Creating protein-imprinted self-assembled monolayers with multiple binding sites and biocompatible imprinted cavities

Article abstract of DOI:10.1021/ja402423r

Imprinted monolayers have several advantages over bulk imprinted polymers such as excellent mass transfer of molecules into and out of imprinted sites and transduction of binding signals detected in real time. Protein-imprinted self-assembled monolayers (SAMs) were created with multiple binding sites and biocompatible imprinted cavities from functional thiols and novel disulfide compounds containing an oligoethylene glycol (OEG) terminal moiety and two amide groups incorporated in the chain (DHAP) in a biologically benign solution. DHAP played an important role in the formation of multiple binding sites and biocompatible cavities in addition to resisting nonspecific protein binding. The created protein-imprinted SAMs exhibited the excellent ability of specific binding of target proteins determined by multiple binding sites and imprinted cavities. The strategy generates tailor-made monolayer surfaces with specific protein binding and opens the possibility of controlled assembly of intellectual biomaterials and preparation of biosensors.

Full text of DOI:10.1021/ja402423r

Communication
pubs.acs.org/JACS
Creating Protein-Imprinted Self-Assembled Monolayers with
Multiple Binding Sites and Biocompatible Imprinted Cavities
Xianfeng Zhang, Xuezhong Du,* Xuan Huang, and Zhongpeng Lv
Key Laboratory of Mesoscopic Chemistry (Ministry of Education), State Key Laboratory of Coordination Chemistry, and School of
Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, People’s Republic of China
S
* Supporting Information
out of imprinted sites.3e,f This is especially important for large
ABSTRACT: Imprinted monolayers have several advan-
tages over bulk imprinted polymers such as excellent mass
transfer of molecules into and out of imprinted sites and
transduction of binding signals detected in real time.
Protein-imprinted self-assembled monolayers (SAMs)
were created with multiple binding sites and biocompatible
imprinted cavities from functional thiols and novel
disulfide compounds containing an oligoethylene glycol
(OEG) terminal moiety and two amide groups incorpo-
rated in the chain (DHAP) in a biologically benign
solution. DHAP played an important role in the formation
of multiple binding sites and biocompatible cavities in
addition to resisting nonspecific protein binding. The
created protein-imprinted SAMs exhibited the excellent
ability of specific binding of target proteins determined by
multiple binding sites and imprinted cavities. The strategy
generates tailor-made monolayer surfaces with specific
protein binding and opens the possibility of controlled
assembly of intellectual biomaterials and preparation of
biosensors.
templates such as proteins, which can be encapsulated and
cannot be removed completely even from thin polymer
matrices.3e Furthermore, rebinding of the templates is typically
fast, and sensing can be further enhanced by the monolayer
surfaces that facilitate transduction of binding signals detected
in real time.3e Protein-imprinted monolayers from binary
Langmuir monolayers containing positively charged lipids or
glycolipids at the air−water interface have been constructed
with the horizontal Langmuir−Blodgett (LB) technique by us
and collaborators.4 However, there are questions about the
practical application of LB films because of their long-term
stability.
Self-assembled monolayers (SAMs) through chemisorptions
of surface-active molecules at a liquid−solid interface can
directly build stable sensor platforms and modify physical or
chemical properties of surfaces.5 Crooks and co-worker6 were
the first to describe the concept of imprinted SAMs composed
of nanoporous molecular assemblies. The imprinted SAMs on
gold-coated chips using preadsorption7 or preimmobilization8
of small-template molecules followed by covalent attachment of
alkanethiols were built, but the methods are unsuitable for
protein imprinting because of unavailable specific binding sites
except for hydrophobic imprinted cavities/monolayer surfaces
for hydrophobic interactions. Rafailovich and co-workers9 built
protein-imprinted SAMs by mixing hydroxyl-terminated
alkanethiols and template proteins in a solution of water and
acetic acid followed by coadsorption of thiols and proteins on
gold-coated chips. The hydrophobic interactions still played a
dominant role. However, no progress on molecular imprinting
with SAMs for protein recognition has been made since then.
Herein, we designed a disulfide molecule containing an
oligoethylene glycol (OEG) terminal group and two amide
groups (DHAP) (synthesis procedures are in the Supporting
Information [SI]). OEG terminal moieties are known to be able
to resist nonspecific protein binding,10 and the amide groups
incorporated in the chains not only can function on protein
binding sites but also can strengthen the interactions between
neighboring DHAP molecules for development of imprinted
cavities. The strategy of a protein-imprinted SAM is illustrated
in Figure 1. Template proteins, DHAP, and water-soluble
functional thiols, such as glutathione (GSH, pI 5.93),
mercaptoethylamine (MEA), or thioglycolic acid (TGA),
were mixed in a water−acetic acid solution, and supramolecular
olecular imprinting allows creation of artificial recog-
Mnition sites in synthetic polymers and has received much
attention in the past four decades.1 Molecularly imprinted
polymers have considerable potential for applications in the
areas of clinic analysis, medical diagnostics, environmental
monitoring, and catalysis.2 While successful for small-template
molecules, the molecular imprinting of proteins faces several
problems related to their size, structural complexity, conforma-
tional flexibility, and compatibility with solvents.3 These
primarily include reduced mass transfer and permanent
retention of protein templates in polymer matrices and
restricted selection of aqueous media.3c,e The use of water as
solvent provides a biologically benign environment for proteins
although water can reduce hydrogen bonding and electrostatic
interactions between the template molecules and functional
monomers. Highly desirable for economical, stable, and
recyclable synthetic biological materials, protein imprinting as
synthetic antibody mimics, exhibiting excellent chemical,
mechanical, and thermal stability, could be substituted for
expensive biological antibodies used in isolation and extraction
of proteins and biosensing. Imprinting of proteins represents
one of the most challenging tasks.2c The benefits of imprinted
monolayers provide several advantages over bulk imprinted
polymers such as excellent mass transfer of molecules into and
Received: March 8, 2013
© XXXX American Chemical Society
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Communication
molar ratio of BHb to DHAP was far less than 0.1, which could
avoid protein aggregation.9 A cleaned gold disk electrode was
dipped into the solution mixture for 2.5 h to build a protein-
imprinted SAM. Imprinted sites and cavities complementary to
the template proteins were created in the SAM after removal of
the templates with 1 M NaCl.13 The differential pulse
voltammograms (DPVs) of the BHb-imprinted SAM showed
that reduction peak current of electroactive probe Fe-
4−/3−
(CN)6
significantly increased and then reached a constant
value after 21 h of elution (Figure S3a in SI). This indicated the
highly concentrated NaCl solution could efficiently remove the
templates from the protein-imprinted SAM, while a small
amount of proteins was only desorbed when washing with
double-distilled water (Figure S3b in SI). The cyclic voltammo-
grams (CVs) of the imprinted SAM exhibited typical redox
4−/3−
peaks of Fe(CN)6
after template removal (Figure 2A).
Figure 1. Preparation of a protein-imprinted SAM on a gold electrode
(not drawn to scale) and chemical structures of relevant thiols.
Figure 2. (A) CVs of the imprinted electrode (a) before and (b) after
washing and (c) upon addition of BHb (24 μg/mL): scan rate, 0.1 V/
s. (B) CVs of the nonimprinted electrode (a) before and (b) after
addition of BHb (24 μg/mL); scan rate, 0.1 V/s. (C) EIS of different
electrodes: (a) bare gold electrode; (b) imprinted electrode before
BHb removal; (c) imprinted electrode after BHb removal; (d)
imprinted electrode upon addition of BHb (24 μg/mL); (e)
nonimprinted electrode. The frequency range was between 0.01 and
100000 Hz with the signal amplitude of 5 mV. (Inset) EIS of the bare
gold electrode. (D) DPVs of different electrodes: (a) bare gold
electrode; (b) imprinted electrode after BHb removal; (c) imprinted
electrode upon addition of BHb (24 μg/mL); (d) nonimprinted
electrode; (e) DHAP-modified electrode; (f) GSH-modified electrode;
(g) imprinted electrode from BHb and DHAP after BHb removal. All
structures of the proteins with DHAP and functional thiols
were developed through hydrogen bonds and electrostatic
interactions (Figures S1 and S2 in SI, NMR and saturation
transfer difference (STD) NMR spectra). A gold-coated chip
was then immersed for coassembly of DHAP and functional
thiols together with the proteins through multiple interactions.
Free DHAP in the solution preferentially bound to surrounding
unoccupied zones of bound proteins on the chip surface for the
development of imprinted cavities relative to the functional
thiols, but could not displace the immobilized functional thiols
interacting with the proteins through multiple binding sites.
The protein-imprinted binary SAMs with multiple binding sites
and biocompatible imprinted cavities were created after
desorption of the template proteins together with removal of
unimmobilized DHAP and functional thiols. The imprinted
SAMs exhibited the ability to recognize target proteins with
excellent sensitivity and selectivity.
of the electrodes were measured in 10 mM PB (pH 7.4) containing 2.5
4−/3−
mM Fe(CN)6
and 0.1 M KCl.
The nonimprinted SAM (Figure 2B), which was prepared
exactly like the imprinted one but without protein and formed
only from DHAP and GSH, did not exhibit any electrochemical
signal. These behaviors indicate that imprinted cavities were
created in the imprinted SAM. Upon addition of BHb, the
redox currents of the imprinted electrode decreased, which
indicated BHb binding to the complementary sites and cavities
in the imprinted SAM, and the CVs of the nonimprinted
electrode remained unchanged.
Bovine hemoglobin (BHb, pI 7.0, 5.0 × 5.5 × 6.5 nm3)11 and
DHAP were dissolved in 10 mM phosphate buffer without
NaCl (PB, pH 7.4) and acetic acid, respectively. BHb, DHAP,
and GSH solutions (DHAP:GSH molar ratio of 40) were put
into a small test tube and mixed homogenously. In this
compromising dissolving process, a good solubility was
available for DHAP, and the concentration of DHAP was
about 0.1 mM, which was enough to form a stable SAM.12 The
Electrochemical impedance spectroscopy (EIS) was used to
test the performance of the imprinted SAM. The semicircle
B
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Journal of the American Chemical Society
Communication
diameter corresponds to the electron transfer resistance, Ret.
This resistance controls the electron transfer kinetics of the
redox probes at the electrode interface (Figure 2C). A bare gold
electrode exhibited an almost straight line, characteristic of a
mass diffusion-limited electron transfer process. Ret was
remarkably increased after the BHb-imprinted SAM was
created on the electrode but significantly decreased after
protein removal, whereas an increase in Ret was observed upon
binding of BHb. However, the nonimprinted counterpart
showed a maximum Ret. These results are consistent with those
of the CV measurements.
Compared with the bare gold electrode, the imprinted SAM
displayed a significant decrease in differential pulse voltamme-
try (DPV) of the reduction peak current (Figure 2D). In the
presence of BHb, the peak current was further diminished
because of protein binding to the imprinted sites and cavities.
The nonimprinted electrode showed the minimal peak current,
very comparable to that of the DHAP-modified electrode,
which indicates that the nonimprinted SAM was nearly
constituted by DHAP. This is because DHAP preferentially
bound to the gold electrode to form a stable SAM, considering
the large difference in molecular structure between DHAP and
GSH. Moreover, the GSH-modified electrode showed a larger
peak current than the imprinted SAM. It is confirmed that the
imprinted SAM was constituted by DHAP and GSH with the
imprinted cavities, which was different from the nonimprinted
one. In addition, a control SAM imprinted only from BHb and
DHAP without GSH was also prepared for comparison. The
control imprinted one without GSH displayed a larger peak
current than the imprinted SAM with GSH. This indicates that
GSH was involved in the formation of both multiple binding
sites and biocompatible imprinted cavities in the imprinted
SAM to prevent direct contact of BHb with the gold electrode
to a great extent.
target proteins could be recognized by the imprinted SAMs
with complementary binding sites and cavities. The recognition
of BHb by the imprinted SAM was further studied using
surface-enhanced resonance Raman scattering (SERRS). BHb
binding to the imprinted SAM followed by spreading of silver
nanoparticles (AgNPs) (Figure S7 in SI) modified with 2-
(iminodiacetic acid)ethanethiol in the presence of Cu2+ was
subjected to SERRS (Figure S8 in SI).
To prove recognition specificity of the BHb-imprinted
SAMs, myoglobin (Mb, pI 7.0, 2.5 × 3.5 × 4.5 nm3),14
ovalbumin (Ova, pI 4.6, 7.0 × 4.5 × 5.0 nm3),15 and lysozyme
(Lyz, pI 11.0, 3.0 × 3.0 × 4.5 nm3)16 were chosen for
comparison. These proteins have large differences in isoelectric
point and dimension. The imprinted SAMs showed a very
sensitive response to the original template BHb but were
almost insensitive to nontemplate proteins (Mb, Ova, and Lyz)
(Figure 3A). Mb and Lyz have smaller dimensions than BHb
To prove the role of the amide groups of DHAP in the
imprinted sites and cavities, the electrochemical responses of
the BHb-imprinted SAMs before and after protein removal
were compared. A significant increase in reduction peak current
was observed for the imprinted SAM prepared from BHb,
GSH, and DHAP, while a small increase occurred for the
imprinted SAM from BHb, GSH, and MDTG (amide-free in
the chain) (Figure S4 in SI). It is mostly likely that BHb could
not be easily removed from the protein-imprinted SAM due to
the hydrophobic interactions between the proteins and MDTG
hydrocarbon chains and that denaturation of proteins might be
induced by the hydrophobic interactions. This verifies that the
amide groups of DHAP were involved in the formation of
hydrogen-bonded binding sites and biocompatible imprinted
cavities.
The DPV peak current of the imprinted SAM decreased
upon addition of BHb with increasing concentration (Figure S5
in SI). Electrochemical response was defined as a change in
reduction peak current before and after protein binding (Δi).
Δi increased with the increase of BHb concentration and then
gradually approached a constant value up to about 100 μg/mL
(Figure S6a in SI). However, very subtle changes in peak
current were only observed for the nonimprinted electrode in
the range of protein concentrations, which were very similar to
those for the DHAP-modified electrode. Accordingly, the
imprinted SAMs prepared from BHb, DHAP, and other
functional thiols (MEA or TGA) and the corresponding
nonimprinted ones showed similar electrochemical response
behaviors (Figure S6b,c in SI). These results indicate that the
Figure 3. Decrease of DPV reduction peak current of the imprinted
electrodes (template protein, GSH, and DHAP) in 10 mM PB (pH
7.4) containing 2.5 mM Fe(CN)64−/3− and 0.1 M KCl as a function of
concentration of different proteins: (A) BHb; (B) no template
(nonimprinted); (C) Mb; (D) Ova; (E) Lyz; (F) BHb with FcDM as
a probe (2.5 mM). The error bars represent standard deviations for
triplicate tests.
and could enter the imprinted cavities, and Ova has dimensions
comparable to those of BHb with a slight difference and could
enter the imprinted cavities at a right orientation mode.
However, the nontemplate proteins could not be efficiently
recognized or captured by the imprinted SAMs, similar to the
nonimprinted counterparts in the presence of these proteins
(Figure 3B). These results unambiguously demonstrate that
there were binding sites on the surfaces of the imprinted SAMs
C
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Journal of the American Chemical Society
Communication
Notes
that specifically recognized the template protein. When Mb,
Ova and Lyz were respectively imprinted, similar recognition
selectivity was observed dependent on the template protein
(Figure 3C−E). In order to avoid possible influence of the
charge on the proteins on accessibility of electroactive probes
to imprinted cavities, the noncharged probe ferrocene
dimethanol (FcDM) was used for further identification of
protein recognition specificity (Figure S9 in SI and Figure 3F).
Similarly, the BHb-imprinted SAMs exhibited recognition
selectivity of BHb in comparison with nontemplate proteins.
The number of imprinted sites6b on the surfaces of the BHb-
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Natural Science
Foundation of China (No. 21273112) and Natural Science
Foundation of Jiangsu Province (BK2012719). We thank Dr.
Xiaoliang Yang at Nanjing University for her tests of 600 MHz
NMR spectra.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis details, experimental details, and supporting figures
and illustrations. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
xzdu@nju.edu.cn
D
dx.doi.org/10.1021/ja402423r | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX