Bioelectronic sniffer with a diaphragm flow-cell for acetaldehyde vapor

Sensors and Actuators B 95 (2003) 303–308

Bioelectronic sniffer with a diaphragm flow-cell for acetaldehyde vapor Kohji Mitsubayashi∗ , Hirokazu Amagai, Hidenori Watanabe, Yoshihiro Nakayama Department of Human and Information Science, Tokai University, 1117 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan

Abstract A bioelectronic sniffer (bio-sniffer) for gaseous acetaldehyde was constructed by incorporating an aldehyde dehydrogenase immobilized electrode (calibration range to acetaldehyde solution: 0.01–1.0 mmol/l) into a reaction cell with both gas- and liquid-phase compartments, separated by a porous diaphragm membrane. The bio-sniffer devices with two types of the diaphragm (pore size: 1–2 and 20–30 ␮m) were used to measure gaseous acetaldehyde from 0.525 to 20.0 and 0.105 to 5.25 ppm, respectively, with good gas-selectivity being attributed to enzyme specificity. The buffer circulation into the liquid compartment was effective for removing acetaldehyde diffusing from the gas compartment, thus permitting the continuous monitoring of acetaldehyde in the gas phase. The characteristics of the sniffer such as the calibration range and the response time can be adjusted by selecting the porous diaphragm for purpose of use. © 2003 Elsevier B.V. All rights reserved. Keywords: Bioelectronic sniffer; Acetaldehyde; Acetaldehyde dehydrogenase

1. Introduction Gaseous acetaldehyde is one of chemical malodors. The maximum permitted concentrations of gaseous acetaldehyde as defined by the American Conference of Governmental Industrial Hygienists (ACGIH) and by the Environment Agency in Japan are 100 and 50 ppm, respectively [1,2]. The convenient measurement of acetaldehyde concentration in the gas phase is required in the fields of alcohol fermentation process (brewery, winery) and physiological research of alcohol metabolism as well known. The existing semiconductor sensors are still at present inadequate for sensing acetaldehyde vapor in the air, including various kinds of chemical substances such as dietary odorants, body scents and chemical solvents in the gas phase, because the detection principle of the device is based on only changes in electrical conductivity of the device following adsorption of gaseous substances [3–6]. Although an improvement in semiconductor sensors has been reported [7–11], it is still far from the selectivity achievable using biological recognition materials such as enzymes. On the other hand, many kinds of biosensors have been extensively developed for the measurement of chemicalanalytes with good selectivity in the liquid phase. The biosensor for acetaldehyde solution has also been investi-

∗ Corresponding author. E-mail address: [email protected] (K. Mitsubayashi).

0925-4005/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0925-4005(03)00428-3

gated and applied widely for the measurement of acetaldehyde concentration in the food, medical, physiological and environmental fields. Aldehyde dehydrogenase (ALDH) is commonly used in the construction of the acetaldehyde biosensor with diaphorase and electrochemical mediator [12]. In the previous issues [13–16], we reported some biochemical gas sensor (bioelectronic sniffer; bio-sniffer) for ethanol, trimethylamine, methyl mercaptan, formaldehyde, ammonia, etc. In this study, a bioelectronic sniffer for detecting and monitoring acetaldehyde vapor was constructed using a hand-made reaction cell with a porous diaphragm membrane.

2. Experimental 2.1. Biosensor for acetaldehyde solution Prior to the sniffer fabrication, the acetaldehyde biosensor in the liquid phase was constructed by immobilizing enzyme into a Pt electrode-deposited polytetrafluoroethylene (PTFE) membrane. Fig. 1 illustrates the structure of the biosensor and its fabrication process. The Pt layer (3000 Å) as working electrode was formed by sputtering (CFS-4ES-231, Shibaura Engineering Works Co. Ltd.) onto one side of a hydrophilic polytetrafluoroethylene (H-PTFE) membrane (JGWP14225, thickness = 80 ␮m, pore size = 0.2 ␮m, Nihon Millipore

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Fig. 1. Construction process of an ALDH immobilized electrode with H-PTFE membrane.

Ltd., Tokyo, Japan). This electrode membrane offers chemical stability, strength, and flexibility. As previously reported [14], the Pt-deposited membrane retained their flexibility after vacuum deposition. The electrode membrane was cut using a scalpel into 6 mm wide stick (50 mm in length). In order to isolate a sensitive area at the center, the electrode membrane was covered with two heat-weldable polyethylene films with a circle hole (diameter = 4 mm) in a sandwich configuration, and then insulated electrically by a heat sealing machine (SURE Sealer, NL-201P, Ishizaki Electricity Manufacturing Co. Ltd., Tokyo, Japan) except for the circle hole and the both edges of the stick electrode. The stick electrode was thus separated into three discrete areas: sensitive area, lead area and electrical terminal area. The biosensor for acetaldehyde was constructed by immobilizing aldehyde dehydrogenase (ALDH, EC 1.2.1.5, 171832, 20 units/mg, from yeast, Boehringer Mannheim, France) and diaphorase (D5540, EC 1.8.1.4, 5–20 units/mg, from clostridium, Kluyveri, Sigma Chemical Co., St. Louis, MO, USA) into the central sensitive region of the stick electrode. Two kinds of enzyme (ALDH and diaphorase) was mixed with distilled water and photocrosslinkable poly(vinyl alcohol) with stilbazolium groups (PVA-SbQ (Stilbazole Quaternized); PVA-SbQ, SPP-H-13 (Bio), Toyo Gosei Kogyo Co. Ltd., Tokyo, Japan) in a weight ratio of 1:2:60. PVA-SbQ (developed at the Research Institute for Polymers and Textiles, the Japanese Ministry of International Trade and Industry) is a biocompatible, non-hazardous material that has been assessed by a range off toxicity tests (oral, eye and skin surface) [17,18]. The enzyme/PVA-SbQ mixture was placed onto the non-deposited side of the central sensitive area and spread over the surface of the membrane until it had permeated (observed as a darkening of the membrane). The membrane stick was then placed in the dark at room temperature for 1 h to allow for complete permeation then irradi-

ated with a fluorescent light for 30 min. The device was immediately rinsed in phosphate buffer solution (50.0 mmol/l, pH 7.5) and any mixture on the surface of the electrode was removed. Because the mixture swells when wetted, it peels away from the surface and could be easily removed by gentle rubbing. The membrane electrode was stored below 10 ◦ C until required. Acetaldehyde is dehydrogenated by ALDH using oxidized NAD as electron acceptor. Then NADH is dehydrogenated by diaphorase using potassium ferricyanide (1.0 mmol/l) as an electrochemical mediator, thus obtaining the oxidizing current of its reduction form at the Pt electrode [12] as shown Fig. 2. A computer controlled potentiostat (Potentiostat, Model 1112, BAS Inc., Tokyo, Japan) was electrically connected to the edge of the Pt-deposited membrane. A fixed voltage of 81 mV (versus a Pt-wire counter electrode) was applied to the Pt working electrode coated on the H-PTFE membrane [19]. The output current was monitored graphically on a continuous computer display and saved on hard disk for later analysis. The biosensor behavior in the liquid phase was evaluated by a flow injection assessment using a hand-made flow-cell which was fabricated with two stainless steel tubes (o.d. 2.41 mm, i.d. 1.99 mm), two plastic plates (thickness: 5 mm) and two silicon sheets (thickness: 1 mm) as illustrated in Fig. 3.

Fig. 2. Enzymatic and electrochemical reactions for measuring acetaldehyde.

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Fig. 3. Reaction cell with the ALDH immobilized electrode for the flow injection analysis in the liquid phase.

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Fig. 5. Schematic diagram of a gas-flow measurement system for the bioelectronic device.

2.2. Construction of a bioelectronic sniffer The ALDH immobilized biosensor was incorporated into the reaction cell with both gas- and liquid-phase compartments separated by the PTFE diaphragm membrane [13], thus obtaining a bioelectronic sniffer for acetaldehyde vapor (Fig. 4). The reaction unit was constructed using the PTFE materials. Two hollow PTFE tubes (o.d. 40 mm, i.d. 20 mm) were cut to a length of 20 mm and the surfaces of the tubes were polished. Two 6 mm diameter tapped holes were drilled across the entire diameter of each tube and four PTFE tube connectors (o.d. 6 mm, i.d. 2 mm) were screwed into the outside of all of the tapped holes. A porous PTFE membrane and the enzyme electrode were sandwiched between the two tube blocks, which were then held firmly together using a mechanical clamp. In this way, the PTFE membrane acted as a separating diaphragm between the two hollow compartments of the tubes. The enzyme immobilized side was faced to the diaphragm membrane. In order to investigate the effect of the diaphragm membrane, two types of porous PTFE

membranes (PTFE membrane, A-135: pore size 20–30 ␮m, thickness 0.13 mm, or G-110: pore size 1–2 ␮m, thickness 0.25 mm, Zitex Co. Ltd., Nagano, Japan) were used in the reaction cell. A cylindrical rubber stop cock (o.d. 20.5 mm) was inserted into the large hole of one of the tubes, thus forming the gas compartment of the reaction unit. The volume of the compartment was adjusted to 0.5 ml. A rubber o-ring was placed around the cylindrical rod (o.d. 20.5 mm) and inserted into the hole of the other tube, thus producing the liquid compartment of the reaction unit. The sensitive area of the enzyme electrode was immersed in the liquid compartment filled with phosphate buffer (50.0 mmol/l, pH 7.5) and adjusted so as to directly touch the surface of the diaphragm membrane by pushing by the rod. Fig. 5 shows the experimental setup for monitoring acetaldehyde vapor. In the system, sample gas and phosphate buffer solution could be flowed individually through each inlet tube connector to the gas- and liquid-compartments of the reaction cell, respectively. The sensitive area of the biosensor

Fig. 4. Bioelectronic sniffer with the ALDH immobilized electrode and a reaction cell with liquid- and gas-phase compartments separated by porous PTFE membrane.

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was placed onto the surface of the diaphragm membrane. Gaseous substances in the gas compartment could diffuse through into the liquid compartment through the PTFE diaphragm. Buffer solution including electric mediator (potassium ferricyanide, 1.0 mmol/l) and co-enzyme in a carrier reservoir was flowed and circulated into the liquid compartment of the bioelectronic sniffer using a peristaltic pump.

3. Results and discussion 3.1. Evaluation of the biosensor in the liquid phase The performance of the biosensor in the liquid phase was assessed with the flow injection system prior to use for the gas measurement. The effect of the buffer flow rate on the peak current for the acetaldehyde measurement was shown in Fig. 6. The experimental conditions (buffer flow rate, ␤-NAD, pH) for flow injection analysis were evaluated by the injection (100 ␮l) of 1 mmol/l acetaldehyde. As the figure indicates, the peak current after the injection of acetaldehyde was flat between 0.4 and 0.8 ml/min but decreased by increasing the buffer flow rate above 1.0 ml/min. Since the output current was unstable below 0.4 ml/min, a buffer flow rate of 0.45 ml/min was generally used in all experiments with buffer flow. Fig. 7 indicates the effect of ␤-NAD (oxidized form) as co-enzyme in the buffer solution on the sensor output to 1.0 mmol/l acetaldehyde. As the figure indicates, the sensor output was increased by increasing the concentration of ␤-NAD. An amount of 1.0 mmol/l ␤-NAD in buffer solution was, however, generally used in all experiments since there was little enhancement of the sensor output of the biosensor for the concentration of ␤-NAD above 1.0 mmol/l. The effect of pH in buffer solution on the sensor output to 1.0 mmol/l acetaldehyde was also illustrates in Fig. 8. As the product data sheet from Boehringer Mannheim is reported that an optimal pH value of ALDH is from 8.0 to

Fig. 7. Effect of the concentration of ␤-NAD in buffer solution for the flow injection analysis on the peak output of the ALDH immobilized biosensor.

Fig. 6. Effect of buffer flow rate for the flow injection analysis on the peak output of the ALDH immobilized biosensor.

Fig. 8. Effect of pH in buffer solution for the flow injection analysis on the peak output of the ALDH immobilized biosensor.

9.0, the peak value of the sensor current was observed at pH 8.5. Then, this pH value was used in all experiments too. On the basis of those experimental conditions, the characteristics of the biosensor were assessed. Fig. 9 shows the typical responses to various concentration of acetaldehyde in the liquid phase. As the figure indicates, the sensor current was increased by injecting standard solutions of acetaldehyde (100 ␮l) and the output peak was regularly observed at 40 s after the injection. The peak current was related to the concentration of acetaldehyde in the liquid phase over the range 0.01–1.0 mmol/l (correlation coefficient of 0.997), deduced from exponential regression analysis of the log–log plot by a method of least squares according to the following equation: peak current (nA) = 48.0[acetaldehyde (mmol/l)]0.793 Then, the biosensor was applied for gaseous acetaldehyde using the reaction cell with both gas- and liquid-phase compartments.

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As Fig. 10 also indicates, the sensor output was related to the concentration of acetaldehyde in the gas phase over the range 0.525–20.0 ppm using the 1–2 ␮m pore size (correlation coefficient of 0.999) and 0.105–5.25 ppm using the 20–30 ␮m (correlation coefficient of 0.999), respectively, deduced from exponential regression analysis of the log–log plot by a method of least squares according to the following equations. For pore size: 1–2 ␮m, output current (nA) = 97.8[acetaldehyde (mmol/l)]0.935 For pore size: 20–30 ␮m, Fig. 9. Typical responses of the ALDH immobilized electrode to various concentrations of acetaldehyde in the liquid phase (0.1, 0.3, 0.5, 1.0 and 5.0 mmol/l) for the flow injection analysis.

3.2. Evaluation of the bioelectronic sniffer Fig. 10 shows the calibration curves of the bio-sniffer with using two types of diaphragm membranes (pore size: 1–2 or 20–30 ␮m) to acetaldehyde in the gas phase. The typical response for acetaldehyde vapor was also illustrated in the inset in Fig. 10. As the inset indicates, the amperometric current of the bio-sniffer with 1–2 ␮m diaphragm membrane increased rapidly (approximately 2 min to 90% of steady current) following application of acetaldehyde vapor, followed by a steady state current which gradually decreased to the initial output following standard air application. The buffer flow was effective for removing acetaldehyde through the diaphragm from the gas compartment, thus monitoring the concentration change of acetaldehyde in the gas phase.

Fig. 10. Calibration curves of the bioelectronic sniffer for acetaldehyde vapor (filled circle (pore size): 20–30 ␮m, open circle (pore size): 1–2 ␮m). Inset: typical response of the bioelectronic sniffer by applying gaseous acetaldehyde.

output current (nA) = 226[acetaldehyde (mmol/l)]0.979 By use of the more porous type of diaphragm membrane, it is possible to improve the detection limit from 0.525 to 0.105 ppm of gaseous acetaldehyde. The detection limits of the sniffer is equal to the sensitive level 3 (0.15 ppm) for sense of smell in human [20]. The calibration range of the bio-sniffer covered the maximum permitted concentrations of acetaldehyde vapor in USA (100 ppm) and Japan (50 ppm) as described above. 3.3. Gas-selectivity of the bioelectronic sniffer The selectivity of the bio-sniffer for several gases (acetaldehyde: 10.0 ppm, methanol: 11.2 ppm, ethanol: 10.0 ppm, benzene: 10.4 ppm, acetone: 10.0 ppm) and blends of gases (acetaldehyde: 10.0 ppm, ethanol: 10.0 ppm) in shown in Fig. 11. The bio-sniffer using an ALDH electrode gave negligible responses to all the chemicals other than acetaldehyde in the gas phase. The sensor output to a mixture of acetaldehyde (10 ppm) and ethanol (21.5 ppm) was nearly equal to the response to acetaldehyde alone (10 ppm). Thus, the bio-sniffer possessed good gas-selectivity being attributed ALDH specificity.

Fig. 11. Gas-selectivity of the ALDH sniffer for various substances in the gas phase.

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4. Conclusions The bioelectronic sniffer for acetaldehyde vapor was constructed by incorporating the ALDH immobilized electrode into the reaction cell with the liquid- and gas-phase compartments by separated with the porous diaphragm membrane. The bio-sniffer was used to measure the concentration of acetaldehyde in the gas phase (pore size: 1–2 ␮m from 0.525 to 20.0 ppm, pore size: 20–30 ␮m from 0.105 to 5.25 ppm, respectively) with good gas-selectivity. The sensor would be applied for the environmental assessment, the food process control, and breath-air analysis after drinking non-invasive to allow the physiological research of alcohol metabolism. The potential application of the bio-sniffer includes to construct a new intelligent nose for monitoring odorants with chemical multi-analytes.

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Biographies Kohji Mitsubayashi has received his PhD from The University of Tokyo and is an associate professor at Tokai University (Department of Human and Information Science). His research interests include a newly olfactometric system using biological materials, wearable chemical sensors for human monitoring, biomolecular devices, microsystem technology, etc. Hirokazu Amagai is a graduate student at Tokai University (Mitsubayashi Lab.). He has newly done research on sniffer devices for an artificial nose. Hidenori Watanabe was an undergraduate student at Tokai University (Mitsubayashi Lab.) from 2000 to 2001. He had done research on a bio-sniffer for aldehyde chemicals in the gas phase. Yoshihiro Nakayama was an undergraduate student at Tokai University (Mitsubayashi Lab.) from 1999 to 2000. He had done research on a bio-sniffer for aldehyde chemicals in the gas phase.