| Abstract |
The proposed one-year project will seed the development of the concept and the demonstration of a three-dimensional (3-D) nano-needle array technology for rapid and massively parallel screening of infectious diseases based on fluorescent in-situ hybridization (FISH). It will help extend the reach and power of FISH in infectious disease screening and in AIDS and cancer research and in understanding the transfection or genetic basis of the disease process, by scaling up the FISH technique and making it massively parallel and massively multiplexed. The project itself will also serve as an effective vehicle to enable us to begin the collaborations with multiple members of CFAR, with whom we have been brainstorming ideas over the past months, and do so in a disciplined manner. If approved, it would represent the first opportunity for us to contribute to the CFAR success after being invited to become a CFAR member in the summer.
This project has two specifics aims. The first is to design and develop the nano-fabrication processes for constructing the 3-D nano-needle array out of carbon nanotubes or silicon nanopillars through the application of nano-fabrication techniques that have been developed in our laboratory over the years.
The second aim is to demonstrate the feasibility of intracellular delivery and controlled release of probe DNA or RNA by the nano-needles into the intact cell nucleus, followed by excess probe DNA removal by the proposed "solid-state washing" technique. To accomplish this goal, we will functionalize the nano-needles by tip-coating each nano-needle with gold to which a single DNA strand is covalently linked and then hybridized with a complementary probe strand carrying the fluorescent label. The release of the probe DNA carried by each nano-needle will be induced by electric heating of the needle tip, or laser illumination, or voltage induced change of the local PH level, causing dehybridization of the strands.
For the spectroscopic measurements required for the large number of simultaneous assays, our lab is well equipped with a 3-D micro-Raman scanning microscope system. This system can be adapted for real-time ultra-sensitive detection, spatial imaging of sub-micron resolution, and sub-micron spot spectroscopic measurements of distributed fluorescence marks. Moreover, it features automated sub-micron spatial resolution in three dimensions, a spectral range from deep blue to IR, and sub-second scanning time. The time required for complete spectroscopic measurement of a hypothetical 2000-spot array could be less than an hour, once the system is adapted for this study.
While the focus of the first-year effort will necessarily be on developing the nano-needle array, experiments will be conducted in parallel to test and investigate the FISH protocols, and their adaptation to the nano-needle platform, for detection and subcellular localization of HIV virus or therapeutic monitoring of mtDNA depletion in patients treated with the NRTI etc.. |
