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Amperometric urea biosensor based on covalently immobilized urease on an electrochemically polymerized film of polyaniline containing MWCNTs
Synthetic Metals 194 (2014) 1–6
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Synthetic Metals journal homepage: www.elsevier.com/locate/synmet
Amperometric urea biosensor based on covalently immobilized urease on an electrochemically polymerized film of polyaniline containing MWCNTs Azam S. Emami Meibodi ∗ , Sara Haghjoo Department of Chemistry, Karaj Branch, Islamic Azad University, Karaj, Iran
a r t i c l e
i n f o
Article history: Received 28 January 2014 Received in revised form 3 April 2014 Accepted 11 April 2014 Keywords: Electrochemical biosensor MWCNTs Polyaniline Enzyme Amperometry Urea
a b s t r a c t In this work, polyaniline-multiwalled carbon nanotubes (PANI/MWCNTs) composite were fabricated by electropolymerization method, as a matrix for entrapment of enzyme. Urease has been immobilized by using physical adsorption and electrochemical entrapment technique. A conventional three-electrode system was used. Pencil graphite disk electrode (PGDE), Ag/AgCl (3 M KCl) and platinum wire were used as working electrode, reference electrode and counter electrode respectively. The influence of several experimental parameters such as applied potential, type and concentrate of dopant and enzyme loading was explored to optimize the electroanalytical performance of the biosensor. The responses of the enzyme electrodes were measured via monitoring amperometric current at +0.3 V (vs. Ag/AgCl). Kinetic parameters operational and storage stabilities were determined. The value of the apparent Michaelis–Menten constant, Imax and sensitivity (Imax /Km ) experimentally determined to be 2.02 mM, 2.5 × 10−5 A cm−2 and 12 × 10−5 A mM−1 cm−2 , respectively. The optimized urea biosensor shows a good sensitivity from 10−2 M to 10−5 M urea concentration range and a response time of about 50 s. The detection limit was 0.04 mM. The proposed biosensor retained 50% of its original response after 15 days. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Urea is one of the final metabolite of proteins and it has great significance in clinical chemistry where blood urea nitrogen is an important indicator of possible kidney malfunction [1]. Moreover, urea is widely distributed in nature and its analysis is necessary in food chemistry and environmental monitoring [2]. Urea can stress the environment in a different way because it acts as a nitrogenous fertilizer. It decomposes to ammonia, which is very toxic, and so it can pollute the streams and rivers into which it drains. Several methods have been reported to detect urea, such as colorimetric, flow injection analysis, enzyme electrode, and electrochemical methods [3–7]. Many biosensors have been developed for the determination of urea in the biological samples namely spectrometry [8], potentiometry with application of pH-sensitive electrode or an ion-selective electrode or an ion-sensitive field effective transistor [9], conductometry [10], coulometry [11], amperometry [12] and inductometry [13]. Urease in urea biosensor, catalyses
∗ Corresponding author. Tel.: +98 26 34332315. E-mail address: [email protected] (A.S. Emami Meibodi). http://dx.doi.org/10.1016/j.synthmet.2014.04.009 0379-6779/© 2014 Elsevier B.V. All rights reserved.
the hydrolysis of urea producing ammonium and bicarbonate ions, causing a pH increase in the aqueous reaction medium. Specific transducers can detect ammonium ions [14], ammonia gas [15] and carbon dioxide [21], or pH changes [17,18]. Immobilization of urease onto a suitable matrix is crucial in developing an electrochemical urea biosensor [19]. Many electrode materials, such as polymers, sol–gels, conducting polymers, Langmuir–Blodgett films, nanomaterials, and self-assembled monolayers have been used to obtain enzyme biosensors. Recently, composite materials based on conducting polymers, redox mediators, metal nanoparticles, nanocomposites, and nanoclusters have been used to combine the properties of the individual components with a synergistic performance in biosensor fabrication [20]. However, biosensors containing electrochemical transducers are the most used and in these cases urea is detected via either potentiometric [21,22] or amperometric methods [18,23]. This last one has attracted a lot of attention due to both its effectiveness and simplicity of preparation. Amperometric biosensors appear promising in determining urea concentration due to their effectiveness and simplicity. In this study, a new biosensor based on single walled carbon nanotube and conducting polymer composite film was developed
2
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and used for the detection of urea, which exhibited acceptable sensitivity, selectivity and stability. 2. Experimental
working graphite electrode in the above dispersion was subjected between 0.1 V and −1.0 V, with a sweep rate 40 mV s−1 for 30 cycles. The composite-coated electrode surface which shined with a light green color was rinsed with pure water several times to remove the aniline residues on the electrode surface.
2.1. Materials All chemicals were of analytical reagent grade, and used without further purification except aniline. Aniline (95%), urea, perchloric acid, potassium dihydrogen phosphate and glutaraldehyde were obtained from Merck. Multiwall carbon nanotubes [95%, L: 20–30 nm, 1–2 nm, 0.5–2 m]. Urease type III (EC 3.5.1.5, from jack bean, 5000 U g−1 ) was obtained from Aldrich. Phosphate buffer was prepared from potassium dihydrogen phosphate (0.1 M, pH 7.2) and urea stock solution (1000 ppm) was prepared by dissolving an appropriate amount in double distilled water. The working solutions were prepared by diluting the stock solution to appropriate volumes. 2.2. Apparatus PGDE (2 mm diameter), platinum wire and Ag/AgCl (3 M KCl) was used as working electrode, counter electrode and reference electrode respectively. Laboratory measurements were performed in a glass cell with a three-electrode configuration by using an Autolab Electrochemical Analyzer PGSTAT 30 (Ecochemie, Utrecht, and The Netherlands) controlled by a PC computer via GPES software interface. FTIR spectra were obtained using a Perkin Elmer spectrophotometer by making pellet with dehydrated KBr in reflectance mode. 2.3. Preparation of the electrodes A pencil graphite cylinder (4 mm di), modeled with epoxy resin to expose the basal plane, with a circular geometric area of approximately 0.5 cm2 were polished with emery paper and 0.05 m alumina slurry to a mirror-like surface, cleaned under ultrasonic bath for 10 min, and then thoroughly rinsed with distilled water. 2.4. Purification and functionalization of MWCNTs In order to be used in biology and medicinal chemistry, carbon nanotubes need to be purified and soluble in physiological media. Various methods have been developed to purify CNTs, such as oxidation, microfiltration and chromatography and microwave irradiation [24]. In this work, to eliminate metal oxide catalysts, 100 mg of MWCNTs were dispersed in 60 mL of 3 mol L−1 of nitric acid and were refluxed for 24 h at 80 ◦ C. Then, the purified MWCNTs were rinsed to neutral with double-distilled water. To functionalized carbon particles, after drying in a vacuum drying oven at 60 ◦ C, the purified MWCNTs were dispersed by sonication in a mixture of concentrated sulfuric and nitric acids (3:1, v/v) for 6 h. Then, the pretreated MWCNTs were rinsed to neutral with double-distilled water and dried at 60 ◦ C in a vacuum drying oven. 2.5. Preparation of PANI/MWCNTs/PGDE Aniline was distilled twice under atmospheric pressure and stored in dark at low temperature prior to use. A suspension of 2 mg of the acid-treated MWCNTs (functionalizing with carboxylic acid groups) was prepared by sono-dispersing for 2 h, into 30 mL of solution containing 0.2 M aniline and 0.8 M perchloric acid as dopant. The electrolyte was purged with N2 for 15 min before use. The three-electrode cell included the Pt-wire as counter electrode, an Ag/AgCl (3 M KCl) electrode as reference electrode and a treated PGDE as working electrode was used. Cyclic voltammetry on the
2.6. Immobilization of enzyme Later the modified electrode PANI/MWCNTs/PGDE was thoroughly washed with deionized water. For covalent coupling of urease to the PANI/MWCNTs composite electrode, a solution of glutaraldehyde (0.5%, w/w) was used. In this way, 10 L of glutaraldehyde solution 0.5% were dropped on the PANI/MWCNT modified electrode surface and allowed to dry at room temperature. Then electrode was rinsed twice with deionized water to remove the unbound glutaraldehyde molecules. The electrode was immersed into 3 mL of a urease solution which was prepared by dissolving 15 mg of urease in 3 mL of phosphate buffer (pH 7.2) and kept it for 90 min. Then the PANI/MWCNTs/Urease electrode was washed with water and kept in a phosphate buffer (pH 7.2) and stored at 4 ◦ C until to use. 2.7. Amperometric measurements Amperometric measurements were performed using a threeelectrode cell consisting of PANI/MWCNTs/Urease/PGDE as working electrode, platinum wire as counter electrode and Ag/AgCl (3 M KCl) as reference electrode. Electroanalysis was performed in a 5 mL (phosphate buffer, pH 7.2) by using chronoamperometry under a constant potential (+0.3 V vs. Ag/AgCl) at room temperature. Amperometric response was measured as a function of urea concentration.
3. Result and discussion 3.1. Electrochemical polymerization of PANI/MWCNTs composites Electrically conducting polymers (CPs) have considerable flexibility in chemical structures and MWCNTs have a p-conjugative structure with a highly hydrophobic surface responsible for pp electronic and hydrophobic interaction with the many organic compounds. Chemical or electrochemical polymerization has been mainly used to develop polymer/MWCNTs composites. Covalent sidewall functionalization of MWCNT provides a feasible route to incorporate carbon nanotubes in polymer. CPs/MWCNTs composites can lead to new nanocomposites with enhanced electrical and mechanical properties and have shown mechanical stability, sensitivity for different techniques and for electrocatalysis [25]. An oxidant affects the properties of the CPs in chemical approach, but electrochemical polymerization can control the film morphology, thickness and other characteristics of the polymer coating without any interference. Polymer can be prepared easily in a rapid one-step procedure in electrochemical polymerization. Cyclic voltammetry electro-polymerization of PANI/MWCNTs composite film (Fig. 1) onto the PGDE, have been used in this work to fabrication of a biosensor. There are three redox peaks in discrete electroactive regions, a redox pair with peak potentials at 0.04 and 0.28 V and the accompanying proton elimination–addition process (Fig. 1). Redox couples with peak potentials at 0.68 and 0.78 V were related to the redox processes of PANI in proton-deficient solutions. The redox pair with peak potentials at approximately 0.5 V was suggested to be due to the higher oxidation level of aniline. The redox peak currents increased with increasing scan rate while the peak potentials showed slight increase in positive potential. These
A.S. Emami Meibodi, S. Haghjoo / Synthetic Metals 194 (2014) 1–6
Fig. 1. Cyclic voltammograms recorded during electrochemical deposition of PANI/MWCNTs composites on PGDE, by 5, 10, 20, 30, 40 cycle between potential −0.1 V and 1.0 V (vs. Ag/AgCl) at a scan rate of 30 mV s−1 , in solutions containing 0.2 M aniline and 0.8 M perchloric acid.
observations show that the polymer is electroactive and the peak currents are diffusion controlled [26]. 3.2. Optimization of electropolymerization condition The effect of scan rate () on the peak currents of the PANI/MWCNTs nanocomposite in the range from 10 to 50 mV s−1 is shown in Fig. 2. The redox peak currents increased with increasing scan rate while the peak potentials showed slight increase in positive potential, the anodic peak potential shifts toward a more positive value and the cathodic peak potential shifts toward a more negative value. A is the surface area of the electrode (0.5 cm2 ), is the scan rate (V s−1 ), R is the gas constant (8.314 J mol K−1 ), and T is the absolute temperature of the system (298 K). Ip =
∗ A n2 F 2 comp
(1)
4RT
The Randles–Sevcik (Eq. (2)) was used to calculate the diffusion coefficient of the electrons within the nanocomposite film, where ip is the peak current (A), n is the number of electrons appearing in half-reaction for redox couple (n = 2), A is the area of the electrode (0.5 cm2 ), D is the diffusion coefficient (cm2 s−1 ), C is the concentration of analyte (2 × 10−4 mol cm−3 ) and v is scan rate (V s−1 ). A plot of peak current vs. the square root of the scan rate was obtained (Fig. 3) and the slope of the linear regression was used to estimate the diffusion coefficient of the electrons within the polymer (De ) as 3.5 × 10−7 cm2 s−1 . 1/2
ip = 2.69 × 105 n3/2 ADe
C v1/2
(2)
Fig. 2. Current peak in redox couples of cyclic voltammetry, anodic peak at ∼0.68 (. . .. . .) and cathodic peak at ∼0.78 V (–䊉–) in different scan rate 10, 20, 30, 40 and 50 mV s−1 , in 0.2 M aniline and 0.8 M perchloric acid.
3
Fig. 3. Current peak in redox couples of cyclic voltammetry, anodic peak at ∼0.68 () and cathodic peak at ∼0.78 V (䊉) in vs. square root of different scan rates (0.01, 0.02, 0.03, 0.04 and 0.05 V s−1 ), in 0.2 M aniline and 0.8 M perchloric acid.
3.3. Spectroscopic characterization The FT-IR spectrum of PANI/MWCNTs/Urease is shown in Fig. 4. There are two peaks at 1585 and 1136 cm−1 indicated the stretching vibrations of C C and C O C respectively, which verified the presence of the carboxylic acid groups on the functionalized MWCNTs. The peak at 1495 cm−1 due to the stretching vibration of benzenoid ring band (C C). The peak at 809 cm−1 is attributed to out of plane bending vibration of C H on 1,4-di-substituted rings. In addition, the characteristic peak at 1297 cm−1 is ascribed to the stretching vibration of C N. The stretching vibrations of N H appeared at 3434 cm−1 . 3.4. Immobilization of urease on PANI/MWCNTs The biosensors can be classified by nature of mediator and the immobilization method. Supplementary step in the biosensor fabrication for increasing selectivity and sensitivity is selection of bio-recognition element in the presence of an intermediary compound (mediator). The mediator should integrate together with the bio-recognition element and the electrode, to avoid of sample contamination by artificial mediator. Enzymes are biocatalysts that often used in biosensors, and often electrochemically deposited conducting polymers films used for the bio-molecule immobilization on the electrode. Conducting polymers such as polyaniline can apply as mediators in biosensors, shuttles redox equivalents between the recognition element and transducer. The most widely used biosensors are the enzyme-based amperometric electrodes. In this work, urease was selected as enzyme for recognition of urea and polyaniline was selected as mediator. 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. In amperometric biosensors the recorded current is proportional to the substrate concentration. Application of large voltage and small oxygen concentration in real samples, can be limiting factors in these measurements. There are a few methods to retain the bio-molecule on the electrode surface such as adsorption, covalent binding, mixing as carbon paste, or entrapment into a polymer matrix, in order to assure a proper electron transfer pathway. In this work, a two-step chemical enzyme immobilization was used, by means of cross-linking with glutaraldehyde, followed by the covalent binding of the enzyme to the composite surface. Because of the large size of urease as compared with the pore size of polymers, enzyme is exclusively bound to the composite. 3.4.1. Cross-linking of urease with glutaraldehyde Urease (200 L, 150 mg mL−1 ) was mixed with glutaraldehyde (10 L of glutaraldehyde 0.5% in 390 L of buffer) was then added
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Fig. 4. FTIR; PANI/MWCNTs/Urease.
Table 1 A summary of analytical response and linear regression characteristics of calibration curves for urea. Response time (s)
Stability (50%)
Km (mM)
Sensitivity (A/mM)
DL (mM)
Linear rang (mM)
Matrix
50
15 day
2.02
12
0.04
0.07–10
PANI/MWCNTs/Urease
(200 L) and different aliquots of the mixture placed on the electrode surface in order to immobilize 5 U of urease. A gel was formed after 1.5 min, and the urease activity then monitored accordingly. The gel obtained after urease cross-linkage showed a significant thickness and therefore immobilization of glutaraldehyde by the same procedure was not considered in order to minimize electrode fouling. The decrease in urease activity after immobilization was evaluated in terms of the Km value (Section 3.6) calculated for each method following the Hanes–Woolf approximation [30]. The urease activity obtained after immobilization by amperometric response of PANI/MWCNTs/Urease working electrode (Table 1).
3.5. Electrochemical measurements
3.6. Enzyme kinetics In bioanalysis chrono-amperometric measurements are often employed for studying the kinetics of enzymatic reaction. By increasing in substrate concentration, the calibration curve tends to level off, thus it shows the characteristics of the Michaelis–Menten kinetic mechanism. In this work, with the increase in substrate concentration, there was an increase in amperometric current signal of biosensor. This current reached a steady state after addition of 66 mM substrate. Kinetic parameters Km and Imax for the enzyme biosensor were found from the Lineweaver–Burk (Eq. (3)) plot at room temperature and phosphate buffer (pH 7.2) while varying the substrate concentration (Fig. 7). 1 1 Km 1 + = Imax [S] Imax I
(3)
By immersion of enzymic modified electrode in substrate solution, analyte migrates toward the interior of the layer and converses by reaction with the immobilized enzyme [32]. Chronoamperometric measurements consist of applying a fixed potential at the working electrode and monitoring the current resulting, which is directly proportional to the analyte concentration (Fig. 5). Amperometry responses of biosensor to a solution of 1 mM urea in constant potential (+0.3 V vs. Ag/AgCl) in phosphate buffer 0.1 M at different pH values were shown in Fig. 6. Optimum response of biosensor was obtained at pH 7.2. Urease can denature and hence reduces enzyme activity in lower or higher of pH 7.2.
where Iss is the steady-state current by addition of substrate, Imax is the maximum current measured under saturated substrate conapp dition and C is the bulk concentration of the substrate. The Km is determined by analyzing the slope and intercept for the plot of the reciprocals of the cathodic current vs. urea concentration. app The value of the apparent Michaelis–Menten constant (Km ) of the urea biosensor is determined by the steady-state amperometric app response. The small value of Km indicates that the immobilized urease has higher enzymatic activity and high affinity of Urease to nanocomposite PANI/MWCNTs matrix over the electrode surface.
Fig. 5. Current response of chronoamperometry vs. urea concentration (mM).
Fig. 6. A typical amperometric response of the PANI/MWCNTs/Urease under 1 mM concentration of urea at +0.3 V (vs. Ag/AgCl) in stirred phosphate buffer at different pH.
A.S. Emami Meibodi, S. Haghjoo / Synthetic Metals 194 (2014) 1–6
Fig. 7. Lineweaver–Burk plots of the immobilized urease enzyme in nanocomposite. 1/current response vs. 1/concentration for the enzyme electrode (pH 7.2, 4 ◦ C).
5
Fig. 10. Storage stabilities of urease immobilized on PANI/MWCNTs electrode. The amperometric responses of this enzyme electrode were regularly checked during 35 days (pH 7.2, 4 ◦ C).
3.8. Calibration curve
Fig. 8. A typical amperometric response of the PANI/MWCNTs/Urease under continuous stirring after successive additions of 0.1 M concentrations of urea at +0.3 V (vs. Ag/AgCl) in stirred phosphate buffer (pH 7.2).
Fig. 8 shows the typical chronoamperometry of different concentrations of urea at the PANI/MWCTs/urease modified electrode under the optimum experimental conditions described above. All measurements were performed in triplicate. A linear relationship between the anodic peak current and the urea concentration was obtained covering a concentration range from 0.2 mM to 100 mM (linear range). The linear regression equation was obtained i (A) = 9.528 + 7.495 [urea] (mM), with a correlation coefficient (r) of 0.985 (n = 3). A detection limit of 0.2 mM (S/N = 3) was obtained. The biosensor exhibited a rapid and sensitive response to the change urea concentration, which indicated the nice electrocatalytic behavior of PANI/MWCNTs/Urease/PGDE. The response time is less than 50 s. 3.9. Operational and storage stabilities
Km , Imax and sensitivity (Imax /Km ) were calculated as 2.02 mM, 2.5 × 10−5 A cm−2 and 1.23 × 10−5 A mM−1 cm−2 , respectively. 3.7. Urea measurements A potential of 0.3 V (vs. Ag/AgCl) was applied in PANI/MWCNTs/Urease/PGDE. The chronoamperometry procedure accompanied continued stirring with a stirrer. Different concentrations (from 0.01 to 300 mM) of urea solution were added to the electrochemical cell (Fig. 8). Correlation between response currents (background current was deducted) and different concentrations of urea solution was obtained. The resulting current was plotted as a function of urea concentrate (Fig. 9) and the slope of the linear portion of the plot used as analytical data.
The stability of the modified electrode has been investigated by measuring the current response to 0.6 mM urea in PHOSPHAT buffer (pH 7.2) every few days. The chronoamperometry response currents of the modified electrode decreased gradually to 50% of the initial values after being stored 15 days in the refrigerator at 4 ◦ C (Fig. 10). The reproducibility of the modified electrode for the determination of urea was also investigated. The relative standard deviation (RSD) was 2.6% for 5 successive determinations of 0.6 mM urea solution. These results suggested that the modified electrode possessed a good stability and repeatability for the detection of urea. 4. Conclusion From the above results we have concluded that the composite system developed with conductive polyaniline and MWCNTs provide a hybrid system and suitable platform for the fabrication of amperometric urea biosensor. The detection limit and sensitivity of these electrodes was found to be 10 mM and 12 A/mM, respectively. The shelf-life of this electrode was approximately 15 days when kept in phosphate buffer, pH 7.2 at 4 ◦ C. References
Fig. 9. Calibration curve of biosensor for urea determined in phosphate buffer (pH 7.2) with working potential of +0.3 V vs. Ag/AgCl.
[1] M. Gutierrez, S. Alegret, M. Valle, Potentiometric bioelectronic tongue for the analysis of urea and alkaline ions in clinical samples, Biosens. Bioelectron. 22 (2007) 2171–2178. [2] B. Lakard, G. Herlem, S. Lakard, A. Antoniou, B. Fahys, Urea potentiometric biosensor based on modified electrodes with urease immobilized
6
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10] [11] [12] [13]
A.S. Emami Meibodi, S. Haghjoo / Synthetic Metals 194 (2014) 1–6 on polyethylenimine films, Biosens. Bioelectron. 19 (2004) 1641– 1647. P.S. Francis, S.W. Lewis, K.F. Lim, Analytical methodology for the determination of urea current practice and future trends, Trends Anal. Chem. 21 (2002) 389–400. Z.Y. Fan, H.C. Liu, C. Zhu, Detection of urea in milk by spectrophotometry to p-dimethylaminobenzaldehyde as chromogenic reagent, Chin. J. Anal. Lab. 28 (Suppl.) (2009) 313–315. M.T. Knorst, R. Neubert, W. Wohlrab, Analytical methods for measuring urea in pharmaceutical formulations, J. Pharm. Biomed. Anal. 15 (11) (1997) 1627–1632. F. Manea, A. Pop, C. Radovan, P. Malchev, A. Bebeselea, G. Burtica, S. Picken, J. Schoonman, Voltammetric detection of urea on an Ag-modified zeoliteexpanded graphite–epoxy composite electrode, Sensors 8 (2008) 5806– 5819. M.J. Syu, Y.S. Chang, Ionic effect investigation of a potentiometric sensor for urea and surface morphology observation of entrapped urease/polypyrrole matrix, Biosen. Bioelectron. 24 (2009) 2671–2677. D. Liu, K. Ge, K. Cheng, L. Nie, S. Yao, Clinical analysis of urea in human blood by coupling a surface acoustic wave sensor with urease extracted from pumpkin seeds, Anal. Chim. Acta 307 (1995) 61. D. Liu, M.E. Meyerhoff, H.D. Goldberg, R.B. Brown, Potentiometric ion and bioselective electrodes based on asymmetric polyurethane membranes, Anal. Chim. Acta 274 (1993) 37. H. Sangodkar, S. Sukeerthi, R.S. Srinivasa, R. Lal, A.Q.A. Contractor, Biosensor array based on polyaniline, Anal. Chem. 68 (1996) 779. R.E. Adams, P.W. Carr, Coulometric flow analyzer for use with immobilized enzyme reactors, Anal. Chem. 50 (1978) 944. S.B. Adeloju, S.J. Shaw, G.G. Wallace, Polypyrrole-based amperometric flow injection biosensor for urea, Anal. Chim. Acta 323 (1996) 107. W.O. Ho, S. Krause, C.J. McNeil, J.A. Pritchard, R.D. Armstrong, D. Athey, et al., Electrochemical sensor for measurement of urea and creatinine in serum based on ac impedance measurement of enzyme-catalyzed polymer transformation, Anal. Chem. 71 (1999) 1940.
[14] S. Anne, J.R. Nicolo, M. Claude, C. Serge, Aminiaturized urea sensor based on the integration of both ammonium based urea enzyme field effect transistor and a reference field effect transistor in a single chip, Talanta 50 (1999) 219–226. [15] G.G. Guilbault, M. Trap, A specific enzyme electrode for urea, Anal. Chim. Acta 73 (1974) 355–365. [17] R. Sahney, S. Anand, B.K. Puri, A.K. Srivastava, A comparative study of immobilization techniques for urease on glass-pH-electrode and its application in urea detection in blood serum, Anal. Chim. Acta 578 (2006) 156–161. [18] F. Kuralay, H. Özyörük, A. Yildiz, Amperometric enzyme electrode for urea determination using immobilized urease in poly(vinylferrocenium) film, Sens. Actuators B: Chem. 114 (2006) 500–506. [19] A. Maaref, H. Barhoumi, M. Rammah, C. Martelet, N. Jaffrezic-Renault, C. Mousty, S. Cosnier, Sens. Actuators B: Chem. 123 (2007) 671. [20] G. Dhawan, G. Sumana, B.D. Malhotra, Biochem. Eng. J. 44 (2009) 42. [21] B. Lakard, G. Herlem, S. Lakard, A. Antoniou, B. Fahys, Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films, Biosens. Bioelectron. 19 (2004) 1641–1647. [22] F. Kuralay, H. Özyörük, A. Yildiz, Potentiometric enzyme electrode for urea determination using immobilized urease in poly(vinylferrocenium) film, Sens. Actuators B: Chem. 109 (2005) 194–199. [23] S.B. Adeloju, S.J. Shaw, G.G. Wallace, Pulsed-amperometric detection of urea in blood samples on a conducting polypyrrole–urease biosensor, Anal. Chim. Acta 341 (1997) 155–160. [24] P.M. Ajayan, Nanotubes from carbon, Chem. Rev. 99 (1999) 1787–1799. [25] V. Rajesh, W. Bisht, K. Takashima, Kaneto, An amperometric urea biosensor based on covalent immobilization of urease onto an electrochemically prepared copolymer poly (N-3-aminopropyl pyrrole-co-pyrrole) film, Biomaterials 26 (2005) 3683–3690. [26] N.G.R. Mathebe, A. Morrin, E.I. Iwuoha, Electrochemistry and scanning electron microscopy of polyaniline/peroxidase-based biosensor, Talanta 64 (2004) 115–120. [30] B.A. Lawton, Z.-H. Lu, R. Pethig, Y. Wei, J. Mol. Liquids 42 (1989) 83–96. [32] T.M. Canh, Biosensors, Chapman & Hall, London, 1993, pp. 46.
Contents lists available at ScienceDirect
Synthetic Metals journal homepage: www.elsevier.com/locate/synmet
Amperometric urea biosensor based on covalently immobilized urease on an electrochemically polymerized film of polyaniline containing MWCNTs Azam S. Emami Meibodi ∗ , Sara Haghjoo Department of Chemistry, Karaj Branch, Islamic Azad University, Karaj, Iran
a r t i c l e
i n f o
Article history: Received 28 January 2014 Received in revised form 3 April 2014 Accepted 11 April 2014 Keywords: Electrochemical biosensor MWCNTs Polyaniline Enzyme Amperometry Urea
a b s t r a c t In this work, polyaniline-multiwalled carbon nanotubes (PANI/MWCNTs) composite were fabricated by electropolymerization method, as a matrix for entrapment of enzyme. Urease has been immobilized by using physical adsorption and electrochemical entrapment technique. A conventional three-electrode system was used. Pencil graphite disk electrode (PGDE), Ag/AgCl (3 M KCl) and platinum wire were used as working electrode, reference electrode and counter electrode respectively. The influence of several experimental parameters such as applied potential, type and concentrate of dopant and enzyme loading was explored to optimize the electroanalytical performance of the biosensor. The responses of the enzyme electrodes were measured via monitoring amperometric current at +0.3 V (vs. Ag/AgCl). Kinetic parameters operational and storage stabilities were determined. The value of the apparent Michaelis–Menten constant, Imax and sensitivity (Imax /Km ) experimentally determined to be 2.02 mM, 2.5 × 10−5 A cm−2 and 12 × 10−5 A mM−1 cm−2 , respectively. The optimized urea biosensor shows a good sensitivity from 10−2 M to 10−5 M urea concentration range and a response time of about 50 s. The detection limit was 0.04 mM. The proposed biosensor retained 50% of its original response after 15 days. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Urea is one of the final metabolite of proteins and it has great significance in clinical chemistry where blood urea nitrogen is an important indicator of possible kidney malfunction [1]. Moreover, urea is widely distributed in nature and its analysis is necessary in food chemistry and environmental monitoring [2]. Urea can stress the environment in a different way because it acts as a nitrogenous fertilizer. It decomposes to ammonia, which is very toxic, and so it can pollute the streams and rivers into which it drains. Several methods have been reported to detect urea, such as colorimetric, flow injection analysis, enzyme electrode, and electrochemical methods [3–7]. Many biosensors have been developed for the determination of urea in the biological samples namely spectrometry [8], potentiometry with application of pH-sensitive electrode or an ion-selective electrode or an ion-sensitive field effective transistor [9], conductometry [10], coulometry [11], amperometry [12] and inductometry [13]. Urease in urea biosensor, catalyses
∗ Corresponding author. Tel.: +98 26 34332315. E-mail address: [email protected] (A.S. Emami Meibodi). http://dx.doi.org/10.1016/j.synthmet.2014.04.009 0379-6779/© 2014 Elsevier B.V. All rights reserved.
the hydrolysis of urea producing ammonium and bicarbonate ions, causing a pH increase in the aqueous reaction medium. Specific transducers can detect ammonium ions [14], ammonia gas [15] and carbon dioxide [21], or pH changes [17,18]. Immobilization of urease onto a suitable matrix is crucial in developing an electrochemical urea biosensor [19]. Many electrode materials, such as polymers, sol–gels, conducting polymers, Langmuir–Blodgett films, nanomaterials, and self-assembled monolayers have been used to obtain enzyme biosensors. Recently, composite materials based on conducting polymers, redox mediators, metal nanoparticles, nanocomposites, and nanoclusters have been used to combine the properties of the individual components with a synergistic performance in biosensor fabrication [20]. However, biosensors containing electrochemical transducers are the most used and in these cases urea is detected via either potentiometric [21,22] or amperometric methods [18,23]. This last one has attracted a lot of attention due to both its effectiveness and simplicity of preparation. Amperometric biosensors appear promising in determining urea concentration due to their effectiveness and simplicity. In this study, a new biosensor based on single walled carbon nanotube and conducting polymer composite film was developed
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and used for the detection of urea, which exhibited acceptable sensitivity, selectivity and stability. 2. Experimental
working graphite electrode in the above dispersion was subjected between 0.1 V and −1.0 V, with a sweep rate 40 mV s−1 for 30 cycles. The composite-coated electrode surface which shined with a light green color was rinsed with pure water several times to remove the aniline residues on the electrode surface.
2.1. Materials All chemicals were of analytical reagent grade, and used without further purification except aniline. Aniline (95%), urea, perchloric acid, potassium dihydrogen phosphate and glutaraldehyde were obtained from Merck. Multiwall carbon nanotubes [95%, L: 20–30 nm, 1–2 nm, 0.5–2 m]. Urease type III (EC 3.5.1.5, from jack bean, 5000 U g−1 ) was obtained from Aldrich. Phosphate buffer was prepared from potassium dihydrogen phosphate (0.1 M, pH 7.2) and urea stock solution (1000 ppm) was prepared by dissolving an appropriate amount in double distilled water. The working solutions were prepared by diluting the stock solution to appropriate volumes. 2.2. Apparatus PGDE (2 mm diameter), platinum wire and Ag/AgCl (3 M KCl) was used as working electrode, counter electrode and reference electrode respectively. Laboratory measurements were performed in a glass cell with a three-electrode configuration by using an Autolab Electrochemical Analyzer PGSTAT 30 (Ecochemie, Utrecht, and The Netherlands) controlled by a PC computer via GPES software interface. FTIR spectra were obtained using a Perkin Elmer spectrophotometer by making pellet with dehydrated KBr in reflectance mode. 2.3. Preparation of the electrodes A pencil graphite cylinder (4 mm di), modeled with epoxy resin to expose the basal plane, with a circular geometric area of approximately 0.5 cm2 were polished with emery paper and 0.05 m alumina slurry to a mirror-like surface, cleaned under ultrasonic bath for 10 min, and then thoroughly rinsed with distilled water. 2.4. Purification and functionalization of MWCNTs In order to be used in biology and medicinal chemistry, carbon nanotubes need to be purified and soluble in physiological media. Various methods have been developed to purify CNTs, such as oxidation, microfiltration and chromatography and microwave irradiation [24]. In this work, to eliminate metal oxide catalysts, 100 mg of MWCNTs were dispersed in 60 mL of 3 mol L−1 of nitric acid and were refluxed for 24 h at 80 ◦ C. Then, the purified MWCNTs were rinsed to neutral with double-distilled water. To functionalized carbon particles, after drying in a vacuum drying oven at 60 ◦ C, the purified MWCNTs were dispersed by sonication in a mixture of concentrated sulfuric and nitric acids (3:1, v/v) for 6 h. Then, the pretreated MWCNTs were rinsed to neutral with double-distilled water and dried at 60 ◦ C in a vacuum drying oven. 2.5. Preparation of PANI/MWCNTs/PGDE Aniline was distilled twice under atmospheric pressure and stored in dark at low temperature prior to use. A suspension of 2 mg of the acid-treated MWCNTs (functionalizing with carboxylic acid groups) was prepared by sono-dispersing for 2 h, into 30 mL of solution containing 0.2 M aniline and 0.8 M perchloric acid as dopant. The electrolyte was purged with N2 for 15 min before use. The three-electrode cell included the Pt-wire as counter electrode, an Ag/AgCl (3 M KCl) electrode as reference electrode and a treated PGDE as working electrode was used. Cyclic voltammetry on the
2.6. Immobilization of enzyme Later the modified electrode PANI/MWCNTs/PGDE was thoroughly washed with deionized water. For covalent coupling of urease to the PANI/MWCNTs composite electrode, a solution of glutaraldehyde (0.5%, w/w) was used. In this way, 10 L of glutaraldehyde solution 0.5% were dropped on the PANI/MWCNT modified electrode surface and allowed to dry at room temperature. Then electrode was rinsed twice with deionized water to remove the unbound glutaraldehyde molecules. The electrode was immersed into 3 mL of a urease solution which was prepared by dissolving 15 mg of urease in 3 mL of phosphate buffer (pH 7.2) and kept it for 90 min. Then the PANI/MWCNTs/Urease electrode was washed with water and kept in a phosphate buffer (pH 7.2) and stored at 4 ◦ C until to use. 2.7. Amperometric measurements Amperometric measurements were performed using a threeelectrode cell consisting of PANI/MWCNTs/Urease/PGDE as working electrode, platinum wire as counter electrode and Ag/AgCl (3 M KCl) as reference electrode. Electroanalysis was performed in a 5 mL (phosphate buffer, pH 7.2) by using chronoamperometry under a constant potential (+0.3 V vs. Ag/AgCl) at room temperature. Amperometric response was measured as a function of urea concentration.
3. Result and discussion 3.1. Electrochemical polymerization of PANI/MWCNTs composites Electrically conducting polymers (CPs) have considerable flexibility in chemical structures and MWCNTs have a p-conjugative structure with a highly hydrophobic surface responsible for pp electronic and hydrophobic interaction with the many organic compounds. Chemical or electrochemical polymerization has been mainly used to develop polymer/MWCNTs composites. Covalent sidewall functionalization of MWCNT provides a feasible route to incorporate carbon nanotubes in polymer. CPs/MWCNTs composites can lead to new nanocomposites with enhanced electrical and mechanical properties and have shown mechanical stability, sensitivity for different techniques and for electrocatalysis [25]. An oxidant affects the properties of the CPs in chemical approach, but electrochemical polymerization can control the film morphology, thickness and other characteristics of the polymer coating without any interference. Polymer can be prepared easily in a rapid one-step procedure in electrochemical polymerization. Cyclic voltammetry electro-polymerization of PANI/MWCNTs composite film (Fig. 1) onto the PGDE, have been used in this work to fabrication of a biosensor. There are three redox peaks in discrete electroactive regions, a redox pair with peak potentials at 0.04 and 0.28 V and the accompanying proton elimination–addition process (Fig. 1). Redox couples with peak potentials at 0.68 and 0.78 V were related to the redox processes of PANI in proton-deficient solutions. The redox pair with peak potentials at approximately 0.5 V was suggested to be due to the higher oxidation level of aniline. The redox peak currents increased with increasing scan rate while the peak potentials showed slight increase in positive potential. These
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Fig. 1. Cyclic voltammograms recorded during electrochemical deposition of PANI/MWCNTs composites on PGDE, by 5, 10, 20, 30, 40 cycle between potential −0.1 V and 1.0 V (vs. Ag/AgCl) at a scan rate of 30 mV s−1 , in solutions containing 0.2 M aniline and 0.8 M perchloric acid.
observations show that the polymer is electroactive and the peak currents are diffusion controlled [26]. 3.2. Optimization of electropolymerization condition The effect of scan rate () on the peak currents of the PANI/MWCNTs nanocomposite in the range from 10 to 50 mV s−1 is shown in Fig. 2. The redox peak currents increased with increasing scan rate while the peak potentials showed slight increase in positive potential, the anodic peak potential shifts toward a more positive value and the cathodic peak potential shifts toward a more negative value. A is the surface area of the electrode (0.5 cm2 ), is the scan rate (V s−1 ), R is the gas constant (8.314 J mol K−1 ), and T is the absolute temperature of the system (298 K). Ip =
∗ A n2 F 2 comp
(1)
4RT
The Randles–Sevcik (Eq. (2)) was used to calculate the diffusion coefficient of the electrons within the nanocomposite film, where ip is the peak current (A), n is the number of electrons appearing in half-reaction for redox couple (n = 2), A is the area of the electrode (0.5 cm2 ), D is the diffusion coefficient (cm2 s−1 ), C is the concentration of analyte (2 × 10−4 mol cm−3 ) and v is scan rate (V s−1 ). A plot of peak current vs. the square root of the scan rate was obtained (Fig. 3) and the slope of the linear regression was used to estimate the diffusion coefficient of the electrons within the polymer (De ) as 3.5 × 10−7 cm2 s−1 . 1/2
ip = 2.69 × 105 n3/2 ADe
C v1/2
(2)
Fig. 2. Current peak in redox couples of cyclic voltammetry, anodic peak at ∼0.68 (. . .. . .) and cathodic peak at ∼0.78 V (–䊉–) in different scan rate 10, 20, 30, 40 and 50 mV s−1 , in 0.2 M aniline and 0.8 M perchloric acid.
3
Fig. 3. Current peak in redox couples of cyclic voltammetry, anodic peak at ∼0.68 () and cathodic peak at ∼0.78 V (䊉) in vs. square root of different scan rates (0.01, 0.02, 0.03, 0.04 and 0.05 V s−1 ), in 0.2 M aniline and 0.8 M perchloric acid.
3.3. Spectroscopic characterization The FT-IR spectrum of PANI/MWCNTs/Urease is shown in Fig. 4. There are two peaks at 1585 and 1136 cm−1 indicated the stretching vibrations of C C and C O C respectively, which verified the presence of the carboxylic acid groups on the functionalized MWCNTs. The peak at 1495 cm−1 due to the stretching vibration of benzenoid ring band (C C). The peak at 809 cm−1 is attributed to out of plane bending vibration of C H on 1,4-di-substituted rings. In addition, the characteristic peak at 1297 cm−1 is ascribed to the stretching vibration of C N. The stretching vibrations of N H appeared at 3434 cm−1 . 3.4. Immobilization of urease on PANI/MWCNTs The biosensors can be classified by nature of mediator and the immobilization method. Supplementary step in the biosensor fabrication for increasing selectivity and sensitivity is selection of bio-recognition element in the presence of an intermediary compound (mediator). The mediator should integrate together with the bio-recognition element and the electrode, to avoid of sample contamination by artificial mediator. Enzymes are biocatalysts that often used in biosensors, and often electrochemically deposited conducting polymers films used for the bio-molecule immobilization on the electrode. Conducting polymers such as polyaniline can apply as mediators in biosensors, shuttles redox equivalents between the recognition element and transducer. The most widely used biosensors are the enzyme-based amperometric electrodes. In this work, urease was selected as enzyme for recognition of urea and polyaniline was selected as mediator. 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. In amperometric biosensors the recorded current is proportional to the substrate concentration. Application of large voltage and small oxygen concentration in real samples, can be limiting factors in these measurements. There are a few methods to retain the bio-molecule on the electrode surface such as adsorption, covalent binding, mixing as carbon paste, or entrapment into a polymer matrix, in order to assure a proper electron transfer pathway. In this work, a two-step chemical enzyme immobilization was used, by means of cross-linking with glutaraldehyde, followed by the covalent binding of the enzyme to the composite surface. Because of the large size of urease as compared with the pore size of polymers, enzyme is exclusively bound to the composite. 3.4.1. Cross-linking of urease with glutaraldehyde Urease (200 L, 150 mg mL−1 ) was mixed with glutaraldehyde (10 L of glutaraldehyde 0.5% in 390 L of buffer) was then added
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Fig. 4. FTIR; PANI/MWCNTs/Urease.
Table 1 A summary of analytical response and linear regression characteristics of calibration curves for urea. Response time (s)
Stability (50%)
Km (mM)
Sensitivity (A/mM)
DL (mM)
Linear rang (mM)
Matrix
50
15 day
2.02
12
0.04
0.07–10
PANI/MWCNTs/Urease
(200 L) and different aliquots of the mixture placed on the electrode surface in order to immobilize 5 U of urease. A gel was formed after 1.5 min, and the urease activity then monitored accordingly. The gel obtained after urease cross-linkage showed a significant thickness and therefore immobilization of glutaraldehyde by the same procedure was not considered in order to minimize electrode fouling. The decrease in urease activity after immobilization was evaluated in terms of the Km value (Section 3.6) calculated for each method following the Hanes–Woolf approximation [30]. The urease activity obtained after immobilization by amperometric response of PANI/MWCNTs/Urease working electrode (Table 1).
3.5. Electrochemical measurements
3.6. Enzyme kinetics In bioanalysis chrono-amperometric measurements are often employed for studying the kinetics of enzymatic reaction. By increasing in substrate concentration, the calibration curve tends to level off, thus it shows the characteristics of the Michaelis–Menten kinetic mechanism. In this work, with the increase in substrate concentration, there was an increase in amperometric current signal of biosensor. This current reached a steady state after addition of 66 mM substrate. Kinetic parameters Km and Imax for the enzyme biosensor were found from the Lineweaver–Burk (Eq. (3)) plot at room temperature and phosphate buffer (pH 7.2) while varying the substrate concentration (Fig. 7). 1 1 Km 1 + = Imax [S] Imax I
(3)
By immersion of enzymic modified electrode in substrate solution, analyte migrates toward the interior of the layer and converses by reaction with the immobilized enzyme [32]. Chronoamperometric measurements consist of applying a fixed potential at the working electrode and monitoring the current resulting, which is directly proportional to the analyte concentration (Fig. 5). Amperometry responses of biosensor to a solution of 1 mM urea in constant potential (+0.3 V vs. Ag/AgCl) in phosphate buffer 0.1 M at different pH values were shown in Fig. 6. Optimum response of biosensor was obtained at pH 7.2. Urease can denature and hence reduces enzyme activity in lower or higher of pH 7.2.
where Iss is the steady-state current by addition of substrate, Imax is the maximum current measured under saturated substrate conapp dition and C is the bulk concentration of the substrate. The Km is determined by analyzing the slope and intercept for the plot of the reciprocals of the cathodic current vs. urea concentration. app The value of the apparent Michaelis–Menten constant (Km ) of the urea biosensor is determined by the steady-state amperometric app response. The small value of Km indicates that the immobilized urease has higher enzymatic activity and high affinity of Urease to nanocomposite PANI/MWCNTs matrix over the electrode surface.
Fig. 5. Current response of chronoamperometry vs. urea concentration (mM).
Fig. 6. A typical amperometric response of the PANI/MWCNTs/Urease under 1 mM concentration of urea at +0.3 V (vs. Ag/AgCl) in stirred phosphate buffer at different pH.
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Fig. 7. Lineweaver–Burk plots of the immobilized urease enzyme in nanocomposite. 1/current response vs. 1/concentration for the enzyme electrode (pH 7.2, 4 ◦ C).
5
Fig. 10. Storage stabilities of urease immobilized on PANI/MWCNTs electrode. The amperometric responses of this enzyme electrode were regularly checked during 35 days (pH 7.2, 4 ◦ C).
3.8. Calibration curve
Fig. 8. A typical amperometric response of the PANI/MWCNTs/Urease under continuous stirring after successive additions of 0.1 M concentrations of urea at +0.3 V (vs. Ag/AgCl) in stirred phosphate buffer (pH 7.2).
Fig. 8 shows the typical chronoamperometry of different concentrations of urea at the PANI/MWCTs/urease modified electrode under the optimum experimental conditions described above. All measurements were performed in triplicate. A linear relationship between the anodic peak current and the urea concentration was obtained covering a concentration range from 0.2 mM to 100 mM (linear range). The linear regression equation was obtained i (A) = 9.528 + 7.495 [urea] (mM), with a correlation coefficient (r) of 0.985 (n = 3). A detection limit of 0.2 mM (S/N = 3) was obtained. The biosensor exhibited a rapid and sensitive response to the change urea concentration, which indicated the nice electrocatalytic behavior of PANI/MWCNTs/Urease/PGDE. The response time is less than 50 s. 3.9. Operational and storage stabilities
Km , Imax and sensitivity (Imax /Km ) were calculated as 2.02 mM, 2.5 × 10−5 A cm−2 and 1.23 × 10−5 A mM−1 cm−2 , respectively. 3.7. Urea measurements A potential of 0.3 V (vs. Ag/AgCl) was applied in PANI/MWCNTs/Urease/PGDE. The chronoamperometry procedure accompanied continued stirring with a stirrer. Different concentrations (from 0.01 to 300 mM) of urea solution were added to the electrochemical cell (Fig. 8). Correlation between response currents (background current was deducted) and different concentrations of urea solution was obtained. The resulting current was plotted as a function of urea concentrate (Fig. 9) and the slope of the linear portion of the plot used as analytical data.
The stability of the modified electrode has been investigated by measuring the current response to 0.6 mM urea in PHOSPHAT buffer (pH 7.2) every few days. The chronoamperometry response currents of the modified electrode decreased gradually to 50% of the initial values after being stored 15 days in the refrigerator at 4 ◦ C (Fig. 10). The reproducibility of the modified electrode for the determination of urea was also investigated. The relative standard deviation (RSD) was 2.6% for 5 successive determinations of 0.6 mM urea solution. These results suggested that the modified electrode possessed a good stability and repeatability for the detection of urea. 4. Conclusion From the above results we have concluded that the composite system developed with conductive polyaniline and MWCNTs provide a hybrid system and suitable platform for the fabrication of amperometric urea biosensor. The detection limit and sensitivity of these electrodes was found to be 10 mM and 12 A/mM, respectively. The shelf-life of this electrode was approximately 15 days when kept in phosphate buffer, pH 7.2 at 4 ◦ C. References
Fig. 9. Calibration curve of biosensor for urea determined in phosphate buffer (pH 7.2) with working potential of +0.3 V vs. Ag/AgCl.
[1] M. Gutierrez, S. Alegret, M. Valle, Potentiometric bioelectronic tongue for the analysis of urea and alkaline ions in clinical samples, Biosens. Bioelectron. 22 (2007) 2171–2178. [2] B. Lakard, G. Herlem, S. Lakard, A. Antoniou, B. Fahys, Urea potentiometric biosensor based on modified electrodes with urease immobilized
6
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10] [11] [12] [13]
A.S. Emami Meibodi, S. Haghjoo / Synthetic Metals 194 (2014) 1–6 on polyethylenimine films, Biosens. Bioelectron. 19 (2004) 1641– 1647. P.S. Francis, S.W. Lewis, K.F. Lim, Analytical methodology for the determination of urea current practice and future trends, Trends Anal. Chem. 21 (2002) 389–400. Z.Y. Fan, H.C. Liu, C. Zhu, Detection of urea in milk by spectrophotometry to p-dimethylaminobenzaldehyde as chromogenic reagent, Chin. J. Anal. Lab. 28 (Suppl.) (2009) 313–315. M.T. Knorst, R. Neubert, W. Wohlrab, Analytical methods for measuring urea in pharmaceutical formulations, J. Pharm. Biomed. Anal. 15 (11) (1997) 1627–1632. F. Manea, A. Pop, C. Radovan, P. Malchev, A. Bebeselea, G. Burtica, S. Picken, J. Schoonman, Voltammetric detection of urea on an Ag-modified zeoliteexpanded graphite–epoxy composite electrode, Sensors 8 (2008) 5806– 5819. M.J. Syu, Y.S. Chang, Ionic effect investigation of a potentiometric sensor for urea and surface morphology observation of entrapped urease/polypyrrole matrix, Biosen. Bioelectron. 24 (2009) 2671–2677. D. Liu, K. Ge, K. Cheng, L. Nie, S. Yao, Clinical analysis of urea in human blood by coupling a surface acoustic wave sensor with urease extracted from pumpkin seeds, Anal. Chim. Acta 307 (1995) 61. D. Liu, M.E. Meyerhoff, H.D. Goldberg, R.B. Brown, Potentiometric ion and bioselective electrodes based on asymmetric polyurethane membranes, Anal. Chim. Acta 274 (1993) 37. H. Sangodkar, S. Sukeerthi, R.S. Srinivasa, R. Lal, A.Q.A. Contractor, Biosensor array based on polyaniline, Anal. Chem. 68 (1996) 779. R.E. Adams, P.W. Carr, Coulometric flow analyzer for use with immobilized enzyme reactors, Anal. Chem. 50 (1978) 944. S.B. Adeloju, S.J. Shaw, G.G. Wallace, Polypyrrole-based amperometric flow injection biosensor for urea, Anal. Chim. Acta 323 (1996) 107. W.O. Ho, S. Krause, C.J. McNeil, J.A. Pritchard, R.D. Armstrong, D. Athey, et al., Electrochemical sensor for measurement of urea and creatinine in serum based on ac impedance measurement of enzyme-catalyzed polymer transformation, Anal. Chem. 71 (1999) 1940.
[14] S. Anne, J.R. Nicolo, M. Claude, C. Serge, Aminiaturized urea sensor based on the integration of both ammonium based urea enzyme field effect transistor and a reference field effect transistor in a single chip, Talanta 50 (1999) 219–226. [15] G.G. Guilbault, M. Trap, A specific enzyme electrode for urea, Anal. Chim. Acta 73 (1974) 355–365. [17] R. Sahney, S. Anand, B.K. Puri, A.K. Srivastava, A comparative study of immobilization techniques for urease on glass-pH-electrode and its application in urea detection in blood serum, Anal. Chim. Acta 578 (2006) 156–161. [18] F. Kuralay, H. Özyörük, A. Yildiz, Amperometric enzyme electrode for urea determination using immobilized urease in poly(vinylferrocenium) film, Sens. Actuators B: Chem. 114 (2006) 500–506. [19] A. Maaref, H. Barhoumi, M. Rammah, C. Martelet, N. Jaffrezic-Renault, C. Mousty, S. Cosnier, Sens. Actuators B: Chem. 123 (2007) 671. [20] G. Dhawan, G. Sumana, B.D. Malhotra, Biochem. Eng. J. 44 (2009) 42. [21] B. Lakard, G. Herlem, S. Lakard, A. Antoniou, B. Fahys, Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films, Biosens. Bioelectron. 19 (2004) 1641–1647. [22] F. Kuralay, H. Özyörük, A. Yildiz, Potentiometric enzyme electrode for urea determination using immobilized urease in poly(vinylferrocenium) film, Sens. Actuators B: Chem. 109 (2005) 194–199. [23] S.B. Adeloju, S.J. Shaw, G.G. Wallace, Pulsed-amperometric detection of urea in blood samples on a conducting polypyrrole–urease biosensor, Anal. Chim. Acta 341 (1997) 155–160. [24] P.M. Ajayan, Nanotubes from carbon, Chem. Rev. 99 (1999) 1787–1799. [25] V. Rajesh, W. Bisht, K. Takashima, Kaneto, An amperometric urea biosensor based on covalent immobilization of urease onto an electrochemically prepared copolymer poly (N-3-aminopropyl pyrrole-co-pyrrole) film, Biomaterials 26 (2005) 3683–3690. [26] N.G.R. Mathebe, A. Morrin, E.I. Iwuoha, Electrochemistry and scanning electron microscopy of polyaniline/peroxidase-based biosensor, Talanta 64 (2004) 115–120. [30] B.A. Lawton, Z.-H. Lu, R. Pethig, Y. Wei, J. Mol. Liquids 42 (1989) 83–96. [32] T.M. Canh, Biosensors, Chapman & Hall, London, 1993, pp. 46.