| AVS 55th International Symposium & Exhibition | |
| Biomaterial Interfaces | Wednesday Sessions |
| Session BI+NC-WeM |
| Session: | Quantitative Nanoscale Sensing and Single Molecule Techniques |
| Presenter: | D. Klenerman, Cambridge University, UK |
| Correspondent: | Click to Email |
One major challenge in biology is to understand how the individual molecules and complexes of the cell are organised and interact to form a functional living cell. To address this problem new biophysical tools are needed that are capable of studying single molecules in complexes both in the test-tube and on or in living cells. To determine the oligomerisation state of proteins we have used two colour single molecule coincidence detection based on the excitation of two distinct fluorophore labels on proteins with two lasers focussed to the same spot.1 This method requires no prior knowledge of the structure of any complex formed or control of fluorophore position on the molecule. We show that this method can be used to characterise the protein oligomers formed during protein misfolding, ultimately resulting in amyloid fibril formation, and can distinguish between protein monomers and dimers on the cell surface.2 Working together with Professor Yuri Korchev at Imperial College, we have developed a method for functional nanoscale mapping of the cell surface that is based on a scanned nanopipette. This allows high resolution, non-contact imaging of the soft and responsive cell surface using the ion current that flows between an electrode in the nanopipette and bath for distance feedback control.3 Recently we have made a major advance in the resolution of the topographic images, by scanning with fine quartz pipettes, so we can directly visualise protein complexes on the surface of live cells.4 The pipette can also be use to perform local nanoscale assays on the cell surface so as to perform single channel recording5 or apply pressure to probe the mechanical properties. We have also combined high resolution topographic imaging with simultaneous recording of the fluorescence from the cell surface.6 In addition the pipette can be used for controlled voltage driven delivery and deposition of biomolecules down to the single molecule level3 and this is being used to probe the structure of the cell membrane using single molecule fluorescence tracking.
References
1. Anal.Chem. 78, 7707-7715 (2006).
2. PNAS 104,17662-17667 (2007)
3. Phys. Chem. Chem. Phys. 7 , 2859-2866 (2005)
4. Angewandte Chemie-International Edition 45, 2212-2216 (2006)
5. Biophysical Journal 83, 3296-3303 (2002).
6. PNAS 99 , 16018-16023 (2002)