Detection of chemical species in subject's sweat

Abstract

A detection device for being placed on an investigation zone of the epidermis of a human or animal subject for detecting at least nitric oxide dissolved in sweat, comprising a structure (1) defining a microfluidic circuit (8) for directing a sweat flow and including an inlet orifice (4) allowing the passage of sweat from the epidermis, said microfluidic circuit (8) comprising at least one microfluidic channel (9) communicating with said inlet orifice, and at least one electrochemical sensor (10) comprising at least four electrodes arranged in succession in the longitudinal direction of the microfluidic channel, said at least four electrodes including a reference electrode, at least two working electrodes and a counter electrode, configured to generate a signal representative of the concentration of nitric oxide and further performing depletion and / or generating a signal representative of the flow rate of the sweat flow.

Description

[Technical field]

[0001] The present invention relates to methods and apparatus for detecting and measuring dissolved chemical species, more specifically nitric oxide, in the sweat of a human or animal subject. [Background technology]

[0002] Nitric oxide is a gas that constitutes an intercellular messenger. It is an important cardiovascular messenger of stress by mechanical transmission. More specifically, it is released during muscular exercise to stimulate vasodilation of the vascular system. The changes in the flow of gas produced in the blood and thus in the liquids in equilibrium with the blood, such as sweat, therefore constitute a particularly relevant indicator of the patient's cardiovascular ability to adapt to the muscular forces required during an exercise test.

[0003] Thus, there is a real need to develop an apparatus for detecting nitric oxide (NO) that is easy to manufacture, reliable, and easy to use.

[0004] WO 2019 / 229380 describes a device that can instantaneously measure nitric oxide in a subject's epidermal sweat and its clinical application. Summary of the Invention

[0005] Certain aspects of the present invention are based on the idea that quantitative measurement of changes in nitric oxide concentration in sweat provides a non-invasive technique for monitoring cardiovascular fitness during preventive examinations or for establishing a diagnosis.

[0006] Certain aspects of the invention relate to the spontaneous reaction of nitric oxide in the presence of molecular oxygen to produce nitrite ions (NO2 -In other words, changes in the concentration of nitric oxide represent the current state of the cardiovascular response to a particular exercise, whereas changes in the concentration of nitrite constitute a temporal record of these responses.

[0007] Certain aspects of the present invention are based on the idea of ​​combined detection of multiple compounds by an integrated electrochemical device.

[0008] Certain aspects of the present invention are based on the idea that changes in the concentrations of nitric oxide and nitrite ions can be detected and quantified in a coupled manner by an integrated electrochemical device.

[0009] Certain aspects of the present invention are based on the observation that nitric oxide is generated by a specialized enzyme (NO synthase) from the breakdown of intracellular L-arginine in the presence of molecular oxygen (O2) and an electron source. When the availability of L-arginine is reduced, either due to high levels of intake (e.g., after prolonged exercise) or chronic deficiency, NO synthase continues to react with oxygen, converting molecular oxygen into superoxide ions (O22 - ), which spontaneously and very rapidly transforms into hydrogen peroxide (H2O2) via an overall 2:1 stoichiometric reaction. Certain aspects of the invention are based on the idea that the presence of detectable concentrations of hydrogen peroxide in blood, and thus in sweat, etc., provides an indication of the level of distress in the cardiovascular network. Furthermore, in the presence of metal salts, hydrogen peroxide decomposes to form highly toxic radical species (HO·, HO2·, etc.), which can be very damaging to cells of the cardiovascular system, including the heart. Certain aspects of the invention are based on the idea that detecting the production of hydrogen peroxide in parallel with the detection of the production of nitric oxide and / or nitrite ions is relevant for the assessment of the cardiovascular capacity of a patient.

[0010] A particular embodiment of the present invention is based on the idea of ​​measuring at least the amount of nitric oxide by an electrochemical device and the amount of nitrite ions by colorimetric techniques.

[0011] Certain aspects of the present invention are based on the observation that the physiological system of a subject is dynamic, since the volumetric flow rate of sweat can change to adjust the capacity of removal of the generated heat energy as a function of the transmitted muscle force. The exchange flow of each chemical species at the blood-sweat interface can change as a function of the subject's exercise. Certain aspects of the present invention are based on the idea of ​​quantitatively and dynamically detecting the production of one or more chemical species selected from, for example, nitric oxide, nitrite ions, hydrogen peroxide, and optionally peroxynitrite, for example, during exercise testing or medical monitoring of a subject.

[0012] For this purpose, the invention relates to a detection device, which is placed on an investigation zone of the epidermis of a human or animal subject, for detecting at least nitric oxide dissolved in sweat, comprising: a structure defining a microfluidic circuit for directing sweat flow and including an inlet orifice for permitting the passage of sweat from the epidermis, said microfluidic circuit comprising at least one microfluidic channel, said microfluidic channel in communication with said inlet orifice; at least one electrochemical sensor arranged in series along the length of the microfluidic channel and comprising at least four electrodes including a reference electrode, at least two working electrodes, and a counter electrode; Equipped with the electrochemical sensor is configured to generate at least one signal indicative of a concentration of nitric oxide dissolved in the sweat stream; and depleting chemical species in said sweat stream having an oxidation potential lower than that of nitric oxide; generating a signal representative of the sweat flow rate; The present invention provides a detection device configured to perform at least one additional operation among the above.

[0013] These properties make it possible to reliably measure the concentration of nitric oxide (NO) dissolved in sweat.

[0014] The epidermis refers to the top layer of the skin of humans and animals.

[0015] The detection device is non-invasive and does not require application to a wound.

[0016] According to embodiments, a device of this kind may comprise one or more of the following features:

[0017] According to one embodiment, the structure is a multi-layer structure comprising a lower layer and at least one layer above the lower layer, the microfluidic circuit extending parallel to the lower layer, the lower layer comprising the inlet orifice.

[0018] According to one embodiment, the, or each, electrochemical sensor, or at least one electrochemical sensor, is configured to generate a signal representative of a sweat flow rate in the microfluidic channel, i.e. a volumetric flow rate of sweat in the microfluidic channel.

[0019] According to one embodiment, the multi-layer structure comprises an upper layer and at least one intermediate layer located between the lower layer and the upper layer, the microfluidic circuit being formed within the thickness of the at least one intermediate layer. The layers can be secured together by any suitable technique, such as adhesives, welding, mechanical clamps, etc.

[0020] These properties make the sensing device easy to manufacture, assemble and therefore industrialize.

[0021] According to one embodiment, the at least one intermediate layer includes a first intermediate layer and a second sealing intermediate layer located between the first intermediate layer and the upper layer, the second sealing intermediate layer having an opening in the electrode.

[0022] These properties allow the detection device to adapt to any curvature when applied to the epidermis. Moreover, the one or more intermediate layers also make it possible to create a thickness that can compensate for the thickness of at least four electrodes, thus ensuring the hermeticity of the detection device.

[0023] According to one embodiment, the at least one intermediate layer comprises an exit orifice that allows for the exit of one or more electrochemically measured sweat streams.

[0024] According to one embodiment, the multi-layer structure includes an upper layer and an exit orifice across the upper layer, the at least one microfluidic channel communicating with the exit orifice.

[0025] According to one embodiment, the underlayer is coated with an adhesive made of a flexible biocompatible material. According to one embodiment, the underlayer is adhesive on a first side intended for placement on the skin and on a second side intended for receiving an overlying layer.

[0026] These properties allow the detection device to measure at least the concentration of nitric oxide without interfering with the circulation of sweat in the investigation zone of the subject's epidermis, meaning that the detection device operates without affecting the passage of sweat through the epidermis in the investigation zone of the subject's epidermis.

[0027] According to one embodiment, the multi-layer structure is made from one or more of the following materials: -Inorganic materials such as silica, glass, and photosensitive glass. -Elastomeric materials such as polydimethylsiloxane (PDMS), modified polydimethylsiloxane (PDMS) (poly(acrylic acid), poly(ethylene oxide), TiO2, aluminum film, polycations, polyanions, mPEG, sol-gel, amine-thiol-carboxylalkoxysilanes, surfactants), poly(methyl methacrylate) (PMMA), and polycarbonate (PC). - Thermosetting materials such as the photosensitive resin SU-8. Thermoplastic materials such as polystyrene (PS), polyethylene terephthalate (PET), polyvinyl chloride (PVC), perfluoroalkoxy (Teflon-PFA®), fluoroethylene propylene (Teflon-FEP®). - Hydrogel materials such as Matrigel®, collagen, chitosan, alginate, agarose, PEG, polyacrylamide. -Cellulose and other paper materials.

[0028] According to one embodiment, the multi-layer structure includes at least one layer made of a polymeric material, for example, membrane cutting or molding techniques can be used to trace the microfluidic circuitry within the polymeric material.

[0029] According to one embodiment, the multi-layer structure is made of a polymeric material.

[0030] According to one embodiment, the multi-layer structure includes at least one layer made of a fibrous material, such as paper or a nonwoven fabric. For example, to trace a microfluidic circuit within a fibrous material, a coating of hydrophobic ink or hydrophobic resin can be used that defines the contours of the microfluidic circuit.

[0031] These properties make the multi-layer structure lightweight, flexible and pliable, which among other things allows the detection device to be used on many parts of the body, such as the back, arms, shoulders, legs and neck, without risk of damaging the device or reducing detection reliability.

[0032] According to one embodiment, the multi-layer structure includes at least one layer made of glass.

[0033] Preferably, the multi-layer structure is made of polyethylene terephthalate (PET), polyethylene naphthalate (PEN), an acrylic adhesive, polybutyrate adipate terephthalate (PBAT), a polyurethane layer and a polyacrylate layer (MPU).

[0034] According to one embodiment, the at least four electrodes are arranged on an inner surface of the upper layer closing the microfluidic channel at the top and / or on an upper surface of the lower layer closing the microfluidic channel at the bottom.

[0035] These properties ensure the location of the electrodes of the sensing device and also facilitate the fabrication of multi-layer structures containing these electrodes in that the electrodes can be fabricated on planar layers when microfluidic circuits are formed in the intermediate layers.

[0036] According to one embodiment, the electrodes consist of an applied metal.

[0037] According to one embodiment, the applied metal is selected from the group consisting of silver (Ag), gold (Au), platinum (Pt), platinum black. Graphite or carbon can also be used. According to one embodiment, the electrochemical sensor is composed of a silver / silver chloride (Ag / AgCl) or other reference electrode.

[0038] According to one embodiment, the electrochemical sensor is configured to deplete chemical species having an oxidation potential lower than that of nitric oxide. To this end, in one embodiment, in said direction of flow, said at least four electrodes comprise, in order, said first working electrode in the form of a depletion electrode, said second working electrode for measuring the concentration of nitric oxide, and said counter electrode, said reference electrode being placed at a position immediately upstream of said first working electrode or at a position immediately downstream of said second working electrode.

[0039] These properties can increase the accuracy of the electrochemical sensor by significantly reducing spurious signals that can result from compounds that oxidize at potentials lower than the oxidation potential of nitric oxide. Thus, the electrode set works synergistically to produce accurate results.

[0040] According to one embodiment, the depletion electrode is wider, for example at least four times wider, than the second working electrode needed to measure the concentration of nitric oxide.

[0041] Due to the large surface area of ​​the depletion electrode, electrolysis of the sweat stream passing over the depletion electrode can be substantially completed, and furthermore, the passivation phenomenon of the electrode is reduced.

[0042] According to one embodiment, one or more of the electrodes, for example the depletion electrode, is covered with platinum (Pt) black.

[0043] These properties allow the electrode to exploit the incomplete reactivity of the Pt black dendrites to catalyze electrochemical reactions and prevent passivation of the bare metal surface.

[0044] According to one embodiment, the electrochemical sensor detects hydrogen peroxide (H2O2), peroxynitrite (ONOO - ), and other species that oxidize at these potentials.

[0045] According to one embodiment, the counter electrode of the electrochemical sensor has a width at least equal to the sum of the widths of a set of electrodes arranged upstream of the counter electrode.

[0046] According to one embodiment, the electrochemical sensor is configured to measure the flow rate of a sweat stream in a microfluidic channel. For this purpose, according to one embodiment, in the direction of the flow, the at least four electrodes comprise, in order, the first working electrode for measuring the concentration of nitric oxide, the second working electrode for measuring the concentration of nitric oxide, and the counter electrode, the reference electrode being arranged at a position immediately upstream of the first working electrode or at a position immediately downstream of the second working electrode.

[0047] These properties allow the flow rate of sweat flow between the first and second working electrodes to be measured.

[0048] According to one embodiment, the electrochemical sensor is configured to generate a signal representative of the flow rate by measuring a delay between a change in current at the first working electrode and a change in current at the second working electrode.

[0049] According to one embodiment, the distance between the upstream and downstream working electrodes is preferably less than the distance covered by the flow in one minute. These characteristics allow accurate flow measurements to be made without interference from physiological changes in the subject.

[0050] According to one embodiment, the electrochemical sensor is configured to generate a signal representative of the instantaneous production of nitric oxide in the investigation zone based on a signal representative of the concentration of nitric oxide and a signal representative of the flow rate of the sweat flow.

[0051] "Instantaneous generation" means measurements taken over a time period that is very short compared to the characteristic time for a change in the physiological response of the subject, which for human subjects is typically on the order of one to several minutes.

[0052] According to one embodiment, the electrochemical sensor is configured to generate a signal representative of the concentration of nitric oxide by electrical measurement, in particular amperometric measurement, between at least one of said working electrodes and said counter electrode.

[0053] According to one embodiment, the electrochemical sensor is configured to polarize at least one of the working electrodes to a potential for the oxidation of nitric oxide.

[0054] According to one embodiment, the electrochemical sensor is configured to measure the concentration of one or more other chemical species in the sweat stream in addition to measuring the concentration of nitric oxide, and for this purpose, the electrochemical sensor is configured to generate a signal representative of the concentration of the one or more chemical species of interest by amperometry.

[0055] According to one embodiment, the electrochemical sensor is further configured to generate a signal representative of the concentration in the sweat stream of at least one of the following compounds dissolved in sweat: nitrite ion, hydrogen peroxide, and peroxynitrite.

[0056] To this end, according to one embodiment, the electrochemical sensor comprises a third working electrode between the first or second working electrode and the counter electrode for measuring a compound.

[0057] According to one embodiment, the electrochemical sensor is configured to measure the concentration of multiple compounds in the sweat stream sequentially in order of increasing potential of oxidation of the compounds.

[0058] According to one embodiment, the detection device further comprises a colorimetric detection device connected to the microfluidic circuit.

[0059] According to one embodiment, a colorimetric detection device is connected to the channel downstream of the electrochemical sensor, the colorimetric detection device comprising a hydrophilic porous body impregnated with a chemical reagent capable of reacting with one of the following compounds dissolved in the sweat to provide a colored indicator of the amount of said compound in the sweat stream.

[0060] According to one embodiment, the hydrophilic porous body is selected from a microporous membrane, paper, cloth, cellulose cotton, nonwoven fabric, and the like.

[0061] According to one embodiment, the chemical reagent comprises a Grease reagent capable of reacting with nitrite ions dissolved in the sweat stream.

[0062] According to one embodiment, the colorimetric detection device is disposed at the exit orifice.

[0063] According to one embodiment, a microfluidic circuit comprises a plurality of microfluidic channels connected to inlet orifices spaced apart from one another.

[0064] According to the embodiments described below, the detection device is implemented to detect multiple chemical species, such as 2, 3, 4 or 5, simultaneously or sequentially, including nitric oxide, using one or more electrochemical sensors. According to one embodiment, the detection device is implemented to detect the concentration of a chemical species selected from nitric oxide NO, nitrite ion NO2 and hydrogen peroxide HO2.

[0065] According to this embodiment, the detection device comprises three parallel microfluidic channels fed in parallel by the same inlet orifice.

[0066] According to one embodiment, the lower layer comprises a plurality of inlet orifices and the microfluidic circuit comprises a plurality of independent microfluidic channels each connected to a respective inlet orifice.

[0067] According to one embodiment, the detection device comprises a fibrous body for conducting sweat from the investigation zone to or towards the inlet orifice by capillary action. Such a fibrous body may be a woven or non-woven material.

[0068] According to one embodiment, the plurality of microfluidic channels includes an additional microfluidic channel comprising an electrochemical sensor, the electrochemical sensor comprising at least three electrodes arranged consecutively in a longitudinal direction of the additional microfluidic channel, the at least three electrodes including a reference electrode, a counter electrode and at least one working electrode, the additional electrochemical sensor configured to polarize electrodes to a potential for the oxidation of a compound selected from nitrite ions, hydrogen peroxide and peroxynitrite, the additional electrochemical sensor configured to generate at least one signal representative of the concentration of the compound dissolved in the sweat stream.

[0069] According to one embodiment, the electrochemical sensor is configured to repeatedly and cyclically polarize at least one of the working electrodes for a predetermined period of time.

[0070] These properties make it possible to measure the concentration of nitric oxide dissolved in the sweat stream while reducing electrode passivation phenomena.

[0071] According to one embodiment, the electrodes are polarized for a time period between 1 second and 500 seconds, which is repeated over a period of between 1 minute and 60 minutes.

[0072] These properties allow the concentration of multiple compounds to be measured.

[0073] According to one embodiment, a layer of polyeugenol (4-allyl-2-methoxyphenol), another polyphenol, or a similar polymer is applied to at least one working electrode of an electrochemical sensor, preferably electrochemically applied.

[0074] Other embodiments of the electrochemical sensor are described below.

[0075] According to one embodiment enabling sequential detection, the, or each, electrochemical sensor, or at least one electrochemical sensor, is configured for successively detecting a plurality of chemical species during a plurality of measurement steps, the electrochemical sensor being configured for polarizing an electrode to a potential for the oxidation of hydrogen peroxide HO during a first step and for polarizing an electrode to a potential for the oxidation of nitric oxide NO during a second step, and the electrochemical sensor is configured for generating a signal representative of the concentration of nitric oxide NO based on a first amperometric signal obtained in the first step and a second amperometric signal obtained in the second step.

[0076] In this case, advantageously, the electrochemical sensor, or each electrochemical sensor, or at least one electrochemical sensor, is adapted to detect nitrite ions NO during the third step. - The electrochemical sensor is configured to polarize the electrodes to a potential for oxidation of nitrite ions NO2 based on the first and second amperometric signals and the third amperometric signal obtained in the third step. - The device is configured to generate a signal representative of the concentration of

[0077] According to one embodiment, the detection device is implemented to be able to detect three of the aforementioned chemical species consecutively with a single electrochemical sensor during multiple steps of a time measurement sequence. According to this embodiment, the electrochemical sensor detects, in a given sequence, during a first time step of a few seconds (e.g. 5 seconds), a platinum-plated (platinum black) platinum electrode for the oxidation of hydrogen peroxide H2O2, then during a second time step of the same time for the oxidation of nitric oxide NO, and optionally during a third time step of the same time for the oxidation of nitrite ions NO2. - The electrochemical sensor is configured to generate a signal representative of the concentration of nitric oxide (NO) based on the first amperometric signal obtained in the first step and the second amperometric signal obtained in the second step. This sequence is repeated as many times as necessary during the exercise test. - Solving a system of three equations with the concentrations of , and , (the currents measured successively on electrodes polarized to each potential) gives the values ​​of each of the three concentrations at the moment each sequence was performed based on the three measurements.

[0078] According to another embodiment allowing simultaneous detection, the detection device comprises: a first microfluidic channel coupled to the inlet orifice for directing a first flow of sweat from the investigation zone; and a first electrochemical sensor comprising an electrode disposed within the first microfluidic channel, the first electrochemical sensor being configured to polarize the electrode to a potential for the oxidation of hydrogen peroxide, H2O2; a second microfluidic channel coupled to the inlet orifice for directing a second flow of sweat from the investigation zone; and a second electrochemical sensor comprising an electrode disposed in the second fluid circuit, the second electrochemical sensor being configured to polarize the electrode to a potential for the oxidation of nitric oxide (NO); Equipped with The electrochemical sensor is configured to generate a signal representative of a concentration of nitric oxide, NO, based on a first amperometric signal generated by the first electrochemical sensor and a second amperometric signal generated by the second electrochemical sensor.

[0079] In this case, the detection device advantageously further comprises: a third microfluidic channel coupled to the inlet orifice for directing a third flow of sweat from the investigation zone, and a third electrochemical sensor including an electrode disposed in the third fluid circuit, the third electrochemical sensor detecting nitrite ions, NO2 - a third electrochemical sensor configured to polarize the electrode to a potential for the oxidation of Equipped with The electrochemical sensor determines the amount of nitrite ions (NO2) based on the first and second amperometric signals and a third amperometric signal generated by a third electrochemical sensor. - The device is configured to generate a signal representative of the concentration of

[0080] According to one embodiment, the detection device further comprises: A further microfluidic channel, e.g., a fourth microfluidic channel, coupled to the inlet orifice for directing a further sweat stream, e.g., a fourth sweat stream, from the investigation zone, and a further electrochemical sensor, e.g., a fourth electrochemical sensor comprising an electrode disposed in the fourth microfluidic channel.

[0081] According to this embodiment, the further or fourth electrochemical sensor is configured to polarize the electrodes to a potential for the oxidation of nitric oxide, and the fourth microfluidic channel comprises a coating layer of polyeugenol on the working electrode of the fourth electrochemical sensor, in particular for removing hydrogen peroxide.

[0082] According to one embodiment, the device further comprises a layer of adhesive material covering the lower surface of the lower layer of the multi-layer structure without covering the entrance orifice, to form an impermeable barrier around the investigation zone by contacting the epidermis of the subject.

[0083] These properties ensure that gases, liquids, and microorganisms such as bacteria and viruses that are outside the investigation zone cannot enter the investigation zone. The impermeable contact between the casing and the skin ensures that any chemical species detected are from biological liquids produced by the investigation zone and not from external flows.

[0084] According to one embodiment, the plurality of microfluidic channels includes an additional microfluidic channel comprising a colorimetric detection device comprising a hydrophilic porous body impregnated with a chemical reagent capable of reacting with one of the following compounds: nitrite ion, hydrogen peroxide, peroxynitrite, sulfur dioxide, hydrogen sulfide, nitric oxide, carbon monoxide, and hypochlorous acid to provide a colored indicator of the concentration or amount of said compound dissolved in the sweat stream.

[0085] These properties allow for easy read-out of usage as concentrations are monitored over time, and further allow for the integration of results obtained via electrochemical sensors.

[0086] According to one embodiment, the additional channel comprises a chronosampling system connected to the inlet orifice, the chronosampling system including a plurality of chambers configured to be filled sequentially with sweat, and a plurality of colorimetric detection devices disposed within the chambers, each of the plurality of colorimetric detection devices including a chemical reagent capable of reacting with a compound, whereby the colorimetric detection devices disposed within the chambers provide a colored indicator indicative of the cumulative amount of the compound in the sweat stream.

[0087] One suitable chronosampling system is described in particular in Choi et al., “A thin, soft, skin-attached microfluidic network with capillary rupture valves for sweat chronosampling, Adv. Healthcare Mater. 2017.”

[0088] The structures of the detection device described above can be realized in different ways, for example in the form of multi-layer structures, which can also be obtained by additive manufacturing, 3D printing, lamination or by adding materials in successive layers.

[0089] According to one embodiment, the detection device further comprises a light sensor configured to generate a measurement signal representative of the color intensity of the chemical reagent in the visible or ultraviolet spectrum.

[0090] According to one embodiment, the detection device is configured to periodically perform and transmit measurements, for example at a parameterizable frequency or at a frequency dependent on the activity state detected by the device. The device may, for example, comprise a gyro module and / or an accelerometer for detecting the subject's activity state. This allows the subject's activity state to be detected during the sweat analysis, facilitating the analysis of the correlation between the subject's activity state and the production of the chemical species to be analyzed.

[0091] According to one embodiment, the device comprises a geolocation module.

[0092] According to one embodiment, the system comprises a communication device configured to transmit one or more measurement signals generated by the detection device to a storage or post-processing device.

[0093] According to a second subject, the invention relates to a mobile device comprising a detection device as described above, implemented in the form of a watch, a phone, a piece of fabric, a headband, clothing or underwear.

[0094] According to one embodiment, the measurements generated by the detection device are received, read and analyzed via a connected watch or smartphone. The measurements may be received by wired, infrared, Bluetooth, Wi-Fi, or 3G, 4G or 5G wave connection.

[0095] According to a second subject, the invention relates to a method for determining the production of at least nitric oxide dissolved in sweat by a human or animal subject, comprising: selecting an investigation zone of the subject's epidermis; applying said detection device for a period of time necessary to generate a signal representative of the concentration of nitric oxide (NO) dissolved in the sweat stream and a signal representative of the flow rate of the sweat stream; A measurement of the production of nitric oxide (NO) by the subject is determined from a signal representative of the concentration of nitric oxide (NO) dissolved in the sweat stream. The present invention relates to a method comprising the steps of:

[0096] According to one embodiment, the method includes previously disinfecting the investigation zone.

[0097] These properties make the measurement of nitric oxide in sweat more accurate, as it does not include nitric oxide production caused by bacteria or viruses present on the subject's skin. In other words, this ensures that the detected compound comes from sweat produced in the investigation zone and not from an external stream.

[0098] Measuring the production of one or more of the aforementioned chemical species by a subject can be used for a variety of purposes, such as, for example, to assess damage to the subject's vascular tissue based on these measurements, or to assess the subject's cardiovascular performance based on these measurements.

[0099] Other possible applications are in the diagnosis, medical treatment, and monitoring of diseases such as cardiovascular diseases, neurodegenerative diseases, pulmonary arterial hypertension, cancer, hypercholesterolemia, diabetes, systemic endothelial dysfunction, arteriosclerosis, thrombotic or ischemic diseases, platelet accumulation inhibitory dysfunction or leukocyte adhesion dysfunction or smooth muscle fibrocyte cell proliferation dysfunction, bronchial inflammation, asthma, and Alzheimer's disease.

[0100] Other possible applications include, for example, monitoring the development and / or muscle soreness of individuals undergoing physical training, preventing injuries due to overtraining, and / or improving muscle performance in a subject.

[0101] In order that the subject of the present invention may be better understood, embodiments thereof will now be described, purely by way of illustrative and non-limiting example, as illustrated in the accompanying drawings, in which: [Brief description of the drawings]

[0102] [Figure 1] FIG. 1 shows a schematic rear view of a subject with a detection device placed thereon according to one embodiment. [Diagram 2] FIG. 2 is a perspective view partially illustrating a multi-layer structure of a detection device according to one embodiment. [Diagram 3] FIG. 3 represents a cross-sectional view of the multi-layer structure along line II-II of the figure. [Figure 4] FIG. 4 is an expanded view of a multi-layer structure according to one embodiment. [Diagram 5] FIG. 5 is an expanded view of a multi-layer structure according to another embodiment. [Figure 6] FIG. 6 is an enlarged perspective view of a multi-layered electrochemical sensor according to one embodiment. [Figure 7] FIG. 7 is an expanded view of a multi-layer structure according to another embodiment. [Figure 8] FIG. 8 is a functional schematic diagram of a microfluidic circuit that can be used in the detection device. [Figure 9] FIG. 9 is another functional schematic diagram of another microfluidic circuit that can be used in the detection device. [Figure 10] FIG. 10 is another functional schematic diagram of another microfluidic circuit that can be used in the detection device. [Figure 11] FIG. 11 is a schematic perspective view of an electrochemical sensor that can be used in the microfluidic circuits of FIGS. [Figure 12] FIG. 12 is a chronogram illustrating a detection method that can be implemented with the electrochemical sensor of FIG. [Figure 13]FIG. 13(A) shows a scheme of an electrochemical sensor that can be used in the microfluidic circuits of FIGS. 2-10 of a detection device according to one embodiment, and FIG. 13(B) shows a method of detecting sweat flow rate that can be performed using the electrochemical sensor of FIG. 13(A). [Figure 14] FIG. 14 shows a scheme of an electrochemical sensor of a detection device according to one embodiment with depletion function. [Figure 15] FIG. 15 is an illustration of the depletion function according to one embodiment. [Figure 16] FIG. 16 is a chronogram illustrating a detection method that can be implemented with the electrochemical sensor of FIG. [Figure 17] FIG. 17 is a schematic diagram of an electrochemical sensor according to one embodiment having five electrodes. [Figure 18] FIG. 18 is a schematic diagram of an electrochemical sensor according to one embodiment having six electrodes. [Figure 19] FIG. 19 is a schematic diagram of an embodiment of a multi-layer structure further comprising a colorimetric detection device. [Figure 20] FIG. 20 is an exploded view of a multi-layer structure according to another embodiment having multiple channels. [Figure 21] FIG. 21 is a functional schematic diagram of a detection device that can be used in the apparatus of FIG. [Figure 22] FIG. 22 is a diagram of steps illustrating a method that can be implemented in the apparatus of FIG. [Diagram 23] FIG. 23 is a graph showing the measurement results obtained with the device of FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0103] 1 shows a detection device 100 placed on the skin 2 of a subject, for example on the subject's back, for the purpose of making a quantitative measurement of dissolved chemical species in sweat, including nitric oxide and optionally nitrite or hydrogen peroxide. Detection device 100 may also be placed on other parts of the body, for example on the neck, shoulders, arms, legs, etc.

[0104] The detection device 100 comprises a microfluidic part and other functional parts which are further described below, in particular a control device 40 (FIG. 21).

[0105] 2, the multi-layer structure 1 may take the form of, for example, a thin casing, and comprises a bottom layer 3 made of a flexible, biocompatible, preferably self-adhesive material, which may also be placed directly on the skin of a subject, and the multi-layer structure 1 further comprises a second layer 6 located on top of the bottom layer 3. The multi-layer structure 1 is made of a waterproof material, for example a polymer.

[0106] The second layer 6 is hollowed out through its thickness to form a microfluidic channel 9 and a sampling dome 99 located over the opening 4 formed in the lower layer 3 .

[0107] With reference to Fig. 3, the underlayer 3 is attached to the skin 2 by an adhesive layer 96. The central part of the underlayer 3 and the adhesive layer 96 comprises a circular opening 4, for example with a diameter of a few mm to a few cm, which defines an investigation zone 97 on the subject's skin 2. The circular opening 4 may also have other shapes, for example oval, triangular, rectangular, square, polygonal, etc. The circular opening 4 is an inlet orifice through which a sweat flow 98 can be directed, in particular to introduce sweat into the microfluidic channel 9. The sweat flow 98 passes from the subject's skin 2 into the microfluidic channel 9 and passes through the circular opening 4.

[0108] In the embodiment of Figure 3, a top layer 7 covers the second layer 6 to form the microfluidic circuitry on top. The microfluidic circuitry can thus be formed throughout the entire thickness of the second layer 6, thereby facilitating manufacture, for example by cutting or engraving.

[0109] A hydrophilic collector element (not shown), e.g. a fibrous body such as cotton or a nonwoven material, may be placed within the circular opening 4 and dome 99. The function of the collector element is to transport sweat generated in the interrogation zone to the microfluidic circuit.

[0110] As shown in FIG. 4, the multi-layer structure is a lower layer 3 provided with an inlet orifice 4 allowing the passage of sweat; an upper layer 7 provided with an outlet orifice 13; an intermediate layer 6 located between the lower layer 3 and the upper layer 7, the intermediate layer 6 having a microfluidic circuit formed in at least one thickness of the intermediate layer 6 and extending parallel to the lower layer 3; Equipped with. The microfluidic circuit consists of a microfluidic channel 9, which communicates at a first end with an inlet orifice 4 and at a second end with an outlet orifice 13. Thus, sweat flow from the subject's skin 2 travels through the microfluidic channel 9, which directs the sweat from the inlet orifice 4 to the outlet orifice 13 by capillary action. The electrochemical sensor 10 comprises four electrodes arranged on the inner surface of the top layer 7, which closes the microfluidic channel at the top. The electrodes are therefore arranged in the interior space of the microfluidic channel.

[0111] Example dimensions include an inlet orifice with a diameter between 1 mm and 15 mm, a microfluidic channel with a length between 0.5 cm and 5 cm and a width between 25 μm and 500 μm, an intermediate layer with a thickness between 10 μm and 200 μm, and each layer of the multi-layer structure with a width between 1 cm and 5 cm and a length between 2 cm and 15 cm.

[0112] For example, the inlet orifice 4 has a diameter of 5 mm, the microfluidic channel 9 is 1.8 cm long and 100 μm wide, the intermediate layer has a thickness of less than 70 μm, e.g. 20 μm, and the layers of the multi-layer structure are 3 cm wide and 9 cm long.

[0113] Referring to Fig. 5, the multi-layer structure is similar to Fig. 4. However, in this embodiment, the exit orifice 13 is located at one end of the intermediate layer in the intermediate layer 6. The detection device 100 comprises electrochemical sensors 10 each comprising four electrodes, each comprising two parts arranged opposite each other, a first part arranged on the inner surface of the upper layer 7 and closing the microfluidic channel 9 at the top, and a second part arranged on the upper surface of the lower layer 3 and closing the microfluidic channel at the bottom. Each electrode part comprises a connector, indicated by a black rectangle, through which the electrodes may be electrically connected.

[0114] According to one embodiment, not shown, the detection device may comprise a single electrochemical sensor 10 comprising four electrodes arranged on the upper surface of the lower layer 3 closing the microfluidic channel 9 at the bottom.

[0115] FIG. 6 shows four electrodes arranged in a microfluidic channel, which may be for example the microfluidic channel 9 shown in FIGS. 4 and 5. The four electrodes are metal applications arranged on the inner surface of the top layer 7 closing the microfluidic channel 9 at the top. The four electrodes are arranged successively in the longitudinal direction of the microfluidic channel and include a working electrode 20, here implemented in the form of a depletion electrode operating in a manner described in more detail below, a second working electrode 23, a reference electrode 21 and a counter electrode 30. The height of the electrodes is between 1 and 50 nanometers (nm), the spacing between the electrodes is between 10 and 10,000 micrometers (μm) and the width of the electrodes is between 1 and 1000 μm. The electrodes may be made of, for example, platinum (Pt), gold (Au), silver (Ag) or silver chloride (AgCl).

[0116] At least one of the electrodes may be fully or partially covered with polyeugenol, platinum black, or polyphenols and is configured to perform one or more of the following actions: depleting, measuring nitric oxide concentration, measuring the concentration of at least one other chemical component, and measuring the flow rate of the sweat stream through the microfluidic channel 9.

[0117] Figure 7 shows a multi-layer structure 1 similar to that of Figure 4, with a second sealing interlayer 26 located between the first intermediate layer 6 and the top layer 7, the second sealing interlayer 26 providing an opening 27 for the electrode to allow the electrode to come into contact with the sweat stream circulating in the microfluidic channel 9. The second sealing interlayer 26 further provides an intermediate opening 28 in communication with the exit orifice 13 of the top layer 7 to allow the escape of sweat. For example, the opening 27 is rectangular, 5 mm long and 200 μm wide.

[0118] With reference to FIG. 8, the intermediate layer 6 comprises a microfluidic circuit 8 to which a sweat flow is supplied. The sweat is received towards the microfluidic circuit 8 from an inlet orifice in the lower layer. The microfluidic circuit 8 may comprise one or more microfluidic channels 9, in particular four parallel microfluidic channels 9 in the example shown. However, the microfluidic circuit 8 may adopt a different form. For example, FIG. 9 represents a microfluidic circuit 8 with parallel microfluidic channels 9, while FIG. 10 represents four radially distributed microfluidic channels 9. The microfluidic channels 9 are formed, for example, in the thickness of the intermediate layer 6 and are separated by partitions 11. Each microfluidic channel 9 is separated from the other microfluidic channels, and sweat can circulate independently therein. The number of microfluidic channels 9 may be more or less than in these figures.

[0119] Each fluid circuit 9 is equipped with a sensor 10A, 10B, 10C or 10D. Arrows 12 indicate the direction of sweat flow in the microfluidic channels 9. Via an exit orifice 13, the microfluidic channels 9 preferably end in a drainage reservoir that holds the analyzed fluid, to prevent the reaction products of the electrolysis from contacting the subject's skin again.

[0120] The sensors 10A, 10B, 10C and 10D arranged in the microfluidic circuit 9 for analyzing sweat are preferably electrochemical sensors, the working principle of which is the total or partial electrolysis of the solution present in the fluid channel 9 between a working electrode and a counter electrode. Electrochemical sensors of this kind may be implemented in various ways, in particular in miniaturized form with dimensions of the order of millimeters.

[0121] Some example embodiments of electrochemical sensors will now be described with reference to FIG.

[0122] Example 1 The sensor 10A is intended to detect hydrogen peroxide. Therefore, the oxidation potential E H2O2 The sensor 10B is intended to detect nitric oxide. Thus, nitric oxide E NO The sensor 10C is for detecting nitrite ions. Therefore, the nitrite ions E NO2- It operates at a potential difference equal to the oxidation potential of

[0123] The sensors 10A, 10B, and 10C measure the instantaneous intensity of the faradaic current (i oxdn The sensors 10A, 10B, 10C thus enable the detection and quantification of the instantaneous concentrations of the aforementioned chemical species.

[0124] Each of the three aforementioned species can be detected amperometrically using a microelectrode; the latter can be detected, for example, by the platinum salt, Pt(Cl)6 4-It consists, for example, of a platinum strip covered with a thin layer of platinum black of micrometer dimensions, which is applied by electrochemical reduction of the anions of

[0125] Three chemical species (NO, NO2 - , and H2O2) are at the oxidation potential of E H2O2 <E NO <E NO2- can be distinguished by the fact that the faradaic currents are clearly separated in the order of oxidation potential, i.e., 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 ...1, 1.2, 1.3, 1.4, 1.5,

[0126] Therefore, the oxidation potential E H2O2 Only the H2O2 species can be oxidized at the oxidation potential E NO The three species can be oxidized at oxidation potential E NO2- Therefore, each of i oxdn (E H2O2 ), i oxdn (E NO ), and i oxdn (E NO2- The currents measured by the sensors 10A to 10C indicated by the arrows 10A to 10C satisfy the following formula:

[0127] i oxdn (E H2O2 ) = a1 i H2O2

[0128] i oxdn (E NO ) = a2 i H2O2 + a3 i NO

[0129] i oxdn (E NO2- ) = a4 i H2O2 +a5 i NO + a6 i NO2- where coefficients a1 through a6 represent the calibration constants of the sensors and can be measured experimentally.

[0130] Therefore, by subtraction, which can be easily implemented in electronic circuits, we obtain:

[0131] i H2O2 = (1 / a1) i oxdn (E H2O2 )

[0132] i NO = (1 / a3) i oxdn (E NO ) - (a2 / a1).(1 / a3) i oxdn (E H2O2 )

[0133] i NO2- = (1 / a6) i oxdn (E NO2- ) - (a4 / a6) i H2O2 - (a5 / a6) i NO

[0134] At any time t, the instantaneous intensity i of the faradaic oxidation current of each chemical species S S (t) is the concentration C of the fluid in the volume above the electrode that detects it S (t). The proportionality factor depends on the form factor, denoted γ, which is a function of the shape of the sensor and the n S It is a function of the Faraday constant,

[0135] n H2O2 = n NO2- = 2 and n NO = 1

[0136] Note that F stands for Faraday, or the value of the charge of one mole of electrons, which is 96,500 coulombs.

[0137] The form factor γ is a constant coefficient that depends on the geometry of the electrochemical sensor and can be evaluated theoretically or measured experimentally by calibration. For simplicity, in the following, the three sensors 10A-10C are considered to have the same geometric shape, such that the form factor is the same for all sensors.

[0138] As a result, the concentrations of chemical species can be obtained from the currents measured by sensors 10A-10C using the following equation with time variable t:

[0139] C H2O2 (t) = i oxdn (E H2O2 ,t) / (2Fγ)

[0140] C NO (t) = [i oxdn (E NO ,t) - i oxdn (E H2O2 ,t)] / (Fγ)

[0141] CNO 2- (t) = [i oxdn (E NO2- ,t) - i oxdn (E NO ,t)] / (2Fγ)

[0142] Therefore, in the first embodiment, the three sensors 10A to 10C each have a constant oxidation potential, i.e., E H2O2 , E NO , and E NO2- can operate in parallel.

[0143] In an alternative embodiment, NO and NO - This embodiment is particularly advantageous when the measurement of H2O2 is not critical and does not affect the intended outcome. H2O2 (t) is uniformly zero, that is, C H2O2 It is assumed that (t) = 0. Thus, the system of equations is simplified.

[0144] Example 2 In example 2, a single microfluidic channel 9 and a single sensor 10A are used, the others being optional.

[0145] In this case, the sensor 10A operates sequentially to detect the aforementioned species during three successive steps. Thus, the oxidation potential varies between three potential stages, e.g., E H2O2 →E NO →E NO2- →E H2O2 →E NO →E NO2- →It switches periodically according to the etc. sequence.

[0146] In this case, each oxidation potential is maintained for a period that is very long compared to the time constant of the working electrode, which is, for example, a few milliseconds for microelectrodes used in microfluidic channels, and a current is implemented at the end of each potentiostatic stage.

[0147] The remaining measurement signals can be processed using the same equations as in Example 1.

[0148] Example 3 Nitric oxide is a small molecule that is both hydrophilic and lipophilic, so it is much more effective than the other two species, H2O2 and NO2. - In contrast to , it can easily pass through thin layers of organic polymers and can therefore be detected alone using electrochemical sensors protected with a layer of this type, for example using a platinized platinum working electrode coated with a thin layer of polyeugenol (4-allyl-2-methoxyphenol) applied by electropolymerization.

[0149] Thus, in Example 3, the working electrode of sensor 10D is coated with a layer generally designated by the numeral 19. The instantaneous concentration of nitric oxide is thus determined by the chemical species H2O2 and NO2 - can be measured independently of the concentration according to the following formula:

[0150] C NO (t) = [i oxdn (ENO ,t)] eugenol / (Fγ).

[0151] where i oxdn (E NO ,t)] eugenol indicates the current measured by sensor 10D.

[0152] The other sensors 10A-10C and the other microfluidic channels 9 can be omitted. Thus, the method can be advantageously used with a single sensor when only the concentration of NO is required.

[0153] Example 4 In this case, sensor 10D of Example 3 is fused with sensors 10A-10C of Example 1 or sensor 10A of Example 2. This configuration can be used to obtain two measurements independent of the concentration of dissolved nitric oxide and check the consistency of the measurements, in particular by making sure that the sensor does not exhibit drift, which may be associated for example with partial deactivation of the surface of one of the electrodes.

[0154] In this case, the electrochemical electronic controller 40 (FIG. 21) is preferably configured to compare the two measurements of nitric oxide concentration and issue an alarm if the result of the comparison meets a predefined criterion, for example exceeds a predefined threshold.

[0155] In the above examples 1-4, the measured instantaneous Faradaic current can be used to measure the concentration of a chemical species in the analytical solution. As a result, in a static system, the intensity of the current is sufficient to record the production of the detected species.

[0156] However, when the detection device 100 is applied to physiological systems that are dynamic in nature, such as during exercise testing or medical monitoring, it is also desirable to have quantitative access to the kinetics of production of each chemical species by the cardiovascular system. In dynamic situations, access to the instantaneous amount of a chemical species, denoted ΔQ(t), produced over a short period of time, denoted Δt(t), can be achieved by calculating the average concentration C of the chemical species, as shown below:S It is desirable to know (t) and the volumetric flow rate of the analyte fluid simultaneously.

[0157] d(t) = (ΔV / Δt)

[0158] where ΔV denotes the volume scanned during the time interval Δt. Thus, P S The intensity of the productive flow of species S at time t, denoted (t), is given by:

[0159] P S (t) = [ΔQ / Δt](t) = C S (t).d(t)

[0160] Here, the average concentration C S (t) is obtained from the average intensity of the electrochemical oxidation current measured between times t and t+Δt.

[0161] Thus, in the context of the envisaged dynamic application, the detection device 100 simultaneously and at each time t necessary for the desired accuracy of monitoring the physiological condition of the patient over time, for example once per minute, measures the average intensity i of the faradaic current associated with the electrochemical oxidation of one or more chemical species to be monitored. av It is desirable to measure the value of (t) and the corresponding volumetric flow rate of sweat d(t) in the fluid circuit at time t.

[0162] FIG. 11 shows an embodiment of an electrochemical sensor 10 that can meet this dual requirement in an integrated way. This electrochemical sensor 10 comprises at least a pair of working electrodes 20, 23. This type of strip microelectrode can be manufactured from platinized (platinum black) platinum, which may or may not be covered with a layer of electropolymerized eugenol of micrometer dimensions. This type of strip microelectrode can be embedded by microfabrication, for example CVD and / or lithography. One or more such strip microelectrodes can be used to electrochemically oxidize selected chemical species.

[0163] The microfluidic circuit 9 of Fig. 11 further comprises a reference electrode 21, for example manufactured in the form of an Ag / AgCl microstrip and arranged upstream of the pair of working electrodes 20, 23. Finally, the fluidic circuit 9 comprises a counter electrode 30 made of platinized platinum and arranged downstream of the pair of working electrodes 20, 23. Although a functional schematic diagram is shown in Fig. 11, the surface area of ​​the counter electrode 30 is in fact 2-3 times larger than the surface areas of the other electrodes.

[0164] The assembly of the microfluidic channel 9 with the electrodes 20, 21, 23, 30 is immersed in a layer of sweat (not shown) and thus constitutes a microfluidic electrochemical cell with four electrodes. Each of the electrodes 20, 21, 23, 30 is connected to an electrochemical electronic controller 40 (FIG. 21) by electrical contacts insulated from sweat.

[0165] This embodiment of the electrochemical sensor 10 may be used in one or more of the microfluidic circuits 9 described above.

[0166] To measure the volumetric flow rate d(t), the electrochemical sensor 10 must be equipped with a pair of working electrodes 20, 23. The solution described here is simple and easily industrializable, since it has no moving parts and does not insist on fluid dynamics. No intervention is required to regulate the flow rate of the fluid, and at the same time, it is suitable for reasonable physiological flow rates.

[0167] Two working electrodes 20, 23, e.g. two strips of platinized platinum, can function as working microelectrodes, are electrically independent and are spaced apart by a distance L along the path of the fluid to be analyzed in the microfluidic circuit 9. The two working electrodes 20, 23 are, for example, placed at the bottom of a straight channel with a constant cross-sectional area A.

[0168] The working electrode 23 placed downstream is used according to the method shown in Fig. 12, which comprises two steps. Graph 81 represents the potential applied to the working electrode 20 as a function of time. Graph 82 represents the potential applied to the working electrode 23 as a function of time. The potentials shown as "0" on graphs 81 and 82 actually mean the disconnection (open circuit) of the corresponding electrode. Graph 83 represents the faradaic current measured at the working electrode 20 as a function of time. Graph 84 represents the faradaic current measured at the working electrode 23 as a function of time.

[0169] During a first step, which is carried out over a period of time prior to time t0, the potential E applied to the working electrode 20 oxdn is sufficient to allow oxidation of one or more target species, while the downstream working electrode 23 is not connected. The upstream positioned working electrode 20 provides an instantaneous electrochemical current i oxdn (t), which, according to the calculations further given above, indicates the concentration C(t) of one or more target chemical species in the analyzed fluid.

[0170] During a second step, which is carried out for a certain period of time from time t0, the working electrode 20 is disconnected and a potential E oxdn is applied to the downstream working electrode 23.

[0171] At time t0, the sweat stream passing over the working electrode 23 has already been electrolyzed (fully or partially) while passing over the upstream working electrode 20 in such a way that the concentration of the target species is zero or at least significantly lower than before it entered the electrochemical sensor. Thus, the intensity of the current detected by the working electrode 23 i oxdn (Graph 84) is zero (or at least the current i detected at the working electrode 20 before time t0). oxdn much lower).

[0172] At time t0+Δt, the working electrode 23 starts analyzing the non-electrolyte solution and detects a current intensity i oxdnis the current intensity i detected by the working electrode 20 before time t0 oxdn The current increase, schematized in the steps of FIG. 12, is detected by an ad-hoc electronic circuit. The delay between this increase and the moment t0 of disconnection of the working electrode 20, the period Δt, represents the time required for the sweat flow to travel between the two working electrodes 20 and 23. The period Δt is represented by a double-headed arrow at the bottom of FIG. 12. For simplicity of presentation, it is assumed in FIG. 12 that the electrolysis of the target species is complete when the working electrode 20 is connected. The same measurement principle applies if this electrolysis is only partially performed.

[0173] Therefore, the flow rate v(t) and the flow rate d(t) can be estimated as follows:

[0174] v(t) = L / Δt

[0175] d(t) = Av(t)

[0176] The potential E applied to the working electrode 23 oxdn is sufficient to allow oxidation of one or more target species while the working electrode 20 is disconnected. Concentration measurements by the working electrode 23 can therefore continue for an optionally fixed period of time. The second step ends with the disconnection of the working electrode 23 at time t1. The working electrode 20 can then be reconnected and the method repeated as many times as necessary to continuously evaluate the flux d(t).

[0177] The distance L between the two working electrodes 20 and 23 is preferably small enough that the change in the patient's physiological response over the period Δt is negligible, for example on the order of 1 mm.

[0178] A second method for measuring the volumetric sweat flow rate is shown in Fig. 13. In contrast to the above explanation, the measurement principle here is to detect a drop in the measurement current of a compound such as nitric oxide. Fig. 13A shows a schematic representation of an assembly of four electrodes arranged in a microfluidic channel 9 according to one embodiment. The electrodes are arranged consecutively in the longitudinal direction of the microfluidic channel 9. The electrodes are arranged as follows: a reference electrode 21, placed upstream and first receiving the sweat flow 98, a first and a second working electrode 20, 23, spaced apart by a distance g, represented by a double-headed arrow, and a counter electrode 30. The measurement principle is illustrated in FIG. 13(B) and involves the simultaneous connection and polarization of the two working electrodes 20, 23 and then monitoring over time the change in current at the second working electrode 23, located downstream in the microfluidic channel 9. From the start of polarization (t=0), the two working electrodes 20, 23 oxidize the same species. The first upstream working electrode 20 causes a depletion of these species, which after a period dt causes a decrease in the current at the second working electrode 23, located downstream. dt is the time required for the depletion zone containing the species oxidized by the first working electrode 20 to reach the second electrode 23 by convection. Thus, after simultaneous polarization of the first working electrode 20 and the second working electrode 23 at t=0, the current at the downstream second working electrode 23 is observed to decrease with duration dt. As an example, duration dt in FIG. 13(B) is estimated to be 5 seconds. The linear velocity of flow v is then given by the simple relationship v=g / dt.

[0179] This principle can be utilized in combination with the depletion function described with reference to Figures 14 to 16 below.

[0180] FIG. 14 shows a microfluidic circuit with an electrochemical sensor with four electrodes arranged consecutively in the longitudinal direction of a microfluidic channel 9. A depletion electrode 20 is arranged upstream of the working electrode 23 to deplete interfering species whose downstream detection is undesirable. The polarization of the depletion electrode 20 selectively oxidizes the interfering species. The depletion electrode 20 is wide to optimize the depletion and to remove substantially all of one or more interfering species, for example hydrogen peroxide H2O2. The species of interest is oxidized by the working electrode 20 located downstream. This type of configuration can be used to deplete one or more interfering species that have an oxidation potential lower than that of nitric oxide and to improve the measurement of the concentration of a compound, for example nitric oxide. The counter electrode 30 and the reference electrode 20 are necessary to control the potential and circulate the current in the measurement zone of the electrochemical sensor.

[0181] In this microfluidic channel 9 equipped with a depletion electrode 20, the concentration of nitric oxide can be obtained directly without the need to solve the system of linear equations presented further above.

[0182] NO2 in Microfluidic Channel 9 - In an alternative embodiment where it is desirable to obtain a measurement of the concentration of nitric oxide, the depletion electrode 20 may be configured to remove nitric oxide.

[0183] FIG. 15 shows an example of hydrogen peroxide depletion by the sensor of FIG. 14. Reference and counter electrodes are not shown. The depletion electrode 20 and the working electrode 23 are polarized independently of each other under constant sweat flow conditions. The direction of sweat flow is represented by the arrows in the microfluidic channel 9. The depletion electrode 20 is very large, for example 8 times larger, compared to the working electrode 23 in order to remove all upstream interfering species, here hydrogen peroxide (H2O2), by oxidation. The species whose concentration it is desired to measure, here nitric oxide, is not oxidized by the depletion electrode 20. Nitric oxide is oxidized downstream by the working electrode 23. The depletion electrode 20 and the working electrode 23 are therefore polarized at a constant E DE <E WE The potential is polarized such that

[0184] As mentioned above, the flow rate of sweat flow may be measured, for example, via the working electrode 20 performing depletion and the working electrode 23 oxidizing nitric oxide. Figure 16 shows the measurement of the flow rate of sweat flow 98 in a manner similar to that described in Figures 13A and B. Thus, the depletion electrode 20 oxidizing a species whose detection is undesirable, for example hydrogen peroxide, and the working electrode 23 oxidizing nitric oxide are connected and polarized simultaneously. Thus, during a first period, the amount of sweat located between the depletion electrode 20 and the working electrode 23 is not depleted by the depletion electrode 20, so that the working electrode 23 detects nitric oxide NO and hydrogen peroxide. During a second period, the depletion electrode 20 causes a depletion of hydrogen peroxide, thereby inducing a decrease in the current of the working electrode 23 after a duration dt, in a manner similar to that described in Figure 13. In this embodiment, the current at working electrode 23 decreases, but does not go to zero or substantially zero, because working electrode 23 selectively detects nitric oxide following depletion.

[0185] The flow measurement methods described above can be used simultaneously on all of the parallel microfluidic channels. However, if these channels are configured and fed in a similar manner, a single flow measurement may be sufficient. In that case, the flow measurement methods described above may be employed on a single microfluidic channel 9. Furthermore, these flow measurement methods can be combined with various example sensors.

[0186] 18 and 19, an electrochemical sensor using a larger number of electrodes is described.

[0187] The electrodes shown in Figures 17-19 may be arranged on the inner surface of an upper layer closing the microfluidic channel at the top and / or on the upper surface of a lower layer closing the microfluidic channel at the bottom. In the schematic drawings, no distinction is made between the various layers.

[0188] 17 depicts an electrode configuration according to an embodiment, where the electrochemical sensor comprises, from left to right in FIG. 17, a reference electrode 21, a depletion electrode 20, a first working electrode 23, a second working electrode 24, and a counter electrode 30. In this configuration, the following can be performed in that order: depletion of undesirable species such as hydrogen peroxide via polarization of the depletion electrode 20 to the oxidation potential of hydrogen peroxide; oxidation of nitric oxide via the first working electrode 23, which has an oxidation potential higher than that of hydrogen peroxide; oxidation of nitrite via the second working electrode 24, which has an oxidation potential higher than that of hydrogen peroxide and that of nitric oxide; and measurement of sweat flow rate by the delay between the first working electrode 23 and the second working electrode 24. The flow rate measurement may be measured by the delay between the depletion electrode 20 and the first working electrode 23.

[0189] Figure 18 shows a similar embodiment to the previous figures. This embodiment differs in that the electrochemical sensor comprises a third working electrode 25 located between the second working electrode 24 and the counter electrode 30. The flow rate of sweat flow 98 is measured between the second working electrode 24 and the third working electrode 25. By spacing the electrodes 24 and 25 apart, in other words by moving the electrodes 25 and 30 further towards the end of the channel 9, it is possible to improve the resolution of the flow rate measurement of sweat flow 98.

[0190] FIG. 19 represents a variant embodiment in which the detection device comprises a microfluidic circuit 8 including two parallel microfluidic channels 9 and 109. The first microfluidic channel 9 comprises an electrochemical sensor. An assembly of electrochemical sensors as disclosed herein may be integrated into this first microfluidic channel 9. The second microfluidic channel 109 comprises a colorimetric detection device 18. The colorimetric detection device 18 comprises a hydrophilic microporous membrane. The hydrophilic microporous membrane contains at least one chemical reagent capable of reacting with a detection species, such as, for example, nitrite ions, hydrogen peroxide, peroxynitrite, sulfur dioxide, hydrogen sulfide, nitric oxide, carbon monoxide, and hypochlorous acid. In proportion to the accumulation of the detection species in the porous body impregnated with the reagent, the reagent changes color and increases in intensity of its inherent color. The color intensity can be detected to quantitatively measure the amount of the chemical species dissolved in the sweat stream. For example, the reagent used is the Griess reagent, which allows the detection of nitrite ions by providing a red indicator.

[0191] According to a variant embodiment shown in figure 20, a colorimetric detection device 18 is placed at the outlet of the microfluidic channel 9 in series with the electrochemical sensor 10. Figure 20 thus shows the same multi-layer structure 1 as in figure 4. The colorimetric detection device 18 is placed downstream of the electrochemical sensor and therefore receives the solution electrolyzed by the electrochemical sensor 10. However, colorimetric measurements are possible, provided that the species to be detected is not substantially adversely affected by the operation of the electrochemical sensor 10.

[0192] The methods for detecting concentrations and flow rates described above can be carried out in an automated manner with the aid of an electronic controller 40, which is preferably integrated into the detection device 100.

[0193] With reference to FIG. 21, one embodiment of an electronic controller 40 that may be incorporated into the detection device 100, for example in the form of an electronic circuit board, will now be described.

[0194] The or each electrochemical sensor 10 described above is connected to an analog-to-digital converter 14, which in turn provides power to a processor 15. The processor 15 is programmed to carry out the methods for detecting concentrations and flow rates, for example, as described above.

[0195] An energy source 16, e.g., a battery, powers the electronic controller 40. A communication module 17, which may be wired or wireless, may also be provided for communicating concentration, flow rate, and / or quantitative mass flow measurements for one or each target species to a storage or post-processing device.

[0196] FIG. 22 illustrates a method that may be performed by processor 15 in one embodiment.

[0197] In step 31, the instantaneous concentration Cs(t) of a chemical species S is determined from an electrochemical measurement.

[0198] In step 32, the volumetric flow rate d(t) in the corresponding fluid circuit is determined.

[0199] In step 33, the quantitative material flow of the species under consideration is determined based on Cs(t) and d(t), for example as follows:

[0200] P S (t) = C S (t).d(t)

[0201] FIG. 23 is a graph showing a quantitative mass flow measurement signal as a function of time that may be obtained using the detection device 100 during exercise testing of a subject, for example for the species NO.

[0202] The electronic control device 40 may optionally include other functional modules, such as a gyro and / or accelerometer module for detecting the subject's orientation and movement and the subject's activity level, and a temperature sensor for measuring the subject's epidermal temperature, which is useful for investigating the correlation between skin temperature and vasodilation.

[0203] Certain elements of the detection device 100, in particular the electronic controller 40, can be realized in various forms, in a single or distributed manner, using physical and / or software components. Physical components that can be used are application-specific integrated circuits (ASICs), field programmable gate arrays (FPGAs) or microprocessors. Software components can be written in various programming languages, such as C, C++, Java, VHDL, etc. This list is not exhaustive.

[0204] Although the invention has been described in relation to a number of specific embodiments, it is clear that the invention is in no way limited to the specific embodiments, but encompasses all equivalents of the described means, and combinations thereof, provided they fall within the scope of the invention. For example, the described detection devices may include additional microfluidic channels, or may include different electrochemical sensors and / or sensors that include a different number of electrodes.

[0205] Use of the verbs "comprise", "encompass" or "include" and their conjugations does not exclude the presence of elements or steps other than those stated in a claim.

[0206] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

Claims

1. A detection device (100) for placement on an investigation zone (97) of the epidermis of a human or animal subject for detecting at least nitric oxide dissolved in sweat, comprising: a structure defining a microfluidic circuit (8) and including an inlet orifice (4) allowing the passage of sweat from the epidermis, said microfluidic circuit (8) comprising at least one microfluidic channel (9) for directing a sweat flow (98), said microfluidic channel (9) communicating with said inlet orifice (4); at least one electrochemical sensor (10) configured to generate at least one signal representative of a concentration of nitric oxide dissolved in the sweat stream (98) within the microfluidic channel (9); Equipped with The electrochemical sensor (10) comprises at least four electrodes arranged in succession in the longitudinal direction of the microfluidic channel (9), the at least four electrodes including a reference electrode (21), at least two working electrodes (20, 23) and a counter electrode (30); The electrochemical sensor further comprises: depleting chemical species contained in the sweat stream in the microfluidic channel (9), the chemical species having an oxidation potential lower than that of nitric oxide; generating a signal representative of a flow rate of said sweat stream in said microfluidic channel (9); 13. A detection device (100) configured to perform at least one additional operation of:

2. The structure is a multilayer structure (1) comprising a sublayer (3) and at least one layer on the sublayer (3), The microfluidic circuit (8) extends parallel to the lower layer (3); The lower layer (3) includes the inlet orifice (4).

2. The detection device (100) of claim 1.

3. The multilayer structure (1) comprises an upper layer (7) and at least one intermediate layer (6) located between the lower layer (3) and the upper layer (7), The microfluidic circuit (8) is formed within the thickness of at least one intermediate layer (6). Detection device (100) according to claim 2.

4. The at least one intermediate layer includes a first intermediate layer (6) and a second sealing intermediate layer (26) located between the first intermediate layer (6) and the top layer (7); The second sealing interlayer (26) includes an opening (28) in the electrode. Detection device (100) according to claim 3.

5. The multi-layer structure (1) comprises an upper layer (7) and an exit orifice (13) across the upper layer (7), The at least one microfluidic channel (9) communicates with the outlet orifice (13). Detection device (100) according to any one of claims 2 to 4.

6. The at least four electrodes are arranged on the inner surface of the upper layer (7) closing the microfluidic channel (9) at the top and / or on the upper surface of the lower layer (3) closing the microfluidic channel (9) at the bottom. Detection device (100) according to any one of claims 3 to 4.

7. In the direction of the flow (98), the at least four electrodes said first working electrode (20) in the form of a depletion electrode; said second working electrode (23) for measuring the concentration of nitric oxide; The counter electrode; In order, The reference electrode (21) is located immediately upstream of the first working electrode (20) or immediately downstream of the second working electrode (23). Detection device (100) according to any one of claims 1 to 4.

8. In the direction of the flow (98), the at least four electrodes said first working electrode (20) for measuring the concentration of nitric oxide; said second working electrode (23) for measuring the concentration of nitric oxide; The counter electrode; In order, The reference electrode (21) is located immediately upstream of the first working electrode (20) or immediately downstream of the second working electrode (23). Detection device (100) according to any one of claims 1 to 4.

9. The electrochemical sensor (10) is configured to generate a signal representative of the flow rate by measuring a delay (Δt) between a change in current at the first working electrode (20) and a change in current at the second working electrode (23). Detection device (100) according to claim 7.

10. The electrochemical sensor (10) is configured to generate a signal representative of the instantaneous production of nitric oxide in the investigation zone (97) based on a signal representative of the concentration of nitric oxide and the signal representative of the flow rate of the sweat flow (98). Detection device (100) according to any one of claims 1 to 4.

11. The electrochemical sensor (10) is configured to generate a signal representative of the concentration of nitric oxide by electrical measurement, in particular amperometric measurement, between at least one of the working electrodes (20, 23) and the counter electrode (30). Detection device (100) according to any one of claims 1 to 4.

12. The electrochemical sensor (10) is configured to polarize at least one of the working electrodes (20, 23) to a potential for the oxidation of nitric oxide. Detection device (100) according to any one of claims 1 to 4.

13. The electrochemical sensor (10) is configured to generate a signal representative of the concentration in the sweat stream of at least one of the following compounds dissolved in sweat: nitrite ion, hydrogen peroxide, and peroxynitrite. Detection device (100) according to any one of claims 1 to 4.

14. The electrochemical sensor (10) comprises a third working electrode (25) between the first or second working electrode (20, 23) and the counter electrode for measuring a compound. Detection device (100) according to claim 13.

15. a colorimetric detection device (18) connected to the channel (9) downstream of the electrochemical sensor (10); The colorimetric detection device (18) comprises a hydrophilic porous body impregnated with a chemical reagent capable of reacting with one of the following compounds dissolved in the sweat: nitrite ion, hydrogen peroxide, peroxynitrite, sulfur dioxide, hydrogen sulfide, nitric oxide, carbon monoxide, and hypochlorous acid, to provide a colored indicator of the amount of the compound in the sweat stream (98). Detection device (100) according to any one of claims 1 to 4.

16. The chemical reagent includes a greasing reagent capable of reacting with nitrite ions dissolved in the sweat stream (98). Detection device (100) according to claim 15.

17. A colorimetric detection device (18) connected to the channel (9) downstream of the electrochemical sensor (10), the colorimetric detection device (18) comprises a hydrophilic porous body impregnated with a chemical reagent capable of reacting with one of the following compounds dissolved in the sweat: nitrite ion, hydrogen peroxide, peroxynitrite, sulfur dioxide, hydrogen sulfide, nitric oxide, carbon monoxide, and hypochlorous acid, to provide a colored indicator of the amount of the compound in the sweat stream (98); The colorimetric detection device (18) is disposed at the exit orifice (13). Detection device (100) according to claim 5.

18. The electrochemical sensor (10) is configured to periodically and repeatedly polarize at least one of the working electrodes (20, 23, 25) for a predetermined period of time. Detection device (100) according to any one of claims 1 to 4.

19. The microfluidic circuit (8) comprises a plurality of microfluidic channels (9) each for directing a sweat flow; The microfluidic channels (9) are connected to the inlet orifice (4) so ​​as to be spaced apart from one another. Detection device (100) according to any one of claims 1 to 4.

20. The plurality of microfluidic channels (9) includes an additional microfluidic channel (9) that includes an electrochemical sensor (10); The electrochemical sensor (10) comprises at least three electrodes arranged successively in the longitudinal direction of the additional microfluidic channel (9), The at least three electrodes include a reference electrode (21), a counter electrode (30), and at least one working electrode (20, 23, 25); the additional electrochemical sensor (10) is configured to polarize an electrode to a potential for the oxidation of a compound selected from nitrite ions, hydrogen peroxide, and peroxynitrite; The additional electrochemical sensor (10) is configured to generate at least one signal representative of the concentration of the compound dissolved in the sweat flow in the additional microfluidic channel (9).

20. Detection device (100) according to claim 19.

21. the plurality of microfluidic channels includes an additional microfluidic channel (109) that includes a colorimetric detection device (18); The colorimetric detection device (18) comprises a hydrophilic porous body impregnated with a chemical reagent capable of reacting with one of the following compounds: nitrite ion, hydrogen peroxide, peroxynitrite, sulfur dioxide, hydrogen sulfide, nitric oxide, carbon monoxide, and hypochlorous acid, and providing a colored indicator of the concentration or amount of said compound dissolved in the sweat stream in the additional microfluidic channel (109).

20. Detection device (100) according to claim 19.

22. said additional channel (109) comprising a chronosampling system connected to said inlet orifice (4); The chrono-sampling system includes a plurality of chambers configured to be sequentially filled with sweat; A plurality of colorimetric detection devices (18) are disposed within the chamber; Each of the plurality of colorimetric detection devices (18) includes a chemical reagent capable of reacting with a compound such that the colorimetric detection devices disposed within the chamber provide a colored indicator of the cumulative amount of the compound in the sweat stream within the additional microfluidic channel (109). Detection device (100) according to claim 21.

23. an optical sensor configured to generate a measurement signal representative of the color intensity of the chemical reagent in the visible or ultraviolet spectrum; Detection device (100) according to claim 15.

24. a communication device (17) configured to transmit one or more measurement signals generated by said detection device (100) to a storage or post-processing device. Detection device (100) according to any one of claims 1 to 4.

25. A portable device comprising a detection device (100) according to any one of claims 1 to 4, A portable device that is implemented in the form of a watch, a phone, fabric, a headband, clothing, or underwear.

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