In the last few decades, multifunctional magnetic nanoarchi-
tectures have attracted increasing attention due to their
wide range of applications in the biomedical fi eld. [ 1–3 ] They
allow intricate micromanipulation to be easily performed in
Benzoboroxole-Functionalized Magnetic Core/Shell Microspheres for Highly Specifi c Enrichment of Glycoproteins under Physiological Conditions
Yuting Zhang , Wanfu Ma , Dian Li , Meng Yu , Jia Guo , Changchun Wang *
Effi cient enrichment of specifi c glycoproteins from complex biological samples is of great importance towards the discovery of disease biomarkers in biological systems. Recently, phenylboronic acid-based functional materials have been widely used for enrichment of glycoproteins. However, such enrichment was mainly carried out under alkaline conditions, which is different to the status of glycoproteins in neutral physiological conditions and may cause some unpredictable degradation. In this study, on-demand neutral enrichment of glycoproteins from crude biological samples is accomplished by utilizing the reversible interaction between the cis -diols of glycoproteins and benzoboroxole-functionalized magnetic composite microspheres (Fe 3 O 4 /PAA-AOPB). The Fe 3 O 4 /PAA-AOPB composite microspheres are deliberately designed and constructed with a high-magnetic-response magnetic supraparticle (MSP) core and a crosslinked poly(acrylic acid) (PAA) shell anchoring abundant benzoboroxole functional groups on the surface. These nanocomposites possessed many merits, such as large enrichment capacity (93.9 mg/g, protein/beads), low non-specifi c adsorption, quick enrichment process (10 min) and magnetic separation speed (20 s), and high recovery effi ciency. Furthermore, the as-prepared Fe 3 O 4 /PAA-AOPB microspheres display high selectivity to glycoproteins even in the E. coli lysate or fetal bovine serum, showing great potential in the identify of low-abundance glycoproteins as biomarkers in real complex biological systems for clinical diagnoses.
Microspheres
complex biological systems, which is accomplished simply
by an external magnetic fi eld and is especially applicable to
proteomics research. [ 4,5 ] Glycosylation is recognized as one
of the most ubiquitous post-translational modifi cations of
proteins, which plays a vital role in many physiological pro-
cesses, including protein folding, [ 6 ] signal transduction, [ 7 ] cell
recognition, [ 8 ] as well as tumorigenesis. [ 9 ] To date, more than
half of the discovered cancer biomarkers are glycosylated
proteins, and the carbohydrate changes are related to the
progression of tumors. [ 10 ] Therefore, the specifi c profi ling
of endogenous glycoproteins in serum are highly valued
towards the discovery of disease biomarkers. Despite the
awareness on the importance of detecting glycoproteins
have greatly increased, it is diffi cult to analyze them due
to the co-existence of high-abundance non-glycoproteins in
complex biological samples. Thus, effi cient strategies of iso-
lation and enrichment of low-abundance glycoproteins are DOI: 10.1002/smll.201302841
Y. T. Zhang, W. F. Ma, D. Li, M. Yu, J. Guo, Prof. C. C. Wang State Key Laboratory of Molecular Engineering of Polymers Department of Macromolecular Science and Laboratory of Advanced Materials Fudan University Shanghai , 200433 , People’s Republic of China E-mail: [email protected]
posite microspheres as carrier which contain higher surface
area and easier micromanipulation should be much more
advantageous. Furthermore, magnetic microspheres would
be more convenient and practical due to the easy magnetic
separation. Consequently, combining magnetic unit and
benzoboroxoles into an integrated system for glycoprotein
enrichment in biological systems should become an indis-
pensable and ideal candidate.
Herein, a new kind of benzoboroxole-functionalized
magnetic composite microspheres (Fe 3 O 4 /PAA-AOPB) was
prepared by a novel refl ux-precipitation polymerization [ 18 ]
and a post-grafting modifi cation, and then was applied to
the enrichment of glycoproteins under neutral physiolog-
ical condition for the fi rst time. The well-designed magnetic
microspheres possess the following attractive features: (1)
The enrichment process could be completed under neutral
physiological condition, which is highly suitable for complex
biological systems because the traditional basic enrichment
gives rise to the risk of degradation of labile glycoproteins;
(2) The high grafting density of benzoboroxoles, along with
highly hydrophilic and uncharged surface leads to remark-
able selectivity and a large enrichment capacity (92.9 mg/g)
towards glycoproteins; (3) The reversible adsorption and
desorption mechanism has excellent sensitivity towards pH,
ensuring the effective enrichment with short time (10 min.)
and high recovery effi ciency; (4) A high-magnetic-response
Fe 3 O 4 core allows separation to be conveniently performed,
by simply using an external magnet (less than 20 s); (5) The
whole fabrication procedure of Fe 3 O 4 /PAA-AOPB micro-
spheres is robust, facile and time-saving. The experimental
results have demonstrated the excellent performance of
these Fe 3 O 4 /PAA-AOPB microspheres in separation of gly-
coproteins from biological systems and show great potentials
in further practical applications.
2 . Results and Discussion
The preparation procedure of composite microspheres con-
taining a Fe 3 O 4 magnetic supraparticle (MSP) core and a
polymer shell with AOPB was schematically illustrated in
Scheme 1 . Firstly, MSPs (about 200 nm) were prepared by
a modifi ed solvothermal reaction; Secondly, the MSPs were
modifi ed with MPS to form abundant available double bonds
in order to facilitate the polymer coating in the next step;
Thirdly, a layer of PAA/MBA was coated on the Fe 3 O 4 /MPS
surface by refl ux-precipitation polymerization (RPP) of AA
(monomer) and MBA (cross-linker) to form Fe 3 O 4 /PAA
core/shell microspheres; Finally, AOPB was added to react
with the carboxyl groups of AA through amidation reaction,
and the benzoboroxoles of AOPB provide abundant binding
sites to glycoproteins under physiological environment.
2.1 . Preparation of Magnetic Core/Shell Fe 3 O 4 /PAA Micro-spheres by RPP Method
Fe 3 O 4 /MPS was prepared similar with our previous
report. [ 19 ] PAA shell coated on Fe 3 O 4 /MPS was prepared by
Scheme 1. Schematic illustration of the fabrication procedures of Fe 3 O 4 /PAA-AOPB microspheres and their application in selective enrichment of glycoproteins.
small 2013, DOI: 10.1002/smll.201302841
Benzoboroxole-Functionalized Magnetic Core/Shell Microspheres
( Figure 2 a) were carried out to determine the composition
of MSPs. All the diffraction peaks in the XRD patterns
were indexed and assigned to the typical cubic structure of
Fe 3 O 4 (JCPDS 75–1609). After coated with PAA/MBA, the
composite microspheres have a smoother surface with hydro-
philic polymer shell. Meanwhile, the crystallinity of Fe 3 O 4
remains very well (Figure 2 a (ii)). The hydrodynamic diam-
eter and size distribution were determined by dynamic light
scattering (DLS). The hydrodynamic diameter (D h ) of Fe 3 O 4
is about 302 nm (Figure 1 e), which is close to the size meas-
ured by TEM. After encapsulation, the D h of Fe 3 O 4 /PAA
increased to about 621 nm, which is much larger than the size
from TEM. The dispersion of Fe 3 O 4 /PAA was demonstrated
to have excellent stability, which facilitates its further post-
modifi cation with benzoboroxoles in water. The polydisper-
sity indexes (PDI) of Fe 3 O 4 and Fe 3 O 4 /PAA are 0.123 and
0.077, respectively, indicating that all the particles are nearly
uniform.
2.2 . Fabrication of Magnetic Fe 3 O 4 /PAA-AOPB Microspheres
The as-prepared Fe 3 O 4 /PAA microspheres were further
applied to react with AOPB through amidation reaction,
forming a layer of functional groups which have high affi nity
to glycoproteins under physiological environment. The whole
synthesis process was monitored by FT-IR spectra. As shown
in Figure 2 b, the peak at 584 cm −1 appearing in all curves was
attributed to the typical stretching vibration modes of Fe-O
in Fe 3 O 4 . The new peaks at 1720 and 1530 cm −1 in Figure 2 b
(iii) were attributed to the stretching vibration of the C = O
carboxyl groups of PAA and the bending vibration of N-H in
MBA, which further proved the coating of PAA/MBA layer.
After reaction with AOPB, the appearance of bands at 1592
and 1308 cm −1 was derived from the vibrations of benzene
rings and B-O bonds, and the weakened peak of 1720 cm −1
was due to the change of carboxyl groups to amido bonds
after amidation reaction, which clearly proved that AOPB-
functionalized microspheres have been obtained. Moreover,
the “graft-on” process was characterized by monitoring the
zeta potential of microspheres in water as well. The Fe 3 O 4 /
PAA microspheres had a highly negatively charged surface
(−38 mV) and the benzoboroxole-modifi ed Fe 3 O 4 /PAA-
AOPB microspheres were almost uncharged (+0.7 mV),
which matches our expectation because the electron-neg-
ative carboxyl groups of AA were replaced by uncharged
benzoboroxole.
To quantitatively estimate the related composition of
the microspheres, thermogravimetric analysis (TGA) was
executed. The TGA curves displayed the grafted amount of
organic component in each step. As shown in Figure 2 c, the
weight loss of Fe 3 O 4 (6 wt.%) was attributed to the stabilizer
sodium citrate, and the weight loss of Fe 3 O 4 /MPS (13.6 wt.%)
was caused by sodium citrate and MPS. Their distinct mass-
loss proved the formation of MPS layer on the surface of
Fe 3 O 4 . After coating the PAA/MBA layer onto the magnetic
core, the weight loss noticeably increased to 60.2 wt.%. It is
worth mentioning that the TGA profi le for Fe 3 O 4 /PAA had
a pronounced step around 650 °C, which did not appear for
Fe 3 O 4 /MPS. We guess this peak may be attributed to the
weight loss of partial PAA chains that are directly attached
to the surface of MSPs, and the remarkably increased tem-
perature refl ects the strong interactions between the polymer
Figure 1. Representative TEM and SEM images of (a, c) Fe 3 O 4 and (b, d) Fe 3 O 4 /PAA. The scale bars for (a, b) are 200 nm, and for (c, d) are 1 μ m; DLS histograms of the microspheres (e) Fe 3 O 4 and (f) Fe 3 O 4 /PAA.
chains and the surface of MSPs. [ 19,20 ] Through post-grafting
process with AOPB, the weight loss could mainly be respon-
sible for PAA/MBA and AOPB (68.7 wt.%), and the post-
grafting AOPB was calculated to be around 21 wt.%. The
content of boron of the as-prepared Fe 3 O 4 /PAA-AOPB
microspheres was also measured by inductively coupled
plasma-atomic emission spectrometry (ICP-AES), and the
B element concentration was estimated to be 0.85 mmol g −1 ,
corresponding to 0.92 wt.%, which basically matches the
TGA results. The magnetic properties of the microspheres
were determined using vibrating sample magnetometer
(VSM). The magnetic hysteresis curves (Figure 2 d) dem-
onstrated that all the microspheres have no obvious rema-
nence or coercivity at 300 K, indicating that they are all
superparamagnetic. As a comparison, the saturation mag-
netization (Ms) value of Fe 3 O 4 was measured, and it reached
74.3 emu/g. After modifi ed by MPS, the Ms value decreased
slightly to about 69.5 emu/g. Upon the encapsulation of
PAA/MBA layer and the post-grafting of AOPB, the Ms
value strikingly reduced to 33.3 and 24.2 emu/g, respectively.
These results supplementally verifi ed the magnetic content
from TGA analysis. The magnetic susceptibility of the fi nal
product Fe 3 O 4 /PAA-AOPB is strong enough to facilitate the
quick separation of particles from solution (within 20 s) using
a magnet (Supporting Information Figure S1).
2.3 . Applications in Separation and Enrichment of Model Glycoproteins
Benzoboroxoles are a unique class of boronic acids which
display excellent water solubility and improved sugar binding
capacity in neutral water. [ 22 ] They have been used as binding
ligands for the detection of saccharide and oligosaccharide. [ 23 ]
Fe 3 O 4 /PAA with a low crosslinking degree
(20%) possessed high water-dispersity and
had abundant carboxyl group sites for
water-soluble AOPB to graft on, therefore
a highly hydrophilic surface with suffi cient
benzoboroxoles was obtained, and it may
greatly promote the adsorption of glyco-
proteins. This thought was then testifi ed
through the following adsorption experi-
ments, and the enrichment process was
shown in Scheme 2 . Actually, the enrich-
ment was performed based on the syner-
gistic effect of hydrophilic interaction and
targeting ligands of benzoboroxoles, the
enrichment mechanism and binding of
benzoboroxoles to glycan was illustrated
in Scheme 1 . The pKa of benzoboroxoles
is around 7.0, when the enrichment pH
was higher than 7.0, the benzoboroxole
can react with the cis -diol to form fi ve-
membered cyclic ester, and the captured
glycoprotein can be eluted from the
microspheres while lower the pH to acidic
condition. [ 16a ] The standard glycoprotein HRP and
the standard non-glycoproteins BSA, β -casein and MYO
were used to investigate the specifi city of the composite
microspheres to glycoproteins. We fi rst tested the enrich-
ment ability of Fe 3 O 4 /PAA-AOPB with purifi ed glycopro-
tein HRP, which has a molecular weight of 44 kDa. The HRP
concentration of the stock solution was 40 μ g/mL (1 mL PBS
solution containing 40 μ g HRP, pH = 7.4), and 1 mg Fe 3 O 4 /
PAA-AOPB was incubated with 500 μ L stock solution. After
incubation and magnetic separation, 10 μ L elution solution
(50% AN containing 1% TFA) was applied to elute HRP
from the microspheres. Then 10 μ L stock solution, 10 μ L
supernatant and 10 μ L eluent were collected and lyophilized
for SDS-PAGE analyses. As shown in Figure 3 a, the glyco-
protein concentration from the eluent far surpassed that in
the stock solution by comparing the difference in densities on
the SDS-PAGE gel, which meant that the glycoprotein could
be enriched and concentrated by the magnetic microspheres
from a stock solution with low-abundance glycoproteins,
Scheme 2. Schematic illustration of the detailed selective enrichment process for the glycoproteins using Fe 3 O 4 /PAA-AOPB microspheres under physiological conditions.
Figure 2. (a) XRD patterns of (i) Fe 3 O 4 , (ii) Fe 3 O 4 /PAA-AOPB; (b) FT-IR spectra, (c) TGA curves and (d) Magnetic hysteresis curves of (i) Fe 3 O 4 , (ii) Fe 3 O 4 /MPS, (iii) Fe 3 O 4 /PAA and (iv) Fe 3 O 4 /PAA-AOPB.
small 2013, DOI: 10.1002/smll.201302841
Benzoboroxole-Functionalized Magnetic Core/Shell Microspheres
which will greatly facilitate the subsequent detection. Our
experimental results proved that the binding capacity was
around 92.9 mg/g (protein/beads) measured by a microplate
reader at the wavelength of 402 nm [ 24 ] (the standard curve
was shown in Figure 2S). In addition, recycling experiments
were carried out for six times. The recyclability of Fe 3 O 4 /
PAA-AOPB was evaluated by comparing the difference in
densities of HRP each time with that in the fi rst cycle on the
SDS-PAGE gel. After six cycles, the separation capability
remained (Figure 3 b), which showed excellent recyclability of
the composite microspheres in glycoprotein separation. To further inspect the specifi city of Fe 3 O 4 /PAA-AOPB
for the enrichment of glycoproteins in the presence of large
amount of non-glycoproteins, a mixture of 5 μ g BSA, 5 μ g
MYO, 5 μ g β -casein and 5 μ g HRP were dissolved in loading
buffer (50 mM ABC containing 20% AN, pH = 9, 100 μ L)
or (10 mM PBS, pH = 7.4, 100 μ L). The enrichment results
of Fe 3 O 4 /PAA and Fe 3 O 4 /PAA-AOPB to glycoprotein HRP
and non-glycoproteins were shown in Figure 4 . When the
pH of the incubation solution was 9, the adsorption capa-
bility of Fe 3 O 4 /PAA-AOPB to HRP is excellent as can be
seen in Lanes (2–5) of Figure 4 a, only with little non-specifi c
adsorption of non-glycoproteins. However, the Fe 3 O 4 /PAA
microspheres hardly adsorb any proteins before modifi cation
with AOPB (Lanes 6–8 in Figure 4 a). Thus, the big difference
conveyed that AOPB modifi cation was very important for
the enrichment of glycoprotein with high selectivity. More-
over, when the pH decreased to 7.4, HRP was still captured
with comparable high selectivity to that under alkaline condi-
tion, showing great potential in direct separation of glycopro-
tein from complex biological samples (Figure 4 b). Then the
enrichment pH was set to 7.4 for the subsequent experiments.
In order to test the applicability of this material to different
glycoproteins, another glycoprotein RNB was added to the
mixture, and it was also effectively enriched (Figure 3S),
exhibiting the universality of this material in enriching dif-
ferent glycoproteins. To further evaluate their ability in capturing glycopro-
tein from complex samples, a mixture of HRP and BSA with
different ratio was used to test the selectivity ( Figure 5 ).
In these experiments, the HRP amount was maintained to
be 3 μ g in the stock solution, and BSA was 30, 60, 120 and
240 μ g, respectively. Due to the content of BSA was too high,
we need to adjust the concentration of each stock solution
to do SDS-PAGE (maintaining BSA to be approximated
5 μ g). As shown in Figure 5 , when the ratio of HRP to BSA
reached 1:40, the highly specifi c enrichment of glycoprotein
could be achieved. When the ratio increased to 1:80, the HRP
could still be enriched with only a little non-specifi c adsorbed
BSA, exhibiting remarkable selectivity.
2.4 . Applications in Separation and Enrichment of Glycopro-teins from Biological Samples
In addition, in order to demonstrate the practical applica-
tions of Fe 3 O 4 /PAA-AOPB, we use HRP to mix with crude
E. coli lysate, and then the separation and purifi cation of gly-
coproteins in the presence of lysate was accomplished under
physiological condition. E. coli lysate belongs to prokaryotes,
the structure of which is too simple and lack of organelles
Figure 3. SDS-PAGE analysis of (a) the enrichment of pure glycoprotein HRP (Lane 1: marker; Lane 2: HRP before enrichment with Fe 3 O 4 /PAA-AOPB; Lane 3: HRP in supernate after enrichment with Fe 3 O 4 /PAA-AOPB; Lane 4: eluate with acidic solution) and (b) the recycling property of the Fe 3 O 4 /PAA-AOPB in separation of HRP (Lane 1: marker; Lane 2–7: HRP released from the Fe 3 O 4 /PAA-AOPB reused up to six times).
Figure 4. SDS-PAGE analysis of the model proteins before and after treatment with Fe 3 O 4 /PAA-AOPB or Fe 3 O 4 /PAA in the condition of (a) pH = 9 and (b) pH = 7.4, respectively. (Lane 1: marker; Lane 2: protein mixture (BSA + HRP + β -casein + MYO) before treatment; Lane 3: the supernate of protein mixture (BSA + HRP + β -casein + MYO) after treatment with Fe 3 O 4 /PAA-AOPB; Lane 4: washing solution with the incubation solution; Lane 5: eluate with acidic solution; Lane 6: the supernate of protein mixture (BSA + HRP + β -casein + MYO) after treatment with Fe 3 O 4 /PAA; Lane 7: washing solution with the incubation solution; Lane 8: eluate with acidic solution).
Figure 5. SDS-PAGE analysis of glycoproteins separated using Fe 3 O 4 /PAA-AOPB from a mixture of HRP and BSA with different ratios (Lane 1: marker; Lane 2: the stock solution with HRP to BSA ratio of 1:10; Lane 3: eluate with acidic solution; Lane 4: the stock solution with HRP to BSA ratio of 1:20; Lane 5: eluate with acidic solution; Lane 6: the stock solution with HRP to BSA ratio of 1:40; Lane 7: eluate with acidic solution; Lane 8: the stock solution with HRP to BSA ratio of 1:80; Lane 9: eluate with acidic solution).
Figure 6. SDS-PAGE analysis of glycoproteins separated from (a) crude E. coli lysate and (b) FBS containing glycoproteins (Lane 1: marker; Lane 2: crude E. coli lysate or FBS mixed with HRP before treatment; Lane 3: the supernate of crude E. coli lysate or FBS mixed with HRP after treatment with Fe 3 O 4 /PAA-AOPB; Lane 4: washing solution with the incubation solution; Lane 5: eluate with acidic solution; Lane 6: the supernate of crude E. coli lysate or FBS mixed with HRP after treatment with Fe 3 O 4 /PAA; Lane 7: washing solution with the incubation solution, Lane 8: eluate with acidic solution).
small 2013, DOI: 10.1002/smll.201302841
Benzoboroxole-Functionalized Magnetic Core/Shell Microspheres
(NaOH) and aqueous ammonia solution (25%) were purchased from Shanghai Chemical Reagents Company and used as received. γ -methacryloxypropyltrimethoxy-silane (MPS) was supplied by Jiangsu Chen Guang Silane Coupling Reagent Co., Ltd. N,N′-methylenebisacrylamide (MBA) was obtained from Fluka and recrystallized from acetone. 2,2-azobisisobutyronitrile (AIBN) was supplied by Sinopharm Chemical Reagents Company and recrys-tallized from ethanol. 5-amino-2-hydroxymethylphenylboronic acid (AOPB) was purchased from Energy Chemical Company. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was purchased from Aladdin. Myoglobin (MYO, 95%), horse-radish peroxidase (HRP, 98%), β -casein (98%), bovine serum albumin (BSA, 95%), ribonuclease B (RNB, 95%), ammonium bicarbonate (ABC, 99.5%), acetonitrile (ACN, 99.9%) and trif-luoroacetic acid (TFA, 99.8%) were purchased from Sigma-Aldrich. Deionized water (18.4 M Ω cm) used for all experiments was obtained from a Milli-Q system (Millipore, Bedford, MA).
Preparation of Fe 3 O 4 Supraparticles : Fe 3 O 4 magnetic suprapar-ticles (MSPs) were prepared by a modifi ed solvothermal reaction according to our previous work. [ 19 ] Typically, 1.350 g of FeCl 3 ·6H 2 O, 3.854 g of NH 4 Ac and 0.4 g of sodium citrate were dissolved in 70 ml of ethylene glycol. The mixture were stirred vigorously for 1 h at 170 °C to form a homogeneous black solution, and then transferred into a 100 ml Tefl on-lined stainless-steel autoclave. The autoclave was heated to 200 °C and maintained for 16 h. Then it was cooled to room temperature. The product was washed with ethanol for several times and re-dispersed in ethanol for subsequent use.
The Fe 3 O 4 microspheres were then modifi ed with γ -methacryloxypropyltrimethoxy-silane (MPS) to form abundant double bonds on the surface. The modifi cation is generally as follows: 40 ml of ethanol, 10 ml of deionized water, 1.5 ml of NH 3 ·H 2 O and 0.6 g of MPS were added into the Fe 3 O 4 ethanol sus-pension, then the mixture were vigorously stirred for 24 h at 70 °C. The obtained product was separated by a magnet and washed with ethanol to remove excess MPS. The resultant Fe 3 O 4 /MPS were dried in a vacuum oven at 40 °C till constant weight.
Fabrication of Fe 3 O 4 /PAA Core/Shell Composite Microspheres by RPP Method : The core/shell Fe 3 O 4 /PAA microspheres were syn-thesized by a one-step refl ux-precipitation polymerization (RPP) of AA, with MBA as the cross-linker and AIBN as the initiator, in acetonitrile. Specifi cally, 200 mg of Fe 3 O 4 /MPS seed microspheres were dispersed in 160 ml acetonitrile in a dried 250 ml single-necked fl ask under ultrasonic condition for 3 min. Then a mixture of 0.8 ml of AA, 200 mg of MBA and 20 mg of AIBN were added to the fl ask to initiate the polymerization. The reaction mixture were heated from ambient temperature to the boiling state within 30 min and maintained at 110 °C for 1 h. The obtained Fe 3 O 4 /PAA microspheres were collected by magnetic separation, washed with ethanol and water for fi ve times.
Synthesis of Fe 3 O 4 /PAA-AOPB Microspheres : 5-amino-2-hy-droxymethylphenylboronic acid (AOPB) was used to react with Fe 3 O 4 /PAA microspheres through amidation reaction in water. Typ-ically, 50 mg Fe 3 O 4 /PAA was dispersed in 25 ml water containing 60 mg EDC, and the mixture were stirred for 1 h at room tempera-ture. Subsequently, 30 mg AOPB was added into the solution and the pH was adjusted to 9 with NaOH solution (0.1 M ). After stir-ring for 12 h at room temperature, the resultant Fe 3 O 4 /PAA-AOPB microspheres were collected by magnetic separation, washed with water and stored for further use.
Separation and Enrichment of Glycoprotein from Model Proteins or Real Biological Samples : The obtained Fe 3 O 4 /PAA-AOPB mag-netic microspheres were fi rst washed with ethanol for three times and then suspended in deionized water (10 mg/mL). A mixture of 5 μ g BSA, 5 μ g MYO, 5 μ g β -casein and 5 μ g HRP were dissolved in loading buffer (50 mM ABC containing 20% AN, pH = 9, 100 μ L) or (10 mM PBS, pH = 7.4, 100 μ L), then 1 mg Fe 3 O 4 /PAA-AOPB were added and incubated at room temperature for 10 min. After that, Fe 3 O 4 /PAA-AOPB with captured glycoproteins were separated from the mixed solution using an external magnet, and the protein-bonded composite microspheres were washed twice with loading buffer (100 μ L) to remove the non-specifi cally adsorbed proteins. Subsequently, the trapped glycoproteins were directly eluted from the microspheres with 50 μ L of elution solution (50% AN containing 1% TFA). The enrichment of glycoprotein from real biological samples (in the E. coli lysate or fetal bovine serum) has the same procedure as that from the model proteins. The protein solutions in each step (including the stock, supernatant, wash and elute solutions) were all collected and lyophilized for SDS-PAGE analyses. The relative amount of the proteins in supernatants before and after adsorption was compared by the difference in densities on the SDS-PAGE gel.
Characterization : High-resolution transmission electron micro-scopy (HR-TEM) images were taken on a JEM-2100F transmission electron microscope at an accelerating voltage of 200 kV. Samples dispersed at an appropriate concentration were cast onto a carbon-coated copper grid. Field-emission scanning electron microscopy (FE-SEM) was performed on a Hitachi S-4800 Scanning electron microscope at an accelerating voltage of 20 kV. Sample dispersed at an appropriate concentration was cast onto a glass sheet at room temperature and sputter-coated with gold. Magnetic characteriza-tion was carried out on a VSM on a Model 6000 physical property measurement system (Quantum, USA) at 300 K. Hydrodynamic diameter (Dh) measurements were conducted by dynamic light scat-tering (DLS) with a ZEN3600 (Malvern, UK) Nano ZS instrument using He-Ne laser at a wavelength of 632.8 nm. Fourier transform infrared spectra (FT-IR) were recorded on a Magna-550 (Nicolet, USA) spec-trometer. Spectra were scanned over the range of 400–4000 cm −1 . All of the dried samples were mixed with KBr and then compressed to form pellets. Thermogravimetric analysis (TGA) measurements were performed on a Pyris 1 TGA instrument. All measurements were taken under a constant fl ow of nitrogen of 40 mL/min. The temperature was fi rst increased from room temperature to 100 °C and held until constant weight, and then increased from 100 to 800 °C at a rate of 20 °C/min. Inductively coupled plasma-atomic emission spectrometry (ICP-AES) measurement was taken on a P-4010 instrument. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4–15% pre-cast polyacrylamide gels and Mini-Protean Tetra cell (Tanon, China). Protein concentration was obtained by measuring absorbance at 402 nm using BioTek Power Wave XS2 microplate reader.
Acknowledgements
This work was supported by National Science and Technology Key Project of China (2012AA020204), National Science Foundation of China (Grant No. 21034003, 21128001 and 51073040), and
full papersScience and Technology Commission of Shanghai (Grant Nos. 13JC1400500 and 13520720200).
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