DE29505707U1 - Lid for closing vessels - Google Patents

Description

BOEHRINGER MANNHEIM GMBH 4136/OG/DE

Lids for closing containers

The invention relates to a lid for closing an opening of a vessel.

Lids are usually used to close vessels in order to reduce the penetration of environmental influences into the vessel or to prevent the contents of a vessel from escaping into the environment. In particular, if the vessel is intended to hold a sample whose components are to be further processed or analyzed later, a lid is mandatory in many cases. This applies to a greater extent in the field of analytics in the healthcare sector, food analysis, environmental analysis and molecular biology. In particular, in the case of particularly low-concentration analytes and analytes that are very similar to other analytes, contamination of the samples from the environment has a lasting effect on the analysis result. Nucleic acid analysis is particularly affected here. Avoiding contamination, both in its formation (active avoidance) and in its entry into the sample (passive avoidance), has proven to be a key requirement for nucleic acid diagnostics.

Nucleic acid diagnostic tests are currently carried out in particular in vessels that are based either on the general design of Eppendorf vessels (single vessels) or on the shape of microtiter plates (multiple wells). They are generally round and can be closed with a lid. The lids proposed so far for such vessels are either firmly attached to the sample vessel body or firmly connected to lids for adjacent vessels. This makes it easier to handle the lids manually when opening and closing the vessel. It must also be borne in mind that the vessels are quite small due to the small sample quantities available. This applies in particular to formats in which a large number of reaction vessels are accommodated next to one another in a very small space, such as the 96-well microtiter plate format. The lid designs proposed so far were therefore not or only with difficulty suitable for automation, i.e. opening and closing with the help of devices was difficult, if not impossible.

The subject of the invention is now a lid for closing an opening of a sample vessel containing a component which corresponds to the opening of the sample vessel to be closed

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and a component for gripping a shaped body that can be inserted into the sample vessel.

Regardless of the presence of the component for gripping the molded body, it was found that gripping lids in a particularly small space can be achieved particularly favorably by providing a component that extends essentially perpendicular to the opening of the vessel. This component preferably does not protrude beyond the lateral outer wall of the vessel. The lids used to date have components that protrude horizontally (laterally) far beyond the outer walls of the vessel.

Preferably, the lid is a single closure that can be completely and reversibly separated from the vessel.

Vessels that can be closed by the lid according to the invention are limited in their nature only by the presence of a closable opening (AlO). This opening is expediently positioned in an easily accessible location.

The advantages of the lid according to the invention will be particularly evident in vessels that are used in the analysis of ingredients in samples, such as body fluids. Therefore, the vessel to be closed is preferably a sample vessel A.

Sample vessels (A) within the meaning of the invention are vessels in which samples can be stored, in particular in liquid form, but also in a form bound to a solid matrix, but also vessels in which samples can be processed in their original or already purified form. They have at least one opening (AlO), but can also have further openings. A further opening can be, for example, an outlet opening (Al 1). The outlet opening (Al 1) can preferably also be closed. The opening (AlO) is preferably an opening through which a sample can be introduced into the vessel.

Sample vessels in the sense of the invention can have practically any external shape. However, sample vessels with a hollow cylindrical shape are particularly preferred, as are found in microtiter plates, for example. Preferred sample vessels have an essentially cylindrical shape with a total length of between 100 and 10 mm, particularly preferably of about 50 mm, an external diameter of between 20 and 5 mm, particularly preferably of about 9 mm and an internal diameter of 5-1 mm less than the external diameter, particularly preferably of about 7 mm. The opening (AlO), which is formed by the

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which can be closed with a lid according to the invention, is preferably located at one end of this hollow cylinder, particularly preferably at the upper end.

In order to ensure that the vessel is sealed as tightly as possible, the component (BIO) is designed so that it either tightly encloses the part of the vessel that surrounds the opening (e.g. the edge) from the outside, or, in the preferred case, is tightly fitted to the part of the vessel that surrounds the opening (AlO) inside the vessel. For this purpose, the component (BIO) is preferably a circumferential web that protrudes into the sample vessel, i.e. a ring, starting from the horizontal part (B 17) of the lid that covers the opening. Sealing lips (B 16) can also be provided on this web, which ensure a further improvement in the seal towards the sample vessel. The dimensions of the component (BIO) are preferably selected so that the lid can be removed from the sample vessel again after a desired time without the sample vessel or the lid being destroyed.

The component (B 11) is preferably located within the space surrounded by the component (B 10). Like the component (B 10), the component (B 11) is also preferably designed in such a way that it is adapted to the inner contour (C16) of the molded body (C) in such a way that the molded body automatically remains on the component after the component is pressed onto an opening in the molded body. Here, too, it is preferably a ring-shaped, circumferential web that is tightly adapted from the inside or outside to the part of the molded body surrounding the opening of the molded body. The outer surface of the circumferential web is preferably slightly conical, so that the conicity causes the molded body to be gripped and held. The opening in the molded body is therefore also closed by the cover according to the invention.

One finding of the invention is that the opening and closing of sample vessels is particularly dangerous for the introduction and removal of contamination. It has also proven to be disadvantageous that the lid must be removed during processes that involve the removal of liquids or gases from the sample vessel. During this time, the risk of contamination is particularly high. An advantage of a special embodiment of the lid according to the invention is that the lid can remain on the sample vessel even during processes that involve gas exchange with the environment, e.g. suction and cooling/heating processes. For this purpose, air passage openings (B 13) are provided in the lid, in particular on its upper side. It has proven to be particularly useful to cover these ventilation channels with a filter element (B 14) in the interior of the lid from the interior of the sample vessel or the interior of the molded body. These filters are preferably made of a sintered plastic material made of polyethylene, polypropylene or polyethersulfone or poly-

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sulfone. Such filters are available, for example, as sterile filters. The filter serves in particular to prevent aerosols that may arise during ventilation and deaeration processes during a sample processing process, e.g. a sample preparation process (during heating, mixing and suctioning as well as forcing liquids into the interior of the molded body) from escaping into the environment. At the same time, the introduction of undesirable aerosols/components of the ambient air into the sample vessel is prevented. The installation of the filter in the lid is particularly expediently carried out in such a way that the cross-sections of the ventilation channels only take up a maximum of 10% of the total usable area of the filter. It is particularly preferred that the filter material does not lie completely on the part of the lid covering the opening (B 17), so that a gap remains between the surface of the lid with the openings (B 13) and the top (usable area) of the filter. For this purpose, spacers (B 18) can be manufactured on the surface. This prevents excessive pressure loss and yet the entire surface of the sintered material can be used for flow.

In a second embodiment, the lid as a molded body (C) grips and closes a stamp (E), which also has an interior (E 12). Since no heating/cooling/extraction processes are carried out when using the stamp (E), no air openings closed by aerosol filters are required. The component (B11) can therefore also close from the outside via the removal opening (E 14) of the stamp. The stamp (E) is designed for a process for isolating nucleic acids from a porous, compressible matrix in such a way that it protrudes into a vessel containing the porous, compressible matrix. In this case, the vessel is called an elution vessel (D). The matrix is preferably part of a molded body (C), into the interior of which the stamp protrudes. By pressing on the lid (B), the stamp is pressed against the matrix and the liquid containing the nucleic acids penetrates into the interior of the stamp.

The closure and gripping of the vessels with the lid according to the invention is preferably reversible. If two different vessels are to be closed at the same time, the frictional connections to each vessel are preferably different. In the above-mentioned first embodiment, the frictional connection to the molded body (C) is greater than to the sample vessel (A). In the second embodiment, the frictional connection to the elution vessel (D) and the stamp (E) is so small that the lid detaches from both before the stamp detaches from the elution vessel.

In a particularly preferred embodiment, a component (B 12) extending essentially perpendicular to the closure opening for gripping the lid is located on the part of a lid facing away from the sample vessel (outside). The term

"essentially vertical" in the sense of the invention means that the component (B 12) has an axis of symmetry or plane that has an angle of between 85 and 90° to an imaginary plane above the closure opening. The component particularly preferably protrudes exactly vertically (with the exception of manufacturing tolerances) from the imaginary plane. The component (B 12) can lie outside the vessel to be closed when the vessel (A) is closed, but it is preferably positioned inside the vessel to be closed. The component is preferably rotationally symmetrical, in particular cylindrical, with the length of the cylinder being greater than its diameter. The diameter of this cylinder is preferably chosen to be as small as possible. In particular, the diameter of the cylinder is smaller than half the diameter of the part of the lid that covers the opening (AlO). This ensures that the lid is securely held over the component (B 12) by a gripping tong in the form of a so-called lid handle. This gripping tong can, for example, be used to hold the lid in place. B. the design of the mechanical pencils that are commonly used, in which the gripper opens when pressure is applied to a mechanical part provided for this purpose to hold the component and closes around the cylinder when the pressure is reduced from the outside. It has been proven that this type of gripping of components is particularly simple for the use of the lid to close sample containers and can even be implemented in the smallest of spaces, such as in 96-well microtiter plate formats. For reliable function (holding, positioning), it is particularly useful if the component (B 12) has a comparatively long guide in the gripper. The component (B 12) can have grooves that prevent the gripper from slipping on the component (B 12) and ensure a reliable holding function.

A particularly preferred embodiment 1 of the lid, which is shown in longitudinal section in Figure 1, consists of a cylindrical cap (B) with a cylindrical pin (B12) attached and several ventilation channels (B13), which are sealed from the inside with an aerosol filter (B14). The ring-shaped webs (B11) also serve to attach the filter (B14). The walls of a sample vessel (A) with an inner shape (A17) and an outer shape (A19) and a molded body (C) with an inner contour (C16) and an outer contour (C12) are indicated.

In Figure 2, a lid is shown in a state in which it closes a sample vessel (A) and a molded body (C) (embodiment 1),

Figure 3 shows schematically the intermediate steps of a nucleic acid isolation process.

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In Figure 4, a lid according to the invention is shown in a state in which it closes a molded body (C) and an elution vessel (D) (embodiment 2). The state in which the stamp (E) is also closed and gripped can be seen in Figure 3 (second to last partial drawing) or Figure 8.

Figure 5 shows a lid in which the component (B 12) is located inside the sample vessel.

Figure 6 shows a device for gripping the lid according to the invention schematically. Components for locking the respective operating state (empty, position B; gripping, position A) are also shown schematically.

Figure 7 shows a longitudinal section of a more advanced device for gripping the lid. It works on the principle of a mechanical pencil.

In the following, dimensions are given which have proven to be useful for the cover according to the invention in the first embodiment.

Outer diameter of the lid: 8.8 mm

Inner diameter of the lid: 7.2 mm

Height of the cover including component (B 12): 14.6 mm

Diameter of ventilation channels (B 13): 0.8 mm

Height of ventilation channels (B 13): 0.315 mm

Thickness of the filter (B 14): 1.6 mm

Radius of the component (B 11): 2.5 mm

Wall thickness of the component (B 11): 0.4 mm

Height of component (B 12): 7.0 mm

Diameter of the component (B 12): 1.9 mm

The corresponding internal dimensions of the sample vessel (A) and the molded body (C) are adapted to the dimensions mentioned.

Lids (B) according to the invention can be easily manufactured by injection molding from thermoplastics, preferably polypropylene (type Novolen® 1100 U.CX). The vessels mentioned here can also be manufactured in this way. Any filter materials used can be pressed into the designated places and/or glued or welded.

The lid according to the invention can preferably be used in processes for isolating nucleic acids. Such a process is described below with reference to

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FIG 3. Lids according to the invention can even be used twice here, namely for closing and handling the sample vessel (A) and the molded body (C) (embodiment 1) as well as the elution vessel (D) and the stamp (E) (embodiment 2).

In the special embodiment for the processing of sample solutions containing nucleic acids, the following work steps are carried out. In a first step (Γ), a sample liquid containing cells is incubated in a sample vessel (A) with a material to which the cells from which nucleic acids are to be obtained are bound. For this purpose, this material can either have specific binding properties for the surface of the cells, ζ. e.g. by immobilizing antibodies against surface antigens or an absorber material (Al 6), but a material with filter properties (Al 5) can also be provided, through which the cells are retained when the liquid passes through the material, e.g. is removed from the sample vessel. Conditions for the immobilization of cells on surfaces are known to the person skilled in the art, e.g. B. from Methods in Enzymology Vol. 171, Biomembranes / Part R Transport Theory: Cell and Model Membranes, Edited by Sidney Fleischer, Becca Fleischer, Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee.

During incubation, the sample vessel is preferably closed by a lid (B) according to the invention in order to ensure active or passive protection against contamination. The design of this lid corresponds to FIG. 1. It has air passage openings (B 13) which allow air to escape when the lid is placed and pressed down, so that the resulting excess pressure does not force any liquid out of the sample vessel (A) (e.g. via an outlet opening below).

In a further step, the liquid is removed from the sample vessel, while cells whose nucleic acids are to be isolated remain in the sample vessel in a state bound to the material. If the cell-binding material is a particulate material, retention can also be achieved by the material being magnetic and a magnetic field being applied to the sample vessel from the outside, which is so strong that the particulate material remains in the sample vessel when the liquid is removed. The liquid can be removed in a variety of ways. For example, the liquid can be removed through an outlet opening (A12) that is spatially separated from the inlet opening (AlO). If the outlet opening is located in the lower part of the sample vessel and below the retained cells, the liquid can be sucked off, ζ. β. by applying a slight vacuum. For this purpose, a valve can be provided at the outlet opening, which opens when negative pressure is applied. When the liquid is sucked out through the valve, the lid can

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(B) remain on the vessel due to the air passage openings (B 13). The air that is sucked into or through the openings in the vessel is largely freed of contamination by the filter (B 14). The webs (B18) keep the pressure loss low.

To remove as much of the sample components as possible from the cells, one or more washing steps can be carried out. To do this, the lid (B) is removed and a washing liquid is poured into the sample vessel, in which any impurities are dissolved, but which do not significantly impair the binding of the cells to the surface of the cell-binding material. Such washing solutions are known to the expert, for example, from cell separation protocols or from corresponding cleaning kit protocols for nucleic acids. They depend essentially on the type of binding of the cells to the material.

After the last washing solution has been removed from the sample vessel (A), if necessary, the cleaned, enriched cells are brought into contact with a suitable lysis liquid to release the nucleic acids from the cells. The reagents in this lysis solution depend largely on the type of immobilized cells. If the cells are bacteria, the lysis solution preferably contains proteinase K to break down the cell wall. If desired, the lysis is supported by heating or cooling and mixing the reaction mixture. If the cell-binding material is magnetic particles, mixing can also be carried out using magnets. Mixing by shaking the sample vessel is also possible. At the end of this digestion, the nucleic acids to be isolated are free in the solution.

During lysis, the reaction vessel is preferably closed with a lid to prevent contamination from the environment. After lysis, the lid is removed, preferably using a suitable mechanical device. A shaped body (C) is then introduced into the sample vessel, which contains a mixture of cell degradation products and nucleic acids, the outer contour (C 12) of which is matched to the inner contour (Al 7) of the sample vessel. This shaped body is hollow and is closed by a filter (Cl 1) in the direction of the sample vessel and the reaction mixture. The shaped body (C) is preferably introduced using a component (B 11) of the lid (B), which also contains a component (B 10) suitable for closing the sample vessel. In this case, the shaped body is grasped with the lid (B) (�Pgr;) and introduced into the sample vessel at the same time as the sample vessel is closed (UI). During this process, the reaction mixture will also penetrate through the filter (Cl 1) into the cavity (C14) of the molded body (JV). By providing the filter, large particles can be prevented from entering the

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cavity can be prevented and, on the other hand, if the filter has nucleic acid binding properties, the nucleic acids can bind to the filter while the reaction mixture is passing through. In this case, it is advisable to choose a filter material containing glass fibers.

In a next step, the remaining lysis reaction mixture is removed from the device formed by A and C, e.g. by suction through an outlet opening (Al 1) at the bottom of the sample vessel. The solution that has penetrated into the hollow body (C14) of the molded body is also removed so that the filter contains as few liquid residues as possible. The previously used lid (B) is then removed, with the molded body (C) initially remaining in the sample vessel (snapped in) (V).

At the same time or subsequently, an elution vessel (D) is prepared to receive the shaped body (C). A lid (B, embodiment 2) that may be on this vessel is removed (VI). Preferably, before the shaped body (C) is transferred to the elution vessel (D), an elution solution is placed in the elution vessel, e.g. pipetted in. It contains reagents under the action of which the immobilized (bound) nucleic acids are eluted from the material, i.e. dissolved. The composition of the elution solution and the conditions of the elution depend on the type of binding of the nucleic acids to the material in the filter (C). Suitable conditions are known to the person skilled in the art. The lid (B) that originally closed the elution vessel is placed on the sample vessel (A) with the shaped body (C) (VII).

To remove the shaped body (C) from the sample vessel (A), the shaped body (C) is removed with the lid (B) (VET). The combination of lid and shaped body is then introduced into the elution vessel (JX). The shaped body (C) preferably contains means (C 13) for fixing the shaped body in the elution vessel (D), which ensure that the shaped body can only be removed from the vessel (D) by destroying the shaped body (C) or the vessel (D) or with a force that is greater than the force required to detach the lid (B) from the shaped body (C). Removal of the shaped body from the elution vessel is not intended.

As the molded body (C) enters the elution vessel, any elution solution that may have been added penetrates the filter (Cl 1) and thereby dissolves the immobilized nucleic acid from the solid matrix. Depending on the amount of elution solution added, either only the filter is saturated with the elution solution or/and the elution solution with the dissolved nucleic acids penetrates the hollow body (C 14). To ensure that the elution of the nucleic acids is as complete as possible, the inner contour of the elution vessel should be adapted as closely as possible to the outer contour of the molded body.

In a subsequent step, the lid (B) is removed (X) from the combination of molded body (C) and elution vessel (D). It is used to hold a stamp (E) (XS) and to insert it into the cavity of the molded body (C) (XH). This lid grips the stamp (E) from the inside. The stamp is pressed so forcefully against the filter (Cl 1) that liquid from the filter penetrates into the interior of the stamp through an opening in the pressure surface. This process is particularly effective if the pressure surface is adapted in its outer contour to the inner contour of the molded body (C), at least in the area in which the pressing is to take place. The stamp (E) can be fixed in this position, e.g. by snapping it into place. The solution containing nucleic acids can be stored in the device thus formed.

To remove a desired amount of nucleic acid solution, the lid (if present) can be removed and the desired amount of solution can be removed through an opening in the interior of the plunger, e.g. in a pipetting process (XIV). The lid can then be replaced.

The lid according to the invention can be used extremely advantageously. It reduces the risk of contamination for samples that are in a vessel closed by the lid and can be used in processes that work with mechanical devices for opening and closing the vessels. In addition, manual processes using simple hand-held devices for opening/closing are of course also suitable.

The invention also relates to a method for closing and reopening the vessel, characterized by

Inserting a shaped body (C) which is gripped by a lid (B) into the vessel, so that the lid closes the vessel, and

Removing the lid from the vessel and the molded body (C), leaving the molded body in the vessel.

For this purpose, the molded body preferably has means which engage with a corresponding means of the vessel, so that the force required to remove the lid is less than that required to remove the molded body from the vessel.

A particularly preferred method involves the subsequent removal of the molded body from the vessel by means of a lid which grips the molded body with a force that is greater than the force with which the molded body is held by the vessel.

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List of reference symbols: 10 B 10 Sample vessel &Phi;&Phi; III ·· Il A 11 11 Inlet opening 15 12 Exhaust opening 17 13 Filter material 19 14 Inner form 15 External shape 16 Lid 17 Component for closing the sample vessel A 18 Component for gripping the molded body C Components for gripping the lid Air passage opening Filter element Element for locking B10 into A Sealing lip Part of the lid that covers the opening Grooves

C molded body

11 porous matrix

12 outer contour

13 Means for fixing the shaped body in the elution vessel

14 hollow bodies

15 Means for attaching a lid

16 inner contour

D Elution vessel

E Stamp

12 Interior

14 Removal opening

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Claims (8)

Expectations 1. Lid for closing an opening of a vessel, characterized by - a component (B 10) which is adapted to the opening of the vessel to be closed and - a component (B 11) for gripping a shaped body that can be inserted into the vessel 2. Lid according to claim 1, characterized in that the component (BIO) is adapted to the inside of the opening. 3. Lid according to claim 1, characterized in that the lid has a component (B 12) extending substantially perpendicular to the closure opening for gripping the lid. 4. Lid according to claim 1, characterized in that it contains air passage openings (B 13) which are closed by means of a filter element (B 14). 5. Lid according to claim 1, characterized in that the component (BIO) contains an element (BIS) which can snap into a corresponding element of the vessel when the vessel is in the closed state. 6. Lid according to claim 3, characterized in that the component (B 12) is essentially long and cylindrical. 7. Lid according to claim 4, characterized in that spacers (B 18) are attached to the part (B 17) of the lid covering the opening, in which the air passage openings are located, so that a gap remains between the part (B 17) and the top of the filter element. 8. Lid for closing an opening of a vessel, characterized by a component (BIO) which is adapted to the opening of the vessel to be closed, and a component (B 12) extending essentially perpendicular to the opening for gripping the lid. AOTOR-OTVPATENMIMOGOEJCN