C-Reactive protein-directed immobilization of phosphocholine ligands on a solid surface

Article abstract of DOI:10.1039/c1cc15079k

The complexes of C-reactive protein (CRP) with its polymerizable phosphocholine ligands adsorbed at the styrene-water interface were polymerized. The molecularly imprinted polymer (MIP) exhibited a binding affinity for CRP comparable to that of immobilize

Full text of DOI:10.1039/c1cc15079k

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COMMUNICATION
C-Reactive protein-directed immobilization of phosphocholine ligands on
a solid surfacew
Eunjoo Kim, Hyun-Chul Kim, Se Geun Lee, Sung Jun Lee, Tae-Jung Go, Chul Su Baek and
Sang Won Jeong*
Received 16th August 2011, Accepted 27th September 2011
DOI: 10.1039/c1cc15079k
The complexes of C-reactive protein (CRP) with its polymerizable
phosphocholine ligands adsorbed at the styrene–water interface
were polymerized. The molecularly imprinted polymer (MIP)
exhibited a binding affinity for CRP comparable to that of
immobilized anti-CRP antibody. The determination of human
serum CRP using the MIP-based sandwich immunoassay has been
demonstrated.
by proteins in the subphase through electrostatic and multivalent
interactions. The rearranged fluid monolayer was immobilized
by transfer onto a hydrophobic support, but an additional
stabilization step like polymerization may be required to
ensure long-term performance because of the possible lateral
diffusion of the lipids on solid supports.4,5
Here, we describe a facile approach to the surface molecular
imprinting of proteins under biologically benign conditions by
photopolymerization of the complexes of a multivalent protein
with its polymerizable ligands adsorbed at the oil–water inter-
face. C-Reactive protein (CRP) was selected as a target protein.
CRP is a homopentameric protein of five identical non-
covalently bound subunits with each subunit possessing a
calcium-dependent binding site for phosphocholine (PC).6
It is produced in the liver and binds to PC in damaged
or pathogenic cell membranes inducing the innate immune
system response. Due to the large increase in serum CRP
levels upon inflammation, CRP has been used as a general
marker of health.7 CRP levels in human blood are usually
below 1 mg Lꢀ1 for individuals without inflammation, whereas
these values are as high as 100 mg Lꢀ1 or even higher for
patients with bacterial infections, autoimmune diseases, and
cancer. Its role as an indicator for cardiovascular diseases
is prompting much research interest.7,8 The current methods
for human CRP detection utilize immunological techniques,
such as ELISA, turbidimetry, SPR, etc.9 Owing to the high
binding affinity of PC to CRP and the well-defined multivalent
structure, several kinds of artificial CRP receptors using
PC-conjugated molecules have been prepared, including
exposed PC groups on supported lipid monolayers, an MIP
by a micro-contact method, and a PC-appended supramolecu-
lar assembly.9c,10,11
The preparation of molecularly imprinted polymers (MIPs)
with high binding affinity and selectivity is of great interest
due to their potential applications in the areas of clinical
analysis, medical diagnostics, environmental monitoring, and
drug delivery.1 In particular, considerable attention has been
focused on the molecular imprinting of biomacromolecules
like proteins as the synthetic antibody mimics exhibiting
excellent chemical, mechanical, and thermal stability could
be substituted for expensive biological antibodies used in
isolation, extraction of proteins, biosensors, and the develop-
ment of biological materials.2 In contrast to small molecules,
the molecular imprinting of proteins faces several problems
related to their size, structural complexity, conformational
flexibility, and compatibility with solvents.2b Two-dimensional
surface molecular imprinting and the epitope approach offer
routes to overcome low mass transport and poor accessibility
to the binding sites caused by the large molecular size of the
proteins.
The use of water as solvent provides a biologically friendly
environment to proteins; however, water reduces hydrogen
bonding and electrostatic interactions between the template
molecule and the functional monomer. In addition, the choice
of monomers for the preparation of MIPs is highly restricted
in aqueous solution. While most surface imprinting methods
for proteins utilize the techniques developed for bulk imprinting,
recently protein imprinting in the supported lipid monolayers
has been proposed as a biomimetic approach.3 The lateral
rearrangement of functional lipids in monolayers was directed
A novel PC-containing polymerizable ligand, 3(4)-vinylbenzyl
12-phosphorylcholinedodecanoate (VPC), was designed to
have an appropriate spacer between the ligand (PC) and the poly-
merizable group (styrene). As shown by the structural model
of the CRP–VPC complex in Fig. 1(a), the spacer would allow
the protein parts to remain in the aqueous environment when
the pendant styrene groups are adsorbed to the water-insoluble
monomer phase (e.g., a mixture of styrene and divinylbenzene).
The hydrophobicity of the spacer would facilitate the adsorption
of the CRP–VPC complex. The synthesis of VPC was straight-
forward starting from 12-hydroxydodecanoic acid (Fig. 1(b)).
Nano & Bio Research Division, Daegu Gyeongbuk Institute of Science
and Technology (DGIST), Daegu 711-873, Korea.
E-mail: sjeong@dgist.ac.kr; Fax: +82-53-785-3559;
Tel: +82-53-785-2510
w Electronic supplementary information (ESI) available: Experimental
details and data. See DOI: 10.1039/c1cc15079k
c
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Fig. 2 CRP rebinding to the MIP, the NIP, and the CP determined
by ELISA (a) and MSIA-1 (b) ([CRP] = 6.7 nM). Each column
represents the mean value of three experiments with error bars
indicating ꢁSD.
the absorbance of the MIP, the NIP, and the CP at 450 nm
was 0.749 ꢁ 0.094, 0.478 ꢁ 0.090, and 0.433 ꢁ 0.087, respectively
(Fig. 2(b)). Although BSA was reported to be a good blocking
agent to prevent protein adsorption on polystyrene surfaces,12
there appeared to be nonspecifically bound CRP and anti-
CRP-HRP on the surfaces that could cause the background
signal. After background correction by subtraction of the
absorbance of the CP, the absorbance ratio of the MIP to
the NIP was obtained as 7.0, which almost coincided with the
IF value by ELISA.
Fig. 1 (a) A structural model of the CRP–VPC complex generated
using CAChe. (b) Synthetic scheme of VPC. (c) Schematic illustration
of preparation of MIP.
The PC moiety was introduced by use of 2-chloro-2-oxo-
1,3,2-dioxaphospholane (COP), followed by ring-opening with
trimethylamine. The chemical structure was confirmed by
1H NMR, 13C NMR, FT-IR, and ESI-MS (ESIw). The MIP
for CRP was prepared by photopolymerization of a mixture of
styrene and DVB in the presence of the CRP–VPC complex in
standard buffer (Fig. 1(c)). CRP was reacted with VPC in a
1 : 5 ratio at the concentration of VPC (0.5 mM) lower than
its critical micelle concentration (45 mM) (Fig. S1, ESIw).
The polymerization was performed initially at 37 1C for 2 h
to maintain physiological conditions and proceeded further at
90 1C for 2 h to get a solid film. The template CRP was
dissociated from the MIP by EDTA in elution buffer (0.1 M
Tris/HCl, 150 mM NaCl, 10 mM EDTA, pH 8.0). The
polystyrene surface was then blocked with BSA to prevent
nonspecific adsorption of proteins.12 A non-imprinted polymer
(NIP) was prepared in the same manner without CRP, and a
control polymer (CP) was synthesized without both CRP and
VPC to correct background changes caused by any possible
nonspecific binding.
The imprinting effect was investigated by the release of
bound CRP to the MIP and NIP upon the addition of free
PC in solution (Fig. S2, ESIw).6a At the PC concentration of
0.1 mM and 100 mM in binding buffer, CRP binding to the
MIP was decreased to 42% and 33% of the original binding,
respectively. In sharp contrast, only 6% of the original bound
CRP remained on the NIP at 0.1 mM PC and almost all the
bound CRP was dissociated from the NIP at 100 mM PC.
Although CRP binding to the MIP and the NIP was decreased
in the presence of free PC, the IF value was increased up to 66
at 100 mM PC, indicating the strong and multivalent interactions
between CRP and surface PC groups on the MIP. To examine
the selectivity of the MIP for CRP, competitive binding with
other serum proteins was demonstrated (Fig. S3, ESIw). When
excess of other serum proteins (approximately 35-fold to CRP
in weight) was added, CRP binding to the NIP dropped
remarkably to 10% of the original binding. At the same
experimental conditions, the amount of bound CRP on the
MIP was not affected within the experimental error range.
The binding constant of CRP to the MIP was calculated
using the Langmuir adsorption model.13 The correlation of the
The CRP rebinding to the MIP was performed in binding
buffer (0.1 M Tris/HCl, 150 mM NaCl, 5 mM CaCl2, pH 8.0)
containing CRP (6.7 nM) for 30 min at room temperature.
After washing with TBST buffer (0.1% Tween 20, 10 mM
Tris/HCl, 150 mM NaCl, pH 7.5) three times and PBS twice,
CRP was liberated from the MIP in elution buffer before
ELISA analysis. The amount of CRP rebound to the MIP and
the NIP was 6.06 ꢁ 2.79 ng cmꢀ2 and 0.88 ꢁ 0.68 ng cmꢀ2
,
respectively (Fig. 2(a)). The imprinting factor (IF, CRP bound
to the MIP/CRP bound to the NIP) was calculated as 6.9.
Assuming that the outer diameter of a pentameric CRP
molecule was 10.2 nm and CRP molecules were adsorbed on
the polymer surface with a face-on orientation,6 the surface
coverage of CRP on the MIP was estimated to be about 3%.
To determine CRP bound to the MIP directly, an MIP-based
sandwich-type immunoassay (MSIA) was constructed using
the MIP instead of the capture anti-CRP antibody. With the
MSIA-1 method using anti-CRP-HRP as a detection antibody,
Fig. 3 Plot of C/R vs. C for CRP binding to the MIP. Each point
represents the mean value of three experiments with error bars
indicating ꢁSD.
c
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Table 1 Binding constants of CRP to various receptors
Receptor
Detection method
Binding constant (KA, Mꢀ1
)
Ref.
Anti-CRP antibody (immobilized)
Anti-CRP antibody (immobilized)
Exposed PC on supported lipid monolayersa
PC-appended supramolecular assembly
The MIP in this study
SPR
Fiber-optic detection
SPR
ELISA
MSIA
1.4 ꢃ 106
3.8 ꢃ 108
9.9 ꢃ 106
7.1 ꢃ 106
3.0 ꢃ 109
9a
9b
9c
11
—
a
Rabbit CRP was used.
CRP concentration and the response from the MSIA-2 is
A and avidin, and the pentavalent serum amyloid P component,
Shiga and cholera toxins.3b,15,16
expressed as the following equation (ESIw):
This work was supported by the DGIST R&D Program
(11-NB-01) of the Ministry of Education, Science and Technology
of Korea.
C
R
C
1
¼
þ
ð1Þ
Rmax Rmax ꢂ KA
Notes and references
where R is the background-corrected absorbance (Abs405nm
)
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high binding constant.
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11902 Chem. Commun., 2011, 47, 11900–11902
This journal is The Royal Society of Chemistry 2011