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Amperometric enzyme electrode with the use of dehydrogenase and NAD(P)H oxidase
Sensorsand Achcators 8, 13-14 (1993) 57~575
514
Amperometric enzyme electrode with the use of dehydrogenase and NAD(P)H oxidase Fumio Mizutani, Soichi Yabuki and Tatsuo Katsura NationalInstiluteof Bioscience and Human-Technology,1-I Higashi, Twkuba, Ibaraki 305 (Japan)
Introduction Intensive research effort continues for the development of bi-enzyme electrodes, i.e., electrodes utilizing coupled enzymatic reactions [l, 21.The b&enzyme system of dehydrogenase/NAD(P)H oxidase equipped with an oxygen electrode can be used to determine the substrate of the dehydrogenase [3, 41. The dehydrogenase catalyzes the oxidation of analyte by NAD(P)+ to produce NAD(P)H, and NAD(P)H oxidase catalyzes the oxidation of NAD(P)H by the use of oxygen: RH2 &hydrogsnmc
RH,+NAD(P)+
battery type with a platinumcathode,3 mm in diameter). Each enzyme electrode thus prepared was immersed in an air-saturated test solution (10 ml), whose temperature was kept at 30 “C. The solution usually used for testing the ADHMADH oxidase-based electrode (electrode I) was 0.1 M potassium phosphate buffer (pH 7.4) containing 0.2 mM FAD (flavin adenine dinucleotide), and that for the G-6-PDH/NADPH oxidasebased electrode (electrode II), e same buffercontaining 0.2 mM FMN (flavin monon Jdcleotide). FAD and FMN have to be added to the test solutions as activators for NADH oxidase and NADPH oxidase, respectively.
. R+NAD(P)H+H+
(1) Results and discussion
NAD(P)H+O,+H+
m NAD(P) + + Hz02
(2)
where, RH, and R denote the analyte and its oxidized form, respectively. Such a bi-enzyme electrode can be applied to the highly-sensitive measurement of NAD(P)H: the cyclic enzymatic reactions for NAD(D)H regeneration/oxidation in the presence of RH, cause an enhancement of electrode response [5, 61. The present paper discussed the construction and the use of two kinds of bi-enzyme electrodes: one based on alcohol dehydrogenase (ADH) and NADH oxidase and the other on glucose&phosphate dehydrogenase (G-4-PDH) and NADPH oxidase.
Experimental A layer containing ADH (Sigma) and NADH oxidase (Asahi Chemical Industry) and another containing G-6-PDH (Toyobo) and NADPH oxidase (Asahi Chemical Industry) were prepared from a mixture of the corresponding dehydrogenase, oxidase, and an aqueous solution of photocross-linkable poly(viny1 alcohol) (PVA-SbQ) [5, 71. Each enzyme layer (thickness: 50 w, diameter 5 mm) was attached on the PTFF! membrane of a Clark oxygen electrode (Able, DG-SG,
09254005/93/$6.00
ActivilylpHprojiles of NASH oxkiase and NADPH oxidase Tbe effect of the pH on the responses of electrode
I for NADH and of electrode II for NADPH were first examined. In the pH range of 5.0-9.4, the response of electrode I for NADH was essentially independent of the PH. NADH oxidase can therefore be coupled with a variety of NAD+-dependent dehydrogenases having pH optima in this wide pH range. On the other hand, the response for NADPH on electrode II depended strongly on the pH: it showed a maximum around pH 5 and decreased with the increase in pH. One should use NADPH oxidase with such NADP+dependent dehydrogenases as exhibiting high activities in acidic or neutral media, in order to prevent sacrificing sensitivity of NADPH detection. Hence G-6-PDH, which shows a high activity in neutral solutions, has been used with NADPH oxidase in the present study. Response of ekdmde I Curve I in Fig. 1 shows the response of electrode
I for the successive addition of 0.2 mM NADH and 0.2 mM ethanol to the test solution containing 0.2 mM FAD and 1 mM NAD’. The electrode response for ethanol was almost equal to that for NADH, which showed that ADH reaction proceeded efficiently in the
Q 1993 - Elsevier Sequoia. All rights reserved
515
5-
DI
Time
Fig. 1. Response-time curves (I) on electrode I for the successive addition of NADH (0.2 mM, at T,) and ethanol (0.2 mM, at T2)and (II) on electrode II for the successiveaddition of NADPH (0.2 mIvf, at Tr) and G-6-P (0.2 mM, at T,).
solution. Ethanol in the concentration range of 5 PM to 1.5 mM could be measured. The addition of NADH into the test solution contaming0.1 M ethanol brought about muchlarger current response than the case of the ethanol-free test solution, owing to the substrate regeneration by ADH. The electrode response in the presence of ethanol was proportional to the concentration of NADH only up to 5 FM. In the linear region, however, the NADH response was amplified by a factor of 100 compared with that in the ethanol-free solution. The detection limit for NADH in the presence of ethanol was 50 nM (S/N=5). Responseof electrodeII Curve II in Fig. 1 shows the response of electrode II for the successiveaddition of 0.2 mM NADPH and
0.2mMglucose&phosphate (G-6-P)to the test solution containing 0.2 mM FMN and 1 mM NADP+. The current decrease for G-6-P was almost equal to that for NADPH. The calibration curve on electrode II for G-6-P in the NADP’-containing solution was almost linear up to 2 n&f, and the detection limit for the analyte was 10 PM. A linear relationship was obtained between the current response for NADPH and its concentration up to 10 PM in the test solution containing 4 mM G-6-P. In the linear region,the NADPHresponsewas amplified by a factor of 50 compared with that in G-6-P-free solution. References 1 F. W. Scheller, F. Schubert, R. Remreberg, H.-G. Muiler. M. Jan&en and H. Weise, Biosensors: trends and commercialization, Biosenso~ I (1985)135-160. 2 F. Miitani and M. Asai, in D. L. Wiie (ed.), Bioinstmmmiztim, Buttenuarths, Boston, USA, 1990, Cb. 13, pp. 317-353. 3 F. Miitani, S. Yabuki and hf. Asai, tMaIate-sensing electrode based on malate dehydrogenase and NADH oxidase, AnaL Ch Acta, 245 (1991) 145-150. 4 P. Mizutani, S. Yabuki andT. Katsura, Amperomatric ensyme electrode based on dehydrogenase and NADH oxidase,/lnol sci, 7 (Suppl.) (1991) 871-874. 5 F. Mizutani, T. Yamanaka, Y. Tanabe and K. Tsuda, An enzyme electrode. for Llactate with a chemically-amplified response, Anal. Chim. Acta, 177 (1985) 15346. 6 F. hfizutani and If_Tsuda, Highly-sensitiveenzyme electrode for NADH. NknwnKkaku K&hi. 1198n531-533. 7 K. Ichimtk, i conveiient photochemical method to immobilize ensymes, I. PO&~.Se& P@n. Chem Ed, 22 (1984) 2817-2828.
514
Amperometric enzyme electrode with the use of dehydrogenase and NAD(P)H oxidase Fumio Mizutani, Soichi Yabuki and Tatsuo Katsura NationalInstiluteof Bioscience and Human-Technology,1-I Higashi, Twkuba, Ibaraki 305 (Japan)
Introduction Intensive research effort continues for the development of bi-enzyme electrodes, i.e., electrodes utilizing coupled enzymatic reactions [l, 21.The b&enzyme system of dehydrogenase/NAD(P)H oxidase equipped with an oxygen electrode can be used to determine the substrate of the dehydrogenase [3, 41. The dehydrogenase catalyzes the oxidation of analyte by NAD(P)+ to produce NAD(P)H, and NAD(P)H oxidase catalyzes the oxidation of NAD(P)H by the use of oxygen: RH2 &hydrogsnmc
RH,+NAD(P)+
battery type with a platinumcathode,3 mm in diameter). Each enzyme electrode thus prepared was immersed in an air-saturated test solution (10 ml), whose temperature was kept at 30 “C. The solution usually used for testing the ADHMADH oxidase-based electrode (electrode I) was 0.1 M potassium phosphate buffer (pH 7.4) containing 0.2 mM FAD (flavin adenine dinucleotide), and that for the G-6-PDH/NADPH oxidasebased electrode (electrode II), e same buffercontaining 0.2 mM FMN (flavin monon Jdcleotide). FAD and FMN have to be added to the test solutions as activators for NADH oxidase and NADPH oxidase, respectively.
. R+NAD(P)H+H+
(1) Results and discussion
NAD(P)H+O,+H+
m NAD(P) + + Hz02
(2)
where, RH, and R denote the analyte and its oxidized form, respectively. Such a bi-enzyme electrode can be applied to the highly-sensitive measurement of NAD(P)H: the cyclic enzymatic reactions for NAD(D)H regeneration/oxidation in the presence of RH, cause an enhancement of electrode response [5, 61. The present paper discussed the construction and the use of two kinds of bi-enzyme electrodes: one based on alcohol dehydrogenase (ADH) and NADH oxidase and the other on glucose&phosphate dehydrogenase (G-4-PDH) and NADPH oxidase.
Experimental A layer containing ADH (Sigma) and NADH oxidase (Asahi Chemical Industry) and another containing G-6-PDH (Toyobo) and NADPH oxidase (Asahi Chemical Industry) were prepared from a mixture of the corresponding dehydrogenase, oxidase, and an aqueous solution of photocross-linkable poly(viny1 alcohol) (PVA-SbQ) [5, 71. Each enzyme layer (thickness: 50 w, diameter 5 mm) was attached on the PTFF! membrane of a Clark oxygen electrode (Able, DG-SG,
09254005/93/$6.00
ActivilylpHprojiles of NASH oxkiase and NADPH oxidase Tbe effect of the pH on the responses of electrode
I for NADH and of electrode II for NADPH were first examined. In the pH range of 5.0-9.4, the response of electrode I for NADH was essentially independent of the PH. NADH oxidase can therefore be coupled with a variety of NAD+-dependent dehydrogenases having pH optima in this wide pH range. On the other hand, the response for NADPH on electrode II depended strongly on the pH: it showed a maximum around pH 5 and decreased with the increase in pH. One should use NADPH oxidase with such NADP+dependent dehydrogenases as exhibiting high activities in acidic or neutral media, in order to prevent sacrificing sensitivity of NADPH detection. Hence G-6-PDH, which shows a high activity in neutral solutions, has been used with NADPH oxidase in the present study. Response of ekdmde I Curve I in Fig. 1 shows the response of electrode
I for the successive addition of 0.2 mM NADH and 0.2 mM ethanol to the test solution containing 0.2 mM FAD and 1 mM NAD’. The electrode response for ethanol was almost equal to that for NADH, which showed that ADH reaction proceeded efficiently in the
Q 1993 - Elsevier Sequoia. All rights reserved
515
5-
DI
Time
Fig. 1. Response-time curves (I) on electrode I for the successive addition of NADH (0.2 mM, at T,) and ethanol (0.2 mM, at T2)and (II) on electrode II for the successiveaddition of NADPH (0.2 mIvf, at Tr) and G-6-P (0.2 mM, at T,).
solution. Ethanol in the concentration range of 5 PM to 1.5 mM could be measured. The addition of NADH into the test solution contaming0.1 M ethanol brought about muchlarger current response than the case of the ethanol-free test solution, owing to the substrate regeneration by ADH. The electrode response in the presence of ethanol was proportional to the concentration of NADH only up to 5 FM. In the linear region, however, the NADH response was amplified by a factor of 100 compared with that in the ethanol-free solution. The detection limit for NADH in the presence of ethanol was 50 nM (S/N=5). Responseof electrodeII Curve II in Fig. 1 shows the response of electrode II for the successiveaddition of 0.2 mM NADPH and
0.2mMglucose&phosphate (G-6-P)to the test solution containing 0.2 mM FMN and 1 mM NADP+. The current decrease for G-6-P was almost equal to that for NADPH. The calibration curve on electrode II for G-6-P in the NADP’-containing solution was almost linear up to 2 n&f, and the detection limit for the analyte was 10 PM. A linear relationship was obtained between the current response for NADPH and its concentration up to 10 PM in the test solution containing 4 mM G-6-P. In the linear region,the NADPHresponsewas amplified by a factor of 50 compared with that in G-6-P-free solution. References 1 F. W. Scheller, F. Schubert, R. Remreberg, H.-G. Muiler. M. Jan&en and H. Weise, Biosensors: trends and commercialization, Biosenso~ I (1985)135-160. 2 F. Miitani and M. Asai, in D. L. Wiie (ed.), Bioinstmmmiztim, Buttenuarths, Boston, USA, 1990, Cb. 13, pp. 317-353. 3 F. Miitani, S. Yabuki and hf. Asai, tMaIate-sensing electrode based on malate dehydrogenase and NADH oxidase, AnaL Ch Acta, 245 (1991) 145-150. 4 P. Mizutani, S. Yabuki andT. Katsura, Amperomatric ensyme electrode based on dehydrogenase and NADH oxidase,/lnol sci, 7 (Suppl.) (1991) 871-874. 5 F. Mizutani, T. Yamanaka, Y. Tanabe and K. Tsuda, An enzyme electrode. for Llactate with a chemically-amplified response, Anal. Chim. Acta, 177 (1985) 15346. 6 F. hfizutani and If_Tsuda, Highly-sensitiveenzyme electrode for NADH. NknwnKkaku K&hi. 1198n531-533. 7 K. Ichimtk, i conveiient photochemical method to immobilize ensymes, I. PO&~.Se& P@n. Chem Ed, 22 (1984) 2817-2828.