Date of Award
Doctor of Philosophy (PhD)
Mechanical Engineering & Applied Mechanics
Haim H. Bau
Carbon nanopipettes (CNPs) consist of a pulled-quartz micropipette with a thin layer of amorphous carbon deposited along its entire interior surface via chemical vapor deposition. The micropipette maintains a continuous fluidic pathway from its nanoscopic tip to its distal macroscopic end, while the insulated carbon film provides an electrical path to the tip that can be used as a working electrode. The quartz at the tip of the CNP can be chemically etched to expose a desired length of a carbon pipe to control the size and characteristics of the electrode. CNPs are inexpensive, batch- fabricated, and can be made hollow or solid. They can be used as nanoelectrodes, nanoinjectors, or both simultaneously, with improved durability and biocompatibility compared with glass micropipettes. Here, I will describe work stemming from CNP technology.
We have developed an impedimetric AC technique for detecting cellular and nuclear penetration during microinjection and cellular probing with CNPs. The technique has submicron spatial and millisecond temporal resolution. Signal magnitude can be used to discern between penetration into the cytoplasm or nucleus, and using the CNPs as nanoelectrodes. We find a monotonic dependence of the signal on penetration depth. The behavior of this system is well-predicted by an equivalent circuit model, and could be used to provide electrical feedback during single-cell microinjection, nanosampling, or electrochemical studies. Using solid CNP electrodes (CNPEs), we have also characterized CNPs for use in fast-scan cyclic voltammetry to measure neurotransmitter concentrations in the brain of Drosophila melanogaster (fruit fly). CNPEs are sharper and smaller than commonly used carbon-fiber microelectrodes (CFMEs), allowing them to penetrate the tough glial sheath of the fly brain and perform more localized measurements. CNPEs are also easier to batch fabricate and have better dimensional control than CFMEs.
As a target biological application of microinjection, we are using injection of fluorescently labeled tRNA to monitor subcellular tRNA dynamics in real time. We have developed a simple model to capture trafficking dynamics, and fit our model to experimental data for the measurement of nuclear/cytoplasmic trafficking kinetics of tRNA during nutrient deprivation of mouse embryonic fibroblasts. This data confirms that cells have mechanisms for the regulation of tRNA transport, and suggests that we can use our microinjection technique to perform quantitative studies of tRNA trafficking.
In order to facilitate microinjection studies such as these, we have developed an adaptable Matlab-based semi-automated injection system. We have incorporated our electrical feedback signal, with the goal of improving success rates and throughput of microinjection, while minimizing difficulty and user error.
Anderson, Sean Eric, "Carbon Nanopipettes for Advanced Cellular Probing and Microinjection" (2015). Publicly Accessible Penn Dissertations. 1005.