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Rother J.,Institute of Physical Chemistry | Noding H.,Institute of Physical Chemistry | Mey I.,Institute of Organic and Biomolecular Chemistry | Janshoff A.,Institute of Physical Chemistry
Open Biology | Year: 2014

Mechanical phenotyping of cells by atomic force microscopy (AFM) was proposed as a novel tool in cancer cell research as cancer cells undergo massive structural changes, comprising remodelling of the cytoskeleton and changes of their adhesive properties. In this work, we focused on the mechanical properties of human breast cell lines with different metastatic potential by AFM-based microrheology experiments. Using this technique,we are not only able to quantify the mechanical properties of living cells in the context of malignancy, but we also obtain a descriptor, namely the loss tangent, which provides model-independent information about the metastatic potential of the cell line. Including also other cell lines from different organs shows that the loss tangent (G00/G0) increases generally with the metastatic potential from MCF-10A representing benign cells to highly malignant MDA-MB-231 cells. © 2014 The Authors.

Lazzara T.D.,Institute of Organic and Biomolecular Chemistry | Behn D.,Institute of Organic and Biomolecular Chemistry | Kliesch T.-T.,Institute of Organic and Biomolecular Chemistry | Janshoff A.,Institute of Physical Chemistry | Steinem C.,Institute of Organic and Biomolecular Chemistry
Journal of Colloid and Interface Science | Year: 2012

Anodic aluminum oxide (AAO) substrates with aligned, cylindrical, non-intersecting pores with diameters of 75. nm and depths of 3.5 or 10 μm were functionalized with lipid monolayers harboring different receptor lipids. AAO was first functionalized with dodecyl-trichlorosilane, followed by fusion of small unilamellar vesicles (SUVs) forming a lipid monolayer. The SUVs' lipid composition was transferred onto the AAO surface, allowing us to control the surface receptor density. Owing to the optical transparency of the AAO, the overall vesicle spreading process and subsequent protein binding to the receptor-doped lipid monolayers could be investigated in situ by optical waveguide spectroscopy (OWS). SUV spreading occurred at the pore-rim interface, followed by lateral diffusion of lipids within the pore-interior surface until homogeneous coverage was achieved with a lipid monolayer. The functionality of the system was demonstrated through streptavidin binding onto a biotin-DOPE containing POPC membrane, showing maximum protein coverage at 10. mol% of biotin-DOPE. The system enabled us to monitor in real-time the selective extraction of two histidine-tagged proteins, PIGEA14 (14. kDa) and ezrin (70. kDa), directly from cell lysate solutions using a DOGS-NTA(Ni)/DOPC (1:9) membrane. The purification process including protein binding and elution was monitored by OWS and confirmed by SDS-PAGE. © 2011 Elsevier Inc.

Lazzara T.D.,Institute of Organic and Biomolecular Chemistry | Mey I.,Institute of Organic and Biomolecular Chemistry | Steinem C.,Institute of Organic and Biomolecular Chemistry | Janshoff A.,Institute of Physical Chemistry
Analytical Chemistry | Year: 2011

Porous substrates have gained widespread interest for biosensor applications based on molecular recognition. Thus, there is a great demand to systematically investigate the parameters that limit the transport of molecules toward and within the porous matrix as a function of pore geometry. Finite element simulations (FES) and time-resolved optical waveguide spectroscopy (OWS) experiments were used to systematically study the transport of molecules and their binding on the inner surface of a porous material. OWS allowed us to measure the kinetics of protein adsorption within porous anodic aluminum oxide membranes composed of parallel-aligned, cylindrical pores with pore radii of 10-40 nm and pore depths of 0.8-9.6 μm. FES showed that protein adsorption on the inner surface of a porous matrix is almost exclusively governed by the flux into the pores. The pore-interior surface nearly acts as a perfect sink for the macromolecules. Neither diffusion within the pores nor adsorption on the surface are rate limiting steps, except for very low rate constants of adsorption. While adsorption on the pore walls is mainly governed by the stationary flux into the pores, desorption from the inner pore walls involves the rate constants of desorption and adsorption, essentially representing the protein-surface interaction potential. FES captured the essential features of the OWS experiments such as the initial linear slopes of the adsorption kinetics, which are inversely proportional to the pore depth and linearly proportional to protein concentration. We show that protein adsorption kinetics allows for an accurate determination of protein concentration, while desorption kinetics could be used to capture the interaction potential of the macromolecules with the pore walls. © 2011 American Chemical Society.

Lazzara T.D.,Institute of Organic and Biomolecular Chemistry | Aaron Lau K.H.,Northwestern University | Knoll W.,AIT Austrian Institute of Technology | Janshoff A.,Institute of Physical Chemistry | Steinem C.,Institute of Organic and Biomolecular Chemistry
Beilstein Journal of Nanotechnology | Year: 2012

Layer-by-layer (LbL) deposition of polyelectrolytes and proteins within the cylindrical nanopores of anodic aluminum oxide (AAO) membranes was studied by optical waveguide spectroscopy (OWS). AAO has aligned cylindrical, nonintersecting pores with a defined pore diameter d 0 and functions as a planar optical waveguide so as to monitor, in situ, the LbL process by OWS. The LbL deposition of globular proteins, i.e., avidin and biotinylated bovine serum albumin was compared with that of linear polyelectrolytes (linear-PEs), both species being of similar molecular weight. LbL deposition within the cylindrical AAO geometry for different pore diameters (d 0 = 25-80 nm) for the various macromolecular species, showed that the multilayer film growth was inhibited at different maximum numbers of LbL steps (n max). The value of n max was greatest for linear-PEs, while proteins had a lower value. The cylindrical pore geometry imposes a physical limit to LbL growth such that n max is strongly dependent on the overall internal structure of the LbL film. For all macromolecular species, deposition was inhibited in native AAO, having pores of d 0 = 25-30 nm. Both, OWS and scanning electron microscopy showed that LbL growth in larger AAO pores (d 0 > 25-30 nm) became inhibited when approaching a pore diameter of d eff,n_max = 25-35 nm, a similar size to that of native AAO pores, with d 0 = 25-30 nm. For a reasonable estimation of d eff,n_max, the actual volume occupied by a macromolecular assembly must be taken into consideration. The results clearly show that electrostatic LbL allowed for compact macromolecular layers, whereas proteins formed loosely packed multilayers. © 2012 Lazzara et al.

Hofer I.,Institute of Organic and Biomolecular Chemistry | Steinem C.,Institute of Organic and Biomolecular Chemistry
Soft Matter | Year: 2011

Pore-spanning planar membranes on highly ordered porous silicon substrates were shown to be well suited to monitor the calcium ion mediated fusion of large unilamellar vesicles by means of confocal laser scanning fluorescence microscopy and scanning ion conductance microscopy in real-time. © 2011 The Royal Society of Chemistry.

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