Preparation of magnetic molecularly imprinted polymer for selective recognition of resveratrol in wine
The magnetic molecularly imprinted polymers (MMIPs) for resveratrol were prepared by using sur- face molecular imprinting technique with a super paramagnetic core–shell nanoparticle as a supporter. Rhapontigenin, which is the analogues of resveratrol, was selected as dummy template molecules to avoid the leakage of trace amount of resveratrol. Acrylamide and ethylene glycol dimethacrylate were chosen as functional monomers and cross-linker, respectively. The obtained MMIPs were characterized by using scanning electron microscopy, Fourier transform infrared spectrum, X-ray diffraction and vibrating sam- ple magnetometer. High performance liquid chromatography was used to analyze the target analytes. The resulting MMIPs exhibited high saturation magnetization of 53.14 emu g−1 leading to the fast sep- aration. The adsorption test showed that the MMIPs had high adsorption capacity for resveratrol and contained homogeneous binding sites. The MMIPs were employed as adsorbent of solid phase extraction for determination of resveratrol in real wine samples, and the recoveries of spiked samples ranged from 79.3% to 90.6% with the limit of detection of 4.42 ng mL−1 . The prepared MMIPs could be employed to selectively pre-concentrate and determine resveratrol from wine samples.
1. Introduction
Resveratrol is a polyphenol compound which has beneficial effects on human health, such as antioxidative [1], anticancer [2], anti-inflammatory [3], antiestrogenic [4], and platelet aggregation inhibitory effects [5]. The concentrations of resveratrol contained in wine vary from one kind to another, according to the vari- ety [6], origin [7], and growing conditions of the grapes [8]. In view of the increasing interest in resveratrol, various analytical methods were developed for the measurement of resveratrol in wine [9–11]. However, in some case, quantitation for resveratrol is influenced by the complex nature of the sample matrix. Therefore, it is necessary to develop a selective and practicable enrichment material for separation and determination of such an important bioactive compound.
Molecular imprinted polymers (MIPs) are functional porous materials with molecular-specific recognition sites to a particu- lar target molecule [12,13]. They have been widely applied in the fields of catalysis [14], environmental analysis [15], pharmaceuti- cal analysis [16], food analysis [17], chromatography [18], chemical sensors [19], and solid-phase extraction (SPE) etc. [12,13,20]. MIPs as SPE sorbents have attracted considerable attention for being able to selectively recognize the target molecules from a mixture of chemical species. With the development of molecular imprinted- SPE (MISPE), a technique that is based on magnetic polymer has received increasing attention. The magnetic MIPs (MMIPs) can be dispersed into the solution directly and then easily separated from the matrix using an external magnetic field without additional centrifugation or filtration. The MMIPs, which have already been prepared in some works [21–23], also display higher adsorption ability and excellent recognition selectivity.
The potential risk for leakage of the residual template molecules during the adsorption process is a common issue for the MIPs including MMIPs in SPE. To completely avoid the interference from residual template, the structural analogue of the target molecules as dummy template was used to prepare MIPs [24]. In the preparation process, the interaction of template molecules with functional monomer, and the conformation effects between tem- plate molecules and rebinding sites may affect the properties of the MIPs [25]. Therefore, the screening of the dummy template is important to obtain proper affinity and sufficient recovery of MIPs. The dummy template of MIPs should not interfere with the analysis of target molecule, either.
MISPE was applied to selectively extract and determine resver- atrol in previous work, in which resveratrol was used as the template molecule [26–30]. In this study, it was the first attempt to synthesize the dummy template MMIPs for the recognition of resveratrol in wine sample. Especially, rhapontigenin (RH) was used as the dummy template for the preparation of MMIPs using surface molecular imprinting technique with a super paramagnetic core–shell nanoparticle as the supporter. The characterization, adsorption capacity and selectivity of MMIPs and magnetic non imprinted polymers (MNIPs) were investigated. The resveratrol recognition in real wine samples was realized by using MMIPs as SPE sorbents with high selectivity and good recovery.
2. Experimental
2.1. Reagents and apparatus
Rhapontigenin (RH), desoxyrhapontigenin, desoxyrhaponticin, and rhaponticin were provided by our lab [31]. Resveratrol and pro- tocatechuic acid were provided by the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). Ethylene glycol dimethacrylamide (EGDMA), 2,2∗-azobisissobutyronitrile (AIBN), 3-methacryloxypropyltrimethoxysilane (MPS), acrylamide (AM), and methacrylic acid were obtained from Alfa Aesar (Tian- jin, China). Ferric chloride (FeCl3·6H2O) and ferrous chloride (FeCl2·4H2O) were purchased from Beijing Chemicals Corporation (Beijing, China). Anhydrous toluene and ethanol were purchased from Lianlong Bohua Pharmaceutical Chemical Co., Ltd. (Tianjin, China). Chromatographic grade methanol and acetonitrile were purchased from Merck Co. (Darmstadt, Germany). Tetraethoxysi- lane (TEOS), isopropanol, acetic acid, ammonium hydroxide and the other chemicals were supplied from Tianjin Chemical Reagent Co. (Tianjin, China). Deionized water (18 M▲ cm) was prepared with a water purification system (Shanghai, China). The following wines were obtained from a local wine shop (Gansu, China): Qilian ice white wine (Qianlian, Gansu), Mogao dry red wine (Wuwei, Gansu), red wine (Yantai, Shandong). All the solutions used for HPLC were filtered through a 0.45 µm filter before use.
Scanning electron microscopy (SEM) images were obtained via JEM-5600LV scanning electron microscope (Tokyo, Japan). The Fourier transform infrared (FT-IR) spectra were obtained via a Nicolet Nexus-670 FT-IR spectrometer. The wave numbers of FT-IR measurement range were controlled from 500 cm−1 to 4000 cm−1. The magnetic properties were measured using Lake Shore 7304 vibrating sample magnetometer (VSM) (Lakeshore, USA). X-ray diffraction (XRD) pattern were carried out by an X-ray diffraction using Cu-Ka1 radiation (PANalytical X’Pert, Holland). Sample analysis was performed by using liquid chro- matographic system equipped with Agilent 1200 HPLC system and diode array detection (DAD) system. The analytical column was a 250 mm × 4.6 mm, 5 µm C18 column (Agilent, USA). The mobile phase consisted of acetonitrile and water (65:35, v/v) with a flow rate at 1.0 mL min−1. DAD monitoring was at 321 nm for resveratrol [29].
2.2. Preparation of MMIPs
The preparation protocol is shown in Fig. 1. At first Fe3O4@SiO2–MPS nanoparticles were prepared. The Fe3O4 parti- cles were prepared using chemical co-precipitation according to Zhang et al. with small modifications [32]: 15 mmol FeCl3·6H2O and 10 mmol FeCl2·4H2O were dissolved in 80 mL of deoxygenated water stirring at 300 rpm under nitrogen gas. Ammonium hydrox- ide solution (28%, weight percent) of 50 mL was dropwise added into the clear yellow solution. With the addition of ammonium hydroxide solution, the solution turned black. The reaction was maintained at 80 ◦C for 30 min. The black precipitates obtained (Fe3O4 nanoparticles) were collected and washed repeatedly with deionized water until the pH of the washings became neutral and finally dried under vacuum at 60 ◦C for 24 h. Then the Fe3O4 parti- cles were modified with SiO2 according to the work of Zeng et al. [21]. The obtained Fe3O4@SiO2 nanoparticles were dried under vacuum at 60 ◦C, and then modified with MPS-introduced polymer- izable double bonds [33]. Briefly, 1 g of Fe3O4@SiO2 nanoparticles were dispersed in 50 mL of anhydrous toluene containing 5 mL of MPS, and the mixture was reacted with reflux at 90 ◦C for 24 h under dry nitrogen. The products were collected and washed with toluene and ethanol for several times. Finally, surface-modified magnetic particles (Fe3O4@ SiO2–MPS nanoparticles) were dried under vac- uum at 60 ◦C.
The MMIPs were prepared via surface-imprinted polymeriza- tion method as follows: RH (0.1 mmol) as the dummy template and AM (0.5 mmol) as the functional monomer were dissolved in 20 mL acetonitrile and stored in dark for 12 h at room temperature. Then, 100 mg of Fe3O4@SiO2–MPS nanoparticles was added into the mixture, and stirred for 2 h. Subsequently, cross-linker EGDMA (3.0 mmol) and initiator AIBN (20 mg) were added into the system and the mixture was degassed in an ultrasonic bath for 15 min. After filled with nitrogen gas for 10 min to remove oxygen, the polymerization was performed at 60 ◦C with nitrogen protection for 24 h. The MMIPs were collected magnetically, and washed by a mixture of methanol/acetic acid (9:1, v/v) to remove the tem- plates and then washed by methanol until no RH absorption was detected by HPLC. Finally, the particles were dried in vacuum. The MNIPs were prepared by the same method as MMIPs without the addition of template.
2.3. Adsorption equilibrium and selectivity evaluation
The adsorption equilibrium experiment was performed to eval- uate the recognizing and binding capacity of MMIPs towards resveratrol. MMIPs or MNIPs of 20 mg were suspended in 1 mL of the initial resveratrol acetonitrile concentrations ranging from 10 µg mL−1 to 150 µg mL−1 in 2 mL -centrifuge tube. After the tubes were shaken in SHA-B incubator (Jintan Zhengji Instrument Co., Ltd., Jiangsu, China) for 24 h at 25 ◦C, the MMIPs or MNIPs were magnetically separated from the solution. Then, the supernatant was measured using HPLC. The equilibrium adsorption amounts of resveratrol (Q µmol g−1) were calculated according to the following equation: (C − C )V.
3. Results and discussion
3.1. Preparation of MMIPs
As illustrated in Fig. 1, the synthesis of the MMIPs is a mul- tistep procedure. First, super paramagnetic Fe3O4 nanoparticles were prepared by the coprecipitation method and the nanopar- ticles had to be less than 25 nm in order to ensure the super paramagnetism. Secondly, the surface of Fe3O4 nanoparticles was encapsulated with silica to avoid oxidation and provide a bio- compatible and hydrophilic surface. Furthermore, surface silanol groups could offer many possibilities for surface modification through covalent attachment of specific ligands on the surface of Fe3O4@SiO2 nanoparticles [34]. Then, the Fe3O4@SiO2 nanoparticles were surface-modified with MPS which introduced double bonds on to the surface of Fe3O4@SiO2 nanoparticles to form poly-where Ci and Cf (µg mL−1) are the concentrations of resveratrol at initial and final equilibrium, respectively. V (mL) is the volume of resveratrol solution, M is the molar mass of resveratrol, and W (g) is the weight of polymers. All the experiments were carried out at least twice, and the mean values were used in data analysis.
The selectivity of the magnetic imprinted sorbent towards structurally resveratrol-related (RH, desoxyrhapontigenin, des- oxyrhaponticin, and rhaponticin) and a reference compound (protocatechuic acid) was investigated. 1 mL of a 20 µg mL−1 solution prepared in acetonitrile, individually and as a mixture (without reference compound) of the studied compounds was incubated with 20 mg MMIPs or MNIPs. The extraction procedure was then conducted as described earlier in adsorption equilibrium experi- ment.
The interrelated absorbed coefficient was evaluated by the fol- lowing equations: merizable sites. During the synthesis procedure of the MMIPs, the double bonds on the surface of the Fe3O4@SiO2 nanoparticles can react with EGDMA and AM [35].The molecular recognition capability of MMIPs was affected by many factors such as porogen, the type and amount of monomer. The MMIPs should be synthesized in a suitable solvent, in which the template RH and functional monomer could dissolve well. Acetonitrile was selected as the porogen, because RH is a strong polar molecule that does not possess any hydrophobic functional group, and the weak polarity solvent is suitable for non-covalent molecular recognition of MMIPs. Molecular recognition of the tem- plate molecule and structurally related compound by imprinted polymers is based on the intermolecular interaction between the template molecule and functional groups in the polymer [36]. In this study, different monomers such as AM and methacrylic acid, were selected to evaluate the specific recognition ability of MMIPs for resveratrol molecule. The resulted polymers prepared using AM had better molecular recognition in polar conditions. In addition, the amount of AM functional monomer was optimized. Three molar ratios of the monomer AM to the template of 4:1, 5:1 and Ca is the concentration of the absorbed medium and Cf is the final free concentrations of the solution; Ca = (Ci − Cf) × V/W, where Ci (µg mL−1), Cf (µg mL−1), V (mL), and W (g) are as described pre- viously. For comparison of the obtained MMIPs selectivity for the resveratrol-to-competitive molecule, the selectivity coefficient (k), and relative selectivity coefficient (k∗) were calculated according to the following formula: 6:1 were tested. The optimum ratio of functional monomer to cross-linker was 1:6 according to previous reports [12,13]. The experimental results indicated that the optimum molar ratio of template:AM:EGDMA was 1:5:30 to prepare MMIPs for resveratrol. The RSD% of batch-to-batch reproducibility of the prepared MMIPs was 6.4%, showing that the synthesis process was satisfactory.
2.4. Analysis of resveratrol in wine samples
0.2 mL of Mogao dry red wine diluted to 1 mL and 1 mL of Qilian ice white wine and red wine real samples were prepared, respec- tively. 40 mg of MMIPs were added into the solution and shook at room temperature for 30 min. A magnet was used to separate MMIPs from the solution followed by washing the MMIPs with 1 mL of methanol/acetic acid (9:1, v/v) for 20 min. 0.5 mL supernatants were evaporated to dryness and dissolved in 0.1 mL of methanol for further HPLC–UV analysis.
3.2. Characterization of MMIPs
The MMIPs were characterized by a series of physical tests. Fig. 2 shows SEM images of the obtained MMIPs and MNIPs. The results indicated that almost all of these obtained MMIPs were regular spheres. The shape of the MMIPs were more round than that of MNIPs, which demonstrated that the template molecules had some influence on the spherical particles’ growth during the synthesis procedure [12].
The saturation magnetization of MMIPs decreased in comparison with the Fe O nanoparticles, but remained strong magnetism and peak of C H stretching vibration at 2924 cm−1, suggesting that MPS was successfully modified onto the surface of Fe3O4@SiO2. The absorbance peak of C O at 1729 cm−1 showed that the MMIPs and MNIPs were synthesized through the polymerization of EGDMA and AM.
3.3. Adsorption isotherms
The experimental equilibrium isotherms for the adsorption of resveratrol onto the MMIPs and MNIPs with different initial con- centrations are shown in Fig. 6. As it can be seen in Fig. 6, the amount of resveratrol binding to the polymers increases along with its ini- tial concentration until it reaches a saturation level, and the MMIPs has higher affinity for resveratrol than MNIPs. It suggested that the resveratrol binding to MMIPs was caused by the specific binding to a Dubinin–Radushkevich model, it was not suitable for the fitting of experiment data of resveratrol on MMIPs.
3.4. Binding specificity and selectivity of the MMIPs
The specific adsorption capability of MMIPs was evaluated by using resveratrol (3,5,4∗-trihydroxystilbene), its structurally similar compound of RH, desoxyrhapontigenin, rhaponticin, and desoxyrhaponticin and a reference compound of protocatechuic acid. Fig. 7 illustrates the adsorption ability of MMIPs and MNIPs for five structurally similar compounds and a reference compound (protocatechuic acid). It is obvious that the adsorption capacity of MMIPs for the five polyphenols is much higher than that of MNIPs. The adsorption capacity of the MMIPs for resveratrol is slightly higher than RH. It may be explained by its close structural homology to RH. Molecular recognition ability of MMIPs was mainly depend- ent on the hydrogen bonding between the polymeric matrix and the analytes and complementary to the analytes in size and shape [41]. The molecular size of resveratrol was smaller than RH as they had the same number of H-bond donors but an extra methoxyl group existed in RH. Thus, resveratrol may form the same number of hydrogen bonds and easily enter the MMIPs cavity than RH. Thus, a better match of resveratrol in the cavities could be explained.
This result indicated that RH was an ideal dummy template for preparation of MMIPs for the adsorption of resveratrol.There was one less phenolic OH group in desoxyrhaponti- genin than RH which resulted in reduced binding to MMIPs. For rhaponticin, and desoxyrhaponticin, the presence of the bulky glucose group presumably prevented the interaction of the meta- positioned OH group with the binding cavities. As a result, weak interactions were expected and a reduction binding was achieved. Low adsorption capability of MMIPs for protocatechuic acid was observed due to the different structure in comparison with RH. This indicated that the MMIPs had no specific site to the compound with
significantly different structures.
The adsorption of polyphenol analogues to MMIPs was also investigated under competitive conditions. In order to reduce the complexity of the mixture, the reference compound was not included in this mixture because of the low affinity for MMIPs (as discussed above). The selectivity of MMIPs for resveratrol was eval- uated using three parameters: kd, k, and k∗. The measured values of the three parameters for the tested compounds are shown in Table 3. Kd suggests the adsorption capacity. The larger the value of Kd is, the stronger the adsorption capability of a substance would be. In addition, it was calculated to be 182 mL g−1of resveratrol for MMIPs and 9.3 mL g−1 for MNIPs. The parameter k indicated the selectivity between the target molecule and the reference com- pound. It can be seen from Table 3 that high k value of 9.7 and 8.9 of MMIPs can be achieved, which indicated high discrimination property of the MMIPs between resveratrol and other compounds. Then imprinting effect of the MMIPs could make sense for selective rebinding resveratrol from real complex samples.
3.5. Extraction and desorption time
The experimental results (Fig. 8a) indicate that the extrac- tion time has an obvious effect on the resveratrol adsorption. The adsorption of resveratrol should achieve equilibrium for enough time to obtain satisfactory recoveries. In our study, the extraction process was conducted in 1 mL resveratrol solution of 100 ng mL−1, which was close to the concentration in real sample. The equilib- rium adsorption amounts of resveratrol were calculated according to Eq. (1). Then 0.5 mL supernatants were evaporated to dryness and dissolved in 0.1 mL of methanol for further HPLC–UV analy- sis. The extraction time was investigated from 5 min to 90 min. The adsorption equilibrium was reached after approximately 30 min. Further increase of extraction time could not obviously affect the bounding amounts of resveratrol. Therefore, the optimal extraction time for the extraction of resveratrol was 30 min in the following experiments.To obtain the desorption profile of resveratrol, different time intervals (i.e., 5, 10, 15, 20, 30, 45, and 60 min) were evaluated. Fig. 8b illustrates that 20 min is sufficient to accomplish desorption process. Therefore, desorption time was set at 20 min.
3.6. Validation of the method
The linear range obtained for analyzing resveratrol in wine samples ranged from 15 ng mL−1 to 750 ng mL−1. The regression equation was y = 1.7403x − 1.3483 with correlation coefficient of 0.9998 for resveratrol. The limit of detection (LOD) and limit of quantification (LOQ), defined as 3 and 10 times of the signal to noise ratio were 4.42 and 15.06 ng mL−1, respectively.
The recovery test using standard addition method was used to evaluate the repeatability and accuracy of the MMIPs-HPLC extrac- tion process. The recoveries, which were calculated by spiking resveratrol standard into the sample solutions at three levels, range from 79.3% to 90.6% with the RSD% values ranging from 2.8% to 8.2% (Table 4). This result demonstrated that the proposed method was a suitable method for the determination of resveratrol in wine samples.
3.7. Analysis in real samples
The present study aims to provide selective and practical MMIPs as adsorption materials, which can avoid the residual template leakage and apply in the determination of analytes from compli- cated samples. Our study had verified that RH and resveratrol could be well separated by HPLC. Three wine samples, Qilian ice white wine, Mogao dry red wine, and red wine, were extracted by MMIPs and MNIPs (Section 2.4). 40 mg MMIPs were used to avoid the possi- ble interferences from the wine samples and increase the number of available binding sites. The chromatograms of the initial wine (Qil- ian ice white wine), extracted by MNIPs and MMIPs, and standard addition in wine (40 ng mL−1) extracted by MMIPs are exhibited in Fig. 9. Resveratrol could not be directly determined because of the interference of wine matrix (Fig. 9a). No apparently resvera- trol peak is observed in Fig. 9b in the solutions extracted by MNIPs, and the selective adsorption of resveratrol from wine sample is obtained in the solutions extracted by MMIPs (Fig. 9c and d). The contents of resveratrol in the three wine samples are found to be 59.6, 196.0, and 44.8 ng mL−1, respectively. These results demon- strated that the prepared MMIPs were selective sorbents for the extraction of resveratrol in wine, which could supply some guid- ance for the determination and separation of active compounds with low concentration in real samples.
4. Conclusions
In this study, magnetic surface molecularly imprinted polymers for adsorbing and recognizing resveratrol were prepared using RH as dummy templates. The obtained MMIPs were characterized via SEM, FT-IR, XRD, and VSM. The selectivity recognition properties of the MMIPs were evaluated and the results showed that the MMIPs had high adsorption capacity and selectivity for resveratrol. The extraction procedure took a short time to reach the equilibrium of adsorption and desorption. The MMIPs were easily collected using an external magnetic field, avoiding the steps of making packed columns as the traditional SPE. Furthermore, the MMIPs could effectively avoid the potential risk of residual templates leakage and ensured the reliability. The analytical method of resveratrol in real wine samples by using RH MMIPs as sorbents was developed, and the results showed that the MMIPs could selectively recognize and effectively extract the analyte from wine samples. The MMIPs were efficient adsorbent sorbents for resveratrol, and had great potential application in enrichment and determination of resveratrol from real samples.