Electrochemical Estrogen Receptor a based Biosensor for Label-Free Detection of Estradiol

Zhenzhong Guo,a Nadia Zine,a Patrick Balaguer,b Aidong Zhang,c Philippe Namour,a Florence Lagarde,a

Nicole Jaffrezic-Renault*a

a University of Lyon, Institute of Analytical Sciences, UMR-CNRS 5280, 5, Rue de La Doua, 69100 Villeurbanne, Francephone: +33437423558

b IRCM, Institut de Recherche en Canc�rologie de Montpellier, Montpellier, F-34298, Francec Key Laboratory of Pesticides and Chemical Biology, Huazhong Normal University, Luoyu Str 152, Hongshan District, 430079

Wuhan, P. R. China*e-mail: nicole.jaffrezic@univ-lyon1.fr

Received: April 9, 2013Accepted: April 29, 2013Published online: June 14, 2013

AbstractWe describe a new electrochemical biosensor based on estrogen receptor a (ER-a) for label-free detection of 17b-estradiol, a model of endocrine-disrupting compounds. ER-a is coupled onto the gold electrode through its 6-Histag and NTA-copper complex. After interaction of estradiol with ER-a, the biosensor presents a well-defined peakat +500 mV due to estradiol oxidation (E17 peak). The linear range of detection is from 1 fM to 1 nM and the de-tection limit is 1 fM. Good selectivity was obtained for interfering substances at nanomolar level, for concentrationof E17 up to 0.1 pM. The E17 was detected in hospital effluents.

Keywords: Estradiol, Estrogen receptor a, Square-wave voltammetry, NTA-copper complex

DOI: 10.1002/elan.201300163

1 Introduction

A large number of natural and synthetic chemicals havebeen identified as disruptors of the normal functioning ofthe endocrine system and produce untoward effects inhormone-responsive target tissues and organs in bothhumans and animals. These chemicals are referred to asendocrine disrupting chemicals (EDCs), and include al-kylphenols, bisphenol A, and a number of pesticides andsome chlorinated compounds, the contraceptive 17-a-ethi-nylestradiol, as well as naturally occurring phytoestrogens(e.g. some coumarin derivatives, flavonoids and isofla-vones) and mycoestrogens (e.g. F-2 mycotoxin) and othersubstances.There is considerable concern about the envi-ronmental occurrence of these EDCs. In addition, earlylife exposure to EDCs may alter gene expression andconsequently transmit these effects to future generations[1–5].

So, public interest in possible health threats posed bychemicals in general and EDCs in particular, is on therise. To limit environmental threats to human and envi-ronmental health, the EU has taken action and madeprogress in various fields during the last few years, suchas the implementation of the REACH (Registration,Evaluation, Authorization and Restriction of Chemicals,EC 1907/2006), the Water Framework Directive (WFD),and the agreement reached on the Industrial Emissions

Directive. The public-spirited conscience of citizens, com-bined with more visible responsibility of public authori-ties, stimulates demand for more stringent water monitor-ing. Indeed the WFD Daughter Directive lists two estro-gens – 17b-estradiol (17E) and 17-a-ethinylestradiol(EE2) – and several EDCs as priority pollutants (e.g.,nonylphenols) and some other candidate substances arealso EDCs as polybrominated (PBB) and polychlorinated(PCB) biphenyls. European countries might will requireby 2021 to limit EE2 in water bodies to an annual aver-age of no more than 0.035 ng/L [6].

As recently as 2003, the International Programme onChemical Safety (IPCS) of the United Nations sponsoreda joint workshop on “Endocrine Disruptors: ResearchNeeds and Future Directions,” in Tokyo, and the work-shop identified as a research need the development ofhighly sensitive and selective chemical and biological ana-lytical methods for EDCs and their metabolites, includingnovel technologies such as biosensors and nanotechnolo-gy. At present, only OECD (Organization for EconomicCooperation and Development) tests on the embryonicstages of aquatic organisms are recognized. However, thisprocedure is extremely long and very time consuming.Also, there is an urgent need for tools for rapid screeningof endocrine disrupters, not only in environmental andfood samples, but also in order to select substitutes toknown EDCs such as bisphenol A, phthalates (DBP,

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DEHP, BBP) or nonylphenol and to designate any poten-tial EDC. Analytical techniques such as GC/MS and LC/MS/MS have been used to determine the amounts ofEDCs in effluents before and after water treatmentplants [7] and in surface waters and sediments [8].

Endocrine compounds (hormones) travel throughout thebody searching for cells that contain matching receptorswithin the target cell or located on the surface of thetarget cell. The hormone binds with the receptor, likea key in a lock. Some exogenous compounds (EDCs) canmimic hormonal structures (key) and interact with the re-ceptor (lock) which over-responds to the stimulus or re-sponds at inappropriate times, inducing some undesirableendocrinal effects (e.g.: developmental and reproductiveproblems). Indeed, EDC effects are dependent on receptorbinding, which depends on configurational parameters, andmost progress has been made with structures that bind tothe estrogen receptor (ER). Then, genetically producedand purified ERs can be used for the recognition of EDCsand can serve as a base for toxicological assays [9,10].

Based on this huge societal demand, several electro-chemical sensors have already been reported for the de-tection of 17b-estradiol (17E) as a model of EDC mole-cules. Electrochemical sensors are good candidates fordetection in real water samples without any preconcentra-tion or any separation steps, and moreover they are asso-ciated with light and low-cost instrumentation. Some pro-posed biosensors are based on receptor molecules such asa specific aptamer or antibody or estrogen receptora (ER-a) and the irreversible interaction of 17b-estradiolwith the receptor molecule influences the electron chargetransfer with a redox probe present in the measuringmedium. Based on this principle and using electrochemi-cal impedance spectroscopy, detection limits in the orderof picomolar were obtained [11–16] (cf. Table 1).

Likewise, it is well known that 17b-estradiol presentsan oxidation peak due to oxidation of the phenol functioninto phenoxy radical and then into a phenoxonium ion,this reaction being followed by the formation of ketoneafter loss of one proton [17]. The direct detection of 17b-estradiol through its oxidation peak after accumulationhas been exploited and a detection limit as low as fewtens of nM has been achieved [18–22] (cf. Table 1).

In this work, a new approach is proposed for the con-ception of a biosensor for the detection of the natural es-trogen 17b-estradiol, as a model of EDC, based on thespecific interaction of 17b-estradiol with the ER-a -recep-tor, as shown in Reference [23] that reported the first useof the NTA-Cu2+�histidine-tagged protein system for thedesign of an electrochemical biosensor and its electro-chemical characterization. The His tag- ER-a-receptor isimmobilized on a gold electrode using the 6His-copperinteraction. Monitoring of the intensity of the Cu(I)/Cu(II) peak depends on ER-a-receptor conformation, asalready shown in the SWV immunosensor [24]. Throughintensity monitoring, the immobilization of the ERa-re-ceptor can be checked (decrease of Cu(I)/Cu(II) peak) aswell as the specific interaction with the ER-a-receptor(increase of Cu(I)/Cu(II) peak). Sensitive and selectivedetection of 17b-estradiol is obtained through SWV, mon-itoring the Cu(I)/Cu(II) peak and 17E oxidation peak.

2 Experimental

2.1 Reagents and Materials for Estradiol ElectrochemicalDetection

b-Estradiol (�98%), Estrone (�99%), Estriol (�97%)were purchased from Sigma. Bisphenol A (BPA) (97 %),6-mercaptohexanoic acid (�90%,C6), Na,Na-bis(carbox-

Table 1. Relevant electrochemical biosensors for detection of 17b-estradiol.

Type ofprobe

Type of electrode Electrochemical technique Detectionlimit

Dynamic range Reference

Aptamer Gold chip SWV with ferri/ferrocyanide probe 1 pM 10 pM–1 nM [11]Aptamer Gold electrode Impedance with ferri/ferrocyanide probe in-

crease of RCT

2 pM 10 pM–10 nM [16]

Estrogenreceptor a

Gold electrode Impedance with ferri/ferrocyanide probe in-crease of RCT

0.1 pM 0.1 pM–1 nM [15]

antibody Au NPs/gold electrode Impedance and SWV with ferri/ferrocyanideprobe decrease of RCT, increase of current

SWV: 66 pMEIS: 95 pM

SWV: 0–4.4 nMEIS: 0–3.6 nM

[13]

ER/Lipidbilayer

Au NPs Impedance with ferri/ferrocyanide probe de-crease of RCT

3.7 pM 18 pM–0.55 nM [12]

MIP PtNPs/GCE Impedance and DPV with ferri/ferrocyanideprobe

16 nM 30 nM–50 mM [14]

No Nano-Al2O3/GCE+CTAB 2 minaccumulation

Voltammetry oxidation peak at 0.45 V 80 nM 0.4 mM–40 mM [18]

No Pt nanoclusters/MWCNTs/GCE Voltammetry oxidation peak at 0.45 V 0.5 mM 0.5 mM–15 mM [19]No CNTs/Ni cyclam/GCE Voltammetry oxidation peak at 0.85 V 60 nM 0.5 mM–40 mM [20]No Poly(l-serine) film/GCE 2 min

accumulationVoltammetry oxidation peak at 0.6 V 20 nM 0.1 mM–30 mM [21]

No LbL (MWCNTs/AuNP)/graphiteelectrode 200 s accumulation

Voltammetry oxidation peak at 0.9 V 10 nM 70 nM–42 mM [22]

ER-a Au/thiol SAM/NTA Cu/6His-ER-a

SWV oxidation peak at 0.5 V 1 fM 1 fM–1 nM This work

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ymethyl)-l-lysine hydrate (�97%,NTA), N-hydroxysucci-nimide (98 %,NHS), sodium acetate, copper acetate werepurchased from Aldrich. Acetic acid, N-(3-dimethylami-nopropyl)-N’-ethylcarbodiimide hydrochloride (�99%,EDC), ethanolamine were purchased from Fluka.Carbon tetrachloride and heptane were purchased fromSigma-Aldrich. Copper acetate solution was obtained bydiluting 10 mM copper(II) acetate in 10 mM acetatebuffer solution (pH 4.6). The buffer solution used for allthe experiments was 10 mM phosphate buffer saline(PBS) at pH 7. All solutions were made in ultrapurewater produced by a Millipore Milli-Q system.

2.2 ER-a Production and Purification [25]

His6-ER (302–552) construction was inserted into theNdeI-BamHI sites of pET15b recombinant plasmid (No-vagen) using PCR, leading to fusion proteins with a Histag. Escherichia coli BL21 (DE3) cells were electroporat-ed with 20 mg pET15b recombinant plasmid. The trans-formed cells were grown in a Lysogeny broth mediumsupplemented with estradiol and sucrose. Introductionwas carried out for 4 hrs at 25 8C. The cell disruptionbuffer contained 2 M of nondetergent sulfobetaines(NDSB).

The cells were lysed by sonification, then the extractswere centrifuged and the expression of soluble proteinwas estimated on SDS-PAGE after passing througha Zn2+ affinity chromatography column. Then the purifi-cation steps were: ion-exchange, gel filtration and recrys-tallization.

2.3 Biofunctionalization of Gold Electrodes

2.3.1 Pretreatment

Gold substrates were provided by the Laboratoire d�Ana-lyse et d�Architecture des Syst�mes (LAAS, CNRS Tou-louse). They were fabricated using standard silicon tech-nologies. (100)-oriented, P-type (3–5 Wcm) silicon waferswere thermally oxidized to grow a 800 nm-thick fieldoxide. Then, a 30 nm-thick titanium layer followed bya 300 nm thick gold top layer were deposited by evapora-tion under vacuum. Gold electrodes were first sonicatedfor 15 min at room temperature in acetone using a Bran-son 5210 ultrasonic bath (Branson Ultrasonics Corpora-tion, Danbury, USA), rinsed with ultrapure water anddried under nitrogen flow. Then the gold electrodes werecleaned for 5 min with freshly prepared “piranha” mix-ture (piranha: H2O2/H2SO4, 3 :7, v/v), then rinsed with ul-trapure water, ethanol and dried under nitrogen flow.

2.3.2 Biofunctionalisation Steps

Tagged ER-a was anchored on the gold electrode surfaceas presented in Scheme 1. The pretreated electrodes wereimmersed overnight at �4 8C in 1 mM 6-mercaptohexano-ic acid solution in ethanol to give alkanethiol SAM bear-

ing carboxylic terminal groups. The modified electrodeswere then rinsed with ethanol. An activation step wasperformed by reaction with a 150 mL drop of 10 mMEDC/10 mM NHS mixture in 10 mM PBS solution for1 h. Carboxyl groups of gold electrodes were convertedinto active carbodiimide esters which can then react withNTA terminal amine groups to form covalent bonds(7 mM NTA in 10 mM PBS solution for 1 h). Then theunreacted carbodiimide esters were passivated by rinsingwith ethanolamine. After this, the gold electrodes wereimmediately rinsed with sodium acetate/acetic acid buffersolution and immersed in 10 mM copper(II) acetate in10 mM acetate buffer solution at pH 4.6 for 45 min, inorder to coordinate the Cu2 + ions with grafted NTAgroups and form a Cu2+ metal complex. Finally, 150 mLof cell extract containing 6-His tagged ER-a were drop-ped onto the surface for 1 h and then thoroughly rinsedwith PBS to remove excess ER-a and other nonspecifical-ly adsorbed species, before measurements were made.

2.4 Microcontact Printing for Fluorescence Microscopy

A glass slide electrode was used for fluorescence micros-copy. The PDMS stamp (1 cm2) with holes of 50 mm diam-eter was used for contact printing of octadecyltrichlorosi-lane on a glass slide electrode (cf. Scheme 2). First, thePDMS stamps were cleaned with ethanol and were ultra-sonicated for 30 min. Then the glass slide was cleaned for30 min in piranha mixture, rinsed with ultrapure waterand then dried with nitrogen. The PDMS stamp was thenimmersed in octadecyltrichlorosilane (OTS) (5 mM OTSand 0.4 mM of carbon tetrachloride in heptane solution)for 30 seconds and dried under nitrogen flow. Next, theglass was stamped with the inked PDMS for a few sec-

Scheme 1. Schematic representation of the 6-His tagged ER-a anchored on gold surface.

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onds. The glass substrates needed to be left in the oven at100 8C for one hour in order to enhance the chemicalbonding of OTS to the glass substrate.

The glass substrates were then immersed in 10-(carbo-methoxy)decyl-dimethylchlorosilane (CMDCS) solution(10 mM CMDCS and 0.4 mM of carbon tetrachloride inheptane solution at 4 8C for 4 hours. Silanized glass slideswere thoroughly and slowly rinsed with heptane, thenwater, and dried under a nitrogen flow. The glass sub-strates needed to be left one more time in the oven at100 8C for one hour in order to enhance the chemicalbonding of CMDCS to the glass substrate. In the end, thesubstrates were immersed in 1N HCl (37 %) overnight atambient temperature, then rinsed with plenty of water,and finally dried under nitrogen flow. After these treat-ments, ER-a was immobilized by following the steps de-scribed in Section 2.3.2.

After the ER-a immobilization step, 2 mL of FITCconjugated anti-His antibody was maintained in contactwith the biofunctionalized glass slide. Then the modifiedglass slide was observed by fluorescence microscopy(Zeiss Axioplan 2 Imaging apparatus, Carl Zeiss MicroI-maging GmbH, Jena, Germany).

2.5 Electrochemical Measurements

Square wave voltammetry (SWV) was performed usinga Voltalab 80 (Hach Lange, France). A three-electrodecell was used, including a saturated calomel electrode(SCE) as reference electrode, a platinum plate as counterelectrode (0.54 cm2) and a modified gold working elec-trode (0.19 cm2). The procedure of electrochemical detec-tion were carried out at 4 8C using a Minichiller thermo-stated bath (Huber, company) and used a PBS solution atpH 7.0. The square wave voltammetry measurementswere carried out in the potential range from �400 mV to+600 mV after maintaining potential at �400 mV for5 min for the total reduction of Cu(II) to Cu(I).The scanrate, duration, amplitude were 100 mV/s, 0.05 s, 5 mV, re-spectively [24].

2.6 Incubation of Estradiol

The 17b-estradiol should first be dissolved in organic sol-vents such as methanol. A 10�3 M mother solution of 17Ein methanol was prepared and then diluted in PBS con-taining methanol for each concentration. The concentra-tion range from 10�15 M to 10�9 M of 17E was chosen foranalysis. The ER-a functionalized gold electrodes wereexposed to increasing concentrations of 17E at 4 8C. Foreach concentration, the incubation time was 1 hour at4 8C.

3 Results

3.1 Control of Copper Complexation by Voltammetry

In order to check that NTA/Cu2+ complex has successful-ly been formed on the gold electrode, square wave vol-tammetry was performed. After maintenance at �400 mVfor 5 min, the oxidation peak Cu(I) to Cu(II) appears at+33 mV (data not shown). This result demonstrates thatcopper ions are well immobilized as NTA complex at thegold electrode surface and its peak surface area allowsthe determination of a copper surface coverage of 1 �10�9 molcm2, with an RSD of 20%, showing that a densemonolayer of chelated copper ion is available on graftedelectrode surface.

3.2 Control of ER-a Immobilization by Voltammetry andFluorescence Microscopy

3.2.1 Square Wave Voltammetry

As shown in Figure 1 (black curve), after ER-a is immo-bilized, the Cu(II)/Cu(I) peak (Cu peak) shifted to285 mV, showing that a different Cu(II) complex isformed. Its peak maximum decreased by a factor of 10,showing that the electron transfer rate greatly decreasedafter ER-a immobilization. This phenomenon has alreadybeen observed in the case of 6 His-tagged reduced anti-body immobilization [24]. The Cu(II)/Cu(I) peak maxi-

Scheme 2. Microcontact Printing and fluorescence detection of ER-a.

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mum is correlated to the surface density of immobilizedER-a.

3.2.2 Fluorescence Control of ER-a Immobilization

Due to the use of FITC conjugate anti His-antibody, it ispossible to reveal the position of the immobilization ofER-a. As shown in Figure 2, where OTS was stamped

there is no fluoresecence (black part). Green spots of50 mm diameter appear, due to the presence of 6-Histagged ER-a immobilized on Cu-NTA. This result dem-onstrates that ER-a was successful immobilized on sur-face Cu-NTA, and so it can be used for further target de-tection.

3.3 Electrochemical Detection of Estradiol

3.3.1 Impedance Control

After 17E was incubated for 1 h with ER-a functionalizedgold electrode, a decrease of the impedance of the elec-trode/electrolyte interface is observed (cf. Figure 3), re-flecting a decrease of the charge transfer resistance of thebiolayer after ER-a/17E interaction. This observation isdue to a change of conformation of ER-a after its inter-action with 17E. This phenomenon has already been ob-served during olfactory binding protein/odorant interac-tion [26].

3.3.2 SWV Measurements

After incubation of E17, typical SWV curves obtainedare presented in Figure 1. A typical E17 oxidation peakappears at 500 mV. The position of this peak has alreadybeen observed in the range of 450 mV–600 mV[18,19, 21]. The E17 peak maximum increases with suc-

Fig. 1. SVW of ER-a biolayer before and after interaction with 17b-estradiol.

Fig. 2. Fluorescence image of FITC conjugate anti His-antibodyon the ER-a grafted on microcontact printed glass substrate.

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cessive increases of E17 concentration increases. This ob-servation is in agreement with the decrease of the chargetransfer resistance of the biolayer after ER-a/E17 interac-tion.

When incubated E17 concentration increases, the Cupeak maximum is shifted to lower potential (from285 mV to 125 mV when concentration varies from10�15 M to 10�9 M) and it increases in intensity. This phe-nomenon must be probably due to the change of confor-mation of Cu surrounding when ERa-E17 complex isformed.

As a calibration curve, the E17 peak/Cu peak ratio wasplotted as a function of �logarithm of concentration of17b-estadiol, as presented in Figure 4. Through this repre-sentation, a standard deviation of 10% was obtained withthree different electrodes.

The selectivity of the ER-a biosensor was evaluated byincubating solutions of different concentrations of 17b-es-tradiol (10�15 M to 10�9 M) containing a mixture of inter-fering substances (EE2, estrone, estradiol, BPA) at a con-centration of 10�9 M. The measured points are presentedin Figure 4. For concentrations of E17, up to 10�13 M, nointerfering effect is observed. When E17 concentration ishigher than 10�13 M, in the presence of interfering sub-stances, the E17 oxidation peak does not increase, due toa competition effect on ER-a.

The performances of the ER-a biosensor developed inthis work are compared to those of the relevant electro-chemical biosensors for detection of 17b-estradiol, inTable 1. The lowest detection limit was obtained with ourER-a biosensor. The obtained limited dynamic range isquite compatible with the observed concentrations in realwaters.

3.3.3 Detection of 17b-Estradiol in Hospital Effluents

The method of standard additions was applied to realwater samples, using the ER-a biosensor. Hospital efflu-ents before and after the Bellecombe water treatmentplan were sampled from the SIPIBEL pilot site (http://www.graie.org/graie/sipibelpublic/a-sipibel.htm). The re-sults are presented in Figure 5. It is noticeable that theE17/Cu ratio is lower for input effluents, compared tooutput effluents. This is due to the fact that the Cu peakmaximum is higher for input effluents, the concentrationof EDC substances being higher. The measurement of the

Fig. 3. Nyquist diagram of the ER-a functionalized gold elec-trode/electrolyte interface before and after 17b-estradiol interac-tion.

Fig. 4. Ratio of E17 peak/Cu peak as a function of �logarithm of concentration of 17b-estadiol. (�) E17 in 10 mM PBS solution ,(*) E17+10�9 M of interfering substances (estrone, estriol, BPA).

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Cu peak could be used in an early warning system for theassessment of a global endocrine disrupting effect of aneffluent.

The determined concentration of 17b-estradiol in theinput effluent was 262�30 fM and 14�0.2 fM in theoutput effluent. These samples were analysed using theLC-MS/MS technique, whose detection limit is 0.4 pM for17b-estradiol. No estradiol was detected.

4 Conclusions

In summary, a sensitive, selective, rapid and simple bio-sensor for the detection of 17b-estradiol has been devel-oped using ER-a immobilized on gold electrodes. The de-tection was based on SWV of the E17 oxidation peak andthe Cu peak, the calibration curve being presented asE17/Cu ratio versus �log[E17]. This device has beendemonstrated to be able to detect E17 in the linear rangefrom 1 fM to 1 nM, with a detection limit of 1 fM. More-over, it is shown that the measurement of the Cu peakcould be used in an early warning system for the assess-ment of a global endocrine disrupting effect of an efflu-ent.

Acknowledgements

The authors thank EGIDE for financial support throughPHC XU GUANG QI (No 25982SG), the China Scholar-ship Council for Zhenzhong Guo�s doctoral grant and theEuropean Communities for IRSES Project NANODEV.Dr A. Baraket is acknowledged for fluorescence measure-ments. Dr H. Korri-Youssoufi is warmly thanked for fruit-ful discussions.

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