Indirect immunoassay on functionalized silicon surface: molecular arrangement, composition and orientation examined step-by-step with multi-technique and multivariate analysis
PBN-AR
Instytucja
Wydział Fizyki i Informatyki Stosowanej (Akademia Górniczo-Hutnicza im. Stanisława Staszica w Krakowie)
Źródłowe zdarzenia ewaluacyjne
Informacje podstawowe
Główny język publikacji
EN
Czasopismo
Colloids and Surfaces. B, Biointerfaces (35pkt w roku publikacji)
ISSN
0927-7765
EISSN
1873-4367
Wydawca
Elsevier Science BV
DOI
Rok publikacji
2017
Numer zeszytu
Strony od-do
437--444
Numer tomu
150
Link do pełnego tekstu
Identyfikator DOI
Liczba arkuszy
0.57
Autorzy
Pozostali autorzy
+ 5
Słowa kluczowe
EN
TOF-SIMS
Principal Component Analysis
biosensor functionalization
antibody orientation
surface analysis
indirect immunoassay
Streszczenia
Język
EN
Treść
The arrangement, composition and orientation of immunoreagents employed in an indirect immunoassay for determination of mycotoxin OchraToxin A (OTA) are specified for Si3N4 substrate, aiming to imitate biosensor transducers made of the same material. Si3N4 surfaces 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 6.9 mg/m(2). 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 similar to 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. (C) 2016 Elsevier B.V. All rights reserved.
Cechy publikacji
original article
peer-reviewed
Inne
System-identifier
idp:105530
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