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A novel polymeric ionic liquid-coated magnetic multiwalled carbon nanotubes for the solid-phase extraction of Cu, Zn-superoxide dismutase
Analytica Chimica Acta 939 (2016) 54e63
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A novel polymeric ionic liquid-coated magnetic multiwalled carbon nanotubes for the solid-phase extraction of Cu, Zn-superoxide dismutase Qian Wen, Yuzhi Wang*, Kaijia Xu, Na Li, Hongmei Zhang, Qin Yang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A strategy for the magnetic solidphase extraction of Cu, Zn-SOD based on polymeric ionic liquid has been developed. The Cu, Zn-SOD remained high specific activity after extraction. The magnetic adsorbent could be recycled and successfully employed in the extraction of Cu, Zn-SOD from real samples.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 June 2016 Received in revised form 10 August 2016 Accepted 18 August 2016 Available online 21 August 2016
A novel magnetic adsorbent, benzyl groups functionalized imidazolium-based polymeric ionic liquid (PIL)-coated magnetic multiwalled carbon nanotubes (MWCNTs) (m-MWCNTs@PIL), has been successfully synthesized and applied for the extraction of Cu, Zn-superoxide dismutase (Cu, Zn-SOD). The mMWCNTs@PIL were characterized by X-ray diffraction (XRD), Fourier transform infrared spectrometry (FT-IR), thermal gravimetric analysis (TGA), field emission scanning electron microscopy (FESEM), vibrating sample magnetometer (VSM) and zeta-potential nanoparticles. In this method, the mMWCNTs@PIL could interact with Cu, Zn-SOD through hydrogen bonding, p-p and electrostatic interactions. The extraction performance of the m-MWCNTs@PIL in the magnetic solid-phase extraction (MSPE) procedure was investigated, coupled with the determination by UVevis spectrophotometer. Compared with m-MWCNTs@IL and m-MWCNTs, the m-MWCNTs@PIL exhibited the highest extraction capacity of 29.1 mg/g for Cu, Zn-SOD. The adsorbed Cu, Zn-SOD remained high specific activity after being eluted from m-MWCNTs@PIL by 1 moL/L NaCl solution. Besides, the m-MWCNTs@PIL could be easily recycled and successfully employed in the extraction of Cu, Zn-SOD from real samples. Under the optimal conditions, the precision, repeatability and stability of the proposed method were investigated and the RSDs were 0.29%, 1.68% and 0.54%, respectively. Recoveries were in the range of 82.7e102.3%, with the RSD between 3.47% and 5.35%. On the basis of these results, the developed method has great potential in the extraction of Cu, Zn-SOD or other analytes from biological samples. © 2016 Elsevier B.V. All rights reserved.
Keywords: Cu Zn-superoxide dismutase Magnetic solid-phase extraction Magnetic multiwalled carbon nanotubes Polymeric ionic liquid
* Corresponding author. E-mail address: [email protected] (Y. Wang). http://dx.doi.org/10.1016/j.aca.2016.08.028 0003-2670/© 2016 Elsevier B.V. All rights reserved.
Q. Wen et al. / Analytica Chimica Acta 939 (2016) 54e63
1. Introduction Superoxide dismutase (SOD) is well-known as a significant antioxidant metalloenzyme, which is widely existed in prokaryotic and eukaryotic cells [1]. It is able to keep the balance of reactive oxygen species (ROS) in vivo, protecting cells from lipid peroxidation and oxidative damage [2]. Cu, Zn-SOD is the first discovered and the fullest studied among them, which plays a key role in catalyzing the disproportionation of the superoxide anion radicals O 2 to O2 and H2O2 for the cells equilibrium [3]. So developing a convenient and effective method for the extraction of Cu, Zn-SOD has a profound significance in our lives. To date, the common methods for extraction of proteases are salting out method [4], organic solvent method [5], isoelectric precipitation method [6], thermal denaturalization precipitation method [7] and chromatography method [8]. In addition, a novel magnetic solid-phase extraction (MSPE) method was proposed to be used for purifying protein [9]. MSPE method is an improved solid-phase extraction method, in which magnetic materials are dispersed into the sample solution as adsorbents in the extraction process [10e13]. Noticeably, the adsorbent has a great impact on MSPE process, because it directly affects the selectivity, affinity and capacity of the method [14]. Nanoscale zero-valent iron (nZVI) has large surface area and high intrinsic reactivity of surface sites [15,16]. With the borohydride reduction method, the oxidization of Fe particles is inevitable even being synthesized under inert environment [17]. The generated iron oxides (Fe2O3, Fe3O4 and FeOOH) are able to accumulate on the nZVI surface, which prevent the nZVI corrosion [18]. Owing to these advantages, the nZVI shows a great prospect in MSPE. Whereas, the nZVI is likely to aggregate that limit its application [19]. Carbon based materials are often involved to obtain more stable materials, which can improve the performance of nZVI [20]. Multiwalled carbon nanotubes (MWCNTs) are a new type of carbon based materials. They have unique properties which make them suitable for extraction application, such as highly porous and hollow structure, large specific surface area, light mass density and strong interaction between carbon and hydrogen molecules [21,22]. The large delocalized p-electron system of MWCNTs is capable of establishing p-p interaction with analytes [23]. Furthermore, modifying nZVI with MWCNTs (m-MWCNTs) is not only capable of improving the specific surface area and the extraction capacity of adsorbent, but also preventing nZVI from being oxidized and agglomerating [24,25]. Ionic liquids (ILs) are organic salts with a melting point of 100 C or less. The useful physicochemical properties of ILs include high chemical stability, non-flammability, negligible vapour pressure, high ionic conductivity and the capability to attract dissolves molecules by a variety of salvation interactions [26,27]. One of the most interesting advantages of ILs is that functional groups can be introduced to form task-specific ILs, which are able to interact analytes in specific ways [28]. More importantly, polymeric ionic liquid (PIL) is a subclass of polyelectrolyte while largely maintaining the unique properties of IL [29]. The PIL has already been applied in many analytical extraction processes, presenting high extraction selectivity and efficiency for analytes [30,31]. Benzyl groups functionalized imidazolium-based PIL was selected because of the p-p interactions of benzene and imidazole rings as well as hydrogen bonding via the N and O atom. In this manuscript, benzyl groups functionalized imidazoliumbased PIL-coated m-MWCNTs (m-MWCNTs@PIL) were applied as the adsorbent, which could interact with Cu, Zn-SOD through hydrogen bonding, p-p and electrostatic interactions in the extraction procedure. The extraction performance of the mMWCNTs@PIL in the MSPE procedure was investigated, coupled
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with UVevis spectrophotometer. Subsequently, Cu, Zn-SOD was eluted from the adsorbent with 1 mol/L NaCl solution. The activity analysis of Cu, Zn-SOD after extraction and the reusability of the mMWCNTs@PIL were examined. Finally, the analysis of real sample presented good selectivity and favorable applicability of this method. The whole MSPE procedure was presented in Scheme 1. 2. Experimental 2.1. Apparatus All products were dried by a DZF-6051 vacuum drying oven (Shanghai, China) and a FD-1C-50 vacuum-freezing drier (Beijing, China). The MSPE procedure was conducted in a QYC200 incubator shaker (Shanghai, China). Prepared NH2-IL monomer was investigated by INOVA 400NB NMR (Varian, America). The magnetism of sorbents was studied by EV11 Vibrating Sample Magnetometer (MicroSense, USA). The morphology of MWCNTs and magnetic composites were obtained using a MIRA3 LMU field emission scanning electron microscopy (FESEM, TESCAN, Czech). Infrared spectrum was recorded using a Spectrum One FT-IR spectrometer (PerkinElmer, USA). Thermogravimetric analyses were studied by a STA 449C thermal gravimetric analyzer (Netzsch, Germany). X-ray diffraction pattern was achieved using a D/Max 2500 X-ray diffraction (Rigaku, Japan). Zeta potentials were studied by a Zetasizer Nano-ZS90 dynamic light scattering (Malvern, Britain). Ultraviolet absorption spectrum was studied by a UV2450 UVevis spectrophotometer (Shimadzu, Japan). 2.2. Chemicals and reagents All chemical reagents in this work were of analytical grade. HNO3, H2SO4, FeSO4$7H2O, polyethylene glycol (PEG-4000), ethanol, methanol, Na2HPO4, NaH2PO4, ammonium persulfate, Span 80, tetramethylethylenediamine (TEMED), acetone, sodium chloride, pyrogallol, hydrochloric acid and disodium edetate dihydrate (EDTA$2Na) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). KBH4 and NaOH were supplied by Taishan Chemical Co., Ltd. (Guangdong, China). N-(3Aminopropyl)-imidazole, n-dodecane and Ethylene dimethacrylate (ED) were acquired from Adamas Reagent Co., Ltd. (Shanghai, China). 4-Vinylbenzyl chloride was obtained from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). MWCNTs were purchased from Xian Feng nano-materials Technology Co., Ltd. (Nanjing, China). Cu, Zn-SOD (from porcine blood), 1,1,1-tris (hydroxymethyl)-methanamin, Lysozyme (Lys), porcine whole blood, bovine hemoglobin (BHb) were got from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). Dicyclohexylcarbodiimide (DCC) and N,N-dimethylformamide (DMF) were purchased from Titan Scientific Co., Ltd. (Shanghai, China). 2.3. Synthesis of m-MWCNTs According to literature [32], 0.5 g of MWCNTs was pretreated by a mixture of concentrated HNO3 and concentrated H2SO4 (V/V 1:3), refluxed with magnetic stirring at 70 C for 12 h. After cooling, the resultant of COOH-MWCNTs was centrifuged. They were washed with ultrapure water for several times to make product neutral, and dried in a freeze-dryer. COOH-MWCNTs powders were used in the subsequent synthesis of the m-MWCNTs. 2.78 g of FeSO4$7H2O and 0.5 g of PEG-4000 were completely dissolved in 30 mL alcohol-water system (V/V 4:1), then 0.5 g of MWCNTs-COOH was added. High-purity nitrogen was passed through the system and mixed with a stirring speed of 2000 rpm for 20 min. 21 mL of KBH4 mixed solution (20 mL 0.02 mol/L KBH4
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Scheme 1. Preparation of m-MWCNTs@PIL and its application for the MSPE of Cu, Zn-SOD.
Fig. 1. Synthesis of NH2-IL monomer.
and 1 mL 0.5 mol/L NaOH) was added into the above mixture at the rate of 2 drops per second. Finally, after reaction for 30 min, the product was washed with water and ethanol three times respectively, and then dried in a vacuum oven. Finally, m-MWCNTs composites were obtained. 2.4. Synthesis of NH2-IL monomer NH2-IL monomer was synthesized on the basis of Fig. 1. 6.26 g of N-(3-Aminopropyl)-imidazole (0.05 mol) and 7.63 g of 4Vinylbenzyl chloride (0.05 mol) were mixed in 50 mL of methanol. Run the reaction at room temperature for 12 h under vigorous stirring conditions. Removed residual reactant by suction filtration,
the clear solution was washed with ultrapure water for several times by rotary evaporator. In the end, NH2-IL monomer (1-(3aminopropyl)-3-(4-vinylbenzyl) imidazolium chloride) was gained by vacuum drying at 80 C for 12 h. The 1H-NMR spectra of the NH2-IL monomer: (DMSO-d6, 400 MHz; d, ppm, relative to TMS): 7.66 (s, 1H), 7.42 (d, 2H), 7.31 (d, 2H), 7.15 (d, 1H), 6.89 (d, 1H), 6.88e6.75 (m, 1H), 5.77e5.82 (m, 1H), 5.53 (s, 2H), 5.21e5.25 (m, 1H), 4.09 (t, 2H), 3.51 (t, 2H), 2.42e2.46 (m, 2H), 1.83e1.87 (m, 2H). 2.5. Synthesis of m-MWCNTs@IL According to the literature [33], 0.1 g of m-MWCNTs, 0.1 g of NH2-IL monomer and 0.1 g of DCC were ultrasonically dispersed
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into 50 mL of DMF solution for 20 min. The whole system was placed into 50 C water bath for 24 h under vigorous stirring conditions. The IL-functionalized m-MWCNTs (m-MWCNTs@IL) were obtained by magnetic separation and washed with ultrapure water for 3 times, and dried in vacuum. 2.6. Synthesis of m-MWCNTs@PIL The preparation of m-MWCNTs@PIL was referencing a classical W/O emulsion polymerization method [34]. Firstly, 100 mg of mMWCNTs@IL, 1 mL of ED and 10 mg of ammonium persulfate were dispersed into 5 mL sodium phosphate buffer (0.2 mol/L, pH 6.0) at a 50 mL centrifuge tube. 1 ml of continuous organic phase (0.75 mL n-dodecane, 0.25 mL Span 80) was added into centrifuge tube dropwise. High-purity nitrogen bubbled into the emulsion system while 63 mL of TEMED was added. And then the mixture was shaken for 1 h at 25 C with a shaking speed of 200 rmp in an incubator shaker. After the reaction, 15 mL of acetone was added. The product was separated by external magnet, washed with ultrapure water and freeze-dried. Ultimately, m-MWCNTs@PIL composite were acquired. 2.7. MSPE procedure In the MSPE procedure, m-MWCNTs@PIL was investigated to extract Cu, Zn-SOD from sample solution. Firstly, 10 mg of mMWCNTs@PIL and 1 mL of Cu, Zn-SOD solution (0.5 mg/mL) were added into a 2 mL centrifuge tube. Subsequently, the mixture was shaken for 1 h at room temperature with a shaking speed of 200 rmp. After that, the adsorbents were separated by an external magnet and the limpid supernatant was detected by UVevis spectrophotometer at 205 nm to acquire the concentration of Cu, Zn-SOD. The extraction capacity (Q) of Cu, Zn-SOD was calculated as following:
Q¼
ðC0 CÞV m
where Q (mg/g) is the quantity of Cu, Zn-SOD adsorbed on a unit amount of adsorbent, C0 (mg/mL) is the initial concentration of Cu, Zn-SOD solution, C (mg/mL) is the concentration of Cu, Zn-SOD in supernatant, V (mL) is the volume of Cu, Zn-SOD solution, m (mg) is the dose of adsorbent. The absorbed Cu, Zn-SOD was eluted from the m-MWCNTs@PIL with 1 mL of NaCl (1 mol/L) solution for 1 h at room temperature via shaking. The activity analysis of Cu, Zn-SOD after extraction was examined. Finally, the adsorbents were collected and reused.
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3. Results and discussion 3.1. Characterization of the m-MWCNTs@PIL The XRD studies of MWCNTs, m-MWCNTs, m-MWCNTs@IL and m-MWCNTs@PIL were presented in Fig. 2. In Fig. 2a, peaks at 2q angle of 25.78 (002), 43.36 (100) and 53.94 (004) were attributed to MWCNTs which had a graphite-like structure. As shown in Fig. 2bed, m-MWCNTs m-MWCNTs@IL and m-MWCNTs@PIL had similar diffraction peaks. It could be found that two peaks appeared at 2q angle of 43.6 (110), and 63.4 (200), which confirmed the existence of Fe0 (JCPDS, No. 89-4186). In addition, peaks at 21.2 , 30.2 , 35.8 and 53.1 suggested the presence of lepidocrocite (FeOOH), magnetite (Fe3O4) and/or maghemite (Fe2O3) [35]. The results were corresponding to previous studies which have reported a characteristic core shell structure of nZVI [36,37]. The XRD analysis indicated that m-MWCNTs@PIL contained MWCNTs and magnetic particles. The FT-IR spectra of NH2-IL monomer, m-MWCNTs, mMWCNTs@IL and m-MWCNTs@PIL were performed in Fig. 3. In the spectrum of NH2-IL monomer (Fig. 3a), double peaks of amino group at 3410 and 3097 cm1 (stretching vibrations of NeH) were presented. The OeH stretching vibration for carboxyl group obviously occurred at 3055 cm1 in the m-MWCNTs (Fig. 3b), which was obviously disappeared from the m-MWCNTs@IL and mMWCNTs@PIL (Fig. 3c and d). Furthermore, the peak at 1627 cm1 (C]O stretching vibration) could be accredited to the formation of amide linkage between m-MWCNTs and NH2-IL monomer. The thermal stabilities of m-MWCNTs, m-MWCNTs@IL and mMWCNTs@PIL were investigated by TGA, which were conducted in argon atmosphere at 10 K/min. The TGA data were demonstrated in Fig. 4, manifesting that m-MWCNTs, m-MWCNTs@IL and mMWCNTs@PIL had unlike weight loss. All of them were exhibited a bit of mass loss (1%) below 100 C, which were due to the removal of absorbed water molecules. Further heating of samples to 800 C, oxygen-containing groups were decomposed. However, the mMWCNTs@PIL presented 12.4% and 2.4% higher weight loss than mMWCNTs and m-MWCNTs@IL, respectively. The phenomena were due to the dissociation of PIL from the surface of m-MWCNTs@PIL. Based on above discussion, we could confirm that PIL modified the m-MWCNTs successfully. The morphologies of MWCNTs, m-MWCNTs, m-MWCNTs@IL and m-MWCNTs@PIL were analyzed by FESEM. As shown in Fig. 5a, the MWCNTs were intertwined and had smooth surface. It was clear that the surface of MWCNTs was covered by a mass of nanoparticles in the electron micrograph of m-MWCNTs (Fig. 5b). Hence, during the synthesis of m-MWCNTs, the magnetic
Fig. 2. XRD patterns of MWCNTs (a), m-MWCNTs (b), m-MWCNTs@IL (c) and m-MWCNTs@PIL (d).
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Fig. 3. FT-IR spectra of NH2-IL monomer (a), m-MWCNTs (b), m-MWCNTs@IL (c) and m-MWCNTs@PIL (d).
of PIL that covered the m-MWCNTs. Besides, this result was in good corresponding to the FESEM view as mentioned before. From Fig. 6b, the black suspension of m-MWCNTs@PIL could be removed from a solution by an external magnet. The magnetic behavior of mMWCNTs@PIL proved that it owned sufficient saturation magnetization for magnetic separation. The isoelectric points (IEPs) of m-MWCNTs and mMWCNTs@PIL were surveyed by zeta-potential nanoparticles at different pH values. As shown in Fig. 7, we could find that the IEP of m-MWCNTs and m-MWCNTs@PIL were 2.07 and 4.05, respectively. The IEP of the m-MWCNTs was much less than previously reported value for pristine MWCNTs (6.8) [38]. This might be due to the carboxylic groups on MWCNTs were partially substituted in the mMWCNTs. Similarly, the surface groups of m-MWCNTs were changed after polymerization process. Thus high IEP for mMWCNTs@PIL was a conclusive evidence to prove that PIL coated on the m-MWCNTs. Fig. 4. Weight loss curves of m-MWCNTs (a), m-MWCNTs@IL (b) and m-MWCNTs@PIL (c).
nanoparticles was successfully wrapped around the surface of MWCNTs. In Fig. 5c, the surface of m-MWCNTs@IL was rougher when compared with m-MWCNTs. From Fig. 5d, we could observe that the m-MWCNTs were coated by PIL uniformly and entirely. Moreover, the tube morphology of MWCNTs nearly maintained unchanged, manifesting that the polymerization process wouldn't damage the tube structure. The energy-dispersive spectrometry (EDS) analysis of the mMWCNTs@PIL was shown in Fig. 5e. There were 70.77 wt% of C, 13.84 wt% of Fe, 8.11 wt% of O, 6.57 wt% of N and 0.7 wt% of Cl in the m-MWCNTs@PIL composite. Consequently, the m-MWCNTs@PIL was prepared successfully. The magnetic properties of m-MWCNTs and m-MWCNTs@PIL were measured by VSM technique at room temperature. Magnetic hysteresis curves for them were displayed in Fig. 6a. The saturation magnetization intensities of m-MWCNTs and m-MWCNTs@PIL were 12.84 and 2.8 emu g1. It was worth noting that the saturation magnetization of m-MWCNTs@PIL was lower than that of mMWCNTs. This could be mainly ascribed to the nonmagnetic phase
3.2. Optimization of MSPE conditions 3.2.1. Effect of the extraction time In order to reach the equilibrium of MSPE procedure, extraction time was investigated in the range of 0.25e4 h. As can be seen in Fig. 8a, the extraction capacities of Cu, Zn-SOD increased when extraction time ranged from 0.25 to 1.5 h and maintained unchanged with a further extension of extraction time from 1.5 to 4 h. Obviously, the extraction equilibrium was achieved in 1.5 h, and the m-MWCNTs@PIL indeed possessed commendable extraction capacity for analytes. Ultimately, 1.5 h was selected as the optimum extraction time. 3.2.2. Effect of the extraction temperature To assess the influence of temperature on the extraction capacity, batch experiments were conducted from 20 to 45 C. Fig. 8b revealed the relationship between the extraction capacity and extraction temperature, indicating that an increase in extraction temperature resulted in an advanced extraction capacities. The reason for this result could be that the relative motion between the molecules was accelerated by heating up, which was helpful for extraction. According to literature [39], Cu, Zn-SOD has high thermal stability below 75 C. However, in order to maintain mild
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Fig. 5. FESEM images of MWCNTs (a), m-MWCNTs (b), m-MWCNTs@IL (c), m-MWCNTs@PIL (d) and EDS patterns of m-MWCNTs@PIL (e).
Fig. 6. Magnetic hysteresis loops of m-MWCNTs, m-MWCNTs@PIL (a), and the magnetic response of m-MWCNTs@PIL to external magnetic field (b).
conditions for MSPE procedure, 45 C was set as the extraction temperature for subsequent experiments.
3.2.3. Effect of the amount of m-MWCNTs@PIL To evaluate the effect of the amount of adsorbent for the MSPE, different quantities of m-MWCNTs@PIL were surveyed from 4 to 12 mg. As shown in Fig. 8c, the extraction capacity reached the maximum when 10 mg of m-MWCNTs@PIL was applied. The extraction capacities increased with the increasing mass of mMWCNTs@PIL because of active sites raised. However, the extraction capacities reduced when the amount of adsorbent exceeded 10 mg, indicating that excess m-MWCNTs@PIL were helpless with
Fig. 7. Zeta potentials of m-MWCNTs and m-MWCNTs@PIL in different pH solutions.
such small volume of the extraction solvent. Based on these results, 10 mg of m-MWCNTs@PIL were employed for further researches. 3.2.4. Effect of the pH value The pH of the sample solution played a key role in MSPE process, which can affect the surface charge of both analytes and adsorbent. The pH of the sample solution was conducted in 5 mmol/L phosphate buffer solution in the range of 4e9. Based on the IEP characterization of m-MWCNTs@PIL (IEP 4.05), when the solution pH value was higher than 4.05, the surface charge of m-MWCNTs@PIL became negative. The Cu, Zn-SOD (IEP 7.12) was protonated when the solution pH was below 7.12. As presented in Fig. 8d, extraction capacities improved when pH varied from 4 to 5 and after that,
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Fig. 8. Effect of extraction time (a), temperature (b), amount of m-MWCNTs@PIL (c), pH value (d), initial Cu, Zn-SOD concentration (e) and ionic strength (f).
extraction capacities displayed an absolute downward trend. The maximum extraction capacity appeared at pH 5 where the surface of m-MWCNTs@PIL became negative and enable to extract positive Cu, Zn-SOD. This phenomenon revealed that electrostatic interaction was indeed existed in adsorption. Consequently, the sample solution was set as pH 5. 3.2.5. Effect of the initial Cu, Zn-SOD concentration The effect of initial Cu, Zn-SOD concentration in the sample solution was examined in the range of 0.25e2 mg/mL. In Fig. 8e, the extraction capacities increased rapidly with increasing the initial Cu, Zn-SOD concentration from 0.25 to 1.5 mg/mL, and remained constant when further increased the initial Cu, Zn-SOD concentration to 2 mg/mL. Obviously, a higher initial Cu, Zn-SOD concentration provided more possibilities for m-MWCNTs@PIL to capture analytes. However, the limited active reaction sites on the adsorbent should be responsible for the stagnant growth. To make full use of the active reaction sites of m-MWCNTs@PIL, 1.5 mg/mL initial Cu, Zn-SOD concentration was used.
Fig. 9. Extraction capacities of m-MWCNTs@PIL, m-MWCNTs@IL and m-MWCNTs for three different analytes.
3.2.6. Effect of the solution ionic strength In order to evaluate the effect of ionic strength in extraction performance, the concentration of NaCl was varied from 0 to 0.5 mol/L. As shown in Fig. 8f, the extraction capacities decreased obviously as the NaCl concentration was increased. It could be inferred that the role of electrostatic interaction in MSPE process was significant, with which sodium ions and positively charged Cu, Zn-SOD were competitively extracted by m-MWCNTs@PIL. Ultimately, no salt was added in the MSPE procedure. After optimizing six effective factors for extraction process, the maximum extraction capacity of Cu, Zn-SOD was 85.28 mg/g.
analytes (Cu, Zn-SOD at 205 nm, BHb at 406 nm and Lys at 278 nm) were compared with those of both m-MWCNTs@IL and mMWCNTs. As manifested in Fig. 9, the extraction capacity of mMWCNTs@PIL for Cu, Zn-SOD was higher than those acquired by mMWCNTs@IL and m-MWCNTs. It was also found that mMWCNTs@PIL showed the best extraction capacity for Cu, Zn-SOD than BHb and Lys. This indicated that m-MWCNTs@PIL might possess specific adsorption for Cu, Zn-SOD. Therefore, mMWCNTs@PIL was a promising adsorbent for MSPE of Cu, Zn-SOD.
3.3. Comparison of the extraction capacity
3.4. Desorption of Cu, Zn-SOD
In order to check out the adsorption property of mMWCNTs@PIL, their extraction capacities for three different
Desorption of analytes from adsorbents had a significant impact on the reusability of the m-MWCNTs@PIL and the retrieve of Cu,
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Fig. 10. The autoxidation rate of pyrogallol (a), The autoxidation rate of pyrogallol with the initial Cu, Zn-SOD was added (b), The autoxidation rate of pyrogallol with the Cu, Zn-SOD after extraction was added (c), the specific activities of the initial Cu, Zn-SOD and the Cu, Zn-SOD after extraction.
Zn-SOD. After extraction, the m-MWCNTs@PIL were magnetically separated from aqueous solution and then eluted by 1 mL of 1 mol/ L NaCl solution. The suspension was shaken in a 2 mL centrifuge tube for 1 h at 25 C. In this way, the desorption efficiency of Cu, ZnSOD was 84.35%, indicating the adsorbed analytes could be eluted from m-MWCNTs@PIL successfully. Based on the result of the effect of ionic strength in extraction performance, the high desorption efficiency might attribute to the increase of iron strength which could weaken the electrostatic interaction between mMWCNTs@PIL and Cu, Zn-SOD.
Zn-SOD and the Cu, Zn-SOD after extraction were calculated according to the following formula:
specific activity ðU=mgÞ ¼
a 50%
V m
where a is inhibitory rate for pyrogallol autoxidation, V is the total volume, m is the mass of Cu, Zn-SOD. It was found in Fig. 10d that the specific activities of the initial Cu, Zn-SOD and the Cu, Zn-SOD after extraction were high enough. Therefore, this MSPE method could extract Cu, Zn-SOD effectively without inactivating it.
3.5. Activity analysis of Cu, Zn-SOD 3.6. Reusability of the m-MWCNTs@PIL The examination of Cu, Zn-SOD activity was conducted by pyrogallol autoxidation method [40,41]. 0.015 mL of 45 mmol/L pyrogallol solution was added into 4.5 mL of 50 mmol/L Tris-HCl buffer solution (pH 8.2, 1 mmol/L EDTA) at 25 C. The absorbance at 325 nm of the mixture was detected by UVevis spectrophotometer for 3 min. As can be seen in Fig. 10a, the autoxidation rate of pyrogallol was 0.07216/min. Afterwards, 0.005 mL of the initial Cu, Zn-SOD (0.1 mg/mL) and the Cu, Zn-SOD after extraction (0.1 mg/mL) were added into above mixture, respectively. Their autoxidation rates of pyrogallol were 0.04812/min and 0.05071/ min (Fig. 10b and c). Finally, the specific activities of the initial Cu,
To examine reusability of the m-MWCNTs@PIL, the adsorbent was subjected to different extraction-desorption cycles. After desorption of Cu, Zn-SOD, the collected adsorbent was washed twice with ultrapure water, and then dried for next analysis run. As shown in Fig. 11, extraction capacities of Cu, Zn-SOD decreased in the second and third run and then remained constant in the following runs. The residue of Cu, Zn-SOD on the adsorbent might be responsible for the reduction of extraction capacity. Nevertheless, the m-MWCNTs@PIL showed an acceptable reusability in MSPE procedure.
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Q. Wen et al. / Analytica Chimica Acta 939 (2016) 54e63 Table 1 The results of the precision, repeatability and stability experiments. Precision measurement results (n ¼ 5) Repeats Extraction capacities (mg g1) RSD (%)
1 87.01 0.29
2 87.01
3 87.36
4 87.03
5 87.56
2 85.04
3 87.4
4 85.25
5 85.67
2 87.22
3 86.68
4 86.75
5 86.25
Repeatability measurement results (n ¼ 5) Sample number Extraction capacities (mg g1) RSD (%)
1 83.4 1.68
Stability measurement results (n ¼ 5) Day number Extraction capacities (mg g1) RSD (%)
Fig. 11. Extraction capacities of Cu, Zn-SOD for different runs.
3.7. Analysis of real sample To study the practical extraction performance of the proposed method in real samples, the porcine whole blood was selected and analyzed. Under the optimum experimental conditions as mentioned above, 1 mL of the porcine whole blood which was diluted 50 times with phosphate buffer solution (pH 5) was applied for the MSPE procedure. The analytical result was illustrated in Fig. 12, which was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). It could be found that the specific pattern of Cu, Zn-SOD in the porcine whole blood became fade after extraction. Consequently, the proposed MSPE method based on m-MWCNTs@PIL could extract Cu, Zn-SOD from porcine whole blood sample effectively.
3.8. Method validation After the optimal experimental factors were confirmed, the precision, repeatability and stability experiments were conducted to evaluate the proposed method. The results were summarized in Table 1. The relative standard deviation (RSD) of the apparatus
1 87.44 0.54
precision exhibited to be 0.29%, which was examined by detecting a sample for 5 times coupled with UVevis spectrophotometer. Five sets of parallel experiments were performed to investigate the repeatability and the RSD was 1.68%. The stability of the method was investigated by surveying the same sample for five consecutive days and the RSD was 0.54%. The recovery of the method was investigated by standard addition method. As observed from Table 2, the recoveries were calculated after spiking three concentration levels (0.1, 1 and 10 mg/mL) of Cu, Zn-SOD. Recoveries were in the range of 82.7e102.3%, with the RSD between 3.47% and 5.35%. The results evidenced that this method was reliable for receiving precise experimental data and existed excellent repeatability and stability. 4. Conclusions It is well known that the common magnetic particles are Fe3O4,
g-Fe2O3, Fe, Co, MnFe2O4, MgFe2O3, CoFe2O3, FePt and CoPt3.
Compared with Co, MnFe2O4, MgFe2O3, CoFe2O3, FePt and CoPt3, the Fe nanoparticles are more easily synthesized and have the advantage that the small particle size of nZVI results in large specific surface area and great intrinsic reactivity of surface sites. In addition, the Fe3O4 are the most widely used magnetic particles due to its good stability. However, nZVI technology has been widely investigated for the treatment of environmental pollutants, so applying the Fe nanoparticles to the MSPE method is a very meaningful attempt. In this way, we can promote the diversification of science. In the present work, a novel magnetic adsorbent, mMWCNTs@PIL has been successfully synthesized and applied for the extraction of Cu, Zn-SOD. The m-MWCNTs@PIL could interact with Cu, Zn-SOD through hydrogen bonding, p-p and electrostatic interactions and showed its good extraction capacity to Cu, Zn-SOD. The adsorbed Cu, Zn-SOD remained high specific activity after being eluted from m-MWCNTs@PIL by 1 mol/L NaCl solution. Besides, the m-MWCNTs@PIL could be easily recycled and successfully employed in extraction of Cu, Zn-SOD from real samples. Under the optimal conditions, the precision, repeatability and stability of the proposed method were investigated and the RSDs were 0.29%, 1.68% and 0.54%, respectively. Recoveries were in the range of
Table 2 Recovery for Cu, Zn-SOD from blood samples.
Fig. 12. SDS-PAGE of protein molecular weight maker (a), 1.5 mg/mL Cu, Zn-SOD (b), porcine whole blood sample (c) and porcine whole blood after extraction (d).
Analyte (n ¼ 5)
Spiked (mg/mL)
Recovery (%)
RSD (%)
Cu, Zn-SOD
0 0.1 1 10
82.7 94.4 102.3
3.47 3.89 5.35
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82.7e102.3%, with the RSD between 3.47% and 5.35%. All these indicated that the developed method has great potential on the extraction of Cu, Zn-SOD or other analytes from biological samples.
[14] [15] [16] [17] [18]
Acknowledgements
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The authors greatly appreciate the financial supports by the National Natural Science Foundation of China (No. 21375035; No. J1210040) and the Foundation for Innovative Research Groups of NSFC (Grant 21521063). References [1] F. Johnson, C. Giulivi, Mol. Asp. Med. 26 (2005) 340e352. s, E. Sala, M. Vaque , A. Arde vol, M. Blay, J. Fernandez-Larrea, [2] F. Puiggro , J. Agr. Food Chem. 57 (2009) 3934e3942. M.J. Salvado [3] A.F. Miller, Curr. Opin. Chem. Biol. 8 (2004) 162e168. lisle, D. Garant, F. Pelletier, [4] L. Haroune, R. Cassoulet, M.P. Lafontaine, M. Be J.P. Bellenger, Anal. Chim. Acta 891 (2015) 160e170. [5] M. Gupta, A. Jain, K.K. Verma, Talanta 80 (2009) 526e531. [6] L. Anta, M.L. Marina, M.C. García, J. Chromatogr. A 1217 (2010) 7138e7143. [7] L. Zhang, G. Xue, Huang, Nat. Prod. Res. Dev. 16 (2003) 334e337. [8] R. Nian, W. Zhang, L. Tan, J. Lee, X. Bi, Y. Yang, P. Gagnon, J. Chromatogr. A 1431 (2016) 1e7. [9] Y. Huang, Y. Wang, Y. Wang, Q. Pan, X. Ding, K. Xu, Q. Wen, RSC Adv. 6 (2016) 5718e5728. [10] Q.L. Li, L.L. Wang, X. Wang, M.L. Wang, R.S. Zhao, J. Chromatogr. A 1449 (2016) 39e47. [11] J. Ding, L.J. Mao, N. Guo, L. Yu, Y.Q. Feng, J. Chromatogr. A 1446 (2016) 103e113. [12] N. Li, J. Chen, Y.P. Shi, J. Chromatogr. A 1441 (2016) 24e33. [13] K. Xu, Y. Wang, X. Ding, Y. Huang, N. Li, Q. Wen, Talanta 148 (2016) 153e162.
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A novel polymeric ionic liquid-coated magnetic multiwalled carbon nanotubes for the solid-phase extraction of Cu, Zn-superoxide dismutase Qian Wen, Yuzhi Wang*, Kaijia Xu, Na Li, Hongmei Zhang, Qin Yang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A strategy for the magnetic solidphase extraction of Cu, Zn-SOD based on polymeric ionic liquid has been developed. The Cu, Zn-SOD remained high specific activity after extraction. The magnetic adsorbent could be recycled and successfully employed in the extraction of Cu, Zn-SOD from real samples.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 June 2016 Received in revised form 10 August 2016 Accepted 18 August 2016 Available online 21 August 2016
A novel magnetic adsorbent, benzyl groups functionalized imidazolium-based polymeric ionic liquid (PIL)-coated magnetic multiwalled carbon nanotubes (MWCNTs) (m-MWCNTs@PIL), has been successfully synthesized and applied for the extraction of Cu, Zn-superoxide dismutase (Cu, Zn-SOD). The mMWCNTs@PIL were characterized by X-ray diffraction (XRD), Fourier transform infrared spectrometry (FT-IR), thermal gravimetric analysis (TGA), field emission scanning electron microscopy (FESEM), vibrating sample magnetometer (VSM) and zeta-potential nanoparticles. In this method, the mMWCNTs@PIL could interact with Cu, Zn-SOD through hydrogen bonding, p-p and electrostatic interactions. The extraction performance of the m-MWCNTs@PIL in the magnetic solid-phase extraction (MSPE) procedure was investigated, coupled with the determination by UVevis spectrophotometer. Compared with m-MWCNTs@IL and m-MWCNTs, the m-MWCNTs@PIL exhibited the highest extraction capacity of 29.1 mg/g for Cu, Zn-SOD. The adsorbed Cu, Zn-SOD remained high specific activity after being eluted from m-MWCNTs@PIL by 1 moL/L NaCl solution. Besides, the m-MWCNTs@PIL could be easily recycled and successfully employed in the extraction of Cu, Zn-SOD from real samples. Under the optimal conditions, the precision, repeatability and stability of the proposed method were investigated and the RSDs were 0.29%, 1.68% and 0.54%, respectively. Recoveries were in the range of 82.7e102.3%, with the RSD between 3.47% and 5.35%. On the basis of these results, the developed method has great potential in the extraction of Cu, Zn-SOD or other analytes from biological samples. © 2016 Elsevier B.V. All rights reserved.
Keywords: Cu Zn-superoxide dismutase Magnetic solid-phase extraction Magnetic multiwalled carbon nanotubes Polymeric ionic liquid
* Corresponding author. E-mail address: [email protected] (Y. Wang). http://dx.doi.org/10.1016/j.aca.2016.08.028 0003-2670/© 2016 Elsevier B.V. All rights reserved.
Q. Wen et al. / Analytica Chimica Acta 939 (2016) 54e63
1. Introduction Superoxide dismutase (SOD) is well-known as a significant antioxidant metalloenzyme, which is widely existed in prokaryotic and eukaryotic cells [1]. It is able to keep the balance of reactive oxygen species (ROS) in vivo, protecting cells from lipid peroxidation and oxidative damage [2]. Cu, Zn-SOD is the first discovered and the fullest studied among them, which plays a key role in catalyzing the disproportionation of the superoxide anion radicals O 2 to O2 and H2O2 for the cells equilibrium [3]. So developing a convenient and effective method for the extraction of Cu, Zn-SOD has a profound significance in our lives. To date, the common methods for extraction of proteases are salting out method [4], organic solvent method [5], isoelectric precipitation method [6], thermal denaturalization precipitation method [7] and chromatography method [8]. In addition, a novel magnetic solid-phase extraction (MSPE) method was proposed to be used for purifying protein [9]. MSPE method is an improved solid-phase extraction method, in which magnetic materials are dispersed into the sample solution as adsorbents in the extraction process [10e13]. Noticeably, the adsorbent has a great impact on MSPE process, because it directly affects the selectivity, affinity and capacity of the method [14]. Nanoscale zero-valent iron (nZVI) has large surface area and high intrinsic reactivity of surface sites [15,16]. With the borohydride reduction method, the oxidization of Fe particles is inevitable even being synthesized under inert environment [17]. The generated iron oxides (Fe2O3, Fe3O4 and FeOOH) are able to accumulate on the nZVI surface, which prevent the nZVI corrosion [18]. Owing to these advantages, the nZVI shows a great prospect in MSPE. Whereas, the nZVI is likely to aggregate that limit its application [19]. Carbon based materials are often involved to obtain more stable materials, which can improve the performance of nZVI [20]. Multiwalled carbon nanotubes (MWCNTs) are a new type of carbon based materials. They have unique properties which make them suitable for extraction application, such as highly porous and hollow structure, large specific surface area, light mass density and strong interaction between carbon and hydrogen molecules [21,22]. The large delocalized p-electron system of MWCNTs is capable of establishing p-p interaction with analytes [23]. Furthermore, modifying nZVI with MWCNTs (m-MWCNTs) is not only capable of improving the specific surface area and the extraction capacity of adsorbent, but also preventing nZVI from being oxidized and agglomerating [24,25]. Ionic liquids (ILs) are organic salts with a melting point of 100 C or less. The useful physicochemical properties of ILs include high chemical stability, non-flammability, negligible vapour pressure, high ionic conductivity and the capability to attract dissolves molecules by a variety of salvation interactions [26,27]. One of the most interesting advantages of ILs is that functional groups can be introduced to form task-specific ILs, which are able to interact analytes in specific ways [28]. More importantly, polymeric ionic liquid (PIL) is a subclass of polyelectrolyte while largely maintaining the unique properties of IL [29]. The PIL has already been applied in many analytical extraction processes, presenting high extraction selectivity and efficiency for analytes [30,31]. Benzyl groups functionalized imidazolium-based PIL was selected because of the p-p interactions of benzene and imidazole rings as well as hydrogen bonding via the N and O atom. In this manuscript, benzyl groups functionalized imidazoliumbased PIL-coated m-MWCNTs (m-MWCNTs@PIL) were applied as the adsorbent, which could interact with Cu, Zn-SOD through hydrogen bonding, p-p and electrostatic interactions in the extraction procedure. The extraction performance of the mMWCNTs@PIL in the MSPE procedure was investigated, coupled
55
with UVevis spectrophotometer. Subsequently, Cu, Zn-SOD was eluted from the adsorbent with 1 mol/L NaCl solution. The activity analysis of Cu, Zn-SOD after extraction and the reusability of the mMWCNTs@PIL were examined. Finally, the analysis of real sample presented good selectivity and favorable applicability of this method. The whole MSPE procedure was presented in Scheme 1. 2. Experimental 2.1. Apparatus All products were dried by a DZF-6051 vacuum drying oven (Shanghai, China) and a FD-1C-50 vacuum-freezing drier (Beijing, China). The MSPE procedure was conducted in a QYC200 incubator shaker (Shanghai, China). Prepared NH2-IL monomer was investigated by INOVA 400NB NMR (Varian, America). The magnetism of sorbents was studied by EV11 Vibrating Sample Magnetometer (MicroSense, USA). The morphology of MWCNTs and magnetic composites were obtained using a MIRA3 LMU field emission scanning electron microscopy (FESEM, TESCAN, Czech). Infrared spectrum was recorded using a Spectrum One FT-IR spectrometer (PerkinElmer, USA). Thermogravimetric analyses were studied by a STA 449C thermal gravimetric analyzer (Netzsch, Germany). X-ray diffraction pattern was achieved using a D/Max 2500 X-ray diffraction (Rigaku, Japan). Zeta potentials were studied by a Zetasizer Nano-ZS90 dynamic light scattering (Malvern, Britain). Ultraviolet absorption spectrum was studied by a UV2450 UVevis spectrophotometer (Shimadzu, Japan). 2.2. Chemicals and reagents All chemical reagents in this work were of analytical grade. HNO3, H2SO4, FeSO4$7H2O, polyethylene glycol (PEG-4000), ethanol, methanol, Na2HPO4, NaH2PO4, ammonium persulfate, Span 80, tetramethylethylenediamine (TEMED), acetone, sodium chloride, pyrogallol, hydrochloric acid and disodium edetate dihydrate (EDTA$2Na) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). KBH4 and NaOH were supplied by Taishan Chemical Co., Ltd. (Guangdong, China). N-(3Aminopropyl)-imidazole, n-dodecane and Ethylene dimethacrylate (ED) were acquired from Adamas Reagent Co., Ltd. (Shanghai, China). 4-Vinylbenzyl chloride was obtained from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). MWCNTs were purchased from Xian Feng nano-materials Technology Co., Ltd. (Nanjing, China). Cu, Zn-SOD (from porcine blood), 1,1,1-tris (hydroxymethyl)-methanamin, Lysozyme (Lys), porcine whole blood, bovine hemoglobin (BHb) were got from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). Dicyclohexylcarbodiimide (DCC) and N,N-dimethylformamide (DMF) were purchased from Titan Scientific Co., Ltd. (Shanghai, China). 2.3. Synthesis of m-MWCNTs According to literature [32], 0.5 g of MWCNTs was pretreated by a mixture of concentrated HNO3 and concentrated H2SO4 (V/V 1:3), refluxed with magnetic stirring at 70 C for 12 h. After cooling, the resultant of COOH-MWCNTs was centrifuged. They were washed with ultrapure water for several times to make product neutral, and dried in a freeze-dryer. COOH-MWCNTs powders were used in the subsequent synthesis of the m-MWCNTs. 2.78 g of FeSO4$7H2O and 0.5 g of PEG-4000 were completely dissolved in 30 mL alcohol-water system (V/V 4:1), then 0.5 g of MWCNTs-COOH was added. High-purity nitrogen was passed through the system and mixed with a stirring speed of 2000 rpm for 20 min. 21 mL of KBH4 mixed solution (20 mL 0.02 mol/L KBH4
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Scheme 1. Preparation of m-MWCNTs@PIL and its application for the MSPE of Cu, Zn-SOD.
Fig. 1. Synthesis of NH2-IL monomer.
and 1 mL 0.5 mol/L NaOH) was added into the above mixture at the rate of 2 drops per second. Finally, after reaction for 30 min, the product was washed with water and ethanol three times respectively, and then dried in a vacuum oven. Finally, m-MWCNTs composites were obtained. 2.4. Synthesis of NH2-IL monomer NH2-IL monomer was synthesized on the basis of Fig. 1. 6.26 g of N-(3-Aminopropyl)-imidazole (0.05 mol) and 7.63 g of 4Vinylbenzyl chloride (0.05 mol) were mixed in 50 mL of methanol. Run the reaction at room temperature for 12 h under vigorous stirring conditions. Removed residual reactant by suction filtration,
the clear solution was washed with ultrapure water for several times by rotary evaporator. In the end, NH2-IL monomer (1-(3aminopropyl)-3-(4-vinylbenzyl) imidazolium chloride) was gained by vacuum drying at 80 C for 12 h. The 1H-NMR spectra of the NH2-IL monomer: (DMSO-d6, 400 MHz; d, ppm, relative to TMS): 7.66 (s, 1H), 7.42 (d, 2H), 7.31 (d, 2H), 7.15 (d, 1H), 6.89 (d, 1H), 6.88e6.75 (m, 1H), 5.77e5.82 (m, 1H), 5.53 (s, 2H), 5.21e5.25 (m, 1H), 4.09 (t, 2H), 3.51 (t, 2H), 2.42e2.46 (m, 2H), 1.83e1.87 (m, 2H). 2.5. Synthesis of m-MWCNTs@IL According to the literature [33], 0.1 g of m-MWCNTs, 0.1 g of NH2-IL monomer and 0.1 g of DCC were ultrasonically dispersed
Q. Wen et al. / Analytica Chimica Acta 939 (2016) 54e63
into 50 mL of DMF solution for 20 min. The whole system was placed into 50 C water bath for 24 h under vigorous stirring conditions. The IL-functionalized m-MWCNTs (m-MWCNTs@IL) were obtained by magnetic separation and washed with ultrapure water for 3 times, and dried in vacuum. 2.6. Synthesis of m-MWCNTs@PIL The preparation of m-MWCNTs@PIL was referencing a classical W/O emulsion polymerization method [34]. Firstly, 100 mg of mMWCNTs@IL, 1 mL of ED and 10 mg of ammonium persulfate were dispersed into 5 mL sodium phosphate buffer (0.2 mol/L, pH 6.0) at a 50 mL centrifuge tube. 1 ml of continuous organic phase (0.75 mL n-dodecane, 0.25 mL Span 80) was added into centrifuge tube dropwise. High-purity nitrogen bubbled into the emulsion system while 63 mL of TEMED was added. And then the mixture was shaken for 1 h at 25 C with a shaking speed of 200 rmp in an incubator shaker. After the reaction, 15 mL of acetone was added. The product was separated by external magnet, washed with ultrapure water and freeze-dried. Ultimately, m-MWCNTs@PIL composite were acquired. 2.7. MSPE procedure In the MSPE procedure, m-MWCNTs@PIL was investigated to extract Cu, Zn-SOD from sample solution. Firstly, 10 mg of mMWCNTs@PIL and 1 mL of Cu, Zn-SOD solution (0.5 mg/mL) were added into a 2 mL centrifuge tube. Subsequently, the mixture was shaken for 1 h at room temperature with a shaking speed of 200 rmp. After that, the adsorbents were separated by an external magnet and the limpid supernatant was detected by UVevis spectrophotometer at 205 nm to acquire the concentration of Cu, Zn-SOD. The extraction capacity (Q) of Cu, Zn-SOD was calculated as following:
Q¼
ðC0 CÞV m
where Q (mg/g) is the quantity of Cu, Zn-SOD adsorbed on a unit amount of adsorbent, C0 (mg/mL) is the initial concentration of Cu, Zn-SOD solution, C (mg/mL) is the concentration of Cu, Zn-SOD in supernatant, V (mL) is the volume of Cu, Zn-SOD solution, m (mg) is the dose of adsorbent. The absorbed Cu, Zn-SOD was eluted from the m-MWCNTs@PIL with 1 mL of NaCl (1 mol/L) solution for 1 h at room temperature via shaking. The activity analysis of Cu, Zn-SOD after extraction was examined. Finally, the adsorbents were collected and reused.
57
3. Results and discussion 3.1. Characterization of the m-MWCNTs@PIL The XRD studies of MWCNTs, m-MWCNTs, m-MWCNTs@IL and m-MWCNTs@PIL were presented in Fig. 2. In Fig. 2a, peaks at 2q angle of 25.78 (002), 43.36 (100) and 53.94 (004) were attributed to MWCNTs which had a graphite-like structure. As shown in Fig. 2bed, m-MWCNTs m-MWCNTs@IL and m-MWCNTs@PIL had similar diffraction peaks. It could be found that two peaks appeared at 2q angle of 43.6 (110), and 63.4 (200), which confirmed the existence of Fe0 (JCPDS, No. 89-4186). In addition, peaks at 21.2 , 30.2 , 35.8 and 53.1 suggested the presence of lepidocrocite (FeOOH), magnetite (Fe3O4) and/or maghemite (Fe2O3) [35]. The results were corresponding to previous studies which have reported a characteristic core shell structure of nZVI [36,37]. The XRD analysis indicated that m-MWCNTs@PIL contained MWCNTs and magnetic particles. The FT-IR spectra of NH2-IL monomer, m-MWCNTs, mMWCNTs@IL and m-MWCNTs@PIL were performed in Fig. 3. In the spectrum of NH2-IL monomer (Fig. 3a), double peaks of amino group at 3410 and 3097 cm1 (stretching vibrations of NeH) were presented. The OeH stretching vibration for carboxyl group obviously occurred at 3055 cm1 in the m-MWCNTs (Fig. 3b), which was obviously disappeared from the m-MWCNTs@IL and mMWCNTs@PIL (Fig. 3c and d). Furthermore, the peak at 1627 cm1 (C]O stretching vibration) could be accredited to the formation of amide linkage between m-MWCNTs and NH2-IL monomer. The thermal stabilities of m-MWCNTs, m-MWCNTs@IL and mMWCNTs@PIL were investigated by TGA, which were conducted in argon atmosphere at 10 K/min. The TGA data were demonstrated in Fig. 4, manifesting that m-MWCNTs, m-MWCNTs@IL and mMWCNTs@PIL had unlike weight loss. All of them were exhibited a bit of mass loss (1%) below 100 C, which were due to the removal of absorbed water molecules. Further heating of samples to 800 C, oxygen-containing groups were decomposed. However, the mMWCNTs@PIL presented 12.4% and 2.4% higher weight loss than mMWCNTs and m-MWCNTs@IL, respectively. The phenomena were due to the dissociation of PIL from the surface of m-MWCNTs@PIL. Based on above discussion, we could confirm that PIL modified the m-MWCNTs successfully. The morphologies of MWCNTs, m-MWCNTs, m-MWCNTs@IL and m-MWCNTs@PIL were analyzed by FESEM. As shown in Fig. 5a, the MWCNTs were intertwined and had smooth surface. It was clear that the surface of MWCNTs was covered by a mass of nanoparticles in the electron micrograph of m-MWCNTs (Fig. 5b). Hence, during the synthesis of m-MWCNTs, the magnetic
Fig. 2. XRD patterns of MWCNTs (a), m-MWCNTs (b), m-MWCNTs@IL (c) and m-MWCNTs@PIL (d).
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Fig. 3. FT-IR spectra of NH2-IL monomer (a), m-MWCNTs (b), m-MWCNTs@IL (c) and m-MWCNTs@PIL (d).
of PIL that covered the m-MWCNTs. Besides, this result was in good corresponding to the FESEM view as mentioned before. From Fig. 6b, the black suspension of m-MWCNTs@PIL could be removed from a solution by an external magnet. The magnetic behavior of mMWCNTs@PIL proved that it owned sufficient saturation magnetization for magnetic separation. The isoelectric points (IEPs) of m-MWCNTs and mMWCNTs@PIL were surveyed by zeta-potential nanoparticles at different pH values. As shown in Fig. 7, we could find that the IEP of m-MWCNTs and m-MWCNTs@PIL were 2.07 and 4.05, respectively. The IEP of the m-MWCNTs was much less than previously reported value for pristine MWCNTs (6.8) [38]. This might be due to the carboxylic groups on MWCNTs were partially substituted in the mMWCNTs. Similarly, the surface groups of m-MWCNTs were changed after polymerization process. Thus high IEP for mMWCNTs@PIL was a conclusive evidence to prove that PIL coated on the m-MWCNTs. Fig. 4. Weight loss curves of m-MWCNTs (a), m-MWCNTs@IL (b) and m-MWCNTs@PIL (c).
nanoparticles was successfully wrapped around the surface of MWCNTs. In Fig. 5c, the surface of m-MWCNTs@IL was rougher when compared with m-MWCNTs. From Fig. 5d, we could observe that the m-MWCNTs were coated by PIL uniformly and entirely. Moreover, the tube morphology of MWCNTs nearly maintained unchanged, manifesting that the polymerization process wouldn't damage the tube structure. The energy-dispersive spectrometry (EDS) analysis of the mMWCNTs@PIL was shown in Fig. 5e. There were 70.77 wt% of C, 13.84 wt% of Fe, 8.11 wt% of O, 6.57 wt% of N and 0.7 wt% of Cl in the m-MWCNTs@PIL composite. Consequently, the m-MWCNTs@PIL was prepared successfully. The magnetic properties of m-MWCNTs and m-MWCNTs@PIL were measured by VSM technique at room temperature. Magnetic hysteresis curves for them were displayed in Fig. 6a. The saturation magnetization intensities of m-MWCNTs and m-MWCNTs@PIL were 12.84 and 2.8 emu g1. It was worth noting that the saturation magnetization of m-MWCNTs@PIL was lower than that of mMWCNTs. This could be mainly ascribed to the nonmagnetic phase
3.2. Optimization of MSPE conditions 3.2.1. Effect of the extraction time In order to reach the equilibrium of MSPE procedure, extraction time was investigated in the range of 0.25e4 h. As can be seen in Fig. 8a, the extraction capacities of Cu, Zn-SOD increased when extraction time ranged from 0.25 to 1.5 h and maintained unchanged with a further extension of extraction time from 1.5 to 4 h. Obviously, the extraction equilibrium was achieved in 1.5 h, and the m-MWCNTs@PIL indeed possessed commendable extraction capacity for analytes. Ultimately, 1.5 h was selected as the optimum extraction time. 3.2.2. Effect of the extraction temperature To assess the influence of temperature on the extraction capacity, batch experiments were conducted from 20 to 45 C. Fig. 8b revealed the relationship between the extraction capacity and extraction temperature, indicating that an increase in extraction temperature resulted in an advanced extraction capacities. The reason for this result could be that the relative motion between the molecules was accelerated by heating up, which was helpful for extraction. According to literature [39], Cu, Zn-SOD has high thermal stability below 75 C. However, in order to maintain mild
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Fig. 5. FESEM images of MWCNTs (a), m-MWCNTs (b), m-MWCNTs@IL (c), m-MWCNTs@PIL (d) and EDS patterns of m-MWCNTs@PIL (e).
Fig. 6. Magnetic hysteresis loops of m-MWCNTs, m-MWCNTs@PIL (a), and the magnetic response of m-MWCNTs@PIL to external magnetic field (b).
conditions for MSPE procedure, 45 C was set as the extraction temperature for subsequent experiments.
3.2.3. Effect of the amount of m-MWCNTs@PIL To evaluate the effect of the amount of adsorbent for the MSPE, different quantities of m-MWCNTs@PIL were surveyed from 4 to 12 mg. As shown in Fig. 8c, the extraction capacity reached the maximum when 10 mg of m-MWCNTs@PIL was applied. The extraction capacities increased with the increasing mass of mMWCNTs@PIL because of active sites raised. However, the extraction capacities reduced when the amount of adsorbent exceeded 10 mg, indicating that excess m-MWCNTs@PIL were helpless with
Fig. 7. Zeta potentials of m-MWCNTs and m-MWCNTs@PIL in different pH solutions.
such small volume of the extraction solvent. Based on these results, 10 mg of m-MWCNTs@PIL were employed for further researches. 3.2.4. Effect of the pH value The pH of the sample solution played a key role in MSPE process, which can affect the surface charge of both analytes and adsorbent. The pH of the sample solution was conducted in 5 mmol/L phosphate buffer solution in the range of 4e9. Based on the IEP characterization of m-MWCNTs@PIL (IEP 4.05), when the solution pH value was higher than 4.05, the surface charge of m-MWCNTs@PIL became negative. The Cu, Zn-SOD (IEP 7.12) was protonated when the solution pH was below 7.12. As presented in Fig. 8d, extraction capacities improved when pH varied from 4 to 5 and after that,
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Fig. 8. Effect of extraction time (a), temperature (b), amount of m-MWCNTs@PIL (c), pH value (d), initial Cu, Zn-SOD concentration (e) and ionic strength (f).
extraction capacities displayed an absolute downward trend. The maximum extraction capacity appeared at pH 5 where the surface of m-MWCNTs@PIL became negative and enable to extract positive Cu, Zn-SOD. This phenomenon revealed that electrostatic interaction was indeed existed in adsorption. Consequently, the sample solution was set as pH 5. 3.2.5. Effect of the initial Cu, Zn-SOD concentration The effect of initial Cu, Zn-SOD concentration in the sample solution was examined in the range of 0.25e2 mg/mL. In Fig. 8e, the extraction capacities increased rapidly with increasing the initial Cu, Zn-SOD concentration from 0.25 to 1.5 mg/mL, and remained constant when further increased the initial Cu, Zn-SOD concentration to 2 mg/mL. Obviously, a higher initial Cu, Zn-SOD concentration provided more possibilities for m-MWCNTs@PIL to capture analytes. However, the limited active reaction sites on the adsorbent should be responsible for the stagnant growth. To make full use of the active reaction sites of m-MWCNTs@PIL, 1.5 mg/mL initial Cu, Zn-SOD concentration was used.
Fig. 9. Extraction capacities of m-MWCNTs@PIL, m-MWCNTs@IL and m-MWCNTs for three different analytes.
3.2.6. Effect of the solution ionic strength In order to evaluate the effect of ionic strength in extraction performance, the concentration of NaCl was varied from 0 to 0.5 mol/L. As shown in Fig. 8f, the extraction capacities decreased obviously as the NaCl concentration was increased. It could be inferred that the role of electrostatic interaction in MSPE process was significant, with which sodium ions and positively charged Cu, Zn-SOD were competitively extracted by m-MWCNTs@PIL. Ultimately, no salt was added in the MSPE procedure. After optimizing six effective factors for extraction process, the maximum extraction capacity of Cu, Zn-SOD was 85.28 mg/g.
analytes (Cu, Zn-SOD at 205 nm, BHb at 406 nm and Lys at 278 nm) were compared with those of both m-MWCNTs@IL and mMWCNTs. As manifested in Fig. 9, the extraction capacity of mMWCNTs@PIL for Cu, Zn-SOD was higher than those acquired by mMWCNTs@IL and m-MWCNTs. It was also found that mMWCNTs@PIL showed the best extraction capacity for Cu, Zn-SOD than BHb and Lys. This indicated that m-MWCNTs@PIL might possess specific adsorption for Cu, Zn-SOD. Therefore, mMWCNTs@PIL was a promising adsorbent for MSPE of Cu, Zn-SOD.
3.3. Comparison of the extraction capacity
3.4. Desorption of Cu, Zn-SOD
In order to check out the adsorption property of mMWCNTs@PIL, their extraction capacities for three different
Desorption of analytes from adsorbents had a significant impact on the reusability of the m-MWCNTs@PIL and the retrieve of Cu,
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Fig. 10. The autoxidation rate of pyrogallol (a), The autoxidation rate of pyrogallol with the initial Cu, Zn-SOD was added (b), The autoxidation rate of pyrogallol with the Cu, Zn-SOD after extraction was added (c), the specific activities of the initial Cu, Zn-SOD and the Cu, Zn-SOD after extraction.
Zn-SOD. After extraction, the m-MWCNTs@PIL were magnetically separated from aqueous solution and then eluted by 1 mL of 1 mol/ L NaCl solution. The suspension was shaken in a 2 mL centrifuge tube for 1 h at 25 C. In this way, the desorption efficiency of Cu, ZnSOD was 84.35%, indicating the adsorbed analytes could be eluted from m-MWCNTs@PIL successfully. Based on the result of the effect of ionic strength in extraction performance, the high desorption efficiency might attribute to the increase of iron strength which could weaken the electrostatic interaction between mMWCNTs@PIL and Cu, Zn-SOD.
Zn-SOD and the Cu, Zn-SOD after extraction were calculated according to the following formula:
specific activity ðU=mgÞ ¼
a 50%
V m
where a is inhibitory rate for pyrogallol autoxidation, V is the total volume, m is the mass of Cu, Zn-SOD. It was found in Fig. 10d that the specific activities of the initial Cu, Zn-SOD and the Cu, Zn-SOD after extraction were high enough. Therefore, this MSPE method could extract Cu, Zn-SOD effectively without inactivating it.
3.5. Activity analysis of Cu, Zn-SOD 3.6. Reusability of the m-MWCNTs@PIL The examination of Cu, Zn-SOD activity was conducted by pyrogallol autoxidation method [40,41]. 0.015 mL of 45 mmol/L pyrogallol solution was added into 4.5 mL of 50 mmol/L Tris-HCl buffer solution (pH 8.2, 1 mmol/L EDTA) at 25 C. The absorbance at 325 nm of the mixture was detected by UVevis spectrophotometer for 3 min. As can be seen in Fig. 10a, the autoxidation rate of pyrogallol was 0.07216/min. Afterwards, 0.005 mL of the initial Cu, Zn-SOD (0.1 mg/mL) and the Cu, Zn-SOD after extraction (0.1 mg/mL) were added into above mixture, respectively. Their autoxidation rates of pyrogallol were 0.04812/min and 0.05071/ min (Fig. 10b and c). Finally, the specific activities of the initial Cu,
To examine reusability of the m-MWCNTs@PIL, the adsorbent was subjected to different extraction-desorption cycles. After desorption of Cu, Zn-SOD, the collected adsorbent was washed twice with ultrapure water, and then dried for next analysis run. As shown in Fig. 11, extraction capacities of Cu, Zn-SOD decreased in the second and third run and then remained constant in the following runs. The residue of Cu, Zn-SOD on the adsorbent might be responsible for the reduction of extraction capacity. Nevertheless, the m-MWCNTs@PIL showed an acceptable reusability in MSPE procedure.
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Q. Wen et al. / Analytica Chimica Acta 939 (2016) 54e63 Table 1 The results of the precision, repeatability and stability experiments. Precision measurement results (n ¼ 5) Repeats Extraction capacities (mg g1) RSD (%)
1 87.01 0.29
2 87.01
3 87.36
4 87.03
5 87.56
2 85.04
3 87.4
4 85.25
5 85.67
2 87.22
3 86.68
4 86.75
5 86.25
Repeatability measurement results (n ¼ 5) Sample number Extraction capacities (mg g1) RSD (%)
1 83.4 1.68
Stability measurement results (n ¼ 5) Day number Extraction capacities (mg g1) RSD (%)
Fig. 11. Extraction capacities of Cu, Zn-SOD for different runs.
3.7. Analysis of real sample To study the practical extraction performance of the proposed method in real samples, the porcine whole blood was selected and analyzed. Under the optimum experimental conditions as mentioned above, 1 mL of the porcine whole blood which was diluted 50 times with phosphate buffer solution (pH 5) was applied for the MSPE procedure. The analytical result was illustrated in Fig. 12, which was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). It could be found that the specific pattern of Cu, Zn-SOD in the porcine whole blood became fade after extraction. Consequently, the proposed MSPE method based on m-MWCNTs@PIL could extract Cu, Zn-SOD from porcine whole blood sample effectively.
3.8. Method validation After the optimal experimental factors were confirmed, the precision, repeatability and stability experiments were conducted to evaluate the proposed method. The results were summarized in Table 1. The relative standard deviation (RSD) of the apparatus
1 87.44 0.54
precision exhibited to be 0.29%, which was examined by detecting a sample for 5 times coupled with UVevis spectrophotometer. Five sets of parallel experiments were performed to investigate the repeatability and the RSD was 1.68%. The stability of the method was investigated by surveying the same sample for five consecutive days and the RSD was 0.54%. The recovery of the method was investigated by standard addition method. As observed from Table 2, the recoveries were calculated after spiking three concentration levels (0.1, 1 and 10 mg/mL) of Cu, Zn-SOD. Recoveries were in the range of 82.7e102.3%, with the RSD between 3.47% and 5.35%. The results evidenced that this method was reliable for receiving precise experimental data and existed excellent repeatability and stability. 4. Conclusions It is well known that the common magnetic particles are Fe3O4,
g-Fe2O3, Fe, Co, MnFe2O4, MgFe2O3, CoFe2O3, FePt and CoPt3.
Compared with Co, MnFe2O4, MgFe2O3, CoFe2O3, FePt and CoPt3, the Fe nanoparticles are more easily synthesized and have the advantage that the small particle size of nZVI results in large specific surface area and great intrinsic reactivity of surface sites. In addition, the Fe3O4 are the most widely used magnetic particles due to its good stability. However, nZVI technology has been widely investigated for the treatment of environmental pollutants, so applying the Fe nanoparticles to the MSPE method is a very meaningful attempt. In this way, we can promote the diversification of science. In the present work, a novel magnetic adsorbent, mMWCNTs@PIL has been successfully synthesized and applied for the extraction of Cu, Zn-SOD. The m-MWCNTs@PIL could interact with Cu, Zn-SOD through hydrogen bonding, p-p and electrostatic interactions and showed its good extraction capacity to Cu, Zn-SOD. The adsorbed Cu, Zn-SOD remained high specific activity after being eluted from m-MWCNTs@PIL by 1 mol/L NaCl solution. Besides, the m-MWCNTs@PIL could be easily recycled and successfully employed in extraction of Cu, Zn-SOD from real samples. Under the optimal conditions, the precision, repeatability and stability of the proposed method were investigated and the RSDs were 0.29%, 1.68% and 0.54%, respectively. Recoveries were in the range of
Table 2 Recovery for Cu, Zn-SOD from blood samples.
Fig. 12. SDS-PAGE of protein molecular weight maker (a), 1.5 mg/mL Cu, Zn-SOD (b), porcine whole blood sample (c) and porcine whole blood after extraction (d).
Analyte (n ¼ 5)
Spiked (mg/mL)
Recovery (%)
RSD (%)
Cu, Zn-SOD
0 0.1 1 10
82.7 94.4 102.3
3.47 3.89 5.35
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82.7e102.3%, with the RSD between 3.47% and 5.35%. All these indicated that the developed method has great potential on the extraction of Cu, Zn-SOD or other analytes from biological samples.
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Acknowledgements
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