Device for filtering a sample in aqueous solution and associated detection system

Abstract

The invention relates to a device (2) for filtering a sample (28) in aqueous solution, comprising a support member (12) having two opposite faces (20, 22), and a porous membrane (14) designed to receive at least one microorganism (38) contained in the sample (28). The membrane (14) is received in a cavity (24) formed by a recess in one of the two opposite faces (20, 22) of the support member (12), the support member (12) further having a plurality of through-holes (24) connecting the two opposite faces (20, 22) at the cavity (24) in order to allow the extraction of a liquid phase. The device further comprises a microporous heat sink (18) arranged between the membrane (14) and the plurality of holes (24).

Description

[0001] Description

[0002] Title: Filtering device for a sample in aqueous solution and associated detection system

[0003] The invention relates to the field of microorganism detection and filtration. Microorganism detection and filtration are used in numerous technical fields such as petrochemicals, pharmaceuticals, organic chemistry, biology, and food processing.

[0004] In the agri-food sector, the detection and quantification of microorganisms is a major challenge. All stakeholders are subject to extremely rigorous hygiene standards to prevent the sale of food containing problematic microorganisms such as bacteria, regardless of its degree of processing, and at the very least to control their quantity. Similar issues exist in the pharmaceutical and cosmetic sectors.

[0005] Indeed, when the presence of bacteria is detected too late, it is common for many consumers to suffer the consequences, which can range from more or less significant digestive discomfort to poisoning and / or infections that can cause death.

[0006] The detection of the presence of microorganisms relies, in the state of the art, on carrying out environmental tests which involve taking samples at regular intervals on the production lines as well as in the vicinity thereof, and counting the bacteria present in the samples after culturing.

[0007] This conventional approach involves culturing bacteria for several days in a nutrient medium, followed by manual colony counting to determine the number of bacteria, usually measured in colony-forming units per milliliter (CFU / mL). However, while considered the gold standard in microbiology, this method has several drawbacks, notably the significant time required to obtain results, which can range from two to five days depending on the specific microorganisms being quantified. This duration is even longer for sterility testing, which can take up to 14 days in the pharmaceutical and cosmetic industries.This duration is extremely restrictive in terms of logistics and generates numerous constraints for the supply chain, particularly in terms of storage and production, which can sometimes lead to quarantines or even recalls.

[0008] In recent years, alternative methods have been explored to try to replace the Petri dish culture technique: polymerase chain reaction (PCR), most probable number approach, ATP measurement, flow or solid phase cytometry, microscopy-based methods, methods based on the analysis of hyperspectral measurements, or the use of excitation and emission matrices, etc.

[0009] Solid-phase cytometry classically includes various steps involving membrane filtration to retain the cells to be quantified in a sample, labeling of the retained cells to allow their counting and identification, and scanning of the membrane to perform these operations.

[0010] For the membrane filtration step, black-membrane filters are generally the most commonly used. This results in a higher contrast between the fluorescently labeled cells and the black background of the filter membrane, making them easier to identify during membrane scanning.

[0011] The Applicant's patent application FR2400617 describes a system for detecting bacteria at very low detection levels and within a timeframe compatible with an industrial production line. This is achieved by using a membrane to retain microorganisms, and one or more imaging operations followed by computer processing are performed to measure the quantity of bacteria present in the sample filtered through the membrane.

[0012] Initially, the Applicant therefore tested the compatibility with membranes developed for solid-phase cytometry solutions. It thus tested the compatibility of its method with various black membranes, typically made of cellulose esters, polycarbonate, or polyester.

[0013] The use of these membranes is described in various patents. For example, US patent 11,643,677 from Rapid Micro Biosystems describes the use of a black cellulose ester membrane for rapid detection based on autofluorescence measurement after a growth step. US patent application 2021,0277,440 from Redberry describes the use of black polyester membranes for direct enumeration and black cellulose ester membranes for microcolony growth, for fluorescence imaging detection of the presence of unamplified microorganisms or microorganisms in microcolony form.

[0014] The tests carried out by the Applicant with these membranes were unsuccessful. Indeed, the system described in patent FR2400617 requires significant illumination, which generates considerable heat and causes the membrane to burn, thus invalidating the measurement.

[0015] The Applicant therefore became interested in other types of membranes, such as white PC or white cellulose polyester (compliant with standard NF EN ISO 9308-1 for example).

[0016] The tests carried out by the Applicant were again unsuccessful. Indeed, the use of white membranes results in excessive transmission of spurious signals under the membrane, unless a glass support is used. However, the use of a glass support is incompatible with the system described in patent FR2400617 because it prevents proper filtration. The Applicant therefore turned its attention to metallic and metallized membranes. Generally speaking, these proved unsuitable for the following reasons:

[0017] - Metallic membranes posed compatibility problems with the optical system used in patent FR2400617, - Metallized membranes, which are made by depositing a metallic layer on the surface of the membrane, have been shown to pose stability problems of this metallic layer during filtration in the research carried out by the Applicant.

[0018] Furthermore, these two types of membranes have a significant cost and are therefore poorly suited for "consumable" use.

[0019] In general, the Applicant noted the difficulty of creating a filtering device that could be used directly to perform detection in a system of the type described in patent FR2400617. Indeed, the detection method is based on the detection of fluorescence, and the autofluorescence of the majority of conventional membranes presents a problem by "polluting" the signal collected on the sample.

[0020] In addition, it is desirable that the filtering device be of the "consumable" type, meaning that it is easy to produce and reasonably priced, and that it allows both the sample to be filtered through the membrane and can be used directly in the detection system without complex handling or the risk of contaminating the sample. This last point is all the more critical given that the detection levels of microorganisms targeted by the Applicant's system are extremely low.

[0021] The invention improves the situation. To this end, it proposes a device for filtering a sample in aqueous solution, comprising a support with two opposite faces and a porous membrane arranged to receive at least one microorganism contained in the sample. The membrane is received in a recess formed by a cavity in one of the two opposite faces of the support. The support further has a plurality of through holes connecting the two opposite faces at the level of said recess to allow the extraction of a liquid phase. The device also includes a microporous heat sink disposed between the membrane and said plurality of holes.

[0022] The filtering device, and more particularly the presence of a microporous heat sink arranged between the membrane and the plurality of holes, is particularly advantageous in the context of the invention.

[0023] Indeed, the heat sink reduces membrane heating even when the sample is illuminated. Furthermore, the fact that the heat sink is microporous ensures efficient sample filtration.

[0024] According to various embodiments, the invention may have one or more of the following features: - a retaining element is arranged to hold the membrane and the heat sink in the housing, - another of the two opposite faces has at least one protruding part around the plurality of holes, the support being suitable for being connected to a filtering system through a groove conforming to a predefined shape of the protruding part,

[0025] - The membrane is made of polyester, PET,

[0026] - the membrane has primary pores with a diameter between 0.1 pm and 5 pm,

[0027] - the membrane is black,

[0028] - the support is made of plastic or metal,

[0029] - The heat sink and the membrane are arranged so that the flatness gradient of the membrane in the contact plane with the heat sink is less than 15 pm.mnr 1 ,

[0030] - the heat sink has a thermal conductivity greater than or equal to 0.1 W / m*K,

[0031] - the heat sink has secondary pores with a diameter between 1 pm and 200 pm, and - the sample in aqueous solution corresponds to a volume between 100 pL and 1 L.

[0032] The invention also relates to a system for detecting the presence or absence of at least one microorganism in a sample, the detection system comprising:

[0033] - a filtration system capable of recovering a liquid phase from the sample filtered through a filtration device according to the invention;

[0034] - an optical bench arranged to receive the filtering device; and

[0035] - an analyzer allowing the quantification of the number of at least one microorganism;

[0036] the optical bench comprising:

[0037] - an illumination unit, suitable for illuminating the filtering device; and

[0038] - a measuring block, capable of scanning the illuminated filtering device.

[0039] Other features and advantages of the invention will become clearer upon reading the following description, drawn from illustrative and non-limiting examples taken from the drawings shown:

[0040] - Figure 1 is a functional block diagram representation of a detection system,

[0041] - Figure 2 is a functional block representation of an optical bench from Figure 1,

[0042] - Figure 3 is a top view of a filtering device included in the optical bench of Figure 1,

[0043] - Figure 4 is a bottom view of the filtering device included in the optical bench of Figure 1, and

[0044] - Figure 5 is a side view of the filtering device included in the optical bench of Figure 1.

[0045] The drawings and description below contain, for the most part, elements of a definite nature. They can therefore not only serve to better understand the present invention, but also contribute to its definition, if necessary. As can be seen in Figure 1, a detection system 1 is shown. The detection system 1 comprises an optical bench 3, an analyzer 8, and a filtering system 9.

[0046] Optical bench 3 is shown in Figure 2. Optical bench 3 includes a filtering device 2, an illumination block 4 and a measuring block 6.

[0047] According to various embodiments, filtration can be carried out upstream of the measurement by the optical block 3. In this case, the filtration system 9 is a separate element from the optical bench 3. The filtration system 9 conventionally comprises a pump, a housing to receive the filtration device 2 and all the elements necessary to carry out the filtration of the sample so that the filtration device 2 retains only the phase of the sample useful for measurement by the analyzer 8.

[0048] Indeed, as we will see below, the filtering device 2 is designed to be used from the beginning to the end of the detection process:

[0049] - initially, the filtering device 2, which is received in the filtering system 9, receives a liquid containing the microorganisms that the detection system 1 aims to detect, and filters the liquid in which these microorganisms are suspended,

[0050] - in a second step, the filtering device 2 is introduced into the optical bench 3 to carry out the measurement(s) allowing the detection of the microorganisms which have been filtered on a membrane of the filtering device 2.

[0051] The filtering device 2 will now be described with reference to Figures 3, 4 and 5.

[0052] The filtering device 2 comprises a support 12, a membrane 14, a clamping ring 16, and a heat sink 18, as shown in Figure 3. The filtering device 2 is, for example, partially or totally single-use. Those skilled in the art will understand that the term "single-use" refers to the filtering of a single sample 28 from a predefined volume. The support 12 has two opposing faces 20, 22. The support 12 also has a plurality of through holes 34 connecting the two opposing faces 20, 22. Alternatively, the plurality of holes 34 could be replaced by one or more channels opening onto the face 20 and connected to one or more discharge openings opening onto the face 22.

[0053] The support 12 is preferably made of plastic, such as polycarbonate (PC). Alternatively, the support 12 can be made of any other plastic or of metal, or even of ceramic or wood. Generally, the material of the support 12 is chosen so that it does not exhibit autofluorescence in the excitation wavelength range between 200 nm and 500 nm, preferably between 280 nm and 450 nm, and more restrictively between 350 nm and 400 nm. The support 12 is, for example, manufactured using a molding technique or by additive manufacturing, which makes it easier to produce in large quantities compared to other materials besides plastic.

[0054] Alternatively, the 12-inch holder is not single-use. The 12-inch holder is made of metal to facilitate reuse.

[0055] Support 12, for example, has a length between 10 and 200 millimeters, a width between 5 and 200 millimeters, and a thickness between 1 and 10 millimeters.

[0056] One of the two opposite faces 20, 22 corresponds to a top face 20 and another of the two opposite faces 20, 22 corresponds to a bottom face 22 of the support 12 respectively.

[0057] The plurality of holes 34 are several orifices 34 spaced uniformly and ensuring good filtration of a liquid phase 30 from a sample 28. The orifices 34 designate openings in the support 12 and are used to allow the passage of the liquid phase 30.

[0058] Sample 28 designates an aqueous solution which is poured into a housing 24 provided in the filtering device 2. Sample 28 comprises the liquid phase 30 and a residual phase 36. Advantageously, the volume of sample 28 is between 10 microlitres and 1 litre, so that the housing 24 provided in the filtering device 2 is suitable for receiving such a volume of sample 28 between 10 microlitres and 1 litre.

[0059] The housing 24 connects the two opposite faces 20, 22 by means of the orifices 34 connecting the upper face 20 to the lower face 22. This connection between the two faces 20, 22 allows the extraction of the liquid phase 30.

[0060] The shape of the recess forming the housing 24 is, for example, a circle. Alternatively, the shape of the recess is a rectangle or any type of closed polygon allowing the support 12 to be manufactured industrially.

[0061] The liquid phase 30 corresponds to the phase of sample 28 extracted by the filtration system 9. As will be seen below, the quantity of microorganisms present in the liquid phase 30 depends on the type of detection required. When the aim is to detect small microorganisms, the liquid phase 30 (for example, water) contains a negligible quantity of microorganisms, that is, a quantity advantageously less than 10 CFU / sample. When the desired detection involves larger microorganisms such as yeasts, then filtration by the filtering device 2 is appropriate, and the liquid phase can contain a significant quantity of microorganisms, provided their size is less than 3 µm, for example.

[0062] The residual phase 36 corresponds to the phase of the sample 28 remaining in the membrane 14 and containing a number of microorganisms 38, if any.

[0063] The upper face 20 is the face on which the illumination block 4 illuminates the support 12 containing a sample 28 in solid or semi-solid phase, the filtering device 2 being left to dry after the filtration operation. The upper face 20 has a recess forming the housing 24 into which the membrane 14 is inserted. The upper face 20 is adapted to the arrangement of the support 12 with the illumination block 4 and the measuring block 6 in the detection system 1. The upper face 20 advantageously has a positioning surface 29A and a gripping surface 29B.

[0064] The upper positioning surface 29A conforms to a predefined shape of the recess, facilitating installation for the filtration operation. The shape of the positioning surface 29A will therefore depend on the housing into which the filtering device 2 must be received to perform the filtration operation.

[0065] The gripping surface 29B is designed to facilitate the handling of the support 12 by an operator. The gripping surface 29B is shown here in the shape of a rectangle. Alternatively, any other shape suitable for facilitating the handling of the support 12 by an operator may be considered.

[0066] The lower face 22 is adapted for an arrangement in the filtering system 9, which will be described later. The lower face 22 optionally has at least one projecting portion 27 around the orifices 34. For example, in Figure 4, the lower face 22 has two projecting portions 27A, 27B curved around the orifices 34.

[0067] The lower face 22 advantageously presents a lower positioning surface 31 A and a lower support surface 31 B.

[0068] The lower positioning surface 31A, for example, has two projecting circles 33A and 33B that define the surface of the recess forming the housing 24 and creating a hollow cylinder. The circles 33A and 33B are, for example, shorter in height than the projecting portion 27.

[0069] The lower bearing surface 31 B has for example a plurality of ribs 35. The ribs 35 are particularly useful in stiffening the gripping portion of the support 12 when it is made of plastic, for example by molding.

[0070] Figure 5 shows the protruding portion 27 relative to the lower face 22 more clearly. The membrane 14 is preferably made of plastic, for example polyester (PET). The membrane 14 has the same shape as the recess forming the housing 24 in which it is received. For example, the membrane 14 has a circular shape.

[0071] Membrane 14 is advantageously black. The reason for this choice is that the Applicant explored several membrane colors and determined that black has low autofluorescence, minimizing interference during analysis performed by analyzer 8. The use of this type of membrane is made possible by the heat sink, which will be described below.

[0072] Alternatively, the membrane 14 could be made of any other plastic, ceramic or metallized membrane material which does not generate radiation in the wavelength range for the autofluorescence measurement considered for detection when illuminated by the illumination block 4, while structurally supporting the heat release induced by this illumination.

[0073] The color black naturally retains heat and can degrade membrane 14 very quickly. Therefore, a black PET membrane 14 initially appeared to be a poor choice, given its significantly higher heating under illumination by the light block 4 compared to other colored membranes, such as the gray membranes tested in the interior art. However, the Applicant unexpectedly discovered that the heat sink 18, which will be described later, also mitigates the heating of membrane 14 below a critical temperature, below which its performance is not impaired.

[0074] The membrane 14 also includes primary pores 42, enabling the membrane 14 to retain one or more types of microorganisms 38. The membrane 14 is typically for single use. However, the membrane 14 can be reused under certain conditions. For example, when the membrane 14 has been used in the detection system 1, confirming the absence of the types of microorganisms of interest 38, and the membrane 14 remains functional, the membrane 14 can then be reused to retain the same types of microorganisms of interest 38.

[0075] The primary pores 42 preferably have a maximum diameter of 0.5 pm. This diameter ensures that most types of microorganisms 38 are retained by the membrane 14. Alternatively, the maximum diameter is less than 0.5 pm, for example 0.1 pm, depending on the microorganisms 38 to be retained. The microorganisms 38 may be pathogens contaminating the sample 28, such as bacteria. As mentioned above, the detection may target larger microorganisms, such as yeasts. In this case, it may be advantageous to increase the diameter of the primary pores 42 so that the membrane 14 retains only larger elements, for example 3 pm, 5 pm, or larger. In this case, smaller microorganisms will be released into the liquid phase, as desired.

[0076] The clamping ring 16 is used to secure the membrane 14. The clamping ring 16 is positioned above the membrane 14 and around the recess. For example, the clamping ring 16 is a ring made of plastic. Generally speaking, the clamping ring 16 is a retaining element: its role is to hold the membrane 14 in place within the support housing.

[0077] The heat sink 18 is positioned between the membrane 14 and the orifices 34. The heat sink 18 is microporous. By definition, the heat sink 18 comprises secondary pores 44 with a maximum diameter of approximately 1 µm. Preferably, the maximum diameter is between 1 µm and 200 µm. Advantageously, this diameter may be between 5 µm and 50 µm, as this allows for faster filtration while maintaining the flatness of the membrane 14. The heat sink 18 preferably has a thermal conductivity of at least 0.1 W / m·K. The maximum value of this thermal conductivity will depend on the material used. Obviously, a higher value is more desirable.

[0078] The heat sink 18 further comprises a contact surface 46 in contact with the membrane 14. This contact surface 46 can, for example, be the upper or lower part of a rigid or semi-rigid layer covered with a metallic layer. Any other material (alloy, glass, ceramic, etc.) may be considered provided that the desired thermal conductivity is achieved and that the secondary pores 44 can be formed.

[0079] The metallic layer, for its part, limits the temperature increase, and therefore limits the heating of membrane 14. It is thanks to this function that the Applicant discovered that it was possible to use black membranes with the detection system of patent FR2400617 without having to fear that they would be burned during illumination.

[0080] The contact surface 46 is uniformly flat. Preferably, the contact surface 46 is arranged so that, when the membrane 14 is received on the contact surface 46, the flatness gradient of the membrane 14 in this contact plane is less than 15 m.mnr 1 , preferably less than 10 m.mnr 1 , and even more preferably less than 2 m.mnr 1 This can be achieved by working the surface condition of the contact surface 46 so that the membrane 14 does not remain "sucked" into the secondary pores 44 after the filtration operation (and / or the subsequent drying operation where applicable).

[0081] Illumination block 4 is suitable for illuminating sample 28. Illumination block 4 is, for example, a white light source. Generally, a white light source can have an emission spectrum including wavelengths between 300 nm and 1000 nm. Illumination block 4 therefore emits incident radiation 50, enabling autofluorescence between 300 nm and 1000 nm. Illumination block 4 includes, for example, one or more filters (not shown). Each filter absorbs a specific range of wavelengths from Illumination block 4. Illumination block 4 also includes a magnifying lens and an autofocus system (not shown).

[0082] The presence or absence of microorganisms 38 is determined by scanning the residual phase 36, after the liquid phase 30 has been filtered. The scan of the illuminated residual phase 36 is performed by the measurement unit 6. The measurement unit 6 comprises a collection optic 48 and a measurement optic 49. The measurement unit 6 is configured, for example, to perform multispectral imaging on a sample 28 for which a detection value for microorganisms 38 is sought.

[0083] More specifically, the measurement block 6 is responsible for collecting autofluorescence and reflectance radiation 52 from the sample 28 and redirecting it to the analyzer 8 via the collection optics 48 and the measurement optics 49. The measurement block 6 acquires, for example, the plurality of multispectral images 26 that allow scanning the residual phase 36 of the sample 28. The measurement block 6 is, for example, a multispectral measurement block (from the English "multispectral imaging" or "MSI"). Those skilled in the art will understand that the multispectral approach is advantageous due to the imaging rate for a given resolution.More specifically, the multispectral approach, with a faster acquisition rate, allows, at a constant time budget, to produce higher resolution images, and therefore to detect lower levels of colony-forming units per milliliter (CFU / mL) than with other imaging methods such as the hyperspectral approach.

[0084] The collection optics 48 advantageously include separators and lenses that each redirect autofluorescence and reflectance radiation 52 from the sample 28 to the measurement optics 49. The measurement optics 49 preferably include cameras. At the output of the measurement optics 49, a plurality of images 26 are transmitted to the analyzer 8, as shown in Figure 1, to determine detection data for microorganisms 38, such as a bacterial count value.

[0085] Analyzer 8 implements a machine learning model (ML). For example, during a training phase, a Petri dish is used. The bacteria present in sample 28 are conventionally cultured in this dish and then counted to provide data for training Analyzer 8. Once the training phase is complete, Analyzer 8 quantifies the number of microorganisms 38 using a first analysis operation and a second analysis operation.Analyzer 8 receives, for example, data from a deep neural network (DNN) and more particularly from a convolutional neural network (CNN) and the plurality of multispectral images 26 to perform the first analysis operation as well as a computer not shown to perform a second analysis operation allowing to estimate the concentration of microorganisms 38. The Applicant has filed patents describing analyzer 8 (see for example application FR2400617).

[0086] The filtering system 9 is used to extract the liquid phase 30 from the sample 28 when the filtering device 2 is received into it. The filtering system 9 is therefore arranged to receive the filtering device 2 and guides its placement by means of a groove conforming to a predefined shape of the protruding portion 27. For example, the filtering system 9 has two curved grooves identical in shape to the two protruding portions 27A, 27B. This facilitates the placement of the filtering device 2 and ensures that the sample 28 flows correctly onto the membrane 14 for filtering the liquid phase 30. Advantageously, the filtering system 9 may include a vacuum flask (not shown) located below the housing for the filtering device 2, which allows the liquid phase 30 that has passed through the filtering device 2 to be evacuated.Thanks to the filtering device 2, and more particularly to the smooth and flat contact surface of the heat sink 18, the detection system 1 makes it possible to limit digital noise or parasitic fluctuation during image acquisition by the illumination block 4 without heating of the membrane 14. Thus the detection system 1 quantifies more precisely the presence or absence of microorganisms 38.

Claims

Demands

1. A filtering device (2) for a sample (28) in aqueous solution comprising a support (12) having two opposite faces (20, 22), a porous membrane (14) arranged to receive at least one microorganism (38) contained in the sample (28), said membrane (14) being received in a housing (24) formed by a recess in one of the two opposite faces (20, 22) of the support (12), the support (12) further having a plurality of through holes (24) connecting the two opposite faces (20, 22) at the level of said housing (24) to allow the extraction of a liquid phase, the device further comprising a microporous heat sink (18) disposed between the membrane (14) and said plurality of holes (24).

2. Device according to claim 1, wherein a retaining member (16) is arranged to retain the membrane (14) and the heat sink (18) in the housing (24).

3. Device according to claim 1 or 2, wherein one other of the two opposite faces (20, 22) has at least one projecting part (27) around the plurality of holes (24), the support (12) being able to be connected to a filtering system (9) through a groove conforming to a predefined shape of the projecting part (27).

4. Device according to any one of the preceding claims, wherein the membrane (14) is made of polyester, PET.

5. Device according to any one of the preceding claims, wherein the membrane (14) has primary pores (42) having a diameter between 0.1 pm and 5 pm.

6. Device according to any one of the preceding claims, wherein the membrane (14) is black.

7. Device according to any one of the preceding claims, wherein the support (12) is made of plastic or metal.

8. A device according to any one of the preceding claims, wherein the heat sink (18) and the membrane (14) are arranged such that the flatness gradient of the membrane (14) in the contact plane with the heat sink (18) is less than 15 m.mnr 1 .

9. Device according to any one of the preceding claims, wherein the heat sink (18) has a thermal conductivity greater than or equal to 0.1 W / m*K.

10. Device according to any one of the preceding claims, wherein the heat sink (18) has secondary pores (44) with diameters between 1 pm and 200 pm.

11. A detection system (1) for the presence or absence of at least one microorganism (38) in a sample (28), the detection system comprising: - a filtering system (9) capable of recovering a liquid phase of the sample (28) filtered through a filtering device (2) according to one of the preceding claims; - an optical bench (3) arranged to receive the filtering device (2); and - an analyzer (8) allowing the number of at least one microorganism (38) to be quantified; the optical bench (3) comprising: - an illumination block (4), suitable for illuminating the filtering device (2); and - a measuring block (6), capable of scanning the illuminated filtering device (2).

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