FR3120126A1 - Analysis cartridge and device for analyzing a liquid - Google Patents

Abstract

The invention relates to an analysis cartridge (1) for the detection and/or analysis of a species likely to be present in a liquid, the analysis cartridge (1) being provided with a front face and a rear face opposite the front face, the cartridge further comprises: - at least one microfluidic analysis chamber (5) intended to receive the liquid, the at least one microfluidic chamber being formed in the cartridge, and includes a bottom (5a); - at least one first cluster (9) formed of magnetic nanoparticles held together, and on which capture agents are grafted, said capture agents being configured to bind specifically with the species, the at least one first cluster ( 9) adhering to the bottom (5a). Figure 4b

Description

Analysis cartridge and device for analyzing a 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 a species (an analyte) in a liquid, in particular a biological liquid. The invention relates more particularly to a cartridge comprising at least one micro-fluidic chamber intended to receive a liquid to be analyzed.

The cartridge is preferably intended to be used in a device (for example a portable device) for immunological analysis of the “Point of Care” type, that is to say making it possible to carry out and interpret a test on site to take 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 process for capturing and detecting a species, often referred to as an “analyte”, in a liquid, in particular a biological liquid. The principles of pattern capture and detection implemented by this process are also exposed in the article by Fratzl et al “Magnetophoretic induced convective capture of highly diffusive superparamagnetic nanoparticles”, Soft Matter, 14. 10.1039/C7SM02324C.

This method comprises in particular a step which consists in mixing a sample, formed of a liquid to be analyzed, with magnetic particles. These particles have, in this respect, a nanometric or more generally sub-micrometric size, and are coupled to capture elements capable of binding specifically to the species to be detected and/or quantified. This species, the analyte, can be an antigen and the element an antibody, but the reverse configuration is also possible.

During this step, detection elements are also introduced into the sample. The detection elements can in particular comprise an antibody or a detection antigen carrying a photoluminescent label, for example fluorescent.

At the end of this step, complexes formed of the capture element, the species, and the detection element are thus formed in the solution. These complexes are then immobilized on a support comprising magnetic micro-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) make it possible to 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 bearing the photoluminescent labels remain dispersed in suspension in the solution. They help to form a relatively homogeneous luminous background. The (spatially) average intensity of this bright 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 the detection element are also immobilized on the support, but since they do not carry labels, they do not contribute to the light pattern or the light 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 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 image plane) depending on 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 to 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, for example a single-use 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 conduct several analyzes from a sample of biological fluid, each analysis being able to be independently conducted 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 to fluidically connect the opening to the analysis chambers. The sample of biological liquid poured into the opening spreads by capillarity in the network of channels to fill the chambers.

The implementation of this cartridge, although undeniably presenting numerous advantages, requires a number of manipulations which it is desirable to reduce.

An object of the present invention is therefore to propose an integrated analysis cartridge making it possible to limit the manipulations necessary for carrying out the analysis of a fluid.

Another object of the present invention is to provide an analysis cartridge as well as an analysis device that is easy to implement.

BRIEF DESCRIPTION OF THE INVENTION

The aims of the present invention are, at least in part, achieved by an analysis cartridge for the detection and/or analysis of a species likely to be present in a liquid, the analysis cartridge being provided with a front face and a rear face opposite the front face, the cartridge further comprises:

- at least one microfluidic analysis chamber intended to receive the liquid, the at least one analysis chamber being formed in the cartridge, and comprises a bottom;

- at least one first cluster formed of magnetic nanoparticles held together, and on which capture agents are grafted, said capture agents being configured to bind specifically with the species, the at least one first cluster being adherent on the bottom.

According to one embodiment, the magnetic nanoparticles forming the first cluster are dried and/or freeze-dried and/or encapsulated in an encapsulation material.

According to one embodiment, the at least one analysis chamber comprises side walls surmounting the bottom, and in which a gas is trapped, said gas being capable of being released under the action of a mechanical stress applied to the rear face, the release of the gas leading to the generation of gas bubbles on the side wall, advantageously, the gas is initially trapped in slots opening onto the side walls.

According to one embodiment, said cartridge comprises at least one second cluster adhering to the bottom of the at least one chamber, the at least one second cluster comprises detection agents, held together, and binding specifically with the species, said detection agents also forming photoluminescent markers.

According to one embodiment, said analysis cartridge comprises a magnetic layer arranged to immobilize the magnetic nanoparticles of the at least one chamber on the bottom of said chamber, said layer being advantageously microstructured so that the magnetic nanoparticles, when they are immobilized on the bottom of the chamber, form a predefined pattern.

According to one embodiment, the magnetic layer comprises a repeated juxtaposition of at least a first region and a second region, the first region having a magnetic polarization in a first direction, and the second region having either zero magnetic polarization or a magnetic polarization in a second direction different from the first direction.

According to one embodiment, said analysis cartridge further comprises a non-magnetic layer covering the magnetic layer and intended to screen at least in part the magnetic interaction between the magnetic layer and the magnetic nanoparticles.

According to one embodiment, said cartridge comprises at least one liquid discharge opening, said opening being in fluid communication with the at least one analysis chamber, advantageously, the fluid communication between the at least one inlet and the at least one analysis chamber being ensured by at least one microfluidic channel.

The invention also relates to an analysis device which comprises:

- a support intended to receive the analysis cartridge according to the present invention for the purpose of analyzing a liquid contained in the at least one chamber of said analysis cartridge;

- piezoelectric vibration means arranged to impose a vibration on the bottom of the analysis chamber so as to generate an acoustic pressure field in a liquid likely to be present in the at least one analysis chamber, the field of acoustic pressure allowing the resuspension and mixing of the first cluster in said liquid.

According to one embodiment, the piezoelectric vibration means are associated with at least one finger resting against the rear face and intended to transmit a vibration generated by said piezoelectric vibration means to the bottom of the analysis chamber.

According to one embodiment, the at least one finger is also configured to exert a pressing force against the rear face when it transmits a vibration generated by the piezoelectric vibration means.

According to one embodiment, the at least one analysis chamber comprises a plurality of analysis chambers, and the analysis device is configured to move the analysis cartridge and/or the piezoelectric vibration means, so to be able to put in correspondence, successively, each of the analysis chambers and the piezoelectric vibration means and thus to impose successively, and specifically, at the bottom of each of the analysis chambers a vibration.

According to one embodiment, said analysis device comprises complementary magnetic means intended to impose a complementary magnetic field in the at least one analysis chamber of the analysis cartridge.

According to one embodiment, said analysis device further comprises means for analyzing the liquid present in the at least one analysis chamber.

According to one embodiment, the analysis means comprise a detector, and a radiation source configured to, when said radiation interacts with the detection agents, generate a photoluminescent signal capable of being detected by the detector.

The invention also relates to a method for detecting a species likely to be present in a liquid, the method comprising:

a) a step of injecting a sample of liquid into the at least one chamber of the cartridge according to the present invention, the species being capable of binding with the capture agents and the detection agents so as to form magnetic nanoparticle agent/species/capture agent complexes;

b) a step which comprises the resuspension and the dispersion of the first cluster with the means of piezoelectric vibration of the analysis device according to the present invention, and thus allows the formation of the complexes;

c) a complex detection step.

According to one mode of implementation, step c) is preceded by a step c0) of immobilizing the complexes on the bottom of at least one analysis chamber, step c) comprises the implementation of the detector and the radiation source.

Other characteristics and advantages will appear in the following description of an analysis cartridge and an analysis device according to the invention, given by way of non-limiting examples, with reference to the appended drawings in which:

There is a schematic representation, in perspective, of an analysis cartridge capable of being implemented according to the principles of the present invention;

There is a schematic representation of the fluidic section according to a cutting plane of said fluidic section parallel to the upper face, the represents in particular the at least one analysis chamber in fluid communication on the one hand with the opening via the channels and on the other hand with the at least one vent via the at least one vent channel;

There is a schematic representation of the cartridge shown in in perspective and in exploded view;

There is a schematic representation according to a cross-sectional plane (perpendicular to the main faces) of the cartridge of the at the level of the at least one analysis chamber;

There is another schematic representation according to a cross-sectional plane (perpendicular to the main faces) of the cartridge of the at the level of at least one analysis chamber, said cartridge implementing a non-magnetic layer;

There is a representation in perspective of an interlayer film likely to be implemented for the assembly of the cartridge of the ;

There is a schematic representation in top view of 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;

There is a schematic representation of an analysis chamber filled with a liquid which comprises in suspension the analysis, the magnetic nanoparticles on which are grafted capture agents, detection agents, the illustrates in particular the mechanism of complex formation;

There is a representation of an analysis device implemented according to the principles of the present invention;

FIGS. 7a and 7b are representations of the interior of the device for analyzing the , the finger of the piezoelectric vibration means being in a retracted position, the being a sectional view;

There is a representation of the interior of the device for analyzing the , the finger of the piezoelectric vibration means being in an engaged position;

There is an image taken under a microscope of a deposit of clusters on the bottom of an analysis chamber in accordance with the present invention;

There is an image taken under a microscope of the bottom of an empty analysis chamber in accordance with the present invention;

There is an observation made under a microscope at the bottom of a chamber at the end of the sequence of a step of resuspension and capture of magnetic nanoparticles.

DETAILED DESCRIPTION OF THE INVENTION

There represents an analysis cartridge 1 for the analysis of a species (hereinafter “analyte”) likely to be present in a liquid, and more particularly a biological liquid.

The analysis cartridge 1 is, in this respect, adapted to receive a sample of a liquid, for example a biological liquid, in order to detect the presence of a given analyte in said liquid.

The analysis cartridge 1 thus comprises at least one microfluidic analysis chamber 5. In particular, the analysis cartridge can comprise between 1 and 10, for example 5, analysis chambers 5.

The analysis cartridge 1 can comprise a gripping end 1a allowing it to be handled. The gripping end 1a can carry a label, for example provided with a bar code or a two-dimensional code, allowing identification and traceability of the analyzes carried out by means of the analysis cartridge 1 in question. The means of identification may alternatively comprise an “RFID” chip.

The analysis cartridge 1 also comprises an active section 1b formed, for example, in the extension of the gripping end 1a.

The active section 1b is of generally planar shape and comprises two main faces called, respectively, upper face and lower face.

The analysis cartridge 1 can comprise an opening 2 for pouring which allows the introduction of a liquid into the analysis cartridge 1. This opening 2 leads in particular to the upper face of the active section 1b.

The opening 2 is in particular in fluidic communication with the at least one analysis chamber 5. In particular, the active section comprises at least one microfluidic channel 4 ensuring the fluidic communication between the opening 2 and the at least one analysis chamber 5. In other words, the at least one microfluidic channel 4 ensures the flow and the distribution of the liquid poured into the opening 2 towards the at least one analysis chamber.

The analysis cartridge 1 can also comprise at least one vent 3 in fluid communication with the at least one analysis chamber 5 (FIGS. 1 and 2). The at least one vent 3 is in particular configured to allow an evacuation of air likely to be present in the at least one analysis chamber 5 during the filling by the liquid of the latter. In particular, fluid communication between at least one analysis chamber 5 and at least one vent 3 is provided by at least one vent channel 4'.

Thus, in operation, a sample of liquid is introduced, for example by means of a pipette, into the opening 2. The sample then flows into the at least one analysis chamber 5 via the at least a microfluidic channel 4. The air likely to be present in the at least one analysis chamber 5 is expelled during the flow of the liquid in said analysis chamber 5 towards the at least one vent 3 .

In the case of a plurality of analysis chambers 5, the microfluidic channels 4 can be arranged so that the flow of the liquid sample is simultaneous in each of the chambers 5 or sequential. By "sequential flow" is meant a filling of the analysis chambers 5 according to a predetermined order. In particular, according to this principle, the flow in a given chamber only begins when the analysis chamber which precedes it in the order of filling is completely filled.

Still in the case of a plurality of analysis chambers 5, provision can also be made for the analysis cartridge to comprise a plurality of openings, for example an opening dedicated to each chamber of the cartridge.

The analysis cartridge 1 shown in may further comprise a reservoir 2' surmounting the opening 2 whose volume equals that of the microfluidic network formed by the at least one microfluidic channel 4, the at least one analysis chamber 5 and the at least one 4' vent channel. In this respect, this volume can be between 5 mm3 and 500 mm3, and more precisely between 20 mm3 and 100 mm3.

Equivalently, the at least one vent 3 is surmounted by a peripheral wall in order to retain an excess volume of liquid, according to the principle of communicating vessels. Advantageously, the peripheral wall has a height at least equal to the height of the tank 2 'in order to prevent the liquid from escaping from the analysis cartridge 1. This arrangement makes it possible to limit the problems of sanitary origin, even damage to an analysis device (described below) in which the analysis cartridge 1 is intended to be inserted.

By way of illustration, the analysis 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. The at least one analysis chamber 5 can have a volume typically between 1 mm ³ and 50 mm ³ to receive the sample, advantageously between 5 mm ³ and 25 mm ³ .

According to a non-limiting example shown in , the analysis cartridge 1 can be formed of a support 6 and an upper cover 7 covering the support 6. The support 6 and the upper cover 7 are in particular assembled to each other by putting opposite their so-called “main” surfaces.

The at least one analysis chamber 5, the at least one microfluidic channel 4 and the at least one vent channel 4' form a microfluidic network of the analysis cartridge 1. This microfluidic network is in particular 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 one and/or the other of the faces of these two elements which are intended to be joined together.

Each channel 4, 4' is delimited, on the one hand, by the main surfaces of the support 6 and of the cover 7 forming, respectively, a bottom and a vault, and on the other hand, by side walls connecting the bottom and the vaulted. The distance separating the bottom and the top of a channel 4, 4' defines a channel height, while that separating two opposite side walls defines a channel width.

Equivalently, the at least one analysis chamber 5 is also delimited by the main surfaces of the support 6 and of the cover 7 forming, respectively, a chamber bottom 5a and a chamber vault 5b (FIGS. 4a and 4b). The at least one chamber 5 further comprises chamber side walls 5c which extend from the chamber bottom 5a towards the chamber vault 5b. It is however understood that the at least one analysis chamber 5a may have no vault, and for example form an open well at the level of the active section.

It is also understood that the side walls of the at least one chamber can be deformable.

The upper cover 7, at least for the part which overhangs the at least one analysis chamber 5, can be formed of a material transparent to a photoluminescence signal capable of being emitted by detection agents described below. of the statement. The material forming the upper cover 7 may comprise at least one of the materials chosen from: a plastic material, for example based on polycarbonate, cycloolefinic copolymer or polystyrene, glass.

The outer surface of the cover 7 can be optically polished at least in line with the at least one analysis chamber 5.

The microfluidic network, such as the one presented on the , therefore extends in the main plane of the analysis cartridge 1. It is of millimetric dimension, that is to say that the width of the channels 4, 4' and of the analysis chambers 5 is typically between 0.1mm and 10mm. The height of these elements, that is to say the distance separating the bottom of an arch, is also between 0.1 mm and 10 mm. The liquid capable of being introduced at the level of the opening 2 propagates in the microfluidic network by capillarity.

The analysis cartridge 1 according to the present invention also comprises at least one first cluster 9 adhering to the bottom of the at least one analysis chamber 5 ( ). The at least one first cluster 9 is in particular formed of magnetic nanoparticles 9a held together, and onto which capture agents 9b are grafted ( ).

Alternatively, it may be considered to arrange the at least one first cluster on the vault 5b of the at least one chamber 5. This configuration can in particular facilitate the resuspension (described below) of said first cluster when the latter is subjected to magnetic forces due to the presence of a magnetic layer 6b described below.

A first cluster can advantageously have a volume comprised between 0.1 μl and 5 μl and advantageously between 0.5 μl and 2 μl.

By “maintained together”, is meant a set of nanoparticles linked together. This cohesion between the nanoparticles can be direct or indirect. Direct cohesion can in particular be ensured by dry or freeze-dried nanoparticles, while indirect cohesion can be ensured by an encapsulation material. In this respect, the encapsulation material may comprise sugar (trehalose, glucose, etc.) or viscous solutions (for example Tween), or glycerol.

Maintaining the nanoparticles together, and in the form of clusters, ensures better stability of the latter over time.

The implementation of an encapsulation material facilitates the suspension of nanoparticles presented in the rest of the description.

The magnetic nanoparticles 9a can be of nanometric size, typically between 25 nm and 500 nm, and preferentially between 100 and 300 nm. The magnetic nanoparticles 9a can be generally spherical in shape. The latter have superparamagnetic characteristics and are biocompatible. They can also be covered with a polymer (of the polystyrene type) having a surface treatment which allows them to be functionalized, for example, by proteins of the Ac or Ag type. The controlled quantity of the particles is such that their concentration in the volume of the chamber once filled with the liquid to be analyzed is between 10 ⁶ particles/ml and 10 ^{1 2} particles/ml, advantageously between 10 ⁹ particles/ml and 10 ^{1 1} particles/ml.

9b capture agents are able to bind specifically to the analyte likely to be present in the liquid. In this regard, the analyte can be an antigen, while the capture agent 9b comprises an antibody (the reverse configuration is also possible).

The analysis cartridge 1 can also comprise at least one second cluster 10 adhering to the bottom of the at least one analysis chamber 5 (FIGS. 4a and 4b). The at least one second cluster 10 is in particular formed of detection agents 10a linked together ( ).

A second cluster can advantageously have a volume comprised between 0.1 μl and 5 μl and advantageously between 0.5 μl and 2 μl.

The at least one first cluster 9 and the at least one second cluster 10 are both intended to allow, respectively, a capture, even an immobilization, of an analyte 11 present in a liquid, and to detect, even quantify, the presence of said analyte 11. To this end, the analyte 11 present in a liquid, and in the presence of detection agents 10a and of magnetic nanoparticles 9a on which the capture agents 9b are grafted, comes to form complexes 12 with these elements ( ).

The magnetic nanoparticles are intended to isolate the complexes, while the detection agents make it possible to detect them.

Thus, the implementation of the analysis cartridge 1 for the detection and/or the quantification of an analyte in a liquid involves pouring a sample of said liquid into the at least one analysis chamber via the opening 2 .

The formation of the complexes 12 also requires suspending, in the liquid sample present in the analysis chamber 5, the elements forming the first cluster 9 and the second cluster 10.

This suspension can in particular comprise a separation of the first and the second cluster from the bottom of the chamber 5a as well as a separation of the elements from each other in order to disperse them in the sample. To this end, piezoelectric vibration means 110 described later in the statement can be implemented.

These piezoelectric vibration means 110 are in particular suitable for imposing a vibration on the bottom of the chamber 5a. This vibration makes it possible to generate an acoustic pressure field in the liquid present in the analysis chamber 5, and thus to suspend the elements forming the first cluster 9 and the second cluster 10.

According to a first variant, the vibration can be imposed at fixed frequencies, for example between 5 KHz and 2 MHz, advantageously between 20 KHz and 200 KHz. Still according to this first variant, the vibration can be close to a resonance frequency of the analysis chamber 5 (by "close to the resonance frequency" is meant a frequency at +/- 15%, advantageously at +/- - 10%, even more advantageously at +/- 5% of the resonance frequency of the analysis chamber). Advantageously, the resonance frequency of the analysis chamber 5 is of the order of 50 KHz, or of the order of 80 KHz or of the order of 110 KHz.

An analysis chamber exhibiting such resonant frequencies may comprise, along a section plane parallel to the cartridge, a rectangular section terminated at each of these two ends by a mouth whose section, along the same plane, is triangular. . The length and width of the rectangular section are 8.4mm and 2.4mm respectively, while the base and height of the triangular section are 2.4mm and 4mm respectively. The height of the analysis chamber is 390 μm.

According to a second variant, the vibration can be imposed in the form of cycles, and for example in the form of repeated cycles. In this respect, a cycle can comprise an increasing or decreasing frequency sweep, and more particularly a frequency sweep around the resonance frequency of the analysis chamber. The full frequency sweep can last between 10 seconds and 200 seconds. In particular, this frequency sweep comprises an incrementation or a decrementation by step of the frequency. In particular, each step corresponds to maintaining the frequency for a duration which may be between 2 seconds and 10 seconds so as to allow the liquid to move sufficiently for a minimum mixing effect.

For example, a cycle can involve a frequency sweep from 110KHz to 120KHz, or conversely a frequency sweep from 120 KHz to 110 KHz. This sweep can be performed in steps of 1 KHz, and maintenance, at each step, of the vibration for a period of a few seconds and in particular 2 seconds.

Still by way of example, the vibratory sequence may include the repetition of an elementary sequence which includes a first cycle and a second.

The first cycle can include a frequency sweep from 120 KHz to 111 KHz in steps of 1 KHz and maintaining the vibration at each step for 2 seconds.

The second cycle can comprise a frequency sweep from 110 KHz to 119 KHz in steps of 1 KHz and maintaining the vibration at each step for 2 seconds.

This elementary sequence can, for example, be repeated from 1 to 4 times.

In a particularly advantageous manner, the side walls 5c can be structured so as to create vortices favorable to mixing in the sample when the latter circulates in the analysis chamber. This structuring of the side walls 5c can comprise a slotted surface.

In a particularly advantageous manner, gas, and more particularly air, is initially trapped in the side wall 5c of the at least one chamber 5.

The trapped gas can be released under the action of a mechanical stress applied to the at least one analysis chamber and thus generate gas bubbles on the side wall of the at least one chamber as soon as the sample of liquid is present in at least one chamber 5. This mechanical stress can, in particular, be applied to the rear face of the analysis cartridge 1.

According to an advantageous embodiment, the side walls 5c of the at least one chamber 5 can be made of a porous material. The gas is initially in the pores of the porous material.

According to an alternative or complementary embodiment, the gas can be initially trapped in fifth slots opening onto the side walls ( ).

The 5th slots can have a length of between 100 μm and 1000 μm, and a cross section whose largest dimension is between 100 μm and 400 μm.

The cross section of a slot can include at least one of the shapes chosen from: square, rectangle, round.

According to the present invention, as soon as the gas bubbles are generated (in particular under the effect of the stress described above), the acoustic pressure field, generated by vibration of the bottom of the chamber, makes it possible to make them vibrate in the liquid. present in the analysis chamber.

These vibrating gas bubbles then act as additional vibrating agents that improve the mixing of the liquid present in the analysis chamber.

This effect can nevertheless be optimized by requiring the gas bubbles to vibrate at a frequency close to their resonance frequency (by "close to the resonance frequency", we mean a frequency at +/- 15%, advantageously at +/- 10%, even more advantageously at +/- 5% of the resonance frequency of the gas bubbles). Indeed, under these conditions, the contribution of the gas bubbles to the mixture is exacerbated, all the more so since certain bubbles are likely to transmit energy by their implosion and the resulting shock wave.

For this purpose, the pores and/or the slits trapping the gas can be configured so that the gas bubbles likely to be generated have a resonance frequency close to that of the analysis chamber.

In particular, for the specified slit sizes, the bubble sizes can be between 50 μm and 500 μm. For this range of bubble sizes, the resonant frequencies are between 20 kHz and 500 kHz.

Still according to the present invention, the analysis cartridge 1 can comprise a magnetic layer 6b ( ) arranged to immobilize the magnetic nanoparticles 9a of the at least one analysis chamber 5 on the bottom of said chamber. It is therefore understood that this magnetic layer also makes it possible to immobilize the complexes 12. The magnetic layer 6b is advantageously microstructured so that the magnetic nanoparticles immobilized on the bottom of the analysis chamber 5 form a predefined pattern.

The magnetic layer 6b can in this respect comprise a repeated juxtaposition of at least a first region 6b1 and a second region 6b2. More particularly, the first region 6b1 can present a magnetic polarization according to a first direction, and the second region 6b2 can present either a null magnetic polarization or a magnetic polarization according to a second direction different, preferentially at 180°, from the first direction ( ).

The support 6 can in particular be arranged so that it comprises, from its main face towards the rear face, an interlayer film 6d, the magnetic layer 6b and a rigid substrate 6a. It is understood that the magnetic layer 6b does not necessarily extend over the entire surface of the substrate 6a (FIGS. 3, 4a and 4b).

The rigid substrate 6a can comprise a plastic material. The magnetic layer 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 microfluidic network.

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

Substrate 6a may also include a non-magnetic layer 6c (or a plurality of such films) interposed between magnetic layer 6b and interlayer film 6d. This non-magnetic layer 6c, which is optional, aims to distance the magnetic layer 6b from the bottom of the analysis chamber 5.

By "non-magnetic layer" is meant a layer whose magnetic susceptibility is zero or even less (in absolute value) than 10 ^-3 .

The non-magnetic layer 6c can, for example, comprise a plastic material, such as a polypropylene on an acrylic.

In the example presented above, the interlayer film 6d defines the microfluidic network. In particular, the 6d interlayer film shown in has a cutout according to a pattern corresponding to the microfluidic network. When this interlayer film 6d is assembled to the substrate 6a to form the support 6, the latter therefore has recesses reproducing the cutting pattern of the film 6d. These recesses, possibly in combination with complementary recesses formed in the upper cover 7, constitute the microfluidic network of the cartridge 1.

In a particularly advantageous manner, the intermediate film 6d is an adhesive film, also making it possible to assemble and hermetically retain to one another the upper cover 7 to the support 6 at the level of their surfaces in contact, that is to say say surrounding recesses. It may, for example, be a double-sided adhesive film, then simultaneously ensuring its assembly to the substrate 6a, and to the upper cover 7. As is well known per se, such an interlayer film 6d may consist of a strip, for example plastic, the two faces of which are coated with an adhesive material.

Still advantageously, the bubbles of gas or air, during the application of a mechanical stress on the bottom of the chamber can be generated by the release of gas trapped in the interlayer film. Thus, this film may be porous and/or comprise slits, for example formed during the cutting of the pattern corresponding to the microfluidic network.

Returning to the description of the magnetic character of the analysis cartridge, the magnetic layer 6b comprises a succession of regions 6b1 and 6b2 polarized in two different directions (opposite in FIGS. 4a and 4b). As shown in the which shows the magnetic layer 6b in top view, the magnetically polarized regions extend in a line along a main direction P in the example shown.

Regions of relatively strong magnetic intensity, referred to as zones of attraction, are observed at the interfaces between zones of different polarization. The attraction zones are in particular 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. An analysis 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.

The Bc field generated by the magnetic layer 6b as well as its norm are also represented on the . As will be explained later in this presentation, it may be useful to add an additional (or complementary) external field Bext to the field produced by layer 6b. We represented on the this external field Bext which combines 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 an analysis chamber 5 having the dimensions indicated above, it is possible to envisage forming a detection pattern comprising between 2 and 50 lines, these having a thickness of between 1 μm and 150 μm (advantageously between 5 μm and 30 μm) and separated from each other by a spacing of between 5 μm and 300 μm, advantageously between 25 μm and 150 μm.

On the basis of this description, the inventors calculated the surface gradient on the surface of a non-magnetic layer with a thickness close to 55 μm and resting on a magnetic layer.

The micro magnets of the magnetic layer, on which rests the non-magnetic layer, has a width of 50 µm and a height of 10 µm. These micro magnets have in-plane polarization and are alternating with each other. In this calculation, a NdFeB magnetic field source below this magnetic layer is also implemented. The latter imposes a magnetic field of 1.2 Tesla.

In the context of this calculation, the inventors have been able to demonstrate that the gradients on the surface of the non-magnetic layer are between 50 T/m and 150 T/m, and can be extended to more or less an order of magnitude. These gradients result in a magnetic force exerted on the particles. This force is likely to retain the magnetic particles (in particular the clusters), and also participates in the immobilization of the latter as illustrated in the (described at the end of this application).

Thus, when a sample of liquid is introduced into the analysis cartridge 1, it flows into the microfluidic channels 4 to fill the at least one analysis chamber 5 and to spread in the channels of 'vent 4'. A vibration as well as a mechanical stress are imposed on the rear face of the analysis cartridge 1. Gas bubbles are then generated by the release of gas under the effect of the mechanical stress. The acoustic pressure field resulting from the vibration imposed on the bottom makes it possible to suspend the elements forming the first cluster and the second cluster. This acoustic pressure field also causes the gas bubbles to vibrate.

The vibration of the latter also contributes, to a certain extent, to the suspension and mixing of the detection agents 10a and the capture agents 9b associated with the magnetic nanoparticles 9a in the liquid sample present in the analysis chamber 5 During the reaction time which follows, and when the analyte is present in the sample, complexes comprising at least one capture element, at least one magnetic nanoparticle, at least one analyte, and at least one detection element are forming. These complexes are immobilized on the support 6 of the analysis chamber 5 by agglomerating in a privileged manner at the level of the maxima of the norm of the intensity of the magnetic field (induced by the micro-sources and possibly the external magnetic field) , and therefore to arrange according to the detection pattern defined by the magnetic layer 6b. Excess detection elements remain suspended in the sample.

Provision can be made for each analysis 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 a sample of liquid introduced into the cartridge. analysis 1. Provision can also be made for the detection pattern encoded by the portion of the magnetic layer 6b which is arranged at the level of a chamber 5 to be different from one chamber to another.

It is also possible to consider a magnetic layer which includes a zone devoid of magnetic zones, and above which the first cluster is formed (the resuspension of the magnetic nanoparticles of the first cluster can thus be facilitated).

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.

The invention also relates to an analysis device 100 intended to cooperate with the analysis cartridge ( ).

The analysis device 100 notably comprises a support 101 intended to receive an analysis cartridge 1 for the purpose of analyzing the liquid sample contained in the at least one analysis chamber 5 (FIGS. 7a to 7c) .

The analysis device 100 also comprises piezoelectric vibration means 110 arranged to impose a vibration on the bottom of the analysis chamber so as to generate an acoustic pressure field in a liquid likely to be present in the at least one analysis chamber 5.

The piezoelectric vibration means 110 may comprise a transducer, in particular an elliptical transducer of the Elliptec™ transducer type.

Alternatively, the piezoelectric vibration means 110 may comprise a piezoelectric stack (Piezostack), in particular a so-called Langevin transducer (A Langevin transducer is produced by a stack of ceramics held between two metal parts which ensure clamping of the assembly ).

In this regard, the inventors were able to demonstrate that these linear transducers exhibited good mixing dynamics.

Bolt-clamped Langevin ultrasonic transducers are also very efficient.

As specified above, the acoustic pressure field allows the resuspension and mixing of the first cluster 9 and the second cluster 10 in the liquid sample.

In a particularly advantageous manner, the piezoelectric vibration means 110 are associated with at least one finger 111 which can bear against the cartridge and in particular against the rear face. This at least one finger 111 is in particular intended to transmit the vibration generated by said piezoelectric vibration means to the bottom of the analysis chamber 5. Still advantageously, the at least one finger 111 is also configured to exert a force of bearing against the rear face when it transmits the vibration generated by the piezoelectric vibration means. Even more advantageously, this pressing force (or mechanical stress) makes it possible to release the gas trapped in the interlayer film to generate gas bubbles on the side wall of the analysis chamber. These gas bubbles when in resonance with the sound pressure field allow better mixing.

Advantageously, the support force can be between 1 N and 50 N, advantageously between 5 N and 25 N. Furthermore, the support force can be exerted on a surface with an extent of between 4 mm ² and 64 mm ² .

Thus, for a support force of 1 N on a surface with an area of 4 mm ² or 64 mm ² , the pressure exerted is of the order of 5000 Pa and 156.25 Pa respectively.

Equivalently, for a support force of 50 N on a surface with an area of 4 mm ² or 64 mm ² , the pressure exerted is of the order of 125 KPa and 7812.5 Pa respectively. .

The displacement of the bottom of the chamber under the effect of the pressing force to start the mixing, and possibly the generation of bubbles, can be between 0.2 and 4 μm, more advantageously between 2 and 4 μm.

The analysis device 100 can also be configured to move the analysis cartridge 1 and/or the piezoelectric vibration means 110, so as to be able to bring into correspondence, successively, each of the analysis chambers and the piezoelectric vibration means and thus impose successively, and specifically, at the bottom of each of the analysis chambers a vibration. Thus, lateral movements of the analysis cartridge can be provided to bring the latter opposite the piezoelectric vibration means. In a complementary manner, the piezoelectric vibration means can be arranged to be moved in a horizontal direction so as to bring the finger of said means into contact with the rear face of the analysis cartridge.

The analysis device 100 can comprise additional magnetic means intended to impose an additional magnetic field in the at least one analysis chamber of the analysis cartridge. The complementary means are advantageously implemented when a non-magnetic layer 6c is considered.

The analysis device 100 can further comprise means for analyzing the sample present in the at least one analysis chamber 1. These analysis means 120 cooperate with the detection agent 10a. The analysis means 120 can in this respect comprise optical means, and more particularly an epifluorescence microscope.

More particularly, the analysis means 120 are configured to locate/detect the complexes 12 immobilized on the bottom of the analysis chamber 5 by the magnetic layer 6b. In particular, these immobilized complexes form a pattern imposed by the arrangement of the magnetic regions of the magnetic layer 6b.

Also, when the detection agents associated with the immobilized complexes 12 carry a label, it is possible to reveal said immobilized complexes 12.

In particular, the label carried by the detection agents can be photoluminescent, for example fluorescent.

Thus, the analysis means of the analysis device can advantageously comprise a radiation source 121 and a detector 122. The radiation source is in particular intended to induce the emission of a photoluminescence signal by the markers, while the detector is configured to collect said photoluminescence signal.

The present invention also relates to a method for detecting a species likely to be present in a liquid.

The method according to the present invention comprises a step a) of injection into at least one chamber of the cartridge according to the present invention. The injection can be carried out by means of a pipette pouring the liquid into the opening 2 of the analysis cartridge 5.

The injection cartridge is then positioned on the support of the analysis device 100.

The species is likely to bind with the capture agents and the detection agents so as to form complexes agent of magnetic nanoparticles/species/capture agent.

However, the formation of the complexes is preceded by a step b) of dispersion and suspension in the sample of the elements forming the first cluster and the second cluster.

This step b) is carried out in particular by imposing a vibration on the bottom of the analysis chamber with the piezoelectric vibration means so as to generate an acoustic pressure field in the sample.

During this step b), this acoustic pressure field causes the suspension of the elements forming the first cluster and the second cluster and thus makes it possible to promote the formation of the complexes.

Advantageously, the generation of gas bubbles on the side wall of the analysis chamber can also be considered during the execution of step b). The generation of gas bubbles is in particular induced by a mechanical stress exerted by the finger of piezoelectric vibration means on the rear face of the analysis cartridge.

Finally, the method according to the present invention comprises a step c) of detection of the complexes. The person skilled in the art, depending on the conditions imposed on him, may or may not resort to a washing step before performing step c). In any case, this washing step, which is not strictly necessary, can advantageously not be considered.

The method according to the present invention may also comprise the execution of a step c0) before step c). Step c0) leads to the immobilization of the complexes on the bottom of at least one analysis chamber so that said complexes form a pattern defined by the magnetic layer 6b.

When the means of piezoelectric vibration are interrupted, the formation of the complexes can continue. These are then immobilized on the bottom of the analysis chamber.

The vibration imposed by the piezoelectric vibration means can, alternatively, be maintained throughout the period of formation of the complexes. If a non-magnetic film is present, it is also necessary to apply the Bext magnetic field.

Finally, if the detection agent carries a photoluminescent marker, the detection step is performed by photoluminescence.

The invention also relates to a method for studying the kinetics of complex formation. This process incorporates all the characteristics presented in relation to the analysis cartridge and the analysis device. In particular, the implementation of this method may involve an integrated system formed by the analysis cartridge and the analysis device described above. Alternatively, these two elements can be independent.

To this end, it may be considered to proceed successively to the execution, in a cyclical manner, of steps b), c0), and c). In particular, step b) is during each cycle executed for a duration less than the duration necessary for the formation of all the complexes liable to form.

The execution of these cycles can advantageously be implemented to study the kinetics of antibody reaction with a given antigen (or vice versa), and thus characterize the antibody considered.

In the remainder of the description, a process for suspending particles contained in an analysis chamber of a cartridge according to the present invention is described.

More specifically, this process is intended to suspend dried fluorescent beads forming a cluster on the bottom of the analysis chamber.

It is thus proposed a method of preparation of the first clusters then a resuspension of the latter.

The preparation of a cluster involves in particular the drying of Chemicell 100 nm green ARA (fluorescent in the green) magnetic nanoparticles (hereinafter "NP") diluted to 1/40th (compared to a stock solution which has an initial concentration of 45 10 ¹² NP/mL) in order to obtain a concentration of 1.1x10 ¹² NP/mL.

A drop of 2 ml of these magnetic nanoparticles is deposited in each of the analysis chambers of a cartridge in accordance with the invention (FIGS. 8a and 8b respectively representing the deposition of clusters in the analysis chamber, and the chamber empty analysis). There is in particular an image taken by means of a microscope of a first cluster formed at the bottom of an analysis chamber. One can observe on this figure a multitude of clear points (representative of the magnetic nanoparticles) dispersed. Within this first, these magnetic nanoparticles seem however to be organized, within the first cluster, along the zones of strong magnetic gradient (and in the present case in the form of lines spaced 55 μm. These lines spaced 55 μm , are an imprint of the magnetic zones of the magnetic layer revealed by the deposition of the nanoparticles. The image of the first cluster nevertheless remains diffuse. , associated with an empty analysis chamber, reveals no lines and remains dark.

These drops are then dried at 37° C. for approximately one hour.

The cartridge is then closed.

The resuspension procedure then includes the following steps:

1- introduction of PBS liquid into the cartridge analysis chambers;

2- Resuspension with elliptec™ type transducer associated with a finger placed vertically in contact with the center of the cartridge, and exerting a preload of 20N;

3- Magnetic capture of magnetic nanoparticles for 2 minutes using a cylindrical magnet with a diameter of 8mm and a height of 8mm placed 3mm below two chambers for 90 seconds respectively;

4- Observation using a microscope equipped with a 10X magnification lens, an Alexa 488 filter, the exposure being 500ms - Gain 20.

In this regard, the represents an observation made under the microscope at the level of the analysis chamber. In this figure, one can clearly observe clear lines, spaced 110 μm, representative of a fluorescence signal emitted by the magnetic nanoparticles. These images clearly demonstrate the efficiency of the resuspension of the magnetic nanoparticles followed by their immobilization at the end of the magnetic capture step.

The analysis cartridge and the analysis device according to the present invention make it possible to limit manipulations during the analysis of a liquid, and in particular of a biological liquid. Indeed, the pre-positioning of the first and the second cluster on the bottom of at least one chamber, according to predefined quantities, simplifies the analysis process.

The implementation of the piezoelectric vibration means, in association with the release of gas bubbles in the sample of liquid to be analyzed, allows an effective dispersion of the elements forming the first and the second cluster in the sample of liquid.

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 (17)

Analysis cartridge (1) for the detection and/or analysis of a species likely to be present in a liquid, the analysis cartridge (1) being provided with a front face and a rear face opposite the front face, the cartridge (1) further comprises:
- at least one microfluidic analysis chamber (5) intended to receive the liquid, the at least one analysis chamber (5) being formed in the cartridge (1), and comprises a bottom (5a);
- at least one first cluster (9) formed of magnetic nanoparticles held together, and on which capture agents are grafted, said capture agents being configured to bind specifically with the species, the at least one first cluster ( 9) adhering to the bottom (5a). Analytical cartridge (1) according to Claim 1, in which the magnetic nanoparticles forming the first cluster (9) are dried and/or freeze-dried and/or encapsulated in an encapsulating material. Analytical cartridge (1) according to Claim 1 or 2, in which the said analytical cartridge (1) comprises at least one second cluster (10) adhering to the bottom (5a) of the at least one chamber, the at least a second cluster (10) comprises detection agents, held together, and binding specifically with the species, said detection agents also forming photoluminescent markers. Analysis cartridge (1) according to one of Claims 1 to 3, in which the said analysis cartridge (1) comprises a magnetic layer (6b) arranged to immobilize the magnetic nanoparticles of the at least one chamber on the bottom (5a) of said chamber, said magnetic layer (6b) being advantageously microstructured so that the magnetic nanoparticles, when immobilized on the bottom (5a) of the chamber, form a predefined pattern. Analytical cartridge (1) according to claim 4, in which the magnetic layer (6b) comprises a repeated juxtaposition of at least a first region (6b1) and a second region (6b2), the first region (6b1) having a magnetic polarization along a first direction, and the second region (6b2) having either zero magnetic polarization or a magnetic polarization along a second direction different from the first direction. Analysis cartridge (1) according to Claim 4 or 5, in which the said analysis cartridge (1) further comprises a non-magnetic layer (6d) covering the magnetic layer (6b) and intended to screen at least in part the magnetic interaction between the magnetic layer (6b) and the magnetic nanoparticles. Analysis cartridge (1) according to one of Claims 1 to 6, in which the said cartridge comprises at least one opening (2) for discharging the liquid, the said opening being in fluid communication with the at least one analysis chamber (5), advantageously, the fluidic communication between the at least one inlet and the at least one analysis chamber (5) being ensured by at least one microfluidic channel (4). Analysis cartridge (1) according to one of Claims 1 to 7, in which the at least one analysis chamber (5) comprises side walls (5c) surmounting the bottom (5a), and in which a gas is trapped, said gas being capable of being released under the action of a mechanical stress applied to the rear face, the release of the gas leading to the generation of gas bubbles on the side wall, advantageously, the gas is initially trapped in slots opening on the side walls. Analysis device (100) which comprises:
- a support (101) intended to receive the analysis cartridge (1) according to one of Claims 1 to 8 for the purposes of analyzing a liquid contained in the at least one chamber of the said analysis cartridge (1);
- piezoelectric vibration means (110) arranged to impose a vibration on the bottom (5a) of the analysis chamber (5) so as to generate an acoustic pressure field in a liquid likely to be present in the at least an analysis chamber (5), the acoustic pressure field allowing the resuspension and the mixing of the first cluster (9) in the said liquid. Analysis device according to Claim 9, in which the piezoelectric vibration means (110) are associated with at least one finger (111) resting against the rear face and intended to transmit a vibration generated by the said piezoelectric vibration means (110 ) at the bottom (5a) of the analysis chamber (5). Analysis device according to Claim 10, in which the at least one finger (111) is also configured to exert a pressing force against the rear face when it transmits a vibration generated by the piezoelectric vibration means (110) . Analysis device according to one of Claims 9 to 11, in which the at least one analysis chamber (5) comprises a plurality of analysis chambers, and the analysis device (100) is configured to move the analysis cartridge (1) and/or the piezoelectric vibration means, so as to be able to put in correspondence, successively, each of the analysis chambers (5) and the piezoelectric vibration means (110) and thus impose successively, and specifically, at the bottom (5a) of each of the analysis chambers a vibration. Analysis device according to one of Claims 9 to 12, in which the said analysis device (100) comprises complementary magnetic means intended to impose a complementary magnetic field in the at least one analysis chamber (5) of the analysis cartridge (1). Analysis device according to one of Claims 9 to 13, in which the said analysis device (100) further comprises means (120) for analyzing the liquid present in the at least one analysis chamber (5 ). An analysis device according to claim 14, wherein the analysis means comprises a detector (121), and a radiation source (122) configured to, when said radiation interacts with the detection agents of claim 3, generate a photoluminescent signal capable of being detected by the detector. A method of detecting a species likely to be present in a liquid, the method comprising:
a) a step of injecting a sample of liquid into the at least one chamber (5) of the analysis cartridge (1) according to one of Claims 1 to 8 in combination with Claim 3, the species being capable of binding with the capture agents and the detection agents so as to form complexes agent of magnetic nanoparticles/species/capture agent;
b) a step which comprises resuspending and dispersing the first cluster (9) with the means of piezoelectric vibration of the analysis device (100) according to claim 15, and thus allowing the formation of the complexes;
c) a complex detection step. Detection method according to claim 16, in which step c) is preceded by a step c0) of immobilization of the complexes on the bottom (5a) of the at least one analysis chamber (5), the step c) includes operating the detector (121) and the radiation source (122).