title: Model immunoassay on silicon surfaces: vertical and lateral nanostructure vs. protein coverage.
abstract: To provide complete characterization of immunoassay on silicon biosensor surfaces, atomic force microscopy, (angle-resolved) X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry were applied to examine Si(3)N(4) surfaces modified with (3-aminopropyl)triethoxysilane, coated with gamma globulins (IgG), blocked with bovine serum albumin and then reacted with anti-IgG antibody for two complementary pairs (rabbit and mouse IgG) at various concentrations (from 0.3 nM to 330 nM). Protein coverage, as reflected in (amine to total N1s) XPS signal ratio and determined from ARXPS, decreases slightly due to blocking and then increases monotonically for anti-IgG antibody concentrations higher than 1 nM. AFM images reveal hardly any change of lateral nanostructure due to blocking but response to antibody solutions, based on both the mean size (from autocorrelation) and dominant spacing (from Fourier analysis) of surface features, similar to that given by ARXPS. AFM height histograms provided information about the vertical nanostructure and the parameters of height distribution (average height, spread - roughness and skewness) were distinctly influenced by coating, blocking and immunoreaction. Average protein layer thickness values determined based on protein structure (molecular weight, dimensions) and surface coverage provided from ARXPS were in accord with average height of protein layer determined from AFM. TOF-SIMS analysis indicated that BSA blocks free surface sites and in addition replaces some already adsorbed IgGs.
authors.affiliation: M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, 30-059 Kraków, Poland.
authors.forename: K
authors.initials: K
authors.lastname: Awsiuk
authors.forename: A
authors.initials: A
authors.lastname: Budkowski
authors.forename: P
authors.initials: P
authors.lastname: Petrou
authors.forename: A
authors.initials: A
authors.lastname: Bernasik
authors.forename: M M
authors.initials: MM
authors.lastname: Marzec
authors.forename: S
authors.initials: S
authors.lastname: Kakabakos
authors.forename: J
authors.initials: J
authors.lastname: Rysz
authors.forename: I
authors.initials: I
authors.lastname: Raptis
mesh_terms: D000818:Animals; D002417:Cattle; D007118:Immunoassay / Q000379:methods*; D051379:Mice; D018625:Microscopy, Atomic Force; D049329:Nanostructures / Q000737:chemistry*; D056951:Photoelectron Spectroscopy; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000032:analysis*; D012825:Silicon / Q000737:chemistry*; D013499:Surface Properties
chemical_list: D012710:Serum Albumin, Bovine; D012825:Silicon