Zsolt SZEGLETES
research associate

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Attila Gergely VÉGH
research associate

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BIOLOGICAL APPLICATION OF THE ATOMIC FORCE MICROSCOPE

With the invention of the atomic force microscope (AFM) imaging surface structures at high resolution in air or liquid became possible. A small tip at the end of a µm-size cantilever scans the studied surface. During the scan the deflection of the cantilever is proportional to the force acting between the tip and the surface. The spatial resolution of the instrument is determined by the sharpness of the tip. Biological samples can be studied in fluids, in their natural environment. One of these methods is the analysis of surface forces.

The objective of our work is the micro- and nanometer scale characterization of different samples such as peptides and oligonucleotides, photosynthetic reaction centers, membrane proteins (i.e. bacteriorhodopsin) and the investigation of membranes or living cell’s surface and their responses to different extracellular stimuli.

By studying oligonucleotides we observed that short sequences of nucleic acids self-assemble on mica surface in a long chain-like formation. This self-assembly could have a role in the origin of life, by forming DNA or RNA, the information carrying molecules. On protein level we studied the interaction of the photosynthetic reaction center with carbon nanotubes. The complex formed is a promising material for biotechnological applications. Carbon nanotubes are efficient in biomatter-based devices. The working hypothesis was that the SWNTs could play the role of electrodes by evacuating the photoexcited electrons from the RCs.



AFM Group (from left to right): György Váró (retired former group leader), Zsolt Szegletes,
Béla Varga and Attila Gergely Végh. (2)


Single walled carbon nanotube covered with bacterial photosynthetic reaction centers. (3)


The effect of the antimicrobial peptides on supported membranes was studied. The purple membrane was less sensitive to indolicidin treatment compared to DPPC, most likely due to the high membrane protein content. For these membranes, indolicidin tended to bind to the border of membrane disks, where the lipids are easier to interact with.



Effect of indolicidin on purple membrane of Halobacterium salinarum. (4)

The AFM’s possibility to work in liquid environment and especially the design of the MFP 3D type AFM made possible to study living cells or even living tissues on a Petri dishes’ surface, in their physiological circumstances. As subject of this study the cerebral endothelial cells were chosen. They are important components of the blood brain barrier, which is the interface between the peripheral circulation and the central nervous system. The tight junctions between the endothelial cells restrict diffusion of water-soluble substances from blood to brain. Disruption of the tight junctions can lead to improper blood brain barrier function. In certain cases the blood brain barrier can be an impediment for the chemical treatment of diseases of the central nervous system. High concentration of mannitol was successfully used for reversible opening of the BBB both experimentally and clinically, although the mechanism of osmotic disruption is not well understood. During the study of endothelial cells we observed that mannitol treatment influences their volume and elasticity. Upon prolonged calcium treatment a change in cell shape could be detected. We demonstrated a difference in shape and elasticity between wild-type and mutant bacteria.



Endothelial cells before (left image) and after mannitol treatment (right image). (5)

The force measurement with AFM has great importance in determining the micromechanical properties of biological objects. There are two representation modes of the measured signal: as a function of the distance of the tip from the probe and as a function of time. The direct measurement of the strong binding between melanoma cells and living endothelial cells and the decrease of the binding to fixed cells reflects that a specific interaction takes place between the two cell types. By monitoring mechanical parameters of the cells, information can be collected about their health state.



Elastic properties of melanoma and endothelial cells. (6)

Not only confluent cell cultures, but individual cells, like bacteria or tissues can be measured by AFM.

Selected publications

Wilhelm I, Fazakas C, Molnár J, Haskó J, Végh AG, Cervenak L, Nagyőszi P, Nyúl-Tóth A, Farkas AE, Bauer H, Guillemin GJ, Bauer HC, Váró G, Krizbai IA: Role of Rho/ROCK signaling in the interaction of melanoma cells with the blood-brain barrier. PIGMENT CELL MELANOMA RES. 24(1): 113 124 (2014) DOI: 10.1111/pcmr.12169

Szabó Tibor, Bencsik Gábor, Magyar Melinda, Visy Csaba, Gingl Zoltán, Nagy Krisztina, Váró György, Hajdu Kata, Kozák Gábor, Nagy László: Photosynthetic reaction centers/ITO hybrid nanostructure. MATERIALS SCIENCE AND ENGINEERING C-BIOMIMETIC AND SUPRAMOLECULAR SYSTEMS 33(2): 769 774. (2013) DOI: 10.1016/j.msec.2012.10.031

Nagy K, Váró G, Szalontai B: κ-Casein terminates casein micelle build-up by its "soft" secondary structure. EUR BIOPHYS J. 41(11):959-68 (2012) DOI: 10.1007/s00249-012-0854-0

Vegh AG, Fazakas C, Nagy K, Wilhelm I, Molnar J, Krizbai IA, Szegletes Z, Varo G: Adhesion and stress relaxation forces between melanoma and cerebral endothelial cells. EUROPEAN BIOPHYSICS JOURNAL 41: pp. 139-145. (2012) DOI: 10.1007/s00249-011-0765-5

Vegh AG, Fazakas C, Nagy K, Wilhelm I, Krizbai IA, Nagyoszi P, Szegletes Z, Varo G: Spatial and temporal dependence of the cerebral endothelial cells elasticity: JOURNAL OF MOLECULAR RECOGNITION 24:(3) pp. 422-428. (2011) DOI: 10.1002/jmr.1107

Vegh AG, Nagy K, Balint Z, Kerenyi A, Rakhely G, Varo G, Szegletes Z: Effect of Antimicrobial Peptide-Amide: Indolicidin on Biological Membranes. JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY 2011: pp. 1-6. (2011) DOI: 10.1155/2011/670589

Balint Z, Krizbai IA, Wilhelm I, Farkas AE, Parducz A, Szegletes Z, Varo G: Changes Induced by Hyperosmotic Mannitol in Cerebral Endothelial Cells: an Atomic Force Microscopic Study. EUROPEAN BIOPHYSICS JOURNAL 36:(2) pp. 113-120. (2007) DOI: 10.1007/s00249-006-0112-4

Balint Z, Nagy K, Laczko I, Bottka S, Vegh GA, Szegletes Z, Varo G: Adsorption and self-assembly of oligodeoxynucleotides ontoa mica surface. JOURNAL OF PHYSICAL CHEMISTRY C - NANOMATERIALS AND INTERFACES 111:(45) pp. 17032-17037. (2007) DOI: 10.1021/jp0744001

Balint Z, Vegh GA, Popescu A, Dima M, Ganea C, Varo G: Direct Observation of Protein Motion During The Photochemical Reaction Cycle of Bacteriorhodopsin. LANGMUIR 23:(13) pp. 7225-7228. (2007)

Wilhelm I, Farkas AE, Nagyoszi P, Varo G, Balint Z, Vegh GA, Couraud PO, Romero IA, Weksler B, Krizbai IA: Regulation of cerebral endothelial cell morphology by extracellular calcium. PHYSICS IN MEDICINE AND BIOLOGY 52:(20) pp. 6261-6274. (2007)