Diazonium-protein adducts for graphite electrode microarrays modification: Direct and addressed electrochemical immobilization

Article abstract of DOI:10.1021/ja056946w

Diazonium cation electrodeposition was investigated for the direct and electro-addressed immobilization of proteins. For the first time, this reaction was triggered directly onto diazonium-modified proteins. Screen-printed (SP) graphite electrode microarr

Full text of DOI:10.1021/ja056946w

Published on Web 12/02/2005
Diazonium-Protein Adducts for Graphite Electrode
Microarrays Modification: Direct and Addressed
Electrochemical Immobilization
Benjamin P. Corgier, Christophe A. Marquette,* and Lo¨ıc J. Blum
Contribution from the Laboratoire de Ge´nie Enzymatique et Biomole´culaire, UMR 5013 EMB2,
CNRS UniVersite´ Claude Bernard Lyon 1, Baˆt CPE, 43, bouleVard du 11 NoVembre 1918,
69622 Villeurbanne, Cedex, France
Received October 11, 2005; E-mail: christophe.marquette@univ-lyon1.fr
Abstract: Diazonium cation electrodeposition was investigated for the direct and electro-addressed
immobilization of proteins. For the first time, this reaction was triggered directly onto diazonium-modified
proteins. Screen-printed (SP) graphite electrode microarrays were studied as active support for this
immobilization. A 10-microelectrode (eight working electrodes, 0.2 mm2 each; one reference; and one
auxiliary) setup was used to study the addressing possibilities of the method. These electrode microarrays
were shown to be able to covalently graft diazonium cations through electrochemical reduction. Cyclic
voltammetry and X-ray photoelectron spectroscopy were used to characterize the electrochemical grafting
onto our SP graphite surface and suggested that a diazonium monolayer was deposited. Rabbit and human
immunoglobulins (IgGs) were then chemically coupled to an aniline derivative (4-carboxymethylaniline),
followed by diazotation to form an aryl diazonium function available for the electrodeposition. These modified
proteins were both successfully electro-addressed at the surface of the graphite electrodes without cross-
talk or interference. The immuno-biochip obtained using this novel approach enabled the specific detection
of anti-rabbit IgG antibodies with a detection limit of 50 fmol of protein. A promising strategy to immobilize
markedly different biological entities was then presented, providing an excellent spatial specificity of the
electro-addressing.
Introduction
always require a physical addressing of the molecules of interest
following the surface modification, i.e., spotting of nanodrops
Chemical modification of surfaces has proven to be one of
the best strategies to ensure the covalent immobilization of a
large scale of either organic or biological molecules. Indeed,
following the extended use of molecule adsorption onto activated
polystyrene surfaces (96-well plate material), the coming of
microarray developments has generated a dramatic increase in
surface chemistry research programs. Thus, silane on glass1-5
or silicon6,7 and thiol on gold8-11 were the first to be brought
to the forefront because of their well-known chemistry. How-
ever, these techniques, when used for microarray developments,
or photolithography technologies.
Other solutions to obtain the precise localization of im-
mobilized molecules on a surface include the use of active
materials such as electrode arrays acting as catalyst of the
immobilization reaction. This is mainly obtained through the
electropolymerization of insoluble polymers (polypyrrole,12-14
polyaniline15,16 and derivatives), entrapping or supporting the
molecule of interest.
A step further could be achieved by the use of aryl diazonium
cations to perform a direct electrochemical grafting at the surface
of a conducting electrode17-19 (Scheme 1). Diazonium-based
procedures offer the advantages of simplicity, efficiency, and
speed of the chemistry involved: (i) the diazotation reaction of
an aniline derivative leads easily to the formation of an aryl
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Diazonium−Protein Adducts for Electrode Microarrays
A R T I C L E S
Scheme 1. (i) Diazotation of Aniline Derivative, (ii)
Electroreduction of Diazonium Cation, and (iii) Covalent Linkage to
the Electrode Surface
printing (SP) technique. This technology is presented as a
promising tool for achieving marketable biosensors, taking
advantage of low-cost and mass-production possibilities.44,45
Herein, a biological model is presented based on rabbit
immunoglobulin (IgG) first functionalized with 4-carboxym-
ethylaniline (CMA) and then diazotated and electro-addressed
on target electrodes of the microarray (Figure 1). X-ray
photoelectron spectroscopy (XPS) and cyclic voltammetry were
used to characterize the diazonium reduction and grafting on
this electrode material.
Validation of the quality of the addressed immobilization was
performed through the recognition of the immobilized proteins
by peroxidase-labeled anti-IgG antibodies and the measurement,
with a CCD camera imaging system, of the chemiluminescent
signals emitted.
diazonium, (ii) the electrochemical reduction of this latter species
generates an aryl radical, which attacks the surface and forms
an X-C bond (where X is the electrode material, namely, Au,
C, Cu, Si).
This technique of derivatization has already been demon-
strated on a wide range of conducting materials such as
carbon,19-28 carbon nanotubes,29 silicon,30-33 metals,34-38 and
diamond.39 In most cases, the electrochemical addressing was
performed in organic solvent (acetonitrile), and the modified
electrode was subsequently grafted with an interesting molecule
(in some cases a protein).
An innovative approach is presented herein based on the
modification of proteins with aniline derivatives (NH2-Ar-
R) and the subsequent diazotation and electro-addressing of the
modified proteins at a graphite electrode surface (Figure 1). This
approach benefits the possibility of diazonium reduction in acidic
aqueous medium28,40-42 and enables, to our knowledge, for the
first time, a true addressed electrochemical covalent grafting
of proteins at an electrode surface. The conducting material used
is a 10-electrode microarray, developed and characterized
previously by our group14,43 and obtained through a screen-
Experimental Section
Reagents. 4-Aminophenylacetic acid (4-carboxymethylaniline; CMA),
4-bromobenzenediazonium tetrafluoroborate (BrDz), bovine serum
albumin (BSA), immunoglobulin from human serum (human IgG),
luminol (3-aminophthalhydrazide), N-hydroxysuccinimide (NHS), N,N′-
dicyclohexylcarbodiimide (DCC), and peroxidase-labeled polyclonal
anti-rabbit IgG antibodies developed in goat and polyoxyethylenesor-
bitan monolaureate (tween 20) were purchased from Sigma (Lyon,
France). Sodium nitrite and Veronal (diethylmalonylurea sodium) were
purchased from Prolabo (Fontenay Sous Bois, France). Immunoglo-
bulins from rabbit serum (rabbit IgG) were obtained from Life Line
Lab (Pomezia, Italy). Peroxidase-labeled polyclonal anti-human IgG
(H + L) antibodies developed in goat were purchased from Jackson
ImmunoResearch (West Grove, PA). All buffers and aqueous solutions
were made with distilled demineralized (dd) water.
Electrode Microarray Preparation. A DEK 248 screen-printing
machine (DEK, Lyon, France) was used to produce the graphite
electrode microarrays. A polyester monofilament fiber screen (DEK,
Dorset, U.K.) characterized by a mesh size of 260 counts per inch and
a thickness of 13 µm was used to print the graphite ink (Electrodag
423 SS, Acheson, Erstein, France) onto a polyester flexible foil. After
being printed, the polyester foils supporting 16 electrode arrays were
baked for 10 min at 100 °C to cure the thermoplastic carbon ink.
A second layer, composed of insulating polymer (MINICO M 7000,
Acheson, Erstein, France) was then printed onto the microarrays to
define a window (easily covered with a 35-µL drop of solution)
delimiting the active area composed of eight 0.2-mm2 working
electrodes, one ring-shaped reference electrode, and one central auxiliary
electrode (Figure SI 1, Supporting Information).
X-ray Photoelectron Spectroscopy (XPS). XPS spectra were
realized on a 1-mm analysis window with an ESCALAB instrument
(VG Scientific) using an Al anode (KR X-rays at 1486.6 eV) and an
analysis energy of 50 eV. Larger screen-printed electrodes (25 mm2),
prepared as described above, were used to achieve convenient XPS
experiments. The atom percentages were determined from the peak
areas, the sensitivity factors given by the VG software (inelastic mean
free path and transmission function), and the cross section for X-ray
excitation as calculated by Scofield.46
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Protein Modifications. A two-step reaction protocol was elaborated
to modify the proteins. First, 10 mg of 4-carboxymethylaniline (CMA)
was activated in the presence of 20.6 mg of DCC and 11.5 mg of NHS
in 1 mL of DMSO for 30 min under stirring. Then, 20 µL of the
activated CMA was added to 500 µL of a 5 mg/mL protein solution in
0.1 M carbonate buffer, pH 11. The protocols were identical for both
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6865.
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A R T I C L E S
Corgier et al.
Figure 1. Strategy for direct electro-addressing of modified antibody onto SP graphite electrode surface. CMA, 4-carboxymethylaniline; DDC, N,N′-
dicyclohexylcarbodiimide.
rabbit and human IgG modification. This coupling solution was left
for 2 h under stirring at room temperature before being purified on a
P-10 Sephadex G-25M 5 mL column (Supelco, Bellefonte, PA). The
desalted modified protein obtained was then concentrated to 10 mg/
mL under centrifugation using MicroconYM-3 (Millipore, Billerica,
MA). The obtained retentate was recovered in 100 µL of dd water and
stored at +4 °C.
Diazotation of 4-Carboxymethylaniline. The aniline derivative
CMA, either free or coupled to protein, was diazotated in an aqueous
solution of 20 mM HCl and 20 mM NaNO2 for 10 min under stirring
in ice-cold water. The diazonium solution formed was then immediately
used to perform electro-addressing.
Electrodeposition of Diazonium Derivatives. Electro-addressing
was achieved by directly depositing on the SP microarray surface a
35-µL drop of either 5 mM free diazonium solution or 0.1 mg/mL
Figure 2. Cyclic voltammograms of 4-bromobenzenediazonium (5 mM)
modified proteins. Therefore, three cyclic voltammograms were
in 0.1 M HCl and 0.1 M KCl. Potential scanned from 0 to -1000 mV vs
obtained from 0 to -1000 mV, on the chosen electrodes of the
microarray at a scan rate of 200 mV/s. The cyclic voltammograms were
obtained with a Voltalab PGZ 100 potentiostat (Radiometer Analytical).
A specially designed 10-path connector was used to control the potential
application on the electrodes of the SP microarray. An external platinum
counter electrode and a ring-shaped carbon reference electrode of the
screen-printed chip were used to achieve a three-electrode setup. After
deposition, the carbon electrodes were fully rinsed with distilled water
and sonicated to remove unbound molecules.
Protein Detection Procedure. Following electro-addressing, soni-
cation, and washing, the SP microarrays were saturated for 20 min at
37 °C with VBSTA (30 mM Veronal, 0.2 M NaCl, pH 8.5, with
addition of 1% Tween and 1 g/L BSA). Peroxidase-labeled anti-rabbit
or anti-human IgG antibodies at various concentrations in VBSTA were
incubated for 1 h at 37 °C on the microarrays and then washed with
VBS (30 mM Veronal, 0.2 M NaCl, pH 8.5) for 20 min. Finally, the
SP microarrays were covered with a 35-µL drop of VBS containing in
addition 200 µM luminol, 20 µM p-iodophenol, and 500 µM hydrogen
peroxide, and introduced into a charge-coupled device (CCD) cooled
camera light measurement system (Las-1000 Plus, Intelligent Dark Box
II, Fujifilm, Tokyo, Japan). The emitted light was then collected and
integrated for 10 min. The numeric micrographs obtained were
quantified with a Fujifilm image analysis program (Image Gauge 3.122).
graphite pseudoreference electrode. Three cycles performed at a scan rate
of 200 mV/s.
4-Bromobenzenediazonium (BrDz) was used to study this
electroreduction process at our SP graphite electrode surface.
This derivative was chosen for its bromide residue, which is
easily evidenced through XPS measurements. Optimized depo-
sition of BrDz was shown to be obtained using three potential
scans from 0 to -1000 mV in aqueous solutions of 0.1 M HCl
and 0.1 M KCl.
The three consecutive cyclic voltammograms obtained are
presented in Figure 2. They are characterized by a first cycle
exhibiting a well-defined, reproducible, and irreversible reduc-
tion peak at -750 mV. This feature corresponds to the typical
electroreduction wave of the diazonium function, leading to the
elimination of a nitrogen molecule and the production of an
aryl radical (Scheme 1, steps i and ii). This radical was
previously shown to attack the surface and to form a covalent
bond between the aryl group and the electrode material (Scheme
1, step iii).39,42,47
More information could be obtained from Figure 2 about the
deposited layer. Very low currents were observed during the
second and third voltammetric cycles, evidencing surface
saturation and suggesting that a monolayer of fixed molecules
was achieved. Indeed, very fast deposition processes (30 s), as
used herein, usually lead to the grafting of only one molecular
layer.48,49
Results and Discussion
Screen-printed (SP) microarrays, previously described and
characterized,14,43 were used as robust and single-use supports
to study the possibilities of electrochemically addressing dia-
zonium cations and diazonium-cation-modified proteins. First,
the reduction and deposition of aryl diazonium cations at the
surface of our SP graphite microarrays was investigated to
validate the possibility of electro-addressing these molecules.
(47) Vakurov, A.; Simpson, C. E.; Daly, C. L.; Gibson, T. D.; Millner, P. A.
Biosens. Bioelectron. 2004, 20 (6), 1118-1125.
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3837.
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Diazonium−Protein Adducts for Electrode Microarrays
A R T I C L E S
Figure 3. X-ray photoelectron spectra of (a) electro-addressed BrDz, (b)
control experiment without BrDz electroreduction, and (c) bare SP graphite
electrode.
Figure 4. Dose response curve of anti-rabbit IgG antibodies. Error bars
are the standard deviation of four microarrays. Inset: chemiluminescent
micrograph of a SP graphite microarray incubated with peroxidase-labeled
anti-rabbit IgG antibodies (2 µg/mL). Electrodes 2 and 7 were electro-
addressed with CMA-rabbit IgG.
XPS experiments were performed on our SP graphite surface
to demonstrate the electro-addressed deposition of the bromo
aryl. Three SP graphite electrodesseither unmodified, modified
with adsorbed BrDz, or modified with electrodeposited BrDzs
were scanned at 50 eV, particularly focusing the analysis on
the binding energies corresponding to the Br 3d and the N 1s
electrons.
The electrodeposited electrode (Figure 3, curve a) exhibited
a peak at 1412 eV, indicating the presence of bromide atoms at
the surface with a relative composition of 2.6%. Conversely,
no signal from the presence of Br could be found either on the
electrode modified by dipping in a BrDz solution (Figure 3,
curve b) or on the bare unmodified electrode (Figure 3,
curve c).
Exclusively bromo aryl seems then to be present at the surface
of the graphite electrode, following the electroreduction of BrDz.
Moreover, no detectable signal was obtained for the N 1s
decomposition at the electrodeposited electrode (data not
shown), confirming the elimination of nitrogen and the agree-
ment of the present reaction with the commonly accepted
mechanism presented in Scheme 1.
rabbit IgG was elaborated to produce proteins ready to be
addressed (CMA-rabbit IgG). The rabbit IgG primary sequence
suggests that up to 80 primary amines (essentially lysine) are
available for grafting. Attempting as much as possible to keep
the protein in its native conformation after immobilization, CMA
was reacted with IgG molecules using stoichiometric reagent
conditions (moles of CMA vs moles of primary amine).
The CMA-rabbit IgG conjugates obtained were diazotated
according to the procedure optimized above with free CMA
and electrodeposited onto the SP graphite microarray. Neverthe-
less, the low protein concentration used (0.1 mg/mL) did not
enable the recording of any diazonium reduction peak as the
corresponding maximum concentration of coupled diazonium
was found to be 100 times lower than the CMA concentration
used above (<50 µM).
An indirect demonstration was then performed to detect the
electrodeposited rabbit IgG. Peroxidase-labeled anti-rabbit IgG
antibodies were used to specifically detect the immobilized
proteins through chemiluminescent imaging of the SP microar-
ray surface. As the inset of Figure 4 shows, a clear distinction
between the addressed electrodes (nos. 2 and 7) and the six
unaddressed electrodes was obtained. Moreover, no nonspecific
adsorption of the proteins onto either the graphite material or
the SP support (PVC or insulating layer) was observed,
demonstrating extremely interesting potentialities for analytical
applications.
The use of this electrochemical deposition to address proteins
at the surface of our SP microarrays requires, first, the use of an
aniline derivative that can be easily linked to the proteins and,
second, the achievement of its diazotation under gentle condi-
tions. 4-Carboxymethylaniline (CMA) was found to be such a
potential candidate as it contains a carboxylic acid function that
is easily activated and grafted through carbodiimide reaction.
Diazotation reaction conditions of the CMA aniline residue,
adapted from ref 50, were optimized to fit with the pH and salt
concentration requirements for biocompatibility. The cyclic
voltammograms obtained during the electrodeposition of freshly
diazotated CMA were found to be characteristic of an aryl
diazonium grafting (Figure SI 2, Supporting Information), with
behaviors similar to those obtained during BrDz deposition onto
the SP graphite electrodes. Thus, a clear reduction wave was
observed in the presence of diazotated CMA, whereas no current
at all could be obtained from the nondiazotated aniline deriva-
tive.
This possibility of using the addressed electrodeposited
protein as an analytical tool was studied by applying to the SP
microarrays a range of peroxidase-labeled anti-rabbit IgG
antibody concentrations from 0.25 to 10 µg/mL and determining
a dose-response curve (Figure 4). A classical ligand/receptor
binding curve was obtained with a pseudolinear relation for
antibody concentrations ranging from 0.25 to 2 µg/mL, followed
by a plateau at higher concentrations. Assuming that the
immobilization of the proteins occurred as a monolayer, a
limited number of macromolecules are accessible for recogni-
tion, leading to saturation at high antibody concentrations. These
performances compared well with those obtained using SP
The diazotation and electrodeposition of CMA was then
shown to be easily obtained, and subsequently, its coupling to
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A R T I C L E S
Corgier et al.
To validate the possibility of producing eight electro-
addressed sensing layers on the different electrodes of a
microarray, eight different solutions of diazotated CMA-rabbit
IgG were sequentially electrodeposited. Eight sensing layers
composed of electro-addressed rabbit IgG were then obtained
on a single SP microarray, with good reproducibility (data not
shown), and characterized by a consistent chemiluminescent
signal emitted from each electrode.
Conclusion and Perspectives
Screen-printed (SP) graphite electrode microarrays demon-
strated their ability to be modified with electro-addressed
diazonium cations. Thus, an innovative approach was elaborated
to electro-address proteins. Biomolecules were first modified
with the aniline derivative 4-carboxymethylaniline (CMA) and
then successfully electrodepositedsi.e., without parasitic reduc-
tion of the protein partsat the graphite surface through an
optimized protocol compatible with biomolecules such as rabbit
and human antibodies (IgG).
For the first time ever, such direct electro-addressing of
multiple proteins was then described. The immuno-biochip
obtained was shown to exhibit interesting detection properties
and very low nonspecific binding.
Figure 5. (a) Illustration of the SP carbon electrode microarray supporting
two distinct sensing layers covalently immobilized by electrochemical
addressing. Chemiluminescent micrograph of an SP microarray modified
with both rabbit and human CMA-IgG and incubated with peroxidase-
labeled (b) anti-rabbit IgG antibodies (2 µg/mL), (c) anti-human antibodies
(0.1 µg/mL), and (d) both anti-rabbit IgG antibodies (2 µg/mL) and anti-
human antibodies (0.1 µg/mL). Electrode 2 was electro-addressed first with
CMA-rabbit IgG, and then electrode 7 was electro-addressed with CMA-
human IgG.
The present system appeared to offer a promising strategy
for strongly immobilizing different biological entitiessproteins,
nucleic acid strands, and peptidesswith excellent spatial ad-
dressing of the interesting molecules onto electrode microarrays.
Indeed, the CMA coupling procedure is compatible with the
majority of the biomolecules that could be kept in their native
configuration in rather acidic solutions. This is the case, for
example, for DNA sequences, carbohydrate structures, peptides,
and numerous proteins. Moreover, less drastic conditions could
be examined for saving protein activity such as enzyme catalytic
activity.
support and protein trapped in an electropolymer14 or using a
standard support such as coated glass slides.51
To be fully useful for analytical use, the present addressing
system needs to be applied to the sequential immobilization of
different proteins.
Two CMA proteins, rabbit and human IgGs, were then
synthesized, diazotated, and sequentially electro-addressed to
different electrodes of the SP microarray, as presented in Figure
5a.
Despite the fact that, in the addressing protocol, rabbit IgG
was deposited first and was then in contact with the diazotated
solution of CMA-human IgG, the reactivity of the immobilized
rabbit IgG was preserved, and a fully acceptable chemilumi-
nescent signal was obtained (Figure 5b, when compared to the
dose-response curve presented above). As shown in Figure 5c,
peroxidase-labeled anti-human IgG antibodies also react properly
with the immobilized human IgG without cross-reactivity with
the immobilized rabbit IgG. Moreover, the two sensing layers
could be used concomitantly to detect anti-rabbit and anti-human
IgG (Figure 5d).
Thus, the consecutive electro-addressing of two different
proteins did not alter the specificity and the sensitivity of the
detection, and no cross-talk between the two protein layers was
observed, indicating that only the specificity of the antigen/
antibody interaction was involved in the detection process.
Interesting possibilities should also be obtained by concomi-
tantly using the present modification system with poly(lysine)-
tagged proteins. Thus, grafting CMA preferentially onto the
polyamine tag52 will lead to the electro-addressing of oriented
proteins at the surface, enhancing the protein immobilization
quality and thus the immuno-biochip performances.
Acknowledgment. The authors acknowledge P. Delichere
from the Institut de Recherches sur la Catayse (Lyon, France)
for his useful help in the recording and interpretation of XPS
spectra.
Supporting Information Available: Diagram of the SP
graphite electrode microarray and electrochemical (cyclic vol-
tammetry) data for the electrodeposition of 4-carboxymethyla-
niline (CMA). This material is available free of charge via the
Internet at http://pubs.acs.org.
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18332 J. AM. CHEM. SOC. VOL. 127, NO. 51, 2005