GB2343949A

GB2343949A - Handling magnetic beads during assays

Info

Publication number: GB2343949A
Application number: GB9927309A
Authority: GB
Inventors: Thomas Moore; Peter Zimmermann
Original assignee: Jenoptik Jena GmbH; Jenoptik AG
Current assignee: Jenoptik AG
Priority date: 1998-11-18
Filing date: 1999-11-18
Publication date: 2000-05-24
Also published as: GB9927309D0; DE19854003A1

Abstract

Device for automated handling and separation of magnetic beads or spheres during assay procedures comprises a multipipette arrangement 1, a multicavity plate typically a microtitre plate 2, magnetic rods 4 and plate 3 and a handling mechanism(not shown). Utility is in purification of RNA/DNA products, solid phase sequencing, cell separation, mRNA isolation, RNA/DNA hybridisation and ELISA type assays. Higher throughput of microtitration plates is obtained.

Description

2343949 Simultaneous handling of magnetic beads in a two-dimensional

arrangement Magnetic beads have been used in molecular biology/biochemistry since the end of the 1970s. In many instances, microscopically small, polymer-coated spheres which contain magnetic material in the form of, for example, iron oxide are used to secure other molecules at the surface and to transport these molecules.

The advantage of these microscopically small spheres - "magnetic beads" consists in the huge surface area of only a few milligrams of material and the simplicity of producing homogenous suspensions of beads which can be pipettea, metered, dispensed, diluted and mixed using standard liquidhandling appliances. The number of possible applications cannot be described in full here, being very extensive and including: Purification of RNA/DNA products mRNA isolation DNA/RNA hybridization Solid-phase sequencing Cell separation techniques and also standard ELISA processes can alternatively be carried out using magnetic beads, since these can be washed very well using standard laboratory equipment.

Another very important step is the concentration of suspension volumes, which is also possible by magnetic- bead binding.

Various appliances are in use for separating the solid and liquid phase. For example, t heDYNAL company, for an Eppendorf microcentrifuge tube, offers a holder which secures the tube using a spring and presses it against a permanent magnet, so that virtually all the magnetic beads which are in suspension move towards this magnet.

2 It is very easy to remove the liquid using a standard manual pipette, so that only the magnetic particles then remain behind on the wall of the vessel. If the tube is removed from the holder and is again filled with liquid, followed by thorough mixing, the beads are washed, so that ultimately only the bonded product remains on the particles. The separation of product and magnetic beads takes place in the same way as that mentioned above. The prior art also includes electrically controllable magnetic fields (DYNAL-MPC-auto 96) or permanent magnets which can be moved by means of a motor; AGOWA magnetic separator (DE-U 29614623).

The magnet holders MPC-96 or MPC-9600 produced by DYNAL can be used to handle magnetic beads in the microtitation plate format, in particular for PCR preparation work in so-called thin wall tube plates. The magnetic-bead holder MPC-9600 is also incorporated in the abovementioned AGOWA magnetic separator. The two-part magnetic separator produced by PROMEGA also operates using the 8 x 12 well format. Iron pins penetrate into the microtitration plate and hold the magnetic beads in place, so that the solid phase becomes fixed to these pins. This functions for as long as the iron pins are coupled to a permanent magnet. If the latter is removed, the magnetic particles can be resuspended. To carry out screening experiments, it is important, in the microtitration plate format, to achieve a high throughput of test points per unit time.

As with all liquid-handling steps, this can only be achieved by suitable automation.

For this reason, the pipetting machines which are in extremely widespread use in screening, such as for example those produced by TECAN, BECKMAN, HAMILTON and ROSYS, have been retrofitted with magnetic separators produced by AGOWA or DYNAL. However, complete test sequences (e.g. mRNA isolation from cell culture or viral IRNA from whole blood for PCR detection) still last several hours using these 3 appliances. A characteristic feature of these machines is that they carry out the liquid-handling steps using one pipette tip or 2 to 8 pipette tips which are arranged in a one-dimensional row.

Therefore, the object of the invention is to propose a solution which can be automated in order to achieve a higher throughput of microtitration plates per unit time.

Such an increase in throughput can be achieved by combining the magnet holder arrangements which are compatible with microtitration plates and are known per sel and the simultaneous metering appliances with 8 x 12 well tips in the microtitration plate format, which are likewise known, with a correspondingly designed microtitration-plate and magnet-holder presentation mechanism. Using these devices for handling liquids and magnetic beads, which are in each case two-dimensional, with the addition of a suitably designed plate- handling system, produces a completely new tool for i solation/puri f i cation which works on the scale of minutes.

Embodiments of the invention will now be described, by way of example, with reference to the drawings of which:

Figs. I to 4 show two embodiment forms of arrangements according to the invention for simultaneous magnetic particle handling. Fig. I shows a first embodiment form with a comb4ike arrangement of permanent magnet rods as a magnet holder arrangement, Fig. 2 shows the embodiment form according to Fig. 1 with an offset cavity arrangement; Fig. 3 shows a third embodiment form with a magnet carrier plate as a magnet holder arrangement; and Fig. 4 shows the; embodiment form according to Fig. 3 with an offset cavity arrangement.

4 The first embodiment form shown in Fig. 1 essentially comprises a AWdimen'sl6n'altip arrangement 1, a two-dimensional cavity arrangement 2, a ma gnet holder arrangement in this case constructed as a comb-like arrangement of permanent magnet rods 3, and a carder plate 4.

The tip arrangement 1 comprises 96 tips. for example, (arran 8 l' Eis M 12 ge!n - in.

columns) which are arranged in a given grid dimension (spaclng-6f int6r- s sed6n of the lines and colutrihs).

The cavity arrangement 2 is a special, commercially available mi n plate with and,"ich is a small wall thickness, whose cavities are arranged identically to the ti's V arranged below the t I ip arrangement 1 in such a way that the tips ariguided into. the cavities and an ex6ange of liquid can take place.

The carder plate 4 is a plane plate with a hole arrangement having the same grid dimension as the cavity arrangement 2- and the tip arrangement 1 The diameters of the holes are so selected with respect to size that the cavities of the cavity arrangement 2 supported on the carrier plate 4 project into the holes. However, the carrier plate 4 has an additional raw of holes, so that an offset of the cavity arrangement 2 arranged on the carrier plate 4 is poss;ible. The cavity arrangement 2 can accordingly.be arranged in two different rela positions with respect to the carrier plate 4.

Fig. 1 and Fig. 2 show the described first embodiment form in one of the two possible reiative piositions. As is dear from Fig. 1 when considered in combination with Fig. 2, the permanent magnet rods 3 are located in the tovo relative positions, each in a position' located opposite to an indi'vidual pipette tip. This is achieved in that the permanent magnet rods 3 are arranged in the column direction between every second column. By means of a handling mechanism, not shown, the permanent magnet rods 3 are guided out of nd into the tip arrangement 1 in the column direction and the cavity arrangement is moved in the line back and forth between the two relative positions. The magnetic particles of a suspension located in the cavities are thOefore moved back and forth, i.e., they move to the side of the cavities where the permanent magnet rod 3 is located.

A second embod iment form not shown in the Figures differs from the first embodiment form'essentially with regard to the handling mechanism- In this case, the latter is conceived in such a way that only a relative movement of the comb-like arrangement of permanent magnet rods 3 is carded out in that the latter are guided out of the tip arrangement in the column direction, subsequently displaced in the line direction and introduced into the tip arrangement again in the column directi on. Since the offsetting of the cavity arrangement 2 is therefore omitted, the carrier plate 2 must also not have any additional raw of holes.

In a third embod,iment form, shown in Figs. and 4, the functions that are carried out in the first and second embodiment forms of the carrier plate 4,and comb-like arrangement of permanent magnet rods 3, are taken over by a magnet carrier plate 5. The magnet arrier plate 5 resembles the carrier plate 4 only outwardly. A permanent magnet strip is introduced into the plate body between every second row of holes arranged in the column diredion. In this embodiment form, the handling mechanism is designed only for the offsetting of the cavity arrangement..

The views in the individual Figures are limited to the features essential for an understanding of the invention. Accordingly, it is clear for the person skilled in the art that the tip arrangement is connected with a simultaneous dosing device and that the quantity of the bps, cavities and holes arranged in the. columns and rows are adapted to one another, but can be optionally selected in principle..

Simultaneous metering appliance (DD Patent 260571) These appliances allow the simultaneous uptake/dispensing of liquids in the two-dimensional 8 x 12 or 16 x 24 well grid which is standard for microtitration plates, by means of pipette tips, needles or similar devices.

Magnet holders:

Two-dimensional 8 x 13 hole arrangement in the microtitration plate format, which are able to accommodate, for example, the wells of so-called thin wall tube plates, and between the columns of holes or rows in which permanent magnets are arranged in such a way that, when the abovementioned plates are inserted, 6 the magnetic beads located in the wells of the plates are attracted by these magnets and are fixed to the wall of the wells. An additional column or row on this perforated plate makes it easy, by transferring the thin wall tube plates, to fix the magnetic particles to the right-hand or left-hand side of the wells. By changing the position, it is possible to wash the particles in the liquid phase. In the following example, it is assumed that there is an additional column. Or:

A 'comb arrangement of permanent -magnet bars, oriented in rows or columns, the distance between which allows them to be positioned between the pipette tips of the simultaneous metering appliance. In this way, it is possible to hold the magnetic beads in the pipette tip and to take up or dispense liquid.

Thin wall tube plates (TWP):

Special 8 x 12 = 96 well microtitration plates (192 and 384 well also standard) of small wall thickness, usually made from PP or PC, which are usually dimensioned in such a way that they are used in standard commercial thermocyclers, and thus provide the possibility of also being positioned in a perforated plate with magnets.

Microtitration plate and magnet-holder handling mechanism:

Device for positioning microtitration plates (MTPs), TWPs and storage and washing vessels in relation to a magnet holder, and also for positioning the magnet holder in relation to a two-dimensional pipette tip arrangement which is in the form of a matrix, in such a manner that it is possible, for example, to deposit TWPs alternately, beginning with column 1 or 2, in the magnet holder or to position a magnet holder with respect to the pipette tips, with, in a first variant, means for picking up the TWPs or other vessels with a - 7 geometry similar to that of microtitration plates from a conveyor device and putting them down on the magnet holder, and positioning the latter with respect to the pipette tips in such a manner that, with a pipette-tip arrangement in the form of a matrix, it is possible to exchange liquid between tips and cavities.

Alternatively, in a second variant, a magnetic comb is arranged between the tips in such a way that the magnetic comb is arranged alternately, beginning in the first or second row of tips, and it becomes possible to exchange liquid between pipette tips and th'e cavities of the microtitration plate.

The means for achieving this technical object can be described as follows:

To move the microtitration plates and the magnetic holder in the vertical direction, a suitable motor drive, e.g. in the form of a rack drive with a stepper motor, is provided. Positioning in the two horizontal directions is effected using an electrically controllable mechanical stage. The plates can be fixed beneath the pipette tips using a gripper mechanism. The plates are transported to the simultaneous metering appliance by means of a carria ge, for example on a - rod guide mechanism. To move the magnet holders between the pipette tips, a horizontally running linear drive is provided, which pulls the magnets out of the space between the tips and pushes them back in a position which has been shifted by one grid. The prior art reveals all these drive and handling means. It is easy to use a computer control system to automate these various sequences of movements.

A simple sequence for washing the beads is described below:

The particles are in a homogenous suspension in a TWP and have, for example, nucleic acid bonded to their surface. By inserting the thin wall plates intothe magnetic adaptor, the beads are fixed to the walls of the wells, and the two-dimensional pipette arrangement can be used to remove the liquid phase and, at the same time, to add washing solutions from a reservoir which may, inter alia, be a MTP or a similar vessel. As a result of the position of the plate being changed by one column with respect to the magnets, the particles move from one side of the well to the other and are washed. This operation can be supplemented outside the magnet holder by the simultaneous suction/dispensing of the liquid, including the beads, using the two-dimensional pipette tip arrangement.

Working with such an arrangement leads to a considerable increase in the processing rate, so that it' becomes possible to carry out the purification of nucleic acids, using adding reagents, lyses, elution and addition of the PCR mix, within a few minutes.

Claims

9 Claims

1. Arrangement for handling liquids, magnetic beads and vessels, characterized in that it has a two-dimensional pipette arrangement, a two-dimensional magnet holder arrangement and a two-dimensional arrangement of cavities, in combination with a handling mechanism.

2. Arrangement according to 1, characterized in 10 that the twodimensional pipette arrangement is a simultaneously operating automatic pipetting/metering appliance with at least 4 tips/needles.

3. Arrangement according to 1, characterized in that the two-dimensional magnet holder arrangement is a carrier plate for a multiplicity of permanent magnets which are arranged in the form of a matrix.

4. Arrangement according to 1, characterizedin that the two-dimensional magnet holder arrangement is a carrier plate for a multiplicity of permanent -magnet bars which are arranged in rows.

5. Arrangement according to 1, characteriz'ed in that the two-dimensional magnet holder arrangement is a carrier plate for a multiplicity of permanent -magnet bars which are arranged in.columns.

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6.: Arrangement according to 1, characterized in that the twodimensional magnet holder arrangement is a comb-like arrangement of permanent-magnet bars.

7. Arrangement according to 1, characterized in that the two-dimensional arrangement of cavities is oriented to the microtitration plate format.

8. Arrangement according to 2, characterized in that the geometric arrangement of the tips/needles follows the standard microtitration plate format.

9. Arrangement according to 4, characterized in that the arrangement of the permanent magnets is oriented to the cavities of the microtitration plate.

10. Arrangement according to 6, characterized in that the grid of the comb arrangement results from the grid of tips of the simultaneous metering appliance, so that the magnet bars fit into the spaces between the tips.

11. A method of handling liquids, magnetic beads and vessels comprising a two-dimensional array of pipettes arranged to cooperate with a thin wall tube plate having a corresponding array of wells, and a magnetic plate comprising an array of cavities arranged to receive respective wells, the array of cavities being greater in number in at least one dimension than the array of wells, the method comprising the step of inserting the wells into corresponding cavities, carrying ou t a processing operation using the pipette array, displacing the thin wall tube plate and pipette array relative to the magnetic plate, inserting the wells into respective different cavities of the cavity array and carrying out a processing operation using the pipette array.