WO2000050171A1

WO2000050171A1 - Device for preparing samples for analysis

Info

Publication number: WO2000050171A1
Application number: PCT/FR2000/000462
Authority: WO
Inventors: Christophe Fraudeau; Michel Kubacki
Original assignee: Gilson, S.A.
Priority date: 1999-02-26
Filing date: 2000-02-24
Publication date: 2000-08-31
Also published as: FR2790315A1

Abstract

The invention concerns a device for preparing samples for analysis, comprising a plate (2) with several columns (4) each comprising a product (6), a vacuum source, an injection needle. The device comprises a sheet (8) for covering the plate (2) sealing a top end of the columns (4) when a bottom end of the columns is communicating with the vacuum source, even after the sheet has been pierced with the needle.

Description

"DEVICE FOR PREPARING SAMPLES FOR PURPOSES

ANALYSIS ". The invention relates to the preparation of samples for analysis by means of an extraction plate.

In a known manner, the extraction plates are consumables for preparing samples composed for example of ninety-six minicolumns assembled in the geometry of a microtitration plate (8 × 12, center distance 9 mm). The mini-columns contain a stationary phase in the form of powder or disc. In current sample preparation protocols, the solvents (conditioning, washing, elution) and the sample must pass through the stationary phase, with a controlled flow rate.

The manual use of these plates can be done by means of a vacuum box, which seals with the edges of the plate, by applying a vacuum at the foot of the ninety-six columns simultaneously. The different liquids (solvents or samples) are distributed in the columns and sucked through the stationary phase by this depression. In the vacuum box, the space under the extraction plate allows to place a receptacle which collects the solvents and the samples having passed through the phase.

When the packaging solvents, washing solvents and the samples (in most cases) are discarded, a disposable receptacle is placed having only one large cavity (sink). When elution solvents and samples (in a few rare cases) are collected for processing and / or analysis, a collection plate is placed fitted with ninety-six individual wells of 0.3, 1 or 2 ml.

The disadvantages are:

Since the vacuum is applied simultaneously to the ninety-six columns, the air preferably flows through the empty columns or those having received a liquid of lower viscosity. Thus, in practice, it is impossible to pass a serum or plasma sample through the phase if the plate is only partially used. This is why some manufacturers recommend selectively covering unused wells with a sheet, but this is not very practical. Likewise, if the ninety-six wells are loaded with samples of variable viscosity (which is often the case: serum / plasma coming from different subjects or having been taken more or less recently), the least viscous pass faster, and when these columns are empty and the air passes freely through the phase, the other columns remain blocked. In this case, you cannot use a sheet. To compensate for this phenomenon, it would be necessary to manage to maintain the vacuum level under the plate despite the numerous "leaks" represented by the empty columns, which would require very high pumping power (in flow).

Furthermore, when the liquid has passed through the stationary phase, a stream of air continues to pass through it until the step is completed for all the columns. This tends to dry out the phase. This phenomenon can be sought in certain cases

(drying before elution for example), but in the in most cases this is a drawback (deconditioning of the phase). The increase in pumping rate (mentioned as a possible remedy in the previous point) would worsen this phenomenon. An object of the invention is to provide a device for preparing samples allowing the extraction through the columns whatever the differences between the substances contained in the columns, and allowing the extraction when certain columns are empty, and this without risking drying the phase.

In order to achieve this goal, there is provided according to the invention a device for preparing samples for analysis purposes, comprising a plate having several columns each comprising a product, a vacuum source, and a needle. injection, and comprising a sheet capable of covering the plate by sealing an upper end of the columns in a leaktight manner when a lower end of the columns is in communication with the vacuum source, even after the sheet has been pierced by the needle.

The term sheet used in the context of the present invention designates a kind of relatively thin plate, made of a material ensuring it sufficient flexibility and elasticity to be able to be easily pierced one or more times by a needle, and of which the perforation orifice is able to close tightly after withdrawal of the needle. This type of material is for example used to make plugs which often bear the name of septum. Under the action of the vacuum, the sheet 'is strongly pressed on the extraction plate and comes to join with the top of the columns (self-sealing) in an almost waterproof way, a small leak being admissible. The vacuum at the foot of the columns can thus be maintained at a high level without very high pumping power.

To inject a liquid into a column, the needle pierces the sheet then enters the column and injects the liquid into it. The needle has on its outer face a groove allowing the passage of air through the sheet, so that the liquid is sucked through the phase by vacuum. Everything then happens as if the vacuum was only applied to the columns in which the device is dispensing liquid. It should be noted that this pressure balancing groove can be replaced by a central cylindrical or annular peripheral duct connected to a gas source making it possible to fulfill the same function.

The advantages are as follows: samples of high viscosity (for example undiluted bovine plasma) can be loaded without difficulty; - the performances are exactly the same whether the plate is used partially or totally; and once the solvent has passed, the air flow through the phase is stopped (no unconditioning). After dispensing the liquid, the needle withdraws from the sheet, which closes in a substantially sealed manner despite the depression in the column. The sheet will preferably be a consumable, discarded at the end of the sample preparation protocol.

Advantageously, the device comprises a support element having orifices and capable of covering the sheet so that the orifices are in coincidence with the columns.

This element improves the sealing of the sheet on the plate and prevents the exit of the needle from lifting the sheet.

Advantageously, the plate and the support element comprise mutual centering means.

Advantageously, the device is arranged to put a column in communication with the ambient air through the sheet crossed by the needle.

For example, the device could be arranged to connect the needle to an air source to bring air into the column after the injection of liquid.

Advantageously, the needle is arranged so that the communication takes place outside the needle.

Ambient air thus serves as a direct air source.

Advantageously, the needle has a longitudinal groove. This groove allows the ambient air to be brought into the column, putting it at atmospheric pressure, with a view to suction of the liquid by the vacuum source.

Advantageously, the device comprises a support capable of supporting the plate above at least one container capable of communicating with the source of vacuum. Advantageously, the plate is mounted ^'to vertical sliding on the support between a low position in which an upper mouth of the container is in air communication only with the plate, and an upper position wherein the mouthpiece is in communication with the ambient air between the container and the plate.

This container can be a sink or a collection container with individual columns. Advantageously, the device comprises means for returning the plate from the low position to the high position, these means being capable of generating a return force of sufficiently low intensity that these means are inoperative when the plate in the low position. is in communication with the vacuum source via the container.

The plate is therefore kept in the low position by the vacuum source. The suppression of the communication with this one produces in the long term the rise of the plate. Advantageously, the device comprises an arm capable of passing the plate from the high position to the low position against the return means.

Advantageously, the device comprises two containers, the plate being mounted movable with horizontal sliding so as to be able to extend over any one of the containers.

Advantageously, the device comprises an arm capable of urging the horizontally sliding plate to place it above any of the containers.

We can thus automate the entire protocol. Advantageously, the arm comprises the needle. There is also provided according to the invention a method for preparing samples for analysis purposes using the device of the invention comprising the steps consisting in: covering the plate with the sheet; placing the lower end of the columns in communication with the vacuum source; pierce the sheet opposite one of the columns using the needle, inject a second product into the column and put the column in communication with an air source.

Advantageously, the second product is injected into the column through the sheet by means of the needle and the needle is held through the sheet after the injection.

Atmospheric air is thus introduced into the column through the groove of the needle, which ensures that all the liquid is sucked in by the vacuum source. According to the invention, a method of preparing samples for analysis is further provided, in which: a plate having several columns each comprising a first product is available; - the plate is covered with a sheet; placing a lower end of the columns in communication with a vacuum source; and the sheet is pierced opposite one of the columns by means of an injection needle, a second product is injected into the column and the upper end of the column is placed in communication with an air source, the sheet being able to seal the upper end of the columns tightly, even after the sheet has been pierced when the columns are in communication with the vacuum source.

Other characteristics and advantages of the invention will become apparent in the following description of a preferred embodiment and of a variant given by way of nonlimiting example. In the accompanying drawings: FIG. 1 is an exploded perspective view of a device according to the invention; Figure 2 is a longitudinal sectional view of the device of Figure 1; Figures 3 and 4 are enlarged views of a detail of Figure 2, respectively with the plate in the high position and in the low position; Figure 5 is a cross-sectional view of the device of Figure 1; Figure 6 is a local longitudinal sectional view of the device of Figure 1 at the plate and the sheet; Figure 7 is a cross-sectional view of the needle of the device of Figure 1; Figure 8 is a view similar to Figure 3 showing an alternative embodiment; and - Figure 9 is a view of a detail of Figure 8.

In the embodiment illustrated in FIGS. 1 to 7, the device according to the invention comprises an extraction plate 2 of a type known per se, having columns 4, here ninety-six in number, arranged according to an 8 x 12 matrix, each column comprising a stationary phase 6 under the shape of a powder or a disc. This plate 2 ′ has the general shape of a rectangular parallelepiped slightly less high than wide and three times longer than high. The device comprises a sheet or elastic flexible sheet 8 of elastomer and / or of a material of the septum type. This sheet 8 has in plan a rectangular shape. The device comprises a needle 10 mounted on an arm not shown and arranged to be movable along three axes X, Y and Z perpendicular to each other. The arrangement for giving the needle 10 this mobility is known per se, for example from document EP-0 180 511. The device will advantageously include two, four or eight needles 10 of this type movable in one piece and parallel between them in order to work simultaneously. The device also comprises a vacuum source 11 illustrated diagrammatically in FIG. 2 and which can provide a vacuum of up to 0.65 × 10 ⁵ Pa.

The sheet 8 is capable of being pierced by the needles 10 by means of a moderate force which can be supplied by the arm of the device. It is here chosen so that even after five or six needle passages, it closes and remains sufficiently airtight when it is subjected to a vacuum of 0.65 × 10 ⁵ Pa. Its chemical resistance is sufficient for it '' it resists the time required for a standard solvent protocol for sample preparation. Sheet 8 is here a consumable discarded at the end of the protocol. The device further comprises a support plate 12, here made of metal to be sufficiently heavy. This support plate 12 has in plan the same general shape as the extraction plate 2 and has cylindrical conduits 14 of the same shape and the same arrangement as the columns 4 so that the conduits 14 coincide coaxially with the columns 4 when the support plate 12 covers the sheet 8. The support plate 12 has flanges 16 laterally covering the external faces of the extraction plate 2 for their mutual centering. The support plate 14 holds the sheet 8 in place on the extraction plate 2 and improves the tightness of their connection. Referring to Figure 7, each needle 10 has a tapered tip not shown for piercing the sheet 8. It further has a longitudinal groove or groove 18 extending along the needle. When the needle 10 crosses the sheet 8, this groove ensures air communication between the interior of the column 4 and the ambient air, thus bringing the interior of the column to atmospheric pressure.

To inject a liquid such as a reagent or a sample into a column 4, the device controls the needle 10 so that its tip enters the associated conduit 14, then pierces the sheet 8 and enters the column. The vacuum source 11 is in operation and connected to the lower ends of all the columns 4 as will be seen below. It creates a depression in all the columns. However, this depression does not generate an air flow or liquid in all columns. Indeed, for the columns opposite which the sheet 8 has not been pierced, the sealed closure of the sheet on the upper end of the column prohibits any flow of fluid. For columns 4 opposite which the sheet 8 has already been pierced but the needle of which is absent, the sheet has closed resiliently so that we find ourselves in the aforementioned sealed closure situation. For each column in which a needle extends, the groove 18 of the needle cooperates with the depression to produce a flow of atmospheric air entering the column 4 through its upper end. Thus, when the needle injects a liquid into the column, it passes through the solid phase 6 as expected and then the quantity of excess liquid, or even all of the liquid, is evacuated by being sucked by the vacuum towards the source of vacuum. 11. After the injection, the needle 10 remains a few moments in the column while the liquid finishes crossing the phase, in order to continue to produce the air flow.

The device comprises a lower rack 20 and an upper rack 22. The upper rack 22 comprises two horizontal longitudinal slides 24. A carriage 26 or slide has two longitudinal grooves 27 illustrated in FIG. 5 and allowing it to be mounted with horizontal longitudinal sliding on the slides 24.

The device comprises a frame 28 of generally rectangular shape extending under the carriage 26 and suspended from the latter by springs 30, here four in number, forming return springs. The framework 28 has a shoulder 32 extending towards the center of the frame and on which the extraction plate 2 rests by its underside with the interposition of a seal 34 of the frame. The extraction plate 2, sheet 8, support plate 12 assembly is thus mobile with horizontal longitudinal sliding and vertical sliding.

The lower rack 20 comprises two containers, a collection container 36 and a sink 38, following each other longitudinally, having the same general dimensions and located at the same vertical level. The collection container 36 has columns homologous to those of the extraction plate 2. The sink 38 has a single cavity. Both are connected to the vacuum source 12 by conduits 40 which can be closed at will on command of the device.

It is assumed that the extraction plate 2 is located as in FIGS. 3 to 5 above the collection container 36. The extraction plate 2 returned by the springs 30 is then in the high position.

To carry out the preparation, the vacuum source 11 is switched on. Then the needles 10 are placed above the center of the support plate 12 and push them vertically down against the force. return spring 30 (displacement Z). This tightens the springs and separates the frame 28 from the carriage 26. The frame 28 seals with the upper face of a compartment 36 of the lower rack surrounding the collection container 36, the frame 28 being provided for this purpose with a seal. sealing 38. The extraction plate 2 is then in the low position. A depression is created very quickly in the compartment which firmly holds the frame 28 and the plate 2 on the collection container 36 although the needles cease to act and then move away from the support plate 12. The force of the springs 30 is chosen so that they are thus inoperative for returning the extraction plate 2 to the high position. It is then possible to distribute liquids in the plate 2 for their reception in the collection tank 36. At the end of the distribution step, the communication of the collection container 36 with the vacuum source 11 is interrupted and a vent valve, not shown, putting this container in communication with atmospheric air. This produces the ascent of the extraction plate 2 in the high position under the effect of the return springs 30.

In the high position, the extraction plate 2 can be biased in horizontal sliding by the needles 10 pushing the carriage 26 to put the extraction plate as desired above one of the two containers.

When the columns 4 of the extraction plate have a lower point projecting from the plate, in the collection position, the downward movement of the plate and of the frame also makes it possible to make the point of the columns penetrate into the collection wells, which avoids splashing and contamination from one column to another.

The two containers 36, 38 can be connected to the vacuum source by means of a solenoid valve which makes it possible to select the compartment to which the vacuum is applied. As can be seen, the device makes it possible to fully automate the execution of the protocol. The sample can be processed by the device without manual operation. The device can even bring the extraction plate 2 back above the sink 38, and in this position the collection container 36 is in the open air and accessible to the needles 10.

The method according to the invention has been described as implemented automatically by an automaton. However, it can also be implemented manually by an operator having a container 36, a vacuum source 11, an extraction plate 2, the sheet 8, the support plate 12 and of the groove needle 10. The invention saves significant time. It is applicable to solid-liquid, liquid-liquid extraction and filtration.

Figures 8 and 9 show an alternative embodiment essentially differing from the mode described above by the shape of the profile of the frame 28. While in the mode of Figures 1 to 7, the seal 34 extends from an outer edge to an internal edge of the frame, in this variant, the seal 34 only extends in the vicinity of the internal edge.

Claims

1. Device for preparing samples for analysis, comprising a plate (2) having several columns (4) each comprising a product (6), a vacuum source (11), and an injection needle ( 10), characterized in that it comprises a sheet (8) capable of covering the plate (2) by sealing an upper end of the columns (4) in leaktight manner when a lower end of the columns is in communication with the source of empty (11), even after the sheet (8) has been pierced by the needle (10) facing at least one of the columns.

2. Device according to claim 1, characterized in that it comprises a support element

(12) having holes (14) and capable of covering the sheet (8) so that the holes are in coincidence with the columns (4).

3. Device according to claim 2, characterized in that the plate (2) and the support element (12) comprise mutual centering means (16).

4. Device according to any one of claims 1 to 3, characterized in that it is arranged to put a column (4) in communication with the ambient air through the sheet (8) traversed by the needle (10 ).

5. Device according to claim 4, characterized in that the needle (10) is arranged so that the communication takes place outside the needle.

6. Device according to claim 4 or ^' 5, characterized in that the needle (10) has a longitudinal groove (18).

7. Device according to any one of claims 1 to 6, characterized in that it comprises a support (22) capable of supporting the plate (2) above at least one container (36, 38) capable of communicate with the vacuum source (11).

8. Device according to claim 7, characterized in that the plate (2) is mounted so as to slide vertically on the support (22) between a low position in which an upper mouth of the container (36, 38) is in communication with air only with the plate (2), and a high position in which the mouthpiece is in communication with the ambient air between the container and the plate.

9. Device according to claim 8, characterized in that it comprises return means (30) of the plate (2) from the low position to the high position, these means being capable of generating a return force d intensity low enough that these means are inoperative when the plate (2) in the low position is in communication with the vacuum source (11) via the container (36, 38).

10. Device according to claim 9, characterized in that it comprises an arm (10) capable of passing the plate (2) from the high position to the low position against the return means (30).

11. Device according to any one of claims 7 to 10, characterized in that it comprises two containers (36, 38), the plate (2) being mounted movable with horizontal sliding in order to be able to extend above any of the containers.

12. Device according to claim 11, characterized in that it comprises an arm (10) capable of urging the plate (2) to slide horizontally to place it above any of the containers (36, 38).

13. Device according to claim 10 or 12, characterized in that the arm (10) comprises the needle.

14. A method of preparing samples for analysis by means of the device of one of claims 1 to 13, characterized in that it comprises the steps consisting in: covering the plate (2) by means of the sheet (8); putting the lower end of the columns (4) in communication with the vacuum source (11); pierce the sheet (8) opposite one of the columns (4) using the needle (10), inject a second product into the column and put the column in communication with an air source.

15. Method according to claim 14 and according to claim 5 or 6, characterized in that the second product is injected into the column (4) through the sheet (8) by means of the needle (10) and is maintained the needle through the sheet after injection.

16. Method for preparing samples for analysis, characterized in that: there is a plate (2) having several columns (4) each comprising a first product (6); the plate is covered with a sheet (8); putting a lower end of the columns (4) in communication with a vacuum source (11); and the sheet (8) is pierced opposite one of the columns (4) by means of an injection needle (10), a second product is injected into the column and an upper end of the column is placed in communication with an air source, the sheet (8) being capable of sealing the upper end of the columns (4) in leaktight manner after the sheet has been pierced when the columns are in communication with the vacuum source (11).