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An ultrasensitive electrochemical immunosensor for the detection of salbutamol based on Pd@SBA-15 and ionic liquid
Electrochimica Acta 69 (2012) 79–85
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An ultrasensitive electrochemical immunosensor for the detection of salbutamol based on Pd@SBA-15 and ionic liquid Zhentao Cui, Yanyan Cai, Dan Wu, Haiqin Yu, Yan Li, Kexia Mao, Huan Wang, Haixia Fan, Qin Wei ∗ , Bin Du ∗∗ Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China
a r t i c l e
i n f o
Article history: Received 23 November 2011 Received in revised form 17 February 2012 Accepted 21 February 2012 Available online 3 March 2012 Keywords: Sandwich-type immunosensor Salbutamol antigen Graphene sheets Pd@SBA-15 Ionic liquid
a b s t r a c t A sandwich-type electrochemical immunosensor for the ultrasensitive detection of the salbutamol (SAL) antigen based on sodium dodecylbenzene sulfonate-functionalized graphene sheets (SDBS-GS), Pd nanoparticles in functionalized SBA-15 (Pd@SBA-15) and ionic liquid (IL) is described. The primary SAL antibody (Ab1 ) can be covalently attached to the sulfonic groups of SDBS-GS through the amines of Ab1 with an acyl chloride cross-linking reaction. Pd@SBA-15 was prepared by adsorption of H2 PdCl4 onto the SBA-15 surface; the adsorbed H2 PdCl4 was then reduced to Pd nanoparticles with sodium borohydride. Pd@SBA-15 was conjugated to the secondary SAL antibody (Ab2 ) through glutaraldehyde. IL was added to the mixture of Pd@SBA-15 and Ab2 to promote electron transport. The synergistic effect between IL and Pd@SBA-15 retained the bioactivity of Ab2 . The sensitivity of the sandwich-type immunosensor using Pd@SBA-15/Ab2 /IL for the detection of SAL was much higher than those that used either SBA-15/Ab2 or Pd@SBA-15/Ab2 . Under optimal conditions, the electrochemical immunosensor exhibited a wide working range from 0.02 to 15.0 ng/mL with a detection limit of 7 pg/mL. The precision, reproducibility and stability of the immunosensor were acceptable. The proposed strategy could be easily extended to fabricate immunosensors for other tumor markers. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Salbutamol (SAL) is widely used in the clinical treatment of bronchial asthma, chronic obstructive pulmonary disease and other allergic diseases associated with the respiratory pathway [1]. SAL has also been used as a growth-promoting agent in poultry and cattle. However, it accumulates easily in animal organs and can enter the human body through the food chain; thus, it is harmful to human health. The detection of SAL in food is not permitted in China or the European Union. The consumption of high levels of SAL can lead to nausea, dizziness, aches, quivering hands and other symptoms of poisoning, especially in patients with heart disease or hypertension; long-term exposure through food can lead to chromosomal aberration, malignant tumors and other disorders [2]. Thus, devices or methods for the detection of SAL are particularly important. In recent years, many analytical methods have been reported for the determination of SAL, including high performance liquid chromatography, spectrophotometry, capillary electrophoresis, gas chromatography–mass spectrome-
∗ Corresponding author. Tel.: +86 531 82765730; fax: +86 531 82765969. ∗∗ Co-corresponding author. E-mail addresses: [email protected] (Q. Wei), [email protected] (B. Du). 0013-4686/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2012.02.073
try, flow injection analysis–chemiluminescence and the use of an immunosensor [3–5]. Although these methods are proven and widely accepted, they require advanced technical expertise and are expensive and time-consuming. In contrast with conventional immunoassay techniques, electrochemical immunosensors with a sandwich-type structure have a simple pre-treatment procedure, a short analytical time, precise current measurement and miniaturizable instrumentation [6,7]. Therefore, electrochemical immunosensors are of great interest. Here, we have developed a convenient sandwich-type immunosensor for the sensitive detection of SAL. For the sandwich-type immunosensor, the labeling and immobilization of antibodies are very important. Various types of labels have been used to amplify the electrochemical signal for such immunosensors, including enzymes, quantum dots, carbon nanotubes, and electroactive component-loaded nanoparticles [8]. Nanostructured materials are a very attractive option due to their unique optical, electrical, catalytic and magnetic properties; they have been used for a wide variety of applications in the fabrication of immunosensors. SBA-15, which is a mesoporous silica with uniform tubular channels, adjustable pore size (from 5 to 30 nm) and good biocompatibility [9], has been widely used for the construction of
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immunosensors [10–12]. To date, SBA-15 has been used for immobilizing glucose oxidase [13], nanoparticles [14], and antibodies [15]. The highly ordered porous structures, large surface areas and huge pore volume render it an ideal host for the immobilization of nanoparticles. Pd nanoparticles on functionalized SBA-15 (Pd@SBA-15) could retain both the good biocompatibility of the SBA-15 and the excellent catalytic ability of Pd to H2 O2 [16]. Graphene sheets (GS), a monolayer of carbon atoms packed into a close, honeycombed, two-dimensional lattice, have shown some intriguing attributes that may be very beneficial in designing electrochemical sensors, such as high conductivity, a high surface area-to-volume ratio, electronic properties and good biocompatibility [17]. GS tends to form irreversible agglomerates or even restack to form graphite through strong – stacking and van der Waals interactions [18]. Thus, great efforts have been made to increase graphene solubility through covalent [19,20] or noncovalent [21,22] functionalization. Thus, utilizing the advantages of functionalized GS to immobilize a primary antibody should obtain good results. Recently, ionic liquid has been used as a modifier for the fabrication of immunosensors due to its high ionic conductivity and good biocompatibility for an enhanced electrochemical response [23–25]. Li’s group obtained a dramatically enhanced activity of ionic, liquid-based, sol–gel silica matrix [26]. Other groups [27,28] have also successfully applied ionic liquids to fabricate electrochemical immunosensors and have achieved satisfactory results. For the development of sandwich-type electrochemical immunoassays, the high capture concentration of the secondary antibody (Ab2 ) can often improve the sensitivity of the immunoassay [29]. In this study, Pd@SBA-15 was used in a sandwich-type immunosensor for capturing Ab2 . In addition, hydrophilic IL 1butyl-3-methylimidazolium bromide (BMIM·Br) was also included in the labels to provide a favorable microenvironment and promote electron transport. The synergistic effect between BMIM·Br and Pd@SBA-15 retained the bioactivity of Ab2 . This new method using Pd@SBA-15/Ab2 /BMIM·Br should improve the electrochemical signal of the immunosensor for the detection of SAL.
2. Materials and methods 2.1. Apparatus and reagents Primary Anti-SAL (Ab1 ), SAL and the Ab2 were purchased from Wanger Biotechnology Co., Ltd. (Beijing, China). Bovine serum albumin (BSA, 96–99%), PdCl2 , and tetraethylorthosilicate (TEOS) were obtained from Sigma–Aldrich (Beijing, China). Graphite, sodium dodecylbenzene sulfonate (SDBS) and ammonia solution (28 wt%) were purchased from Sigma–Aldrich (Beijing, China). EO20 PO70 EO20 (P123) was obtained from ACROS (Beijing, China). BMIM·Br was purchased from the Lanzhou Institute of Chemical Physics (Lanzhou, China). All other chemicals were of analytical reagent grade and were used without further purification. Phosphate buffered saline (PBS, 0.1 mol/L containing 0.1 mol/L NaCl, pH 7.4) was used as an electrolyte for all the electrochemistry measurements. Ultra-pure water (18.25 M cm, 24 ◦ C) was used for all of the experiments. All the electrochemical measurements were performed on a CHI 760D electrochemical workstation (Chenhua Instrument Shanghai Co., Ltd., China). A conventional three-electrode system was used for all the electrochemical measurements: a glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode. Transmission electron microscope (TEM) images were obtained from an H-800 microscope
(Hitachi, Japan). Scanning electron microscope (SEM) images were obtained using a field emission SEM (Zeiss, Germany). Surface area measurements were performed on a Micromeritics ASAP 2020 surface area and porosity analyzer (Quantachrome, United States). 2.2. Synthesis of SDBS-functionalized GS For the improved method [30], a 9:1 mixture of concentrated H2 SO4 /H3 PO4 (360:40 mL) was added to a mixture of graphite flakes (3.0 g, 1 wt equiv.) and KMnO4 (18.0 g, 6 wt equiv.), which produces a slightly exothermic reaction, increasing the temperature to 35–40 ◦ C. The reaction was then heated to 50 ◦ C and stirred for 12 h. Subsequently, the reaction was cooled to room temperature and poured over ice (∼400 mL) with 30% H2 O2 (3 mL). For the workup, the filtrate was centrifuged (8000 rpm for 1 h), and the supernatant was decanted. The remaining solid material was then washed in succession with 200 mL of water, 200 mL of 0.1 mol/L HCl, and 200 mL of ethanol. The material remaining after this extended, multiple-wash process was coagulated with 200 mL of ether; the resulting suspension was filtered over a polytetrafluoroethene (PTFE) membrane with a 0.45 m pore size. The solid retained on the filter was vacuum-dried overnight at room temperature, which resulted in 5.8 g of product. The as-purified graphite oxide suspension was then dispersed in water by ultrasonication for at least 0.5 h, and then the graphite oxide was reduced with hydrazine at 100 ◦ C for at least 24 h in the presence of sodium dodecylbenzene sulfonate (SDBS) (30.6 g). Finally, the black precipitates were filtered and washed with ultra-pure water. The resulting solid was dried to obtain SDBS-functionalized GS (SDBS-GS) [31]. 2.3. Synthesis of mesoporous Pd@SBA-15 SBA-15 was synthesized following the method reported previously [32,33]. In brief, 1 g of P123 was dissolved in 30 mL of 2 mol/L HCl, followed by the addition of 2.215 g tetraethyl orthosilicate (TEOS). The mixture was stirred for 5 h at 35 ◦ C and then transferred to an autoclave and heated for 24 h at 100 ◦ C. After filtration, the particles obtained were air-dried and calcined at 550 ◦ C for 5 h to remove the templates. Subsequently, SBA-15 (1 g) was refluxed for 2 h in 80 mL of anhydrous toluene with 1 mL of 3-aminopropyltrimethoxysilane to yield the 3-aminopropylfunctionalized SBA-15 material. The amino-group-functionalized SBA-15 (16 mg) and H2 PdCl4 (16 mg) were mixed at room temperature with stirring for 24 h; HCl was then added until the pH < 2. Stirring continued for at least half an hour longer, and sodium borohydride (50.0 mmol/L) was added until the mixture turned black. The resulting Pd@SBA-15 was obtained by centrifuging and subsequent vacuum drying at room temperature. 2.4. Preparation of the Pd@SBA-15/Ab2 /BMIM·Br, Pd@SBA-15/Ab2 , SBA-15/Ab, Pd/Ab2 labels The procedures for the immobilization of antibodies and IL on Pd@SBA-15 are shown in Fig. 1a. Pd@SBA-15 (0.4 mg) was dispersed in 0.2 mL of phosphate buffer at pH 7.4; 0.1 mL of 2.5% glutaraldehyde solution was added into this SBA-15 buffer solution and stirred for 1 h. Then, 0.01 mg of Ab2 was added to the solution, and the mixture was allowed to react at 4 ◦ C with stirring for 24 h. Next, 0.1 mL of 12.0 mg/mL BMIM·Br solution was added to the mixture, stirred for 1 h, and then centrifuged. The resulting Pd@SBA-15/Ab2 /BMIM·Br was stored at 4 ◦ C when not in use. For the Pd@SBA-15/Ab2 label, Pd@SBA-15 (0.3 mg) was dispersed in 0.2 mL of phosphate buffer at pH 7.4, and 0.1 mL of 2.5% glutaraldehyde solution was added to this SBA-15 buffer solution and stirred for 1 h. Then, 0.01 mg of Ab2 was added to the solution, and the mixture was allowed to react at 4 ◦ C with stirring for 24 h.
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Fig. 1. Schematic diagram for the fabrication of the immunosensor.
The preparation of the SBA-15/Ab2 label was the same as that of the Pd@SBA-15/Ab2 label, except that SBA-15 was used instead of Pd@SBA-15. Similarly, to prepare the Pd/Ab2 label, Pd nanoparticles were used instead of Pd@SBA-15.
Pd@SBA-15/Ab2 /BMIM·Br buffer solution was dropped onto the electrode surface and incubated for an additional 1 h. After washing, the electrode was ready for measurement. 2.7. Detection of SAL
2.5. Preparation of SDBS-GS-Ab1 The primary SAL antibody (Ab1 ) was immobilized onto the SDBS-GS through an acyl chloride cross-linking reaction between the sulfonic groups attached to SDBS-GS and the available amine groups of Ab1 . In a 10 mL flask, 100 mg SDBS-GS and 200 mg PCl5 were mixed well, and 5 mL acetone was added; after refluxing in a water bath for 30 min, the excess PCl5 and acetone were evaporated to obtain the product, SDBS-GS-SO2 Cl. Typically, 1 mL of Ab1 solution (10 g/mL) was added to a 1 mL SDBS-GS-SO2 Cl solution (2.0 mg/mL). After 12 h of reaction, the SDBS-GS-SO2 Cl solution was centrifuged and washed. The resulting SDBS-GS-Ab1 conjugates were stored at 4 ◦ C in phosphate buffer solution before use [34–36]. 2.6. Fabrication of the immunosensor Fig. 1b shows the fabrication procedure for the immunosensor. A glassy carbon electrode was polished repeatedly using alumina powder and then thoroughly cleaned before use. Subsequently, 6 L of prepared SDBS-GS-Ab1 solution (2.0 mg/mL) was dropped onto the GCE surface to form SDBS-GS-modified GCE. After drying, the electrode was incubated in 1 wt% BSA solution for 1 h to eliminate nonspecific binding between the SAL and the electrode surface. Subsequently, SAL buffer solution with a different concentration was applied to the electrode surface and incubated for 1 h at room temperature, and the electrode was washed extensively to remove unbounded SAL molecules. Finally, the prepared
The pH 7.4 PBS buffer solution was used for all the electrochemical measurements. Cyclic voltametry (CV) was recorded in PBS at 100 mV/s. For amperometric measurement of the immunosensor, a detection potential of −0.4 V was selected. After the background current was stabilized, 5.0 mmol/L H2 O2 was added to the buffer solution, and the current change was recorded. 3. Results and discussion 3.1. Characterization of the Pd@SBA-15/BMIM·Br nanoparticles and graphene Fig. 2(a and c) shows the TEM and SEM images of the prepared mesoporous SBA-15, which illustrate that the prepared SBA-15 contained ordered silica channels with a uniform size of approximately 600 nm. The Pd@SBA-15/BMIM·Br nanoparticles that were dispersed in the buffer solution were characterized by TEM (Fig. 2b). The ordered silica channels of SBA-15 became indistinct, and a mass of Pd nanoparticles hybridized to the SBA-15. Energy-dispersive Xray spectroscopy (Fig. 3) analyses showed that the sample consisted of Pd, Si, and O elements, indicating the formation of a Pd@SBA15 composite. The specific surface area and pore volume obtained by the N2 adsorption isotherm and calculated by the Brunauere Emmette Teller (BET) method [37] were 515 m2 /g and 0.68 cm3 /g for SBA-15 and 229.4 m2 /g and 0.33 cm3 /g for Pd@SBA-15 (Fig. 4a), respectively. As shown in Fig. 4b, the SBA-15 and Pd@SBA-15 had median pore size distributions of approximately 6 nm and 5 nm,
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Fig. 2. TEM images of SBA-15 (a) and Pd@SBA-15/BMIM·Br (b); SEM image of SBA-15 (c).
Fig. 5. IR spectroscopy of SBA-15 (a) and amino-group-functionalized SBA-15 (b).
Fig. 3. Energy-dispersive X-ray spectroscopy characterization of Pd@SBA-15.
respectively, indicating that Pd nanoparticles hybridized to SBA15. Using the ninhydrin test [38], 1.4 mg/g of the amino group was grafted onto SBA-15, and the cross-linking agent glutaraldehyde was used to conjugate Ab2 to the fabricated immunosensor to enhance its sensitivity. To further analyze the amino group grafted onto SBA-15, IR characterization was employed (Fig. 5); the double peaks of 3300–3500 cm−1 revealed the N H stretching vibration, indicating successful functionalization of the amino group on SBA15. Therefore, due to the high pore volume, large surface area and the abundant amino groups of SBA-15, it is possible to achieve
high loading levels of guest molecules on SBA-15 for use in various biomedical applications [39]. The morphology of SDBS-GS is shown in Fig. 6a: the SDBS-GS is rippled and it resembles waves of crumpled silk veils. Fig. 6b–d shows the comparison of SDBS-GS and pure graphene sheets at different time intervals. Pure graphene sheets lost their water dispersability and eventually precipitated after 15 min and 30 min, but SDBS-GS could readily be redispersed in water. The SDBS-GS dispersions were homogeneous and stable. While more concentrated dispersions generated a small amount of precipitate after a few days, these precipitates never completely settled, even after months of storage.
Fig. 4. (a) N2 adsorption–desorption isotherm and (b) pore size distribution of mesoporous SBA-15 and Pd@SBA-15.
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Fig. 6. (a) TEM image of SDBS-GS. (b–d) SDBS-GS (2.0 mg/mL) (right) and pure GS (2.0 mg/mL) (left). Pure GS-generated precipitates after 15 min and 30 min; however, SDBS-GS could be readily redispersed in water.
3.2. Optimization of experimental conditions To achieve an optimal electrochemical signal, the experimental conditions were optimized. The pH value of the substrate solution was an important factor for the current response. As shown in Fig. 7a, the optimal amperometric response was achieved at pH 7.4; this is likely because highly acidic or alkaline surroundings could damage the immobilized protein [40]. Thus, PBS at pH 7.4 was used as electrolyte for all electrochemistry measurement. In addition, the amperometric response of the measuring system was related to the concentration of SDBS-GS, Pd@SBA-15 and BMIM·Br. As shown in Fig. 7b, the peak current tended to plateau at a SDBS-GS concentration of 2.0 mg/mL; this optimal concentration of SDBS-GS was utilized for subsequent experiments. The concentrations of Pd@SBA-15 and BMIM·Br in the electrode modification were also important parameters that affected the performance of the immunosensor. Higher or lower concentrations of Pd@SBA-15 affected the catalytic performance for the reduction of H2 O2 . If the
concentration of BMIM·Br is higher than 12.0 mg/mL, the formed antigen and antibody conjugates may partly cleave due to the high ionic strength. Therefore, 2.5 mg/mL Pd@SBA-15 and 12.0 mg/mL BMIM·Br were chosen for subsequent experiments (Fig. 7c and d). 3.3. Characterization of the immunosensor As the controls, immunosensors using three different labels, Pd@SBA-15/Ab2 /BMIM·Br, Pd@SBA-15/Ab2 and Pd/Ab2 , SBA15/Ab2 , were also prepared. As shown in Fig. 8, the H2 O2 detection sensitivity of the immunosensor using Pd@SBA-15/Ab2 /BMIM·Br as the label increased with the decrease of the detection potential. Thus, for amperometric measurements of the immunosensor, a detection of −0.4 V was selected. The amperometric responses of the immunosensors prepared with different labels for the detection of 10 ng/mL SAL are shown in Fig. 9. These data were compared to the amperometric current change of the immunosensor for the detection of H2 O2 . As expected, the immunosensor using
Fig. 7. (a) The effect of pH and (b) the concentration of SDBS-GS, (c) Pd@SBA-15 and (d) BMIM·Br on the response of the immunosensor for the detection of 10 ng/mL SAL in 10 mL PBS (pH 7.4) with 5.0 mmol/L H2 O2 .
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Z. Cui et al. / Electrochimica Acta 69 (2012) 79–85 Table 1 A comparison of the performance of the described and referenced immunosensors for the detection of SAL.
Fig. 8. Cyclic voltammograms of the immunosensor using Pd@SBA15/Ab2 /BMIM·Br for the detection of 2 ng/mL SAL as a label before (a, black) and after (b, red) the addition of 5 mmol/L H2 O2 . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Linear range (ng/mL)
Detection limit (ng/mL)
Reference
5–100 12–67 3–1000 0.02–15
3.5 3.2 1 0.007
[41] [42] [2] This work
facilitates the electron transfer of H2 O2 at the electrode surface. Hence, using Pd@SBA-15/Ab2 /BMIM·Br as a label enhances the sensitivity of the immunosensor. An immunosensor using Pd@SBA-15/Ab2 /BMIM·Br as a label was used to detect different concentrations of SAL. The relationship between the current response with 5.0 mmol/L H2 O2 and the SAL concentration is shown in Fig. 10a. The equation of the calibration curve is: Y = 7.852 + 4.608 X, r = 0.9992. The catalytic current increased linearly with an increasing SAL concentration, within the range of 0.02–15 ng/mL. The low detection limit (7 pg/mL) might be attributed to two factors. First, a relatively large amount of Ab2 had been conjugated to the SBA-15-based label. Generally, when the SAL concentration was low, the amount of SAL captured by the Ab1 immobilized onto the electrode surface was also low. However, the relatively large amount of Ab2 immobilized onto the label increases the probability of Ab2 –antigen interactions, which leads to higher sensitivity. Secondly, as discussed earlier, the large amounts of Pd nanoparticles and BMIM·Br immobilized onto the labels provided strong catalytic effects and a good electron transfer ability for the reduction of H2 O2 . The performance of the immunosensor was compared with previously described electrochemical immunosensors for the detection of SAL; Table 1 illustrates that the immunosensor described here has a lower detection limit than previously described immunosensors. 3.4. Precision, reproducibility, selectivity and stability of the immunosensor
Fig. 9. Amperometric responses of the immunosensor for the detection of 10 ng/mL SAL with different labels at −0.4 V with the successive addition of 5.0 mmol/L H2 O2 ; (a) SBA-15/Ab2 , (b) Pd/Ab2 , (c) Pd@SBA-15/Ab2 and (d) Pd@SBA-15/Ab2 /BMIM·Br.
Pd@SBA-15/Ab2 /BMIM·Br as a label displayed the highest current change, which was approximately 2, 9, and 200 times higher than those using Pd@SBA-15/Ab2 , Pd/Ab2 and SBA-15/Ab2 , respectively. In addition, the response was quite rapid, with the catalytic current reaching a steady-state value within 10 s. The high sensitivity can mainly be ascribed to the following factors: (1) the large amount of immobilized Pd nanoparticles could increase the loading of Ab2 onto the SBA-15 surface, and (2) the good conductivity of BMIM·Br
To evaluate the reproducibility of the immunosensor, a series of seven electrodes was prepared for the detection of 2 ng/mL SAL. The relative standard deviation (RSD) of the measurements for the seven electrodes was 2.6%. These results indicated that the precision and reproducibility of the devised immunosensor was quite good. To investigate the specificity of the fabricated immunosensor, interference studies were performed using glucose, ractopamine and ascorbic acid. A 2 ng/mL solution of SAL containing 200 ng/mL of an interfering substance was measured with the immunosensor; the results are shown in Fig. 10b. The variation in the current due to the presence of interfering substances was less than 4% com-
Fig. 10. (a) Calibration curve of the immunosensor for different concentrations of SAL. Error bar = RSD (n = 5). (b) Amperometric response of the immunosensor to 2 ng/mL SAL (1), 2 ng/mL SAL + 200 ng/mL glucose (2), 2 ng/mL SAL + 200 ng/mL ractopamine (3), 2 ng/mL SAL + 200 ng/mL ascorbic acid (4). Error bar = RSD (n = 5).
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Table 2 The results of the SAL determination in serum samples. Content of SAL in the sample (ng/mL)
The addition content (ng/mL)
The detection content (ng/mL)
RSD (%)
Recovery (%)
0.3
5.0 10.0 15.0
5.5, 5.3, 5.6, 5.7, 5.5 10.2, 10.4, 10.5, 10.1, 10.2 15.0, 15.4, 15.8, 14.5, 15.0
2.4 1.5 3.6
103.6 99.4 98.7
pared to the SAL solution alone, indicating that the selectivity of the immunosensor was acceptable. The stability of the immunosensor was also examined by periodically checking its current response. When the immunosensor was not in use, it was stored in air at 4 ◦ C. The amperometric response of the immunosensor for the detection of 10 ng/mL SAL in 10 mL PBS (pH 7.4) at −0.4 V with the successive addition of 5.0 mmol/L H2 O2 was 53 A. Under the same conditions, the catalytic current of the immunosensor using Pd@SBA-15/Ab2 /BMIM·Br as a label was measured after 2 weeks and after 1 month; the catalytic current decreased approximately 92% and 84%, respectively, from its initial value. This result indicates that the immunosensor has a high stability. Therefore, the developed immunosensor has a good reproducibility, selectivity and stability and could be applied for the detection of real samples.
Environmental Protection Industry of Shandong Province (2010) and the Science and Technology Development Plan Project of Jinan City (201004015). All of the authors express their deepest thanks to their funding sources. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
3.5. Real sample analysis [12]
To demonstrate the practical application of the immunosensor, the detection of SAL in serum samples was performed using standard addition methods, 5 ng/mL, 10 ng/mL and 15 ng/mL SAL solution were added into serum samples, respectively. The average recovery of the immunosensor was 103.6% (n = 5), 99.4% (n = 5) and 98.7% (n = 5), respectively (Table 2). Hence, the developed immunoassay methodology could be satisfactorily applied to the clinical determination of SAL levels in serum samples. 4. Conclusions In conclusion, this paper has reported the development of an immunosensor using the Pd@SBA-15 nanoparticles and BMIM·Br as label for the detection of SAL. This immunosensor can be easily prepared based on a sandwich-type protocol, with the primary antibody immobilized onto the surface of an SDBS-GS-modified electrode. The immunosensor displayed a linear response for the detection of SAL within a wide range (15.0 ng/mL). The proposed immunosensor shows a low detection limit (7 pg/mL), good reproducibility, satisfactory selectivity and acceptable stability. The simplicity of the fabrication procedure and the ultrasensitivity of the immunosensor may provide many potential applications for the detection of SAL in clinical diagnostics. Acknowledgments This study was supported by the Natural Science Foundation of China (Nos. 21075052 and 21175057), the Natural Science Foundation of Shandong Province (Nos. ZR2010BM030 and ZR2010EM063), the Key Subject Research Foundation of Shandong Province (XTD1105), the Science and Technology Key Plan Project of Shandong Province (2010GSF10628), the National Water Pollution Control and Management Technology Major Projects (2008ZX07422-001-5-1), the Special Research and Development
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
An ultrasensitive electrochemical immunosensor for the detection of salbutamol based on Pd@SBA-15 and ionic liquid Zhentao Cui, Yanyan Cai, Dan Wu, Haiqin Yu, Yan Li, Kexia Mao, Huan Wang, Haixia Fan, Qin Wei ∗ , Bin Du ∗∗ Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China
a r t i c l e
i n f o
Article history: Received 23 November 2011 Received in revised form 17 February 2012 Accepted 21 February 2012 Available online 3 March 2012 Keywords: Sandwich-type immunosensor Salbutamol antigen Graphene sheets Pd@SBA-15 Ionic liquid
a b s t r a c t A sandwich-type electrochemical immunosensor for the ultrasensitive detection of the salbutamol (SAL) antigen based on sodium dodecylbenzene sulfonate-functionalized graphene sheets (SDBS-GS), Pd nanoparticles in functionalized SBA-15 (Pd@SBA-15) and ionic liquid (IL) is described. The primary SAL antibody (Ab1 ) can be covalently attached to the sulfonic groups of SDBS-GS through the amines of Ab1 with an acyl chloride cross-linking reaction. Pd@SBA-15 was prepared by adsorption of H2 PdCl4 onto the SBA-15 surface; the adsorbed H2 PdCl4 was then reduced to Pd nanoparticles with sodium borohydride. Pd@SBA-15 was conjugated to the secondary SAL antibody (Ab2 ) through glutaraldehyde. IL was added to the mixture of Pd@SBA-15 and Ab2 to promote electron transport. The synergistic effect between IL and Pd@SBA-15 retained the bioactivity of Ab2 . The sensitivity of the sandwich-type immunosensor using Pd@SBA-15/Ab2 /IL for the detection of SAL was much higher than those that used either SBA-15/Ab2 or Pd@SBA-15/Ab2 . Under optimal conditions, the electrochemical immunosensor exhibited a wide working range from 0.02 to 15.0 ng/mL with a detection limit of 7 pg/mL. The precision, reproducibility and stability of the immunosensor were acceptable. The proposed strategy could be easily extended to fabricate immunosensors for other tumor markers. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Salbutamol (SAL) is widely used in the clinical treatment of bronchial asthma, chronic obstructive pulmonary disease and other allergic diseases associated with the respiratory pathway [1]. SAL has also been used as a growth-promoting agent in poultry and cattle. However, it accumulates easily in animal organs and can enter the human body through the food chain; thus, it is harmful to human health. The detection of SAL in food is not permitted in China or the European Union. The consumption of high levels of SAL can lead to nausea, dizziness, aches, quivering hands and other symptoms of poisoning, especially in patients with heart disease or hypertension; long-term exposure through food can lead to chromosomal aberration, malignant tumors and other disorders [2]. Thus, devices or methods for the detection of SAL are particularly important. In recent years, many analytical methods have been reported for the determination of SAL, including high performance liquid chromatography, spectrophotometry, capillary electrophoresis, gas chromatography–mass spectrome-
∗ Corresponding author. Tel.: +86 531 82765730; fax: +86 531 82765969. ∗∗ Co-corresponding author. E-mail addresses: [email protected] (Q. Wei), [email protected] (B. Du). 0013-4686/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2012.02.073
try, flow injection analysis–chemiluminescence and the use of an immunosensor [3–5]. Although these methods are proven and widely accepted, they require advanced technical expertise and are expensive and time-consuming. In contrast with conventional immunoassay techniques, electrochemical immunosensors with a sandwich-type structure have a simple pre-treatment procedure, a short analytical time, precise current measurement and miniaturizable instrumentation [6,7]. Therefore, electrochemical immunosensors are of great interest. Here, we have developed a convenient sandwich-type immunosensor for the sensitive detection of SAL. For the sandwich-type immunosensor, the labeling and immobilization of antibodies are very important. Various types of labels have been used to amplify the electrochemical signal for such immunosensors, including enzymes, quantum dots, carbon nanotubes, and electroactive component-loaded nanoparticles [8]. Nanostructured materials are a very attractive option due to their unique optical, electrical, catalytic and magnetic properties; they have been used for a wide variety of applications in the fabrication of immunosensors. SBA-15, which is a mesoporous silica with uniform tubular channels, adjustable pore size (from 5 to 30 nm) and good biocompatibility [9], has been widely used for the construction of
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immunosensors [10–12]. To date, SBA-15 has been used for immobilizing glucose oxidase [13], nanoparticles [14], and antibodies [15]. The highly ordered porous structures, large surface areas and huge pore volume render it an ideal host for the immobilization of nanoparticles. Pd nanoparticles on functionalized SBA-15 (Pd@SBA-15) could retain both the good biocompatibility of the SBA-15 and the excellent catalytic ability of Pd to H2 O2 [16]. Graphene sheets (GS), a monolayer of carbon atoms packed into a close, honeycombed, two-dimensional lattice, have shown some intriguing attributes that may be very beneficial in designing electrochemical sensors, such as high conductivity, a high surface area-to-volume ratio, electronic properties and good biocompatibility [17]. GS tends to form irreversible agglomerates or even restack to form graphite through strong – stacking and van der Waals interactions [18]. Thus, great efforts have been made to increase graphene solubility through covalent [19,20] or noncovalent [21,22] functionalization. Thus, utilizing the advantages of functionalized GS to immobilize a primary antibody should obtain good results. Recently, ionic liquid has been used as a modifier for the fabrication of immunosensors due to its high ionic conductivity and good biocompatibility for an enhanced electrochemical response [23–25]. Li’s group obtained a dramatically enhanced activity of ionic, liquid-based, sol–gel silica matrix [26]. Other groups [27,28] have also successfully applied ionic liquids to fabricate electrochemical immunosensors and have achieved satisfactory results. For the development of sandwich-type electrochemical immunoassays, the high capture concentration of the secondary antibody (Ab2 ) can often improve the sensitivity of the immunoassay [29]. In this study, Pd@SBA-15 was used in a sandwich-type immunosensor for capturing Ab2 . In addition, hydrophilic IL 1butyl-3-methylimidazolium bromide (BMIM·Br) was also included in the labels to provide a favorable microenvironment and promote electron transport. The synergistic effect between BMIM·Br and Pd@SBA-15 retained the bioactivity of Ab2 . This new method using Pd@SBA-15/Ab2 /BMIM·Br should improve the electrochemical signal of the immunosensor for the detection of SAL.
2. Materials and methods 2.1. Apparatus and reagents Primary Anti-SAL (Ab1 ), SAL and the Ab2 were purchased from Wanger Biotechnology Co., Ltd. (Beijing, China). Bovine serum albumin (BSA, 96–99%), PdCl2 , and tetraethylorthosilicate (TEOS) were obtained from Sigma–Aldrich (Beijing, China). Graphite, sodium dodecylbenzene sulfonate (SDBS) and ammonia solution (28 wt%) were purchased from Sigma–Aldrich (Beijing, China). EO20 PO70 EO20 (P123) was obtained from ACROS (Beijing, China). BMIM·Br was purchased from the Lanzhou Institute of Chemical Physics (Lanzhou, China). All other chemicals were of analytical reagent grade and were used without further purification. Phosphate buffered saline (PBS, 0.1 mol/L containing 0.1 mol/L NaCl, pH 7.4) was used as an electrolyte for all the electrochemistry measurements. Ultra-pure water (18.25 M cm, 24 ◦ C) was used for all of the experiments. All the electrochemical measurements were performed on a CHI 760D electrochemical workstation (Chenhua Instrument Shanghai Co., Ltd., China). A conventional three-electrode system was used for all the electrochemical measurements: a glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode. Transmission electron microscope (TEM) images were obtained from an H-800 microscope
(Hitachi, Japan). Scanning electron microscope (SEM) images were obtained using a field emission SEM (Zeiss, Germany). Surface area measurements were performed on a Micromeritics ASAP 2020 surface area and porosity analyzer (Quantachrome, United States). 2.2. Synthesis of SDBS-functionalized GS For the improved method [30], a 9:1 mixture of concentrated H2 SO4 /H3 PO4 (360:40 mL) was added to a mixture of graphite flakes (3.0 g, 1 wt equiv.) and KMnO4 (18.0 g, 6 wt equiv.), which produces a slightly exothermic reaction, increasing the temperature to 35–40 ◦ C. The reaction was then heated to 50 ◦ C and stirred for 12 h. Subsequently, the reaction was cooled to room temperature and poured over ice (∼400 mL) with 30% H2 O2 (3 mL). For the workup, the filtrate was centrifuged (8000 rpm for 1 h), and the supernatant was decanted. The remaining solid material was then washed in succession with 200 mL of water, 200 mL of 0.1 mol/L HCl, and 200 mL of ethanol. The material remaining after this extended, multiple-wash process was coagulated with 200 mL of ether; the resulting suspension was filtered over a polytetrafluoroethene (PTFE) membrane with a 0.45 m pore size. The solid retained on the filter was vacuum-dried overnight at room temperature, which resulted in 5.8 g of product. The as-purified graphite oxide suspension was then dispersed in water by ultrasonication for at least 0.5 h, and then the graphite oxide was reduced with hydrazine at 100 ◦ C for at least 24 h in the presence of sodium dodecylbenzene sulfonate (SDBS) (30.6 g). Finally, the black precipitates were filtered and washed with ultra-pure water. The resulting solid was dried to obtain SDBS-functionalized GS (SDBS-GS) [31]. 2.3. Synthesis of mesoporous Pd@SBA-15 SBA-15 was synthesized following the method reported previously [32,33]. In brief, 1 g of P123 was dissolved in 30 mL of 2 mol/L HCl, followed by the addition of 2.215 g tetraethyl orthosilicate (TEOS). The mixture was stirred for 5 h at 35 ◦ C and then transferred to an autoclave and heated for 24 h at 100 ◦ C. After filtration, the particles obtained were air-dried and calcined at 550 ◦ C for 5 h to remove the templates. Subsequently, SBA-15 (1 g) was refluxed for 2 h in 80 mL of anhydrous toluene with 1 mL of 3-aminopropyltrimethoxysilane to yield the 3-aminopropylfunctionalized SBA-15 material. The amino-group-functionalized SBA-15 (16 mg) and H2 PdCl4 (16 mg) were mixed at room temperature with stirring for 24 h; HCl was then added until the pH < 2. Stirring continued for at least half an hour longer, and sodium borohydride (50.0 mmol/L) was added until the mixture turned black. The resulting Pd@SBA-15 was obtained by centrifuging and subsequent vacuum drying at room temperature. 2.4. Preparation of the Pd@SBA-15/Ab2 /BMIM·Br, Pd@SBA-15/Ab2 , SBA-15/Ab, Pd/Ab2 labels The procedures for the immobilization of antibodies and IL on Pd@SBA-15 are shown in Fig. 1a. Pd@SBA-15 (0.4 mg) was dispersed in 0.2 mL of phosphate buffer at pH 7.4; 0.1 mL of 2.5% glutaraldehyde solution was added into this SBA-15 buffer solution and stirred for 1 h. Then, 0.01 mg of Ab2 was added to the solution, and the mixture was allowed to react at 4 ◦ C with stirring for 24 h. Next, 0.1 mL of 12.0 mg/mL BMIM·Br solution was added to the mixture, stirred for 1 h, and then centrifuged. The resulting Pd@SBA-15/Ab2 /BMIM·Br was stored at 4 ◦ C when not in use. For the Pd@SBA-15/Ab2 label, Pd@SBA-15 (0.3 mg) was dispersed in 0.2 mL of phosphate buffer at pH 7.4, and 0.1 mL of 2.5% glutaraldehyde solution was added to this SBA-15 buffer solution and stirred for 1 h. Then, 0.01 mg of Ab2 was added to the solution, and the mixture was allowed to react at 4 ◦ C with stirring for 24 h.
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Fig. 1. Schematic diagram for the fabrication of the immunosensor.
The preparation of the SBA-15/Ab2 label was the same as that of the Pd@SBA-15/Ab2 label, except that SBA-15 was used instead of Pd@SBA-15. Similarly, to prepare the Pd/Ab2 label, Pd nanoparticles were used instead of Pd@SBA-15.
Pd@SBA-15/Ab2 /BMIM·Br buffer solution was dropped onto the electrode surface and incubated for an additional 1 h. After washing, the electrode was ready for measurement. 2.7. Detection of SAL
2.5. Preparation of SDBS-GS-Ab1 The primary SAL antibody (Ab1 ) was immobilized onto the SDBS-GS through an acyl chloride cross-linking reaction between the sulfonic groups attached to SDBS-GS and the available amine groups of Ab1 . In a 10 mL flask, 100 mg SDBS-GS and 200 mg PCl5 were mixed well, and 5 mL acetone was added; after refluxing in a water bath for 30 min, the excess PCl5 and acetone were evaporated to obtain the product, SDBS-GS-SO2 Cl. Typically, 1 mL of Ab1 solution (10 g/mL) was added to a 1 mL SDBS-GS-SO2 Cl solution (2.0 mg/mL). After 12 h of reaction, the SDBS-GS-SO2 Cl solution was centrifuged and washed. The resulting SDBS-GS-Ab1 conjugates were stored at 4 ◦ C in phosphate buffer solution before use [34–36]. 2.6. Fabrication of the immunosensor Fig. 1b shows the fabrication procedure for the immunosensor. A glassy carbon electrode was polished repeatedly using alumina powder and then thoroughly cleaned before use. Subsequently, 6 L of prepared SDBS-GS-Ab1 solution (2.0 mg/mL) was dropped onto the GCE surface to form SDBS-GS-modified GCE. After drying, the electrode was incubated in 1 wt% BSA solution for 1 h to eliminate nonspecific binding between the SAL and the electrode surface. Subsequently, SAL buffer solution with a different concentration was applied to the electrode surface and incubated for 1 h at room temperature, and the electrode was washed extensively to remove unbounded SAL molecules. Finally, the prepared
The pH 7.4 PBS buffer solution was used for all the electrochemical measurements. Cyclic voltametry (CV) was recorded in PBS at 100 mV/s. For amperometric measurement of the immunosensor, a detection potential of −0.4 V was selected. After the background current was stabilized, 5.0 mmol/L H2 O2 was added to the buffer solution, and the current change was recorded. 3. Results and discussion 3.1. Characterization of the Pd@SBA-15/BMIM·Br nanoparticles and graphene Fig. 2(a and c) shows the TEM and SEM images of the prepared mesoporous SBA-15, which illustrate that the prepared SBA-15 contained ordered silica channels with a uniform size of approximately 600 nm. The Pd@SBA-15/BMIM·Br nanoparticles that were dispersed in the buffer solution were characterized by TEM (Fig. 2b). The ordered silica channels of SBA-15 became indistinct, and a mass of Pd nanoparticles hybridized to the SBA-15. Energy-dispersive Xray spectroscopy (Fig. 3) analyses showed that the sample consisted of Pd, Si, and O elements, indicating the formation of a Pd@SBA15 composite. The specific surface area and pore volume obtained by the N2 adsorption isotherm and calculated by the Brunauere Emmette Teller (BET) method [37] were 515 m2 /g and 0.68 cm3 /g for SBA-15 and 229.4 m2 /g and 0.33 cm3 /g for Pd@SBA-15 (Fig. 4a), respectively. As shown in Fig. 4b, the SBA-15 and Pd@SBA-15 had median pore size distributions of approximately 6 nm and 5 nm,
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Fig. 2. TEM images of SBA-15 (a) and Pd@SBA-15/BMIM·Br (b); SEM image of SBA-15 (c).
Fig. 5. IR spectroscopy of SBA-15 (a) and amino-group-functionalized SBA-15 (b).
Fig. 3. Energy-dispersive X-ray spectroscopy characterization of Pd@SBA-15.
respectively, indicating that Pd nanoparticles hybridized to SBA15. Using the ninhydrin test [38], 1.4 mg/g of the amino group was grafted onto SBA-15, and the cross-linking agent glutaraldehyde was used to conjugate Ab2 to the fabricated immunosensor to enhance its sensitivity. To further analyze the amino group grafted onto SBA-15, IR characterization was employed (Fig. 5); the double peaks of 3300–3500 cm−1 revealed the N H stretching vibration, indicating successful functionalization of the amino group on SBA15. Therefore, due to the high pore volume, large surface area and the abundant amino groups of SBA-15, it is possible to achieve
high loading levels of guest molecules on SBA-15 for use in various biomedical applications [39]. The morphology of SDBS-GS is shown in Fig. 6a: the SDBS-GS is rippled and it resembles waves of crumpled silk veils. Fig. 6b–d shows the comparison of SDBS-GS and pure graphene sheets at different time intervals. Pure graphene sheets lost their water dispersability and eventually precipitated after 15 min and 30 min, but SDBS-GS could readily be redispersed in water. The SDBS-GS dispersions were homogeneous and stable. While more concentrated dispersions generated a small amount of precipitate after a few days, these precipitates never completely settled, even after months of storage.
Fig. 4. (a) N2 adsorption–desorption isotherm and (b) pore size distribution of mesoporous SBA-15 and Pd@SBA-15.
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Fig. 6. (a) TEM image of SDBS-GS. (b–d) SDBS-GS (2.0 mg/mL) (right) and pure GS (2.0 mg/mL) (left). Pure GS-generated precipitates after 15 min and 30 min; however, SDBS-GS could be readily redispersed in water.
3.2. Optimization of experimental conditions To achieve an optimal electrochemical signal, the experimental conditions were optimized. The pH value of the substrate solution was an important factor for the current response. As shown in Fig. 7a, the optimal amperometric response was achieved at pH 7.4; this is likely because highly acidic or alkaline surroundings could damage the immobilized protein [40]. Thus, PBS at pH 7.4 was used as electrolyte for all electrochemistry measurement. In addition, the amperometric response of the measuring system was related to the concentration of SDBS-GS, Pd@SBA-15 and BMIM·Br. As shown in Fig. 7b, the peak current tended to plateau at a SDBS-GS concentration of 2.0 mg/mL; this optimal concentration of SDBS-GS was utilized for subsequent experiments. The concentrations of Pd@SBA-15 and BMIM·Br in the electrode modification were also important parameters that affected the performance of the immunosensor. Higher or lower concentrations of Pd@SBA-15 affected the catalytic performance for the reduction of H2 O2 . If the
concentration of BMIM·Br is higher than 12.0 mg/mL, the formed antigen and antibody conjugates may partly cleave due to the high ionic strength. Therefore, 2.5 mg/mL Pd@SBA-15 and 12.0 mg/mL BMIM·Br were chosen for subsequent experiments (Fig. 7c and d). 3.3. Characterization of the immunosensor As the controls, immunosensors using three different labels, Pd@SBA-15/Ab2 /BMIM·Br, Pd@SBA-15/Ab2 and Pd/Ab2 , SBA15/Ab2 , were also prepared. As shown in Fig. 8, the H2 O2 detection sensitivity of the immunosensor using Pd@SBA-15/Ab2 /BMIM·Br as the label increased with the decrease of the detection potential. Thus, for amperometric measurements of the immunosensor, a detection of −0.4 V was selected. The amperometric responses of the immunosensors prepared with different labels for the detection of 10 ng/mL SAL are shown in Fig. 9. These data were compared to the amperometric current change of the immunosensor for the detection of H2 O2 . As expected, the immunosensor using
Fig. 7. (a) The effect of pH and (b) the concentration of SDBS-GS, (c) Pd@SBA-15 and (d) BMIM·Br on the response of the immunosensor for the detection of 10 ng/mL SAL in 10 mL PBS (pH 7.4) with 5.0 mmol/L H2 O2 .
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Z. Cui et al. / Electrochimica Acta 69 (2012) 79–85 Table 1 A comparison of the performance of the described and referenced immunosensors for the detection of SAL.
Fig. 8. Cyclic voltammograms of the immunosensor using Pd@SBA15/Ab2 /BMIM·Br for the detection of 2 ng/mL SAL as a label before (a, black) and after (b, red) the addition of 5 mmol/L H2 O2 . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Linear range (ng/mL)
Detection limit (ng/mL)
Reference
5–100 12–67 3–1000 0.02–15
3.5 3.2 1 0.007
[41] [42] [2] This work
facilitates the electron transfer of H2 O2 at the electrode surface. Hence, using Pd@SBA-15/Ab2 /BMIM·Br as a label enhances the sensitivity of the immunosensor. An immunosensor using Pd@SBA-15/Ab2 /BMIM·Br as a label was used to detect different concentrations of SAL. The relationship between the current response with 5.0 mmol/L H2 O2 and the SAL concentration is shown in Fig. 10a. The equation of the calibration curve is: Y = 7.852 + 4.608 X, r = 0.9992. The catalytic current increased linearly with an increasing SAL concentration, within the range of 0.02–15 ng/mL. The low detection limit (7 pg/mL) might be attributed to two factors. First, a relatively large amount of Ab2 had been conjugated to the SBA-15-based label. Generally, when the SAL concentration was low, the amount of SAL captured by the Ab1 immobilized onto the electrode surface was also low. However, the relatively large amount of Ab2 immobilized onto the label increases the probability of Ab2 –antigen interactions, which leads to higher sensitivity. Secondly, as discussed earlier, the large amounts of Pd nanoparticles and BMIM·Br immobilized onto the labels provided strong catalytic effects and a good electron transfer ability for the reduction of H2 O2 . The performance of the immunosensor was compared with previously described electrochemical immunosensors for the detection of SAL; Table 1 illustrates that the immunosensor described here has a lower detection limit than previously described immunosensors. 3.4. Precision, reproducibility, selectivity and stability of the immunosensor
Fig. 9. Amperometric responses of the immunosensor for the detection of 10 ng/mL SAL with different labels at −0.4 V with the successive addition of 5.0 mmol/L H2 O2 ; (a) SBA-15/Ab2 , (b) Pd/Ab2 , (c) Pd@SBA-15/Ab2 and (d) Pd@SBA-15/Ab2 /BMIM·Br.
Pd@SBA-15/Ab2 /BMIM·Br as a label displayed the highest current change, which was approximately 2, 9, and 200 times higher than those using Pd@SBA-15/Ab2 , Pd/Ab2 and SBA-15/Ab2 , respectively. In addition, the response was quite rapid, with the catalytic current reaching a steady-state value within 10 s. The high sensitivity can mainly be ascribed to the following factors: (1) the large amount of immobilized Pd nanoparticles could increase the loading of Ab2 onto the SBA-15 surface, and (2) the good conductivity of BMIM·Br
To evaluate the reproducibility of the immunosensor, a series of seven electrodes was prepared for the detection of 2 ng/mL SAL. The relative standard deviation (RSD) of the measurements for the seven electrodes was 2.6%. These results indicated that the precision and reproducibility of the devised immunosensor was quite good. To investigate the specificity of the fabricated immunosensor, interference studies were performed using glucose, ractopamine and ascorbic acid. A 2 ng/mL solution of SAL containing 200 ng/mL of an interfering substance was measured with the immunosensor; the results are shown in Fig. 10b. The variation in the current due to the presence of interfering substances was less than 4% com-
Fig. 10. (a) Calibration curve of the immunosensor for different concentrations of SAL. Error bar = RSD (n = 5). (b) Amperometric response of the immunosensor to 2 ng/mL SAL (1), 2 ng/mL SAL + 200 ng/mL glucose (2), 2 ng/mL SAL + 200 ng/mL ractopamine (3), 2 ng/mL SAL + 200 ng/mL ascorbic acid (4). Error bar = RSD (n = 5).
Z. Cui et al. / Electrochimica Acta 69 (2012) 79–85
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Table 2 The results of the SAL determination in serum samples. Content of SAL in the sample (ng/mL)
The addition content (ng/mL)
The detection content (ng/mL)
RSD (%)
Recovery (%)
0.3
5.0 10.0 15.0
5.5, 5.3, 5.6, 5.7, 5.5 10.2, 10.4, 10.5, 10.1, 10.2 15.0, 15.4, 15.8, 14.5, 15.0
2.4 1.5 3.6
103.6 99.4 98.7
pared to the SAL solution alone, indicating that the selectivity of the immunosensor was acceptable. The stability of the immunosensor was also examined by periodically checking its current response. When the immunosensor was not in use, it was stored in air at 4 ◦ C. The amperometric response of the immunosensor for the detection of 10 ng/mL SAL in 10 mL PBS (pH 7.4) at −0.4 V with the successive addition of 5.0 mmol/L H2 O2 was 53 A. Under the same conditions, the catalytic current of the immunosensor using Pd@SBA-15/Ab2 /BMIM·Br as a label was measured after 2 weeks and after 1 month; the catalytic current decreased approximately 92% and 84%, respectively, from its initial value. This result indicates that the immunosensor has a high stability. Therefore, the developed immunosensor has a good reproducibility, selectivity and stability and could be applied for the detection of real samples.
Environmental Protection Industry of Shandong Province (2010) and the Science and Technology Development Plan Project of Jinan City (201004015). All of the authors express their deepest thanks to their funding sources. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
3.5. Real sample analysis [12]
To demonstrate the practical application of the immunosensor, the detection of SAL in serum samples was performed using standard addition methods, 5 ng/mL, 10 ng/mL and 15 ng/mL SAL solution were added into serum samples, respectively. The average recovery of the immunosensor was 103.6% (n = 5), 99.4% (n = 5) and 98.7% (n = 5), respectively (Table 2). Hence, the developed immunoassay methodology could be satisfactorily applied to the clinical determination of SAL levels in serum samples. 4. Conclusions In conclusion, this paper has reported the development of an immunosensor using the Pd@SBA-15 nanoparticles and BMIM·Br as label for the detection of SAL. This immunosensor can be easily prepared based on a sandwich-type protocol, with the primary antibody immobilized onto the surface of an SDBS-GS-modified electrode. The immunosensor displayed a linear response for the detection of SAL within a wide range (15.0 ng/mL). The proposed immunosensor shows a low detection limit (7 pg/mL), good reproducibility, satisfactory selectivity and acceptable stability. The simplicity of the fabrication procedure and the ultrasensitivity of the immunosensor may provide many potential applications for the detection of SAL in clinical diagnostics. Acknowledgments This study was supported by the Natural Science Foundation of China (Nos. 21075052 and 21175057), the Natural Science Foundation of Shandong Province (Nos. ZR2010BM030 and ZR2010EM063), the Key Subject Research Foundation of Shandong Province (XTD1105), the Science and Technology Key Plan Project of Shandong Province (2010GSF10628), the National Water Pollution Control and Management Technology Major Projects (2008ZX07422-001-5-1), the Special Research and Development
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