EP3962647A1 - Closure element for a microplate well having vents and a slot, and method for the use thereof - Google Patents

Abstract

The invention relates to a closure element (1) for a well for receiving a chemical or biological solution to be analysed, comprising, for each well, a plug (4) comprising an insertion skirt (5) taking the form of a cylinder provided with two truncated regions (15) formed in a symmetrically opposite manner on the outside of the skirt to form vents, this insertion skirt (5) being provided with an end wall (9) in which there is formed a through-cutout (18) oriented radially in the direction of the two truncated regions (15).

Description

Description

Title of the invention: BLOCKING ELEMENT FOR WELLS

MICROPLATE PRESENTING VENTS AND A SLOT AND METHOD FOR ITS USE

Technical area

[0001] The object of the invention relates to the technical field of the handling of fluid samples in chemical or biological processes in the general sense and it relates more specifically to the field of diagnostic kits for chemical or biological solution to be analyzed. Prior art

[0002] US Pat. No. 6,500,390 relates to the technical field of microplates intended to receive and prevent the loss of fluid samples during the various processing steps. This microplate has containers each fitted with a suitable stopper to confine the liquid sample inside the container, regardless of the position of the microplate. Each stopper is also provided with a central opening forming a vent to allow communication of the containers with the outside. By the presence of the vents, such a microplate does not make it possible to guarantee complete sealing, which constitutes a risk of contamination of the liquid samples.

[0003] Patent application WO 2015/059254 describes a lysis kit and a method for preparing a sample, such as, for example, a sample of biological material to be analyzed. This kit includes a tube intended to contain beads facilitating lysis and a lysis buffer solution. An elastically deformable cap is fitted to the open end of the tube to close it

hermetically. This cap has a slot allowing the passage of the tip of a pipette which allows the introduction of a sample of biological material and the aspiration of the lysate obtained. Such a kit is not suitable for allowing in particular a lyophilization operation which requires placing the tube in communication with the outside for the evacuation of the water. Removal of the cap is conducive to contamination of the sample material contained in the container or the working environment. Hood itself placed on the work surface can be a source of contamination. In addition, it is commonly observed that the sample expels when caps on small containers are removed. Finally, the operations of fitting and removing the caps are constraints for operators with regard to the number of analyzes they must perform.

[0004] Patent FR 2 416 848 describes a stopper for medicaments, in

particularly for products intended to be subjected to a treatment such as lyophilization. This stopper has a skirt intended to be inserted into a well by elastic deformation. This skirt is provided from its free end with an opening forming a vent when the plug occupies a partial recessed position in the well. This skirt is surmounted by a head cooperating with the well to ensure its leaktight closure when the stopper is pushed completely into the well. To use the product, the stopper is pierced with a hollow needle. In practice, such a stopper is difficult to penetrate by relatively blunt tips such as disposable plastic pipette tips or disposable flexible plastic pipette tips.

[0005] The present invention aims to remedy the drawbacks of the prior art by providing a closure element for a receiving well for a chemical or biological solution to be analyzed, compatible on the one hand with the implementation of a operation requiring communication with the outside such as a freeze-drying operation and, on the other hand, a sealed storage operation, this closure element also allowing rapid and reliable access to the contents of the reception well to the using a plastic pipette.

Disclosure of the invention

To achieve such an objective, the invention relates to a closure element for a well for receiving a chemical or biological solution to be analyzed, comprising for each well, a plug comprising a skirt for inserting into the well by deformation elastic, this insertion skirt delimiting from the free end of the skirt, at least one vent when the stopper occupies a partial depression position, the plug comprising a sealed part positioned so that in a more depressed position of the stopper by With respect to the partial depression position, this part cooperates with the well to ensure its tight closure.

According to the invention, the insertion skirt is in the form of a cylinder provided with two truncated areas arranged symmetrically opposite on the outside of the skirt to form vents, this insertion skirt being provided with a bottom wall in which is arranged an emerging cutout, oriented radially in the direction of the two truncated zones, this cutout extending between two passage faces of the bottom wall, in contact with one another for ensure a seal and allow by elastic deformation, the passage of a tip of a pipette.

[0008] In addition, the closure element according to the invention may further include in combination at least one and / or the other of the following additional characteristics:

- cutting is performed without removing material;

- each truncated zone extends over an angular range of the skirt, with the cutout extending in a direction intersecting each of the truncated zones substantially in its middle;

- each truncated zone extends over an angular range of the insertion skirt of between 40 ° and 160 °;

- the two truncated areas serving as vents are flats formed from the free end of the insertion skirt and diametrically opposed in the extension of the cutout;

- the truncated areas leave between them, an area of the skirt having an outer diameter greater than the diameter of the well to ensure, when the stopper occupies its closed position of the well, a deformation of the skirt directing the mechanical stresses in a substantially perpendicular direction with the cutout tending to support the passage faces against one another;

- the waterproof part is part of the insertion skirt overhanging the truncated areas;

- the sealed part is a cylindrical head connected to the insertion skirt by a shoulder which cooperates in a sealed manner with the edge of the well;

- each stopper has an internal cavity delimited by the inside of the skirt insertion and opening at the level of the cylindrical head, by a guide chamfer for the pipette tip;

- each cap is provided with a removable attachment system to a backing sheet from which protruding the insertion skirts of a series of caps.

Another object of the invention is to provide a kit for a chemical or biological solution to be analyzed, comprising a container provided with at least one well each receiving a reagent, a cap of a sealing element ensuring the closure of each well.

[0010] According to an advantageous characteristic of the kit, the truncated areas of the

stopper leave between them, a zone of the skirt having an outside diameter greater than the diameter of the well to ensure, when the stopper occupies its closed position of the well, a deformation of the skirt directing the mechanical stresses in a direction substantially perpendicular to the cutout tending to support the passage faces against one another.

[0011] Another object of the invention is to provide a method of using a kit for a chemical or biological solution to be analyzed, comprising a container provided with at least one well, the method consisting of:

- pre-fill each well with an aqueous reagent;

- Prior to a treatment step, place in each well, in its partial depression position, a plug of a sealing element according to the invention;

- Perform the treatment step during which a gaseous medium is evacuated from each well through the vents of the plugs;

- and lower each cap to ensure the tight closure of each well after the treatment operation.

[0012] Advantageously, the treatment step is a lyophilization step.

[0013] According to an alternative embodiment, the method consists, after the tight closure by a plug of each well after the treatment operation, in:

- Introduce the tip of a pipette for injecting an aqueous solution into the cutout of each stopper; - deliver the aqueous solution into the well using the pipette;

- remove the pipette tip from each stopper;

- and ensure an amplification operation, before a detection operation.

[0014] Advantageously, the amplification operation is preceded by a centrifugation operation.

[0015] According to a characteristic of the invention, the aqueous solution contains a

chemical or biological sample to be analyzed.

[0016] According to another characteristic of the invention, the caps are removed to be replaced by an optical cap prior to an amplification operation preceding a detection operation.

Various other characteristics emerge from the description given below with reference to the accompanying drawings which show, by way of non-limiting examples, embodiments of the object of the invention.

Brief description of the drawings

[0018] [Fig. 1] Figure 1 is a perspective view of an exemplary embodiment of the closure element according to the invention.

[0019] [Fig. 2] Figure 2 is a perspective view from below, showing the slit of the closure element according to the invention.

[0020] [Fig. 3] Figure 3 is a sectional view of the closure element according to the invention, taken along a plane perpendicular to the slot.

[0021] [Fig. 4] Figure 4 is a sectional view of the closure element according to the invention, taken along the lines IV-IV of Fig. 3.

[0022] [Fig. 5] Figure 5 shows a closure element according to the invention, partially engaged inside a well.

[0023] [Fig. 6] Figure 6 shows a closure element according to the invention, fully engaged inside a well.

[0024] [Fig. 7] Figure 7 is a sectional view of a closure member taken perpendicular to the direction of the slot of the stopper and showing the tip of a pipette engaged through the slot of a closure member according to invention, mounted inside a well. [0025] [Fig. 8] Figure 8 is a partial view of a kit comprising a support plate of several wells equipped with plugs of a closure element according to the invention, capable of being held together by a support sheet.

Description of embodiments

As emerges more specifically from the Figures, the object of the invention

relates to a closure element 1 intended for use with a receptacle 2 for receiving a chemical or biological solution to be analyzed. According to a preferred application illustrated in FIG. 8, the container 2 is in the form of a microplate having a series of wells 3 each intended to receive a chemical or biological solution to be analyzed. The closure element 1 comprises for each well 3, a plug 4 in accordance with the invention which is illustrated more precisely in FIGS. 1 to 4.

Each plug 4 has an insertion skirt 5 intended to be engaged in a well 3 by elastic deformation. This insertion skirt 5 is in a tubular form or in the form of a cylinder of circular section, the diameter of which is adapted to the diameter of the well 3 to ensure the tight fitting and by elastic deformation of the stopper inside the well. . The insertion skirt 5 thus has an outer face 5e and an inner face 5i.

[0028] The insertion skirt 5 is connected to a cylindrical head 6 by means of a shoulder 7 forming a sealed part intended in the complete insertion position of the plug, to cooperate with the edge of the well 3.

This insertion skirt 5 is provided opposite the head 6, with a bottom wall 9. The stopper 4 thus has an internal cavity 11 delimited by the inside of the skirt 5 and more precisely by the internal face 5i. This internal cavity 1 1 is open at the level of the cylindrical head 6 and closed by the bottom wall 9. In the example illustrated, the bottom wall 9 is provided at the level of the free end of the insertion skirt 5 but it should be noted that the bottom wall 9 may extend back from the free end of the insertion skirt 5.

According to an advantageous characteristic of embodiment, the internal cavity 1 1 opens at the level of the cylindrical head 6, by a chamfer 13 which like this will be explained in the remainder of the description serves as a guide for the tip of a pipette.

[0031] According to one characteristic of the invention, the insertion skirt 5 defines from the free end of the skirt two vents 14 when the stopper occupies a partial depression position as illustrated in FIG. 5. For this purpose, the insertion skirt 5 is provided with two truncated zones 15 arranged in a symmetrically opposed manner on the outside of the insertion skirt 5 and from the free end of the insertion skirt. As appears more precisely in Figs. 1, 2 and 4, each truncated zone 15 is a flat formed on the external face 5e of the insertion skirt 5 so that the insertion skirt has a frustoconical profile on the outside at the locations of the flats. Each flat 15 corresponds to a removal of material, the thickness of which, removed in the example illustrated, gradually decreases from the free end of the insertion skirt. Each flat 15 is arranged from the free end of the insertion skirt 5 extending along a determined height of the insertion skirt to stop behind the head 6 so as to leave a part sealed 16. The insertion skirt 5 thus has a sealed part 16 which has a circular section overhanging the flats 15. It should be understood that each truncated zone 15 corresponds to a material removal made on the outer face 5e of the skirt d The insertion 5 so that the section of the insertion skirt 5 passing through these truncated areas is a symmetrically truncated circular section, smaller than the circular section of the sealed part 16.

It should be understood that when this sealed part 16 cooperates with the

well 3, the stopper 4 ensures the tight closing of the well 3. When the tight part 16 cooperates with the well 3, the stopper 4 is more pushed in than in the partial push-in position for which the vents 14 provide communication with the outside. The tight closure of well 3 by the

stopper 4 is obtained either for a partial depression position for which the sealed part 16 cooperates with the well 3 or either for a complete depression position for which this sealed part 16 cooperates with the well and the head 6 by its shoulder 7 rests tightly on the edge of the well 3. It should be understood that each flat 15 extends over a limited angular range of the insertion skirt so as to leave between them, two contact zones 5c delimited by the external face 5e. In the partial depression position (Fig. 5), the stopper 4 cooperates with the well 3 via these contact zones 5c, ensuring that the stopper is held in position inside the well.

Advantageously, each truncated zone 15 extends over an angular range of the insertion skirt 5 of between 40 ° and 160 °, this angular range being taken at the level of the free end of the insertion skirt 5.

[0034] According to another characteristic of the invention, the bottom wall 9 has an emerging cutout 18 which passes right through the bottom wall 9, extending between two passage faces 9a of the bottom wall. This cutout 18 thus communicates with the internal cavity 1 1 and with the environment delimited by the external face of the bottom wall 9. According to an advantageous characteristic of embodiment, this cutout 18 is made without removing material using for example of a sharp tool. This cutout 18 is rectilinear in a dimension suitable for allowing the passage, by elastic deformation of the passage faces 9a, of a tip 19 of a pipette (Fig. 7). This cutout is arranged diametrically or radially with respect to the insertion skirt 5.

[0035] According to a characteristic of the invention, this cutout 18 is oriented

radially or diametrically in the direction of the two truncated zones 15. Thus, the cutout 18 extends in a direction which intersects each of the truncated zones 15. Advantageously, the cutout 18 has a direction which intersects each of the truncated zones 15 substantially in its middle. According to a preferred variant embodiment, the cutout 18 does not extend as far as the flats 15. In other words, the cutout 18 does not open onto the external face 5e of the skirt 5. This cutout 18 allows by elastic deformation , the passage of the tip 19 of a pipette and ensures, in the absence of the tip 19 of the pipette, a seal by the two passage faces 9a of the bottom wall in contact with one of the other. It should be noted that when the plug 4 occupies its closed position of the well, the insertion skirt 5 is deformed by orienting the mechanical stresses in a direction substantially perpendicular to the cutout 18 tending to support the passage faces 9a l 'against each other, in because of the orientation of the cutout 18 in the direction of the flats 15 and the presence of these flats. The presence of the flats 15 avoid creating mechanical stresses due to the compression of the insertion skirt 5 directed along the axis of the cutout 18 which would tend to open this cutout.

The contact zones 5c defined between the flats 15 have an outside diameter adapted to the diameter of the well 3 to ensure elastic deformation of the insertion skirt ensuring the tight fitting of the plug. Typically, the diameter of the insertion skirt 5 is greater than the diameter of the well 3. It follows that this deformation of the insertion skirt 5 causes the support between them, of the passage faces 9a of the cutout which fits. thus found hermetically sealed.

[0037] According to an advantageous embodiment characteristic, each cap 4 is provided with a removable fastening system 22 to a support sheet 23. In the example illustrated in FIG. 8, each stopper 4 comprises, as a removable fixing system 22, studs or lugs arranged on the cylindrical head 6 of the stopper and intended to cooperate with the housings 25 provided on the support sheet 23. Of course, the shapes of the studs 22 and housings 25 are congruent and the dimensions of the nipples 22 and housings 25 are adapted to obtain a fitting by elastic deformation during the insertion of each stud 22 in a complementary housing 25. In the mounted position of the caps 4 on the sheet support 23, the insertion skirts 5 of the plugs 4 protrude from the support sheet 23. The closure element 1 according to the invention thus comprises a series of plugs 4 connected to each other. by the support sheet 23 allowing simultaneous handling of the series of caps 4. The caps 4 can thus be simultaneously engaged in the wells 3 of a microplate. Likewise, the plugs 4 can be detached from the backing sheet 23 while remaining inserted in the wells 3 when applying a lifting force on the backing sheet 23.

Of course, each plug 4 can be provided with a removable fixing system 22 to a support sheet 23, different from that illustrated by way of example in FIG. 8. For example, each plug 4 can be provided at its head 6, with a peripheral groove in which the edge of an opening made in the support sheet 23 engages. The implementation of the closure element 1 according to the invention follows directly from the above description. The closure element 1 comprises one or more plugs 4 each intended to equip a well 3 with a container 2, for example in the form of a microplate. The method

of use of this closure element 1 with a microplate 2 consists in carrying out the following steps:

- Pre-fill each well 3 with an aqueous reagent in the general sense. The term “reagent” is understood to mean any substance used for the preparation of the chemical or biological solution to be analyzed. In the case of a sample of biological material to be analyzed, the reagents are PCR mix reagents (enzymes, nucleotides and other components necessary for amplification);

- Equip each well 3 with a plug 4 according to the invention in a partial depression position in which the plug 4 delimits with the well 3, two vents 14. As illustrated in FIG. 5, the stopper 4 is held in a stable position inside the well 3 by the contact zones 5c of the insertion skirt 5. The flats 15 of the insertion skirt 5 allowing communication through the vents 14, between the interior of well 3 and the exterior environment;

carrying out a step of processing the contents of each well 3, during which a gaseous medium is evacuated from each well 3 through the vents 14 of the stoppers 4. Typically, such a processing operation can be an operation of freeze-drying the contents. of well 3. In a preferred embodiment, each well 3 is pre-filled with aqueous PCR reagents;

- Lower each stopper 4 to ensure the tight closure of each well 3 after the treatment operation. Typically, in the closed position illustrated in FIG. 6, the cylindrical head 6 of each stopper 4 comes to rest on the edge of the well 3. In this complete closed position, each stopper 4 seals off the well 3 by the cylindrical head 6 and the sealed part 16 of the skirt overhanging the truncated zones 15. Moreover, as explained above, in this position of insertion of the skirt 5 inside the well 3, the cutout 18 is hermetically closed by the passage faces 9a bearing the one against the other, undergoing mechanical stresses tending to close the cutout. It is thus possible to have a kit for a chemical or biological solution to be analyzed comprising a container 2 provided with at least one well 3 each receiving a reagent, a plug 4 of a sealing element 1 in accordance with the invention ensuring the closure of each well 3.

[0041] Such a kit can be used to allow the continuation of a method of use aimed at ensuring the analysis of an aqueous chemical or biological solution and advantageously, a sample of biological material. The method consists of introducing the tip 19 of a pipette to allow the injection of an aqueous solution through the cutout 18 of each stopper 4. Typically, this aqueous solution is a biological sample to be analyzed. This aqueous solution or biological sample is also used for rehydration of the reagent contained in the well, and in particular of the PCR mix reagents.

[0042] After the injection of this aqueous solution, the tip 19 of the pipette is removed from each stopper 4 before carrying out an amplification operation that may occur before a detection operation. The possibility of keeping the same cap throughout the process is particularly advantageous for the operators given the number of analyzes to be carried out. Keeping such a plug having a slit goes against the habits of operators in molecular biology.

[0043] It should be noted that the amplification operation can be preceded by a

centrifugation operation. Centrifugation is necessary to thoroughly mix the lyophilized reagents and the aqueous solution and to drive the whole to the bottom of the container. By dragging the mixture to the bottom of the container, centrifugation allows the stopper to be clean and prevent contamination. This operation also makes it possible to avoid the formation of bubbles. It should be noted that the caps 4 of the kit can be replaced by optical caps prior to an amplification operation preceding a detection operation.

The invention is not limited to the examples described and shown because various modifications can be made to it without departing from its scope.

Claims

Claims [Claim 1] Sealing element (1) for receiving well (3) of a chemical or biological solution to be analyzed, comprising for each well, a plug (4) comprising an insertion skirt (5) in the well by elastic deformation, this insertion skirt (5) delimiting from the free end of the skirt, at least one vent (14) when the stopper occupies a partial depressed position, the stopper (4) comprising a sealed part (7, 16) positioned so that in a more depressed position of the stopper, relative to the partial depression position, this part cooperates with the well to ensure its tight closure, characterized in that the insertion skirt (5) is in the form of a cylinder provided with two truncated areas (15 ) arranged symmetrically on the outside of the skirt to form vents (14), this skirt insert (5) being provided with a bottom wall (9) in which is arranged an emerging cutout (18), oriented radially in the direction of the two truncated zones (15), this cutout (18) extending between two passage faces (9a) of the bottom wall, in contact with one another to ensure a seal and to allow by elastic deformation, the passage of a tip (19) of a pipette. [Claim 2] A sealing element according to the preceding claim, according to which the cutout (18) is made without removing material. [Claim 3] Sealing element according to one of the claims previous ones, according to which each truncated zone (15) extends over an angular range of the skirt, with the cutout (18) extending in a direction intersecting each of the truncated zones (15) substantially in its middle. [Claim 4] Sealing element according to one of the claims previous ones, according to which each truncated zone (15) extends over an angular range of the insertion skirt (5) of between 40 ° and 160 °. [Claim 5] Sealing element according to one of the claims above, according to which the two truncated zones (15) serving as vents (14) are flats formed from the free end of the skirt insertion (5) and diametrically opposed in the extension of the cutout (18). [Claim 6] Sealing element according to one of the claims above, according to which the sealed part (16) is a part of the insertion skirt (5) overhanging the truncated areas (15). [Claim 7] Sealing element according to one of the claims above, according to which the sealed part (7) is a cylindrical head connected to the insertion skirt (5) by a shoulder which cooperates in a sealed manner with the edge of the well. [Claim 8] Sealing element according to one of the claims previous ones, according to which each plug (4) has a cavity internal (11) delimited by the inside of the insertion skirt (5) and opening at the level of the cylindrical head, by a guide chamfer (13) for the tip (19) of the pipette. [Claim 9] Sealing element according to one of the claims previous ones, according to which each plug (4) is provided with a system of removable attachment (22) to a support sheet (23) from which projecting the insert skirts (5) of a series of caps (4). [Claim 10] Kit for a chemical or biological solution to be analyzed, comprising a container provided with at least one well (3) each receiving a reagent, a stopper (4) of a sealing element (1) according to one of claims 1 to 10, ensuring the closure of each well (3). [Claim 11] Kit according to the preceding claim according to which the truncated zones (15) of the stopper leave between them, a zone (5c) of the skirt having an outer diameter greater than the diameter of the well to ensure when the stopper occupies its position of closure of the well, a deformation of the skirt directing the mechanical stresses in a direction substantially perpendicular to the cutout (18) tending to support the passage faces (9a) against one another. [Claim 12] A method of using a kit for a chemical or biological solution to be analyzed, comprising a container (2) provided with at least one well (3), the method consisting of: - pre-fill each well (3) with an aqueous reagent; - prior to a treatment step, placing in each well (3), in its partial depression position, a plug (4) of a sealing element (1) according to one of claims 1 to 10; - Performing the treatment step during which a gaseous medium is evacuated from each well (3) through the vents (14) of the plugs (4); - And lower each stopper (4) to ensure the tight closure of each well (3) after the treatment operation. [Claim 13] A method according to the preceding claim, wherein the treatment step is a lyophilization step. [Claim 14] Method according to one of claims 12 to 13, consisting after the tight closure by a plug (4) of each well (3) after the treatment operation, to: - Introducing the tip of a pipette (19) for injecting an aqueous solution into the cutout (18) of each stopper (4); - delivering the aqueous solution into the well (3) using the pipette (19); - remove the tip (19) of the pipette from each stopper (4); - And ensure an amplification operation, before a detection operation. [Claim 15] A method according to the preceding claim, wherein the amplification operation is preceded by a centrifugation operation. [Claim 16] A method according to claim 14 or 15, wherein the aqueous solution contains a chemical or biological sample to be analyzed. [Claim 17] Method according to one of claims 12 to 16, according to which the plugs are removed to be replaced by an optical plug prior to an amplification operation preceding a detection operation.