Development of Uric Acid Sensor Based on Molecularly Imprinted Polymer-Modified Hanging Mercury Drop Electrode

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Development of Uric Acid Sensor Based on Molecularly ImprintedPolymer-Modified Hanging Mercury Drop Electrode

Dhana Lakshmi, Piyush Sindhu Sharma, Bhim Bali Prasad*

Analytical Division, Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi – 221005, India*e-mail: [email protected]

Received: January 06, 2006Accepted: February 16, 2006

AbstractUric acid (UA) sensor based on molecularly imprinted polymer-modified hanging mercury drop electrode wasdeveloped for sensitive and selective analysis in aqueous and blood serum samples. The uric acid-imprinted polymerwas prepared from melamine and chloranil and coated directly onto the surface of a hanging mercury drop electrode,under charge-transfer interactions at þ0.4 V (vs. Ag/AgCl), in model 303A electrode system connected with apolarographic analyzer/stripping voltammeter (PAR model 264A). The binding event of uric acid was detected in theimprinted polymer layer through differential pulse, cathodic stripping voltammetry (DPCSV) at optimizedoperational conditions [accumulation potential þ0.4 V (vs. Ag/AgCl), accumulation time 120 s, pH 7.0, scan rate10 mV s�1, pulse amplitude 25 mV]. The limit of detection for UA was found to be 0.024 mg mL�1 (RSD¼ 0.64%, S/N¼ 3). Under the optimized operational conditions, the sensor was able to differentiate between uric acid and otherclosely structural-related compounds and interfering substances. Ascorbic acid (AA), a major interferent in UAestimation, was not adsorbed on the surface of sensor electrode. The present sensor is, therefore, UA-selective at allconcentrations of AA present in human blood serum samples. The precised and accurate quantification of UA havebeen made in the dilute as well as concentrated regions varying within limits 0.1 – 4.0 and 9.8 – 137.0 mg mL�1,respectively.

Keywords: Uric acid sensor, Molecularly imprinted polymer-modified hanging mercury drop electrode, Differentialpulse, Cathodic stripping Voltammetry, Human blood serum

DOI: 10.1002/elan.200603478

1. Introduction

Molecular imprinting is a technique for the fabrication ofbiomimetic polymeric recognition sites or plastic antibod-ies/receptors, which is attracting rapidly increasing interest[1]. It has become nowadays a well-established analyticaltool [2, 3] to produce artificial recognition elements of lowerprice and higher thermal/chemical stability. The techniqueinvolves copolymerization of functional and cross-linkingmonomers, where template molecules (test analytes) arebound via hydrogen bonding, electrostatic forces or hydro-phobic interaction [1]. Alternatively, a template –monomeradduct is preformed via reversible covalent bonds beforepolymerization [4]. On subsequent extraction of the tem-plate, the molecularly imprinted polymer (MIP) retains“molecular memory” of the template, representing as anideal candidate for sensor development [5, 6]. Majority ofthe MIP-based sensors involves mass accumulation in theMIP coating with the transduction principle of a quartzmicrobalance [7] or a surface acoustic wave resonator [8].The detection of template utilizes corresponding physico-chemical property [3] like fluorescence, absorbance, lumi-nescence and induced scintillation or surface plasmonresonance.

Despite the increasing numbers of MIP reports onseparations and mass-transduction sensors, a very limitedwork have been carried out on the design of electrochemicalsensors based on molecular imprinting technology. Severalpiezoelectric transducers are known, which rely exclusivelyon theMIP element for the selective detection in capacitive,conductometric, amperometric, field-effect and voltammet-ric transduction systems [3]. However, selectivity may beenhanced if voltammetry is used as transducer because thepotential necessary for generation of the analytical signal ischaracteristics of the species that is oxidized or reduced.This obviates interferences from structurally similar com-pounds as usually encountered during the use of MIP insolid-phase extraction (SPE). A sensor is, therefore, de-signed with enhanced selectivity and added advantages oflow cost, small size, lowdetection limit and easy automation.The diffusion of analyte is governed by controlling poly-meric layer thickness, particularly in the case of a non-conducting and highly cross-linked MIP grafted onto theelectrode surface, and the selective measurement of tem-plate is tested in complex matrices of natural samples. TheMIP-film should also have a membrane forming propertywith hydrophobic-hydrophilic character. If layer thickness isexcessive, the recognition sites far away from the electrodesurface may cause a strong diffusion impediment.

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Electroanalysis 18, 2006, No. 9, 918 – 927 I 2006 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

In the present investigation, the voltammetric sensor wasprepared by drop coating on hanging mercury dropelectrode (HMDE) surface with a solution of preformedMIP as recognition element [9]. The evaporation of solvent(porogen) from the electrode surface resulted in a rigidlayer, whichwas found to be insoluble in thewater and otherorganic solvents. The modified HMDE with a tiny hangingmercury drop has been considered a method of choice tomitigate the major problems associated with solid electro-des. These problems are saturation of sites, nonspecificbinding, electrode fouling and swelling/shrinkage of film.Moreover, if the solid sensor is to be re-used, a cumbersomeprocedure for surface renewal such as potential cycling,mechanical polishing and thorough washing has to befollowed. On the other hand, the proposed modifiedHMDE is reproducible and easily renewable. The MIPpreparation is simple and cost-effective and has no problemof deformation of cavities during template extraction andsubsequent coating of polymer onto electrode surface. Theselection of MIP-modified HMDE in the present study isappropriate for analysis in a laboratory setting and can betaken as an alternative to the low-cost disposable trans-ducers in electrochemical sensors.Uric acid (UA) is a principal end product of purine

metabolism [10]. Therefore, its determination serves as amarker for the detection of disorders associated with purinemetabolism such as Gout and Lesch –Nyhan Syndrome[11]. The physiological UA serum levels range from 4.1 to8.8 mg dL�1 and urinary excretion is typically 250 – 750 mgdL�1 [10]. Although the abnormal UA in urine and bloodserum is symptom of several diseases such as gout, hyper-uricemia and heavy hepatitis, its rapid and selectivedetermination with low detection limit is required in thecomplex matrices of biological fluids. Finally, low detectionlimits are required because of the low concentration of UAin biological systems such as heart microdialyzates. Therapid determinations of UA are used in real-time monitor-ing in order to determine fast concentration changes,particularly in the myocardial interstitial space whereadenosine and its metabolites such as UA are importantmarkers of ischemia and regulators of blood flow, and mayproduce cardioprotection against ischemia.Electrochemical methods have received much attention

for UA analysis because of more selective, less expensiveand less time consuming measurements than colorimetricand enzymatic methods [12, 13]. One of the major problemsin the determination of UA by electrochemical methods isthe presence of AA as a main interferent in biologicalsamples.Electrodes modified with poly (4-vinyl pyridine), over-

oxidized polypyrrole, poly (N,N’-dimethylaniline), polygy-cine, osmium complex Nafion bilayer, clay-Nafion, methyl-ene blue/sol – gel and electrodes modified with self-assem-bledmonolayers of thiols havebeenused successfully for thedetermination of UA in the presence of AA [14]. However,these modified electrodes involved tedious preparationprocedures or sufferings from instability and lack ofsensitivity. As far as carbon-based electrodes are concerned

for the determination of UA, only carbon paste electrodecould be used to detect low concentration of UA in thepresence of AA in alkaline medium though it was foundto be swollen after repeat measurements [14, 15]. Recently,the selective determination of UA in the presence ofhigh concentration of AA in 0.1 M HClO4 with the useof oxidized diamond film electrode has been reported[14].Although electrochemists succeeded in developing sev-

eral sensitive techniques of UA detection in biologicalfluids, the selectivemeasurements in the presence of severalinterferents are still limited. Insofar as MIP-based solidelectrode sensor is concerned, the relatively poor sensitivityowing to slow analyte permeability across the film is stillcrucial owing to intensive interferences and nonspecificbindings caused by the complex matrices of the samples.This could be the reason that the solid sensors reported todate have been found to be tested with standard samplesonly. However, there are a few reports of MIP sensors foranalysis in real samples [16 – 18]. The present work meritsspecial mention because of the fact that this is the first workdescribing the UA analysis in blood serum samples with aMIP-modified HMDE. This primarily involved an electro-chemical oxidation of UA at pre-anodized electrode fol-lowed by cathodic stripping to generate DPCSV signals.

2. Experimental

2.1. Chemicals and Materials

The reagents melamine (mel), chloranil (chl) and uric acid(UA) were purchased from Loba Chemie, India. Allchemicals were of analytical grade and the solvent dimethylformamide (DMF), was of HPLC quality. The stock stand-ard solution ofUric acidwas prepared by dissolving 0.1681 gof UA and 0.0800 g of lithium carbonate in about 20 mLwater (triple distilled, deionized), heating at 80 8C, anddiluting after cooling with water to 100 mL in a volumetricflask. The working UA standard solutions were prepared byappropriate dilutions and the desired pH values weremaintained with the help of aqueous solutions of sodiumhydroxide or nitric acid.

2.2. Instrumentation

Voltammetricmeasurementswereperformedwith apolaro-graphic analyzer/stripping voltammometer (Model 264AEG&GPrinceton Applied Research, USA) in conjunctionwith a X–Y recorder (PAR Model RE 0089). In the threeelectrode cell of a 303A static mercury drop unit (EG & GPrinceton Applied Research), a hanging mercury dropelectrode (HMDE, surface area 0.0092 cm2), a saturatedAg/AgCl electrode with porous Vycor frit, and a platinumwire electrodewereused asworking, reference andauxiliaryelectrodes, respectively. IRandNMRspectrawere recordedusing JASCO FT/IR5300, and JEOL AL 300 FT NMR

919Uric Acid Sensor Based on Hanging Mercury Drop Electrode

Electroanalysis 18, 2006, No. 9, 918 – 927 www.electroanalysis.wiley-vch.de I 2006 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

www.electroanalysis.wiley-vch.de

(JAPAN), respectively. Elemental analyses were performedusing Heraeus Vario EL111 Carlo Erba 1108.

2.3. Synthesis of UA-Imprinted Polymer

The preparation of UA imprinted polymer [abbreviatedhereafter as P(UA)] involved two steps: i) polymerizationbased on the TakagishiPs system approach [19] and ii)extraction of the template (Fig. 1). In TakagishiPs approach,instead of mixing the template (UA) with functionalmonomers (mel and chl) prior to polymerization, linearpolymeric chain is mixed with the template, and the pre-existing polymer is either cross-linked or further polymer-ized. However, in the present study, cross-linking wasavoided in the polymer chain so as to overcome any sterichindrance toward mass-transfer via template diffusionacross the linear polymeric membrane. The binding forcebetween the templatemolecules and imprinted polymerwasa kind of noncovalent or more specifically multiple hydro-gen bondings in DMF porogen. However, these hydrogen

bondings were basically hydrophobically driven, on rebind-ing of the template in aqueousmedium, as a consequence ofeffective hydrophobic interactions between imprinted pol-ymer and template molecules. Despite the fact that polarsolvent does not favor hydrogen bonding, the imprintingprotocol (Fig. 1) in the present instance of hydrophobicenvironment necessarily involvedmultiple hydrogen-bonds[20] for template recapture.Normally in a single batch, the equimolar DMF solutions

of mel (1.26 g/10 mL) and chl (2.46 g/10 mL) were mixedtogether and heated at ca. 160 8C for an hour followed by theaddition of an equimolar DMF solution of the template(1.68 g UA/10 mL) into the reaction mixture. A brownish-black colored mel-chl-UA adduct, in the form of DMFslurry, was obtained after the complete evaporation of theDMFat 160 8C.The residualmonomers, if any,werewashed-off from this slurry with ethanol. The polymer adduct wascompletely dried over rota vapor for approximately 10hours and finally ground to powder. The non-imprintedreference (blank) polymer, mel-chl copolymer [P (Rf)], wasprepared in the similar manner in the absence of UAtemplate. Deionized hot water was used to extract UAtemplate from the adduct. The extractions were carried outbatch-wise (5.0 mL hot water portions, n¼ 7, shaking time10 min) until no further UA in the filtration was measured.BothP (UA)andP (Rf) polymerswere found tobe insolublein water. These polymers, with identical structures [m.p.300 8C (distortion)], correspond to the following elementalanalyses: [found (%) C¼ 36.15, H¼ 4.10, N¼ 24.76; calcu-lated (%) C¼ 36.57, H¼ 3.26, N¼ 24.88 for P (UA) (nþ 1)DMF.H2O; n¼ 9] and the spectral data: IR (KBr, lmax, cm

�1)at 3429 (aryl –NH2 stretching), 1500(�NH bending), 1390(�CNstretching vibration), 1643 (>C¼Ostretching), 779.31(terminal>C�Cl), and 1H NMR (DMSO d6, 300 MHz,TMS,dppm): 3.44 (aromatic (18) amine), 2.51 (aromatic (28)amine).While removing template UA from the mel-chl-UA

adduct, chloride ions were also washed off. Thus, anyestimation of chloride ions had been possible only from theadduct sample. The conductometric titration inDMF-watermedium indicated 3.79 mol Cl�/1000 g which correspondedto the proposed structures shown in Figure 1.

2.4. Voltammetric Method

In the present investigation, themodification ofHMDEwasmade using imprinted polymer [P(UA)] in all measure-ments. For this, an optimized amount (200 mg mL�1) of P(UA) in 10 mL DMF was taken into a voltammetric cellcontainer allowing HMDE to be submerged for 120 s atþ0.4 V (vs. Ag/AgCl) for electrode coating. During thisperiod, the stirring was on which later brought to equilibra-tion suspending the potential scan with the help of a holdpushbuttonprovidedwith the instrument. The cell containerwas then removed and the electrode was rinsed by insertingin a water-containing cell to avoid any carry over of theDMF solution, if any, in the surrounding. The hanging

Fig. 1. Schematic representation of the preparation of molecu-larly imprinted polymer for UA.

920 D. Lakshmi et al.



mercury drop was not dislodged from siliconized capillaryorifice after this treatment. This is because of a solenoid-actuated valve control mechanism provided with stationarymercury drop unit, even though there is no liquid in the celland that too at open circuit for a moment. At this stage,another cell container, containing 10 mLofUA(prepared inLi2CO3 aqueous solution), was brought under the modifiedelectrode for an optimized accumulation period (tacc) of120 s atþ 0.4 V (vs. Ag/AgCl) under quiescent condition.As the hold pushbutton of the instrument was released free,the polarographic analyzer was all set to record strippingvoltammogram through a cathodic, differential pulse scan(scan rate 10 mV s�1, pulse amplitude 25 mV, equilibrationtime 15 s). The scan was terminated at � 0.6 V (vs. Ag/AgCl). Since themercury drop automatically drips out fromthe capillary orifice, while the run pushbutton is on forsubsequent measurement, the quantification of the record-ed DPCSV peak was made by the method of RstandardadditionP in the same cell but with a freshmercury drop dulymodified with the imprinted polymer, under identicalconditions of operation. Each concentration of UA testanalyte was measured as stated above with a fresh MIP-modified HMDE at 25� 1 8C. The deaeration of the cellcontent was avoided in order to retain hanging drop at theorifice of capillary electrode. The above voltammetricprocedure was also performed using the reference polymer[P(Rf)]-modified HMDE, under identical operational con-ditions.

3. Results and Discussion

3.1. Sensor Development

Imprinted polymer, P(UA), is a linear polymer with back-bone and pendant functional groups consisting of alternat-ing amine and carbonyl moieties. On evaporation of DMFsolution of P(UA) from the surface of preanodized HMDEat þ0.4 V vs. Ag/AgCl, a water-insoluble polymer filmsurrounding the electrode surface was firmly coated as arigid layer owing to the interaction between electron-richpolymer backbone and positively charged electrode. Ad-justing the concentration of polymer in the casting solutioncould control the porosity and permeability of the film. Thependant functional groups in the monolayer film wereexposed outwardly providing all binding cavities accessiblefor template recapture, via multiple hydrogen bondingmechanism, in the diffused part of an electropositiveelectrical double layer.Any attempt to modify a glassy carbon electrode with

P(UA) was a total failure owing to its non-adherence andelectrode fouling risk in the complex media of real samples.The mercury thin film electrode, whether bare or modified,has equal possibility of contamination in complex matricesand requires a critical regeneration step for reuse. It shouldbe born in mind that MIP-interfacing with transducerusually requires MIP-particle entrapment either in gel orin membrane and sometimes in MIP suspension with the

help of an inert and soluble plasticizer and as such, criticalproblems of longer response time, nonspecific analytebinding, and diminishing binding capacities are likely toemerge. As a distinct measure, in the present investigation,the recognition property of the imprinted polymer wasutilized over the surface of modified HMDE so as totranslate the binding event of UA directly into the corre-sponding voltammetric signal. The DPCSV resulted insuperior peaks with baseline better than the linear sweepvoltammetry. This entails an effective preconcentration stepvia facilitatedmultiple hydrogen-bonding inwater than thatin non-aqueous porogen (DMF) medium [18, 20]. The film-encapsulated UAmolecules are instantaneously oxidized atpositive potential prior to DPCSV scan.The drop coating on HMDE surface with an optimized

amount of the preformed polymer as recognition element isan easier and fast technique [9] before performing a solutionexchange for every experiment. The entiremeasurement foreach sample can be accomplished with a single modifiedmercury drop with PAR model 303 A static mercury dropsystem. The modified mercury drop electrode can berenewed in reproducible manner for further experiments.The uniform MIP layer at positive potential does not allowUAmolecules to make a direct contact with the mercury toform corresponding salt [21]. This obviates nonspecificsorptions and electrode fouling as usually encountered withnonmodified mercury and other solid electrodes.The mechanical stability of the modified HMDE was

favored even at high positive potential. This was because ofthe mercury-MIP charge-transfer complex formation caus-ing an overall electron-deficiency in the MIP-film. Thus,preanodization of the electrode promotes a type of electro-catalytic effect in the P(UA)-modified layer.The conformational changes (cavity deformations) during

the film solidification on the electrode have been ruled out[22]. The shape of the binding sites of MIP is not lost afterdissolution in DMFas P(UA) is instantly soluble and can berecovered intact without any deformation of bindingcavities after solvent evaporation. This was supportedfrom a chromatographic experiment in which MIP as suchand MIP obtained after redissolution in DMF had demon-strated the imprinting with identical efficiency followingsimilar retention mechanism at optimized conditions (flowrate, 5 mL min�1; pH, 7.0; UA extraction volume, 10 mLwater; column, 13.0 cm long and 1.4 cm id) of columnoperation. For further substantiation, HMDEwas modifiedwith mel-chl-UA adduct polymer/DMF solution at þ0.4 Vvs. Ag/AgCl. After UA retrieval by multiple hot waterextractions (n¼ 5, 10 mL), directly from the electrodesurface, the DPCSV current (0.69 mA) similar to that witha P(UA)-modified HMDE was observed for the knownconcentration (28.30 mg mL�1) of analyte.The hydrogen bonding interactions between P(UA) and

UAhave been supported from the fact the 1HNMR spectralpeaks for amines (mel) in the MIP were observed to beshifted to 0.38 ppm (for�NH2) upfield and 0.024 (for�NH)ppm downfield and UA peaks appeared at 7.55 (s) for�NHCO group upon UA binding. A very sharp peak for




quaternized nitrogens in mel-chl-UA adduct was noticed at8.25 ppm. Similarly, FT-IR frequencies were found to beshifted downwardly by 44 and 24 cm�1 for mel amines(broad) and chl carbonyl groups, respectively. All peakscorresponding UA at 1665 cm�1 (�CO), 3070 cm�1 (lactamring) and 577, 995, 774 cm�1 (finger print peaks) were foundto be appeared along with the characteristic polymerskeletonpeaks [3385, 3312 (amine) and 2096 cm�1 (aromaticamine salt)] in the hydrogen bonded mel-chl-UA adduct[20]. Interestingly, the imprinted polymer, P(UA), obtainedafter UA removal, reassumed all corresponding peaksidentical to the reference polymer [P(Rf)]. Moreover, 13CNMR spectra (DMSO) of mel-chl-UA adduct revealed theappearance of the�NHCO characteristic peak of UA. Thisreadily disappeared on hot water extraction in P(UA)corroborating the successful imprinting and extraction stepsin MIP synthesis.

3.2. Electrochemical Behavior

The imprinted polymer [P(UA)]-modified HMDE was setat þ0.4 V vs. Ag/AgCl in UA/Li2CO3 aqueous solution foraccumulation time 120 s and cyclic voltammograms (CV,Fig. 2) were recorded in cathodic strippingmode. The use ofsupporting electrolytes (KCl, NaNO3, borax, acetate, diso-dium hydrogen phosphate) was deliberately avoided tocheck anionic oxidation at positive potential. It is furtherknown that borate and chlorate buffers, which are report-edly reactive particularly toward chloranil>C¼Oof P(UA)polymer, cannot be used as a supporting electrolyte [18].However, one may note that the physiological amount ofsuch salts, if any, in the blood samples is insignificant tohamper our investigation. In the present study, lithiumcarbonatewas found to be inactive toward anionic oxidationin an aqueousmediumof pH 7.0 adjustedwith diluteHNO3.In between the pH 5.3 and 8.5, UA exists as anionic form,which has an apparent tendency to accumulate over thepositively charged MIP-modified HMDE. A higher magni-tude of UA current obtained at pH> 8.5 were presumablydue to the interference of OH� ion [23]. In all measure-

ments, the pH value of the cell content was maintainedneutral by adjusting with dilute HNO3 in order to avoid anycomplication toward hydrogenbonding and also to facilitatethe oxidation of UA at positive potential.The film entrapped UA molecules were readily preoxi-

dized during accumulation which subsequently, after 15 sequilibration time, demonstrated a broad cathodic strippingpeak at �0.3 V followed with no anodic peak on reversescan. This indicated slow electron-transfer kinetics at thescan rate of 10 mV s�1 (Fig. 2). However, the cathodicstripping peak at �0.4 V and a small re-oxidation peak atþ0.15 V with a pre-adsorption anodic peak were observedat higher scan rate of 50 mV s�1. The appearance of acathodic stripping peak at higher negative potential mightbe attributed to the hydrophobically driven hydrogenbonding of UA molecules in MIP cavities which required arelatively high energy for the cathodic stripping. The slightlyeasier stripping with smaller current at low scan rate couldbe attributed to the lesser extent of hydrophobicity andrestricted hydrogen binding as a consequence of somehydration of oxidized form ofUAbefore cathodic stripping.It is to be noted in this context that water promotes poorhydrogen bonding because of hydration effect. Despite thefact that the re-reduction peak of UA oxidation is notreported in the literature [10, 24]. The increased reactionreversibility on cathodic scan and reduced rate of hydrationafter pre-oxidation of UA at þ0.4 V vs. Ag/AgCl revealedthe presence of strong electrocatalytic effect in the MIP-modified layer over HMDE surface [24]. The charge-transport through redox centers is believed to occur via anelectron hopping process between redox centers incorpo-rated in the MIP film and the positively charged electrodesurface [25] under the net effect of electropositive charac-teristic of diffusion layer. The electron-transfer mechanism(Fig. 3) as reported earlier [26] is apparently most plausibleto explain the aforesaid voltammetric behavior. Accord-ingly, the encapsulated UA (I, Fig. 3) is initially electro-chemically oxidized in a 2e�� 2Hþ process to give a bis-imine species designated as II (Fig. 3). The bis-imine formedfromUAcould exist in two tautomeric forms (IIa, IIb). Sucha system of conjugated double bonds is expected to be

Fig. 2. Typical cathodic stripping cyclic voltammograms of UA with MIP-modified hanging mercury drop electrode [(run a) [UA]:100 mg mL�1, scan rate: 10 mV s�1; (run b) [UA]: 100 mg mL�1, scan rate: 50 mV s�1 MIP concentration : 200 mg mL�1, MIP and UAaccumulation potential : þ 0.4 V (vs. Ag/AgCl); deposition time of polymer (td): 120 s; accumulation time of UA (tacc): 120 s, pH 7.0].




electrochemically reducible. The expected ease of reductionof structures IIa and/or IIb along with their expected facilehydration across the imine �N¼C�double bonds mayaccount nicely the observed cyclic voltammetry of UA(Fig. 2). The bis-imine was readily reduced on cathodic scanand re-oxidized toUA to give peaks Ic and Ia, at higher scanrate. The appearance of a prewave to Ia is presumablybecause of strong adsorption ofUA(anionic format pH 7.0)in the anodic scan. However, at slow scan rate, the bis-iminetautomers (IIa and IIb) were hydrated upon consuming onemolecule of water to give III (Fig. 3). This compoundrevealed an irreversible cathodic peak (I’c) (Fig. 3) as aconsequence of reduction from III to IV (Fig. 3) at slow scanrate. Further addition of water molecule to III to form uricacid 4,5-diol (V, Fig. 3) is apparently restricted due to thehydrophobic environment of MIP film at the modifiedelectrode surface.The DPCSV runs of UA at P(UA)-coated HMDE are

shown in Figure 4. Accordingly, DPCSV peak at higherconcentration of UA> 10 mg mL�1 appeared at�0.15 V vs.Ag/AgCl. In the lower range of concentrations of UA, thiswas found to be shifted toward negative potential presum-ably because of predominating hydrophobically driven

strong hydrogen bond interactions between UA andP(UA) in dilute aqueous conditions and that required thehigher energy for stripping [27]. On the other hand, P(Rf)failed to bind UA at lower level of concentration under theoptimizedDPCSV conditions (Fig. 4, g). The blank polymerrevealed some nonspecific binding (Fig. 4, h) while theMIPsensor responded a symmetric and quantitative peak (Fig. 4,e) for UA concentration 19.23 mg mL�1. This confirms thesignificance of imprinting in a molecular receptor. AllDPCSVpeaks in the present investigation correspond to themechanism discussed (Fig. 3) for the cathodic peak in CVmode at 10 mV s�1. The negative shift (ca. 150 mV) withbroader CV stripping peak as compared to correspondingDPCSV signal reflects a slow electron-transfer kinetics at10 mV s�1 scan rate. However, DPCSV peaks are relativelysharp and quantifiable than CV, in spite of some asymme-tricity inDPCSV peak in the higher range of concentrationsof UA due to the partial contributions of oxygen andadsorption currents in the foot of cathodic wave.

3.3. Optimization of Analytical Parameters

The modified HMDE was examined for its imprintingperformance by recording an optimised DPCSV responsefor two kinds of saturation experiments. In the first experi-ment, the current response for analyte (100 mg mL�1, fixed)was measured against increasing concentration of P(UA)-DMF solution. The accumulation time (tacc¼ 120 s) anddeposition potential of [Eacc¼þ0.40 V (vs. Ag/AgCl)] ofUA over modified HMDE were same as those adopted forpolymer coating. The Eacc was preferred as þ0.4 V (vs. Ag/AgCl) since any potential lesser than this may leadreduction in DPCSV current owing to the less favoredelectrostatic encapsulation of the test analyte (prevalent inanionic form at pH 7.0) into the cavities of MIP film. Thepoor analyte diffusion at Eacc�þ0.2 V revealed drasticculmination in electrocatalytic action of themonolayer film.The concentration of P(UA) in the voltammetric cell usedfor HMDEmodification, which responded an optimumUAbinding by revealing a maximum current, was found to be200 mg mL�1. The drastic fall in UA current beyond thisconcentration of polymer was probably due to some sort ofstack aggregations of the polymer molecules throughintermolecular electrostatic interactions under the pool ofelectron-withdrawing effect of modified HMDE condi-tioned at þ0.4 V vs. Ag/AgCl. This consequently blockedthe vacant recognition cavities and thereby UA entrapmentresulting in a diminished current.In the second kind of saturation experiments, the HMDE

was modified with fixed amount of imprinted polymer[P(UA), 200 mgmL�1] andDPCSV responseswere recordedfor varying concentrations of template analyte (UA). Thisrevealed complete saturation in the binding sites due tooptimum analyte uptake at 137 mg mL�1 when UA concen-trationswere varied from9.8 to 150 mgmL�1. Surprisingly anearlier saturation in the current response in the dilute rangeof analyte concentrations was observed at 4.0 mgmL�1 when

Fig. 3. Primary electrochemical oxidation and reduction cyclicvoltammetric peaks observed for UA.




UA concentrations were initially measured from 0.10 to4.5 mg mL�1 in a new set of experiment. This could bepossible in an aqueous-rich media, where hydrophobically-driven hydrogen bonding was dominant to cause an earlyoptimization of UA uptake. However, in the UA concen-trations beyond 9.8 mg mL�1, there were initially somewhatcounterbalanced hydrophobic and electrostatic interactionsand later an enhanced charge repulsion between electron-deficient membrane and bis-imine at film/analyte interfaceduring cathodic scan. Consequently the cathodic stripping isfacilitated enough to give rise to a longer linearity in thecurrent response. The reference polymer [P(Rf)] –modifiedHMDE responded a negligible binding of UA below 10 mgmL�1 but showed about one half of the current responses ascompared to the MIP-modified electrode for UA concen-trations varying from 9.8 to 137 mg mL�1. The imprinting

factor, that is the ratio of current response of MIP to thecurrent response of P(Rf), could be considered as ill-definedowing to the contributions from nonspecific binding at theP(Rf) –modified HMDE, in the higher concentrationregion of the test analyte. Under the optimized conditions[Eacc¼þ0.4 V, tacc¼ 120 s, pH 7.0 (UA/lithium carbonateaqueous solution), scan rate 10 mV s�1, Pulse amplitude25 mV], one may opt either of the concentration regions asand when the situation demands. Because, correspondingbinding isotherms followedwith excellent linearity betweenthe cathodic peak current (Ipc,mA)andUAconcentration (c,ppm) [Ipc¼ (0.3122� 0.0002)cþ (0.0037� 0.0011] for con-centration 0.1 – 4.0 mg mL�1; Ipc¼ (0.0212� 0.00006)cþ(0.0856� 0.0003) for concentration 9.8 – 137.0 mg mL�1,with correlation coefficient 1.0 and confidence limit 95%]for both sets of aliquots prepared in distilledwater. The limit

Fig. 4. Differential pulse cathodic stripping voltammograms of uric acid (UA) with MIP-modified hanging mercury drop electrode [UAconcentration (mg mL�1): a) 0.099; b) 0.398; c) 0.74; d) 9.8; e) 19.23; f) 57.52] and with reference polymer P(Rf)-modified hangingmercury drop electrode [UA concentrations (mg mL�1): g) 0.74; h) 19.23], MIP concentration: 200 mg mL�1, MIP deposition time (td) andaccumulation time of UA (tacc): 120 s, MIP and UA accumulation potential: þ0.4 V. (vs. Ag/AgCl), pH 7.0, scan rate: 10 mV s�1.




of detection (LOD) is calculated as 0.024 mg mL�1 (RSD0.64%)on thebasis ofminimumdistinguishable signals (sm)and the slope of linear regression for lower concentrationfollowing the equation (28)LOD¼ (Sm� S�bl) /m, where Smis equivalent to the sum of mean blank signal S�bl plus amultiple 3 of the standard deviation of the blank (Sbl).In order to check the validity of the proposed method, an

additional LC-DPCSV experiment involving SPE proce-dure was followed for the selective and quantitative analysisof UA at optimized conditions [pH 7.0, flow rate 5 mLmin�1, column 13.0� 1.4 cm, packing material P(UA)-modified silica-gel] and the corresponding results are shownin Table 1. The studentPs RtP test and correlation coefficient(v) for comparison of both methods (tcal� ttab, confidencelevel 95%, v¼ 1.0) revealed the similar precision within theconcentration range studied. However, the MIP-basedHMDE sensors appears to be superior than LC-DPCSVand other spectrophotometric methods [29] for UA analysisinsofar as solvent and time consumption as well as theeconomy of the method are concerned.

3.4. Effect of Interferences

The binding specificity of UA-imprinted polymer wasstudied for individual interferents and UA analogues viz.,ascorbic acid, cytosine, uracil, urea, histidine, adenine,theophylline and glucose in test solution (Fig. 5). Themodified HMDE sensor was found to be responsivequantitatively forUAand less responsive for urea, histidine,uracil and cytosine (Fig. 5). However, P(UA)-modifiedHMDEhad shownno recognition for caffeine, theophylline,glucose, thiourea, ascorbic acid and adenine. In the case ofblank polymer [P(Rf)], all interferents (except glucose andthiourea) produced significant responses when presentalone in the test solution (Fig. 5). As regard to the mixture

analysis, where most of the interferents coexist with UA inlots of samples, the UA uptake was found to be highlyselective and quantitative at optimized conditions ofanalysis (Table 2) using P(UA)-modified HMDE.Although caffeine, adenine, theophylline are of nine-

member ring molecules, which are similar to UA, they aredifferent in the number and types of functional groups. TheP(UA) prepared was known to be of a noncovalent type. Asa result, the ability to form hydrogen bonds with the MIPwas certainly different. In addition, it could be expected thatthe major detection interferences of UA by MIP in clinical

Table 1. Analytical results of DPCSV measurements of uric acid (UA) in distilled water at imprinted [P(UA)] modified hangingmercury drop electrode (HMDE). Recovery: amount of analyte detected/amount of analyte taken; nd: not detected; t : StudentPs t-testfor comparison of two methods at confidence level of 95%; v : correlation coefficient.

Uric acid (UA) Analyte concen-tration (mg mL�1)

Determined value [a] mean� SD (mg mL�1) Recovery (%) RSD (%)(n¼ 3)

t V

with P(Rf)- with MIP-modifiedmodified HMDEHMDE

Lower 0.10 nd 0.11� 0.01 (0.11) 110.0 9.09concentration 0.20 nd 0.21� 0.01 (0.20) 105.0 4.76Range 0.40 nd 0.40� 0.01 (0.39) 100.0 2.50

0.57 nd 0.57� 0.01 (0.58) 100.0 1.75 0.40 (cal) 1.000.74 nd 0.74� 0.01 (0.75) 100.0 1.35 2.44 (tab)1.15 nd 1.15� 0.02 (1.16) 100.0 1.742.00 nd 2.02� 0.02 (2.01) 101.0 0.99

Higher 9.80 9.05� 0.18 9.80� 0.01 (9.71) 100.0 0.10concentration 19.23 6.61� 0.04 19.23� 0.09 (18.89) 100.0 0.47 0.85 (cal) 1.00Range 93.49 57.71� 0.75 93.30� 0.27 (93.73) 99.8 0.29 2.77 (tab)

124.06 77.60� 0.32 124.16� 0.20 (125.55) 100.1 0.16137.68 nd 137.60� 0.70 (138.23) 99.9 0.50

[a] Average of three determination, S/N¼ 3, values in parentheses denote UA concentration (mg mL�1) determined by solid-phase extraction (SPE)method with P (UA)-modified silica gel sorbent at flow rate 5 mL min�1 and pH 7.0.

Fig. 5. Sensor response for 9.8 mg mL�1 solution of UA and itsinterfering molecules. Caffeine: Cff, Theophylline: Theo, Glucose:Glu, Urea, Histidine: His) Thiourea: Thio U, Uracil, Ascorbicacid: AA, Adenine: Ad, Cytosine: Cy, and Uric acid: UA.




applications might come fromAA, urea, cytosine, thiourea,uracil and histidine [30]. The stereoshapes of AA andhistidine (five-member ring) as well as cytosine, uracil andglucose (six-member ring) are quite different from UAdespite some common functional groups present in theirstructures. It is interesting that even thiourea and urea,which are small enough to “fit” to virtually any binding sites,did not produce a responseof comparablemagnitude toUA.Therefore, the binding results elucidated the stereoshaperecognition ability of the MIP toward UA. We can assumethat it is not only the size of molecule that governs theinteraction between the analyte and polymer; the sensorresponse is probably a summation of two processes, abinding event with higher affinity to the polymer followedby transduction to produce a voltammetric signal at specificredox potential of UA.The proposed sensor was used for UA analysis in human

blood serum samples. The results are portrayed in Table 2.In order to ascertain the correctness of the results, allsamples were spiked with certain amount of UAwhich ledquantitative recoveries of UA from blood samples. Thedistortion and asymmetricity in voltammetric peak asusually encountered due to the cosorption ofmanypotentialinterferents in human serum sample have not been observedin DPCSV runs obtained with the proposed P(UA)-basedsensor (Fig. 6). However, the corresponding broadness ofpeak (sample without spike) might have some contributiondue to the anionic oxidation of anticoagulant species (e.g.

fluoride and citrate). Nevertheless, this did not affect theaccuracy of the result as symmetricity of the peaks wasprogressively improved after UA spiking to the bloodsamples under investigation.

4. Conclusions

The simplicity of preparation and versatility, with a rapidlyrenewable and reproducible active surface, of the imprintedpolymer-modified HMDE reported here are the mainattractive features of the described analytical techniques.The reported MIP-modified HMDE sensor in DPCSVmode allows the sensitive and selective determination ofUA in aqueous environment in the presence of potentialinterfering species without any cross-reactivity or matrixeffect. The DPCSV results of mixture solutions suggestedthat P(UA) demonstrated stereoshape recognition abilityand hydrophobically driven hydrogen binding force todetect UA from the aqueous and blood samples. Thepresent investigation may be considered as a novel step indevising sensor based on molecular imprints with theultimate goal of giving rise to an immediate strippingvoltammetry signal on binding into the cavity.

Table 2. Analytical results of DPCSV measurements of uric acid (UA) in human blood serum and interferents at imprinted [P(UA)]modified hanging mercury drop electrode (HMDE). Recovery: amount of analyte determined/amount of analyte taken; cy: cytosine;Ad: adenine; AA: ascorbic acid; His: histidine; Theo: theophylline; Glu: glucose.

Sample Analyte concentration(mg mL�1)

Determined value [a](mean� SD) (mg mL�1)with MIP modified (HMDE)

Recovery (%) RSD (n¼ 3)

Dilute human blood serum (unspiked) [b] – 0.41� 0.01 (68.58) – 2.4Dilute human blood serum (spiked) 9.33 9.42� 0.07 101.0 0.7

17.94 17.52� 0.16 97.7 0.922.14 22.29� 0.29 100.7 1.3

Interferents [c] 18.86 (19.43 cy) 18.79� 0.20 99.6 1.032.11 (39.17 cy) 32.11� 0.03 100.0 0.118.86 (19.43 Ad) 18.90� 0.05 100.2 0.332.11 (39.17 Ad) 32.11� 0.08 100.0 0.318.86 (19.43 Uracil) 18.79� 0.04 99.6 0.232.11 (39.17 Uracil) 32.09� 0.01 99.9 0.018.86 (19.43 AA) 18.88� 0.02 100.1 0.140.54 (58.00 AA) 40.50� 0.20 99.9 0.518.86 (19.43 His) 18.90� 0.09 100.2 0.532.11 (39.17 His) 32.10� 0.06 100.0 0.218.86 (19.43 Urea) 18.82� 0.11 99.8 0.632.11 (39.17 Urea) 32.13� 0.04 100.1 0.118.86 (19.43 Theo) 18.79� 0.11 99.6 0.632.11 (39.17 Theo) 32.13� 0.04 100.1 0.118.86 (19.43 Glu) 18.99� 0.13 100.7 0.732.11 (39.17 Glu) 32.11� 0.01 100.0 0.0

[a] Average of three determination, S/N¼ 3.[b] Value in parenthesis indicates total concentration in UA in original blood sample (undiluted) as obtained by multiplication with dilution factor of166.67.[c] Values in parentheses indicate concentration (mg mL�1) of various interferents taken with UA in aqueous mixture solution.




5. Acknowledgement

Instrumental support from UGC-DSA program and anUGC-JRF fellowship to one of the authors (DL) aregratefully acknowledged.

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Fig. 6. Differential pulse, cathodic stripping voltammograms of uric acid (UA) with MIP-modified hanging mercury drop electrode inhuman blood serum sample [UA concentration (mg mL�1): a) 0.405; b) 9.33; c) 17.94; d) 22.14; e) 25.87] and with reference polymer[P(Rf)] modified hanging mercury drop electrode [UA concentrations (ppm): f) 17.94], other conditions as in Figure 4.




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Development of Uric Acid Sensor Based on Molecularly Imprinted Polymer-Modified Hanging Mercury Drop Electrode