P1 − Combined AFM-SECM with Insulated Conductive Cantilevers

Poster .
P1 − Combined AFM-SECM with Insulated Conductive Cantilevers
Keywords: AFM, conductive cantilevers, OmpF, porin, SECM
Patrick D. Bosshart1, Patrick L.T.M. Frederix1, Mohamed Chami1, Terunobu Akiyama2, Maurizio R.
Gullo2, Nicolaas F. de Rooij2, Urs Staufer2, Andreas Engel1
1: NCCR Nanoscale Science, M.E. Müller Institute, Biozentrum, University of Basel
Klingelbergstrasse 70, CH-4056 Basel; Switzerland
2: NCCR Nanoscale Science, Institute for Microtechnology, University of Neuchâtel
Rue Jacquet-Droz 1, CH-2007 Neuchâtel; Switzerland
Email: [email protected]
Homepage: http://www.mih.unibas.ch; http://www-samlab.unine.ch
With the atomic force microscope (AFM) biomolecules and membranes can be imaged at
nanometer lateral resolution once all conditions are optimized[1]. With cantilevers that
incorporate a small electrode near or at the tip apex scanning electrochemical microscopy
(SECM) can be carried out at the same time and functional data can be recorded in parallel.
This has been used to study enzyme activity [2, 3]. Functional experiments can also be carried
out on thin layers, like porous membranes [4], which are adsorbed on or above a conductive
support. Of particular interest for structural biology is the study of structural and conductive
properties of membrane proteins embedded in lipid bilayers.
Here, we present electrochemical experiments where a conductive cantilever [5-7] approaches
platinum supports covered with two types of membrane patches that are packed with different
proteins: outer membrane porin F (OmpF) from E. coli, and the bacterial proton pump
bacteriorhodopsin (bR) from H. salinarum. OmpF is an unspecific pore, which allows
particles with a molecule mass MR<550Da to diffuse through [8] and shows voltage gating [9].
Consequently, the open pores of OmpF should be sufficiently large to pass redox molecules
(like Ru(NH3)63+/2+, MR=203Da). On the other hand, the membrane patches containing bR
only transport protons and are impermeable for the redox molecules. This expected difference
in conductivity could be confirmed by combined AFM-SECM. In addition, by applying a
potential across membranes that incorporate OmpF a change in conductivity is observed that
can be ascribed to voltage-gating.
References
1. Müller, et al. 1999; Biophys. J. 76: 1101
2. Kueng, et al. 2003; Angew. Chem. Int. Ed. Engl. 42: 3238
3. Kranz, et al. 2004; Ultramicrosc. 100: 127
4. Kueng, et al. 2005; Angew. Chem. Int. Ed. Engl. 44: 3419
5. Akiyama, et al. 2004; Jap. J. Appl. Phys. 43: 3865
6. Frederix, et al. 2005; Nanotechn. 16: 997
7. Gullo, et al. 2006; Anal. Chem. 78: 5436
8. Nakae 1976; Biochem. Biophys. Res. Comm. 71: 877
9. Schindler and Rosenbusch 1978; PNAS 75: 3751
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6 Int. Workshop − Scanning Probe Microscopy in Life Sciences − 9 October 2007 in Berlin
www.spm-workshop.jpk.com
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Poster .
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th
6 Int. Workshop − Scanning Probe Microscopy in Life Sciences − 9 October 2007 in Berlin
www.spm-workshop.jpk.com
2/2