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Amperometric uric acid sensors based on polyelectrolyte multilayer films
Talanta 61 (2003) 363 /368 www.elsevier.com/locate/talanta
Amperometric uric acid sensors based on polyelectrolyte multilayer films Tomonori Hoshi *, Hidekazu Saiki, Jun-ichi Anzai Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki Aoba-ku, Sendai 980-8578, Japan Received 10 July 2002; received in revised form 23 April 2003; accepted 23 April 2003
Abstract Uricase (UOx) and polyelectrolyte were used for preparation of a permselective multilayer film and enzyme multilayer films on a platinum (Pt) electrode, allowing the detection of uric acid amperometrically. The polyelectrolyte multilayer (PEM) film composed of poly(allylamine) (PAA) and poly(vinyl sulfate) (PVS) were prepared via layer-bylayer assembly on the electrode, functioning as H2O2-selective film. After deposition of the permselective film (PAA/ PVS)2PAA, UOx and PAA were deposited via layer-by-layer sequential deposition up to 10 UOx layers to prepare amperometric sensors for uric acid. Current response to uric acid was recorded at /0.6 V vs. Ag/AgCl to detect H2O2 produced from the enzyme reaction. The response current increased with increasing the number of UOx layers. Even in the presence of ascorbic acid, uric acid can be detected over the concentration range 10 6 /10 3 M. The response current and deposited amount of UOx were affected by deposition bath pH and the addition of salt. The deposition of PAA/UOx film prepared in 2 mg ml 1 solution (pH 11) of PAA with NaCl (8 mg ml 1) and 0.1 mg ml 1 solution (pH 8.5) of UOx with borate (100 mM) resulted in an electrode which shows the largest response to uric acid. The response of the sensor to uric acid was decreased by 40% from the original activity after 30 days. # 2003 Elsevier B.V. All rights reserved. Keywords: Polyelectrolyte; Uric acid sensors; Amperometric measurement
1. Introduction Reliable and simple method for uric acid detection has been expected in a physician’s office or clinical laboratory because uric acid level in blood is an important index of disorders such as gout, Lesch-Nyhan syndrome and Fanconi syndrome [1 /3]. Uric acid sensors have been developed since
1970s, in which uricase (UOx) was employed as a recognition element for uric acid [4,5]: uric acidO2 0 allantoinCO2 H2 O2
In those reports, uric acid was detected based on decrease of oxygen level or increase of CO2 level. As an alternative strategy, amperometric detection of H2O2 is also available: H2 O2 0 O2 2H 2e
* Corresponding author. Fax: /81-22-217-6840. E-mail address: [email protected] (T. Hoshi).
(1)
(2)
To detect oxidation current of H2O2, one concomitant problem must be overcome: uric
0039-9140/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0039-9140(03)00303-5
364
T. Hoshi et al. / Talanta 61 (2003) 363 /368
acid is oxidized as easy as H2O2. For this reason, this type of uric acid sensor has not been developed yet. One possible solution is that electrodes are covered with a membrane in which H2O2 can pass through freely but uric acid cannot permeate. Therefore, we tried to prepare such a membrane using polyelectrolyte multilayer (PEM) method and found it possible [6]. Recently, much attention has been devoted to PEM method because of its simplicity in procedure and wide choice of materials [7 /11]. This technique involves an alternate deposition of anionic and cationic polyelectrolytes from solution onto an electrode. PEM formation depends mainly on ionic attraction between the oppositely charged polyelectrolytes. Poly(allylamine) (PAA) and poly(vinyl sulfate) (PVS) are often used as positively charged polyelectrolyte and negatively charged polyelectrolyte, respectively. As a merit of the PEM method, some molecular recognition elements are also deposited with electrostatic force without further modification or derivatization. Additionally, the PEM method can be applicable to prepare permselective films. We reported a permselective film composed of PAA/PVS for detecting H2O2, in which interferences such as ascorbic acid cannot penetrate by size-exclusion mechanism. Glucose sensors based on H2O2 oxidation were fabricated with the permselective film and found useful for detection of glucose in the presence of typical interferences, ascorbic acid, uric acid and acetaminophen. In the present paper, we prepared amperometric sensors for uric acid using PEM method. We deposited UOx and PAA alternately on the Pt electrode modified with the permselective films. The proposed uric acid sensor is based on the direct amperometric measurement of the increase of H2O2 by the UOx-catalyzed oxidation of uric acid.
Japan). A 20% aqueous solution of PAA (average molecular weight (MW), /10 000) and PVS (average MW, 242 000) were obtained from Nitto Boseki (Tokyo, Japan). The chemical structures of the polymeric materials are shown in Fig. 1. An alumina suspension for fine polishing of electrodes was obtained from Marumoto Struers (Tokyo, Japan). All other chemicals were highest grade of commercially available reagents and were used without further purification. 2.2. Apparatus A quartz crystal microbalance (QCM; QCA917, Seiko EG&G, Tokyo, Japan) was employed for the gravimetric analysis for the formation of multilayer films. A 9 MHz AT-cut quartz resonator coated with a thin platinum layer was used as a probe, which sensitivity is 0.91 Hz ng 1. 2.3. Gravimetric measurement with QCM The surface of the quartz resonator was washed thoroughly with distilled water before use. After the resonance frequency had reached to a stable value in air, the resonator was immersed in a polyelectrolyte solution or UOx solution for 30 min. The resonator was raised and washed in water and dried in air until the frequency became unchangeable, followed by recording its frequency. The resonator was then immersed in a polyelectrolyte solution or UOx solution for 30 min. The deposition and measurement with QCM were repeated. 2.4. Preparation of the electrodes modified with permselective film The permselective film was prepared on the surface of a platinum disk electrode (3 mm diameter) according to the reported procedure
2. Experimental sections 2.1. Materials UOx (uric acid oxidase; EC 1.7.3.3 from Candida sp.) were purchased from Toyobo (Osaka,
Fig. 1. Chemical structures of polyelectrolytes used.
T. Hoshi et al. / Talanta 61 (2003) 363 /368
[6]. Two bathing solutions were employed for the permselective film: a PAA solution and a PVS solution. These polyelectrolytes were dissolved in Dulbecco’s phosphate-buffered saline to be 2 mg ml 1 solution. The Pt electrode was polished thoroughly using an alumina suspension and sonicated in water before use. The electrode was immersed in the PAA solution for 30 min and rinsed in a working buffer for 5 min. The PAAmodified electrode was then immersed in the PVS solution for 30 min to deposit PAA/PVS bilayer. To deposit further layers, the above procedure was repeated five times. The formed multilayer, (PAA/ PVS)2PAA, functions well as a permselective film, in which H2O2 can pass through while interferences such as uric acid and ascorbic acid cannot [6]. 2.5. Preparation of uric acid sensors For depositing UOx on the electrode modified with the permselective film, a series of UOx solutions (0.1 mg ml 1) in borate buffer (100 mM) and PAA solutions (2 mg ml 1) were used. The electrode was immersed in the UOx solution for 30 min, followed by washing with the working buffer. This allowed UOx to be deposited on the permselective film. The electrode was then immersed in the PAA solution for 30 min to deposit the UOx/PAA bilayer on the permselective film. In this way, UOx and PAA were deposited repeatedly on the permselective film up to 10 UOx layers. The resulting electrode modified with the polyelectrolyte and UOx is expected as follows: ‘‘Ptj(PAA/ PVS)2PAAj(UOx/PAA)9UOx’’. A schematic illustration of a uric acid sensor was also shown in Fig. 2. 2.6. Electrochemical measurements The electrochemical response of the uric acid sensors was measured with a conventional threeelectrode system using Ag/AgCl as a reference electrode. A Pt wire (0.5 mm diameter) was used as a counterelectrode. An amperometric measurement of the sensors was carried out at /0.6 V vs. the reference electrode. A phosphate buffer solution (0.1 M, pH 6.8) was used for the
365
Fig. 2. Layer-by-layer deposition of UOx and polyelectrolytes.
measurement and a series of uric acid solutions including Li2CO3 as a solubilizer were injected under stirring. All measurements were carried out at room temperature (/20 8C). The sensors detect oxidation current of enzymatically generated H2O2 from uric acid and O2 (Eqs. (1) and (2)).
3. Results and discussion 3.1. Optimum conditions for alternate deposition of UOx and PAA Because electrostatic attraction between UOx and PAA is thought to drive the depositions, the amount of these adsorbed components depends on ionic strength and pH of the deposition baths. In addition, the enzyme activity of UOx is pHdependent [12]. Therefore, we checked the effect of pH and additives on formation of UOx/PAA assembly. Since UOx is relatively stable in borate buffer (pH 8.5), we selected borate as an additive in UOx bath (Table 1). As shown in Table 1, PEM formation from fully charged UOx in borate buffer (100 mM, pH 8.5) with barely charged PAA in NaCl solution (8 mg ml 1, pH 11) resulted in a uric acid sensor which showed highest sensitivity to uric acid. Because the isoelectric point for UOx is 5.4 [13], the enzyme carries a net negative charge above this
T. Hoshi et al. / Talanta 61 (2003) 363 /368
366
Table 1 Effect of additives and pH of polyelectrolyte solution on the amperometric response to uric acid (0.1 mM); configuration: Pt electrodej(PAA/PVS)2PAAj(UOx/PAA)9UOx PAA (2 mg ml 1)
UOx (0.1 mg ml 1)
pH
Additives
pH
Additives
11 7.5 8.5 9.5 11 11
NaCl (8 mg ml 1) NaCl (8 mg ml 1), HCl NaCl (8 mg ml 1), HCl NaCl (8 mg ml 1), HCl NaCl (8 mg ml 1) None
6.5 7.5 8.5 9.5 8.5 8.5
Borate Borate Borate Borate Borate Borate
a
Response current (nA)a
(100 mM), (100 mM), (100 mM), (100 mM), (100 mM), (100 mM),
HCl HCl HCl HCl HCl HCl
1100 1000 1200 100 2500 1800
The average values of three preparations are listed.
pH under the experimental condition (pH 8.5) as well. Accordingly, the first UOx layer was deposited on the surface of the positively charged permselective film, resulting in Ptj(PAA/ PVS)2PAAjUOxn . On the other hand, at pH 11, less than 5% of the amine groups are protonated in PAA [14]. For this reason, PAA carries limited amount of positive charge in the PAA bath (pH 11). This means that a larger amount of PAA could be adsorbed, although the electrostatic attraction between the deposited UOxn layer and PAA in the deposition bath is fairly weak. If PAA was fully ionized, linear-shaped PAA would be deposited on the UOxn layer to form a thin PAAm layer because of intramolecular electrostatic repulsion. In contrast, nonlinear-shaped PAA in the bath (pH 11) could form a rather thicker layer which has many loops because the repulsive interaction is much smaller in a weakly charged PAA. Conformation of PAA is also dependent on ionic strength. The addition of NaCl to the PAA bath could be also involved in reduction of the repulsive interaction within PAA. After deposition of PAA, the loops within PAA carries a positive charge in the UOx bath (pH 8.5), followed by adsorption of UOxn via electrostatic force. Fig. 3 shows a relationship between the number of UOx layers and the response current to uric acid. The zero layer represents the electrode modified with only the permselective membrane, Ptj(PAA/PVS)2PAA. As shown in Fig. 3, response current to uric acid was nearly zero. It means that uric acid was not oxidized directly on the electrode
Fig. 3. Amperometric response to uric acid (0.1 mM) dependent on the number of UOx layers deposited on the electrode modified with permselective membrane. The average values of three preparations are plotted.
surface, showing that the permselective membrane functioned well. The potential at /0.6 V vs. Ag/ AgCl at which oxidation of H2O2 takes place is positive enough to cause direct oxidation at the electrode of uric acid. The response current derived from the oxidation of H2O2 increased with increasing the number of UOx layers. This is the first uric acid sensor based on direct oxidation of H2O2 at the electrode. Though there are some reports for amperometric detection of H2O2, horseradish peroxidase (HRP) or mediators were employed [15 /17]. Compared to the reported procedure, the present method was much simpler: the sensor can be fabricated using only polyelectrolytes and UOx.
T. Hoshi et al. / Talanta 61 (2003) 363 /368
367
As shown in Fig. 3, the incremental current became small gradually with increasing the number of layers. It seems to be caused by low permeability of uric acid in sensing layers, (UOx/ PAA)n UOx. Ascorbic acid and acetaminophen could hardly pass through the sensing layers (data not shown). As another possibility, the amount of UOx deposited with each proceeding layer may decrease gradually. Therefore, we checked the deposition behavior of the sensing layers using QCM. In QCM measurement, since it is well-known that thicker membrane formed on the probe could not be detected characteristically, we prepared (PAA/UOx)10 film on the Pt-coated probe without the permselective membrane. Both sides of the probe were in touch with the PAA or the UOx solutions, implying that the (PAA/ UOx)10 film was formed on the both sides of the probe. As shown in Fig. 4, the resonance frequency changed almost linearly up to (PAA/ UOx)10, suggesting that the same amount of UOx was deposited at each deposition step. Because the sensitivity of QCM is 0.91 Hz ng 1, the mass of 2 /(PAA/UOx)10 may be evaluated as ca. 4500 ng. However, the frequency change should be understood as qualitative analysis because H2O in (PAA/UOx)n did not seemed to be removed completely. It was also found that the frequency increased after the deposition of PAA, suggesting that UOx was partially removed when
PAA was adsorbed on the (PAA/UOx)n film. It is probably caused by the salt added in the PAA bath or pH change. Because the outmost PAA layer in the PEM film is thought to be barely ionized when it is immersed in the PAA bath (pH 11), a part of deposited UOx could be released from the PEM film due to attenuated electrostatic attraction. Fig. 5 shows calibration curve of the uric acid sensor prepared under the best conditions: PAA in NaCl solution (8 mg ml 1, pH 11) and UOx in borate buffer (100 mM, pH 8.5). The concentration range covers that of uric acid in serum: the levels were 0.3059/0.066 (S.D.) mM in 2283 men and 0.2399/0.056 (S.D.) mM in 2844 women [1]. In the presence of ascorbic acid, response current decreased by ca. 20% at 10 3 M solution of uric acid. This is most likely caused by reduction of H2O2 with ascorbic acid. This phenomenon is often observed in other detection methods based on H2O2 generation [18,19]. Oxidation current of ascorbic acid (0.1 mM) was ca. 3000 nA when unmodified Pt electrode (3 mm diameter) was used at /0.6 V vs. Ag/AgCl. This means positive offset of ca. 3000 nA should be observed when H2O2 was oxidized. The 20% reduction of signal is less effective compared to the 3000 nA offset. Because the permselective film is located between the electrode and UOx layers, it is possible to eliminate direct oxidation of such interferences on the electrode. However, it is impossible to inhibit such
Fig. 4. Frequency response for adsorption of PAA (odd) and UOx (even) on the both sides of Pt electrodes. UOx bath: 0.1 mg ml 1 solution of UOx containing 100 mM borate (pH 8.5). PAA bath: 2 mg ml 1 solution of PAA containing 8 mg ml 1 NaCl (pH 11).
Fig. 5. Typical calibration curve with or without interferences: with 0.1 mM ascorbic acid ("), 0.1 mM acetaminophen (2), 0.1 mM ascorbic acid and 0.1 mM acetaminophen (/), without interferences (k). The average values of three preparations are plotted.
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T. Hoshi et al. / Talanta 61 (2003) 363 /368
ing in the uric acid sensor. At /0.6 V vs. Ag/AgCl, the response current to uric acid increased with increasing the number of UOx layers. Even in the presence of ascorbic acid, uric acid can be detected over the concentration range 10 6 /10 3 M.
Acknowledgements This work was financially supported in part by a Grant-in-Aid (No. 10584) from Ministry of Education, Science, Sports and Culture of Japan. Fig. 6. Lifetime of the sensor. The concentration of uric acid was 0.1 mM.
References interferences from consuming H2O2. Therefore, as a preventive measure against H2O2 consumption, it is required to deposit another permselective film on UOx layers. Regarding ascorbic acid, ascorbate oxidase (AOx) is eminently suitable for use in the second permselective film [20]. AOx could be readily deposited with a polyelectrolyte via layerby-layer deposition. We also checked a long-term stability of the sensors for 30 days (Fig. 6). The sensors were stored in borate buffer (100 mM, pH 8.5) when not in use. During 12 days after the sensor was built up, the response to uric acid decreased. This seems to be caused by deactivation of UOx and/or release of UOx from the sensing layers. After 13 days, the response current increased gradually. It is caused probably by partial deterioration of the permselective film. After storage for 30 days, the sensor still retained ca. 60% activity to uric acid of the original value.
4. Conclusions We prepared uric acid sensors successfully based on the direct amperometric measurement of H2O2 produced by the UOx-catalyzed oxidation of uric acid, employing a permselective film composed of (PAA/PVS)2PAA. UOx and PAA were deposited alternately up to (UOx/PAA)9UOx on the Pt electrode modified with permselective film, result-
[1] A.P. Hall, P.E. Barry, T.R. Dawber, P.M. McNamara, Am. J. Med. 42 (1967) 27. [2] A.D. Meisei, H.S. Diamond, Am. J. Med. Sci. 273 (1977) 109. [3] W.L. Nyhan, Arch. Intern. Med. 130 (1972) 186. [4] M. Nanjo, G.G. Guilbault, Anal. Chem. 46 (1974) 1769. [5] T. Kawashima, G.A. Rechnitz, Anal. Chim. Acta 83 (1976) 9. [6] T. Hoshi, H. Saiki, S. Kuwazawa, C. Tsuchiya, Q. Chen, J. Anzai, Anal. Chem. 73 (2001) 5310. [7] Y. Lvov, K. Ariga, I. Ichinose, T. Kunitake, J. Am. Chem. Soc. 117 (1995) 6117. [8] F. Caruso, K. Niikura, D.N. Furlong, Y. Okahata, Langmuir 13 (1997) 3427. [9] Y.M. Lvov, Z. Lu, J.B. Schenkman, X. Zu, J.F. Rusling, J. Am. Chem. Soc. 120 (1998) 4073. [10] F. Caruso, H. Mo¨hwald, J. Am. Chem. Soc. 121 (1999) 6039. [11] J. Anzai, T. Hoshi, N. Nakamura, Langmuir 16 (2000) 6306. [12] K. Itaya, T. Yamamoto, J. Fukumoto, Agric. Biol. Chem. 31 (1967) 1256. [13] K. Itaya, J. Fukumoto, T. Yamamoto, Agric. Biol. Chem. 35 (1971) 813. [14] J. Hodak, R. Etchenique, E. Calvo, K. Singhal, P.N. Bartlett, Langmuir 13 (1997) 2708. [15] M.A.T. Gilmartin, J.P. Hart, Analyst 119 (1994) 833. [16] T. Tatsuma, T. Watanabe, Anal. Chim. Acta 242 (1991) 85. [17] E. Miland, A.J.M. Ordieres, P.T. Blanco, M.R. Smyth, ´ . Fa´ga´in, Talanta 43 (1996) 785. C.O [18] P. Sharp, Clin. Chim. Acta 40 (1972) 115. [19] L.N.W. Daae, A. Juell, Scand. J. Clin. Lab. Invest. 43 (1983) 747. [20] J. Anzai, H. Takeshita, T. Hoshi, T. Osa, Denki Kagaku 63 (1995) 1141.
Amperometric uric acid sensors based on polyelectrolyte multilayer films Tomonori Hoshi *, Hidekazu Saiki, Jun-ichi Anzai Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki Aoba-ku, Sendai 980-8578, Japan Received 10 July 2002; received in revised form 23 April 2003; accepted 23 April 2003
Abstract Uricase (UOx) and polyelectrolyte were used for preparation of a permselective multilayer film and enzyme multilayer films on a platinum (Pt) electrode, allowing the detection of uric acid amperometrically. The polyelectrolyte multilayer (PEM) film composed of poly(allylamine) (PAA) and poly(vinyl sulfate) (PVS) were prepared via layer-bylayer assembly on the electrode, functioning as H2O2-selective film. After deposition of the permselective film (PAA/ PVS)2PAA, UOx and PAA were deposited via layer-by-layer sequential deposition up to 10 UOx layers to prepare amperometric sensors for uric acid. Current response to uric acid was recorded at /0.6 V vs. Ag/AgCl to detect H2O2 produced from the enzyme reaction. The response current increased with increasing the number of UOx layers. Even in the presence of ascorbic acid, uric acid can be detected over the concentration range 10 6 /10 3 M. The response current and deposited amount of UOx were affected by deposition bath pH and the addition of salt. The deposition of PAA/UOx film prepared in 2 mg ml 1 solution (pH 11) of PAA with NaCl (8 mg ml 1) and 0.1 mg ml 1 solution (pH 8.5) of UOx with borate (100 mM) resulted in an electrode which shows the largest response to uric acid. The response of the sensor to uric acid was decreased by 40% from the original activity after 30 days. # 2003 Elsevier B.V. All rights reserved. Keywords: Polyelectrolyte; Uric acid sensors; Amperometric measurement
1. Introduction Reliable and simple method for uric acid detection has been expected in a physician’s office or clinical laboratory because uric acid level in blood is an important index of disorders such as gout, Lesch-Nyhan syndrome and Fanconi syndrome [1 /3]. Uric acid sensors have been developed since
1970s, in which uricase (UOx) was employed as a recognition element for uric acid [4,5]: uric acidO2 0 allantoinCO2 H2 O2
In those reports, uric acid was detected based on decrease of oxygen level or increase of CO2 level. As an alternative strategy, amperometric detection of H2O2 is also available: H2 O2 0 O2 2H 2e
* Corresponding author. Fax: /81-22-217-6840. E-mail address: [email protected] (T. Hoshi).
(1)
(2)
To detect oxidation current of H2O2, one concomitant problem must be overcome: uric
0039-9140/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0039-9140(03)00303-5
364
T. Hoshi et al. / Talanta 61 (2003) 363 /368
acid is oxidized as easy as H2O2. For this reason, this type of uric acid sensor has not been developed yet. One possible solution is that electrodes are covered with a membrane in which H2O2 can pass through freely but uric acid cannot permeate. Therefore, we tried to prepare such a membrane using polyelectrolyte multilayer (PEM) method and found it possible [6]. Recently, much attention has been devoted to PEM method because of its simplicity in procedure and wide choice of materials [7 /11]. This technique involves an alternate deposition of anionic and cationic polyelectrolytes from solution onto an electrode. PEM formation depends mainly on ionic attraction between the oppositely charged polyelectrolytes. Poly(allylamine) (PAA) and poly(vinyl sulfate) (PVS) are often used as positively charged polyelectrolyte and negatively charged polyelectrolyte, respectively. As a merit of the PEM method, some molecular recognition elements are also deposited with electrostatic force without further modification or derivatization. Additionally, the PEM method can be applicable to prepare permselective films. We reported a permselective film composed of PAA/PVS for detecting H2O2, in which interferences such as ascorbic acid cannot penetrate by size-exclusion mechanism. Glucose sensors based on H2O2 oxidation were fabricated with the permselective film and found useful for detection of glucose in the presence of typical interferences, ascorbic acid, uric acid and acetaminophen. In the present paper, we prepared amperometric sensors for uric acid using PEM method. We deposited UOx and PAA alternately on the Pt electrode modified with the permselective films. The proposed uric acid sensor is based on the direct amperometric measurement of the increase of H2O2 by the UOx-catalyzed oxidation of uric acid.
Japan). A 20% aqueous solution of PAA (average molecular weight (MW), /10 000) and PVS (average MW, 242 000) were obtained from Nitto Boseki (Tokyo, Japan). The chemical structures of the polymeric materials are shown in Fig. 1. An alumina suspension for fine polishing of electrodes was obtained from Marumoto Struers (Tokyo, Japan). All other chemicals were highest grade of commercially available reagents and were used without further purification. 2.2. Apparatus A quartz crystal microbalance (QCM; QCA917, Seiko EG&G, Tokyo, Japan) was employed for the gravimetric analysis for the formation of multilayer films. A 9 MHz AT-cut quartz resonator coated with a thin platinum layer was used as a probe, which sensitivity is 0.91 Hz ng 1. 2.3. Gravimetric measurement with QCM The surface of the quartz resonator was washed thoroughly with distilled water before use. After the resonance frequency had reached to a stable value in air, the resonator was immersed in a polyelectrolyte solution or UOx solution for 30 min. The resonator was raised and washed in water and dried in air until the frequency became unchangeable, followed by recording its frequency. The resonator was then immersed in a polyelectrolyte solution or UOx solution for 30 min. The deposition and measurement with QCM were repeated. 2.4. Preparation of the electrodes modified with permselective film The permselective film was prepared on the surface of a platinum disk electrode (3 mm diameter) according to the reported procedure
2. Experimental sections 2.1. Materials UOx (uric acid oxidase; EC 1.7.3.3 from Candida sp.) were purchased from Toyobo (Osaka,
Fig. 1. Chemical structures of polyelectrolytes used.
T. Hoshi et al. / Talanta 61 (2003) 363 /368
[6]. Two bathing solutions were employed for the permselective film: a PAA solution and a PVS solution. These polyelectrolytes were dissolved in Dulbecco’s phosphate-buffered saline to be 2 mg ml 1 solution. The Pt electrode was polished thoroughly using an alumina suspension and sonicated in water before use. The electrode was immersed in the PAA solution for 30 min and rinsed in a working buffer for 5 min. The PAAmodified electrode was then immersed in the PVS solution for 30 min to deposit PAA/PVS bilayer. To deposit further layers, the above procedure was repeated five times. The formed multilayer, (PAA/ PVS)2PAA, functions well as a permselective film, in which H2O2 can pass through while interferences such as uric acid and ascorbic acid cannot [6]. 2.5. Preparation of uric acid sensors For depositing UOx on the electrode modified with the permselective film, a series of UOx solutions (0.1 mg ml 1) in borate buffer (100 mM) and PAA solutions (2 mg ml 1) were used. The electrode was immersed in the UOx solution for 30 min, followed by washing with the working buffer. This allowed UOx to be deposited on the permselective film. The electrode was then immersed in the PAA solution for 30 min to deposit the UOx/PAA bilayer on the permselective film. In this way, UOx and PAA were deposited repeatedly on the permselective film up to 10 UOx layers. The resulting electrode modified with the polyelectrolyte and UOx is expected as follows: ‘‘Ptj(PAA/ PVS)2PAAj(UOx/PAA)9UOx’’. A schematic illustration of a uric acid sensor was also shown in Fig. 2. 2.6. Electrochemical measurements The electrochemical response of the uric acid sensors was measured with a conventional threeelectrode system using Ag/AgCl as a reference electrode. A Pt wire (0.5 mm diameter) was used as a counterelectrode. An amperometric measurement of the sensors was carried out at /0.6 V vs. the reference electrode. A phosphate buffer solution (0.1 M, pH 6.8) was used for the
365
Fig. 2. Layer-by-layer deposition of UOx and polyelectrolytes.
measurement and a series of uric acid solutions including Li2CO3 as a solubilizer were injected under stirring. All measurements were carried out at room temperature (/20 8C). The sensors detect oxidation current of enzymatically generated H2O2 from uric acid and O2 (Eqs. (1) and (2)).
3. Results and discussion 3.1. Optimum conditions for alternate deposition of UOx and PAA Because electrostatic attraction between UOx and PAA is thought to drive the depositions, the amount of these adsorbed components depends on ionic strength and pH of the deposition baths. In addition, the enzyme activity of UOx is pHdependent [12]. Therefore, we checked the effect of pH and additives on formation of UOx/PAA assembly. Since UOx is relatively stable in borate buffer (pH 8.5), we selected borate as an additive in UOx bath (Table 1). As shown in Table 1, PEM formation from fully charged UOx in borate buffer (100 mM, pH 8.5) with barely charged PAA in NaCl solution (8 mg ml 1, pH 11) resulted in a uric acid sensor which showed highest sensitivity to uric acid. Because the isoelectric point for UOx is 5.4 [13], the enzyme carries a net negative charge above this
T. Hoshi et al. / Talanta 61 (2003) 363 /368
366
Table 1 Effect of additives and pH of polyelectrolyte solution on the amperometric response to uric acid (0.1 mM); configuration: Pt electrodej(PAA/PVS)2PAAj(UOx/PAA)9UOx PAA (2 mg ml 1)
UOx (0.1 mg ml 1)
pH
Additives
pH
Additives
11 7.5 8.5 9.5 11 11
NaCl (8 mg ml 1) NaCl (8 mg ml 1), HCl NaCl (8 mg ml 1), HCl NaCl (8 mg ml 1), HCl NaCl (8 mg ml 1) None
6.5 7.5 8.5 9.5 8.5 8.5
Borate Borate Borate Borate Borate Borate
a
Response current (nA)a
(100 mM), (100 mM), (100 mM), (100 mM), (100 mM), (100 mM),
HCl HCl HCl HCl HCl HCl
1100 1000 1200 100 2500 1800
The average values of three preparations are listed.
pH under the experimental condition (pH 8.5) as well. Accordingly, the first UOx layer was deposited on the surface of the positively charged permselective film, resulting in Ptj(PAA/ PVS)2PAAjUOxn . On the other hand, at pH 11, less than 5% of the amine groups are protonated in PAA [14]. For this reason, PAA carries limited amount of positive charge in the PAA bath (pH 11). This means that a larger amount of PAA could be adsorbed, although the electrostatic attraction between the deposited UOxn layer and PAA in the deposition bath is fairly weak. If PAA was fully ionized, linear-shaped PAA would be deposited on the UOxn layer to form a thin PAAm layer because of intramolecular electrostatic repulsion. In contrast, nonlinear-shaped PAA in the bath (pH 11) could form a rather thicker layer which has many loops because the repulsive interaction is much smaller in a weakly charged PAA. Conformation of PAA is also dependent on ionic strength. The addition of NaCl to the PAA bath could be also involved in reduction of the repulsive interaction within PAA. After deposition of PAA, the loops within PAA carries a positive charge in the UOx bath (pH 8.5), followed by adsorption of UOxn via electrostatic force. Fig. 3 shows a relationship between the number of UOx layers and the response current to uric acid. The zero layer represents the electrode modified with only the permselective membrane, Ptj(PAA/PVS)2PAA. As shown in Fig. 3, response current to uric acid was nearly zero. It means that uric acid was not oxidized directly on the electrode
Fig. 3. Amperometric response to uric acid (0.1 mM) dependent on the number of UOx layers deposited on the electrode modified with permselective membrane. The average values of three preparations are plotted.
surface, showing that the permselective membrane functioned well. The potential at /0.6 V vs. Ag/ AgCl at which oxidation of H2O2 takes place is positive enough to cause direct oxidation at the electrode of uric acid. The response current derived from the oxidation of H2O2 increased with increasing the number of UOx layers. This is the first uric acid sensor based on direct oxidation of H2O2 at the electrode. Though there are some reports for amperometric detection of H2O2, horseradish peroxidase (HRP) or mediators were employed [15 /17]. Compared to the reported procedure, the present method was much simpler: the sensor can be fabricated using only polyelectrolytes and UOx.
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As shown in Fig. 3, the incremental current became small gradually with increasing the number of layers. It seems to be caused by low permeability of uric acid in sensing layers, (UOx/ PAA)n UOx. Ascorbic acid and acetaminophen could hardly pass through the sensing layers (data not shown). As another possibility, the amount of UOx deposited with each proceeding layer may decrease gradually. Therefore, we checked the deposition behavior of the sensing layers using QCM. In QCM measurement, since it is well-known that thicker membrane formed on the probe could not be detected characteristically, we prepared (PAA/UOx)10 film on the Pt-coated probe without the permselective membrane. Both sides of the probe were in touch with the PAA or the UOx solutions, implying that the (PAA/ UOx)10 film was formed on the both sides of the probe. As shown in Fig. 4, the resonance frequency changed almost linearly up to (PAA/ UOx)10, suggesting that the same amount of UOx was deposited at each deposition step. Because the sensitivity of QCM is 0.91 Hz ng 1, the mass of 2 /(PAA/UOx)10 may be evaluated as ca. 4500 ng. However, the frequency change should be understood as qualitative analysis because H2O in (PAA/UOx)n did not seemed to be removed completely. It was also found that the frequency increased after the deposition of PAA, suggesting that UOx was partially removed when
PAA was adsorbed on the (PAA/UOx)n film. It is probably caused by the salt added in the PAA bath or pH change. Because the outmost PAA layer in the PEM film is thought to be barely ionized when it is immersed in the PAA bath (pH 11), a part of deposited UOx could be released from the PEM film due to attenuated electrostatic attraction. Fig. 5 shows calibration curve of the uric acid sensor prepared under the best conditions: PAA in NaCl solution (8 mg ml 1, pH 11) and UOx in borate buffer (100 mM, pH 8.5). The concentration range covers that of uric acid in serum: the levels were 0.3059/0.066 (S.D.) mM in 2283 men and 0.2399/0.056 (S.D.) mM in 2844 women [1]. In the presence of ascorbic acid, response current decreased by ca. 20% at 10 3 M solution of uric acid. This is most likely caused by reduction of H2O2 with ascorbic acid. This phenomenon is often observed in other detection methods based on H2O2 generation [18,19]. Oxidation current of ascorbic acid (0.1 mM) was ca. 3000 nA when unmodified Pt electrode (3 mm diameter) was used at /0.6 V vs. Ag/AgCl. This means positive offset of ca. 3000 nA should be observed when H2O2 was oxidized. The 20% reduction of signal is less effective compared to the 3000 nA offset. Because the permselective film is located between the electrode and UOx layers, it is possible to eliminate direct oxidation of such interferences on the electrode. However, it is impossible to inhibit such
Fig. 4. Frequency response for adsorption of PAA (odd) and UOx (even) on the both sides of Pt electrodes. UOx bath: 0.1 mg ml 1 solution of UOx containing 100 mM borate (pH 8.5). PAA bath: 2 mg ml 1 solution of PAA containing 8 mg ml 1 NaCl (pH 11).
Fig. 5. Typical calibration curve with or without interferences: with 0.1 mM ascorbic acid ("), 0.1 mM acetaminophen (2), 0.1 mM ascorbic acid and 0.1 mM acetaminophen (/), without interferences (k). The average values of three preparations are plotted.
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ing in the uric acid sensor. At /0.6 V vs. Ag/AgCl, the response current to uric acid increased with increasing the number of UOx layers. Even in the presence of ascorbic acid, uric acid can be detected over the concentration range 10 6 /10 3 M.
Acknowledgements This work was financially supported in part by a Grant-in-Aid (No. 10584) from Ministry of Education, Science, Sports and Culture of Japan. Fig. 6. Lifetime of the sensor. The concentration of uric acid was 0.1 mM.
References interferences from consuming H2O2. Therefore, as a preventive measure against H2O2 consumption, it is required to deposit another permselective film on UOx layers. Regarding ascorbic acid, ascorbate oxidase (AOx) is eminently suitable for use in the second permselective film [20]. AOx could be readily deposited with a polyelectrolyte via layerby-layer deposition. We also checked a long-term stability of the sensors for 30 days (Fig. 6). The sensors were stored in borate buffer (100 mM, pH 8.5) when not in use. During 12 days after the sensor was built up, the response to uric acid decreased. This seems to be caused by deactivation of UOx and/or release of UOx from the sensing layers. After 13 days, the response current increased gradually. It is caused probably by partial deterioration of the permselective film. After storage for 30 days, the sensor still retained ca. 60% activity to uric acid of the original value.
4. Conclusions We prepared uric acid sensors successfully based on the direct amperometric measurement of H2O2 produced by the UOx-catalyzed oxidation of uric acid, employing a permselective film composed of (PAA/PVS)2PAA. UOx and PAA were deposited alternately up to (UOx/PAA)9UOx on the Pt electrode modified with permselective film, result-
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