Single-use patch for measuring oxidative stress in an individual

Abstract

The invention relates to the field of single-use patches. More particularly, the invention relates to a single-use device for measuring the total antioxidant status of an individual. This device is suited for use outside the laboratory, in field environments. It is easy to use, cost-effective and enables rapid measurement in less than 5 min.

Description

SINGLE-USE PATCH INTENDED FOR MEASURING AN INDIVIDUAL'S OXIDATIVE STRESS

[0001] The invention relates to the field of single-use patches. More specifically, the invention concerns a single-use device for measuring an individual's oxidative stress. This device is suitable for use outside the laboratory, in mobile environments. It is easy to use, economical, and allows for rapid measurement in less than 5 minutes. Scope of the invention

[0002] The present invention relates to the field of medical devices and methods for measuring oxidative stress status in a biological matrix. Oxidative stress results from an imbalance between the production of reactive oxygen species and the antioxidant capacity of the matrix. This phenomenon is involved in numerous biological processes, including cellular aging and various pathologies, such as neurodegenerative diseases.

[0003] Document WO2020 / 109736 describes an electrochemical device for measuring the total antioxidant / oxidizing power of a biological matrix comprising a measurement medium containing at least one compound AB chosen from NADH, NADPH, Cyt C (Fe 2+or H2O2, the compound AB being available in two forms, A and B, capable of interacting and / or forming complexes respectively with antioxidants and oxidants present on the surface or in the matrix, and not interacting with each other. This device comprises a working electrode and a reference electrode, the working electrode being a complex electrode comprising at least gold and platinum. This device allows the measurement of the total antioxidant status of a biological matrix. It is used in the laboratory. The working electrode is made of precious and expensive metals. The measurement is performed in the laboratory.

[0004] Available redox status measurement technologies are expensive, complex and confined to a laboratory, with analysis times often exceeding 10 minutes.

[0005] The present invention addresses the need for a device for measuring total antioxidant status (PAOT), reflecting oxidative stress, which is simple, fast and offered at a reasonable cost, usable at home or in the field.

[0006] The invention relates to a device for measuring the total antioxidant status of an individual, comprising: A rigid support (6); A flexible layer (4) with a thickness of between 0.5 and 3 mm, said layer comprising a housing in which a solid hydrogel (3) is disposed; Two silver / silver chloride (Ag / AgCl) electrodes; A first electrode inserted in the housing (3) of the flexible layer (4) and covered with the solid hydrogel, acting as a reference electrode; A second electrode inserted in the flexible layer (4) outside the housing (3), acting as a working electrode; The distance between said electrodes being between 1 and 2 cm; A gel film (2) comprising a gelled mediator covering said flexible layer (4) and said electrodes.

[0007] It also relates to the use of such a device to measure PAOT status. Advantages of the invention

[0008] The device according to the invention combines reliability and speed thanks to the use of two identical silver / silver chloride electrodes separated by an optimized distance which allows to create a suitable measurement surface and allows a rapid electrochemical equilibrium, improving both the speed and sensitivity of the measurement of the PAOT status, and the presence of an oxidoreducing agent and a gelled mediator allowing to increase the sensitivity and reduce the response time.

[0009] The device's sensitivity is enhanced by the combination of different agents / mediators and the specific electrode configuration: the device is capable of measuring small molecules present on the skin's surface that contribute to oxidative status, which are not usually considered in PAOT measurements. Furthermore, this measurement is rapid, as equilibrium is quickly reached, allowing results to be obtained in a short time (approximately 1 minute). This device has a high measurement capacity, therefore it is not saturated and exhibits high potential.

[0010] Thus, the device is: Fast: Results are obtained in less than 5 minutes. Reliable: The reliability of the result is comparable to, or even greater than, equivalent devices used specifically in laboratories. Affordable: The cost is moderate thanks to standard materials. Portable: It is easy to use anywhere.

[0011] This patch is used with a portable point-of-care device, democratizing health monitoring outside of medical facilities.

[0012] Single-use devices including silver / silver chloride electrodes are in common use for electrocardiograms, which makes the innovation industrially feasible and compatible with regulatory requirements.

[0013] This device aims to democratize skin health monitoring, outside of medical facilities and in an independent manner. It can be used, in particular, in dermatological diagnostic methods to assess the biological age of the skin, quantify the rate of skin aging, and / or evaluate the effectiveness of dermato-cosmetic and cosmetic treatments.

[0014] The device may include, in addition to the two Ag / AgCl electrodes, a complex electrode allowing for additional measurements of oxidative stress for a more personalized cosmetic diagnosis. DETAILED DESCRIPTION OF THE INVENTION

[0015] The device consists of a rigid support (6), a flexible layer (4) comprising a housing in which a solid hydrogel (3) is disposed and two Ag / AgCl electrodes covered by a gel film.

[0016] A first object of the invention relates to a device for measuring the total antioxidant status of an individual, comprising: A rigid support (6); A flexible layer (4) with a thickness of between 0.5 and 3 mm, said layer comprising a housing in which a solid hydrogel (3) is disposed; Two silver / silver chloride (Ag / AgCl) electrodes: A first electrode inserted in the housing (3) of the flexible layer (4) and covered with the solid hydrogel, acting as a reference electrode; A second electrode inserted in the flexible layer (4) outside the housing (3), acting as a working electrode; The distance between said electrodes being between 1 and 2 cm; A gel film (2) comprising a gelled mediator covering said flexible layer (4) and said electrodes.

[0017] Each electrode consists of a male connector (5) and a female connector (1) assembled by interlocking, thus forming a standard format push button.

[0018] The rigid support (6) is designed to hold the various components of the device. A flexible layer (4), with a thickness between 0.5 and 3 mm, preferably between 1 and 2 mm, is attached to the support (6). This flexible layer (4) has a compartment containing a solid hydrogel (3), which acts as a reservoir to facilitate electrochemical reactions, in particular by initiating the measured redox reaction.

[0019] This solid hydrogel is of the type "hydrogel used for electrocardiograms".

[0020] As an example, such a hydrogel may comprise (i) an aqueous phase (40-80%) including purified water and conductive electrolytes such as NaCl, KCl, CaCl2 and MgCl2 in a proportion of 3 to 15%, (ii) crosslinking polymers (10-30%), (iii) crosslinking polymers (10-30%) such as sodium polyacrylate, polyvinylpyrrolidone, or polyethylene glycol (PEG), (iv) crosslinking agents (1-5%), (v) humectants (5-15%), (vi) pH buffers (less than 5%), and (vii) antimicrobial / preservative agents (less than 1%).

[0021] In the context of the invention, the solid hydrogel comprises at least one redox agent such as ascorbic acid, reduced glutathione.

[0022] Glutathione is a tripeptide that plays a key role in biological redox reactions. Reduced glutathione (GSH) can be used to assess redox status by providing a substrate for the measured electrochemical reactions.

[0023] Ascorbic acid (vitamin C) is a natural antioxidant capable of participating in redox reactions.

[0024] The PAOT measurement is performed using two identical silver / silver chloride (Ag / AgCl) electrodes.

[0025] A first electrode is positioned in the housing of the flexible layer (4) and acts as a reference electrode. It is covered with a solid hydrogel (3).

[0026] A second electrode is placed outside the housing of the flexible layer (4) and acts as a working electrode.

[0027] The distance between these electrodes is optimized to create a suitable measurement surface. This distance is between 1 and 2 cm, preferably between 1.3 and 1.9 cm. It can be 1.5 cm.

[0028] The electrodes interact with body fluids, and the mediator contained in the gel film (2) accelerates the transfer of electrons between the redox agent and the electrodes for a rapid measurement of the total redox status.

[0029] A gel-film covers the flexible layer and forms a protective layer on the surface of the device (top surface). It can be made, for example, of a polyethylene-paper-polyethylene sheet or a silicone layer. The mediator of the gel-film can be chosen from potassium ferrocyanide, blue methylene, quinones, or iron, particularly Fe 2+ Nicotinamide Adenine Dinucleotide, polyphenols.

[0030] Potassium ferrocyanide / ferricyanide (Fe(CN)₆³⁻ / Fe(CN)₆⁴⁻) is a well-known mediator for its electron transfer properties, often used in electrochemical sensors to improve conductivity and response. In oxidative stress measurement applications, it helps to measure oxidation / reduction reactions by capturing electrons produced by the oxidation of reactive oxygen species or antioxidants.

[0031] Blue methylene is a colored compound used as a mediator in many electrochemical reactions. It is both stable and efficient, capable of transferring electrons between analytical species (ROS and antioxidants) and electrodes. In oxidative stress detection systems, it is used to interact with species such as glutathione, ascorbic acid, and oxidative stress products to facilitate their electrochemical detection.

[0032] Quinone / hydroquinone is a classic redox couple used to improve reactivity in electrochemical systems.

[0033] Iron (Fe²⁺ / Fe³⁺), particularly in its Fe²⁺ (ferrous) form, is a common electrochemical mediator in systems where reactive oxygen species, such as hydroxyl radicals, are involved. This mediator is used to detect changes in the redox state of cells in response to oxidative stress, in particular to measure the reduction or oxidation of reactive species.

[0034] Nicotinamide Adenine Dinucleotide (NAD+ / NADH): NAD+ and NADH are important biological mediators in electron transfer processes. NAD+ is an oxidant, and NADH is a reductant. In oxidative stress reactions, they can be used to measure antioxidant activity or the redox state of the cell by enabling electrochemical interaction with reactive oxygen species.

[0035] Polyphenols are natural antioxidants and can also act as mediators in electrochemical systems. They can participate in ROS reduction reactions under oxidative stress, helping to amplify electrochemical signals for improved redox status detection.

[0036] In a preferred embodiment of the invention, the redox agent is reduced glutathione and the mediator is potassium ferrocyanide.

[0037] The electrodes are made with materials compatible with medical applications. The connectors are advantageously made of stainless steel.

[0038] The device includes on its lower face a rigid support (6) which is a protective layer made up, for example, of a polyethylene-paper-polyethylene sheet, or of a silicone layer.

[0039] In one particular embodiment, the device further comprises a third electrode (7) inserted in the flexible layer (4), said third electrode being a complex electrode made up of at least two materials selected from gold, platinum and tungsten.

[0040] Thus, in this configuration, the device comprises: A rigid support (6); A flexible layer (4) with a thickness between 0.5 and 3 mm, said layer comprising a housing in which a solid hydrogel (3) is disposed; Two silver / silver chloride (Ag / AgCl) electrodes: A first electrode disposed in the housing of the flexible layer (4) and covered with the solid hydrogel (3), acting as a reference electrode; A second electrode inserted in the flexible layer (4) outside the housing (3), acting as a working electrode; The distance between said electrodes being between 1 and 2 cm; A gel film (2) comprising a gelled mediator covering said flexible layer (4) and said electrodes; A third electrode (7) inserted in the flexible layer (4), said third electrode being a complex electrode made up of at least two materials chosen from gold, platinum and tungsten.

[0041] The third electrode is inserted into the flexible layer (4) outside the housing (3), preferably equidistant from the two Ag / AgCl electrodes. It acts as a working electrode. This electrode is a complex electrode, made of at least two materials chosen from gold, platinum, and tungsten.

[0042] In alternative embodiments, this electrode may be made of gold and platinum, or gold and tungsten, or platinum and tungsten. It may also be made of a gold, platinum and tungsten alloy; such an electrode may, for example, be made of 60% gold, 10% platinum and 30% tungsten or of 50% gold, 27% platinum and 33% tungsten.

[0043] Noble metal alloys, such as gold and platinum, are the preferred materials for skin-applied working electrodes in cosmetics due to their biocompatibility, chemical and electrochemical stability, and high durability. Unlike Ag / AgCl electrodes, which can release irritating silver ions and degrade upon contact with certain cosmetic components, these alloys offer increased resistance to chemical reactions and product buildup. Their excellent conductivity ensures precise and consistent electrochemical measurements, even in the presence of creams, serums, or other formulations. Furthermore, their chemical inertness minimizes interactions with substances applied to the skin, thus ensuring reliable and long-lasting performance in applications such as skin diagnostics or electrostimulation.These properties make alloys based on noble metals preferred materials for the development of innovative cosmetic devices, combining safety and effectiveness.

[0044] The other elements of this embodiment are as described previously.

[0045] A second object of the invention relates to the use of a device as defined above for measuring the total antioxidant status of an individual.

[0046] Oxidative stress refers to the measurement of the balance between free radicals (or oxidants) and antioxidants. It is therefore possible to measure antioxidant status, oxidative status, or the resultant of these two parameters: oxidative stress. These parameters all allow for the assessment of the level of oxidation in skin tissue.

[0047] In the present invention, the device provided allows preferential measurement of antioxidant status, also called PAOT status or PAOTscore.

[0048] Total oxidizing power and total antioxidant power are measured according to the following formulas:

[0049]

[0050] Where PE Peau10 corresponds to the electrochemical potential measured at time 10 min.

[0051] PE control t0 corresponds to the electrochemical potential measured at time t0.

[0052] And PE peau 3 min corresponds to the highest electrochemical potential.

[0053] The device is brought into contact with the biological sample, here the skin, and the PAOT status is measured in the biological fluids present on the skin such as sweat or interstitial fluid.

[0054] The measurement time is less than or equal to 5 min, it is typically between 3 and 5 min.

[0055] The present invention will be better understood by reading the following examples, which are provided by way of illustration and should in no way be considered as limiting the scope of the present invention. DESCRIPTION OF THE FIGURES

[0056] : Exploded view representation of a two-electrode device for measuring oxidative stress with (1) female connector, (2) film-gel, (3) solid hydrogel, (4) flexible layer, (5) male connector and (6) rigid support.

[0057] : Top view representation of a two-electrode device for measuring oxidative stress as defined in the.

[0058] : Electrochemical potential measured using a two-electrode patch-type device according to the invention (grey curve, upper) versus a laboratory device (black curve, lower) for a period of 5 min.

[0059] : Electrochemical potential measured using a two-electrode patch-type device according to the invention (grey curve, upper) versus a laboratory device (black curve, lower) for a period of 10 min.

[0060] : Exploded view representation of a 3-electrode device for measuring oxidative stress with (1) female connector, (2) film-gel, (3) solid hydrogel, (4) flexible layer, (5) male connector, (6) rigid support, (7) female connector of the third electrode and (8) male connector of the third electrode.

[0061] : Top view representation of a three-electrode device for measuring oxidative stress as defined in the. EXAMPLE

[0062] EXAMPLE 1: Description of the device in a particular embodiment of the invention

[0063] The PAOT measurement device comes in the form of a single-use patch that is applied to the skin.

[0064] In the prototype tested in this example, the patch comprises a flexible layer (4) 1 mm thick and the electrodes are spaced 1.5 cm apart. The redox agent present in the hydrogel (3) is reduced glutathione and the mediator present in the gel film is ferrocyanide.

[0065] The concentrations of reduced glutathione (redox agent) and ferrocyanide (mediator) in the hydrogel and film-gel are consistent with those of similar devices using redox mediators for electrochemical applications, namely: Reduced glutathione (GSH): Typically between 1 and 10 mM in the hydrogel, to ensure effective redox activity without causing premature oxidation or skin toxicity. Ferrocyanide (Fe(CN)₆⁴⁻): Generally used between 0.5 and 5 mM, depending on the conductivity and electron transfer requirements in the film-gel.

[0066] When the patch is applied to the skin, the electrodes and the gel-like mediator interact with bodily fluids such as sweat or interstitial fluid. The identical Ag / AgCl electrodes, in contact with the gel-like mediator present in the gel film, enable an efficient electrochemical reaction.

[0067] The protective film on a PAOT patch plays a crucial role in preserving the device and ensuring its effectiveness. First and foremost, it provides a seal, protecting the internal components, including the electrodes and conductive gel, from moisture and external contaminants such as dust and bacteria. By maintaining the gel's integrity and preventing evaporation, it guarantees optimal transmission of the heart's electrical signals. Furthermore, this film protects the patch's adhesive surface, preventing it from drying out or becoming contaminated before application, thus ensuring effective adhesion to the skin. Finally, it facilitates handling and application of the patch by maintaining optimal conditions until use. Therefore, this film is an essential element for ensuring the reliability and durability of the PAOT patch.

[0068] The integration of the mediator and redox compounds increases the reactivity and sensitivity of the electrodes. This allows for efficient and reliable measurement of PAOT and POT status.

[0069] EXAMPLE 2: Evaluation of the functional properties of the device

[0070] The functional characteristics of the patch were compared to those of a device used in the laboratory (prior art). For this purpose, the evaluations were carried out using a patch with two Ag / AgCl electrodes.

[0071] The differences between the two devices are presented in Table 1.

[0072] Criteria Laboratory apparatus Device according to the invention Reference electrode Mixed: Silver / Silver chloride (Ag / Cl) + Gold, Platinum, Tungsten alloy Silver / Silver chloride (Ag / AgCl) Working electrode Identical to the reference electrode: Ag / AgCl Presence of a protective film on the electrodes No Yes Electrode configuration Distance between electrodes Random Distance between electrodes Optimized Mediator Liquid or pre-gelled Gelified

[0073] Table 1: Comparative description of oxidative stress measurement devices

[0074] Two types of measurement were carried out to evaluate the performance of the two devices: short measurement and long measurement.

[0075] The potential between the two electrodes was measured for each device to assess the antioxidant status ("PAOT score") and the oxidative status ("POTscore") at the skin level of the same individual. Short measurement

[0076] Device Type PAOTscore POTscore Laboratory device (prior art) 61,390,87 Device according to the invention 17,690,042

[0077] Table 2: Comparative measurement of PAOTscore and POTscore after 5 min.

[0078] It is observed that the PAOTscore measured with the device according to the invention is 3.47 times higher than that measured with an existing device. Similarly, the POTscore is higher, by a factor of 20.7.

[0079] This result shows the improved sensitivity of the patch according to the invention for a short measurement lasting 5 minutes.

[0080] La represents the difference in sensitivity between the tested devices over a period of 5 minutes. It is observed that the device according to the invention is significantly more sensitive than the laboratory device from the very first seconds of measurement, and that this difference persists over time. This result confirms that the device according to the invention is particularly well-suited for short measurements. Long measurement

[0081] The same type of measurement was carried out over a period of 10 minutes.

[0082] Device Type PAOTScore POTScore Laboratory device (prior art) 65,320,41 Device according to the invention 34,20,167

[0083] Table 3: Comparative measurement of PAOTscore and POTscore after 10 min.

[0084] It is observed that the PAOTscore measured with the device according to the invention is 1.9 times higher than that measured with an existing device. Similarly, the POTscore is higher, by a factor of 2.5.

[0085] This result shows the improved sensitivity of the patch according to the invention for a long measurement lasting 10 minutes.

[0086] Thus, even when lengthening the measurement time, the device according to the invention is more sensitive.

[0087] La represents the difference in sensitivity between the tested devices over a 10-minute period. It is observed that the device according to the invention is significantly more sensitive than the laboratory device from the very first seconds of measurement, and that this difference persists over time. This result confirms that obtained in the previous section. Even when increasing the measurement time, the device according to the invention remains more sensitive.

[0088] The advantages of the single-use patch-type device that is the subject of the invention are summarized in Table 4.

[0089] Evaluation Criteria: Speed: Results in less than 5 minutes; Reliability: Comparable to or even superior to laboratory devices; Accessibility: Standard materials, moderate cost; Portability: Compatible with point-of-care devices, easy to use outside of a medical setting; Industrialization: Based on electrodes used in ECG, facilitating regulatory compliance

[0090] Table 4: Description of the advantages of the device according to the invention

[0091] EXAMPLE 3: Using PAOT to assess an individual's age and rate of skin aging

[0092] In this example, the device used to measure PAOT status is a device according to the invention which comprises two Ag / AgCl electrodes, a hydrogel and a film-gel.

[0093] PAOT measurement can be used to estimate the skin's biological age and its rate of aging. Indeed, the skin's biological age does not always correspond to an individual's chronological age. Exposure to external aggressors, oxidative stress, and genetic characteristics all influence skin aging. To assess this age and its rate of progression, biomarkers such as PAOT are used. This parameter measures the oxidation state of skin tissues and, by extension, estimates the progressive degradation of the skin. Furthermore, pH balance, hydration levels, and the concentration of vitamins C, E, and GSH play a key role in protecting against premature aging. Phototype and skin type also influence susceptibility to aging and must be considered in the analysis.

[0094] Skin age is estimated by applying the following equation:

[0095] in which: Apeau is the estimated age of the skin. Achrono is chronological age, PAOTT is the value of PAOT after a certain time. PAOT0H is the initial value of PAOT, pHopt is the optimal pH for the skin (approximately 5.5), pHmeasured is the measured pH. Hydmesuré is the optimal hydration level. Hydopt is the measured hydration level.

[0096] Phototype facteur is a coefficient adjusted according to skin phototype (I to VI according to the Fitzpatrick classification),

[0097] Skin Type facteur is a coefficient reflecting the skin type (dry, combination, oily, sensitive).

[0098] To estimate the rate of skin aging, the first step is to measure the factor antioxVitamins C, E, and GSH are powerful antioxidants that directly influence cellular protection against oxidative stress. A correction factor is applied based on the levels of these molecules in the skin, and the equation is:

[0099] in which: Facteurantiox is the measured antioxidant factor. VitCmesuré is the measured vitamin C level. VitEmesuré is the measured level of vitamin E.

[0100] GSH mesuré is the measured level of reduced glutathione, VitCopt is the optimal level of vitamin C. VitEopt is the optimal level of vitamin E.

[0101] GSH opt is the optimal reduced glutathione level.

[0102] Finally, the aging rate is measured using the factor antiox calculated previously, as well as with the PAOT by applying the equation:

[0103] To estimate skin age, a process using an algorithm was employed. It comprises the following steps:

[0104] 1. Data acquisition: Extraction of PAOT, pH, hydration, vitamin, GSH, phototype and skin type values ​​at different time intervals.

[0105] 2. Calculation of skin age: Application of the adjusted equation taking into account skin parameters.

[0106] 3. Evaluation of the rate of aging: Use of the rate of evolution formula.

[0107] 4. Interpretation of results: Detection of accelerated aging if a critical threshold is exceeded.

[0108] The results obtained from a sample of 54,000 volunteers show a significant correlation between PAOT, skin parameters, and chronological age. The integration of factors such as hydration, pH, antioxidants, phototype, and skin type improves the accuracy of the estimates and allows for better identification of profiles at risk of premature aging.

[0109] The proposed approach allows for a robust estimation of the skin's biological age as well as a quantification of the skin aging rate. This methodology could be integrated into dermatological diagnostic tools and tools for evaluating the effectiveness of anti-aging treatments. Conclusion

[0110] The new device surpasses the laboratory device in sensitivity, speed, and cost. Thanks to its simplified and optimized design, it enables more efficient antioxidant analysis while facilitating viable industrial production. Its use in a portable device enhances its potential for democratizing health monitoring outside of medical facilities. It is significantly more efficient and better suited to widespread medical use than the current device, which, moreover, must be used in a laboratory.

Claims

Device for measuring the total antioxidant status of an individual, comprising: A rigid support (6); A flexible layer (4) with a thickness between 0.5 and 3 mm, said layer comprising a housing in which a solid hydrogel (3) is disposed; Two silver / silver chloride (Ag / AgCl) electrodes, A first electrode disposed in the housing (3) of the flexible layer (4) and covered with the solid hydrogel, acting as a reference electrode; A second electrode inserted in the flexible layer (4) outside the housing (3), acting as a working electrode; The distance between said electrodes being between 1 and 2 cm; A gel film (2) comprising a gelled mediator covering said flexible layer (4) and said electrodes. Device according to claim 1 further comprising a third electrode (7) inserted in the flexible layer (4), said third electrode being a complex electrode. Device according to claim 2 characterized in that said complex electrode is made of a gold, platinum and tungsten alloy. Device according to any one of the preceding claims characterized in that said mediator comprises an agent promoting increased redox reactivity selected from reduced glutathione and ascorbic acid. Device according to any one of the preceding claims characterized in that said mediator is selected from potassium ferrocyanide, blue methylene or quinones, nicotinamide adenine dinucleotide, iron, and polyphenols. Device according to any one of the preceding claims characterized in that said redox agent is reduced glutathione and said mediator is potassium ferrocyanide. Device according to one of the preceding claims characterized in that the distance between said two Ag / AgCl electrodes is between 1.3 and 1.9 cm. Use of a device as defined in any one of claims 1 to 7 for measuring the total antioxidant status of an individual. Use according to claim 8 wherein said measurement is carried out in a biological fluid present on the surface of the skin of said individual. Use according to claim 9 wherein said biological fluid is sweat or interstitial fluid.

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