Analytica Chimica Acta 542 (2005) 157161

Enzyme coated glass pH-electrode: Its fabrication and applications in the determination of urea in blood samples
Rachana Sahney a, , B.K. Puri a , S. Anand b
b a Indian Institute of Technology, Department of Chemistry, New Delhi-110016, India Indian Institute of Technology, Centre for Bio-medical Engineering, New Delhi-110016, India

Received 16 February 2005; received in revised form 30 March 2005; accepted 30 March 2005 Available online 11 May 2005

Abstract A new enzyme coated electrode for the determination of urea in blood samples has been developed. It is based on the encapsulation of urease enzyme in the porous silicate matrix by the solgel technique on a glass electrode for the purpose of sensing urea in blood samples. Various parameters like the effect of pH, selection of a suitable buffer of appropriate concentration and interference of common substances in blood samples have been evaluated to optimize the conditions for the determination of urea. The electrode can be used for the determination of urea in the concentration range 0.0330.0 mM in a solution. The detection limit of the present enzyme-coated electrode is found to be 52 g/ml of urea. The relative standard deviation for the electrode-to-electrode reproducibility is found to be 2.4% for the determination of 0.1 mM of urea (six replicate electrodes). Solgel matrix containing immobilized enzyme was stable for about 25 days at 4 C with 80% urease activity. Urea content in various clinical blood samples has been estimated using this electrode and the results are found to be in good agreement with the standard clinical methods as reported in the literature. 2005 Elsevier B.V. All rights reserved.
Keywords: Enzyme-coated electrode; Urea sensor; Solgel technique; Urea determination; Blood samples

1. Introduction Assay of blood urea is of great clinical importance in the diagnosis of malfunctioning of kidney [1]. Its determination in human blood is one of the most frequent types of analysis carried out in a routine clinical laboratory. Several methods for the estimation of urea are documented in the literature [29]. In most of the urea biosensors, the immobilized urease enzyme catalytically converts urea into ammonium and hydrogen carbonate ions as described below:

Corresponding author. Present address: Indian Institute of Technology, Centre for Bio-medical Engineering, hauzkaz, New Delhi-110016, India. Tel.: +91 11 25469547; fax: +91 11 26515616. E-mail addresses: sahneyrachanas@rediffmail.com, rachanasahney 4@hotmail.com (R. Sahney).

Various methods have been developed for monitoring the products formed in the enzymatic reaction, such as UVvis spectrophotometry [2,3], coulometry [4] and amperometry [5,6]. The potentiometric determination of urea belongs to the best-known class of biosensors. Here, the transducer developed, monitors the potential change due to the generation of ammonium- or hydrogen- carbonate ions in the course of an enzymatic reaction. In the fabrication of biosensors, immobilization of relevant enzyme at the surface of a sensing electrode is one of the crucial steps. Solgel silica matrix offers a new and interesting technique in the eld of biosensors [1012]. It provides an alternative way to immobilize and stabilize active enzymes on an electrode. In these matrices, enzymes retain their functional characteristics to a large extent [11]. It is a low temperature technique by which heat sensitive biological entities (enzymes, proteins, antibodies etc.) can easily be immobilized. This class of solgel silicate matrix possesses chemical inertness, physical rigidity, negligible swelling in aqueous solution, tunable porosity,

0003-2670/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.03.069

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high photochemical and thermal stability and transparency. These attractive features have led to an intense research in this area mainly based on optical and electrochemical techniques. In the present work, we have tried to fabricate a urea sensor by dip-coating the immobilized urease enzyme in a solgel silicate matrix derived from tetramethyl orthosilicate (TMOS) on a glass electrode specic for pH measurements. It has been observed that when substrate active urease enzyme immobilized in silica solgel matrix is placed directly on the sensing surface of the glass electrode, it can be conveniently used for sensing urea in a sample. A survey of the literature reveals that a glass electrode has rarely been used for this purpose [13,14] probably owing to the difculties in immobilization of the enzyme on its sensing surface. The effect of pH and buffer concentration on the function of this enzyme-coated electrode is also investigated. The results of clinical analysis of urea in human blood serum samples using this sensor has been reported and compared with the standard method used in clinical laboratories.

room temperature. This process hardly takes 23 min. Three successive coatings of casting solution were done on the sensing surface of the electrode. The glass electrode coated with solgel lm was kept in 50 ml of phosphate buffer at pH 7.0 and at 4 C overnight before using it. After each use, the enzyme-coated electrode was rinsed and stirred in water for l min. The electrode was stored in phosphate buffer at 4 C when it was not in use. 2.3. Test for urease leaching from solgel layer We tested urease leaching from the enzyme-coated layer of the pH-electrode by storing it in 50 ml of phosphate buffer solution for overnight and then assaying the buffer solution for urease enzyme. After removing the enzyme-coated electrode from the buffer solution, this buffer solution was mixed with l00 L (0.1 M) of urea solution and the resulting solution was stirred. Ammonia released was assayed using the well-known Nesslers reagent (K2 HgI4 ) method. Blank determinations were carried out under identical conditions. 2.4. Experimental setup and measurements

2. Experimental 2.1. Materials and apparatus Urea (enzyme grade), urease-active meal (Jack-beans CDH, India), tetramethylorthosilicate (TMOS, 98% purity, Milwaunkee, WI). Nesslers reagent, phosphate buffer and all others chemicals used in this work were of analytical reagent grade obtained from commercial sources. All solutions were prepared in double distilled water. A combined glass electrode (Elico India Limited, No. CL 5 IB) and a pHmeter (Elico India Limited, No. L1612 India) were used in the present work. 2.2. Preparation of enzyme coated electrode A stock solution of urease enzyme (5 mg/ml) was prepared in phosphate buffer (5 mM, pH 7.0) and kept in a refrigerator. A solution of silicate matrix was prepared by mixing 10 ml of tetramethylorthosilicate (TMOS), 3 ml of de-ionized water and l00 L of 0.1 M HC1 in a glass vial (TMOS/water (v/v) ratio 3). The mixture was stirred vigorously until a clear solution was obtained. This solution was used throughout the present work for the preparation of solgel matrix and was diluted as and when required. Specic casting solution was prepared by mixing 0.5 ml of the stock solution of TMOS with 0.5 ml of 5 mM phosphate buffer containing urease enzyme (5 mg/ml) prior to lm casting. A combined glass electrode was rinsed with de-ionized water and then treated with acetone to ensure that the sensing surface is thoroughly clean. The lm was cast on the sensing surface of the pH-electrode by dip-coating method at the rate of 10 cm/min. The casting solution was allowed to polymerize till it became a gel at In the conical ask, 50 ml of phosphate buffer of appropriate concentration and pH containing 0.1 M NaCl was taken and l ml of urea solution of desired concentration was added to it through the side tube. This solution was thoroughly stirred for a few seconds. The potential difference across the two leads of the composite glass electrode was measured using a pH meter at a time interval of 60 s until a steady value was obtained. For constructing a calibration curve, the experiment was repeated with different urea solutions of known concentrations. To estimate urea in a clinical blood sample l ml of blood plasma/serum was added to the same buffer solution instead of urea solution. This enzyme-coated electrode was used in the urea measurements roughly for 5 h a day. To optimize the reaction conditions for urea determinations, the effect of pH on the electrode response was studied in 5 mM phosphate buffer between pH 6.08.0. Calibrations were made in l, 5, 10 and 15 mM phosphate buffer solutions at a xed pH 7.

3. Result and discussion 3.1. Characterization of solgel matrix TMOS derived solgel lms were optically transparent and porous and thus found to be suitable for the immobilization of urease enzyme. The entrapment of enzyme was conrmed with the help of a Fourier transform infrared (FTIR) spectrometer. The prominent peaks at 3100 and 3190 cm1 are due to the presence of N H linkage and N H stretching of free amide, respectively. The broad peak in the range 14001600 cm1 conrms the presence of SiO4 unit.

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3.2. Optimization of enzyme coating on the electrode In the solgel derived biosensors, the solgel preparation conditions have signicant effect on the response of biosensor [10]. Higher TMOS/Water (v/v) ratio in the stock solgel solution results into a denser solgel layer with small pore size and consequently high enzyme loading [15,16]. Therefore, the solgel stock solution of TMOS/water ratio 3 as given in literature [15,16] was prepared for casting silicate matrix on the pH-sensitive surface of a glass electrode. The gellation time for sol was found to be 23 minutes in a closed chamber with relative humidity 84%. Three successive coating of casting solution was done on the sensing surface of a glass electrode to obtain a moderately thick lm. Usually the large size and high molecular weight of proteins prevent their leaching from the solgel matrix [12]. But to ensure the complete enzyme loading, we tested the electrode for urease leaching. In 50 ml buffer solution (5 mM, pH 7.0) the enzyme coated electrode was kept for 1216 h. The electrode was removed and the buffer solution was tested for urease enzyme. The assay was carried out by using Nesslers Reagent to remove the ammonia produced by the enzymatic hydrolysis of urea [17]. It was found that there was no leaching of urease enzyme. 3.3. Effect of pH of buffer solution Since pH has considerable effect on the response of a biosensor, therefore, it was necessary to optimize the working condition for the present enzyme coated-electrode. The response of the electrode has been studied within the maximum urease activity range i.e. from pH 68. The variation of magnitude of response with l.0 mM urea solution at different pH of buffer is depicted in Fig. 1. The maximum biosensor response in terms of potential difference was obtained at pH 7.0. 3.4. Effect of concentration of buffer solution The inuence of buffer concentration on the response of the sensor was studied by calibrating the enzyme-coated electrode for different concentrations of buffer solution i.e. l, 5,

Table 1 Parameters for the calibration graphs of enzvme-coated electrode Conditions CB = l, pH 7 CB = 5, pH 7 CB = 10, pH 7 CB = 15, pH 7 Range (mM) 0.015.0 0.0130 1.0030 2.5030 Sensitivity (mV/log (M)) 16.200 11.066 1.720 1.050

Range: linear range, CB : buffer concentration (mM).

10 and 15 mM at constant pH 7. A comparison of the parameters of the calibration graph obtained for the enzymecoated electrode is given in Table 1 which indicates that the response for urea determination in the concentration range 0.015 mM in 15 mM buffer concentration was quite high (16 mV/log (Urea)). The response of the enzyme-coated electrode above 5 mM buffer concentration was considerably decreased which, may be due to the limited capacity of phosphate buffer to allow the local pH change within the enzyme coated membrane. This type of local pH effect in potentiometric urea sensor has also been observed by Mascini and Guibault [18]. Therefore, in consideration of magnitude of response, sensitivity and linear dynamic range 5.0 mM phosphate buffer at pH 7.0 was preferred in all measurements in the present work. 3.5. Electrode response characteristics In the use of encapsulated enzymes, the response of the enzyme coated glass electrode depends on the mass transportation of the substrate or product through the solgel lm. The response time with the present experiments was never found to be more than 5 min. The response time curve in one particular case is shown in Fig. 2 as a sample. The electrodeto-electrode reproducibility of six replicate biosensors in term of response was carried out in 1.0 mM urea. A relative standard deviation of (R.S.D.) 2.4% was obtained. The response of a single solgel urease coated electrode to successive urea determination at this concentration was also carried out. It was observed that the response was moderately constant for six replicates. Relationship between the urea concentration and the change in the potential difference at a xed time and

Fig. 1. Potential change (in mV) at different pH of buffer solutions.

Fig. 2. Response-time (time vs. potential difference (mV)) in one particular case (l mM-urea concentration).

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Fig. 3. Calibration curve showing variation of electrical potential difference (mV) with the negative logarithm of the concentration of urea (M). The straight line is the best t line.

Fig. 4. The stability prole of biosensor showing decrease in analytical signal (in mV) with time (in days). Table 2 List of different interferring species affecting the response of the enzvmecoated electrode Interferents None Glucose FeSO4 Pb(NO3 )2 HgCl2 Thiourea Concentrations 0.00 5.0 mg/ml 0.20 mM 0.20 mM l.00 mM 2.00 mM Relative response (%) 100 97 98 94 64 84

temperature 5 min and 25 C, respectively, in the present work were studied using the enzyme-coated-electrode system. The calibration curve obtained at pH 7 (5 mM buffer) by plotting the potential difference (mV) across the two leads of the combined glass electrode against the logarithm of the concentration of urea (M) is shown in Fig. 3. The calibration graph was found to be linear over a urea concentration of 0.0130 mM using the present enzyme-coated electrode. The straight line shown in Fig. 3 is the best-t line, y = 11.066 X + 71.508 (regression = 0.9967) passing through the experimental data points. This range includes the concentration of urea that is found in the clinical samples. The sensitivity obtained from the calibration curve was 11.066 mV/log (Conc.) while the detection limit of the present system based on three times the signal-to-noise ratio (S/N = 3) was found to be 52 g/ml. Apparent MichaelisMenten constant (KM app ) was calculated for urea to show the suitability of the enzyme in the silicate matrix to the substrate. LineweawerBurk plot (1/ E versus 1/(S)) was constructed for this purpose at optimum working conditions and it was found to be 1.32 105 M for the immobilized enzyme. This small value of KM app indicates maximal catalytic activity of the enzyme at low substrate concentration [19]. 3.6. Electrode storage, stability and applications The biosensor electrode prepared under optimum working conditions was tested with respect to its stability in l mM urea solution. The life-time of the present enzyme-coated electrode was studied by calibrating the electrode in 5 mM phosphate buffer containing 0.1 M NaCl at pH 7. The response was found to be more or less constant for 4 weeks even using the electrode continuously for 5 h/day. The electrode was dipped in 5 mM phosphate buffer and kept in a refrigerator when not in use. The analytical signal (in mV) was taken as a measure of the electrode life-time. The response of the electrode was found to be decreased by 20% in the initial value of the signal in 4 weeks of operation as shown in Fig. 4.

3.7. Interferences Many substances may alter the activity of an enzyme electrode by different ways of its interaction with the surface of the biosensor. To decide whether the electrode could be applicable to real samples or not, the interference of various species found in blood serum was studied under the optimum working conditions. A denite amount of interfering species were introduced into the test solution containing l mM urea and the response of the biosensor was measured. Among the various species studied (Table 2) only mercury ions interfered signicantly, indicating that the biosensor is highly selective for the determination of urea in complex samples. 3.8. Clinical analysis of urea in human blood serum The concentration of urea in the clinical blood samples obtained from the Laboratory of the department of medicine at All India Institute of Medical Sciences, Delhi, India were estimated using the proposed biosensor. The results obtained
Table 3 Determination of urea in blood serum samples Samples number 1 2 3 4 5 Concentration of urea using clinical auto-analyzer method (mg % w/v) 43 30 98 28 110 Concentration of urea using the present method (mg % w/v) 44.96 31.18 99.18 29.16 111.92 1.15 1.00 1.00 1.50 1.20

Values reported as average of 10 reports + S.D.

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(Table 3) compare favorably well with that obtained by the usual clinical method using an auto-analyzer.

One of us (Rachana Sahney) is grateful to the Council of Scientic and Industrial Research, New Delhi, for the award of research Associate ship. References

4. Conclusion Several urea sensors are documented in the literature, which may be used for the determination of urea but most of them cannot be used for continuous determination of urea in different samples since the surface in some of them has to be renewed even after a single run. It is worth mentioning here that the composite glass electrode as suggested in the present work can be reusable after several estimations of urea in solution. The improvised one described in the present communication seems to have a few advantages like: 1. The ability of glass electrode to act as a sensing electrode is an advantage in itself for obvious reason. 2. The present urea biosensor as compare to other biosensors reported in the literature is very simple in terms of fabrication, better sensor-to-sensor reproducibility with no chemical modication of substrate and enzyme and have low detection limit (52 g/ml). 3. It can be satisfactorily applied for the determination of urea in small volumes of blood samples.
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Acknowledgement We thank Dr. A.K. Srivastava, Laboratory of Medicine, All India Institute of Medical Science, Delhi, India, for providing us blood serum samples and their urea analysis, which allowed us to compare our results with the clinical methods.