Method for manufacturing an analysis chip and analysis chip

Abstract

A method of manufacturing an analytical chip (1) for the analysis of a biological sample is disclosed, the method comprising providing a matrix (10) formed in a support material in solid state, at least one through hole (11) having been formed in the matrix and providing at least one pad (3) cut from a piece (6) of an analytical material in solid and porous state; inserting the at least one pad (3) into the at least one through hole (11) of the matrix (10) by translating the pad (3) in a direction perpendicular to the lower and upper surfaces of the matrix (10); mechanically assembling by applying a compacting force to the upper surface of the matrix in a right angle manner at a temperature lower than the melting point of the support material and the analytical material.

Description

Technical Field

[0001] This invention relates to the field of bioanalysis, and more specifically to the field of biochemical analysis.

[0002] More specifically, the present invention relates to a process for manufacturing a filter chip, which may optionally be functionalized for bioanalysis. Background Technology

[0003] In the field of bioanalysis, protein chips (“protein microarrays”) are commonly used to study the biochemical activity of proteins. In such chips, 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 deposited on the matrix.

[0004] They also developed a bioanalytical device that allows for the parallel analysis of multiple samples.

[0005] Document WO2014 / 053237 is an example in which a miniaturized device allows for the simultaneous analysis of multiple biological samples (“multiplex” analysis). Each sample can also be continuously exposed to multiple different probes. In other words, the device allows for “3D analysis” type of analysis.

[0006] The device described in this document includes multiple channels, each of which can be used to inject a sample into the liquid phase independently of the other channels.

[0007] Each channel can be formed from multiple tubular sections. Between two consecutive sections, an approximately cylindrical analytical region formed in a suitable matrix is ​​inserted.

[0008] In particular, the analytical region can be formed in a flat nitrocellulose matrix, and the entire surface of the nitrocellulose matrix, except for the analytical region, becomes hydrophobic through wax impregnation.

[0009] The analytical region can simply consist of untreated nitrocellulose so that it constitutes the filtration region, or the analytical region can consist of nitrocellulose functionalized, for example, by probe molecules.

[0010] The wax impregnation process makes it possible to define the analytical region and restrict the lateral diffusion of molecules of interest (probe molecules or sample molecules) within the matrix (i.e., from the given analytical region toward other adjacent analytical regions).

[0011] For this immersion operation, a solid ink printing process is employed to deposit a layer of wax onto the substrate in areas where hydrophobicity is required. At the end of printing, the substrate is heated to a temperature above the melting point of the wax used, and then cooled, allowing the wax to diffuse throughout the substrate thickness in both the lateral and depth directions, thereby limiting its undesirable distribution into the analytical areas.

[0012] However, this process cannot precisely control the volume of a given analytical region or the shape of the surface defining that volume, as shown in the figures in this document. In particular, the perimeter of the upper surface of the test location varies at different locations and is generally not circular; therefore, in the flow direction, the cross-section of the analytical region is not exactly the same as the internal cross-section of the channel into which the analytical region will be inserted.

[0013] Therefore, the accuracy of such devices is limited due to the manufacturing process used to form the analytical region, and the limits of quantification remain too high for certain bioanalyses involving particularly low concentrations (or variations thereof).

[0014] Document US2004 / 0115707 discloses a biochemical analysis unit comprising a substrate having a plurality of pores filled with a porous adsorbent material, thereby forming a plurality of analytical regions.

[0015] The holes can be filled by laminating sheets of absorbent material onto a pre-drilled substrate.

[0016] During lamination, the sheet of analytical material is subjected to anisotropic stress due to the tension applied in the lamination direction. Therefore, the properties of the adsorbent material after insertion into the pore are anisotropic. The thickness of the adsorbent material may even vary within the same pore.

[0017] Furthermore, lamination does not disrupt the continuity of the adsorbent sheet. Therefore, the adsorbent forms a continuous surface under the plate or between two channels on the plate, as shown in Figure 2b of document US2004 / 0115707. Due to this continuity, molecules of interest (from the sample or probe) are therefore at risk of diffusing from one channel 3 to another.

[0018] Therefore, the accuracy and sensitivity of quantitative analysis using this type of plate are limited.

[0019] In another embodiment described in US2004 / 0115707, the adsorbent material can be dissolved in a solvent. The resulting solution is then injected into the pores, and the solvent evaporates. This liquid-phase injection technique also cannot precisely control the isotropic properties of the test area, especially because the gas flow that causes the solvent to evaporate must be directional.

[0020] Furthermore, trace amounts of solvent may remain in the adsorbent material, potentially interacting with probe molecules or the molecules being analyzed. Additionally, the use of solvents, particularly organic solvents in the case of nitrocellulose, can cause process contamination.

[0021] Finally, once the adsorbent solidifies, the bond between the adsorbent and the substrate cannot be definitively guaranteed. The quality of this bond depends particularly on the chemical composition of the adsorbent and the substrate. Therefore, the connection between a given analytical region and the plate can be considered fragile. Under forced liquid circulation, these analytical regions may detach and be carried away by the circulating liquid due to the relative vacuum. Therefore, it is impossible to analyze the analytical regions obtained through this embodiment using forced liquid circulation.

[0022] Other processes following the first lamination step, using different chemicals or heating or radiation steps, are also described in document WO01 / 19502A2.

[0023] In addition to the previously disclosed drawbacks of lamination, all these embodiments have the disadvantage of causing physicochemical changes in the filter membrane, which alter properties that are critical to analysis and thus change the sensitivity and accuracy of the analysis.

[0024] Since the chemical composition and physical structure of the membrane used for analysis affect the performance of the analytical method, especially the quantitative limit of the method, the present invention aims to provide a method for manufacturing a chip for analyzing biological samples, which allows for precise control of this chemical composition and physical structure.

[0025] In particular, the present invention aims to provide a method for manufacturing low-cost analytical chips that does not require thermal, chemical, or radiation treatment steps for forming test sites in a matrix (except during possible biochemical functionalization of these sites, either before or after site formation), and enables high-precision and high-sensitivity quantitative analysis and / or analysis of biological samples or simple filtration of liquid biological samples. Summary of the Invention

[0026] Therefore, the present invention relates to a method for manufacturing a biological sample analysis chip, comprising:

[0027] - Provides a matrix formed in a solid support material having a lower surface and an upper surface, and at least one through-hole extending between the lower surface and the upper surface has been formed in the matrix;

[0028] - Provide at least one pad cut from a sheet of solid and porous analytical material, the pad having a lower surface and an upper surface.

[0029] - Insert at least one pad into at least one through-hole in the substrate by translating at least one pad in a direction perpendicular to the upper and lower surfaces of the substrate;

[0030] - Mechanical assembly is performed at a temperature below the melting temperature of the support material and the analytical material, during which a clamping force perpendicular to the lower and upper surfaces of the matrix is ​​applied to at least a portion of the matrix adjacent to at least one pad inserted into the matrix, and / or applied to at least one of the lower and upper surfaces of at least one pad inserted into the matrix.

[0031] Due to these measures, the resulting analytical chip comprises at least one pad of analytical material inserted through a hole in the support material. The assembly between the pad and the support material is not achieved through a chemical process, nor through melting either material; therefore, the physical and chemical properties of the support and analytical materials do not change or change very little (including changes near the interface between the two materials) before and after assembly. Assembly is achieved solely by mechanical means and by applying a clamping force perpendicular to the upper and lower surfaces of the matrix, resulting in uniform deformation of the material in a plane perpendicular to the direction of the clamping force.

[0032] Therefore, unlike methods that perform lamination steps, this method makes it possible to prevent anisotropy from being introduced into the support material and / or analysis material in a direction perpendicular to the direction of the clamping force.

[0033] For example, in immunoassays using fluorescent reagents, this anisotropy can cause uneven fluorescence on the surface of the analytical pad, which can lead to inaccurate quantitative analysis of the fluorescence signal.

[0034] Due to all these arrangements, the sensitivity and reproducibility of the analytical chip obtained by the method according to the invention are thus improved compared to chips obtained by methods according to the prior art.

[0035] Furthermore, the method of implementation is purely mechanical, as it does not contain solvents and is easy to implement, so the pollution will not be severe.

[0036] Depending on the aspects, one and / or another of the following features may be provided individually or in combination.

[0037] According to one embodiment, in a method of manufacturing a biological sample analysis chip, a clamping force is applied to a portion of a matrix adjacent to at least one pad inserted into the matrix.

[0038] Due to this arrangement, this portion of the matrix can be folded above and / or below the pad, allowing the pad to be at least partially pressed against the matrix. Therefore, the matrix into which the pad is assembled into the analytical chip will exhibit good mechanical resistance and will not be affected by the flow of the liquid sample to be analyzed in directions perpendicular to the upper and lower surfaces of the matrix, or even by a relative vacuum applied to the side of one of these surfaces to accelerate the flow of the liquid sample.

[0039] According to one embodiment of a method for manufacturing a biosample analysis chip, a clamping force is applied to at least one of the lower and upper surfaces of at least one pad inserted into a matrix.

[0040] Due to this arrangement, the pad presses against the matrix at least partially, so that the assembly of the pad to the matrix will present a certain mechanical resistance, and it will not be affected by the flow of the liquid sample to be analyzed in the direction perpendicular to the upper and lower surfaces of the matrix, or even by the application of a relative vacuum on the side of one of these surfaces to accelerate the flow of the liquid sample.

[0041] According to a specific embodiment, in a method for manufacturing a biosample analysis chip, the support material is hydrophobic, while the analytical material is hydrophilic, and vice versa. In this way, for example, a sample of a liquid phase to be tested can be deposited on the pad, and if the support material is hydrophobic, the sample will not diffuse into the support material. Conversely, if the support material is hydrophilic and the analytical material is hydrophobic, then a sample of an organic phase to be tested can be deposited on the pad without diffusing into the support material.

[0042] According to a specific embodiment, in a method for manufacturing a biological sample analysis chip, in order to insert at least one pad into at least one through hole, at least one pad is translated into at least one through hole by a punch, and prior to insertion, at least one pad has been cut from an analytical material sheet by the same punch, and at least one through hole has been pre-formed in a matrix by the same punch.

[0043] Because of this arrangement, fabricating the analytical chip requires a single tool, namely a tool with one or more punches whose size and shape are adapted to the desired well shape. This tool is easy to design and implement and can potentially allow for process automation, making it possible to obtain analytical chips with high and controllable accuracy in a repeatable, rapid, and low-cost manner.

[0044] According to a specific embodiment, in a method for manufacturing a biological sample analysis chip, at least one pad is functionalized after mechanical assembly.

[0045] For example, biochemical functionalization could be considered by adsorbing antibodies or antigens onto the pad.

[0046] In this way, the bioanalytical chips obtained by this method enable analytical testing (e.g., immunological tests) using reagents for functionalization. Due to this functionalization step, the analytical chips can thus be adapted to analytical needs. The analytical chips can be manufactured continuously, for example, prior to the functionalization step, and each analytical chip can be functionalized arbitrarily during analysis.

[0047] According to a specific embodiment, in a method for manufacturing a biological sample analysis chip, the analytical material is functionalized before inserting at least one pad into the matrix.

[0048] This arrangement allows for functionalization of the entire analytical material on the wafer prior to dicing the pads. This saves time in the continuous fabrication of analytical chips. Better control over functionalization, particularly over the amount of analytical reagent deposited on each pad, ultimately results in better accuracy and reproducibility for tests performed with a given series of analytical chips.

[0049] According to a specific embodiment, in a method for manufacturing a biological sample analysis chip, at least one pad is inserted into a matrix at least once, with each new insertion using a functionalized analytical material different from that used in the previous insertion and a punch different from that used in the previous insertion and corresponding to at least one through-hole in the matrix.

[0050] This arrangement allows for the formation of multiple different functionalized analytical pads on the same analytical chip. This makes it possible to perform multiple different tests simultaneously on the same chip, the same sample, or multiple different samples. The process remains simple to implement, requiring only different punches or equivalent single tools equipped with several punches positioned at different locations, which can be activated individually or in groups. According to a specific embodiment, in a method of manufacturing a biosample analytical chip, the mechanical assembly of at least one pad with the matrix results in at least one pad being pressed against the matrix on at least a portion of its lower and upper surfaces.

[0051] Due to this arrangement, under normal operating conditions, at least on one side of the matrix, the pad will not be pushed outside the matrix. Therefore, the assembly of the matrix and pad can withstand relative vacuum or the pressure applied to one side of the pad during sample deposition.

[0052] According to a specific embodiment, in a method for manufacturing a biological sample analysis chip, the temperature of at least one pad is lower than the temperature of the matrix before inserting at least one pad into the matrix. Due to this arrangement, the analytical material shrinks before insertion, which facilitates its insertion into the via by reducing contact forces, and the analytical material expands after insertion, such that the contact between the pad and the matrix is ​​stable after insertion, and allows the pad to be fixed in place within the matrix.

[0053] This invention also relates to a biological sample analysis chip, comprising:

[0054] - A matrix formed in a solid support material, the matrix having a lower surface and an upper surface, and having at least one through-hole extending between the lower surface and the upper surface formed in the matrix;

[0055] - At least one pad, cut from a sheet of solid and porous analytical material and inserted into at least one through-hole, the at least one pad having a lower surface and an upper surface, the biosample analysis chip being characterized in that at least one pad is pressed onto at least one of its upper and lower surfaces by a matrix.

[0056] The advantage of this analytical chip is that it contains no solvent, molten, or solder residues that could alter the accuracy of tests performed using this chip. The press-fit design allows for the preservation of the natural physicochemical properties of both the support and analytical materials. It also enables sample flow along the axis of the via from one side of the pad to the other for testing, due to the good mechanical resistance of the assembly between the pad and the substrate.

[0057] According to one embodiment of a biosample analysis chip sample, the support material includes at least one component selected from metals, plastic materials, and cellulose, and the analytical material forming at least one pad includes at least one component selected from nitrocellulose, cellulose, and organic polymers.

[0058] This material is inexpensive and possesses the necessary biochemical inertness and adsorption properties to perform analyses such as biochemical tests.

[0059] According to one embodiment of the biosample analysis chip, at least one pad and matrix component is resistant to a relative vacuum of at least 0.100 bar.

[0060] This arrangement allows for the analysis of samples that are forced to flow through the pad without the pad separating from the matrix due to locally applied overpressure.

[0061] The present invention also relates to an apparatus for analyzing biological samples, comprising at least two stacked biological sample analysis chips according to one of the foregoing embodiments, wherein at least one pad of one of the at least two chips is configured to perform a filtering function, and said pad is stacked with at least one functionalized pad of the other of the at least two chips.

[0062] Therefore, multiple analytical chips can be stacked to obtain a three-dimensional analytical device, and the analyses performed are different in the stacking direction and / or within a given analytical chip, from one analytical position to another, and a first filtration step can be performed before analysis, in particular to separate serum from red blood cells to analyze blood samples.

[0063] In the latter case, the device makes it possible to avoid the centrifugation step.

[0064] The present invention also relates to a diagnostic kit comprising at least one biological sample analysis according to one of the foregoing embodiments and at least one analytical reagent.

[0065] Therefore, one or more analytical reagents, especially buffers, solvents, antigens, and antibodies, can be provided to enable standardized testing, such as immunological testing.

[0066] The present invention also relates to the use of a biological sample analysis chip for diagnostic purposes or for immune testing according to one of the foregoing embodiments.

[0067] Finally, the present invention relates to an apparatus for manufacturing a biological sample analysis chip according to one of the foregoing embodiments, the manufacturing apparatus comprising:

[0068] - An insertion system adapted to insert at least one pad into at least one through-hole of a substrate by translating the pad in a direction perpendicular to the lower and upper surfaces of the substrate;

[0069] - A mechanical assembly system, the temperature of which is below the melting temperature of the support material and the analytical material, the mechanical assembly system being adapted to apply a clamping force on at least a portion of the matrix adjacent to at least one pad inserted into the matrix in a direction perpendicular to the lower and upper surfaces of the matrix, and / or to apply a clamping force on at least one of the lower and upper surfaces of at least one pad inserted into the matrix.

[0070] This fabrication apparatus is easy to implement and introduces only minimal and isotropic deformation of the support material and / or analytical material in any plane parallel to the lower and upper surfaces of the matrix. Therefore, it enables the low-cost fabrication of analytical chips while preserving the natural physicochemical properties of the support and analytical materials. Attached Figure Description

[0071] Embodiments of the present invention will now be described with reference to the accompanying drawings, which are briefly described below:

[0072] Figure 1 shows an embodiment of a support substrate used for analysis chips.

[0073] Figure 2 shows a support strip in which three base components have just been cut, each of which allows the formation of a support matrix.

[0074] Figure 3a1 shows the support matrix portion at the start of drilling a through hole according to a specific embodiment (viewed in cross section along the plane containing the axis of the hole).

[0075] Figure 3a2 shows the support matrix portion of the drilled through hole according to a specific embodiment (viewed in cross section along the plane containing the axis of the hole).

[0076] Figure 3b shows a portion of the support matrix (viewed in cross section along the plane containing the axis of the hole) after drilling the through hole and at the beginning of the step of inserting the pad for analysis material, according to a specific embodiment.

[0077] Figure 3c shows a portion of the support matrix during the insertion of the analytical material pad according to a specific embodiment (viewed in cross section along the plane containing the axis of the hole).

[0078] Figure 3d shows a portion of the support matrix at the end of the step of inserting the pad for analysis material according to a specific embodiment (viewed in cross section along the plane containing the axis of this hole).

[0079] Figure 4a shows a cross-sectional view of the same support matrix portion as in Figure 3d at the start of the assembly step in a particular embodiment.

[0080] Figure 4b shows a cross-sectional view of the same support matrix portion as in Figure 3d during the assembly steps of a particular embodiment.

[0081] Figure 4c shows a cross-sectional view of the same support matrix portion as in Figure 3d at the end of the assembly steps in a particular embodiment.

[0082] Figure 5 shows a top view of one embodiment of the analysis chip.

[0083] Figure 6 shows a multi-analysis device that includes two analysis chips.

[0084] In the accompanying drawings, the same reference numerals denote the same or similar objects.

[0085] Figure 7 shows a top view of an embodiment of the support substrate for the analysis chip, wherein the vias have different shapes.

[0086] Figure 8a shows a photograph of a biosample analysis chip 1 obtained using a binocular magnifying glass (Zeiss, Stemi SV8 type). The biosample analysis chip 1 is cut along a plane orthogonal to the upper and lower surfaces of the chip and has a circular cross-section with a cylindrical through-hole.

[0087] Figure 8b reproduces a photograph of the analysis chip obtained using a method based on existing technology, employing a Xerox® solid ink printer, with the analysis pad having a diameter on the order of 500 micrometers.

[0088] Figure 8c shows a photograph of the analysis chip in Figure 8b obtained using a binocular magnifying glass (Zeiss, Stemi SV8, magnification x64).

[0089] Figure 8d shows a photograph of the analytical chip of Figure 8a obtained by means of the method according to the invention using a binocular magnifying glass (Zeiss, Stemi SV8 type, magnification x64), wherein the analytical material is nitrocellulose, the backing material of the black paper is coated with wax, and the diameter of the analytical pad is equal to 500 micrometers.

[0090] Figure 9 shows a photograph of the analytical chip obtained by means of the method according to the invention using a binocular magnifying glass (Zeiss, Stemi SV8 type, magnification x64), wherein the analytical material is nitrocellulose, the support material is brass, and the diameter of the analytical pad is 500 micrometers. Detailed Implementation

[0091] This invention relates to a method for manufacturing a biological sample analysis chip 1, which is intended to be implemented alone or in an analytical device 7. The analytical device 7—or the biological sample analysis chip 1 itself—makes it possible to analyze, for example, biological fluids such as blood or liquid portions of blood (plasma, serum), urine, saliva, etc.

[0092] The liquid to be analyzed can also be a reaction medium containing biomolecules such as antibodies or proteins.

[0093] Therefore, the concept of biological sample analysis must be understood in a broad sense, that is, it is the analysis involving at least one biomolecule among reagents and / or analytes.

[0094] The biosample analysis chip 1 can therefore be used to detect and quantify complex biomolecules in biological media: blood, plasma, serum, organs or organ extracts, and reaction media (antibodies, proteins) in which complex biomolecules are produced.

[0095] In particular, bioanalysis can be an immunological test, such as an ELISA ("enzyme-linked immunosorbent assay") test.

[0096] The biological sample analysis chip 1 can also be implemented in the food industry for example, to search for pathogens during hygiene inspections.

[0097] The method for manufacturing a biological sample analysis chip 1 according to the present invention includes:

[0098] - A support matrix 10 is provided, which is formed of a solid material called "support material" having a lower surface and an upper surface, and at least one through hole 11 extending between its lower surface and upper surface has been formed in the support matrix 10.

[0099] - Provide at least one pad 3 cut from a sheet of a second porous solid material called "analytical material", the at least one pad 3 having a lower surface and an upper surface;

[0100] - By translating at least one pad 3 in a direction perpendicular to the lower and upper surfaces of the substrate 10, at least one pad 3 is inserted into at least one through hole 11 of the supporting substrate 10;

[0101] - Cold mechanical assembly is performed at a temperature below the melting temperature of the support material and the analytical material, during which a clamping force is applied to at least a portion of the matrix 10 in a direction perpendicular to the lower and upper surfaces of the matrix 10, the at least portion of the matrix 10 being adjacent to at least one pad 3 inserted into the matrix 10, and / or the clamping force is applied to at least one of the lower and upper surfaces of the at least one pad 3 inserted into the matrix 10.

[0102] The biosample analysis chip 1 obtained at the end of this process includes:

[0103] - A matrix 10, which is formed in a solid support material, wherein at least one through-hole 11 has been formed in the matrix 10;

[0104] - At least one pad 3, which is cut from a sheet of solid and porous analytical material and inserted into at least one through hole 11, wherein at least one pad 3 is pressed onto at least one of its upper and lower surfaces by a matrix 10.

[0105] Therefore, the biosample analysis chip 1, as observed in the specific embodiment of FIG5, includes a support matrix 10 formed in a solid material of thickness e1, wherein one or more through-holes 11 are formed. FIG1 shows a specific embodiment of the support matrix 10. Another specific embodiment of the support matrix 10 is shown in FIG7. As shown in FIG2, the support matrix 10 is formed by cutting out a substrate component 21, the shape of which is suitable for use in an analytical device or for use alone in an analytical device. The substrate component 21 is, for example, a rectangular or square parallelepiped cut from a support strip 2 of solid material, referred to hereinafter as the "support material," having a lower surface parallel to each other and a flat upper surface.

[0106] The base component 21 cut from the support strip 2 to form the support matrix 10 can be a parallelepiped and has a width L1 between 5 mm and 50 mm and a length L2 between 5 mm and 50 mm.

[0107] In a particular embodiment, the support matrix 10 is formed between the lower and upper surfaces by an analytical material of constant thickness e1, in which case these surfaces are flat and parallel to each other.

[0108] The thickness e1 of the support strip 2 is constant and is the same as the thickness of the base component 21. It is preferably smaller than the other dimensions (length L1 and width L2) of the base component 21, for example, at least one-tenth of the other dimensions of the base component 21.

[0109] For example, the width L3 of the support strip 2 can be equal to or slightly larger than the width L1 of the support matrix 10, or for example, twice the width L1.

[0110] In the latter case, multiple base components 21 can be cut out along the width of the support bar 2.

[0111] Therefore, the width L3 of the support bar 2 is, for example, between 5 mm and 50 mm.

[0112] The length L4 of the support bar 2 can be greater than or even much greater than the length L2 of the base component 21. For example, the length L4 is greater than 1 m or even greater than 10 m.

[0113] In this way, multiple base components 21 can be continuously cut from the support bar 2. The cutting of the base components 21 can be performed, for example, by a cutting machine, and the support bar 2 is inserted into the cutting machine.

[0114] If the support bar 2 is long enough, the base component 21 can be cut automatically, and the support bar 2 can be translated a sufficient distance between two consecutive cuts of the base component 21.

[0115] The thickness e1 of the support strip 2 (and the thickness of the base component 21 cut from the support strip 2) can be less than 1 mm, less than 0.15 mm, or even less than 0.1 mm. For example, the thickness e1 of the support strip can be equal to 0.06 mm.

[0116] In a particular embodiment, the support strip 2 has, for example, a width of 20 mm and a length of 25 m. The width and length can vary depending on the type of analytical chip 1 to be manufactured. The thickness of the support strip 2 can be equal to 0.12 mm, which is the current thickness of the filter membrane (typically made of nitrocellulose) formed in the analytical material, but it can also be on the order of 0.10 mm.

[0117] In a particular embodiment, the substrate component 21 is a square filter membrane with a side length of 20 mm.

[0118] Specifically, the support strip 2 can be made of metal, such as steel, copper, or brass. In alternative embodiments, the support strip 2 can be made of plastic. As a non-limiting example, the plastic material can be polyethylene, polyvinyl chloride, polystyrene, polymethyl methacrylate, polypropylene, or any other plastic material commonly used in the field of biochemical analysis. It may be surface-treated or have UV-resistant properties.

[0119] The supporting material may also contain plant fibers, such as cellulose. In particular, it can be paper.

[0120] The support material is robust, but not necessarily rigid. Therefore, the support strip 2 can have a degree of flexibility, as long as the support strip 2 or the support matrix 10 formed by it can be manipulated and moved to prepare the analytical chip 1, especially without tearing, including when the preparation of the support matrix 10 is automated. For example, a material distributed by Mondi® with a weight of 80 g / m² is available as of the priority date of this patent application. 2 Rex Copy A4 copy paper is suitable for this invention.

[0121] When the support bar 2 is flexible, the material has sufficient rigidity to allow the upper and lower surfaces to be flat when the lower surface is simply placed on a flat support, at least partially.

[0122] In one embodiment, the support material is rigid enough to allow one or more base components 21 to be cut by a cutting machine. In the base component 21 of the support material, at least one through hole 11 is formed through the material over its thickness, i.e., along a direction perpendicular to the lower and upper surfaces of the base component 21.

[0123] In a particular embodiment, as shown in Figures 3a1 (at the start of drilling), 3a2 (during drilling), and 3b (just before the injection step following the drilling step, described later), a through hole 11 is formed by a punch 42. In this embodiment, the punch 42 translates along the axis of the future through hole 11 to penetrate the support matrix 10. A dedicated cutting guide 4 can be placed below the support bar 2. As shown in Figures 3a1 and 3a2, the stroke of the punch 42 through the cutting guide 4 is adjusted to allow the pad 10b to pop out of the support bar 2.

[0124] Then, the punch can move in the opposite direction to release the support matrix 10, which includes one or more through holes 11.

[0125] In a particular embodiment, before one or more base components 21 are cut out, through holes 11 are formed in the support strip 2 at positions corresponding to the future base components 21.

[0126] In another embodiment, the through hole 11 is formed in the already cut base component 21.

[0127] Alternatively, the through hole 11 may be formed simultaneously with the base component 21, for example by cutting with a suitable-shaped cutting machine.

[0128] The shape of the through hole 11 can be selected according to the needs of the analysis. For example, the internal surface of the through hole 11 can be defined as a cylinder with its generatrix parallel to the direction perpendicular to the lower and upper surfaces of the base component 21. This direction will be referred to below as the "axis of hole 11". For example, the through hole 11 is a cylinder of revolution.

[0129] In the embodiment shown in Figure 7, one of the through-holes 11 can be analyzed as consisting of two sub-components 11a and 11b with circular cross-sections, connected by a channel 11c. Once the pad of analytical material is inserted as described below, it is possible to place the sample to be analyzed in the well corresponding to the first "sub-hole" and allow the sample to diffuse from sub-component 11a to sub-component 11b. In this case, an analytical chip can be used for lateral flow testing.

[0130] The feature dimension of the through hole 11 in the direction of the upper or lower surface of the base component 21 forming the through hole 11 can be less than 1 mm.

[0131] For example, the support matrix 10 may include 9, 12, 24, 48 or 96 holes (or wells) 11 having the shape of a rotating cylinder with a diameter d1 on the order of 300 to 800 μm (micrometers), and two consecutive through holes 11 being separated by a distance d2 on the order of 100 to 250 μm.

[0132] Optionally, cuts or reference marks 12 are formed on the support matrix 10 to allow for identification of their orientation, particularly during analysis to be performed later. When the support matrix 10 has symmetrical elements, this arrangement makes it possible to distinguish the through-holes 11 from one another.

[0133] At the end of the step of drilling the through hole 11, the lower and upper surfaces of the support material are no longer perfectly flat near the lower and upper bases of the through hole 11. Instead, due to the resistance of the support material to being cut, a protrusion 10a of the support material is formed above the entire circumference of the through hole 11 on the lower side of the support material. This protrusion 10a (see Figure 3a2) will be used during the subsequent assembly steps.

[0134] Therefore, in the first step of the method according to the invention, a support matrix 10 is provided, which is formed in a support material of constant thickness e1 between a lower surface and an upper surface, wherein one or more holes 11 are formed in the support material through the support material at its thickness.

[0135] In the second step, a sheet 6 of a second porous solid material, referred to as "analytical material", with a constant thickness (denoted as e2) is provided, having an upper surface and a lower surface.

[0136] An analytical material is designed to receive a liquid sample to be analyzed or filtered on one of its lower and upper surfaces, and the liquid sample must then be able to flow spontaneously to the other of these surfaces by simple diffusion, or to the other of these surfaces due to forced circulation of the liquid. Therefore, the analytical material can be a porous material, such as paper, especially filter paper, i.e., paper with a high α-cellulose content (particularly over 90%, 95%, or even 98% α-cellulose).

[0137] The analytical material can also be nitrocellulose.

[0138] Nitrocellulose has a good affinity for small molecules such as proteins, peptides, or nucleic acids. Therefore, it is particularly suitable for bioanalysis. However, these examples should not be considered limiting.

[0139] Materials used for analysis can be selected specifically based on their moisture resistance, filtration rate, tensile strength, capillary rise rate, or air passage resistance.

[0140] When the liquid to be analyzed mainly contains water, the analytical material is preferably hydrophilic so that the liquid to be analyzed wets the surface of the analytical material. In this case, the support material can be hydrophobic.

[0141] In the following text, if water cannot wet the material, that is, if the angle between the water droplet and the material surface where the water droplet is deposited is strictly greater than 90°, we will consider the material to be hydrophobic. Otherwise, the material is analyzed to be hydrophilic.

[0142] Alternatively, the analytical material can be hydrophobic, while the supporting material can be hydrophilic.

[0143] The analytical material can be an isotropic or anisotropic filter membrane. In particular, it can be an organic filter membrane, i.e., a membrane comprising organic polymers such as cellulose acetate, polysulfone, or polyamide.

[0144] The thickness e2 of the analyzed material can be close to the thickness e1 of the supporting material. The thickness e2 can be greater than, equal to or less than the thickness e1.

[0145] When the support material is nitrocellulose, the thickness e2 of the analytical material can therefore be on the order of hundreds of micrometers, or even tens of micrometers, for example, on the order of 50 μm to 150 μm.

[0146] In a third step, referred to as injection, a portion of the analytical material, referred to as pad 3, is inserted into at least one through-hole 11 of the support matrix 10, such that pad 3 closes the hole 11.

[0147] Therefore, the pad 3 is complementary to the hole 11, and the pad 3 must be inserted into the hole 11 at least a portion of the thickness of the supporting material. In other words, if the surface defining the interior of the through hole 11 is a cylinder whose generatrix is ​​parallel to the normal direction of the lower and upper surfaces of the base component 21, then the pad 3 inserted therein is a cylinder whose generatrix is ​​parallel to the axis of the through hole 11 after insertion, and the bottom of the pad 3 has the same shape as the bottom of the through hole 11.

[0148] Therefore, the term "pad" should not be interpreted as a limitation on shape. It was chosen relative to the most easily implemented embodiment, namely, the embodiment where the through hole 11 and the pad 3 are rotating cylinders.

[0149] Therefore, in the embodiment shown in FIG7, the pad 3 inserted into the through hole 11 formed by the two sub-components 11a, 11b and the channel 11c will have a complementary shape suitable for filling the sub-components 11a, 11b and the channel 11c, while the pad 3 fitted into the cylindrical through hole 11 will be cylindrical.

[0150] The height of pad 3 can in all cases be equal to the height of hole 11 (see cross-sectional view along the plane containing the axis of through hole 11 as shown in Figure 4c), or different from it (see Figure 9, which shows the analytical chip 1 according to the invention, whose support material is brass coated with Le Parfait® food-grade paraffin (reference 365 EMB 44 026, package 250g) and materials for nitrocellulose analysis) (Reference: Amersham Protran® Premium pores 0.45pm NitroCellulose, GE Healthcare Life Science Nitrocellulose Blotting Membrane Nucleic acid and Protein application Catalog No 10600008)

[0151] The interlocking performed during the insertion step is achieved solely by translating the pad 3 along the axis of the through hole 11. For example, if a punch is used to form the through hole 11 in the base material, the support matrix 10 can be held in place below the punch 42 after the hole 11 is drilled.

[0152] Then, as shown in Figure 3b, the sheet 6 of the analytical material is placed above the penetrated support matrix 10, and the punch 42 is moved again along the axis of the hole a distance that is at least slightly lower than the distance that can be drilled out of the hole 11.

[0153] In this way, the punch 42 cuts out the pad 3 to be inserted and drives the pad 3 along its path in the through hole 11, but the pad 3 does not come out completely from the through hole 11 on the lower surface side of the support matrix 10, so that the pad 3 is positioned above at least a portion of the protrusion 10a.

[0154] Therefore, at the end of this insertion step, the pad 3 is well fitted into the through hole 11, at least at a portion of its height.

[0155] The choice of punch stroke makes it possible to position the pad 3 at a selected height in the relevant through hole 11, for example, such that the lower base of the pad 3 is in the same plane as the lower surface of the support matrix 10 or at least the lowest point of the protrusion 10a, as shown in FIG3c.

[0156] For example, in embodiments where the substrate manufacturing is automated and where the time interval between the insertion step and substrate manufacturing is long, a punch specifically designed for the insertion step may also be used.

[0157] One or more pre-cut pads 3 may also be provided, for example, cut by a cutting machine or any other precise cutting tool, and the pads 3 are inserted into the corresponding through holes 11 by a vertical translational motion.

[0158] The embodiment of continuously cutting and inserting with the same punch has the advantages of simple pad positioning and fast speed in performing this step.

[0159] In the latter case, a punch tool and two corresponding mating parts are provided to allow proper perforation of the support strip 2, thereby initially forming a well (or even a "dot", or even a through hole 11) on the support strip 2. Specifically, this tool can be made of steel to ensure its rigidity and long-term resistance. The dimensions of this tool will be suitable for the type of analytical chip 1 to be manufactured.

[0160] In a particular embodiment, 25 through holes 11 with a diameter of 500 micrometers are formed in a 6mm x 6mm square at intervals of 200 micrometers, the square being placed at the center of a square (20 x 20mm) shaped base component 21 of the supporting material.

[0161] Therefore, the punch tool will have 25 punches with a diameter of 500 micrometers. For other configurations of the biosample analysis chip 1, the punches used for all or a portion of these vias 11 can have different diameters. Thus, the diameter of the punches (or the characteristic dimension in the case where the cross-section of the via 11 is not circular) can 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, or less than 100 micrometers.

[0162] The "punch and two mating parts" assembly can be fixed under the press and between the jaws 5a and 5b of the press. The support bar 2 automatically unfolds below the first mating part and adjusts in the middle of the "punch, first mating part, second mating part" assembly to automatically form a well (through hole 11) through a simple movement from top to bottom at a predetermined position. Once the first punch is completed and the punch returns to the "high" position, a strip of analytical material is introduced above the second mating part. A second punch is then performed (this time for the analytical material, forming a filter membrane) to allow the cutting of the pad 3 of this analytical material, such as the filter membrane.

[0163] Then, the downward stroke of the punch of the cutting machine can be adjusted, for example, for this second punch, so that at the low position, the punch stops at the starting point of the already perforated support strip 2. In this way, the punch will push the newly cut analytical material (e.g., nitrocellulose) pads 3 and insert them into the through holes 11 so as to at least partially fill these through holes 11 of the support matrix (or membrane).

[0164] Once completed, the strip can advance under a second press, the function of which is to press the pad of the analytical material (e.g., nitrocellulose) into the support strip 2 or at least into the support matrix 10 by applying impact (pressure) to the entire surface of the membrane, so as to properly secure the pad in the support strip, as described below. The intensity of this pressure or (impact) can be determined by testing.

[0165] Regardless of the embodiment chosen for the injection step, if possible, insertion is accomplished solely by translating the pad 3 along the axis of the associated through-hole 11, so as to maintain the properties of the analytical material unchanged during this step. In particular, an advantage of the method according to the invention is that it avoids any steps that could introduce anisotropic properties into the material, thereby reducing the accuracy and sensitivity of the analysis, such as the lamination step discussed above.

[0166] Furthermore, in cases where the support matrix 10 includes at least two pads 3 inserted into at least two different through-holes 11, these pads 3 are not connected to any part of the analytical material. Therefore, if the selected analytical material is sufficiently different from the support material, molecules adsorbed on a given pad 3 are unlikely to risk migrating to an adjacent pad 3.

[0167] Similarly, if the liquid to be analyzed wets pad 3, by selecting a support material with a hydrophobicity different from that of the analytical material, the lateral diffusion of the liquid to be analyzed (from pad 3 to the support material - and possibly to another pad 3) can be limited or even avoided.

[0168] Therefore, if the sample to be analyzed is an aqueous solution, hydrophilic analytical materials and hydrophobic support materials can be selected.

[0169] When the sample to be analyzed is an organic phase that is immiscible with water, it is also possible to envision a pad 3 consisting of a hydrophilic support material and a hydrophobic analytical material.

[0170] This injection step, which involves translational movement along the axis of the hole 11, makes it possible to obtain a biosample analysis chip 1 capable of highly sensitive quantitative analysis at the end of the entire process, while taking into account the physicochemical properties of the support and analytical materials.

[0171] In a particular embodiment, the support matrix 10 has at least two through holes 11, and the first pad 3 is inserted into one of the through holes 11 before the other pad 3 is inserted into the other through hole 11.

[0172] In this case, at least two different cutting machines should be used consecutively.

[0173] This embodiment makes it possible to insert two pads 3 made of different analytical materials into two different through holes 11.

[0174] For example, at least two pieces of initially identical analytical material can be prepared, but each piece undergoes different biofunctionalization steps (specifically by adsorbing two different antigens).

[0175] The pad 31a, on which the first antigen has been adsorbed, can be inserted into the first through-hole 11 of the support matrix 10, and the other pad 31b, on which the second antigen has been adsorbed, can be inserted into the second through-hole 11 of the support matrix 10.

[0176] In this case, the cuts or reference marks 12 optionally formed on the support matrix 10 can make it possible to identify the location of different test sites.

[0177] When biofunctionalization is performed on the scale of the material sheets used for analysis, rather than on a given chip and / or one pad after another on a series of chips, it is possible to continuously produce identical analytical chips in high yields that exhibit the same analytical quality, making it possible to operate under conditions of satisfactory reproducibility and even reproducibility. Under the experimental conditions described in this method, for a series of analytical chips automatically produced from sheets of the same analytical material, the limit of quantification (i.e., the minimum concentration or amount of an analyte that can be quantified with acceptable uncertainty) can be considered constant.

[0178] Compared to the case of a single pad 3, it is easier to control this quantitative limit in the case of a sheet because the edge effect will play an important role in the case of a single pad 3.

[0179] The probe molecules can also be oriented for functionalization, allowing the binding sites of the molecule to be tested to be aligned along the axis of the pore. This arrangement enables further improvements in analytical sensitivity (or quantitation limit). The probe molecules can be, in particular, those described in patent EP3591024B1 (filed July 5, 2018, by inventors Wong Ka-Leung, Goetz Joan, et al.), namely ultra-bright luminescent lanthanide nanoparticles containing terbium. This achieves quantitation limits on the order of several atoms per microliter of the liquid being tested.

[0180] In a particular embodiment, the analytical material is not functionalized and retains its natural structure at the level of pad 3. In this way, a so-called "filter" pad 32 is formed, whose sole function is filtration.

[0181] If a biosample analysis chip 1 including filter pad 32 is superimposed with a biosample analysis chip 1 including functionalized pads 31 (31a, 31b, etc.), such that each filter pad 32 is placed on top of a functionalized pad 31, all fluid passing through the filter pad 32 reaches the corresponding functionalized pad 31, blood samples can be analyzed without prior centrifugation, wherein red blood cells are retained by the filtered biosample analysis chip 1, while serum or plasma passes through this chip and is then analyzed by the functionalized biosample analysis chip 1.

[0182] Therefore, this arrangement saves a significant amount of time and materials for this analysis. In a particular embodiment, one or more pads 3 may be calibration pads 33 of the biological sample analysis chip 1.

[0183] In a particular embodiment of the injection step, the pad 3 is cooled to a temperature slightly below that of the support matrix 10 into which it will be inserted before injection. In this way, insertion is convenient, but during insertion, the pad 3 heats up and thus expands, preferably sufficiently to ensure that it remains in place at the end of the injection step.

[0184] This embodiment is advantageous when the supporting material has a specific rigidity, such as in the case of certain plastic materials. As shown in FIG3d, after the injection step, the pad 3 is nested in the through hole 11 such that it is positioned above at least a portion of the protrusion 10a.

[0185] If the biosample analysis chip 1 is in a static state, the pad 3 remains in the proper position within the through-hole 11. Therefore, the biosample analysis chip 1 can be used as is.

[0186] However, unless chemical or heat treatment is performed during the injection step, it cannot be determined whether the pad 3 will remain in the proper position, for example, due to the flow, force, or gravity of the sample liquid.

[0187] Therefore, the fourth step, known as assembly, is implemented by ensuring the assembly of the pad 3 with the support matrix 10.

[0188] For this purpose, a clamping force along the axis of the through hole 11 is applied to the analysis chip through two jaws 5a and 5b of the clamping system, which are positioned below and above the bottom of the pad 3, and at least a portion of the support matrix 10 is adjacent to the pad 3.

[0189] The portion of the support substrate 10 adjacent to the pad 3 refers to the part of the support substrate immediately adjacent to the pad 3, which defines the through hole 11 into which the pad 3 is inserted. In particular, the portion of the support substrate 10 adjacent to the pad 3 may include all or part of the protrusion 10a.

[0190] In a particular embodiment, the portion of the support matrix 10 adjacent to the cylindrical pad 3 along the axis of the through-hole 11 and having a cross-section S can be at least a portion located within a cylindrical volume with an axis of the through-hole 11 and a cross-section S', obtained by an expansion ratio greater than 1, centered at the intersection of the axis of the through-hole and the cross-section S. For example, if the pad 3 is a cylinder with a diameter equal to 100 micrometers, it is possible to apply a clamping force to the portion of the support matrix located within the cylinder, which has the same axis as the inserted pad 3, and the diameter of the cylinder is: 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, or 150 micrometers.

[0191] If multiple pads 3 are inserted into the support matrix 10, the same idea applies to each pad 3.

[0192] In a particular embodiment, the clamping force is applied to the entire upper and / or lower surface of the support substrate 10 via a clamping system.

[0193] Then, the clamping force can be applied by a clamping system that clamps at least a portion of the support matrix 10 when the jaws 5a and 5b of the clamping system come close together, with the support matrix 10 adjacent to the pad 3, such that a portion of the support matrix 10 presses against the upper and / or lower surfaces of the pad 3.

[0194] In this embodiment, it should be understood that the clamping force may not include a component in the direction perpendicular to the axis of the through-hole 11. Therefore, the direction of the clamping force is collinear with the axis of the through-hole 11, thus preventing the introduction of unnatural anisotropy into the support and analytical materials in directions not collinear with the axis of the through-hole 11. In particular, this arrangement allows for precise control of the quantitative limits of the analytical chip.

[0195] As a variation, the clamping force can then be applied by a clamping system that, when the jaws of the clamping system approach, clamps at least a portion of the lower and / or upper surface of the pad 3, causing the pad 3 to protrude from the supporting substrate 10, such that the upper and / or lower surface of the pad 3 folds over the substrate 10 and clamps the substrate 10.

[0196] Therefore, the mechanical assembly step can result in the pressing of at least one pad 3 onto at least one of its lower and upper surfaces via the substrate 10. For simplicity, we consider the preceding statement in this invention to cover two possible cases: pressing the substrate 10 with the pad 3 or pressing the pad 3 with the substrate 10. In both cases, the technical effect is the same, i.e., at least one pad 3 is assembled onto the substrate 10 to withstand the stress applied along the axis of the through hole 11.

[0197] If the initial height e2 of the pad 3 is lower than the height e1 of the through hole 11, assuming the lower base of the pad 3 is positioned above at least a portion of the protrusion 10a, the clamping force applied along the axis of the hole makes it possible to perform the pressing as shown in Figures 4a (at the beginning of the assembly step), 4b (during assembly), and 4c (at the end of the assembly step): at the end of the assembly step, the thickness e'1 of the support material and the thickness e'2 of the pad 3 are less than their thicknesses e1 and e2 before this step, and the protrusion 10a has been folded over the entire circumference of the pad 3, such that the support material forms flanges above and below the pad 3. In a particular embodiment, the pad 3 is pressed over the entire circumference of its lower bottom by the support matrix. In a particular embodiment, the pad 3 is pressed over the entire circumference of its upper bottom by the support matrix. The pad 3 can be pressed over the entire circumference of its lower bottom and its upper bottom simultaneously.

[0198] Therefore, after this assembly step, the pad 3 is more firmly attached to the support matrix 10 than before, and is more resistant to tearing caused by forces applied from the upper surface of the pad towards the lower surface. During the assembly step, since only mechanical action is applied, and this mechanical action is applied in the direction of the axis of the through-hole 11, and can be uniformly distributed on the base of the pad 3, it is possible to maintain the homogeneity and isotropy of the physicochemical properties of the analytical material in a plane perpendicular to the axis of the associated through-hole 11.

[0199] The pressure applied during this assembly step can be selected based on the mechanical resistance of the components required for analysis.

[0200] For example, a biosample analysis chip 1 can be obtained in which, when a pressure difference of less than 100 mbar between the upstream and downstream surfaces of the pad, the pad 3 remains in place as fluid is forced through the biosample analysis chip 1, and the pressure difference is either less than 200 mbar; less than 300 mbar; less than 400 mbar; less than 500 mbar; less than 600 mbar; less than 650 mbar; less than 700 mbar; less than 750 mbar; less than 800 mbar; less than 850 mbar; less than 900 mbar; less than 950 mbar; or less than 1.00 bar.

[0201] In this paper, the upstream and downstream surfaces are understood relative to the direction and orientation of fluid flow.

[0202] If, at the end of the analysis, the analytical pad 3 remains completely closed to its inserted through-hole 11, the analytical pad 3 is considered to have “remained in place.” In particular, movement of the pad 3 in the axial direction of the through-hole due to the pressure difference between its upstream and downstream surfaces can occur without affecting the quality of the analysis performed through the biological sample analysis chip 1.

[0203] If the analysis pad 3 "remains in place" when there is a pressure difference between its upstream and downstream surfaces, then it can be said that the biological sample analysis chip 1 is "tolerant" to the corresponding relative vacuum.

[0204] In a particular embodiment, the upper surface of the pad 3 is readily subjected to atmospheric pressure, while the lower surface is placed under low pressure. In this way, the analytical device including the biosample analysis chip 1 can be implemented using forced circulation of fluid, which makes it possible to control the contact time between the sample to be tested and the pad 3, and thus control the reproducibility of the analysis.

[0205] This arrangement also makes it possible to reduce the duration of the analysis.

[0206] In particular, the forced circulation of the test sample avoids or at least accelerates the typically necessary washing step, which removes unreacted portions of the test sample and molecules that are adsorbed onto the membrane in a nonspecific manner. For example, blood tests can be performed within 30 minutes between sample deposition (without centrifugation) and the analysis results. Traditional ELISA tests require much longer times, typically 12 to 24 hours.

[0207] Mechanical assembly takes place in the solid phase at temperatures below the melting temperatures of the supporting and analytical materials. Therefore, this assembly does not involve processes such as welding, which would deform or alter the physical structure of the materials.

[0208] Because of the assembly method according to the present invention, the support material or solvent cannot migrate to the analytical material, and vice versa; therefore, the analytical material retains its natural properties, i.e., its properties prior to assembly with the support material. Furthermore, the interface between the support material and the analytical material is clean, as shown in Figure 8a, which presents a photograph of a cross-section of the biosample analysis chip 1 in a plane containing the axis and cross-sectional diameter of the cylindrical through-hole 11. In this case, as in Figure 8d, the support material available at the priority date of this patent application is black paper distributed by Mondi®, with a weight of 80 g / m³. 2It had been impregnated with Le Parfait® food-grade paraffin (reference 365 EMB 44 026, package 250 g), therefore, the supporting matrix pierced with 9 pores weighed 55 mg before impregnation and 77 mg after impregnation. The analytical material was nitrocellulose (reference: Amersham Protran® Premium pores 0.45 pm NitroCellulose, GE Healthcare Life Science Nitrocellulose Blotting Membrane Nucleic acid and Protein application Catalog No 10600008).

[0209] The diameter of the via is 500 micrometers. The photographs in Figures 8a, 8c, and 8d were obtained using a binocular magnifying glass (Zeiss, STEMISV8, magnification x64). It is also noted that in Figures 8d and 9, under binocular magnification, the analytical material and the support material do not diffuse towards each other. Finally, it can be observed in Figures 8a, 8d, and 9 that the process used to fabricate the analytical chip makes it possible to obtain wells with clean edges and dimensions of tens or hundreds of micrometers.

[0210] In Figures 8b and 8c, the situation is different. Figures 8b and 8c show photographs of the analytical chip obtained using a solid ink printer printing process, where the diameter of the wells is 500 micrometers. In these photographs, it can be observed that the ink used to form the wells diffuses into the analytical material, therefore the cross-section of the wells is not truly circular. This affects the accuracy and reproducibility of the analysis; the contours of two different wells will never be exactly the same.

[0211] The white spots in the support material of Figure 8c (except for pad 3) correspond to areas where the ink forming the particles has diffused. Due to the printing process, the support material loses its natural properties, and the amount of ink forming a given pad is therefore unknown. Consequently, it is difficult to control the reproducibility and accuracy of pad analysis using this method with existing techniques.

[0212] As can be seen in Figure 8d, its magnification is basically the same as that of Figure 8c, and the grains of the supporting material can be observed, but the diffusion of the analytical material into the supporting material is not analyzed. The same is true in the case of Figure 9.

[0213] Therefore, compared with the methods of the prior art, the method according to the present invention can achieve more precise control over the analytical pad 3.

[0214] At the end of the assembly step, one or more functionalization steps for pad 3 can be performed.

[0215] For example, a selected volume of probe molecule solution can be deposited onto one or more pads 3, optionally automatically, using a pipette or micropipette.

[0216] Specifically, the biofunctionalization of the biosample analysis chip 1 includes attaching capture molecules (e.g., antibodies for detecting antigens) to the biofluid to be analyzed, targeting and quantifying complex biomolecules.

[0217] In a particular embodiment, a roll of analytical chip 1 can be placed on a "spotting" machine, with pad 3 already placed within analytical chip 1. The roll is unrolled to roll a strip of analytical chip 1 onto a filter plate connected to a vacuum pump. The injection head of the spotting machine deposits a solution on the order of 10 µL in, for example, two or three injections, the solution containing, for example, trap molecules at a concentration of 10 to 30 µg / mL.

[0218] A vacuum can be applied to allow 10 µL of solution to be slowly filtered over approximately 20 seconds. Therefore, all pads 3 of each biosample analysis chip 1 can be processed in the same manner.

[0219] A second application can then be performed under the same conditions, but using, for example, a BSA (bovine serum albumin) solution at a concentration of approximately 100 µg / mL. This solution allows for saturation filtration of the polar sites of the biosample analysis chip 1, avoiding nonspecific binding between the biomolecules to be detected and the analytical surface of the biosample analysis chip 1 (e.g., nitrocellulose).

[0220] After the rolls of analytical chip 1 have been cultured, for example, at 37°C for 30 minutes, the analytical chips 1 can be separated from each other using a cutting tool to obtain separate analytical chips of all the same size. At the end of the assembly step, and possibly after functionalization, if they have not been separated before, the substrate component 12 can therefore be cut to separate the analytical chips 1 from the support strip 2. The biological sample analytical chips 1 obtained by the method according to the invention can be stored at room temperature for several months, preferably in a dry atmosphere (e.g., under airtight and watertight protection). In particular, the analytical 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, or at least 12 months without altering their analytical properties. Specifically, the reference test for the reference biological sample will statistically provide the same concentration (same mean and same standard deviation) of the analyte required on a batch of biological sample analysis chips, wherein the biological sample analysis chip 1 is stored after manufacturing under airtight and watertight protection (e.g., blister packaging) 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, and at least 12 months.

[0221] Unlike existing methods, the last two steps (injection and assembly) make it possible to control the properties of the support material independently of analyzing the properties of the material, and vice versa.

[0222] Typically, if the support material is formed of a metal sheet, this metal sheet can be pre-treated to be hydrophobic. For example, a surface treatment can be performed (e.g., coating with natural or synthetic wax).

[0223] In known methods, this process limits the analytical quality of the chip because wax can migrate uncontrollably from the support material to the analytical material, for example, during heating, chemical treatment, or curling steps. Wax (or any other compound used for surface treatment) can interfere with the analysis. Among other things, fluorescence quenching has been observed, which reduces the sensitivity of the analysis when using fluorescent probe molecules. In this invention, the assembly steps do not lead to this uncontrolled diffusion or migration of wax. Some embodiments even prevent the uncontrolled diffusion or migration of chemicals from or toward the analytical pads 3. Therefore, the method according to the invention makes it possible to obtain a biological sample analysis chip 1 whose test area (i.e., analytical pad 3) is formed with better precision than prior art methods.

[0224] This analysis also applies to cases where pad 3 is functionalized prior to the injection step.

[0225] Therefore, we can see the advantages of the injection steps according to the present invention, which makes it possible to limit interference between the support material and the analytical materials constituting the support material, thereby obtaining a biosample analysis chip 1 with a low limit of quantitation. Furthermore, no solvent or heat treatment is involved in the injection steps between the test area and the support matrix, as well as the assembly steps. These steps can be performed using simple tools. Therefore, the process is low-cost, fast, and low-contamination.

[0226] Provided that one of the support material and the analytical material is hydrophilic, in a particular embodiment, for one or more steps of the process, it is possible to operate under controlled humidity measurement conditions in order to maintain precise control over the geometry and volume of the support matrix 10 and / or pad 3 of the biosample analysis chip 1.

[0227] The biosample analysis chip 1 obtained by the method according to the invention can be implemented independently. In this case, the sample to be analyzed can be deposited on one or more pads 3 of the chip. Alternatively, several samples to be analyzed can be deposited simultaneously or sequentially on one or more pads 3, which are different from the pads used for other samples.

[0228] For this purpose, the biological sample analysis chip 1 can be placed horizontally, so that the given liquid sample to be analyzed flows from the upper surface of the pad 3 already deposited thereon to the lower side of the pad 3 under the action of gravity or pressure gradient, and a relative vacuum is applied to the lower side of the pad 3.

[0229] As described in application WO2014 / 053,237A1, several analytical chips 1 can be stacked, particularly analytical chips 1 with different functions to form different channels, each channel containing a single pad 3 or multiple pads 3, each pad 3 belonging to a different biological sample analytical chip 1.

[0230] This three-dimensional multiplex analysis device is schematically shown in Figure 6. The analysis device 7 consists of a stack of solid support plates 72 (e.g., made of polymethyl methacrylate (PMMA) or another plastic material), forming microchannels 71 in the analysis device 7, and the analysis chip 1 is inserted between the microchannels 71.

[0231] The microchannels are aligned with each other, and the analysis sites (i.e., pads 3) of the analysis chip 1 are inserted between two microchannels of two consecutive support plates 72. Multiple analysis chips 1 can also be stacked between two consecutive support plates 72. In this case, 3D multiplexing can be performed if different samples to be analyzed are tested in different channels.

[0232] More simply, an analytical device 7 comprising four pillars, with the biosample analysis chip 1 fixed to the pillars at its four corners, can be provided. These two examples are not limiting. Detection of the analyte of interest can be accomplished using an immunological assay of the ELISA type: once a complex of the capture molecule / biomolecule of interest has formed at the analytical site (or equivalent well) of the biosample analysis chip 1, a revealing antibody that specifically binds to the capture molecule / biomolecule complex is added. Fluorescence or color appearing in each well is measured using a device such as a photomultiplier or a CMOS camera, in conjunction with a computer program that performs calculations.

[0233] Therefore, the present invention also relates to an analytical device 7 comprising at least one biological sample analysis chip 1.

[0234] As described above, the analysis device 7 may include multiple analysis chips 1, particularly stacked analysis chips.

[0235] The present invention also relates to a diagnostic kit comprising at least one biological sample analysis chip 1.

[0236] The diagnostic kit may also include a support for the biosample analysis chip 1 and / or at least one analytical reagent. For performing immunological tests, the analytical reagent may specifically contain one or more antibodies or one or more antigens. The analytical reagent may also be a display agent.

[0237] In the context of this invention, the term immunological test (“immunoassay”) should be understood as a test that uses at least one antigen to detect antibodies against pathogens in a sample or uses at least one antibody to detect antigens of pathogens in a sample.

[0238] The analytical reagent can also be a buffer solution, such as phosphate-buffered saline (PBS) or another solution, such as bovine serum albumin (BSA) solution.

[0239] This invention relates to using a biosample analysis chip 1 for diagnostic purposes or for performing immunological tests. Specifically, after functionalizing the biosample analysis chip 1 with appropriate antigens, quantitative serological studies of immunoglobulin G or M antibodies (IgG or IgM) can be performed. The biosample analysis chip 1 can also be functionalized to search for and quantify heat shock proteins (e.g., proteins of the HSP60 family) using specific antibodies (e.g., fluorescent antibodies). Apolipoprotein ApoA1 or even inflammatory mediators such as C-reactive protein (CRP) or pancreatic stabilizing protein PSP (“pancreatic stone protein”) can be identified by performing an enzyme immunoassay on the biosample analysis chip 1.

[0240] Finally, the present invention relates to an apparatus for manufacturing a biological sample analysis chip 1 according to any embodiment, comprising:

[0241] - An insertion system adapted to insert at least one pad 3 into at least one through hole 11 of the substrate 10 by translating the pad 3 in a direction perpendicular to the upper and lower surfaces of the substrate 10.

[0242] - A mechanical assembly system, at a temperature below the melting temperature of the support material and the analytical material, is adapted to apply a clamping force perpendicular to the lower and upper surfaces of the matrix 10 to at least a portion of the matrix 10 adjacent to at least one pad 3 inserted into the matrix 10, and / or to at least one of the lower and upper surfaces of the at least one pad 3 inserted into the matrix 10.

[0243] Specifically, the apparatus for manufacturing the biological sample analysis chip 1 may include one or more punches, each punch including one or more punches and one or more mating parts, the punches may be the same or different, and their stroke is adjustable.

[0244] The device used to manufacture biological sample analysis chip 1 can be fully automated.

[0245] Figure Labels

[0246] 1: Analyze the chip

[0247] 10: Supporting matrix

[0248] 10a: Protrusions of supporting material

[0249] 11: Holes passing through the support matrix 10

[0250] 11a, 11b: Sub-components of through hole 11

[0251] 11c: Channel connecting two sub-components 11a and 11b

[0252] 12: Cutting / Reference Marks

[0253] 2: Support bar

[0254] 21: Base Components

[0255] 3: Pad for analyzing materials

[0256] 31a, b, c: Functionalized pads 3

[0257] 32: Filter pad

[0258] 33: Calibration Pad

[0259] 4: Cutting guide components

[0260] 42: The punch of the cutter

[0261] 5a, 5b: Press jaws

[0262] 6: Analysis of the material slices

[0263] 7: Multiplex Analysis Device

[0264] 71: Microchannel

[0265] 72: Support plate

Claims

1. A method for manufacturing a biological sample analysis chip (1), comprising: - A matrix (10) is formed in a solid support material having a lower surface and an upper surface, and at least one through hole (11) extending between the lower surface and the upper surface has been formed in the matrix, wherein the through hole (11) is a rotating cylinder; - Provide at least one pad (3) cut from a sheet (6) of solid and porous analytical material, the pad (3) having a lower surface and an upper surface. - Insert at least one pad (3) into at least one through hole (11) of the substrate (10) by translating at least one pad (3) in a direction perpendicular to the lower and upper surfaces of the substrate (10); - Mechanical assembly is performed at a temperature below the melting temperature of the support material and the analytical material. During the mechanical assembly, a clamping force perpendicular to the lower and upper surfaces of the matrix (10) is applied to at least a portion of the matrix (10) adjacent to at least one pad (3) inserted into the matrix (10) to cause the matrix (10) to fold or be deformed in a controlled manner around at least one pad (3), or applied to at least one of the lower and upper surfaces of at least one pad (3) inserted into the matrix (10) to cause the peripheral portion of at least one pad (3) to fold toward the matrix (10). The mechanical assembly of at least one pad (3) with the matrix (10) results in at least one pad (3) being pressed against the matrix (10) on at least a portion of its lower and upper surfaces. The pressing is performed by applying a clamping force collinear with the axis of the through hole (11).

2. The method for manufacturing a biological sample analysis chip (1) according to claim 1, characterized in that, The clamping force is applied to a portion of the substrate (10), which is adjacent to at least one pad (3) inserted into the substrate (10).

3. The method for manufacturing a biological sample analysis chip (1) according to claim 1, characterized in that, The clamping force is applied to at least one of the lower and upper surfaces of at least one pad (3) inserted into the matrix (10).

4. The method for manufacturing a biological sample analysis chip (1) according to claim 1, characterized in that, The supporting material is hydrophobic, while the analytical material is hydrophilic, and vice versa.

5. The method for manufacturing a biological sample analysis chip (1) according to claim 1, characterized in that, In order to insert at least one pad (3) into at least one through hole (11), at least one pad (3) is translated into at least one through hole (11) by a punch, and before insertion, at least one pad (3) has been cut from the sheet (6) of analytical material by the same punch, such that at least one through hole (11) has been pre-formed in the matrix (10) by the same punch.

6. The method for manufacturing a biological sample analysis chip (1) according to claim 1, characterized in that, After mechanical assembly, at least one pad (3) is functionalized.

7. The method for manufacturing a biological sample analysis chip (1) according to claim 1, characterized in that, The analytical material is functionalized before inserting at least one pad (3) into the matrix (10).

8. The method for manufacturing a biological sample analysis chip (1) according to claim 1, characterized in that, Before inserting at least one pad (3) into the substrate (10), the temperature of at least one pad (3) is lower than the temperature of the substrate (10).

9. The method for manufacturing a biological sample analysis chip (1) according to claim 5, characterized in that, Before inserting at least one pad (3) into the matrix (10), the analytical material is functionalized, and the insertion of at least one pad (3) into the matrix (10) is repeated at least once, each new insertion using a different functionalized analytical material than the previous insertion and a punch that is different from the previous insertion and corresponds to at least one through hole (11) of the matrix (10).

10. 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 having formed in the matrix at least one through-hole (11) extending between the lower surface and the upper surface, the through-hole (11) being a rotating cylinder; - At least one pad (3), cut from a sheet of solid and porous analytical material and inserted into at least one through-hole (11), the at least one pad (3) having a lower surface and an upper surface, Characterized by the fact that at least one pad (3) is pressed against a matrix (10) on at least one of its upper and lower surfaces, The pressing is performed by applying a pressing force that is collinear with the axis of the through hole (11) to cause the substrate (10) to fold or deform in a controlled manner around at least one pad (3), or to fold the peripheral portion of at least one pad (3) toward the substrate (10).

11. The biological sample analysis chip (1) according to claim 10, characterized in that, The support material includes at least one component selected from metals, plastic materials and cellulose or combinations thereof, and the analytical material forming at least one pad (3) includes at least one component selected from nitrocellulose, cellulose and organic polymers.

12. The biological sample analysis chip (1) according to claim 10, characterized in that, The combination of at least one pad (3) and matrix (10) can withstand a relative vacuum of at least 0.100 bar.

13. An analytical apparatus comprising at least two stacked biological sample analysis chips (1) according to claim 10, wherein at least one pad (3) of one of the chips is configured to perform a filtering function, and the at least one pad (3) is stacked with at least one functionalized pad (3) of the other of the chips.

14. A diagnostic kit comprising at least one biosample analysis chip (1) according to claim 10 and at least one analytical reagent.

15. The use of the biological sample analysis chip (1) according to claim 10 for in vitro analysis of biological samples for non-diagnostic purposes.

16. An apparatus for manufacturing the biosample analysis chip (1) according to claim 10, the apparatus comprising: - An insertion system adapted to insert at least one pad (3) into at least one through hole (11) of the substrate (10) by translating the pad (3) in a direction perpendicular to the lower and upper surfaces of the substrate (10); - A mechanical assembly system, at a temperature below the melting temperature of the support material and the analytical material, the mechanical assembly system being adapted to apply a clamping force on at least a portion of the matrix (10) adjacent to at least one pad (3) inserted into the matrix (10) in a direction perpendicular to the lower and upper surfaces of the matrix (10), or to apply a clamping force on at least one of the lower and upper surfaces of the at least one pad (3) inserted into the matrix (10), the mechanical assembly of the at least one pad (3) with the matrix (10) resulting in at least one pad (3) being pressed against the matrix (10) on at least a portion of its lower and upper surfaces.

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