Analytical card for analyzing biological samples and production and quality control method - Patents.com

Abstract

The present invention relates to an analytical card (1) for analyzing biological samples using an in-vitro diagnostic device, comprising a plurality of wells (2) formed in a board (3), each well (2) containing at least one reagent (4), the analytical card (1) comprising a channel (5) for conveying a liquid sample to the wells (2), characterized in that each well (2) forms in the board (3) an interior space defined by a lateral surface, the lateral surface comprising at least one wall, and each well (2) has at least one reagent (4) deposited only on its lateral surface.

Description

[Technical field]

[0001] The present invention relates to the field of biological sample analysis, and more particularly to an assay card for the analysis of biological samples using an in vitro diagnostic instrument, in particular for the detection of endotoxins, as well as a method for producing the assay card and a method for the quality control of the plates of the assay card. [Background technology]

[0002] The analysis of biological samples, such as endotoxin detection tests, is based on one or more reactions between the biological sample and one or more reagents. There are microfluidic systems and methods for performing these biological sample analyses. The reagents are deposited in wells and the biological sample is introduced, for example, via a supply channel. This requires the preparation of multiple standard dilutions and internal controls. In the context of endotoxin detection, for example, the test is laborious and requires many steps of handling by the operator. These manual preparation steps can be time-consuming and can lead to variable or even invalid results. In addition, there is no solution to monitor the preparation of the test and therefore to quickly determine whether an error has been made. Thus, any problem can only be detected at the end of a measurement period, which is preselected to be long enough to allow the complete completion of the various reactions that may occur with different dynamics.

[0003] There are microplates such as the "GOPLATE™" system, which have 96 wells pre-filled with the required standard amounts of reagents whose concentrations have been verified. This device reduces the handling time by more than 50% compared to conventional microplate endotoxin tests. However, many additional accessories are required to perform biological sample analysis using this device, and its execution still involves multiple manual steps. There are also microplates integrated into consumable systems such as the "FilmArray®", which require little manual manipulation. These pre-filled microplates also make it possible to limit human intervention in the biological sample analysis process, thus reducing the risk of human error.

[0004] However, the pre-filled microplates found in the prior art are not suitable for certain reactions that require the use of different reagents that must not react with each other and therefore must not be mixed before the biological sample is introduced into the wells of the microplate. Furthermore, in the prior art, the reagents are most often deposited on the bottom surface of the wells of the microplate. This requires multiple steps, including, among others, depositing the reagents on a film that acts as the bottom surface of the wells and is adhesively bonded to one of the two sides of the microplate, attaching a double-sided adhesive film to each of the two sides of the microplate, and then inserting the plate between the two films of an analysis bag. These various layers of plastic films contribute to the increased cost of the consumables and their complex production.

[0005] Thus, there is no solution that allows adjacent droplets (<1 μL) to be deposited without contact between them during drying in small microplates that do not already contain a deposit support perpendicular to the axis of reagent deposition. Summary of the Invention

[0006] The present invention therefore aims to enable more reliable, faster and cheaper analysis of biological samples, in particular for the detection of endotoxins.

[0007] For this purpose, the present invention provides an assay card for analyzing biological samples using an in vitro diagnostic device, the assay card comprising a plurality of wells formed in a plate, the wells containing at least one reagent, the assay card comprising a supply channel for supplying a liquid sample to the wells, each well defines an interior space within the plate defined by a lateral surface, the lateral surface comprising at least one wall, and reagents within a well are deposited and dried only on the lateral surface of the well. Suggest an analysis card.

[0008] The invention is advantageously complemented by various characteristics which can be implemented alone or in their various possible combinations:

[0009] Each well extends through the plate from one side of the plate to another.

[0010] Each well preferably has several different reagents deposited only on its lateral surfaces, said different reagents including a first reagent and a second reagent.

[0011] The first reagent is activated by the second reagent and is then capable of reacting with the liquid sample.

[0012] Each well contains multiple lobes and multiple junctions connecting the lobes.

[0013] Each well has several different reagents deposited only on its lateral surfaces, the different reagents including a first reagent and a second reagent, and the well contains at least the first reagent deposited on a wall of a first lobe of the well and the second reagent deposited on a wall of a second lobe of the well.

[0014] The lobes of the wells have an elliptical shape and the junctions of the wells are straight in the direction of the junctions.

[0015] One junction connects only two lobes of one well; all lobes and junctions of one well form an open chain.

[0016] The analysis card is associated with an analysis direction which is imposed on the analysis card during the analysis of a biological sample using an in vitro diagnostic device, said analysis direction being characterized in that the surface of the plate extends in a predetermined direction, preferably vertically, a first lobe of a well is connected to a second lobe of said well by a junction in a junction direction, the angle between the predetermined direction and said junction direction being preferably greater than 10°.

[0017] The well lobe has a diameter greater than 0.1 mm, the junction width is less than 1 mm, and the junction length is greater than 0.05 mm.

[0018] Each side of the plate is covered with a transparent film, at least on the side that is to allow for analysis of the assay card.

[0019] The assay reagent is adapted to produce a luminescent reaction in the presence of endotoxin.

[0020] The present invention relates to a method for producing an assay card, comprising the steps of: a) providing a plate comprising a plurality of wells; b) positioning the plate in an analysis orientation with the plane of the plate extending in a predetermined direction; c) depositing at least one droplet of a reagent liquid in contact with a lateral surface of the well; d) drying at least one droplet of reagent to obtain the reagent deposited on the lateral surface walls of the well. The present invention also relates to a method, comprising:

[0021] This production method is advantageously complemented by the various following features, which can be implemented alone or in their various possible combinations:

[0022] The production method includes step e) of inserting the plate between two films and gluing the films to the plate.

[0023] The step c) of depositing a droplet of a reagent comprises: c1) placing a needle with one end of the needle within the interior space of the well; c2) forming a droplet on the end of the needle until it comes into contact with the wall of the lateral surface; c3) Removing the needle Includes.

[0024] The present invention relates to a method for quality control of a plate comprising a plurality of wells, each well forming an interior space in said plate defined by lateral surfaces, the wells having a plurality of deposits of liquid reagents separately deposited on said lateral surfaces, the method comprising: Q1) acquiring an image of the plate; Q2) Verifying the absence of liquid between the deposits of liquid reagent The present invention also relates to a method, comprising:

[0025] This quality control method is advantageously complemented by the following various features, which can be implemented alone or in their various possible combinations:

[0026] The quality control method comprises a step Q0) of acquiring an image of the blank plate, and a step Q2) comprises comparing pixels of the image acquired in step Q1) with pixels of the image acquired in step Q0).

[0027] The image of the plate is a shadowgraphy image.

[0028] The quality control method comprises a step a1) of recognizing by an algorithm the wells of the plate to be detected and, for each well of the plate, identifying an area of ​​interest in which the absence of liquid is verified.

[0029] Each well comprises a number of lobes and a number of junctions connecting the lobes, the lobes being for receiving a deposit of a liquid reagent, the region of interest including the junctions, and the absence of liquid within the junctions is verified.

[0030] Other characteristics, objects and advantages of the present invention will become apparent from the following description which is given by way of example and is non-limiting and which should be read in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 illustrates an exemplary analysis card. [Diagram 2] FIG. 2 is a schematic depiction of a well of an assay card according to one possible embodiment of the invention. [Diagram 3] FIG. 2 is a schematic representation of a three-lobed well of an assay card according to one possible embodiment of the invention. [Figure 4] FIG. 2 is a schematic depiction of an assay card arranged in an assay orientation according to one possible embodiment of the invention, the assay card comprising wells with three lobes. [Diagram 5] 1 is a diagram showing steps of a method for producing an assay card according to one possible embodiment of the invention. [Figure 6a] FIG. 13 depicts two needles positioned just inside the interior space of a well. [Figure 6b] FIG. 1 illustrates the formation of two droplets of reagent on the end of a needle. [Figure 6c] FIG. 1 illustrates the deposition of two droplets of reagent onto the lateral surfaces of the lobes of the same reaction well. [Figure 6d] FIG. 1 illustrates the deposition of two droplets of reagent onto the lateral surfaces of the lobes of the same reaction well, after which the two needles are withdrawn from the interior space of the well. [Figure 7] 1 is a diagram showing the steps of a method for quality control of a plate having multiple wells, according to one possible embodiment of the invention. [Figure 8] 1 is a diagram of a control system. [Figure 9] FIG. 2 shows the plate of the assay card after the deposition of droplets of reagents. [Figure 10] 2 is an image acquired in step Q0 of the control method according to the present invention. [Figure 11] 1 is an image acquired in step Q1 of the control method according to the present invention. [Figure 12a] 13 is an image showing how an algorithm identifies areas of interest in the lobes of each well where liquid is examined for presence. [Figure 12b] 13 is an image showing how an algorithm identifies regions of interest at the junctions of each well to verify whether liquid is present within those junctions. [Figure 13] FIG. 13 shows the extraction of a lobe region of interest and the algorithmic verification of the presence of liquid. [Figure 14] FIG. 13 shows the extraction of a region of interest at the junction and the presence of liquid verified by an algorithm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Analysis Card With reference to FIG. 1, an assay card 1 comprises a number of wells 2 that can be used to introduce one or more reagents 4. Typically, an assay card comprises more than 20 wells 2. In the illustrated example, the assay card 1 comprises 35 wells 2. The wells 2 are formed in a plate 3, each well 2 penetrating the plate 3 from a first face 3a to a second face 3b opposite the first face 3a. The plate 3 is generally defined as an element with a flat thin surface. In other words, the plate 3 comprises at least two flat faces 3a, 3b opposite each other and separated by a slight thickness, i.e. the thickness is at most 10 times smaller than the width and length of the faces 3a, 3b. The length and width of the faces 3a, 3b of the plate 3 of the assay card 1 are preferably greater than 2 cm, preferably less than 10 cm. The thickness of the plate 3 is preferably less than 5 mm, more preferably less than 3 mm, and the thickness of the plate 3 is preferably greater than 1 mm. Preferably, the thickness of the plate 3 is constant across the assay card 1 except where there are wells 2 or other functional elements that form cavities within the plate 3 .

[0033] The plate 3 can be made of materials such as, for example, polypropylene, polyethylene, polystyrene, polycarbonate, PMMA, COP, POM, ABS, or any thermoplastic that can be processed by injection molding. Preferably, the wells 2 are regularly distributed over the faces 3a, 3b of the plate 3, forming a grid, and can be arranged, for example, in various rows and columns, in this case, for example, in five columns in the width direction of the plate 3 and seven rows in the length direction of the plate 3. An angular distribution of the reaction wells is also possible. The assay card 1 also comprises supply channels 5a to 5d, configured to supply the wells 2 with a liquid biological sample or another liquid, for example a reference fluid used for control wells, in order to fill the wells 2 with liquid.

[0034] Each well 2 has at least one reagent 4. At least some of the reagents 4 are capable of generating a luminescent reaction in the presence of an analyte, in particular in the presence of endotoxin. The assay card 1 can therefore be used to detect the presence of endotoxin in a biological sample. Even though the invention relates more specifically to the analysis of biological samples to detect the possible presence of endotoxin, it can also relate to the analysis of biological samples to detect other analytes, such as for example the assay of analytes in biochemistry or immunology, the assay of the amount of RNA or DNA in molecular biology, the detection of the presence of microorganisms in trace amounts, antibiogram analysis in trace amounts, the detection and quantification of microorganisms for agro-food applications, cosmetic applications, pharmaceutical applications or veterinary applications.

[0035] With reference to FIG. 2, each well 2 forms in the plate 3 an internal space 6 defined by a lateral surface 7. The lateral surface 7 extends along the well 2 from the first face 3a to the second face 3b. The lateral surface 7 forms an interface between the material of the plate 3 and the internal space 6. The lateral surface 7 comprises at least one wall 8. In the case shown in FIG. 2, the internal space 6 is of oval cross section and therefore the lateral surface 7 comprises a single wall 8. The lateral surface 7 may also be made of several walls 8, as shown below in another embodiment. When traversing the lateral surface 7, these walls 8 are encountered in turn. The wall 8 is therefore defined as part of the lateral surface 7. If the lateral surface 7 comprises several walls 8, these walls 8 are separated from the remaining walls 8 by an edge. An edge is a line of intersection between two walls 8 and represents a discontinuity. For example, the edge represents an angular discontinuity or a geometric discontinuity. For example, the edge can represent the intersection between a circular wall 8 and a straight wall 8. Or, for example, the edge can represent the intersection between two adjacent circular walls 8, in which case it is possible to envisage an interior space taking the shape of a hollow number 8, and thus a lateral surface 7 taking the shape of a ribbon forming the number 8.

[0036] Each well 2 has at least one reagent 4a, 4b or 4c deposited on its lateral surface 7. Preferably, after deposition, the reagent 4 is dry (dehydrated) and therefore not liquid. The reagent 4 therefore forms a deposit of dry material on the lateral surface 7. An assay card 1 with the reagent 4 deposited on the lateral surface 7 of the well 2 has several advantages. First of all, it allows a better control of the position of the reagent 4 in each well 2. In particular, the reagent 4 is precisely located on the lateral surface 7 of the well 2 compared to a reagent deposited in the bottom surface of the well 2, which has a certain tendency to spread due to the absence of an angular wall that allows the droplet of the reagent to be held in place by capillary action. Thus, for example, by positioning the reagent 4 on the lateral surface 7 in a location opposite to the direction of propagation of an air bubble, the reagent 4 can be positioned in such a way as to ensure an interaction between the reagent 4 and the biological sample even in the presence of an air bubble. Furthermore, this better control of the position of reagent 4 means that there is better interaction between the biological sample and reagent 4, since reagent 4 is concentrated on a portion of lateral surface 7 of well 2. Furthermore, because the position of reagent 4 on lateral surface 7 of well 2 is known, it is possible to know whether the biological sample has come into contact with reagent 4 by ascertaining where in well 2 the biological sample was positioned.

[0037] Finally, in the case where several reagents 4 are present in each well 2, the reagents 4 are deposited on the lateral surfaces 7 of the wells 2, making it possible to avoid mixing between them. In particular, if the reagents 4 in the same well are deposited on the bottom surface of the well 2, they may spread out during the deposition operation or during the drying period before complete dehydration until they come into contact, whereas deposition on the lateral surfaces 7 of the wells 2 prevents this. First of all, the length of the lateral surfaces 7 of the wells 2 is much larger than the diameter (longest length) of the bottom surface of the well 2, thereby making it possible to space the reagents much farther apart from each other. This aspect becomes all the more important when the reagents 4 are generally deposited in the form of droplets before being dried, and these droplets tend to spread out, with the risk of mixing if they are too close. The separation of the reagents becomes all the more effective when two reagents 4 are deposited on two different walls 8 of the same lateral surface. Take for example a well 2 whose lateral surface 7 is made of two communicating circular walls 8, a first wall 8 and a second wall 8, such that the internal space 6 has the shape of the number 8. If a first reagent 4 is deposited in the form of droplets (before drying) on ​​the first wall 8 and a second reagent 4 is deposited in the form of droplets (before drying) on ​​the second wall 8, these droplets of reagent 4 will be separated by the edge separating the two walls 8 and will not mix. Conversely, if the reagents are placed on the bottom surface of the well 2, they will be closer without being separated by an edge and will therefore possibly mix.

[0038] The fact that the reagents 4 are deposited on the lateral surfaces 7 of the wells 2 also means that each well 2 of the plate 3 does not need a bottom surface when the reagents 4 are deposited in each well 2 of the assay card 1. The absence of a bottom surface firstly removes major constraints regarding the depth of the assay card 1 and its respective positioning in terms of the equipment used for deposition. The absence of a bottom surface also facilitates the quality control of the plate 3 filled with the reagents 4 by the shadowgraphic method described below, since there is no layer preventing the light beam from passing through the empty space of the inner space 6 of the well 2. The absence of a bottom surface on the plate 3 of the assay card 1 also allows a reduction in costs, since the assay card 1 can be inserted directly between two transparent films. Advantageously, in order to protect the reagents 4 in the wells 2, after the deposition of the reagents 4 in the wells 2 and their drying therein, each side 3a, 3b of the plate 3 of the assay card 1 is covered on each side by a transparent film or inserted into a consumable that is already equipped with two films. In conclusion, the presence of reagents 4 on the lateral surfaces 7 of the wells 2 greatly simplifies the method for producing the assay card 1 and the cost of the assay card 1 .

[0039] In an alternative embodiment, not shown, it is possible to deposit the reagents on the lateral surfaces of the wells, even if the plate comprises a bottom surface. In this case, the instrument used for the deposition is partially inserted into the well 2 without touching the bottom surface, and the reagents are deposited only on the lateral surfaces of the wells, i.e. depending on the viscosity of the deposit, the reagents may touch the bottom surface of the plate, but only to a negligible extent. Advantageously, the bottom surface of the plate may be opaque or transparent. When the bottom surface is opaque, the emission analysis is carried out on the side of the assay card opposite to the side on which the bottom surface of the plate is located. The rest of the features described above with respect to the plate or assay card remain unchanged in this alternative embodiment, only the feature that the plate comprises a bottom surface differs from the remaining embodiments described, the deposition method being the same.

[0040] According to a particular embodiment, each well 2 has several different reagents 4 deposited only on its lateral surface 7, said different reagents 4 including a first reagent 4a, 4b, 4c and a second reagent 4a, 4b, 4c. The reagents 4 did not come into contact and therefore did not mix when they were in liquid form before being dried. To avoid unintentionally triggering a reaction between the different reagents before the use of the consumable with this plate 3, the reagents 4 are dried to ensure that they do not mix. The reagents 4 in the wells 2 must not be mixed so that they do not react together before the biological sample is brought to the wells 2 via the supply channel 5. In particular, to analyze the biological sample, it may be essential that a cascade reaction is performed and that the reagents 4 present in each well 2 do not react with each other beforehand. For example, in the case of endotoxin detection, the well 2 may contain three different reagents 4a, 4b, 4c: a detection agent 4a that is inactive unless there is endotoxin-free activation, an activating agent 4b that comprises an enzyme and a fluorogenic substrate for activating the detection agent, and a control reagent 4c that is adapted to control the functionality of the detection reagent. In this example, the detection agent and the activating agent must not react with each other prior to the introduction of the liquid biological sample via the supply channel 5. Thus, the reagents 4a, 4b, 4c are only brought into contact when the liquid biological sample is introduced into the well 2.

[0041] The geometry of the well 2 is adapted to avoid mixing of the reagents 4a, 4b, and 4c before the introduction of the reference liquid. Firstly, when the lateral surface 7 of the well 2 comprises a single wall 8, the well 2 can be wide enough to allow a separation space between each of the reagents 4 deposited on the wall 8, and therefore the lateral surface 7 can be long enough to allow a separation space between each of the reagents 4 deposited on the wall 8. For example, in FIG. 2 depicting a well 2 whose lateral surface 7 comprises a single wall 8, the well 2 is wide enough to leave a space between the reagents 4a, 4b, 4c on the lateral surface 7, thereby ensuring the separation of the reagents 4a, 4b, 4c. Furthermore, the lateral surface 7 of the well 2 can have a shape that allows a separation between each of the reagents 4a, 4b, 4c by including a discontinuity, such as an edge between the two reagents 4a, 4b, 4c. The lateral surface 7 can comprise several walls 8 as explained above, with discontinuities between the walls 8 of the same lateral surface 7 allowing for separation spaces between the reagents 4a, 4b, 4c and also boundaries between them. It is possible to envisage a well 2 with an internal space 6 with three circles, one of which communicates with the other two. The lateral surface 7 comprises three continuous circular walls 8 across the lateral surface 7 and a discontinuity when going from one circle to another and thus from one wall 8 to another. Each reagent 4a, 4b, 4c can be placed on one of the three walls 8 of the lateral surface 7, respectively, such that each reagent 4a, 4b, 4c is placed on a different wall 8 and thus inside a separate part of the internal space 6 constituted by a circle. The reagents 4a, 4b, 4c are thus separated from one another within the same well 2.

[0042] According to a preferred embodiment, as shown on the plate 3 portion of Fig. 3, each well 2 comprises a number of lobes 9, e.g. three lobes 9a, 9b, 9c, and a number of junctions 10, e.g. two junctions 10a, 10b connecting the lobes 9a, 9b, 9c. In other words, each well 2 comprises several distinct locations, referred to as lobes 9, which communicate with each other via the junctions 10. In this embodiment, the lateral surface 7 comprises several walls 8a, 8b, 8c, 8d, 8e, 8f, 8g, each wall 8 associated with a lobe 9a, 9b, 9c or a junction 10a, 10b, 10c.

[0043] Preferably, each lobe 9 comprises 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 shown in FIG. 3, a first reagent 4a and a second reagent 4b. The first reagent 4a is deposited on the wall 8a of the first lobe 9a of said well 2, and the second reagent 4b is deposited on the wall 8b of the second lobe 9b of said well 2. In the example shown in FIG. 3, a third reagent 4c is deposited on the wall 8c of the third lobe 9c. In an embodiment suitable 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 the first lobe 9a carrying the detection agent 4a, the wall 8b of the second lobe 9b carrying the activating agent 4b and the wall 8c of the third lobe 9c carrying the control reagent 4c. Advantageously, the reagents 4a, 4b, 4c are deposited only on the walls 8a, 8b, 8c of the lobes 9, and there is no reagent 4 on the walls 8d to 8g of the junction 10, since if there is a reagent 4 on the walls 8d to 8g of the junction 10, this may mean that it will come into contact with another reagent 4 placed in the lobes 9, which is undesirable. Moreover, as mentioned, after deposition the reagent 4 is preferably dry and not in liquid form.

[0044] Preferably, the lobes 9 of each well 2 have an elliptical shape and the junctions 10 of each well 2 are straight in the direction of the junctions. Even more preferably, the lobes 9 of each well 2 have a circular shape. Thus, as shown, the walls 8a, 8b, 8c of the lobes 9a, 9b, 9c are generally circular or curved, while the walls 8d, 8e, 8f, 8g of the junctions 10a, 10b, 10c are flat.

[0045] The assay 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 junction 10 is preferably less than 1 mm and preferably greater than 0.05 mm. The length of the junction 10 is preferably less than 5 mm and preferably greater than 0.05 mm. These dimensions are large enough to allow circulation of a liquid, such as a biological sample, but small enough that the surface tension effects of the liquid allow the droplets to be contained outside the junction 10, which also facilitates mixing of the reagents 4 after the sample is introduced and captures the dried reagents 4, triggering the assay reaction.

[0046] As explained, this structure consisting of lobes 9 and joints 10 makes it possible to isolate the different reagents 4 and prevent them from mixing before drying. Nevertheless, with the aim of preventing mixing of the reagents 4 as much as possible, the lobes 9 of each well 2 are preferably arranged in a specific way relative to one another in order to optimize the imaged surface area. An analytical direction is imposed on the analysis card 1 during the analysis of a biological sample with an in vitro diagnostic device 12. With reference to FIG. 4, the analytical direction corresponds to the vertical direction of the analysis card 1, in which the analysis card 1 is placed when it is analyzed with an in vitro diagnostic device 12 comprising an imager 13, typically a fluorometer, that defines a field of view 14. However, this direction, called vertical, is merely indicative and is used as an example, and therefore there is no technical connection between the card orientation and the analysis itself. When the analysis card 1 is analyzed, it is placed in this analytical direction in the in vitro diagnostic device 12, in 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 having a wavelength capable of causing fluorescence to appear, i.e., causing the emission of fluorescent light after excitation of a fluorophore.

[0047] The analytical direction of the assay card 1 preferably corresponds to the direction in which the assay card 1 is placed in FIG. 4. The axis y corresponds to the vertical direction and the axis x corresponds to the horizontal direction. FIG. 4 illustrates an assay card 1 with a number of wells 2 with several lobes 9, in this case three lobes 9, according to a particular embodiment. In this case, the analytical direction of the assay card 1 is the direction in which the axis of each cylinder defined by each lobe 9 is perpendicular to the vertical direction. Thus, returning to the relative positioning of the lobes 9 of one and the same well 2, when the assay card 1 is placed in the analytical direction, the angle between the vertical direction and the joining direction of the joint 10 between the two lobes 9 of one well 2 is preferably greater than 10° and preferably less than 180° (or 0°). The vertical direction and the angle between the vertical direction and the direction of the joint 10 are shown in FIG. 3 by dotted lines. Moreover, this is made possible by the fact that the reagents 4 are deposited on the lateral surface 7 of each well 2. Indeed, returning to the example of endotoxin detection, the reagents 4 used have a high wettability (contact angle between 75° and 90°). As a result, if droplets of reagents 4 are deposited on the bottom surface of the lobes 9 of a well 2, they are very likely to move and spread in such a way that they mix with each other before drying, which is undesirable. The structure imposed by the different lobes 9 of one well 2, and the fact that the reagents are deposited on the lateral surfaces 7 of each well 2, therefore guarantees as best as possible the isolation of the reagents 4 deposited in the different lobes 9 from each other.

[0048] To allow a coherent cascade reaction and to prevent mixing between the various reagents 4 in the wells 2, each junction 10 connects only two lobes 9 of one well 2, and all lobes 9 and junctions 10 of one well 2 form an open chain. By coherent cascade reaction we mean a reaction in which the order in which the sub-reactions of the reaction are carried out corresponds to the optimal order to obtain a usable result. In other words, in the context of a reaction involving several sub-reactions and therefore several reagents 4, it may be essential that a certain reagent 4 reacts with a biological sample before reacting with another reagent 4. In the example of endotoxin detection, it is preferred that the biological sample is contacted with an activating agent 4b before it is contacted with a detecting agent 4a. The open chain formed by all lobes 9 and junctions 10 of one well 2 therefore makes it possible to control the order in which the sub-reactions of the cascade reaction are carried out. The term "open chain" describing all lobes 9 and junctions 10 means that two lobes 9 of all 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 two ends of the chain. Obviously, the lobe 9 at one end of these lobes 9, in addition to being connected to a single junction 10, can also be connected to a supply channel 5.

[0049] Analysis card production method The invention also relates to a method for producing an assay card 1. This method is presented in Figure 5. In step a), a plate 3 is provided, preferably having a number of wells 2 passing through it. By "provided" we mean that the production method requires that a plate 3 with a number of wells 2, preferably free of any reagents 4, is available.

[0050] The method may also include a preliminary step 0) of plastic injection moulding the plate 3 .

[0051] In step b), the plate 3 is placed in an analysis direction, which in this example corresponds to the direction in which the faces 3a, 3b of the plate 3 are perpendicular.

[0052] Then, in step c), a droplet of the reagent 4 is deposited in contact with the lateral surface 7 of the well 2. This step c) is depicted in Fig. 6a to 6d, which show the simultaneous deposition of two droplets. Advantageously, each droplet is deposited using a needle 11. Preferably, the step c) of depositing the droplets of the reagent 4 comprises three sub-steps c1), c2), c3), sub-step c1) being the placement of the needle 11, one end 11a of the needle being placed just inside the interior space 6 of the well 2, as shown in Fig. 6a. Preferably, the end 11a of the needle does not protrude beyond the interior space 6. Thus, the end 11a of the needle preferably penetrates only one of the two faces 3a, 3b of the plate 3, and is therefore preferably located between the first face 3a and the second face 3b. In Fig. 6a, for example, the end 11a of the needle penetrates only the first face 3a.

[0053] Substep c2) corresponds to the formation of a droplet of the reagent 4 at the end 11a of the needle by supplying the channel 11b of the needle with the reagent 4 in liquid form. As shown in FIG. 6b, a droplet forms and grows until it comes into contact with the wall 8 of the lateral surface 7 of the well 2. The position of the needle 11, and more specifically the distance of the end 11a of the needle from the lateral surface 7, defines the size of the droplet when it touches the wall. The end 11a of the needle is preferably offset relative to the center of the interior space 6 and is thus closer to the part of the lateral surface 7 on which it is desired to deposit the droplet of the reagent 4, thus allowing the droplets to be collected and held in a lobe as soon as they form at the end 11a of the needle 11.

[0054] Each droplet of reagent 4 formed usually has a diameter greater than 0.8 mm, more preferably greater than 1.2 mm. The diameter of each droplet of reagent 4 is, for example, approximately equal to 1 mm. After each droplet has come into contact with the wall 8 of the lateral surface 7 of the well 2, it is deposited on the wall 8, as shown in FIG. 6c. Each deposited droplet of reagent 4 preferably has a volume of less than 1 μL, more preferably less than 0.65 μL. Preferably, the volume of each deposited droplet of reagent 4 is greater than 0.1 μL, more preferably greater than 0.35 μL. For example, each droplet of reagent 4 has a volume approximately equal to 0.5 μL.

[0055] After each droplet has been deposited, each needle 11 or plate support is withdrawn out of the interior space 6 of the well 2 according to sub-step c3), as shown in Fig. 6d. Then, referring again to Fig. 5, in step d), each droplet of reagent 4 is dried so as to obtain a dried reagent 4 on the wall 8 of the lateral surface 7 of the well 2. It will of course be understood that the droplet depositing step c) and the drying step d) are carried out simultaneously or sequentially for a number of wells 2 of the plate 3, preferably for all the wells 2 of the plate 3.

[0056] In a particular embodiment, in which the wells 2 accordingly comprise several lobes 9, each lobe 9 of each well 2 comprises at least one droplet of the reagent 4, preferably a single droplet of the reagent 4. Preferably, the droplet depositing step c) and the drying step d) are performed simultaneously for each lobe 9 of several wells 2 of the plate 3, preferably for each lobe 9 of each well 2 of the plate 3. This droplet depositing mode therefore allows several droplets to be deposited simultaneously in a microplate, in other words in the plate 3. In particular, by using a support comprising several needles 11 and bringing this support to the plate 3 so that its end 11a of each needle 11 is within the interior space 6 of a well 2 of the plate 3, it is possible to deposit droplets of the reagent 4 simultaneously on the walls 8 of the lateral surfaces 7 of several wells 2. Moreover, this method allows several droplets to be deposited simultaneously on the walls 8 of each lobe 9 of each well 2 of the plate 3, without the droplets mixing with each other in the same well 2. As a result, tens or even hundreds of droplets of reagent 4 in amounts on the order of microliters can be deposited in a short time without human intervention. Taking as an example an assay card 1 with 35 wells 2 having three lobes 9 as shown in FIG. 4, the claimed production method may allow droplets of reagent 4 to be deposited simultaneously on the walls 8 of each lobe 9, thus allowing 105 droplets of reagent 4 to be deposited simultaneously in the plate 3 of the assay card 1.

[0057] Preferably, the end 11a of the needle used for deposition is covered with a non-stick coating. For example, this coating can be a hydrophobic coating based on Teflon, such as polytetrafluoroethylene (PTFE). In this way, the droplets forming on the end 11a of the needle do not deform or flow along the needle 11, but on the contrary maintain a circular shape that allows a good control of the amount deposited in the plate 3. Furthermore, in the case where several needles 11 are arranged next to each other in the support, the covering with a non-stick coating makes it possible to avoid the droplets coming into contact and mixing, as shown in Figures 6a to 6d. In addition, the use of this coating makes the solution more universal, allowing the deposition of liquids with very different surface tensions.

[0058] Preferably, the production method comprises a step e) of inserting the plate 3 between two films, preferably transparent films, and gluing the films to the plate 3 in such a way as to protect the reagents 4 and to close the fluidic network of the wells 2. The films form the edges of the wells 2 which are closed and which are free of the reagents 4.

[0059] Quality Control Methods To ensure that the plate 3 is correctly provided with the reagents 4, i.e. that each well 2 contains a droplet of the reagent 4 and, in the case where the well 2 comprises several lobes 9, that the droplets present in the different lobes 9 of the same well 2 are not mixed, a method is proposed for quality controlling the plate 3 before drying the reagents 4 (substep c3). More specifically, a method is proposed for quality controlling a plate 3 comprising a number of wells 2 passing through it, each well 2 having a number of liquid reagents 4 separately deposited on its walls 8. The method is illustrated in FIG. 7.

[0060] In step Q1) an image of the plate 3 whose wells 2 have a plurality of droplets of a liquid reagent 4 is acquired as shown in FIG. 9 or FIG. 11. Preferably, the quality control method comprises a preliminary step Q0) of acquiring an image of an empty plate 3 as shown in FIG. 10. In other words, step Q0) consists in acquiring an image of the plate 3 when no reagent 4 is deposited on the walls 8 of the wells 2 of the plate 3. Advantageously, the images acquired in steps Q1) and Q0) of the quality control method are shadowgraphic images. With reference to FIG. 8, the shadowgraphic images can be acquired using a control system 16 comprising a light source 17, for example 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 direction (usually with the faces 3a, 3b perpendicular) between the light source 17 and the image acquisition device 18. A coaxial ray of light is emitted by the light source 17 in the direction of the plate 3. These light rays pass through the internal space 6 of the wells 2 of the plate 3 or are absorbed by the faces 3a, 3b of the plate 3. The light rays passing through the internal space 6 of the wells 2 are deflected by the liquid reagent 4 present in the wells 2 or pass undeflected through the empty parts of the internal space 6 if there is no liquid reagent 4 on their path. 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, i.e. only the light rays that have passed through the empty parts of the internal space 6. By this control system 16, a black and white image of the plate 3 is obtained. The wells 2 are recognizable by those parts of the empty internal space 6 that are white in the image. Still referring to FIG. 8, the control system 16 can include mirrors 20, 21, which allows further miniaturization. For example, the control system 16 can include two mirrors 20, 21, namely a fully reflective mirror 21 and a semi-reflective mirror 20. The light beam emitted by the light source 17 passes through a semi-reflecting mirror 20 and then through the plate 3 positioned in its analysis direction.The undeflected or absorbed light rays reach a fully reflective mirror 21, which reflects them. They pass through the plate 3 again and finally reach a semi-reflective mirror 20, which reflects them towards the image capture device 18.

[0061] Furthermore, the quality control method preferably comprises a step Q1') of recognizing by an algorithm for detecting the wells 2 of the plate 3 and identifying for each well 2 of the plate 3 an area of ​​interest. Detection of the wells 2 means that the wells 2 of the plate 3 are precisely located in the acquired image. In the case where each well 2 comprises a lobe 9 and a junction 10, the detection may involve precisely locating the lobes 9 and the junction 10. The detection algorithm of the wells 2 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 the 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 may be used, as shown in FIG. 12a.

[0062] Next, the 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 the region of interest can for example correspond to the identification of a junction 10 as shown in FIG. 12b. In particular, in the case where it is desired to determine that the reagents 4 present in the different lobes 9 of one well 2 are not mixed, it can be verified whether the junction 10 is completely free of reagent. In this case, the junction 10 therefore constitutes the region of interest. The region of interest can also be a lobe 9, in order to be particularly sure of the presence of a reagent. It goes without saying that several approaches can be combined.

[0063] The quality control method therefore comprises a step Q2) of verifying whether liquid is absent between each deposit of liquid reagent 4. As explained, this may for example correspond to verifying whether there is no presence of reagent 4 in the junction 10 of the well 2. Preferably, this step Q2) comprises comparing the pixels of the image acquired in step Q1) with the pixels of the image acquired in step Q0). It is also possible to carry out a comparison for groups of pixels. This comparison makes it possible to determine where in the well 2 the reagent 4 is present and, consequently, to identify an absence of reagent 4, an excess of reagent 4 or an undesired presence of reagent 4 in a particular part of the well 2. Preferably, comparing the pixels means comparing their light intensity values, more preferably comparing their grey levels. Moreover, preferably, each compared pixel of the images acquired in step Q1) is compared with a pixel located at the same location in the image acquired in step Q0). Advantageously, only the pixels corresponding to the region of interest of each well are taken into account in the context of this comparison. Specifically, the region of interest of a well 2 of a plate 3 is the exact location where it is desired to detect the presence or absence of a reagent 4. For example, the region of interest may be a junction 10 of a well 2, and it may be desired to detect whether a reagent 4 is present in the junction 10. The presence of a reagent 4 in the junction 10 probably means that the reagents 4 from two different lobes 9 of one well 2 have mixed. Furthermore, this comparison step Q2) can be likened to a subtraction of two images. If a pixel of the image of the plate 3 injected with a reagent 4, acquired in step Q1), has a different light intensity value than the same pixel of the image of the empty plate 3, acquired in step Q0), this may mean that a reagent 4 is present in the plate 3 at the position corresponding to said pixel, as shown in FIG. 13 for the region of interest at the lobe 9, and in FIG. 14 for the region of interest at the junction 10. A threshold value for the difference in the light intensity values ​​of the pixels, as well as a threshold value for the difference in the number of pixels whose light intensity values ​​differ between the images acquired in steps Q1) and Q0), can be defined.Thus, starting from a certain number of pixels whose light intensity values ​​differ from a certain difference in the light intensity values ​​of the pixels between the two images, it can be determined that reagent 4 is present at the location corresponding to said pixels in plate 3. Based on the number of pixels identified as corresponding to the space injected with reagent 4, the surface area of ​​the pixels corresponding to the space injected with reagent 4 can be extracted. This surface area can be used to extrapolate to obtain an estimate of the amount of reagent 4 present in well 2 of plate 3.

[0064] The invention is not limited to the embodiments described and shown in the attached drawings: modifications are possible, in particular in terms of the nature of the various technical features or in terms of the substitution of technical equivalents, but without departing from the scope of protection of the invention.

Claims

1. 1. An assay card (1) for analyzing biological samples using an in vitro diagnostic device, comprising a plurality of wells (2) formed in a plate (3), each well containing at least one reagent (4), said assay card (1) comprising a supply channel (5) for supplying a liquid sample to said wells (2), Each well (2) forms an internal space (6) in the plate (3) defined by a lateral surface (7), the lateral surface (7) having at least one wall (8), and the reagent (4) of a well is deposited and dried only on the lateral surface (7) of the well (2), each well (2) comprising a plurality of lobes (9) and a plurality of junctions (10) connecting said lobes (9); Each well (2) has several different reagents (4) deposited only on its lateral surface (7), said different reagents (4) including a first reagent (4a) and a second reagent (4b), and the well (2) contains at least the first reagent (4a) deposited on a wall (8a) of a first lobe (9a) of the well (2) and the second reagent (4b) deposited on a wall (8b) of a second lobe (9b) of the well (2). Analysis card (1).

2. 2. An assay card (1) according to claim 1, wherein each well (2) penetrates the plate (3) from one side (3a) to another side (3b) of the plate (3).

3. 2. An assay card (1) according to claim 1, wherein each well (2) has several different reagents (4) deposited preferably only on its lateral surface (7), said different reagents (4) including a first reagent (4a) and a second reagent (4b).

4. 4. The assay card (1) of claim 3, wherein the second reagent (4b) is an activator for activating the first reagent (4a), and when the activated first reagent (4a) is brought into contact with the liquid sample, the activated first reagent (4a) is capable of reacting with the liquid sample.

5. 2. An assay card (1) according to claim 1, wherein the lobes (9) of the wells (2) have an elliptical shape and the joints (10) of the wells (2) are linear in the direction of the joints.

6. 2. The assay card (1) of claim 1, wherein one junction (10) connects only two lobes (9) of one well (2), and all the lobes (9) and junctions (10) of one well (2) form an open chain.

7. 2. The assay card (1) according to claim 1, wherein the assay card (1) is associated with an assay direction imposed on the assay card (1) during the analysis of the biological sample using the in vitro diagnostic device, the assay direction being characterized in that the faces (3a, 3b) of the plate (3) extend in a predetermined direction, preferably vertically, and a first lobe (9a) of a well (2) is connected to a second lobe (9b) of the well (2) by a joint (10) in a joint direction, the angle between the predetermined direction and the joint direction being preferably greater than 10°.

8. 2. The assay card (1) of claim 1, wherein the diameter of the lobe (9) of the well (2) is greater than 0.1 mm, the width of the junction (10) is less than 1 mm, and the length of the junction (10) is greater than 0.05 mm.

9. 2. An assay card (1) according to claim 1, wherein each face of the plate (3) is covered with a transparent film at least on the face intended to allow the assay of the assay card.

10. 2. The assay card (1) according to claim 1, wherein the assay reagent (4) is adapted to produce a luminescent reaction in the presence of endotoxin.

11. A method for producing an assay card (1) according to any one of claims 1 to 10, comprising: a) providing a plate (3) comprising 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 a predetermined direction; c) depositing at least one droplet of a reagent liquid (4) in contact with the wall (8) of said lateral surface (7) of said well (2); d) drying said at least one droplet of reagent (4) to obtain a reagent (4) deposited on said walls (8) of said lateral surfaces (7) of said wells (2). A method comprising:

12. 12. The method according to claim 11, comprising step e) of inserting said plate (3) between two films and gluing said films to said plate (3).

13. The step c) of depositing said droplets of reagent (4) comprises: c1) placing a needle with one end (11a) of said needle in said interior space (6) of the well (2); c2) forming a drop at the end (11a) of the needle until it comes into contact with the wall (8) of the lateral surface (7); c3) withdrawing the needle (11) The method of claim 11 , comprising:

14. 1. A method for quality control of a plate (3) comprising a plurality of wells (2), each well (2) forming an interior space (6) in said plate (3) defined by a lateral surface (7), the wells (2) having a plurality of deposits of liquid reagents (4) separately deposited on said lateral surfaces (7), comprising: Q1) acquiring an image of said plate (3); Q2) Verifying the absence of liquid between the deposits of liquid reagent (4) A method comprising:

15. 15. A quality control method according to claim 14, comprising a step Q0) of acquiring an image of the empty plate (3), and a step Q2) of comparing the pixels of the image acquired in step Q1) with the pixels of the image acquired in step Q0).

16. 16. A quality control method according to claim 14 or 15, wherein the image of the plate is a shadowgraphic image.

17. 16. A quality control method according to claim 14 or 15, comprising a step a1) of recognizing by an algorithm the wells (2) of the plate (3) for detection and identifying for each well (2) of the plate (3) an area of ​​interest in which the absence of liquid is verified.

18. 18. The quality control method of claim 17, wherein each well (2) comprises a plurality of lobes (9) and a plurality of junctions (10) connecting the lobes (9), the lobes being for receiving the deposit of liquid reagent (4), the region of interest including the junctions (10), and the absence of liquid in the junctions (10) is verified.

Images