EP1123160B1 - Device for conducting biochemical and microbiological reactions - Google Patents

Abstract

The invention relates to a device for conducting biochemical and microbiological reactions, comprising a support (2) for receiving at least one reaction vessel (3) and a device (5) for pressing a closing means (11, 12) sealing the reaction vessel (3), wherein the closing means (11, 12) project in closed position in the area surrounded by the reaction vessel (3). According to the invention, in order to accelerate and simplify reaction, an inner wall of the closing means opposite the reaction vessel (3) is coated with a molecule (14) on which a molecule to be detected can be bonded or deposited, wherein the molecule (14) contains a nucleic acid, an amino acid or a synthetic derivative of a nucleic or amino acid.

Description

The invention relates to a device for performing biochemical and microbiological reactions according to the generic term of claim 1.

Such a device is known from WO 93/20240. To it is further known in the prior art for simultaneous Performing a variety of molecular biological reactions, e.g. the polymerase chain reaction (PCR), a 96-well Use microtiter plate. After filling the Wells of the microtiter plate with the reaction solution these covered with a film. The slide is during the Perform the PCR with a heated lid against the Microtiter plate pressed. - The known method is relative time consuming because of relatively large amounts of reaction solution at the same time an exactly observable temperature treatment have to undergo.

A so-called thermal cycler is known from US Pat. No. 5,455,175, with which a plurality of liquid biological samples to perform the PCR recurring a given Temperature profile can be exposed. To the required Shortening time for the heat treatment is here in each case a small volume of biological samples in one thin-walled glass capillary added. Every sample has to do this individually filled into the capillary and then sealed become. Sample preparation is time consuming.

DE-A1-OS 44 12 286 describes a system for contamination-free Processing of reaction processes known. The system includes reaction vessels, individual closures for the reaction vessels and a device for automatically opening the individual closures. When closed, the lid jumps into the space surrounded by the reaction vessel. This allows an intervention of the device and thus an automatic Opening the individual locks.

US-A-4,599,314 discloses a carrier in which a plurality of reaction vessels can be used. The to lock the Reaction vessels provided can be closed by means of a Adhesive tape plate together from the reaction vessels be lifted off. The time required to complete the This does not significantly reduce reactions.

The object of the present invention is to overcome the disadvantages to eliminate the state of the art. In particular, a alternative device can be specified with the low Time spent doing a variety of biochemical or microbiological reactions can be carried out.

This object is solved by the features of claim 1. Appropriate configurations result from the features of claims 2 to 21.

According to the invention, it is provided that the closure means at least in the area of the coating from one electrically conductive plastic is made. So that is it is possible e.g. B. to create a potential above the sample solution and charged molecules to be detected contained in the sample thereby moving in the direction of the coating. It is but it is also possible to bind or add evidence Molecules attached to the molecule of the coating using voltametric Evidence of methods.

The molecule used for coating can be for example a nucleic acid probe, a gene, a peptide, Protein, PNA (= peptide nucleic acid), PTO (phosphorothioate) or the like. The bindability of the The molecule to be detected exists when using a Nucleic acid probe in a hybridization with a Nucleic acid probe complementary nucleic acid. In case of Use of a peptide or protein as a molecule may change an antigen / antibody bond with the molecule to be detected form. The molecule has the property of being specific bind the molecule to be detected.

The device according to the invention enables a particularly rapid and efficient implementation of microbiological reactions. Since the lid is in the one enclosed by the reaction vessel Projecting space, only small sample volumes needed. A molecule to be detected, if any, contained therein can quickly and efficiently e.g. amplified by PCR become.

The lid can be made of a preferably transparent plastic, become. Such a Lid is inexpensive. It can be designed as a single-use item his.

The device for pressing can be used to shape the cavity have corresponding projection. So it becomes simple Way tight sealing of the cavity is achieved.

According to a further design feature, the device for pressing a holding device for the lid (s) on. So an automated placement of the lid (s) can be achieved.

The receiving device can expediently for frictional Inclusion of a separate cover as a tube or Rod be trained. But it can also be that in the Set up a device near the free end of the pipe or rod is provided for releasable attachment of the lid. there can be a radially projecting U-profile act, one leg connected to the tube and whose other leg is shorter than one leg is. Such a facility allows simple Way the inclusion of a lid. This is convenient an outwardly projecting edge provided on the lid corresponding to the U-profile. The edge and that corresponding U-profiles advantageously extend over two circumferential sections of approximately 90 °. The receiving element can be rotated by about 90 °. By such Rotational movement can be done using the design described above of the edge and the corresponding U-profile a simple inclusion of the lid can be achieved.

Each receiving element can also be axially movable. The enables a separate pressing and pulling of certain Cover.

According to a further embodiment, the receiving element coaxially surrounding and movable relative to the receiving element Ejector element may be provided. That enables one Discard e.g. frictionally held on the receiving element Lids.

Of course, the lids can not only be frictionally engaged, but also by means of a detachable snap connection on the Receiving device are held. This can be done on the outer circumference a receiving device designed as a rod or tube a radially circumferential bead can be provided, which with a corresponding groove cooperates on the inner wall of the lid.

It is advantageous between a plurality of receiving elements at least one movable relative to the receiving elements Stamp provided. That makes the simultaneous easier Loosen several lids.

It is considered particularly advantageous that the projection, the tube or rod for introducing optical fibers Light in or for the observation of formed in the cavity Has light. The optical fibers can with a Light source and / or a device for the detection of Fluorescence. This enables a special fast and efficient process management.

A plurality of reaction vessels is advantageous provided and the reaction vessels are part of a microtiter plate. Reaction vessels designed in this way can be carried out for example in conventional thermal cyclers the PCR can be used. With such training the reaction vessels can contain a plurality of lids a cover plate. That makes closing easier and opening.

It is considered particularly advantageous that the electrical conductive plastic made of an electrically conductive Polycarbonate is made. Such an electrically conductive Polycarbonate can be added, for example, by adding graphite or graphite fibers are produced. But it can also intrinsically conductive plastics, such as polyaneline or Polyacetylene can be used. The electrically conductive area can be contacted.

Fig. 1

eine schematische Querschnittsansicht eines Reaktionsgefäßes mit Verschlussmittel in einer ersten Stellung,

Fig. 2

eine schematische Querschnittsansicht gemäß Fig. 1 in einer zweiten Stellung,

Fig. 3

eine schematische Querschnittsansicht eines weiteren Reaktionsgefäßes mit Deckel,

Fig. 4

Draufsicht auf den Deckel nach Fig. 3,

Fig. 5

eine schematische Querschnittsansicht eines Ausführungsbeispiels in einer ersten Stellung und

Fig. 6

eine schematische Querschnittsansicht gemäß Fig. 5 in einer zweiten Stellung.

Exemplary embodiments of the invention are explained in more detail below with reference to the drawing. Show here:

Fig. 1

2 shows a schematic cross-sectional view of a reaction vessel with closure means in a first position,

Fig. 2

2 shows a schematic cross-sectional view according to FIG. 1 in a second position,

Fig. 3

2 shows a schematic cross-sectional view of a further reaction vessel with a lid,

Fig. 4

Top view of the lid according to FIG. 3,

Fig. 5

is a schematic cross-sectional view of an embodiment in a first position and

Fig. 6

a schematic cross-sectional view of FIG. 5 in a second position.

1 and 2 is preferably made of polycarbonate manufactured transparent microtiter plate 1 in a carrier 2 added. Cylindrical reaction vessels or Cavities 3 of the microtiter plate 1 engage in corresponding ones Recesses in the carrier 2. As a closure a separate cover 12 is provided for each cavity 3. On the underside of the lid opposite the cavity 3 13 is a nucleic acid probe 14. The lid 12 is received on a receiving element designed as a tube 15. A circumferential bead 16 provided on the tube 15 engages into an annular groove 17 on an inner wall of the cover 12. in the Tube 15 accommodates the first optical fiber 7. The pipe 15 is coaxially surrounded by another tube 18. The further Tube 18 is axially movable. One end of a second Optical fiber 9 is located in a bottom surface of the Recess.

A reaction solution taken up in the cavities 3 is included R denotes. 2 shows the closed state. The underside of the lid 13 is in contact with the reaction solution R.

The lid is in the reaction vessel shown in FIGS. 3 and 4 12 in turn is essentially cylindrical and provided with a nucleic acid probe on the underside of the lid 13.

The cover has two radial radial segments Edge projections 19, at one end of which one axially extending stop 20 is molded. At the end of the pipe 15 are two U-profiles 21 encircling in sections for engagement provided in the edge projections 19.

5 and 6, a fourth embodiment is shown. The cover 12 is made in sections from one electrically conductive plastic. The conductive Areas B1, B2 are designed in the form of pipe sections, the one transparent between the end of the first Optical fiber and the nucleic acid probe 14 located area surrounded by a jacket. The electrically conductive first Area B1 is at the end of one worked into the tube 15 first line 22 with a contact (not shown here) Mistake. The contact becomes when the cover is put on 12 on the pipe 15 against the first electrically conductive Area B1 pressed. The same is also in the bottom of the cavity 3 of the microtiter plate 1 is a tubular second electrically conductive area B2 provided with a transparent plastic is filled. The second electric conductive area B2 is connected to a second line 23 electrically connected.

The function of the devices is as follows:

First, a predetermined amount of reaction solution R pipetted into the cavities 3. The one on the reaction solution facing underside of the lid 12 provided nucleic acid probe 14 is brought into contact with the reaction solution R.

The one made of an at least partially transparent plastic manufactured cover 12 can on an axially movable Tube 15 by means of a locking connection 16, 17 or a frictional connection Be kept connected. The lid 12 will by an axial movement of the tube 15 into that in FIG. 4 and 8 shown locking position brought. To open the Cavity 3, the lid 12 by an opposite Axial movement of the tube 15 lifted off. He can then thrown by an axial movement of the further tube 18 become.

In the third embodiment shown in FIGS. 5 and 6 the tube 15 moves axially with the end of it provided U-profiles 21 in between the edge projections 19 formed gaps of the lid 12. By rotation by about 90 °, the U-profiles 21 in one of the edge projections 19 brought comprehensive position. Ensure the stops 20 a proper fit of the lid 12 relative to Tube 15. The lid 12 is loosened in reverse.

The carrier can be part of a thermal cycler for implementation the PCR. To carry out a biochemical detection reaction becomes a containing the molecule to be detected Reaction solution R in each cavity 3 of a microtiter plate Pipette. Each of the cavities 3 is by means of a lid 12 closed. The lid 12 are advantageously part a one-piece cover plate. Everyone the lid 12 is facing the reaction solution R. Coated on the inside with another nucleic acid probe.

The microtiter plate closed with the cover plate is then inserted into the carrier 2. The carrier 2 becomes cyclical heated and cooled. This results in a PCR through which a molecule to be detected contained in the reaction solution R. is amplified.

The molecule binds to the corresponding nucleic acid probe 14. In the case of binding to nucleic acid probe 14, this can either be detected using fluorescence. For this purpose, the fluorescence radiation that may occur is by means of the first 7 and / or second optical fiber 9 to a detector transferred and evaluated.

To accelerate the enrichment of the molecule to be detected on the nucleic acid probe 14 is via the in the lid 12th and provided in the bottom of the cavity electrically conductive Areas also have a potential above the reaction solution R created. This can e.g. negatively charged nucleic acid molecules moved in the direction of the nucleic acid probe 14 and there be enriched.

But it is also possible using the electrically conductive Areas or electrodes e.g. using voltammetric methods binding of nucleic acids to be detected or Molecules to the nucleic acid probe 14 or molecular coating to detect.

It may also be that the cover 12 is only in the first area B1 the molecular coating of an electrically conductive Plastic is made or in this area Electrode made of metal, preferably gold is incorporated. The electrically conductive area B1, B2 can be from an area made of insulating plastic be surrounded. In this case, the electrical conductive area B1, B2 one with the molecular coating electrode in contact. If a variety of Lids 12 are provided in the form of a cover plate in this case, each cover 12 has a separate electrode. With the electrodes it is possible to simultaneously change the potential to measure on each lid 12.

The optical fibers 7, 9 alternatively allow the irradiation e.g. of UV light and / or the detection of one in the Reaction solution R occurring fluorescence. With that too the presence of the molecule to be detected in the reaction solution R can be demonstrated.

Stamps (not shown here) can be between the tubes 15 be provided. The stamp can be removed during the peeling the cover 12 can be moved against the microtiter plate 1 and hold them in position on the carrier 2.

LIST OF REFERENCE NUMBERS

1

microtiter plate

2

carrier

3

cavity

7

first optical fiber

8th

projection face

9

second optical fiber

11

foil

12

cover

13

Cover underside

14

nucleic acid probe

15

pipe

16

bead

17

ring groove

18

another pipe

19

edge projections

20

attack

21

U-profile

R

reaction solution

H

height

B1

first area

B2

second area

Claims (21)

1. Device for conducting biochemical and microbiological reactions comprising a support (2) for receiving at least one reaction vessel (3) and a device (15) for pressing a cover (12) sealing the reaction vessel (3), the cover (12) projecting in its closed position in the space surrounded by the reaction vessel (3), an inner wall of the cover (12) that faces the reaction vessel (3) being coated with a molecule (14) on which a molecule to be detected can be bonded or accumulated, the molecule (14) containing a nucleic acid, an amino acid or a synthetic derivate of a nucleic or amino acid, characterised in that the cover (12), at least in the zone of the coating, is made of an electrically conductive synthetic material.
2. Device according to claim 1 wherein the cover (12) is made of a transparent plastic.
3. Device according to one of the previous claims wherein the pressing device (15) is provided with a projection that conforms to the shape of the reaction vessel (3).
4. Device according to one of the previous claims wherein the pressing device (15) is provided with a receiving facility for the cover(s) (12).
5. Device according to one of the previous claims wherein the receiving facility is designed as a tube (15) or a rod.
6. Device according to one of the previous claims wherein a facility for removably fastening the cover (12) is provided in the vicinity of the free end of the tube (15) or rod.
7. Device according to one of the previous claims wherein the facility for removably fastening the cover (12) is provided with an U-shaped profile (21) that projects radially outward, one of its legs being connected to the tube (15) and the other leg being shorter than the one leg.
8. Device according to one of the previous claims wherein the cover (12) is provided with an outward projecting rim (19) that corresponds to the U-shaped profile (21).
9. Device according to one of the previous claims wherein the rim (19) and the U-shaped profile (21) each extend over two peripheral sections of about 90°.
10. Device according to one of the previous claims wherein the receiving element is rotatable about approximately 90°.
11. Device according to one of the previous claims wherein each receiving element is axially movable.
12. Device according to one of the previous claims wherein a discarding element (18) is provided, said throwing off element coaxially surrounding the receiving element and being movable relative to said receiving element.
13. Device according to one of the previous claims wherein a receiving facility designed as a rod or a tube (15) is provided on its outer periphery with a radially contouring bulge (16) that co-operates with a corresponding groove on the inner wall of the cover (12).
14. Device according to one of the previous claims wherein there is provided between a plurality of location elements at least one punch that is movable relative to the receiving elements.
15. Device according to one of the previous claims wherein the projection, the tube (15) or the rod is provided with optical waveguides (7, 9) for introducing light into or for observing light produced in the cavity (3), respectively.
16. Device according to one of the previous claims wherein the optical waveguides (7, 9) are connected to a source of light.
17. Device according to one of the previous claims wherein the optical waveguides (7, 9) are connected to a facility for detecting fluorescence.
18. Device according to one of the previous claims wherein a plurality of reaction vessels (3) are provided and the reaction vessels are part of a microtiter plate.
19. Device according to claim 18 wherein a plurality of covers (3) are part of a cover plate.
20. Device according to one of the previous claims wherein the electrically conductive synthetic material is made of an electrically conductive polycarbonate.
21. Device according to one of the previous claims wherein the electrically conductive zone is contacted.

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