- Email: [email protected]
Bioelectronic sniffer with monoamine oxidase for methyl mercaptan vapor
Sensors and Actuators B 108 (2005) 639–645
Bioelectronic sniffer with monoamine oxidase for methyl mercaptan vapor Takeshi Minamidea , Kohji Mitsubayashib,∗ , Hirokazu Saitob a
Graduate School of Engineering, Tokai University, 1117 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan b Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan Received 13 July 2004; received in revised form 25 November 2004; accepted 26 November 2004 Available online 19 January 2005
Abstract A bioelectronic sensor for methyl mercaptan (MM) was developed. The biosensor consists of Clark-type dissolved oxygen electrode and a monoamine oxidase type-A (MAO-A) immobilized membrane. In order to amplify the biosensor output, a substrate regeneration cycle caused by coupling the monooxygenase with l-ascorbic acid (AsA) as reducing reagent system was applied. The AsA 10 mmol/l concentration could optimally amplify the sensor output more than 10 times. The MAO-A biosensor was used with AsA to measure MM solution from 0.004 to 4.0 mmol/l, and had good selectivity attributed to enzyme specificity obtained for several substances (triethyl amine, trimethyl amine, dimethyl sulfoxide, dimethyl sulfide, etc.). The biosensor was applied to detect gaseous MM as a bioelectronic sniffer (bio-sniffer) with a reaction unit which had liquid–gaseous compartments separated by a hydrophobic porous polytetrafluoroethylene (PTFE) diaphragm membrane. The tip of the biosensor was placed into the liquid compartment as touching to the PTFE diaphragm membrane. The results of MM vapor measurements showed that the calibration range of the bio-sniffer for MM vapor was from 0.01 to 10 ppm (correlation coefficient: 0.983) and included the human sense of smell level 5 (0.2 ppm). © 2004 Elsevier B.V. All rights reserved. Keywords: Bioelectronic sniffer; Halitosis diagnosis; Methyl mercaptan; Monoamine oxidase type-A; l-Ascorbic acid
1. Introduction Halitosis diagnosis is important in the medical and dental fields. Methyl mercaptan (MM) is one of typical causation for halitosis [1,2]. The American Conference of Governmental Industrial Hygienists (ACGIH) and the Environment Agency Government of Japan had specified the MM as a typical volatile organic compound (VOC) and malodorous substance. The maximum permissible concentration of gaseous MM in the work place is 5.0 ppm (threshold limit value-time weighted average concentration, TLV-TWA) and 0.5 ppm as defined by ACGIH, respectively. In addition, Occupational Safety and Health Administration (OSHA) defined the MM
∗
Corresponding author. Tel.: +81 3 5280 8091; fax: +81 3 5280 8094. E-mail address: [email protected] (K. Mitsubayashi).
0925-4005/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2004.11.091
concentration to 10 ppm as permissible exposure limit time weighted average concentration (PEL TWA). However, there are no convenient approaches to measure the MM vapor for evaluating the halitosis. Many types of gas sensors have been investigated and developed. For example, semiconductor type gas sensors have improved the gas selectivity and sensitivity [3–5]. Nevertheless, semiconductor sensors are still inadequate to sense multi-substances such as those included in expiratory gas, because the sensor outputs the change in electrical conductivity by adsorption of gaseous substances [3–7]. On the other hand, a xenobiotic metabolizing enzyme has good selectivity of producing certain chemical changes in organic substances by catalytic action. For example, flavincontaining monooxygenase (FMO) and monoamine oxidase type-A (MAO-A) are reported to catalyze the oxidation of nitrogen- and sulfur-compounds including the MM for a
640
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
Fig. 1. Construction process of an enzyme immobilized membrane and biosensor for methyl mercaptan solution.
xenobiotic metabolism, and are polymorphic family with several kinds of isoform, which has a dissimilar specificity for the enzyme substrates [8–10]. A biosensor using the enzyme would realize volatile vapor measurement with high selectivity [11–13]. In the previous study, there is biochemical gas sensor (bioelectronic sniffer; bio-sniffer) using the FMO enzyme for MM measurement [14]. The FMO bio-sniffer had gas-selectivity for some substances and measuring range from 1.0 to 4000 ppm of MM vapor. In this study, MAO-A biochemical sensors for MM in the liquid and gas phases were developed and their characteristics were evaluated. 2. Experimental 2.1. Biosensor for MM solution Prior to the sniffer fabrication, the MM biosensor in the liquid phase was constructed using a commercially available Clark-type dissolved oxygen electrode and a MAO-A
immobilized membrane. Fig. 1 shows the process of immobilizing the enzyme to a membrane and a schematic of the MM biosensor. The MAO-A solution (E.C.1.4.3.4., 142 nmole/min mg protein, from adult human liver, Gentest Corp., MA, USA) was mixed with photocrosslinkable polyvinyl alcohol containing stilbazolium groups (PVA-SbQ, type: SPP-H-13 (Bio), Toyo Gosei Co. Ltd., Tokyo, Japan) [15] and was coated onto a dialysis membrane (part no. 1570144-02, thickness: 15 m, Technicon Chemicals Co., S.A., Oecq, Belgium) and was desiccated in the dark place below 2 ◦ C for 1 h. And then, the membrane was irradiated with a fluorescent light for 30 min. The MAO-A membrane was cut into 7 mm square, and was placed onto the sensing area of the Clark-type dissolved oxygen electrode (Model BO-P, ABLE Corp., Tokyo, Japan) covered with a supporting nylon mesh net, and was secured with a silicone O-ring (P-4, o.d. 7.6 mm, i.d. 3.8 mm, FLON Industry Co. Ltd., Tokyo, Japan). The MAO-A immobilized biosensor was kept at 2 ◦ C in a refrigerator when not in use. The tip of the enzyme electrode was immersed in phosphate buffer solution that regulated the pH to 8.5 by mixing of disodium hydrogenphosphate and sodium dihydrogenphosphate. The MAO-A immobilized biosensor was evaluated by using MM solutions in a batch flow measurement system. The tip of the biosensor was dipped into 50 ml of the buffer solution. The MM solutions regulated the concentration to 1, 10 and 100 mmol/l delivered by drop into the buffer solution. During the measurement, a stirrer (TR-100, AS ONE Corp., Osaka, Japan) agitated the buffer solution. An external voltage of −700 mV was maintained on Pt working electrode by a computer-controlled potentiostat (Model 1112, BAS Inc., Tokyo, Japan) relative to Ag/AgCl counter electrode. The sensor output of oxygen consumption induced by MAO-A enzymatic reaction was monitored by a personal computer (PCG-FX11V, SONY, Tokyo, Japan) via the potentiostat and analogue to digital converter (ADC-16, Pico Technology Co. Ltd., Cambridgeshire, UK). In order to amplify the biosensor output, a substrate regeneration cycle caused with l-ascorbic acid (AsA; 196-01252, Wako pure Chemical Industries Ltd., Osaka, Japan) by coupling the monooxygenase as reducing reagent system was applied for MM measurement (Fig. 2) [14,16–19]. And then, the biosensor evaluated the selectivity for several substances (2 mmol/l concentration of triethyl amine, trimethyl amine, ammonia, dimethyl sulfoxide, acetone, ethanol, hexane, diethyl ether and dimethyl sulfide) in the liquid phase.
Fig. 2. Principle of methyl mercaptan measurement using MAO-A enzymatic reaction and substance regeneration [14,16–19].
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
641
Fig. 3. Bioelectronic sniffer with the MAO-A immobilized Clark-type oxygen electrode (MM biosensor) and a reaction cell with liquid and gas phase compartments separated by a porous PTFE membrane.
2.2. Construction of a bioelectronic sniffer The MAO-A immobilized biosensor was applied to measure the MM vapor as a biochemical gas-sensor (bio-sniffer). Fig. 3 shows the structure and the assembly parts of the MAO-A immobilized bio-sniffer. The enzyme biosensor was built into a reaction unit with liquid and gaseous compartments separated by a hydrophobic porous polytetrafluoroethylene (PTFE) diaphragm membrane (type
A-105, pore size: 30–60 m, thickness: 0.20 mm, ZITEX, NORTON KK, Nagano, Japan). The tip of the enzyme biosensor was placed into the liquid compartment as touching to the PTFE diaphragm membrane. The phosphate buffer solution and the MM vapor were individually flowed to the liquid- and gas-compartments of the reaction unit through the each inlet, respectively. The gaseous MM permeating through the micropores of the diaphragm membrane would be detected by the MAO-A immobilized biosensor.
Fig. 4. Schematic diagram of a gas-flow measurement setup for the bioelectronic sniffer.
642
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
2.3. Gas flow measurement using bioelectronic sniffer
The performance of the biosensor in the liquid phase was assessed with the batch flow measurement system prior to use for the gas measurement. Fig. 5 shows a typical response curve of the MAO-A biosensor to 3.0 mmol/l concentration of MM solution without AsA (a) and with 10 mmol/l of AsA (b). The
negative currents of the biosensor output decreased since MM was oxidized by the MAO-A reaction and induced the dissolved oxygen consumption and then reached to steady-state within 7.0 min (90% response). In this paper, the changes in negative current of the biosensor are illustrated. The steady-state currents of the biosensor with and without AsA were 0.76 and 0.07 nA, respectively. Thus, the substrate regeneration cycle caused by coupling the monooxygenase with AsA amplified the sensor outputs more than 10 times. The effect of AsA in buffer solution on the steady-state currents for the MM measurement is shown in Fig. 6. The experimental conditions of AsA concentration for the batch flow measurement were evaluated by the injection of 3 mmol/l MM solution. The sensor outputs were increased by addition of AsA into the buffer solution; the maximum output was obtained at 10 mmol/l of AsA concentration. Therefore, AsA concentration of 10 mmol/l was generally used in all experiments with buffer solution. At high concentrations more than 11 mmol/l, the output was decreased from the maximum and was independent of the AsA concentration at more than 20 mmol/l. As the reason for behavior of the biosensor output shown in Fig. 6, it seemed that the concentration of dissolved oxygen was increased since the MM reduction by AsA exceeded the oxidation by MAO-A, and then balanced in a condition dependent on a thickness, an area and permeability of the enzyme membrane. The reason of decreased sensor output under higher concentration of AsA was seemed decreasing of dissolved oxygen by auto-oxidation of AsA by oxygen. Fig. 7 shows the calibration curve of the biosensor with MAO-A immobilized membrane to MM in the liquid phase with AsA. The changes in output current of the biosensor related to the concentration of the MM over the range from 0.004 to 4.0 mmol/l with correlation coefficient of 0.993, deduced from exponential regression analysis of the log–log plot by a method of a least squares according to the following
Fig. 5. Typical response of the MM biosensor with 10 m/mol of AsA (a) and without AsA (b). MM solution (3 mmol/l) was injected at the point of the arrow on the graph.
Fig. 6. Effect of l-ascorbic acid (AsA) concentration in buffer solution. AsA amplified the sensor output by a reducing reagent system. The maximum output was obtained at 10 mmol/l of AsA concentration.
The MAO-A immobilized bio-sniffer was applied to a gas flow measurement for evaluating the characteristics of MM detection. Fig. 4 shows a schematic diagram of a gas flow measurement setup for the bio-sniffer. Standard gaseous substances were supplied from a gas generator (PERMEATER PD-1B-2, Gastec Corp., Yokohama, Japan). The flow rate of the standard gaseous substances was regulated to 100 ml/min using a mass flow controller with a needle valve (RK1200, Koflok, Tokyo, Japan). A peristaltic pump (type: MP-3N, Tokyo Rikakikai Co. Ltd., Tokyo, Japan) circulated phosphate buffer solution (100 mmol/l, pH 8.5) between a carrier reservoir and the liquid compartment of the reaction unit. The circulation of phosphate buffer solution was applied to realize a continuous measurement of the gaseous substances by supplying the dissolved oxygen and AsA as the reducing reagent to the enzyme and the substrate regeneration reactions, and by removing the enzyme products and surplus gaseous substances which were diffused through the diaphragm membrane into the liquid compartment and the enzyme membrane at the tip of the biosensor. The flow rate of the buffer circulation was set to 1.68 ml/mim. The bio-sniffer output was monitored by the personal computer via the potentiostat and the A/D converter.
3. Results and discussion 3.1. Evaluation of the biosensor in liquid phase
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
Fig. 7. Calibration curve of the biosensor for MM solution.
643
Fig. 9. Typical response of the MM bio-sniffer with 10 mmol of AsA. MM vapor (0.29 ppm) was flowed during arrow line on the graph.
equations:
3.3. Evaluation of the bioelectronic sniffer
sensor output for MM solution (A)
The MAO-A biosensor was applied into the reaction unit for the gas analysis of MM. Typical response of the MAO-A bio-sniffer for MM vapor is shown in Fig. 9. The MM vapor (0.29 ppm) was flowed from 3 to 11 min. Fig. 10 shows the calibration curves of the MAO-A immobilized bio-sniffer for MM. As the figure illustrates, the changes in output of the bio-sniffer were found to be related to the MM concentrations in the gas phase, since gaseous chemicals that diffuse through the enzyme membrane were oxidized by MAO-A using oxygen as electron acceptor, causing a decrease in the concentration of dissolved oxygen. The calibration range of the bio-sniffer for MM vapor was from 0.01 to 10 ppm (correlation coefficient: 0.983) and included the human sense of smell level 5 (0.2 ppm). The sensor output deduced from exponential regression analysis of the log–log plot by a method of a least squares was according to the following equation:
= 0.220 [MM (mmol/l)]0.811 The steady-state output signal over 4.0 mmol/l of MM concentration was represented for saturated dissolved oxygen solution. 3.2. Response selectivity of the biosensor The selectivity of the biosensor for several substances solution (trimethyl amine, ammonia, dimethyl sulfoxide, acetone, ethanol, hexane, diethyl ether and dimethyl sulfide) of 2 mmol/l concentration is shown in Fig. 8. The MAO-A biosensor indicated good selectivity being attributed to the enzyme specificity for chemical substrates because the MAO-A could not catalyze the oxidation of the organic compounds which had no thiol and amino such as dimethyl sulfoxide, acetone, ethanol, hexane, diethyl ether and dimethyl sulfide [2–4].
Fig. 8. Selectivity of the MM biosensor for various substance solutions.
sensor output (nA) = 13.7 [MM (ppm)]0.13 (with 10.0 mmol/l AsA)
Fig. 10. Calibration curve of the biosensor for MM vapor.
644
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
In this study, the MAO-A as oxidation catalysis enzyme was used for construction of the MM bio-sniffer. As same example of MM vapor measurement, the enzyme bio-sniffer using a flavin-containing monooxygenase type 3 (FMO3) was reported [14]. The F10MO3 immobilized bio-sniffer also had high gas-selectivity caused with substrate specificity of enzyme catalysis and the calibration curve for the MM vapor was according to the following equation: sensor output (nA) = 1.21 [MM (ppm)]0.50 (with 10.0 mmol/l AsA) The sensitivity of MAO-A immobilized bio-sniffer was more than 4.8 times compared with the FMO immobilized bio-sniffer as shown in the calibration curve for 10 mmol/l of MM vapor with 10.0 mmol/l AsA. In both bio-sniffers, the structure and materials of dialysis membrane for enzyme immunization and of diaphragm membrane for liquid–gaseous phase separation were similar. Therefore, it seemed that the difference of the sensitivity between MAO-A and FMO immobilized bio-sniffer was depending on the activity of oxidation catalysis of the enzymes. In the measurement of MM vapor using the MAOA immobilized bio-sniffer, the buffer circulation (flow rate = 1.68 ml/min) was applied to realize the continuous measurement. Namely, the buffer flow removes gaseous substances that diffused through the diaphragm membrane and the enzyme membrane in the liquid compartment, thus recovering the sensor output to the initial current. As mentioned above, MAO-A that was one of the xenobiotic metabolizing enzyme in living organisms catalyzed oxidation of the MM, and was possible to use as a recognizable material for the measurement of the malodor substances.
4. Conclusions The bioelectronic sniffer for gaseous methyl mercaptan was constructed of Clark-type dissolved oxygen electrode and a monoamine oxidase type-A immobilized membrane. In order to amplify the biosensor output, a substrate regeneration cycle caused by coupling the monooxygenase with l-ascorbic acid as reducing reagent system was applied. The AsA 10 mmol/l concentration could optimally amplify the sensor output more than 10 times. The MAO-A biosensor was used with AsA to measure MM solution from 0.004 to 4.0 mmol/l, and had good selectivity being attributed to enzyme specificity was obtained for several substances. The bio-sniffer consisted of the MAO-A biosensor and a reaction unit with liquid–gaseous compartments was used to measure MM vapor. The bio-sniffer was calibrated against MM vapor from 0.01 to 10 ppm (correlation coefficient: 0.983) and included the human sense of smell level 5 (0.2 ppm). The bio-sniffer would provide the application including not only the environmental assessment but also a diagnosis
of metabolizing diseases such as a fish-odor syndrome and the process control in food industries.
Acknowledgements This study was supported in part by The Hoso-Bunka Foundation Inc. Assistance Grants, by Tokyo Ohka Foundation Grants-in-Aid for promotion of science and technology and by SECOM Science and Technology Foundation for Research Grants.
References [1] J. Tonzetich, P.A. Carpenter, Production of volatile sulphur compounds from cysteine, cystine and methionine by human dental plague, Arch. Oral Biol. 16 (1971) 599–607. [2] N.F. Schmidt, S.R. Missan, W.J. Tarbet, The correlation between organoleptic mouth-odor ratings and levels of volatile sulfur compounds, Oral Surg. Oral Med. Oral Pathol. 45 (1978) 560–567. [3] Z. Chen, K. Colbow, MgO-doped Cr2 O3 : solubility limit and the effect of doping on the resistivity and ethanol sensitivity, Sens. Actuators B 9 (1992) 49–53. [4] Y. Hiranaka, T. Abe, H. Murata, Gas-dependent response in the temperature transient of SnO2 gas sensors, Sens. Actuators B 9 (1992) 177–182. [5] T. Maekawa, J. Tamaki, N. Miura, N. Yamazoe, Development of SnO2 -based ethanol gas sensor, Sens. Actuators B 9 (1992) 63–69. [6] M. Tonoike, Current status and future view of development for odorant sensors, Bull. Electrotech. Lab. 52 (1988) 63–79. [7] X. Wang, S. Yee, P. Carey, An integrated array of multiple thinfilm metal oxide sensors for quantification of individual components in organic vapor mixtures, Sens. Actuators B 13-14 (1993) 458– 461. [8] H. Hidaka, B. Hartman, S. Udenfriend, Comparison of mitochondrial monoamine oxidases from bovine brain and liver using antibody to purified liver monoamine oxidase, Arch. Biochem. Biophys. 147 (1971) 805–809. [9] R. McCauley, E. Raeker, Separation of two monoamine oxidases from bovine brain, Mol. Cell Biol. 1 (1973) 73–81. [10] S. Aoki, T. Manabe, T. Okuyama, Molecular weight estimation of bovine brain mitochondrial monoamine oxidase, J. Biochem. 82 (1977) 1533–1539. [11] K. Mitsubayashi, K. Yokoyama, T. Takeuchi, I. Karube, Gas-phase biosensor for ethanol, Anal. Chem. 66 (1994) 3297–3302. [12] K. Mitsubayashi, Y. Hashimoto, Development of a gas-phase biosensor for trimethylamine using a flavin-containing monooxygenase 3, Electrochemistry 68 (2000) 901–903. [13] K. Mitsubayashi, H. Amagai, H. Watanabe, Y. Nakayama, Bioelectronic sniffer with a diaphragm flow-cell for acetaldehyde vapor, Sens. Actuators B 95 (2003) 303–308. [14] K. Mitsubayashi, Y. Hashimoto, Bioelectronic nose for methyl mercaptan vapor using xenobiotic metabolizing enzyme: flavincontaining monooxygenase, Sens. Actuators B 83 (2002) 35–40. [15] K. Ichimura, A convenient photochemical method to immobilize enzymes, J. Polym. Sci. 22 (1984) 2817–2828. [16] Y. Hasebe, K. Oshima, O. Takise, S. Uchiyama, Chemically amplified kojic acid responses of tyrosinase-based biosensor, based on inhibitory effect to substrate recycling driven by tyrosinase and lascorbic acid, Talanta 42 (19952079-2085). [17] K.E. Seefeldt, B.C. Weimer, Diversity of sulfur compound production in lactic acid bacteria, J. Dairy Sci. 83 (2000) 2740–2746.
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
645
[18] H.-W. Chin, R.C. Lindsay, Ascorbate and transition-metal mediation of methanethiol oxidation to dimethyl disulfide and dimethyl trisulfide, Food Chem. 49 (1994) 387–392. [19] T.H. Parliment, M.G. Kolor, D.J. Rizzo, Volatile components of Limburger cheese, J. Agric. Food Chem. 30 (1982) 1006–1008.
Kohji Mitsubayashi is a professor of Tokyo Medical and Dental University (Department of Biomedical Devices and Instrumentation). His research interests include wearable chemical sensors for human monitoring, a newly olfactometric system using biological materials, biomolecular and medical devices, microsystem technology, etc.
Biographies
Hirokazu Saito is a research official of Tokyo Medical and Dental University (Department of Biomedical Devices and Instrumentation). His research interests include an automatic blood sucking system and biosniffers.
Takeshi Minamide has been a graduate student at Tokai University. He has investigated electrical bio-sniffers and their applications for biomedical analysis.
Bioelectronic sniffer with monoamine oxidase for methyl mercaptan vapor Takeshi Minamidea , Kohji Mitsubayashib,∗ , Hirokazu Saitob a
Graduate School of Engineering, Tokai University, 1117 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan b Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan Received 13 July 2004; received in revised form 25 November 2004; accepted 26 November 2004 Available online 19 January 2005
Abstract A bioelectronic sensor for methyl mercaptan (MM) was developed. The biosensor consists of Clark-type dissolved oxygen electrode and a monoamine oxidase type-A (MAO-A) immobilized membrane. In order to amplify the biosensor output, a substrate regeneration cycle caused by coupling the monooxygenase with l-ascorbic acid (AsA) as reducing reagent system was applied. The AsA 10 mmol/l concentration could optimally amplify the sensor output more than 10 times. The MAO-A biosensor was used with AsA to measure MM solution from 0.004 to 4.0 mmol/l, and had good selectivity attributed to enzyme specificity obtained for several substances (triethyl amine, trimethyl amine, dimethyl sulfoxide, dimethyl sulfide, etc.). The biosensor was applied to detect gaseous MM as a bioelectronic sniffer (bio-sniffer) with a reaction unit which had liquid–gaseous compartments separated by a hydrophobic porous polytetrafluoroethylene (PTFE) diaphragm membrane. The tip of the biosensor was placed into the liquid compartment as touching to the PTFE diaphragm membrane. The results of MM vapor measurements showed that the calibration range of the bio-sniffer for MM vapor was from 0.01 to 10 ppm (correlation coefficient: 0.983) and included the human sense of smell level 5 (0.2 ppm). © 2004 Elsevier B.V. All rights reserved. Keywords: Bioelectronic sniffer; Halitosis diagnosis; Methyl mercaptan; Monoamine oxidase type-A; l-Ascorbic acid
1. Introduction Halitosis diagnosis is important in the medical and dental fields. Methyl mercaptan (MM) is one of typical causation for halitosis [1,2]. The American Conference of Governmental Industrial Hygienists (ACGIH) and the Environment Agency Government of Japan had specified the MM as a typical volatile organic compound (VOC) and malodorous substance. The maximum permissible concentration of gaseous MM in the work place is 5.0 ppm (threshold limit value-time weighted average concentration, TLV-TWA) and 0.5 ppm as defined by ACGIH, respectively. In addition, Occupational Safety and Health Administration (OSHA) defined the MM
∗
Corresponding author. Tel.: +81 3 5280 8091; fax: +81 3 5280 8094. E-mail address: [email protected] (K. Mitsubayashi).
0925-4005/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2004.11.091
concentration to 10 ppm as permissible exposure limit time weighted average concentration (PEL TWA). However, there are no convenient approaches to measure the MM vapor for evaluating the halitosis. Many types of gas sensors have been investigated and developed. For example, semiconductor type gas sensors have improved the gas selectivity and sensitivity [3–5]. Nevertheless, semiconductor sensors are still inadequate to sense multi-substances such as those included in expiratory gas, because the sensor outputs the change in electrical conductivity by adsorption of gaseous substances [3–7]. On the other hand, a xenobiotic metabolizing enzyme has good selectivity of producing certain chemical changes in organic substances by catalytic action. For example, flavincontaining monooxygenase (FMO) and monoamine oxidase type-A (MAO-A) are reported to catalyze the oxidation of nitrogen- and sulfur-compounds including the MM for a
640
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
Fig. 1. Construction process of an enzyme immobilized membrane and biosensor for methyl mercaptan solution.
xenobiotic metabolism, and are polymorphic family with several kinds of isoform, which has a dissimilar specificity for the enzyme substrates [8–10]. A biosensor using the enzyme would realize volatile vapor measurement with high selectivity [11–13]. In the previous study, there is biochemical gas sensor (bioelectronic sniffer; bio-sniffer) using the FMO enzyme for MM measurement [14]. The FMO bio-sniffer had gas-selectivity for some substances and measuring range from 1.0 to 4000 ppm of MM vapor. In this study, MAO-A biochemical sensors for MM in the liquid and gas phases were developed and their characteristics were evaluated. 2. Experimental 2.1. Biosensor for MM solution Prior to the sniffer fabrication, the MM biosensor in the liquid phase was constructed using a commercially available Clark-type dissolved oxygen electrode and a MAO-A
immobilized membrane. Fig. 1 shows the process of immobilizing the enzyme to a membrane and a schematic of the MM biosensor. The MAO-A solution (E.C.1.4.3.4., 142 nmole/min mg protein, from adult human liver, Gentest Corp., MA, USA) was mixed with photocrosslinkable polyvinyl alcohol containing stilbazolium groups (PVA-SbQ, type: SPP-H-13 (Bio), Toyo Gosei Co. Ltd., Tokyo, Japan) [15] and was coated onto a dialysis membrane (part no. 1570144-02, thickness: 15 m, Technicon Chemicals Co., S.A., Oecq, Belgium) and was desiccated in the dark place below 2 ◦ C for 1 h. And then, the membrane was irradiated with a fluorescent light for 30 min. The MAO-A membrane was cut into 7 mm square, and was placed onto the sensing area of the Clark-type dissolved oxygen electrode (Model BO-P, ABLE Corp., Tokyo, Japan) covered with a supporting nylon mesh net, and was secured with a silicone O-ring (P-4, o.d. 7.6 mm, i.d. 3.8 mm, FLON Industry Co. Ltd., Tokyo, Japan). The MAO-A immobilized biosensor was kept at 2 ◦ C in a refrigerator when not in use. The tip of the enzyme electrode was immersed in phosphate buffer solution that regulated the pH to 8.5 by mixing of disodium hydrogenphosphate and sodium dihydrogenphosphate. The MAO-A immobilized biosensor was evaluated by using MM solutions in a batch flow measurement system. The tip of the biosensor was dipped into 50 ml of the buffer solution. The MM solutions regulated the concentration to 1, 10 and 100 mmol/l delivered by drop into the buffer solution. During the measurement, a stirrer (TR-100, AS ONE Corp., Osaka, Japan) agitated the buffer solution. An external voltage of −700 mV was maintained on Pt working electrode by a computer-controlled potentiostat (Model 1112, BAS Inc., Tokyo, Japan) relative to Ag/AgCl counter electrode. The sensor output of oxygen consumption induced by MAO-A enzymatic reaction was monitored by a personal computer (PCG-FX11V, SONY, Tokyo, Japan) via the potentiostat and analogue to digital converter (ADC-16, Pico Technology Co. Ltd., Cambridgeshire, UK). In order to amplify the biosensor output, a substrate regeneration cycle caused with l-ascorbic acid (AsA; 196-01252, Wako pure Chemical Industries Ltd., Osaka, Japan) by coupling the monooxygenase as reducing reagent system was applied for MM measurement (Fig. 2) [14,16–19]. And then, the biosensor evaluated the selectivity for several substances (2 mmol/l concentration of triethyl amine, trimethyl amine, ammonia, dimethyl sulfoxide, acetone, ethanol, hexane, diethyl ether and dimethyl sulfide) in the liquid phase.
Fig. 2. Principle of methyl mercaptan measurement using MAO-A enzymatic reaction and substance regeneration [14,16–19].
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
641
Fig. 3. Bioelectronic sniffer with the MAO-A immobilized Clark-type oxygen electrode (MM biosensor) and a reaction cell with liquid and gas phase compartments separated by a porous PTFE membrane.
2.2. Construction of a bioelectronic sniffer The MAO-A immobilized biosensor was applied to measure the MM vapor as a biochemical gas-sensor (bio-sniffer). Fig. 3 shows the structure and the assembly parts of the MAO-A immobilized bio-sniffer. The enzyme biosensor was built into a reaction unit with liquid and gaseous compartments separated by a hydrophobic porous polytetrafluoroethylene (PTFE) diaphragm membrane (type
A-105, pore size: 30–60 m, thickness: 0.20 mm, ZITEX, NORTON KK, Nagano, Japan). The tip of the enzyme biosensor was placed into the liquid compartment as touching to the PTFE diaphragm membrane. The phosphate buffer solution and the MM vapor were individually flowed to the liquid- and gas-compartments of the reaction unit through the each inlet, respectively. The gaseous MM permeating through the micropores of the diaphragm membrane would be detected by the MAO-A immobilized biosensor.
Fig. 4. Schematic diagram of a gas-flow measurement setup for the bioelectronic sniffer.
642
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
2.3. Gas flow measurement using bioelectronic sniffer
The performance of the biosensor in the liquid phase was assessed with the batch flow measurement system prior to use for the gas measurement. Fig. 5 shows a typical response curve of the MAO-A biosensor to 3.0 mmol/l concentration of MM solution without AsA (a) and with 10 mmol/l of AsA (b). The
negative currents of the biosensor output decreased since MM was oxidized by the MAO-A reaction and induced the dissolved oxygen consumption and then reached to steady-state within 7.0 min (90% response). In this paper, the changes in negative current of the biosensor are illustrated. The steady-state currents of the biosensor with and without AsA were 0.76 and 0.07 nA, respectively. Thus, the substrate regeneration cycle caused by coupling the monooxygenase with AsA amplified the sensor outputs more than 10 times. The effect of AsA in buffer solution on the steady-state currents for the MM measurement is shown in Fig. 6. The experimental conditions of AsA concentration for the batch flow measurement were evaluated by the injection of 3 mmol/l MM solution. The sensor outputs were increased by addition of AsA into the buffer solution; the maximum output was obtained at 10 mmol/l of AsA concentration. Therefore, AsA concentration of 10 mmol/l was generally used in all experiments with buffer solution. At high concentrations more than 11 mmol/l, the output was decreased from the maximum and was independent of the AsA concentration at more than 20 mmol/l. As the reason for behavior of the biosensor output shown in Fig. 6, it seemed that the concentration of dissolved oxygen was increased since the MM reduction by AsA exceeded the oxidation by MAO-A, and then balanced in a condition dependent on a thickness, an area and permeability of the enzyme membrane. The reason of decreased sensor output under higher concentration of AsA was seemed decreasing of dissolved oxygen by auto-oxidation of AsA by oxygen. Fig. 7 shows the calibration curve of the biosensor with MAO-A immobilized membrane to MM in the liquid phase with AsA. The changes in output current of the biosensor related to the concentration of the MM over the range from 0.004 to 4.0 mmol/l with correlation coefficient of 0.993, deduced from exponential regression analysis of the log–log plot by a method of a least squares according to the following
Fig. 5. Typical response of the MM biosensor with 10 m/mol of AsA (a) and without AsA (b). MM solution (3 mmol/l) was injected at the point of the arrow on the graph.
Fig. 6. Effect of l-ascorbic acid (AsA) concentration in buffer solution. AsA amplified the sensor output by a reducing reagent system. The maximum output was obtained at 10 mmol/l of AsA concentration.
The MAO-A immobilized bio-sniffer was applied to a gas flow measurement for evaluating the characteristics of MM detection. Fig. 4 shows a schematic diagram of a gas flow measurement setup for the bio-sniffer. Standard gaseous substances were supplied from a gas generator (PERMEATER PD-1B-2, Gastec Corp., Yokohama, Japan). The flow rate of the standard gaseous substances was regulated to 100 ml/min using a mass flow controller with a needle valve (RK1200, Koflok, Tokyo, Japan). A peristaltic pump (type: MP-3N, Tokyo Rikakikai Co. Ltd., Tokyo, Japan) circulated phosphate buffer solution (100 mmol/l, pH 8.5) between a carrier reservoir and the liquid compartment of the reaction unit. The circulation of phosphate buffer solution was applied to realize a continuous measurement of the gaseous substances by supplying the dissolved oxygen and AsA as the reducing reagent to the enzyme and the substrate regeneration reactions, and by removing the enzyme products and surplus gaseous substances which were diffused through the diaphragm membrane into the liquid compartment and the enzyme membrane at the tip of the biosensor. The flow rate of the buffer circulation was set to 1.68 ml/mim. The bio-sniffer output was monitored by the personal computer via the potentiostat and the A/D converter.
3. Results and discussion 3.1. Evaluation of the biosensor in liquid phase
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
Fig. 7. Calibration curve of the biosensor for MM solution.
643
Fig. 9. Typical response of the MM bio-sniffer with 10 mmol of AsA. MM vapor (0.29 ppm) was flowed during arrow line on the graph.
equations:
3.3. Evaluation of the bioelectronic sniffer
sensor output for MM solution (A)
The MAO-A biosensor was applied into the reaction unit for the gas analysis of MM. Typical response of the MAO-A bio-sniffer for MM vapor is shown in Fig. 9. The MM vapor (0.29 ppm) was flowed from 3 to 11 min. Fig. 10 shows the calibration curves of the MAO-A immobilized bio-sniffer for MM. As the figure illustrates, the changes in output of the bio-sniffer were found to be related to the MM concentrations in the gas phase, since gaseous chemicals that diffuse through the enzyme membrane were oxidized by MAO-A using oxygen as electron acceptor, causing a decrease in the concentration of dissolved oxygen. The calibration range of the bio-sniffer for MM vapor was from 0.01 to 10 ppm (correlation coefficient: 0.983) and included the human sense of smell level 5 (0.2 ppm). The sensor output deduced from exponential regression analysis of the log–log plot by a method of a least squares was according to the following equation:
= 0.220 [MM (mmol/l)]0.811 The steady-state output signal over 4.0 mmol/l of MM concentration was represented for saturated dissolved oxygen solution. 3.2. Response selectivity of the biosensor The selectivity of the biosensor for several substances solution (trimethyl amine, ammonia, dimethyl sulfoxide, acetone, ethanol, hexane, diethyl ether and dimethyl sulfide) of 2 mmol/l concentration is shown in Fig. 8. The MAO-A biosensor indicated good selectivity being attributed to the enzyme specificity for chemical substrates because the MAO-A could not catalyze the oxidation of the organic compounds which had no thiol and amino such as dimethyl sulfoxide, acetone, ethanol, hexane, diethyl ether and dimethyl sulfide [2–4].
Fig. 8. Selectivity of the MM biosensor for various substance solutions.
sensor output (nA) = 13.7 [MM (ppm)]0.13 (with 10.0 mmol/l AsA)
Fig. 10. Calibration curve of the biosensor for MM vapor.
644
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
In this study, the MAO-A as oxidation catalysis enzyme was used for construction of the MM bio-sniffer. As same example of MM vapor measurement, the enzyme bio-sniffer using a flavin-containing monooxygenase type 3 (FMO3) was reported [14]. The F10MO3 immobilized bio-sniffer also had high gas-selectivity caused with substrate specificity of enzyme catalysis and the calibration curve for the MM vapor was according to the following equation: sensor output (nA) = 1.21 [MM (ppm)]0.50 (with 10.0 mmol/l AsA) The sensitivity of MAO-A immobilized bio-sniffer was more than 4.8 times compared with the FMO immobilized bio-sniffer as shown in the calibration curve for 10 mmol/l of MM vapor with 10.0 mmol/l AsA. In both bio-sniffers, the structure and materials of dialysis membrane for enzyme immunization and of diaphragm membrane for liquid–gaseous phase separation were similar. Therefore, it seemed that the difference of the sensitivity between MAO-A and FMO immobilized bio-sniffer was depending on the activity of oxidation catalysis of the enzymes. In the measurement of MM vapor using the MAOA immobilized bio-sniffer, the buffer circulation (flow rate = 1.68 ml/min) was applied to realize the continuous measurement. Namely, the buffer flow removes gaseous substances that diffused through the diaphragm membrane and the enzyme membrane in the liquid compartment, thus recovering the sensor output to the initial current. As mentioned above, MAO-A that was one of the xenobiotic metabolizing enzyme in living organisms catalyzed oxidation of the MM, and was possible to use as a recognizable material for the measurement of the malodor substances.
4. Conclusions The bioelectronic sniffer for gaseous methyl mercaptan was constructed of Clark-type dissolved oxygen electrode and a monoamine oxidase type-A immobilized membrane. In order to amplify the biosensor output, a substrate regeneration cycle caused by coupling the monooxygenase with l-ascorbic acid as reducing reagent system was applied. The AsA 10 mmol/l concentration could optimally amplify the sensor output more than 10 times. The MAO-A biosensor was used with AsA to measure MM solution from 0.004 to 4.0 mmol/l, and had good selectivity being attributed to enzyme specificity was obtained for several substances. The bio-sniffer consisted of the MAO-A biosensor and a reaction unit with liquid–gaseous compartments was used to measure MM vapor. The bio-sniffer was calibrated against MM vapor from 0.01 to 10 ppm (correlation coefficient: 0.983) and included the human sense of smell level 5 (0.2 ppm). The bio-sniffer would provide the application including not only the environmental assessment but also a diagnosis
of metabolizing diseases such as a fish-odor syndrome and the process control in food industries.
Acknowledgements This study was supported in part by The Hoso-Bunka Foundation Inc. Assistance Grants, by Tokyo Ohka Foundation Grants-in-Aid for promotion of science and technology and by SECOM Science and Technology Foundation for Research Grants.
References [1] J. Tonzetich, P.A. Carpenter, Production of volatile sulphur compounds from cysteine, cystine and methionine by human dental plague, Arch. Oral Biol. 16 (1971) 599–607. [2] N.F. Schmidt, S.R. Missan, W.J. Tarbet, The correlation between organoleptic mouth-odor ratings and levels of volatile sulfur compounds, Oral Surg. Oral Med. Oral Pathol. 45 (1978) 560–567. [3] Z. Chen, K. Colbow, MgO-doped Cr2 O3 : solubility limit and the effect of doping on the resistivity and ethanol sensitivity, Sens. Actuators B 9 (1992) 49–53. [4] Y. Hiranaka, T. Abe, H. Murata, Gas-dependent response in the temperature transient of SnO2 gas sensors, Sens. Actuators B 9 (1992) 177–182. [5] T. Maekawa, J. Tamaki, N. Miura, N. Yamazoe, Development of SnO2 -based ethanol gas sensor, Sens. Actuators B 9 (1992) 63–69. [6] M. Tonoike, Current status and future view of development for odorant sensors, Bull. Electrotech. Lab. 52 (1988) 63–79. [7] X. Wang, S. Yee, P. Carey, An integrated array of multiple thinfilm metal oxide sensors for quantification of individual components in organic vapor mixtures, Sens. Actuators B 13-14 (1993) 458– 461. [8] H. Hidaka, B. Hartman, S. Udenfriend, Comparison of mitochondrial monoamine oxidases from bovine brain and liver using antibody to purified liver monoamine oxidase, Arch. Biochem. Biophys. 147 (1971) 805–809. [9] R. McCauley, E. Raeker, Separation of two monoamine oxidases from bovine brain, Mol. Cell Biol. 1 (1973) 73–81. [10] S. Aoki, T. Manabe, T. Okuyama, Molecular weight estimation of bovine brain mitochondrial monoamine oxidase, J. Biochem. 82 (1977) 1533–1539. [11] K. Mitsubayashi, K. Yokoyama, T. Takeuchi, I. Karube, Gas-phase biosensor for ethanol, Anal. Chem. 66 (1994) 3297–3302. [12] K. Mitsubayashi, Y. Hashimoto, Development of a gas-phase biosensor for trimethylamine using a flavin-containing monooxygenase 3, Electrochemistry 68 (2000) 901–903. [13] K. Mitsubayashi, H. Amagai, H. Watanabe, Y. Nakayama, Bioelectronic sniffer with a diaphragm flow-cell for acetaldehyde vapor, Sens. Actuators B 95 (2003) 303–308. [14] K. Mitsubayashi, Y. Hashimoto, Bioelectronic nose for methyl mercaptan vapor using xenobiotic metabolizing enzyme: flavincontaining monooxygenase, Sens. Actuators B 83 (2002) 35–40. [15] K. Ichimura, A convenient photochemical method to immobilize enzymes, J. Polym. Sci. 22 (1984) 2817–2828. [16] Y. Hasebe, K. Oshima, O. Takise, S. Uchiyama, Chemically amplified kojic acid responses of tyrosinase-based biosensor, based on inhibitory effect to substrate recycling driven by tyrosinase and lascorbic acid, Talanta 42 (19952079-2085). [17] K.E. Seefeldt, B.C. Weimer, Diversity of sulfur compound production in lactic acid bacteria, J. Dairy Sci. 83 (2000) 2740–2746.
T. Minamide et al. / Sensors and Actuators B 108 (2005) 639–645
645
[18] H.-W. Chin, R.C. Lindsay, Ascorbate and transition-metal mediation of methanethiol oxidation to dimethyl disulfide and dimethyl trisulfide, Food Chem. 49 (1994) 387–392. [19] T.H. Parliment, M.G. Kolor, D.J. Rizzo, Volatile components of Limburger cheese, J. Agric. Food Chem. 30 (1982) 1006–1008.
Kohji Mitsubayashi is a professor of Tokyo Medical and Dental University (Department of Biomedical Devices and Instrumentation). His research interests include wearable chemical sensors for human monitoring, a newly olfactometric system using biological materials, biomolecular and medical devices, microsystem technology, etc.
Biographies
Hirokazu Saito is a research official of Tokyo Medical and Dental University (Department of Biomedical Devices and Instrumentation). His research interests include an automatic blood sucking system and biosniffers.
Takeshi Minamide has been a graduate student at Tokai University. He has investigated electrical bio-sniffers and their applications for biomedical analysis.