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Nanoporous PtCo-based ultrasensitive enzyme-free immunosensor for zeranoldetection
Biosensors and Bioelectronics 42 (2013) 367–372
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Nanoporous PtCo-based ultrasensitive enzyme-free immunosensor for zeranol detection Rui Feng a, Yong Zhang b, Haiqin Yu b, Dan Wu b, Hongmin Ma b, Baocun Zhu a, Caixia Xu b, He Li b, Bin Du a,n, Qin Wei b,n a
School of Resources and Environmental Sciences, University of Jinan, Jinan 250022, PR China Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b
a r t i c l e i n f o
abstract
Article history: Received 12 August 2012 Received in revised form 8 October 2012 Accepted 8 October 2012 Available online 17 October 2012
Nanoporous PtCo alloy was designed as an antibody carrier for preparation of a highly sensitive immunosensor. The immunosensor was constructed by assembling the capture zeranol antibody on thionine decorated graphene nanosheets modified glassy carbon electrode. With an enzyme-free immunosensor mode, the nanoporous PtCo alloy, synthesized by dealloying method, had shown strong electrocatalytic activity toward antigen–antibody reaction. The use of PtCo alloy carrier offered a high amount of antibody on each immunoconjugate, hence amplified the detectable signal from the electroreaction of dissolved oxygen. Cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the recognition of zeranol. Due to the poor conductivity of zeranol, a small amount of zeranol immobilized onto the electrode could result in great change in the electron-transfer resistance. Some factors that would affect the performance of the immunosensor were studied, such as concentration of PtCo, pH, and the ratio of TH to GS. With zeranol concentration range (0.05 to 5.0 ng/mL), the immunosensor exhibited a highly sensitive response to zeranol with a detection limit of 13 pg/mL. The immunosensor was evaluated for bovine urine sample, receiving satisfactory results. & 2012 Elsevier B.V. All rights reserved.
Keywords: Nanoporous PtCo alloy Zeranol Enzyme-free immunosensor Environment-friendly determination
1. Introduction Zeranol (a-zearalanol), a resorcyl lactone derived from the mycotoxin zearalenone, has been widely used as a growth promoter for cattle and sheep in various countries (Nsahlai et al., 2002). However, its residue in animal tissues affects human health, which can cause human sex function disorder, or even lead to cancer (Haschek et al., 2002). Furthermore, zeranol could still be detectable in some animal derived food even though it is prohibited by China and European Community for several years (The Ministry of Agriculture Bulletin of PRC176, 2001; Commission of the European Communities. 657/2002/EC.) (Launay et al., 2004). Nowadays, different methods have been used to detect zeranol such as liquid chromatography–tandem mass spectrometry (LC–MS) (Kaklamanos et al., 2009) and gas chromatography–mass spectrometry (GC–MS) (Dickson et al., 2009). Great attentions have been attributed to fabricating immunosensors for ultrasensitive and fast detecting. However, the immunosensor, based on the highly specific antibody–antigen recognition, has never been used in the sensitive quantitative detection of zeranol.
n
Corresponding authors. Tel.: þ 86 531 82767370; fax: þ 86 531 82767370. E-mail addresses: [email protected] (B. Du), [email protected] (Q. Wei).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.10.031
Among previous researches, different kinds of enzymes have been used to improve sensitivity in various immunosensors (Tang et al., 2010a; Hong et al., 2012). But the enzyme is expensive and could be easily inactivated. Enzyme-free immunosensor, as an effective approach for selective and sensitive analysis, has recently attracted great interests and has been developed in different fields, including food safety, environmental monitoring, and clinical diagnosis (Wei et al., 2012; Wang et al., 2012; Lin et al., 2012; Gan et al., 2010). Compared with enzyme-labeled immunosensors (Tang et al., 2008a; Yang et al., 2011), enzymefree immunosensors have the advantages of low-cost, good stability, and simplicity (Wei et al., 2011; Huang et al., 2010). Therefore, there is an urgent need for seeking materials with excellent stability and catalytic activity. The combination of electrochemical immunosensors and nanoporous structured metal materials opens new horizons for highly sensitive detection of molecules. Nanoporous PtCo alloy (NP-PtCo), prepared using dealloying method, shows excellent catalytic activity, which can be used to replace enzyme for fabricating immunosensors. The catalytic activity of Pt nanomaterials have been extensively investigated (Luo et al., 2008; Li and Somorjai, 2010), and bimetallic systems are believed to offer unprecedented benefits in catalysis design. Xu et al. reported that PtCo showed dramatically enhanced catalytic activity and structure stability toward electro-oxidation of
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small molecules (Xu et al., 2011a; Liu et al., 2011; Xu et al., 2011b). Recent publications have also indicated that the activity of Pt catalysts can be improved by alloying Pt with other transition metals such as Co (Wang et al., 2011). Graphene sheets (GS), a single layer of carbon atoms bonded together in a hexagonal lattice, have attracted considerable attentions to fabricate immunosensors attribute to its excellent electronic properties and large surface area (Zhong et al., 2010; Du et al., 2010). Thionine (TH), a kind of conventional dye, was widely used as mediator in immunosensor preparation (Tang et al., 2010b; Chen et al., 2010; Yuan et al., 2010). Due to the large specific surface area of GS, high quantity of TH can be adsorbed onto GS through strong p–p stocking force between these two kinds of conjugated frames (Zhao et al., 2007; Liu et al., 2009). The aim of this work is to exploit advanced nanoporous materials to prepare enzyme-free electrochemical immunosensor for the detection of zeranol. NP-PtCo, due to its abundant porous structures and admirable catalytic activity, not only favors the bioactivity of antibody but also facilitates the electron transfer. Greatly amplified sensitivity was achieved by using GS as the electron transfer medium. In this study, using zeranol as a model analyte, the sensitive detection of zeranol was demonstrated based on the peak current change of TH before and after the antigen–antibody reaction. This immunosensing method is simple, which may provide potential applications for the ultrasensitive detection of different contaminants in animal derived food.
2. Experimental section 2.1. Materials and methods 2.1.1. Reagents TH used in this study was purchased from Sinopharm Chemical Reagent Ltd Co. (Beijing, China). Bovine serum albumin (BSA) and chitosan (CS) were obtained from Sigma–Aldrich. Zeranol antibody (anti-zeranol, Ab) were purchased from Beijing Wanger Biotechnology Co., Ltd (Beijing, China). Phosphate-buffered solutions (PBS, 0.13 mol/L) at various pH values were prepared by mixing the stock solutions of 0.1 mol/L KH2PO4 and 0.1 mol/L Na2HPO4 at different volume ratios to appropriate pH value. PBS was used as electrolyte for all electrochemistry measurements. Double distilled water was used throughout the experiment. All other chemicals were of analytical reagents grade and used without further purification. 2.1.2. Apparatus All electrochemical measurements were performed on a CHI760D electrochemical workstation (Shanghai CH Instruments Co., China). The Electrochemical impedance spectroscopy (EIS) of the electrode membrane was measured with a Model IM6e (ZAHNER Elektrik, Germany). Transmission electron microscope (TEM) images were obtained from a Hitachi H-800 microscope (Japan). Scanning electron microscope (SEM), and Energy Dispersive X-Ray Spectroscopy (EDX) were recorded by JEOL JSM-6700F microscope (Japan). All SEM specimens were sputter-coated with a thin layer of gold under vacuum in an argon atmosphere prior to examination. UV/Vis measurements were carried out using a Lambda 35 UV/Vis Spectrometer (PerkinElmer, USA).
Typically, a 9:1 mixture of concentrated H2SO4/H3PO4 (360: 40 mL) was added into a mixture of graphite flakes (3.0 g, 1 wt%) and KMnO4 (18.0 g, 6 wt%), producing a slight exotherm to 35– 40 1C. And then, the reaction was heated to 50 1C and stirred for 12 h. The reaction was cooled to room temperature and poured onto ice ( 400 mL) with 30% H2O2 (3 mL). Subsequently, the filtrate was centrifuged (8000 rpm for 30 min), and the supernatant was decanted away. Successively, the remaining solid material was washed in succession with 200 mL of water, 200 mL of 30% HCl, and 200 mL of ethanol. The material remaining after this extended, multiple-wash process was coagulated with 200 mL of ether, and the resulting suspension was filtered over a polytetrafluoroethylene membrane with a 0.45 mm pore size. The solid obtained on the filter was vacuum-dried overnight at room temperature, obtaining 5.8 g product. As-purified GO suspension was then dispersed in water by ultrasonication for over 0.5 h and then the GO suspension was reduced with hydrazine at 100 1C for over 24 h. Finally, the black precipitates were filtrated and washed with water. The resulting solid was dried to obtain GS. 2.3. Synthesis of NP-PtCo NP-PtCo was prepared according to reported method (Qiu and Zou, 2012). Ternary PtCoAl alloy foils were made by refining highpurity ( 499.99%) Pt, Co, and Al at high temperatures under the protection of high-purity argon in a furnace, followed by meltspinning. The structures were obtained after dealloying PtCoAl alloy in 2 mol/L NaOH solution for 72 h at 30 1C. After re-dispersed in water, dealloyed material was then washed until the pH ¼7 nearby. The resulting solid fresh product was obtained by sedimentation, and was dried under vacuum. 5 mg of NP-PtCo dispersed in 5 mL of 1 wt% CS was ultrasonically to be given a black suspension (CS–PtCo). 2.4. Preparation of CS–PtCo–Ab The Ab was immobilized onto the CS–PtCo through acid amides cross-linking reaction between the amine groups attached to CS and the available carboxyl groups of Ab. Typically, 10 mg of Ab was added into the CS–PtCo solution. The mixture was allowed to react at 4 1C under stirring for 24 h, followed by centrifugation. Ab could be loaded into channel of the CS–PtCo through adsorption since it has been proven that amino groups in Ab can be bound to Pt (Nakabayashi et al., 2009). The resulting CS–PtCo–Ab conjugates were stored at 4 1C before use. 2.5. Fabrication of the immunosensor Fig. 1 displayed the preparation process of the immunosensor. Glassy carbon electrode (GCE) was polished with 1.0, 0.3, and
2.2. Synthesis of GS GS were prepared from graphite oxide (GO) through a thermal exfoliation method (McAllister et al., 2007). GO powders were synthesized from graphite by a modified Hummer’s method (Liu et al., 2008).
Fig. 1. Illustration of fabrication process of modified immunosensor.
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0.05 mm alumina powder sequentially and then washed ultrasonically in ethanol for a few minutes. Afterwards, solutions of GS and TH were mixed, sonicated, and then dropped onto the electrode surface to prepare the GS (Zeng et al., 2010; Daniela et al., 2010) and TH composite film modified electrode. The electrode was then thoroughly rinsed with PBS to remove unbounded particles. Next, the CS–PtCo (Xu et al., 2010; Saikat et al., 2004; Li et al., 2011) was beneficial to immobilize the Ab onto GS surface, 6 mL of prepared excessive anti-zeranol solution (10 mg/mL) was added onto electrode surface and incubated for 1 h. The electrode was then washed and incubated in 1 wt% BSA solution to block nonspecific binding sites. Finally, the electrode was incubated with different concentrations of zeranol solution. After washing, the electrode was stored at 4 1C prior to use.
nanoarchitectures. Fig. 2(b) displayed the pore diameter of NP-PtCo was about 6.0 nm, and the dark skeletons further confirm the formation of a three-dimensional (3D) interconnected network structure on the nanoscale. Furthermore, the different mesoporous nanomaterials were obtained from different atomic ratios of Pt and Co. The atom ratio between Pt and Co (1:1) was examined by Fig. 2(c). Consequently, NP-PtCo had been synthesized successfully. The morphology of GS was shown in Fig. 2(d) and (e): the GS was rippled, transparent with irregular size, and it resembled waves of crumpled silk veils.
2.6. Detection of zeranol
The interactions in the composite system based on CS, PtCo, and Ab are complicated, containing electrostatic, hydrogen bond, charge–charge interaction, van der Waals force, and hydrophilic and hydrophobic interactions (Lu et al., 2006). The good stability of the CS–PtCo–Ab/GCE in buffer solution could be mainly ascribed to the good biocompatibility of the composite system and the strong interactions within the composite system. The CS– PtCo–Ab nanoparticles were characterized using UV–Vis spectroscopy (Fig. S1). According to the UV–Vis spectral analysis, no absorption peak was observed for the CS–PtCo (curve a, Fig. S1). Taken together, a distinct adsorption peak at 276 nm from the pure Ab was observed (curve b, Fig. S1). According to the curve of pure Ab, the peak at 276 nm in curve c was mainly ascribed to Ab (Tang et al., 2008b). EIS has been employed to characterize the interface properties of surface-modified immunosensors. It is well known that the high frequency region of the impedance plot shows a semicircle related to the redox probe Fe(CN)3–/4– , followed by a Warburg line 6 in the low frequency region which corresponds to the diffusion step of the overall process (Panagopoulou et al., 2010). The semicircle portion at higher frequencies corresponds to the electron–transfer limited process, and the linear portion at lower frequencies represents the diffusion-limited process. The resistance (Ret) can be calculated from the semicircle diameter at higher frequencies in the nyquist plot of impedance spectroscopy. Fig. 3 shows nyquist diagrams of electrochemical impedance spectra. Clearly, the GCE presented a very small semicircle domain implying a very fast electron-transfer process with a diffusional limiting step (curve a). After the bare electrode was modified by GS–TH, the semicircle became larger (curve b). Then, the Ret of the resultant CS–PtCo–Ab film surprisingly increased
A conventional three-electrode system was used for all electrochemical measurements: a GCE (3 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode and a platinum wire electrode as the counter electrode. The pH¼7.4 PBS was used for all the electrochemical measurements. Cyclic voltammetry was recorded in PBS at 100 mv/s. After the background current was stabilized, the change in the current response (DI) before and after antigen–antibody reaction was recorded.
3. Results and discussion 3.1. Characterization of the NP-PtCo and GS Onto the electrode, TH–GS solution was added. After the TH–GS coated electrode was dried and washed, CS–PtCo–Ab was added onto electrode surface. After 1 h of incubation, the electrode was washed with buffer and incubated in 1 wt% BSA solution. Subsequently, zeranol buffer solution with a varying concentration was added onto the electrode surface and incubated, and then the electrode was washed and ready for measurement. To characterize NP-PtCo and GS, several methods were used during this work, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray spectrometry (EDX). Fig. 2(a) shows the SEM image of the NP-PtCo. From the image, it can be seen the inner, bright regions were the bicontinuous hollow channels embedded in the solid
3.2. Characterization of the immunosensor fabrication
Fig. 2. SEM (a), TEM (b) and EDX (c) images of NP-PtCo materials; SEM (d) and TEM (e) images of GS.
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(curve c), indicating that Ab was immobilized on the electrode and the hydrophobic layer of protein insulted the conductive support and hindered the interfacial electron transfer. Ret rose in the same way after 1 wt% BSA was used to block nonspecific sites (curve d), which may attribute to the same reason with loading the protein. After the capture of 2 ng/mL zeranol, the Ret elevated again (curve e), which indicated the successful capture of zeranol and the formation of hydrophobic immunocomplex layer embarrassing
Fig. 3. EIS obtained for different modified electrodes in PBS (pH¼ 7.4) containing 5 mmol/L Fe(CN)3–/4– (a) GCE, (b) TH–GS/GCE, (c) CS–PtCo–Ab/TH–GS/GCE, 6 (d) BSA/CS–PtCo–Ab/TH–GS/GCE, (e) Zeranol(2.5 ng/mL)/BSA/CS–PtCo–Ab/ TH– GS/GCE.
the electron transfer. As a result, immunosensor had been fabricated successfully. 3.3. Optimization of experimental conditions In order to obtain the best analytical performance for zeranol, experimental conditions have been optimized. For the enzyme-free immunosensor, CS–PtCo has a great influence on the sensitivity of the fabricated immunosensor. Fig. 4A shows the cyclic voltammetry curves of prepared immunosensors. As can be seen, the immunosensor with CS–PtCo showed a quite higher current response than the immunosensors without CS–PtCo. The fact indicated that CS–PtCo enhanced the redox activity of the mediator. As a result, it was necessary to use CS–PtCo–Ab for the following experiments. Since the antigen–antibody linkage would be broken under drastic conditions (e.g., in alkalinic or acidic solutions), the pH of the working buffer (0.13 mol/L PBS) has a great effect on the electrochemical behavior of immunosensor. In order to optimize the pH, a series of PBS with the pH from 4.5 to 9.4 were prepared and the immunosensor was tested by cyclic voltammetry (CV). The experimental results (Fig. 4B revealed that the maximum current response occurred at pH ¼7.4. Thus, pH¼7.4 PBS was appropriate for subsequent tests. Polymer film of TH has a function of electrode mediator (Edna and Robert, 1993). To achieve an optimal electrochemical signaling, the TH concentration plays an important role. Different concentrations of TH between 0.0 and 1.0 mg/mL were prepared, and used to fabricate the immunosensors. CV of these immunosensors was recorded. The effect of different concentrations of TH used for immunosensors on the current response (DI) before and
Fig. 4. Effect of PtCo (A, (a) CS–PtCo–Ab/TH–GS/GCE, (b) zeranol/BSA/CS–PtCo–Ab/TH–GS/GCE, (c) Ab/TH–GS/GCE, (d) zeranol/BSA/Ab/TH–GS/GCE, 2.5 ng/mL zeranol), the concentration of pH (B, cGS ¼ 0.5 ng/mL, cTH ¼0.5 ng/mL, 2 ng/mL zeranol), TH (C, cGS ¼ 0 ng/mL, 2 ng/mL zeranol) and GS (D) on current change (DI) of the immunosensor for the detection of zeranol in 5 mL PBS (pH ¼7.4), Error bar¼ RSD (n¼ 5).
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Table 1 Results for the determination of zeranol in bovine urine by the prepared immunosensor. Bovine urine The addition The detection RSD Recovery (%) sample (ng/mL) content (ng/mL) content (ng/mL) (%, n¼5) 0.60 0.60 0.60
Fig. 5. Calibration curve of the immunosensor toward different concentrations of zeranol, Error bar ¼RSD (n ¼5).
after antigen–antibody reaction for the detection of 2 ng/mL zeranol was shown in Fig. 4C. The response increased and reached a maximum value at 0.6 mg/mL. Therefore, 0.6 mg/mL was used as the optimal concentration of TH. In addition, the effect of the ratio of GS and TH in the GS–TH film was investigated on the electrochemical response of the immunosensor. As seen in Fig. 4D, the current response of the electrode to 2 ng/mL of zeranol increased highest at 0.6 mg/mL of GS. Therefore, 0.6 mg/mL of GS was used as the performed concentration. Under the optimum conditions, immunosensors based on CS–PtCo–Ab were used to detect different concentrations of zeranol. The relationship between the current changes and the concentrations of zeranol had shown in Fig. 5. The catalytic current increased linearly by the zeranol concentration within the range of 0.05 to 5.0 ng/mL (n ¼8). The equation of the calibration curve was Y ¼86.80þ22.76 X, r ¼0.9849. To the development of many further techniques, lower limits of detection are a major criterion of successful application (James and Jane, 2005). Based on S/N¼3, a low detection limit (13 pg/mL) was obtained. The low detection limit may be attributed to three factors. First, a relatively large amount of Ab has been conjugated onto the NP-PtCo. Generally, when the zeranol concentration is low, the amount of zeranol captured by the Ab immobilized onto the electrode surface is also low; however, the relatively large amount of Ab immobilized onto the labels can greatly increase the probability of Ab interactions, thereby leading to higher sensitivity. Second, the NP-PtCo as immobilization matrices, fabricates an enzyme-free electrochemical immunosensor, with high active surface area, through which the immobilization of Ab is enhanced, are quite simple, economical, and environmental friendly. Third, as discussed earlier, GS have high conductivity, good biocompatibility, electronic property to transfer electron. The mixture of TH and GS on the electrode surface, which has high space area, was used to capture more PtCo and amplify the signal. The electrochemical immunosensor based on NP-PtCo and GS exhibited excellent sensitivity and selectivity, with the low detection limit compared with previous detection for zeranol. The proposed strategy provides a catalytical immobilization and sensitized recognition platform for analytes as micromolecules and possesses promising application in food sample diagnosis. 3.4. Selectivity, stability, and reproducibility of immunosensors Selective determination of target analytes plays an important role in analyzing biological samples (Perrotta et al., 2012). The effect of possible inhibitors that might interfere with the response of immunosensor was investigated. The evaluation selectivity of the immunosensor was carried out by incubating
1.00 2.00 4.00
1.59 7 0.02 2.61 7 0.08 4.63 7 0.12
1.4 3.0 2.5
99.9 101 101
the immunosensor in 2 ng/mL of zeranol containing some potential co-existed species. 2 ng/mL of zeranol solution containing 200 ng/mL of interfering substance (norethisterone, kanamycin, BSA, glucose, and vitamin C, respectively) was measured by the immunosensor and the results are shown in Fig. S2. The current variation due to the interfering substances was less than 5.7% of that without interferences. The above results demonstrated that the selectivity of the immunosensor was acceptable. The stability of the immunosensor was also checked by periodically checking the current responses. First, when the immunosensor was prepared and not in use, it was stored in refrigerator at 4 1C. The current response of the as-prepared immunosensor decreased 3.6% after 7-day storage in PBS (pH¼7.4). After 26 days, the catalytic current of the immunosensor using NP-PtCo as labels decreased to about 95% of its initial value. Therefore, molecules can be firmly immobilized on the surface of electrode and showed long life time (Li et al., 2003). The slow decrease in the current response may be due to the gradual denature of Ab. The relative standard deviation (RSD) of the measurements for the five as-prepared immunosensors for the detection of 2 ng/mL zeranol was 4.5%, suggesting the precision of the proposed immunosensor was quite good. The selectivity, stability, and reproducibility of immunosensors were acceptable, thus it was suitable for the determination of zeranol in real sample.
3.5. Real sample analysis In order to investigate the feasibility of the immunosensors for practical analysis, the proposed immunosensors were used to detect the recoveries of different concentrations (1.00, 2.00, and 4.00 ng/mL) of zeranol in bovine urine by standard addition methods. Bovine urine was extracted by 0.2 mm micropore filter. The extract was then diluted with PBS (pH¼7.4). The recovery was in the range of 99.9–101% and RSD was in the range of 1.4–3.0% (Table 1).
4. Conclusions In conclusion, the electrochemical immunosensor based on NP-PtCo exhibited excellent sensitivity and selectivity, with the limit of detection for zeranol being as low as 13 pg/mL (n ¼8). The main advantages of the presented immunosensor contributed to two aspects. First, the NP-PtCo as immobilization matrices with high active surface area and excellent catalytic, through which the loading of anti-zeranol was enhanced. Second, GS are characterized as the ‘thinnest material in our universe’, with high electrical conductivity and high surface area, which could enhance the electron transfer between solution and electrode. The proposed strategy provides an immobilization and sensitized recognition platform for analytes as micromolecules and possesses promising application in food sample analysis.
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Acknowledgments This study was supported by the Natural Science Foundation of China (No. 21075052, 21175057), Special Research and Development Environmental Protection Industry of Shandong Province, and all the authors express their deep thanks.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.10.031.
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Nanoporous PtCo-based ultrasensitive enzyme-free immunosensor for zeranol detection Rui Feng a, Yong Zhang b, Haiqin Yu b, Dan Wu b, Hongmin Ma b, Baocun Zhu a, Caixia Xu b, He Li b, Bin Du a,n, Qin Wei b,n a
School of Resources and Environmental Sciences, University of Jinan, Jinan 250022, PR China Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b
a r t i c l e i n f o
abstract
Article history: Received 12 August 2012 Received in revised form 8 October 2012 Accepted 8 October 2012 Available online 17 October 2012
Nanoporous PtCo alloy was designed as an antibody carrier for preparation of a highly sensitive immunosensor. The immunosensor was constructed by assembling the capture zeranol antibody on thionine decorated graphene nanosheets modified glassy carbon electrode. With an enzyme-free immunosensor mode, the nanoporous PtCo alloy, synthesized by dealloying method, had shown strong electrocatalytic activity toward antigen–antibody reaction. The use of PtCo alloy carrier offered a high amount of antibody on each immunoconjugate, hence amplified the detectable signal from the electroreaction of dissolved oxygen. Cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the recognition of zeranol. Due to the poor conductivity of zeranol, a small amount of zeranol immobilized onto the electrode could result in great change in the electron-transfer resistance. Some factors that would affect the performance of the immunosensor were studied, such as concentration of PtCo, pH, and the ratio of TH to GS. With zeranol concentration range (0.05 to 5.0 ng/mL), the immunosensor exhibited a highly sensitive response to zeranol with a detection limit of 13 pg/mL. The immunosensor was evaluated for bovine urine sample, receiving satisfactory results. & 2012 Elsevier B.V. All rights reserved.
Keywords: Nanoporous PtCo alloy Zeranol Enzyme-free immunosensor Environment-friendly determination
1. Introduction Zeranol (a-zearalanol), a resorcyl lactone derived from the mycotoxin zearalenone, has been widely used as a growth promoter for cattle and sheep in various countries (Nsahlai et al., 2002). However, its residue in animal tissues affects human health, which can cause human sex function disorder, or even lead to cancer (Haschek et al., 2002). Furthermore, zeranol could still be detectable in some animal derived food even though it is prohibited by China and European Community for several years (The Ministry of Agriculture Bulletin of PRC176, 2001; Commission of the European Communities. 657/2002/EC.) (Launay et al., 2004). Nowadays, different methods have been used to detect zeranol such as liquid chromatography–tandem mass spectrometry (LC–MS) (Kaklamanos et al., 2009) and gas chromatography–mass spectrometry (GC–MS) (Dickson et al., 2009). Great attentions have been attributed to fabricating immunosensors for ultrasensitive and fast detecting. However, the immunosensor, based on the highly specific antibody–antigen recognition, has never been used in the sensitive quantitative detection of zeranol.
n
Corresponding authors. Tel.: þ 86 531 82767370; fax: þ 86 531 82767370. E-mail addresses: [email protected] (B. Du), [email protected] (Q. Wei).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.10.031
Among previous researches, different kinds of enzymes have been used to improve sensitivity in various immunosensors (Tang et al., 2010a; Hong et al., 2012). But the enzyme is expensive and could be easily inactivated. Enzyme-free immunosensor, as an effective approach for selective and sensitive analysis, has recently attracted great interests and has been developed in different fields, including food safety, environmental monitoring, and clinical diagnosis (Wei et al., 2012; Wang et al., 2012; Lin et al., 2012; Gan et al., 2010). Compared with enzyme-labeled immunosensors (Tang et al., 2008a; Yang et al., 2011), enzymefree immunosensors have the advantages of low-cost, good stability, and simplicity (Wei et al., 2011; Huang et al., 2010). Therefore, there is an urgent need for seeking materials with excellent stability and catalytic activity. The combination of electrochemical immunosensors and nanoporous structured metal materials opens new horizons for highly sensitive detection of molecules. Nanoporous PtCo alloy (NP-PtCo), prepared using dealloying method, shows excellent catalytic activity, which can be used to replace enzyme for fabricating immunosensors. The catalytic activity of Pt nanomaterials have been extensively investigated (Luo et al., 2008; Li and Somorjai, 2010), and bimetallic systems are believed to offer unprecedented benefits in catalysis design. Xu et al. reported that PtCo showed dramatically enhanced catalytic activity and structure stability toward electro-oxidation of
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small molecules (Xu et al., 2011a; Liu et al., 2011; Xu et al., 2011b). Recent publications have also indicated that the activity of Pt catalysts can be improved by alloying Pt with other transition metals such as Co (Wang et al., 2011). Graphene sheets (GS), a single layer of carbon atoms bonded together in a hexagonal lattice, have attracted considerable attentions to fabricate immunosensors attribute to its excellent electronic properties and large surface area (Zhong et al., 2010; Du et al., 2010). Thionine (TH), a kind of conventional dye, was widely used as mediator in immunosensor preparation (Tang et al., 2010b; Chen et al., 2010; Yuan et al., 2010). Due to the large specific surface area of GS, high quantity of TH can be adsorbed onto GS through strong p–p stocking force between these two kinds of conjugated frames (Zhao et al., 2007; Liu et al., 2009). The aim of this work is to exploit advanced nanoporous materials to prepare enzyme-free electrochemical immunosensor for the detection of zeranol. NP-PtCo, due to its abundant porous structures and admirable catalytic activity, not only favors the bioactivity of antibody but also facilitates the electron transfer. Greatly amplified sensitivity was achieved by using GS as the electron transfer medium. In this study, using zeranol as a model analyte, the sensitive detection of zeranol was demonstrated based on the peak current change of TH before and after the antigen–antibody reaction. This immunosensing method is simple, which may provide potential applications for the ultrasensitive detection of different contaminants in animal derived food.
2. Experimental section 2.1. Materials and methods 2.1.1. Reagents TH used in this study was purchased from Sinopharm Chemical Reagent Ltd Co. (Beijing, China). Bovine serum albumin (BSA) and chitosan (CS) were obtained from Sigma–Aldrich. Zeranol antibody (anti-zeranol, Ab) were purchased from Beijing Wanger Biotechnology Co., Ltd (Beijing, China). Phosphate-buffered solutions (PBS, 0.13 mol/L) at various pH values were prepared by mixing the stock solutions of 0.1 mol/L KH2PO4 and 0.1 mol/L Na2HPO4 at different volume ratios to appropriate pH value. PBS was used as electrolyte for all electrochemistry measurements. Double distilled water was used throughout the experiment. All other chemicals were of analytical reagents grade and used without further purification. 2.1.2. Apparatus All electrochemical measurements were performed on a CHI760D electrochemical workstation (Shanghai CH Instruments Co., China). The Electrochemical impedance spectroscopy (EIS) of the electrode membrane was measured with a Model IM6e (ZAHNER Elektrik, Germany). Transmission electron microscope (TEM) images were obtained from a Hitachi H-800 microscope (Japan). Scanning electron microscope (SEM), and Energy Dispersive X-Ray Spectroscopy (EDX) were recorded by JEOL JSM-6700F microscope (Japan). All SEM specimens were sputter-coated with a thin layer of gold under vacuum in an argon atmosphere prior to examination. UV/Vis measurements were carried out using a Lambda 35 UV/Vis Spectrometer (PerkinElmer, USA).
Typically, a 9:1 mixture of concentrated H2SO4/H3PO4 (360: 40 mL) was added into a mixture of graphite flakes (3.0 g, 1 wt%) and KMnO4 (18.0 g, 6 wt%), producing a slight exotherm to 35– 40 1C. And then, the reaction was heated to 50 1C and stirred for 12 h. The reaction was cooled to room temperature and poured onto ice ( 400 mL) with 30% H2O2 (3 mL). Subsequently, the filtrate was centrifuged (8000 rpm for 30 min), and the supernatant was decanted away. Successively, the remaining solid material was washed in succession with 200 mL of water, 200 mL of 30% HCl, and 200 mL of ethanol. The material remaining after this extended, multiple-wash process was coagulated with 200 mL of ether, and the resulting suspension was filtered over a polytetrafluoroethylene membrane with a 0.45 mm pore size. The solid obtained on the filter was vacuum-dried overnight at room temperature, obtaining 5.8 g product. As-purified GO suspension was then dispersed in water by ultrasonication for over 0.5 h and then the GO suspension was reduced with hydrazine at 100 1C for over 24 h. Finally, the black precipitates were filtrated and washed with water. The resulting solid was dried to obtain GS. 2.3. Synthesis of NP-PtCo NP-PtCo was prepared according to reported method (Qiu and Zou, 2012). Ternary PtCoAl alloy foils were made by refining highpurity ( 499.99%) Pt, Co, and Al at high temperatures under the protection of high-purity argon in a furnace, followed by meltspinning. The structures were obtained after dealloying PtCoAl alloy in 2 mol/L NaOH solution for 72 h at 30 1C. After re-dispersed in water, dealloyed material was then washed until the pH ¼7 nearby. The resulting solid fresh product was obtained by sedimentation, and was dried under vacuum. 5 mg of NP-PtCo dispersed in 5 mL of 1 wt% CS was ultrasonically to be given a black suspension (CS–PtCo). 2.4. Preparation of CS–PtCo–Ab The Ab was immobilized onto the CS–PtCo through acid amides cross-linking reaction between the amine groups attached to CS and the available carboxyl groups of Ab. Typically, 10 mg of Ab was added into the CS–PtCo solution. The mixture was allowed to react at 4 1C under stirring for 24 h, followed by centrifugation. Ab could be loaded into channel of the CS–PtCo through adsorption since it has been proven that amino groups in Ab can be bound to Pt (Nakabayashi et al., 2009). The resulting CS–PtCo–Ab conjugates were stored at 4 1C before use. 2.5. Fabrication of the immunosensor Fig. 1 displayed the preparation process of the immunosensor. Glassy carbon electrode (GCE) was polished with 1.0, 0.3, and
2.2. Synthesis of GS GS were prepared from graphite oxide (GO) through a thermal exfoliation method (McAllister et al., 2007). GO powders were synthesized from graphite by a modified Hummer’s method (Liu et al., 2008).
Fig. 1. Illustration of fabrication process of modified immunosensor.
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0.05 mm alumina powder sequentially and then washed ultrasonically in ethanol for a few minutes. Afterwards, solutions of GS and TH were mixed, sonicated, and then dropped onto the electrode surface to prepare the GS (Zeng et al., 2010; Daniela et al., 2010) and TH composite film modified electrode. The electrode was then thoroughly rinsed with PBS to remove unbounded particles. Next, the CS–PtCo (Xu et al., 2010; Saikat et al., 2004; Li et al., 2011) was beneficial to immobilize the Ab onto GS surface, 6 mL of prepared excessive anti-zeranol solution (10 mg/mL) was added onto electrode surface and incubated for 1 h. The electrode was then washed and incubated in 1 wt% BSA solution to block nonspecific binding sites. Finally, the electrode was incubated with different concentrations of zeranol solution. After washing, the electrode was stored at 4 1C prior to use.
nanoarchitectures. Fig. 2(b) displayed the pore diameter of NP-PtCo was about 6.0 nm, and the dark skeletons further confirm the formation of a three-dimensional (3D) interconnected network structure on the nanoscale. Furthermore, the different mesoporous nanomaterials were obtained from different atomic ratios of Pt and Co. The atom ratio between Pt and Co (1:1) was examined by Fig. 2(c). Consequently, NP-PtCo had been synthesized successfully. The morphology of GS was shown in Fig. 2(d) and (e): the GS was rippled, transparent with irregular size, and it resembled waves of crumpled silk veils.
2.6. Detection of zeranol
The interactions in the composite system based on CS, PtCo, and Ab are complicated, containing electrostatic, hydrogen bond, charge–charge interaction, van der Waals force, and hydrophilic and hydrophobic interactions (Lu et al., 2006). The good stability of the CS–PtCo–Ab/GCE in buffer solution could be mainly ascribed to the good biocompatibility of the composite system and the strong interactions within the composite system. The CS– PtCo–Ab nanoparticles were characterized using UV–Vis spectroscopy (Fig. S1). According to the UV–Vis spectral analysis, no absorption peak was observed for the CS–PtCo (curve a, Fig. S1). Taken together, a distinct adsorption peak at 276 nm from the pure Ab was observed (curve b, Fig. S1). According to the curve of pure Ab, the peak at 276 nm in curve c was mainly ascribed to Ab (Tang et al., 2008b). EIS has been employed to characterize the interface properties of surface-modified immunosensors. It is well known that the high frequency region of the impedance plot shows a semicircle related to the redox probe Fe(CN)3–/4– , followed by a Warburg line 6 in the low frequency region which corresponds to the diffusion step of the overall process (Panagopoulou et al., 2010). The semicircle portion at higher frequencies corresponds to the electron–transfer limited process, and the linear portion at lower frequencies represents the diffusion-limited process. The resistance (Ret) can be calculated from the semicircle diameter at higher frequencies in the nyquist plot of impedance spectroscopy. Fig. 3 shows nyquist diagrams of electrochemical impedance spectra. Clearly, the GCE presented a very small semicircle domain implying a very fast electron-transfer process with a diffusional limiting step (curve a). After the bare electrode was modified by GS–TH, the semicircle became larger (curve b). Then, the Ret of the resultant CS–PtCo–Ab film surprisingly increased
A conventional three-electrode system was used for all electrochemical measurements: a GCE (3 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode and a platinum wire electrode as the counter electrode. The pH¼7.4 PBS was used for all the electrochemical measurements. Cyclic voltammetry was recorded in PBS at 100 mv/s. After the background current was stabilized, the change in the current response (DI) before and after antigen–antibody reaction was recorded.
3. Results and discussion 3.1. Characterization of the NP-PtCo and GS Onto the electrode, TH–GS solution was added. After the TH–GS coated electrode was dried and washed, CS–PtCo–Ab was added onto electrode surface. After 1 h of incubation, the electrode was washed with buffer and incubated in 1 wt% BSA solution. Subsequently, zeranol buffer solution with a varying concentration was added onto the electrode surface and incubated, and then the electrode was washed and ready for measurement. To characterize NP-PtCo and GS, several methods were used during this work, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray spectrometry (EDX). Fig. 2(a) shows the SEM image of the NP-PtCo. From the image, it can be seen the inner, bright regions were the bicontinuous hollow channels embedded in the solid
3.2. Characterization of the immunosensor fabrication
Fig. 2. SEM (a), TEM (b) and EDX (c) images of NP-PtCo materials; SEM (d) and TEM (e) images of GS.
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(curve c), indicating that Ab was immobilized on the electrode and the hydrophobic layer of protein insulted the conductive support and hindered the interfacial electron transfer. Ret rose in the same way after 1 wt% BSA was used to block nonspecific sites (curve d), which may attribute to the same reason with loading the protein. After the capture of 2 ng/mL zeranol, the Ret elevated again (curve e), which indicated the successful capture of zeranol and the formation of hydrophobic immunocomplex layer embarrassing
Fig. 3. EIS obtained for different modified electrodes in PBS (pH¼ 7.4) containing 5 mmol/L Fe(CN)3–/4– (a) GCE, (b) TH–GS/GCE, (c) CS–PtCo–Ab/TH–GS/GCE, 6 (d) BSA/CS–PtCo–Ab/TH–GS/GCE, (e) Zeranol(2.5 ng/mL)/BSA/CS–PtCo–Ab/ TH– GS/GCE.
the electron transfer. As a result, immunosensor had been fabricated successfully. 3.3. Optimization of experimental conditions In order to obtain the best analytical performance for zeranol, experimental conditions have been optimized. For the enzyme-free immunosensor, CS–PtCo has a great influence on the sensitivity of the fabricated immunosensor. Fig. 4A shows the cyclic voltammetry curves of prepared immunosensors. As can be seen, the immunosensor with CS–PtCo showed a quite higher current response than the immunosensors without CS–PtCo. The fact indicated that CS–PtCo enhanced the redox activity of the mediator. As a result, it was necessary to use CS–PtCo–Ab for the following experiments. Since the antigen–antibody linkage would be broken under drastic conditions (e.g., in alkalinic or acidic solutions), the pH of the working buffer (0.13 mol/L PBS) has a great effect on the electrochemical behavior of immunosensor. In order to optimize the pH, a series of PBS with the pH from 4.5 to 9.4 were prepared and the immunosensor was tested by cyclic voltammetry (CV). The experimental results (Fig. 4B revealed that the maximum current response occurred at pH ¼7.4. Thus, pH¼7.4 PBS was appropriate for subsequent tests. Polymer film of TH has a function of electrode mediator (Edna and Robert, 1993). To achieve an optimal electrochemical signaling, the TH concentration plays an important role. Different concentrations of TH between 0.0 and 1.0 mg/mL were prepared, and used to fabricate the immunosensors. CV of these immunosensors was recorded. The effect of different concentrations of TH used for immunosensors on the current response (DI) before and
Fig. 4. Effect of PtCo (A, (a) CS–PtCo–Ab/TH–GS/GCE, (b) zeranol/BSA/CS–PtCo–Ab/TH–GS/GCE, (c) Ab/TH–GS/GCE, (d) zeranol/BSA/Ab/TH–GS/GCE, 2.5 ng/mL zeranol), the concentration of pH (B, cGS ¼ 0.5 ng/mL, cTH ¼0.5 ng/mL, 2 ng/mL zeranol), TH (C, cGS ¼ 0 ng/mL, 2 ng/mL zeranol) and GS (D) on current change (DI) of the immunosensor for the detection of zeranol in 5 mL PBS (pH ¼7.4), Error bar¼ RSD (n¼ 5).
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Table 1 Results for the determination of zeranol in bovine urine by the prepared immunosensor. Bovine urine The addition The detection RSD Recovery (%) sample (ng/mL) content (ng/mL) content (ng/mL) (%, n¼5) 0.60 0.60 0.60
Fig. 5. Calibration curve of the immunosensor toward different concentrations of zeranol, Error bar ¼RSD (n ¼5).
after antigen–antibody reaction for the detection of 2 ng/mL zeranol was shown in Fig. 4C. The response increased and reached a maximum value at 0.6 mg/mL. Therefore, 0.6 mg/mL was used as the optimal concentration of TH. In addition, the effect of the ratio of GS and TH in the GS–TH film was investigated on the electrochemical response of the immunosensor. As seen in Fig. 4D, the current response of the electrode to 2 ng/mL of zeranol increased highest at 0.6 mg/mL of GS. Therefore, 0.6 mg/mL of GS was used as the performed concentration. Under the optimum conditions, immunosensors based on CS–PtCo–Ab were used to detect different concentrations of zeranol. The relationship between the current changes and the concentrations of zeranol had shown in Fig. 5. The catalytic current increased linearly by the zeranol concentration within the range of 0.05 to 5.0 ng/mL (n ¼8). The equation of the calibration curve was Y ¼86.80þ22.76 X, r ¼0.9849. To the development of many further techniques, lower limits of detection are a major criterion of successful application (James and Jane, 2005). Based on S/N¼3, a low detection limit (13 pg/mL) was obtained. The low detection limit may be attributed to three factors. First, a relatively large amount of Ab has been conjugated onto the NP-PtCo. Generally, when the zeranol concentration is low, the amount of zeranol captured by the Ab immobilized onto the electrode surface is also low; however, the relatively large amount of Ab immobilized onto the labels can greatly increase the probability of Ab interactions, thereby leading to higher sensitivity. Second, the NP-PtCo as immobilization matrices, fabricates an enzyme-free electrochemical immunosensor, with high active surface area, through which the immobilization of Ab is enhanced, are quite simple, economical, and environmental friendly. Third, as discussed earlier, GS have high conductivity, good biocompatibility, electronic property to transfer electron. The mixture of TH and GS on the electrode surface, which has high space area, was used to capture more PtCo and amplify the signal. The electrochemical immunosensor based on NP-PtCo and GS exhibited excellent sensitivity and selectivity, with the low detection limit compared with previous detection for zeranol. The proposed strategy provides a catalytical immobilization and sensitized recognition platform for analytes as micromolecules and possesses promising application in food sample diagnosis. 3.4. Selectivity, stability, and reproducibility of immunosensors Selective determination of target analytes plays an important role in analyzing biological samples (Perrotta et al., 2012). The effect of possible inhibitors that might interfere with the response of immunosensor was investigated. The evaluation selectivity of the immunosensor was carried out by incubating
1.00 2.00 4.00
1.59 7 0.02 2.61 7 0.08 4.63 7 0.12
1.4 3.0 2.5
99.9 101 101
the immunosensor in 2 ng/mL of zeranol containing some potential co-existed species. 2 ng/mL of zeranol solution containing 200 ng/mL of interfering substance (norethisterone, kanamycin, BSA, glucose, and vitamin C, respectively) was measured by the immunosensor and the results are shown in Fig. S2. The current variation due to the interfering substances was less than 5.7% of that without interferences. The above results demonstrated that the selectivity of the immunosensor was acceptable. The stability of the immunosensor was also checked by periodically checking the current responses. First, when the immunosensor was prepared and not in use, it was stored in refrigerator at 4 1C. The current response of the as-prepared immunosensor decreased 3.6% after 7-day storage in PBS (pH¼7.4). After 26 days, the catalytic current of the immunosensor using NP-PtCo as labels decreased to about 95% of its initial value. Therefore, molecules can be firmly immobilized on the surface of electrode and showed long life time (Li et al., 2003). The slow decrease in the current response may be due to the gradual denature of Ab. The relative standard deviation (RSD) of the measurements for the five as-prepared immunosensors for the detection of 2 ng/mL zeranol was 4.5%, suggesting the precision of the proposed immunosensor was quite good. The selectivity, stability, and reproducibility of immunosensors were acceptable, thus it was suitable for the determination of zeranol in real sample.
3.5. Real sample analysis In order to investigate the feasibility of the immunosensors for practical analysis, the proposed immunosensors were used to detect the recoveries of different concentrations (1.00, 2.00, and 4.00 ng/mL) of zeranol in bovine urine by standard addition methods. Bovine urine was extracted by 0.2 mm micropore filter. The extract was then diluted with PBS (pH¼7.4). The recovery was in the range of 99.9–101% and RSD was in the range of 1.4–3.0% (Table 1).
4. Conclusions In conclusion, the electrochemical immunosensor based on NP-PtCo exhibited excellent sensitivity and selectivity, with the limit of detection for zeranol being as low as 13 pg/mL (n ¼8). The main advantages of the presented immunosensor contributed to two aspects. First, the NP-PtCo as immobilization matrices with high active surface area and excellent catalytic, through which the loading of anti-zeranol was enhanced. Second, GS are characterized as the ‘thinnest material in our universe’, with high electrical conductivity and high surface area, which could enhance the electron transfer between solution and electrode. The proposed strategy provides an immobilization and sensitized recognition platform for analytes as micromolecules and possesses promising application in food sample analysis.
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Acknowledgments This study was supported by the Natural Science Foundation of China (No. 21075052, 21175057), Special Research and Development Environmental Protection Industry of Shandong Province, and all the authors express their deep thanks.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.10.031.
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