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Sensors and Actuators B 123 (2007) 671–679

Comparative study between organic and inorganic entrapment matricesfor urease biosensor development

A. Maaref a, H. Barhoumi a,b,∗, M. Rammah a, C. Martelet b, N. Jaffrezic-Renault b,C. Mousty c, S. Cosnier c

a Laboratoire de Physique et Chimie des Interfaces, Faculte des Sciences de Monastir, Tunisiab CEGELY, UMR 5005, Ecole Centrale de Lyon, 69134 Ecully Cedex, France

c LEOPR, UMR 5630, ICMG FR 2607, Universite Joseph Fourier, 38041Grenoble, France

Received 11 July 2006; received in revised form 17 September 2006; accepted 3 October 2006Available online 15 November 2006

bstract

An essential step in biosensors development is the bonding of the biological components to the transduction element. Here, we present someatrices used as support materials for urease immobilization based on organic and inorganic matrices including in many cases glutaraldehyde

s cross-linking agent. Organic matrices were designed through the use of four latex polymers; neutral hydroxy polymer, three polycationicerivatives polynorbornene with different non-cytotoxic counter ions and a biopolymer obtained under chemical oxidation step of biotinylatedyrrole monomers. Inorganic matrices were based on Zn–Al layered double hydroxide nanohybrid materials (LDH). UV–vis and permeabilityechniques were carried out, respectively, in order to control the immobilized urease activity and to determine the permeability of the variousiomembranes. All these support matrices containing urease biomolecules were deposited on insulator semiconductor (IS) transducers for furtherrea biosensors development. Biochemical responses of these modified IS electrodes to urea addition were examined using the electrochemical

apacity-potential measurements C(V). Developed urea electrodes exhibit a linear response in the range from 10−4 to 10−1 M. For all matrices theichaelis–Menten constant, Km (0.67 ≤ Km (mM) ≤ 5) was obtained from the Lineweaver–Burk representation and was compared with values

btained using other immobilization strategies.2006 Elsevier B.V. All rights reserved.

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eywords: Latex polymer; Zn–Al nanohybrid; Biotinylated pyrrole; Capacitan

. Introduction

Biosensors are consisting of one part containing the biorecog-ition element (enzyme, antibodies, whole cells, nucleiccids. . .) coupled to a physical transducer (metallic, semi-onductor, optical, piezoelectric. . .). Historically, research oniosensors really began in 1962 with the pioneering work oflark [1]. Earlier biosensors were simple enzymes being suc-essfully immobilized through chemical or physical methods2]. However, these methods presented some drawbacks, suchs partial enzyme deactivation. To overcome such disadvantages

ntrapment of enzyme in polymers was proposed [3].

Urease can be considered as one of the most importantnzyme for uses in biomedical applications. As it is implicated

∗ Corresponding author. Tel.: +216 73 500 274; fax: +216 73 500 278.E-mail address: barhoumih@yahoo.fr (H. Barhoumi).

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925-4005/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2006.10.010

asurements; Urease biosensor

n the degradation process of urea to ammonium and hydrogenarbonate anions as shown in reaction (1):

H2CONH2 + H+ + 2H2Ourease−→2NH4

+ + HCO3− (1)

In recent years, extensive efforts have been done inrder to find an optimized strategy for urease immobiliza-ion in the aim to develop commercial urea biosensors. Untilow, a lot of matrices were used for urease immobiliza-ion as phosphine oxide polyether [4], polygalacturonate [5],olyurethane-acrylate [6], acrylonitrile [7], hydroxyapatite [8],lginate [9], polyaniline [10], albumine serum bovine [11],oly(vinylferrocenium) [12], poly(N-3-aminopropylpyrrole-co-yrrole) [13], polyethylenimine [14], synthetic latex polymer

15], . . ..

Such immobilization studies in several matrices have led tolot of works in relation to urease biosensors and artificial

idneys. Immobilized urease was also used for blood detoxi-

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72 A. Maaref et al. / Sensors and

cation [16] and measurement of urea in serum [17] and milkuring milking [18]. In literature, there is a variety of technolo-ies including photometric methods [19], conductimetric [20],mperometric [21] and potentiometric [22] electrodes have beensed to analyse urea in solution. Among these characterizationethods, potentiometric measurements were extensively used

or urea biosensors characterization. The basic principle drivingotentiometric measurements is the development of a significantotential at the electrode by accumulation of charge and hencencreased charge density at an electrode surface. The potential iselated to the analyte activity ai in the sample through the Nernstelation:

= E0 ±(

RT

nF

)log ai

here E0 is the standard electrode potential, R is the universal gasonstant, F is the Faraday constant, T is the absolute temperaturen Kelvin and n is the total number of charges on analyte i.

The aim of the present study is the development and evalua-ion of analytical performance of different organic and inorganic

atrices used for urease immobilization. In this work we havesed a neutral polymer functionalized by hydroxyl groups andhree polycationic derivatives of polynorbornene with differentounter ions (chloride, acetate and lactobionate). In addition, aiopolymer matrix was used for urease immobilization based

n the avidin-biotin interaction. The interaction system wasased on the assembled layer by layer between biotinylatedolypyrrole polymer layer obtained by chemical polymerisationtep, avidin interlayer and a biotinylated urease biomolecules

au

Fig. 1. Support mate

ators B 123 (2007) 671–679

ayer. This interaction process was used as the biotin/avidin sys-em has a quite high affinity (Ka = 1015 M−1) resulting in thetrongest receptor-ligand interaction found in nature [23]. Inddition, Zn–Al layered double hydroxide nanohybrides weresed as inorganic matrices for urease encapsulation. Ureaseas incorporated in the inorganic host matrices through steps

onsisting in exchange and coprecipitation methods [24]. Thehoice of Zn–Al hybrid as host matrix is dictated by somedvantages as well as, high charge density, high specific areand best change anionic capacity [25]. UV-vis spectroscopy andermeability measurements were carried out, respectively, onany urease/matrix composites to control the immobilized ure-

se activity and the permeability of biomembranes for analyteiffusion.

All matrices containing urease biomolecules were depositedn insulator semiconductor (IS) structures by a spin-coating pro-ess allowing a good reproducibility for further urea biosensorsevelopment. The biochemical biosensor signal resulting fromrea hydrolysis by urease was determined using electrochemicalapacitance C(V) measurements.

. Experimental

.1. Reagents

Urease from Jack Beans with an activity of 80 units mg−1

nd avidin (from egg white) were purchased from Sigma,rease-biotin from Biomeda, glycerol and glutaraldehyde from

rial structures.

Actuators B 123 (2007) 671–679 673

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A. Maaref et al. / Sensors and

luka. The latex polymers shown in Fig. 1 were synthesizedccording to the procedures described previously [26]. Biotiny-ated pyrrole (Fig. 1) was synthesized according to the proce-ure described elsewhere [27]. The layered double hydroxiden3Al(OH)8SDS·2H2O and ZnAl-urease matrices were synthe-ized by the coprecipitation method developed by de Roy [24].ll other reagents were of pure analytical grade.

.2. Insulator semiconductor (IS) substrate

The IS substrate (Si/SiO2/Si3N4) was purchased from LAAS-NRS Toulouse. These heterostructures were based on a p-type

ilicon substrate, 400 �m thickness, with a 10 � cm resistivity,overed with a 50 nm layer of thermally grown silicon dioxidend a 100 nm layer of silicon nitride prepared by Low Pressurehemical Vapor Deposition (LPCVD). The ohmic contact wasbtained by deposition of aluminium on the silicon unpolishedace.

Before depositing an urease/matrix layer on the IS devicesurface a cleaning procedure was necessary in order to improveydrophilicity and to obtain a reproducible clean surface.riefly, IS substrate was cleaned using three successive ultra-

onic baths of trichloroethylene (15 min), acetone (10 min)nd 2-propanol (10 min). Then, IS structure was immersed for5 min into a sulfochromic mixture and washed with ultra-pureater, dried under nitrogen atmosphere at room temperature andlaced at 70 ◦C for 10 min.

.3. Preparation of different urease-matrix biomembranes

.3.1. Urease/latex matricesThe latex suspensions (5%, w/v) were obtained by dissolv-

ng the polymers in an ethanol/water mixture using ultrasonicath for 1 h. The ethanol/water ratio was 67/33 (v/v) for theydroxy polymer and 20/80 (v/v) for the acetate, the lactobion-te and the chloride polymers. These latex suspensions wereixed with urease solution (10 g L−1) obtained by dispersing

he enzyme in 20 mM of KH2PO4 buffer at pH 7.4. Ten per-ent of glycerol was added to the mixtures of latex-urease (1/1,/v) and kept at 4 ◦C for 4 h. The surface of the Si/SiO2/Si3N4lectrode (1 cm2) was coated with 20 �L of urease-latex mix-ures (0.09 mg urease and 0.450 mg latex) using the drop coating

ethod (v = 1000 rpm). Next, these functionalized electrodesere placed for 15 or 30 min in an atmosphere saturated withlutaraldehyde vapor as a cross-linking agent [28]. After that,he functionalized IS structures were dried at room temperaturevernight and the non-linked enzyme molecules were washedut by vigorously stirred phosphate buffer solution.

.3.2. Polypyrrole-biotin/avidin/biotin-urease multilayersIS structure was spin-coated (2000 rpm) by 20 �L of pyrrole-

iotin solution (1 mM) dissolved in acetonitrile. After, the struc-ure was dried for 20 min at ambient temperature before being

oaked for 2 h in 0.1 mol L−1 FeCl3 dissolved in 0.1 mol L−1

ydrochloric acid to achieve the chemical oxidation polymeri-ation of the adsorbed pyrrole-biotin monomers on the IS struc-ures [29]. After the chemical oxidation step, IS structure was

2

d

Fig. 2. Conceptual picture of assembled layers system.

ashed with deionized water. Then, a drop of 20 �L of avidin0.5 mg mL−1) was deposited on the biotinylated polypyrroleayer. Finally, avidin layer was coated with a drop (20 �L) ofrease-biotin (1 mg mL−1). The building up of successive lay-rs system is given by the conceptual representation shown inig. 2. The modified IS structures was rinsed with phosphateuffer solution before the electrical measurements.

.3.3. ZnAl-urease nanohybride materialsWere obtained under two exchange mechanisms.

Stepwise exchange: was performed with a colloidal solutioncontaining delaminated ZnAl-DS nanoparticles. The delam-inated ZnAl-DS LDH (1 g L−1) were first transferred intodeionized water overnight under vigorous stirring then afterliquid/liquid separation, urease (1 g L−1) dissolved in Phos-phate buffer (20 mM KH2PO4, pH 7.4) was added to this LDHaqueous suspension for the exchange reaction. 3.5% of glycerolwas added to this solution. Twenty microlitre of this solutionwas deposited by spin coating (1000 rpm) on the IS substrate.Coprecipitation method: ZnAl-urease matrices with a ure-ase/LDH ratio Q from 1/3 to 3 were prepared by the copre-cipitation method. The bioinorganic materials were obtainedusing mixed aqueous solution (23 mL) of ZnCl2 and AlCl3,with a Zn2+/Al3+ molar ratio R = 3 and a total concentrationof metallic cation of 0.02 M, was introduced with a constantflow into a reactor containing a 30 mL urease solution. Duringthe coprecipitation step, the pH was maintained constant at avalue of 8.0 by addition of a 0.04 M NaOH solution. The finalproduct was centrifuged and washed several times with decar-bonated water, and finally dried in air. All experiments werecarried out under a stream of N2, in order to avoid or at leastminimize the contamination by atmospheric CO2. IS transduc-ers were spin-coated (1000 rpm) by 20 �L of Zn–Al-urease(2 g L−1) dissolved in Phosphate buffer (20 mM KH2PO4, pH7.4) solution containing 3.5% of glycerol.

.4. UV–vis spectroscopy and permeability measurements

The enzymatic activity of free and immobilized urease inifferent matrices were investigated by UV–vis measurements

6 Actuators B 123 (2007) 671–679

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74 A. Maaref et al. / Sensors and

arried out with a Varian Cary 1 UV–vis spectrophotometer.ll composite urease-polymer films were prepared on glassy

arbon electrodes. According to Worthington method [30], themmobilized urease activity was given by the decrease of NADHbsorbance signal in presence of NH4

+ ions generated by therea hydrolysis reaction, NADH being �-nicotinamide adeninei-nucleotide and GLDH being l-glutamic dehydrogenase:

NH4+ + 2α − ketoglutarate + 2NADH

GLDH−→ 2 − glutamate

+ 2NAD+ + 2H2O

The permeability measurements were performed with aotentiostat (Autolab, PGSTAT 100, Eco-Chemie, the Nether-ands) by controlling current and potential with GPES software.ll experiments were carried out in a three-electrode cell. Theorking electrodes were carbon glassy electrodes with a diam-

ter of 5 mm connected to a Tacussel EDI 101 T. The experi-ents were conducted in hydroquinone solution (2 mM) and the

otential were measured versus the aqueous saturated calomellectrode (SCE). The permeability Pm of different films useds supported membranes for urease immobilization was investi-ated by the use of rotating disk electrode (RDE) voltammetry.he permeability technique was previously described in detail

15].

.5. Electrochemical measurements

Electrochemical capacitance measurements were performedn a conventional electrochemical cell containing a three elec-rode system: the IS device as working electrode, a platinumlate electrode and a standard Ag/AgCl (saturated KCl) elec-rodes were used as counter and reference electrode, respec-ively. The electrochemical cell was connected to an amplifierystem Voltalab 40 (Radiometer Analytical SA Villeurbane,rance). The capacitance measurements were carried out at arequency of 10 kHz with a signal amplitude of 10 mV.

Capacitance method was based on the measurement of theocal pH change when the H+ ion concentration decreases nearhe interface silicon nitride and biomembrane. pH variationesulting from urea hydrolysis modifies the IS flat band poten-ial (�VFB). This pH change induces the shift of the capacity-otential characteristic along the voltage axis for different ureaoncentrations (Fig. 3). In this case the Nernst equation takeshe form as follows:

VFB = VFBi + α

(RT

F

)log[urea]

here VFBi is the initial flat band potential of the functionalizedS structure and α a coefficient introduced in a previous work31].

Capacitance measurements were performed in 5 mM Phos-hate buffer (ionic strength from 70 mM NaCl) at pH 7.4. Thisuffer concentration was selected after an optimized step car-

ied out on the different urease-polymer membranes [15]. Alleasurements were performed in a dark Faraday cage at room

emperature. When not in use, the samples were stored in 5 mMhosphate buffer, pH 7.4 at 4 ◦C.

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ig. 3. Capacitance spectra of poly(pyrrole-biotin)/avidin/biotin-urease/IS elec-rode for different urea concentrations performed in 5 mM Phosphate buffer, atH 7.4.

. Results and discussion

.1. Performances of IS/urease-latex biomembranes forrea detection

Optimization of the deposition procedure for different latexolymers and urease on Si/SiO2/Si3N4 was investigated via theesponse of the resulting biosensor to urea. To obtain optimalechanical adsorption of the latex film and preserve the enzyme

ctivity an additional cross-linking step by glutaraldehyde vaporor 15 min was used and 10% of glycerol as a plasticiser agentas added to the deposited mixtures to increase the adhesion of

he biomembrane to the surface of the transducer. The presencef this additive, moreover, preserved the native enzyme con-ormation and reduced the urease-polymer aggregation in thenzymatic membrane [32].

The potentiometric responses versus urea concentration ofll composite urease-polymer film coated electrodes preparednder the same conditions were recorded for concentrationsanging between 10−4 and 10−1.5 M urea (Fig. 4). The urease-ydroxy polymer and urease lactobionate polymer compos-te films led to similar biosensor sensitivities, namely 22 and4 mV/p[urea], respectively. In contrast, the host matrices basedn acetate and chloride derivatives exhibited a markedly lowerensitivities: 10 and 7 mV/p[urea], respectively, for concentra-ions ranging from 10−4 to 10−2 M and from 10−4 to 10−1 M,espectively. This may be ascribed to a more important deac-ivation process of urease during its immobilization in acetatend chloride polymers than within the two other latexes. Asconsequence, the enzymatic activity of urease encapsulated

n the organic polymer matrices was evaluated through thebsorbance decrease of NADH consumed in the presence ofH4

+ by an enzymatic coupled reaction and compared to the

ctivity of a free urease. The resulting immobilized activities:.6, 2.4 and 1.2 U cm−2 for hydroxy, lactobionate and acetateolymeric films, corroborate the previous difference observed.he presence of hydroxy groups or biocompatible oligosaccha-

A. Maaref et al. / Sensors and Actuators B 123 (2007) 671–679 675

Fig. 4. Calibration curves for urea obtained at IS electrodes coated with differ-ent urease-latex films in 5 mM Phosphate buffer solution, pH 7.4. Transducermpa

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Fig. 5. Effect of the cross-linking process by glutaraldehyde on the sensitivityof urease-acetate and urease-chloride polymer composite films: (�) urease-acetate and (�) urease-chloride films exposed for 30 min to glutaraldehydevapor, (�) urease-acetate and (�) urease-chloride films exposed for 15 min togg

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odified by adsorption of 0.25 mg of (�) hydroxy polymer, (�) lactobionateolymer, (�) acetate or (�) chloride polymer in the presence of urease (0.25 mg)nd glycerol followed by a cross-linking step by glutaraldehyde for 15 min.

ide group for hydroxy polymer or lactobionate polymer shouldreserve the enzyme activity during the film formation and coun-erbalances the negative effect of the chemical cross-linking bylutaraldehyde. Moreover, the enzymatic activity of the ure-se electrode was examined as a function of time indicatingbetter retention of the enzyme activity and a stronger adhe-

ion of the cationic polymers to the transducers compared tohe hydroxy one. Owing to the strong anionic nature of ure-se at pH 7.4 (isoelectric point = 5), the latter should exhibittrong electrostatic interactions with the cationic latex films.ince glutaraldehyde may exert a possible negative effect on

he immobilized enzyme activity, the mechanical stability ofcetate and chloride polymers elaborated without glutaralde-yde was confirmed by the stable and the reproducible responsebtained by repetitive measurements. It appears that acetate andhloride polymer-enzyme coatings are stable without any use oflutaraldehyde whereas both hydroxy and lactobionate polymer-rease coatings required a cross-linking step for their adhesionn Si/SiO2/Si3N4. This different behaviour between cationicatexes having different counter ions may be attributed to thearger steric effect of the lactobionate compared to the one ofcetate and chloride. The effect of the cross-linking process bylutaraldehyde on the performance of the acetate and chlorideolymer-urease sensors was investigated. It clearly appeared thatensitivity increased without cross-linking step whereas a highecrease of the biosensor response was observed if the expo-ure time to glutaraldehyde was extended up to 30 min (Fig. 5).his indicates either a denaturation of the enzyme activity orhighly limited diffusion of urea due to the reticulation pro-

ess [33]. Furthermore, we observe a stable signal and goodeproducible response in the case of the urease-acetate, urease-hloride and urease-lactobionate biosensors associated to quite

igh stability leading to a life-time greater than 1 month. Thesenteresting performances could not be achieved in the case ofrease-hydroxy biosensors. These results were attributed to theigh hydrophilic character of the cationic polymers that can give

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lutaraldehyde vapor, (�) urease-acetate and (�) urease-chloride films withoutlutaraldehyde vapor.

ufficient interaction with the urease biomolecules. So, it can beoncluded that the interaction process between urease and themmonium group of the cationic polymer depends of the counteron size. In a previous work [34], it was shown that the interac-ion between ammonium groups and DNA biomolecule increase,espectively, with the counter ion for lactobionate, acetate andhloride polymers. In addition, to control the reticulation processffect of glutaraldehyde on the biomembrane hydrophobicityontact angle measurements were performed. This measurementechnique consisted of the sessile drop method with an apparatusrovided by GBX scientific instrument (Romans, France). Imagef the drop deposit on the biomembrane surface was registeredy a video camera and an image analysis system calculates theontact angle (θ) from the shape of the drop. Ultra-pure wateras used as liquid test to determine the hydrophilic (low contact

ngle) or hydrophobic (high contact angle) surface character.In a previous work [35], the contact angle measurements

ere used to characterize silicon nitride surfaces functional-zed with polymeric membrane. We have demonstrated that isossible to obtain some information concerning the depositedembrane surface proprieties. In this present work, all urease-

olymer composites deposited on the silicon nitride surface wereeticulated with glutaraldehyde vapors for different times. Fig. 6hows the variation of the contact angle as a function of theeticulation time. Without glutaraldehyde vapor we observe aow contact angle (θ < 15◦) for the biomembrane layers basedn cationic polymers (chloride, acetate and lactobionate poly-er) in comparison with the biomembrane based on the hydroxy

olymer (θ ∼ 40◦). This result demonstrates that charged poly-ers are more hydrophilic than the hydroxy polymer. So, when

he reticulation time increases we observed a high increase of

ontact angle in the case of the biomembranes based on the chlo-ide and acetate polymers to give a high hydrophobic surface.his behavior was not observed in the case of the biomembranes

676 A. Maaref et al. / Sensors and Actuators B 123 (2007) 671–679

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ig. 6. Effect of the cross-linking time on the biomembrane hydrophobic char-cter: (�) urease-acetate, (�) urease-chloride, (�) urease-hydroxy and (�)rease-lactobionate.

ased on the hydroxy and lactobionate polymers. Furthermore,t can be concluded that urease-polymer biomembranes affinityoward glutaraldehyde is a quite important parameter in the casef chloride and acetate polymers. These results obtained fromhe contact angle measurements contribute to explain the poornalytical response obtained for the reticulated urea biosensorsased on chloride and acetate polymers.

.2. Performances of IS/poly(pyrrole-biotin)/avidin/iotin-urease biosensor

In general, polypyrrole polymer can be easily formed throughlectropolymerization on metal surface electrodes [36]. In ourork, IS structures are not conductive and for this reason weave used a simple chemical polymerisation based on the oxida-ive polymerisation of pyrrole-biotin with FeCl3. However, theyrrole-biotin captures Cl− anions and the reaction can beescribed by the stoichiometric equation [37–38]:

C4H5N + (2 + y)nFeCl3 → (C4H5N)nnyCl−+ (2 + y)nFeCl2 + 2nHCl

here y is the degree of polypyrrole oxidation, which defines itson exchange properties.

Urease-biotin binds to the poly(pyrrole-biotin) polymerhrough the avidin elements. The electrochemical response ofhe assembled multilayers system was studied by capacitance

easurements. Fig. 7 shows the potentiometric response ofS/poly(pyrrole-biotin)-avidin-biotin-urease biosensor for dif-erent urea concentrations. The modified electrode shows a lin-ar response between 10−4 and 10−1 M with a good sensitivity of0 mV/p[urea]. The good IS/poly(pyrrole-biotin)-avidin-biotin-rease biosensor performaces were obtained using a storage

edium (5 mM phosphate buffer, pH 7.4) containing 10−3 MDTA. According to the EDTA effect on urease biomoleculesuoted in many works [39], it can be concluded that the usef EDTA in the storage medium stabilizes and ensures a bet-

aact

ig. 7. Urea calibration curves of poly(pyrrole-biotin)/avidin/biotin-urease/ISlectrode obtained by varying the poly(pyrrole-biotin) layer number (�) 2, (�)and (�) 4 layers. Performed in 5 mM Phosphate buffer, at pH 7.4.

er operation of the IS/poly(pyrrole-biotin)-avidin-biotin-ureaserea biosensor and permits its re-use for several times. The effectf the poly(pyrrole-biotin) thickness on the biosensors responseas investigated by varying the number of pyrrole-biotin layerseposition on the IS transducer. Fig. 7 illustrates the calibrationurves for pyrrole-biotin/avidin/biotin-urease matrix obtainedor two, three and four pyrrole-biotin layers. A net decrease ofhe urea biosensors response was observed when the number ofhe pyrrole-biotin layers increased. This result can be explainedy diffusion limit effects of the OH− to the insulator/membranenterface for quite thick poly(pyrrole-biotin) layer. The good per-ormances demonstrated by the developed urea biosensor giveise to a novel immobilization strategy of urease using the highffinity between avidin-biotin. In addition this immobilizationethod excludes the cross-linking process with glutaraldehyde

eagent and prevents enzyme from inhibition effects. For theyrrole-biotin/avidin/biotin-urease assembled system the cal-ulated Michaelis–Menten constants, Km = 5 mM was obtainedrom the Lineweaver–Burk representation. This value of Kmhows that the immobilized urease activity was kept after themmobilization process in comparison with the free urease.

.3. Performances of IS/ZnAl-urease biosensors

In previous works using [Zn–Al–Cl] LDH as immobilizationatrix for enzymes, we had shown that a chemical cross-linkingith glutaraldhyde appeared as a neccessary step for the stabilityf the biosensors [40]. Chemical cross-linking reduced markedlyhe slow release of enzymes into solution [41]. The usefulnessf this step was also investigated in the case of ZnAl-ureaseDH biomembranes. Exposure time to glutaraldehyde was opti-ized, it was observed that the electrode response depends and

ecreases with the increase of cross-linking time. This may be

scribed to a decrease in the activity of the immobilized urease orn increase in steric hindrance in the coating related to the highross-linking degree of the entrapped enzymes. Consequently,he exposure time was fixed at 8 min. A sharp decrease of the

Actuators B 123 (2007) 671–679 677

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A. Maaref et al. / Sensors and

rea sensor response was observed after three days soaking in amM Phosphate buffer solution and afterwards it becomes sta-le for more than ten days. Moreover after a conditioning steperformed in 0.1 M phosphate buffer solution for 1 h, the ureaiosensor response also decreased to 60% of its initial value.

The calculated Michaelis–Menten constants (Km) ofhe ZnAl-urease biomembranes conditioned in buffer solu-ion at different concentrations were determined using theineweaver–Burk representation. The estimated Km values are.67, 1.16 and 1.52 mM, respectively, for the conditioning inmM PB solution for 1 h, in 5 mM PB solution for 3 days and

n 100 mM PB solution for 1 h. It can be noticed that Km valuef 0.67 is less than Km values reported in the literature for freerease (1–5 mM) [42]. The increase of the Km constant after theonditioning step for longer time or in more concentrated bufferolution can be attributed to a partial exchange of the ZnAl-rease nanocomposite by electrolyte phosphate anions inducinghanges in the swelling properties of the LDH nanoparticlesnd consequently modifications of the accessibility of urease torea substrate. Other biomembranes of Zn–Al-urease compos-te synthesized by coprecipitation method were tested for ureaseiosensor development. Fig. 8 illustrates the calibration curvesbtained for different Zn–Al/urease ratio (1/3, 1/2, 1/1, 2/1, 3/1)owards urea additions. A decrease of the biosensor responseas observed as the urease/HDL ratio decrease. The use oflutaraldehyde as reticulation agent for these biomembranesbtained by coprecipitation method was attempted and the resultead to a counter effect on the biomembrane sensitivity. Theseotentiometric responses were lower than those obtained with anrease/HDL biomembrane prepared by the stepwise exchangeethod (Fig. 8). These potentiometric responses of different ure-

se/HDL matrix were in good agreement with the permeabilityesults. So, the estimated permeability values shown in Table 1ndicate a high permeability for such urease/HDL matrices. Inddition, the activity values obtained for different Zn–Al/urease

iebd

able 1haracteristics of some developed urea biosensors using different matrices for urease

iomembrane Sensitivity (mV/p[urea]) Li

ydroxy polymer/urease 22actobionate polymer/urease 24 1cetate polymer/urease 26hloride polymer/urease 17oly(pyrrole-biotin)/avidin/urease-biotin 50 1n3Al-urease (stepwise exchange) 70 2n3Al-urease (coprecipitation; 1/3) 17 1n3Al-urease (coprecipitation; 1/2) 14n3Al-urease (coprecipitation; 1/1) 16 1n3Al-urease (coprceipitation; 2/1) 9n3Al-urease (coprecipitation; 3/1) 3olyethylenimine/urease 50.24olypyrrole/urease 110 1oly(vinylferrocenium)/urease 9aponite/urease 53.22 3olyurethane-acrylate/urease 58 2VAc-PE/urease 52 2VC-NH2/urease 48 1ovine serum albumin/urease 8.43 1VA/Sbc/urease 20 1

ig. 8. Calibration curves for urea of the urease-LDH composite films: (�)tepwise exchange and coprecipitation method for different urease/HDL ratio�) 3/1, (�) 2/1, (�) 1/1, (�)1/2 and (�) 1/3.

atio 1/3, 1/2, 1/1, 2/1 and 3/1 were 0.23, 0.34, 0.47, 0.29 and.96 U mg−1, respectively.

.4. Features of such matrices for urea biosensorevelopment

In Table 1 we report the analytical biosensors performancesbtained for such matrices: sensitivity, Michaelis–Menten con-tant (Km), permeability coefficient (P), and linear rangepUrea). In the same table results obtained for potentio-etric urea biosensors from various works using different

mmobilization techniques and different matrices are gath-red [6,12,14,36,40,43–46]. The main drawbacks of most ureaiosensors is their narrow dynamic range rendering them notirectly usable for direct detection in human blood where normal

immobilization

near range (p[urea]) Km (mM) Pm (cm s−1) Reference

2–3.92 1.14 0.93 × 10−2 This work.69–3.92 1.12 0.83 × 10−2 ′′

2–3.55 1.25 1.06 × 10−2 ′′2–3.39 1.4 – ′′

.41–2.92 5 – ′′

.69–4 0.67 1.2 × 10−2 ′′

.92–3.92 1.18 1.6 × 10−2 ′′1.9–3.69 1.42 1.3 × 10−2 ′′.91–4 1.15 1.6 × 10−2 ′′

1–4.5 1.06 0.95 × 10−2 ′′1–2.69 6 0.7 × 10−2 ′′

1.5–2.5 – – [14].52–3.52 – – [36]

1–4.3 – – [12].25–5.3 2.8 3.1 × 10−3 [40].44–5.39 – – [6].67–5.14 – – [43].30–3.30 – [44].92–3.52 79.3 – [45].09–2.30 200 – [46]

678 A. Maaref et al. / Sensors and Actu

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ig. 9. The long-term urea sensitivity of the (�) poly(pyrrole-biotin)/vidin/biotin-urease, (�) acetate-urease and urease-HDL ((�) stepwisexchange, (�) coprecipitation 3/1 ratio) biosensors.

rea concentrations are ranging from 6 to 8 mM. To increase suchrange different strategies have been used as specific buffers act-

ng as competitive inhibitor [45] or utilization of recombinantrease [46].

In this study the best response for urea addition was obtainedn the case of ZnAl-urease biomembrane obtained by stepwisexchange. This good result was limited by the swelling phe-omenon that decreases the biosensors response as functionf the time. In addition, the poly(pyrrole-biotin)-avidin-biotin-rease biomembrane was characterized by a stable and highesponse for urea addition. The advantage of this immobilizationethod based on the avidin-biotin affinity was given by the sim-

licity of the biomembrane fabrication. Also, the biotinylatedurface of the IS electrode can be regenerated to be used foreveral time using SDS surfactant to dissociate the avidin-biotinomplex [47]. These good results obtained in the case of bothnAl-urease and (pyrrole-biotin)-avidin-biotin-urease biomem-ranes are not better than those obtained with the cationicolymers for urease encapsulation such as the acetate and theactobionate polymers. For example, in Fig. 9 we report the ureaiosensors stability as function of the time for the different usedatrices. However, among all tested matrices we observe a sta-

le and more reproducible response in the case of lactobionate,cetate, and poly(pyrrole-biotin) polymers used as entrapmentatrices for urease.

. Conclusion

This paper has demonstrated the interests for developingrease biosensor for using organic and inorganic materials foronitoring urea in aqueous media. All tested matrices for ure-

se immobilization were deposited on insulator semiconductorevices to be characterized by electrochemical capacitance mea-

urements in presence of urea. Among these tested matrices forrea biosensor development poly(pyrrole-biotin) represent annnovative procedure and suitable matrix for urease immobiliza-ion. The efficient non covalent bonding of urease-biotin to the

[

ators B 123 (2007) 671–679

iotinilyted coated IS devices ensured by the high avidin-biotinffinity leads the enzyme electrode to exhibit a good perfor-ance in term of dynamic range, fast response and long lifetime

tability. In addition, the cationic polymers and ZnAl doubleayered hydroxide nanohybrid materials can be considered asew trends for urease immobilization with low cost and simpleethod for making enzymatic sensor.

cknowledgments

This work was supported by the CMCU contractrant No. 03S1205. Authors thank the GDR CNRS 2619Microcapteurs», Rhone-Alpes MIRA cooperation program andCI Nanosciences No. NR0005 for partial support.

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