Sensors and Actuators B 140 (2009) 260–266
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Potentiometric biosensor for urea determination in milk U.B. Trivedi a , D. Lakshminarayana a , I.L. Kothari b , N.G. Patel c , H.N. Kapse d , K.K. Makhija a , P.B. Patel a , C.J. Panchal e,∗ a
Department of Electronics, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India Department of Bio-Sciences, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India c Department of Chemistry and Physics, University of North Florida, Jacksonville, FL, USA d Institute of Science and Technology for Advanced Studies and Research (ISTAR), Vallabh Vidyanagar 388120, Gujarat, India e Applied Physics Department, Faculty of Technology and Engineering, M.S. University of Baroda, Vadodara 390001, Gujarat, India b
a r t i c l e
i n f o
Article history: Received 3 March 2009 Received in revised form 3 April 2009 Accepted 9 April 2009 Available online 24 April 2009 Keywords: Potentiometry Biosensor Urea Ion sensitive electrode Urease Polymer matrix Milk
a b s t r a c t A potentiometric urea sensitive biosensor, using a NH4 + ion sensitive electrode in double matrix membrane (DMM) technology as the transducer, has been described. Thick ﬁlm screen printing technique was utilized for the fabrication of the basal conducting track of the potentiometric electrode and also for the development of Ag/AgCl reference electrode. The ion sensitive polymer matrix membrane could be formed in the presence of an electrochemically inert ﬁlter paper. The electrochemical response behavior of the NH4 + ion sensitive electrode has been studied initially. Later, a urea biosensor has been developed by immobilizing the urease enzyme, through entrapping, onto the ion sensitive membrane using a polymer matrix of poly(carbamoylsulphonate) (PCS) and polyethyleneimine (PEI). The urea biosensor responded rapidly and in a stable manner to the changes in the test urea concentrations. The sensor exhibited a detection limit of 2.5 × 10−5 mol/L. The average slope in the linear range has been found to be 51.7 ± 0.5 mV/decade. The biosensor system has, then, been tested for the detection of urea levels in the real samples of milk. A good agreement could be observed between the urea concentrations detected by the developed biosensor and the spectrophotometric technique. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Urea, [CO(NH2 )2 ], an end product of nitrogen metabolism, has great signiﬁcance in clinical chemistry and dairy industry [1–6]. The food industry has the requirement of real time and accurate analysis of dairy products during manufacture and quality control. In this regard, urea biosensors could prove to be a valuable tool for monitoring the urea content of milk [7,8]. Milk is an emulsion of fat in watery solution of sugars, mineral salts and proteins in colloidal solutions. The typical concentration of urea in milk is 18–40 mg/dl [9,10]. High urea content in milk, which occurs from the unbalanced feeding of cows, is known to inﬂuence the milk production and fertility [11,12]. Excessive nitrogen (N) in the feed causes high systemic urea N without a corresponding increase in milk protein . Determination of values of urea and true protein in milk may be useful to assess the nutritional program of cows and particularly lactating dairy cows . It is reported [15,16] that when the milk urea N (MUN) reaches above 20 mg/dl or so, it may indicate the underlying pathological problems in cows. Milk urea level is also linked to the thermal stability of milk at higher temperatures .
∗ Corresponding author. Tel.: +91 9825094761; fax: +91 265 2423898. E-mail address: cjpanchal [email protected]
(C.J. Panchal). 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.04.022
Urea, as mentioned above, is a normal constituent of milk and it forms a major part (55%) of the non-protein nitrogen of milk . However, for commercial beneﬁts chemicals like urea, caustic soda, reﬁned oil and detergents are sometimes used to adulterate the milk. The adulteration decreases the nutritive value of the milk and may pose a great threat to the human health. The mixture of chemicals gives the solutions the milk like properties and is termed as ‘Synthetic Milk’ [8,18–20]. A cut off limit for urea concentration in milk is normally accepted at 70 mg/dl. The presence of urea above this cut off limit in milk can cause severe health problems for human beings, such as, indigestion, acidity, ulcers, cancer, malfunctioning of kidneys etc. Hence, urea detection and its estimation have great signiﬁcance in dairy industries, clinical analyses and food processing technology. Moreover urea is widely distributed in the nature and its analysis is necessary in food chemistry and environmental monitoring . Because urea concentrations in human blood and urine are both important indicators of renal health, many sensors including biosensor types, speciﬁc to urea, have been developed in the biomedical industry. Numerous types of urea sensors have, thus, been proposed based on conduct metric [22–24], potentiometric [25–30], thermometric , and optical methods . Most of these sensors are too delicate for use with raw milk without an extensive pretreatment [13,33–35]. The biosensors speciﬁc to urea detection in milk will have to be simple, reliable and robust enough
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to withstand repeated exposure to the high lipid and protein concentrations in milk. There are only a few reports available in the literature over the development of sensors for the speciﬁc application of urea detection in milk [8,18]. The objective of the present investigation is to develop a low cost screen printed and disposable type urea sensitive enzymatic biosensor system, based on potentiometry, for the detection and estimation of urea levels in milk samples. Potentiometry is selected for the purpose as it allows the determination of a wide spectrum of ions and also as it applies portable and inexpensive equipment. This technique is especially useful for carrying out the tests (urea detection) at remote sites (‘in ﬁeld’ measurements at rural milk collection centers, house hold consumer locations etc.). The sensor designed in the present investigation consisted of a screen printed disposable NH4 + ion sensitive electrode developed on the basis of double matrix membrane (DMM) technology  as the transducer with a layer of immobilized urease. The urease enzyme has been immobilized by entrapping in a unique polymer matrix of poly(carbamoylsulphonate) (PCS) and polyethyleneimine (PEI). A screen printed Ag/AgCl reference electrode has formed the stand alone urea biosensor system for the detection of urea in milk samples. 2. Experimental 2.1. Chemicals and reagents The various solutions utilized in the present investigation were all prepared with analytical reagent grade chemicals and double distilled water. The various enzymes, polymers, pastes and chemicals used in this investigation are listed below with their makes. 2.1.1. Enzyme • Urease, Jack bean source [E.C. 22.214.171.124] (Sigma, St. Louis, USA). 2.1.2. Polymers • Polyethyleneimine (50% (w/v) aqueous solution) (PEI) (Sigma, USA). • PCS hydrogel (Sens Lab, Germany). 2.1.3. Pastes for screen printed fabrication of the electrodes • Silver/silver chloride paste R-414 (DPM-68) (Ag/AgCl) (Ercon, USA). • Silver paste (Ted Pella Inc., USA). 2.1.4. Polyester sheets, screen and chemicals • Polyester foil Melinex type 505 (thickness: 175 m) (Putz, Germany). • Polyester/polyethylene sheet (thickness: 150 m) (Haltem, Germany). • Polyester sheet, 68.110/H1, 300 m thick opaque sheet size 297 mm × 420 mm, mesh size 1/77.5 cm (77 wires in 1 cm). • Screen (MonolenPuls-77T) mesh size 68 m (Berlin, Germany). • Urea (extra pure, ultra) (Sigma, Deisenhofen, Germany). • Tetrahydrofuran (THF) (99.5+%, spectrophotometric grade) (Sigma–Aldrich, St. Louis, USA). • Cyclohexanone (extra pure) (Merck, Darmstadt, Germany). • Polyvinylchloride (PVC), high molecular weight (Fluka, Switzerland). • Bis(2-ethylsebacate) (Sigma, Deisenhofen, Germany). • Disodium hydrogen phosphate 67 mM (Merck, Darmstadt, Germany). • Sodium dihydrogen phosphate 67 mM (Merck, Darmstadt, Germany).
• • • •
Potassium chloride 0.1 M KCl (Merck, Darmstadt, Germany). Sodium chloride (Merck, Darmstadt, Germany). Sodium hydroxide (Merck, Darmstadt, Germany). Tris(hydroxymethyl)-aminomethane (analytical grade) (Sigma, St. Louis, USA). • Ethylenediaminetetraacetic acid (EDTA) (disodium salt) (analytical grade) (Sigma, St. Louis, USA). • Phenol (Fluka, Switzerland). • Sodium hypochloride dihydrate (Fluka, Switzerland). 2.1.5. Apparatus • Micropipette (Eppendorf, Germany). • Micro ﬁber matrix (MFM) material ﬁlter paper (110 mm diameter) (Whatman, USA). • Multi-Channel ISE/pH/mV/ORP/Temperature bench top meter (Thermo Orion, USA). • Vacuum Coating Unit (Hind Hi Vacuum, India). 2.2. NH4 + ion sensitive electrode fabrication A potentiometric biosensor system was developed in the present investigation for the detection of urea. The potentiometric biosensors make use of ion selective electrodes in order to transduce the biological reaction into an electrical signal. Initially, a NH4 + ion sensitive electrode was fabricated and it was, later, used for the detection of urea by immobilizing urease enzyme on the NH4 + ion sensitive membrane of the electrode. The method of screen printing that was utilized for the fabrication of amperometric electrodes and reported earlier by the authors  was again adopted for the development of NH4 + ion sensitive electrodes and also for the Ag/AgCl reference electrode. Silver (Ag) was selected as the base conducting track of the potentiometric electrode. The silver paste was screen printed using screens having a screen mesh size of 68 m. The base transducer used was, accordingly, a screen printed silver electrode on a Melinex sheet of 150 m thickness. The basic steps for the preparation of screen or mask normally involved the processes of layout, artwork generation, mask frame preparation, image transfer and mounting . The electrodes were produced in a batch process and after each screen printing the sheets were kept at 100 ◦ C for 1 h annealing. Then, in a separate experimental process, high purity thin silver ﬁlm was thermally deposited on one side of a ﬁlter paper in a high vacuum (10−4 Pa). From the silver coated ﬁlter paper, circular shaped 3 mm diameter discs were separated. The ﬁlter paper disc with the silver deposited side was then attached to one end of the previously screen printed silver basal conducting track. The contact between the silver conducting track and the silver coated side of the ﬁlter paper disc was made with a high purity silver paste. Later, the silver conducting track with the polyester sheet base was laminated using a laminating machine (SANON, CR-309, India), leaving the 3 mm diameter unsilvered ﬁlter paper without any lamination. Later, a NH4 + ion sensitive membrane was prepared on the unlaminated and unsilvered ﬁlter paper, following the procedure described below. The other end of the conducting track was utilized for the electrical connection purpose. The entire lamination process was carried out at 413 K. A large number of silver based ﬁlter paper tipped electrodes were fabricated in a similar way. The NH4 + sensitive DMM electrode was then prepared following the procedure described by Eggenstien et al. [36,39]. A mixture of 1% nonactine, 66.8% bis(2-ethylsebacate) and 32.2% PVC was prepared ﬁrst. From this mixture, 400 mg was dissolved in 1.5 ml THF (99.5%, spectrophotometric grade) and 0.5 ml cyclohexanone. The components were allowed to dissolve overnight and shaken vigorously until a homogenous solution was obtained. A micropipette
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(Eppendorf, Germany) was used to dispense 10 l solution over the ﬁlter paper tip. The NH4 + ion sensitive electrodes thus prepared, in a batch of 60, were all stored overnight at 4 ◦ C. These working electrodes were now ready on one hand for the NH4 + measurements and on the other hand, for the immobilization of the urease enzyme for urea detection. 2.3. Urea detection electrode fabrication For urea detection, the urease enzyme was immobilized by entrapping it in a PCS + PEI polymer matrix on the surface of the NH4 + ion sensitive membrane electrode. For the urease immobilization, a PCS solution was prepared ﬁrst, by dissolving 150 mg PCS pre-polymer in 400 l deionized water. Next, a PEI solution was prepared by dissolving 2.5 mg PEI pre-polymer in 60 l deionized water. A mixture of PCS and PEI solution was prepared later by slowly adding PEI solution using a micropipette (Eppendorf, Germany) into the acidic PCS solution. The PEI (2.5% in aqueous solution) was added till the pH value of the mixture solution reached 6.8. A urease enzyme stock solution was prepared by using 60 mg/ml of the enzyme with the buffer solution and then stored at −10 ◦ C. Then, 30 l of urease stock solution was added to the mixed solution of PCS and PEI in a proportion of 1:1 (urease:PCS + PEI) and gently stirred at the room temperature of 303 K. Using the micropipette, 2 l mixture of urease and PCS + PEI was dropped on the NH4 + ion sensitive membrane of the working electrode at the room temperature of 303 K. A batch of 30 working electrodes was prepared accordingly and were all kept at 4 ◦ C overnight. Fig. 1 shows a schematic of a typical working electrode for urea biosensor for use in potentiometric measurements. In the ﬁgure, the layer formation at the electrochemically active end is shown on the right hand side with arrows (i.e. ﬁlter paper/NH4 + membrane/urease:PCS + PEI). 2.4. Ag/AgCl reference electrode fabrication Eggenstien et al.  reported the preparation of the Ag/AgCl reference electrodes following the way described by Diekmann et al. , leading to a combination of the reference electrode with the urea sensitive working electrode. In the present investigation, the authors, however, adopted the simple thick ﬁlm screen printing technique for the fabrication of a separate Ag/AgCl reference electrode for use with the working electrode in the potentiometric setup. The Ag/AgCl paste was screen printed on a Melinex sheet of 150 m thickness using a screen with a screen mesh size of 68 m. The screen printing procedure was similar to the above described way of the fabrication of the silver conducting basal track.
2.5. Measurements The potentiometric measurements were made at room temperature (303 K) using a Multi-Channel ISE/pH/mV/ORP/Temperature ion sensitive meter (Thermo Orion-920A, USA). The standard stock solutions of NH4 Cl and urea were duly prepared to check the ion selective nature of the stand alone NH4 + electrode and the urea detection capability of the urease immobilized enzyme electrode (having the NH4 + sensitive membrane) respectively. 2.6. Principle of sensor operation The urea sensor operation is based on the enzymatic decomposition of urea by urease: Urease
CO(NH2 )2 + H2 O −→ CO2 + 2NH3 In the pH region where the enzyme is active (around pH 7), the products of the above enzymatic reaction dissociate as: CO2 + H2 O HCO3 − + H+ NH3 + H2 O NH4 + + OH− Thus, it is possible to determine the urea concentration potentiometrically using a variety of transducers such as the pCO2 electrode, the pNH3 electrode, the pH electrode, and the pNH4 + electrode. Urease can be physically entrapped in a polymer gel and placed on the tip of an electrode, which is sensitive to ammonium ions. This basic transducer has the drawback of being highly sensitive to H+ , K+ , and Na+ ions, which considerably restricts the use of the sensor in environments containing monovalent cations. A buffer solution is therefore, needed such that its constituents do not interfere with the response of the enzyme electrode . The potentiometric urea biosensor, therefore, needs an ion sensitive electrode in combination with an immobilized urease enzyme. Hence, a NH4 + ion sensitive electrode was ﬁrst prepared and characterized. Later, this NH4 + sensitive electrode was used for the detection of urea by immobilizing the urease enzyme onto the NH4 + ion sensitive membrane of the electrode. 3. Results and discussion 3.1. Characterization of NH4 + ion sensitive electrode At ﬁrst, the NH4 + sensitive electrodes were characterized to evaluate whether they could serve as transducers in sensing urea. Fig. 2 shows the response behavior of the NH4 + sensitive electrode against the Ag/AgCl reference electrode. The electrode responded rapidly and in a stable manner to the additions of the ammonium chloride stock solution. The response time was found to be the in the range of a few seconds. The detection limit determined from the calibration graph was found to be 3.0 × 10−5 mol/L. The inset of Fig. 2 shows the calibration curve of the NH4 + electrode. The average slope in the linear range was found to be 56.7 ± 0.4 mV/decade, indicating a high reproducibility. It could, therefore, be concluded that the developed NH4 + ion sensitive electrode was a suitable transducer for the potentiometric urea sensor fabrication. The next step was then, the immobilization of the urease enzyme onto the NH4 + sensitive electrode. 3.2. Urea biosensor
Fig. 1. Schematic of a typical fabricated working electrode for potentiometric urea detection.
The fabricated enzyme based urea biosensors were used as the working electrodes with the screen printed Ag/AgCl as the reference electrodes. The electrodes were suitably dipped into 30 ml of potassium phosphate buffer solutions of varied pH values and then
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Fig. 2. Response curve of the NH4 + ion sensitive electrode. The inset shows the calibration graph.
Fig. 4. Response curve of the urea biosensor with screen printed reference electrode. The inset shows the calibration graph.
connected to the Multi-Channel ISE/pH/mV/ORP/Temperature ion sensitive meter (Thermo Orion-920A, USA). 1 M urea stock solution of 10 ml was added to the buffer solutions of different pH values. The solutions were then continuously stirred for about 15–18 min to get the stable base line. Then, solutions of known urea concentrations were carefully added, as the test samples, to the above buffers to study the urea testing ability of the sensor and its pH dependence. The measurements were all made at the room temperature of about 303 K.
cerned. The inhibition of urease by phosphate buffer is of partially mixed type while that by citrate is uncompetitive . The inhibition by phosphate increases rapidly with decreasing pH . Below pH 7.0, urease activity was not inﬂuenced by the presence of electrolytes in a wide range of both concentration and type of ions. At pH value higher than 7.0, urease activity was reported to decrease more signiﬁcantly with increasing electrolyte concentration than at low pH values . In order to obtain urea sensors which would show Nernstian potentiometric response over a wide pH range, different organic functional groups as the hydrogen ion carriers were studied by Yuan et al. . These functional groups were found to be capable of creating pH response in acidic and alkaline regions. In the present investigation, as mentioned above, a pH 7.5 was found to offer the optimum response.
3.2.1. pH dependence Fig. 3 shows the pH dependence of buffer solutions on the potentiometric response of the fabricated urea biosensor. In the present investigation, the highest response could be observed at pH 7.5 which was subsequently utilized in further experimental investigations. It has been reported  that there is no consistent pattern so far as the effect of buffer ions on the urease kinetics are con-
Fig. 3. Inﬂuence of buffer solution pH on the response of urea biosensor.
3.2.2. Response behavior of urea biosensor The response time of biosensor is normally the time taken for it to reach the steady state when there is no further variation in the signal. The response time depends upon the analyte, co-substrate, product transport through different membranes, membrane permeability etc. The response time also depends upon the activity of the molecular recognition system. Fig. 4 shows the electrochemical response behavior of the fabricated urea biosensor system. The system responded rapidly and in a stable manner to the additions of known concentration of urea from the stock solution. The behavior shows that the screen printed Ag/AgCl reference electrode provides a suitable reference electrode leading to the development of a complete low cost and disposable type of system. The present simple urea biosensor system exhibited a detection limit of 2.5 × 10−5 mol/L, on par with the detection limit reported by Eggenstien et al.  with their more sophisticated version. The urea biosensor system was also found to function in the analytically useful range with a response time of 30–40 s. The inset in Fig. 4 shows the calibration curve obtained with the disposable type of electrode system. The average slope in the linear range was found to be 51.7 ± 0.5 mV/decade with a high degree of reproducibility. The urea biosensor exhibited a good linear least squares ﬁt (linear regression analysis) with a good correlation coefﬁcient (R2 = 0.989, n = 8). The noise range amounted to about 1.0 mV. The slope showed
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Fig. 5. Effect of temperature on the response of urea biosensor.
Fig. 6. Response curves for biosensors in batch process.
no signiﬁcant decline over a period of more than 24 h of operation. This shows that the fabrication technology utilized here is suitable for the development of a disposable urea biosensor. Also, the investigation clearly indicates the suitability of PCS + PEI polymer matrix for the immobilization of urease enzyme.
3.2.5. Storage stability and shelf life Most enzymes lose their activity when not stored in refrigerator, and therefore, storage at low temperatures is one of the most important parameters to retain the stability of enzyme based biosensors. Moreover, in order to attain high water content in the immobilized layer, the sensors were kept in a closed bottle together with wet cotton to achieve moisture condition. Fig. 6 compares the storage stability of the two enzyme sensors stored at two different temperatures. The operational stability of a biosensor response may vary considerably depending upon the sensor geometry, method of preparation, biological recognition reactions etc. From Fig. 7 it can be seen that the performance of the urea sensor stored at low temperature is higher. A slight decrease in the electrode activity and selectivity was observed over a week of operation. Thereafter, the decrease in the activity was more signiﬁcant. The life time of the enzyme based urea biosensor in the present investiga-
3.2.3. Temperature dependence Urea biosensors use enzymatic reactions and so an increase in temperature will also increase the catalytic activity and hence, the rate of reaction. The effect of temperature of the buffer solution on the response of urea biosensor was studied in the range of 280–330 K. Fig. 5 shows the response of urea biosensor based on DMM technology against the buffer solution temperature. It is seen that the potentiometric response initially increases with the temperature and then decreases later. The response reached a maximum at about 313 K and the decrease of response after 313 K may be due to the thermal inactivation of the urease enzyme or the enhanced disproportionate kinetics of electrochemical reactions. Fig. 5, thus, indicates a maximum working temperature of 313 K. 3.2.4. Reproducibility Reproducibility is a measure of the scatter or the drift in a series of observations or results performed over a period of time. It is generally determined for the analyte concentration within a usable range. During the present investigation, a large number of urea biosensors were fabricated in a batch process. From a batch of 30 screen printed DMM based urea sensitive electrodes, the authors randomly selected ﬁve electrodes and subjected them for obtaining the potentiometric response. Fig. 6, thus, shows the response curves for biosensors fabricated in four batches, with each batch having a total of 30 biosensors. It is to be noted that each point of the curve, corresponding to each batch, is an average value of ﬁve measurements. It is seen from the ﬁgure that the results are reproducible for each batch of process. Hence, the present low cost screen printing technology can be effectively implemented for the mass/batch production of disposable type of urea biosensors comprising of enzyme and screen printed Ag/AgCl electrode.
Fig. 7. Storage stability of urea biosensor: curve (a) corresponds to urea biosensor stored at 303 K and curve (b) corresponds to the biosensor stored at 270 K.
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tion was found to be in the range of 7–8 days when operated at room temperature (303 K) and below. This life time is quite suitable for disposable type  of urea biosensors for use in food and clinical analysis. An untested urea biosensor was found to remain intact for about 3 months also. A prudent use (maximum 12 h/day operation and storage at very low temperatures) however, enabled the sensor system to function properly up to about 25–30 days only. Whenever the sensor was not in operation, the electrodes after disconnecting from the Multi-Channel ISE/pH/mV/ORP/Temperature ion sensitive meter (Thermo Orion-920A, USA) were kept at about 4 ◦ C in a refrigerator, along with the buffer solution. That is, the electrodes were always placed in a buffer solution and connected to the ion sensitive meter whenever the measurements were carried out. For any subsequent measurements over the time, the electrodes were cleaned with the deionized water ﬁrst and later, were placed into a freshly prepared phosphate buffer solution. 3.2.6. Effect of interferents on the biosensor response The use of enzyme based potentiometric biosensors for determining substrates in complex media like biological ﬂuids poses interference problems. For the determination of urea, especially in human blood samples for the medical diagnosis purpose, it is necessary to take into account the interference by Na+ and K+ ions [36,47]. These ions are abundantly present in human and animal body ﬂuids. The general problem of NH4 + ion selective electrode based urea biosensors is that the potentiometric signal can be fouled by the interferents like Na+ and K+ ions. Hence, in order to gauge the inﬂuence of the interfering Na+ and K+ ions on the response behavior of the developed urea biosensors, the electrodes were dipped in 20 ml of the sample buffer solution containing various combinations of NaCl and KCl solution concentrations. The calibration curves were then obtained by the addition of standard urea stock solutions. Fig. 8 shows the inﬂuence of increasing Na+ and K+ concentrations and the response of the fabricated urea biosensors. It can be seen that as the concentration of these ions increased, the lower detection limit of urea moved towards higher concentrations. Concentrations up to a level of 140 mmol/L for Na+ and 4.5 mmol/L for K+ ions were tested because these are the normal levels of the ions in human blood and serum. The observed response in the presence of the interference ions indicates that the fabricated biosensors can be applied for the urea detection in real samples of milk, blood,
Table 1 Urea detection in milk samples. Milk sample no.
Urea concentration measured by biosensor (mg/dl)
Urea concentration measured by spectrophotometric technique (mg/dl)
1. Sample-1 2. Sample-2 3. Sample-3 4. Sample-4 (67 mg/dl natural urea presence detected by the biosensor + 20 mg/dl added urea) 5. Sample-5 (48 mg/dl natural urea presence detected by the biosensor + 30 mg/dl added urea)
23.4 54.9 30.7 84.6
24.0 55.5 31.4 86.3
serum, and urine for clinical and food analysis. Probably the size exclusion and/or ion exclusion capabilities of the unique PCS + PEI matrix might have resulted in the suppression of the interferents. The DMM is also known to prevent the interference of Na+ and K+ ions towards urea estimation . 3.3. Urea biosensor’s applicability to milk samples In the present investigation, the raw milk samples of cows were collected from the cattle farms situated around the countryside. The samples were all skimmed in the laboratory by centrifugation and then were subjected to urea detection by the above developed urea biosensor. A few samples of milk were deliberately mixed with known amounts of urea to test the efﬁcacy of the sensor system. The urea concentrations detected by the present biosensor system were compared with the concentrations obtained by the spectrophotometric technique. The results obtained are summarized in Table 1. The table shows clearly that the fabricated enzyme based biosensors are capable of detecting urea presence in milk samples in general and more importantly, the biosensors can detect if there is any adulteration in milk with urea. Since urea is one of the ingredients in the manufacturing of ‘Synthetic Milk’ [8,18–20,48], the present ammonium ion sensitive based and urease immobilized screen printed biosensor system can detect the urea levels and the adulteration of milk. Milk samples with and without added urea were preserved with 0.2–0.4% of the commonly used preservative of hydrogen peroxide. The samples were stored at 4 ◦ C in the refrigerator overnight and then investigated to observe for the changes in urea concentrations utilizing the DMM based urea biosensor. The initial results indicated that there was no signiﬁcant change in the urea contents of the samples, similar to the observations made by Bector et al.  by their chemical method. However, further work is still in progress in this aspect and also with other preservatives. Sharma et al.  recently reported a method for the estimation of urea in milk using a standard ammonia electrode. The present biosensor system adopted low cost and disposable screen printed ammonium ion sensitive electrodes and the concentrations of the urea detected were found to be in good agreement with those obtained by the sophisticated and expensive spectrophotometric technique. The average urea levels in skimmed milk were generally found to be higher than those in the whole milk samples. Also, the preliminary results of the present investigation indicated that the urea concentrations present in the buffalo milk samples were lower than that of cow milk samples. 4. Conclusions
Fig. 8. Inﬂuence of Na+ and K+ concentrations on the response of urea biosensor.
A potentiometric NH4 + ion sensitive electrode and Ag/AgCl reference electrode system has been developed by a simple screen printing technology and characterized. The ion sensitive polymer
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matrix membrane was formed in the presence of an additional electrochemically inert ﬁlter paper, with silver coating on one side, leading to a DMM technology based transducer. A detection limit of 3.0 × 10−5 mol/L could be observed with a response time in the range of a few seconds against the reference electrode for the test additions from ammonium chloride stock solution. To obtain the urea biosensor, the urease enzyme has been immobilized using a unique PCS + PEI polymer matrix. Subsequently, a potentiometric urea sensitive biosensor using the NH4 + ion sensitive disposable electrode in DMM technology as the transducer could also be developed. The developed urea biosensor responded rapidly and in a stable manner to the changes in the test concentrations of urea with a detection limit of 2.5 × 10−5 mol/L. The biosensor exhibited a good linear least squares ﬁt (linear regression analysis) with a good correlation coefﬁcient (R2 = 0.989, n = 8). The noise range amounted to about 1.0 mV. The maximum buffer solution working temperature has been found to be 313 K. The present double matrix membrane sensor system has been found to offer a reasonably effective barrier to the interferents like Na+ and K+ ions. The studies showed a reasonably good storage stability and shelf life. The designed urea biosensor system has, then, been tested for the detection of urea levels in the real samples of milk. The results indicated a close agreement between the urea levels detected in the milk samples by the present biosensor system with those obtained by the spectrophotometric technique. The reported biosensor system, the authors believe, should also be able to detect any possible contamination of milk by synthetic milk of a kind.
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I.L. Kothari received his M.Sc. degree in botany in 1968 from M.S. University of Baroda, Vadodara, Gujarat and later, a Ph.D. degree in Botany from Sardar Patel University, Vallabh Vidyanagar, Gujarat, in 1974. Since 1997 he has been a professor at the Department of Biosciences, Sardar Patel University and has guided doctoral theses in the ﬁelds of botany, microbiology and biotechnology. His current areas of research include plant morphogenesis, environmental & agricultural microbiology and biosensors.
                                    
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Biographies U.B. Trivedi received his M.Sc. degree in electronics in 1999 from Sardar Patel University, Vallabh Vidyanagar, Gujarat. He later secured a Ph.D. in electronics in 2007 from the same university, for his research work on the development of biosensors for food and clinical analyses. He is presently working as a senior technician in the Department of Electronics, Sardar Patel University. His current areas of research interest are biosensors, bioelectronics and instrumental methods of analysis. D. Lakshminarayana received his M.Sc. and Ph.D. degrees in Physics from Sardar Patel University, Vallabh Vidyanagar, Gujarat in 1976 and 1983 respectively. He is presently working as a professor in the Department of Electronic Science, Sardar Patel University and has guided doctoral theses in the ﬁelds of semiconductor thin ﬁlms and biosensors. He has several research publications in the ﬁelds of semiconductor single crystal growth, thin ﬁlm science, gas sensors and biosensors. His current research interests are mainly connected with electronic materials science and devices, biosensors and nanoscience.
N.G. Patel received his M.Sc. degree in physics in 1978 from Sardar Patel University, Vallabh Vidyanagar, Gujarat, India and later, a Ph.D. degree in physics from the same university in 1984. He functioned as the professor and head of the Department of Electronics, Sardar Patel University, Vallabh Vidyanagar during the period 1990–2002. He later moved to University of North Florida, USA and is presently associated with Department of Chemistry and Physics as a faculty. His areas of research interest are in the ﬁelds of semiconductor thin ﬁlm devices, instrumentation, gas sensors and biosensors. H.N. Kapse received his M.Sc. degree in electronics in 1999 from Sardar Patel University, Vallabh Vidyanagar, Gujarat. He later secured a Ph.D. in electronics in 2004 from the same university. He is presently working as a lecturer in instrumentation at the Institute of Science and Technology for Advanced Studies and Research (ISTAR), Vallabh Vidyanagar, Gujarat. His current research interests include gas sensors and biosensors. K.K. Makhija received his M.Sc. degree in physics in 1968 from the Vikram University, Ujjain (M.P.) and later, a Ph.D. degree in electronics from the Sardar Patel University, Vallabh Vidyanagar, Gujarat in 1996. Since 1996 he worked as a reader at Department of Electronics, Sardar Patel University, Vallabh Vidyanagar, before his retirement from university service in October 2008. He is currently associated with the Department of Electronics, Sardar Patel University as a visiting faculty. His current areas of research include semiconductor thin ﬁlm active and passive devices, gas sensors and biosensors. P.B. Patel received his M.Sc. degree in electronics in 1993 from Sardar Patel University, Vallabh Vidyanagar, Gujarat. He is presently working as a senior lecturer in the Department of Electronic Science, Sardar Patel University. His current research interests include electronic materials science and devices, sensors and microprocessor interfacing circuits. C.J. Panchal received his M.Sc. degree in applied physics in 1990 from M.S. University of Baroda, Vadodara, Gujarat and Ph.D. degree in electronics in 1995 from Sardar Patel University, Vallabh Vidyanagar, Gujarat. He is presently working as a reader in the Applied Physics Department of M.S. University of Baroda, Vadodara. He worked extensively in the areas of semiconductor thin ﬁlm active and passive devices. His current areas of research include semiconducting thin ﬁlm hetero-structures, sensors and development of high power diode lasers.