly, LCs was used to image ssDNA immobilized on solid surfaces. Interestingly, a clear transition of the optical appearance of LCs from dark to bright at a threshold ssDNA concentration was observed. This enables us to correlate the LCs interference colors with ssDNA concentrations, and it also serves as a basis for the quantification of immobilized ssDNA on solid surface. Later on, we hybridized the ssDNA with complementary targets and used LCs to image the dsDNA. However, because the color contrast between ssDNA and dsDNA is not significant, we added the surfactants, CTAB, to the DNA targets to aid the re-organization of LCs because the hydrocarbon tail of surfactants has strong orientational effect on LCs molecules. This approach is further explored to show the ability to discriminate the complementary strands from the non-complementary strands even at low hybridization efficiency (33%).

Even though the detection of DNA targets can be carried out by using the LC-based method above on a solid surface, the long duration of DNA hybridization makes this method not feasible for real-time detection. Thus, in our final part of this thesis, we developed an LC-based method which can detect DNA targets in a real-time manner. We self-assembled cholesterol-labeled DNA probes at the LC-aqueous interface. When the system is exposed to perfect match DNA targets, the optical appearance of LC shows a continuous change from dark to bright under the crossed polars within 15 min. No obvious change can be observed when the system is exposed to 1 or 2 base-pair mismatch DNA targets.