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mass spectrometry of urease-immobilized sol–gel silica and the application of such a urease-modified electrode to the potentiometric determination of urea
Analytica Chimica Acta 384 (1999) 219±225
Thermogravimetry/mass spectrometry of urease-immobilized sol±gel silica and the application of such a urease-modi®ed electrode to the potentiometric determination of urea K. Ogura*, K. Nakaoka, M. Nakayama, M. Kobayashi, A. Fujii Department of Applied Chemistry, Yamaguchi University, Ube 755-8611, Japan Received 27 August 1998; received in revised form 3 November 1998; accepted 15 November 1998
Abstract Urease has been immobilized in a sol±gel silica matrix, and the enzyme-®xed silica characterized by means of thermogravimetry/mass strectrometry and Fourier transform infrared (FTIR) spectroscopy. The former indicated that the urease immobilized in the sol±gel silica is thermally more stable than free urease. The FTIR spectrum of the urease-®xed silica results from the superimposition of the silica and urease spectra, con®rming that the enzyme can be immobilized in the sol±gel silica without any chemical deterioration. The urease-®xed sol±gel silica electrode has been applied to the potentiometric determination of urea. A Nernstian relationship (slope 59 mV decadeÿ1) was obtained for 110ÿ5±510ÿ2 M urea at pH 8. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Thermogravimetry; Sol±gel silica; Urease
1. Introduction Enzymes have been extensively used as analytical reagents [1]. In practical use, however, the high cost of enzymes is a disadvantage. To overcome this, enzymes have been repeatedly used by immobilizing them in a water-insoluble matrix without loss of activity. Urease is an enzyme of consequence, catalysing the conversion of urea to ammonia and carbon dioxide. The determination of NH 4 or protons formed permits the determination of urea. In early work, a urease-
*Corresponding author. Tel.: +81-836-35-9417; fax: +81-83632-2886; e-mail: [email protected]
immobilized layer of acrylamide gel was placed on the surface of a glass electrode which is responsive to ammonium ions [2,3]. Commercially available polymer membranes were later used to immobilize urease [4±8]. Recently, the incorporation of an enzyme into a conducting polymer was accomplished during the electrodeposition of the polymer; this method was extensively applied to the immobilization of glucose oxidase [9±11]. The many advantages of this method of immobilization include better electric properties due to the conducting polymer, ease of polymerization and incorporation of the enzyme, a relatively fast and inexpensive polymerization procedure and readily available fresh polymer ®lm [12]. However, the use of this procedure for the incorporation of urea is rare [12±14], and the sensitivity is not very satisfactory
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K. Ogura et al. / Analytica Chimica Acta 384 (1999) 219±225
because the potentiometric response never exceeds 35 mV decadeÿ1. The sol±gel process is a low-temperature method for the production of ceramic materials [15,16], providing a very convenient route for the immobilization of biomolecules in the silica matrix [17±20]. The sol± gel inorganic matrix is highly porous, showing physical rigidity, chemical inertness, high thermal stability and only a small degree of leaching of the immobilized species from the matrix. These properties are particularly advantageous for the development of an electrochemical biosensor. In this paper, a platinum electrode was modi®ed with the urease-immobilized sol±gel silica, and this electrode was applied for the potentiometric determination of urea. 2. Experimental A platinum plate (1 cm2) was used as the substrate electrode. The electrode was pretreated by repeated potential cycling between ÿ0.6 and 2.0 V (vs. Ag/ AgCl) in 0.1 M H2SO4. The pretreatment was continued until a reproducible voltammogram was obtained in the region from ÿ0.2 to 1.25 V. One ml of tetraethylorthosilicate (TEOS) and 5 ml of 2.4 mM HCl were mixed and stirred at 08C using a magnetic stirrer for about 2 h. A 4 ml portion of a phosphate buffer solution of urease (5 g lÿ1, pH 7) was added to the solution, and the mixture was further stirred at 08C for 2 min. Fifty ml of the ®nal solution was cast on the pretreated Pt electrode, and the ureaseimmobilized electrode was stored for a given time at room temperature. All chemicals including urease were obtained from Wako Chemical. Potentiometric measurements of the response for the determination of urea were carried out in the usual manner. A stock solution of urea, 1 M, was prepared in a phosphate buffer solution. pH values of the buffer were adjusted by changing the volume ratio of 1/15 M Na2HPO4 and 1/15 M KH2PO4 solutions. An aliquot of the urea stock solution was pipetted into a beakertype cell containing an appropriate amount of the phosphate buffer. The urease-immobilized electrode and an Ag/AgCl reference electrode were immersed in the sample solution which were magnetically stirred until the potential reached a steady state. The potential of the working electrode was measured with an elec-
trometer. The enzyme electrode was stored in the phosphate buffer solution at 258C between measurements. Thermogravimetry/mass spectral analyses of sol± gel silica, urease and urease-immobilized silica were carried out on a Jeol 220 TG/DTA-MS system under a helium atmosphere. The temperature was changed from 408C to 5008C at the programmed heating rate of 58C minÿ1. Urease was applied as received. Sol±gel silica and urease-immobilized silica were cut into pieces, and dried under vacuum. A given amount of these pieces was put in a sample holder to measure the TG/MS spectra. Fourier transform infrared (FTIR) spectroscopic measurements were made on a Shimadzu FTIR (type 8100 M) spectrometer. FTIR spectra were obtained in the transmission mode by using KBr tablets. 3. Results and discussion 3.1. FTIR spectra of sol±gel silica, urease and urease-immobilized silica FTIR spectra of sol±gel silica (a), urease (b) and urease-immobilized silica (c) are shown in Fig. 1. The frequencies for sol±gel silica and urease were assigned in comparison with the literature values. As listed in Table 1, the formation of the silica network is con®rmed by the Si±O stretching vibration at 1220, 1090 and 800 cmÿ1 and the deformation vibration of Si±O± Si±O at 465 cmÿ1. In addition to these peaks the frequencies at 1640 and 960 cmÿ1 are indicative of the existence of surface silanol groups. The assignment of urease was achieved by examining the absorption bands due to the polypeptide bands and the R groups (CH3, ±CH(CH3)2, etc.) belonging to the amino acids. As shown in Table 2, the absorption bands at 1650, 1545, 1270, 630, 730 and 600 cmÿ1 are attributed to the C=O stretching mode (amide I), NH deformation mode (amide II), mixed vibrations involving C±N and NH (amide III), skeletal vibration (amide IV), out of plane N±H bend (amide V) and skeletal vibration (amide VI), respectively. The doublet around 2925 cmÿ1 and the 1435, 1380 and 1340 bands are due to CH stretching and CH deformation vibrations of the CH2 group, respectively. Hence, as seen from Fig. 1, the spectrum (c) of the urease-
K. Ogura et al. / Analytica Chimica Acta 384 (1999) 219±225
221
Fig. 1. FTIR spectra of sol±gel silica (a), urease (b) and ureaseimmobilized silica (c) in KBr.
immobilized silica results from the superimposition of the silica and urease spectra, and it is con®rmed that urease can be immobilized in the sol±gel silica without any chemical deterioration. 3.2. TG/MS spectra of sol±gel silica, urease and urease-immobilized silica Fig. 2(a) shows the thermogravimetric (TG) and differential thermogravimetric (DTG) curves of the
Fig. 2. TG and DTG plots (a) for sol±gel silica, and the corresponding mass spectra at 1008C (b) and 3608C (c).
sol±gel silica. The rate of weight loss of the sol±gel silica was maximum at 808C (Ic), and became sluggish beyond 1408C. The corresponding mass spectra (MS)
Table 1 Assignment of the frequencies for sol±gel silica in KBr cmÿ1
Assignment
Reported cmÿ1
Reference
1640 1220 1090 800 960 465
(O±H) of water adsorbed on silica gel
1640 1200 1100 800 880±830 448
[21]
Si±O stretching vibrations of silica network Bending vibration of SiOH group Deformation vibration of Si±O±Si±O
[22] [22] [23]
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K. Ogura et al. / Analytica Chimica Acta 384 (1999) 219±225
Table 2 Assignment of the frequencies for urease in KBr cmÿ1
Reported cmÿ1 [24]
Assignment
(i) Frequencies of the polypeptide bonds 1650 C=O stretching mode (Amide I) 1545 NH deformation mode (Amide II)
1700±1600 1550±1500
1300 1270 1200
Mixed vibrations involving C±N and NH (Amide III)
630 730 600
Skeletal vibration (Amide IV) Out of plane N±H bend (Amide V) Skeletal vibration (Amide VI)
700±600 730 600
cmÿ1
Assignment
Reported cmÿ1 [25]
(ii) Frequencies of the R groups 2925 2850
CH stretching of CH2 group
292610 285310
CH deformation of CH2 group
1450±1340
1435 1380 1340
at 1008C and 3608C are shown in Fig. 2(b) and (c), respectively. Three major peaks (m/z: 18, 31, 45) are observed at 1008C; m/z18 is due to H2O, showing the detachment of the physically adsorbed water molecules from the silica surface. The m/z values of 31 and 45 are ascribed to CH3O and C2H5O species, respectively, which are due to fragments from the starting material, TEOS. The peak at m/z18 was observed even at high temperature (3608C, Fig. 2(c)), suggesting that some water molecules generated during the dehydration process of TEOS stay in the interior of the sol±gel network. The weight loss upto 5008C was only about 8%, showing a favorable heat-resisting property of the sol±gel silica. A TG spectrum for urease is shown in Fig. 3(a), and the corresponding mass spectra shown in Fig. 3(b)± (e). The rate of weight loss in the heating process is a maximum at 1408C (Iu), 1708C (IIu) and 2108C (IIIu). The mass spectrum at 1008C (Fig. 3(b)) indicates the detachment of adsorbed water molecules from the urease surface. The thermal decomposition of urease is considered to occur at a higher temperature than that corresponding to Iu because the TG peaks at m/z 43 and 29 attributable to the fragments CONH and CHO, respectively, from the urease skeleton, appeared from 1408C (Fig. 3(c)). The decomposition products with
1320±1200
higher molecular weight were observed at a higher temperature (Fig. 3(e)), which result from the thermal decomposition of residual amino acids of urease. In Fig. 4, the TG curve for the urease-immobilized silica is exhibited. As seen from the DTG curve, peak IIu, corresponding to the thermal decomposition of urease, appears at about 1908C. This temperature was higher by about 208C than that observed for free urease (see Figs. 3 and 4), which suggests that the urease ®xed in the sol±gel silica matrix is thermally stabilized. This can also be rationalized by comparing the DTG intensity of peak IIIu in Figs. 3 and 4; i.e., the DTG value for the immobilized urease was about 300 g minÿ1 while that for free urease was about 850 g minÿ1, meaning that the thermal decomposition of urease was suppressed by the protection of the sol± gel silica. The generation of the decomposition species with higher molecular weight from the immobilized species took place at a higher temperature than 3008C (Fig. 4(e)), in a similar way to that for free urease. 3.3. Response of the urease electrode The response of the urease-immobilized sol±gel silica electrode to urea is shown in Fig. 5, where the same electrode was repeatedly used and the elec-
K. Ogura et al. / Analytica Chimica Acta 384 (1999) 219±225
223
Fig. 3. TG and DTG plots (a) for urease, and the corresponding mass spectra at 1008C (b), 1608C (c), 2058C (d) and 4008C (e).
Fig. 4. TG and DTG plots (a) for urease immobilized in sol±gel silica, and the corresponding mass spectra at 1008C (b), 1608C (c), 2108C (d) and 3308C (e).
trode was stored in the phosphate buffer solution between measurements. The steady-state response increases as the number of repeats is increased, and the steady-state potential became constant after the electrode was used three times. The steady-state electrode potential was therefore measured three times under the same conditions, and the ®nal value was adopted in the following data.
The calibration graphs for the urea response of the urease-immobilized electrode are shown at three different pH values in Fig. 6. A Nernstian relationship (slope 59 mV decadeÿ1) was obtained in the concentration range of urea from 110ÿ5±510ÿ2 M at pH 8. At pH 6 and 7, however, such a linear relationship held only in the limited concentration range from 510ÿ4 to 510ÿ2 M; in the region below
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K. Ogura et al. / Analytica Chimica Acta 384 (1999) 219±225
Fig. 5. Urea response for a urease-immobilized sol±gel silica electrode in a phosphate buffer solution of pH 8 containing 1 mM urea. The same electrode was used firstly (*), secondly (&), thirdly (*) and fourthly (~).
Fig. 7. Steady-state calibration graphs of potential vs. urea concentration at pure sol±gel silica (*), bright platinum (&) and urease-immobilized (*) electrodes in a phosphate buffer solution of pH 8.
The catalytic hydrolysis of urea by urease is known to proceed by the following reaction urease
ÿ ÿ NH2 2 CO 3H2 O ! 2NH 4 OH HCO3
(1) The progress of this reaction leads to the change in pH, and a potentiometric measurement of pH permits us to determine the urea concentration. The equilibrium constant for reaction (1) is given by K Fig. 6. Steady-state calibration graphs of potential vs. urea concentration for a urease-immobilized electrode in a phosphate buffer solution of pH: 6 (*), 7 (&) and 8 (*).
210ÿ4 M the slope dropped to 40 mV decadeÿ1. Hence, the urea-immobilized electrode would be useful as a chemical urea sensor in a buffer solution adjusted to around pH 8 since the urea concentration in human serum is in the order of 10ÿ4±10ÿ3 M. In Fig. 7, the potential vs. urea concentration graphs are plotted for the urease-immobilized, pure sol±gel silica and bright platinum electrodes. As seen from this ®gure, there is no Nernstian relationship for the pure sol±gel silica and bright platinum electrodes, con®rming that the urease is involved in the potential-determining reaction.
2 ÿ ÿ NH 4 OH HCO3 : NH2 2 CO
(2)
As described above, the sol±gel silica has silanol groups on the surface, and the electrode potential of the pure sol±gel silica should be related to the following reaction: j
j
j
j
ÿS iO H eÿ !ÿS iOH
(3)
The electrode potential of this reaction is given by E E0 RT=nF lnH ;
(4)
0
where E is the standard potential of reaction (3). From Eqs. (2) and (4), the following relationship can be deduced at 258C: E mV ' ÿ59 log NH4 2 CO:
(5)
K. Ogura et al. / Analytica Chimica Acta 384 (1999) 219±225
Therefore, the steady-state potential of the ureaseimmobilized electrode in the urea solution should shift negatively by 59 mV when the urea concentration is increased by a factor of 10. Such a Nernstian relationship (slope of 59 mV decadeÿ1) was obtained de®nitely at pH 8, but the linear relationship was not valid over the whole range of urea concentration examined at lower pH, as noted above. This is probably due to the occurrence of a neutralization reaction of OHÿ ions generated by reaction (1) in bulk solution without involving reaction (3). 4. Conclusions The characterization of the urease-immobilized sol±gel silica electrode was revealed by FTIR, TG and MS methods. This electrode was applied to the potentiometric determination of urea. The formation of a silica network was con®rmed from the Si±O stretching vibrations at 1220, 1090 and 800 cmÿ1 and the deformation vibration of Si±O±Si± O at 465 cmÿ1. The assignment of urease was achieved by examining the absorption bands due to the polypeptide bonds and the R groups belonging to amino acids. The FTIR spectrum of the urease-immobilized silica resulted from the superimposition of the silica and urease spectra. The immobilization of urease in the sol±gel silica was con®rmed from the TG and MS results, and the immobilized urease was suggested to be more thermally stable than free urease. The potentiometric determination of urea was conducted with the urease-immobilized sol±gel silica electrode in a phosphate buffer solution. A Nernstian relationship (slope of 59 mV decadeÿ1) was obtained for 110ÿ5±510ÿ2 M urea at pH 8, but the linear relationship was valid only over a more limited range of urea concentration at pH values lower than 8.
225
References [1] G.G. Guilbault, Anal. Chem. 38 (1966) 527R. [2] G.G. Guilbault, J.G. Montalvo Jr., , J. Am. Chem. Soc. 91 (1969) 2164. [3] G.G. Guilbault, J.G. Montalvo Jr., , Anal. Lett. 2 (1969) 283. [4] M. Mascini, G.G. Guilbault, Anal. Chem. 49 (1977) 795. [5] G. Palleschi, M. Mascini, Anal. Lett 21 (1988) 1115. [6] Y.J. Wang, C.H. Chen, G.H. Hsiue, B.C. Yu, Biotechnol. Bioeng. 40 (1992) 446. [7] S. Alegret, J. Bartroli, C. JimeÏnez, E. Martinez-FaÆbregas, D. Matorell, F. ValdeÏz-Perezgasga, Sensors and Actuators B 15 (1993) 453. [8] I. Walcerz, R. Koncki, E. LeszynÏska, S. Glab, Anal. Chim. Acta 315 (1995) 289. [9] M. Umana, J. Waller, Anal. Chem. 58 (1986) 2979. [10] J.R. Li, M. Cai, T.F. Chen, L. Jiang, Thin Solid Films 180 (1989) 205. [11] W. Schuhmann, R. Lammert, B. Uhe, H.L. Schmidt, Sensors and Actuators B 1 (1990) 537. [12] S.B. Adeloju, S.J. Shaw, G.G. Wallace, Anal. Chim. Acta 281 (1993) 611. [13] S.B. Adeloju, S.J. Shaw, G.G. Wallace, Anal. Chim. Acta 281 (1993) 621. [14] S. Komada, M. Seyama, T. Homma, T. Osaka, Electrochim. Acta 42 (1997) 383. [15] C. Brinker, G. Scherer, Sol±Gel Science, Academic Press, New York, 1990. [16] L. Hench, J. West, Chem. Rev. 90 (1990) 33. [17] S. Braun, S. Rappoport, R. Zusman, D. Avnir, M. Ottolenghi, Mater. Lett. 10 (1990) 1. [18] O. Lev, Analusis 20 (1992) 543. [19] B. Dave, B. Dunn, J.S. Valentine, J.I. Zenk, Anal. Chem. 66 (1994) 1120A. [20] O. Lev, M. Tsionsky, L. Rabinovich, V. Glezer, S. Sampath, I. Pankratov, J. Gun, Anal. Chem. 67 (1995) 22A. [21] M.L. Hair, Infrared Spectroscopy in Surface Chemistry, Marcel Dekker, New York, 1967, p. 79. [22] H.A. Benesi, A.C. Jones, J. Phys. Chem. 63 (1959) 179. [23] C.W. Wung, Y. Pang, P.N. Prasad, F.E. Karasz, Polymer 32 (1991) 605. [24] T. Miyazawa, T. Shimanouchi, S. Mizushima, J. Chem. Phys. 24 (1956) 408. [25] L.J. Bellamy, The Infrared, Spectra of Complex Molecules, 3rd ed., Chapman & Hall, London, 1975, p. 13.
Thermogravimetry/mass spectrometry of urease-immobilized sol±gel silica and the application of such a urease-modi®ed electrode to the potentiometric determination of urea K. Ogura*, K. Nakaoka, M. Nakayama, M. Kobayashi, A. Fujii Department of Applied Chemistry, Yamaguchi University, Ube 755-8611, Japan Received 27 August 1998; received in revised form 3 November 1998; accepted 15 November 1998
Abstract Urease has been immobilized in a sol±gel silica matrix, and the enzyme-®xed silica characterized by means of thermogravimetry/mass strectrometry and Fourier transform infrared (FTIR) spectroscopy. The former indicated that the urease immobilized in the sol±gel silica is thermally more stable than free urease. The FTIR spectrum of the urease-®xed silica results from the superimposition of the silica and urease spectra, con®rming that the enzyme can be immobilized in the sol±gel silica without any chemical deterioration. The urease-®xed sol±gel silica electrode has been applied to the potentiometric determination of urea. A Nernstian relationship (slope 59 mV decadeÿ1) was obtained for 110ÿ5±510ÿ2 M urea at pH 8. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Thermogravimetry; Sol±gel silica; Urease
1. Introduction Enzymes have been extensively used as analytical reagents [1]. In practical use, however, the high cost of enzymes is a disadvantage. To overcome this, enzymes have been repeatedly used by immobilizing them in a water-insoluble matrix without loss of activity. Urease is an enzyme of consequence, catalysing the conversion of urea to ammonia and carbon dioxide. The determination of NH 4 or protons formed permits the determination of urea. In early work, a urease-
*Corresponding author. Tel.: +81-836-35-9417; fax: +81-83632-2886; e-mail: [email protected]
immobilized layer of acrylamide gel was placed on the surface of a glass electrode which is responsive to ammonium ions [2,3]. Commercially available polymer membranes were later used to immobilize urease [4±8]. Recently, the incorporation of an enzyme into a conducting polymer was accomplished during the electrodeposition of the polymer; this method was extensively applied to the immobilization of glucose oxidase [9±11]. The many advantages of this method of immobilization include better electric properties due to the conducting polymer, ease of polymerization and incorporation of the enzyme, a relatively fast and inexpensive polymerization procedure and readily available fresh polymer ®lm [12]. However, the use of this procedure for the incorporation of urea is rare [12±14], and the sensitivity is not very satisfactory
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(98)00821-6
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K. Ogura et al. / Analytica Chimica Acta 384 (1999) 219±225
because the potentiometric response never exceeds 35 mV decadeÿ1. The sol±gel process is a low-temperature method for the production of ceramic materials [15,16], providing a very convenient route for the immobilization of biomolecules in the silica matrix [17±20]. The sol± gel inorganic matrix is highly porous, showing physical rigidity, chemical inertness, high thermal stability and only a small degree of leaching of the immobilized species from the matrix. These properties are particularly advantageous for the development of an electrochemical biosensor. In this paper, a platinum electrode was modi®ed with the urease-immobilized sol±gel silica, and this electrode was applied for the potentiometric determination of urea. 2. Experimental A platinum plate (1 cm2) was used as the substrate electrode. The electrode was pretreated by repeated potential cycling between ÿ0.6 and 2.0 V (vs. Ag/ AgCl) in 0.1 M H2SO4. The pretreatment was continued until a reproducible voltammogram was obtained in the region from ÿ0.2 to 1.25 V. One ml of tetraethylorthosilicate (TEOS) and 5 ml of 2.4 mM HCl were mixed and stirred at 08C using a magnetic stirrer for about 2 h. A 4 ml portion of a phosphate buffer solution of urease (5 g lÿ1, pH 7) was added to the solution, and the mixture was further stirred at 08C for 2 min. Fifty ml of the ®nal solution was cast on the pretreated Pt electrode, and the ureaseimmobilized electrode was stored for a given time at room temperature. All chemicals including urease were obtained from Wako Chemical. Potentiometric measurements of the response for the determination of urea were carried out in the usual manner. A stock solution of urea, 1 M, was prepared in a phosphate buffer solution. pH values of the buffer were adjusted by changing the volume ratio of 1/15 M Na2HPO4 and 1/15 M KH2PO4 solutions. An aliquot of the urea stock solution was pipetted into a beakertype cell containing an appropriate amount of the phosphate buffer. The urease-immobilized electrode and an Ag/AgCl reference electrode were immersed in the sample solution which were magnetically stirred until the potential reached a steady state. The potential of the working electrode was measured with an elec-
trometer. The enzyme electrode was stored in the phosphate buffer solution at 258C between measurements. Thermogravimetry/mass spectral analyses of sol± gel silica, urease and urease-immobilized silica were carried out on a Jeol 220 TG/DTA-MS system under a helium atmosphere. The temperature was changed from 408C to 5008C at the programmed heating rate of 58C minÿ1. Urease was applied as received. Sol±gel silica and urease-immobilized silica were cut into pieces, and dried under vacuum. A given amount of these pieces was put in a sample holder to measure the TG/MS spectra. Fourier transform infrared (FTIR) spectroscopic measurements were made on a Shimadzu FTIR (type 8100 M) spectrometer. FTIR spectra were obtained in the transmission mode by using KBr tablets. 3. Results and discussion 3.1. FTIR spectra of sol±gel silica, urease and urease-immobilized silica FTIR spectra of sol±gel silica (a), urease (b) and urease-immobilized silica (c) are shown in Fig. 1. The frequencies for sol±gel silica and urease were assigned in comparison with the literature values. As listed in Table 1, the formation of the silica network is con®rmed by the Si±O stretching vibration at 1220, 1090 and 800 cmÿ1 and the deformation vibration of Si±O± Si±O at 465 cmÿ1. In addition to these peaks the frequencies at 1640 and 960 cmÿ1 are indicative of the existence of surface silanol groups. The assignment of urease was achieved by examining the absorption bands due to the polypeptide bands and the R groups (CH3, ±CH(CH3)2, etc.) belonging to the amino acids. As shown in Table 2, the absorption bands at 1650, 1545, 1270, 630, 730 and 600 cmÿ1 are attributed to the C=O stretching mode (amide I), NH deformation mode (amide II), mixed vibrations involving C±N and NH (amide III), skeletal vibration (amide IV), out of plane N±H bend (amide V) and skeletal vibration (amide VI), respectively. The doublet around 2925 cmÿ1 and the 1435, 1380 and 1340 bands are due to CH stretching and CH deformation vibrations of the CH2 group, respectively. Hence, as seen from Fig. 1, the spectrum (c) of the urease-
K. Ogura et al. / Analytica Chimica Acta 384 (1999) 219±225
221
Fig. 1. FTIR spectra of sol±gel silica (a), urease (b) and ureaseimmobilized silica (c) in KBr.
immobilized silica results from the superimposition of the silica and urease spectra, and it is con®rmed that urease can be immobilized in the sol±gel silica without any chemical deterioration. 3.2. TG/MS spectra of sol±gel silica, urease and urease-immobilized silica Fig. 2(a) shows the thermogravimetric (TG) and differential thermogravimetric (DTG) curves of the
Fig. 2. TG and DTG plots (a) for sol±gel silica, and the corresponding mass spectra at 1008C (b) and 3608C (c).
sol±gel silica. The rate of weight loss of the sol±gel silica was maximum at 808C (Ic), and became sluggish beyond 1408C. The corresponding mass spectra (MS)
Table 1 Assignment of the frequencies for sol±gel silica in KBr cmÿ1
Assignment
Reported cmÿ1
Reference
1640 1220 1090 800 960 465
(O±H) of water adsorbed on silica gel
1640 1200 1100 800 880±830 448
[21]
Si±O stretching vibrations of silica network Bending vibration of SiOH group Deformation vibration of Si±O±Si±O
[22] [22] [23]
222
K. Ogura et al. / Analytica Chimica Acta 384 (1999) 219±225
Table 2 Assignment of the frequencies for urease in KBr cmÿ1
Reported cmÿ1 [24]
Assignment
(i) Frequencies of the polypeptide bonds 1650 C=O stretching mode (Amide I) 1545 NH deformation mode (Amide II)
1700±1600 1550±1500
1300 1270 1200
Mixed vibrations involving C±N and NH (Amide III)
630 730 600
Skeletal vibration (Amide IV) Out of plane N±H bend (Amide V) Skeletal vibration (Amide VI)
700±600 730 600
cmÿ1
Assignment
Reported cmÿ1 [25]
(ii) Frequencies of the R groups 2925 2850
CH stretching of CH2 group
292610 285310
CH deformation of CH2 group
1450±1340
1435 1380 1340
at 1008C and 3608C are shown in Fig. 2(b) and (c), respectively. Three major peaks (m/z: 18, 31, 45) are observed at 1008C; m/z18 is due to H2O, showing the detachment of the physically adsorbed water molecules from the silica surface. The m/z values of 31 and 45 are ascribed to CH3O and C2H5O species, respectively, which are due to fragments from the starting material, TEOS. The peak at m/z18 was observed even at high temperature (3608C, Fig. 2(c)), suggesting that some water molecules generated during the dehydration process of TEOS stay in the interior of the sol±gel network. The weight loss upto 5008C was only about 8%, showing a favorable heat-resisting property of the sol±gel silica. A TG spectrum for urease is shown in Fig. 3(a), and the corresponding mass spectra shown in Fig. 3(b)± (e). The rate of weight loss in the heating process is a maximum at 1408C (Iu), 1708C (IIu) and 2108C (IIIu). The mass spectrum at 1008C (Fig. 3(b)) indicates the detachment of adsorbed water molecules from the urease surface. The thermal decomposition of urease is considered to occur at a higher temperature than that corresponding to Iu because the TG peaks at m/z 43 and 29 attributable to the fragments CONH and CHO, respectively, from the urease skeleton, appeared from 1408C (Fig. 3(c)). The decomposition products with
1320±1200
higher molecular weight were observed at a higher temperature (Fig. 3(e)), which result from the thermal decomposition of residual amino acids of urease. In Fig. 4, the TG curve for the urease-immobilized silica is exhibited. As seen from the DTG curve, peak IIu, corresponding to the thermal decomposition of urease, appears at about 1908C. This temperature was higher by about 208C than that observed for free urease (see Figs. 3 and 4), which suggests that the urease ®xed in the sol±gel silica matrix is thermally stabilized. This can also be rationalized by comparing the DTG intensity of peak IIIu in Figs. 3 and 4; i.e., the DTG value for the immobilized urease was about 300 g minÿ1 while that for free urease was about 850 g minÿ1, meaning that the thermal decomposition of urease was suppressed by the protection of the sol± gel silica. The generation of the decomposition species with higher molecular weight from the immobilized species took place at a higher temperature than 3008C (Fig. 4(e)), in a similar way to that for free urease. 3.3. Response of the urease electrode The response of the urease-immobilized sol±gel silica electrode to urea is shown in Fig. 5, where the same electrode was repeatedly used and the elec-
K. Ogura et al. / Analytica Chimica Acta 384 (1999) 219±225
223
Fig. 3. TG and DTG plots (a) for urease, and the corresponding mass spectra at 1008C (b), 1608C (c), 2058C (d) and 4008C (e).
Fig. 4. TG and DTG plots (a) for urease immobilized in sol±gel silica, and the corresponding mass spectra at 1008C (b), 1608C (c), 2108C (d) and 3308C (e).
trode was stored in the phosphate buffer solution between measurements. The steady-state response increases as the number of repeats is increased, and the steady-state potential became constant after the electrode was used three times. The steady-state electrode potential was therefore measured three times under the same conditions, and the ®nal value was adopted in the following data.
The calibration graphs for the urea response of the urease-immobilized electrode are shown at three different pH values in Fig. 6. A Nernstian relationship (slope 59 mV decadeÿ1) was obtained in the concentration range of urea from 110ÿ5±510ÿ2 M at pH 8. At pH 6 and 7, however, such a linear relationship held only in the limited concentration range from 510ÿ4 to 510ÿ2 M; in the region below
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Fig. 5. Urea response for a urease-immobilized sol±gel silica electrode in a phosphate buffer solution of pH 8 containing 1 mM urea. The same electrode was used firstly (*), secondly (&), thirdly (*) and fourthly (~).
Fig. 7. Steady-state calibration graphs of potential vs. urea concentration at pure sol±gel silica (*), bright platinum (&) and urease-immobilized (*) electrodes in a phosphate buffer solution of pH 8.
The catalytic hydrolysis of urea by urease is known to proceed by the following reaction urease
ÿ ÿ NH2 2 CO 3H2 O ! 2NH 4 OH HCO3
(1) The progress of this reaction leads to the change in pH, and a potentiometric measurement of pH permits us to determine the urea concentration. The equilibrium constant for reaction (1) is given by K Fig. 6. Steady-state calibration graphs of potential vs. urea concentration for a urease-immobilized electrode in a phosphate buffer solution of pH: 6 (*), 7 (&) and 8 (*).
210ÿ4 M the slope dropped to 40 mV decadeÿ1. Hence, the urea-immobilized electrode would be useful as a chemical urea sensor in a buffer solution adjusted to around pH 8 since the urea concentration in human serum is in the order of 10ÿ4±10ÿ3 M. In Fig. 7, the potential vs. urea concentration graphs are plotted for the urease-immobilized, pure sol±gel silica and bright platinum electrodes. As seen from this ®gure, there is no Nernstian relationship for the pure sol±gel silica and bright platinum electrodes, con®rming that the urease is involved in the potential-determining reaction.
2 ÿ ÿ NH 4 OH HCO3 : NH2 2 CO
(2)
As described above, the sol±gel silica has silanol groups on the surface, and the electrode potential of the pure sol±gel silica should be related to the following reaction: j
j
j
j
ÿS iO H eÿ !ÿS iOH
(3)
The electrode potential of this reaction is given by E E0 RT=nF lnH ;
(4)
0
where E is the standard potential of reaction (3). From Eqs. (2) and (4), the following relationship can be deduced at 258C: E mV ' ÿ59 log NH4 2 CO:
(5)
K. Ogura et al. / Analytica Chimica Acta 384 (1999) 219±225
Therefore, the steady-state potential of the ureaseimmobilized electrode in the urea solution should shift negatively by 59 mV when the urea concentration is increased by a factor of 10. Such a Nernstian relationship (slope of 59 mV decadeÿ1) was obtained de®nitely at pH 8, but the linear relationship was not valid over the whole range of urea concentration examined at lower pH, as noted above. This is probably due to the occurrence of a neutralization reaction of OHÿ ions generated by reaction (1) in bulk solution without involving reaction (3). 4. Conclusions The characterization of the urease-immobilized sol±gel silica electrode was revealed by FTIR, TG and MS methods. This electrode was applied to the potentiometric determination of urea. The formation of a silica network was con®rmed from the Si±O stretching vibrations at 1220, 1090 and 800 cmÿ1 and the deformation vibration of Si±O±Si± O at 465 cmÿ1. The assignment of urease was achieved by examining the absorption bands due to the polypeptide bonds and the R groups belonging to amino acids. The FTIR spectrum of the urease-immobilized silica resulted from the superimposition of the silica and urease spectra. The immobilization of urease in the sol±gel silica was con®rmed from the TG and MS results, and the immobilized urease was suggested to be more thermally stable than free urease. The potentiometric determination of urea was conducted with the urease-immobilized sol±gel silica electrode in a phosphate buffer solution. A Nernstian relationship (slope of 59 mV decadeÿ1) was obtained for 110ÿ5±510ÿ2 M urea at pH 8, but the linear relationship was valid only over a more limited range of urea concentration at pH values lower than 8.
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