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Materials Chemistry and Physics 139 (2013) 107e112

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Materials Chemistry and Physics

journal homepage: www.elsevier .com/locate/matchemphys

Paraoxon imprinted biopolymer based QCM sensor

Ebru Birlik Özkütük a,*, Sibel Emir Diltemiz b, Elif Özalp a, Tevfik Gedikbey a, Arzu Ersöz b

aDepartment of Chemistry, Eskisehir Osmangazi University, Kimya Bölümü, Eskisehir, TurkeybDepartment of Chemistry Anadolu University, Eskisehir, Turkey

h i g h l i g h t s

* Corresponding author. Tel./fax: þ90 222 239 35 7E-mail address: ebirlik@ogu.edu.tr (E.B. Özkütük).

0254-0584/$ e see front matter � 2013 Elsevier B.V.http://dx.doi.org/10.1016/j.matchemphys.2012.12.068

g r a p h i c a l a b s t r a c t

< New and simple method hasimproved for designing and prepa-ration for paraoxon determination.

< We have proposed molecularimprinted polymer (MIP) film forthe detection of paraoxon.

< We have investigated measurementof binding interaction of paraoxonimprinted quartz crystal microbal-ance (QCM) sensor.

a r t i c l e i n f o

Article history:Received 1 June 2012Received in revised form3 December 2012Accepted 22 December 2012

Keywords:Surface propertiesShape memory effectsElectronic characterisationMicroporous materialsThin films

a b s t r a c t

In this study, a novel quartz crystal microbalance (QCM) based on the modification of paraoxonimprinted polymer (TCM-Cd(II)-paraoxon) film onto a quartz crystal sensor has been developed for thedetermination of paraoxon. The sensor is based on a molecular imprinted polymer (MIP) which can besynthesized using paraoxon as a template molecule, Thiourea Modified Chitosan-Cd(II) (TCM-Cd(II)) asthe metal-chelate monomer, ephychlorohydrin as a crosslinking agent. The MIP particles have beencharacterized by FTIR measurements and QCM sensor has characterized using AFM and ellipsometer. Theperformance of the paraoxon imprinted sensor has indicated that a selective and sensitive paraoxonimprinted sensor could be fabricated. The sensor is able to discriminate paraoxon in solution owing tothe specific binding of the imprinted sites. The obtained paraoxon imprinted sensor has 0.02e1 mMlinear range and low detection limit (0.02 mM). The selectivity studies have shown that the selectivity ofprepared paraoxon imprinted sensor has found as being very high in the presence of parathion which issimilar in structure with paraoxon. The paraoxon imprinted sensor has been repeatedly used for morethan 7 months in many continuous experiments.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Organophosphorus insecticides are very toxic compounds,intensively used in agriculture and they irreversibly inhibit thecatalytic active sites of acetylcholinesterase (AChE). Themechanismof pesticide action, their high toxicity and their wide use inagriculture to protect crops from pests, represent a general hazard

8.

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for environmental welfare and could become a real threat to life inthe biosphere, so that their continuous monitoring in the envi-ronment is required. The need for such monitoring devices hasintensified the work in the field of biosensors, in particular thosebased on the activity or inhibition of AChE [1,2].

Due to its wide use and toxicity, several methods like titri-metric [3], voltametric [4], gas chromatography [5], HPLC [6] spec-trophotometric [7,8] and sensor [9e18] have been reported in theliterature for the determination of organophosphates. Erbahar et al.have investigated the pesticide sensing inwater with phthalocyaninebasedquartz crystalmicrobalance (QCM) sensors. Thephthalocyanine

Scheme 1. The structure of TCM.

E.B. Özkütük et al. / Materials Chemistry and Physics 139 (2013) 107e112108

core structure was chemically modified to increase sensor sensitivityand create sensors with widely diverging analyte responses [19].Mulchandani et al. have improved the biosensor for direct determi-nation of organophosphate nevre agents [9]. The molecularlyimprinted conducting polymer based electrochemical sensor fordetection of atrazine [20] and themolecularly imprinted polymerfilmwhich has characterized by a scanning electronmicroscope (SEM), ACimpedance and cyclic voltammetry for the detection of profenofosbased on a QCM sensor [21] have also prepared. Marx et al. [22] haveimproved theparathion sensorbasedonmolecularly ımprinted sol gelfilms. Gas-phase binding measurements were performed on coatedQCMresonators. Thebindingofparathion to the imprintedfilms in theliquid phase was investigated by steady-state experiments withanalysis by GC-FPD and cyclic voltammetry.

AT-cut thickness-shear mode QCM sensors are commonly usedas gravimetric elements due to their good mass resolution atcomparatively low operation frequency. The QCM is a simple, costeffective, high-resolution mass sensing technique, which has beenfavorably adopted for analytical chemistry, and electrochemistryapplications due to its sensitive solutionesurface interface meas-urement capability [23]. QCM can be applied as a transducer inanalytical chemistry [24], biology [25], pharmaceutical detection[26], environmental assays [27], and life science [28].

The fabrication of MIP films to detect certain compounds viaa QCM transducer has been accomplished in recent years [29e33].Molecular imprinting is a promising technique for the preparationof polymers which possess specific recognition sites. By means ofa synthetic organic polymer matrix, the imprints of the templatemolecule are created in the polymer. After the template is eluatedfrom the rigid polymer network, recognition sites complementaryto the template molecule in shape and size can be obtained. A MIPhas unique advantages over natural biological receptors in terms ofphysical and chemical stability, ease of preparation, low cost andapplication in harsh environmental conditions. Based on thesefavorable properties, the MIP has been successfully used in manyfields, such as chromatographic separation [34e36], immunoassays[37,38], solid-phase extraction [39], chiral separation [40], phar-maceutical analysis [41], recognition of elements in chemical orbiological sensors [42e44] and so on.

In this study, we have proposed novel phosphotriesterasemimick surface imprinted polymeric QCM sensor for the selectivedetermination of the nerve agent by thiourea modified chitosan(TCM-Cd(II)) as a new metal-chelating monomer via metal coord-inationechelation interactions and paraoxon (diethyl-4-nitro-phenyl phosphate, target nevre agent) templates. We haveattempted to combine the selective adsorption of MIP withmicromass determinations by QCM to analyze paraoxon, paraoxonimprinted biopolymer. These are coated onto piezoelectric crystals,and then the binding characteristics have been investigated.

2. Experimental

2.1. Chemicals

High molecular weight chitosan and epichlorohydrin weresupplied from Aldrich Chemical (USA). All other chemicals wereanalytical reagent grade and purchased from Merck (Darmstadt,Germany). All glasswares were extensively washed with dilutenitric acid before use. All other chemicals were of analytical gradepurity and purchased from Merck AG (Darmstadt, Germany). Allwater used in the experiments was purified using a Barnstead(Dubuque, IA) ROpure LP� reverse osmosis unit with a high flowcellulose acetate membrane (Barnstead D2731) followed bya Barnstead D3804 NANO pure� organic/colloid removal and ionexchange packed-bed system.

2.2. Instrumentation

Fourier transform infrared (FTIR) spectrum was recorded usingKBr plate on PerkineElmer Spectrum 2000 Spectrometer betweenthe range of 4000e400 cm�1.

Binding events were followed using a Research quartz crystalmicrobalance (RQCM) with phase-lock oscillator, Kynar crystalholder, and 1-in., Ti/Au, AT-cut, polished, 5-MHz quartz crystals, allpurchased from Maxtek, Inc, New York, USA. The holder wasmounted with crystal face positioned 90� to ground to minimizegravity precipitation onto the surface. The RQCM phase-lockoscillator provided loading resistance measurements and allowedfor the examination of crystal damping resistance during frequencymeasurements. All measurements were made at room tempera-ture. Sensitivity is known to be 56.6 Hz cm2 mg�1 for a 5-MHzcrystal.

The characteristics of the thiol monolayer and MIP film on thesurface of the QCMwere studied by atomic forcemicroscopy (AFM).

2.3. Synthesis of thiourea modified chitosan (TCM)

Three grams of thiourea was dissolved in 60 mL of distilledwater. A 17.1 mL of 50 wt. % glutaraldehyde solutionwas added intothiourea solution in a round flask. The mixture was stirred andheated on a water bath for 3 h at 50 �C. After completion of thereaction, 1.36 g chitosan was dissolved in 30 mL of distilled water,and the solution was then added into the mixture in the flask andheated for 8 h at 70 �C. A large quantity of resin was formed andwashed several timeswith dilute NaOH, distilledwater and acetone.The resinwas dried at 60 �C for 2 h. FTIR spectra is given below: FTIR(KBr, cm�1); 3435 cm�1 (eOH band), 1402 cm�1 (NeC]S band),1156 cm�1 (C]S band), 1658 cm�1 (C]N). The FTIR informationconfirms the suggested structure of the ThioureaModified Chitosan(TCM) given in Scheme 1.

2.4. Synthesis of TCM-Cd(II) chelate monomer

TCM (1 mmol) was dissolved in 30 mL of CH3COOH (1%, v/v),followed by addition of 1 mmol Cd(NO3)2 into the solution by stir-ring. After the mixture was stirred for 3 h at room temperature, theprecipitate obtained by the addition of 200 mL of acetone into themixture was collected by filtration, successively washed with 95%ethanol, and dried to get thiourea chitosan-Cd(II) complex. FTIRspectra is given the following: FTIR (KBr, cm�1); 3436 cm�1 (eOHband), 1384 cm�1 (NeC]S band), 1077 cm�1 (C]S band),1636 cm�1 (C]N).

As a result, when the possible interactions between Cd(II) and“S” atoms were considered, it has been concluded that Cd(II) ionhas mainly coordinated to the “S” atom of TCM; because the con-siderable changes in the infrared frequencies were observed onlyfor those bands containing (NeC]S) groups.

Cd+2

O P O

OEt

OEt

NO2

CH(CH2)3CH=N-C-N=CH(CH2)3CH=ChitosanChitosan=

SH

Scheme 2. The recognition sites of paraoxon imprinted biopolymer.

E.B. Özkütük et al. / Materials Chemistry and Physics 139 (2013) 107e112 109

2.5. Preparation of paraoxon imprinted QCM sensors

Gold surfaces were cleaned in piranha solution for 1 h (1:3 30%H2O2/concentrated H2SO4) before coating. The cleaned gold sur-faces were immersed into allyl mercaptan (0.30 mM) for 24 h, inorder to introduce thiol groups on to gold surface of QCM electrode.The electrode was then washed with ethanol and deionized waterfor 10 min to remove excess of thiols.

For the polymerization, allyl-activated crystals were immersedin the reaction mixture containing the metal-chelate monomer[TCM-Cd(II)] (0.01 mmoL), paraoxon (0.01 mmol) template, andephyclorohidryn crosslinking monomer (0.5 mmoL). Pure nitrogengas was purged into the cell for 5 min to evacuate the air com-pletely since the presence of oxygen would prohibit polymer-ization. The polymerization was carried out at room temparatureapplying UV light irradiation for 4 h under nitrogen atmosphere.The recognition sites of paraoxon imprinted biopolymer was givenin Scheme 2. The non-imprinted polymer-coated QCM sensors, asa reference, were also prepared in a similar manner with [TCM-Cd(II)]. The electrodes were finally washed with 0.5 M NaOH forthe template extraction. Fig. 1 shows the AFM images of MIP coatedgold electrodes. As seen in the figure, the surface roughness anddeepness have been increased after the polymerization on the QCM

Fig. 1. AFM images of the MIP coated gold surface

electrode compared to uncoated QCM electrode. The surfaceroughness and deepness values of the paraoxon imprinted elect-rode were determined as 6.0 nm and 12.61 nm, respectively. Theroughness of surface is well distributed through whole surface ofthe electrode. This result indicates that paraoxon imprinting on theQCM electrode has been homogeneously achieved. This property isone of the important parameters controlling the specificity, selec-tivity and recognition rate of the sensor.

2.6. Monitoring of paraoxon imprinted QCM sensor response

The paraoxon imprinted QCM sensor was used for realtimedetection of paraoxon. The paraoxon was dissolved in 2-propanoland the frequencyof thesensorwasmonitoreduntil it becamestable.The frequency shift (Δf) for each concentration (0.1e1.0 mM) of par-aoxon was determined and the evaluation was performed in tripli-cate. The desorption process was applied using 10 mL of 1 M NaOHsolution. After the desorption step, the paraoxon imprinted QCMsensor was washedwith deionizedwater. For each paraoxon sampleapplication, adsorptionedesorption-cleaning steps were repeated.

2.7. Selectivity of MIP coated QCM sensor

The selectivity of the prepared sensor for paraoxon was esti-mated using parathion which is similar in chemical structure withparaoxon. The QCM sensor was treated with these competitivemolecules. After the equilibrium, the frequency shift of parathionmeasured by prepared paraoxon imprinted sensor and the Δm andQ (nmol) values were calculated according to Eq. (1).

DF ¼ �2F20 ðrqmqÞ�1=2Dm=A (1)

where ΔF is the measured frequency shift due to the added mass inhertz, F0 is the fundamental oscillation frequencyof thedrycrystal,mis the surface mass loading in grams, mq is the density of quartz

on wavemode in the scale of 20 mm� 20 mm.

Fig. 3. Scathard plot of paraoxon imprinted sensor.

E.B. Özkütük et al. / Materials Chemistry and Physics 139 (2013) 107e112110

(2.65 g cm�3), rq is the shearmodulus (2.95�1011 dyn cm�2), andAis the electrode area (0.19� 0.01 cm2). For 5 MHz quartz crystalsused in this work, Eq. (1) predicted that a frequency change of 1 Hzcorresponds to amass increase of 1.03 ng cm�2 on the electrode [45].

The selectivity coefficient (k) for the binding of paraoxon in thepresence of competitor species can be obtained from equilibriumbinding data consistent to

k ¼ Qtemplate molecule=Qcompetitor species

The relative selectivity coefficient (k0 ¼ kimprinted/knon-imprinted)results from the comparison of the k values of the imprinted pol-ymer with non-imprinted polymers allow an estimation of the ef-fect of imprinting on selectivity.

3. Results and discussion

3.1. Measurement of binding interaction of molecularly imprintedQCM sensor via ligand interaction

The binding of paraoxon to the metal-chelate imprinted poly-mer on gold quartz crystals causes a mass change, Dm that isreflected in the crystal frequency. The relationship between Dm andΔf can be expressed by the Sauerbrey’s equation [46].

The imprinted [TCM-Cd(II)-paraoxon] polymer is expected tobind the paraoxon sensing. The frequency of the sensor decreasedafter addition of paraoxon solution, then reached the constantvalue in 90 min (Fig. 2). It can be seen that the reaction reachedequilibrium and these frequency changes strongly indicated thatthe paraoxon molecules were bound to the imprinted polymer onthe quartz crystal. When the non-imprinted polymer was used theparaoxon binding to non-imprinted polymer was much weaker.

Marx and Zaltsman [47] investigated the molecular imprintingof sol gel polymers for the detection of paraoxon in water. The ki-netic profile of paraoxon binding to the polymer matrix was stud-ied, and the saturation was found to occur after ca. 2 h.

The binding interaction and equilibrium information betweenthe imprinted polymer and paraoxon template can be obtained byScathard analysis (Fig. 3) and a tool has already applied [48e50].This analysis employs the following equation:

Q=C ¼ Qmax=KD � Q=KD (2)

where Q is the amount of paraoxon bound to the polymer, as cal-culated by the mass frequency variation upon the addition of an-alyte and C is the concentration of free paraoxon. Qmax representsthe apparent maximum number of binding sites, and KD is theequilibrium dissociation constant of the metal-chelate copolymer

Fig. 2. QCM response of paraoxon imprinted and non-imprinted films(Cparaoxon¼ 0.1 mM).

based on ligand exchange. The obtained Scathard regressionequation for Fig. 3 is

Q=C ¼ �1:0446Q þ 4:9336

So, the association constant (Ka) for the binding of paraoxon toTCM-Cd(II)-paraoxon based MIP sensor is 1.04�103 M�1 and themaximum number of ligand-exchange interaction site, Qmax, is4722 nmol. The value of Ka suggests that affinity of the binding sitesis strong.

3.2. Effect of pH on frequency shift

Molecular imprinting with paraoxon gives a cavity that is se-lective for paraoxon. The paraoxon can simultaneously chelate tometal ion and fit into the shape-selective cavity. So, this interactionbetween Cd(II) ion and free coordination spheres has an effect onthe binding ability of the quartz sensor. The pH value of thedetection medium, one of the effecting parameters during molec-ular recognition processes, also plays an important role for thebinding ability of the quartz crystal sensor. The pH response profilefor paraoxon imprinted sensor was investigated in a 0.1 mM para-oxon solution over the pH range 6.0e10.0 by adjusting with HNO3and NaOH. The sensor responses of paraoxon imprinted QCMsensor at different pH values (pH 6e10) are shown in Fig. 4. As itwas seen in the figure, the frequency shift for the imprinted poly-mer decreased with increasing pH. The apparent binding affinitywas observed at pH 7.0. The increase in pH value causes theincrease in hydroxide concentration which might compete with/inhibit the binding of the template paraoxon molecules. Further,increase in pH value, or hydroxide concentration, promoted theoccupation of coordination sphere of Cd(II) ions with hydroxide

Fig. 4. The effect of pH on binding ability (Cparaoxon¼ 0.1 mM).

Fig. 5. Response of the paraoxon imprinted sensor (pH¼ 7.0).

Table 1Comparison of dedection limit of QCM/MIP sensor.

Organophosphate detection limits

Ref. [19] Ref. [9] Ref. [20] Ref. [21] Ref. [22] Thiswork

0.03 mg L�1 2 mM 10�7 mol L�1 2� 10�7 mgmL�1 0.005 mgmL�1 0.02 mM

E.B. Özkütük et al. / Materials Chemistry and Physics 139 (2013) 107e112 111

molecules instead of paraoxon molecules. This inhibiting effectcauses lower frequency shift in imprinted QCM sensor response.

Alizadeh [51] has also developed the paraoxon imprinted vol-tametric sensor. He has synthesized a molecularly imprinted pol-ymer composed of methacrylic acid and ethylene glycoldimethacrylate as a functional monomer and cross-linker agent,respectively. This polymer has used as the recognition part of anelectrochemical sensor. The pH of parathion solution has noticedand its effect on the paraoxon extraction has investigated. The re-sults of the experiment have showed that in the pH range of 3e7,the paraoxon related electrochemical signals were high andattained about fixed amounts. It has seen that the extraction hasstarted to decrease when the pH is lower than 3 and higher than 7.According to these results the pH 5 has chosen as an optimum pHfor parathion extraction in the electrode.

3.3. Recognition selectivity of imprinted polymer on QCM sensor

The adsorption of parathion that is similar in structures withparaoxon on the imprinted quartz crystal sensor was investigatedfor the better understanding of the specificity of the interactionsbetween the binding sites of the MIPeQCM sensors and its tem-plate molecules, and the ability of this imprinted polymer to rec-ognize the paraoxon molecules. The initial slopes in the sorptionisotherms (Fig. 5) indicate that the frequency changes are directlyproportional to the concentration of biomolecules.

In the sorption isotherms obtained, the initial slope forparathion is 632 Hzmmol L�1, while the initial slope of the curveis 1396 Hzmmol L�1 for paraoxon. The selectivity coefficient of theparaoxon imprinted QCM with respect to parathion is 2.20(1396/632). From this point; we can get the following conclusion:

Fig. 6. Calibration curve for paraoxon.

The TCM-Cd(II)-paraoxon monomer has been incorporated intoa polymer matrix in order to increase the selectivity of paraoxonrecognition on the QCM sensors. The selectivity coefficients ofparaoxon with respect to parathion, is high, because paraoxon cansimultaneously fit into the shape-selective cavity.

3.4. Analytical performances

Fig. 6 shows the calibration curve obtained plotting thefrequency shifts versus the concentration (0.02e1 mM) of paraoxonimprinted quartz crystal. A linear curve was obtained between 0.02and 1 mM of paraoxon. As shown in the figure, when the concen-tration of paraoxon increased, the frequency shift of the QCMwas also increased. The sensitivity was calculated as the slopeat the linear part of low concentration range and found as184 Hzmmol L�1. The detection limit, defined as the concentrationof analyte giving frequency shift equivalent to three standarddeviation of the blank plus the net blank frequency shift, was0.02 mM. The experiments were performed in replicates of threeand the samples were analyzed in replicates of three as well.

Table 1 shows comparison of detection limit of QCM/MIP sensor.The detection limit for our MIP/QCM sensor is 0.02 mM. Our studiesprovide superior to studies in literature.

3.5. Reproducibility

The regeneration of the coated QCM is of critical importance forsensor application. Reproducibility of the same paraoxon imprintedsensor was evaluated by measuring the response signal 10 times ina continuous manner and the obtained results were presented inFig. 7. After each injection of paraoxon and the attainment ofequilibrium, the paraoxon imprinted sensor was recovered bysequential washes with 1 M NaOH solution and deionized wateruntil the frequency of QCM reached a steady value. The sensor canbe stored in deionized water at room temperature when not in use.It was observed from Fig. 7 that the paraoxon imprinted sensorexhibited better reproducibility, and the response signal of the

Fig. 7. Reproducibility of frequency response using the same paraoxon imprintedsensor (Cparaoxon¼ 0.1 mM).

E.B. Özkütük et al. / Materials Chemistry and Physics 139 (2013) 107e112112

sensor was about 4.2% loss at the 7th cycle. The sensor has provedits stability for more than 7 months with a slight loss of sensitivity,which is a significant advantage of the molecular imprintingtechnique employed here. It can be concluded that the paraoxonimprinted sensor can be used many times without the significantdecrease of the response signal.

4. Conclusions

The experimental results have showed that the combination ofa molecular imprinting technique and a piezoelectric quartz crystalmicrobalance is able to function as a simple, specific, and reusablebio-sensing system. The sensor had an excellent storage stabilityand the sensing unit could be regenerated for reuse for a long timeperiod. In this study, we have shown that MIP coated gold sensorscan be used for the selective detection of paraoxon in the liquidphase with a detection limit of 0.02 mM. The natural extension ofthis work is to show a selective paraoxon imprinted sensor whichwas improved using metal coordination (metaleligand chelate)binding mode. The frequency shift for the paraoxon imprinted andnon-imprinted QCM decreased with increasing pH. The value of Ka(1.04�103 M�1) that was obtained using Scathard graph suggeststhat the affinity of the binding sites is strong. In addition, theselectivity experiments showed that the selectivity coefficients ofthe TCM-Cd(II)-paraoxon complex with respect to parathion isquite high.

Acknowledgments

This work has been supported by Eskisehir OsmangaziUniversity, Commission of Scientific Research Projects (Project No:200619004).

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