Anal. Chem. 1995,67,3922-3927
GaslPhase Microbiosensor for Vapor at ppb Levels Manus J. Dennison, Jennifer M. Hall, and Anthony P. F. Tumer* Cranfield Biotechnology Centre, Cranfield Univem'ty, Cranfield, BeuYodshire MK43 OAL, U.K.
A microbiosensor capable of measuring very low levels of phenol vapor directly in the gas phase has been constructed. The microbiosensoris based on the enzyme polyphenol oxidase, which catalyzes the oxidation of phenols to catechols and then to quinones. Polyphenol oxidasewas immobilized in a glycerol-basedgel which did not dehydrate sigd?cantly over time. An interdigitated microelectrode array was used as transducer. Phenol vapor partitioned into the glycerol gel, where it was enzymatically oxidized to quinone. Signal amplification was achieved by redox recycling of the quinone/catechol couple. This redox recycling produced a biosensor capable of measuring phenol vapor concentrations of 30 ppb. The biosensor produced a constant signal after 5 days of continuous use at room temperature and has potential application in the field of health and safety monitoring, where its ease of use, selectivity, and realtime monitoring would provide personnel with accurate data. Gas-phase sensing has been dominated by nonbiological sensors, such as electrochemical, semiconducting, and pellistertype sensors. Commercial semiconductingsensors exist for many gases,' while chemical sensors based on colorimetric principles are commercially available for over 100 different gases.2 There is a great variety of applications for sensors which can detect the presence of hazardous gases in industrial environments, and current equipment suffers from a lack of portability and the inability to determine cumulative exposure? Potentiometric and amperometric gas sensors are, in general, limited to a narrow range of electroactivegases! Semiconductinggas sensors, while able to detect a wide range of gases, have high power consumption and suffer from a serious lack of specificity. Pellister-type sensors are used for volatile organic carbons and cannot effectively distinguish between different gases. There exists a need for gas sensors with low power consumption and which are selective for unreactive gases or vapors, gas-phase biosensors could fulfill a niche requirement here. Biological recognition proteins, such as enzymes and antibodies, have high inherent selectivity. These proteins, when incorporated into sensors, confer this property of selectivity on the biosensor. Biosensors have been developed for a large range of (1) Bo& B.; Thorpe, S. C. In Techniquesand mechanisms in gas sensing; Moseley, P. T., Noms, J. 0. W., Williams, D. E., Eds.; Adam Hilger: Bristol. UK, 1991; pp 139-160. (2) Draegertube handbook, 8th ed.; Draeger Ltd.: Lubeck, Germany, 1992. (3) Hollingum, J. Sens. Reu. 1993,13, 32-33. (4) Hobbs. B. S.; Tantram, A D. S.; Chan-Henry, R In Techniques and mechanisms in gas sensing Moseley, P. T., Noms, J. 0. W., Williams, D. E., Eds.; Adam Hilger: Bristol, UK, 1991; pp 161-181.
3922 Analytical Chemisty, Vol. 67, No. 21, November 1, 1995
analytes, including glu~ose,~ cholesterol,6 alcoh~l,~ and lactate,* and have made their mark mainly in the field of clinical analysis, although recently many have been developed for environmental analysis? Biosensors can also operate in certain organic phases,10 provided that some water is available to the enzyme. Certain problems are involved with applying biosensors for gasphase sensing. As all enzymes need water for activity, and as the gas phase is usually a drier environment than the aqueous phase, water from the biosensor will evaporate to the gas phase. This loss of water will eventually affect enzyme activity and will also change the concentrations of the substrates and products. Hence, biosensor response, stability, and lifetime will be affected by the relative humidity. The fact that enzyme activity is dependent on the availability of water has been the largest limitation on the advancement of biosensors into the field of gas monitoring. Early gas-phase biosensors were essentially bioreactors containing the sensing element (usually bacteria or enzymes) in an aqueous phase, into which was pumped the gas in question. The gas then dissolved in the aqueous phase, where it was detected by the sensing element. Using a bioreactor format overcomes the problem of water evaporation by avoiding direct interfacing with the gas phase. Biosensors based on this principle include the earliest reported gas-phase biosensor.11 This biosensor was based on a methane oxidizing bacterium, Methylomonasfiagellata, dissolved in byfkr which, when exposed to methane in solution, oxidized the &as, reducing aqueous levels of 02, which were detected by a Clark-type oxygen electrode. A similar biosensor has also been constructed for nitrogen dioxide.12 A carbon monoxide sensor incorporating CO oxidoreductase similarly involved dissolution of the analyte in a layer retained in a reactor or as a probe.13 A mediator was used to effect electron transfer from the enzyme to the electrode. Guilbault14 produced a biosensor for formaldehyde based on formaldehyde dehydrogenase immobilized on a piezoelectric crystal detector which could (5) C a s , A E. G.; Davis, G.; Francis, G. D.; Hill, H. A 0.;Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A P. F. Anal. Chem. 1984,56, 667-671. (6) Ball, M. R; Frew, J. E.; Green, M. J.; Hill, H. A 0. Proc. Electrochem. Soc. 1986,86, 14-25. (7) Wang, J.; Romero, E. G.; Reviejo, A J. 1.Electroanal. Chem. 1993,353, 113-120. (8)Mullen, W. H.; Churchouse, S. J.; Freedy, F. H.; Vadgama, P. M. Clin. Chim. Acta 1986,157, 191-198. (9) Dennison, M. J.; Turner, A P. F. Biotechnol. Adu. 1995,13, 1-12. (10) Hall, G.; Best, D.; Turner, A. Enzyme Microb. Technol. 1988,10, 543-546. (11) Karube, I.; Okada, T.; Sumki, S. Anal. Chim. Acta 1982,135,6147. (12) Okada, T.; Karube, I.; Suzuki, S. Biotechnol. Bioeng. 1983,25 (6), 16411651. (13) Turner, A P. F.; Aston, W. J.; Higgins, I. J.; Bell, J. M.; Colby, J.; Davis, G.; Hill, A 0. Anal. Chem. Acta 1984,163, 161-174. (14) Guilbault, G. G. Anal. Chem. 1983,455, 1682-1684.
0003-2700/95/0367-3922$9.00/0 0 1995 American Chemical Society
detect 10 ppm formaldehyde. Guilbault did not report investigating the effect of humidity on sensor response. Further gas-phase biosensors include a potentiometric biosensor for COJ5 based on the enzyme carbonic anhydrase dissolved in a commercial hydrogel. The stability and response of this biosensor were dependent on the relative humidity of the test gas. A gas-phase biosensor for ethanoP was based on immobilized alcohol oxidase with an oxygen electrode. A circulating buffer system was necessary to prevent dehydration of the enzyme. Spectral changes observed on binding of HCN to hemoglobin were used as the basis for a gas-phase HCN biosensor.17 Air humidity was found to have a significant effect on response, but the authors found they could compensate for it by measurement at a third wavelength. Biosensors offer a number of important advantages over conventional analytical techniques: specificity, low cost, and portability. Their biological base also makes them exquisitely sensitive to toxins and ideal for health and safety applications, and also for pollution monitoring. Biosensors are unsuitable for use at high temperatures (due to biological inactivation) or at low temperatures. These high- and low-temperature areas will probably remain the domain of chemical sensors. Gas-phase biosensors could function admirably in the areas of health and safety monitoring and clinical sensing. Monitoring of clinically signifcant gases and vapors in the breath in a noninvasive fashion is particularly appealing. Phenol is one of the most important and most widely used industrial chemicals,’* being used in the manufacture of products ranging from plastic resins to pesticides. Studies have shown the existence of phenols as pollutants of air, water, and s0il.19-21 Studies in factories using phenol have shown the presence of low levels of background phenol v a p ~ r . ’ ~Phenol , ~ ~ is easily adsorbed by humans, regardless of the type of exposure, and high levels of phenols have been shown to have detrimental effects on animal The effect of long-term exposure to low levels of phenols in the atmosphere is as yet unclear. However, natural phenols present in plants have been shown to have estrogenic properties.23324 A phenol present in certain plastics, pnonylphenol, has also been shown to have estrogenic pr0perties,2~as have alkylHighly sensitive biosensors for monitoring phenols using the enzyme polyphenol oxidase have been described for organic solutions27and for aqueous solutions.28 Polyphenol oxidase catalyzes the oxidation of phenol to catechol and then to quinone (15) Tiemey, J. J.; Kim, H. L. Anal. Chem. 1993,65, 3435-3440. (16) Mitsubayashi, K; Yokoyama, K; Takeuchi, T.; Karube, I. Anal. Chem. 1994, 66, 3297-3302. (17) Moss, D. A; Sans, J.; Ache, H. J. Abstracts from the World Congress on Biosensors, New Orleans, LA, 1994; p 3.12 (18) Gilbert, J. Sci. Tot. Environ. 1994,143, 103-111. (19) Drugov, Y. S.; Murav’eva, G. V. Zh. Anal. a i m . 1991,46, 2014-2020. 1381-1388. (20) Shah, J. J.; Singh, H. B. Environ. Sci. Technol. 1988,22, (21) Ciccoli, P.; Cecinato, A; Brancaleoni, E.; Frattoni, M. Fresenius Environ. Bull. 1992,1 , 73-78. (22) Cleghom, H. P.; Fellin, P. Toxic. Enuiron. Chem. 1992,34, 85-98. (23) VanOettingen. Phenol and its derivatives: m e relationship between their chemical constitution and their effect on the organism; National Institute of Health: The Netherlands, 1949. (24) Stob, M. Handbook of naturally occum’ng food toxicants; CRC Press Ltd.: Boca Raton, FL, 1983. (25) Soto, A M.; Justica, H.; Wray, J. W.: Sonnenschein, C. Enuiron. Health Persp. 1991,92,167-173. (26) Colbom, T.: vonSaal, F. S.; Soto, A M. Envir. Health Perspect. 1993,101, 378-384. (27) Wang, J.; Lin, Y.; Chen, L. Analyst 1993,118, 277-280.
using molecular oxygen. Quinones can be electrochemically reduced at approximately -150 mV (vs Ag/AgCl). To date, no gas-phase biosensor for phenol vapor has been reported. As phenol is very volatile (mp 41 “C) and one of the most widely used industrial chemicals, and legislation requires a maximum exposure limit of no more than 5 ppm over 8 h,29there exists a need for a selective, sensitive phenol vapor sensor providing realtime results. This paper reports on the development of a microbiosensor that uses polyphenol oxidase incorporated in a water-retaining gel with a microelectrodefunctioning as transducer for measurement of phenol directly in the vapor phase. EXPERIMENTAL SECTION
Reagents. Chemicals. Analar grade chemicalswere employed without further purification. Sodium dihydrogen orthophosphate and potassium chloride were supplied by BDH (Poole, UK). Disodium hydrogen orthophosphate was supplied by Fisons (Loughborough, UK) Enzyme “Gel”. Mushroom polyphenol oxidase (EC 1.14.18.1) (1mg) with an activity of 6300 units/mg from Sigma (Poole, UK) was dissolved in a “gel” of 80% (v/v) glycerol (BDH) and 20%0.1 M sodium phosphate buffer, pH 7, containing 0.1 M potassium chloride. Although a solution of glycerol is not technically a gel, but rather a visous solution, for brevity the viscous glycerol solution will be refered to as a gel. Biosensor Construction. Enzyme gel (4 pL) was deposited onto the interdigitated area of a SAW-302 interdigitated microelectrode (MicrosensorSystems Inc., Bowling Green, Ky) (Figure 1, inset). The SAW-302 interdigitated microelectrode is a gold two-electrode system with arrays of 50 microelectrodes each (15 pm x 4 mm). The macrosections of the electrodes were insulated with red conformal coating (RS Components, Corby, UK), leaving the microelectrode area and the electrical contact area free of insulation. Each biosensor was based on one interdigitated microelectrode in which one microelectrode array acted as a working electrode and the other array acted as a combined counter and quasi-reference electrode (CCSQRE). SAW-302 interdigitated microelectrodes were used unless otherwise stated. Gold microdisk electrodes with a combined Ag/AgCl reference and counter electrode incorporated into the electrode design were used in certain experiments and were kindly supplied by Ecossensors Ltd. (Long Hanborough, UK). Gas Rig. A gas rig capable of generating phenol vapor under different humidity conditions was constructed (Figure 1). Phenol highemission permeation tubes (Vici Metronics Inc., Santa Clara, CA), which penetrates phenol at a rate which is temperature dependent, were sealed in air-tight glass U-tube and immersed in an oil bath. Low relative humidity air was then passed through the U-tube over the permeation tubes at a known flow rate. This yielded low relative humidity air containing phenol vapor, which was then mixed with air which had been humidifled by passing through a Dreschel bottle containing water, generating air containing phenol vapor at the required concentration and relative humidity. (28) Ortega, F.: Dominguez, E.; Jonsson-Pettersson, G.; Gorton, L J. Biotechnol. 1993,31, 289-300. (29) Lenga, R E. Library of chemical safety data, 2 ed.; Sigma-Aldrich Corp.: Milwaukee, WI, 1988.
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on
LOW
hunldlty
mldny
ak
boneway wive *Glass beads fw heat exchange llned
I!&\&
Test C h a m b e r vith b l o s s n s o r and temp & humid I t Y uonitor
U
Hicrorlcdrodc
Phon01
perneation t u b e
Figun I. Schematic diagram of gas rig constructed for generating dfterent concentrations d phenol vapor at different humidities. Arrows refer to direction of air flow. Left-hand side shows schematic diagram of biosensor.
; i-0.5
E -1.0 4
-
Y
-1.5
L
a 0 -2.0
-2.5
-0.8
-0.6
-0.4
-0.2
0.0
Voltage (volts) Figure 2. Cyclic voltammagram of phenol biosensor in the absence (A) and presence (6) of -8.5 ppm phenol vapor at 75% RH and 25 "C. Scan rate, 0.005 V/s vs CC+QRE. Inset shows an amperometric response of the phenol biosensor on exposure to 1.6 ppm phenol vapor for 100 s at 40% RH, 25 OC. Poise potential, -700 mV vs
CCCORE. The phenol permeation rate of the gas rigwas measured using the method of Ihna"anno An impinger was used to trap the phenol vapor in 0.1 N NaOH at point B F i r e 2). The phenol was then determined spectrophotometrically (UV-visible spectrophotometer) with gnitroaniline at 530 nm. The permeation rate was then back calculated. This measured phenol permeation rate agreed closely with the estimated permeation rates calculated from data provided by Vici Metronics Inc. It was not possible to use the entrapment method to measnre phenol vapor concentrations in the ppb range due to time limitations, and concentrations in this range were estimated using data supplied by Vici Metronics Inc. Actual phenol vapor concentrations were calculated using (30)Leithe. W. Awlysis of drpdl*tm!x Ann Arbor-HumphreyScience Publishers: Ann Arbor, MI,1970: p 247. 3924 Ana/yiica/Chemistry, Vol. 67, No. 21, November 1, 1995
the measured phenol permeation rate at the set temperature and the input flow rates of the lowhumidity air containing phenol vapor and the dilutant humidified air. Concentrations were calculated as parts per million by volume (ppm) or parts per billion by volume (ppb). Two three-way valves were used so that the low relative humidity input air could be switched between clean air and air containing phenol vapor without affecting humidity or flow rate. This prevented fluctuations in the flow rate and relative humidity. All interconnecting tubing on the gas rig was composed of short length FlTFilined tubing @Idrich, Poole, W. AU work was carried out in a fume hood. Apparatus and Measurements. AU electrochemical measurements were carried out using an Autolab Pstat 10 electre chemical analyzer (EcoChemie, Utrecht, the Netherlands). Pmcedum. The biosensor was inserted in a flow-through chamber Wdrich) which was thermostated at 25 "C by a circulating water bath. A poised potential of -700 mVwas applied between the worldng electrode and the CC+QRE. A steady base line current was established in air of the appropriate humidity with no phenol vapor, and the biosensor was then exposed to air containing phenol vapor for a measured time period (100 s unless otherwise stated) using the two three-way valve control system. The input air was then switched back to non-phenol air. Temperature and humidity measurements were made by a Vaisala HM34 rela!ive humidity and temperaturemeter (RS Components), inserted into the test chamber near the biosensor. The response to phenol vapor was evaluated by calculating the difference between the base line current and the amperometric response. The activity of the biosensor was defined as the amperometric response (minus background current) recorded after 100 s of exposure to phenol vapor and was measured in nanoamperes per 100 s. The maximum amperometric response was defined as the level (minus background current) the current reached after the biosensor had been exposed to phenol vapor for 100 s. This is similar to peak response. This measnrement was independent
Table 1. Response Times and Maximimum Amperometric Responses of Phenol Blosensors as a Functlon of Gel Thickness.
gel thickness (mm)
response timeb
maximum responsec (A)
0.14 f 0.025 0.23 f 0.039 0.34 f 0.059 0.45 f 0.079
135 f 13 143 f 40 191 f 37 253 f 36
68.5 f 5.4 41.3 f 10 9.7 f 2.8 2.4 f 1
70 60 50 40
30
In column 1, f figures are the limits of measurement; in columns 2 and 3, f figures are 1standard deviation. Biosensors were exposed to 0.53 ppm phenol vapor for 100 s at 40%RH, 25 "C. Time to reach
20 10 0
maximum amperometric response. Average of four measurements.
0
of time and was measured in nanoamperes. The response time was defined as the time taken for the biosensor to reach its maximum amperometric response, with T = 0 when phenol exposure had just begun. As phenol exposure was normally 100 s, the response time cannot be less than 100 s. RESULTS AND DISCUSSION Cyclic Voltammetry. Cyclic voltammograms of the biosensor system in the presence and absence of phenol vapor (Figure 2) were recorded. A large increase in cathodic current at a p proximately -700 mV (vs CC+QRE) was observed when the biosensor was exposed to phenol vapor (8.5 ppm). When a gold microdisk electrode with a Ag/AgCl reference electrode was used as the transducer instead of the gold two-electrode microband array, a large increase in current at -150 mV (vs Ag/AgCl) was observed. This reduction potential for benzoquinone agrees with previously published value^,^*-^^ indicating that benzoquinone is being reduced at -700 mV (vs CC+QRE) on the interdigitated gold microband electrode. This would seem to indicate that the QRE is operating at -550 mV vs Ag/AgCl when measuring quinones. AmperometricResponse. The Figure 2 inset shows a typical amperometric response of the biosensor on exposure to phenol vapor. The biosensor showed no response to exposure to a range of solvent vapors including isopropyl alcohol, chloroform, and acetone. The response time and magnitude of the amperometric response depend on the gel thickness (Table l),relative humidity 0 ,and phenol vapor concentration. The response time is shortest for a thin gel and a high relative humidity. As glycerol is hygr0scopic,3~a high relative humidity would increase the water content in the gel, increasing diffusion coefficients and decreasing the response time. Calibration. The sensor was calibrated over a range of phenol vapor concentrations at three different humidities. Figure 3 shows a calibration curve at 40%RH,and Table 2 summarizes details of the biosensor response at different humidities. The biosensor response to phenol vapor is dependent on the relative humidity. This is not a limitation, because as the background current is also dependent on the relative humidity (Table 2), the biosensor is able to measure the relative humidity and take its affect into account. Many commercial humidity sensors are based on a similar principle: that the conductivity of electrolyte in a hygro(31) Skladal, P. Collect. Czech. Chem. Commun. 1990,56, 1427-1433. (32) Cosnier, S.; Innocent, C. 1.Electroanul. Chem. 1992,328, 361-366. (33) Cosnier, S.; Innocent, C. Bioelectrochem. Bioenerg. 1993,31, 147-160. (34) The Merck Index, 9th ed.; Merck & Co., Inc.: Rahway, NJ, 1976.
2
4
6
8 1 0 1 2 1 4
Phenol Concentration (ppm) Flgure 3. Amperometric response of phenol biosensors on exposure to a range of phenol vapor concentrations. A minimum of five measurements were recorded at each phenol vapor concentration. Error bars are f l standard deviation. Conditions: 40% RH, 25 "C; poise potential, -700 mV vs CC+QRE. Table 2. Effect of Relative Humidity on Phenol Biosensor Sensitivity and Background Current.
RH (%)
sensitivity (nA/ppm)
correlation coefficient
background current (nA)
28.5 5.86 2.39
0.977 0.994 0.950
9.0 3.5 1.6
64 44 27
nEach sensitivity value is the slope of a line through 5 x 5 measurements over the phenol vapor range 0.5- 14 ppm. Temperature, 25 "C.
14 n
*n
-
12-
E
4 10-
d c
5
8 6;
4-
0
20
40
60
80
100
Time (Minutes) Flgure 4. Amperometric response of phenol biosensors on repeated to 1.4 ppm phenol vapor. Exposure time was 100 s. Conditions: 40%-47% RH, 22-26.5 "C;poise potential, -700 mV vs CC+QRE.
scopic medium will be directly dependent on the relative humidity of the atm0sphere.3~ Repeatability. The biosensor could be used for successive measurements of phenol vapor (Figure 4). The maximum amperometric response declined in direct proportion to the assay number. This is thought to be due to the fact that repeat exposures to phenol vapor were performed before the biosensor current had reached its original background level, indicatingthat (35) Yamozoe, N. Sens. Actuators 1986,10, 379-398.
Analytical Chemistty, Vol. 67, No. 21, November 1, 1995
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20 I 18 16 1412108642-
1
-
n
8 0
a d E
3 -
18
'v
600
9
f
500
28
n
Y
400
F 9)
300 0 0
4 2 0
0 0
20
40
60
80 100 120
200 100
0
20
Time (hours) Figure 5. Amperometric response of phenol biosensors on exposure to 0.5 ppm phenol vapor over a continuous 5 day period. Exposure time was 100 s. Conditions: 43% RH, 25 "C; poise potential, -700 mV vs CC+QRE.
the electrode was still reducing quinones at the electrode surface. Quinone polymers 'are thought to result in enzyme inactivation36-38 and could react with phenol and catechol to form complexes. Darkening of the biosensor gel (indicative of melanin formation) after exposure to phenol vapor was apparent. The presence of quinone polymers could affect activity and electrode surface activity (by adsorpsion), which could account for the 17.5%decline in maximum amperometric response after nine consecutive exposures.. However, if the biosensor is repeatedly exposed to phenol vapor, but with a time period between subsequent exposures long enough to ensure complete reduction of quinone species (Figure 5), then little or no decline in the maximum amperometric response occurs. A time period stdicient to allow complete or near complete reduction of quinone species to inert melanin polymers would avoid enzyme inactivation due to quinone species or cross reactions between quinone species and phenol or catechol. Adsorption of quinone species is thought to account for the increase in background current apparent in Figure 4. Stability. The maximum amperometric response to 0.5 ppm phenol vapor remained approximately constant over the 5 day period (Figure 6). Fluctuations in peak height corresponded to uncontrollable fluctuations in temperature and relative humidity over the course of the experiment. The time required for the current to reach its maximum amperometric response (the response time) increased over the course of the experiment. The water content of the gel remained ,constant over a 5 day period, so the increase in response time is most likely due to enzyme inactivation. As enzyme inactivation proceeds, the rate of product (benzoquinone) production decreases, corresponding to a d e crease in activity (as shown in Figure 6). However, the final amount of benzoquinone produced for a given quantity of phenol is not affected by enzyme inactivation; hence, the maximum amperometric response does not change with time. Only the rate of production of product and not the concentration of product is affected by enzyme inactivation. Hence, for the phenol vapor (36)Ingraham, L.L.;Corse, J.; Makower, B. J. Am. Chem. SOC.1952,74,26232630. (37)Asimov, A; Dawson, C. R Anal. Chem. 1950,72,820-828. (38)Vanneste, W. H.;Zuberbuhler, 2. In Molecular mechanisms of oxygen actiuation; Hayaishi, 0.; Ed.; Academic Press: New York, 1974; pp 371404.
3926 Analytical Chemistry, Vol. 67, No. 21, November 7, 7995
8
40
60
80
100 120
Time (hours) Figure 6. Profiles of the maximum amperometric response, response time, and activity on exposure to 0.5 ppm phenol vapor for 100 s over a 5 day period. Conditions as in Figure 5.
gr
0
1
2
3
4
5
6
Time (hours) Figure 7. Amperometric response of a phenol biosensor to 30 ppb phenol vapor. Exposure time, 45 min. Conditions as in Figure 5.
biosensor, although enzyme activity apparently declines over time and results in a slower conversion of phenol to catechno, the magnitude of the maximum amperometric response is not affected. Limit of Detection. The limit of detection GOD) depends on the relative humidity and the exposure time to phenol vapor. At 40%RH,the LOD was calculated to be 29 ppb phenol for an initial exposure time of 100 s (a signal-to-noise ratio of 3). For repeated exposures, the LOD would be somewhat less, as the drift in background current observable on multiple exposures (Figure 4) would affect the LOD. If the exposure time is increased, then the LOD will depend mainly on the exposure time, as the glycerol gel will concentrate the phenol vapor until suffcient phenol has been trapped to generate a signal. Experiments showed that phenol is very soluble in glycerol, more so than water: glycerol will dissolve up to 50%of its own weight in phenol, compared to 10%for water. Time is the main limiting factor. Figure 7 shows the response of the phenol biosensor on exposure to 30 ppb phenol for a period of 45 min. Although 30 ppb phenol is the lowest vapor level generatable at present, it is possible that the sensor will measure phenol at lower concentrations if suffcient time is allowed.
Anode
OH
OH
0
Cathode Figure 8. Redox recycling of the catechoWquinone couple at the
electrode surface. CONCLUSIONS The sensitivity of the microbiosensor to phenol is postulated to be due to redox recycling. Redox recycling has been reported in the literature at microband arrays" and has been spedcally reported for the catechol/qninoneredox couple." The oxidation of catechol to quinone at an anodic microelectrode is followed by diffusion of the quinone to the neighboring cathodic microelectrode, where it is reduced back to the catechol F i r e 8). This recycling prohably continnes until quinone polymerization products are formed. The factthat both microelectrode arrays are (39)Johnson, D. C.; Ryan, M. D.; Wilson. G. S. Anal. Cham. 1988,60,14m167R ___._
(40)Hintsche, R F'aesehke. M.; Wollenbqer, U.;Sehnakenkg, U.;Wagner, B.: Us=, T.Biosm. Bioelcchon. 1994.9,697-705. (41) Bond, A M.; Feldberg, S. W.; Greenhill, H. B.; Mahon, P. I.; Colton, R Whyte, T.Anal. C h m . 1992,64,1014-1021. (42)Dressman. S.F.; Michael, A C. Anol. C h n . 1955, 67,1339-1345. (43)Michael, A C.; Wright", R M. Anal. Chcn. 1989.61.272-275. (44)Sullenberger, E.F.; Michael. A C. A i d . Ckm. 1993,65.2304-2310. (45)Bond, A M.: Lay.P. A I. ElemoamI. Chen. 1986,199,285-295.
involved in redox recycling suggests that the potential of the QRE will change as the catechol/quinone couple replaces the original species. We believe that this !ransition period is very short and that the catechol/quinone couple reaches equilibrium quickly. Cyclic voltammograms at different times show very little change in peak potential, indicating that either the catechol/quinone couple establishes itself very quickly or the shift in potential of the QRE is small. Although the use of a QRE is somewhat unorthodox, there are many reports of their use in electroRedox recycling amplifies the signal considerably. In one repott, it was calculated that the signal was amplified by a factor of 30 for benzoquinone redox recycling," although a strict comparison is not possible here, as the authors controlled the reference potentials of their microelectrode relative to a &/&I in this report This amplification system, in conjunction with a hydrogel which can concenhte phenol from the vapor phase, results in a biosensor capable of measuring low levels of phenol vapor which could function admirably in the field of health and safety applications. ACKNOWLEWMENT The authors are grateful to the European Commission Directorate-General XII, Science, Research and Development Environmental Research program for generous sponsorship of thisproject Many thanks to Dr. W.J. Aston and Dr. B. Hobbs of City Technology Ltd.,to Dr. J. MacAleer of Ecossensors Ltd., and to Miss C. O'Sullivan, Dr. S. M s , Dr. S. Saini, and Dr. G. Pilidis of ELVIM for their very helpful advice and assistance.
Received for review May 9, 1995. Accepted August 4, 1995."
AC9.504432 e Abshact
published in Adunncc ACS Ab$fmch, September 15, 1595.
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