Nanopipettes as a Tool to Study Single Biological Entities

Nanopores offer a fast, convenient label-free platform for the detection and analysis of single biological entities, revealing data unseen in bulk analysis due to averaging. The ribosome is a biological entity of particular interest, due to its key role in protein synthesis. However, one challenge associated with this macromolecule is their typical small sample size, particularly when one is selecting ribosomes from specific tissues, or time points in development. Here, a nanopipette platform for single ribosome analysis is reported, enabling the fingerprinting of 80S ribosomes and polysomes from Drosophila melanogaster cultured cells and ovaries.
When the nanopipette is filled with a solution containing the ribosome or polysome sample of interest, an application of a positive voltage to the nanopipette electrode causes the translocation of the macromolecules across the nanopipette pore. Within this thesis, it is demonstrated that the peak amplitude of 80S ribosomes is significantly different to those of polysomes, and can be used to distinguish them in a mixed isolate. Furthermore, it is reported within this work that the nanopipette platform can be used to distinguish large polysomes from smaller polysomes, and as large polysomes represent mRNAs which are being actively translated, this is of great biological significance. Moreover, this thesis demonstrates the successful detection of a Chikungunya virus (CHIKV) RNA replicon, which will enable future work for the injection of viral genomes into living cells.