Controlling orientation, conformation, and biorecognition of proteins on silane monolayers, conjugate polymers, and thermo-responsive polymer brushes: investigations using TOF-SIMS and principal component analysis

Abstract

Control over orientation and conformation of surface-immobilized proteins, determining their biological activity, plays a critical role in biointerface engineering. Specific protein state can be achieved with adjusted surface preparation and immobilization conditions through different types of protein-surface and protein-protein interactions, as outlined in this work. Time-of-flight secondary ion mass spectroscopy, combining surface sensitivity with excellent chemical specificity enhanced by multivariate data analysis, is the most suited surface analysis method to provide information about protein state. This work highlights recent applications of the multivariate principal component analysis of TOF-SIMS spectra to trace orientation and conformation changes of various proteins (antibody, bovine serum albumin, and streptavidin) immobilized by adsorption, specific binding, and covalent attachment on different surfaces, including self-assembled monolayers on silicon, solution-deposited polythiophenes, and thermo-responsive polymer brushes. Multivariate TOF-SIMS results correlate well with AFM data and binding assays for antibody-antigen and streptavidin-biotin recognition. Additionally, several novel extensions of the multivariate TOF-SIMS method are discussed.

Biocompatibility Evolves: Phenomenology to Toxicology to Regeneration

“…One example worth highlighting here is the application of multivariate statistical techniques, such as principal component analysis, to the analysis of time-of-flight secondary ion mass spectrometry (ToF-SIMS) data to determine the impact of various biomaterial properties on protein adsorption. [262][263][264]…”

Characterization of polymeric surfaces and interfaces using time‐of‐flight secondary ion mass spectrometry

“…Spectral analysis is the simplest and most common and involves acquiring a high‐resolution mass spectrum for the surface under analysis. This can be used for a variety of purposes including identifying contaminants, 40,41 determining the relative abundances of elements or molecules, 38,39,42,43 studying the orientations of molecules on a surface, 44,45 or distinguishing between block and random copolymers 37 . For example, Liu et al analyzed poly(styrene– co –4‐vinyl phenol) (PS‐P4VP) random and block copolymers matched in size and composition 37 .…”

Comparison of Physical Adsorption and Covalent Coupling Methods for Surface Density-Dependent Orientation of Antibody on Silicon

“…The arrangement of immobilized antibodies is a complex phenomenon that involves molecule-molecule and molecule-surface interactions (reviewed in [8][9][10]). The dominant antibodies orientation is determined by the amount of adsorbed molecules Γ [11,12] as well as by physical (electrostatic, hydrophobic, van der Waals) forces and the reactivity of amine groups on the protein molecule, which depends on immobilization conditions, e.g., the pH of solution [9,13].…”

“…In contrast, antibody orientations can be directly deduced from microscopic visualizations of single molecules by AFM [16,17] compared with insights from Monte Carlo and molecular dynamics (MD) simulations [16,18]. In turn, direct analysis of the dominant IgG orientation can be provided by Time-of-Flight Secondary Ion Mass Spectroscopy (TOF-SIMS) combined with Principal Component Analysis (PCA) [3,5,6,8,12,19,20]. While TOF-SIMS examines the outermost region of adsorbed molecules with regard to their amino acid composition, which is different for the exposed Fc and Fab domains (Figure 1a,b), PCA enhances the chemical specificity of TOF-SIMS and enables a comparison of different samples (as explained in Figure 1c-e).…”

“…While TOF-SIMS examines the outermost region of adsorbed molecules with regard to their amino acid composition, which is different for the exposed Fc and Fab domains (Figure 1a,b), PCA enhances the chemical specificity of TOF-SIMS and enables a comparison of different samples (as explained in Figure 1c-e). TOF-SIMS supported with PCA has been applied to compare the orientations adopted by antibodies immobilized on different surfaces of various types (e.g., gold or silicon modified with SAMs [5,6,20,21], polymer layers [20,22,23] and brushes [24], protein monolayers [21,25]), in order to analyze the factors determining molecular orientation (other than surface amount Γ of molecules) and to correlate them with protein biorecognition (as extensively reviewed in [8,26]). In fact, the above approach provides the relation between different samples of the average composition of the outermost regions of immobilized antibodies expressed by the PCA scores, reflecting the average ratio f Fc of the exposed area of Fc domain to the footprint area of the entire molecule (that is, the sum of exposed Fc and F(ab) 2 ).…”