WO2016189259A1

WO2016189259A1 - Device for the preparation of biological samples and use thereof

Info

Publication number: WO2016189259A1
Application number: PCT/FR2016/051255
Authority: WO
Inventors: Patrick Broyer; Marie-Hélène Charles; Laurent Drazek; Dominique LANET; Hervé ROSTAING; Laurent Veron
Original assignee: bioMérieux
Priority date: 2015-05-28
Filing date: 2016-05-27
Publication date: 2016-12-01
Also published as: FR3036799A1

Abstract

Device comprising at least one part (20) intended to accept at least one tube (21) able to contain a biological sample, said part (20) comprising a support (22) able to accept and hold the fixed part (14) of an ultrasound probe, a vibrating part (24) able to be coupled to the sonotrode (16) of an ultrasound probe, a mobile part (26) secured to the support (22) able to move, preferably translationally, relative to the support (22) from a rest position to a clamping position (28) and vice versa, by means of a clamping means (28), a means of heating the contents of the tube (21) so that when the mobile part (26) is in the rest position a space is left between said mobile part (26) and the vibrating part (24) allowing said at least one tube (21) to be positioned or removed, whereas when the mobile part (26) is in the clamping position (28) in the presence of at least one tube (21), said mobile part (26) presses against said at least one tube (21) and applies force to the wall thereof, which is passed on to the vibrating part (24), thereby trapping said at least one tube (21), said force being generated by the clamping means (28) on the mobile part (26).

Description

DEVICE FOR THE PREPARATION OF BIOLOGICAL SAMPLES

TECHNICAL AREA

The present invention relates to the field of methods and devices for preparing samples, preferably biological samples, and more particularly to methods and devices for sonication and incubation of biological samples, in particular for the preparation of biological samples. intended to be analyzed by a molecular biology protocol or by a mass spectrometry device.

As a preferred application, the invention relates more particularly to the preparation of samples containing microorganisms. PRIOR ART

Traditionally, ultrasonic sonication probes used with plastic tubes have blind holes formed in the sonotrode to receive the tubes. These sonication probes require a good control of the force of engagement of the tubes in the blind holes in order to be able to control the amount of ultrasound transmitted to the sample contained in the tube. Thus, during a sonication step, for the same delivered power and if this engagement force is poorly controlled, it is possible to achieve the melting of the plastic constituting the tube. On the other hand, the control of the insertion height of the tubes in the blind holes shows significant variations along the longitudinal axis of the tube. This variability can be particularly troublesome for use with an automatic pipettor.

An example sonotrode having six blind tubes receiving holes is given in the international patent application W096 / 19301 or the commercial product Sonotrode VialTwetter marketed by the company Hielscher. This type of sonotrode presents blind holes drilled in the sonotrode and whose diameter is fixed. The positioned tubes are thus held by a tight fit between the walls of the tube and the diameter of the blind hole and therefore only by friction forces. The ultrasonic coupling, or ultrasonic coupling, between the wall of the tube and the sonotrode is therefore achieved by contact forces maintaining the tube after insertion, these forces being variable as a function of the adjustment of the outside diameter of the tube in the hole and the force applied during insertion. A major disadvantage of this device is that it forces the operator to insert the tube with force, which makes it difficult manual or automatic removal of the tubes. It follows that the introduction and positioning of the tube in the blind hole is difficult to achieve and reproducible, especially since the outer diameter of the tubes has large variations. These variations are observable for the same model of tube in the same batch, especially because of the injection tube manufacturing process and the polymer used, generally polypropylene. In addition, this large tolerance of variation of the outer diameter of the tubes induces significant variations in the positioning of the tube in the blind hole along an axis parallel to the longitudinal axis of the tube (in the Z axis), in particular for tubes of frustoconical shape. This positioning constraint is all the more problematic during pipetting operations in tubes, for example for the recovery of a lysate without loss at the end of the sonication process, or for the automatic manipulation of the tubes (loading and unloading in the sonotrode) by a pipettor robot. Indeed, it is essential in this type of application that the flange of the tubes, which is a gripping element, is located at a position along an axis parallel to the longitudinal axis of the tube (along the Z axis) which is reproducible from one tube to another. In the context of a pipetting robot, the Z axis corresponds to one of the three translation axes of the automated pipetting device, the Z axis being substantially vertical or vertical. Another disadvantage of this type of device is that it can be used with a small number of types of tube, having a slightly variable outside diameter and cylindrical longitudinal shape.

On the other hand, after a step of sonication of the tubes on this type of device, heating caused on the plastic wall of the tube may cause expansion / deformation thereof which may interfere with the extraction / unloading of the tube or prevent the tube from being removed from the blind hole. In more extreme cases of warming can even result in the formation of a hole in the wall of the tube and thus the effusion of the sample content which is unacceptable.

This type of device is therefore not usable in a fully automated system, such as a pipettor robot, where it is desirable that the operator can insert / remove tubes in the system, the controller taking care of transporting the tubes and insert them on the sonotrode and then remove them automatically at the end of the sonication to submit them following a biological or chemical protocol. On the other hand, this type of sonotrode can only be used with tubes of shapes, external diameters and known sizes and low form tolerance to hope for maintenance and ultrasonic coupling reproducible according to the tubes and therefore a good sonication the contents of each tube. International patent application WO 2006/060566 discloses an automated sample preparation device. The device comprises a tube holder, a sonication probe and a cooling system. The sonication probe is placed opposite the bottom of the tube when it is positioned in the tube holder so as to ultrasonically lube the sample contained in the tube. During the lysis step, the cooling system cooled the sample through the walls of the tube so as to prevent degradation of the biological constituents of the sample.

In order to apply a defined force on the bottom of the tube, the sonication probe is constrained by a compression spring. However, the force of engagement of the tube in the tube holder is not controlled, so that the energy transmitted to the sample during the sonication step is uncertain. Thus, in order to compensate for this uncertainty on the transmitted energy is to prevent the melting of the tube during the sonication step, this device is forced to continuously regulate the activation energy of the sonication probe and ship a tube cooling system.

On the other hand, this device has a large footprint for the treatment of a single tube and does not allow a loading / unloading of the tube automatically. On the other hand, the blind hole for receiving the tube does not adapt to different outside diameter tubes and variables.

International patent application WO 2012/125708 discloses a device for the preparation of nucleic acids from biological samples.

This device comprises a sonication probe comprising a tube holder. A tube containing a sample is positioned in the tube holder by one of its ends. The tube is constrained by its other end by a plate subjected to the action of return springs so as to ensure pressure and a constant and reproducible orientation of the tube during the sonication step.

This device however does not allow its geometry rapid insertion and automated tube in the tube holder. In addition, the geometry of the tube holder does not allow to adapt to tubes of different external diameters and variables.

Ultrasonic devices (generally called sonifiers) that can perform simultaneous sonications in tubes of different diameters exist but require to contact the sonotrode of diameter adapted to the diameter of the tube with the product to be lysed contained in the tube. In the case of infectious applications in microbiology, the risk for the operator related to spraying and aerosol generation containing microorganisms and the cleaning stress of the end of the sonotrode at each treatment of a new sample makes this type of device unsuitable. It appears that no sonication sample preparation device of the state of the art is entirely satisfactory for treating a sample or a series of several samples automatically or semi-automatically, while minimizing the steps of transfer and pipetting. On the other hand, no device providing a reproducible ultrasonic coupling, simple to implement and can adapt to a wide variety of tubes is currently available.

SUMMARY OF THE INVENTION

The object of the invention is to overcome the aforementioned drawbacks by providing a device for preparing samples, especially biological samples, allowing sonication and possibly incubation in a more controlled and reproducible manner.

Another object of the invention is to provide a device for ultrasonic coupling tubes effective, reversible, easily manipulated and suitable for a wide variety of tubes.

It also aims to achieve a simple design device safe and rational, comfortable to use and having a small footprint. It also aims at providing a device that can make it possible to perform a sonication and incubation step by limiting the transfer of liquid or the displacement of tubes.

It also aims to achieve a device that can automate all or part of the steps of loading (insertion) tubes, positioning, sonication, incubation and unloading (withdrawal) tubes, while allowing access to tube by a pipettor robot during and / or upstream and / or as a result of these different steps.

To achieve these objectives, it has been developed a device comprising at least one part intended to receive at least one tube capable of containing a sample, said part comprising:

· A support adapted to receive and maintain the fixed part of a sonication probe a vibrating part capable of being coupled with the sonotrode of a sonication probe, a movable part secured to the support, able to move, preferably in translation relative to the support, from a rest position to a clamping position and conversely, by means of a clamping means,

means for heating the contents of the tube,

so that when the mobile part is in the rest position, a space is provided between said movable part and the vibrating part for positioning or removing said at least one tube, whereas when the mobile part is in the clamping position in the presence of at least one tube, said movable part abuts against said at least one tube and exerts a force against the wall of the latter, reflected on the vibrating part, thus trapping said at least one tube, said force being generated by the means tightening on the moving part.

Preferably, when the mobile part is in the clamping position in the presence of at least one tube, said movable part bears against said at least one tube and exerts a force against the wall of the tube along a transverse axis of the latter , reflected on the vibrating part, thereby trapping said at least one tube, said force being generated by the clamping means on the movable part. More preferably, said support is able to receive and hold the fixed part of a sonication probe according to at least one sonication axis and said force is exerted by the mobile part parallel to the sonication axis, along the sonication axis. or perpendicular to the sonication axis. By sample is meant a biological sample and / or a mixture of one or more products, in liquid, viscous form and / or in the form of solid elements in liquid suspension.

By biological sample is meant a liquid or viscous sample capable of containing one or more microorganisms of interest. A biological sample may also have a solid or semi-solid matrix, such as tissues, respiratory specimens, sputum, pus or stool, suspended in a liquid.

For the purposes of the present invention, the term "microorganism" covers Gram-positive or Gram-negative bacteria, yeasts, molds, amoebae and, more generally, unicellular organisms, invisible to the naked eye, which can be handled and multiplied. in laboratory.

According to a preferred embodiment of the invention, the microorganism is a bacterium, Gram negative or positive, a yeast, or a mold. By way of example of Gram-positive bacteria, mention may be made of the following genera: Enterococcus, Streptococcus, Lactobacillus, Bifidobacterium, Staphylococcus, Bacillus, Listeria, Clostridium, Mycobacteria, Nocardia, Corynebacteria, Micrococcus and Deinococcus. As examples of Gram-negative bacteria, mention may be made of the following genera: Escherichia, Enterobacter, Klebsiella, Salmonella, Proteus, Serratia.

As examples of yeasts, mention may be made of the following genera: Candida, Cryptococcus, Saccharomyces and Trichosporon.

As examples of molds, mention may be made of the following genera: Aspergillus, Penicillium, Cladosporium.

The device according to the invention is of interest for the preparation of samples in many areas of in vitro diagnostics: clinical diagnosis, contamination tests of pharmaceutical products, tests on food matrices.

The device according to the invention also allows the preparation of biological samples for many analysis protocols, in particular in molecular biology or mass spectrometry (MALDI-ToF MS or LC-ESI-MS). These protocols require, for example, to perform a generic and efficient microorganism lysis followed, for analysis by LC-ESI-MS (MS), of a reduction / alkylation / digestion protocol that can be carried out using the device according to the invention. and as described in the French patent application FR2990217A1. Advantageously, in the context of applications in the field of chemistry, the sample may be a mixture comprising one or more chemicals. The device according to the invention can thus be used for accelerating the kinetics of mixtures or the study and the realization of reactions by sonochemistry. A tube that can be used with the methods and devices according to the invention can have different shapes and a great variability in terms of geometric tolerance and more particularly form tolerance, in particular on the outside diameter of the tube coming into contact with the sonotrode. A tube is generally in the form of a wall having a section, generally circular, elliptical or square, extending along a longitudinal axis. The surface of the section may be constant, thus forming a cylindrical portion, or variable so as to form a truncated cone or truncated pyramid. Advantageously, the wall has a symmetry along an axis of revolution coincides with its longitudinal axis.

A tube comprises two ends on its longitudinal axis, one open end and the other closed, so as to receive a liquid or viscous content. The closed end corresponds to the bottom of the tube, also called the lower part of the tube.

The term transverse axis is any axis perpendicular to the longitudinal axis defined by the wall of a tube. The tubes that can be used with the invention are generally made of plastic and may have wide dimensional tolerances, in particular on the outside diameter of the wall of the tubes, coming into contact with the mobile part and the vibrating part of the device. The outer diameter is the largest value of the section of a tube measured along a transverse axis. This value being measured in the parts of the wall of the tube able to come into contact with the sonotrode of a sonication probe.

Examples of tubes that can be used with the device according to the invention are so-called Eppendorf tubes that can receive 1.5 ml or 2 ml of liquid content, made of plastic or even 50 ml tubes. These tubes have a cylindrical section extending along a longitudinal axis, one end of the cylindrical portion emerging on a circular opening adapted to receive a stopper, the other end being closed by a frustoconical, hemispherical or conical portion, extending according to the same longitudinal axis. These tubes have a flange in contact with the circular opening, the latter can serve as gripping means of the tube.

The circular opening of a tube is able to receive a plug. This can also receive a septum to be crossed by a needle or the end portion of a pipetting device.

The invention has the advantage of being easily adapted to tubes of outside diameters and different heights along the longitudinal axis. In addition, the invention has the advantage of adapting to the tube shape tolerances of the commonly available tubes, in particular their outside diameter.

In all the embodiments of the invention, a space is provided between said movable part and the vibrating part when the mobile part is in the rest position. This space makes it possible to position or withdraw said at least one tube in the device according to the invention. This space also has a geometry adapted to cooperate with the wall of the tube, so as to insert the tube. This space can thus be adapted according to the geometry of the tube, and more particularly of the wall of the tube, that one wants to insert. By way of example, for use with a tube whose section of the wall is oval or cylindrical, said space may have an oblong hole shape extending along a longitudinal axis, this axis being substantially parallel or parallel with that of the tube when it is positioned in said space.

According to a particular embodiment, this space also provides access to the opening portion of said tube when it is positioned therein and / or trapped by the device, thereby allowing the addition or removal of liquid at any time. In all the embodiments of the invention, the moving part as well as the vibrating part of the device according to the invention each have one or more contact surfaces capable of cooperating with at least one tube, preferably with the wall of at least one a tube so as to exert a force along the transverse axis or the longitudinal axis of the tube when the movable portion is in the clamping position. These contact surfaces can thus be adapted according to the geometry of the wall of the tube and, if appropriate, of its plug or septum.

However, it is not necessary for the shape of the contact surfaces to be perfectly adapted to the wall of the tube, this being due to the capacity for elastic deformation of the wall of the plastic tubes and / or to the force generated by the clamping means on the moving part, for example along the axis of sonication, when the mobile part is in the clamping position in the presence of at least one tube, the latter being able to be applied by an elastic return means or by means of an elastic return means, such as a spring, a resilient support (silent block in English) or a pocket of compressed air. By way of example, for use with a tube whose section of the wall is oval or cylindrical and where the moving part when it is in the clamping position exerts a force along a transverse axis of the tube, the contact surfaces may exhibit a cylindrical portion of diameter slightly greater than the outer diameter of the wall of the tube. For example, the movable portion may have at least one portion of cylindrical shape and the vibrating portion at least a portion of cylindrical shape. In this way, when the mobile and vibrating parts are in the clamping position, they form two parts of the same diameter cylinder adjusted to the outside diameter of the wall of the tube to ensure the clamping thereof. Advantageously, when the mobile and vibrating parts are in the clamping position, the opening portion of the tube remains accessible thus allowing the addition or withdrawal of liquid at any time.

Alternatively, the movable portion may have a planar contact surface. For example, the mobile part may have a flat contact surface so as to cooperate with a tube plug, the vibrating part having a contact surface adapted to cooperate with the bottom of a tube, for example a groove , this to ensure the clamping of a tube and its plug along its longitudinal axis when the movable portion and in the clamping position.

Alternatively, the vibrating portion may have a contact surface adapted to cooperate with the wall of a tube or with the lower part of a tube. By way of example, the vibrating part may have a contact surface comprising at least one part of cylindrical shape so that when the mobile part is in the clamping position, the latter can exert a force along a transverse axis of the tube which allows to imprison him. Said contact surface also comprising at least one part capable of cooperating with the lower part a tube, for example a groove, so that when the movable portion is in the clamping position, it can exert a force along a longitudinal axis of the tube which allows to imprison. In this way, a tube may be trapped by the moving and vibrating parts in the clamping position by a force exerted along a transverse axis of the tube or a longitudinal axis of the tube.

A sonication probe, well known to those skilled in the art, comprises a fixed part and a vibrating part, also called sonotrode. The fixed part must be held on a support. The sonotrode is able to transmit the ultrasonic vibrations produced by a transducer. In the context of our invention, the ultrasonic vibrations have a frequency of between 20 kHz and 100 kHz. Preferably, the ultrasonic vibration frequencies are between 20 and 40 kHz and more preferably at 30 kHz.

The vibrating part of the device according to the invention is able to be coupled with the sonotrode of a sonication probe. The coupling may be carried out by any means known to those skilled in the art, preferably rigid so as not to dissipate the sonication energy, preferably using a rod rod / threaded torque.

According to an alternative mode of the device according to the invention, the vibrating part of the device is included in the sonotrode of a sonication probe. By way of example, the sonotrode of a sonication probe may be machined in such a way as to ensure the function of the vibrating part according to the invention, so that a coupling with another part is not necessary. The sonotrode performing the function of vibrating part according to the invention, thus cooperates with a mobile part according to the invention. In this alternative embodiment, the invention relates to a device for the preparation of biological samples comprising at least one sonication probe, comprising a fixed part and a sonotrode, at least one part intended to receive at least one tube containing the biological sample, said part comprising:

a support secured to the sonication probe by its fixed part a movable part integral with the support, able to move, preferably in translation, relative to the support, from a rest position to a clamping position and vice versa, through a clamping means, o means for heating the contents of the tube

so that when the moving part is in the rest position, a space is provided between said movable part and the sonotrode for positioning or removing said at least one tube, whereas when the movable part is in the clamping position in the presence at least one tube, said movable part is in abutment against said at least one tube and exerts a transverse force against the wall of the latter, echoed on the sonotrode, thus trapping said at least one tube, said force being generated by the clamping means on the moving part, preferably along the axis of sonication of the sonotrode or parallel to the sonication axis or perpendicular to the sonication axis.

By the configuration of a sonication probe, the sonotrode oscillates along a main axis called the sonication axis. Any object in contact or coupled to the sonotrode when it is in oscillation receives a maximum of mechanical energy along this axis. According to one embodiment of the invention, the device comprises several moving parts, each movable part being able to move, preferably in translation, relative to the support, from a rest position to a clamping position and vice versa, by by means of a corresponding clamping means, so that when one of the moving parts is in the rest position, a space is provided between said movable part and said vibrating part for positioning or removing said at least one tube, whereas when one of the moving parts is in the clamping position in the presence of at least one tube, said movable part bears against said at least one tube and exerts a force against the wall of the latter, reflected on the vibrating part, thus trapping said at least one tube, said force being generated by the clamping means on the movable part.

Preferably, the device comprises a plurality of moving parts, each movable part being able to move, preferably in translation, relative to the support, from a rest position to a clamping position and vice versa, by means of a means corresponding clamping means, so that when one of the moving parts is in the rest position, a space is provided between said movable part and said vibrating part for positioning or removing said at least one tube, whereas when one of the moving parts is in the clamping position in the presence of at least one tube, said movable part bears against said at least one tube and exerts a force against the wall of the tube along a transverse axis of the latter, reflected on the vibrating part, trapping thus said at least one tube, said force being exerted by the clamping means on the moving part parallel to the sonication axis, according to the sonication axis or perpendicular stretching to the sonication axis.

The device according to the invention can thus provide a plurality of tube receiving spaces, each constituted by a portion of the vibrating part and a corresponding moving part. The interest and ability to perform sonication steps on more than one tube simultaneously and homogeneously while allowing the positioning and removal of each tube independently. For this purpose and regardless of the embodiment of the invention, the mobile part may be provided to be easily removable and replaced with a movable portion having a shaped contact surface adapted to the tubes so as to increase or decrease the number of tubes that can be received and / or receive and trap tubes of different shapes and sizes. This in order to subject them to an efficient and homogeneous ultrasonic lysis.

In order to provide a plurality of tube receiving spaces, each consisting of a portion of the vibrating part and a corresponding moving part, the vibrating part has a plurality of contact surfaces capable of cooperating with the wall of at least one tube so as to exert therein a transverse or longitudinal force when the corresponding movable part is in the clamping position and as defined above.

These tube receiving spaces may for example be distributed along an axis perpendicular to the sonication axis and so that each of the tubes receive a similar amount of energy during the sonication step, whatever the reception space in which it has been placed. These reception spaces of tubes may preferably be placed along an axis perpendicular to the sonication axis and regularly distributed, so as to be able to use a multi-position pipette offering the possibility of delivering and sucking a liquid simultaneously in several tubes received or imprisoned. in the device according to the invention.

These tube receiving spaces may also be distributed radially, preferably equidistant from the sonication axis. In the case where these receiving spaces are distributed radially, the longitudinal axis of each of the tubes when they are positioned in the receiving spaces of the device according to the invention and that the mobile part is in the clamping position, is preferentially parallel to the sonication axis. In this particular embodiment, the vibrating part may comprise a disk having, in cooperation with corresponding moving parts, a plurality of tube receiving spaces. In this particular embodiment, the sonotrode which can be coupled to the vibrating part according to the invention can be arranged vertically, the disc being centered on the sonication axis, the tubes being receptive in receiving spaces radially distributed around the sonication axis. Preferably, the tubes are received and maintained vertically. One of the advantages of this embodiment is to present a horizontal footprint, also called footprint, reduced.

Advantageously and whatever the embodiment, the heating means may be included, in particular inserted, in a mobile part according to the invention. By heating means any means capable of raising or lowering the temperature of the device and therefore the temperature of the tube and its contents when it is trapped in the device. By way of example, the heating means may be a heating cartridge or a resistor. This characteristic makes it possible in particular to increase the temperature of the device in order to perform a step of incubating the contents of the tube. Alternatively, this heating means makes it possible to maintain a temperature that is constant or lower than a threshold of the device in order to limit the degradation of the contents of the tube during the sonication step. By way of example, the heating means may be a Peltier regulator thus allowing the heating and / or cooling of the device. Such a regulator also makes it possible to maintain the temperature of the device, and in particular the content of the tube, between 10 ° C. and 90 ° C., thus making it possible to produce a large variety of experimental protocols. This or these heating or cooling means are advantageously inserted into the movable part (s). Alternatively and regardless of the embodiment, the device may comprise means for measuring the temperature of the tube and / or its contents and / or the device, in communication with means for regulating the heating means in order to improve heating and / or cooling the tube and / or its contents and / or the device. Alternatively, said means for measuring the temperature of the tube and / or its contents and / or the device are included in one or more mobile part (s). Alternatively, said means for measuring the temperature of the tube and / or its contents are placed in one or possibly several tubes in order to measure the temperature of the tube and / or its contents at any time, in particular during a sonication step. or incubation and this to allow regulation by feedback loop.

Whatever the embodiment, the device may also comprise means for adjusting the position of the tube relative to the mobile and vibrating parts along a longitudinal axis defined by the tube

The advantage of this adjustment means is in particular to allow the tube to be positioned along a longitudinal axis relative to the moving and vibrating parts so as to optimize the sonication or incubation step, whatever the size of the tube according to this axis. In fact, if a tube is trapped at a height different from the other tubes placed in the same device, relative to the mobile and vibrating parts and along this longitudinal axis, when the mobile part is in the clamping position, the ultrasonic coupling for this tube will be differ (because the clamping force will be different). As a result, this difference in positioning can induce a different and variable ultrasonic lysis performance in this tube compared to other tubes placed in the same device, which is not desired by the user. Another advantage of this adjustment means is to use tubes of different sizes along their longitudinal axis on the same device. Another advantage of this adjustment means and of being able to position along a longitudinal axis, preferably vertical, the collar being able to serve as a means of gripping each tube relative to the mobile part and to the vibrating part of the device according to the invention, especially when it is used with a pipettor robot.

Another advantage of this adjustment means is to allow, when the moving part is in the rest position, to retain and position the tube positioned in the space between the corresponding mobile and vibrating part along a longitudinal axis and relative to the moving parts. and vibrant. This allows in particular to perform pipetting operations on a pipettor robot, these operations requiring very precise positioning of the tube.

Adjusting means that can be used are for example a screw, a threaded rod, a pin fitted into a bore or any other means well known to those skilled in the art.

Advantageously, the adjustment means is a thermal conductor, in order to optimize heat transfer, especially during an incubation step.

Since the adjustment means are in contact with the bottom of the tube, also called the lower part of the tube, when it is positioned or trapped in the device, the latter may advantageously be a thermal conductor so as to transmit the energy of heating means to the tube and its contents, in particular during an incubation step. This characteristic therefore makes it possible to improve the incubation and / or temperature maintenance step of the tube contents. This characteristic also makes it possible to dissipate the heat stored by the tube during the sonication step by allowing it to be evacuated from the bottom of the tube to the device, advantageously to the mobile part without the lower part of the tube is mechanically constrained and reduces the ultrasound transmission effect to the contents of the tube. Preferably, the clamping means comprises an electric jack and / or a pneumatic jack and / or an elastic return means such as a compression spring. The device can easily be adapted for manual, automatic or semi-automatic operation depending on the elements constituting the clamping means. In a particular embodiment, the clamping means comprises a pusher in connection with the movable part. The pusher can be moved in translation relative to the support thereby causing the movable portion of a rest position to a clamping position. In the case of manual use, the pusher may be moved by a handle operated by an operator. In the case of semi-automatic use, the pusher can be moved by a cylinder controlled by an operator. In the case of automatic use, an automaton will actuate the actuator according to a pre-established protocol.

In a particular embodiment, the clamping means comprises a pusher in connection with the movable part and an elastic return means such as a compression spring disposed in abutment on the support and the pusher. The elastic return means, when compressed, exerts a force on the pusher and on the support. In this embodiment, when the moving part is in the clamping position, the elastic return means exerts on the pusher a force reflected on the moving part and the walls of the tube, thus trapping the tube between the mobile part and the vibrating part. . The imprisoning force thus generated is directly proportional to the stiffness of the elastic return means. Still in this embodiment, when the movable part is in the rest position, the elastic return means is forced by the pusher against the support so as to release the movable part, thus allowing the tube to be removed from the space arranged between the moving and vibrating parts. The advantage of using an elastic return means is to ensure elastic clamping of the tube thus allowing a greater geometric tolerance and more particularly of shape tolerance, in particular of the outside diameter of the tube which can be received and tightened so to ensure a suitable and reproducible ultrasonic coupling when the mobile part is in the clamping position.

Alternatively, the clamping means comprises a pusher in connection with the movable part and an elastic return means such as a compression spring disposed in abutment on the pusher and on the movable part so as to ensure an elastic clamping of the tube. In this embodiment, when the moving part is in the clamping position, the elastic return means exerts on the pusher a force reflected on the moving part and the walls of the tube, thus trapping the tube between the mobile part and the vibrating part. in an elastic way. The imprisoning force thus generated is directly proportional to the stiffness of the elastic return means. Advantageously, the movable portion comprises an elastic return means disposed on its contact surfaces so as to ensure an elastic clamping of the tube. In this embodiment, when the moving part is in the clamping position, the elastic return means exerts a force on the walls of the tube, thus trapping the tube between the elastic return means and the vibrating part in an elastic manner. The imprisoning force thus generated is directly proportional to the stiffness of the elastic return means.

By elastic return means means a compression spring, a resilient support (silent block in English) or a compressed air pocket. According to a particular embodiment, at least one movable part is secured to the support by means of a mobile part carriage adapted to move in translation relative to a slideway secured to the support. In this embodiment, the movable portion carriage can be disposed and held between two positions, a proximal position and a distal position, to change the distance between the corresponding movable portion and the vibrating portion of the device.

The advantage of this movable part carriage is to adapt the size of the space between the corresponding movable and vibrating parts when the corresponding movable part is in the rest position. As a result, the outer diameter of the tube and / or the size of the tube along its longitudinal axis, able to be received in this space is modified by the movement of the mobile part carriage.

Said movable portion carriage can be arranged and held in a first position of the so-called proximal movable portion carriage, the outer diameter of the tube being receptive in this space being then minimal.

Said movable portion carriage can also be arranged and held in a second position of the distal mobile part carriage, the size of the tube, preferably assembled with its plug or septum, along the longitudinal axis of the tube, and can be received in this space is then maximum.

Between these two positions of the moving part carriage, proximal and distal, the carriage can be arranged and maintained according to the desired spacing between the corresponding movable and vibrating parts (the spacing corresponding to the size of the space between the movable parts and corresponding vibrating) so as to receive the tube that is desired to use with the device when the corresponding movable portion is in the rest position. The longitudinal axis of the positioned tube being perpendicular, parallel or disposed along the sonication axis when the corresponding movable part is in the rest position. The longitudinal axis of the trapped tube being perpendicular, parallel or disposed along the sonication axis when the corresponding movable part is in the clamping position.

For this purpose, the movable portion carriage may be disposed and held by a holding means, such as a clamping screw, in one of the positions between the proximal and distal positions.

In this embodiment, the mobile part is secured to the corresponding movable part carriage and able to move, preferably in translation relative to the corresponding moving part carriage, from a rest position to a clamping position and vice versa, through a clamping means.

A major advantage of this embodiment comprising a mobile part trolley is to adapt the device according to the invention to tubes of very different diameters and capacity, including tubes that can contain between 1mL and 50mL. This embodiment makes it possible in particular to position and imprison a tube by applying a clamping force along the transverse axis of the tube or along its longitudinal axis. In a first configuration of this embodiment and in order to lyse small volumes (from 50μί to ImL) of sample in the bottom of a small volume tube, for example containing up to 1.5mL or up to 2mL, the movable portion carriage is disposed and maintained in its proximal position or near its proximal position so as to apply a clamping force along a transverse axis of the tube to the wall of the tube, perpendicular to the longitudinal axis of the tube when the moving part is in the clamping position.

In a second configuration of this embodiment and in order to lyse small volumes (from 50μί to ImL) of sample in the bottom of a large volume tube, for example containing up to 50mL, the mobile part carriage is disposed and held in a position between its proximal and distal so as to apply a clamping force along a transverse axis of the tube on the wall of the tube, perpendicular to the longitudinal axis of the tube when the movable portion is in the clamping position.

In a third configuration of this embodiment and in order to lyse large volumes (from ImL to 50mL) of sample in a large volume tube, for example containing up to 50mL, the movable part carriage is arranged and maintained in its distal position or near its distal position so as to apply a clamping force along a longitudinal axis of the tube on the wall of the tube, when the movable part is in the clamping position. Advantageously, in this third configuration, the device according to the invention is inclined or placed vertically so as to incline or place vertically the longitudinal axis of the tube when it is positioned or imprisoned.

In these three configurations, the sonication axis may be perpendicular, parallel or along the axis in which the clamping force is applied. In other words, the sonication axis can be perpendicular, parallel or along the longitudinal axis of the tube.

An advantage of this embodiment is therefore to be able to propose a device that can receive tubes so as to orient the longitudinal axis of the tube parallel to the sonication axis, perpendicular to the sonication axis or along the sonication axis , applying a clamping force along the longitudinal axis of the tube or perpendicular to the longitudinal axis of the tube when the movable portion is in the clamping position.

In order to receive and imprison the same type of tube along the longitudinal axis of the tube or perpendicular to the longitudinal axis of the tube, the contact surface of the part mobile may be substantially flat in order to cooperate with the plug of a tube, the contact surface of the vibrating portion having a shape adapted to cooperate with both the bottom of said tube and with the wall of said tube. In this embodiment, the device comprises

At least one part intended to receive at least one tube capable of containing a biological sample, said part comprising:

a support able to receive and maintain the fixed part of a sonication probe

a vibrating part capable of being coupled with the sonotrode of a sonication probe,

a movable part secured to the support by means of a mobile part carriage adapted to be displaced in translation relative to a slideway integral with the support, from a position proximal to a distal position and to be held in this position by a holding means,

said mobile part being able to move, preferably in translation relative to the mobile part carriage, from a rest position to a clamping position and vice versa, by means of a clamping means,

so that when the mobile part is in the rest position, a space is provided between said movable part and the vibrating part for positioning or removing said at least one tube, whereas when the mobile part is in the clamping position in the presence of at least one tube, said movable part is in abutment against said at least one tube and exerts a force against the wall of the latter applied along the longitudinal axis of the tube or along the transverse axis of the tube, reflected on the vibrating part thus trapping said at least one tube, said force being generated by the clamping means on the movable part. In this embodiment, the device according to the invention may comprise several moving parts and one or more mobile part carriages. Several movable parts that can be secured to the support by means of a movable portion carriage adapted to be displaced in translation relative to a slideway integral with the support, from a position proximal to a distal position and to be held in this position by a holding means,

In this embodiment, the device according to the invention allows the sonication of one or more tubes along their transverse axes or along their longitudinal axes. In this embodiment, the device may be disposed horizontally, vertically or inclined relative to the horizontal. The advantage of having the device vertically or inclined with respect to the horizontal is to limit the size in a horizontal plane of the device, for use in an analyzer or robot pipettor. In addition, arranging the device vertically or inclined relative to the horizontal facilitates the loading / unloading of the tubes between the mobile and vibrating part when the / the corresponding moving parts is / are in the rest position.

Whether the device is disposed horizontally, vertically or inclined relative to the horizontal, the (s) trolley (s) mobile part (s) may be arranged independently between the distal and proximal positions to adapt the size space between the corresponding moving and vibrating parts.

Another subject of the invention relates to a system for the preparation of biological samples comprising

A device according to the invention

A sonication probe, comprising a fixed part and a sonotrode, the sonotrode defining a sonication axis,

the support of the device being secured to the sonication probe by its fixed part, the vibrating part of the device being coupled to the sonotrode

Another subject of the invention relates to the use of a system according to the invention for the preparation of biological samples comprising the following steps:

a) Insert a tube containing a biological sample in the space of the device, the mobile part being in the rest position

b) moving the moving part from its rest position to its clamping position so that the tube is trapped in the space of the device,

c) Perform a step of sonication of the biological sample by activating the sonotrode

Advantageously, an additional step prior to step c) and of adding beads into the tube is carried out. The interest of these beads and to improve the disruption of the walls of microorganisms or matrices contained in the biological sample.

Another subject of the invention relates to the use of a system according to the invention for the preparation of biological samples, the device comprising a heating means, according to the following steps:

b) moving the moving part from the rest position to the clamping position so that the tube is trapped in the space of the device,

c) Perform a step of sonication of the biological sample by activating the sonotrode d) Perform a step of incubating the biological sample by activating the heating means

Advantageously, an additional step prior to step c) and of adding beads into the tube is carried out.

Beads that can be used with the invention have a diameter of between 0.01 mm and 2 mm, preferably between 0.1 mm and 1 mm and even more preferably a ball mixture with diameters of between 0.1 mm and 1 mm. Balls that can be used with the invention are made of glass, zirconium or ceramic.

Advantageously and whatever the embodiment, the clamping means may comprise an elastic return means, a handle, and a pusher. The handle is rotatable about an axis relative to the pusher. The pusher moves in translation relative to the support. The elastic return means is disposed in support on the support and on the pusher.

The handle can be moved from a first rest position to a clamping position to move the movable portion from a rest position of the movable portion to a clamping position of the movable portion. The handle can also be moved from said clamping position to said first rest position of the handle.

Preferably, the handle can be moved from a second rest position to said clamping position in order to move the movable portion of said mobile part rest position to said clamping position of the movable part. The handle can then also be moved from said clamping position to said second rest position of the handle.

For this purpose, the handle of the clamping means comprises a contact surface with the support configured so as to move the axis of rotation of the handle away from the support, thus causing the pusher when the handle is moved from the clamping position to the first position. resting and so as to block the pusher in this position, the resilient return means being then constrained.

If appropriate, said contact surface with the support is also configured to move the axis of rotation of the handle away from the support relative to the pusher when the handle is moved from the clamping position to the second position of rest and maintained in this second rest position. Said contact surface is configured so that the pusher resumes its rest position under the action of the elastic return means when the force applied to move and hold the handle in this second rest position is removed. Thus, when the handle is in the clamping position, the axis approaches the support, moving the pusher so that it is constrained by the force exerted by the resilient biasing means in the direction of the vibrating part. In the case where a tube is positioned in the device in space, the movable part thus bears against the tube and exerts a force against the latter, echoing on the vibrating part, thus trapping the tube.

When the handle is moved from its clamping position to its first rest position, the axis is remote from the support, moving the pusher so that it forces the elastic return means towards the support. In the case where a tube was trapped between the corresponding moving and vibrating parts, it can then be removed / discharged from the device, a space being provided between these two parts.

When the handle is moved from its clamping position to its second rest position, the axis is removed from the support, moving the pusher so that it forces the elastic return means towards the support. A space for loading / unloading a tube is thus provided between the corresponding mobile part and the vibrating part. In the case where the handle is maintained in said second rest position, if a tube was trapped between the corresponding moving and vibrating parts, it can then be removed / discharged from the device, a space being provided between these two parts. If the force maintaining the handle in its second rest position is removed, the handle naturally resumes its clamping position under the action of the resilient biasing means.

Alternatively, the invention also relates to a device for the preparation of biological samples comprising at least one sonication probe, comprising a fixed part and a sonotrode, the sonotrode defining a sonication axis, at least one part intended to receive at least one tube containing the biological sample, said part comprising:

a support secured to the sonication probe by its fixed part o a vibrating part coupled to the sonotrode,

a movable part secured to the support, able to move, preferably in translation relative to the support, from a rest position to a clamping position and vice versa, by means of a clamping means, heating the contents of the tube

so that when the mobile part is in the rest position, a space is provided between said movable part and the vibrating part for positioning or removing said at least one tube, whereas when the movable part is in the clamping position in the presence of at least one tube, said movable part bears against said at least one tube and exerts a force along a transverse axis of the tube and against the wall of the latter, which is reflected on the vibrating part, thus trapping said at least one tube, said force being generated by the clamping means on the moving part, along the sonication axis of the sonotrode

Advantageously, the heating means may be included in a moving part.

The device may also comprise means for adjusting the position of the tube relative to the mobile and vibrating part along a longitudinal axis defined by the tube

Advantageously, the adjustment means is a thermal conductor and is adapted to the lower part of the tube shape in contact with said means without mechanically constraining the tube so as not to attenuate the ultrasonic coupling.

Preferably, the clamping means comprises a jack or an electromechanical actuator controllable manually, pneumatically or electrically.

Preferably, the clamping means comprises an elastic return means such as a compression spring

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be well understood and other features and advantages of the invention will become apparent from the description which is given below, by way of indication and in no way limiting, with reference to the drawing, in which: FIG. perspective representation of a first embodiment of the device according to the invention;

FIG. 2 is a side view representation of a sonication probe that may be used with said preferred embodiment of the device according to the invention;

Figure 3 is a side view of said embodiment of the device according to the invention when the movable part is in the rest position;

Figure 4 is a side view of said embodiment of the device according to the invention when the movable part is in the clamping position;

- Figure 5 is a top view of said embodiment of the device according to the invention when the movable part is in the rest position;

Figure 6 is a sectional representation, along the plane B defined in Figure 5, of the movable portion of said embodiment of the device according to the invention when the movable part is in the clamping position;

- Figure 7 is a sectional representation, according to the plane B defined in Figure 5, the movable portion of said embodiment of the device according to the invention when the movable part is in the rest position;

Figure 8 is a top view of a second particular embodiment of the device according to the invention when the movable part is in the clamping position and the mobile part carriage is in the proximal position, the device being disposed horizontally.

Figure 9 is a front view of said second particular embodiment of the device according to the invention having the movable part carriage distal, the device being arranged vertically.

FIG. 10 is a side view representation of said second particular embodiment of the device according to the invention having the mobile portion carriage in a distal position, the device being inclined

FIG. 11 is a top view of the vibrating part of said particular embodiment of the device according to the invention shown in FIGS. 8, 9, 10. FIG. 12 is a front view of the vibrating part of said particular embodiment of the device according to the invention visible in FIGS. 8, 9, 10.

FIG. 13 is a representation in plan view of a third particular embodiment of the device according to the invention.

FIG. 14 is a side view representation of a fourth particular embodiment of the device according to the invention;

Figure 15a shows the results of Example 1 for tubes containing Escherichia coli strains.

Figure 15b shows the results of Example 1 for tubes containing Candida Albicans strains.

FIG. 15c shows the results of example 1 for the tubes containing strains of Staphylococcus Epidermidis

Figure 16a shows the results of Example 2 for tubes containing Escherichia coli strains.

- Figure 16b shows the results of Example 2 for tubes containing strains of Candida Albicans.

Figure 16c shows the results of Example 2 for tubes containing Staphylococcus Epidermidis strains. For simplicity, parts or elements of an embodiment that are identical or similar in another embodiment will be identified by the same reference numerals and will not be described again.

DETAILED DESCRIPTION OF THE INVENTION

Figures 1 to 7 show a first embodiment of the device (10) according to the invention, this example being in no way limiting as to the scope of the invention.

The device (10) includes a portion (20) for receiving at least one tube (21).

The portion (20) also comprises a vibrating part (24) capable of being coupled with the sonotrode (16) of a sonication probe. The part (20) intended to receive at least one tube (21) also comprises a mobile part (26) integral with the support (22), able to move, preferably in translation relative to the support, a rest position to a clamping position and vice versa, by means of a clamping means (36).

The device can be used with tubes of volumes, sizes and diameters slightly different for the same embodiment of the device. When the diameters of the tubes are very different, the mobile part and the vibrating part of the device can easily be disassembled and replaced by moving parts and vibrating parts having contact surfaces adapted to the walls of the desired tubes, in particular the shapes and diameters of these contact surfaces. The moving and vibrating parts can also be defined to treat tubes of different shapes and diameters by specific machining of the moving and vibrating parts adapted to the desired tubes. The tube (21) presented in this example is an Eppendorf type plastic tube 1.5mL. This tube comprises an oval section extending along a longitudinal axis. The bottom of the tube comprises a truncated cone portion, the opening portion of the tube is closable by a cap or a resealable septum through which a pipettor can come pipette the contents of the tube. Advantageously and so as to be able to independently receive and trap more than one tube, the part (20) also comprises a second movable part (28), integral with the support (22), able to move, preferably in translation, relative to the support, from a rest position to a clamping position and vice versa, by means of a clamping means (38).

More specifically, and with reference to FIG. 2, the part (20) comprises a support (22) able to receive and hold the fixed part (14) of a sonication probe (12) according to at least one sonication axis. In the embodiment shown, the portion (20) can accommodate up to eight tubes (21). The number of tubes that can be received is easily adaptable by modifying the portion (20).

With reference to FIG. 3, the mobile part (26) and the vibrating part (24) are arranged so that when the mobile part (26) is in the rest position, a space (46) is provided between said movable portion (26) and the vibrating portion (24). This space makes it possible to position or withdraw the at least one tube (21) in the part (20). The tube (21) can thus be positioned along the arrow A in the space (46). With reference to FIG. 4, when the mobile part (26) is in the clamping position in the presence of at least one tube (21), said movable part (26) bears against said at least one tube (21) and exerts a transverse force against the wall of the latter, reflected on the vibrating part (24), thus trapping said at least one tube (21), said force being generated by the clamping means (28) on the movable part (26) , along a transverse axis of the tube and along the axis of sonication

Figure 5 shows the same embodiment of the device in plan view. In this embodiment, each tube can be positioned and trapped independently. For this, the device comprises eight moving parts, in correspondence with a single vibrating part. When each of the moving parts is in the rest position, a space adapted to receive a tube is formed between said movable part and the vibrating part. The device can easily be modified so that a mobile part can provide two spaces able to receive two tubes and imprison them.

A cutting plane B along a sonication axis is defined.

Figure 6 shows a sectional view along the sectional plane B of the movable part (26) of the device according to the invention when the latter is in the clamping position.

The tube (21) has a flange (23) that can be used as a gripping means. In this clamping position, the tube is in contact with the contact surfaces (25a, 25b) of the movable part (26) and the vibrating part (24) on part of its wall.

In this embodiment, the movable portion (26) comprises means for heating (40) the contents of the tube in the form of a heater cartridge comprising a resistor. The movable portion (26) also includes means (50) for adjusting the position of the tube relative to the movable portion (26) when it is positioned or trapped in the device. For example, the adjusting means here comprises an assembled screw in a threaded bore made in the movable part (26). The screw extends along an axis parallel to the longitudinal axis of the tube when it is positioned or trapped in the device. The screw used is made of metal so as to transmit the heat stored by the tube and its wall and vice versa so as to transmit the heat from the moving part, especially when the heating means that comprises is activated. The adjustment means, here a screw, may have a shape adapted to its end in contact with the lower part of the tube, allowing optimum heating of the volume of liquid in the tube, it may be low. This heating function is ensured while maintaining a functional mechanical play sufficient to ensure efficient transmission of ultrasound into the contents of the tube, thus ensuring adequate and reproducible ultrasonic coupling. The screw can be moved between a high position and a low position over a range of about two centimeters in order to receive tubes of different sizes along their longitudinal axis. The positioning of the tube relative to the moving and vibrating part is thus optimized as well as the automatic pipetting or gripping of the tube, guaranteeing positioning in the longitudinal axis of the reproducible tube and, where appropriate, guaranteeing access to the collar of the tube for a pipettor robot. The size of the range in which the adjusting means can move can easily be adapted by those skilled in the art to the size of the tube used with the invention.

Alternatively, the device comprises means for measuring the temperature of the tube and / or its contents and / or the device, in communication with means for regulating the heating means in order to improve the heating and / or cooling of the tube and / or its contents and / or the device. Alternatively, said means for measuring the temperature of the tube and / or its contents and / or the device are included in a moving part. Alternatively, said means for measuring the temperature of the tube and / or its contents are placed in one or, where appropriate, several tubes in order to measure the temperature of the tube and / or its contents at any time, in particular during a sonication step. or incubation. A means for measuring the temperature of the tube such as a temperature sensor (not shown) may be disposed in one of the moving parts so as to record an effective heating temperature of the tube and / or its contents. This temperature information may advantageously be used by a regulating means such as a thermal regulation box to adjust the heating setpoint of the heating cartridge, depending on the desired heating temperature. The temperature of each tube may also be individually controlled by determining different desired heating temperatures and advantageously by providing a temperature probe in each heating cartridge. The temperature of each tube can also be regulated simultaneously using the same set point for the eight tubes.

Still according to Figure 6, the clamping means (36) comprises a compression spring (60), a handle (70), and a pusher (80). The handle (70) is rotatable about an axis (90) relative to the pusher (80). The pusher (80) moves in translation relative to the support (22). A compression spring (60) with a stiffness of 8.92N / mm is supported on the support (22) at one end and on a shoulder (82) formed on the pusher (80) at its other end. The compression spring (60) thus exerts a force on the shoulder along a transverse axis, corresponding to the main axis of the pusher. In order to constrain translation displacement of the movable portion (26), a guide pin (90) is fitted into the movable portion (26) and fitted movable in translation relative to the support (22).

The handle (70) can be moved from a first rest position to a clamping position to move the movable portion (26) from a home position to a clamping position.

The handle (70) can be moved from a second rest position to said clamping position to move the movable portion (26) from a home position to a clamping position.

The handle (70) can also be moved from said clamping position to said first rest position of the handle (70) or moved from said clamping position to said second rest position of the handle (70).

The handle (70) of the clamping means (36) for this purpose comprises a contact surface (71) with the support (22) configured so as to move the axis of rotation (90) of the handle away from the support (22). (70) when the handle (70) is moved from the clamping position to the first rest position. Said contact surface with the support (22) is also configured to move the axis of rotation (90) of the handle (70) away from the support (22) when the handle (70) is moved from the clamping position to the second rest position and maintained in this second rest position, for example by an operator.

Thus, when the handle (70) is in the clamping position, the axis (90) approaches the support (22), displacing the pusher (80) so that it is constrained by the force exerted by the spring of compression (60) towards the vibrating part (24). In the case where a tube is positioned in the device in the space (46), the mobile part (26) thus bears against the tube (21) and exerts a force against the wall of the latter along a transverse axis of the tube, echoed on the vibrating part (24), thus trapping the tube (21).

When the handle (70) is moved from its clamping position to its first resting position, the axis (90) is moved away from the support (22), displacing the pusher (80) so that it pushes the spring of compression (60) towards the support (22) along a transverse axis of the tube and parallel to the sonication axis. In the case where a tube (21) was trapped between the corresponding moving parts (26) and vibrators (24), it can then be removed / discharged from the device, a space (46) being provided between these two parts.

When the handle (70) is moved and held from its clamping position to its second rest position, the axis (90) is moved away from the support (22), displacing the pusher (80) so that it pushes the spring compression device (60) towards the support (22) along a transverse axis of the tube and parallel to the sonication axis. A space (46) for loading / unloading a tube (21) is thus provided between the moving part (26) corresponding and the vibrating part (36). In the case where the handle (70) is held in said second rest position, if a tube (21) is trapped between the corresponding movable (26) and vibrating (24) parts, this can then be removed / discharged from the device , a space (46) being formed between these two parts. If the force maintaining the handle in its second rest position is removed, the handle (70) naturally returns to its clamping position, under the action of the elastic return means, here a compression spring (60). The corresponding moving part (26) also resumes its clamping position.

7 shows a sectional view along the sectional plane B of the movable part (26) of the device according to the invention when the latter is in said first rest position. When the movable portion (26) is in said first rest position, the axis (90) is moved away from the support (22) by the handle (70) thus driving the pusher (80). The compression spring (60) is then compressed and constrained more than when the movable portion (26) is in the clamping position. As a result, the movable portion (26) moves away from the vibrating portion (24) so that a tube (21) can be withdrawn or inserted into the space (46) between the fixed portion and the movable portion. When the tube is positioned in the space (46), it is supported on the adjustment means (50). The contact surface (71) has a particular shape so as to block the pusher (80) in this rest position. Figures 8, 9, 10, 11 and 12 show a second embodiment of the device (110) according to the invention, this example being in no way limiting as to the scope of the invention.

The device (110) includes a portion (120) for receiving at least one tube (121). According to this particular embodiment, at least one movable portion (126) is integral with the support by means of a movable portion carriage (190) adapted to move in translation relative to a slide (200) integral with the support. In this embodiment, the movable portion carriage can be disposed and held between two positions, a proximal position and a distal position, to change the distance between the corresponding movable portion and the vibrating portion of the device.

The portion (120) comprises a vibrating portion (124) adapted to be coupled with the sonotrode (116) of a sonication probe. The part (120) intended to receive at least one tube (121) also comprises a movable part (126) integral with the support (122) via a movable part carriage (190) able to move in translation relatively a slide (200) secured to the support (122). The movable part (126) is able to move, preferably in translation, relative to the corresponding moving part carriage (136) and to the support (122), from a rest position to a clamping position and vice versa, by means of a clamping means (136).

Advantageously and so as to be able to independently receive and trap more than one tube, the portion (120) also includes a second movable portion (128). Said second movable portion (128) is secured to the support (122) via the movable portion carriage (190). Said second moving part (128) is able to move, preferably in translation relative to the mobile part carriage (190) and the support (122), from a rest position to a clamping position and vice versa, by intermediate of a second clamping means (138).

The device (110) can be used with tubes of volumes, sizes and diameters very different for the same embodiment of the device. When the diameters of the tubes are very different, the mobile part and the vibrating part of the device can easily be dismantled and replaced by moving part and vibrating part having contact surfaces adapted to the walls of the desired tubes, in particular the shapes and diameters of these contact surfaces. The mobile and vibrating part can also be defined to treat tubes of different shapes and diameters by specific machining of the mobile and vibrating parts adapted to the desired tubes.

The tube (121) presented in this example is a Becton Dickinson Falcon plastic tube with a capacity of 50 ml. This tube comprises a round section extending along a longitudinal axis. The bottom of the tube comprises a lower truncated cone portion, the opening portion of the tube is closable by a plug or resealable septum through which a pipettor can come pipette the contents of the tube.

More precisely and still with reference to FIG. 8, the part (120) comprises a support (122) able to receive and hold the fixed part (114) of a sonication probe according to at least one sonication axis. In the embodiment shown, the portion (120) can accommodate up to four tubes. The number of tubes that can be received is easily adaptable by modifying the portion (120).

The movable part (126) and the vibrating part (124) are arranged so that when the movable part (126) is in the rest position, a space (not shown) is provided between the said movable part (126) and the vibrating part (124). This space makes it possible to position or withdraw said at least one tube (121) in the part (20).

Still with reference to FIG. 8, the movable portion carriage (190) is held in a position close to its proximal position in order to receive a Becton tube. Dickinson Falcon with a capacity of 50mL when the mobile part (126) is in the rest position. When the movable part (126) is in the clamping position in the presence of at least one tube (121), said movable part (126) bears against said at least one tube (121) and exerts a transverse force against the wall of the latter, reflected on the vibrating part (124), thus trapping said at least one tube (121), said force being generated by the clamping means (128) on the movable part (126), along the sonication axis . The longitudinal axis of the tube (121) is thus maintained perpendicular to the sonication axis. This position / configuration corresponds to the large volume sonication configuration (lmL at 50mL) in a high capacity tube such as a Falcon 50mL tube.

In this embodiment, each tube can be positioned and trapped independently. For this, the device comprises four moving parts, in correspondence with a single vibrating part. When each of the movable parts is in the rest position, a space adapted to receive a tube is formed between said movable part and the vibrating part. The device can easily be modified so that a mobile part can provide two spaces able to receive two tubes and imprison them. All movable parts are secured to the movable portion carriage (190) so that movement of the movable portion carriage causes movement of all moving parts. The device can easily be modified so that a mobile part can be secured to a moving part carriage independently of other moving parts.

With reference to FIG. 9, the movable portion carriage (190) is moved and held in a position close to its distal position in order to be able to receive a Becton Dickinson Falcon tube with a capacity of 50mL when the movable part (126) is in the rest position. When the movable part (126) is in the clamping position in the presence of at least one tube (121), said movable part (126) bears against said at least one tube (121) and exerts a force along the axis longitudinal of the latter, reflected on the vibrating part (124), thus trapping said at least one tube (121), said force being generated by the clamping means (128) on the movable part (126), along the axis of sonication. The longitudinal axis of the tube (121) is thus maintained parallel to the sonication axis. The device is arranged vertically, so as to limit the footprint of the latter, in particular for use in a pipettor robot, and to facilitate loading unloading by an operator. This position / configuration corresponds to the low volume (50μί to lmL) sonication configuration in a high capacity tube such as a Becton Dickinson Falcon 50mL tube.

Advantageously and in order to improve the holding of at least one tube (121), the contact surface of the movable part (126) is flat so as to cooperate with the part upper tube cap (121). The contact surface of the vibrating part (124) comprises a groove, so as to cooperate with the lower part of the tube (121).

With reference to FIG. 10, the movable portion carriage (190) is moved and held in a position close to its distal position in order to be able to receive a Becton Dickinson Falcon tube with a capacity of 50mL when the movable part (126) is in the rest position. When the movable part (126) is in the clamping position in the presence of at least one tube (121), said movable part (126) bears against said at least one tube (121) and exerts a force along the axis longitudinal of the latter, reflected on the vibrating part (124), thus trapping said at least one tube (121), said force being generated by the clamping means (128) on the movable part (126), along the axis of sonication. The longitudinal axis of the tube (121) is thus maintained parallel to the sonication axis. The device is inclined relative to the horizontal, so as to limit the footprint of the latter while facilitating the loading / unloading by an operator, the tubes being more easily positioned in this position. This position / configuration corresponds to the low volume sonication configuration (50μί to lmL) in a large capacity tube such as a 50mL bottle.

Advantageously and in order to improve the holding of at least one tube (121), the contact surface of the movable part (126) is flat so as to cooperate with the upper part of the plug of the tube (121). The contact surface of the vibrating portion (124) includes a groove so as to cooperate with the lower portion of the tube (121).

With reference to FIG. 11, the contact surface (125) of the vibrating part (124) comprises a groove (127) so as to cooperate with the lower part of the tube (121, partially shown) and to trap the tube according to FIG. longitudinal axis of the tube when the movable part is in the clamping position.

With reference to FIG. 12, the contact surface (125) of the vibrating part (124) comprises a part of cylindrical shape so as to be able to cooperate with the wall of the tube (121, partially shown) and to imprison the tube along an axis transverse of the tube when the movable part is in the clamping position.

FIG. 13 shows a third embodiment of the device (310) according to the invention, this example being in no way limiting as to the scope of the invention. The device (310) includes a portion (320) for receiving at least one tube (321, 323). The portion (320) also comprises a vibrating portion (324) capable of being coupled with the sonotrode (316) of a sonication probe (not shown). The portion (320) for receiving at least one tube (321,323) also includes four movable portions (326a, 326b, 326c, 326d) integral with the support (not shown), able to move in translation, relative to the support (not shown), from a rest position to a clamping position and vice versa, via a means of tightening (not shown). In this embodiment, the receiving spaces (346) of tubes (321,323) are distributed radially equidistant from the sonication axis. The longitudinal axis of each of the tubes (321,323) when positioned in the receiving spaces of the device according to this embodiment and that the corresponding moving part is in the clamping position, is parallel to the sonication axis. In this particular embodiment, the vibrating part (324) comprises a disk having, in cooperation with corresponding moving parts, several receiving spaces (346) of tubes. In this particular embodiment, the sonotrode (not shown) which can be coupled to the vibrating part according to the invention is arranged vertically, the disc being centered on the sonication axis, the tubes being receivable in distributed reception spaces. radially around the sonication axis. The sonication axis is perpendicular to the plane of the figure and thus passes through the center of the disc. Preferably, the tubes are received and maintained vertically.

FIG. 14 shows a fourth embodiment of the device (410) according to the invention, this example being in no way limiting as to the scope of the invention.

The device (410) includes a portion (420) for receiving at least one tube (421, 423). The portion (420) also comprises a vibrating portion (424) capable of being coupled with the sonotrode (416) of a sonication probe (414). The part (420) intended to receive at least one tube (421, 428) also comprises a plurality of movable parts (426, 428) integral with the support (422), able to move in translation, relative to the support (422), of a position resting to a clamping position and vice versa, by means of a clamping means (436,438).

In this embodiment, the receiving spaces (446) of tubes (421, 428) are distributed radially equidistant from the sonication axis. The longitudinal axis of each of the tubes (321,328) when positioned in the receiving spaces of the device according to this embodiment and that the corresponding moving part is in the clamping position, is parallel to the sonication axis. In this particular embodiment, the vibrating part (424) may comprise a disc having, in cooperation with corresponding moving parts, several receiving spaces (446) of tubes, the number of moving parts being easily adapted to the geometry of the vibrating part in order to receive the desired number of tubes. In this particular embodiment, the sonotrode (416) which can be coupled to the vibrating part (424) according to the invention is arranged vertically, the vibrating part (424) being advantageously centered on the sonication axis, the tubes being able to be received in reception spaces distributed radially around the sonication axis. Preferably, the tubes are received and maintained vertically.

The clamping means (436), (438) advantageously take up the characteristics of the clamping means (36), (38) of the first embodiment illustrated in FIGS. 6 and 7 and previously described. The vibrating portions comprise means for heating (440) the contents of the tube in the form of a heating cartridge comprising a resistor.

EXAMPLES

A device according to the invention, and capable of receiving up to eight Eppendorf tubes of 1.5mL or 2mL is used. This device incorporates the features of the embodiment described in Figures 1 to 7. Each tube can be positioned or removed independently in the device. For this, the device comprises eight moving parts and a vibrating part. The vibrating part thus comprises eight housings each cooperating with a movable part to form eight spaces each capable of receiving a tube. Each tube can be trapped independently by moving the corresponding movable part, via a handle, from a rest position to a clamping position.

The sonication probe is connected to a generator box for exciting the sonotrode, coupled to the vibrating part, at a frequency of 30kHz. The power generated and delivered to the tubes is in a range of 0 to 80% of the maximum power of the generator box; 40%> of max power corresponding to 89W dissipated.

A thermal regulation box is associated with eight heating cartridges, each moving part comprising a heating cartridge consisting of a resistor. A temperature sensor is disposed in one of the heating cartridges to record an effective heating temperature used by the thermal control box to adjust the heating setpoint of the heating cartridge, depending on the desired heating temperature.

An adjustment screw makes it possible to adjust the position of each tube relative to the vibrating part and the corresponding moving part along the longitudinal axis of the tube. The adjusting screw is made of metal so as to conduct the heat generated by the corresponding heating cartridge.

Three ATCC (American Type Culture Collection) strains of microorganisms are selected to represent Gram-negative, Gram-positive and yeast species. These strains are Escherichia coli (ATCC No. 11775T), Staphylococcus epidermidis (ATCC No. 14990), Candida albicans (ATCC No. 18804).

Gram-negative and Gram-positive microorganisms are cultured on an agar plate Columbia with sheep blood and yeasts on a Dextrose Sabouraud agar, both marketed by the plaintiff. After culture, some colonies are collected with an oμΙ oesse, and resuspended. These suspensions are then calibrated at 10 ⁸ CFU / mL (Colony Forming Unit or Colony Forming Unit) by adjusting their concentration to reach a defined turbidity value. The turbidity values are measured spectrophotometrically at a wavelength of 550 nm. The optical density values corresponding to suspensions at 10 ⁸ CFU / mL are as follows for the strains studied:

0.2 (E.coli, ATCC 11775T)

0.43 (S.epidermidis, ATCC 14990)

0.67 (C.albicans, ATCC 18804, for a suspension at 10 ⁷ CFU / mL)

One milliliter of each of these suspensions at 10 ⁸ CFU / mL is then introduced into tubes of 1.5mL of reference "protein Low Bind" marketed by the company Eppendorf. These tubes are then centrifuged at 10000 g for 5 minutes in order to obtain, after removal of the supernatant, bacterial pellets containing 10 ⁸ CFU.

A sonication probe of the state of the art VialTweeter ^m , excited by an ultrasonic generator UIS250v with a power of 250W at 24kHz, both marketed by the company Hielscher are used in comparison with the device according to the invention. Only the 6 distal positions of the VialTweeter ^Tm sonotrode are used because the two positions closest to the generator receive an inhomogeneous power from the other positions during the sonication. Example 1

In a first protocol, each pellet containing 10 ⁸ CFU of microorganism is dispersed in 150 μl of 50 mM NH ₄ HCO ₃ buffer, pH 8.0. After rapid mixing, the volume of the liquid is transferred to a 1.5 ml tube containing a mixture containing 50 mg of zirconium beads coated with a silica layer of 0.1 mm in diameter (reference 11079102 marketed by Biospec Products) and 200mg of glass beads 1mm in diameter (reference 11079110 marketed by Biospec Products). 3.5 DTT to 150mM are also added. For each of the strains, 9 tubes are thus prepared and then closed by plugs. For each of the strains, eight tubes are subsequently placed in the receiving spaces of the the tubes of the device according to the invention called "Si" device in the corresponding figures, the ninth tube is placed in one of the reception spaces of the sonication probe of the state of the art, called probe "Hi" in the figures corresponding. A sonication step resulting in a lysis-reduction is then performed on each of the devices according to the invention "Si" and the state of the art "Hi".

For the device of the state of the art "Hi", the amplitude is set to 100 and cycle 1 for 5 minutes. At the end of this sonication step, the temperature of the tube reaches about 95 ° C. After cooling in ice for 1 min, an alkylation step is performed by adding 9μΙ. 150mM IAA in 50mM NH ₄ HCO ₃ buffer pH 8.0. Trypsin at 1 μg.μL ^-1 is then added, after which the tube is transferred to a thermo-mixer to carry out a digestion step of 15 minutes at 50 ° C. and 850 rpm. then stopped.

For the device according to the invention "Si", the amplitude is set to 40% of the power or 89W for 5 minutes. At the end of this sonication step, the temperature of the tube reaches about 95 ° C. After cooling in ice for 1 min, an alkylation step is carried out by adding 9 i of IAA to 150 mM in a buffer of NH ₄ HCO ₃ at 50 mM pH 8.0. Trypsin at 1 μg.μL ^-1 is then added, after which the eight tubes are transferred to a thermo-mixer to carry out a digestion step of 15 minutes at 50 ° C. and 850 rpm. is then stopped.

This protocol is reproduced successively for the following two strains, ie a total of three sets of nine tubes.

Peptides resulting from the sequence of these stages of Lyse-Reduction-Alkylation and Digestion were then injected into a liquid chromatography system coupled to an electrospray mass spectrometer. These LC-ESI-MS / MS analyzes, carried out in Multiple Reaction Monitoring (MRM) mode with 3 different methods depending on the microorganisms in question, are so-called targeted methods in which a defined number of peptides of known masses are sought in the US. 'sample. In the case where the identification of the micro-organism of the sample would have been obtained by a preliminary analysis, this type of targeted analysis would make it possible to search for and quantify the presence of resistance marker peptides, typing, virulence or confirmation of identification. In the context of the present invention, this type of analysis has been used to quantitatively compare different lysis protocols. digestion conducted on different ultrasound probes. The methods target about 60 peptides. Each peptide is searched through the detection of 3 transitions. When these three transitions meet two criteria of intensity and area, the peptide is considered present and the areas of its three transitions are summed. The performance indicators of lysis-digestion protocols are the total number of correctly detected peptides and the sum of their areas.

Figure 15a shows the results of tubes containing Escherichia coli strains. The results of the tube processed by the sonication probe of the state of the art are noted P2A_Hi_ECl. The results of the eight tubes having been treated with the device according to the invention are noted P2A_Si_EC1, P2A_Si_EC2, P2A_Si_EC3, P2A_Si_EC4, P2A_Si_EC5, P2A_Si_EC6, P2A_Si_EC7, P2A_Si_EC8.

Figure 15b shows the results of tubes containing strains of Candida Albicans. The results of the tube processed by the sonication probe of the state of the art are noted P2A_Hi_CAl. The results of the eight tubes having been treated with the device according to the invention are noted P2A_Si_CAl, P2A_Si_CA2, P2A_Si_CA3, P2A_Si_CA4, P2A_Si_CA5, P2A_Si_CA6, P2A_Si_CA7, P2A_Si_CA8. Figure 15c shows the results of tubes containing strains of Staphylococcus Epidermidis. The results of the tube processed by the sonication probe of the state of the art are denoted SE_P2A_Hi_01. The results of the eight tubes having been treated with the device according to the invention are denoted SE_P2A_Si_l, SE_P2A_Si_2, SE_P2A_Si_3, SE_P2A_Si_4, SE_P2A_Si_5, SE_P2A_Si_6, SE_P2A_Si_7, SE_P2A_Si_8.

These results show excellent inter-tube reproducibility. The results of the eight tubes treated simultaneously for the same species on the device according to the invention indeed has accumulated areas and comparable numbers of peptides detected for each of the positions.

These results also demonstrate the ability of the device according to the invention to perform a sonication step comparable to the device of the state of the art, and whether the treated species is Gram-positive, Gram-negative or a yeast. Example 2 In a second protocol, each pellet of 10 8 CFU is dispersed in 150 μl of 50 mM NH ₄ HCO ₃ buffer, pH 8.0. After rapid mixing, the volume of the liquid is transferred into a 1.5 ml tube containing a mixture containing 50 mg of zirconium beads coated with a 0.1 mm diameter silica layer (reference 11079101z marketed by Biospec Products) and 200 mg. glass beads 1mm in diameter (reference 11079110 marketed by Biospec Products). DTT to 150mM are also added.

2 tubes for EC and SE, 3 tubes for CA, are subsequently placed in the receiving spaces of the tubes of the device according to the invention called "Si" device, 2 other tubes per species are placed in two of the reception spaces of the sonication probe of the state of the art, called "Hi" probe.

For the device of the state of the art "Hi", an embodiment using a single tube is realized as follows. The alkylating agent is directly introduced before initiating the sonication (13.5 μΕ of IAA to 150 mM in a buffer of NH ₄ HCO ₃ at 50 mM pH 8.0) as well as ΙΟ μΙ of trypsin at 1 μg.μL ^-1 . The sonication step is then initiated by adjusting the amplitude to 80 and 0.5 cycle for 30 minutes.The 4 stages (lysis-reduction-alkylation-digestion) are therefore conducted simultaneously.The digestion is then stopped by adding formic acid.

For the device according to the invention "Si", an embodiment using a single tube can be conducted by sequentially adding the reagents taking advantage of the possibility of easily accessing and opening the tubes and to be able to use the heating function of the probe.

A sonication step is carried out directly by adjusting the amplitude to 40% of the power, ie 89W for 5 minutes. At the end of this sonication step, the tubes are opened to add 13.5 μl of IAA to 150 mM in a buffer of NH ₄ HCO ₃ at 50 mM pH 8.0. as well as ΙΟμΙ of trypsin at 1 μg.μL ^"1. A digestion step of 25 minutes at 50 ° C. is then performed by deactivating the sonication and heating the tubes by means of the heating cartridges of each of the eight positions. digestion is then stopped.

Subsequently, the contents of the tubes are analyzed on the same LC-ESI-MS / MS system as previously described.

Figure 16a shows the results of tubes containing Escherichia coli strains. The results of two tubes processed by the sonication probe of the state of the art are noted OP Hi ECl and OP_Hi_EC2. The results of two tubes having been treated with the device according to the invention are denoted OP_Si_EC5_l and OP_Si_EC5_2.

Figure 16b shows the results of tubes containing strains of Candida Albicans. The results of two tubes by the sonication probe of the state of the art are noted OP Hi CAl and OP_Hi_CA2. The results of two of the eight tubes having been treated with the device according to the invention are denoted OP_Si_CA5_7 and OP_Si_CA5_8.

Figure 16c shows the results of tubes containing strains of Staphylococcus Epidermidis. The results of two tubes processed by the sonication probe of the state of the art are noted SE OP Hi l and SE_OP_Hi_2. The results of two of the eight tubes having been treated with the device according to the invention are denoted SE_OP_Si_5_3 and SE_OP_Si_5_4.

These results demonstrate the ability of the device according to the invention to simplify a process comprising a sonication step and a heating step in the same device without transferring tubes or liquids. These results also demonstrate this ability regardless of the species being treated. The execution times of a protocol comprising a sonication step are thus shortened by eliminating manipulations such as transfers of tubes or liquids. The risks of contamination related to pipetting steps are also reduced.

Claims

1. Device comprising:

a support able to receive and maintain the fixed part of a sonication probe,

a movable part integral with the support, able to move, preferably in translation relative to the support, from a rest position to a clamping position and vice versa, by means of a clamping means,

a means for heating the contents of the tube,

so that when the mobile part is in the rest position, a space is provided between said movable part and the vibrating part for positioning or removing said at least one tube, whereas when the mobile part is in the clamping position in the presence of at least one tube, said movable part abuts against said at least one tube and exerts a force against the wall of the latter, reflected on the vibrating part, thus trapping said at least one tube, said force being generated by the means clamping on the moving part

2. Device according to claim 1 characterized in that it comprises a plurality of movable parts each movable part being able to move, preferably in translation, relative to the support, a rest position to a clamping position and vice versa, by through a corresponding clamping means,

so that when one of the moving parts is in the rest position, a space is provided between said movable part and said vibrating part for positioning or removing said at least one tube, whereas when one of the moving parts is in the position of clamping in the presence of at least one tube, said movable part bears against said at least one tube and exerts a force against the wall of the latter, reflected on the vibrating part, thus trapping said at least one tube, said force being generated by the clamping means on the moving part

3. Device according to claim 1 or 2 characterized in that when the movable part is in the clamping position in the presence of at least one tube, said movable part is in bearing against said at least one tube, and exerts said force against the wall of the tube, along a transverse axis thereof, reflected on the vibrating part, thus trapping said at least one tube, said force being generated by the clamping means on the moving part.

Device according to any one of claims 1 to 3 characterized in that said support is adapted to receive and maintain the fixed portion of a sonication probe according to at least one sonication axis and said force is exerted parallel to the axis of sonication, along the sonication axis or perpendicular to the sonication axis.

Device according to any one of claims 1 to 4, characterized in that a movable part is secured to the support by means of a movable part carriage adapted to move in translation relative to a slideway secured to the support and to be disposed and maintained between two positions, a proximal position and a distal position.

6. Device according to any one of claims 1 to 5, characterized in that at least one movable part comprises a heating means

7. Device according to one of claims 1 to 6 characterized in that it comprises a means for adjusting the position of the tube relative to the movable and vibrating part along a longitudinal axis defined by the tube

8. Device according to claim 7 characterized in that the adjusting means is a thermal conductor

9. Device according to one of claims 1 to 8, characterized in that at least one clamping means comprises a jack

10. Device according to one of claims 1 to 9, characterized in that at least one clamping means comprises an elastic return means such as a compression spring

11. System for the preparation of biological samples comprising

A device according to one of claims 1 to 10

A sonication probe, comprising a fixed part and a sonotrode

The support being secured to the sonication probe by its fixed part, the vibrating part being coupled to the sonotrode

12. Use of a system according to claim 11 for the preparation of biological samples comprising the following steps:

a) Insert a tube containing a biological sample in the reception space, the mobile part being in the rest position

b) move the moving part from its rest position to its clamping position so that the tube is trapped,

13. Use of a system according to claim 11 for the preparation of biological samples, according to the following steps:

a) Insert a tube containing a biological sample in the space, the mobile part being in the rest position

b) move the moving part from the rest position to the clamping position so that the tube is trapped in the space,

c) Perform a step of sonication of the biological sample by activating the sonotrode,

d) Perform a step of incubating the biological sample by activating the heating means