Ultra-sensitive DNA Assay Based On Single-molecule Detection Coupled With Fluorescent Quantum Dot-labeling

In chapter one of this thesis, an ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The limit of detection (LOD) of the method was 1×10-14 mol L-1. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.In chapter two, we developed an ultrasensitive quantitative single-molecule assay for quantification of DNA using a combination of hybridization accumulation and TIRFM. In this method,1 mL of target DNA (tDNA) molecules were accumulated on the silanized coverslip. then, they were captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin, Next, the QD-labeled probe DNA was hybridized to the tDNA. Finally, fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The LOD of the method was as low as 8×10-18 mol/L.