Parallel intracellular integration of nanoscale structures for delivery and expression of free and scaffolded DNA

Abstract

We report on the parallel integration of nanoscale structures with the intracellular domains of viable cells to provide long-term, controlled cellular biochemical manipulation. Vertically-aligned carbon nanofibers (VACNF) are self-assembling structures that feature nanoscale diameter tips on vertical supports that can be grown to tens of microns in length. Using wafer-scale processing techniques, VACNFs may be synthesized and incorporated as functional elements of microscale devices and thus provide a bridge between molecular-scale phenomena (at nanofiber tips) and the microscale architecture of the underlying device. The vertical orientation and needlelike aspect of VACNFs facilitate their implementation as molecular-scale interfaces that potentially can be introduced, on a massively parallel basis, to the intracellular domains of viable cell matrices. To demonstrate this parallel introduction, and to observe cell response to such manipulations, VACNFs were modified with either adsorbed or covalently-linked plasmid DNA and such arrays were interfaced with cellular matrices. Following VACNF/cell interfacing, cells were found to proliferate on the nanofiber substrate and to express delivered plasmid DNA (a fluorescent reporter construct) from both desorbed and adsorbed, immobilized plasmid, as well as from plasmid that was covalently tethered to the penetrant nanofiber scaffold. Post interfacing viability of these cells was observed for relatively long durations (>22 day) by tracking expression of the introduced plasmid. Of particular interest, covalently bound plasmids appeared to remain tethered to nanofibers and were expressed in interfaced cells but were not partitioned into noninterfaced progeny. Expression from such immobilized plasmids ceased when the nanofiber was no longer retained within a cell. In addition to demonstrating parallel microinjection of macromolecules, these results provide a method for achieving a genetic modification that is noninheritable and whose extent in time can be directly and precisely controlled. This work also provides basis for application of VACNF device architectures for molecular-scale engineering within whole cells and as a platform for biosensing and clinical diagnostic applications.