WO2004063739A1

WO2004063739A1 - Electrophoresis device and moulding accessories for one such device

Info

Publication number: WO2004063739A1
Application number: PCT/FR2002/004312
Authority: WO
Inventors: Yonathan Benech; Monique Beyneix; Daniel Corti; Gilles Brai
Original assignee: Yonathan Benech; Monique Beyneix; Daniel Corti; Gilles Brai
Priority date: 2002-12-12
Filing date: 2002-12-12
Publication date: 2004-07-29
Also published as: AU2002364921A1

Abstract

The invention relates to a moulding accessory (6) for an electrophoresis device, which is intended to create pits in a gel. According to the invention, the moulding accessory comprises a long bar which extends in one main direction and a plurality of teeth (36) which project out from said bar in a secondary direction which is essentially orthogonal to the main direction. Said teeth (36) are distributed over at least two rows (33, 34) which are spaced apart in a third direction which is essentially orthogonal to the main direction and the secondary direction, the teeth (36) in one row (33) being offset in relation to the teeth in another row (34). The invention also relates to an electrophoresis device comprising the above-mentioned moulding accessories (6).

Description

ELECTROPHORESIS DEVICE AND MOLDING ACCESSORIES

FOR SUCH A DEVICE

The present invention relates to an electrophoresis device and molding accessories for such a device.

The development of technical platforms within research establishments or in industry leads them to equip themselves more and more in robotic stations for programs of screening or manufacturing of high flow biological molecules. Several centers are thus engaged in the robotization of the amplification processes of nucleotide molecules by PCR (“Polymerase Chain Reaction”) on 96-well plates (generally four plates per automated cycle). Nevertheless, amplified products must necessarily be checked, generally for both their quality and their quantity. To do this, the quality of the products is visualized by electrophoresis on agarose gel and the quantity evaluated by optical density. This prerequisite requires the development of a methodology and devices for processing a large number of samples quickly and standardized at an affordable cost. However, at present, there is no electrophoresis system on the market whose size allows, on a single agarose gel support, the deposit of a high number of samples (for example 384 samples, representing 4 96-well microplates), their efficient migration as well as their analysis and quantification in a single manipulation using a digital camera placed below or above the gel.

In the frame. of an electrophoresis, particles having a net electric charge are subjected to the action of an electric field and move in the direction of the field towards the pole of sign opposite to their charge, at a speed proportional to this charge. This operation, applied to a heterogeneous set of charged particles, results in the separation of the particles. Different types of electrophoresis are defined, including analytical electrophoresis and separative electrophoresis.

The first aims to separate the greatest possible number of constituents from a mixture. The supports often used are starch, polyacrylamide and acrylamide-agarose gels, that is to say gels having the filtering properties which make it possible to separate the particles not only according to their electrical charge but also of their size.

The second aims to obtain more or less large quantities of one or more constituents of a mixture; therefore the performance of the operation must be taken into account, possibly at the expense of the finesse of the separation. The carriers used in this case are cellulose, starch powder and acrylamide gels.

The production of a horizontal agarose gel (in an independent support or in the electrophoresis tank itself) indeed requires its molding as well as that, at the same time, of the wells which must receive the samples. Creating wells requires the use of one or more comb units containing a varied number of teeth. Once the gel has solidified, the combs are removed and the impression of the teeth constitutes a receptacle in which the sample is deposited. Thus the dimensions of the gel and, consequently, of the tank which contains it, will be conditioned by the constraint of preserving a spacing between each tooth, preferably of approximately 9 mm. Indeed, these dimensions must be compatible with the available working surface of the robotic platform as well as with the safe transport of the gel, the texture of which is fragile and its subsequent analysis on light tables, the sizes of which are limited.

The invention relates in particular to a molding accessory for an electrophoresis device intended to create wells in a gel, said accessory comprising an elongated bar in a main direction and a O 2004/063739

plurality of teeth projecting from said bar in a secondary direction substantially orthogonal to the main direction.

The present invention aims in particular to solve the various problems essentially encountered in the context of analytical electrophoresis, and proposes a device and accessories for electrophoresis making it possible to produce a high number of wells on a reduced gel surface, for simultaneous analysis of a large number of samples (up to 384), and suitable for manual, automated or semi-automated use.

The invention is notably based on the development of new combs, making it possible to generate a high number of wells on a limited surface of gel.

To this end, a molding accessory according to the invention is characterized in that the teeth are distributed in at least two rows spaced from one another in a third direction substantially orthogonal to the main direction and to the secondary direction, the teeth of one row being offset from the teeth of the other row.

According to other characteristics: α said rows are spaced at least 4 mm apart; α the teeth of the same row are spaced at a regular interval, in particular substantially equal to 9 mm; α each row has a number of teeth between 10 and 30; o the teeth of the same row are identical; α each tooth has a length along the main direction between 2.5 and -3.1 mm, a width along the third direction between 1 and 1.5 mm, and a height along the secondary direction between 8 and 9 mm ; and the accessory is made of a rigid material, in particular delrin, metal, plastic, plexiglass. The invention further relates to an electrophoresis device comprising a molding tank intended to contain a gel, molding accessories adapted to create wells in the gel contained in the tank, as described above, and means for support for said accessories, adapted to hold the accessories parallel to each other.

According to other characteristics: α said support means comprise a frame disposed on an outer periphery of the tank, said frame comprising two opposite parallel beams provided with anchoring housings for the accessories; α two consecutive housings of the same spar are at least 2 cm apart, preferably by a distance of about 2.5 cm; α the device further comprises a pipette support, and the frame comprises means for supporting and guiding the pipette support allowing it to slide in the direction of the side members; α the support and gufdage means comprise a rod made of a rigid material, for example metal, said rod comprising positioning elements adapted to cooperate with complementary elements of the pipette support; α the positioning elements are recesses formed in the rod at locations corresponding respectively to the locations of the rows of teeth of the molding accessories during the formation of the wells in the gel, while the complementary elements comprise at least one ball resiliently biased towards one of the recesses; α pipette support is mounted on the rod with a clearance in the main direction of the accessories, the clearance corresponding at least to the offset between the teeth of the two rows; the positioning elements comprise pairs of recesses spaced along the secondary direction and along the main direction, respectively by a distance corresponding to the spacing of the rows of teeth and by a distance corresponding to the offset of the teeth by two rows, so that the pipette holder can be positioned at the right a row of wells formed in the gel, or the consecutive row offset; and α the accessories support means comprise means for moving said accessories, adapted to simultaneously lift all the accessories in place so as to at least partially free their teeth from the gel contained in the tank.

The electrophoresis device according to the invention thus advantageously makes it possible to migrate, with good separation, up to 384 samples over a relatively small area of gel and in particular of agarose gel, while maintaining a spacing between the samples ( from center to center) corresponding to that of the tips of multi-channel pipettes or pipetting arms of robotic platforms from all manufacturers. It also greatly facilitates the deposition of samples in the gel wells during manual deposition.

In addition, the system of the invention incorporates features which allow easy release of the gel and manual deposition of the samples which do not require a constant effort on the part of the user to maintain the multi pipette at the sole force of his wrist. -channels in an axis which ensures positioning of the tips at the center of the agarose gel wells.

A particular embodiment of the invention, given by way of nonlimiting example, will now be described with reference to the accompanying drawings, in which: α in Figure 1 is a perspective view of an electrophoresis device according to the invention; Figure 2 is an exploded perspective view of the device of Figure 1; α Figure 3 is a schematic sectional view along a vertical plane, on a larger scale, of a detail of the device -represented in the previous figures, in a peripheral region; α in Figure 4 is an elevational view of a molding accessory or comb of the device of the preceding Figures; α in Figure 5 is a top view of the comb of Figure 4; a Figure 6 is a schematic top view of the device of Figure 1, further equipped with a pipette holder; and α in Figure 7 is a schematic perspective view, on a larger scale, of the pipette support of Figure 6 and of a support and guide part of this support. α Figure 8 is a schematic sectional view of the electrophoresis device and the pipette support of Figure 7 guiding the tips of multi-channel pipettes.

In Figures 1 and 2, there is shown, respectively in ready-to-use assembled form and exploded form, an electrophoresis device according to the invention.

Figure 1 illustrates the normal arrangement of the device in its conditions of use, namely that the device, which has a very flattened shape along a reference plane, rests on a horizontal parallel plane support. In all that follows, the terms “horizontal”, “vertical” will be understood with reference to this orientation of the device.

The device comprises an electrophoresis tank 1, resting on supports 2 for anchoring to a platform (not shown), a series of molding accessories or combs 6, only one of which has been shown in FIG. 2 for reasons of clarity, and a frame 8 forming a support for the combs 6. The frame 8 has a shape corresponding to the peripheral edge of the tank 1, so that it can be arranged in superposition on the edge of the tank. Conventionally, the tank 1 and the frame 8 are rectangular. The frame 8 comprises two beams 10A, 10B corresponding to two opposite sides, intended to support the respective ends of the combs 6. For this purpose, each beam 10A, 10B is provided with the same number of anchoring housings 12 regularly spaced, and arranged opposite corresponding anchoring housings on the opposite spar, so that the frame 8 can horizontally support several combs above the tank 1, parallel to each other.

Preferably, said housings are arranged to ensure a distance between each comb of at least 2 cm, advantageously about 2.5 cm.

As will be better seen in FIG. 3, the tank 1 has an internal face 15 in which a peripheral shoulder 17 is formed. It comprises a molding container 19 intended to contain a gel 20, and a plexiglass plate 21 resting on the shoulder 17 in order to support the molding tray 19 itself. In FIG. 3, a comb has been shown in its functional position, bearing on the frame 8, its ends being received in the corresponding anchoring housings 12 .

We will now describe in detail, with reference to Figures 4 and 5, the structure of a comb according to the invention, and used in the device described above.

The comb shown essentially consists of an elongated horizontal bar 31, the two ends of which 31 A, 31 B form the support parts on the frame 8 engaging in the anchoring housings 12, and two rows of teeth vertical 33, 34 spaced transversely. The rows of teeth are cut from vertical flat blades, which are elongated in the longitudinal direction of the bar 31, and fixed to the latter by a longitudinal edge. The teeth 36 of one row are offset longitudinally relative to the teeth of the other row.

Preferably, as shown, the teeth 36 are all identical, of square shape, and spaced apart from one another by a regular or "pitch" interval, for example equal to approximately 9 mm, which commonly corresponds to the spacing of the tips of multi-channel pipettes ^' or of pipetting arms of robotic platforms. This spacing is not only compatible with the main commercial instruments (pipettes), but also ensures good use of gel space. It is understood that this interval can be slightly smaller or larger, for example between 8 and 20 mm.

The teeth have a length, in the longitudinal direction of the comb, between 2.5 and 3.1 mm, a width, in the transverse direction, between 1 and 1.5 mm, and a height between 8 and 9 mm , the height being taken vertically between the top and the base of the niche.

More preferably, the offset between the teeth of the two rows is equal to half a step. It is not essential that the offset is perfectly centered, that is to say that the tooth of the first row is exactly in the center of the gap between two teeth of the second row.

However, to ensure better separation of the wells, it is advantageous for this offset to be as large as possible.

In the embodiment shown, the rows are spaced about 4 mm apart, which corresponds to a minimum spacing for correct molding of wells in a commonly used gel.

In the embodiment shown, the rows of teeth are two in number, but one could conceive of combs comprising a greater number of offset rows. Similarly, a comb with two rows of 27 teeth has been shown, but it is estimated that each row can contain from 10 to 30 teeth to meet the needs commonly encountered.

In the specific example shown, each comb therefore contains a total of 54 teeth, distributed over a distance equal to 24.6 cm, of which 6 teeth can be used for depositing size markers.

The combs can be made of any material strong enough to create wells in a gel without otherwise altering said gel, for example delrin, metal, plastic, plexiglass, etc.

Referring now to Figure 6, which shows a device according to what has been described above, further comprising a pipette holder 40. Such a pipette holder 40 is necessary for use manual of the electrophoresis device, during which a sample deposition is carried out using in particular a multi-channel pipette. The frame 8 is, in this embodiment, provided with a lining 42 also in the form of a frame, two sides of which are superimposed on the side members 10A, 10B, above them, and constitute rods 44A, 44B of support and guide of the pipette support 40..

Although the anchoring housings 12 have been shown in FIG. 6, it can be understood that the latter, formed in the longitudinal members 10A, 10B, are located below the rods 44A, 44B The rods 44A, 44B cooperate with complementary parts respective pipette support 40 so as to allow its horizontal sliding in the direction of the side members 10A, 10B.

The rods 44A, 44B are made of a rigid material and sufficiently resistant to support pipette supports, for example metal. Like the pipette support 40, they are symmetrically shaped with respect to the median vertical plane of the device, which will make it possible to describe only one of these rods and only part of the pipette support 40, this which will be done later with reference to Figure 7. As it appears in Figure 6, the device comprises eight pairs of anchoring housings 12, of which only four have been shown, corresponding to eight combs 6 (represented schematically by the imprints of their teeth). These eight combs represent a total of 432 teeth, distributed for example as follows: 384 for the samples and 48 for the markers. The number of combs, which depends on the needs of the user, can differ and be between 2 and 10. The combs can be embedded on the longitudinal members 10A, 10B of the tank so as to have between the first row of the first comb and the first row of the next comb, a distance of at least 2.5 cm. This distance allows a separation much greater than that currently offered for electrophoresis devices designed for deposits of 91 samples. The length of the gel, in the direction of migration, will therefore be 21.4 cm if the example described above is taken into account. The gel, the dimensions of which are 21.4 cm × 24.6 cm (LxW), corresponds to a standard size gel that can be analyzed on light tables (called “transilluminators”) sold by most manufacturers. A rod comprises positioning elements 46, 47 of the pipette support 40, formed by holes adapted to cooperate with a complementary ball 49 (Figure 7), linked to said support 40 and urged elastically towards one of the holes. The ball 49 and the holes 46, 47 thus define notches on the rod 44A, 44B corresponding to the position of the pipettes in line with the wells formed in the gel by a given row of teeth. For this, the holes 46, 47 of a rod 44A, 44B are associated in pairs with the position of a comb 6 marked by the anchoring housings 12. With respect to the main direction of the side members 10A, 10B, the holes 46, 47 of the same pair are offset longitudinally by a distance equal to the spacing of the two rows of teeth of the same comb 6, and laterally by an interval corresponding to the offset of the rows of teeth.

As shown in Figure 7, the pipette holder 40 has a bar 50 transverse to the direction of the side members 10A, 10B and the rods 44A, 44B. The crossbar 50 is, for its part, secured to several (three in number in the previous figure) parts 51 with notches and pipette holding holes. Each end of the crossbar 50 is provided with a U-shaped guide part 52, the opening of which faces the rod 44A, 44B and the outside of the tank, fedite guide part 52 cooperating with sliding with the rod, with a lateral clearance making it possible to position the ball 49 in line with one or the other of the holes 46, 47 of the same pair.

Thanks to this system, an operator can bring and maintain the row of pipettes in line with any row of channels, and carry out a deposit of samples there.

Referring again to FIG. 6, means are provided for simultaneous vertical displacement of all the combs arranged in their housings anchoring, during the demolding phase, in order to at least partially free the teeth from the gel. These means comprise lever elements actuated by a manual pressure control member 60, arranged in frame 8. The means described above are intended to extract the combs from the gel without altering the structure thereof, with force. homogeneous on all the combs.

The frame 8 can therefore have a recess, with a width of four fingers for example, making it possible to engage, advantageously, at the push of a button, a lever element which causes a simultaneous vertical rise of all the combs with a homogeneous distribution. forces. This system prevents the wells from collapsing during the comb release phase and therefore makes it possible to preserve the integrity of the gel and the structure of the wells formed. Many modifications can be made to the invention without departing from the scope thereof.

The electrophoresis device can be implemented with the aforementioned support means for the molding accessories without these accessories having the two rows of teeth. Likewise, it is possible to use the means for supporting and guiding a pipette support without the molding accessories having the two rows of teeth.

Claims

1. Molding accessory for an electrophoresis device intended to create wells in a gel, said accessory (6) comprising an elongated bar (31) in a main direction and a plurality of teeth (36) projecting from said bar (31 ) in a secondary direction substantially orthogonal to the main direction, characterized in that the teeth (36) are distributed in at least two rows (33, 34) spaced from each other in a third direction substantially orthogonal to the main direction and the secondary direction, the teeth (36) of one row (33) being offset with respect to the teeth of the other row (34).

2. Molding accessory according to claim 1, characterized in that said rows (33, 34) are spaced at least 4 mm.

3. Molding accessory according to claim 1 or 2, characterized in that the teeth (36) of the same row (33) are spaced at a regular interval, in particular substantially equal to 9 mm.

4. Molding accessory according to one of claims 1 to 3, characterized in that each row (33, 34) has a number of teeth (36) between 10 and 30.

5. Molding accessory according to any one of the preceding claims, characterized in that the teeth (36) of the same row (33,

34) are identical.

6. molding accessory according to any one of the preceding claims, characterized in that each tooth (36) has a length in the main direction between 2.5 and 3.1 mm, a width in the third direction between 1 and 1.5 mm, and a height in the secondary direction of between 8 and 9 mm.

7. Molding accessory according to any one of the preceding claims, characterized in that it is made of a rigid material, in particular delrin, metal, plastic, plexiglass.

8. Electrophoresis device comprising a molding tray (19) intended to contain a gel (20), molding accessories (6) adapted to create wells in the gel contained in the tray, and support means (8 , 12) of said accessories (6), characterized in that said accessories (6) conform to any one of the preceding claims, and the support means (8, 12) are adapted to hold the accessories (6) parallel to each other to others.

9. Electrophoresis device according to claim 8, characterized in that said support means (8, 12) comprise a frame (8) disposed on an outer periphery of the tank (19), said frame (8) comprising two parallel beams (10A, 10B) opposite provided with housings (12) for anchoring the accessories (6).

10. Electrophoresis device according to claim 9, characterized in that two consecutive housings (12) of the same spar (10A, 10B) are at least 2 cm apart, preferably by a distance of about 2 , 5 cm.

11. An electrophoresis device according to claim 9 or 10 further comprising a pipette support (40), characterized in that said frame (8). further comprises support and guide means (44A, 44B) of the pipette support (40) allowing it to slide in the direction of the side members (10A, 10B).

12. An electrophoresis device according to claim 11, characterized in that the support and guide means comprise a rod (44A, 44B) made of a rigid material, for example metal, said rod (44A, 2004/063739

14

44B) comprising positioning elements (46, 47) adapted to cooperate with complementary elements (49) of the pipette support (40).

13. An electrophoresis device according to claim 11 or 12, characterized in that the positioning elements are recesses (46, 47) formed in the rod (44A, 44B) at locations corresponding respectively to the locations of the rows of teeth ( 33, 34) molding accessories (6) during the formation of the wells in the gel (20), while the complementary elements comprise at least one ball

(49) urged elastically towards one of the recesses (46, 47).

14. Electrophoresis device according to claim 13, characterized in that the pipette support (40) is mounted on the rod (44A, 44B) with a clearance in the main direction of the accessories (6), the clearance corresponding at least offset between the teeth (36) of the two rows (33, 34).

15. Electrophoresis device according to claim 14, characterized in that the positioning elements comprise pairs of recesses (46, 47) spaced in the secondary direction and in the main direction, respectively by a distance corresponding to the spacing of the rows of teeth (33, 34) and a distance corresponding to the offset of the teeth of two rows (33, 34), so that the pipette holder (40) can be positioned in line with a row of wells formed in the gel (20), or of the consecutive row offset.

16. An electrophoresis device according to any one of claims 8 to 15, characterized in that the support means (8, 12) of the accessories (6) comprise means for moving said accessories (6), suitable for lifting simultaneously all the accessories (6) in place so as to at least partially free their teeth (36) from the gel (20) contained in the tank (19).