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Electrochimica Acta 85 (2012) 131– 138

Contents lists available at SciVerse ScienceDirect

Electrochimica Acta

jou rn al hom epa ge: www.elsev ier .com/ locate /e lec tac ta

ynthesis of cyclodextrin-reduced graphene oxide hybrid nanosheets forensitivity enhanced electrochemical determination of diethylstilbestrol

aban Lu, Shaoxiong Lin, Letao Wang, Xuezhao Shi, Chunming Wang ∗, Yan Zhang ∗∗

ollege of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China

r t i c l e i n f o

rticle history:eceived 18 June 2012eceived in revised form 19 July 2012ccepted 19 July 2012vailable online 10 August 2012

a b s t r a c t

�-Cyclodextrin functionalized reduced graphene oxide hybrid nanosheets (�-CD-RGO) were success-fully prepared by using l-ascorbic acid (l-AA) as the reducing agent under a mild condition and usedas enhanced material for sensitivity determination of diethylstilbestrol (DES). In 0.1 M PBS (pH 7.0), theredox peak currents of DES increased significantly on �-CD-RGO modified glassy carbon electrode (�-CD-RGO/GCE), suggesting that the composite film not only shows excellent electronic properties of RGO

eywords:raphene-Cyclodextrin-Ascorbic acidiethylstilbestroletermination

sheets but also exhibits high supramolecular recognition capability of �-CD. The experimental conditionswere optimized and the kinetic parameters were investigated. Under the optimal conditions, the reduc-tion peak current of DES increased linearly with increasing the concentration in the range from 0.01 to13 �M with the detection limit of 4 nM (S/N = 3). The developed electrochemical sensor exhibited highselectivity, good stability and reproducibility, and also successfully determined DES in milk samples withsatisfactory results.

. Introduction

Diethylstilbestrol (DES) is a synthetic non-steroidal estrogenubstance that is widely used in clinical medicine. The moleculetructure is shown in Fig. 1. The pharmacological and therapeuticffects of DES are the same as natural estrogen. While, in contrasto natural estrogen, DES is more stable and remain in body longer1]. Recently, numerous unfortunate effects have shown that DESs one of the most potential carcinogens [2]. Therefore, the use ofrowth-promoting drugs for fattening livestock has been banned inSA and many European countries [3], yet DES is still illegally abuse

n animal production in pursuit of economical interests. Thus, theetermination of the residual DES is very important.

Several methods have been used for determining DES, suchs, high performance liquid chromatography (HPLC) [4–6], gashromatography–mass spectrometry (GC–MS) [7,8], pressurizedapillary electrochromatography (pCEC) [9], chemiluminescence10] and enzyme-linked immunosorbent assay (ELISA) [11]. How-ver, these techniques are either time-consuming or expensive.

herefore, electrochemical techniques, which are often simple andess expensive, have been used to determine DES [12–16]. How-ver, DES exhibits slow electron transfer at unmodified electrodes,

∗ Corresponding author. Tel.: +86 931 8911895; fax: +86 931 8912582.∗∗ Co-corresponding author. Tel.: +86 931 7971879; fax: +86 931 8912582.

E-mail addresses: wangcm@lzu.edu.cn (C. Wang), zhangyan01@lzu.edu.cnY. Zhang).

013-4686/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.electacta.2012.07.071

© 2012 Elsevier Ltd. All rights reserved.

which lead to low sensitivity for its detection. So, some functionalmaterials have been synthesized to develop sensitive electrochem-ical sensors, while the design of new functional materials to achievesensitive, fast and facile detection of DES remains a challenge.

Graphene, a new two-dimensional (2D) structure consistingof sp2-hybridized carbon, has attracted enormous attention inconstructing electrochemical sensors because of its unique prop-erties, such as good mechanical strength, large specific surfacearea and high conductivity [17–20]. However, due to van derWaals interactions and strong �–� stacking, high-quality graphenetends to agglomerate in water, limiting their further applicationsin designing sensors [21]. This problem has been initially over-come by the attachment of other molecules or polymers, such aspoly(diallyldimethylammonium chloride) [22], DNA [23] or aminoacid [24] onto the graphene nanosheets to improve solubility inaqueous medium. Moreover, functionalized graphene reported inour previous work [25,26] exhibited good conductivity and solubil-ity. Therefore, it is highly desirable and technologically importantto design or introduce appropriate functional groups for effectivelydispersing graphene and meanwhile bringing in enhanced func-tions.

Cyclodextrins (CDs) are oligosaccharides composed of six, seven,or eight glucose units (named �, �, or �-CD, respectively), whichare toroidal in shape with a hydrophilic exterior and a hydropho-

bic internal cavity. CDs and its derivatives can form complexes withmany compounds through various kinds of intermolecular interac-tions (van der Waals force, hydrophobic interaction, electrostaticaffinity, dipole–dipole interaction, and hydrogen bond interaction)

132 D. Lu et al. / Electrochimica A

[tmclwtobCpab

gtut[ws

swCswp

2

2

jP(wrfw

eertU(shiiw

Fig. 1. Chemical structure of DES.

27]. Therefore, the interesting characteristics can enable themo bind selectively various organic, inorganic and biological guest

olecules into their cavities to form stable host–guest inclusionomplexes, showing high molecular selectivity and enantiose-ectivity [28]. Additionally, CDs are environmentally friendly,

ater-soluble, and can improve the solubility and stability of func-ional materials. Furthermore, CDs can be attached on the surfacef reduced graphene oxide (RGO) sheets by the strong hydrogenonding to make graphene more hydrophilic [28]. Based on that,Ds functionalized RGO sheets simultaneously possess the uniqueroperties of RGO sheets (large surface area and high conductivity)nd CDs (high supramolecular recognition and enrichment capa-ility), which can be used as electrochemical sensor.

In most cases, the reductant used in the chemical reduction ofraphene oxide (GO) was usually hydrazine or hydrazine deriva-ives. Unfortunately, they are highly poisonous and dangerouslynstable. While l-ascorbic acid (l-AA), a nontoxic and mild reduc-ant, is an ideal material and naturally employed as a reducing agent29], many reactive oxygen species can be reduced by l-AA intoater. Thus, l-AA was considered as an ideal material to reduce GO

heets in an aqueous solution.In the present work, a simple method for water-phase synthe-

is of �-CD modified RGO reduced by l-AA at room temperatureas demonstrated. Then, an electrochemical sensor based on �-D-RGO modified glassy carbon electrode (GCE) was fabricated forensitive detection of DES. The electrochemical behaviors of DESere studied in detail at the �-CD-RGO/GCE. Moreover, the pre-ared method was applied to determine DES in real milk samples.

. Experimental

.1. Materials and apparatus

Graphite flake (nature, −325 mesh) was from Alfa Aesar (Bei-ing, China). Diethylstilbestrol (DES) was purchased from Ying Faeng Na (Ningbo, China). �-Cyclodextrin (�-CD) and l-ascorbic acidl-AA) were from Sigma. 0.1 M phosphate buffer solution (PBS)as prepared with 0.1 M Na2HPO4 and 0.1 M NaH2PO4. All other

eagents and solvents were of analytical grade and used withouturther purification. All chemicals were prepared with deionizedater purified via Milli-Q unit.

Electrochemical measurements were performed on a CHI 832lectrochemical workstation (CH Instrument, China) with a three-lectrode system consisting of a platinum wire auxiliary, an Ag/AgCleference and a bare or modified GCE ( ̊ = 3 mm) working elec-rode. UV–vis spectra were acquired with a Beijing Puxi TU-1810V-vis spectrometer (Beijing China). Fourier transform infrared

FT-IR) spectra were run on an FT-IR spectrometer (Thermo Matt-on, Madison, WI). Transmission electron microscopy (TEM) and

igh-resolution TEM (HRTEM) were carried out by a JEM1200EX

nstrument (JEOL Tokyo). Raman spectra were conducted on a Ren-shaw inVia Raman microscope equipped with a ×50 objective

ith 514.5 nm diode laser excitation on 1800-line grating. Atomic

cta 85 (2012) 131– 138

force microscopic (AFM) measurements were performed using aNanoscope III MultiMode SPM (Digital Instruments) operating intapping mode.

2.2. Synthesis of ˇ-CD-RGO

GO was prepared from graphite by a modified Hummers method[30]. �-CD-RGO hybrid nanosheets were synthesized according tothe literature [28]. But l-AA was used as the reducing agent instead.Briefly, GO (10 mg) was dispersed in water (20.0 mL) and ultrason-ically treated in a flask for 2 h to yield a yellow-brown dispersion.Subsequently, the homogeneous GO dispersion was mixed with20.0 mL of �-CD aqueous solution (80 mg) followed by stirring forseveral minutes. Then, 100 mg of l-AA was added to the resultingdispersion. The mixture was stirred at room temperature for 48 h.Finally, the resulting stable black dispersion was centrifuged andwashed with water for three times and then dried in vacuum. Thepreparation of RGO sheets was similar with �-CD-RGO except noaddition of �-CD.

2.3. Fabrication of electrochemical sensor

Before modification, the bare GCE was polished to a mirror-likesurface with �-alumina down to 0.05 �m, then cleaned ultrason-ically in deionized water for 2 min and finally cleaned in turn inan ultrasonic cleaner with 1:1 nitric acid, alcohol and deionizedwater. �-CD-RGO composite (0.1 mg mL−1) was obtained by ultra-sonication for 2 h to form a homogeneous mixture. Then, 7 �L of themixture was dropped on the pretreated GCE and dried in a desic-cator. For comparison, the RGO/GCE was prepared with the similarprocedure.

2.4. Electrochemical measurements

The electrochemical properties of DES were measured by cyclicvoltammetry (CV) in a standard three-electrode cell. About 10 mLPBS (pH 7.0) containing an appropriate amount of DES standardsolution was added into the electrochemical cell. The CVs wererecorded in the potential range from −0.20 V to 0.60 V with a scanrate of 50 mV s−1 after accumulating for 200 s under stirring. Thedifferential pulse voltammetry (DPV) was carried out to obtaina calibration curve with the parameters of increment potential,0.004 V; pulse amplitude, 0.05 V; pulse width, 0.05 s; sample width,0.0167 s; pulse period, 0.2 s; quiet time, 2 s. Chronocoulometry wasperformed to determine the electrochemically effective surfaceareas of the bare and modified GCE.

3. Results and discussion

3.1. Characterization of the ˇ-CD-RGO

Scheme 1 shows the procedure for preparing �-CD func-tionalized RGO hybrid nanosheets. Briefly, the homogeneousyellow-brown GO dispersion was mixed with �-CD aqueous solu-tion. After being vigorously stirred for several minutes, l-AA wasadded into the aqueous dispersion. Then the mixture was stirredat room temperature for 48 h, and the stable black dispersion of�-CD-RGO was obtained.

The obtained composite was confirmed by the UV–vis absorp-tion spectrum. The GO dispersion (curve a in Fig. 2A) exhibits astrong absorption peak at 231 nm corresponding to the � → �*

transition of aromatic C C bonds and a shoulder peak at 300 nmwhich is attributed to the n → �* transition of the C O bond [31].After reduced by l-AA, the absorption peak of the GO dispersion at231 nm shifts to 264 nm (curve b), suggesting that GO is reduced

D. Lu et al. / Electrochimica Acta 85 (2012) 131– 138 133

repar

al

R1oaCbd(�7ctaws

CTco

tgb

Fi

Scheme 1. Illustration of the p

nd the electronic conjugation within the RGO is restored upon-AA reduction [32].

The FT-IR spectra of GO, unmodified RGO, �-CD and �-CD-GO are shown in Fig. 2B. GO (curve a) shows a typical peak at724 cm−1 due to the C O stretching, indicating the presence ofxygen functionalities at GO surface [29]. The spectrum of GOlso exhibits the presence of O H (3415 cm−1), C C (1621 cm−1),

O C (1223 cm−1), and C O (1050 cm−1). As can be seen in curve, the characteristic absorption bands of oxide groups decreasedramatically in spectrum of RGO except the C C conjugation1570 cm−1). The spectrum of �-CD-RGO (curve d) exhibited typical-CD absorption features (curve c) of the ring vibrations at 579, 708,57, 944 cm−1 and the C C conjugation of RGO at 1562 cm−1, theoupled C O C stretching/O H bending vibrations at 1152 cm−1,he coupled C O/C C stretching/O-H bending vibrations at 1033nd 1076 cm−1, and CH2 stretching vibrations at 2924 cm−1 [27],hich clearly confirmed that �-CD molecules were attached to the

urface of RGO sheets.The TEM image (Fig. 2C) illustrates the flake-like shapes of �-

D-RGO, which corrugated and scrolled like crumpled silk veils.he HRTEM image shows that the spacing of �-CD-RGO layers islose to 0.34 nm, corresponding to the expected value (0.335 nm)f crystal plane (0 0 2) of graphite.

Raman spectroscopy is one of the most effective techniqueso characterize the ordered and disordered crystal structures ofraphene. G band is usually assigned to the E2g mode of sp2 car-on atoms (usually observed at ∼1575 cm−1), while D band is a

ig. 2. (A) UV–vis absorption spectra of (a) GO and (b) �-CD-RGO. (B) FT-IR spectra of (a) Gmage of �-CD-RGO. (D) Raman spectra of (a) GO and (b) �-CD-RGO. (E) AFM image of (a)

ation procedure of �-CD-RGO.

breathing mode of K-point phonons of A1g symmetry (∼1350 cm−1)[33]. As shown in Fig. 2D, it is obviously seen that GO (curve a) dis-plays a D band at 1347 cm−1 and a G band at 1589 cm−1. While thecorresponding Raman spectrum of �-CD-RGO (curve b) also con-tained both G and D bands at 1598 and 1350 cm−1, respectively. Itis also found that �-CD-RGO shows relative higher intensity of Dto G band than that of GO. These observations suggest a decreasein the average size of the sp2 domains from �-CD-RGO and furtherconfirm the formation of new graphitic domains after the reductionprocess [34].

Fig. 2E is the AFM image of GO and �-CD-RGO. As can be seen, anumber of flake-like nanostructures are obviously found. The thick-ness of the well-exfoliated GO sheets obtained is about 0.99 nm(Fig. 2Ea), matching well with the reported apparent thickness ofsingle-sheet GO [35]. Compared with GO sheets, the thickness of as-prepared �-CD-RGO measured from the height profile was about1.31 nm (Fig. 2Eb). This could be attributed to the presence of the�-CD molecules covered onto both sides of RGO sheets.

3.2. Electrochemical behavior of DES

The electrochemical responses of DES at different electrodeswere studied using CV. As shown in Fig. 3A, no redox peaks

are observed on �-CD-RGO/GCE in blank buffer solution (inset ofFig. 3A), indicating that the �-CD-RGO/GCE is non-electroactive inthe selected potential region. When DES was added into pH 7.0PBS, DES showed rather poor redox current peaks at the bare GCE

O, (b) RGO, (c) �-CD and (d) �-CD-RGO. (C) TEM image of �-CD-RGO. Inset: HRTEM GO and (b) �-CD-RGO.

134 D. Lu et al. / Electrochimica A

Fig. 3. (A) CVs of 10 �M DES on the (a) bare GCE, (b) RGO/GCE, and (c) �-CD-RGO/GCE in 0.1 M PBS (pH = 7.0) at scan rate of 50 mV s−1. Inset: the CV of�-CD-RGO/GCE in 0.1 M PBS (pH = 7.0) without DES. (B) CVs of (a) the first scana�

(leigtCswcRloioetnecei

aS

−1

nd (b) the following scan after 200 s accumulation under stirring of 10 �M DES on-CD-RGO/GCE in 0.1 M PBS (pH = 7.0) at scan rate of 50 mV s−1.

curve a) within the potential window from −0.20 to 0.60 V, revea-ing that DES underwent a quasi-reversible redox process on thelectrode. On the RGO/GCE (curve b), the redox peak currents arencreased, which are clear evidences of excellent conductivity andood catalytic activity of RGO sheets. Compared with the RGO/GCE,he redox peak currents show a remarkable increase on the �-D-RGO/GCE (curve c). These results might be attributed to theynergistic effect of �-CD and RGO sheets, in which �-CD moleculesith high supramolecular recognition and enrichment capability

an form host–guest inclusion complexes with DES [28] and theGO sheets provides a large specific surface area to increase the

oading amount of DES [36]. Therefore, the concentration of DESn the surface of the �-CD-RGO/GCE is increased, which resultsn pronounced enhancement of the redox peak currents. More-ver, RGO sheets could accelerate the electron transfer on thelectrode surface to amplify the electrochemical signal because ofheir outstanding electric conductivity. Thus, the composite filmot only shows the excellent properties of RGO sheets but alsoxhibits the excellent supramolecular recognition and enrichmentapability of �-CD. Therefore, the composite film could provide anlectron-transfer microenvironment to promote the electrochem-

cal reaction.

As can be observed from Fig. 3B, at the first CV cycle (curve a) in fresh solution of 10 �M DES, a pair of redox peaks is obtained.ubsequent CV (curve b) in the same solution without cleaning

cta 85 (2012) 131– 138

the GCE surface was carried out after another 200 s accumulationunder stirring. While a new oxidation peak appeared in the secondpositive scan. That is because the oxidation product of DES formsan adsorbed layer, and the free energy of adsorption of productmakes oxidation of DES to adsorbed product easier than to productin solution [37]. Therefore, the oxidation occurs at more negativepotential. The peak for oxidation of dissolved DES to dissolved prod-uct is followed by the pre-peak, and the redox peaks resemble thatobserved in the absence of adsorption. In contrast to the oxidationpeaks of DES, the reduction peak has higher sensitivity and stability.Hence, we chose the reduction peak to analyze and detect DES.

3.3. Optimization of the experimental conditions

3.3.1. Effect of the amount of ˇ-CD-RGOThe effect of the amount of �-CD-RGO cast onto the GCE surface

on the reduction peak current of 10 �M DES in 0.1 M PBS (pH 7.0)was investigated. Obviously, the reduction peak current of DES isdependent on the cast amounts. As shown in Fig. 4A, the reduc-tion peak current increases with the amount increasing from 4.0 to7.0 �L on the electrode and reaches the maximum at 7.0 �L, thendecreases when the cast amounts increase further, which could beattributed to the increase of film thickness, leading to an increaseof interface electron transfer resistance, and making the electrontransfer more difficult [38]. Therefore, 7.0 �L �-CD-RGO cast ontothe electrode is chosen for all subsequent experiments.

3.3.2. Effect of the accumulation timeAccumulation can improve the amount of DES absorbed on

the electrode surface, and then improve sensitivity determination.Therefore, the influence of accumulation time was investigated in10 �M DES solution on �-CD-RGO/GCE in 0.1 M PBS (pH 7.0). Itcan be observed from Fig. 4B, the reduction peak current of DESincreases gradually with time and reaches a plateau after 200 s,indicating that 200 s is sufficient to reach the DES saturation on �-CD-RGO/GCE. With further increase of the accumulation time, thereduction peak current increases slightly. Therefore, 200 s is usedas the accumulation time.

3.3.3. Effect of pHFurthermore, the influence of pH values on the redox reaction

of DES on the �-CD-RGO/GCE was also studied in the pH rangefrom 5.0 to 9.0 using 0.1 M PBS. As can be seen in Fig. 4C, thereduction peak current of DES increases with increasing pH valueuntil it reached 7.0, and then decreases when the pH increasedfurther. Considering the sensitivity for determining DES, pH 7.0 ischosen for the subsequent analytical experiments. Moreover, withpH value of the solution increasing, the reduction peak potential(Epc) shifts negatively, indicating that protons have taken part inthe electrode reaction process of DES [39]. It is found that the valueof the reduction peak potential changed linearly with pH values,and that it obeys the following equation: Epc = 0.6204 − 0.0616 pH(R = 0.9912). The absolute value of the slope is approximately closeto the theoretical value of 59 mV pH−1, indicating that the num-ber of proton and electron involved in the electrochemical redoxprocess of DES is equal [40].

3.4. Effect of scan rate on the peak currents of DES

The kinetics of the electrode reactions was investigated bystudying the effect of scan rate on the redox of DES on the �-CD-RGO/GCE. The CVs for 10 �M DES in 0.1 M PBS (pH = 7.0) with

scan rates ranging from 20 to 900 mV s are shown in Fig. 5A.The CVs was carried out in the same solution without chang-ing modified electrode, the new oxidation peak appeared in thesecond scan rate. That is because of the formation of a layer of

D. Lu et al. / Electrochimica A

Fp

apiIirsa(rdF(ltE

ig. 4. The effects of (A) the amount of �-CD-RGO, (B) accumulation time, and (C)H on the reduction peak current of DES. Other conditions are the same as Fig. 3.

dsorbed oxidation product. As scan rate increases both of the redoxeak currents enhance. As shown in Fig. 5B, the pre-peak current

ncreases linearly with the scan rates with regression equation:pa1 (�A) = 2.840 + 0.09559v (mV s−1) (R = 0.9933). These results aren agreement with Bard report [37]. Another oxidation peak cur-ent and the reduction peak current increase linearly with thequare root of scan rate (Fig. 5C). The linear regression equationre Ipa2 (�A) = −4.443 + 1.346v1/2 (mV s−1)1/2 (R = 0.9979) and Ipc

�A) = 24.29 − 4.604v1/2 (mV s−1)1/2 (R = 0.9976), respectively. Theesults indicate that the redox of DES on �-CD-RGO/GCE is pre-ominantly diffusion-controlled process. In addition, as shown inig. 5D, with increasing scan rate, the anode (Epa2) and cathode

Epc) peak potential have a linear relationship with the Napierianogarithm of scan rate (ln v). In the scan rates ranging from 50o 900 mV s−1, the linear regression equations are expressed aspa2 = 0.1949 + 0.04219 ln v (mV s−1) and Epc = 0.3645 − 0.04863 ln v

cta 85 (2012) 131– 138 135

(mV s−1) with R = 0.9984 and 0.9967, respectively. According toLaviron’s model [41], the slope of the line for Epa and Epc couldbe expressed as 2.303RT/(1 − ˛)nF and −2.303RT/˛nF, respectively.Therefore, the value of the electron-transfer coefficient (˛) andthe electron-transfer number (n) are calculated as 0.46 and 2,respectively. Considering that the number of electron and protoninvolved in the DES oxidation process is equal (see Section 3.3.3),the electrooxidation of DES on �-CD-RGO/GCE is a two-electronand two-proton process, which can be described in Scheme 2.

The apparent heterogeneous electron transfer rate constant (ks)can also be obtained according to reference [40] based on Eq. (1):

log ks = ̨ log(1 − ˛) + (1 − ˛)log ̨ − log

(RT

nFv

)− ˛(1 − ˛)nF�Ep

2.303RT(1)

where n is the number of electrons involved in the reaction, �Ep

is the peak-to-peak potential separation, ̨ is the electron-transfercoefficient, and v is the scan rate. According to this equation, the ks

is calculated to be 0.050 s−1.

3.5. Chronocoulometry

The electrochemically effective surface areas (A) of bare GCEand �-CD-RGO/GCE can be determined by chronocoulometry using0.1 mM K3[Fe(CN)6] as model complex (the diffusion coefficient ofK3[Fe(CN)6] in 1 M KCl is 7.6 × 10−6 cm2 s−1 [42]) based on Ansonequation [43]:

Q (t) = 2nFAcD1/2t1/2

�1/2+ Qdl + Qads (2)

where A is surface area of working electrode, c is concentrationof substrate, D is diffusion coefficient, Qdl is double layer chargewhich could be eliminated by background subtraction, and Qads isFaradic charge. Other symbols have their usual meanings. Based onthe slopes of the linear relationship between Q and t1/2 (Fig. 6A),A is calculated to be 0.062 cm2 and 0.335 cm2 for bare GCE and �-CD-RGO/GCE, respectively. The results indicate that the electrodeeffective surface area is increased obviously after electrode modifi-cation, which would increase the electrochemical active site of DES,enhance the electrochemical response, and decrease the detectionlimit.

Furthermore, the chronocoulometry experiments were carriedout on �-CD-RGO/GCE in 0.1 M PBS (pH 7.0) in the absence andpresence of 0.1 mM DES. As can be seen in Fig. 6B, the plot of Qversus t1/2 shows a linear relationship after background subtrac-tion. The slope is 9.460 × 10−5 C s−1/2 and the intercept (Qads) is1.172 × 10−5 C. As n = 2, A = 0.335 cm2, and c = 0.1 mM, D is calcu-lated to be 1.23 × 10−4 cm2 s−1 at 25 ◦C. According to the equationQads = nFA� s, the adsorption capacity (� s) can be obtained as1.813 × 10−10 mol cm−2.

3.6. Determination of DES

As a highly sensitive and a low detection limit electrochem-ical method, DPV was performed to investigate the relationshipbetween the reduction peak current and the concentration of DES atthe proposed electrochemical sensor under the optimal conditions.The DPV responses for different concentrations of DES in 0.1 M PBS(pH 7.0) are illustrated in Fig. 7. As can be seen in the insert of Fig. 7,the reduction peak current has a good linear relationship with theDES concentration in the range from 0.01 to 13 �M. The regres-sion equation is Ipc (�A) = 0.05738 + 1.251c (�M) with a correlationcoefficient of 0.9986. The detection limit is 4 nM (S/N = 3), which is

compared with the values reported by other reported electrochem-ical methods in Table 1. This wide linear range and low detectionlimit can be attributed to the synergetic effect of RGO sheets and�-CD, in which RGO sheets provides a large specific surface area to

136 D. Lu et al. / Electrochimica Acta 85 (2012) 131– 138

Fig. 5. (A) CVs of 10 �M DES on �-CD-RGO/GCE in 0.1 M PBS (pH = 7.0) at different scan

in 0.1 M PBS (pH = 7.0). (B) The plot of the pre-peak current versus scan rate. (C) The plot

between the peak potentials and the Napierian logarithm of scan rate.

Table 1Comparison of the proposed sensor for determination of DES with others.

Electrode Linear range (�M) LOD (�M) References

SWNTsa/Ptnano/GCE 0.1–20 0.015 [12]CPBb/CPEc 0.1–15 0.01 [13]GCE 2–100 0.08 [14]CCBPEd 0.016–0.465 0.008 [15]PEIe/GCE 0.112–17.9 0.02162 [16]�-CD-RGO/GCE 0.01–13 0.004 This work

a Single-wall carbon nanotube.b Cetylpyridine bromide.

ishte

c Carbon paste electrode.d Conductive carbon black paste electrode.e Polyethylenimine.

ncrease the loading amount of DES and �-CD molecules with high

upramolecular recognition and enrichment capability can formost–guest inclusion complexes with DES. Meanwhile, the elec-ron transfer on the electrode surface can be accelerated and thelectrochemical signal is amplified due to the outstanding electric

Scheme 2. Proposed reaction mechanism for

rates (20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 mV s−1)of the redox peak currents versus the square root of scan rate. (D) The relationship

conductivity of RGO sheets. Therefore, sensitive detection of DES isachieved.

Furthermore, the fabrication reproducibility of the modifiedelectrode was evaluated by repeating the determination of a 10 �MDES solution. The relative standard deviation (RSD) was 4.19% forsix successive measurements, suggesting the good reproducibilityand precision. After the modified electrode was stored in refrig-erator at 4 ◦C for 2 weeks, the current kept 93.56% of its originalresponse. The results demonstrate that the sensor exhibits excel-lent stability.

The influences of some normal anions and cations, some estro-gens and some other organic compounds were examined in thepresence of 10 �M DES, and the results were given in Table 2.The result shows that the species investigated almost have nointerference in the analyses carried out, showing changes in the

analytical signal for DES of less than 5%. Under the existence ofthose interferents, the recovery of DES changes between 97.9% and104.4%, suggesting that the proposed method has excellent selec-tivity toward DES.

the electrochemical oxidation of DES.

D. Lu et al. / Electrochimica Acta 85 (2012) 131– 138 137

Fig. 6. (A) Plot of Q–t curves of (a) bare GCE and (b) �-CD-RGO/GCE in 0.1 mMK3[Fe(CN)6] containing 1.0 M KCl. Insert: plot of Q–t1/2 curves on (a) bare GCE and((b

3

utdT

FDo

Table 2Interference of different species to the determination of DES with the proposedsensor.

Interferents Interferentsconcentration (mol L−1)

Recovery (%)

Na+, Ca2+, Mg2+, Fe3+, Al3+, Zn2+,Cu2+, Cl− , NO3

− , SO42− , PO4

3−5.0 × 10−3 101.3

Dopamine 1.0 × 10−5 104.4Ascorbic acid 1.0 × 10−4 102.9Uric acid 1.0 × 10−4 97.9Glucose 1.0 × 10−3 102.2l-Histidine 1.0 × 10−3 98.6Phenylalanine 1.0 × 10−3 100.9Tyrosine 1.0 × 10−3 99.8Estradiol 1.0 × 10−3 102.7Estriol 1.0 × 10−3 103.1Estrone 1.0 × 10−3 101.4

Table 3Determination of DES in milk samples.

Samples Measured(�M)

Added(�M)

Found (�M) RSD (%) Recovery (%)

1 0 1.00 1.05 ± 0.03 3.44 105.02.00 2.07 ± 0.07 3.91 103.55.00 5.10 ± 0.09 2.31 102.0

2 0.02 1.00 1.06 ± 0.04 5.38 103.92.00 1.95 ± 0.05 3.48 96.55.00 5.05 ± 0.11 3.17 100.6

3 0 1.00 0.99 ± 0.04 4.78 99.02.00 2.05 ± 0.05 3.42 102.55.00 4.90 ± 0.13 3.53 98.0

4 0 1.00 1.06 ± 0.03 3.77 106.0

b) �-CD-RGO/GCE. (B) Plot of Q–t curves of �-CD-RGO/GCE in 0.1 M PBS (pH = 7.0)a) in the absence and (b) presence of 0.1 mM DES. Insert: plot of Q–t1/2 curve afterackground subtraction.

.7. Application in real samples analysis

The proposed method with the above optimal conditions wassed to determine DES in milk products from four manufac-

ures purchased from a supermarket in Lanzhou (China). Theetermination was performed by the standard addition method.hese analyses were performed for three times under the same

ig. 7. DPVs of 0.01, 0.1, 0.3, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 �MES on �-CD-RGO/GCE in 0.1 M PBS (pH = 7.0) at scan rate of 50 mV s−1. Inset: plotf the reduction peak current against the concentration of DES.

2.00 2.04 ± 0.04 2.96 102.05.00 5.09 ± 0.08 2.16 101.8

conditions, and the results obtained are shown in Table 3. Therecoveries of DES are in the range from 96.5% to 106.0%, indicat-ing that the sensor in this work is suitable for DES determinationin real samples with high sensitivity and precision.

4. Conclusions

In this work, �-CD functionalized RGO sheets was successfullyprepared by using l-AA as the reducing agent and applied to deter-mine DES. The composite combining the advantages of RGO sheetsand �-CD dramatically improved the electrochemical response ofDES and enhanced the sensitivity. Moreover, the fabricated elec-trode had wider linear range and lower detection limit, and it alsoexhibited good stability and reproducibility. In practical applicationinvestigations, the fabricated electrode showed good recoveriesand could be applied to determine DES in milk samples with sat-isfactory results. Therefore, �-CD-RGO composite was a promisingmodified electrode material for the determination of DES by elec-trochemical technique.

Acknowledgement

This work was supported by the National Natural Science Foun-dation of China (No. 20775030).

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