Selective protein trapping within hybrid nanowells

Abstract

Nanostructured surfaces offer a great deal in view of the control of biological processes at subcellular level. An innovative methodology has been developed to fabricate large-scale hexagonally close-packed arrays of polymer/gold nanowells of tunable diameter and depth, ranging between about 70 and 100 nm (diameter) and 15 and 40 nm (depth). Nanowell volumes down to 0.3 attolitres and nanowell densities as high as ∼10(9) wells per cm(2) could also be demonstrated. The present paper investigates the main features of protein trapping processes within the obtained nanowell arrays. Selective protein trapping, also involving orientation and biofunctionality changes, appears to be induced by the nanoconfinement. Nanomorphology measurements and antibody preferential linkages are demonstrated for human serum albumin versus lysozyme, the first being efficiently trapped within the nanocavities and the second being preferentially deposited outside them. The selective protein-dependent trapping/untrapping within the nanowells is discussed in terms of the variation in the out-diffusion coefficients of the biomolecules entering the nanowells, either as a function of the matching/mismatching of the biomolecules and nanocavity dimensions, or, alternatively, owing to the drastic conformational changes due to nanoconfinement. In this case, the trapping of large and soft human serum albumin is privileged with respect to the small and hard lysozyme. Furthermore, the observed peculiar antibody response to the confined proteins is accounted for in terms of the enhancement of their biological response following the modified accessibility of the key epitopes, which in turn suggests drastic conformational changes.

Controlling Protein Adsorption through Nanostructured Polymeric Surfaces

“…[118] In a follow-up study, the hexagonally ordered well arrays were shown to selectively confine albumin and lysozyme molecules within and outside the 100 nm diameter wells, respectively (Figure 5a). [10] The implication of a further decrease in surface topographical features, pit size below 100 nm, on protein confinement, conformation as well as elasticity has been emphasized in recent studies by Wang and Akcora. [117] It has been shown that fibrinogen undergoes less structural changes and behaves less stiff when the size of confining pits is close to the protein size, i.e., 50 nm (Figure 5b).…”

“…Adapted with permission. [10] Copyright 2016, The Royal Society of Chemistry. b) Schematic representation of rod-shaped (fibrinogen) and globular (lysozyme) protein confinement inside PMMA pores of varying sizes (left).…”

Spatially Resolved Spectroscopic Characterization of Nanostructured Films by Hyperspectral Dark-Field Microscopy

“…The adsorption of protein on the membrane surface is influenced by the molecular properties of proteins and surface physicochemical properties of the membrane . Surface wettability and nanoscale morphology comparable to the physical size of the protein greatly affects the adsorbed amount of protein molecules. , The stability of the BSA molecules adsorbed on the surface of the TiO 2 film is affected by the pore size, the hydrophilicity difference in the inner and outer of the pore, and the density of the hydroxyl group in the pore. − As shown in Figure , surface scattering of the TiO 2 thin film was enhanced due to the adsorbed BSA molecules. However, the averaged scattering spectra of the local regions can only reveal the macroscopical protein adsorption characteristics of the nanoporous TiO 2 thin films.…”

Chelating Surfaces for Oriented Human Serum Albumin Molecules

“…It is also known to form binary and ternary structures which can involve histidine and/or the copper binding region of the albumin molecule. − The origins of this behavior are not yet understood, but their unravelling would prompt the possible extension of the methodology as a general method to drive protein adsorption in a desired conformation by carefully selecting the metal cation and, in turn, the coordination structure of interest. The multistep construction of the GHK–Cu complex and the related human serum albumin (HSA)-adsorption features have been studied by means of quartz crystal microbalance with dissipation monitoring (QCM-D), , providing unique semiquantitative information on the formation and viscoelastic properties of the chelation layer and the subsequent protein adsorption processes, , and dynamic force spectroscopy, which allowed determination of the strength of interaction between the tip and the protein adsorbed on the chelating layer. , Finally, we have implemented a simple kinetic model, based on random sequential adsorption (RSA), , accounting for the formation of a protein film with a native-like character.…”