PROGRESS TOWARDS DEVELOPMENT OF A SYSTEM FOR INTRACELLULAR EXTRACTION FROM LIVING CELLS UTILIZING CARBON NANOFIBERS. STEPHEN JONES (Earlham College Richmond, IN 47374) TIM MCNIGHT (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

Do to their size and mechanical strength, vertically aligned carbon nanofibers (VACNF) provide an excellent platform for intracellular manipulation and probing. By attaching antibodies specific for an intracellular molecule of interest to a VACNF array and then impaling cells with the VACNFs, it may be possible to extract or probe for a molecule of interest in a living cell. The goal of the project is to confirm that this system will work by extracting and identifying an abundantly available species, tubulin, from tissue cultures of the Chinese Hamster Ovary (CHO) cell line. Initial attempts employed an EDC-mediated condensation of avidin protein onto the VACNF array and subsequent binding of biotinylated antibodies specific for tubulin. Cells could then be impaled on the VACNF's upon which the antibodies would putatively bind unpolymerized tubulin dimers. The tubulin would then be identified on VACNF's using a fluorescently tagged taxol compound, which has affinity for the tubulin dimer. This procedure was attempted in-vitro with bovine tubulin, but it was difficult to qualify success via taxol fluorescence because of native fluorescence of the fibers. To overcome this problem, the original procedure has been modified so that quantum dot conjugated anti tubulin antibodies will now be used for identifying fibers with bound tubulin. This procedure was attempted and succeeded in showing a clear difference between samples and negative controls. However the protocol was not reproducible to an acceptable extent, so further tests must be performed. However the preliminary results suggest that the basic protocol is feasible as proposed, and give some indication as to problems that must be overcome in the future. The biggest flaws in this procedure are the native fluorescence of the fibers (single to noise ration), and the sensitivity of fluorescent techniques. Future experiments may use radioactively labeled antibodies to resolve these problems.