EP2483689A2

EP2483689A2 - Disposable device for the detection of particles of interest, such as biological entities, detection system comprising said device and method for using same

Info

Publication number: EP2483689A2
Application number: EP10773650A
Authority: EP
Inventors: Bruno François Marcel WACOGNE; Christian Gérard Daniel PIERALLI; Wilfrid Hervé BOIREAU; Thomas Mangeat; Alain Léon René COAQUETTE; Lionel Pazart; Christian Louis Davrinche; Serge Piranda; Jean-François Maurice Louis DELFORGE
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: Centre National de la Recherche Scientifique CNRS
Priority date: 2009-09-29
Filing date: 2010-09-28
Publication date: 2012-08-08
Also published as: US20120264114A1; WO2011039430A2; FR2950699A1; WO2011039430A3

Abstract

The invention relates to a disposable device comprising all of the reagents necessary for the detection of one or more particles of interest, and which can be incorporated in a detection system including simple means for the automatic management of fluids. For this purpose, the invention relates to a disposable device for the detection of one or more particles of interest present in a liquid sample, said device comprising a substrate provided with: a chamber for capturing the particle(s) of interest to be detected; a fluid channel connecting, upstream, the capture chamber to a buffer solution container, a liquid sample injection means and a container of labelling probes that can be secured to the particle(s) of interest to be detected; and a fluid channel connecting, downstream, the capture chamber to a container for the recovery of liquids which, during use, can flow from the capture chamber.

Description

SINGLE USE DEVICE FOR DETECTION OF PARTICLES OF INTEREST SUCH AS BIOLOGICAL ENTITIES. DETECTION SYSTEM COMPRISING SAID DEVICE AND METHOD FOR IMPLEMENTING

IMPLEMENTATION. The invention relates to a disposable device for the detection of particles of interest, such as biological entities, to a detection system comprising said device and to a method of implementation.

The disposable device includes a portion specific to the biological entity to be detected.

The detection system includes a detection portion adaptable to the type of disposable device used.

Currently, the development of embedded systems, allowing rapid detection of particles of interest such as pathogens or other biological entities, is in full swing.

An embedded device is a standalone device that performs a predefined task. It must be easily transportable, consume enough energy to run on a standalone battery, and must have the shortest possible response time.

In addition, its implementation must be economical while ensuring impeccable hygiene and reliable results.

Currently, the detection of biological entities (mainly proteins and viruses), is carried out by means of massive devices to be located in specific laboratories. This detection takes several days and makes certain tasks very restrictive.

The techniques mainly used are ELISA (Enzyme-linked immunosorbent assay), PCR (Polymerase Chain Reaction or Polymerase Chain Reaction in French) or immuno-PCR and resonance systems. plasmon type Biacore. These systems are massive and / or expensive and they should in no way be used nomadically. Nevertheless, some miniaturization tests have been proposed.

Among these tests, mention may be made of the system developed by Ymeti and his colleagues (A. YMETI, J. GREVE, LAMBECK PV et al., "Fast, ultrasensitive virus detection using a Young interferometer sensor." Nano letters [online] 2006, vol 0, no 0, pp AD.)

This system is based on the phenomenon of interference of two light rays coming from the same source (monochromatic laser).

The device described in this article comprises at least two waveguides. On the surface of one of the guides, antibodies complementary to the virus to be detected are arranged. Then, the two light beams are sent into the waveguides. One of the guides is not modified by the presence of viruses, while in the other, the presence of virus attached to the antibodies slightly modifies the speed of propagation of the light. Interference at the output is then imaged on a camera and analyzed (Fourier transformation).

This device is fast, responsive and easy to use. It also has the advantage of not requiring the labeling of the molecules.

However, the interferometric technique proposed is based on the preparation of optical waveguides whose manufacture is delicate and expensive (micro-technology in clean room). In addition, the analysis of the interferometric signal recorded on the CCD sensor of the camera can be extremely delicate in the case of a simultaneous multi-detection system. Finally, no mention is made of the management of the different fluids required for detection.

This article therefore describes a principle device, expensive and incompatible with the requirements of a high-performance onboard device.

The objective of the present invention is therefore to provide a reliable, economical on-board apparatus which enables rapid detection and precise, without requiring special technical knowledge to use it.

For this, the invention proposes to provide a sufficiently economical device to be disposable, comprising all the reagents necessary for the detection of one or more particles of interest, and can be integrated into a detection system incorporating simple means of automatic fluid management.

To this end, the invention relates to a disposable device for the detection of one or more particles of interest present in a liquid sample, comprising a support provided with:

a capture chamber of the particle or particles of interest to be detected;

a fluidic channel connecting, upstream with respect to the direction of flow in use, the capture chamber:

o a pre-filled tank with a predefined volume of buffer solution;

o means for injecting the liquid sample;

o a pre-filled tank with a predefined volume of marking probes capable of attaching to the particle or particles of interest to be detected; and

- A fluid channel connecting, downstream with respect to the direction of flow in use, the capture chamber to a liquid recovery tank may flow, in use, from the capture chamber.

Thanks to the embedded system according to the invention, the unique use ensures impeccable hygiene and reliable results. In addition, automatic fluid management pre-integrated into the device allows for rapid implementation, including by non-specialized personnel.

The device and system described hereinafter may be used for any pathogen attached to a surface by a specific ligand-bound link and revealed by attachment of a probe allowing measurement (optical, magnetic, etc.). For each agent, it will be necessary to adapt the system (type of fluid, type of probe (s), fluid volumes, fluid management, type of detection, ligand-bound, etc.).

According to other embodiments:

The support may further comprise a reservoir of the liquid sample, arranged downstream of the sample injection means and upstream of the capture chamber;

The buffer solution reservoir and the marking probe reservoir may have a pressurization structure adapted to allow the flow, in use, of the buffer solution and the labeling probes to the capture chamber via the upstream fluid channel ;

• the sample reservoir may have a pressurizing structure adapted to allow the flow, in use, of the sample to the capture chamber via the upstream fluid channel;

The buffer solution tank and the marking probe tank may be made of a material that is sufficiently flexible to be deformed by an external pressure, between a storage volume and an ejection volume;

The reservoir of the sample may be of a material that is sufficiently flexible to be deformed by an external pressure, between a storage volume and an ejection volume;

The buffer solution reservoir and the marking probe reservoir may be of a sufficiently rigid material to allow, in use, an internal overpressure sufficient to respectively expel the buffer solution and the marking probes towards the capture chamber;

The reservoir of the sample may be of a sufficiently rigid material to allow, in use, an internal overpressure sufficient to drive the sample towards the capture chamber; the sufficiently rigid material may comprise a membrane made of a leaktight material which retains its seal after being pierced, preferably chosen from a silicone polymer, such as polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyvinyl chloride (PVC) , and Tygon®;

the injection means of the liquid sample may comprise a membrane made of a leaktight material which retains its leak-tightness, preferably selected from a silicone polymer, such as polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyvinyl chloride (PVC), and Tygon®;

the buffer solution tank, the sample tank and the marking probe tank may have a pressure-sensitive anti-return valve, allowing, in use, an opening of the valve when a threshold pressure is applied;

the recovery tank may have a volume at least equal to the sum of the volumes of the buffer solution reservoir, the secondary reservoir of the liquid sample and the reservoir of marking probes;

the capture chamber can present:

at least one functionalized surface with ligands, fixed on said surface, and capable of associating specifically with the particle or particles of interest to be detected; and

at least one surface adapted to allow, in use, the detection of the marking probes attached to the particle or particles of interest which have specifically associated with the ligands;

the support may further comprise at least one reagent reservoir of the liquid sample, arranged downstream of the sample injection means and upstream of the capture chamber;

the support may further comprise at least one reagent reservoir, arranged downstream of the reservoir of the liquid sample and upstream of the capture chamber; and or The device may further comprise a suction channel opening into the reservoir of the liquid sample and connectable to a pump for sucking the liquid sample from the injection means to the reservoir of the liquid sample.

The invention also relates to a system for detecting one or more particles of interest present in a liquid sample, said system being provided with a housing comprising:

a receptacle adapted to receive a previous single-use device;

means for pressurizing the pre-filled tank with a predefined volume of buffer solution and the tank pre-filled with a predefined volume of marking probes; and

a means for detecting the marking probes.

According to other embodiments:

The detection system may further comprise means for pressurizing the reservoir of the sample;

The detection system may comprise a receptacle adapted to receive a previous single-use device, and wherein the pressurizing means is a mechanical, electromechanical, pneumatic or hydraulic device adapted to deform the reservoirs;

• the detection system may comprise a receptacle adapted to receive a previous single-use device, and wherein the pressurizing means comprises means for injecting a pressurized fluid into the tanks; and or

• the detection system may further comprise a removable cassette adapted to receive the support of a previous single-use device, and intended to be inserted into the receptacle.

The invention also relates to a method for implementing a previous single-use device, comprising the following steps: a) injection of the sample to the capture chamber, to trap the particles of interest to be detected;

b) evacuate the sample to the liquid recovery tank; c) causing the flow of buffer solution from its reservoir to the capture chamber to flush said chamber of non-trapped particles;

d) evacuate the buffer solution to the liquid recovery tank;

e) causing the flow of the probes, from their reservoir to the capture chamber, so that they attach to the particles of interest trapped in the capture chamber;

f) evacuate the probes to the liquid recovery tank; g) causing the buffer solution to flow from its reservoir to the capture chamber to flush said chamber of unfixed probes;

h) evacuate the buffer solution to the liquid recovery tank;

i) measure the presence or absence of the probes, and therefore the particles of interest to be detected.

According to other embodiments:

Steps a) to d) can be performed sequentially until all the sample has been injected into the capture chamber;

Steps e) to h) can be performed sequentially until all the probes have been injected into the capture chamber;

Steps a), c), e) and g) can be performed by deforming, respectively, the reservoir of the liquid sample, the buffer solution reservoir and the probe reservoir; and or Steps a), c), e) and g) can be performed by pressurizing, respectively, the reservoir of the liquid sample, the buffer solution tank and the probe reservoir.

Other features of the invention will be set forth in the detailed description below, made with reference to the appended figures which represent, respectively:

FIG. 1, a schematic plan view of a first embodiment of a disposable device according to the invention;

Figure 1a, a schematic plan view of a variant of the embodiment of Figure 1;

FIG. 2 is a diagrammatic plan view of a second embodiment of a disposable device according to the invention;

Figure 3 is a schematic plan view of a third embodiment of a single-use device according to the invention;

- Figure 3a, a schematic plan view of the embodiment of Figure 3 and provided with a liquid suction means to the liquid sample reservoir;

Figures 4 to 9, schematic perspective views of the implementation of a first embodiment of a detection system according to the invention; and

Figures 10 to 12, schematic perspective views of the implementation of a second embodiment of a detection system according to the invention.

Figure 13 is a schematic perspective view of a fourth embodiment of a disposable device according to the invention;

Figure 13a is a schematic perspective view of the embodiment of Figure 13 in a bottom view;

Figure 14 is a schematic perspective view of a variant of the fourth embodiment of Figure 13;

FIG. 15, a schematic perspective view of an enlargement of the device of FIG. 14 Figure 16 is a schematic perspective view of the embodiment of Figure 14 in a bottom view;

Figure 17 is a schematic perspective view of a fifth embodiment of a device according to the invention and comprising a reagent reservoir;

Figure 18 is a schematic perspective view of the embodiment of Figure 17 in a bottom view; and

Figure 19 is a schematic perspective view of a variant of the embodiment of Figure 17 in bottom view, and including a liquid suction means to the liquid sample reservoir.

The detection system according to the invention is illustrated by the detection of Cytomegalovirus in infants but it can be used for the detection of any particle capable of binding specifically to a ligand attached to a capture surface and to be revealed by fixation. a specific marking probe.

The detection system according to the invention is not limited to immuno-capture reactions alone, but it can also be used to generate and detect other types of reactions: chemical, enzymatic, etc.

Thus, the detection system according to the invention can advantageously be used for the detection of biological entities such as bacteria, viruses, proteins, DNA or RNA strands, etc.

The detection is advantageously a fluorescence detection.

The first embodiment of a disposable device 100 according to the invention, illustrated in FIG. 1, comprises a support 101 provided with a chamber 110 for capturing the particle or particles of interest to be detected. The support 101 comprises a fluid channel 120 connecting, upstream with respect to the direction of flow F1, the capture chamber 10 to a pre-filled reservoir of a predefined volume of buffer solution 130, to a means of injection of the liquid sample 140 and a tank 150 pre-filled with a predefined volume of marking probes. These probes are included in a transport liquid and are likely to specifically bind to the particle or particles of interest to be detected.

The support 101 also comprises a fluid channel 160 connecting, downstream with respect to the direction of flow F 2, the capture chamber 1 0 to a tank 170 for recovering liquids that may flow, in use, from the capture chamber 1 10.

Advantageously, the support 101 is made of a material suitable for producing microfluidic channels. The characteristic dimensions of these channels are of the order of a few hundred microns but can be up to a few tens of nanometers. To achieve these channels, lithography and etching of a material such as glass, silicon or quartz can be used. It is also possible by molding or stamping polymeric materials.

The upstream fluidic channel 120 comprises three fluidic channels 120a, 120b and 120c connecting, respectively, the reservoir of buffer solution

130, the injection means 140 and the marking probe reservoir 150.

In the embodiments of FIGS. 1 to 3, the channel 120 has a main channel 120a into which the secondary channels 120b and

120c. Alternatively, in one embodiment illustrated in FIG. 1a, the three channels 120a, 120b and 120c of the fluidic channel 120 may be independent and each lead into the capture chamber 1 10.

Other conformations are conceivable.

In the embodiment of FIG. 1, the disposable device comprises buffer solution volumes and predefined probes, determined by the volume of the reservoirs 130 and 150. The volume of the liquid sample is, for its part, determined by the user who injects this sample by the injection means 140.

The buffer solution and probe tanks are pre-filled during manufacture, so that the user does not have to handle the reagents. It is sufficient for him to select the single-use device adapted to the reaction it wants to perform, to inject a predefined volume of the test solution, and to perform the measurement (as described later).

Advantageously, the disposable device according to the invention further comprises a reservoir of the liquid sample arranged downstream of the sample injection means and upstream of the capture chamber. This embodiment is illustrated in FIGS. 2 and 3.

This sample reservoir 145 makes it possible to inject the sample of liquid into the capture chamber 1 0 with a predetermined volume, that of the reservoir 145. In other words, before the actual detection and the injection of the sample towards the capture chamber, the user fills the reservoir 145 with the liquid sample. For example, it can fill the reservoir 145 with a piston device such as a syringe (see Figure 8).

More generally, the injection means 140 of the liquid sample, whether it is directly connected to the upstream fluid channel 120 or that it is connected via the reservoir 145, comprises a membrane in one waterproof material retaining its seal after being pierced. Preferably, this material is chosen from a silicone polymer such as polydimethylsiloxane (PD S), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), Tygon® (manufactured by Saint-Gobain), etc. .

In the embodiment of FIG. 2, the injection means 140 and the probe tank 150 are arranged on the same side of the support 101. The embodiment of FIG. 3 has an injection means 140 and a reservoir of probes 150 arranged on either side of the support 101.

The injection means may be directly connected to a source of liquid to be sampled for analysis: for example the gastric aspiration circuitry of a newborn. Advantageously, as represented in FIG. 3a, the device according to the invention further comprises a suction channel 141 opening into the reservoir of the liquid sample 145 and intended to be connected to a pump P for sucking up the sample of liquid from the injection means 140 connected to the source of the liquid to be sampled, towards the reservoir of the liquid sample 145.

Such an arrangement is particularly interesting since it prevents a nurse from having to perform additional actions compared to those currently practiced. Indeed, the gastric suction circuitry comprises a tubular connecting the newborn to a bottle, itself connected by a tubular to a pump P. The device according to the invention can therefore replace the bottle and allow an analysis of the gastric fluid simple to implement and does not require additional gesture. Of course, a non-return valve (not shown) is provided to prevent the depression generated by the pump from drawing other liquids contained in the device through the microfluidic channels to the sample reservoir.

Alternatively or in combination, the disposable device according to the invention may also comprise at least one reagent reservoir arranged downstream of the injection means 140 of the sample and upstream of the capture chamber 110 (see the description of embodiments of figures 17 to 19 by analogy). In one embodiment having a sample reservoir 145, said at least one reagent reservoir is arranged downstream of the reservoir of the liquid sample 145 and upstream of the capture chamber 110. In this case, the channel 120b connects the reagent reservoir to the main track 120a or to the capture chamber 110 if each reservoir is connected independently to this chamber (by analogy with the embodiment of Figure 1a). Of course, non-return valves (not shown) are provided to ensure the flow of different liquids to the capture chamber 1 0.

This reagent reservoir may be provided with a filling nozzle, but, advantageously, the reservoir is pre-filled with a predefined volume of reagent. This avoids the person who implements the disposable device according to the invention to have an additional gesture to make, may cause analysis errors in case the reagent filled by the user is not suitable for analysis.

In all the embodiments of a disposable device according to the invention, the reservoirs (130, 145 and 150), as well as the insertion means 140, can be arranged anywhere upstream with respect to the capture chamber 10. Nevertheless, it will be avoided to arrange the channels 120a, 120b and 120c so that they face one opposite the other. Indeed, in this case, a liquid ejected from a tank could flow to the path opposite it and not to the capture chamber 110.

Advantageously, the buffer solution tank 30, the reservoir of marking probes 150 and, for the embodiments of FIGS. 2 and 3, the reservoir 145 of the sample, have a structure allowing them to be pressurized. This pressurizing structure makes it possible, in use, for the flow of the different liquids (buffer solution, marking probes and, possibly, liquid sample) to the capture chamber via the fluidic channel 120.

The reservoirs containing the buffer solution and the marking probes are filled during the manufacture of the disposable device. The reservoir for collecting the sample is filled by the operator.

The recovery tank 170 has a volume at least equal to the sum of the storage volumes of the buffer reservoir, the secondary reservoir of the liquid sample and the reservoir of marking probes.

In order to make it possible to store the volume of the different liquids, the recovery tank 70 can be kept under vacuum, or initially crushed by a pressurizing means which is actuated in the opposite direction (expansion) of the other pressurizing means. and as their action (compression tanks 130, 145, 150 by means of pressurizing 420, see below, with reference to Figures 9, 11 and 12). An alternative would be to provide an air exhaust, either by a valve or by the use of a porous material in air, but not liquids. The fact that the disposable device according to the invention incorporates all the reagent reservoirs necessary for the detection of particles of interest has many advantages. Thus, each device comprises calibrated volumes of reagents (buffer solution, marking probes and, optionally, liquid sample) which allows an optimal reaction without the user having complex handling to do. In addition, these predefined volume tanks ensure the unique use of each device.

The recovery tank 170 makes it possible to avoid rejecting the reagents (buffer solution, marking probes and liquid sample) out of the device. In particular, it avoids providing such a reservoir in the detection system (see below), tank in which all the reagents from the detection would be mixed on several disposable devices.

According to a first preferred embodiment, the buffer solution tank 130, the marking probe reservoir 150 and, for the embodiments of FIGS. 2 and 3, the reservoir of the sample 145 are made of a material that is sufficiently flexible to be deformed by external pressure applied to the tanks. Thus, each reservoir has a first volume, called "storage", in which all the liquid fills the reservoir, and a second volume, called "ejection", less than the storage volume, and in which the liquid is ejected from the tank to the capture chamber.

The capture chamber 110 has at least one functionalized surface with ligands, fixed on said surface, and capable of specifically associating with the molecule or molecules of interest to be detected.

The operating principle of such a capture chamber is based on an immuno-capture reaction on the functionalized surface of the chamber. This surface is covered with ligands such as antibodies specific for the particles to be detected, such as, for example, Cytomegalovirus. Then, the liquid biological sample (blood, saliva, urine, gastric juice, etc.) is injected via the injection means 140 to the capture chamber. Thus, the sample is brought into contact with the surface covered with ligands. If particles of interest are present in the sample, they are trapped by the ligands. In the case of Cytomegalovirus or, more generally, biological molecules, this trapping step is carried out by immuno-capture using antibodies (the ligands). When the sample has remained in the capture chamber long enough, it is discharged to the liquid recovery tank 170. Advantageously, the capture chamber is rinsed with buffer solution flowing from the tank 130. evacuate the non-trapped particles. The rinse buffer solution is then discharged to the liquid recovery tank.

After this step, it causes the flow of the marking probes from their reservoir to the capture chamber. These probes are adapted to bind specifically to the particles of interest trapped by the ligands in the capture chamber 110. In the case of biological particles to be detected, the probes are preferably composed of labeled specific antibodies. This labeling is preferably carried out by grafting fluorescent molecules onto the antibodies. Thus, if biological particles were trapped in the previous step, the probes are fixed on the trapped particles. Otherwise, the probes are rinsed with buffer solution to the liquid recovery tank 170. Other types of markings may be used such as magnetic markers, radioactive markers, etc.

Then, the presence or absence of the probes and therefore the particles of interest to be detected are measured. For this purpose, the capture chamber 10 has at least one surface adapted to allow this detection of the marking probes. In the case of a fluorescence detection, the capture chamber has a transparent cover at the excitation and emission wavelengths of the fluorescent particles used.

The aforementioned method of implementing the disposable device according to the invention can be realized in a single step. In other words, the sample can be injected at one time, and the marking probes can be injected equally at one time.

However, preferably, the implementation method of the device according to the invention is carried out sequentially. In this case, the sample is injected in several phases, between each of which the chamber can optionally be rinsed with buffer solution. Likewise, the marking probes can be injected sequentially, that is to say they are injected in several phases, between each of which the capture chamber is rinsed with buffer solution.

This implementation in a sequential manner ensures a better capture of the particles of interest and therefore a better sensitivity of the device.

In the embodiments of Figures 1 to 3, the device according to the invention has a substantially rigid support 101 incorporating the fluidic channels and the capture chamber. The various tanks 130, 150, 170, and possibly 145, are reported on this support 101 and in fluid connection 120 and 160. The manufacture of such a structure is very advantageous in terms of manufacturing cost in the case where it uses molding techniques, hot stamping etc. However, it can be fragile at the connections between the tanks and the support.

For this purpose, provision is made to incorporate this device 100 in a removable cassette 200, as shown in Figure 4, to stiffen the device. This removable cassette 200 comprises means for holding the device 210. In the embodiment of FIG. 4, these holding means are constituted by a cavity 210 complementary to the device 100. Preferably, as illustrated in FIG. 200 includes a cover 300 holding the device 100 in the cassette 200.

To implement the above method, the invention proposes a detection system of which two embodiments are illustrated, respectively, in FIGS. 6 to 9 and 10 to 12. In the first embodiment, the detection system comprises a housing 400 comprising a receptacle 410 adapted to receive a disposable device 100 according to the invention. In FIG. 6, this receptacle 410 is constituted by an additional imprint of the cassette 200. Once the cassette 200 is positioned in the receptacle 410 (FIG. 7), the sample is injected into the disposable device according to the invention. via the injection means 140 of the sample (Figure 8). The injection is performed, in this example, with a syringe S.

The detection system also comprises a pressurizing means 420 of the different reservoirs of the disposable device. In FIG. 9, the pressurizing means 420 comprises a buffer solution pressurizing mechanism 420a, and two pressurizing mechanisms 420b and 420c, respectively, of the sample and probe reservoir. marking.

The detection system also comprises a detection means 430 of the marking probes. It is thus clear that the removable cassette is able to allow the pressurization of the reservoirs of the device that it contains.

The pressurizing means can be mechanical, electromechanical, pneumatic or hydraulic devices, adapted to deform the tanks by crushing. For example, these pressurizing means are pistons.

A second embodiment of a detection system according to the invention is illustrated in Figures 10 to 12. In this embodiment, the housing 500 comprises a receiving slot 510a adapted to receive a disposable device according to the invention . In the illustrated embodiment, this disposable device 100 is combined, as in FIGS. 4 and 5, with a removable cassette 200. It is therefore this cassette 200 which is inserted into the receiving slot 510a. More generally, the cassette 200 can be placed horizontally or vertically and even in any orientation required by the internal structure of the detection system. Figures 1 1 and 12 illustrate the interior of the system of Figure 10. Thus, this system comprises a receptacle 510b facing the slot 510a (not shown in these figures). This receptacle is provided, moreover, with holding means 520 of a detection means 530 of the marking probes. Of course, these holding means 520 could be independent of the receptacle 510a and fixed elsewhere in the system, for example on the frame 501.

Advantageously, these holding means allow a removable attachment of the detection means 530. In the example illustrated, the holding means 520 are cylindrical pins cooperating, slightly in force, with grooves 531 disposed on the detection means 530. detection means 530 can therefore be removed from the housing via a cover 540 (FIG. 10), and replaced by another detection means.

This structure makes it possible either to replace a defective detection means or to change the type of detection means.

The detection system also comprises means for pressurizing the various reservoirs (buffer solution, marking probes and, optionally, sample). In FIG. 12, these pressurizing means 550 consist of pistons 551 activated by motors 552. Depending on the control program chosen by the user or direct commands made by the user, the pistons 551 apply a pressure on the tanks and deform them, so that the liquid they contain is ejected by the fluid channels to the capture chamber.

An alternative to the pressure means described is to provide studs positioned inside the housing, so that they cause the sequential crushing of the tanks during the introduction (manual or automatic) of the cassette in the housing.

According to a non-illustrated alternative, the buffer solution reservoirs, marking probes and, possibly, the sample are made of a sufficiently rigid material to allow, in use, a internal overpressure sufficient to flush liquids (buffer solution, labeling probes, and possibly the sample) to the capture chamber.

To implement such a device, the detection system according to the invention comprises a means for injecting a fluid under pressure into the tanks. For example, this injection means may be constituted by several needles connected to a pneumatic system and inserted into the tanks. To this end, it can be provided that the sufficiently rigid material comprises a membrane of a sealed material, retaining its seal after being pierced. This material may be chosen from a silicone polymer such as polydimethylsiloxane, polymethylmethacrylate, polyvinyl chloride or Tygon®.

In this embodiment, the method of implementation is carried out by pressurizing the buffer solution reservoir, the probe reservoir and, optionally, the reservoir of the liquid sample.

In all of the previously described embodiments, the buffer solution reservoir, the sample reservoir and the reservoir for marking probes are advantageously provided with a pressure-sensitive, non-return valve allowing opening of the valve when a threshold pressure is applied. This pressure can be either an external pressure or an internal pressure.

It can also be provided that the liquid recovery tank 170 is also provided with a non-return valve. In this way, the evacuated fluids can not return to pollute the capture chamber.

A detection system according to the invention can be fully automatic. In this case, it detects the insertion of a device according to the invention and controls, according to a preprogrammed sequence, the fluid management members (the means for pressurizing the reservoir of buffer solution of the reservoir of marking probes and possibly the sample reservoir). The detection system can be adapted to different cassettes 200, each adapted to a specific pathology. The detection system recognizes the pathology in question (reading a bar code present on the cassette for example) and automatically adapts fluid management (flow, timing, etc.). Thus, in the case of an automated procedure, the same detection system can be used for different pathologies.

However, a detection system according to the invention preferably comprises a user interface 560 (FIG. 10). In this case, the user controls the implementation of the detection. We can choose from:

• an automatic mode, in which the system manages fluid management and detection,

• a semi-automatic mode, in which the user chooses the fluid management program, or

A manual mode in which the experienced user controls the ejection of the fluids out of their respective tanks and controls the detection.

The detection system according to the invention may comprise a temperature control means of the cassette. Indeed, in the case of a fluorescence detection, if the temperature is too low, the fluorescence efficiency is low and the detection is difficult. Conversely, if the temperature is too high, the anti-bodies may be deteriorated and the capture of entities of interest may be inefficient.

FIG. 13 illustrates a fourth embodiment of a disposable device 600 according to the invention. In this embodiment, the buffer solution reservoir 630, the marker probe reservoir 650, the liquid recovery reservoir 670 and, optionally, the sample reservoir 645 and / or at least one reagent reservoir are directly incorporated in the support 601 of the device. This embodiment avoids the use of a cassette to stiffen the device. This embodiment may consist in etching or molding in a support the capture chamber 610, the fluidic channels 620, 620a, 620b and 620c, as well as the reservoirs, and then disposing on the support thus etched or molded, a structured layer of a deformable material 700.

This layer 700 preferably has compression-deformable structures arranged facing the different reservoirs and the injection means 640 of the sample.

Moreover, this layer 700 has an opening or a transparent portion 720 facing the capture chamber, adapted to allow, in use, the detection of the marking probes attached to the particle or particles of interest that have specifically associated with the ligands in said capture chamber.

As shown in FIG. 13a which illustrates the layer 700 seen from below with respect to FIG. 13, the face of the layer 700 intended to be in contact with the support 601 carries no fluidic channel, but only tanks. The liquids circulate in the channels carried by the support 601 and closed by the layer 700 when the tanks of the layer 700 are pressurized.

A particularly advantageous variant is illustrated in FIGS. 14 to 16.

In this variant, all the structures (reservoirs and fluidic channels) are molded in a layer of a deformable material 700 ', while the support 601' is not structured.

As shown in Figure 16, which illustrates the layer 700 'seen from below with respect to Figure 14, the face of the layer 700' intended to be in contact with the support 601 'carries the fluidic channels and tanks.

It is then sufficient to deposit the layer 700 'on the support 601' to obtain the device according to the invention. When the layer 700 'is in contact with the support 601', the liquids can circulate in the channels carried by the layer 700 'and closed by the support 601' when the tanks of the layer 700 'are pressurized.

Unlike the embodiment of FIG. 13, this variant does not require any particular alignment between the tanks of the layer 700 and the channels carried by the support 601 because, in the embodiment of FIGS. 14 to 16, the support 601 ' only serves to close the channels when the layer 700 'is associated with the support 601'. The implementation is simplified.

A cover 300 is then deposited on the device according to the invention, by analogy with FIG.

An additional seal (not shown) can be added between the layer of deformable material 700 or 700 'and the support 601 or 601'.

Alternatively or in combination, a disposable device according to the invention may also comprise a reagent reservoir 646 'arranged downstream of the injection means 640' of the sample and upstream of the capture chamber 720 '(see FIGS. 17 to 19). In one embodiment having a sample reservoir 645 ', said at least one reagent reservoir 646' is arranged downstream of the reservoir of the liquid sample 645 'and upstream of the capture chamber 610'. In this case, the channel 120b connects the reagent reservoir 646 'to the main track 20a or the capture chamber 720' if each tank is connected independently to this chamber (by analogy with the embodiment of Figure 1a). Of course, non-return valves (not shown) are provided to ensure the flow of different liquids to the capture chamber 720 '.

This reagent reservoir 646 'may be provided with a filling tip, but, advantageously, the reservoir is pre-filled with a predefined volume of reagent. This avoids the person who implements the disposable device according to the invention to have an additional action to be performed, which may cause analysis errors in case the reagent filled by the user is not suitable for analysis. As for the embodiments of FIGS. 1 to 12, the embodiments of FIGS. 13 to 16 may also comprise an injection means connected directly to a source of liquid to be sampled for analysis: for example the gastric suction circuitry of FIG. 'a newborn. This is illustrated in Figure 19. Advantageously, as shown in Figure 3a, the disposable device according to the invention comprises a suction channel 641 'opening into the reservoir of the liquid sample 645' and intended to be connected to a pump P for drawing the liquid sample from the injection means 640 'connected to the source of liquid to be sampled (not shown) to the reservoir of the liquid sample 645'. Of course, a non-return valve (not shown) is provided to prevent the depression generated by the pump from drawing other liquids contained in the device through the microfluidic channels to the sample reservoir.

An exemplary embodiment of a device according to the invention is given below:

- Size of the cassette: 185 x 50 x 6 mm

- Capture chamber: Chrome-Gold deposit by spraying on the capture surface, then functionalization by fixation of trapping antibodies (IgG type)

The tanks are cylinders of height h = 3mm.

Many viruses are not affected by this kind of detection system as part of a standard screening. Indeed, in practice a serological test is largely sufficient, as for enteroviruses (adenovirus, rotavirus, etc.), the HIV virus, hepatitis A, B and C, etc. On the other hand, in some emergency contexts, it is necessary to have a fast and efficient way to detect any trace of the virus. Before a transplant, the graft undergoes multiple tests to ensure that it does not carry viruses such as retroviruses (HIV, HTLV), hepatitis viruses, herpes virus, Epstein virus Barr, etc. However, the success of the transplant depends greatly on the time interval between the time when the donor sample is taken and the time when the transplant is performed in the recipient. To reduce this time interval is therefore a considerable stake, which can be solved by the device and the detection system according to the invention.

The liquid sample used may be a pure or diluted sample of blood, saliva, urine, gastric juice, or other body fluid. It can also be achieved by buffering a support (inside the cheek, clothing, luggage, etc.) and then rinsing with an inert liquid and recovery of the rinsing liquid.

The device, the system and the method according to the invention will, for example, enable patient bed detection or emergency medicine in the field.

In addition, a disposable device according to the invention may be retained as evidence or for a counter-expertise.

Claims

A disposable device (100, 600) for the detection of one or more particles of interest present in a liquid sample, characterized in that it comprises a support (101, 601) provided with:

a capture chamber (110, 610) of the particle or particles of interest to be detected;

a fluidic channel (120, 120a-120b-120c, 620, 620a-620b-620c) connecting, upstream with respect to the direction of flow (F1) in use, the capture chamber (110, 610):

a pre-filled reservoir (130, 630) of a predefined volume of buffer solution;

o injection means (140, 640) of the liquid sample; o a pre-filled tank (150, 650) with a predefined volume of marking probes capable of being fixed to the particle or particles of interest to be detected; and

- A fluidic channel (160) connecting, downstream with respect to the direction of flow (F2) in use, the capture chamber (110, 610) to a reservoir (170, 670) for recovering liquids likely to flow, in use, from the capture chamber (110, 610).

The disposable device according to claim 1, wherein the carrier (101, 601) further comprises a reservoir (145, 645) of the liquid sample, arranged downstream of the injection means (140, 640) of the sample and upstream of the capture chamber (110, 610).

The disposable device according to one of claims 1 or 2, wherein the reservoir (130, 630) of buffer solution and the reservoir (150, 650) of marking probes have a pressurizing structure adapted to allow the flow, in use, of the buffer solution and the labeling probes to the capture chamber via the upstream fluid channel.

4. Disposable device according to one of claims 1 or 2, wherein the reservoir (145, 645) of the sample has a pressurizing structure adapted to allow the flow, in use, of the sample to the capture chamber via the upstream fluid channel.

A disposable device according to claim 3, wherein the reservoir (130,630) of buffer solution and the reservoir (150,650) of marking probes are of a material sufficiently flexible to be deformed by external pressure between a storage volume and an ejection volume.

A disposable device according to claim 4, wherein the reservoir (145, 645) of the sample is of a material sufficiently flexible to be deformed by an external pressure, between a storage volume and an ejection volume.

A disposable device according to claim 3, wherein the buffer solution reservoir and the marking probe reservoir are of sufficiently rigid material to allow, in use, sufficient internal overpressure to drive out the buffer solution and the probes respectively. marking to the capture chamber.

The disposable device according to claim 4, wherein the sample reservoir is of a sufficiently rigid material to allow, in use, sufficient internal overpressure to drive the sample to the capture chamber.

9. disposable device according to one of claims 7 or 8, wherein the sufficiently rigid material comprises a membrane of a sealed material retaining its seal after being drilled, preferably selected from a silicone polymer, such as polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyvinyl chloride (PVC), and Tygon®.

10. Single-use device according to any one of claims 1 to 9, wherein the injection means of the liquid sample comprises a membrane of a waterproof material retaining its sealing after being pierced, preferably selected from a silicone polymer, such as polydimethylsiloxane (PD S), polymethylmetacrylate (PMMA), polyvinyl chloride (PVC), and Tygon®.

A disposable device according to any one of claims 3 to 10, wherein the buffer solution reservoir, the sample reservoir and the marking probe reservoir have a pressure-sensitive anti-return valve, enabling, in use, an opening of the valve when a threshold pressure is applied.

A disposable device according to any of claims 2 to 1, wherein the recovery reservoir has a volume at least equal to the sum of the volumes of the buffer solution reservoir, the secondary reservoir of the liquid sample and the tank of marking probes.

13. Disposable device according to any one of claims 1 to 12, wherein the capture chamber has

at least one surface adapted to allow, in use, the detection of the marking probes attached to the particle or particles of interest which have specifically associated with the ligands.

The disposable device according to any one of claims 1 to 13, wherein the carrier (101, 601) further comprises at least one reservoir (646 ') of reagent of the liquid sample, arranged in downstream of the injection means (140, 640) of the sample and upstream of the capture chamber (110, 610)

15. Disposable device according to any one of claims 2 to 13, wherein the support (101, 601) further comprises at least one reservoir (646 ') of reagent, arranged downstream of the reservoir. of the liquid sample (145, 645) and upstream of the capture chamber (110, 610).

16. A disposable device according to any one of claims 2 to 15, further comprising a suction channel (141, 641 ') opening into the reservoir of the liquid sample (145, 645') and capable of being connected to a pump (P) for drawing the liquid sample from the injection means (140, 640 ') to the reservoir of the liquid sample (145, 645').

17. System for detecting one or more particles of interest present in a liquid sample, characterized in that it is provided with a housing (400, 500) comprising:

a receptacle (410, 510a, 510b) adapted to receive a disposable device (100, 600) according to any one of the preceding claims;

means (420, 420a-420c, 550, 551-552) for pressurizing the pre-filled tank with a predefined volume of buffer solution and the pre-filled tank with a predefined volume of marking probes; and

a detection means (430, 530) for the marking probes.

18. A detection system according to claim 17, comprising a receptacle adapted to receive a disposable device according to claim 2 and further comprising means (420b) for pressurizing the reservoir of the sample.

19. Detection system according to one of claims 17 or 18, comprising a receptacle adapted to receive a disposable device according to one of claims 5 or 6, and wherein the pressurizing means is a mechanical device, electromechanical, pneumatic or hydraulic, adapted to deform the tanks.

20. The detection system according to one of claims 17 or 18, comprising a receptacle adapted to receive a disposable device according to one of claims 7 or 8, and wherein the means of pressurizing comprises means for injecting a fluid under pressure into the tanks.

The detection system of any one of claims 17 to 20, further comprising a removable cassette (200-300) adapted to receive the holder of a disposable device according to any one of the preceding claims, and intended to be inserted into the receptacle.

22. A method of implementing a disposable device according to any one of claims 1 to 16, characterized in that it comprises the following steps:

a) injection of the sample to the capture chamber, to trap the particles of interest to be detected;

d) evacuate the buffer solution to the liquid recovery tank;

h) evacuate the buffer solution to the liquid recovery tank;

23. The method of implementation of claim 22, wherein steps a) to d) are performed sequentially until all the sample has been injected into the capture chamber.

24. The method of implementation according to one of claims 22 or 23, wherein steps e) to h) are performed sequentially until all the probes have been injected into the capture chamber.

25. The method of implementation according to any one of claims 22 to 24, wherein the steps a), c), e) and g) are performed by deformation, respectively, of the reservoir of the liquid sample, the buffer solution tank and probe tank.

26. The method of implementation according to any one of claims 22 to 24, wherein the steps a), c), e) and g) are performed by pressurizing, respectively, the reservoir of the liquid sample. , the buffer solution tank and the probe tank.