Facile Preparation of Phenyboronic-Acid-Functionalized Fe 3 O 4 Magnetic Nanoparticles for the Selective Adsorption of Ortho-Dihydroxy-Containing Compounds

: A new facile strategy was designed to prepare the phenyboronic acid-functionalized Fe 3 O 4 magnetic nanoparticles (Fe 3 O 4 @PBA) via direct silanization and thiol-ene click chemistry for the selective adsorption of ortho-dihydroxy-containing compounds. The three kinds of Fe 3 O 4 @PBA nanoparticles obtained showed excellent adsorption capacity and selectivity for ortho-dihydroxy-containing compounds including adenosine and o -dihydroxybenzene. Among them, the Fe 3 O 4 @MPS@MPBA exhibited the highest adsorption capacity and selectivity for adenosine and o -dihydroxybenzene, followed by Fe 3 O 4 @MPTES@AAPBA and Fe 3 O 4 @MPTES@VPBA. A synthesis method of superparamagnetic and boronate afﬁnity nanocomposites with mild reaction conditions and simple process has been developed, which also provides a novel way for the synthesis of other types of enrichment materials of ortho-dihydroxy-containing compounds


Introduction
Due to the advantages of superparamagnetism, stability and low cytotoxicity [1][2][3], magnetic nanomaterials are widely used in various fields, such as pollutant detection and separation [4][5][6][7], drug delivery [8][9][10], magnetic resonance imaging [11][12][13] and biosensing [14][15][16]. Such a wide range of applications is attributed to the various functionalizations of magnetic nanomaterials, and phenylboric acid is one of the important functional modification groups. Phenylboric acid can selectively adsorb ortho-dihydroxy-containing compounds through the formation of reversible five-or six-membered cycloesters. The adsorption and desorption can be conveniently controlled by adjusting the pH value, which gives boric acid affinity materials incomparable advantages in the recognition and separation of biomolecules [17][18][19][20][21][22][23][24][25]. Accordingly, it has become a challenge to develop a facile modification strategy for the preparation of magnetic nanomaterials modified by phenylboric acid. Click chemistry has become a powerful means to solve the above challenge because of its high reaction efficiency, high reliability and good selectivity [26][27][28][29].
In this study, the direct silylation and thiol-ene click reaction were used for functionalized modification of Fe 3 O 4 magnetic nanoparticles. The adsorption capacity and selectivity of the three kinds of prepared phenyboronic-acid-functionalized Fe 3 O 4 magnetic nanoparticles (Fe 3 O 4 @PBA) toward ortho-dihydroxy-containing compounds were evaluated by adsorption experiments of adenosine and o-dihydroxybenzene.

Synthesis of Fe3O4 Nanoparticles
A solution containing anhydrous sodium acetate (7.2 g), polyethylene FeCl3•6H2O (2.7 g) and ethylene glycol (80.0 mL) was stirred at 50 °C for 15 solution was transferred to a PTFE reactor and heated at 200 °C for 8 h. The p collected with magnets and washed repeatedly using deionized water an spectively. Finally, the products were dried overnight in a vacuum at 50 °C

Synthesis of Fe3O4@MPTES
The Fe3O4 nanoparticles (200.0 mg), ethanol (25.0 mL) and deionized w were added into a 250 mL two-neck round-bottom flask, and the mixture w cally dispersed for 30 min. Then, 1.0 mL of glacial acetic acid was slowly

Synthesis of Fe 3 O 4 Nanoparticles
A solution containing anhydrous sodium acetate (7.2 g), polyethylene glycol (2.0 g), FeCl 3 ·6H 2 O (2.7 g) and ethylene glycol (80.0 mL) was stirred at 50 • C for 15 min. Then the solution was transferred to a PTFE reactor and heated at 200 • C for 8 h. The products were collected with magnets and washed repeatedly using deionized water and ethanol, respectively. Finally, the products were dried overnight in a vacuum at 50 • C.

Synthesis of Fe 3 O 4 @MPTES
The Fe 3 O 4 nanoparticles (200.0 mg), ethanol (25.0 mL) and deionized water (20.0 mL) were added into a 250 mL two-neck round-bottom flask, and the mixture was ultrasonically dispersed for 30 min. Then, 1.0 mL of glacial acetic acid was slowly added under mechanical agitation (350 rpm) to adjust the pH value of the solution to about 5, and stirring continued for 30 min. Subsequently, the direct silanization reaction was initiated by the dropwise addition of 5.0 mL MPTES, which was maintained in a water bath at 50 • C for 24 h. The products were Separations 2023, 10, 4 3 of 12 separated with magnets, and washed with deionized water and ethanol for several times. Then, the products were dried overnight at 50 • C to obtain Fe 3 O 4 @MPTES nanoparticles.

Synthesis of Fe 3 O 4 @MPS
The Fe 3 O 4 nanoparticles (200.0 mg), ethanol (40.0 mL), deionized water (10.0 mL) and ammonium hydroxide (1.5 mL) were added into a 250 mL two-neck round-bottom flask, and the mixture was ultrasonically dispersed for 30 min. Whereafter, 500 µL of MPS was dropwise added under mechanical agitation (350 rpm) to initiate the direct silanization reaction. After reacting at 50 • C for 24 h, the obtained Fe 3 O 4 @MPS was repeatedly washed using deionized water and ethanol and dried overnight at 50 • C.

Binding Experiments
A static adsorption method was used to evaluate the adsorption capacity of the three kinds of Fe 3 O 4 @PBA nanoparticles: Briefly, Fe 3 O 4 @PBA (2.0 mg) was dispersed by ultrasound in test solution (0.2 mL) with different concentrations (0.1~1.0 mg/mL) which was prepared with an ammonium bicarbonate (50 mM, pH = 8.5, containing 500 mM NaCl) as buffer solution, and shaken at room temperature (1000 rpm) for 20~140 min. The concentration of the adsorbate in the test solution after adsorption was measured by UV-Vis spectrophotometry. Then the adsorption capacity (Q e ) was calculated according to the equation below: where C 0 (mg/mL) is the initial concentration of the adsorbate in the test solution; C e (mg/mL) is the equilibrium concentration of the adsorbate; V (mL) is the volume of the adsorbate solution; m (mg) is the mass of Fe 3 O 4 @PBA. Contrast adsorption experiments were also performed with Fe 3 O 4 @MPTES and Fe 3 O 4 @MPS under the same conditions. The Scatchard equation was employed to investigate the binding properties of the Fe 3 O 4 @PBA nanoparticles: where Q (mg/g) is the equilibrium adsorption capacity of the material to the adsorbate, C (mg/mL) is the adsorbate concentration remaining in the test solution after adsorption equilibrium, Q max (mg/g) is the maximum apparent binding amount, K D is the equilibrium dissociation constant.

Selectivity Experiments
The selective adsorption capacity of Fe 3 O 4 @PBA nanoparticles can be investigated by adsorption experiments on mixed solutions of two or more adsorbates with similar structure but different in the presence of cis-diol. Two mixed solutions (1.0 mg/mL) with equal mass ratios of adsorbates were used in the selective adsorption experiments of Fe 3 O 4 @PBA nanoparticles, including adenosine and deoxyadenosine, and three dihydroxybenzene isomers. The procedure of adsorption experiments is the same as in Section 2.3, except that the Fe 3 O 4 @PBA nanoparticles were eluted by acetic acid buffer solution (0.05 mL, pH = 3.0) for 60 min after adsorption and rinsed. The eluent was analysed by HPLC. The Chromatographic analysis was carried out using a P230II HPLC with a Shim-pack C18 column (150 mm × 4.6 mm, 5 µm), and the mobile phase was methanol-water (12:88, v/v) at the flow rate of 1.0 mL/min. The column temperature was maintained at 30 • C, and the wavelength of the UV detector was 260 nm for adenosine and deoxyadenosine (280 nm for dihydroxybenzene isomers). Fe3O4@PBA nanoparticles were eluted by acetic acid buffer solution for 60 min after adsorption and rinsed. The eluent was analysed by H tographic analysis was carried out using a P230II HPLC with a Shim (150 mm × 4.6 mm, 5 μm), and the mobile phase was methanol-wat flow rate of 1.0 mL/min. The column temperature was maintained at 3 length of the UV detector was 260 nm for adenosine and deoxyade dihydroxybenzene isomers).  The results of the scanning transmission electron microscopy (ST ment distribution ( Figure 3) shows that B, Si, N and S are uniformly the core composed of Fe and O, indicating that the modified layer nosilane and phenylboric acid has been successfully introduced on th The results of the scanning transmission electron microscopy (STEM) analysis of element distribution ( Figure 3) shows that B, Si, N and S are uniformly distributed around the core composed of Fe and O, indicating that the modified layer composed of organosilane and phenylboric acid has been successfully introduced on the particle surface.

Characterization of Fe3O4@PBA Nanoparticles
The above elements' distribution of Fe 3 O 4 @PBA nanoparticles was also confirmed by X-ray photoelectron spectroscopy (XPS). As shown in Figure 4, strong signal peaks at 530 eV and 284.8 eV indicate that there are abundant oxygen-containing groups and carbon elements on the surface of the nanoparticles, and the peaks at 710.5 eV, 399.6 eV, 285.0 eV, 191.4 eV, 163.2 eV, and 102.1 eV correspond to Fe2p, N1s, C1s, B1s, S2p 3 and Si2p 2 , respectively. The signal peaks of S in Figure 4a,b shows that the hydrosulphonyl groups were introduced to the surface of Fe 3 O 4 nanoparticles by the coating of MPTES, and the signal peak of N in Figure 4b and the signal peak of B in Figure 4a-c all indicate that phenylboric acid has been successfully modified on the particle surface.
The weight loss of different modified nanoparticles can provide more supporting evidence for the modification process under nitrogen atmosphere with a heating rate of 10 • C/min. As can be seen from Figure 5, the Fe 3 O 4 nanoparticles had no significant weight loss during the heating process. However, with the modification of organosilane and the introduction of phenylboronic acid groups, the weight loss rate of the modified nanoparticles increased.  The above elements' distribution of Fe3O4@PBA nanoparticles was also confirmed by X-ray photoelectron spectroscopy (XPS). As shown in Figure 4, strong signal peaks at 530 eV and 284.8 eV indicate that there are abundant oxygen-containing groups and carbon elements on the surface of the nanoparticles, and the peaks at 710.5 eV, 399.6 eV, 285.0 eV, 191.4 eV, 163.2 eV, and 102.1 eV correspond to Fe2p, N1s, C1s, B1s, S2p 3 and Si2p 2 , respectively. The signal peaks of S in Figure 4a,b shows that the hydrosulphonyl groups were introduced to the surface of Fe3O4 nanoparticles by the coating of MPTES, and the signal peak of N in Figure 4b and the signal peak of B in Figure 4a-c all indicate that phenylboric acid has been successfully modified on the particle surface.  The weight loss of different modified nanoparticles can provide more supporting evidence for the modification process under nitrogen atmosphere with a heating rate of 10 °C/min. As can be seen from Figure 5, the Fe3O4 nanoparticles had no significant weight loss during the heating process. However, with the modification of organosilane and the idence for the modification process under nitrogen atmosphere with a heating ra °C/min. As can be seen from Figure 5, the Fe3O4 nanoparticles had no significant loss during the heating process. However, with the modification of organosilane introduction of phenylboronic acid groups, the weight loss rate of the modified na ticles increased. The room temperature magnetism of the prepared Fe3O4@PBA nanoparticles alysed by using the vibration sample magnetometer (VSM). As can be seen from F The room temperature magnetism of the prepared Fe 3 O 4 @PBA nanoparticles was analysed by using the vibration sample magnetometer (VSM). As can be seen from Figure 6, the room temperature saturation magnetic curves of the three kinds of Fe 3 O 4 @PBA nanoparticles have no obvious hysteresis loop and coercivity. In addition, nanoparticles dispersed in water can be reaggregated within 15 s under an applied magnetic field. The above analysis results and experimental phenomena shown that the prepared Fe 3 O 4 @PBA nanoparticles have excellent superparamagnetism.

Thermodynamics of Adsorption
The adsorption isotherms of the Fe3O4@MPTES@VPBA toward different ads are shown in Figure 7. As can be seen from the adsorption isotherms, when the tration of adsorbate is low, the adsorption capacity of the Fe3O4@MPTES@VPBA adsorbate increases synchronously with the concentration of the adsorbate. Howev to the high concentration of adsorbate, the boric acid binding site on the surfac material tends to saturate, which makes the increasing trend of adsorption capaci down gradually. When the initial concentration of adsorbed substance was 1.0 the adsorption capacity basically reaches the maximum. Similarly, the isothermal tion curves of Fe3O4@MPTES@AAPBA and Fe3O4@MPS@MPBA shown the same t

Thermodynamics of Adsorption
The adsorption isotherms of the Fe 3 O 4 @MPTES@VPBA toward different adsorbates are shown in Figure 7. As can be seen from the adsorption isotherms, when the concentration of adsorbate is low, the adsorption capacity of the Fe 3 O 4 @MPTES@VPBA on the adsorbate increases synchronously with the concentration of the adsorbate. However, due to the high concentration of adsorbate, the boric acid binding site on the surface of the material tends to saturate, which makes the increasing trend of adsorption capacity slow down gradually. When the initial concentration of adsorbed substance was 1.0 mg/mL, the adsorption capacity basically reaches the maximum. Similarly, the isothermal adsorption curves of Fe 3 O 4 @MPTES@AAPBA and Fe 3 O 4 @MPS@MPBA shown the same trend of adsorption capacity as that of Fe 3 O 4 @MPTES@VPBA. are shown in Figure 7. As can be seen from the adsorption isotherms, when the concen tration of adsorbate is low, the adsorption capacity of the Fe3O4@MPTES@VPBA on the adsorbate increases synchronously with the concentration of the adsorbate. However, due to the high concentration of adsorbate, the boric acid binding site on the surface of the material tends to saturate, which makes the increasing trend of adsorption capacity slow down gradually. When the initial concentration of adsorbed substance was 1.0 mg/mL the adsorption capacity basically reaches the maximum. Similarly, the isothermal adsorp tion curves of Fe3O4@MPTES@AAPBA and Fe3O4@MPS@MPBA shown the same trend o adsorption capacity as that of Fe3O4@MPTES@VPBA. The adsorption isotherm of Fe3O4@MPS, Fe3O4@MPTES, and three kinds o Fe3O4@PBA nanoparticles for the adsorption of adenosine and o-dihydroxybenzene were analysed by Scatchard equation [33,34]. The fitting curves of Fe3O4@MPTES@VPBA and Fe3O4@MPTES are shown in Figure 8. As can be seen from Figure 8, due to the introduction The adsorption isotherm of Fe 3 O 4 @MPS, Fe 3 O 4 @MPTES, and three kinds of Fe 3 O 4 @PBA nanoparticles for the adsorption of adenosine and o-dihydroxybenzene were analysed by Scatchard equation [33,34]. The fitting curves of Fe 3 O 4 @MPTES@VPBA and Fe 3 O 4 @MPTES are shown in Figure 8. As can be seen from Figure 8, due to the introduction of phenylboric acid high-affinity sites, the fitting curves of Fe 3 O 4 @MPTES@VPBA are composed of two straight lines representing high-affinity sites and low-affinity sites, respectively (Figure 8a,c). In contrast, the fitting curves of Fe 3 O 4 @MPTES show a linear relationship over the whole concentration range due to the absence of high-affinity sites (Figure 8b,d). Actually, the three kinds of Fe 3 O 4 @PBA nanoparticles have similar fitting analysis results. According to Table 1, there are two binding sites of high affinity and low affinity on the three kinds of Fe 3 O 4 @PBA nanoparticles. The equilibrium dissociation constant (K d ) of the high-affinity binding sites for the adsorption of adenosine and o-dihydroxybenzene was significantly lower than those of the low-affinity sites, indicating that the specific adsorption caused by boric acid affinity had a higher affinity than the non-specific adsorption caused by hydrogen bonding and electrostatic interaction.

Kinetics of Adsorption
As can be seen from the adsorption kinetics curve of Fe3O4@MPTES@VPBA ( Figure  9), at the initial stage of adsorption, a relatively stable complex was formed between the  Boric acid groups can specifically form ester rings with ortho-dihydroxy-containing compounds, resulting in selective adsorption. Unlike adenosine, deoxyadenosine does not have adjacent hydroxyl groups. The adsorption of deoxyadenosine by Fe 3 O 4 @PBA depends only on hydrogen bonding and electrostatic interaction, which results in the adsorption capacity of deoxyadenosine being significantly lower than that of adenosine.
Of the three isomers of dihydroxybenzene, o-Dihydroxybenzene is the only one that has adjacent hydroxyl groups, and it forms ester rings with boric acid much more easily than m-dihydroxybenzene and p-dihydroxybenzene. Therefore, the adsorption capacity and selectivity of Fe 3 O 4 @PBA for o-dihydroxybenzene are greater than that of m-dihydroxybenzene and p-dihydroxybenzene.

Kinetics of Adsorption
As can be seen from the adsorption kinetics curve of Fe 3 O 4 @MPTES@VPBA (Figure 9), at the initial stage of adsorption, a relatively stable complex was formed between the adsorbate and the boric acid binding site, which causes the adsorption capacity to rise rapidly. When most of the boric acid binding sites on the surface are occupied, it becomes difficult for the adsorbate to bind to the deeper binding sites, resulting in a slow increase in the amount of adsorption. With the continuous progress of the adsorption, all binding sites in the material are saturated, and the adsorption capacity tends to be stable. As shown in Figure 9, all the three kinds of prepared Fe 3 O 4 @PBA nanoparticles showed the same trend of adsorption capacity and reached their maximum adsorption capacity at 80~100 min.

Kinetics of Adsorption
As can be seen from the adsorption kinetics curve of Fe3O4@MPTES@VPBA ( Figure  9), at the initial stage of adsorption, a relatively stable complex was formed between the adsorbate and the boric acid binding site, which causes the adsorption capacity to rise rapidly. When most of the boric acid binding sites on the surface are occupied, it becomes difficult for the adsorbate to bind to the deeper binding sites, resulting in a slow increase in the amount of adsorption. With the continuous progress of the adsorption, all binding sites in the material are saturated, and the adsorption capacity tends to be stable. As shown in Figure 9, all the three kinds of prepared Fe3O4@PBA nanoparticles showed the same trend of adsorption capacity and reached their maximum adsorption capacity at 80~100 min.

Selectivity
A solution of adenosine or deoxyadenosine (1.0 mg/L) was adsorbed by the three prepared Fe3O4@PBA nanoparticles, respectively. The adsorption selectivity factor (α =

Selectivity
A solution of adenosine or deoxyadenosine (1.0 mg/L) was adsorbed by the three prepared Fe 3 O 4 @PBA nanoparticles, respectively.
The adsorption selectivity factor (α = Q adenosine /Q deoxyadenosine ) was calculated. Similarly, the adsorption selectivity factors of dihydroxybenzene isomers were also obtained by the same method. The calculation results are shown in Table 2. Table 2.
Absorption capacity (Q e ) and selectivity factor (α) of different adsorbates by Fe 3 O 4 @PBA nanoparticles. According to the Table 2, the adsorption capacity of the three kinds of Fe 3 O 4 @PBA nanoparticles toward adenosine was significantly higher than that of deoxyadenosine, and the adsorption selectivity factor reached more than 4.5. Furthermore, the adsorption selectivity factor for o-dihydroxybenzene ranged from 2.18 to 3.17, indicating that all three kinds of Fe 3 O 4 @PBA nanoparticles offered excellent adsorption selectivity toward adenosine and o-dihydroxybenzene. Particularly, the Fe 3 O 4 @MPS@MPBA exhibited the highest adsorption capacity and selectivity factor for adenosine and o-dihydroxybenzene.

Adsorbate
The adsorption selectivity was further confirmed by HPLC analysis of the eluents after adsorption of the mixed solutions. Figure 10 presents the HPLC chromatograms of the eluents of the mixed solution (1.0 mg/mL) with equal mass ratio of adenosine and deoxyadenosine adsorbed by three kinds of Fe 3 O 4 @PBA nanoparticles, respectively. As can be seen from Figure 10, the chromatographic peak area of adenosine in the eluents was significantly higher than that of deoxyadenosine, which proved that three kinds of Fe 3 O 4 @PBA nanoparticles also had obvious adsorption selectivity for adenosine in the mixed solution of adenosine and deoxyadenosine.  According to the Table 2, the adsorption capacity of the three kinds of Fe3O4@PBA nanoparticles toward adenosine was significantly higher than that of deoxyadenosine and the adsorption selectivity factor reached more than 4.5. Furthermore, the adsorption selectivity factor for o-dihydroxybenzene ranged from 2.18 to 3.17, indicating that all three kinds of Fe3O4@PBA nanoparticles offered excellent adsorption selectivity toward adeno sine and o-dihydroxybenzene. Particularly, the Fe3O4@MPS@MPBA exhibited the highes adsorption capacity and selectivity factor for adenosine and o-dihydroxybenzene.
The adsorption selectivity was further confirmed by HPLC analysis of the eluents after adsorption of the mixed solutions. Figure 10 presents the HPLC chromatograms o the eluents of the mixed solution (1.0 mg/mL) with equal mass ratio of adenosine and deoxyadenosine adsorbed by three kinds of Fe3O4@PBA nanoparticles, respectively. As can be seen from Figure 10, the chromatographic peak area of adenosine in the eluents was significantly higher than that of deoxyadenosine, which proved that three kinds o Fe3O4@PBA nanoparticles also had obvious adsorption selectivity for adenosine in the mixed solution of adenosine and deoxyadenosine.  The HPLC chromatograms of the eluents of the mixed solution (1.0 mg/mL) with an equal mass ratio of o-dihydroxybenzene, m-dihydroxybenzene and p-dihydroxybenzene adsorbed by the three kinds of Fe 3 O 4 @PBA nanoparticles, respectively, are shown in Figure 11. Obviously, the peak area of o-dihydroxybenzene was the largest in all the eluents. It was also significantly different from that of m-dihydroxybenzene and p-dihydroxybenzene, further indicating that the three kinds of Fe 3 O 4 @PBA nanoparticles also showed the adsorption selectivity of o-dihydroxybenzene in the mixed solution of the dihydroxybenzene isomers.

Conclusions
In this study, a new modification strategy for Fe3O4 magnetic nanoparticles with phe nylboric acid was designed using the direct silanizing method and thiol-ene click reaction Based on this new strategy, three kinds of phenylboric-acid-modified magnetic nanopar ticles (Fe3O4@PBA) were prepared by a simple process under mild reaction conditions The three kinds of Fe3O4@PBA nanoparticles obtained showed excellent adsorption capac ity and selectivity for ortho-dihydroxy-containing compounds, including adenosine and o-dihydroxybenzene. The reactivity of acrylates was higher than that of acrylamide and styrene among the double-bond monomers that produce thiol-ene click chemistry, which resulted in a relatively larger number of phenylboronic acid binding sites on the surface of magnetic nanoparticles modified with 4-mercaptophenylboronic acid (MPBA). There fore, Fe3O4@MPS@MPBA exhibited the highest adsorption capacity and selectivity factor for adenosine and o-diphenol among the three kinds of Fe3O4@PBA.
Furthermore, boric acid with low pka value should be used in the modification o nanoparticles to reduce the bonding pH value of Fe3O4@PBA, which enables Fe3O4@PBA to selectively adsorb glycopeptides and glycoproteins in biological samples.

Conclusions
In this study, a new modification strategy for Fe 3 O 4 magnetic nanoparticles with phenylboric acid was designed using the direct silanizing method and thiol-ene click reaction. Based on this new strategy, three kinds of phenylboric-acid-modified magnetic nanoparticles (Fe 3 O 4 @PBA) were prepared by a simple process under mild reaction conditions. The three kinds of Fe 3 O 4 @PBA nanoparticles obtained showed excellent adsorption capacity and selectivity for ortho-dihydroxy-containing compounds, including adenosine and o-dihydroxybenzene. The reactivity of acrylates was higher than that of acrylamide and styrene among the double-bond monomers that produce thiol-ene click chemistry, which resulted in a relatively larger number of phenylboronic acid binding sites on the surface of magnetic nanoparticles modified with 4-mercaptophenylboronic acid (MPBA). Therefore, Fe 3 O 4 @MPS@MPBA exhibited the highest adsorption capacity and selectivity factor for adenosine and o-diphenol among the three kinds of Fe 3 O 4 @PBA.
Furthermore, boric acid with low pk a value should be used in the modification of nanoparticles to reduce the bonding pH value of Fe 3 O 4 @PBA, which enables Fe 3 O 4 @PBA to selectively adsorb glycopeptides and glycoproteins in biological samples.