Krishna K Dey

S Prasad

Jason Ash

Michael Deschamps

Philip J Grandinetti
email: grandinetti.1@osu.edu.

Spectral Editing in Solid-State MAS NMR of Quadrupolar Nuclei using Selective Satellite Inversion

Keywords: Quadrupolar Nuclei, Sensitivity enhancement, Inversion, Spectral editing

A sensitivity enhancement method based on selective adiabatic inversion of a satellite transition has been employed in a (π/2) CT -(π) ST 1 -(π/2) CT spectral editing sequence to both enhance and resolve multisite NMR spectra of quadrupolar nuclei. In addition to a total enhancement of 2.5 times for spin 3/2 nuclei, enhancements up to 2.0 times is reported for the edited sites in a mixture of rubidium salts.

Rotor Assisted Population Transfer (RAPT) is a method [START_REF] Yao | Sensitivity enhancement of the central transition NMR signal of quadrupolar nuclei under magic-angle spinning[END_REF][START_REF] Kwak | Enhanced sensitivity in RIACT/MQ-MAS NMR experiments using rotor assisted population transfer[END_REF] for enhancing the solid-state NMR central transition (CT) sensitivity of half-integer quadrupolar nuclei by transferring populations from the (unobserved) satellite transitions (ST) through selective saturation of the satellite transitions. An improved RAPT sequence using Frequency-Switched Gaussian pulses (FSG-RAPT) was later designed [START_REF] Prasad | A simple technique for determining nuclear quadrupole coupling constants using RAPT solid-state NMR spectroscopy[END_REF] to provide not only a more robust experimental enhancement but also the ability to measure quadrupolar coupling constants, C q . This approach combines the sensitivity advantage of the central transition with the C q measurement precision advantage of the satellites. The dependence of the FSG-RAPT enhancement on offset frequency for nuclei with different C q values was also exploited to design the (π/2) CT -RAPT-(π/2) CT and RAPT-(π) CT -RAPT-(π/2) CT schemes, for the selective excitation or suppression, respectively, of nuclei with large quadrupolar couplings [START_REF] Kwak | Selective suppression and excitation of solid-state NMR resonances based on quadrupole coupling constants[END_REF]. These methods, specifically (π/2) CT -RAPT-(π/2) CT , have been successfully utilized to understand the complex structure of Pyrex r by 11 B NMR [START_REF] Prasad | Solidstate multinuclear magnetic resonance investigation of pyrex r[END_REF]. More recently, Smith and Seith [START_REF] Smith | Site-selective QPASS for the isolation of large quadrupolar coupling environments[END_REF] used (π/2) CT -RAPT-(π/2) CT to simplify a complex 93 Nb spectrum in a layered perovskite, and, additionally, appended the (π/2) CT -RAPT scheme to QPASS [START_REF] Massiot | Order-resolved sideband separation in magic angle spinning NMR of half integer quadrupolar nuclei[END_REF] to edit 93 Nb NMR spectrum of KCa 2 Nb 3 O 10 that contains two overlapping niobium sites. Also, the RAPT editing schemes have been applied to selective 23 Na-1 H cross polarization experiments, and to simplfy heteronuclear correlation spectra in various Na salts [START_REF] Eden | Quadrupolar coupling selective cross-polarization in solid state NMR[END_REF].

Recently, Wasylishen and coworkers [START_REF] Siegel | Signal enhancement of NMR spectra of half-integer quadrupolar nuclei in solids using hyperbolic secant pulses[END_REF] demonstrated that a hyperbolic secant pulse [START_REF] Silver | Selective spin inversion in nuclear magnetic resonance and coherent optics through an exact solution of the bloch-riccati equation[END_REF] can provide even greater central transition enhancements through a selective inversion of the satellite transitions [START_REF] Vega | Triple quantum NMR on spin systems with I = 3/2 in solids[END_REF]. Many theoretical details behind this approach have yet to be worked out, but the successful use of a hyperbolic secant pulse to invert the full manifold of satellite spinning sidebands represents an exciting possibility for central transition sensitivity enhancements, selective excitation or suppression of sites, and C q measurements, as was the case for RAPT.

In this communication, we report a new scheme for spectral editing based on the selective inversion obtained using the WURST pulse shape for adiabatic inversion [START_REF] Kupce | Adiabatic pulses for wideband inversion and broadband decoupling[END_REF][START_REF] Kupce | Optimized adiabatic pulses for wideband spin inversion[END_REF]. WURST stands for Wideband, Uniform rate, Smooth Truncation: WURST operates at a lower peak radiofrequency field level than a hyperbolic secant pulse [START_REF] Silver | Selective spin inversion in nuclear magnetic resonance and coherent optics through an exact solution of the bloch-riccati equation[END_REF], prevents out-of-band excitation and inverts uniformly on a wide frequency range. The amplitude, phase and the excitation profile for the shaped pulse are shown in Fig. 1. We show that (1) a single WURST inversion pulse applied to single satellite transition spinning sideband can invert the full manifold of satellite spinning sidebands, and (2) selective inversion of resolved satellite spinning sidebands can be used to perform spectral editing of overlapping sites in a central transition spectrum. At the same time, substantial sensitivity enhancements of up to 2.0 times for the edited sites are reported.

In Fig. 2A is the CT resonance and part of the ST spinning sideband resonances for the three crystallographically distinct 87 Rb sites in RbNO 3 . With similar quadrupolar coupling constants [START_REF] Baltisberger | Pines, 87 Rb dynamic-angle spinning NMR spectroscopy of inorganic rubidium salts[END_REF], all three sites are overlapping in the CT and ST resonances. As shown in Fig. 2B, by applying a WURST inversion pulse on a single ST spinning sideband, which contains all three sites, an enhancement of all three sites in the central transition spectrum is obtained. The en- Excitation width (kHz) hanced spectrum was obtained by applying a 5 ms inversion pulse on the n = 6 ST spinning sideband, located at an offset of 122.3 kHz from the CT. An enhancement of 2.5 times is observed, and equally as important, the enhancement is uniform though out the line shape. The enhancement factor was identical when applied to individual spinning sidebands ranging from n = 5 to 17 (offset range 100-300 kHz). Again, we emphasize that the adiabatic WURST inversion pulse was applied to a single spinning sideband. This method differs from that of Siegel et al. [START_REF] Siegel | Signal enhancement of NMR spectra of half-integer quadrupolar nuclei in solids using hyperbolic secant pulses[END_REF][START_REF] Siegel | Sensitivity enhancement of solid-state NMR spectra of half-integer spin quadrupolar nuclei using hyperbolic secant pulses: Applications to spin-5/2 nuclei[END_REF] who applied double frequency adiabatic inversion pulses at frequencies far off resonance with single site systems such as 87 RbClO 4 , 27 Al(acac) 3 and 55 Mn 2 (CO) 10 [START_REF] Siegel | Signal enhancement of NMR spectra of half-integer quadrupolar nuclei in solids using hyperbolic secant pulses[END_REF][START_REF] Siegel | Sensitivity enhancement of solid-state NMR spectra of half-integer spin quadrupolar nuclei using hyperbolic secant pulses: Applications to spin-5/2 nuclei[END_REF].

2.0 -2.0 -0.8 -0.6 -0.4 -0.2 0.8 0.6 0.4 0.2 -4.0 0 τ t (π/2) CT (π) ST 1 0 1 -1 p τ w (A) (B) (C) (D) ν 0 +ν off
The (π/2) CT -RAPT-(π/2) CT approach [START_REF] Prasad | A simple technique for determining nuclear quadrupole coupling constants using RAPT solid-state NMR spectroscopy[END_REF][START_REF] Kwak | Selective suppression and excitation of solid-state NMR resonances based on quadrupole coupling constants[END_REF] for selective excitation of resonances with the largest C q values also exists with adiabatic inversion pulses. However, there is an additional possibility with adiabatic inversion pulses, not available with RAPT, for selective excitation amongst sites with similar C q values, provided the satellite transitions resonances of different sites are resolved. In fact, this possibility is enhanced for higher spin nuclei where the secondorder anisotropic width of the innermost satellite is narrower than the central transition under MAS (assuming the rotor angle is accurately set). To illustrate this aspect, simulated spectra of central and satellite transitions as a function of nuclear spin I are shown in Fig. 3. The spectra were simulated with a spinning frequency of 10 kHz All experiments were performed on a Bruker DMX 400 spectrometer using a Bruker 2.5 mm MAS probehead. The magic-angle was set using K 79 Br, and further calibrated by maximizing the ST-CT echo intensity in 87 RbNO 3 [START_REF] Ashbrook | Satellitetransition MAS NMR of spin I=3/2, 5/2, 7/2, and 9/2 nuclei: Sensitivity, resolution, and practical implementation[END_REF]. Solid RbCl was used to calibrate the rf-field strengths and used as chemical shift reference. The equilibrium magnetization recovery delay used was approximately 500 ms. Typically, the WURST excitation widths used were slightly larger than the anisotropic linewidth of the satellite spinning sidebands.

For creating a WURST pulse, the Bruker ShapeTool r was used.

and using a dwell time of half a rotor period in order to separate the even and odd spinning sidebands. The odd sidebands contain little to no contribution from the central transition. For spin I = 3/2 one can see that the second-order anisotropic broadening of the satellites are slightly narrower than the central transition. For spin I = 5/2 the inner satellite pair is significantly narrower, whereas for spins I = 7/2 and 9/2 it is the 2nd inner satellite pair, ±5/2, ±3/2 that is narrowest. This well-known narrowing [START_REF] Samoson | Satellite transition highresolution NMR of quadrupolar nuclei in powders[END_REF][START_REF] Jäger | How to get more from 27 Al MAS NMR by high-speed satellite-transition spectroscopy[END_REF][START_REF] Massiot | DOR and MAS NMR study of 27 Al : Reexamination of the aluminum borate 9 Al 2 O 3 -2 B 2 O 3[END_REF][START_REF] Ashbrook | Rotorsynchronized acquisition of quadrupolar satellite-transition NMR spectra: practical aspects and double-quantum filtration[END_REF][START_REF] Amoureux | Resolution enhancement in 1D solid-state NMR spectra of spin-9/2 quadrupolar nuclei[END_REF][START_REF] Massiot | Two-dimensional one pulse MAS of half-integer quadrupolar nuclei[END_REF], can be readily exploited with adiabatic inversion pulses to perform spectral editing of the central transition region. Thus, our approach is to use a (π/ Fig. 3. Simulated central and satellite transitions spectra for spin I = 3/2, 5/2, 7/2, and 9/2 nuclei with C q values of 1.0, 2.04, 3.13, and 4.24 MHz, respectively using half-rotor synchronized acquisitions to separate even and odd sidebands. The C q value for each spin I was adjusted for constant central transition line widths (η q =0.0 and ν L =131.45 MHz). The second-order shifts [START_REF] Samoson | Satellite transition highresolution NMR of quadrupolar nuclei in powders[END_REF] of the central (δ 2Q CT ) and satellite transitions (δ 2Q ST ) are given by

δ 2Q m,m-1 = -3[I(I + 1) -9m(m -1) -3] 40 ν 2 L I 2 (2I -1) 2 C 2 q 1 + η 2 q 3
× 10 6 (ppm). For spin 7/2 and 9/2 nuclei, the central and the inner satellite transitions (ST 1 ) overlap.

As an example we use the with edited site enhancements of 2.0, 1.8, and 1.2 for Rb 2 SO 4 -I, RbClO 4 and Rb 2 SO 4 -II, respectively. The approach works well for two of the three sites, with the inversion being more efficient for smaller C q sites. Some residual Rb 2 SO 4 -I resonance is obtained in the Rb 2 SO 4 -II edited spectrum because slight spectral overlap of ST resonances prevents the inversion pulse from being completely selective on the Rb 2 SO 4 -II ST resonance. A lower than expected CT enhancement may arise from the (π/2) CT pulse not being completely CT selective, thus affecting the ST populations to some small degree. The spectral editing based on selective inversion is not limited to spin 3/2 nuclei; significant signal enhancements of the edited sites could be achieved for higher spin systems.

Fig. 1 .

 1 Fig. 1. (A) Amplitude and (B) phase as a function of the pulse length of the WURST pulse. (C) magnetization profile as a function of frequency offset. The pulse length and the excitation width are as shown. (D) The (π) ST 1 -(π/2) CT sequence, where the selective π rotation of the innermost satellite is accomplished by adiabatic inversion using the WURST pulse.

Fig. 2 .

 2 Fig. 2. (A) 87 Rb spectrum of RbNO 3 (10 k scans) acquired at a spinning rate of 20 kHz showing CT and ST signals. Only one side of the spinning sideband envelope is shown. (B) Comparison of CT intensity obtained from WURST[START_REF] Kupce | Adiabatic pulses for wideband inversion and broadband decoupling[END_REF][START_REF] Kupce | Optimized adiabatic pulses for wideband spin inversion[END_REF] adiabatic inversion pulse (τ = 5 ms, excitation width = 3 kHz, ν rf =5.5 kHz) with no enhancement. All experiments were performed on a Bruker DMX 400 spectrometer using a Bruker 2.5 mm MAS probehead. The magic-angle was set using K 79 Br, and further calibrated by maximizing the ST-CT echo intensity in 87 RbNO 3[START_REF] Ashbrook | Satellitetransition MAS NMR of spin I=3/2, 5/2, 7/2, and 9/2 nuclei: Sensitivity, resolution, and practical implementation[END_REF]. Solid RbCl was used to calibrate the rf-field strengths and used as chemical shift reference. The equilibrium magnetization recovery delay used was approximately 500 ms. Typically, the WURST excitation widths used were slightly larger than the anisotropic linewidth of the satellite spinning sidebands. For creating a WURST pulse, the Bruker ShapeTool r was used.

FrequencyFig. 4 .

 4 Fig. 4. (A) 87 Rb spectrum of a 1:1 mixture of polycrystalline RbClO 4 and Rb 2 SO 4 [15,23] (40 k scans) acquired at a spinning rate of 20 kHz showing CT and ST signals. Only one side of the spinning sideband envelope is shown. (B) Single pulse spectrum (512 scans) (and (C)-(E) (π/2) CT -(π) ST 1 -(π/2) CT edited spectra (512 scans) for each site (CT region only). In each case, the inversion pulse was applied on one spinning sideband at the chosen offset as shown in (A). The adiabatic pulse parameters for the different sites are as follows: (C) τ = 2 ms, excitation width = 8 kHz, ν rf = 8.5 kHz. (D) τ = 5 ms, excitation width = 3 kHz, ν rf = 5.5 kHz. (E) τ = 5 ms, excitation width = 3 kHz, ν rf = 5.5 kHz. The spectrometer offset was placed at the middle of the CT resonances to minimize the off-resonance effects that are caused by the low power read pulse.

  87 Rb spectrum of a mixture of Rb 2 SO 4 and RbClO 4 , shown in Fig.4. Rb 2 SO 4 contains two Rb sites, while RbClO 4 contains one (see Table1). In the mixture spectrum, the CT resonance of Rb 2 SO 4 -II overlaps with the CT resonance of the RbClO 4 site. In contrast, the ST resonances of all three sites are better resolved, although there still is partial overlap of the Rb 2 SO 4 -I and Rb 2 SO 4 -II ST resonances. Using the (π/2) CT -(π) ST 1 -(π/2) CT sequence we obtained the edited spectra shown in Fig.4C, D, and E for each site,

	Site	C q	η q	δ 2Q CT	†	δ 2Q ST	†
		(MHz)	-	Hz		Hz
	Rb 2 SO 4 -I	2.67	0.89 -1718 3436
	RbClO 4	3.2	0.21 -1979 3959
	Rb 2 SO 4 -II	5.28	0.13 -5343 10686
	Table 1
	Quadrupolar parameters and relative seond-
	order quadrupolar shifts of rubidium salts. †
	Represents second-order shifts for the CT and
	ST, respectively.

Acknowledgments

This material is based upon work supported by the National Science Foundation under Grants CHE 0111109. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. PJG also acknowledges Le Studium, Orléans, France.


A Cazalé

W Sant

F Ginot

J-C Launay

G Savourey

F Revol-Cavalier

J M Lagarde

D Heinry

P Temple-Boyer

PHYSIOLOGICAL STRESS MONITORING USING SODIUM ION POTENTIOMETRIC MICROSENSORS FOR SWEAT ANALYSIS

Keywords: potentiometric sensor, ISE, ISFET, Na + ion detection, sweat analysis, physiological stress

In the frame of sweat analysis, two technologies, based on either ISE or ISFET devices, were developed for the implementation of pNa potentiometric microsensors. Both of them demonstrated good sodium ion Na + detection properties with a global sensitivity of around 110 mV/pNa in NaClbased solutions due to the use of an integrated "Ag/AgCl ink" pseudo-reference electrode. Then, in order to deal with in-vivo analysis of sweat natremia, a physiological sweatband prototype was developed, consisting of pNa-ISE and pNa-ISFET electronic detection modules as well as a textilebased sweat pump. Finally, sweating process was studied during series of experiments on twentyfive healthy consenting subjects. The sodium ion concentration [Na + ] was successfully monitored in sweat during various heat exposures, demonstrating a global increase with exercise trial duration. Furthermore, a strong correlation was found between the sweat [Na + ] concentration and the subject's internal temperature θ, allowing monitoring the subject's heat stress state. All in all, the relevance of the Na + ion analysis was demonstrated for the physiological stress monitoring and pNa potentiometric microsensors were shown to be very promising for the development of smart sweatbands.

Introduction

In the human body, the balance between heat production and heat losses is continuously being regulated by sweat evaporation on the skin surface in order to prevent heat stroke [START_REF] Parsons | Human thermal environment: the effects of hot, moderate and cold environments on human health, comfort and performance[END_REF]. As a result, sweating is essential for thermoregulation. Highly variable sweating rates were observed, mainly depending on physical activity and climate conditions [START_REF] Robinson | Chemical composition of sweat[END_REF][START_REF] Adolph | Physiology of man in the desert[END_REF][START_REF] Sawka | Fluid and electrolyte supplementation for exercise heat stress[END_REF]. Sweat consists mostly in water (99%) but contains many different chemical and biochemical compounds: water-based and mineral ions, lactate ion, ammonia, urea, uric and ascorbic acids, amino-acids, lipids and/or proteins [START_REF] Robinson | Chemical composition of sweat[END_REF][START_REF] Shirreffs | The sweating response of elite professional soccer players to training in the heat[END_REF][START_REF] Fisberg | Acid-base balance in sweat[END_REF][START_REF] Mickelsen | The composition of sweat with special reference to the vitamins[END_REF][START_REF] Costill | Sweating: its composition and effects on body fluids[END_REF][START_REF] Czarnowki | Sweat ammonia excretion during submaximal cycling exercise[END_REF][START_REF] Latzka | Water and electrolyte requirements for exercise[END_REF][START_REF] Huang | Uric acid and urea in human sweat[END_REF][START_REF] Saat | Effects of exercise in the heat of thermoregulation of Japanese and Malaysian males[END_REF][START_REF] Meyer | Effect of age and gender on sweat lactate and ammonia concentrations during exercise in the heat[END_REF].

According to these different works, sweat composition varies considerably from individual to individual. Influential parameters were associated to age, sex, race, genotype and of course heat stress, either resulting from climate conditions and/or levels of physical exercise. Among the different chemical and biochemical species present in sweat and/or on the skin surface, sodium (Na + ) and chloride (Cl -) ions, i.e. NaCl salts, were more specifically studied since both ions play key roles in the ionic equilibrium of body fluids. Focusing on sodium ion Na + , it was shown that sweat [Na + ] concentration depends widely on individual physiologies and varies with sweating conditions [START_REF] Robinson | Chemical composition of sweat[END_REF][START_REF] Shirreffs | The sweating response of elite professional soccer players to training in the heat[END_REF][START_REF] Fisberg | Acid-base balance in sweat[END_REF][START_REF] Costill | Sweating: its composition and effects on body fluids[END_REF][START_REF] Maughan | Fluid and electrolyte loss and replacement in exercise[END_REF][START_REF] Shirreffs | Whole body sweat collection in humans: an improved method with preliminary data on electrolyte content[END_REF][START_REF] Montain | Sweat mineral-element responses during 7h of exercise heat stress[END_REF]. In fact, assuming that sweat conductivity depends mainly on Na + /Cl -ion concentrations, strong correlations were evidenced between heart rate, sub-lingual temperature, sweat rate and sweat sodium concentration [START_REF] Shamsuddin | Changes in the index of sweat ion concentration with increasing sweat during passive heat stress in humans[END_REF]. Consequently, the sodium ion Na + was considered as a potential marker for heat stress monitoring.

In the frame of point of care applications, Na + potentiometric sensors, such as ion-sensitive electrodes (ISE) and ion-sensitive field effect transistors (ISFET), were preferentially studied using different ion-sensitive membranes. They can be either inorganic, focusing on the use of sodium aluminosilicate Na-AlSi 3 O 8 [START_REF] Baccar | Sodium microsensors based on ISFET/REFET prepared through an ion implantation process fully compatible with a standard silicon technology[END_REF][START_REF] Shin | H + , Na + and K + ion sensing properties of sodium and aluminium co-implanted LPCVD silicon oxynitride thin films[END_REF], either organic, emphasizing the use of sodium ionophores and polymers [START_REF] Oesch | Ion-selective membrane electrodes for clinical use[END_REF][START_REF] Abramova | Photocurable polymers for ion selective field effect transistors. 20 Years of Applications[END_REF]. As far as polymers are concerned, poly-vinyl-chloride (PVC) [START_REF] Oesch | Field effect transistors sensitive to sodium and ammonium ions[END_REF][START_REF] Brunink | Chemically modified field effect transistors; a sodium ion selective sensor based on calix[4]arene receptor molecules[END_REF][START_REF] Tsujimura | Comparison between silicone rubber membranes and plasticized poly(vinyl chloride) membranes containing calix[4]arene ionophores for sodium-selective field-effect transistors in applicability to sodium assay in human body fluids[END_REF][START_REF] Pijanowska | Na + -selective ChemFETs based on a novel ionophore: bis(phenylbenzo)-13-azocrown-5[END_REF][START_REF] Chudy | Multi-ion analysis based on versatile sensor head[END_REF], polyurethane (PY) [START_REF] Muñoz | Photosensitive polyurethanes applied to the development of ChemFET and EnFET devices for biomedical sensing[END_REF][START_REF] Ipatov | Integrated multisensor chip with sequential injection technique as a base for "electronic tongue" devices[END_REF], polypyrolle (PPy) [29], polysiloxane (PSX) [START_REF] Harris | Chemically modified field effect transistors; a sodium ion selective sensor based on calix[4]arene receptor molecules[END_REF][START_REF] Brunink | The design of durable Na + selective ChemFETs based on polysiloxane membranes[END_REF][START_REF] Reinhoudt | Durable chemical sensors based on field-effect transistors[END_REF][START_REF] Högg | Novel membrane material for ion-selective field-effect transistors with extended lifetime and improved selectivity[END_REF] and fluoropolysiloxane (FPSX) [START_REF] Dumschat | Filled fluorosiloxane as matrix for ion-selective membranes[END_REF][START_REF] Cazalé | Study of field effect transistors for the sodium ion detection using fluoropolysiloxane-based sensitive layers[END_REF] were studied, demonstrating fundamental differences in terms of physical properties and integration processes. Alternatively, the use of numerous sodium ionophores enabled the development of Na + -sensitive ISE and ISFET microdevices with quasinernstian potentiometric responses.

Even if sweat samples collection is relatively easy, measurement in sweat remains uncertain because of physiological discrepancy between human subjects as well as differences between sampling techniques and/or experimental conditions. Therefore, in order to attain skin chemical analysis, several approaches were proposed [START_REF] Bandodkar | Non-invasive wearable electrochemical sensors: a review[END_REF]. Integrated electrochemical microcells were developed for skin antioxidant properties but, as epidermal contact was limited to low durations, skin hydrolipidic film, rather than sweat, was deliberately analysed [START_REF] Christophe | Elaboration of integrated microelectrodes for the detection of antioxidant species[END_REF]. In order to activate sweat production, iontophoresis was successfully used for the transdermal ethanol measurement due to a miniaturized amperometric biosensor [START_REF] Gamella | A novel non-invasive electrochemical biosensing device for in situ determination of the alcohol content in blood by monitoring ethanol in sweat[END_REF]. Nevertheless, such technique is not compatible with sweat ambulatory monitoring systems. Indeed, in order to thoroughly deal with sweat analysis, long duration epidermal contact is required. This goal was reached by developing flexible/textile technologies and smart tattoos. Thus, using a stretchable impedimetric microsensor incorporating a porous substrate to collect sweat, the in-situ monitoring of sweat dielectric properties and production rate was demonstrated [START_REF] Huang | Stretchable, wireless sensors and functional substrates for epidermal characterization of sweat[END_REF]. Epidermal tattoos were also developed for the continuous sweat monitoring, based on amperometric detection of antioxidant species [START_REF] Windmiller | Electrochemical sensing on printable temporary transfer tattoos[END_REF][START_REF] Jia | Electrochemical tattoo biosensors for real-time non-invasive lactate monitoring in human perspiration[END_REF], pH measurement [START_REF] Bandodkar | Tattoo-based potentiometric ion-selective sensors for epidermal pH monitoring[END_REF] and potentiometric pNa detection [START_REF] Bandodkar | Epidermal tattoo potentiometric sodium sensors with wireless signal transduction for continuous non-invasive sweat monitoring[END_REF]. This last work should be highlighted since it showed that sweat Na + concentration increases during physical exercise. However, since these technologies are qualified as temporary, their capacity to deal with high sweat production rates and volumes for long-term skin analysis was not fully demonstrated. This is not the case for smart physiological sweatbands. Consequently, a wearable sensing system was developed, integrating a textile-based fluid handling system for sweat collection and transport as well as conductivity, pH and pNa sensors [START_REF] Coyle | BIOTEX -Biosensing textiles for personalised healthcare management[END_REF][START_REF] Schazmann | A wearable electrochemical sensor for the real-time measurement of sweat sodium concentration[END_REF]. As previously demonstrated, the sweat natremia was shown to increase during an exercise trial, highlighting the vital role of Na + ion in human physiology.

Our interest lies in the safety of first responders (soldiers, fire fighters,...) in their everyday rescue actions. In this frame, the internal temperature monitoring is crucial for preventing hyperthermia and similar physiological threats. However, the use of rectal or tympanic temperature probes is not conceivable and standard body or skin temperature sensors do not give effective results for obvious reasons: heavy gears, external heat stresses, physiological inadequacies,... As a result, an indirect measurement of internal temperature should be established, and sweat natremia monitoring appears to be the most promising solution for this matter. Thus, this paper presents the development of a wearable sweatband integrating a textile-based pump to collect and deliver sweat samples as well as ISE and ISFET microsensors to monitor sodium ion Na + concentration in sweat during heat stress tests. pNa microsensors detection performances are analysed, the fabrication procedure of a complete Na + -monitoring sweatband is described, sweat monitoring is demonstrated during in-vivo experiment on human subjects, and correlations between sweat sodium ion concentration [Na + ] and internal temperature θ are finally examined.

Experimental

Two technologies based on potentiometric detection principles were compared for the development of pNa potentiometric microsensors: ion-sensitive electrodes (ISE) at CEA-LETI (Grenoble, France) and ion-sensitive field effect transistors (ISFET) at LAAS-CNRS (Toulouse, France).

pNa-ISE microsensors fabrication

The developed electrochemical sensor consists in a host ionophore incorporated in a conducting polymeric membrane. The targeted ion complexation with the host molecule leads to a modification of the electrochemical potential. This modification is measured by potentiometry through the evolution of the open circuit potential (OCP). In this application, the electrochemical measurement cell was comprised of three silver working electrodes (ET1, ET2 and ET3) to assure measurement reproducibility [START_REF] Schazmann | A wearable electrochemical sensor for the real-time measurement of sweat sodium concentration[END_REF]. These ISE microdevices used an "Ag/AgCl ink" pseudoreference electrode integrated on the pNa-ISFET detection module (see section 2.2 hereafter).

To fabricate the pNa-ISE sensor (Fig. 1), a poly-vinyl-chloride (PVC) platform (volume: 16 × 8 × 4 mm 3 ), containing a cavity, was designed and glued on a dedicated substrate. Then, the empty cavity was filled with a mix of 0.1 M NaCl solution and glycerol (25% and 75% in weight respectively). Finally, 90 mg PVC, 20 mg ETH500, 20 mg 4-tert-butylcalix [START_REF] Sawka | Fluid and electrolyte supplementation for exercise heat stress[END_REF]arene-tetra-acetic acid tetraethyl ester and 201 µL of bis(2-ethylhexyl)adipate solution were dissolved in 1.5 ml of tetrahydrofuran (all the compounds were purchased from Sigma Aldrich). This solution was dropcasted directly on the filling solution at the top of the PVC platform. Over a night at room temperature, after solvent evaporation, a Na + -sensitive PVC membrane was automatically sealed on the PVC platform. So, detection modules involving three pNa-ISE with a common solid/liquid interface were successfully developed (Fig. 2).

pNa-ISFET microsensors fabrication

The SiO 2 /Si 3 N 4 -pH-ChemFET technology used was already presented in previous works, demonstrating quasi-nernstian pH responses (sensitivity around 53 mV/pH), detection accuracy around 1 mV or 0.02 pH, maximal drift lower than 5 mV/day and lifetime higher than four months [START_REF] Sant | On-line monitoring of urea using enzymatic field effect transistors[END_REF]. This technology was successfully adapted to sodium Na + ion detection using fluoropolysiloxane (FPSX) based ion-sensitive layers [START_REF] Cazalé | Study of field effect transistors for the sodium ion detection using fluoropolysiloxane-based sensitive layers[END_REF]. Consequently, P-well, N-channel, SiO 2 /Si 3 N 4 -gate, pH-sensitive chemical field effect transistors (pH-ChemFET) were fabricated on 6-inch, (100)-oriented, N-type (500 Ω.cm) silicon wafers in a silicon foundry in order to assure mass fabrication at low cost, with high reproducibility and high fabrication yield. Then, "Smart cards" techniques were used in order to reach automated, mass fabrication/packaging of detection microdevices (called "ISFET detection module" hereafter) integrating two pH-ChemFET chips (size: 2.1 × 1.6 mm 2 ) and an "Ag/AgCl ink" (purchased from Gwent group) pseudo-reference electrode (surface: ~ 5 mm 2 ). Finally, "drop on demand" ink jet process was used for the controlled deposition of FPSX-based, Na + -sensitive membranes using 10 mg of "4-tert-butylcalix [START_REF] Sawka | Fluid and electrolyte supplementation for exercise heat stress[END_REF]arenetetra-acetic acid tetraethyl ester" sodium ionophore and 5 mg of "sodium tetrakis [3,5-bis(1,1,1,3,3,3-hexafluoro-2-methoxy-2-propyl)phenyl]borate trihydrate" ionic additive (all the chemical compounds were purchased from Sigma-Aldrich) [START_REF] Cazalé | Study of field effect transistors for the sodium ion detection using fluoropolysiloxane-based sensitive layers[END_REF]. Consequently, detection modules involving a pNa-ISFET, a pH-ChemFET and "Ag/AgCl ink" pseudo-reference electrode were successfully developed (Fig. 3).

Development of a physiological sweatband for Na + ion concentration analysis in sweat

To deal with the final objective related to sweat in-vivo analysis, a physiological sweatband prototype was conceived and fabricated. First of all, the ISE and ISFET detection modules and their associated EPROM (SOIC8 from Conrad company) measurement interfaces were connected on a Kapton-based flexible substrate. An Analog Device ADT7301 temperature probe was finally connected on the electronic platform to take into account sweat temperature during the electrochemical measurement (Fig. 4). Then, a hydrophilic, textile-based sweat pump was also developed. It involves a sampling zone (surface: 5 × 3 cm 2 , thickness: 2.4 mm) and a transfer zone (surface: 2 × 1 cm 2 , thickness: 0.8 mm) in order to collect and transport sweat towards sensor sensitive surfaces using capillary forces, as well as a storage zone (surface: 5 × 3 cm 2 , thickness: 5 mm) to assure an efficient pumping capacity (Fig. 5). The sampling and transfer zones were both made of non-woven polypropylene with a trilobal fiber structure. Their absorption properties were characterized by an initial "dry" regime with a high capillary velocity (~ 1.5 mm/s) and a steady state "wet" regime with a lower one (~ 0.2 mm/s), enabling a global capillarity absorption of around 0.11 meter in four minutes. For the storage zone, the material used was based on polyester/polyacrylate fibers, assuring a water absorption rate of 300% in weight (or 240% in thickness). According to these characteristics, delay associated to the sweat transport towards the sensor sensitive surface was estimated at around two minutes, i.e. significantly lower than the sweating physiological delay (10-15 minutes depending on subjects). Finally, the functional integration of the different sweat analysis elements was performed on the basis of a chest strap for heart rate monitoring (Fig. 6).

In vitro electrochemical measurements

A portable electronic board was developed as an interface for the electrochemical sensors. This electronic part is responsible for signal acquisition and signal processing in order to convert the electrical information to sodium ion Na + concentration. This prototype consists of analogical parts (regulation, generators, commutation matrix) and control blocks (microcontroller MSP430F1611 pulsated at 8 MHz), integrating software for communication and signal processing.

In-vitro electrical measurements were performed by biasing the solution to the mass (V reference = 0 V) due to the "Ag/AgCl ink" pseudo-reference electrode integrated on the pNa-ISFET detection module [START_REF] Cazalé | Study of field effect transistors for the sodium ion detection using fluoropolysiloxane-based sensitive layers[END_REF]. Both detection modules, i.e. pNa-ISE and pNa-ISFET, were biased in the same way.

Concerning the ISE microsensor, the open circuit potential (OCP) was measured by

determining the potential difference between the Na + -sensitive working electrode and the Ag/AgCl pseudo-reference electrode when no current is applied. An electronic board (input impedance: > 0.3 GΩ) recorded finally this OCP value in order to compare it with calibration curves using a 12-bit analog-to-digital converter.

For the ISFET microsensor, the gate-source voltage V GS of the pNa-ISFET microdevice was monitored using the "source-drain follower" electronic measurement interface, i.e. working with constant drain-source voltage V DS and drain-source current I DS (typically V DS = 2 V and I DS = 0.1 mA) in order to reach saturation mode. In both cases, a specific data treatment was developed: an analog-to-digital converter sampled the output voltage useful signal at 1 Hz and a microprocessor was finally responsible for an average procedure every five seconds. The latter was introduced to improve the signal to noise ratio (measurement accuracy: 1 mV), leading finally to a measurement display with a 0.2 Hz sampling frequency.

Sodium ion Na + detection properties were studied for each electrochemical microsensor using these measurement setups. Concerning ISE devices, pNa measurements performances were only tested in solutions with sodium chloride NaCl concentrations corresponding to the physiological range in sweat (10 -2 to 10 -1 M). For ISFET, they were tested in lithium acetate solutions (CH 3 COOLi 0.1 M) with various concentrations of sodium chloride NaCl (range: 10 -5 to 10 0 M).

In vivo physiological measurements

Twenty-five young healthy subjects participated in the study after they went through clinical exam and signed an official consent to the experimental protocol (approved by the Ethics committee of Grenoble University). Their biometrical characteristics are given in Table 1. They underwent three heat stress tests in climatic chamber (IRBA -Antenne CRSSA, La Tronche, France) in order to stimulate sweating at different levels and through different ways: sweating test (SWT), passive controlled hyperthermia test (PCHT) and active controlled hyperthermia test (ACHT).

SWT is a passive heat test [START_REF] Launay | Anthropological and thermoregulatory changes induced by a survical sojourn in a tropical climate[END_REF]. The subject at rest was submitted during 1.5 hour to an external dry heat load (dry air temperature = radiant temperature = ambient temperature = 45-50°C; relative humidity: < 10%; air speed: = 0.8 m/s) in order to stimulate the sweating response at a level that assures a new thermal equilibrium as heat losses (due to evaporation of sweat) counterbalance the external heat load of the climatic chamber. The aim of the PCHT is to induce a rise in internal temperature up to 39°C in 1.5 hour only by changes in the climatic environment (ambient temperature: 45-50°C and 20% < relative humidity < 80%; air speed: = 0.8 m/s) without any physical exercise. The subject was strictly at rest, wearing airtight protective clothing (purchased from TYVEK ® Classic, DuPont France, Puteaux, France). Such high ambient temperature induces an external heat load and a high relative humidity microclimate, impairing thermolysis that finally leads to a rise in internal temperature. Lastly, ACHT leads to the same rise in internal temperature for the same duration as in PCHT (internal temperature of 39°C reached in 1.5 hour). Here, the subject, dressed as in PCHT, underwent a walk on a treadmill (speed: 4 km/h, slope: 4 %) inducing an internal heat load combined with an external heat load due to the environmental climatic conditions (ambient temperature: 30-40°C and 20% < relative humidity < 80%). Before each test (see above), subjects lied down and rested during 1.5 hour inside the climatic chamber at thermoneutrality (ambient temperature: 28-30°C and relative humidity: 40-50%) in order to be in the same thermo-physiological state before each test, especially without sweating. The target internal temperature of 39°C was chosen since it is the highest possible safety level for the subjects in the specified experimental heat exposure conditions. This target level also allowed each subject to produce enough sweat for samples collection, taking into account differences between physiological capabilities.

During ACHT test, ten subjects wore also the physiological sweatband dedicated to continuous monitoring of sodium ion concentration [Na + ] in sweat (see section 2.3) (Fig. 6).

Internal temperature θ was continuously measured by a rectal probe equipped with a calibrated Cu-Ct thermocouple (measurement accuracy: ± 0.05°C, see hereafter) and recorded every 10 seconds.

Sweat was collected with the modified Brisson method [START_REF] Brisson | of humans submitted to climatic constraints, especially heat, cold, terrestrial altitudes and protective[END_REF] into two hand-made pockets made of Opsite transparent adhesive film (Smith & Nephew, London, GB) and impermeable Parafilm M (Bemis, Wisconsin, USA) placed on the abdomen at either side of the umbilicus. The skin was carefully cleaned with alcohol, deionized water and sterile compresses before sweat pocket positioning. The surface of each pocket in contact with skin was of 10 × 10 cm 2 . Each of them was equipped with a miniature pipe that is used to withdraw sweat easily. Sweat was first collected after 35 minutes and then every 20 minutes in order to have sufficient volumes for biochemical analyzes.

In each sample, a mix of sweat was collected from the two pockets (volume: 1 to 2.5 mL every 20 minutes) and Na + and Cl -ion concentrations were measured online using a calibrated automated analyzer (Roche Hitachi 912 chemistry analyzer) that consisted of three ion-sensitive electrodes (measurement accuracy: ± 0.02 pNa or pCl). The subjects were also equipped with eleven Cu-Ct thermocouples and other specific sensors to measure continuously skin temperature in ten different sites, tympanic temperature (i.e. internal temperature θ) as well as heart rate and metabolic heat production. The different thermocouples used were T-type, copper-constantan (Cu-Ct) (JD Mesures, La Seyne sur Mer, France) calibrated using a specific bath (WIKA CTB9210, WIKA France, Eragny sur Oise, France), and a Pt100 reference thermometer (PHP602, Ris Orangis, France), with measurement accuracy of ± 0.05°C. Lastly, the body mass of the subjects was continuously measured during all three tests, not only during exercise, but also before and after.

Results and discussion

Since both potentiometric microsensors, i.e. pNa-ISE and pNa-ISFET were associated with an "Ag/AgCl ink" pseudo-reference electrode whose potential is also sensitive to chloride ion Clconcentration (see hereafter), a global detection sensitivity s 0 will be defined while taking into the different sensitivities interfering in the measurement chain.

In-vitro measurement

The validation of the pNa-ISE detection module consisted in determining its sensitivity towards a Na + ion concentration variation (estimation for three different devices). As shown in Fig. 7, we observed a linear correlation between pNa and OCP values. Due to the use of an "Ag/AgCl ink" pseudo-reference electrode and NaCl-based test solutions (see hereafter), the pNa-ISE sensor response was characterized by a global sensitivity s 0 of around 110 ± 5 mV/pNa in the range of 10 to 100 mM, corresponding to the typical sodium ion concentration in sweat [START_REF] Coyle | BIOTEX -Biosensing textiles for personalised healthcare management[END_REF]49].

The pNa-ISFET microsensor was already studied and optimized, demonstrating linear responses (detection sensitivity of 55 mV/pNa) in the [0 -4] pNa range, good selectivity towards potassium ion (K Na+/K+ ≈ 10 -3 ) as well as no pH interferences in the [START_REF] Robinson | Chemical composition of sweat[END_REF][START_REF] Adolph | Physiology of man in the desert[END_REF][START_REF] Sawka | Fluid and electrolyte supplementation for exercise heat stress[END_REF][START_REF] Shirreffs | The sweating response of elite professional soccer players to training in the heat[END_REF][START_REF] Fisberg | Acid-base balance in sweat[END_REF][START_REF] Mickelsen | The composition of sweat with special reference to the vitamins[END_REF][START_REF] Costill | Sweating: its composition and effects on body fluids[END_REF][START_REF] Czarnowki | Sweat ammonia excretion during submaximal cycling exercise[END_REF] pH range [START_REF] Cazalé | Study of field effect transistors for the sodium ion detection using fluoropolysiloxane-based sensitive layers[END_REF]. As a result, its characterization was here limited to its analytical response to pNa. As previously shown, pNa-ISFET detection modules were characterized by a linear response (global sensitivity s 0 estimated for ten different sensors: 110 ± 5 mV/pNa) in the [0 -5] pNa range (Fig. 8).

Thus, both pNa microsensors were characterized by a global sensitivity of 110 mV/pNa. Such high sensitivity is only related to the use of an "Ag/AgCl ink" pseudo-reference electrode and NaCl-based solutions. Indeed, from a theoretical point of view and according to our measurement interface, the detection module output voltage V out is given by: V out = V offset + s pNa-sensor pNa + s Ag/AgCl pCl [START_REF] Parsons | Human thermal environment: the effects of hot, moderate and cold environments on human health, comfort and performance[END_REF] where V offset is the constant parameter depending on the sensor technology and the measurement interface, s pNa-sensor is the pNa-sensor sensitivity to sodium ions and s Ag/AgCl is the sensitivity to chloride ions of the Ag/AgCl integrated electrode.

Since NaCl-based solutions were used, the concentrations of sodium Na + and chloride Cl - ions should be equal, i.e. pNa = pCl. As a result, the output voltage global sensitivity s 0 is expected to be roughly the sum of s pNa-sensor and s Ag/AgCl according to:

V out = V offset + (s pNa-sensor + s Ag/AgCl )pNa (2)
Since the s pNa-sensor and s Ag/AgCl sensitivities were estimated to 55 mV per decade in previous works [START_REF] Cazalé | Study of field effect transistors for the sodium ion detection using fluoropolysiloxane-based sensitive layers[END_REF], a global detection sensitivity of 110 mV/pNa should be obtained. Experimental results confirmed this theoretical approach (Fig. 7 and8). According to them, the potentiometric analytical responses of the different pNa-sensor technologies were finally given by:

-pNa-ISE: V out (mV) ≈ 110pNa -218

-pNa-ISFET: V out (mV) ≈ 110pNa +1230 (4)

Correlation between Na + ion concentration in sweat and internal temperature

For different subjects and considering the SWT, PCHT and ACHT tests, a correlation was established between the sweat sodium ion concentration [Na + ] measured in-vitro and the internal temperature θ (Fig. 9). In most cases (23 out of 25 subjects), a linear relation was demonstrated:

[Na + ] = aθ + b (5)
Nevertheless, since a and b parameters were different for each subject, a relationship between them was investigated. According to Fig. 10, it appeared that they are linearly correlated:

-b = aθ * with: θ * ≈ 37.0°C

This linearity is not related to human physiology but to a mathematical artefact. It is due to the very low internal temperature relative variations Δθ/θ compared to the sodium concentration variations Δ[Na + ]/[Na + ]. Nevertheless, according to equations ( 5) and ( 6), a direct correlation between the sweat sodium ion concentration [Na + ] and the internal temperature θ can be written as following:

[Na + ] = a(θ -θ * ) (7)
Whatever the subject, sweating does not occur immediately but 10-15 minutes after the beginning of the exercise trial. Consequently, it takes approximately 15-20 minutes to collect 1 mL sweat volume. For these very first sweat samples, physiological differences are responsible for a wide range of sodium concentration [Na + ] 0 (from 14 to 76 mM). On the contrary, the corresponding internal temperature θ 0 was estimated at around 37.5 ± 0.2 °C. Considering these two parameters, equation ( 7) becomes: [START_REF] Costill | Sweating: its composition and effects on body fluids[END_REF] else:

Since the output voltage global sensitivity was found to depend on both sodium and chloride ions concentrations [Na + ] and [Cl -] (cf. equation 1), the two parameters were studied independently using all different sweat samples (Fig. 11). Consequently, it appeared that they are linearly correlated [50]:

[Cl -] ≈ 0.92[Na + ] (10)
Thus, according to equations ( 1) and ( 10), it can be written:

V out = V cte + s o pNa [START_REF] Huang | Uric acid and urea in human sweat[END_REF] with: V cte = V offset -s Ag/agCl log(0.92) and s 0 = s pNa-sensor + s Ag/AgCl

As previously stated, by introducing the detection module output voltage V 0 obtained for the very first sweat sample, equation ( 11) becomes: [START_REF] Saat | Effects of exercise in the heat of thermoregulation of Japanese and Malaysian males[END_REF] Finally, according to equations ( 9) and ( 12), the sodium ion concentration [Na + ] in sweat and the internal temperature θ can be simply calculated: [START_REF] Meyer | Effect of age and gender on sweat lactate and ammonia concentrations during exercise in the heat[END_REF] or: [START_REF] Maughan | Fluid and electrolyte loss and replacement in exercise[END_REF] where [Na + ] 0 and θ 0 are the sodium ion concentration and the internal temperature for the subject very first sweat sample respectively ([Na + ] 0 depends mainly on subject's physiology but θ 0 ≈ 37.5°C), s 0 is the sodium sensor potentiometric sensitivity (s 0 ≈ 110 mV/decade) and θ * is a constant parameter (θ * ≈ 37.0°C).

Such equation system is very interesting since it makes simpler the sensor calibration procedure. Of course, in the frame of the natremia monitoring in sweat, the pNa sensor will have to be pre-calibrated previously in order to determine its analytical response. Thus, the very first sweat sample concentration [Na + ] 0 , which mainly depends on subject's physiology, will be estimated and the sweat [Na + ] concentration will be finally monitored using equation [START_REF] Meyer | Effect of age and gender on sweat lactate and ammonia concentrations during exercise in the heat[END_REF]. However, in the frame of the internal temperature θ monitoring, since the first sweat temperature θ 0 can be considered as a constant parameter (θ 0 ≈ 37.5°C) for all subjects, the knowledge of the pNa-sensor global sensitivity s 0 alone (~ 110 mV/decade) will be sufficient, with no need of any calibration.

Thus, according to our experimental results and associated empirical studies, it can be concluded that (i) the natremia level in sweat is a good marker for the human heat stress analysis, (ii) potentiometric detection principles are well fitted to the measurement of the sodium ion concentration [Na + ] in sweat, and (iii) pNa-ISE and/or pNa-ISFET potentiometric results can be extended to internal temperature θ monitoring.

In-vivo measurement

In-vivo experiments were finally performed for ten subjects, during an ACHT test, using the physiological sweatband prototype with both pNa-sensor detection modules (Fig. 6). Half of them gave comprehensive results and, among them, the experiment of subject n°7 was finally chosen for discussion. In such real conditions, both detection modules gave similar voltage variations, allowing us to study them globally (result not shown). Then, using the previous pNa-ISE and pNa-ISFET calibrations (equations 3 and 4 respectively), we were able to measure in real time the sodium ion concentration [Na + ] in sweat (Fig. 12). It takes roughly a quarter of an hour after the beginning of the test to obtain a first comprehensive answer. As shown previously (cf. section 3.2), this delay should be associated to the time required for sweating phenomena to start (10-15 minutes) and for the textile-based pump to transport sweat towards the sensor sensitive surface (~ 2 minutes). Then, the sodium concentration in sweat was monitored continuously. Thus, in agreement with standard chemical analysis (Roche Hitachi 912 chemistry analyzer) performed on samples collected from sweat pockets, a similar trend was obtained for both detection modules, ~ 64 mV/pNa) for sweat analysis [START_REF] Bandodkar | Epidermal tattoo potentiometric sodium sensors with wireless signal transduction for continuous non-invasive sweat monitoring[END_REF]. Thus, they were able to monitor the skin/sweat natremia before and after perspiration, always demonstrating a final [Na + ] concentration increase with time.

Compared to our results, these improved performances should be associated to a more functional integration of the potentiometric detection system. Nevertheless, such tattoo-like technology can be qualified as temporary and its capacity to handle high sweat production rates and volumes for longterm skin analysis was not fully demonstrated. Compared to these works, the proposed technology, either based on ISE or ISFET sensors (global detection sensitivity: ~ 110 mV/pNa), is fully compatible with standard sweatbands and confirmed the sweat [Na + ] concentration increase with time during an exercise trial. Moreover, its main achievement should also be associated to the monitoring of physiological hyperthermia. Indeed, considering the initial temperature θ 0 associated to the first sweat sample (θ 0 = 37.5°C) and according to equation ( 14), we were also able to determine the internal temperature variations during the ACHT test, evidencing a global increase in agreement with rectal temperature measurements (Fig. 13). Such result demonstrates that the indirect internal temperature measurement technique based on sweat natremia analysis has similar performances to the rectal probe (known as the best one). Of course, it will be argued that measurement accuracy will have to be more precisely calculated. According to physiological discrepancy between subjects, it could be estimated to ± 0.2°C (compared to 0.05°C for the rectal or tympanic probe). Such measurement accuracy should be sufficient for physiological applications. Moreover, even if rectal and tympanic probes were used in our sweating tests (SWT, HTCP and HTCA), their use for physiological monitoring in real life is not conceivable for obvious reasons.

All in all, these experiments allow a better understanding of human sweating phenomenon during a mixed internal and external heat-load. Starting at neutral internal temperature (37.0 ± 0.2°C), muscular activity is responsible for a physiological heat production and therefore an internal temperature increase. However, no sweating occurs up to 37.5 ± 0.2°C. As soon as the internal temperature reaches this threshold value, sweating is triggered in order to dissipate heat due to sweat evaporation. In the case of a sweating test (SWT), thermoregulation is efficient to maintain (or even decrease) the internal temperature θ as well as the sodium concentration [Na + ] constant. In the case of an ACHT test, wearing of a CBRN protection suit prevents evaporation of sweat and therefore any thermal regulation. As a result, the internal temperature θ as well as the sweat sodium concentration [Na + ] increase gradually in a correlated way.

Conclusion

Two technologies, based on pNa-ISE and pNa-ISFET potentiometric devices, were developed and compared for the in-vivo monitoring of natremia in sweat. In order to do so, they were integrated in a physiological sweatband prototype incorporating a textile-based sweat pump to collect sweat in real time. Characterized by similar Na + ion global detection sensitivities, i.e. 110 mV/pNa due to the use of an integrated "Ag/AgCl-ink" pseudo-reference electrode, both pNa microsensors enabled to determine in real time the [Na + ] concentration in sweat, demonstrating a global increase during hard physical exercises.

Finally, potentiometry was found to be well-fitted with human physiology. Indeed, according to an experimental campaign on twenty-five healthy consenting subjects, [Na + ] concentration in sweat was linearly related to the corresponding [Cl -] concentration as well as to subject's internal temperature θ. Thus, through the analysis of the first sweat sample, the use of pNa potentiometric microsensors (ISE and/or ISFET) allowed to monitor the sweat natremia as well as the internal temperature, independently of the subject physiology and without any complex calibration procedure. All in all, the relevance of the Na + ion analysis was therefore demonstrated for the physiological stress analysis.

Lastly, coming back to our core interest, i.e. the safety of first responders (soldiers, fire fighters,...) in their everyday rescue actions, the developed pNa-sensitive sweatband is finally fully compatible with others already-worn for heart rate monitoring. Thus, through sweat natremia analysis and associated internal temperature measurement, it could be used to prevent hyperthermia and related physiological threats. Furthermore, potentiometric detection could be applied to the detection of other chemical and/or biochemical species in sweat, still improving physiological diagnosis and associated safety. Finally, in a less lethal context, developments can be extended to physiological monitoring in the frame of sport practice.

Tables and figures caption

Table 1: biometrical characteristics of the twenty-five male subjects participating in the sweating tests ( : maximal oxygen consumption)

  demonstrating a [Na + ] global increase during the ACHT test. The use of integrated sensors for the sweat natremia monitoring during physical exercise was already shown in literature. S. Coyle et al. developed a "pNa-ISE -gold reference electrode" sweat patch and were able to demonstrate the pNa-ISE open circuit potential (OCP) increase with time, assuming the correlation with the sweat [Na + ] concentration [44]. B. Schazmann et al. developed a"pNa-ISE -Ag/AgCl/KCl reference electrode" sweat sensor belt (sensitivity: ~ 50 mV/pNa) and were able to monitor effectively the sweat [Na + ] concentration increase with exercise trial duration[START_REF] Schazmann | A wearable electrochemical sensor for the real-time measurement of sweat sodium concentration[END_REF]. Nevertheless, due to the use of a non-integrated solid/liquid reference electrode, this microsensor was finally not fully compatible with standard sweatbands. Finally, A. Bandodkar et al. developed "pNa-ISE -Ag/AgCl reference electrode" epidermal tattoos (detection sensitivity:

Fig. 1 :Fig. 2 :Fig. 3 :Fig. 4 :Fig. 5 :Fig. 6 :Fig. 7 :Fig. 8 :Fig. 9 :Fig. 10 :Fig. 11 :Fig. 12 :Fig. 13 :

 12345678910111213 Fig. 1: Fabrication scheme of the pNa-ISE detection module: a) gluing PVC platform on electronic circuit, b) filling solution into the cavity, c) drop-casting of the Na + -sensitive PVC solution,

Fig. 1 :Fig. 2 :Fig. 3 :Fig. 4 :Fig. 5 :Fig. 6 :Fig. 7 :Fig. 8 : 5 Fig. 9 :Fig. 10 :Fig. 11 :Fig. 12 :Fig. 13 :

 123456785910111213 Fig. 1: Fabrication scheme of the pNa-ISE detection module: a) gluing PVC platform on electronic circuit, b) filling solution into the cavity, c) drop-casting of the Na + -sensitive PVC solution,

Acknowledgements

The project was financially supported by the French "Agence Nationale de la Recherche" (ANR CSOSG, SWEAT project, n°ANR-08-SECU-010) in partnership with the French "Direction de la Police Nationale (DPN)" and "Direction Générale de l'Armement (DGA)". The authors would like to thank Y. Besnard, N. Clerc, C. Bourrilhon, J. Denis, P. Colin, J. Bompard and A. Alonso from IRBA-CRSSA for in-vivo physiological measurements and associated analysis. Finally, technological realizations were partly supported by the French RENATECH network.

French "Unité de thermophysiologie" then "Pôle tolérance climatique et vêtements" of the French "Centre de recherches du service de santé des armées" (CRSSA) in 1995, before taking the direction of the "Unité Environnements extrêmes" of the Institute of biomedical researches of the French Army (IRBA-CRSSA) for four years. During all these years, he studied human physiological tolerance and adaptation to heat, cold, altitude and protective clothing. Since then, after 18 years in biomedical research on tolerance of man in extreme environments (mainly in heat, cold and altitude and also on protective clothing) he has been working as an occupational practitioner at STMicroelecronics company (Crolles -France).

Gustave Savourey was born on February 1956. Medicine doctor in 1981, he received his PhD degree in "Biology of physical exercise" in 1987 from Claude Bernard University in Lyon (France) and his French "Habilitation à diriger des recherches" at the University of Lyon in 1993. He joined the French "Unité de thermophysiologie" of the French "Centre de recherches du service de santé des armées" (CRSSA) in 1986 and became its head director in 1997, studying the physiological