A molecularly-imprinted polymer decorated on graphene oxide for the selective recognition of quercetin

ZHAO Xiao-feng, DUAN Fei-fei, CUI Pei-pei, YANG Yong-zhen,3, LIU Xu-guang, HOU Xiang-lin

(1. Key Laboratory of Interface Science and Engineering in Advanced Materials (Taiyuan University of Technology), Ministry of Education, Taiyuan030024, China;2. College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan030024, China;3. Research Center on Advanced Materials Science and Technology, Taiyuan University of Technology, Taiyuan030024, China;4. Shanxi Engineering Research Center of Biorefinery, Institute of Coal Chemistry, Chinese Academy of Sciences, Taiyuan030001, China)

Abstract: A molecularly-imprinted polymer decorated on graphene oxide (GO/MIP) was designed with the aid of molecular modeling to select an appropriate functional monomer and to optimize its ratio to an imprinting molecule of quercetin (Qu) for the selective adsorption of Qu. The GO/MIP was prepared by free radical polymerization on the surface of GO using a functional monomer of 4-vinylpyridine at its optimal molar ratio to Qu of 4∶1. The GO/MIP was characterized by Fourier transform infrared spectroscopy, elemental analysis, Raman spectroscopy, thermogravimetric analysis, scanning electron microscopy, atomic force microscopy and adsorption measurements. Results indicate that the equilibrium time and adsorption capacity of the GO/MIP towards Qu are 30 min and 30.61 mg g-1 at 298 K, respectively and the adsorption data are well described by a pseudo-second-order kinetic model and the Langmuir isotherm model. From competing adsorption tests in a solution containing three flavonoids (Qu, kaempferol and rutin), the GO/MIP displays an excellent recognition ability for Qu with faster adsorption kinetics than bulk MIP without GO, and has highest adsorption capacity and selectivity for Qu among the three flavonoids, which is superior to un-imprinted polymer.

Key words: Graphene oxide; Molecularly-imprinted polymers; Quercetin; Flavonoids; Density functional theory

1 Introduction

Flavonoids are a class of naturally occurring polyphenolic compounds among the most prevalent classes of compounds in the plant kingdom. They possess many useful properties, including anti-inflammatory activity, antimicrobial activity, antioxidant activity, et al.[1]. Among flavonoids, quercetin (3, 3′, 4′, 5, 7-penta-hydroxy flavone, Qu) is a typical member in the family of flavonoids, widely existing in the leaves, fruits, and flowers of many plants[2]. Besides antioxidant properties, Qu possesses antitumor and antiviral properties as well as aiding in adjusting the immune system[3]. Therefore, the determination and separation of Qu from food and plants are of great importance.

At present, the high-performance liquid chromatography (HPLC) and/or mass spectrometric detection have been used for the determination of Qu at minor or trace levels[4,5]. Beyond that, the techniques, such as instantly heated reflux extraction, microwave-assisted extraction, ultrasonication, maceration, pressurized fluid extraction, matrix solid-phase dispersion and solid phase microextraction, have been used to extract and separate Qu from complex samples[6,7,8]. Although the techniques have unique advantages, their applications are limited owing to the interference of the complexity of the sample matrices and the poor affinity and selectivity for the target compounds. So, it is urgent to further improve the selectivity of these methods to determine and separate Qu from complex samples.

An effective way to attain this objective is based on molecularly-imprinted polymers (MIPs), which are synthesized and artificial cross-linked polymers with stable cavity size, shape, and a certain sequence of functional groups complementary to the target analyte[9]. Since MIPs have many excellent characteristics, such as high selectivity, good stability and long life, they have been widely used in catalysis[10], solid phase extraction, chromatography separation[11,12]and sensors[13,14]. For the determination and separation of Qu, MIPs were mainly applied to the enrichment process prior to the determination by HPLC and MS by selective adsorption from complex matrices in solid phase extraction. Recently, some MIPs have been fabricated for the determination and separation of Qu[15,16]. Although the MIPs prepared by bulk polymerization exhibit high selectivity, some disadvantages still exist, such as time-consuming and complicated preparation process, low binding affinity, high diffusion barrier, low mass transfer rate, and poor site accessibility[17].

Currently, surface imprinting techniques combining with nanomaterials are effective in solving the fore-mentioned problems, in which most imprinted sites are located at or near the material surface, thus enabling the surface-imprinted polymers on nanomaterials to possess remarkable performance over the bulk imprinted materials, such as the complete removal of templates, good accessibility to the target species, and low mass-transfer resistance[18]. Obviously, the support material is a crucial factor determining the performance of surface MIPs. At present, molecular imprinting strategy at the surface of some solid supports for adsorption of polyphenols typically uses silica beads[19,20], Fe3O4nanoparticles[21], and chitosan beads[22]as supports. However, some shortcomings involving low adsorption capacity and slow adsorption rate still exist in the MIPs based on above solid supports due to their low surface areas. Therefore, it is indispensable to look for a support to overcome the above shortcomings.

Graphene and its derivative graphene oxide (GO), as novel two-dimensional (2D) carbon nanomaterials with a monolayer structure and honeycomb lattice, have been regarded as prospective candidates as supports for fabricating surface MIPs owing to their small dimension with a higher surface-to-volume ratio and larger specific surface area than three-dimensional silica and other nanoparticles, which are beneficial to the generation of the composites of graphene or GO with thin MIP layers. Because the MIPs are located on the surface of graphene or GO, the resulting MIPs decorated on graphene oxide or graphene (GO/MIP or graphene/MIP composite) not only possess a faster adsorption and desorption dynamics but also exhibit a high binding capacity and high selectivity towards template molecules, demonstrating the promising application of graphene or GO as supporting material in the preparation of MIPs. Moreover, the largeπ-electron system of graphene or GO also has a strong affinity for carbon-based ring structures (e.g. Qu), which are ubiquitous in drugs, pollutants, and biomolecules[23]. Therefore, these attractive properties of graphene or GO in the preparation of MIPs make it possible to prepare GO/MIP composites for improving the selectivity, the binding affinity and kinetics of surface MIPs for adsorption of polyphenols.

Actually, the MIP layer has been grafted on the 2D structure of graphene or GO[24-29]. Although remarkable progress has been made to the various preparation methods and different practical applications of GO/MIP composites, the study of imprinted preassembly system has been rarely involved, which plays a decisive role in the performance of GO/MIP composites. Obviously, computer simulation seems to be rapid and effective for understanding molecular imprinting mechanism in depth and designing corresponding MIPs[30,31]. Therefore, the purpose of this study is to prepare a GO/MIP composite for Qu adsorption on the basis of the investigation of imprinted preassembly system including the selection of optimum monomer and its ratio to Qu to improve the selectivity and binding kinetics of surface MIPs and provide the guidance for the preparation of surface MIPs for adsorption of polyphenols.

Herein, GO was used as a carrier to fabricate the MIPs by surface free radical polymerization (FRP) in consideration of its easy operation and applicability to almost all of vinyl monomers. Qu was selected as the template for this investigation and three popular functional monomers and their ratios to the template were theoretically evaluated. A typical procedure for preparing GO/MIP composites is illustrated in Fig. 1, which involves three stages. First, GO was synthesized from natural graphite by the Hummers method[32]. Subsequently, the silane coupling agent,γ-methacryloxypropyl trimethoxysilane (MPS), with vinyl groups was introduced onto the surface of GO to obtain vinyl-functionalized graphene oxide sheets (MPS-GO). Finally, GO/MIP composites were fabricated by thermal polymerization using 4-vinylpridine (4-VP) as the functional monomer, Qu as the template molecule, Ethylene glycol dimethacrylate (EGDMA) as the cross-linker. The prepared GO/MIP composites were systematically characterized by Fourier transform infrared spectroscopy (FT-IR), elemental analysis (EA), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), atomic force microscopy (AFM) and Raman spectroscopy. The prepared GO/MIP composites exhibited fast binding kinetics, decent binding capacity, and good selectivity towards the template Qu at low concentrations in methanol.

Fig. 1 Schematic procedure of the GO/MIP composite preparation.

2 Experimental

2.1 Materials and chemicals

Graphite powder, quercetin (Qu, 97%), rutin (Ru, 97%), kaempferol (Ka, 97%) and 4-vinylpridine (4-VP, 96%) were purchased from Aladdin Chem. Co., Ltd. (Shanghai, China) and Fig. 2 shows the chemical structures of Qu, Ru and Ka. H2SO4(98%) and H2O2(30%) were obtained from Xilong (Guangdong, China) and Dongfang (Beijing, China) Chem. Co., Ltd., respectively. KMnO4and NaNO3were obtained from Kermel Chem. Reagent Co., Ltd. (Tianjin, China). Acetic acid andγ-methacryloxypropyl trimethoxysilane (MPS) were obtained from Dongli Chemical Reagent Factory (Tianjin, China). Hydrochloric acid, phosphoric acid, methanol, ethanol, N, N-dimethylformamide (DMF) and 2, 2-azobisisobutyronitrile (AIBN) were obtained from Guangfu Fine Chemical Research Institute (Tianjin, China). Ethylene glycol dimethacrylate (EGDMA, 98%) was obtained from Alfa Aesar (Tianjin, China). All the reagents were used as received without further purification.

Fig. 2 Chemical structures of Quercetin (Qu), Kaempferol (Ka) and Rutin (Ru).

2.2 Apparatus

The morphologies and structures of the products were characterized by scanning electron microscopy (SEM; JSM-6700F, operating at 10 kV, Japan) and atomic force microscopy (AFM; SPI 3800N). The GO and functionalized GO were characterized by Fourier transformation infrared spectroscopy (FT-IR; Agilent, Cary 630, USA), Raman spectrometry (Jobin-Yvon HR 800, with laser excitation at 532 nm), elemental analysis (EA; Elementar, Hanau, Germany) and thermogravimetric analysis (TGA; Netzsch TG 209 F3, operating from 100 to 800 ℃ with a heating rate of 10 ℃ min-1under nitrogen atmosphere, Germany). The adsorption properties were determined by using a Shimadzu high-pressure liquid chromatograph (LC-10AT) equipped with a UV-Vis detector (SPD-10A) and a Shim-pack VP-ODS C18 column (5 μm, 150 mm × 4.6 mm). The mobile phase was a methanol/water mixture solution (Vmethanol∶Vwater= 6∶4) containing 0.4% phosphoric acid with a flow rate of 0.6 mL min-1. The injection sample volume was 5.0 μL and the wavelength for UV detector was 256 nm. All solutions were filtered through a 0.22 μm polytetrafluoroethylene (PTFE) membrane before detection.

2.3 Molecular modeling

In a non-covalent molecular imprinting, complexes assembled by non-covalent interactions are formed in the pre-polymerization mixture, which govern the resulting binding site distribution and the recognition properties of the imprinted polymer matrix[33]. Therefore, analysis of the monomer-template complexes at molecular level can offer a deep insight into the imprinting based on self-assembly.

The density functional theory (DFT) simulation was introduced and all calculations were performed using DMol3code available from Materials Studio 5.5 to investigate the properties of the pre-polymerization complexes[34]. Herein, molecular geometries of Qu, 4-VP, methacrylic acid (MAA) and acrylamide (AM) were optimized on the basis of the Kohn-Sham equation within the generalized gradient approximation (GGA) with Perdew-Burke-Ernzerhof (PBE) functional for the exchange and correlation effects of the electrons. All electron calculations were performed with a double numerical polarization (DNP) basis set. The plane wave basis set was restricted by global orbital cutoff energy of 0.37 nm. The criterion of the self-consistent field procedure was converged to 10-5a.u. for population analysis. To improve the accuracy of the simulation, calculation of weak interactions, Tkatchenko and Scheffler (TS) method was used for DFT with dispersion corrections (DFT-D)[34].

The optimal monomer and optimal ratio of monomer to template were investigated according to the structural parameters of the hydrogen-bond model and the binding energy of the complex. The main procedures of computational simulation are as follows. First, the minimum energy conformations of Qu, 4-VP, MAA and AM were optimized, and the information containing the energy and Millikan charge was obtained. Second, the hydrogen-bonded complex model was built by analyzing the Millikan population, from which it was possible to decide the position easiest to form hydrogen bond. Then, the functional monomer and the template molecule were arranged in the same plane to simplify the complex model. The structural parameters of the hydrogen-bond model were calculated at the O-H…N (O-H…O) atom distances of 0.25 nm between the hydrogen atom of hydroxy of Qu in position 3′ and the nitrogen atom of the pyridine ring of 4-VP (the oxygen atom of carbonyl of the MAA and AM) with the fixed O-H…N (O-H…O) angle of 180°[35]. Finally, on the basis of the modeling, various complexes formed between different functional monomers and template were considered to select the optimal monomer. Simultaneously, the ratio of the above optimal monomer to the template in the complexes was further optimized in the range from 1∶1 to 5∶1.

The binding energy (ΔE) of the complex can be obtained by the following formula:

ΔE=Ecomplex-Etemplate-nEmonomer

(1)

where,Ecomplex(kJ mol-1) is the potential energy of the complex,Etemplate(kJ mol-1) is that of template,Emonomer(kJ mol-1) is that of monomer and thenvalue represents the number of monomer among monomer-template complexes.

2.4 Preparation of GO and MPS-GO

Graphene oxide (GO) was prepared from graphite powder according to the Hummers method[32]. Typically, 5.0 g of graphite powder, 2.5 g of NaNO3and 115 mL of H2SO4were mixed under magnetic stirring for 30 min in an ice bath, then 15 g of KMnO4was slowly added to the above mixture. Next, the mixture was transferred to a water bath of 35 ℃ and kept stirring for 30 min. Later, 230 mL of water was slowly added to the above reaction system, which was kept stirring for 15 min at 98 ℃. Subsequently, 700 mL of water was added and then 50 mL of H2O2(30%) was slowly added to the reaction system. Finally, the overall reaction was stopped when the color of the mixture was transformed from dark brown to yellow, which suggested the formation of graphite oxide. Then, HCl (1 L, 1 mol·L-1) and sufficient amount of distilled water were consecutively used to wash the obtained yellow mixture. The graphite oxide was separated from the mixture by spray drying method and the obtained graphite oxide (0.5 g) was dispersed into 500 mL of deionized water and exfoliated by ultrasonic irradiation at 40 kHz for 180 min. Finally, the GO was dried by the freeze-drying method.

The MPS-GO was prepared by grafting the silane coupling agent 3-methacryloxypropyl trimethoxysilane (MPS) onto GO surface. Specific steps are as follows. 0.3 g of GO was dispersed in a mixed solution containing 20 mL of water and 40 mL of ethanol, after 30 min of ultrasonic dispersion, 2 mL of MPS was slowly added dropwise to the mixture solution. Simultaneously, the pH of the solution was adjusted to 5 by acetic acid and the solution was kept stirring for 30 min at room temperature. Finally, the solution was heated to 65 ℃ and kept stirring for 4 h, and the obtained product was washed with ethanol three times and dried at 50 ℃ for 12 h to get vinyl-functionalized GO sheets (MPS-GO).

2.5 Preparation of GO/MIP composite

GO/MIP composite was synthesized by forming MIP layer onto the MPS-GO surface via polymerization using Qu as the template molecule and 4-vinylpridine as the functional monomer. Concisely, 80 mg of MPS-GO was dispersed in 20 mL of DMF, and then 302.24 mg (1 mmol) of Qu and 430 μL (4 mmol) of 4-VP were consecutively added to the above solution. After self-assembly of 4-VP and Qu for 60 min in the solution, 1.885 mL (10 mmol) of EGDMA and 33 mg of AIBN dissolved in 5 mL of DMF were added into the solution, and stirred for 30 min under nitrogen atmosphere. Subsequently, the reaction system was heated to 60 ℃ and the reaction proceeded for 24 h in an oil bath. Finally, the resulting product was thoroughly washed with ethanol to remove unreacted reagents and then transferred to a Soxhlet extractor to elute template molecules with a mixed acid (Vaceticacid∶Vmethanol= 1∶9) for 24 h. The obtained product was first washed with ethanol and then deionized water until neutral and freeze-dried for 24 h to gain the GO/MIP composite.

For comparison, the synthesis protocol of blank non-imprinted polymers (GO/NIP) was adopted with the same procedure and conditions above except that no template Qu was added in polymerization.

2.6 Adsorption test of GO/MIP composite

All the binding experiments were carried out in centrifuge tubes using a batch technique. Before binding experiments, a calibration curve was obtained from the HPLC-UV measurement of the Qu solutions at different concentrations.

In order to investigate the adsorption kinetics of the GO/MIP composite, seven aliquots of 2 mg of the GO/MIP composite were separately dispersed in 2.0 mL of 100 mg L-1Qu solution in methanol. Then, the mixture was continuously stirred on a shaker with a constant speed of 100 r/min at 25 ℃. Finally, the seven aliquots were withdrawn at different times: 1, 2, 5, 10, 20, 30 and 60 min and filtered through a 0.22 μm membrane filter. The concentration of the solution at different times was measured by HPLC-UV at 256 nm and the adsorption capacityQt(mg g-1) was calculated according to the following equation:

(2)

where,C0is the initial concentration of Qu solution (mg L-1),Ctis the Qu concentration at different times (mg L-1),Vis the volume of Qu solution (mL), andmis the mass of GO/MIP composite (mg).

For the adsorption isotherm experiments, seven aliquots of 2 mg of the GO/MIP composite were separately added to 2 mL of Qu solution in methanol with different initial concentration (5-200 mg L-1). After static adsorption for 60 min at 25 ℃, the saturated polymer was separated and filtered by a syringe filter. The concentration of the filtrate was determined by HPLC-UV at 256 nm and the equilibrium adsorption capacityQe(mg g-1) was calculated according to the following equation:

(3)

where,C0is the initial concentration of Qu solution (mg L-1),Ceis the equilibrium concentration of Qu at different initial concentrations (mg L-1);Vis the volume of Qu solution (mL), andmis the mass of the GO/MIP composite (mg).

2.7 Selectivity

Considering the complexity of the matrix in real samples, a mixture solution containing three flavonoids (Qu, Ka, Ru) was used to evaluate the selectivity of the GO/MIP composite towards Qu. Meanwhile, before binding experiments, a mixed calibration curve was obtained from HPLC-UV measurement of the mixed solutions of flavonoids at different concentrations in consideration of the mutual interference of the flavonoid components.

The selectivity of the GO/MIP composite was estimated using Ru and Ka as competitive molecules with an initial concentration of 100 mg L-1for each. The GO/MIP or GO/NIP composite (2 mg) was added to a centrifuge tube containing 2 mL of flavonoid solution, and the centrifuge tubes were subjected to shaking for 60 min at 25 ℃. Finally, the amounts of remaining flavonoids were tested by HPLC-UV under the above-mentioned conditions.

The selectivity of the GO/MIP composite was evaluated by the distribution constant (Kd), selectivity coefficient (k), and relative selectivity coefficient (k′)[21,28,36]. The distribution constant (Kd, L g-1) for each substance was calculated according to the equation:

(4)

where,Qe(mg g-1) andCe(mg L-1) are the same as mentioned in the equation 3.

The selectivity coefficient (k) of the GO/MIP composite for Qu in regard to the competing substances Ru and Ka can be defined by the following equation:

(5)

Thekvalue gives an estimation on the recognition ability and selectivity of the GO/MIP composite for Qu with respect to other competing compounds.

Finally, a relative selectivity coefficientk′ can be calculated as illustrated in the equation 6, where the value ofk′ shows the imprinting effect on binding affinity and selectivity of the GO/MIP composite against GO/NIP for Qu.

(6)

3 Results and discussion

3.1 Molecular simulation of the imprinted preassembly system

3.1.1 Screening of functional monomers

To screen functional monomers, three monomers, 4-VP, MAA and AM were theoretically selected to interact with Qu, and the binding energy, hydrogen-bond type and structural parameters of complex were studied. Obviously, the interactions of three complexes mainly come from hydrogen bond (A-H…B), which can be primarily defined by the parameters such as H…B length and A-H-B angle of the hydrogen bond[37]. The hydrogen bond is principally dominated by electrostatic interactions. Although other interactions are involved, such as charge-transfer, exchange-repulsion, induction and dispersion interaction, these interactions are much weaker than electrostatic interactions. According to the experimental results, the hydrogen-bond length is less than the sum of the A-H covalent-bond length and the Van der Waals radius of hydrogen and B atoms[38]. Besides, the more linear the hydrogen-bond angles, the stronger the hydrogen bond. Table 1 shows the energy and structural parameters of the studied monomer-template complexes calculated by DFT calculations in vacuum. All the bond lengths of complexes are in the range of hydrogen bond length and the smallest bond angles of complexes is 152.021°, which suggest the hydrogen-bond interaction between the selected functional monomers and template. With regard to the 4-VP and Qu complexes, the bond length is the minimum and the bond angle is the maximum among the three complexes. Thereby, the functional monomer 4-VP can form the strongest hydrogen-bond interaction with template Qu. To the best of our knowledge, the monomer giving the highestΔEcan be capable of forming the strongest complex with the specific template and thus be the most suitable monomer to prepare MIPs. As can be seen from Table 1, 4-VP presents the highest binding energy with Qu, -36.877 kJ mol-1. This result is consistent to the order of the bond length and angle between monomers and template. Hence, 4-VP was chosen as the functional monomer for synthesizing MIPs.

Table 1 Energy and structural parameters of the studied monomer-template complexes calculated by DFT calculation in vacuum.

Note: (1) Interatomic separation (O-H, H…X and O-X, X represents the nitrogen or oxygen atom)， (2) Valence angles (∠(OHX)), In the above hydrogen-bond type, all the hydroxy group comes from the hydroxyl of Qu in position 3′and the oxygen atom belongs to the carbonyl group of MAA or AM. Van der Waals radius:ro= 0.14 nm;rH= 0.12 nm;rN= 0.15 nm.

3.1.2 Optimization the ratio of 4-VP to Qu

The binding energy of complexes formed at different ratios of 4-VP to Qu was analyzed by DFT calculations (The energy parameters are listed in Table 2). The modeling of 4-VP and Qu complexes at the different ratios (5∶1 to 1∶1) was based on the Millikan charge on the hydrogen atom of the hydroxyl groups in Qu molecule (The size of its Millikan charge is in the order of position 3′, 4′, 3, 7, 5). With the increase in the ratio of 4-VP to Qu, the binding energy becomes large, indicating that the complex system becomes stable. However, the change in binding energy becomes the maximum (63.451 kJ mol-1) when the ratio of 4-VP to Qu changes from 3∶1 to 4∶1. As the ratio of 5∶1 is reached, the binding energy is

reduced to 35.528 kJ mol-1. This result can be explained by the formation of intramolecular hydrogen bond between the hydrogen atom of the hydroxyl group in position 5 and the oxygen atom in position 4, which allows an enhanced electronic delocalization and makes the molecular structure of complex more stable than the structure of complex formed by the intermolecular hydrogen bond between the hydrogen atom of the hydroxyl group in position 5 and the nitrogen atom of the 4-VP[39,40]. Hence, the most stable complex conformation is achieved when the ratio of 4-VP to Qu is 4∶1. In a word, the most suitable functional monomer is 4-VP for Qu and the optimum ratio of 4-VP to Qu is 4∶1, which were used for the synthesis of MIPs in following sections.

Table 2 Binding energies (ΔE) of complexes at different 4-VP to Qu molar ratios by DFT calculation in vacuum.

3.2 The morphology and structure of the GO/MIP composite

As shown in Fig. 3, the morphology of GO, MPS-GO and the GO/MIP composite was examined by SEM. In Fig. 3a or 3b, GO sheets exhibit a fairly smooth surface and layer-like structure with typical wrinkles, which prevent the aggregation of GO and maintain a high surface area[41]. In comparison with the GO, MPS-GO shows shallower wrinkles (Fig. 3c) and rougher surface (Fig. 3d) because of the MPS coating on GO surface. Quite differently, the GO/MIP composite have no significant folding or overlapping (Fig. 3e) but rather rough surface (Fig. 3f), which is an indication of grafting MIP layer on the GO surface.

Fig. 3 SEM images of (a, b) GO, (c, d) MPS-GO and (e, f) the GO/MIP composite.

The cross section analysis of AFM images was used to characterize the 2D surface morphology and the thickness of GO, MPS-GO and the GO/MIP composite. As displayed in Fig. 4a, the typical AFM image of GO with a flat structure shows an average thickness of 4.99 nm, roughly corresponding to 4-5 layers of GO nanosheets[42]. After GO is modified by MPS, the thickness is increased to about 15.88 nm for MPS-GO (Fig. 4b), implying that the MPS layer of about 5.44 nm was coated onto the two sides of GO surface. After grafting MIPs on MPS-GO, the the GO/MIP composite present an intensive and uniform coverage of MIPs onto the MPS-GO sheet and with an overall thickness of about 31.46 nm, consequently, an MIP layer with a thickness of 7.79 nm (31.46-15.88)/2=7.79) is attached to each side of the MPS-GO surface.

Fig. 4 AFM images and surface profiles of (a) GO, (b) MPS-GO and (c) GO/MIP composite.

Fig. 5 FT-IR spectra of (a) GO, (b) MPS-GO and (c) GO/MIP composite.

The existence of imprinted layer on the GO was further verified by elemental analysis (Table 3). From Table 3, the carbon content in GO is 47.44%, which is suitable for the further modification because of high oxidation degree, and the nitrogen content is 0.29%, which is mainly caused by the oxidation for preparing GO. After MPS modification, the carbon content of MPS-GO increases from 47.44% to 48.96% and the C/N atomic ratio increases from 190.85 to 228.48. Furthermore, the carbon and nitrogen contents in the GO/MIP composite increase to 51.45% and 1.23%, respectively, therefore, the C/N atomic ratio has a dramatical decrease, which manifests that the imprinted layer made of 4-VP and EGDMA is anchored onto the surface of MPS-GO.

Table 3 Elemental compositions of GO, MPS-GO and the GO/MIP composite.

TGA can reveal the composition and thermal stability of samples[47]. Fig. 6 exhibits the TGA curves of GO, MPS-GO and the GO/MIP composite. For GO (curve a), a sharp decrease in weight occurs below 250 ℃, which is attributed to the pyrolysis of oxygen-containing groups from the surface of GO (～26 wt%)[48], and a total weight loss of 45 wt% is observed within 800 ℃. Compared with GO, MPS-GO (curve b) is more stable and shows a lower weight loss (～39 wt%) within 800 ℃, revealing that MPS-GO has an improved thermal stability after the chemical modification. In contrast with MPS-GO, the GO/MIP composite (curve c) shows a more weight loss below 214 ℃, which results from the decomposition of labile oxygen functional groups. Besides, the weight loss of the GO/MIP composite is more dramatical between 214 and 800 ℃ in comparison with GO and MPS-GO, which might be attributed to the degeneration of carbon skeleton for MIPs. the GO/MIP composite shows a 57 wt% weight loss in nitrogen atmosphere until 800 ℃, whereas GO and MPS-GO have weight losses of 45 wt% and 39 wt% until 800 ℃, respectively. Therefore, the difference in thermal stability among GO, MPS-GO and the GO/MIP composite reveals the grafting of the MIP layer onto the surface of GO.

Fig. 6 TGA curves of (a) GO, (b) MPS-GO and (c) the GO/MIP composite.

As shown in Fig. 7, GO (a), MPS-GO (b) and the GO/MIP composite (c) were evaluated by Raman spectroscopy. The three samples haveD(～1 361 cm-1) andG(～1 587 cm-1) bands, which correspond to the disordered sp3carbon structures and sp2ordered crystalline graphite-like structures, respectively[49]. The intensity ratios (ID/IG) of GO, MPS-GO and the GO/MIP composite are 0.964, 0.972 and 1.003, respectively. In general, the increase ofID/IGratio reflects the increase of disorder present within materials[50]. It has been reported that silica, when non-covalently attached to carbon nanotubes, showed no significant band shift or relative intensity variation after the coating process[49]. Therefore, the increase ofID/IGin GO, MPS-GO and the GO/MIP composite indicates that MPS is consolidated on the surface of GO by covalent linkage and the MIP layer is also deposited on the surface of MPS-GO by covalent linkage. In addition, theDandGbands of the GO/MIP composite are located at 1 348 and 1 578 cm-1, which are downshifted by 13 and 9 cm-1with respect to GO, respectively. The Raman shifts ofDandGbands for the GO/MIP composite offer a strong argument for the charge transfer between GO and MIPs, demonstrating that there is covalent interaction between GO and MIPs[24,51]. The results of Raman spectroscopy reveal that MIPs are grafted onto the surface of GO.

Fig. 7 Raman spectra of (a) GO, (b) MPS-GO and (c) the GO/MIP composite.

3.3 Adsorption ability of the GO/MIP composite

3.3.1 Adsorption kinetics

Adsorption kinetics of the GO/MIP composite, GO/NIPs and GO towards Qu were investigated to evaluate the adsorption performance of the GO/MIP composite, as represented in Fig. 8. It can be seen that the adsorption rate of the GO/MIP composite is quite high within 20 min, then it decreases and the adsorption equilibrium is reached within 30 min. The adsorption processes of GO/NIPs and GO are also completed within 30 min. The results reveal a remarkable rapid adsorption process of Qu molecule onto the GO/MIP composite, which is faster than that on conventional bulk MIPs with an adsorption equilibrium time of about 6 h[15]and the surface imprinted SiO2/MIP with an adsorption equilibrium time of about 3 h[19,20]. This is because the MIP layer is anchored on the surface of GO with a large surface-to-volume ratio, generating a high ratio of surface-imprinted sites, easily accessible to the template molecules in short adsorption time. Furthermore, the adsorption capacity of the GO/MIP composite (30.61 mg g-1) is much higher than those of GO/NIPs (19.08 mg g-1) and GO (11.58 mg g-1), manifesting the formation of specific recognition sites on the surface of GO.

Fig. 8 Adsorption kinetic curves of GO, GO/NIPs and the GO/MIP composite for Qu.

The dash lines are the pseudo-first-order kinetics model; The solid lines are the pseudo-second-order kinetics model. Testing conditions: 2 mg of adsorbent (GO, GO/NIPs or the GO/MIP composite), 2 mL of

Qu solution of 100 mg L-1in methanol, temperature 298 K. Data are represented as the mean ± standard deviation (SD) (n= 3).

To evaluate the adsorption process of the GO/MIP composite towards Qu, including mass transfer and possible chemical reaction, the experimental data for the adsorption kinetics of the GO/MIP composite, GO/NIPs and GO towards Qu were further analyzed by pseudo-first-order and pseudo-second-order kinetic models[46].The pseudo-first-order kinetic model is generally expressed as follows：

ln(Qe-Qt)=lnQe-k1t

(7)

where,tis the adsorption time (min),Qt(mg g-1) is the adsorption capacity at timet,Qe(mg g-1) is the equilibrium adsorption capacity,k1(min-1) is the first-order rate constant.

The pseudo-second-order kinetic model is expressed as:

(8)

where,QeandQtare defined as the same in the above pseudo-first-order model, k2is the rate constant of pseudo-second-order (g mg-1min-1).

The pseudo-first-order and pseudo-second-order curves of the GO/MIP composite, GO/NIPs and GO towards Qu are represented in Fig. 8, and the corresponding parameters are summarized in Table 4. The pseudo-second-order model describes better the adsorption process for the higher correlation coefficient value and closerQe,caltoQe,expthan the pseudo-first-order model, revealing that the pseudo-second-order mechanism is predominant and chemisorption might be the rate-limiting step that controls the adsorption process[52].

Table 4 Kinetics parameters for the adsorption of Qu by the GO/MIP composite, GO/NIPs and GO.

Note:Qe,expis the experimental value andQe,calis the value calculated by kinetic model.

3.3.2 Adsorption isotherms

To further investigate the adsorption capacity, the adsorption isotherms of the GO/MIP composite, GO/NIPs and GO were obtained by static equilibrium experiments. As represented in Fig. 9, the GO/MIP composite exhibits a highest adsorption capacity among the three, which is attributed to the specific binding sites complementary to Qu within the GO/MIP composite. Besides, it can be noticed that the adsorption capacity of the GO/MIP composite increases with increasing equilibrium concentration of Qu, which can be explained by the increasing driving force of the concentration gradient because the increase in Qu concentration can accelerate the diffusion of Qu molecules within the GO/MIP composite[53].

The solid lines are the Langmuir model simulation; The dash lines are the Freundlich model simulation. Testing conditions: 2 mg of adsorbent (GO, GO/NIPs or The GO/MIP composite), 2 mL of Qu solution of 5-200 mg L-1in methanol, temperature 298 K. Data are represented as the mean ± standard deviation (SD) (n= 3).

Fig. 9 Adsorption isotherms of GO, GO/NIPs and the GO/MIP composite towards Qu.

In order to elucidate the adsorption mechanism of the GO/MIP composite towards Qu, the data obtained from static adsorption experiments were processed by two commonly used equilibrium isotherm models, Langmuir and Freundlich models. The Langmuir model assumes that the adsorption takes place on a homogeneous surface with monolayer coverage and uniform energies. The equation of Langmuir adsorption model is described as[46]:

(9)

where,Qe(mg g-1) andQmax(mg g-1) are the equilibrium adsorption amount and theoretical maximum adsorption capacity of Qu, respectively,Ce(mg L-1) is the concentration of Qu in equilibrium solutions, andKL(L mg-1) is the Langmuir adsorption equilibrium constant towards Qu. The values ofQmaxandKLare calculated from the slope and intercept of the linear plot ofCe/QeagainstCe.

The Freundlich adsorption model is an empirical model under the precondition of multilayer adsorption on a heterogeneous surface. The equation of the Freundlich adsorption model is expressed as[46]:

(10)

where,CeandQeare defined the same as in the Langmuir adsorption model andnandKFare the Freundlich constants that represent adsorption favorability and adsorption capacity, respectively. Ifn>1, adsorption is favorable.KFandncan be obtained by a linear plot of lnQeversus lnCe.

The fitting data with the above two different isotherm models are exhibited in Fig. 9 and the corresponding parameters are summarized in Table 5. By comparison of the correlation coefficient (R2) values between the Langmuir and Freundlich models, it can be seen that the Langmuir isotherm model fits the equilibrium data better than the Freundlich model, suggesting that the imprinting sites for Qu molecules are homogeneously distributed onto the GO/MIP composite surface. In addition, according to the Langmuir adsorption model, the value ofQmaxfor the GO/MIP composite towards Qu (32.15 mg g-1) is greatest among the three, GO/NIPs (11.99 mg g-1) and GO (20.24 mg g-1), which is ascribed to the special recognition sites within the GO/MIP composite.

Table 5 Isothermal parameters for the adsorption of Qu by the GO/MIP composite, GO/NIPs and GO.

3.3.3 Adsorption selectivity

In order to verify the selectivity of the GO/MIP composite towards Qu, Ka and Ru were selected as the competing compounds taking into account their structural similarity with Qu to a certain extent (their structures are shown in Fig. 2). The selective recognition of the GO/MIP composite was investigated by equilibrium adsorption tests in a mixture solution containing these three flavonoids, and the results are shown in Fig. 10. From Fig. 10, it can be observed that the adsorption capacity of the GO/MIP composite towards Qu is the highest among the three flavonoids, suggesting that the GO/MIP composite has a strongest affinity for the template Qu. The imprinting effect of molecularly-imprinted materials can be further evaluated byKd,kandk′ from the equations (4), (5) and (6) in section 2.7, as summarized in Table 6. Thekvalues of the GO/MIP composite for Qu over Ru and Ka are 4.765 and 2.017, respectively, which are highly larger than 1.0, revealing that the GO/MIP composite has a significant selectivity towards the template Qu against the other two flavonoids. In contrast, the k values of GO/NIPs for Qu over Ru and Ka are only 1.339 and 1.026, respectively, which are all close to 1.0, meaning that the GO/NIPs have no selectivity. All these results further elucidate the origin of good selectivity of

Fig. 10 Binding capacities of the three different flavonoids onto the GO/MIP composite and GO/NIPs.

Test compoundsGO/MIP Kd (L g-1)kGO/NIPsKd (L g-1)kk'Qu0.296/0.117//Ka0.1472.0170.1141.0261.966Ru0.0624.7650.0881.3393.557

the GO/MIP composite from the imprinting effect.

The high adsorption selectivity and specificity of the imprinted GO/MIP composite towards the template Qu could be attributed to two major roles[17,54]. First, the adsorption specificity of the GO/MIP composite is contingent on the multiple weak interactions (such as the hydrogen-bond interaction,π-πbonds and Van der Waals forces) within the imprinted cavities for interaction with the template Qu. As for Ka molecule, although it has a similar structure or even smaller size than Qu, thekvalue of the GO/MIP composite for Qu over Ka reaches 2.017, indicating that the shape memory effect of microenvironment formed by multiple weak interactions plays an important role in the conformation memory. Second, the sterically complementary imprinted structure within the GO/MIP composite just fitting for the unique molecular structure of Qu also makes a great contribution to the adsorption specificity of Qu. As for Ru molecule, it has a similar structure to Qu except for the bulky glucoside group in position 3 with a large steric hindrance, which induces the difficulty for Ru molecule to enter the complementary cavities of the GO/MIP composite. Therefore, thekvalue of the GO/MIP composite for Qu over Ru reaches 4.765, indicating the importance of the steric complementarity of imprinted cavity. In short, the imprinted cavity with the microenvironment and steric complementarity specific to Qu is not suitable for Ru and Ka, which allows as-prepared GO/MIP composite to possess a potential application in terms of extraction and determination of Qu from complex matrix.

4 Conclusions

A computational model based on the DFT was established and used to screen the functional monomers and the optimal ratio of the template to functional monomer for polymerization of MIPs on GO surface. On this basis, the GO/MIP composite was prepared by surface free radical polymerization with the functional monomer 4-VP in a 4∶1 molar ratio towards Qu. A series of adsorption experiments in terms of kinetics, isotherms and selective recognition adsorption were conducted to evaluate the adsorption and recognition properties of the GO/MIP composite. As-prepared GO/MIP composite displays a fast adsorption kinetics, in which the adsorption equilibrium time and binding capacity were 30 min and 30.61 mg g-1at 298 K, respectively. In addition, the kinetics and isotherm data are well fitted with the pseudo-second-order kinetic model and Langmuir isotherm, respectively. In a mixed solution of flavonoids, the estimated relative selectivity coefficients (k′) of the GO/MIP composite for Qu over structural analogues Ka and Ru are 1.966 and 3.557, respectively, suggesting an excellent selectivity. Therefore, the GO/MIP composite exhibits fast binding kinetics, decent binding capacity, and excellent selective recognition capability towards template Qu at low concentrations of Qu in methanol solution, making the GO/MIP composite attractive for the specific determination and fast separation of Qu from flavonoids in natural products.