System for dilution within a device and method for manufacturing this device

Abstract

The present invention relates to a system (1) for diluting a sample (Fe) of biological material, comprising a fluid circuit, characterized in that the fluid circuit comprises at least a first container (11) configured to contain a sample (Fe) of biological material containing the biological material to be diluted, said sample (Fe) being a fluid, a second container (12) configured to contain a first dilution fluid (Fd1), and at least one first metering member (16) for metering a predetermined amount of fluid, said metering member having a first wall (101) and a second wall (102) and comprising a metering part configured to change at least from an initial state in which the first wall (101) and the second wall (102) are in contact with each other to an operating state in which the first wall (101) and the second wall (102) are separated from each other.

Description

Technical Field

[0001] The present invention relates to the technical field of a system used to perform metering for precise dilution in order to carry out a biological test for quantifying endotoxin present in a sample. The present invention can also be applied to metering in immunoassay tests or the preparation of samples or reagents for subsequent analysis.

Background Art

[0002] There are numerous biological tests capable of quantifying endotoxin. Currently, the detection of endotoxin mainly involves the use of Limulus amebocyte lysate (LAL), and this LAL test is an in vitro test for quantifying the concentration of endotoxin present in a sample.

[0003] In other tests, recombinant enzyme chemistry is used to dispense with the use of horseshoe crab blood, limit the number of invalid results associated with the variability inherent in the blood extraction process, and also protect this endangered species.

[0004] These other tests include an endotoxin detection test in bacteria based on an enzyme / substrate reaction and the reading of a fluorescence signal. The advantage of this method is that it uses recombinant factor C (rFC), which is generated without using any horseshoe crab blood at all. Recombinant factor C (rFC) is used together with a synthetic fluorescence-generating substrate to detect endotoxin.

[0005] However, commercially available test kits based on this method still require numerous operations (pipetting, mixing), and quite a lot of techniques are needed, which inevitably increases the risk of human error. The collection of data and the calculations for obtaining results are also limiting factors for users seeking very specific performance.

[0006] For several years now, there has been a test developed by the applicant called ENDOZYME (R) II GO, which uses a system called "GOPLATE (TM)", which is a microplate containing 96 wells pre-filled with the required standard amounts and a positive control at the concentration of the product.

[0007] However, this system, which requires additional accessories (pipettes, vortexes, cones, etc.) and allows several samples to be analyzed "in batch", is not suitable for "online" single-sample tests, i.e., tests performed directly using samples in the laboratory and on the production line of the product to be tested. The line operators and the working environment require a very simple and rapid new test concept.

Summary of the Invention

[0008] The object of the present invention is to propose a new and compact disposable device, including an integrated, precise, reproducible, autonomous, economical, and disposable metering system (specifically capable of serving for dilution), to improve all or some of the aforementioned drawbacks, specifically by saving time by eliminating the upstream preparation of samples. The present invention can also be applied to immunoassays and molecular diagnostics, or complete quantitative tests for bacterial endotoxins, in a short time, with a sensitivity of 0.005 EU / ml, while guaranteeing the performance level and conforming to the standards imposed by the pharmacopoeia.

[0009] Towards this goal, the present invention relates to a dilution system for diluting a sample of a biological substance, including a fluid circuit, wherein the fluid circuit of the dilution system is characterized by including at least the following. - A first container configured to contain a sample of a biological substance containing the biological substance to be diluted, the sample being a fluid, the first container. - A second container configured to contain a first dilution fluid, the first container and the second container being fluidly connected by at least one fluid path, the second container. - At least a first metering member for metering a fluid of a determined volume, comprising a first wall and a second wall, configured to change at least from an initial state in which the first wall and the second wall are in contact with each other to an operating state in which the first wall and the second wall are spaced apart from each other so as to delimit a determined metering space, the metering part being configured to realize the operating state by transporting a sample and / or a diluting fluid into the metering part, and the first metering member being arranged on a fluid path connecting a first container and a second container between the first container and the second container.

[0010] Advantageously, the first metering member enables precise and rapid separation of the fluid to be diluted and / or the diluting fluid, and moreover, in a reproducible manner, the walls of the metering member move apart from each other only when the metered fluid is transported into the metering member. Intentionally, the metering part of the metering member remains in a stable position (operating state) and guarantees a reproducible volume without applying excessive pressure to the upstream container. Furthermore, if there is no air in the metering part in the initial state, and thus also in the operating state, there will necessarily be no air bubbles in the downstream dilution system, which is highly advantageous.

[0011] According to one feature of the present invention, there is no air in the metering part in the initial state. Furthermore, according to one feature of the present invention, there is no air in the metering part in the operating state.

[0012] According to one feature of the present invention, in the initial state, the first wall and the second wall of the metering member form a concave surface, such as a hemispherical concave cap, at the level of the metering part. In fact, each wall constituting the metering member is in the form of a hemispherical concave cap and is stacked in order in the same direction, thus forming a cavity.

[0013] According to one feature of the present invention, in the operating state, the space of the metering part is substantially spherical like a bubble.

[0014] According to one feature of the present invention, the dilution system is formed by a fluid circuit that integrates a container, one or more mixing chambers, one or more metering members, and one or more fluid channels that carry one or more fluids between the container, one or more mixing chambers of the channels, one or more metering members, and the reaction chamber.

[0015] According to one feature of the present invention, the fluid circuit is manufactured by laser welding, or thermal welding, or ultrasonic welding of the film that makes up the device in the form of a flexible bag.

[0016] According to one feature of the present invention, the metering section is delimited by welding the wall of the metering section at its periphery. This welding makes it possible to enclose the fluid inside the metering section and also to obtain a reproducible space.

[0017] According to one feature of the present invention, the determined volume of the metering section in the operating state is invariant and reproducible, thereby ensuring the accuracy of metering and the robustness of the dilution system.

[0018] Advantageously, the first metering member is arranged upstream of the first mixing chamber and downstream of the second container, thereby making it possible to carry the fluid that has been metered so far, such as the dilution fluid or the sample, into the first mixing chamber.

[0019] According to one feature of the present invention, the first metering member comprises at least one fluid inlet connected to the first fluid path supplying the first and second containers. Preferably, the first metering member comprises a fluid inlet directly connected to the first container and a fluid inlet directly connected to the second container.

[0020] According to one feature of the present invention, each fluid inlet of the first metering member is closed by a frangible valve so as to be sealed in the initial state of the metering part of the first metering member, and the frangible valve is preferably irreversibly opened by the pressure of the fluid between the sample carried to the first metering member or the first diluent fluid.

[0021] According to one feature of the present invention, the dilution system comprises at least a first mixing chamber configured to contain a first mixture of fluid resulting from mixing a portion of the sample and at least a portion of the first diluent fluid, and the first mixing chamber is fluidly connected to the first container and the second container.

[0022] According to one feature of the present invention, the second container contains the first diluent fluid and is configured to serve as the first mixing chamber.

[0023] According to one feature of the present invention, the first mixing chamber is fluidly connected to the first container and the second container by the same fluid path or by a fluid path different from the fluid path connecting the first container to the second container.

[0024] According to one feature of the present invention, the first mixing chamber is configured to receive a determined and metered amount of sample coming from the first container and metered by the first metering member, and a determined and metered amount of the first diluent fluid coming from the second container.

[0025] According to one feature of the present invention, the first metering member comprises at least one fluid outlet connected to the first mixing chamber. Advantageously, each fluid outlet of the first metering member is closed by a frangible valve so as to be sealed in the initial state of the metering part of the first metering member, and the frangible valve is preferably irreversibly opened by the pressure of the fluid between the sample carried to the first mixing chamber or the first diluent fluid or the first mixture of fluids.

[0026] According to one feature of the present invention, at least one fluid outlet opens directly into the first mixing chamber.

[0027] According to one feature of the present invention, the dilution system comprises a third container configured to contain a second dilution fluid.

[0028] According to one feature of the present invention, the dilution system comprises at least a second chamber for mixing the second dilution fluid and the first mixture, the second mixing chamber being fluidly connected to the first mixing chamber via a second metering member and also fluidly connected to the third container.

[0029] According to one feature of the present invention, the second container contains a first dilution fluid and is configured to serve as the second mixing chamber.

[0030] According to one feature of the present invention, the dilution system comprises a second metering member disposed upstream of the second mixing chamber, preferably between the second mixing chamber and the third container.

[0031] According to one feature of the present invention, the second metering member is identical to the first metering member in terms of its operation.

[0032] According to one feature of the present invention, the dilution system comprises a single first metering member.

[0033] According to one feature of the present invention, this single first metering member is disposed downstream of the container.

[0034] According to one feature of the present invention, the second metering member is configured to meter a first mixture of the fluid entering from the first mixing chamber and the fluid diluted by the second dilution fluid entering from the third container.

[0035] Advantageously, the second diluent fluid can be metered by a second metering member, or the volume of the second diluent fluid can be determined and metered in advance before it is injected into the device.

[0036] According to one feature of the invention, the second metering member comprises at least one fluid inlet directly or indirectly connected to a fluid path supplying a third container, and a fluid inlet directly or indirectly connected to the first mixing chamber.

[0037] According to one feature of the invention, each fluid inlet of the second metering member is closed by a frangible valve so as to be sealed in the initial state of the metering part of the second metering member, and the frangible valve is configured to be opened, preferably irreversibly, by the pressure of the fluid carried to the second metering member.

[0038] According to one feature of the invention, the second metering member comprises at least one fluid outlet opening directly into the second mixing chamber. Advantageously, each fluid outlet of the second metering member is closed by a frangible valve so as to be sealed in the initial state of the metering part of the second metering member, and the frangible valve is configured to be opened, preferably irreversibly, by the pressure of the fluid contained in the second mixing chamber.

[0039] Advantageously, in order to dilute the sample by a factor of 10, the first metering member has a volume of, for example, 10 μl, whereby 10 μl of the sample is taken from the first container and injected into the first mixing chamber. Then, 90 μl of the first diluent fluid is taken from the second container and injected into the first mixing chamber containing 10 μl of the sample.

[0040] Advantageously, to dilute the sample 100-fold, the second metering member has, for example, a volume of 10 μl, whereby 10 μl of the first mixture (10-fold dilution) obtained in advance is taken out from the first mixing chamber and injected into the second mixing chamber. Then, 90 μl of the second diluent fluid is taken out from the third container and injected into the second mixing chamber containing 10 μl of the first mixture. Thus, by diluting the sample 100-fold, a second mixture is obtained.

[0041] The maximum capacity of the first container is 500 μl, preferably 200 μl. According to the present invention, the first container contains a sample of 20 μl to 200 μl, preferably about 100 μl of the sample.

[0042] The maximum capacity of the second container is 500 μl, preferably 180 μl. According to the present invention, the second container contains a diluent fluid of 20 μl to 200 μl, even more preferably 90 μl to 180 μl, preferably about 90 μl of the diluent fluid.

[0043] The maximum capacity of the third container is 500 μl, preferably 180 μl. According to the present invention, the second container contains a diluent fluid of 20 μl to 200 μl, even more preferably 90 μl to 180 μl, preferably about 90 μl of the diluent fluid.

[0044] Preferably, and according to the present invention, the capacities of the second container and the third container are the same.

[0045] Preferably, the first diluent fluid and / or the second diluent fluid is a liquid. For example, in applications where the analyte required is endotoxin, the first diluent fluid and / or the second diluent fluid is preferably sterile water (water without endotoxin) without any trace of endotoxin, or a dilution buffer without any trace of endotoxin.

[0046] Advantageously, the second diluent fluid is the same as the first diluent fluid. Alternatively, the second diluent fluid is different from the first diluent fluid.

[0047] The present invention also relates to an apparatus in the form of a flexible bag comprising at least a first film and a second film, at least partially laminated to each other, the apparatus comprising a dilution system according to the present invention, and a reaction chamber, characterized in that the dilution system is fluidly connected to the reaction chamber.

[0048] Advantageously, with this apparatus according to the present invention, it is possible to detect endotoxins in the range of 0.005 EU / ml to 50 EU / ml in about 20 minutes by means of the dilution system and the associated reaction chamber. Furthermore, this apparatus enables the automation of the entire detection process, and thus reduces human intervention (load-and-go system) in collecting the sample to be analyzed and injecting it into the first container of the dilution system.

[0049] According to one feature of the present invention, the reaction chamber of this apparatus is preferably a plastic component.

[0050] According to one feature of the present invention, the reaction chamber comprises a plurality of wells configured to contain at least one reagent.

[0051] According to one feature of the present invention, the apparatus uses a chemical reaction based on the recombinant factor rFC for the reagent to detect whether the sample contains endotoxins. Of course, the present invention is applicable to any type of analysis that requires at least one dilution and a search by chemical reaction, and if necessary, the reagent will be adapted to the elements sought in the sample.

[0052] According to one feature of the present invention, the apparatus is configured to cooperate with a first plurality of mechanical valves arranged upstream of the fragile valve of the first metering member.

[0053] Each of the first plurality of mechanical valves is arranged at the fluid inlet or fluid outlet of the first metering member and is configured to allow / prevent fluid from entering the first metering member or to allow / prevent fluid from exiting the first metering member.

[0054] Advantageously, according to the present invention, a first mechanical valve disposed at the fluid inlet of the first metering member is connected to a mechanical valve disposed at the fluid outlet of the first metering member.

[0055] According to one feature of the present invention, the device is configured to cooperate with a second plurality of mechanical valves disposed upstream of the vulnerable valve of the second metering member.

[0056] Each of the second plurality of mechanical valves is disposed at the fluid inlet or fluid outlet of the second metering member and is configured to allow / prevent fluid from entering the second metering member or allow / prevent fluid from exiting the second metering member.

[0057] According to one feature of the present invention, the vulnerable valve of the dilution system is disposed transversely to the fluid channel so as to allow or prevent the flow of fluid in the fluid channel.

[0058] According to one feature of the present invention, the vulnerable valves are each formed in a laminate of two films.

[0059] According to one feature of the present invention, the mechanical valve is disposed transversely to the fluid channel so as to allow or prevent the flow of fluid in the fluid channel.

[0060] The present invention also relates to a measurement system including the device according to the present invention, an analytical instrument including a first plurality of mechanical valves and / or a second plurality of mechanical valves and / or a third plurality of mechanical valves, and at least one insertion area into which the device is inserted and which cooperates with each of the mechanical valves.

[0061] The present invention also relates to a method for manufacturing a device according to the present invention incorporating a dilution system according to the present invention, the manufacturing method including the following. - By welding the film of the device, a fluid circuit of the dilution system according to the present invention is produced on the film, wherein the film is at least partially pre-laminated to produce the fluid circuit. - Forming at least a first metering member, wherein (i) at least the metering part of the first metering member produced in the step of producing the fluid circuit is arranged in a mold, the mold including at least two mold parts, each having at least one mold cavity, the mold cavity of the first mold part being arranged at least partially facing the mold cavity of the second mold part and being at least partially complementary to the mold cavity of the second mold part, and (ii) by closing the two parts of the mold towards each other, two films of the device are deformed together in a single deformation direction on one side or the other side of the device at the level of the metering part by a deformation element, the deformation element being arranged between the device and the second mold part or between the device and the first mold part, to form at least the first metering member.

[0062] By this method, the metering member remains in a stable position, ensuring a determined and reproducible volume without applying excessive pressure to the original upstream container from which the fluid is conveyed to the metering member. If there is no air due to the prior lamination and the subsequent production of the fluid circuit by welding, there will necessarily be no air bubbles either in the downstream dilution system that guarantees the quality and accuracy in fluid metering and dilution.

[0063] According to one feature of the present invention, the deformation element preferably contacts the outer surface of one of the two films of the device and deforms the two films simultaneously in a single direction.

[0064] Advantageously, only the (one or more) metering members are produced by the forming step, and the other chambers or containers are produced in different ways, for example, by blowing between the two films of the device or by symmetric plastic deformation.

[0065] Advantageously, the deformed metering part is in the form of a multi-layer hemispherical concave cap, i.e., it consists of various films that have been laminated and deformed corresponding to each wall of the metering member, which contains no creases or air.

[0066] Advantageously, when the metering part of the first metering member and / or the second metering member changes to the operating state, i.e., when the fluid reaches one of the metering parts and the space of this metering part is filled, a characteristic popping noise is generated, which is related to the separation of each wall of the metering member and the deformation of one of its concave surfaces in the other direction.

[0067] According to one feature of the present invention, the deformation of the metering part is a plastic deformation.

[0068] According to one feature of the present invention, the deformation of the metering part of each metering member of the dilution system is carried out by embossing with a deformation element.

[0069] According to one feature of the present invention, the deformation element is integrated into one of the mold parts.

[0070] According to one feature of the present invention, each mold part is heated. Therefore, the deformation element itself provided on one of the mold parts is then heated.

[0071] According to one feature of the present invention, each metering part is formed by deformation with a dedicated deformation element.

[0072] Preferably, the deformation element is a protruding lug formed on the first mold part or the second mold part, and this lug protrudes from the surface of the mold cavity of the first mold part or the second mold part respectively. Even more preferably, the deformation element is a ball.

[0073] According to one feature of the present invention, the lug protrudes from the surface of the mold cavity in an intersecting direction, preferably in a vertical direction.

[0074] According to one feature of the present invention, each deformable element is integrated into the second mold part, each deformable element projects from the surface of the second mold part and is configured to cooperate with a complementary mold cavity provided in the first mold part. Thus, when the second mold part approaches the first mold part, one or more balls arranged facing one or more metering parts to be deformed push each film of the device into the complementary mold cavity of the first mold part, thereby deforming one or more metering parts of the device.

[0075] Alternatively, the deformable element is a fluid, preferably a gas, and the deformation of the metering part is performed by blowing the fluid.

[0076] Advantageously, this blowing is performed on the outer surface of one of the two films of the device in a single deformation direction, so that the two films are deformed simultaneously.

[0077] According to one feature of the present invention, this blowing can be performed at a high temperature.

[0078] According to one feature of the present invention, the fluid can be even more preferably pressurized air, preferably pressurized air between 4 and 10 bar.

[0079] Advantageously, the second mold part or the first mold part is provided with an opening channel on the surface facing the first mold part or the second mold part respectively, and the fluid configured to deform one or more metering parts is blown towards and guided to the opening channel.

[0080] According to one feature of the present invention, this blowing fluid can be heated. Thus, the fluid softens and pushes the two films towards one of the mold parts, specifically into the mold cavity of the mold part adapted to the shape of the metering part.

[0081] According to one feature of the present invention, this method includes a cooling step called passive cooling.

[0082] According to one feature of the present invention, before deforming the bag in the mold, the laminated film of the device is preheated for a determined time, preferably between 2 seconds and 6 seconds, between 25°C and 100°C, preferably between 40°C and 80°C. The preheated laminated film is then carried between two mold parts, and the mold itself is preferably temperature-controlled at a temperature between 25°C and 80°C. The film is maintained at the temperature due to contact when the mold is closed, and a deformation element in the form of a fluid is injected for 2 to 6 seconds, enabling the film to deform at the level of each metering part.

[0083] In the present invention, the term "sample" is understood to mean a sample of a biological substance.

[0084] In the present invention, the terms upstream and downstream are used according to the direction of fluid flow.

[0085] In the present invention, the term "flexible bag" is understood to mean a bag having the property of being foldable without plastic deformation and, after losing its shape or volume by compression or elongation, partially or entirely recovering its shape or volume.

[0086] In the present invention, the term "diluent fluid" is understood to mean a fluid, preferably a liquid, that enables dilution of a substance by adding it to the substance.

[0087] In the present invention, the term "biological substance" is understood to mean any material containing biological information.

[0088] In the present invention, the term "biological information" is understood to mean any element that constitutes the biological substance or is produced by the biological substance, such as microorganisms, nucleic acids (DNA, RNA), proteins, peptides, or membrane elements of metabolites. Biological information can specifically be contained within the biological substance or excreted / secreted by the biological substance.

[0089] In the present invention, the term "fragile valve" is understood to mean a weld disposed transversely to a fluid channel that blocks / permits the flow of fluid within said channel, and this valve is called "fragile" because this valve opens as soon as fluid is carried to contact this valve at a pressure of about 10 N to 20 N, and in some cases, a pressure exceeding that, specifically depending on factors such as lamination, the materials used to manufacture the device, and the geometric shape of the fluid circuit. The fragile valve is a single-acting valve that cannot close again once it has opened.

[0090] In the present invention, the term "mechanical valve" is understood to mean a valve disposed transversely to a fluid channel so as to permit or prevent the flow of fluid within the fluid channel, and said mechanical valve is operable and reversible, i.e., it can be opened and closed by command. In the present invention, the mechanical valve supports the fragile valve when the fragile valve is closed and replaces the fragile valve when the fragile valve is permanently open.

[0091] In the present invention, the term "welding" is understood to mean the permanent welding of a film, which restricts the circulation of fluid and enables the enclosure of fluid within the fluid circuit thus produced. "Welding" can be produced by a laser, thermal welding, or any other method capable of achieving equivalent results.

[0092] The present invention will be better understood from the following description of embodiments of the present invention, given by way of non-limiting example and described with reference to the accompanying schematic drawings. The accompanying schematic drawings are shown below.

Brief Description of the Drawings

[0093]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Embodiments for Carrying Out the Invention

[0094] Next, the present invention will be described with reference to FIGS. 1 to 35.

[0095] The dilution system 1 according to the present invention is specifically shown in FIG. 1 according to the first configuration, the second configuration in FIG. 2, the third configuration in FIG. 3, and the fourth configuration in FIG. 4, and then shown in more detail in FIGS. 5 to 29. An apparatus 100 according to the present invention incorporating any one of the dilution systems 1 according to the present invention is shown in FIGS. 5 and 6. Further, each step of the method for manufacturing the dilution system is shown in FIGS. 30 to 35.

[0096] The apparatus 100 according to the present invention is configured to enable dilution of a sample to be analyzed and to demonstrate an analyte (e.g., endotoxin) that may be present in the sample for diagnostic purposes. According to the present invention, regardless of the configuration of the dilution system, the apparatus 100 includes a dilution system 1 and a reaction chamber 103 shown in FIGS. 5 and 6 that is fluidly connected to the dilution system 1. The apparatus 100 according to the present invention is manufactured in the form of a flexible bag including at least a first film 101 and a second film 102 that are at least partially laminated to each other.

[0097] In the example shown in FIG. 5, the apparatus 100 incorporates a dilution system 1 with a third configuration. In the example shown in FIG. 6, the apparatus 100 incorporates a dilution system 1 with a second configuration. Of course, this apparatus can incorporate a dilution system according to the first configuration or according to another configuration including more metering members and containers and mixing chambers, and thus without departing from the scope of the present invention.

[0098] According to the present invention, the dilution system 1 is connected to the reaction chamber by fluid channels 21 and 22 in the first configuration, and by fluid channels 21, 22, and 23 in the second and third configurations. Each of the fluid channels 21, 22, 23 opens onto one or more rows of dedicated wells 104 of the reaction chamber 103, as specifically shown in FIGS. 5, 6, and 25 - 27. This aspect will be described in more detail later in this description.

[0099] Next, with reference to FIGS. 1, 2, 3, and 4, the dilution system 1 according to the present invention will be described. The only difference between the first configuration (FIG. 1) of the dilution system 1 and the third configuration (FIG. 3) of the dilution system 1 is the fact that the dilution system 1 according to the first configuration enables only one dilution since it comprises only a single dilution member 16 and a single dilution fluid container. The only difference between the second configuration (FIG. 2) and the third configuration (FIG. 3) of the dilution system 1 is the fact that there is no separate mixing chamber in the second configuration. In fact, in the second configuration, the container in which the dilution fluid is disposed serves as the mixing chamber. The difference between the fourth configuration (FIG. 4) and the other configurations is the fact that the latter have only a single dilution member that enables several dilutions to be performed.

[0100] Regardless of the configuration of the dilution system 1 according to the present invention, the dilution system 1 comprises a fluid circuit connecting a fluid container and a fluid mixing chamber. Preferably, this fluid circuit is manufactured by welding two films of the apparatus 100 that are laminated together.

[0101] Regardless of the configuration of the dilution system 1 according to the present invention, this dilution system 1 includes a first container 11 configured to contain a sample of a biological substance, which contains the biological substance to be diluted, and this sample is a fluid indicated as Fe in each figure.

[0102] Furthermore, regardless of the configuration of the dilution system 1 according to the present invention, the dilution system 1 includes at least a second container 12 configured to contain a first dilution fluid indicated as Fd1 in each figure.

[0103] Advantageously, this dilution system 1 includes the same number of dilution fluid containers as there are dilution liquids and / or various dilution fluids to be executed.

[0104] Regardless of the configuration of the dilution system 1 according to the present invention, the first container 11 is configured to receive a sample to be analyzed in fluid form or a member that receives the sample to be analyzed, and includes a fluid inlet, such as a pipette. Advantageously, the fluid inlet of the first container 11 can be sealed when the sample is injected into the first container 11 or can remain open, as shown in each figure. Furthermore, the first container 11 includes a fluid outlet.

[0105] Regardless of the configuration of the dilution system 1 according to the present invention, the second container 12 is configured to contain a determined amount of the first dilution fluid Fd1 and includes a fluid inlet and a fluid outlet. Similar to the first container 11, it is preferable that the fluid inlet of the second container 12 is sealed when the dilution fluid is injected into the second container 12.

[0106] Regardless of the configuration of the dilution system 1 according to the present invention, this dilution system 1 includes a first metering member 16. The first metering member 16 is arranged on the fluid path connecting the first container 11 and the second container 12, specifically between the first container 11 and the second container 12, as can be seen specifically by looking at FIGS. 1, 2, 3, and 4.

[0107] According to the present invention, regardless of the configuration of the dilution system 1, as can be seen from FIGS. 9 and 10, each metering member 16, 17 includes a first wall 101 and a second wall 102 corresponding to a part of the first film 101 and a part of the second film 102 that constitute the apparatus 100 according to the present invention, respectively.

[0108] Furthermore, regardless of the configuration of the dilution system 1, each metering member 16, 17 includes a metering part configured to change from an initial state (see FIG. 9) where the first wall 101 and the second wall 102 are in contact with each other to an operating state (see FIG. 10) where the first wall 101 and the second wall 102 are spaced apart from each other by a certain distance so as to delimit a determined space. This metering part realizes the operating state by transporting the sample Fe and / or the dilution fluids Fd1, Fd2, or the mixed fluid Fm1 into the space of the metering members 16, 17. The deformation of the metering part is reversible, and this metering part can be reset to its initial state.

[0109] According to the present invention, and according to the first, third, and fourth configurations of the dilution system 1, this dilution system 1 includes a first mixing chamber 14. In this first mixing chamber 14, the sample Fe to be analyzed and the first dilution fluid Fd1 are mixed so as to be diluted at a predetermined ratio according to a desired dilution degree. The first mixing chamber 14 includes a fluid inlet through which the sample Fe and the first dilution fluid Fd1 enter, and at least one fluid outlet through which the first fluid mixture Fm1 (for example, shown in FIGS. 17 and 18) exits.

[0110] According to the first, third, and fourth configurations of the dilution system 1, as can be seen from FIGS. 1, 3, and 4, the first metering member 16 is disposed upstream of the fluid inlet of the first mixing chamber 14.

[0111] According to the second configuration of the dilution system 1 shown in FIGS. 2 and 6, the containers 12, 13 containing the first dilution fluid Fd1 and the second dilution fluid Fd2 serve as a mixing chamber.

[0112] According to the third configuration, specifically as shown in FIGS. 3A and 3B, the dilution system includes a second metering member 17. Further, the dilution system 1 includes a third container 13 configured to contain a second dilution fluid Fd2. Advantageously, the second metering member 17 is disposed on a fluid path connecting the first mixing chamber 14 and the third container 13. Specifically, the second metering member 17 is disposed between the first mixing chamber 14 and the third container 13.

[0113] According to the third configuration, the dilution system 1 includes a second mixing chamber 15. In this second mixing chamber 15, the first fluid mixture Fm1 and the second dilution fluid Fd2 are mixed so as to be diluted at a predetermined ratio according to a desired dilution degree.

[0114] As shown in FIGS. 3A and 3B, the second mixing chamber 15 includes a fluid inlet through which the first fluid mixture Fm1 and the second dilution fluid Fd2 enter, and at least one fluid outlet through which a second fluid mixture Fm2 (not shown) exits.

[0115] In FIG. 3B, the first metering member 16 and the second metering member 17 are fluidly directly connected to each other.

[0116] In the example shown in the figure, each metering member 16, 17 is disposed upstream of the fluid inlet of the mixing chambers 14, 15. In such an example, the metering members are configured to continuously meter the fluid coming from several containers. Of course, it is also possible to consider that each container has a dedicated metering member, and the metering of each fluid may still be continuous or simultaneous (in this case, the mixing chamber should be provided with several fluid inlets).

[0117] According to the third configuration of the dilution system 1, as shown in FIG. 27, the first container 11 includes two fluid outlets, specifically, a first fluid outlet connected to the fluid inlet of the first metering member 16, and a second fluid outlet connected to a fluid channel 21 that directly transports a part of the sample Fe to the reaction chamber 103 of the device 100.

[0118] According to a third configuration of the dilution system 1, as shown in FIG. 28, the first mixing chamber 14 has two fluid outlets, specifically, a first fluid outlet connected to the fluid inlet of the second metering member 17, and a second fluid outlet connected to a fluid channel 22 that directly conveys a part of the first fluid mixture Fm1 to the reaction chamber 103 of the apparatus 100.

[0119] According to a third configuration of the dilution system 1, as shown in FIG. 29, the second mixing chamber 15 has a fluid outlet connected to a fluid channel 23 that directly conveys a second fluid mixture Fm2 of fluid to the reaction chamber 103 of the apparatus 100.

[0120] According to a fourth configuration shown in FIG. 4, the first metering member 16, which is the only metering member of the dilution system 1, has a first fluid inlet connected to the first container 11, a second fluid inlet connected to the second container 12, a third fluid inlet connected to the third container 13, a fourth fluid inlet connected to the first mixing chamber 14 that also serves as a first fluid outlet, and a second fluid outlet connected to the second mixing chamber 15.

[0121] According to the present invention, regardless of the configuration, in the initial state of the metering parts of the metering members 16 and 17, each fluid inlet and each fluid outlet of each of the metering members 16 and 17 are sealed by a fragile valve, which is shown by a dotted line and is arranged laterally with respect to the fluid channels connecting the containers to the metering members. Each fragile valve is configured to be opened by the pressure of the fluid flowing from a container or mixing chamber arranged upstream in the fluid direction of the metering member in the fluid flow direction towards a mixing chamber arranged downstream in the fluid direction of the metering member.

[0122] FIG. 7 shows the positioning of the fragile valves at the levels of each fluid inlet 16a, 16b, 17a, 17b of each of the metering members 16 and 17 and at the levels of each fluid outlet 16c, 17c of each of the metering members 16 and 17 in the third configuration. Of course, a similar configuration can be applied in each configuration.

[0123] Each of the fragile valves is connected to the mechanical valves V1 to V6. Therefore, when such a fragile valve is open, the mechanical valves V1, V2, V3, V4, V5, and V6 alternate to close or reopen the fluid inlets 16a, 16b, 17a, 17b, and the outlets 16c, 17c. FIG. 8 shows the positioning of the mechanical valves V1, V2, V3, V4, V5, and V6 with respect to the fragile valves when the metering parts of the first metering member 16 and the second metering member 17 are in the initial state in the third configuration. Of course, the same configuration can be applied in each configuration.

[0124] In this case, as shown in FIG. 11, the device 100 is configured to cooperate with a first plurality of valves V1, V2, and V3 configured to close the first fluid inlet 16a of the first metering member 16, the second fluid inlet 16b of the first metering member 16, and the fluid outlet 16c of the first metering member 16, respectively.

[0125] Furthermore, the device 100 is configured to cooperate with a second plurality of mechanical valves V4, V5, and V6 configured to close the first fluid inlet 17a of the second metering member 17, the second fluid inlet 17b of the second metering member 17, and the fluid outlet 17c of the second metering member 17, respectively.

[0126] Furthermore, the device 100 is configured to cooperate with a third plurality of mechanical valves V7, V8, and V9 respectively arranged at the inlet of the first fluid channel 21, the inlet of the second fluid channel 22, or the outlet of the first mixing chamber 14, and the inlet of the third fluid channel 23, or the outlet of the second mixing chamber 15.

[0127] In this description, the mechanical valves V1 to V9 are shown in two positions: an open position indicated by an empty / white rectangle and a closed position indicated by a dark / black rectangle.

[0128] Next, with reference to FIGS. 11 to 26, the dilution principle according to the present invention will be described. This principle will be described using the dilution system 1 according to the third configuration. Of course, this principle is also applicable to the dilution system 1 according to other configurations.

[0129] When the apparatus 100 is first used, as shown in FIG. 4, the dilution system 1 has not yet been used, and each measuring part of the first measuring member 16 and the second measuring member 17 is in an initial state, and all the fragile valves are sealed. Further, when the apparatus 100 is inserted into the equipment enabling the implementation of dilution via the dilution system 1, the first plurality of valves V1 to V3, the second plurality of mechanical valves V4 to V6, and the third plurality of mechanical valves V7 to V9 are closed and arranged as shown in FIG. 8.

[0130] First, the first mechanical valve V1 is opened, and at least the mechanical valves V2 and V3 are closed. Then, pressure is applied to the first container 11 containing the sample Fe in the form of a fluid. Then, as shown in FIG. 11, the sample Fe passes through the fluid circuit of the dilution system 1 up to the first inlet 16a of the first measuring member 16 at the level of the fragile valve arranged at the inlet 16a of the first measuring member 16. As shown in FIG. 12, the fragile inlet valve 16a opens under the pressure applied by the arrival of the sample fluid Fe. This opening is sudden, and as shown in FIG. 13, the sample Fe fills the entire determined internal space of the measuring part of the first measuring member 16, and the valve V1 is closed immediately when the measuring part is filled. As shown in FIG. 10, the measuring part of the first measuring member 16 is in an operating state because its walls 101, 102 are at a distance from each other so as to delimit the measuring space, and as shown in FIG. 13, all the fragile valves arranged at the fluid inlets 16a, 16b, and the fluid outlet 16c of the first measuring member 16 are opened and replaced by the closed mechanical valves V1, V2, and V3 respectively.

[0131] According to the first operating mode shown in FIG. 15, although the first metering member 16 contains the metered sample Fe and the mechanical valve V1 is closed, the mechanical valves V2 and V3 are open. Then, the first dilution fluid Fd1 contained in the second container 12 is moved back and forth between the first metering member 16 and the mixing chamber 14 to mix the first dilution fluid Fd1 and the metered sample Fe. This first operating mode has the advantage of ensuring that the entire metered sample Fe is well diluted with the entire first dilution fluid Fd1. When the first fluid mixture Fm1 is obtained and collected in the first mixing chamber 14, the mechanical valves V2 and V3 are closed.

[0132] Alternatively, according to the second operating mode, when the sample Fe is metered, the mechanical valve V3 is opened so that the metered sample Fe flows into the first mixing chamber 14 (FIG. 14). Then, the mechanical valve V2 arranged at the second fluid inlet 16b of the first metering member 16 is opened, the mechanical valve V3 is opened, the mechanical valve V1 is closed, and as shown in FIG. 15 and as described by the first operating mode, the back-and-forth movement is executed. As shown in FIG. 16, when the first fluid mixture Fm1 is obtained and collected in the first mixing chamber 14, the mechanical valves V2 and V3 are closed.

[0133] Alternatively, according to the third operating mode shown in FIG. 14, when the sample Fe is metered, the mechanical valve V3 arranged at the fluid outlet 16c of the first metering member 16 is opened. As a result, the metered sample Fe flows into the first mixing chamber 14, and the mechanical valves V1 and V2 are closed. Then, the metering part of the first metering member 16 is reset to its initial state, but the fragile valve becomes inactive. This reset can be achieved by the pushing element pushing back and rearranging the device walls with each other at the level of the metering part.

[0134] Next, the mechanical valve V2 disposed at the second fluid inlet 16b of the first metering member 16 is opened, while the mechanical valves V1 and V3 remain closed. When the mechanical valve V3 is opened, the metering part of the first metering member 16 changes to an operating state, whereby, as shown in FIG. 17, it becomes possible to sample only a precise quantity of the first dilution fluid Fd1 accommodated in the second container 12. When the space of the metering part is filled, the mechanical valve V2 is closed so as to separate the quantity of the first dilution fluid Fd1 in the first metering member 16, and the mechanical valves V1 and V3 are also closed.

[0135] In this third operating mode, the first metering member 16 can be emptied by the pressure applied to its metering part and this pressure can be applied by the same pushing element that serves to reset it, or by another element.

[0136] Next, as shown in FIG. 18, the mechanical valve V3 is opened, and as a result, the first metered dilution fluid Fd1 flows into the first mixing chamber 14 that already contains the metered sample Fe, and the resulting mixture forms the first fluid mixture Fm1. The steps of resetting the first metering member 16 and metering the first dilution fluid Fd1 are executed the necessary number of times according to the required dilution degree. For example, with a determined capacity of 10 μl for the first metering member, when diluting 10 μl of the sample Fe by a factor of 10, 90 μl of the dilution fluid is obtained and mixed with 10 μl of the sample, and thus nine doses of the first dilution fluid Fd1 are required to form the first fluid mixture Fm1.

[0137] Figures 19 to 26 show the second part of the dilution process, which consists of diluting the first mixture Fm1 of the fluid obtained so far. Therefore, in Figure 19, the mechanical valves V3, V5, V6, and V8 are closed, and the mechanical valve V4 is opened. Then, pressure is applied to the first mixing chamber 14, and as a result, a part of the first mixture Fm1 of the fluid present in the first mixing chamber 14 is carried into the second metering member 17 and metered. When the first mixture Fm1 of the fluid fills the second metering member 17, the fragile valve arranged at the level of the first fluid inlet 17a of the second metering member 17 opens, and the fragile valves arranged at the levels of the second fluid inlet 17b and the fluid outlet 17c also open under the action of the metering part that deforms to the fluid Fm1 and the operating state.

[0138] As shown in Figure 20, under the pressure applied by the arrival of the first mixture Fm1 of the fluid, the fragile valve arranged at the level of the first inlet 17a of the second metering member opens, and the metering part of the second metering member 17 is completely filled in the same way as the first metering member 16 in Figure 13. The valve V4 is closed immediately when the metering part is filled. As shown in Figure 10, the metering part of the second metering member 17 is in the operating state because its walls 101, 102 are at a distance from each other so as to define the metering space, and all the fragile valves arranged at the fluid inlets 17a, 17b, and the fluid outlet 17c of the second metering member 17 are opened and replaced by the closed mechanical valves V4, V5, and V6 respectively to separate the precise amount of the required fluid mixture Fm1.

[0139] According to the first operating mode shown in Figure 22, although the second metering member 17 still contains the first mixture Fm1 of the fluid and the mechanical valve V4 is closed, the mechanical valves V5 and V6 are open. Then, the second dilution fluid Fd2 accommodated in the third container 13, the second metering member 17, and the second mixing chamber 15 are moved back and forth to mix the second dilution fluid Fd2 and the first metered mixture Fm1 of the fluid to obtain the second mixture Fm2 of the fluid.

[0140] As shown in Fig. 23, when the second mixture Fm2 of the fluid is obtained and collected in the second mixing chamber 15, the mechanical valves V5 and V6 are closed.

[0141] Alternatively, as shown in Fig. 21, according to the second operation mode, when the first mixture Fm1 of the fluid is metered and enters into the second metering member 17, the mechanical valve V6 disposed at the fluid outlet 17c of the second metering member 17 opens. As a result, the first mixture Fm1 of the fluid flows into the second mixing chamber 15, and the mechanical valves V4 and V5 are closed. Then, the mechanical valve V5 disposed at the second fluid inlet 17b of the second metering member 17 opens, the mechanical valve V6 opens, the mechanical valve V4 is closed, and the back-and-forth movement shown in Fig. 22 and described by the aforementioned first operation mode is executed. As shown in Fig. 23, when the second mixture Fm2 of the fluid is obtained and collected in the second mixing chamber 15, the mechanical valves V5 and V6 are closed.

[0142] Alternatively, as shown in Fig. 21, according to the third operation mode, when the first mixture Fm1 of the fluid is metered in the second metering member 17, the mechanical valve V6 disposed at the fluid outlet 17c of the second metering member 17 opens. As a result, the first mixture Fm1 of the fluid flows into the second mixing chamber 15, and the mechanical valves V4 and V5 are closed. Then, since the opening of the fragile valve is irreversible, the metering part of the second metering member 17 is in the initial state but is reset so that the fragile valve becomes inactive. This reset can be achieved by the pushing elements driving the walls of the device against each other at the level of the metering part. Then, the mechanical valve V5 disposed at the second fluid inlet 17b of the second metering member 17 opens, and the mechanical valves V4 and V6 remain closed. When the mechanical valve V6 opens, the metering part of the second metering member 17 shifts to the operating state, whereby, as shown in Fig. 24, it becomes possible to sample only a precise quantity of the second dilution fluid Fd2 accommodated in the third container 13. When the space of the metering part is filled, the mechanical valve V5 is closed so as to separate the quantity of the second dilution fluid Fd2 in the second metering member 17, and the mechanical valves V4 and V6 are also closed.

[0143] In this third operating mode, the second metering member 17 can be emptied by the pressure applied to its metering section and this pressure can be applied by the same pushing element or by another element that serves to reset it.

[0144] Then, as shown in FIG. 25, the machine valve V6 is opened, and as a result, the second metered dilution fluid Fd2 flows into the second mixing chamber 15 that already contains the first metered mixture Fm1 of the fluid, and as shown in FIG. 26, the resulting mixture forms the second mixture Fm2 of the fluid.

[0145] The step of resetting the second metering member 17 and the step of metering the first dilution fluid Fd1 are carried out the required number of times according to the required dilution factor. In this case, to obtain a 100-fold dilution of the sample Fe, when the determined volume of the second metering member 17 is 10 μl, the second dilution fluid Fd2 is metered 9 times for one dose of the first mixture Fm1 of the fluid.

[0146] As shown in FIG. 29, when the dilution process is complete and the volume of the second mixture Fm2 of the fluid is obtained, the valve V9 is opened at the outlet of the second mixing chamber 15, and the second mixture Fm2 of the fluid flows through the fluid channel 23 into one or more dedicated rows 104 of wells in the reaction chamber 103.

[0147] According to the present invention, when selecting an operating mode with opposite movements, which is described according to the first operating mode of the first part of the dilution process and the second part of the dilution process, and also described according to the second operating mode of the first part of the dilution process and the second part of the dilution process, the dilution fluid (Fd1 or Fd2) needs to be metered first before being injected into the container (12 or 13), and thus when the fluid mixture is obtained, the container remains empty.

[0148] According to the present invention, when selecting an operation mode involving metering of the dilution fluid, in accordance with the third operation mode of the first and second parts of the dilution process, it is possible to avoid metering before injecting the dilution fluid into the container, which is less restrictive.

[0149] To ensure that the detection of the analyte(s) sought in the collected sample is complete and reliable, it is necessary to compare the second mixture Fm2 of the fluid, which is the final result of the dilution, with the fluid from the previous step. Thus, as shown in FIG. 27, a portion of the undiluted sample Fe is collected in one or more rows 104 of dedicated wells and is conveyed via the fluid channel 21. As shown in each figure, the fluid outlet of the first container 11 comprises a two-way with two branches, the first branch being connected to the first fluid inlet 16a of the metering member 16 and the second branch constituting the channel 21 that directly connects the first container 11 to the reaction chamber 103. A valve V7 is arranged downstream of the two-way on the channel 21, so that when the sample Fe is conveyed to the first metering member 16, the fluid is directed only to the first branch.

[0150] Furthermore, the first mixing chamber 14 comprises a second fluid outlet directly connected to the reaction chamber 103 via the channel 22. The valve V8 separates this channel 22 when the channel 22 is not in use. Thus, as shown in FIG. 28, a portion of the first mixture Fm1 of the fluid conveyed via the fluid channel 22 is also collected in one or more rows 104 of dedicated wells. Such collection can be performed during or after the dilution process.

[0151] In the dilution system according to the first configuration, it is possible to proceed according to any of the three aforementioned operation modes.

[0152] In the dilution system according to the second configuration, the first operation mode is recommended, i.e., the amounts of the dilution fluids Fd1 and Fd2 must first be measured before injecting them into the dilution system.

[0153] In the dilution system according to the fourth configuration, in order to enable at least the metering of the sample fluid Fe and the first mixture Fm1 of the fluids, as described in connection with the third operating mode, the metering member 16 must be reset between at least two dilutions, and for the other fluids (Fd1, Fd2), it is possible to proceed according to any one of the three operating modes involving metering or reciprocating movement.

[0154] Next, with reference to FIGS. 30 to 35, a manufacturing method of the apparatus 100 according to the present invention will be described. The manufacturing method to be described is effective regardless of the configuration of the dilution system integrated into the apparatus according to the present invention.

[0155] As described so far, the apparatus 100 is in the form of a flexible bag composed of at least two films 101, 102. This "bag" includes several compartments corresponding to the arrangement for inserting the containers 11, 12, 13, the mixing chambers 14, 15, the metering members 16, 17, the fluid channels, and the reaction chamber 103.

[0156] To form the apparatus 100, two films 101, 102 are laminated in the form of a bag over a part of the height of the apparatus, and then a fluid circuit including various compartments (containers, mixing chambers, metering members, channels) of the bag is welded by permanent welding. Fragile valves are also arranged at the fluid inlets and fluid outlets of the metering members 16, 17.

[0157] To fabricate the containers 11, 12, 13, a fluid, preferably a gas such as compressed air which may be heated in some cases, is blown between the two films 101 and 102 at the level of each marking of each container 11, 12, 13, and the upper part of each container is not laminated, thus leaving an opening between the two films 101 and 102. During the blowing, the bag is inserted into a mold having a mold cavity with a mold for each container so that the mold cavity conforms to the engraving pattern during this blowing.

[0158] The production of the metering members 16 and 17 is independent of the production of the container, that is, the production can be carried out even if the container is not formed. To produce the metering members, the procedure is as follows.

[0159] In the first step, as shown in FIGS. 30 and 33, by marking each of the metering members 16 and 17 like a ring, a deformed area D on the bag is produced, and this deformed area D delimits the positions of the metering members 16 and 17 to be formed.

[0160] In the second step, at least this produced deformed area D is placed in the mold 200. As shown in FIGS. 30 and 33, the mold 200 includes at least two mold parts 201 and 202 each having at least one mold cavity 201a and 202a respectively, and the cavity 201a of the first mold part 201 is arranged at least partially facing the cavity 202a of the second mold part 202.

[0161] In the third step, as shown in FIGS. 31 and 34, the two films 101 and 102 of the bag 100 are both deformed toward the first mold part 201 by the deformation elements 203 and 204 at the level of the deformed area D, and these deformation elements 203 and 204 are arranged between the bag 100 and the second mold part 202.

[0162] According to the first embodiment, the deformation of the bag 100 is carried out by indenting with the external deformation element 203, which is a lug protruding from the surface of the cavity 202a of the second mold part 202. Specifically, as shown in FIGS. 30 to 32, the shape of the protruding lug 203 is adapted to the shape of the metering members 16 and 17 to be produced. For example, this protruding lug is in the shape of a ball, and at least one hemispherical part thereof protrudes from the second mold part 202.

[0163] As can be seen with reference to Fig. 32, according to the first embodiment, each metering member 16, 17 is manufactured by external deformation by a dedicated external deformation element 203. Specifically, as shown in Fig. 32, preferably two protruding lugs 203 in the shape of balls are arranged on the second mold part 202. Such lugs are configured to face the deformation area D so that when the bag is inserted into the mold, each lug produces one metering member respectively. Advantageously, the first mold part 201 includes an inverse shape within its mold cavity 201a so as to accompany the deformation of the deformation area D.

[0164] According to the second embodiment shown in Figs. 33 to 35, the deformation of the bag is realized by blowing, and the external deformation element 204 is a fluid. Preferably, this external deformation element 204 is a gas, and even more preferably air.

[0165] Advantageously, a fluid useful for forming the container is used, which is reused or a part of it is diverted to deform the metering member. As can be seen with reference to Fig. 34, the fluid passes between the second mold part 202 and one of the films 102 of the bag 100.

[0166] In Fig. 35, the cavity 202a of the second mold part 202 is provided with an opening channel 205 intended to be arranged opposite the first mold part 201, and a fluid 204 configured to deform the bag 100 is blown into the opening channel 205. In fact, the opening channel 205 is configured to guide the fluid 204 constituting the external deformation element 204 to the deformation area D of each metering member 16, 17. In the example shown in the figure, the opening channel 205 distributes the deformation area, and nevertheless, other forms of channels can be conceived without departing from the scope of protection of the present invention.

[0167] Of course, the present invention is not limited to the embodiments described and illustrated in each of the accompanying drawings. Specifically, with respect to the configurations of various elements or by substitution of technical equivalents, modifications can still be realized without departing from the scope of protection of the present invention.

Claims

1. A dilution system (1) for diluting a sample (Fe) of a biological substance, comprising a fluid circuit, wherein the fluid circuit comprises at least - a first container (11) configured to contain a sample (Fe) of a biological substance containing the biological substance to be diluted, wherein the sample (Fe) is a fluid, the first container (11); - a second container (12) configured to contain a first dilution fluid (Fd1), wherein the first container (11) and the second container (12) are fluidly connected by at least one fluid path, the second container (12); - at least a first metering member (16) comprising a first wall (101) and a second wall (102) for metering a fluid of a determined volume, wherein the first wall (101) and the second wall (102) are spaced apart from each other from an initial state in which they are in contact with each other to a working state in which they delimit a determined metering space, and the metering part is configured to at least change, and the metering part realizes the working state by transporting the sample (Fe) and / or the dilution fluid (Fd1) into the metering part, and the first metering member (16) is arranged on the fluid path connecting the first container (11) and the second container (12) between the first container (11) and the second container (12), at least the first metering member (16); A dilution system (1), characterized by comprising the above.

2. The dilution system according to claim 1, wherein the first metering member (16) comprises a fluid inlet (16a) directly connected to the first container (11) and a fluid inlet (16b) directly connected to the second container (12).

3. Each fluid inlet (16a, 16b) of the first metering member (16) is closed so as to be sealed by a frangible valve in the initial state of the metering part of the first metering member (16), and each frangible valve is configured to be opened by the fluid pressure from between the sample or the dilution fluid carried to the first metering member. The dilution system according to claim 2.

4. Comprising at least a first mixing chamber (14) configured to contain a first mixture (Fm1) of fluid resulting from mixing a part of the sample (Fe) and at least a part of the first dilution fluid (Fd1), the first mixing chamber (14) being fluidly connected to the first container (11) and the second container (12). The dilution system according to any one of claims 1 to 3.

5. The first metering member (16) comprises at least one fluid outlet (16c) connected to the first mixing chamber (14), and each fluid outlet (16c) of the first metering member (16) is closed so as to be sealed by a frangible valve in the initial state of the metering part of the first metering member (16), and each frangible valve is configured to be opened by the fluid pressure from between the sample or the first dilution fluid carried to the first mixing chamber (14). The dilution system according to claim 4.

6. A third container (13) configured to contain a second dilution fluid (Fd2), and at least a second mixing chamber (15) configured to contain a second mixture (Fm2) of fluid resulting from mixing a part of the first mixture (Fm1) of fluid and at least a part of the second dilution fluid (Fd2), the second mixing chamber (15) being fluidly connected to the first mixing chamber (14) and the third container (13). The dilution system according to claim 4 or 5.

7. Comprising a second metering member (17) arranged upstream of the second mixing chamber (15), the second metering member (17) being configured to meter the first mixture (Fm1) of fluids, which is intended to flow in from the first mixing chamber (14) and be diluted by the second dilution fluid (Fd2) flowing in from the third container (13). The dilution system according to claim 6.

8. An apparatus (100) in the form of a flexible bag comprising at least a first film (101) and a second film (102) that are at least partially laminated to each other, the apparatus (100) comprising the dilution system (1) according to any one of claims 1 to 7 and a reaction chamber (103), the dilution system (1) being fluidly connected to the reaction chamber (103), and the reaction chamber (103) of the apparatus (1) comprising a plurality of wells (104) configured to accommodate at least one reagent.

9. The apparatus is configured to cooperate with a first plurality of mechanical valves (V1, V2, V3) arranged upstream of each of the fragile valves of the first metering member (16), each of the mechanical valves (V1, V2, V3) being arranged at the fluid inlet (16a, 16b) or fluid outlet (16c) of the first metering member (16), and being configured to allow / prevent fluid (Fe, Fd1) from entering the first metering member (16) or allow / prevent fluid (Fe, Fd1) from exiting the first metering member (16). The apparatus according to claim 8.

10. The apparatus is configured to cooperate with a second plurality of mechanical valves (V4, V5, V6) arranged upstream of the fragile valve of the second metering member (17), each of the plurality of mechanical valves (V4, V5, V6) being arranged at the fluid inlet (17a, 17b) or fluid outlet (17c) of the second metering member (17), and being configured to allow / prevent fluid (Fm1, Fd2) from entering the second metering member or allow / prevent fluid (Fm1, Fd2) from exiting the second metering member (17). The apparatus according to claim 8 or 9.

11. A method of manufacturing an apparatus according to any one of claims 8 to 10, incorporating the dilution system according to any one of claims 1 to 7, comprising: - By welding the films (101, 102) of the apparatus (100), at least one step of creating the fluid circuit of the dilution system according to any one of claims 1 to 7 on the film, wherein the films (101, 102) are at least partially pre-laminated, at least one step of creating the fluid circuit of the dilution system; - At least one step of forming at least the first metering member (16), wherein (i) at least the metering part of the first metering member (16) created in the step of creating the fluid circuit is disposed within a mold (200), the mold (200) including at least two mold parts (201, 202), each having at least one mold cavity (201a, 202a), the mold cavity (201a) of the first mold part (201) being disposed at least partially facing the mold cavity (202a) of the second mold part (202), and being at least partially complementary to the mold cavity (202a) of the second mold part (202), and (ii) by closing the two mold parts (201, 202) of the mold (200) towards each other, the films (101, 102) of the apparatus (100) are deformed together on one or the other side of the apparatus (100) in a single deformation direction at the level of the metering part by deformation elements (203, 204), the deformation elements (203, 204) being disposed between the apparatus (100) and the second mold part (202) or between the apparatus (100) and the first mold part (201), at least one step of forming at least the first metering member (16); A method comprising the above.

12. The method according to claim 11, wherein the deformation of the metering part of each metering member (16, 17) of the dilution system (1) is brought about by embossing by the deformation element (203).

13. The deformation element (203) is a protruding lug (203) provided on the first mold part (201) or the second mold part (202), and the lug (203) protrudes from the surfaces of the mold cavities (201a, 202a) of the first mold part (201) or the second mold part (202), respectively. The method according to claim 12.

14. The deformation element (204) is a fluid, and the deformation of the metering part is brought about by blowing the fluid (204). The method according to claim 13.

15. The second mold part (202) or the first mold part (201) is provided with an opening channel (205) on the surface facing the first mold part (201) or the second mold part (202), respectively, and the fluid (204) configured to deform one or more of the metering parts is blown toward and guided to the opening channel (205). The method according to claim 14.

Images