EP4247557A1

EP4247557A1 - Cartridge comprising a plurality of analysis chambers for receiving a biological liquid

Info

Publication number: EP4247557A1
Application number: EP21819917.2A
Authority: EP
Inventors: Mario FRATZL; Sarah DELSHADI; Paul KAUFFMANN
Original assignee: Magia Diagnostics SAS
Current assignee: Magia Diagnostics SAS
Priority date: 2020-11-17
Filing date: 2021-11-09
Publication date: 2023-09-27
Also published as: FR3116215A1; ZA202305210B; KR20230117365A; US20230415156A1; MX2023005824A; CA3196987A1; CN116600897A; FR3116215B1; JP2023550376A; WO2022106770A1

Abstract

The invention relates to a cartridge (1) for analysing a biological fluid, comprising an array of channels (4, 4') defining a plurality of analysis paths. Each channel is defined by at least two channel walls facing each other and defining a channel height. According to the invention, a wall of at least one channel has a step (M) for defining, on either side of the step: - a first segment (S1) of the channel in which the wall has a first surface energy (E1) and a first elevation (e1) defining a first height of the channel (h1); - a second segment (S2) of the channel in which the wall has a second surface energy (E2) and a second elevation (e2) defining a second height of the channel (h2). The first height (h1) of the channel and the first surface energy (E1) of the wall are greater than the second height (h2) of the channel and the second surface energy (E2), respectively.

Description

DESCRIPTION

TITLE: CARTRIDGE COMPRISING A PLURALITY OF ANALYSIS CHAMBERS FOR RECEIVING A BIOLOGICAL LIQUID

FIELD OF THE INVENTION

The technical field of the invention is that of biological analysis with a view to detecting the presence and/or the concentration of an analyte in a sample of biological fluid. The invention relates more particularly to a cartridge comprising a plurality of analysis chambers for receiving the biological fluid. The cartridge is preferably intended to be used in a portable immunological analysis device of the “Point of Care” type, that is to say making it possible to carry out and interpret a test on site in order to make an immediate clinical decision, at the bedside rather than in a central laboratory. It can also be used in any other type of biological analysis, for example for molecular biological analyzes or cell analyses.

TECHNOLOGICAL BACKGROUND OF THE INVENTION

Document EP3447492 discloses a method for capturing and detecting a molecule, often referred to as an “analyte”, in a sample of a biological fluid. The principles of pattern capture and detection implemented by this method are also set out in the article by Fratzl et al. "Magnetophoretic induced convective capture of highly diffusive superparamagnetic nanoparticles", Soft Matter, 14.10.1039/C7 SM02324C. According to this process, the sample is mixed with magnetic particles of nanometric or more generally submicrometric size respectively coupled to capture elements capable of binding to the molecule which it is desired to detect or quantify presence. The molecule to be detected, the analyte, can be an antigen and the element an antibody, but the reverse configuration is also possible. Detection elements are also introduced into the sample, for example a detection antibody bearing a photoluminescent marker, for example fluorescent. Complexes formed of the capture element, the analyte, and the detection element are thus formed in the solution, which are then immobilized on a support comprising micro magnetic sources ordered according to a determined spatial pattern. The pattern is defined by areas of strong magnetic field and areas of weak magnetic field inducing significant magnetic field gradients. The complexes entrained by the magnetic particles tend to agglomerate on the support at the level of the zones where the norm of the magnetic field is maximum. Photoluminescent markers (and in particular fluorescent markers) can make the determined spatial pattern apparent, which indicates the presence of the analyte in the solution. The (spatially) average intensity of this light pattern is usually referred to as the "specific signal".

In most cases, and particularly when the analyte is absent from the sample or when its quantity is limited in the sample, the unbound detection elements carrying the photoluminescent markers remain dispersed in suspension in the solution. They help to form a relatively homogeneous luminous background. The (spatially) average intensity of this luminous background forms a signal called the “supernatant signal”. In addition to the unbound photoluminescent markers, this luminous background is also constituted by the light intensity emitted by all the photoluminescent materials of the sample. The capture elements not bound to the analyte and to the detection element are also immobilized on the support, but do not bearing no markers, they do not contribute to the luminous pattern or luminous background.

The spatial ordering in the plane of the support of the magnetic field micro sources and the light intensity of the patterns made apparent by the photoluminescent markers make it possible to carry out detection and quantification of the analyte in the sample without washing, it is that is to say without removing the liquid solution after having immobilized the complexes on the surface of the support, which is particularly advantageous. To allow this detection, the sample and the surface of the support are illuminated to allow the detection of the photoluminescent markers and a digital image is acquired. This digital image therefore has a spatially variable intensity (in the plane of the image) according to the intensity of the magnetic field produced by the support. The image is processed to identify this spatial variation, and to determine the specific signal and the supernatant signal, and the specific signal/supernatant signal ratio makes it possible to conclude that the analyte is present in the sample or even estimate the concentration.

The simplicity of this approach, and in particular the absence of a washing step, allows its integration into an autonomous, portable or transportable immunological analysis device "at the patient's bedside", in the field and without pump or valve, whereas traditionally this type of analysis is carried out in a central laboratory.

To allow the application of the detection method, the biological liquid is introduced into a cartridge comprising a plurality of analysis chambers, this cartridge being intended to be introduced into the analysis device. The plurality of analysis chambers makes it possible to carry out several analyzes at from a sample of biological fluid, each analysis being able to be carried out independently on samples respectively held in each of the chambers.

The cartridge comprises a liquid discharge opening, a plurality of vents arranged downstream of the analysis chambers and a network of channels for fluidly connecting the opening to the analysis chambers. The sample of biological liquid poured into the opening propagates by capillarity in the network of channels to fill the chambers. However, it was observed that the propagation of the liquid in the network of channels was not identical from one channel to another. The flow may favor certain channels, which may lead to an overflow of liquid from the cartridge. More generally, some chambers may fill up more slowly, or even not fill up at all. As a result of this phenomenon, the analyzes are delayed, or even sometimes impossible to carry out on at least some of the analysis chambers of the cartridge.

Document US2004/028566 relates to a microfluidic device and aims to control the flow of fluid in this device by combining a displacement of this fluid with pressure forces exerted by an external pump. More specifically, this document aims to make it possible to inject and control the flow of a plurality" of fluids in an analysis cartridge in several directions. One flow is produced by capillarity, the other is forced by an effort of outside pressure.

OBJECT OF THE INVENTION

An object of the invention is therefore to provide an at least partial solution to this problem. More specifically, an object of the invention is to provide a cartridge intended to be used in a portable immunological analysis device of the “Point of Care” type and comprising a plurality of analysis chambers, these chambers being able to be reliably filled by capillarity with a biological fluid, repeatable and over a fill period of controlled duration. An object of the invention is therefore to better control the propagation by capillarity of the biological liquid in the cartridge. Advantageously, the immunological analysis is of the magnetic type, using magnetic particles to mark the presence of the analyte in a sample of biological fluid.

BRIEF DESCRIPTION OF THE INVENTION

With a view to achieving this object, the object of the invention proposes an optical analysis cartridge having a step and in accordance with claim 1.

Advantage is taken of the claimed characteristics of the step to control the propagation of the biological liquid in the fluidic network of the cartridge and in particular to promote the loading of biological liquid into the chambers of the network in a reliable , repeatable manner and during a filling period . whose duration is controlled.

According to other advantageous and non-limiting characteristics of the invention, taken alone or according to any technically feasible combination:

• the wall facing the structured wall is planar and has a third surface energy greater than the first surface energy and the second surface energy; • the step has a flank, and the surface energy of the flank of the step is closer to the first energy than to the second energy;

• the main surfaces of the support and of the upper cover (7) have a hydrophilic character;

• the network of channels comprises an upstream channel for fluidly connecting the opening to the analysis chamber, and a venting channel for fluidly connecting the analysis chamber to a vent;

• a step is arranged in at least one venting channel and tends to reduce the height of this channel in the direction of fluid flow;

• the analysis cartridge comprises at least one incubation chamber fluidly arranged upstream of the analysis chamber of an analysis channel;

• the analysis chamber and/or the incubation chamber comprises at least one reagent;

• the reagent comprises photoluminescent markers, and the upper cover, at least for the part which overhangs the analysis chambers, is formed of a transparent material in the range of wavelengths of the photoluminescent markers;

• the top cover has at least part of an optically polished outer surface;

• the opening is surmounted by a reservoir and the vents are respectively surmounted by peripheral walls having a height at least equal to a height of the reservoir. • the interlayer film is an adhesive film, advantageously a double-sided adhesive film;

• the magnetic layer comprises magnetically polarized regions defining a determined detection pattern.

According to another aspect, the invention relates to a method of manufacturing this analysis cartridge, the method comprising the supply of the support and the upper cover and the assembly of the support to the upper cover by placing their surfaces respective main ones and thus form the facing walls which define the channels.

BRIEF DESCRIPTION OF FIGURES

Other characteristics and advantages of the invention will emerge from the detailed description of the invention which will follow with reference to the appended figures in which:

[Fig. the]

[Fig. 1b] Figures la and 1b show a cartridge according to the invention respectively in perspective and in exploded view;

[Fig. le] FIG. le schematically represents an example of a fluidic network of a cartridge according to the invention;

[Fig. 2a] FIG. 2a represents a longitudinal section of a channel implementing a characteristic making it possible to slow down or accelerate the flow of the biological fluid; [Fig. 2b] Figure 2b shows a longitudinal section of a venting channel having a height restriction, in the form of a step;

[Fig. 3] Figure 3 shows a cross section of the cartridge at the analysis chambers;

[Fig. 4] Figure 4 schematically shows in top view a detection pattern defined by the magnetization produced by a magnetic layer integrated in the support of a cartridge, the magnetic field present in an analysis chamber and the norm of this field ;

[Fig. 5] Figure 5 shows an analysis device for using a cartridge according to the invention;

[Fig. 6] Figure 6 shows other views of the elements making up a cartridge according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 1a and 1b represent a cartridge 1 for receiving samples of a biological fluid which is likely to contain an analyte which it is desired to detect or whose concentration it is desired to determine. This cartridge 1 has a gripping end 1a, which allows it to be handled. The gripping end of the cartridge here bears a label, arranged on the side of the upper face of the cartridge and making it possible in particular to identify it using an identification mark, for example a QR code, or for carry any other type of information. The cartridge 1 also comprises a microfluidic part 1b. This part extends along a main plane intended to be positioned horizontally. As shown schematically in Figure 1c, it comprises an overflow opening 2 allowing the introduction of the biological liquid into the cartridge 1, for example by means of a pipette. The opening 2 opens into a network of channels 4, 4' extending in the main plane of the cartridge 1 and allowing the flow and the distribution of the biological liquid in a plurality of analysis chambers 5 via channels 4, called “upstream”, of the channel network.

The network of channels of the cartridge 1 also comprises venting channels 4' which fluidically and respectively connect the analysis chambers 5 to the vents 3, these vents making it possible to expel the air from the fluidic network of the cartridge 1 to the as the biological fluid progresses through this network.

The sample analyzed is formed from the biological fluid which fills a chamber 5, and the illustrated cartridge 1 therefore makes it possible to conduct a plurality of analyzes on the biological fluid, an analysis being able to be independently conducted on the samples respectively held in the chambers 5. The opening 2, the vents 3 and the network of channels 4, 4' connecting the opening 2 to the vents 3 define a plurality of channels for analyzing the cartridge 1.

In the example shown in Figures la and 1b, the opening 2 is surmounted by a reservoir 2 'projecting on an upper face of the cartridge 1. The reservoir has sufficient capacity to hold a volume of biological fluid at least equal to the volume of the fluidic network of the cartridge 1 (that is to say the network of channels 4, the analysis chambers 5 and the venting channels 4' as shown in Figure 1e). This volume can typically be between 5 mm ^A 3 and 500 mm ^A 3, and more precisely between 20 mm ^A 3 and 100 mm ^A 3.

Similarly, the vents 3 are respectively surmounted by peripheral walls in order to retain an excess volume of biological liquid, according to the principle of communicating vessels. Advantageously, these walls having a height at least equal to the height of the tank 2 'in order to prevent the liquid from escaping from the cartridge, which could pose health problems, or even damage an analysis device in which the cartridge is intended to fit.

By way of illustration, the cartridge 1 can have a dimension of between 2 cm and 10 cm in width and in length, and have a thickness of between 4 mm and 10 mm. Each chamber 5 can have a volume typically between 1 and 50 mm ^A 3 to receive the sample, advantageously between 5 and 25 mm ^A 3.

The cartridge 1 is formed by a support 6 and an upper cover 7 covering the support. The support 6 and the upper cover 7 are assembled to one another by placing their so-called “main” surfaces face to face. The fluidic network of the cartridge 1 is defined by recesses formed on the main surface of the support 6 and/or on the main surface of the upper cover 7, that is to say on the faces of these two elements which are intended to be assembled together. Each channel of the network 4, 4', is defined by two channel walls, these walls facing each other and defining a channel height, and by two side walls defining a channel width. The walls are formed from the main surfaces of the support 6 and of the upper cover 7, at the level of their recesses. It is the same for the analysis chambers 5 of the cartridge 1 and for any other element of the fluidic network of this cartridge 1.

The upper cover 7, at least for the part which overhangs the analysis chambers 5, is formed of a transparent material in the range of emission wavelengths of the photoluminescent markers when the cartridge is used for immunological analysis. presented in the introduction to this application. It may be a plastic material, for example based on polycarbonate, on cycloolefinic copolymer or on polystyrene. It can still be glass. The outer surface of the cover 7 is optically polished at least in line with the analysis chambers 5. These characteristics allow and promote the optical analysis of the samples of biological fluid contained in the chambers 5, as will be explained in a later section of this description.

The fluidic network, such as that presented in FIG. 1c by way of illustration, therefore extends in the main plane of the cartridge. It is of millimetric dimension, that is to say that the width of the channels of the network 4, 4' and of the analysis chambers 5 is typically between 0.1 and 10 mm. The height of these elements, that is to say their extent in a direction perpendicular to the main plane of the cartridge 1, is also millimetric, between 0.1 and 10 mm. The biological liquid spreads in this network by capillarity.

According to an important characteristic of the cartridge 1, one of the walls defining the height of at least one channel, called "structured wall", has a step and the energy of the surfaces upstream and downstream of the step is controlled to, at choice, accelerate or slow down the propagation of the fluid in the channel. By "surface energy" is meant, for simplicity of expression, the surface density of energy between the surface considered and the biological liquid.

A cartridge 1 according to the present invention takes advantage of this characteristic to promote the loading of biological fluid into the chambers 5 of the fluidic network, and more generally to control the propagation of this liquid in the fluidic network of the cartridge.

More specifically, and with reference to Figure 2a, at least one channel of the cartridge 1 comprises a step M which defines a first segment S I of the channel in which the structured wall (here formed by the main surface of the support 6) has a first surface energy El and a first elevation el defining a first height of the channel hl. The step M also defines a second segment S2 of the channel in which the structured wall has a second surface energy E2 and a second elevation e2 defining a second height of the channel h2. The first height of the channel h1 and the first surface energy El of the structured wall are respectively greater than the second height of the channel h2 and the second surface energy E2 of the second segment S2.

The variation in height of the channel, combined with the variation in the energy of the surfaces upstream and downstream of this variation, makes it possible to influence the flow of the fluid in the channel.

Thus, when the fluid encounters an "uphill" course, that is to say that it flows by capillarity from the first section SI of the channel to the second S2, its flow is slowed down by the height restriction formed by walking combined with the lesser surface energy of the second segment. Conversely, when the fluid encounters a "downward" course, that is, that is to say that it flows by capillarity from the second section S2 of the channel to the first SI, its progression is accelerated.

It is recalled that the surface energy density is a positive quantity which characterizes an interface, here the interface between the surface of the structured wall and the biological liquid. It can be determined, as is well known per se, by measuring the contact angle of a drop of water placed on the surface whose energy it is desired to measure. A high contact angle, greater than 90° indicates a low surface energy density, and the surface is said to be hydrophobic. Conversely, a contact angle of less than 90° indicates a high surface energy density is the so-called hydrophilic surface. A relatively more hydrophobic surface will tend to slow the capillary progression of a fluid in comparison to the progression of fluid over a relatively more hydrophilic surface.

By way of illustration of these principles, FIG. 2b shows an "ascending" step arranged in a venting channel 4' of the cartridge, that is to say a channel arranged between an analysis chamber 5 and a vent 3. The fluid therefore progresses in this channel towards the vent 3. The step formed on the support 6, defines an upstream section of the venting channel (the first segment of the channel, to use the terms of the general principles of the invention) which has, on the support 6, a greater surface energy than the surface energy of the downstream section (on the side of the vent 3, the second segment) of the channel 4'. Advantageously, provision can be made to have such a step in each venting channel 4' of the cartridge 1, in each of the analysis channels of the cartridge.

These characteristics of the cartridge 1 have the effect of slowing down the progression of the biological liquid when it encounters the walking, after it has progressed by capillarity along a channel of the network 4 and through the analysis chamber 5. This braking effect, when it occurs in a venting channel 4' which fills more quickly than the others, leads to forcing the flow of the liquid in the other channels of the network (ie supplying the other analysis chambers 5 of the cartridge 1) and to balancing the progression of the liquid in each of these channels. These characteristics of the venting channel or channels 4' therefore ensure the filling of the analysis chambers 5 with biological fluid in a reliable, repeatable manner and during a filling period whose duration is controlled. Provision could be made for the “rising” step(s) to be arranged in an upstream channel 4 or in a plurality of upstream channels 4, rather than in the venting channels 4′. Some of these steps can be arranged in an upstream channel 4, and others in a downstream venting channel 4'.

More generally, a cartridge 1 in accordance with the invention may have one or a plurality of steps in each analysis channel, ascending and/or descending. Provision can thus be made for a "downward" step to be arranged in the upstream channel 4 supplying a chamber 5 of an analysis channel, and for an "upward" step to be arranged, in this same analysis channel or in another, in the venting channel 4' connecting this chamber 5 to the vent 3.

Advantageously, and as illustrated in FIGS. 2a and 2b, the step causes a sudden variation in the height of the channel in which it is located, the side of the step forming an angle of 90° with the wall. But more generally, this flank can form an angle of between 60° and 90°. This step can be formed on the support 6, as illustrated in FIGS. 2a and 2b, but it is possible to envisage forming it entirely or in part on the opposite wall, of the side of the top cover 7. It may have a height of between 0.1 and 0.5 mm to effectively slow down or accelerate the progression of the liquid. Advantageously, the side of the step has a surface energy closer to the first surface energy E1 than to the second surface energy E2.

The main surfaces of the support 6 and of the upper cover 7 (and therefore the walls of the channels 4, 4' and of the chambers 5 of the cartridge 1) are preferably chosen or treated so that they have a hydrophilic character. This character generally makes it possible to facilitate the progression of the fluid in the fluidic network by capillarity. Surprisingly, it is not necessary for the difference between the first surface energy El of the first segment SI and the second surface energy E2 of the second segment S2 of the channel to be very large. In particular, it is not necessary for the second surface energy E2, on the "high" section of the step, to be of a hydrophobic nature in order to retain the progress of the liquid. Measured in contact angle, the difference between these two energies El, E2 can be 5° or more, or 10° or more and these energies confer a hydrophilic nature on the surfaces, that is to say that the angle of contact remains less than 90°. By way of example, the first segment SI may have a contact angle, qualifying the first energy El, of between 50° and 80°, and the second segment S2 may have a contact angle of between 65° and 89° (all keeping a distance of at least 5°) . A detailed example of the preparation of a support 6 allowing the first and second surface energies E1, E2 to be controlled in accordance with what has just been presented will be given in the rest of this description.

Advantageously, the wall opposite the structured wall has a third surface energy greater than the first energy El and at the second surface energy E2. It can be between 15 and 65° (while remaining advantageously greater than the first and second surface energies), and advantageously less than 50°, or even close to or smaller than 35°. This energy results in the formation on this opposite wall of a more hydrophilic surface than that of the structured wall and generally makes it possible to entrain the liquid by capillarity in the fluidic network. The variable energies of the structured wall in the different segments SI, S2 of the channel make it possible to modulate this progression. When the wall exhibiting this third energy E3 is supplied by the main surface of the upper cover 7, the latter may have been treated with a surfactant, for example based on poloxamer, tending to increase the hydrophilic character of this surface.

Of course, it is possible to provide a cartridge comprising a more complex fluidic network than that taken as an example. Thus, an analysis channel of the cartridge can include other chambers than the analysis chamber 5, such as for example one or a plurality of incubation chambers arranged upstream of the analysis chamber 5. These chambers incubation may comprise distinct reagents with which the fluid mixes before being transported into the analysis chamber 5. The network of channels 4, 4' may therefore also be more complex than that represented in the figures, and extend in each analysis channel, from the opening 2 to the vent 3, by fluidly connecting the different chambers according to any conceivable configuration.

With reference to FIGS. 1b, 6 and 3, this last figure representing a cross section of a cartridge 1 at the level of the analysis chambers 5, an embodiment of a cartridge 1 in accordance with the invention is described more precisely. The cartridge 1 of this embodiment makes it possible to apply a magnetic immunological analysis, therefore implementing magnetic particles to mark the presence of one analyte in a sample of biological fluid. This embodiment also makes it possible to simply control the surface energies on the various upstream and downstream segments of the steps (or of the step) present in the fluidic network of this cartridge.

The support 6 here is composed of a rigid substrate 6a comprising a layer or a magnetic zone 6b. Substrate 6a may be formed from a plastic material. The magnetic layer/area 6b can be arranged on the substrate 6a, or integrated into this substrate, at least at the level of the analysis chambers 5 of the fluidic network. It does not necessarily cover the entire surface of the substrate 6a.

The magnetic layer 6b is typically composed of magnetic composite materials, such as ferrites, randomly distributed in a polymer or else oriented along a pre-orientation axis. This magnetic layer may be similar to a conventional magnetic recording tape.

Substrate 6a may also include a non-magnetic film 6c (or a plurality of such films) covering magnetic layer 6b, and more generally substrate 6a. This non-magnetic film 6c is optional, it aims to distance the magnetic layer 6b from the bottom of the analysis chamber 5, when the cartridge 1 has been formed by assembling the support 6 to the upper cover 7 . In order not to disturb the measurement, the non-magnetic film 6a exhibits weak autofluorescence. For the sake of clarity, “amagnetic” designates a material whose magnetic susceptibility is very low, such as a paramagnetic or diamagnetic material. The non-magnetic film 6c can for example be formed from a plastic material, such as polypropylene. In all cases, the substrate 6a has an exposed surface Al which may consist of the substrate 6a itself or of the non-magnetic layer 6c when the latter is present. This exposed surface Al is intended to form the first section SI of the structured wall of a channel of the fluidic network of the cartridge. The exposed surface Al is designed or has been treated to exhibit the first surface energy El, which is hydrophilic in nature. This first surface energy El can result from the choice of the material forming the exposed surface, or from its texturing or from a treatment, for example with plasma or via a surfactant, aimed at making this surface particularly hydrophilic. As already specified, this first surface energy El can be characterized by a contact angle comprised between 50° and 80°.

In addition to the substrate 6a, the support 6 also comprises an intermediate film 6d placed on the exposed surface Al of the substrate 6a. The interlayer film 6d of FIG. 1b has a cutout according to a pattern corresponding to the network of upstream channels 4 and to the analysis chambers 5 and advantageously to the opening 2. In general, the interlayer film 6d has cutouts D aimed at defining part of the fluidic network of the cartridge. When this interlayer film 6d is assembled superficially to the substrate 6a to form the support 6, the latter therefore has recesses reproducing the cutting pattern D of the film 6d. These recesses, in combination with complementary recesses formed in the upper cover 7, constitute the network of channels 4, 4', the analysis chambers 5, and any other element of the fluidic network of the cartridge 1. It is noted that the pattern cutting D of the interlayer film 6d of Figure 1b does not take the imprint of the venting channels 4 ', and that these are therefore exclusively constituted by recesses formed in the upper cover 7 and not in the support 6. This arrangement leads to the formation of a rising step at the entrance of the venting channels 4', as was previously taken as an example. More generally, the cutting pattern D of the interlayer film 6d corresponds to only part of the fluidic network of the cartridge 1 in order to constitute the steps of this network.

The exposed surface A2 of the intermediate film 6d (comprising the face opposite that brought into contact with the substrate 6a) is intended to form the second section S2 of the structured wall of a channel of the fluidic network of the cartridge 1. It is designed or has been treated to present the second surface energy E2, also of hydrophilic nature, but lower than the first surface energy El. This second surface energy E2 can result from the choice of the material forming the interlayer film or its surface, or from its texturing or a specific treatment. As already specified, this second surface energy E2 can be characterized by a contact angle comprised between 65° and 89° (while being greater than the first surface energy El).

In this configuration, and when the interlayer film 6d having the cutouts D has been assembled to the substrate 6a, the main surface of the support 6 then consists of an exposed surface A2 of the interlayer film 6d having the second surface energy E2 and of the exposed surface Al of the substrate 6c, at the level of the cutouts D of the intermediate film 6d, having the second surface energy E2.

Advantageously, the interlayer film 6d is an adhesive film, also making it possible to assemble and hermetically retain to each other the upper cover 7 to the support 6 at the level of their surfaces in contact, that is to say surrounding the recesses. It may be a double-sided adhesive film, thus simultaneously ensuring its assembly to the substrate 6a, and to the top cover 7 . As is well known per se, such a film consists of a strip, for example plastic, the two faces of which are coated with an adhesive material. By its nature, this adhesive material can naturally have a surface energy lower than that of the exposed surface of the substrate 6a and therefore constitute the second surface energy.

Whether it is formed of a double-sided adhesive interlayer film or not, the cartridge 1 is assembled by providing the substrate 6a provided with the magnetic zone 6b, by placing the interlayer film 6d on this substrate 6a and thus forming the support 6, then by assembling this assembly to the upper cover 7 . This assembly is made by aligning the complementary recesses arranged on the main surface of this cover 7 with those defined by the cutting pattern D of the intermediate layer 6d of the support 6 . The fluidic network of the cartridge 1 is defined in this way.

It is naturally possible to arrange, on the intermediate film 6d, other films having other cutting patterns so as to form in the fluidic network a plurality of consecutive rising or falling steps in a channel. Care will be taken in this case to preserve the relationship according to which , at the level of each step , the surface energy of the segment of the channel having the greatest height is greater than the surface energy of the segment of the channel having the greatest height . smaller .

Returning to the description of the magnetic character of the cartridge, the magnetic layer 6b comprises a succession of regions polarized in two different directions (opposite in FIG. 3). As shown in Figure 4 which shows a top view of the magnetic layer 6b, the magnetically polarized regions extend in line in a main direction P in the example shown. At the interfaces between two zones of different polarizations, regions of relatively high magnetic intensity are created, designated zones of attraction in the remainder of this description. The attraction zones are therefore arranged in the form of a plurality of lines Za oriented along the main direction P. The particular arrangement of these lines defines, in combination, a detection pattern.

It is understood that the line arrangement taken as an example only forms a particular case of a detection pattern. A cartridge 1 is more generally provided with magnetically polarized regions and defining a well-determined detection pattern, but the configuration of which can be freely chosen.

Also shown in Figure 4 respectively the field Bc generated by the magnetic layer 6b and the norm of this field. As will be explained later in this presentation, it may be useful to add an external additional field Bext to the field produced by layer 6b. FIG. 4 shows this external field Bext which is combined with the field Bc produced by the layer and the norm of this combined field. It is observed that the application of this external magnetic field Bext can lead to the elimination of certain attraction zones Za produced when only the field supplied by the magnetic layer 6b is present. But in all cases, these attraction zones are arranged along lines Za oriented along the main direction P, or more generally along a detection pattern whose characteristics are perfectly determined.

In the case of a chamber 5 having the dimensions indicated previously, it is possible to envisage forming a detection pattern comprising between 2 and 50 lines, these having a thickness of between 1 and 150 microns (advantageously between 5 and 30 microns) and separated from each other by a spacing of between 5 and 300 microns, advantageously between 25 and 150 microns.

Returning to the description of FIG. 3, the cartridge 1 has been prepared to place in each chamber 5 a controlled quantity of magnetic particles 9 of nanometric dimensions, typically between 25 nm and 500 nm, and preferentially between 100 and 260 nm. These particles are typically in the form of beads with superparamagnetic characteristics and are biocompatible. They can in particular be covered with a polymer (of the polystyrene type) having a surface treatment which allows them to be functionalized by proteins of the Ac or Ag type. The magnetic particles are linked to capture elements capable of s associate with the analyte. The controlled quantity of the particles is such that their concentration in the volume of the chamber once filled with the biological fluid is between 10 ^A 6 and 10 ^A 11 particles/ml. The controlled quantity of nanoparticles and capture elements is here arranged in the form of a dry mass 9 resting on the support 6 of the chamber 5.

Similarly, the chambers 5 also each contain a dry cluster 10 of sensing elements. These detection elements are also capable of binding to the analyte and carry photoluminescent markers, for example fluorescent.

The dry clusters 9, 10 of capture and detection elements are also visible in FIG. 1b. They can be placed on the support 6 at locations corresponding to the position of the analysis chambers 5, before the upper cover 7 is placed on the support 6. We can use the recesses of the support 6 which define in particular the footprint of the chambers 5, to identify these locations. When the biological liquid to be analyzed is introduced into the cartridge 1, it flows in the network of channels 4 to fill the analysis chambers 5 and to spread in the venting channels 4'. The presence of at least one step (and of the variation in surface energy upstream and downstream of this step) in these channels 4, 4' ensures the correct filling, in a determined time, of all the chambers of analysis 5, as specified in a previous paragraph. The detection elements 10 and the capture elements associated with the magnetic particles 9 are respectively resuspended in the sample of each chamber 5 in order to mix therewith. During the reaction time which follows, and when the analyte is present in the sample, complexes are formed comprising a capture element, a magnetic particle, the analyte, and a detection element. These complexes are immobilized on the support 6 of each chamber 5 by agglomerating in a privileged manner at the level of the magnetic field intensity maxima, and therefore to arrange themselves according to the detection pattern defined by the magnetic layer 6b. Excess detection elements remain suspended in the sample.

It is possible to provide for each chamber 5 of a cartridge 1 to be prepared to receive capture elements and detection elements of different natures, so as to carry out multiple analyzes of the biological liquid introduced into the cartridge 1. It is also possible to provide that the detection pattern encoded by the portion of the magnetic layer 6b which is arranged at the level of a chamber 5 is different from one chamber to another.

In all cases, the presence of an analyte in the sample retained in an analysis chamber 5 leads to the formation of a detection pattern defined by the magnetic layer 6b. To perfectly control the phenomena which take place in an analysis chamber during the reaction period and to detect the presence and the intensity of the detection pattern at the end of this period, it is advantageous to place the cartridge 1 in or on a analysis device E shown schematically in Figure 5.

The device E comprises reception elements for the cartridge 1 to position it as precisely as possible in an analysis position. In this position, at least one chamber 5 of the cartridge 1 is placed in the field of a shooting device 11, such as an image sensor. This chamber 5 is also placed in the illumination field of a light source 12, for example a light-emitting diode-based source. It is also possible to provide on the optical path between the source 12, the chamber 5 and the shooting device 11 optical elements 13 such as separators, filters, objectives in order to improve the quality of the shooting and in particular to choose an appropriate magnification and depth of field. It is possible with this arrangement to acquire a digital image of the sample and of the support 6 of the chamber 5, in order to reveal on the image the light intensity produced by the fluorescent markers. The cartridge 1 is of course arranged in the analysis device so that the upper cover 7, transparent in line with the chambers 5 at least, is in the optical path in order to allow this shooting.

In operation, the photoluminescent markers in solution in the sample or immobilized on the support 6 of the illuminated chamber 5 are activated using the light source 12 and made visible in the image plane of the imaging device.

11. We can thus proceed to the acquisition of a digital image of the distribution in the plane of the support of the photoluminescent markers.

Continuing the description of the analysis device E of FIG. 3, the latter may also comprise a mechanical actuator 14, for example a piezoelectric actuator, capable of coming into contact with the support 6 of the cartridge in order to apply vibratory forces thereto. The actuator 14 can be activated after the cartridge has been introduced into or onto the device E so as to allow the effective resuspension of the capture elements, the magnetic particles and the detection elements 9, 10 in the sample.

Finally, the device E can comprise a source of magnetic field 15, for example an electromagnet, which can be activated so as to exacerbate the magnetic field produced by the magnetic layer 6b. The magnetic field produced by the source 15 can be between 1 and 400 mT at the level of the chamber 5 , but it must in all cases remain of an intensity lower than the value of the coercive field of the magnetic layer 6b so as to preserve its magnetization, and the detection pattern that this magnetization defines. The field produced by the source 15 is preferably oriented orthogonal to the surface of the support 6 to add to the field generated by the magnetic layer 6b, and thus increase the intensity of the magnetic field in the zones of attraction Za, and reinforce the detection pattern. As seen previously, the presence of this field can lead to modifying the row arrangement Za of the attraction zones, or more generally to redefining the detection pattern. The field produced by the source 15 can be continuous or pulsed, in this case with a pulse duration typically greater than 1 ms. The field produced by the source 15 also makes it possible to magnetize the superparamagnetic particles of the sample. This promotes the migration of these particles and complexes when these are present towards the surface of the support 6 to immobilize them.

To put the analysis device E into operation and carry out the digital processing of the image which is acquired, the device E also comprises a calculation device 16. This may be a microcontroller, a microprocessor, an FPGA circuit. In addition to the computing means strictly speaking, the computing device 16 also comprises memory components making it possible to store data and computer programs making it possible to operate the device E. The computing device 16 can also comprise interface components making it possible to for exchanging data (of the USB interface type) or making it possible to connect the analysis device E to maintenance equipment. The interface components can also include a screen and control buttons to allow the use of the device E by an operator. The computing device 16 is connected, for example via an internal bus, to the camera device 11, to the light source 12, to the mechanical actuator 14, to the magnetic field source 15 to coordinate their actions and/or collect the data produced, for example the digital images supplied by the shooting device 11.

Thus, once the cartridge 5 has been placed in or on the analysis device E by an operator, retained by the receiving elements in the analysis position, the following sequence of actions can be implemented by the calculation 16, after for example that the device E has been actuated via a control button:

- activation of the mechanical actuator 14 to apply a vibratory force to the cartridge 1 and engage or promote the resuspension of the capture elements, the particles magnets and sensing elements in the sample. This instant of activation marks the start of the capture period;

- activation of the magnetic source 15 to promote the immobilization of the magnetic particles, complexed or not with analytes, on the support 6 of the chamber 5;

- Switching on of the light source 12, then at the expiration of the capture period, activation of the imaging device 11 to carry out the acquisition of an image of the sample and of the support. The image can be transferred from the image capture device 11 to a memory component of the calculation device 16 in order to be able to carry out processing there.

- image processing to determine a detection signal representative of the presence or concentration of the analyte in the sample.

- processing of the detection signal to provide information indicating the presence, absence and/or concentration of the analyte in the sample in a standard measure, for example in parts per ml.

Of course, the invention is not limited to the mode of implementation described and variant embodiments can be added thereto without departing from the scope of the invention as defined by the claims.

Claims

28 CLAIMS

1. Optical analysis cartridge (1) of a biological fluid comprising an opening (2) for pouring the liquid, a plurality of vents (3) and a network of channels (4, 4') fluidly connecting the opening (2) to the vents (3), the opening (2), the vents (3) and the network of channels (4, 4') defining a plurality of analysis channels each comprising an analysis chamber (5 ), the cartridge being formed of a support (6) having a main surface and of an upper cover (7) formed at least in part of a transparent material and also having a main surface, the upper cover (7) and the support (6) being assembled to one another by their main surface, at least part of the network of channels (4, 4') being constituted by complementary recesses formed in part on the main surface of the support (6 ) and partly on the main surface of the upper cover (7), each channel then being defined by a channel wall called "structured wall" fo urned by the support (6) and by an opposite channel wall provided by the upper cover (7), the two walls facing each other and defining a channel height, the structured wall, having a step (M) to define on both sides and on the other side of the walk:

- a first segment (SI) of the channel in which the structured wall has a first surface energy (El) and a first elevation (el) defining a first height of the channel (hl);

- a second segment (S2) of the channel in which the structured wall has a second surface energy (E2) and a second elevation (e2) defining a second height of the channel (h2); the first height of the channel (hl) and the first surface energy (El) of the structured wall being respectively greater than the second height (h2) of the channel and the second surface energy (E2); the support (6) further comprising: - a rigid substrate (6a) having the first surface energy (El), the substrate incorporating a magnetic layer (6b), placed under a non-magnetic film;

- an interlayer film (6d) having the second surface energy (E2) arranged on the substrate (6a), the interlayer film (6d) having a cutting pattern (D) to form the recesses of the support (6) and to discover a exposed surface (Al) of the substrate (6a), the main surface of the support (6) then consisting of an exposed surface (A2) of the interlayer film (6d) having the second surface energy (E2) and of the exposed surface (Al) of the substrate (6a) having the first surface energy (El).

2. Analysis cartridge (1) according to the preceding claim, in which the wall facing the structured wall is planar and has a third surface energy greater than the first surface energy (El) and the second surface energy ( E2).

3. Analysis cartridge (1) according to one of the preceding claims, in which the step has a flank, and the surface energy of the flank of the step is closer to the first energy (El) than to the second energy. (E2) .

4. Analysis cartridge (1) according to one of the preceding claims wherein the main surfaces of the support (6) and the upper cover (7) have a hydrophilic nature.

5. Analysis cartridge (1) according to one of the preceding claims, in which the network of channels comprises an upstream channel (4) for fluidly connecting the opening (2) to the analysis chamber (5), and a venting channel (4') for fluidly connecting the analysis chamber (5) to a vent (3). Analytical cartridge (1) according to the preceding claim, in which a step (M) is arranged in at least one venting channel (4') and tends to reduce the height of this channel in the direction of the flow of the fluid . Analysis cartridge (1) according to one of the two preceding claims comprising at least one incubation chamber fluidly arranged upstream of the analysis chamber (5) of an analysis channel. Analysis cartridge (1) according to the preceding claim, in which the analysis chamber (5) and/or the incubation chamber comprises at least one reagent. Analysis cartridge (1) according to the preceding claim, in which the reagent comprises photoluminescent markers, and the upper cover (7), at least for the part which overhangs the analysis chambers (5), is formed of a material transparent in the wavelength range of photoluminescent markers. Analysis cartridge (1) according to one of Claims 7 to 10, in which the upper cover has at least part of an optically polished outer surface. Analysis cartridge (1) according to one of the preceding claims, in which the opening (2) is surmounted by a reservoir (2') and the vents (3) are respectively surmounted by peripheral walls having a height at least equal at a height of the reservoir (2'). Analysis cartridge (1) according to one of the preceding claims, in which the intermediate film (6d) is an adhesive film, advantageously a double-sided adhesive film. Analysis cartridge (1) according to one of the preceding claims, in which the magnetic layer (6b) includes magnetically biased regions defining a determined detection pattern. Method of manufacturing a cartridge according to one of the preceding claims, comprising the supply of the support (6) and the upper cover (7) and the assembly of the support (6) to the upper cover (7) by putting -vis their respective main surfaces and thus form the facing walls which define the channels (4, 4 ').