Supporting information
Synthesis of magnetic dual-template molecularly imprinted nanoparticles for
specific removal of two high-abundant proteins simultaneously in blood plasma
Ruixia Gao1,*, Siqi Zhao1, Yi Hao1,2, Lili Zhang1,2, Xihui Cui1,2,
Dechun Liu3, Min Zhang4, Yuhai Tang1,2,*
1
Institute of Analytical Science, School of Science, Xi’an Jiaotong University, Xi’an
710049, China.
2
3
College of Pharmacy, Xi’an Jiaotong University, Xi’an 710061, China.
Department of Hepatobiliary Surgery, First Hospital of Xi’an Jiaotong University
Xi’an 710061, China
4
College of Chemistry and Chemical Engineering, Shanghai University of
Engineering Science, Shanghai 201620, China
* Corresponding authors: Tel.: +86 29 82655399; fax: +86 29 82655399.
E-mail: ruixiagao@mail.xjtu.edu.cn (R. Gao); tyh57@mail.xjtu.edu.cn (Y. Tang).
Materials
BHb, BSA, cytochrome C (Cyt c), and lysozyme (Lyz) were purchased from
Shanghai Lanji Chemicals Ltd. (3-aminopropyl)triethoxysilane (AMEO) and
octyltrimethoxysilane (OTMS) were obtained from Alfa Aesar Chemical Company.
1,6-Diaminohexane (DAH), ferric chloride hexahydrate (FeCl3·6H2O), anhydrous
sodium acetate (NaOAc), ethylene glycol (EG), ethanol, and sodium hydroxide
(NaOH) were provided by Tianjin Fuyu Chemicals Ltd. Trihydroxymethyl
aminomethane (Tris) and hydrochloric acid (HCl) were provided by Xi’an Chemicals
Ltd. The fresh bovine blood sample was the gift of a local market, which was
collected in an anticoagulant tube and stored at -20 oC. The sample should be thawed
at room temperature and mixed well before use. The highly purified water (18.0 MΩ
cm-1) was obtained from a Water-Pro water system (Axlwater Corporation,
TY10AXLC1805-2, China) and used throughout the experiments. All the chemicals
were of at least analytical grade and used as received.
Characterization
The morphology and structure of the Fe3O4-NH2 and DM-MIPs were examined
using a JEM-2100 (JEOL Co., Japan) transmission electron microscope (TEM).
Fourier transform infrared (FT-IR) analysis was recorded using a Nicolet AVATAR
330 FT-IR spectrophotometer. The identifications of the crystalline phase of
Fe3O4-NH2 and DM-MIPs were investigated by a Rigaku D/max/2500v/pc (Japan)
X-ray diffractometer with a Cu Kα source. The 2θ angles probed were from 25° to 70°
at a rate of 4° min-1. Magnetic properties were measured using a vibrating sample
magnetometer (VSM) (LDJ 9600-1, USA). Electrophoretic analysis of protein
samples was performed using regular SDS-PAGE (Bio-Rad, Hercules, CA) with 10%
running and 5% stacking gels. Proteins were stained with Coomassie Brilliant Blue
R-250.
Binding experiments
A series of binding experiments were carried out to examine the adsorption
isotherms, adsorption kinetics, and the selectivity of DM-MIPs and M-NIPs. In all the
experiments, the mass of polymers was 10 mg and the volume of the adsorption
solution was 5 mL. The mixture was incubated on a shaker, and then the polymers
were separated using an external magnetic field after adsorption. The concentrations
of protein before and after adsorbed by the resultant imprinted nanomaterials were
measured by a UV-vis spectrophotometer. The adsorption capacity (Q, mg g-1) of
protein bound to DM-MIPs or M-NIPs was calculated using equation (S1).
Q=
(Ci - Cf )V
W
(S1)
where Ci and Cf (mg mL-1) are initial and equilibrium concentration of protein, V
(mL) is the volume of protein mixture solution, and W (mg) is the weight of polymers.
The isothermal study was conducted through using different initial concentrations
(0.05 to 0.50 mg mL-1) of the mixed solution of BSA and BHb and kept shaking the
mixture for 35 min at room temperature. The kinetic study was performed by
changing the adsorption time at regular intervals from 0 to 50 min while maintaining
the protein mixture at a concentration of 0.35 mg mL-1. For selectivity experiments,
the polymers were added to different protein solutions of 0.35 mg mL-1 (BSA and
BHb, Cyt c, and Lyz) with incubating for 35 min at room temperature. The imprinting
factor (IF) and selectivity coefficient (SC) were used to evaluate the specificity of
DM-MIPs and M-NIPs towards the template proteins and competitive proteins, which
are calculated by equations (S2) and (S3).
IF =
QMIPs
QNIPs
SC =
IFTEM
IFCOM
(S2)
(S3)
Where QMIPs and QM-NIPs (mg g-1) represent the adsorption capacity of proteins for
DM-MIPs and M-NIPs. IFTEM and IFCOM are the imprinting factors for the template
and competitive protein.
Reusability and repeatability
To evaluate the reusability of DM-MIPs and M-NIPs, the procedure of
adsorption-desorption was repeated six times by using the same polymers. Typically,
10 mg of polymers were added to 5 mL of BSA and BHb mixed solution at a
concentration of 0.35 mg mL-1 (pH=7.0, 10 mM Tris-HCl) and incubated at room
temperature for 35 min. Then, DM-MIPs or M-NIPs were separated by a magnet and
the concentrations of BSA and BHb in supernatant were quantified by a UV-vis
spectrophotometer. The adsorbed polymers were eluted by 0.1 M NaOH for 3 h to
ensure complete removal of BSA and BHb. The recovered nanomaterials were used
for other five adsorption-desorption cycles, and the supernatant of every cycle was
collected and determined.
To investigate the repeatability of the resultant nanomaterials, 10 mg of each of
six batches of DM-MIPs or M-NIPs prepared on different days were added to 5 mL of
BSA and BHb mixed solution with a concentration of 0.35 mg mL-1 (pH=7.0, 10 mM
Tris-HCl). After incubation for 35 min at room temperature, DM-MIPs or M-NIPs
were isolated by a magnet and the adsorption capacity was measured by a UV-vis
spectrophotometer.
Effect of the mass ratio and amount of template and monomer
The binding capability of imprinted polymers is largely dependent on the
functional monomers which can recognize the templates through hydrogen bonds, van
der Waals forces, and hydrophobic interactions, etc. In this work, two kinds of silane
coupling agents were selected as co-monomers to interact with template proteins. One
is AMEO, whose amino groups can form hydrogen bonds with amino or carboxyl
groups of proteins. The other is OTMS which produces hydrophobic interaction
between its alkyl chains and hydrophobic part of proteins. To obtain the optimal
recognition performance, the effect of the volume ratios of AMEO and OTMS (1:0,
2:1, 1:1, 1:2, or 0:1) was investigated. As shown in Table S1, when the volume ratio
of AMEO and OTMS was 1:1, DM-MIPs exhibited the best adsorption ability to two
templates in adsorption capacity (Q) and imprinting factor (IF). Moreover, the
adsorption performance of imprinted polymers with AMEO and OTMS as
co-monomers was better than that with one monomer. This might result from that the
recognition ability of the obtained imprinted nanomaterials greatly relied on the
cooperative effect of hydrogen bonds and hydrophobic interactions from two
monomers, and the imprinting performance could achieve the best condition when
AMEO and OTMS were in the same volume.
To further optimize the conditions of polymerization, the Q and IF of DM-MIPs
and M-NIPs with different volumes of functional monomers ranging from 30 µL to
110 µL were compared. It was observed from Table 1 that the Q and IF increased
along with the increasing of the volumes of functional monomers from 30 µL to 70
µL for the augment of the amounts of recognition cavities on the imprinted layers.
However, excessive monomer (90 µL or 110 µL) might have homogeneous
self-condensation and lead to the formation of fewer recognition sites, which made a
decrease of Q and IF. Therefore, 70 µL of AMEO and 70 µL of OTMS were chosen
for preparing imprinted nanoparticles in the experiment.
The mass ratio of BHb and BSA affects the recognition capability of DM-MIPs.
It was observed from Table S2 that the QMIPs and imprinting factor (IF) of DM-MIPs
towards BHb or BSA were dependent on the mass ratio of BHb and BSA. With
increasing the mass ratio of BHb and BSA, the QMIPs and IF of DM-MIPs towards
BHb and BSA increased, suggesting the formation of larger amount of specific
recognition cavities complementary to template BHb and BSA. We also found that the
mass ratio of BHb and BSA of 1:1 had the highest QT and better QMIPs and IF towards
BHb and BSA. Therefore, the mass ratio of two template proteins of 1:1 was adopted
for preparation of imprinted nanomaterials.
The amount of template proteins was also investigated. The QMIPs increased with
the increasing of MBHb/BSA from 20 mg to 40 mg, indicating that the recognition ability
of the MIPs towards template proteins was enhanced. A higher MBHb/BSA might react
adequately with functional monomers and create enough recognition cavities, thus
QMIPs increased distinctly. However, the QMIPs remained almost unchanged with the
increasing of MBHb/BSA from 40 mg to 60 mg. The possible reason was that the space
of the MIPs layer was restricted whose thickness was approximately 5 nm. Excessive
MBHb/BSA could not increase the number of recognition cavities. The MBHb/BSA of 40
mg was chosen in this work.
Fig. S1 The size distribution histograms of Fe3O4-NH2 (A) and DM-MIPs (B).
Fig. S2
M-NIPs.
The specific adsorption capability (A) and reusability (B) of DM-MIPs and
Fig. S3 SDS-PAGE analysis to evaluate the applicability of DM-MIPs towards BSA
and BHb. Lane 0, protein molecular weight marker; Lane 1, bovine blood diluted
150-fold; Lane 2, remaining bovine blood after adsorption by DM-MIPs; Lane 3, the
elution of absorbed DM-MIPs eluted by 0.1 M sodium hydroxide.
Table S1 Effect of volume ratios and amounts of functional monomers on the
imprinting performance of DM-MIPs and M-NIPsa
Analytes
QMIPs (mg g-1)
QM-NIPs (mg g-1)
IF
BSA
28.11
8.097
3.47
BHb
40.16
12.48
3.22
BSA
45.19
9.453
4.78
BHb
49.78
12.94
3.85
BSA
44.25
8.893
4.98
BHb
73.12
13.21
5.54
BSA
25.76
8.014
3.21
BHb
74.22
13.99
5.31
BSA
21.09
7.851
2.69
BHb
46.77
13.01
3.59
BSA
21.55
7.182
3.00
BHb
35.88
11.84
3.03
BSA
32.98
7.961
4.14
4.14
BHb
49.98
12.42
4.02
BSA
44.25
8.893
4.98
BHb
73.12
13.21
5.54
BSA
34.32
8.453
4.06
BHb
62.66
12.98
4.83
BSA
28.01
7.972
3.51
BHb
41.22
12.41
3.32
VAMEO/VOTMS
1:0
2:1
1:1
1:2
0:1
VAMEO/OTMS (µL)
30
50
70
90
110
a
In this experiment, 10 mg of DM-MIPs or M-NIPs were incubated in the mixed solution of BSA
and BHb at a concentration of 0.35 mg mL-1 for 35 min.
Table S2 Effect of the mass ratio and amount of two template proteins on the
imprinting performance of DM-MIPs and M-NIPs.a
Analytes
QMIPs (mg g-1)
BSA
50.62
QT (mg g-1)c
MBSA/MBHbb
2:1
BHb
37.33
BSA
44.25
8.893
5.69
13.21
2.83
8.893
4.97
13.21
5.54
8.893
2.27
13.21
6.52
8.893
2.98
13.21
2.64
8.893
4.15
13.21
4.38
8.893
4.97
13.21
5.54
8.893
4.91
13.21
5.52
8.893
5.01
13.21
5.59
117.4
BHb
73.12
BSA
20.21
1:2
106.3
BHb
86.13
BSA
26.56
20
61.44
BHb
34.88
BSA
36.98
30
94.81
BHb
57.83
BSA
44.25
40
117.4
BHb
73.12
BSA
43.67
50
116.6
BHb
72.96
BSA
44.56
60
118.4
BHb
a
IF
87.95
1:1
MBHb/BSA
(mg)
QM-NIPs (mg
g-1)
73.81
In this experiment, 10 mg of DM-MIPs or M-NIPs were incubated in the mix solution of BSA
and BHb at a concentration of 0.35 mg mL-1 for 35 min.
bM
BSA (mg) and MBHb (mg) are the mass of the added BSA and BHb during the polymerization of
MIPs.
c Q (mg g-1) is the total adsorption capacity of DM-MIPs towards BSA and BHb.
T
Table S3 The parameters of Langmuir and Freundlich isothermal models for
DM-MIPs and M-NIPs.
DM-MIPs
Isothermal models
Langmuir
Freundlich
M-NIPs
Parameters
BHb
BSA
BHb
BSA
Qmax (mg g1)
74.07
45.31
14.38
9.713
KL (mL mg-1)
159.2
88.32
25.75
24.40
r
0.9995
0.9992
0.9979
0.9978
KF (mg g-1)
135.8
74.44
22.39
14.16
m
0.4132
0.4170
0.5182
0.4720
r
0.9417
0.9563
0.9656
0.9693
Table S4 Repeatability of DM-MIPs and M-NIPs.
Polymers
Batch
1
2
3
4
5
6
Average
QBSA (mg g-1)
44.46
44.20
44.31
44.24
44.12
44.17
44.25
RSDBSA (%)
3.5
5.9
4.6
4.1
6.6
3.6
4.7
QBHb (mg g-1)
73.01
74.54
71.98
72.65
73.42
73.12
73.12
RSDBHb (%)
4.8
5.7
3.2
2.9
5.1
6.5
5.3
QBSA (mg g-1)
8.795
8.624
8.398
9.106
9.197
9.238
8.893
RSDBSA (%)
7.1
5.3
2.2
6.0
3.7
4.8
4.6
QBHb(mg g-1)
13.11
13.32
13.16
13.04
13.41
13.22
13.21
RSDBHb (%)
5.6
4.3
3.7
6.2
6.9
3.8
5.1
DM-MIPs
M-NIPs