WO2025088055A1 - Handling apparatus, handling assembly and handling method - Google Patents

Abstract

A handling apparatus (5) for handling a sample of biological material via capture elements, comprising: - a tube support configured to receive a tube (3); - an aggregation member (15) configured to generate a magnetic attraction force on the capture elements (2), and intended to cooperate with an interface sheath (20), - an interface-sheath carriage (30) movable between an upper position and a lower position, - a biasing system (45) configured to be placed in contact with the interface sheath (20) and to generate, in the interface sheath (20), vibrations suitable for separating the capture elements from the interface sheath (20) after the aggregation member (15) and the interface sheath (20) have passed from an active state, in which the aggregation member (15) exerts the magnetic attraction force on the capture elements, to an inactive state, in which the aggregation member (15) does not exert the magnetic attraction force on the capture elements.

Description

DESCRIPTION TITLE: MANIPULATION APPARATUS, MANIPULATION ASSEMBLY AND MANIPULATION METHOD

SCOPE OF DISCLOSURE

The present disclosure relates to an apparatus for manipulating a sample of biological material by means of capture elements as well as to a manipulation assembly and to a manipulation method implementing such a manipulation apparatus.

Without being limited thereto, the present disclosure applies in particular to the manipulation of a sample of nucleic acids, proteins, antibodies or other biological material, by means of capture elements, generally in the form of beads, magnetic and configured to be attached to the sample of biological material.

STATE OF THE ART

During processing, a sample of biological material is manipulated, in particular by being successively immersed in different solutions. This is the case, for example, of a purification treatment of a sample of nucleic acids as biological material.

To recover the sample of biological material dispersed in each of the solutions and to be able to release it into one of the other solutions, it is known to use a handling device of the type comprising:

- a frame extending along a vertical axis,

- a tube holder mounted on the frame and comprising at least one receiving space configured to receive at least one tube adapted to contain a solution comprising the sample of biological material and the capture elements;

- an aggregation member configured to generate a magnetic attraction force on the capture elements, the aggregation member being intended to cooperate with a sheath interface such that, in an active state of the aggregating member and the interface sheath, the aggregating member is received in the interface sheath and exerts the magnetic attraction force on the capture elements to aggregate said capture elements on the interface sheath and, in an inactive state of the aggregating member and the interface sheath, the aggregating member does not exert a magnetic attraction force on the capture elements.

An example of a handling apparatus of the aforementioned type is described in WO 2021/056005. The aggregation member comprises a permanent magnet which, in the inactive state of the aggregation member and the interface sheath, is placed at a distance from the interface sheath. Even in the absence of magnetic attraction force, capture elements may remain attached to the interface sheath. These capture elements and residues of biological material attached to them may not follow one of the processing steps and may risk, during a subsequent step, contaminating a solution or the part of the sample of biological material which has followed all the processing steps.

To release the capture elements when the aggregation member and the interface sheath are immersed in the tube solution, the manipulation apparatus of document WO 2021/056005 provides for moving the aggregation member apart and rotating the interface sheath in the tube solution.

However, capture elements may remain attached to the interface sheath and the turbulence generated in the solution by the rotation of the interface sheath may damage the sample of biological material.

It is also known, in particular from documents CN 203 174 103, CN 206 594 187 and US 2020/392481, to generate vibrations in the interface sheath suitable for separating the capture elements from the interface sheath. The stressing systems implemented for this purpose do not, however, offer satisfactory control for effectively separating the capture elements without damaging the sample of biological material. SUMMARY OF DISCLOSURE

The invention aims to solve the problem stated above.

To do this, according to a first aspect, the invention proposes an apparatus for handling a sample of biological material by means of capture elements, the capture elements being magnetic and configured to be secured to the sample of biological material, the handling apparatus comprising: a frame extending along a vertical axis,

- a tube holder mounted on the frame and comprising at least one receiving space configured to receive at least one tube adapted to contain a solution comprising the sample of biological material and the capture elements;

- an aggregation member configured to generate a magnetic attraction force on the capture elements, the aggregation member being intended to cooperate with an interface sheath in such a way that, in an active state of the aggregation member and the interface sheath, the aggregation member is received in the interface sheath and exerts the magnetic attraction force on the capture elements to aggregate said capture elements on the interface sheath and, in an inactive state of the aggregation member and the interface sheath, the aggregation member does not exert a magnetic attraction force on the capture elements, the interface sheath having an access end by which the aggregation member is inserted into the interface sheath, and a free end opposite the access end and in the vicinity of which the capture elements are aggregated in the active state of the aggregation member and the interface sheath,

- an interface sheath carriage comprising a holding device configured to hold the interface sheath opposite the receiving space, the interface sheath carriage being movable along the vertical axis relative to the frame between a high position in which said interface sheath carriage places the interface sheath at a distance from the receiving space, and a low position in which said interface sheath carriage places the interface sheath in the receiving space,

- a biasing system configured to be placed in contact with the interface sheath and to generate in the interface sheath vibrations adapted to separate the capture elements from the interface sheath after the aggregation member and the interface sheath are switched from the active state to the inactive state, wherein the biasing system is mounted on the interface sheath carriage and is arranged to generate the vibrations in the vicinity of the access end, and wherein the holding device is configured to allow propagation of the vibrations from the access end to the free end of the interface sheath.

Thus, the handling device according to the invention imposes a stress on the interface sheath only, which makes it possible to make the detachment of the capture elements more reliable while preserving the sample of biological material.

According to specific provisions, the handling device can allow one or more steps in the treatment of a single sample of biological material to be carried out in a simple and intuitive manner. It can be adapted to different types of treatment of different types of biological material sample. It can offer compactness and robustness allowing it to be easily transported.

The holding device may have a holding surface configured to contact the interface sheath at a distance from the access end along the vertical axis, such that the interface sheath has a proximal sheath portion between the access end over the distance, the proximal sheath portion being projecting relative to the holding surface, and wherein the biasing system is spaced from the holding surface along the vertical axis in a direction opposite the tube support, so as to come into contact with the proximal sheath portion.

The holding device may comprise a ring along a holding axis parallel to the vertical axis and having the holding surface configured to be in peripheral contact with the interface sheath.

The ring may be configured to receive the interface sheath comprising a side wall along a sheath axis and at least two lugs projecting transversely in an equally distributed manner from an outer surface of the side wall, the ring comprising a groove provided on the holding surface around the holding axis and configured to receive the lugs, and at least two grooves extending axially on the holding surface from a passage end to the groove, so as to allow passage of the lugs of the interface sheath in an angular position of the interface sheath relative to the ring and maintenance of the lugs of the interface sheath in the groove in other angular positions of the interface sheath relative to the ring.

The holding device may further comprise a flange movable relative to the interface sheath carriage between an open position allowing the ring to be placed in position and a closed position ensuring that the ring is held in position relative to the interface sheath carriage.

The biasing system may be configured to perform at least one shock on the interface sheath.

The biasing system may comprise a cam driven in rotation about a pivot axis and comprising a radial contact portion arranged to come into contact with the interface sheath in an angular position range of the cam relative to the frame, the cam being at a distance from the interface sheath outside the angular position range.

The cam may have a biased state in which said cam is rotated and a stopped state in which said cam is fixed relative to the frame, and the biasing system may include a detection device configured to stop the cam outside the angular position range when the cam transitions from the biased state to the stopped state.

The handling apparatus may further comprise an aggregation member carriage carrying the aggregation member, the aggregation member carriage being movable along the vertical axis relative to the frame between a disassembled position remote from the interface sheath carriage such that the aggregation member is spaced from the interface sheath, and an assembled position proximate to the interface sheath carriage such that the aggregation member is received in the interface sheath.

The aggregating member may include a permanent magnet at a distal end for receipt in the interface sheath, the aggregating member, and the sheath. interface being in the inactive state when the aggregation organ carriage is in the disassembled state.

The tube holder may include a barrel having a plurality of receiving spaces about a barrel axis, the barrel being rotatable about the barrel axis to selectively position one of the receiving spaces opposite the interface sheath.

The holding device may be configured to hold a single interface sheath, the biasing system being arranged to generate the vibrations in the vicinity of the access end of the single interface sheath. The handling apparatus is then suitable for handling small series of samples of biological material, in which each sample of biological material is handled individually.

According to a second aspect, the invention proposes a handling assembly comprising a handling apparatus as defined previously and at least one interface sheath configured to receive the aggregation member and to be held in the holding device of the interface sheath carriage.

According to a third aspect, the invention proposes a method for handling a sample of biological material, the handling method implementing a handling assembly as defined previously and comprising steps of:

- placing in the receiving space of the tube holder a tube containing a solution comprising the sample of biological material and capture elements, the capture elements being magnetic and configured to be secured to the sample of biological material,

- place the interface sheath in the interface sheath carriage holding device in the high position,

- placing the interface sheath carriage in the low position and the aggregation member and the interface sheath in the active state, the aggregation member being received in the interface sheath and exerting the magnetic attraction force on the capture elements to aggregate said capture elements on the interface sheath,

- put the interface sheath carriage in the high position, the aggregation organ and the interface sheath remaining in the active state,

- place the interface sheath carriage in the low position, the aggregation member and the interface sheath remaining in the active state,

- put the aggregation organ and the interface sheath in the inactive state,

- activate the stress system to generate vibrations in the interface sheath suitable for separating the capture elements from the interface sheath.

BRIEF DESCRIPTION OF THE FIGURES

Other objects and advantages of the invention will appear on reading the following detailed description of a particular embodiment of the invention made in relation to the appended drawings in which:

- Figure 1 is a perspective representation of a manipulation assembly comprising a manipulation apparatus and an interface sheath for manipulating a sample of biological material by means of capture elements, the capture elements configured to be secured to the sample of biological material, the interface sheath being configured to receive an aggregation member of the manipulation apparatus in such a way that, in an active state of the aggregation member and the interface sheath, the aggregation member is received in the interface sheath and exerts the magnetic attraction force on the capture elements to aggregate them,

- Figures 2 and 3 are partial representations, respectively in front and side view, of the handling assembly of Figure 1, illustrating the aggregation member and the interface sheath mounted respectively on an aggregation member carriage and an interface sheath carriage movable along a vertical axis on a frame, the interface sheath carriage being in the high position in which it places the interface sheath at a distance from a tube adapted to contain a solution comprising the sample of biological material and the capture elements, the aggregation member carriage being in the disassembled position at a distance from the interface sheath carriage in such a way that the aggregation member is spaced from the interface sheath, - Figure 4 is an enlarged side view representation of a portion of the handling assembly of Figure 1, illustrating the interface sheath carriage in the lowered position in which it places the interface sheath in the tube, the aggregation member carriage being in the disassembled position

- Figure 5 is an enlarged side view representation of a portion of the handling assembly of Figure 1, illustrating the interface sheath carriage in the lowered position and the aggregator member carriage in the assembled position proximate the interface sheath carriage such that the aggregator member is received in the interface sheath,

- Figure 6 is an enlarged side view representation of a portion of the handling assembly of Figure 1, illustrating the interface sheath carriage in the upper position and the aggregation member carriage in the assembled position,

- Figure 7 is an enlarged perspective view of a portion of the handling assembly of Figure 1, illustrating the interface sheath carriage comprising a holding device configured to hold the interface sheath opposite the tube, and a biasing system configured to be placed in contact with the interface sheath and generate vibrations in the interface sheath suitable for separating the capture elements from the interface sheath after the aggregation member and the interface sheath have passed from the active state to an inactive state,

- Figure 8 is an exploded perspective view from above of the holding device of Figure 7 and the interface sheath, the holding device comprising a ring into which the interface sheath is inserted and a flange configured to receive the ring,

- figure 9 is an exploded perspective view from below of the ring of the holding device of figure 8,

- figure 10 is a sectional representation along the orientation referenced X-X in figure 8 of the ring of the holding device of figure 8,

- Figures 11 and 12 are enlarged front view representations of the biasing system of Figure 7, the biasing system comprising a cam driven in rotation to produce at least one shock on the interface sheath generating vibrations in the vicinity of an access end of the interface sheath by which the aggregation member is inserted into the interface sheath,

- Figure 13 is an exploded perspective representation of the biasing system of Figure 7, illustrating a detection device configured to stop the cam outside an angular position range in which the cam contacts the interface sheath,

- figures 14 and 15 are enlarged representations in front view of a variant of the detection device of the stress system of figure 7,

- figure 16 is a schematic representation of a particular, non-limiting example of implementation of a manipulation method with the manipulation assembly of figure 1 in a binding step of a purification treatment of a nucleic acid sample,

- figures 17a and 17b are schematic representations of a particular, non-limiting example of implementation of a handling method with the handling assembly of figure 1 in a step of washing the nucleic acid sample of the purification treatment of a nucleic acid sample,

- figure 18 is a schematic representation of a particular, non-limiting example of implementation of a handling method with the handling assembly of figure 1 in a drying step of the purification treatment of a nucleic acid sample,

- figures 19a and 19b are schematic representations of a particular, non-limiting example of implementation of a manipulation method with the manipulation assembly of figure 1 in an elution step of the purification treatment of a nucleic acid sample,

- Figure 20 is a schematic representation of a particular, non-limiting example of implementing a manipulation method with the manipulation assembly of the Figure 1 in a capture element washing step of the purification treatment of a nucleic acid sample.

DETAILED DESCRIPTION

Figures 1 to 3 represent an embodiment of an apparatus 5 for manipulating a sample of biological material 1. The manipulation is carried out by means of capture elements 2, generally in the form of magnetic beads and configured to be secured to the sample of biological material 1.

In a particular example, the manipulation apparatus 5 may be used to manipulate a sample of nucleic acids, proteins, antibodies, or other biological material.

The handling device 5 comprises a frame 6 extending along a vertical axis A from a base 7 configured to rest on a support surface.

A tube holder 8 is mounted on the base 7 of the frame 6 and comprises one or more receiving spaces 9 each having a receiving axis R parallel to the vertical axis A. Each receiving space 9 is configured to coaxially receive a tube 3 adapted to contain a solution comprising the sample of biological material 1 and the capture elements 2. Each tube 3 has a side wall around a tube axis T and which extends from a closed bottom 3a to an upper opening 3b.

In the embodiment shown, the tube support 8 comprises a barrel 10 having several receiving spaces 9, for example eight, around a barrel axis B parallel to the vertical axis A. The barrel 10 is driven in rotation along the barrel axis B by a drive system, such as an electric motor placed in the base 7 of the frame 6.

The manipulation apparatus 5 comprises an aggregation member 15 configured to generate a magnetic attraction force on the capture elements 2. In the embodiment shown, without being limited thereto, the aggregation member 15 comprises a rod 16 which extends along a rod axis I between a proximal end and a distal end. The aggregating member 15 also comprises a permanent magnet 17 extending coaxially to the distal end of the rod 16.

The aggregation member 15 is configured to cooperate with an interface sheath 20. The interface sheath 20 constitutes a consumable avoiding soiling and damaging the aggregation member 15. The interface sheath 20 comprises a side wall around a sheath axis G and which extends between an access end 20a by which the distal end of the aggregation member 15 can be inserted into the interface sheath 20 and a free end 20b, closed. In the embodiment shown, the side wall of the interface sheath 20 comprises lugs 22 projecting transversely relative to the sheath axis G from an outer surface. The lugs 22 are arranged at a distance D from the access end 20a so as to define a proximal portion of sheath 21 which extends, opposite the free end 20b, from the access end 20a over the distance D. In FIG. 8, the interface sheath 20 comprises two diametrically opposed lugs 22. Any other arrangement of equally distributed lugs 22 could however be provided.

The interface sheath 20 is mounted on an interface sheath carriage 30. The interface sheath carriage 30 extends perpendicular to the vertical axis A from a guide end 31 slidably mounted on a vertical mast 35. The interface sheath carriage 30 is movable along the vertical axis A between high and low positions by means of a drive device comprising, in the embodiment shown, a worm screw 36 extending along the vertical axis A and driven in rotation by an electric motor placed in the base 7 of the frame 6.

In Figure 3, the interface sheath carriage 30 is in the high position in which it places the interface sheath 20 at a distance from and opposite one of the receiving spaces 9 with the sheath axis G aligned with the receiving axis R. When a tube 3 is placed in the receiving space 9, the sheath axis G is aligned with the tube axis T and the free end 20b of the interface sheath 20 is opposite the upper opening 3b of the tube 3. The rotation of the barrel 10 makes it possible to selectively place one of the receiving spaces 9 and, where appropriate, the tube 3 which it receives, opposite the interface sheath 20. In Figure 4, the interface sheath carriage 30 is in the low position in which it places the interface sheath 20 in the receiving space 9. The interface sheath 20 can then coaxially penetrate the tube 3 received in the receiving space 9 through the upper opening 3b until the free end 20b of the interface sheath 20 is located in the vicinity of the bottom 3a of the tube 3.

Similarly, the aggregating member 15 is mounted on an aggregating member carriage 40. The aggregating member carriage 40 extends perpendicular to the vertical axis A from a guide end 41 slidably mounted on the vertical mast 35, above the interface sheath carriage 30. The aggregating member carriage 40 is movable along the vertical axis A between disassembled and assembled positions by means of a drive device comprising, as for the interface sheath carriage 30, another worm screw 36 extending along the vertical axis A and driven in rotation by an electric motor placed in the base 7 of the frame 6.

In Figures 3 and 4, the aggregator member carriage 40 is in the disassembled position away from the interface sheath carriage 30 such that the aggregator member 15 is spaced from the interface sheath 20, with the rod axis T aligned with the sheath axis G and the distal end of the aggregator member 15 facing the access end 20a of the interface sheath 20.

In Figures 5 and 6, the aggregator member carriage 40 is in the assembled position near the interface sheath carriage 20 such that the aggregator member 15 is received coaxially in the interface sheath 20, with the permanent magnet 17 near the free end 20b of the interface sheath 20.

In Figure 5, the interface sheath carriage 30 in the lower position and the aggregating member carriage 40 in the assembled position place the aggregating member 15 in the interface sheath 20 itself placed in the tube 3 in the vicinity of the bottom 3a of the tube 3. This arrangement constitutes an active state of the aggregating member 15 and the interface sheath 20 in which the aggregating member 15 exerts the magnetic attraction force on the capture elements 2 to aggregate them on the interface sheath 20. In Figures 3 and 4, the disassembled position of the aggregation member carriage 40 places the aggregation member 15 at a distance from the interface sheath 20 where it does not exert a magnetic attraction force on the capture elements 2. The aggregation member 15 and the interface sheath 20 are then in an inactive state.

Figures 7 to 10 illustrate a holding device 24 arranged on the interface sheath carriage 30, opposite the guide end 31, and configured to hold a single interface sheath 20.

The holding device 24 comprises a ring 25 along a holding axis M parallel to the vertical axis A and having a holding surface intended to be in peripheral contact with the interface sheath 20, in the vicinity of the access end 20a.

In the embodiment shown, the ring 25 comprises a groove 26 formed on the holding surface around the holding axis M and configured to receive the lugs 22. Two grooves 27 extend along the holding axis M on the holding surface from a passage end to the groove 26, so as to allow the lugs 22 of the interface sheath 20 to pass in an angular position of the interface sheath 20 relative to the ring 25 and to hold the lugs 22 of the interface sheath 20 in the groove 26 in other angular positions of the interface sheath 20 relative to the ring 25.

The holding device 24 further comprises a flange 28 provided with a housing along a housing axis L parallel to the vertical axis A and configured to receive the ring 25 with the holding axis M aligned with the housing axis L. The flange 28 is movable relative to the interface sheath carriage 30 between an open position allowing the ring 25 to be placed in position and a closed position ensuring that the ring 25 is held in position relative to the interface sheath carriage 30. The flange 28 may have a stop surface arranged to, in the closed position, come into contact with a stop surface extending radially relative to the holding axis M of the ring 25, for example an upper transverse surface of the ring 25. These arrangements make it possible to ensure that the ring 25 is held in the flange 28 in the closed position when the interface sheath 20 is removed from the ring 25. Figures 11 and 12 represent a stressing system 45 configured to be placed in contact with the interface sheath 20 and to generate in the interface sheath 20 vibrations adapted to separate the capture elements 2 from the interface sheath 20 after the aggregation member 15 and the interface sheath 20 have passed from the active state to the inactive state.

In the embodiment shown, the biasing system 45 is mounted on the interface sheath carriage 30 and comprises a cam 46 pivotally mounted about a pivot axis P perpendicular to the vertical axis A on the interface sheath carriage 30. The cam 46 comprises a radial contact portion 47 arranged to come into contact with the proximal sheath portion 21 of the interface sheath 20, in the vicinity of the access end 20a, in an angular position range of the cam 46 relative to the interface sheath carriage 30. The cam 46 is at a distance from the interface sheath 20 outside the angular position range.

The cam 46 has a stop state in which it is fixed relative to the frame 6 and to the interface sheath carriage 30. It also has a stress state in which it is rotated along the pivot axis P so as to intermittently stress, in the form of shocks, the interface sheath 20 and generate vibrations in the proximal portion of the sheath 21, in the vicinity of the access end 20a.

The holding device 24 is then configured to allow propagation of the vibrations from the access end 20a to the free end 20b of the interface sheath 20. In particular, the ring 25 may be configured, in particular in terms of material, shape and/or dimensions, to ensure holding of the interface sheath 20 while allowing relative movement between the interface sheath 20 and the ring 25 to allow propagation of the vibrations. The ring 25 may, for example, be made of an elastomer such as silicone or Polyurethane (TPU). It may have a Shore A hardness of between 40 and 50 so as to have sufficient flexibility to allow movement of the interface sheath 20 relative to the ring 25, and sufficient hardness to realign the interface sheath 20 when no stress is applied to it. Additionally or alternatively, the shape or dimensions of the ring 25 may be provided to provide clearance between the holding surface of the ring 25 and the sheath. interface sheath 20. The flange 28 is also designed to, in the closed position, ensure the retention of the ring 25 without hindering the relative movement between the interface sheath 20 and the ring 25. In addition to the aforementioned provisions concerning the ring 25 and the interface sheath 20 or alternatively, a clearance could also be provided between the ring 25 and the flange 28 to allow relative movement between them.

In order to position the cam 46 in a stop position outside the angular position range when the cam 46 transitions from the biased state to the stop state, the biasing system 45 comprises a detection device 50.

In the embodiment shown in Figures 11 to 13, the detection device 50 implements a REED type sensor 51 mounted on the interface sheath carriage 30 outside the angular position range of the cam 46. The cam 46 comprises a flange 48 which extends over an angular sector perpendicular to the pivot axis P and parallel to the contact portion 47 of the cam 46, being centered on this contact portion 47. The flange 48 is placed at a distance from the contact portion 47 of the cam

46 along the pivot axis P so as not to interfere with the interface sheath 20 when the cam 46 is in the angular position range. On a surface facing the REED type sensor 51, the flange 48 comprises a magnetic member 52, in the form of two magnets in the embodiment shown. Thus, when the cam 46 passes from the biased state to the stopped state, it is driven by pulses until the magnetic member 52 is detected by the REED type sensor 51 and stops the cam 46 in the stopped position, outside the angular position range of the cam 46.

In a variant shown in figures 14 and 15, the flange 48' extends generally radially relative to the pivot axis P while being centered on the contact portion

47 of the cam 46, at a distance from the latter. The flange 48 is configured to cooperate with a detection device 50', for example electromagnetic, in particular optical, in order to detect the stop position, for example vertical.

In the state of stress of the cam 46, the flange 48, 48' passes behind the interface sheath 20 without interacting with it. At the end of stress, the device detection 50, 50' detects the flange 48, 48' aligned with the contact portion 47 of the cam 46 and the cam can enter the stop state.

The handling apparatus 5 can be implemented in a handling method providing for placing in the reception space 9 of the tube holder 8 a tube 3 containing a solution comprising the sample of biological material 1 and capture elements 2.

The interface sheath carriage 30 is in the high position and the flange 28 of the holding device 24 is placed in the open position. The ring 25 is placed in the housing of the flange 28. The interface sheath 20 is inserted into the ring 25 of the holding device 24 in the angular position putting the lugs 22 of the interface sheath 20 in correspondence with the grooves 27. When the lugs 22 reach the groove 26, the interface sheath 20 is pivoted about its sheath axis G to lock the interface sheath 20 in the ring 25. The flange 28 is placed in the closed position. The interface sheath 20 is then held in the holding device 24 by allowing the proximal portion of the sheath to protrude relative to the flange 28 so that the proximal portion of the sheath is free.

The interface sheath carriage 30 is moved to the lower position and then the aggregating member carriage 40 is placed in the assembled position. The aggregating member 15 and the interface sheath 20 are then in the active state with the aggregating member 15 received in the interface sheath 20 and exerting the magnetic attraction force on the capture elements 2 to aggregate them on the interface sheath 20. The biological material sample 1 can thus be collected on the interface sheath 20.

The interface sheath carriage 30 is moved into the high position, the aggregation member 15 and the interface sheath 20 remaining in the active state to extract the biological material sample 1 from the solution contained in the tube 3.

The barrel 10 can be pivoted to place another tube 3 with another solution opposite the interface sheath 20. The interface sheath carriage 30 can then be placed in the lower position, the aggregation member 15 and the interface sheath 20 remaining in the active state, then the aggregation member carriage 40 is moved to the position disassembled to put the aggregation member 15 and the interface sheath 20 in the inactive state.

The stressing system 45 can then be activated to generate vibrations in the interface sheath 20 suitable for separating the capture elements 2 from the interface sheath 20.

In relation to Figures 16 to 20, a particular, non-limiting example of implementation of the manipulation method during different stages of a purification treatment of a DNA sample is now described.

The experiment proposes a comparison of results of a "manual" extraction and an "automated" extraction, i.e. using the manipulation device, of Listeria monocytogenes DNA from a culture in the presence or absence of an agri-food matrix. Both types of extraction are carried out using the same extraction kit and on the same sample.

Protocol followed

Culture of Listeria monocytogenes

Strain used: Listeria monocytogenes American Type Culture Collection (ATCC) 13932

Off-matrix culture: Deposit of a cryobead in 10ml of Buffered Peptone Water (BPE)

Culture with matrix: Deposit of a cryobead in 9ml of EPT + 1ml of semi-skimmed milk Incubation at 37°C (±2°C) for 18 hours.

Sample lysis (release of genetic material)

1- Taking 200pl of sample using a single-channel pipette

2- Deposit in 200pl of lysis buffer. Vortex

3- Incubate for 5 minutes at 95°C. Allow to cool to room temperature.

Manual extraction:

1 - Deposit of 400pl of lysed sample in the binding buffer mixture + magnetic capture elements 2- Homogenize the capture elements in the sample by suction-discharge (10x)

3- Incubate for 1 minute at room temperature.

4- Place the aggregation organ + interface sheath in the capture elements. Leave to incubate until the capture elements are captured (approximately 1 min)

5- Transfer the capture elements into the first wash tube (wash buffer no. 1).

6- Remove the aggregation organ from the interface sheath and homogenize the capture elements in wash buffer 1.

7- Replace the aggregation organ in the interface sheath until all the capture elements are captured (approximately 1 min).

8- Let the capture elements dry at room temperature for 5 minutes.

9- Transfer the capture elements into the elution tube.

10- Remove the aggregation organ from the interface sheath and homogenize the capture elements in the elution buffer.

11- Incubate for 1 minute at room temperature.

12- Replace the aggregation organ in the interface sheath until all the capture elements are captured (approximately 1 min).

13- Remove the aggregation organ from the interface sheath of the elution tube. The DNA is in the elution tube.

Duration of the manipulation = 15 min

Operator handling time = 10 min

Automated extraction:

See description below.

Duration of the manipulation = 8 min

Operator handling time = 2 min Amplification of extracted DNA samples:

The extracted DNA samples are amplified by a qPCR method with DNA intercalators and specific for Listeria monocytogenes. The qPCR analysis is carried out on a CFX 96 thermocycler.

In order to be able to quantify the number of genome units present in each reaction, a specific Listeria monocytogenes quantification range for amplification is launched at the same time as the samples.

A negative control (sample free of any trace of Listeria monocytogenes DNA - here Molecular Biology quality water) is also analyzed.

The amplification conditions are as follows:

Initial denaturation: 95°C 2 min

Cycling: 40x [95°C-5s / 60°C-10s / 72°C-20] + FAM reading

Melt: ramping of 0.3°C ranging from 75°C to 95°C

A sample is considered positive if, following amplification, it shows a sigmoidal curve crossing the positivity threshold (threhsold) defined for this analysis. Positive curves are confirmed by the melting value of the samples.

Compliance criteria:

The qPCR is considered compliant if:

The first range point has a CT (Cycle Threshold = Value of the crossing of the amplification curve and the positivity threshold) equal to the reference value ±1.

The amplification range used has: o A slope between -2.8 and -4.1 o An r ² > 0.95 o Each point in the range has a melt value equal to the reference value ±2.

The negative control shows a negative result: o No amplification curve o Presence of an amplification curve but melt value outside the acceptable range.

The measured accuracy of this qPCR analysis is 30%. The two methods will therefore be considered as giving equivalent results if the genomic unit values calculated following amplification of one compared to the other are within a range of 70% to 130%.

Description of the automated extraction method:

This analysis is a 4-step DNA sample purification analysis:

- binding in English,

- washing or washing in English,

- drying or drying in English

- elution or elution in English.

For this analysis, the barrel contains 8 tubes distributed as follows:

Tube No. 1: Binding buffer + magnetic beads (600pl)

Tube No. 2: Wash Buffer 1 (1000pl)

Tube No. 3: empty

Tube No. 4: empty

Tube No. 5: empty

Tube No. 6: empty

Tube No. 7: Wash Buffer 4 (1000pl)

Tube No. 8: Elution buffer (400pl)

Sample preparation:

Taking 200p I of sample with a single-channel pipette

Deposit in 200p I of lysis buffer. Vortex

Incubate for 5 minutes at 95°C. Allow to cool to room temperature.

Preparation of the handling device:

Placing the interface sheath in the ring of the holding device Deposit 400pl of extracted sample into reception space no. 1 of the barrel containing the binding buffer

Installing the barrel on the frame

Turning on the manipulation device

Launch of the purification sequence

DNA purification:

Initialization of the sequence:

The initial position of barrel 10 presents tube No. 3 under the aggregation member 15.

During initialization, barrel 10 rotates to position tube No. 1 under aggregation member 15.

Binding step (duration 90s)

In a preliminary phase, the interface sheath 20 can be outside the tube No. 1 (interface sheath carriage 30 in the high position) and the aggregation member 15 can be outside the interface sheath 20 (aggregation member carriage 40 in the disassembled position). The interface sheath 20 can descend alone into the tube No. 1 (interface sheath carriage 30 in the low position and aggregation member carriage 40 in the disassembled position) then make back and forth movements along the tube axis T to homogenize the solution.

In Figure 16, the interface sheath 20 is outside the tube No. 1 (interface sheath carriage 30 in the high position) and the aggregation member 15 is outside the interface sheath 20 (aggregation member carriage 40 in the disassembled position) (a). The aggregation member 15 enters the interface sheath 20 (aggregation member carriage 40 in the assembled position) (b). The interface sheath 20 descends into tube No. 1 with the aggregation member 15 inside (interface sheath carriage 30 in the lower position and aggregation member carriage 40 in the assembled position) until the interface sheath 20 is near the bottom 3a of the tube 1 with the aggregation member 15 in the vicinity of its free end 20b (c). In this position, the aggregation member 15 can exert the magnetic attraction force to aggregate the capture elements 2 to which the DNA sample 1 is bound on the interface sheath 20. The interface sheath 20 rises to exit the tube 1 with the aggregation member 15 in the interface sheath 20 ...). interface 30 in the high position and aggregation organ carriage 40 in the assembled position) so that the capture elements 2 and the DNA sample 1 are extracted from the solution of tube No. 1 (d).

Transfer of capture elements

The barrel 10 rotates to position the tube No. 2 under the interface sheath 20 and the aggregation member 15.

Washing step (duration 90s)

In Figures 17a and 17b, the aggregating member 15 is in the interface sheath 20 (aggregating member carriage 40 in the assembled position) with the interface sheath 20 outside the tube No. 2 (interface sheath carriage 30 in the upper position) (a). The capture elements 2 and the DNA sample 1 are aggregated on the interface sheath 20. The interface sheath 20 descends into the tube No. 2 (interface sheath carriage 30 in the lower position) with the aggregating member 15 inside (aggregating member carriage 40 in the assembled position) and then the aggregating member 15 comes out of the interface sheath 20 (aggregating member carriage 40 in the disassembled position) (b). The biasing device 45 is activated so that the cam 46 periodically comes into contact with the access end 20a of the interface sheath 20 (biased state) and thus produces shocks generating vibrations which propagate to the free end 20b of the interface sheath 20 (c and d). The capture elements 2 detach from the interface sheath 20 which can make back and forth movements along the tube axis T. The interface sheath 20 ends its movement outside the tube No. 2 (interface sheath carriage 30 in the high position) (e). The aggregation member 15 is inserted into the interface sheath 20 (aggregation member carriage 40 in the assembled position) (f) then the interface sheath 20 and the aggregation member 15 descend into the tube No. 2 (interface sheath carriage 30 in the lower position and aggregation member carriage 40 in the assembled position) (g). The interface sheath 20 and the aggregation member 15 are in the active state. The interface sheath 20 and the aggregation member 15 exit the tube 2 with the capture elements 2 and the DNA sample 1. Transfer of capture elements

Barrel 10 rotates to position tube No. 5 under the interface sheath and the aggregation member.

Drying stage (duration 120s)

In Figure 18, the aggregation member 15 is in the interface sheath 20 (aggregation member carriage 40 in the assembled position) with the interface sheath 20 outside the tube No. 5 (interface sheath carriage 30 in the upper position) (a). The capture elements 2 and the DNA sample 1 are aggregated on the interface sheath 20. The interface sheath 20 descends into the empty tube No. 5 (interface sheath carriage 30 in the lower position) with the aggregation member 15 inside (aggregation member carriage 40 in the assembled position) (b). The cam 46 of the biasing system 45 passes from the stop state to the biasing state to move the interface sheath 20 and the aggregation member 15 into the empty tube No. 5 (c). The interface sheath 20 rises to exit the tube No. 5 with the aggregation member 15 in the interface sheath 20 (interface sheath carriage 30 in the high position and aggregation member carriage 40 in the assembled position).

Transfer of capture elements

The barrel rotates to position tube No. 8 under the interface sheath 20 and the aggregation member 15.

Elution step (duration 90s)

In Figures 19a and 19b, the aggregating member 15 is in the interface sheath 20 (aggregating member carriage 40 in the assembled position) with the interface sheath 20 outside the tube No. 8 (interface sheath carriage 30 in the upper position) (a). The capture elements 2 and the DNA sample 1 are aggregated on the interface sheath 20. The interface sheath 20 descends into the tube No. 8 (interface sheath carriage 30 in the lower position) with the aggregating member 15 inside (aggregating member carriage 40 in the assembled position) and then the aggregating member 15 comes out of the interface sheath 20 (aggregating member carriage 40 in the disassembled position) (b). The biasing device 45 is activated so that the cam 46 periodically comes into contact with the access end 20a of the interface sheath 20 (stress state) and thus produce shocks generating vibrations which propagate to the free end 20b) of the interface sheath 20 (c and d). The capture elements 2 detach from the interface sheath 20 which can make back and forth movements along the tube axis T. The interface sheath 20 ends its movement at the bottom 3a of the tube No. 8 (interface sheath carriage 30 in the low position) and remains in position for a determined duration (e). The aggregation member 15 is inserted into the interface sheath 20 (aggregation member carriage 40 in the assembled position) and maintained in this position for a determined duration (f). The DNA sample 1 is detached from the capture elements 2. The interface sheath 20 is extracted from the tube No. 8 with the aggregation member 15 inside (interface sheath carriage 30 in the high position and aggregation member carriage 40 in the assembled position), so as to extract the capture elements 2 while leaving the DNA sample 1 in the tube No. 8 (g).

Transfer of capture elements

The barrel rotates to position tube No. 7 under the interface sheath 20 and the aggregation member 15.

System cleaning step (duration 60s)

In Figure 20, the aggregating member 15 is in the interface sheath 20 (aggregating member carriage 40 in the assembled position) with the interface sheath 20 outside the tube No. 7 (interface sheath carriage 30 in the upper position) (a). The capture elements 2 are aggregated on the interface sheath 20. The interface sheath 20 descends into the tube No. 7 (interface sheath carriage 30 in the lower position) with the aggregating member 15 inside (aggregating member carriage 40 in the assembled position) then the aggregating member 15 comes out of the interface sheath 20 (aggregating member carriage 40 in the disassembled position) (b). The biasing device 45 is activated so that the cam 46 periodically comes into contact with the access end 20a of the interface sheath 20 (biased state) and thus produces shocks generating vibrations which propagate to the free end 20b of the interface sheath 20 (c and d). The capture elements 2 detach from the interface sheath 20. The interface sheath 20 rises to exit the tube No. 7 (interface sheath carriage 30 in the high position) (e) Return to the initial position:

The barrel 10 rotates to position the tube No. 3 under the interface sheath 20 and the aggregation member 15.

Following the end of purification: The operator directly takes the volume required for analysis (here 5pl) from tube 8. He can then take the entire elution volume and place it in a suitable tube to store this extracted sample.

Results obtained:

Validation of qPCR conformity Results obtained with the DNA quantification range:

Conclusion: As the qPCR range shows consistent results, the samples can be analyzed. Sample results:

Conclusion: The presence of Listeria monocytogenes was detected in all samples. The quantification results obtained show a deviation within the tolerable range of accuracy. General conclusion:

Automated extraction using the sample preparer allows for similar results to be obtained with manual extraction. However, automated extraction has two advantages: significantly reduced operator handling time and difficulty, and significantly reduced overall analysis time.

Claims

1. Apparatus (5) for handling a sample of biological material (1) by means of capture elements (2), the capture elements (2) being magnetic and configured to be secured to the sample of biological material, the handling apparatus (5) comprising: - a frame (6) extending along a vertical axis (A), - a tube holder (8) mounted on the frame (6) and comprising at least one receiving space (9) configured to receive at least one tube (3) adapted to contain a solution comprising the sample of biological material (1) and the capture elements (2); - an aggregation member (15) configured to generate a magnetic attraction force on the capture elements (2), the aggregation member (15) being intended to cooperate with an interface sheath (20) in such a way that, in an active state of the aggregation member (15) and the interface sheath (20), the aggregation member (15) is received in the interface sheath (20) and exerts the magnetic attraction force on the capture elements (2) to aggregate said capture elements (2) on the interface sheath (20) and, in an inactive state of the aggregation member (15) and the interface sheath (20), the aggregation member (15) does not exert a magnetic attraction force on the capture elements (2), the interface sheath (20) having an access end (20a) through which the aggregation member (15) is inserted into the interface sheath (20), and a free end (20b) opposite the access end (20a) and in the vicinity of which the capture elements (2) are aggregated in the active state of the aggregation member (15) and of the interface sheath (15), - an interface sheath carriage (30) comprising a holding device (24) configured to hold the interface sheath (20) opposite the receiving space (9), the interface sheath carriage (30) being movable along the vertical axis (A) relative to the frame (6) between a high position in which said interface sheath carriage (30) places the interface sheath (20) at a distance from the receiving space (9), and a low position in which said interface sheath carriage (30) places the interface sheath (20) in the receiving space (9), - a biasing system (45) configured to be placed in contact with the interface sheath (20) and generate in the interface sheath (20) vibrations adapted to separate the capture elements (2) from the interface sheath (20) after the aggregation member (15) and the interface sheath (20) have passed from the active state to the inactive state, the handling apparatus (5) being characterized in that the biasing system (45) is mounted on the interface sheath carriage (30) and is arranged to generate the vibrations in the vicinity of the access end (20a), and in that the holding device (24) is configured to allow propagation of the vibrations from the access end (20a) to the free end (20b) of the interface sheath (20). 2. Handling apparatus (5) according to claim 1, wherein the holding device (24) has a holding surface configured to be in contact with the interface sheath (20) at a distance (D) from the access end (20a) along the vertical axis (A), such that the interface sheath (20) has a proximal sheath portion between the access end (20a) over the distance (D), the proximal sheath portion being projecting relative to the holding surface, and wherein the biasing system (45) is spaced from the holding surface along the vertical axis (A) in a direction opposite to the tube support, so as to come into contact with the proximal sheath portion. 3. Handling apparatus (5) according to any one of claims 1 and 2, in which the holding device (24) comprises a ring (25) along a holding axis (M) parallel to the vertical axis (A) and having the holding surface configured to be in peripheral contact with the interface sheath (20). 4. Handling apparatus (5) according to claim 3, wherein the ring (25) is configured to receive the interface sheath (20) comprising a side wall along a sheath axis (G) and at least two lugs (22) projecting transversely in an equally distributed manner from an outer surface of the side wall, the ring (25) comprising a groove (26) provided on the holding surface around the holding axis (M) and configured to receive the lugs (22), and at least two grooves (27) extending axially on the holding surface from a passage end to the groove (26), so as to allow passage of the lugs (22) of the interface sheath (20) in an angular position of the interface sheath (20) relative to the ring (25) and holding of the lugs (22) of the interface sheath (20) in the groove (26) in other angular positions of the interface sheath (20) relative to the ring (25). 5. Handling apparatus (5) according to any one of claims 3 and 4, in which the holding device (24) further comprises a flange (28) movable relative to the interface sheath carriage (30) between an open position allowing the ring (25) to be placed in position and a closed position ensuring that the ring (25) is held in position relative to the interface sheath carriage (30). 6. Handling apparatus (5) according to any one of claims 1 to 5, in which the stressing system (45) is configured to carry out at least one impact on the interface sheath (20). 7. Handling apparatus (5) according to any one of claims 1 to 6, in which the stressing system (45) comprises a cam (46) driven in rotation about a pivot axis (P) and comprising a radial contact portion (47) arranged to come into contact with the interface sheath (20) in an angular position range of the cam (46) relative to the frame (6), the cam (46) being at a distance from the interface sheath (20) outside the angular position range. 8. A handling apparatus (5) according to claim 7, wherein the cam (46) has a biasing state in which said cam (46) is rotated and a stopping state in which said cam (46) is fixed relative to the frame (6), and wherein the biasing system (45) comprises a detection device (50) configured to stop the cam (46) outside the angular position range when the cam (46) passes from the biasing state to the stopping state. 9. A handling apparatus (5) according to any one of claims 1 to 8, further comprising an aggregation member carriage (40) carrying the member aggregation (15), the aggregation member carriage (40) being movable along the vertical axis (A) relative to the frame (6) between a disassembled position at a distance from the interface sheath carriage (30) such that the aggregation member (15) is spaced from the interface sheath (20), and an assembled position near the interface sheath carriage (30) such that the aggregation member (15) is received in the interface sheath (20). 10. A handling apparatus (5) according to claim 9, wherein the aggregating member (15) comprises a permanent magnet (17) at a distal end intended to be received in the interface sheath (20), the aggregating member (15) and the interface sheath (20) being in the inactive state when the aggregating member carriage (40) is in the disassembled state. 11. Handling apparatus (5) according to any one of claims 1 to 10, in which the tube support (8) comprises a barrel (10) comprising a plurality of receiving spaces (9) around a barrel axis (B), the barrel (10) being movable in rotation along the barrel axis (B) so as to selectively place one of the receiving spaces (9) opposite the interface sheath (20). 12. Handling apparatus (5) according to any one of claims 1 to 11, in which the holding device (24) is configured to hold a single interface sheath (20), the stressing system (45) being arranged to generate the vibrations in the vicinity of the access end (20a) of the single interface sheath (20). 13. Handling assembly comprising a handling apparatus (5) according to any one of claims 1 to 12 and at least one interface sheath (20) configured to receive the aggregation member (15) and to be held in the holding device (24) of the interface sheath carriage (30). 14. Method for handling a sample of biological material (1), the handling method implementing a handling assembly according to claim 13 and comprising steps of: - placing in the receiving space (9) of the tube support (8) a tube (3) containing a solution comprising the sample of biological material (1) and capture elements (2), the capture elements (2) being magnetic and configured to be secured to the sample of biological material (1), - placing the interface sheath (20) in the holding device (24) of the interface sheath carriage (30) in the high position, - putting the interface sheath carriage (30) in the low position and the aggregation member (15) and the interface sheath (20) in the active state, the aggregation member (15) being received in the interface sheath (20) and exerting the magnetic attraction force on the capture elements (2) to aggregate said capture elements (2) on the interface sheath (20), - place the interface sheath carriage (30) in the high position, the aggregation member (15) and the interface sheath (20) remaining in the active state, - place the interface sheath carriage (30) in the low position, the aggregation member (15) and the interface sheath (20) remaining in the active state, - putting the aggregation member (15) and the interface sheath (20) in the inactive state, - activate the stress system (45) to generate vibrations in the interface sheath (20) suitable for separating the capture elements (2) from the interface sheath (20).