Bioconjugate Chem. 2007, 18, 2197–2201
2197
Facile and Rapid Direct Gold Surface Immobilization with Controlled
Orientation for Carbohydrates
Jeong Hyun Seo,† Kyouichi Adachi,‡,§ Bong Kuk Lee,‡,§ Dong Gyun Kang,† Yeon Kyu Kim,† Kyoung Ro Kim,†
Hea Yeon Lee,*,‡,§ Tomoji Kawai,‡,§ and Hyung Joon Cha*,†
Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea, Institute for
Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan, and Core Research for Evolutional Science and
technology, Japan Science and Technology Agency, Saitama 332-0012, Japan. Received July 30, 2007;
Revised Manuscript Received August 15, 2007
Effective surface immobilization is a prerequisite for numerous carbohydrate-related studies including carbohydrate–biomolecule interactions. In the present work, we report a simple and rapid modification technique for
diverse carbohydrate types in which direct oriented immobilization onto a gold surface is accomplished by coupling
the amine group of a thiol group-bearing aminophenyl disulfide as a new coupling reagent with an aldehyde
group of the terminal reducing sugar in the carbohydrate. To demonstrate the generality of this proposed reductive
amination method, we examined its use for three types of carbohydrates: glucose (monosaccharide), lactose
(disaccharide), and GM1 pentasaccharide. Through successful mass identifications of the modified carbohydrates,
direct binding assays on gold surface using surface plasmon resonance and electrochemical methods, and a terminal
galactose-binding lectin assay using atomic force microscopy, we confirmed several advantages including direct
and rapid one-step immobilization onto a gold surface and exposure of functional carbohydrate moieties through
oriented modification of the terminal reducing sugar. Therefore, this facile modification and immobilization method
can be successfully used for diverse biomimetic studies of carbohydrates, including carbohydrate–biomolecule
interactions and carbohydrate sensor or array development for diagnosis and screening.
INTRODUCTION
Carbohydrates encode information for specific molecular
recognition, help determine protein folding, stability, and
pharmacokinetics, play critical roles in determining biological
functions, and affect diverse physiological processes (1–4). In
addition, carbohydrate–protein interactions have been used to
elucidate fundamental biochemical processes and identify new
pharmaceutical substances in living cell systems (1–4). In order
to fully study specific carbohydrate interactions with biomolecules such as proteins, DNA, and other carbohydrates in the
cell, researchers need effective methods for carbohydrate
immobilization (5–8).
To date, modification for carbohydrate immobilization has
been attempted using several techniques, including copolymerization, coupling with divinyl sulphone, coupling to CNBractivated substrates, and reductive amination (9, 10). However,
although these modification methods have demonstrated promising results, general usefulness for diverse carbohydrate types
has not been proven (11, 12). Previous studies have mainly
focused on mono and/or disaccharides, with a few attempts made
to immobilize oligo or polysaccharides, because of their
structural complexity and problems with severe structural
deformity during modification (13–15). Therefore, more advanced methods including alternative surface modification have
been developed for diverse carbohydrate types (6, 16–20).
However, these strategies also have tended to be multistep
protocols aimed at indirect immobilization. Especially, for
biosensor applications, it is desirable to directly and rapidly
immobilize various carbohydrates on surfaces by one-step protocols.
Successful surface immobilization of carbohydrates has been
further complicated by the need to retain the original structure and
expose the functional sites so that the immobilized molecule can
mimic the specific biomolecular interactions occurring on the cell
surface. Because carbohydrates do not have functional groups for
orientation, it has proven technically challenging to immobilize
carbohydrates (especially oligo and polysaccharides) in an oriented
fashionwhileretainingtheirinherentstructuresandfunctionalities(11,12).
Therefore, we sought to develop a method for orienting and
immobilizing various carbohydrate types directly and rapidly onto
a gold surface that will allow specific carbohydrate–biomolecule
recognition. Immobilization of carbohydrates on gold surface can
be easily applicable for gold-based biosensors, such as electrochemical (EC) sensors, surface plasmon resonance (SPR), and
quartz crystal microbalances (QCM).
We herein report a simple and rapid reductive amination
technique by using aminophenyl disulfide as a new coupling
reagent for diverse carbohydrate types in which orientation is
accomplished by coupling a thiol (-SH) group to the terminal
reducing sugar of the carbohydrate, and the oriented carbohydrate is directly immobilized onto a bare gold surface (Scheme
1). To show the generality of our proposed modification/
immobilization method, we examined its use for three types of
carbohydrates having terminal reducing sugar moieties, namely,
glucose (monosaccharide), lactose (disaccharide), and GM1
pentasaccharide. We also demonstrated the interaction of a
representative protein with carbohydrates surface-immobilized
using our method (Scheme 1).
EXPERIMENTAL PROCEDURES
* Corresponding author. E-mail: [email protected] (H.J.C.). Email: [email protected] (H.Y.L.).
†
Pohang University of Science and Technology.
‡
Osaka University.
§
Japan Science and Technology Agency.
Carbohydrate Modification. Glucose (Sigma), lactose
(Sigma), and GM1 pentasaccharide (Alexis Biochemicals) at
100 mM each were separately dissolved in water, and 50 mM
aminophenyl disulfide (Aldrich) was dissolved in acetic acid.
10.1021/bc700288z CCC: $37.00  2007 American Chemical Society
Published on Web 10/05/2007
2198 Bioconjugate Chem., Vol. 18, No. 6, 2007
Seo et al.
Scheme 1. Strategy for the Modification and Direct Immobilization of Various Carbohydrate Typesa
a
Modified SH-saccharides can be directly immobilized on gold and interact specifically with lectin. R1, glucose (monosaccharide); R2, lactose
(disaccharide); R5, GM1 pentasaccharide.
The carbohydrate and aminophenyl disulfide solutions were mixed
well and incubated in sealed tubes for 1 h at 20 °C (glucose and
lactose) or 30 °C (GM1 pentasaccharide) for the amination reaction
step. Then, freshly prepared reducing reagent, 100 mM dimethylamine borane (Fluka), was added to each reaction, and the tubes
were incubated unsealed for 1 h at 20 °C for the reduction step.
The tubes were then resealed and heated for 1 h at 50 °C under
nitrogen gas steaming for the condensation step. All modified
carbohydrates were dissolved in 5% acetic acid solution and
preserved at -20 °C under light-blocking conditions. Without
further purification steps, modified samples were used for MALDITOF MS analysis and immobilization.
Mass Identification Using MALDI-TOF MS. To improve
the ionization efficiency of MALDI-TOF MS, the samples were
desalted using Zip-tip C18 (Millipore) and then eluted onto
MALDI target plates using matrix solution (10 mg/mL; R-cyano4-hydroxycinnamic acid dissolved in a solution consisting of
50% acetonitrile and 0.5% trifluoroacetic acid). All mass spectra
were acquired in reflection mode using a 4700 Proteomic
Analyzer (Applied Biosystems) at Korea Basic Science Institute.
Spectra were obtained in the mass range between 100 to 2000
Da with ∼200 laser shots. Internal calibration was performed
with 4700 Cal Mix (Applied Biosystems).
SPR-Based Immobilization Analysis. All SPR experiments
were performed using a Biacore 2000 instrument (Biacore AB)
with a flow system that loaded the solutions as discrete channels.
The SIA Kit Au (Biacore AB) was used as the bare gold surface
for the direct immobilization of modified SH-containing carbohydrates. The flow rate for binding onto the gold surface was
5 µL/min, and a noninjected channel was used as the baseline.
Immobilization was performed by injecting 60 µL of each
sample onto the gold surface, and immobilized carbohydrate
amounts were measured as RU. All measurements were
performed in HEPES-EP buffer (Biacore AB; 10 mM HEPES
at pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant
F20).
EC-Based Immobilization Analysis. EC experiments were
performed using a BAS 100 B/W potentiostat (Bioanalytical
System) at room temperature. The biosensor consisted of an
8-multielectode chip comprising an array of 200-nm diameter
gold electrodes on glass (21–23). Modified GM1 pentasaccharide (1 µL at 5 mM in HEPES-buffered saline) was directly
immobilized onto the multigold electrodes for 1 h at room
temperature, and unmodified GM1 pentasaccharide (1 µL at 5
mM) was loaded as a negative control. Electrode pretreatment
was carried out for 3 min at 1.8 V in 10 mM H2SO4. All CV
measurements were performed in solutions containing 5 mM
K3Fe(CN)6 at a scan rate of 100 mV/s. A solid bar of Ag/AgCl
in 100 mM KCl and a 5-mm diameter platinum wire were used
as the reference and the counter electrode, respectively. The
SWV curves were recorded at a 10 mV/s scan rate with a pulse
height of 25 mV and a step time of 0.2 s.
Lectin Interaction Analysis Using AFM. We deposited a
layer of 99.5% pure Au(111) on a cleaved mica surface using
a high speed vacuum (base pressure, 5 × 10-7 Pa) at 450 °C.
After the immobilization of SH-GM1 pentasaccharide onto the
gold surface, we added 20 mg/mL of galectin (Biovision) and
incubated the samples for 1 h. Topology was imaged using
Multimode DI AFM (Veeco Instruments).
RESULTS AND DISCUSSION
For the efficient immobilization of diverse carbohydrate types
onto a bare gold surface, we used an amination reaction to
couple the aldehyde (-CHO) group of the carbohydrate’s
terminal reducing sugar with the amine (-NH2) group of a thiolbearing aminophenyl disulfide (Scheme 1). A reductive amination reaction, namely, coupling the aldehyde group of a
carbohydrate’s terminal reducing sugar with the amine group
of 2-aminopyridine, is commonly used for pyridylamination
tagging of glycans with 2-aminopyridine in order to facilitate
their purification and molecular weight analysis (13, 14, 24, 25).
In the present work, we found that mono and disaccharides
could be modified at 20 °C reaction temperature, whereas
modification of GM1 pentasaccharide was successful at 30 °C.
This may suggest that a comparatively higher reaction temper-
Bioconjugate Chem., Vol. 18, No. 6, 2007 2199
Figure 2. SPR binding assays of SH-modified monosaccharide glucose
(A), disaccharide lactose (B), and GM1 pentasaccharide (C).
Figure 1. MALDI-TOF MS analysis of SH-modified monosaccharide
glucose (A), disaccharide lactose (B), and GM1 pentasaccharide (C).
ature is required to give a higher ratio of an acyclic form of the
terminal reducing sugar of complex carbohydrates. We further
found that the modification efficiency depended on the concentration of reactants, with experiments containing 100 mM GM1
pentasaccharide showing a higher efficiency than those containing 10 mM of the same carbohydrate at the same concentration
(50 mM) of aminophenyl disulfide (data not shown).
To check the feasibility of the modification of target
carbohydrates using the proposed reductive amination reaction,
we first performed nuclear magnetic resonance spectroscopy
(NMR). The NMR spectrum contained features consistent with
a -CH2-NH- moiety (for glucose, 1H NMR [600 MHz, D2O] δ
3.42–3.48 [3H, m], 3.58–3.62 [5H, m], 6.48 [2H, d, J)8.0 Hz],
7.09 [2H, d, J)8.0 Hz]). We then used matrix-assisted laser
desorption–ionization mass spectrometry with time-of-flight
(MALDI-TOF MS) analysis to investigate the molecular weights
of the modified carbohydrates (Figures 1A–C). Although we
used aminophenyl disulfide as the coupling reagent, the
modification primarily occurred through the aminophenyl sulfide
that might be formed from disulfide bond breakage under acidic
(pH 2–3) conditions or reduction of its bond by dimethylamine
borane. Therefore, the molecular weights of the modified
glucose, lactose, and GM1 pentasaccharide were 290, 452, and
1108 (1130: +Na), respectively. Interestingly, all of the
examined samples yielded a double mass value, representing a
small peak of modified carbohydrates that had reacted directly
with the aminophenyl disulfide. In addition, we also performed
the modification of four other types of carbohydrates and
confirmed their modifications using mass analyses (data not
shown). Overall, these mass analyses confirmed that all of the
target carbohydrates were successfully modified.
Without further purification, we then directly immobilized
the SH-modified carbohydrates onto a gold surface. All of the
SH-modified carbohydrates showed successful binding on the
gold surface, as assessed by SPR binding assays (Figure 2A–C).
Since the mass of lactose is somewhat higher than that of
glucose at the same mole concentration, the resonance unit (RU)
value of SH-lactose (950 RUs) was slightly higher than that of
SH-glucose (900 RUs). In the case of GM1 pentasaccharide,
although we used a carbohydrate concentration of 100 mM for
the modification step, we diluted the SH-GM1 pentasaccharide
10-times for immobilization because of its limited available
amount. Although GM1 pentasaccharide has a much higher
2200 Bioconjugate Chem., Vol. 18, No. 6, 2007
Seo et al.
Figure 3. EC response of gold electrodes bound with unmodified GM1 pentasaccharide (red line) and modified SH-GM1 pentasaccharide (green
line) compared to that of the bare gold electrode (black line). Inset, CV; main figure, SWV. The schematic illustration represents immobilization
of modified SH-GM1 pentasaccharide on the gold surface.
molecular weight than glucose or lactose, the RU value was
lower (800 RUs) because the utilized concentration was onetenth that used in the other experiments. Collectively, these
findings indicate that all of the SH-modified carbohydrates were
successfully immobilized onto the gold surface without requiring
additional treatment steps.
To further evaluate the direct immobilization of SH-modified
carbohydrates onto the gold surface, we used EC analysis, which
has the advantages of easy detection and high sensitivity at low
concentrations (19–21) and which might be a useful tool for
practical application as a carbohydrate sensor that needs high
sensitivity due to very low portion of carbohydrates in the
cellular materials. EC analysis was used to obtain the cyclic
voltammetry (CV) and square wave voltammetry (SWV) curves
from SH-modified and unmodified GM1 pentasaccharide exposed to 200-µm diameter gold electrodes (Figure 3). The CV
measurements showed that the EC responses were significantly
enhanced for immobilized SH-GM1 pentasaccharide (Figure 3,
inset). The peaks and dips in the redox current of the CV signal
showed the typical oxidation and reduction responses in the
electrolyte of K3Fe(CN)6. The SWV curves (Figure 3, main
figure) revealed that the limiting redox current decreased from
1.05 on the bare gold electrode to 0.2 µA in the presence of
immobilized SH-GM1 pentasaccharide. The change ratios of
the redox currents of bare gold electrodes in the presence or
absence of unmodified or SH-modified GM1 pentasaccharide
were ∼45% and ∼80%, respectively. This clearly showed that
the EC signal ratio changed in response to carbohydrate
immobilization, indicating that EC could have potential carbohydrate sensor applications. The reduced redox current is due
to the decrease in reactive surface area exposed to the electrolyte.
Notably, while the current ratio decreased to some extent in
the presence of unmodified GM1 pentasaccharide, likely due
to physical adsorption from the relatively small carbohydrate
size, the redox current was almost completely blocked by the
binding of SH-GM1 pentasaccharide to the gold electrode.
Future systematic studies may be warranted to examine additional levels of control that may be exerted over this physical
adhesion. Therefore, these results collectively indicate that our
proposed method is a highly efficient strategy for immobilizing
carbohydrates to the tested substrate.
Although the structure of the terminal reducing sugar is
changed to the acyclic form by coupling of the aldehyde and
amine groups (see Scheme 1), this should not be a problem in
long chain carbohydrates such as GM1 pentasaccharide because
the generation of the acyclic form only occurred at the surfacelinked terminal sugar. A previous study showed that galactose
and sialic acid, two terminal sugars of the branched GM1
pentasaccharide, exhibited substantial specific binding interactions (∼39% from galactose and ∼43% from sialic acid), with
a smaller contribution (∼17%) arising from the N-acetyl
galactosamine residue (26). However, the central galactose and
terminal reducing glucose residues do not appear to make direct
interactions with the receptor protein (26), indicating that the
terminal reducing and central sugars are more likely to act as
bridges or linkers. Accordingly, we examined the binding of
GM1 pentasaccharide to its terminal galactose-binding lectin,
galectin, by topology analysis using atomic force microscopy
(AFM) (Figure 4). Our results revealed that galectin did not
bind in the case of unmodified, nonimmobilized GM1 pentasaccharide (Figure 4A), whereas galectin was clearly detected as a
white spot on SH-GM1 pentasaccharide-immobilized gold
Figure 4. AFM analysis of the GM1 pentasaccharide–galectin interaction on Au(111). (A) Unmodified pentasaccharide, (B) modified SHpentasaccharide, (C) magnified image of box in B, and (D) line profile
of the SH-modified GM1 pentasaccharide–galectin interaction.
Bioconjugate Chem., Vol. 18, No. 6, 2007 2201
(Figure 4B and C). Line profile analysis revealed that the protein
spots were 3–4 nm in size (Figure 4D). Importantly, since
galectin should interact with only the terminal galactose, these
findings confirmed that the modified carbohydrate had been
successfully immobilized with the proper orientation.
In the present work, we kept our method simple by using
aminophenyl disulfide as both the coupling reagent and the
linker. In the future, the use of a longer coupling reagent might
help increase the flexibility and receptor binding of the target
carbohydrate. However, since increasing the length of the alkyl
chain group can change the hydrophobicity of the molecule,
the use of an overly long chain could inhibit carbohydrate
modification. One of the advantages of our immobilization
method is that only the terminal reducing sugar is specifically
coupled with the linker reagent, so that the functional sites (e.g.,
biomolecule recognition or binding sites) of the carbohydrate
can be preserved.
In summary, we herein demonstrated that the amine group
of thiol-bearing aminophenyl disulfide was successfully coupled
with the aldehyde group of the terminal reducing sugar in three
types of carbohydrates, namely, glucose (monosaccharide),
lactose (disaccharide), and GM1 pentasaccharide. The proposed
modification method has several advantages over previously
reported methods, including direct and rapid one-step immobilization onto a gold surface without surface pretreatment(s)
by thiol group coupling in a mild reaction environment and
exposure of functional carbohydrate moieties through oriented
immobilization of the terminal reducing sugar. This modification
and immobilization method should prove useful for diverse
biomimetic studies in carbohydrates, including carbohydrate–
biomolecule interaction and carbohydrate sensor or array
development for diagnosis and screening.
ACKNOWLEDGMENT
This work was supported by National R&D Project for Nano
Science and Technology and Advanced Environmental Biotechnology Research Center at POSTECH from the Korea
Science and Engineering Foundation and the Brain Korea 21
Program from the Ministry of Education, Korea.
LITERATURE CITED
(1) Jelinek, R., and Kolusheva, S. (2004) Carbohydrate biosensors.
Chem. ReV. 104, 5987–6015.
(2) Bertozzi, C. R., and Kiessling, L. L. (2001) Chemical glycobiology. Science 291, 2357–2364.
(3) Zachara, N. E., and Hart, G. W. (2002) The emerging
significance of O-GlcNAc in cellular regulation. Chem. ReV. 102,
431–438.
(4) Ritchie, G. E., Moffatt, B. E., Sim, R. B., Morgan, B. P., Dwek,
R. A., and Rudd, P. M. (2002) Glycosylation and the complement
system. Chem. ReV. 102, 305–320.
(5) Nyquist, R. M., Eberhardt, A. S., Silks, L. A., Li, Z., Yang,
X., and Swanson, B. I. (2000) Characterization of self-assembled
monolayers for biosensor applications. Langmuir 16, 1793–1800.
(6) Park, S. J., and Shin, I. J. (2002) Fabrication of carbohydrate
chips for studying protein-carbohydrate interactions. Angew.
Chem., Int. Ed. 41, 3180–3182.
(7) Lazcka, O., Campo, F. J. D., and Munoz, F. X. (2007) Pathogen
detection: A perspective of traditional methods and biosensors.
Biosens. Bioelectron. 22, 1205–1217.
(8) Ni, J. H., Singh, S., and Wang, L. X. (2003) Synthesis of
maleimide-activated carbohydrates as chemoselective tags for
site-specific glycosylation of peptides and proteins. Bioconjugate
Chem. 14, 232–238.
(9) Hermanson, G. T., Mallia, A. K., and Smith, P. K. (1992)
Immobilized Affinity Ligand Techniques, Vol. 1, pp 137–200,
Academic Press, London.
(10) Hatanaka, K., Takeshige, H., and Akaike, T. (1994) Synthesis
of a new polymer containing uridine and galactose as pendant
groups. J. Carbohydr. Biochem. 13, 603–610.
(11) Shin, I., Park, S., and Lee, M. R. (2005) Carbohydrate
microarrays: An advanced technology for functional studies of
glycans. Chem.–Eur. J. 11, 2894–2901.
(12) de Paz, J. L., and Seeberger, P. H. (2006) Recent advances in
carbohydrate microarrays. QSAR Comb. Sci. 25, 1027–1032.
(13) Kondo, A., Suzuki, J., Kuraya, N., Hase, S., Kato, I., and
Ikenaka, T. (1990) Improved method for fluorescence labeling
of sugar chains with sialic acid residues. Agric. Biol. Chem. 54,
2169–2170.
(14) Hase, S., Ibuki, T., and Ikenaka, T. (1984) Reexamination of
the pyridylamination used for fluorescence labeling of oligosaccharides and its application to glycoproteins. J. Biochem. (Tokyo)
95, 197–203.
(15) Hase, S., Koyama, S., Daiyasu, H., Takemoto, H., Hara, S.,
Kobayashi, Y., Kyogoku, Y., and Ikenaka, T. (1986) Structure
of a sugar chain of a protease inhibitor isolated from barbados
pride (Caesalpinia pulcherrima Sw.) seeds. J. Biochem. (Tokyo)
100, 1–10.
(16) (a) Wang, D. N., Liu, S. Y., Trummer, B. J., Deng, C., and
Wang, A. L. (2002) Carbohydrate microarrays for the recognition
of cross-reactive molecular markers of microbes and host cells.
Nat. Biotechnol. 20, 275–281.
(17) Lee, M., and Shin, I. (2005) Facile preparation of carbohydrate
microarrays by site-specific, covalent immobilization of unmodified carbohydrates on hydrazide-coated glass slides. Org. Lett.
7, 4269–4272.
(18) Fukui, S., Feizi, T., Galustian, C., Lawson, A. M., and Chai,
W. G. (2002) Oligosaccharide microarrays for high-throughput
detection and specificity assignments of carbohydrate-protein
interactions. Nat. Biotechnol. 20, 1011–1017.
(19) Zhi, Z., Powell, A. K., and Turnbull, J. E. (2006) Fabrication
of carbohydrate microarrays on gold surfaces: Direct attachment
of nonderivatized oligosaccharides to hydrazide-modified selfassembled monolayers. Anal. Chem. 78, 4786–4793.
(20) Zhou, X., and Zhou, J. (2006) Oligosaccharide microarrays
fabricated on aminooxyacetyl functionalized glass surface for
characterization of carbohydrate-protein interaction. Biosens.
Bioelectron. 21, 1451–1458.
(21) Lee, H. Y., Park, J. W., and Kawai, T. (2004) SNPs feasibility
of nonlabeled oligonucletides on using electrochemical sensing.
Electroanalysis 16, 1999–2002.
(22) Kim, J. M., Jung, H. S., Park, J. W., Yukimasa, T., Oka, H.,
Lee, H. Y., and Kawai, T. (2005) Spontaneous immobilization
of liposomes on electron-beam exposed resist surfaces. J. Am.
Chem. Soc. 127, 2358–2362.
(23) Lee, H. Y., Park, J. W., Kim, J., Jung, H., and Kawai, T.
(2006) Well-oriented nanowell array metrics for integrated digital
nanobiosensors. Appl. Phys. Lett. 89, 113901.
(24) Takahashi, C., Nakakita, S., and Hase, S. (2003) Conversion
of pyridylamino sugar chains to corresponding reducing sugar
chains. J. Biochem. (Tokyo) 134, 51–55.
(25) Kuraya, N., and Hase, S. (1992) Release of O-linked sugar
chains from glycoproteins with anhydrous hydrazine and pyridylamination of the sugar chains with improved reaction
conditions. J. Biochem. (Tokyo) 112, 122–126.
(26) Merritt, E. A., Sarfaty, S., Vandenakker, F., Lhoir, C., Martial,
J. A., and Hol, W. (1994) Crystal-structure of cholera-toxin
B-pentamer bound to receptor G(M1) pentasaccharide. Protein
Sci. 3, 166–175.
BC700288Z