WO2002087760A1

WO2002087760A1 - Method and device for storing and dosing small quantities of liquid

Info

Publication number: WO2002087760A1
Application number: PCT/CH2002/000218
Authority: WO
Inventors: Ernst BÜRGISSER
Original assignee: Epr Labautomation Ag
Priority date: 2001-04-30
Filing date: 2002-04-19
Publication date: 2002-11-07

Abstract

According to the invention, dosing plates (2) with capillary openings (3) are used for storing and dosing small quantities of liquid, particularly for substance libraries and for the parallel withdrawal of samples from substance libraries. Said dosing plates are stacked one on top of the other so that the capillary openings (3) of the individual dosing plates (2) are aligned with one another whereby forming capillary openings (3') that pass through the plate stack (1). These capillary openings (3') that pass through the plate stack (1) are filled with liquid and form the liquid reservoir. For effecting dosing, the dosing plates (2) are separated individually from the plate stack (1), and the quantities of liquid located inside the capillary openings (3) are ejected. The capillary openings (3) of the dosing plates (2) are arranged, for example, in a pattern that corresponds to the pattern of the cavities (5) of standardized test plates (4), and the quantities of liquid are ejected into these cavities (5). The method enables a parallel, precise dosing of a large number of quantities of liquid of 0.1 νl or less.

Description

METHOD AND ARRANGEMENT FOR STORING AND DOSING SMALL LIQUID AMOUNTS

The invention is in the field of chemical / biological analysis and relates to a method and an arrangement according to the preambles of the corresponding claims. The method and arrangement serve for the storage and metering of small amounts of liquid, in particular for the parallel metering of small amounts of different liquids, as is necessary, for example, for the provision of samples by taking dissolved substances from substance libraries.

Substance libraries contain large numbers of different substances, for example chemical compounds produced by combinatorial chemistry. A specific, usually very small amount (aliquot) of each substance or of a selected number of substances is taken from a substance library of this type, in which the substances are usually stored in dissolved form, for specific, biological or pharmaceutical tests and subjected to the test ,

Usually, the tests or at least the sample preparations necessary for the tests are carried out in parallel on the largest possible number of substances. For this purpose, the substances are placed in cavities of microtest plates and in these, for example, by adding reaction partners, by incubation and / or treated by separation and then subjected to physical measurements. The plates preferably have a standardized shape and have 96, 384 or 1536 cavities (according to standards of the Society for Biomolecular Screening, SBS). For the parallel treatment of samples contained in the cavities, arrangements of instruments are usually used, which are matched to the test plates or to the grid of the cavity arrangement on the test plates and with which all cavities of a plate simultaneously or different groups of cavities or individual cavities in succession and treated after a corresponding shift of the instrument arrangement relative to the test plate.

To remove liquids from cavities from test plates, for example arrangements of pipettes or arrangements of capillary-like or needle-like instruments are immersed in the samples, the liquids being actively sucked into the pipettes, passively rising into the capillaries or simply wetting the needle-like instruments. To dispense the liquids removed with the instrument arrangements, they are ejected from pipettes or capillaries or dispensed from needle-like instruments by contact with a surface that is as adhesive as possible. For the removal of liquids from capillaries, it is also proposed to accelerate the capillaries or entire capillary arrangements and then to brake them in a shock-like manner such that the liquid is thrown out of the capillaries. Pipette arrangements of the type mentioned are described for example in the publication EP-0363450 or US-5193403 (Bürgisser), arrangements with capillary-like instruments in the publication US-5957167 (Pharmacopeia Inc.).

Pipette arrangements, as briefly described above, are also used to introduce dissolved substances from a substance library into cavities on test plates. To do this, it is necessary for the substance library to be at the test plates and the instrument arrangement has a coordinated format that the substance library thus consists, for example, of a plurality of so-called library plates, a substance, for example dissolved in dimethyl sulfoxide, being stored in each cavity of each library plate. Such libraries thus consist, for example, of test plates with 96 wells each, from which aliquots are taken using pipette arrangements with 96 pipettes and placed on test plates with 96 cavities (one test plate from a library plate), 384 cavities (one test plate from four library plates) or 1536 cavitates (a test plate consisting of 16 library plates) can be transferred, whereby the pipettes must be cleaned after each dosing step. The library plates are usually stored at low temperatures and / or in a protective gas atmosphere. To remove substances, the library plates are removed from the protective gas atmosphere and warmed up at least to such an extent that the substance solutions are liquid.

Known pipette arrangements allow non-contact dosing (dispensing an aliquot) down to liquid quantities of approx. 5μl. Library plates are usually used, in the cavities of which about 100 μl of the solutions are introduced when the library is created. In other words, this means that the amount of substance in the library plates is sufficient to create a large number of test plates. On the one hand, this reduces the complex production of library plates, but on the other hand it harbors the risk that sensitive substances in particular can change, because the library plates are removed and heated so many times from the protective gas atmosphere. A pipette must also be immersed in the library solutions for each withdrawal. Even after delivery, solution remains on the pipettes, which is washed away. Not only does this mean that something is lost from the library substances with every intake / release, it also means that mutual contamination of the library substances cannot be completely ruled out. The object of the invention is now to demonstrate a method and an arrangement for storing and dosing small amounts of liquid, the method and the arrangement being intended to make it possible to meter amounts of liquid down to 0.1 μl or even less precisely and without contact and to carry out large numbers of such doses in parallel. The loss of liquids and the mutual contamination of successively metered liquids should be significantly smaller than is the case when using the known pipette arrangements. The mutual pollution mentioned should become negligible. The method and arrangement according to the invention should make it possible to set up and use substance libraries, it being possible for relatively large amounts of substance to be stored in the substance libraries, so that a large number of samples can be prepared from each substance without this affecting the substances. The method and arrangement according to the invention should, however, not only be usable in connection with substance libraries and with the taking of sample quantities from substance libraries, but also for any application in which the smallest quantities of liquid are to be metered, in particular metered in parallel.

This object is achieved by the method and the arrangement as defined in the patent claims.

According to the invention, for the storage and metering of liquids, Dosieφlatten each with at least one capillary opening running through the Dosieφlatte with a given volume, advantageously with a plurality of such capillary openings, are used. A plurality of such Dosieφlatten is stacked such that the capillary openings of the individual plates are exactly aligned. The aligned capillary openings of such a plate stack are then filled with liquid, for example only the one outer opening of the capillary openings running through the plate stack on the stack surface being brought into contact with the liquid and the liquid by the capillary action, possibly supported by a pressure difference, is drawn into the opening and through the stack.

For storage, the Dosieφlatten are stored as a stack, in which stacks the capillary openings of the individual Dosieφlatten are aligned with each other and the capillary openings formed in this way, completely penetrating the stack, are filled with liquid. Each plate closes the capillary openings of the adjacent plates and it is only necessary to use, for example, a blank plate on the end faces of the plate stack to cover the capillary openings of the dosage plates which are extremely positioned in the stack.

For the dosing one Dosieφla te is separated from the stack and the amounts of liquid positioned in the capillary openings of this Dosieφlatte are expelled from the capillary openings by a pressure difference between the two sides of the plate, by a shock-like deceleration of the plate, by acceleration (centrifugation) of the plate, by displacement with another liquid or by expelling it by mechanical means. The quantities of liquid dispensed in this way correspond to the volume of the capillary openings in the plate. For multiple dosing, the dosing step is repeated with further individual dosing plates or a corresponding number of dosing plates is subjected to removal as a stack.

For the separation of Dosieφlatten from a plate stack, it is not relevant whether the substances originally introduced into the liquid in the capillary openings are in a liquid or in a solid (frozen) state. In order for the dosage to be reproducible, it is important to carry out the separation at a constant temperature. In a substance library, according to the method according to the invention, the substances are no longer stored in cavities of test plates, but in capillary openings of stacked dose plates aligned with one another. Individual dosage plates are advantageously removed from the library for each dosing step, so that each dose pre-formed in the library only has to be removed once from the protective gas atmosphere of the library and / or brought to a higher temperature environment, which also applies to libraries in the case of sensitive substances makes relatively large amounts of substance possible.

The Dosieφlatten are, for example, 0.5 to 2 mm thick, the capillary openings have, for example, a round cross section from 0.05 to 1mm. With a plate thickness of 1mm, capillary openings with a diameter of 0.36mm should be provided for liquid quantities of 0.1 μl.

In the case of passive filling of the capillary openings of a plate stack from below, the height of the plate stack is limited by the capillary rise height, which in turn depends on the cross section of the capillary openings and on the ratio of the cohesion in the liquid surface and the adhesion between liquid and capillary wall, the capillary wall must be wettable by the liquid. For a given pairing of liquid and capillary wall, the rise height is inversely proportional to the radius of the capillary opening. For the water / glass system, the proportionality factor is approximately 15.

The Dosieφlatten consist for example of a suitable plastic, glass, metal, ceramic or a semiconductor material. The capillary openings are, for example, drilled or etched with a laser (plasma etching or wet etching). However, the plates can also be produced, for example, in an injection molding process, for example from polypropylene, with the capillary openings. the. If necessary, the inner surfaces of the capillary openings must be treated for good wettability or coated with another material.

Those surfaces of the Dosieφlatten, with which they lie against each other in the plateau stacks, are to be designed in such a way that there is as little as possible a capillary action between plates placed on top of one another, through which substances positioned in adjacent capillary openings could come into contact with one another. Such capillary braking can be achieved, for example, in that the surfaces of the dosing plates mentioned have a very small surface roughness and are advantageously not wettable or very little wettable by the liquid to be dosed. The desired low wettability of these surfaces can also be achieved with a suitable surface treatment or coating. Mechanical capillary brakes in the form of, for example, sharp edges can also be provided around the mouths of the capillary openings.

For an exact separation of the columns of substances running in the capillary openings through a stack of Dosieφlatten when separating the Dosieφlatten it is particularly advantageous for frozen such columns of substance if a kind of predetermined breaking points are generated in the columns by appropriate design of the mouths of the capillary openings.

The arrangement of the capillary openings on the plates advantageously corresponds to the arrangement of cavities on test plates into which the liquids are to be dosed. For dosing in test plates, for example in accordance with SBS standards, the dosing plates therefore have a grid-like arrangement of 12x8, 24x16 or 48x32 capillary openings with grid dimensions of 9 mm, 4.5 mm or 2.25 mm. The method and arrangement according to the invention are described in more detail with reference to the following figures. Show:

1 shows the use of methods and apparatus according to the invention for storing substances in substance libraries and for dosing small amounts of the library substances in cavities of test plates;

Figures 2 to 4 sections through exemplary embodiments of Dosieφlatten on a larger scale;

FIGS. 5 and 6 show exemplary arrangements for filling the aligned capillary openings of a plate stack;

Figures 7 to 10 exemplary arrangements for dispensing the liquid cans from capillary openings of Dosieφlatten.

FIG. 1 shows a partially sectioned, three-dimensional representation of a plate stack 1 of Dosieφlatten 2, in which plate stack 1 the capillary openings 3 of the Dosieφlatten 2 are aligned with one another and form capillary openings 3 'which penetrate the plate stack 1 and are filled with liquid. Furthermore, the figure shows a Dosieφlatte 2 lifted from the plate stack 1, which is positioned for dispensing the amounts of liquid from its capillary openings 3 on a test plate 4 with cavities 5.

The plate stack 1 is positioned, for example, on a correspondingly perforated base 10. As alignment means, for example, alignment bolts 11 are fastened on the base 10 and the dosage plates 2 have, in addition to the capillary openings 3, alignment bores 12 into which the alignment bolts 11 engage. So that the Dosieφlatten 2 can easily be separated from the plate stack 1 (arrow S), they advantageously have separating means, for example, as shown in FIG. 1, beveled edge regions 13 which allow each plate to be gripped under with a suitable tool. Alternating edge patterns can also be provided as separating means.

Figures 2 to 4 show on a larger scale than Figure 1 sections through Dosieφlatten 2 with differently designed capillary openings 3rd

Figure 2 shows a Dosieφlatte 2, which consists of a base material 20 and which is coated on its surfaces as a capillary brake with a non or poorly wettable material 21, in the capillary openings 3 with a wettable material 22. Depending on the wetting properties of the base material 20 and depending on the application, one or the other of the coatings 21 or 22 or both coatings 21 and 22 may also be missing. A corresponding surface treatment can also take the place of a surface coating.

In any case, it is advantageous to design the Dosieφlatten 2 such that they can be manufactured as cheaply as possible and can therefore be used as disposable items.

Figure 3 shows a Dosieφlatte with sharp edges 23 around the mouths of the capillary openings 3 on one side of the plate. These sharp edges 23 serve as a capillary brake. The Dosieφlatten 2 are only in this case directly in the region of the mouths 25 of the capillary openings. Dosieφlatten 2 lying on top of each other are separated by a distance 24 between the mouths, which distance is advantageously so large that it does not have any capillary action. generated. The formations required to form the sharp edges 23 on one side of the Dosieφlatten advantageously correspond to corresponding recesses on the opposite side of the Dosieφlatten, such that formations and recesses can take over the alignment function for aligning the capillary openings of the individual plates.

Figure 4 shows on a single Dosieφlatte 2 two ways of designing the capillary openings 3, such that in the frozen liquid columns at the transition from one plate to the next a narrowing serving as a predetermined breaking point. For this purpose, the capillary openings 3 shown on the left taper from one side of the dosage plate to the other, so that each capillary opening has two orifices 25 and 25 'of different sizes. The capillary openings shown on the right have narrowed orifices 25.

FIG. 5 shows a simple arrangement for filling the capillary openings 3 'penetrating a plate stack 1. It is an arrangement of filling pipettes 30 which carry seals 31 at their open ends directed towards the plate stack 1 and which can be pressed against the plate stack 1 by means of a support plate 32, for example. The filling pipettes 30 are charged from their upper, likewise open ends with the corresponding liquids 33, which are then drawn into the filling pipettes with an overpressure (arrows 34) or passively through the capillary action of the capillary openings 3 '. In order to prevent mutual contamination, the plate which comes into contact with the filling cylinders is advantageously not used for metering. This top plate in the plate stack 1 can also be designed as a press plate, which is thicker, heavier and / or stiffer than the Dosieφlatten 2 arranged below it in the plate stack 1 for a pressing action on the other Dosieφlatten and for this reason also not used as Dosieφlatte becomes. FIG. 6 illustrates a further method for filling capillary openings 3 ′ which penetrate a stack of dose plates 1. This method consists in positioning a liquid drop 35 on each capillary opening 25 on the top of the stack, which liquid drop 35 is then drawn into the capillary opening by the capillary action.

FIGS. 7 to 10 show in a very schematic manner exemplary, simple arrangements for ejecting amounts of liquid from capillary openings 3 of dosage plates 2.

FIG. 7 shows a device for ejecting the quantities of liquid with the aid of a pressure difference between the two sides of the dosage plate 2, that is to say by slightly increasing the pressure on one side of the dosage plate or slightly reducing the pressure on the other side. A Dosieφlatte 2 is positioned on a corresponding test plate 4 such that each capillary opening 3 of the metering plate 2 is aligned with a cavity 5 of the test plate 4. A pressure chamber 40 is positioned above the dosage plate 2, which can be connected to a pressure source (arrow 41), in such a way that a very slight excess pressure can be created in the pressure chamber, with the aid of which the amounts of liquid are expelled from the capillary openings 3 into the cavities 5.

FIG. 8 shows a device for ejecting the quantities of liquid by means of a shock-like deceleration of the dosage plate 2. The dosage plate 2 is operatively connected to compression springs 42, the compression springs being kept in a compressed state by means of suitable locking means (not shown). The test plate 4, in the cavities 5 of which the amounts of liquid are to be transferred, is arranged on the side of the metering plate 2 opposite the compression springs 42. The locking means are then deactivated so that the dosage plate 2 is accelerated against the test plate 4. nigt and is braked by the test plate or by other suitable braking means, the amounts of liquid are thrown into the cavities 5. ,

FIG. 9 shows a device for mechanically ejecting the quantities of liquid. For this purpose, an arrangement of needles 43 is provided, which are pushed into the capillary openings 3 (arrows 44). A hydraulic or pneumatic linear motor or a piezoelectric drive is suitable for driving the needle arrangement.

Figure 10 shows an application of Dosieφlatte 2 in a closed fluid system. Fluid connections 45 are tightly connected to the mouths of the capillary openings 3 on both sides of the dosing plate 2. By moving the fluid through the capillary openings 3 of the dosage plate 2, the amounts of liquid positioned in the capillary openings 3 are expelled or flushed out.

Claims

PATENT CLAIMS

1. A method for storing and dosing small amounts of liquid, characterized in that a plurality of Dosieφlatten (2) with at least one of the Dosieφlatte (2) essentially parallel to their thickness penetrating capillary opening (3) with a predetermined volume, such as one Plate stack (1) is layered on top of one another such that the capillary openings (3) of the individual dosage plates (2) are aligned with one another and at least one capillary opening (3 ') penetrates the plate stack (1) such that the at least one capillary penetrates the plate stack (1) - The opening (3 ') is filled with liquid so that a dosage plate (2) is separated from the plate stack (1) and that the amount of liquid positioned in the at least one capillary opening (3) of the separated dosage plate (2) is pushed out of the capillary opening (3) becomes.

2. The method according to claim 1, characterized in that each Dosieφlatte (2) has a plurality of capillary openings (3) and that in each one

Plate stack (1) penetrating capillary opening (3 ') another liquid is filled.

3. The method according to claim 2, characterized in that the different liquids are solutions of different substances in a substance library.

4. The method according to any one of claims 1 to 3, characterized in that plate stacks (1) with filled capillary openings (3 ') are kept in an inert gas atmosphere.

A method according to claim 4, characterized in that the liquids in the capillary openings (3 ') penetrating the plate stack (1) are in a frozen state when a dosing plate (2) is separated from the plate stack (1) for dosing.

6. The method according to any one of claims 2 to 5, characterized in that the arrangement of the capillary openings (3) of the Dosieφlatten (2) corresponds to an arrangement of cavities (5) on test plates (4) and that the amounts of liquid from the capillary openings (3) of Dosieφlatten (2) in the cavities (5) of test plates (4) are ejected.

7. The method according to any one of claims 1 to 6, characterized in that in the at least one capillary opening (3) of Dosieφlatten (2) positioned amount of liquid by a pressure difference, by abrupt braking of a movement of the Dosieφlatte (2), by accelerating the metering plate (2), ejected by mechanical means or by rinsing with a fluid.

8. Arrangement for storing and metering small amounts of liquid characterized by a plurality of Dosieφlatten (2) with at least one substantially parallel to the thickness of the Dosieφlatte (2) aligned, the Dosieφlatte (2) penetrating capillary opening (3), which Dosieφlatten (2 ) can be stacked into plate stacks (1) in such a way that the at least one capillary opening (3) of the stacked dosage plates (2) are aligned with one another and form a capillary opening (3 ') penetrating the plate stack (1), by means of filling the plate stack ( 1) penetrating at least one capillary opening (3 ') with liquid, by means for separating Dosieφlatten (2) with liquid-filled capillary opening (3) from the plate pel (1) and by means for ejecting the amount of liquid positioned in the capillary opening (3) of the dose plate (2) separated from the plate stack (1).

9. Arrangement according to claim 8, characterized in that the Dosieφlatten (2) have an arrangement of capillary openings (3) which corresponds to the arrangement of cavities (5) on standardized test plates (4).

10. Arrangement according to one of claims 8 or 9, characterized in that the Dosieφlatten (2) are 0.5 to 2 mm thick.

11. Arrangement according to one of claims 8 to 10, characterized in that the capillary openings (3) of the Dosieφlatten (2) have a volume of 0.1 ul or more.

12. Arrangement according to one of claims 8 to 11, characterized in that the Dosieφlatten (2) consist of plastic, metal, glass, ceramic or a semiconductor material.

13. Arrangement according to one of claims 8 to 12, characterized in that the Dosieφlatten (2) have coated or treated surfaces for the smallest possible wettability.

14. Arrangement according to one of claims 8 to 13, characterized in that the inner surfaces of the capillary openings (3) of the Dosieφlatten (2) are coated or treated for the best possible wettability.

15. Arrangement according to one of claims 8 to 14, characterized in that the mouths (25) of the capillary openings (3) are formed at least on one side of the Dosieφlatten (2) as formations with sharp edges (23).

16. Arrangement according to one of claims 8 to 15, characterized in that the mouths (25) of the capillary openings (3) of the Dosieφlatten (2) are designed as alignment means.

17. Arrangement according to one of claims 8 to 16, characterized in that the capillary openings (3) of the Dosieφlatten (2) are designed such that in the a plate stack (1) penetrating capillary openings

(3 ') between constricting Dosieφlatten (2) form constrictions.

18. Arrangement according to one of claims 8 to 17, characterized in that the means for filling the at least one, the plate stack (1) penetrating capillary opening (3 ') at least one, against the plate stack (1) compressible filling pipette (30) or Means for positioning liquid drops (35) on upper mouths (25) of capillary openings (3 ') penetrating a plate stack (1).

19. Arrangement according to one of claims 8 to 18, characterized in that the means for ejecting the amount of liquid from the at least one capillary opening (3) of the Dosieφlatte (2) separated from the plate stack (1) for generating a pressure difference between the two Sides of the

Dosieφlatte (2) is equipped.

20. Arrangement according to one of claims 8 to 18, characterized in that the means for ejecting the amount of liquid from the at least one capillary opening (3) of the Dosieφlatte (2) separated from the plate stack (1) at least one needle insertable into the capillary opening (3) (43).

21. Arrangement according to one of claims 8 to 18, characterized in that the means for ejecting the amount of liquid from the at least one capillary opening (3) of the plate stack (1) separated Dosieφlatte (2) equipped for a plate acceleration and / or an abrupt plate braking is.

22. Arrangement according to one of claims 8 to 18, characterized in that the means for ejecting the amount of liquid from the at least one capillary opening (3) of the Dosieφlatte (2) separated from the plate stack (1) for rinsing out the capillary opening (3) with a Fluid is equipped.

23. Dosieφlatten (2) for storing and metering liquids, which Dosieφlatten have at least one substantially parallel to their thickness penetrating capillary opening (3), characterized in that the Dosieφlatten (2) have alignment means for stacking the same Dosieφlatten (2) such that the at least one capillary opening (3) of the stacked Dosieφlatten (2) are aligned with each other, and that the metering plates have means for capillary braking between stacked Dosieφlatten.

24. Dosieφlatten (2) according to claim 23, characterized in that it has an arrangement of capillary openings (3) which corresponds to an arrangement of cavities (5) on standardized test plates (4).

25. Dosieφlatten (2) according to any one of claims 23 or 24, characterized in that the mouths (25) of the capillary openings (3) to form constrictions in a stack (1) of Dosieφlatten (2) penetrating capillary opening (3 ') are trained.