Development of new bioanalysis techniques based on confocal fluorescence spectroscopy and nanotechnology for nucleic acid research.

摘要

Although we are in the post-genomic era, we still face many unsolved questions in functional genomics and proteomics. One major challenge is due to the fact that we lack research tools to elucidate the sophisticated genetic processing and cellular signaling mechanisms that are often obscured by cellular heterogeneity and the stochastic nature of molecular processes. Among the tools that are currently being developed to facilitate biomedical studies, it is increasingly evident that probes in the nanometer size-scale and tools with single-molecule sensitivity are necessary to characterize this inherent variability in biological systems. For modern analytical chemistry, an important goal is thus to develop highly sensitive and low-volume molecular analysis systems that enable the quantification of low-copy number biomolecules in minute amount of samples. This dissertation presents my work in combining three innovative technologies including nanomaterial-based probes, microfluidics and single-molecule detection in pursuing this analytical goal.; The analytical strategies and tools demonstrated in this dissertation primarily focus on the aspect of nucleic acid research, which is of great importance in disease diagnostics, disease treatment, as well as understanding the fundamental cell regulation mechanisms. Two analytical tasks often required in nucleic acid study are the identification and quantification of particular nucleic acids or point mutations that have critical biological implications, and the characterization of binding energy landscapes between DNA-binding proteins and DNA under various chemical conditions that directly link to the development of new therapeutic methods for treating certain diseases. To address the requirements for nucleic acid sensing, novel quantum-dot-based nanoprobes for specific nucleic acid detection and mutational analysis of oncogenes were demonstrated (Nucleic Acids Research 2006, 34(5), e35; Nanomedicinc: Nanotechnology, Biology, and Medicine 2005, 1(2), 115-121; and Natural Materials 2005, 4(11), 826-831; in collaboration with researchers in the Department of Pathology at Johns Hopkins). The fascinating optical properties of quantum dots enabled the development of nanoprobes with ultrahigh sensitivity and detection specificity in a homogeneous, separation-free detection formal. To facilitate the study of the binding of the transcription factor Sp1 to its DNA cognate sites (GC box) under the influence of the chemotherapeutic agent doxorubicin, a microfluidic titration chip was designed and fabricated to interface with a confocal fluorescence technique, the fluorescence correlation spectroscopy, to determine the IC50 (the concentration required for 50% inhibition) of the drug (Nucleic Acids Research 2006, 34(21). e144; in collaboration with researchers in the Department of Biology at Johns Hopkins). The merger of fluorescence fluctuation analysis techniques that have single-molecule sensitivity and microfluidics targets the needs for drug discovery in the pharmaceutical industry, particularly in the aspect of assay miniaturization.; While working with fluorescence fluctuation spectroscopy, I discovered that when labeled on a DNA probe, the commonly used cyanine dye Cy5, acquires an intriguing fast-blinking phenomenon (fluorescence intermittency on the order of microseconds) that can be "tuned" upon probe-target hybridization. The tunable blinking kinetics were then used as an indicator in differentiating DNA targets with single-nucleotide differences, as a proof of concept for application of this blinking phenomenon in bioanalysis of (manuscript accepted by Biophysical Journal lot publication). This finding may open the door to a whole new type of fluorescence-based detection technique that is not dependent on fluorescence intensity, lifetime, anisotropy, or spectral properties, but on the rate of the fluorophore being switched "on" and "off".; The major contribution of this dissertation is tw