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Anal. Chem. 1983, 55,1582-1585
minescence (7)and these should also be applicable to analytical methods. A report from this laboratory on an analogous luminescent reaction involving reduction of Ru(bpy),2+in the presence of peroxydisulfate has appeared recently (16). ECL and chemiluminescence reactions based on this reaction also appear possible and are under investigation. Registry No. R~(bpy)3~+, 18955-01-6;oxalic acid, 144-62-7.
LITERATURE CITED Hodgkinson, A. “Oxallc Acid in Blology and Mediclne”; Academic Press: New York, 1977; pp 230-253. Hodgkinson, A. ”Oxalic Acid In Blology and Medlclne”; Academic Press: New York, 1977; pp 83-96. Murray, J. F., Jr.; Nolen, H. W., 111; Gordon G. R.; Peters, J. H. Anal. Blochem. 1982, 121, 301-309. Thrlvlkraman, K. V.; Keller, R. W., Jr.; Wolfson, S. K., Jr.: Yao, S.J.; Morgenlander, J. C. Bloelectrochem. Bloenerg. 1982, 9 , 357-364. Chang, M. M.; Saji, T.; Bard, A. J. J. Am. Chem. SOC. 1977, 99, 5339.
(6) Rublnsteln, I.; Bard, A. J. J . Am. Chem. SOC. 1981, 103, 512. (7) Rublnsteln, I.; Bard, A. J. J. Am. Chem. SOC. 1981, 103, 5007. ( 8 ) Nonldez, W. K.; Leyden, D. E. Anal. Chim. Acta 1978, 96, 401. (9) Keszthelyl, C. P.; Tachikawa, H.; Bard, A. J. J. Am. Chem. SOC. 1972, 94, 1522. (10) Tokel-Takvoryan, N. E.; Hemingway, R. E.; Bard, A. J. J. Am. Chem. SOC. 1973, 95, 6582. (11) Auses, J. P.; Cook, S. L.; Maloy, J. T. Anal. Chem. 1975, 47, 244. (12) Lytle, F.; Hercules, D. M. Photochem. Photobiol. 1971, 13, 123. (13) Kobos, R. K.; Ramsey, T. A. Anal. Chlm. Acta 1980, 721, 111. Hodgkinson, A. “Oxallc Acid In Biology and Medicine”; Academic Press: New York, 1977; pp 173-174. Levlnson, S. A,; MacFate, R. P. “Clinical Laboratory Diagnosis”. 4th ed.; Lea and Febiger: Philadelphla, PA, 1951; pp 387-388. Whlte, H. S.;Bard, A. J. J. Am. Chem. SOC. 1982, 104, 6891.
RECEIVED for review December 27, 1982. Accepted April 26, 1983. The support of this research by the Army Research Office and the National Science Foundation (Grant No. CHE7903729) is gratefully acknowledged.
Enzyme Electrode and Thermistor Probes for Determination of Alcohols with Alcohol Oxidase
*‘ Bengt Danlelsson, Carl Frederlk Mandenlus, and Klaus Mosbach
George 6. Gullbault,
Pure and Appiied Biochemistry, Chemical Center, University of Lund, Box 470, 5-220-07,Lund, Sweden
Enzyme electrode and thermistor probes have been prepared by chemically blndlng alcohol oxidase from Candlda bo/d/n//. By use of the electrode probe (enzyme lmmoblllzed onto an O2 electrode) from 1 to 100 mM methanol, ethanol, or butanol can be assayed wlth a preclslon of about 2.0-2.5%. With an enzyme thermistor probe (enzyme lmmoblllzed onto glass beads) in a flow system, as llttle as 0.2 mM, with llnearlty to 2.0 mM, of methanol, ethanol, and butanol can be assayed wlth a preclslon of about 1.5%. The thermlstor probe system is quite stable for several hundred assays and the electrode probe, for about I 0 0 assays.
Blood alcohol analysis has been the subject of considerable interest, particularly in legal cases where there is a need for simple, quick analysis. Alcohols have been det,ermined by gas chromatography (1-3) and spectrophotometry ( 4 , 5 ) . Some assays have used enzymes like alcohol dehydrogenase and a solution spectrophotometric procedure ( G , 7 ) . Guilbault and Lubrano (8) have attempted to build an enzyme electrode probe, amperometricdy by detecting the hydrogen peroxide formed during the enzymatic reaction. Because of low activity, the enzyme could not be immobilized. Guilbault and Nanjo (9) described an enzyme electrode that could be used in concentrations as low as 1 mg %, but the electrode used an enzyme that was not commercially available. In recent years bioanalytical devices have been developed, in which the %ensing” enzymes are placed in close proximity to the transduceran electrode or a thermistor probe (IO,11). Apart from the obvious possibility of repeated use of the On Sabbatical leave from the University of New Orleans, Chemistry Department, New Orleans, Louisiana 70148. Address all correspondence t,o this address.
immobilized biocatalyst, the advantages gained by such an arrangement include higher sensitivity, quicker response, stabilization of the enzyme, and the possibility of applying such devices in continuous flow operations. The calorimetric principle of analysis possesses unique universality, since most enzyme reactions are accompanied by considerable heat evolution in the range of 5-100 kJ/mol ( 1 0 , I I ) . A number of applications to the analysis of biochemical systems using calorimetric measurements have been described (10-14). In this paper we report on the use of a new alcohol oxidase enzyme, Candida boidinii, immobilized by chemical bonding, for the assay of methanol, ethanol, and butanol. Both an enzyme electrode probe and a continuous flow approach, using a column of bound enzyme and a thermistor probe, are described and compared. The use of this enzyme in a column with an O2electrode for assay of ethanol has been reported by Gulberg and Christian (16).
EXPERIMENTAL SECTION Reagents. Catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase E.C. 1.11.1.6, beef liver, Boehringer, a suspension in water of 1300 000 U/mL). Alcohol oxidase (a1cohol:oxygen oxidoreductase, E.C. 1.1.3.13), from Candida boidinii, obtained from Boehringer Mannheim, had a specific activity of 6.5 U/mg of enzyme protein. The enzyme (50 units) was dialyzed against 0.1 M phosphate buffer, pH 7.0, overnight. The enzyme solution was then freeze-dried. Methanol, ethanol, l-propanol, and 1-butanolwere all obtained from Merck and were C.P. analytical grade. Water was doubly distilled. Immobilized Enzyme Reagents. The enzyme electrode probe was prepared by placing a layer of pig intestine (obtained from Universal Sensors) onto a Universal Sensors O2 electrode (Universai Sensors, P.O. Box 736, New Orleans, LA 70148), and affixing with the rubber O-ringprovided for the O2electrode. The intestine is wet with doubly distilled water, then 10 p L of a 5% albumin solution is added to the membrane. Into this 10 pL is then dissolved 2 mg of the freeze-dried alcohol oxidase, and
0003-2700/83/0355-1582$01.50/00 1983 American Chemical Society
ANALYTICAL
towheat
CHEMISTRY, VOL. 55, NO. 9,
AUGUST 1983
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AT (m'C1 6
0.6
n
--
waste inlet
I
4
0.4
i I
il
10 min
I 2
I
I
I
0 Time
Flgure 2. Recorder tracing In the enzyme thermlstor probe determi-
nation of ethanol with an alcohol oxidase/catalase column.
Figure 1. Enzyme thermistor with aluminum constant temperature jacket: (1)polyurethane Insuilation; (2) Plexiglas tube with bayonet lock for column insertion; (3) thermostated aluminum cyllnder; (4) heat exchangers; (5) enzyme column; (6) thermistor attached to a gold capillary; (7) column outlet (in this thermostated metal block the temperature Is controlled to withln f0.002 " C on 'equilibratlon).
following dissolution 0.5 ILLof 12.5% glutairaldehyde is added. Following 30 s of stirring on the surface of the membrane with a glass rod (Caution: be careful not to rupture the membrane) the enzyme electrode is pliaced on the bench to dry for about 2-4 h. Between use, the electrode is stored in phosphate buffer, pH 7.0, at room temperature. In practical preparation of the electrode, it is suggested that onto the outer jacket of the O2electrode be placed first the gas membrane, then the pig intestine, followed by affixing with rubber O-ring. The attachment of the enzyme is effected, and only then is the outer jacket screwed onto the base electrode, after filling with the solution provided with the Universal Sensors electrode. When the enzyme layer becomes useless following prolonged usage, it can be replaced with a new membrane. The enzyme column used with the thermistor probe is prepared as follows: To 3 g of alkyllamine glass beads (CPG-10, Corning 1350 A pore diameter, BDH, Poole, England) is added 15 mL of 2.5% (25% diluted 1:lO with 0.1 M phosphate buffer, pH 7.0) glutaraldehyde. Swirl and let stand 1 h with periodic swirling. Filter on a glass filter and wash the activated beads with 500 mL of doubly distilled water. To 1 mL of beads add 3 mL of 0.1 M phosphate buffer containing 50 units of the dialyzed enzyme with 100 rL of catalase solutioii. Mix well and put on a shaker in a cold room overnight. Wash the beads with 200 mL of 0.1 M phosphate buffer, pH 7.5, faillowed by 200 mL of 0.5 M NaC1. This preparation is loaded in a small plastic column (1 mL volume) which is then mounted inta a Plexiglas holder (Figure 1)that can be inserted in the enzyme thermistor unit. Similarly, a second enzyme column is prepwed exactly as above but without the catalase solution. Apparatus. Enzyme Electrode. The electrode probe, prepared as described above, can be plugged into a Universal Sensors adaptor, which is a small tubular device designed both to apply the constant potential required by the O2probe and to convert the output current resulting into a voltage that can be read directly on any pH-voltmeter. In this case an Orion Research pH/millivolt meter 811 was used. Thermistor Probe. The column of immobilized enzyme is mounted into the enzyme thermistor device (10)as shown in Figure 1. A thermostated (30 "C)aluminum cylinder contains
the heat exchanger capillary tubes and provides a constant tem"C) environment to the enzyme column (0.2-1 perature (h2 x mL). There are two parallel fluid lines, which could be used either independently or with one of them as a reference system. The sample/buffer is pumped through the enzyme thermistor unit with a peristaltic pump at a flow rate of 0.70 mL/min; a 0.5-mL sample size loop was used to introduce the sample. The temperature at the column outlets is continuously registered by a thermistor connected to a sensitive and stable Wheatstone bridge. At the highest amplification the recorder output is 100 mV for "C. In most studies a full scale a temperature change of sensitivity of "C has been found sufficient. Electrical and other calibrations have shown that as much as 80% of the total heat evolved in this "semiadiabatic" device can optimally be registered as a change in temperature (IO). This implies that for a given substrate present at a concentration of 1 mM and with a molar enthalpy change for the enzymic reaction of 80 kJ/mol, a peak height corresponding to 1V2"C or higher will be obtained and requires a temperature resolution of "C to give 1% accuracy in the measurement. This unit allows two different columns to be used (provided no reference column is required), thus permitting the simultaneous analysis of two components in a sample. The enzyme thermistor can quickly (i.e., within 15 min) be recharged with a new enzyme column when required. Procedure. Electrode Probe. Immerse the enzyme electrode into 3 mL of phosphate buffer, 0.1 M, pH 7.5, containing the sample to be analyzed. Record the total potential change, AE, with time. Prepare a calibration plot of mV/min vs. alcohol concentration. Thermistor Probe. Plug the column of either alcohol oxidase or alcohol oxidase/catalase into the apparatus. Equilibrate by pumping phosphate buffer 0.1 M, pH 7.5, at a flow rate of 0.7 mL/min for 30 min. When a stable base line is reached, introduce via a sample loop valve 500 pL of sample and record the AT with time (Figure 2). Prepare calibration plots of AT/min (recording at high speed and estimating the maximum slope) and also AT vs. alcohol concentration. RESULTS AND DISCUSSION Specificity. Alcohol oxidase is specific for lower primary alcohols (16-18), according to the equation
RCHZOH
+02
alcohol
RCHO
+ HZOP
(1)
In a previous study Guilbault and Lubrano (8)showed that the substrate selectivity data published by Janssen et al. (16, 17) were in error, due to an interaction between the aldehyde and the H20zproduced in the primary enzymatic reaction (eq 1). Hence, the data calculated on the basis of the HzOz-dye indicator reaction showed incorrectly that methanol was a better substrate for the Basidiomycetes enzyme. Data obtained on the basis of dissolved O2 uptake gave the correct pattern, ethanol being a 4000 times better substrate (8). In this study we attempted to show the substrate selectivity of this new enzyme from another organism, Candida boidinii,
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
Table I. Substrate Specificity of Alcohol Oxidases
alcohols
re1 vela
methanol ethanol 1-propanol 1-butanol
0.025 100 11 21
Basidiomycetes re1 vel b 357 100 19 7.5
Poria
Candida boidiniid
contigua Km,CmM
Km,bmM 1.52 10 54.6 133
Km, mM
re1 vel
0.2
125
1.0
100 7.1 45
8.3 21.3
0.4 2.1 65.0 2.8
a Calculated on basis of dissolved 0, uptake at -0.6 V, ref 9. Data of Janssen, ref 16 and 17. Data of Bringer et al., ref 19, measurement of H,O,, no re1 vel measurements reported. This study calculated on basis of 0, uptake.
I
L .P‘
20
40 Alcohol
60 Conc
80
100
and to calculate the Km values from Lineweaver-Burke plots, since no formal published values have appeared. (The Km values were determined by using the initial slopes of the temperaturetime curves. This method was found to correlate well with more “orthodox” procedures for Km calculations.) Gulberg and Christian (15) have described the use of this enzyme for assay of ethanol but gave no data on substrate selectivity of the biocatalyst. As shown in Table I and Figures 3 and 4, methanol is indeed the best substrate for the Candida enzyme (unlike the Basidiomycetes), the order of reactivity being methanol > ethanol > butanol > propanol. These data were calculated both from the enzyme electrode studies, in which the rate of dissolved O2uptake is measured, and also from the heat data, in which the primary reaction of the alcohol with enzyme is monitored. Thus, the data reported should reflect the true reactivity of the substrate with the Candida enzyme. Sensitivity. Thermistor Probe. Linearity was obtained between the concentration of the four different alcohols tested by using both the rate of change in the temperature with time method (Figure 3) and the total heat change in m “C procedure (Figure 4), with both the alcohol oxidase columns and the alcohol oxidase/catalase mixed column. With either column linearity extends from 0.1 to 2.0 mM concentrations, and assays can be performed with a CV (RSD) of about 1.5%. The alcohol oxidase/catalase columns have the advantage of better stability and are recommended (see section on Stability). Enzyme Electrode. The electrode probe is best used in the rate mode, with a measurement of the rate of change in the dissolved O2content with time, AmV/min. Poor correlation was found between alcohol concentration and the total potential (current) change. Linearity was obtained between the rate, AmV/min (actually the current change Ai/min), and ethanol from 1to 10 mM, methanol from 2 to 100 mM, and butanol from 10 to 100 mM, with a CV of 2.0%. Poor results were obtained with propanol, the same as obtained with thermistor probe. Ev-
0
d
-
(mM)
Flgure 3. Calibration plots for methanol (a), ethanol (b), butanol (c), and propanol (d) obtained with an alcohol oxidase column measuring the rate of reaction (AT/min).
,+d: .........................................................
....................
1.0
2.0 Alcohol
3.0 Conc.
4.0
(mM)
Figure 4. Calibration plots for methanol (a),ethanol (b), butanol (c), and propanol ( d ) obtained with an alcohol oxidase/catalase column measuring the total heat change.
~
100
> I
4 u
ds 50
I 0
30
;
10
BO 90 Number of assays
20
30
120
40
Number of d a y s
Flgure 5. Stability curves for an alcohol oxidasekatalase (a) and an alcohol oxidase (b) column. Response to 1.O mM ethanol plotted as percent activity vs. number of assays. idently the alcohol oxidase shows little reactivity with this alcohol. Stability. The operational stability of the coimmobilized alcohol oxidase/catalase column was quite good, as shown in Figure 5. The alcohol oxidase columns were less stable, probably due to the destruction of alcohol oxidase activity by the peroxide produced. It has previously been found that coimmobilizing catalase or including activated carbon or manganese oxide in the enzyme column substantially increases the stability of immobilized alcohol oxidase (20). A similar effect was noted for another sulfhydryl group containing enzyme, glucose, by Prenosil(21). The catalase present destroys the peroxide produced in the enzyme layer, and hence prolongs the stability. Curve b of Figure 5 would be expected to begin at -70% because the temperature from the alcohol oxidase reaction alone is lower than the alcohol oxidase/catalase reaction.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
The sudden jump in activity from 33 to 40 days in Figure 5 is unexpected, and greater than the cvithin-day and dayto-day deviations ( - 2 4 % ) . Perhaps this is due to a creation of additional diffusional channels in the immobilized enzyme that allows greater reactivity. The storage stability is excellent for both alcohol oxidase cdumns-over a period of 3 months h o s t no loss of activity is observed. The electrode probe was less stable than the column because it contains (1) much less active enzyme, being a thin membrane, and (2) no catalase was added, since the rate of O2 uptake is monitored. Only about 100 assays were possible; however, when not in w e the electrode probe was active over several months. Interferences. An assay of blood ethanol was attempted to demonstrate a typical application of the new probe described. Blood is a complex matrix, containing several hundreds of compounds. IJnlike the alcoholl oxidase from Basidiomycetes (9), lactic acid and other hydroxy acids are not oxidized by the Candida enzyme and do not present a problem. Likewise, none of the compounds present in blood that are electrooxidizable (ascorbic acid, cysteine, glucose, uric acid) react,with the systems described. Since only a small amount of serum (0.025 mL) is used, the dissolved oxygen level already present in the 3.0 mL of buffer remains ess,entially undisturbed on addition of sample. Unfortunately, the enzyme does show some activity with methanol, and this coisld cause a problem in some assays. To test the validity of the results, five samples of blood serum, with ethanol added to levels of P 2 5 mg % (1-5 mM), were assayed. Values obtained by GC were almost identical with the results obtained with the electrode probe (within about 2.5%). In assay of blood serum samples; however, it is necessary that a calibration curve be made using a freezedried serum preparation like Monitrol (Dade, Miami) with alcohol added. Since the normal concentration of alcohol in blood in legal questions of “drunk-driving” is 40-400 mg per 100 mL (about 10-100 mM) very small samples of bloocl (10-25 pL) are required for excellent results. Results obtained using the enzyme thermistor probe and both serum from freshly drawn blood (veneous), as well as DADE serum spiked with various amounts of EtOH, showed excellent results analogous to those of the electrode approach. Since 5 mg % (or 1mM) lies in the upper linear range, samples
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can be diluted 10-100 in the thermistor approach. Only 10-50 in required, the same as the electrode method. Comparison of Techniques. Greater sensitivity is obtained with the thermal probe (0.1 mM vs. 1mM of alcohol with the electrode) and both methods have about the same precision 1.5-2 % ). The enzyme thermistor is suitable for continuous monitoring and in automated systems and has a better operational stability, while the electrode method utilizes simpler equipment and simpler analytical procedures with discrete samples. p L of sample
ACKNOWLEDGMENT George G. Guilbault is grateful for a guest appointment at the University of Lund that permitted a cooperative effort to be affected. We thank Lena Persson for her technical assistance in support of the project. Registry No. Alcohol oxidase, 9073-63-6;methanol, 67-56-1; ethanol, 64-17-5; butanol, 71-36-3. LITERATURE CITED (1) Kung, J. T.; Whltney, J. E. Anal. Chem. 1966, 33, 1505-1507. (2) Bluestein, C.; Postmanter, H. N. Anal. Chem. 1966, 38, 1865-1869. (3) Lyons, H.; Bard, J. Clin. Chem. (Winston-Salem, N.C.) 1964, 10, 429-432. (4) Reid, V. W.; Salmon, D. G. Ana/yst (London) 1855, 8 0 , 704-707. (5) Mantel, M.; Anbar, M. Anal. Chem. 1964, 36, 936-937. (6) Bonnichsen, R. K.; Theorell, H. Scand. J. Clin. Lab. Invest. 1951, 3 , 58-80. (7) Bonnichsen, R. K.; Lundgren, G. T. Acta Pharmacol. Toxlcol. 1957, 13, 256-260. (8) Gulibault, G. 6.; Lubrano, G. J. Anal. Chim. Acta 1974, 69,189-195. (9) Nanjo, M.; Gullbault, G. G. Anal. Chim. Acta 1975, 75, 169-180. (10) . . Danielsson, H.: Mattlasson. B.: Mosbach.. K. ADD/. Blochem. Bloena. 1981, 3 , 97-143. (11) Mosbach, K.; Danielsson, B. Anal. Chem. 1981, 53, 83A-94A. (12) Spink, C.; Wadso, I. Methods Biochem. Anal. 1976, 23, 1-159. (13) Martin, C. J.; Marini, M. A. CRC Crit. Rev. Anal. Chem. 1977, 8 , 221-286. (14) G i m e i J . K. Anal. Chlm. Acta 1980, 718, 191-225. (15) Gulberg, E. L.; Christian, G. D. Anal. Chim. Acta 1981, 123, 125-131. (16) Janssen, F. W.; Ruelius, H. W. Blochim. Biophys. Acta 1868, 151, 330-342. (17) Janssen, F. VV.; Kerwln, R. M.; Ruelius, H. W. Blochem. Biophys. Res. COmm~n.1965, 20, 630-635. (18) Tanl, Y . ; Mlyu, T.; Nishlkawa, H.; Ogata, K. Agric. Biol. Chem. 1972, 36, 68-71. (19) Bringer, S.; Sprey, B.; Sahm, H. Eur. J. Biochem. 1979, 701, 563-566. ( 2 0 ) Mandenius, C . F.; Johnson, D.; Danlelsson, B.; Mosbach, K., unpublished results, University of Lund, 1983. (21) Prenosil, J. Blotechnol. Bioeng. 1879, 27, 89-109.
..
I
RECEIVED for review March 4,1983. Accepted April 25, 1983. This project was in part financed by Grant No. 79-3887 from the National Swedish Board for Technical Development.
