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stearic acid Langmuir–Blodgett films for application to urea biosensor
Biosensors and Bioelectronics 17 (2002) 697 /703 www.elsevier.com/locate/bios
Immobilization of urease on poly(N-vinyl carbazole)/stearic acid Langmuir Blodgett films for application to urea biosensor /
Rahul Singhal a,b, Anamika Gambhir a, M.K. Pandey a, S. Annapoorni b, B.D. Malhotra a,* a
Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012, India b Department of Physics and Astrophysics, University of Delhi, Delhi 110007, India Received 30 January 2001; received in revised form 26 November 2001; accepted 11 January 2002
Abstract Urease was immobilized in mixed monolayers of poly(N -vinyl carbazole) (PNVK) and stearic acid (SA) formed at an air /water interface. The monolayers were transferred onto indium-tin-oxide (ITO) coated glass plates using Langmuir /Blodgett (LB) film deposition technique. Urease immobilized on PNVK/SA LB films, characterized using FTIR and UV /visible spectroscopy, was found to exhibit increased stability over a wide pH (6.5 /8.5) and temperature (25 /50 8C) range. Potentiometric measurements on these urease electrodes were carried out using an ammonium ion analyzer. Two values for K app m were obtained at lower and higher concentrations of substrate urea. # 2002 Published by Elsevier Science B.V. Keywords: Langmuir /Blodgett films; Enzyme; Urease; Immobilization; Urea biosensor
1. Introduction The metabolic function of kidney is reflected in the concentration of organic compounds such as urea in blood or urine. Therefore, the estimation of urea is frequently performed in the medical field. The use of urease as a biocatalyst for the development of urea biosensor has attracted continued interest from biochemical and clinical analysts. The general principle for fabricating a urea biosensor is based on immobilization of urease onto a membrane or support in which urea is catalytically converted into ammonium and bicarbonate ions: (NH 2 )2 CO3H2 O
UREASE
0
2NH (1) 4 OH HCO 3
For monitoring the enzymatic products, various techniques such as spectrophotometry, potentiometry (Senillou et al., 1999; Koncki et al., 2000), flow injection technique (Milardovic et al., 1999), coulometry (Lobanov et al., 1995) and amperometry (Malitesta et al.,
* Corresponding author. Tel.: 91-11-582-4620; fax: 91-11-5852678. E-mail address: [email protected] (B.D. Malhotra).
1990) have been proposed. Table 1 lists the characteristics of some urea biosensors. Potentiometric and amperometric methods of determination are however frequently used. Enzymatic urea electrodes are the most studied potentiometric biosensors (Mascini et al., 1983). Some of the potentiometric transducers used to construct urea biosensors are ion-selective membrane electrodes (H or NH4) or gas potentiometric electrodes (NH3 or CO2), which maintain the combination of these potentiometric transducers with enzyme-based membranes. More recently, enzyme-catalyzed polymer transformation with electrochemical ac-impedance detection (Ho et al., 1999) has been employed for the measurement of urea. An essential prerequisite for the development of a biosensor is the immobilization of a biomolecule by a method wherein the enzyme remains active. Immobilization of enzyme/protein has been attempted in conducting polymeric matrices which are currently drawing much attention. Conducting polymers possess the ability to bind oppositely charged complex entities in their oxidized conducting states and release them in their neutral insulating states (Hailin and Wallace, 1989). Immobilization of a biomolecule in conducting polymers can be achieved by several techni-
0956-5663/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 9 5 6 - 5 6 6 3 ( 0 2 ) 0 0 0 2 0 - 9
R. Singhal et al. / Biosensors and Bioelectronics 17 (2002) 697 /703
698 Table 1 Characteristics of some urea biosensors Matrix
Method
Linearity range (mM)
Response time (min)
Shelf-life (days)
Reference
PVC ammonium electrode Screen printed electrode Porous PVC Polypyrrole PNVK/SA/urease LB film
Amperometry Conductometry Potentiometry FIA Potentiometry
15 /80 5 /25 0.16 /12.5 0.01 /30 0.5 /68
1 1 10 4 2
12 /14 / 120 / 35
Campanella et al. (1990) Ho et al. (1999) Hirose et al. (1983) Osaka et al. (1999) Present work
ques. These include physical adsorption, electrochemical entrapment, chemical cross-linking and covalent coupling etc. For optimum activity of an immobilized enzyme, it is important that its active sites are oriented towards the free surface of the immobilizing matrix. Morphological studies have revealed that the surface of a conducting polymer is critically dependent on the method of preparation (Diaz and Bargon, 1986) and plays an important role in the effective immobilization of a desired enzyme (Ramanathan et al., 1994, 1995a,b). Fig. 1 shows a schematic for the biosensor configuration. Langmuir /Blodgett technique for monolayer deposition is known to facilitate the desired orientation of a biomolecule (Fig. 2). Only a few reports have appeared on this subject. Formation of LB films of lipid /enzyme mixtures by using a conventional constant-perimeter barrier trough has been described (Sriyudthsak et al., 1988; Wan et al., 2000; Choi et al., 1998; Barmin et al., 1994; Zhu et al., 1989; Dubrovsky et al., 1994; GirardEgrot et al., 1997; Paddeu et al., 1995). Immobilization of Glucose oxidase (GOD) on a phospholipid analogous vinyl polymer has been recently reported by Yasuzawa et al. (2000). LB films of polyemeraldine base of polyaniline with GOD entrapped between the layers was deposited on indium-tin-oxide (ITO) glass plate by Ramanathan et al. (1995b). Guiomar et al. (1997) found that glucose oxidase can be immobilized on LB films of cellulose acetate propionate deposited on a self-assembled coated substrate. In the present paper we report the studies on technical development of urea biosensor based on the immobilization of urease within an LB film of poly(N -vinyl carbazole)/stearic acid (PNVK/SA). When the immobilized urease is placed in a solution containing urea, urea
Fig. 1. Schematic of a biosensor.
diffuses into the PNVK/SA LB film of the immobilized enzyme. The enzyme urease then catalyzes the decomposition of urea into ammonium ions. The potentiometric response as a result of hydrolysis of urea has been measured by an ammonium ion analyzer.
2. Materials and methods Poly(N -vinyl carbazole) (Aldrich, USA) was used as received. Stearic acid was recrystallized three times using acetone prior to being used. Monolayers of PNVK and stearic acid dissolved in chloroform were spread onto the aqueous subphase containing 2 /10 4 M CdCl2. The resulting solution was sonicated for about 30 min prior to being dispensed onto the subphase of the Joyce/Loebl LB (Model Trough 4) trough. Deionized water from a Millipore water purification system was used as the subphase. The pressure/area (p /A ) isotherms of PNVK/SA system were obtained at a compression rate of 0.5 cm/min. LB film deposition was carried out at a surface pressure of 30 mN/m onto the ITO coated glass plates at pH 7. The temperature of the subphase was maintained at 27 8C using a refrigerated water circulator (BioRad E4870). The speed of the dipping head was 5 mm/min. For entrapment about 7.35 mg of urease was mixed with 0.5 ml solution of stearic acid and PNVK in choloroform (1 IU/ml) just before LB deposition. The solution was carefully loaded onto the surface of deionized water (Millipore 10 RTS) in the LB trough with a microliter syringe. For physical adsorption a stock solution of urease was made using 1.46 mg/100 ml of phosphate buffer to give a concentration of 1 IU/ml. Different aliquots from the stock were applied to the LB films of PNVK deposited on ITO glass plates. The activity of urease in the adsorbed and entrapped states was measured in a measuring cell (Fig. 3) containing phosphate buffer (0.01 M, pH 7.0) by an ammonium ion analyzer (AR 25, Fisher Scientific). The distance between ammonium ion sensitive electrode and PNVK/SA/UREASE electrode was 1 cm. Different concentrations of substrate (urea) were added in the presence of immobilized urease on ITO glass. The concentration of NH4 ion produced was measured in parts per million (ppm) and the
R. Singhal et al. / Biosensors and Bioelectronics 17 (2002) 697 /703
699
Fig. 2. Schematic of monolayers of PNVK/SA/UREASE obtained on a water surface: (a) expanded; (b) partially compressed; (c) closed packed molecules.
Fig. 3. Schematic of the measuring cell used for the estimation of urea concentration.
resulting change in potential was measured in millivolts (mV). The thermal stability of urease immobilized on PNVK/SA LB films was investigated by measuring the urease activity as a function of temperature by holding the film in a 1 ml phosphate buffer at varying temperature between 25 and 60 8C maintained in a hot water bath for about 10 min. The films were then tested for urease activity by the method described earlier. Leaching of urease from adsorbed and entrapped states on PNVK/SA LB films was monitored by placing the film in a 2 ml phosphate buffer solution and periodically withdrawing 100 ml of buffer for testing the presence of urease. Response studies of the PNVK/ SA/urease LB films were conducted as a function of pH and concentration of urea.
3. Results and discussion 3.1. Characteristics of LB films of PNVK/stearic acid Fig. 4 shows the pressure/area isotherm of PNVK/ SA mixed monolayer obtained at 30 8C. It can be seen that the gas /liquid phase transition occurs at about a surface pressure of 2.6 mN/m at a molecular area of 41.3 ˚ 2. The liquid /solid phase transition occurs at a surface A ˚ 2. The pressure of 26 mN/m at a molecular area of 31 A compressibility of gas to liquid phase transition changes from 0.03 to 0.02 m/mN and for liquid to solid phase transition, it changes from 0.008 to 0.005 m/mN.
Fig. 4. Pressure /area isotherm of mixed monolayers of PNVK and SA at a subphase temperature of 30 8C and pH 7.0.
3.2. FTIR spectra of urease immobilized PNVK/SA LB films FTIR studies conducted on urease entrapped PNVK/ SA LB films show (Fig. 5) bands at 795 and 840 cm 1 ascribed to C /H bending of 1,2,4-trisubstitution indicating that carbazole ring is intact in the polymer. The bands at 693 and 745 cm 1 have been ascribed to C /H bending of 1,2-disubstituted rings in PNVK. The peak at 1230 cm 1 has been attributed to C /H stretching. The absorbance at 722 cm 1 is attributed to the intrachain dication transition. These results are in agreement with the values reported in literature (Verghese, 1997). The peptide groups of urease (protein) backbone cause five vibrations in the CONH plane and five outof-plane (CONH) vibrations. A strong band observed at about 3300 cm 1 and a less stronger band at about 3100 cm 1 have been assigned to amide A and amide B bands, respectively. The amide A band is caused by the NH stretching vibration whereas the amide B band arises due to the first overtone of the amide II vibration, that becomes intensified by Fermi resonance with the amide A vibration. At about 1650 cm 1 the amide I band has been understood to arise due to C /O stretching vibrations (Belanger et al., 1989). Suppressed C /N stretching and NH bending vibrations at 1540 cm 1 can be assigned to partial shielding effect of deposited monolayers of PNVK and stearic acid. The presence of vibrations corresponding to a-helix and bpleated sheet structure indicates that urease immobilized
700
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Fig. 5. FTIR spectra of urease entrapped in LB film of PNVK and SA: (a) active urease (inset shows the expended region 600 /800 cm 1); (b) denatured urease.
on LB films of PNVK/SA has not lost its secondary structure.
3.3. Response characteristics
3.3.1. Enzyme loading Fig. 6 shows the results of experiments carried out for estimating the minimum enzyme units required for activity. The experiments for assessing minimum enzyme units required for activity was carried out using 68 mM of urea substrate. It is clear from figure that no detectable response was obtained below 5 IU of urease activity. However, as the concentration approaches 10 IU the enzyme remains unsaturated even after 15 min of the reaction. Thus this concentration was used for further experiments. The increased requirement for enzyme can be attributed to the fact that urease was dissolved in chloroform for entrapment in LB films of PNVK/SA.
Fig. 6. Effect of enzyme concentration immobilized per cm2 of PNVK/ SA LB films on activity at a urea concentration of 93 mM in phosphate buffer at pH 7.0.
3.3.2. pH profile of urease immobilized PNVK/SA LB films Fig. 7 shows optimum pH requirement of free and immobilized urease at 30 8C at a substrate concentration of 10 mM. The pH optimum of immobilized urease is comparable to that for free urease which shows a pH optimum of 7.2. It can be noted that the fall in activity in the immobilized state is more gradual on both sides of optimum pH as compared to free urease. A broadening of profile towards both acidic and alkaline range was observed implying that the enzyme becomes less sensitive to pH changes when it is immobilized. Such an
Fig. 7. Effect of pH on the activity of urease (10 IU/cm2) in (") solution and in (j) immobilized PNVK/SA LB films.
R. Singhal et al. / Biosensors and Bioelectronics 17 (2002) 697 /703
effect is anticipated when neither proton partitioning nor diffusion limitation is present. It appears that the effect is due to substrate diffusion limitation alone. An immobilized enzyme preparation that has a high enzyme loading (large quantity of enzyme activity per unit polymer) may be subject to substrate diffusion limitation. Since in LB films the enzyme is deposited symmetrically in monolayers along with the polymer, the enzyme present in the first few PNVK/SA monolayers is likely to come into contact with the substrate. If the enzyme has high intrinsic specific activity, which is the case when the enzyme is immobilized in the PNVK/ SA LB monolayers, the substrate concentration gradient through the particle will be steep and consequently the substrate may not penetrate to the center of the immobilized enzyme (urease) molecules. If some constraint like a change in pH is subsequently applied leading to the reduced enzyme activity, the substrate concentration gradient will become less steep, thus allowing the substrate to penetrate further into the immobilized enzyme molecules. Effectively the enzyme concentration increases resulting in the availability of increased number of urease molecules to the substrate. These two antagonistically factors tend to moderate the effect of pH change (Trevan, 1980). The fact that the immobilized enzyme is more stable in varying pH has also been reported by other researchers (Ramanathan et al., 1997; Gambhir et al., in press, Carr and Bowers, 1980). 3.3.3. Determination of enzyme activity and substrate kinetics A good linear correlation between potential sensed by an ammonium ion selective electrode and urea concentration was obtained in the range from 0.5 to 93 mM when this electrode was used. Two linear ranges were obtained viz., 0.5 /10 and 10 /68 mM (Fig. 8a and b). Lineweaver/Burke plots (Fig. 9) for the immobilized urease gave two values of K app m . Sensitivity was higher in the 10/68 mM range and K app m was 9 mM. The value of K app m for the lower range was justifiably higher (30 mM). Various reports available on the effect of immobilization on enzyme kinetic parameters investigating the relationship between substrate concentration (S ) and activity (V ) over a narrow range of substrate concentration indicate the reciprocal plots (Lineweaver /Burke) as linear and a single K app is obtained by extrapolating m the plot. The two K app values obtained in this report m suggest that K app is perhaps dependent upon the m substrate concentration used. The higher Km at low substrate concentration can be attributed to the higher substrate diffusion limitation at lower substrate concentrations. Besides this, partitioning effects will be greater at lower substrate concentrations. Both these factors are likely to influence the apparent ease with which enzyme and substrate can associate and will thus affect K app m .
701
Fig. 8. (a) Response curve of PNVK/SA/urease LB films as a function of urea concentration (0 /10 mM). (b) Response curve of PNVK/SA/ urease LB films as a function of urea concentration (0 /93 mM).
Fig. 9. Lineweaver /Burke plots for immobilized urease on PNVK/SA LB films: (a) 1 for 0 /10 mM urea; (b) 10 /68 mM urea concentration.
3.3.4. Sensitivity, repeatability and detection limit The response time of the urease/PNVK/SA LB electrode was about 2 min. The detection limit and the sensitivity for this urease electrode have been experimentally determined as 5 mM and 10 mV/mM, respectively. This urease electrode could be used about 10 times. 3.3.5. Storage and stability The observed leaching of about 5% and shelf-life of about 5 weeks has been attributed to very thin and stable architecture of PNVK/SA LB films. It is important to point out here that urease immobilized on 60/70 monolayers of PNVK/SA obtained by the physical
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R. Singhal et al. / Biosensors and Bioelectronics 17 (2002) 697 /703
References
Fig. 10. Effect of temperature (25 /50 8C) on the activity of urease immobilized on PNVK/SA LB films in phosphate buffer at pH 7.0.
adsorption technique shows high leaching (75%) and a short shelf-life (2 days). A very thin and smooth surface of LB film is perhaps unable to retain the physically adsorbed enzyme on the surface. A higher temperature (41 8C) optimum (Fig. 10) was obtained for PNVK/SA/urease electrode, which could perhaps be due to higher intermolecular interactions. About 75% activity was recorded at 45 8C after which a steady decrease was observed which is likely to be due to the denaturation of protein at higher temperatures. This decrease in activity was reversible until 49 8C indicating that the enzyme perhaps attains its near original conformation after returning to normal temperature. The value of activation energy before and after critical temperature calculated from the Arrhenius plot was found to be 4027 and 7595 cal, respectively.
4. Conclusions It can be seen that stable PNVK/SA monolayers can be formed at the air /water interface. Further, urease can be immobilized onto these PNVK/SA LB monolayers. These PNVK/SA/UREASE monolayers transferred onto ITO coated glass plates can be utilized for urea sensing. The detection limit and sensitivity of these electrodes was found to be 5 mM and 10 mV/mM, respectively. The shelf-life of these electrodes was found to be 5 weeks at 4 8C.
Acknowledgements We are grateful to Dr K. Lal, Director, NPL for his interest in this work. The financial support received under the DST funded project (SP/S2/M-52/96) and the Indo-Polish project (INT/POL/P015/2000) are gratefully acknowledged. A.G. and R.S. are grateful to CSIR for award of Research Associateship (RA) and Senior Research Fellowship (SRF), respectively.
Belanger, D., Nadreau, J., Fortier, G. 1989. Electrochemistry of polypyrrole GOD electrode. J. Electroanal. Chem. 274, 143 /155. Barmin, A.V., Eremenko, A.V., Moskvitina, T.A., Kurochkin, I.N. 1994. Langmuir /Blodgett films of monoamine oxidase with amphiphilic polyelectrolytes. Study of catalytic properties of the enzyme. Biol. Membr. 7 (6), 633 /640. Campanella, L., Mazzei, F., Sammartino, M.P., Tommassetti, M. 1990. New enzyme sensor for urea and creatinine analysis. Bioelectrochem. Bioenerg. 23, 195 /202. Carr, P.W., Bowers, L.D. 1980. Immobilized Enzymes in Analytical and Clinical Chemistry. Wiley, New York. Choi, J.W., Min, J., Jung, J.W., Rhee, H.W., Lee, W.H. 1998. Fiberoptic biosensor for the detection of organophosphorus compound using AchE-immobilized viologen LB films. Thin Solid Films 327 / 329, 676 /680. Diaz, A.F., Bargon, J. 1986. Electrochemical synthesis of conducting polymers. In: Skotheim, T.P. (Ed.), Handbook of Conducting Polymers, vol. 1. Marcel Dekker, New York. Dubrovsky, T., Vakula, S., Nicolini, C. 1994. Preparation and immobilization of Langmuir /Blodgett films of antibodies conjugated to enzymes for potentiometric sensor applications. Sens. Actuators B 22, 69 /73. Gambhir, A., Gerard, M., Mulchandani, A.K., Malhota, B.D. Coimmmobilization of urease and glutamate dehydrogenase on electrochemically prepared polypyrole-polyvinyl sulphonate films. Appl. Biochem. & Biotechnol. 96 (1 /3), 249 /257. Girard-Egrot, A.P., Morelis, R.M., Coulet, P.R. 1997. Bioactive nanostructure with glutamate dehydrogenase associated with LB films: protecting role of the enzyme molecules on the structural lipidic organization. Thin Solid Films 292, 282 /289. Guiomar, A.J., Evans, S.D., Guthrie, J.T. 1997. Immobilization of glucose oxidase onto Langmuir /Blodgett ultrathin film of a cellulose derivative deposited on a self-assembled monolayer. Supramol. Sci. 4, 279 /291. Hailin, G., Wallace, G.G. 1989. Electrosynthesis of chromatographic stationary phases. Anal. Chem. 61, 198 /201. Hirose, S., Hayashi, M., Tamura, N., Kamidate, T., Karube, I., Suzuki, S. 1983. Determination of urea in blood serum with use of immobilized urease and a microwave cavity ammonia monitor. Anal. Chim. Acta 151, 377 /382. Ho, W.O., Krause, S., McNeil, C.J., Pritchard, J.A., Armstrong, R.D., Athey, D., Rawson, K. 1999. Electrochemical sensor for measurement of urea and creatinine in serum based on ac impedance measurement of enzyme catalyzed polymer transformation. Anal. Chem. 71, 1940 /1946. Koncki, R., Radomska, A., Glab, S. 2000. Potentiometric determination of dialysate urea nitrogen. Talanta 52, 13 /17. Lobanov, O.V., Dubrovsky, T.B., Savitsky, A.P., Akindinov, D.E., Alexeev, I.V., Savransky, V.V. 1995. Optical methods of investigation of the protein Langmuir /Blodgett films. Thin Solid Films 259, 85 /90. Malitesta, C., Palmisano, F., Torsi, L., Zambonin, P.G. 1990. Glucose fast-response amperometric sensor based on glucose oxidase immobilized in an electropolymerized poly(o -phenylenediamine) film. Anal. Chem. 62, 2735 /2740. Mascini, M., Iannello, M., Palleschi, G. 1983. Enzyme electrode with improved mechanical and analytical characteristics obtained by binding enzyme on nylon nets. Anal. Chim. Acta 146, 135 /148. Milardovic, S., Kruhak, I., Grabaric, B.S. 1999. Urea determination in FIA mode by a newly designed urease based biosensor. LRA 11, 266 /271. Osaka, T., Komaba, S., Fujino, Y., Matsuda, T., Satoh, I. 1999. High sensitivity flow injection analysis of urea using composite electro-
R. Singhal et al. / Biosensors and Bioelectronics 17 (2002) 697 /703 polymerized polypyrrole /polyion complex film. J. Electrochem. Soc. 146 (2), 615 /619. Paddeu, S., Fanigliulo, A., Lanzi, M., Dubrovsky, T., Nicolini, C. 1995. LB-based PAB immunosystem: activity of an immobilized urease monolayer. Sens. Actuators B 24/25, 876 /882. Ramanathan, K., Annapoorni, S., Malhotra, B.D. 1994. Application of polyaniline as a glucose biosensor. Sens Actuators B (Chemical) 21, 165 /169. Ramanathan, K., Sundaresan, N.S., Malhotra, B.D. 1995. Ion exchanged PPY based glucose biosensor: enhanced loading and response. Electroanalysis 7, 579 /582. Ramanathan, K., Ram, M.K., Malhotra, B.D., Murthy, A.S.N. 1995b. Application of polyaniline-Langmuir /Blodgett films as glucose biosensor. Mater. Sci. Eng. C3, 159 /163. Ramanathan, K., Kamalasanan, M.N., Malhotra, B.D., Pradhan, D.R., Chandra, S. 1997. J. Sol /Gel Sci. Technol. 10, 309 / 316. Senillou, A., Jaffrezic-Renault, A., Martelet, C., Cosnier, S. 1999. A miniaturized urea sensor based on the integration of both
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ammonium based urea enzyme field effect transistor and a reference field effect transistor in a single chip. Talanta 50, 219 / 226. Sriyudthsak, M., Yamagishi, H., Moriizumi, T. 1988. Enzyme immobilized Langmuir-Blodgett film for a biosensor. Thin Solid Films 160, 463 /469. Trevan, M.D. (Ed.),Immobilized Enzyme. Wiley, New York 1980., pp. 11 /53. Verghese, M.M., 1997. PhD Thesis, Jamia Millia Islamia, New Delhi, India. Wan, K., Chovelon, J.M., Jaffrezic, N., Enault, R. 2000. Enzyme octadecyamine Langmuir /Blodgett membranes for ENFET biosensor. Talanta 52 (4), 663 /670. Yasuzawa, M., Hashimoto, M., Fujii, S., Kunugi, A., Nakaya, T. 2000. Preparation of glucose sensors using the Langmuir /Blodgett technique. Sens. Actuators B 65, 241 /243. Zhu, D.G., Petty, M.C., Ancelin, H., Yarwood, J. 1989. On the formation of Langmuir /Blodgett films containing enzymes. Thin Solid Films 176, 151 /156.
Immobilization of urease on poly(N-vinyl carbazole)/stearic acid Langmuir Blodgett films for application to urea biosensor /
Rahul Singhal a,b, Anamika Gambhir a, M.K. Pandey a, S. Annapoorni b, B.D. Malhotra a,* a
Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012, India b Department of Physics and Astrophysics, University of Delhi, Delhi 110007, India Received 30 January 2001; received in revised form 26 November 2001; accepted 11 January 2002
Abstract Urease was immobilized in mixed monolayers of poly(N -vinyl carbazole) (PNVK) and stearic acid (SA) formed at an air /water interface. The monolayers were transferred onto indium-tin-oxide (ITO) coated glass plates using Langmuir /Blodgett (LB) film deposition technique. Urease immobilized on PNVK/SA LB films, characterized using FTIR and UV /visible spectroscopy, was found to exhibit increased stability over a wide pH (6.5 /8.5) and temperature (25 /50 8C) range. Potentiometric measurements on these urease electrodes were carried out using an ammonium ion analyzer. Two values for K app m were obtained at lower and higher concentrations of substrate urea. # 2002 Published by Elsevier Science B.V. Keywords: Langmuir /Blodgett films; Enzyme; Urease; Immobilization; Urea biosensor
1. Introduction The metabolic function of kidney is reflected in the concentration of organic compounds such as urea in blood or urine. Therefore, the estimation of urea is frequently performed in the medical field. The use of urease as a biocatalyst for the development of urea biosensor has attracted continued interest from biochemical and clinical analysts. The general principle for fabricating a urea biosensor is based on immobilization of urease onto a membrane or support in which urea is catalytically converted into ammonium and bicarbonate ions: (NH 2 )2 CO3H2 O
UREASE
0
2NH (1) 4 OH HCO 3
For monitoring the enzymatic products, various techniques such as spectrophotometry, potentiometry (Senillou et al., 1999; Koncki et al., 2000), flow injection technique (Milardovic et al., 1999), coulometry (Lobanov et al., 1995) and amperometry (Malitesta et al.,
* Corresponding author. Tel.: 91-11-582-4620; fax: 91-11-5852678. E-mail address: [email protected] (B.D. Malhotra).
1990) have been proposed. Table 1 lists the characteristics of some urea biosensors. Potentiometric and amperometric methods of determination are however frequently used. Enzymatic urea electrodes are the most studied potentiometric biosensors (Mascini et al., 1983). Some of the potentiometric transducers used to construct urea biosensors are ion-selective membrane electrodes (H or NH4) or gas potentiometric electrodes (NH3 or CO2), which maintain the combination of these potentiometric transducers with enzyme-based membranes. More recently, enzyme-catalyzed polymer transformation with electrochemical ac-impedance detection (Ho et al., 1999) has been employed for the measurement of urea. An essential prerequisite for the development of a biosensor is the immobilization of a biomolecule by a method wherein the enzyme remains active. Immobilization of enzyme/protein has been attempted in conducting polymeric matrices which are currently drawing much attention. Conducting polymers possess the ability to bind oppositely charged complex entities in their oxidized conducting states and release them in their neutral insulating states (Hailin and Wallace, 1989). Immobilization of a biomolecule in conducting polymers can be achieved by several techni-
0956-5663/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 9 5 6 - 5 6 6 3 ( 0 2 ) 0 0 0 2 0 - 9
R. Singhal et al. / Biosensors and Bioelectronics 17 (2002) 697 /703
698 Table 1 Characteristics of some urea biosensors Matrix
Method
Linearity range (mM)
Response time (min)
Shelf-life (days)
Reference
PVC ammonium electrode Screen printed electrode Porous PVC Polypyrrole PNVK/SA/urease LB film
Amperometry Conductometry Potentiometry FIA Potentiometry
15 /80 5 /25 0.16 /12.5 0.01 /30 0.5 /68
1 1 10 4 2
12 /14 / 120 / 35
Campanella et al. (1990) Ho et al. (1999) Hirose et al. (1983) Osaka et al. (1999) Present work
ques. These include physical adsorption, electrochemical entrapment, chemical cross-linking and covalent coupling etc. For optimum activity of an immobilized enzyme, it is important that its active sites are oriented towards the free surface of the immobilizing matrix. Morphological studies have revealed that the surface of a conducting polymer is critically dependent on the method of preparation (Diaz and Bargon, 1986) and plays an important role in the effective immobilization of a desired enzyme (Ramanathan et al., 1994, 1995a,b). Fig. 1 shows a schematic for the biosensor configuration. Langmuir /Blodgett technique for monolayer deposition is known to facilitate the desired orientation of a biomolecule (Fig. 2). Only a few reports have appeared on this subject. Formation of LB films of lipid /enzyme mixtures by using a conventional constant-perimeter barrier trough has been described (Sriyudthsak et al., 1988; Wan et al., 2000; Choi et al., 1998; Barmin et al., 1994; Zhu et al., 1989; Dubrovsky et al., 1994; GirardEgrot et al., 1997; Paddeu et al., 1995). Immobilization of Glucose oxidase (GOD) on a phospholipid analogous vinyl polymer has been recently reported by Yasuzawa et al. (2000). LB films of polyemeraldine base of polyaniline with GOD entrapped between the layers was deposited on indium-tin-oxide (ITO) glass plate by Ramanathan et al. (1995b). Guiomar et al. (1997) found that glucose oxidase can be immobilized on LB films of cellulose acetate propionate deposited on a self-assembled coated substrate. In the present paper we report the studies on technical development of urea biosensor based on the immobilization of urease within an LB film of poly(N -vinyl carbazole)/stearic acid (PNVK/SA). When the immobilized urease is placed in a solution containing urea, urea
Fig. 1. Schematic of a biosensor.
diffuses into the PNVK/SA LB film of the immobilized enzyme. The enzyme urease then catalyzes the decomposition of urea into ammonium ions. The potentiometric response as a result of hydrolysis of urea has been measured by an ammonium ion analyzer.
2. Materials and methods Poly(N -vinyl carbazole) (Aldrich, USA) was used as received. Stearic acid was recrystallized three times using acetone prior to being used. Monolayers of PNVK and stearic acid dissolved in chloroform were spread onto the aqueous subphase containing 2 /10 4 M CdCl2. The resulting solution was sonicated for about 30 min prior to being dispensed onto the subphase of the Joyce/Loebl LB (Model Trough 4) trough. Deionized water from a Millipore water purification system was used as the subphase. The pressure/area (p /A ) isotherms of PNVK/SA system were obtained at a compression rate of 0.5 cm/min. LB film deposition was carried out at a surface pressure of 30 mN/m onto the ITO coated glass plates at pH 7. The temperature of the subphase was maintained at 27 8C using a refrigerated water circulator (BioRad E4870). The speed of the dipping head was 5 mm/min. For entrapment about 7.35 mg of urease was mixed with 0.5 ml solution of stearic acid and PNVK in choloroform (1 IU/ml) just before LB deposition. The solution was carefully loaded onto the surface of deionized water (Millipore 10 RTS) in the LB trough with a microliter syringe. For physical adsorption a stock solution of urease was made using 1.46 mg/100 ml of phosphate buffer to give a concentration of 1 IU/ml. Different aliquots from the stock were applied to the LB films of PNVK deposited on ITO glass plates. The activity of urease in the adsorbed and entrapped states was measured in a measuring cell (Fig. 3) containing phosphate buffer (0.01 M, pH 7.0) by an ammonium ion analyzer (AR 25, Fisher Scientific). The distance between ammonium ion sensitive electrode and PNVK/SA/UREASE electrode was 1 cm. Different concentrations of substrate (urea) were added in the presence of immobilized urease on ITO glass. The concentration of NH4 ion produced was measured in parts per million (ppm) and the
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Fig. 2. Schematic of monolayers of PNVK/SA/UREASE obtained on a water surface: (a) expanded; (b) partially compressed; (c) closed packed molecules.
Fig. 3. Schematic of the measuring cell used for the estimation of urea concentration.
resulting change in potential was measured in millivolts (mV). The thermal stability of urease immobilized on PNVK/SA LB films was investigated by measuring the urease activity as a function of temperature by holding the film in a 1 ml phosphate buffer at varying temperature between 25 and 60 8C maintained in a hot water bath for about 10 min. The films were then tested for urease activity by the method described earlier. Leaching of urease from adsorbed and entrapped states on PNVK/SA LB films was monitored by placing the film in a 2 ml phosphate buffer solution and periodically withdrawing 100 ml of buffer for testing the presence of urease. Response studies of the PNVK/ SA/urease LB films were conducted as a function of pH and concentration of urea.
3. Results and discussion 3.1. Characteristics of LB films of PNVK/stearic acid Fig. 4 shows the pressure/area isotherm of PNVK/ SA mixed monolayer obtained at 30 8C. It can be seen that the gas /liquid phase transition occurs at about a surface pressure of 2.6 mN/m at a molecular area of 41.3 ˚ 2. The liquid /solid phase transition occurs at a surface A ˚ 2. The pressure of 26 mN/m at a molecular area of 31 A compressibility of gas to liquid phase transition changes from 0.03 to 0.02 m/mN and for liquid to solid phase transition, it changes from 0.008 to 0.005 m/mN.
Fig. 4. Pressure /area isotherm of mixed monolayers of PNVK and SA at a subphase temperature of 30 8C and pH 7.0.
3.2. FTIR spectra of urease immobilized PNVK/SA LB films FTIR studies conducted on urease entrapped PNVK/ SA LB films show (Fig. 5) bands at 795 and 840 cm 1 ascribed to C /H bending of 1,2,4-trisubstitution indicating that carbazole ring is intact in the polymer. The bands at 693 and 745 cm 1 have been ascribed to C /H bending of 1,2-disubstituted rings in PNVK. The peak at 1230 cm 1 has been attributed to C /H stretching. The absorbance at 722 cm 1 is attributed to the intrachain dication transition. These results are in agreement with the values reported in literature (Verghese, 1997). The peptide groups of urease (protein) backbone cause five vibrations in the CONH plane and five outof-plane (CONH) vibrations. A strong band observed at about 3300 cm 1 and a less stronger band at about 3100 cm 1 have been assigned to amide A and amide B bands, respectively. The amide A band is caused by the NH stretching vibration whereas the amide B band arises due to the first overtone of the amide II vibration, that becomes intensified by Fermi resonance with the amide A vibration. At about 1650 cm 1 the amide I band has been understood to arise due to C /O stretching vibrations (Belanger et al., 1989). Suppressed C /N stretching and NH bending vibrations at 1540 cm 1 can be assigned to partial shielding effect of deposited monolayers of PNVK and stearic acid. The presence of vibrations corresponding to a-helix and bpleated sheet structure indicates that urease immobilized
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Fig. 5. FTIR spectra of urease entrapped in LB film of PNVK and SA: (a) active urease (inset shows the expended region 600 /800 cm 1); (b) denatured urease.
on LB films of PNVK/SA has not lost its secondary structure.
3.3. Response characteristics
3.3.1. Enzyme loading Fig. 6 shows the results of experiments carried out for estimating the minimum enzyme units required for activity. The experiments for assessing minimum enzyme units required for activity was carried out using 68 mM of urea substrate. It is clear from figure that no detectable response was obtained below 5 IU of urease activity. However, as the concentration approaches 10 IU the enzyme remains unsaturated even after 15 min of the reaction. Thus this concentration was used for further experiments. The increased requirement for enzyme can be attributed to the fact that urease was dissolved in chloroform for entrapment in LB films of PNVK/SA.
Fig. 6. Effect of enzyme concentration immobilized per cm2 of PNVK/ SA LB films on activity at a urea concentration of 93 mM in phosphate buffer at pH 7.0.
3.3.2. pH profile of urease immobilized PNVK/SA LB films Fig. 7 shows optimum pH requirement of free and immobilized urease at 30 8C at a substrate concentration of 10 mM. The pH optimum of immobilized urease is comparable to that for free urease which shows a pH optimum of 7.2. It can be noted that the fall in activity in the immobilized state is more gradual on both sides of optimum pH as compared to free urease. A broadening of profile towards both acidic and alkaline range was observed implying that the enzyme becomes less sensitive to pH changes when it is immobilized. Such an
Fig. 7. Effect of pH on the activity of urease (10 IU/cm2) in (") solution and in (j) immobilized PNVK/SA LB films.
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effect is anticipated when neither proton partitioning nor diffusion limitation is present. It appears that the effect is due to substrate diffusion limitation alone. An immobilized enzyme preparation that has a high enzyme loading (large quantity of enzyme activity per unit polymer) may be subject to substrate diffusion limitation. Since in LB films the enzyme is deposited symmetrically in monolayers along with the polymer, the enzyme present in the first few PNVK/SA monolayers is likely to come into contact with the substrate. If the enzyme has high intrinsic specific activity, which is the case when the enzyme is immobilized in the PNVK/ SA LB monolayers, the substrate concentration gradient through the particle will be steep and consequently the substrate may not penetrate to the center of the immobilized enzyme (urease) molecules. If some constraint like a change in pH is subsequently applied leading to the reduced enzyme activity, the substrate concentration gradient will become less steep, thus allowing the substrate to penetrate further into the immobilized enzyme molecules. Effectively the enzyme concentration increases resulting in the availability of increased number of urease molecules to the substrate. These two antagonistically factors tend to moderate the effect of pH change (Trevan, 1980). The fact that the immobilized enzyme is more stable in varying pH has also been reported by other researchers (Ramanathan et al., 1997; Gambhir et al., in press, Carr and Bowers, 1980). 3.3.3. Determination of enzyme activity and substrate kinetics A good linear correlation between potential sensed by an ammonium ion selective electrode and urea concentration was obtained in the range from 0.5 to 93 mM when this electrode was used. Two linear ranges were obtained viz., 0.5 /10 and 10 /68 mM (Fig. 8a and b). Lineweaver/Burke plots (Fig. 9) for the immobilized urease gave two values of K app m . Sensitivity was higher in the 10/68 mM range and K app m was 9 mM. The value of K app m for the lower range was justifiably higher (30 mM). Various reports available on the effect of immobilization on enzyme kinetic parameters investigating the relationship between substrate concentration (S ) and activity (V ) over a narrow range of substrate concentration indicate the reciprocal plots (Lineweaver /Burke) as linear and a single K app is obtained by extrapolating m the plot. The two K app values obtained in this report m suggest that K app is perhaps dependent upon the m substrate concentration used. The higher Km at low substrate concentration can be attributed to the higher substrate diffusion limitation at lower substrate concentrations. Besides this, partitioning effects will be greater at lower substrate concentrations. Both these factors are likely to influence the apparent ease with which enzyme and substrate can associate and will thus affect K app m .
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Fig. 8. (a) Response curve of PNVK/SA/urease LB films as a function of urea concentration (0 /10 mM). (b) Response curve of PNVK/SA/ urease LB films as a function of urea concentration (0 /93 mM).
Fig. 9. Lineweaver /Burke plots for immobilized urease on PNVK/SA LB films: (a) 1 for 0 /10 mM urea; (b) 10 /68 mM urea concentration.
3.3.4. Sensitivity, repeatability and detection limit The response time of the urease/PNVK/SA LB electrode was about 2 min. The detection limit and the sensitivity for this urease electrode have been experimentally determined as 5 mM and 10 mV/mM, respectively. This urease electrode could be used about 10 times. 3.3.5. Storage and stability The observed leaching of about 5% and shelf-life of about 5 weeks has been attributed to very thin and stable architecture of PNVK/SA LB films. It is important to point out here that urease immobilized on 60/70 monolayers of PNVK/SA obtained by the physical
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References
Fig. 10. Effect of temperature (25 /50 8C) on the activity of urease immobilized on PNVK/SA LB films in phosphate buffer at pH 7.0.
adsorption technique shows high leaching (75%) and a short shelf-life (2 days). A very thin and smooth surface of LB film is perhaps unable to retain the physically adsorbed enzyme on the surface. A higher temperature (41 8C) optimum (Fig. 10) was obtained for PNVK/SA/urease electrode, which could perhaps be due to higher intermolecular interactions. About 75% activity was recorded at 45 8C after which a steady decrease was observed which is likely to be due to the denaturation of protein at higher temperatures. This decrease in activity was reversible until 49 8C indicating that the enzyme perhaps attains its near original conformation after returning to normal temperature. The value of activation energy before and after critical temperature calculated from the Arrhenius plot was found to be 4027 and 7595 cal, respectively.
4. Conclusions It can be seen that stable PNVK/SA monolayers can be formed at the air /water interface. Further, urease can be immobilized onto these PNVK/SA LB monolayers. These PNVK/SA/UREASE monolayers transferred onto ITO coated glass plates can be utilized for urea sensing. The detection limit and sensitivity of these electrodes was found to be 5 mM and 10 mV/mM, respectively. The shelf-life of these electrodes was found to be 5 weeks at 4 8C.
Acknowledgements We are grateful to Dr K. Lal, Director, NPL for his interest in this work. The financial support received under the DST funded project (SP/S2/M-52/96) and the Indo-Polish project (INT/POL/P015/2000) are gratefully acknowledged. A.G. and R.S. are grateful to CSIR for award of Research Associateship (RA) and Senior Research Fellowship (SRF), respectively.
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