Electrochemical immunosensor for salbutamol detection based on CS-Fe3O4-PAMAM-GNPs nanocomposites and HRP-MWCNTs-Ab bioconjugates for signal amplification

Sensors and Actuators B 156 (2011) 71–78

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Electrochemical immunosensor for salbutamol detection based on CS-Fe3 O4 -PAMAM-GNPs nanocomposites and HRP-MWCNTs-Ab bioconjugates for signal amplification Su Liu a , Qing Lin b , Xiuming Zhang b , Xiaorui He b , Xianrong Xing b , Wenjing Lian b , Jiadong Huang c,∗ a

College of Resources and Environment, University of Jinan, Jinan 250022, PR China College of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China c College of Medicine and Life Sciences, University of Jinan, Jinan 250022, PR China b

a r t i c l e

i n f o

Article history: Received 20 January 2011 Received in revised form 23 March 2011 Accepted 30 March 2011 Available online 8 April 2011 Keywords: Electrochemical immunosensor CS-Fe3 O4 -PAMAM-GNPs nanocomposites HRP-MWCNTs-Ab bioconjugates Signal amplification Salbutamol detection

a b s t r a c t An ultrasensitive electrochemical immunosensor based on chitosan-iron oxide-poly(amino-amine) dendrimers-gold nanoparticles (CS-Fe3 O4 -PAMAM-GNPs) nanocomposites and horseradish peroxidasemultiwall carbon nanotubes-antibody (HRP-MWCNTs-Ab) bioconjugates was developed for the detection of salbutamol (SAL). CS-Fe3 O4 -PAMAM-GNPs nanocomposites as immobilization matrix were used to enhance the electroactivity and stability of the electrode. HRP-MWCNTs-Ab bioconjugates as label were used to improve catalytic activity for hydrogen reduction of the electrode. Under the optimized conditions, a calibration plot for SAL was obtained with a linear range between 0.11 ng/mL and 1061 ng/mL (r = 0.9984). The detection limit was 0.06 ng/mL. The immunosensor was examined in real samples for the analysis of SAL. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Salbutamol (SAL) is one of the ␤2 -agonists, which has been used in human and veterinary medicine for the treatment of pulmonary disorders. It is also extensively misused in farm animals, where high doses give rise to a preferential muscle to fat ratio, resulting in financial gain for the farmer. The remained SAL in animal food will do harm to people’s health [1,2], leading the most countries to forbid the use of some or all of these substances in animals raised for human consumption [3,4]. However, SAL is still used illegally in several countries, while the extent of this illicit use is uncertain. Therefore, the ultrasensitive method to monitor therapeutic use as well as to control the illegal use of SAL is essential. Among the currently available analytical methods, electrochemical immunosensors have become the predominant analytical technique for the quantitative detection of biomolecules due to their high sensitivity, low cost, fast analysis and ease of miniaturization [5–7]. In the process of immunosensor design, immobilization of biomolecules on the sensing electrode surface has been considered to be one of the most important points [8] and so there have been

∗ Corresponding author. Tel.: +86 531 89736122; fax: +86 531 82769122. E-mail address: chm [email protected] (J. Huang). 0925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2011.03.074

many reports about immunosensor based on different immobilization matrix. Chitosan (CS) has been used to construct biosensor due to its excellent film-forming ability, mechanical strength, biocompatibility, nontoxicity and the presence of reactive amino and hydroxyl functional groups for biomolecules immobilization. Considering its relatively poor conductivity, many attempts have been made to improve the disadvantage of CS by combining with carbon nanotubes (CNTs), metal nanoparticles or metal oxide nanoparticles to form nanocomposites for the fabrication of biosensing platform [9]. Iron oxide (Fe3 O4 ) nanoparticles, due to their unique properties including biocompatibility, superparamagnetic property, low toxicity and high electron efficiency, can provide a favourable microenvironment for biomolecules immobilization. CS-Fe3 O4 nanocomposites have recently aroused much interest for the fabrication of biosensor since surface functionalization of magnetic nanoparticles allows the immobilization of biomolecules on their surface by covalent attachment, self-assembly or embedment method [10–13]. In addition, dendrimers are spheroidal or globular nanostructures that are precisely engineered to carry molecules encapsulated in their interior void spaces or attached to the surface. Size, shape and reactivity are determined by generation (shells) and chemical composition of the core, interior branching and surface functionalities [14,15]. The conductivity of dendrimer is not good, which limits its extensive application in the electrochemical biosensors. However, various nanoparticles including Cu, Pt, Pd

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and Au have successfully formed at the surface of the dendrimer or encapsulated in the dendrimer due to the higher generations of dendrimers [16,17]. Metal-dendrimers have good conductivity, which can further amplify the electrochemical signal. In addition, a bioactive enzyme molecule is usually conjugated to an antibody (Ab), and the amount of labeled enzyme is limited on each Ab, which may affect the sensitivity of the immunosensor [18–20]. To increase the amount of immobilized enzyme, various nanomaterials are used as carriers for constructing sensitive biosensors due to their large specific surface area and good biocompatibility [21–25]. CNTs have been widely used in the preparation of biosensors [26], owing to their high surface area, high electrical conductivity and good chemical stability. In this work, electrochemical immunosensor for salbutamol detection based on CS-Fe3 O4 -PAMAM-GNPs nanocomposites and horseradish peroxidase-multiwall carbon nanotubes-antibody (HRP-MWCNTs-Ab) bioconjugates for signal amplification was developed. Gold nanoparticles (GNPs) with good conductivity and biocompatibility were used for the preparation of GNPspoly(amino-amine) dendrimers (PAMAM) nanocomposites. The CS-Fe3 O4 -PAMAM-GNPs nanocomposites were synthesized to immobilize biomolecules via the large specific area and strong adsorption ability of GNPs, and further enhance the conductibility of CS-Fe3 O4 nanocomposites. MWCNTs with the high surface area, high electrical conductivity and good chemical stability were used for the preparation of HRP-MWCNTs-Ab bioconjugates in order to increase the amount of immobilized HRP. The immunosensor was fabricated by three main steps: firstly, CS-Fe3 O4 -PAMAM-GNPs nanocomposites and HRP-MWCNTs-Ab bioconjugates were synthesized before the experiment. Secondly, CS-Fe3 O4 -PAMAM-GNPs nanocomposites were modified onto the electrode surface in order to immobilize the antigen molecule and enhance the current response of the electrode. Thirdly, a multilabeled HRP-MWCNTs-Ab bioconjugates were used as a second single amplification strategy, and a competitive-type protocol was used to prepare the immunosensor with HRP-MWCNTs-Ab immobilized on the CS-Fe3 O4 -PAMAM-GNPs nanocomposites modified electrode. 2. Materials and methods 2.1. Reagent and materials SAL kit (SAL standard solution and antibody working solution) was obtained from Beijing Wanger Biotechnology Co., Ltd. (China). Cysteamine, chloroauric acid, poly(amino-amine) dendrimers (PAMAM), 1-(3-(dimethylamino)-propyl)-3-ethyl-carbodiimide, hydrochloride (EDC), N-hydroxysulfo-succinimide (NHS), ractopamine and clenbuterol were obtained from Sigma–Aldrich Company (USA). Fe3 O4 nanoparticles were obtained from Aladdin Chemistry Co., Ltd. (China). Horseradish peroxidase (HRP) was obtained from Sangon Biotech Co., Ltd. (China). Bovine serum albumin (BSA), sodium citrate, hydrogen peroxide and chitosan were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). All other chemicals were analytical grade. Double distilled water was used throughout this study. 2.2. Apparatus All electrochemical experiments were carried out using a Model VersaSTAT 3 electrochemical workstation (Princeton Applied Research, USA). Electrochemical measurements were performed using a three-electrode system composed of a platinum foil as auxiliary electrode, an Ag/AgCl as reference electrode and a modified gold electrode as working electrode. Scanning electron microscope

(SEM) images were obtained using field emission SEM (ZEISS, Germany). UV/VIS measurements were carried out using a Lambda 35 UV/VIS Spectrometer (PerkinElmer, USA). 2.3. Preparation of the HRP-MWCNTs-Ab bioconjugate The preparation of HRP-MWCNTs-Ab bioconjugates was carried out according to the previous report [27]. Briefly, 1.5 mg of the functionalized MWCNTs were dispersed in 2 mL pH 7.2 PBS and sonicated for 10 min to obtain a homogeneous dispersion, which indicated that the MWCNTs were well activated with hydrophilic carboxylate groups. This dispersion was mixed with 1 mL mixture including 400 mM 1-(3-(dimethylamino)propyl)-3-ethyl-carbodiimide (EDC) and 100 mM N-hydroxysulfosuccinimide (NHS) under vigorous mechanical stirring for 5 min at room temperature (RT). The resulting mixture was centrifuged at 15,000 rpm for 5 min and the supernatant was discarded. In order to remove excessive EDC and NHS, the above centrifugation procedure was repeated. Then, 150 ␮L Ab (2.5 ␮g mL−1 ) and 150 ␮L HRP (1 mg mL−1 ) were added to the activated MWCNTs and stirred for 6 h at RT. The resulting mixture was centrifuged at 12,000 rpm for 10 min and the supernatant was removed. Washing was crucial to remove free Ab and HRP and was repeated four times. Finally, 1 mL pH 7.2 PBS was added into the bioconjugate precipitate to form a homogeneous dispersion and stored in the refrigerator at 4 ◦ C before use. The synthesis process of HRP-MWCNTs-Ab bioconjugates was shown in Scheme 1A. 2.4. Preparation of PAMAM-GNPs doped chitosan-iron oxide nanocomposites (CS-Fe3 O4 -PAMAM-GNPs) Our approach for the preparation of dendrimer-encapsulated Au metal particles is similar to the previously reported [28]. PAMAMGNPs nanocomposites were prepared as follows: 2.5 mL HAuCl4 solution (0.3 mM) was added into 2.5 mL PAMAM (0.1 mM) solution with vigorous stirring for 20 min. Then, 2.5 mL of sodium citrate (0.1 mM) was incrementally added (with at least 15 min between additions) into the previous solution. When zero valent Au complex was formed, the color changed from yellow to fuchsia. This reaction took over 4 h. To obtain the different sizes of PAMAM-GNPs particles, various volumes of HAuCl4 were added to the same volume of PAMAM together with an excess of reducing agent to make sure that most of the HAuCl4 was reduced to Au. The CS-Fe3 O4 -PAMAM-GNPs nanocomposites were prepared by following a previously reported procedure [29]. Initially, 5 mg Fe3 O4 nanoparticles were added to 5 mL 1 wt% CS aqueous solution with mechanical stirring at RT and a highly dispersed black solution was formed. Subsequently, the PAMAM-GNPs were mixed with the resulting black suspension to form PAMAM-GNPs doped CS-Fe3 O4 nanocomposites. The synthesis process of CS-Fe3 O4 -PAMAM-GNPs nanocomposites was shown in Scheme 1B. 2.5. Preparation of the modified electrode The gold electrode was first polished using fine emery paper and then polished to a mirror finish with 0.3 and 0.05 ␮m alumina slurry. The schematic diagram of the stepwise procedure of the preparation of the immunosensor was shown in Scheme 1C. After it was thoroughly washed ultrasonically in ethanol and double distilled water, 10 ␮L CS-Fe3 O4 -PAMAMGNPs nanocomposites solution was dropped on the activated (0.5% cysteamine) electrode (Scheme 1C(a)) and air-dried for 2 h (Scheme 1C(b)). Subsequently, 20 ␮L 100 ng/mL SAL solution was applied to the surface of CS-Fe3 O4 -PAMAM-GNPs modified electrode and air-dried for 5 h (Scheme 1C(c)). Then, 0.5% HRP was used to block the nonspecific binding sites on the surface

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Scheme 1. (A) The synthesis process of the HRP-MWCNTs-Ab bioconjugates; (B) the synthesis process of the CS-Fe3 O4 -PAMAM-GNPs nanocomposites; (C) schematic illustration of the stepwise immunosensor fabrication process.

of electrode (Scheme 1C(d)). Finally, the SAL/CS-Fe3 O4 -PAMAMGNPs modified electrode was covered with 20 ␮L mixture solution including HRP-MWCNTs-Ab bioconjugates and free SAL to perform the immunocomplex for 40 min (Scheme 1C(e)). Each incubation procedure was performed at RT in order to maintain the immobilization and immune reaction under the same experimental condition.

2.6. Preparation of animal feed and pork samples A 5 g sample of finely swine feed was accurately weighed and 40 mL of freshly prepared phosphate acid–methanol extraction solution (0.2 M) was added. The mixture was shaken vigorously for 20 min and then centrifuged at 3000 rpm for 10 min. Next, the supernatant was diluted with dilution buffer (1:3, v/v) for

S. Liu et al. / Sensors and Actuators B 156 (2011) 71–78

immunoassay determination. The pork sample (5 g) was accurately weighted into a centrifuge tube and 6 mL hydrochloric acid (0.1 M) was added with vortex mixing for 1 min. The preparation was incubated at RT for 30 min. Then, the resulting mixture was centrifuged at 3000 rpm for 10 min and the supernatant was diluted with dilution buffer (1:3, v/v) for further immunoassay determination. 2.7. Measurement protocol During testing, amperometric responses of the immunosensor were recorded using cyclic voltammetry (CV) in pH 7.2 PBS containing H2 O2. The electrochemical measurements were carried out in an unstirred electrochemical cell at RT.

6 5 4

absorbance

74

3 2

0

3. Results and discussion 3.1. Characterization of CS-Fe3 O4 -PAMAM-GNPs nanocomposites

a

1

100

C 200

b

300

400

500

600

700

800

Wave / nm Fig. 1. UV–vis spectra of PAMAM-GNPs (a), PAMAM (b), and HAuCl4 (c).

UV–vis spectroscopy was used to characterize the formation of PAMAM-Au nanocomposites (Fig. 1). An absorption peak of the GNPs was red-shifted at about 590 nm in the PAMAM-GNPs solution (Fig. 1, curve a), confirming the formation of GNPs and PAMAM-GNPs. The absorption peak at 285 nm for the PAMAMGNPs solution was attributed to PAMAM in the nanocomposites since the pure PAMAM solution showed an absorption peak at 290 nm (Fig. 1, curve b). The HAuCl4 solution did not show obvious absorption peak (curve c). The observation of two bands at 590 and 285 nm for the PAMAM-GNPs solution thus indicated the formation of PAMAM-GNPs nanocomposites, which proves that the gold nanoparticles are encapsulated in dendrimer molecules. As shown in Fig. 2, SEM images confirmed that the PAMAM-

GNPs complex formed a layer on the surface of the CS-Fe3 O4 nanocomposites. 3.2. Electrochemical characteristics of the modified electrode The ferricyanide was chosen as a marker to investigate the changes of the electrode behavior by using CV. Fig. 3A was the CVs of Fe(CN)6 3−/4− at the bare gold electrode (curve a), CS-Fe3 O4 GNPs modified electrode (curve b) and CS-Fe3 O4 -PAMAM-GNPs modified electrode (curve c). It was obviously that the current response of CS-Fe3 O4 -PAMAM-GNPs modified electrode became

Fig. 2. SEM aspects of (A) CS-Fe3 O4 nanocomposites and (B) PAMAM-GNPs doped CS-Fe3 O4 nanocomposites.

S. Liu et al. / Sensors and Actuators B 156 (2011) 71–78

40

60

A

20

I/µA

40

c b a

0

c'

-20

-40

-40

-60

-60 0.0

0.2

a'

0.4

0.6

b'

0

-20

-0.2

B

20

I/µA

60

75

-0.2

0.0

E/V

0.2

0.4

0.6

E/V

Fig. 3. A CVs of (a) bare gold electrode, (b) CS-Fe3 O4 -GNPs modifed electrode; (c) CS-Fe3 O4 -PAMAM-GNPs modifed electrode; (B) CVs of (a ) CS-Fe3 O4 -PAMAM-GNPs modified electrode; (b ) SAL/CS-Fe3 O4 -PAMAM-GNPs modified electrode; (c ) HRP-MWCNTs-Ab/SAL/CS-Fe3 O4 -PAMAM-GNPs modified electrode. CV conditions: pH 7.2 PBS solution containing 0.2 M KCl and 5 mM K3 [Fe(CN)6 ]; scan rate, 50 mV/s.

Fig. 4. (A) Effect of the incubation time between the Ab and SAL on peak current; (B) Effect of the substrate (H2 O2 ) concentration on peak current.

largest and increased about 29 ␮A than that of bare electrode due to the good conductivity of PAMAM-GNPs. In addition, CS-Fe3 O4 PAMAM-GNPs modified electrode had better current response compared with that of the CS-Fe3 O4 -GNPs modified electrode. The reason might be the fact that the PAMAM-GNPs display the larger surface area and the better conductivity compared with GNPs due to the three-dimensional network of PAMAM. Fig. 3B was the CVs of Fe(CN)6 3−/4− at the CS-Fe3 O4 -PAMAM-GNPs modified electrode (curve a ), SAL/CS-Fe3 O4 -PAMAM-GNPs modified electrode (curve b ) and HRP-MWCNTs-Ab/SAL/CS-Fe3 O4 -PAMAMGNPs modified electrode (curve c ). As shown in Fig. 3B, when SAL was immobilized on the CS-Fe3 O4 -PAMAM-GNPs modified electrode surface, the peak currents decreased about 20 ␮A (curve b ). When SAL/CS-Fe3 O4 -PAMAM-GNPs modified electrode reacted with HRP-MWCNTs-Ab, the peak current further decreased about 6 ␮A (curve c ). These results illustrated that the SAL and Ag–Ab insulated the electrode and hindered the diffusion of the redox marker toward the electrode surface. And it also showed that the SAL and HRP-MWCNTs-Ab were successfully immobilized on the electrode surface.

3.3. Optimisation of the immunoassay conditions In order to achieve maximum electrochemical response, we studied the effect of Ab incubation time and substrate (H2 O2 ) concentration on the current response. The incubation time between Ab and antigen (Ag) was an important parameter for the immunoassay. The influence of the Ab incubation time was investigated and the results obtained were shown in Fig. 4A. The incubation time was 10, 20, 30, 40 and 50 min, respectively. The current response decreased with the increasing of the Ab

incubation time till 30 min. That was because the formation of insulating immunocomplex (Ab–Ag) hindered the diffusion of the redox marker toward the electrode surface. With further increase in Ab incubation time, the decrease of the current response was nearly not observed. The results suggested that the immunocomplex would not form when Ab incubation time was more than 30 min. Thus, the Ab incubation time of 30 min was selected for the subsequent assays. The effect of substrate concentration was also studied and the results were shown in Fig. 4B. The current response decreased with the increasing of the H2 O2 substrate concentration till 1.2 mM. The reason is that all the enzyme molecules become saturated with substrate, so addition of more substrate does not make much difference. Thus, the substrate concentration of 1.2 mM was used as an optimal condition. 3.4. Detection of salbutamol For amperometric immunosensor, the electrochemical response mainly derives from surface amperometric response by CV. After SAL molecules were immobilized on the surface, they specifically bound to Ab, followed by reaction with HRP-labeled AAb. With the addition of H2 O2 , the catalytic mechanism may be as follows: HRP

2H2 O2 −→2H2 O + O2 CV of electrode in PBS buffer (pH 7.2 PBS) containing 1.2 mM H2 O2 was recorded in the range of −0.6 V and 0.6 V at a scan rate of 50 mV/s. As shown in Fig. 5, the immunosensor displayed reduction at −110 mV in blank PBS. And a dramatic increase of the reduction current was observed after the addition of H2 O2 , demonstrating the high sensitivity of the immunosensor.

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S. Liu et al. / Sensors and Actuators B 156 (2011) 71–78 Table 1 Comparison of the analytical methods for the detection of salbutamol.

0

Analytical method Capillary electrophoresis Ion chromatography Enzyme-linked immunosorbent assay Electrochemical immunosensor

I / µA

-20

a -40

LR (ng/mL)

b

LOD (ng/mL)

Reference

2.39

[30]

3–1000

1.00

[31]

1–80

1.00

[32]

0.11–1061

0.06

This work

16.46–71.7

-60

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

E/V Fig. 5. CV of the electrode in pH 7.2 PBS without (a) and with 1.2 mM H2 O2 (b).

of SAL following the same protocols, and it exhibited a less steep decline within the concentration range of 1.2–143 ng/mL with a correlation coefficient of 0.9947 (Fig. 6b). These results revealed that the CS-Fe3 O4 -PAMAM-GNPs modified electrode for their electrochemical assay possessed the advantages of wider measurement range and higher sensitivity than that of the CS-Fe3 O4 -GNPs electrode. Meanwhile, the linear response range and the detection limit of the proposed immunoassay were compared with those of other SAL analytical methods [30–32] reported as shown in Table 1. The comparative data suggested superiority of the present sensor was over some earlier reported methods. 3.5. Reproducibility, selectivity and stability of the immunosensor To investigate the reproducibility of the immunosensors, intraassay and inter-assay precision were studied. The inter-assay precision of the immunosensor was estimated by determining the SAL levels with the five immunosensors made using the same conditions independently. The relative standard deviations (RSDs) were found to be 4.6%, 4.9% and 4.1% for 1.8, 5.4 and 16.2 ng/mL SAL, respectively. The intra-assay precision was estimated by assaying

16 Fig. 6. Calibration plots for SAL solution with the CS-Fe3 O4 -PAMAM-GNPs modified immunosensor (a) and CS-Fe3 O4 -GNPs modified immunosensor (b) under optimal conditions. SAL standard solution: 0.2 ng/mL, 0.6 ng/mL, 1.8 ng/mL, 5.4 ng/mL, 16.2 ng/mL, 100 ng/mL, and 1000 ng/mL.

I/ µA

The electrochemical detection of SAL was based on direct competitive-type immunoassay, which was controlled by the formation of antigen–antibody complex on the electrode surface. With increasing standard concentration of SAL in the incubation solution, the peak current of the immunosensor after the Ag–Ab reaction showed a decrease. The reason was that the formation of an immunocomplex acted as an inert block layer to hinder the electron transfer. As presented in Fig. 6a, a linear relationship between the response signal and logarithm of SAL concentration was obtained from 0.11 to 1061 ng/mL with a correlation coefficient of 0.9984. The detection limit was estimated to be 0.06 ng/mL at a signal/noise ratio of 3. As controlled experiment, we also fabricated a second immunosensor without PAMAM for the detection

12

8

4

0

a

b

c

d

e

f

Fig. 7. CVs peak currents of the immunosensor for the detection of (a) without salbutamol, (b) salbutamol (10 ng/mL), (c) clenbuterol interference (100 ng/mL), (d) clenbuterol interference (1000 ng/mL), (e) ractopamine interference (100 ng/mL), and (f) ractopamine interference (1000 ng/mL).

Table 2 Recoveries of salbutamol from the spiked samples determined by electrochemical immunosensor. Amount added (ng/mL)

The detection content of spiked swine feed (ng/mL)

1.0 2.0 10 20

0.98 1.85 9.65 208.2

± ± ± ±

0.45 0.32 0.85 0.47

Recovery (%)

The detection content of spiked pork (ng/mL)

98.0 92.5 96.5 104.1

1.02 19.06 10.84 20.76

± ± ± ±

0.35 0.62 0.58 0.73

Recovery (%)

102.0 95.3 108.4 103.8

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three SAL levels for five replicate measurements. The RSDs were found to be 4.0%, 3.8% and 4.2% (n = 5) for 1.8, 5.4 and 16.2 ng/mL SAL solutions, respectively. All results indicated that developed immunosensor had good reproducibility. The selectivity of the immunosensor for SAL was examined against two other non-target compounds, clenbuterol (CLB) and ractopamine (RAC), which potentially co-exist with SAL in biological systems. As shown in Fig. 6, despite in the presence of excess CLB (100 or 1000 ng/mL) and RAC (100 or 1000 ng/mL) in the sample solution, the current responses were not significantly affected (Fig. 7c–f), compared to the controlled experiment (Fig. 7a) in the absence of SAL. However, the current response decreased dramatically with 10 ng/mL SAL in the sample solution (Fig. 7b). Such selectivity reflected the highly specific binding affinity of the antigen–antibody immunoreactions, as well as the minimization of non-specific adsorption by the efficient blocking. It confirmed that the developed immunosensor had excellent selectivity. The stability of the immunosensors was examined by the property of the HRP-MWCNTs-Ab. When the HRP-MWCNTs-Ab bioconjugates were not in use, they were stored in PBS (pH 7.2) at 4 ◦ C. They retained 92.5% of the initial response after a storage period of 7 days indicated that the HRP-MWCNTs-Ab bioconjugates had long storage life. Therefore, the developed immunosensor had long-term stability. 3.6. Accuracy and analysis of real samples To investigate the utility of this immunosensor for real sample analysis, various concentrations of SAL (1.0, 2.0, 10 and 20 ng/mL) were added into the real sample solution and then were analyzed. As shown in Table 2, the mean recoveries of the spiked samples detected by the immunosensor ranged from 92.5% to 108.4%, which was acceptable. It showed that developed immunosensor had high accuracy. 4. Conclusions The immunosensors based on the CS-Fe3 O4 -PAMAM-GNPs nanocomposites as immobilization matrix and the HRP-MWCNTsAb bioconjugates as labels displayed a linear response for detection SAL within a wide range. The proposed biosensor showed low detection limit, excellent selectivity, good reproducibility, longterm stability and high accuracy. More importantly, this method is well suited for the rapid and effective determination of SAL in real samples. Acknowledgments This work was supported by the National Natural Science Foundation of the People’s Republic of China (Nos. 30972056 and 30911140278), the Foundation for Outstanding YoungScientist in Shandong Province (No. BS2009NY001), the Scientific and Technological Development Plan in Shandong Province (No. 2010GNC10960), the Natural Science Foundation of Shandong Province (No. ZR2010DQ025) and the Shandong Province Higher Educational Science and Technology Program (No. J10LB14). References [1] J.H.W. Lau, C.S. Khoo, Determination of clenbuterol, salbutamol, and cimaterol in bovine retina by electrospray ionization-liquid Chromatography-tandem mass spectrometry, J. AOAC Int. 87 (2004) 31–38. [2] D.R. Doerge, M.I. Churchwell, C.L. Holder, L. Rowe, S. Bajic, Detection and confirmation of ␤-agonists in bovine retina using LC–APCI/MS, Anal. Chem. 68 (1996) 1918–1923. [3] H.A. Kuiper, M.Y. Noordam, M.M.H. Dooren-Flipsen, R.R. Schilt, A.H. Roos, Illegal use of ␤-adrenergic agonist: European community, J. Anim. Sci. 76 (1998) 195–207.

77

[4] G.A. Mitchell, G. Dunnavan, Illegal use of beta-adrenergic agonists in the United States, J. Anim. Sci. 76 (1998) 208–211. [5] F. Tsagkogeorgas, M. Ochsenkuhn-Petropoulou, R. Niessner, D. Knopp, Encapsulation of biomolecules for bioanalytical purposes: preparation of diclofenac antibody-doped nanometer-sized silica particles by reverse micelle and sol–gel processing, Anal. Chim. Acta 573 (2006) 133–137. [6] X.D. Su, F.T. Chew, S.F.Y. Li, Piezoelectric quartz crystal based label-free analysis for allergic disease, Biosens. Bioelectron. 15 (2000) 629–639. [7] A.L. Ghindilis, P. Atanasov, M. Wilkins, E. Wilkins, Immunosensors: electrochemical sensing and other engineering approaches, Biosens. Bioelectron. 13 (1998) 113–131. [8] T. Hianik, M. Snejdárková, L. Sokolıíková, E. Meszár, R. Krivánek, V. Tvarózek, et al., Immunosensors based on supported lipid membranes, protein films and liposomes modified by antibodies, Sens. Actuators B Chem. 57 (1999) 201–212. [9] J.H. Lin, W. Qu, S.S. Zhang, Disposable biosensor based on enzyme immobilized on Au-chitosan modified ITO electrode with flow injection amperometric analysis, Anal. Biochem. 36 (2007) 288–293. [10] A. Kaushika, R.J. Khana, P.R. Solanki, P. Pandey, J. Alam, S. Ahmad, et al., Iron oxide nanoparticles–chitosan composite based glucose biosensor, Biosens. Bioelectron. 24 (2008) 676–683. [11] M.S. Lin, H.J. Leu, A Fe3 O4 -based chemical sensor for cathodic determination of hydrogen peroxide, Electroanalysis 17 (2005) 2068–2073. [12] A. Kaushik, P.R. Solanki, A.A. Ansari, S. Ahmad, B.D. Malhotra, Chitosan-iron oxide nanobiocomposite based immunosensor for ochratoxin-A, Electrochem. Commun. 10 (2008) 1364–1368. [13] S.F. Wang, Y.M. Tan, A novel amperometric immunosensor based on magnetic nanoparticles/chitosan composite film for determination of ferritin, Anal. Bioanal. Chem. 387 (2007) 703–708. [14] Y. Zeng, Y. Li, J. Chen, G. Yang, Y. Li, Dendrimers: a mimic natural lightharvesting system, Chem. Asian J. 5 (2010) 992–1005. [15] G. Franc, A.K. Kakkar, Click methodologies: efficient, simple and greener routes to design dendrimers, Chem. Soc. Rev. 39 (2010) 1536–1544. [16] M.Q. Zhao, R.M. Crooks, Homogeneous hydrogenation catalysis with monodisperse dendrimer-encapsulated Pd and Pt nanoparticles, Angew. Chem. Int. Ed. 38 (1999) 364–366. [17] D.P. Tang, J. Tang, B.L. Su, G.N. Chen, Gold nanoparticles-decoratedamineterminated poly(amidoamine)dendrimer for sensitive electrochemical immunoassay of brevetoxins in food samples, Biosens. Bioelectron. 26 (2011) 2090–2096. [18] G.D. Liu, Y.H. Lin, Nanomaterial labels in electrochemical immunosensors and immunoassays, Talanta 74 (2007) 308–317. [19] J. Tang, B.L. Su, D.P. Tang, G.N. Chen, Conductive carbon nanoparticles-based electrochemical immunosensor with enhanced sensitivity for ␣-fetoprotein using irregular-shaped gold nanoparticles-labeled enzyme-linked antibodies as signal improvement, Biosens. Bioelectron. 25 (2010) 2657–2662. [20] Y. Yun, A. Bange, W.R. Heineman, H.B. Halsall, V.N. Shanov, Z.Y. Dong, et al., A nanotube array immunosensor for direct electrochemical detection of antigen–antibody binding, Sens. Actuators B Chem. 123 (2007) 177–182. [21] S.Q. Liu, H.X. Ju, Reagentless glucose biosensor based on direct electron transfer of glucose oxidase immobilized on colloidal gold modified carbon paste electrode, Biosens. Bioelectron. 19 (2003) 177–183. [22] D.P. Tang, R. Yuan, Y.Q. Chai, Biochemical and immunochemical characterization of the antigen–antibody reaction on a non-toxic biomimetic interface immobilized red blood cells of crucian carp and gold nanoparticles, Biosens. Bioelectron. 22 (2007) 1116–1120. [23] S. Pozzi Mucelli, M. Zamuner, M. Tormen, G. Stanta, P. Ugo, Nanoelectrode ensembles as recognition platform for electrochemical immunosensors, Biosens. Bioelectron. 23 (2008) 1900–1903. [24] D.P. Tang, B.L. Su, J. Tang, J.J. Ren, G.N. Chen, Nanoparticle-based sandwich electrochemical immunoassay for carbohydrate antigen 125 with signal enhancement using enzyme-coated nanometer-sized enzyme-doped silica beads, Anal. Chem. 82 (2010) 1527–1534. [25] D.P. Tang, J. Tang, B.L. Su, G.N. Chen, Ultrasensitive electrochemical immunoassay of staphylococcal enterotoxin B in food using enzyme-nanosilica-doped carbon nanotubes for signal amplification, J. Agric. Food Chem. 58 (2010) 10824–10830. [26] J. Wang, Carbon-nanotube based electrochemical biosensors: a review, Electroanalysis 17 (2005) 7–14. [27] R. Malhotra, V. Patel, J.P. Vaqué, J.S. Gutkind, J.F. Rusling, Ultrasensitive electrochemical immunosensor for oral cancer biomarker IL-6 using carbon nanotube forest electrodes and multilabel amplification, Anal. Chem. 82 (2010) 3118–3123. [28] K. Esumi, K. Miyamoto, T. Yoshimura, Comparison of PAMAM-Au and PPI-Au nanocomposites and their catalytic activity for reduction of 4-nitrophenol, J. Colloid Interface Sci. 254 (2002) 402–405. [29] Z.J. Song, R. Yuan, Y.Q. Chai, J.F. Wang, X. Che, Dual amplification strategy for the fabrication of highly sensitive amperometric immunosensor based on nanocomposite functionalized interface, Sens. Actuators B Chem. 145 (2010) 817–825. [30] J. Ouyang, J.L. Duan, W.R.G. Baeyens, J.R. Delanghe, A simple method for the study of salbutamol pharmacokinetics by ion chromatography with direct conductivity detection, Talanta 65 (2005) 1–6. [31] F.S. Felix, M.S.M. Quintino, A.Z. Carvalho, L.H.G. Coelho, C.L. DoLago, L. Angnes, Determination of salbutamol in syrups by capillary electrophoresis with contactless conductivity detection (CE-C4D), J. Pharm. Biomed Anal. 40 (2006) 1288–1292.

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S. Liu et al. / Sensors and Actuators B 156 (2011) 71–78

[32] H.X. Sun, H.L. Ling, L. Wang, H.H. Wang, Y.H. Zhao, R. Dong, Development of ELISA method for residue of salbutamol, Chin. J. Ani. Health Insp. 26 (2009) 44–47.

Biographies Su Liu received her BSc in Biotechnology in 2004 and PhD in Environmental Toxicology in 2009 from Ocean University of China, Qingdao, China. Now she is serving as appointed lecturer in University of Jinan. She has long been engaged in the research fields of Environmental Toxicology and biological monitoring. Qing Lin received her BSc in Food Science and Engineering in 2008 from Harbin University, Harbin, China. She is a master course student in University of Jinan. Her current interests are biosensors and electrochemistry. Xiaorui He received her BSc in Chemical Engineering and Technology Speciality in 2008 from Henan Institute of Science and Technology, Xinxiang, China. She is a

master course student in University of Jinan. Her current interests are biosensors and electrochemistry. Xiuming Zhang received her BSc in Materials Chemistry in 2008 from Inner Mongolia University for the Nationalities, Tongliao, China. She is a master course student in University of Jinan. Her current interests are biosensors and electrochemistry. Xianrong Xing received her BSc in Biology in 2008 from University of Jinan, Jinan, China. She is a master course student in University of Jinan. Her current interests are biosensors and electrochemistry. Wenjing Lian is an undergraduate course student in College of Quan-cheng, University of Jinan. Her current interests are biosensors and electrochemistry. Jiadong Huang received his BSc in Biology in 1996 and MS in Biosensor in 2002 from Shandong Normal University, Jinan, China. He received his PhD (in 2006) in Biosensor from Nankai University, Tianjin, China. Now he is serving as a specially appointed professor in University of Jinan. He has long been engaged in the research fields of biosensors and biological electrochemistry.