Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films

Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films

Biosensors and Bioelectronics 19 (2004) 1641–1647 Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenim...

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Biosensors and Bioelectronics 19 (2004) 1641–1647

Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films Boris Lakard∗ , Guillaume Herlem, Sophie Lakard, Alexandros Antoniou, Bernard Fahys LCMI, Université de Franche-Comté, 16 route de Gray, 25030 Besançon Cédex, France Received 7 October 2003; received in revised form 17 December 2003; accepted 17 December 2003

Abstract The development of a new electrochemical sensor consisting in a glass-sealed metal microelectrode coated by a polyethylenimine film is described. The use of polymers as the entrapping matrix for enzymes fulfils all the requirements expected for these materials without damaging the biological material. Since enzyme immobilization plays a fundamental role in the performance characteristics of enzymatic biosensors, we have tested four different protocols for enzyme immobilization to determine the most reliable one. Thus the characteristics of the potentiometric biosensors assembled were studied and compared and it appeared that the immobilization method leading to the most efficient biosensors was the one consisting in a physical adsorption followed by reticulation with dilute aqueous glutaraldehyde solutions. Indeed, the glutaraldehyde immobilized urease sensor provides many advantages, compared to the other types of sensors, since this type of urea biosensor exhibits short response times (15–30 s), sigmoidal responses for the urea concentration working range from 1 × 10−2.5 to 1 × 10−1.5 M and a lifetime of 4 weeks. © 2003 Elsevier B.V. All rights reserved. Keywords: Polyethylenimine; Urea biosensor; Electrochemical polymerization; Enzyme immobilization

1. Introduction Urease is an important enzyme in biological systems, where it catalyses the conversion of urea to carbon dioxide and ammonia as follows: urease

NH2 CONH2 + H2 O −−→ CO2 + 2NH3 The development of a sensor of urea based on this catalytic reaction is of significant interest since urea is one of the bioproducts which is monitored in blood as an indicator of renal function. Indeed, elevated levels of urea are pathognomic of renal insufficiency (Spencer, 1986) and it has been shown that inadequacy of dialysis can be directly linked to mortality (Held et al., 1991). Moreover, urea is widely distributed in nature and its analysis is of considerable interest in agro-food chemistry and environmental monitoring. So, many different sensors have been developed: thermal (Xie et al., 1995; Xie and Danielsson, ∗ Corresponding author. Tel.: +33-381-666-294; fax: +33-381-666-288. E-mail address: [email protected] (B. Lakard).

0956-5663/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2003.12.035

1996), amperometric (Bertocchi et al., 1996; Adeloju et al., 1996), conductimetric (Chen et al., 1994; Sheppard et al., 1996), optical (Stamm et al., 1993; Li and Wolfbeis, 1993), piezoelectric (Xu et al., 1996) or potentiometric (Mascini and Palleschi, 1983; Alegret and Mart´ınez-Fàbregas, 1989; Gil et al., 1992; Adeloju et al., 1993; Glab et al., 1994; Gracia et al., 1996) biosensors. Potentiometric biosensors, based on the detection of ammonium ion (Alegret and Mart´ınez-Fàbregas, 1989; Gil et al., 1992; Gracia et al., 1996; Zamponi et al., 1996), ammonia gas (Mascini and Palleschi, 1983; Narinesingh et al., 1991) or pH change (Glab et al., 1994; Walcerz et al., 1995) produced by the enzymatic reaction, seem to be the most attractive biosensors for urea because of the general availability of the instrumentation required for their utilization. Indeed, these sensors only need ion-selective electrodes, ion-sensitive field effect transistors or pH-sensitive electrodes. The aim of the present work is to develop a potentiometric urea biosensor, based on urease immobilized on a chemically modified electrode. Indeed, the sensor is derived from polyethylenimine modified electrode obtained from electrochemical polymerization of pure ethylenediamine. Indeed, we have already showed that polyethylenimine modified

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electrodes can be used as pH, chemical and biochemical sensors since this polymer film coated on platinum electrodes showed good linear potentiometric responses to pH changes from 3 to 10 pH units. More, resulting electrodes present both good reversibility and good stability versus time (Lakard et al., 2002, in press). This phenomenon is due to the protonation and deprotonation of nitrogen atoms in the polymer films. This mechanism of protonation and deprotonation has been described in details in a previous paper (Herlem et al., 2001). Enzyme immobilization plays a fundamental role in the performance characteristics of biosensors since a large amount of the active enzyme should be directly attached to the surface of the electrode. So we have tested four different procedures of enzyme immobilization to determine which methods lead to polymer films with high enzyme loadings. The techniques tested were called: (P) for physical adsorption of urease on polyethylenimine films, (P ) for urease entrapment in polymer films, (C) for physical adsorption followed by reticulation with dilute aqueous glutaraldehyde solutions, and (C ) for activation with glutaraldehyde followed by contact with the enzyme solution. The characteristics of the biosensors assembled were studied and compared.

2. Experimental 2.1. Materials Ethylenediamine (EDA) and lithium trifluoromethanesulfonate (LiCF3 SO3 ) were from Sigma–Aldrich, and used as received in a glove box (Jacomex, France) under Ar stream of electronic grade (Argon U, Air Liquide, France). Urease (EC 3.5.1.5, from jack bean, 50 U/mg) was obtained from Sigma. We used glass-sealed platinum microdisk electrodes. These microdisk electrodes have an area of 0.785 mm2 . The preparation procedure of the electrodes involved pulling platinum wires into glass pipets. Immediately after pulling, the metal core is covered with glass and the next step is to expose the tip of the wire by polishing using a slowly rotating abrasive disk covered with 0.5 ␮m alumina (ESCIL). 2.2. Apparatus All cyclic voltammetry experiments were performed using an Autolab, model PGSTAT 20 (Ecochemie, The Netherlands), controlled by a PC computer via GPES software interface. The electrochemical cell consists in a classical three-electrode setting with an Ag wire as the reference electrode, noted SRE for Silver Reference Electrode, a platinum disk as the counter-electrode and a smooth platinum disk electrode, 0.785 mm2 area, as the working electrode. Electrode potential in the different buffered solutions were measured versus a saturated calomel electrode (SCE), XR100 model from Radiometer Analytical-France, via a pH meter,

PHN130T model (Tacussel electronics, France), used as a high impedance voltmeter (>10 M). Each potentiometric measurement was performed at about ±1 mV. 2.3. Preparation of polyethylenimine films Before each anodic passivation, all electrodes were polished with 0.5 ␮m alumina and washed with water. We used our new way of synthesis to electrodeposit PEI films onto the smooth Pt surface by means of cyclic voltammetry in 0.01 mol/l LiCF3 SO3 solutions of pure EDA (Herlem et al., 1997). Five cycles between 0 and +3V/SRE at 50 mV/s were performed and during the first scan the electrode was biased at +3V/SRE for 15 min (Fig. 1a). Following four scans were performed to check the stability and the insulating property of the coating. Then the modified electrodes were washed with water and acetone, then dried in an oven at 40 ◦ C, and tested in different urea solutions. 2.4. Enzyme immobilization Formation of functionalized modified electrode surfaces by means of electrochemical polymerization procedures can be applied for immobilization of enzymes or other biological recognition elements on electrode surfaces. In principle, there are two different approaches to immobilize enzymes using polymers. The first and most widely used is to entrap the enzyme within the polymer chain network during its electrochemical polymerization (Umana and Waller, 1986; Foulds and Lowe, 1986; Bartlett and Whitaker, 1987). The second is to use a two-step procedure consisting of the formation of a functionalized polymer film followed by the covalent binding of the biocomponents to the functionalities at the polymer surface (Schuhmann, 1994). Because of the large size of urease as compared with the pore size of polymers, urease is exclusively bound to the outer polymer surface whereas they should be evenly distributed within the film after the first approach. The procedures described below were used. 2.4.1. Procedure P (physical adsorption) (Roosevear et al., 1987a) After coating of the polyethylenimine films on the platinum electrode surfaces, the electrodes were left overnight at +5 ◦ C, in contact with an urease solution containing 2 mg of the enzyme per ml of a pH 5.6 phosphate buffer. The next day, the electrodes were washed with water and with a pH 7.0 phosphate buffer solution. Then, electrodes were kept, at +25 ◦ C, in a pH 7.0 phosphate buffer solution until use. 2.4.2. Procedure P (enzyme entrapment in polymer films) Two milligrams per ml of the enzyme were added to the 0.01 mol/l LiCF3 SO3 solutions of pure EDA. Five scans were performed between 0 and +3V/SRE at 20 mV/s and, during the first scan the electrode was biased at +3V/SRE for 15 min (Fig. 1b). Following four scans were performed

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Fig. 1. (a) Cyclic voltammograms of 0.01 M LiCF3 SO3 in pure EDA at a smooth platinum electrode for a bias time of 15 min. (b) Cyclic voltammograms of 0.01 M LiCF3 SO3 in EDA + urease (2 mg/ml) at a smooth platinum electrode for a bias time of 15 min.

to check the stability and the insulating property of the coating. Then, the modified electrodes were rinsed in water and acetone, then dried in an oven at 40 ◦ C and stored at +5 ◦ C until use. 2.4.3. Procedure C (adsorption followed by reticulation) (Roosevear et al., 1987b) Type P electrodes were reticulated with a 0.01% glutaraldehyde solution for 60 min and then washed with water. Then, electrodes were kept in a pH 7.0 phosphate buffer solution until use. 2.4.4. Procedure C (activation with glutaraldehyde) (Roosevear et al., 1987c) After coating of the polyethylenimine films on the electrode surfaces, the electrodes were activated with 0.02 ml of a 1% glutaraldehyde solution and were allowed to dry. Then, 0.02 ml of an urease solution with 2 mg/ml in a pH 5.6 phosphate buffer was spread on the same surface and left until dry. The electrode was washed with water and kept in a pH 7.0 phosphate buffer until use.

3. Results and discussion The development of a biosensor for biological analysis not only requires that the sensing polymer has adequate functional moieties for stable enzyme immobilization, but also that this polymeric material shows good biocompatibility. Polyethylenimine has this latter characteristic and is often used for biological or medical applications (Lee and Chu, 1997; Ruardij et al., 2000; Gaumann et al., 2000; Putnam et al., 2001; Tiyaboonchai et al., 2001). Moreover, polyethylenimine is a good polymer to modify electrode surfaces since the active –NH2 groups on the surface can covalently bind urease (Caras et al., 1985). Although the amine

groups are active in forming the covalent bond with protein, the electrode surface is still pH sensitive and consequently potential sensitive using the lone electron pairs of nitrogen atoms. Moreover, we can notice that most urea biosensors are enzymatically modified potentiometric sensors. Indeed, as the products of the urea hydrolysis are alkaline, an increase of urea concentration in the analyzed solution causes an increase of pH inside the enzyme layer of the biosensor. Then this variation of pH causes a variation of the electrode potential which can be detected. Therefore, many urea biosensors are potentiometric ones. 3.1. Response characteristics of the urea biosensors Besides equilibria the sigmoidal shape of calibration curves depends on the kinetic parameters of the enzyme reaction (Walcerz et al., 1995). The upper determination limit observed for all calibrations (see Fig. 2) results from the change of the order of the biocatalytic reaction. For high urea concentrations the enzyme process is a zero order reaction. This means that the increase of urea concentration does not raise the concentration of reaction products (causing changes of the pH at the electrode and changes of the electrode potential). However, for low concentrations of analyte it is possible to find experimental conditions for a sigmoidal response of the biosensor (Koncki et al., 1999). Indeed we can observe that the calibration curves of the urea biosensors showed a sigmoidal response (response potential versus logarithm of urea concentration) for the urea concentration working range from 1 × 10−2.5 to 1 × 10−1.5 M with satisfactory regression coefficient r since r > 0.977 whatever is the procedure of immobilization used (see Table 1). Moreover we can notice that all types of urea biosensors exhibit short response times. Indeed, for all measurements, the equilibria were reached after roughly 15–30 s.

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Fig. 2. Calibration curves of urea biosensors of types P, P , C and C .

3.2. Sensitivity of the urea biosensors The sensitivity of the biosensor strongly depends on the magnitude of the analytical signal (potential changes of the biosensor). Moreover, this latter mainly depends on the procedure for urease immobilization on electrochemically modified electrodes. Thus, so as to determine which biosensor is the most sensitive, we have fitted the curves giving the potentiometric responses versus pUrea = −log([Urea]). We found that the potentiometric response is sigmoidal and governed by the following Boltzmann equation: y=

A1 − A 2 + A2 1 + e(x−x0 )/dx

Table 1 Regression coefficients of the sigmoidal curves obtained for the urea biosensors P, P , C and C on different days Days

Biosensor P

Biosensor P

Biosensor C

Biosensor C

1 8 15 22 29

0.98599 0.99088 0.97532 0.94406 0.91469

0.97762 0.99479 0.99113 0.96944 0.93574

0.98053 0.97882 0.99420 0.98146 0.97898

0.98193 0.96656 0.95429 0.96539 0.93354

where x0 : center of the sigmoid, dx: width, A1 : initial E value = E(−∞) and A2 : final E value = E(+∞). Consequently, the potential sensitivity of the biosensor, noted S, can be calculated as: S = E(−∞) − E(+∞) = A1 − A2 . The values obtained for the different urea biosensors are given Table 2. We can observe that urea biosensors C is the most sensitive since the sensitivity obtained, during the first test of the sensor, was: S = 50.24 mV. Consequently, the activation of the polymer film with glutaraldehyde leads to very sensitive biosensors thanks to the formation of covalent bonds between glutaraldehyde and urease. Electrodes P shows a good sensitivity (S = 28.59 mV) thanks to the reaction between urea solutions and urease fixed on the polymer by physical adsorption. Electrodes P and C show less sensitive potentiometric responses. Table 2 Potential sensitivity S of the urea biosensors of type P, P , C and C Days

Biosensor P

Biosensor P

1 8 15 22 29

S = 28.59 S = 16.45 S = 12.27 – –

S S S S –

= 15.48 = 11.57 = 10.59 = 8.53

Biosensor C S S S S S

= 13.20 = 12.80 = 11.39 = 12.66 = 13.00

Biosensor C S S S S –

= 50.24 = 26.95 = 13.68 = 8.36

S was calculated as: E(+∞) − E(−∞) = A2 − A1 (sensitivities corresponding to regression coefficients <0.95, in Table 1, are not given).

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3.3. Stability of the urea biosensors The analytical signal of urea biosensors C is constant in time when the signals of sensors C, P and P decrease

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slowly in time. Indeed, the sensitivity of biosensors C is the same after 4 weeks: S = 13.20 during the first test and S = 13.00 after 4 weeks. More, S remains in the range between 11.39 and 13.20 during 4 weeks (see Table 2). On

Fig. 3. Calibration curves of urea biosensors of type C on different days.

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the contrary, the analytical signals of modified electrodes P and P decrease after 4 weeks to approximately 50% of their original values. Concerning, electrodes C the same trend is observed since the signal of these electrodes decrease after 4 weeks to approximately 17% of their original values. This trend is confirmed by the regression coefficients of the sigmoidal curves obtained on different days. Indeed, these coefficients remain superior to 0.978 for more than 30 days for biosensors of type C but only 15–22 days for biosensors C , P and P . So contrary to electrode C, electrodes C , P and P seem to have a poor long-term stability. 3.4. Comparison between physical and chemical urease immobilization methods When using enzymes as biological elements in biosensors, two important considerations have to be taken into account: operational stability which can be linked here to the potentiometric signal to changes in urea concentration, and long-term use which can be studied by repeating potentiometric measurements during some weeks. Our application of polyethylenimine modified electrodes enabled the use of chemical immobilization methods. Our results confirm the advantages of chemical immobilization over the physical ones. Indeed, it is generally accepted that physical methods of enzyme immobilization, such as adsorption (electrodes P) and entrapment (electrodes P ), have the benefit of providing small perturbation of the enzyme native structure and function. In general, chief disadvantages of these techniques are lack of mechanical strength and diffusional limitations encountered by substrates and products. Consequently, entrapment is a method which generates a continuous loss of enzyme. Moreover, direct physical adsorption of enzymes on a surface leads generally to poor long-term stability of the sensor because of enzyme leakage from the surface even if the surface has already been coated by a polymer film. So our study confirms that these methods lead to poor long-term stability of the sensors since it was shown that electrodes P and P are efficient only during 2 or 3 weeks, respectively. So, the low efficiency of electrodes P and P can be explained by the continuous loss of urease and by the leakage from the surface, respectively. The chemical methods of enzyme immobilization, such as reticulation (electrodes C) and activation (electrodes C ), have the benefit of providing low diffusional resistance, giving strong binding force between enzyme and matrix and so reducing loss of enzyme. That is why this kind of electrodes is more stable in time than physical ones. In particular, we have seen that the potentiometric response of modified electrodes C is stable during more than 4 weeks. This can also be seen in Fig. 3 which represents the potentiometric responses of urea biosensors C on different days. We can see that the shape of these calibration curves remains the same all over the 30 days and that the potential range is roughly constant.

4. Conclusion We have tested a new electrochemical potentiometric and enzymatic biosensor consisting in a glass-sealed platinum electrode coated by a thin polyethylenimine film. We have tested four different protocols for enzyme immobilization, two chemical techniques and two physical ones and we found that the chemical enzyme immobilization methods lead to a weak loss of enzyme. Consequently, the sensors using these techniques are more efficient and especially stabler in time than sensors using other immobilization methods. More, it appeared that the immobilization method leading to the most efficient urea biosensors was the physical adsorption of urease on polyethylenimine films followed by reticulation with a dilute aqueous glutaraldehyde solution. Indeed, these latter biosensors have a sigmoidal shape and are stable in time during at least 4 weeks. We want now to develop interdigitated microarray electrodes, using the latter urease immobilization method, to use them as miniaturized urea biosensors. We want to use photolithography, sputtering and lift-off process to develop these microelectrodes since we have already developed such sensors, pH or NH3 sensors for example, using microsystem technologies (Lakard et al., 2002, in press). References Adeloju, S.B., Shaw, S.J., Wallace, G.G., 1993. Polypyrrole-based potentiometric biosensor for urea part 1. Incorporation of urease. Anal. Chim. Acta 281, 611–620. Adeloju, S.B., Shaw, S.J., Wallace, G.G., 1996. Polypyrrole-based amperometric flow injection biosensor for urea. Anal. Chim. Acta 323, 107–113. Alegret, S., Mart´ınez-Fàbregas, E.M., 1989. Biosensors based on conducting filled polymer all-solid-state pvc matrix membrane electrodes. Biosens. Bioelectron. 4, 287–297. Bartlett, P.N., Whitaker, R.G., 1987. Electrochemical immobilisation of enzymes: Part II. Glucose oxidase immobilised in poly-N-methylpyrrole. J. Electroanal. Chem. 224, 37–48. Bertocchi, P., Compagnone, D., Palleschi, G., 1996. Amperometric ammonium ion and urea determination with enzyme based probes. Biosens. Bioelectron. 11, 1–10. Caras, S.D., Janata, J., Saupe, D., Schmitt, K., 1985. pH-based enzyme potentiometric sensors, Part 1. Theor. Anal. Chem. 57, 1917–1920. Chen, K., Liu, D., Nie, L., Yao, S., 1994. Determination of urea in urine using a conductivity cell with surface acoustic wave resonator-based measurement circuit. Talanta 41, 2195–2200. Foulds, N.C., Lowe, C.R., 1986. Enzyme entrapment in electrically conducting polymers. Immobilization of glucose oxidase in polypyrrole and its application in amperometric glucose sensors. J. Chem. Soc., Faraday Trans. 82, 1259–1263. Gaumann, A., Laudes, M., Jacob, B., Pommersheim, R., Laue, C., Vogt, W., Schrezenmeir, J., 2000. Effect of media composition on long-term in vitro stability of barium alginate and polyacrylic acid multilayer microcapsules. Biomaterials 21, 1911–1917. Gil, M.H., Piedade, A.P., Alegret, S., Alonso, J., Martinez-Fábregas, E., Orellana, A., 1992. Covalent binding of urease on ammonium-selective potentiometric membranes. Biosens. Bioelectron. 7, 645–652. Glab, S., Koncki, R., Kopczewska, E., Walcerz, I., Hulanicki, A., 1994. Urea sensors based on PVC membrane pH electrode. Talanta 41, 1201–1205.

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