Facile Preparation of Phenyboronic-Acid-Functionalized Fe₃O₄ Magnetic Nanoparticles for the Selective Adsorption of Ortho-Dihydroxy-Containing Compounds

Abstract

A new facile strategy was designed to prepare the phenyboronic acid-functionalized Fe₃O₄ magnetic nanoparticles (Fe₃O₄@PBA) via direct silanization and thiol-ene click chemistry for the selective adsorption of ortho-dihydroxy-containing compounds. The three kinds of Fe₃O₄@PBA nanoparticles obtained showed excellent adsorption capacity and selectivity for ortho-dihydroxy-containing compounds including adenosine and o-dihydroxybenzene. Among them, the Fe₃O₄@MPS@MPBA exhibited the highest adsorption capacity and selectivity for adenosine and o-dihydroxybenzene, followed by Fe₃O₄@MPTES@AAPBA and Fe₃O₄@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.

1. 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 Fe₃O₄ magnetic nanoparticles. The adsorption capacity and selectivity of the three kinds of prepared phenyboronic-acid-functionalized Fe₃O₄ magnetic nanoparticles (Fe₃O₄@PBA) toward ortho-dihydroxy-containing compounds were evaluated by adsorption experiments of adenosine and o-dihydroxybenzene.

2. Materials and Methods

2.1. Reagents and Material

Ferric trichloride hexahydrate (FeC1₃·6H₂O), ammonium hydrogen carbonate, sodium chloride, ethylene glycol, anhydrous sodium acetate, glacial acetic acid, methanol, ethanol and aqueous ammonia (25 wt%) were obtained from FengChuan Fine Chemical Research Institute (Tianjin, China). Adenosine, deoxyadenosine, azodiisobutyronitrile (AIBN), 3-mercaptopropyltrimethoxysilane (MPTES) and 4-vinylphenylboronic acid (VPBA) were obtained from Adamas-beta (Basel, Switzerland). 3-methacryloxypropyltrimethoxysilane (MPS) and 3-acrylamidophenylboronic acid (AAPBA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4-Mercaptophenylboronic acid (MPBA), o-dihydroxybenzene, m-dihydroxybenzene, and p-dihydroxybenzene were obtained from Aladdin (Shanghai, China). Deionised water was used to prepare all buffer and analyte solutions.

2.2. Synthesis of Fe₃O₄@PBA Nanoparticles

The general scheme for the synthesis of magnetic nanoparticles modified by phenylboronic acid (Fe₃O₄@PBA) is illustrated in Figure 1. The Fe₃O₄ nanoparticles was synthesized by a previously reported solvothermal method [30]. The Fe₃O₄ nanoparticles were functionalized with the sulfydryl and vinyl groups through the direct silanization using MPTES and MPS [31]. Subsequently, the functionalization of the nanoparticles based on phenylboric acid was achieved by the thiol-ene click reaction using 4-vinylphenylboronic acid (VPBA), 3-acrylamidophenylboronic acid (AAPBA) and 4-mercaptophenylboronic acid (MPBA) [32].

2.2.1. Synthesis of Fe₃O₄ Nanoparticles

A solution containing anhydrous sodium acetate (7.2 g), polyethylene glycol (2.0 g), FeCl₃·6H₂O (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.

2.2.2. Synthesis of Fe₃O₄@MPTES

The Fe₃O₄ 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 separated with magnets, and washed with deionized water and ethanol for several times. Then, the products were dried overnight at 50 °C to obtain Fe₃O₄@MPTES nanoparticles.

2.2.3. Synthesis of Fe₃O₄@MPS

The Fe₃O₄ 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 Fe₃O₄@MPS was repeatedly washed using deionized water and ethanol and dried overnight at 50 °C.

2.2.4. Phenyboronic-Acid-Functionalized Fe₃O₄@MPS and Fe₃O₄@MPTES

200.0 mg of Fe₃O₄@MPS or Fe₃O₄@MPTES was ultrasonically dispersed in a solution containing 50 mL of ethanol and 200.0 mg of MPBA (400.0 mg of AAPBA or 200.0 mg of VPBA for Fe₃O₄@MPTES) for 30 min. Then the thiol-ene click reaction was initiated by the addition of AIBN (20.0) mg under nitrogen protection, mechanical stirring (400 rpm) and 50 °C for 24 h. The products were separated with magnets, and washed using deionized water and ethanol for several times. The vacuum drying process was maintained at 50 °C for 12 h. Finally, the obtained products were named as Fe₃O₄@MPTES@VPBA, Fe₃O₄@MPTES@AAPBA and Fe₃O₄@MPS@MPBA, respectively.

2.3. Binding Experiments

A static adsorption method was used to evaluate the adsorption capacity of the three kinds of Fe₃O₄@PBA nanoparticles: Briefly, Fe₃O₄@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:

$$Q_e=\frac{V\left(C_0-C_e\right)}{m}\times 1000$$

(1)

where C₀ (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 Fe₃O₄@PBA. Contrast adsorption experiments were also performed with Fe₃O₄@MPTES and Fe₃O₄@MPS under the same conditions.

The Scatchard equation was employed to investigate the binding properties of the Fe₃O₄@PBA nanoparticles:

$$\frac{Q}{C}=\frac{\left(Q_{max}-Q\right)}{K_D}$$

(2)

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.

2.4. Selectivity Experiments

The selective adsorption capacity of Fe₃O₄@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 Fe₃O₄@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 Fe₃O₄@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).

3. Results and Discussion

3.1. Characterization of Fe₃O₄@PBA Nanoparticles

Figure 2 is the transmission electron microscope (TEM) images of Fe₃O₄@PBA nanoparticles. As shown in Figure 2, three kinds of nanoparticles formed a core-shell structure including a core composed of Fe₃O₄ and a modified layer about 5 nm thick.

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.

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, S2p³ and Si2p², 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 introduction of phenylboronic acid groups, the weight loss rate of the modified nanoparticles increased.

The room temperature magnetism of the prepared Fe₃O₄@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 Fe₃O₄@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 Fe₃O₄@PBA nanoparticles have excellent superparamagnetism.

3.2. Binding Properties of Fe₃O₄@PBA Nanoparticles

3.2.1. Thermodynamics of Adsorption

The adsorption isotherms of the Fe₃O₄@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 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 adsorption curves of Fe₃O₄@MPTES@AAPBA and Fe₃O₄@MPS@MPBA shown the same trend of adsorption capacity as that of Fe₃O₄@MPTES@VPBA.

The adsorption isotherm of Fe₃O₄@MPS, Fe₃O₄@MPTES, and three kinds of 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 of phenylboric acid high-affinity sites, the fitting curves of Fe₃O₄@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 Fe₃O₄@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 Fe₃O₄@PBA nanoparticles have similar fitting analysis results. According to

Table 1. K_D of different Fe₃O₄@PBA for the adsorption of adenosine and o-dihydroxybenzene.

Adsorbate	Adsorbent	KD (mg/mL)
		High-Affinity Binding Sites	Low-Affinity Binding Sites
Adenosine	Fe3O4@MPTES@VPBA	0.13	1.29
	Fe3O4@MPTES@ AAPBA	0.05	1.95
	Fe3O4@MPTES	—	3.68
	Fe3O4@MPS@MPBA	0.05	1.33
	Fe3O4@MPS	—	3.45
o-Dihydroxybenzene	Fe3O4@MPTES@VPBA	0.41	2.50
	Fe3O4@MPTES@ AAPBA	0.33	2.64
	Fe3O4@MPTES	—	5.15
	Fe3O4MPS@MPBA	0.13	2.73
	Fe3O4@MPS	—	4.17

, there are two binding sites of high affinity and low affinity on the three kinds of Fe₃O₄@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.

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 Fe₃O₄@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 Fe₃O₄@PBA for o-dihydroxybenzene are greater than that of m-dihydroxybenzene and p-dihydroxybenzene.

3.2.2. 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.

3.3. 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 = 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. Absorption capacity (Q_e) and selectivity factor (α) of different adsorbates by Fe₃O₄@PBA nanoparticles.

Adsorbate	Fe3O4@MPTES@VPBA		Fe3O4@MPTES@AAPBA		Fe3O4@MPS@MPBA
	Qe (mg/g)	α	Qe (mg/g)	α	Qe (mg/g)	α
Adenosine	91.9	-	92.7	-	94.6	-
Deoxyadenosine	18.5	4.97	19.1	4.85	17.3	5.47
o-Dihydroxybenzene	80.6	-	79.3	-	83.1
m-Dihydroxybenzene	34.6	2.33	36.4	2.18	28.5	2.92
p-Dihydroxy-benzene	31.1	2.59	32.6	2.43	26.2	3.17

.

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 adenosine and o-dihydroxybenzene. Particularly, the Fe₃O₄@MPS@MPBA exhibited the highest 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 of 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 of 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 Fe₃O₄@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 Fe₃O₄@PBA nanoparticles also showed the adsorption selectivity of o-dihydroxybenzene in the mixed solution of the dihydroxybenzene isomers.

4. Conclusions

In this study, a new modification strategy for Fe₃O₄ 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 (Fe₃O₄@PBA) were prepared by a simple process under mild reaction conditions. The three kinds of Fe₃O₄@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, 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 pk_a value should be used in the modification of nanoparticles to reduce the bonding pH value of Fe₃O₄@PBA, which enables Fe₃O₄@PBA to selectively adsorb glycopeptides and glycoproteins in biological samples.