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Food Control 42 (2014) 109e115

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Food Control

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Development of an aptasensor for electrochemical detection oftetracycline

Dan Chen a,b, Dongsheng Yao a,b, Chunfang Xie a,c,d, Daling Liu a,c,d,*

a Institute of Microbial Technology, Guangzhou 510632, ChinabNational Engineering Research Center of Genetic Medicine, Guangzhou 510632, ChinacBiological Engineering, Jinan University, Guangzhou 510632, ChinadDepartment of Bio-engineering, Jinan University, Guangzhou 510632, China

a r t i c l e i n f o

Article history:Received 27 September 2013Received in revised form12 January 2014Accepted 13 January 2014Available online 4 February 2014

Keywords:TetracyclineAptamerIsothermal Titration CalorimetryElectrochemicalDetection

* Corresponding author. Institute of Microbial TecChina. Tel.: þ86 (0)20 85228422; fax: þ86 (0)20 852

E-mail addresses: tldl@jnu.edu.cn, dalingliu@263.n

0956-7135/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2014.01.018

a b s t r a c t

Developing the rapid, simple and sensitive biosensor system for tetracycline detection is very importantin food safety. In this paper, we developed a label-free aptasensor for electrochemical detection oftetracycline. The reorganization of tetracycline binding aptamer was confirmed by Isothermal TitrationCalorimetry, Kd ¼ 5.18 � 10�5 mol L�1. According to the electrochemical impendence spectroscopy (EIS)analysis, there was a linear relationship between the log concentration of tetracycline and the chargetransfer resistance (DRet) from 5.0 to 5.0 � 103 ng mL�1 of the tetracycline conc. The detection limit was1.0 ng mL�1 within a detection time of 15 min. The average of assemble rate Q was at 82.4% with adifferential batches’ RSD of 4.6%. The current change of this aptasensor lies within at 8.5% after a storageof 15 days under 4 �C. The result aptasensor had shown a good reproducibility with an acceptablestability in tetracycline detection. The recoveries of TET in spiked milk samples were in the range of 90.0e95.7%.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Tetracyclines (TCs) are a group of broad-spectrum antibioticscontaining four condensed aromatic rings. According to the dif-ference of sources, TCs can be divided into two types: naturaltetracycline (tetracycline, oxytetracycline, chlortetracycline anddemethylation aureomycin) and semisynthetic tetracycline(methacycline, doxycycline and minocycline) (Dai, 2003). Mostcommonly used TCs have oxytetracycline (OTC), doxycycline (DOX)and tetracycline (TET). They have been widely used in animalbreeding industry as antibiotics and growth promoters (Wang,Zhao, Quan, & Chen, 2011). However, their widespread use hasled to TC residues in animal foods, which becomes one of the mostnoticeable problems for food safety. Therefore, it is very importantto develop a rapid, simple, sensitive and specific detection methodto detect TCs in food products.

The common techniques used to detect TCs include microbio-logical assay (Nagel, Molina, & Althaus, 2011), enzyme-linkedimmunoassay (ELISA) (Chafer-Pericas et al., 2010), high-performance thin-layer chromatography (HPTLC) (Meisen et al.,

hnology, Guangzhou 510632,26223.et (D. Liu).

All rights reserved.

2010), capillary electrophoresis (CE) (Ibarra, Rodriguez, Miranda,Vega, & Barrado, 2011), electrochemical (Kim, Kim, Niazi, & Gu,2010), liquid chromatography-mass spectrometry (LC/MS)(Bousova, Senyuva, & Mittendorf, 2013), high performance liquidchromatography (HPLC) (Yang, Yang, & Yan, 2013). Microbiologicalassay is simple with low cost, but time-consuming, and lack ofsensitivity and specificity. ELISA has high specificity, but is time-consuming (multiple incubations and washing steps) with highbackground absorption and susceptible to being influenced bysample matrix. CE has the advantages of rapid separation, analysis,and low cost, but not suitable for small molecular detection. LC/MS,HPTLC and HPLC need expensive and large instrument. The elec-trochemical technologies have gained a great deal of attention,mainly because of their ease in operation, high specificity andsensitivity, easy to miniaturization and amenability to automation.

Wang et al. (2011) developed a tetracycline sensor usingMolecularly Imprinted Polymer Modified Carbon Nanotube-GoldNanoparticles Electrode which has high specificity, but the linearrange is narrow and the sensitivity can not meet the requirements.The linear range is from 0.1 to 40.0 mg mL�1, and the detection limitwas 40.0 ng mL�1. Que et al. (2013) developed a sensitive electro-chemical immunoassay of tetracycline residues by using platinum-catalyzed hydrogen evolution reaction (HER) on an anti-TC anti-body modified immunosensor, which has high sensitivity andspecificity. The detection limit is 6 pg mL�1 However, this

D. Chen et al. / Food Control 42 (2014) 109e115110

immunosensor is complex and has some difficulty for the realsamples analysis in the complex system by using platinum nano-particle based HER (platinum nanoparticle is relatively active in theaqueous solution, and the catalytic property is easily affected by theexternal conditions (Que et al., 2013)).

Aptamers are single-stranded RNA or DNA oligonucleotidesscreened from synthetic DNA/RNA libraries which rely on hydrogenbonding, electrostatic and hydrophobic interactions for high affin-ity, specific recognition to their target. The selection of theaptamers for the specific target is based on the SELEX procedure(systematic evolution of ligands by exponential enrichment)(Ellinglon & Szostak, 1990; Tuerk & Gold, 1990). Comparing totraditional immunological and chemical recognition molecules,aptamers have better target versatility, stronger affinity, and higherspecificity. In addition, aptamers are more stable because of theirtolerancewith temperature and other physical conditions (Citartan,Gopinath, Tominaga, Tan, & Tang, 2012; Shangguan et al., 2006).Aptamers are easy to produce through chemical synthesis. And themodification process is simple and the molecule is resistant todenaturation and degradation (Kirby et al., 2004). Moreover,aptamers are taking advantages in small molecular weights, non-toxicity, immunogenicity, and good osmosis in tissue (White,Sullenger, & Rusconi, 2000). So far, aptamers have been widelyused in analytical chemistry, biochemistry, molecular biology,medicine, food, environment and other fields, which provides anew, efficient and fast identification platform for basic research,drug analysis, medical inspection, environmental monitoring andfood safety (Fischer, Tarasow, & Tok, 2007; Lares, Rossi, & Ouellet,2010; Tombelli, Minunni, & Mascini, 2005).

Kim et al. (2010) developed a label-free electrochemical apta-sensor on a screen printed gold electrode for the detection of TETusing square-wave voltammetry (SWV) technique. The detectionlimit is 10.0 nmol L�1 (4.4 ng mL�1), but the stability and repro-ducibility of the method were not discussed. Zhou, Li, Gai, Wang,and Li (2012) developed an electrochemical TET aptasensor withmulti-walled carbon nanotubes (MWCNTs) modification, moni-tored by cyclic voltammetry (CV) and differential pulse voltam-metry (DPV). The detection limit is 5.0 nmol L�1 (2.2 ng mL�1).However, these aptasensor is relatively complex and needs 30 minfor reaction. At present, more attentions have focused on theresearch of TET aptasensor.

In this work, we developed a simple label-free electrochemicalaptasensor for the specific detection of TET using TET-bindingaptamer, which was screened by Isothermal Titration Calorimetry,as bio-recognizer. The interaction between TET and aptamer wasinvestigated by the electrochemical probe of ferricyanide andmonitored by electrochemical impedance spectroscopy (EIS).

2. Materials and methods

2.1. Reagents and apparatus

Tetracycline (purity: 98%) was purchased from Sigma Company(St. Louis, MO, USA). Single-strand DNA library of random sequencesynthesized by Sangon Biotech Company (Shanghai, China). Allother chemicals were of analytical grade, purchased fromGuangzhou Chemical Reagent Company (Guangdong, China).All solutions were prepared with ultrapure water (resistivity:18.2 MU/cm).

The ITC experiment was carried out on a MicroCal� Auto iTC200system (GE Corporation, USA). AFM was performed using Multi-mode AFM (Nanoscope III, Thermo Co., USA). Electrochemicalanalysis was performed at room temperature using an electro-chemical analyzer Epsilon Autolab (BAS Corporation, USA) for CVand DPV analysis, CHI660C electrochemical workstation (Shanghai

Chenhua Instrument Corporation, China) for EIS analysis. Allexperiments were carried out by using a conventional three-electrode system which was consisted of an aptamer modifiedgold electrode or gold electrode (2 mm) as the working electrode, aplatinum wire as the auxiliary electrode, and a Ag/AgCl referenceelectrode. The impedance measurements were performed in thepresence of a K3[Fe(CN)6]/K4[Fe(CN)6] (1:1, 5 mmol L�1) mixture, ina solution that contained KCl (0.1 mol L�1). They were recorded byapplying a potential equivalent to that of the open circuit (OCP),0.22 V (vs. Ag/AgCl) over the frequency range of 0.1 Hze105 Hz. TheDPV measurements were performed in 80 mmol L�1 Na2HPO4e

C6H10O8 (citric acid) buffer, pH 7.0, containing 50 mmol L�1 NaCland 2 mmol L�1 K4[Fe(CN)6] in the potential range from þ0.3 Vto þ0.7 V with a pulse amplitude of 50 mV.

2.2. Preparation of aptamer

The 13 bp single-strand DNA with random sequence were syn-thesized, respectively. After dissolved this single-strand DNA intoan 8 mmol L�1 Na2HPO4eC6H10O8 (citric acid) buffer (pH 5.0),respectively, which contained 1molL�1 NaCl, a 0.2 mmolL�1 single-strand DNA solution was obtained as titrant solution. With thesame buffer, the TET solution of 5.0 � 10�3 mmol L�1 was preparedas ligand solution. Prior to use, the buffer, titrant solution andligand solution were degassed for 30 min. Afterwards, MicroCal�Auto iTC200 was used to analyze the affinity between single-strandDNA and TET. According to the affinity analysis, a single-strandDNA, which has a high affinity to TET was screened and namedAP. Finally, a fixed groups (50-NH2e(CH2)6e) was introduced intothe 50 end of AP, which was used as bio-recognizer to developaptasensor. The Auto iTC200 running parameters: cell temperature25 �C, number of injections 20, reference power 5 mCal/s, the vol-ume (mL) of titrant 2, stirring speed 750 rpm, duration time 4 s (thetime that the instrument should take to inject), injection spacingtime 180 s, filter period 5 s.

2.3. Fabrication of aptamer-based electrochemical sensor

The gold electrode was sequentially polished using 1.0 mm,0.3 mm, 0.05 mm alunima slurry, and then sonicated in ultrapurewater for 5 min. After that, the gold electrode was immersed inPiranha solution [30% H2O2/98% H2SO4 (3:7, v/v)] for 10 min andwashed twice with ultrapure water and ethanol for 5 min,respectively. After washing, EIS responses of the gold electrodewere recorded.

After the analysis of EIS, the active gold electrode was rinsedwith ultrapure water and immersed into 2 mmol L�1 AP solutioncontaining 1 mol L�1 NaCl to self-assemble for 24 h at 4 �C. At thispoint, the AP modified gold electrode was obtained. To remove thenon-fixed AP, the modified electrode was rinsed with ultrapurewater after incubated in AP solution. After washing, EIS responsesof the modified electrode were recorded.

2.4. Electrochemical measurements

The prepared aptasensor was incubated in 1.0 ng mL�1 con-centration of TET solution at room temperature for 15 min. Afterwashingwith ultrapurewater to remove the nonspecific combiningof TET, EIS responses of the aptasensor were recorded.

In the same way, the prepared aptasensor was sequentiallyincubated in 5.0 ng mL�1, 10.0 ng mL�1, 50.0 ng mL�1,1.0 � 102 ng mL�1, 5.0 � 102 ng mL�1, 1.0 � 103 ng mL�1 and5.0 � 103 ng mL�1 concentration of TET solution at room temper-ature for 15 min. After washing with ultrapure water to remove thenonspecific combining of TET, EIS responses of the aptasensor were

D. Chen et al. / Food Control 42 (2014) 109e115 111

recorded. To evaluate the sensitivity of aptasensor, the relationshipbetween concentration of TET and electron transfer resistance (Ret)was analyzed.

2.5. Optimization the performance of aptasensor

The different electrolytes of pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 wereprepared in a solution containing 20 mmol L�1 Na2HPO4eC6H10O8(citric acid) buffer, 50 mmol L�1 NaCl and 2 mmol L�1 K4[Fe(CN)6].The prepared aptasensor was sequentially immersed in the elec-trolyte solutions to record the DPV responses in the evaluation ofpH effect to the peak current (Ip).

The prepared aptasensor was immersed in 10.0 ng mL�1 TETsolution and sequentially incubated in 20 �C, 25 �C, 30 �C, 35 �C,40 �C, 45 �C, 50 �C for 15 min, respectively. After washing withultrapure water to remove the nonspecific combining of TET, DPVresponses of the aptasensor were recorded. To evaluate the influ-ence of temperature (T), the relationship between the peak current(Ip) and T was analyzed.

2.6. Detection of TET in food samples

Three concentrations of TET in spiked milk were analyzed.Samples were prepared as follows: firstly, aliquots of 1 mL of milksamples spiked with TET at 60 mg, 16 mg and 6 mg, respectively, werediluted with 5 mL 0.1 mol L�1 C6H10O8eNa2HPO4 containing0.1 mol L�1 Na2EDTA and centrifugated at 8000 rpm for 10 min at4 �C. After the centrifugation, casein was separated from the milkserum. After filtered through filter paper, 3 mL of the milk serumwas transferred to 1cc/30 mg Waters Oasis HLB cartridge (Waters,Milford, Massachusetts) which was previously conditioned with1 mL of methanol and 1 mL of DI water. The cartridge was thenwashed with 1 mL ddH2O and 1 mL 5% (v/v) methanol aqueoussolution. The analytes were eluted with 1 mL 10% (v/v) methanoleethyl acetate solution. The eluate was evaporated to dryness with anitrogen stream and the dried residue was redissolved in 1 mLddH2O. After a 100-fold dilution, the sample was ready for elec-trochemical analysis.

3. Results and discussion

3.1. Preparation of aptamer

In this study, Isothermal Titration Calorimetry was used toevaluate the affinity between the single-strand DNA and the TET,and to screen the DNA that has high affinity to TET. Such DNA wasnamed as AP which was later used as bio-recognizer to develop

Fig. 1. The diagram of iTC result. A: the raw date plot of titration

biosensor. The length of AP was 13 base-pairs with a molecularweight of 3.95 KDa. The percentage of guanine and cytosine (GC)was 61.54, and the dissociation constant (Kd) to TET was5.18 � 10�5 mol L�1. Fig. 1 shows the results of iTC experiment.According to Fig. 1, TET binding with AP was an endothermic pro-cess. The enthalpy change (DH) wasþ1.30� 105 cal/mol, suggestingthat some intramolecular hydrogen-bond was broken when TETboundwith AP. It also indicated that AP self-folded into a high-leverstructure in the buffer before titration. According to the formula:DG ¼ RT ln Kd, DG was equal to �5.84 � 103 cal/mol. Since DG wasnegative, which suggests TET binding with AP was a spontaneousreaction. For small molecular targets, they tend to bury within thebinding pockets of aptamer structure and often be assayed usingthe single-site binding configuration (Song, Wang, Li, Zhao, & Fan,2008). Thus, we surmise that TET molecule was inserted into theself-folded AP structure to binding with AP, as Fig. 3 shows.

3.2. Atomic force microscope (AFM)

In order to characterize the surface of electrode and to investi-gate the orientation change of AP when binding with TET, AtomicForce Microscope (AFM) images of AP modified gold electrode (Au-AP) and Au-AP-TET (Au-AP incubationwith TET) were analyzed, theresult was shown in Fig. 2. The depth of AP on the surface ofelectrode was range from 0.3 to 3.5 nm, the pitch diameter of DNAdouble helix was 2 nm, indicating that AP was lay or reclined on thesurface. After incubating aptasensor with TET for 15 min, the depthof AP increased about 5.5 nm, equal to the height of 16 base pairs inDNA double helix, indicating that the orientation of AP changedfrom lying flat or reclining to upright after binding with TET.

3.3. Sensing mechanisms of the aptasensor

The aptasensor was constructed by covalently attaching anamino-modified AP to an activated gold electrode. The ferricyanidesolution was used as an electrochemical indicator to generateelectron flow between bulk solution and work electrode (Fig. 3).According to the results of iTC experiment, AP was at a high-levelstructure of double-stranded form on the surface of electrode. Inthe absence of TET, the nitrogen moieties within the nucleotidebases prompted AP nonspecifically bound to the gold electrodesurface, and formed a disordered orientation (Herne & Tarlov,1997). The negative charge of AP rejected the anionic redox probe[Fe(CN)6]3�/4� in the bulk solution and hindered the electron-transfer of [Fe(CN)6]3�/4�, which led to increased electron transferresistance (Ret). The TET molecules were inserted into the double-stranded area of AP which resulted in the spatial structure of

, B: the fitting curve of the time integral of the peak yields.

Fig. 2. AFM images of AP and APeTET deposited onto Auemica. After deposition onto Auemica, the samples were rinsed with ultrapure water and nitrogen dried. The images wereacquired in air with Multimode AFM (Nanoscope III) operating in tapping mode. A: AFM image of Au-AP; B: AFM image of Au-AP-TET.

D. Chen et al. / Food Control 42 (2014) 109e115112

high-level structure with further extension which forced AP tochange from lying flat or reclining into upright (Fig. 3), whichinduced the electron-transfer of [Fe(CN)6]3�/4� between bulk so-lution and work electrode with a detectable signal response.

3.4. EIS analysis

Electrochemical impedance spectroscopy (EIS) was a powerfultechnique to characterize the interface properties of the modifiedelectrode. Fig. 4(A) shows the variation tendency of aptasensorresponding to the increased concentrations of TET. As shown inFig. 4(A), increased concentrations corresponded to the increasedresponses at concentrations of 1.0e5.0 ng/mL and decreased re-sponses at concentrations of 5.0 to 5.0 � 103 ng/mL, respectively.This might be due to that the orientation of AP on the surface ofelectrode was changed from lying flat or reclining to upright afterTET bound with AP. The upright orientation of APeTET compoundwas able to reduce the electron transfer resistance (Ret) and in-crease the conductivity of [Fe(CN)6]3�/4 of the gold electrode.

Figs. 4(B) and 5 showed the Nyquist plots of aptasensorresponding to the concentration change of TET. The semicirclediameter at higher frequencies correlated to the electron transferresistance (Ret). The linear part at lower frequencies associatedwith the Warburg resistance of diffusion process (Baranski,Krogulec, Nelson, & Norouzi, 1998). As the figure shown Nyquistplot of bare Au was a straight line. After AP was cast on the surfaceof Au electrode, the Ret increased dramatically (1316 U). Retdecreased after incubating of this aptasensor with various con-centration of TETover the range of 5.0 to 5.0� 103 ngmL�1. The plotof DRet (RetieRet0) versus the logarithmic value of TET concen-trationwas linear over the interval of 5.0 to 5.0� 103 ngmL�1 (insetplot of Fig. 5, [DRet (KU) ¼ �0.32 lg C (ng mL�1) þ 0.67,R2 ¼ 0.9879]), where Ret0 and Reti refer to the electron transferresistance of Au-AP and Au-AP after incubating with the varyingconcentrations of TET solution, respectively. Limit of Quantitation

Fig. 3. The schematic diagram of the electrochemical aptasensor detecting TET.

(LOQ) was 1.0 ng/mL (3S/N) and significantly lower than theMaximum Residue Limit of TET (6.0 � 102 ng mL�1, in kiyney) (TheBulletin No. 235, 2002).

3.5. Effect of solution pH

The electrolytes at various pH influenced the performance ofaptasensor which was related to the charge of AP, directly affectedthe current response of the aptasensor. As shown in Fig. 6(A), thepeak current (Ip) decreased with a linear relationship over therange of pH 3.0 to pH 8.0. The linear regression equation was ︱Ip︱(mA) ¼ �1.8 pH þ 17.7 with R2 ¼ 0.9512. The changes of Ip indi-rectly reflected the changing of AP charge formed on the electrodeinterface. As pH increased, the more negative charge of AP formedon the electrode interface, the more repulsion to FeðCNÞ64�.However, the relatively unchanged peak voltage indicated that theAP had good stability on the bare gold electrode.

3.6. Effect of incubation temperature

The incubation temperature was optimized to obtain a highsensitivity of the fabricated electrochemical aptasensor. The effectof incubation temperature on Ip was investigated from 20 �C to50 �C as shown in Fig. 6(B). The results showed that the peak cur-rent beganwith increasing to reach its maximum around 25 �C, andthen decreased as Ip rose further (Fig. 6(B), insert A). There was amaximum value at about 25 �C. Therefore, the incubation of roomtemperature was adopted in this work. The logarithmic value ofDPV Ip (ln I) was linear against to the reciprocal of temperature(1/T) over 30 �Ce50 �C (inset plot B of Fig. 6(B), [ln I(mA) ¼ 3.1 � 103 1/T (K) ¼ 7.8, R2 ¼ 0.9950]).

3.7. The specificity of the aptasensor to TET

In order to evaluate the selectivity of Au-AP to TET in the pres-ence of interferents, chloramphenicol, streptomycin sulphate,ampicillin and hydrochloric acid ractopamine were selected asinterferents in the electrochemical analysis with TET. Firstly, fourinterferents were mixed to a mixture shown in Table 1. The testresults indicated that no signal had changed when Au-AP apta-sensor was incubated with TET in the mixture. Then, when TETwith final concentration of 1.0 ng mL�1 (the Maximum ResidueLimit diluted 600-mole) was added into the mixture, the signalchanged significantly (Fig. 7). These tests indicated that the

Fig. 4. A: the variation of aptasensor responded to the increased concentrations of TET; B: Nyquist plots obtained in the presence of 5.0 mmol L�1 [Fe(CN)6 ]3�/4� as a redox probe,after the aptasensor incubating in TET solutions of various concentrations.

Fig. 5. Nyquist plots was obtained in the presence of 5.0 mmol L�1 [Fe(CN)6 ]3�/4�as a redox probe, after the aptasensor incubating in TET solution with various concentrations. Insetplot shown the dependence of DRet on the log concentration of TET. DRet ¼ Reti e Ret0, where Ret0 and Reti refer to the electron transfer resistance of aptasensor before and afterincubating with various concentrations of TET solution, respectively.

Fig. 6. DPV analysis for the effect of pH and temperature. (A) aptasensor at various pH electrolyte. Inset plot shows the dependence of peak current value (Ip) on the pH. (B)aptasensor after incubating in TET solution at various temperature. Inset plot A shows the dependence of peak current value (Ip) on various temperatures (T). Inset plot B shows thedependence of ln peak current value (ln Ip) on the reciprocal of temperature (1/T).

D. Chen et al. / Food Control 42 (2014) 109e115 113

Table 1The concentration of elements in mixture.

Interferents The concentrationin mixture(ng mL�1)

Maximum residue limit (ng mL�1)(The Bulletin No. 235, 2002)

Tetracycline 1.0 6.0 � 102 (kiyney)a

Chloramphenicol 0.1 Negative (less than 1.0)a

Streptomycinsulphate

20.0 2.0 � 102 (milk)

Ampicillin 1.0 10.0 (milk)Hydrochloric acid

ractopamine3.0 30.0 (beef)

a All in animals.

Table 2Recoveries of TET from spiked milk samples.

Sample Standard value ofTET (ng/mL)

Found(ng/mL)

Recovery(%)

RSD (%)

1 30.0 28.7 95.7 3.12 80.0 72.0 90.0 7.43 3.0 � 102 2.8 � 102 93.3 4.9

D. Chen et al. / Food Control 42 (2014) 109e115114

developed strategy could be used to identify TET with highspecificity.

3.8. Reproducibility and stability of the aptasensor

The binding of TET with AP produced a detectable signal. Themore AP assembled on the bare gold, themore TETwas bound. Thatwas to say the number of AP assembling on the bare gold directlydetermined the detection sensitivity of aptasensor. In order todisplay the effect of self-assemble of AP intuitively and reflect thereproducibility of aptasensor simultaneously, we set a standard forevaluation called assemble rate Q.

assemble rate Q as : Q ¼ IpðAuÞ � IpðAPÞIpðAuÞ

Where Ip (Au) was the peak current of bare gold electrode, Ip (AP)was the peak current of AP modified electrode. Ip (Au) and Ip (AP)were obtained by DPV scanned.

The reproducibility of the aptasensor was examined by calcu-lating the relative standard deviation (RSD) of assemble rate Q. Theaverage and the RSD of assemble rate Qwas at 82.4% and 4.6% for 10electrodes of 4 batches, respectively. It indicated an acceptablereproducibility of the fabrication protocol.

A storage stability of the aptasensor was studied on a 15-dayperiod. After keeping it in 8 mmol L�1 Na2HPO4eC6H10O8 (citric

Fig. 7. Selective evaluation of aptasensor: bare gold (black); Au-AP (green); mixture of 4 iractopamine (red); mixture of TET and 4 interferents (violet). Inset figure (A) shows electrpretation of the references to colour in this figure legend, the reader is referred to the web

acid) buffer, containing 50mmol L�1 NaCl, pH 5.0 at 4 �C for 15 days,the peak current only shows a decline of 8.5% comparing with theinitial response signal. It demonstrated the good stability ofaptasensor.

3.9. Detection of TET in spiked food samples

In order to examine the ability of this aptasensor for thedetermination of TET in food samples, the recoveries of threedifferent concentrations of TET in spiked milk samples weredetermined using aptasensor. As shown in Table 2, the recoverieswere in the range of 90.0e95.7%, which indicated that the devel-oped aptasensor is adequate for the determination of TET in foodsamples.

4. Conclusions

In this study, we developed a label free electrochemical apta-sensor for specific detection of TET. The bio-recognizer, TET bindingaptamer (Kd ¼ 5.18 � 10�5 mol L�1), was screened out from the 13bp single-strand DNA with random sequence by Isothermal Titra-tion Calorimetry (ITC). The interaction between TET and aptamerwas investigated by the electrochemical probe of ferricyanide andmonitored by electrochemical impedance spectroscopy (EIS). Thevarious experimental factors influencing on the aptasensor’s per-formance are investigated as well.

The aptasensor was sensitive to TET and showed a detectionlimit of 1.0 ng mL�1 with linear range from 5.0 to 5.0 � 103 ng mL�1

nterferents: chloramphenicol, streptomycin sulphate, ampicillin and hydrochloric acidochemical impedance spectra. Inset figure (B) shows cyclic voltammogram. (For inter-version of this article.)

D. Chen et al. / Food Control 42 (2014) 109e115 115

within a detection time of 15 min. The electrochemical aptasensordescribed here offered several advantages:

(1) TET binding aptamer screened by ITC. As a means foraptamer selecting, ITC was different from the existing SELEXtechnique. This method had several advantages such assimplicity of operation with visual results displaying. Afterdata analysis, we were able to obtain such information asdissociation equilibrium constant Kd, and enthalpy changeDH. The affinity of aptamer toTETcould directly display as Kdvalue. Therefore, using ITC to screen TET binding aptamerprovided a high reliability.

(2) Unlike antibody-based immunoassays, the aptasensorresponded directly to the presence of TET without the needfor multiple labeling and washing steps.

(3) The aptasensor in our study had a high sensitive and speci-ficity with fast response. Comparing to the electrochemicalaptasensor for TET reported by Kim et al. (2010) and Zhouet al. (2012), this aptasensor had a higher sensitive, shorterincubation time and simpler steps to develop (a detectionlimit of: 1.0 ng mL�1 in this work, 4.6 ng mL�1 in Kim’s and2.2 ng mL�1 in Zhou’s; incubation time: 15 min in this work,30 min in Kim’s and Zhou’s). Furthermore, this aptasensorshows good stability and reproducibility.

Future work will focus on: 1) improving the stability of aptamerin real samples, such a chemical modification to enhance thenuclease resistance; 2) trying to quantitative detection of TET in avariety of samples.

Acknowledgements

The authors gratefully appreciate the financial support of thisproject by Guangdong province Agricultural Science & TechnologyProgram (No. 2012A020200003) and by Scientific Cultivation andInnovation Fund Program of Jinan University (No. 21612453). Welike also to express sincere acknowledgements to GuangdongProvincial Key Laboratory of Bioengineering Medicine and NationalEngineering Research Center of Genetic Medicine for offering usfacility.

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