Urease immobilization on biotinylated polypyrrole coated ChemFEC devices for urea biosensor development

Urease immobilization on biotinylated polypyrrole coated ChemFEC devices for urea biosensor development

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ITBM-RBM 29 (2008) 192–201

Original article

Urease immobilization on biotinylated polypyrrole coated ChemFEC devices for urea biosensor development Immobilisation de l’uréase sur des dispositifs ChemFEC recouvert d’un film de polypyrrole biotinylé en vue du développement d’un biocapteur d’urée H. Barhoumi a,∗ , A. Maaref a , S. Cosnier b , C. Martelet c , N. Jaffrezic-Renault c a

Laboratoire de physique et chimie des interfaces, faculté des sciences de Monastir FSM, rue de Kairouan-Monastir, 5000 Elomrane, Tunisia Département de chimie moléculaire, UMR, CNRS 5250, FR CNRS 2607, université Joseph-Fourier, B.P. 53, 38041 Grenoble cedex 9, France c Laboratoire de sciences analytiques, UMR, CNRS 5180, université Claude-Bernard-Lyon-1, bâtiment Raulin, 69622 Villeurbanne cedex, France b

Received 27 October 2007; accepted 12 November 2007 Available online 25 April 2008

Abstract In this report, we describe a novel strategy for the design of a clinical urea biosensor using a process based on assembled multilayer systems. Biotinylated enzyme (urease–subiotin) was immobilized on the biotinylated polypyrrole coated Chemical field effect capacitance (ChemFEC) devices using the high avidin–biotin affinity. The immobilized enzyme activity was checked by its catalytic conversion of urea into carbon dioxide and ammonia. Electrochemical response of the bridge system constructed on Si/SiO2 /Si3 N4 electrodes to urea addition was evaluated using the capacity–potential measurements. In addition, contact-angle measurements were carried out to control the change of surface energy and their components before and after each layer formation. The developed urea biosensor demonstrates high performances: a good sensitivity of 50 mV/pUrea in the linear urea concentration range from 10−4 to 10−1 M and an excellent operational stability after three weeks of continuous use. The immobilized urease was characterised through its apparent Michaelis–Menten constant (5 mM) and the activation energy of the enzymatic reaction (20 kJ mol−1 ). It was also shown that capacitive measurements can be used to quantify the interaction between molecular systems, based on avidin and biotin molecules. © 2007 Elsevier Masson SAS. All rights reserved. Résumé Dans cet article, nous décrivons une nouvelle stratégie pour la conception d’un biocapteur clinique pour la détection de l’urée, basée sur un système de multicouches assemblées. L’enzyme biotinylée (biotine–uréase) est immobilisée sur des dispositifs Chemical Field Effect Capacitance (ChemFEC), recouverts d’un film de polypyrrole biotinylé, en utilisant la forte affinité avidine–biotine. L’activité de l’enzyme immobilisée a été contrôlée par la conversion catalytique de l’urée en dioxyde de carbone et en ammoniaque. La réponse électrochimique du système multicouche, construit sur des électrodes Si/SiO2 /Si3 N4 à l’addition, d’urée a été évaluée grâce à des mesures capacité–potentiel. De plus, des mesures d’angle de contact ont été menées pour contrôler le changement de l’énergie de surface et de ses composantes avant et après la formation de chaque couche. Le biocapteur d’urée développé présente de très bonnes performances : une bonne sensibilité de 50mV/pUrée, dans une gamme linéaire de concentration de 10–4 à 10–1 M et une excellente stabilité opérationnelle après trois semaines d’utilisation continue. L’uréase immobilisée a été caractérisée par sa constante apparente de Michaelis-Menten (5mM) et l’énergie d’activation de la réaction enzymatique (20kJ mol–1 ).



Corresponding author. E-mail address: [email protected] (H. Barhoumi).

1297-9562/$ – see front matter © 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.rbmret.2007.11.004

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Il a également été montré que les mesures capacitives peuvent être utilisées pour quantifier l’interaction entre les systèmes moléculaires basés sur les molécules avidine et biotine. © 2007 Elsevier Masson SAS. All rights reserved. Keywords: Urea biosensor; ChemFEC devices; Poly(pyrrole–biotin); Avidin–biotin interaction; Contact-angle; Capacitance measurements Mots clés : Biocapteur d’urée ; Dispositifs ChemFEC ; Polypyrrole biotinylé ; Interaction avidine–-biotine ; Angle de contact ; Mesures capacitives

1. Introduction The basic approach for biosensor development is the simple adsorption, covalent attachment or the entrapment of biorecognition molecules in organic and inorganic matrices [1–4]. Recently, a novel strategy was extensively employed to immobilize biomolecules using the non-covalent binding avidin–biotin interaction. The latter immobilization method generally maintains biomolecule activity more successfully than other regularly-used methods because biomolecules are situated on the outside of the support matrix and this improves access of the substrate to them. Consequently, a lot of biotinylated biospecies such as bacteria, enzyme, ADN. . . were successfully immobilized using the robust way that is highly compatible with a lot of biological function and have non-denaturant effect [5,6]. The avidin–biotin strategy presents a variety of specific advantages over other immobilization techniques. In particular, the extremely specific and high affinity interactions between biotin and the glycoprotein-avidin (association constant Ka = 1015 M−1 ) lead to strong associations similar to the formation of covalent binding [7]. Hence, several studies have been carried out using the coupling avidin–biotin system to develop electrochemical biosensors, biochips and bioreactors. For example, biotinylated cholera toxin B was immoblized on polypyrrole–biotin coated optical fiber to construct a biosensor for anticholera toxin antibody detection [8]. Glucose oxidase (GOD) and peroxidase (POD) have successfully been immobilized in a polypyrrole matrix by an avidin–biotin molecular recognition process on carbon felt for bioreactors development [9]. DNA probes were immobilized on the polypyrrole–biotin film deposit on silicon substrate via an intermediate avidin layer. The developed biorecognition multilayer was successfully applied to the genotyping of Hepatitis C virus in blood samples [10]. Biotinylated bacteria were immobilized onto biotin–avidin modified gold electrode and a glassy carbon macro-electrode surfaces for nitrate sensor development [11]. In the present work, we report the development of urea enzymatic biosensor using a layer-by-layer assembly process. The sandwich system was constructed onto the chemical field effect capacitance (ChemFEC) (Si/SiO2 /Si3 N4 ) electrode surface respecting the regular procedure succession · · ·biotin/avidin/biotin· · ·. So, biotinylated urease biomolecules were grafted through avidin layer deposit on the biotinylated polypyrrole film prepared by chemical polymerization on the semiconductor electrode surface. The hydrophobic–hydrophilic character and surface thermodynamic properties of such deposit layer as well as surface free energy, acid (γH+ ) and base (γOH− ) components were determined by contact-angle measurements. In addition, the layer-by-layer system was constructed

at the solide–liquid interface and monitoring using capacitance measurements to quantify the high interaction between avidin and biotin. The biosensor signal resulting from urea hydrolysis by immobilized biotinylated urease was checked using electrochemical capacitance measurements. Furthermore, the adsorption isotherms of avidin and biotinylated urease biomolecules onto the biotinylated ChemFEC electrode surface were fitted with the Langmuir model to determine the energy of adsorption. 2. Experimental section 2.1. Reagents Biotinylated Urease from Biomeda and avidin (from egg white) were purchased from Sigma. Biotinylated pyrrole (Fig. 1) was synthesized according to the procedure described elsewhere [12]. Formamide (Sigma chemical co) and diiodomethane (Sigma chemical co, St. Louis, MO, USA) and all other reagents were of pure analytical grade. 2.2. ChemFEC substrates The ChemFEC substrates (Si/SiO2 /Si3 N4 ) were purchased from LAAS–CNRS Toulouse. These heterostructures were based on a p-type silicon substrate, 400 ␮m thickness, with a 10  cm resistivity, covered with a 50 nm layer of thermally grown silicon dioxide and a 100 nm layer of silicon nitride prepared by low pressure chemical vapor deposition (LPCVD). The ohmic contact was obtained by deposition of aluminum on the silicon unpolished face. Before depositing the biotinylated pyrrole layer on the ChemFEC device surface a cleaning procedure was necessary in order to improve hydrophilicity and to obtain a reproducible clean surface [13]. Briefly, surface substrate was cleaned using three successive ultrasonic baths of trichloroethylene (15 min), acetone (10 min) and propan-2-ol (10 min). Then, ChemFEC structure was immersed for 15 min into a sulfochromic mixture and washed with ultra-pure water, dried under nitrogen atmosphere at room temperature and placed at 70 ◦ C for 10 min. 2.3. Preparation of the ChemFEC/polypyrrole–biotin/avidin/biotin–urease system ChemFEC structure was spin-coated (2000 rpm) with 20 ␮L of pyrrole–biotin solution (1 mM) dissolved in acetonitrile. The structure was then dried for 20 min at ambient temperature before being soaked for two hours in 0.1 mol L−1 FeCl3 , dissolved in 0.1 mol L−1 hydrochloric acid to achieve, by chemical

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Fig. 1. Chemical polymerization of biotinylated polypyrrole polymer.

oxidation, the polymerisation of the adsorbed pyrrole–biotin monomers on the ChemFEC structure [14,15]. After the chemical oxidation step, ChemFEC structure was washed with deionised water. Then, a drop of 20 ␮L of avidin (0.5 mg/mL) was deposited on the biotinylated polypyrrole layer. Finally, avidin layer was coated with a drop (20 ␮L) of biotin–urease (1 mg/mL). The building up of successive layers system is given by the conceptual representation shown on Fig. 2. The modified ChemFEC structure was rinsed with phosphate buffer solution before the electrical measurements in order to remove adsorbed biomolecules. 2.4. Electrochemical measurements Electrochemical capacitance measurements were performed in a conventional electrochemical cell containing a three electrode system [16]. The ChemFEC device was the working electrode while a platinum plate electrode and a standard Ag/AgCl (saturated KCl) electrodes were used as counter and reference electrode, respectively. The electrochemical cell was connected to an amplifier system Voltalab 40 (radiometer analyt-

ical, SA, Villeurbane, France). The capacitance measurements were carried out at a frequency of 10 kHz with a signal amplitude of 10 mV. Capacitance method was based on the measurement of the local pH change when the H+ ion concentration decreases near the silicon nitride–biomembrane interface. pH variation resulting from urea hydrolysis (equation (1)) modifies the ChemFEC flat band potential (VFB ). urease

(NH2 )2 CO + 2H2 O + H+ −→ NH4 + + HCO3 −

(1)

This pH change induces the shift of the capacity–potential characteristic along the voltage axis for different urea concentrations and the VFB is given by equation (2) as follows:   RT Log[urea] (2) VFB VFBi + α F where VFBi is the initial flat band potential of the functionalized ChemFEC structure before adding urea. R is the universal gas constant, F is the Faraday constant, T is the absolute temperature in Kelvin and α is a coefficient introduced in a previous work [17].

Fig. 2. Schematic representation and synthetic procedure used for the multilayer system construction: (a) chemical polymerization of the polypyrrole–biotin film at the electrode surface, (b) immobilization of the avidin layer via the biotin entities linked to the polypyrrole network and (c) anchoring of the urease–biotin entities on the polypyrrole–biotin/avidin double layer.

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All measurements were performed in a dark Faraday cage at room temperature. When not used, the samples were stored in 5 mM phosphate buffer, pH = 7.4 at 4 ◦ C. 2.5. Contact-angle measurements and surface energy determination Contact-angle measurements consisted of the sessile drop method with an apparatus provided by GBX scientific instrument (Romans, France). Image of the drop deposit on the modified electrode surface was recorded by a video camera and an image-analysis system calculates the contact-angle (θ) from the shape of the drop. 3. Results and discussion 3.1. Chemical polymerization and atomic force microscope (AFM) characterisation of the biotinylated polypyrrole layer In general, polypyrrole polymers can be easily formed following two conventional preparation methods, namely electropolymerization on conductive surfaces and chemical oxidation step [18–20]. Since ChemFEC structures are not conductive, the oxidative polymerization of biotinylated pyrrole monomers was carried out by a simple chemical oxidation in aqueous solution of FeCl3 . The polymerization of biotinylated pyrrole, thus, proceeds through the formation of radical cations along the polymer backbone. These radical cations couple to

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form oligomers, which are further oxidized to form additional radical cations. The chemical polymerization reaction can be described by the stoichiometric equation [21,22]: nC4 H5 N + (2 + y)n FeCl3 → [(C4 H5 N)n nyCl− ] + (2 + y)nFeCl2 + 2nHCl where y is the degree of polypyrrole oxidation, which defines its ion exchange properties. AFM technique was carried out to obtain a topographical map of the poly(pyrrole–biotin) surface layer after the chemical polymerisation step. The AFM images of the poly(pyrrole–biotin) layer show a nodular structure, typical of conducting polymer, including polypyrrole (Fig. 3) [23]. The nodes or grains, whose size is ranging from 17.5 to 22.5 nm in radius, are uniformly distributed to give homogeneous surface characterised by low roughness (root-mean-square [RMS] = 6.807 nm). 3.2. Contact-angle and free energy parameters of such deposit layer Contact-angle measurements were performed to control the wettability change of the modified electrode surface before and after such deposited layer. Ultra-pure water was used as liquid test to determine the hydrophilicity (low contact-angle) or the hydrophobicity (high contact-angle) of the modified electrode surface. According to the Van-Oss approach [24], the surface free energy and Lewis acid-base parameters were calculated from the contact-angles of sessile drops of apolar

Fig. 3. An AFM image of biotinylated polypyrrole film obtained by chemical oxidation step of pyrrole–biotin deposited on ChemFEC electrode surface.

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Fig. 4. Camera images of small water droplet (2 ␮L) deposited on silicon nitride electrode surface before and after the anchoring of different layers.

(diiodomethane), less polar (formamide) and high polar (water) liquids deposited on the electrode surface. In Fig. 4, we report the camera images of small water droplet (2 ␮L) deposited on silicon nitride substrate surface before and after such modification step. We note that untreated silicon nitride surface presents superhydrophobic character predicted by high contact-angle value (80◦ ). After the cleaning treatment process, ChemFEC electrode shows a highly hydrophilic surface (23◦ ), once treated successively with organic solvents, hot sulfochromic mixture. After the deposit of the biotinylated pyrrole layer, the coated silicon nitride surface becomes more hydrophobic due to the organic nature of the coating. Then, when the biotinylated pyrrole layer was oxidised by FeCl3 solution, we observe that the hydrophobicity of the electrode surface increases. This behaviour reflects the formation of a more compact organic layer via the formation of long alkyl chains due to the polymerization step. Nevertheless, the contact-angle value (42◦ ) obtained for the biotinylated polypyrrole surface layer was less important than these values given in many other works, including unmodi-

fied polypyrrole polymer [25]. The subsequent avidin anchoring leads to the coverage by an avidin monolayer exhibiting a superhydrophilic character shown by a low contact-angle (10◦ ). Then, the formation of biotinylated urease layer on the ChemFEC electrode via an avidin bridge induces an increase in surface hydrophobicity (72◦ ). This may be ascribed to the hydrophobic part of the immobilized urease. To confirm the high affinity between the different assembled layers of the sandwich system, free surface energy, acid and base components were calculated according to the Van-Oss model. All resuls are gathered in Table 1. The high free-surface energy values (46.5 < free energy (mJ/m2 ) < 58.3) demonstrate the best adhesion between different biotinylated layers and the intercalated avidin layer. After the functionnalization steps, the base component remains important, in particular for the avidin layer. This result can be explained by the basic character of avidin (isoelectric point ∼11) at neutral pH. A high decrease in the basic component was observed for the biotinylated urease layer. Also, urease contains both hydrophobic and hydrophilic amino acids and their spatial arrangement near the surface membrane can be

Table 1 Contact-angle, total free energy and their components obtained from unmodified and modified ChemFEC electrode surface using the Van-Oss approach Electrode

ΘWater (◦ )

ΘForm (◦ )

ΘDiiod (◦ )

Energy (mJ/m2 )

γOH− (mJ/m2 )

γH+ (mJ/m2 )

Untreated Si3 N4 surface Cleaned Si3 N4 surface Si3 N4 /pyrrole–biotin Si3 N4 /poly(pyrrole–biotin) Si3 N4 /poly(pyrrole–biotin)/avidin Si3 N4 /poly(pyrrole–biotin) /avidin/biotin–urease

80.0 23.0 34.3 42.0 12.2 73.7

62.1 17.6 25.3 29.6 28.5 44.9

65.4 21.2 38.2 35.9 33.7 34.0

32.1 58.3 52.4 51.1 47.0 46.5

6.4 44.6 40.3 34.6 63.1 5.6

1.1 5.5 0.9 0.6 0.1 0.7

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biosensor give rise to a novel immobilization strategy of urease using the high affinity between avidin and biotin. In addition, this immobilization method excludes the use of cross-linking chemical reagent and prevents enzyme from inhibition effects. For the pyrrole–biotin/avidin/biotin–urease assembled system the calculated Michaelis–Menten constants, Km = 5 mM was obtained from the Lineweaver–Burk representation. This value of Km shows that the immobilized urease activity was kept after the immobilization process in comparison with the free urease (1 ≤ Km ≤ 5). 3.4. Thermal stability of the multilayer system

Fig. 5. Urea calibration curves of ChemFEC/poly(pyrrole–biotin)/ avidin/biotin–urease electrode, obtained by varying the poly(pyrrole–biotin) amount: (䊉) 1 × 10−7 , (), 1.5 × 10−7 and () 2 × 10−7 mol cm−2 . Performed in phosphate buffer, 5 mM and pH = 7.4.

influenced by the presence of the grafted biotin entity. The first types of amino acid are oriented towards the membrane volume to accomplish the formation of avidin–biotin complex. Also, the second types of amino acid are distributed at urease surface and oriented outside, leading to a more hydrophobic membrane surface [26].

The activity of immobilized enzyme can be influenced by some thermodynamic parameters, as well as pH and temperature. For this reason, the effect of temperature on the biosensor response to urea addition was also studied. The measurements were carried out in thermostatic three electrode cell between 15 and 55 ◦ C (Fig. 6). It was shown that immobilized enzyme activity increases with temperature and presents an optimum value at 35 ◦ C. For higher temperature, we observed a decrease in biosensor response and the immobilized enzyme losses its

3.3. Application of the ChemFEC modified electrode to urea detection The electrochemical response of the assembled multilayers system was studied by capacitance measurements. Fig. 5 shows the potentiometric response of ChemFEC/ poly(pyrrole–biotin)/avidin/biotin–urease biosensor for different urea concentrations. The modified electrode shows a linear response to urea addition from 10−4 to 10−1 M with a good sensitivity of 50mV/pUrea. The good ChemFEC/ poly(pyrrole–biotin)/avidin/biotin–urease biosensor performances were obtained using a storage medium (phosphate buffer 5 mM, pH = 7.4) containing ethylenediaminetetraacetic acid (EDTA) (10−3 M). According to the EDTA effect on urease biomolecules quoted in many works [27], it can be concluded that the use of EDTA in the storage medium stabilises and ensures a better operation of the constructed urea biosensor and permit his re-use for several times. The effect of the poly(pyrrole–biotin) thickness on the biosensors response was investigated by varying the amount of pyrrole–biotin monomer deposited and polymerized on the ChemFEC transducer. Fig. 5 illustrates the calibration curves for polypyrrole–biotin/avidin/biotin–urease matrix obtained for pyrrole–biotin coatings of 1 × 10−7 , 1.5 × 10−7 and 2 × 10−7 mol cm−2 . A net decrease of the urea biosensor response was observed when the amount of pyrrole–biotin and hence, the polymer thickness increased. This result can be explained by diffusion limit effects of the OH− to the insulatormembrane interface for quite thick poly(pyrrole–biotin) layer. The good performances demonstrated by the developed urea

Fig. 6. (A) Relation between temperature and the biosensor response in 5 mM phosphate buffer (pH = 7.4). (B) Arrhenius plot for poly(pyrrole– biotin)/avidin/biotin–urease membrane.

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Fig. 7. Schematic description of the multilayer deposition process monitored by capacitance measurements.

activity. This result is in agreement with that obtained for free or immobilized urease in another matrix [28]. The activation energy (Ea ) was estimated for immobilized biotinylated urease, through the Arrhenius equation (Eq. (3)): k = Ae−Ea /RT

(3)

where k is the constant rate, A is the frequency factor for the catalytic reaction, R is the universal gas constant and T is the absolute temperature in Kelvin. In electrochemical, process it was noted that is possible to use equivalent parameters in order to estimate constant rate k such as current, conductivity, optical density or potential. In our work, k was estimated by the flat band potential shift (ΔVFB ). The activation energy was obtained by plotting ln ΔVFB versus 1/T, which gives a straight line whose slope is –Ea /R, inside the temperature range where enzyme activity increases. The activation energy found for the immobilized biotinylated urease was 20.0 kJ mol−1 . This calculated value differs significantly from those reported for the free urease (12 kJ mol−1 ). The higher value of activation energy obtained for immobilized urease indicated that the applied immobilization procedure introduced changes in the structure of the urease molecules, which impeded the enzyme-catalyzed reaction [29]. Our result was similar and in best agreement with those reported for this enzyme entrapped in a different other matrix ∼20 kJ mol−1 [30]. 3.5. Avidin–biotin interaction at a biotinylated ChemFEC solid–liquid interface In literature, different techniques have been used to evaluate the specific interaction between avidin and biotin as well as AFM [31], impedance spectroscopy [32], cyclic voltammetry [33], Fourrier transform infrared reflection absorption spectra (FT-IRRAS) spectroscopy [34]. . . In the present work, an electrochemical method was used to evaluate the avidin–biotin interaction at a biotinylated polypyrrole coated ChemFEC elec-

trodes. The specific interaction was investigated by the shift of the flat band potential of the ChemFEC modified electrode when avidin bound to biotin. Fig. 7 shows in detail the different steps of the multilayer system formed at the solid–liquid interface. After the chemical polymerization step of the biotinylated polypyrrole layer, the ChemFEC coated electrode (working electrode) was transferred into the three electrode cells, in presence of a platinum auxiliary electrode and Ag/AgCl reference electrode for capacitive measurements in 5 mM phosphate buffer electrolyte. Then, the deposition of avidin and biotinylated urease layerby-layer on the ChemFEC biotinylated electrode surface was attempted by injection of avidin and biotinylated urease (diluted in phosphate buffer) in the cell. The sensorgram of layer-bylayer construction of avidin/biotin–urease multilayer is shown in Fig. 8(A). The results demonstrate that each injection of avidin and biotinylated urease caused the shift of the flat band potential of the ChemFEC electrode. During the formation of the first layer due to the grafting of avidin molecules onto the biotinylated polypyrrole surface, a high shift of the flat band potential was observed. When the biotinylated polypyrrole surface was completely covered by an avidin layer, we observed no change in flat band potential, which remains constant. The same results were observed during the formation of the second layer, when biotinylated urease was anchored on the avidin layer. A higher shift of the flat band potential was observed in the case of the avidin layer, compared to the deposited biotinylated urease layer. This result can be attributed to the field effect in the ChemFEC device. Indeed, the avidin biomolecules are in direct contact with the biotinylated polypyrrole surface whereas the biotinylated urease biomolecules are located at a longer distance. Moreover, the urease effect was masked by the avidin layer. 3.6. Adsorption isotherms The experimental adsorption isotherms of avidin and biotinylated urease from aqueous solutions onto the biotinylated

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Fig. 8. (A) Adsorption isotherms of avidin and biotinylated urease at different concentrations (mg/L) onto the biotinylated polypyrrole surface. (B) Experimental data () and Langmuir isotherm plot (—).

ChemFEC electrode surface are modeled using the Langmuir model [35]. The latter assumes uniform energies of adsorption onto the surface and no transmigration of adsorbate in the plane of the surface. In our work, we suppose that the shift of the flat band potential (ΔVFB ) resulting from the modified surface charge is proportional to the amount (qm ) of adsorbed biomolecules. The Langmuir equation may be written as qe = A + qm

bCe 1 + bCe

(4)

where qe is the amount of solute charge adsorbed per unit weight of adsorbent at equilibrium, A is a constant, Ce the equilibrium concentration of the solute in the bulk solution, qm the maximum adsorption capacity and b is the constant related to the free energy of adsorption and may be written as b = Ke Ead /RT

(5)

where K is the ratio of the adsorption constant (ka ) to the desorption constant (kd ), Ead is the free energy of adsorption, R is the universal gas constant and T is the absolute temperature in Kelvin.

Fig. 8(B) shows the best-fit obtained for such adsorption isotherm with a high coefficient of correlation R2 greater than 0.996 and with low chi-square (χ2 ∼10−6 ) showing a good linearity. Adsorption isotherms modeling shows that the interaction of avidin and biotinylated urease biomolecules with activated ChemFEC surface is a localized monolayer adsorption, that is, adsorbed biomolecules are adsorbed at definite, localized sites. Each site can receive only one molecule. The calculated energies of adsorption for avidin and biotinylated urease layers are respectively E1 = −327.49 J mol−1 and E2 = −2502.09 J mol−1 . Nevertheless, the thermochemistry of the avidin–biotin reaction demonstrates that the binding energy of avidin to biotin has been determined at 25 ◦ C (pH = 9, ammonium acetate buffer) to be equal to 94 kJ mol−1 [36]. The latter value of binding energy, found in literature for the native avidin–biotin complex, is greater in comparison with both values obtained in our work. This behaviour can be attributed to the presence of organic (pyrrole) and bioactive (urease) moieties linked to the biotin biomolecules. So, the interactions between biotin and avidin are characteristically associated with strong shape complementarity and subsequent changes in conformation would serve to reduce

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the affinity of biotin towards avidin [37,38]. Finally, we conclude that low accessibility of biotin could be responsible for the decrease of avidin–biotin binding energy system. 4. Conclusion Urea biosensors have been elaborated by the successive non-covalent immobilization of avidin and biotinylated urease biomolecules on a biotinylated polypyrrole film, chemically generated onto an insulated semiconductor electrode surface. The constructed multilayer system exhibits a good sensitivity to urea addition and high operational stability leading to urea biosensors with satisfactory analytical performances. In addition, we have demonstrated the feasibility to control the avidin–biotin interaction by constructing the multilayer system at the solid-liquid interface under electrochemical monitoring. Adsorption isotherms were established using flat-band potential shift. The flat-band potential shift allows to characterise the catalytic rate and to determine activation energy, showing the potentialities of this type of direct capacitance–voltage (C–V) measurements. Acknowledgments This work was supported by the CMCU contract grant no 03S1205 and by Rhone-Alpes MIRA cooperation program. Drs. S. Cosnier, C. Martelet and N. Jaffrezic-Renault would like to thank the GDR CNRS 2619 Microbiocapteurs for partial support References [1] Cosnier S. Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. Biosens Bioelectron 1999;14(5):443–6. [2] Barhoumi H, Maaref A, Rammah M, Martelet C, Jaffrezic-Renault N, Mousty C, et al. Insulator semiconductor structures coated with biodegradable latexes as encapsulation matrix for urease. Biosens Bioelectron 2005;20:2318–23. [3] Barhoumi H, Maaref A, Rammah M, Martelet C, Jaffrezic N, Mousty C, et al. Urea biosensor based on Zn3 Al-Urease layered double hydroxides nanohybrid coated on insulated silicon structures. Mater Sci Eng C 2006;26:233–328. [4] Soldatkin AP, Volotovsky V, El’skaya AV, Jaffrezic-Renault N, Martelet C. Improvement of urease based biosensor characteristics using additional layers of charged polymers. Anal Chim Acta 2000;403:25–9. [5] Ouerghi O, Touhami A, Jaffrezic-Renault N, Martelet C, Ben Ouada H, Cosnier S. Impedimetric immunosensor using avidin-biotin for antibody immobilization. Impedimetric immunosensor using avidin-biotin for antibody immobilization. Bioelectrochemistry 2002;56:131–3. [6] Cosnier S, Galland B, Gondran C, Le Pellec A. Electrogeneration of biotinylated functionalized polypyrroles for the simple immobilization of enzymes. Electroanalysis 1998;10:808–13. [7] Green NM. Avidin. Adv Protein Chem 1975;29:85–133. [8] Marks RS, Bassis E, Bychenko A, Levine MM. Chemiluminescent optical fiber immunosensor for detecting cholera antitoxin. Opt Eng 1997;36:3258–64. [9] Amounas M, Innocent C, Cosnier S, Seta P. A membrane based reactor with an enzyme immobilized by an avidin-biotin molecular recognition in a polymer matrix. J Membr Sci 2000;176:169–76. [10] Bidan G, Billon M, Livache T, Mathis G, Roget A, Torres-Rodriguez ML. Conducting Polymers as a link between biomolecules and microelectronics. SyntheticSynth Met 1999;102:1363–5.

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