Lactate sensor

Abstract

A wearable device comprising a sweat biosensor (1) for lactate, comprising: a sweat collection inlet (2) arranged in the device for collecting sweat when the device is sworn by a user, a microfluidic channel for conveying the collected sweat from the inlet to the sweat biosensor (1), a lactate biosensor (3), which operates by means of amperometry comprising a working electrode (4) comprising an immobilized lactate oxidase and a redox molecule film under a constant potential to reduce the said redox molecule, wherein the measuring electrode (4) of the said lactate biosensor (3) comprises a diffusion-limiting membrane comprising PVC and dioctyl sebacate in a weight ratio 1:4 to 2:1 and wherein the processing unit is hermetically sealed (6).

Description

[0001] LACTATE SENSOR

[0002] Technical Field

[0003] The present invention relates to a wearable device for measuring lactate concentration in sweat.

[0004] Prior art

[0005] It is known in the prior art to assess the lactate levels to reflect the anaerobic metabolic activity. During exercise, especially in high-intensity activities, the body utilizes the anaerobic pathway to generate energy. In this process, when the energy demand exceeds the capacity of aerobic metabolism, pyruvate (one of the products of glycolysis) is converted into lactate through the action of the enzyme lactate dehydrogenase, producing ATP in the process. This lactate is not simply a waste byproduct; rather, it acts as an alternative fuel and an indicator of the balance between production and clearance in the muscle, which influences the onset of fatigue and the training response.

[0006] Lactate measurement is key to determining the aerobic and anaerobic thresholds, fundamental parameters for planning training and improving athletic performance. Traditionally, it has been done using a blood sample, but lactate monitoring in sweat offers a non-invasive and continuous alternative. This allows real-time tracking of the athlete's metabolic response, enabling immediate adjustments in training intensity, preventing overtraining, and optimizing individualized planning.

[0007] Similarly, tumors may be hypoxic, which will, again, produce higher lactate levels, at least locally.

[0008] The measurement of sweat lactate levels has been proposed as a more practical alternative: this could be an indicator for both sport performance and physiology. However, according to Xuan et al., Analysis and Sensing, 2022, doi.org / 10.1002 / anse.202200047, among the ten studies identified, four have found an inverse relationship between sweat lactate levels, one study was unconclusive and five suggested a positive relationship between lactate and exercise.To overcome these discrepancies, Xuan et al. developed a wearable sweat lactate sensor, where sweat is continuously measured and the usefulness of measuring sweat as an indicator of sport performance has been suggested. In practice Xuan et al. have developed an epidermal patch comprising a lactate biosensor, a sweat inlet connected to a microfluidic channel, in which the electrodes needed for the amperometry readout (working, reference, and counter) were placed, followed by the outlet. With such a design, once the inlet is full, the sweat flows through the channel until it reaches the outlet. The sweat is continuously replenished in the inlet, and thus in the channel due to active perspiration. The working electrode is a lactate biosensor based on measuring the peroxide (H2O2) formed as a subproduct in the reaction of lactate with the lactate oxidase enzyme.

[0009] A stable linear range for lactate measurement in sweat has been reported between 1 and 20 mM, with a correlation coefficient of 0.813 to blood lactate levels. However, this correlation weakens as exercise intensity increases, leading to spikes in lactate levels in both sweat and blood: a significant portion of valuable exercise data might be overlooked, particularly at higher intensities where lactate surpasses the 20 mM threshold.

[0010] This limitation highlights a significant drawback of the device. Despite its potential, it fails to capture the full range of lactate concentrations, especially during high-intensity exercise, where levels can reach up to 40-45 mM. Consequently, crucial physiological information could be missed, reducing the device's effectiveness in accurately monitoring exercise performance.

[0011] Brief summary of the invention

[0012] A first aspect of the present invention relates to a wearable device 1 to be fixed on the skin of a user for providing lactate measurement in real-time, the said device 1 comprising:

[0013] a sweat collection inlet 2 arranged in the device 1 for collecting sweat when the device 1 is sworn by the said user,

[0014] a microfluidic channel 9 for conveying the collected sweat from the inlet 2 to a lactate biosensor 3, which operates by means of amperometry, said lactate biosensor 3 comprising a working electrode 4 comprising a redox molecule layer and a chitosan-immobilized lactate oxidase,a processing unit 5 adapted for receiving and processing data provided by the lactate biosensor 3,

[0015] wherein the working electrode 4 of the said lactate biosensor 3 comprises a diffusion-limiting membrane comprising polyvinyl chloride (PVC) and dioctyl sebacate in a weight ratio from 1 :4 to 2:1 and wherein the processing unit 5 is hermetically sealed 6 and wherein the working electrode 4 measures the current generated upon oxidation of the said redox molecule.

[0016] Preferably, this device is further comprising a reference electrode 7 comprising a polyvinyl butyral layer, and being preferably an Ag / AgCI electrode.

[0017] Preferably, the diffusion membrane of the working electrode 4 consists essentially of PVC, dioctyl sebacate, and

[0018] tetradodecylammonium tetrakis(4-chlorophenyl) borate (ETH500), or tridodecylmethylammonium chloride (TDMACL).

[0019] In one preferred alternative development, the electrodes are screen-printed electrodes (SPE), advantageously fabricated by screen-printing technology. Preferably, the working electrode 4, and the counter electrode are printed using a carbon-based conductive ink, and the reference electrode 7 is printed using a silver / silver chloride (Ag / AgCI) ink.

[0020] In the other preferred alternative, all the electrodes, including the counter electrode, the reference electrode 7, and the working electrode 4, are made of graphene, advantageously produced by laser-induced graphene (LIG) formation on a polyimide film using a CO2laser. Preferably, in this alternative, the working electrode and the counter electrode are directly formed as LIGs, while the reference electrode is subsequently functionalized by deposition or printing of a silver / silver chloride (Ag / AgCI) ink over the graphene surface, thereby forming a stable reference electrode.

[0021] As mentioned, preferably one, two, or the three electrodes including, the counter, the reference 7 and the working electrode 4 are made of graphene, advantageously produced by a CO2 laser over a polyimide film. The laser-induced graphene is obtained by irradiating the polyimide substrate with a CO2laser. Preferably, in this use, the electrode(s) are obtained using a laser wavelength comprised between about 8000 and 15000 nm (or 13000 nm), preferably between about 9000 and 12000 nm, more preferably is of (about) 10,600 nm.

[0022] The laser-induced graphene is generated by irradiating the polyimide substrate under conditions delivering a sufficient energy density to locally convertthe polyimide into conductive graphene while avoiding ablation or excessive degradation of the substrate. The laser system may have a nominal output power comprised between 10 and 200 W, more preferably between 20 and 60 W. The laser power applied during irradiation is preferably controlled as a percentage of the nominal laser power, typically between 1% and 30%, depending on the laser system used and the focal conditions.

[0023] The laser scan speed is preferably comprised between 1 and 1,500 mm / s, more preferably between 10 and 300 mm / s (or 10 and 100 mm / s), the selected value being adjusted in combination with the applied laser power and focal distance.

[0024] Preferably, the laser scan speed is comprised between 1 and 300 mm / s, more preferably between 10 and 150 mm / s (or 100 mm / s).

[0025] Advantageously, the laser focus may be adjusted by applying a z-offset relative to the focal plane, preferably between -10 mm and +5 mm, in order to modulate the energy density delivered to the polyimide surface and to control the morphology and conductivity of the resulting graphene layer.

[0026] It is understood that the absolute values of laser power, scan speed and focus depend on the laser platform used; however, equivalent LIG electrodes are obtained by selecting combinations of these parameters that deliver comparable energy densities at the substrate surface.

[0027] Advantageously, this device is to be applied to the skin in a sealed fashion, preferably wherein the processing unit 5 is sealed with a silicon, adhesive and / or a neoprene barrier 6.

[0028] Preferably, the lactate oxidase is immobilized in a chitosan network, advantageously obtained upon addition of chitosan solution (1%).

[0029] Preferably, the redox molecule comprises a redox-active material selected from Prussian Blue, tetrathiafulvalene, or grafted-polymerized MgO-templated carbon.

[0030] More preferably, the redox molecule is Prussian Blue, also referred to as iron(lll) hexacyanoferrate(ll), wherein the redox layer may reversibly operate between the Prussian Blue and Prussian White redox states. Advantageously, the Prussian Blue layer is formed on the working electrode surface either by electrodeposition or by in situ formation from precursor solutions. Preferably, the in situ formation of the Prussian Blue layer is performed by deposition of precursorsolutions onto the electrode surface, said deposition being carried out either manually by drop-casting or, more preferably, by automated liquid dispensing.

[0031] Automated dispensing allows increased reproducibility across lactate biosensors, especially the layer of the redox molecule (preferably Prussian Blue), the lactate oxidase and chitosan, and / or the layer for the diffusion limiting membrane.

[0032] Preferably, in this device, the working electrode 4 is placed at less than 2 cm from the sweat collection inlet 2, preferably less than 1 cm, more preferably less than 5 mm or less than 3 mm.

[0033] Preferably, this device comprises no pH and / or temperature probe(s).

[0034] A related aspect of the present invention is the use of one or several graphene electrode(s) for measuring lactate concentration in sweat.

[0035] A related aspect of the present invention is a process for the large-scale production of this device 1 , comprising the following steps:

[0036] deposition of an aqueous composition comprising the precursors of the redox molecule layer or the redox molecule layer on the working electrode 4, followed by drying, being preferably air drying and annealing at 100 °C, preferably wherein the precursors of the redox molecule (Prussian Blue) are potassium hexacyanoferrate(lll) and iron(lll) chloride,

[0037] deposition of an aqueous composition, possibly having a viscosity of less than 1000 cP (25°C), comprising lactate oxidase and chitosan on the working electrode 4 coated with the said redox molecule layer and air-drying, and deposition of the diffusion-limiting membrane on the lactateoxidase coated working electrode 4 being solved in tetra hydrofuran (THF) / cyclohexanone, preferably in a volume ratio comprised between 25:75 and 100:0 (THF:cyclohexanone), and of evaporating the said THF / cyclohexanone.

[0038] Preferably, in this process, the diffusion-limiting membrane is deposited in an automatic fashion, and / or in a computer- and / or camera-controlled 3D fashion. Advantageously, this (automatic) deposition is contact-free.

[0039] Preferably, in this process, the diffusion-limiting membrane consists essentially of PVC, dioctyl sebacate and tetradodecylammonium tetrakis(4-chlorophenyl) borate (ETH500).Preferably, in this process, the diffusion-limiting membrane is deposited in an automatic fashion, and / or in a computer- and / or camera-controlled 3D fashion. Advantageously, this (automatic) deposition is contact-free.

[0040] Preferably this process further comprises the step of applying a polyvinyl butyral layer on a reference electrode 7, the said polyvinyl butyral to be applied being in a dispersion in an organic phase, the said dispersion having a viscosity of less than 500 cP, and of evaporating the organic phase after application of the said composition on the said reference electrode 7.

[0041] Preferably, the deposition of the diffusion-limiting membrane and / or of other functional layers is performed using an automated liquid dispensing system functionally, such as Nordson EFD EV2 series jetting dispenser, defined as a system capable of non-contact dispensing of controlled liquid volumes onto a substrate surface. Such a system preferably provides high positioning precision, allowing the deposition location and geometry to be accurately and reproducibly defined by computer-controlled motion systems and / or camera-based alignment.

[0042] Advantageously, the system enables high volumetric accuracy and repeatability, for micro- and sub-microlitre volumes, and is suitable for dispensing liquids over a broad viscosity range, including aqueous compositions and organic solvent-based formulations. Preferably, the system is programmable to perform repeated and sequential deposition steps on multiple devices, thereby enabling series production or mass production with consistent deposition times, volumes and covered areas.

[0043] The automated liquid dispensing system is further preferably configured to control process parameters including inlet pressure, dispensing speed, pulse duration, stroke or actuation force, and valve closing voltage, said parameters being adjustable in relation to the tip or nozzle geometry, in particular the tip or nozzle orifice diameter.

[0044] The inventors have found it to be especially useful when THE solvent is used.

[0045] Advantageously, said parameters are adjusted as a function of the geometry of the dispensing nozzle, in particular the diameter of the nozzle orifice, in order to ensure stable jetting, accurate volume delivery and reproducible deposition on the electrode surface.

[0046] Advantageously, the dispensing valve is positioned at a controlled distance from the electrode surface, and the deposition location and area arereproducibly defined, optionally by computer-controlled positioning and / or camera-based alignment.

[0047] Preferably, the inlet pressure is comprised between 0.1 and 5 bar, more preferably between 0.3 and 2 bar, still more preferably between 0.5 and 1.5 bar, still more preferably between 0.8 and 1.2 bar.

[0048] Preferably, the dispensing speed is comprised between 0.1 and 100 mm / s, more preferably between 1 and 30 mm / s, still more preferably between 5 and 20 mm / s, such as between 10 and 15 mm / s.

[0049] Preferably, the pulse duration is comprised between 0.25 ms and 10 ms, more preferably between 0.5 ms and 5 ms, still more preferably between 1 and 2 ms, thereby controlling the amount of composition dispensed through the nozzle.

[0050] Preferably, the stroke parameter is adjusted to provide a sufficient dispensing force to ensure reproducible droplet formation without splashing or spreading beyond the working electrode area. Preferably, the stroke parameter is comprised between 60 and 110, more preferably between 65 and 80.

[0051] Preferably, a closing voltage is applied to the dispensing valve in order to prevent leakage of the composition, said closing voltage being comprised between 80 and 120 V.

[0052] Preferably, the dispensing valve is positioned at a distance from the electrode surface comprised between 2 and 5 mm, more preferably about 3 mm.

[0053] As above-mentioned, in the preferred alternative, the electrodes are fabricated by screen-printing technology. Preferably, the working electrode 4, and the counter electrode are printed using a carbon-based conductive ink, and the reference electrode 7 is printed using a si Iver / silver chloride (Ag / AgCI) ink.

[0054] Preferably, the redox molecule is Prussian Blue and / or the deposition method of the redox molecule is automated.

[0055] Brief description of the Drawings

[0056] Figure 1 is a schematic view of the wearable device according to the invention

[0057] Figure 2 shows the current / concentration relationship of different formulations of the diffusion-limiting membrane.

[0058] In the drawings, the same reference numbers have been allocated to the same or analogue element.Detailed description of the invention

[0059] The inventors have developed a unique expertise in wearable devices for lactate sensors in sweat. In their development work, they have found a diffusionlimiting membrane allowing (i) a broad linear response rate, (ii) a reasonably good sensitivity, (iii) a reasonable limit of detection and (iiii) an acceptable response time.

[0060] They have also found a process to apply the different layers on the working (measuring) electrode at an industrial scale and how to arrange the different elements so as to avoid leakage while ensuring a correct flux of sweat from the user's body through the working (measuring) electrode.

[0061] A first object of the present invention is a wearable device 1 for measurement in real-time of lactate concentration in sweat, the said device 1 comprising:

[0062] a sweat collection inlet 2 arranged in the device for collecting sweat when the device 1 is sworn by a user,

[0063] a microfluidic channel 9 for conveying the collected sweat from the inlet 2 to a lactate biosensor 3,

[0064] the said lactate biosensor 3, which operates by means of amperometry, comprising a working electrode 4 comprising an immobilized lactate oxidase within a chitosan network and a redox molecule film (or a film comprising a redox molecule),

[0065] a processing unit 5 adapted for receiving and processing data provided by the sweat lactate sensor,

[0066] wherein the working (measuring) electrode 4 of the said lactate biosensor 3 comprises a diffusion-limiting membrane and wherein the said lactate biosensor3 provides lactate measurement in real-time, wherein the diffusion-limiting membrane of the working (measuring) electrode comprises PVC and dioctyl sebacate in a weight ratio comprised between 1 :4 to 2:1, preferably between 1 :3 and 1 :1 and wherein the processing unit is hermetically sealed 6.

[0067] This wearable device is to be applied on the skin.

[0068] Preferably, the working (measuring) electrode 4 is placed at less than 2 cm from the inlet 2.

[0069] This ensures a rapid and unbiased measurement of the lactate concentration in sweat.Preferably, a current is applied to reduce the redox molecule and / or to ensure a minimal amount of the redox molecule in an oxidized state.

[0070] This allows to reflect the H2O2 production generated by the immobilized lactate oxidase when contacted to sweat lactate.

[0071] Preferably the device of the present invention does not comprise pH probe and / or pH sensor.

[0072] The inventors have found that these additional sensors, although appearing to provide interesting complementary information, are difficult to combine with the lactate sensor (working, measuring electrode 4) since the collected sweat does not reach all the sensors at the same time.

[0073] Preferably, the diffusion-limiting membrane of the working (measuring) electrode 4 consists essentially of PVC, dioctyl sebacate (DOS) and either tetradodecylammonium tetrakis(4-chlorophenyl) borate (ETH500) or tridodecylmethylammonium chloride (TDMACI), preferably the ETH500 or the TDMACI being incorporated in a weight percent between 2 and 10% ((weight ETH500 orTDMACI):(weight ETH500 orTDMACI+weight PVC+weight DOS)), preferably between 2.2 and 8, more preferably between 2.4 and 7 %; ETH500 is preferred over TDMACI.

[0074] The inventors have found that the combined use of PVC and dioctyl sebacate at the right relative ratios allows a good optimum between the abovelisted parameters.

[0075] Preferably, the diffusion-limiting membrane is around 10 m thick and / or has a weight comprised between 200 and 1000 g.

[0076] Preferably, this device 1 further comprises a reference electrode 7 comprising a polyvinyl butyral (PVB) layer. A PVB membrane saturated with chloride in the reference electrode ensures stable potential and accurate measurements in amperometric lactate detection in sweat. It enhances durability, especially reference electrode durability, and protects against contaminants, ensuring reliable long-term performance.

[0077] Preferably, the lactate oxidase is immobilized on, or in, a porous network or (porous) resin. The inventors have identified that chitosan is advantageous to act as a porous network to anchor the enzyme.This allows to gently immobilize the enzyme as well as the diffusion of lactate.

[0078] Preferably, the redox molecule is Prussian Blue (iron hexa cyanoferrate; CAS 14038-43-8), tetrathiafulvalene, or grafted-polymerized MgO-templated carbon. The most preferred redox molecule is Prussian Blue; the inventors have found that the molecule is easily reduced by an electric source into Prussian White, then oxidized by reacting with the co-product of the lactate oxidase, hydrogen peroxide.

[0079] Advantageously, this device is applied to the skin in a sealed fashion, preferably the processor is isolated from the sweat through a tight junction 6, preferably with an additional silicone or adhesive barrier and / or with an additional neoprene barrier, as well as O-ring or an adhesive foam.

[0080] Preferably, all the electrical connections are protected by insulating material such as rubber.

[0081] A related aspect of the present invention is a process for the large-scale production of the above device 1 comprising the following steps:

[0082] deposition of an aqueous composition comprising the precursors of the redox molecule layer on the working (measuring) electrode 4, followed by air-drying and annealing,

[0083] deposition of an aqueous composition having a viscosity of less than 1000 cP (25°C), preferably of less than 500 cP, more preferably of less than 100 cP, still more preferably of less than 50 cP comprising lactate oxidase and chitosan on the working (measuring) electrode 4 and air-drying, and

[0084] deposition of the diffusion-limiting membrane in a composition comprising THF / cyclohexanone on the working (measuring) electrode 4, preferably in a volume ratio comprised between 25:75 and 100:0 (THF:cyclohexanone), and of evaporating the said THF / cyclohexanone.

[0085] Usually, between 0.5 and 5 l of the composition comprising THF and the membrane is applied. The inventors have found an important risk to block the dispensing head for the solution / suspension, especially the suspension for the membrane. The inventors have found that the risk is major when the membrane is dissolved in THF or in THF / cyclohexanone and when the application of the membrane in THF is applied manually or too slowly: due to the rapid evaporation,local heterogeneities are formed, directly affecting the end-product. The automated system, allowing a rapid and ultra-precise deposition, without any air contact, of the composition comprising the membrane, has overcome this issue.

[0086] Preferably, in such process, the diffusion-limiting membrane consists essentially of, PVC, dioctyl sebacate and tetradodecylammonium tetrakis(4-chlorophenyl) borate (ETH500).

[0087] This process preferably further comprises the step of applying a polyvinyl butyral layer on a reference electrode 7, the said polyvinyl butyral to be applied being in a dispersion in an organic phase, the said dispersion having a viscosity of less than 500 cP, preferably of less than 300 cP, still more preferably of less than 200 cP and of evaporating the organic phase after application of the said composition on the said reference electrode.

[0088] Such a process is advantageously performed at room temperature (e.g. between 15°C and 30°C, preferably at about 20°C) and at atmospheric pressure.

[0089] Other characteristics and advantages of the present invention will be derived from the non-limitative following description, and by making reference to the drawings and the examples.

[0090] It should be understood that the present invention is not limited to the described embodiments and that variations can be applied without going outside of the scope of the claims.

[0091] Examples

[0092] The inventors have started from their own publication of Xuan et al. Example 1 - Components of the barrier membrane.

[0093] The working (measuring) electrode to be incorporated in the wearable device is firstly recovered with a layer where a redox molecule is embedded; here, Prussian Blue (Prussian white, to be oxidized into Prussian Blue), then with a layercomprising immobilized lactate oxidase and chitosan, and finally with a diffusionlimiting membrane.

[0094] The Prussian Blue (Prussian white) layer is applied in a water suspension of its precursors, so as the lactate oxidase suspension (after the first layer has been dried and annealed). The diffusion-limiting membrane is then applied in an organic solvent, such as DMF (dimethylformaldehyde), THF and cyclohexanone. In this Example, the inventors used THF as the organic solvent.

[0095] For the diffusion-limiting membrane, the inventors have selected PVC membranes, to be formulated with either ETH500 or TDMACL (tridodecylmethylammonium chloride), and with dioctyl sebacate (DOS), according to the Table 1 here below.

[0096] Table 1: Composition of the membranes assayeda

[0097] Weight (mg) of the components

[0098] Membrane

[0099] PVC / mg DOS / mg ETH500 / mg TDMACI / mg M1 33 66

[0100] M2 33 66 3

[0101] M3 33 66 6

[0102] M4 33 66 9

[0103] M5 33 66 3

[0104] M6 33 66 6

[0105] M7 33 66 9

[0106] M8 50 50 6

[0107] M9 66 33 6

[0108] aAll components are prepared and dissolved in 1 mL of THF.

[0109] Then the inventors have tested the wearable devices with the different diffusion-limiting membranes (see Figure 2); the l values indicate the amount of the composition used to form the diffusion-limiting membrane.

[0110] More into details, dynamic responses and corresponding calibration curves (insets) have been carried out in phosphate buffer solution 0.1 M at increasinglactate concentration using A) no diffusion limiting membrane, B) Ml membrane, C) M2 membrane, D) M3 membrane, E) M4 membrane, F) M3 membrane, prepared with 6 L membrane cocktail, G) M3 membrane, prepared with 7.5 pL membrane cocktail, H) M5 membrane, I) M6 membrane, J) M7 membrane, K) M8 membrane, and L) M9 membrane. 3 pL of membrane cocktail was used for drop-casting preparation of each membrane, except for the conditions F and L (6 pl) and G (7.5 pl).

[0111] The polymeric membranes act as diffusion-limiting elements in the lactate biosensor, expanding the sensing range to the millimolar level (>10 mM) compared to the case without membrane, Figure 2a (<0.5 mM). However, the dynamic responses of the biosensor are significantly influenced by the composition of the membrane. Particularly noteworthy was the effect of introducing a lipophilic salt (Figure 2c - I), which enhanced the linear range as compared (>15 mM) to membranes without the lipophilic salt (Figure 2b, <10 mM).

[0112] The main results are summarized at the Table 2

[0113] ,, , Linear range / . ,aSensitivity / LOD / Membrane mM t 95% I s nA . mM-,1m^|

[0114] meWmib“rane 0.05 - 0.5 28 ± 6

[0115]

[0116] A0 033

[0117] M1 1 - 10 198 ± 8 11.5 ± 0.5 0.51 M2 1 - 20 123 ± 8 10.3 ± 0.4 0.24 M3 (3 pL) 1 - 20 107 ± 8 14.6 ± 0.5 0.29 M3 (6 pL) 1 - 25 177 ± 36 8.1 ± 0.3 0.66 M3 (7.5 pL) 1 - 40 187 ± 11 1.18 ± 0.3 1.51 M4 1 - 20 56 ± 10 22.8 ± 1.2 0.31 M5 1 - 20 188 ± 10 9.5 ± 1.2 0.35 M6 1 - 15 126 ± 18 17.0 ± 0.7 0.39 M7 1 - 10 91 ± 23 21.6 ± 1.4 0.23 M8 1 - 15 223 ± 21 9.3 ± 0.8 0.33M9 1 - 10 210 ± 15 7.4 ± 0.5 0.47aThe response time (tgs%) was calculated with the highest concentration within the linear range.

[0118] As shown in Table 2 or Figure 2, the dynamic range can be expanded, at the expense of the detection limit or of the sensitivity. However, the presence of the diffusion-limiting membrane also prolonged the response time.

[0119] In a subsequent run of experiments, the inventors have developed a membrane with 33% PVC, 66% DOS and 2.5 mg / mL ETH550 with good results.

[0120] Example 2; Process for scaling-up

[0121] The inventors have found that THE, which is used in Example 1 for manual dispensing of the diffusion-limiting membrane, evaporates extremely quickly and risks blocking the dispensing head of the composition for the diffusion-limiting membrane. When combined with the very small dispensing volumes required (typically between 0.5 and 10 L), this rapid evaporation leads to significant variations in the covered electrode area and membrane thickness, making the deposition process irreproducible On the other hand, the inventors have found that the mixture of THE with cyclohexanone, together with an automated liquid dispensing system such as a Nordson EFD EV2 series jetting dispenser, overcomes these limitations by reducing the evaporation rate while maintaining a viscosity compatible with automated dispensing overcomes this issue.

[0122] Regarding the dispensing area and the amount of solution dispensed, experimental trials revealed that the size, shape and reproducibility of the deposited solution are influenced by several adjustable dispensing parameters, including the viscosity of the solution, the pressure exerted on the syringe barrel introducing the solution into the jetting dispensing valve, and the jetting valve operating parameters, such as pulse duration, closing voltage and stroke. Control of these parameters allows reproducible deposition patterns, including circular or linear dispensing geometries, with a fixed and well-defined deposition area across multiple electrodes.Furthermore, the use of automated dispensing ensures identical dispensing times for each electrode, resulting in uniform evaporation conditions and significantly improved reproducibility in membrane thickness and surface coverage, which is essential for large-scale manufacturing.

[0123] On the other hand, the inventors have compared electrochemical deposition of Prussian Blue and drop-casting of Prussian Blue; the latter allows an expanded linear range, reduced response time and detection limit, while maintaining sensitivity.

Claims

CLAIMS1. A wearable device ( 1 ) to be fixed on the skin of a user for providing lactate measurement in real-time, the said device (1) comprising:a sweat collection inlet (2) arranged in the device ( 1 ) for collecting sweat when the device (1) is sworn by the said user,a microfluidic channel (9) for conveying the collected sweat from the inlet (2) to a lactate biosensor (3), which operates by means of amperometry, said lactate biosensor (3) comprising a working electrode (4) comprising an immobilized lactate oxidase, in a chitosan network, and a redox molecule film,a processing unit (5) adapted for receiving and processing data provided by the lactate biosensor (3),wherein the working electrode (4) of the said lactate biosensor (3) comprises a diffusion-limiting membrane comprising PVC and dioctyl sebacate in a weight ratio from 1 :4 to 2:1 and wherein the processing unit (5) is hermetically sealed (6) and wherein the working electrode (4) measures the current generated upon oxidation of the said redox molecule.

2. The device of claim 1 further comprising a reference electrode (7) comprising a polyvinyl butyral layer, and being preferably an Ag / AgCI electrode.

3. The device of claim 1 or 2, wherein the diffusion membrane of the working electrode (4) consists essentially of PVC, dioctyl sebacate, and tetradodecylammonium tetrakis(4-chlorophenyl) borate (ETH500), or tridodecylmethylammonium chloride (TDMACL).

4. The device according to any one of the preceding claims, wherein the working electrode (4) comprises a carbon ink screen-printed electrode or a graphene electrode, preferably a laser-induced graphene electrode.

5. The device according to any one of the preceding claims being applied to the skin in a sealed fashion, preferably wherein the processing unit (5) is sealed with a silicone or adhesive and / or a neoprene barrier (6).

6. The device according to any one of the preceding claims, wherein the redox molecule is Prussian Blue, tetrathiafulvalene or grafted-polymerized MgO- templated carbon, preferably Prussian Blue, preferably wherein the Prussian Blue layer has been applied by drop-casting.

7. The device according to any one of the preceding claims wherein the working electrode (4) is placed at less than 2 cm from the sweat collection inlet (2).

8. The device according to any one of the preceding claims comprising no pH and / or temperature probe(s).

9. A process for the large-scale production of the device (1) according to any one of the preceding claims comprising the following steps:deposition of an aqueous composition comprising the redox molecule, or potassium hexacyanoferrate(lll) and iron(lll) chloride, on the working electrode (4) and of air-drying and annealing,deposition of an aqueous composition having a viscosity of less than 1000 cP (25°C) comprising lactate oxidase and chitosan on the measuring electrode coated with the said redox molecule and of air-drying, and deposition of the diffusion-limiting membrane on the lactate-oxidase coated working electrode (4) being in a composition comprising THF / cyclohexanone, preferably in a volume ratio comprised between 25:75 and 100:0 (THF:cyclohexanone), and of evaporating the said THF / cyclohexanone.

10. The process of claim 10, wherein the diffusion-limiting membrane consists essentially of PVC, dioctyl sebacate and tetradodecylammonium tetrakis(4- chlorophenyl) borate (ETH500), and / or wherein the redox molecule is Prussian Blue and / or wherein the deposition method of the redox molecule is by drop casting.

11. The process of claims 10 or 11, wherein one, two or the three the deposition steps is (are) automated, preferably wherein the step of depositing the diffusion-limiting membrane is automated and / or the step of depositing the composition comprising the lactate-oxidase and the chitosan is automated.

12. The process of claim 11, wherein one or several of the parameter(s) is (are) controlled, said parameters being selected from inlet pressure, dispensing speed, pulse duration, stroke or actuation force, and valve closing voltage, said parameters being adjustable in relation to the tip or nozzle geometry, in particular the tip or nozzle orifice diameter, preferably wherein the inlet pressure is comprised between 0.1 and 5 bar, and / or the dispensing speed is comprised between 0.1 and 100 mm / s, and / or the pulse duration is comprised between 0.25 ms and 10 ms, and / or the stroke parameter iscomprised between 60 and 110, and / or a closing voltage is applied to the dispensing valve in order to prevent leakage of the composition, said closing voltage being comprised between 80 and 120 V, and / or the dispensing valve is positioned at a distance from the electrode surface comprised between 2 and 5 mm.

13. The process according to any one of the preceding claims 9 to 12, wherein the drying steps comprise air-drying, preferably at room temperature and / or at 100 °C.

14. The process according to any one of the preceding claims 9 to 13 further comprising the step of applying a polyvinyl butyral layer on a reference electrode (8), the said polyvinyl butyral to be applied being in a dispersion in an organic phase, and of evaporating the organic phase after application of the said composition on the said reference electrode (8).

15. The process according to any one of the preceding claims 9 to 14, wherein the pressure exerted on the syringe barrel introducing the solution into the device is controlled and is within 0.1 - 5 bar.

16. The process according to any one of the preceding claims 9 to 15, wherein one or all the electrode(s) is (are) made of graphene produced by a CO2 laser over a polyimide film.

17. The process according to any one of the preceding claims 9 to 15, wherein at least one electrode is made of carbon by screen-printing technology and / or one electrode is made of Ag / AgCI ink by screen-printing technology.

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