Anal. Chern. 1980, 52, 2109-2112
2109
Reagentless Amperometric Lactate Electrode H613ne Durliat and Maurice Comtat * Laboratoire de Chirnie Physique et Electrochirnie, Laboratoire associ6 au CNRS No. 192, Universit6 Paul Sabatier, 118, Route de Narbonne, 3 1077 Toulouse Cedex, France
The lactate-specific, amperometric, enzyme electrode based on the redox couple hexacyanoferrate(II)/hexacyanoferrate( 111) has been modifled by replaclng thls couple by the ferriferrocytochrome c couple. It makes possible the measurement of lactate concentrations In the range 0.05-7 mM, with an accuracy of about 2 % and a measurement tlme of about 1 min. The regeneration, between two readings, of the ferricytochrome, by electrolyds at constant potentlal, requires 2-5 mln and so does not allow readings to be made at such a rapid pace as with the hexacyanoferrate(II1). The results of thls work led to the development of a new lactate-lonspeciflc electrode which uses no cofactor and Is based on the direct electron transfer between the enzyme molecule and the metal forming the electrode. A study of the effects of temperature and the use of this electrode In a devlce for contlnuous lactate monltorlng show that In vivo measurement of blood lactate by this method is a practical posslblllty.
The measurement of blood lactate is of interest in numerous pathological conditions (including state of shock, respiratory insufficiency, and heart disease). The concentration of this ion in the blood is undoubtedly the best biochemical criterion for judging the severity of serious circulatory deficiencies. The most widely used measurement technique brings into play the oxidation of t h e lactate ion by the NAD+ cofactor in the presence of muscle lacticodehydrogenase (1)
CH3CHOHCOO-
+ NAD’
-CH3COCOO+ NADH + H+ LDH
,
(1)
together with a spectrophotometric measurement of the amount of NAD+ coenzyme reduced in this reaction. This method, which requires a preliminary preparation of the blood sample, is tedious and expensive. In recent years, a certain number of lactate-ion-specific enzyme electrodes have been developed. Most are based on the reaction in which the lactate is oxidized by potassium hexacyanoferrate (111) CH3CHOHCOO-
-
+ 2Fe(CN)63CH,COCOO- + 2Fe(CN)64- + 2H+ (2)
the hexacyanoferrate(I1) formed being detected either by constant-potential amperometry (2-4) or by zero-current potentiometry (5). An electrode qualified as “reagentless” was proposed elsewhere by Blaedel and Jenkins (6): it brings into play reaction 1and the reoxidation of the NADH at a vitreous carbon electrode. T h e electrode developed in this laboratory is based on reaction 2, with constant-potential amperometric detection of the hexacyanoferrate(I1) formed (7). I t makes use of a lacticodehydrogenase extracted from the Hansenula anomala strain of aerobic yeast which, when purified (8),displays a high stability and a high specific activity for the oxidation of lactate. After optimization of the various parameters, the use of this enzyme allows lactate concentrations to be measured in the range from 0.05 to 7 m M with an accuracy of about 2% and
with response times of less than 1min (9). The electrode has been used for determining lactate concentration in various biologically derived fluids such as milk, wine, and blood (IO), and it has been incorporated into a device allowing continuous lactate monitoring (11) which is a t present being applied t o checking the level of this metabolite in arteritic patients whose lower limbs are subjected to controlled muscular efforts (12, 13). Other medical applications are possible in the domain of obstetrics and in cardiovascular surgery. Even so it would be useful, particularly for applications in cardiology, to have an electrode which could be used in vivo. It was this concern which led us t o carry out the present work. We first of all sought t o replace the hexacyanoferrate(II)/ hexacyanoferrate(II1) couple by a redox couple made up of large molecules which would be kept out of contact with the blood by a semipermeable membrane. In a second step, we have made use of the electrochemical properties of the lacticodehydrogenase itself in developing a new reagentless lactate electrode.
EXPERIMENTAL SECTION Apparatus. The linear chronoamperometry was performed by using a traditional setup consisting of a potentiostat (Tacussel PIT), a triangular-wave signal generator (Tacussel GSATP),and an X-Y pen recorder (Sefram Luxytrace). The cell contains a working electrode of either polished platinum, gold, or vitreous carbon, with a surface area of a few square millimeters, a platinum auxiliary electrode of large surface area, shut off in a compartment separated from the working electrode compartment by a finely porous glass frit, and a saturated-calomel reference electrode with respect to which all potentials are measured. A Luggin capillary filled with the buffered phosphate solution used in the cell acts as junction between the reference electrode and the working electrode. The selective enzyme electrode previously described ( 9 )is a platinum disk 7 mm2in surface area. The thickness of the reaction chamber enclosed by the platinum surface and the semipermeable membrane is of the order of a few tens of microns. The membrane is a dialysis membrane (Hoeffer Scientific Instruments Type EMD 104) with a cutoff threshold of about 6000. This electrode is incorporated into a three-electrode potentiostatic circuit identical with the one described above. The circulation of the liquids for the continuous assay is ensured by a system of LKB peristaltic pumps previously described (11). The absorbance measurements were performed by using a UVvisible spectrophotometer (Beckman Acta IV). Reagents. All the solutions were prepared from analytical grade reagents from various manufacturers and doubly distilled water. The buffer solutions are mixtures of monopotassium phosphate and disodium phosphate of pH 7.2 and ionic strength 0.5 M. The enzyme is lacticodehydrogenase (L(+)-lactate: ferricytochrome c oxidoreductase, EC 1.1.2.3) extracted from the aerobic yeast, Hansenula Anomala, and purified according to the procedure of Baudras and Spyridakis (8). Such pure enzyme preparations have a specific activity of about 555 units/mg. The ferrocytochrome c present in commercially prepared ferricytochrome (10% approximately) is oxidized by a solution of potassium hexacyanoferrate(II1) and the hexacyanoferrate(I1) is separated by passing the solution through a column of Sephadex G25. Procedure. Before current-potential curves can be obtained, the working electrode immersed in the buffer solution must un-
0003-2700/80/0352-2109$01.00/00 1980 American Chemical Society
2110
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
1
I
1 / , E applled V SCEO
1
0 5
YS
Flgure 1. Current-potential curve obtained with a platinum electrode for a solution containing 1 p M cytochrome c , 0.86 p M lacticodehydrogenase, and 3.3 mM lactate, speed of scan of the potential scale 50 mV min-’. dergo a preliminary treatment. The electrode is subjected to a linear sweep in potential varying at the rate of 3 V/min, within the following limits: +0.20, -0.20, 0.00, +1.20, 0.00 V. This treatment is repeated until reproducible current-potential characteristics are obtained. A measurement of the residual current is then performed between the limits quoted above, with a rate of variation of the potential identical with that chosen for tracing the current-potential characteristics when the reagents are introduced into the buffer solution. The preparation of the solution of ferrocytochrome c is performed by electrolysis a t an imposed potential using a gold electrode of large surface area maintained at a potential of 4 . 1 0 V. This value was deduced from our own measurements and from the work of Anderson et al. (14). The concentration of ferrocytochrome is checked by measurements of optical density at a wavelength of 418 nm (8). The reduced form of the lacticodehydrogenase is obtained by mixing a 10 mM lactate solution with a 1 1M enzyme solution in the electrolysis cell. The enzyme electrode used in the various lactate measurements contains, in its reaction chamber, either 3 mM ferricytochromewith 2 p L of an enzyme solution of strength 1800 units/mL or else 2 pL of the same enzyme solution on its own. The precautions to be taken have already been described
Eapplied
V vs
SCE
Flgure 2. Current-potential curve for the oxidation of the reduced form of lacticodehydrogenase: concentration 0.8 pM, speed of scan of the potential scale 1 v min-’.
o 30
t,me,
60
5
Flgure 3. Graph of the response i ( t )of the electrode without cofactor. Enzyme concentration in the reaction chamber was 115 pM, with an activity of 1800 units/mL. Lactate concentration was 2 mM.
(9,10).
It should be noted that the arrangement of soluble enzyme held by a membrane seems to be the one best suited to the operation of the reagentless electrode; in this arrangement both the oxidized and the reduced forms of the enzyme are in close contact with the metal on which they undergo the electron transfer. Also both forms diffuse easily from the bulk of the layer to the metal/solution interface.
RESULTS AND DISCUSSION T h e current-potential curves obtained in the stationary state (after subtraction of the residual current) for the oxidation of ferrocytochrome c and the reduced formed of the enzyme are shown in Figures 1 and 2, respectively. They indicate the existence of plateaus in the range of potential lying between 0.40 and 0.55 V for the ferrocytochrome c and between 0.42 and 0.50 V for the lacticodehydrogenase. In both cases, a potential of +0.50 V was applied to the ion-selective electrode during lactate measurements; this potential corresponds to the diffusional limiting current for the oxidation of the ferricytochrome or of the reduced lacticodehydrogenase. An example of a response curve is shown in Figure 3 for a platinum electrode without cofactor in the reaction chamber, immersed in a 2 mM lactate solution. The same type of curve is obtained when ferricytochrome is used in the reaction chamber or when the platinum is replaced by gold. With a carbon electrode the time required to reach stationary state (indicated by a constant current) is longer (about 5 min).
/
/
I
I
I
1
2
6 lactate
7
concentration,mM
Flgure 4. Calibration curves: (a)without cofactor, pM; (b) with ferricytochrome c , [enzyme] = 115 pM,
[enzyme] = 115 [ferricytochrome]
= 2.8 mM; (c) with ferricyanide.
Between two measurements the electrode is rinsed and then immersed in a buffer solution and held at a potential of +0.50 V until the electrolysis current falls below 0.1 FA. Depending on the concentration of the lactate solutions under study, the rinsing time varies from 2 to 10 min; this rinsing time reduces the rate of measurement in comparison with the electrode using hexacyanoferrate(II1).
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
injection
0
;
1 6
I 10 t i m e , mtn
2111
of n o r a d r e n a l i n e
I
I
14
18
Figwe 6. Variation in lactate level in the coronary sinus of a dog during injection of noradrenaline, directly implanted electrode.
Two calibration curves measured (b) by use of ferricytochrome c or (a) with no cofactor present are given in Figure 4;they are compared with a typical line obtained by using hexacyanoferrate(II1) (c). For the electrode using ferricytochrome or the one without cofactor, the zone of linearity is somewhat narrower (0-6 mM) than in the case of the electrode with hexacyanoferrate(II1) as cofactor (0-7 mM) (7, 9). The reagentless electrode may thus be used in a range of lactate concentrations lying between 0.05 and 6 mM with an accuracy of about 2% and response times of about 1min. It can be used in place of the electrode previously described (7, 9) in applications requiring discontinuous measurements of lactate. It should be noted, in Figure 4,that a t a given lactate concentration, the current is greater for the reagentless electrode than for the ferricytochrome electrode; this may be due to the adsorption of the ferricytochrome c which when occurring together with the adsorption of the lacticodehydrogenase, makes the electron transfer between platinum and protein more difficult. This observation deserves closer study. With this electrode we were able to perform about 30 titrations per day with a reproducibility of the order of 2 % . Using one particular electrode, we performed 200 titrations, spread out over a period of 1 month, without any deviation greater than 3% being observed. To obtain such a result, it is important to store the electrode after use in a buffer solution a t about 4 "C. To be able to propose this electrode for biomedical applications in which continuous monitoring is found necessary, we first of all studied the effect of temperature; the results show a mean variation of 3% /deg (Figure 5 ) thus requiring either a calibration to be performed a t the measurement temperature or else a correction to be made on the basis of the curve shown in Figure 4 without necessarily implying a strict temperature control during the course of the experiment. T h e fact that the amplitude of the signal a t the stationary state depends relatively little on temperature, as already observed with the electrode using hexacyanoferrate(II1) as cofactor, is undoubtedly related to the nature of the ratedetermining step of the process, the diffusion of the lactate through the semipermeable membrane. Using the arrangement described in ref 11 and using the same procedures, we have determined the limiting rate of variation in lactate concentration which is measurable by the electrode without cofactor. It is possible to follow variations in lactate in the concentration range already indicated, with the same precision, if the rate of variation is less than 1 mM/min.
These results justified testing the electrode by using it directly in a biological fluid containing lactate. So the electrode was used to follow the variation in lactate concentration in the coronary sinus of a dog subjected to an injection of noradrenaline. T h e results of the test are shown in Figure 6 where the lactate concentration is given in arbitrary units; as a matter of fact it is not possible to carry out a calibration under hydrodynamic conditions absolutely identical with those encountered in the coronary sinus. T h e principle behind this reagentless electrode can undoubtedly be transposed to any measurement system in which an oxidoreductase is associated with a redox reaction of a substrate and amperometric detection of one of the reactants or products. We have thus considered measuring glucose concentrations by using a glucose oxidase supplied by Boehringer (100 units/mg) enclosed in the reaction chamber of the electrode a t a concentration of approximately 4 mM. The electrode potential deduced from a preliminary electrochemical study is +0.50 V. By adding 10 mM NaN3to the sample to be assayed (15),it is possible to obtain a t the stationary state values of the current proportional to the glucose concentration in the range 0.5 to 6 mM; the response times for these first attempts are of the order of 10 min. These measurements have not yet been optimized and in particular no differential system allowing for the electrochemical oxidation of glucose a t the potential indicated to be taken into account (16-18) has been employed. Even so this second example shows how it is possible t o extend the methodology and the principle of electrochemical enzymatic assay without cofactor. It is important to keep under control the electrochemical behavior of the enzyme which must not be denatured by the electric field but must retain its activity and its specificity. In the case of the lactate, an earlier study of the adsorption of the enzyme has made it possible to estimate its surface concentration (19);thus it is possible to consider applications other than simple lactate w a y s : e.g., indirect electrochemical conversion of lactate into pyruvate so as to obtain a desired level of lactate in a solution; one simply has to adapt the electrochemicalreactor to the quantity of lactate to be treated (electrode surface area, enzyme concentration, thickness of the semipermeable membrane), Other systems of electrochemical production might be devised after studying the electrochemical behavior of redox enzymes.
ACKNOWLEDGMENT We wish to thank Alain Baudras of the Centre de GBnetique et Biologie Cellulaire at the Universitti Paul Sabatier for the supply of enzyme and Henri Boccalon of the Service de
2112
Anal. Chem. 1980, 52, 2112-2116
Chirurgie Cardiovasculaire of the Centre Hospitalier Universitaire of Toulouse-Rangueil for his help with the experiments on animals.
LITERATURE CITED (1) (2) (3) (4)
(5) (6) (7) (8) (9) (10) (1 I )
Hess, B. Biochem. 2. 1956, 328, 110-116. Williams. D. L.; Doig, A. R.; Korosi, A. Anal. Chem. 1970, 42, 118-121. Racine, P.; Mindt. W. Experientia. Suppl. 1971, 18, 525-529. Racine, P.; Engelhardt, R.; Higelin, J. C.; Mindt, W. Med. Instrum. 1975, 9 , 11-14. Shinbo, T.; Sugiura, M.; Kamo, N. Anal. Chem. 1979, 51, 100-104. Biaedei, W. J.; Jenkins, R . A. Anal. Chem. 1976, 48, 1240-1247. Durliat. H.; Comtat, M.; Mahenc, J.; Baudras, A. J . Electroanal. Chem. 1975, 66, 73-76. Baudras, A.; Spyridakis, S. Biochimie 1971, 53, 943-955. Duriiat, H.; Corntat, M.; Mahenc, J.; Baudras, A. Anal. Chim. Acta 1976, 85, 31-40. Duriiat. H.; Comtat, M.; Baudras, A. Clin. Chem. 1976, 22, 1802-1805. Duriiat, H.; Corntat, M.; Mahenc, J. ~ n a l .chim. Acta 1979, 106, 13 1-135.
(12) Boucays, A. These Madecine, UniversRB Paul Sabatier, Touiouse, 1978. (13) Boccaion, A.; Puel, P.; Comtat, M.; Mahenc, J. Communication to the 2nd Congress on Hemodynamics of the Limbs, Scottsdale, AZ, Feb 1979. (14) Betso, S. R.; Kbpper, M. H.; Anderson, L. B. J. Am. Chem. Soc. 1972, 94, 8197-8204. (15) Mor, J. R.; Guarnaccia, R. Anal. Biochem. 1977, 79, 319-328. (16) Lerner. H.; Giner, J.; SoeMner, J. S.; Cotton. C. K. J . Electrochem. Soc. 1979, 726 (2), 237-242. (17) Marincic, L.; Soeldner, S.; Giner, J.; Cotton, C. K. J. Electrochem. Soc. 1979. 126 (10). 1687-1692. (18) Skou; E. €/ect&him. Acta 1977, 22(4), 313-318. (19) Duriiat, H.; Comtat, M. J. Elecfroanal. Chem. 1978, 89, 221-229.
RECEIVED for review February 5,1980. Accepted July 9,1980. This research was financially supported by the Ddegation GBnBrale la Recherche Scientifique et Technique within the framework of Contract No. 78.7.2932 of the Biomedical Engineering Committee.
Corrections for Systematic Errors from Analog Integration in Controlled-Potential Coulometry Thomas L. Frazzini, Michael K. Holland,* Jon R. Weiss, and Charles E. Pietri New Brunswick Laboratoty, U S . Department of Energy, 9800 South Cass Avenue, Argonne, Illinois 60439
The output signal from an ideal analog integrator should be proportional to the integrated current. Deviation from this ideal analog integrator response in a state-of-the-art controiledpotential coulometer has introduced systematlc errors of up to -0.1 % In the measurement of electroactive species. An extenslve evaluation of a controlled-potentlal coulometry system, widely used In the nuclear fleld, resulted in the identification of the sources of these errors. The analog integrator studied suffered from voltage output signal drift and offset caused by the operational amplifier. Furthermore, shlfts In this voltage output signal occurred because of leakage and dielectric absorption by the Integrating capacitor. Equatlons which describe the leakage and offset phenomena have been derived. Instrument operating methods which compensate for the drift and dielectrlc absorption effects have been developed. Corrections based on these equations and on the modlfled instrumental operatlng methods have slgnlflcantly decreased these systematic errors. These improvements permit the coulometer to be used in coulometric measurements whlch are directly based on a derived physical constant, the Faraday. The proposed corrections are applicable to other systems using analog lntegratlon circults.
Theoretical discussions of controlled-potential coulometry typically state its application permits the determination of electroactive species directly from the quantity of charge used to electrolyze the desired species and that chemical standard reference materials are not required ( I , 2). In application, the use of controlled-potentialcoulometry based on the Faraday is not simple. Many of the special considerations required for this application have been well researched: accurate corrections for background current ( 3 , 4 ) ,correction for the fraction of the sample electrolyzed within the selected potential span ( 5 ) ,the uncertainty in the value of the Faraday
( 6 ) , and the general requirements of an integrator for controlled-potential coulometry (7, 8). Although analog integrators have been designed which, in practice, are close to meeting the integration requirements, no corrections for the significant deviations from ideal integrator response in controlled-potential coulometry have been proposed. A stateof-the-art controlled-potentid coulometer widely used in the nuclear field contains a highly reliable solid-state analog integrator with nearly ideal response which should behave according to
where Ek and Eoutare the integrator input and output signal voltages, respectively, and R and C are the integrating resistor and capacitor values as shown in Figure 1. Our evaluation of this integrator resulted in the development of corrections for previously recognized integrator behavior (8,9):(1)the voltage output signal is subject to amplifier drift and offset; (2) the output signal which is held on the integrating capacitor is subject to dielectric absorption in the capacitor; (3) the output signal is subject to leakage. These deviations from ideal response have introduced a systematic error of approximately -0.1% in the measurement of electroactive species such as plutonium. We report, in this paper, an improved methodology which dramatically reduces these systematic errors and permits the coulometer to be used for measurements directly based on Faraday’s constant. The impact of drift,offset, dielectric absorption, and leakage phenomena upon analog integration of the exponentially decaying dc electrolysis current produced during a controlled-potential coulometry determination can be predicted. Drift is the rate of change of integrator output, measured under conditions where the input voltage from the potentiostat is zero. Since uncompensated input offset in the integrator will cause drift, the operational amplifier of the integrator must be adjusted to minimize the drift which may, in turn,
This article not subject to U S . Copyright. Published 1980 by the American Chemical Society
