WO2023011950A1

WO2023011950A1 - Improved biological test support

Info

Publication number: WO2023011950A1
Application number: PCT/EP2022/070688
Authority: WO
Inventors: Charlotte LEGER; Alice EPAULE
Original assignee: Withings
Priority date: 2021-08-04
Filing date: 2022-07-22
Publication date: 2023-02-09
Also published as: FR3126046A1

Abstract

Single-use biological test support (1) comprising: - an upstream reservoir (11) to receive a liquid-form biological sample that is to be tested (ET), - an extraction membrane (2) positioned in a first housing (24), in fluidic communication with the upstream reservoir (11), - a reaction zone (31) comprising a reaction housing (72, 74) containing a test reaction mixture (33, 34), - a transfer duct (44) connecting the first housing (24) to the reaction housing (72, 74), - a withdrawal membrane (4) positioned in a third housing (73), the third housing being placed in fluidic communication with the transfer duct (44).

Description

ENHANCED BIOTESTING SUPPORT

This document generally relates to diagnostic devices by nucleic acid amplification, in particular, from a biological sample such as drops of blood, urine, saliva and sweat. More specifically, the present invention relates to test plates, used in an automated biological test system intended to identify the presence of an infectious microorganism (bacterium, virus, etc.). However, the proposed device and method can be applied to the detection of other biological markers that can reveal other types of any pathology, including cancer.

With regard to diagnostic tests based on the detection by amplification of the DNA/RNA of an infectious microorganism, we know the PCR test methods (PCR for Polymerization Chain Reaction) or the test methods known as LAMP (Loop -mediated isothermal amplification) or RT-LAMP (Reverse transcriptase Loop-mediated isothermal amplification).

Generally, a biological sample to be tested undergoes a lysis operation, then the lysed sample is incubated with an extraction membrane. Then, after rinsing, possibly drying and eluting the molecules of interest from the extraction membrane, the resulting eluate is directed to one or more first amplification reaction zone(s).

According to an embodiment of interest, the operations of rinsing, elution and reading of the result are carried out on a single-use cartridge.

The following documents give examples of existing technologies.

Document US2009325276 teaches various cartridge configurations for carrying out such test methods.

Document CA3100263 describes a microfluidic device for the separation, purification and concentration of components in a fluid medium.

The document "An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids" by Chen et al., Biomed Microdevices (2010) 12 705-719 [doi: 10.1007/s10544-010-9423-4] describes an integrated and self-sufficient cassette for the isolation, amplification and detection of nucleic acids.

Disclosure of Invention

It has been observed by the inventors that the eluted sample which is transported from the extraction membrane to the reaction/amplification zone may contain some undesirable residues which will hinder or even prevent the amplification reactions to occur. These impurities are more commonly found on point-of-care devices, which automate many steps of analytical methods, than on laboratory devices.

The inventors have also observed that this type of impurity is generally found in a front portion (i.e. the start of the stream) of the eluted sample stream, while the following portion is devoid of this type of undesirable impurity.

To this end, there is proposed here a biological test support, for single use, intended to be used with a test station to form a biological test system (ST) for detecting the presence of one or more pathogenic species and/or a nucleic acid sequence to be revealed, the test medium comprising:

- an upstream tank, to receive a biological sample to be tested in liquid form,

- an extraction membrane, arranged in a first housing, in fluid communication with the upstream reservoir,

- a reaction zone comprising a reaction housing containing a test reaction mixture,

- a transfer channel connecting the first housing to the reaction housing,

- a sampling membrane arranged in a third housing, the third housing being placed in fluid communication with the transfer channel.

The sampling membrane thus makes it possible to trap the front of the eluate, which may contain impurities and prevent it from reaching the reaction mixture(s).

In particular, the absorption capacity of the sampling membrane is chosen so that a flow of liquid passing through the transfer channel is partially sampled by the sampling membrane.

A front portion of the eluate is captured and retained by the sample membrane without reaching the first test reaction mixture, while a subsequent portion of the eluate reaches the first test reaction mixture. We can consider that the sampling membrane acts here as a 'depollution' membrane. Such a membrane makes it possible to effectively manage the impurities without any particular intervention on the test wafer and in particular without piloting the biological test system. This is passive trapping.

In particular, said fluidic communication is direct.

The term “placed in fluid communication” covers in particular an arrangement where the sampling membrane is placed adjacent to the transfer channel. However, it is not excluded to have a diversion channel specific functioning as a connection, for example capillary, between the transfer channel and the sampling membrane. When the sampling membrane is saturated, it no longer captures additional liquid and lets the incoming fluid pass without capturing more.

The term “extraction membrane” designates here a porous member making it possible to momentarily absorb the nucleic acids contained in the sample to be tested, to retain these nucleic acids during one or more rinsing operations intended to eliminate chemical species such as proteins or debris, including lysis buffer residues. Following this, an eluent with a stronger ionic affinity with said nucleic acids is introduced, making it possible to capture the nucleic acids of interest and to carry them, in the form of an eluate, towards the reaction/amplification zones.

The biological test support forms a cartridge, that is to say a consumable object (i.e. single-use) intended to be used with a test station which receives the cartridge and which interacts with the latter. The test station makes it possible to successively analyze a plurality of cartridges.

The aforementioned reaction housing may comprise one or more housings, called second and/or fourth housing.

In various embodiments of the invention, it is also possible to resort to one and/or the other of the following provisions, taken in isolation or in combination.

According to one aspect, the sampling membrane may have an absorption capacity representing a volume of between 10% and 90% of the predefined volume of eluent. The predefined volume of eluent is typically embedded in a housing of the test plate (in a blister for example). Advantageously, the start of the flow is detected thanks to the sampling membrane, but it becomes saturated at a given moment and there is still some eluate which will continue on its way to the reaction zone.

According to an optional aspect, the sampling membrane can have an absorption capacity representing a volume of between 20% and 70% of the predefined volume of eluent, or even a volume of between 30% and 60% of the predefined volume of eluent. According to various possibilities, it is possible to predict quite precisely the volume which will be trapped by the sampling membrane and also consequently the volume which remains available to react in the reaction mixture zones. In addition, a sufficient volume of the eluate which could contain undesirable elements is removed by trapping.

According to one aspect, the sampling membrane can rub shoulders with the transfer channel over a distance greater than 4 mm or else N times a diameter or a dimension (width or height) of the cross section of the transfer channel, N preferably being greater than 3, 5, 10 or even 15.

Thus we obtain a suction by blotting effect (capillarity), with a sufficient interface surface to capture the precursory portion (ie of forehead) flow over a wide range of possible speeds and flow rates.

According to one aspect, the test medium may further comprise at least a first volume of first rinsing liquid, and/or a predefined volume of eluent. The volumes can be contained directly in housings of the test medium or contained in bags arranged in housings of the test medium.

The liquids are housed on board the test stand, and thus the only interface needed for fluid transmission between the test wafer and the test station is a pneumatic interface. Transfers of fluid between the test station and the test support are thus minimized and, in fact, the risks of leakage and contamination of the test station.

In one aspect, the test reaction mixture is in lyophilized form. The freeze-dried form allows the test support to be kept ready for use over a long period before its actual use. The mixture can allow the amplification and detection of at least one nucleic acid sequence of interest.

According to one aspect, the reaction zone comprises a first reaction zone which can contain a porous reaction medium arranged in a second housing of the reaction housing and containing the first test reaction mixture in lyophilized form, the first reaction mixture allowing the amplification and detection of at least one nucleic acid sequence of interest.

According to one aspect, the test support may further comprise at least one recovery tank, to recover the liquids resulting from the rinsing, and the overflow of eluate.

No liquid comes out, we keep the station clean and we avoid cross contamination.

According to one aspect, the test support may further comprise at least one air outlet, to facilitate the filling of said reservoir. As a result, the air that was in the recovery tank can easily escape when pushing liquids towards the recovery tank.

According to one aspect, the test support may further comprise a valve system for directing a flow of fluid (liquid and/or air) from the extraction membrane selectively to the recovery tank or to the reaction zone via the channel of transfer. One can reliably direct the liquid flows inside the test medium by controlling the valve system. Said valve system can be activated from the test station which processes the test medium.

In one aspect, the valve system may include a first bypass valve and a second bypass valve for directing fluid flow from the extraction membrane selectively to the recovery tank or to the reaction zone(s) through the transfer channel. THE diverter valves can be membrane-type valves. This kind of membrane-type valve can be easily controlled thanks to pushers arranged in the test station which processes the test support(s).

According to one aspect, the sampling membrane (or the third housing) is located fluidically between the valve system and the reaction zone. In other words, the sampling membrane (or the third housing) is located fluidically on the path that leads from the valve system to the reaction zone. Thus, the sampling membrane is arranged at the most relevant location of the microfluidic circuit, i.e. just upstream of the reaction mixing zones and after the valve system, which prevents the sampling membrane from sampling, for example, liquids from rinses which are used to clean the extraction membrane.

According to one aspect, the reaction zone may further comprise a second reaction zone containing a fourth housing and containing a second control reaction mixture. This makes it possible to verify that the eluate has indeed arrived at its destination in the reaction mixture zones; because in fact the control reaction mixture reacts to the presence of molecules always present in the eluate, whether or not the latter contains the nucleic species of interest.

According to one aspect, the test medium may further comprise a result reading zone comprising at least the reaction zone and in particular the first reaction zone and/or the second reaction zone. For example, it may be an optical reading of the result. The result can be obtained quickly and directly on site while the test medium has not been handled or transported, in a logic of point-of-care accessibility and speed.

According to one aspect, the test medium (more precisely the reaction zone) may also comprise a distribution reservoir arranged adjacent to the first, and if necessary the second, reaction zone(s) so as to be able to supply eluting the first and second reaction zones by capillary pumping. This makes the supply of eluate to the reaction zones more reliable.

According to one aspect, the sampling membrane can be formed from a material comprising at least one of: cellulose, polyvinylidene fluoride ('PVDF'). These materials have adequate porosity to capture and retain the eluate.

According to one aspect, the absorption capacity of the sampling membrane is determined so that a flow of liquid passing through the transfer channel is partially sampled by the sampling membrane. By partially withdrawn, it is meant that only a portion of the flow is withdrawn and not all of it (for example between 5 and 90%, or 10 and 90%, or between 10 and 50% of the flow). The absorption capacity is notably defined by the dimensions of the sampling membrane, its material, the interface with the transfer channel, etc.

According to one aspect, the sampling membrane can be formed from a material having a water absorption of between 50 mg/cm ² and 150 mg/cm ² , or even between 95 and 105 mg/cm ²

According to one aspect, the biological test support is configured to bring the biological sample to be tested into the extraction membrane, then after rinsing, drying and eluting said extraction membrane, directing the resulting eluate towards at least the first reaction zone, a precursor portion of the eluate being trapped by the sampling membrane without reaching the test reaction mixture, a following portion of the eluate reaching the test reaction mixture. Thus, multiple fluidic operations are carried out inside the test support so as to be able to deduce, after amplification reaction, a result through the reading zone.

According to one aspect, the reaction housing is located in a thermal interaction zone configured to be heated by the analysis station, and the third housing is located outside the thermal interaction zone. The thermal interaction zone is typically less than 5cm ² .

This avoids possible optical disturbances, on the one hand because the sampling membrane is not heated (or less heated), so that the reactions allowing optical visualization are not activated. In addition, since the sampling membrane is far from the reaction zone (in which the optical reading zone is included), the risks of interference are further reduced.

According to one aspect, the first housing is arranged in the thermal or optical surface interaction zone at most equal to 5 cm ² . This makes it possible to use the same heating system for the extraction membrane and for the reaction zone, which reduces the complexity of the station and increases the compactness of the assembly. Incidentally, this can accelerate the drying of the extraction membrane.

According to one aspect, the test support may be presented as a wafer with a body in which channels and housings are formed, which are generally isolated from the external environment. Such a test support can be mass-produced at well-controlled costs.

According to one aspect, the support may have a generally flat parallelepipedal shape, with two main faces. An air inlet may be provided and an inlet in fluid communication with the upstream reservoir may be provided to receive a biological sample to be tested. These two entrances can be arranged on a single main face.

Preferably, all the interfaces can be arranged on a single main face. There is thus a particularly simple interface with the test station, which makes it possible to make the tests more reliable and to increase productivity. In one aspect, the test holder is configured to cooperate with a biological test station to form the biological test system.

In one aspect, the eluent contained in the eluent volume is RNase- and/or DNase-free distilled water and the eluent volume has a volume of between 30 microliters and 100 microliters.

According to one aspect, there may be provided at least one air inlet, merged with or separate from the inlet for receiving the biological sample. The separate air inlet or the combined inlet can additionally be fitted with a non-return valve.

According to one aspect, the inlet for receiving the biological sample can preferably be sealable or reclosable, or even be equipped with a non-return valve

In one aspect, a second volume of a second rinse liquid may be provided, the first and second volumes of rinse liquid having a volume between 100 microliters and 300 microliters.

The present invention also relates to a biological test system comprising an analysis station and one or more biological test supports as described previously, the analysis station comprising at least one pneumatic connector configured to be coupled to the input of air from the biological test medium.

Other aspects, aims and advantages of the invention will appear on reading the following description of several embodiments of the invention, given by way of non-limiting example. The invention will also be better understood with regard to the attached drawings in which:

- Figure 1 is a schematic plan view of a first embodiment of test support according to the present invention

- Figure 2 is a plan view of a second embodiment of test support according to the present invention.

- Figure 3 schematically illustrates in elevation the coupling between the transfer channel and the sampling membrane.

- Figure 4 schematically illustrates in cross section detail the coupling between the transfer channel and the sampling membrane.

- Figure 5 schematically illustrates the interface between the analysis station and the biological test support in the fluid activation zones.

- Figure 6 schematically illustrates the interface between the analysis station and the biological test support, in the result reading zone.

- Figure 7 illustrates an analysis station with a biological test support.

- Figures 8A and 8B are detail views illustrating a membrane-type valve.

- Figure 9 is a detail view illustrating a sachet of liquid on board the wafer and pushed by a piston of the analysis station.

- Figure 10 is a schematic plan view of another embodiment of test support according to the present invention. - Figure 11 is a plan view of another embodiment of test support according to the present invention.

- Figure 12 is a three-dimensional view of the example of Figure 11.

- Figure 13 is a view similar to Figure 4 and shows a section relating to the embodiment of Figures 11 and 12.

DETAILED DESCRIPTION

In the various figures, the same references designate identical or similar elements. For reasons of clarity of the presentation, certain dimensions may not be represented to scale.

System - general

According to the synoptic view shown in Figure 7, a biological test system ST is proposed which includes an analysis station 8 which will be commented on later, and a test support 1.

The test support 1, which can be called in practice the “cartridge” or the “wafer” is intended for single use; there are therefore as many test supports as there are tests to be carried out.

A biological sample is collected, for example saliva, a nasopharyngeal swab, or urine, or even sweat. Although the proposed system is intended in particular for the processing of human samples, it is not excluded to use the proposed system to process samples taken from animals. Optionally, for certain types of tests, it may be necessary to collect blood or lymph as a biological sample to be tested.

The collection of the biological sample can be introduced into a collection container where a lysis buffer is added (to break the membranes of the biological cells). The collection container can be agitated for a sufficient time of one to several minutes, after which a predefined amount of ethanol is poured into the container to freeze the sample in a prepared sample state. Note that it is sufficient to collect a small amount of raw body fluid, typically a volume between 0.1 milliliter and 1 milliliter is sufficient for multiple types of tests.

The biological sample in the collection container thus prepared (denoted ET) is then poured into an inlet orifice of the test support marked 12. Following this, this inlet orifice is sealed, ie this inlet is sealed by a plug or even sealing of a watertight closure membrane. The inlet 12 is fluidically connected to an upstream reservoir 11, which receives the biological sample. For example, the upstream reservoir 11 can store the biological sample temporarily (for a few minutes). The upstream reservoir 11 can also be a simple passage channel.

A non-return valve can also be used, which also allows this inlet to be used as an air inlet for subsequent operations. This valve allows entry into the test support but prohibits reverse movement. The valve can be mounted on the inlet port by a retaining ring, for example. The valve can be a silicone valve, such as a “duckbill valve”, “dome valve”, “umbrella valve”, etc.

The test support 1 now containing the biological sample to be treated is placed on a test station in the analysis station 8 which then has its cover 80 open. Then the door (the cover) is closed and a user triggers a processing cycle, i.e. a test cycle. At the end of said cycle, a test result is given by the analysis station, in a form which will be described later. The test stand and analysis station may have various functions and features; exemplary embodiments are presented below.

The result is obtained in a time ranging from a few minutes to a few tens of minutes.

Such a system can be used in the context of an analysis laboratory, but, given its simplicity and the speed of its implementation, this biological test system can be used beyond, in a very broad context. , point-of-care type, i.e. in a pharmacy, in a doctor's office, in a general care establishment, in a quarantine and quarantine release system, etc. This biological test system can also be used in a veterinary vehicle for testing animals.

In general, the term "test support" must be taken here in the very broad sense, the test support is here a support which can take any geometric shape, for example a wafer shape, but any other shape is not however, is not excluded.

By misuse of language when speaking of the test medium, it can also be called 'chip' or 'chip', a term that comes from the logic 'Lab-on-a-chip'.

All the processing operations carried out on the biological sample to be tested are carried out in this plate, in particular the extraction of DNA/RNA molecules, rinsing, elution, the plate also comprising at least one zone for reading the result.

In the example illustrated, the test support/the test wafer has a generally flat parallelepipedal shape, with two main faces, namely a first main face 10A called the interaction face and a second main face 10B called the rear face. In addition, a peripheral slice 10C forming the other four faces extends between the two main faces. In the example illustrated, the thickness of the wafer denoted H1 is between 3 mm and 20 mm. The length L1 is between 20 mm and 80 mm and the width W1 is between 10 mm and 60 mm. The geometric configuration can similar to a credit card format with however a slightly greater thickness.

The test wafer 1 can however have another shape. For reasons of optical readings, shapes comprising a small thickness and flat main faces are preferred.

The test support can be identified by an identification element, which can be arranged on the support (i.e. QR Code) or in the support as in the case of an RFID chip, NFC or equivalent. The identifier can be even simpler, such as color coding or mechanical coding. The identification element, when it is digital, makes it possible to uniquely identify the test medium, and incidentally to store the result in the identification element at the end of the test before opening the door /hood.

Furthermore, the biological sample ET being associated with an individual from which this biological sample comes, which makes it possible to associate the biological sample, the support and/or the donor individual in a one-to-one way.

The RFID or NFC chip also makes it possible to identify that a support is properly mounted in the station. This may condition the activation of the station lock.

In the example illustrated, the test support formed as a wafer is obtained by removal in such a way as from a block of homogeneous parallelepipedal material. Milling and drilling are performed from the rear face 10B to obtain the channels and housings which will be discussed later.

Optionally, milling and drilling can be done from the front side as well.

The body of the wafer is formed from translucent plastic material 100, such as polymethyl methacrylate (PMMA), polycarbonate (PC), cyclo-olefin copolymer (COC) or Cyclo Olefin Polymers (COP).

Instead of starting from a homogeneous parallelepipedal block, one can start from a molded shape where the main housings are already molded and only small diameter holes remain to be made.

It is also possible to obtain the insert directly from molding with all its channels and housings, without re-machining.

In addition, instead of a material removal method, a 3D printing-type additive manufacturing process can be used.

It should be noted that the chosen substrate forms an effective barrier to contain the fluids considered, it does not let through or absorb the air and the rinsing liquids which will be discussed later.

On the rear face 10B, to close the channels and the housings, we come glue a closure membrane 59, as illustrated in Figure 4, for example a bio-compatible adhesive plastic film. Alternatively, provision may be made to weld a rear cover, for example by ultrasonic welding.

A closure membrane may also be provided on part of the front face of the wafer.

Thus the channels and housings are generally isolated from the external environment.

Figures 1, 2 and 10 illustrate several embodiments of the test support. In the channels (generally identified by 13) and the housings circulates the fluid which can be liquid or air. The channels generally have a small cross-section and/or a small length with regard to the dimensions of the housings. Among the housings, there is a so-called upstream reservoir 11 which forms a reservoir for the temporary storage of the biological sample to be tested ET; this reservoir is a widened channel in the form of a serpentine in the example illustrated II can nevertheless have another shape. There is also a downstream tank also called recovery tank 18. This recovery tank 18 is downstream of the channels transporting liquid fluids and this recovery tank 18 is sized to contain: the volume of the biological sample to be tested + the volumes of rinsing liquid + the volume of eluate. The volume of this recovery tank 18 is for example between 0.1 milliliter and 2 milliliters.

According to an optional configuration, the recovery tank 18 contains an absorbent pad 180 (see Fig 10). This absorbent pad has a blotting effect and captures liquids. It is expected that the absorbent pad does not occupy all the available volume in the recovery tank, and to leave at least one air passage between the pad and the walls of the tank so that air can escape through an outlet orifice 16.

To return to the channels and with particular reference to FIG. 2, a channel (known as the injection channel 14a) connects the air inlet 14 to the space forming the upstream tank 11. The upstream tank 11 can be a housing in the form of a channel with a length and/or a section greater than those of the communication channels 13. The biological sample typically comprises saliva and lysis buffer.

Another channel (known as connecting channel 11a) connects the space forming the upstream reservoir 11 to a first housing 24 containing an extraction membrane 2 which will be discussed later.

Another channel (known as the first intermediate channel 24a) connects the housing 24 to a bypass valve 52 (called second bypass valve). From there, another channel, called transfer channel 44, connects the outlet of the bypass valve 52 to the zone containing the reactants, in particular to a distribution tank 38. We will speak of reaction housing or reaction zone. to signify the location in the cartridge where the molecules of interest react and are optically analyzed.

Another channel (called second intermediate channel 24b) connects the housing 24 (or the diverter valve inlet 52) to another diverter valve 51 (known as the first diverter valve) which authorizes or stops the passage of fluid to the downstream in the direction of the recovery tank 18, via an evacuation channel 18a. The evacuation channel 18a therefore opens into the recovery tank 18 and may include an optional air outlet 17 (Figure 2).

As illustrated in Figures 1, 2 and 10, the first intermediate channel 24a and the second intermediate channel 24b can have a common portion and then be divided into two sub-portions, the sub-portions respectively integrating the first bypass valve 52 and the second bypass valve 51 . The two bypass valves 51, 52 are then arranged in parallel at the outlet of the first housing 24.

Alternatively (not shown), a single valve can be provided, instead of the valves 51, 52. The single valve then comprises an inlet and two outlets, which can be selected selectively.

Other channels 61a, 62a, 63a extend respectively from housings 61, 62, 63, which contain treatment liquids, to housing 24 of the extraction membrane (typically the channels 61a, 62a, 63a join the connecting channel 11a). Figures 1 and 2 illustrate three housings 61, 62, 63 (two rinsing liquids and one eluent); FIG. 10 illustrates two housings 61, 63 (a rinsing liquid and an eluent).

The treatment liquids are arranged upstream of the housing 24 of the extraction membrane 2, and are configured to be activated selectively by the analysis station.

It is noted that for each of the channels inside the test support, the liquids flow in a single direction. Unless otherwise specified, channels are used one way.

There is provided a pneumatic interface 66, represented schematically in FIG. 6, at the level of the air inlet 14. Thanks to this pneumatic inlet, and the control of a valve system already presented but described in more detail below , the analysis station can push the liquids inside channels of the test pad.

Extraction membrane, treatment liquids and valves

As already mentioned, the test medium 1 comprises an extraction membrane marked 2. The extraction membrane 2 can be a silica membrane or a cellulose membrane. The extraction membrane has a porous structure, i.e. an alveolar structure. The function of the extraction membrane is the isolation of DNA/RNA molecules and the removal of proteins and other short molecule waste products as will be described below.

In one example, the silica matrix of the extraction membrane is capable of selectively fixing DNA/RNA under conditions of high ionic strength and at pH < 7. Impurities are eliminated by rinsing and after drying, the molecules of DNA and RNA sequences are eluted in a solution at low ionic strength and at pH > 7. The silica matrix can be in the form of glass powder, glass/silica particles or glass fibers.

For more details on the extraction membranes used here, reference may be made to document EP1463744.

It is noted that the extraction membrane 2 occupies the entire housing 24 in which it is arranged in the direction transverse to the flow of the fluids which are passed through the extraction membrane 2. In other words, the flow of fluid flow (rinsing, drying, elution) cannot bypass the extraction membrane 2.

The Whatman GF/F membrane from Sigma-Aldrich™ source can be chosen for the extraction membrane. According to another example, the GF/D Whatman membrane from Sigma-Aldrich™ source can be chosen for the extraction membrane.

For example, the length of the membrane can be of the order of 10 mm, its width can be between 3 mm and 5 mm and its thickness can be between 0.3 mm and 0.6 mm.

In addition, the test medium includes processing liquids that are predisposed on the test medium, i.e. present on the test medium itself. We can say that they are embedded or on board the test medium.

In the example illustrated, there is provided a first volume of rinsing liquid R1 arranged in the housing 61, and optionally a second volume of a second rinsing liquid R2 arranged in the other housing 62. In addition, there is provided a volume of eluent 23 arranged in the other housing 63, the capacity of which is predetermined and known in advance.

These volumes/products are contained directly in housings of the body 10 or contained in sachets which are arranged in housings of the body. In the sachet/blister configuration, these sachets are prepared in advance and have a very specific capacity. The liquid of interest is contained in this sachet, for example in aluminum film, and there is no direct contact between the body of the wafer and the liquid of interest before actual use by piercing/tearing of the sachet .

The content of these volumes of liquid can be poured out and directed towards the extraction membrane 2 through the aforementioned channels 61a, 62a, 63a as will be seen later, in response to pneumatic activation.

In the example shown, the first and second volumes V1, V2 of rinsing liquid R1, R2 of the housings 61, 62 have a volume of between 100 microliters and 300 microliters. The invention also works for smaller flushing volumes and also for larger volumes.

Regarding the volume (denoted V3) of eluent 23 from housing 63, it can be between 30 microliters and 50 microliters.

Regarding the eluent, we will preferably choose a particular water, namely distilled water free of RNase and Dnase, i.e. devoid of Rnase and Dnase, called “Dnase/Rnase free water”. In another example, a Tris/EDTA buffer is used. For the eluent, one can also choose as eluent a compound known as Ibuffer™ from Optigene™.

The eluent has a low ionic strength and has a pH > 7.

For more details on the type of eluent used here, reference may be made to document EP1463744.

Furthermore, in the example illustrated, the test support comprises a system of valves 5 to direct a flow of fluid from the outlet of the extraction membrane selectively towards the recovery tank 18 or towards the zone(s) feedback via transfer channel 44.

In one example as shown, the test stand comprises two diverter valves which serve to direct or stop a flow of fluid circulating in the channels 13.

More precisely, the two valves make it possible to direct a flow of fluid from the extraction membrane 2 selectively towards the recovery tank 18 or towards the reaction zone.

The first bypass valve 51 can selectively close access to the recovery tank 18. The second bypass valve 52 can selectively open access to the reaction zones. The two bypass valves 51, 52 are arranged in parallel at the outlet of the housing 24, so that the activation of one or the other makes it possible to selectively guide the fluids towards the recovery tank 18 or the reaction zones. .

As described previously, the valve system 5 can comprise different configurations, such as a single valve which makes it possible to selectively send an input to the two channels previously described.

The diverter valves 51, 52 are in the example illustrated valves of the membrane type. In fact, a deformable membrane selectively opens or closes a passage between two neighboring orifices, the membrane being placed in a closed housing, the two orifices being the only passageways available for the fluid. Alternatively, the bypass valves 51, 52 are tap-type valves, the rotation of which can be activated pneumatically.

The diaphragm valve has been shown in a normally open configuration, but it is also possible to use a normally closed configuration diaphragm.

The air outlet 16 is arranged at the bottom or in communication with the bottom of the recovery tank, while the optional intermediate air outlet 17 is arranged near the inlet of the recovery tank. Both can be equipped with a Goretex™ capsule or equivalent which prevents liquid spillage while allowing air to pass through.

For the air outlet 16 from the bottom of the tank, depending on the configuration of the tank and in particular if it has an excess volume compared to the storage requirement, the Goretex™ cap is optional.

Transfer channel, reaction zones and read zone

In addition, the test support 1, and in particular the reaction zone, comprises a first reaction zone 31 with a first test reaction mixture 33 positioned in a reaction housing. More specifically, the first reaction zone contains a porous media 35 containing the first test reaction mixture 33.

Optionally, there is provided in the reaction zone a second reaction zone 32 a second control reaction mixture 34. In practice, the second reaction zone contains a porous media 35 containing a second control reaction mixture 34. The first and second reaction zones are respectively arranged in a second housing 72 and in a fourth housing 74. The second housing 72 and the fourth housing 74 form part of the reaction housing.

We will therefore speak more generally of a reaction housing (or zone), whether it comprises one, two or more reaction housings (or zones), and/or the distribution reservoir 38.

According to the configuration promoted here, the transfer channel 44 connects the first housing 24 to the reaction housing 72, 74. There is provided a sampling membrane 4 arranged in a third housing 73, the third housing 73 being placed in fluid communication with transfer channel 44.

The sampling membrane 4 is sized so that only part of the fluid flow passing through the transfer channel 44 in normal use is sampled by the sampling membrane 4. More specifically, the sampled part comprises the front of the flow. Concretely, when a flow of fluid passes through the transfer channel 44, the front of the flow is picked up by the sampling membrane 4. Once the latter is saturated, it stops absorbing and the rest of the flow joins the reaction zone. .

In particular, the dimensioning of the sampling membrane 4 is done with respect to the volumes of fluid present on the wafer. More specifically, the dimensioning of the sampling membrane 4 is done with respect to the volume of eluent alone, and even more specifically with respect to the volume of eluent passing through the extraction membrane. The volume of eluent can be the volume of eluent V3 stored in the housing 63. In one embodiment, the sampling membrane 4 can absorb between 5% (or 10%) and 90% of the volume of eluent stored on the test plate 1.

By sizing, we mean in particular the size of the sampling membrane 4 and/or the surface of the interface with the transfer channel 44, and/or the absorption capacity of the material of the sampling membrane 4.

In an embodiment illustrated in the figures and particularly visible in FIG. 11, the third housing 73 takes the form of an enlargement of the transfer channel 44 and the sampling membrane 4 is positioned in said enlargement. By the term enlargement is meant in practice an enlargement of the cross section of the transfer channel 44.

In this way, the transfer channel 44 is not obstructed by the sampling membrane 4 but the fluidic interface between the transfer channel 44 and the sampling membrane 4 is enlarged. In other words, a wall of a portion of the transfer channel 44 is formed by the sampling membrane 4. This allows the fluid front passing through the channel to be effectively captured, until the sampling membrane 4 is saturated. In one example, the interface between the internal volume of transfer channel 44 and sampling membrane 44 extends at least 3mm, or 5mm or even 8mm in length.

The sampling membrane 4 can have a shape substantially similar to that of the third housing 73, or even slightly larger (for example homothetically), so that the sampling membrane 4 can be inserted by slightly deforming it into the third housing 73, this which holds it in place.

In one embodiment of interest, the sampling membrane 4 is arranged downstream of the valve system 5 and upstream of the first reaction zone 31. More particularly, in the case of a valve system 5 with the first valve of bypass 51 and the second bypass valve 52, the sampling membrane 4 is arranged downstream of the second valve 52.

The sampling membrane 4 can be obtained from a cellulose material (for example cotton fibers, such as cotton linter). Alternatively, the sampling membrane 4 can be obtained from a material of polyvinylidene fluoride ('PVDF'). Other porous materials can also be selected. The water absorption of the material can be between 50 mg/cm ² and 150 mg/cm ² Conclusive tests have been obtained for an absorption of the material of 99.2 mg/cm ² (with a thickness of 954 µm). The material used for the tests was cotton linter (“cotton linter”).

With reference to FIGS. 3 and 4, the sampling membrane 4 can rub shoulders with the transfer channel 44 over a distance L4 greater than N times a diameter (if circular section) or a dimension (width or height, if rectangular section) of the section cross section of the transfer channel 44, N preferably being greater than 3, or 5 or even more. In one embodiment, the transfer channel 44 has a section of 0.5mmx0.3mm for an interface extending over almost 10mm, i.e. an N approximately equal to 20 (the entire length of the membrane can be interface). This makes it possible to determine the surface of the aforementioned interface. This characteristic is notably permitted when the third housing 73 is formed by enlarging the transfer channel 44. The transverse dimensions of the transfer channel are denoted D3 and H3.

H3 can be between 0.3 mm and 2 mm. D3 can be between 0.2 mm and 0.5 mm.

According to an embodiment illustrated in Figure 4, the depth H3 of the transfer channel may be greater than the depth H4 of the third housing 73. It is noted that the depths are along the Z axis.

According to another embodiment illustrated in Figures 12 and 13, the depth H3 of the transfer channel 44 may be lower than the depth H4 of the third housing 73.

The height H2 represents the height of the insert body. According to one embodiment (FIG. 4), H3 can be between 50% and 90% of H2. According to another embodiment (FIGS. 12 and 13) H3 can be between 20% and 50% of H2.

As indicated above, the sampling membrane 4 can have an absorption capacity representing a volume of between 10% and 90% of the predefined volume V3 of eluent of the housing 63.

In some embodiments, the sampling membrane may even have an absorption capacity representing a volume of between 20% and 70% of the predefined volume of eluent, or even a volume of between 30% and 60% of the predefined volume of eluent V3.

In one embodiment, the sampling membrane 4 can have a parallelepipedal shape, as illustrated in FIGS. 1 and 2, 3 and 4. In another embodiment, the sampling membrane 4 can have another shape, in particular to optimize the available space (L-shape for example, as illustrated in FIGS. 11 and 12).

The height H4 represents the height of the sampling membrane 4,

The volume generally occupied by the sampling membrane is defined by the product H4 x L4 x D4. D4 is the dimension of the sampling membrane in the direction transverse to the canal, denoted Y. (cf Fig 4).

Depending on the pore diameter and the void ratio, the absorption capacity of the membrane can be expressed by K x H4 x L4 x D4.

K being a coefficient between 0.5 and 0.8. K can be considered as capture rate.

It is possible to ensure that K×H4×L4×D4 makes it possible to absorb between 5 and 90, or even 20% and 70% or else 10 and 50µ of the predefined volume V3 of eluent. In an example of interest, K×H4×L4×D4 is chosen so that the sampling membrane absorbs between 30% and 60% of the volume V3.

In one embodiment, sampling membrane 4 is in fluid communication only with transfer channel 44. This means that housing 73 forms a cul-de-sac or dead end when viewed from transfer channel 44. Fluid communication from transfer channel 44 to third housing 73 is a fluid impasse.

The reaction housing 72, 74 of the test support 1 is included in a thermal interaction zone 126 with the analysis station 8. The thermal interaction zone 126 corresponds to a region of small surface area which is preferentially heated by the analysis station heater (described below). In particular, the thermal interaction zone 126 can be at most 5 cm ² , or even 4 cm ² . In one embodiment, the thermal interaction zone 126 further comprises the first housing 24 (which comprises the extraction membrane 2).

In particular, to limit optical interactions, the sampling membrane 4 is located outside the thermal interaction zone 126, but also outside an optical reading zone (see the optical devices described below). Indeed, the sampling membrane 4 can comprise molecules of interest, captured during sampling, which react during heating.

As visible in Figure 12, the third housing 73 does not extend over the entire thickness of the test wafer 1 but only over less than half. This allows you to arrange channels and components on either side of the wafer.

In the examples illustrated, the reaction housing 72, 74 comprises a small distribution reservoir 38 arranged adjacent to the second and fourth housings 72, 74 to indirectly feed the porous media of the housings 72, 74. Thus the porous media of the zones of reaction 31, 32, i.e. the discs of the first and second reaction mixtures 33, 34 are supplied with eluate by the arrival of this eluate from the transfer channel 44 in the distribution tank 38, without this eluate passing through the discs.

According to an advantageous optional arrangement, in each disc of reaction mixture 33, 34, a radial projection is provided for the capillary pumping function. Only the radial projection is bathed directly by the flow of eluate. The discs are not bathed by the flow of eluate, they are supplied with eluate by capillary pumping. It is noted that the second and fourth housings 72,74 which contain the discs have a single input/output port through which the radial projection passes, in other words said housings are in a dead-end configuration. It is noted that the contact interfaces between the discs and the reservoir are minimized, as are the dead volumes around the discs so as to limit the diffusion of the reagents contained on the discs.

Both test and control discs can be chosen from Whatman GF/DVA references from Cytiva™ (previously GE Healthcare™ Life Sciences™). For example, the discs have a diameter between 5 mm and 6 mm and a thickness between 0.7 mm and 1 mm.

According to one example, each disk occupies the entire slot in which it is placed; there is no clearance or space available for eluate to lodge. According to a variant, provision is made for the air initially contained in the disc to be able to escape during the rehydration of the disc. For example, a groove is provided under the discs and an escape passage to facilitate this air escape.

Included reagents are in one example: H2O, dNTPs, 10x isothermal buffer, MgSO4, betaine, intercalating agent, primer mix, Bst 2.0 W S enzyme, and AMV-RT enzyme. In one example, this may be the WarmStart LAMP Kit from New England Biolabs™.

The test reaction mixture contained in the porous media thus comprises, in one example, the reverse transcriptase which makes it possible to transcribe the RNA into DNA, the set of primers specific to the DNA of the pathogen, the DNA polymerase, a buffer isotherm containing deoxynucleotide triphosphates (dNTPs), necessary for DNA amplification, MgSO4 acting as a cofactor and reaction catalyst, as well as betaine, an additive often used to improve the amplification of DNA sequences. Finally, a fluorophore or a colorimetric probe is included to reveal the amplification reaction when it takes place.

The first and second reaction mixtures 33, 34 are in freeze-dried form. This shape is stable and allows long storage of new test media before use.

The first and second reaction mixtures 33, 34 are presented as a general disc shape.

According to an advantageous optional arrangement, the porous medium 35 is formed from paper. Its ability to absorb RNAse- and/or DNase-free water (eluent) is known, controlled and repeatable, which contributes to the reliability of the test.

The result is read by optical measurement, in particular a first optical device 98 arranged in the analysis station 8 opposite the first reaction zone 31 and a second optical device 99 arranged in the analysis station. analysis 8 vis-à-vis the second reaction zone 32. There is provided in the analysis station 8 an illumination element, common or not, and an imaging device, an optical sensor or photodiodes to receive radiation reflected (or transmitted) by the porous media of the first and second reaction mixtures 33, 34.

In the example shown, the fluorescence properties of the porous media where the nucleic acids have multiplied are used.

Said first and second reaction mixtures allowing the amplification and detection of one or more nucleic acid sequences.

It is noted that a result reading zone is provided, comprising at least the first and second reaction zones 31, 32. In the case where the body is formed of translucent material, it can be considered that the reading zone is also wider than the wafer itself. If the substrate used for the body is not transparent or not sufficiently translucent, a specific transparent window can be provided for reading the result.

The fluorescence response of the first and second reaction zones is determined to derive a test result therefrom.

More specifically, the fluorescence response of each disc before and after heating is compared. If the fluorescence response of the control disc is weak then the test is declared invalid.

If the fluorescence response of the control disc is high and the fluorescence response of the test disc is low, then the test is declared negative.

If the fluorescence response of the control disc is high and the fluorescence response of the test disc is also high, then the test is declared positive.

Indeed, the first test zone amplifies the DNA specific to the pathogen sought. The second area (control disc) is to amplify non-specific RNA from the sample. In fact, the two mixtures reactions are identical except for the set of primers in place. In the case of the control, these are primers not specific to the virus sought (pathogen sought) but cooperating with an RNA sequence always present in the sample/incident eluate. The purpose of the control is to confirm that both the extraction step and the amplification step have taken place, thus eliminating the option of a false negative.

It is not excluded to deliver a richer result than a binary result. Provided that the fluorescence response of the control disc is high, the fluorescence response intensity of the test disc can be translated into an index ranging from 0 to 1.

It should be noted that instead of a fluorescence response measurement, other optical methods could also be used, such as a colorimetric method, a photometric method, a transparency coefficient method.

It should also be noted that the reaction mixtures can be analyzed optically after reactions, either by measuring reflectivity or by measuring transmissivity (transmitter and receiver on either side of the wafer).

It should also be noted that a reference optical reading is carried out before heating, to allow the "zero" of the optical system to be taken, under ambient radiation conditions and with the wafer as it is in transparency. After reaction heating and cooling, the new optical measurements are taken with the reference measurement before heating as a reference (differential method).

Analysis station and interface

Analysis station 8 looks like a parallelepipedic box. In one example, this box is close to a cubic shape. In one example, the side of this cubic shape has a length of between 25 cm and 40 cm. The analysis station could however have any shape.

The analysis station 8 comprises a cover 80 mounted by means of a hinge, and a cover locking system is provided. Indeed, when the test operations are in progress on a test wafer, the cover is closed and locked. When the test is finished and the result is obtained, the locking system unlocks the bonnet. An operator can then open the cover, remove the test wafer and place a new test wafer to be processed on the base. The analysis station 8 includes an air pump. This can be a pressure-generating pump or a vacuum-generating pump, depending on the possible configurations with regard to the station's test support.

The analysis station 8 comprises a heating device 96. The heating device 96 heats the test wafer 1 in a thermal interaction zone 126. A first heating portion 96a heats up opposite the housing 24 of the membrane of extraction 2 and a second heating portion 96 opposite the reaction zones, in particular below the housings 72, 73.

Note that the heating device 96 is not located opposite the sampling membrane 4, which is outside the thermal interaction zone 126.

The heating device can be complemented by a cooling device. The analysis station 8 includes a temperature sensor to regulate the control of the heater. The heating device can be a heating resistor, a Peltier effect cell, or even comprise one or more black body(ies) arranged near the reaction zones and infrared lighting to heat the said ) black body(ies).

The station controls the heating device to have a plateau at a certain temperature and over a certain duration. For example, the bearing temperature is between 50°C and 70°C. In one example, the plateau temperature is 65°C. The duration of the stage is between 20 minutes and 40 minutes. The analysis station can operate with a predetermined dwell time (parameter). In an alternative mode, the end of the stage can depend on the optical analysis of the reaction zones, for example the result can be read before the predefined duration, or in the opposite case, the optical analysis of the reaction zones can allow to identify an error.

Several actuators are provided in the test station to push on the volumes 61, 63 and/or activate the controlled valves, in particular the bypass valves, according to the various embodiments. More precisely, a first actuator 91 makes it possible to exert a mechanical action on the volume of the first rinsing volume 61. A second actuator 92 makes it possible to exert a mechanical action on the volume of the second rinsing volume 62. A third actuator 93 makes it possible to exert a mechanical action on the volume of eluent 63. A fourth actuator makes it possible to exert a mechanical action on the first bypass valve 51. A fifth actuator makes it possible to exert a mechanical action on the second diverter valve 52. The actuators can be called 'push-buttons'.

An LED illuminates the disc 35 first reaction zone 31 and the radiation received in return is picked up by one or more photodiodes 98.

On the other disc, the same process takes place with one or more photodiodes 99, for disc 35 second reaction zone 32.

The heating device 96 can be combined with the optical cells 98,99 (transmission and/or reception) to form a combined heating and reading device 120.

The document PCT/FR2021/050545 describes in more detail the operating logic of the test wafer 1, in particular the activations of bypass valves.

Figures 8A and 8B illustrate a bypass valve 51 in the form of a deformable membrane, which selectively opens or closes a passage between two channels 131, 132.

The deformable membrane delimits a chamber 151 from below. The body of the wafer delimits the chamber 151 on the sides and on the top, except at the places where the channels open out, with a first port 131a (first mouth) and a second port 132a (second mouth). In FIG. 8A the deformable membrane 51 is at rest (flat shape at rest) and the passage between the two mouths 131a, 132a is open (circulation in dotted arrow). In FIG. 8B under the effect of a mechanical pushing action by the element denoted 94, the deformable membrane 51 is bent and bears on the ports (two mouths) 131a, 132a; the passage is between the two mouths 131a, 132a is then closed.

With reference to FIG. 9, the pouring of a rinsing liquid contained in a sachet 37 (rinsing liquid or eluent) has been illustrated. The upper end of the actuator 94 pushes on the pouch/bag. There are pins 36 in the bottom of the housing 74. When the bag 37 is pressed, the said pins 36 sink into it and tear the bag, thus releasing the liquid contained therein which flows into the channel 131.

General operation The ST test station works as follows. The first step of interest consists of:

S1 - pass the biological sample to be tested ET in liquid form through the extraction membrane 2. In practice, the air pump pushes the biological sample ET from the upstream reservoir 11 towards the reservoir recovery 18, through the extraction membrane 2. The first bypass valve 51 is open while the second bypass valve 52 is closed. The nucleic acids and other molecular species are retained by the extraction membrane 2. It is noted that the air outlet 16 allows the air present in the recovery tank to escape as the recovery tank fills with fluid. Then we will proceed to rinsing, in a step denoted S2.

52- the test system passes a first rinsing liquid R1 through the extraction membrane 2, and directs it downstream towards the recovery tank 18. The first bypass valve 51 is open while the second bypass valve 52 is closed

Optionally, a second rinsing pass can be used. Then we will proceed to the drying, in a step denoted S3.

53- the test station passes air through the extraction membrane 2, to dry it. The station sends pressurized air into the air inlet 14. The air can exit at the bottom of the tank through the outlet 16 but also the air can exit through the intermediate air outlet 17. The step S3 can also include: heating the membrane, to accelerate the drying, in particular in the embodiments where it is in the thermal interaction zone. This heating step can be alternative or complementary to the step of passing air through the extraction membrane to dry it. The extraction membrane is then eluted, in a step denoted S4.

54- pass a volume of eluent through the extraction membrane 2, and direct the eluate which comes out downstream towards the transfer channel 44. The first bypass valve 51 is then closed while the second bypass valve 52 is open. The front of the eluate stream is then captured by the sampling membrane 4, as described before, and the rest of the eluate stream is directed to the reaction zone, for analysis. The analysis consists in particular of heating the reaction zone to a predefined temperature then waiting for the cooling to finally illuminate the reaction zone (the first and second reaction zones) to receive in return a level of fluorescence (using the optical devices 98, 99).

Claims

26 CLAIMS

1. Biological test support (1) for single use, intended to be used with a test station to form a biological test system (ST) for detecting the presence of one or more pathogenic species and/or of a sequence nucleic acid to be revealed, the test support comprising:

- an upstream reservoir (11) for receiving a biological sample to be tested (ET) in liquid form,

- an extraction membrane (2), arranged in a first housing (24), in fluid communication with the upstream reservoir (11),

- a reaction zone (31) comprising a reaction housing (72, 74) containing a test reaction mixture (33, 34),

- a transfer channel (44) connecting the first housing (24) to the reaction housing (72, 74),

- a sampling membrane (4) arranged in a third housing (73), in which the third housing is placed in fluid communication with the transfer channel (44) and the sampling membrane is placed adjacent to the transfer channel.

2. Biological test support according to claim 1, further comprising a predefined volume of eluent, and in which the sampling membrane (4) has an absorption capacity representing a volume of between 10% and 90% of the predefined volume of eluent.

3. Biological test support according to one of claims 1 to 2, in which the sampling membrane (4) borders the transfer channel (44) over a distance (L4) greater than 5 mm, or even 8 mm, or a distance greater than N times a diameter or a dimension of the cross section of the transfer channel (44), N being greater than 3, or 5, or 10 or 15.

4. Biological test support according to one of claims 1 to 3, further comprising at least a first volume of first rinsing liquid (R1).

5. Biological test support (1) according to any one of the preceding claims, in which the reaction zone (31) contains a porous reaction medium (35) arranged in a second housing (72) and containing the reaction mixture of test (33) in freeze-dried form.

6. Biological test medium according to one of claims 1 to 5, further comprising:

- a recovery tank (18), to recover the liquids resulting from the rinsing, and the overflow of eluate, and, for example - At least one air outlet (16,17), to facilitate filling the tank.

7. Biological test support according to one of claims 1 to 6, further comprising a valve system (5) for directing a flow of fluid from the extraction membrane (2) selectively towards the recovery tank (18) or to the transfer channel (44).

8. Biological test support according to claim 7, in which the third housing is located fluidically between the valve system (5) making it possible to direct the flow towards the reaction zone, and the reaction zone.

9. Biological test support according to any one of claims 1 to 8, in which the sampling membrane is formed of a material comprising at least one of: cellulose, polyvinylidene fluoride.

10. Biological test support according to any one of claims 1 to 9, in which the absorption capacity of the sampling membrane (4) is determined so that a flow of liquid passing through the transfer channel is partially collected by the sampling membrane.

11 . Biological test support according to any one of claims 1 to 10, in which the biological test support is configured to bring, via a connecting channel (11a), the biological sample to be tested (ET) into the membrane of extraction (2), then after rinsing, drying and eluting said extraction membrane, directing the resulting eluate, via the transfer channel (44), towards at least the reaction zone, a front portion of the eluate being trapped by the sample membrane without reaching the test reaction mixture, a next portion of the eluate reaching the test reaction mixture (35).

12. Biological test support according to any one of claims 1 to 11, in which the reaction housing (72, 74) is located in a thermal interaction zone (126) configured to be heated by the analysis station. , and wherein the third housing is located outside the thermal interaction zone (126).

13. Biological test support according to claim 12, in which the first housing (24) is arranged in the thermal interaction zone.

14. Biological test support according to one of claims 1 to 13, wherein the third housing (73) is formed by an enlargement of the transfer channel (44), the sampling membrane (4) being positioned in said enlargement.

15. Biological test medium according to any one of claims 1 to

14, in which the transfer channel (44) is not obstructed by the sampling membrane (4).

16. Biological test support according to any one of claims 1 to 15, in which a wall of a portion of the transfer channel (44) is formed by the sampling membrane (4).

17. Biological test support according to one of claims 1 to 16, having a generally flat parallelepipedic shape, with two main faces (10A, 10B) with an air inlet (14) and an inlet (12) in fluid communication with the upstream tank (11) to receive a biological sample to be tested (ET) and, preferably, all the interfaces are arranged on a single main face (10A).

18. Biological test system, comprising:

- an analysis station (8),

- one or more biological test supports (1) according to one of the preceding claims, the analysis station comprising at least one pneumatic connector configured to be coupled to the air inlet (14) of the biological test support.