Molecularly Imprinted Polymer-Hybrid Electrochemical Sensor forthe Detection of β‑EstradiolSonia Des Azevedo, Dhana Lakshmi, Iva Chianella,* Michael J. Whitcombe, Kal Karim,Petya K. Ivanova-Mitseva, Sreenath Subrahmanyam, and Sergey A. Piletsky

Cranfield Health, Vincent Building, Cranfield University, Cranfield, Bedfordshire, MK43 0AL, U.K.

*S Supporting Information

ABSTRACT: This paper discusses the construction of a novel electrochemical sensor for 17β-estradiol (E2) based on amolecularly imprinted polymer (MIP)-conducting polymer modified hybrid electrode. A bifunctional monomer, N-phenylethylene diamine methacrylamide (NPEDMA), was used for the construction of the electrochemical sensor. Conductingfilms were prepared on the surface of a gold electrode by electropolymerization of the aniline moiety of NPEDMA. A layer ofMIP was photochemically grafted over the polyaniline, via N,N-diethyldithiocarbamic acid benzyl ester (iniferter) activation ofthe methacrylamide groups. Computational modeling was used to select the most suitable monomer for preparation of MIPs forE2. Experimental parameters such as deposition time, cyclic voltammetric (CV) scan cycles, and conditions for polymeraccumulation were optimized. The detection limit of the resulting sensor, determined by CV, was 6.86 × 10−7 M. Furthermore,the hybrid electrode was successfully used to analyze E2 in water without complex sample pretreatment. These results reveal thatthe MIP hybrid sensor has potential to be an effective technique for the electrochemical determination of E2 in real-time incomplicated matrices.

1. INTRODUCTION

Naturally occurring conjugates of estrogens, such as theendocrine disruptor 17β-estradiol (E2) and its primemetabolites estriol (E3) and estrone (E1) are largely excretedfrom the human body as sulfates and glucuronide conjugates,via urine. The fact that the free parent compounds arefrequently found in either sewage effluents or in environmentalwaters is a significant issue, causing concern in relation to theireffect on both aquatic life and public health, such as alterationsin reproductive function and development, and implications inbreast tumors and other endocrinal disorders.1−3 Therefore,development of an inexpensive biosensor for the real timedetection of estrogen is of significant importance. Electro-chemical detection of E2 offers great advantages because it israpid, inexpensive, and easy to perform. Major drawbacks ofthis method however are the undesirably high detection limitand lack of selectivity of unmodified electrodes. Combiningmolecularly imprinted polymers (MIPs), which can selectivelycapture and concentrate analytes, with electrochemicaldetection can enhance selectivity of the sensor and enable itto operate at detection limits low enough for practical use.MIPs are synthetic polymers possessing binding sites specific

for a target compound, used as template during synthesis of thematerial. A functional monomer, which interacts chemicallywith the template, is copolymerized with cross-linkingmonomers to create a rigid polymer network. After polymer-ization the template is removed and binding sites, comple-mentary in size, shape, and chemical functionality, are created.The sites are capable of specifically and selectively rebinding thecompound used as template.4−7 MIPs have successfully beenapplied in solid phase extraction,8,9 as drug delivery vehicles,10

in bioassays,11 to induce crystallization,12 and as the recognitionelement in biosensors.13−16 One of the most attractive features

of MIPs as the capture agent of biosensors is the increasedlifetime of the sensor. This not only helps in the regenerationand reuse of the sensor, but also makes it more useful for fielduse due to its robust nature.Polymers imprinted with E2 have been successfully

synthesized and showed high absorption capacities in solidphase extraction, rapid binding kinetics, and good selectivity totrace levels of E2 in water and in biological samples.17−21

Molecularly imprinted solid-phase extraction was applied to thedetection of 17β-estradiol from fish and prawn samples by highperformance liquid chromatography.22 The detection andquantification limits were 0.023 and 0.076 mg L−1. Morerecently a MIP-based “immunosensor” for estradiol wasfabricated on a quartz crystal microbalance and incorporatedinto a microfluidic chip by pressing a “stamp” coated with MIPnanoparticles, imprinted with specific antibodies, into a layer ofanother polymer, creating secondary imprints capable ofbinding the original antigen.23

These few examples show the benefits of sensing technology,when combined with MIPs. The limitations of theseapplications are the inability of sensing in the field and thecomplex preparation procedures. The lack of a direct path forconduction from the polymer active sites to the electrodeseriously limits their use as receptors in electrochemical sensors.As we have previously demonstrated, a way to mitigate this

Special Issue: Recent Advances in Nanotechnology-based WaterPurification Methods

Received: October 31, 2012Revised: February 1, 2013Accepted: February 1, 2013

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problem is to incorporate polymers based on N-phenylethylenediamine methacrylamide (NPEDMA) as part of the sensor.24

NPEDMA combines two orthogonal polymerizable function-alities, an aniline group (for the conductive polyaniline layer)and a methacrylamide (for further attachment of polymers),which are able to form an electrical contact between the MIPcomponent and the sensor element (Figure 1).24,25 Using thisapproach we integrated an estradiol MIP with the sensor viagrafting to poly(NPEDMA) for the construction of a hybridsensor for E2. In this work we report a convenient method forthe construction of a novel electrochemical sensor for β-estradiol based on a computationally designed MIP.

2. MATERIALS AND METHODS

2.1. Chemicals. 17β-Estradiol (E2), progesterone, estriol,and α-estradiol, methacrylic acid (MAA), and ethylene glycoldimethacrylate (EGDMA) were purchased from Sigma Ltd.,UK. N,N′-Diethyldithiocarbamic acid benzyl ester, the iniferteror living initiator was from TCI Europe, Belgium. Gold-coatedglass electrodes were acquired from NanoSPR Devices, USA,and screen-printed carbon/platinized electrodes were fromDropSens, Spain. All other chemicals were of analytical orHPLC grade and utilized as received (e.g., acetonitrile, acetone,hydrochloric acid (HCl), ethanol (EtOH), dimethylformide(DMF)).2.2. Contact Angle Measurements. Sessile water contact

angle (CA) measurements were made using Cam 100 opticalAngle Meter (KSV Instruments Ltd., Finland) with thesoftware provided.2.3. Computational Molecular Modeling. The rational

design of MIPs was carried out on a PC running Linuxexecuting the software package SYBYL 7.3 (Tripos Inc.) usedto simulate monomer−template interactions.2.4. Gold Electrodes Preparation. Gold-coated glass

slides were cut with a gold-cutter into 1 cm × 2 cm rectangles.These were then cleaned by immersion in DMF for about 30min, rinsed with deionized water and then placed into a plasmachamber, and nitrogen plasma was applied for 5 to 15 min with

an energy of 75 W. The gold sensors were immersed inethanol/methanol solution, then dried and prepared forpolymerization by placing a small line (horizontal) of Aralditeglue leaving about 1 cm × 1 cm of electrode working area.

2.5. Electrochemical Setup. Three electrodes were usedfor the electrochemical procedure: a working (gold electrode),an Ag/AgCl reference, and a platinum counter electrode. Thesewere set up using a mountable stand. The working, reference,and counter electrodes were then connected to the electro-chemical instrument, a potentiostat, Autolab 4.9, usingcrocodile clips AGS20. The three electrodes were accom-modated and submerged in a 10 mL beaker containing theelectrolyte solution, such that most of the surface area of theworking electrode was covered with the electrochemicalsolution.

2.6. Baseline Measurement. Prior to adding the E2analyte, a baseline was acquired for each electrode (and later forMIP and NIP sensors, respectively). Baseline measurementswere obtained from a solution of ethanol/PBS (2.5/7.5 mL) oracetonitrile/water (9.68/320 mL) for the polymerization ofNPEDMA. Cyclic voltammetry (CV) was then performedbetween different ranges of potentials according to the type ofexperiment using the Autolab 4.9 interface connected to a PC.

2.7. Electrochemical Testing of 17β-Estradiol. For thetesting of E2, after recording the baseline, a range of increasingvolumes (5−15 μL) of a stock solution of 17β-estradiol (3.67 ×10−3 M) were added in the testing solution. After each addition,CV was performed using the same range and conditions as thebaseline signal.

2.8. Electrochemical Polymerization of NPEDMA onGold Electrodes. The polymerization of NPEDMA wascarried out in accordance with a previously published report.24

A 2.44 mM stock solution of the monomer was prepared bydissolving 30 mg of NPEDMA in 2 mL of acetonitrile. A 320μL aliquot of this solution was taken and diluted to 10 mL bythe addition of 9.18 mL of water and 500 μL of 1 M perchloricacid. Polymerization was then performed by CV between −0.2V and +1 V with a scan rate of 50 mV/s. Before NPDEMA

Figure 1. Schematic diagram of steps followed during the development of MIP hybrid sensor for E2. N-phenylethylene diamine methacrylamide(NPEDMA) was used as a functional monomer for the synthesis of a conducting graftable polymer on the surface of the electrode.

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polymerization, a baseline was recorded in 2.5/7.5 mL EtOH/PBS. This was repeated also before E2 testing.2.9. Immobilization of Iniferter to Pendant Double

Bonds of NPEDMA Layer over Gold Electrode. A solutionof N,N′-Diethyldithiocarbamic acid benzyl ester, the iniferter orliving initiator, was prepared in hexane (10 mL, 0.63 mM). Thegold electrodes containing the NPEDMA thin film wereimmersed in the iniferter solution with the help of crocodileclips AGS20 mounted on a clamp. After the removal of oxygenby purging nitrogen for 10 min, the system was irradiated underdark for 60 min using a fiber optic UV lamp delivering a 50 Wcm−2, mounted at a distance of 4 cm from the beaker. Theelectrode was then washed with water and dried in a stream ofnitrogen gas. Contact angle measurements were performed toconfirm the grafting of iniferter as described in section 2.2.2.10. Grafting of MIP and NIP Thin Films onto the

Iniferter-Activated NPEDMA. The protocol for the prepara-tion of the gold electrodes and the production of the 17β-estradiol MIP was similar to the procedure reported before.24

EGDMA was used as cross-linking monomer, acetonitrile asporogen, and MAA was used as the best suited functionalmonomer. For the grafting of MIP and NIP, a solution of E2(17 mg, 62.5 μmol), MAA (27 mg, 319 μmol), and EGDMA(1.2 g, 6.06 mmol) was prepared in acetonitrile (2.5 g) in a 30mL capacity vial. The reaction mixture was sonicated for 10min. The ratio of template to monomer was 1:4. The iniferter-activated polymer-coated electrode was immersed in thereaction mixture and clamped using a crocodile clip. After theremoval of oxygen by purging nitrogen for 10 min, the systemwas irradiated under dark for 1 h using a fiber optic UV lampdelivering 50 W cm−2, mounted at a distance of 4 cm from thebeaker. The electrode was then washed with water and dried ina stream of nitrogen gas. A similar procedure was also followedto prepare the (nonimprinted) control-grafted electrodes. Inthis case the same steps were carried out with the exclusion ofthe template E2 from the polymerization mixture.2.11. Extraction of Template. After polymer grafting,

template removal was carried out by flashing the electrode with5 mL of water, followed by 5 mL of 5% acetic acid in methanoland finally 5 mL of 0.1 M NaOH prepared in methanol untilthe electrochemical signal for E2, as monitored by CV,disappeared (5−6 washing cycles). Electrodes were then rinsedthree times with ethanol and water. These were then dried withnitrogen gas and stored in the dark to prevent lightdecomposition. NIP sensors were subjected to the samewashing conditions used for the MIP material.2.12. Rebinding and Measurement of 17β-Estradiol

with MIP and NIP. Electrochemical experiments wereperformed using an Autolab 4.9 connected to a PC with thepolymer-modified working electrode, a platinum auxiliaryelectrode, and an Ag/AgCl reference electrode. CV wasperformed from −0.2 V to +1 V at 50 mV s−1 scan rate.Briefly, a blank solution made of EtOH/PBS (2.5/7.5 mL) wasprepared and polymer-modified electrodes were immersed inthis solution, to which aliquots (5 μL at the time) of differentstock solution of E2 prepared in 50% DMF/50% water wereadded in an ascending order (from 3.67 × 10−4 M stocksolution, 1.84 × 10−7, 3.67 × 10−7, 5.49 × 10−7, 7.33 × 10−7

M), (from 7.34 × 10−3 M stock solution, 3.67 × 10−6, 7.33 ×10−6, 1.11 × 10−5, 1.47 × 10−5 M), and (from 3.67 × 10−2 Mstock solution, 1.82 × 10−5, 3.67 × 10−5, 5.5 × 10−5, 7.33 ×10−5 M).

2.13. Cross-Reactivity Study. Cross-reactivity studieswere performed using the same protocol utilized for E2rebinding to MIP, with 17α-estradiol (α-E2), estriol (E3), andprogesterone (P4) stock solutions being prepared instead ofE2. Briefly, MIP sensors, specific for E2 were tested usingincreasing concentrations (from 3.67 × 10−4 M stock solution,1.84 × 10−7, 3.67 × 10−7, 5.41 × 10−7, 7.33 × 10−7, 9.15 × 10−7,1.10 × 10−6, 1.46 × 10−6 M) of each of the hormones (α-E2,E3, P4), and CV was recorded as for the testing of E2.

3. RESULTS AND DISCUSSIONScreen printed carbon/platinized electrode (Pt) as well as goldelectrodes were initially considered as working electrodes forthe study. Preliminary investigations revealed that a much morepronounced signal for E2 was seen on the Pt electrode ascompared with the gold electrodes (data not shown). This wasprobably because E2 was capable of accumulating more on theporous carbon layer than on the smooth gold metal surfaceHowever, because of difficulty in polymerizing NPDEMA on Ptelectrodes, gold electrodes were preferred and finally selected asworking electrodes.Before the polymer grafting, the electrochemical behavior of

17β-estradiol (E2) at the poly-NPEDMA gold-modifiedelectrode was investigated and no peak for E2 was observed.This was not surprising, as the conductivity of the poly-NPEDMA gold modified electrodes is not as high as the baregold electrode, indicating that the use of a MIP layer on theconductive polymer should be able to increase the amount ofE2 immobilized (adsorbed) producing a detectable signal.

3.1. Polymerization Procedure. Surface confined graftingof all polymers was readily initiated upon UV irradiation of theelectro-polymerized poly-NPEDMA layer, containing theiniferter in the presence of deoxygenated solutions of cross-linkers and monomers such as EGDMA and MAA, respectively.This permitted precise control of the macromoleculararchitectures of the grafted surfaces. Figure 1 presents theschematic representation of the polymerization process. It waspreviously shown that NPEDMA can be polymerized usingeither the free radical initiator (via the double bond) or byoxidative polymerization/electro-polymerization mechanism(via the aniline part).24 However, for this work, we employedelectro-polymerization to create a conductive layer followed byUV-activation of double bonds for attachment of the livinginitiator, diethyl dithiocarbamic acid benzyl ester, followed byMIP and NIP grafting using the initiator attached to theconductive polymer.

3.2. Electrochemical Polymerization of NPEDMA onGold Electrodes. As it is shown in Figure S1 reported inSupporting Information, anodic and cathodic peaks around+0.6 V and +0.5 V, respectively, were seen growing up to 15CV scans confirming successful polymerization of NPDEMA.Furthermore, successful electrochemical polymerization ofNPEDMA onto the gold electrodes was confirmed by theformation of a green layer of poly-NPEDMA (formed on theworking part of the electrodes). Nevertheless furtherconfirmation of successful polymerization was also done usingcontact angle measurements (Table 1).

3.3. Computational Molecular Modeling. Computa-tional molecular modeling (in silico method for rational designof polymers) was used to screen the most promising monomersfor synthesis of MIPs for E2. The rational design protocol,which has been already extensively explained elsewhere,26

involved four steps. Briefly the first step was the design of a

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functional monomer database or virtual library. The secondstep was the design of a molecular model of template E2. Thethird step was the screening of the library of functionalmonomers for their possible interactions with the template E2using the LEAPFROG algorithm. Monomers giving the highestbinding scores (see Table S1 in Supporting Information) arethose forming the strongest complexes with the template andtherefore they represented the best candidates for polymerpreparation.For E2, the best binding monomers were identified as

bisacrylamide, itaconic acid, 2-hydroxyethyl methacrylate,acrylamide, EGMP, and methacrylic acid. Finally methacrylicacid, which has been extensively investigated in the literature,was the preferred monomer for the preparation of MIPs for E2(Figure 2B). The last and fourth step of the modeling involvedrefinement using Molecular Mechanics (MM) and MolecularDynamics simulations for optimization of the polymercomposition. These were performed as described elsewhere,27

and the best monomer/template ratio (Figure 2C) wasidentified.3.4. Sensor Responses of MIP and NIP. Figure 3A

presents CVs of the MIP sensor straight after polymerization(solid line) and after removal of the template E2 (dashed line).The solid curve confirms that E2 is still trapped inside thepolymer matrix, as indicated by the oxidation peak at +0.4 V,while the dashed lined curve shows that the template wassuccessfully removed following extensive washing (no peak at+0.4 V). The rebinding studies were performed at severalconcentrations (Figure 3B). The anodic peak of E2 (solid line)suggested that oxidation of E2 takes place at around +0.40 V.

After rigorous washing with a combination of solvents (asexplained in section 2.11), the second curve (dashed line) wasobtained. The absence of an oxidation peak for E2 at +0.40 Vindicates that E2 has been removed completely. The CVsconfirm both a successful grafting of the specific MIP sensorand successful E2 rebinding after the template extraction.At higher concentrations of template (from 1.82 × 10−5 to

7.33 × 10−5 M), the electrochemical oxidation of E2 could notbe observed indicating the influence of sample saturation on theE2 oxidation peak (data not shown). Therefore, rebindingstudies were carried out at lower concentrations, ranging from1.84 × 10−7 to 7.33 × 10−7 M (Figure S2 in Supporting

Table 1. Water Contact Angle Measurement Data, Prior andPost Each Modification Step of the Electrode Surface:Electropolymerization of NPEDMA; grafting of Iniferter,MIP, and NIP Layersa

surfaces average ± STDEV (degrees ± STDEV)

bare electrodes 64.31 ± 1.69poly-NPEDMA 66.79 ± 1.70iniferter layer 70.56 ± 1.46MIP layer 60.76 ± 5.72NIP layer 61.28 ± 3.38

aStandard deviations (STDEV) were calculated using three replicatesfor each measurement.

Figure 2. Minimum-energy conformations of (A) E2; (B) complex of E2 with methacrylic acid; (C) 2:1 complex of methacrylic acid monomer withthe E2 template, as determined by computer modeling. Oxygen atoms are shown in red, carbon atoms are white, and the light blue atoms arehydrogen.

Figure 3. CVs of MIP sensor: (A) after polymer synthesis, containingthe template (solid line) and post template removal (dashed line); (B)rebound template (solid line, 3.67 × 10−7 M E2) and post-templateremoval (dashed line).

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Information). The anodic peak current of E2 was enhancedlinearly as the concentration of E2 increased. When the NIPelectrode was tested at the same conditions, no template peakwas observed (Figure S3 in Supporting Information), whichconfirms the specificity of the synthesized MIP. Preliminarycalibration curves for MIP and NIP sensors are shown in FigureS4 in Supporting Information. From the calibration curve adetection limit of 6.89 × 10−7 M was calculated for the MIPsensor. A detection limit could not be calculated for the NIPsensor as a real response to E2 was not recorded.The selectivity of the MIP sensors was assessed using 17α-

estradiol (α-E2), progesterone (P4), and estriol (E3), which arestructurally related to E2 (Figure 4). When the MIP sensor was

exposed to solutions of different concentrations of both, α-E2and progesterone, no binding was observed (Figure 5A,B). Thisfact demonstrates excellent selectivity and specificity for E2over both α-E2 and progesterone. When E3 was tested in asimilar manner, it did not show any response for the lowerconcentrations (1.84 × 10−7 M and 3.67 × 10−7 M), but startedto show a response with the two highest concentrations tested(5.49 × 10−7 M and 7.33 × 10−7 M) (Figure 5C). This showsthat the MIP sensor lacks selectivity for E3 at highconcentrations, which is not surprising due to the similarityof the two molecular structures, with the only differencebetween the two being an extra hydroxyl group in E3 whichseem to interfere only slightly with the binding.

4. CONCLUSIONSTraditional methods of β-estradiol detection in food andenvironmental samples suffer from a number of disadvantagessuch as a need for long detection time and are expensive andrequire trained personnel. Furthermore, some methods havelimitations that arise from a lack of stability, sensitivity, orspecificity of the assay components. Although the developmentof electrochemical sensing devices based on MIPs can havedrawbacks due to nonconductive nature of the polymers, theirintegration with electrochemical transducers would producerobust devices capable of working in complicated matrices,overcoming many of the problems found in clinical, food, andenvironmental analysis. The development of an electrochemicalsensor for 17β-estradiol (E2) based on a MIP-conductingpolymer modified hybrid electrode, constructed using thebifunctional monomer NPEDMA, has been described here.Conducting films were prepared on the surface of Au electrodes

by electropolymerization of the aniline moiety of NPEDMA.The detection limit of the resulting MIP sensor assessed bycyclic voltammetry was found to be 6.86 × 10−7 M with alinearity range between 1 × 10−7 M and 8 × 10−7 M. Animprovement of the detection limit could easily be achieved byreplacing cyclic voltammetry with a more sensitive electro-chemical technique such as for example chronoamperometry.Further improvements of the detection limit and a widerlinearity range could be obtained by increasing the sensorsurface area using gold or platinum nanoparticles28 beforeNPEDMA deposition. The MIP sensor developed here alsodemonstrated high selectivity, showing a lack of cross-reactivity

Figure 4. Molecular structure of 17β-estradiol (E2), 17α-estradiol (α-E2), estriol (E3), and progesterone.

Figure 5. CVs of MIP sensor in the presence of differentconcentrations of (A) α-E2; (B) progesterone and (C) E3. In panelA the CVs at concentrations of 3.67 × 10−7, 5.49 × 10−7, 7.33 × 10−7,and 9.15 × 10−7 M are the same as that with 1.10 × 10−6 M, and arenot shown for clarity of the figure. In panel B the CVs atconcentrations of 3.67 × 10−7 M and 5.49 × 10−7 M are overlappingwith that at concentration of 1.84 × 10−7 M and are not shown forclarity of the figure. In panel C the CVs for concentrations at 1.84 ×10−7 and 3.67 × 10−7 M are very similar to that at 0 M, and are notshown for clarity of the figure.

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with α-E2 and progesterone and only a limited cross-reactivitywith the highest concentrations of E3. This highlights that theβ-hydroxyl group present both in E2 and E3 is the mainfunctionality responsible for the binding to the MIP, as was alsoshown by the results of the computer modeling study, asreported in Figure 3. In conclusion these findings confirm thatthe MIP-electrochemical sensor developed here has potential tosuccessfully determine E2 in real-time in complicated matrices.

■ ASSOCIATED CONTENT*S Supporting InformationThe electropolymerization of NPEDMA on the gold sensor;the binding energies of various template-functional monomerscomplexes; the sensorgrams for the rebinding of severalconcentrations of E2 both to MIP and NIP sensors; and theresulting calibration curves. This material is available free ofcharge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: i.chianella.1998@cranfield.ac.uk.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thank the European Union for funding this workunder the Framework 7 programme, CP-FP 226524, Watertreatment by molecularly imprinted materials (WATERMIM),and http://mipdatabase.com/ for the useful article analysis inthe MIP field.

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membranes at platinum nanoparticles-modified electrode for determi-nation of 17β-estradiol. Biosens. Bioelectron. 2011, 29, 29.

Industrial & Engineering Chemistry Research Article

dx.doi.org/10.1021/ie302999j | Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXXG