WO2007017443A1

WO2007017443A1 - Apparatus for filling receptacles in parallel

Info

Publication number: WO2007017443A1
Application number: PCT/EP2006/064972
Authority: WO
Inventors: Bastian Rapp; Dirk Herrmann; Andreas Guber; Anette Jacob; Marc Dauber; Jörg HOHEISEL
Original assignee: Forschungszentrum Karlsruhe Gmbh; Deutsches Krebsforschungszentrum
Priority date: 2005-08-05
Filing date: 2006-08-02
Publication date: 2007-02-15
Also published as: DE102005036967A1

Abstract

The invention relates to an apparatus for filling receptacles in parallel (e.g. microtiter plates of any size, Eppendorf reaction vessels, or cartridges) or for coating planar surfaces (optionally comprising cavities or wells) such as glass supports, e.g. object supports, membranes, wafers, or similar. The inventive apparatus encompasses a frame (1) with outlets (1a), a movable portioning plate (2) with bores (3), and a bottom plate (4) with outlets (4a). Also disclosed is a method for filling receptacles.

Description

Device for parallel filling of vessels

The subject of the present invention is an apparatus and a method for filling vessels. The device may also be referred to as a "scoop" or "microbalance".

In the last decade, biotechnology, genetic engineering or organic synthesis have seen miniaturization of reaction volumes and an attempt to reduce the quantities of chemicals and substances used in the laboratory. The handling of small volumes, for example in the range of 0.1 .mu.l to 1000 .mu.l, is of great importance in cell culture, when working with DNA, proteins or the peptide synthesis. For example, parallel solid-phase synthesis, purification or enzymatic reactions are often carried out on beads in microtiter plates.

In this work, solid substances in the form of powders or similar bulk state must be portioned in small quantities and filled into suitable containers. There is the problem that the solids used (e.g.

Resins, granules) can be weighed in small quantities only with great inaccuracy or high workload. This happens mostly for each well individually, so can not be spoken of a parallel process. Particularly when working with microtiter plates, the fast and, if possible, parallel loading of the reaction vessels (wells) is important.

So far, the handling of very small amounts of solid substances has been made possible by first preparing a suspension of the material to be portioned, which was subsequently transferred to microtiter plates using commercially available pipettes. A further approach to portioning resins as a suspension in parallel by means of a syringe is described by Lebl et al. (J.Chem. Chem., 7, 42-45, 2005).

Disadvantage of these methods is the suspension, in addition, care must be taken that the distribution in the suspension remains constant over the time of portioning. In addition, pipetting of the suspension can easily lead to blockage of the pipette tip by particles. In addition, devices for portioning resins into microtiter plates or cartridges are offered by various companies, such as Radleys Discovery Technologies, Essex, UK or aappTec (advanced automated peptide protein technologies, Louisville, U.S.A.). An essential part of these devices is a plate with holes that specify the volume to be measured. Between this plate and the microtiter plate is a bottom plate, which closes the holes from below. In the Radleys Discovery Technologies product, the bottom plate has openings and is displaced to fill the microtiter plate so that the contents of the holes fall through the openings of the bottom plate in the wells. In the aappTec brand, the bottom plate is pulled away so that the contents of the holes fall into cartridges. Common to both products is the filling of the holes, which is done manually by gently brushing the resin with a slider.

=> These two devices have no integrated reservoir and are still filled manually, that is to implement the

Plates, brushing the beads, etc. manual work is necessary. Thus, microtiter plates can be filled only consuming.

Therefore, these devices are unsuitable for use in fully automated series production.

The object of the present invention is to provide a device for filling microtiter plates which does not have these disadvantages of the prior art. Furthermore, the device should allow accurate portioning, regardless of individual steps, such as manual brushing or steps that provide for an even distribution of particles in a suspension. In addition, the device should allow easy handling and thus a fast loading of vessels. In this connection, the device must be usable in an automation process.

Accordingly, a novel apparatus for filling vessels has been found comprising

A frame with outlet openings,

• a sliding portioning plate with holes

• and a bottom plate with outlet openings. The frame can also serve as a storage container.

The bores of the portioning plate define the volume to be portioned and are accordingly also referred to as filling or portioning volume. The vessels to be filled may, according to the invention, be e.g. Microtiter plates of any size, Eppendorf tubes or cartridges act. Further, planar surfaces (possibly with pits or wells), such as glass slides, e.g. Slides, membranes, wafers or similar in the context of the present invention to be filled as vessels to understand. In a preferred embodiment, the subject of the invention is a device for parallel filling of vessels.

In one embodiment are in the Portionierplatte per frame outlet openings and bottom plate outlet openings two holes, which are arranged in pairs so that if one hole of the pair is below the frame outlet openings, the other hole of the pair above the floor panels -Agress ports is located. In this way it is possible to carry out a filling operation with each movement of the portioning plate. There are no more vacuums.

If the portioning plate is displaceable not only along an axis but also along the perpendicular to this axis, then a plurality of patterns of portioning volume can be arranged on a portioning plate. Thus it is possible to choose between different portioning volumes. First of all, a pattern of portioning volume of the portioning plate over the outlet openings of the bottom plate (indicated as circles in FIG. 7) comes to rest. If the portioning plate is now displaced by a small amount along the vertical axis, it is possible to change to another pattern of holes. In this way it is possible to fill a plurality of filling volumes by a portioning plate without retrofitting the device. The arrangement of the different filling volumes and thus the patterns are freely selectable depending on the requirement. In another embodiment, it is a device in which the number of holes is equal to the number of outlet openings of the frame and / or the bottom plate and / or in which the number of holes equal to the number of sample containers (wells) on the microtiter plate , Contrary to previously described apparatus, in this embodiment, the portioning plate must be moved back and forth once to perform a filling operation.

Preferably, the device according to the invention can be made larger or smaller than the vessel to be filled. Thus, either only a portion of the vessel (e.g., a portion of a multi-well plate) or multiple vessels (e.g., multiple multi-well plates) may be filled.

In a preferred embodiment of the present invention, all parts of the device are made of teflon (polytetrafluoroethylene, [C ₂ F ₄ ] _n ) or other material (eg brass) having a low coefficient of friction of 0.05. Preferably, all those parts of the device according to the invention are to be made of materials that must slide on each other during the necessary relative movement of two components of the device and thus subject to effects of friction and abrasion. These materials can have a coefficient of friction of 0.02 to 0.15; preferably from 0.03 to 0.10; from 0.04 to 0.08; particularly preferably from 0.045 to 0.06, preferably 0.05.

In a particularly preferred embodiment, at least the portioning plate is made of ceramic. Preferably, however, all those parts of the device according to the invention are made of ceramic, which come into contact with the material to be filled, since in this way the effect of static charge caused adhesion to the components of the device is reduced. Suitable for this example, alumina, zirconia or similar materials. Apart from ceramics, it is also possible to use all surface-derivatized materials which reduce the adhesion of the material to be filled, for example the bulk material. This can be done, for example, by utilizing the lotus effect or the like. In principle, all materials can be used for the construction of the device according to the invention, which meet the mechanical requirements in the production. However, it is important to ensure that there is no interaction between material to be portioned and device for reproducible results. Such interactions may be more mechanical, for example, by interdigitation of the particles or friction, or electrostatic nature, such as electrostatic charging. Furthermore, the device according to the invention may include a thrust rail which receives the portioning plate.

In a preferred embodiment, the frame may be constructed to act as a receiver for the other components. First, the thrust rail is inserted into the space provided for the frame. The push rail in turn forms the receptacle for the portioning plate. As a last part of the bottom plate is placed on the Portionierplatte and bolted to the frame.

Another object of the present invention is a method for parallel filling of vessels with the device according to the invention.

The process according to the invention is carried out in the following steps:

(a) Fill the reservoir with material

(b) moving the portioning plate so that the bores are located below the outlet openings of the storage space, (c) filling the bores,

(D) moving the portioning plate, so that the holes are located above the outlet openings of the bottom plate.

In a preferred embodiment, the method according to the invention is a method for parallel filling of vessels or settling on or covering of planar surfaces (possibly with wells) in which the device according to the invention is used. This process can also be carried out fully automatically in series.

In one embodiment, when the portioning plate is displaced, a pulse is triggered which causes a shock, through which the material from the reservoir falls through the outlet openings into the bores and / or out of the bores into the microtiter plate through the outlet openings of the bottom plate.

In the simplest case, the pulse is caused by the movement of the portioning plate. This can be done manually or in automated form by means of a motor. In the manual version, the user holds with one hand the frame (and thus also the bottom plate screwed therewith) firmly and pushes the push bar (and the portioning plate lying in it) back and forth.

Preferably, the pulse is triggered by a zipper mechanism. In the impulse caused by a fangular mechanism in the system, a tooth placed in the frame first engages in a groove on the portioning plate. Now, when the portioning plate is moved relative to the frame, the tooth jumps from this groove to another, parallel groove, wherein the biasing and sudden release of the tooth, a short pulse is generated. In another embodiment, a sequential pulse is impressed. This can be caused by resonant vibration by, for example, acoustic sounder or resonant vibration by means of a piezoelectric element.

In an alternative embodiment, the holes of the portioning plate are mechanically emptied. This can be done by flushing the holes by means of compressed air or liquid or the emptying can be done by inserting pins into the holes.

Of course, an automated embodiment of the present invention is possible in which the individual steps are performed without manual intervention, by connecting the device according to the invention to a corresponding apparatus which is easy to produce for the skilled person.

In another embodiment of this automated method, at least two or more arrays of vessels (eg, microtiter plates) may be positioned, for example, over a conveyor line below the devices of the invention and filled in sequence.

The device according to the invention is used for filling vessels with flowable materials. The term "flowable materials" according to the invention means liquids, liquids containing solids, dispersions, as well as solids, for example, bulk material. Bulk solids are powders, granules, capsules, resins, preferably beads, particularly preferably beads, as used in the solid-phase synthesis of, for example, peptides. These include beads or resins or solid phases based on polystyrene. All commercial resins (resins) used for peptide synthesis can be used. These are offered for example in the catalog Novabiochem, Sigma or glschina, matrix-innovation.com or company Rapp. In addition, resins for "solid phase organic synthesis" (also Novabiochem), including the combinatorial organic synthesis falls. In general, there are resins with different surface modifications, ie different linker molecules and different cleavable linker molecules on the surface. Furthermore, beads according to the invention are to be understood as bulk material for oligonucleotide synthesis, for example Controlled Pore Glass (CPG) (from, for example, purebiotech or Proligo or glenresearch). So-called Dynabeads (Dynal, Hamburg) which are magnetically and surface modified and carry streptavidin on the surface, as well as any other commercially available type of surface modified and unmodified beads used, inter alia, for the selective binding of reagents. The latter are used, for example, in affinity chromatography or affinity separation and purification.

As bulk material, further beads are to be classified which are based on silica particles, surface-modified or unmodified (modifications: for example C ₂ -C 18, linkers, linkers with functional groups such as -NH ₂ , -OH, -COOH, epoxy, maleimides, etc. ). In general, this includes all materials which are available for separation and chromatography, including HPLC (column material, for example from Gram Analytik + HPLC GmbH, Merck, Fluka, Sigma, etc.).

As bulk material according to the invention also all resins and / or beads are referred to, which serve as cation exchangers, anion exchangers, and all powdery materials for enrichment, separation and also "filtering" (size exclusion chromatography) of substances.

Also included are cryobeads that are used to freeze cells.

Furthermore, the cells coated with cryobeads, cells or encapsulated, for example encapsulated in beads according to the invention are to be understood as bulk material. It also includes resins to which nucleic acids, oligonucleotides, peptides, proteins and similar biomolecules are attached. Since the individual components of the system are always connected to each other during use and no conversion of plates is necessary, the individual components can be coated with commercial sealants and sealed against each other. The device can now be used efficiently for the microdosing of liquids. The liquid can be stored in the storage container and portioned in the same manner.

The device according to the invention makes it possible to introduce defined quantities of flowable materials in parallel directly into suitable vessels, e.g. Portion wells from multi-well plates. In contrast to conventional, commercially available systems, neither carrier media (such as liquids or gas streams) nor other handling steps (such as redeployment of the plates, coating of the resins, etc.) are necessary.

With the device according to the invention, the portioning, for example, of beads in one step can be carried out in parallel for all wells of a microtiter plate. The portioning plate removes the beads from a storage container, weighs them to a required volume and then makes them ready for distribution. The devices are designed to weigh the amount of beads through a given volume. The loading of the microtiter plates does not take place sequentially for each well but for, for example, 384 wells in a parallel step.

In an alternative embodiment, the device can be used to fill only individual rows, whereby the entire device can either be made smaller, or the storage container is equipped with an additional module. This module only covers individual rows.

The invention is explained in more detail below with reference to the figures:

Description of the figures:

Fiq. 1 : The device consists of

• a frame 1, which serves as a storage container for the bulk material used

A displaceable portioning plate 2, which determines the portioning quantity to be weighed via defined bores 3

• and a bottom plate 4, which sits on the actual microtiter plate (or slightly above it).

Fig. 2: Embodiment of the device as in Fig. 1 with the difference that in the Portionierplatte 2 next to the holes 3 further holes 3 a, which are arranged in pairs so that if holes 3 are below the outlet openings 1 a, the Holes 3a are located above the outlet openings 4a and if holes 3a are located below the outlet openings 1a, then the holes 3 are above the outlet openings 4a. In this way, during the emptying of the portioning volume, the filling of the volumes for the next portioning can already take place.

3:

Components of the portioning: above - the frame plate 1 with outlet opening 1a, second row - push rail 1 ^1, third line - bottom plate having outlet opening 4a, below - portioning plate 2 with holes 3 each top view (left column) and bottom (right column)

Fig .: 4

Sketch of the device according to the invention with Fang mechanism.

The scoop consists of four components. The frame plate 1, which provides the reservoir for the beads available (see also Fig. 1-

3), the thrust rail, which receives the required for the portioning plate (not shown in the sketch, see also Fig. 3), the portioning plate 2, the ceramic component, which performs the actual portioning of the beads and the bottom plate 4, which sits directly on the microtiter plate (or slightly above it) and ensures that the beads are filled in the correct wells.

The uniform filling and emptying of the beads is ensured by the imposition of a pulse (red arrows, for example Fang mechanism).

5: preliminary test (micrograph of the bore before, during and after filling)

Holes 3 in the portioning plate 2 made of ceramic, Portionierbohrung 1000 .mu.m diameter thus filling volume 100 micrograms above - volume before filling, middle - filling with beads, below - emptying after impressing a pulse.,

Fig. 6:

Quantity distribution of the beads according to Example 4 on 25 wells in a square arrangement.

Fig. 7: filling of vessels with different Portioniervolumina in a Portionierplatte. The portioning plate comprises two patterns each with different holes (see a), small and large circles). These are arranged in a line pattern. The portioning plate is inserted so that one of the two patterns comes to lie over the outlet openings during the filling process for emptying the filling volumes, but the other pattern remains permanently concealed and closed (see b). Will the Portionierplatte now by one shifted small amount in the XXX direction (here referred to as x), so you can change to another pattern of holes. Subsequently, the other pattern of portioning volume (small circles) of the portioning plate comes to rest over the exit openings of the bottom plate (indicated as large circles) during operation. Now the other pattern is permanently closed (see c).

The invention is explained in more detail below with reference to the examples:

Examples:

1. Structure of the device according to the invention

The frame 1 is designed to act as a receiver for the other components. First, the thrust rail V is inserted in the space provided for the frame. The push rail V in turn forms the receptacle for the portioning plate 2. As a last part, the bottom plate 4 is placed on the portioning plate 2 and screwed to the frame. The user now holds with one hand the frame 1 (and thus also screwed with her bottom plate 4) and pushes the push rail V (and lying in her portioning plate 2) back and forth.

An exemplary embodiment of the components is described in FIG. An exemplary tearing mechanism embodiment is described in FIG.

2. Preliminary examination

In the present prototype, a parallel filling of five times five (25) wells was attempted for the demonstration of the Prinziptauglichkeit. The degree of filling corresponds to the divider filling volume and actually filled by beads volume. It is exactly 74% assuming a cubic closest packing. However, this value can not be achieved in reality, since there is no reason for the beads to be arranged in the tightest packing. In the experiment outlined above, the degree of filling was 19.25%. 3. Experiments on the reproducibility of the loading and unloading process The main problem here is the adhesion of the beads to the surface of the bore as a result of electrostatic interaction. Therefore, it is important to adjust the surface of the bore accordingly.

3.a) Examination with the Light Microscope First, it was examined whether and if so how well beads attach to the holes and if they can be removed again. For this purpose, beads were sprinkled on the holes, the plate is acted upon by briefly tapping with a pulse and the volumetric filling subsequently examined in a light microscope. The holes used in the preliminary test had a diameter of 1000 μm, 500 μm and 150 μm.

The test series was carried out with a ceramic portioning plate. In Fig. 5, the filling volume is shown with 1000 microns diameter. The holes were made with a laser. It can be seen clearly that for a filling volume in ceramic with a diameter of 1000 microns, a sufficiently accurate and almost complete emptying can be achieved. In the following system, a ceramic plate with holes of diameter 1000 μm was thus selected as a portioning unit. The filling volume can thus be varied over the height of the ceramic plate.

In a laboratory test, the princess capability was tested on a 1000 μm hole in a ZrO2 wafer. It was examined whether the beads can be portioned through a suitable bore and also removed by an impulse from this bore. First, beads of diameter 100 μm were scattered onto the hole and the filling was examined in a light microscope. in the

Following the wafer was raised and tapped to impart a pulse. The emptying of the bore was checked by light microscopy and was quantitative (see FIG. 5).

3.b) Reproducibility of the filling in microtiter plates

With the device according to the invention as shown in FIGS. 1 and 3, a test series for the examination of the fitness was carried out. The scoop was filled with beads, placed on a microtiter plate, the pusher was manually operated. To test the reproducibility of the portioning from well to well, the portioner was placed on a microtiter plate and filled 5 times 5 wells (25). For this purpose, beads were used which are used for the solid phase synthesis of peptides, and a Fmoc protective group (9-Fluorenylmethoxycarbonyl) wear. Since the concentration of the cleaved Fmoc groups can be detected by extinction in the UV spectrum, it was possible to determine the amount of beads weighed in via this. In order to detect a sufficient amount of Fmoc groups, the scoop was operated 10 times. Per actuation, approximately 0.1 mg of beads per well should be dispensed with the portioning plate used, which means that after 10 actuations the amount should be exactly 1 mg per well. As shown in FIG. 6, the mean value determined by the cleavage of the Fmoc group is 0.97 mg per well, the standard deviation being 0.13 mg.

Claims

claims:

1. Apparatus for filling vessels which

A frame (1) with outlet openings (1a), a sliding portioning plate (2) with holes (3)

• And a bottom plate (4) with outlet openings (4a).

2. Apparatus according to claim 1, characterized in that the frame (1) serves as a reservoir (1).

3. Apparatus according to claim 1, characterized in that an additional module within the frame serves as a storage container.

4. Apparatus according to claim 1, wherein all parts are made of Teflon (polytetrafluoroethylene, [C2F4] n) or other low-material material

Friction value OVERRUN VALUE !!! (E.g., brass, etc.).

5. Apparatus according to claim 1 wherein the at least the portioning plate (2) consists of ceramic.

6. Apparatus according to claim 1, characterized in that the portioning plate per outlet openings (1 a) and / or (4 a) in pairs has two holes (3) and (3 a).

7. Apparatus according to claim 1, characterized in that the

Portionierplatte per outlet openings (1a) and / or (4a) each have at least in pairs two holes of different diameters (3) and (3a)

8. A method for filling vessels in which a device according to

Claim 1 is used.

9. The method of claim 8 which is carried out in the following steps: (a) filling the storage space (1) with material

(b) moving the portioning plate (2) so that the bores (3) are located below the outlet openings (1a) of the storage space (1),

(c) filling the bores (3) (d) moving the portioning plate (2) so that the bores (3) are located above the outlet openings (4a) of the bottom plate.

10. The method of claim 9 in which the displacement of the portioning plate (2) triggered a pulse causing a vibration, through which the material from the storage space (1) through the outlet openings (1 a) in the

Holes (3) or (3a) and / or from the holes (3) or (3a) in the microtiter plate through the outlet openings (4a) falls.

11. The method of claim 10, wherein the pulse is triggered by a fangle mechanism.

12. The method of claim 10 wherein a sequential pulse is impressed.

13. The method of claim 9 in which the holes (3) and / or (3a) are emptied mechanically.

14. The method according to claim 8, characterized in that a plurality of vessel units are filled in succession by the respective following vessel unit is positioned below the device according to claim 1.

This can be done manually or fully automatically (mass production).

15. Use of the device according to claim 1 for filling vessel units with flowable materials.

16. Use of the device according to claim 1 for portioning of liquids