1 Test sample card filled in combination with at least a buffer supply DESCRIPTION 5 The invention concerns a test sample card including at least an opening for the introduction of a fluid sample into the card, a receiving compartment which can receive (directly or indirectly) all or part of the introduced sample, and a primary transfer channel for bringing said sample from the input opening to the receiving compartment (direct transfer), or from an intermediate compartment to said receiving compartment 10 (indirect transfer). More specifically, the invention relates to means for filling each compartment more effectively. Document US-A-5,230,866 describes a card which substantially meets the technicalfield of the applicant's invention but in which the transfer of the sample into the receiving compartment is performed indirectly since there is always a compartment 15 between the input opening and the relevant receiving compartment. Only one feature of this invention can be interpreted as a way of enhancing filling efficiency (like the use of a buffer supply according to our invention), namely the wide internal chamber (reference 102). However, this compartment is linked to the exterior via a capillary channel and an opening. Moreover, the point of intersection between the channel which 20 links the wide internal chamber compartment and said wide internal chamber does not meet the specifications of the applicant's invention. The applicant's invention includes at least one opening for introduction of the sample. The exterior surface can be provided with an optional opening which can be optionally closed by means of a valve. 25 In other documents, e.g. US-A-5,288,463, the contents of a compartment is connected to the exterior through a simple vent. This therefore does not describe a true buffer supply inside the card with the advantages of this invention when it comes to automatically filling several compartments with equal volumes of fluid. 30 2 The background art is given in patents US-A-5,746,980, US-A-5,766,553 and US-A-5,843,380 granted to the applicant. These documents describe an improvement for lest sample cards. Thus, the test compartments of this card are associated with a bubble trap which consists in a small receptacle linked to the compartment via a 5 channel. However, the volume of this bubble trap is much smaller than that of the compartment with which it is associated. Therefore, the role of the bubble trap is not to enhance filling of said compartment but to prevent bubbles in the compartment well and thereby improve optical performance when results are being read. Moreover, any 10 analytes sequestered in bubbles burst in said trap would not participate in the reaction which is supposed to take place in the compartment and this may compromise the accuracy of the result. In accordance with this invention, at least one of the compartments is associated 15 with a receptacle which is not a bubble trap since it does not contain any liquid. On the other hand, it increases the overall volume of the compartment in order to enhance filling efficiency. Of course, it is quite feasible to incorporate a bubble trap (as defined above) between the compartment and the receptacle. 20 To this effect, this invention concerns a test sample card comprising, at least: - an opening for the introduction of a fluid sample into the card, - a receiving compartment which can receive (directly or indirectly) all or part of the introduced sample, and - a primary transfer channel for bringing said sample from the input opening to the 25 receiving compartment (direct transfer), or from an intermediate compartment to said receiving compartment (indirect transfer), characterized in that each intermediate or receiving compartment is connected to a buffer supply located inside the card which is so arranged and configured as to prevent the card from being filled with the introduced sample. 30 Preferably, there is a channel between the compartment and the buffer supply.
3 According to an alternate embodiment, the card is positioned vertically or on a slope during use so that, during transfer of the fluid sample, the point of intersection between the compartment and the channel is positioned in the upper part, and preferably at the uppermost point of this compartment. 5 According to another complementary embodiment, the card is positioned vertically during use so that, during transfer of the fluid sample, the point of intersection between the compartment and the channel is positioned in the upper part, and preferably at the uppermost point of this buffer supply. In the case in which the card is substantially flat and comprises two 10 substantially parallel surfaces, the compartment is located on one side of the card and the buffer supply on the opposite side, said compartment and said buffer supply being interconnected by a transverse channel. In a particular embodiment, the capacity of the compartment and that of the buffer supply are substantially the same. 15 According to another embodiment, an apparatus which can burst any bubbles which might form in the fluid sample is included in or replaces the channel. Such an apparatus has already been described in the background art given above and in two patent applications submitted by the applicant on the same day as this 20 invention and entitled "Dispositif et proc6d6 de positionnement d'un liquide" (A device and method for positioning a liquid) and "Dispositif de pompage permettant de transf6rer au moins un fluide dans un consommable" (A pumping device for transferring at least one fluid into a consumable). The contents of the descriptions in these patent applications are considered as 25 being included in this invention. The buffer supply can be a very important feature in the pumping device dealt with in the second patent application mentioned above. Thus, when pressure is applied to the flexible film to reduce the volume of one of the compartments, it is possible to apply pressure to the buffer supply associated with that compartment at the same time. 30 Therefore, it is of particular value to have a compartment and a buffer supply at the 4 same level but on opposite sides of the card so that the two forces are additive. Of course, every receptacle has to be covered with a flexible film which may be the same as long as said film accepts said card in sandwich configuration. More specifically, the bubble-bursting apparatus consists of a chamber with a S cross-sectional area greater than that of the opening between the compartment and the chamber. In the case in which the card has two opposite sides, at least one of which is covered with flexible film, partitioning the card, the buffer supply includes at least one reinforcing strip to insure that the film does not come into contact with the bottom of 10 said buffer supply. Whatever the embodiment, the card includes at least: - two receiving compartments which can receive, directly or indirectly, part of the introduced sample, and - two primary transfer channels to bring said sample from the input opening to the 15 receiving compartments (direct transfer) or from at least one intermediate compartment to said receiving compartments (indirect transfer), each primary transfer channel only functioning with one receiving compartment and the whole unit, together with its associated buffer supply, constituting one reaction line. Said card is characterized in that the arrangement and configuration of the reaction lines 20 lead to a pressure drop similar to the one that takes place in each other, parallel reaction lines so that each compartment is filled with exactly the same volume. According to a first embodiment, the buffer supplies are interconnected. According to a second embodiment, each buffer supply consists of a channel 'which includes at least one opening at the exterior surface of the card. 25 According to a third alternate embodiment, the opening is located at the free end of the channel that makes up the buffer supply. According to a fourth alternate embodiment, all the buffer supplies share a single opening. According to a fifth alternate embodiment, the opening is fitted with a valve. 30 According to a particular embodiment, the card comprises at least: 5 - two receiving compartments which can receive (directly or indirectly) part of the introduced sample, and - two primary transfer channels to bring said sample from the input opening to the receiving compartments (direct transfer) or from at least one intermediate compartment 5 to said receiving compartments (indirect transfer), each primary transfer channel only functioning with one receiving compartment and the whole unit, together with its associated buffer supply, constituting one reaction line. Said card is characterized in that all the reaction lines share a single buffer supply. According to a another alternate embodiment, one buffer supply is associated 10 with each intermediate compartment and/or receiving compartment. Such a card can be used for the analysis of one or more liquid samples to identify one or more analytes, using any method, be it a simple or complex method and be it based on one or more types of reagent, depending on the chemical, physical or biological nature of the analyte being tested. The technical principles defined hereafter 15 are not restricted to any single, specific analyte; the only required condition being that the analyte must either be dissolved or in suspension in the test sample. In particular, the test process being used can be performed on a homogenous, heterogeneous or mixed forn. One particular, non restricted mode of such a device, concerns biological tests 20 for the detection and/or quantitative determination of one or more ligands, in which the assay involves one or more anti-ligands. The word ligand is taken to mean any biological species, e.g. an antigen, a fragment of an antigen, a hapten, a nucleic acid, a fragment of nucleic acid, a hormone or a vitamin. One example of an application of the test methods concerns immunoassays, 25 whatever their particulars and whether the assay is direct or based on competition. Another example of an application concerns the detection and/or quantitative determination of nucleic acids, including all operations required for such detection and/or quantitation in any kind of sample containing the target nucleic acid species. Among such diverse operations, the following could be specified: lysis, melting, 30 concentration, enzyme-mediated nucleic acid amplification, and detection modalities 6 which include a hybridization step using, for example, a DNA chip or a labeled probe. Patent application WO-A-97/02357 stipulates the various stages involved in the case of nucleic acid analysis. 5 The figures herewith are given by way of example and are not to be taken as in any way limiting. They are intended to make the invention easier to understand. Figure 1 shows one of the sides of a test sample card according to this invention. Figure 2 shows the other side of this test sample card. Figure 3 shows a cross-section through A-A in Figures 1 and 2. 10 Finally, Figure 4 shows the other side of a test sample card according to this invention, but in a second embodiment. This invention concerns a device to aid the filling of a test sample card (1), more particularly of compartment 3, as shown in the Figures. 15 All the Figures correspond to a particular embodiment in which a test sample card (1) contains five receiving compartments (3). Of course, this number of compartments (3) is in no way limiting-there might be just one compartment (3) or a very large number. In terms of production, the card is manufactured by the machining of special 20 plastic material, e.g. impact polystyrene (reference: R540E from the Goodfellow company) which is compatible with the liquids being processed. For industrial-scale production, the card could be manufactured by precision molding, but any other manufacturing method (including those used in the semi-conductor industry as stipulated in patent application WO-A-97/02357) may be used for test card production. 25 The essential purpose of this invention is to associate each receiving compartment (3) with at least one buffer supply (5) which increases the volume available for aspiration of the liquid sample which should be introduced via the input opening (2) or an input port. Such a port (2) is clearly shown in Figure 1. It can be seen that from this input port (2), five primary transfer channels (4) 30 bring the introduced biological sample to five different receiving compartments (3).
7 The buffer supplies (5) are not shown in Figure 1; these will be deal with later. Actually, the card (1) is usually used in a vertical position or sometimes on a slope. It can be used in a horizontal position although this is not a preferred method. In order to transfer the biological fluid from the input opening (2) to the 5 receiving compartments (3), a depression must first be created throughout the network of channels. These techniques are familiar to those skilled in the art, e.g. a vacuum might be induced in the network as described in previous patent application PCT/FR99/02082 filed by the applicant as a priority on September 1, 1998. Each receiving compartment (3) is fitted with an apparatus which bursts any 10 bubbles formed when the introduced liquid enters the compartment (3) through the opening (13). This apparatus (11) essentially consists of a chamber (12) which is separated from the main volume of the receiving compartment (3) through an opening (13), which is smaller than both the compartment (3) and the chamber (13). As a result, any bubble that forms at this opening (13) includes a quantity of liquid which is not 15 large enough to fit the increase in the cross-section of the chamber (12) . As a result, the bubble bursts inside the chamber (12). If the card is being used in a vertical position, the liquid contained in the bubble falls to the bottom of the compartment (3) when the bubble bursts. Therefore, there is no line loss and the accuracy with which the compartment (3) is filled is enhanced. This 20 is particularly relevant to biological samples which contain detergents such as Triton XI 00 (registered trademark), Tween 20 (registered trademark), SDS (Sodium Dodecyl Sulfate) and Lithium or Sodium Lauryl Sulfate, which tend to create foam. Figure 1 clearly shows that there are sharp edges around the opening (13) between the compartment (3) and the chamber (12) in order to help bubble bursting. 25 Also in said chamber, there is a channel, actually marked as 6 in Figure 3, of which the only part which can be seen is the point of intersection (7) between this channel (6) and the compartment (3), or more accurately between the channel (6) and the bubble-bursting apparatus (11). Nevertheless, it can readily be imagined that not every compartment (3) is fitted with a bubble-bursting apparatus (11) and that this point 30 o f intersection (7) is directly present in the compartment (3).
8 As explained above, when the card is used in a vertical position, the apparatus (11) is found at the top of the compartment (3). Similarly, the point of intersection (7) is found at the top of the apparatus (11). This configuration makes it possible not to fill the associated buffer supply when filling a compartment (3). Finally, the Figure shows 5 that, in the lower part of every compartment (3), there is an outlet opening, also referred to as an outlet port (19). Ports suitable for use in this invention exist already in the background art and also in patent application FR98/11383 filed by the applicant on September 8, 1998 and entitled: "Dispositif permettant des r6actions, systeme de transfer entre dispositifs et proc6d6 de mise en ouvre d'un tel systbme" (A device in 10 which reactions can be performed, a transfer system between devices and a method for implementing such a system). The contents of the description of this patent application are considered as being included in this invention The presence of the port, be it at the input (2) or the outlet (19), makes it 15 possible to control the movement of the introduced liquid sample. In a particular embodiment, when it is necessary to heat one of the compartments in order to favor some chemical or biological process (e.g. to a temperature of between 30 and 120 'C, advantageously between 40 and 95C), the presence of ports will inhibit evaporation phenomena which might affect the volume inside the compartment and thereby lead to 20 unregulated heating of the reagents or problems of cross-contamination between different compartments. In this particular embodiment which involves a heating step, another advantage of the buffer supply is that any pressure variations caused by the heating are absorbed, making it unnecessary to include a vent in the test sample card and thereby reducing the risk of contamination. For this, the buffer supply is preferably 25 thermally insulated or alternatively located at a position on the card where little heat can be transferred from its associated reaction compartment. Of course, one or more channels to bring the liquid to the input opening (2) can be imagined, as could be at least one drain channel at the outlet opening (19) as these associated channels could be integrated into said test sample card (1), and could be used 9 to send the liquid to other parts of the card or to other cards where other processes and/or reactions could be carried out. Figure 2 shows the other side of the test sample card. The point in common S between Figures 1 and 2 is the presence of the channel (6) which is represented on this side by the point of intersection (8). Point 8 is the point of intersection between the buffer supply (5) and the channel (6). This transverse channel (6) is clearly shown in Figure 3. In Figure 2, it can be seen that the volume of the buffer supply is fairly large, at least as great as that of each receiving compartment (3), even if the volume of this 10 compartment (3) is considered together with that of its associated primary transfer channel (4). In Figure 2, the shape of the buffer supply is substantially rectangular, but other shapes could be chosen without affecting the way the buffer supply works. The v!olune of buffer could range from 1 to 5000 microliters (advantageously between 5 and 2000 microliters and preferably between 10 and 1000 microliters). The volume of 15 the compartment (3) can vary substantially in the same range. By way of example, Figure 2 shows a buffer supply defined by a rectangular parallelepiped of 30 millimeters (mm) by 10 mm and with a depth of 1 mm. The reinforcing strips do not appreciably affect the volume. The cross-sectional profile of the transverse channel (6) or the transfer channel (4) may be square, rectangular, oval or circular, e.g. a circular 20 transverse channel (6) with a diameter of between 0.1 and 4 mm (advantageously between 0.3 and 2 mm) would be suitable for the buffer supply shown in Figure 2. In this configuration (and still by way of example), the size of the primary transfer channel (4) could vary in the same range. 25 When referring to Figure 3, it can be seen that the test sample card (1) is flat, i.e. that it has two sides, with the compartment (3) on one side (9) and the buffer supply (5) on the other (10). These two elements (3 and 5) are linked by the transverse channel (6). The channel is joined to the compartment (3)-more specifically, the bubble-bursting apparatus-via a point of intersection (7), and to the buffer supply (5) via another point 10 of intersection (8) (dealt with above). Both sides (9 and 10) are covered by a wall or flexible film (14) which defines the various volumes. The nature of the flexible film may vary according to the nature of the test card and of the fluids being tested, especially when compatibility is at issue. For example, 5 TPX (polymethyl pentene copolymer) or BOPP (bi-oriented polypropylene) films are suitable for biological assays. These films can be fixed in place either using an adhesive, (with the adhesive applied to the film, e.g. a silicon-based adhesive) or by heat-sealing. An example of a BOPP adhesive is available from BioMdrieux Inc. (St. Louis, MO, USA) (reference: 022004-2184). 10 Since the dimensions of the buffer supply (5) are fairly large, it may be necessary to add reinforcing strips (16) as shown in Figures 2 and 3. It should be noted that liquid is introduced into the compartment (3) via a drainage mechanism (20) which consists of a slope built into the form of the bottom of said compartment (3). This particular shape maximizes capillary action when the liquid enters said compartment 15 thereby promoting the flow of the liquid into the compartment (3). As can be seen in Figure 4, the buffer supply can be formed in various shapes. With reference to Figure 2, again there are points of intersection (8) between the buffer supply (15) and the transverse channel (6) (not shown in this Figure). 20 In the case of Figure 4, each buffer supply (15) consists of a channel of suitable length, volume and shape to be able to control the pressure drop in each compartment (3). As a result, it is possible to vary either the diameter of the channel, the number of times it turns, or finally, the length of each channel to control the pressure drop. The dimensions of the channel constituting the buffer supply (15) are defined by those 25 skilled in the art. The dimensions given above for channels 6 and 4 can be used but, advantageously, the size of part or all of the channel may be reduced in order to enhance the pressure drop, e.g. a channel, semicircular in cross-section, with a diameter of between 0.02 and 0.6 mm (preferably between 0.2 and 0.4 mm). The same way of thinking underlies the shape of the primary transfer channels 30 (4) depicted in Figure 1.
I1 In practice, a reaction chain consists of a primary transfer channel (4) associated with a receiving compartment (3) plus, in some cases, a bubble-bursting apparatus (11), a transverse channel (6) or a buffer supply (5 or 15) which may have various shapes. 5 The purpose of this invention is also to allow identical filling of each compartment (3). To achieve this, it is necessary that the shape of each primary transfer channel 4 together with the buffer supply (5) or the volume and shape of the buffer supply (15) be calculated to insure that the reduction in liquid volume is the same in every compartment (3). As a result, when the liquid is introduced via the opening (2), 10 exactly the same amount will be sent to each compartment (3). This is particularly relevant to biological applications and fluid micromanipulations on test sample cards in which problems can arise due to the retention of fluid at certain spots. Obtaining identical volumes in each compartment (3) can be guaranteed by performing precise calculations and taking into account both capillarity and pressure drop phenomena. 15 According to the embodiments shown in Figure 4, it is possible to interconnect the different buffer supplies. Thus, the free ends of the buffer supplies (15) meet at the opening (17) of the buffer supply (15) at the exterior surface where the port (18) is located. 20 According to an embodiment not shown in any of the Figures, it is also possible that the channels (6) all meet in a common buffer supply.
12 REFERENCES 1. Test sample card 5 2. Input opening or input port 3. Receiving compartment 4. Primary transfer channel 5. Buffer supply 6. Channel or transverse channel 10 7. Point of intersection between the compartment (3) and the channel (6) 8. Point of intersection between the buffer supply (5) and the channel (6) 9. Side of the card (1) with the compartment (3) 10. Side of the card (1) with the buffer supply (5) 11. Bubble-bursting apparatus 15 12. Chamber of the apparatus (11) 13. Opening between the compartment (3) and the chamber (12) 14. Flexible film 15. Buffer supply 16. Reinforcing strip 20 17. Opening of the buffer supply (15) at the exterior surface 18. Input port (17) 19. Outlet opening or port 20. Liquid drainage mechanism