Device used for the filtration and resuspension of biological material contained in a liquid sample

Abstract

The invention relates to a device used for the filtration and resuspension of biological material contained in a liquid sample, said device comprising: A housing made of at least a first part and a second part assembled in a hermetic manner, said housing defining an internal volume; A filter (50) arranged in said internal volume, said filter having two opposing faces; Said housing having a first fluidic connection (C1) and a second fluidic connection (C2) positioned on either side of the filter and transversely with respect to the two opposing faces of said filter; The first part and / or the second part of the housing having a first cavity (104); The device comprising a first ultrasonic source having a first emitting head (31) housed in said first cavity (104). Figure to be published with the abbreviation: Figure 1 A

Description

Title of the invention: Device used for the filtration and resuspension of biological material contained in a liquid sample. Technical field of the invention

[0001] The invention relates to a device used for the filtration and resuspension of biological material contained in a biological sample. State of the art

[0002] In the context of environmental analyses for the search for biological contaminants (for example the search for Escherichia Coli bacteria or Enterococcus bacteria for the European standard for bathing water or for the protection of oyster farming), the targets to be sought are present in low concentrations within the samples to be analyzed.

[0003] At present, laboratory analysis techniques, from a sample taken in the field, are based on the amplification of the number of targets, either by incubation for 24h at 37°C (amplification by bacterial growth), or by multiplication of the genetic material of the target via a PCR (“Polymerase Chain Reaction”) type protocol.

[0004] At the end of the amplification, the targets are sufficiently enriched to be detected and identified by laboratory equipment. None of the known techniques allow for a response time of less than 6 hours for bacterial concentrations, between the time of sample collection and the time of the laboratory result. This processing time constraint prevents rapid decision-making in the event of contamination.

[0005] Conventional techniques, known as pre-concentration techniques, make it possible to artificially increase the concentration of bacterial targets.

[0006] These techniques consist of taking a large volume (1 to 10 liters, for example) of the sample to be analyzed, this sample containing N target bacteria. This sample is filtered through a substrate in order to isolate the bacteria.

[0007] Next, a resuspension protocol is applied to these bacteria using a smaller volume resuspension solution. At the end of the process, a sample is obtained with a higher concentration of bacterial targets than that of the initial sample taken.

[0008] The known techniques for carrying out filtration and resuspension have certain drawbacks: - They often require manipulation of the filter, to switch from the filtration stage to the resuspension stage. - Bacteria are often attached to the surface of the filter, also requiring manual operations to remove them effectively.

[0009] However, these manipulations can cause loss of biological material, its degradation and contamination.

[0010] There is therefore a need for a reliable, easy-to-use and suitable device to perform filtration of a liquid sample, possibly of a large volume, and to resuspend filtered biological material, while limiting the operator's handling as much as possible. Description of the invention

[0011] This objective is achieved by a device used for the filtration and resuspension of biological material contained in a liquid sample, said device comprising: - A casing made of at least a first part and a second part assembled in a watertight manner one on top of the other, said casing delimiting an internal volume, - A filter arranged within said internal volume, said filter comprising two opposing faces, - Said housing comprising a first fluidic connection and a second fluidic connection positioned on either side of the filter and transversely with respect to the two opposite faces of said filter, said first fluidic connection being intended to receive the fluidic sample and the second fluidic connection being intended to receive the fluidic sample, after filtration by the filter, - The first part and / or the second part of the housing comprising a first cavity, - The device comprising a first ultrasonic source equipped with a first emitting head which fits into said first cavity.

[0012] According to one particular feature, the first part is in the form of a first flange and the second part is in the form of a second flange, the first flange and the second flange being assembled coaxially on top of each other.

[0013] According to another feature, the first flange and the second flange are of identical shapes.

[0014] According to another feature, said first cavity is formed on the first flange and the device comprises a second cavity formed on the second flange, said second cavity being adapted to house said first head of the first ultrasonic source or a second head of a second ultrasonic source of the device.

[0015] According to another feature, the device includes a main sealing gasket positioned between the two flanges when they are assembled one on top of the other.

[0016] According to another feature, the device comprises a first grid and a second grid positioned on either side of the filter.

[0017] According to another feature, the device includes a first sealing gasket positioned between said first grid and said first flange.

[0018] According to another feature, the device includes a second sealing gasket positioned between said second grid and said second flange.

[0019] The invention also relates to a fluidic system for processing a liquid sample containing biological material, comprising a fluidic circuit in which are placed: - A first reservoir containing the said liquid sample, - A second reservoir containing a resuspension solution, - A filtration device for said liquid sample to filter said biological material and for resuspension of said biological material after filtration. - Controlled pumping means for pumping said liquid sample present in the first reservoir through said filtration and resuspension device and for pumping said resuspension solution through said filtration and resuspension device, - The device includes a control unit configured to control the pumping means according to a predetermined liquid sample processing sequence, - The filtration and resuspension device being as defined above.

[0020] According to a particular feature, the fluidic circuit includes a filtrate recovery unit.

[0021] According to another feature, the fluidic circuit includes a retentate recovery unit. Brief description of the figures

[0022] Other features and advantages will become apparent in the detailed description that follows, given in relation to the accompanying drawings in which: - Figures IA and IB show the device of the invention, seen in exploded view according to two distinct orientations; - Fig. 2 schematically shows a fluidic system in which the device of the invention is inserted and illustrates its operating principle;

[0023] Detailed description of at least one embodiment

[0024] For the remainder of the description, an orthonormal coordinate system (X, Y, Z) is defined.

[0025] Terms such as "superior", "inferior", "above" and "below" are to be understood taking into account the Z direction which is chosen to be vertical.

[0026] The invention relates to a device used for the filtration of biological material and for the resuspension of this biological material, once it has been retained on the filter of the device.

[0027] The device is inserted into a fluidic system ([Fig.2]) intended to treat said liquid sample in order to filter it, to isolate the biological material, and to allow the resuspension of this biological material after filtration in order to recover it.

[0028] With reference to [Fig. 1A] and [Fig. 1B], the device comprises a body in two parts that are assembled one on top of the other. The first part consists of a first flange 10 and the second part consists of a second flange 20.

[0029] The two flanges 10, 20 have a general disc shape. Around its axis (along Z), each flange 10, 20 has an internal annular surface by which it is held against the other flange. The two flanges 10, 20 are thus assembled coaxially against each other. The assembly of the two flanges is, for example, achieved using screws 30.

[0030] The device comprises an internal volume delimited by the two flanges. Each flange thus comprises an internal face 100, 200 oriented towards the inside and an external face 101, 201 oriented towards the outside.

[0031] Between the two flanges, the device includes a main annular sealing gasket 40, designed to ensure a watertight assembly of the two flanges against each other. The gasket 40 is positioned at the interface between the two internal annular surfaces of the two flanges 10, 20.

[0032] Within its internal volume, the device comprises the filter 50. The filter is in the form of a part with a small thickness (along Z) relative to its shape in the other two dimensions (along X, Y). The filter advantageously has a disc shape, having two opposing faces 500, 501. The two opposing faces of the filter 50 are positioned transversely with respect to the axis (along Z) of the device and thus interpose themselves in the fluidic flow. The filter 50 has pores capable of capturing biological material against one of its faces when the sample is injected through its thickness.

[0033] The device also includes two retaining grids 60, 70 positioned on either side of the filter 50, against its two opposing faces 500, 501. These two grids allow a fluidic flow to pass through.

[0034] To ensure a leak-proof fluid passage through the filter 50, the device also includes two sealing gaskets 80, 90, a first gasket 80 being positioned between the first grid 60 and the first flange 10, and a second seal 90 being positioned between the second grid 70 and the second flange 20. These two seals are annular in shape.

[0035] Each joint 80, 90 rests on one side against an annular bearing surface 102, 202 made on the inner face of the flange and on the other side against the grid 60, 70 located opposite.

[0036] When the two flanges 10, 20 are assembled one on top of the other using the screws 30, the filter 50 is held in place by compression of the various seals.

[0037] According to a particular aspect of the invention, the device comprises two fluid connections. The first fluid connection C1 is made on the body of the first flange 10, and the second fluid connection C2 is made on the body of the second flange 20. The two fluid connections are advantageously identical and each comprises a fitting 103, 203 for connecting a conduit of the fluid system. Each fitting 103, 203 is, for example, arranged along the axis of the device on its external face.

[0038] A first fluidic flow can be created from the first fluidic connection Cl to the second fluidic connection C2, passing through the filter 50 in the first direction (flow), and a second fluidic flow can be created from the second fluidic connection C2 to the first fluidic connection Cl of the device, passing through the filter 50 in the opposite direction (reflux). In the flow direction, for example, biological material is trapped, and in the reflux direction, the biological material is detached for recovery.

[0039] According to another aspect of the invention, the device also includes at least one ultrasonic source. This ultrasonic source has an ultrasonic emitting head 31 that connects to the body of the device to cause it to vibrate sufficiently to contribute to the detachment of the biological material trapped by the filter 50. The ultrasonic treatment is directed towards the surface of the filter to aid in the detachment of the biological material during resuspension.

[0040] Advantageously, the first flange 10 has a first cavity 104 made on its external face, said first cavity 104 being arranged to accommodate said emitting head 31 of the ultrasonic source.

[0041] Advantageously, the second flange 20 can also include a second cavity (not shown in the figures), identical to the first cavity, also arranged to accommodate the emitting head 31 of the ultrasonic source or an emitting head of a second ultrasonic source.

[0042] According to a particular aspect, the assembly consisting of the device housing and the filtering elements housed in the housing can be chosen to be symmetrical with respect to a plane (along X, Y) passing through filter 50. Thanks to this symmetry, the device could be used indifferently in both directions.

[0043] Furthermore, it should be noted that: - The dimensions of the internal volume are optimized to reduce dead volumes and minimize the dilution of biological material captured by the filter during resuspension; - The internal volume architecture allows for optimal flow of the sample / resuspension solution, without loss and in a homogeneous manner over the filter.

[0044] With reference to [Fig.2], the device of the invention is intended to be inserted into the fluidic circuit of a fluidic system.

[0045] Without limitation, the fluidic system and its operating principle are described below.

[0046] A first RI reservoir containing the liquid sample, the liquid sample itself containing the target biological material. This first RI reservoir is connected via a first fluidic connection to the first fluidic connection Cl of the device, through a first valve VL

[0047] A second reservoir R2 containing a resuspension solution is connected via a second fluidic link to the second fluidic connection C2 of the device, through a second fluidic valve V2.

[0048] A filtrate recovery unit U1 is connected to the second fluidic connection C2, via the second fluidic valve V2.

[0049] The second fluidic valve V2 is controlled to connect the second fluidic connection C2 to the second tank R2 or to the filtrate recovery unit U1.

[0050] A retentate recovery unit U2 is connected to the first fluidic connection Cl, via the first fluidic valve VL

[0051] The first fluidic valve V1 is controlled to connect the first connection fluidic Cl to the first RI tank or to the U2 retentate recovery unit.

[0052] The system includes pumping means PI, P2 arranged in its fluidic circuit. The pumping means are controlled to: - Allow the liquid sample to be injected into the device and passed through filter 50 to the filtrate recovery unit U1. - Allow the injection of the resuspension solution into the device and pass it through the filter (in reflux) to the U2 retentate recovery unit.

[0053] The pumping means may include, for example, two peristaltic pumps PI, P2, a first pump PI positioned on the fluidic circuit between the first reservoir RI and the first fluidic valve VI, a second pump P2 positioned on the fluidic circuit between the second reservoir R2 and the second fluidic valve V2. Any other pumping system could be provided.

[0054] The system advantageously includes a control unit adapted to execute a sequence of system operation according to the principles described below.

[0055] The operating principle of the system is thus as follows: - El: The control unit controls the first fluidic valve VI in order to connect the first reservoir RI containing the fluidic sample to the first fluidic connection Cl of the device. - The control unit controls the second fluidic valve V2 in order to connect the second fluidic connection C2 of the device to the filtrate recovery unit U1. - The control unit controls the first PI pump in order to inject the fluidic sample into the device. - E2: The fluidic sample passes through filter 50 to reach the filtrate recovery unit U1. - The targeted biological material, present in the liquid sample, remains trapped by the filter 50 of the device. - The control unit controls the first fluidic valve V1 to connect the first fluidic connection Cl on the retentate recovery unit U2. - The control unit controls the second fluidic valve V2 to connect the second fluidic connection C2 to the second tank R2 containing the resuspension solution. - E3: The control unit controls the second pump P2 in order to inject the resuspension solution into the device, in reflux, through the filter 50 of the device, so as to detach the previously trapped biological material. - According to the invention, the control unit advantageously controls at least one ultrasonic source of the device to assist in the detachment of biological material from the filter. - E4: The detached biological material is eluted by the resuspension solution towards the U2 retentate recovery unit.

[0056] The solution of the invention has many advantages. In particular, it allows for improved resuspension of biological material, thanks to the use of the ultrasonic source, its head connecting directly to the body of the device.

[0057] The invention allows the recovery, in a small volume, of microorganisms or any other particles retained on the surface of a filter. Filtration / concentration being a very widespread method of sample preparation for microbiological analysis.

Claims

Demands

1. A device used for the filtration and resuspension of biological material contained in a liquid sample, said device comprising: - A housing made of at least a first part and a second part assembled in a hermetically sealed manner one on top of the other, said housing delimiting an internal volume, - A filter (50) arranged in said internal volume, said filter having two opposite faces, - Said housing having a first fluidic connection (Cl) and a second fluidic connection (C2) positioned on either side of the filter and transversely with respect to the two opposite faces of said filter, said first fluidic connection (Cl) being intended to receive the fluidic sample and the second fluidic connection being intended to receive the fluidic sample, after filtration by the filter (50),- Characterized in that: - The first part and / or the second part of the housing comprises a first cavity (104), - The device comprises a first ultrasonic source having a first emitting head (31) which fits into said first cavity (104).

2. Device according to claim 1, characterized in that the first part is in the form of a first flange (10) and in that the second part is in the form of a second flange (20), the first flange (10) and the second flange (20) being assembled coaxially on top of each other.

3. Device according to claim 2, characterized in that the first flange and the second flange are of identical shapes.

4. Device according to claim 2 or 3, characterized in that said first cavity (104) is made on the first flange and in that the device comprises a second cavity made on the second flange, said second cavity being adapted to house said first head of the first ultrasonic source or a second head of a second ultrasonic source of the device.

5. Device according to any one of claims 2 to 4, characterized in that it comprises a main sealing gasket (40) positioned between the two flanges when they are assembled one on top of the other.

6. Device according to any one of claims 2 to 5, characterized in that it comprises a first grid (60) and a second grid (70) positioned on either side of the filter (50).

7. Device according to claim 6, characterized in that it comprises a first sealing gasket (80) positioned between said first grid and said first flange.

8. Device according to claim 7, characterized in that it comprises a second sealing gasket (90) positioned between said second grid and said second flange.

9. A fluidic system for processing a liquid sample containing biological material, comprising a fluidic circuit in which are placed: - A first reservoir (RI) containing said liquid sample, - A second reservoir (R2) containing a resuspension solution, - A filtration device for said liquid sample to filter said biological material and for resuspension of said biological material after filtration, - Controlled pumping means (PI, P2) for pumping said liquid sample present in the first reservoir (RI) through said filtration and resuspension device and for pumping said resuspension solution through said filtration and resuspension device, - The device comprising a control unit configured to control the pumping means according to a determined liquid sample processing sequence,- Characterized in that the filtration and resuspension device is as defined in any one of claims 1 to 8.

10. System according to claim 9, characterized in that the fluidic circuit comprises a filtrate recovery unit (Ul).

11. System according to claim 9 or 10, characterized in that the fluidic circuit comprises a retentate recovery unit (U2).