Surface density dependent orientation and immunological recognition of antibody on silicon: TOF-SIMS and surface analysis of two covalent immobilization methods

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

“…1a). At the same time, the "escape" depth, determined as the thickness where a particular ion intensity is 3 standard deviations from the background [43], is about 3 nm for ions derived from organic films [34,43]. This value corresponds to the protein surface density of~3.8 mg/m 2 and reveals the TOF-SIMS ability to probe the complete protein monolayers.…”

“…In this operating regime, the dose density of primary ions is kept below 10 12 ions/cm 2 to ensure that secondary ions originate only from intact sample areas [42]. TOF-SIMS surface sensitivity is characterized by the mean emission (attenuation) depth of secondary ions λ~0.6 nm [34] (this value for Bi 3 + (30 keV) primary ions), indicating a much greater sensitivity for the outermost region of the protein layer ( Fig. 1a).…”

“…Therefore, the access to binding sites and antigen binging efficiency depends on antibody orientation involving flat-on (all domains attached to a surface), side-on (Fc and one Fab domain attached to a surface), head-on (both Fab domains attached to surface and Fc facing up), and tail-on (Fc domain attached to a surface and both Fab facing up) orientations [22]. TOF-SIMS with PCA was applied for the examination of the dominant orientation adopted by antibodies on a SAM modified gold [33,37,73] and silicon surface [34,74], polymer layers [28,33,35,55], and polymer brushes [75] as well as to determine the orientation of bioaffinity-bound antibodies [74,76]. The studies performed so far have provided a comparison of a dominant antibody orientation on different surfaces and a definition of the factors determining protein orientation, the being latter discussed in detail in "Factors affecting protein orientation: examination illustrated with the antibody on self-assembled monolayers" of this review.…”

“…Furthermore, time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an especially attractive method for protein state analysis: due to its excellent chemical specificity, it can directly probe the amino acid concentration of surface-immobilized proteins with a sensitivity peaked at their outermost region [29]. As a result, TOF-SIMS, supported by multivariate principal component data analysis, enables a comparison of the orientation and conformation of proteins immobilized not only on simple model substrates but also on functional molecular and polymer surfaces [24,[30][31][32][33][34][35]. Such a chemical glimpse at protein conformation and orientation can be provided, although the ultra-high vacuum conditions required by TOF-SIMS induce additional protein denaturation [36] that causes a potential drawback [24].…”

Using a lactadherin-immobilized silicon surface for capturing and monitoring plasma microvesicles as a foundation for diagnostic device development

“…ToF-SIMS, with sensitivity enhanced for the outermost region of the protein layer, probes surface amino acid concentration, enabling examination of protein orientation in a more direct manner than other surface analysis methods [36,37]. ToF-SIMS analysis of dominant orientation of surfaceimmobilized proteins is well-established for IgG antibody [38][39][40][41][42] and demonstrated also for other proteins [43][44][45][46]. Therefore, we performed PCA of ToF-SIMS data to provide an insight into the orientation of LACT immobilized following two approaches, namely, covalent binding to a GAmodified surface and site-directed binding to an αvβ3 integrin-functionalized surface.…”