Recognition of lectin with a high signal to noise ratio: Carbohydrate-tri(ethylene glycol)-alkanethiol co-adsorbed monolayer

Article abstract of DOI:10.1039/b809481k

Densely packed co-adsorbed ultrathin mono molecular layers of short tri(ethylene glycol)-alkanethiolate (for repelling proteins) and maltoside terminated alkanethiolate (for capturing lectin) provided an extremely high signal to noise ratio surface: the r

Full text of DOI:10.1039/b809481k

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Recognition of lectin with a high signal to noise ratio:
carbohydrate-tri(ethylene glycol)-alkanethiol co-adsorbed monolayerw
Yukari Sato,* Kyoko Yoshioka, Mutsuo Tanaka, Teiichi Murakami,
Miho Neide Ishida and Osamu Niwa
Received (in Cambridge, UK) 4th June 2008, Accepted 4th August 2008
First published as an Advance Article on the web 9th September 2008
DOI: 10.1039/b809481k
Densely packed co-adsorbed ultrathin mono molecular layers of
short tri(ethylene glycol)-alkanethiolate (for repelling proteins)
and maltoside terminated alkanethiolate (for capturing lectin)
provided an extremely high signal to noise ratio surface: the
repelling molecules, which had two different functions (highly
flexible-hydrophilic arm and rigid packing tail group), worked as
‘‘nano barriers’’ in the recognition monolayer.
effectively to suppress the non-specific protein adsorption.
When we constructed a co-adsorbed mono molecular layer
on the substrate using this amphoteric molecule and the
protein recognition molecule, target molecules could be cap-
tured effectively because the non-specific adsorption on the
substrate was greatly suppressed. As a result, the signal to
noise ratio was effectively increased.
We synthesized and used tri(ethylene glycol) terminated
alkanethiols (EGCnSH, n = 2, 4, 6, 8, 11, Fig. 1) as protein
repelling modifiers. EGCnSH consisted of a flexible repellent
arm and a closely packed tail group as shown in Fig. 1.
(1-Mercaptoundec-11-yl)tri(ethylene glycol) (EGC11SH) was
purchased from SensoPath Technologies and also used in the
experiment. 12-Mercaptododecyl b-maltoside (Mal-C12SH,
Fig. 1) was synthesized and used for the specific recognition
of the lectin Concanavalin A (ConA).11–13 The interface
structure and the number of adsorbed Con A molecules were
monitored by using surface plasmon resonance (SPR) mea-
surement (Biacore T-100, 2000 and NTT-AT Handy SPR
system). The packing ratio of the EGCnSH monolayers was
evaluated from their amount on the substrate by measuring
the current, peak area, and peak potential of the electro-
chemical desorption.14 Fig. 2(a) shows the specific recognition
of ConA on the MalC12SH and EGCnSH mixed monolayer
surfaces (MalC12SH : EGCnSH = 1 : 9 (in surface modifi-
cation solution); signal response). The real MalC12SH ratio is
about 30% by the electrochemical reductive desorption
method, although this ratio slightly changes depended on the
alkylchain length of EGCnSH. The non-specific adsorption of
ConA on the 100% EGCnSH monolayers is also shown on the
left of the figure (red bars; noise response). ConA recognized
the a-1,4-bond of the maltoside part of MalC12SH.11 Large
signal responses caused by ConA adsorption were observed on
There is widespread interest in the formation of nanostruc-
tured biological membranes for new applications in sensing
and for fundamental studies of interfacial phenomena. The
non-specific adsorption of biological species on solid surfaces
is a ubiquitous problem as regards to device and biochip
design. The prevention of non-specific biomolecule adsorption
is linked to a low noise response and the achievement of bio-
recognition surfaces with high signal to noise ratios.
The suppression of non-specific adsorption using molecules
including oligo(ethlene glycol), and its derivatives, with long
alkanethiols has been investigated in terms of layer formation
and basic characteristics.1–7 Protein adsorption has actually
been prevented by using polymer layers with oligo(ethylene
glycol) terminals.8 The membrane structure of the oligo(ethyl-
ene glycol) polymer is hydrophilic and flexible, but its bulky
volume occasionally becomes a problem. It has also been
reported that the use of polymer layers with different molecular
weights is very effective in suppressing non-specific adsorp-
tion.9,10 From these important reports, we learn that several
requirements must be met if we are to realize an excellent
biomolecule-repelling characteristic and
a
good bio-
recognition surface; the repellent modifier must have (1) highly
flexible and highly hydrophilic parts and (2) structures for
forming condensed packing layers. A modifier that provides
both of these functions appears to be a contradiction.
Resistance to non specific protein adsorption was increased
by increasing the unit number of the terminal oligo(ethylene
glycol) group.4 We confirmed that tri(ethylene glycol) termi-
nated short alkanethiol is a tiny amphoteric modifier that
includes a highly flexible-bulky-hydrophilic part and a short
alkane backbone to provide dense packing. Tri(ethylene
glycol) terminated short alkanethiols (n = 2–8) worked
National Institute of Advanced Industrial Science and Technology
(AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566,
Japan. E-mail: yukari-sato@aist.go.jp; Fax: +81-29-861-6177;
Tel: +81-29-861-6158
w Electronic supplementary information (ESI) available: Experimen-
tal. See DOI: 10.1039/b809481k
Fig. 1 Capturing ConA molecule (MalC12SH) and repelling molecules
(EGCnSH).
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MalC12SH–EGCnSH monolayer. In particular, the EGCnSH
packing density was the key to improving the ability to prevent
adsorption. The number of adsorbed EGCnSH monolayers on
the gold surface was evaluated with the electrochemical re-
ductive desorption method. An alkanethiolate monolayer was
desorbed from the gold substrate in an alkaline solution
through a one-electron reductive path (RS-Au + eÀ
-
RSÀ + Au).14–17 Each EGCnSH molecule was reduced at
around À1.0 V (vs. Ag/AgCl(NaCl)) in 0.5 M KOH. As shown
in Fig. 2(c), the number of attached EGCnSH molecules
became larger as the alkylchain became longer. The reductive
potential was shifted in a negative direction as the alkylchain
became longer (EGC2SH: À0.86 V, EGC11SH: À1.08 V
(vs. Ag/AgCl (3 M NaCl)), indicating that a higher packing
density was achieved by lengthening the alkylchain. The
position of the reductive peak depends on the length of the
alkane chain. The attractive interaction between the alkyl-
chains increases with increasing alkyl chain length and the
reductive desorption potential shifts in a negative direction.
Moreover, the full width at half-maximum (fwhm) of the
reductive desorption peaks indicated the strength of the inter-
action between the adsorbed molecules. In fact, the fwhm
became smaller as the alkylchain of EGCnSH became longer
(EGC2SH: 46.0 mV, EGC11SH: 37.4 mV). The fwhm of the
ideal case was around 20 mV for normal alkanethiol
(CH3(CH2)nSH; n 4 10), which formed a rigid self-assembled
monolayer.16 These experimental results supported the idea
that (1) longer alkyl-EGCnSH was adsorbed significantly on
the surface and (2) EGCnSH with longer alkylchains inter-
acted more strongly. In fact, a densely adsorbed EGCnSH
monolayer can prevent non-specific adsorption almost
Fig. 2 Relationship between the alkylchain length of EGCnSH and
(a) the number of adsorbed ConA (right (blue) bars: MalC12SH
(10%)–EGCnSH mixed monolayer; left (red) bars: 100% EGCnSH
monolayer), (b) the signal to noise ratio (signal response : noise
response; S : N ratio) of ConA adsorption, and (c) the number of
adsorbed EGCnSH molecules evaluated by electrochemical reduction.
each MalC12SH–EGCnSH mixed interface. In particular,
when EGC2SH, EGC4SH, and EGC6SH were used as nano
barriers, the ConA adsorption signals became higher. When
we focused on the results of the ConA non-specific adsorption,
the amounts of nonspecific adsorbates decreased gradually as
the alkylchain became longer. In fact, EGCnSH with a longer
alkylchain was much more effective in preventing ConA
adsorption as shown in Fig. 2(a). The repellant ability as
regards to non-specific adsorption reached its maximum value
around C6 to C8 of EGCnSH. With the EGC11SH monolayer,
it exhibited a repellant ability similar to that for C6–C8.
The signal to noise (S : N) ratio data is shown in Fig. 2(b)
(signal response (S): specific recognition of ConA–maltoside
on the MalC12SH–EGCnSH monolayer; noise response
(N): non-specific ConA adsorption on the EGCnSH mono-
layer). The S : N ratio was improved significantly when using
longer EGCnSH molecules such as EGC6SH and EGC8SH.
Preventing non-specific molecular adsorption and achieving a
high S : N ratio were attributable to a densely packed
Fig. 3 Models of ConA recognition on MalC12SH–EGCnSH mixed
monolayers. (a) MalC12SH–EGC2SH mixed monolayer and
(b) MalC12SH–EGC6SH.
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Table 1 Repulsion of proteins and peptides on a surface modified with EGCnSH (n = 2,4,6,8) and polymer (PEG2000) layer (RU is a resonance
unit in the Biacore system (1000RU = 0.1 degrees resonance angle shift))
Molecular adsorption/RU
PEGC2SH (C2)
C4
C6
C8
PEG2000
Proteins
Peptides
Concanavalin A (MW 104 000, 1 mM)
18.9
9.2
7.7
6.3
9.6
9.7
5.7
6.4
7.0
6.9
6.3
6.5
5.1
5.8
5.0
2.9
1.8
1.5
1.1
0.8
11.5
7.8
10.0
11.4
29.1
BSA (MW 66 300, 1 mM)
Angiotensin (MW 1296.5, 0.1 mg mlÀ1
)
Bradykinin (MW 1060.2, 0.1 mg mlÀ1
)
RGDS (MW 433.42, 0.1 mg mlÀ1
)
completely. Thus, the S : N ratio of ConA recognition was
improved significantly. The mixed monolayer model is shown
in the Fig. 3. The combination of EGCnSH with a longer
alkylchain and a MalC12SH mixed layer was effective with
regards to the specific recognition of lectin with a high S : N
ratio.
peptide such as RGDS was completely repelled (EGC2SH: 9.6
RU (1.0 ng cmÀ2), EGC8SH: 0.8 RU (80 pg cmÀ2)). With a
polymer PEG layer, 36 times more RGDS was adsorbed than
on the EGC8SH surface. EGCnSH nanolayers showed excellent
repellent effects for all other small peptides.
The most important result from this work lies in the fact
that the nano level short EGCnSH molecules provided a
completely repellent gold surface. Even though the alkanethiol
unit (–(CH2)n–) was short (n = 2–8), EGCnSH molecules
could form a well packed monolayer, and then we could create
an inert surface for protein adsorption by using flexible-
hydrophilic tri(ethylene glycol) units. EGCnSH is a small
amphoteric molecule and is an excellent tool for forming a
‘‘nano barrier’’ on a substrate surface.
EGC11SH can form a very densely packed self-assembled
monolayer because EGC11SH had a longer alkylchain. The
non specific adsorption of ConA on the EGC11SH layer was
also suppressed effectively (Fig. 2(a)). Unfortunately, with a
mixed MalC12SH–EGC11SH layer, the amount of ConA
adsorption was decreased to about one-sixth that of
MalC12SH–EGC2SH, EGC4SH, and EGC6SH because the
ConA recognition site, a-1,4-bond of maltoside, was hindered
in the arms of the EGC11SH molecules. Consequently, the
S : N ratio of MalC12SH–EGC11SH was unsatisfactory.
These nano scale controlled monolayers (EGCnSH)
completely prevented the non-specific adsorption of biomole-
cules. In particular, small biomolecules, whose adsorption was
difficult to suppress, were prevented from reaching the gold
surface. Our molecules, which consist of highly mobile ethyl-
ene glycol units and densely packed alkyl-chain units, function
as nano barrier molecules. The non specific adsorption of the
proteins (ConA, BSA (Bovine Serum Albumin)), and peptides
(angiotensin, bradykinin, and RGDS (Arg-Gly-Asp-Ser)) on
the each EGCnSH surface is shown in Table 1. A PEG2000
(mPEG-thiol, M.W. 2000) polymer layer was used for com-
parison. Our EGCnSH layers can prevent the non specific
adsorption of smaller proteins and peptides. The repelling
effect became clearer as the alkylchain became longer, parti-
cularly for the tetrapeptide RGDS, which has the smallest
MW(433.42).
Notes and references
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There have been many reports on protein repellent properties
and methods.8 A polymer PEG layer (PEG2000) is very useful
for preventing protein adsorption. ConA and BSA are adsorbed
slightly on a PEG2000 surface (ConA: 11.5 RU (1.2 ng cmÀ2),
BSA: 7.8 RU (0.8 ng cmÀ2); 1RU as 0.1 ng cmÀ2 14,15). Mixed
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can simply form a rigid repellent monolayer and effectively
prevent the adsorption of small molecules. For example, a small
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