1. Raster image correlation spectroscopy as a novel tool to study interactions of macromolecules with nanofiber scaffolds
- Author
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Martin Hof, Evžen Amler, David Lukas, Sam C. P. Norris, Matej Buzgo, Radek Macháň, Martina Huranova, and Jana Humpolíčková
- Subjects
Materials science ,Nanofibers ,Biomedical Engineering ,Nanotechnology ,02 engineering and technology ,Biochemistry ,Diffusion ,Biomaterials ,03 medical and health sciences ,chemistry.chemical_compound ,Molecule ,Diffusion (business) ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Fluorescence recovery after photobleaching ,General Medicine ,021001 nanoscience & nanotechnology ,chemistry ,Nanofiber ,Self-healing hydrogels ,Biophysics ,Agarose ,0210 nano-technology ,Two-dimensional nuclear magnetic resonance spectroscopy ,Protein Binding ,Biotechnology ,Macromolecule - Abstract
Dynamic processes such as diffusion and binding/unbinding of macromolecules (e.g. growth factors or nutrients) are crucial parameters for the design and application of effective artificial tissue materials. Here, dynamics of selected macromolecules were studied in two different composite tissue engineering scaffolds containing an electrospun nanofiber mesh (polycaprolactone or hydrophobically plasma modified polyvinylalcohol–chitosan) encapsulated in agarose hydrogels by a conventional approach fluorescence recovery after photobleaching (FRAP) and a novel technique, raster image correlation spectroscopy (RICS). The two approaches are compared, and it is shown that FRAP is unable to determine processes occurring at low molecular concentrations, especially accurately separating binding/unbinding from diffusion, and its results depend on the concentration of the studied molecules. RICS measures processes of single molecules and, because of its multiple adjustable timescales, can distinguish whether diffusion or binding controls molecular movement and separates fast diffusion from slow transient binding. In addition, RICS provides a robust read-out parameter quantifying binding affinity. Finally, the combination of FRAP and RICS helps to characterize diffusion and binding of macromolecules in tested artificial tissues better, and therefore predicts the behavior of biologically active molecules in these materials for medical applications.
- Published
- 2011