Detection of chemicals in the subjects' sweat

Abstract

The detection device detects at least one chemical substance in the sweat of a human or animal subject, wherein the at least one chemical substance is selected from the group consisting of nitric oxide (NO), nitrite ions (NO2-) and hydrogen peroxide (H2O2). The device comprises a collecting element and a detection device, wherein the collecting element is used to be placed in a study area of ​​the subject's epidermis, and the detection device comprises at least one fluid passage (9) and an electrochemical sensor (10) comprising electrodes arranged in the fluid passage, wherein the at least one fluid passage is coupled to the collecting element to guide at least one sweat flow (12) from the study area, and the electrochemical sensor is configured to generate a signal representing the concentration of the at least one chemical substance in the sweat flow and a signal representing the flow rate of the sweat flow.

Description

Technical Field

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

[0002] Nitric oxide is a gas that constitutes an intercellular messenger. It is an important cardiovascular messenger that transmits stresses by mechanotransduction. In particular, its release is intended to stimulate vasodilation of the vascular system during muscle exercise. The resulting flow changes in the blood and therefore in the fluids that balance the blood, such as sweat, therefore constitute a particularly relevant indicator of the patient's cardiovascular capacity to adapt to the muscle forces required during exercise testing.

[0003] In the case of cardiovascular disease, existing devices and tools for prevention and prediction are either limited to indirect measurements of nitric oxide while the patient is at rest or to direct measurements taken hours apart in order to observe pathological problems. In all cases, measurements can only be made 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 nitric oxide concentration in sweat provides a non-invasive method for monitoring cardiovascular capacity during preventive monitoring or for diagnosis.

[0005] Certain aspects of the present invention are based on the observation that in the presence of molecular oxygen, nitric oxide reacts to produce nitrite ions (NO2 - ). In other words, while changes in nitric oxide concentration represent the current state of the cardiovascular system's response to a given exercise, changes in nitrite ion concentration constitute a temporal record of these responses.

[0006] Certain aspects of the present invention are based on the concept that changes in nitric oxide and nitrite ion concentrations can be detected and quantified in a manner coupled to an integrated electrochemical device.

[0007] Certain aspects of the present invention are based on the observation that nitric oxide is produced by the degradation of intracellular L-arginine by a specialized enzyme (NO synthase) in the presence of molecular oxygen (O2) and an electron source. When the availability of L-arginine is reduced due to high consumption (e.g., after prolonged exercise) or chronic deficiency, NO synthase reduces molecular oxygen to superoxide ions (O2) by limiting itself. -) to continue reacting with oxygen. The ion is spontaneously and very rapidly converted to hydrogen peroxide (H2O2) by a reaction with a stoichiometric ratio of 2:1. Certain aspects of the present invention are based on the concept that the presence of detectable concentrations of hydrogen peroxide in the blood and therefore in the sweat provides an indicator of the degree of distress in the cardiovascular system. In addition, in the presence of metal salts, hydrogen peroxide decomposes into highly toxic free radical species (HO*, HO2*, etc.), which can cause extensive damage to cells of the cardiovascular system, including heart cells. Certain aspects of the present invention are based on the concept that detecting hydrogen peroxide production while detecting nitric oxide production and / or nitrite ion production is relevant to assessing the cardiovascular capacity of a patient.

[0008] Certain aspects of the present invention are based on the concept of detecting the three aforementioned chemical species coupled to 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 volume flow of sweat can vary to adjust the ability to remove heat generated according to the transmitted muscle force. The exchange flow of each chemical substance at the blood-sweat interface can vary with the movement of the subject. Certain aspects of the invention are based on the idea of ​​detecting in a quantitative and dynamic manner one or more of the above-mentioned chemical substances produced by the cardiovascular system, for example during exercise testing or medical monitoring of the subject.

[0010] To this end, according to one embodiment, the present invention provides a detection device for detecting at least one chemical substance in a biological fluid such as sweat of a subject such as a human or an animal, wherein the at least one chemical substance is selected from nitric oxide NO, nitrite ion NO2 - and hydrogen peroxide H2O2, and preferably nitric oxide NO, the device comprising:

[0011] a collection element for placement on a subject's epidermis at a study area;

[0012] and a detection device comprising at least one fluid pathway and at least one electrochemical sensor comprising electrodes disposed in the fluid pathway, the at least one fluid pathway being coupled to a collecting element to direct at least one sweat flow originating from an investigation area, the electrochemical sensor being configured to generate a signal representative of a concentration of the at least one chemical substance in the sweat flow.

[0013] The term "chemical substance in a biological fluid" means that the chemical substance, in particular nitric oxide, is dissolved in the biological fluid.

[0014] The term "epidermis" refers to the surface layer of plant tissue that forms a protective covering for aerial parts of plants or the surface layer of the skin of humans and animals. For example, biological fluids are exudates in plants or sweat in humans and animals.

[0015] According to some embodiments, a device of this type may include one or more of the following features.

[0016] According to one embodiment, the or each or at least one electrochemical sensor is configured to additionally generate a signal representative of the flow rate of the sweat flow or the volumetric flow of sweat.

[0017] According to one embodiment, the detection device is configured to generate a signal representing the instantaneous generation of the at least one chemical substance in the investigation zone based on a signal representing the concentration of the at least one chemical substance and a signal representing the flow rate of the sweat flow.

[0018] The term "transiently generated" refers to measurements made over a very short period of time compared to the characteristic time of a change in a subject's physiological response. For human subjects, this characteristic time is usually about one to several minutes.

[0019] According to one embodiment, the electrochemical sensor or each electrochemical sensor or at least one electrochemical sensor comprises at least one working electrode and a counter electrode arranged in the fluid path, and the electrochemical sensor is configured to generate a signal representing the concentration of the at least one chemical substance by electrical measurement, in particular amperometric measurement, between the at least one working electrode and the counter electrode.

[0020] According to one embodiment, the electrochemical sensor or each electrochemical sensor or at least one electrochemical sensor comprises an upstream working electrode and a downstream working electrode spaced apart in a fluid path in the direction of flow of the sweat flow, the electrochemical sensor being configured to generate a signal representing the flow rate by measuring the delay between a change in current or potential in the upstream working electrode and a change in current or potential in the downstream working electrode.

[0021] According to one embodiment, the upstream working electrode is spaced apart from the downstream working electrode by a distance L between 0.05 mm and 1 cm.

[0022] According to one embodiment, the electrochemical sensor or at least one of the electrochemical sensors is configured to polarize an electrode to a potential for oxidizing nitric oxide, and the detection device comprises a filter configured to filter the sweat flow reaching the electrochemical sensor in particular to eliminate hydrogen peroxide.

[0023] This type of filter can be produced in different ways, for example using a membrane made of polytetrafluoroethylene (PTFE) or a membrane of the eugenol type, which can be arranged in the fluid path. According to one embodiment, the filter comprises a layer of eugenol (4-allyl-2-methoxyphenol) deposited on at least one electrode of the electrochemical sensor.

[0024] This configuration specifically for measuring NO, based on a detection electrode modified with a filter of this type, generates a signal representing the concentration of nitric oxide NO directly from the current measurement signal.

[0025] According to the embodiments described below, the detection device is produced in such a way that it can detect two or three of the above-mentioned chemical substances simultaneously or sequentially using one or more electrochemical sensors.

[0026] According to one embodiment, the at least one chemical substance comprises nitric oxide NO and nitrite ions NO2 - According to one embodiment, the at least one chemical substance comprises nitric oxide NO and hydrogen peroxide H2O2. According to one embodiment, the at least one chemical substance comprises nitric oxide NO, nitrite ions NO2 - and hydrogen peroxide H2O2.

[0027] According to one embodiment for achieving sequential detection, the electrochemical sensor or each electrochemical sensor or at least one electrochemical sensor is configured to detect several chemical substances sequentially during a plurality of measurement steps, the electrochemical sensor is configured to polarize the electrode to a potential for oxidizing hydrogen peroxide H2O2 during a first step and to polarize the electrode to a potential for oxidizing nitric oxide NO during a second step, and the detection device is configured to generate a signal representing the concentration of nitric oxide NO based on a first current measurement signal obtained during the first step and a second current measurement signal obtained during the second step.

[0028] Advantageously, in this case, the or each or at least one electrochemical sensor is configured to polarize the electrode during the third step to oxidize nitrite ions NO2 - The detection device is configured to generate a signal representing nitrite ions NO2 based on the first current measurement signal, the second current measurement signal and the third current measurement signal obtained during the third step. - Concentration signal.

[0029] According to one embodiment, the detection device is produced in such a way that the three above-mentioned chemical substances can be detected sequentially with a single electrochemical sensor during multiple steps of a time sequence of measurements. According to this embodiment, during a given sequence, the electrochemical sensor is configured to sequentially polarize a platinum-plated platinum electrode (coated with platinum black) to an electrochemical potential for oxidation of hydrogen peroxide H2O2 during a first time step of a few seconds (e.g. 5 seconds), then for oxidation of nitric oxide NO during a second time step of the same duration, and optionally for oxidation of nitrite ions NO2 during a third time step of the same duration. -The detection device is configured to generate a signal representing the concentration of nitric oxide (NO) based on a first current measurement signal obtained during the first step and a second current measurement signal obtained during the second step. This sequence is repeated as often as possible throughout the exercise test. - concentrations), solving a series of three equations (the current measured sequentially on an electrode polarized to each potential in a sequence) provides the value of each of the three concentrations at the moment each sequence is generated from the three measurements.

[0030] According to another embodiment of realizing simultaneous detection, the detection device comprises:

[0031] a first fluid pathway and a first electrochemical sensor including an electrode disposed in the first fluid pathway, the first fluid pathway being coupled to the collecting element to direct a first flow of sweat originating from the investigation zone, the first electrochemical sensor being configured to polarize the electrode to a potential for oxidizing hydrogen peroxide H2O2, and

[0032] a second fluid pathway and a second electrochemical sensor including an electrode disposed in the second fluid pathway, the second fluid pathway being coupled to the collecting element to direct a second sweat flow originating from the investigation area, the second electrochemical sensor being configured to polarize the electrode to a potential for oxidizing nitric oxide (NO),

[0033] And the detection device is configured to generate a signal representing the concentration of nitric oxide (NO) based on a first current measurement signal generated by the first electrochemical sensor and a second current measurement signal generated by the second electrochemical sensor.

[0034] Advantageously, in this case, the detection device additionally comprises:

[0035] a third fluid pathway and a third electrochemical sensor including electrodes disposed in the third fluid pathway, the third fluid pathway coupled to the collecting element to direct a third sweat flow originating from the investigation area, the third electrochemical sensor configured to polarize the electrodes to oxidize nitrite ions NO2 - The detection device is configured to generate a signal representing nitrite ions NO2 based on the first current measurement signal, the second current measurement signal and the third current measurement signal generated by the third electrochemical sensor. - Concentration signal.

[0036] According to this embodiment, the detection device comprises three parallel microfluidic pathways supplied in parallel by the same sweat collecting element.

[0037] According to one embodiment, the detection device additionally comprises:

[0038] Another fluid pathway, such as a fourth fluid pathway, is coupled to the collection element to direct another sweat flow, such as a fourth sweat flow, originating from the investigation area, and another electrochemical sensor, such as a fourth electrochemical sensor, includes an electrode disposed in the fourth fluid pathway.

[0039] According to this embodiment, the further or fourth electrochemical sensor is configured to polarize an electrode to a potential for oxidizing nitric oxide, and the fourth fluid pathway includes a filter configured to filter the sweat flow reaching the fourth electrochemical sensor to specifically eliminate hydrogen peroxide.

[0040] According to one embodiment, the collecting element comprises a fibrous body to conduct biological fluids, in particular sweat, from the investigation area by capillary action. A fibrous body of this type can be a woven or nonwoven material.

[0041] According to one embodiment, the device additionally comprises an envelope surrounding the collection element in such a manner as to form an impermeable barrier around the investigation area, such as by contact with the epidermis of said subject.

[0042] Due to these characteristics, gases, liquids and microorganisms such as bacteria or viruses located outside the study area cannot enter the study area. The impermeability of the contact between the envelope and the epidermis ensures that the chemical substances detected originate from the biological fluids generated in the study area and not from external fluids.

[0043] According to one embodiment, the apparatus additionally comprises a wired or wireless communication device configured to transmit one or more measurement signals generated by the detection device to a post-processing device.

[0044] According to one embodiment, the or each or at least one fluid passage is formed in an insulating support and the electrodes of the or each or at least one electrochemical sensor consist of a metal deposit on the insulating support. This type of insulating support may be made of a material selected from elastomers such as polydimethylsiloxane (PDMS), polyimide, epoxy resin and parylene.

[0045] According to one embodiment, the metal deposit is selected from the group consisting of silver (Ag), gold (Au), platinum (Pt) and platinum black. According to one embodiment, the electrochemical sensor comprises a reference electrode made of silver chloride (AgCl).

[0046] In an interesting embodiment, the electrode consists of a graphene deposit doped with silver (Ag) or gold (Au) nanoparticles functionalized with a binder for nitric oxide, in particular guanylate cyclase or porphyrins.

[0047] According to one embodiment, the device is configured to perform and periodically transmit measurements, for example at a customizable frequency or at a frequency that depends on the activity state detected by the device. As an example, the device may include a gyroscope module and / or an accelerometer to detect the activity state of the subject. Thus, the activity state of the subject may be detected during sweat analysis to assist in analyzing the correlation between the activity state of the subject and the production of the chemical being analyzed.

[0048] According to one embodiment, the device comprises a geo-location module.

[0049] According to one embodiment, the present invention also provides a method for determining the production of at least one chemical substance by a human or animal subject, wherein the at least one chemical substance is selected from the group consisting of nitric oxide NO, nitrite ions NO2 - and hydrogen peroxide H2O2, and preferably nitric oxide NO, the method comprising:

[0050] selecting a study area of ​​the subject's epidermis,

[0051] applying the device for a period of time required to generate a signal representative of the concentration of the at least one chemical substance in the sweat flow and, if appropriate, the flow rate of the sweat flow, and

[0052] The measurement value of the production of the at least one chemical substance by the subject is determined from the signal representing the concentration of the at least one chemical substance in the sweat flow and, if appropriate, the flow rate of the sweat flow.

[0053] Measurements of one or more of the above-described chemical substances produced by a subject can be used for a variety of applications, such as to assess distress in the subject's vascular tissue from these measurements or to assess the subject's cardiovascular capacity from these measurements.

[0054] Other possible applications are diagnosis, healthcare and monitoring of diseases such as cardiovascular diseases, neurodegenerative diseases, pulmonary hypertension, cancer, hypercholesterolemia, diabetes, systemic endothelial dysfunction, atherosclerosis, thrombotic or ischemic diseases, platelet aggregation inhibition dysfunction or leukocyte adhesion defects or cell proliferation dysfunction of smooth muscle fiber cells, bronchial inflammation, asthma, Alzheimer's disease.

[0055] Other possible applications are monitoring growth and / or muscle distress in individuals, such as individuals undergoing physical training, preventing damage due to overtraining and / or enhancing the subject's muscle performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to better understand the subject matter of the invention, the embodiments shown in the accompanying drawings will now be described by way of purely illustrative and non-limiting examples. In the drawings:

[0057] [ Figure 1 ] Figure 1 A schematic diagram is shown of the back of a subject on which a device according to one embodiment is placed.

[0058] [ Figure 2 ] Figure 2 is a perspective view showing a detection device according to an embodiment.

[0059] [ Figure 3 ] Figure 3 Show Figure 2 Exploded view of the device.

[0060] [ Figure 4 ] Figure 4 According to the first embodiment, it can be used Figure 2 Schematic cross-sectional view of a collecting element in a device.

[0061] [ Figure 5 ] Figure 5 According to the second embodiment, it can be used Figure 2 Schematic cross-sectional view of a collecting element in a device.

[0062] [ Figure 6 ] Figure 6 It is available for Figure 2 Functional schematic diagram of the fluid pathways in the device.

[0063] [ Figure 7 ] Figure 7 It is available for Figure 6 Schematic perspective view of an electrochemical sensor in a fluid pathway.

[0064] [ Figure 8 ] Figure 8 This means that Figure 7 Figure 2. Time diagram of the detection method used together with the electrochemical sensor.

[0065] [ Fig. 9 ] Fig. 9 Yes, you can Figure 2 Functional schematic diagram of the detection equipment used in the device.

[0066] [ Fig.10 ] Fig.10 It means that you can use Figure 2 A diagram of the steps of a method implemented by an apparatus.

[0067] [ Fig.11 ] Fig.11 It means that you can use Figure 2 A diagram of the measurement results obtained with the device. DETAILED DESCRIPTION

[0068] Figure 1 A detection device 1 is shown, which is arranged on the skin of a human subject 2, for example, on the back of the subject, for quantitatively measuring chemical substances dissolved in sweat, including nitric oxide dissolved in sweat, and optionally nitrite ions and hydrogen peroxide.

[0069] refer to Figure 2 As an example, the detection device 1 is shown in the form of a tight shell, which includes a base plate 3 and an upper cover 7 made of a biocompatible flexible material, the base plate 3 is preferably self-adhesive and can be directly positioned on the skin of the subject, and the upper cover 7 is preferably waterproof, such as silicone, which surrounds the other components of the detection device 1.

[0070] Reference now Figure 3 , the central part of the substrate 3 comprises a circular recess 4 which delimits the area of ​​interest on the subject's skin and whose diameter may be, for example, a few millimeters to a few centimeters. The circular recess 4 contains a collecting element 5 which is then positioned directly on the subject's skin 2 when the device is in operation. The recess 4 may have other shapes, such as elliptical, triangular, rectangular, square, polygonal or other shapes.

[0071] As an 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 carrying one or more fluid pathways and one or more sensors, as described below. The support 6 can be a rigid or flexible support, for example made of polyimide. The collecting element 5 implements the function of bringing the sweat produced in the study area to the fluid pathway of the support 5, so that one or more chemical substances can be detected with the aid of the sensor. To this end, the collecting element 5 can be arranged in a variety of ways.

[0072] exist Figure 4 In the example of , the collecting element 5 has a portion in contact with the skin 2 on one side of the support 6 and a portion to cover the upper surface of the support 6. In other words, the collecting element is installed between the skin and the support 6. On the upper surface of the support 6, the collecting element 5 is connected to the fluid passage where the sensor is provided.

[0073] exist Figure 5In the example of the embodiment of the present invention, the collecting element 5 will form a sandwich structure around the support 6. As a result, a portion of the collecting element 5 arranged below the support 6 is arranged against the skin. A portion of the collecting element 5 extending the portion arranged on the skin is folded over the support 6 and will therefore cover the support 6. On the upper surface of the support 6, the collecting element 5 is connected to the fluid passage in which the sensor is arranged.

[0074] refer to Figure 6 , the support 6 carries a microfluidic system 8, which is supplied via the collecting element 5 by capillary action. The sweat is collected by the part of the collecting element 5 placed in contact with the skin 2 and then transferred by capillary action so that it moves into the microfluidic system 8. The microfluidic system 8 may include one or more fluid pathways, namely four parallel fluid pathways 9 in the example shown. The fluid pathways 9 are, for example, formed in the thickness of the support 6 and separated by partitions 11. The fluid pathways 9 can thus form separate channels in which sweat can move independently. The number of fluid pathways 9 can be higher or lower than the number shown in the figure.

[0075] Each fluid pathway 9 is provided with a sensor 10A, 10B, 10C or 10D. Arrow 12 shows the direction of sweat flow in fluid pathway 9. Preferably, fluid pathway 9 is drained into a drain reservoir containing analysis fluid to prevent reaction products from electrolysis from contacting the subject's skin again.

[0076] The sensors 10A, 10B, 10C and 10D arranged in the fluid path 9 for sweat analysis are preferably electrochemical sensors. The working principle of an electrochemical sensor is the electrolysis of a solution present in the fluid path 9 between a working electrode and a counter electrode. Sensors of this type can be produced in a variety of ways, in particular in a miniaturized way with millimeter-scale dimensions.

[0077] Now refer to Figure 6 Several examples of electrochemical sensor implementations are described.

[0078] Example

[0079] Example 1

[0080] The sensor 10A is used to detect hydrogen peroxide. Therefore, it has a potential difference E equal to the oxidation potential of hydrogen peroxide. H2O2 The sensor 10B is intended for the detection of nitric oxide. Therefore, it is operated at a potential difference E equal to the oxidation potential of nitric oxide. NO The sensor 10C is intended for detecting nitrite ions. Therefore, it is operated at a potential difference E equal to the oxidation potential of nitrite ions. NO2- run.

[0081] The sensors 10A, 10B, 10C perform instantaneous intensity measurements of the Faradaic current associated with the electrochemical oxidation of the chemical species, denoted as i oxdn The sensors 10a, 10B, 10C can therefore be used to detect and quantify the instantaneous concentration of the above-mentioned chemical substances.

[0082] Each of the three chemical species mentioned above can be detected by amperometric measurement with the aid of microelectrodes. They consist, for example, of a platinum strip covered with a thin layer of platinum salt anions Pt(Cl)6 obtained by electrochemical reduction in an aqueous medium. 4- A deposited platinum black thin layer, such as a micron-sized platinum black thin layer.

[0083] These three chemicals (NO, NO2 - and H2O2) can be distinguished by the fact that their oxidation potentials at these electrodes are clearly separated, occurring in the following order: E H2O2 <E NO <E NO2 -. However, the Faradaic current is additive. Therefore, the current measured at the oxidation potential of each chemical species is added to the base current associated with the oxidation of that chemical species and with the oxidation of all chemical species with lower oxidation potentials.

[0084] Therefore, only the substance H2O2 can be oxidized at the oxidation potential EH2O2. The substances H2O2 and NO can be oxidized at the oxidation potential E NO These three substances can be oxidized at the oxidation potential E NO2 - is oxidized. The currents measured by sensors 10A to 10C are respectively denoted as i oxdn (EH2O2),i oxdn (E NO ) and i oxdn (E NO2 -), so the following equation is satisfied:

[0085] i oxdn (E H2O2 )=a1 i H2O2

[0086] i oxdn (E NO )=a2 i H2O2 +a3 i NO

[0087] i oxdn (E NO2 -)=a4 i H2O2 +a5 i NO +a6 i NO2 -

[0088] The coefficients a1 to a6 represent the calibration constants of the sensor, which can be measured experimentally.

[0089] So, after doing a simple subtraction using an electronic circuit, we get the following result:

[0090] i H2O2 =(1 / a1)i oxdn (E H2O2 )

[0091] i NO =(1 / a3)i oxdn (E NO )-(a2 / a1).(1 / a3)i oxdn (E H2O2 )

[0092] i NO2 - = (1 / a6)i oxdn (E NO2 -)-(a4 / a6)i H2O2 –(a5 / a6)i NO

[0093] At any time t, the instantaneous intensity i of the oxidation Faraday current of each chemical species S is S (t) and its concentration C in the fluid volume above the electrode that detects it S (t). The proportionality factor depends on the shape factor, denoted γ, which is a function of the sensor geometry, and on the Faraday constant per mole of chemical consumed, denoted n S ,Right now:

[0094] n H2O2 =n NO2 - = 2, and n NO =1

[0095] It should be noted that F stands for Faraday, which is 96,500 coulombs, the value of the charge carried by one mole of electrons.

[0096] The form factor γ is a constant imposed by the geometry of the electrochemical device, which can be estimated theoretically and measured experimentally by calibration. For simplicity, the following three sensors 10A to 10C are considered to have the same geometry, so the form factor γ is the same for all sensors.

[0097] As a result, the concentration of the chemical substance can be obtained from the current measured by the sensors 10A to 10C by means of the following expression, in which the time variable t is defined:

[0098] C H2O2 (t) = i oxdn (E H2O2,t) / (2Fγ)

[0099] C NO (t) = [i oxdn (E NO ,t)-i oxdn (E H2O2 ,t)] / (Fγ)

[0100] C NO2 -(t)=[i oxdn (E NO2 -,t)-i oxdn (E NO ,t)] / (2Fγ)

[0101] In Example 1, the three sensors 10A to 10C can therefore be operated in parallel, each with a constant oxidation potential, namely E H2O2 、E NO and E NO2 -.

[0102] According to a variant embodiment, only NO and NO2 are detected. - This embodiment is particularly advantageous when the measurement result of H2O2 is not significant and does not affect the result of the desired target. Then it is considered that the above concentration C H2O2 (t) is uniformly zero, that is, C H2O2 (t) = 0. Therefore, a series of equations are simplified.

[0103] Example 2

[0104] In Embodiment 2, a single fluid passage 9 and a single sensor 10A are used; the others may be omitted.

[0105] In this case, the sensor 10A operates sequentially to detect the above chemical substances during three consecutive steps. The oxidation potential is thus switched between three constant potential stages respectively equal to the above three oxidation potentials, for example according to E H2O2 →E NO →E NO2- →E H2O2 →E NO →E NO2- →The order is switched periodically.

[0106] In this case, each oxidation potential is maintained for a very long time compared to the time constant of the working electrode, for example a few milliseconds for microelectrodes used in microfluidic channels, and the current is measured at the end of each constant potential stage.

[0107] The remaining measurement signals can be processed using the same equations as in Example 1.

[0108] Example 3

[0109] Because nitric oxide is a small molecule that is both hydrophilic and lipophilic, it can easily pass through thin layers of organic polymers, unlike the other two substances, H2O2 and NO2. - It can therefore be detected in isolation by means of an electrochemical sensor protected by a filter layer of this type, for example using a working electrode made of platinum coated with platinum covered with a thin layer of eugenol (4-allyl-2-methoxyphenol) deposited by electropolymerization.

[0110] In embodiment 3, the sensor 10D is therefore protected by a filter layer, which is schematically indicated by the number 19. It is thus possible to be independent of the chemical substances H2O2 and NO2 - The instantaneous concentration of nitric oxide was measured according to the following expression:

[0111] C NO (t) = [i oxdn (E NO ,t)] 丁香酚 / (Fγ).

[0112] where i oxdn (E NO ,t)] 丁香酚 Refers to the current measured by sensor 10D.

[0113] The other sensors 10A to 10C and the other fluid passages 9 may be omitted. Thus, when only the concentration of NO is required, the method may be advantageously used with a single sensor.

[0114] In a variant, the filter 19 can be arranged at another position 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-) or hydrogen peroxide (H2O2).

[0115] Example 4

[0116] In this case, the sensor 10D of Example 3 is combined with the sensors 10A to 10C of Example 1 or with the sensor 10A of Example 2. This configuration can be used to obtain two measurements independent of the concentration of dissolved nitric oxide, thereby checking the consistency of the measured values, in particular by verifying that the sensor is not drifting, for example related to partial passivation of the surface of one of the electrodes.

[0117] In this case, the electrochemical electronic control device 40 ( Fig. 9) is preferably configured to compare two measurements of nitric oxide concentration and to issue an alarm when the comparison meets predetermined criteria, for example if it exceeds a predetermined threshold.

[0118] In the above-mentioned Examples 1 to 4, the measured instantaneous Faraday current can be used to measure the concentration of the chemical substance in the analytical solution. Therefore, in a static system, the current intensity is sufficient to prove the generation of the detected substance.

[0119] However, when the detection device 1 is applied to physiological systems that are dynamic in nature, it is also desirable to be able to obtain the quantitative dynamics of each chemical produced by the cardiovascular system, for example during exercise testing or medical monitoring. Under dynamic conditions, in order to obtain the instantaneous amount of a chemical produced in a short period of time (expressed as Δt(t)), expressed as ΔQ(t), it is desirable to simultaneously know the average concentration C of the chemical. S (t) and the chemical substance and volume flow rate of the fluid being analyzed, namely:

[0120] d(t)=(ΔV / Δt)

[0121] where ΔV represents the volume scanned during the time interval Δt. Therefore, the production flow rate of chemical substance S at time t (denoted as PS(t)) is given by:

[0122] P S (t) = [ΔQ / Δt] (t) = C S (t).d(t)

[0123] The average concentration C S (t) obtained from the average intensity of the electrochemical oxidation current measured between time t and t+Δt.

[0124] In the context of the envisaged dynamic application, it is therefore desirable that the detection device 1 measures the average intensity i of the Faradaic current associated with the electrochemical oxidation of the monitored chemical substance simultaneously and every time t (for example once per minute) with the accuracy required for monitoring the patient's physiological state over time. av (t) and the value of the volume flow rate d(t) of sweat in the corresponding fluid pathway at time t.

[0125] Figure 7An embodiment of an electrochemical sensor 10 that can meet this dual requirement in an integrated manner is shown. The electrochemical sensor 10 includes at least one strip microelectrode forming a working electrode 20 or a working electrode pair 20, 23. This type of strip microelectrode can be made of platinum plated with platinum (platinum black), which can be covered or not covered with a micron-sized electropolymerized eugenol layer. This type of strip microelectrode can be implanted by micromachining, for example by CVD and / or photolithography. The strip microelectrode or these strip microelectrodes can be used for electrochemical oxidation of selected chemicals.

[0126] also, Figure 7 The fluid passage 9 is equipped with a reference electrode 21, which is produced, for example, in the form of an Ag / AgCl microstrip and is placed upstream of the working electrode 20 or the working electrode pair 20, 23. Finally, the fluid passage 9 is equipped with a counter electrode 30 made of platinum-plated platinum, which is placed downstream of the working electrode 20 or the working electrode pair 20, 23. Figure 7 Schematic diagram of the function, the surface of the counter electrode 30 is actually two to three times larger than that of the other electrodes.

[0127] The entire fluid path 9 with electrodes 20, 21, 23, 30 is immersed in the sweat layer, not shown, thus constituting a microfluidic electrochemical cell with three or four electrodes. Each of the electrodes 20, 21, 23, 30 is connected to an electrochemical electronic control device 40 ( Fig. 9 ).

[0128] This embodiment of the electrochemical sensor 10 may be used in one or more of the aforementioned fluid passages 9 .

[0129] In order to measure the volume flow rate d(t), the electrochemical sensor 10 must include a working electrode pair 20, 23. The solution described here is simple and easy to industrialize, because it has no moving parts and it does not claim to be fluid dynamics. It does not require any intervention aimed at regulating the fluid flow rate, but is applicable to any reasonable physiological flow rate.

[0130] Two working electrodes, for example two platinum-coated platinum strips, can be used as working microelectrodes, are electrically independent, and are spaced apart by a distance L along the fluid analysis path in the fluid passage 9. The two working electrodes 20 and 23 are, for example, mounted at the bottom of a linear channel, whose cross section has a constant area A.

[0131] according to Figure 8The method shown uses a working electrode 23 located downstream, and the method includes two steps. Curve 81 represents the potential applied to the working electrode 20 as a function of time. Curve 82 represents the potential applied to the working electrode 23 as a function of time. The potential indicated as "0" on Figures 81 and 82 actually represents the disconnection (open circuit) of the corresponding electrode. Curve 83 represents the Faraday current measured at the working electrode 20 as a function of time. Curve 84 represents the Faraday current measured at the working electrode 23 as a function of time.

[0132] During the first step, which is performed some time before time t0, the potential E applied to the working electrode 20 is oxdn The current i is sufficient to allow oxidation of the target chemical species, while the downstream working electrode 23 is disconnected. The working electrode 20 positioned upstream can then be used to continuously record the transient electrochemical current i oxdn (t), based on any calculations that may be required as above, then shows the concentration C(t) of the target chemical in the analyzed fluid.

[0133] During the second step, which is performed within a certain time range starting from time t0, the working electrode 20 is disconnected and the potential E oxdn Applied to the downstream working electrode 23.

[0134] At time t0, the sweat flow passing over the working electrode 23 has been electrolyzed (completely or partially) during its passage over the upstream working electrode 20 in such a way that the concentration of the target chemical is zero here, or at least much lower than before it entered the electrochemical device. The current intensity i detected by the working electrode 23 oxdn (curve 84) is therefore zero (or at least well below the current i detected at the working electrode 20 before time t0). oxdn ).

[0135] At time t0+Δt, the working electrode 23 starts analyzing the non-electrolytic solution, and the current intensity i oxdn becomes the same order of magnitude as the current intensity detected by the working electrode 20 before time t0. The increase in current (given by Figure 8 The duration Δt is the delay between this growth and the moment t0 when the working electrode 20 is disconnected, representing the time required for the sweat flow to be transmitted between the two working electrodes 20 and 23. The duration Δt is determined by Figure 8 The double-headed arrow at the bottom indicates that Figure 8 It is assumed that the electrolysis of the target chemical is complete when the working electrode 20 is connected. When this electrolysis is only partial, the same measurement principle is applicable.

[0136] Therefore, the flow velocity v(t) and rate d(t) can be estimated as follows:

[0137] v(t)=L / Δt

[0138] d(t)=Av(t)

[0139] The potential E applied to the working electrode 23 oxdn It is sufficient to oxidize the target chemical while the working electrode 20 is disconnected. Thus, the concentration can be measured optionally using the working electrode 23 for a period of time. The second step ends with disconnecting the working electrode 23 at time t1. The working electrode 20 can then be reconnected, and the method can be repeated as many times as necessary to evaluate the rate d(t) at successive times.

[0140] The distance L between the two working electrodes 20 and 23 is preferably small enough, for example, about 1 mm, so that the change of the patient's physiological response within the period Δt can be ignored.

[0141] The above method for measuring the rate can be used simultaneously in all parallel fluid paths. However, if these circuits are configured and powered in a similar manner, it may be sufficient to measure the rate once. In this case, the above method for measuring the rate can be used in a single fluid path 9. In addition, this method for measuring the rate can be combined with sensors from various embodiments 1 to 4 described above.

[0142] The above-described method for detecting concentration and rate can be implemented in an automated manner with the aid of an electronic control device 40 , which is preferably integrated into the detection apparatus 1 .

[0143] refer to Fig. 9 , an embodiment of the electronic control device 40 which can be integrated into the detection device 1 will now be described, for example in the form of Figure 3 The form of the electronic circuit board 13 is shown.

[0144] The or each electrochemical sensor 10 is connected to an analogue to digital converter 14 which in turn powers a processor 15. The processor 15 is for example programmed to carry out the methods for detecting the concentrations and rates described above.

[0145] An energy source 16 such as a battery powers the electronic control device 40. A communication module 17, which may be wired or wireless, may also be provided to communicate measurements of the concentration, rate and / or flow of one or each target chemical material quantity to a storage or post-processing device.

[0146] Fig.10 A method that may be performed by processor 15 in one embodiment is shown.

[0147] In step 31 , the instantaneous concentration Cs(t) of the chemical species S is determined by electrochemical measurement.

[0148] In step 32, the volumetric velocity d(t) in the corresponding fluid path is determined.

[0149] In step 33, the flow rate of the material amount of the chemical substance under consideration is determined based on Cs(t) and d(t), for example:

[0150] P S (t) = C S (t).d(t)

[0151] Fig.11 is a diagram showing a measurement signal of a material flow as a function of time, which can be obtained with the detection device 1 , for example during an exercise test carried out on a subject for the substance NO.

[0152] The electronic control device 40 optionally includes other functional modules, such as a gyroscope module and / or an accelerometer module for detecting the orientation and movement of the subject and the subject's activity level, and a temperature sensor for measuring the temperature of the subject's epidermis. For the correlation between temperature and vascular dilation, it is useful to know the temperature of the skin.

[0153] Some elements of the detection device 1, in particular the electronic control device 40, can be produced in different forms, in a single or distributed manner using physical and / or software components. The physical components that can be used 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 exhaustive.

[0154] Although the present invention has been described in conjunction with several specific embodiments, it is apparent that the present invention is in no way limited to these specific embodiments and encompasses all technical equivalents of the described means and combinations thereof if they fall within the scope of the present invention.

[0155] Use of the verbs "comprise", "comprises" or "includes" and their conjugations does not exclude the presence of other elements or steps than those defined in a claim.

[0156] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

Claims

1. A detection device (1) for detecting at least one chemical substance dissolved in sweat of a human or animal subject, the at least one dissolved chemical substance comprising nitric oxide (NO), the device comprising: a collecting element (5) for placement on an investigation area of ​​the epidermis (2) of a subject, and A detection device, comprising at least one fluid passage and at least one electrochemical sensor comprising electrodes arranged in the fluid passage, the at least one fluid passage being coupled to the collecting element to guide at least one sweat flow originating from the study area, wherein the electrodes comprise at least one working electrode (20) and a counter electrode (30), the electrochemical sensor being configured to generate at least a signal representing the concentration of nitric oxide (NO) in the sweat flow and a signal representing the flow rate of the sweat flow in an integrated manner, wherein the electrochemical sensor is configured to generate the signal representing the concentration of nitric oxide (NO) by electrical measurement between the at least one working electrode and the counter electrode, The at least one working electrode (20) comprises an upstream working electrode (20) and a downstream working electrode (23) spaced apart in the flow direction (12) of the sweat flow in the fluid pathway, and the electrochemical sensor is configured to generate a signal representing the flow rate by measuring a delay (Δt) between a current change in the upstream working electrode and a current change in the downstream working electrode.

2. Apparatus according to claim 1, wherein the detection device is configured to generate a signal representing the instantaneous production of nitric oxide (NO) in the investigation zone based on a signal representing the concentration of nitric oxide (NO) and a signal representing the flow rate of the sweat flow.

3. The device according to claim 1 or claim 2, wherein the electrochemical sensor is configured to generate the signal representing the concentration of nitric oxide (NO) by amperometric measurement between the at least one working electrode and the counter electrode.

4. The apparatus according to any one of claims 1 to 2, wherein the electrochemical sensor is configured to polarize an electrode to a potential for oxidizing nitric oxide (NO), and wherein the detection device comprises a filter (19) configured to filter the sweat flow reaching the electrochemical sensor to eliminate hydrogen peroxide.

5. The device according to any one of claims 1 to 2, wherein the at least one chemical substance additionally comprises nitrite ions NO2 - and / or hydrogen peroxide H2O2.

6. An apparatus according to claim 5, wherein the electrochemical sensor is configured to continuously detect several chemical substances during multiple measurement steps, the electrochemical sensor is configured to polarize the electrode to a potential for oxidizing hydrogen peroxide H2O2 during a first step and to polarize the electrode to a potential for oxidizing nitric oxide NO during a second step, and wherein the detection device is configured to generate the signal representing the concentration of nitric oxide NO based on a first current measurement signal obtained during the first step and a second current measurement signal obtained during the second step.

7. The device according to claim 6, wherein the electrochemical sensor is configured to polarize the electrode during the third step to oxidize nitrite ions NO2 - and wherein the detection device is configured to generate a signal representing nitrite ions NO2 based on the first current measurement signal and the second current measurement signal and a third current measurement signal obtained during the third step. - Concentration signal.

8. The apparatus according to claim 5, wherein the detection device comprises: a first fluid pathway and a first electrochemical sensor including an electrode disposed in the first fluid pathway, the first fluid pathway being coupled to the collecting element to direct a first sweat flow originating from the investigation zone, the first electrochemical sensor being configured to polarize the electrode to a potential for oxidizing hydrogen peroxide H2O2, and a second fluid pathway and a second electrochemical sensor including an electrode disposed in the second fluid pathway, the second fluid pathway being coupled to the collecting element to direct a second sweat flow originating from the investigation zone, the second electrochemical sensor being configured to polarize the electrode to a potential for oxidizing nitric oxide (NO), The detection device is configured to generate a signal representing the concentration of nitric oxide (NO) based on a first current measurement signal generated by the first electrochemical sensor and a second current measurement signal generated by the second electrochemical sensor.

9. The apparatus according to claim 8, wherein the detection device (5, 6) additionally comprises: A third fluid pathway and a third electrochemical sensor including electrodes disposed in the third fluid pathway, the third fluid pathway coupled to the collecting element to direct a third sweat flow originating from the investigation area, the third electrochemical sensor configured to polarize the electrodes to oxidize nitrite ions NO2 - The potential of the nitrite ion NO2 is generated based on the first current measurement signal, the second current measurement signal and the third current measurement signal generated by the third electrochemical sensor. - Concentration signal.

10. The apparatus according to claim 8, wherein the detection device additionally comprises: A fourth fluid pathway and a fourth electrochemical sensor comprising electrodes disposed in the fourth fluid pathway, the fourth fluid pathway being coupled to the collecting element to direct a fourth sweat flow originating from the investigation area, wherein the fourth electrochemical sensor is configured to polarize the electrodes to a potential for oxidizing nitric oxide (NO), and wherein the fourth fluid pathway comprises a filter (19) configured to filter the sweat flow reaching the fourth electrochemical sensor to eliminate hydrogen peroxide (H2O2).

11. The device according to claim 10, wherein the filter (19) comprises a layer of eugenol (4-allyl-2-methoxyphenol) deposited on at least one electrode of the electrochemical sensor.

12. The apparatus of claim 11, wherein the detection device is configured to compare the nitric oxide concentration measurement value obtained using the fourth electrochemical sensor with the nitric oxide concentration measurement values ​​obtained using the first electrochemical sensor and the second electrochemical sensor, and to issue an alarm when the comparison result meets a predetermined criterion.

13. The device according to any one of claims 1 to 2, wherein the collecting element (5) comprises a fibrous body for conducting sweat by capillary action.

14. Device according to any one of claims 1 to 2, additionally comprising an envelope (7) surrounding the collecting element (5) in such a way as to form an impermeable barrier around the investigation area by contact with the epidermis (2) of the subject.

15. The apparatus according to any one of claims 1 to 2, further comprising a wired or wireless communication device (17), wherein the wired or wireless communication device (17) is configured to transmit one or more measurement signals generated by the detection device to a storage or post-processing device.

16. The device according to any one of claims 1 to 2, wherein the fluid passage (9) is formed in an insulating support (6) and wherein the electrodes of the electrochemical sensor are constituted by metal deposits on the insulating support, the metal deposits being selected from the group consisting of silver, gold, platinum and platinum black.

17. The device according to any one of claims 1 to 2, additionally comprising a gyroscope module and / or at least one accelerometer for detecting the activity state of the subject.

18. The device according to any one of claims 1 to 2, additionally comprising a temperature sensor for measuring the skin temperature of a subject.

19. A method for determining the production of at least one chemical substance in a human or animal subject (P S ), wherein the at least one chemical substance comprises nitric oxide (NO), the method comprising: selecting a study area of ​​the subject's epidermis, the time required for the continuous generation of a signal representing the concentration of nitric oxide NO in the sweat flow and a signal representing the flow rate of the sweat flow using the device (1) according to any one of claims 1 to 2, and The generation of (P) by the subject is determined by a signal representing the concentration of nitric oxide (NO) in the sweat flow and a signal representing the flow rate of the sweat flow. S )Measurement value of nitric oxide NO.

20. The method according to claim 19, additionally comprising: Distress in the subject's vascular tissue is assessed by measuring the production of nitric oxide (NO).

21. The method of claim 19, additionally comprising: The cardiovascular capacity of the subject is assessed by measuring the production of nitric oxide (NO).

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