Fluorescence microscopy of biophysical protein dynamics in nanoporous hydrogels

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.

In-situ Neutron Reflectometry Study on Adsorption of Glucose Oxidase at Mesoporous Aluminum Oxide Film

“…The protein distribution on and in nanoporous materials is thus an important subject to be clarified, and in-situ spectroscopic techniques for protein adsorption have been developed. [4][5][6][7][8][9][10][11][12][13][14][15][16][17] The distribution of adsorbate on a single nanoporous particle has been often observed by microspectroscopy techniques. 4,5 For a nanoporous film on a solid substrate, spectroscopic methods utilizing optical interference [6][7][8][9][10][11][12][13][14] and waveguide mode [15][16][17] are available for the in-situ observation of the molecular adsorption.…”

Imaging Switchable Protein Interactions with an Active Porous Polymer Support

“…27 Super-resolution fluorescence microscopy techniques have been applied for the elucidation of nanoscale properties of various soft polymer materials. 28,29 Three-dimensional structures of block copolymers and polymers blends have been studied using various forms of stimulated emission depletion (STED) microscopy, 30−32 localization-based photoactivated localization microscopy (PALM) 33,34 and stochastic optical reconstruction microscopy (STORM). 35−41 In the present work, we use single-particle tracking (SPT) 42 and fluorescence correlation spectroscopy Super-resolution Optical Fluctuation Imaging (fcsSOFI) 43 to characterize the porous structure and diffusion properties of allylamine functionalized poly-N-isopropylacrylamide (pNIPAM-co-AA) pH-sensitive hydrogels.…”

Polymeric Composite of Magnetite Iron Oxide Nanoparticles and Their Application in Biomedicine: A Review

“…Electrons are promoted to an excited state, and the molecule will be in an excited vibrational energy state. A photon will be emitted when the molecule comes back to its ground electronic state [125].…”