Rapid construction of an effective antifouling layer on a Au surface via electrodeposition

Article abstract of DOI:10.1039/c4cc01329h

A new approach to immobilize zwitterionic molecules rapidly and highly efficiently on a gold surface applies aniline-based electrodeposition. The zwitterion-functionalized antifouling surface enables a decrease of the adsorption of non-specific proteins by 95% from fetal bovine serum (FBS, 10%). This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc01329h

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Rapid construction of an effective antifouling
layer on a Au surface via electrodeposition†
Bor-Ran Li,‡a Mo-Yuan Shen,‡a Hsiao-hua Yub and Yaw-Kuen Li*a
Cite this: Chem. Commun., 2014,
50, 6793
Received 20th February 2014,
Accepted 6th May 2014
DOI: 10.1039/c4cc01329h
www.rsc.org/chemcomm
A new approach to immobilize zwitterionic molecules rapidly and
Goda et al.16 demonstrated that thiol-functionalized zwitter-
highly efficiently on a gold surface applies aniline-based electro- ions were chemisorbed to form a self-assembled monolayer
deposition. The zwitterion-functionalized antifouling surface enables (SAM) on the Au surface of a QCM chip via a Au–S bond.
a decrease of the adsorption of non-specific proteins by 95% from Via ionic solvation, the zwitterionic layer can strongly trap water
fetal bovine serum (FBS, 10%).
molecules on the surface of the chip. The expulsion of water
molecules from between the protein and the zwitterion surface
Biosensors have drawn much attention because of their becomes difficult leading to the reduction of non-specific
potential to greatly improve biomedical research,1–4 drug dis- protein adsorption on the chip. The presence of the inert
covery,5,6 environmental monitoring,7 and diagnosis of many zwitterionic layer decreased the non-specific binding up to
diseases.8–10 In particular, a biosensor applied to the clinical 60–70% from the dilute serum (10% fetal bovine serum, FBS),
diagnosis of epidemic diseases,11 long-term nursing of chronic indicating that modifying a QCM surface with zwitterions is an
diseases,12 and early diagnosis of cancers13 could save many ideal strategy to create antifouling platforms for sensitive and
lives around the world. To improve various properties of specific detection involving a clinical sample.17,18 Although a
biosensors with the latest technology is hence of continual thiol-SAM is readily prepared on the Au surface of a QCM chip
interest to engineers and scientists. The quartz crystal micro- on immersing the substrate in a dilute solution of zwitterionic
balance (QCM),14 an ultra-sensitive balance capable of measur- thiols, the thiol-SAM process requires 12 to 72 h at room
ing changes in mass at the molecular level, is one of the most temperature.19,20 A Au surface of high quality and pure reagents
attractive biosensors that has been commercialized. The struc- are essential to avoid defects in the monolayer formation.21
ture of a QCM chip is robust; the measurement is processed in Unlike the smooth Au surface on SPR, a bumpy sub-micrometre
real time, free of labels, and almost independent of the ionic structure of a Au surface on a QCM chip is commonly observed
strength. A QCM is thus supposed to be an ideal system for because of its advantage of enlarging the binding area to
biological sensing, and has been established to detect various enhance sensitivity.22 Defects (e.g. monatomic vacancy islands)
biomolecules via the specific recognition of target analytes on the rough Au surface are easily formed upon modification of
with the corresponding receptors (i.e. antibodies) immobilized a zwitterionic layer using thiol-SAM approaches. Intermolecular
on the Au surface of the device.15 The nonspecific binding electrostatic repulsion between zwitterionic molecules might
between the proteins in a patient’s serum and the Au surface also cause the formation of a non-uniform SAM layer that
of a QCM is a critical problem that might greatly decrease consequently produces a poor zwitterion density and results
the sensitivity and reliability of the detection. To decrease the in a limited antifouling effect. The development of a new approach
nonspecific interaction between the components of plasma and to decrease the duration of reaction and to achieve a uniform
the Au surface is thus an important issue for sensitive and antifouling layer on the Au surface is hence highly desirable.
specific sensing of biological matrices.
Here, we demonstrate a new approach to directly modify the
zwitterionic molecule, dimethyl-ammonio propane-1-sulfonate
(sulfobetaine or SB),23,24 on the Au surface of the QCM chip
via electrodeposition. In the first step, sulfobetaine (SB) is conju-
gated with an aniline functional group to from 3-[(4-aminophenyl)-
dimethylammonio]propane-1-sulfonate (referred to as aminophenyl
sulfobetaine or AP-SB in this report). Upon optimizing the condi-
tions, AP-SB can be efficaciously synthesized in a yield 470%.
a Department of Applied Chemistry, National Chiao Tung University, Hsinchu,
Taiwan. E-mail: ykl@cc.nctu.edu.tw
b Institute of Chemistry, Academia Sinica, Taipei, Taiwan
† Electronic supplementary information (ESI) available: Fig. S1–S6 and Section
S1–S7. See DOI: 10.1039/c4cc01329h
‡ Bor-Ran Li and Mo-Yuan Shen made equal contributions to this work.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 6793--6796 | 6793

View Article Online
Communication
ChemComm
PBS (phosphate buffered saline, containing 137 mM NaCl,
2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4, pH 7.2) at
a scan rate of 100 mV sꢀ1. When the Au electrodes were coated
with an octyl moiety (as insulated layer) by either thiol-SAM
(Fig. 1A) or aniline-based electrodeposition (Fig. 1B) for varied
durations, the exposed areas of Au electrodes were decreased,
consequently resulting in smaller redox peaks. Whereas the
electrode modified with aniline-based electrodeposition for 1 to
3 min greatly decreased the Ip peaks (blue and cyan lines in
Fig. 1B), the electrode modified with a thiol-SAM approach for
1 h, 3 h or even 3 days induced only smaller effects on the Ip
peak changes (red, orange and violet lines in Fig. 1A). Similar
results were obtained when RuHex, [Ru(NH3)6]3+, was used as
the redox indicator (Fig. S4, ESI†).
A relationship between the electrode area and Ip (Ae) is
described using the Randles–Sevcik eqn (1):
Scheme 1 Schematic illustration of the synthesis and deposition of anti-
fouling zwitterions on the gold electrode.
rffiffiffiffiffiffiffiffiffiffiffi
nFvD
Ip ¼ 0:4463 nFAeC
/ Ae
(1)
RT
Unlike thiol-sulfobetaine, (HS-SB) can automatically chemisorb to
form a SAM on the QCM chip through chemical binding between
the mercapto group and Au; the AP-SB modification operates
through the in situ generation of the aminophenyl diazonium cation
in aqueous HCl (0.5 M) and NaNO2 at an equivalent concentration,
and subsequently coating onto the Au surface by electrochemical
reduction via cyclic voltammetry (Scheme 1),25,26 which can be
completed in a few minutes. The details of thiol self-assembly,
electrodeposition, and organic synthesis are provided in Fig. S1
and S2, and Section S2, S3, S7 and S8 of the ESI.† The surface
modification is confirmed by ESCA (Fig. S3, ESI†).
in which Ip = measured peak current/A; n = number of electrons
transferred in the redox event, F = faraday constant/C molꢀ1
,
Ae = exposed area of the Au electrode/cm2, C = concentration of
ferricyanide/mol cmꢀ3, n = scan rate/V sꢀ1, D = diffusion
coefficient/cm2 ꢀ1, R = gas constant, and T = temperature/
s
deg F. In addition, the relative density of surface modification
(RD) is defined as Ae.mod/Ae.t (eqn (2)). Therefore,
Ae:mod Ae:t ꢀ Ae:unmod Ip:max ꢀ Ip
DIp
Ip:max
RD
¼
¼
¼
¼
(2)
Ae:t
Ae:t
Ip:max
Cyclic voltammetry is utilized to evaluate the efficiency of
thiol-SAM and the aniline electroplating modification. As the
results in Fig. 1A and B show, the bare gold electrode (black
line) reveals higher oxidation and reduction current peaks (Ip)
in a solution of 10 mM [Fe(CN)6]3ꢀ as the indicator dissolved in
where Ae.mod is the area of the Au electrode modified, and Ae.t is
the total surface area of the Au electrode. Ip.max is the Ip of the
current peak of CV scanning by a bare Au electrode. RD is
utilized to quantify the immobilization. Fig. 1C shows the RD of
the electrodes modified with 1-octanethiol and 4-octylaniline
for varied durations. The histogram reveals that 99 ꢁ 1%
surface area of the Au electrode is efficiently modified by the
aniline-based electrodeposition in 3 min, but the electrode
modified with thiol-SAM for 3 days still leaves 30 ꢁ 5%
unmodified area. These results indicate that aniline-based
electrodeposition is a rapid and highly efficient method for
chemical modification of a bumpy Au surface.
As shown in the illustrations in the upper panel of Fig. 2A, a
higher SB density on the QCM chip might result in effective
antifouling ability. To confirm the antifouling properties of a
Au surface modified with SB by thiol-SAM and aniline-based
electrodeposition, we employed the QCM chips modified by
these two methods to compare the non-specific adsorption of
dilute serum in real-time monitoring. Whereas fetal bovine
serum (10% FBS, in PBS, pH 7.2) caused a prominent decrease
of the resonance frequency (130 ꢁ 10 Hz) on the bare QCM
chip, the same sample induced a smaller frequency change
(45 ꢁ 10 Hz) on the HS-SB modified chip and a minor frequency
change (6 ꢁ 3 Hz) on the AP-SB electrodeposited QCM chip. The
frequency change can serve to estimate the accumulated
change in mass based on the Sauerbrey eqn (3),27 in which
f0 = resonant frequency/Hz, Df = frequency change/Hz, Dm = mass
Fig. 1 (A) Cyclic voltammogram of Au electrodes bare (black) and mod-
ified with 1-octanethiol for 1 h (red), 3 h (orange), and 3 days (violet) in
a solution of ferricyanide (10 mM, dissolved in 1 ꢂ PBS) at a scan rate of
100 mV sꢀ1. (B) CV curves measured with Au electrodes bare (black) and
electrodeposited with 4-octylaniline in 1 (blue) and 3 min (cyan). (C) The
histogram shows a comparison of the relative density of surface modifica-
tion (RD) of Au electrodes modified with 1-octanethiol (red) for 0 h, 1 h, 3 h
and 3 days and 4-octylaniline (blue) for 0 min, 1 min, and 3 min. The error
bars represent the standard deviations of three measurements.
6794 | Chem. Commun., 2014, 50, 6793--6796
This journal is ©The Royal Society of Chemistry 2014

View Article Online
ChemComm
Communication
Fig. 2 (A) Real-time recording of the D frequency responses to FBS (10%)
with AP-SB-electrodeposited (blue), HS-SB SAM-coated (red), and unmodi-
fied (black) QCM devices in PBS buffer (pH 7.2). (B) Comparison of responses
of bare (black) and electroplated SB QCM devices to FBS (10%) at pH 6.0, 6.5,
7.0, 7.5 and 8.0. Green arrows indicate the point of FBS injection.
Fig. 3 Images of adhesion of the mCherry fluorescent protein on a
micro-patterned gold electrode on a photoresist-(LOR-5B)-preserved
silicon substrate. (A) Fluorescent (i) and bright-field (ii) images of a bare
Au electrode without mCherry treatment. (B) Images of a bare Au elec-
trode incubated with the mCherry fluorescent protein (10 mg mLꢀ1) for 1 h.
(C) Images of the AP-SB-modified Au electrode incubated with the
mCherry fluorescent protein (10 mg mLꢀ1) for 1 h.
change/g, A = area of electrodes/cm2, rq = density of quartz and
mq = shear modulus of quartz.
2
2f0
pffiffiffiffiffiffiffiffiffiffi
Df ¼ ꢀ
Dm
(3)
A
rqmq
shown in these bright-field images resulted from the reflection of
incident light.
As the results in Fig. 2A show, QCM with the zwitterionic layer
immobilized by electrodeposition is demonstrated to adsorp non-
specific bound proteins (0.04 mg cmꢀ2) (blue line) to a lesser extent
than a bare (1.6 mg cmꢀ2) (black line) surface or the one modified
by the thiol-SAM approach (0.6 mg cmꢀ2) (red line). As the pKa
of sulfonic acid is relatively low, the SB is expected to maintain
a zwitterionic form in a wide range of pH. For this reason, a
SB-modified QCM chip exhibits pH-independent antifouling abil-
ity, particularly in the pH range 6–8, the common conditions
of bio-analysis or diagnosis (Fig. 2B). Since low ionic strength
In summary, we present a synthesis of an aniline-based
zwitterionic molecule (AP-SB) in a high yield (470%) that is
demonstrated to be a key reagent to improve the surface
modification of Au chips by electrodeposition. Relative to a
conventional thiol-SAM approach, electrodeposition has the
advantages of decreasing the duration of reaction from hours
or even days to 3 min and increasing the SB modification
density, resulting in significantly decreased non-specific bind-
ing up to 95 ꢁ 3% from the dilute FBS. The SB-modified chip
clearly demonstrated much better antifouling properties than
those of bare gold and the hydrophobic surface (Fig. S6, ESI†).
The platform demonstrated herein is highly useful to improve
the sensitivity and reliability of a biosensor using Au as the
sensory chip. It is also useful for carbon,30 ITO,31 and other
electrodes.32
(o100 mM) may increase non-specific protein adsorption,28,29
a
high buffer concentration (150 mM) is employed in this study.
The stability of the SB-modified chip is monitored for more
than 2 weeks with no significant loss of the antifouling ability
(Fig. S5, ESI†).
We utilized fluorescence images to display the antifouling
ability of Au electrodes with and without the SB modification.
We dropped a red fluorescent protein (mCherry) onto the
silicon substrate with the micro-patterned Au electrodes. As
the control experiment shows (Fig. 3A), a bare substrate without
mCherry treatment revealed no fluorescence emission. After
incubation for 1 h with the concentrated mCherry protein
(10 mg mLꢀ1, approximately equivalent to the concentration
of total proteins in 10% serum), non-specific binding, resulting
in red fluorescence, is clearly revealed on the bare Au electrodes
(Fig. 3B), but almost no mCherry protein adhered to the electrode
selectively coated with SB by electrodeposition (Fig. 3C). The
relative fluorescence intensities in Fig. 3A(i), B(i), and C(i) quan-
tified by the Photoshop program, is 0, 116, and 4, respectively.
The bottom panels (Fig. 3A(ii), B(ii), and C(ii)) show bright-field
images at the same positions. The red colour (not fluorescence)
The Ministry of Science and Technology of Taiwan (NSC 101-
2120-M-009-011-CC1) and the Center for Interdisciplinary
Science (CIS) of National Chiao Tung University of Taiwan
under MOE 5Y50B project financially supported this research.
Notes and references
1 J. Wang, Chem. Rev., 2008, 108, 814–825.
2 J. T. Sheu, C. C. Chen, K. S. Chang and Y. K. Li, Biosens. Bioelectron.,
2008, 23, 1883–1886.
3 Y. Liu, X. Dong and P. Chen, Chem. Rev., 2012, 41, 2283–2307.
4 B. R. Li, C. C. Chen, R. U. Kumar and Y. T. Chen, Analyst, 2014, 139,
1589–1608.
5 M. A. Cooper, Nat. Rev. Drug Discovery, 2002, 1, 515–528.
6 M. F. Templin, D. Stoll, M. Schrenk, P. C. Traub, C. F. Vohringer and
T. O. Joos, Drug Discovery Today, 2002, 7, 815–822.
7 F. Long, A. Zhu and H. Shi, Sensors, 2013, 13, 13928–13948.
8 K. I. Chen, B. R. Li and Y. T. Chen, Nano Today, 2011, 6, 131–154.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 6793--6796 | 6795

View Article Online
Communication
ChemComm
9 B. R. Li, C. W. Chen, W. L. Yang, T. Y. Lin, C. Y. Pan and Y. T. Chen, 21 P. Fenter, A. Eberhardt and P. Eisenberger, Science, 1994, 266,
Biosens. Bioelectron., 2013, 45, 252–259. 1216–1218.
10 B. R. Li, Y. J. Hsieh, Y. X. Chen, Y. T. Chung, C. Y. Pan and 22 N. L. Rosi and C. A. Mirkin, Chem. Rev., 2005, 105, 1547–1562.
Y. T. Chen, J. Am. Chem. Soc., 2013, 135, 16034–16037.
11 R. B. Belshe, N. Engl. J. Med., 2005, 353, 2209–2211.
23 Z. Zhang, J. Borenstein, L. Guiney, R. Miller, S. Sukavaneshvar and
C. Loose, Lab Chip, 2013, 13, 1963–1968.
12 H. M. So, K. Won, Y. H. Kim, B. K. Kim, B. H. Ryu, P. S. Na, H. Kim 24 G. Li, G. Cheng, H. Xue, S. Chen, F. Zhang and S. Jiang, Biomaterials,
and J. O. Lee, J. Am. Chem. Soc., 2005, 127, 11906–11907.
13 M. J. Barry, N. Engl. J. Med., 2001, 344, 1373–1377.
14 W. H. King, Anal. Chem., 1964, 36, 1735–1739.
15 C. I. Cheng, Y. P. Chang and Y. H. Chu, Chem. Soc. Rev., 2012, 41,
1947–1971.
2008, 29, 4592–4597.
´
25 J. Lyskawa and D. Belanger, Chem. Mater., 2006, 18, 4755–4763.
26 I. Jabin, C. Mangeney, C. Roux, O. Reinaud, L. Santos, J. F.
Bergamini, P. Hapiot and C. Lagrost, Nat. Commun., 2012, 3,
1130.
16 T. Goda, M. Tabata, M. Sanjoh, M. Uchimura, Y. Iwasaki and 27 G. Sauerbrey, Phys. Z., 1959, 155, 206–222.
Y. Miyahara, Chem. Commun., 2013, 49, 8683–8685.
17 H. Zhao, B. Zhu, S. C. Luo, H. A. Lin, A. Nakao, Y. Yamashita and
H. H. Yu, ACS Appl. Mater. Interfaces, 2013, 5, 4536–4543.
18 L. Zhang, Z. Cao, T. Bai, L. Carr, J. R. Ella-Menye, C. Irvin, B. D. Ratner
and S. Jiang, Nat. Biotechnol., 2013, 31, 553–556.
19 J. C. Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo and G. M. Whitesides,
Chem. Rev., 2005, 105, 1103–1169.
28 R. E. Holmlin, X. Chen, R. G. Chapman, S. Takayama and
G. M. Whitesides, Langmuir, 2001, 17, 2841–2850.
29 E. Ostuni, R. G. Chapman, M. N. Liang, G. Meluleni, G. Pier,
D. E. Ingber and G. M. Whitesides, Langmuir, 2001, 17, 6336–6343.
30 C. Dalmolin, S. C. Canobre, S. R. Biaggio, R. C. Rocha-Filho and
N. Bocchi, Electrochem. Commun., 2005, 578, 9–15.
31 E. Venancio, C. Costa, S. Machado and A. Motheo, Electrochem.
Commun., 2001, 3, 229–233.
20 H. B. Akkerman, P. W. Blom, D. M. de Leeuw and B. de Boer, Nature,
2006, 441, 69–72.
32 J. Pinson and F. Podvorica, Chem. Soc. Rev., 2005, 34, 429–439.
6796 | Chem. Commun., 2014, 50, 6793--6796
This journal is ©The Royal Society of Chemistry 2014