One-step preparation of phenyl boron-modified magnetic mesoporous silica for selective enrichment of cis-diol-containing substances

Article abstract of DOI:10.3390/molecules23030603

For enrichment and separation of cis-diol-containing compounds from biomatrix, a new type of magnetic nanoparticles named MS-48-PBSC, whichwas facilely prepared in a one-step heterogeneous reaction. The morphology results demonstrated that the MS-48-PBSC

Full text of DOI:10.3390/molecules23030603

molecules
Article
One-Step Preparation of Phenyl Boron-Modified
Magnetic Mesoporous Silica for Selective Enrichment
of cis-Diol-Containing Substances
Hua Fu 1,2, Jing Hu , Min Zhang *, Yuerong Wang , Hongyang Zhang and Ping Hu 1,* ID
1
3,
1
1
1
Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry & Molecular Engineering,
East China University of Science and Technology, Shanghai 200237, China; fuhua8711@126.com (H.F.);
hjhuali2012@163.com (J.H.); wangyuerong@ecust.edu.cn (Y.W.); hongyang_zhang@ecust.edu.cn (H.Z.)
School of Chemical Engineering, Qinghai University, Xining 810016, China
2
3
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science
and Technology, Shanghai 200237, China
*
Correspondence: zhangm@ecust.edu.cn (M.Z.); huping@ecust.edu.cn (P.H.); Tel.: +86-021-6425-2844 (M.Z. & P.H.)
Received: 1 February 2018; Accepted: 2 March 2018; Published: 7 March 2018
Abstract: For enrichment and separation of cis-diol-containing compounds from biomatrix, a new
type of magnetic nanoparticles named MS-48-PBSC, whichwas facilely prepared in a one-step
heterogeneous reaction. The morphology results demonstrated that the MS-48-PBSC was a spherical
nanomaterial containing a core of silica-coated magnetic particle with a diameter of about 200 nm,
and a cover layer of mesoporous silica with a thickness of approximate 50 nm. The characterization
2
−1
,
results showed that MS-48-PBSC presented a pore size of 4.2 nm, a surface area of 548 m
·
g
·
and a pore volume of 0.30 cm3
·
g
−1
−1
. The MS-48-PBSC also exhibited magnetism of 42 emu g
that contributed to the easy separation of magnetic nanomaterial within 30 s from the matrix with
the aid of the external magnetic field. In addition, the MS-48-PBSC exhibited high adsorption
capacity for adenosine, xanthosine, uridine, sialic acid, and teicoplanin with 0.60, 0.51, 0.42, 0.75,
and 1.26 mg/g, respectively, and showed a high selectivity for the cis-diol structure compounds,
relative to interferences of bovine serum albumin, guanine, uric acid, and xanthine. The recoveries of
adenosine, xanthosine, uridine, sialic acid, and teicoplanin were 71.8–114.1% with relative standard
deviation (RSD)
≤
8.6%, and the enrichment factors of them were 8–11. MS-48-PBSC exhibited quick
separation capability from matrix, high adsorption capacity and size exclusion for bovine serum
albumin, which could meet the requirements of separation and enrichment for substances with
a cis-diol structure.
Keywords: magnetic solid-phase extraction; one-step preparation; phenylboronic acid; cis-diol-
containing substance; selective enrichment
1
. Introduction
Cis-diol-containing biomolecules, such as sugars, nucleosides, and catecholamines, are closely
related to physiological functions and diseases and participate in various biochemical reactions and
life activities [ ]. The identification of these molecules is of considerable significance. Molecules
of medicine, including aminoglycoside antibiotics, flavonoid glycosides, and saponins from natural
sources, also contain the cis-diol function group [ ]. The biomolecules and drug metabolites are widely
distributed in tissue, blood, and urine [ ]. Some of these substances are present at trace levels
1,2
3
4–6
in complex matrices and difficult to detect directly. Thus, pretreatment technologies for enriching
target compounds with cis-diol structures and eliminating matrix interference should be developed.
Boron-group-functionalized materials have become the main solid-phase extractants in the separation
Molecules 2018, 23, 603; doi:10.3390/molecules23030603
www.mdpi.com/journal/molecules

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and enrichment of cis-diol compounds [7]. Given the boronic acid group is at equilibrium between the
undissociated and dissociated forms by a changing pH, the group can be used as selective adsorbent
for cis-diol compounds. Under alkaline conditions, the boron atom is complexed with oxygen and
2
3
changes from the original planar sp hybrid to the tetrahedral sp hybrid. The cis-boric acid ions
simultaneously form a five- or six-membered cyclic ester with 1,2-cis-diols. When the condition
3
2
becomes acidic, sp hybridizes back to the sp hybrid, and the cyclic ester dissociates [8–10]. Therefore,
the separation and enrichment of cis-diol compounds from complex samples by using boron-affinity
materials are highly selective and easy to operate.
To date, a variety of boron-affinity materials, such as boron-based polymer resins, molecularly
imprinted materials, magnetic nanoparticles, silica particles, monolith columns, and graphenes
have been developed. The materials have been widely applied in sample pretreatment, such as
solid-phase extraction (SPE) and dispersive extraction–adsorption. Compared with other materials,
the prominent feature of boron-based magnetic nanomaterials is that the solid–liquid separation
can be simplified by applying a magnetic field. In addition, a higher extraction efficiency and
faster desorption time can be achieved on nanoparticles compared with micron-sized particles due
to the large surface area [11–13]. Xiong and coworkers [14] developed one type of boron-affinity
magnetic nanoparticles. Ferroferric oxide magnetic nanoparticles, ethyl orthosilicate hydrolysates,
and bifunctional-molecule-modified epoxy groups were used as core, cover layer, and surface
functional groups, respectively. Amino phenylboronic acid was finally immobilized on the surface of
the magnetic nanoparticles through a ring-opening reaction. This method utilized the easy modification
of silica gel groups and thus became one of the most commonly used methods for the preparation of
boron-affinity magnetic materials. The solvothermal method is another commonly used method for
the preparation of magnetic nanoparticles by direct modification of specific active groups. Materials
with specific active groups were subsequently directly grafted using boronic acid groups for the
determination of biomolecules. Deng et al. [15] prepared amino-group-modified magnetic iron oxide
nanoparticles by using the solvothermal method and then obtained the magnetic boron-affinity
material by using a sequential two-step amide reaction with oxalyl chloride and amino phenylboronic
acid. This magnetic boron-affinity material was suitable for glycoprotein separation. In recent years,
in order to improve the extraction capacity of boron-based nanomaterials, researchers have increased
the specific surface area of nanoparticles and the surface grafting rate of the functional group.
Magnetic mesoporous nanosilica (MMNS) materials contain a core of silica-coated magnetic
practical and a cover layer of mesoporous nanosilica. As functional nanomaterials used for SPE,
MMNS possesses an easily modifiable Si–OH on the surface and uniform mesopore structure for
carrying targets. Pore size of MMNS is also easily adjustable for various applications. We propose
that modification of the mesoporous silica surface by using phenylboron to increase the loading
capacity of boron-affinity materials. In addition, the exterior mesoporous silica shell can also keep
the macromolecular interference (such as bovine serum albumin (BSA)) in the matrices out of the
interior pore walls through the adjustable mesopore diameters. This method improves the selectivity
of boron-affinity materials to the small-molecule cis-o-dihydroxy compounds.
The most common MMNS were prepared using MCM-41 and SBA-15 as their exterior shells.
The former material contains an exterior mesoporous shell within a confined molecular pore size
of 2.3–3.8 nm. Such small pore sizes were unfavorable for immobilizing phenylboronic acid groups
onto the surface of the magnetic mesoporous layer. The SBA-15 contains a mesoporous pore size
within 8–16 nm. The pore size allowed phenylboronic acid groups to be immobilizated, but such
large dimensions result in the absence of size exclusion capability for certain macromolecular
compounds, such as chorionic gonadotropin (36.7 kDa, 7.5 nm
×
3.5 nm
×
3 nm), luteinizing hormone
(
approximately 30 kDa), and a thyroid stimulating hormone (approximately 25–28 kDa). This drawback
lead to inevitable interference on the selective adsorption for small-molecule cis-diol compounds.
For the modification of phenylboron functions on the silicon surface, the common method was to
heterogeneously modify the phenylboron to the silicon material through multi-step heterogeneous

Molecules 2018, 23, 603
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reaction. However, the yield of phenylboron-based modification is low. Obviously, various factors
including reaction kinetics would affect heterogeneous reactions. In order to meet the challenge,
Xu et al. [8] repeated the grafting reaction twice to improve the yield of the boronic acid functionalized
FDU-12 mesoporous material. Xu and coworkers [16] modified the polyethyleneimine on the silicon
surface to increase the amount of modification of the boron group with a sequential four-step
heterogeneous amidation reaction. However, the purification process of the product consumed
a lot of solvent and time to clean and centrifuge the nanometer-sized product, which would easily lead
to the loss of materials.
In our previous work, a facile method was developed to prepare magnetic mesoporous silica
nanoparticles with an average pore size of ~5.0 nm by using polyethylene glycol–polylactic acid
copolymer as a template. Size exclusion for macromolecular compounds with a molecular weight
of more than 20 kDa can be performed on this nanomaterial [17]. If these magnetic mesoporous
nanoparticles are used to immobilize a phenylboronic acid silane coupling (PBSC) agent onto the
surface of a magnetic mesoporous layer, then the obtained new magnetic nanoparticles will have
appropriate pore size for size exclusion, large surface area for loading capacity, and magnetic
performance for simple process of separation of high selectivity and high capacity adsorption for
small-molecule cis-diol compounds in a complex matrix.
To prepare boron-affinity materials, the phenylboron group is normally immobilized on the silicon
material surface step-by-step by heterogeneous reaction. The low yield limited the application of
this method. In this work, we aimed to develop a simple and convenient approach to preparation of
boronic-modified MMNS named MS-48-PBSC based on a coupled reaction of a prepared PBSC agent
with Si–OH on the surface of MMSN. Then, we applied MS-48-PBSC to the adsorption and separation
of small-molecule-containing cis-diol group.
2
. Results and Discussion
2
.1. Synthesis of MS-48-PBSCP
The preparation and application of MS-48-PBSC is illustrated in Scheme 1.
Scheme 1. Cont.

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Scheme 1. Scheme of the preparation of mesoporous boronic acid-functionalized MS-48-PBSC and its
application in magnetic solid-phase extraction. Target; macromolecular compound; interfering
substance; PBA, phenylboronic acid; MS-48-PBSCP, phenylboron modified magnetic mesoporous silica
nanoparticles extractant.
Scheme 1 demonstrates the approach to preparation of MS-48-PBSC and its application process.
First, we synthesized a silane-coupling agent PBSCP by hydrosilylation reaction with triethoxysilane
and pinacol-protected 4-vinylbenzeneboronic acid as a starting reagent. The synthesis steps of PBSCP
are shown at the top of the Scheme 1. Then, we applied the Fe O particles as magnetic core and
3
4
mesoporous material MS-48 as the outer cover, which has appropriate pore size with 4.7 nm to
conjugate benzene boron silane coupling agent PBSCP. Finally, we used this new magnetic mesoporous
material to extract and enrich several cis-diol-containing substances. The separation process is shown
in the bottom of Scheme 1.
To prevent the introduced silane from affecting the stability of boron hydroxyl, we used a predesigned
pinacol-protected 4-vinyl phenylboronic acid as an initial reactant to synthesize a benzene boron silane
coupling agent with triethoxysilane through hydrosilylation reaction. Finally, MS-48-PBSCP was prepared
using a coupled reaction that immobilized the PBSCP agent onto the surface of a magnetic mesoporous layer.
The product of PBSCP contained two substitution isomers, which can react with hydroxyl on MS-48.
Hence, it is not necessary to separate them after the reaction. PBSCP was then grafted using a coupling
reaction between triethoxysilane and Si–OH in one step. The first advantage of the coupling reaction is
the extremely mild conditions and facile operation. The second advantage is that the one-step method
significantly improved the synthetic efficiency for the heterogeneous reaction, In addition, the approach can
reduce the posttreatment steps, which can not only make the product treatment simple but also reduce the
usage of organic regents.
The infrared (IR) spectra (Figure 1a) of PBSCP and MS-48-PBSCP show bands centered at 2979,
−
921, and 1356 cm , which are ascribed to the vibrations of adsorbed –CH and –CH . The vibration
3 2
at 3429 cm is ascribed to –OH, and the vibration at 1611 cm is ascribed to C=C from the benzene
rings. These results imply that the PBSCP-bonded molecules are successfully grafted onto the
MS-48 materials [18].
1
2
−
1
−1
The X-ray photoelectron spectroscopy (XPS) spectra (Figure 1b) of MS-48-PBSCP (top) and MS-48
(bottom) showed peaks centered at 714, 533, 285, 191, and 104 eV, which are the characteristic peaks
of Fe, O, C, B, and Si, respectively. The peak heights represent their relative content at the material
surface. Evidently, MS-48-PBSCP displays a higher C peak than MS-48; the peak was derived from
unhydrolyzed TEOS. The C and B content ratio in MS-48-PBSCP was in agreement with that of PBSCP,
thus demonstrating the successful combination of PBSCP with the MS-48 surface.

Molecules 2018, 23, 603
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Figure 1.
(a) Infrared spectroscopy of MS-48 (bottom), PBSCP (middle), and MS-48-PBSCP (top);
(
b) X-ray photoelectron spectroscopy curves of the mesoporous silica MS-48 and MS-48-PBSCP.
2
.2. Morphology of MS-48-PBSCP
The surface profiles of MS-48 and MS-48-PBSCP are illustrated in Figure 2a,b. According to
the scanning electron microscopy (SEM) images, the obtained microspheres exhibited a narrow
size distribution with a mean diameter of 250 nm, a spherical particle profile, uniform dispersion,
and identical bubble size.
The transmission electron microscopy (TEM) images of MS-48 and MS-48-PBSCP (Figure 2a,b,
respectively) show a magnetic core with a diameter of approximate 200 nm and porous silica shells with
a thickness of approximate 50 nm. The TEM images further show that the mesostructure is retained
after functionalization. The pore sizes are approximate 4.7 and 4.2 nm for MS-48 and MS-48-PBSCP,
respectively, as derived from the adsorption branch by using the Barrett–Joyner–Halenda (BJH)
method. MS-48-PBSCP exhibited a narrower pore size distribution than MS-48 by approximate
0.5 nm (Figure 3). This result implied that the size exclusion was effective for molecules with
a size of approximate 4 nm, such as the hormone glycoproteins mentioned in the introduction.
2
−1
The Brunauer–Emmett–Teller (BET) surface area and total pore volume were 705 cm
·
g
and
−1
g for MS-48-PBSCP, respectively. This result
.38 cm3
·
g
−1
for MS-48 and 548 cm2
·
g
−1
and 0.30 cm3
·
0
implied that even after functionalization, the MS-48-PBSCP material retained its high porosity
compared with MS-48. Moreover, the result suggested that the dense pore channels in the shell
were exits to the microsphere surface, which improved the extraction capacity of the material.
Figure 2. Scanning electron microscopy and Transmission electron microscopy (inset) images of
(
a) MS-48 and (b) MS-48-PBSCP.

Molecules 2018, 23, 603
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Figure 3. N sorption isotherm curves and pore size distribution plots for MS-48 and MS-48-PBSCP.
2
The saturation magnetization values of MS-48 and MS-48-PBSCP were approximate 44 and
−
1
42 emu
·
g
(Figure 4). When suspended homogeneously in water, both MS-48 and MS-48-PBSCP
exhibited fast sedimentation under the applied magnetic field in 30 s. Hence, it was indicated that
MS-48-PBSC had the fast separation performance.
Figure 4. Room-temperature magnetic hysteresis loops of MS-48 and MS-48-PBSCP.
2
.3. pH Investigation of Adsorption and Elution
Binding pH is an essential factor for boronate to selectively adsorb or elute cis-diol structures.
Boronate and cis-diol can complex to form a lactone structure under alkaline conditions and release
cis-diols under acidic conditions. Notably, a low pH is a critical factor for the boronate affinity of
cis-diol-containing biomolecules to ensure biological stability. The absorption capacities of MS-48-PBSC
for adenosine at different pH values were therefore measured (Figure 5). Phosphate buffer saline (PBS)
was used to adjust the pH in the 6.0–8.5 range, and hydrochloric acid–Tris buffer was used to adjust the
pH in the 8.5 to 10.0 range. Upon the addition of adenosine (Figure 5a), the adsorption capacity during
supernatant adsorption increased with increasing pH. However, when the pH exceeded 7.8, no evident
change was observed. We therefore selected pH = 7.8 as the best binding pH. The alkalescence
adsorption condition indicated that MS-48-PBSC was fit for binding cis-diol-containing biomolecules,
which usually exist in a neutral environment. During desorption (Figure 5b), we used formic acid
from pH 1.0 to 4.0 to elute the samples. Formic acid at pH 1.5 produced optimum results. Therefore,
we selected a formic acid solution with pH = 1.5 plus 40% methanol as the optimum elution conditions.

Molecules 2018, 23, 603
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Figure 5. Effect of pH on MS-48-PBSC adsorption (n = 3). (a) Loading with adenosine in alkalinity
buffer and (b) Eluting adenosine in acidic solution (n = 3).
2
.4. Adsorption Capacity Investigation
We selected uridine, xanthosine, adenosine, sialic acid, teicoplanin as model compounds for
nucleosides, monosaccharides and glycopeptide antibiotics in cis-diol-containing biomolecules to test
the extraction performance of MS-48-PBSC. The adsorption capacity for targets was determined using
high-performance liquid chromatograph (HPLC) and ultraviolet (UV)-visible (vis) spectrophotometry.
All testing results are shown in Figure 6a–d. The loading time for the binding of uridine, xanthosine,
adenosine, sialic acid, teicoplanin, and BSA to MS-48-PBSC was investigated. The results are shown in
Figure 6a. Figure 6b shows the HPLC of elution of uridine, xanthosine, and adenosine with loading
times of 2, 20, and 30 min. Figure 6c shows the UV absorption spectrum of eluted sialic acid with
loading times of 2, 20, and 30 min, and Figure 6d is the HPLC of eluted teicoplanin with loading
times of 2, 20, and 30 min. Following the addition of the tested samples to MS-48-PBSC, (Figure 6a–d),
the absorption of all samples except for BSA rapidly increased and reached a maximum value
within 30 min. This result implied the rapid and stable bindings between cis-diols and MS-48-PBSC.
Such characteristics are important features for high-throughput detection of cis-diol structures.
−
1
The maximum adsorption amount of MS-48-PBSC reached 0.60, 0.54, 0.42, 0.75, and 1.26 mg·g
for adenosine, xanthosine, uridine, sialic acid, and teicoplanin, respectively.
Figure 6.
sialic acid, teicoplanin, and bovine serum albumin; (
of the elution of xanthosine, uridine, and adenosine with different loading times; (
absorption spectrum of eluted sialic acid with different loading times; ( ) High-performance liquid
chromatograph of the elution of teicoplanin with different loading times.
(a
) Adsorption capacity at different loading times for uridine, xanthosine, adenosine,
) High-performance liquid chromatograph
) Ultraviolet
b
c
d

Molecules 2018, 23, 603
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We applied a commercial magnetic nanoparticle prepared by coating a boron-based polymer
onto the outer layer of the magnetic core for comparison. The maximum adsorption amount of
−
1
commercial nanoparticle reached 0.36, 0.48, 0.51, 0.54, and 1.02 mg
·
g
for adenosine, xanthosine,
uridine, sialic acid, and teicoplanin, respectively. These results showed that the adsorption capacity of
MS-48-PBSC was similar to that of the commercial material.
2
.5. Selectivity Investigation
We also selected BSA as biological macromolecule interference and guanine, uric acid, and xanthine as
small biological molecule interferences. The selectivity test results showed the MS-48-PBSC exhibited no
adsorption toward BSA (Figure 6a) because BSA cannot enter the channel of MS-48-PBSC owing to the size
exclusion effect. This outcome implied that the small pore entrance size of approximate 4.2 nm enabled the
exclusion of macromolecule proteins.
The HPLC of the supernatants of uridine, xanthosine, adenosine, guanine, uric acid, and xanthine
during loading, washing, and elution are shown in Figure 7. The small biological molecule interferences
of guanine, uric acid, and xanthine without cis-diol structures existed in the loading and washing
supernatants but were absent in the elution solution. Thus, the interferences were weakly adsorbed
by the porous structure and easily desorbed during the washing step. By contrast, the targets of
uridine, xanthosine, and adenosine with cis-diol structures were detected in the elution solution but
not in the loading and washing supernatants. Thus, the materials exhibited high selectivity toward
cis-diol structures due to the complex reaction. High selectivity was also derived from the mesoporous
structure exclusion. This feature is beneficial for the application of MS-48-PBSC in complex biological
samples, such as blood, urine, and tissue.
Figure 7. Selectivity toward uridine, xanthosine, adenosine, guanine, uric acid, and xanthine.
2
.6. Recovery and Enrichment Times
The recovery of the test objects in Section 2.4 enriched by the MS-48-PBSC was investigated and
the results are listed in Table 1. The recovery of three concentration levels of uridine, xanthosine,
adenosine, sialic acid and teicoprazole were 71.8–114.1% with RSD 1.5–8.6%. The results suggested
that the MS-48-PBSC could be used for separation and enrichment of cis-diol-containing compounds.
The concentrations of the five model compounds were increased after enrichment by MS-48-PBSC.
The enrichment factor reached 10, 11, 9, 10, and 9 corresponding to uridine, xanthosine, adenosine,
sialic acid and teicoplanin respectively. After enrichment, the detection limits of cis-diol-contaning
substances were decreased.

Molecules 2018, 23, 603
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Table 1. Recovery of the uridine, xanthosine, adenosine, sialic acid, and teicoplanin.
Sample
The Added Amount (µg)
.533
5.421
The found Amount (µg)
Recovery (%)
RSD (%, n = 3)
0
0.407
4.315
16.19
76.4
79.6
80.6
4.4
6.5
5.7
Uridine
2
0.09
0
.521
0.374
3.765
14.97
71.8
72.1
73.7
4.4
1.5
5.7
Xanthosine
Adenosine
Sialic acid
Teicoplanin
5.222
2
0.31
0
.493
0.423
4.413
18.01
85.9
83.6
89.2
4.4
6.5
5.7
5.279
2
0.20
0
.516
0.524
4.287
19.73
101.6
82.8
96.9
8.4
6.7
7.7
5.178
2
0.36
0
.547
0.482
6.078
19.13
88.2
114.1
94.9
7.9
8.6
6.2
5.327
0.16
2
2
.7. Comparative Results for the Different Methods
The preparation method for MS-48-PBSC proposed in this paper has advantages in minimization
of processing time and solvent consumption compared to those of heterogeneous reaction products
containing at least two steps. This was attributed to the one-step preparation approach of MS-48-PBSC
reduce the posttreatment steps. In extraction and enrichment processes, the MSPE method only used
an external magnetic field for separation and enrichment. On one hand, it reduced the costs of the
solid-phase extraction cartridge. On the other hand, the solvent usage of 1 mL for MS-48-PBSC method
was less than that of the SPE method with at least 3–5 mL. Therefore, the MS-48-PBSC was more
promising than the nanomaterials prepared with numerous heterogeneous reaction steps for extraction
and enrichment of cis-diol-containing compounds.
3
. Materials and Methods
FeCl (99.9%), ethylene glycol (98%), and sodium acetate (99.9%) were obtained from Sinopharm
3
Chemical Reagent Co. Ltd., Shanghai, China. Pluronic polyethylene glycol–polylactic acid
(
(
PEG4000-PLA800 (P848)) was obtained from Jinan Daigang Co. Ltd., Jinan, China; tetraethoxysilane
TEOS, 98%), triethoxysilane (97%), pinacol (99%) and 4-vinyl phenylboronicacid (98%) bovine
serum albumin (98%), sialic acid (98%), teicoplanin (98%), uridine, xanthosine, adenosine, guanine,
uric acid, and xanthine standards were obtained from Energy Chemica, Shanghai, China; pyrocatechol
(99.9%), hydroquinone (99.9%), and trifluoroacetic acid were obtained from Shanghai Ling Feng
Chemical Reagent Co. Ltd., Shanghai, China; Karstedt’s catalyst was obtained from Alfa Aesar,
Shanghai, China, commercial phenylboron modified magnetic nanomaterials were obtained from
Enriching Biotechnology Ltd., Shanghai, China. All chemicals and solvents were used without
◦
further purification. N adsorption-desorption isotherms were measured at 196 C using ASAP
2
2
010 gas adsorption apparatus (Micromeritics Instrument Co., Norcross, GA, USA). Field emission
scanning electron microscopy (FE-SEM) was performed on an FEI Magellan 400 electron microscope
Hillsboro, OR, USA). Transmission electron microscopy (TEM) images were taken with a JEOL
(
JEM-2100 microscope operated at 200 kV (Tokyo, Japan). The magnetic properties were determined
with a physical property measurement system (PMS-9T (EC-II), Quantum Design Company of
USA, San Diego, CA, USA). Fourier-transform infrared (FT-IR) spectrometry was carried out using
a Nicolet 380 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Nuclear magnetic
resonance (NMR) spectra was measured using an Agilent Technologies 400 MR (Santa Clara, CA, USA)
.
Exact mass was measured with a Waters ACQUITY TQD Electrospray ionization mass spectrometry

Molecules 2018, 23, 603
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(
ESI-MS) (Milford, MA, USA). Chromatography detection was carried out using a Shimadzu LC-20AT
high-performance liquid chromatograph (Tokyo, Japan).
3
.1. Synthesis of Mesoporous Silica-Coated Magnetic Oxide (MS-48)
Highly water-dispersible Fe O nanospheres were prepared using the solvothermal reaction
3
4
reported by Zhao et al. [19] Fe O @SiO microspheres were prepared using sol–gel method [20].
3
4
2
The core–shell-structured MS-48 composites were prepared using the nonionic diblock copolymer
®
Pluronic PEG(4000)–PLA(800) (P48), developed by our laboratory [16] as template. MS-48 particles
◦
with ~4.7 nm pore diameters were obtained after being refluxed in ethanol solution at 60 C for 48 h to
remove the templates, and the reflux was repeated for three times.
3
.2. Synthesis of Mesoporous Boronic-Acid-Functionalized Magnetic Oxide (MS-48-PBSCP)
-Vinyl benzene boronic acid pinacol ester was prepared according to the literature [21,22].
4
Specifically, a mixture of 4-vinyl phenylboronic acid (0.5 g) and pinacol (0.44 g) in 100 mL of
◦
dichloromethane in the presence of 20 g of 4A molecular sieves was stirred at 40 C for 3 h to give a near
1
quantitative yield of 4-vinyl benzene boronic acid pinacol ester. Analytical data for H-NMR of 4-vinyl
1
benzene boronic acid pinacol ester is given as H-NMR (CDCl ):
5
δ
: 1.36 (s, 12H), 5.31 (d, 1H, J = 8 Hz),
3
13
.83 (d, 1H, J = 16.0 Hz), 6.7 (dd, 1H), 7.41 (d, 2H, J = 8 Hz), 7.77 (d, 2H, J = 8 Hz); C-NMR (CDCl3):
36.84, 134.98, 125.46, 114.79, 114.77, 83.71, 77.00, 24.82; ESI-MS (M + H) : m/z = 231.1. Analytical data
for H-NMR of the PBSCP is given as H-NMR (CDCl ):
δ: 140.17,
+
1
1
1
δ: 0.91 (m, 2H), 1.17 (dt, 9H, J = 14 Hz and
3
J = 6 Hz), 1.38 (s, 12H), 2.71 (m, 2H), 3.68 (dm, 6H), 7.22 (m, 2H, J = 8 Hz), 7.68 (d, 2H, J = 12 Hz);
1
3
C-NMR (CDCl3):
δ
: 134.93, 136.84, 129.02, 128.21, 127.35, 127.24, 83.58, 77.06, 58.82, 58.41, 29.13, 24.85,
+
1
8.33, 12.44. ESI-MS (M + Na) : m/z = 417.2.
PBSCP was prepared according to the literature [23–25]. Specifically, 0.95 g of 4—vinyl benzene
boric acid pinacol ester and 300 ppm of Karstedt’s catalyst were dissolved in 5 mL of drying
◦
N,N-dimethylformamide (DMF). After the mixture was activated for 20 min at 50 C, 0.82 g of
◦
triethoxysilane was added dropwise to the solution and stirred for 5 h at 60 C under the protection of
nitrogen. Finally, the DMF and excess triethoxysilane were removed under vacuum, and the nearly
equivalent amount of PBSCP as a colorless oil was obtained.
MS-48 (0.15 g) and 0.2 g of Na CO were dispersed in 20 mL of a mixed solution of toluene and
2
3
DMF (v:v = 2:3). After ultrasonic treatment for 30 min, 0.5 g of PBSCP was added to the solution.
◦
The mixture was heated to 60 C for 24 h with magnetic stirring under the protection of nitrogen.
The products were collected with a magnet and washed twice by DMF followed by three washings
◦
with toluene. Finally, product MS-48-PBSCP was dried at 60 C overnight.
3
.3. Removal of the Pinacol Protecting Group from the MS-48-PBSCP
Prior to extraction, MS-48-PBSCP was used to remove the pinacol-protecting group. In brief,
−
1
1
00 mg of MS-48-PBSCP was added into 3 mL of 1 mol L formic acid and methanol mix solution
(v:v = 1:1) for 30 min of sonication dispersion and vortex mixing. After isolation using a magnet,
the supernatant with the pinacol protecting group was abandoned. Finally, the unprotected magnetic
nanoparticle product MS-48-PBSC was washed with 3 mL of 50% methanol solution several times and
1
mL of phosphate-buffered saline (PBS) (pH = 7.8) several times.
3
.4. pH Optimization of the Extraction and Desorption
The pH optimization of the extraction and desorption were investigated according to the
−
1
literature [16]. Typically, an adenosine solution at 20
µg mL was selected as the test sample.
·
In the loading process, pH of the adenosine solution was adjusted with PBS buffer or hydrochloric
acid–tris buffer. After interacted with MS-48-PBSC nanoparticles for 30 s with the aids of ultrasonic
and vortex mixing, the magnetic nanoparticles were isolated by a magnet and the supernatant was
detected via HPLC. During the elution, adenosine-adsorbed MS-48-PBSC was eluted with different

Molecules 2018, 23, 603
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concentrations of formic acid solution and 40% methanol as elution solvent. The desorption time
varies from 2 min to 3 h, and the elutes were detected via HPLC.
3
.5. Enrichment of cis-Diol-Containing Compounds
Adenosine, xanthosine, and uridine solutions were used as the typical nucleoside samples,
sialic acid as the monosaccharide sample, teicoplanin as the antibiotic sample, and BSA as the
macromolecular sample. Small biological molecules guanine, uric acid, and xanthine were selected as
−
1
interferences samples. All samples were prepared as stock solutions at 1 mg·mL
.
For evaluation of the adsorption capacity of MS-48-PBSC, the above-mentioned cis-diol
structure-containing samples except teicoplanin and BSA were formulated into 100
−
1
µg
·
mL
−
1
(The concentration of teicoplanin was 200
µg
·
mL ) solutions with PBS (pH = 7.8) buffer, respectively,
and then 100 mg MS-48-PBSC was added. The loading and elution processes were performed using the
optimal conditions listed in Section 3.4. For the washing process, 3 mL of PBS (pH = 7.8) and acetonitrile
solution (v:v = 90:10) were added, and ultrasonic and magnetic separation steps were repeated.
Finally, the supernatants were diluted 3-fold and detected using HPLC and UV-Vis spectrophotometer.
Benzene boron modified commodity nanomaterials was activated with PBS buffer (pH = 8.5) and
5
% of cetyltrimethylammonium bromide in methanol/H O (v/v, 1:1). The following operation was
2
similar to the method used for MS-48-PBSC.
For evaluation of the selectivity to cis-diol-containing compounds, the 1 mL solution included all
−
µg mL
1
of the above nucleoside samples, and small biological molecules interferences samples at 1.7
·
with pH = 7.8, and 60 mg MS-48-PBSC was added. The eluate is finally concentrated to 100
µL.
The loading, washing, and eluting processes were performed using the above optimal conditions.
Then, the concentrated eluates were detected using HPLC and UV-Vis spectrophotometer.
For evaluation of the recovery and enrichment times, we used three nucleoside standards, sialic acid
and teicoplanin as the test samples. All of them were mixed in the three quality levels at 0.5, 5, and 20 µg
respectively. Finally, the mixed cis-diol structure sample supernatants were detected using HPLC and
UV-Vis spectrophotometer.
3
.6. HPLC and UV-Vis Methods
The chromatographic column type was a VP-ODS C18 (5
The mobile phase was methanol: 0.2% acetic acid solution (v:v = 30:70), the wavelength of diode array
µ
m, 4.6 mm
×
200 mm, Shimadzu).
−
1
detector for HPLC was 260 nm, the sample size was 10 µL, and the flow rate was 1 mL·mL
.
BSA sample supernatants were diluted to 10:1 and detected using ultraviolet (UV)–visible (vis)
spectrophotometry at 280 nm.
Sialic acid sample supernatants were detected using a procedure reported in the literature [26].
In brief, 100 µL of supernatant was added to 2 mL of a resorcinol–hydrochloric acid solution. After
boiling for 30 min, the mixture was treated in an ice-bath for 3 min. Then, 4 mL of butyl acetate–butanol
extract solution (v:v = 4:1) was added to extract the derivative. The derivative was detected using
UV–vis spectrophotometry at 580 nm. The resorcinol–hydrochloric acid solution was prepared as
−
1
−1
follows: 20 mL of 8 mol
1
·
L
HCl solution, 62.5
µL of 0.1 mol
·
L CuSO solution, and 2 mL of
4
−
1
81.6 mmol·L resorcinol solution were mixed together and diluted with deionized water to 25 mL.
Teicoplanin sample supernatants were detected according to the literature [27] by HPLC.
The chromatographic column type was VP-ODS C18 (5
phase A was PBS (pH = 6.0) and acetonitrile solution at 85:15 (v:v), while mobile phase B was PBS
pH = 6.0) and acetonitrile solution at 30:70 (v:v). The diode array detector wavelength selection of
HPLC was 260 nm, the sample size was 10 L, and the flow rate was 1 mL/min. The elution gradient
conditions for the LC mobile phase were based on A/B from 100/0 (v/v) to 70/30 (v/v) at 32 min and
2.5/37.5 (v/v) at 32 min. The total chromatographic run duration was 35 min.
µm, 4.6 mm
×
200 mm, Shimadzu). Mobile
(
µ
6

Molecules 2018, 23, 603
12 of 13
4
. Conclusions
A new mesoporous material (MS-48-PBSC) for detecting cis-diol biological substances was
developed. MS-48-PBSC with mesoporous silicon-coated Fe O @SiO as the nucleus resulted applying
3
4
2
in a material with a high specific surface area. The mesoporous size exclusion effect enabled the
exclusion of macromolecular proteins, which is beneficial for complex biological samples. Magnetism
allows the easy separation of MS-48-PBSC from a complex matrix through an external magnetic field
and reuse of material. During material preparation, the one-step immobilization of phenylboronic
acid groups on the mesoporous wall allowed for an easy, time-efficient operation. Moreover,
the phenylboronic acid group-modified mesoporous material can function at a relatively low pH range
and improve the binding selectivity and affinity toward cis-diol structures. The method can be further
applied to enrich of sugar complexes with cis-diol structures in biological samples.
Acknowledgments: This research was supported financially by the National Natural Science Foundation of China
(
Nos. 81273481 and 81202493). This is an open project of the State Key Laboratory of Quality Research in Chinese
Medicine (Macau University of Science and Technology, MUST-SKL-2016-06) funded by the Macao Science and
Technology Development Fund, Macau special administrative region.
Author Contributions: P.H. M.Z. and H.F. conceived and designed the experiments; H.F. and J.H. performed the
experiments and wrote the paper; H.Z., and Y.W. analyzed the data; M.Z. contributed to the writing of the paper.
Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Sample of the compound MS-48-PBSC is available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).