Ec

La

b

a

ARRAA

KTAMD

1

tdmm[hheM

tmt[msss

ZT

(

0d

Sensors and Actuators B 162 (2012) 201– 208

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

journa l h o mepage: www.elsev ier .com/ locate /snb

lectrochemical aptasensor for the detection of tetracycline with multi-walledarbon nanotubes amplification

ing Zhoua, Du-Juan Lia, Ling Gaia, Jian-Ping Wanga,∗, Yan-Bin Lia,b

College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, ChinaDepartment of Biological & Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA

r t i c l e i n f o

rticle history:eceived 21 October 2011eceived in revised form 6 December 2011ccepted 19 December 2011vailable online 27 December 2011

a b s t r a c t

Herein, we present a simple electrochemical tetracycline (TET) aptasensor with multi-walled carbonnanotubes (MWCNTs) modification. MWCNTs were dropped on the glassy carbon electrode (GCE) toimmobilize the anti-TET aptamer and to construct the aptasensor. The stepwise assembly process ofthe aptasensor was characterized by cyclic voltammetry. Results demonstrated that the peak currentsof Fe(CN)6

3−/Fe(CN)64− redox pair decreased due to the formation of anti-TET/TET complexes on the

eywords:etracyclineptasensorulti-walled carbon nanotubesifferential pulse voltammetry

modified electrode. The optimization of the loading amount of MWCNTs, the incubating conditions ofaptamer and the detection time of TET were investigated in details. Under optimal conditions, the peakcurrents change obtained by DPV increased linearly with the increasing TET concentrations in the rangefrom 1 × 10−8 M to 5 × 10−5 M with a linear coefficiency of 0.995. This electrochemical aptasensor hasa detection limit of 5 × 10−9 M and was successfully applied to the determination of TET in spiked milk

samples.

. Introduction

Tetracycline (TET) is one of the most common tetracyclines (TCs)hat are widely used for treatment of infectious diseases in fod-er animals. The extensive use of TCs including TET in veterinaryedicine as antibiotics and growth promoters has led to their accu-ulation in diary food products, such as meat [1,2], milk [3], honey

4], eggs/chicken [5], and found to cause serious threats to humanealth [6]. To safeguard human health, the European Union (EU)as set safe maximum residue limits (MRL) [7] for residues of vet-rinary drugs in animal tissues entering the human food chain. TheRL of TET in milk for example is 100 �g/kg.There have been lots of efforts to develop an analysis tool for

he detection of tetracycline antibiotics in food products, phar-aceutical preparations, and water. The most classical techniques

hat have been used so far are HPLC [8,9], capillary electrophoresis10], and chemiluminescence [11]. However, these detection

ethods for TCs are often time-consuming and expensive that lack

pecificity and always need the authentic samples as referencetandards. Several other analysis methods for detection TCs inerum, meat, milk samples, such as colorimetric sensors [12–14],

∗ Corresponding author at: College of Biosystems Engineering and Food Science,hejiang University, 866 Yuhangtang Road, Hangzhou 310058, China.el.: +86 571 88982350; fax: +86 571 88982350.

E-mail addresses: 10913016@zju.edu.cn (L. Zhou), jpwang@zju.edu.cnJ.-P. Wang).

925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2011.12.067

© 2011 Elsevier B.V. All rights reserved.

bioluminescent biosensors [15] and amperometric detectionmethods [16,17], were found to be neither specific nor sensitive(Table 1), due to the high structural similarity of TC derivatives(Fig. 1), which could be a hurdle in their selective recognitions.

In recent decades, numerous biosensors using ssDNA or RNAaptamers as alternatives to antibodies have been reported. Analo-gous to protein-based antibodies, aptamers are nucleic acid-basedmolecules that can be selected to bind to any molecule of choice[18–20]. However, aptamers possess more advantages over anti-bodies such as chemical synthesis, selection through the SELEX(systematic evolution of ligands by exponential enrichment)process, easy modification, high stability, target versatility, easy-to-stock, and resistant to denaturation and degradation. Theseproperties make aptamers ideal candidates as molecule recognitionelements in a wide range of bioassays. A variety of aptamer-basedbiosensors have been illustrated in connection to colorimetric[21,22], fluorescence [23,24], quartz crystal microbalance [25], andelectrochemical systems [26,27], and so on.

To the best of our knowledge, electrochemical aptasensors havebeen rarely applied to detect TET at solid electrodes. Kim et al.[28] have developed a label-free electrochemical aptasensor ona screen printed gold electrode for the detection of TET usingsquare-wave voltammetry (SWV) technique. The linearity wasfound between the SW voltammetric peak currents and TET con-

centrations in the 10 nM to 10 �M range. However, the detectionlimit and the stability and reproducibility of the method were notdiscussed. And it suffered the drawbacks of low sensitivity. Electro-chemical aptasensors have been extensively used in environmental

202 L. Zhou et al. / Sensors and Actuators B 162 (2012) 201– 208

Table 1Analytical characteristics of methods for the detection of tetracyclines.

Tetracyclines Method Linear range Limit of detection Cross-reactivity Ref.

Tetracycline, Amperometric detection 1–500 �M 0.96 �M [17]chlortetracycline, 5–50 0.58oxytetracycline 1–500 0.35Tetracycline, Amperometric detection 2.5–100 �M 0.12 �M [16]chlortetracycline, 1–100 0.31oxytetracycline 2.5–100 0.09Tetracycline, Bioluminescent biosensor 25 ng/g [15]chlortetracycline, 7.5doxycycline 5Oxytetracycline Colorimetric biosensor 0.025–1 �M 0.025 �M [12]Tetracycline Colorimetric biosensor 0.4 ng mL−1 Oxytetracycline

(30%),chlortetracycline(10%),

[13]

Tetracycline Colorimetric biosensor 0.316–316 nM 0.1 nM Oxytetracycline(10%),chlortetracycline

[14]

mlT

dtefpsbsa

fttatvewt

FfCt

onitoring and food safety applications due to their simplicity,ow-cost, specificity. Therefore, electrochemical aptasensors forET detection have great applications in the analysis of food safety.

Carbon nanotubes (CNTs) have been attracted much attentionue to their high chemical stability, high surface area, unique elec-ronic properties and relatively high mechanical properties. Aslectrode materials, CNTs can be used for promoting electron trans-er between the electroactive species and the electrode and canrovide a novel method for fabricating chemical sensors or biosen-ors [29–32]. On the other hand, carboxyl functionalized CNTs haveeen widely used for the construction of electrochemical aptasen-ors [33,34] due to the fact that they can increase the loadingmount of the biomolecules and then amplify the current response.

In this work, we proposed a novel electrochemical aptasensoror TET based on the advantages of MWCNTs. The carboxyl func-ionalized MWCNTs prepared by acid treatment were coated onhe GCE surface to immobilize the anti-TET aptamer. The inter-ction between the anti-TET/TET complexes was investigated byhe electrochemical probe of ferricyanide and monitored by cyclicoltammetry (CV) and differential pulse voltammetry (DPV). Thexperiment variables influencing the aptasensor’s performance

ere investigated in details. The proposed aptasensor was applied

o determine TET in milk samples with satisfactory results.

ig. 1. General structure of tetracyclines (TCs). The inset table shows the uniqueunctional groups on different positions on tetracycline backbone. The letters A, B,, and D, respectively, denote the rings from right to left and the numbers representhe positions of carbon atoms.

(13.7%),

2. Experimental

2.1. Chemicals and reagents

The synthetic anti-TET oligonucleotides (sequence designedby Niazi [35]): 5′-NH2-(CH2)-CGT ACG GAA TTC GCT AGC CCCCCG GCA GGC CAC GGC TTG GGT TGG TCC CAC TGC GCG TGGATC CGA GCT CCA CGT G-3′ were obtained from Takara Biotech-nology (Dalian, China) Co. Ltd. Multi-walled carbon nanotubes(MWCNTs) were purchased from Chengdu Organic Chemicals Co.Ltd., Chinese Academy of Sciences (Chengdu, China). Tetracyclinehydrochloride (TET), doxycycline hydrochloride (DOX), oxytetra-cycline hydrochloride (OTC), diclofenac sodium salt (DCF), andN, N-dimethylformamide (DMF) were purchased from Sigma.Ethanolamine, potassium ferricyanide and ferrocyanide, andTween 20 were purchased from Wenzhou Chemical Reagents Co.Ltd. (Wenzhou, China). Salts for buffer solutions (NaCl, MgCl2,KCl, CaCl2) were purchased from Sinopharm Chemical Reagent Co.Ltd. (Shanghai, China). All the reagents were used without furtherpurification. Ultrapure water from a Milli-Q plus system (MilliporeCo.) was exclusively used throughout the experiments.

2.2. Apparatus

Cyclic voltammetry (CV) and differential pulse voltammetry(DPV) measurements were performed with CHI 440a electro-chemical workstation (Shanghai Chenhua Instrument Corporation,China). All experiments were carried out using a conventionalthree-electrode system with a GCE ( ̊ = 3 mm) as the working elec-trode, a platinum wire as the auxiliary electrode, and a Ag/AgClreference electrode. All potentials were referred to this refer-ence electrode. Scanning electron microscopic (SEM) images wereacquired with a JSM-6380 scanning electron microscope (JEOL Ltd.,Japan).

2.3. Preparation of MWCNTs COOH

The carboxyl functionalized MWCNTs (MWCNTs COOH) wereprepared as reported in the literatures [36,37]. Briefly, 100 mgof MWCNTs were dispersed in 100 mL of a nitric acid and sulfu-ric acid (1:3) solution. The mixture solution was ultrasonically

agitated for 30 min and then refluxed for 4 h at 80 ◦C. After that,the mixture solution was filtered and rinsed with distilled wateruntil the filtrate became neutral. Then the precipitate was driedunder an infrared lamp. That was, the MWCNTs COOH. The

L. Zhou et al. / Sensors and Actuators B 162 (2012) 201– 208 203

ectroc

MM

2

doFaooauodspaTMadftaqccwavrfwrw

2

iT1twcpFmtw

Fig. 2. The mechanism of the el

WCNTs COOH suspension was prepared by dispersing 20 mgWCNTs COOH in 10 mL DMF with the aid of ultrasonic agitation.

.4. Fabrication of the aptasensor

The fabrication processes of the proposed aptasensor wereescribed as follows: the pretreatment of a GCE, the immobilizationf MWCNTs COOH, and the self-assembly of anti-TET aptamer.irstly, A GCE was polished on chamois leather with 1.0, 0.3,nd 0.05 �m Al2O3 slurry successively followed by rinsing thor-ughly with doubly-distilled water until a mirror-like surface wasbtained. Then it was washed ultrasonically in absolute ethanolnd doubly-distilled water for 5 min, respectively, and finally driednder a stream of nitrogen at room temperature. On the sec-nd step, 14 �L of 2 mg mL−1 suspension of MWCNTs COOH wasropped onto a freshly smoothed GCE surface uniformly, and theolvent was then evaporated under an infrared lamp. The pre-ared electrode was thoroughly rinsed, the first with ethanol,nd then with ultrapure water to remove excessive nanotubes.he last step was the self-assembly of anti-TET aptamer onto theWCNTs COOH modified GCE surface. Before use, the anti-TET

ptamer was firstly prepared as follows: the anti-TET aptamer wasenatured by heating at 90 ◦C for 10 min, quickly cooled at 4 ◦Cor 15 min and incubated at 25 ◦C for 5 min to allow renatura-ion of aptamer to attain its most stable conformation, which is

perquisite condition for its binding to target molecule. Subse-uently, the MWCNTs/GCE was immersed in 10 mM PBS (pH = 7.4)ontaining 5 mM NHS and 2 mM EDC in order to activate thearboxyl of MWCNTs COOH. After 1 h, the resulting electrodeas rinsed again and transferred in 30 �L of 2.5 �M anti-TET

ptamer for 3 h to form the sensing interface and then passi-ated by 1 M ethanolamine for 1 h (ethanolamine was used toemove non-specific adsorption on the anti-TET/MWCNTs/GCE sur-ace). Finally, the ethanolamine/anti-TET/MWCNTs/GCE was rinsedith deionized water, followed by drying under a N2 dream. The

esulting electrode was then employed as the aptasensor in thisork.

.5. Electrochemical measurements

The formation of anti-TET/TET complexes was performed bymmersing the prepared aptasensor into binding buffer (20 mMris–HCl, pH 7.6 with 100 mM NaCl, 2 mM MgCl2, 5 mM KCl,

mM CaCl2, 0.02% Tween 20) containing a given TET concen-ration for 30 min at room temperature, followed by thoroughlyashing with binding buffer to remove unbound TET. The electro-

hemical measurements of the modified GCE and bare GCE wereerformed in detection buffer (20 mM Tris–HCl, pH 7.6 with 5 mM

e(CN)6

3−/Fe(CN)64− and 100 mM KCl) by CV and DPV. The DPV

easurements were performed in the potential range from +0.05 Vo +0.4 V with pulse amplitude of 50 mV. The concentrations of TETere quantified by oxidation peak currents at +0.225 V.

hemical aptamer-based sensor.

3. Results and discussion

3.1. Sensing mechanisms of the aptasensor

In this work, the aptasensor was constructed by covalentlyattaching an amino-modified anti-TET aptamer to the MWC-NTs coated GCE surface. The aptamer is selected through anin vitro selection process known as SELEX [35]. It contains 76-basesequence and specifically binds to TET with high affinity (63.6 nM).The MWCNTs are employed as the carriers of the electrochemicalcapture probe to amplify the change of peak currents upon com-bining of TET. The ferricyanide solution is used as a mediator togenerate the electron flow between bulk solution and work elec-trode as shown in Fig. 2. In the absence of TET, the anti-TET aptamerpresents a stable and free configuration on the GCE surface to givea significantly strong Faradaic current. In the presence of TET, theformation of anti-TET/TET complex hinders the electron-transfer ofFe(CN)6

3−/Fe(CN)64−, thus producing a detectable signal [38]. The

electrochemical data analysis was carried out and the decreasingpercent of anodic peak currents before and after the sample treat-ment (�I = (I0 − I1)/I0 × 100%) were measured. Where �I is relativecurrent change, I0 and I1 represent the anodic peak current beforeand after TET treatment, respectively.

3.2. Characterization of the aptasensor

CV is an effective and convenient technique for probing thefeature of the modified electrode surface. Here, CV was used toinvestigate the electrochemical behaviors of Fe(CN)6

3−/Fe(CN)64−

after each assemble step. The redox-label Fe(CN)63−/Fe(CN)6

4−

revealed a reversible CV at the bare GCE with a peak-to-peak sep-aration �Ep of 72 mV (Fig. 3, curve a). After the pretreated GCEwas modified with MWCNTs, the shape of the CV changed dra-matically with a �Ep of 165 mV, which was as twice as the bareGCE (�Ep = 72 mV), but the peak current (Ia = 60 �A) markedly sur-passed that of the bare GCE (Ia = 19 �A) (Fig. 3, curve b), suggestingthat the introduction of MWCNTs played a role in the increase ofthe electroactive surface area and provided the conducting bridgesfor the electron-transfer of Fe(CN)6

3−/Fe(CN)64− [39]. When the

surface of the MWCNTs/GCE was further coated with anti-TET andethanolamine, the peak currents decreased obviously (Fig. 3, curvesc and d), which indicating that the anti-TET and ethanolamineseverely reduced effective area and active sites for electron transfer.In addition, we also characterized the aptasensor after incubatedwith 10 �M TET for 30 min. Compared curve e with curve d, a dra-matically decrease in current was observed (Fig. 3, curve e). Thiswas attributed to the fact that the formation of anti-TET/TET com-plexes hindered the diffusion of Fe(CN)6

3−/Fe(CN)64− towards the

electrode surface.The surface morphology of the MWCNTs/GCE was character-

ized by SEM. The SEM images of a bare GCE and MWCNTs modifiedGCE were shown in Fig. 4(A) and (B), respectively. MWCNTs were

204 L. Zhou et al. / Sensors and Actuators B 162 (2012) 201– 208

Fig. 3. CV obtained in 20 mM Tris–HCl buffer (pH 7.6) containing 5 mMFe(CN)6

3−/Fe(CN)64− and 100 mM KCl at a scan rate of 100 mV s−1 at (a) a bare GCE,

(b) the MWCNTs/GCE, (c) the anti-TET/MWCNTs/GCE, (d) the ethanolamine/anti-TET/MWCNTs/GCE, and (e) the TET/ethanolamine/anti-TET/MWCNTs/GCE.

Fig. 4. SEM images of (A) a bare GCE and (B) the MWCNTs/GCE.

Fig. 5. (A) CV analysis for the effect of MWCNTs loading (a–f: the loading amount ofMWCNTs are 16 �g, 20 �g, 24 �g, 28 �g, 32 �g, 36 �g) obtained in 20 mM Tris–HCl

3− 4−

buffer (pH 7.6) containing 5 mM Fe(CN)6 /Fe(CN)6 and 100 mM KCl at a scan rateof 100 mV s−1, and (B) shows the relationship between the anodic peak currents andMWCNTs qualities.

homogeneously dispersed into the GCE surface and no significantamorphous carbon particles were observed. That meant that theMWCNTs would act as a high conductivity nanowire between theGCE surface and sample solution. From the SEM images we can alsofind that the surface of MWCNTs/GCE was rougher in Fig. 4(B) thanin Fig. 4(A). The higher roughness of the MWCNTs modified GCEwill increase the loading amount of the anti-TET. The modificationof GCE with MWCNTs to give good electrochemical responses with-out destroying co-immobilized biomolecules had been reported byDemirkol and Timur [40] and Wang et al. [41].

3.3. Optimization the performance of aptasensor

3.3.1. Influence of the loading amount of MWCNTsThe loading amount of MWCNTs influenced the performance

of aptasensor. The thickness, compactness of the MWCNTs layer,which was related to the loading amount, directly affected thecurrent response of the aptasensor [42]. Therefore, the MWCNTsloading were optimized from 16 to 32 �g through CV studies (see

Fig. 5(A)). With the increase of loading amount, more MWCNTswere immobilized on the electrode, which led to load more anti-TET aptamer. Fig. 5(B) shows the effect of loading amount on anodicpeak current (Ipa) at MWCNTs modified GCE. It exhibited a rapid

L. Zhou et al. / Sensors and Actuators B 162 (2012) 201– 208 205

er con

iswa

3

tftFttawahoraiiaacaut

3

cotsi

Fig. 6. Optimization of the experimental parameters: effects of (A) aptam

ncrease of current response during 28 �g followed by a muchlower increase over the next 4 �g, revealing that the MWCNTsere saturated on the GCE surface. Therefore, 28 �g was selected

s the optimum loading amount in this study.

.3.2. Influence of the incubating conditions of anti-TETThe incubating conditions of anti-TET involving in concentra-

ion and time were optimized to obtain a high sensitivity of theabricated electrochemical aptasensor. The effect of the concentra-ion of anti-TET on �I was investigated from 500 nM to 5 �M (seeig. 6(A)). The results showed that �I increased from the concentra-ion 500 nM to 2.5 �M to reach the maximum and decreased fromhe concentration 2.5 �M to 5 �M. The mechanism was presumeds follows: at low concentrations, the modified electrode surfaceas not fully occupied by aptamer but closed by ethanolamine. So

small current change was obtained when adding 10 �M TET; atigh concentrations, the anti-TET cannot have a good combinationn the electrode surface due to the steric effect and electrostaticepulsion [43]. Considering the peak current response and anti-TETctivity, we chose the concentration 2.5 �M as optimum exper-ment parameter. As shown in Fig. 6(B), the dependence of TETmmobilization time on the peak current response was studied. Theptasensor was incubated with a constant concentration of 2.5 �Mnti-TET and then 10 �M TET for different time intervals. The peakurrent change (�I) increased with increasing immobilization timend reached a platform at 2.5 h, indicating that the aptamer was sat-rated on the modified GCE surface. Therefore, the immobilizationime of 2.5 h was adopted in this work.

.3.3. Influence of the detection time of TETBeside the effect of MWCNTs loading and the incubating

onditions of anti-TET, it was found that a different detection time

f TET caused a visible difference in the increase of �I. Therefore,he dependence of TET detection time on the increase of �I wastudied to determine the optimum detection time of TET. As shownn Fig. 6(C), the �I increased immediately when 10 �M TET was

centration, (B) aptamer immobilization time, and (C) TET reaction time.

introduced and then tended to stabilize after 30 min, indicating thatthe anti-TET/TET complexes were saturated on the modified GCEsurface. Therefore, 30 min was chosen as the TET detection time.

3.4. DPV measurements of TET

Under the optimal experimental conditions, DPVs for TETdetection were obtained. Fig. 7(A) depicts the DPV profiles forthe ethanolamine/anti-TET/MWCNTs/GCE after reacting with var-ious concentrations of TET ranging from 0.01 �M to 50 �M.From Fig. 7(A), it can be seen that the peak currents decreasedwith increasing concentrations of TET as a consequence of theefficient capture of TET by the aptasensor. Additionally, a cali-bration plot was obtained for TET between 0.01 �M and 50 �M.The linear regression equation was expressed as follows: �I(�A) = 80.601 + 9.3995 log C (M), with a correlation coefficient of0.995. The limit of detection (LOD) of TET was calculated as threetimes the standard deviation for the average measurements ofblank samples (LOD = 3 × RSD/slope) [44]. Therefore, the LOD wasdetermined to be as low as 0.005 �M, lower than that of TET detec-tion using the anti-TET antibody chip [45].

Experiment was also performed without WMCNTs under thesame conditions. Fig. 7(B) showed the calibration plot, which wasobtained by plotting DPV current responses of TET concentrationsin logarithmic coordinates. The current responses based on theMWCNTs modified GCE surface were obviously larger than thebare GCE surface in the concentration range of 10−7–10−3 �M. Thelarger current responses could increase the differentiation betweenthe non-specific interference signal and TET detection signal in thecomplex samples. Thus the specificity of the aptasensor would beimproved. In addition, the detection limit obtained without MWC-NTs (green dots) was 0.015 �M which is ∼3 times less sensitive

than that obtained with the anti-TET/MWCNTs system (red dots).The reason might be as follows: the excellent electrical conduc-tivity of MWCNTs enhanced the charge transport and the uniquephysical and chemical features could increase the surface loading

206 L. Zhou et al. / Sensors and Actuators B 162 (2012) 201– 208

Fig. 7. (A) DPV of the aptasensor incubated with different concentrations of TET(a-f: the concentrations of TET are 5 × 10−5 M, 1 × 10−5 M, 1 × 10−6 M, 1 × 10−7 M,1 × 10−8 M, and 0 M). Inset shows the linear relationship between the anodic peakcurrent changes obtained by DPV and the logarithm of TET concentrations. (B) Thepeak current changes (�I) in the DPV as a function of logarithm of TET concentra-tions over the range from 1 × 10−10 M to 5 × 10−4 M at the anti-TET/MWCNTs/GCE(red dot) and anti-TET/GCE (green dot). Error bars show the standard deviations ofmeasurements taken from three independent experiments. The parameters of DPV:pulse amplitude 50 mV, pulse width 50 ms, pulse period 0.2 s. (For interpretation ofto

dspe

3

lmicsdsctT

Fig. 8. Specificity of the aptasensor to 50 �M TET by comparing it to the inter-fering agents, including two structurally similar tetracycline derivatives (OTC andDOX) and a structurally distinct molecule (DCF) at the same concentration. Errorbars show the standard deviations of measurements taken from three independentexperiments. The DPV conditions were the same as Fig. 7.

Table 2Recoveries of TET from spiked milk samples (n = 3).

Sample Standard valueof TET (M)

Found (M) Recovery (%) Standarddeviation

1 5 × 10−8 4.6 × 10−8 92 0.0922 5 × 10−7 4.4 × 10−7 88 0.084

element was consisted of a label-free ssDNA aptamer. GCE mod-

he references to color in this figure legend, the reader is referred to the web versionf this article.)

ensity (amount) of the anti-TET [29,30]. The data obtained alsouggested that the anti-TET/MWCNTs offer the possibility of a sim-le and realistic biosensor platform to diagnosis of food safety usinglectrochemical methods.

.5. The specificity of the aptasensor

The specificity of the aptasensor plays an important role in ana-yzing complex samples. The effect of possible interferences that

ight interfere with the determination of target analyte TET wasnvestigated. The aptasensor was incubated with 50 �M TET byomparing it to the interfering agents, including two structurallyimilar tetracycline derivatives (OTC and DOX) and a structurallyistinct molecule (DCF) (Fig. 8). It was found that only the TETample gave significant peak current change, while the same con-

entration of the other three chemicals had slight emissions. Theseests indicated that the developed strategy could be used to identifyET with high specificity.

3 5 × 10−6 4.8 × 10−6 96 0.1054 2.5 × 10−5 2.2 × 10−5 88 0.056

3.6. Stability and reproducibility of the aptasensor

A long-time stability of the aptasensor was studied on a 14-dayperiod. After keeping it in refrigerator (4 ◦C) for two weeks, theaptasensor was used to detect the same TET concentration (1 �M).The current change (�I) was 26% before and 22% later. It did notshow an obvious change (lower than 5%), demonstrating that theaptasensor had good stability.

The reproducibility of the aptasensor was examined by deter-mining 10 �M of TET with six detections. The relative standarddeviation was 1% for six independent determinations. The exper-imental results indicated good reproducibility of the fabricationprotocol.

3.7. Preliminary application of the aptasensor

In order to evaluate the feasibility of the aptasensor system forpossible applications, the aptasensor was used for determining therecoveries of four different concentrations of TET in whole milk.The processes of the sample preparation were briefly described asfollows: the milk was firstly diluted 1:10 with the binding bufferand ultracentrifugated at 30,000 × g for 90 min at 4 ◦C. In such away, fat and casein are separated (upper and lower layers) fromthe milk serum (intermediate layer). Then we added TET to the col-lected TET-free serum to a final concentration of 0.05 �M, 0.5 �M,5 �M and 25 �M. As shown in Table 2, the recovery was in the rangeof 88–96%, which indicated that the developed aptasensor could bepreliminarily applied for the determination of TET for routine use.

4. Conclusions

In this paper, we described the development of an electro-chemical aptasensor for the detection of TET. The biorecognition

ified with anti-TET/MWCNTs showed Faradaic current signaturewas consistent with redox potential of Fe(CN)6

3−/Fe(CN)64−. The

binding of TET caused current to decrease, thus providing a basis

Actuat

fa5[eatlmoo

bschutwsTc

A

o

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

L. Zhou et al. / Sensors and

or TET detection. The aptasensor was specific to TET and showed limit of detection of 0.005 �M with linear range extending to0 �M. While traditional ELISA was 5–10 times more sensitive4], the electrochemical aptasensor described here offered sev-ral advantages: (1) unlike antibody-based immunoassays, theptasensor responded directly to the presence of TET withouthe need for multiple labeling/washing steps and (2) the aptamerayer was chemically stable so that the aptasensor may be reused

ultiple times. In addition, further modifications in the geometryr length of aptamer molecules may lead to future developmentf more sensitive TET aptasensors.

Compared to the electrochemical aptasensor for TET reportedy Kim et al. [28], the aptasensor exhibited higher sensitivity andtability. The use of MWCNTs on the GCE surface can increase theonductibility and biocompatibility of the aptasensor describedere [46], thus increased the Faradaic current changes that occurredpon analyte binding. Under the optimal experimental conditions,he present aptasensor displayed long term stability up to twoeeks and was successfully used to detect TET in spiked milk

amples. In future, this method will be useful for the detection ofET in milk samples of bacterial infection cows undergoing TEThemotherapy.

cknowledgement

This research was supported by Science Technology Departmentf Zhejiang Province, China (No. 2011C21062).

eferences

[1] F.K. Muriuki, Tetracycline residue levels in cattle meat from Nairobi salughterhouse in Kenya, J. Vet. Sci. 2 (2001) 97–101.

[2] K. De Wasch, L. Okerman, H. De Brabander, J. Van Hoof, P. De Backer, Detectionof residues of tetracycline antibiotics in pork and chicken meat: correla-tion between results of screening and confirmatory tests, Analyst 123 (1998)2737–2741.

[3] A.L. Pena, C.M. Lino, I.N. Silveira, Determination of oxytetracycline, tetracy-cline, and chlortetracycline in milk by liquid chromatography with postcolumnderivatization and fluorescence detection, J. AOAC Int. 82 (1999) 55–60.

[4] M. Jeon, I.R. Paeng, Quantitative detection of tetracycline residues in honey bya simple sensitive immunoassay, Anal. Chim. Acta 626 (2008) 180–185.

[5] S.M. Croubels, K.E. Vanoosthuyze, C.H. VanPeteghem, Use of metal chelateaffinity chromatography and membrane-based ion-exchange as clean-up pro-cedure for trace residue analysis of tetracyclines in animal tissues and egg, J.Chromatogr. B 690 (1997) 173–179.

[6] M.C.E. Gwee, Can tetracycline-induced fatty liver in pregnancy be attributed tocholine deficiency? Med. Hypotheses 9 (1982) 157–162.

[7] European Commission, Commission Regulation (EU), No. 37/2010 of 22ndDecember, 2009, Off. J. Eur. Union L 15 (2010) 1–72.

[8] L. Monser, F. Darghouth, Rapid liquid chromatographic method for simul-taneous determination of tetracyclines antibiotics and 6-epi-doxycycline inpharmaceutical products using porous graphitic carbon column, J. Pharm.Biomed. Anal. 23 (2000) 353–362.

[9] M. Ng, S.W. Linder, HPLC separation of tetracycline analogues: comparisonstudy of laser-based polarimetric detection with UV detection, J. Chromatogr.Sci. 41 (2003) 460–466.

10] P. Kowalski, Capillary electrophoretic method for the simultaneous determina-tion of tetracycline residues in fish samples, J. Pharm. Biomed. Anal. 47 (2008)487–493.

11] S.Q. Han, E.B. Liu, H. Li, Determination of tetracycline, chlortetracycline andoxytetracycline by flow injection with inhibitory chemiluminescence detectionusing copper(II) as a probe ion, Luminescence 21 (2006) 106–111.

12] Y.S. Kim, J.H. Kim, I.A. Kim, S.J. Lee, J. Jurng, M.B. Gu, A novel colorimetricaptasensor using gold nanoparticle for a highly sensitive and specific detectionof oxytetracycline, Biosens. Bioelectron. 26 (2010) 1644–1649.

13] N.P. Navarro, S. Morais, A. Maquieira, R. Puchades, Synthesis of haptens anddevelopment of a sensitive immunoassay for tetracycline residues applicationto honey samples, Anal. Chim. Acta 594 (2007) 211–218.

14] M. Jeon, J. Kim, K.J. Paeng, S.W. Park, I.R. Paeng, Biotin–avidin mediated compet-itive enzyme-linked immunosorbent assay to detect residues of tetracyclinesin milk, Microchem. J. 88 (2008) 26–31.

15] N.E. Virolainen, M.G. Pikkemaat, J.W.A. Elferink, M.T. Kapp, Rapid detection of

tetracyclines and their 4-epimer derivatives from poultry meat with biolumi-nescent biosensor, J. Agric. Food Chem. 56 (2008) 11065–11070.

16] D. Vega, L. Agui, A. Gonzalez-Cortes, P. Yanez-Sedeno, J.M. Pingarron, Voltam-metry and amperometric detection of tetracyclines at multi-wall carbonnanotube modified electrodes, Anal. Bioanal. Chem. 389 (2007) 951–958.

[

ors B 162 (2012) 201– 208 207

17] P. Masawat, J.M. Slater, The determination of tetracycline residues in food usinga disposable screen-printed gold electrode (SPGE), Sens. Actuators B 149 (2010)110–115.

18] P.L. Sazani, R. Larralde, J.W. Szostak, A small aptamer with strong and specificrecognition of the triphosphate of ATP, J. Am. Chem. Soc. 126 (2004) 8370–8371.

19] W. Yoshida, E. Mochizuki, M. Takase, H. Hasegawa, Y. Morita, H. Yamazaki, K.Sode, K. Ikebukuro, Selection of DNA aptamers against insulin and constructionof an aptameric enzyme subunit for insulin sensing, Biosens. Bioelectron. 24(2009) 1116–1120.

20] R. Joshi, H. Janagama, H.P. Dwivedi, T.M.A.S. Kumar, L.A. Jaykus, J. Schefers, S.Sreevatsan, Selection, characterization, and application of DNA aptamers forthe capture and detection of Salmonella enterica serovars, Mol. Cell. Probes 23(2009) 20–28.

21] C. Yang, Y. Wang, J.L. Marty, X.R. Yang, Aptamer-based colorimetric biosens-ing of Ochratoxin A using unmodified gold nanoparticles indicator, Biosens.Bioelectron. 26 (2011) 2724–2727.

22] J.L. Chávez, W. Lyon, N.K. Loughnane, M.O. Stone, Theophylline detection usingan aptamer and DNA–gold nanoparticle conjugates, Biosens. Bioelectron. 26(2010) 23–28.

23] J. Zhang, L.H. Wang, H. Zhang, F. Boey, S.P. Song, C.H. Fan, Aptamer-based mul-ticolor fluorescent gold nanoprobes for multiplex detection in homogeneoussolution, Small 6 (2010) 201–204.

24] L.F. Sheng, J.T Ren, Y.Q. Miao, J.H. Wang, E.K. Wang, PVP-coated graphene oxidefor selective determination of ochratoxin A via quenching fluorescence of freeaptamer, Biosens. Bioelectron. 26 (2011) 3494–3499.

25] L. Michael, P. Birgit, W. Hans, P. Elke, An aptamer-based quartz crystal proteinbiosensor, Anal. Chem. 74 (2002) 4488–4495.

26] Z.X. Zhang, W. Yang, J. Wang, C. Yang, F. Yang, X.R. Yang, A sensitive impedi-metric thrombin aptasensor based on polyamidoamine dendrimer, Talanta 78(2009) 1240–1245.

27] L. Bonel, J.C. Vidal, P. Duato, J.R. Castillo, An electrochemical competitive biosen-sor for ochratoxin A based on a DNA biotinylated aptamer, Biosens. Bioelectron.26 (2011) 3254–3259.

28] Y.J. Kim, Y.S. Kim, J.H. Niazi, M.B. Gu, Electrochemical aptasensor for tetracyclinedetection, Bioprocess. Biosyst. Eng. 33 (2010) 31–37.

29] W. Zheng, Y.F. Zheng, Gelatin-functionalized carbon nanotubes for the bioelec-trochemistry of hemoglobin, Electronchem. Commun. 9 (2007) 1619–1623.

30] Q. Cao, H. Zhao, Y.M. Yang, Y.J. Hea, N. Ding, J. Wang, Z.J Wu, K.X. Xiang, G.W.Wang, Electrochemical immunosensor for casein based on gold nanoparticlesand poly(l-arginine)/multi-walled carbon nanotubes composite film function-alized interface, Biosens. Bioelectron. 26 (2011) 3469–3474.

31] X.R. Liu, Y. Li, J.B. Zheng, J.C. Zhang, Q.L. Sheng, Carbon nanotube-enhancedelectrochemical aptasensor for the detection of thrombin, Talanta 81 (2010)1619–1624.

32] M.P. Siswana, K.I. Ozoemena, T. Nyokong, Electrocatalysis of asulam on cobaltphthalocyanine modified multi-walled carbon nanotubes immobilized on abasal plane pyrolytic graphite electrode, Electrochim. Acta 52 (2006) 114–122.

33] J.Y. Qu, Y. Shen, X.H. Qu, S.J. Dong, Electrocatalytic reduction of oxygen atmulti-walled carbon nanotubes and cobalt porphyrin modified glassy carbonelectrode, Electroanalysis 16 (2004) 1444–1450.

34] L.J. Bai, R. Yuan, Y.Q. Chai, Y.L. Yuan, L. Mao, Y. Zhuo, Highly sensitive electro-chemical label-free aptasensor based on dual electrocatalytic amplification ofPt-AuNPs and HRP, Analyst 136 (2011) 1840–1845.

35] J.H. Niazi, S.J. Lee, M.B. Gu, Single-stranded, DNA aptamers specific for antibi-otics tetracyclines, Bioorg. Med. Chem. 16 (2008) 7245–7253.

36] S.Y. Niu, M. Zhao, L.Z. Hu, S.S. Zhang, Carbon nanotube-enhanced DNA biosensorfor DNA hybridization detection using rutin-Mn as electrochemical indicator,Sens. Actuators B 135 (2008) 200–205.

37] L.Y. Jiang, R.X. Wang, X.M. Li, L.P. Jiang, G.H. Lu, Electrochemical oxidationbehavior of nitrite on a chitosan-carboxylated multiwall carbon nanotube mod-ified electrode, Electrochem. Commun. 7 (2005) 597–601.

38] Y.S. Kim, J.H. Niazi, M.B. Gu, Specific detection of oxytetracycline using DNAaptamer-immobilized interdigitated array electrode chip, Anal. Chim. Acta 634(2009) 250–254.

39] N.Chauhan, J. Narang, C.S. Pundir, Fabrication of multiwalled carbon nano-tubes/polyaniline modified Au electrode for ascorbic acid determination,Analyst 136 (2011) 1938–1945.

40] D.O. Demirkol, S. Timur, Chitosan matrices modified with carbon nanotubes foruse in mediated microbial biosensing, Microchim. Acta 173 (2011) 537–542.

41] S.G. Wang, Q. Zhang, R.L. Wang, S.F. Yoon, A novel multi-walled carbonnanotube-based biosensor for glucose detection, Biochem. Biophys. Res. Com-mun. 311 (2003) 572–576.

42] J.M. Nugent, K.S.V. Santhanam, A. Rubio, P.M. Ajayan, Fast electron transferkinetics on multiwalled carbon nanotube microbundle electrodes, Nano Lett. 1(2001) 87–91.

43] L.Ying, T. Nazgul, R. Erlan, R. Alexander, Aptamer-based electrochemical biosen-sor for interferon gamma detection, Anal. Chem. 82 (2010) 8131–8136.

44] Y.H. Kim, J.P. Kim, S.J. Han, S.J. Sim, Aptamer biosensor for lable-free detection ofhuman immunoglobulin E based on surface plasmon resonance, Sens. ActuatorsB 139 (2009) 471–475.

45] P. Su, N. Liu, M.X. Zhu, B.A. Ning, M. Liu, Z.H. Yang, X.J. Pan, Z.X. Gao, Simultane-

ous detection of five antibiotics in milk by high-throughput suspension arraytechnology, Talanta 85 (2011) 1160–1165.

46] S. Carrara, V.V. Shumyantseva, A.I. Archakov, B. Samori, Screen-printed elec-trodes based on carbon nanotubes and cytochrome P450scc for highly sensitivecholesterol biosensors, Biosens. Bioelectron. 24 (2008) 148–150.

2 Actuat

B

LEo

DiSf

Lei

Yan-Bin Li obtained his Ph.D. in agricultural engineering from Penn State Universityin 1989. He is a professor of biological engineering at the University of Arkansas. Hisresearch is focused on biosensors for the detection of foodborne pathogens andchemical residues and microbial prediction modeling and simulation.

08 L. Zhou et al. / Sensors and

iographies

ing Zhou is currently a Ph.D. student of Zhejiang University majoring in Biosystemsngineering. Her research interests cover aptamer-based sensors for rapid detectionf antibiotic residues in agricultural products.

u-Juan Li received her Ph.D. in Biosystems Engineering from Zhejiang Universityn 2010. She is currently working at College of Biosystems Engineering and Food

cience, Zhejiang University, as a postdoctor. Her research is focused on biosensorsor rapid detection of bacteria and virus with agriculture, food, and environment.

ing Gai is currently working at college of Biosystems Engineering and Food Sci-nce, Zhejiang University, as an associate professor. Her research is focused onmmunosensors.

ors B 162 (2012) 201– 208

Jian-Ping Wang made his Ph.D. in 1991 at Northeast Agricultural University. He hasbeen working as a professor at the college of biosystems Engineering and Food Sci-ence, Zhejiang University since 2002. His research interests cover immunosensors,aptasensors and molecular simulation for the detection of pathogens, pesticide andantibiotic residues.