Urea biosensor based on Zn3Al–Urease layered double hydroxides nanohybrid coated on insulated silicon structures

Urea biosensor based on Zn3Al–Urease layered double hydroxides nanohybrid coated on insulated silicon structures

Materials Science and Engineering C 26 (2006) 328 – 333 www.elsevier.com/locate/msec Urea biosensor based on Zn 3Al–Urease layered double hydroxides ...

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Materials Science and Engineering C 26 (2006) 328 – 333 www.elsevier.com/locate/msec

Urea biosensor based on Zn 3Al–Urease layered double hydroxides nanohybrid coated on insulated silicon structures H. Barhoumi a,b, A. Maaref a, M. Rammah a, C. Martelet b,*, N. Jaffrezic b, C. Mousty c,*, S. Vial d, C. Forano d a

Laboratoire de Physique et Chimie des Interfaces Faculte´ des Sciences de Monastir, Tunisia b CEGELY, UMR 5005, Ecole Centrale de Lyon 69134 Ecully Cedex, France c LEOPR, UMR 5630, ICMG FR 2607, Universite´ Joseph Fourier, 38041 Grenoble, France d L M I, UMR CNRS 6002, Universite´ Blaise Pascal, 63177 Aubie`re, France Available online 28 November 2005

Abstract Urea biosensors for medical diagnostic monitoring were developed based on the immobilization of urease within layered double hydroxides (LDH). The urease – LDH material was obtained by a stepwise exchange reaction by urease of a Zn3Al – dodecyl sulphate (ZnAl – DS) colloidal suspension. XR diffraction and FTIR analysis show that this method gives rise to a Zn3Al – Urease LDH nanohybrid material with urease dispersion and textural properties. An aqueous suspension of this urease – LDH nanohybrid material was deposited on an insulated semiconductor (IS) structure. Biosensor responses to urea additions were obtained using capacitance (C vs. V) and impedance (Z vs. x) measurements. An enhanced maximum limit of the dynamic range was observed in the case of the impedance measurements (110 mM) compared to (5.6 mM) the capacitive urea biosensor. The Michaelis – Menten constant was also calculated according to the Lineweaver – Burk plot. It was found that the K m value with immobilized enzymes was lower (K m = 0.67 mM) in comparison with free enzymes. This K m value obtained from the capacitance measurements indicates that the urea degradation is performed within any inhibition action on the IS/Zn3Al – Urease LDH electrode. A comparative study was carried out between these results and those obtained previously, using urease/ZnAl – Cl layered double hydroxides mixture coated on the pH-ISFET transducer. D 2005 Elsevier B.V. All rights reserved. Keywords: IS transducers; Layered double hydroxide; Electrochemical measurements; Urea; Urease biosensor

1. Introduction In the last few decades, the development of enzyme biosensor devices has been a topic of considerable interest due to their potential applications. Applications can touch a large variety of fields including medicine, drug discovery, environment, food and process industries. Among a large number of enzymes used for biosensor construction, urease takes a large part in enzyme sensor development due to the large demand for urea determination [1]. Urea is one of the byproducts monitored in blood as an indicator of renal function. Indeed, high levels of urea are pathologic signs of renal inefficiency [2] and can be directly linked to mortality [3]. Moreover, urea is widely distributed in nature and its analysis * Corresponding authors. E-mail address: [email protected] (C. Martelet). 0928-4931/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2005.10.042

is of considerable interest in agro-food chemistry and environmental monitoring. A biosensor is a device containing two principal parts: the biological receptor for analyte recognition (i.e. enzyme) and the transducer for conversion of the specific biorecognition event to an optical or electrical signal. For this reason, the major step in the biosensors construction is to find the best strategy to immobilize biorecognition macromolecules on the transducer without any change of their structural conformation and of their activity [4]. Consequently, the choice of the matrix for the bioreceptor immobilization plays a decisive role in the sensor architecture. This immobilization feature will govern the ultimate reliability and performance of the obtained biosensor. Among the various host materials used for urease biosensor elaboration, clays occupy a privileged position [5,6]. Indeed, these inorganic materials which present an important thermal stability, a chemical inertia, a well-defined layered structure, ion-

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exchange properties, a high accessible surface and a low cost, can find applications in electroanalysis as sensors and biosensors [7]. In particular, layered double hydroxides (LDH), also called anionic clays, were reported to be an attractive material for electrochemical biosensor design [6,8 – 11]. In particular ZnAl – Cl LDH was already used successfully for biosensor fabrication with urease and polyphenol oxidase [6,8,10]. For urea determination, the biosensor detection was based on field effect transistor (ENFETs) [6]. Specific electrostatic interactions with anionic inhibitors within LDH membranes allow the development of sensitive inhibitor biosensors [6,10]. Moreover, it is known that layered double hydroxide materials were already used as host materials to encapsulate some organic molecule guests to make inorganic– organic intercalate systems such as catalysts [12,13], catalyst precursors or catalyst supports [14,15], adsorbents [16,17], anion exchangers [18,19] and in medical applications [20] among other uses. Their use is nowadays enlarged using LDH materials for protein encapsulation to obtain inorganic – biological intercalate biosystems [21]. For example, LDH matrix was used to incorporate many biological species such as adenosine monophosphate [22], DNA [23,24], and biopolymer such as poly(a, h-aspartate) [25], alginic acid [26], cyclodextrin [27] and enzyme [28]. We report here the use of a ZnAl – Urease nanohybrid for the development of urea biosensor. This new approach of enzyme immobilization was based on an original urease entrapment within layered double hydrides by stepwise exchange reaction with a ZnAl – dodecyl sulphate LDH colloidal solution. The resulting hybrid material was deposited by spin coating method on Si/SiO2/Si3N4 used as physical transducers. The response of the resulting biosensors to urea additions was measured by capacitance and impedance measurements. 2. Experimental section 2.1. Materials Urease from jack beans with an activity of 80 U/mg was obtained from Sigma, urea, glycerol and glutaraldehyde (GA) from Fluka. All other reagents were of pure analytical grade. Zn3Al(OH)8DS.nH2O (abbreviated as ZnAl – DS) layered double hydroxide, was synthesised by the co-precipitation method developed by De Roy [29]. A mixed aqueous solution of ZnCl2 and AlCl3 with a Zn2+ / Al3+ molar ratio R = 3 was introduced with a constant flow into a reactor containing a sodium dodecyl sulfate solution (DS/Al3+ = 2). The pH was maintained constant at 8.0 by a simultaneous addition of a 1 M NaOH solution. The suspension was aged at room temperature with stirring for 24 h. The final product was centrifuged, washed several times with decarbonated water, and finally dried in air at room temperature. All experiments were carried out under a stream of N2, to avoid atmospheric CO2 contamination. Colloidal solution of ZnAl –DS in butanol was obtained after stirring a ZnAl –DS/Butanol suspension (1 g/L) under reflux at 90 -C during 48 h according Adachi et al. [30]. The exchange reaction was performed with this colloidal


solution containing delaminated ZnAl – DS nanoparticles. The delaminated ZnAl –DS LDH (1 g/L) were first transferred into deionised water by an overnight vigourous stirring then after liquid/liquid separation, urease (1 g/L) dissolved in phosphate buffer (KH2PO4, 20 mM at pH = 7.4) was added to this LDH aqueous suspension for the exchange reaction. 3.5% of glycerol was added to this solution in order to reduce the aggregation between urease and host HDL nanoparticles and to enhance the membrane adhesion onto the transducer surface [31]. 2.2. Sensor insulator– semiconductor (IS) transducer The pH-sensitive IS system (Si/SiO2/Si3N4) were purchased from the Institute of Microtechnology of the University of Neuchatel (Switzerland). The studied Si/SiO2/Si3N4 structures were based on a p-type silicon substrate, 400 Am thickness, with 10 V cm resistivity, covered with a 50 nm layer of thermally grown silicon dioxide and a 100 nm layer of silicon nitride prepared by LPCVD (Low Pressure Chemical Vapour Deposition). The ohmic contact was obtained by deposition of a gallium –indium mixture on the back side of the Si/SiO2/ Si3N4 structures. Before the deposition of the biomembrane, the IS structures were treated using an optimized cleaning procedure [32]. Surface transducers were cleaned in successive baths of trichloroethylene, acetone, isopropanol and sulphochromic mixture (sulphuric acid and chromic acid), washed with ultrapure water, dried under nitrogen atmosphere at room temperature and placed at 70 -C for 10 nm. 2.3. Urease immobilization The surface of the Si/SiO2/Si3N4 electrode (1 cm2) was coated by the spin-coating process (m = 1000 rpm) with a drop (20 AL) of the suspension containing the ZnAl – Urease nanohydrid (1 g/L). Then, the ZnAl –Urease LDH coated IS device was placed for maximum 16 min in saturated glutaraldehyde vapour for the cross-linking of the biomembrane [33]. After the cross-linking step the biomembrane was dried at room temperature for 30 min and then washed with a 5 mM phosphate buffer solution (PB, pH 7.4) to remove the noncross-linked urease. After the first measurement, the biosensors were stored for long term stability study in a 5 mM PB solution at 4 -C. 2.4. Electrochemical measurements The biosensor response to urea addition was measured at ambient temperature using the capacitance (C vs. V) and the impedance (Z vs. x) electrochemical measurements. The urease/LDH coated pH-IS electrodes were connected to an amplifier system Voltalab 40 (Radiometer Analytical SA Villeurbane, France). The electrochemical measurements were performed in a conventional electrochemical cell containing a three-electrode system, ensuring stable positioning of the electrodes and an agitation of the solution. The coated electrode


H. Barhoumi et al. / Materials Science and Engineering C 26 (2006) 328 – 333

was the working electrode, a platinum plate the auxiliary electrode and a saturated Ag/AgCl/KCl electrode served as reference electrode. All measurements were performed in darkness to avoid photoinduction of charges in the semiconductor transducer. Capacitance measurements were carried out at a frequency of 10 kHz with signal amplitude of 10 mV. Impedance measurements were performed in the frequency range from 0.1 to 100,000 Hz at the imposed bias of 1500 mV (accumulation zone of the IS structure).

Refluxing Zn3Al – DS in butanol ensures the dispersion of LDH platelets in the organic solvent, giving a colloidal solution containing anionic clays with enhanced surface properties. To perform the exchange reaction with urease, this delaminated phase was first transferred into water and put in the presence of urease solution. This stepwise procedure prevents any possible denaturation of the enzyme by the organic phase. The stepwise exchange reaction is monitored by XR diffraction and FTIR analysis. Fig. 1 shows the X-ray diffraction patterns and FTIR spectra of precursors and hybrid LDH materials. The diffractogram of the Zn3Al(OH)6ClI2H2O LDH phase is presented as a reference diffraction pattern, indexed in the R –3 m hexagonal system. As expected for this preparation method, the hybrid materials display a low crystallinity due to a high dispersion of platelets and a low degree of ordered restacking. However, the occurrence of the (012) and (110) diffraction lines is an evidence of the structural stability of the inorganic matrix during the synthesis. We must point out the intercalation of phosphate anions during the contact step with the buffered urease solution. Highly charge HPO42 groups have a high affinity for anionic exchanger LDH and intercalation can be easily promoted when charge compensation is needed. Indeed, large negative biomolecules such as urease with low surface charge density will not succeed to totally neutralize the positive charge of the layers and coexchange of smaller anions must occur. FTIR spectra of Zn2Al –Urease materials (Fig. 1B, curve c) exhibit vibration bands of both phosphate and urease. However, the vibration bands of urease are intense indicating that the presence of phosphate does not affect the immobilization of urease.

3. Results and discussion 3.1. Characterization of the ZnAl –Urease hybrid material The development of biosensors based on LDH matrixes not only requires an adequate microenvironment created by LDH nanoparticles for enzyme immobilisation without any conformational change, but also this host material must have a good biocompatibility without any inhibition effect. Due to their anion exchange properties, the Layered Double Hydroxides constitute the most suitable layered inorganic host structures for the intercalation/adsorption of biomolecules. Urease with an isoelectric point equal to 5.1 displays an overall negative charge in the standard pH conditions of the work and can be easily exchanged on LDH. Moreover, amino acids units of enzymes with polar functions favor strong hydrogen bonding interactions with the LDH layer surfaces entirely covered by OH groups. This high charge compatibility between both biomolecule and inorganic matrix leads to efficient hybrid organic – inorganic aggregations. With the objective to optimise the biocompatibility and the stabilization of urease on the biosensor electrode, a specific preparation method of hybrid Zn3Al – Urease LDH was developed. Intercalation of large biomolecules in layered materials is not possible using a standard exchange method due to strong layer – layer interactions. First, hydrophobic Zn3Al LDH is obtained by intercalation of an anionic hydrophobic surfactant (namely dodecylsulfate, DS) by the coprecipitation method.

3.2. Capacitance measurements The IS surface was modified with 20 AL of the hybrid ZnAl –Urease LDH. The intercalated bio-inorganic system is given by the conceptual picture of urease LDH composite film deposit on the pH-sensitive structure (Fig. 2). Electrochemical measurements were performed in a 5 mM PB (ionic strength from 70 mM NaCl) solution, at pH 7.4. The potentiometric


B interlayer H2O interlayer anion M-O-H

interlayer anion


c b


Relative intensity



(003) (006)



(012) (015) (018) (101)




(110) (113)


b a

a 70









Wavenumber cm-1

Fig. 1. X-ray diffraction patterns (A) and Fourier Transform Infrared spectra (B) for (a) Zn3Al – Cl LDH reference material, (b) Zn3Al – DS LDH, (c) Zn3Al – Urease LDH from stepwise exchange, and (d) urease.

H. Barhoumi et al. / Materials Science and Engineering C 26 (2006) 328 – 333 anion


Table 1 Analytical characteristics of IS/ZnAl – Urease biomembrane as a function of reticulation conditions


Without GA With GA (8 min) With GA (16 min)



Èe urea


Maximal response (mV) 132 Sensitivity (mV/pUrea) 90 Linear range (mM) 0.1 – 3.2


X Urease



126 70 0.1 – 5.6

30 21 0.56 – 5.6


Si // SiO SiO22//SiSi3N 44 3N Fig. 2. Conceptual picture of IS/Zn – Al – Urease LDH biomembrane.

response vs. urea concentration was recorded in the concentration range between 1 10 4 and 1 10 1 M (Fig. 3). The biosensors response can be explained by the local pH change of the electrolyte near the silicon nitride surface, which originates from the enzymatic reaction of urea hydrolysis catalysed by urease according to the reaction (1):  NH2 CONH2 þ Hþ þ 2H2 O urease 2NHþ 4 þ HCO3 :



The pH variation, resulting from the urea hydrolysis, causes the change of the flat band potential (DV FB) of the pH –IS transducer at different urea concentrations inducing position shifts of the C curves along the voltage axis. In the previous works using [Zn– Al –Cl] LDH as immobilization matrix for enzymes, we have shown that a chemical cross linking with glutaraldhyde appeared as an indispensable step for the stability of the biosensors [6,8,10]. Indeed, chemical cross-linking, reduced markedly the slow release of enzymes into solution [7,34]. The usefulness of this step was also investigated in the case of ZnAl – Urease LDH biomembrane. Table 1 shows the biosensor characteristics obtained with unreticulated and reticulated biomembranes. The unreticulated biomembrane was characterized by a high response (90 mV/pUrea), but the high response observed in the first measurement is not reproducible and the urea biosensor loses its sensitivity after a few days. This confirms that unreticulated

biomembrane leads to poor long-term stability of the sensor because of slow enzyme leakage from the LDH matrix into the electrolyte solution. When the exposure time to glutaraldehyde increased from 8 to 16 min, it was observed that the electrode response was reduced three or four times in sensitivity and the maximal response, respectively (Fig. 3, Table 1). This may be ascribed to a decrease in the activity of the immobilized urease or an increase in steric hindrances in the coating related to the high reticulation degree of the entrapped enzymes. Consequently, the exposure time was fixed at 8 min and the stability of the resulting biosensor was investigated (Fig. 4). A sharp decrease of the urea sensor response was observed after three days soaking in 5 mM PB solution and afterwards it becomes stable for more than ten days. Moreover, after a conditioning step performed in 0.1 M PB solution for 1 h, the urea biosensor response was also decreased to 60% of the initial value. The calculated Michaelis– Menten constants (K m) of the ZnAl – Urease biomembranes conditioned in buffer solution at different concentrations were determined using the Lineweaver –Burk representation. The estimated K m values are respectively 0.67, 1.16 and 1.52 mM for the conditioning in 5 mM PB solution for 1 h, in 5 mM PB solution for 3 days and in 100 mM PB solution for 1 h. It is remarkable that K m value of 0.67 is less than K m values reported in the literature for the free urease (1– 5 mM) [35]. The increase of the K m constant after the conditioning step for longer time or in more concentrated buffer solution can be attributed to a partial exchange of the ZnAl – Urease nanocomposite by electrolyte phosphate anions causing changes in the swelling properties of the LDH nanoparticles and consequently modifications of the accessibility of urease to urea substrate. Indeed in the previous work

160 140

Biosensor response (mV)

120 ∆VFB(mV)

100 80 60 40 20 0 -20 1,0


2,0 2,5 3,0 3,5 pUrea = - Log[urea (M)]



Fig. 3. Calibration curve for urea in 5 mM PB (pH 7.4) at the IS/ZnAl – Urease LDH biomembrane (without reticulation (n), with reticulation 8 nm (R) and 16 mn (r).

120 110 100 90 80 70 60 50 40 30 20 10 0 -10 0


100 150 Time (h)



Fig. 4. Storage stability in 5 mM PB (pH 7.4) of the IS/ZnAl – Urease LDH biomembrane (8 min GA).


H. Barhoumi et al. / Materials Science and Engineering C 26 (2006) 328 – 333









∆VFB (mV)


1,2 0,8




2,64 2,62



0,0 0,6






Yreal(mS) Fig. 5. Nyquist spectra in admittance representation for IS bare electrode (n) and IS/ZnAl – Urease LDH biomembrane (R) (5 mM PB, pH 7.4 E app = 1.5 V).

[6], the urease/ZnAl– Cl LDH mixture coated on the pH-FET transducers were used as sensing biomembranes for urea determination under the same conditions (5 mM PB solution pH 7.4). In this case, K m value was found to be 2.4 mM. This value was higher than that obtained with the ZnAl – Urease LDH biomembrane. Moreover, for the 1 : 1 and 2 : 1 ratios of urease/ZnAl – Cl LDH mixture, the urea biosensors were characterized by sensitivities of 6.06 and 12.42 mV/pUrea and maximum responses of 47 and 72 mV, respectively. These urea pH-FET biosensor performances were not so good in comparison with our results obtained with urea pH-IS biosensors using hybrid ZnAl –Urease LDH. Our results show the positive role played by the exchange reaction to prepare hybrid enzyme LDH material for biosensor development. 3.3. Impedance measurements The electrochemical impedance spectroscopy (EIS) measurements were presented in the complex plan as a plot of YW vs. YV which represents the imaginary and real part of the admittance, respectively. Fig. 5 shows the Nyquist plots of the bare IS electrode and of the IS/ZnAl–Urease LDH biomem-














3 mM 30 mM 60 mM 80 mM 100 mM 110 mM

1.0 0.8 0.6 0.4 0.2

Log |Z (Mohm)|





40 60 [Urea]/mM



2,58 120

Fig. 7. Calibration curves of the urea biosensors obtained from impedance (A) and capacitance (B) measurements for IS/Zn – Al – Urease LDH biomembrane (8 min GA).

brane performed in the frequency range from 0.1 to 100,000 Hz. We observe a decrease of admittance after the functionalization step. This change in the admittance was attributed to the capacitive term induced by the deposited ZnAl – Urease composite film. Moreover, in the presence of urea, we have noticed a small change in the admittance for IS/ZnAl– DS LDH electrode, this low response can be explained by the adsorption of the urea molecules on the LDH matrix that can induce a change of the LDH layer conductivity. Fig. 6 shows the Nyquist spectra of the ZnAl – Urease LDH composite film coated on IS electrode at different urea concentrations. We observe a decrease in admittance when the urea concentration increases. This variation was attributed to changes in concentration of OH ions, generated by the hydrolysis urea reaction, and responsible for the conductivity changes near the biomembrane/transducer interface. In addition, we observed clearly that the change in conductivity decreases the charge transfer resistance characterized by the decrease of the semi circle diameter. The calibration curves for urea obtained from impedance measurements represented by the log |Z| vs. the urea concentration. The curve shows the enhanced dynamic range of the impedance method (3 to 110 mM) in comparison with the small dynamic range obtained in the case of the capacitance method (0.1 to 5.6 mM) (Fig. 7). In human blood, urea concentration varies between 6 and 8 mM for a healthy person. For a person with a kidney dysfunction, urea concentration is higher, up to 40 mM [36]. This justifies greatly the use of IS/ ZnAl –Urease LDH biomembrane by impedance measurements for urea determination in clinical applications. It should be noted that, for the pH-FET –urease/[Zn –Al – Cl] LDH device, the linear range remains smaller even in the presence of tetraborate, an urease inhibitor inducing the linear range expansion (0.1 to 12 mM) [6].

0.0 0.0


1.0 1.5 Yreal(mS)



Fig. 6. Nyquist spectra of IS/ZnAl – Urease LDH biomembrane (8 min GA) at different urea concentrations (5 mM PB, pH 7.4 E app = 1.5 V).

4. Conclusion The construction of bio-inorganic host – guest system based on hybrid ZnAl –urease LDH materials coated on the pH-IS electrode is of substantial fundamental and practical impor-

H. Barhoumi et al. / Materials Science and Engineering C 26 (2006) 328 – 333

tance for urea determination. We have used capacitance and non faradaic impedance spectroscopy for urea biosensors characterizations. The optimized urea biosensor shows a high sensitivity to urea detection. In addition, impedance spectroscopy measurements allowed extending the dynamic range of urea concentration useful for blood analysis and more precisely for hemodialysis monitoring. The same order of magnitude K m values were obtained for ZnAl – Urease composite with respect to the value reported for the free enzyme. This indicates the high biocompatibility produced by LDH matrix to enhance the urease – urea complex stability. This work will be continued by studying new urease – LDH nanocomposites prepared by the coprecipitation method varying the urease amount entrapped within the material.

[10] [11] [12] [13] [14] [15]



This work was supported by the CMCU contract with grant No. 03S1205. Authors thank the GDR CNRS 2619 ‘‘Microcapteurs,’’ Rhone-Alpes MIRA cooperation program and ACI Nanosciences No. NR0005 for partial support. References [1] B. Krajewska, M. Leszko, W. Zaborska, J. Chem. Technol. Biotechnol. 48 (1990) 337. [2] K. Spencer, Ann. Clin. Biochem. 23 (1986) 1. [3] P.J. Held, N.W. Levin, R.R. Bovbjerg, M.V. Pauly, L.H. Diamond, J. Am. Med. Assoc. 265 (1991) 871. [4] S. Vitalii, W. Howard, V.J. David, J. Colloid and Interface Sci. 185 (1997) 94. [5] A. Senillou, N. Jaffrezic, C. Martelet, S. Cosnier, Anal. Chim. Acta 401 (1999) 117. [6] J.V. De Melo, S. Cosnier, C. Mousty, C. Martelet, N. Jaffrezic-Renault, Anal. Chem. 74 (2002) 4037. [7] C. Mousty, Appl. Clay Sci. 27 (2004) 159. [8] D. Shan, S. Cosnier, C. Mousty, Anal. Chem. 75 (2003) 3872. [9] D. Shan, S. Cosnier, C. Mousty, Anal. Lett. 36 (2003) 909.

[16] [17] [18] [19] [20] [21] [22]

[24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]


D. Shan, C. Mousty, S. Cosnier, Anal. Chem. 76 (2004) 178. D. Shan, S. Cosnier, C. Mousty, Biosens. Bioelectron. 20 (2004) 390. A. Vaccari, Appl. Clay Sci. 14 (1999) 161. E. Suzuki, Y. Ono, Bull. Chem. Soc. Jpn. 61 (1988) 1008. H. Schaper, J.J. Berg-Slot, W.H.J. Stork, Appl. Catal. 54 (1989) 79. L. Barloy, J.P. Lallier, P. Battioni, D. Mansuy, Y. Piffard, M. Tournoux, J.B. Valim, W. Jones, New J. Chem. 16 (1992) 71. P.C. Pavan, G.D. Gomes, J.B. Valim, Microporous Mesoporous Mater. 21 (1998) 659. P.C. Pavan, E.L. Crepaldi, G.D. Gomes, J.B. Valim, Colloids Surf., A Physicochem. Eng. Asp. 154 (1999) 399. S. Miyata, T. Kumura, Chem. Lett. (1973) 843. M. Meyn, K. Beneke, G. Lagaly, Inorg. Chem. 29 (1990) 5201. A. Schmassmann, A. Tarnawski, B. Flogerzi, M. Sanner, L. Varga, F. Halter, Eur. J. Gastroenterol. Hepatol. 5 (1993) 111. A.I. Khan, D. O’Hare, J. Mater. Chem. 12 (2002) 3191. J.H. Choy, S.Y. Kwak, J.S. Park, Y.J. Jeong, J. Mater. Chem. 11 (2001) 1671. J.H. Choy, S.Y. Kwak, Y.J. Jeon, J.S. Park, Angew. Chem., Int. Ed. Engl. 39 (2000) 4042. J.H. Choy, S.Y. Kwak, J.S. Park, Y.J. Jeon, J. Portier, J. Am. Chem. Soc. 121 (1999) 1399. N.T. Whilton, P.J. Vickers, S. Mann, J. Mater. Chem. 7 (1997) 1623. F. Leroux, J. Gachon, Jean-Pierre Besse, J. Solid State. Chem. 177 (2004) 245. J.-H. Choy, E.-Y. Jung, Y.-H. Son, M. Park, J. Phys. Chem. Solids 65 (2004) 509. L. Ren, J. He, S. Zhang, D.G. Evans, X. Duan, J. Mol. Catal., B Enzym. 18 (2002) 3. A. De Roy, C. Forano, k. El Molki, J.P. Besse, M.L. Occelli, H.E. Robson, Eds: Van Nostrand Reinhold: New York, (1992) 108. M. Adachi-Pagano, C. Forano, J.-P. Besse, Chem. Commun. (2000) 91. H. Barhoumi, A. Maaref, R. Mlika, C. Martelet, N. Jaffrezic-Renault, L. Ponsonnet, Mater. Sci. Eng., C, Biomim. Mater., Sens. Syst. 25 (2005) 61. N. Jaffrezic-Renault, Sensors 1 (2001) 60. D. Pijanowska, W. Torbicz, Sens. Actuators, B 44 (1997) 370. S. Poyard, N. Jaffrezic-Reanault, C. Marelet, S. Cosnier, P. Labbe´, Anal. Chim. Acta 364 (1998) 165. D. Liu, K. Ge, K. Chen, L. Nie, S. Yao, Anal. Chim. Acta 307 (1995) 61. A.P. Soldatkin, O.A. Burbriak, N.F. Starodub, A.E. El’skaya, A.K. Sandrovskii, A.A. Shul’ga, V.I. Strikha, Russ. J. Electrochem. 29 (1993) 279.