Detection of chemical species in the subject's sweat

Abstract

The detection device detects at least one chemical species in the sweat of a human or animal subject, including nitrogen oxide, NO, and nitrite, NO2. - and hydrogen peroxide, H2O2. The detection device includes a collecting element disposed in an investigation zone of the epidermis of the subject, a detection portion including at least one fluid circuit (9) coupled to the collecting element to conduct at least one sweat stream (12) from the investigation zone, and an electrochemical sensor (10) with electrodes disposed in the fluid circuit, the electrochemical sensor configured to generate a signal representative of the concentration of the at least one chemical species in the sweat stream and a signal representative of the sweat stream flow rate.

Description

[Technical field]

[0001] The present invention relates to a method for detecting at least one chemical species, in particular nitric oxide (NO) and nitrite ion (NO2), in the sweat of a human or animal subject. - and hydrogen peroxide, H2O2. [Background technology]

[0002] Nitric oxide is a gas that constitutes an intercellular messenger. It is an important circulatory messenger for stress by mechanotransduction. It is released in particular during muscular exercise to stimulate the dilation of the vascular system. Therefore, the changes in the flow of blood, and thus of the fluids that balance with it, such as sweat, are particularly suitable indicators of the ability of the circulatory system to match the muscular forces required during exercise testing.

[0003] In the case of cardiovascular diseases, existing devices and instruments for prevention and prediction are limited to indirect measurement of nitric oxide in the patient's body at rest, or direct measurement several hours after the onset of pathological problems, in either case only possible in a clinical setting. Summary of the Invention

[0004] Certain aspects of the present invention are based on the concept that quantitative measurement of changes in the concentration of nitric oxide in sweat provides a non-invasive method for monitoring cardiovascular performance during preventive monitoring or for making a diagnosis.

[0005] A particular aspect of the present invention is the reaction of nitric oxide in the presence of molecular oxygen to produce nitrite ions (NO - ), with the overall reaction having a 1:1 stoichiometry. In other words, the change in concentration of nitric oxide represents the current state of the cardiovascular response to a given exercise, whereas the change in concentration of nitrite provides a time record of that response.

[0006] Certain aspects of the present invention are based on the concept that changes in the concentrations of nitric oxide and nitrite ions can be detected and quantified in conjunction with an integrated electrochemical device.

[0007] Certain aspects of the present invention are based on the observation that nitric oxide is produced by a specific enzyme (NO synthase) from the breakdown of intracellular L-arginine in the presence of molecular oxygen (O2) and an electron source. When L-arginine is reduced by high consumption (e.g., after prolonged exercise) or chronic deficiency, NO synthase converts molecular oxygen into superoxide ions (O2 - ), which spontaneously and very rapidly converts to hydrogen peroxide (HO) with a 2:1 overall stoichiometry. Certain aspects of the present invention are based on the concept that the presence of detectable concentrations of hydrogen peroxide in blood and therefore in sweat provides an indicator of the level of distress in the circulatory system. Furthermore, hydrogen peroxide, in the presence of metal salts, can be converted to highly toxic radical species (HO * , HO2 * etc.), which radical species can cause significant damage to cells of the circulatory system, including cells of the heart. Certain aspects of the present invention are based on the concept that detecting hydrogen peroxide production, simultaneously with detecting nitric oxide and / or nitrite ion production, is relevant to assessing the performance of the circulatory system of a patient.

[0008] Certain aspects of the present invention are based on the concept of detecting the above three chemical species in conjunction with an integrated electrochemical device.

[0009] Certain aspects of the invention are based on the observation that the physiological system of a subject is dynamic since the volumetric flow of sweat may change to adjust the capacity to dissipate the heat generated depending on the muscle force exerted. The exchange rate of each chemical species at the blood-sweat interface may change depending on the exercise performed by the subject. Certain aspects of the invention are based on the idea of ​​quantitatively and dynamically detecting the production of one or more of the aforementioned chemical species by the circulatory system, such as during exercise testing or medical monitoring of a subject.

[0010] To achieve the above, in one embodiment, the present invention provides a detection device for detecting at least one chemical species in a biological fluid, such as sweat, of a subject, such as a human or animal, the at least one chemical species being nitric oxide (NO) and nitrite ion (NO2). - and a collecting element selected from the group consisting of hydrogen peroxide H2O2, preferably containing nitric oxide NO, which is placed in an investigation zone of the epidermis of the subject; a detection unit including at least one fluid circuit coupled to the collection element for conducting at least one sweat stream originating from the investigation zone; The detection unit further comprises: at least one electrochemical sensor comprising an electrode disposed in the fluid circuit, the at least one electrochemical sensor configured to generate a signal representative of a concentration of the at least one chemical species in the sweat stream; The present invention provides a detection device comprising:

[0011] "Chemical species in biological fluids" refers to chemical species dissolved in the biological fluids, particularly nitric oxide.

[0012] "Epidermis" refers to the superficial layer of plant tissue that provides a protective layer for the above-ground parts of plants, or the superficial layer of skin of humans or animals. For example, a biological fluid is a plant exudate or a human or animal sweat.

[0013] In some embodiments, the apparatus of the above configuration may include one or more of the following features.

[0014] In an embodiment, the or each electrochemical sensor or at least one electrochemical sensor is further configured to generate a signal representative of sweat flow rate or sweat volumetric flow rate.

[0015] In one embodiment, the detection unit is configured to generate a signal representative of the instantaneous production of the at least one chemical species in the investigation zone based on a signal representative of the concentration of the at least one chemical species and a signal representative of the sweat flow velocity.

[0016] "Instantaneous generation" refers to measurements obtained over a time period that is very short compared to the time characteristic of a change in the physiological response of the subject, which for human subjects is typically on the order of one to several seconds.

[0017] In an embodiment, the or each electrochemical sensor or at least one electrochemical sensor comprises at least one working electrode and a counter electrode arranged in the fluid circuit, and the electrochemical sensor is configured to generate a signal representative of the concentration of the at least one chemical species by electrical measurement, in particular amperometry, between the at least one working electrode and the counter electrode.

[0018] In one embodiment, the or each electrochemical sensor or at least one electrochemical sensor comprises an upstream working electrode and a downstream working electrode spaced apart in the flow direction of the sweat flow in the fluid circuit, and the electrochemical sensor is configured to generate a signal representative of the flow rate by measuring a delay between a change in current or potential at the upstream working electrode and a change in current or potential at the downstream working electrode.

[0019] In one embodiment, the distance L between the upstream working electrode and the downstream working electrode is 0.05 mm to 1 cm.

[0020] In one embodiment, the or at least one of the electrochemical sensors is configured to polarize the electrodes to a potential that oxidizes nitric oxide, and the detection unit comprises a filter that filters the sweat flow reaching the electrochemical sensor, in particular to eliminate hydrogen peroxide.

[0021] This type of filter can be made in various ways, for example using a polytetrafluoroethylene (PTFE) membrane or a eugenol-based membrane, and can be placed in the fluid circuit. In one embodiment, the filter comprises a layer of eugenol (4-allyl-2 methoxyphenol) placed on at least one electrode of the electrochemical sensor.

[0022] The above described arrangement based on a detection electrode modified with such a filter, in particular for measuring NO, directly generates a signal representative of the concentration of nitric oxide NO on the basis of the amperometric measurement signal.

[0023] In the embodiments described below, the detection unit is fabricated to be capable of simultaneously or sequentially detecting two or three of the above chemical species using one or more electrochemical sensors.

[0024] In one embodiment, the at least one chemical species is nitric oxide, NO, and nitrite, NO. - In one embodiment, the at least one chemical species comprises nitric oxide, NO, and hydrogen peroxide, H2O2. In one embodiment, the at least one chemical species comprises nitric oxide, NO, and nitrite, NO2 - and hydrogen peroxide H2O2.

[0025] In one embodiment capable of sequential detection, the or each electrochemical sensor or at least one electrochemical sensor is configured to detect a plurality of chemical species in a plurality of sequential measurement steps, the electrochemical sensor being configured to polarize the electrode to a potential for oxidizing hydrogen peroxide HO in a first step and to polarize the electrode to a potential for oxidizing nitric oxide NO in a second step, and the detection unit being configured to generate a signal representative of the concentration of nitric oxide NO based on a first amperometric measurement signal obtained in the first step and a second amperometric measurement signal obtained in the second step.

[0026] Advantageously, the or each electrochemical sensor or at least one electrochemical sensor is adapted to convert the electrode into the nitrite ions NO2 in a third step. - The detection unit is configured to polarize the nitrite ion NO2 based on the first amperometric measurement signal, the second amperometric measurement signal, and the third amperometric measurement signal obtained in the third step. - The device is configured to generate a signal representative of the concentration of

[0027] In one embodiment, the detection unit is constructed so that three of the above chemical species can be detected sequentially in multiple steps of a time sequence of measurements using one electrochemical sensor. In this embodiment, the electrochemical sensor polarizes a platinized platinum electrode (platinum black coated) in a first time step of several seconds (e.g., 5 seconds) during a specific sequence to an electrochemical potential that oxidizes hydrogen peroxide H2O2, and then in a second time step of the same time to a potential that oxidizes nitric oxide NO, and optionally in a third time step of the same time to detect nitrite ions NO2. -The detection unit is configured to generate a signal representing the concentration of nitric oxide (NO) based on a first amperometric measurement signal obtained in the first step and a second amperometric measurement signal obtained in the second step. The above sequence is repeated as many times as necessary over the entire duration of the exercise test. Two or three unknowns (H2O2, NO and nitrite ion (NO2)) are measured. - By solving a series of three equations with the concentrations of 1, 2, and 3 (the currents measured in sequence at the electrodes polarized to each potential in one sequence), the values ​​of each of the three concentrations at the time each sequence was generated based on the three measurements are obtained.

[0028] In another embodiment capable of simultaneous detection, the detector comprises: a first fluid circuit coupled to the collection element for conducting a first sweat stream originating from the investigation zone; a first electrochemical sensor including an electrode disposed in the first fluid circuit, the first electrochemical sensor being configured to polarize the electrode to a potential that oxidizes hydrogen peroxide, H2O2; a second fluid circuit coupled to the collection element for conducting a second sweat stream originating from the investigation zone; 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 that oxidizes nitric oxide (NO); The detection unit is configured to generate a signal representative of a concentration of nitric oxide (NO) based on a first amperometric measurement signal generated by the first electrochemical sensor and a second amperometric measurement signal generated by the second electrochemical sensor.

[0029] In this case, it is advantageous that the detection unit further comprises: a third electrochemical sensor including a third fluid circuit coupled to the collecting element for conducting a third sweat stream originating from the investigation zone; and an electrode disposed in the third fluid circuit, the electrode being adapted to detect the nitrite ions, NO2 -and a third electrochemical sensor configured to be polarized to a potential that oxidizes nitrite ions NO2 based on the first amperometric measurement signal, the second amperometric measurement signal, and a third amperometric measurement signal generated by the third electrochemical sensor. - The device is configured to generate a signal representative of the concentration of

[0030] In this embodiment the sensing portion comprises three parallel microfluidic circuits fed in parallel by the same sweat collecting element.

[0031] In one embodiment, the detection unit further comprises another fluid circuit, e.g., a fourth fluid circuit, etc., coupled to the collecting element for conducting another sweat flow originating from the investigation zone, e.g., a fourth sweat flow, etc., and another electrochemical sensor, e.g., a fourth electrochemical sensor, etc., having an electrode disposed in the fourth fluid circuit.

[0032] In this embodiment, the further or fourth electrochemical sensor is configured to polarize the electrodes to a potential that oxidizes nitric oxide, and the fourth fluid circuit includes a filter that filters the sweat flow reaching the fourth electrochemical sensor, in particular to dissipate hydrogen peroxide.

[0033] In one embodiment, the collecting element comprises a fibrous body that transmits biological liquids, in particular sweat, from the investigation zone by capillary action. Such a fibrous body can be a woven or non-woven material.

[0034] In one embodiment, the device further comprises an envelope surrounding the collecting element to contact the epidermis of the subject, thereby forming an impermeable barrier around the investigation zone.

[0035] This arrangement prevents gases, liquids and microorganisms such as bacteria or viruses from entering the investigation zone outside the investigation zone. The impermeable contact between the envelope and the epidermis ensures that the species detected originate from the biological liquids produced by the investigation zone and not from external flows.

[0036] In one embodiment, the device further comprises a wired or wireless communication unit configured to transmit one or more measurement signals generated by the detection unit to a post-processing unit.

[0037] In one embodiment, the or each fluid circuit or at least one of the fluid circuits is formed on an insulating support and the electrodes of the or each electrochemical sensor or at least one of the electrochemical sensors are formed by metal deposition on the insulating support, such an insulating support may be made of a material selected from the elastomer family, for example polydimethylsiloxane (PDMS), polyimide, epoxy resin and parylene.

[0038] In one embodiment, the metal deposit is selected from the group consisting of silver (Ag), gold (Au), platinum (Pt) and platinum black. In one embodiment, the electrochemical sensor comprises a reference electrode made of silver chloride (AgCl).

[0039] In one interesting embodiment, the electrode consists of a deposit of graphene doped with silver (Ag) or gold (Au) nanoparticles functionalized with a binder for nitric oxide, in particular guanylyl cyclase or a porphyrin.

[0040] In one embodiment, the device is configured to take measurements and transmit periodically, such as at a customizable frequency or at a frequency dependent on the activity state detected by the device. For example, the device may include a gyroscope module and / or an accelerometer for detecting the activity state of the subject, which may facilitate the detection of the subject's activity state during sweat analysis and the analysis of the correlation between the subject's activity state and the production of the analyte species.

[0041] In one embodiment, the device may include a geolocation module.

[0042] In one embodiment, the present invention also provides a method for measuring the production of at least one chemical species by a human or animal subject, the at least one chemical species being nitric oxide (NO) and nitrite ion (NO2). - and hydrogen peroxide H2O2, preferably nitric oxide NO, said method comprising: selecting an investigation zone of the epidermis of the subject; - applying said device for a time required to generate a signal representative of the concentration of said at least one chemical species in the sweat stream and, where appropriate, a signal representative of the flow rate of said sweat stream; obtaining a measure of the production of said at least one chemical species by said subject from a signal representative of the concentration of said at least one chemical species in said sweat flow and, where appropriate, from a signal representative of the sweat flow rate; has.

[0043] Measurement of the production of one or more of the above chemical species by a subject can be used in a variety of applications, for example, to assess distress in the subject's vascular tissue or to assess the performance of the subject's cardiovascular system.

[0044] Other possible applications include diagnosis, medical treatment, and monitoring of diseases such as cardiovascular diseases, neurodegenerative diseases, pulmonary arterial hypertension, cancer, hypercholesterolemia, diabetes, systemic endothelial dysfunction, arteriosclerosis, thrombosis or ischemia, cell proliferation or platelet accumulation inhibition or leukocyte adhesion defects in smooth muscle fibrocytes, bronchitis, asthma, Alzheimer's disease, etc.

[0045] Other possible applications include monitoring the growth and / or muscle distress of individuals, such as individuals undergoing physical training, preventing injuries caused by overtraining a subject, and / or improving muscle performance in a subject.

[0046] In order to make the subject matter of the invention easier to understand, the following describes an embodiment illustrated in the attached drawings, which is given by way of example only and is not intended to limit the invention. [Brief description of the drawings]

[0047] [Figure 1] 1 is a schematic diagram of a subject's back with an embodiment of a device placed thereon. [Diagram 2] FIG. 1 is a perspective view showing a detection device according to an embodiment. [Diagram 3] FIG. 3 is an exploded view of the device of FIG. 2. [Figure 4] 3 is a schematic cross-sectional view of a first embodiment of a collecting element that can be used in the device of FIG. 2. [Diagram 5] 3 is a schematic cross-sectional view of a second embodiment of a collecting element that can be used in the device of FIG. 2. [Figure 6] FIG. 3 is a schematic functional diagram of a fluid circuit usable in the device of FIG. 2. [Figure 7] FIG. 7 is a schematic perspective view of an electrochemical sensor that can be used in the fluid circuit of FIG. 6. [Figure 8] FIG. 8 is a timing diagram illustrating a detection method usable with the electrochemical sensor of FIG. 7. [Figure 9] FIG. 3 is a schematic functional diagram of a detector usable in the device of FIG. 2; [Figure 10] 3A-3D show the steps of a method that can be carried out using the apparatus of FIG. 2; [Figure 11] 3 is a graph showing the results of measurements obtained using the device of FIG. 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] FIG. 1 shows a detection device 1 placed on the skin of a human subject 2, for example on the subject's back, which performs a quantitative measurement of chemical species dissolved in sweat, including nitric oxide and, optionally, nitrite and hydrogen peroxide dissolved in sweat.

[0049] Referring to Figure 2, the detection device 1 is illustrated in the form of a compact housing comprising a base plate 3 made of a biocompatible flexible material, preferably a self-adhesive material, that can be placed directly on the skin of a subject, and an upper envelope 7 that encloses the other components of the detection device 1, the envelope 7 being preferably impermeable to water, for example silicone.

[0050] 3, the center of the base plate 3 has a circular recess 4 that defines an investigation zone on the subject's skin, the diameter of the recess 4 can be, for example, a few mm to a few cm. The circular recess 4 contains a collecting element 5 which is then placed directly on the subject's skin 2 upon operation of the device. The recess 4 can have other shapes, such as an oval, a triangle, a rectangle, a square, a polygon or other shapes.

[0051] By way of example, the collecting element 5 comprises a fibrous body such as cotton or a nonwoven material. The collecting element 5 is connected to a support 6 which supports one or more fluid circuits and one or more sensors, as described below. The support 6 can be a rigid or flexible support, for example made from polyimide. The collecting element 5 serves to transport sweat generated in the interrogation zone to the fluid circuits of the support 6 so that one or more chemical species can be detected using the sensors. To achieve this, the collecting element 5 can be arranged in a variety of ways.

[0052] In the example embodiment of Figure 4, the collecting element 5 has a portion that contacts the skin 2 next to the support 6 and a portion that covers the upper surface of the support 6. In other words, the collecting element 5 is attached between the skin and the support 6. At the upper surface of the support 6, the collecting element 5 communicates with a fluid circuit in which a sensor is provided.

[0053] In the example embodiment of Figure 5, the collecting element 5 forms a sandwich around the support 6, such that the portion of the collecting element 5 below the support 6 is positioned to contact the skin. The portion of the collecting element 5 that extends beyond the portion that is positioned on the skin is folded over and cradles the support 6. On top of the support 6, the collecting element 5 is in communication with a fluid circuit in which a sensor is provided.

[0054] With reference to FIG. 6, the support 6 is provided with a microfluidic system 8, which is fed by capillary action via the collecting element 5. Sweat is collected by the part of the collecting element 5 that is placed in contact with the skin 2 and is then transported by capillary action to the microfluidic system 8. The microfluidic system 8 may comprise one or more fluid circuits, and in the example shown may comprise four parallel fluid circuits 9. The fluid circuits 9 are formed, for example, in the thickness of the support 6 and are separated by separators 11. The fluid circuits 9 may thus form separate flow paths through which sweat can move independently. The number of fluid circuits 9 may be more or less than that shown in the drawing.

[0055] Each fluid circuit 9 is provided with a respective sensor 10A, 10B, 10C or 10D. Arrows 12 indicate the direction of sweat flow in the fluid circuits 9. Preferably, the fluid circuits 9 drain to a drain reservoir which retains the fluid to be analysed and prevents reaction products from the electrolyte from re-contacting the subject's skin.

[0056] The sensors 10A, 10B, 10C and 10D arranged in the fluid circuit 9 for sweat analysis are preferably electrochemical sensors. The operating principle of electrochemical sensors is to electrolyze the solution present in the fluid circuit 9 between a working electrode and a counter electrode. Sensors of this kind can be manufactured in various ways and in particular can be manufactured to be miniaturized with dimensions of the order of millimeters.

[0057] Several example embodiments of electrochemical sensors will now be described with reference to FIG.

[0058] <Example of embodiment> [Embodiment Example 1] The sensor 10A detects hydrogen peroxide. Therefore, the sensor 10A detects a potential difference E H2O2 The sensor 10B detects nitric oxide. Therefore, the sensor 10B detects a potential difference E NO The sensor 10C detects nitrite ions. Therefore, the sensor 10C detects a potential difference E NO2- Works with.

[0059] The sensors 10A, 10B, and 10C measure the instantaneous intensity of the faradaic current associated with the electrochemical oxidation of the above-mentioned chemical species. oxdn Thus, the sensors 10A, 10B, and 10C can be used to detect and quantify the instantaneous concentrations of the above-mentioned chemical species.

[0060] Each of the three species mentioned above can be detected by amperometric measurements using a microelectrode, such as the platinum salt Pt(Cl)6. 4-It consists of a platinum strip covered by a thin layer of platinum black, deposited by electrochemical reduction of the anions of 1,2-dichloro-1,3,4-tetraphenylene in an aqueous medium, this thin layer having, for example, micrometer dimensions.

[0061] Three chemical species (NO, NO2 - and H2O2) can be distinguished by their distinct oxidation potentials at the above-mentioned electrodes, which occur in the following order: E H2O2 <E NO <E NO2 - However, faradaic currents are additive, so the measured current at the oxidation potential of each species is added to the elementary currents associated with the oxidation of this species and to the elementary currents associated with the oxidation of all species with lower oxidation potentials.

[0062] Therefore, only the chemical species H2O2 can be oxidized at the oxidation potential EH2O2, and the oxidation potential E NO The chemical species H2O2 and NO can be oxidized at the oxidation potential E NO2 - can oxidize three species. oxdn (EH2O2), i oxdn (E NO ), i oxdn (E NO2 The current measured by each of the sensors 10A to 10C indicated by -) satisfies the following formula:

[0063] i oxdn (E H2O2 )=a1 i H2O2

[0064] i oxdn (E NO )=a2 i H2O2 +a3 i NO

[0065] i oxdn (E NO2 -)=a4 i H2O2 +a5 i NO +a6 i NO2 - In the above formula, coefficients a1 to a6 are calibration constants of the sensor, which can be measured by experiment.

[0066] So, using a simple electronic subtraction we get:

[0067] i H2O2 =(1 / a1)i oxdn (E H2O2 )

[0068] i NO =(1 / a3)i oxdn (E NO )-(a2 / a1)·(1 / a3)i oxdn (E H2O2 )

[0069] i NO2 -=(1 / a6)i oxdn (E NO2 -)-(a4 / a6)i H2O2 -(a5 / a6)i NO

[0070] At any time t, the oxidation faradaic current i of each species S S The instantaneous intensity of (t) is the concentration C of each chemical species S in the volume of fluid above the electrode that detects each species S. S (t) is proportional to the shape factor (symbol γ) that depends on the geometric characteristics of the sensor, and the Faraday constant (symbol n S ), and depends on . That is,

[0071] n H2O2 =n NO2 -=2 and n NO =1

[0072] F stands for Faraday, or 96,500 coulombs, which is the amount of charge carried by one mole of electrons.

[0073] The shape factor γ is a constant imposed by the geometrical characteristics of the electrochemical device, which can be evaluated theoretically and measured experimentally by calibration. In the following, for simplicity, it is assumed that the three sensors 10A-10C have the same geometrical characteristics, and therefore the shape factor γ is the same for all sensors.

[0074] The concentration of the chemical species can then be determined from the current measured by the sensors 10A-10C using the following equation, which defines the time variable t:

[0075] C H2O2 (t)=i oxdn (E H2O2 ,t) / (2Fγ)

[0076] C NO (t) = [i oxdn (E NO ,t)-i oxdn (E H2O2 ,t)] / (Fγ)

[0077] C NO2 -(t)=[i oxdn (E NO2 -,t)-i oxdn (E NO ,t)] / (2Fγ)

[0078] Therefore, in the first embodiment, the three sensors 10A to 10C are E H2O2 , E NO , and E NO2 - can be operated in parallel at a constant oxidation potential of 0.1 -.

[0079] In one variant, only NO and NO2- are detected. This variant is particularly advantageous when the measurement of H2O2 is not important and does not affect the intended result. In that case, the above-mentioned concentration C H2O2 (t) is uniformly zero, i.e., C H2O2 (t) = 0. Therefore, the above sequence of equations is simplified.

[0080] [Embodiment Example 2] In the second embodiment, one fluid circuit 9 and one sensor 10A are used, and other fluid circuits and sensors can be omitted.

[0081] In this example, the sensor 10A is operated sequentially to detect the above species in three successive steps. Thus, the oxidation potential is switched in three potentiostatic steps equal to each of the three oxidation potentials, e.g., E H2O2 →E NO →E NO2 -→E H2O2 →E NO →E NO2 -→ etc. in this order.

[0082] In this case, each oxidation potential is maintained for a time much longer than the time constant of the working electrode (e.g., a few milliseconds for microelectrodes used in microfluidic channels) and current measurements are made at the end of each potentiostatic step.

[0083] The remaining measurement signals can be processed using the same formulas as in the first embodiment.

[0084] [Embodiment Example 3] Nitric oxide is a small molecule that is both hydrophilic and lipophilic, so it is different from the other two species, H2O2 and NO2. - Unlike nitric oxide, which can easily pass through a thin layer of an organic polymer, electrochemical sensors protected by such a filtration layer can be used to isolate and detect nitric oxide, for example, using a working electrode made from platinized platinum covered by a thin layer of eugenol (4-allyl-2-methoxyphenol) deposited by electropolymerization.

[0085] Therefore, in the third embodiment, the sensor 10D is protected by a filtering layer, indicated generally by the numeral 19. The instantaneous concentration of nitric oxide is then calculated by the following equation: - Can be measured independently of:

[0086] C NO (t) = [i oxdn (E NO ,t)] eugenol / (Fγ)

[0087] In the same formula, [i oxdn (E NO ,t)] eugenol denotes the current measured by sensor 10D.

[0088] The other sensors 10A-10C and the other fluid circuits 9 can be omitted. Thus, the method can be advantageously used with one sensor when only the concentration of NO is desired.

[0089] In a variant, the filter 19 can be placed at another location between the collecting element 5 and the sensor 10D. The function of the filter 19 is to filter the sweat to prevent certain elements contained therein from interfering with the measurement of NO dissolved in the sweat. Examples of interfering elements are peroxynitrite (ONOO-) and hydrogen peroxide (H2O2).

[0090] [Embodiment Example 4] In this case, sensor 10D of embodiment 3 is fused with sensors 10A-10C of embodiment 1 or sensor 10A of embodiment 2. This configuration can be used to obtain two measurements that are independent of the concentration of dissolved nitric oxide and to check the consistency of both measurements, in particular by making sure that there is no drift between the two sensors due to, for example, partial inactivation of the surface of one of the electrodes.

[0091] In the present case, the electrochemical electronic controller 40 (FIG. 9) is preferably configured to compare the two measurements of the concentration of nitric oxide and to issue an alarm if the result of the comparison meets a predetermined criterion, for example if it exceeds a predefined threshold.

[0092] In the above embodiments 1-4, the measured instantaneous faradaic current can be used to measure the concentration of the chemical species in the solution being analyzed, so in a static system, the current intensity is sufficient to know the formation of the detected chemical species.

[0093] However, when the detection device 1 is applied to a substantially dynamic physiological system, for example during exercise testing or medical monitoring, it is also desirable to be able to obtain a quantified dynamic characteristic of the production of each chemical species by the circulatory system. In order to obtain the instantaneous amount of a chemical species produced in a short time period denoted Δt(t) under dynamic conditions, denoted ΔQ(t), the average concentration C S It is desirable to simultaneously know (t) and the volumetric flow rate of the fluid being analyzed (equation below).

[0094] d(t)=(ΔV / Δt)

[0095] In the above formula, ΔV denotes the volume scanned during time Δt. Thus, the production flow rate (symbol P S The intensity of (t) is given by:

[0096] P S (t) = [ΔQ / Δt](t) = C S (t) d(t)

[0097] Here, the average concentration C S (t) is determined from the average magnitude of the electrochemical oxidation current measured between times t and t+Δt.

[0098] Thus, in the dynamic application envisaged here, at each time instant t necessary to achieve the desired accuracy in monitoring the physiological state of the patient over time (e.g., once per minute), the detection device 1 measures the average intensity i of the faradaic current associated with the electrochemical oxidation of the chemical species being monitored. av It is desirable to simultaneously measure the sweat volume flow rate d(t) of the corresponding fluid circuit at time t.

[0099] FIG. 7 shows an embodiment of an electrochemical sensor 10 that can meet the above-mentioned dual requirements in an integrated manner. This electrochemical sensor 10 comprises at least one strip microelectrode that forms a working electrode 20 or a pair of working electrodes 20, 23. This type of strip microelectrode can be made of platinized (platinum black) platinum, which can be covered or not with a layer of electropolymerized eugenol of micrometer dimensions. This type of strip microelectrode can be embedded by microfabrication techniques, for example, by chemical vapor deposition and / or lithography. The strip microelectrode or strip microelectrodes can be used to electrochemically oxidize selected chemical species.

[0100] Furthermore, the fluid circuit 9 of Fig. 7 is provided with a reference electrode 21, for example made in the form of an Ag / AgCl microstrip, which is placed upstream of the working electrode 20 or the pair of working electrodes 20, 23. Finally, the fluid circuit 9 comprises a counter electrode 30 made of platinized platinum, which is placed downstream of the working electrode 20 or the pair of working electrodes 20, 23. Despite the schematic functional diagram of Fig. 7, the surface area of ​​the counter electrode 30 is in fact two to three times larger than the surface area of ​​the other electrodes.

[0101] The fluid circuit 9 with the electrodes 20, 21, 23, 30 is entirely immersed in a layer of sweat (not shown), so that the fluid circuit 9 becomes a microfluidic electrochemical cell with three or four electrodes. Each electrode 20, 21, 23, 30 is connected to an electrochemical electronic controller 40 (FIG. 9) by an insulated electrical contact with the collecting element 5 and the sweat.

[0102] The electrochemical sensor 10 of this embodiment may be used in one or more of the fluid circuits 9 described above.

[0103] To measure the volumetric flow rate d(t), the electrochemical sensor 10 must comprise a pair of working electrodes 20, 23. The solution described here has no moving parts and does not need to be hydrodynamic, making it simple and easy to industrialize. It also does not require any intervention aimed at regulating the fluid flow rate and is suitable for all reasonable physiological flow rates.

[0104] The two working electrodes 20, 23, e.g. two strips of platinized platinum, can act as micro-working electrodes, are electrically independent and are separated by a distance L along the path of the fluid to be analyzed in the fluid circuit 9. The two working electrodes 20 and 23 are, for example, placed at the bottom of a straight flow channel with a constant cross-sectional area A.

[0105] The downstream working electrode 23 is used in a two-step method as shown in FIG. 8. Plot 81 represents the potential applied to the working electrode 20 as a function of time. Plot 82 represents the potential applied to the working electrode 23 as a function of time. The potentials marked "0" on plots 81 and 82 actually refer to the unconnected state (open circuit) of the corresponding electrode. Plot 83 represents the faradaic current measured at the working electrode 20 as a function of time. Plot 84 represents the faradaic current measured at the working electrode 23 as a function of time.

[0106] In the first step, which takes place over a time range before time t0, a potential E oxdn is sufficient to allow the target species to be oxidized while leaving the downstream working electrode 23 unconnected. The upstream working electrode 20 is then used to generate an instantaneous electrochemical current i oxdn (t) can be continuously recorded, and this instantaneous electrochemical current i oxdn (t) will represent the concentration C(t) of the target species in the fluid being analyzed, after performing any of the above calculations as necessary.

[0107] In a second step, which takes place over a time range from time t0, the working electrode 20 is disconnected and a potential E oxdn is applied.

[0108] At time t0, the sweat flow passing over the working electrode 23 has already been electrolyzed (in whole or in part) when passing over the upstream working electrode 20, so that the concentration of the target species is now zero, or at least is much lower than before it entered the electrochemical device. Thus, the current intensity i detected by the working electrode 23 is oxdn (plot 84) becomes zero (or at least, the current i detected at the working electrode 20 before time t0). oxdn (The strength of the

[0109] At time t0+Δt, the working electrode 23 starts analyzing the non-electrolyzed solution, and the working electrode 23 detects a current intensity i oxdn will be comparable to the current intensity detected by the working electrode 20 before time t0. The current increase, outlined by the steps in FIG. 8, is detected by the ad-hoc electronic circuit. The time difference Δt between this increase and time t0 when the working electrode 20 is unconnected represents the time required for the sweat flow to travel between the two working electrodes 20 and 23. Time Δt is represented by the double arrow at the bottom of FIG. 8. For simplicity of presentation, it is assumed in FIG. 8 that the electrolysis of all the target species is completed when the working electrode 20 is connected. The same measurement principle is applicable when this electrolysis is only partially performed.

[0110] Therefore, the flow velocity v(t) and flow rate d(t) can be estimated as follows:

[0111] v(t)=L / Δt

[0112] d(t)=A v(t)

[0113] The potential E applied to the working electrode 23 oxdnis sufficient to allow the target species to be oxidized while the working electrode 20 is left unconnected. Optionally, the concentration measurements can then be continued for a certain period of time using the working electrode 23. 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 evaluate the flow rate d(t) at successive time points.

[0114] The distance L between the two working electrodes 20 and 23 is preferably small enough, such as on the order of 1 mm, so that the change in the patient's physiological response over the period Δt is negligible.

[0115] The above-mentioned flow rate measurement method can be used simultaneously in all of the multiple parallel fluid circuits. However, if these multiple circuits have the same configuration and are supplied in the same manner, one measurement of the flow rate may be sufficient. In this case, the above-mentioned flow rate measurement method can be used for one fluid circuit 9. Furthermore, this flow rate measurement method can also be combined with multiple sensors of the above-mentioned embodiment examples 1 to 4.

[0116] The above-described methods for detecting concentration and flow rate can be carried out automatically using an electronic control unit 40 , which is preferably integrated into the detection device 1 .

[0117] Next, one embodiment of the electronic control device 40 will be described with reference to Fig. 9. The electronic control device 40 can be incorporated into the detection device 1, for example in the form of an electronic circuit board 13 shown in Fig. 13.

[0118] The or each electrochemical sensor 10 is connected to an analogue to digital converter 14 which provides an input to a processor 15. The processor 15 is programmed to carry out, for example, the methods for detecting concentration and flow rates described above.

[0119] An energy source 16, for example a battery, powers the electronic controller 40. A communication module 17 can be provided, which can be wired or wireless, for transmitting the concentration, flow rate and / or mass flow measurements for one or each target species to a storage or post-processing unit.

[0120] FIG. 10 illustrates a method executable by processor 15 in one embodiment.

[0121] In step 31, the instantaneous concentration Cs(t) of the chemical species S is determined from the electrochemical measurement results.

[0122] In step 32, the volumetric flow rate d(t) in the corresponding fluid circuit is determined.

[0123] In step 33, the flow of the amount of substance of the investigated chemical species is calculated based on Cs(t) and d(t), for example by the following formula:

[0124] P S (t)=C S (t) d(t)

[0125] FIG. 11 is a graph illustrating a measurement signal for material flow as a function of time that may be obtained with the detection device 1 during a kinetic test performed, for example, on an analyte of the chemical species NO.

[0126] The electronic controller 40 may optionally include other functional modules, such as a gyroscope 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, etc. Knowing the skin temperature is useful for correlating temperature with vasodilation.

[0127] Certain elements of the detection device 1, in particular the electronic controller 40, can be manufactured in various forms, unitary or distributed, using physical and / or software components. Possible physical components are application specific integrated circuits, ASICs, field programmable gate arrays, FPGAs, or microprocessors. The software components can be written in various programming languages, such as C, C++, Java, or VHDL. This list is not limiting.

[0128] Although the present invention has been described with reference to some specific embodiments, it is clear that the present invention is not limited to these embodiments, and that the present invention encompasses all equivalents of the means described herein and combinations thereof, when falling within the scope of the present invention.

[0129] Use of the verbs "comprise" or "have" and their conjugations does not exclude the presence of elements or steps other than those stated in a claim.

[0130] In the claims, any signs placed in parentheses shall not be construed as limiting the claim.

Claims

1. 1. A detection device (1) for detecting at least one dissolved chemical species in sweat of a human or animal subject, the dissolved chemical species comprising nitric oxide (NO), comprising: a collecting element (5) placed in an investigation zone of the epidermis (2) of the subject; a detection section comprising at least one fluid circuit (8, 9) coupled to said collection element for conducting at least one sweat stream originating from said investigation zone; Equipped with The detection unit further includes: At least one electrochemical sensor (10) comprising electrodes including at least one working electrode (20) and a counter electrode (30) arranged in the fluid circuit, the at least one electrochemical sensor being configured to generate at least a signal representative of the concentration of nitric oxide (NO) in the sweat stream by electrical measurement between the at least one working electrode (20) and the counter electrode (30). Equipped with The electrodes further include an upstream working electrode (20) and a downstream working electrode (23) spaced apart in the direction of sweat flow (12) in the fluid circuit; The electrochemical sensor is configured to generate a signal representative of a sweat flow rate by measuring a delay (Δt) between a change in current at the upstream working electrode and a change in current at the downstream working electrode.

2. The detector is configured to generate a signal representative of an instantaneous production of nitric oxide (NO) in the investigation zone based on the signal representative of the concentration of nitric oxide (NO) and the signal representative of the sweat flow rate.

2. The detection device according to claim 1.

3. The electrochemical sensor is configured to generate the signal representative of the concentration of nitric oxide (NO) by amperometry between the at least one working electrode and the counter electrode.

3. The detection device according to claim 1 or 2.

4. The electrochemical sensor (10D) is configured to polarize the electrode to a potential that oxidizes nitric oxide (NO); The detection unit includes a filter (19) for filtering the sweat flow reaching the electrochemical sensor to eliminate hydrogen peroxide.

4. A detection device according to claim 1.

5. The at least one chemical species further comprises nitrite ion, NO ２ － and / or hydrogen peroxide H ２ O ２ It includes, 4. A detection device according to claim 1.

6. the electrochemical sensor (10A) is configured to detect a plurality of chemical species in a plurality of sequential measurement steps; In the first step, the electrochemical sensor is ２ O ２ and in a second step polarizing the electrode to a potential that oxidizes nitric oxide, NO; The detection unit is configured to generate the signal representing the concentration of nitric oxide (NO) based on a first amperometric measurement signal obtained in the first step and a second amperometric measurement signal obtained in the second step.

6. The detection device according to claim 5.

7. In the third step, the electrochemical sensor (10A) converts the electrode into the nitrite ion NO ２ － and configured to polarize the electrode to a potential that oxidizes the electrode, The detection unit detects the nitrite ion NO based on the first amperometric measurement signal, the second amperometric measurement signal, and the third amperometric measurement signal obtained in the third step. ２ － and configured to generate a signal representative of the concentration of 7. The detection device according to claim 6.

8. The detection unit is a first fluid circuit (9) coupled to the collection element for conducting a first sweat flow originating from the investigation zone; A first electrochemical sensor (10A) having an electrode disposed in the first fluid circuit, the electrode being connected to a sensor for detecting hydrogen peroxide H ２ O ２ a first electrochemical sensor (10A) configured to be polarized to a potential that oxidizes a second fluid circuit (9) coupled to the collection element for conducting a second sweat stream originating from the investigation zone; a second electrochemical sensor (10B) having an electrode disposed in the second fluid circuit, the second electrochemical sensor (10B) being configured to polarize the electrode to a potential that oxidizes nitric oxide (NO); Equipped with The detection unit is configured to generate a signal representative of a concentration of nitric oxide (NO) based on a first amperometric measurement signal generated by the first electrochemical sensor (10A) and a second amperometric measurement signal generated by the second electrochemical sensor (10B).

6. The detection device according to claim 5.

9. The detection unit (5, 6) further includes a third fluid circuit (9) coupled to the collection element for conducting a third sweat stream originating from the investigation zone; A third electrochemical sensor (10C) comprising an electrode disposed in the third fluid circuit, the electrode being adapted to detect the nitrite ions NO ２ － a third electrochemical sensor (10C) configured to be polarized to a potential that oxidizes Equipped with The detection unit detects the nitrite ion NO based on the first amperometric measurement signal, the second amperometric measurement signal, and a third amperometric measurement signal generated by the third electrochemical sensor. ２ － and configured to generate a signal representative of the concentration of 9. The detection device according to claim 8.

10. The detection unit further includes: a fourth fluid circuit (9) coupled to the collection element for conducting a fourth sweat stream originating from the investigation zone; a fourth electrochemical sensor (10D) comprising an electrode disposed in the fourth fluid circuit, the fourth electrochemical sensor (10D) being configured to polarize the electrode to a potential that oxidizes nitric oxide (NO); Equipped with The fourth fluid circuit is a hydrogen peroxide H ２ O ２ a filter (19) for filtering the sweat flow reaching the fourth electrochemical sensor to dissipate 10. The detection device according to claim 8 or 9.

11. The filter (19) comprises a layer of eugenol (4-allyl-2 methoxyphenol) disposed on at least one electrode of the electrochemical sensor (10D).

11. The detection device according to claim 4 or 10.

12. the detection unit is configured to compare a measurement result of the concentration of nitric oxide obtained by the fourth electrochemical sensor (10D) with a measurement result of the concentration of nitric oxide obtained by the first and second electrochemical sensors (10A, 10B), and to issue an alarm when the result of the comparison satisfies a predetermined criterion. The detection device according to claim 10.

13. The collecting element (5) is provided with a fibrous body that transmits sweat by capillary action. Detector device according to any one of claims 1 to 12.

14. an envelope (7) surrounding the collecting element (5) so as to contact the epidermis (2) of the subject, thereby forming an impermeable barrier around the investigation zone; Detector device according to any one of claims 1 to 13.

15. The device further includes a wired or wireless communication unit (17) configured to transmit one or more measurement signals generated by the detection unit to a storage unit or a post-processing unit. Detector device according to any one of claims 1 to 14.

16. The fluid circuit (9) is formed in an insulating support part (6), the electrodes of the electrochemical sensor (10) are formed by metal depositions on the insulating support, the metal depositions being selected from the group consisting of silver, gold, platinum and platinum black; Detector device according to any one of claims 1 to 15.

17. Further comprising a gyroscope module and / or at least one accelerometer for detecting an activity state of the subject. Detector device according to any one of claims 1 to 16.

18. and a temperature sensor for measuring a temperature of the epidermis of the subject. Detector device according to any one of claims 1 to 17.

19. A method for measuring the production of at least one chemical species (P ｓ ) by a human or animal subject, comprising: said at least one chemical species comprising nitric oxide, NO; The method comprises: selecting an investigation zone of the epidermis of the subject; - applying a detection device (1) according to any one of claims 1 to 18 for a time required to generate a signal representative of the concentration of nitric oxide NO in the sweat flow and a signal representative of the sweat flow velocity; From the signal representing the concentration of nitric oxide NO in the sweat flow and the signal representing the flow rate of the sweat flow, the production of nitric oxide NO by the subject (P ｓ ) measuring the The method according to claim 1, further comprising:

20. The method further comprises a step of evaluating distress in the vascular tissue of the subject based on the measurement result of the production of nitric oxide (NO); 20. The method of claim 19.

21. Further comprising a step of evaluating the circulatory system capacity of the subject based on the measurement result of the production of nitric oxide (NO); 20. The method of claim 19.

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