| Translating genome-based knowledge into clinical health benefits is one of the critical elements in the future nanomedicine. As the study of biology becomes more quantitative, understanding the genetic contribution to disease is becoming more readily accessible. However, subpopulations within mixtures of molecules under both equilibrium and non-equilibrium conditions complicate the dissection of complex dynamics of biological processes. We developed a single particle quantum dot (QD) based biosensing platform which integrates nanophotonics with single molecule fluorescence spectroscopy, to investigate multiple individual events in situ. Single particle biosensing renders better understanding of biophysical properties or dynamics at the single molecules/complexes level, allowing heterogeneity in the populations to be directly observed. This platform was evaluated specifically on three aspects: (1) identification of multiple disease-related genes for medical diagnostics, (2) study of barriers during gene delivery, and (3) characterization of DNA nanocomplexes for non-viral vector based gene therapy.;The QD-nanoprobe system identifies DNA hybridization based on the colocalization of QD nanoprobes at the single molecule/particle level and is therefore ideal for analyzing low-abundant targets. Simultaneous detection of multiple anthrax pathogenicity-relevant genes was demonstrated through a single-tube colorimetric assay. Additionally, QD-mediated FRET (QD-FRET) was introduced to provide a quantitative and sensitive measure for molecular interactions with subcellular resolution. Herein, single particle biosensing was applied on the study of non-viral vectors based gene delivery. Non-viral vectors are attractive alternatives to viral vectors due to their low toxicity and immunogenicity. Critical barriers during delivery and the stability of DNA nanocomplexes, major challenges for non-viral vector-based delivery, were investigated through QD-FRET or two-step QD-FRET system. Better understanding of the biophysical properties of DNA nanocomplexes and mechanistic barriers during delivery is expected to facilitate rational design of optimized gene carriers. Furthermore, the integration of micro- (microfluidics) and nano-technology (QD-FRET) was utilized to monitor the DNA nanocomplex self-assembly process in real-time. Kinetic aspects of DNA nanocomplexes self-assembly are particularly valuable for the future production of customizable nanocomplexes, which allows accurate assessment on structure-function relationships of gene carriers.;The single particle QD platform, serving as a generic biosensing scheme to investigate a variety of biological entities and their interactions, is expected to aid modern biology by elucidating various biological interactions and to facilitate genetic medicine by contributing fundamentally to therapeutic design. |
