Supporting information
One
step
preparation
of
magnetic
imprinted
nanoparticles
adopting
dopamine-cupric ion as co-monomer for specific recognition of bovine
hemoglobin
Ruixia Gao1,*, Lili Zhang1,2, Yi Hao1,2, Xihui Cui1,2, Dechun Liu 3, Min Zhang4
and Yuhai Tang1,2,*
1
Institute of Analytical Science, School of Science, Xi’an Jiaotong University, Xi’an
710049, China.
2
College of Pharmacy, Xi’an Jiaotong University, Xi’an710061, China.
3
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 author information:
Associate prof. Ruixia Gao, Prof. Yuhai Tang, Institute of Analytical Science, School
of Science, Xi’an Jiaotong University, Xi’an710049, China.
Tel.: +86 2982655399; fax: +86 2982655399.
E-mail: ruixiagao@mail.xjtu.edu.cn (R. Gao); tyh57@mail.xjtu.edu.cn (Y. Tang).
Materials
Dopamine (DA) was purchased from Alfa Aesar Chemical Company. Sodium
acetate anhydrous (NaOAc), ferric chloride hexahydrate (FeCl3·6H2O), ethylene
glycol
(EG),
polyethylene
glycol
(PEG;
Mw
=
4000),
ethanol,
trihydroxymethylaminomethane (Tris), copper sulfatepentahydrate (CuSO4·5H2O),
L-histidine, potassium hydroxide (KOH), and acetic acid (HAc) were provided by
Xi’an Chemicals Ltd. Lysozyme (Lyz; Mw = 13.4KDa), bovine serum albumin (BSA;
Mw = 66.0KDa), and bovine hemoglobin (BHb; Mw = 64.0KDa) were obtained from
Sigma. The highly purified water (18.0 MΩ cm-1) was prepared with a WaterPro water
system (Axlwater Corporation, TY10AXLC1805-2, China) and used throughout the
experiments. The bovine blood sample was kindly gifted from the local market. All
reagents were of at least analytical grade and used without further treatment.
Experiments of adsorption capacity
To test the kinetics adsorption of MIP1 and NIP1, 10 mg of polymers were
suspended in 10 mL of BHb Tris-HCl buffer (pH = 7.0, 10 mM) solution at a
concentration of 0.50 mg mL-1. The mixture was incubated at regular time intervals
from 30 min to 300 min at room temperature, and the polymers were magnetically
isolated from the solution. Then, the concentration of BHb in the supernatant was
measured by UV-vis spectrophotometer at 405 nm.
To determine the isothermal binding capability of the adsorbents, 10 mg of MIP1 or
NIP1 was added into 10 mL of BHb Tris-HCl buffer (pH = 7.0, 10 mM) solution with
varied initial concentration (0.10-0.80 mg mL-1) of BHb and the mixture was shaked
for 180 min at room temperature. Then the adsorbents were magnetically separated
from the solution and the residue BHb was determined by UV-vis spectrometry. The
adsorption capacity (Q) of polymers was calculated by the Eq. S1.
The rebinding selectivity experiments were investigated by using BSA and Lyz as
the reference proteins during the binding process. 10 mg of MIP1 or NIP1 was placed
in a 10 mL of Tris-HCl buffer (pH = 7.0, 10 mM) solution containing 0.50 mg mL-1 of
different proteins, respectively. The mixture was stirred at room temperature for 180
min, and then the separation and detection were conducted as described earlier in the
kinetic experiment.
To estimate the reusability of MIP1 and NIP1, BHb adsorption-regeneration
procedure was repeated 5 times by using the same polymers. In brief, 10 mg of
polymers were added to 10 mL of BHb solution at a concentration of 0.50 mg mL-1
(pH = 7.0, 10 mM Tris-HCl) and incubated at room temperature for 180 min. Then,
MIP1 or NIP1 were removed by external magnetic field and the bound amount of
BHb was quantified by UV-vis spectrophotometer. The reused polymers were eluted
with L-histidine, 60/40 (v/v) ethanol/ water containing 3 M KOH, and highly purified
water to remove BHb and L-histidine. The recovered products were then reused for
adsorption of BHb for another 4 cycles, and every cycle of supernatant was collected
and determined by UV-vis spectrophotometer at about 405 nm.
Characterization
The morphology of the obtained nanomaterials was studied using a JEM-2100
(JEOLCo., Japan) transmission electron microscope (TEM). Energy dispersive X-ray
(EDX) was performed on a SS-550scanningelectronmicroscopy. Fourier transform
infrared (FT-IR) spectra were obtained via a Nicolet AVATAR 330FT-IR
spectrophotometer. The identification of the crystalline phase was carried out by a
Rigaku D/max/2500v/pc (Japan) X-ray diffractometer with Cu Kα radiation. The
magnetic properties were analyzed with a vibrating sample magnetometer (VSM)
(LDJ 9600-1, USA). The data of adsorption were measured by using a UV-2450
spectrophotometer (Shimadzu, Japan). 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 Coomasie Brilliant Blue R-250.
Equations
Q 
(C0  C )V
m
（S1）
Where C0 and C (mg mL-1) represent the initial and the free solution concentration
of the adsorbed protein, V(mL) represents the volume of the solution, and m (mg) is
the weight of the imprinted polymers.
IF 
QMIPs
QNIPs
（S2）
SC 
IFtem
IFcom
（S3）
Where QMIP and QNIP (mg g-1) represent the adsorption capacity of protein for
MIP1 and NIP1, IFtem and IFcom are the imprinting factors of template protein and
competitive protein.
t
1
t


2
Qt K 2Qe Qe
（S4）
Where Qe and Qt (mg g-1) are the amounts of BHb adsorbed onto MIP1 at
equilibrium and at time t (min), respectively; K2 (g mg-1 min-1) represents the rate
constant of pseudo-second-order adsorption, which can be calculated from the slope
and intercept of the plot of t/Qt versust.
Ce
Ce
1

+
Q
Qmax K L
Qmax
（S5）
log Q = log K F + n log Ce
（S6）
Where Q(mg g-1) is the amount of BHb bound to MIP1 at equilibrium, Qmax(mg g-1)
is the apparent maximum adsorption capacity, Ce (mg mL-1) is the free analytical
concentration at equilibrium, KL (mL mg-1) and KF (mg g-1) are the Langmuir and
Freundlich constant respectively, and n is the Freundlich exponent which represents
the heterogeneity of the system. The values of KL, Qmax and n, KF can be calculated
from the slope and intercept of the linear plotted in Ce/Q versus Ce and log Q versus
log Ce, respectively.
Fig. S1 Effect of the amount of DA (A), BHb (B), the concentration of Cu2+ (C) and
the polymerization time (D) on the imprinting performance of MIP1 and NIP1.
Fig. S2 EDX of MIP1.
Fig. S3 FT-IR spectra of Fe3O4 and MIP1.
Fig. S4 XRD patterns of Fe3O4 and MIP1.
Fig. S5 Magnetization curves of Fe3O4 and MIP1.
Fig. S6 SDS-PAGE analysis to evaluate the applicability of MIP1 towards BHb. Lane
0, marker; lane 1, bovine blood diluted 150-fold; lane 2, remaining bovine blood after
adsorption by MIP1; lane 3, the eluted solution of absorbed bovine blood.
Table S1 Comparation of this work with the reference of [27].
items
this work
[27]
Q1 (mg g-1)a
206.7
181.1
Q2 (mg g-1)b
10.80
73.89
Q3 (mg g-1)c
10.20
105.7
MM 1d
19.14
2.45
MM 2 e
20.26
1.71
Reusability (%)
91.7 f
92.9 g
a
Q1: Adsorption capacity of MIPs (BHb as template) towards BHb.
b
Q2: Adsorption capacity of MIPs (BHb as template) towards BSA.
c
Q3: Adsorption capacity of MIPs (BHb as template) towards Lyz.
d
MM 1= Q1/Q2.
eMM
2=
Q1/Q3.
f
The MIPs was reused for five times.
g
The MIPs was reused for six times.
Table S2 The adsorption capacity, imprinting factors, and selectivity coefficients of
BHb, Lyz and BSA for MIP1 and NIP1.a
a
Proteins
QMIP1 (mg g-1)
QNIP1 (mg g-1)
IF
SC
BHb
206.7 ± 10.9
48.81 ± 3.5
4.23 ± 0.49
—
Lyz
10.20 ± 0.1
6.72 ± 0.1
1.52 ± 0.04
2.78 ± 0.38
BSA
10.80 ± 0.3
7.45 ± 0.1
1.45 ± 0.06
2.92 ± 0.44
In this experiment, 10 mg of MIP1 and NIP1 was placed in a 10 mL of Tris-HCl buffer (pH = 7.0,
10 mM) solution of BHb, Lyz and BSA at a concentration of 0.50 mg mL-1 respectively at room
temperature for 180 min (n=3).