DEVICE FOR THE ANALYSIS OF A BIOLOGICAL SAMPLE, MANUFACTURING PROCESS AND QUALITY CONTROL

Description

technical field

[0001] The present invention relates to the field of biological sample analysis, and more specifically concerns an analysis card for the analysis of a biological sample, in particular for the detection of endotoxins, by means of an in vitro diagnostic instrument and the methods of manufacturing the analysis card and quality control of a plate of an analysis card. Technological background

[0002] Biological sample analysis, such as endotoxin testing, relies on one or more reactions between the biological sample and one or more reagents. Microfluidic systems and processes exist for performing these biological sample analyses. Reagents are loaded into wells, and the biological sample is introduced, for example, via a feed channel. This necessitates the preparation of multiple standard dilutions and internal controls. Endotoxin testing, for instance, is demanding and requires numerous operator handling steps. These manual preparation steps are time-consuming and can lead to variable or even invalid results. Furthermore, there is no way to monitor test preparation and thus quickly determine if an error has occurred.Thus, a possible problem can only be detected at the end of a measurement period, previously chosen to be long enough to allow the complete completion of the various reactions that may occur with different dynamics, when there is an attempt to exploit erroneous results.

[0003] Microplates such as the GOPLATE™ system, which includes 96 pre-filled wells containing standard quantities of reagents and undergoing concentration checks, are available. This device reduces handling time by more than 50% compared to traditional microplate endotoxin tests. However, performing biological sample analysis with this device requires numerous additional accessories and still involves multiple manual steps. Microplates integrated into consumable systems such as the FilmArray® system are also available, requiring minimal manual operation. These pre-filled microplates also limit human intervention in the biological sample analysis process, thus reducing the risk of human error.WO2019 / 116209 A1 discloses a microfluidic chip comprising a main chip body, which has a center of rotation, a sample reservoir, a liquid channel, multiple reaction chambers, a first inlet channel and multiple second inlet channels, and a sealing membrane connected to the main chip body. The reagents are attached to the walls of the reaction chamber, such as the bottom or side walls. US2016195524 A1 discloses a cassette comprising a fluid conduit and one or more chambers containing reagents in the fluid conduit, a sample measurement being obtainable from these chambers using a cassette reader. US2010209927 A1 discloses a microfluidic processing device comprising a tablet containing a reagent.

[0004] However, pre-filled microplates of the prior art are not suitable for certain reactions requiring the use of different reagents that must not react with each other and therefore must not mix before a biological sample is introduced into the microplate wells. Furthermore, in the prior art, a reagent is most often deposited at the bottom of the microplate wells. This necessitates multiple steps, including, among others, the application of reagents to a film adhered to one side of the microplate, which serves as the base for the wells; the application of double-sided adhesive films to each side of the microplate; and finally, the insertion of the plate between two films of an analysis pocket. These various layers of plastic film contribute to increasing the cost of the consumable and complicate its manufacture.

[0005] Thus, there is no solution allowing the deposit of adjacent drops (< 1µL) without contact between the drops during drying in a miniature microplate which does not previously have a deposition support perpendicular to the axis of reagent deposition. Presentation of the invention

[0006] The invention therefore aims to enable the analysis of a biological sample, in particular for the detection of endotoxins, in a more reliable, rapid and less expensive way.

[0007] To this end, the invention proposes an analysis card for the analysis of a biological sample using an in vitro diagnostic instrument according to claim 1. The analysis card comprises a plurality of wells formed in a plate, wells containing at least one reagent, the analysis card comprising a channel for supplying a liquid sample to the well, characterized in that each well forms in said plate an internal space defined by a lateral surface, said lateral surface comprising at least one wall, and in that the reagent of a well is deposited and dried only on the lateral surface of said well.

[0008] The invention is advantageously complemented by the following various features taken alone or according to their various possible combinations: Each well passes through the plate from one face to another face of said plate.

[0009] Each well has several different reagents deposited only on its lateral surface, said different reagents comprising a first reagent and a second reagent.

[0010] The first reagent is likely to be activated by the second reagent and then react with the liquid sample.

[0011] Each well comprises a plurality of lobes and a plurality of junctions connecting the lobes.

[0012] Each well has several different reagents deposited only on its lateral surface, said different reagents comprising a first reagent and a second reagent, and wherein a well contains at least the first reagent deposited on a wall of a first lobe of said well and the second reagent deposited on a wall of a second lobe of said well.

[0013] The lobes of a well have an elliptical shape and the junctions of a well are straight along a junction direction.

[0014] A junction connects only two lobes of a well, and the set of lobes and junctions of a well forms an open chain.

[0015] The analysis card is associated with an analysis orientation imposed on the analysis card during the analysis of the biological sample using the in vitro diagnostic instrument, this analysis orientation being characterized in that the faces of the plate extend in the determined direction, preferably a vertical direction, and in which a first lobe of a well is connected to a second lobe of said well by a junction along a junction direction and the angle between the determined direction and said junction direction is preferably greater than 10°.

[0016] The diameter of the well lobes is greater than 0.1 mm, the width of the junctions is less than 1 mm and the length of the junctions is greater than 0.05 mm.

[0017] Each side of the plate is covered with a transparent film, at least on one side, to allow analysis of the analysis card.

[0018] The analytical reagents are capable of causing a luminescence reaction in the presence of endotoxins.

[0019] The invention also relates to a method for manufacturing an analysis card according to claim 10 comprising the following steps: a) provision of a plate having a plurality of wells, b) arrangement of the plate in an analysis orientation in which the faces of the plate extend in the determined direction, c) deposition of at least one drop of reagent liquid in contact with a lateral wall of the lateral surface of the well, d) drying of the at least one drop of reagent and obtaining reagent deposited on the wall of the lateral surface of the well.

[0020] This manufacturing process is advantageously complemented by the following different characteristics taken alone or according to their different possible combinations: The manufacturing process includes a step e) of inserting the plate between two films, and adhering the films to the plate.

[0021] Step c) of depositing the reagent drop includes the following steps: c1) placement of a needle with one end of the needle in the internal space of a well, c2) formation of a drop at the end of the needle, until contact with a wall of the lateral surface, c3) withdrawal of the needle.

[0022] The invention also relates to a quality control method for a plate having a plurality of wells according to claim 13, each well forming in said plate an internal space defined by a lateral surface, the wells having a plurality of liquid reagent deposits placed separately on its lateral surface, comprising steps of: Q1) acquisition of an image of said plate, Q2) verification of absence of liquid between deposits of liquid reagent.

[0023] This quality control process is advantageously complemented by the following various characteristics taken alone or according to their various possible combinations: The quality control process includes a step Q0) of acquiring an image of said empty plate, and step Q2) includes comparing pixels of the image acquired in step Q1) and pixels of the image acquired in step Q0).

[0024] The image of the plate is an ombroscopic image.

[0025] The quality control process includes a step a1) of recognition by a detection algorithm of the wells of the plate and identification of regions of interest for each well of the plate for which the absence of liquid is verified.

[0026] Each well comprises a plurality of lobes and a plurality of junctions connecting the lobes, the lobes being intended to receive the deposits of liquid reagents, and the regions of interest include the junctions, the absence of liquid in said junctions being verified. Presentation of the figures

[0027] Other features, purposes and advantages of the invention will become apparent from the following description, which is purely illustrative and not limiting, and which should be read in conjunction with the accompanying drawings on which: Figure 1 : there Figure 1shows an example of an analysis map that does not fall within the scope of the claims; Figure 2 : there Figure 2 schematically illustrates a well in the analysis map according to an example not falling within the scope of the claims; Figure 3 : there Figure 3 schematically illustrates a well of the analysis map comprising three lobes according to a possible embodiment of the invention; Figure 4 : there Figure 4 schematically shows an analysis map arranged in an analysis orientation, the analysis map comprising three-lobed wells according to a possible embodiment of the invention; Figure 5 : there Figure 5 is a diagram showing steps in the manufacturing process of the analysis card according to a possible embodiment of the invention; Figure 6 : there Figure 6a illustrates two needles positioned right into the internal space of a well; Figure 6b : there Figure 6billustrates the formation of two drops of reagents at the tip of the needles; Figure 6c : there Figure 6c illustrates the deposition of two drops of reagent on the lateral surface of the lobes of the same reaction well; Figure 6d : there Figure 6d illustrates two needles withdrawn from the internal space of a well after depositing two drops of reagent on the lateral surface of the lobes of the same reaction well; Figure 7 : there Figure 7 is a diagram showing steps in the quality control process of a plate equipped with a plurality of wells according to a possible embodiment of the invention, Figure 8 : there Figure 8 is a diagram of the control system, Figure 9 : there Figure 9 is a representation of the analysis card plate, after the application of reagent drops. Figure 10 : there Figure 10 is an image acquired at step Q0 of the inspection process according to the invention, Figure 11 : there Figure 11is an image acquired at step Q1 of the inspection process according to the invention, Figure 12a : there Figure 12a is an image representing the algorithmic identification of regions of interest for the lobes of each well, where the presence of liquid will be checked, Figure 12b : there Figure 12b is an image representing the algorithmic identification of regions of interest at the junctions for each well, where the presence of liquid in these junctions will be checked. Figure 13 : there Figure 13 represents the extraction of regions of interest from the lobes and the algorithmic verification of the presence of liquid, Figure 14 : there Figure 14 represents the extraction of regions of interest from the junctions and the algorithmic verification of the presence of liquid. Detailed description Analysis map

[0028] With reference to the Figure 1 ,The analysis chart 1 has a plurality of wells 2 that can be used for the placement of one or more reagents 4. Typically, an analysis chart has more than 20 wells 2. In the illustrated example, the analysis chart 1 has 35 wells 2. The wells 2 are formed in a plate 3, and each well 2 passes through the plate 3 from a first face 3a to a second face 3b opposite the first face 3a. A plate 3 is generally defined as a thin, flat surface element. In other words, a plate 3 comprises at least two opposite, flat faces 3a and 3b separated by a small thickness, i.e., the thickness is at least 10 times less than the widths and lengths of the faces 3a and 3b. The lengths and widths of faces 3a, 3b of plate 3 of analysis card 1 are preferably greater than 2 cm, and preferably less than 10 cm.The thickness of plate 3 is preferably less than 5 mm, preferably even less than 3 mm, and preferably greater than 1 mm. Preferably, the thickness of plate 3 is constant on the analysis chart 1, except in the presence of a well 2 or other functional elements that erode plate 3.

[0029] Plate 3 can be made of materials such as polypropylene, polyethylene, polystyrene, polycarbonate, PMMA, COP, POM, ABS, or any thermoplastic that can be manufactured by injection molding. Preferably, the wells 2 are evenly distributed on faces 3a and 3b of plate 3, forming a grid, and can, for example, be aligned in different rows and columns, here for example, five columns across the width of plate 3 and seven rows across its length. An angular arrangement of the reaction wells is also possible. The analysis chart 1 also includes supply channels 5a to 5d configured to supply the wells 2 with liquid biological sample or another liquid, such as a reference fluid used, for example, for control wells, in order to fill the wells 2 with liquid.

[0030] Each well 2 contains at least one reagent 4. At least some of the reagents 4 are capable of causing a luminescence reaction in the presence of analytes, and in particular in the presence of endotoxins. The analysis card 1 can thus be used to detect the presence of endotoxins in the biological sample. Although the primary focus is the analysis of biological samples to detect the possible presence of endotoxins, the invention may also relate to the analysis of biological samples to detect other analytes, such as the quantification of analytes in biochemistry or immunology, the quantification of RNA or DNA in molecular biology, the detection of microorganisms in micro-volumes, the analysis of antibiograms in micro-volumes, and the detection and quantification of microorganisms for agri-food, cosmetic, pharmaceutical, or veterinary applications.

[0031] With reference to the Figure 2 ,Each well 2 forms an internal space 6 in the plate 3, defined by a lateral surface 7. The lateral surface 7 extends from the first face 3a to the second face 3b, along the well 2. The lateral surface 7 forms the interface between the material of the plate 3 and the internal space 6. The lateral surface 7 includes at least one wall 8. In the case illustrated in Figure 2 ,The internal space 6 has an oval cross-section, so the lateral surface 7 comprises a single wall 8. A lateral surface 7 can also be composed of several walls 8, as will be illustrated later in another embodiment. These walls 8 are encountered successively when traversing the lateral surface 7. A wall 8 is therefore defined as a portion of the lateral surface 7. If the lateral surface 7 comprises several walls 8, these walls 8 are separated from the other walls 8 by edges. An edge is a line of intersection between two walls 8 and marks a discontinuity. For example, the edge marks an angular or shape discontinuity. For example, the edge can mark the intersection between a circular wall 8 and a straight wall 8.For example, the edge can mark the intersection between two circular walls that meet, we can then imagine an empty 8-shaped internal space, and therefore a lateral surface in the shape of a ribbon forming the number eight.

[0032] Each well 2 has at least one reagent 4a, 4b, or 4c deposited on its lateral surface 7. Preferably, the reagent 4, once deposited, is dry (dehydrated) and therefore not in liquid form. A reagent 4 thus forms a deposit of dry material on the lateral surface 7. An analysis chart 1 in which the reagents 4 are deposited on the lateral surface 7 of the wells 2 offers several advantages. First, it allows for better control of the position of the reagents 4 in each well 2. Indeed, the reagents 4 are more precisely located on the lateral surface 7 of the wells 2 compared to reagents that would be deposited at the bottom of the wells 2 and would have a certain tendency to spread because there is then no angular wall to hold the reagent droplets by capillary action.Thus, it is possible to position the reagents 4 in such a way as to ensure interaction between them and the biological sample, even in the presence of air bubbles, for example, by placing the reagents 4 on a point on the lateral surface 7 opposite to the direction of air bubble propagation. Furthermore, this better control of the position of the reagents 4 results in better interaction between the biological sample and the reagent 4, since the reagent 4 is concentrated on a portion of the lateral surface 7 of a well 2. In addition, since the position of a reagent 4 on the lateral surface 7 of a well 2 is known, it is possible to determine whether the biological sample has been in contact with the reagent 4 by checking where the biological sample is positioned within the well 2.

[0033] Finally, if several reagents 4 are present in each well 2, depositing them on the lateral surface 7 of the wells 2 prevents mixing. If the reagents 4 in the same well are deposited on the bottom of the well 2, they can spread out and come into contact during the depositing operation or during the drying process before complete dehydration. Depositing them on the lateral surface 7 of the well 2 prevents this. Firstly, the lateral surface 7 of the well 2 is significantly longer than the diameter (greatest length) of the well 2's bottom, allowing the reagents to be kept much further apart. This is especially important because the reagents 4 are generally deposited as liquid droplets before drying, and these droplets tend to spread out, potentially mixing if they are too close together.The separation of reactants is all the more efficient when two reactants 4 are deposited on two different walls 8 of the same lateral surface. Consider, for example, a well 2 whose lateral surface 7 is composed of two communicating circular walls 8, a first wall 8 and a second wall 8, such that the internal space 6 takes the shape of the number 8. If a first reactant 4 is deposited as a droplet (before drying) on ​​the first wall 8 and a second reactant 4 is deposited as a droplet (before drying) on ​​the second wall 8, the droplets of reactants 4 will not mix, being separated by an edge delimiting the two walls 8. Conversely, if the reactants were placed at the bottom of the well 2, they would be closer together, not separated by an edge, and therefore probably mixed.

[0034] Depositing reagent 4 onto the lateral surface 7 of a well 2 also means that each well 2 of the plate 3 does not require a bottom when depositing reagent 4 into each well 2 of the analysis card 1. This firstly relieves a significant constraint on the respective depth arrangement of the analysis card 1 and the tool used for deposition. Secondly, it facilitates quality control of a plate 3 filled with reagents 4 by shadowcopy, as will be described later, since no layer prevents light rays from passing through the voids in the internal space 6 of the wells 2. A plate 3 of an analysis card 1 without a bottom also reduces costs, since the analysis card 1 can be directly inserted between two transparent films.Advantageously, to protect the reagents 4 in wells 2, each face 3a, 3b of the plate 3 of the analysis card 1 is covered with a transparent film on both sides or inserted into a consumable already containing both films after the reagents 4 have been deposited and dried in wells 2. In conclusion, the presence of reagents 4 on a lateral surface 7 of wells 2 significantly simplifies the manufacturing process of the analysis card 1 and reduces the cost of the analysis card 1.

[0035] In an alternative embodiment not shown, it is possible to deposit the reagent onto the lateral surface of a well even though the plate has a bottom. In this case, the tool used for deposition is partially inserted into well 2 without touching the bottom, and the reagent is deposited only on the lateral surface of the well. Depending on the viscosity of the deposit, the reagent may touch the bottom of the plate, but only negligibly. Advantageously, the bottom of the plate can be opaque or transparent. When the bottom is opaque, the luminescence analysis is performed on the side of the analysis card opposite to the side on which the bottom of the plate is positioned.The remaining characteristics described above on the plate or analysis card remain unchanged for this variant embodiment, only the characteristic that the plate is provided with a background differs from the other embodiments described and the deposition method is identical.

[0036] According to the invention, each well 2 has several different reagents 4 deposited only on its lateral surface 7, said different reagents 4 comprising a first reagent 4a, 4b, 4c and a second reagent 4a, 4b, 4c. The reagents 4 are not in contact and were therefore not mixed when they were in liquid form, before being dried. The reagents 4 are dried to ensure they do not mix and thus prevent uncontrolled reactions between the different reagents from starting before use of the consumable implementing this plate 3. The reagents 4 in a well 2 must not be mixed to prevent them from reacting together before a biological sample has been introduced into the well 2 via the feed channel 5. Indeed, in order to analyze the biological sample, it may be necessary for a cascade reaction to take place and for the reagents 4 present in each well 2 not to have reacted with each other beforehand.For example, in the case of endotoxin detection, wells 2 can contain three different reagents 4a, 4b, 4c: a detection agent 4a in an inactive state (without activation) free of endotoxins, an activation agent 4b for the detection agent comprising an enzyme and a fluorogenic substrate, and a control reagent 4c suitable for monitoring the functionality of the detection reagent. In this example, the detection and activation agents must not react with each other prior to the introduction of a liquid biological sample via the feed channel 5. Therefore, reagents 4a, 4b, and 4c are only brought into contact when a liquid biological sample is introduced into well 2.

[0037] The geometry of wells 2 is adapted to prevent mixing of reagents 4a, 4b, and 4c before the introduction of the reference liquid. First, if the lateral surface 7 of a well 2 comprises a single wall 8, the well 2 can be wide enough, and therefore the lateral surface 7 long enough, to allow a separation space between each reagent 4 deposited on the wall 8. For example, in the Figure 2This illustrates a well 2 whose lateral surface 7 comprises a single wall 8. Well 2 is wide enough to allow a space between reactants 4a, 4b, and 4c on the lateral surface 7, which ensures the separation of reactants 4a, 4b, and 4c. Furthermore, the lateral surface 7 of wells 2 can have a shape that allows separation between each reactant 4a, 4b, and 4c, by including a discontinuity such as an edge between two reactants 4a, 4b, and 4c. The lateral surface 7 can include several walls 8, as explained above. The discontinuity between the walls 8 of the same lateral surface 7 allows a separation space between reactants 4a, 4b, and 4c, and even a boundary between them. One can imagine a well 2 whose internal space 6 comprises three circles, one of which communicates with the other two circles.The lateral surface 7 comprises three successive rounded walls 8 as one traverses the lateral surface 7, with a discontinuity at the transition from one circle to the next, and therefore from one wall 8 to the next. Each reagent 4a, 4b, 4c can be placed respectively on one of the three walls 8 of the lateral surface 7 such that each reagent 4a, 4b, 4c is located on a different wall 8, and therefore within a distinct portion of the internal space 6 constituted by a circle. The reagents 4a, 4b, 4c are thus separated from each other within the same well 2.

[0038] According to the invention, as illustrated on plate part 3 in Figure 3 ,Each well 2 comprises a plurality of lobes 9, such as for example three lobes 9a, 9b, 9c, and a plurality of junctions 10, such as for example two junctions 10a, 10b, connecting the lobes 9a, 9b, 9c. In other words, each well 2 comprises several distinct locations, called lobes 9, which communicate with each other via junctions 10. In this embodiment, the lateral surface 7 comprises several walls 8a, 8b, 8c, 8d, 8e, 8f, 8g, each wall 8 being associated with a lobe 9a, 9b, 9c or with a junction 10a, 10b, 10c.

[0039] Preferably, each lobe 9 is provided with a different reagent 4. Each well 2 has several different reagents 4 deposited only on its lateral surface 7, said different reagents 4 comprising, in the example illustrated in Figure 3 ,a first reagent 4a and a second reagent 4b. The first reagent 4a is deposited on the wall 8a of a first lobe 9a of said well 2 and the second reagent 4b is deposited on the wall 8b of a second lobe 9b of said well 2. In the example illustrated in Figure 3 ,a third reagent 4c is deposited on a wall 8c of a third lobe 9c. In an embodiment adapted for the detection of endotoxins, each well 2 comprises three lobes 9a, 9b and 9c and two junctions 10a and 10b, the wall 8a of a first lobe 9a presenting a detection agent 4a, the wall 8b of a second lobe 9b presenting an activation agent 4b and the wall 8c of a third lobe 9c presenting a control reagent 4c. Advantageously, reagents 4a, 4b, and 4c are deposited only on the walls 8a, 8b, and 8c of lobes 9, and there is no reagent 4 on the walls 8d to 8g of junctions 10. Indeed, if a reagent 4 is present on a wall 8d-8g of a junction 10, it could come into contact with another reagent 4 located in a lobe 9, which is undesirable. Furthermore, it should be noted that the reagents 4, once deposited, are preferably dry and not in liquid form.

[0040] Preferably, the lobes 9 of each well 2 have an elliptical shape and the junctions 10 of each well 2 are straight along a junction direction. Even more preferably, the lobes 9 of each well 2 have a circular shape. Thus, and as illustrated, the walls 8a, 8b, 8c of a lobe 9a, 9b, 9c are typically rounded or curved, while the walls 8d, 8e, 8f, 8g of a junction 10a, 10b, 10c are planar.

[0041] Analysis card 1 is a microfluidic system. The diameter of the lobes 9 is preferably less than 3 mm and preferably greater than 0.1 mm. The width of the junctions 10 is preferably less than 1 mm and preferably greater than 0.05 mm. The length of the junctions 10 is preferably less than 5 mm and preferably greater than 0.05 mm. These dimensions are, on the one hand, large enough to allow the circulation of a liquid such as the biological sample, and on the other hand, small enough that the surface tension effects of the liquid allow droplets to be contained outside the junctions 10 and also promote the mixing of the reagents 4 once the sample has been introduced, thus facilitating the retrieval of the dried reagents 4 and the initiation of the analysis reaction.

[0042] As explained, this structure, composed of lobes 9 and junctions 10, isolates the different reagents 4 to prevent them from mixing before drying. Also, to minimize mixing of the reagents 4, the lobes 9 of each well 2 are preferably arranged in a specific way relative to each other to optimize the imaged area. An analytical orientation is imposed on the analysis card 1 during the analysis of the biological sample using the in vitro diagnostic instrument 12. With reference to the Figure 4 ,The analysis orientation corresponds to a vertical orientation of the analysis card 1, in which the analysis card 1 is positioned when analyzed using an in vitro diagnostic instrument 12 comprising an imager 13, typically a fluorometer, defining a field of view 14. However, this so-called vertical orientation is purely indicative and used as an example; there is no technical link between the card's orientation and the analysis itself. During the analysis of the analysis card 1, the analysis card 1 is inserted into the in vitro diagnostic instrument 12 in this analysis orientation, within the field of view 14 of the imager 13.The in vitro diagnostic instrument 12 may also include a light source 15 configured to illuminate the field of view 14 with light whose wavelength is likely to highlight a fluorescence phenomenon, i.e. to cause the emission of fluorescence light after excitation of a fluorescent phore.

[0043] The analysis orientation of analysis map 1 preferably corresponds to that in which analysis map 1 is placed in Figure 4 . The y-axis corresponds to the vertical direction and the x-axis corresponds to the horizontal direction. Figure 4illustrates an analysis chart 1 according to a certain embodiment, comprising a plurality of wells 2 with several lobes 9, here three lobes 9. In this case, the analysis orientation of the analysis chart 1 is such that the axis of each cylinder defined by each lobe 9 is perpendicular to the vertical direction. Thus, to return to the relative arrangement of the lobes 9 of the same well 2, when the analysis chart 1 is arranged in the analysis orientation, the angle between the vertical direction and the junction direction of the junction 10 between two lobes 9 of a well 2 is preferably greater than 10°, and is preferably less than 180° (or 0°). The vertical direction and the angle between the vertical direction and the direction of a junction 10 are shown in dashed lines on the Figure 3 .Moreover, this is made possible by the fact that the reagents 4 are deposited on the lateral surface 7 of each well 2. Indeed, taking the example of endotoxin detection, the reagents 4 used have high wettability (contact angle between 75° and 90°). Consequently, if drops of reagent 4 were deposited on the bottom of the lobes 9 of a well 2, they would be very likely to move and spread out, mixing with each other before drying, which is undesirable. Thus, the structure imposed by the different lobes 9 of a well 2 and the fact that the reagents are deposited on the lateral surface 7 of each well 2 best ensures the isolation of the reagents 4 deposited in different lobes 9 from each other.

[0044] To enable a coherent cascade reaction and prevent mixing between the different reagents 4 in a well 2, each junction 10 connects only two lobes 9 of a well 2, and all the lobes 9 and junctions 10 of a well 2 form an open chain. A coherent cascade reaction is one in which the order in which the subreactions occur is optimal for obtaining usable results. In other words, in a reaction involving several subreactions and therefore several reagents 4, it may be necessary for a certain reagent 4 to react with the biological sample before reacting with another reagent 4. In the example of endotoxin detection, it is preferable for the biological sample to come into contact with the activating agent 4b before coming into contact with the detecting agent 4a.Thus, the open chain formed by all the lobes 9 and junctions 10 of a well 2 allows the order in which the sub-reactions of a cascade reaction will occur to be controlled. The term "open chain" to characterize all the lobes 9 and junctions 10 means that two lobes 9 of the set of lobes 9 are each connected to a single junction 10. These two lobes 9 are in fact the first lobe 9 and the last lobe 9 of the chain; in other words, these two lobes 9 constitute the ends of the chain. Obviously, one of the lobes 9 located at an end can also, in addition to being connected to a single junction 10, be connected by a feed channel 5. Analysis card manufacturing process

[0045] The invention also relates to the manufacturing process for the analysis card 1. This process is presented in Figure 5 .In step a), a plate 3 provided with a plurality of wells 2, which preferably pass through said plate 3, is supplied. By supplied, it is understood that the manufacturing process requires having at its disposal a plate 3 comprising a plurality of wells 2, the wells 2 being preferably empty of any reagent 4.

[0046] The present process may also include a preliminary step a0) of plastic injection molding of plate 3.

[0047] In step b), plate 3 is arranged in an analysis orientation. In this example, the analysis orientation corresponds to the orientation in which faces 3a, 3b of plate 3 are vertical.

[0048] Then, in step c), a liquid drop of reagent 4 is deposited in contact with the lateral surface 7 of well 2. This step c) is illustrated in figures 6a to 6daccording to which a simultaneous deposition of two drops is shown. Advantageously, each drop is deposited using a needle 11. Preferably, step c) of depositing the reagent drop 4 comprises three substeps c1), c2), and c3), substep c1) being the placement of the needle 11, one end of the needle 11a being positioned up to the internal space 6 of the well 2 as illustrated in Figure 6a . Preferably, the end 11a of the needle does not protrude from the internal space 6. The end 11a of the needle therefore preferentially passes through only one of the two faces 3a, 3b of the plate 3, and is thus preferably located between the first face 3a and the second face 3b. On the Figure 6a , for example, the end 11a of the needle only passes through the first face 3a.

[0049] Substep c2) corresponds to the formation of a droplet of reagent 4 at the tip 11a of the needle, by supplying a channel 11b of the needle with reagent 4 in liquid form. A droplet forms and grows until it contacts a wall 8 of the lateral surface 7 of a well 2 as illustrated in Figure 6b . The position of the needle 11, and more specifically the distance of the needle tip 11a from the lateral surface 7, dictates the size of the droplet at which the droplet touches the wall. The needle tip 11a is preferably offset from the center of the internal space 6, and is therefore closer to the part of the lateral surface 7 on which the reagent droplet 4 is to be deposited, thus allowing the droplets to be collected and retained in the lobes as soon as they form at the tip 11a of the needles 11.

[0050] Each drop of reagent 4 formed typically has a diameter greater than 0.8 mm and, preferably, a diameter greater than 1.2 mm. The diameter of each drop of reagent 4 is, for example, approximately 1 mm. Once each drop comes into contact with the wall 8 of the lateral surface 7 of the well 2, it deposits on the wall 8 as illustrated Figure 6c . Each drop of reagent 4 deposited preferably has a volume less than 1 µL, and even more preferably, a volume less than 0.65 µL. Preferably, the volume of each drop of reagent 4 deposited is greater than 0.1 µL, and even more preferably, is greater than 0.35 µL. For example, each drop of reagent 4 has a volume of approximately 0.5 µL.

[0051] Once each drop is deposited, each needle 11 or plate holder is removed according to substep c3) from the internal space 6 of well 2 as illustrated in Figure 6d . Then, again referring to the Figure 5, in a step d), each drop of reagent 4 is dried so that dried reagent 4 is obtained on the wall 8 of the lateral surface 7 of the well 2. It will be understood of course that the steps c) and d) of drop deposition and drying are carried out on a plurality of wells 2 of the plate 3, preferably all of the wells 2 of the plate 3 simultaneously or sequentially.

[0052] In a certain embodiment in which the wells 2 comprise several lobes 9, each lobe 9 of each well 2 is provided with at least one drop of reagent 4, and preferably with only one drop of reagent 4. Preferably, the droplet deposition and drying steps c) and d) are performed simultaneously for each lobe 9 of several wells 2 of the plate 3, and preferably for each well 2. Thus, this droplet deposition method allows the simultaneous deposition of a plurality of drops in a microplate, i.e., the plate 3. Indeed, by using a support comprising a plurality of needles 11 and bringing this support close to a plate 3 such that each needle 11 has its tip 11a in the internal space 6 of a well 2 of the plate 3, it is possible to simultaneously deposit drops of reagent 4 onto a wall 8 of the lateral surface 7 of several wells 2.Furthermore, this method allows the simultaneous deposition of multiple drops onto the walls 8 of each lobe 9 of each well 2 of a plate 3 without the drops mixing within the same well 2. Consequently, dozens, or even hundreds, of reagent drops 4 with a volume on the order of microliters can be deposited without human intervention in a short time. Consider, for example, the analysis chart 1 shown in Figure 1. Figure 4 comprising 35 wells 2 of three lobes 9, the claimed manufacturing process can allow to simultaneously deposit on the wall 8 of each lobe 9 a drop of reagent 4 and therefore to simultaneously deposit 105 drops of reagent 4 in the plate 3 of the analysis card 1.

[0053] Preferably, the tip 11a of a needle used for deposition is coated with a non-stick coating. For example, this coating can be a hydrophobic Teflon-based coating such as polytetrafluoroethylene (PTFE). In this way, the droplet that forms at the tip of the needle 11a does not deform or run down the needle 11, but instead retains a round shape, allowing for better control of the volumes deposited in the plate 3. Furthermore, when several needles 11 are arranged side by side in a holder, this prevents the droplets from coming into contact and mixing, as illustrated in figures 6a to 6d Furthermore, the coating makes this solution more generic, allowing the deposition of liquids with highly varied surface tensions.

[0054] Preferably, the present manufacturing process includes a step e) of inserting the plate 3 between two films, preferably transparent, and of adhering the films to the plate 3 so as to protect the reagents 4 and to close the fluidic network of the wells 2. The films form the ends of the closed wells 2 and are devoid of reagents 4. Quality control process

[0055] To ensure that the plate 3 is correctly supplied with reagents 4, i.e., that each well 2 contains a drop of reagent 4 and that, in the case where the wells 2 comprise several lobes 9, the drops present in different lobes 9 of the same well 2 have not mixed, a quality control method for a plate 3 is proposed before the drying of the reagents 4 (substep c3). More specifically, a quality control method for a plate 3 having a plurality of wells 2 passing through said plate 3, each well 2 having a plurality of liquid reagents 4 deposited separately on its walls 8, is proposed. The method is described by the Figure 7 .

[0056] In step Q1), an image of the plate 3, whose wells 2 contain a plurality of drops of liquid reagents 4, is acquired as illustrated in Figure 9 or in Figure 11 .Preferably, the quality control process includes a preliminary step Q0) of acquiring an image of the empty plate 3 illustrated in Figure 10 . In other words, step Q0) consists of acquiring an image of plate 3 when no reagent 4 has been deposited on the walls 8 of its wells 2. Advantageously, the images acquired in steps Q1) and Q0) of the quality control process are shadow images. With reference to the Figure 8 ,An ombroscopic image can, for example, be acquired using a control system 16 comprising a light source 17 such as a telecentric lamp and an image acquisition device 18, for example equipped with a telecentric lens 19. The plate 3 is positioned in its analysis orientation (typically with faces 3a, 3b vertical) between the light source 17 and the image acquisition device 18. Coaxial light rays are emitted by the light source 17 towards the plate 3. These light rays pass through the internal spaces 6 of the wells 2 of the plate 3 or are absorbed by the faces 3a, 3b of the plate 3. The light rays that pass through the internal spaces 6 of the wells 2 are deflected by liquid reagents 4 present in the wells 2 or pass without deflection through the empty parts of the internal spaces 6, in the absence of liquid reagents 4 in their paths.The image acquisition device 18 receives only the light rays that are neither deflected nor absorbed by the faces 3a, 3b of the plate 3, that is, the light rays that have passed through an empty portion of an internal space 6. Thanks to this control system 16, a black and white image of the plate 3 is obtained. The wells 2 are recognizable by their empty internal spaces 6, which appear white in the image. (Continuing with reference to...) Figure 8 ,The control system 16 can include mirrors 20, 21, which allows for a more compact design. For example, the control system 16 can comprise two mirrors 20, 21, one of which is a fully reflective mirror 21 and the other a semi-reflective mirror 20. The light rays emitted by the light source 17 pass through the semi-reflective mirror 20 and then through the plate 3, which is positioned in its analysis orientation. The undeflected or unabsorbed light rays reach the fully reflective mirror 21, which reflects them. These rays then pass through the plate 3 again, until they reach the semi-reflective mirror 20, which reflects the rays back towards the image acquisition device 18.

[0057] Furthermore, the quality control process preferably includes a step Q1') of recognition by a detection algorithm of the wells 2 of the plate 3 and identification of regions of interest for each well 2 of the plate 3. By well 2 detection, it is understood that the wells 2 of the plate 3 are located in an acquired image. In the case where each well 2 comprises lobes 9 and junctions 10, the detection may involve the localization of lobes 9 and junctions 10. The well 2 detection algorithm may implement a detection function that depends on the known shape of the wells 2, adapted to the geometry of the wells 2 of the plate 3 and / or to a distribution of the wells 2 on the plate 3. For example, if it is desired to detect the lobes 9 and the lobes 9 have a circular shape, a circular edge detection function can be used, as illustrated in Figure 12a .

[0058] Next, regions of interest are identified. Advantageously, at least one region of interest is identified for each well 2 of the plate 3. The identification of regions of interest may, for example, correspond to the identification of the junctions 10 as illustrated in Figure 12b . Indeed, if we wish to determine that the reagents 4 present in the different lobes 9 of a well 2 have not mixed, we can verify that the junctions 10 are free of any reagent. Consequently, in this case, the junctions 10 constitute regions of interest. The lobes 9 can also be regions of interest, particularly to ensure the presence of reagents. It is, of course, possible to combine several approaches.

[0059] The quality control process therefore includes a step Q2) to verify the absence of liquid between each application of liquid reagent 4. As explained, this can, for example, involve verifying that no reagent 4 is present in the junctions 10 of a well 2. Preferably, this step Q2) includes comparing pixels from the image acquired in step Q1) with pixels from the image acquired in step Q0). It is also possible to perform comparisons on groups of pixels. This comparison makes it possible to determine where the reagents 4 are present within a well 2 and, consequently, to identify a lack of reagents 4, an excess of reagents 4, or the unwanted presence of reagents 4 in a certain part of a well 2. Preferably, comparing pixels means comparing their light intensity values ​​and, even more preferably, comparing their gray levels.Furthermore, preferably, each pixel compared in an image acquired in step Q1) is compared to the pixel at the same location in the image acquired in step Q0). Advantageously, only the pixels corresponding to the regions of interest for each well are considered in this comparison. Indeed, the regions of interest of the wells 2 of the plate 3 are the precise locations where one wishes to detect the presence or absence of reagent 4. For example, the regions of interest could be the junctions 10 of the wells 2, and one might wish to detect whether reagent 4 is present in the junctions 10. This would likely mean that reagents 4 from two different lobes 9 of a well 2 have mixed. Moreover, this comparison step Q2) can be likened to a subtraction of the two images.If pixels in the image of plate 3 filled with reagents 4 acquired in step Q1) have a different value of the light intensity value of the same pixels in the image of the empty plate 3 acquired in step Q0), this may mean that a reagent 4 is present in plate 3 at the position corresponding to said pixels as illustrated in . Figure 13 for regions of interest at the level of lobes 9 and in Figure 14For regions of interest at junctions 10, threshold values ​​for differences in pixel light intensity and the number of pixels with different light intensity values ​​between the images acquired in steps Q1) and Q0) can be defined. Thus, based on a certain number of pixels whose light intensity value differs by a certain difference in pixel light intensity value between the two images, it can be determined that reagent 4 is present in plate 3 at the position corresponding to said pixels. From the number of pixels identified as corresponding to spaces filled with reagent 4, a surface area of ​​pixels corresponding to spaces filled with reagent 4 can be extracted. This surface area can be used and extrapolated to obtain an estimated volume value of reagent 4 present in the wells 2 of plate 3.

[0060] The invention is not limited to the embodiment described and shown in the accompanying figures. The invention is limited only by the attached claims.

Claims

1. An analysis card (1) for analysing a biological sample by means of an in vitro diagnostics instrument, the analysis card (1) comprising a plurality of wells (2) formed in a plate (3) having a first face (3a) and a second face (3b) opposite to the first face (3a), wells (2) containing at least one reagent (4), the analysis card (1) comprising a supply channel (5) for supplying a liquid sample to the well (2), characterized in that each well (2) forms in said plate (3) an internal space (6) defined by a lateral surface (7) extending from the first face (3a) to the second face (3b) along the wells, said lateral surface (7) comprising at least one wall (8), and in that the reagent (4) of a well is deposited and dried only on the lateral surface (7) of said well (2), wherein each well (2) includes a plurality of lobes (9) and a plurality of junctions (10) connecting the lobes (9), wherein each well (2) has several different reagents (4) deposited only on its lateral surface (7), said different reagents (4) comprising a first reagent (4a) and a second reagent (4b), and wherein a well (2) contains at least the first reagent (4a) deposited on a wall (8a) of a first lobe (9a) of said well (2) and the second reagent (4b) deposited on a wall (8b) of a second lobe (9b) of said well (2).

2. The analysis card (1) as claimed in claim 1, wherein each well (2) passes through the plate (3) from one face (3a) to another face (3b) of said plate (3).

3. The analysis card (1) as claimed in claim 1 or 2, wherein the second reagent (4b) is an activation agent for activating the first reagent (4a) and the activated first reagent (4a) is capable of reacting with the liquid sample when the activated first reagent (4a) is brought into contact with the liquid sample.

4. The analysis card (1) as claimed in one of claims 1 to 3, wherein the lobes (9) of a well (2) have an elliptical shape and the junctions (10) of a well (2) are rectilinear in a junction direction.

5. The analysis card (1) as claimed in one of claims 1 to 4, wherein a junction (10) connects only two lobes (9) of a well (2) and all of the lobes (9) and junctions (10) of a well (2) form an open chain.

6. The analysis card (1) as claimed in any one of claims 1 to 5, wherein the analysis card (1) is associated with an analysis orientation imposed on the analysis card (1) during the analysis of the biological sample by means of the in vitro diagnostics instrument, this analysis orientation being characterized in that the faces (3a, 3b) of the plate (3) extend in the predetermined direction, preferably a vertical direction, and wherein a first lobe (9a) of a well (2) is connected to a second lobe (9b) of said well (2) by a junction (10) in a junction direction and the angle between the predetermined direction and said junction direction is preferably greater than 10°.

7. The analysis card (1) as claimed in any one of claims 1 to 6, wherein the diameter of the lobes (9) of the wells (2) is greater than 0.1 mm, the width of the junctions (10) is less than 1 mm, and the length of the junctions (10) is greater than 0.05 mm.

8. The analysis card (1) as claimed in any one of the preceding claims, wherein each face of the plate (3) is covered with a transparent film at least on a face intended to allow analysis of the analysis card.

9. The analysis card (1) as claimed in any one of the preceding claims, wherein the analysis reagents (4) are adapted to cause a luminescence reaction in the presence of endotoxins.

10. A method for manufacturing an analysis card (1) as claimed in any one of the preceding claims, comprising the following steps: a) supplying a plate (3) provided with a plurality of wells (2), b) positioning the plate (3) in an analysis orientation in which the faces (3a, 3b) of the plate (3) extend in the predetermined direction, c) depositing at least one drop of reagent liquid (4) in contact with a wall (8) of the lateral surface (7) of the well (2), d) drying the at least one drop of reagent (4) and obtaining reagent (4) deposited on the wall (8) of the lateral surface (7) of the well (2).

11. The method as claimed in the preceding claim, comprising a step e) of inserting the plate (3) between two films, and adhesion of the films to the plate (3).

12. The method as claimed in either of claims 10 and 11, wherein step c) of depositing the drop of reagent (4) comprises steps of: c1) placing a needle with one end of the needle (11a) in the internal space (6) of a well (2), c2) forming a drop at the end of the needle (11a), until contact is made with a wall (8) of the lateral surface (7), c3) withdrawing the needle (11).

13. A method for quality control of a plate (3) manufactured according to any of the claims 10 to 12, and comprising steps of: Q1) acquisition of an image of said plate (3), Q2) verification of the absence of liquid between deposits of liquid reagent (4).

14. The quality control method as claimed in the preceding claim, comprising a step Q0) of acquisition of an image of said empty plate (3), and step Q2) comprises the comparison of pixels of the image acquired in step Q1) and of pixels of the image acquired in step Q0).

15. The quality control method as claimed in either of claims 13 and 14, wherein the image of the plate is a shadowgraphy image.

16. The quality control method as claimed in any one of claims 13 to 15, comprising a step a1) of recognition by an algorithm for detecting the wells (2) of the plate (3) and identification of regions of interest for each well (2) of the plate (3) for which the absence of liquid is verified.

17. The quality control method as claimed in claim 16, wherein each well (2) comprises a plurality of lobes (9) and a plurality of junctions (10) connecting the lobes (9), the lobes being intended to receive the deposits of liquid reagents (4), and the regions of interest include the junctions (10), the absence of liquid in said junctions (10) being verified.