- Email: [email protected]
Amperometric biosensor for determination of lactate in sweat
35
Analytica &mica Acta, 278 (1993) 35-40 Ekevier Science Publishers B.V., Amsterdam
Amperometric biosensor for determination of lactate in sweat Mohammad H. Faridnia, Giuseppe Palleschi ’ and Glenn J. Lubrano Universal Sensors, Inc., 5258 Veterans Blvd., Suite D, iUetai&, LA 70006 (USA)
George G. Guilbault Department of Chemistry Universi@ of New Orbzans, 2ooOLakeshore Dr., New Orleans, LA 70148 (USA) (Received 2nd January 1992; revised manuscript received 29th December 1992)
An amperometric hydrogen peroxide based biosensor has been developed for non-invasive determination of L-lactate. The biosensor utilizes lactate oxidase immobilized between a polycarbonate membrane and a polytetrafluoroethylene (PTFE) blocking membrane to effectively eliinate electrochemical interferences. Both the steady state current and maximum rate of current change were measured. The response times were 2 min and 10 s, respectively. Because of the addition of polycarbonate and PTFE membranes, the linear range of lactate was extended up to 140 mg dl-’ and the apparent Michaelis-Menten constant was almost two orders of magnitude higher than that of the free enzyme. The biosensor was applied to the analysis of sweat L-lactate content of healthy subjects during physical exercise. Keywords: Amperometry; Biosensors; Body fluids; Lactate
Increasing demand for non-invasive, fast and reliable methods for metabolite determination in body fluids resulted in extensive research in this area. L-Lactate is one of the most important metabolites in clinical analysis for medical patients with respiratory failures [ll and in sport medicine 12-81, in which the blood L-lactate level is used to monitor the maximum performance level of an athlete. We have previously reported development of an amperometric L-lactate biosensor for determination of lactate in saliva samples [9]. There are two major limitations in using salvia as a medium for lactate determina-
Corresmndence to: M.H. Faridnia, Universal Sensors Inc., 5258 Veterans Blvd., Suite D, Metairie, LA 70006 (USA). ’ Present address: DISTAAM, Universit% de1 Molise, Via Tiberio 21, 86100 Campobasso (Italy). 0003-2670/93/$06.00
tion; the low saliva to blood L-lactate ratio [lo] and the presence of bacteria in saliva [11,12]. To overcome these limitations, very sensitive and highly selective electrodes are required and the analysis must be carried our promptly after sample collection. Sweat, on the other hand, has a much higher sweat to blood L-lactate ratio [lo]. Electrochemical biosensors have been shown to be specific, selective and easy to use and many L-lactate selective electrodes have been reported [l-9]. Presently, there are four enzymes available for detection of L-lactate: lactate dehydrogenase (LDH) [13-U], cytochrome 6, [16], lactate monooxygenase (LMO) and lactate oxidase (LOD) [17-221. Bienzyme systems such as LOD/ LDH [23,24] and cytochrome b,/LDH [25] have also been proposed. Although dehydrogenases are highly selective in many cases, the difficulty in oxidation of coen-
Q 1993 - Elsevier Science Publishers B.V. All rights reserved
M.H. Fariakia et al. /Anal
36
zyme (NADH or NADPH) poses a problem as an additional parameter to be optimized. Biosensors utilizing immobilized lactate monooxygenase and lactate oxidase attached to oxygen or hydrogen peroxide electrodes measure the rate of oxygen consumption or hydrogen peroxide production [17-221. In addition, some biosensors utilize microbial cells [26,27]. Microbial cells, although functional as L-lactate biosensors, suffer from limited operating life time due to deactivation. We have developed an amperometric biosensor for L-lactate determination. The sensor utilizes lactate oxidase chemically immobilized on a polycarbonate membrane. The electrochemical interferents have been eliminated by the use of a hydrophobic blocking membrane that was permeable to hydrogen peroxide. The biosensor was applied to the analysis of sweat samples of healthy subjects, before and after physical exercise. The electrochemical lactate biosensor operates according to the following reaction: Lactate
L-Lactate + 0, + H,O *
Chiti Acta 278 (1993) 35-40
lactate kit (No. 826-W) were purchased from Sigma. Bovine serum albumin (BSA, 98-99%, Catalog No. A-7906, lot No. 18F-0409) and 25% glutaraldehyde, grade II (Catalog No. G6257, lot No. 116F-50271, were also purchased from Sigma. All other chemicals not listed were reagent grade. Materials Microporous polycarbonate membrane (0.015 ~1 was obtained from Nuclepore. The hydrophobic blocking membrane, polytetrafhroroethylene (PTFE), 4 mil thickness (Catalog No. 4106) was from Universal Sensors. Solutions Phosphate buffered saline (PBS) was used for all measurements and electrode storage. The PBS contained the following salt concentrations in deionized water: 137 mM NaCl, 2.7 mM KCl, 8.0 mM Na,HPO, and 1.5 mM KI-I,PO, (pH 7.45 f 0.10). BSA and glutaraldehyde immobilization solutions were prepared in 0.10 M phosphate buffer, pH 7.5.
Pyruvate + H,O,
This reaction is catalyzed by the enzyme lactate oxidase and the hydrogen peroxide generated is measured at a platinum electrode poised at + 650 mV vs. Ag/AgCl electrode.
EXPERIMENTAL
znstrumentution An amperometric biosensor detector (ABD) (Catalog No. 2001) and a hydrogen peroxide electrode with combined working (Pt) and reference (Ag/AgCl) electrode (Catalog No. 4006) were from Universal Sensors (New Orleans, LA). For temperature studies, a jacketed glass wall beaker was thermostated by an MS-20 Lauda heating circulator from Brinkmann Instruments. The sample temperature was monitored with a Thermometrics 10 kR thermistor (MC50F103A) connected to a digital voltmeter. Chemicals Lactate oxidase from Pediococcus sp. (EC 1.1.3.2) 30 units mg-’ and a spectrophotometric
Enzyme WrwbiIization A 2-cm2 piece of polycarbonate membrane was attached to the electrode jacket with its dull side facing out and secured in place with an O-ring. A lo-p1 volume of a solution of 5% BSA in 0.1 M phosphate buffer (pH 7.5) was used to dissolve 10 units of lactate oxidase. The enzyme solution was then placed on the polycarbonate membrane and 1 ~1 of 2.5% glutaraldehyde was added and the reaction mixture quickly stirred and spread out over the membrane surface with a small glass rod to form a 6-mm diameter disk. The enzyme membranes were air dried (approximately 2 h). They were then washed once with PBS, soaked for a few minutes in PBS containing 0.1 M glycine, then washed and stored in PBS. Electrode construction The biosensor was constructed with the PTFE membrane innermost, and the immobilized lactate oxidase sandwiched between it and the polycarbonate membrane. It has the following configuration: Pt/PTFE/LOD/polycarbonate.
MH. Faridnia et aL /Anal
37
Chim. Acta 278 (1993) 35-40
Test method The applied potential was set at 0.650 V vs. Ag/AgCl. A 5000 mg dl-’ L-lactate stock solution in PBS was used to calibrate the biosensor. Appropriate volumes of the stock solution were pipetted into 5 ml of PBS and the current monitored with a strip chart recorder. The maximum, initial rate of reaction was read from the display of the ABD. The change in current was calculated by subtraction of background current from the steady state current. The steady state current was taken as the current at 2 min. All measurements were carried out at room temperature and no adjustments for temperature were made.
RESULTS AND DISCUSSION
Electrode response Typical response curves of the lactate electrode are given in Fig. 1. The time to 95% or better of the steady state current was 2 min. The maximum initial rate of current change occurred at 10 s. A typical calibration curve of steady state current vs. concentration is given in Fig. 2. The membranes used extended the linear range of the lactate up to 140 mg dl-‘. Conventional lactate probes based on oxygen or hydrogen peroxide electrodes assembled with the same enzyme C f
0
40
120 200 (mg /dl) Fig. 2. Calibration curve of L-lactate biosensor, given as the steady state current vs. the L-lactate concentration. LACTATE
[l&28] used in this work have been used in clinical medicine. In these papers a lactate probe has a linear range up to 5 mg dl-‘. With this electrode, the linearity goes up to 140 mg dl-‘. We did not use the initial rate for calibration because the steady state current method was more reproducible (5 vs. 10%). Thus, the steady state method was used throughout. The initial rate method is good enough to measure the lactate variation in sweat within 10 s, but for our purposes, this fast procedure was unnecessary. Because the enzyme is immobilized and not used in a homogeneous solution and because there is a substrate diffusion restricting membrane between the sample and the enzyme, the K, of the electrode system (140 mg dl-’ lactate) is referred to as an apparent K,, which is almost two orders of magnitude larger than the K, of corresponding homogeneous systems. This is consistent with the results of other biosensors.
smin B
d A
M
Fig. 1. Response curves of the L-lactate biosensor. (A) 20 mg cl-‘, (B) 40 mg dl-‘, and (0 80 mg dl-‘.
Interference study Effects of the following interferents on the L-lactate biosensor were investigated: ascorbic acid, acetaminophen, acetylsalicylic acid, uric acid, valine, lysine, histidine, leucine, tyrosine, threonine, arginine, sodium, chloride, calcium and potassium. The electrode was highly selective against these compounds. For all these interferents, the electrode response was less than detectable (i.e., less than 0.01 nA) at concentrations 10 times higher that present in sweat.
h4.H. Fariahia et al /Anal
Chim Acta 278 (1993) 35-40
tures, the analyses were carried
out at ambient temperature, since extended exposure to high temperature results in more rapid denaturation of the enzyme. Denaturation due to extreme temperature is usually irreversible, resulting in permanent loss of enzyme activity.
0:
5
6
7
8
9
10
PH Fig. 3. Steady state response of the L-lactate biosensor to 20 mg dl-’ L-lactate at different pH values: (0) = 0.1 M phosphate buffer; (*) = 0.1 M Tris buffer.
pH study The response to 20 mg dl-’ L-lactate was measured at different pH values. Fig. 3 is a plot of steady state current vs PH. Phosphate buffer (0.1 M) and Tris buffer (0.1 MI were used for pH 6-9 and 8-11, respectively. Within this range, there was no noticeable change in response. Temperature study The response to 20 mg dl-’ L-lactate was measured at 5°C intervals from 25 to 45°C. The effect of temperature on response is given in Fig. 4. Although response is higher at higher tempera-
50 I
40 2 0’
‘530 w
4.
=20 k
/
zio 0
’
20
30
40
50
Temperature Fig. 4. Steady state current response of L-lactate biosensor to 20 mg dl-’ L-lactate at different temperatures.
Apglication Subjects. The participants in this study were males, ages 24-36 years, accustomed to moderate exercise and were not professional athletes. Sample collection and analysis. Sweat samples, l-2 ml, were collected in 5-ml disposable vials, capped and either immediately assayed or kept at 0°C until assay. During each experiment, samples were collected from upper parts of legs and arms, area covering Gluteus Maximas and Biceps respectively. To establish a baseline response, at least three samples were collected before exercise, while the subject was at rest. The subject then ran at a speed of 4-6 miles h-l for 30 min. Sweat samples were collected immediately after the run and thereafter at 5-min intervals, until the sweat lactate level returned to normal. The biosensor was calibrated before the analysis and 20 mg dl-’ L-lactate controls were run throughout the study. 100 ~1 of each sample was pipetted into 5.00 ml of stirring PBS and the steady state current and maximum initial rate responses were monitored. The steady state current was measured within 2 min and the initial rate of reaction was measured in less than 10 s. A typical plot of sweat L-lactate vs. time is given in Fig. 5. The values of lactate concentration reported in the figure were calculated considering the dilution of 100 ~1 sweat sample injected into 5 ml of buffer solution. The values calculated according with the dilution attained were always in the linearity range of the probe. All subjects had an increase in sweat lactate after the physical exercise and the concentration decreased during he resting period. This finding is in agreement with that reported by many researchers on L-lactate metabolism after a physical exercise [2-81. Location of sample collection is crucial and the concentration of L-lactate depends on the type of exercise. The samples collected from subjects’ arms and legs showed the
39
M.H. Fariahia et at!/Anal. Chim. Acta 278 (1993) 35-40
Sottoprogetto Biostrumentazione, !30,0061,PF 70
Contratto
No.
REFERENCES
0
TIME lmin)
Fig. 5. L-lactate metabolism curve presented as sweat L-lactate concentration (mg dl-‘1 vs. time (min), samples were collected from the subject’s legs (0) and arms (0) before and after a physical exercise.
same trend, but the former showed much lower baseline level and peaked at a lower maximum value. Stability. The short term stability of sweat Llactate was investigated by storage at 2-4°C for 22 h and 25°C for 5 h. The latter’s L-lactate concentration was periodically measured over the 5-h period. No decrease in L-lactate concentration was observed for either storage condition. This allowed the samples to be collected and stored at 2-4°C and analyzed at a later time. No preservatives were added to enhance stability. Conclusion We have developed an amperometric hydrogen peroxide based L-lactate biosensor, that has shown good characteristics in terms of its selectivity, indifference to pH change and extended linearity. The biosensor was applied to the non-invasive determination of sweat L-lactate of subjects before and after physical exertion. The results were comparable to that reported by others doing the same type of experiment, but measuring blood L-lactate content. Dr. Palleschi gratefully acknowledges the CNR (Conciglio Nazionale delle Ricerche) Progetto Finalizzato Biotecnologie e Biostrumentazione,
1 A.L. Herrick, B.M. Fisher, M.R. Moore, S. Cathcart, K.E.L. McCoIl and A. Goldberg, CIin. Chim. Acta, 190 (1990) 157. 2 G. PaIlesclhi, R. Pilloton, M. Mascini, L. Bemardi, G. Bombardieri, A.M. Deluca and P. Zippilli, J. Biosensors, 14 (1989) 3. K. Wasserman and M.B. Mcilroy, Am. J. Cardiol., 14 (1964) 844. J. Karlsson, L.O. Nordesio, L. Jiorfeldt and B. Sahin, J. Appl. Physiol., 33 (1972) 199. J. KarIsson and I. Jacobs, Int. Sports Med., 3 (1982) 190. G. Palleschi, M. Mascini, L. Bemardi and P. Zeppilli, Med. Biol. Eng. Comput., 38 (1990) B25. C. Juel, J. Bang&o, T. Graham and B. Saltin, Physiol. Stand., 140 (1990) 147. N. Shimojo, K. Fujino, S. Kitahashi, M. Nakao, K. Naka and K. Okuda, Clin. Chem., 37 (1991) 1978. G. Palleschi, M.H. Faridnia, G.J. Lubrano and G.G. GuiIbault, Anal. Chim. Acta, 245 (1991) 151. D.S. Dittmer (Ed.), Blood and Other Body Fluids, Federation of American Societies for Experimental Biology, Washington DC, 1961, p. 480. 11 J.O. Tenovuo, Human Saliva: Clinical Chemistry & Microbiology, Vol. I, CRC Press, Boca Raton, FL, 1989. 12 J.O. Tenovuo, Human Saliva: Clinical Chemistry & Microbiology, Vol. II, CRC Press, Boca Raton, FL, 1989. 13 W.J. Blaedel and R.A. Jenkins, Anal. Chem., 48 (1976) 1240. 14 W.J. Blaedel and R.C. Engstrom, Anal. Chem., 52 (1986) 1691. 15 M.F. Suaud-Chagny and F.G. Gonon, Anal. Chem., 58 (1986) 412. 16 J.J. Kulys and G.J.S. Svirmickas, Anal. Chim. Acta, 117 (1980) 115. 17 N. Shimozo, K. Naka, C. Nakajima, C. Yoshikawa, K. Okuda and K. Okada, Clin. Chem., 35 (1989) 1992. 18 M. Mascini, S. Fortunati, D. Moscone, G. Palleschi, M. Massi-Benedetti and P. Fabietti, Clin. Chim., 31 (1985) 451. 19 J. Wandrup, K. Tvede, J. Grinsted and H. Jordening, Chn. Chem., 35 (1989) 1740. 20 M. Mascini, F. Maxxel, D. Moscone, G. Calabresel and M. Massi-Benedetti, Chn. Chem, 33 (1987) 591. 21 G. Bardeletti, F. Sechaud and R. Coulet, Anal. Chim. Acta., 187 (1986) 47. 22 R. Pilloton, T.N. Nwosou and M. Mascini, Anal. Lett., 21 (1988) 747. 23 F. Mixutani, T. Yamanaka and Y. Tanabe, Chem. Lett., 2 (1984 199. 24 F. Mitxutani, Y. Yamanda, Y. Tanabe and K. Tsuda, Anal. Chim. Acta, 177 (1985) 153.
40 25 F. Schubert, D. Khtein, K.L. Schoder and F.W. Scheller, Anal. Chim. Acta, 169 (1985) 391. 26 J.H.T. Luong, M. Mulchandani and CA. Groom, J. Biotechnol., 10 (1989) 241.
M.H. Fadnia et aL /Am!
Chim. Acta 278 @93) 35-40
27 J.H.T. Luong, M. Mulchandani and C.A. Groom, J. Biotechnol. Stand., 140 (1990) 147. 28 G. Palleschi, M. Mascini, L. Bemardi, G. Bombardieri and A.M. De Luca, Anal. L&t., 22 (1989) 1209.
Analytica &mica Acta, 278 (1993) 35-40 Ekevier Science Publishers B.V., Amsterdam
Amperometric biosensor for determination of lactate in sweat Mohammad H. Faridnia, Giuseppe Palleschi ’ and Glenn J. Lubrano Universal Sensors, Inc., 5258 Veterans Blvd., Suite D, iUetai&, LA 70006 (USA)
George G. Guilbault Department of Chemistry Universi@ of New Orbzans, 2ooOLakeshore Dr., New Orleans, LA 70148 (USA) (Received 2nd January 1992; revised manuscript received 29th December 1992)
An amperometric hydrogen peroxide based biosensor has been developed for non-invasive determination of L-lactate. The biosensor utilizes lactate oxidase immobilized between a polycarbonate membrane and a polytetrafluoroethylene (PTFE) blocking membrane to effectively eliinate electrochemical interferences. Both the steady state current and maximum rate of current change were measured. The response times were 2 min and 10 s, respectively. Because of the addition of polycarbonate and PTFE membranes, the linear range of lactate was extended up to 140 mg dl-’ and the apparent Michaelis-Menten constant was almost two orders of magnitude higher than that of the free enzyme. The biosensor was applied to the analysis of sweat L-lactate content of healthy subjects during physical exercise. Keywords: Amperometry; Biosensors; Body fluids; Lactate
Increasing demand for non-invasive, fast and reliable methods for metabolite determination in body fluids resulted in extensive research in this area. L-Lactate is one of the most important metabolites in clinical analysis for medical patients with respiratory failures [ll and in sport medicine 12-81, in which the blood L-lactate level is used to monitor the maximum performance level of an athlete. We have previously reported development of an amperometric L-lactate biosensor for determination of lactate in saliva samples [9]. There are two major limitations in using salvia as a medium for lactate determina-
Corresmndence to: M.H. Faridnia, Universal Sensors Inc., 5258 Veterans Blvd., Suite D, Metairie, LA 70006 (USA). ’ Present address: DISTAAM, Universit% de1 Molise, Via Tiberio 21, 86100 Campobasso (Italy). 0003-2670/93/$06.00
tion; the low saliva to blood L-lactate ratio [lo] and the presence of bacteria in saliva [11,12]. To overcome these limitations, very sensitive and highly selective electrodes are required and the analysis must be carried our promptly after sample collection. Sweat, on the other hand, has a much higher sweat to blood L-lactate ratio [lo]. Electrochemical biosensors have been shown to be specific, selective and easy to use and many L-lactate selective electrodes have been reported [l-9]. Presently, there are four enzymes available for detection of L-lactate: lactate dehydrogenase (LDH) [13-U], cytochrome 6, [16], lactate monooxygenase (LMO) and lactate oxidase (LOD) [17-221. Bienzyme systems such as LOD/ LDH [23,24] and cytochrome b,/LDH [25] have also been proposed. Although dehydrogenases are highly selective in many cases, the difficulty in oxidation of coen-
Q 1993 - Elsevier Science Publishers B.V. All rights reserved
M.H. Fariakia et al. /Anal
36
zyme (NADH or NADPH) poses a problem as an additional parameter to be optimized. Biosensors utilizing immobilized lactate monooxygenase and lactate oxidase attached to oxygen or hydrogen peroxide electrodes measure the rate of oxygen consumption or hydrogen peroxide production [17-221. In addition, some biosensors utilize microbial cells [26,27]. Microbial cells, although functional as L-lactate biosensors, suffer from limited operating life time due to deactivation. We have developed an amperometric biosensor for L-lactate determination. The sensor utilizes lactate oxidase chemically immobilized on a polycarbonate membrane. The electrochemical interferents have been eliminated by the use of a hydrophobic blocking membrane that was permeable to hydrogen peroxide. The biosensor was applied to the analysis of sweat samples of healthy subjects, before and after physical exercise. The electrochemical lactate biosensor operates according to the following reaction: Lactate
L-Lactate + 0, + H,O *
Chiti Acta 278 (1993) 35-40
lactate kit (No. 826-W) were purchased from Sigma. Bovine serum albumin (BSA, 98-99%, Catalog No. A-7906, lot No. 18F-0409) and 25% glutaraldehyde, grade II (Catalog No. G6257, lot No. 116F-50271, were also purchased from Sigma. All other chemicals not listed were reagent grade. Materials Microporous polycarbonate membrane (0.015 ~1 was obtained from Nuclepore. The hydrophobic blocking membrane, polytetrafhroroethylene (PTFE), 4 mil thickness (Catalog No. 4106) was from Universal Sensors. Solutions Phosphate buffered saline (PBS) was used for all measurements and electrode storage. The PBS contained the following salt concentrations in deionized water: 137 mM NaCl, 2.7 mM KCl, 8.0 mM Na,HPO, and 1.5 mM KI-I,PO, (pH 7.45 f 0.10). BSA and glutaraldehyde immobilization solutions were prepared in 0.10 M phosphate buffer, pH 7.5.
Pyruvate + H,O,
This reaction is catalyzed by the enzyme lactate oxidase and the hydrogen peroxide generated is measured at a platinum electrode poised at + 650 mV vs. Ag/AgCl electrode.
EXPERIMENTAL
znstrumentution An amperometric biosensor detector (ABD) (Catalog No. 2001) and a hydrogen peroxide electrode with combined working (Pt) and reference (Ag/AgCl) electrode (Catalog No. 4006) were from Universal Sensors (New Orleans, LA). For temperature studies, a jacketed glass wall beaker was thermostated by an MS-20 Lauda heating circulator from Brinkmann Instruments. The sample temperature was monitored with a Thermometrics 10 kR thermistor (MC50F103A) connected to a digital voltmeter. Chemicals Lactate oxidase from Pediococcus sp. (EC 1.1.3.2) 30 units mg-’ and a spectrophotometric
Enzyme WrwbiIization A 2-cm2 piece of polycarbonate membrane was attached to the electrode jacket with its dull side facing out and secured in place with an O-ring. A lo-p1 volume of a solution of 5% BSA in 0.1 M phosphate buffer (pH 7.5) was used to dissolve 10 units of lactate oxidase. The enzyme solution was then placed on the polycarbonate membrane and 1 ~1 of 2.5% glutaraldehyde was added and the reaction mixture quickly stirred and spread out over the membrane surface with a small glass rod to form a 6-mm diameter disk. The enzyme membranes were air dried (approximately 2 h). They were then washed once with PBS, soaked for a few minutes in PBS containing 0.1 M glycine, then washed and stored in PBS. Electrode construction The biosensor was constructed with the PTFE membrane innermost, and the immobilized lactate oxidase sandwiched between it and the polycarbonate membrane. It has the following configuration: Pt/PTFE/LOD/polycarbonate.
MH. Faridnia et aL /Anal
37
Chim. Acta 278 (1993) 35-40
Test method The applied potential was set at 0.650 V vs. Ag/AgCl. A 5000 mg dl-’ L-lactate stock solution in PBS was used to calibrate the biosensor. Appropriate volumes of the stock solution were pipetted into 5 ml of PBS and the current monitored with a strip chart recorder. The maximum, initial rate of reaction was read from the display of the ABD. The change in current was calculated by subtraction of background current from the steady state current. The steady state current was taken as the current at 2 min. All measurements were carried out at room temperature and no adjustments for temperature were made.
RESULTS AND DISCUSSION
Electrode response Typical response curves of the lactate electrode are given in Fig. 1. The time to 95% or better of the steady state current was 2 min. The maximum initial rate of current change occurred at 10 s. A typical calibration curve of steady state current vs. concentration is given in Fig. 2. The membranes used extended the linear range of the lactate up to 140 mg dl-‘. Conventional lactate probes based on oxygen or hydrogen peroxide electrodes assembled with the same enzyme C f
0
40
120 200 (mg /dl) Fig. 2. Calibration curve of L-lactate biosensor, given as the steady state current vs. the L-lactate concentration. LACTATE
[l&28] used in this work have been used in clinical medicine. In these papers a lactate probe has a linear range up to 5 mg dl-‘. With this electrode, the linearity goes up to 140 mg dl-‘. We did not use the initial rate for calibration because the steady state current method was more reproducible (5 vs. 10%). Thus, the steady state method was used throughout. The initial rate method is good enough to measure the lactate variation in sweat within 10 s, but for our purposes, this fast procedure was unnecessary. Because the enzyme is immobilized and not used in a homogeneous solution and because there is a substrate diffusion restricting membrane between the sample and the enzyme, the K, of the electrode system (140 mg dl-’ lactate) is referred to as an apparent K,, which is almost two orders of magnitude larger than the K, of corresponding homogeneous systems. This is consistent with the results of other biosensors.
smin B
d A
M
Fig. 1. Response curves of the L-lactate biosensor. (A) 20 mg cl-‘, (B) 40 mg dl-‘, and (0 80 mg dl-‘.
Interference study Effects of the following interferents on the L-lactate biosensor were investigated: ascorbic acid, acetaminophen, acetylsalicylic acid, uric acid, valine, lysine, histidine, leucine, tyrosine, threonine, arginine, sodium, chloride, calcium and potassium. The electrode was highly selective against these compounds. For all these interferents, the electrode response was less than detectable (i.e., less than 0.01 nA) at concentrations 10 times higher that present in sweat.
h4.H. Fariahia et al /Anal
Chim Acta 278 (1993) 35-40
tures, the analyses were carried
out at ambient temperature, since extended exposure to high temperature results in more rapid denaturation of the enzyme. Denaturation due to extreme temperature is usually irreversible, resulting in permanent loss of enzyme activity.
0:
5
6
7
8
9
10
PH Fig. 3. Steady state response of the L-lactate biosensor to 20 mg dl-’ L-lactate at different pH values: (0) = 0.1 M phosphate buffer; (*) = 0.1 M Tris buffer.
pH study The response to 20 mg dl-’ L-lactate was measured at different pH values. Fig. 3 is a plot of steady state current vs PH. Phosphate buffer (0.1 M) and Tris buffer (0.1 MI were used for pH 6-9 and 8-11, respectively. Within this range, there was no noticeable change in response. Temperature study The response to 20 mg dl-’ L-lactate was measured at 5°C intervals from 25 to 45°C. The effect of temperature on response is given in Fig. 4. Although response is higher at higher tempera-
50 I
40 2 0’
‘530 w
4.
=20 k
/
zio 0
’
20
30
40
50
Temperature Fig. 4. Steady state current response of L-lactate biosensor to 20 mg dl-’ L-lactate at different temperatures.
Apglication Subjects. The participants in this study were males, ages 24-36 years, accustomed to moderate exercise and were not professional athletes. Sample collection and analysis. Sweat samples, l-2 ml, were collected in 5-ml disposable vials, capped and either immediately assayed or kept at 0°C until assay. During each experiment, samples were collected from upper parts of legs and arms, area covering Gluteus Maximas and Biceps respectively. To establish a baseline response, at least three samples were collected before exercise, while the subject was at rest. The subject then ran at a speed of 4-6 miles h-l for 30 min. Sweat samples were collected immediately after the run and thereafter at 5-min intervals, until the sweat lactate level returned to normal. The biosensor was calibrated before the analysis and 20 mg dl-’ L-lactate controls were run throughout the study. 100 ~1 of each sample was pipetted into 5.00 ml of stirring PBS and the steady state current and maximum initial rate responses were monitored. The steady state current was measured within 2 min and the initial rate of reaction was measured in less than 10 s. A typical plot of sweat L-lactate vs. time is given in Fig. 5. The values of lactate concentration reported in the figure were calculated considering the dilution of 100 ~1 sweat sample injected into 5 ml of buffer solution. The values calculated according with the dilution attained were always in the linearity range of the probe. All subjects had an increase in sweat lactate after the physical exercise and the concentration decreased during he resting period. This finding is in agreement with that reported by many researchers on L-lactate metabolism after a physical exercise [2-81. Location of sample collection is crucial and the concentration of L-lactate depends on the type of exercise. The samples collected from subjects’ arms and legs showed the
39
M.H. Fariahia et at!/Anal. Chim. Acta 278 (1993) 35-40
Sottoprogetto Biostrumentazione, !30,0061,PF 70
Contratto
No.
REFERENCES
0
TIME lmin)
Fig. 5. L-lactate metabolism curve presented as sweat L-lactate concentration (mg dl-‘1 vs. time (min), samples were collected from the subject’s legs (0) and arms (0) before and after a physical exercise.
same trend, but the former showed much lower baseline level and peaked at a lower maximum value. Stability. The short term stability of sweat Llactate was investigated by storage at 2-4°C for 22 h and 25°C for 5 h. The latter’s L-lactate concentration was periodically measured over the 5-h period. No decrease in L-lactate concentration was observed for either storage condition. This allowed the samples to be collected and stored at 2-4°C and analyzed at a later time. No preservatives were added to enhance stability. Conclusion We have developed an amperometric hydrogen peroxide based L-lactate biosensor, that has shown good characteristics in terms of its selectivity, indifference to pH change and extended linearity. The biosensor was applied to the non-invasive determination of sweat L-lactate of subjects before and after physical exertion. The results were comparable to that reported by others doing the same type of experiment, but measuring blood L-lactate content. Dr. Palleschi gratefully acknowledges the CNR (Conciglio Nazionale delle Ricerche) Progetto Finalizzato Biotecnologie e Biostrumentazione,
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