A Nanoengineering Approach for Investigation and Regulation of Protein Immobilization

Abstract

It is known that protein attachment to surfaces depends sensitively upon the local structure and environment of the binding sites at the nanometer scale. Using nanografting and reversal nanografting, both atomic force microscopy (AFM) - based lithography techniques, protein binding sites with well-defined local environments are designed and engineered with nanometer precision. Three proteins, goat-anti-biotin Immunoglobulin G (IgG), lysozyme and rabbit-Immunoglobulin G, are immobilized onto these engineered surfaces. Strong dependence on the dimension and spatial distribution of protein binding sites are revealed in antibody recognition, covalent attachment via primary amine residues and surface bound aldehyde-groups. This investigation indicates that AFM based nanolithography enables the production of protein nanostructures and more importantly, protein-surface interactions at a molecular level can be regulated by changing the binding domains and their local environment at nanometer scale.

Targeted drug distribution in tumor extracellular fluid of GD2-expressing neuroblastoma patient-derived xenografts using SN-38-loaded nanoparticles conjugated to the monoclonal antibody 3F8

“…These results were supported by HR-SEM that showed the spherical morphology of all the NPs and the change of the surface topography from a very smooth for R-NPs to a patterned one for IgG3-NPs and 3F8-NPs (Figure 1). In the latter two, the conjugated antibodies were visualized as small dots (<20 nm), as previously reported upon the conjugation of other proteins to polymeric NPs [40] and in line with the reported size of IgG antibodies [41]. Upon analysis of the non-conjugated fraction of antibody in the supernatant of the reaction, the calculated efficiency of the conjugation process was 96.7 ± 3.0%, i.e., most of the antibody reacted with the NPs in the selected conditions.…”

Effect of the Protein Corona on Antibody–Antigen Binding in Nanoparticle Sandwich Immunoassays

“…Ab coverage was determined to be 4.3 ± 2.2 Abs/NP, equivalent to an Ab footprint of 232.6 nm 2 per Ab using the calculated surface area of a NP of 1000 nm 2 . 24 The dimensions of a typical IgG antibody of 14.5, 8.5, and 4 nm, 33 which has an average footprint of 81.3 nm 2 , suggest that the coverage is less than a monolayer. However, it is of note that even if the Ab had a high coverage resulting in a multilayer on the NPs, it does not preclude the NP-Ab from being able function in an immunoassay.…”

Optical Characterization of the Antigen-Antibody Thin Layer Using the Whispering Gallery Mode

“…Furthermore, the size of the immobilized antibody is similar to that of the "Y" model size of the IgG antibody (Peace III et al and Tan et al). 40,41 Therefore, we conclude that antibody "Y" immobilized on the PS microsphere surface is a monolayer.…”