The main goal of this Phase I SBIR project is to demonstrate the feasibility of using a novel carbon-based nanopipette (CNP) for precise and controlled microinjection of macromolecules into single cells and allow electrical measurement of variations in membrane potential upon penetrating a cell. The ultimate goal is to develop a unique and versatile nanopipette device for concurrent microinjection and electrical measurements of changes in cell membrane potential upon penetration of a cell. CNP fabrication does not require any cumbersome assembly. The CNP consists of a pulled glass capillary terminating with an exposed carbon tip with a diameter ranging from tens to hundreds of nanometers. A thin carbon film coated inside the glass capillary of the CNP provides an electrically conducting path that can be used as an electrode for measurement of changes in membrane potential in cells. In addition, these devices can inject chemicals and macromolecules into single cells and extract fluids and specific proteins from cell's interior. The CNPs offer significant advantages over the commonly used pulled glass pipettes such as smaller size (minimal damage to cells and the ability to probe organelles), better mechanical properties, higher durability (they do not break or clog easily), potential for automation (the cell's penetration can be sensed through an electric signal), potential to carry out electrophysiological measurements concurrently with injection, and multifunctional analytic capabilities while being competitive in price with the glass micropipettes. Moreover, given their durability, the CNPs offer higher efficiency and lower cost (on a per cell basis) than their glass counterparts. The feasibility of the CNP to inject into the cytoplasm and inject into the nucleus with higher cell survival rates will be tested and compared to that of commercially available micropipettes to demonstrate the advantages of CNPs in microinjection. Furthermore, the unique characteristic of CNPs as nanoelectrodes will be demonstrated by measuring the change in electrical signal upon cell penetration. Feasibility studies will be performed using CNPs with inner diameter in the ~100 nm range to achieve less damage to cell membrane upon cell penetration.
Public Health Relevance: The proposed CNP devices will have applications in manipulation and study of individual cells including facilitating controlled injection of proteins and nucleic acids, drug delivery, biomedical diagnostics, and cellular engineering research. Direct comparison of microinjection using CNP to commercially available glass micropipettes will allow advancement in single cell microinjection applications by replacing the conventional glass micropipettes with CNPs.
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