AnalyticalMethods

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The Key Lab of Health Chemistry and Mo

Chemistry, Chemical Engineering and M

Suzhou, Jiangsu 215123, PR China. E-m

65880089; Tel: +86 512 65880354

Cite this: Anal. Methods, 2013, 5, 5222

Received 15th June 2013Accepted 22nd July 2013

DOI: 10.1039/c3ay40976g

www.rsc.org/methods

5222 | Anal. Methods, 2013, 5, 5222–

Development of a label-free and reagentless plasmonicimmunosensor for the detection of salbutamol

Yiting Wu, Peipei Dong, Anping Deng and Junwei Di*

A label-free and reagentless immunosensor based on localized surface plasmon resonance (LSPR) sensing

was developed for the detection of salbutamol (Sal). Silver triangular nanoparticles deposited on

transparent indium tin oxide film glass were used as the sensing platform. The immunosensor was

prepared by direct adsorption of antibody against Sal on the surface of silver triangular nanoparticles.

LSPR bands were obtained by a basic UV-vis spectrophotometer in the transmission mode. The analytical

performance of the biosensor for the detection of Sal was examined. Under the optimized conditions, a

calibration plot for Sal was obtained with a linear range between 0.02 mg mL�1 and 0.8 mg mL�1 (r ¼0.991). The detection limit was 0.01 mg mL�1. The proposed biosensor has been used to detect the

concentration of Sal in pig complex feed and pork liver samples. It is believed that the plasmonic

immunosensor is simple, cost-effective and can be easily used for the detection of Sal in cases of its

abuse in animal feed for field analysis.

1 Introduction

Salbutamol (Sal), [1-(4-hydroxy-3-hydroxymethylphenyl)-2-(tbu-tylamino) ethanol], also known as albuterol, is a b2-adrenergicreceptor agonist primarily used in the treatment of bronchialasthma and other forms of allergic airway disease in humanbeings. It also is used as a growth promoter and fatteningagent which reduces fat deposition in cattle, sheep, pigsand poultry.1 However, residues of Sal which accumulate inanimal tissues can cause symptoms of serious poisoning inhumans.2 Therefore, the application of Sal has been banned asa repartitioning agent in meat-producing animals in manycountries.3 It is necessary to develop a quick andsimple method for the detection of Sal in animal feeds andresidues.

Up to now, many analytical methods for the determination ofSal in pharmaceuticals, feeds, animal tissues, and body uidshave been developed. They include spectrophotometry,4,5 high-performance liquid chromatograph (HPLC),6,7 capillary elec-trophoresis,8 electrochemical method,9–11 electrogeneratedchemiluminescence,12 enzyme-linked immunosensor13 etc.Many of these techniques require expensive instrumentation orare time-consuming. On the other hand, visible spectropho-tometry is simple but the sensitivity is poor, and thus it is notsuitable for on-site measurements.

lecular Diagnosis of Suzhou, College of

aterial Science, Soochow University,

ail: djw@suda.edu.cn; Fax: +86 512

5226

Recently, label-free immunosensors based on localizedsurface plasmon resonance (LSPR) have attracted considerableinterest.14–16 The LSPR peak position is highly dependent on thesize, shape, or composition of the nanoparticles, as well as therefractive index of the dielectric medium around them. Therefractive index (RI) change in the environment near metalnanoparticles has been demonstrated to be an effective plat-form for highly sensitive detection techniques. The metalnanoparticles deposited on transparent substrates (mostly glassslides) can be measured by the transmission mode. This LSPRsensing requires a simple experimental setup and is low costbecause the experiments can be reduced to record absorptionspectra in a visible spectrophotometer. The common experi-mental scheme for LSPR sensing includes immobilization ofreceptor molecules on the surface of noble metal nanoparticlesand monitoring variations in the optical response resultingfrom LSPR peak changes for binding of the target specic bio-logical analyte from solution.

In our previous study, a simple, sensitive and specic label-free LSPR immunosensor based on an antibody–antigen reactionwas developed.17 Taking advantage of sensitivity, specicity,rapidity, simplicity and economy, the aim of this study is todevelop an LSPR immunosensor for the rapid detection of Sal ineld analysis. In this work, silver triangular nanoprisms(AgTNPs) were used as an LSPR platform. Antibody against Salwas immobilized on the AgTNP surface and employed as acapture probe for Sal. Thus, a simple label-free LSPR immuno-sensor was fabricated for the detection of Sal (Scheme 1). To ourknowledge, this is the rst report of a label-free LSPR immuno-sensor for the detection of an organic small-molecule drug.

This journal is ª The Royal Society of Chemistry 2013

Scheme 1 Scheme representation of the label-free LSPR immunosensor basedon AgTNPs immobilized on a transparent ITO substrate.

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2 Experimental2.1 Materials and apparatus

ITO transparent glass (1.1 mm of thickness, less than 100 U)was purchased from Suzhou NSG Electronics Co., Ltd. (Suzhou,China). Antibody (Ab) against Sal was prepared in our labora-tory. Briey, the polyclonal antibodies against Sal wereproduced by immunizing New Zealand rabbits using Sal-bovineserum albumin (BSA) conjugate as an immunogen in ourlaboratory. The Sal was chemically modied, so that the Salderivative contained a space arm with a carboxylic group at theend. The Sal derivative was coupled to carrier protein (BSA) toform the immunogen which was injected into rabbits for theproduction of Ab. Tetrachloroauric acid (HAuCl4) and silvernitrate (AgNO3) were obtained from Guoyao Chemical ReagentCo, Ltd., China. All the chemicals were of analytical grade andwere used as received without further purication. All thesolutions were made up with double-distilled deionized water.

Electrodeposition was performed using a CHI 830 electro-chemical workstation with a conventional three-electrodesystem (CH Instruments, Shanghai, China). All the potentialsare cited with respect to a saturated calomel electrode (SCE)reference electrode. Scanning electron microscopy (SEM) wascarried out with an S-4700 instrument (Hitachi, Japan). UV-visspectra were recorded with a TU-2810 spectrophotometer (Bei-jing Purkinje General Instrument Co., Ltd., China).

2.2 Fabrication of label-free LSPR immunosensor

The preparation of AgNPs immobilized on ITO substrate wasdescribed in a previous report.18 Briey, an ITO glass strip (0.6 �3 cm) was cleaned with NH3–H2O (1 : 20), ethanol, and water for10 min consecutively in an ultrasonic bath. The gold seed wasdeposited on the ITO substrate using an RST 5100 electro-chemical work station (Suzhou Risetest Instrument Co., Ltd.,China) with a conventional three-electrode system. The electro-lyte included 0.02 mmol L�1 of HAuCl4 and 0.1 mol L�1 ofphosphate buffer solution (pH 2.0). Electrolysis was performed atan applied potential of �0.9 V for 8 s in N2 saturated solution.

This journal is ª The Royal Society of Chemistry 2013

Then, the strip was dipped in 10 mL of 1% PVP solution. 0.3 mLof 0.01mol L�1 sodium citrate, 1.5 mL of 0.01 mol mL�1 ascorbicacid, and 0.5 mL of 1 mmol L�1 AgNO3 solutions were addedconsecutively with stirring. Next, AgTNPs was grown by placingthe strip in a 30 �C water bath for 12 h. The AgTNPs were char-acterized using an S-4700 scanning electronmicroscope (Hitachi,Japan). Prior to functionalization, the AgTNPs were cleaned withDMSO and water, respectively. Finally, the strips were incubatedin 7 mg mL�1 of Ab and PBS solution (pH 7.0) for 10 h at 4 �C andthen the remaining active sites were blocked with bovine serumalbumin (BSA) for 1 h. Aer rinsing with water, the resultingimmunosensors were stored at 4 �C before use.

2.3 Sensing measurements

The LSPR band of the AgTNPs was recorded using a TU-2810spectrophotometer in the transmission mode (Beijing PurkinjeGeneral Instrument Co., Ltd.). All the measurements werecarried out using a bare ITO glass as a reference. For Saldetection, the immunosensor was rst rinsed thoroughly withwater and dried with nitrogen gas. The LSPR band of theimmunosensor was recorded prior to detection. Then theimmunosensor was incubated in Sal and phosphate buffersolution (pH 7.0) for 30 min at 30 �C. Aer rinsing with waterand drying with nitrogen gas, the LSPR band of the immuno-sensor was monitored again. Thus, the changes of the LSPRpeak band were monitored and were used for measurement ofSal concentration.

2.4 Sample treatments

Pig complex feed was obtained from a local feedstuff market(Suzhou). Pork liver was purchased from a supermarket (Suzhou).The samples were used directly for the experiments. A 3.0 gportion of pig complex feed was ground and then transferred intoa glass tube with a glass stopper. For pork liver, 3.0 g of sample,which was prepared by mincing and homogeneous mixing, wasalso transferred into a glass tube with a glass stopper. Then, 10mL of 0.1 mol L�1 HCl was added into the tube. The sampleswere treated in an ultrasonic vibration system for 0.5 h and keptat room temperature overnight. The samples were ultrasonic-mixed for 15 min, and then centrifuged at 12 000 rpm. The clearsupernatants were collected for analysis.

3 Results and discussion3.1 Characterization of AgTNPs deposited on transparentITO substrate

The growth of AgTNPs on transparent ITO substrate wasmonitored by UV-visible absorption spectrometry and SEM(Fig. 1). As shown in Fig. 1A, the spectrum exhibited two char-acteristic peaks. One strong resonance was located at �700 nm,which was attributed to a dipole plasmon resonance of AgTNPs.The other weak peak was located at �440 nm, representative ofquadrupole plasmon resonance of AgTNPs.18–22 The SEM imagedisplays triangular nanoparticles formed on the ITO substrate(Fig. 1B). These results demonstrated that AgTNPs weresuccessfully grown on the transparent ITO substrate.

Anal. Methods, 2013, 5, 5222–5226 | 5223

Fig. 1 UV-visable absorption spectrum (A) and SEM images (B) of AgTNPs grownon the ITO substrate.

Fig. 2 Calibration curve of the LSPR peak on the refractive index for AgTNPssensing platform. Inset: absorption spectra of the AgTNPs platform in differentsolvents: a, air; b, ethanol; c, cycloexane; d, chloroform, and e, carbontetrachloride.

Fig. 3 LSPR peak wavelength shift of AgTNPs vs. concentration of Ab.

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We examined the ability of the AgTNPs deposited on ITOsubstrate as a sensing platform to transduce changes in thesurrounding refractive index (RI) into the LSPR band. We foundthat the LSPR band of AgTNPs exhibited a red shi in the peakwavelength along with an increase of the medium RI (Fig. 2).Inset in Fig. 2 shows the dependence of the LSPR peak on the RIof the surrounding medium. From the slope of linear t, the RIsensitivity of the sensing platform could be calculated to be251 nm per RIU.

3.2 Preparation and optimization of immunosensor for Saldetection

The principle of the label-free LSPR biosensor for detecting Salis shown in Scheme 1. Ab against Sal was rst immobilized onAgTNPs as the sensor surface by direct adsorption. It couldselectively capture Sal from the aqueous solution. This causedenhancement of the RI value on the sensor surface and resultedin a red-shi of the LSPR peak. Thus, a label-free and reagent-less LSPR immunosensor for Sal with very simple and highselectivity was designed.

The immobilization of Ab on the AgTNP surface is a criticalstep. Since the AgTNPs grown on the ITO substrate were cleanedby DMSO, protein could be directly adsorbed on their surface.The immobilization of Ab on the AgTNPs surface was demon-strated by the changes of the LSPR peak. Fig. 3 shows the red-shi of the AgTNP LSPR peak incubated in Ab solution at 4 �C

5224 | Anal. Methods, 2013, 5, 5222–5226

for 10 h. It was observed that the red-shi of the LSPR peakincreased with an increase of Ab concentration. A plateau wasobtained when the concentration of Ab was over 5 mg mL�1.Therefore, 7 mg mL�1 of Ab was employed in the nextexperiments.

Fig. 4 shows the red-shi of the LSPR peak at differentincubation times in Sal solution at 30 �C. It can be seen that thered-shi of the LSPR peak increased withan increase of incu-bation time. When the incubation time was over 25 min, thepeak shi reached a plateau. In the control experiments, nomarked peak changes were obtained in the LSPR band aerincubation in buffer solution. Therefore, an incubation time of30 min was selected for the detection.

3.3 Calibration curve of the LSPR biosensor

Fig. 5 shows the red-shis of the LSPR immunosensors to theresponse aer incubation in Sal solution. It indicated that a

This journal is ª The Royal Society of Chemistry 2013

Fig. 4 LSPR peak wavelength shift of AgTNPs immobilized Ab vs. incubationtime in 0.5 mg mL�1 of Sal solution at 30 �C.

Fig. 5 Relationship between the red-shift of LSPR peak and the concentrationof Sal.

Table 1 Comparison of some different methods for the determination of Sal

MethodLinerange/mg mL�1

LOD/mg mL�1 Ref.

Spectrophotometry 0.5–5.0 — 5HPLC 0.025–0.3 — 6HPLC–MS 0.01–2 0.01 7Differential pulse voltammetry 0.72–29.3 0.12 9Osteryoung square wavevoltammetry

0.1–2 0.04 10

Capillary electrophoresis 10–50 1.30 8Electrogeneratedchemiluminescence

2.4–23.8 0.1 12

Enzyme-linked immunosensor 8 � 10�5 to 1 4 � 10�5 13LSPR immunosensor 0.02–0.8 0.01 This

work

Table 2 Determination of Sal in real samples (n ¼ 3)

SampleSpiked/mg mL�1

Founded/mg mL�1 Recovery (%)

Pig complex feed 0.2 0.186 93Pork liver 0.2 0.18 90

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good linear relationship was obtained for the determination ofSal concentration in the range of 0.02 to 0.8 mg mL�1, with acorrelation coefficient of 0.991. The detection limit was esti-mated to be 0.01 mg mL�1 at a signal-to-noise ratio of 3.

A comparison of the detection limits and linear ranges for theprevious methods reported for Sal detection and the presentedwork is shown in Table 1. Based on these data, the presentimmunosensor represents a rather high sensitivity and lowdetection limit. However, the sensitivity of the LSPR immuno-sensor was lower than that of the enzyme-linked immunosensor.

3.4 Reproducibility, stability and selectivity of the LSPRbiosensor

Reproducibility of the LSPR immunosensor was very importantfor developing a practical immunosensor. To evaluate thereproducibility of the LSPR immunosensor, ve biosensors werefabricated for the detection of 0.4 mg mL�1 Sal. The relative

This journal is ª The Royal Society of Chemistry 2013

standard deviation (RSD) for the detection was 6.2%. This resultindicated that the immunosensor exhibited good reproducibility.The stability of the immunosensor was also evaluated by exam-ining the response wavelength shi. Aer storing at 4 �C in arefrigerator for 20 days, the response of the immunosensorshowed no marked changes, suggesting good long-term stability.

Selectivity is one of the most important parameters for abiosensor. The possible interference from other species in thedetection of Sal was also tested. Experimental results showedthat 10 mg mL�1 of chloromycetin, 10 mg mL�1 of ractopamine,and 10 mg mL�1 of BSA yielded no signicant changes of peakwavelength shi (less than 1 nm), indicating a good selectivityof the immunosensor. However, 0.5 mg mL�1 of clenbuterolcaused amarked red-shi of the peak wavelength. It is fortunatethat clenbuterol is another b2-adrenergic receptor agonistbanned in meat-producing animals.

3.5 Recovery of Sal from real samples

To investigate the applicability of the proposed immunosensorfor the detection of Sal, the immunosensor was used to estimatethe recovery of Sal from spiked pig complex feed and pork liversamples. Sal was not detected in both samples. The resultsobtained are presented in Table 2. Recovery of Sal from realsamples was 90–93%, which demonstrated that this immuno-sensor can be used for the detection of Sal in animal feedsamples. However, the immunosensor currently is not suffi-ciently sensitive for the analyses of serum and urine samples.

4 Conclusion

A label-free and reagentless immunosensor was developed forthe detection of Sal. This LSPR sensor was based on the use of

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silver nanoparticles as a sensing platform and immunologicaltechnique. It has highly desirable properties such as lowdetection limit, high selectivity, and simple instrumentation forthe quantitative analysis of Sal. This LSPR immunosensor couldbe used for the simple and fast identication of Sal in cases ofits abuse in animal feed for eld analysis.

Acknowledgements

This work was nancially supported by the National NaturalScience Foundation of China (no. 21075086), the Project ofScientic and Technologic Infrastructure of Suzhou(SZS201207) and the Priority Academic Program Developmentof Jiangsu Higher Education Institutions.

References

1 S. Sheu, Y. Lei, Y. Tai, T. Chang and T. Kuo, Anal. Chim. Acta,2009, 654, 148–153.

2 S. E. Libretto, Arch. Toxicol., 1994, 68, 213–216.3 C. Chai, G. Liu, F. Li, X. Liu, B. Yao and L. Wang, Anal. Chim.Acta, 2010, 675, 185–190.

4 G. G. Mohamed, S. M. Khalil, M. A. Zayed and M. A. E. El-Shall, J. Pharm. Biomed. Anal., 2002, 28, 1127–1133.

5 K. Basavaiah and H. C. Prameela, Anal. Bioanal. Chem., 2003,376, 879–883.

6 S. H. R. A. Mazhara and H. Chrystyn, J. Pharm. Biomed. Anal.,2009, 50, 175–182.

7 J. Zhang, Y. Xu, X. Di and M. Wu, J. Chromatogr., B: Anal.Technol. Biomed. Life Sci., 2006, 831, 328–332.

5226 | Anal. Methods, 2013, 5, 5222–5226

8 P. Mikus, I. Valaskova and E. Havraanek, Arch. Pharm., 2005,338, 498–501.

9 Y. Wang, Y. Ni and S. Kokot, Anal. Biochem., 2011, 419, 76–80.10 R. N. Goyal, D. r. Kaur, S. P. Singh and A. K. Pandey, Talanta,

2008, 75, 63–69.11 K. Lin, C. Hong and S. Chen, Sens. Actuators, B, 2013, 177,

428–436.12 C. A. Lindino and L. O. S. Bulh~oes, Talanta, 2007, 72, 1746–

1751.13 J. Huang, Q. Lin, X. Zhang, X. He, X. Xing, W. Lian, M. Zuo

and Q. Zhang, Food Res. Int., 2011, 44, 92–97.14 K. M. Mayer and J. H. Hafner, Chem. Rev., 2011, 111, 3828–

3857.15 B. Sepulveda, P. C. Angelome, L. M. Lechuga and L. M. Liz-

Marzan, Nano Today, 2009, 4, 244–251.16 Y. Shon, H. Y. Choi, M. S. Guerrero and C. Kwon, Plasmonics,

2009, 4, 95–105.17 J. Deng, Y. Song, Y. Wang and J. Di, Biosens. Bioelectron.,

2010, 26, 615–619.18 P. Dong, Y. Wu, W. Guo and J. Di, Plasmonics, DOI: 10.1007/

s11468-013-9574-2.19 R. Jin, Y. Cao, C. A. Mirkin, K. L. Kelly, G. C. Schatz and

J. G. Zheng, Science, 2001, 294, 1901–1903.20 L. J. Sherry, R. Jin, C. A. Mirkin, G. C. Schatz and R. P. Van

Duyne, Nano Lett., 2006, 6, 2060–2065.21 R. Morarescu, F. Trager and F. Hubenthal, Int. J. Circ. Syst.

Signal Pr., 2011, 5, 407–414.22 D. E. Charles, D. Aherne, M. Gara, D. M. Ledwith,

Y. K. Gun'ko, J. M. Kelly, W. J. Blau and M. E. Brennan-Fournet, ACS Nano, 2010, 4, 55–64.

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