Method for manufacturing analysis chips and analysis chip thus obtained

Abstract

Disclosed is a method for manufacturing an analysis chip for analysing a biological sample (1), which method comprises providing a matrix (10) formed in a solid support material in which at least one through hole has been formed (11) and at least one pellet (3), cut out of a sheet (6) of solid and porous analysis material; inserting at least one pellet (3) into the at least one through hole (11) of the matrix (10) by translation of the pellet (3) in a direction at right angles to the lower and upper surfaces of the matrix (10); mechanically assembling at a temperature lower than the melting points of the support and analysis materials by means of a pressing force applied at right angles to the upper surface of the matrix.

Description

[0001] Method of manufacturing an analysis chip and analysis chip

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of biological analyses, in particular biochemical analyses.

[0004] More specifically, the invention relates to a method for manufacturing filter chips, which can optionally be functionalized to carry out biological analyses.

[0005] TECHNOLOGICAL BACKGROUND

[0006] In the field of biological analysis, protein microarrays are used to study the biochemical activity of proteins. In such microarrays, a library of antibodies or protein fragments ("probes") - or even entire proteins - is placed on a matrix such as a glass slide. In this case, a single sample is tested on all the probes placed on the matrix.

[0007] Biological analysis devices allowing the parallel analysis of several samples have also been developed.

[0008] Document WO2014 / 053237 is an example in which a miniaturized device allows analysis of several biological samples simultaneously (“multiplex” analysis). Each sample can also be exposed to several different probes successively. In other words, the device allows for “3D analysis” type analysis.

[0009] The device described by this document comprises a plurality of channels, into each of which a liquid phase sample can be injected independently of the other channels.

[0010] Each channel can be formed of several tubular portions. Between two successive portions is inserted an approximately cylindrical analysis zone formed in a suitable matrix.

[0011] The analysis zones can in particular be formed in a flat nitrocellulose matrix, the entire surface of which except the analysis zones is made hydrophobic by wax impregnation.

[0012] The analysis zones can be simply made of untreated nitrocellulose, so that they constitute federation zones, or of nitrocellulose functionalized for example by means of a probe molecule.

[0013] The wax impregnation operation makes it possible to delimit the analysis zones, but also to limit the lateral diffusion inside the matrix (i.e. outside a given analysis zone towards the other neighboring analysis zones) of the molecules of interest (probe molecules or molecules of a sample).

[0014] For this impregnation operation, a solid ink printing process is implemented, so as to deposit a layer of wax on the matrix in the areas which must be made hydrophobic. After printing, the matrix is ​​heated to a temperature higher than the melting temperature of the wax used, then cooled, so that the wax diffuses laterally and deeply, in the thickness of the matrix, so as to limit its subsequent undesirable diffusion towards the analysis areas.

[0015] However, such a method does not allow fine control of the volume of a given analysis zone or the shape of the surface that delimits this volume, as shown in the figures presented in this document. In particular, the circumference of the upper surface of a test site varies from one site to another and is generally not circular, so that, in the direction of flow, the cross-section of the analysis zone is not precisely identical to the internal cross-section of the channel in which the analysis zone is to be inserted.

[0016] Consequently, the precision of such a device is limited due to the manufacturing process used to form the analysis zones, and concomitantly, the limit of quantification remains too high for certain biological analyses in which the concentrations involved (or their variations) are particularly low.

[0017] Document US2004 / 0115707 discloses a biochemical analysis unit comprising a base plate having a plurality of holes filled with a porous and adsorbent material so as to form a plurality of analysis zones.

[0018] Filling of the holes can be achieved by rolling a sheet of adsorbent material onto the previously drilled base plate.

[0019] During rolling, the sheet of analysis material is stressed anisotropically due to the tensile force exerted in the rolling direction. The properties of the adsorbent material after insertion into the holes are therefore anisotropic. It is even possible for the thickness of the adsorbent material to vary within the same hole.

[0020] Furthermore, the rolling does not break the continuity of the adsorbent material sheet. The adsorbent material therefore forms a continuous surface between two channels under or on the plate, as can be observed in Figure 2b of document US2004 / 0115707. The molecules of interest (from the sample or probes) therefore risk diffusing from one channel 3 to the other due to this continuity.

[0021] The accuracy and sensitivity of a quantitative analysis carried out with such a plate are therefore limited.

[0022] In another embodiment described by US2004 / 0115707, the adsorbent material can be dissolved in a solvent. The resulting solution is then injected into the holes and the solvent evaporated. This liquid-phase injection technique also does not allow precise control of the isotropy of the properties of the test area, in particular because the air flow allowing the evaporation of the solvent is necessarily directional.

[0023] Furthermore, traces of solvent may remain in the adsorbent material, which may interact with the probe molecules or molecules to be analyzed. In addition, the use of solvents, particularly organic solvents in the case of nitrocellulose, makes the process polluting.

[0024] Finally, the bond between the adsorbent material, once solidified, and the base plate is not guaranteed with certainty. The quality of this bond depends in particular on the chemical compositions of the adsorbent material and the base plate. The bond between a given analysis zone and the plate may therefore prove fragile. In the event of forced circulation of liquid, by means of a relative vacuum, these analysis zones would risk detaching and being carried away by the circulating liquid. It is therefore not possible to carry out an analysis with forced circulation of liquid through the analysis zones obtained by this embodiment.

[0025] Other processes, using different chemical products or a heating step or an irradiation step for example, are also described in document W001 / 19502A2, after a first rolling step.

[0026] In addition to the disadvantages of lamination previously explained, all these embodiments have the disadvantage of causing physicochemical modifications of the filter membrane which alter its essential properties for the analysis and therefore the sensitivity and precision of the analysis.

[0027] To the extent that the chemical composition and physical structure of the membrane on which the analysis is carried out influence the performance of the analysis method, and in particular the quantification limit of this method, the invention therefore aims to propose a method for manufacturing a chip for analyzing a biological sample making it possible to finely control this chemical composition and this physical structure.

[0028] In particular, the invention aims to propose a method for manufacturing a low-cost analysis chip, not requiring a thermal, chemical or irradiation treatment step for the formation of the test sites in the matrix (apart from during a possible biochemical functionalization of these sites after or before the formation of the sites) and making it possible to carry out a quantitative analysis of high precision and sensitivity and / or an analysis of a biological sample or a simple filtration of a liquid biological sample.

[0029] SUMMARY OF THE INVENTION

[0030] Thus, the invention relates to a method of manufacturing a chip for analyzing a biological sample comprising:

[0031] - a matrix formed from a solid support material is provided, having a lower surface and an upper surface and having at least one hole formed therethrough between said lower and upper surfaces;

[0032] - at least one pellet is provided, cut from a sheet of solid and porous analysis material, the pellet having a lower surface and an upper surface,

[0033] - at least one pellet is inserted into at least one through hole of the die by translation of at least one pellet in the direction normal to the lower and upper surfaces of the die;

[0034] - a mechanical assembly is carried out at a temperature lower than the melting temperatures of the support and analysis materials, during which a pressing force in a direction normal to the lower and upper surfaces of the matrix is ​​exerted on at least one portion of the matrix which adjoins the at least one pellet inserted into the matrix and / or on at least one of the lower and upper surfaces of the at least one pellet inserted into the matrix.

[0035] Thanks to these arrangements, an analysis chip is obtained comprising at least one pellet of analysis material inserted into a hole passing through a support material. The assembly between the pellet and the support material is not obtained by a chemical process, nor by melting one of the materials so that the physical and chemical properties of the support and analysis materials before assembly are not or at least are very little altered after assembly, including near the interface between these two materials. The assembly is obtained solely mechanically and by the exercise of a pressing force normal to the upper and lower surfaces of the matrix, so that the deformation of the materials is uniform in a plane normal to the direction of the pressing force.The method therefore makes it possible, unlike methods implementing a rolling step, not to introduce anisotropy into the support material and / or the analysis material in a direction normal to the direction of the pressing force. Such anisotropy would lead, for example, in the case of immunological tests using fluorescent reagents to inhomogeneous fluorescence on the surface of an analysis pellet, which would make the quantitative analysis of the fluorescence signal imprecise.

[0036] Thanks to all of these provisions, the sensitivity and reproducibility of an analysis chip obtained by the method according to the invention are therefore improved compared to chips obtained according to the methods of the prior art.

[0037] Furthermore, the means necessary for implementation are solely mechanical, therefore not very polluting in that they do not include solvents and they are simple to implement. According to different aspects, it is possible to provide one and / or the other of the characteristics below taken alone or in combination.

[0038] According to one embodiment, in the method for manufacturing a chip for analyzing a biological sample, the pressing force is exerted on a portion of the matrix which adjoins the at least one pellet inserted in the matrix. Thanks to this arrangement, this portion of the matrix can be folded above and / or below the pellet so that the pellet can be at least partially crimped by the matrix. Thus, the assembly of the pellet to the matrix in the analysis chip will have good mechanical strength and will not be affected by the flow of a liquid sample to be analyzed in the direction normal to the upper and lower surfaces of the matrix, or even by a relative vacuum applied on the side of one of these surfaces in order to accelerate the flow of the liquid sample.

[0039] According to one embodiment of the method for manufacturing a chip for analyzing a biological sample, the pressing force is exerted on at least one of the lower and upper surfaces of the at least one pellet inserted into the matrix. Thanks to this arrangement, the pellet at least partially crimps the matrix so that the assembly of the pellet to the matrix will have a certain mechanical resistance and it will not be affected by the flow of a liquid sample to be analyzed in the direction normal to the upper and lower surfaces of the matrix, or even by a relative vacuum applied on the side of one of these surfaces in order to accelerate the flow of the liquid sample.

[0040] According to a particular embodiment, in the method for manufacturing a chip for analyzing a biological sample, the support material is hydrophobic and the analysis material is hydrophilic or vice versa. In this way, it is for example possible to deposit on the pad a sample to be tested in aqueous phase without it diffusing towards the support material if the latter is hydrophobic. Conversely, if the support material is hydrophilic, the analysis material is hydrophobic and it is then possible to deposit on the pad a sample to be tested in organic phase without it diffusing towards the support material.

[0041] According to a particular embodiment, in the method for manufacturing a chip for analyzing a biological sample, for inserting the at least one pellet into the at least one through-hole, the at least one pellet is translated into the at least one through-hole by means of a punch, the at least one pellet having been cut out from the sheet of analysis material before its insertion by means of this same punch and the at least one through-hole having been previously formed in the matrix by means of this same punch. Thanks to this arrangement, only one tool is necessary to prepare an analysis chip, namely a punch having one or more punches of sizes and shapes adapted to the shapes of the desired wells.Such a tool is simple to design and implement and possibly allows automation of the process, which makes it possible to obtain high and controlled precision analysis chips in a reproducible, rapid and low-cost manner. According to a particular embodiment, in the method for manufacturing a chip for analyzing a biological sample, at the end of the mechanical assembly, the at least one pellet is functionalized. For example, biochemical functionalization can be envisaged, using an antibody or an antigen that adsorbs onto the pellet.

[0042] In this way, the analysis chip of a biological sample obtained by the method makes it possible to implement an analysis test using the reagent used for functionalization, for example an immunological test. The analysis chip can therefore be adapted to the analysis needs thanks to this functionalization step. The analysis chips can for example be mass-produced before the functionalization step and each functionalized at will at the time of analysis. According to a particular embodiment, in the method for manufacturing a chip for analyzing a biological sample, the functionalization of the analysis material is carried out before the insertion of the at least one pellet into the matrix. Thanks to this arrangement, the functionalization can be carried out on the entire sheet of analysis material before the cutting of the pellet. This saves time when the analysis chips are prepared in series.The control of functionalization, and in particular of the quantity of analysis reagent deposited on each pellet, is also better, which ultimately allows for better precision and better reproducibility of tests carried out with a given series of analysis chips.

[0043] According to a particular embodiment, in the method for manufacturing a chip for analyzing a biological sample, the insertion of the at least one pellet into the matrix is ​​repeated at least once using for each new insertion a functionalized analysis material different from that used for the previous insertion and a punch corresponding to at least one through hole in the matrix different from that used for the previous insertion.

[0044] Thanks to this arrangement, it is possible to form several differently functionalized analysis pellets on the same analysis chip. It is then possible to carry out several different tests simultaneously on the same chip, on the same sample or on several different samples. The method remains simple to implement since it only requires different punches or equivalently a single punch equipped with several punches positioned at different locations and which can be activated separately or in groups. According to a particular embodiment, in the method for manufacturing a chip for analyzing a biological sample, the mechanical assembly of at least one pellet with the matrix results in crimping of at least one pellet on at least a portion of its lower and upper surfaces by the matrix.

[0045] By virtue of this arrangement, the pellet cannot be pushed, under normal conditions of use, outside the die at least on one side of this die. The assembly of the die and the pellet therefore resists a relative vacuum or the pressure exerted on one side of the pellet by the sample to be tested when it is deposited.

[0046] According to a particular embodiment, in the method for manufacturing a chip for analyzing a biological sample, before inserting the at least one pellet into the matrix, the at least one pellet is brought to a temperature lower than that of the matrix. Thanks to this arrangement, the analysis material shrinks before its insertion, which facilitates its insertion into the through hole by reducing the contact forces and it expands after insertion, so that after insertion, contact between the pellet and the matrix is ​​ensured and allows the pellet to be held in place in the matrix. The invention also relates to a chip for analyzing a biological sample comprising:

[0047] - a matrix formed in a solid support material, having a lower surface and an upper surface and in which at least one hole has been formed passing through it between said lower and upper surfaces;

[0048] - at least one pellet, cut from a sheet of solid and porous analysis material and inserted into the at least one through hole, the at least one pellet having a lower surface and an upper surface, the chip for analyzing a biological sample being characterized in that the at least one pellet is crimped on at least one of its upper and lower surfaces by the matrix.

[0049] Such an analysis chip has the advantage of not containing any solvent residue or fusion or solder zones that could alter the accuracy of a test carried out with this chip. Crimping allows both the native physicochemical properties of the support material and the analysis material to be preserved. It also allows a test to be carried out with flow of a sample along the axis of the through hole from one side of the chip to the other, since the assembly between the chip and the matrix has good mechanical resistance.

[0050] According to one embodiment of the chip for analyzing a biological sample, the support material comprises at least one component chosen from a metal, a plastic material and cellulose and in that the analysis material from which the at least one pellet is formed comprises at least one component chosen from nitrocellulose, cellulose, and an organic polymer.

[0051] Such materials are inexpensive and have the necessary qualities of biochemical inertness and adsorption to carry out analyses such as biochemical tests.

[0052] According to one embodiment of the chip for analyzing a biological sample, the assembly of the at least one pellet and the matrix resists at least a relative vacuum equal to 0.100 bar.

[0053] Thanks to this arrangement, it is possible to carry out an analysis on a sample flowing forcibly through the pellet without the pellet separating from the matrix due to the overpressures which are exerted locally.

[0054] The invention also relates to a device for analyzing a biological sample comprising at least two chips for analyzing a biological sample according to one of the preceding embodiments, superimposed and in which at least one pellet of one of the at least two chips is configured to provide a filtration function and is superimposed with at least one functionalized pellet of another chip of the at least two chips.

[0055] It is thus possible to stack several analysis chips to obtain a three-dimensional analysis device, the analysis carried out varying from one analysis site to another in the direction of the stacking and / or within a given analysis chip and to carry out a first filtration step before the analysis, in particular to separate the serum from the red blood cells for the purpose of analyzing a blood sample. In the latter case, the device makes it possible to avoid a centrifugation step. The invention further relates to a diagnostic kit comprising at least one chip for analyzing a biological sample according to one of the preceding embodiments and at least one analysis reagent. One or more analysis reagents, in particular a buffer, a solvent, an antigen, an antibody, can thus be provided in order to carry out a test, such as a standardized immunological test.

[0056] The invention also relates to the use of a chip for analyzing a biological sample according to one of the preceding embodiments for diagnostic purposes or for an immunological test.

[0057] The invention finally relates to a device for manufacturing a chip for analyzing a biological sample according to one of the preceding embodiments, the manufacturing device comprising:

[0058] - an insertion system adapted to insert the at least one pellet into the at least one through hole of the die by translation of the pellet in the direction normal to the lower and upper surfaces of the die;

[0059] - a mechanical assembly system at a temperature lower than the melting temperatures of the support and analysis materials, adapted to exert a pressing force in a direction normal to the lower and upper surfaces of the matrix on at least a portion of the matrix which adjoins the at least one pellet inserted into the matrix and / or on at least one of the lower and upper surfaces of the at least one pellet inserted into the matrix.

[0060] Such a manufacturing device is simple to implement and introduces only minimal and isotropic deformation of the support and / or analysis material in any plane parallel to the lower and upper surfaces of the matrix. It therefore makes it possible to form analysis chips at low cost while preserving the native physicochemical properties of the support and analysis materials.

[0061] BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Embodiments of the invention will be described below with reference to the drawings, briefly described below:

[0063] Figure 1 shows an embodiment of a test chip support matrix.

[0064] Figure 2 shows a support strip from which three basic pieces, each of which can form a support matrix, have just been cut out.

[0065] Figure 3al represents a portion of support matrix at the start of drilling a through hole, seen in section along a plane containing the axis of this hole, according to a particular embodiment.

[0066] Figure 3a2 represents a portion of support matrix in which a through hole is being drilled, seen in section along a plane containing the axis of this hole, according to a particular embodiment. Figure 3b represents a portion of support matrix, after drilling a through hole and at the start of a step of inserting a pellet of analysis material, seen in section along a plane containing the axis of this hole, according to a particular embodiment.

[0067] Figure 3c represents a portion of support matrix in the process of inserting a pellet of analysis material, seen in section along a plane containing the axis of this hole, according to a particular embodiment.

[0068] Figure 3d represents a portion of support matrix at the end of a step of inserting a pellet of analysis material, seen in section along a plane containing the axis of this hole, according to a particular embodiment.

[0069] Figure 4a shows a sectional view of the same portion of support die as in Figure 3d at the start of the assembly step in a particular embodiment.

[0070] Figure 4b shows a sectional view of the same portion of support die as in Figure 3d during the assembly step in a particular embodiment.

[0071] Figure 4c represents a sectional view of the same portion of support matrix as in Figure 3d at the end of the assembly step in a particular embodiment.

[0072] Figure 5 shows a top view of one embodiment of an analysis chip.

[0073] Figure 6 shows a multiplexed analysis device comprising two analysis chips.

[0074] In the drawings, like references designate identical or similar objects.

[0075] Figure 7 shows a top view of an embodiment of an analysis chip support matrix, in which the through holes have different shapes.

[0076] Figure 8a shows a photograph obtained with a binocular loupe (Zeiss, model Stemi SV8) of a chip for analyzing a biological sample 1 cut along a plane orthogonal to the upper and lower faces of the chip and containing a diameter of the circular section of a cylindrical through hole.

[0077] Figure 8b reproduces a photograph of an analysis chip obtained with a prior art method using a Xerox® Solid Ink printer, the diameter of the analysis pellets being of the order of 500 micrometers.

[0078] Figure 8c shows a photograph of the analysis chip of Figure 8b obtained with a binocular loupe (Zeiss, model Stemi SV8, magnification x64).

[0079] Figure 8d shows a photograph of the analysis chip of Figure 8a obtained with a binocular loupe (Zeiss, model Stemi SV8, magnification x64) obtained by the method according to the invention, in which the analysis material is nitrocellulose, the support material is black paper coated with wax, the diameter of the analysis pellets being equal to 500 micrometers.

[0080] Figure 9 shows a photograph of an analysis chip obtained with a binocular loupe (Zeiss, model Stemi SV8, magnification x64) obtained by the method according to the invention, in which the analysis material is nitrocellulose, and the support material is brass, the diameter of the analysis pellets being equal to 500 micrometers. DETAILED DESCRIPTION

[0081] The invention relates to a method for manufacturing a chip for analyzing a biological sample 1 intended to be implemented in isolation or in an analysis device 7. The analysis device 7 - or the chip for analyzing a biological sample 1 alone - makes it possible, for example, to carry out analyses of biological liquids such as blood or a liquid fraction of blood (plasma, serum), urine, saliva, etc.

[0082] The liquid analyzed can also be a reaction medium comprising biomolecules such as antibodies or proteins.

[0083] The notion of analysis of a biological sample must therefore be understood in the broad sense, that is to say that it is an analysis involving at least one biomolecule among the reagent(s) and / or the analyte(s).

[0084] The biological sample analysis chips 1 can thus be used to detect and quantify complex biomolecules in biological media: blood, plasma, serum, organs or organ extract, reaction medium in which complex biomolecules are produced (antibodies, proteins).

[0085] In particular, the biological analysis can be an immunological test such as an ELISA test (“Enzyme-Linked ImmunoSorbent Assay”).

[0086] Biological sample analysis chips 1 can still be used in the food industry to search for pathogenic agents, for example during health checks.

[0087] The method for manufacturing a chip for analyzing a biological sample 1 according to the invention comprises:

[0088] - providing a support matrix 10 formed from a solid material, called “support material”, having a lower surface and an upper surface and in which at least one hole 11 has been formed passing through it between its lower and upper surfaces;

[0089] - providing at least one pellet 3 cut from a sheet of a second porous solid material, called “analysis material”, the at least one pellet 3 having a lower surface and an upper surface;

[0090] - the insertion of the at least one pellet 3 into the at least one through hole 11 of the support matrix 10 by translation of the at least one pellet 3 in the direction normal to the lower and upper surfaces of the matrix 10;

[0091] - a cold mechanical assembly, at a temperature lower than the melting temperatures of the support and analysis materials, during which a pressing force in a direction normal to the lower and upper surfaces of the matrix 10 is exerted on at least a portion of the matrix 10 which adjoins the at least one pellet 3 inserted in the matrix 10 and / or on at least one of said lower and upper surfaces of the at least one pellet 3 inserted in the matrix 10. The chip for analyzing a biological sample 1 obtained at the end of the method comprises:

[0092] - a matrix 10 formed in a solid support material, in which at least one hole 11 has been formed passing through it;

[0093] - at least one pellet 3, cut from a sheet of solid and porous analysis material and inserted into the at least one through hole 11, the at least one pellet 3 being crimped on at least one of its upper and lower surfaces by the die 10.

[0094] The chip for analyzing a biological sample 1, which can be seen in a particular embodiment in FIG. 5, therefore comprises a support matrix 10 formed in a solid material of thickness el in which one or more through holes 11 have been formed. A particular embodiment of the support matrix 10 is shown in FIG. 1. Another particular embodiment of the support matrix 10 is shown in FIG. 7. As shown in FIG. 2, the support matrix 10 is formed by cutting out a base part 21, of a suitable shape for the analysis device in which it is intended to be used or for its use in isolation. The base part 21 is for example a rectangular or square parallelepiped cut out from a support strip 2 of a solid material, hereinafter called "support material", having a lower surface and a plane upper surface parallel to each other.

[0095] A base piece 21 cut from the support strip 2 to form a support matrix 10 may be a parallelepiped and have a width L1 of between 5 mm (5 millimeters) and 50 mm and a length L2 of between 5 mm and 50 mm.

[0096] In a particular embodiment, the support matrix 10 is formed in an analysis material of constant thickness el between the lower surface and the upper surface, these surfaces being in this case flat and parallel to each other.

[0097] The thickness el of the support strip 2 is then constant and identical to that of the cut-out part 21. It is preferably less, for example by at least a factor of ten, than the other dimensions (length L1 and width L2) of the cut-out part 21.

[0098] The support strip 2 may for example have a width L3 either identical to, or slightly greater than, the width L1 of the support matrix 10, or for example greater than twice the width LL.

[0099] In the latter case, it is possible to cut several basic pieces 21 in the width of the support strip 2.

[0100] The width L3 of the support strip 2 is thus, for example, between 5 mm and 50 mm.

[0101] The length L4 of the support strip 2 may be greater, or even much greater, than the length L2 of the part 21. The length L4, for example, may be greater than 1 m or even 10 m.

[0102] In this way, it is possible to successively cut out several base pieces 21 from the support strip 2. The cutting of a base piece 21 can for example be carried out by means of a punch into which the support strip 2 is inserted.

[0103] If the support strip 2 is long enough, the cutting of the base pieces 21 can be automated, the support strip 2 being translated by an adequate distance between two successive cuts of a base piece 21.

[0104] The thickness el of the support strip 2 (and of a base piece 21 cut from this support strip 2) may be less than 1 mm, less than 0.15 mm, or even less than 0.1 mm. For example, the thickness el of the support strip may be equal to 0.06 mm.

[0105] In a particular embodiment, the support strip 2 has a width of 20 mm and a length of 25 m for example. The width and length can be changed depending on the type of analysis chips 1 to be manufactured. The thickness of the support strip 2 can be equal to 0.12 mm, i.e. the current thickness of the filter membranes (generally nitrocellulose) formed in the analysis material, but it could also be of the order of 0.10 mm.

[0106] In a particular embodiment, a base part 21 is a square filter membrane with a side of 20 mm.

[0107] The support strip 2 may in particular be made of metal, for example steel, copper or brass. The support strip 2 may, in an alternative embodiment, be made of plastic. By way of non-limiting example, the plastic material may be polyethylene, polyvinyl chloride, polystyrene, polymethyl methacrylate, polypropylene or any other plastic material commonly used in the field of biochemical analyses. It may have undergone a surface treatment or be UV-resistant.

[0108] The carrier material may also contain plant fibers, for example cellulose. This may include paper.

[0109] The support material is solid but not necessarily rigid. The support strip 2 may thus have a certain flexibility, provided that the support strip 2 or a support matrix 10 formed from this support strip 2 can be handled and moved for the preparation of the analysis chips 1, in particular without tearing, including in the case where the preparation of the support matrix 10 is automated. For example, the Rex Copy A4 photocopier paper distributed by the company Mondi®, with a weight of 80g / m 2 , available on the priority date of this patent application, is suitable for the invention.

[0110] In the case where the support strip 2 is flexible, the material is sufficiently rigid so that the upper and lower surfaces are effectively flat when the lower surface is, at least locally, simply placed on a flat support.

[0111] In one embodiment, the support material is rigid enough to allow one or more base pieces 21 to be cut out using a die cutter. In a base piece 21 of support material, at least one through hole 11 is formed passing through the material in its thickness, i.e. in the direction normal to the lower and upper surfaces of the piece 21.

[0112] In a particular embodiment, a through hole 11 is formed by means of a punch 42, as shown in Figures 3al (at the start of drilling), 3a2 (during drilling), and 3b (just before the injection step which follows the drilling step, described later). In this embodiment, the punch 42 is translated along the direction of the axis of the future through hole 11, so as to pierce the support die 10. A dedicated cutting guide 4 can be placed under the support strip 2. The stroke of the punch 42 through the cutting guide 4 is adjusted so as to allow the ejection of a pellet 10b from the support strip 2, as can be seen in Figures 3al and 3a2.

[0113] The punch can then be moved in the opposite direction so as to release the support die 10 then comprising one or more through holes 11.

[0114] In a particular embodiment, the through holes 11 are formed on the locations corresponding to the future base pieces 21 in the support strip 2 before one or more base pieces 21 are cut out.

[0115] In another embodiment, the through holes 11 are formed in an already cut base piece 21.

[0116] Alternatively, the through holes 11 are formed at the same time as the part 21 is cut out, for example by means of a suitably shaped die.

[0117] The shape of the through holes 11 can be chosen according to the needs of the analysis. For example, the surface delimiting the interior of a through hole 11 is a cylinder whose generatrix is ​​parallel to the direction normal to the lower and upper surfaces of the part 21, a direction which will be called in the following “axis of the hole 11”.

[0118] For example, the through holes 11 are cylinders of revolution.

[0119] In the embodiment shown in Figure 7, one of the through holes 11 can be analyzed as formed by two through sub-portions of circular section 11a and 11b, connected by a channel 11c. Once the analysis material pellets are inserted as described below, it will thus be possible to deposit the sample to be analyzed in the well corresponding to the first “sub-hole” and to let the sample diffuse from the sub-portion 11a to the sub-portion 11b. In this case, it is possible to use the analysis chip to carry out a lateral flow type test.

[0120] The characteristic dimensions of a through hole 11 in a direction of the upper or lower surface of the part 21 in which the through hole 11 is formed may be less than 1 mm.

[0121] For example, a support matrix 10 may comprise 9, 12, 24, 48 or 96 holes (or wells) 11 having the shape of cylinders of revolution with a diameter dl of the order of 300 to 800 pm (micrometers), two successive through holes 11 being spaced apart by a distance d2 of the order of 100 to 250 pm.

[0122] Optionally, a cutout or reference mark 12 is formed on the support matrix 10 so as to be able to identify its orientation, in particular during an analysis which will be carried out subsequently. This arrangement makes it possible to differentiate the through holes 11 from each other when the support matrix 10 has elements of symmetry.

[0123] At the end of the step of drilling a through hole 11, the lower and upper faces of the support material are no longer strictly flat near the lower and upper bases of the through hole 11 but an overhang 10a of support material is formed over the entire periphery of a through hole 11 on the side of the lower face of the support material, due to the resistance that the support material opposes to its cutting. This overhang 10a, which can be observed in figure 3a2, will be used to advantage during the subsequent assembly step.

[0124] In the first step of the method according to the invention, a support matrix 10 is therefore provided, formed in a support material of constant thickness el between a lower surface and an upper surface in which one or more holes 11 have been formed passing through its thickness.

[0125] In a second step, a sheet 6 of constant thickness, denoted e2, of a second porous solid material called “analysis material”, having an upper surface and a lower surface, is provided.

[0126] The analytical material is intended to receive on one of its lower and upper surfaces a liquid sample to be analyzed or filtered, which must then be able to flow towards the other of these surfaces, either spontaneously by simple diffusion, or due to forced circulation of the liquid. The analytical material can therefore be a porous material such as paper, in particular filter paper, that is to say paper with a high alpha-cellulose content (in particular more than 90%, 95%, or even 98% alpha-cellulose).

[0127] The analysis material can still be nitrocellulose.

[0128] Nitrocellulose has a good affinity for small proteins, peptides, or nucleic acids. It is therefore particularly well suited for biological analyses. However, these examples should not be considered limiting.

[0129] The analysis material can be chosen in particular according to its resistance to humidity, its filtration speed, its breaking force, its capillary rise speed or even its resistance to the passage of air.

[0130] In the case where the liquid to be analyzed contains mainly water, the analysis material is preferably hydrophilic, so that the liquid to be analyzed wets the surface of the analysis material. In this case, the support material can be hydrophobic.

[0131] Throughout the following, a material will be considered hydrophobic if water does not wet the material, that is, if the angle between a drop of water and the surface of the material on which the drop is deposited is strictly greater than 90°. Otherwise, the material under analysis is hydrophilic.

[0132] Alternatively, the assay material may be hydrophobic and the support material hydrophilic.

[0133] The analytical material may be an isotropic or anisotropic filter membrane. In particular, it may be an organic filter membrane, i.e., a membrane comprising an organic polymer such as cellulose acetate, a polysulfone, or a polyamide.

[0134] The thickness e2 of the analysis material can be close to the thickness el of the support material. The thickness e2 can be greater than, equal to or less than the thickness el.

[0135] In the case where the support material is nitrocellulose, the thickness e2 of the analysis material could thus be of the order of a few hundred, or even a few tens of micrometers, for example 50 pm to 150 pm.

[0136] In a third step, called injection, a portion of the analysis material, called pellet 3, is inserted into at least one through hole 11 of the support matrix 10, so that the pellet 3 closes this hole 11.

[0137] A pellet 3 is therefore complementary to a hole 11 in which it must be inserted over at least part of the thickness of the support material. In other words, if the surface delimiting the interior of a through hole 11 is a cylinder whose generatrix is ​​parallel to the direction normal to the lower and upper surfaces of the part 21, a pellet 3 which can be inserted therein is a cylinder whose generatrix is ​​parallel, after insertion, to the axis of the through hole 11 whose base has the same shape as the base of the through hole 11.

[0138] The term "lozenge" should therefore not be interpreted in a limiting manner in terms of shape. It was chosen in relation to the easiest embodiment to implement, that is to say the one for which the through hole 11 and the lozenge 3 are cylinders of revolution.

[0139] Thus, in the embodiment shown in Figure 7, the pellet 3 inserted into the through hole 11 formed by two sub-parts 11a, 11b and a channel 11c will have the complementary shape adapted to fill the sub-parts 11a, 11b and 11c, while the pellet 3 inserted into the cylindrical through hole 11 will be cylindrical.

[0140] The height of the pellet 3 may in any case be equal to the height of the hole 11 (as seen in the sectional view along a plane containing the axis of the through hole 11 shown in Figure 4c) or different from it (see Figure 9 which shows an analysis chip 1 according to the invention whose support material is brass coated with Le Parfait® food paraffin (reference 365 EMB 44 026, packaging 250g) and the analysis material nitrocellulose (Reference: Amersham Protran® Premium pores 0.45pm NitroCellulose, GE Healtcare Life Science Nitrocellulose Bloting Membrane Nucleic acid and Protein application Catalogue No 10600008).

[0141] The fitting carried out in the insertion step is obtained solely by translation of the pellet 3 along the axis of the through hole 11. For example, if the through hole 11 has been formed in the support material by means of a punch, the support die 10 can remain in place under the punch 42 after drilling the hole 11.

[0142] A sheet 6 of analysis material is then placed above the pierced support die 10, as shown in Figure 3b and the punch 42 is again moved along the axis of the hole by a distance at least slightly less than that which made it possible to pierce the hole 11.

[0143] In this way, the punch 42 cuts out the pellet 3 to be inserted and drives it in its path inside the through hole 11, but without it completely emerging from the through hole 11 on the side of the lower surface of the support die 10 and in such a way that it is positioned above at least part of the overhang 10a.

[0144] At the end of this insertion step, the pellet 3 is therefore well fitted, at least over part of its height, in the through hole 11.

[0145] The choice of the stroke of the punch makes it possible to position the pellet 3 at a chosen height in the through hole 11 concerned, for example so that the lower base of the pellet 3 is in the same plane as the lower surface of the support die 10 or at least the lowest points of the overhang 10a, as shown in Figure 3c.

[0146] It is also possible to use a die dedicated to the insertion step, for example in an embodiment in which the production of the dies is automated and carried out well before the insertion step.

[0147] It is also possible to provide one or more pellets 3 cut in advance, for example by means of a punch or any other precision cutting tool and to insert them into the through hole 11 which corresponds to them by a vertical translation movement.

[0148] The embodiment in which cutting and insertion are carried out consecutively with the same die has the advantage of the simplicity of positioning the pellets and the speed of carrying out this step.

[0149] In the latter case, it is necessary to produce a punch tool and two corresponding counter parts in order to be able to properly perforate the support strip 2 and thus produce the wells (or "spots", or through holes 11) in the support strip 2 initially. This tool may in particular be made of steel so that its rigidity and durability over time are guaranteed. The dimensions of this tool will be adapted to the types of analysis chips 1 to be produced.

[0150] In a particular embodiment, 25 through holes 11 of 500 micrometers in diameter are formed, spaced 200 micrometers apart, contained in a 6mm x 6mm square placed in the center of a base piece 21 in the shape of a square of support material (20x20mm).

[0151] The punch tool will therefore have 25 punches with a diameter of 500 micrometers. For other configurations of the biological sample analysis chip 1, the punches used for all the through holes 11 or for some of these through holes 11 may have different diameters. The diameter (or a characteristic dimension in the case where the section of the through hole 11 is not circular) of the punch may thus be less than 1000 micrometers, less than 900 micrometers, less than 800 micrometers, less than 700 micrometers, less than 600 micrometers, less than 500 micrometers, less than 400 micrometers, less than 300 micrometers, less than 200 micrometers, less than 150 micrometers, less than 100 micrometers.

[0152] The "dies and two counterpieces" assembly can be fixed under a press, between the jaws 5a and 5b of this press. The support strip 2 is automatically unwound in the lower part of the first counterpiece and adjusted in the middle of this "dies; first counterpiece; second counterpiece" in order to produce wells (through holes 11) automatically by simple up and down movement in the intended place. As soon as this first stamping is finished, the strip of analysis material is introduced above the second counterpiece, once the die has returned to the "high" position. A second stamping (this time of the analysis material, forming the filter membrane) is then carried out, allowing the cutting of pellets 3 of this analysis material, for example a filter membrane.

[0153] The downward stroke of the punches of the die can then, for example, be adjusted for this second stamping in such a way that in the lower position, the punch stops at the start of the already pierced support strip 2. In this way, the punches will push the freshly cut pellets 3 of analysis material, for example nitrocellulose, and insert them into the through holes 11 so as to fill, at least partially, these through holes 11 of the support matrix (or membrane).

[0154] This being done, the strip can advance under a second press which has the function of crimping the pellets of analysis material, for example nitrocellulose, in the support strip 2, or at least in the support matrix 10, by a shock (pressure) which can be exerted on the entire surface of the membrane in order to properly block the pellets in the support strip, as described later. The force of this pressure or (shock) can be determined by tests.

[0155] Whatever the embodiment chosen for the injection step, the insertion is done if possible only by translation of the pellet 3 along the axis of the through hole 11 concerned, so as to maintain the properties of the analysis material unchanged during this step. In particular, the method according to the invention has the advantage of not implementing any step which could introduce anisotropy of the properties of this material and thus degrade the precision and sensitivity of the analysis, as discussed previously for a rolling step.

[0156] Furthermore, in the case where the support matrix 10 comprises at least two pellets 3 inserted into at least two different through holes 11, these pellets 3 are not connected by a portion of analysis material. Consequently, if the analysis material is chosen to be sufficiently different from that of the support material, it is unlikely that the molecules which adsorb on a given pellet 3 risk migrating towards a neighboring pellet 3.

[0157] In the same way, if the liquid to be analyzed wets the pellet 3, by choosing a support material with a hydrophobicity different from that of the analysis material, it is possible to limit, or even avoid, the lateral diffusion of the liquid to be analyzed from a pellet 3 towards the support material - and possibly towards another pellet 3.

[0158] Thus, if the sample to be analyzed is an aqueous solution, a hydrophilic analysis material and a hydrophobic support material can be chosen.

[0159] A hydrophilic support material and hydrophobic analysis material pellets 3 may also be considered in the case where the sample to be analyzed is an organic phase immiscible with water.

[0160] This injection step, solely by a translational movement along the axis of the hole 11, allows, while respecting the physicochemical properties of the support and analysis materials, to obtain at the end of the complete process an analysis chip of a biological sample 1 allowing qualitative analyses of high sensitivity.

[0161] In a particular embodiment, the support matrix 10 has at least two through holes 11 and a first pellet 3 is inserted into one of the through holes 11 before another pellet 3 is inserted into another through hole 11.

[0162] In this case, at least two different cookie cutters are placed in play consecutively.

[0163] This embodiment makes it possible to insert two pellets 3 formed from different analysis materials into two different through holes 11.

[0164] For example, at least two sheets of analysis material can be prepared, initially identical but each having undergone a different biofunctionalization step, in particular by adsorption of two different antigens.

[0165] A pellet 31a on which a first antigen has been adsorbed may be inserted into a first through hole 11 of a support matrix 10 and another pellet 31b on which a second antigen has been adsorbed may be inserted into a second through hole 11 of the support matrix 10.

[0166] In this case, a cutout or reference mark 12 optionally formed on the support matrix 10 may make it possible to identify the positions of the different test sites.

[0167] In the case where the management of bio-functionalization is done at the scale of the sheet of analytical material, rather than pellet 3 by pellet 3 on a given chip and / or on successive chips, it is possible to mass-produce with a high yield identical analytical chips with identical analytical qualities, making it possible to work under conditions of satisfactory repeatability, or even reproducibility. The limit of quantification, i.e. the smallest concentration or content of the analyte that can be quantified, with an acceptable uncertainty, under the experimental conditions described in the method, can be considered constant for a series of analytical chips produced in an automated manner from the same sheets of analytical material.

[0168] This limit of quantification is easier to control in the case of a sheet than in the case of a single pellet 3 in which edge effects will play an important role.

[0169] It is also possible to orient the probe molecules used for functionalization so that the sites to which the molecules to be tested can bind are oriented along the axis of the hole. This arrangement makes it possible to further increase the sensitivity (or the limit of quantification) of the analysis. The probe molecules may in particular be those described in patent EP3591024B1 (inventors Wong Ka-Leung, Goetz Joan et al.) filed on 05 / 07 / 2018, namely ultrabright luminescent lanthanide nanoparticles comprising terbium. This achieves limits of quantification of the order of a few atomoles per microliter of liquid to be tested.

[0170] In a particular embodiment, the analysis material is not functionalized and is preserved in its native structure at the level of the pellets 3. In this way, a so-called “filtering” pellet 32 ​​is formed, the sole function of which is a filtration function.

[0171] If a biological sample analysis chip 1 comprising filter pellets 32 and a biological sample analysis chip 1 comprising functionalized pellets 31 (31a, 31b, etc.) are superimposed so that each filter pellet 32 ​​is placed above a functionalized pellet 31, so that all the fluid which passes through a filter pellet 32 ​​reaches the corresponding functionalized pellet 31, it is thus possible to analyze a blood sample without prior centrifugation, the red blood cells being retained by the filtering biological sample analysis chip 1 while the serum or plasma passes through this chip to then be analyzed by the functionalized biological sample analysis chip 1.

[0172] This arrangement therefore allows a significant saving of time and material for such analyses. In a particular embodiment, one or more pellets 3 may be calibration pellets 33 of the analysis chip of a biological sample 1.

[0173] In a particular embodiment of the injection step, a pellet 3 is cooled just before injection to a temperature slightly lower than that of the support matrix 10 into which it is to be inserted. In this way, insertion is facilitated but simultaneously with insertion, the pellet 3 heats up and therefore expands, preferably sufficiently to ensure that it is held in place at the end of the injection step.

[0174] This embodiment is advantageous when the support material has a particular rigidity, as is the case for certain plastic materials. At the injection step, a pellet 3 is fitted into a through hole 11, so that it is above at least a fraction of the overhang 10a, as shown in Figure 3d.

[0175] If the biological sample analysis chip 1 is at rest, the pellet(s) 3 remain in place in the through-hole(s) 11. The biological sample analysis chip 1 could therefore possibly be used as is.

[0176] However, since no chemical or thermal treatment is implemented at the injection stage, it is not certain that the pellets 3 remain in place, for example due to the flow of a liquid sample, forced or under the effect of gravity.

[0177] A fourth step, called assembly, is therefore implemented so as to secure the assembly of the pellet(s) 3 with the support matrix 10.

[0178] To do this, a pressing force of direction Taxe of the through hole 11 is exerted on the analysis chip by means of two jaws 5a, 5b of a clamping system placed below and above the bases of the pellet 3 and at least a fraction of the support matrix 10 which adjoins it.

[0179] By fraction of the support matrix 10 which adjoins a pellet 3 is meant the fraction of the support matrix which is in the immediate vicinity of this pellet 3 and delimits the through hole 11 in which it is inserted. In particular, the fraction of the support matrix 10 which adjoins a pellet 3 may include all or part of an overhang 10a.

[0180] In a particular embodiment, the fraction of support matrix 10 which adjoins a cylindrical pellet 3 with axis the axis of a through hole 11 and section S can be at least that which is located in the cylindrical volume with axis Ax of the through hole 11 and section S', S' being obtained by a homothety of ratio greater than 1 and center the intersection of Ax of the through hole and section S. For example, if a pellet 3 is cylindrical with a diameter equal to 100 micrometers, a pressing force can be exerted on the portion of support matrix located in the cylinder with the same axis as the pellet 3 once inserted and with a diameter at least equal to 101 micrometers, at least equal to 102 micrometers, at least equal to 103 micrometers, at least equal to 104 micrometers, at least equal to 104 micrometers, at least equal to 110 micrometers, at least equal to 120 micrometers, at least equal to 130 micrometers, at least equal to 140 micrometers, 150 micrometers.

[0181] If several pellets 3 are inserted into the support matrix 10, the same reasoning is applied to each of the pellets 3.

[0182] In a particular embodiment, the pressing force is exerted by means of the clamping system on the entire upper surface and / or lower surface of the support matrix 10.

[0183] The pressing force can then be exerted by means of the clamping system whose jaws 5a, 5b, when they come together, grip at least a portion of the support die 10 which adjoins a pellet 3 so that the portion of the support die 10 crimps the upper surface and / or the lower surface of the pellet 3.

[0184] In this embodiment, it is understood that the pressing force may not include a component in a direction normal to the axis of a through-hole 11. The direction of the pressing force is then collinear with the axis of the through-hole 11, so that no non-native anisotropy is introduced into the support and analysis materials in a direction not collinear with the axis of the through-hole 11. This arrangement makes it possible in particular to precisely control the quantification limit of the analysis chip.

[0185] Alternatively, the pressing force can then be exerted by means of the clamping system whose jaws 5a, 5b, when they come together, grip at least a portion of the lower surface and / or the upper surface of a pellet 3 which protrudes from the support die 10, so that the upper surface and / or the lower surface of the pellet 3 folds back onto the die 10 and crimps it.

[0186] The mechanical assembly step can therefore result in crimping of at least one pad 3 on at least one of its lower and upper surfaces by the die 10. For simplicity, in this document, the formulation of the preceding sentence covers the two possible scenarios: crimping of the die 10 by the pad 3 or crimping of the pad 3 by the die 10, the technical effect being the same in both cases, namely an assembly of at least one pad 3 to the die 10 resistant to a stress exerted along the axis of the through hole 11.

[0187] If a pad 3 is initially of height e2 less than the height e1 of the through hole 11, assuming that the lower base of the pad 3 was placed higher than at least a fraction of the overhang 10a, the pressing force exerted along the axis of the hole makes it possible to carry out crimping as shown in figures 4a (at the start of the assembly step), 4b (during assembly) and 4c (at the end of the assembly step): the thicknesses e'1 of the support material and e'2 of the pad 3 at the end of the assembly step are less than their thicknesses e1 and e2 before this step, and the overhang 10a has been folded over the entire periphery of the pad 3 so that the support material forms a collar above and below the pad 3. In a particular embodiment, the pad 3 is crimped over the entire periphery of the pad 3 so that the support material forms a collar above and below the pad 3. the perimeter of its lower base by the support matrix.In a particular embodiment, the pellet 3 is crimped around the entire circumference of its upper base by the support die. The pellet 3 can be simultaneously crimped around the entire circumference of its lower base and around the entire circumference of its upper base.

[0188] As a result, the pellet 3 is more firmly assembled to the support matrix 10 after this assembly step than before and is more resistant to tearing due to a force exerted from the upper face to the lower face of this pellet. During the assembly step, the fact of only exerting a mechanical action, this being furthermore exerted in the direction of the axis of a through hole 11 and possibly distributed uniformly on the bases of a pellet 3, makes it possible to maintain the uniformity and isotropy of the physicochemical properties of the analysis material in the planes normal to the axis of the through hole 11 concerned.

[0189] The pressure exerted during this assembly step can be chosen according to the mechanical resistance of the assembly required for the analyses.

[0190] For example, it is possible to obtain a chip for analyzing a biological sample 1 whose pellets 3 remain in place when a fluid passes through them in a forced manner by means of a pressure difference between the upstream face and the downstream face of the pellet of less than 100 mbar.

[0191] (millibar); less than 200 mbar; less than 300 mbar; less than 400 mbar; less than

[0192] 500 mbar; less than 600 mbar; less than 650 mbar; less than 700 mbar; less than

[0193] 750 mbar; less than 800 mbar; less than 850 mbar; less than 900 mbar; less than

[0194] 950 mbar; less than 1.00 bar.

[0195] The upstream and downstream faces are here understood relative to the direction and direction of flow of the fluid.

[0196] An analysis pellet 3 is considered to “remain in place” if, at the end of the analysis, this analysis pellet still completely blocks the through-hole 11 into which it was inserted. In particular, a shift of the pellet 3 in the direction of the axis of the through-hole due to the pressure difference between its upstream and downstream faces can occur without compromising the quality of the analysis carried out by means of the analysis chip of a biological sample 1.

[0197] If the analysis chip 3 “stays in place” when a pressure difference exists between its upstream and downstream faces, we will then say that the analysis chip of a biological sample 1 “resists” the corresponding relative vacuum.

[0198] The upper face of a pellet 3 can, in a particular embodiment, simply be subjected to atmospheric pressure and the lower face placed in depression. In this way, an analysis device comprising a chip for analyzing a biological sample 1 can be implemented with forced circulation of fluid, which makes it possible to control the contact time of the sample to be tested with a pellet3 and therefore the reproducibility of the analysis.

[0199] This arrangement also makes it possible to reduce analysis times.

[0200] In particular, the forced circulation of the test sample avoids, or at least accelerates, the washing steps generally necessary to remove the fraction of the test sample that has not reacted as well as the molecules that have adsorbed non-specifically on the membrane. For example, it is possible to perform a blood test over a period of 30 minutes between the deposit of the sample (not centrifuged) and the result of the analysis. A conventional ELISA test requires a much longer time, generally 12 to 24 hours.

[0201] The mechanical assembly is carried out in the solid phase, and at a temperature lower than the melting temperatures of the support and analysis materials. This assembly therefore does not use any welding-type processes, for example, which could distort the materials or modify their physical structure.

[0202] Thanks to the assembly method according to the invention, there is no possibility of migration of the support material or of a solvent towards the analysis material and vice versa, so that the analysis material retains its native properties at the end of the properties, that is to say its properties before assembly with the support material. In addition, the interface between the support material and the analysis material is clear, as can be seen in Figure 8a on which is presented a photograph of a section of an analysis chip of a biological sample 1 in a plane containing the axis of a cylindrical through hole 11 and a diameter of its section. In this case, as well as in the case of Figure 8d, the support material is the black paper distributed by the company Mondi®, with a weight of 80g / m 2, available on the priority date of this patent application. It was impregnated with Le Parfait® food paraffin (reference 365 EMB 44 026, packaging 250g) so that a support matrix pierced with 9 holes weighs 55mg before impregnation and 77mg after impregnation. The analytical material is nitrocellulose (Reference: Amersham Protran® Premium pores 0.45pm NitroCellulose, GE Healtcare Life Science Nitrocellulose Blotting Membrane Nucleic acid and Protein application Catalogue No 10600008).

[0203] The diameter of the through holes is 500 micrometers. The photographs in Figures 8a, 8c and 8d were obtained with a binocular loupe (Zeiss, model STEMI SV8, magnification x64). It can also be seen in Figures 8d and 9 that at the magnification of the binocular loupe, the analysis material and the support material do not diffuse towards each other. Finally, it can be seen in Figures 8a, 8d and 9 that the preparation process of the analysis chip makes it possible to obtain wells with clean edges and this with dimensions of the order of tens or hundreds of micrometers.

[0204] The situation is different in the case of figures 8b and 8c, which present a photograph of an analysis chip obtained with a printing process using a solid ink printer whose wells have a diameter of 500 micrometers. It can be seen in this photograph that the ink used to form the wells diffuses towards the analysis material, so that the section of a well is not truly circular, which affects the precision of the analysis as well as its reproducibility, the contours of two different wells never being strictly the same.

[0205] The white spots present (other than the pellets 3) in the support material of Figure 8c correspond to areas in which the ink forming the pellets has diffused. The support material has therefore lost its native properties due to printing and the quantity of ink forming a given pellet is therefore not known. The reproducibility and precision of an analysis on a pellet is therefore difficult to control with this prior art method.

[0206] It will be noted in Figure 8d, whose magnification is substantially equal to that of Figure 8c, that the grain of the support material is observed but no diffusion of the analysis material towards the support material. The same is true in the case of Figure 9.

[0207] The method according to the invention therefore makes it possible to obtain finer control of the analysis tablets 3 than the methods of the prior art.

[0208] At the end of the assembly step, it is possible to carry out a functionalization step of one or more pellets 3. For example, it is possible to deposit with a pipette or a micropipette, possibly in an automated manner, a chosen volume of a solution of probe molecules on one or more pellets 3.

[0209] The biofunctionalization of a chip for analyzing a biological sample 1 consists in particular of fixing a capture molecule (for example an antibody to detect an antigen) targeting the complex biomolecule to be detected and quantified in the biological fluid to be analyzed.

[0210] In a particular embodiment, a roll of analysis chips 1, in which the pellets 3 are already in place, can be placed on a “spotting” machine. The roll is unrolled to scroll the strips of analysis chips 1 on a filter tray connected to a vacuum pump. The injection head of the deposition (“spotting”) machine deposits, for example, in 2 or 3 injections, a volume of the order of 10 pL of a solution containing the capture molecule, for example, at a concentration of 10 to 30 pg / mL.

[0211] The suction vacuum can be chosen to allow slow filtration over a time of approximately 20 seconds of the 10 pL of solution. All the pellets 3 of each analysis chip of a biological sample I can thus be treated in the same way.

[0212] A second application can then be carried out under the same conditions but with a BSA (Bovine Serum Albumin) solution, for example at a concentration of around 100 pg / mL. This solution saturates the polar sites of the filtering biological sample analysis chip 1 to avoid non-specific bonds between the biomolecule that will be detected and the analysis surface, for example nitrocellulose, of the biological sample analysis chip 1.

[0213] After incubating the roll of analysis chips 1, for example at 37 degrees Celsius for 30 minutes, the analysis chips 1 can be separated from each other with a cutting tool so as to obtain isolated analysis chips all of the same dimensions.

[0214] At the end of the assembly step, and possibly after functionalization, it is therefore possible, if this has not already been done previously, to cut the base parts 12 to detach the analysis chip(s) 1 from the support strip 2. An analysis chip of a biological sample 1 obtained by the method according to the invention can be stored for several months at room temperature, preferably in a dry atmosphere (for example under airtight and waterproof protection). In particular, analysis chips 1 can be stored at 20°C + / - 5°C for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months without alteration of their analytical properties.In particular, a reference test on a reference biological sample will statistically give the same concentration of the analyte sought (same mean and same standard deviation) on a batch of analysis chips of a biological sample 1 just after manufacture and after storage at 20°C + / - 5°C under airtight and waterproof protection (for example in a blister pack) for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months.

[0215] The last two steps (injection and assembly) allow the properties of the support material to be controlled independently of the properties of the analysis material and vice versa, unlike the methods of the prior art.

[0216] Typically, if the support material is formed from a metal plate, this metal plate may be made hydrophobic beforehand. For example, a surface treatment, such as a coating with a natural or synthetic wax, may be implemented.

[0217] In known methods, such treatment limits the analytical qualities of the chip, since the wax can migrate in an uncontrolled manner from the support material to the analysis material, for example during a heating or chemical treatment or rolling step. The wax (or any other chemical compound used for surface treatment) can then interfere with the analysis. Among other things, fluorescence quenching phenomena are observed, which reduce the sensitivity of the analysis when fluorescent probe molecules are used. In the invention, the assembly step does not cause such uncontrolled diffusion or migration of the wax. Some embodiments even make it possible to avoid uncontrolled diffusion or migration of chemical species from the analysis pellets 3 or towards these pellets 3.The method according to the invention therefore makes it possible to obtain a chip for analyzing a biological sample 1 whose test zones (in other words the analysis pellets 3) are formed with better precision than with the methods of the prior art.

[0218] This analysis is also valid for the case where the pellets 3 are functionalized before the injection step.

[0219] We can therefore see the advantage of the injection step according to the invention, which makes it possible to limit interference between the support and analysis materials that constitute it, and thus to obtain a chip for analyzing a biological sample 1 with a low quantification limit. In addition, no solvent or heat treatment is involved in the injection and assembly steps of the test zones with the support matrix. These steps can be carried out using simple tools. The process is therefore inexpensive, rapid, and low-polluting.

[0220] To the extent that one of the materials among the support and analysis materials can be hydrophilic, it will be possible in a particular embodiment to work under controlled hygrometry conditions for one or more steps of the method, so as to maintain precise control over the geometry and volume of the support matrix 10 and / or the pellets 3 of a chip for analyzing a biological sample 1.

[0221] A chip for analyzing a biological sample 1 obtained by the method according to the invention can be implemented in isolation. In this case, a sample to be analyzed can be deposited on one or more of the pads 3 of the chip. Or several samples to be analyzed can each be deposited on one or more pads 3 different from those used for the other samples, simultaneously or successively.

[0222] The chip for analyzing a biological sample 1 can, to do this, be placed horizontally, so that a given liquid sample to be analyzed flows from the upper face of the pellet 3 on which it has been deposited towards the lower face of this same pellet 3, either under the effect of gravity, or under the effect of a pressure gradient, a relative vacuum being applied on the side of the lower face of the pellet 3.

[0223] Several analysis chips 1, in particular functionalized differently from one another, can be superimposed as described in application WO2014 / 053,237A1, so that different channels are formed, each channel containing a single pellet 3 or several pellets 3, each of the latter belonging to a different analysis chip of a biological sample 1.

[0224] Such a three-dimensional multiplexed analysis device is schematically represented in Figure 6. The analysis device 7 consists of a stack of solid support plates 72, for example made of Polymethyl methacrylate (PMMA) or other plastic material, in which microchannels 71 are formed and between which analysis chips 1 are interposed.

[0225] The microchannels are aligned with each other and the analysis sites (i.e., the pads 3) of the analysis chips 1 are inserted between two microchannels of two consecutive support plates 72. It is also possible to superimpose several analysis chips 1 between two consecutive support plates 72. In this case, if different samples to be analyzed are tested in the different channels, it is possible to perform a 3D multiplexed analysis.

[0226] More simply, it is possible to provide an analysis device 7 comprising four pillars on which the analysis chip of a biological sample 1 is fixed by its four corners. These two examples are not limiting. The detection of an analyte of interest can be done by an immunological analysis of the ELISA type: once the capture molecule / biomolecule of interest complex is formed on the analysis sites (or equivalently wells) of the analysis chip of a biological sample 1, a revealing antibody is added which binds specifically to the capture molecule / biomolecule complex. The fluorescence or the color which appears in each well is measured using a device such as a photomultiplier or a CMOS type camera, coupled with a computer program which carries out the calculations.

[0227] The invention therefore also relates to an analysis device 7 comprising at least one analysis chip for a biological sample 1. The analysis device 7 may contain several analysis chips 1, in particular superimposed, as described above.

[0228] The invention further relates to a diagnostic kit comprising at least one chip for analyzing a biological sample 1. The diagnostic kit may also comprise a support for the chip for analyzing a biological sample 1 and / or at least one analysis reagent. The analysis reagent may in particular contain one or more antibodies or one or more antigens for the purpose of carrying out an immunological test. The analysis reagent may also be a developer.

[0229] In the case of the present application, an immunoassay is understood to mean a test using at least one antigen to detect antibodies directed against a pathogenic agent in a sample or at least one antibody to detect an antigen of a pathogenic agent in a sample.

[0230] The analytical reagent can also be a buffer, for example phosphate buffered saline (PBS) or another solution, for example bovine serum albumin (BSA) solution.

[0231] The invention relates to the use of a chip for analyzing a biological sample 1 for diagnostic purposes or for carrying out an immunological test. In particular, serological tests for searching for and quantifying antibodies of the immunoglobulin G or M type (IgG or IgM) can be implemented after functionalizing the chip for analyzing a biological sample 1 using the appropriate antigen. The chip for analyzing a biological sample 1 can also be functionalized to search for and quantify heat shock proteins such as proteins of the HSP60 family using a specific antibody, for example fluorescent. Apolipoprotein ApoAl or even inflammatory mediators such as C-reactive protein (CRP) or pancreatic stabilizing protein PSP (pancreatic stone protein) can be searched for by implementing an enzymoimmunological method on the chip for analyzing a biological sample 1.

[0232] The invention finally relates to a device for manufacturing a chip for analyzing a biological sample 1 according to any one of the embodiments comprising:

[0233] - an insertion system adapted to insert at least one pellet 3 into at least one through hole 11 of the matrix 10 by translation of the pellet 3 in the direction normal to the lower and upper surfaces of the matrix 10

[0234] - a mechanical assembly system at a temperature lower than the melting temperatures of the support and analysis materials, adapted to exert a pressing force in a direction normal to the lower and upper surfaces of the matrix 10 on at least a portion of the matrix 10 which adjoins the at least one pellet 3 inserted in the matrix 10 and / or on at least one of said lower and upper surfaces of the at least one pellet 3 inserted in the matrix 10.

[0235] The device for manufacturing a chip for analyzing a biological sample 1 may in particular comprise one or more punches, each comprising one or more identical or different punches and the stroke of which is adjustable, and one or more counter-pieces.

[0236] The device for manufacturing a chip for analyzing a biological sample 1 can be fully automated.

[0237] LIST OF REFERENCE SIGNS

[0238] I: analysis chip

[0239] 10: support matrix

[0240] 10a: overhang of support material

[0241] II: hole through the support matrix 10

[0242] 1 la, 11b: sub-part of a through hole 11

[0243] 1 le: canal connecting two sub-parts 1 la and 11b

[0244] 12: cutout / reference mark

[0245] 2: support strip

[0246] 21: basic part

[0247] 3: analysis material pellet

[0248] 31 a, b, c: functionalized pellet 3

[0249] 32: filter tablet

[0250] 33: calibration tablet

[0251] 4: cutting guide

[0252] 42: punch of a cookie cutter

[0253] 5a, 5b: jaws of a press

[0254] 6: Analysis material sheet

[0255] 7: multiplexed analysis device

[0256] 71: microchannel

[0257] 72: support plate

Claims

29 DEMANDS

1. A method for manufacturing a biological sample analysis chip (1) comprising: - a matrix (10) formed in a solid support material is provided, having a lower surface and an upper surface and in which at least one hole (11) has been formed passing through it between said lower and upper surfaces; - at least one pellet (3) is provided, cut from a sheet (6) of solid and porous analytical material, said pellet having a lower surface and an upper surface, - at least one pellet (3) is inserted into at least one through hole (11) of the matrix (10) by translating at least one pellet (3) along the direction normal to the lower and upper surfaces of the matrix (10); - a mechanical assembly is carried out at a temperature below the melting temperatures of the support and analysis materials, during which a pressing force in the direction normal to the lower and upper surfaces of the matrix (10) is exerted on at least one portion of the matrix (10) which adjoins at least one pellet (3) inserted in the matrix (10) and / or on at least one of the lower and upper surfaces of at least one pellet (3) inserted in the matrix (10).

2. Method of manufacturing a biological sample analysis chip (1) according to claim 1 characterized in that said pressing force is exerted on a portion of the matrix (10) which adjoins at least one pellet (3) inserted in the matrix (10).

3. Method of manufacturing a biological sample analysis chip (1) according to claim 1 or claim 2 characterized in that said pressing force is exerted on at least one of the lower and upper surfaces of at least one pellet (3) inserted in the matrix (10).

4. Method of manufacturing a biological sample analysis chip (1) according to any one of claims 1 to 3 characterized in that the support material is hydrophobic and the analysis material is hydrophilic or vice versa.

5. A method for manufacturing a biological sample analysis chip (1) according to any one of claims 1 to 4, characterized in that, for the insertion of at least one pellet (3) into at least one through hole (11), at least one pellet (3) is translated into at least one through hole (11) by means of a punch, in that at least one pellet (3) has been cut from the sheet (6) of analysis material before its insertion by means of this same punch, and in that at least one hole 30 through (11) was previously formed in the die (10) using the same cutter.

6. Method of manufacturing a biological sample analysis chip (1) according to any one of claims 1 to 5 characterized in that after mechanical assembly, at least one pellet (3) is functionalized.

7. Method of manufacturing a biological sample analysis chip (1) according to any one of claims 1 to 6 characterized in that the analysis material is functionalized before the insertion of at least one pellet (3) into the matrix (10).

8. Method of manufacturing a biological sample analysis chip (1) according to any one of claims 1 to 7 characterized in that said mechanical assembly of at least one pellet (3) with the die (10) results in crimping of at least one pellet (3) on at least a portion of its lower and upper surfaces by the die (10).

9. Method of manufacturing a biological sample analysis chip (1) according to any one of claims 1 to 8 characterized in that before the insertion of at least one pellet (3) into the matrix (10), the at least one pellet (3) is brought to a temperature lower than that of the matrix (10).

10. A method for manufacturing a biological sample analysis chip (1) according to claims 5 and 7 characterized in that the insertion of at least one pellet (3) into the matrix (10) is repeated at least once using for each new insertion a functionalized analysis material different from that used for the previous insertion and a punch corresponding to at least one through hole (11) of the matrix (10) different from that used for the previous insertion.

11. A biological sample analysis chip (1) comprising: - a matrix (10) formed in a solid support material, having a lower surface and an upper surface and in which at least one hole (11) has been formed passing through it between said lower and upper surfaces; - at least one pellet (3), cut from a sheet of solid and porous analysis material and inserted into at least one through hole (11), at least one pellet (3) having a lower surface and an upper surface, and characterized in that at least one pellet (3) is set on at least one of its upper and lower surfaces by the matrix (10).

12. A biological sample analysis chip (1) according to claim 11, wherein the support material comprises at least one component selected from a metal, a plastic material and cellulose or a combination thereof, and wherein the analysis material from which at least one pellet (3) is formed comprises at least one component selected from nitrocellulose, cellulose and an organic polymer.

13. A biological sample analysis chip (1) according to any one of claims 11 to 12 wherein the assembly of at least one pellet (3) and the matrix (10) is resistant to at least a relative vacuum of 0.100 bar.

14. Analytical device comprising at least two biological sample analysis chips (1) according to any one of claims 11 to 13 superimposed and wherein at least one pellet (3) of one of the at least two chips is configured to perform a filtration function and is superimposed with at least one functionalized pellet (3) of another chip of the at least two chips.

15. Diagnostic kit comprising at least one biological sample analysis chip (1) according to any one of claims 11 to 13 and at least one analysis reagent.

16. Use of a biological sample analysis chip (1) according to any one of claims 11 to 13 for diagnostic purposes or in an immunological test.

17. Device for manufacturing a biological sample analysis chip (1) according to any one of claims 10 to 13, said manufacturing device comprising: - an insertion system adapted to insert at least one pellet (3) into at least one through hole (11) of the matrix (10) by translation of the pellet (3) along the direction normal to the lower and upper surfaces of the matrix (10); - a mechanical assembly system at a temperature below the melting temperatures of the support and analysis materials, adapted to exert a pressing force in a direction normal to the lower and upper surfaces of the matrix (10) on at least a portion of the matrix (10) which adjoins the at least one pellet (3) inserted in the matrix (10) and / or on at least one of said lower and upper surfaces of the at least one pellet (3) inserted in the matrix (10).

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