Time-of-Flight
Secondary Ion Mass Spectrometry Investigation
of the Orientation of Adsorbed Antibodies on SAMs Correlated to Biorecognition
Tests
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Abstract
The
adsorption of an antiglutamate dehydrogenase (Anti-GDH) antibody
on different surfaces was studied to probe its orientation and bioactivity.
Three different situations were investigated: physisorption on a −COOH-terminated
thiols self-assembled monolayer (SAM) on gold, covalent grafting on
the same SAM using NHS-EDC activation, and physisorption on a −CH<sub>3</sub> SAM. The orientation of the antibody was investigated combining
time-of-flight secondary ion mass spectrometry and principal component
analysis. Several orientations are proposed for each case and compared
to the results of biorecognition measurements with the antigen (GDH).
At each step, protein layers were characterized ex-situ with polarization-modulated
infrared reflection absorption spectroscopy and in situ (i.e., in
the liquid phase) with quartz crystal microbalance with dissipation
monitoring. Biorecognition measurements showed interesting correlations
with proposed protein orientations. The role of hydrophobic and/or
electrostatic interactions and that of covalent bonding are discussed
to underline the influence of the orientation on the bioactivity of
adsorbed Anti-GDH