urease materials: Synthesis and application to urea biosensors

urease materials: Synthesis and application to urea biosensors

Materials Science and Engineering C 26 (2006) 387 – 393 www.elsevier.com/locate/msec Nanohybrid-layered double hydroxides/urease materials: Synthesis...

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Materials Science and Engineering C 26 (2006) 387 – 393 www.elsevier.com/locate/msec

Nanohybrid-layered double hydroxides/urease materials: Synthesis and application to urea biosensors S. Vial a , C. Forano a,⁎, D. Shan b , C. Mousty b , H. Barhoumi c , C. Martelet c , N. Jaffrezic c a

b

Laboratoire de Matériaux Inorganiques, CNRS UMR 6002, FR 2404, Université Blaise Pascal, Aubière, France Laboratoire d'Electrochimie Organique et de Photochimie redox UMR CNRS 5630, ICMG FR 2607, Université Joseph Fourier, Grenoble, France c CEGELY UMR 5005, Ecole Centrale de Lyon, Ecully, France Available online 27 December 2005

Abstract Nanohybrid [ZnAl]-layered double hydroxides/urease were prepared for the first time using the coprecipitation of enzyme and inorganic matrix. By varying the respective amount of urease and LDH, we obtained hybrid materials with various amount and dispersion rate of active biomolecules. X-ray diffraction and infrared spectroscopy confirm the preservation of the structure of each partner while the morphology properties are in good agreement with the permeability study. These new nanohybrids were applied for the development of urea biosensors. Biosensor responses to urea additions were obtained using capacitance (C vs. V) measurements at urease–LDH biofilm deposited on an insulated semiconductor (IS) structure. © 2005 Elsevier B.V. All rights reserved. Keywords: Biosensors; Hybrid materials; Urease; Layered double hydroxides; Hydrotalcite-like compounds

1. Introduction Bioinorganic hybrid materials constitute a new generation of materials, at the interface of biology and material science, able to display functionalities as complex as that of natural systems such as drug vectorisation and delivery, molecular machinery functions or sensing properties [1]. The development of an efficient biomaterial must answer to drastic constraints such as a high density of biomolecules immobilized in the host inorganic structure, an opened structure allowing the easy accessibility of the molecular target, the maintenance of the biological activity and a good stability under storage. Soft chemistry processes appear then to be the only synthetic preparation methods that are able to preserve the integrity of the biological properties. Indeed, immobilization of biomolecules in inorganic matrix has been obtained either by the Sol–Gel process [2] in silica matrices or by adsorption on clays [3]. Layered inorganic solids have opened structures due to their low-dimensional character favourable for the intercalation of a large diversity of organic molecules and macromole⁎ Corresponding author. E-mail address: [email protected] (C. Forano). 0928-4931/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2005.10.069

cules. These inorganic structures are known to exhibit high specific surface area for biomolecule immobilisation. The expansion of their interlayer domains is favoured by an easy breaking of weak van der Waals or hydrogen bonding, which allows the interlamellar confinement of active molecules. Moreover, bidimensional materials are easily nanostructured as thin films with biomolecules sandwiched between inorganic layers, allowing oriented properties. The intercalation or ionic exchange properties of layered inorganic solids can be advantageously coupled with redox or acido-basic properties of the framework leading to a synergy of both guest and host properties. Among these bioinorganic structures, enzyme–clay complexes [3,4] have already demonstrated to play a key role in the biochemistry of soil and to find applications as biosensors [5] or bioreactors [6]. However, biomolecules often bear an overall negative charge, which limits their attraction by positively charged layers of clays or cationic clays. Layered double hydroxides (LDHs also referred as anionic clays) appear to be very appropriate alternative materials due to their anionic exchange properties. LDHs display a layered structure built on a stackII ing of positive layers ([M1−x MxIII(OH)2]x+), separated by interlamellar domains constituted of anions and water molecules

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([Xx/q·nH2O]x−) (Fig. 1) [7]. The main properties of LDH arise from

2. Experimental details 2.1. Materials

– their wide layer and interlayer chemical compositions, based on the multiple combination of [MII–MIII]–X and R = MII/ MIII, – their high and tunable layer charge density, determined by R = MII/MIII and leading to variable anion exchange capacities (for [Zn–Al]), – their opened structure which can accommodate large anionic molecules, – their adjustable textural properties controlled by the synthesis process and conditions. All these suitable properties make the LDH very favourable host structures for the preparation of bio-LDH hybrid materials [8] and particularly for the confinement of proteins [9,10]. Compared to clay, they display higher guest–host interactions due to the association of ionic and hydrogen bonding. It was reported recently in the literature the intercalation of nucleoside monophosphate [11], DNA [12] and Penicillin G Acylase [9] in LDH matrix. One of the most interesting field of applications for layered double hydroxides as host structure for enzymes is the development of biosensors. Biosensors are under strong investigations due to their large potentiality of applications, more particularly for medical diagnosis or continuous monitoring in environment [13]. Such devices contend a biological sensing element connected to a transducer that converts the specific biorecognition event to an electrical signal. One of the key steps in the fabrication of biosensors consists of the effective immobilization of biomolecules onto the transducer surface. Urea biosensors are developed for the determination of urea in human bodies in order to diagnose diabetes or uremie pathology and dysfunction of liver or kidney organs [14]. We present in this paper the preparation of nanohybrid urease–LDH materials for the elaboration of urea biosensors. The strategy of urease immobilization in [Zn–Al] LDH explores the coprecipitation process at different synthesis conditions. Structural and textural characterizations have been studied in order to assess the dispersion of the biomolecules in the inorganic matrix. Enzymatic activity of immobilized urease biosensors has been measured and compared to the free enzyme.

For all preparations, the aluminium chloride (Acros), zinc chloride (Acros) were of analytical grade. Jack beans urease (EC 3.5.1.5) with an activity of 54.3 U/mg was purchased from Sigma. 2.2. Preparation of ZnAl–Urease materials by the coprecipitation method Hybrid [ZnRAlCl]/Urease phases with an urease/LDH ratio Q from 1/3 to 3 were prepared by the coprecipitation method according to procedure already published [15]. A 23-ml mixed aqueous solution of ZnCl2 and AlCl3, with a Zn2+/Al3+ molar ratio R = 2, 3, 4, and a total concentration of metallic cations of 0.02 M, was introduced with a constant flow into a reactor containing a 30-ml urease solution. The pH was maintained constant at a value of 8.0 during all the coprecipitation by the simultaneous addition of a 0.04 M NaOH solution. The suspension was aged at room temperature with stirring for 24 h. The final product was centrifuged and washed several times with decarbonated water and finally dried in air at room temperature. The amount of immobilized urease was determined by UV spectroscopy. The results indicates a total precipitation of urease/LDH ratio for Q = 1/3 to 1 and a slight decrease of the ratio for Q = 2 and 3. Moreover, no release of urease was observed during the washing process, showing the strong affinity of urease of the LDH matrix. All experiments were carried out under a stream of N2, to avoid or at least minimize the contamination by atmospheric CO2. 2.3. Spectroscopic test procedure The enzymatic activity was determined following a protocol described by Worthington-Biochem and adapted from Kaltwasser et al. [16]. The hydrolysis of urea by urease is measured by coupling ammonia production to the glutamate dehydrogenase (GLDH) reaction. The UV–Vis measurements were performed on a Varian Cary spectrophotometer. The cell contains 2.4 ml 0.1 M phosphate buffer (pH 7.6), 0.1 ml 0.03 M adenosine-5′ diphosphate, 0.1 ml 0.01 M NADH, 0.1 ml 0.03 M α-ketoglutarate, 0.1 ml 1 M urea, 0.1 ml GLDH (500 U/ml). After incubation at 30 °C, 0.1 ml of urease or urease LDH samples

Fig. 1. Structural model of layered double hydroxides.

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was added into the cell. The decrease in NDH absorbance was recorded at 340 nm for 10 min. The equivalent activities of 1 mg [Zn–Al–Urease] nanohybrids (Ueq/mg) were determined several times by comparison with a well-known amount of free urease. It should be noted that these values of equivalent activity could not be used as an absolute urease activity within the nanohybrid since the absorbance measurements were disturbed by the LDH particles within UV cell. We use these values as an indicator of the presence of urease within the LDH nanohybrids related to the urease/LDH ratios (Table 1). 2.4. Electrochemical measurements All electrochemical measurements were performed in a conventional electrochemical cell containing a three-electrode system. The [Zn–Al–Urease]-coated electrode was the working electrode, a platinum plate or wire the auxiliary electrode and a saturated Ag/AgCl/KCl electrode served as reference electrode. Permeability measurements were performed with a potentiostat (Autolab, PGSTAT 100, Eco-Chemie, the Netherlands). The rotating disk electrode was glassy carbon electrode of 5 mm connected to a Tacussel EDI 101T/CTV 101T. Clay colloidal suspension (1 g.l− 1) was prepared by dispersing LDH in deionized water stirring overnight. A defined amount 20 μg LDH was spread on the surface of glassy carbon electrode. The coating was dried in air at room temperature. Before use, the modified electrode was incubated into phosphate buffer solution for 20 min. The reference electrode used was an Ag/AgCl/ KClsat electrode and the counter electrode was a Pt wire. The electrochemical probe was 2 mM hydroquinone dissolved in phosphate buffer solution pH 7.4. The biosensor response to urea addition was measured at ambient temperature using the capacitance (C vs. V) electrochemical measurements. The pH-sensitive IS field effect system (Si/SiO2/Si3N4) was 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 μm thickness, with 10 Ω 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 (3/8–5/8) mixture on the back side of the Si/SiO2/Si3N4 structures. Before the deposition of the biomembrane, the surface transducer was cleaned in successive baths of trichloroethylene, acetone, isoTable 1 Electrochemical characterisation of [ZnAl–Urease] films: permeability and biosensor performance Material

Q = Urease/ Activity Pm (cm/s) [ZnAl] (Ueq/mg)

[Zn3Al–Urease] [Zn3Al–Urease] [Zn3Al–Urease] [Zn4Al–Urease] [Zn3Al–Cl]/Urease [22]

3 2 0.33 0.33 2

0.96 0.29 0.23 0.12 –

1.6 × 10− 2 1.3 × 10− 2 0.70 × 10− 2 1.7 × 10− 2 1.4 × 10− 2

Sensitivity Vmax (mV/decade) (mV) 17 14 3 12 12

42 26 10 19 72

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propanol and sulphochromic mixture (sulphuric acid and chromic acid), washed with ultra-pure water, dried under nitrogen atmosphere at room temperature and placed at 70 °C for 10 min. The [Zn–Al–Urease] nanohybrids (2 g/L) were dispersed in 20 mM phosphate buffer solution (pH 7.4) by overnight stirring. The surface of the Si/SiO2/Si3N4 electrode (1 cm2) was coated by the spin-coating process (v = 1000 rpm) with a drop (20 μL) of this colloidal suspension. Then, the [Zn–Al– Urease] LDH-coated IS device was dried at room temperature for 30 min and then washed with a 5 mM phosphate buffer solution (PB, pH 7.4). All measurements were performed in darkness to avoid photoinduction of charges in the semiconductor transducer. The capacitance measurements were carried out at a frequency of 10 kHz with signal amplitude of 10 mV. The response of the biomembrane to urea can be explained by the local pH change of the electrolyte near the silicon nitride surface, which originates from the enzymatic reaction as follows: COðNH2 Þ2 þ 3H2 O

Y urease

− CO2 þ 2NHþ 4 þ 2OH :

Depending on the resulting pH value in the enzymatic membrane, the ΔVFB (flat band potential) of the semiconductor transducer changes and the position of the C–V curve shifts along the voltage axis. 2.5. Instruments Powder X-ray diffraction patterns were collected on a Siemens D500 diffractometer using CuKα radiation. Patterns were recorded over the 2θ range 2–70 in steps of 0.04° and a counting time of 8 s per stepscan. FT-infrared spectra of the samples in KBr matrix were recorded on a Perkin-Elmer 2000 FT in the range 4000–400 cm− 1. Scanning electron micrographs were recorded with LEO Stereoscan 360 at 15 kV at Casimir Technique, Aubière, France. 3. Results and discussion 3.1. Characterisations of hybrid materials With an isoelectric point of 5.0, urease is negatively charged at a pH = 8.0 and consequently displays appropriate ionic properties to be adsorbed or intercalated by anionic exchangers such as Layered Double Hydroxides. LDH exhibit great affinity towards organic anions and more particularly towards polar organic anions such as amino acids. Narita et al. [17] have recently shown by XRD study that a large series of amino acids (glutamate, aspartate, hystidine, typtophane, phenylglycine) are easily intercalated in ZnAl LDH. Intercalation is favoured not only by charge compensation between guest and host partners but also by hydrogen bonding between polar function of the biomolecules. Poly-amino acids such as proteins or enzymes must display a great affinity to the positive hydroxylated layers of LDH. LDH also referred as anionic clays are then better candidates for the immobilization of enzymes than cationic clays. Moreover, its bidimensional framework is

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opened for both a high confinement of urease and easy accessibility for molecular diffusion. This novel generation of hybrid bio-inorganic materials must display new properties based on a synergic effect of both inorganic layers and biomolecules functions which must develop at the nanoscale interface. High dispersion of enzymes and nano-inorganic layers must be reached in order to obtain a real homogeneous hybrid material. Soft chemistry preparation routes are well adapted to prepare such fragile materials. We then realized the coprecipitation of the LDH matrix at the surface of the biomolecules in a similar way than some bioorganisms (cocolytes, diatomee) developed their biomineralisation [18]. Nucleation and growing are supposed to occur at the negative surface of urease and to favor the association of the enzymes and the inorganic layers. Urease is also used as a template agent in the reaction medium, preventing the crystallization and the aggregation of the LDH particles. The coprecipitation of [Zn1−xAlx(OH)2]x+ layers at constant pH, with pH values chosen between 7.0 and 8.0 allows to prevent any deactivation of the urease. The effect of the anion exchange capacity and the urease/ZnAl mass ratio on the structure, morphology and activity of the hybrid biomaterials were investigated. The X-ray diffraction patterns of the coprecipitated [Zn–Al– Urease] samples are compared to the diffractogram of the reference [Zn–Al–Cl] LDH material (Fig. 2). This later compound shows structural properties in good agreement with that of the hydrotalcite-like compounds [19] and indicate that the solids consist of a well-crystallized single phase. The lattice parameters, refine on the hexagonal setting with rhombohedral symmetry (space group: R−3m), are a = 3.08 Å and c = 23.39 Å (d = c/3).The interlayer distance defined by (003) line, d = 7.80 Å, indicates the presence of the chloride anion between the sheets. The urease containing LDH coprecipitated phases show a strong decrease of crystallinity. The number of diffraction lines is lowered compared to the reference material. Their enlargement confirms both a reduction of the particle size according the Laue–Scherrer law, and a greater disorder of the structure

relative intensity

Q=2

Q=1

30

40

2θ °

1010 0111

018

012

20

015

101

10

50

110 113

Q=0

006

003

Q = 1/2

Q=0

60

70

Fig. 2. PXRD patterns of [Zn3Al–Urease] coprecipitated at different Q = Urease/ [Zn3Al] mass ratio.

absorbance

390

Interlayer H2O Interlayer anion MO-H

Interlayer anion

Latice

Q=0

urease

Q=1/3 Q=1 Q=2 Q=3 4000

3500

3000

2500

2000

1500

1000

500

Wavenumber cm-1 Fig. 3. Infrared spectra of [Zn3Al–Urease] coprecipitated at different Q = Urease/[Zn3Al] mass ratio.

with a net turbostratic effect. These XRD patterns are characteristic of highly disordered Cl- or carbonate-containing LDH phases. When increasing the urease over ZnAl mass ratio from 1/2 to 2, we observe a strong decrease of the (00l) diffraction lines intensities due to a loss of a well-ordered layer stacking. This structural change arises from a better dispersion of the inorganic layers in the bioorganic matter. The number of stacked layers is reduced from typically about 60 to 100 for highly crystallized [ZnAl–Cl] LDH to 15. Such exfoliation involves an increase of the surface area of the inorganic matrix and of the interface with urease. However, the large size of the urease monomer (9.6 nm × 5.4 nm) prevents any layer–interlayer ordering. We must then admit that the charge compensation between host structure and enzyme cannot be insured only by urease and co-intercalation of smaller inorganic anions must arise. The preservation of the (012) and (110) diffraction lines for all the hybrid materials evidences the formation of the layer structure. These structural observations show the successful presence of a LDH, although the presence of urease influence the precipitation of the inorganic compounds. We must mention that the XRD patterns of samples prepared by exchange reaction show that the structural feature of the [Zn–Al–Cl] precursor is preserved, indicating that no exfoliation/intercalation process takes place. The immobilisation of urease is also confirmed by FTIR (Fig. 3). All stretching and bending vibration modes of the urease [20] are observed in the spectra of the various hybrid samples beside the low-frequency bands characteristic of the [ZnAl] LDH matrix (νM–O at 841–647 cm− 1 and δO–M–O at 427 cm− 1). The bands observed at 1652 and 1543 cm− 1 correspond to the stretching and deformation mode of C_O (amide I) and N–H (amide II), respectively. The bands between 1306 and 1228 cm− 1 can be assigned to the C–N and N–H vibrations (amide III). The bands at 1440 and 1394 cm− 1 are due to C–H vibrations of the CH2 group. The increase of the relative intensity of the urease vibration bands with increase in Urease/ [ZnAl] mass ratio confirms the increase of amount of urease

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Fig. 4. Ideal representation of the structure of hybrid bioinorganic [ZnAl– Urease] materials.

immobilised by the inorganic support. The vibration bands of the confined enzymes are at the same wavelength position than those of the free enzyme. The structure of the protein coprecipitated in the inorganic materials seems to be preserved. However, the urease bands can also mask the contribution of charge compensating CO32− anions. Fig. 4 displays a realistic model of the hybrid materials structure in which non-exfoliated and exfoliated layers coexist in interactions with urease and compensating charge anions. 3.2. Textural and diffusion properties of the hybrid materials Scanning electron microscopy images of the reference [ZnAl–Cl] material and the coprecipitated [ZnAl–Urease] phases show the morphology of the samples with regard to the coprecipitation conditions (Fig. 5). The micrograph of [Zn3 AlCl] indicates a homogeneous morphology of hexagonalshaped primary particles forming “sand roses” aggregations where individual platelets are well identified. Such classical texture [21] for hydrotalcite like materials is due to the isotropic intergrowth of well-crystallized platelets caused by strong surface energies. When [ZnAl] LDH particles are coprecipitated with urease, strong textural changes are observed. Hybrid

3 µm

Q=0

10 µm

Q=1

391

[ZnAl–Urease] particles undergo a great tendency of preferential orientation. Platelets aggregate face to face stuck by urease enzymes. Thick films are formed by a stacking of parallel particles. The increase of Q = urease/[ZnAl] ratio increases the density of the film. For a ratio Q = 1, individual platelets are still observed with a preferential parallel orientation in the film and a random orientation at the surface of the aggregates. Urease appears to be the minor component in the hybrid material. In contrast, for Q = 2, the inorganic layers are no more identified. They are totally dispersed and strongly orientated in an urease medium. Both morphologies are in favour of a strong association between urease and [ZnAl] layers. The permeability Pm of the [Zn–Al–Urease] films was investigated by the use of linear sweep voltammetry at rotating disk electrode. The voltammograms were recorded at different rotation rates (ω) in an aqueous solution of a neutral molecule, hydroquinone as electroactive permeant, to avoid any interference with the charges of the membrane. These experiments were based on the typical equations introduced by Gough and Leypoldt in 1979 [22], relating the variation of limiting current ilim with the mass transport for a rotating disk electrode functionalized with an electro-inactive membrane. To estimate the permeability value (Pm), a plot of 1/ilim vs. 1/ω1/2 presents a linear behavior with the same slope as for a bare electrode (Levich current) with a positive intercept that depends on the permeability Pm of the biomembrane. The values of permeability for the different [ZnAl–Urease] films are given in Table 1. It clearly appears that the host matrices presented good permeability (1.6–0.7 × 10− 2 cm/s). It should be noted that the incorporation of urease biomolecule with the LDH structure does not affect drastically the permeability of the resulting biofilms compared to the permeability obtained with the [ZnxAl–Cl] LDH (2≤ x ≤ 4, Pm = 2.2–2.5 × 10− 2 cm s− 1). Moreover, these permeability values are higher than those obtained at a cationic clay modified electrode such as laponite without (2 × 10− 3 cm s− 1) or in the presence of urease (3 × 10− 3 cm s− 1) [23].

50 µm

10 µm

Q=1

Q=2

Fig. 5. Scanning electron micrographs of [Zn3Al–Urease] coprecipitated at different Q = Urease/[Zn3Al] mass ratio.

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decreases of 76%, whereas the free urease stored under the same conditions decreased of 62%. These observations show that the inorganic materials preserve and protect the enzyme and so evidence the stabilization of the enzyme when immobilized in the LDH matrix.

a

∆VFB (mV)

50

b

40

4. Conclusion

30

d 20

c

10 0 0

20

40

60

80

100

120

[Urea]/mM Fig. 6. Calibration curves for urea obtained at IS electrodes coated with different [Zn3Al–Urease] LDH Q = 3 (a), 2 (b), 0.33 (c) and [Zn4Al–Urease] Q = 0.33 (d) in 5 mM phosphate buffer solution, pH = 7.4.

3.3. Biosensor performances In order to validate this synthetic method, we ensured that the urease activity was not affected by the contact of Zn2+ and Al3+ metallic cations, added during the precipitation. The calibration curves for urea were obtained from the potentiometric responses of the [ZnAl–Urease] nanohybrid films prepared on IS under the same conditions. They were recorded in a 5 mM phosphate buffer at pH 7.4 (Fig. 6). As expected, the sensitivity and maximum potential response (Vmax) of the biosensors vary with the urease loading within the nanohybrids but also with the permeability of the biomembrane (Table 1). Indeed, biosensor characteristics depend on the activity of immobilized enzyme depending on the biocompatibility of the matrix and on steric hindrance in the coating limiting the diffusion of the substrate to reach the enzyme active site. For example, for the urease–LDH ratio 0.33, the increase of permeability for the [Zn4Al–Urease] LDH compared to the [Zn3Al–Urease] causes a significant increase of the sensitivity and Vmax. Moreover, biomembrane with higher urease loading gives rise to an extended dynamic range from 3 × 10− 3–0.1 M for 0.33 to 10− 4–0.1 M for 3. These biomembrane characteristics to urea are similar to that previously reported with other urease biosensors [23,24]. Indeed, we have used previously urease/[ZnAl–Cl] LDH mixture (1/2) coated on pH-ISFET transducer. In this case the sensitivity was 12 mV/decade [23]. Similarly IS coated with biodegradable latexes as encapsulation matrix for urease showed a linear response for urea concentration in the range 10− 4–10− 2 M with sensitivities of 10–24 mV/decade [24]. It should be noted that in both these cases, the stabilization of the biomembrane was necessary by using an additional chemical cross-linking step with gluraldehyde vapour. This confirms the good chemical inertia of LDH towards urease, since for similar permeability for urease–latexes films, the same sensibility and dynamic range are obtained. The long-term stability of urease within the nanohybrid was analysed after 6 months storing in freeze. The urease activity

The use of the coprecipitation method at fixed pH for the preparation of hybrid [ZnAl–Urease] materials allows a high dispersion of the enzyme in the inorganic matrix. In such soft chemistry conditions, the structure and part of the activity of the enzyme is preserved. Even though ordered urease intercalated LDH are not obtained, structural and textural characterizations show a great affinity between both partners. The preparation method used favours the immobilisation of a great and tunable amount of urease. The enzyme quantity affects strongly the morphology of these organo-mineral complexes. All these preliminary results show the very promising future of these new bio-nanohybrid materials. After a careful optimization of the preparation procedures, eventually with other enzymes, these new synthetic biomaterials will certainly find applications in the development of biosensors as well as of bioreactors. Acknowledgments This work was financially supported by the ACI Program Nanohybrids Enzymes-HDL 2003-NR0005 from the Research Ministry of France and from CMCU No. 03S1205. The authors acknowledge Arielle Lepellec for technical assistance. References [1] C.N.R. Rao, J. Mater. Chem. 9 (1999) 1. [2] W. Jin, J.D. Brennan, Anal. Chim. Acta 461 (2002) 1. [3] H. Quiquampoix, S. Servagent-Noinville, M.H. Baron, in: R.G. Burns, R.P. Dick (Eds.), Enzymes in the Environment, Marcel Dekker, New York, 2002, p. 285. [4] L. Gianfreda, M.A. Rao, F. Sannino, F. Saccomandi, A. Violante, Dev. Soil Sci. 28B (2002) 301. [5] K. Katsuki, K. Hamamato, Y. Yagi, F. Fukuoka, PCT Int. Appl., 2001 37 pp. [6] J.G. Miller, R.H. Bates, T.A. Boyer, D.R. Durham, PCT Int. Appl., 2000 22 pp. [7] V. Rives, Layered Double Hydroxides: Present and Future, Nova Science Publishers, New York, 2001. [8] J.H. Choy, J. Phys. Chem. Solids 65 (2004) 373. [9] L. Ren, J. He, S. Zhang, D.G. Evans, X. Duan, J. Mol. Catal., B Enzym. 18 (2002) 3. [10] Shan Dan, S. Cosnier, C. Mousty, Anal. Chem. 75 (2003) 3872. [11] S.-Y. Kwak, Y.-J. Jeong, J.-S. Park, J.-H. Choy, Solid State Ion. 151 (2002) 229. [12] J.-H. Choy, S.-Y. Kwak, J.-S. Park, Y.-J. Jeong, J. Portier, J. Am. Chem. Soc. 121 (1999) 1399. [13] G. Ramsey, Commercial Biosensors: Applications to Clinical, Bioprocess, and Environmental Samples, Wiley-Interscience, 1998. [14] A.P. Soldatkin, O.A. Burbriak, N.F. Starodub, A.E. El'skaya, A.K. Sandrovskii, A.A. Shul'ga, V.I. Strikha, Elektrokhimiya 29 (1993) 279. [15] A. de Roy, C. Forano, M. El Malki, J.P. Besse, in: M.L. Occelli, H.E. Robson (Eds.), Synthesis of Microporous Materials, Expanded Clays and Other Microporous Solids, Van Nostrand Reinhold, New York, 1992, p. 108.

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