title: Indirect immunoassay on functionalized silicon surface: Molecular arrangement, composition and orientation examined step-by-step with multi-technique and multivariate analysis.
abstract: The arrangement, composition and orientation of immunoreagents employed in an indirect immunoassay for determination of mycotoxin OchraToxin A (OTA) are specified for Si3N4substrate, aiming to imitate biosensor transducers made of the same material. Si3N4surfaces are examined after modification with (3-aminopropyl)triethoxysilane, spotting with OTA-ovalbumin conjugate (probe), blocking with bovine serum albumin, reaction with a mouse monoclonal antibody against OTA and, finally, reaction with a goat anti-mouse secondary antibody. Atomic force micrographs, their autocorrelation and height histogram parameters, show the stepwise development of a multi-component monolayer covered by groups of secondary antibody molecules. Time-Of-Flight Secondary Ion Mass Spectrometry reveals the composition of probe and blocking protein, as well as their partial desorption during the primary immunoreaction. Ellipsometry provides surface amount of all proteins, increasing step-by-step from 0.7 to 6.9mg/m2. In addition, ellipsometry combined with TOF-SIMS reveals the mass loadings of different molecules in the intermediate and the final overlayer. Based on this, some orientations of the immobilized molecules are proposed and a molar ratio of ∼2.5 for secondary to primary antibody is calculated. The orientations of the primary and secondary antibody are further clarified by Principal Component Analysis of TOF-SIMS data, through which a side-on and a head-on orientation is deduced for the primary and the secondary antibody, respectively. These findings demonstrate how the combination of multiple surface analysis techniques can provide insight on the arrangement, composition and orientation of biomolecules in the course of multi-step procedures employed in biosensors.
authors.affiliation: M. Smoluchowski Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland. Electronic address: katarzyna.gajos@doctoral.uj.edu.pl.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Gajos
authors.affiliation: M. Smoluchowski Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Budkowski
authors.affiliation: Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety NCSR Demokritos, P. Grigoriou & Neapoleos St., Aghia Paraksevi 15310, Athens, Greece.
authors.forename: Varvara
authors.initials: V
authors.lastname: Pagkali
authors.affiliation: Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety NCSR Demokritos, P. Grigoriou & Neapoleos St., Aghia Paraksevi 15310, Athens, Greece.
authors.forename: Panagiota
authors.initials: P
authors.lastname: Petrou
authors.affiliation: M. Smoluchowski Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Biernat
authors.affiliation: M. Smoluchowski Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland.
authors.forename: Kamil
authors.initials: K
authors.lastname: Awsiuk
authors.affiliation: M. Smoluchowski Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland.
authors.forename: Jakub
authors.initials: J
authors.lastname: Rysz
authors.affiliation: Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Mickiewicza 30, 30-059 Kraków, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Bernasik
authors.affiliation: Department of Microelectronics, Institute of Nanoscience and Nanotechnology, NCSR Demokritos, P. Grigoriou & Neapoleos St., Aghia Paraksevi 15310, Athens, Greece.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Misiakos
authors.affiliation: Department of Microelectronics, Institute of Nanoscience and Nanotechnology, NCSR Demokritos, P. Grigoriou & Neapoleos St., Aghia Paraksevi 15310, Athens, Greece.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Raptis
authors.affiliation: Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety NCSR Demokritos, P. Grigoriou & Neapoleos St., Aghia Paraksevi 15310, Athens, Greece.
authors.forename: Sotirios
authors.initials: S
authors.lastname: Kakabakos
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002417:Cattle; D007118:Immunoassay / Q000379:methods; D007074:Immunoglobulin G / Q000737:chemistry; D051379:Mice; D018625:Microscopy, Atomic Force; D015999:Multivariate Analysis; D009793:Ochratoxins / Q000737:chemistry*; D025341:Principal Component Analysis; D011437:Propylamines / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012821:Silanes / Q000737:chemistry; D012825:Silicon / Q000737:chemistry*; D018629:Spectrometry, Mass, Secondary Ion; D055672:Static Electricity; D013499:Surface Properties
chemical_list: D007074:Immunoglobulin G; D009793:Ochratoxins; D011437:Propylamines; D012821:Silanes; C025589:ochratoxin A; D012710:Serum Albumin, Bovine; C477625:amino-propyl-triethoxysilane; D012825:Silicon
keywords: Antibody orientation; Biosensor functionalization; Indirect immunoassay; Principal Component Analysis; Surface analysis; TOF-SIMS