Many important analytical studies of biomolecules are not feasible because current technology is both too insensitive and at a scale too gross for experimentation with extremely small sample volumes. The overall objective of this proposal is to explore the potential of micro- and nanometer scale technology to overcome these limitations. Two underlying principles motivate this work: molecular detection capability can be greatly enhanced in micro/nanometer scale devices. small volumes, from micro- to pico-liters or less, can be more appropriately handled in small devices. The ultimate objective in these endeavors is to realize integrated instruments that permit in situ extraction and analysis of targeted molecular components from an individual cell. In these initial efforts, the immediate goal is to study a specific class of molecules involved in the process of sea urchin embryogenesis using prototypical microdevices specifically designed for this research. Transcription factor (TF) proteins regulate differential gene expression in temporally and spatially restricted regions of the embryo by binding with high affinity to specific DNA sequences. With existing technology, functional TF/DNA binding studies can only be achieved using hundreds of thousands of embryos. To study TF/DNA binding from small regions within an embryo, or from individual cells, microdevices that provide the following functions will be sequentially constructed, evaluated, and optimized in this research pro~ram: Manipulation and transport of cells, small tissue fragments, and solutions. Lysis of cells/organelles to open their contents for further analysis. Reagent mixing, to combine synthetic, fluorescent oligonucleotides and unlabeled, non-specific DNA with cell extracts. Electrical conductivity measurements/optical absorption spectroscopy upon picoliter solutions. Electrophoretic separation and detection of fluorescent molecules. Many materials systems widely employed in contemporary nanostructure research are chosen to optimize electronic or photonic properties, but these are not necessarily ideally suited, nor perhaps even compatible, with biological systems. An important thrust in this initial program is the development of materials/processing combinations, especially highly-anisotropic dry etching procedures, which permit high resolution microfabrication while retaining biological compatibility. Devices will be constructed using both high resolution optical and electron beam lithography, dry and wet etching processes, and both thermallyand sputter- deposited thin film materials. At each step of their development, the instruments created will be applied to the aforementioned TF/DNA binding assays. This iterative and interactive research, carried out on both nanofabrication and biological fronts, will drive the practical attainment of new classes of high resolution instruments for biological science. It is anticipated that this work will spawn new research efforts at the nanometer scale in both the science of biological phenomena and the technology of bioinstrumentation.
