Liquid analysis cartridge and analysis device

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) is provided with a front face and a rear face opposite the front face. The cartridge further comprises: - at least one microfluidic analysis chamber (5) for receiving the liquid, the at least one microfluidic chamber being formed within the cartridge and comprising a base (5a); - at least one first cluster (9) formed of magnetic nanoparticles held together and onto which capture agents are grafted, said capture agents being configured to bind specifically with the species, the at least one first cluster (9) being adherent to the base (5a). Figure 4b

Description

Title of the invention: Analytical cartridge and device for analyzing a liquid. FIELD OF THE INVENTION

[0001] The technical field of the invention is that of biological analysis for the purpose of detecting the presence and / or concentration of a species (an analyte) in a liquid, in particular a biological fluid. The invention relates more particularly to a cartridge comprising at least one microfluidic chamber for receiving a liquid to be analyzed.

[0002] The cartridge is preferably intended for use in a point-of-care immunoassay device (for example, a portable device), i.e., one that allows for on-site testing and interpretation to enable immediate clinical decision-making at the patient's bedside rather than in a central laboratory. It can also be used in any other type of biological analysis, for example, for molecular or cell analyses. TECHNOLOGICAL BACKGROUND OF THE INVENTION

[0003] A method for capturing and detecting a species, often referred to as an "analyte," in a liquid, particularly a biological liquid, is known from document EP3447492. The principles of pattern capture and detection implemented by this method are also described in the article by Fratzl et al., "Magnetophoretic induced convective capture of highly diffusive superparamagnetic nanoparticles," Soft Matter, 14. 10.1039 / C7SM02324C.

[0004] This process includes, in particular, a step in which a sample, consisting of a liquid to be analyzed, is mixed with magnetic particles. These particles are nanometric or, more generally, sub-micrometric in size and are coupled to capture elements capable of binding specifically to the species to be detected and / or quantified. This species, the analyte, may be an antigen and the element an antibody, but the reverse configuration is also possible.

[0005] Detection elements are also introduced into the sample during this step. These detection elements may include, in particular, a detection antibody or antigen bearing a photoluminescent marker, for example, a fluorescent one.

[0006] At the end of this step, complexes are formed in the solution, consisting of the capture element, the species, and the detection element. These complexes are then immobilized on a support comprising magnetic micro-sources. ordered according to a specific spatial pattern. The pattern is defined by areas of strong and weak magnetic fields, inducing significant magnetic field gradients. The complexes carried by the magnetic particles tend to aggregate on the support in the areas where the magnetic field strength is greatest. Photoluminescent (and particularly fluorescent) markers make the specific spatial pattern visible, thus indicating the presence of the analyte in the solution. The average (spatially) intensity of this luminous pattern is usually referred to as the "specific signal."

[0007] 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 markers remain dispersed in suspension in the solution. They contribute to forming a relatively homogeneous luminous background. The average (spatially) intensity of this luminous background forms a signal called the "supernatant signal." In addition to the unbound photoluminescent markers, this luminous background also consists of the light intensity emitted by all the photoluminescent materials in 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 bear markers, they do not contribute to the luminous pattern or the luminous background.

[0008] The spatial arrangement of the micro-magnetic field sources in the plane of the support and the luminous intensity of the patterns made apparent by the photoluminescent markers allow for the detection and quantification of the analyte in the sample without washing, i.e., without removing the liquid solution after immobilizing the complexes on the surface of the support, which is particularly advantageous. To enable 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 exhibits a spatially variable intensity (in the plane of the image) 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; the specific signal / supernatant signal ratio allows us to conclude whether the analyte is present in the sample and even to estimate its concentration.

[0009] 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 a pump or valve, whereas traditionally this type of analysis is carried out in a central laboratory.

[0010] To enable the application of the detection method, the biological fluid is introduced into a cartridge, for example a single-use cartridge, comprising The device consists of multiple analysis chambers, with the cartridge designed to be inserted into the analysis unit. These multiple chambers allow for several analyses to be performed on a single biological fluid sample, with each analysis independently conducted on samples held in separate chambers.

[0011] The cartridge includes a liquid discharge opening, a plurality of vents arranged downstream of the analysis chambers, and a network of channels to fluidly connect the opening to the analysis chambers. The biological fluid sample discharged into the opening spreads by capillary action through the network of channels to fill the chambers.

[0012] The implementation of this cartridge, although undeniably presenting many advantages, requires a number of manipulations which it is desirable to reduce.

[0013] One object of the present invention is therefore to provide an integrated analysis cartridge that limits the manipulations necessary to carry out the analysis of a fluid.

[0014] Another object of the present invention is to provide an analysis cartridge and an analysis device that is easy to implement. BRIEF DESCRIPTION OF THE INVENTION

[0015] The objectives 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 comprising:

[0016] - at least one microfluidic analysis chamber intended to receive the liquid, the less an analysis chamber being formed in the cartridge, and includes a bottom;

[0017] - at least a first cluster formed of magnetic nanoparticles held between them, and on which capture agents are grafted, said capture agents being configured to bind specifically with the species, at least a first cluster being adhering to the bottom.

[0018] According to one embodiment, the magnetic nanoparticles forming the first cluster are dried and / or lyophilized and / or encapsulated in an encapsulation material.

[0019] According to one embodiment, at least one analysis chamber comprises side walls above the bottom, 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.

[0020] According to one embodiment, said cartridge comprises at least a second cluster adhering to the bottom of at least one chamber, the at least a second cluster comprises detection agents, held together, and binding specifically with the species, said detection agents also forming photoluminescent markers.

[0021] According to one embodiment, said analysis cartridge includes a magnetic layer arranged to immobilize the magnetic nanoparticles of at least one chamber on the bottom of said chamber, said layer being advantageously micro-structured so that the magnetic nanoparticles, when immobilized on the bottom of the chamber, form a predefined pattern.

[0022] 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 along a first direction, and the second region having either a zero magnetic polarization or a magnetic polarization along a second direction different from the first direction.

[0023] 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.

[0024] According to one embodiment, said cartridge includes at least one liquid discharge opening, said opening being in fluidic communication with at least one analysis chamber, advantageously, the fluidic communication between at least one inlet and at least one analysis chamber being ensured by at least one microfluidic channel.

[0025] The invention also relates to an analysis device which comprises:

[0026] - a support for receiving the analysis cartridge according to the present invention for the purpose of analyzing a liquid contained in at least one chamber of said analysis cartridge;

[0027] - piezoelectric vibration means arranged to impose a vibration on bottom of the analysis chamber so as to generate an acoustic pressure field in a liquid likely to be present in at least one analysis chamber, the acoustic pressure field enabling the resuspension and mixing of the first cluster in said liquid.

[0028] 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.

[0029] According to one embodiment, 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.

[0030] According to one embodiment, 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 as to be able to successively match each of the analysis chambers and the piezoelectric vibration means and thus successively and specifically impose a vibration on the bottom of each of the analysis chambers.

[0031] According to one embodiment, said analysis device includes complementary magnetic means for imposing a complementary magnetic field in at least one analysis chamber of the analysis cartridge.

[0032] According to one embodiment, said analysis device further includes means for analyzing the liquid present in at least one analysis chamber.

[0033] According to one embodiment, the analysis means include 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.

[0034] The invention also relates to a method for detecting a species likely to be present in a liquid, the method comprising:

[0035] a) a step of injecting a sample of liquid into 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;

[0036] b) a step which includes the resuspension and dispersion of the first cluster with the piezoelectric vibration means of the analysis device according to the present invention, and thus allows the formation of the complexes;

[0037] c) a complex detection step.

[0038] According to one embodiment, step c) is preceded by a step c0) of immobilizing the complexes on the bottom of at least one analysis chamber; step c) includes the implementation of the detector and the radiation source. Brief description of the drawings

[0039] Other features and advantages will become apparent 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 accompanying drawings in which:

[0040] [Fig.1] Fig.1 is a schematic, perspective representation of an analysis cartridge that can be implemented according to the principles of the present invention;

[0041] [Fig.2] [Fig.2] is a schematic representation of the fluidic section according to a cross-sectional plane of said fluidic section parallel to the upper face, [Fig.2] represents in particular at least one analysis chamber in fluidic communication on the one hand with the opening via the channels and on the other hand with at least one vent via at least one vent channel;

[0042] [Fig.3] Fig.3 is a schematic representation of the cartouche shown in the [Fig.l] in perspective and exploded view;

[0043] [Fig.4a] Fig.4a is a schematic representation along a cross-sectional plane transverse (perpendicular to the main faces) of the cartridge of [Fig.1] at the level of at least one analysis chamber;

[0044] [Fig.4b] Fig.4b is another schematic representation along a cross-sectional plane transverse (perpendicular to the main faces) of the cartridge of [Fig.1] at the level of at least one analysis chamber, said cartridge implementing a non-magnetic layer;

[0045] [Fig.4c] Fig.4c is a perspective representation of an interleaving film likely to be implemented for the assembly of the cartridge of [Fig.1];

[0046] [Fig.4d] The [Fig.4d] is a schematic top view representation of a detection pattern defined by the magnetization produced by a magnetic layer integrated into the support of a cartridge, the magnetic field present in an analysis chamber and the magnitude of this field#;

[0047] [Fig.5] [Fig.5] is a schematic representation of an analysis chamber filled with a liquid which includes in suspension the analysis, the magnetic nanoparticles on which capture agents, detection agents are grafted, [Fig.5] illustrates in particular the mechanism of formation of the complexes;

[0048] [Fig.6] Fig.6 is a representation of an analysis device implemented according to the principles of the present invention;

[0049] [Fig.7a]

[0050] [Fig. 7b] Figures 7a and 7b are representations of the interior of the device analysis of [Fig.6], the finger of the piezoelectric vibration means being in a retracted position, [Fig.7b] being a cross-sectional view;

[0051] [Fig.7c] Fig.7c is a representation of the interior of the analysis device [Fig.6], the finger of the piezoelectric vibration means being in an engaged position;

[0052] [Fig.8a] [Fig.8a] is a microscopic image of a deposit of clumps on the background of an analysis chamber conforming to the present invention;

[0053] [Fig.8b] Fig.8b is a microscopic image of the bottom of a chamber empty analysis according to the present invention;

[0054] [Fig.8c] Fig.8c is a microscopic observation of the bottom of a chamber at the end of the sequence of a resuspension step and capture of magnetic nanoparticles. DETAILED DESCRIPTION OF THE INVENTION

[0055] Fig. 1 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.

[0056] 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.

[0057] The analysis cartridge 1 thus comprises at least one microfluidic analysis chamber 5. In particular, the analysis cartridge may comprise between 1 and 10, for example 5, analysis chambers 5.

[0058] The analysis cartridge 1 may include a gripping end for handling it. The gripping end may bear a label, for example with a barcode or a two-dimensional code, enabling identification and traceability of analyses performed using the analysis cartridge 1 in question. Alternatively, the identification means may include an RFID chip.

[0059] The analysis cartridge 1 also includes an active section 1b formed, for example, in the extension of the gripping end la.

[0060] The active section 1b is generally planar in shape and comprises two principal faces called, respectively, upper face and lower face.

[0061] The analysis cartridge 1 may include a pouring opening 2 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.

[0062] The opening 2 is in particular in fluidic communication with at least one analysis chamber 5. In particular, the active section includes at least one microfluidic channel 4 ensuring fluidic communication between the opening 2 and at least one analysis chamber 5. In other words, at least one microfluidic channel 4 ensures the flow and distribution of the liquid poured into the opening 2 towards at least one analysis chamber.

[0063] The analysis cartridge 1 may also include at least one vent 3 in fluidic communication with at least one analysis chamber 5 (Figures 1 and 2). The at least one vent 3 is specifically configured to allow air to be evacuated likely to be present in at least one analysis chamber 5 during the filling of the latter with liquid. In particular, fluid communication between at least one analysis chamber 5 and at least one vent 3 is ensured by at least one vent channel 4'.

[0064] 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 at least one analysis chamber 5 via at least one microfluidic channel 4. The air likely to be present in at least one analysis chamber 5 is expelled during the flow of the liquid in said analysis chamber 5 towards at least one vent 3.

[0065] In the case of multiple 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. "Sequential flow" means that the analysis chambers 5 are filled in a predetermined order. In particular, according to this principle, the flow in a given chamber does not begin until the analysis chamber preceding it in the filling order is completely filled.

[0066] Still in the case of plurality of analysis chambers 5, it can also be provided that the analysis cartridge includes a plurality of openings, for example an opening dedicated to each chamber of the cartridge.

[0067] The analysis cartridge 1 shown in [Fig. 1] may further include a reservoir 2' above the opening 2, the volume of which is equal to that of the microfluidic network formed by at least one microfluidic channel 4, at least one analysis chamber 5 and at least one vent channel 4'. In this respect, this volume may be between 5 mm3 and 500 mm3, and more precisely between 20 mm3 and 100 mm3.

[0068] Equivalently, at least one vent 3 is surmounted by a peripheral wall 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 reservoir 2' to prevent liquid from escaping from the analysis cartridge 1. This arrangement helps to limit health problems, or even damage to an analysis device (described later in the statement) into which the analysis cartridge 1 is intended to be inserted.

[0069] By way of illustration, the analysis cartridge 1 may have dimensions between 2 cm and 10 cm in width and length, and a thickness between 4 mm and 10 mm. The at least one analysis chamber 5 may have a volume typically between 1 mm³ and 50 mm³ for receiving the sample, advantageously between 5 mm³ and 25 mm³.

[0070] According to a non-limiting example shown in [Fig.3], 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 placing their so-called "main" surfaces opposite each other.

[0071] At least one analysis chamber 5, at least one microfluidic channel 4 and at least one vent channel 4' form a microfluidic network of the analysis cartridge 1. This microfluidic network is defined in particular 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 assembled to each other.

[0072] Each channel 4, 4' is delimited, on the one hand, by the main surfaces of the support 6 and the cover 7, forming, respectively, a bottom and an arch, and on the other hand, by side walls connecting the bottom and the arch. The distance separating the bottom and the arch of a channel 4, 4' defines a channel height, while the distance separating two opposing side walls defines a channel width.

[0073] Equivalently, the at least one analysis chamber 5 is also delimited by the main surfaces of the support 6 and the cover 7, forming, respectively, a chamber bottom 5a and a chamber vault 5b (Figures 4a and 4b). The at least one chamber 5 further comprises chamber side walls 5c extending from the chamber bottom 5a to the chamber vault 5b. It is understood, however, that the at least one analysis chamber 5a may lack a vault, and, for example, form an open well at the active section.

[0074] It is also understood that the side walls of at least one chamber can be deformable.

[0075] The upper hood 7, at least for the part that overhangs at least one The analysis chamber 5 may be made of a material transparent to a photoluminescence signal emitted by detection agents described later in the statement. The material forming the upper cover 7 may comprise at least one of the following materials: a plastic material, for example based on polycarbonate, cycloolefin copolymer or polystyrene, or glass.

[0076] The outer surface of the hood 7 can be optically polished at least at the right of at least one analysis chamber 5.

[0077] The microfluidic network, such as that shown in [Fig. 2], therefore extends in the principal plane of the analysis cartridge 1. It is millimeter-sized, meaning that the width of the channels 4, 4' and the analysis chambers 5 is typically between 0.1 mm and 10 mm. The height of these elements, i.e., the distance separating the bottom of an arch, is also between 0.1 mm and 10 mm. The liquid that can be introduced at opening 2 spreads through the microfluidic network by capillarity.

[0078] The analysis cartridge 1 according to the present invention also comprises at least a first cluster 9 adhering to the bottom of at least one analysis chamber 5 ([Fig. 4a]). The at least first cluster 9 is in particular formed of magnetic nanoparticles 9a held together, and onto which capture agents 9b are grafted ([Fig. 5]).

[0079] Alternatively, it may be considered to place at least one first cluster on the vault 5b of at least one chamber 5. This configuration may 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 later in the statement.

[0080] A first cluster may advantageously have a volume between 0.1 pl and 5 pl and advantageously between 0.5 pl and 2 pl.

[0081] By "held together," we mean a set of nanoparticles bonded together. This cohesion between the nanoparticles can be direct or indirect. Direct cohesion can, in particular, be ensured by dry or lyophilized nanoparticles, while indirect cohesion can be ensured by an encapsulating material. In this respect, the encapsulating material can include sugar (trehalose, glucose, etc.), viscous solutions (for example, Tween), or glycerol.

[0082] Maintaining the nanoparticles together, and in the form of clusters, ensures better stability of the latter over time.

[0083] The implementation of an encapsulation material makes it easier to suspend nanoparticles as presented in the rest of the description.

[0084] Magnetic nanoparticles 9a can be nanometric in size, typically between 25 nm and 500 nm, and preferably between 100 and 300 nm. Magnetic nanoparticles 9a can generally be spherical in shape. These nanoparticles exhibit superparamagnetic characteristics and are biocompatible. They can also be coated with a polymer (such as polystyrene) with a surface treatment that allows them to be functionalized, for example, with Ac or Ag type proteins. The controlled quantity of particles is such that their concentration in the volume of the chamber once filled with the liquid to be analyzed is between 10⁶ particles / ml and 10¹² particles / ml, advantageously between 10⁹ particles / ml and 10¹¹ particles / ml.

[0085] Capture agents 9b are capable of binding specifically to the analyte likely to be present in the liquid. In this respect, the analyte may be an antigen, while capture agent 9b includes an antibody (the reverse configuration is also possible).

[0086] The analysis cartridge 1 may also include at least a second cluster 10 adhering to the bottom of at least one analysis chamber 5 (Figures 4a and 4b). The at least one second cluster 10 is notably formed of detection agents 10a bonded together ([Fig. 5]).

[0087] A second cluster may advantageously have a volume between 0.1 pl and 5 pl and advantageously between 0.5 pl and 2 pl.

[0088] At least one first cluster 9 and at least one second cluster 10 are both intended to enable, respectively, the capture, or even the immobilization, of an analyte 11 present in a liquid, and the detection, or even the quantification, of 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 magnetic nanoparticles 9a on which the capture agents 9b are grafted, forms complexes 12 with these elements ([Fig.5]).

[0089] The magnetic nanoparticles are intended to isolate the complexes, while the detection agents enable them to be detected.

[0090] Thus, the implementation of the analysis cartridge 1 for the detection and / or quantification of an analyte in a liquid involves pouring a sample of said liquid into at least one analysis chamber via the opening 2.

[0091] 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.

[0092] This suspension may include, in particular, separating the first and second clusters from the bottom of chamber 5a, as well as separating the elements from each other in order to disperse them within the sample. To this end, piezoelectric vibration means 110 described later in this statement may be used.

[0093] These piezoelectric vibration means 110 are particularly suitable for imposing a vibration on the bottom of 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.

[0094] According to a first embodiment, the vibration can be imposed at fixed frequencies, for example between 5 kHz and 2 MHz, advantageously between 20 kHz and 200 kHz. Also according to this first embodiment, the vibration can be close to a resonance frequency of the analysis chamber 5 (by "close to the resonance frequency" is meant a frequency within + / - 15%, advantageously within + / - 10%, and even more advantageously within + / - 5% of the resonance frequency of the analysis chamber). Advantageously, the resonance frequency of the analysis chamber 5 is on the order of 50 KHz, or on the order of 80 KHz or on the order of 110 KHz.

[0095] An analysis chamber exhibiting such resonance frequencies may comprise, along a cross-sectional plane parallel to the cartridge, a rectangular section terminated at each end by an opening whose cross-section, along the same plane, is triangular. The length and width of the rectangular section are 8.4 mm and 2.4 mm respectively, while the base and height of the triangular section are 2.4 mm and 4 mm respectively. The height of the analysis chamber is 390 sq m.

[0096] According to a second embodiment, 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 include a sweep in increasing or decreasing frequency, and more particularly a frequency sweep around the resonance frequency of the analysis chamber. The complete frequency sweep can last from 10 seconds to 200 seconds. In particular, this frequency sweep includes an increment or decrement of the frequency in steps. Specifically, each step corresponds to holding the frequency for a duration that can be from 2 seconds to 10 seconds so as to allow the liquid to move sufficiently for a minimal mixing effect.

[0097] By way of example, a cycle may involve a frequency sweep from 110 kHz to 120 kHz, or conversely a frequency sweep from 120 kHz to 110 kHz. This sweep may be executed in steps of 1 kHz, and the vibration may be maintained at each step for a duration of a few seconds, and in particular for 2 seconds.

[0098] As another example, the vibrational sequence may include the repetition of an elementary sequence which includes a first cycle and a second.

[0099] The first cycle may include a frequency sweep from 120 KHz to 111 KHz in 1 KHz steps and a holding of the vibration at each step for 2 seconds.

[0100] The second cycle may include a frequency sweep from 110 KHz to 119 KHz in 1 KHz steps and a holding of the vibration at each step for 2 seconds.

[0101] This elementary sequence can, for example, be repeated from 1 to 4 times.

[0102] Advantageously, the side walls 5c can be structured to create vortices that promote mixing in the sample as it flows through the analysis chamber. This structuring of the side walls 5c can include a crenellated surface.

[0103] In a particularly advantageous manner, gas, and more particularly air, is initially trapped in the side wall 5c of at least one chamber 5.

[0104] The trapped gas can be released under the action of a mechanical stress applied to at least one analysis chamber and thus generate gas bubbles on the wall lateral of at least one chamber when the liquid sample is present in at least one chamber 5. This mechanical constraint can, in particular, be applied to the rear face of the analysis cartridge 1.

[0105] According to an advantageous embodiment, the side walls 5c of at least one chamber 5 may be made of a porous material. The gas is initially located in the pores of the porous material.

[0106] According to an alternative or complementary embodiment, the gas can initially be trapped in slots 5e opening onto the side walls ([Fig.4c]).

[0107] The slots 5e may have a length between 100 qm and 1000 qm, and a cross-section whose largest dimension is between 100 qm and 400 qm.

[0108] The cross-section of a slot may include at least one of the shapes chosen from: square, rectangle, round.

[0109] According to the present invention, once the gas bubbles are generated (in particular under the effect of the previously described stress), 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.

[0110] These vibrating gas bubbles then act as additional vibrating agents which help to improve the mixing of the liquid present in the analysis chamber.

[0111] This effect can nevertheless be optimized by forcing the gas bubbles to vibrate at a frequency close to their resonant frequency (by "close to the resonant frequency," we mean a frequency within + / - 15%, advantageously within + / - 10%, and even more advantageously within + / - 5% of the resonant frequency of the gas bubbles). Indeed, under these conditions, the contribution of the gas bubbles to the mixture is exacerbated, especially since some bubbles are likely to transmit energy through their implosion and the resulting shock wave.

[0112] For this purpose, the pores and / or slits trapping the gas can be configured so that the gas bubbles that may be generated have a resonance frequency close to that of the analysis chamber.

[0113] In particular, for the specified slot sizes, the bubble sizes can range from 50 sqm to 500 sqm. For this range of bubble sizes, the resonance frequencies are between 20 kHz and 500 kHz.

[0114] According to the present invention, the analysis cartridge 1 may include a magnetic layer 6b ([Fig. 4a]) arranged to immobilize the magnetic nanoparticles 9a of at least one analysis chamber 5 on the bottom of said chamber. It is therefore understood that this magnetic layer also immobilizes the complexes 12. The magnetic layer 6b is advantageously microstructured so as that the magnetic nanoparticles immobilized on the bottom of the analysis chamber 5 form a predefined pattern.

[0115] The magnetic layer 6b may 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 may have a magnetic polarization along a first direction, and the second region 6b2 may have either a zero magnetic polarization or a magnetic polarization along a second different direction, preferably at 180°, from the first direction ([Fig.4a]).

[0116] The support 6 can in particular be arranged so that it comprises, from its main face to 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 (Figures 3, 4a and 4b).

[0117] The rigid substrate 6a may comprise a plastic material. The magnetic layer 6b may be disposed on the substrate 6a, or integrated into this substrate, at least at the level of the analysis chambers 5 of the microfluidic network.

[0118] The magnetic layer 6b may comprise magnetic composite materials, such as ferrites, randomly distributed in a polymer or oriented along a pre-orientation axis. This magnetic layer 6b may be similar to a conventional magnetic recording tape.

[0119] The substrate 6a may also include a non-magnetic layer 6c (or a plurality of such films) intercalated between the magnetic layer 6b and the intercalating film 6d. This non-magnetic layer 6c, which is optional, is intended to move the magnetic layer 6b away from the bottom of the analysis chamber 5.

[0120] By "non-magnetic layer" is meant a layer whose magnetic susceptibility is zero or even less (in absolute value) than 103.

[0121] The non-magnetic layer 6c may, for example, comprise a plastic material, such as polypropylene on acrylic.

[0122] In the example shown above, the interlayer film 6d defines the microfluidic network. In particular, the interlayer film 6d illustrated in [Fig. 4c] has a cut-out pattern corresponding to the microfluidic network. When this interlayer film 6d is assembled with the substrate 6a to form the support 6, the latter therefore has recesses reproducing the cut-out pattern of the film 6d. These recesses, possibly in combination with additional recesses formed in the upper cover 7, constitute the microfluidic network of the cartridge 1.

[0123] Advantageously, the interlayer film 6d is an adhesive film, which also allows the upper cover 7 to be assembled and hermetically sealed to each other at their contact surfaces, i.e., surrounding the recesses. It can, for example, be a double-sided adhesive film, ensuring then simultaneously its assembly to the substrate 6a, and to the upper cover 7. As is well known in itself, such an interlayer film 6d can be made of a strip, for example plastic, the two sides of which are coated with an adhesive material.

[0124] Advantageously, gas or air bubbles, when mechanical stress is applied to the bottom of the chamber, can be generated by the release of gas trapped in the interlayer film. This film can therefore be porous and / or contain slits, for example formed during the cutting of the pattern corresponding to the microfluidic network.

[0125] Returning to the description of the magnetic character of the analysis cartridge, the magnetic layer 6b comprises a succession of polarized regions 6b1 and 6b2 in two different directions (opposite in Figures 4a and 4b). As shown in [Fig. 4d], which represents a top view of the magnetic layer 6b, the magnetically polarized regions extend in a line along a principal direction P in the example shown.

[0126] Regions of relatively high magnetic intensity, designated as attraction zones, are observed at the interfaces between areas of different polarization. In particular, the attraction zones are arranged in the form of a plurality of lines Za oriented along the principal direction P. The specific arrangement of these lines defines, in combination, a detection pattern.

[0127] It is understood that the inline arrangement taken as an example is only one particular case of a detection pattern. An analysis cartridge 1 is more generally provided with magnetically polarized regions defining a well-defined detection pattern, but whose configuration can be freely chosen.

[0128] The magnetic field Bc generated by the magnetic layer 6b and its magnitude are also shown in [Fig. 4d]. As will be explained later in this discussion, it can be useful to add an additional (or complementary) external field Bext to the field produced by the layer 6b. This external field Bext, which combines with the field Bc produced by the layer, and the magnitude of this combined field are shown in [Fig. 4d]. It can be observed that applying this external magnetic field Bext can eliminate certain areas of attraction Za produced when only the field provided by the magnetic layer 6b is present. However, in all cases, these areas of attraction are arranged along lines Za oriented along the principal direction P, or more generally, along a detection pattern whose characteristics are precisely defined.

[0129] In the case of an analysis chamber 5 having the dimensions indicated above, it is possible to form a detection pattern comprising between 2 and 50 lines, these having a thickness of between 1 pm and 150 pm (advantageously between 5 pm and 30 pm) and separated from each other by a spacing of between 5 pm and 300 pm, advantageously between 25 pm and 150 pm.

[0130] Based on 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.

[0131] The micro-magnets of the magnetic layer, on which the non-magnetic layer rests, have a width of 50 µm and a height of 10 µm. These micro-magnets are plane-polarized and alternate with each other. In this calculation, a magnetic field source made of NdFeB below this magnetic layer is also implemented. This source imposes a magnetic field of 1.2 Tesla.

[0132] In the course of this calculation, the inventors were 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 within approximately an order of magnitude. These gradients result in a magnetic force exerted on the particles. This force is capable of retaining the magnetic particles (in particular clusters), and also contributes to their immobilization, as illustrated in [Fig. 8c] (described at the end of this application).

[0133] Thus, when a liquid sample is introduced into the analysis cartridge 1, it flows into the microfluidic channels 4 to fill at least one analysis chamber 5 and propagates into the vent channels 4'. Vibration and mechanical stress are applied to 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 applied to the bottom suspends the elements forming the first and second clusters. This acoustic pressure field also causes the gas bubbles to vibrate.

[0134] The vibration of these nanoparticles 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 subsequent reaction time, 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 formed. These complexes are immobilized on the support 6 of the analysis chamber 5 by preferentially agglomerating at the maxima of the magnetic field intensity norm (induced by the micro-sources and possibly the external magnetic field), and thus to arrange themselves according to the The detection pattern is defined by the magnetic layer 6b. Excess detection elements remain suspended in the sample.

[0135] It can be provided that each analysis chamber 5 of a cartridge 1 is prepared to receive capture elements and detection elements of different kinds, so as to carry out multiple analyses of a liquid sample introduced into the analysis cartridge 1. It can also be provided that the detection pattern encoded by the portion of the magnetic layer 6b which is disposed at the level of a chamber 5 is different from one chamber to another.

[0136] It is also possible to consider a magnetic layer which includes an area devoid of magnetic areas, and directly above which the first cluster is formed (the resuspension of the magnetic nanoparticles of the first cluster can thus be facilitated).

[0137] 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.

[0138] The invention also relates to an analysis device 100 intended to cooperate with the analysis cartridge ([Fig.6]).

[0139] The analysis device 100 includes in particular a support 101 intended to receive an analysis cartridge 1 for the purpose of analyzing the liquid sample contained in at least one analysis chamber 5 (Figures 7a to 7c).

[0140] The analysis device 100 also includes 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 at least one analysis chamber 5.

[0141] The piezoelectric vibration means 110 may include a transducer, in particular an elliptical transducer of the Elliptec™ transducer type.

[0142] Alternatively, the piezoelectric vibration means 110 may include a piezoelectric stack (Piezostack), in particular a so-called Langevin transducer (A Langevin transducer is made by a stack of ceramics held between two metal pieces which ensure a clamping of the assembly).

[0143] In this respect, the inventors were able to demonstrate that these linear transducers exhibited good mixing dynamics.

[0144] Bolt-clamped Langevin ultrasonic transducer type transducers are also very effective.

[0145] As previously stated, the acoustic pressure field allows the resuspension and mixing of the first cluster 9 and the second cluster 10 in the liquid sample.

[0146] Advantageously, the piezoelectric vibration means 110 are associated with at least one finger 111 which can bear against the cartridge, particularly against its rear face. This at least one finger 111 is specifically designed to transmit the vibration generated by said piezoelectric vibration means to the bottom of the analysis chamber 5. Also advantageously, the at least one finger 111 is also configured to exert a pressing force against the rear face when it transmits the vibration generated by the piezoelectric vibration means. Even more advantageously, this pressing force (or mechanical stress) allows the gas trapped in the interlayer film to be released, generating gas bubbles on the side wall of the analysis chamber. These gas bubbles, when in resonance with the acoustic pressure field, allow for improved mixing.

[0147] 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 area between 4 mm2 and 64 mm2.

[0148] Thus, for a support force of 1 N on a surface of an area of ​​4 mm2 or 64 mm2, the pressure exerted is on the order of, respectively, 5000 Pa and 156.25 Pa.

[0149] Equivalently, for a support force of 50 N on a surface of an area of ​​4 mm2 or 64 mm2, the pressure exerted is on the order of, respectively, 125 kPa and 7812.5 Pa.

[0150] The displacement of the bottom of the chamber under the effect of the support force to start the mixing, and possibly the generation of bubble, can be between 0.2 and 4 pm, more advantageously between 2 and 4 pm.

[0151] The analysis device 100 can also be configured to move the analysis cartridge 1 and / or the piezoelectric vibration means 110, so as to successively align each of the analysis chambers with the piezoelectric vibration means and thus successively and specifically impart a vibration to the bottom of each of the analysis chambers. Lateral displacements of the analysis cartridge can be provided to bring it into contact with the piezoelectric vibration means. Additionally, 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.

[0152] The analysis device 100 may include complementary magnetic means for imposing a complementary magnetic field in at least one analysis chamber of the analysis cartridge. The complementary means are advantageously implemented when a non-magnetic layer 6c is considered.

[0153] The analysis device 100 may further include means for analyzing the sample present in at least one analysis chamber 1. These analysis means 120 cooperate with the detection agent 10a. The analysis means 120 may in this respect include optical means, and more particularly an epifluorescence microscope.

[0154] More specifically, 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.

[0155] Also, when the detection agents associated with the immobilized complexes 12 carry a marker, it is possible to reveal said immobilized complexes 12.

[0156] In particular, the marker carried by the detection agents may be photoluminescent, for example fluorescent.

[0157] Thus, the analysis means of the analysis device may advantageously include a radiation source 122 and a detector 121. 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.

[0158] The present invention also relates to a method for detecting a species that may be present in a liquid.

[0159] 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.

[0160] The analysis cartridge is then positioned on the support of the analysis device 100.

[0161] The species is capable of binding with capture agents and detection agents so as to form magnetic nanoparticle agent / species / capture agent complexes.

[0162] 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.

[0163] This step b) is notably carried out by imposing a vibration on the bottom of the analysis chamber with piezoelectric vibration means so as to generate an acoustic pressure field in the sample.

[0164] During this step b), this acoustic pressure field generates the suspension of the elements forming the first cluster and the second cluster and thus promotes the formation of the complexes.

[0165] Advantageously, the generation of gas bubbles on the side wall of the analysis chamber can also be envisaged during the execution of step b). The generation of gas bubbles is in particular induced by a mechanical constraint exerted by the finger of piezoelectric vibration means on the rear face of the analysis cartridge.

[0166] Finally, the process according to the present invention includes a step (c) of complex detection. A person skilled in the art may, depending on the conditions imposed upon them, use or not use a washing step before carrying out step (c). In any case, this washing step, which is not strictly necessary, may advantageously be omitted.

[0167] The method according to the present invention may also include performing a step cO) before step c). Step cO) 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.

[0168] When the piezoelectric vibration means are interrupted, the formation of complexes can continue. These are then immobilized on the bottom of the analysis chamber.

[0169] The vibration imposed by the piezoelectric vibration means can alternatively be maintained throughout the entire complex formation period. If a non-magnetic film is present, it is also necessary to apply the magnetic field Bext.

[0170] Finally, if the detection agent carries a photoluminescent marker, the detection step is performed by photoluminescence.

[0171] The invention also relates to a method for the kinetic study of complex formation. This method incorporates all the characteristics presented in connection with 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 may be independent.

[0172] 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 likely to form.

[0173] The execution of these cycles can advantageously be implemented to study the reaction kinetics of antibodies with a given antigen (or vice versa), and thus characterize the antibody in question.

[0174] In the following description, a method for suspending particles contained in an analysis chamber of a cartridge according to the present invention is described.

[0175] More particularly, this process is intended to suspend fluorescent dried beads forming a cluster on the bottom of the analysis chamber.

[0176] A method of preparing the first clusters and then resuspension of the latter is thus proposed.

[0177] The preparation of a cluster involves in particular the drying of Chemicell 100 nm green ARA magnetic nanoparticles (hereinafter “NP”) (fluorescent in the green) diluted at l / 40th (relative to a stock solution which has an initial concentration of 45 1012 NP / mL) in order to obtain a concentration in 1.1 x 012 NP / mL.

[0178] A 2 ml drop of these magnetic nanoparticles is deposited in each of the analysis chambers of a cartridge according to the invention (Figures 8a and 8b respectively showing the deposit of clusters in the analysis chamber and the empty analysis chamber). Figure 8a is in particular an image taken with a microscope of a first cluster formed at the bottom of an analysis chamber. A multitude of scattered bright dots (representing the magnetic nanoparticles) can be observed in this figure. Within this first cluster, these magnetic nanoparticles appear to organize themselves along areas of strong magnetic gradient (and in this case, as lines spaced 55 µm apart). These lines, spaced 55 µm apart, are an imprint of the magnetic zones of the magnetic layer revealed by the nanoparticle deposition. The image of the first cluster nevertheless remains diffuse. Figure 8a shows a 2 ml drop of these magnetic nanoparticles in each of the analysis chambers of a cartridge according to the invention.8b], when paired with an empty analysis chamber, reveals no lines and remains dark.

[0179] These drops are then dried at 37°C for about one hour.

[0180] The cartridge is then closed.

[0181] The resuspension procedure then comprises the following steps:

[0182] 1- introduction of PBS liquid into the analysis chambers of the cartridge;

[0183] 2- Resuspension with an elliptec™ type transducer associated with a finger arranged vertically in contact with the center of the cartridge, and exerting a preload of 20N;

[0184] 3- Magnetic capture of magnetic nanoparticles for 2 minutes using of a cylindrical magnet with a diameter of 8mm and a height of 8mm placed 3mm below two chambers for 90 seconds respectively;

[0185] 4- Observation using a microscope equipped with a magnifying lens 10X, with an Alexa 488 filter, the exposure being 500ms - Gain 20.

[0186] In this regard, [Fig.8c] represents a microscopic observation at the level of the analysis chamber. In this figure, lines can be clearly observed Clear, spaced 110 pm apart, representative of a fluorescence signal emitted by magnetic nanoparticles. These images clearly demonstrate the effectiveness of the resuspension of magnetic nanoparticles followed by their immobilization after the magnetic capture step.

[0187] The analysis cartridge and the analysis device according to the present invention make it possible to limit handling during the analysis of a liquid, and in particular a biological liquid. Indeed, the pre-positioning of the first and second clusters on the bottom of at least one chamber, in predefined quantities, simplifies the analysis process.

[0188] The implementation of piezoelectric vibration means, in association with the release of gas bubbles in the liquid sample to be analyzed, allows an efficient dispersion of the elements forming the first and second clusters in the liquid sample.

[0189] Of course the invention is not limited to the described embodiment and alternative embodiments can be made without departing from the scope of the invention as defined by the claims.

Claims

Demands

1. Analytical cartridge (1) for the detection and / or analysis of a species likely to be present in a liquid, the analytical cartridge (1) being provided with a front face and a rear face opposite the front face, the cartridge (1) further comprising: - at least one microfluidic analytical chamber (5) intended to receive the liquid, the at least one analytical chamber (5) being formed in the cartridge (1), and comprising a base (5a); - at least one first cluster (9) formed of magnetic nanoparticles held together, and onto 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 base (5a);- a magnetic layer (6b) arranged to immobilize the magnetic nanoparticles of 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; - 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.

2. Analysis cartridge (1) according to claim 1, wherein the magnetic nanoparticles forming the first cluster (9) are dried and / or lyophilized and / or encapsulated in an encapsulation material.

3. Analysis cartridge (1) according to claim 1 or 2, wherein said analysis cartridge (1) comprises at least a second cluster (10) adhering to the bottom (5a) of 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.

4. Analysis cartridge (1) according to any one of claims 1 to 3, wherein 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 polarization magnetic along a first direction, and the second region (6b2) exhibiting either zero magnetic polarization or magnetic polarization along a second direction different from the first direction.

5. Analytical cartridge (1) according to any one of claims 1 to 4, wherein said cartridge comprises at least one liquid discharge opening (2), said opening being in fluidic communication with at least one analytical chamber (5), advantageously, the fluidic communication between at least one inlet and at least one analytical chamber (5) being ensured by at least one microfluidic channel (4).

6. Analytical cartridge (1) according to any one of claims 1 to 5, wherein at least one analytical chamber (5) comprises side walls (5c) overhanging 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 onto the side walls.

7. Analytical device (100) comprising: - a support (101) on which is disposed the analytical cartridge (1) according to any one of claims 1 to 6 for the purpose of analyzing a liquid contained in at least one chamber of said analytical cartridge (1); - piezoelectric vibration means (110) arranged to impose a vibration on the bottom (5a) of the analytical chamber (5) so as to generate an acoustic pressure field in a liquid likely to be present in at least one analytical chamber (5), the acoustic pressure field enabling the resuspension and mixing of the first clump (9) in said liquid.

8. Analytical device according to claim 7, wherein the piezoelectric vibration means (110) are associated with at least one finger (111) bearing against the rear face and intended to transmit a vibration generated by said piezoelectric vibration means (110) to the bottom (5a) of the analytical chamber (5).

9. Analytical device according to claim 8, wherein at least one finger (111) is also configured to exert a force support against the rear face when it transmits a vibration generated by the piezoelectric vibration means (110).

10. Analytical device according to any one of claims 7 to 9, wherein at least one analytical chamber (5) comprises a plurality of analytical chambers, and the analytical device (100) is configured to move the analytical cartridge (1) and / or the piezoelectric vibration means, so as to be able to successively match each of the analytical chambers (5) and the piezoelectric vibration means (110) and thus successively and specifically impose a vibration on the bottom (5a) of each of the analytical chambers.

11. Analysis device according to any one of claims 7 to 10, wherein said analysis device (100) comprises complementary magnetic means for imposing a complementary magnetic field in at least one analysis chamber (5) of the analysis cartridge (1).

12. Analytical device according to any one of claims 7 to 11, wherein said analytical device (100) further comprises analytical means (120) for analyzing the liquid present in at least one analytical chamber (5).

13. Analytical device according to claim 12, wherein the analytical means comprise 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.

14. 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 at least one chamber (5) of the analysis cartridge (1) according to any one of claims 1 to 6 in combination with claim 3, 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 resuspension and dispersion of the first cluster (9) with the piezoelectric vibration means of the analysis device (100) according to claim 15, and thus enabling the formation of the complexes; 26 c) a complex detection step.

15. A detection method according to claim 14, wherein step c) is preceded by a step c0) of immobilizing the complexes on the bottom (5a) of at least one analysis chamber (5), step c) includes the implementation of the detector (121) and the radiation source (122).

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