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Article
Molecular imprinting and cladding produces antibody mimics with
significantly improved affinity and specificity
Rongrong Xing
1
, Zhanchen Guo
1
, Haifeng Lu, Qi Zhang, Zhen Liu
⇑
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
article info
Article history:
Received 1 August 2021
Received in revised form 2 September 2021
Accepted 27 September 2021
Available online 5 October 2021
Keywords:
Molecular imprinting
Rational design
Controllable engineering
Protein
Epitope
abstract
Molecularly imprinted polymers (MIPs), as important mimics of antibodies, are chemically synthesized
by polymerization in the presence of a target compound. MIPs have found wide applications in important
fileds. However, the current molecular imprinting technology suffers from a dilemma; there is often a
compromise between the best affinity and the best specificity for MIPs prepared under optimized condi-
tions. Herein, we proposed a new strategy called molecular imprinting and cladding (MIC) to solve this
issue. The principle is straightforward; after molecular imprinting, a chemically inert cladding thinlayer
is generated to precisely cover non-imprinted area. We further proposed a special MIC approach for con-
trollably engineering protein binders. The prepared cladded MIPs (cMIPs) exhibited significantly
improved affinity and specificity. The general applicability of the proposed strategy and method was ver-
ified by engineering of cMIPs for the recognition of a variety of different proteins. The feasibility of cMIPs
for real applications was demonstrated by fluorescence imaging of cancer cells against normal cells and
immunoassay of C-peptide in human urine. This study opened up a new avenue for controllably engi-
neering protein-specific antibody mimics with excellent recognition properties, holding great prospective
in important applications such as disease diagnosis and nanomedicine.
Ó2021 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved.
1. Introduction
Molecular imprinting has developed into a practical methodol-
ogy for the preparation of synthetic binders to mimic the binding
properties of antibodies [1–6]. Via polymerization of functional
monomers and a cross-linker in the presence of a template as well
as removal of the template, binding cavities that are complemen-
tary to the template in functionality, size and shape are left behind
in the formed polymers. The obtained molecularly imprinted poly-
mers (MIPs), also called artificial antibodies or plastic antibodies,
exhibit recognition capability towards a wide range of chemical
and biological species. As compared to natural antibodies, MIPs
have demonstrated a few attractive merits, such as easy to prepare,
cost efficiency, stability, reusability, tolerance to harsh conditions
(solvent, high temperature, etc.). Due to these advantages, MIPs
have shown great potential in many application fields, such as sep-
aration [7,8], bioassays [9,10], disease diagnosis [11,12], drug
delivery [13,14], toxin neutralization [15], single cell analysis
[16], bioimaging [17,18], cancer therapy [19–21], and so on.
According to the nature of functional monomers used, the
molecular imprinting methodology can be simply divided into
three categories, including covalent imprinting, non-covalent
imprinting and hybrid imprinting. Covalent imprinting relies on
the use of a limited number of functional monomers capable of
covalently binding the template [22,23]. Non-covalent imprinting
turns to the use of a variety of functional monomers that can
non-covalently bind the template through weak interactions such
as electrostatic attraction, Van der Waals force and hydrogen bond-
ing [24,25]. While hybrid imprinting depends on the combined use
of covalent and non-covalent functional monomers in the imprint-
ing [26,27]. So far, many imprinting approaches have been devel-
oped to enhance the performance of the prepared MIPs. Some of
these methods, to name a few, epitope imprinting [28,29], post-
imprinting modification [30–32], boronate affinity-based imprint-
ing [33–36] and solid phase synthesis approach [37,38], exhibited
particular advantages and have considerably advanced the
imprinting technology and greatly widened its application scope.
However, the current molecular imprinting strategy suffers from
an apparent dilemma. It was generally believed that imprinted
cavities determine the recognition properties of prepared MIPs.
In fact, MIPs are composed of imprinted cavities as well as non-
imprinted area, and the overall binding properties are thereby
https://doi.org/10.1016/j.scib.2021.10.006
2095-9273/Ó2021 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved.
⇑
Corresponding author.
E-mail address: zhenliu@nju.edu.cn (Z. Liu).
1
These authors contributed equally to this work.
Science Bulletin 67 (2022) 278–287
Contents lists available at ScienceDirect
Science Bulletin
journal homepage: www.elsevier.com/locate/scib
determined by not only imprinted cavities but also non-imprinted
area. To obtain the best binding properties toward the template,
imprinting conditions including the nature and ratio of functional
monomers as well as the polymerization time are generally needed
to optimize. However, because non-imprinted surface is also con-
structed under the same imprinting conditions as those for the
construction of binding cavities, non-imprinted surface contains
plentiful functionalities and thereby inevitably results in apparent
non-specific adsorption. Due to this reason, conditional optimiza-
tion in conventional molecular imprinting usually fails to simulta-
neously provide the best affinity and the best specificity, but a
compromise between the two aspects.
Herein, we present a rational design and controllable engineer-
ing strategy called molecular imprinting and cladding (MIC) to
solve above key issue and to provide both the best affinity and
the best specificity simultaneously. The principle is schematically
illustrated in Scheme 1a. Different from conventional molecular
imprinting, in this new strategy, imprinted cavities are first formed
under appropriate imprinting conditions, and after that, an addi-
tional process called cladding is introduced to controllably form
a chemically inert cladding thinlayer to precisely cover the non-
imprinted surface area of the formed MIPs. After the template is
removed, cavities complementary to the template in shape, size
and functionality are well constructed and non-imprinted surface
with greatly reduced non-specific adsorption is simultaneously
formed. As such, MIPs produced by the MIC strategy, termed as
cladded molecularly imprinted polymers (cMIPs) for convenience
of comparison, can provide both the best affinity and the best
specificity simultaneously.
There are two essential prerequisites in this new imprinting
strategy: (1) the imprinting and cladding processes are compatible
to each other so that the latter cladding process will not affect the
early imprinting process, and (2) the two processes are highly con-
trollable so that the structures of imprinted cavities and the clad-
ding layer can be rationally designed and controllably
constructed. To fulfill the two preconditions, we further develop
a special method termed boronate affinity-anchored epitope-
oriented surface imprinting & cladding (BOSIC). Its principle and
procedure are showed in Scheme 1b. A C- or N-terminal
dodecapeptide of a target protein is chose as an epitope. For
C-terminal epitope, an additional lysine is introduced at its
C-terminal and then glycated with a monosaccharide such as fruc-
tose. For N-terminal epitope, its N-terminal amino group is directly
glycated. For the imprinting, a glycated epitope template is first
immobilized onto a boronic acid-functionalized substrate by boro-
nate affinity. Then four silylating reagents, i.e., aminopropyltri-
ethoxysilane (APTES), 3-ureidopropyltriethoxy-silane (UPTES),
benzyltriethoxysilane (BnTES) and isobutyltriethoxysilane (IBTES),
which can interact with amino acids via different kinds of interac-
tions, are used functional monomers while tetraethyl orthosilicate
(TEOS) is used as the cross-linker for the imprinting. The imprint-
ing process is carried out to cover about nine amino acid residuals
from the glycated end, and the imprinting conditions are opti-
mized to ensure well-formed and functionality-complementary
imprinted cavities. After that, a hydrophilic cladding thinlayer is
formed via the polymerization of only TEOS to cover three addi-
tional amino acids of the template. Since the silica thinlayer
formed by the cladding process contains no functionalities, the
non-imprinted surface of the cladding thinlayer can exhibit limited
non-specific adsorption. As compared with conventional MIPs,
cMIPs can exhibit significantly improved affinity and specificity.
In this study, we first demonstrated and verified the effectiveness
of the proposed strategy and approach using b
2
-microglobulin
(B2M) as a target protein and its glycated C-terminal epitope as
the template. To verify the general applicability of the proposed
strategy and approach, we further prepared cMIPs for the recogni-
tion of transferrin (TRF), transferrin receptor (TfR), alpha fetopro-
tein (AFP) and carcinoembryonic antigen (CEA) using their
glycated C-terminal or N-terminal epitopes as the templates. To
prove the feasibility of the cMIPs in challenging practical applica-
tions, fluorescence imaging of cancer cells against normal cells
was accomplished via targeting TfR by fluorophore-encapsulated
cMIP nanoparticles (NPs). Besides, a dual cMIPs-based surface-
enhanced Raman scattering (SERS) assay was developed to detect
C-peptide in human urine for the diagnosis of diabetes.
2. Experimental
2.1. Preparation of epitope-imprinted and cladded magnetic
nanoparticles (MNPs)
The preparation of epitope-imprinted and cladded magnetic
nanoparticles (MNPs) consisted of four steps: (1) template immo-
bilization. A 2 mg of glycated epitope template was dissolved in
2 mL of 50 mmol/L NH
4
HCO
3
buffer (pH 8.5) containing
500 mmol/L NaCl. Then, a 20-mg amount of Fe
3
O
4
@SiO
2
@2,4-
difluoro-3-formyl-phenylboronic acid (DFFPBA) MNPs was ultra-
sonically dispersed into the resulting solution, and then shaken
at room temperature (RT) for 2 h. The obtained glycated epitope-
immobilized Fe
3
O
4
@SiO
2
@DFFPBA MNPs were magnetically sepa-
rated and rinsed with 50 mmol/L NH
4
HCO
3
buffer (pH 8.5) three
times. (2) Imprinting. The collected glycated epitope-bound
Fe
3
O
4
@SiO
2
@DFFPBA MNPs were added to 145.5 mL of anhydrous
C
2
H
5
OH in a 500-mL three-neck round-bottom flask and dispersed
by ultrasonication. Then 4.5 mL of NH
3
H
2
O (28%) and 10 mL of
H
2
O were added to the flask and mechanically stirred at RT for
5 min. After that, different molar ratios of APTES, UPTES, BnTES,
IBTES and TEOS (the total volume of silylating reagents was kept
at 200
l
L) dissolved in 40 mL of anhydrous C
2
H
5
OH were added
to the flask, and then mechanically stirred at RT for an appropriate
duration. To obtain the best recognition performance, the imprint-
ing time was set at 40, 50, 60, 70 or 80 min under different mono-
mer ratios and the best imprinting time and monomer ratio were
optimized. The prepared epitope-imprinted MNPs were magneti-
cally collected. (3) Cladding. The prepared epitope-imprinted
MNPs were added to 157.2 mL of anhydrous C
2
H
5
OH in another
500-mL three-neck round-bottom flask and dispersed ultrasoni-
cally. Then 2.8 mL of NH
3
H
2
O (28%) was added to the flask and
mechanically stirred at RT for 5 min. After that, 10 mmol/L TEOS
dissolved in 40 mL of anhydrous C
2
H
5
OH was added to the flask
and mechanically stirred at RT for 10 min. The prepared epitope-
imprinted and cladded MNPs were magnetically separated, rinsed
with anhydrous C
2
H
5
OH three times, and dried at 40 °C in a vac-
uum oven overnight. (4) Template removal. The obtained
epitope-imprinted and cladded MNPs were added to 2 mL of
ACN:H
2
O:HAc = 50:49:1 (v:v) and dispersed ultrasonically, and
then shaken at RT for 20 min. This step was repeated three times.
Finally, the prepared epitope-imprinted and cladded MNPs were
magnetically collected, rinsed with H
2
O and anhydrous C
2
H
5
OH
three times each, and dried at 40 °C in a vacuum oven overnight.
For epitope-imprinted but non-cladded MNPs, the preparation
steps were the same as described above except that the glycated
dodecapeptide epitope template was replaced by the glycated non-
apeptide epitope template, and there was no cladding step. For the
corresponding non-imprinted but cladded MNPs, the preparation
steps were the same as those for the epitope-imprinted and
cladded MNPs except that no glycated epitope template was
added. For the non-imprinted and non-cladded MNPs, the prepara-
tion steps were the same as that of the non-imprinted but cladded
MNPs except that there was no cladding step.
R. Xing et al. Science Bulletin 67 (2022) 278–287
279
2.2. Preparation of TfR C-terminal epitope-imprinted and cladded
FITC-doped SiO
2
NPs
The preparation steps of TfR C-terminal epitope-imprinted and
cladded FITC-doped SiO
2
NPs were as follows: (1) template immo-
bilization. A 4-mg amount of glycated TfR C-terminal epitope
template was added to 4 mL of 1 mg/mL DFFPBA-functionalized
FITC-doped SiO
2
NPs dispersed into 50 mmol/L NH
4
HCO
3
buffer
(pH 8.5) containing 500 mmol/L NaCl in a 5-mL centrifuge tube
and shaken at RT for 2 h. The obtained glycated TfR C-terminal
epitope-immobilized DFFPBA-functionalized FITC-doped SiO
2
NPs
were separated by centrifugation, and then rinsed with 50 mmol/
LNH
4
HCO
3
buffer (pH 8.5) three times. After that, the glycated
TfR C-terminal epitope-immobilized DFFPBA-functionalized FITC-
doped SiO
2
NPs were separated again by centrifugation. (2)
Imprinting. The glycated TfR C-terminal epitope-immobilized
DFFPBA-functionalized FITC-doped SiO
2
NPs were added
to14.55 mL of anhydrous C
2
H
5
OH in a 50-mL three-neck round-
bottom flask and dispersed ultrasonically. Then 0.45 mL of
NH
3
H
2
O (28%) and 1 mL of H
2
O were added to the flask and stirred
using a magnetic stirrer at RT for 5 min. After that, different molar
ratios of APTES, UPTES, BnTES, IBTES and TEOS (the total volume of
silylating reagents was kept at 20
l
L) dissolved in 4 mL of anhy-
drous C
2
H
5
OH were added to the flask, and then stirred at RT for
an appropriate imprinting time. The prepared TfR C-terminal
epitope-imprinted FITC-doped SiO
2
NPs were separated by
centrifugation. (3) Cladding. The prepared TfR C-terminal
epitope-imprinted FITC-doped SiO
2
NPs were added to 15.72 mL
of anhydrous C
2
H
5
OH in another 50-mL three-neck round-
bottom flask and dispersed ultrasonically. Then 0.28 mL of NH
3
H
2
-
O (28%) was added to the flask and stirred at RT for 5 min. After
that, 10 mmol/L TEOS dissolved in 4 mL of anhydrous C
2
H
5
OH
was added to the flask and stirred at RT for 10 min. The prepared
TfR C-terminal epitope-imprinted and cladded FITC-doped SiO
2
NPs were separated by centrifugation. (4) Template removal. The
TfR C-terminal epitope-imprinted and cladded FITC-doped SiO
2
NPs were added to 20 mL of ACN:H
2
O:HAc = 50:49:1 (v:v) and dis-
persed ultrasonically, and then shaken at RT for 20 min. This step
was repeated three times. Finally, the prepared TfR C-terminal
epitope-imprinted and cladded FITC-doped SiO
2
NPs were sepa-
Scheme 1. Schematic of the principle and process. (a) Schematic of the principle of the molecular imprinting and cladding strategy. (b) Schematic of the principle and process
of the boronate affinity-anchored epitope-oriented surface imprinting & cladding approach.
R. Xing et al. Science Bulletin 67 (2022) 278–287
280
rated by centrifugation, and then rinsed with H
2
O and anhydrous
C
2
H
5
OH three times each. The TfR C-terminal epitope-imprinted
and cladded FITC-doped SiO
2
NPs were re-dispersed into
1PBS. For non-imprinted but cladded FITC-doped SiO
2
NPs, the
preparation steps were the same as those for the TfR C-terminal
epitope-imprinted and cladded FITC-doped SiO
2
NPs except that
no glycated epitope template was added.
2.3. Preparation of C-peptide N-terminal epitope-imprinted and
cladded Ag/PATP@SiO
2
NPs
The preparation steps of C-peptide N-terminal epitope-
imprinted and cladded Ag/PATP@SiO
2
NPs were as follows: (1)
Template immobilization. DFFPBA-functionalized Ag/PATP@SiO
2
NPs were added to 9 mL of 10 mmol/L phosphate buffer (pH 7.4)
in a 50-mL centrifuge tube and dispersed ultrasonically. 2.0 mg
of glycated C-peptide N-terminal epitope was dissolved in 2 mL
of 10 mmol/L phosphate buffer (pH 7.4), and then 1 mL of the
resulting solution was added to the centrifuge tube. After shaken
at RT for 2 h, the obtained glycated C-peptide N-terminal
epitope-immobilized Ag/PATP@SiO
2
NPs were separated by cen-
trifugation and rinsed with 10 mmol/L phosphate buffer (pH 7.4)
three times. (2) Imprinting. The glycated C-peptide N-terminal
epitope-immobilized Ag/PATP@SiO
2
NPs were added to 14.55 mL
of anhydrous C
2
H
5
OH in a 50-mL three-neck round-bottom flask
and dispersed ultrasonically. Then 0.45 mL of NH
3
H
2
O (28%) and
1mLofH
2
O were added to the flask and stirred using a magnetic
stirrer for 5 min. After that, different molar ratios of APTES, UPTES,
BnTES, IBTES, and TEOS (the total volume of silylating reagents was
kept at 20
l
L) dissolved in 4 mL of anhydrous C
2
H
5
OH were added
to the flask, and then stirred at 25 ℃for an appropriate duration.
The obtained C-peptide N-terminal epitope-imprinted Ag/PATP@
SiO
2
NPs were separated by centrifugation. (3) Cladding. The pre-
pared C-peptide N-terminal epitope-imprinted Ag/PATP@SiO
2
NPs were added to 15.72 mL of anhydrous C
2
H
5
OH in another
50-mL three-neck round-bottom flask and dispersed ultrasonically.
Then 0.28 mL of NH
3
H
2
O (28%) was added to the flask and stirred
at RT for 5 min. After that, 10 mmol/L TEOS dissolved in 4 mL of
anhydrous C
2
H
5
OH was added to the flask and stirred at RT for
10 min. The prepared C-peptide N-terminal epitope-imprinted
and cladded Ag/PATP@SiO
2
NPs were separated by centrifugation.
(4) Template removal. The obtained C-peptide N-terminal
epitope-imprinted and cladded Ag/PATP@ SiO
2
NPs were added
to 20 mL of ACN:H
2
O:HAc = 50:49:1 (v:v) and dispersed ultrason-
ically, and then shaken at RT for 20 min. This step was repeated
three times. Finally, the prepared C-peptide N-terminal epitope-
imprinted and cladded Ag/PATP@ SiO
2
NPs were separated by cen-
trifugation, and then rinsed with anhydrous C
2
H
5
OH and H
2
O three
times each. The C-peptide N-terminal epitope-imprinted and
cladded Ag/PATP@SiO
2
NPs were re-dispersed into 10 mmol/L
phosphate buffer (pH 7.4). For non-imprinted but cladded Ag/
PATP@ SiO
2
NPs, the preparation steps were the same as those
for the C-peptide N-terminal epitope-imprinted and cladded Ag/
PATP@SiO
2
NPs except that no glycated epitope template was
added.
3. Results and discussion
3.1. Selection of polymerizing reagents
In order to obtain high affinity, multiple silylating reagents with
different functional groups, which can non-covalently interact dif-
ferent moieties of peptide epitope, were used as functional mono-
mers for the imprinting in this study. These silylating reagents
were selected based on the structural characteristics of amino
acids as classified in Fig. S1 (online). APTES, which has an amino
group, can interact with acidic amino acids (group I) through elec-
trostatic attraction and hydrogen bonding donor or receptor amino
acids (group V) through hydrogen bonding; UPTES, which has a
carbamido group, can interact with basic amino acids (group II)
and group V amino acids mainly through hydrogen bonding;
BnTES, which has a phenyl group, can interact with aromatic
amino acids (group III) through
p
-
p
stacking interaction; IBTES
containing a hydrophobic group can interact with hydrophobic
amino acids (group IV) through hydrophobic interaction. In addi-
tion, TEOS, which does not have any functional groups, was
employed as a crosslinker to form a silica skeleton in the imprint-
ing process and to form a hydrophilic cladding thinlayer to cover
non-specific adsorption sites on the formed polymer.
3.2. Epitope selection and glycation
Previous studies [39,40] on epitope imprinting have indicated
that epitopes containing nine amino acid residuals can well func-
tion as unique characteristic sequences for the recognition of intact
proteins or peptides. In the proposed MIC strategy, since an addi-
tional cladding thinlayer is introduced, we extended the peptide
length to twelve amino acids. In this study, C- or N-terminal dode-
capeptides of target proteins were selected as the epitopes. Accord-
ing to the peptide sequences documented in the UniProt database,
the C-terminal dodecapeptides of human B2M, TRF and TfR, and
the N-terminal dodecapeptides of human AFP and CEA are
SQPKIVKWDRDM, SSLLEACTFRRP, LSGDVWDIDNEF, RTLHRNEY-
GIAS and KLTIESTPFNVA, respectively. These epitopes were
glycated according to the previous method [12]; that is, for the
C-terminal epitopes, a lysine was first introduced at the
C-terminal of the epitope, and then glycation was carried out by
reductive amination of amino groups on the lysine residue with
fructose; for the N-terminal epitopes, the amino group on the
N-terminal of the epitope was directly glycated with fructose.
The structures of glycated C-terminal epitopes of B2M, TRF and
TfR as well as glycated N-terminal epitopes of AFP and CEA are
shown in Fig. S2 (online).
3.3. Optimization of monomer ratio and imprinting time
For the imprinting, the monomer ratio and imprinting time
were optimized. These reagents were selected because silylating
reagents-based imprinting systems have been reported to be
highly controllable [35,36]. Because the hydrolysis speed of differ-
ent silylating reagents is different, the imprinting time was opti-
mized at each monomer ratio. Since only TEOS was used in the
cladding process, no optimization of monomer composition was
needed. According to our previous results [34], the thickness of sil-
ica layer formed by TEOS polymerization under specified condi-
tions increased linearly with polymerization time (y=
(0.04x± 0.001) + (0.51 ± 0.040), R
2
= 0.994, yin nm, xin min) within
the range of 10–60 min. Because the theoretical length of a peptide
bond is approximately 0.36 nm [41] and three additional peptide
bonds were involved in the cladding, 10 min was selected as the
cladding time. As such, the thickness of the cladding layer was
roughly estimated to be 0.91 nm according to above linear depen-
dence, and the coverage of the sequence of three amino acids by
the cladding layer was about 84%.
To compare traditional imprinting and the MIC strategy, boro-
nic acid-functionalized Fe
3
O
4
@SiO
2
MNPs were selected as a sub-
strate. Meanwhile, the C-terminal nonapeptide of the protein
B2M was selected as the epitope for the preparation of B2M-
binding MIP while the C-terminal dodecapeptide of B2M was
selected as the epitope for the preparation of B2M-binding cMIP.
TEM characterization indicated that the epitope-imprinted and
R. Xing et al. Science Bulletin 67 (2022) 278–287
281
cladded MNPs were spherical, uniform in size and about 150 nm in
diameter (Fig. S3 online).
The boronate affinity of boronic acid-functionalized Fe
3
O
4
@SiO
2
MNPs was first verified using adenosine (containing cis-diol moi-
ety), deoxyadenosine (not containing cis-diol moiety), C-terminal
epitopes of B2M, TRF and TfR, N-terminal epitopes of AFP and
CEA (not containing cis-diol moiety) and their corresponding gly-
cated counterparts (containing cis-diol moiety) as test compounds.
As showed in Fig. S4 (online), the boronic acid-functionalized
MNPs exhibited good affinity to cis-diol-containing compounds
but excluded non-cis-diol-containing compounds.
The monomer ratio and imprinting time were optimized
according to imprinting factor (IF), which is an essential parameter
calculated by the ratio of the amount of template bound by an MIP
over that by its corresponding non-imprinted polymer (NIP) pre-
pared under otherwise identical conditions. Ten monomer ratios
and five imprinting times were selected and totally fifty experi-
mental conditions were investigated. As shown in Figs. S5 and S6
(online), the best imprinting time changed when different mono-
mer ratio was used, suggesting different hydrolysis speeds of dif-
ferent monomers. To make the results easy to compare, the
results at each monomer ratio under its best imprinting time are
displayed together in Fig. 1a and b. For conventional imprinting,
as increasing the overall functional monomer ratio in the prepoly-
mer solution, the binding capability of the prepared MIP toward
the template increased within initial eight monomer ratio points,
reached the highest at the monomer ratio of APTES/UPTES/
BnTES/IBTES/TEOS of 20:20:10:30:20, and then deceased there-
after. The binding capability of the corresponding NIP toward the
template molecules, which can function as a rough index of non-
specific binding, increased within initial nine monomer ratio
points, reached the highest at the ninth point (the monomer ratio:
20:30:10:30:10) and then deceased, as increasing the overall func-
tional monomer ratio in the polymerizing solution. The depen-
dence of the binding capability of MIP and NIP on the overall
functional monomer ratio within the initial eight monomer ratio
points were positively correlated with each other, such trends
are consistent with previous observation by Baggiani and col-
leagues [42], but the last two points were exceptional, which
was due to the insufficiency and absence of cross-linker. Clearly,
if the best imprinting condition is determined in terms of the bind-
ing capability of MIP, the eighth composition should be selected;
however, the corresponding NIP will be associated with rather high
non-specific binding. To make a reasonable compromise, the best
imprinting condition is usually determined in terms of the IF value.
Thus, the best imprinting condition in conventional imprinting
strategy was found to be at the fifth point (the monomer ratio:
10:10:10:20:50), which yield the highest IF value (6.2). It is appar-
ent that such optimized imprinting condition is not real optimal,
but pseudo-optimal; it avoids high non-specific adsorption but
sacrifices the binding capability to a great extent. As contrast, for
the MIC strategy, the binding capability of the cMIP obeyed almost
identical dependence on the monomer ratio, but the binding capa-
bility of the corresponding cNIP was almost the same and very lim-
ited under all the monomer ratios. As such, the best imprinting
condition for cMIP shifted to the eighth monomer ratio point,
yielding much improved IF value (16.6), the highest binding capa-
bility (comparable to that of MIP) and significantly reduced non-
specific adsorption (as compared with MIP). Obviously, the
cladding-incorporated imprinting strategy enabled the access to
the real optimal condition, being much advantageous over conven-
tional imprinting strategy.
3.4. Affinity test
The affinity of B2M-binding MIP and cMIP prepared at each
monomer ratio under its optimal imprinting time was character-
ized and compared. For the convenience of affinity characteriza-
tion, FITC-labeled B2M C-terminal nonapeptide (FITC-
KIVKWDRDM) and dodecapeptide (FITC-SQPKIVKWDRDM) were
used as the target compounds. The binding isotherms of epitope-
imprinted MIP and cMIP were established by plotting fluorescence
intensity for the target compounds captured by the MIP or cMIP
against the logarithmic concentration of the fluorescently-labeled
epitopes. Dissociation constant (K
d
) values were determined by fit-
ting the data according to Hill function. The binding isotherms and
the K
d
values for the MIP and cMIP are show in Fig. 2a and b and
Table S1 (online), respectively. Clearly, through changing the
imprinting conditions, the binding strength of the prepared MIP
and cMIP could be improved. The K
d
value for the MIP under the
pseudo-optimal conditions in terms of the IF value was found to
be 2.00 10
7
mol/L, which is not the real best among the condi-
tions optimized (the real best is 5.39 10
9
mol/L). As contrast,
the K
d
value for the cMIP under the optimized conditions was
1.12 10
9
mol/L, which is definitely the real best result among
the conditions optimized. Even compared with the real best affin-
Fig. 1. Optimization and comparison. The absorbance of test compound bound by MIP and NIP (a) as well as cMIP and cNIP (b) prepared at different monomer compositions.
The blue heart indicates the pseudo-optimal monomer composition and the purple broken heart indicates the monomer composition yielding the highest binding capability
but also rather high nonspecific adsorption for MIP. While the red heart indicates the real optimal monomer composition for cMIP. Test compound: KIVKWDRDM (a);
SQPKIVKWDRDM (b).
R. Xing et al. Science Bulletin 67 (2022) 278–287
282
ity for MIP, the affinity for cMIP was also improved, which can be
attributed to the presence of the cladding layer.
3.5. Specificity test
The specificity of B2M-binding MIP and cMIP prepared under
the different optimal imprinting conditions at the peptide and pro-
tein levels was comparatively investigated in terms of cross-
reactivity, which is a parameter for the quantitative evaluation of
the specificity in affinity techniques. It is calculated by the ratio
of the amount of a nontarget analyte bound by an affinity reagent
to that towards its target. For the MIP prepared under the best
imprinting conditions according to the highest affinity (correspond
to the eighth monomer ratio, 20:20:10:30:20), the specificity was
rather poor at both the peptide and protein levels, yielding the
maximum cross-reactivity of 36.6% at the peptide level (Fig. 3a,
purple box) and 46.0% at the protein level (Fig. 3b, purple box).
For the MIP prepared under the pseudo-optimal conditions in
terms of the highest IF value (the fifth monomer ratio,
10:10:10:20:50), the specificity was much better, giving the max-
imum cross-reactivity of 17.9% at the peptide level (Fig. 3a, blue
box) and 19.5% at the protein level (Fig. 3b, blue box). Clearly,
the improved specificity was reached at the price of sacrificed
affinity (Fig. 2a and Table S1 online). As contrast, the specificity
for the cMIP prepared under the real optimal conditions predicted
by the highest IF value was found to be excellent, with the maxi-
mum cross-reactivity of 6.7% at the peptide level (Fig. 3a, red
box) and 6.8% at the protein level (Fig. 3b, red box).
The affinity and specificity of MIP and cMIP shown are in good
agreement with the results of conditional optimization shown in
Fig. 1. Together, these comparative investigations not only con-
firmed the dilemma encountered in conventional imprinting strat-
egy but also verified the significant superiority the MIC strategy
over the conventional strategy. Through introducing an effective
means to significantly reduce non-specific adsorption of non-
imprinted surface, the MIC strategy provided significantly
improved affinity and specificity. Also, it made the IF-based opti-
mization to reflect the real optimal imprinting conditions.
3.6. Generality test
In order to verify the general applicability of the MIC strategy,
cMIPs for the recognition of other four proteins were further
prepared. C-terminal epitopes were used to prepare TRF- and
TfR-binding cMIPs, while N-terminal epitopes were imprinted to
fabricate AFP- and CEA-binding cMIPs. In theory, a comprehensive
optimization of the silylating monomers to best complementary to
the amino acid residuals contained in each epitope peptide can
provide the best binding properties. In practice, however, this is
unnecessary, because the optimization for the imprinting of C-
terminal epitope of B2M can be used as important reference and
particularly the monomer ratios that exhibited high IF can be used
for preliminary trials. Thus, the fifth to the ninth monomer ratio,
i.e., the APTES/UPTES/BnTES/IBTES/TEOS ratios at 10:10:10:20:50,
10:20:10:20:40, 20:20:10:20:30, 20:20:10:30:20 and 20:30:
10:30:10, were selected for further optimization. For the imprint-
ing of the C-terminal epitopes of TRF and TfR, since the lengths
of the fructose-glycated epitope templates are the same as that
for the glycated C-terminal epitope template of B2M, the optimal
imprinting time should be the same under the selected monomer
ratio. Therefore, the corresponding optimal imprinting times were
set as the previous ones. The five monomer ratios and their corre-
sponding optimal imprinting times were used as the initial
imprinting conditions for further optimization for the imprinting
of C-terminal epitopes of these proteins. Fig. S7a and b (online)
show that the optimal monomer ratio for these proteins was differ-
ent, being the ratio of 10:20:10:20:40 (the imprinting time:
60 min) for TRF but the ratio of 20:20:10:30:20 (the imprinting
time: 60 min) for TfR. The specificity of the cMIPs prepared under
these optimal conditions were investigated. The maximum cross-
reactivity at the peptide level was 8.2% and 7.8% for the TRF- and
TfR-binding cMIPs, respectively (Fig. S7c and d online). While max-
imum cross-reactivity at the protein level was 8.4% and 7.9% for
the TRF- and RfR-binding cMIPs, respectively (Fig. S7e and f
online).
For the imprinting of N-terminal epitopes, no additional lysine
residue was required for the glycation process, which was different
from that for C-terminal epitopes. Since fructose-glycated
N-terminal epitopes have less length as compared with their
C-terminal counterparts, the imprinting time should be
re-optimized even the same monomer ratio is used. As shown in
Fig. S8 (online), when above five monomer ratios were used, the
optimal imprinting time for the AFP-binding cMIP was 60, 50, 50,
50 and 40 min, respectively. Because the fructose-glycated N-
terminal epitopes for different proteins are the same in length,
the same optimal imprinting times for the N-terminal epitope of
AFP were taken for the imprinting of the N-terminal epitope of
CEA. As shown in Fig. S9a and b (online), the optimal monomer
Fig. 2. Binding isotherms and affinity measurement. The concentration-dependent binding profiles of MIP (a) and cMIP (b) prepared at different monomer compositions. Test
compound: FITC-KIVKWDRDM (a); FITC-SQPKIVKWDRDM (b). The blue and red curves indicate the binding profiles obtained at the pseudo-optimal optimal monomer
composition for MIP and the real monomer compositions for cMIP, respectively. While the purple curve indicates the monomer composition yielding the highest affinity for
MIP.
R. Xing et al. Science Bulletin 67 (2022) 278–287
283
ratio was found to be 20:20:10:20:30 (the imprinting time was
50 min) and 10:20:10:20:40 (the imprinting time was 50 min)
for the AFP- and CEA-binding cMIPs, respectively. Then, the speci-
ficity of the cMIPs prepared under the optimal conditions was
investigated. The maximum cross-reactivity at the peptide level
was found to be 7.4% and 8.7% for the AFP- and CEA-binding cMIPs,
respectively (Fig. S9c and d online). While the maximum cross-
reactivity at the protein level was found to be 7.6% and 8.9% for
the AFP- and CEA-binding cMIPs, respectively (Fig. S9e and f
online).
3.7. Real-world application
Above experimental evidences well support the general appli-
cability of the proposed strategy and approach as well as excellent
specificity of prepared cMIPs. The MIC strategy and the BOSIC
approach can be easily extended to other proteins. In fact, the affin-
ity and specificity can be further improved through further opti-
mization of the monomer ratios. Since the binding properties of
the currently obtained cMIPs have been sufficient for most of
real-world applications, we did not carry out such in-depth opti-
mization in this study.
TfR is a transmembrane glycoprotein and regulates the trans-
port of iron ions into cells. TfR is usually low-expressed in normal
cells, but high-expressed in proliferative cells, such as cancer cells
[43,44]. Using TfR as a tumor biomarker, targeted fluorescence
imaging of cancer cells with fluorophore-encapsulated nanoscale
TfR-binding cMIP was carried out in this study. For this purpose,
FITC-doped SiO
2
NPs were prepared as substrate for the imprinting.
The TfR C-terminal epitope-imprinted FITC-doped SiO
2
cMIP NPs
exhibited uniform size (about 75 nm) and good dispersion
(Fig. S10 online). Although the fluorescence intensity of FITC-
doped SiO
2
cMIP NPs was reduced due to the encapsulating,
imprinting and cladding processing (Fig. S11 online), it is still
strong enough for cell imaging. The specificity of the TfR-binding
FITC-doped SiO
2
cMIP NPs was investigated. The cMIP NPs exhib-
ited excellent specificity, giving the maximum cross-reactivity of
8.8% (Fig. S12 online). Fluorescence imaging of human breast can-
cer cell MCF-7 and human hepatoma cell HepG-2 as well as their
normal counterparts including MCF-10A and L-02 cells was carried
out. As shown in Fig. 4, after stained with the FITC-doped SiO
2
cMIP
NPs, the cancer cell lines (MCF-7 and HepG-2) showed strong flu-
orescence intensity and displayed the shape profile of each cell,
but the normal cell lines (MCF-10A and L-02) showed nearly no
Fig. 3. Selectivity test at the peptide (a) and protein (b) levels. Selectivity of B2M-binding MIPs prepared at the monomer composition yielding the highest affinity but also
high non-specific adsorption (purple box) and at the pseudo-optimal monomer composition (blue box) as well as B2M-binding cMIP prepared at the optimal monomer
composition (red box).
R. Xing et al. Science Bulletin 67 (2022) 278–287
284
fluorescence signal. Meanwhile, after stained with the FITC-doped
SiO
2
cNIP NPs, both the cancer cell lines and normal cell lines
exhibited almost no fluorescence signal (Fig. S13 online). The
results indicate that the TfR-binding cMIP NPs specifically targeted
the TfR protein high-expressed on the cancer cell lines and enabled
differentiation of cancer cells from normal cells, providing a
promising cancer targeting reagents for fluorescence imaging.
We further investigated the potential of cMIPs for real-world
application by engineering two C-peptide-binding cMIPs and
developing a dual cMIPs-based C-peptide assay. C-peptide, which
is a short polypeptide containing 31 amino acids, is released into
the blood as a byproduct of the formation of insulin by the pan-
creas. The amount of C-peptide in blood or urine samples has been
employed as a more reliable indicator of the real level of insulin,
since C-peptide tends to stay in the body longer than insulin. Using
the BOSIC approach established, two types of cMIP were prepared,
including: (1) MNPs modified with C-terminal epitope of
C-peptide-binding cMIP (MNPs@cMIP), and (2) silver nanoparticles
(AgNPs) modified with the Raman reporter p-aminothiophenol
(PATP) and N-terminal epitope of C-peptide-binding cMIP
(AgNPs/PATP@cMIP). The MNPs@cMIP could specifically extract
and enrich C-peptide from complex real samples such as human
serum and urine while the AgNPs/PATP@cMIP could specifically
label the extracted C-peptide. As shown in Fig. 5a, C-peptide in real
samples was first extracted and enriched by MNPs@cMIPs, and
then labeled with AgNPs/PATP@cMIPs to form MNPs@cMIPs-C
peptide-AgNPs/PATP@cMIP sandwiches. After facile magnetic sep-
aration and removal of free AgNPs/PATP@cMIP, the sandwiches
were detected by a portable Raman spectrometer. The combination
of the dual cMIPs with ultrasensitive SERS detection provided a
straightforward immunosandwich assay. This assay exhibited
excellent specificity (Fig. 5b). C-peptide in human urine samples
was successfully detected, which could distinguish healthy indi-
vidual from diabetes patient (Fig. 5c). This part of work will be
published in details elsewhere later.
4. Conclusion
We have proposed a rational design and controllable engineer-
ing strategy (MIC) and a particular imprinting and cladding
approach (BOSIC) in this study. The MIC strategy eliminated the
key factor that affect the binding properties in conventional molec-
ular imprinting strategy, allowed for achieving the best affinity and
the best specificity at the same time. Using the BOSIC approach, the
effectiveness and general applicability of the MIC strategy have
been confirmed, through producing cMIPs for a total of six different
target proteins. The feasibility of the proposed strategy and
approach was demonstrated by specific fluorescence imaging of
cancer cells through fluorophore-doped cMIP NPs as well as dual
cMIPs-based C-peptide assay. The new strategy and approach can
greatly facilitate the development and applications of the molecu-
lar imprinting technology. Through rational design, the MIC strat-
egy can be adopted to or combined with other molecular
imprinting approach to prepared cMIPs with desirable structure
and/or functions for different target compounds. Functional mono-
mers, crosslinker and cladding reagent to be used are unnecessarily
limited to those used in this study. Therefore, the proposed strat-
egy opened a new access to engineering affinity binders with
excellent binding properties.
Fig. 4. Fluorescence imaging of cancer cells. Confocal fluorescence imaging of MCF-7, MCF-10A, HepG-2 and L-02 cells after staining with DAPI and/or TfR-specific FITC-doped
SiO
2
cMIP NPs. The concentration of the NPs was 200
l
g/mL.
R. Xing et al. Science Bulletin 67 (2022) 278–287
285
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
This work was supported by the Key Program of the National
Natural Science Foundation of China (21834003), the National
Science Fund for Distinguished Young Scholars (21425520), and
the Excellent Research Program (ZYJH004) from Nanjing University
to ZL.
Author contributions
Zhen Liu developed the concept. Rongrong Xing and Zhanchen
Guo performed most of experiments and data analysis. Haifeng
Lu helped with some experiments. Qi Zhang performed experi-
ments on C-peptide assay. Rongrong Xing and Zhanchen Guo pre-
pared draft of the manuscript. Zhen Liu finalized the paper. All
authors discussed the results and approved for the final version
of the manuscript.
Appendix A. Supplementary materials
Supplementary materials to this article can be found online at
https://doi.org/10.1016/j.scib.2021.10.006.
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Rongrong Xing received his B.S. degree (2011) and M.S.
degree (2015) from Shanxi Medical University, and
Ph.D. degree (2019) from Nanjing University. He is now
a professor at School of Pharmacy, Shanxi Medical
University. His current research interest is mainly
focused on the establishment of new molecular
imprinting strategies and their applications in phar-
maceutical analysis and rapid and accurate diagnosis.
Zhanchen Guo received his B.S. degree from Xinjiang
University in 2017. He is now a Ph.D. candidate in Prof.
Zhen Liu’s group at Nanjing University. His research
focuses on advanced functionalized nanomaterials
preparation and their biomedical applications.
Zhen Liu obtained his Ph.D. degree from Dalian Institute
of Chemical Physics, Chinese Academy of Sciences, in
1998. He was appointed as a full professor at Nanjing
University (NJU) in 2005 and promoted to Distinguished
Professor in 2014. His research interest includes
molecular recognition, affinity separation, disease
diagnosis, single cell analysis, and cancer nanotherapy.
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287
