Aptamer/Au nanoparticles/cobalt sulfide nanosheets biosensor for 17β-estradiol detection using a guanine-rich complementary DNA sequence for signal amplification

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Aptamer/Au nanoparticles/cobalt sulfide nanosheets biosensor for17β-estradiol detection using a guanine-rich complementary DNAsequence for signal amplification

Ke-Jing Huang a,b,n, Yu-Jie Liu a, Ji-Zong Zhang a, Jun-Tao Cao a, Yan-Ming Liu a

a College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, Chinab State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China

a r t i c l e i n f o

Article history:Received 27 May 2014Received in revised form6 August 2014Accepted 8 August 2014

Keywords:Cobalt sulfide nanosheetAu nanoparticles17β-EstradiolMethylene blueGuanine-rich complementary DNAsequenceSignal amplification

x.doi.org/10.1016/j.bios.2014.08.01063/& 2014 Elsevier B.V. All rights reserved.

esponding author at: College of ChemistryNormal University, Xinyang 464000, China. Tail address: [email protected] (K.-J. Huan

e cite this article as: Huang, K.-J., et

a b s t r a c t

We have developed a sensitive sensing platform for 17β-estradiol by combining the aptamer probe andhybridization reaction. In this assay, 2-dimensional cobalt sulfide nanosheet (CoS) was synthesized by asimple hydrothermal method with L-cysteine as sulfur donor. An electrochemical aptamer biosensor wasconstructed by assembling a thiol group tagged 17β-estradiol aptamer on CoS and gold nanoparticles(AuNPs) modified electrode. Methylene blue was applied as a tracer and a guanine-rich complementaryDNA sequence was designed to bind with the unbound 17β-estradiol aptamer for signal amplification.The binding of guanine-rich DNA to the aptamer was inhibited when the aptamer captured 17β-estradiol.Using guanine-rich DNA in the assay greatly amplified the redox signal of methylene blue bound to thedetection probe. The CoS/AuNPs film formed on the biosensor surface appeared to be a good conductorfor accelerating the electron transfer. The method demonstrated a high sensitivity of detection with thedynamic concentration range spanning from 1.0�10�9 to 1.0�10�12 M and a detection limit of7.0�10�13 M. Besides, the fabricated biosensor exhibited good selectivity toward 17β-estradiol evenwhen interferents were presented at 100-fold concentrations. Our attempt will extend the application ofthe CoS nanosheet and this signal amplification assay to biosensing areas.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

One of the major classes of environment contaminants are theendocrine disrupting chemicals, which are ubiquitous in theenvironment because of their extensive uses in residential, in-dustrial and agricultural applications (Schiliro et al., 2009).17β-Estradiol, the most active estrogen, has been widely used tofatten animals due to its anabolic effects (Noppe et al., 2008). Itwill enter into the body from outside and interfere with thenormal physiological processes (G.G. Yang et al., 2002; W. Yanget al., 2002). Therefore, there is a need to develop a sensitivemethod for 17β-estradiol detection.

Generally, the methods for determination of 17β-estradiol aremainly based on high-performance liquid chromatography (Huyet al., 2011; Fedeniuk et al., 2004). However, these assays arerather complicated and therefore labor intensive and time con-suming. Alternatively, electrochemical assays are simple andinexpensive. However, an obstacle is that the bare electrode

and Chemical Engineering,el.: þ86 376 6390611.g).

al., Biosensors and Bioelect

cannot offer enough sensitivity to determine 17β-estradiol atultra-trace levels. Selectivity is another challenge for an electro-chemical approach. Therefore, there are some works focused onbiomolecules, such as antibodies, modified on the electrode todetect 17β-estradiol selectively (Jayasena, 1999; Pemberton et al.,2005). However, the specificity of these sensors was compromiseddue to the nonspecific nature of the 17β-estradiol-receptor toother proteins.

Aptamers are artificial single-stranded DNA or RNA oligonu-cleotieds selected from random-sequence nucleic acid libraries byan in vitro evolution process. They can recognize various targetsincluding small species, sugars, proteins and even whole cells(Sharma et al., 2012; Wu et al., 2013). Compared with antibodies,aptamers exhibit several considerable advantages, such as highspecificity, good stability, desirable biocompatibility and signifi-cant chemical simplicity. Therefore, they offer a powerful alter-native to antibodies as recognition molecules. For example, Caoet al. reported an aptamer-based homogeneous protein detectionassay using a cucurbit[7]uril functionalized electrode (Cao et al.,2014). Xie et al. developed an electrochemical method for Ochra-toxin A detection based on aptamer and loop-mediated isothermalamplification (Xie et al., 2014). Chen et al. developed aptamer-

ronics (2014), http://dx.doi.org/10.1016/j.bios.2014.08.010i

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mailto:[email protected]





K.-J. Huang et al. / Biosensors and Bioelectronics ∎ (∎∎∎∎) ∎∎∎–∎∎∎2

based array electrodes for quantitative interferon-γ detection(Chen et al., 2014). Recently, several aptamer-based biosensors(Kim et al., 2007; Olowu et al., 2010; Lin et al., 2012) have alsobeen developed for 17β-estradiol detection. However, there is stillthe need to develop a more sensitive method for 17β-estradiolbecause it usually presents at an ultra-trace level in real samples.To improve the sensitivity in the electrochemical assays, manysignal amplification strategies have been designed based onnanomaterials, which can provide a promising sensing platformbecause of the high surface areas for enhanced mass transport andthe high loading of receptor molecules for synergistic amplifica-tion of the target response (Zhu et al., 2010; Ke et al., 2012).

Driven by the outstanding properties of graphene, such as highsurface area, excellent electrical conductivity and good chemicalstability, two-dimensional (2D) nanomaterials have attracted in-creasing interest. Especially, the successful fabrication of graphenebased devices has stimulated new interests in graphene-like 2Dlayered materials of elements other than carbon to expect toobtain some unusual properties. Most recently, 2D transition-metal chalcogenides such as CuS, WS2, MoS2 and VS2 havereceived significant attention because they offer many advantagesfor fundamental and technological research in a variety of fields,such as energy storage (Feng et al., 2011; Rout et al., 2013), sensing(Li et al., 2013) and catalysis (Wang et al., 2013). They arecomposed of the metal layers and sulfur layers and are stackedtogether by weak van der Waals' interactions. This kind of materialis expected to act as an excellent functional material because the2D electron–electron correlations among metal atoms would behelpful in enhancing planar electric transportation. Recently, wehave prepared different 2D transition-metal chalcogenides basednanomaterials and applied them in supercapacitors (Huang et al.,2013a, 2013b) and biosensors (Huang et al., 2014a, 2014b).

CoS, as an important class of transition metal chalcogenides,are versatile materials with potential applications in supercapaci-tors (Yang et al., 2011), lithium-ion batteries (Wang et al., 2011)and catalysts. The unique layered morphological characteristics of2D CoS will not only facilitate the infiltration of electrolyte but willalso provide a more active surface when it is evaluated as anelectrode material for biosensors; further, it allows self-assemblyof thiolated compounds on its surfaces that indicates an advantagein facile surface modification. However, CoS is not favorable forapplications in electrode materials because it is a semiconductorand has relatively low conductivity. Thus, it is highly desirable toprepare a CoS composite with electronically conductive materialsto facilitate the charge transfer for electrochemical biosensingapplications. Au nanoparticles (AuNPs) are mostly recommendedowing to the fact that they can greatly increase the currentresponse of the electrochemical sensor with a good conductiveability and immobilization of biomolecules by Au–S bond, andhave been widely used to construct aptamer based assays (Haiet al., 2014). Therefore, the combination of CoS nanosheets andAuNPs to prepare CoS/AuNPs composite will not only greatlyimprove its conductivity but also provides a signal amplificationsensing platform for target molecules.

Methylene blue (MB) is an organic dye which belongs to thephenothiazine family, and it has been widely used in DNAhybridization detection as an electrochemical indicator (Wanget al., 2010). In general, the interaction of MB with ssDNA occursspecifically through the guanine bases of ssDNA (G.G. Yang et al.,2002; W. Yang et al., 2002). Thus, the redox signal of MB will begreatly enhanced if the number of guanine bases in ssDNA isincreased intentionally.

In this work, 2D CoS nanosheets were prepared by a hydro-thermal method using L-cysteine as sulfur donor, and then anovel electrochemical sensing platform was fabricated based onCoS nanosheets and AuNPs modified glassy carbon electrode

Please cite this article as: Huang, K.-J., et al., Biosensors and Bioelect

(GCE). The CoS/AuNPs/GCE possessed low background current,good conductivity and large electroactive surface area. Anelectrochemical aptasensor was then proposed for 17β-estradioldetection using MB as an indicator and cDNA (the sequencefeaturing rich guanine) as a signal amplifier. The cDNA had asequence complementarity to the 17β-estradiol aptamer and itmay compete with 17β-estradiol for the immobilized aptamer onthe electrode surface. When 17β-estradiol hybridizes with apta-mer, it will decrease the degree of binding of cDNA to theaptamer, in turn decreasing the signal of MB bound to the cDNA.On the other hand, the hybridization of cDNA will enhance theselectivity of the aptasensor.

2. Experimental

2.1. Apparatus

Electrochemical measurements were performed on a CHI660E Electrochemical Workstation (Shanghai CH Instruments,China) with a conventional three-electrode system composed ofplatinum wire as auxiliary electrode, saturated calomel elec-trode (SCE) as reference electrode and modified GCE as workingelectrode. A JEM 2100 transmission electron microscope (TEM)and a Hitachi S-4800 scanning electron microscope (SEM) wereused to record the morphologies of the nanocomposite. An X-raypowder diffraction (XRD) pattern was operated on a Japan RigakuD/Maxr-A X-ray diffractometer. Raman spectra were conducted on aRenishaw Raman system model 1000 spectrometer with a 200 mWargon-ion laser (λ¼514.5 nm) in ambient air. The composition ofCoS was carried out using X-ray photoelectron spectroscopy (XPS,VG Multilab 2000, Thermo Scientific, UK). The residual pressure inthe spectrometer during the acquisitionwas always lower than 10�7

Pa. Al Kα (1486.6 eV, 15 mA, 20 keV) and Mg Kα (1253.6 eV, 15 mA,20 keV) sources were used for the experiments. The spherical sectoranalyzer was operated in the fixed analyzer transmission mode,with the pass-energy set at 20 eV for the acquisition of high-resolution spectra.

2.2. Reagents

KNO3, CoCl2 �6H2O, MB, L-cysteine and ethylene glycol werepurchased from Shanghai Chemical Reagent Corporation(Shanghai, China). Chitosan (Chit, M.W. 100,000–300,000, dea-cetylation degree Z95%) and HAuCl4 �4H2O were purchasedfrom Sigma-Aldrich (St. Louis, USA). 17β-Estradiol, 1-aminoan-thraquinone, naphthalene, polychlorinated biphenyl, bisphenolA, phthalic acid ester, testosterone and cholesterol were pur-chased from Shanghai Sangon Biological Engineering Techno-logical Co. Ltd. (Shanghai, China). The 17β-estradiol aptamerand the segmental complementary DNA with rich G content(cDNA) were synthesized by Shanghai Sangon Biological En-gineering Technological Co. Ltd. (Shanghai, China). The basesequences are listed below:

17β-Estradiol aptamer: 5ʹ-SH-(CH2)6-TTT TTT TTT T GCT TCCAGC TTA TTG AAT TAC ACG CAG AGG GTA GCG GCT CTG CGCATT CAA TTG CTG CGC GCT GAA GCG CGG AAG C-3ʹcDNA: 5ʹ-GGA GGA GGA GGA GGA GGA GGA GGA GGA GGA GGAGGA GGA G G CTT CCG CGC TTC AGC GCG CAG CAA-3ʹThe italics denote the complementary sequences.

DNA sequences and 17β-estradiol samples were dissolved in10 mM Tris–HCl buffer solution (pH 8.0, containing 200 mM NaCl,25 mM KCl, 10 mM MgCl2 and 5% ethanol).





Scheme 1. Schematic diagram of the electrochemical aptamer biosensor.

K.-J. Huang et al. / Biosensors and Bioelectronics ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

2.3. Preparation of CoS nanosheets

The preparation of CoS nanosheets was conducted by a facilehydrothermal method: 0.3 mM CoCl2 �6H2O and 80 mg L-cysteinewere dissolved in an 80 mL mixed solution of pure water andethylene glycol (3:1 in volume). The mixed solution was thenstirred for half an hour and transferred to a 100 mL Teflon-linedautoclave, sealed and heated in an oven at 160 °C for 6 h. Aftercooling, the product was collected by filtration, washed with waterand ethanol for several times and dried in vacuum at 60 °C.

2.4. Preparation of aptamer biosensor

Prior to modification, the surface of the GCE was polished to amirror-like smoothness with 0.5 and 0.05 mM alumina slurry, andthen washed successively with ethanol and water. After beingdried with nitrogen gas, the GCE was coated with 5 mL 0.5 mgmL�1 CoS suspension to obtain CoS/GCE, which was prepared bydispersing CoS in 0.1% Chit aqueous solution containing 1% aceticacid. Then, the CoS/GCE was immersed in 0.1% HAuCl4 solutioncontaining 0.1 M KNO3, and AuNPs were electrodeposited on theelectrode by applying a fixed potential at �0.2 V for 80 s. Subse-quently, 8 mL of aptamer (0.1 μM) was covalently immobilized ontothe AuNPs/CoS/GCE by the Au–thiol chemistry for 12 h at roomtemperature. After that, the electrode was incubated in 1.0 mM 6-mercapto-1-hexanol (MCH) for 30 min to block the nonspecificbinding sites of AuNPs. The different concentrations of17β-estradiol and 2.0�10�7 M cDNA were dissolved in 100 mMTris–HCl buffer. Then, the 8 mL mixture was applied on theaptamer/AuNPs/CoS/GCE at 60 °C for 8 h. Finally, 8 mL MB solution(20 mM) was dropped on the modified GCE surface for 10 min.

The procedure for the biosensor fabrication is illustrated inScheme 1.

2.5. Electrochemical measurements

The electrochemical measurements were performed by thedifferential pulse voltammetry (DPV) in a 50.0 mM Tris–HCl buffersolution (pH 7.0) containing 20 mM KCl with instrumental para-meters as: 0.005 V of pulse amplitude, 0.05 s of pulse width and0.2 s of pulse period. Electrochemical impedance spectroscopy(EIS) experiments were carried out in a 10.0 mL aqueous solutioncontaining 1.0 mmol L�1 [Fe(CN)6]3� /4� and 0.1 mol L�1 KCl at apotential of 0.2 V over the frequency range from 0.1 Hz to 100 kHz.

3. Results and discussion

3.1. Characterization of CoS sample

The morphologies of CoS samples were investigated by SEMand TEM. The typical SEM image in Fig. 1A reveals the sheetsubunits' structure of CoS. This 2D architecture of the sample ishelpful in increasing the specific area of the sample and in formingan interconnected conducting network between the sheets, whichfacilitates rapid electronic transport in electrode reactions. Fig. 1Bshows the TEM images of AuNPs/CoS, which displays the AuNPswell dispersed on the CoS nanosheets with sizes of about15–35 nm. As shown in the inset of Fig. 1B, the TEM image of arepresentative CoS product further reveals that the morphology isa 2D thin-layer nanosheet approximately 20 nm in thickness.

Fig. 1C reveals the XRD patterns of the as-synthesized CoS. Ascan be seen, the crystalline peaks that appear at 35° and 48.7° arecorresponding to (200) and (220) planes of CoS, respectively. Nocharacteristic peaks from other impurities are observed in the XRDpattern, indicating the sample is highly pure.


To further investigate the structure of the CoS sample, werecorded Raman spectra (Fig. 1D). The sample exhibits threeRaman characteristics of CoS at 478, 522 and 685 cm�1, whichare assigned to Eg, F2g and A1g modes of CoS materials, respectively(Hadjiev et al., 1988).

Fig. 1E shows the XPS survey spectrum of the CoS product. Thedetected elements are Co, S, C and O, which are as expected forCoS. Peaks corresponding to the Co 2p3/2 and Co 2p1/2 orbitals areobserved at about 793.8 and 778.8 eV, respectively (Fig. 1F). Thepeak at 170.65 eV is attributed to the S 2p binding energy of S inCoS (Fig. 1G). The appearance of O in the sample may be caused bysurface-adsorbed CO2 and O2.

Fig. 1H shows the nitrogen-adsorption isotherm and the pore-size distribution curve of the as-prepared CoS nanosheets. Theisotherm can be classified as one between type I and type V. Thepore-size distribution ranges between 5 and 7 nm; the total porevolume is 0.138 cm3 g�1. The specific surface area of the CoS wascalculated to be 40.13 m2 g�1.

3.2. Electrochemical characterization

Fig. 2A shows the cyclic voltammetry (CV) of the sequentialmodification processes of the biosensor. The probe[Fe(CN)6]3�/4� reveals a reversible CV at the bare GCE (curvea). After AuNPs are deposited (curve b) or CoS is coated on theGCE (curve c), the peak currents are much higher than for thebare GCE because of their good electro-conductibility. However,after the AuNPs are deposited on CoS/GCE, the peak currentsincrease greatly (curve d) due to the synergistic effect of CoSnanosheets and AuNPs. The assembly of aptamer on the AuNPs/CoS/GCE surface induces an obvious decrease in peak current(curve e). One reason can explain this phenomenon: the electro-static repulsion between the negatively charged phosphate back-bone of oligonucleotides in the aptamer and [Fe(CN)6]3� /4�

controlled by negative charge.EIS is an effective method for studying the interface properties

of surface-modified electrodes and electron-transfer resistance at





Fig. 1. (A) SEM image of CoS nanosheets. (B) TEM image of AuNPs/CoS (E). (C) XRD patterns of CoS nanosheets. (D) Raman patterns of CoS nanosheets. (E) XPS survey spectraof the CoS nanosheets. (F) High-resolution spectra for Co 2p. (G) High-resolution spectra for S 2p. (H) N2-sorption isotherms and the pore-size distribution (inset) curves ofCoS nanosheets.


the electrode surface. The semicircle diameter in EIS equals theelectron transfer resistance (Ret) and the linear part at lowerfrequencies represents the diffusion process. As shown in Fig. 2B,


with AuNPs (curve b) or CoS nanosheets (curve c) assembled onthe electrode surface, the Ret become smaller than for the bare GCE(curve a) due to their good conductivity. When AuNPs are





Fig. 2. CVs (A) and EIS (B) of GCE (a), AuNPs/GCE (b), CoS/GCE (c), AuNPs/CoS/GCE (d) and aptamer/AuNPs/CoS/GCE (e) in 1.0 mM [Fe(CN)6]3�/4� containing 0.1 M KCl. Insetof (B): Equivalent circuit used to model impedance data in the presence of redox couples: R1: electrolyte solution resistance; R2: element of interfacial electron transferresistance; Q: constant phase element; and ZW: Warburg impedance resulting from the diffusion of ions. (C) Plot of Q–t curves of the GCE (a) and AuNPs/CoS/GCE (b) in0.1 mM K3[Fe(CN)6] containing 1.0 M KCl. Inset: Plot of Q–t1/2 curves on GCE (a) and AuNPs/CoS/GCE (b).


electrodeposited on the CoS/GCE, the Ret decreases obviously(curve d), which is ascribed to the excellent conductive ability ofthe AuNPs/CoS film. The Ret value increases significantly after themodification of the aptamer, which can be attributed to theelectrostatic repulsion between aptamer and [Fe(CN)6]3�/4� .

The effective surface areas of GCE and AuNPs/CoS/GCE werecompared by chronocoulumetry in 0.1 mM K3[Fe(CN)6] solutioncontaining 1.0 M KCl, where the standard diffusion coefficient (D0)of K3[Fe(CN)6] at 25 °C is 7.6�10�6 cm2 s�1 (Bard and Faulkner,1980). The effective surface area (A) of electrodes is calculatedaccording to the following equation (Anson, 1964):

π= + +Q nFAcD t Q Q2 / (1)1/2 1/2 1/2dl ads

where n is the number of electron transferred, F (C mol�1) is theFaraday constant, A (cm2) is the area of the electrode, c (mol cm�3)is the concentration of substrate, D (cm2 s�1) is the diffusioncoefficient, Qdl (C) is the double layer charge and Qads (C) is theadsorption charge and other symbols have their usual signifi-cances. According to the results shown in Fig. 2C, A is calculated tobe 0.079 cm2 and 0.160 cm2 for bare GCE and AuNPs/CoS/GCE,respectively. The results indicated that the effective surface area ofthe electrode increased greatly after modification with AuNPs/CoSfilm, which would increase the immobilization amount of theaptamer.


3.3. Optimization of experimental conditions

The effect of aptamer concentration was studied (Fig. 3A). Itshows that the peak current of [Fe(CN)6]3� /4� decreases with theincrease of probe concentration in the range of 0.05–0.15 μM. Thecurrent response reaches a plateau when the probe concentrationsexceed 0.1 μM, indicating that most HS-ssDNA molecules havebeen immobilized on the electrode. So the 0.1 μM aptamer wasused in the further experiments.

The effect of the reaction time between aptamer and 17β-estradiol on the DPV response of [Fe(CN)6]3� /4� was also evalu-ated. As shown in Fig. 3B, the peak current of [Fe(CN)6]3� /4�

obviously decreases on increasing the reaction time from 30 to180 min, and almost stays stable after 180 min, suggesting thereaction is completed. Thus, 180 min of reaction time was used.

The effect of deposition time of AuNPs was further tested in therange of 30–110 s (Fig. 3C). The highest current response of[Fe(CN)6]3�/4� is realized at a deposition time of 80 s. So 80 s ofdeposition time was used for AuNPs preparation.

The effects of adding different amounts of cDNA on the redoxsignal of MB were also evaluated in range of 0.05–0.3 μM. Asshown in Fig. 3D, the DPV response of MB increases with theincrease of cDNA concentration in the range of 0.05–0.2 μM, andthen almost stays stable when it exceeds 0.2 μM. Therefore,0.2 μM cDNA was employed in the subsequent experiments forcompetitive binding with the aptamer.





Fig. 3. Effects of aptamer concentration (A), reaction time (B), deposition time of AuNPs (C) and cDNA concentration (D) on the peak current.


3.4. The amplification effect of the cDNA

In order to verify the amplification effect of the cDNA, wecompared the electrochemical performances of different electrodes.MB is chosen as the electrochemical redox substrate because of itsspecific adsorption onto the guanine units of DNA. As shown inFig. 4A, a significant DPV signal of MB appears when aptamer isapplied on Au/CoS/GCE (curve b) because the MB molecules arestrongly adsorbed by the guanine units on the exposed aptamersequence. When cDNA is immobilized on the aptamer/Au/CoS/GCE,the partial hybridization of cDNA and aptamer leads to a very obviousincrease of DPV response of MB (curve c), which is more than 2 timesthat of the aptamer/Au/CoS/GCE, indicating remarkable signal ampli-fication of the hybridization reaction. When 17β-estradiol is furtherimmobilized on the electrode, the signal of MB decreases (curve d).The competition regarding binding with aptamer between the 17β-estradiol and cDNA decreases the amount of guanine units, whichtherefore reduces the adsorption of MB.

3.5. Analytical performance of designed biosensor

Under the optimized conditions, we examined the analyticalperformances of the proposed method upon the addition ofdifferent concentrations of 17β-estradiol and then measured theMB signal. As shown in Fig. 4B, the DPV peak currents decreasewith the increasing of 17β-estradiol concentration in the range of0.001–1 nM, and a linear relationship between the DPV peakcurrents and the logarithm of concentrations is observed (Inset


of Fig. 4B). The regression equation was i (mA)¼�1.41 log(c/M)�3.11 (i is the peak current and c is the concentration of 17β-estradiol) with a correlation coefficient (R) of 0.997. The detectionlimit was calculated to be 7.0�10�13 M based on three times thestandard deviation of the blank sample measurement. The analy-tical performance of as-fabricated biosensor was compared withpreviously reported sensor. The results are presented in Table 1. Itshows the developed biosensor exhibits better analytical perfor-mance toward 17β-estradiol. The reason might be as follows:firstly, the 2D CoS nanosheets greatly enhanced the effective activearea of electrode; secondly, the formation of the AuNPs film on theCoS nanosheets not only promoted the electron transfer but alsoincreased the immobilization amount of aptamer, which resultedin the lower detection limit; thirdly, using guanine-rich DNA in theassay greatly amplified the redox signal of methylene blue.

3.6. Specificity, repeatability and stability

To investigate the specificity of the as-prepared biosensor, theinfluences of some potential interferents on the determination of 17β-estradiol were investigated. As shown in Fig. 4C, only 17β-estradiolcan cause an obvious change of peak current. This result clearlydemonstrated the high specificity of the proposed sensing system.

The reproducibility of the developed biosensor was investi-gated. The peak currents of eight successive measurements of0.05 nM 17β-estradiol by DPV were determined and a relativestandard deviation (RSD) of 2.4% was obtained. Six parallel-madec-DNA/aptamer/AuNPs/CoS/GCEs were used to detect 0.05 nM





Fig. 4. (A) DPVs of 20 mM MB at AuNPs/CoS/GCE (a), aptamer/AuNPs/CoS/GCE (b), c-DNA/aptamer/AuNPs/CoS/GCE (c) and 17β-estradiol/c-DNA/aptamer/AuNPs/CoS/GCE(d) in 50 mM Tris–HCl buffer solution (pH 7.0) containing 20 mM KCl. (B) DPVs of biosensor after reaction with different concentrations of 17β-estradiol (from a to h: 0,1.0�10�12, 5.0�10�12, 5.0�10�11, 8.0�10�11, 1.0�10�10, 5.0�10�10 and 1.0�10�9 M). Inset: The relationship of the peak current with � log c17β-estradiol. (C) Specificityevaluation of the proposed aptasensor for 0.05 nM 17β-estradiol against 5.0 nM interferences: (a) blank, (b) 17β-estradiol, (c) 1-aminoanthraquinone, (d) naphthalene,(e) polychlorinated biphenyl, (f) bisphenol A, (g) phthalic acid ester, (h) testosterone and (i) cholesterol.

Table 1Comparison between different sensors for 17β-estradiol detection.

Electrodes Analytical technique Linear range (nM) LOD (pM) References

Bare GCE DPV 40,000–1,000,000 10,000,000 Salci and Biryol (2002)Poly-serine/GCE DPV 100–30,000 20,000 Song et al. (2008)Aptamer/Au EIS 0.01–10 2 Lin et al. (2012)Aptamer/AuNPs/PEDOT/Au SWV 0.1–100 20 Olowu et al. (2010)Pt/MWNTs/GCE SWV 500–15,000 180,000 Lin and Li (2006)Ag/PAMAM-Au/GR-PANI/GCE DPV 0.14–25 72 Li et al. (2013)cDNA/Aptamer/AuNPs/CoS/GCE DPV 0.001–1 0.7 This work

SWV, square wave voltammograms; PEDOT, poly(3.4-ethylenedioxylthiopene); MWNTs, multi-walled carbon nanotubes; PAMAM, poly(amino–amine) dendrimers; GR,graphene; and PANI, polyaniline.


17β-estradiol and an RSD of 4.7% was achieved, indicating goodreproducibility.

The stability of the biosensor was estimated by detecting 0.05 nM17β-estradiol. When the electrode was stored in the refrigerator at4 °C for one week, the biosensor retained 94.8% of its initial currentresponse, demonstrating good stability of the biosensor.


3.7. Practical application

The developed biosensor was used to detect 17β-estradiol inurine samples. The urine samples were obtained from threevolunteers and diluted 100 times with PBS and then directlydetected. A standard addition method was used to evaluate the






analytical performance of the sensor. The results are shown inTable S1. The recoveries are between 94.4% and 104.0%, and theRSDs are in the range of 1.6–3.4% (n¼3). The results demonstratedthat the proposed biosensor was sensitive and accurate enough forpractical application.

4. Conclusion

We reported the synthesis of CoS nanosheets by a simple one-step hydrothermal process. A novel electrochemical biosensor for17β-estradiol was developed by assembling an aptamer on AuNPs/CoS modified GCE using MB as indicator and cDNA (the sequencefeaturing rich guanine) as signal amplifier. The introduction of CoSnanosheets and AuNPs in the construction of the biosensorefficiently accelerated the electron transfer and enhanced thedetection signal, which led to a low detection limit. The as-prepared biosensor also exhibited excellent selectivity and goodstability. Therefore, this work would be attractive for environmentcontaminants analysis in bioanalytical and environmentalapplications.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (U1304214 and 21375114) and the State KeyLaboratory of Chemo/Biosensing and Chemometrics (No. 2013013).

Appendix A. Supplementary materials

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.bios.2014.08.010.

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