EP3838409A1 - Microfluidic device produced by embossing a paper-based substrate - Google Patents

Abstract

The invention relates to a method for producing a microfluidic device, said method comprising a step of embossing a substrate (2) composed of at least two superimposed layers, a first layer (20) made of paper and a second layer (21) deposited on the first layer and made of a material based on vinylidene polymer and having a so-called upper face (210) to be embossed, said embossing step being performed on said upper face of the second layer of the substrate to form a pattern thereon, said pattern comprising at least one cavity (22) delimited by at least one bottom wall and one side wall, said method also comprising a step of producing conductive electrodes (RE, WE1, WE2) by screen printing at the bottom of said cavity.

Description

Technical field of the invention

The invention relates to a method for producing a microfluidic device as well as to the microfluidic device which can be produced by said method. The method consists in particular in carrying out an embossing of a substrate which is based on paper.

State of the art

In order to establish a biological or medical diagnosis, there is an increasing interest in handling and analyzing small volume liquid samples in the simplest and least intrusive manner possible.

Microfluidics makes it possible to perform manipulations and analyzes on small samples of liquid, of the order of a few tens of microliters to a few milliliters. In general, microfluidic devices are produced by casting crosslinking materials of the PDMS (poly-dimethyl siloxane) type on previously machined molds. The manufacture of these microfluidic devices requires clean room type equipment involving significant means and manufacturing time.

A less expensive technology currently exists which consists in fabricating microfluidic circuits in a paper-based substrate having a hydrophobic surface treatment.

Such solutions are in particular described in the patent application WO2013 / 181656A1 , in the patent application WO2018 / 197814A1 and in the patent application EP3053652A1 .

In these earlier solutions, the microfluidic circuit produced by embossing can be closed from above with a transparent adhesive film using mainly materials based on poly (ethylene terephthalate) (PET) or ethylene vinyl acetate. (EVA). The flow of biological samples is then possible by imposing a positive pressure at the inlet of the circuit using one or more pumps.

However, current devices do not allow measurements to be carried out directly on a biological sample.

There is therefore a need to have a simple, inexpensive solution for carrying out measurements on a biological sample placed in a microfluidic device provided with a paper-based substrate.

Disclosure of the invention

This object is achieved by a method for producing a microfluidic device, this method comprising a step of embossing a substrate composed of at least two superimposed layers, a first layer made of paper and a second layer deposited on it. the first layer and made of a material based on vinylidene polymer and having a so-called upper face to be embossed, said embossing step being performed on said upper face of the second layer of the substrate to form a pattern thereon, said pattern comprising at least at least one cavity delimited by at least one bottom wall and one side wall, said method also comprising a step of producing conductive electrodes by screen printing at the bottom of said cavity.

According to one feature, the electrodes comprise at least one reference electrode and one or more working electrodes.

According to another feature, each electrode is screen printed with silver ink on the substrate.

According to another feature, the reference electrode is then covered with an Ag / AgCl ink and each working electrode with a carbon ink.

According to another feature, an insulator is deposited around each electrode.

The invention also relates to a microfluidic device, the device comprising a substrate composed of at least two superimposed layers, a first layer made of paper and a second layer deposited on the first layer, made of a material based on a polymer of vinylidene and having a so-called upper face to be embossed, said substrate being embossed on at least part of the upper face of its second layer to form a pattern thereon, said pattern comprising at least one cavity delimited by at least one bottom wall and one side wall, said device also comprising conductive electrodes produced by screen printing at the bottom of said cavity.

According to one feature, the electrodes comprise at least one reference electrode and one or more working electrodes.

According to another feature, each electrode is screen printed with silver ink on the substrate.

According to another feature, the reference electrode is then covered with an Ag / AgCl ink and each working electrode with a carbon ink.

According to another feature, the device comprises an insulator deposited around each electrode.

Brief description of the figures

• La figure 1 représente les différentes étapes de préparation et d'analyse d'un échantillon biologique contenant des espèces biologiques ;
• La figure 2 illustre les différentes étapes de réalisation du substrat employé dans un dispositif micro-fluidique conforme à l'invention ;
• Les figures 3A et 3B représentent deux variantes de fabrication d'un dispositif micro-fluidique conforme à l'invention.
• La figure 4 représente un dispositif micro-fluidique comprenant un substrat à base de papier réalisé selon le procédé conforme à l'invention ;
• La figure 5 représente le dispositif de la figure 4, vu en éclaté, et illustre le principe de réalisation de ce dispositif en plusieurs couches ;
• La figure 6 illustre les différentes étapes de réalisation des électrodes conductrices sur un substrat d'un dispositif conforme à l'invention ;

Other characteristics and advantages will appear in the detailed description which follows, made in conjunction with the figures listed below:

• The figure 1 represents the different stages of preparation and analysis of a biological sample containing biological species;
• The figure 2 illustrates the different stages of production of the substrate used in a microfluidic device according to the invention;
• The figures 3A and 3B show two manufacturing variants of a microfluidic device according to the invention.
• The figure 4 represents a microfluidic device comprising a paper-based substrate produced according to the method according to the invention;
• The figure 5 represents the device of the figure 4 , exploded view, and illustrates the principle of making this device in several layers;
• The figure 6 illustrates the different stages of production of the conductive electrodes on a substrate of a device according to the invention;

Detailed description of at least one embodiment

• E1 : Concentration des espèces biologiques présentes dans l'échantillon biologique,
• E2 : Lavage pour purification, pour élimination des interférents de culture,
• E3 : Apport d'un milieu de culture,
• E4 : Culture des espèces biologiques,
• E5 : Suivi optique de croissance lors de la culture et comptage des colonies,
• E6 : Lavage, pour élimination des inhibiteurs de PCR,
• E7 : Lyse mécanique des espèces biologiques présentes dans l'échantillon en vue d'en extraire un matériel biologique à étudier,
• E8 : Séparation entre le matériel biologique à étudier et les polluants présents,
• E9 : Détection de présence de pathogènes dans le matériel biologique par amplification biomoléculaire de type qPCR ("quantitative Polymerase Chain Reaction"), LAMP ("Loop mediated isothermal amplification"), RPA (Recombinase Polymerase Amplification) et détection optique tel que par exemple fluorescence, colorimétrie, imagerie holographique, turbidimétrie, pHmétrie en liaison avec la réaction d'amplification.

The invention aims to use a paper-based substrate for producing a microfluidic device. The microfluidic device obtained will in particular be able to carry out one or more of the stages of preparation and analysis of a biological sample containing biological species. In a non-limiting manner, with reference to the figure 1 , these steps can in particular be the following:

• E1: Concentration of biological species present in the biological sample,
• E2: Washing for purification, for elimination of culture interferents,
• E3: Contribution of a culture medium,
• E4: Culture of organic species,
• E5: Optical growth monitoring during culture and colony counting,
• E6: Washing, for elimination of PCR inhibitors,
• E7: Mechanical lysis of the biological species present in the sample in order to extract a biological material to be studied,
• E8: Separation between the biological material to be studied and the pollutants present,
• E9: Detection of the presence of pathogens in biological material by biomolecular amplification of the qPCR ("quantitative Polymerase Chain Reaction"), LAMP ("Loop mediated isothermal amplification"), RPA (Recombinase Polymerase Amplification) type and optical detection such as fluorescence, for example , colorimetry, holographic imaging, turbidimetry, pHmetry in connection with the amplification reaction.

The object of the present invention is to provide an easily achievable and multifunctional layer on paper in the field of biological diagnosis.

The microfluidic device comprises a substrate 2 formed of at least two layers 20, 21, advantageously only two layers.

• Bonne résistance mécanique à la traction et bonne cohésion de surface préalable,
• Faible absorption d'eau,
• Énergie de surface permettant l'étalement de la préparation de couchage.

The first layer 20 of the substrate 2 is formed from a paper having the following properties:

• Good mechanical tensile strength and good prior surface cohesion,
• Low water absorption,
• Surface energy allowing the spreading of the coating preparation.

In a non-limiting manner, the paper used is sold under the trademark “Powercoat” (registered trademark) having a basis weight of 219 g / m ² .

The second layer 21 of the substrate 2 is deposited on the first layer of “Powercoat”.

• Former une barrière aux liquides et aux gaz ; En effet, lors d'une réaction d'amplification, l'échantillon étant amené à être chauffé au minimum à 65°C pendant 30 min, cette couche doit être suffisamment étanche pour contenir toute la vapeur générée lors du chauffage ;
• Être déformable par embossage à température ambiante, en particulier pour pouvoir réaliser un circuit fluidique ainsi qu'une surface rugueuse permettant la mise en œuvre de l'étape de lyse des bactéries ;
• Être résistante à la déchirure, notamment lors des différentes étapes de préparation de l'échantillon ;
• Être compatible avec l'amplification d'ADN (PCR, LAMP,...) ;
• Être compatible avec les techniques d'électronique imprimée (ex : impression ou sérigraphie de lignes conductrices/ électrodes) ;
• Disposer d'une forte énergie de surface pour permettre un bon étalement de l'échantillon liquide injecté ;

This second layer should advantageously have the following characteristics:

• Form a barrier to liquids and gases; In fact, during an amplification reaction, the sample being heated to a minimum of 65 ° C. for 30 min, this layer must be sufficiently waterproof to contain all the vapor generated during heating;
• Be deformable by embossing at room temperature, in particular to be able to produce a fluid circuit as well as a rough surface allowing the implementation of the bacteria lysis step;
• Be resistant to tearing, in particular during the various stages of sample preparation;
• Be compatible with DNA amplification (PCR, LAMP, ...);
• Be compatible with printed electronics techniques (eg: printing or screen printing of conductive lines / electrodes);
• Have a high surface energy to allow good spreading of the injected liquid sample;

The second layer 21 is advantageously a vinylidene polymer, advantageously “polyvinylidene chloride” (hereinafter PVDC) or polyvinylidene fluoride (PVDF), advantageously polyvinylidene chloride PVDC. In the remainder of the description, in a non-limiting manner, it is chosen to use a second layer 21 based on PVDC.

• les acrylates de méthyle, d'éthyle ou de butyle ;
• l'acrylonitrile, le méthacrylate de méthyle, le méthacrylonitrile ;
• le chlorure de vinyle ;
• des acides carboxyliques insaturés ;

PVDC denotes various copolymers based on vinylidene chloride which can be combined with other polymers with complementary properties. The copolymerization of vinylidene chloride with different comonomers leads to a range of semi-crystalline polymers with remarkable specific properties (impermeability to oxygen, to different gases and to water vapor, sealability, printability, transparency, etc. ). The main comonomers used are:

• methyl, ethyl or butyl acrylates;
• acrylonitrile, methyl methacrylate, methacrylonitrile;
• vinyl chloride;
• unsaturated carboxylic acids;

The figure 2 shows an example of the process for manufacturing substrate 2.

E10: The layer 21 of PVDC is deposited on the first layer 20 of paper by coating. The PVDC layer is deposited at a thickness between 5 and 20 μm, which corresponds to basis weights between 15 and 25 g / m ² . This technique makes it possible to deposit a preparation based on PVDC on the paper then to scrape the excess to keep only a thin functional layer. Drying is then carried out to evaporate the excess water. Drying can be carried out at room temperature; it is possible to carry it out at temperatures up to 70 ° C or even 90 ° C to speed up the process. The PVDC used can be a commercial aqueous emulsion named Diofan A050 (Solvay-registered trademark).

E11: the substrate 2 obtained thus comprises the two superposed layers 20, 21.

The figure 3A shows a first variant of the method for producing a microfluidic device according to the invention, by embossing the substrate 2 with two layers 20, 21.

E20: This step consists in carrying out an embossing of the substrate 2 obtained in order to functionalize the latter with microfluidic channels. The embossing is carried out on the upper face of the second layer.

In known manner, as illustrated on figure 3A , the embossing can consist in placing the substrate between two dies of a press, a lower die M1 in hollow and an upper die M2 in relief. During the press, the reliefs are duplicated on said upper face of the substrate so as to form an imprint. Other techniques can of course be considered.

E21: A hollow is made by embossing said substrate 2, forming a cavity 22.

E22: A substrate 2 is thus obtained provided with the cavity 22 obtained by embossing.

According to one particular aspect, the embossing can thus make it possible to produce a pattern on the upper face of the second layer of the substrate.

According to one particular aspect, the embossing can thus make it possible to produce a pattern forming a concavity on the upper face of the second layer of the substrate. This concavity can be in any possible form. Several patterns can be made juxtaposed on the same substrate in order to obtain several juxtaposed elements

As shown on the figure 3A , it may be a microfluidic imprint in the form of one or more cavities and one or more channels.

By way of example, the cavity may have a hollow shape of constant section over its entire height, with a circular contour. It can also have a hollow shape in the form of a truncated cone. Any other form can be considered.

With reference to the figure 3B , it may also be a rough bearing surface 23 allowing lysis of the biological species contained in a biological sample. This rough bearing surface is thus produced by embossing using a sandpaper (grit 18 to 800, preferably 400 to 600), for example using a vice coupled to a torque wrench (applied moment 10 Nm ). The pressure of the sandpaper against the surface is exerted at a force between 0.5 and 20 MPa and between 3 and 10 MPa in nominal.

• E200 : partant du substrat 2 déjà obtenu par le procédé représenté sur la figure 2, on amène une matrice M10 portant un papier de verre.
• E201 : La matrice M10 supportant le papier de verre est appliquée et comprimée contre la face supérieure du substrat, formée par sa couche de PVDC, sur au moins une partie cette face.
• E202 : La surface d'appui rugueuse 23 est ainsi réalisée sur la face supérieure du substrat. Elle peut également être réalisée au fond d'une cavité, elle-même réalisée par embossage.

For this, the method may include the following steps:

• E200: starting from substrate 2 already obtained by the process shown on figure 2 , we bring an M10 matrix carrying a sandpaper.
• E201: The matrix M10 supporting the sandpaper is applied and compressed against the upper face of the substrate, formed by its PVDC layer, on at least part of this face.
• E202: The rough bearing surface 23 is thus produced on the upper face of the substrate. It can also be produced at the bottom of a cavity, itself produced by embossing.

To carry out the steps of preparation and analysis of a biological sample which are described above, the microfluidic device can be as shown in the figure. figure 4 and comprising a body having a bottom wall 10, a side wall 11 and a top wall 12.

The device 1 comprises a chamber 13 formed in the body. This chamber represents the location in which both purification / concentration, mechanical lysis, separation and optionally detection in biological species can be performed. The chamber 13 is closed at the bottom by the lower wall.

The device comprises a first channel 14 for injecting fluids into the chamber or for discharging fluids outside the chamber. The first channel 14 has a first end comprising an opening formed for example at through the upper wall 12 of the body and a second end which opens into said chamber 13. The first end of the first channel 14 is for example arranged vertically and its second end opens for example horizontally into the chamber 13. The first end of the first channel is for example for example flared to apply the cone of a pipette thereto or will be adapted to the type of device used to inject the fluid into the device.

The device comprises a second channel 15. This second channel 15 also comprises a first end which communicates with the outside, forming an opening made for example through the upper wall and a second end which communicates with the space formed by the chamber 13. Through this second channel 15, it is also possible to inject fluids into said chamber or to evacuate fluids outside said chamber. Its first end is for example arranged vertically and its second end horizontally. The chamber 13 is placed between the first channel 14 and the second channel 15. In an identical manner, the first end of this second channel is for example flared to apply the cone of a pipette thereto or will be adapted to the type of device used for injecting. the fluid in the device. Upwards, the chamber 13 can be closed by a flexible and stretchable membrane 18, preferably transparent. The upper wall 12 of the device housing thus comprises an opening which is hermetically covered by said membrane 18. This membrane 18 will for example be composed of a film, for example of the MicroAmp, 3M (registered trademarks) type, of thickness. , of dimensions and constitution adapted to deform elastically, with respect to its anchoring points, in particular to the bottom of the chamber 13.

The term “transparent” is understood to mean that the material used is at least partially transparent to visible light, so as to allow at least 80% of this light to pass. It should thus be understood that it will be sufficiently transparent to see the interior of the chamber 13, at least the second space located above the filter.

The device comprises a filter 16 arranged in said chamber 13 and separating said chamber 13 into two spaces. The two spaces are for example superimposed and thus designated lower space 130 located under the filter and upper space 131 located above the filter. This filter 16 is preferably produced in whole or in part in the form of a flexible and thin film, drawn in the space formed by the chamber so as to allow passage from one space to another only through the pores of the chamber. filter 16. The film has an elastic deformability allowing it to stretch during the exercise of a support force in a substantially vertical direction, this elastic deformability having a sufficient level to reach the lower surface of the chamber 13. Filter 16 has a average pore diameter as defined above. The diameter of the pores is of course adapted to ensure separation between different biological species present in the sample. The filter 16 will for example be composed of a film of thickness, dimensions and constitution adapted to deform to the bottom of the chamber 13 relative to its anchoring points. According to a particular embodiment, the filter could also be made of a transparent material, for example with the same transparency characteristics as the membrane.

The device can advantageously comprise a rough bearing surface 17 arranged on the bottom of the chamber 13. This rough bearing surface 17 extends over a majority part of the bottom of the chamber. It includes an average surface roughness parameter between 0.1 μm and 10 μm, preferably between 0.2 μm and 3 μm. This rough bearing surface 17 is intended to allow mechanical lysis of the biological species present in a biological sample placed in the device. Preferably, the mechanical lysis is carried out by grinding said biological species, by abrasion on said rough bearing surface. The grinding operation is carried out by a friction movement of the biological species against the rough bearing surface, using a suitable grinding member. This member will for example be a spatula or a rod, for example made of plastic or metallic material. This member is applied from the outside of the chamber 13 and its end is applied against the outer surface of the membrane 18 so as to stretch the membrane 18 and the filter towards the bottom of the chamber and thus rub the biological species present in a sample against the rough bearing surface.

Preferably, the device can integrate means for heating the internal space of the chamber, for example composed of at least one heating resistor or of a Peltier element. The resistance is for example fixed under the bottom wall. A power source will for example be provided to power the resistance. The power source will for example include one or more electric cells, providing enough energy to heat the chamber to a temperature within the range defined above, that is to say from 20 ° C to 100 ° C. . Of course, other heating means could be used, comprising for example a conductive ink deposited by printing or screen printing under the lower wall of the housing.

The membrane 18 is for example composed of a film made of a hyper-elastic two-component polymer material, for example a silicone or polysiloxane polymer. It may in particular be an elastomer of PDMS type (for Polydimethylsiloxane) or ECOFLEX (trademark registered by the company "Smooth-On" - for example Ecoflex 00-50). Its thickness can be between 20 and 500 μm.

• Une surface inférieure d'appui rugueuse 17,
• Un espace inférieur 130 d'une chambre 13 situé au-dessus de la surface d'appui rugueuse 17,
• Un filtre 16 souple et étirable situé au-dessus de l'espace inférieur 130,
• Un espace supérieur 131 de la chambre 13 situé au-dessus du filtre 16,
• Une membrane 18 souple et étirable située au-dessus de l'espace supérieur 131, fermant hermétiquement la chambre et accessible depuis l'extérieur du dispositif.

Thus, to summarize, the device can advantageously comprise the following "multilayer" structure:

• A rough lower bearing surface 17,
• A lower space 130 of a chamber 13 located above the rough bearing surface 17,
• A flexible and stretchable filter 16 located above the lower space 130,
• An upper space 131 of the chamber 13 located above the filter 16,
• A flexible and stretchable membrane 18 located above the upper space 131, hermetically closing the chamber and accessible from outside the device.

With reference to the figure 5 , in a nonlimiting manner, such a device can be produced in several superimposed layers.

It thus comprises a first lower layer C1 which consists of a substrate in accordance with the invention, that is to say comprising two layers, a layer of paper and a layer of PVDC.

A rough bearing surface 30 is produced on the upper face of the substrate 2 formed by its layer of PVDC, on at least part of this face. This rough bearing surface can be produced by embossing using a sandpaper (grit 18 to 800, preferably 400 to 600) at a pressure between 0.5 and 20 MPa, according to the principle detailed below. above. The channel forms said first channel of the device.

Advantageously, only this lower layer C1 is formed from the substrate 2 in accordance with the invention.

It may also include any other microfluidic imprint, formed of one or more cavities and / or one or more channels. This microfluidic impression will be made by embossing, using a matrix with the relief suited to the impression to be created.

The device can then include a second layer C2 formed of an adhesive cut according to the desired structure and bonded to the upper face of the substrate. This adhesive comprises in particular a wide opening 31 delimiting the side walls of the lower space 130 of the chamber 13.

The device may include a third layer C3 forming the filter 16 affixed on the upper face of the second layer to cover the opening. As indicated above, the filter is made of a film.

The device can have a fourth layer C4 formed of an adhesive cut according to the desired structure and bonded to the second layer while maintaining the filter between the two layers. This fourth layer comprises a wide opening 32 forming the upper space 131 of the chamber 13 and a second cutout forming the first channel 14.

The device may comprise a fifth layer C5 comprising the deformable membrane 18 described above and also comprising two orifices.

The device may include a sixth layer C6 formed of an adhesive cut to the desired shape and bonded to the layer C5 formed by the membrane, comprising two orifices each forming the inlet / outlet, respectively of the first channel 14 and of the second channel 15. .

By integrating this rough surface 30 with the sample preparation device, lysis is achieved mechanically by compressing the chamber. This stress induces the rupture of biological membranes on the rough surface and the DNA can then be harvested by rinsing.

• E100 : Un ensemble de trois électrodes RE, WE1, WE2 est sérigraphié à l'encre en Argent Ag sur le substrat 2 de l'invention, composé de la couche de papier et de la couche de PVDC.
• E101 : L'électrode de référence RE est ensuite recouverte d'une encre Ag/AgCl et les deux électrodes de travail WE1, WE2 d'une encre carbone.
• E102 : Un isolant (encre diélectrique) 40 est ensuite déposé en gardant les contacts avec les électrodes. Les pistes conductrices permettent la bonne connexion entre les différents éléments.
• E103 : Un canal est ensuite réalisé par embossage entre 0.5 et 20 MPa dans un étau ce qui permet d'obtenir un relief compris entre 10 et 500 µm.
• E104 : On ajoute ensuite de la polyaniline sensible au pH (PAni) pour former les deux électrodes de travail WE1, WE2.
• E105 : Un capot 42 est enfin déposé pour recouvrir le circuit.

Furthermore, it is also possible to provide the microfluidic device with electrodes, produced by screen printing directly on the paper-based substrate 2. Screen printing is carried out by depositing conductive inks. In a nonlimiting manner, the various stages of manufacture of these electrodes are illustrated on the figure 6 . For each step, the substrate 2 is shown respectively in top view and in side view.

• E100: A set of three electrodes RE, WE1, WE2 is screen-printed in Ag Silver ink on the substrate 2 of the invention, composed of the layer of paper and the layer of PVDC.
• E101: The reference electrode RE is then covered with Ag / AgCl ink and the two working electrodes WE1, WE2 with carbon ink.
• E102: An insulator (dielectric ink) 40 is then deposited while keeping the contacts with the electrodes. The conductive tracks allow the good connection between the different elements.
• E103: A channel is then produced by embossing between 0.5 and 20 MPa in a vice, which makes it possible to obtain a relief of between 10 and 500 µm.
• E104: Then pH sensitive polyaniline (PAni) is added to form the two working electrodes WE1, WE2.
• E105: A cover 42 is finally removed to cover the circuit.

It turns out that the PVDC layer minimizes the impact of warping on the printed tracks during embossing. No drop in conductivity is observed after the embossing of conductive lines deposited on a layer of PVDC whose surface density is> 15 gm ^-2 . At lower densities (10 gm ^-2 ), an increase in resistance is measured.

These electrodes can be integrated into the chamber of the device or at the bottom of a cavity produced by embossing the substrate.

Likewise, on the first layer of the device, formed from the substrate according to the invention, it is possible to integrate any type of microfluidic circuit therein by embossing the upper face of the substrate. It is thus possible to hollow out other cavities and / or microfluidic channels.

It will be understood from the above that it is thus possible to produce microfluidic chips based on an embossed paper substrate with a simple press. It is also possible to add other functionalities (electrodes) to it at a lower cost.

Claims (10)

Method for producing a microfluidic device, characterized in that it comprises a step of embossing a substrate (2) composed of at least two superimposed layers, a first layer (20) made of paper and a first layer (20) made of paper. second layer (21) deposited on the first layer and made of a material based on vinylidene polymer and having a so-called upper face (210) to be embossed, said embossing step being carried out on said upper face of the second layer of the substrate to form a pattern therein, said pattern comprising at least one cavity (22) delimited by at least one bottom wall and one side wall, said method also comprising a step of producing conductive electrodes (RE, WE1, WE2) by screen printing at the bottom of said cavity. Method according to Claim 1, characterized in that the electrodes comprise at least one reference electrode (RE) and one or more working electrodes (WE1, WE2). Process according to Claim 1 or 2, characterized in that each electrode (RE, WE1, WE2) is screen printed with silver ink on the substrate. Method according to Claim 3, characterized in that the reference electrode (RE) is covered with an Ag / AgCl ink and each working electrode (WE1, WE2) with a carbon ink. Method according to Claim 4, characterized in that an insulator (40) is deposited around each electrode. Microfluidic device, characterized in that it comprises a substrate (2) composed of at least two superimposed layers (20, 21), a first layer (20) made of paper and a second layer (21) deposited on the surface. first layer, made of a material based on vinylidene polymer and having a so-called upper face (210) to be embossed, in that said substrate is embossed on at least part of the upper face of its second layer in order to form a pattern thereon , said pattern comprising at least one cavity (22) delimited by at least one bottom wall and one side wall, said device also comprising conductive electrodes (RE, WE1, WE2) produced by screen printing at the bottom of said cavity. Device according to Claim 6, characterized in that the electrodes comprise at least one reference electrode (RE) and one or more working electrodes (WE1, WE2). Device according to Claim 6 or 7, characterized in that each electrode (RE, WE1, WE2) is screen printed with silver ink on the substrate. Device according to Claim 8, characterized in that the reference electrode (RE) is covered with an Ag / AgCl ink and each working electrode (WE1, WE2) with a carbon ink. Device according to Claim 9, characterized in that an insulator (40) is deposited around each electrode.

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