Cap for a biological sample reagent dispensing device

Abstract

A cap (5) for a biological sample reagent diffusion device comprises a body (21) having a first end (12) and a second end (14), said body (21) further comprising a liquid discharge channel (27) opening at the first end (12) and a fastening portion (25) at the second end (14) adapted to be mounted on a container (3) of a biological sample reagent diffusion device, the body (21) further comprising a transfer channel (16) opening at the second end (14) and fluidly connected to said discharge channel (27), so that a liquid contained in said container (3) on which said cap (5) is mounted is adapted to be discharged through the discharge channel (27).The transfer channel (16) further comprises a diffusion portion (18) forming a housing, the diffusion portion (18) comprising a volume, the stopper (5) further comprising a reagent support (39) made of porous material receiving at least one lyophilized reagent (41), which reagent support (39) occupies substantially the entire volume of the diffusion portion (18) so that a liquid from the container and flowing through it towards the discharge channel (27) is forced to pass through the reagent support (39). Figure 2.

Description

Title of the invention: Stopper for a reagent dispensing device for biological samples

[0001] The invention relates to the field of analysis of biological samples, such as for example the detection of nucleic acid sequences by PCR reaction or any other method of amplification of nucleic acids in vitro.

[0002] The analysis of biological samples allows for the performance of diagnostic tests.

[0003] PCR tests are also part of the methods for analyzing biological samples. Conventional methods for preparing biological samples for PCR tests generally require specific equipment and qualified personnel, such as laboratory technicians, particularly for pipetting, mixing, concentration calculation or cartridge filling operations.

[0004] In order to improve the efficiency and speed of these tests, distribution devices equipped with reagents have been developed.

[0005] Such devices are known to us from documents US2010304359 and WO2021066209 but also from documents WO2020257356 and US5454786.

[0006] US2010304359 describes a sample collection device comprising a cap dropper equipped with a compartment containing at least one reagent. The reagents are lyophilized and held inside the compartment by a sieve. The sieve can function as a filter depending on the size or affinity of the extraction mixture.

[0007] WO2021066209 describes a set for cosmetic products. The set The system comprises a first, hollow container of predetermined length, the interior of which is filled with a liquid. The system also includes a second container, removably coupled to the first, into which the liquid contents of the first container can flow. The interior of the second container is filled with dried cosmetic products in powder form.

[0008] WO2020257356 describes devices for detecting target nucleic acids. The The device includes a microfluidic cartridge for the detection of a target nucleic acid equipped with an amplification chamber fluidically connected to a valve, a sample dosing channel, and a detection reagent chamber comprising a programmable nuclease.

[0009] US5454786 describes a cartridge assembly for containing a product lyophilized, forming a disposable part of an injector pen. The assembly includes a cylindrical glass cartridge intended to receive the product, a closing cap, a cartridge case and a piston mechanism.

[0010] It is known to use lyophilized reagents in various forms. Lyophilized reagents are available as powders, pellets, pills, or compressed powders, and also as directly lyophilized reagents. These reagents, regardless of the form in which they are lyophilized, are generally stored in a compartment for rehydration.

[0011] The use of lyophilized reagents offers numerous advantages, such as simplified storage conditions, improved reagent stability, and enhanced reaction with samples in liquid form. However, the use of lyophilized reagents in the form of beads, pills, pellets, or powder presents several disadvantages.

[0012] Handling these reagents is complicated in a production logistics environment because it requires specific equipment and presents an increased risk of losing some of the reagent, which is generally expensive. In the case of reagents directly lyophilized in a compartment, the lyophilization yield is reduced because the container in which they are lyophilized is larger. Compressed pills, tablets, and powders require additional production steps, increasing production time and cost.

[0013] On the other hand, it is difficult to arrange these reagents homogeneously in the mixing compartment. It is often necessary to have a compartment with numerous parts, further complicating the assembly. Moreover, since liquids flow along the path of least fluid resistance, they tend to pass through the spaces between the reagents rather than through the reagents themselves. This results in a reduced reagent recovery rate and therefore reduced performance or increased cost of the device.

[0014] Furthermore, the use of lyophilized reagents in the form of beads, pills, pellets or compressed powders does not allow for great reproducibility of measurements, thus generating a wide variability of results.

[0015] Depending on the field, the reproducibility of measurements does not have the same importance or the same tolerances. In the context of PCR diagnostic tests, for example, the reproducibility of measurements is essential.

[0016] Reagents must also be isolated from the sample liquid so that they do not degrade over time.

[0017] The Applicant sought to improve the situation.

[0018] The invention provides a cap for a reagent diffusion device for biological samples comprising a body having a first end and a second end. Said body further comprises a liquid discharge channel opening at the first end and a fixing portion at the The second end is adapted to be mounted on a container for a biological sample reagent dispensing device. The body further comprises a transfer channel opening at the second end and fluidly connected to said discharge channel, such that a liquid contained in said container on which said stopper is mounted is adapted to be discharged through the discharge channel. The stopper is characterized in that the transfer channel further comprises a diffusion portion forming a housing, the diffusion portion comprising a volume. The stopper further comprises a reagent holder made of porous material receiving at least one lyophilized reagent, which reagent holder occupies substantially the entire volume of the diffusion portion so that a liquid from the container flowing through it towards the discharge channel is forced to pass through the reagent holder. This reduces the number of steps required to prepare biological samples. Since the steps are simple, biological sample preparation can be carried out by individuals without specific technical qualifications.

[0019] In one embodiment, the reagent support is made of cellulose fiber, polyurethane, PVA, or cellulose acetate. These materials allow for good distribution of the reagent and good flow of the sample liquid. They are also biocompatible with the reagent and will not inhibit molecules present in the reagent.

[0020] In one embodiment, the reagent support has a porosity of between 50 μm and 1500 μm. The chosen porosity allows both to support the reagent and to allow good flow of the liquid, thus increasing the recovery rate of the reagent.

[0021] In one embodiment, the reagent is selected from the group consisting of: enzymes, primers, oligos, plasmids, RNA, DNA, dNTPs, bacteria, viruses, antibodies, proteins, stabilizers, lyoprotectants, polyethylene glycol, unbranched polymers, nanoparticles, fluorophores, fluorescent probes and dyes.

[0022] In one embodiment, the cap further comprises a separator disposed in the transfer channel between the second end and the reagent holder, said separator being capable of isolating the reagent from the liquid coming from the container or delaying its migration when the pressure in the transfer channel is less than or equal to the pressure in the discharge channel. The separator makes it possible to isolate the reagent from the sample liquid when the dilution device is not in use.

[0023] In one embodiment, the separator is arranged in the transfer channel, upstream of the reagent support, the separator transversely occupying the entire housing, so that a liquid coming from the container and flowing through the discharge channel is forced to pass through the separator before passing through the reagent support. The isolation of the sample liquid is improved.

[0024] In one embodiment, the separator is made of a porous material, preferably cellulose fiber, polyurethane, PVA or cellulose acetate. These materials make it possible to isolate or delay the dilution of the sample liquid in the absence of overpressure in the container.

[0025] In one embodiment, the separator comprises an elastomeric membrane having orifices capable of opening in the event of overpressure on the side of the fixing portion. The isolation of the sample liquid is improved.

[0026] In one embodiment, the discharge channel has a cylindrical, conical shape or a combination of conical and cylindrical shapes. The volume of the drops is controlled.

[0027] In one embodiment, the body comprises a wall, opposing the flow of liquid from the container, arranged between the discharge channel and the transfer channel, said wall having at least one orifice with a diameter smaller than the diameter of the discharge channel, so that the liquid from the container passes through the wall only through the orifice. The wall makes it possible to interrupt the flow of liquid in the discharge channel if the interior of the container is not pressurized.

[0028] In one embodiment, a reagent delivery device for biological samples comprises a container capable of containing and discharging a liquid, a liquid contained in the container, and is characterized in that it further comprises the cap according to the invention.

[0029] In one embodiment, the reagent diffusion device for biological samples further comprises a sealing cap capable of hermetically sealing the container or wherein the container comprises a heat-sealed cap capable of hermetically sealing said container. The risk of mixing the sample liquid with the reagent outside the sample preparation phase, during transport for example, is eliminated.

[0030] In one embodiment, a method of producing a cap for a reagent delivery device for biological samples comprises the steps of lyophilizing a reagent on the reagent support made of porous material: - apply the reagent in liquid form to the reagent support - dehydrate the reagent at low temperature and low pressure and installation of the reagent holder inside the cap: - place the reagent holder in the housing - keep the reagent holder in the housing.

[0031] In one embodiment, a method of using a bottle cap includes the steps of mounting the cap on a container of liquid, pressurizing the liquid in the container, and applying drops of liquid obtained by release of the cap onto a drop-off location, surface or container. The process of preparing biological samples is simple and quick. Thus, it can be carried out by people without any particular qualifications.

[0032] Other characteristics and advantages of the invention will appear on examining the detailed description below, and the appended drawings, in which:

[0033] [Fig-1] illustrates an exploded view of the reagent diffusion device for bio samples logics comprising a stopper according to one aspect of the invention.

[0034] [Fig.2] illustrates a longitudinal cross-sectional view of the stopper of [Fig.1].

[0035] [Fig.3] illustrates a detailed view of the cross-sectional view of [Fig.2].

[0036] [Fig.4] illustrates a bottom view of the cap of [Fig.l].

[0037] [Fig.5] illustrates the different parts that make up a sample test kit biological using the biological sample reagent diffusion device of [Fig.1].

[0038] [Fig.6] illustrates in side view the first step of using the kit of [Fig.5], namely the taking of a sample.

[0039] [Fig.7] illustrates in exploded view the second step of the use of the kit of [Fig.5].

[0040] [Fig.8] illustrates in side view the third stage of the use of the kit [Fig.5].

[0041] [Fig.9] illustrates in exploded view the fourth step of the use of the kit of [Fig.5].

[0042] [Fig. 10] illustrates in side view the fourth step of using the kit of [Fig.5].

[0043] [Fig. 11] illustrates in perspective the fifth step of using the kit of [Fig.5].

[0044] [Fig. 12] illustrates in side view the fifth step of using the kit of [Fig.5].

[0045] [Fig. 13] illustrates the sixth step of using the kit of [Fig.5].

[0046] [Fig. 14] illustrates the seventh step in using the kit of [Fig.5].

[0047] Reference is made to Figures 1, 2, 3 and 4.

[0048] A reagent dispensing device for biological samples 1 includes a container 3 and a cap 5. The cap 5 is attached to the container 3 by means of a fastener 4. Here, the cap 5 is a dropper cap.

[0049] A sample to be tested is mixed in a carrier liquid 10. The liquid obtained after mixing the sample in the carrier liquid 10 is defined as a sample liquid 101. The carrier liquid 10 may be water, a lysis buffer, a sample discharge buffer or a transport buffer. The sample can be a sample for PCR testing or for another type of analysis.

[0050] The stopper 5 further receives at least one reagent 41 capable of diffusing into the sample liquid 101. The liquid obtained after diffusion of the reagent 41 is defined as being a reaction liquid 102.

[0051] The container 3 is capable of containing and discharging the carrier liquid 10 or the sample liquid 101. The discharging of the carrier liquid 10 or the sample liquid 101 is generally carried out by an increase in pressure in the container 3. The device 1 is configured to generate and deposit drops 19 of reaction liquid 102 on a deposit location 2, a surface or a container which may be a tube or a well plate.

[0052] Here, the container 3 comprises a body 7 of revolution. The body 7 of revolution comprises a reservoir 9 and a fixing portion 11 to the cap 5. The reservoir 9 here has a generally cylindrical shape with a side wall 13 and a bottom 15 at a first end of the reservoir 9. The reservoir 9 is hollow. Here, the reservoir 9 is deformable and flexible. The reservoir 9 has an opening 17 at the fixing portion 11. The opening 17 here has a disc shape.

[0053] An external compression of the reservoir 9 of the container 3 increases the pressure inside the container 3, so that the sample liquid 101 from the reservoir 9 passes through the fixing portion 11 when it is discharged from the container 3, thus discharging the sample liquid 101 contained in the container 3 through the opening 17 of the reservoir 9 and the fixing portion 11. The capacity of the bottle is less than 1 liter, here preferably between 0.5 milliliters and 100 milliliters.

[0054] Here, the container 3 is made of a flexible polymer material.

[0055] Alternatively, the container may be a syringe.

[0056] The cap 5 includes a body 21. The body 21 includes a first end 12 and a second end 14. The body 21 includes an evacuation portion (or nose) 23 at the first end 12 and a fixing portion 25 to the container 3 at the second end 14.

[0057] The body 21 comprises an outlet opening 29 formed in the first end 12. The body 21 comprises an introduction opening 6 formed in the second end 14.

[0058] The discharge portion 23 comprises a discharge channel 27. The discharge channel 27 opens into the outlet opening 29 of the first end 12.

[0059] The outlet opening 29 has a contour forming an edge 30, called a detachment edge, preferably sharp, so as to facilitate the formation of the drops 19.

[0060] The discharge portion 23 is here elongated. Here, the discharge portion 25 is between 0.5 mm and 20 mm.

[0061] The body 21 further comprises a transfer channel 16. The transfer channel 16 opens onto the introduction opening 6 of the second end 14. The transfer channel 16 is fluidically connected to the discharge channel 27.

[0062] The sample liquid 101 from the container 3 can flow into the plug 5 by passing through the transfer channel 16 and be discharged through the discharge channel 27.

[0063] Here, the body 21 of the plug has a shape of revolution, and the evacuation channel 27 has an axis of revolution. The transfer channel 16 has also an axis of revolution. The axis of revolution of the channel 27 and the axis of revolution of the transfer channel 16 are collinear. Alternatively, the axis of revolution of the channel 27 and the axis of revolution of the transfer channel 16 may be angularly offset. Alternatively, the discharge channel has a curved shape.

[0064] The discharge channel 27 is adapted to convey the reaction liquid 102 to the outlet 29. The discharge channel 27 and the detachment edge 30 work together to form the droplets 19 of reaction liquid 102. The discharge channel 27 has a shape of revolution: cylindrical, truncated cone, or a combination of these shapes. Here, the discharge channel has a truncated cone shape, widening in the direction of flow. Here, the diameter of the outlet 29 is between 0.2 mm and 4 mm. The resulting droplets 19 have a precise volume determined according to the dimensions of the discharge channel 27. Thus, the volume of the droplets 19 is reproducible.

[0065] The discharge channel 27 comprises an inner edge 42 and a transverse surface area of ​​between 0.3 and 15 mm2. The discharge channel 27 has a length of between 0.5 mm and 100 mm, preferably between 0.5 mm and 20 mm.

[0066] Preferably, a chamfer 45 is provided on the body 21 between the edge 30 of the outlet opening 29 and the discharge channel 27. Here, the chamfer 45 has an angle of between 0° and 60°. The chamfer 45 and the edge 30 facilitate the separation of the drop 19 from the body 21 of the cap 5.

[0067] The fastener 4 seals the fastening portion 11 of the container 3 to the fastening portion 25 of the cap 5. Generally, the fastener 4 comprises a first part 191 carried by the container 3, and more precisely the fastening portion 11, and a second part 192 carried by the cap 5. The first part 191 is capable of engaging the second part 192 so that the cap 5 and the container 3 are secured when the first part 191 and the second part 192 are engaged.

[0068] Here, the first part 191 of the fastener 4 is a thread located on the outside of the fastener portion 11 of the container 3. The second part 192 of the fastener 4 is a tapped hole located on an inside wall of the transfer channel 16. The fastener 4 is made by screwing the first part 191 of the fastener 4 onto the second part 192 of the fastener 4. Thus, here, the fastener portion 11 of the container 3 enters the fastener portion 25 of the cap 5.

[0069] Alternatively, the first fastening element may be a tapped hole located on the inner wall of the container's fastening portion. The second fastening element may be a thread located on an outer edge of the cap's fastening portion. In this embodiment, the cap's fastening portion penetrates the container's fastening portion.

[0070] Alternatively, the first attachment portion and the second attachment portion may Each groove or smooth surface must be a matching shape. The cap is attached to the container by inserting it into the container.

[0071] Here, the fastening portion 11 of the container 3 includes an annular projecting portion 35 on the outside of the fastening portion 11.

[0072] The stopper 5 comprises a reagent support 39. The reagent support 39 comprises the reagent 41 in dried or lyophilized form. The reagent support 39 equipped with the reagent(s) 41 is capable of diffusing the latter into a liquid passing through it, here the sample liquid 101.

[0073] The use of a porous support to receive the reagent is particularly innovative. The Applicant is not aware of any solution that uses this approach. The difficulty with this solution lies in choosing the substrate for the reagent support 39, as it must allow the fluid to pass through homogeneously, quickly, and efficiently while also being compatible with the reagent(s) 4L. Some conventional substrates are inhibitory and absorb certain molecules of the reagent(s) 4L. Other substrates do not allow for optimization of the fluidic path of the liquid.

[0074] The reagent support 39 is here made of porous material, preferably cellulose fiber or cellulose acetate. The reagent support 39 can also be made of polyurethane or PVA. The reagent support 39 comprises pores distributed uniformly throughout the reagent support 39. The reagent support 39 has a porosity of between 50 μm and 1500 μm. The reagent support 39 is made of compressible material.

[0075] The reagent 41 is deposited on the reagent support 39 before the latter is installed in the stopper 5. The method of depositing the reagent 41 on the reagent support 39 comprises the deposition of the reagent 41 in liquid form on the reagent support 41 and the lyophilization of all of the reagent 41 according to a low temperature and low pressure dehydration process.

[0076] The reagent supports 39 containing the reagent 41 in liquid form are arranged in a freeze dryer on a thermal transfer element for freeze-drying (tray, receptacle, or shelf). The freeze-drying process begins with a freezing phase at a temperature below the glass transition temperature of the reagent 41, generally between -10°C and -80°C, so as to form ice. Freezing is followed by a primary drying phase, allowing the ice to sublimate, at a pressure between 5 pbar and 500 pbar. The freeze-drying process ends with a secondary drying phase at a temperature above 0°C.

[0077] A non-exhaustive list of reagents that can be used is given below: enzymes (polymerase, reverse transcriptase), primers (sense, antisense), oligos, plasmids, RNA, DNA, dNTP, bacteria, viruses, antibodies, proteins, stabilizers, lyoprotectant (Mannitol, Trehalose, Bovine serum albumin), polyethylene glycol, unbranched polymer (Agarose), nanoparticles (gold, silver, silica, polymer), fluorophores (cyanine, fluorescein isothiocyanate, fluorescein, phycoerythrin, green fluorescent protein, rhodamine, SYBR Green), fluorescent probes and dyes.

[0078] The reagent 41 is distributed substantially uniformly in the pores of the reagent support 39. Thus, the reagent support 39 is able to diffuse the reagent 41 homogeneously in the sample liquid 101 during the flow of the latter. The reaction liquid 102 is thus obtained downstream of the reagent support 39 by the diffusion of the reagent 41 in the sample liquid 101.

[0079] The transfer channel 16 of the body 21 further comprises a diffusion portion 18 formed in the latter. The diffusion portion 18 is capable of receiving the reagent support 41 in the manner of a housing. The reagent support 41 occupies substantially the entire volume of the diffusion portion 18, such that the sample liquid 101 from the container 3 is forced to pass through the reagent support 39 to reach the discharge channel 27.

[0080] The stopper 5 may comprise a separator 43. In the mounted state, the separator 43 is configured to fluidically isolate the reagent support 39 from the sample liquid 101 coming from the container 3. The separator 43 is arranged in the transfer channel 16 between the introduction opening 6 of the second end 14 and the reagent support 39.

[0081] In a first variant shown here, the separator 43 is made of a porous material, preferably cellulose fiber or cellulose acetate. The separator 43 can also be made of polyurethane or PVA. Thus, it acts as a dilution retardant. Here, the separator 43 has a porosity of between 50 μm and 1500 μm.

[0082] In a second variant, the separator may be made of an elastomeric material forming a membrane such as a septum. The separator is then equipped with orifices configured to open in the presence of an overpressure on the inside of the fixing portion, making it possible for the liquid to pass through the separator.

[0083] Here, the transfer channel 16 comprises a first portion 24, a second portion 26 and a third portion 28. The first portion 24 and the second portion 26 are fluidly connected. The second portion 26 and the third portion 28 are fluidly connected. The third portion 28 is fluidly connected to the discharge channel 27.

[0084] The first portion 24 is a receiving portion of the container 3. The sample liquid 101 from the container flows into the stopper 5 first through the first portion 24.

[0085] The first portion 24 here has an axis of revolution. The first portion 24 comprises an inner edge 31. The second part 192 of the fixing 4, here a thread, is made in the inner edge 31. The first portion 24 ends with a bottom 8 opposite the insertion opening 6. The bottom 8 is substantially flat and perpendicular to the axis of revolution of the first portion 24. An opening 81 is provided in the body 21 from the bottom 8 for fluid communication with the second portion 26, so that the opening 81 opens onto the second portion 26.

[0086] The second portion 26 here has an axis of revolution. The second portion 26 is cylindrical and includes an inner edge 32. Alternatively, the second portion 26 could be conical. The diffusion portion 18 is formed in the second portion 26. The reagent support 39 has a shape analogous to the diffusion portion 18.

[0087] Like the second portion 26, the diffusion portion 18 can have a cylindrical shape, a truncated cone shape or a combination of these shapes, or even a spherical shape.

[0088] The volume of the second portion 26 is occupied on the one hand by the reagent support 39 and on the other hand by the separator 43. The separator 43 also has a shape similar to the second portion 26.

[0089] The second portion 26 has an inner diameter of between 1 mm and 15 mm. The reagent support 39 and the separator 43 each have an outer diameter. The outer diameter of the reagent support 39 is slightly greater than the inner diameter of the second portion 26. The reagent support 39 is thus tightly mounted in the second portion 26 and more precisely in the diffusion portion 18. The outer diameter of the separator 43 is slightly greater than the inner diameter of the second portion 26. The separator 43 is thus also tightly mounted in the second portion 26.

[0090] Alternatively, the separator and the reagent holder could be glued into the second portion. Alternatively, the separator and the reagent holder could be embedded in the second portion.

[0091] The third portion 28 here presents an axis of revolution. Here, the axis of revolution of the first portion 24, the axis of revolution of the second portion 26 and the axis of revolution of the third portion 28 are collinear.

[0092] The third portion 28 is a connecting portion. The third portion 28 is adapted to channel the reaction liquid 102 exiting the second portion 26 via the reagent support 39 to the discharge channel 27. The third portion 28 comprises a first end adjacent to the second portion 26 and a second end adjacent to the discharge channel 27.

[0093] Here, the third portion 28 has a shape that is first cylindrical and then frustoconical. diameter decreasing in the direction of flow and has an inner edge 44.

[0094] Alternatively, the diffusion portion and the reagent support may have a conical or spherical shape.

[0095] The body 21 may comprise at least one stop for stopping the reagent support 39 which defines the end of the portion 26 opposite the bottom 8. The stop is configured to stop the reagent support 39 in translation during its insertion into the diffusion portion 18.

[0096] Here, the third portion 28 comprises several stops in the form of fins 40. The fins 40 project from the inner edge 44 in the vicinity of the second portion 26. The fins 40 are regularly distributed along the circumference of the inner edge 44. Alternatively, the stop may be a shoulder present over the entire circumference of the second portion.

[0097] Preferably, a wall 51 is arranged between the discharge channel 27 and the transfer channel 16. The wall 51 comprises at least one orifice 52 of diameter smaller than the smallest diameter of the discharge channel 27 so as to form a capillary barrier. The orifice 52 has an edge distant from the edge 42 of the discharge channel 27. The wall 51 makes it possible to interrupt the flow of the reaction liquid 102 in the discharge channel in the absence of a positive pressure difference between the transfer channel 16 and the discharge channel 27. Here, the wall 51 extends perpendicular to the flow and has a thickness of between 0.2 mm and 10 mm, preferably between 0.2 mm and 2 mm.

[0098] In the assembled state, the cap 5 and the container 3 can form a leak-proof connection. Here, the seal is achieved by a flat gasket 47 disposed on the base 8. The flat gasket 47 has a disc shape in which a hole 49 is formed. In the assembled state, the hole 49 is positioned opposite the second portion 26 so as to allow the passage of the sample liquid 101. Here, the flat gasket 47 is compressed between the base 8 and the first end of the container 3.

[0099] Alternatively, the seal may be achieved by a baffle or a radial seal arranged on the fixing portion of the container or on the fixing portion of the cap.

[0100] Here, the stop in translation of the stopper 5 on the container 3 is achieved by the contact of the first end of the container 3 on the flat seal 47. In the absence of a seal, the stop in translation of the stopper 5 on the container 3 is achieved by the contact of the first end of the container 3 on the bottom 8.

[0101] Here, the cap 5 includes a gripping surface 33 on an outer surface. The gripping surface 33 is adapted to facilitate the relative movement of the cap 5 with respect to the container 3, particularly rotation. Here, the gripping surface 33 includes grooves.

[0102] Here, the stopper 5 is made of a polymer material compatible with the reagents for PCR and not an inhibitor of at least one of the reagent components. The stopper 5 is made of a material preferably compatible with injection molding techniques such as PP, HDPE, LDPE, PET or PS.

[0103] Reference is made to [Fig.5].

[0104] The reagent delivery device for biological samples 1 may be part of a biological sample test kit which further comprises a sealing cap 53. The sealing cap 53 is capable of hermetically sealing the container 3. The sealing cap 53 is fixed to the container 3 in the same manner as the cap 5, via a fixing portion 55. The sealing of the container 3 using the sealing cap 53 makes it possible to transport and store the container 3 when it contains sample liquid 101 or carrier liquid 10. The sealing cap 53 may be made of a polymer material (PP, HDPE, LDPE, PET, PS). Alternatively, the sealing cap 53 may take the form of a lid heat-sealed on the container 3. The lid comprises an aluminum film complexed with a heat-sealing resin.

[0105] The biological sample test kit may further comprise a sample recovery instrument, here a swab 57. Alternatively, the sample recovery instrument may be a syringe, a pipette, a biocollector, another container or forceps.

[0106] The biological sample test kit may further comprise a support 59 comprising the deposit location 2 of the reaction liquid 102, here a PoC (Point-of-Care) reaction chip. Alternatively, the support comprising the deposit location may be a container.

[0107] In the biological sample test kit, the cap 5 may be packaged in an individual package that is airtight against oxygen and moisture. In a preferred embodiment, the individual package includes a desiccant element. The package is also capable of protecting the cap from visible and ultraviolet light. The package is generally made of aluminum and is closed by heat sealing.

[0108] The method of using the biological sample test kit comprises the following steps.

[0109] In a first step, illustrated in [Fig.6], the sample is taken using the recovery instrument, here the swab 57.

[0110] Here, in the context of a PCR test, a nasal sample is taken from the patient using swab 57.

[0111] In the context of environmental health control or species monitoring in the food industry, sampling consists of taking a portion of the liquid to be tested.

[0112] In a second step, illustrated in [Fig.7], the container 3 is opened by removing the sealing cap 53, here by unscrewing it.

[0113] In a third step, illustrated in [Fig.8], the sample is introduced into the support liquid 10 contained in the container 3. In this step, the mixture comprising the sample and the support liquid 10 is homogenized so as to distribute the sample homogeneously in the support liquid 10, thus forming the sample liquid 101. For this, the mixing is carried out here using the swab 57.

[0114] In a fourth step, illustrated in Figures 9 and 10, the cap 5 is installed on the container 3 to obtain the biological sample reagent dispensing device 1. The first part 191 of the fastener 4 is engaged with the second part 192 of the fastener 4. The cap 5 is then screwed onto the container 3. The assembly is carried out vertically to avoid emptying the container 3 of its contents. In the variant where the cap is packaged, the cap is first removed from its packaging before being installed on the container 3.

[0115] In a fifth step, illustrated in Figures 11 and 12, the sample liquid 101 is discharged from the container 3 in order to form drops of reaction liquid 102. In a first step, the diffusion device 1 is turned upside down. The stopper 5 is oriented downwards vertically, or at an angle between 0° and 90° with the vertical. In a second step, pressurizing the interior of the container 3 allows the sample liquid 101 to flow through the opening 17 of the fixing portion 11 of the container 3. Here, the pressurization is obtained by compressing the container 3, the container 3 being flexible. Alternatively, the pressurization of the interior of the container can be achieved by moving a piston, for example in the case of a syringe.

[0116] The sample liquid 101 then flows into the plug 5 and passes through the separator 43 and then the reagent holder 39. The sample liquid 101 comes into contact with the reagent 41 arranged on the reagent holder 39. The reagent 41 diffuses into the sample liquid 101, thus forming the reaction liquid 102.

[0117] Here, the reaction liquid 102 passes through the wall 51 via the orifice 52. The reaction liquid 102 flows into the discharge channel 27 in the form of drops 19 of reaction liquid 102. The drops 19 leave the plug 5 via the outlet opening 29. Depending on the pressure exerted on the container 3, one or more drops 19 of reaction liquid 102 pass through the plug 5 and exit via the outlet opening 29.

[0118] Here, the biological sample reagent diffusion device 1 is inclined downwards to emit the droplets 19 of reaction liquid 102. The discharge portion 23 and more particularly the orifice 29 are oriented towards the deposition site 2 of the reaction liquid 102. At least one of the droplets 19 is deposited on the deposition site 2, here on the PoC reaction chip.

[0119] In a sixth step, illustrated in [Fig.13], the support 59 is introduced into an analysis device 60 of the reaction liquid 102 to be analyzed. Here, the device analysis performs thermocycling then optical monitoring.

[0120] In a seventh step, illustrated in [Fig.14], the sample is analyzed and the results are communicated.

[0121] Generally, the results are obtained after a waiting time of a few minutes to a few dozen minutes. All these operations can be carried out by someone without specific technical qualifications.

Claims

Claims

1. Cap (5) for a reagent diffusion device for biological samples comprising a body (21) having a first end (12) and a second end (14), said body (21) further comprising a liquid discharge channel (27) opening at the first end (12) and a fixing portion (25) at the second end (14) suitable for being mounted on a container (3) of a reagent diffusion device for biological samples, the body (21) further comprising a transfer channel (16) opening at the second end (14) and fluidically connected to said discharge channel (27), so that a liquid contained in said container (3) on which said cap (5) is mounted is suitable for being discharged through the discharge channel (27), characterized in that the transfer channel (16) further comprises a diffusion portion (18) forming a housing, the diffusion portion (18) comprising a volume,the stopper (5) further comprising a reagent support (39) made of porous material receiving at least one lyophilized reagent (41), which reagent support (39) occupies substantially the entire volume of the diffusion portion (18) so that a liquid coming from the container and flowing through towards the discharge channel (27) is forced to pass through the reagent support (39).,

2. A cap (5) according to claim 1, wherein the reagent carrier (39) is made of cellulose fiber, polyurethane, PVA or cellulose acetate.

3. Cap (5) according to claim 1 or 2, wherein the reagent support (39) has a porosity of between 50 pm and 1500 pm.

4. Cap (5) according to one of the preceding claims, wherein the reagent (41) is chosen from the group comprising: enzymes, primers, oligos, plasmids, RNA, DNA, dNTPs, bacteria, viruses, antibodies, proteins, stabilizers, lyoprotectants, polyethylene glycol, unbranched polymers, nanoparticles, fluorophores, fluorescent probes and dyes.

5. Cap (5) according to one of the preceding claims, further comprising a separator (43) arranged in the transfer channel (16) between the second end (14) and the reagent support (39), said separator (43) being capable of isolating the reagent from the liquid coming from the container (3) or delay its migration when the pressure in the transfer channel (16) is less than or equal to the pressure in the discharge channel (27).

6. Cap (5) according to claim 5, in which the separator (43) is arranged in the transfer channel (16), upstream of the reagent support (39), the separator (43) transversely occupying the entire housing, so that a liquid coming from the container and flowing through the discharge channel (27) is forced to pass through the separator (43) before passing through the reagent support (39).

7. A stopper (5) according to claim 5 or 6, wherein the separator (43) is made of a porous material, preferably cellulose fiber, polyurethane, PVA or cellulose acetate.

8. Cap (5) according to claim 5 or 6, in which the separator (43) comprises an elastomeric membrane having orifices capable of opening in the event of overpressure on the side of the fixing portion.

9. Plug (5) according to one of the preceding claims, in which the discharge channel (27) has a cylindrical, conical shape or a combination of conical and cylindrical shapes.

10. Cap (5) according to one of the preceding claims, in which the body (21) comprises a wall (51), opposing the flow of liquid from the container (3), arranged between the discharge channel (27) and the transfer channel (16), said wall (51) having at least one orifice (52) of diameter smaller than the diameter of the discharge channel (27), so that the liquid from the container (3) passes through the wall (51) only through the orifice (52).

11. Reagent diffusion device for biological samples comprising: - a container (3) capable of containing and discharging a liquid, - a liquid contained in the container (3), characterized in that it further comprises a stopper (5) according to one of the preceding claims.

12. A reagent delivery device for biological samples according to claim 11 further comprising a sealing cap (53) capable of hermetically sealing the container (3) or wherein the container (3) comprises a heat-sealed cap capable of hermetically sealing said container (3).

13. A method of producing a cap (5) for a reagent diffusion device for biological samples according to one of claims 1 to 10 comprising the steps of lyophilizing a reagent (41) on the reagent support (39) made of porous material: - apply the reagent (41) in liquid form to the reagent support (39), - dehydrating the reagent (41) at low temperature and low pressure, and installing the reagent holder (39) inside the cap (5): - placing the reagent holder (39) in the housing, holding the reagent holder (39) in the housing.

14. A method of using a stopper (5) for a bottle, comprising the steps of: - mounting the cap (5) according to one of claims 1 to 10 on a liquid container, - pressurizing the liquid in the container, - application of drops of liquid obtained at the outlet of the cap to a deposit location, a surface or a container.

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