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Preparation and Application of Core-Shell Structural Carbon Nanotubes-Molecularly Imprinted Composite Material for Determination of Nafcillin in Egg Samples
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 41, Issue 2, February 2013 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2013, 41(2), 161–166.
RESEARCH PAPER
Preparation and Application of Core-Shell Structural Carbon Nanotubes-Molecularly Imprinted Composite Material for Determination of Nafcillin in Egg Samples LIU Yu-Xing1, JIAN Gui-Qin1, HE Xi-Wen1, CHEN Lang-Xing1,*, ZHANG Yu-Kui1,2 1 2
Department of Chemistry, Nankai University, Tianjin, Tianjin 300071, China Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116011, China
Abstract: A novel composite material based on core-shell molecularly imprinted polymers (MIPs) was prepared by combining surface imprinting technique with a sol-gel process based on carbon nanotubes (CNTs) coated with silica. The morphology and structure of the products (CNTs@Naf-MIPs) were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR). The adsorption properties of CNTs@Naf-MIPs were demonstrated by equilibrium rebinding experiments and Langmuir analysis. The maximum adsorption capacity and dissociation constant of CNTs@Naf-MIPs were 9.5 mg g-1 and 56.6 mL mg–1, respectively. The CNTs@Naf-MIPs showed a fast kinetics and reached the equilibrium within only 30 min. The feasibility of determination of nafcillin from real samples was testified in spiked egg samples with concentration of 5 and 10 g kg–1 by using the imprinted polymer as the adsorption material. The recoveries of nafcillin ranged from 61.3% to 84.3% with good accuracy. The MIPs provided a fast and convenient determination platform for nafcillin in egg samples. Key Words: Molecularly imprinted polymers; Carbon nanotubes; Sol-gel; Nafcillin; Egg
1
Introduction
Nafcillin, a member of -lactam antibiotics (BLAs), can be classified into two groups, penicillin and cephalosporins. The BLAs are broad-spectrum antimicrobials, lower toxicity and wider indications antibiotics, which are commonly used as antibacterial drugs in both human and veterinary medicine to fight infectious diseases, or used as growth promoters in animal husbandry, fish farming, and other fields[1]. Nafcillin, a semisynthetic -lactam antibiotic, can be employed in the treatment of serious infectious caused by penicillinaseproducing staphylococci (e.g. septicaemia, osteoncyelitis, pneumonia and endocarditis). As a consequence of the extensive usage of nafcillin, considerable attention has been paid to the potential human health risk and the resistance to
antibiotics by micro-organism, which may have great impact on the ability of humankind to fight infectious diseases. The presence of nafcillin antibiotic in meat and dairy products can also lead to allergic reactions in hypersensitive individuals. To ensure food safety for consumers, the European Union and other countries, including China legislators, have restricted or established a maximum residue levels for the use of antibiotics. There is a constant requirement for accurate, sensitive and selective analytical methods for monitoring nafcillin antibiotic residues to guarantee the safety of food. Usually, the complexity of food matrices and contaminants presented in food at low concentration (ng g–1 to g g–1) levels require analysis performance only after some clean-up and preconcentration steps[2–4], and thus there is a considerable interest in developing new specific and selective methods for
Received 11 June 2012; accepted 15 October 2012 * Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (No. 21275080), and the Natural Science Foundation of Tianjun, China (No. 10JCZDJC17600). Copyright © 2013, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(13)60626-X
LIU Yu-Xing et al. / Chinese Journal of Analytical Chemistry, 2013, 41(2): 161–166
extracting and isolating components from complex food matrices. For this purpose, the solid phase extraction (SPE) technique is the most widely used for sample pre-treatment method due to its advantages such as convenience, low cost, timing saving and simplicity. However, the common solid phase extraction materials show a lack of selectivity except immunoadsorbents which are very selective but expensive and not suitable for harsh environments. As economical, rapid and selective clean-up methods are needed, the application of SPE procedures involving molecularly imprinted polymers (MIPs), called MISPE, offering the advanced specificity in comparison with traditional SPE adsorbents, has received increasing attention over the past decade as an attractive alternative for the analysis of complex samples where analyte selectivity in the presence of very complex samples represents the main problem. The molecular imprinting technique (MIT) is an attractive method for the generation of tailored materials that have the ability to recognize and in some cases respond to biological and chemical agents of interest[5–7]. Molecularly imprinted polymers (MIPs) are tailor-made materials that can exhibit high affinity and selectivity towards a given target or group of target molecules. MIPs are synthesized by polymerization of functional monomers and a cross-linker around a template. Extraction of the template leaves behind recognition sites with functional and shape complementarity to the template. MIPs as biomimetic materials are of interest because they hold the promise of selective recognition, ease of mass preparation and surviving a range of harsh operating conditions not tolerated by their biological counterparts. The high specificity, good stability and low cost of MIPs render them promising alternatives to enzymes, antibodies, and natural receptors for use in areas including chromatographic separation[5], food analysis[6,7], mimicking enzyme[8], selective determination of environmental pollutants[9,10], proteomics[11] and chemical and biochemical sensing[12]. In recent years, unique mechanical properties and extremely large surface area endow carbon nanotubes (CNTs) with more applications. For example, CNTs could serve as the reinforcing element in a polymer in fabricating new advanced materials. Therefore, core-shell nano-structured MIPs composites based on carbon nanotubes (CNTs)[13–16], which combine the advantageous properties of both materials, have enjoyed widespread attention for their unique physicochemical properties and great potential in biomedical, electronic, catalytic and biosensor applications. In comparison with traditional MIPs, the MIPs-CNTs composites based on carbon nanotubes (CNTs having a small dimension with high surface-to-volume ratio) are expected to improve the removal of template molecules, heterogeneous distribution of the binding sites, binding capacity and selectivity, poor site accessibility and slow binding kinetics. Recently, the core@shell nano-structured CNTs@MIPs composites for
estrone or triclosan detection were prepared in our group. The resulting imprinted materials possessed good dispersion in any solvents, favorable selectivity, high capacity and fast kinetics for uptake of the template molecule. The synthetic process is quite simple, and the different batches of imprinted materials demonstrated good reproducibility as a sorbent for estrone or triclosan[15,16]. In this work, core@shell nano-structured MIPs for nafcillin (CNTs@Naf-MIPs) were prepared at the surface of CNTs coated with silica. The synthetic process is quite simple, and the different batches of imprinted materials demonstrated good reproducibility as a sorbent for nafcillin. The resulting CNTs@Naf-MIPs was successfully applied to determine the nafcillin at low concentration in the spiked egg samples.
2 2.1
Experimental Instrumentation and chemicals
Nafcillin, oxacillin and mezlocillin were obtained from Sigma (USA). Tetraethoxysilane (TEOS), 3-aminopropyltriethoxysilane (APTES) and phenyltrimethoxysilane (PTMOS) were purchased from Alfa Aesar Chemical Company (Tianjin, China). Multi-walled carbon nanotubes (MWCNTs, diameter: 60–100 nm, length: 5–15 m) were provided by Shenzhen Nanotech Port Co. Ltd., China. HNO3, cetyltrimethylammonium bromide (CTAB), ammonium hydroxide (25%), were purchased from Tianjin Guangfu Chemicals Ltd. (Tianjin, China). Methanol and acetonitrile of HPLC grade were purchased from Tianjin Concord Technology Co. Ltd. (Tianjin, China). The high purified water (18.0 M cm1) obtained from a WaterPro water system (Aquapro Corporation, AFZ-6000-U, China) was used throughout the experiments. Other chemicals were used as received without further purification. A Tecnai G2 T2 S-TWIN microscope was used to obtain transmission electron microscopy (TEM) images of CNTs, CNTs@SiO2 and CNTs@Naf-MIPs. Fourier transform infrared (FT-IR) spectra were recorded on an AVATAR 360 (Nicolet Corp., USA), and samples were dried at 80 qC in a vacuum oven for at least 12 h prior to fabrication of the KBr pellet. Fifty scans of the region between 400 and 4000 cm1 were collected for each FT-IR spectrum that was recorded. The amount of nafcillin, oxacillin and mezlocillin was analyzed with a Shimadzu (Japan) UV-2450 spectrophotometer at 230 nm or HPLC, and HPLC analyses were performed on a Shimadzu LC-20A HPLC system that included a binary pump and a variable wavelength UV detector (Shimadzu, Kyoto, Japan). The instrument control and data processing were carried out by the LC solution software. A Shimadzu VP-ODS C18 (5 m particle size, 150 mm × 4.6 mm) analytical column was used for the separation of analytes. The eluent was acetonitrile-H2O (40:60, V/V) at a
LIU Yu-Xing et al. / Chinese Journal of Analytical Chemistry, 2013, 41(2): 161–166
flow rate of 1.0 mL min–1. The injection volume was 100 L, and the column effluent was monitored at 230 nm. 2.2
Preparation of nafcillin-imprinted polymers (CNTs@Naf-MIPs) and non-imprinted polymers (CNTs@NIPs)
Impurities such as amorphous carbon and metallic catalyst in the CNTs were removed using an HNO3 solution in a three neck flask with vigorous stirring (500 rpm); the mixture was refluxed for 48 h. The suspension was then filtered through a 0.22-m filter to recover the CNTs, followed by washing repeatedly with highly purified water until the pH reached 7.0, and dried under vacuum at 80 qC for further use[16]. The SiO2 coating on the CNTs was performed according to the method described by Zhu et al[17] and the product CNTs@SiO2 was dried under vacuum for use in further studies. The synthesis of CNTs@Naf-MIPs includes a surface molecular imprinting sol-gel process. In a typical synthesis: 0.1 g of nafcillin were dissolved in 10 mL of ethanol, and mixed with 0.5 mL of APTES and 0.5 mL of PTMOS. The mixture was stirred for 30 min, then, 2 mL of TEOS was added. After stirring for 10 min, 0.5 g of CNTs@SiO2 and 0.6 mL of 1 M HAc (as catalyst) were added. The mixture began to co-hydrolyse and co-condense after stirring for a few minutes, then incubated for 15 h at room temperature. The products were washed with ethanol for three times and then with methanol/acetic acid (80:20, V/V) and subsequently with methanol to remove the residue of template nafcillin. Finally, the product was dried under vacuum at 80 ºC for 12 h. For comparison, the CNTs@NIPs was also prepared using an identical procedure, but without the addition of nafcillin. 2.3
Binding experiment
In kinetic adsorption experiments, 20 mg of CNTs@NafMIPs were added to centrifugal tubes, each containing 4 mL of 60 mg L–1 of nafcillin solution, and incubated at regular time intervals, the supernatants and polymers were separated by centrifugation. The nafcillin concentration of the supernatants was measured by UV-vis spectrometer. The amount of nafcillin bound to the CNTs@Naf-MIPs was
calculated from the difference in the nafcillin concentrations before and after incubation on the rotator. In steady-state binding experiments, the 20 mg of CNTs@Naf-MIPs and CNTs@NIPs were mixed with a 4-mL methanol solution of nafcillin of various concentrations from 20 to 200 mg L–1, respectively. The CNTs@Naf-MIPs and CNTs@NIPs nanocomposites were isolated by centrifugation after incubating for 2 h at room temperature. The nafcillin concentration of the supernatants was measured by UV-vis spectrometer. The cross-reactivity of CNTs@Naf-MIPs and CNTs@NIPs to nafcillin and its analogues oxacillin and mezlocillin (Fig.1) was performed as the following: 20 mg of CNTs@Naf-MIPs and CNTs@NIPs were separately added to mixed solutions of nafcillin, oxacillin and mezlocillin in 4 mL of methanol at the concentration of 160 mg L–1 respectively. After incubation for 2 h at room temperature, the CNTs@Naf-MIPs and CNTs@NIPs were isolated by centrifugation. The filtrate was concentrated to dryness by nitrogen purging and then dissolved in methanol before HPLC analysis. The amount of nafcillin, oxacillin and mezlocillin bound to the CNTs@Naf-MIPs was determined using HPLC method. The mobile phase was acetonitrile-water (40:60, V/V) containing 0.1% acetic acid at a flow rate of 1.0 mL min–1. The injection volume was 100 L, and the column effluent was monitored at 230 nm. Desorption and regeneration of the adsorbents is crucial for all affinity separation techniques. The reusability of CNTs@Naf-MIPs was evaluated. Approximately 20 mg of CNTs@Naf-MIPs were added to the solutions of nafcillin in 4 mL methanol of 160 mg L–1 and then incubated at room temperature while gently stirred on a rocking table for 2 h. Then, the MIP nanocomposites were removed by centrifugation and the bound amount of nafcillin was quantified by HPLC. The recovered CNTs@Naf-MIPs nanocomposites were washed with a mixture of methanol/acetic acid (80:20, V/V) for several times to ensure complete removal of residual nafcillin in the MIPs and washed with methanol for several times, then dried under vacuum at 60 ºC and reused for adsorption of nafcillin. Desorption and regeneration of the adsorbents was repeated for five cycles.
Fig.1 Molecular structure of nafcillin and two analogues, oxacillin and mezlocillin
LIU Yu-Xing et al. / Chinese Journal of Analytical Chemistry, 2013, 41(2): 161–166
2.4
Separation and determination of nafcillin in eggs samples
Eggs were selected for the spiked sample analysis. First, a 25-mL ethanol/water (60:40, V/V) was added to a 5-g eggs sample spiked with the standard nafcillin solution in a 50-mL polypropylene tube. The mixed sample was shaken for 30 min to achieve proteins precipitation. After centrifugation at 3000 rpm for 10 min, the supernatant solution was filtered through a 0.22-m filter. The filtrate was dried with a stream of nitrogen, and then dissolved with 40 mL of methanol. The spiking concentrations for nafcillin were set with two levels of 5 and 10 g kg–1, respectively. About 30 mg of CNTs@Naf-MIPs were added to the above 40 mL methanol solution, and then incubated for 2 h at room temperature. Next, the mixture was centrifugated at 3000 rpm for 10 min. Final, after the supernatant solution was discarded, the CNTs@Naf-MIPs nanocomposites were eluted with a mixing methanol/acetic acid (80:20, V/V) solution, and then the eluate was evaporated to dry under a stream of nitrogen and dissolved with 1 mL of methanol and measured by HPLC. At each spiked concentration of nafcillin, three measurements were performed. The HPLC analyses were performed on a Shimadzu LC-20A HPLC system including a binary pump and a variable wavelength UV detector (Shimadzu, Kyoto, Japan). The instrument control and data processing were carried out by the LC solution software. A Shimadzu VP-ODS C18 (5 m particle size, 150 mm × 4.6 mm) analytical column was used for the determination of analytes. The mobile phase was acetonitrile-water (40:60, V/V) containing 0.1% acetic acid at a flow rate of 1.0 mL min–1. The injection volume was 100 L, and the column effluent was monitored at 230 nm.
3 3.1
CNTs@SiO2 via sol-gel reaction. The thickness of the silica film was observed to increase by repeating the sol-gel reaction. The diameter of the CNTs@Naf-MIPs increased to 100–120 nm after the nafcillin-imprinting process, which corresponds to a 20–30 nm thick imprinted SiO2 layer covering on the CNTs (Fig.2C). The thickness of this imprinted polymer layer will be effective for mass transport between the solution and the surface of the CNTs@Naf-MIPs. The FT-IR spectra of the CNTs, CNTs@SiO2, CNTs@NafMIPs and CNTs@NIPs are shown in Fig.3. There was no apparent peak in the spectrum of the blank CNTs (curve a). The strong peak at about 1069.8 cm1 can be attributed to the stretch of Si-O-Si, indicating the formation of silica film on the surface of CNTs (Fig.3b). A characteristic feature of CNTs@Naf-MIPs in comparison with spectrum of CNTs@SiO2 was N–H bond at 1629.5 and 1557 cm1, and C-H bending vibration of benzene ring at 880–680 cm1 (Fig.3c). These results suggested that functional monomer APTES and PTOMS could be grafted onto the surface of CNTs@SiO2 in the imprinting process. CNTs@Naf-MIPs and CNTs@NIPs showed the similar locations and appearance of the major bands. 3.2
Binding properties of CNTs@Naf-MIPs
Figure 4A presents the adsorption kinetics of 60 mg L–1
Results and discussion Characterization of imprinted materials (CNTs@Naf-MIPs)
TEM images of the CNTs, CNTs@SiO2 and CNTs@Naf-MIPs are shown in Fig.2. The average diameter of the CNTs is observed at 70–80 nm (Fig.2A) before coating; after coating with the SiO2, the diameter of the CNTs@SiO2 increases to 85–95 nm, corresponding to a about 15 nm thickness of SiO2 layer covering the CNTs (Fig.2B). The SiO2 layers were uniformly coated for all of the samples after sol-gel reaction, and there were hardly any free SiO2 particles found in the TEM images. Silica coating can mitigate the difficulty of dispersing CNTs homogeneously in different solvents, and this coating favors the dispersion of CNTs in liquid media. The TEM image of CNTs@Naf-MIPs showed the formation of the core/shell-structured nanocomposites (Fig.2C) after imprinting process on the surface of the
Fig.2 TEM images of CNTs (A), CNTs@SiO2 (B), CNTs@NafMIPs (C)
Fig.3
FT-IR spectra of CNTs (a), CNTs@SiO2 (b), CNTs@NafMIPs (c) and CNTs@NIPs (d)
LIU Yu-Xing et al. / Chinese Journal of Analytical Chemistry, 2013, 41(2): 161–166
Fig.4
Adsorption kinetics of CNTs@Naf-MIPs (A), adsorption isotherm of nafcillin onto CNTs@Naf-MIPs and CNTs@NIPs (B), and Langmuir plot to estimate the binding nature of CNTs@Naf-MIPs (C)
nafcillin onto CNTs@Naf-MIPs. It can be seen that the adsorption capacity increased rapidly in the first 30 min, after 30 min, the adsorption had almost reached equilibrium. For imprinted polymer prepared by traditional bulk polymerizetion, it takes generally 12–24 h to reach the adsorption equilibrium. In this work, template nafcillin reached the surface imprinting cavities of CNTs@Naf-MIPs easily and took less time to reach the adsorption saturation, which implied that CNTs@Naf-MIPs had good mass transport and thus overcame some drawbacks of traditional imprinted materials. The adsorption of naficillin to CNTs@Naf-MIPs and control CNTs@NIPs at different concentrations were investigated by a steady-state binding method. As can be seen in Fig.4B, the amount of nafcillin bound in the CNTs@NafMIPs is higher than that in the CNTs@NIPs. The results clearly confirmed the effectiveness of the molecular imprinting because the CNTs@Naf-MIPs showed more binding sites in comparison with CNTs@NIPs, and higher affinity to nafcillin. The saturation binding data were further processed with a Langmuir equation to estimate the binding properties of the CNTs@Naf-MIPs. The Langmuir equation is as follows: Ce/Q = Ce/Qmax + 1/(KDQmax) (1) –1 where, Ce (mg L ) was free analytical concentration of nafcillin in the solution at equilibrium, Q (mg g–1) was the amount of nafcillin bound to CNTs@Naf-MIPs at equilibrium, Qmax (mg g–1) was the apparent maximum adsorption capacity, and KD was the dissociation constant. The values of KD and Qmax can be calculated from the slope and intercept of the linear line plotted in Ce/Q versus Q (Fig.4C), which indicated that the binding sites of the CNTs@Naf-MIPs were homogeneity. The linear regression equation for the linear region is Ce/Q = 0.1055Ce + 1.864 (r = 0.9875). From the slope and the intercept of the straight line obtained, the values of Qmax and KD were 9.5 mg g–1 and 56.6 mL mg–1, respectively.
binding experiment in a mixed solution of nafcillin, oxacillin and mezlocillin, which are structural analogues (Fig.1). As shown in Fig.5, the binding capacity of nafcillin on the CNTs@Naf-MIPs is 3.1 mg g–1 higher than that of CNTs@NIPs, but the difference of the binding capacity between CNTs@Naf-MIPs and CNTs@NIPs to oxacillin and mezlocillin is 0.4 and 0.7 mg g–1, respectively. The CNTs@Naf-MIPs showed significantly higher specific recognition ability for nafcillin over both oxacillin and mezlocillin in the mixture solution. The CNTs@NIPs displayed the greatest nonspecific adsorption to oxacillin among the three antibiotics. Figure 1 showed the structural difference of nafcillin, oxacillin and mezlocillin. The template of nafcillin have a naphthalene ring, oxacillin have aromatic isoxazole and benzene ring, and mezlocillin have a benzene ring, so there existed the stronger S-S interaction between nafcillin and oxacillin molecule and functional monomer PTMOS. The oxacillin molecules have more N, O atoms which can form more hydrogen bonds in comparison with nafcillin template. The adsorption of CNTs@NIPs towards nafcillin, oxacillin and mezlocillin molecules is non-specific. The CNTs@Naf-MIPs showed the higher selectivity for nafcillin may be attributed to not only the stronger S-S interaction between naphthalene ring and functional monomer PTMOS but also the combined effect of the shape and size complementarities.
3.3 Specific recognition of CNTs@Naf-MIPs and CNTs@NIPs The specific recognition ability of the CNTs@Naf-MIPs and CNTs@NIPs was investigated through a steady-state
Fig.5 Binding selectivity of CNTs@Naf-MIPs and CNTs-NIPs to nafcillin, oxacillin and mezlocillin
LIU Yu-Xing et al. / Chinese Journal of Analytical Chemistry, 2013, 41(2): 161–166
3.4
Repeatability of CNTs@Naf-MIPs
Desorption and regeneration is one of the most important properties for the application of MIPs. As such, the adsorption-desorption cycle experiment was repeated five times using the same CNTs@Naf-MIPs. Figure 6 showed the change of the amount of adsorbed nafcillin on CNTs@NafMIPs after five regeneration cycles experiment. It could be seen that the CNTs@Naf-MIPs lost about 20% of its adsorption capacity on average after two cycles and the adsorption capacity kept constant. It is possible that some recognition sites in the network of CNTs@Naf-MIPs could be blocked after regeneration or destroyed after rewashing, and thus they were no longer fit for the template molecule. They retained their recovery efficiency after three binding/ regeneration cycles by using this treatment. These results demonstrated the reusability of CNTs@Naf-MIPs over several adsorption- desorption cycles, which is a clear advantage over single-use materials. 3.5
Fig.6 Desorption and regeneration of CNTs@Naf-MIPs
Separation and determination of nafcillin in eggs
Generally, the complexity of food matrices and the presence of some potential interference require specific and selective methods of analysis. We investigated the application of CNTs@Naf-MIPs to selective adsorption of nafcillin in the eggs purchased from a local market. The methanol solutions obtained from eggs spiked nafcillin were treated with CNTs@Naf-MIPs. The CNTs@Naf-MIPs was washed with CH3OH/HAc (80:20, V/V) and the eluents were analyzed by HPLC. The chromatograms of the spiked eggs sample with nafcillin at concentration of 5 Pg kg–1 are displayed in Fig.7. The peak of nafcillin cannot be seen from the chromatogram obtained from the spiked eggs sample with concentration of 5 Pg kg–1, however, the peak of nafcillin distinctly appeared after enrichment of spiked eggs sample with CNTs@NafMIPs, which showed that the nafcillin can be enriched after adsorption by the CNTs@Naf-MIPs. To evaluate the accuracy and application of the developed method, the recoveries of spiked nafcillin in eggs with two levels of 5 and 10 g kg1 were measured. At each concentration, three measurements were performed (Table 1). The recoveries of eggs samples were 61.3% and 84.3%, respectively. The relative standard deviations (RSD) were less than 7.8%. These results demonstrated that the CNTs@NafMIPs can be used for the selective enrichment of nafcillin in eggs.
Fig.7
Chromatogram of egg sample spiked with nafcillin at a concentration of 5 g kg–1 (A); chromatogram of elution of the CNTs@Naf-MIPs with CH3OH-HAc (80:20, V/V), followed
by
HPLC
analysis
after
CNTs@Naf-MIPs
extraction of spiked egg sample (B)
imprinting technique with a sol-gel process based on carbon nanotubes coated with silica. The resulting CNTs@Naf-MIPs nanocomposites possessed identical binding sites, favorable selectivity, high capacity, and fast kinetics for uptake of the template molecule. The synthetic process is quite simple, and different batches of the imprinted materials showed good reproducibility in template binding experiments. Additionally, CNTs@Naf-MIPs nanocomposites showed high efficiency as a adsorbent for the selective enrichment of nafcillin in eggs.
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Cite this article as: Chin J Anal Chem, 2013, 41(2), 161–166.
RESEARCH PAPER
Preparation and Application of Core-Shell Structural Carbon Nanotubes-Molecularly Imprinted Composite Material for Determination of Nafcillin in Egg Samples LIU Yu-Xing1, JIAN Gui-Qin1, HE Xi-Wen1, CHEN Lang-Xing1,*, ZHANG Yu-Kui1,2 1 2
Department of Chemistry, Nankai University, Tianjin, Tianjin 300071, China Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116011, China
Abstract: A novel composite material based on core-shell molecularly imprinted polymers (MIPs) was prepared by combining surface imprinting technique with a sol-gel process based on carbon nanotubes (CNTs) coated with silica. The morphology and structure of the products (CNTs@Naf-MIPs) were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR). The adsorption properties of CNTs@Naf-MIPs were demonstrated by equilibrium rebinding experiments and Langmuir analysis. The maximum adsorption capacity and dissociation constant of CNTs@Naf-MIPs were 9.5 mg g-1 and 56.6 mL mg–1, respectively. The CNTs@Naf-MIPs showed a fast kinetics and reached the equilibrium within only 30 min. The feasibility of determination of nafcillin from real samples was testified in spiked egg samples with concentration of 5 and 10 g kg–1 by using the imprinted polymer as the adsorption material. The recoveries of nafcillin ranged from 61.3% to 84.3% with good accuracy. The MIPs provided a fast and convenient determination platform for nafcillin in egg samples. Key Words: Molecularly imprinted polymers; Carbon nanotubes; Sol-gel; Nafcillin; Egg
1
Introduction
Nafcillin, a member of -lactam antibiotics (BLAs), can be classified into two groups, penicillin and cephalosporins. The BLAs are broad-spectrum antimicrobials, lower toxicity and wider indications antibiotics, which are commonly used as antibacterial drugs in both human and veterinary medicine to fight infectious diseases, or used as growth promoters in animal husbandry, fish farming, and other fields[1]. Nafcillin, a semisynthetic -lactam antibiotic, can be employed in the treatment of serious infectious caused by penicillinaseproducing staphylococci (e.g. septicaemia, osteoncyelitis, pneumonia and endocarditis). As a consequence of the extensive usage of nafcillin, considerable attention has been paid to the potential human health risk and the resistance to
antibiotics by micro-organism, which may have great impact on the ability of humankind to fight infectious diseases. The presence of nafcillin antibiotic in meat and dairy products can also lead to allergic reactions in hypersensitive individuals. To ensure food safety for consumers, the European Union and other countries, including China legislators, have restricted or established a maximum residue levels for the use of antibiotics. There is a constant requirement for accurate, sensitive and selective analytical methods for monitoring nafcillin antibiotic residues to guarantee the safety of food. Usually, the complexity of food matrices and contaminants presented in food at low concentration (ng g–1 to g g–1) levels require analysis performance only after some clean-up and preconcentration steps[2–4], and thus there is a considerable interest in developing new specific and selective methods for
Received 11 June 2012; accepted 15 October 2012 * Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (No. 21275080), and the Natural Science Foundation of Tianjun, China (No. 10JCZDJC17600). Copyright © 2013, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(13)60626-X
LIU Yu-Xing et al. / Chinese Journal of Analytical Chemistry, 2013, 41(2): 161–166
extracting and isolating components from complex food matrices. For this purpose, the solid phase extraction (SPE) technique is the most widely used for sample pre-treatment method due to its advantages such as convenience, low cost, timing saving and simplicity. However, the common solid phase extraction materials show a lack of selectivity except immunoadsorbents which are very selective but expensive and not suitable for harsh environments. As economical, rapid and selective clean-up methods are needed, the application of SPE procedures involving molecularly imprinted polymers (MIPs), called MISPE, offering the advanced specificity in comparison with traditional SPE adsorbents, has received increasing attention over the past decade as an attractive alternative for the analysis of complex samples where analyte selectivity in the presence of very complex samples represents the main problem. The molecular imprinting technique (MIT) is an attractive method for the generation of tailored materials that have the ability to recognize and in some cases respond to biological and chemical agents of interest[5–7]. Molecularly imprinted polymers (MIPs) are tailor-made materials that can exhibit high affinity and selectivity towards a given target or group of target molecules. MIPs are synthesized by polymerization of functional monomers and a cross-linker around a template. Extraction of the template leaves behind recognition sites with functional and shape complementarity to the template. MIPs as biomimetic materials are of interest because they hold the promise of selective recognition, ease of mass preparation and surviving a range of harsh operating conditions not tolerated by their biological counterparts. The high specificity, good stability and low cost of MIPs render them promising alternatives to enzymes, antibodies, and natural receptors for use in areas including chromatographic separation[5], food analysis[6,7], mimicking enzyme[8], selective determination of environmental pollutants[9,10], proteomics[11] and chemical and biochemical sensing[12]. In recent years, unique mechanical properties and extremely large surface area endow carbon nanotubes (CNTs) with more applications. For example, CNTs could serve as the reinforcing element in a polymer in fabricating new advanced materials. Therefore, core-shell nano-structured MIPs composites based on carbon nanotubes (CNTs)[13–16], which combine the advantageous properties of both materials, have enjoyed widespread attention for their unique physicochemical properties and great potential in biomedical, electronic, catalytic and biosensor applications. In comparison with traditional MIPs, the MIPs-CNTs composites based on carbon nanotubes (CNTs having a small dimension with high surface-to-volume ratio) are expected to improve the removal of template molecules, heterogeneous distribution of the binding sites, binding capacity and selectivity, poor site accessibility and slow binding kinetics. Recently, the core@shell nano-structured CNTs@MIPs composites for
estrone or triclosan detection were prepared in our group. The resulting imprinted materials possessed good dispersion in any solvents, favorable selectivity, high capacity and fast kinetics for uptake of the template molecule. The synthetic process is quite simple, and the different batches of imprinted materials demonstrated good reproducibility as a sorbent for estrone or triclosan[15,16]. In this work, core@shell nano-structured MIPs for nafcillin (CNTs@Naf-MIPs) were prepared at the surface of CNTs coated with silica. The synthetic process is quite simple, and the different batches of imprinted materials demonstrated good reproducibility as a sorbent for nafcillin. The resulting CNTs@Naf-MIPs was successfully applied to determine the nafcillin at low concentration in the spiked egg samples.
2 2.1
Experimental Instrumentation and chemicals
Nafcillin, oxacillin and mezlocillin were obtained from Sigma (USA). Tetraethoxysilane (TEOS), 3-aminopropyltriethoxysilane (APTES) and phenyltrimethoxysilane (PTMOS) were purchased from Alfa Aesar Chemical Company (Tianjin, China). Multi-walled carbon nanotubes (MWCNTs, diameter: 60–100 nm, length: 5–15 m) were provided by Shenzhen Nanotech Port Co. Ltd., China. HNO3, cetyltrimethylammonium bromide (CTAB), ammonium hydroxide (25%), were purchased from Tianjin Guangfu Chemicals Ltd. (Tianjin, China). Methanol and acetonitrile of HPLC grade were purchased from Tianjin Concord Technology Co. Ltd. (Tianjin, China). The high purified water (18.0 M cm1) obtained from a WaterPro water system (Aquapro Corporation, AFZ-6000-U, China) was used throughout the experiments. Other chemicals were used as received without further purification. A Tecnai G2 T2 S-TWIN microscope was used to obtain transmission electron microscopy (TEM) images of CNTs, CNTs@SiO2 and CNTs@Naf-MIPs. Fourier transform infrared (FT-IR) spectra were recorded on an AVATAR 360 (Nicolet Corp., USA), and samples were dried at 80 qC in a vacuum oven for at least 12 h prior to fabrication of the KBr pellet. Fifty scans of the region between 400 and 4000 cm1 were collected for each FT-IR spectrum that was recorded. The amount of nafcillin, oxacillin and mezlocillin was analyzed with a Shimadzu (Japan) UV-2450 spectrophotometer at 230 nm or HPLC, and HPLC analyses were performed on a Shimadzu LC-20A HPLC system that included a binary pump and a variable wavelength UV detector (Shimadzu, Kyoto, Japan). The instrument control and data processing were carried out by the LC solution software. A Shimadzu VP-ODS C18 (5 m particle size, 150 mm × 4.6 mm) analytical column was used for the separation of analytes. The eluent was acetonitrile-H2O (40:60, V/V) at a
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flow rate of 1.0 mL min–1. The injection volume was 100 L, and the column effluent was monitored at 230 nm. 2.2
Preparation of nafcillin-imprinted polymers (CNTs@Naf-MIPs) and non-imprinted polymers (CNTs@NIPs)
Impurities such as amorphous carbon and metallic catalyst in the CNTs were removed using an HNO3 solution in a three neck flask with vigorous stirring (500 rpm); the mixture was refluxed for 48 h. The suspension was then filtered through a 0.22-m filter to recover the CNTs, followed by washing repeatedly with highly purified water until the pH reached 7.0, and dried under vacuum at 80 qC for further use[16]. The SiO2 coating on the CNTs was performed according to the method described by Zhu et al[17] and the product CNTs@SiO2 was dried under vacuum for use in further studies. The synthesis of CNTs@Naf-MIPs includes a surface molecular imprinting sol-gel process. In a typical synthesis: 0.1 g of nafcillin were dissolved in 10 mL of ethanol, and mixed with 0.5 mL of APTES and 0.5 mL of PTMOS. The mixture was stirred for 30 min, then, 2 mL of TEOS was added. After stirring for 10 min, 0.5 g of CNTs@SiO2 and 0.6 mL of 1 M HAc (as catalyst) were added. The mixture began to co-hydrolyse and co-condense after stirring for a few minutes, then incubated for 15 h at room temperature. The products were washed with ethanol for three times and then with methanol/acetic acid (80:20, V/V) and subsequently with methanol to remove the residue of template nafcillin. Finally, the product was dried under vacuum at 80 ºC for 12 h. For comparison, the CNTs@NIPs was also prepared using an identical procedure, but without the addition of nafcillin. 2.3
Binding experiment
In kinetic adsorption experiments, 20 mg of CNTs@NafMIPs were added to centrifugal tubes, each containing 4 mL of 60 mg L–1 of nafcillin solution, and incubated at regular time intervals, the supernatants and polymers were separated by centrifugation. The nafcillin concentration of the supernatants was measured by UV-vis spectrometer. The amount of nafcillin bound to the CNTs@Naf-MIPs was
calculated from the difference in the nafcillin concentrations before and after incubation on the rotator. In steady-state binding experiments, the 20 mg of CNTs@Naf-MIPs and CNTs@NIPs were mixed with a 4-mL methanol solution of nafcillin of various concentrations from 20 to 200 mg L–1, respectively. The CNTs@Naf-MIPs and CNTs@NIPs nanocomposites were isolated by centrifugation after incubating for 2 h at room temperature. The nafcillin concentration of the supernatants was measured by UV-vis spectrometer. The cross-reactivity of CNTs@Naf-MIPs and CNTs@NIPs to nafcillin and its analogues oxacillin and mezlocillin (Fig.1) was performed as the following: 20 mg of CNTs@Naf-MIPs and CNTs@NIPs were separately added to mixed solutions of nafcillin, oxacillin and mezlocillin in 4 mL of methanol at the concentration of 160 mg L–1 respectively. After incubation for 2 h at room temperature, the CNTs@Naf-MIPs and CNTs@NIPs were isolated by centrifugation. The filtrate was concentrated to dryness by nitrogen purging and then dissolved in methanol before HPLC analysis. The amount of nafcillin, oxacillin and mezlocillin bound to the CNTs@Naf-MIPs was determined using HPLC method. The mobile phase was acetonitrile-water (40:60, V/V) containing 0.1% acetic acid at a flow rate of 1.0 mL min–1. The injection volume was 100 L, and the column effluent was monitored at 230 nm. Desorption and regeneration of the adsorbents is crucial for all affinity separation techniques. The reusability of CNTs@Naf-MIPs was evaluated. Approximately 20 mg of CNTs@Naf-MIPs were added to the solutions of nafcillin in 4 mL methanol of 160 mg L–1 and then incubated at room temperature while gently stirred on a rocking table for 2 h. Then, the MIP nanocomposites were removed by centrifugation and the bound amount of nafcillin was quantified by HPLC. The recovered CNTs@Naf-MIPs nanocomposites were washed with a mixture of methanol/acetic acid (80:20, V/V) for several times to ensure complete removal of residual nafcillin in the MIPs and washed with methanol for several times, then dried under vacuum at 60 ºC and reused for adsorption of nafcillin. Desorption and regeneration of the adsorbents was repeated for five cycles.
Fig.1 Molecular structure of nafcillin and two analogues, oxacillin and mezlocillin
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2.4
Separation and determination of nafcillin in eggs samples
Eggs were selected for the spiked sample analysis. First, a 25-mL ethanol/water (60:40, V/V) was added to a 5-g eggs sample spiked with the standard nafcillin solution in a 50-mL polypropylene tube. The mixed sample was shaken for 30 min to achieve proteins precipitation. After centrifugation at 3000 rpm for 10 min, the supernatant solution was filtered through a 0.22-m filter. The filtrate was dried with a stream of nitrogen, and then dissolved with 40 mL of methanol. The spiking concentrations for nafcillin were set with two levels of 5 and 10 g kg–1, respectively. About 30 mg of CNTs@Naf-MIPs were added to the above 40 mL methanol solution, and then incubated for 2 h at room temperature. Next, the mixture was centrifugated at 3000 rpm for 10 min. Final, after the supernatant solution was discarded, the CNTs@Naf-MIPs nanocomposites were eluted with a mixing methanol/acetic acid (80:20, V/V) solution, and then the eluate was evaporated to dry under a stream of nitrogen and dissolved with 1 mL of methanol and measured by HPLC. At each spiked concentration of nafcillin, three measurements were performed. The HPLC analyses were performed on a Shimadzu LC-20A HPLC system including a binary pump and a variable wavelength UV detector (Shimadzu, Kyoto, Japan). The instrument control and data processing were carried out by the LC solution software. A Shimadzu VP-ODS C18 (5 m particle size, 150 mm × 4.6 mm) analytical column was used for the determination of analytes. The mobile phase was acetonitrile-water (40:60, V/V) containing 0.1% acetic acid at a flow rate of 1.0 mL min–1. The injection volume was 100 L, and the column effluent was monitored at 230 nm.
3 3.1
CNTs@SiO2 via sol-gel reaction. The thickness of the silica film was observed to increase by repeating the sol-gel reaction. The diameter of the CNTs@Naf-MIPs increased to 100–120 nm after the nafcillin-imprinting process, which corresponds to a 20–30 nm thick imprinted SiO2 layer covering on the CNTs (Fig.2C). The thickness of this imprinted polymer layer will be effective for mass transport between the solution and the surface of the CNTs@Naf-MIPs. The FT-IR spectra of the CNTs, CNTs@SiO2, CNTs@NafMIPs and CNTs@NIPs are shown in Fig.3. There was no apparent peak in the spectrum of the blank CNTs (curve a). The strong peak at about 1069.8 cm1 can be attributed to the stretch of Si-O-Si, indicating the formation of silica film on the surface of CNTs (Fig.3b). A characteristic feature of CNTs@Naf-MIPs in comparison with spectrum of CNTs@SiO2 was N–H bond at 1629.5 and 1557 cm1, and C-H bending vibration of benzene ring at 880–680 cm1 (Fig.3c). These results suggested that functional monomer APTES and PTOMS could be grafted onto the surface of CNTs@SiO2 in the imprinting process. CNTs@Naf-MIPs and CNTs@NIPs showed the similar locations and appearance of the major bands. 3.2
Binding properties of CNTs@Naf-MIPs
Figure 4A presents the adsorption kinetics of 60 mg L–1
Results and discussion Characterization of imprinted materials (CNTs@Naf-MIPs)
TEM images of the CNTs, CNTs@SiO2 and CNTs@Naf-MIPs are shown in Fig.2. The average diameter of the CNTs is observed at 70–80 nm (Fig.2A) before coating; after coating with the SiO2, the diameter of the CNTs@SiO2 increases to 85–95 nm, corresponding to a about 15 nm thickness of SiO2 layer covering the CNTs (Fig.2B). The SiO2 layers were uniformly coated for all of the samples after sol-gel reaction, and there were hardly any free SiO2 particles found in the TEM images. Silica coating can mitigate the difficulty of dispersing CNTs homogeneously in different solvents, and this coating favors the dispersion of CNTs in liquid media. The TEM image of CNTs@Naf-MIPs showed the formation of the core/shell-structured nanocomposites (Fig.2C) after imprinting process on the surface of the
Fig.2 TEM images of CNTs (A), CNTs@SiO2 (B), CNTs@NafMIPs (C)
Fig.3
FT-IR spectra of CNTs (a), CNTs@SiO2 (b), CNTs@NafMIPs (c) and CNTs@NIPs (d)
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Fig.4
Adsorption kinetics of CNTs@Naf-MIPs (A), adsorption isotherm of nafcillin onto CNTs@Naf-MIPs and CNTs@NIPs (B), and Langmuir plot to estimate the binding nature of CNTs@Naf-MIPs (C)
nafcillin onto CNTs@Naf-MIPs. It can be seen that the adsorption capacity increased rapidly in the first 30 min, after 30 min, the adsorption had almost reached equilibrium. For imprinted polymer prepared by traditional bulk polymerizetion, it takes generally 12–24 h to reach the adsorption equilibrium. In this work, template nafcillin reached the surface imprinting cavities of CNTs@Naf-MIPs easily and took less time to reach the adsorption saturation, which implied that CNTs@Naf-MIPs had good mass transport and thus overcame some drawbacks of traditional imprinted materials. The adsorption of naficillin to CNTs@Naf-MIPs and control CNTs@NIPs at different concentrations were investigated by a steady-state binding method. As can be seen in Fig.4B, the amount of nafcillin bound in the CNTs@NafMIPs is higher than that in the CNTs@NIPs. The results clearly confirmed the effectiveness of the molecular imprinting because the CNTs@Naf-MIPs showed more binding sites in comparison with CNTs@NIPs, and higher affinity to nafcillin. The saturation binding data were further processed with a Langmuir equation to estimate the binding properties of the CNTs@Naf-MIPs. The Langmuir equation is as follows: Ce/Q = Ce/Qmax + 1/(KDQmax) (1) –1 where, Ce (mg L ) was free analytical concentration of nafcillin in the solution at equilibrium, Q (mg g–1) was the amount of nafcillin bound to CNTs@Naf-MIPs at equilibrium, Qmax (mg g–1) was the apparent maximum adsorption capacity, and KD was the dissociation constant. The values of KD and Qmax can be calculated from the slope and intercept of the linear line plotted in Ce/Q versus Q (Fig.4C), which indicated that the binding sites of the CNTs@Naf-MIPs were homogeneity. The linear regression equation for the linear region is Ce/Q = 0.1055Ce + 1.864 (r = 0.9875). From the slope and the intercept of the straight line obtained, the values of Qmax and KD were 9.5 mg g–1 and 56.6 mL mg–1, respectively.
binding experiment in a mixed solution of nafcillin, oxacillin and mezlocillin, which are structural analogues (Fig.1). As shown in Fig.5, the binding capacity of nafcillin on the CNTs@Naf-MIPs is 3.1 mg g–1 higher than that of CNTs@NIPs, but the difference of the binding capacity between CNTs@Naf-MIPs and CNTs@NIPs to oxacillin and mezlocillin is 0.4 and 0.7 mg g–1, respectively. The CNTs@Naf-MIPs showed significantly higher specific recognition ability for nafcillin over both oxacillin and mezlocillin in the mixture solution. The CNTs@NIPs displayed the greatest nonspecific adsorption to oxacillin among the three antibiotics. Figure 1 showed the structural difference of nafcillin, oxacillin and mezlocillin. The template of nafcillin have a naphthalene ring, oxacillin have aromatic isoxazole and benzene ring, and mezlocillin have a benzene ring, so there existed the stronger S-S interaction between nafcillin and oxacillin molecule and functional monomer PTMOS. The oxacillin molecules have more N, O atoms which can form more hydrogen bonds in comparison with nafcillin template. The adsorption of CNTs@NIPs towards nafcillin, oxacillin and mezlocillin molecules is non-specific. The CNTs@Naf-MIPs showed the higher selectivity for nafcillin may be attributed to not only the stronger S-S interaction between naphthalene ring and functional monomer PTMOS but also the combined effect of the shape and size complementarities.
3.3 Specific recognition of CNTs@Naf-MIPs and CNTs@NIPs The specific recognition ability of the CNTs@Naf-MIPs and CNTs@NIPs was investigated through a steady-state
Fig.5 Binding selectivity of CNTs@Naf-MIPs and CNTs-NIPs to nafcillin, oxacillin and mezlocillin
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3.4
Repeatability of CNTs@Naf-MIPs
Desorption and regeneration is one of the most important properties for the application of MIPs. As such, the adsorption-desorption cycle experiment was repeated five times using the same CNTs@Naf-MIPs. Figure 6 showed the change of the amount of adsorbed nafcillin on CNTs@NafMIPs after five regeneration cycles experiment. It could be seen that the CNTs@Naf-MIPs lost about 20% of its adsorption capacity on average after two cycles and the adsorption capacity kept constant. It is possible that some recognition sites in the network of CNTs@Naf-MIPs could be blocked after regeneration or destroyed after rewashing, and thus they were no longer fit for the template molecule. They retained their recovery efficiency after three binding/ regeneration cycles by using this treatment. These results demonstrated the reusability of CNTs@Naf-MIPs over several adsorption- desorption cycles, which is a clear advantage over single-use materials. 3.5
Fig.6 Desorption and regeneration of CNTs@Naf-MIPs
Separation and determination of nafcillin in eggs
Generally, the complexity of food matrices and the presence of some potential interference require specific and selective methods of analysis. We investigated the application of CNTs@Naf-MIPs to selective adsorption of nafcillin in the eggs purchased from a local market. The methanol solutions obtained from eggs spiked nafcillin were treated with CNTs@Naf-MIPs. The CNTs@Naf-MIPs was washed with CH3OH/HAc (80:20, V/V) and the eluents were analyzed by HPLC. The chromatograms of the spiked eggs sample with nafcillin at concentration of 5 Pg kg–1 are displayed in Fig.7. The peak of nafcillin cannot be seen from the chromatogram obtained from the spiked eggs sample with concentration of 5 Pg kg–1, however, the peak of nafcillin distinctly appeared after enrichment of spiked eggs sample with CNTs@NafMIPs, which showed that the nafcillin can be enriched after adsorption by the CNTs@Naf-MIPs. To evaluate the accuracy and application of the developed method, the recoveries of spiked nafcillin in eggs with two levels of 5 and 10 g kg1 were measured. At each concentration, three measurements were performed (Table 1). The recoveries of eggs samples were 61.3% and 84.3%, respectively. The relative standard deviations (RSD) were less than 7.8%. These results demonstrated that the CNTs@NafMIPs can be used for the selective enrichment of nafcillin in eggs.
Fig.7
Chromatogram of egg sample spiked with nafcillin at a concentration of 5 g kg–1 (A); chromatogram of elution of the CNTs@Naf-MIPs with CH3OH-HAc (80:20, V/V), followed
by
HPLC
analysis
after
CNTs@Naf-MIPs
extraction of spiked egg sample (B)
imprinting technique with a sol-gel process based on carbon nanotubes coated with silica. The resulting CNTs@Naf-MIPs nanocomposites possessed identical binding sites, favorable selectivity, high capacity, and fast kinetics for uptake of the template molecule. The synthetic process is quite simple, and different batches of the imprinted materials showed good reproducibility in template binding experiments. Additionally, CNTs@Naf-MIPs nanocomposites showed high efficiency as a adsorbent for the selective enrichment of nafcillin in eggs.
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