WO2024126449A1

WO2024126449A1 - Device and method for sample concentration

Info

Publication number: WO2024126449A1
Application number: PCT/EP2023/085274
Authority: WO
Inventors: Frederic Olivieri; Francois OBLINGER; Fabrice COMINI
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2022-12-15
Filing date: 2023-12-12
Publication date: 2024-06-20
Anticipated expiration: 2025-06-15
Also published as: EP4633806A1

Abstract

The present invention concerns a device and a method for sample preparation, more specifically for sample concentration, in particular for microorganism sample concentration for microbial testing purposes.

Description

DEVICE AND METHOD FOR SAMPLE CONCENTRATION

Technical Field

Background

For pharmaceutical and food microbial testing, the microorganism concentration in a sample to be tested may be very small. In this case a relatively large sample volume is required to ensure such sample to be representative of the total sample to be tested. Therefore, typical sample volumes may range from about 20 ml to about 100 ml. State of the art detection techniques and processes are based on membrane filtration and subsequent incubation on a culture medium, but this process is slow.

As most rapid detection processes require less than 1 ml of liquid sample, a sample concentration must be performed in order to be able to use a rapid detection process.

Sample concentration techniques are available and comprise the following in particular: Centrifugation: concentration of a microorganism in a sample is effected solely by high centrifugal forces. However, this technique will result in a low viability of the microorganisms collected because of the high stress induced. Dilution and vortexing: a membrane filter is placed on a tube filled with a diluent, and a vortex is created in the diluent to remove microorganisms adhering to a surface of the membrane filter and to suspend them in the solution. This method does not have a good recovery rate and most microorganisms stay on the membrane. Microfluidic devices: there are many microfluidic techniques available to separate and concentrate a sample. However, even if some of them are parallelizable, their flow rate is very low and is not compatible with the processing of tens of millilitres in an acceptable time. Affinity: microorganisms are captured by chemical affinity using chemical bonding of functionalized material on the microorganism surface (for example antibodies and magnet beads). The drawback of this process is that it is not ensured to be fully universal, the bonding may not work for all species of microorganisms.

The present invention aims at providing a device and a method for sample preparation, more specifically sample concentration, in particular for microorganism sample concentration for microbial testing purposes capable of obtaining a sample volume with a microorganism concentration that is compatible to be processed with rapid detection processes.

Summary

To solve that problem, the present invention provides a device for sample concentration as defined by claim 1 and a method for sample concentration as defined by claim 11. Preferred embodiments of the device and the method are defined in the dependent claims.

The present invention in particular provides a device for sample concentration comprising: a filter head including a filter holder configured to hold, preferably removably, a membrane filter and allow a volume of a sample fluid to be guided through a first flow path passing the membrane filter, when the same is held on the filter holder, towards a filtrate outlet; and at least one collection container holder provided on the filter head and configured to removably hold a collection container, wherein the filter head, together with the collection container, when the same is held at the at least one collection container holder, is rotatable about a rotation axis crossing, preferably essentially perpendicularly, a plane defined by a surface of the membrane filter, when the same is held on the filter holder, and wherein the at least one collection container holder is provided on the filter head such that retentate collected on the membrane filter is directed into the collection container, when the same is held at the collection container holder, through a second flow path by a centrifugal force created upon spinning the filter head about the rotation axis.

The present invention in particular also provides a method for sample concentration, preferably using the device for sample concentration according to the invention, the method comprising: filtering a volume of a sample fluid through a membrane filter held on a filter holder of a filter head and removing the filtrate; spinning the filter head with the membrane filter held on the filter holder about a rotation axis crossing, preferably crossing essentially perpendicularly, a plane defined by a surface of the membrane filter such that retentate collected on the membrane filter, preferably on an upstream surface thereof, is directed into a collection container by a centrifugal force created upon rotation of the filter head.

The present invention thus provides a device and method for sample concentration that combines the processes of filtration and centrifugation. The invention thus combines the concentration efficiency of a filter for a large volume of a sample fluid with the concentration efficiency to a small volume of centrifugation but avoiding the drawbacks of processes relying on centrifugation only.

The invention provides a device and a method to concentrate and collect microorganisms present in a large volume of a sample fluid, by initially filtering the entire volume through a membrane filter to collect the microorganisms on the membrane filter, preferably on its surface. Then, the filter head with the membrane filter is centrifuged, preferably by adding some diluent or rinsing liquid to the membrane filter, if necessary, to directly collect the microorganisms on the membrane filter into the collection container, preferably a standard tube or vial.

The present invention brings about the following advantages compared to other collection and concentration techniques: The initial sample volume can be large thanks to the use of a membrane filter for the filtration/concentration step, thereby ensuring that the sample volume is representative of the sample to be tested. The final sample concentration can be efficient, down to 1 ml, preferably down to 0.5 ml. That makes the sample compatible with highly sensitive detection applications (polymerase chain reaction - PCR, microfluidic devices, etc.). The simple workflow with disposable or one-time-use standard components (i.e. the sample and collection containers) makes the sample preparation easy, safe, and cost efficient. Multiple spin cleaning can be performed to prevent membrane clogging and increase the filtration volume. Preferably, the device comprises a membrane filter, preferably one that is configured to retain microorganisms on an upstream surface thereof, preferably a track etched plastic membrane, the membrane filter arranged on the filter holder.

Preferably, the filter head is configured to removably hold a sample container formed to receive the volume of the sample fluid to be filtered, such that the sample container communicates with the first flow path upstream of the filter holder.

Preferably, the device comprises a cover configured to be removably attached in place of the sample container so as to close the first flow path on the upstream side.

Preferably, the second flow path is configured to be blocked during filtration, preferably by a/the sample container, when the same is held on the filter head, and/or by a valve device, and/or by a plug.

Preferably, the filter head includes a port for injecting a diluent towards the membrane filter, when the same is held on the filter holder, preferably towards an/the upstream surface of the membrane filter and/or a downstream surface of the membrane filter.

Preferably, the port for injecting the diluent is displaceable relative to the upstream and/or downstream surface of the membrane filter.

Preferably, the port for injecting the diluent is provided at the cover, preferably aligned with the rotation axis.

Preferably, the at least one collection container holder is configured to releasably hold the collection container in the form of a tube or vial.

Preferably, the device comprises a motor for spinning the filter head about the rotation axis and/or a vacuum pump for pulling the sample fluid through the membrane filter.

Preferably, in the method, the collection container is rotated together with the filter head about the rotation axis.

Preferably, the method further comprises injecting a diluent towards the membrane filter held on the filter holder, before and/or during the spinning of the filter head, preferably towards an/the upstream surface and/or a downstream surface of the membrane filter. The injection of the diluent from the top and/or bottom side(s) of the membrane filter improves and supports the removal of the microorganisms from the membrane surface.

Preferably, the method comprises changing an injecting position of the diluent relative to the upstream and/or downstream surface of the membrane filter held on the filter holder, preferably progressing from a more central position towards a more radial outer position of the membrane filter. The displacement of the injection position from the center to the radial outer side towards the border of the membrane filter improves the recovery rate as it pushes the microorganisms towards the collection container(s).

Though it is not particularly limited and may be adapted depending upon specific conditions and requirements, it is preferred in the present method that the volume of the sample fluid filtered through the membrane filter is 20 to 100 ml.

Brief description of the drawings

Figure 1 shows a schematic cross sectional view of an exemplary preferred embodiment of a filter head of a sample concentration device with a sample container in place.

Figure 2 shows a schematic cross sectional view of the exemplary filter head of Figure 1 with the sample container replaced by a cover.

Detailed description

Preferred exemplary embodiments of the invention will now be described with respect to the attached exemplary schematic drawing in which:

Figures 1 and 2 show the isolated filtration head of an exemplary embodiment of the device 1 for sample concentration of the invention that will be used to describe the concept of the invention without implying that the invention is limited to the specific structure of the exemplary embodiment.

While the filtration head can be used in conjunction with existing filtration and centrifugation periphery, the required functionality for filtration and centrifugation can be integrated in a self-contained functional device or apparatus. However, the filtration and centrifugation periphery are omitted for reasons of clarity. In case of a self-contained functional unit, the device may, for example, include an electrical motor for spinning the filter head about the rotation axis and, optionally, a vacuum pump for pulling the sample through the membrane filter as described later. Instead of the electrical motor, a manually operated drive mechanism including a crank handle may be provided.

The device 1 for sample concentration in the most general configuration comprises a filter head 2 including a filter holder 3 configured to removably hold a membrane filter 4 and allow a volume of a sample fluid to be guided through a first flow path A passing the membrane filter 4, when the same is held on the filter holder 3, towards a filtrate outlet 6. It is noted that, preferably, the first flow path A and the membrane filter 4, more precisely the upper (or lower) surface of the membrane filter 4, are essentially perpendicular to each other.

The device also comprises at least one collection container holder 7 that is provided at or on the filter head 2 and that is configured to removably hold a collection container 8 in a fixed position on the filter head 2. In the exemplary embodiment, two collection container holders 7 are provided at opposite sides of the periphery about a central axis X. The device may have a single collection container holder 7, in which case it is asymmetric, or three or more that are equally distributed about the periphery, in which case it is preferably rotational symmetric. It is, however, preferred that the device comprises an even number (for example, 2 or 4 or 6 or 8 etc.) of collection container holders 7 arranged in symmetry to avoid imbalance during spinning.

The structure of the collection container holder 7 is not particularly limited and is dictated by the structure of the collection container 8 it is to hold and by the functionality of being able to selectively attach and remove the collection container to the filter head. The collection container holder preferably includes means for sealing against a filling opening of the container and means for biasing or fixedly holding the container in place in a sealed state against the gravity and centrifugal forces acting on the container in operation, yet allowing simple removal.

In the exemplary embodiment the collection container holder 7 is specifically designed to releasably hold the collection container s in the form of a tube or vial 8a that is a standard consumable, preferably made from plastics material, and is available in different sizes and forms. The collection container holder 7 includes a cylindrical receptacle 7a surrounding an outlet 3a of the filter head 2 communicating with the second flow path B described later and into which the tube or vial can be inserted with its opening portion ahead, and a releasable locking member 7b that engages the outer periphery of the tube or vial to urge it into the receptacle 7a. The locking member 7b may include a biasing member like a spring or elastic member (not shown) to provide the urging force. It may also include a suitable seal between the opening of the tube or vial 8a and the outlet 3a.

The collection container holder 7 is designed to hold the collection container 8 in an outward (i.e. away from the center of filter head 2) and downward inclined orientation, for example, downwardly inclined by an angle of at least 5° and at most 45° from the plane defined by the surface of membrane filter 4. This will ensure that a defined volume of the diluent (described later) with the microorganisms is collected in the collection container 8 without flowing back into the second flow path B through the outlet 3a of the filter holder when the centrifugation stops.

The filter head 2, together with the collection container(s) 8, when the same is/are held at the one or more collection container holder(s) 7, is rotatable about a rotation axis crossing a plane defined by a surface of the membrane filter 4 and the rotation axis is preferably perpendicular to the plane. The filter head 2 is thus preferably rotational symmetric about the center axis X that is perpendicular to the plane defined by the surface of the membrane filter (except where a single collection container holder is provided) and the center axis X is preferably coaxial with the rotation axis but that is not necessarily so. The use of an even number of containers arranged in symmetry is preferred to avoid imbalance during spinning. Even if the filter head contains plural collection container holders 7, it is possible to attach a limited, again preferably even number of containers to some of the holders during operation in a symmetrical arrangement while plugging the others that are not used.

The one or more collection container holder(s) 7 is/are in any case provided on the filter head 2 such that retentate collected on the membrane filter 4 as described below is directed into the collection container(s) 8, when the same is/are held at the collection container holder(s) 7, through the second flow path B by a centrifugal force created upon spinning the filter head 2 about the rotation axis. As described above, the second flow path B communicates a space adjacent to the membrane filter 4, preferably adjacent its upstream surface, with the one or more outlet(s) 3a respectively opening to the receptacle(s) 7a for the respective collection container(s) 8. The filter holder 3 is provided with a support 3b on which the membrane filter 4 is to be arranged, and that is arranged in the first flow path A that leads to the filtrate outlet 6 such that all sample liquid has to pass the membrane filter 4 to reach the filtrate outlet 6.

While the membrane filter 4 is as such a consumable so that the entity sold must not necessarily include the membrane filter 4, the working entity may include the suitable specific membrane filter 4. This membrane filter 4 is not a classical depth filter but is preferably one that is configured to capture and retain microorganisms present in the large volume sample (i.e. the retentate) predominantly on an upstream surface thereof for the future collection. Preferably the membrane filter is a track etched plastic membrane. As the filtration can sustain a large sample volume and ensure retention of small (e.g. 0.22 pm) microorganisms from the liquid sample, this is the structure of choice for collecting microorganisms, in particular bacteria from the sample.

The filter head 2 is configured to removably hold a sample container 5 that is configured to receive the desired volume of the sample fluid to be filtered in one processing run, such that the sample container 5 communicates with the first flow path A upstream of the filter holder 3 and the membrane filter 4. In Figure 1 the sample container 5 is shown in the position for performing the filtration step at which the device 1 is used as a classical filtration device in that a suction force is applied to the filtrate outlet 6 by suitable means like a vacuum pump (not shown).

The second flow path B, in particular the outlet(s) 3a, is/are configured to be selectively blocked during filtration, preferably by an outer peripheral rim of the sample container 5, when the same is held on its receptacle on the filter head 2, and/or by a valve device, and/or by a plug. The valve device may include a mechanical slider that normally closes the second flow path B in a still stand of the filter head 2 in the filtration step, and that automatically opens the second flow path B due to the action of the centrifugal force when the filter head is spinning in the centrifugation step.

For the centrifugation step the device 1 comprises a cover or lid 9 that is configured to be removably attached in place of the sample container s so as to close or substantially close the first flow path A on the upstream side. The filtrate outlet 6 may, but does not necessarily have to, be closed during the centrifugation as any remaining liquid or diluent or rinsing liquid added for this step (described below) will normally remain on the upstream surface of the membrane filter 4. The filtrate outlet 6 may also be associated to a drain (not shown) for collecting any residue liquid exiting the filtrate outlet 6 during the centrifugation.

In the centrifugation step, after the cover is attached to the filter head 2, the filter head (if provided as a separate entity or device) is placed on a rotating instrument that will make the filter head spin about its rotation axis. If the device includes the motor for spinning, the filter head remains within the device of course.

For the centrifugation step, the filter head 2 includes a port 10 for injecting a diluent or rinsing liquid towards the membrane filter 4 to support the removal of the microorganisms collected on the upstream membrane surface. The diluent is preferably injected towards the upstream surface of the membrane filter 4 and/or a downstream surface of the membrane fi Iter 4. Injection of the diluent towards the downstream surface of the membrane may help to collect bacteria capture in the filter.

During the device spinning only a small amount of diluent (approx. 500 pl) is injected into the device onto the membrane filter. The collection container(s) 8, i.e. the tubes/vials 8a in the example, at the outer peripheral side of the device will collect the diluent together with the microorganisms from the surface of the membrane through the second flow path B by the centrifugal force.

Although not shown, the filter head 3 may include a spillway for directing any excess liquid that cannot be accommodated in the collection container(s) during centrifugation back to the filtrate outlet 6 or to a dedicated separate outlet.

In the example of Figure 2 the port 10 for injecting the diluent is provided at or in the cover 9, and is preferably aligned with the rotation axis, at least in an initial position. However, the port may be provided at or in the filter holder 3 with a channel directing the diluent to the upstream and/or downstream surface of the membrane filter.

The port 10 for injecting the diluent is preferably displaceable relative to the upstream and/or downstream surface of the membrane filter 4, for example in that it is provided on a slider that is movable relative to the filter holder 3 (not shown). The displacement may be automatic under the action of the centrifugal force.

The invention provides a method for sample concentration which may, but does not necessarily have to, utilize the above-described device 1 for sample concentration according to the invention. The volume of the sample fluid filtered through the membrane filter 4 for concentration is typically 20 to 100 ml.

The method in the most general setup comprises the step of filtering a volume of a sample fluid through a membrane filter 4 held on a filter holder 3 of a filter head 2 and removing the filtrate, for example to a drain (i.e. the filtration step), and the step of spinning the filter head 2 with the membrane filter 4 held on the filter holder 3 and the collection container 8 fixedly attached to the filter head 2 about a rotation axis crossing a plane defined by a surface of the membrane filter 4 such that retentate collected on the membrane filter 4, preferably on an upstream surface thereof, is directed into a collection container 8 by a centrifugal force created upon rotation of the filter head 2 (i.e. the centrifugation step).

The method for sample concentration preferably further comprises injecting a diluent or rinsing liquid towards the membrane filter 4 held on the filter holder 3, before and/or during the spinning of the filter head 2, preferably towards an/the upstream surface and/or a downstream surface of the membrane filter 4 to promote and aid the transport of the microorganisms from the membrane surface to the collection container 8.

Preferably, the method may involve changing an injecting position of the diluent or rinsing liquid relative to the upstream and/or downstream surface of the membrane filter 4 held on the filter holder 3, preferably progressing from a more central position towards a more radial outer position of the membrane filter 4.

In the method, the spinning of the filter head can be intermittently performed or repeated multiple times to prevent membrane clogging and increase the filtration volume.

Claims

1. A device (1) for sample concentration comprising: a filter head (2) including a filter holder (3) configured to hold, preferably removably, a membrane filter (4) and allow a volume of a sample fluid to be guided through a first flow path (A) passing the membrane filter (4), when the same is held on the filter holder (3), towards a filtrate outlet (6); and at least one collection container holder (7) provided on the filter head (2) and configured to removably hold a collection container (8), wherein the filter head (2), together with the collection container (8), when the same is held at the at least one collection container holder (7), is rotatable about a rotation axis crossing, preferably essentially perpendicularly, a plane defined by a surface of the membrane filter (4), when the same is held on the filter holder (3), and wherein the at least one collection container holder (7) is provided on the filter head (2) such that retentate collected on the membrane filter (4) is directed into the collection container (8), when the same is held at the collection container holder (7), through a second flow path (B) by a centrifugal force created upon spinning the filter head (2) about the rotation axis.

2. The device (1) for sample concentration according to claim 1, wherein the device (1) comprises a membrane filter (4), preferably one that is configured to retain microorganisms on an upstream surface thereof, preferably a track etched plastic membrane, the membrane filter (4) arranged on the filter holder (3).

3. The device (1) for sample concentration according to claim 1 or 2, wherein the filter head (2) is configured to removably hold a sample container (5) formed to receive the volume of the sample fluid to be filtered, such that the sample container (5) communicates with the first flow path (A) upstream of the filter holder (3).

4. The device (1) for sample concentration according to claim 3, wherein the device (1) comprises a cover (9) configured to be removably attached in place of the sample container (5) so as to close the first flow path (A) on the upstream side.

5. The device (1) for sample concentration according to any one of claims 1 to 4, wherein the second flow path (B) is configured to be blocked during filtration, preferably by a/the sample container (5), when the same is held on the filter head (2), and/or by a valve device, and/or by a plug.

6. The device (1) for sample concentration according to any one of claims 1 to 5, wherein the filter head (2) includes a port (10) for injecting a diluent towards the membrane filter (4), when the same is held on the filter holder (3), preferably towards an/the upstream surface of the membrane filter (4) and/or a downstream surface of the membrane filter (4).

7. The device (1) for sample concentration according to claim 6, wherein the port (10) for injecting the diluent is displaceable relative to the upstream and/or downstream surface of the membrane filter (4).

8. The device (1) for sample concentration according to claim 6 or 7 in combination with claim 4, wherein the port (10) for injecting the diluent is provided at the cover (9), preferably aligned with the rotation axis.

9. The device (1) for sample concentration according to any one of claims 1 to 8, wherein the at least one collection container holder (7) is configured to releasably hold the collection container (8) in the form of a tube or vial (8a).

10. The device (1) for sample concentration according to any one of claims 1 to 9, wherein the device (1) comprises a motor for spinning the filter head (2) about the rotation axis and/or a vacuum pump for pulling the sample fluid through the membrane filter.

11. A method for sample concentration, preferably using the device (1) for sample concentration according to any one of claims 1 to 10, the method comprising: filtering a volume of a sample fluid through a membrane filter (4) held on a filter holder (3) of a filter head (2) and removing the filtrate; spinning the filter head (2) with the membrane filter (4) held on the filter holder (3) about a rotation axis crossing a plane defined by a surface of the membrane filter (4) such that retentate collected on the membrane filter (4), preferably an on an upstream surface thereof, is directed into a collection container (8) by a centrifugal force created upon rotation of the filter head (2).

12. The method for sample concentration according to claim 11, wherein the collection container (8) is rotated together with the filter head (2) about the rotation axis.

13. The method for sample concentration according to claim 11 or 12, further comprising injecting a diluent towards the membrane filter (4) held on the filter holder (3), before and/or during the spinning of the filter head (2), preferably towards an/the upstream surface and/or a downstream surface of the membrane filter (4).

14. The method for sample concentration according to claim 13, comprising changing an injecting position of the diluent relative to the upstream and/or downstream surface of the membrane filter (4) held on the filter holder (3), preferably progressing from a more central position towards a more radial outer position of the membrane filter (4).

15. The method for sample concentration according to claim 11, 12, 13 or 14, wherein the volume of the sample fluid filtered through the membrane filter (4) is 20 to 100 ml.