Accepted Manuscript Selective extraction and determination of chlorogenic acid in fruit juices using hydrophilic magnetic imprinted nanoparticles Yi Hao, Ruixia Gao, Dechun Liu, Gaiyan He, Yuhai Tang, Zengjun Guo PII: DOI: Reference:
S0308-8146(16)30005-X http://dx.doi.org/10.1016/j.foodchem.2016.01.004 FOCH 18578
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
6 August 2015 7 December 2015 1 January 2016
Please cite this article as: Hao, Y., Gao, R., Liu, D., He, G., Tang, Y., Guo, Z., Selective extraction and determination of chlorogenic acid in fruit juices using hydrophilic magnetic imprinted nanoparticles, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2016.01.004
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Selective extraction and determination of chlorogenic acid in fruit juices using hydrophilic magnetic imprinted nanoparticles Yi Haoa,b, Ruixia Gaob*, Dechun Liuc, Gaiyan Hea,b, Yuhai Tanga,b, Zengjun Guo a* a
b
School of Pharmacy, Xi’an Jiaotong University, Xi’an 710061, China.
Institute of Analytical Science, School of Science, Xi’an Jiaotong University, Xi’an 710049, China.
c
Department of Hepatobiliary Surgery, First Hospital of Xi’an Jiaotong University, Xi’an, 710061, China. * Corresponding authors: Tel.: +86 2982655399; fax: +86 2982655399. E-mail: [email protected] (R. Gao); [email protected] (Z. Guo).
1
Abstract In this paper, the novel hydrophilic magnetic molecularly imprinted nanoparticles were developed for selective separation and determination of chlorogenic acid in aqueous fruit juices. The polymers were prepared by using amino-functionalized magnetic nanoparticles as carriers, branched polyethyleneimine as functional monomer, and chlorogenic acid as template molecule. Branched polyethyleneimine with abundant active amino groups could react with template sufficiently, and its unique dendritic structure may amplify the number of the imprinted cavities. Meanwhile, it would improve the hydrophilcity of imprinted materials for attaining high extraction efficiency. The resulted polymers exhibit fast kinetics, high adsorption capacity, and favorable selectivity. In addition, the obtained nanoparticles were used as solid-phase extraction sorbents for selective isolation and determination of chlorogenic acid in peach, apple, and grape juices (0.92, 4.21, and 0.75 µg mL-1, respectively). Keywords: Magnetic separation, branched polyethyleneimine, solid-phase extraction, hydrophilcity, chlorogenic acid
2
1. Introduction Molecular imprinting, proposed by Wuff and Sarhan in 1972 (Wulff, & Sarhan, 1972), has become an attractive technique to create specific cavities complementary to the template molecule in shape, size, and chemical functionality. Molecularly imprinted polymers (MIPs) possess the advantages of predetermination, low cost, easy preparation, mechanical and chemical stability, applicability in harsh environment, and high specificity. Owing to their outstanding merits, MIPs have been widely used in the fields of chromatography (Shao et al., 2015; Santos, Tavares, Boralli, & Figueiredo, 2015), biosensor (Eren, Atar, Yola, & Karimi-Maleh, 2015; Lenain, Saeger, Mattiasson, & Hedström, 2015), drug delivery (Kempe, Pujolràs, & Kempe, 2015; Suksuwan et al., 2015), and solid-phase extraction (SPE) (Lata, Sharma, Naik, Rajput, & Mann, 2015; Yang et al., 2015). Among these applications, MIPs used as SPE adsorbents have exhibited a great prospect for the selective extraction or clean-up of target analytes from various complex matrices.
Traditionally, MIPs have to be packed into SPE cartridge or adopt centrifugation and filtration procedures when used in the process of SPE. The time-consuming and complicated course commonly yield low extraction efficiency. Therefore, magnetic Fe3O4 nanoparticles (NPs), because of their good biocompatibility, magnetic susceptibility, low toxicity, have been encapsulated into MIPs to solve the above problems. The resulting composites not only have magnetic characteristic for rapid separation but also possess high selectivity for recognition of the target molecule (Hao
3
et al., 2015; Huang, Zhou, Chen, Wu, & Lu, 2015; Liu et al., 2015).
Chlorogenic acid (CGA), a bioactive phenolic acid, is extensively distributed in fruit, plant, and used in feed industry. It is reported that CGA has several beneficial biological properties including antibacterial, antiphlogistic, antiviral, mutant resistance, and even inhibitory effects on carcinogenesis in large intestine, liver, and tongue (Bagdas et al., 2015; Heyman et al., 2015; Wang et al., 2015; Naso et al., 2014). CGA is water-soluble and usually coexists with some compounds hold similar functional groups such as caffeic acid, gallic acid, and so on. Thus, selective extraction of CGA from complex matrices prior to its quantification is urgently required. However, CGA-imprinted polymers previously reported were usually prepared in organic media (Gu et al., 2010; Saad, Madbouly, Ayoub, & Nashar, 2015), which could lead to poor recognition ability and high non-specific adsorption in aqueous matrices. Therefore, a facile and efficient approach to prepare hydrophilic CGA-imprinted polymers is highly desirable.
Polyethyleneimine (PEI), a water-soluble and non-toxic polycation (Bahulekar, Ayyangar, & Ponrathnam, 1991), consists of high content of functional groups (primary, secondary, and tertiary amine groups). What is more, PEI could assemble layer-by-layer with glutaraldehyde as cross-linking agent (Zhao et al., 2015). Based on these distinctive features, PEI has been widely applied in gas adsorption (Lee, Lee, & Park, 2015; Sabri, Guillemette, Guermoune, Siaj, & Szkopek, 2012), drug/gene delivery (Jung, Kim, Lee, & Mok, 2015; Ambattu, & Rekha, 2015), enzyme 4
immobilization (Khoobi et al., 2015), and heavy metals removal (Lindén, Larsson, Coad, Skinner, & Nydén, 2014; Chen et al., 2014).
Herein, we attempt to prepare novel hydrophilic MIPs adopting branched PEI as functional monomer and CGA as template molecule. The abundant functional groups on PEI can react with CGA through multiple hydrogen bonds and electronic interactions. Meanwhile, its unique dendritic structure and water solubility could greatly improve the density of recognition sites and hydrophilicity of MIPs. The characteristics and binding properties of the obtained MIPs were investigated. In addition, the resulted polymers were used as SPE sorbents and coupled with HPLC to selective extraction and determination of CGA in fruit juices. The developed approach would exhibit great potential for preparation of hydrophilic MIPs with satisfactory imprinting performance.
2. Experimental 2.1. Chemicals and reagents Ferricchloride hexahydrate (FeCl3·6H2O), 1,6-hexadiamine (HDM), glutaraldehyde, sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), ethylene glycol (EG), anhydrous sodium acetate (NaOAc), phosphoric acid (H3PO4), ethanol, acetonitrile, and acetic acid were provided by Xi’an Chemicals Ltd. CGA, caffeic acid (CA), cichoric acid (CCA), caftaric acid (CFA), protocatechuic acid (PCA), and polyethyleneimine (PEI) (MW=10000, Purity≥99.0%) were obtained from Aladdin Industrial Corporation. All reagents used were of at least analytical grade. 5
The ultrapure water (18.25 MΩ cm-1) was obtained from a WaterPro water system (Axlwater Corporation, TY10AXLC1805-2, China) and used throughout the experiments. 2.2. Instrumentation and analytical conditions JEM-2100 transmission electron microscope (TEM) (JEOL Co., Japan) was used to observe morphology of as-synthesized magnetic nanoparticles. Fourier transform infrared (FT-IR) spectra (4000-400 cm-1) were obtained via a Nicolet AVATAR-330 FT-IR spectrometer (Thermo Electron Co., U.S.A). The magnetic properties were measured at room temperature using an LDJ 9600-1vibrating sample magnetometer (VSM) (LDJ Co., U.S.A). The identification of the crystalline phase was carried out by a Rigaku D/max/2500v/pcX-ray diffractometer (Rigaku Co., Japan) with Cu Kα radiation. The HPLC profiles were recorded using a Shimadzu HPLC system equipped with LC-10AT pump, SPD-M 10A detector, CTO-10AS column oven, and Shimadzu VP-ODS C18 column (5 µm, 150 mm×4.6 mm). The column temperature was 30 °C. The optimized mobile phase was acetonitrile-0.05% phosphoric acid solution (85:15, v/v) delivered at a flow rate of 1.0 mL min−1, the injection volume was 20 µL, and the column effluent was monitored at 323 nm. Sample solutions were filtered through a nylon 0.22 µm filter before determination. 2.3. Preparation of Fe3O4@CGA-MIPs and Fe3O4@NIPs The amino-functionalized Fe3O4 nanoparticles (denoted as Fe3O4@NH2) were synthesized as our previous work (Gao, Zhang, Hao, Cui, & Tang, 2014). The 6
magnetic
molecularly
imprinted
nanoparticles
of
CGA
(designed
as
Fe3O4@CGA-MIPs) were prepared as follows: CGA (40 mg) was dissolved in 20 mL of phosphate buffered solution (PBS, pH=7.4) in a three-necked flask, and combined with Fe3O4@NH2 (200 mg). The mixture was allowed to react for 1h. Then 5 mL of PBS containing 30 mg of PEI (6.0 mg mL-1) and 30 µL of glutaraldehyde were added, the mixture was stirred for 6 h at room temperature. The obtained imprinted polymers were collected by an external magnetic field and washed with ethanol-HAc (95:5, v/v) to remove the embedded template until no adsorption was detected by HPLC. Then, the Fe3O4@CGA-MIPs were washed with ultrapure water and ethanol repeatedly, and dried under vacuum. For comparison, non-imprinted magnetic nanoparticles (designed as Fe3O4@NIPs) were prepared following the same procedure in the absence of the template molecule CGA. 2.4. Adsorption experiment and selectivity evaluation In kinetic adsorption test, 20 mg of Fe3O4@CGA-MIPs or Fe3O4@NIPs were suspended in 10 mL of PBS with CGA at a concentration of 0.40 mg mL-1, and shaken on a reciprocating shaking-table at regular times from 5 min to 50 min. Then the supernatants and polymers were separated by an external magnetic field and the concentration of CGA in the filtrate was measured by HPLC. The adsorption amounts (Q, mg g-1) of Fe3O4@CGA-MIPs or Fe3O4@NIPs to CGA were calculated according to equation (1), and the pseudo-first-order and pseudo-second-order rate kinetic models were applied to fit the kinetic data according to equation (2, 3).
7
(C 0 − C e )V m
(1)
ln ( Qe − Qt ) = ln Qe - ｋ1t
(2)
t 1 t 1 t = + = + 2 Qt k 2Qe Qe v0 Q e
(3)
Q=
Where C0 and Ce (mg mL−1) are the initial and equilibrium concentration of CGA, respectively. V (mL) and m (g) represent the volume of the CGA solution and the mass of the polymers. Qe and Qt are the amount of adsorbate onto sorbent at the equilibrium and time t (min). v0 (mg g-1 min-1) is the initial adsorption rate. k1 and k2 are the equilibrium rate constants of pseudo-first-order and pseudo-second-order equation, respectively.
In equilibrium binding test, 20 mg of Fe3O4@CGA-MIPs or Fe3O4@NIPs were dispersed into 10 mL of PBS with various CGA concentrations (0.050-0.80 mg mL-1), and incubated for 30 min at room temperature. The separation and detection procedures were conducted as described in kinetic adsorption test. For further evaluating the equilibrium adsorption, Langmuir and Freundlich isotherm models were selected to fit the experimental data. They are described as equation (4) and (5). C Ce 1 = + e Q Qmax K L Qmax
(4)
log Q = m log Ce + log K F
(5)
Where Ce (mg mL-1) is equilibrium concentration of adsorbate, Q (mg g-1) is the amount of CGA bound to Fe3O4@CGA-MIPs or Fe3O4@NIPs at equilibrium, Qmax (mg g-1) is the maximum adsorption capacity of the sorbent, KL (mL mg-1) and KF (mg 8
g-1) are the Langmuir and Freundlich constant respectively, and m is the Freundlich exponent which represents the heterogeneity of the system.
The selectivity of Fe3O4@CGA-MIPs was measured using the structure analogues CCA, CFA, CA, and PCA. 10 mL of the mixed standard solution of CGA and analogues at initial concentration of 0.40 mg mL-1 was incubated with 20 mg of Fe3O4@CGA-MIPs or Fe3O4@NIPs for 30 min, and then the operating sequence was the same as kinetic adsorption test. The specific recognition property of Fe3O4@CGA-MIPs was evaluated by imprinting factor (IF), which was calculated according to equation (6), and the selectivity coefficient (SC) was defined as expressed in equation (7).
IF =
SC =
QMIP QNIP
(6)
IFt IFc
(7)
Where QMIP and QNIP (mg g-1) represent the adsorption capacity of CGA on Fe3O4@CGA-MIPs and Fe3O4@NIPs, respectively. IFt and IFc are the imprinting factors for template molecule and competitive molecules. 2.5. Reusability To
evaluate
the
regeneration
of
Fe3O4@CGA-MIPs,
six
cycles
of
adsorption-desorption were conducted using the same polymers. 20 mg of polymers were added to 10 mL of CGA solution at a concentration of 0.40 mg mL-1 and incubated at room temperature for 30 min. Then, Fe3O4@CGA-MIPs were separated 9
by a magnet and the concentration of CGA in supernatant was quantified by HPLC. The adsorbed polymers were eluted by ethanol-HAc (95:5, v/v) for 6 h to ensure complete removal of CGA. The recovered imprinted nanomaterials were used for another five adsorption-desorption cycles, and every cycle of supernatant was collected and determined. 2.6. Selective extraction and determination of CGA in fruit juices Grape juice, apple juice, and peach juice (Huiyuan Co., Ltd., Beijing) were purchased from local market in Xi’an. 30 mL of fruit juice was incubated with 60 mg of Fe3O4@CGA-MIPs for 30 min at room temperature. Then Fe3O4@CGA-MIPs were separated magnetically, and eluted by ethanol-HAc (95:5, v/v) for 6 h. The juice and eluted samples were filtered by 0.22 µm nylon membrane filter and then for HPLC analysis. 3． ．Results and discussions
3.1. Preparation of Fe3O4@CGA-MIPs The synthesis procedure of Fe3O4@CGA-MIPs is depicted in Fig. 1. First, Fe3O4@NH2 with abundant of amino groups on their surface were synthesized through a one-step solvothermal reaction. Next, Fe3O4@NH2 were used as the carriers onto which CGA were immobilized through hydrogen bond and electrostatic interactions. Preimmobilization of template on supporters could not only make the formed imprinted cavities relatively order, but also enhance the number of functional binding sites, thus could improve imprinting effect to some degree compared to the 10
traditional free-template polymerization process (Liu, Zheng, Fang, & Xie, 2012). Then, a thin and adherent imprinted shell embedding CGA was obtained by the reaction between the PEI and
templates-supporter complex.
Finally,
the
Fe3O4@CGA-MIPs with recognition sites were obtained after the removal of the embedded CGA. Notably, PEI is a water-soluble polycation with a branched structure and rich amino groups, which make the prepared polymers possess high density of recognition sites and excellent water compatibility.
The imprinting effect of Fe3O4@CGA-MIPs is affected by many factors. To acquire satisfactory imprinting effect, we optimized typical factors including the selection of carrier, the amount of template, the concentration of PEI, and polymerization temperature and time. As shown in Fig. 2A, Fe3O4@NH2 could immobilize larger amount of templates compared with another three carriers involving pure magnetic nanoparticles (Fe3O4), silica-modified magnetic nanoparticles (Fe3O4@SiO2), and carboxyl-functionalized magnetic nanoparticles (Fe3O4@COOH) (SI). The results illustrated that the amino groups are more suitable to immobilize CGA through forming multi-hydrogen bonds and electrostatic interactions. The amount of template immobilized on Fe3O4@NH2 was investigated ranging from 25 mg to 50 mg, while the amount of Fe3O4@NH2 maintained at a constant value of 200 mg. As displayed in Fig. 2B, the Qim increased with increasing the amount of CGA from 25 mg to 40 mg, and then remained almost unchanged from 40 mg to 50 mg. The results illustrated that the immobilization capacity of Fe3O4@NH2 had been saturated over the 40 mg of CGA. Thus, the 40 mg of CGA was used in the remainder of this work. 11
The adsorption capacity and imprinting effect of Fe3O4@CGA-MIPs towards CGA are greatly influenced by functional monomer. Therefore, the concentration of PEI ranging from 0.8 mg mL-1 to 12.0 mg mL-1 was investigated. The results of the adsorption capacity (Q) and imprinting factor (IF) are shown in Fig. 2C, the Q and IF exhibit an upward trend, manifesting the augment in the number of recognition cavities with increasing the concentration of PEI from 0.8 mg mL-1 to 6.0 mg mL-1. With further increasing the concentration of PEI from 6.0 mg mL-1 to 12.0 mg mL-1, the Q increases slightly while the IF decreases obviously. This may be because excessive functional monomers generate high nonspecific adsorption. Therefore, the concentration of PEI of 6.0 mg mL-1 was adopted in this work.
Polymerization temperature and time play important roles in the morphology and adsorption ability of imprinted composite. Short polymerization time (5 h) or low temperature (50 oC) led to lower adsorption capacity, which would be attribute to that the less imprinted capacities are formed for CGA because of the uncompleted polymerization. With polymerization time and temperature increasing to 6 h and 60 oC, the Fe3O4@CGA-MIPs exhibit the highest adsorption capacity because the optimal recognition sites and the most suitable shell thickness are formed. However, further prolonging the polymerization time (7 h) and increasing the temperature (70 oC), the functional monomers may self-polymerize and block the recognition sites, resulting in low adsorption capacity (Table S1). Therefore, 6 h and 60oC are chosen as the polymerization time and temperature in this work.
12
3.2. Characterization of Fe3O4@CGA-MIPs Representative TEM images of Fe3O4@NH2 and Fe3O4@CGA-MIPs are shown in Fig. 3. It can be obviously seen that Fe3O4@NH2 and Fe3O4@CGA-MIPs exhibit approximately spherical morphology, good dispersibility, and relatively narrow size distribution. The particle diameters of Fe3O4@NH2 and Fe3O4@CGA-MIPs are about 80 nm and 96 nm. It follows that the thickness of imprinted layer is about 8 nm, which would be beneficial for the mass transfer between the solution and the surface of Fe3O4@CGA-MIPs.
Fig. S1A shows the surface groups on Fe3O4@NH2 and Fe3O4@CGA-MIPs analyzed by FT-IR spectrophotometer. The characteristic peaks of Fe-O group for Fe3O4@NH2, and Fe3O4@CGA-MIPs are all observed around 576 cm-1. The typical peaks at 3432 cm-1 and 1630cm-1 assigned to stretching and bending vibrations of N-H indicate that amino groups are strongly anchored on the magnetic nanoparticles through a one-pot solvothermal polymerization (Fig. S1A-a). The increased intensities of peaks of amino groups in Fe3O4@CGA-MIPs compared with that of Fe3O4@NH2 and the asymmetric and symmetric stretching peaks of -CH2 at 2860 cm-1 and 2770 cm-1 confirm that the imprinted shell has been ideally coated onto the surface of Fe3O4@NH2 (Fig. S1A-b).
The XRD patterns of the Fe3O4@NH2 and Fe3O4@CGA-MIPs are illustrated in Fig. S1B. In the 2θ region of 20-70o, six relatively discernible strong diffraction peaks corresponded to Fe3O4 (2θ = 30.2o, 35.6o, 43.2o, 53.5o, 57.2o, and 62.8o) are observed 13
in the curves of two samples, and the peak positions at the corresponding 2θ values are indexed as (220), (311), (400), (422), (511), and (440), respectively, which match well with the database of magnetite in the JCPDS-International Center for Diffraction Data (JCPDS Card: 19-629) file. The results demonstrate that both Fe3O4@NH2 and Fe3O4@CGA-MIPs are composed of Fe3O4.
The magnetic hysteresis loops of Fe3O4@NH2 and Fe3O4@CGA-MIPs are presented in Fig. S1C. The results illustrate that there is no hysteresis, both remanence and coercivity are approximately zero, confirming that samples are super-paramagnetic. The magnetic saturation values are 64.18 and 49.38 emu g-1 for Fe3O4@NH2 and Fe3O4@CGA-MIPs,
respectively.
The
saturation
magnetization
of
Fe3O4@CGA-MIPs is reduced in comparison with Fe3O4@NH2, but the resulted nanoparticles still possess strong magnetism to be isolated within short tine by use of an external magnet. 3.3. Adsorption characteristics of Fe3O4@CGA-MIPs Fig. 4A illustrates the kinetic curves of CGA absorbed on Fe3O4@CGA-MIPs. A rapid increase of the binding capacity in the first 20 min, and then the adsorption rate exhibits a slower increase to reach the adsorption equilibrium after 30 min. The adsorption process just takes about 30 min to approach the equilibrium which is largely shorter than those of other CGA-imprinted polymers (equilibrium time, 1-5 h) (Gu et al., 2010; Li et al., 2013; Li et al., 2005). Therefore, it could be concluded that the formed thin imprinted shell through surface imprinting technology dramatically 14
improve the mass transfer for easy diffusion of CGA to the binding sites. The pseudo-first-order and pseudo-second-order rate kinetic models were used to determine the rate-controlling and mass transfer mechanisms. The related parameters were calculated and listed in Table S2, which shows that the pseudo-second-order model (r＞0.99) fits the experimental data better than pseudo-first-order model (r＜ 0.95). The v0 of imprinted polymers is 15.82 mg g-1 min-1, indicating that the adsorption of CGA onto Fe3O4@CGA-MIPs is a fast process. Moreover, it is assumed that the chemical course could be the rate-limiting step in the adsorption process (Hu et al., 2014).
Fig. 4B displays the isothermal adsorption curves of CGA on Fe3O4@CGA-MIPs and Fe3O4@NIPs at room temperature. It could be seen that the adsorption capacity increase with the increment of initial concentration of CGA, and then become saturated when the concentration attains to 0.40 mg mL-1. Meanwhile, the equilibrium adsorption capacity of CGA onto Fe3O4@CGA-MIPs is 80.1 mg g-1, about 3 times more than that of Fe3O4@NIPs (25.4 mg g-1), which suggests that the recognition sites on the surface of imprinted polymers are suitable for template molecule on spatial position and chemical effect. The Langmuir and Freundlich adsorption models were used to fit the experimental data (Table S3). The Langmuir isotherm model (r＞0.99) is more suitable for CGA adsorption onto Fe3O4@CGA-MIPs than Freundlich isotherm model (r ＜ 0.89). Furthermore, the maximum amount of adsorption calculated from the intercept of Langmuir linear equation (81.3 mg g-1 for Fe3O4@CGA-MIPs and 30.6 mg g-1 for Fe3O4@NIPs) is close to that of experimental 15
results. It could be concluded that the adsorption of CGA onto Fe3O4@CGA-MIPs may be a monolayer adsorption process (Chen, Xie, & Shi, 2013). 3.4. Selectivity of Fe3O4@CGA-MIPs To investigate the selectivity of Fe3O4@CGA-MIPs, four structurally related polyphenolic compounds (CCA, CFA, CA, and PCA) were selected as the competitive analogues whose molecular structures are displayed in Fig. S2. As shown in Table 1, the adsorption capacities of CGA and other four analogues on Fe3O4@NIPs have no distinct difference. In comparison, the adsorption capacity of CGA onto Fe3O4@CGA-MIPs is 58.5 mg g-1, which is much higher than that of four other phenolic acids. The selectivity of Fe3O4@CGA-MIPs is further evaluated using IF and SC, the IF value for CGA is larger than that of the reference polyphenolic, and the SC values are greater than 2.3, illustrating the high selectivity of Fe3O4@CGA-MIPs for CGA. Although CCA, CFA, CA, and PCA are hydroxybenzoic acids, the lower adsorption capacity may due to their different molecular size and stereochemistry. These results demonstrate that the imprinting effect play a significant role in the process of specific recognition. 3.5. Reusability of Fe3O4@CGA-MIPs To test the regeneration of Fe3O4@CGA-MIPs, six consecutive cycles of adsorption-desorption were conducted by using the same polymers and the results are shown in Fig. S3. It could be seen that the adsorption capacity of Fe3O4@CGA-MIPs slightly decreased with increasing the cycle times, which was ascribed to the fact that 16
some recognition sites in the network of Fe3O4@CGA-MIPs could be masked after regeneration or destructed after rewashing, but the adsorption efficiency can be still above 93% in the final recycle, indicating that recognition, interaction, and adsorption processes are occurred reversibly and the method applied for elution is mild. The results manifest that the polymers obtained are stable and may become a promising candidate for scaling-up and economizing the expenditure of sample pretreatment. 3.6. Method validation and real sample analysis The experimental parameters including linear range, limit of detection (LOD), accuracy, and precision were investigated to validate the analytical method. The standard calibration curve was obtained by plotting the peak area versus the corresponding concentrations of CGA. The dada was subjected to least squares regression analysis and the high correlation coefficient (0.9993) is obtained for CGA in the linear concentration range of 0.05-100 µg mL-1. LOD defined as three times ratio of signal to noise is 0.01 µg mL-1. The intra-day and inter-day precisions were investigated by extraction and determination of CGA spiked standard in juice samples at three different spiked levels (1.0, 10.0, and 100.0 µg mL-1). The results show that the RSDs of intra-day precision is 1.9-4.7%, while that of inter-day precision is 2.7-5.9%. Meanwhile, the recoveries of CGA were obtained in the range of 92.8 to 101.7% with RSDs less than 4.7%. The results demonstrate that the proposed method is accurate, sensitive, and selective for determination of CGA in fruit juices.
In order to evaluate the applicability of the proposed method, it was applied to 17
selective extract and determine CGA in three different fruit juices including apple juice, grape juice, and peach juice. As shown in Fig. 5 (red line), there are some other peaks except the peak of CGA, and some interferents even have similar retention time with that of CGA, indicating that the fruit juices are complex matrices. After the pretreatment of fruit juices with Fe3O4@CGA-MIPs and elution by ethanol-HAc (95:5, v/v), it could be seen that the peak of CGA emerges distinctly at about 6.1 min and there are no interfering peaks nearby (black line). The results confirm that CGA in fruit juices could be selectively and efficiently absorbed by Fe3O4@CGA-MIPs with little co-adsorption. Furthermore, the concentrations of CGA in peach juice, apple juice, and grape juice are found to be 0.92, 4.21, and 0.75 µg mL-1. 4． ．Conclusions
Hydrophilic magnetic molecularly imprinted polymers using PEI as functional monomer are found to be effective for selective extraction of CGA in fruit juices. PEI possesses three functions in this work: interacting with template sufficiently, enhancing the density the imprinted cavities, and improving the hydrophilcity of imprinted materials. The as-prepared nanomaterials exhibit fast kinetics, high adsorption capacity, and favorable selectivity under optimized conditions. Meanwhile, the obtained imprinted polymers used as SPE sorbents coupled with HPLC were applied in the specific isolation and determination of CGA in fruit juices. The developed method may enrich the designation strategy for hydrophilic MIPs with outstanding performance, which could provide a feasible and alternative way for fast,
18
efficient, and selective extraction of water-soluble compounds in food, environmental, and biological samples. Acknowledgements
The authors are grateful for financial support from the National Natural Science Foundation of China (No. 21305107), the Fundamental Research Funds for the Central Universities (Nos. xjj2013041, 08142034), and China Postdoctoral Science Foundation (No. 2014M562388). References Ambattu, L. A., & Rekha, M. R. (2015). Collagen synthesis promoting pullulan-PEI-ascorbic acid conjugate as an efficient anti-cancer gene delivery vector. Carbohydrate Polymers, 126, 52-61. Bagdas, D., Etoz, B. C., Gul, Z., Ziyanok, S., Inan, S., Turacozen, O., Gul, N. Y., Topal, A., Cinkilic, N., Tas, S., Ozyigit, M. O., & Gurun, M. S. (2015). In vivo systemic chlorogenic acid therapy under diabetic conditions: Wound healing effects and cytotoxicity/genotoxicity profile. Food and Chemical Toxicology, 81, 54-61. Bahulekar, R., Ayyangar, N. R., & Ponrathnam, S. (1991). Polyethyleneimine in immobilization of biocatalysts. Enzyme and Microbial Technology, 13, 858-868. Chen, F. F., Xie, X. Y., & Shi, Y. P. (2013). Preparation of magnetic molecularly imprinted polymer for selective recognition of resveratrol in wine. Journal of Chromatography A, 1300, 112-118. Chen, J. H., Xing, H. T., Guo, H. X., Weng, W., Hu, S. R., Li, S. X., Huang, Y. H., Sun, X., & Su, Z. B. (2014). Investigation on the adsorption properties of Cr(vi) ions on a novel graphene oxide (GO) based composite adsorbent. Journal of Materials Chemistry A, 2, 12561-12570. Eren, T., Atar, N., Yola, M. L., & Karimi-Maleh, H. (2015). A sensitive molecularly imprinted polymer based quartz crystal microbalance nanosensor for selective determination of lovastatin in red yeast rice. Food Chemistry, 185, 430-436. 19
Gao, R. X., Zhang, L. L., Hao, Y., Cui, X. H., & Tang, Y. H. (2014). Specific removal of protein using protein imprinted polydopamine shells on modified amino-functionalized magnetic nanoparticles. RSC Advances, 4, 64514-64524. Gu, X. H., Xu, R., Yuan, G. L., Lu, H., Gu, B. R., & Xie, H. P. (2010). Preparation of chlorogenic acid surface-imprinted magnetic nanoparticles and their usage in separation of traditional Chinese medicine. Analytica Chimica Acta, 675, 64-70. Hao, Y., Gao, R. X., Shi, L., Liu, D. C., Tang, Y. H., & Guo, Z. J. (2015). Water-compatible magnetic imprinted nanoparticles served as solid-phase extraction sorbents for selective determination of trace 17beta-estradiol in environmental water samples by liquid chromatography. Journal of Chromatography A, 1396, 7-16. Heyman, H. M., Senejoux, F., Seibert, I., Klimkait, T., Maharaj, V. J., & Meyer, J. J. M. (2015). Identification of anti-HIV active dicaffeoylquinic- and tricaffeoylquinic acids in Helichrysum populifolium by NMR-based metabolomic guided fractionation. Fitoterapia, 103, 155-164. Hu, C. H., Deng, J., Zhao, Y. B., Xia, L. S., Huang, K. H., Ju, S. Q., & Xiao, N. (2014). A novel core-shell magnetic nano-sorbent with surface molecularly imprinted polymer coating for the selective solid phase extraction of dimetridazole. Food Chemistry, 158, 366-373. Huang, B. M., Zhou, X. B., Chen, J., Wu, G. F., & Lu, X. Q. (2015). Determination of malachite green in fish based on magnetic molecularly imprinted polymer extraction followed by electrochemiluminescence. Talanta, 142, 228-234. Jung, H., Kim, S. A., Lee, E., & Mok, H. (2015). Linear polyethyleneimine-doxorubicin conjugate for pH-responsive synchronous delivery of drug and microRNA-34a. Macromolecular Research, 23, 449-456. Kempe, H., Pujolràs, A. P., & Kempe, M. (2015). Molecularly imprinted polymer nanocarriers for sustained release of erythromycin. Pharmaceutical Research, 32, 375-388. Khoobi, M., Motevalizadeh, S. F., Asadgol, Z., Forootanfar, H., Shafiee, A., & Faramarzi, M. A. (2015). Polyethyleneimine-modified superparamagnetic Fe3 O4 nanoparticles for lipase immobilization: Characterization and application. Materials Chemistry and Physics, 149-150, 77-86. Lata, K., Sharma, R., Naik, L., Rajput, Y. S., & Mann, B. (2015). Synthesis and application of cephalexin imprinted polymer for solid phase extraction in milk. Food Chemistry, 184, 20
176-182. Lee, M. S., Lee, S. Y., & Park, S. J. (2015). Preparation and characterization of multi-walled carbon nanotubes impregnated with polyethyleneimine for carbon dioxide capture. International Journal of Hydrogen Energy, 40, 3415-3421. Lenain, P., Saeger, S. D., Mattiasson, B., & Hedström, M. (2015). Affinity sensor based on immobilized molecular imprinted synthetic recognition elements. Biosensors and Bioelectronics, 69, 34-39. Li, H., Li, G., Li, Z. P., Lu, C. M., Li, Y. N., & Tan, X. Z. (2013). Surface imprinting on nano-TiO2 as sacrificial material for the preparation of hollow chlorogenic acid imprinted polymer and its recognition behavior. Applied Surface Science, 264, 644-652. Li, H., Liu, Y. J, Zhang, Z. H., Liao, H. P., Nie, L. H., & Yao, S. Z. (2005). Separation and purification of chlorogenic acid by molecularly imprinted polymer monolithic stationary phase. Journal of Chromatography A, 1098, 66-74. Lindén, J. B., Larsson, M., Coad, B. R., Skinner, W. M., & Nydén, M. (2014). Polyethyleneimine for copper absorption: kinetics, selectivity and efficiency in artificial seawater. RSC Advances, 4 25063-25066. Liu, L. J., Zheng, J. J., Fang, G. J., & Xie, W. H. (2012). Improvement of the homogeneity of protein-imprinted polymer films by orientated immobilization of the template. Analytica Chimica Acta, 726, 85-92. Liu M., Li, X. Y., Li, J. J., Su, X. M., Wu, Z. Y., Li, P. F., Lei, F. H., Tan, X. C., & Shi, Z. W. (2015). Synthesis of magnetic molecularly imprinted polymers for the selective separation and determination of metronidazole in cosmetic samples. Analytical and Bioanalytical Chemistry, 407, 3875-3880. Naso, L. G., Valcarcel, M., Roura-Ferrer, M., Kortazar, D., Salado, C., Lezama, L., Rojo, T., González-Baró, A. C., Williams, P. A. M., & Ferrer, E. G. (2014). Promising antioxidant and anticancer (human breast cancer) oxidovanadium(IV) complex of chlorogenic acid. Synthesis, characterization and spectroscopic examination on the transport mechanism with bovine serum albumin. Journal of Inorganic Biochemistry, 135, 86-99. Saad, E. M., Madbouly, A., Ayoub, N., & Nashar, R. M. E. (2015). Preparation and application of molecularly imprinted polymer for isolation of chicoric acid from Chicorium intybus L. 21
medicinal plant. Analytica Chimica Acta, 877, 80-89. Sabri, S. S., Guillemette, J., Guermoune, A., Siaj, M., & Szkopek, T. (2012). Enhancing gas induced charge doping in graphene field effect transistors by non-covalent functionalization with polyethyleneimine. Applied Physics Letters, 100, 113106. Santos, M. G., Tavares, I. M. C., Boralli, V. B., & Figueiredo, E. C. (2015). Direct doping analysis of beta-blocker drugs from urinary samples by on-line molecularly imprinted solid-phase extraction coupled to liquid chromatography/mass spectrometry. Analyst, 140, 2696-2703. Shao, H. K., Zhao, L. G., Chen, J., Zhou, H. T., Huang, S. T., & Li, K. (2015). Preparation, characterization and application of molecularly imprinted monolithic column for hesperetin. Journal of Pharmaceutical and Biomedical Analysis, 111, 241-247. Suksuwan, A., Lomlim, L., Rungrotmongkol, T., Nakpheng, T., Dickert, F. L., Suedee, R. (2015). The composite nanomaterials containing (R)-thalidomide-molecularlyimprinted polymers as a recognition system for enantio selective controlled release and targeted drug delivery. Journal of Applied Polymer Science, 132, 41931-41951. Wang, H. Q., Li, D. L., Du, Z. Y., Huang, M. T., Cui, X. X., Lu, Y. J., Li, C. Y., Woo, S. L., Conney, A. H., Zheng, X., Zhang, K. (2015). Antioxidant and anti-inflammatory properties of Chinese ilicifolius vegetable (Acanthopanax trifoliatus (L) Merr) and its reference compounds. Food Science and Biotechnology, 24, 1131-1138. Wulff, G., & Sarhan, A. (1972). Use of polymers with enzyme-analogous structures for resolution of racemates. Angewandte Chemie International Edition, 11, 341-344. Yang, J. J., Li, Y., Wang, J. C., Sun, X. L., Cao, R., Sun, H., Huang, C. N., & Chen, J. P. (2015). Molecularly imprinted polymer microspheres prepared by Pickering emulsion polymerization for selective solid-phase extraction of eight bisphenols from human urine samples. Analytica Chimica Acta, 872, 35-45. Zhao, J. Q., Lu, C. H., He, X., Zhang, X. F., Zhang, W., & Zhang, X. M. (2015). Polyethylenimine-grafted cellulose nanofibril aerogels as versatile vehicles for drug delivery. ACS Applied Materials & Interfaces, 7, 2607-2615.
22
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Table 1 The adsorption capacities, imprinting factors, and selectivity coefficients of CGA, CCA, CFA, CA, and PCA for Fe3O4@CGA-MIPs and [email protected] Analytes CGA CCA CFA CA PCA a
QMIP（mg g-1） QNIP（mg g-1） 58.5 5.51 9.32 25.7 16.2
9.91 8.27 9.26 10.3 9.52
IF
SC
5.90 0.67 1.01 2.50 1.70
― 8.81 5.84 2.36 3.47
In this experiment, 20 mg of Fe3O4@CGA-MIPs and Fe3O4@NIPs were incubated with 10 mL of the mixed
solution of CGA, CCA, CFA, CA, and PCA at a concentration of 0.40 mg mL-1 for 30 min at room temperature. Fe3O4@CGA-MIPs: magnetic molecularly imprinted nanoparticles of chlorogenic acid. Fe3O4@NIPs: non-imprinted magnetic nanoparticles. CGA: chlorogenic acid; CCA: cichoric acid; CA: caffeic acid; CFA: caftaric acid; PCA: protocatechuic acid
Highlights:
Branched polyethyleneimine used to enhance the density of imprinted cavities and hydrophilcity of MIPs.
Directly amino-functionalized magnetic nanoparticles utilized as supporters to simplify the preparation procedure.
Preparation conditions optimized in detail.
Application of resulted MIPs for extraction and determination of chlorogenic acid in fruit juices.
23
S0308-8146(16)30005-X http://dx.doi.org/10.1016/j.foodchem.2016.01.004 FOCH 18578
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
6 August 2015 7 December 2015 1 January 2016
Please cite this article as: Hao, Y., Gao, R., Liu, D., He, G., Tang, Y., Guo, Z., Selective extraction and determination of chlorogenic acid in fruit juices using hydrophilic magnetic imprinted nanoparticles, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2016.01.004
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Selective extraction and determination of chlorogenic acid in fruit juices using hydrophilic magnetic imprinted nanoparticles Yi Haoa,b, Ruixia Gaob*, Dechun Liuc, Gaiyan Hea,b, Yuhai Tanga,b, Zengjun Guo a* a
b
School of Pharmacy, Xi’an Jiaotong University, Xi’an 710061, China.
Institute of Analytical Science, School of Science, Xi’an Jiaotong University, Xi’an 710049, China.
c
Department of Hepatobiliary Surgery, First Hospital of Xi’an Jiaotong University, Xi’an, 710061, China. * Corresponding authors: Tel.: +86 2982655399; fax: +86 2982655399. E-mail: [email protected] (R. Gao); [email protected] (Z. Guo).
1
Abstract In this paper, the novel hydrophilic magnetic molecularly imprinted nanoparticles were developed for selective separation and determination of chlorogenic acid in aqueous fruit juices. The polymers were prepared by using amino-functionalized magnetic nanoparticles as carriers, branched polyethyleneimine as functional monomer, and chlorogenic acid as template molecule. Branched polyethyleneimine with abundant active amino groups could react with template sufficiently, and its unique dendritic structure may amplify the number of the imprinted cavities. Meanwhile, it would improve the hydrophilcity of imprinted materials for attaining high extraction efficiency. The resulted polymers exhibit fast kinetics, high adsorption capacity, and favorable selectivity. In addition, the obtained nanoparticles were used as solid-phase extraction sorbents for selective isolation and determination of chlorogenic acid in peach, apple, and grape juices (0.92, 4.21, and 0.75 µg mL-1, respectively). Keywords: Magnetic separation, branched polyethyleneimine, solid-phase extraction, hydrophilcity, chlorogenic acid
2
1. Introduction Molecular imprinting, proposed by Wuff and Sarhan in 1972 (Wulff, & Sarhan, 1972), has become an attractive technique to create specific cavities complementary to the template molecule in shape, size, and chemical functionality. Molecularly imprinted polymers (MIPs) possess the advantages of predetermination, low cost, easy preparation, mechanical and chemical stability, applicability in harsh environment, and high specificity. Owing to their outstanding merits, MIPs have been widely used in the fields of chromatography (Shao et al., 2015; Santos, Tavares, Boralli, & Figueiredo, 2015), biosensor (Eren, Atar, Yola, & Karimi-Maleh, 2015; Lenain, Saeger, Mattiasson, & Hedström, 2015), drug delivery (Kempe, Pujolràs, & Kempe, 2015; Suksuwan et al., 2015), and solid-phase extraction (SPE) (Lata, Sharma, Naik, Rajput, & Mann, 2015; Yang et al., 2015). Among these applications, MIPs used as SPE adsorbents have exhibited a great prospect for the selective extraction or clean-up of target analytes from various complex matrices.
Traditionally, MIPs have to be packed into SPE cartridge or adopt centrifugation and filtration procedures when used in the process of SPE. The time-consuming and complicated course commonly yield low extraction efficiency. Therefore, magnetic Fe3O4 nanoparticles (NPs), because of their good biocompatibility, magnetic susceptibility, low toxicity, have been encapsulated into MIPs to solve the above problems. The resulting composites not only have magnetic characteristic for rapid separation but also possess high selectivity for recognition of the target molecule (Hao
3
et al., 2015; Huang, Zhou, Chen, Wu, & Lu, 2015; Liu et al., 2015).
Chlorogenic acid (CGA), a bioactive phenolic acid, is extensively distributed in fruit, plant, and used in feed industry. It is reported that CGA has several beneficial biological properties including antibacterial, antiphlogistic, antiviral, mutant resistance, and even inhibitory effects on carcinogenesis in large intestine, liver, and tongue (Bagdas et al., 2015; Heyman et al., 2015; Wang et al., 2015; Naso et al., 2014). CGA is water-soluble and usually coexists with some compounds hold similar functional groups such as caffeic acid, gallic acid, and so on. Thus, selective extraction of CGA from complex matrices prior to its quantification is urgently required. However, CGA-imprinted polymers previously reported were usually prepared in organic media (Gu et al., 2010; Saad, Madbouly, Ayoub, & Nashar, 2015), which could lead to poor recognition ability and high non-specific adsorption in aqueous matrices. Therefore, a facile and efficient approach to prepare hydrophilic CGA-imprinted polymers is highly desirable.
Polyethyleneimine (PEI), a water-soluble and non-toxic polycation (Bahulekar, Ayyangar, & Ponrathnam, 1991), consists of high content of functional groups (primary, secondary, and tertiary amine groups). What is more, PEI could assemble layer-by-layer with glutaraldehyde as cross-linking agent (Zhao et al., 2015). Based on these distinctive features, PEI has been widely applied in gas adsorption (Lee, Lee, & Park, 2015; Sabri, Guillemette, Guermoune, Siaj, & Szkopek, 2012), drug/gene delivery (Jung, Kim, Lee, & Mok, 2015; Ambattu, & Rekha, 2015), enzyme 4
immobilization (Khoobi et al., 2015), and heavy metals removal (Lindén, Larsson, Coad, Skinner, & Nydén, 2014; Chen et al., 2014).
Herein, we attempt to prepare novel hydrophilic MIPs adopting branched PEI as functional monomer and CGA as template molecule. The abundant functional groups on PEI can react with CGA through multiple hydrogen bonds and electronic interactions. Meanwhile, its unique dendritic structure and water solubility could greatly improve the density of recognition sites and hydrophilicity of MIPs. The characteristics and binding properties of the obtained MIPs were investigated. In addition, the resulted polymers were used as SPE sorbents and coupled with HPLC to selective extraction and determination of CGA in fruit juices. The developed approach would exhibit great potential for preparation of hydrophilic MIPs with satisfactory imprinting performance.
2. Experimental 2.1. Chemicals and reagents Ferricchloride hexahydrate (FeCl3·6H2O), 1,6-hexadiamine (HDM), glutaraldehyde, sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), ethylene glycol (EG), anhydrous sodium acetate (NaOAc), phosphoric acid (H3PO4), ethanol, acetonitrile, and acetic acid were provided by Xi’an Chemicals Ltd. CGA, caffeic acid (CA), cichoric acid (CCA), caftaric acid (CFA), protocatechuic acid (PCA), and polyethyleneimine (PEI) (MW=10000, Purity≥99.0%) were obtained from Aladdin Industrial Corporation. All reagents used were of at least analytical grade. 5
The ultrapure water (18.25 MΩ cm-1) was obtained from a WaterPro water system (Axlwater Corporation, TY10AXLC1805-2, China) and used throughout the experiments. 2.2. Instrumentation and analytical conditions JEM-2100 transmission electron microscope (TEM) (JEOL Co., Japan) was used to observe morphology of as-synthesized magnetic nanoparticles. Fourier transform infrared (FT-IR) spectra (4000-400 cm-1) were obtained via a Nicolet AVATAR-330 FT-IR spectrometer (Thermo Electron Co., U.S.A). The magnetic properties were measured at room temperature using an LDJ 9600-1vibrating sample magnetometer (VSM) (LDJ Co., U.S.A). The identification of the crystalline phase was carried out by a Rigaku D/max/2500v/pcX-ray diffractometer (Rigaku Co., Japan) with Cu Kα radiation. The HPLC profiles were recorded using a Shimadzu HPLC system equipped with LC-10AT pump, SPD-M 10A detector, CTO-10AS column oven, and Shimadzu VP-ODS C18 column (5 µm, 150 mm×4.6 mm). The column temperature was 30 °C. The optimized mobile phase was acetonitrile-0.05% phosphoric acid solution (85:15, v/v) delivered at a flow rate of 1.0 mL min−1, the injection volume was 20 µL, and the column effluent was monitored at 323 nm. Sample solutions were filtered through a nylon 0.22 µm filter before determination. 2.3. Preparation of Fe3O4@CGA-MIPs and Fe3O4@NIPs The amino-functionalized Fe3O4 nanoparticles (denoted as Fe3O4@NH2) were synthesized as our previous work (Gao, Zhang, Hao, Cui, & Tang, 2014). The 6
magnetic
molecularly
imprinted
nanoparticles
of
CGA
(designed
as
Fe3O4@CGA-MIPs) were prepared as follows: CGA (40 mg) was dissolved in 20 mL of phosphate buffered solution (PBS, pH=7.4) in a three-necked flask, and combined with Fe3O4@NH2 (200 mg). The mixture was allowed to react for 1h. Then 5 mL of PBS containing 30 mg of PEI (6.0 mg mL-1) and 30 µL of glutaraldehyde were added, the mixture was stirred for 6 h at room temperature. The obtained imprinted polymers were collected by an external magnetic field and washed with ethanol-HAc (95:5, v/v) to remove the embedded template until no adsorption was detected by HPLC. Then, the Fe3O4@CGA-MIPs were washed with ultrapure water and ethanol repeatedly, and dried under vacuum. For comparison, non-imprinted magnetic nanoparticles (designed as Fe3O4@NIPs) were prepared following the same procedure in the absence of the template molecule CGA. 2.4. Adsorption experiment and selectivity evaluation In kinetic adsorption test, 20 mg of Fe3O4@CGA-MIPs or Fe3O4@NIPs were suspended in 10 mL of PBS with CGA at a concentration of 0.40 mg mL-1, and shaken on a reciprocating shaking-table at regular times from 5 min to 50 min. Then the supernatants and polymers were separated by an external magnetic field and the concentration of CGA in the filtrate was measured by HPLC. The adsorption amounts (Q, mg g-1) of Fe3O4@CGA-MIPs or Fe3O4@NIPs to CGA were calculated according to equation (1), and the pseudo-first-order and pseudo-second-order rate kinetic models were applied to fit the kinetic data according to equation (2, 3).
7
(C 0 − C e )V m
(1)
ln ( Qe − Qt ) = ln Qe - ｋ1t
(2)
t 1 t 1 t = + = + 2 Qt k 2Qe Qe v0 Q e
(3)
Q=
Where C0 and Ce (mg mL−1) are the initial and equilibrium concentration of CGA, respectively. V (mL) and m (g) represent the volume of the CGA solution and the mass of the polymers. Qe and Qt are the amount of adsorbate onto sorbent at the equilibrium and time t (min). v0 (mg g-1 min-1) is the initial adsorption rate. k1 and k2 are the equilibrium rate constants of pseudo-first-order and pseudo-second-order equation, respectively.
In equilibrium binding test, 20 mg of Fe3O4@CGA-MIPs or Fe3O4@NIPs were dispersed into 10 mL of PBS with various CGA concentrations (0.050-0.80 mg mL-1), and incubated for 30 min at room temperature. The separation and detection procedures were conducted as described in kinetic adsorption test. For further evaluating the equilibrium adsorption, Langmuir and Freundlich isotherm models were selected to fit the experimental data. They are described as equation (4) and (5). C Ce 1 = + e Q Qmax K L Qmax
(4)
log Q = m log Ce + log K F
(5)
Where Ce (mg mL-1) is equilibrium concentration of adsorbate, Q (mg g-1) is the amount of CGA bound to Fe3O4@CGA-MIPs or Fe3O4@NIPs at equilibrium, Qmax (mg g-1) is the maximum adsorption capacity of the sorbent, KL (mL mg-1) and KF (mg 8
g-1) are the Langmuir and Freundlich constant respectively, and m is the Freundlich exponent which represents the heterogeneity of the system.
The selectivity of Fe3O4@CGA-MIPs was measured using the structure analogues CCA, CFA, CA, and PCA. 10 mL of the mixed standard solution of CGA and analogues at initial concentration of 0.40 mg mL-1 was incubated with 20 mg of Fe3O4@CGA-MIPs or Fe3O4@NIPs for 30 min, and then the operating sequence was the same as kinetic adsorption test. The specific recognition property of Fe3O4@CGA-MIPs was evaluated by imprinting factor (IF), which was calculated according to equation (6), and the selectivity coefficient (SC) was defined as expressed in equation (7).
IF =
SC =
QMIP QNIP
(6)
IFt IFc
(7)
Where QMIP and QNIP (mg g-1) represent the adsorption capacity of CGA on Fe3O4@CGA-MIPs and Fe3O4@NIPs, respectively. IFt and IFc are the imprinting factors for template molecule and competitive molecules. 2.5. Reusability To
evaluate
the
regeneration
of
Fe3O4@CGA-MIPs,
six
cycles
of
adsorption-desorption were conducted using the same polymers. 20 mg of polymers were added to 10 mL of CGA solution at a concentration of 0.40 mg mL-1 and incubated at room temperature for 30 min. Then, Fe3O4@CGA-MIPs were separated 9
by a magnet and the concentration of CGA in supernatant was quantified by HPLC. The adsorbed polymers were eluted by ethanol-HAc (95:5, v/v) for 6 h to ensure complete removal of CGA. The recovered imprinted nanomaterials were used for another five adsorption-desorption cycles, and every cycle of supernatant was collected and determined. 2.6. Selective extraction and determination of CGA in fruit juices Grape juice, apple juice, and peach juice (Huiyuan Co., Ltd., Beijing) were purchased from local market in Xi’an. 30 mL of fruit juice was incubated with 60 mg of Fe3O4@CGA-MIPs for 30 min at room temperature. Then Fe3O4@CGA-MIPs were separated magnetically, and eluted by ethanol-HAc (95:5, v/v) for 6 h. The juice and eluted samples were filtered by 0.22 µm nylon membrane filter and then for HPLC analysis. 3． ．Results and discussions
3.1. Preparation of Fe3O4@CGA-MIPs The synthesis procedure of Fe3O4@CGA-MIPs is depicted in Fig. 1. First, Fe3O4@NH2 with abundant of amino groups on their surface were synthesized through a one-step solvothermal reaction. Next, Fe3O4@NH2 were used as the carriers onto which CGA were immobilized through hydrogen bond and electrostatic interactions. Preimmobilization of template on supporters could not only make the formed imprinted cavities relatively order, but also enhance the number of functional binding sites, thus could improve imprinting effect to some degree compared to the 10
traditional free-template polymerization process (Liu, Zheng, Fang, & Xie, 2012). Then, a thin and adherent imprinted shell embedding CGA was obtained by the reaction between the PEI and
templates-supporter complex.
Finally,
the
Fe3O4@CGA-MIPs with recognition sites were obtained after the removal of the embedded CGA. Notably, PEI is a water-soluble polycation with a branched structure and rich amino groups, which make the prepared polymers possess high density of recognition sites and excellent water compatibility.
The imprinting effect of Fe3O4@CGA-MIPs is affected by many factors. To acquire satisfactory imprinting effect, we optimized typical factors including the selection of carrier, the amount of template, the concentration of PEI, and polymerization temperature and time. As shown in Fig. 2A, Fe3O4@NH2 could immobilize larger amount of templates compared with another three carriers involving pure magnetic nanoparticles (Fe3O4), silica-modified magnetic nanoparticles (Fe3O4@SiO2), and carboxyl-functionalized magnetic nanoparticles (Fe3O4@COOH) (SI). The results illustrated that the amino groups are more suitable to immobilize CGA through forming multi-hydrogen bonds and electrostatic interactions. The amount of template immobilized on Fe3O4@NH2 was investigated ranging from 25 mg to 50 mg, while the amount of Fe3O4@NH2 maintained at a constant value of 200 mg. As displayed in Fig. 2B, the Qim increased with increasing the amount of CGA from 25 mg to 40 mg, and then remained almost unchanged from 40 mg to 50 mg. The results illustrated that the immobilization capacity of Fe3O4@NH2 had been saturated over the 40 mg of CGA. Thus, the 40 mg of CGA was used in the remainder of this work. 11
The adsorption capacity and imprinting effect of Fe3O4@CGA-MIPs towards CGA are greatly influenced by functional monomer. Therefore, the concentration of PEI ranging from 0.8 mg mL-1 to 12.0 mg mL-1 was investigated. The results of the adsorption capacity (Q) and imprinting factor (IF) are shown in Fig. 2C, the Q and IF exhibit an upward trend, manifesting the augment in the number of recognition cavities with increasing the concentration of PEI from 0.8 mg mL-1 to 6.0 mg mL-1. With further increasing the concentration of PEI from 6.0 mg mL-1 to 12.0 mg mL-1, the Q increases slightly while the IF decreases obviously. This may be because excessive functional monomers generate high nonspecific adsorption. Therefore, the concentration of PEI of 6.0 mg mL-1 was adopted in this work.
Polymerization temperature and time play important roles in the morphology and adsorption ability of imprinted composite. Short polymerization time (5 h) or low temperature (50 oC) led to lower adsorption capacity, which would be attribute to that the less imprinted capacities are formed for CGA because of the uncompleted polymerization. With polymerization time and temperature increasing to 6 h and 60 oC, the Fe3O4@CGA-MIPs exhibit the highest adsorption capacity because the optimal recognition sites and the most suitable shell thickness are formed. However, further prolonging the polymerization time (7 h) and increasing the temperature (70 oC), the functional monomers may self-polymerize and block the recognition sites, resulting in low adsorption capacity (Table S1). Therefore, 6 h and 60oC are chosen as the polymerization time and temperature in this work.
12
3.2. Characterization of Fe3O4@CGA-MIPs Representative TEM images of Fe3O4@NH2 and Fe3O4@CGA-MIPs are shown in Fig. 3. It can be obviously seen that Fe3O4@NH2 and Fe3O4@CGA-MIPs exhibit approximately spherical morphology, good dispersibility, and relatively narrow size distribution. The particle diameters of Fe3O4@NH2 and Fe3O4@CGA-MIPs are about 80 nm and 96 nm. It follows that the thickness of imprinted layer is about 8 nm, which would be beneficial for the mass transfer between the solution and the surface of Fe3O4@CGA-MIPs.
Fig. S1A shows the surface groups on Fe3O4@NH2 and Fe3O4@CGA-MIPs analyzed by FT-IR spectrophotometer. The characteristic peaks of Fe-O group for Fe3O4@NH2, and Fe3O4@CGA-MIPs are all observed around 576 cm-1. The typical peaks at 3432 cm-1 and 1630cm-1 assigned to stretching and bending vibrations of N-H indicate that amino groups are strongly anchored on the magnetic nanoparticles through a one-pot solvothermal polymerization (Fig. S1A-a). The increased intensities of peaks of amino groups in Fe3O4@CGA-MIPs compared with that of Fe3O4@NH2 and the asymmetric and symmetric stretching peaks of -CH2 at 2860 cm-1 and 2770 cm-1 confirm that the imprinted shell has been ideally coated onto the surface of Fe3O4@NH2 (Fig. S1A-b).
The XRD patterns of the Fe3O4@NH2 and Fe3O4@CGA-MIPs are illustrated in Fig. S1B. In the 2θ region of 20-70o, six relatively discernible strong diffraction peaks corresponded to Fe3O4 (2θ = 30.2o, 35.6o, 43.2o, 53.5o, 57.2o, and 62.8o) are observed 13
in the curves of two samples, and the peak positions at the corresponding 2θ values are indexed as (220), (311), (400), (422), (511), and (440), respectively, which match well with the database of magnetite in the JCPDS-International Center for Diffraction Data (JCPDS Card: 19-629) file. The results demonstrate that both Fe3O4@NH2 and Fe3O4@CGA-MIPs are composed of Fe3O4.
The magnetic hysteresis loops of Fe3O4@NH2 and Fe3O4@CGA-MIPs are presented in Fig. S1C. The results illustrate that there is no hysteresis, both remanence and coercivity are approximately zero, confirming that samples are super-paramagnetic. The magnetic saturation values are 64.18 and 49.38 emu g-1 for Fe3O4@NH2 and Fe3O4@CGA-MIPs,
respectively.
The
saturation
magnetization
of
Fe3O4@CGA-MIPs is reduced in comparison with Fe3O4@NH2, but the resulted nanoparticles still possess strong magnetism to be isolated within short tine by use of an external magnet. 3.3. Adsorption characteristics of Fe3O4@CGA-MIPs Fig. 4A illustrates the kinetic curves of CGA absorbed on Fe3O4@CGA-MIPs. A rapid increase of the binding capacity in the first 20 min, and then the adsorption rate exhibits a slower increase to reach the adsorption equilibrium after 30 min. The adsorption process just takes about 30 min to approach the equilibrium which is largely shorter than those of other CGA-imprinted polymers (equilibrium time, 1-5 h) (Gu et al., 2010; Li et al., 2013; Li et al., 2005). Therefore, it could be concluded that the formed thin imprinted shell through surface imprinting technology dramatically 14
improve the mass transfer for easy diffusion of CGA to the binding sites. The pseudo-first-order and pseudo-second-order rate kinetic models were used to determine the rate-controlling and mass transfer mechanisms. The related parameters were calculated and listed in Table S2, which shows that the pseudo-second-order model (r＞0.99) fits the experimental data better than pseudo-first-order model (r＜ 0.95). The v0 of imprinted polymers is 15.82 mg g-1 min-1, indicating that the adsorption of CGA onto Fe3O4@CGA-MIPs is a fast process. Moreover, it is assumed that the chemical course could be the rate-limiting step in the adsorption process (Hu et al., 2014).
Fig. 4B displays the isothermal adsorption curves of CGA on Fe3O4@CGA-MIPs and Fe3O4@NIPs at room temperature. It could be seen that the adsorption capacity increase with the increment of initial concentration of CGA, and then become saturated when the concentration attains to 0.40 mg mL-1. Meanwhile, the equilibrium adsorption capacity of CGA onto Fe3O4@CGA-MIPs is 80.1 mg g-1, about 3 times more than that of Fe3O4@NIPs (25.4 mg g-1), which suggests that the recognition sites on the surface of imprinted polymers are suitable for template molecule on spatial position and chemical effect. The Langmuir and Freundlich adsorption models were used to fit the experimental data (Table S3). The Langmuir isotherm model (r＞0.99) is more suitable for CGA adsorption onto Fe3O4@CGA-MIPs than Freundlich isotherm model (r ＜ 0.89). Furthermore, the maximum amount of adsorption calculated from the intercept of Langmuir linear equation (81.3 mg g-1 for Fe3O4@CGA-MIPs and 30.6 mg g-1 for Fe3O4@NIPs) is close to that of experimental 15
results. It could be concluded that the adsorption of CGA onto Fe3O4@CGA-MIPs may be a monolayer adsorption process (Chen, Xie, & Shi, 2013). 3.4. Selectivity of Fe3O4@CGA-MIPs To investigate the selectivity of Fe3O4@CGA-MIPs, four structurally related polyphenolic compounds (CCA, CFA, CA, and PCA) were selected as the competitive analogues whose molecular structures are displayed in Fig. S2. As shown in Table 1, the adsorption capacities of CGA and other four analogues on Fe3O4@NIPs have no distinct difference. In comparison, the adsorption capacity of CGA onto Fe3O4@CGA-MIPs is 58.5 mg g-1, which is much higher than that of four other phenolic acids. The selectivity of Fe3O4@CGA-MIPs is further evaluated using IF and SC, the IF value for CGA is larger than that of the reference polyphenolic, and the SC values are greater than 2.3, illustrating the high selectivity of Fe3O4@CGA-MIPs for CGA. Although CCA, CFA, CA, and PCA are hydroxybenzoic acids, the lower adsorption capacity may due to their different molecular size and stereochemistry. These results demonstrate that the imprinting effect play a significant role in the process of specific recognition. 3.5. Reusability of Fe3O4@CGA-MIPs To test the regeneration of Fe3O4@CGA-MIPs, six consecutive cycles of adsorption-desorption were conducted by using the same polymers and the results are shown in Fig. S3. It could be seen that the adsorption capacity of Fe3O4@CGA-MIPs slightly decreased with increasing the cycle times, which was ascribed to the fact that 16
some recognition sites in the network of Fe3O4@CGA-MIPs could be masked after regeneration or destructed after rewashing, but the adsorption efficiency can be still above 93% in the final recycle, indicating that recognition, interaction, and adsorption processes are occurred reversibly and the method applied for elution is mild. The results manifest that the polymers obtained are stable and may become a promising candidate for scaling-up and economizing the expenditure of sample pretreatment. 3.6. Method validation and real sample analysis The experimental parameters including linear range, limit of detection (LOD), accuracy, and precision were investigated to validate the analytical method. The standard calibration curve was obtained by plotting the peak area versus the corresponding concentrations of CGA. The dada was subjected to least squares regression analysis and the high correlation coefficient (0.9993) is obtained for CGA in the linear concentration range of 0.05-100 µg mL-1. LOD defined as three times ratio of signal to noise is 0.01 µg mL-1. The intra-day and inter-day precisions were investigated by extraction and determination of CGA spiked standard in juice samples at three different spiked levels (1.0, 10.0, and 100.0 µg mL-1). The results show that the RSDs of intra-day precision is 1.9-4.7%, while that of inter-day precision is 2.7-5.9%. Meanwhile, the recoveries of CGA were obtained in the range of 92.8 to 101.7% with RSDs less than 4.7%. The results demonstrate that the proposed method is accurate, sensitive, and selective for determination of CGA in fruit juices.
In order to evaluate the applicability of the proposed method, it was applied to 17
selective extract and determine CGA in three different fruit juices including apple juice, grape juice, and peach juice. As shown in Fig. 5 (red line), there are some other peaks except the peak of CGA, and some interferents even have similar retention time with that of CGA, indicating that the fruit juices are complex matrices. After the pretreatment of fruit juices with Fe3O4@CGA-MIPs and elution by ethanol-HAc (95:5, v/v), it could be seen that the peak of CGA emerges distinctly at about 6.1 min and there are no interfering peaks nearby (black line). The results confirm that CGA in fruit juices could be selectively and efficiently absorbed by Fe3O4@CGA-MIPs with little co-adsorption. Furthermore, the concentrations of CGA in peach juice, apple juice, and grape juice are found to be 0.92, 4.21, and 0.75 µg mL-1. 4． ．Conclusions
Hydrophilic magnetic molecularly imprinted polymers using PEI as functional monomer are found to be effective for selective extraction of CGA in fruit juices. PEI possesses three functions in this work: interacting with template sufficiently, enhancing the density the imprinted cavities, and improving the hydrophilcity of imprinted materials. The as-prepared nanomaterials exhibit fast kinetics, high adsorption capacity, and favorable selectivity under optimized conditions. Meanwhile, the obtained imprinted polymers used as SPE sorbents coupled with HPLC were applied in the specific isolation and determination of CGA in fruit juices. The developed method may enrich the designation strategy for hydrophilic MIPs with outstanding performance, which could provide a feasible and alternative way for fast,
18
efficient, and selective extraction of water-soluble compounds in food, environmental, and biological samples. Acknowledgements
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Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Table 1 The adsorption capacities, imprinting factors, and selectivity coefficients of CGA, CCA, CFA, CA, and PCA for Fe3O4@CGA-MIPs and [email protected] Analytes CGA CCA CFA CA PCA a
QMIP（mg g-1） QNIP（mg g-1） 58.5 5.51 9.32 25.7 16.2
9.91 8.27 9.26 10.3 9.52
IF
SC
5.90 0.67 1.01 2.50 1.70
― 8.81 5.84 2.36 3.47
In this experiment, 20 mg of Fe3O4@CGA-MIPs and Fe3O4@NIPs were incubated with 10 mL of the mixed
solution of CGA, CCA, CFA, CA, and PCA at a concentration of 0.40 mg mL-1 for 30 min at room temperature. Fe3O4@CGA-MIPs: magnetic molecularly imprinted nanoparticles of chlorogenic acid. Fe3O4@NIPs: non-imprinted magnetic nanoparticles. CGA: chlorogenic acid; CCA: cichoric acid; CA: caffeic acid; CFA: caftaric acid; PCA: protocatechuic acid
Highlights:
Branched polyethyleneimine used to enhance the density of imprinted cavities and hydrophilcity of MIPs.
Directly amino-functionalized magnetic nanoparticles utilized as supporters to simplify the preparation procedure.
Preparation conditions optimized in detail.
Application of resulted MIPs for extraction and determination of chlorogenic acid in fruit juices.
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