Recent developments in electrochemical immunoassays and immunosensors - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 126

Printed carbon working electrode (PCWE)

Dipping in 1 mM
TTCA monomer
solution and dry

Polymer growing
in phosphate
buffered medium
TTCA polymer film on PCWE

anti-Vtg

Immobilization
of HRP and
antibody

Activation by
EDC

HRP

e TTCA polymer film on PCWE

HRP ox

HRP red

Vtg

H 2 O

H 2 O 2

GOx

GOx

O 2

GOx-Vtg

gluconolactone

glucose

FIGURE 5.7 Schematic representation of electrode preparation and signal generation scheme for the
immunoassay based on a fi lm of TTCA. (Reprinted from [35] with permission from Elsevier.)

S-H bond to the gold surface, followed by the reductive elimination of hydrogen, as
shown in Eq. (2) [37].

1
2

——

0

——

0

RSH u RS → u u H

(2)

n n 2

The alkyl chains of the thiols are all in the trans conformation, and the second step
in the formation of a highly ordered monolayer involves van der Waals interactions
between the chains, which results in the chains having a fi nal tilt angle of between
20º and 30º from the normal (Fig. 5.8) [38, 39]. It is the highly ordered nature of
SAMs that is exploited to provide controlled orientation of antibodies in electro-
chemical immunoassay systems. By forming a SAM with an -carboxyfunctional-

ized alkanethiol, the exposed surface of the monolayer consists of free carboxylic acid
groups that, upon activation, can be covalently bound to amine groups from the lysine
residues of the capture antibody or other protein. SAMs with other terminal function-
alities may also be used. For example, in Herrwerth et al. 's work, antibodies were cov-
alently coupled to a SAM of carboxy-functionalized poly(ethylene glycol) alkanethiol
[HOOC ß CH 2 ß (OCH 2 ß CH 2 ) n ß O ß (CH 2 ) 11 ß SH] [40]. This molecule has a dual
function in which the carboxylic acid group is used for coupling to the antibody, and
the poly(ethylene glycol) region provides resistance against non-specifi c binding. The
SAM was formed by immersing thin fi lms of polycrystalline gold in a 10 mL solu-
tion of the alkanethiol (0.5 mM) in absolute N,N -dimethylformamide for 18 h. The
exposed carboxylic acid termini were activated by immersing the SAM-coated fi lms in
a solution containing EDC and N-hydroxysuccinimide (0.2 mol L 1 and 0.05 mol L 1

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