EP3225309A1 - Membranes for analysing microfluidic devices and manufacturing method - Google Patents

Abstract

The invention relates to a method for manufacturing a microfluidic device analysis membrane (10); said analysis membrane being formed from a porous substrate sheet (11) in the thickness of which solid wax forms impermeable borders (12) defining hydrophilic zones (13) and describing, through said sheet substrate (11), a pattern drawn with wax;
characterized in that it comprises the following steps:
- an intermediate pattern (12a, 12b), shaped with wax in the image of said wax pattern formed by the impermeable edges of the analysis membrane (11), is affixed to each of the faces of a sheet of a porous substrate (11) such that, on either side of the thickness of said substrate sheet (11), said mutually symmetrical intermediate units (12a, 12b) are positioned at least substantially opposite to each other; one of the other;
- Maintained at least substantially horizontally, said substrate sheet (11) is subjected to a heat treatment capable of causing at least partial melting of the constituent wax of the intermediate units (12a, 12b) affixed to the faces of said sheet substrate (11), and a mechanical treatment capable of compressing the thickness of all or part of said substrate sheet (11);
said substrate sheet (11) is subjected to a mechanical and thermal expansion phase able to allow said substrate sheet (11) to take up at least part of its initial thickness and the wax to resolidify inside the the thickness of said substrate sheet (11); and

in that the thickness of said substrate sheet (11) is between 200 μm and 1000 μm

Description

The present invention relates to the general field of microfluidics. It relates more particularly to microfluidic devices for diagnostic purposes, as well as the manufacturing processes of such devices.

• pour mélanger, éventuellement pour faire réagir, tout ou partie des constituants d'un ou de plusieurs fluides, entre eux et/ou avec des composés exogènes,
• pour séparer certains constituants d'un fluide, éventuellement en vue d'en analyser les propriétés chimiques et/ou physiques,
• pour détecter, voire pour doser, des agents cibles dont on suspecte la présence.

La microfluidique trouve ainsi des applications dans de nombreux domaines techniques.Microfluidics can be defined as the study of phenomena that govern the movement of small volumes of fluid, especially liquid. It encompasses the development of systems and devices for circulating and / or manipulating small volumes of fluids for a variety of purposes, for example:

• for mixing, optionally to react, all or part of the constituents of one or more fluids, with each other and / or with exogenous compounds,
• to separate certain constituents of a fluid, possibly for the purpose of analyzing the chemical and / or physical properties thereof,
• to detect, or even to dose, target agents whose presence is suspected.

Microfluidics thus finds applications in many technical fields.

In the field of clinical practice, diagnostic tests using microfluidic devices have undergone a rapid evolution since the last two-three decades ( Yetisen et al., Lab Chip, 2013 (13) 2210-2251: "Factor-based microfluidic point-of-care diagnostic devices" ).

Also called "microfluidic sensors", these devices not only make it possible to analyze small volumes of liquid samples, they also make it possible to undertake, on a single platform, a plurality of detection or even quantification tests, targeted analytes and / or pathogens by simple and fast manipulation.

The present invention is more specifically concerned with disposable microfluidic devices, of the type of those which comprise an analysis membrane made of a porous material and operating according to a so-called fluid lateral movement principle, in this case liquids. Because said analysis membrane has long been made from a sheet of cellulose fiber or nitrocellulose, the designation "ΜPAD" (for " microfluidic paper-based analytical devices" , in English) has been widely adopted to refer to this technology. Other porous, hydrophilic and absorbent materials, as long as they can allow lateral movement of the liquids by capillarity, have also been envisaged and then improved in order to be able to act as analysis membranes for the fabrication of these microfluidic devices. These are for example materials made from nitrocellulose fibers, cellulose acetate, fabrics, porous films of polymers.

The general principle of operation of this type of microfluidic device is essentially based on an analysis membrane made up / fabricated from a sheet of porous, hydrophilic and absorbent material, on which and in the bulk of which is formed a hydrophilic network formed areas of interest and channels. Inside and through this network of areas of interest and channels, a liquid to be analyzed (for example a liquid biological sample), once deposited on said analysis membrane, can progress by simple capillarity and be submitted an analysis or a series of qualitative and / or quantitative analyzes of its constituents (immunodetection, molecular detection, affinity test, ligand-receptor coupling test, pH evaluation ...).

• des zones dites zones de dépôt, au niveau desquelles les liquides à analyser sont déposés,
• des zones dites zones de réaction, au niveau desquelles ont lieu les réactions de détection et/ou de dosage,
• éventuellement, des zones mixtes qui font office simultanément de zones de dépôt et de zones de réaction,
• éventuellement, des zones dites zones réservoirs, au niveau desquelles des réactifs et/ou additifs nécessaires aux réactions à mener sont stockés, généralement sous forme sèche avant d'être réhydratés (ou solubilisés) et transférés vers des zones de réaction spécifiques,
• éventuellement, des zones de dépôt secondaires, qui seront alimentées en réactif(s) et/ou en additif(s), extemporanément et/ou au cours du processus d'analyse, et
• des structures formant canaux qui assurent une conduction fluidique entre les zones d'intérêt, et à travers lesquelles les liquides progressent par un simple phénomène de capillarité.

Very schematically, on the analysis membranes of microfluidic devices, it can be distinguished:

• areas called deposition zones, at which the liquid to be analyzed are deposited,
• zones called reaction zones, at which the detection and / or dosing reactions take place,
• possibly mixed zones which simultaneously act as deposition zones and reaction zones,
• optionally, so-called reservoir zones, at which reagents and / or additives necessary for the reactions to be carried out are stored, generally in dry form before being rehydrated (or solubilized) and transferred to specific reaction zones,
• optionally, secondary deposition zones, which will be fed with reagent (s) and / or additive (s), extemporaneously and / or during the analysis process, and
• channel structures that provide fluidic conduction between the areas of interest, and through which the liquids progress by a simple capillarity phenomenon.

With regard to the design of such devices and more particularly of the analysis membrane part, several approaches have been proposed to create, through the thickness of a porous substrate sheet, such hydrophilic zones delimited by borders. waterproof.

Among the methods proposed, that described by WO 2008/049083 takes up the general principle of photolithography. A porous substrate sheet is first impregnated with a negative photoresist (for example, photolithographic resin). SU-8 2010 ), by immersion in a solubilized resin solution in a solvent. After removal of the excess resin, on one of its faces, the substrate sheet is coated with a mask material opaque to light waves. This mask is provided with perforated parts (or simply transparent to ultraviolet) and ultraviolet opaque parts which respectively produce the hydrophobic parts and the hydrophilic parts to be transferred onto the substrate. The assembly is then subjected to ultraviolet irradiation, at wavelengths capable of causing photopolymerization of the negative resin at the exposed areas. At this level, in the thickness of the substrate, hydrophilic zones delimited by impermeable borders are then drawn. An optional additional heat treatment makes it possible to completely evaporate the initial solvent and perfect the crosslinking.

This method, particularly consuming resin, is very expensive to implement, given the high cost of photosensitive resins.

An alternative to the use of photosensitive resins is proposed by WO 2010/003188 . A substrate sheet is first impregnated with a conventional resin by immersion in a solution of resin solubilized with volatile solvent. The substrate sheet is then placed in a heated and ventilated chamber in order to evaporate the solvent and cause the resin to crosslink. The substrate sheet, thus made entirely hydrophobic, is coated on its upper face with an etching mask. This mask has perforated parts and non-perforated parts, which respectively reproduce the hydrophilic parts and impermeable edges to be transferred to the substrate. A plasma or corona treatment is applied to the assembly and makes it possible to remove the resin present on the surface of the substrate, at the level of the exposed zones. At these exposed areas, the upper layers of the substrate find wettability and hydrophilic qualities.

This method is also very consuming in resin. What is more, it requires sophisticated and expensive equipment, whose financial investment is justified. with regard to the possible selling price for single-use diagnostic devices.

More rudimentary, the method described in WO 2008/049083 consists in using an ink-stamp whose print head is specifically shaped to stamp a pattern with well-defined outlines. The ink-pad printing head is coated with hot, liquid wax and then applied to the porous substrate. The liquid wax deposited on the surface deeply impregnates the porous substrate. By hardening, it forms impermeable edges in the thickness of the substrate, which delimit hydrophilic zones. In addition to having to have, for each model of pattern to be realized, a specifically designed print head, this method does not make it possible to produce graphically complex patterns. Moreover, the final rendering is generally imperfect.

Also using wax for the design of impermeable borders defining hydrophilic zones, the method described by WO 2010/102294 makes use of modern IT tools, which makes the creation of highly graphical patterns possible and is significantly simplified.

In this method, the desired pattern is thus first designed by computer. Then, using a solid ink printer, it is printed with the desired image resolution on one side of a porous substrate sheet. With the printed side facing up, the substrate sheet is coated on a hot plate heated to a temperature of the order of 150 ° C, for about 2 minutes. Under the effect of heat, the wax contained in the printed ink melts and, by gravity, penetrates and impregnates the substrate in its thickness. By cooling, the wax resolidifies and forms, in the thickness of the substrate, impermeable edges defining hydrophilic zones, like the pattern initially designed by computer.

The solid ink printers currently available commercially are unfortunately suitable only for printing on printing media (paper, transparent, cardboard) of standard sizes, well defined dimensions and whose thicknesses hardly exceed 250 microns. The thickness of the print medium is a limiting parameter which is however not only related to the computer hardware currently available, it is also related to the method itself. In terms of image resolution and impermeability of the borders formed, the quality of the result decreases progressively with the increase of the thickness of the substrate. The thicker and more porous a printing substrate, the more quantity / density of wax needed to form the impermeable edges will be important. Also, the greater the quantity / density of wax required, the more the lateral diffusion of the melted wax will be marked and deleterious for the fineness of the borders of the pattern initially designed in computer science.

For information, WO 2010/102294 describes an exemplary embodiment in which a XEROX ^® Phaser 8560N printer is used to print on Whatman ™ no. 1. This filter paper used as a porous substrate sheet has a cellulose fiber composition and a thickness of the order of 180 μm. Impermeable functional borders could be obtained by printing a 300 μm wax pattern. thickness (lateral); the impermeable edges thus formed were 850 ± 50 μm thick. With wax plots with a thickness of less than 300 μm, it has not been possible to obtain waterproof borders that are functional.

The way in which the printer is used in this method induces another limiting parameter, that of the constituent material of the porous substrate sheet to be printed. Indeed, it must be able to give the substrate sheet mechanical strength and tensile strength (or tear resistance) sufficient to allow it to be conveyed through the printer, and to be able to withstand all the constraints mechanical and thermal forces inflicted during the printing process. The porous substrate sheets made of flexible material, such as a fabric or a wadding, or a material of high friability, for example a fiberglass filter paper, can not be transformed / reworked by this method.

The object of the present invention is to propose a process for manufacturing microfluidic devices, more specifically a process for making the analysis membrane part, which does not suffer from the abovementioned disadvantages. More specifically, it aims to improve the method described in WO 2010/102294 .

An object of the present invention is therefore to provide a method of manufacturing analysis membranes for microfluidic devices whose implementation is not limited to the processing of sheets of cellulose fiber or nitrocellulose, but also to made sheets. in other porous materials, as well as substrate sheets of relatively large thickness, that is to say with a thickness of at least 200 μm, in particular between 200 μm and 1000 μm. .mu.m.

Another objective of the present invention is also to be able to propose a method that is compatible with the constraints of an industrial operation of a single-use diagnostic device, particularly in terms of production cost and profitability.

Finally, the present invention aims at proposing new analysis membranes whose originality lies not only in their design but also in their structure and / or the nature of the substrates used, as well as in the analytical performance that can be provided.

The present invention therefore relates, firstly, to a method of manufacturing a microfluidic device analysis membrane; said microfluidic membrane being formed from a porous substrate sheet, in the thickness of which solid wax forms impermeable edges defining hydrophilic zones. These impermeable edges thus describe, through said substrate sheet, a pattern drawn with wax.

• un motif intermédiaire, façonné à la cire à l'image dudit motif tracé à la cire que formeront les bordures imperméables de la membrane d'analyse, est apposé sur chacune des faces d'une feuille de substrat poreux de sorte que, de part et d'autre de l'épaisseur de ladite feuille de substrat, lesdits motifs intermédiaires, mutuellement symétriques, se trouvent positionnés au moins sensiblement en regard l'un de l'autre ;
• maintenue au moins sensiblement à l'horizontale, ladite feuille de substrat est soumise à un traitement thermique apte à provoquer une fusion au moins partielle de la cire constitutive des motifs intermédiaires apposés sur les faces de ladite feuille de substrat, et à un traitement mécanique apte à comprimer momentanément l'épaisseur de tout ou partie de ladite feuille de substrat ;
• ladite feuille de substrat est soumise à une phase de détente mécanique et thermique, apte à permettre à ladite feuille de substrat de reprendre au moins en partie son épaisseur initiale et à la cire de se resolidifier à l'intérieur de l'épaisseur de ladite feuille de substrat.

According to the invention, the method comprises the following steps:

• an intermediate pattern waxed in the image of said wax pattern formed by the impermeable edges of the analysis membrane is affixed to each of the faces of a porous substrate sheet so that, on both sides, other than the thickness of said substrate sheet, said intermediate units, mutually symmetrical, are positioned at least substantially opposite one another;
• maintained at least substantially horizontally, said substrate sheet is subjected to a heat treatment capable of causing at least partial melting of the wax constituting the intermediate units affixed to the faces of said substrate sheet, and suitable mechanical treatment temporarily compressing the thickness of all or part of said substrate sheet;
• said substrate sheet is subjected to a mechanical and thermal expansion phase, able to allow said substrate sheet to take up at least part of its initial thickness and the wax to resolidify within the thickness of said sheet of substrate.

Optionally, said substrate sheet thus transformed is re-cut to the final dimensions of the desired analysis membrane. Optionally, before or after this cutting, the lower face is covered with a sealing layer, for example, by spraying or by applying a resin composition or a wax, or by laminating an impermeable plastic film, for example of the poly (ethylene terephthalate) or PET type. Also, intrinsically, this sealing layer may also have the advantage of stiffening and strengthening the structure of said analysis membrane.

In the present text, the term "sheet" is used in a very general and broad sense to designate a part, a piece of substrate whose thickness corresponds to an insignificant value with respect to its surface. In this context, this term is used here to designate a sheet in the strict sense, a ribbon or a disc. In a manner relative to this notion of "sheet", the term "film" will be used hereinafter to denote a piece, a piece of material whose thickness is significantly less than that of a sheet taken in the sense of the present invention.

Also, within the meaning of the present invention, the term "waxes" denotes lipophilic substances, solid at room temperature (25 ° C.) and having a melting point of between 45 ° C. and 120 ° C. Just above the melting point, the waxes are liquid and have a particularly low viscosity. They can be of animal origin (for example, beeswax, Chinese wax, lanolin ...), vegetable (for example, rice wax, mimosa, candellila wax, carnauba, the wax of Japan, the vegetaline ...), mineral (for example, paraffin, ozokerite ...) or synthetic (for example, stearin, ethylene polymers, fatty amines, polyethylene glycol or PEG, ...).

Regarding the heat treatment step of the substrate, care should be taken not to heat the wax too brutally. A gradual rise in temperature will allow the wax deposited on the upper surface of the substrate sheet to melt slowly and gradually descend into the thickness of the substrate. This avoids / limits the saturation at the most superficial layers of the substrate and thereby minimizes the lateral / radial diffusion of the wax.

The wax deposited on the underside of the substrate sheet gradually permeates the substrate in all directions, including upwards.

By compressing the thickness of the substrate sheet, the vertical path of the wax is reduced. In doing so, the deep impregnation is favored and the migration time is shortened. Less freedom is left to the wax to be able to diffuse in one direction radial / lateral; the smoothness of the initial wax pattern is affected less significantly.

Advantageously and according to the invention, the thickness of said substrate sheet is between 200 μm and 1000 μm, preferably between about 250 μm and about 850 μm.

According to a particular embodiment of the process according to the invention, said heat treatment is carried out inside a thermo-controlled heating chamber. As for said mechanical treatment, it is implemented by mechanical means able to apply a pressure or a compressive force sufficient to compress the thickness of said substrate sheet, at least at the intermediate units, installed at the same time. interior of said thermo-controlled enclosure.

By way of example, these mechanical means may consist of one or more plates, having a certain mass, which is applied to the substrate sheet. It may also be a vise device, adapted to maintain, between its jaws, the substrate sheet in a position at least substantially horizontal, and to compress the thickness.

According to an alternative embodiment of the method according to the invention, the heat treatment and the mechanical treatment are carried out simultaneously by means of a thermo-controlled heating compression device, provided with two horizontal heating compression plates. This may include a hot press (or hot press).

Whatever the method of implementation envisaged for carrying out the heat and mechanical treatments, care should be taken to avoid that the wax deposited on the faces of the substrate sheet is in direct contact with the source (s). heat, this is essentially to prevent a rapid and sudden melting of the wax. When a thermo-controlled compression device is used, a layer of flexible and elastic material, at least substantially flat, is interposed between each of the faces of said substrate sheet and the heating compression plates. Said layer of flexible and elastic material then serves as a thermal and mechanical buffer; On the one hand, it makes it possible to slow the transmission of heat substrate and, on the other hand, more evenly distribute the heat and pressure over the entire substrate sheet. As a thermal and mechanical buffer element, one can for example use a piece of rubber or any other similar polymeric material, a piece of tissue, a piece of cork.

Advantageously, it is also possible to insert a layer of absorbent material between each intermediate pattern, affixed to one of the faces of the substrate sheet, and said thermal and mechanical buffer. This layer of absorbent material is intended to quickly capture the molten wax remaining on the surface of the substrate before it slime or run too much on the sides, which could significantly affect the fineness of the wax pattern.

Regarding the thermal and mechanical expansion step, no particular precautions to take is noted. Cooling can be done passively, stopping heating and / or removing and moving the substrate sheet away from any heat source. Similarly, mechanical expansion can be achieved simply by quickly removing the mechanical stresses applied or by gradually reducing their intensity.

Also, the chronological order in which thermal expansion and mechanical expansion are triggered is not very important. Advantageously and according to the invention, the two detents are triggered simultaneously or one after the other.

To affix, on the faces of the substrate sheet, the wax-shaped intermediate patterns, any suitable techniques known to those skilled in the art can be used.

For this purpose, 3D printing can be used. By proceeding face-to-face, the intermediate patterns are traced directly on the substrate sheet by deposition of wax. On the upper face of the substrate sheet, the intermediate pattern reproduces identically the desired final pattern. On the underside, the intermediate pattern appears as an inverted image of the desired final pattern. On either side of the thickness of the substrate sheet, the two intermediate units, mutually symmetrical, are positioned at least substantially opposite one another.

According to another advantageous embodiment, the waxed intermediate units are affixed to each of the faces of the porous substrate sheet with a wax transfer technique. For this purpose, the intermediate patterns are first printed on a transfer film. On this printed transfer film, the intermediate pattern intended to coat the underside of the substrate sheet appears identical to the final pattern to be produced, whereas the intermediate pattern intended to coat the upper face of the substrate sheet appears as an image. inverted of the first intermediate pattern.

These intermediate units are then transferred to each of the faces of the substrate sheet, then in its thickness, by carrying out the thermal and mechanical treatments according to the invention.

By choosing transfer films of relatively small thickness (for example, less than 200 μm thick), a solid ink printer can advantageously be used to print the intermediate patterns. By using a solid ink printer, the amount (per unit area) of wax deposited on the transfer film depends on the print quality chosen, the inks used - more precisely the wax concentration present in these compositions. ink-, and the intensity of the coloring of the patterns to be printed. By playing on these different parameters, an operator will appreciate the best setting to be applied in order to obtain perfectly impermeable borders, which cross the entire thickness of the porous substrate sheet to be transformed / remodeled.

US 2012/190765 , US 2012/287199 , US 2012/287212 , US 2012/309896 and US 2014/123873 provide wax-based solid ink compositions and their method of manufacture. As an indication of solid ink compositions commercially available, mention may be made in particular that of the company XEROX ^® CORP. (Japan) of CQ references 8570 XEVP, 108R00931, 108R00932, 108R00933, 108R00934 and 108R00935.

As transfer film usable for the implementation of the method according to the invention, various materials are possible, since they are printable. This may include a printable plastic sheet (transparent or opaque), a sheet of printing paper, a sheet of parchment paper.

Advantageously and according to the invention, the two intermediate units intended to coat the upper and lower faces of the substrate sheet are printed on two separate transfer films. The substrate sheet is then placed between the two transfer films, the wax patterns being pressed against the faces of the substrate sheet. The assembly is held together and is subjected to heat and mechanical treatments according to the invention.

According to an alternative embodiment, the two intermediate units intended to coat the upper and lower faces of the substrate sheet are printed on one and the same transfer film. On this transfer film, the two intermediate units are arranged side by side, symmetrically with respect to an axis. Once the intermediate patterns are printed and the wax is dried, the transfer film is folded in half along said axis of symmetry with the printed side turned inward. The two intermediate patterns are thus superimposed. The substrate sheet is inserted inside the folded transfer film between the two printed intermediate patterns. The assembly is held together and is subjected to said thermal and mechanical treatments according to the invention.

Advantageously and according to the invention, the transfer film is a paper sheet of cellulose fiber. Slightly absorbent, this film makes it possible, during the thermal and mechanical treatments according to the invention, to capture at least a portion of the molten wax remaining on the surface of the substrate before it slinks / flows on the sides.

The method according to the invention can be implemented from a wide range of porous substrate sheets, in particular from a fibrous composition chosen from: cellulose, nitrocellulose, cotton, linter, glass, silk fiber , viscose, polypropylene, polyester, polyamide (Nylon ^® ), poly (lactic acid) or PLA. Said fibers may optionally be functionalized and / or loaded and / or doped with additives (for example, talc, diatomite, etc.).

Advantageously, the process according to the invention is carried out starting from a porous substrate sheet of the same composition as a filtering medium made of cellulose fiber, nitrocellulose, cotton, linter, silk, viscose, polypropylene, polyester, preferably fiberglass.

• les filtres WHATMAN™ (GE Healthcare Life Sciences, États-Unis), notamment les papiers MF1, LF1, VF1, VF2 et Fusion 5, destinés à la filtration du sang total et à la séparation cellules/plasma, et le filtre GF/C, destiné à la séparation de substances solides en suspension dans un fluide ;
• les filtres de la société SARTORIUS A.G. (Allemagne), notamment ceux destinés au traitement et à l'analyse de fluide.

For the purposes of the present invention and in general, the term "filter media" refers to a filter material conventionally used in laboratories and in industry for the purpose of liquid analysis, and more specifically for purposes of separation and of purification. Depending on their thickness and format, membrane, paper, stamp or disk qualifiers are sometimes assigned to them. Similarly, the term "fiberglass filter medium" means a filter material made from glass microfibers, optionally borosilicate, with or without binder. As examples of filter media currently available in fiberglass fiberglass, these include:

• WHATMAN ™ filters (GE Healthcare Life Sciences, USA), including MF1, LF1, VF1, VF2 and Fusion 5, for whole blood filtration and cell / plasma separation, and the GF / C filter for separating suspended solids in a fluid;
• the filters of the company SARTORIUS AG (Germany), in particular those intended for the treatment and analysis of fluid.

Advantageously and according to the invention, said method of manufacturing a microfluidic device analysis membrane is implemented from a porous fiberglass substrate sheet. Advantageously and according to the invention, said substrate sheet contains the composition of a fiberglass filter medium.

Fiberglass filtering media are characterized by a great fragility in tearing and crumbling. This great structural fragility makes them incompatible with any direct printing by means of solid ink printers currently on the market. The mechanical constraints imposed by them are indeed considerably denaturing.

Thus, in addition to having considered the use of a fiberglass filtering medium as a porous substrate for making analysis membranes of / or for microfluidic devices, it is the merit of the inventors to have succeeded in point a process capable of integrating into the thickness of this particular material, wax to form in fine impervious edges defining so-called hydrophilic zones. Moreover, in addition to the simplicity and low cost of its implementation, the proposed method allows the production, in the thickness of the relatively thick substrate sheets (between 200 microns and 1000 microns), curbs waxes perfectly impermeable to liquids and whose fineness (lateral thickness) can reach of the order of 800 microns.

The present invention relates, secondly, a microfluidic device analysis membrane, capable of being manufactured by a method according to the invention. The said test membrane has a thickness of which solid wax forms impermeable (in this case, liquid impermeable) borders delimiting hydrophilic zones. According to the invention, the thickness of the analysis membrane is between 200 μm and 1000 μm, preferably between about 250 μm and about 850 μm.

Optionally, said analysis membrane comprises a face (in this case, a so-called lower face) covered with a sealing layer. This can be obtained by spraying or by applying a resin composition or a wax, or by laminating a plastic film, for example of the PET type.

Advantageously and according to the invention, the constitutive wax of said impermeable edges is chosen from an animal wax (for example, beeswax, Chinese wax, lanolin, etc.) and vegetable (for example, rice wax). , mimosa, candellila wax, carnauba wax, Japan wax, vegetaline ...), mineral (for example, paraffin, ozokerite ...) and synthetic (for example, stearin, polymers ethylenic, fatty amines, polyethylene glycol or PEG ...).

Advantageously and according to the invention, the analysis membrane is made of cellulose fiber, nitrocellulose fiber or fiberglass, cotton, linter, cellulose wadding, silk fiber, viscose, polypropylene, of polyester. It may optionally be functionalized and / or charged and / or doped with additives (for example, talc, diatomite, etc.).

Advantageously and according to the invention, the analysis membrane is made in a porous substrate whose composition resumes that of a filtering medium of cellulose fiber, nitrocellulose, cotton, linter, glass, silk, viscose , polypropylene, polyester, polyamide or PLA.

• les filtres WHATMAN™ de la compagnie GE HEALTHCARE LIFE SCIENCE, (États-Unis), en particulier les filtres MF1, LF1, VF1 et VF2, qui sont ordinairement destinés à la filtration du sang total et à la séparation cellules/plasma, et du filtre GF/C, et/ou à la séparation de substances solides en suspension dans un fluide ;
• les filtres de la société SARTORIUS A.G. (Allemagne), notamment ceux destinés au traitement et à l'analyse de fluides.

According to a preferred embodiment, the composition of the analysis membrane is fiberglass. It advantageously takes up the composition of a filter media such as:

• WHATMAN ™ filters from GE HEALTHCARE LIFE SCIENCE, (USA), particularly the MF1, LF1, VF1 and VF2 filters, which are typically intended for whole blood filtration and cell / plasma separation, and GF / C filter, and / or separation of suspended solids in a fluid;
• the filters of the company SARTORIUS AG (Germany), in particular those intended for the treatment and analysis of fluids.

Advantageously and according to this preferred embodiment, the analysis membrane has a thickness of between 200 microns and 1000 microns, typically between about 250 microns and about 850 microns.

The analysis membranes made of fiberglass according to the invention allow an analysis of the plasma directly from the whole blood. The blood deposited on the surface of the membrane is fractionated; cells and plasma are separated. The cells remain fixed in the deposition zone while the plasma will diffuse through the membrane, progressing along the hydrophilic channels by lateral diffusion.

In microfluidic devices currently commercially available, the analysis membranes are conventionally made of cellulose fiber or nitrocellulose. These analysis membranes alone are incapable of separating the plasma fraction from the cell fraction. Whole blood deposited on such analysis membranes coagulates rapidly; after the deposition of the blood sample on these supports, the cells block the plasma and prevent its lateral diffusion through the hydrophilic channels. Thus, for the blood tests, the μPADs heretofore proposed comprise an assembly of an analysis membrane with a filtration system (for example, a fiberglass filtering medium) capable of implementing a fractionation of the blood. ; once separated from the cells that remain blocked at the level of the filtration system, the plasma diffuses through the analysis membrane where its components are then analyzed. An example of μPAD comprising such an assembly between an analysis membrane and a filter medium is described by Songjaroen et al., 2012 (Lab Chip, 2012 (12) 3392-3398: "Blood separation on microfluidic paper-based analytical device" ).

For μPAD models that do not have such a filtration system, they must be used with previously purified plasma.

In a particular embodiment, in addition to impermeable edges drawn with wax and delimiting hydrophilic zones, said analysis membrane also comprises, in its thickness, encrustations of wax, present at the level of at least one hydrophilic zone. These wax encrustations do not form impermeable barriers to passage of liquids. Positioned at various hydrophilic zones, they are intended to disrupt and / or slow down the flow of liquid locally and / or to create well-defined reservoir and / or reaction zones. They can also define the contours of wax-free zones, thus very hydrophilic, which can make use of reservoirs or well-defined reaction zones.

These wax encrustations can be made in many ways. A first method consists of taking the previously described method to establish the impermeable edges of the analysis membrane. Localized and localized incrustations of wax are then obtained which extend through the entire thickness of said sheet. They thus form obstacles which the liquids must circumvent by the sides to be able to progress through the membrane of analysis.

Another method is to repeat the process according to the invention but by modifying it somewhat to disperse in the thickness of the porous substrate a small amount of wax, insufficient to completely block the migration of liquids. For this purpose, it is possible, for example, to use a solid ink having a low concentration of wax and / or to affix the solid ink only to one of the two faces of the substrate sheet and / or to print the transfer film with a layer of pale color, weakly intense.

A similar result can be obtained by repeating the process according to the invention, but this time without compressing the thickness of the porous substrate sheet.

Compared to this last embodiment, the previously mentioned modes have the advantage of allowing the realization of wax encrustations concomitantly with those of impermeable edges.

The present invention also extends to a microfluidic device comprising at least one analysis membrane according to the invention.

The present invention also relates to a method for manufacturing a microfluidic device analysis membrane, a microfluidic device analysis membrane and a microfluidic device characterized, in combination, by all or some of the features above or above. -after.

• la Figure 1 est une représentation schématisée d'un mode particulier de mise en oeuvre d'un procédé de fabrication d'une membrane d'analyse de dispositif microfluidique conforme à la présente invention ;
• la Figure 2 présente une photographie d'une membrane WHATMAN™ MF1 à la sortie d'une imprimante à encre solide, après une tentative d'impression directe ;
• la Figure 3 expose en (A), une représentation schématique d'un film de transfert sur lequel est imprimé un exemple de motifs intermédiaires et en (B), une photographie d'une membrane d'analyse obtenue à partir de ce film de transfert ;
• la Figure 4 présente deux photographies de membranes d'analyse pour dispositifs microfluidiques ; la photographie (A) correspond à une membrane d'analyse obtenue par mise en oeuvre d'un procédé selon l'invention ; la photographie (B) correspond à une membrane d'analyse obtenue avec le même procédé mais en omettant le traitement mécanique ;
• la Figure 5 est un graphique mettant en évidence une relation linéaire entre l'épaisseur d'un trait de cire imprimé sur un film de transfert et l'épaisseur de ce même trait après transfert dans l'épaisseur d'une feuille de substrat ;
• la Figure 6 expose des photographies permettant d'apprécier l'imperméabilité des bordures de cire façonnées avec un procédé selon l'invention, en fonction de leur épaisseur ;
• la Figure 7 présente deux photographies prises en microscopie ; la première (A) montre une bordure imprimée à la cire sur un film de transfert en fibre de cellulose, la seconde (B) montre cette même bordure après transfert vers une membrane WHATMAN™ MF1 ;
• La Figure 8 montre des photographies illustrant l'imperméabilité de bordures d'épaisseur de l'ordre de 800 µm, formées sur des substrats de différentes compositions et de différentes épaisseurs, par mise en oeuvre d'un procédé selon l'invention ;
• les Figure 9, Figure 10A et Figure 10B sont des photographies illustrant la mise en application d'une membrane d'analyse selon l'invention à des fins d'immunodétection ; et
• la Figure 11 est une photographie d'une membrane d'analyse selon l'invention façonnée pour le dépistage du virus de la dengue, par une codétection des protéines NS1 et DomIII.

Other objects, features and advantages of the invention will appear on reading the following examples which refer to the appended figures and in which:

• the Figure 1 is a schematic representation of a particular embodiment of a method of manufacturing a microfluidic device analysis membrane according to the present invention;
• the Figure 2 presents a photograph of a WHATMAN ™ MF1 membrane at the exit of a solid ink printer, after a direct print attempt;
• the Figure 3 discloses in (A), a schematic representation of a transfer film on which is printed an example of intermediate units and in (B), a photograph of an analysis membrane obtained from this transfer film;
• the Figure 4 presents two photographs of analysis membranes for microfluidic devices; the photograph (A) corresponds to an analysis membrane obtained by implementing a method according to the invention; the photograph (B) corresponds to an analysis membrane obtained with the same process but omitting the mechanical treatment;
• the Figure 5 is a graph showing a linear relationship between the thickness of a wax line printed on a transfer film and the thickness of this same line after transfer in the thickness of a substrate sheet;
• the Figure 6 discloses photographs to appreciate the impermeability wax edging shaped with a method according to the invention, depending on their thickness;
• the Figure 7 presents two photographs taken in microscopy; the first (A) shows a wax-printed border on a cellulose fiber transfer film, the second (B) shows the same border after transfer to a WHATMAN ™ MF1 membrane;
• The Figure 8 shows photographs illustrating the impermeability of edges of thickness of the order of 800 microns, formed on substrates of different compositions and thicknesses, by implementing a method according to the invention;
• the Figure 9, Figure 10A and Figure 10B are photographs illustrating the application of an analysis membrane according to the invention for immunodetection purposes; and
• the Figure 11 is a photograph of an analysis membrane according to the invention shaped for the detection of the dengue virus, by a co-detection of the NS1 and DomIII proteins.

EXAMPLES 1. Fabrication of a Microfluidic Device Analysis Membrane According to the Invention

The Figure 1 schematically illustrates the implementation of a method of manufacturing an analysis membrane 10, according to the invention, and by which a pattern drawn with wax is integrated into the thickness of a sheet of porous substrate 11; the contours of said pattern being intended to form, in the thickness of said substrate sheet 11, impermeable borders 12 defining hydrophilic zones 13 of determined configuration. In the particular procedure illustrated here, the two intermediate units 12a and 12b are printed on one and the same transfer film 20.

In a first step, an image 12'a corresponding to the pattern to be integrated in the thickness of the substrate sheet 11 is created by computer and by means of a drawing software. This first image 12'a is duplicated in FIG. a symmetrical image 12'b.

By means of a solid ink printer, the two images 12'a and 12'b are printed on a transfer film 20, so as to form two intermediate units 12a and 12b arranged symmetrically with respect to an axis. This axis of symmetry S is also printed on the transfer film 20, as a visual cue.

To do this, the printing is carried out with a XEROX ^® ColorQube ™ type printer, supplied with solid inks of reference XEROX ^® 108R00931 (cyan color), 108R00932 (magenta color), 108R00933 (yellow color) and 108R00934 / 108R00935 ( black color). The transfer film 20 is an ordinary office paper sheet. The contours of the pattern are printed in quality / photo resolution in black ink.

Once this impression is made, the transfer film 20 is folded in two along the axis of symmetry S, the intermediate units 12a and 12b turned inwards. The latter are thus superimposed on one another. A substrate sheet 11 is slid inside the folded transfer film 20, interposed between the two intermediate units 12a and 12b.

The substrate sheet 11, sandwiched between the two flaps of the transfer film 20, is then pressed between two horizontal heating plates and two rubber parts 15 forming a thermal and mechanical buffer.

The assembly is subjected to a pressure of the order of 1 kg / cm ² and at a temperature of 120 ° C for about 3 minutes. During this process, the wax previously printed on the transfer film 20 is transferred onto both sides of the substrate sheet 11 and then impregnates the thickness.

• filtre MF1, d'une épaisseur de 367 µm
• filtre Fusion 5, d'une épaisseur de 370 µm
• filtre VF2, d'une épaisseur de 785 µm.

The process described here applies to many porous substrates, such as for example substrates made of cellulose fiber, nitrocellulose, cotton, interlining, glass, silk, viscose, polypropylene, polyester, polyamide, PLA. This process has been developed primarily on fiberglass substrates and more particularly on WHATMAN ™ (GE HEALTHCARE LIFE SCIENCE, USA) brand filter media of varying thickness, including:

• MF1 filter, with a thickness of 367 μm
• Fusion 5 filter, 370 μm thick
• VF2 filter with a thickness of 785 μm.

These filtration media usually used for the filtration of whole blood samples, for a rapid separation between cellular fraction / liquid fraction, are extremely fragile to the tear and must be handled with care and delicacy.

Given this great fragility, it is not possible to print the intermediate patterns directly on this type of material by means of a solid ink printer. The Figure 2 is a photograph of a WHATMAN ™ MF1 membrane after an unsuccessful direct print attempt. This membrane, previously taped on a sheet of standard size paper to facilitate its passage through the printer and to consolidate somewhat the structure, comes out of the printer completely deteriorated.

2. Exemplary embodiment of an analysis membrane according to the invention, from a fiberglass substrate sheet

The Figure 3 illustrates, in its part (A), a transfer film 20 on which a particular example of intermediate units 12a and 12b is printed. In part (B) is exposed a photograph of an analysis membrane 10 (after use) formed from a fiberglass substrate sheet and using such a transfer film 20.

The intermediate units 12a and 12b, of generally rectilinear shape, have a flared upper part and a narrow lower part with an open end. Their contours are printed in black ink (reference XEROX ^® 108R00934), in photo quality. This solid ink contains a wax concentration which has been found to be sufficient to achieve and obtain impermeable edges in the thickness of relatively thick substrate sheets, including WHATMAN ™ VF2 membranes of 785 μm in thickness.

The upper part of the intermediate units 12a and 12b is flared and is intended to form a hydrophilic zone, in this case a deposition zone 13a able to receive a liquid sample to be analyzed. Once deposited in the deposition zone 13a, the liquid sample will be able to migrate by capillarity towards the other hydrophilic zones of the analysis membrane, namely towards the lower part of the pattern. Narrower, it forms a channel within which four zones of interest 13b, 13c, 13d and 13e are formed. The position of each of these areas of interest is specifically identified by the marking elements 14b, 14c, 14d and 14e, also drawn in colored solid ink.

Due to the great hydrophilicity which characterizes fiberglass filtering media, it may be advantageous to be able to slow down the flow of liquids and / or modify their flow profile, particularly at the level of the zones of interest 13b, 13c, 13d and 13th. To do this, on the lower part of the intermediate units 12a and 12b, around the regions intended to form the areas of interest 13b, 13c, 13d and 13e, a low-wax color layer 14 covers the surface of the transfer film. 20. Unlike the contours of the intermediate units 12a and 12b which are printed in black solid ink, the low-wax layer 14 is obtained by printing, in solid ink, a light-colored layer (weakly intense). The intensity of the color of this layer is proportional to the amount of wax deposited on the transfer film; this color intensity is chosen so that, once in the thickness of the substrate sheet, the amount of wax thus transferred is insufficient to completely block the migration of liquids by capillarity.

The deposition of a pale yellow layer on the transfer film made it possible to reduce and modulate quite accurately the flow velocities of the liquids within the analysis membrane.

Areas of interest 13b, 13c, 13d and 13e were not modified by the wax. They retain the initial porosity and hydrophilicity of the substrate sheet used, and may be functionalized later.

Once the waxes are transferred into the thickness of the substrate sheet, the various more or less hydrophilic zones will be able to be functionalized and / or loaded with reagents according to the desired analyzes and tests.

3. Demonstration of the essential character of the mechanical treatment constituting the process according to the invention

The Figure 4 discloses the photograph of two analysis membranes made from a WHATMAN ™ MF1 membrane-type substrate sheet (367 μm thick) and using a transfer film 20, as set forth in FIG. Figure 3 and as described in the previous example.

The analysis membrane of the photograph (A) was manufactured using a method according to the invention and as illustrated in FIG. Figure 1 . The analysis membrane of the photograph (B) was made using the same method, but omitting the mechanical treatment (compression of the substrate sheet to about 1 kg / cm ² ).

The two analysis membranes are loaded with a colored solution. Unlike the analysis membrane of the photograph (A), with that of the photograph (B), large leaks of the colored solution are observed at the level of the deposition zone. The amount of wax transferred is significantly lower and does not allow to obtain pattern impermeable walls.

4. Analysis of the evolution of the thickness of the wax lines, printed on a transfer film and then transferred to a porous fiberglass substrate

1. a) Circular patterns with variable line widths are printed on a cellulose fiber transfer film (black solid ink printing, photo quality and XEROX ^® ColorQube ™ type printer), then transferred on WHATMAN ™ MF1 membrane (367 microns thick), by thermal and mechanical treatments according to the invention, for 5 minutes at 120 ° C. under a pressure equivalent to 0.5-2 kg.cm ^-2 . The thicknesses of the wax lines before and after transfer are measured under a microscope.

• b) Une solution colorée est ensuite déposée au centre de chacun des motifs réalisés pour évaluer l'étanchéité des bordures crées. La Figure 6 et la Figure 7 présentent les résultats obtenus, sous la forme de photographies.

The results obtained are presented at Figure 5 , in the form of a graph showing a linear relationship between the thickness of a wax line printed on the film of transfer and the thickness of this feature after its transfer into the thickness of a fiberglass substrate sheet.

• b) A colored solution is then deposited in the center of each of the patterns made to evaluate the tightness of the edges created. The Figure 6 and the Figure 7 present the results obtained in the form of photographs.

The impermeability to liquids is obtained for a wax thickness printed at least equal to 260 μm, giving after transfer a wax barrier of the order of 830 μm. Below this thickness, the colorant is no longer contained in the pattern. circular.

5. Transfer of wax to substrate sheets of different compositions and thicknesses, and evaluation of the impermeability of the borders created

Substrates of varying thickness and nature were tested. These are filter media marketed by the company GE HEALTHCARE LIFE SCIENCE (United States), under the trademark WHATMAN ™: MF1 filters, Fusion 5 and VF2, respectively 367 microns, 370 microns and 785 μm thick.

Transfers, by heat and mechanical treatments according to the invention, take place for 3 minutes at 120 ° C. and under a pressure equivalent to about 1 kg / cm ² . The tightness of the patterns is checked by means of a deposit of a colored solution in the flared portion of the pattern (deposition zone).

The results obtained are presented at Figure 8 , in the form of photographs. Whatever the nature and thickness of the substrates tested, barriers with a lateral thickness of the order of 800 microns provides a good seal to the system.

6. Examples of functionalization of an analysis membrane according to the invention a) Functionalization for Hepatitis B Screening

According to a first embodiment of the analysis membrane 10 previously described, it has been functionalized for the detection of hepatitis B, by immunodetection of the HBs antigen contained in the blood.

In this context, particular functions have been assigned to areas of interest 13b, 13c, 13d and 13e.

The zone 13d is functionalized by means of monoclonal antibodies anti-HBs, specific for the reaction; zone 13d forms the "spot test". The 13th zone is functionalized by means of anti-alkaline phosphatase monoclonal antibodies, specific for the detection conjugate; the 13th zone forms the "positive control spot". The zone 13c is functionalized by means of non-specific antibodies of the reaction (for example, anti-rat antibodies); zone 13c forms the "negative control spot".

The functionalization of these different zones by the antibodies is carried out by passive adsorption of the antibodies on the constituent fibers of the substrate.

Above these three spots, zone 13b is dedicated to storage of the second part of the conjugate complex (monoclonal antibodies anti-HBs labeled with biotin); zone 13b forms the "anti-HBs-biot Ac spot".

Optionally, the deposition zone 13a can also be used for storing the conjugate of the enzyme-linked immunosorbent reaction (streptavidin-alkaline phosphatase or STRE-PAL) in dried form. This conjugate will be resolubilized by the liquid phase of the sample to be analyzed.

b) Functionalization for dengue virus screening

According to a second mode of application of the analysis membrane 10 previously described, it has been functionalized for the detection, in blood and plasma, of two proteins of the dengue virus: the NS1 protein and the domain III of the envelope protein of the virus (DomIII).

• zone 13e (zone contrôle anti-PAL) : 0,35 µL d'un anticorps anti-phosphatase alcaline 1 mg/mL en PBS,
• zone 13c (zone de test) : 0,35 µL d'un anticorps anti-NS1 à 1 mg/mL en PBS,
• zone 13b : 1 µL d'une solution d'anticorps anti-NS1 marqués à la phosphatase alcaline à la concentration de 50 µg/mL en PBS-BSA 0,5 %,
• zone 13d (zone de contrôle négatif) : 0,35 µL de PBS-BSA 0,5 %.

Une fois les dépôts effectués, la membrane d'analyse est mise à sécher pendant 3 minutes à 60°C. Elle est alors prête à être utilisée.For the detection of the NS1 protein, the following deposits were made:

• zone 13e (anti-ALP control zone): 0.35 μL of an anti-alkaline phosphatase antibody 1 mg / mL in PBS,
• zone 13c (test zone): 0.35 μl of an anti-NS1 antibody at 1 mg / ml in PBS,
• zone 13b: 1 μl of a solution of anti-NS1 antibodies labeled with alkaline phosphatase at the concentration of 50 μg / ml in PBS-BSA 0.5%,
• zone 13d (negative control zone): 0.35 μL of PBS-BSA 0.5%.

After the deposits have been made, the test membrane is allowed to dry for 3 minutes at 60 ° C. It is then ready for use.

To test this analysis membrane, 20-25 .mu.l of plasma supplemented with 1 .mu.g of NS1 protein are deposited in the reservoir zone 13a. Once the totality of the sample has penetrated and migrated inside the pad, 10 μl of BCIP / NTP ( bromo - 4 - chloro - 3-indol phosphate / 4-nitroblue tetrazolium chloride ) are added to the zone 13b. Very quickly, the anti-PAL control zone and the test zone become positive, the negative control zone remaining colorless (cf. Figure 9 ). The detection is carried out in 5-6 minutes. In parallel, the experiment is carried out with 20-25 μL of plasma without NS1. In this case, only the control zone becomes positive.

Similarly, the previously described assay membrane was functionalized for the detection of the dengue virus envelope protein, using capture and detection antibodies directed against DomIII. The corresponding analysis membrane has been tested. Positive detection of DomIII and negative detection are illustrated by Figure 10A and Figure 10B , respectively.

Finally, the co-detection of the NS1 and DomIII proteins has also been successfully implemented by means of an analysis membrane (multiplexing) whose structure and design are very similar to those of the preceding analysis membranes. The difference lies in the presence of an additional reaction zone at the slower diffusion space. A photograph of this new analysis membrane according to the invention is presented on the Figure 11 .

Claims (15)

A method of manufacturing an analysis membrane (10) of microfluidic device; said analysis membrane (10) being formed from a porous substrate sheet (11) in the thickness of which solid wax forms impermeable borders (12) delimiting hydrophilic zones (13); said impermeable edges (12) describing, through the substrate sheet (11), a pattern patterned with wax;
characterized in that it comprises the following steps: - an intermediate pattern (12a, 12b), shaped with wax in the image of said wax pattern formed by the impermeable edges of the analysis membrane (10), is affixed to each of the faces of a sheet of a porous substrate (11) such that, on either side of the thickness of said substrate sheet (11), said mutually symmetrical intermediate units (12a, 12b) are positioned at least substantially opposite to each other; one of the other; - Maintained at least substantially horizontally, said substrate sheet (11) is subjected to a heat treatment capable of causing at least partial melting of the constituent wax of the intermediate units (12a, 12b) affixed to the faces of said sheet substrate (11), and a mechanical treatment capable of compressing the thickness of all or part of said substrate sheet (11); said substrate sheet (11) is subjected to a mechanical and thermal expansion phase able to allow said substrate sheet (11) to take up at least part of its initial thickness and the wax to resolidify inside the the thickness of said substrate sheet (11); and in that the thickness of said substrate sheet (11) is between 200 μm and 1000 μm. Process according to Claim 1, characterized in that the said process is carried out from a substrate sheet (11) of fibrous composition chosen from: cellulose fiber, nitrocellulose, cotton, linter, glass, silk, viscose, polypropylene, polyester, polyamide, poly (lactic acid). Process according to Claim 1 or 2, characterized in that it is carried out from a substrate sheet (11) containing the composition of a fiberglass filter medium. Process according to any one of Claims 1 to 3, characterized in that the said heat treatment is carried out inside a thermo-controlled heating chamber and the said mechanical treatment is implemented by means of mechanical means, installed within said thermo-controlled enclosure and adapted to apply pressure or compressive force sufficient to compress the thickness of said substrate sheet (11), at least at the intermediate units. Process according to any one of claims 1 to 4, characterized in that said heat treatment and said mechanical treatment are carried out simultaneously by means of a thermo-controlled heating compression device, provided with two horizontal heating compression plates. Process according to claim 5, characterized in that , for the implementation of said thermal and mechanical treatments, a layer of flexible and elastic material, at least substantially planar, is interposed between each of the faces of said substrate sheet (11) and hot compression plates; said layer of flexible material acting as a thermal and mechanical buffer (15). A method according to claim 6, characterized in that a layer of absorbent material is interposed between each intermediate pattern (12a, 12b) affixed to one side of the substrate sheet (11), and said thermal and mechanical buffer. Method according to one of claims 1 to 7, characterized in that the intermediate units (12a; 12b) are affixed to each of the faces of said substrate sheet (11) with a wax transfer technique. Process according to claim 8, characterized in that the intermediate units (12a, 12b) are first printed on a transfer film (20) and are then transferred to each of the faces of said substrate sheet (11), then in its thickness by carrying out said thermal and mechanical treatments. The method of claim 9, characterized in that said transfer film (20) is a cellulose fiber paper sheet. Microfluidic device analysis membrane in the thickness of which solid wax forms impermeable borders (12) delimiting hydrophilic zones (13), and describing through said analysis membrane (10) a pattern drawn with wax ; characterized in that it has a thickness of between 200 μm and 1,000 μm Analysis membrane according to claim 11, characterized in that its composition is made of cellulose fiber, nitrocellulose fiber or fiberglass, cotton, linter, cellulose wadding, silk fiber, viscose, polypropylene, polyester, polyamide or poly (lactic acid). Analysis membrane according to claim 11 or 12, characterized in that it is made of fiberglass. Analysis membrane according to any one of claims 11 to 13, characterized in that it comprises, in its thickness, encrustations of wax, present at the level of at least one hydrophilic zone (13). Analysis membrane according to any one of claims 11 to 14, characterized in that it has a face covered with a sealing layer.

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