Document Type
Article
Publication Date
8-23-2019
Abstract
Proteins within nanoporous hydrogels have important biotechnological applications in pharmaceutical purification, tissue engineering, water treatment, biosensors, and medical implants. Yet, oftentimes proteins that are functional in solution lose activity when in contact with soft, nanostructured, condensed phase materials due to perturbations in the folded state, conformation, diffusion, and adsorption dynamics of the protein by the material. Fluorescence microscopy experimentally measures the biophysical dynamics of proteins within hydrogels at the nanoscale and can overcome the limitations of conventional ensemble techniques. An explanation of the benefits of fluorescence is provided, and principles of fluorescence microscope instrumentation and analysis are discussed. Then several nanoscale fluorescence microscopies that image nanoscale protein dynamics within hydrogels are introduced. First, location-based super-resolution imaging resolves the adsorption kinetics of proteins to charged ligands within hydrogels used in pharmaceutical separations. Next, correlation-based super-resolution techniques image the heterogeneity of the nanoscale pore size of the hydrogels and the diffusion of analytes within the pores simultaneously. Finally, fluorescence resonance energy transfer imaging combined with temperature jump perturbations determines the folding and stability of a protein within hydrogels. A common finding with all three fluorescence microscopies is that heterogeneous nanoporous hydrogel materials cause variability of protein behavior dependent on gel sterics and/or interfacial electrostatic forces. Overall, in situ observations of proteins in hydrogels using fluorescence microscopies can inform and inspire soft nanomaterial design to improve the performance, shelf life, and cost of biomaterials.
Keywords
general procedures and instrumentation, image processing, biomaterials, nanomaterials, adsorption, protein dynamics, fluorescence microscopy, fluorescence resonance energy transfer
Language
English
Publication Title
Journal of Applied Physics
Rights
This article may be downloaded for personal use only. Any other use requires prior permission of the author and AIP Publishing.
Recommended Citation
Anuj Saini, Lydia Kisley; Fluorescence microscopy of biophysical protein dynamics in nanoporous hydrogels. J. Appl. Phys. 28 August 2019; 126 (8): 081101. https://doi.org/10.1063/1.5110299
Manuscript Version
Final Publisher Version