WO2005095940A1

WO2005095940A1 - Cassette for electrophoresis apparatus

Info

Publication number: WO2005095940A1
Application number: PCT/IT2004/000163
Authority: WO
Inventors: Giulia Onorina Caprotti
Original assignee: Delcon S.R.L.
Priority date: 2004-03-31
Filing date: 2004-03-31
Publication date: 2005-10-13
Also published as: EP1730510A1

Abstract

Parallelepiped-shaped cassette for electrophoresis, having the height reduced with respect to the width thereof and consisting of a rigid container (3) able to contain gel and a removable airtight lid (2) which, at the corners of a narrower side thereof, has two chamfers (25, 26) facing the container (3), the latter having on a lateral wall a hole (31) for feeding gel, as well as two protrusions (21, 22) and one comb (23), both being parallel to a shorter side thereof. Such a cassette, while acting as closed until the moment of use, turns instead into the open condition at the moment of the insertion thereof into the apparatus for electrophoresis. It allows the operator to act onto the gel even after the first operation of electrophoretic separation and consequently to carry out further analyses on separated samples.

Description

CASSETTE FOR ELECTROPHORESIS APPARATUS

The present invention relates to the field of products used in molecular biology, and in particular to a closed cassette for electrophoresis, which turns open at the moment of the insertion thereof into the electrophoresis apparatus. In the electrophoretic practice, the closed cassettes provide the advantage of a complete insulation from the outer environment and consequently from every risk of sample contamination. A closed-type cassette for electrophoresis is disclosed for example in US patent 5,865,974. Unfortunately, the known closed cassettes are not free from drawbacks, the main one being the fact that they do not allow further interventions and tests on the gel and consequently, once the electrophoresis has been carried out, they are disposed. An object of the present invention is therefore to provide a cassette that, while behaving as being closed until the moment of use, from that moment on it turns into the open-type, thus providing the relevant advantages, among which that of allowing the operator to act on the gel even after the first operation of electrophoretic separation and consequently to carry out thereon further analyses before disposing the cassette. This object is achieved according to the present invention by means of a cassette provided with a removable¹ lid, the main characteristics of which are specified in Claim 1. Further characteristics of such a cassette are reported in the depending claims. The cassette according to the present invention provides a first remarkable advantage due to automatic removability of the lid, which allows to turn the cassette from a closed-type to an open-type. The lid is embeddable and is automatically removed from the electrophoresis apparatus only when the cassette is inserted therein; accordingly there are not the risks inherent in the open system, as until that moment the cassette is kept airtight closed. The remarkable advantage provided by the present invention is due to the particular structure the cassette has, namely to two chamfers existing at the comers of an embeddable lid side. These chamfers face the inside of the cassette and thus allow the cassette to be opened only when it is inserted into the electrophoresis apparatus, h this way, the operator is preserved from any contact with the content of the cassette . This aspect is particularly important not only for minimizing the risks of sample contamination, but mainly for protecting the operator, since the gels usually used in electrophoretic practice are often toxic and sometimes even carcinogenic. Another advantage provided by the present invention is due to the fact that in its preferred embodiment, the cassette is free from electrodes. The latter are actually directly placed into the gel by the electrophoresis apparatus at the moment of use only, after inserting the cassette therein and removing the lid . This results in a saving from the economic point of view, as the same electrodes can be reused. This is not possible with the traditional closed-type cassettes, in which at the end of usage the electrodes are unavoidably disposed together with the relevant cassette. Further advantages and characteristics of the cassette according to the present invention will be clear to those skilled in the art from the following detailed description of a preferred embodiment thereof taking reference to the annexed drawings, wherein: Figure 1 is an exploded perspective view of the cassette showing the lid removed; Figure 2 shows a plane bottom view of the embeddable lid of the cassette in Figure 1; Figure 3 shows a lateral view by a longer side of the embeddable lid of Figure 2; Figure 4 shows a cross-sectional view of the embeddable lid of Figure 2 taken along line AA'; Figure 5 shows apian top view of the container of the cassette of Figure 1; Figure 6 shows a left lateral view of the container of Figure 5; Figure 7 shows a cross-sectional view of the container of Figure 5 taken along line BB'; Figure 8 shows a right lateral view of the container of Figure 5; and Figure 9 shows a lateral view along a shorter side of the container of Figure 5. Referring to Figure 1, the cassette 1 is shown essentially consisting of a container 3 provided with an embeddable lid 2. It has a substantially very flattened parallelepiped-shaped structure, wherein the embeddable lid 2 is one of the two faces having larger surfaces, whereas the container 3 has a bottom forming the other face with a larger surface. The side walls of the container 3 are very short in height. h Figure 2, the embeddable lid 2, on its wall facing container 3, shows two protmsions 21 and 22 parallel to each other and to the shorter sides of the lid. The protmsions 21 and 22 are useful for forming corresponding grooves into the gel (not shown in figure) contained in the cassette. The first groove, formed by protrusion 21, must be near one of the two electrodes and is useful to promote the evaporation of gases evolving therefrom. The other protrusion 22 will be near the second electrode and is useful for forming a groove intended to collect liquids that can grow inside the gel during the electrophoresis progress. Besides said protrusions facing container 3, the embeddable lid 2 is provided with the so called comb 23 also parallel to the shorter sides of lid 2. In the electrophoresis practice, by comb the row of teeth is meant that form corresponding wells into the gel, which wells axe intended for placing samples by means of a pipette or the like. In Figure 2, on a shorter side of the embeddable lid 2, a recess 28 is also shown having no specific function other than being only the injection point of plastics into the mould where the embeddable lid 2 is manufactured. Referring to Figure 3, the embeddable lid 2 is peripherally provided with a narrow wing 24 slightly raising with respect to plane 27 of the lid. Such a narrow wing is useful for assuring a perfect airtight lockup of container 3 by the embeddable lid 2. hi Figure 3 there is also shown that at both comers of one of the two shorter sides of embeddable lid two chamfers 25 and 26 facing down, i.e. facing container

3, are arranged on the lower face of narrow wing 24. They are useful for promoting lifting and automatic removing of lid 2 and accordingly opening of the cassette at the moment of use, i.e. only at the moment of the insertion thereof into the electrophoresis apparatus, as hereinafter disclosed. Referring to Figure 4, there is shown that comb 23 consists of 12 teeth. Such a number is not binding, as it is possible to manufacture cassettes having a different number of teeth according to the analysis requirements. h Figure 5, container 3 shows on a side wall thereof a gel feeding hole 31. As shown in particular in Figure 6, the feeding hole 31 is preferably pierced on one of the two larger lateral walls, and more preferably at a central point thereof, allowing in this way a feeding of container 3 as homogeneous as possible.

Container 3 too has along its periphery a narrow wing 32 suitably shaped to fit together with narrow wing 24 of embeddable lid 2. Container 3 further shows, still along its periphery but inwardly, a step 33 that at the moment of closing fits in turn together with plane 27 of embeddable lid 2. h this way, the embedding of the embeddable lid 2 on container 3 takes place, which assures a perfect tightness between the two constituents of cassette 1. Referring to Figures 5 and 7, container 3 shows on its bottom 36 two stubbings 34 and 35 that are useful for housing the two electrodes (not shown in the Figures). The surfaces of such stubbings 34 and 35 are preferably rough in order to improve the gel adhesiveness to container 3. It is not unusual actually the fact that the gel sticks to the lid at the moment of removal thereof, thus making unusable the whole cassette 1. Suitable electrodes for this kind of use consist in a known manner of metal elements, preferably aluminium or copper, usually available on the market. Electrodes consisting of other more precious metals, such as silver, gold and platinum, can also be used. In Figure 7, the container 3, on the top rim of its larger lateral wall, shows two recesses 37 and 38 placed in correspondence with stubbings 34 and 35. Each recess acts as a support for the end portion, or tongue, of the metallic electrode in such a way that it can protrude and contact the correspondent electrical contacts of the electrophoresis apparatus. hi the Figure 8, said two recesses 37 and 38 are clearly shown. Since, upon closing cassette 1, the tongues of both electrodes are such as not to completely plug said recesses, the remaining free space allows the air contained in cassette 1 to be evacuated at the moment of gel feeding. Actually, should exist a perfect tightness at both recesses 37 and 38, a complete filling of cassette 1 with gel would be absolutely impossible. In addition, in order to obtain a filling as homogeneous as possible of cassette 1 with gel, it is preferable that said recesses are placed on the lateral wall opposite to that where is feeding hole 31, as shown in Figures 6 and 8. Referring to Figure 9, on a narrower lateral wall of container 3, there are two prominences 39 and 40 having no specific function and being only, similarly to recess 28 of Figure 2, the injection points of plastics into the mould where container 3 is manufactured. hi a first embodiment, the equipping of cassette 1 provides first for both electrodes being placed on stubbings 34 and 35 of container 3. Two shaped elements or sponges (not shown in the Figures) are then superimposed on said electrodes, thus covering them. The sponges consist of a foamed material that may be homogeneously imbibed by gel. The inner surface roughness of both stubbings 34 and 35 and the presence of gel imbibed sponges in cassette 1, before inserting it into the electrophoresis apparatus, are useful for preventing dragging away the gel together with lid 2 when removing the latter. As a matter of fact, this drawback would make cassette 1 completely useless. Furthermore, among the suitable materials for such sponges, the melamine resin is preferable in that it allows to disperse in part the heat being generated within the cassette during the electrophoresis operation. The upper surface of said sponges is preferably provided with numbering ranged along the corresponding wells generated by comb 23, in order to aid working of the operator. If necessary, other inscriptions may be applied on said upper surface of the sponges. Once the embeddable lid 2 is tightly embedded on container 3, the gel is injected through the feeding hole 31 at temperatures preferably about 50-60°C. During this operation, it is important to keep the lid 2 under pressure in order to prevent seal losses, owing to the inner pressure exerted by the inflowing gel. Once the gel is congealed, thereby concurrently clogging the feeding hole 31, the pressure on lid 2 is released without occurring any seal loss between container 3 and embeddable lid 2. The gel is selected among the matrixes for electrophoresis usually employed by those skilled in the art in the case of nucleic acid separation or more generally in similar molecular biology applications, i. e. polyacrylamide gel or agarose gel. The polyacrylamide gels are obtained by vinyl polymerization of acrylamide monomers (CHr=CH-CO-]NIH₂) in long polyacrylamide chains bound to each other by a bifunctional co-monomer, generally N,N'-methylene-bis-acrylamide (CH₂=CH-CO-NΗ-CH₂- H-CO-CH=CH₂), through a radical reaction initiated by ammonium persulphate, that generates free oxygen radicals through a basic catalysis mechanism, wherein the bases are generally aliphatic tertiary amines such as N,N,N,'N'-tetramethylenediamine (TEMED). The average pore size is determined by both the total monomer concentration (T) and the % of bifunctional co-monomer with respect to the total monomers (C). Typically T ranges from 3.5 to 20%, whereas C ranges from 1 to 5%. The optimum polymerization temperature is from 25 to 30°C, in absence of oxygen, a strong polymerization inhibitor; the reaction generally finishes in 60 minutes. Agarose is a linear polysaccharide consisting of agarobiose repeating units between which galactose and 3,6-anhydiOgalactose units alternate. It is one of the constituents of agar, a mixture of polysaccharides isolated from certain seaweed species, and is usually used at concentrations from 0.6% to 4%. Agarose can melt at 80°C and gel at about 40°C. The gel pore sizes (sieve) depend on the initial agarose concentration: large pores are obtained by using low concentrations, vice versa, smaller pores by using higher concentrations. According to the traditional method, the separation travel takes place in electrophoresis cells where the gel is horizontally arranged by bridging two compartments, in each of which there is a buffer solution into which one electrode is let. Unlike what the abovementioned traditional method provides for, a peculiar characteristic of cassette 1 according to the present invention is that the gel is used therein in a "dry" fom , i.e. there is in it no additional buffer solution, besides that already contained in the gel and coming from the preparation thereof, according to known methods. The two compartments are therefore eliminated, that in the traditional method acted as buffer solution containers. Consequently the most is made of the conductive abilities of the only buffer solution contained in the gel, which in this case directly contacts the two electrodes. It has been actually found that the amount of buffer contained in the gel is enough for the electrophoretic separations in molecular biology, for which the cassette 1 is intended. The cassette manufactured according to the present invention is then put into an envelope and sealed under vacuum, to assure the preservation thereof for periods significantly longer than those of nowadays commercially available cassettes; the latter ones being not sealed under vacuum, show disadvantageously high gel deterioration rates. In a preferred embodiment, the equipping of cassette 1 provides for the two shaped elements or sponges (not shown in Figures) only being placed in the stubbings 34 and 35 of container 3. hi this case in fact, both electrodes are directly provided by the apparatus for electrophoresis, after the cassette is inserted therein and the lid removed. Such electrodes, named external, are placed in the apparatus for electrophoresis in such a way as to be opposed to the gel at stubbings 34 and 35. At the moment of closing the apparatus for electrophoresis, the electrodes are brought into contact with the gel and suitably housed in the two grooves previously generated therein by the protrusions 21 and 22. Once the electrophoretic travel is ended, the opening the apparatus involves the simultaneous removal of the electrodes, which can be consequently reused. It is clear that the embodiments of the above disclosed and described cassette for electrophoresis according to the present invention, are just examples susceptible of various modifications. It is actually possible that the chamfers, arranged in order to support the automatic removal of the lid, be present on a lateral wall of the container, instead of the embeddable lid. For the same purpose, it is also possible that such chamfers are arranged on both of them, i.e. on both the lid and the container, thus facing each other. The feeding hole could be made on the bottom of container or on the lid, rather than on the side walls of container, even though the gel feeding operation would be carried out less easily and advantageously from the practical point of view. It can be moreover provided for the possibility of manufacturing cassettes having various sizes, in order to meet the requirements of a number of wells different from that disclosed in the preferred embodiments. In addition, it is possible to use different carrying gels by selecting the matrix accordmg to the biologically interesting molecules contained in the samples. Furthermore, for producing the lid and container of the cassette according to the present invention, it is possible to use any known material deemed suitable and biologically compatible with this kind of application, e.g. thermoplastic or thermosetting polymer materials. Possible changes and/or additions can thus be made to the cassette for electrophoresis of the present invention without departing nevertheless from the scope of the invention.

Claims

1. Cassette for electrophoresis in the shape of a parallelepiped having the height reduced with respect to the width thereof, comprising a rigid container (3) able to contain a gel, characterized in that it is provided with a removable airtight lid (2), which, correspondence of at least one comer thereof, has at least one chamfer (25, 26) facing the container (3), the latter having on a side wall thereof a hole (31) for feeding a gel of the type used in electrophoresis practice.

2. Cassette for electrophoresis according to claim 1, characterized in that the lid (2) is embeddable.

3. Cassette for electrophoresis accordmg to claim 1 or 2, characterized in that the lid (2) has one comb (23) parallel to the narrower side walls thereof, which consists of a row of teeth protruding towards the container (3) and able to generate corresponding wells into the gel, when the latter is present in the container (3).

4. Cassette for electrophoresis according to one or more previous claims, characterized in that the lid (2) has at least one protrusion (21, 22) paralleling the narrower lateral walls thereof and facing the container 3, which is able to generate at least one groove into the gel, when the latter is present in the container (3).

5. Cassette for electrophoresis according to one or more previous claims, characterized in that it contains a gel of the type used in electrophoretic practice.

6. Cassette for electrophoresis according to claim 5, characterized in that, during the electrophoretic travel, the gel contained in the container (3) contacts directly the two electrodes of the apparatus for electrophoresis.

7. Cassette for electrophoresis according to one or more previous claims, characterized in that the bottom (36) of the container (3) has at least one stubbing (34, 35), having rough inner surface and being able to house one of the two electrodes of the apparatus for electrophoresis.

8. Cassette for electrophoresis according to one or more previous claims, characterized in that on the top rim of a side wall of the container (3) at least one recess (37, 38) is present.

9. Cassette for electrophoresis according to one or more previous claims, characterized in that the lid (2), in correspondence of adjacent comers on one shorter side thereof, has two chamfers (25, 26) facing the container (3).

10. Cassette for electrophoresis according to one or more previous claims, characterized in that on the lid (2) two protmsions (21, 22) are present, each one being able to generate one groove into the gel in correspondence of each electrode, both protmsions (21, 22) being parallel to each other and parallel to the comb (23).

11. Cassette for electrophoresis according to one or more previous claims, characterized in that the bottom (36) of the container (3) has two stubbings (34,

35) having rough inner surface, each stubbing (34, 35) being able to house one of the two electrodes and each one being covered with foamed material which may be homogeneously imbibed by gel.

12. Cassette for electrophoresis according to one or more previous claims, characterized in that on the top rim of a larger side wall of the container (3) there are two recesses (37, 38), each one being in correspondence of one stubbing (34, 35).