Use of 1,3-dithiane combined with aryldiazonium cation for immobilization of biomolecules based on electrochemical addressing

Article abstract of DOI:10.1039/b909244g

We report the use of 1,3-dithiane combined with aryldiazonium cation for the immobilization of biomolecules based on electrochemical addressing.

Full text of DOI:10.1039/b909244g

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Use of 1,3-dithiane combined with aryldiazonium cation for
immobilization of biomolecules based on electrochemical addressingw
Al-Monsur Jiaul Haque,a Seung-Ryong Kwon,a Hyejin Park,a Tae-Hyun Kim,a
Young-Seok Oh,a Sung-Yool Choi,b Jong-Dal Honga and Kyuwon Kim*a
Received (in Cambridge, UK) 11th May 2009, Accepted 15th June 2009
First published as an Advance Article on the web 6th July 2009
DOI: 10.1039/b909244g
We report the use of 1,3-dithiane combined with aryldiazonium
cation for the immobilization of biomolecules based on
electrochemical addressing.
1,3-dithiane as an aliphatic electroactive group affords
carbonyl functionality in high yield.11 Scheme 1B displays a
reductive electrodeposition of a DTD cation on ITO electrode
surfaces and a suggested mechanism for the electrochemical
deprotection of aldehydes. Applied anodic potential oxidizes
Electrochemically assisted modification (EAM) of electrode
surfaces with specific chemical end functionalities for the
immobilization of biomolecules has been extensively studied
during the past two decades because the modification can be
completed within a very short time and allows selective
functionalization of closely-spaced electrode surfaces by
application of potential bias.1–9
monosubstituted 1,3-dithiane to
a
positively charged
substituent which is attacked by water present in solution
and subsequently leads to the formation of an aldehyde group
by removing the disulfide as a protecting group. To the best of
our knowledge, this is the first use of 1,3-dithiane for surface
modification although it is well-known as a protecting group
of carbonyl functionality in organic synthesis. An alternative
to the aromatic groups, including hydroquinone moieties
commonly employed as electroactive groups,7–9 the use of
aliphatic 1,3-dithiane is expected to have several merits: a
simpler procedure for synthesis; less change of electrochemical
potential on chemically derivatizing electroactive parts;
lower nonspecific binding of biomolecules on the surfaces.
Differently from the common use of aryldiazonium cation
based on electro-addressing for the selective immobilization,
the electrodeposition of the cation in our work was used only
for the attachment of dithiane functionality.
There are two types of EAMs. The first is a direct electro-
deposition of functional molecules, in which the electro-
chemical reduction of aryldiazonium salts introduced by
Delamar et al.2 became one of the most interesting methods
that offers the advantages of stable covalent attachment of
useful functional groups to various substrates such as
carbon,2,3 silicon,4 metals5 and even indium-tin-oxide
(ITO).6 The second is an electrochemical reaction of electro-
active molecules pre-coated on electrodes. For example,
hydroquinone moieties of self-assembled monolayers on gold
surfaces are electrochemically modified to their corresponding
chemically reactive functions7–9 and nitro groups of nitro-
phenyl derivatized surfaces are electrochemically reduced to
amines.2b Among them, on-demand formation of an aldehyde
group by electrochemical deprotection is a fairly attractive
technique because not only can it be used for the direct
immobilization of amine-terminated molecules, including
proteins, without additional linkers or chemical activation
steps, but also can ensure the stability of the aldehyde during
storage and surface modification.8,10
The electrodeposition of the DTD cation on the ITO
electrode surface was conducted by using cyclic voltammetry
(CV), which produces a large irreversible reduction current
wave at À0.2 V during the first cycle followed by greatly
diminished currents on even the second cycle, as shown in
Fig. 1A. The result exhibits very fast saturation of the
electrode surface, suggesting that the electrodeposition is more
efficient for the molecular layer formation than the previous
reports for other aryldiazonium cations.6 Electrochemical
deprotection of the 1,3-dithiane group on the surface was
investigated with CV and X-ray photoelectron spectroscopy
(XPS). Fig. 1B shows CV for anodic oxidation of the
DTD-modified electrode. An irreversible anodic current peak
was observed at 1.5 V on the first scan, which then disappeared
on the next scans. The surface density calculated from the area
Here we report a combination approach of the two EAMs
as a useful method for the immobilization of proteins. The
combination comprises the aryldiazonium function and the
electroactive function, producing aldehyde groups as their
respective most promising alternatives of the two EAMs. In
this work, we employed in situ generated 4-(1,3-dithian-2-
yl)benzenediazonium (DTD) cations from 4-(1,3-dithian-2-
yl)aniline via diazotation (Scheme 1A). The new aniline
was synthesized in this work. Electrochemical oxidation of
a Department of Chemistry, University of Incheon, Incheon, 402-749,
Korea. E-mail: kyuwon_kim@incheon.ac.kr
Scheme 1 (A) In situ preparation of the DTD cation from its aniline
precursor via diazotation reaction. (B) Electrodeposition of DTD
cation on the ITO electrode surface, followed by electrochemical
conversion of the 1,3-dithiane part on the electrodeposited ITO
surface.
b Electronics and Telecommunications Research Institute, Daejeon,
305-700, Korea
w Electronic supplementary information (ESI) available: Detailed
procedures for synthesis an experiments. XPS data for sulfur. See
DOI: 10.1039/b909244g
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Fig. 2 Fluorescence microscopic image results from a sandwich-type
multianalyte immunoassay for the serial detection of (a) mouse IgG
and (b) rabbit IgG. Scale bar is 150 mm.
EAMs are greatly efficient for the spatially selective immobili-
zation of proteins on the closely spaced microelectrode array.
The immobilization result was applied for a multianalyte
sandwich-type immunoassay based on fluorescence observa-
tion. Two electrodes among four electrodes of the array were
selectively functionalized with two different kinds of anti-
bodies as probes, respectively. The protocol to prepare the
platform for the immunoassay is summarized as below.
Following the simultaneous electrodeposition of DTD on
the entire electrode, the upper electrode only was addressed
for the electrochemical deprotection. The resulting array
surface was exposed to a drop of anti-mouse IgG solution of
PBST for 2 h. After washing the exposed surface, the same
procedure was repeated for the lower electrode and anti-rabbit
IgG. For the immunoassay, the platform of the anti-
body-immobilized array was incubated with a drop of
2 mg mLÀ1 mouse IgG in PBST solution for 1 h. After
washing, the platform surface was exposed to a drop of a
solution mixture of anti-mouse IgG labeled with FITC and
anti-rabbit IgG labeled with TRITC in PBST for 3 h.
Fig. 1 (A) CV for the electrodeposition of the DTD cation (1.5 mM
in 0.1 M HCl) on an ITO microelectrode. Scan rate is 50 mV sÀ1
.
(B) CV for the electrochemical deprotection of the DTD modified
surface. CH3CN : H2O (95 : 5) solution with 0.1 M Bu4NBF4 was used
in the CV. Scan rate is 50 mV sÀ1. C1s XPS results for DTD-modified
surfaces (C) before and (D) after electrochemical deprotection.
(E) Fluorescence microscopic image obtained from the spatially
selective immobilization of antibody. The broken lines indicate the
position of ITO electrodes. Scale bar is 150 mm.
of the peak current with an approximation of the 2-electron
process was found to be 1.0 Â 10À9 mol cmÀ2. Considering the
reaction is not quantitative, the value is greater than the
expected surface density for the monolayer film formed by
electrodeposition of common aryldiazonium cations, implying
partial formation of multilayers due to polymerization of
DTD on the surface.12
XPS for carbon (C1s) atoms was used to examine aldehyde
formation after the electrochemical deprotection of
DTD-modified surfaces. While before the deprotection the
spectra exhibited a peak at 285.3 eV due to the carbon atoms
of DTD (Fig. 1C), after the deprotection a new peak was
detected at 288.0 eV (Fig. 1D) corresponding to the carbonyl
group that might be attributed to the aldehyde.10 Direct
antibody immobilization on the deprotected surfaces was
conducted to verify the aldehyde functionality, reacting with
a primary amine on the protein, producing an imine.10 As an
antibody, anti-rabbit IgG labeled with tetramethylrhodamine
isothiocyanate (TRITC) was employed. We prepared and used
an ITO microelectrode array for this work as previously
reported.13 Following simultaneous electrodeposition of
DTD on all of the electrodes, the resulting surface was
subjected to the electrochemical deprotection of only one
electrode. After that, the whole surface was exposed to
phosphate-buffered saline with a 0.05% Tween (PBST)
solution of the antibody during 1 h. Fig. 1E displays a
fluorescence microscopic image obtained from the exposure.
Extremely contrasted red color is seen exclusively in the
oxidized surfaces. XPS and immobilization results clearly
support that the electrochemical deprotection produces
aldehydes that can directly react with amine containing
proteins. Moreover, Fig. 1E demonstrates that our combined
Fig. 2A shows a fluorescence image obtained from the
immunoassay, where the upper electrode only gives bright
fluorescence due to FITC, indicating that mouse IgG could be
detected with a high specificity and a negligible nonspecific
binding of proteins using our method. To examine multi-
analyte detection, the platform used in mouse IgG detection
was reused for the detection of another antigen, rabbit-IgG
(2 mg mLÀ1). The second immunoassay led also to a highly
contrasted fluorescence image due to TRITC being observed
for the lower electrode (Fig. 2B). Two differently colored
detection images with a high signal to noise ratio imply that
not only cross reaction of two antigens is negligible but also
multianalyte detection is successfully achieved.
In conclusion, we have demonstrated for the first time use of
the combination of 1,3-dithiane with an aryldiazonium cation
for immobilization of proteins. Our work using DTD cations
might present general and promising tools for the covalent
immobilization of biomolecules including DNA, peptides as
well as proteins,14 because it could offer advantages over the
conventional self-assembly process of electroactive thiols or
silanes: (i) saving time to produce an electroactive film due to
rapid electrodeposition of aryldiazonium; (ii) providing the
flexibility of electrode materials for on-demand formation of
aldehyde; (iii) allowing multiple electro-addressing due to the
dually electroactive functionality of the DTD cation.
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This work was supported by a Korea Research Foundation
Grant funded by the Korean Government (KRF-2008-331-
C00186).
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This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 4865–4867 | 4867