Potentiometric Sensor for Non invasive Lactate Determination in Human
Sweat
Massimo Onor1, Stefano Gufoni2, Tommaso Lomonaco3, Silvia Ghimenti3, Pietro Salvo3,4, Fiodor Sorrentino2,5 and Emilia Bramanti1*
1 National Research Council of Italy, C.N.R., Istituto di Chimica dei Composti Organo Metallici-ICCOM- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa (Italy) 2 Marwan Technology Srl, via L. Gereschi 36, 56127 Pisa, Italy
3 Department of Chemistry and Industrial Chemistry, University of Pisa, Via G. Moruzzi 13, 56124 Pisa (Italy)
4 Institute of Clinical Physiology, National Council of Research (IFC-CNR), Via Moruzzi 1, 56124, Pisa, Italy
5 Istituto Nazionale di Fisica Nucleare, sezione di Genova, via Dodecaneso 33, 16146 Genova, Italy
*Corresponding author: Emilia Bramanti; bramanti@pi.iccom.cnr.it
ABSTRACT
The present work describes a non invasive lactate sensing in sweat during workout. The sensing system is based on a non-equilibrium potentiometric measure performed using disposable, chemically modified, screen printed carbon electrodes (SPCEs) that can be wetted with sweat during the exercise. The potentiometric signal, which is proportional to lactate concentration in sweat, is produced by a redox reaction activated by UV radiation, as opposed to the enzymatic reaction employed in traditional, blood-based measuring devices. The sensing system exhibits chemical selectivity toward lactate with linearity from 1 mM up to 180 mM. The dynamic linear range is suitable for measurement of lactate in sweat, which is more than 10 times concentrated than hematic lactate and reaches more than 100 mM in sweat during workout. The noninvasive measure can be repeated many times during exercise and during the recovery time in order to get personal information on the physiological and training status as well as on the physical performance.
The device was successfully applied to several human subjects for the measurement of sweat lactate during prolonged cycling exercise. During the exercise sweat was simultaneously sampled on filter paper and extracted in water, and the lactate was determined by HPLC for method validation. The lactate concentration changes during the exercise reflected the intensity of physical effort. This method has perspectives in many sport disciplines as well as in health care and biomedical area.
1. INTRODUCTION
Lactate (or lactate) is a product of the anaerobic glycolysis resulting from pyruvate by the enzyme lactate dehydrogenase (LDH). The lactate can be found in blood and biological fluids of human beings and animals. A healthy adult normally produces about 120 g of lactate per day. Tissues characterized by an exclusively anaerobic metabolism (e.g. retina and blood red cells) produce about 40 g of lactate. The remaining quota is produced by other tissues (most of all muscles) on the basis of the actual oxygen availability.
The lactate/pyruvate molar ratio in blood is a reliable marker of cell anaerobic metabolism that may occur in inborn errors of the mitochondrial respiratory chain [1], cardiovascular diseases (ischemia, hypoxemia, anemia) [2] and other diseases [3]. The lactate monitoring is also used in diabetes control [4] and rehabilitation [5]. Oxygen deficits (tissue hypoxia) are the most common and often refractory causes of lactic acidosis, including pulmonary problems (low partial pressure of O2), circulatory problems (e.g. poor delivery of O2), and hemoglobin problems (e.g. low O2-carrying capacity) [6]. In cancer cells, the rate of glycolysis increases even under aerobic condition which is commonly known as Warburg Effect [7, 8].
During an intense physical exercise the increases of blood lactate indirectly reflects on the increase of heart rate and oxygen uptake (VO2). The concentration of blood lactate is usually 1–2 mmol L-1 at rest, but can rise to over 20 mmol L-1 during intense exertion because of the switch of muscle cells to an anaerobic metabolism. However, once a certain level of lactate concentration is reached, exhaustion occurs and there is a rapid decline in exercise capacity [9]. Thus, in sport medicine blood lactate is commonly used as an indicator of training adaptation [10, 11] to monitor the maximum performance level of athletes [12-16].
The classical explanation of lactate production was that its increase provides supplementary anaerobically derived energy. Conversely, it has been suggested that the increased lactate
production depends on metabolic adaptations, which primarily activate the aerobic ATP production [17].
On this basis, it is important to determine the amount of lactate in biological fluids using simple, low cost, fast, precise and accurate methods.
Lactate may be determined off line in blood, plasma, sweat or urine using laboratory equipments such as gas chromatography (GC), liquid chromatography both coupled with mass spectrometric detection (LC–MS), high-performance liquid chromatography (HPLC) [18-20]. However, the cost of these instruments is generally high and the analytical procedures for the sample treatment are long and tedious. Alternatively, lactate can be determined using spectrophotometric measurements based on the enzymatic reactions of lactate dehydrogenase (LDH) and lactate oxidase (LO). The main drawbacks of enzymatic methods are the instability of the enzyme over time, especially if immobilized, the need to control the pH and often the need of ancillary reagents (such as the superoxide dismutase reaction coupled to Lactate oxidase reaction) and cofactors (such as NAD+ or NADP+) [21]. Portable lactate analysers equipped with disposable sensor strips are commercially available. These devices are based on the determination of lactate concentration in the blood sampled through a finger-stick or by punching the earlobe and are currently considered the most adequate system to obtain real-time lactate trend during the training session [22-25]. However, this practice is intrusive, it implies a risk of infections for the subject and transmission of potential diseases for the operator and it requires, in principle, the presence of medical staff. From the analytical point of view, during the sampling of the finger-stick test the lactate in sweat can contaminate the blood sample providing artificial high values because of the high concentration of lactate in sweat (about 10 times higher than in blood).
Human sweat can be an excellent alternative matrix for the noninvasive and continuous monitoring of chemical markers for health, fitness or pathological applications (ref. [26-29] and references therein). The sweat sampling is easier, without risks of infections and it can be frequently repeated with much less stress on the patient and athletes. The analysis of metabolites present in sweat is safe and simple.
Recently, several non-invasive methods have been developed to determinate the concentration of lactate in sweat [22, 30-32]. Several methods propose wearable sensor technologies for the non invasive, on line monitoring of lactate and other metabolites in sweat and perspirate [33-40]. Several authors reported a good correlation between the lactate concentration in sweat and in blood [41-43], thus lactate in sweat can be conveniently used for the evaluation of the physical performance [44, 45]. All these methods are based on the enzymatic quantification of lactate concentration (amperometric biosensors) and on electrochemiluminescence (ECL) biosensor and their dynamic linear range is at best 1–30 mM [30]. Roda et al. described the development of a smartphone-based device for non-invasive and simple monitoring of the endurance performance of athletes via lactate detection. This biosensor relies on chemiluminescence (CL) detection and exploits the lactate oxidase catalyzed reaction coupled with the enhanced luminol/H2O2/ horseradish peroxidase CL system. Using this system, a limit of detection (LOD) 0.5 mM and a dynamic linear range 0.5-20 mM were observed in sweat and in oral fluid [46]. Another flexible amperometric lactate biosensor using silver nanoparticle based conductive electrode has been proposed, although not validated yet in vivo [47].
Since the increase in exercise intensity increases the lactate concentration in sweat from ~10 mM up to ~25 mM [48], 62 mM [43] and even more than 100 mM [15, 20, 31], the upper detection limit of biosensors currently proposed limits their use in sweat lactate analysis. To overcome this drawback, several authors have proposed a Prussian Blue based lactate
biosensor capable to detect the lactate concentration continuously up to 70 mM [31]. However, these disposable biosensors showed a limited stability of the enzyme over time.
In this work we have developed a portable and non-invasive analyzer for the electrochemical determination of lactate concentration in sweat. The sweat is deposited on disposable screen printed carbon electrodes (SPCEs), modified with a low cost and stable Fe3+ solution, and the lactate concentration is measured through potentiometry. The potentiometric signal depends on the non-equilibrium potential difference of the Fe(III) /Fe(II) couple [49]. Fe(II) produced depends on the lactate concentration in sweat because of the following redox reaction activated by UV radiation [50, 51]:
CH3CHOH-COOH + 2Fe (III) hυ→ CH3COH + 2Fe(II) + CO2 + 2H+ (1)
The primary photochemical reaction is assumed to be an intramolecular oxidation– reduction caused by an electron transfer from the lactate to Fe(III) [52]. The mechanism proposed for these systems is:
–CO–OFe3+ hυ
→ Fe
2+ + CO2 (2)
Despite this reaction would be faster in the presence of UO22+ [51], we preferred to avoid the
use of UO22+ because of its toxicity [53]. On the other hand the presence of the hydroxyl
group in lactic acid provides an easier oxidation route because the OH group can be transformed into an aldehyde or ketone through a two electron oxidation [54]. Reaction (1) is performed at acid pH in order to obtain reproducible results during the generation of Fe2+ and
to avoid its eventual oxidation by oxygen [51].
The analyzer is equipped with a novel, miniaturized photochemical reaction system emitting between 300 and 400 nm with a maximum at 379 nm, able to irradiate a specific region on the
electrode. After 60 s (irradiation time) the analyzer measures the potential difference of the Fe(III) /Fe(II) couple.
The device was applied to the monitoring of lactate concentration in the sweat of several human subjects during prolonged cycling exercise. During the exercise the sweat was simultaneously sampled on filter paper and extracted in water, and the lactate concentration was also determined by HPLC for method validation and in order to assess the specificity of the method proposed in this work. The temporal lactate profiles demonstrate the utility of the lactate analyser for the noninvasive assessment of lactate levels during the prolonged physical exercise and during the recovery.
2.EXPERIMENTAL SECTION 2.1. Chemicals
L-Lactic acid (L-6402, 98%) was purchased from Sigma-Aldrich-Fluka (Milan, Italy). Stock solutions of L-lactate were prepared in 0.075 M NaCl solution in order to simulate the sweat ionic strength. Ten g/L Fe(III) solution in 0.5 M HNO3 (i.e. 10.000 ppm, 179 mM, 02583 Iron Atomic Spectroscopy Standard Solution) was purchased from Fluka (Milan, Italy).
Phosphate buffer solution (PBS) for reversed phase (RP) HPLC analysis at pH 2.5 was prepared from monobasic monohydrate sodium phosphate (BDH Laboratory Supplies, Poole, England) and phosphoric acid (345245, Sigma-Aldrich-Fluka). Methanol for RP-HPLC was purchased from Carlo Erba (Rodano, MI, Italy). Water deionized with a Milli-Q system (Millipore, Bedford, MA, USA) was used throughout.
The screen printed carbon electrodes (SPCE) were purchased from EcoBioServices &
Researches s.r.l. (Firenze, Italy) (Figure 1A). The SPCEs were composed by three electrodes
printed on a plastic substrate. The electrodes were Ag/AgCl (pseudoreference electrode) and
carbon (working and counter electrodes). Only one carbon electrode (the middle one, 3 mm diameter) is used for the potentiometric measure. The SPCEs were modified in one single
step by casting 8 L of Fe(III) solution, 0.15 M NaCl, onto their surface and drying at room
temperature. After this the SPCEs were ready to use. All voltages were referred to the Ag/AgCl pseudoreference electrode.
2.3. Lactate measurement
Electrochemical measurements were carried out with a portable prototype analyser (Figure
1B) capable to measure the differential potential (P). The analyser was equipped with a
LED (LED370E, ThorLabs, Germany), emitting between 300 and 400 nm with a maximum at 379 nm (Figure 1B); for the measurements described in this work, the LED was driven with 20 mA CW current, and was placed 10 mm over the modified region of SPCEs, providing an
average optical intensity of about 6 mW/cm2, with typical fluctuations of a few percent over
time. For the measurement of the electrochemical potential, one side of the SPCE (left side in Figure 1) is locked into a standard FFC connector, and the potential difference (PD) between the central carbon electrode and the reference Ag/AgCl electrode is read out with 1 mV resolution using a dual rail-to-rail operational amplifier with TeraOhm input resistance. The portable analyser measures the potential difference before the LED is turned on (t=0) and the
PD 60 s after the LED was switched on (PD60). The PD60 value is correlated with the lactate
concentration. The analyser was calibrated using lactate standard solutions with a known concentration and including in the software the calibration parameters of the calibration
curve (see Results). The measurements were performed by pipetting about 5 L onto the
modified SPCE and reading the PD60 value. In the selected operating conditions 4 L is the
minimum volume required for the analysis.
2.3. Epidermal lactate sensing and sweat collection for method validation by RP-HPLC analysis.
Human sweat samples were collected from 5 healthy volunteers (2 males and 3 females) during sport training tests on a cycle ergometer. The participants were non-smoking subjects accustomed to intense exercise, without skin or systemic diseases, and on no systemic medications. All subjects gave written, informed consent before participation in the study. The study was conducted according to the Declaration of Helsinki Principles. Participants were asked not to wash or apply ointments 6 hours prior to the test. Sodium lactate is employed, indeed, in several case as additive in the creation of bath and body products. The subjects were asked to begin cycling at a steady cadence (70-75 rpm) and to maintain this cadence while an increasing resistance was applied at 3 min intervals up to 18 min. This procedure ensures that the anaerobic metabolism was invoked. Afterwards the volunteers were asked to keep the same cadence while the intensity was gradually reduced during a 6 min “cool down” period. The evaluation was prolonged in several cases up to 60-75 min after the exercise.
Sweat lactate samples were taken and measured during and after the 18 min incremental cycle ergometer tests. About 5-8 L of sweat were collected about every 3 min with the SPCEs and measured in situ. Sweat samples were obtained by rapidly applying the strip onto the sweated skin. Fe(III) is fixed onto the electrode in such a way that Fe(III) is not released during sweat sampling. The skin was dried between one measure and the other in order to get
the measure always on fresh sweat samples. Sweat was also collected during exercise on paper filter disks (42 ashless Whatman, n. 1442070, 70 mm diameter), stored at -20 °C and analysed in lab by RP-HPLC as previously reported in detail [20]. This sampling was simultaneous with the measure performed by the SPCEs.
Heart rate was measured at rest and during the exercise using a trans-sternal Polar heart rate monitor (Polar Elecro Oy, Kempele, Finland). Sweat rate was measured volumetrically using a 5 cm2 sweat collector (Macroduct, Wescor, Logan, UT, USA) strapped to the flexor surface of the proximal half of the right forearm. Forearm sweat rate was expressed in milligrams per square centimeter per minute (mg cm-2 min-1 ), and the concentration of lactate in sweat was
expressed in millimoles per liter (mmol l-1 ). Lactate excretion rate (LER, nmol cm-2 min-1 ),
was calculated as the product of forearm sweat rate times.
3. RESULTS and DISCUSSION
3.1. Optimization of Fe(III) concentration and reaction time.
The instrumental dynamic range and response time were characterized by analyzing on unmodified SPCEs different solutions with various concentrations of lactate and of Fe(III). The results were then compared with similar measurements on modified SPCEs .
Figure 2 shows the kinetics obtained by analyzing 0, 10, and 100 mM lactate standard solutions containing 12 mM Fe(III). Error bars represent the standard deviation of n=5 measurements performed using 5 different electrodes randomly selected.
After 60 s in the operating conditions adopted the signal reached more than 95% of the
plateau values. Thus, 60 s were considered the optimum irradiation time.
Figure 3 (A) shows the calibration curve obtained by plotting the potential difference of the Fe(III) /Fe(II) couple, after 60 s of UV irradiation, versus the concentration of lactate (0-250
mM). The calibration plots were performed in the presence of 12 mM Fe(III) (curve a, slope=-68 2 mV mM-1, R2=0.9967) and 3 mM Fe(III) (curve b, slope=-81 5 mV mM-1, R2=0.98161). Error bars represent the standard deviation of n=5 measurements performed using 5 different electrodes randomly selected.
Figure 3 (B) shows the calibration plot performed using SPCEs modified with 3 mM Fe(III) (slope=-58 5 mV mM-1, R2=0.94897).
In both figures the open symbols indicate the signal before switching on the LED in the different operating conditions explored. The limit of quantitation (LOQ) was 1 mM. The LOQ value was calculated in accordance with IUPAC guidelines [55], as ten times the standard deviation of the blank sample. CV% has been calculated as the percentage ratio of the standard deviation and the value of the arithmetic mean value.
This dynamic range covers the concentrations of lactate typically found in human sweat, both at rest and during intense exercise. This range is 10 times more extended than the typical range of commercial blood and sweat lactometer (typical range 0.2-30 mM at most).
The stability of SPCEs was tested by repeating the calibration plot using the same lot of electrodes 1 day after their preparation and every 15 days (7 months currently were tested). No significant changes were observed, the slope varying by 5-6% at most.
It was verified (data not shown for brevity) that the measure was independent on the volume
deposited in the range 4-10 L, and was unaffected by the presence of NaCl in 50-150 mM
concentration range.
The effect of Fe(III) concentration in our operating conditions (UV light intensity of 6 mW/cm2 on SPCE). Figure 4 shows the potential difference of the Fe(III) /Fe(II) couple, after t=0 and t= 60 s UV irradiation, as a function of the Fe(III) concentration (10 mM lactate standard solution pipetted on the SPCE).
The data show that in the conditions investigated higher iron concentrations do not improve the sensitivity and reduce, instead, the difference among the potentiometric signal obtained before and 60 s after light irradiation. This can be due both to a partial absorption of radiation by lactate-Fe(III) complex, which is not available any more for the reaction, and to possible termination reactions due to the recombination of radicals (R-COO, secondary aliphatic radicals, OH radicals) that are formed in the photoreaction of lactate-Fe(III) complexes [56].
Iron concentration optimized in our operating conditions has to be re-optimized whenever LED with different intensities would be employed.
From the calibration curves reported in Figure 3 it results that the concentration of lactate sensed is by far higher than Fe(III) employed for its detection. Thus, the optimized conditions of UV light and Fe(III) concentration allow to operate in non equilibrium condition [49] limiting the Fe(III) consumption.
The photochemistry of Fe(III)-carboxylic acids complexes is rather known [54, 56-58]. Lactate, in particular, can significantly modify the speciation and photochemical reactivity of Fe(III) ions because it may form thermal stable Fe(III)-organo complexes with strong absorptions overlapping the solar spectrum. The complexes between Fe(III) and lactate were studied in ref. [59] and [58]. Fe(III) forms only 1 : 1 Fe(III)-lactate complex. According to previuos studies,[59, 60] in non complexing medium ca. 90% of Fe(III) forms the Fe(III)-lactate complex. However, in our operating conditions also NaCl is present in the medium and Fe(III) is present both as ion and as chloride complexes (FeCl2+ and FeCl2+) [61].
It is reported that first the Fe(III) carboxylate complexes gives Fe(II) and organic radicals. Then, decarboxylation, the formation of secondary aliphatic radicals and their recombination occurs. The interaction of radicals with other compounds (water, OH radicals) may also occur, and with the initial Fe(III) complexes may increase the quantum yield of photolysis. In the specific case of modified SPCEs, the CO2 developed in the photoreaction may diffuse
in a not controlled way from the thin layer. Thus, the study of the reactions occurring on the electrode surface it would be complex [49, 62] and beyond the analytical aim of this work.
Considering that the non-equilibrium potentiometric signal measured has a linear relationship with the log of the lactate concentration within the linear range indicated by the calibration curve, the goodness of analytical application is unquestionable, beyond the reaction mechanisms involved.
Effect of the light source intensity. The effect of the light source intensity was
investigated in a benchtop configuration by varying both the distance between SPCE and LED,(taking care to illuminate the whole reaction area) and the emitted optical power. To this purpose a LEDs with the same spectral distribution as the Thorlabs LED360E, and with different emitted optical power was employed. The light intensity was found to affect mostly the kinetics of the photochemical reaction. After starting the UV illumination on SPCE, the output voltage shows an exponential decay (see Figure 2). When increasing the UV light intensity, the time constant decreases until it reaches a steady minimum value. The time constant also depends on the concentration of lactate and Fe(III); in particular, it decreases for higher lactate concentration up to about 30 mM, and it decreases with Fe(III) concentration. With 10 mM of lactate and 12 mM of Fe(III), the minimum time constant is about 4 s, which is reached at UV light intensities above 20 mW/cm2.
On the contrary, the asymptotic voltage does not significantly depend on the UV light intensity. The typical fluctuations in the UV light emission intensity have thus a limited impact on the measurement, provided that the potential is recorded after the exponential decay has reached the steady state. Such condition is satisfied in the operating conditions described in this work.
3.2. Method validation
The correlation analysis (Figure 5) shows the linear regression obtained by analyzing n= 18 sweat samples collected during several training sessions simultaneously with the in situ measure performed using SPCEs modified with 3 mM Fe(III). The lactate in the real sweat samples was measured by HPLC [20] and its range was 16-200 mM.
The correlation between the lactate analyser and HPLC measure in the same sweat sample was r2=0.96; the slope value was 0.91. The coefficient of variation (CV%) was about 2-3% for HPLC measurements and 5-7% for the measurement with the modified SPCEs. The good correlation confirmed also the specificity of this measure toward sweat lactate.
The correlation found among HPLC lactate measurements and the values found by the lactate analyser also exclude any significant effect of possible interfering compounds present in sweat.
3.3. Application
The device was tested under practical scenarios during training sessions in 5 consenting subjects. Figure 6A, 6B and 6C show the data of three representative subjects. In each plot the left y axis reports the heart rate (beat per minute, bpm), the first right y axis reports the power of the cycle ergometer (watt); the second right y axis shows the lactate values (mM) determined in sweat. Figure 6 D shows the sweat perspiration data measured in subject (C), indicating that the sweat flow rate has a specific trend. After about 5 min the sweat produced is enough for the measurements performed using the modified SPCEs.
The inlet of Figure 6C reports the trend of the lactate excretion rate (LER), in nanomoles per square centimeter per minute (nmol cm-2 min-1) as a funcion of the exercise time. This trend is
analogous to the one of lactate concentration (mM) vs the exercise time, showing that possible effects of lactate dilution are negligible.
From Figure 6 it results that lactate concentration in sweat clearly reflects the intensity of the exercise. The timing of the concentration peak in blood and, thus, in sweat certainly depends on the physical conditions of the subject investigated and on his/her capacity of clearance of lactate. The fine interpretation of these data is beyond the analytical aim of showing the application of the method proposed in this work.
The increase of lactate corresponds to the switch from aerobic to anaerobic metabolism during intense, exhaustive exercise. During the initial period of the cycling exercise the lactate concentration level did not increase and the maximum value of lactate concentration in sweat was delayed with respect to the intensity peak of the exercise, in agreement with Jia et al. [30].
In subjects A and B the lactate decrease was delayed, as well, with respect to the decrease of the intensity of the exercise. In both subjects the increase of the heart rate was simultaneous to the increase of intensity of the exercise and its decrease was approximately simultaneous to the decrease of sweat lactate. Lactate values that increase later during the incremental test suggest well “fit” conditions” because they demonstrate a later condition of transition from aerobic to anaerobic metabolism. Similar profiles were obtained in other 2 subjects. Subject D differs because the decrease of lactate concentration in sweat was simultaneous to intensity of the exercise, despite the decrease of the heart rate was slower.
Although the interpretation of these data from the physiological point of view is beyond the aim of this work, it is known that the recovery rate, i.e. the time required by the subject to reach basal conditions in term of lactate and bpm is an important parameter. The recovery rate is, indeed, correlated with the “fit” conditions of the subject, the proper function of heart and fatigue [63]. Heart in healthy conditions recovers more quickly than an unhealthy or
untrained one. The recovery period can indicate an early warning of potential heart problems [64].
While the recovery of heart rate is well investigated [65, 66] few data are reported related to post-exercise lactate recovery, and lactate is usually determined with an invasive measure in plasma or in muscle (by biopsy) [67-69]. Only one work recently published reports the determination of the lactate concentration in sweat during the recovery using a multisensing wearable platform inside a microfluidic channel based on the lactate oxidase reaction [39]. In fact, other biosensors, which are characterized by 20 -30 mM lactate as upper detection limit [30, 70-73], though suitable for the definition of the “lactate threshold” during the exercise, would eventually saturate and become unsuitable for the analysis of lactate in post-exercise undiluted sweat [30].
It is noteworthy that lactate concentration changes in sweat are fast (min range). This could explain why several works, based on the continuous monitoring of lactate, showed a correlation between the exercise intensity and the sweat lactate concentration [30, 39, 74]. Other authors, instead, did not find any correlation between the exercise intensity and the sweat lactate concentration [75] likely because even small delays in the sweat sampling or relatively long samplings (5-10 min, depending on the collection technique) may give ambiguous results [75].
4. CONCLUSIONS
The measure of the increase of lactate in sweat may contribute to evaluate aerobic endurance performance capacity and to determine the optimal work loading intensities during endurance training [5, 76]. Acidosis results, indeed, in compensatory hyperventilation, although it does not seem to be the unique stimulus [77] and it is a major factor that determines the upper limit of exercise endurance and skeletal muscle fatigue [78, 79]. The timing of this increase during
the exercise and of its decrease during the cool down step is a personal evaluation of the physical status of the subject (sex, age, body mass index, muscle mass percentage and composition [80], muscle mass composition in terms of rapid and slow fibers [81], type of fibers enrolled during exercise [82], starvation [83], illness…), of his/her aerobic capacity. We propose a new, simple procedure and a new portable device for the determination of lactate in sweat. The procedure is based on the photochemical reaction of lactate with Fe(III), which is reduced to Fe(II) when irradiated with UV light. The methodology described in this work is the first that implemented a photochemical reaction performed in situ onto screen printed carbon electrodes in a portable device [50] The Fe(II) produced proportionally to the amount of lactate in the sample, is determined electrochemically with SPCEs. This sensing system is not based on an enzymatic reaction as in traditional measuring devices, and exhibits chemical selectivity towards lactate concentration in sweat with a linear response from 1 mM up to 180 mM. The reagents employed are cheep, no particular precautions that compromise the stability of the solution are required in the modification process of SPCEs, and the modified SPCEs are characterized by short and long term stability.
This method was applied to control the fitness routine of several subjects and it provided the trend of lactate concentration in sweat during controlled physical activity.
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(A) (B)
Figure 1. A) Screen printed carbon electrodes (SPCE). B) device with the SPCE inserted and LED on.
Figure 2. Kinetics obtained by analyzing 0 (a), 10 (b), and 100 mM lactate standard solutions (c) containing 12 mM Fe(III). Error bars represent the standard deviation of n=5 measurements performed using 5 different electrodes randomly selected.
Figure 3. (A) Calibration curves on unmodified SPCEs, obtained by plotting the potential difference of the Fe(III) /Fe(II) couple, after 60 s of UV irradiation, versus the concentration of lactate (0-250 mM). The calibration plots were performed in the presence of 12 (a) and 3 mM Fe(III) (b). Error bars correspond to the standard deviation of N=5 measurements performed using 5 different electrodes randomly selected. Open symbols refer to the potential value at t=0 irradiation time. (B) Calibration plot performed using SPCEs modified with 3 mM Fe(III) (slope=-58 5 mV mM-1, R2=0.94897).
0 5 1 0 1 5 2 0 2 5 3 7 5 4 0 0 4 2 5 4 5 0 4 7 5 5 0 0 5 2 5 S ig n a l, m V [ F e (II I)], m M t = 0 s t = 6 0 s
Figure 4. Potential difference of the Fe(III) /Fe(II) couple, after t=0 and t= 60 s UV irradiation, as a function of the Fe(III) concentration (10 mM lactate standard solution pipetted on the SPCE; UV light intensity of 6 mW/cm on SPCE).
Figure 5. Correlation plot obtained by analyzing N= 18 sweat samples collected during several training sessions simultaneously with the lactate measure performed using SPCEs modified with 3 mM Fe(III) and the photoreaction (slope=0.91 0.05, R2= 0.96).
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 b p m b p m P o w e r ( W a t t ) S w e a t L a c t a t e ( m M ) T i m e , m i n 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 P o w er , W at t 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 S w ea t L ac ta te , m M (A) 0 1 0 2 0 3 0 4 0 5 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 b pm b p m P o w e r S w e a t la c t a t e ( m M ) T i m e , m i n 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 P o w er , W a tt 0 1 0 2 0 3 0 4 0 5 0 S w ea t L ac ta te , m M (B)
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 5 1 0 1 5 2 0 2 5 3 0 0 2 0 4 0 6 0 8 0 n m o l c m -2 m in -1 T i m e , m i n b p m b p m P o w e r ( W a t t ) S w e a t L a c t a t e ( m M ) T i m e , m in 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 P o w er , W at t 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 S w ea t L ac ta te , m M (C) 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 S w ea t, g h -1 m 2 T i m e , m in (D) Figure 6. Monitoring of sweat lactate during 24 min of cycling exercises in 3 different subjects while changing the work intensity and during the successive cool down. A), B) and C) Left y axis reports the heart rate (beat per minute, bpm), the first right y axis reports the power of the cycle ergometer (watt); the second right y axis shows the lactate values (mM) determined in sweat using SPCEs modified with 3 mM Fe(III) and the photoreaction; C) inlet reports the trend of the lactate excretion rate (LER), in nanomoles per square centimeter per minute (nmol cm-2 min-1) as a funcion of the exercise time. D) perspiration data of subject C).
