Recent developments in electrochemical immunoassays and immunosensors - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 127

alkanethiol chains

gold surface

FIGURE 5.8 Schematic representation depicting the 20º to 30º tilt angle of alkanethiol chains in a
SAM on a gold surface. (Reprinted in part with permission from [39]. Copyright 2005 American Chemical
Society.)

respectively). There is a net result of converting the carboxylic acid groups into active
esters. The activated SAM was then exposed to a solution of goat anti-rabbit IgG for
2 h. The resistance of the fi lm towards non-specifi c protein interactions and the abil-
ity of the immobilized capture antibody to partake in specifi c immunoreactions were
investigated. The SAM was exposed to a solution of the protein fi brinogen. After thor-
ough rinsing, the SAMs were characterized by Fourier transform-infrared refl ection
absorption spectroscopy (FT-IRRA). The lack of an amide I or amide II band in the
spectra indicated that, despite the presence of terminal ß COOH groups, the SAM was
resistant to non-specifi c protein interactions. It was also demonstrated that the SAM
was inert towards non-specifi c interactions after antibody immobilization. The immu-
noreactivity of the immobilized capture antibody was investigated by introducing rab-
bit anti-sheep IgG as an antigen. This resulted in signifi cant growth of the amide bands
in the FT-IRRA spectra, indicating the presence of a specifi c interaction.
In addition to covalently attaching antibodies to SAMs, they may also be adsorbed
on charged SAMs via electrostatic interactions. Chen et al. have investigated the
effects of varying surface and solution properties on the orientation of adsorbed anti-
bodies [41]. In this work, the physisorption of anti-human chorionic gonadotrophin
(hCG; which is a hormone used in pregnancy tests and can also act as a tumor marker)
IgG to COOH- and NH 2 -terminated SAMs was studied. Isoelectric focusing was used
to determine the isoelectric points (IEPs) of IgG and its Fc and F(ab ) 2 fragments to be

6.8, 8.3, and 6.0, respectively. Surface plasmon resonance was used to determine the
surface coverage of adsorbed antibody to COOH- (negatively charged) and NH 2 - (pos-
itively charged) terminated SAMs. A buffer with low ionic strength was used (2.1 mM
PBS with 0.3 mM NaCl) in order to maximize electrostatic interaction between IgG
and the SAMs. A much larger quantity of IgG was found to adsorb on the COOH-
terminated SAMs. Maximum adsorption of IgG on COOH- and NH 2 -terminated
SAMs was expected to occur at pH values lower and higher than its IEP, respectively.
The maximum adsorption on the COOH-terminated SAMs of approximately 5 mg
m 2 , occurred at pH 6.25, whereas the maximum adsorption on the NH 2 -terminated
SAMs was approximately 2.3 mg m 2 and was also at pH 6.25. This particular result