A system for measurement of analyte concentrations in bodily secretions and a biosensor including such a system

Abstract

The subject invention pertains to a system, a device, and a method for measuring analyte concentrations in sweat. The system for measurement of analyte concentration in bodily fluids comprises a sweat-inducing device operably coupled with a sweat-measuring device. The method performed by the system and devices comprises applying a system according to the invention to a skin surface, performing a measurement by applying electric current, and obtaining a change of electric current in sensing electrodes that represent a result of a measurement.

Description

[0001] A SYSTEM FOR MEASUREMENT OF ANALYTE CONCENTRATIONS IN BODILY SECRETIONS AND A BIOSENSOR INCLUDING SUCH A SYSTEM CROSS-REFERENCE TO RELATED APPLICATION The present application claims the benefit of U.S. Provisional Application Serial No. 63 / 672,828, filed July 18, 2024, which is hereby incorporated by reference herein in its entirety, including any figures, tables, or drawings. FIELD OF THE INVENTION The present invention is directed to a system for non-invasive electrochemical measurement of analyte concentration in bodily secretions. More specifically, the present invention relates to a system capable of inducing sweat secretion from a skin surface and measuring the analyte concentration, e.g., glucose, in such a collected sweat sample. The present invention is also directed to a biosensor which includes such a system. BACKGROUND OF THE INVENTION Approximately 8.0% of the population in the United States has diabetes (American Diabetes Association). Diabetes is a leading cause of death in the United States. It is a life- threatening disease with broad complications, which include blindness, kidney disease, nerve disease, heart disease, amputation, and stroke. Diabetes results from the inability of the body to produce or properly use insulin, a hormone that regulates the level of glucose in the blood and the movement of glucose into cells. Although the cause of diabetes is not completely understood, it is believed that genetics, environmental factors, and viral causes contribute to the incidence of diabetes in the world population. Insulin is a hormone that regulates the level of glucose in the blood and allows glucose to enter cells. In diabetics, glucose cannot enter the cells, so glucose builds up in the blood to toxic levels. Diabetics using insulin to help regulate their blood sugar levels are at an increased risk for medically dangerous episodes of low blood sugar due to errors in insulin administration, or unanticipated changes in insulin absorption. Insulin-using patients practice self-monitoring of blood glucose. Based upon the level of glucose in the blood, individuals may make insulin dosage adjustments before injection. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS Adjustments are generally necessary since blood glucose levels vary day to day for a variety of reasons, such as exercise, stress, rates of food absorption, types of food, hormonal changes (pregnancy, puberty, etc.) and the like. Despite the importance of self-monitoring, the proportion of individuals who self-monitor at least once a day significantly declines with age. This decrease is likely the result of the typical, most widely used, method of self-monitoring of blood glucose (SMBG), involving obtaining blood from a capillary finger stick. Because the common self-monitoring method can be painful, it would be desirable to provide non-invasive devices, methods and kits for measuring blood glucose levels. Using sweat is attractive at least because it can be collected non-invasively and because sweat glucose level is correlatable to blood glucose level. Sweat may also be a source of other components than glucose, e.g., ammonia, prostaglandins, C12-C22 fatty acids, ethanol, metabolites, such as lactate, urea, cortisol, hydroxybutyrate, electrolytes, such as Na+, K+, and Cl-, trace elements such as Zn2+and Cu2+, nutrients, such as vitamin C and D, and drugs, such as scopolamine, paracetamol, paroxetine [1]. Some are closely related to health status and can be used as biomarkers for in situ non- invasive monitoring of physiological health status. Related art: US20180070866 discloses an ultra-low power, non-invasive in-vivo blood analyte sensor system incorporating multiple sensors including a carbon base and / or carbon base material coated with metallic nanoparticles and / or metallic nanoparticle nanoprobes, as a modified Clark electrode sensor system. The sensor system detects hydrogen peroxide concentrations, pH, and / or glucose concentrations (and other analytes) in bodily secretions (e.g., tears, saliva, sweat). The device consists of multiple chemoreceptive sensors, a microprocessor, a signal amplifier, signal filtering, error correction algorithms, an analog-to- digital converter, and wireless electromagnetic data transmitter to a remote device for further processing and / or data storage (e.g., on a server, on a cloud-based storage system, etc.) and / or visual representation via software. WO / 2010 / 045247 discloses a sweat glucose sensor for determining the glucose concentration in a volume of sweat. The sweat glucose sensor may comprise two or more electrodes in contact with sweat in a container defined by two or more layers of a skin patch. The container may additionally contain a glucose enzyme that reacts with glucose in sweat collected by the container. The sweat glucose sensor may comprise two or more fill electrodes, also positioned to contact sweat collected by the container. The fill electrodes may be used, J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS for example, to determine whether the skin patch contains a requisite volume of sweat to perform an accurate glucose measurement. Devices, methods, and kits for collecting sweat that has come to a skin surface are also disclosed. CN116421184 discloses a full-green degradable flexible laser-induced graphene glucose detection chip matched with sweat detection. According to the method, LIG is used as a three-dimensional electrode, Ag / AgCl is used as a reference electrode, the electrode material is subjected to electric activation treatment and electro-deposition treatment. CN115112738 discloses preparation and glucose sensing application of a laser direct writing graphene / enzyme electrode. A micro flexible electrode is prepared by combining a laser induced graphene (LIG) technology, a hydrophobic end benzene ring of 1-pyrene butyric acid, and a six-membered ring structure of graphene perform carboxylation on the surface of the LIG electrode. Then, glucose oxidase is covalently cross-linked, and the prepared enzyme electrode can be used for glucose detection of human serum, urine samples and sweat. US20180070866 discloses an ultra-low power, non-invasive in-vivo blood analyte sensor system incorporating multiple sensors including a carbon base and / or carbon base material coated with metallic nanoparticles and / or metallic nanoparticle nanoprobes, as a modified Clark electrode sensor system. The sensor system detects hydrogen peroxide concentrations, pH, and / or glucose concentrations (and other analytes) in bodily secretions (e.g., tears, saliva, sweat). The device consists of multiple chemoreceptive sensors, a microprocessor, a signal amplifier, signal filtering, error correction algorithms, analog-to- digital converter, and wireless electromagnetic data transmitter to a remote device for further processing and / or data storage (e.g., on a server, on a cloud-based storage system, etc.) and / or visual representation via software. Given the above, it would be desirable to provide devices, methods, and kits for sensing and measuring the concentration of glucose in sweat, e.g., that has been collected from a skin surface of the subjects. It would be desirable to provide devices, methods, and kits for sensing and measuring the concentration of other substances-analytes present in sweat. It would also be desirable to provide devices, methods, and kits for collecting a volume of sweat from a skin surface that is suitable for measuring the glucose concentration in the sweat, and for collecting small, fixed volumes of sweat without the collected sweat volumes being affected by a variable sweat rate. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS BRIEF SUMMARY OF THE INVENTION A device, a system, and a method for inducing a generation of bodily secretions followed by electrochemical measuring of analyte concentration are provided. The system comprises a sweat-inducing device operably coupled with a sweat-measuring device. The sweat-inducing device comprises chemical and electrical means for inducing sweat generation from a skin surface. A system according to the invention comprises modified LIG (laser- induced graphite / graphene) electrodes. A biosensor according to the invention includes a system according to the invention. BRIEF DESCRIPTION OF THE DRAWINGS Drawing comprises Figure 1 and Figure 2. Figure 1 shows an exploded view of a system according to a preferred embodiment of the invention. Figure 2 shows graphs indicating the change of the glucose concentration over time in two human subjects; the glucose concentration is measured in sweat and in interstitial fluid. DETAILED DISCLOSURE OF THE INVENTION Selected Definitions As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”. The transitional terms / phrases (and any grammatical variations thereof) “comprising”, “comprises”, “comprise”, “consisting essentially of”, “consists essentially of”, “consisting” and “consists” can be used interchangeably. The phrases “consisting essentially of” or “consists essentially of” indicate that the claim encompasses embodiments containing the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim. The term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured, i.e., the limitations of the measurement system. In the context of compositions containing amounts of ingredients where the term “about” is used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0-10% around the value (X J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS ± 10%). In other contexts, the term “about” is providing a variation (error range) of 0-10% around a given value (X ± 10%). As is apparent, this variation represents a range that is up to 10% above or below a given value, for example, X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%. In the present disclosure, ranges are stated in shorthand to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. Values having at least two significant digits within a range are envisioned, for example, a range of 5-10 indicates all the values between 5.0 and 10.0 as well as between 5.00 and 10.00 including the terminal values. When ranges are used herein, combinations and subcombinations of ranges (e.g., subranges within the disclosed range) and specific embodiments therein are explicitly included. By “reduces” is meant a negative alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%. By “increases” is meant as a positive alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%. As used herein, the terms “determining,” “measuring,” and “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations. The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. All references cited herein are hereby incorporated by reference in their entirety. Description and practical uses of the invention The subject invention pertains to a system, a device, and a method for inducing a generation of bodily secretions followed by electrochemical measuring of analyte concentration. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS In one aspect a system for measurement of analyte concentration in bodily fluids comprises a sweat-inducing device operably coupled with a sweat-measuring device as depicted in Figure 1. The sweat-inducing device comprises a first layer, which is in contact with a skin surface, and a second layer. A first layer of a sweat-inducing device comprises a base material (13), preferably a three-dimensional polymeric structure with openings (14) adapted to be filled with a hydrogel carrier (15, 16), such as agarose. The carrier comprises a compound capable of penetrating a skin and / or allowing sweat production resulting in the migration of the sweat and / or an interstitial fluid to the surface of the skin. For example, pilocarpine is used as such a compound. The carrier extends and is in contact with a second layer of a sweat-inducing device, which comprises two planar electrodes, namely a cathode (12) and an anode (11). Preferably the electrodes are of a similar surface area, wherein the cathode is surrounded by the anode. The electrodes are surrounded by the carrier, which inhibits the electrodes from direct contact with the skin. The electrodes are configured to conduct an electric current (DC or AC) to induce sweat generation from the skin surface. The electrodes may be made by thin layer technology, thick layer technology and / or by pyrolysis of the base material resulting in a LIG (laser-induced graphite / graphene) type layer. A specific method for the production of electrodes depends on the kind of base material, wherein the paper is preferably used as an absorbent of the hydrogel carrier. Alternatively, or additionally, polymeric base material may be used. The second layer of a sweat-inducing device is covered with an isolating layer (10), which also acts as a base material for the electrodes. The isolating layer is operably coupled with a sweat-measuring device. The sweat-measuring device comprises several layers. Referring to Figure 1, the sweat-measuring device comprises at least three electrodes, namely a working electrode 4, a counter electrode 3, and a reference electrode 2, forming a system of sensing electrodes, which are surrounded by a paper layer 5 forming a base material for the electrodes. The paper layer is preferably hydrophobic, which allows for forming a space for sample collection 6, a space for the deposition of a biochemical analyte recognition system 7, and a space for the deposition of potassium chloride 8. The sensing electrodes of the sweat-measuring device form a multilayer, planar, elastic electrochemical system comprising the working electrode operating directly or a plurality of J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS electrodes working differentially. The sensing electrodes are covered with an isolating layer 1 with openings 9, which makes evaporation of the liquid from the skin surface easier. The first layer of the working electrode comprises a carbon paste modified with a mixture of graphene and gold nanoparticles. In one embodiment, a mixture of graphene and gold nanoparticles comprises: a) unmodified graphene, and b) gold nanoparticles covered with a thiol-based mediator. In another embodiment, a mixture of graphene and gold nanoparticles comprises: a) graphene modified with a linker, and b) gold nanoparticles covered with a thiol-based mediator or gold nanoparticles covered with thiol-based ferrocene. In another embodiment, a mixture of graphene and gold nanoparticles comprises: a) unmodified graphene, and b) gold nanoparticles covered with a thiol-based linker or gold nanoparticles covered with thiol-based ferrocene. In another embodiment, a mixture of graphene and gold nanoparticles comprises: a) graphene modified with a mediator, and b) gold nanoparticles covered with a thiol-based linker or gold nanoparticles covered with thiol-based ferrocene. Gold nanoparticles preferably have a spherical shape and size ranging from about 2 to about 50 nm. A mediator preferably comprises FAD, NAD, ferrocene, TTF, or osmium pyridyl complexes. Alternatively, or additionally, a mediator may be a thiol base mediator comprising FAD, NAD, ferrocene, TTF, osmium pyridyl complexes, or neutral thiol. A thiol-based linker or a thiol-based mediator for modification of gold nanoparticles is selected from a group comprising: J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS

[0002] A linker or a mediator for modification of graphene is selected from a group 5 comprising: J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS

[0003] A second layer of the working electrode comprises an enzyme of any class of enzymes, preferably oxidoreductase class, mixed with a cross-linking reagent, such as glutaraldehyde. A third layer of the working electrode comprises a film made on the surface of a second layer of the working electrode. The film allows the free flow of ions or other analytes. For example, sulfonated tetrafluoroethylene-based fluoropolymer-copolymer (e.g., NAFION®) is a suitable material for the film. A reference electrode may be a pseudo-reference type electrode, for example, a screen- printed silver chloride layer or in a form of the saturated reference electrode in the form of a screen-printed silver chloride layer in direct contact with a high level of ions, against which the electrode is reversible (Cl-). A counter electrode may be carbon, platinum, or silver electrode. Preferably, the counter electrode is a carbon electrode. A suitable base material for manufacturing an electrochemical system is chromatographic paper or other elastic, porous, or smooth material. Polyamide, polyethylene terephthalate, and the like are suitable materials for manufacturing electrochemical system. Preferably, layers (or sides of layers) of the electrochemical system differ in functional features yielding electrochemical, sensor, reagent, isolating or distributing type layers. The system according to the present invention further comprises battery powered electrical circuit to provide electric current to the electrodes. A measurement of bodily secretions is performed based on a change in the electrical parameters of the sensing electrodes. The resulting change may be further transmitted as electronic data by wire or preferably wireless to another electronic device, such as a mobile phone, tablet, or computer that is capable of storing and processing said data, such as visual processing. A mathematical model to correlate the resulting data representing for example concentration of glucose in sweat with a concentration of glucose in blood may be performed by electronic device and the result may J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS be displayed or stored by such a device. Furthermore, other mathematical models may be applied that take into account the personal characteristics of a subject with other parameters that may be inputted and processed by such an electronic device. In another aspect, a method of measuring analyte concentrations in bodily secretions is provided. The method is performed by the system and devices according to the present inventions and comprises applying a system according to the invention to a skin surface, performing a measurement by applying electric current, and obtaining a change of electric current in sensing electrodes that represent a result of a measurement. In another aspect, a system according to the present invention may be integrated into a skin patch, which may be worn on the limb or glued to the skin surface of any part of the body. In another aspect, a system according to the present invention forms a biosensor intended for attachment to the skin, preferably human skin. In another aspect, a system according to the present invention forming a biosensor may form a kit. The kit may include one or more packaged system and instructions. Typically, the skin patches are individually packaged in sterile containers or wrappings and are configured for a single use. The skin patches further include compounds of the subject invention and pharmaceutical formulations. In further embodiments, a kit includes an article of manufacture, for delivering the compounds of the subject invention into a subject locally and for measuring analyte concentrations in bodily secretions. The term “sweat-measuring device” means a part of the system which measures analyte concentration originating from bodily fluids, preferably from sweat. The term “sweat-inducing device” means a part of the system which induces the generation of bodily fluids from the body of the subject, preferably sweat. The terms “bodily secretions” and “bodily fluids” are used interchangeably herein. These terms mean all substances secreted by the animal body, e.g., saliva, sebum, blood, plasma, semen, urine, sweat. The preferred bodily secretion is sweat. The most preferred bodily secretion is human sweat. The term “analytes” means all components included in bodily secretions. Preferably, it means substances included in human sweat, e.g., glucose, ammonia, prostaglandins, C12- C22 fatty acids, ethanol, lactate, urea, cortisol, hydroxybutyrate, Na+, K+, Cl-, Zn2+, Cu2+, vitamin C and D, scopolamine, paracetamol, paroxetine. The term “subject” means an animal, preferably a human. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS In most preferred embodiments, a biosensor according to the present invention comprises a system including a sweat-measuring device and a sweat-induction device. In the most preferred embodiment, a biosensor according to the present invention comprises a system including a sweat-measuring device and a sweat induction device. The sweat-inducing device comprises a first layer, which is in contact with a skin surface, and a second layer. The first layer of a sweat-inducing device comprises a base material 13 being paper. The base material 13 includes openings 14 adapted to be filled with a hydrogel carrier 15, 16 being agarose. Paper is used as an absorbent of the hydrogel carrier. The carrier comprises also pilocarpine. The carrier extends and is in contact with a second layer of a sweat-inducing device. The second layer comprises two planar electrodes: a cathode 12 and an anode 11. The electrodes are of a similar surface area, wherein the cathode is surrounded by the anode. The electrodes are surrounded by the carrier, which inhibits the electrodes from direct contact with the skin. The electrodes are configured to conduct an electric current (DC or AC) to induce sweat generation from the skin surface. The second layer of a sweat-inducing device is covered with an isolating layer 10, which also acts as a base material for the electrodes. The isolating layer 10 of a sweat-inducing device is operably coupled with a sweat- measuring device. The sweat-measuring device comprises three electrodes: a working electrode 4, a counter electrode 3, and a reference electrode 2. The electrodes form a system of sensing electrodes. The said system of sensing electrodes is surrounded by a paper layer 5 forming a base material for the electrodes. The paper layer is hydrophobic, which allows for forming a space for sample collection 6, a space for the deposition of a biochemical analyte recognition system 7, and a space for the deposition of potassium chloride 8. The sensing electrodes of the sweat-measuring device form a multilayer, planar, elastic electrochemical system comprising the working electrode operating directly. The sensing electrodes are covered with an isolating layer 1 with openings 9. The first layer of the working electrode comprises carbon paste modified with a mixture of graphene having a 3-(5-formyl-thiophen- 2-yl)-benzoic acid linker and gold nanoparticles on its surface. Gold nanoparticles have a spherical shape and size ranging from about 2 to about 50 nm. The second layer of the working electrode comprises an enzyme from the oxidoreductase enzyme class (Glucose Dehydrogenase (PQQ-dependent)) mixed with a crosslinking agent, e.g., glutaraldehyde. The third layer of the working electrode comprises a film formed on the surface of the second layer, wherein the material of the film is sulfonated tetrafluoroethylene-based fluoropolymer- copolymer (NAFION®). The reference electrode 2 is a screen-printed silver chloride layer or J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS is in a form of the saturated reference electrode in the form of a screen-printed silver chloride layer in direct contact with a high level of ions, against which the electrode is reversible (Cl-). The counter electrode 3 is a carbon electrode. The system further comprises battery powered electrical circuit to provide electric current to the electrodes. The system is integrated into a skin patch to be worn on the human skin surface. A measurement of sweat secretion from the human skin surface is performed based on a change in the electrical parameters of the sensing electrodes. The resulting change is further transmitted as electronic data by wire or preferably wireless to another electronic device, such as a mobile phone, tablet, or computer that is capable of storing and processing said data, such as visual processing. The hydrogel carrier can be formed with materials including, but not limited to, agarose, poly(vinyl alcohol) (PVA), sodium alginate (SA), and tannic acid (TA), PEDOT:PSS conductive hydrogel, optionally incorporating Prussian blue nanoparticles, hyaluronic acid (HA), oxidized chitosan (CS), optionally incorporating KCl, Dopamine methacrylate (DMA), methacrylatoethyl trimethyl ammonium chloride (DMC), and acrylic acid (AA). PDDA hydrogel, or any other suitable material and combinations thereof. The above-described devices (a system and a biosensor), methods, and kits are described herein for the purposes of illustration and are not intended to be limiting. Alternative and additional variations may be apparent to those skilled in the art. MATERIALS AND METHODS EXAMPLE 1 ― Tests measuring the change of the glucose concentration over time in sweat and in interstitial fluid A biosensor according to the most preferred embodiment of the present invention was used for glucose concentration measurement in the sweat of two human subjects. The study was conducted in fasting conditions and following an oral glucose load of 75 g. Data were collected with the invention biosensor at 15-minute intervals over three hours starting from the time of glucose ingestion. At the same time, glucose concentration in the interstitial fluid of the given human subject was measured with the commercially available continuous glucose monitoring system - FreeStyle Libre (Abbott). The study also allowed for a comparison of the dynamics of glucose concentration changes in interstitial fluid and sweat in the human subject. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS The obtained results are shown on Figure 2. The results indicate that a biosensor according to the present invention effectively measures glucose concentration in the sweat. Figure 2 shows that the peak glucose concentration in the sweat occurs later in time than the peak concentration observed in the interstitial fluid. This delay in the peak glucose concentration in the sweat is at least 45 minutes, usually between 45 minutes and 75 minutes, against the peak glucose concentration in the interstitial fluid. As the glucose concentrations in the sweat are lower than the corresponding glucose concentrations in interstitial fluid, the glucose concentrations received by a biosensor according to the invention were multiplied by an appropriate factor (10 or 30) to provide better clarity of the results. EXEMPLARY EMBODIMENTS Embodiment 1. A system for non-invasive electrochemical measurement of analyte concentrations in bodily secretions comprising: (a) a sweat-inducing device comprising: (i) a first layer including a base material 13 having openings 14 filled with a hydrogel carrier 15, 16 containing a sweat-inducing compound; (ii) a second layer including at least two planar electrodes, being an anode 11 and a cathode 12, which are configured to apply an electric current to a skin surface through the hydrogel carrier to induce sweat secretion, wherein the second layer is covered with an isolating layer 10; and (b) a sweat-measuring device operably coupled with the sweat-inducing device through the isolating layer 10 of the sweat-inducing device and comprising: (i) a working electrode 4 including a carbon paste modified with graphene and gold nanoparticles; (ii) a counter electrode 3; and (iii) a reference electrode 2, wherein a working electrode 4, a counter electrode 3, and a reference electrode 2, forming a system of sensing electrodes, are surrounded by a base layer 5 creating a space for sample collection 6, a space for the deposition of a biochemical analyte recognition system 7, and a space for the deposition of potassium chloride 8, wherein the electrodes are covered with an isolating layer 1 with openings 9, wherein the electrodes form an electrochemical system configured to detect the presence and concentration of one or more analytes in the induced sweat sample. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS Embodiment 2. The system of embodiment 1, wherein the sweat-inducing compound is pilocarpine. Embodiment 3. The system of any preceding embodiments, wherein the hydrogel carrier is selected from consisting of agarose. Embodiment 4. The system of any preceding embodiments, wherein the electrodes of the sweat-inducing device are manufactured by thin layer technology, thick layer technology and / or by pyrolysis of the base material resulting in a LIG (laser-induced graphite / graphene) type layer. Embodiment 5. The system of any preceding embodiments, wherein the sweat- inducing electrodes are planar and have similar surface areas, with a cathode 12 surrounded by an anode 11. Embodiment 6. The system of any preceding embodiments, wherein the working electrode 4 comprises: (a) a first layer comprising a carbon paste layer modified with a mixture of graphene and gold nanoparticles, wherein graphene is unmodified or modified, and wherein gold particles are covered with a thiol-based mediator or thiol-based ferrocene, (b) a second layer comprising an oxidoreductase enzyme mixed with a cross-linking reagent, such as glutaraldehyde; and (c) a third layer comprising a film made on the surface of a second layer of the working electrode. Embodiment 7. The system of embodiment 6, wherein modified graphene is modified with a mediator or linker which are selected from the group consisting of FAD, NAD, ferrocene, TTF, and osmium pyridyl complexes, a thiol-based mediator comprising FAD, NAD, ferrocene, TTF, osmium pyridyl complexes or neutral thiol, J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS Embodiment 8. The system of embodiment 6, wherein the thiol-based mediator or thiol-based linker for modification of gold nanoparticles is selected from the group consisting of , . Embodiment 9. The system of any preceding embodiments, wherein a reference electrode 2 is a pseudo-reference type electrode being a screen-printed silver chloride layer or in a form of the saturated reference electrode in the form of a screen-printed silver chloride layer in direct contact with a high level of ions, against which the electrode is reversible (Cl-), and a counter electrode 3 is a carbon, platinum or silver electrode. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS Embodiment 10. The system of any preceding embodiments, wherein a base layer 5 for a sweat-measuring electrode is chromatographic paper or other elastic, porous or smooth material selected from the group consisting of polyamide and polyethylene terephthalate. Embodiment 11. The system of any preceding embodiments, further comprising battery powered electrical circuit to provide an electric current to the electrodes. Embodiment 12. The system of any preceding embodiments, further comprising a wireless transmission module configured to communicate measurement data to an external device. Embodiment 13. A biosensor for measuring analyte concentrations in sweat, comprising the system of claim 1 integrated into a skin patch configured to be removably adhered to human skin, wherein the system further includes a battery-powered electrical circuit and a wireless communication module for transmitting analyte measurement data to an external electronic device. Embodiment 14. A method of non-invasive measurement of analyte concentrations in a subject, comprising: (a) applying a system according to claim 1 to a skin surface of the subject; (b) inducing sweat secretion by activating the sweat-inducing device to deliver an electric current via the electrodes through a hydrogel carrier containing a sweat- inducing compound; (c) collecting sweat into a sample chamber of the sweat-measuring device; and (d) measuring an electrical parameter change in the electrochemical system of the sweat-measuring device to determine the concentration of at least one analyte present in the sweat. Embodiment 15. The method of embodiment 14, wherein the analyte is selected from the group consisting of glucose, ammonia, prostaglandins, C12-C22 fatty acids, ethanol, lactate, urea, cortisol, hydroxybutyrate, sodium, potassium, chloride, zinc, vitamin C and D, and drugs. Embodiment 16. A kit for non-invasive electrochemical measurement of analyte concentrations in bodily secretions, comprising one or more packaged system forming a biosensor according to embodiment 1 and instructions, wherein the kit comprises one or more skin patches individually packaged in sterile containers or wrappings configured for a single use. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS REFERENCES 1. Yuan, X. et al., Epidermal Wearable Biosensors for Monitoring Biomarkers of Chronic Disease in Sweat. Biosensors 2023, 13, 313. see worldwide website: doi.org / 10.3390 / bios13030313. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS

Claims

CLAIMS 1. A system for non-invasive electrochemical measurement of analyte concentrations in bodily secretions comprising: (a) a sweat-inducing device comprising: (i) a first layer including a base material 13 having openings 14 filled with a hydrogel carrier 15, 16 containing a sweat-inducing compound; (ii) a second layer including at least two planar electrodes, being an anode 11 and a cathode 12, which are configured to apply an electric current to a skin surface through the hydrogel carrier to induce sweat secretion, wherein the second layer is covered with an isolating layer 10; and (b) a sweat-measuring device operably coupled with the sweat-inducing device through the isolating layer 10 of the sweat-inducing device and comprising: (i) a working electrode 4 including a carbon paste modified with graphene and gold nanoparticles; (ii) a counter electrode 3; and (iii) a reference electrode 2, wherein the working electrode 4, the counter electrode 3, and the reference electrode 2, form a system of sensing electrodes that are surrounded by a base layer 5, creating a space for sample collection 6, a space for the deposition of a biochemical analyte recognition system 7, and a space for the deposition of potassium chloride 8, wherein the electrodes are covered with an isolating layer 1 with openings 9, and wherein the electrodes form an electrochemical system configured to detect the presence and concentration of one or more analytes in the induced sweat sample.

2. The system of claim 1, wherein the sweat-inducing compound is pilocarpine.

3. The system of claim 1, wherein the hydrogel carrier is selected from consisting of agarose.

4. The system of claim 1, wherein the electrodes of the sweat-inducing device are manufactured by thin layer technology, thick layer technology and / or by pyrolysis of the base material resulting in a LIG (laser-induced graphite / graphene) type layer. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS5. The system of claim 1, wherein the sweat-inducing electrodes are planar and have similar surface areas, with a cathode 12 surrounded by an anode 11.

6. The system of claim 1, wherein the working electrode 4 comprises: (a) a first layer comprising a carbon paste layer modified with a mixture of graphene and gold nanoparticles, wherein graphene is unmodified or modified, and wherein gold particles are covered with a thiol-based mediator or thiol-based ferrocene, (b) a second layer comprising an oxidoreductase enzyme mixed with a cross-linking reagent, such as glutaraldehyde; and (c) a third layer comprising a film made on the surface of a second layer of the working electrode.

7. The system of claim 6, wherein modified graphene is modified with a mediator or linker which are selected from the group consisting of FAD, NAD, ferrocene, TTF, and osmium pyridyl complexes, a thiol-based mediator comprising FAD, NAD, ferrocene, TTF, osmium pyridyl complexes or neutral thiol, ., 8. The system of claim 6, wherein the thiol-based mediator or thiol-based linker for modification of gold nanoparticles is selected from the group consisting of J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS.

9. The system of claim 1, wherein a reference electrode 2 is a pseudo-reference type electrode being a screen-printed silver chloride layer or in a form of the saturated reference electrode in the form of a screen-printed silver chloride layer in direct contact with a high level of ions, against which the electrode is reversible (Cl-), and a counter electrode 3 is a carbon, platinum or silver electrode.

10. The system of claim 1, wherein a base layer 5 for a sweat-measuring electrode is chromatographic paper or other elastic, porous or smooth material selected from the group consisting of polyamide and polyethylene terephthalate. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS11. The system of claim 1, further comprising battery powered electrical circuit to provide an electric current to the electrodes.

12. The system of claim 1, further comprising a wireless transmission module configured to communicate measurement data to an external device.

13. A biosensor for measuring analyte concentrations in sweat, comprising the system of claim 1 integrated into a skin patch configured to be removably adhered to human skin, wherein the system further includes a battery-powered electrical circuit and a wireless communication module for transmitting analyte measurement data to an external electronic device.

14. A method of non-invasive measurement of analyte concentrations in a subject, comprising: (a) applying a system according to claim 1 to a skin surface of the subject; (b) inducing sweat secretion by activating the sweat-inducing device to deliver an electric current via the electrodes through a hydrogel carrier containing a sweat- inducing compound; (c) collecting sweat into a sample chamber of the sweat-measuring device; and (d) measuring an electrical parameter change in the electrochemical system of the sweat-measuring device to determine the concentration of at least one analyte present in the sweat.

15. The method of claim 14, wherein the analyte is selected from the group consisting of glucose, ammonia, prostaglandins, C12-C22 fatty acids, ethanol, lactate, urea, cortisol, hydroxybutyrate, sodium, potassium, chloride, zinc, vitamin C and D, and drugs.

16. A kit for non-invasive electrochemical measurement of analyte concentrations in bodily secretions, comprising one or more packaged system forming a biosensor according to claim 1 and instructions, wherein the kit comprises one or more skin patches individually packaged in sterile containers or wrappings configured for a single use. J:\SDP\102XC1PCT\Application\SDP-102XC1PCT-Application.docx / mv / FS

Images