Au composite electrode

Au composite electrode

Biosensors and Bioelectronics 20 (2004) 15–23 Urea biosensor based on PANi(urease)-Nafion®/Au composite electrode Yung-Chien Luo, Jing-Shan Do∗ Depar...

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Biosensors and Bioelectronics 20 (2004) 15–23

Urea biosensor based on PANi(urease)-Nafion®/Au composite electrode Yung-Chien Luo, Jing-Shan Do∗ Department of Chemical Engineering, Tunghai University, Taichung 40704, Taiwan, ROC Received 4 June 2003; accepted 21 November 2003 Available online 05 March 2004

Abstract The polyaniline (PANi)-Nafion® composite film was prepared onto the ceramic plate by the cyclic voltammetry (CV) method with the various cycle numbers. When the PANi-Nafion® /Au/ceramic plate with the preparing cycle number of 5 was as working electrode, the cathodic peak current was achieved as 84.0 ␮A in 60 mg dl−1 NH4 Cl buffer solution. On the other hand, the small cathodic peak currents for buffer solution in the presence of 60 mg dl−1 LiOH, NaCl and KCl, respectively, were found with the same composite electrode as working electrode. The cathodic peak current decreased from 84.0 to 16.3 ␮A in the 60 mg dl−1 NH4 Cl buffer solution when the cycle number for preparing PANi-Nafion® /Au/ceramic plate composite electrode with the CV method increased from 5 to 15. The enzyme of urease was immobilized onto the PANi-Nafion® /Au/ceramic plate composite film by the electrochemical immobilization and the casting methods and used as sensing electrode to detect the concentration of urea in the buffer solution. The sensitivity of composite electrode immobilized with the casting method was greater than that of electrochemical immobilization method. The sensitivity and the detecting limit of the urea sensor were found to be 0.7 and 5.27 ␮A (mg dl−1 )−1 cm−2 , as well as 6 and 0.3 mg dl−1 , respectively, when urease was immobilized by glutaraldehyde (GA) cross-linker and Nafion® network, respectively. © 2004 Elsevier B.V. All rights reserved. Keywords: Urea sensor; Urease; Polyaniline-Nafion® composite electrode; Cyclic voltammetry; Ammonium ion

1. Introduction In general, a biocatalytic biosensor needed a transducer to convert the concentration of sensing target to the digital signal. The combination of enzymatic and electrochemical techniques was widely used for detecting enzymatic products in enzyme-linked assay. The advantages of enzymatic biosensors in the electrochemical field were found as easy operation in vivo and high sensitivity (Deng et al., 2002). For example, pH-sensing electrode was frequently used as a transducer for applications in the urea biosensors (Stred’anský et al., 2000; Vostiar et al., 2002), and other electrochemical transducers such as ion-selective membrane and ISFET were also reported (Kazanskaya et al., 1996). It was very important to monitor the urea level in the blood or urine of human for diagnosing the healthy of kidney. The urea level in the blood of a normal human was located in the range between 10.2 and 49.8 mg dl−1 (Eggenstein et al., 1999). The concentration of urea in serum for people ∗ Corresponding author. Tel.: +86-6-2359-0262; fax: +86-6-2350-0255. E-mail address: [email protected] (J.-S. Do).

0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2003.11.028

with the sharp and chronic kidney diseases were increasing up to 720–900 mg dl−1 and 300–420 mg dl−1 , respectively (Wałerz et al., 1998). Recently, many articles had reported for the detection of urea with enzymatic methods (Komaba et al., 1997; Mizutani et al., 1997; Lee et al., 2000; Stred’anský et al., 2000; Hamlaoui et al., 2002) or immunoassay (Santandreu et al., 1998, Deng et al., 2002). Hamlaoui et al. (2002) had directly immobilized urease by the covalent bond onto the surface of NH4 + -sensitive field effect transistor and the sensitivity was obtained to be 15 mV purea−1 . Electronic conducting polymers were recently to be a interesting field of the new materials for application in biosensors (Gerard et al., 2002). The electronic conducting polymers, such as polypyrrole (PPy) (Adeloju et al., 1996) and polyaniline (PANi) (Strehlitz et al., 2000), have been successfully used in the biosensors for determination of urea. Strehlitz et al. reported a PANi-modified Pt–C electrode for detecting ammonia at a potential of +0.3 V (versus Ag/AgCl) and a linear range up to 6 mg dl−1 . However, the sensitivity and selectivity of the biosensors based on the conducting polymers were promoted by the modification of conducting polymers. Polyaniline modified with

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Nafion® to be PANi/Nafion® /glass carbon electrode (GCE) composite electrode used for sensing the concentration of urea in a flow system exhibited the characteristics of high sensitivity and low detection limit (Cho and Huang, 1998). However, the factors affecting the preparation of composite electrodes and the sensing properties in a batch system were still unclear. The study of the optimal conditions for preparing the PANi on the electrode modified with Nafion® and immobilizing the enzyme on the composite electrode to increase the selectivity and sensitivity of urea sensor was very important for the practical applications. The PANi was electropolymerized by the cyclic voltammetry (CV) method on Nafion® /Au/ceramic plate electrode to form PANi-Nafion® /Au/ceramic plate composite electrode. Using the preparing PANi-Nafion® /Au/ceramic plate as working electrode, the electrochemical characteristics and the selectivity of ammonium ion in the buffer solution were investigated. The composite electrode immobilized with urease was used as the sensing electrode to detect the concentration of urea in a batch system and the factors affected the sensing properties were also studied in this paper.

2. Experimental 2.1. Reagents Urease (EC 3.5.1.5, type III from Jack beans, 26,100 and 16,000 unit g−1 ) was purchased from Sigma. The suitable amount of urease dissolved in phosphate buffer solution prepared by 0.025 M KH2 PO4 (Showa, EP (99%)) and 0.025 M Na2 HPO4 (TEDIA, 99%) was used to immobilize onto the matrix of conducting polymer i.e. polyaniline. Urea (99%, Showa), NH4 Cl (99%, Showa), and glutaraldehyde (GA) (25 wt.% aqueous solution, Lancaster) used in this work was not further purified before usage. 1 wt.% Nafion® solution diluted from 5 wt.% Nafion® solution (Aldrich) with methanol was to be the solid polymer electrolyte (SPE) on the ceramic substrate. 2.2. Preparation of PANi-Nafion® /Au composite electrode Nafion® /Au/ceramic plate was prepared by the casting method. Dropping 8 ␮l 1 wt.% Nafion® solution onto the Au/ceramic plate which was prepared by the sputtering of Au on the ceramic plate with a shadow mask for a desired pattern and drying at 50 ◦ C for 1.0 h. Using the preparing Nafion® /Au/ceramic plate as working electrode, PANi-Nafion® /Au/ceramic plate electrode was prepared by the electropolymerization of PANi with cyclic voltammetry (CHI 614A) in the 0.1 M aniline and 1.0 M HCl aqueous solution. The sweeping potential and rate of CV for preparing PANi were set at the values of −0.3 to 1.0 V (versus Ag/AgCl/3 M NaCl aqueous solution) and 0.02 V s−1 , respectively, for the desired cycle number. Finally, the

PANi-Nafion® /Au/ceramic plate composite electrode was immersed in de-ion water and −0.3 V was applied for 30 min to remove the aniline monomer and the chloride ion existed in the composite film. The composite electrode was then stored in the phosphate buffer solution for usage. 2.3. Immobilization of enzyme Urease was immobilized onto the composite film of the electrode with the electrochemical and casting methods, respectively. The electrochemical method used to immobilize urease on the composite film of PANi-Nafion® /Au/ceramic plate in a solution containing 5.52 ml 50.2 U/ml of urease and 0.48 ml 2 wt.% of glutaraldehyde as cross linker was carried out in the potential of 0.3 V (versus Ag/AgCl/3 M NaCl aqueous solution) for 30 min. The net charge of the urease in the neutral or alkaline solution was found to be negative due to the urease pI value of 4.9 (Komaba et al., 1997). Therefore the urease with negative charge was attracted onto the surface of the composite film and immobilized by the cross linker when a positive potential was applied. Then the PANi-Nafion® /Au composite electrode with immobilization of urease was dried in a vacuum oven at 0 ◦ C. The casting method used to immobilize the urease onto the surface of composite film by dropping 20 ␮l solution with the composition described in the electrochemical method and drying in the room temperature for 30 min. Then the GA(urease)/PANi-Nafion® /Au/ceramic plate electrode was dried at 0 ◦ C in the vacuum condition, and stored at 4 ◦ C pH 6.88 phosphate buffer solution. The Nafion® (urease)/PANi-Nafion® /Au/ceramic plate electrode was prepared by the casting method described in the above with the casting solution prepared by 5.52 ml 50.2 U/ml urease and 0.48 ml 5.0 wt.% Nafion® solution. 2.4. The sensing procedures The detection of NH4 + in buffer solution was implemented on the PANi-Nafion® /Au/ceramic plate electrode using CV method. The reference and counter electrodes used for detecting NH4 + were Ag/AgCl/3 M NaCl aqueous solution and Pt wire, respectively. Changing the working electrode to Nafion® (urease)/PANi-Nafion® /Au/ceramic plate electrode, the monitoring the urea in phosphate buffer solution was carried out by the same procedures.

3. Results and discussion 3.1. Electrochemical properties of PANi-Nafion® /Au/ceramic plate electrode 3.1.1. Doping and undoping of ammonium ion It is well known that the cation-exchange ability of Nafion® film is due to the function of sulfonate groups on the polymer chain. Therefore it is expected to have

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the selectivity for the cation in the presence of anions when PANi-Nafion® /Au/ceramic plate is used as working electrode for sensing cations in the solution. Using the PANi-Nafion® /Au/ceramic plate as working electrode, the cyclic voltammograms in phosphate buffer solution for the absence and presence of 60 mg dl−1 NH4 Cl were shown in Fig. 1. Compared with the cyclic voltammogram for the absence of NH4 + a cathodic reduction peak contributed from NH4 + was found at potential of −0.17 V (versus Ag/AgCl) for solution presence of 60 mg dl−1 NH4 Cl. The cathodic peak was caused by the reduction of PANi in the composite film from the oxidizing state to the reducing state and accompanied with the insertion of NH4 + (doping NH4 + ) from the aqueous phase to the polymer chain to compensate the negative charge of sulfonate ion as described in the following equation: +



+

+



PANi RSO3 + NH4 + e → PANiNH4 RSO3



(1)

PANi+

and PANi were represented the oxidizing and where reducing forms, respectively. An oxidative peak appeared at −0.1 V in Fig. 1 was the oxidation of PANi and caused the undoping of NH4 + that was the reverse reaction of the Eq. (1). The peaks current increased with the cycle number shown in Fig. 2 when the PANi-Nafion® /Au/ceramic plate electrode was immersed into the 30 mg dl−1 NH4 Cl buffer solution. Increasing the cycle number from 1 to 5 the cathodic peak current sharply increased from 38.0 to 80.2 ␮A. Further increase of the cycle number to 10 the cathodic peak current increased slightly to 84.0 ␮A. The experimental results were due to the increase of the concentration of ammonium ion inside of the PANi-Nafion® composite film and the doping rate of ammonium ion with the run time (cycle number) when

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the cycle number increased from 1 to 5. Further increase of the cycle number greater than 5 the steady concentration of ammonium ion inside of PANi-Nafion® composite film and doping rate (cathodic peak current) were obtained. The cyclic voltammograms illustrated in Fig. 3 were obtained by the replacing the 30 mg dl−1 NH4 Cl buffer solution with the buffer solution in the absence of ammonium ion. The back extraction of NH4 + from the inside of the PANi-Nafion® composite film to the bulk solution caused the decrease in the redox peak currents with the increase in cycle number. The cathodic peak current was significantly decreased from 77.1 to 22.5 ␮A with the increase of cycle number from 1 to 50. The steady cathodic peak current of 20.8 ␮A was found for the cycle number of 60. 3.1.2. Selectivity of ammonium ion The relative small redox peaks currents were found from the cyclic voltammograms for the electrolyte in the presence of LiOH, NaCl or KCl as shown in Fig. 4. On the other hand, the obvious redox peaks currents were illustrated in Fig. 4 for the buffer solution containing NH4 Cl and NH4 HCO3 . The experimental results might be due to the hydrogen bond between the ammonium ion and the nitrogen atom on the PANi chains of the composite film. It was deduced that the hydrogen bond would promote the extraction of ammonium ion from the bulk solution into the PANi-Nafion® composite film. Consequently the greater redox peaks currents for the electrolyte in the presence of ammonium ion were obtained. Compared with the redox peaks current of NH4 Cl the smaller redox peaks currents for the solution in the presence of NH4 HCO3 might be caused by larger molecular size of NH4 HCO3 . The experimental results revealed that a high selectivity of NH4 + by using PANi-Nafion® /Au/ceramic plate

150

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E / V vs Ag/AgCl Fig. 1. Cyclic voltammograms on PANi-Nafion® /Au/ceramic plate composite electrode. Counter electrode: Pt wire; reference electrode: Ag/AgCl (3 M NaCl aqueous solution); sweep range: −0.4 to 0.4 V; sweep rate: 0.02 V s−1 ; temperature: 25 ◦ C; buffer solution: 0.025 M KH2 PO4 , 0.025 M Na2 HPO4 aqueous solution. (—): phosphate buffer solution; (- - -): 60 mg dl−1 NH4 Cl in buffer solution.

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Fig. 2. Cyclic voltammograms for changing electrolyte from buffer solution to 5 mM NH4 Cl buffer solution. [NH4 Cl] = 30 mg dl−1 ; working electrode: PANi-Nafion® /Au/ceramic plate composite electrode; counter electrode: Pt wire; reference electrode: Ag/AgCl (3 M NaCl aqueous solution); sweep range: −0.4 to 0.4 V; sweep rate: 0.02 V s−1 ; temperature: 25 ◦ C; buffer solution: 0.025 M KH2 PO4 , 0.025 M Na2 HPO4 aqueous solution.

crease of the sensitivity of the detection of NH4 + from 87.0 to 0.7 ␮A (mg dl−1 )−1 cm−2 and the increase of the detection limit from 0.15 to 15 mg dl−1 , respectively (Table 1). The PANi polymer chain was covered under the Nafion® film when the cycle number for preparing PANi was less than 5. Some of the PANi film was found on the surface of the Nafion® film for the cycle number for preparing PANi greater than 15. When the cycle number for preparing PANi was greater than 15, the diffusion resistance for transferring NH4 + from the bulk phase of the aqueous solution into the

as sensing electrode would be expected in the presence of the other cations. 3.1.3. Effect of the cycle number for preparing PANi The sensing currents (steady cathodic peak currents) in the presence of 60 mg dl−1 NH4 Cl decreased from 84.0 to 16.3 ␮A with the increase of the cycle number for preparing PANi-Nafion® /Au/ceramic plate composite electrode from 5 to 15 as shown in Fig. 5. Increasing the cycle number for preparing PANi-Nafion® from 5 to 15 resulted in the de100 80 60

Current / A

40 20

60th cycle

0 -20 -40

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E / V vs. Ag/AgCl Fig. 3. Cyclic voltammograms for changing electrolyte from 5 mM NH4 Cl buffer solution to buffer solution. Working electrode: PANi-Nafion® /Au/ceramic plate composite electrode; counter electrode: Pt wire; reference electrode: Ag/AgCl (3 M NaCl aqueous solution); sweep range: −0.4 to 0.4 V; sweep rate: 0.02 V s−1 ; temperature: 25 ◦ C; buffer solution: 0.025 M KH2 PO4 , 0.025 M Na2 HPO4 aqueous solution.

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Current / µA

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E / V vs Ag/AgCl Fig. 4. Cyclic voltammograms for various electrolytes. [LiOH] = [KOH] = [NaCl] = [NH4 HCO3 ] = [NH4 Cl] = 60 mg dl−1 ; buffer solution: 0.025 M KH2 PO4 , 0.025 M Na2 HPO4 ; working electrode: PANi-Nafion® /Au/ceramic plate composite electrode; counter electrode: Pt wire; reference electrode: Ag/AgCl (3 M NaCl aqueous solution); sweep range: −0.4 to 0.4 V; sweep rate: 0.02 V s−1 ; temperature: 25 ◦ C.

composite film increased and caused the decrease of the redox peaks currents.

to the enzymatic active site located in the PANi-Nafion® composite film, and the enzymatic reaction was taken place as the following equation (Liu et al., 1996):

3.2. Sensing properties of GA(urease)/PANi-Nafion® /Au/ceramic plate electrode

CO(NH2 )2 + 3H2 O −−→ 2NH4 + + HCO3 − + OH−

Using the composite electrode immobilized with urease as working electrode, urea in the bulk solution was transferred

The product of ammonium ion could be doped into the PANi-Nafion® composite film membrane when the com-

urease

(2)

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Ic

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0 4

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Cycle numbers for preparing PANi Fig. 5. Effect of the cycle number for preparing PANi on the cathodic peak current. Conditions for preparing PANi-Nafion® composite film: [aniline] = 0.1 M, [HCl] = 1.0 M, working electrode: 0.2 cm2 Au/ceramic plate; counter electrode: Au plate; reference electrode: Ag/AgCl (3 M NaCl aqueous solution); sweep range: −0.3 to 1.0 V; sweep rate: 0.02 V s−1 ; temperature: 25 ◦ C. Conditions of cyclic voltammetry in 60 mg dl−1 NH4 Cl buffer solution: working electrode: PANi-Nafion® /Au/ceramic plate composite electrode; counter electrode: Pt wire; reference electrode: Ag/AgCl (3 M NaCl aqueous solution); sweep range: −0.4 to 0.4 V; sweep rate = 0.02 V s−1 ; temperature: 25 ◦ C.

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Table 1 Effect of cycle number for preparing PANi on the sensing properties of NH4 Cl Cycle numbers for preparing PANi

Sensitivitya ␮A (mg dl−1 )−1 cm−2

Linear range of [NH4 + ] (mg dl−1 )

Detection limit (mg dl−1 )

5 10 15

87.0 ± 3.3 4.2 ± 0.4 0.7 ± 0.05

0.15–3 3–30 15–60

0.15 1.5 15

Conditions for preparing PANi-Nafion® : working electrode: 0.2 cm2 Au/ceramic plate; [aniline] = 0.1 M; [HCl] = 1.0 M; counter electrode: Au plate; reference electrode: Ag/AgCl (3 M NaCl aqueous solution); sweep range: −0.3 to 1.0 V; sweep rate: 0.02 V s−1 ; temperature: 25 ◦ C. Conditions of cyclic voltammetry in NH4 Cl buffer solution: working electrode: 0.2 cm2 PANi-Nafion® /Au/ceramic plate; counter electrode: Pt wire; reference electrode: Ag/AgCl (3 M NaCl aqueous solution); sweep range: −0.4 to 0.4 V; sweep rate: 0.02 V s−1 ; temperature: 25 ◦ C. a RSD for cycle number of 5, 10 and 15 are evaluated to be 3.79, 9.52 and 7.14%, respectively.

NH4+

PANi+RSO3- + NH4+

HCO3- + OH-

bulk solution

urease

PANi-Nafion Au

PANi0RSO3-NH4+

urea

Fig. 6. Scheme of the sensing urea on urease/PANi-Nafon® /Au/ceramic plate composite electrode.

posite film was electrochemically reduced from its oxidized state. The scheme of the sensing mechanism of ammonium ion on the PANi(urease)-Nafion® /Au composite electrode was illustrated in Fig. 6. Glutaraldehyde can react with the other molecules such as the amine group of the proteins and plays a role of cross-linker due to high activity of the two OH groups. The amine functional group in protein reacts with OH groups of GA to form a matrix structure and is immobilized

onto the matrix. The urease was cross-linked in the matrix of GA on the surface of PANi-Nafion® /Au/ceramic plate composite electrode to be GA(urease)/PANi-Nafion® /Au/ ceramic plate sensing electrode. Furthermore the urease was also immobilized onto the surface of PANi-Nafion® /Au/ ceramic plate by using the Nafion® matrix to form Nafion® (urease)/PANi-Nafion® /Au/ceramic plate sensing electrode. The characteristics for sensing urea in the buffer solution on both the sensing electrodes were discussed in the following.

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Fig. 7. Effect of concentration of urea on the cathodic peak current on GA(urease)/PANi-Nafion® (urease)/Au/ceramic plate composite electrode. Counter electrode: Pt; reference electrode: Ag/AgCl; sweep rate: 0.02 V s−1 , voltage range: −0.4–0.4 V; temperature: 25 ◦ C. (䊉): electrochemical method; (): casting method.

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Fig. 8. Effect of concentration of urea on the cathodic peak current on Nafion® (urease)/PANi-Nafion® /Au/ceramic plate composite electrode. Counter electrode: Pt; reference electrode: Ag/AgCl; sweep rate: 0.02 V s−1 ; voltage range: −0.4–0.4 V; temperature: 25 ◦ C.

3.2.1. Sensing characteristics on GA(urease)/PANi-Nafion® /Au/ceramic plate As described in the experimental section, urease was immobilized onto PANi-Nafion® /Au/ceramic plate electrode by the electrochemical and casting methods. As shown in Fig. 7, the similar relationships of the cathodic peak current and the concentration of urea on the GA(urease)/PANiNafion® /Au/ceramic plate prepared with electrochemical and casting methods, respectively. However, the sensing current on composite electrode immobilized with electrochemical method was greater than that of casting method. The experimental results might due to the higher immobilize activity of urease by using the electrochemical method. The linear ranges for sensing urea on the composite electrode immobilized with the electrochemical and casting methods were located in the range of 6–60 mg dl−1 and

3–45 mg dl−1 , respectively (Fig. 7). When the urease was immobilized on the surface of composite electrode by using the electrochemical and casting methods, the sensitivities of urea sensors calculated from the slops of the linear relationships shown in Fig. 7 were found to be 0.7 and 0.22 ␮A (mg dl−1 )−1 cm−2 , respectively. The detecting limit found from the cyclic voltammograms of the buffer solution in the presence and absence of urea were 6 and 3 mg dl−1 for the immobilization of urease by the electrochemical and casting methods. 3.2.2. Sensing characteristics on Nafion® (urease)/ PANi-Nafion® /Au/ceramic plate Using Nafion® (urease)/PANi-Nafion® /Au/ceramic plate as sensing electrode, the cathodic peak current increased from 14.7 to 60.9 ␮A with the increase of the concentra-

Table 2 Comparison with the characteristics of urea sensors based on various electrochemical methods Type of electrochemical method

Modifier

Sensitivity

Linear range (mg dl−1 )

Conductometry

PANi-PBMAa



Potentiometry

Polyurethane-acrylate photocurable polymeric membrane PVAc-PE(urease)

58 mV p[urea]−1

Limiting detection (mg dl−1 )

Ref.

20–120



Castillo-Ortega et al. (2002)

0.24–216



Puig-Lleix`a et al. (1999)

44 mV p[urea]−1 52 mV p[urea]−1

1.2–300 0.432–126

0.36 0.12

Eppelsheim et al. (1995) Eggenstein et al. (1999)

Amperometry

PPy-urease (urease)-n-eicosane-graphite PANi/Nafion® /urease

– 0.033 ␮A (mg dl−1 )−1 cm−2 1.81 ␮A (mg dl−1 )−1 cm−2

0.01–0.45 0.15–2.1 0.006–6

0.006 – 0.003

Adeloju et al. (1997) Pizzariello et al. (2001) Cho and Huang (1998)

Cyclic voltammetry

Nafion® (urease)/PANi-Nafion® GA(urease)/PANi-Nafion® (urease)/PANi-Nafion®

5.27 ␮A (mg dl−1 )−1 cm−2 0.22 ␮A (mg dl−1 )−1 cm−2 0.7 ␮A (mg dl−1 )−1 cm−2

3–30 3–45 6–60

0.3 3 6

This work This work This work

a

Polyaniline-poly(n-butyl methacrylate).

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tion of urea from 0 to 30 mg dl−1 (Fig. 8). The cathodic peak current changed slightly with further increase of the concentration of urea greater than 30 mg dl−1 . The sensitivity of the urea sensor found from the linear relationship of the cathodic peak current and the concentration of urea located in the 3–30 mg dl−1 . The sensitivity of urea sensor based on Nafion® (urease)/PANi-Nafion® /Au/ceramic plate electrode was significantly greater than that of GA(urease)/PANi-Nafion® /Au/ceramic plate electrode. The experimental results was due to the decrease of the activity of urease in the detecting period caused by part of urease dissolved from GA(urease)/PANi-Nafion® /Au/ceramic plate electrode. The detecting limit by using Nafion® (urease)/ PANi-Nafion® /Au/ceramic plate electrode found from the cyclic voltammograms to be 0.3 mg dl−1 was less than that of using GA(urease)/PANi-Nafion® /Au/ceramic plate as sensing electrode. The electrochemical methods for detecting the urea level reported in the literatures and this work were summarized and divided into the methods of conductometry, potentiometry, amperometry and cyclic voltammetry, respectively, as illustrated in Table 2. The various modifiers were used to modify the electrodes of the various electrochemical urea sensors. The lower detecting limit was found to be 0.003–0.006 mg dl−1 for the amperometric urea sensors. On the other hand, the sensitivity of urea sensors using CV method developed in this work was obtained to be in the range of 0.22∼5.27 ␮A (mg dl−1 )−1 cm−2 , which was greater than that of the amperometric sensors.

4. Conclusions The PANi-Nafion® /Au/ceramic plate electrode prepared with the CV method exhibited a high sensitivity and selectivity of ammoniium ion in a buffer solution. Using PANi-Nafion® /Au ceramic plate prepared with cycle number of 5 as sensing electrode, the cathodic peak current of 60 mg dl−1 NH4 Cl, LiOH, NaCl and KCl in the buffer solution were obtained to be 84.0, 10.0, 24.4 and 8.8 ␮A, respectively. The electrochemical and casting methods accompanied with GA cross-linker and the Nafion® polymer net work were used to immobilize the urease on the surface of PANi-Nafion® /Au ceramic plate. The sensitivity of urea in the buffer solution was found to be 0.7 ␮A (mg dl−1 )−1 cm−2 when the GA(urease)/PANi-Nafion® / Au/ceramic plate immobilized with the electrochemical method was used as sensing electrode. Furthermore the linear range for detecting urea and the detecting limit were obtained from the cyclic voltammograms to be 6–60 mg dl−1 and 6 mg dl−1 , respectively. When Nafion® (urease)/PANiNafion® /Au/ceramic plate was used the sensing electrode, the sensitivity of urea was significantly increased to 5.27 ␮A (mg dl−1 )−1 cm−2 due to the higher activity of urease on the composite electrode. The detecting limit of 0.3 mg dl−1

and the linear sensing range of 3∼30 mg dl−1 were also obtained from the experimental results.

Acknowledgements The financial support of Ministry of Education of Republic of China (project number: EX-91-E-FA09-5-4) and Tunghai University is acknowledged.

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