2 research outputs found
Time-of-Flight Secondary Ion Mass Spectrometry Investigation of the Orientation of Adsorbed Antibodies on SAMs Correlated to Biorecognition Tests
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
Probing the Orientation of β‑Lactoglobulin on Gold Surfaces Modified by Alkyl Thiol Self-Assembled Monolayers
The adsorption of a globular protein
on chemically well controlled
surfaces was investigated in order to correlate its orientation to
the surface properties. To this end, three different alkyl thiols,
differing by their end group (−COOH, −CH<sub>3</sub>, and −NH<sub>2</sub>), were used to build up self-assembled
monolayers (SAMs) on gold substrates. β-Lactoglobulin (βLG)
was then adsorbed on these SAMs by immersion in a phosphate buffer
solution. The surface modification with alkyl thiols and the subsequent
adsorption of proteins were characterized <i>ex situ</i> by polarization modulated infrared reflection–absorption
spectroscopy (PM-IRRAS) and X-ray photoelectron spectroscopy (XPS).
The adsorption behavior of proteins was also monitored <i>in
situ</i> using quartz crystal microbalance with dissipation measurements
(QCM-D). Direct evidence regarding the protein orientation in the
adsorbed state was obtained by means of time-of-flight secondary ion
mass spectrometry (ToF-SIMS). Principal component analysis (PCA),
performed on the ToF-SIMS results, enables to separate the samples
and shows that the proteins display different distributions of amino
acids at the surface depending on the conditioning thiol layer. Our
results revealed that the adsorption mode of the protein is influenced
by the thiol end groups, and specific orientations of the protein
on the surface are proposed for the different substrates