DE10066431B4 - Micro-titer plate used for growing microorganisms or cells comprises frame made from rigid first plastic having plate with holes, and number of vessels made from second plastic with oxygen permeability connected to plate - Google Patents

Abstract

A micro-titer plate comprises a frame (2) made from a rigid first plastic having a plate (4) with a number of holes; and a number of vessels (3) made from a second plastic with oxygen permeability connected to the plate by directly spraying onto the holes, having a receiving section protruding from the lower side of the plate and accessible through openings (15) from the lower side of the plate. Preferred Features: The vessels have a wall section (10) of low thickness and are connected with the upper wall section of the plate. The vessels have a wall thickness of 0.05-0.25 mm in a wall section.

Description

The invention relates to a microtiter plate.

Microtiter plates are used for a variety of microbiological, cell culture and immunological operations. In particular, microtiter plates are used for the PCR or the cultivation of microorganisms or cells.

There are already known microtiter plates having a frame with a plate to which a plurality of vessels is fixed, which have a projecting from the underside of the plate receiving portion and are accessible through openings from the top of the plate. The vessels are also referred to as "wells". The common 96 'microtiter plates have 8 × 12 = 96 vessels in rows and columns. More and more, however, also microtiter plates are used with a larger number of vessels.

Single-component microtiter plates made of polystyrene are not suitable for the PCR, in particular because the softening temperature of this plastic (about 85 ° C) is exceeded in the PCR.

One-component polypropylene microtiter plates are basically usable for the PCR. However, they are bend-soft, prone to distortion, are uneven and can only be manufactured with large tolerances and are subject to great tolerance fluctuations in use. They are not particularly suitable for machine handling because their softness makes gripping by the machine more difficult. Furthermore, the small dimensional stability may result, so that the metering needles have wall contact during insertion into the vessels. In addition, because of the thick-walledness of the vessels, the heat transfer into the vessels is poor, which is detrimental to the temperature control and the length of the cycle times in the PCR. Two-component microtiter plates are in the DE 600 03 184 T2 (Date of publication: 1 February 2001, priority date: 23 July 1999 and 19 July 2000, filing date: 20 July 2000). The holes of the plate have a circular cylindrical cross-section and the vessels have a constant wall thickness with an outer circumferential bead at the top of the plate.

From the EP 0 844 025 A2 For example, a microtiter plate is known in which each sample receptacle has a receptacle of a first polymer composition having sidewalls and a bottom and disposed in a matrix of an opaque second polymer composition which shields sidewalls of the receptacle. Each receptacle is thermally bonded to the matrix of the second polymer composition to form an integral structure. This microtiter plate is used to analyze samples using analysis techniques based on the emission of light from the sample. They are not suitable for PCR due to the thick-walled nature of the sample holders.

The US 4 599 314 A discloses a carrier and a plurality of sample receptacles removably disposed in the carrier. The sample receptacles have on the periphery a rib 38, with which they are supported in the edges of holes of the carrier, in which the vessels are inserted.

The EP 088 769 B1 discloses a two-piece plastic holder for loosely holding microtiter sample tubes having a shoulder at the circular periphery. The holder has a retaining member with recesses in a plate which loosely receive the tubes, which are supported with the shoulder at the top of the plate. Further, the holder has another holding part with recesses in a plate which receive the tubes above the paragraphs and on the underside of which the heels can be supported. In this way, the tubes are loosely held in the holder. This allows orientation of the tubes when inserting a heatable sample block in the PCR.

In particular, in the breeding of microorganisms or cells, it requires a sufficient supply of oxygen to the sample. This can be ensured in 96 'microtiter plates by the relatively large openings of the vessels. In the case of microtiter plates with a larger number of vessels-for example, 384-the oxygen supply can be greatly impaired by the reduced opening cross-sections. In addition, to avoid cross-contamination between the samples of different vessels, it would be desirable to ensure oxygenation even with the openings closed.

Other applications of microtiter plates also try to avoid cross-contamination. For this there are sealing foils, which are welded on the top of the microtiter plate and must be peeled off again, if the contents of the vessels to be accessed. In addition, there are rubber mats, which have cones on the underside, to engage sealingly in the openings of the vessels when resting on the microtiter plate. Furthermore, there are plastic strips, which are executed at the bottom with plugs to be pressed into the openings of a row of vessels of the microtiter plate.

The known sealing methods are complicated in the application, do not do justice to increased tightness requirements.

Based on this, the object of the invention is to provide a microtiter plate with more favorable application properties.

In addition, a method for producing the microtiter plate is to be made available.

The object is achieved by a microtiter plate with the features of claim 1.

• – einen Rahmen aus einem steifen ersten Kunststoff, der eine Platte mit einer Vielzahl Löcher aufweist,
• – eine Vielzahl Gefäße aus einem für die PCR geeigneten und/oder eine Sauerstoffdurchlässigkeit aufweisenden zweiten Kunststoff, die durch direktes Anspritzen an die Löcher fest mit der Platte verbunden sind, einen von der Unterseite der Platte vorstehenden Aufnahmeabschnitt haben und durch Öffnungen von der Oberseite der Platte aus zugänglich sind,
• – wobei die Gefäße als oberen mit der Platte verbundenen Wandabschnitt einen Kragen mit vergrößerter Wandstärke haben.

The microtiter plate according to the invention has

• A frame of a rigid first plastic having a plate with a plurality of holes,
• - A variety of vessels made of a suitable for PCR and / or oxygen permeability having second plastic, which are connected by direct injection molding on the holes fixed to the plate, a projecting from the underside of the plate receiving portion and through openings from the top of the plate are accessible from
• - The vessels have a collar with an increased wall thickness as the upper wall portion connected to the plate.

Due to its rigidity, the frame of the microtiter plate is particularly suitable for machine handling. Preferably, it is provided at the edge with a protruding from the underside of the enclosure, which increases the stability, can form a footprint and offers a surface for the automatic machine. The frame can be made particularly low distortion and tolerance. The first plastic may be an amorphous or even a partially crystalline, highly filled plastic. This may in particular be polycarbonate, which is in itself unsuitable for the PCR or the oxygen supply. The limitation of this plastic on the frame, however, makes it possible to make use of its advantageous properties even with microtiter plates for the PCR or oxygen supply of samples.

The vessels are made of a different plastic than the frame. It is a suitable for the PCR and / or oxygen permeability having second plastic. The suitability for the PCR can be given in particular by an increased temperature resistance (up to about 90 to 95 ° C). Furthermore, by a reduced affinity or neutrality of the plastic to DNA or other substances of the PCR.

It is preferably a soft and / or semi-crystalline plastic. Preferably, the second plastic may be polypropylene.

Each vessel is molded directly onto the hole assigned to it. The connection with the plate can be done by adhesion and / or by positive engagement and / or by material connection. Preferably, it is made by positive engagement by the vessels are connected to holes with axially variable cross-section and / or at the edge region of the holes on at least one side of the plate form-fitting manner with this.

The direct injection allows very small flow paths of the material during spraying, whereby particularly small wall thicknesses can be achieved, which preferably fall in the range of about 0.05 to 0.25 mm, in particular may be about 0.1 mm. This promotes heat transfer. For this purpose, an injection point is preferably provided on the vessel bottom of each vessel from which the material fills a first wall section with reduced wall thickness and an upper wall section connected to the plate. The upper wall portion is designed as a collar with increased wall thickness, which allows a particularly low-tolerance production of the microtiter plate.

By the execution of the frame of a first plastic and the vessels of a second plastic are thus achieved by means of materials that correspond to the desired functions of frame and vessels, the best possible solutions. Higher rigidity, better flatness, lower draft and smaller tolerances are achieved by an amorphous, rigid and high temperature resistant material for the frame. The extreme thinness for better heat transfer is achieved by direct injection of the gems. About the vessels no frame is filled, so that the entire pressure gradient is available only for one vessel. The vessels can be injected in soft, PCR-suitable materials. The spraying of the frame is not critical. This can advantageously have several edge-side injection points (about four to six).

In view of increased oxygen permeability, the second plastic is preferably a silicone. This may in particular be LSR (Liquid Silicon Rubber).

According to the manufacturing method of the invention, the frame and vessels of the microtiter plate are produced in a multi-component injection molding process. In the simplest case, this is a two-component injection process or "twin-shot" process.

For a particularly low-tolerance production of the frame is preferably sprayed first and then the vessels. This has the advantage that the frame initially has a certain shrinkage can perform before the vessels are injected. The time interval for the spraying of the vessels from spraying the frame can be chosen so that the shrinkage of the frame (by cooling) is substantially complete. After injection of the vessels, the dimensional stability of the microtiter plate is practically no longer impaired by shrinkage processes. In particular, the tolerance of the distance from vessel to vessel can be limited to very low values (about ± 0.15 mm). This favors the insertion of dispensing needles without wall contact.

It is particularly advantageous that the upper wall portion of the vessels is designed as a collar with increased wall thickness, because the collar can compensate for remaining positional tolerances of the holes during spraying.

• – einen starren Rahmen, der eine Platte aufweist,
• – eine Vielzahl Gefäße, die fest mit der Platte verbunden sind, einen von der Unterseite der Platte vorstehenden Aufnahmeabschnitt haben und durch Öffnungen von der Oberseite der Platte aus zugänglich sind,
• – einen auf der Oberseite der Platte lösbar anbringbaren starren Deckel und
• – mindestens eine Dichtung zwischen Deckel und Platte aus einem elastischen Material, das vom Kunststoff der Platte und/oder des Deckels abweicht und fest mit dem Deckel und/oder der Platte verbunden ist, um die Öffnungen zu schließen, wenn der Deckel auf der Platte angeordnet ist.

According to one embodiment, the microtiter plate

• A rigid frame having a plate,
• A plurality of vessels fixedly connected to the plate, having a receiving portion protruding from the underside of the plate and being accessible through openings from the top of the plate,
• - A releasably attachable on the top of the plate rigid lid and
• - At least one seal between the lid and plate of an elastic material which is different from the plastic of the plate and / or the lid and is fixedly connected to the lid and / or the plate to close the openings when the lid is arranged on the plate is.

According to the invention, therefore, the plate and / or the lid with at least one seal made of a deviating from the material of the plate and / or the lid elastic material. The material may in particular be a thermoplastic, an elastomer, a thermoplastic elastomer or a rubber. The connection of the seal with the plate and / or the cover can be made non-positively and / or positively and / or cohesively. In particular thermoplastic elastomers enable a cohesive connection with matching materials of plate or lid. In particular, in the case of a microtiter plate according to claim 1, the seal may be designed on a collar of the vessels. When the vessels are made of an elastic material, the possibility arises here of carrying out the seals integrally with the vessels.

In the case of this microtiter plate, the at least one integrated seal in conjunction with the rigid lid ensures fast and easy sealability of the openings, which meets high sealing requirements. Particularly advantageous for handling and sealing, when the lid is designed so that it can be locked to the frame, in particular by locking with the edge region of the frame. The lid is particularly applicable in execution with a flat seal on its underside with known microtiter plates with thermoplastic sealing collar at the openings of the vessels.

When connecting the at least one seal with the plate, annular contours surrounding the openings are preferred. When connected to the lid may be in particular annular, glandular, mat-shaped or lip-shaped seals.

For the production of this microtiter plate preferably again a multi-component injection method is used, in particular a two-component injection method ("twin-shot" method) or a three-component injection method (three-shot method). A three-component injection method can be used in particular when, in conjunction with claim 1, two different plastics are already used for frames and vessels and a third plastic is used for the at least one seal.

Preferably, the frame is first injected and then sprayed the at least one seal to the frame and / or the lid is first injected and then sprayed the at least one seal to the lid. Optionally, the frame is immediately injected integrally with the vessels. According to claim 1, however, the vessels can be injected in a second step and in a third step, the at least one seal.

The invention will be explained in more detail with reference to the accompanying drawings of exemplary embodiments. In the drawings show:

1 a 96-well microtiter plate with frame and vessels made of different plastics in plan view;

2 the same microtiter plate in a perspective view obliquely from below;

3 the same microtiter plate in a greatly enlarged vertical partial section through the plate of the frame and a vessel;

4 a 96-well microtiter plate integral with integral annular seals in a perspective view obliquely from above;

5 one opposite the execution of 4 through connecting bridges between the Seals modified microtiter plate in a partial perspective view obliquely from above;

6 a microtiter plate with a lid with integrated Pfropenförmigen seals in a perspective partial section.

In the following description of various embodiments, matching elements have the same reference numerals. The associated description is valid for all embodiments.

According to 1 to 3 there is a microtiter plate 1 out of a frame 2 and a variety of vessels 3 , In 8 columns and 12 rows are a total of 96 vessels 3 available.

The frame 2 has a substantially rectangular plate 4 on, around the outer edge of an enclosure 5 revolves, approximately perpendicularly from the underside of the plate 4 stands out, over the vessels 3 out. Below has the mount 5 in a known manner an expansion 6 which has a stackability on top of a corresponding microtiter plate 1 guaranteed.

The frame 2 points in the plate 4 a total of 96 holes 6 on. These each have a cross-sectional profile, which extends to the top of the plate 7 out in two sections of different conicity and to the bottom 8th the plate 4 expanded in a conical section.

The frame 2 is integrally molded in a first injection step from a plastic which is relatively rigid when cured. The injection points are located at the edge of the frame 2 , for example, at the bottom of the enclosure 5 ,

The vessels 3 have a cup-shaped bottom below 9 to which a conical wall section 10 very small wall thickness (about 0.1 mm) adjacent. Above it is a wall section 11 whose wall thickness gradually increases upward. Outside it has the same conicity as the wall section 10 on. Inside, however, it is almost cylindrical, resulting in an approximately wedge-shaped cross-sectional shape.

The wall section 11 ends in a collar 12 , which also faces the wall section 10 has significantly increased wall thickness. In the area of the collar 12 are the vessels 3 to the plate 4 molded. That's what the collar is for 12 outside on the inner circumference of the holes 6 at. He also has at the top 7 and the bottom 8th the plate 4 one supernatant each 13 . 14 , which creates a secure connection with the plate 4 results.

In the area of the collar 12 have the vessels 3 an upwardly extending in two sections of different conicity cross-section. From the top 7 the plate 4 are the vessels through openings 15 accessible.

All vessels are at the same time directly to the frame 1 or to the holes 6 the same injected. Every vessel 6 has its own central injection point at the bottom of the floor 9 , As a result, short spray paths are achieved, which is the very small wall thickness in the wall section 10 allows. The material used in the example is polypropylene or LSR, with regard to the PCR or an oxygen supply to a sample inside the vessel.

4 shows a microtiter plate 1' , in derogation from the previously discussed frame 2 ' and the vessels 3 ' are integrally made in a known manner from a single plastic. The outer shape of the microtiter plate 1' corresponds essentially to the previous example, although the vessels 3 ' have a substantially uniform wall thickness profile and without projections with the plate 4 ' are merged. The plate 4 ' Edge is in the known manner with the enclosure 5 ' connected, which down the expansion 6 having.

The vessels 3 ' are from above through openings 15 ' accessible, with an annular seal around each opening 16 is arranged from an elastic material. In the example, this is a plastic that is capable of cohesively with the plastic of the microtiter plate 1' connect to.

Instead, a positive connection can be created by the fact that the seal 16 in an undercut groove in the top of the plate 4 ' is introduced.

Preferably, the seals 16 in a multi-component injection molding fixed to the microtiter plate 1' connected.

By placing a - not shown - lid of a rigid material, it is now possible, the openings 15 ' seal tightly. The lid can be about the dimensions of the plate 4 ' to have. Preferably, a latching takes place in the edge region of the microtiter plate 1' , The latching can, for example, in the recesses 17 done, which the enclosure 5 ' directly below the plate 4 ' having.

The execution of 5 differs from the above-described in that the adjacent annular seals 16 with each other through straight webs 18 . 19 are connected, which extend in the row and column direction. This can be advantageous in particular for manufacturing reasons, but also for reasons of the fixed connection of the seals with the microtiter plate 1 or for additional sealing.

In the 6 is a microtiter plate 1' shown according to the of 4 made of only one material. However, the annular seals are missing 16 , On the plate 4 ' the microtiter plate 1' there is a lid 20 , This one has a plate 21 that have substantially the same contour as the plate 4 ' Has. The plate 21 is in edge areas 21 ' on the top of the plate 4 ' supported. In one area 21 '' between the border areas 21 ' she is around a small gap from the plate 4 ' spaced. This allows placement on conventional microtiter plates, the sealing collar on the top of the holding plate 4 ' exhibit.

In that area 21 '' has the plate 21 plug-shaped seals 22 protruding from their bottom. These plug-shaped seals 22 have on their outer circumference a circumferential sealing bead 23 ,

Every opening 15 ' of the vessels 3 ' is a seal 22 assigned. The gaskets will grip 22 so in the openings 15 ' that they with their sealing bulges 23 on the inner wall of the vessels 3 ' sealingly abut.

The seals 22 are in corresponding recesses of the plate 21 arranged. They are connected by short bridges 24 . 25 connected, which extend in the row and column direction.

At the edge of the plate 21 stand from the bottom of facing 26 in front, of which inward locking projections 27 which project into the recesses 17 the microtiter plate 1' are latched. Stand up from the borders 26 handles 28 in front. These facilitate gripping the lid 20 , It is also by swiveling the handles 28 possible, the locking between the locking projections 27 and the recesses 17 to dissolve, because the borders 26 be swung with.

The lid 21 with the seals 22 is also preferably produced in a multi-component injection molding process.

Claims (21)

Microplate with - a frame ( 2 ) made of a stiff first plastic, which has a plate ( 4 ) with a large number of holes ( 6 ), - a plurality of vessels ( 3 ) from a suitable for the PCR and / or oxygen permeability having second plastic by direct injection molding on the holes ( 6 ) firmly with the plate ( 4 ), one from the bottom ( 8th ) of the plate ( 4 ) projecting receiving section ( 9 . 10 . 11 ) and through openings ( 15 ) from the top ( 7 ) are accessible from the plate, and - at which the vessels ( 3 ) as upper with the plate ( 4 ) connected wall section ( 12 ) have a collar with increased wall thickness. Microtiter plate according to claim 1 - in which the vessels ( 3 ) by injection molding on the edge region of the holes ( 6 ) on the bottom ( 8th ) of the plate ( 4 ) are positively connected thereto. Microtiter plate according to claim 1 or 2, in which the vessels ( 3 ) by injection molding on holes ( 6 ) with in the axial direction at least in one section to the bottom ( 8th ) of the plate ( 4 ) Expanding cross-section on the bottom ( 8th ) of the plate ( 4 ) are positively connected thereto. Microtiter plate according to one of Claims 1 to 3, in which the vessels ( 3 ) non-positively and / or cohesively with the plate ( 4 ) are connected. Microtiter plate according to one of claims 1 to 4, in which the vessels ( 3 ) by injection molding on holes ( 6 ) with in the axial direction at least in a section to the top ( 7 ) of the plate ( 4 ) widening cross section and / or by injection molding on the edge region of the holes ( 6 ) on the top ( 7 ) of the plate ( 4 ) are positively connected thereto. Microtiter plate according to one of claims 1 to 5, in which the vessels ( 3 ) at least in a wall section ( 10 ) have a wall thickness of 0.05 to 0.25 mm. Microtiter plate according to one of claims 1 to 6, in which the vessels ( 3 ) a cup-shaped bottom ( 9 ) and / or wall sections ( 10 ) are conical with a small wall thickness and / or at an adjoining wall section ( 11 ) have a gradually increasing upward wall thickness. Microtiter plate according to one of claims 1 to 7, in which the frame ( 2 ) on the edge of the plate ( 4 ) one of its underside ( 8th ) above enclosure ( 5 ) having. Microtiter plate according to one of claims 1 to 8, in which the frame ( 2 ) has several edge-side injection points. Microtiter plate according to one of claims 1 to 9, in which the frame ( 2 ) from an amorphous one Plastic or consists of a semi-crystalline, highly filled plastic. Microtiter plate according to one of claims 1 to 10, in which the frame ( 2 ) consists of polycarbonate. Microtiter plate according to one of Claims 1 to 11, in which the vessels ( 3 ) consist of a soft and / or semi-crystalline plastic. Microtiter plate according to one of claims 1 to 12, in which the vessels are made of polypropylene or silicone. Microtiter plate according to Claim 13, in which the vessels consist of liquid silicone rubber. Plastic microtiter plate according to one of claims 1 to 14, with a top side ( 7 ' ) of the plate ( 4 ' ) releasably attachable rigid lid ( 20 ) and at least one seal ( 16 . 22 ) between covers ( 20 ) and plate ( 4 ' ) made of an elastic material made of the plastic of the plate ( 4 ' ) and / or the lid ( 20 ) and fixed with the lid ( 20 ) or the plate ( 4 ' ) connected is. Microtiter plate according to claim 15, wherein the at least one seal ( 16 ) at the top of the plate ( 4 ' ) and the openings ( 15 ' ) surrounds. Microtiter plate according to claim 16, wherein the at least one seal ( 16 ) is annular. Microtiter plate according to Claim 17, in which different openings ( 15 ' ) surrounding seals ( 16 ) by connecting webs ( 18 . 19 ) are connected together from the same material. Microtiter plate according to one of claims 15 to 18, wherein the at least one seal ( 16 ) with a collar of the vessels ( 3 ' ) connected is. Microtiter plate according to claim 15, wherein the at least one seal ( 22 ) on the underside of the lid ( 20 ) and is annular, plug-shaped, mat-shaped or lip-shaped. Microtiter plate according to one of claims 15 to 20, wherein the at least one seal ( 16 . 22 ) is made of a thermoplastic, elastomer, thermoplastic elastomer or rubber.

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