Contactless method for mixing thin fluid layers using acoustic waves

Abstract

The invention relates to a method (100) for mixing fluids using acoustic waves, the method (100) comprising the following steps: (110) providing a planar support (10), a sample (20) of biological material deposited on the support (10), a first fluid (30) and a second fluid (40) including at least one second entity (41), which are capable of interacting; (120) providing at least one emitter (50, 52) capable of converting the electrical energy into acoustic waves (AW); (130) depositing the first and second fluids (30, 40) on the sample (20) so as to form a thin layer (45) of fluids on the planar support (10); (140) emitting the acoustic waves (AW) towards the thin layer (45) so as to mix the first and second fluids, the emitter being separated from a surface (47) of the thin layer by air over a non-zero distance (d), the mixing method (100) being characterised in that, during the emission step (140), the acoustic waves (AW) propagate only through the air so as to generate a pressure wave (PW) capable of striking the surface (47) of the thin layer (45).

Description

[0001] DESCRIPTION

[0002] TITLE: CONTACTLESS MILD FLUIDS MIXING PROCESS USING ACOUSTIC WAVES

[0003] Technical field of the invention

[0004] The invention relates to the technical field of non-contact mixing processes for thin-film fluids using acoustic waves. In particular, the invention relates to non-contact mixing processes for thin-film biological fluids using acoustic waves.

[0005] The invention can be used in any application requiring the non-contact mixing of fluids in a thin film. While the present description uses samples of biological material, other applications are possible, for example in the field of chemistry.

[0006] Technical background

[0007] The state of the art in fluid mixing processes, for example those involving biological materials, offers solutions aimed at improving reactions between certain molecules contained in these fluids, such as antibodies, and / or a biological tissue. Often, the improvement lies in promoting reactions between specific molecules, for example, between antibodies and antigens, while simultaneously seeking to reduce the travel time of the molecules to maximize interactions. Fluid mixing processes, particularly for biological fluids, are generally carried out by dispersion, emulsification, or simply by stirring the fluids.

[0008] Immunohistochemistry allows for the diagnosis of cancers by exposing tissue samples taken from a patient to a first fluid containing primary antibodies with a specific affinity for the antigens of the cancer cells in question, and a second fluid containing other antibodies, called secondary antibodies, which have a specific affinity for the primary antibodies in the first fluid. In this procedure, the biological tissue is placed on a microscope slide, and then the first and second fluids are in turn applied to the slide to form a thin layer. The operator then waits several hours—between 2 and 4.5 hours, with an average of 3 hours—to allow the first and second fluids to mix and for the specific antigen / primary antibody and primary antibody / secondary antibody reactions to occur before examining the tissue.Indeed, the diffusion of antibodies to the sample is very slow and therefore very time-consuming to analyze. Such a mixing process can hardly be deployed on a large scale, even though it allows for the desired diagnosis. Furthermore, the price of antibodies is currently very high, reaching up to €2,000 per vial depending on the complexity of the production.

[0009] Recently, prior art has proposed a method for mixing fluids in a microfluidic reactor. US patent 6948843 discloses a microfluidic reactor used in this method comprising a plurality of microcavities. Biological tissue harvested from a patient is placed in these microcavities, and then fluid(s) containing antibodies capable of specific reactions with any antigens present in the patient's biological tissue are added. The microfluidic reactor is immersed in a first bath, which is itself placed in a second bath. An ultrasonic transducer is positioned at the bottom of the second bath and emits ultrasound waves toward the microcavities, particularly the bottom of the microcavities, of the microfluidic reactor. Each microcavity can contain different biological material(s).

[0010] The mixing of fluids within the microcavities of the microfluidic reactor results from a well-known chain of phenomena. When acoustic waves travel through a liquid medium, cavitation occurs, leading to the formation, growth, and bursting of bubbles within the liquid. When these bubbles burst, they cause the liquid to move, creating a ripple in the vicinity of the bubble, and thus within the liquid itself. It is through this phenomenon that the fluids within the microcavities of the microfluidic reactor mix without any contact with a tool or instrument.

[0011] However, this method presents several drawbacks. The bubbles generated near the surface of the second bath transform, at least partially, the liquid in the second bath into a cloud of extremely fine droplets (down to a few tens of micrometers). Given their size and weight, these highly volatile droplets move easily in the environment and are likely to contaminate the biological material contained in the microcavities. A natural solution would therefore be to close the microcavities to protect the biological material from any contamination. However, it is not always possible to have microcavities closed by windows that allow for optical analysis of the microcavities during the fluid mixing phase. Therefore, it is not advisable to perform fluid mixing using acoustic waves with a liquid as the propagation medium.

[0012] Furthermore, because the biological tissue and fluids are confined within a microcavity, acoustic waves need to propagate through the fluids in the first and second baths for mixing to occur. However, cavitation is highly dependent on the viscosity of the fluid(s). The higher the viscosity, the more difficult cavitation and mixing become. Therefore, it is essential to be able to control the viscosity of the fluid(s) in the baths and the fluid(s) to be mixed, which is very complex. Moreover, this cavitation phenomenon is particularly amplified in cavities when, as in this configuration, the fluids are confined within a micrometer-sized cavity. Consequently, the mixing time can be exceptionally long.

[0013] Besides the fact that the success and / or duration of mixing depends heavily on the viscosity of the fluid(s), acoustic waves are able to use the fluid(s) contained in the microcavities to propagate within the microcavity and thus into the patient's biological tissue. Such an interaction between acoustic waves and the patient's biological tissue is not always desirable because it can degrade the biological tissue or alter the specific reactions between any antigens in the biological tissue and antibodies, particularly when these acoustic waves are ultrasound waves.

[0014] The paper by Draz et al., Lab Chip, 2023, 23, 3258, discloses a method for non-contact mixing of fluids within a microfluidic chip. The microfluidic chip comprises a staining chamber and a top cover, enclosing the staining chamber, on which two piezoelectric transducers capable of generating an acoustic field are arranged. The staining chamber includes a plurality of inlets for one or more fluids containing antibodies and an equal number of outlets for the fluid(s). It further includes a plurality of microfluidic channels, each connecting an inlet to the outlet opposite that inlet. The staining chamber contains a glass slide on which rests a sample of biological tissue or cell pellet taken from a patient. The channels are configured to allow the fluids containing the antibodies to flow through the staining chamber and thus interact with the biological tissue or cell pellet.The antibodies are supplied in volumes of 350 pL each, which allows the staining chamber to be filled.

[0015] The acoustic waves generated by the piezoelectric transducers first propagate through the hood, then through the fluid flowing in the channels, creating vortex-like structures within the fluid. This propagation through the fluid is both surface and volumetric, meaning the fluid acts as a vehicle for the acoustic waves, enabling them to propagate into the patient's biological tissue. However, as mentioned above with reference to document US6948843, such an interaction between acoustic waves and the patient's biological tissue is not always desirable, as it can degrade the tissue or alter the specific reactions between potential antigens and antibodies within the tissue.

[0016] The invention aims to provide a method of mixing fluids by acoustic waves which is efficient, rapid, and which preserves the biological or chemical material, as the case may be, from any contamination as well as from any degradation due to the acoustic waves themselves.

[0017] Summary of the invention

[0018] The invention proposes for this purpose a method for mixing fluids using acoustic waves, the method comprising the following steps:

[0019] (110) provide a flat support, a sample of biological material deposited on the flat support, a first fluid comprising at least one first entity capable of binding with the biological material, and a second fluid comprising at least one second entity capable of binding with the first entity,

[0020] (120) provide at least one emitter capable of converting electrical energy into acoustic waves, (130) deposit the first and second fluids onto the sample so as to form a thin layer of fluids on the flat support,

[0021] (140) emitting acoustic waves in the direction of the thin film so as to mix the first and second fluids, the emitter being separated from a surface of the thin film by air over a non-zero distance, the mixing process being characterized in that, during the emission step, the acoustic waves propagate only through the air so as to generate a pressure wave capable of impacting the surface of the thin film.

[0022] The mixing process according to the invention overcomes the aforementioned drawbacks of the prior art. Indeed, as acoustic waves propagate through air, they create a pressure variation in the air, which gives rise to a discontinuity in air pressure, called a pressure wave. In other words, the pressure wave is a discontinuity in air pressure, formed during the propagation of acoustic waves, which moves like a slip line or a flow. Since this pressure wave travels toward the surface of the thin film only through the air, when it reaches the surface of the thin film, it possesses sufficient kinetic energy to locally impact said surface. The surface height profile varies with the pressure variation induced by the wave over time.This change in shape leads to the circulation of the first and second fluids and an increase in their mixing rate compared to what was previously reported in the prior art. Furthermore, there is no penetration of acoustic waves into the volume of the thin film, meaning that the acoustic waves cannot use the thin film as a vehicle to reach the biological sample. Therefore, during the emission of acoustic waves, there is no risk of sample degradation or alteration of the specific reactions that may occur between the sample and the first and second fluids.

[0023] Furthermore, since acoustic waves travel only through air, there is no risk of sample contamination, as seen in the prior art when acoustic waves are emitted through a liquid and a cloud of fine droplets forms near the sample. In the process according to the invention, the mixing can be carried out on the flat surface without the need for specific protective measures to shield it and the sample from such droplets. Air is the only element impacting the surface of the thin film, making this a contactless process, meaning it involves no contact with a person, no contact with any material other than the first and second fluids and air, and no contact with any instrument or surface.

[0024] According to various features of the invention which may be taken together or separately: during the acoustic wave emission step (140), the emitter is configured so that the pressure wave impacts between 5% and 30% of the area of ​​a surface of the flat support on which the first and second fluids are deposited during the deposition step, the emitter is mobile, during step (140), the acoustic waves are emitted in the direction of the thin film at an angle between 85° and 95°, preferably perpendicular to the surface of the thin film, the emitter is a loudspeaker, said loudspeaker having a frequency between 40 Hz and 160 Hz, preferably between 40 Hz and 60 Hz, during the emission step (140), the acoustic waves are emitted at a duty cycle between 5% and 20%,The method includes a step (125) of providing a waveguide suitable for directing acoustic waves from the loudspeaker to the surface of the thin film. The waveguide includes at least one acoustic wave outlet nozzle opening opposite the surface of the thin film and separated from the surface of the thin film by a distance of 1 cm or less, preferably 0.8 cm or less. The emitter is an ultrasonic transducer, the ultrasonic transducer emitting at a frequency between 16 kHz and 200 kHz. In step (120), an emitter is provided and, if applicable, in step (125), a waveguide including a nozzle is provided. The emitter or the nozzle is located opposite a center of the flat support. In the supply step (120), two emitters are provided or, if applicable, in step (125), a waveguide comprising two nozzles,the two emitters or the two nozzles being located at the same distance from a center of the support and separated by a distance of between 20 mm and 35 mm, preferably equal to 30 mm, the emitter is capable of generating acoustic waves at a frequency between 40 Hz and 200 kHz, the ultrasonic transducer is an air piezoelectric transducer, the thin layer of fluids has, at rest, a thickness of between 50 µm and 200 µm, preferably a thickness of between 70 µm and 100 µm, the support is transparent and comprises or is optionally surmounted by light-emitting diodes, the first entity is a primary antibody capable of binding with antigens of the biological material sample, the second entity comprises a secondary antibody capable of binding with the primary antibody, the second entity comprises an enzyme linked to the secondary antibody, said enzyme being capable of producing a color,distinct from the color of the first and second fluids, when said second entity binds with the first entity.

[0025] Brief description of the figures

[0026] Other objects, features and advantages of the invention will become clearer in the following description, made with reference to the accompanying figures, in which:

[0027] - Figure 1 schematically illustrates a mixing process according to an embodiment of the present invention,

[0028] - Figure 2 schematically illustrates a flat support, a sample of biological material deposited on the support, a first fluid comprising at least one first entity capable of binding with the biological material, and a second fluid comprising at least one second entity capable of binding with the first entity; - Figure 3 schematically illustrates a particular implementation of the mixing process according to a first embodiment of the present invention.

[0029] - Figure 4 schematically illustrates a particular implementation of the mixing process according to a second embodiment of the present invention,

[0030] - Figure 5 schematically illustrates a particular implementation of the mixing process according to a third embodiment of the present invention,

[0031] - Figure 6 is a schematic truncated cross-sectional view of a two-nozzle waveguide,

[0032] - Figure 7 schematically illustrates the principle of an indirect, two-step immunohistochemical reaction,

[0033] - Figure 8 illustrates the mixing of a first and a second fluid during mixing using an ultrasonic transducer,

[0034] - Figure 9 illustrates the mixing of a first and a second fluid during mixing using a loudspeaker,

[0035] - Figure 10 illustrates the 2D profile of the thickness of a thin layer around the impact zone of a pressure wave generated by a loudspeaker,

[0036] - Figure 11 illustrates the 3D profile of the thickness of the thin film whose 2D profile is illustrated in Figure 10.

[0037] In the illustrated figures, optional steps are indicated by dotted rectangles.

[0038] Detailed description of the invention

[0039] 1. Definitions

[0040] The invention relates to a method of mixing fluids by acoustic waves.

[0041] The term "mixture" here refers to the process of combining at least two different fluids to obtain a single fluid, hereinafter referred to as a "thin layer," comprising the constituents of said at least two original fluids. In some applications, this thin layer corresponds to a liquid film.

[0042] The term "acoustic wave(s)" refers to mechanical waves that transmit energy through the movements of atoms and molecules. In the context of this invention, acoustic waves can, for example, be sound waves emitted by a loudspeaker or ultrasonic waves emitted by an ultrasonic transducer. In this invention, an "emitter" is a device capable of converting electrical energy into acoustic waves. Thus, while some electroacoustic emitters can achieve this, not all electroacoustic emitters can. Examples of electroacoustic emitters that convert electrical energy into acoustic waves include loudspeakers and piezoelectric transducers.The transmitter can also be an ultrasonic transducer, that is, a transducer capable of converting electrical energy into ultrasonic acoustic waves.

[0043] A "waveguide," as used in the present invention, is a device for guiding acoustic waves so as to constrain them to travel within a cavity of the waveguide along a predetermined path. This predetermined path is determined by the shape of the cavity itself. In the present invention, the waveguide is filled with air, so that the acoustic waves travel only through the air, as will be described in more detail later in this description.

[0044] The term "biological material" refers to any material originating from a living organism, for example, a human being or individual. The terms "biological material" and "biological material" are synonymous. Biological material may advantageously be collected from the individual prior to any stage of the process. It may consist of biological tissue, biological fluid, cell pellets, or any other biological material that can be analyzed by an operator. Biological material may also be derived from cell cultures, which themselves constitute biological material. For example, monoclonal antibodies are biological material derived from cell cultures, for instance, for the treatment of a specific disease. In the embodiments described below, the biological material typically consists of molecules or macromolecules whose nature depends on the intended application.

[0045] The term "entity" refers to an intermediate substance and / or intermediate biological material whose purpose is to become part of the composition of biological matter.

[0046] A "primary antibody" is an antibody that is capable of binding to both an antigen and another antibody, called a "secondary antibody." A secondary antibody is an antibody that is capable of binding to a primary antibody. Thus, the secondary antibody does not bind directly to the antigen but binds to the primary antibody. In addition to being able to bind to the primary antibody, the secondary antibody can also bind to an enzyme, which confers one or more additional functions upon it. In some applications, primary antibodies are proteins produced by the immune system of animals of specific strains, such as rats, mice, rabbits, or horses, which are exposed to antigens to produce antibodies in their ascites.The secondary antibody is species-specific and targets the primary antibody. It is conjugated to an enzyme such as peroxidase, for example, horseradish peroxidase (HRP). Antibodies of animal origin are being replaced by synthetic antibodies, which are produced without the use of animals and can therefore be manufactured, for example, through molecular engineering.

[0047] A "thin film" is defined as a layer of material with a thickness of micrometers or submicrometers, that is, a layer with a thickness strictly less than 1000 µm. In the context of the present invention, this layer of material is a fluid layer.

[0048] The term "transparent" refers to a medium that transmits more than 90% of visible light, in particular allowing electromagnetic waves with wavelengths between 380 nm and 800 nm to pass through.

[0049] 2. General method of implementation

[0050] The steps of mixing process 100 can advantageously be carried out in the order illustrated in Figure 1. However, the order of implementation of certain steps may be modified. In this case, the description specifies this, as is the case for steps 110, 120, and 125.

[0051] With reference to Figure 1 and Figure 2, a first step of the mixing process 100 according to the invention consists of providing 110 a flat support 10, a sample 20 of biological material deposited on the flat support 10, a first fluid 30 comprising at least a first entity 31 capable of binding with the biological material, and a second fluid 40 comprising at least a second entity 41 capable of binding with the first entity 31.

[0052] The support 10 is flat, meaning it has little to no curvature. Therefore, when placed on a horizontal surface, each point is level, allowing for better control of the mixing dynamics and improving the repeatability of the results. The support 10 is, for example, a microscope slide or a commercially available laboratory glass slide. Prior to the aforementioned supply step 110, the support 10 may be cleaned with a suitable solution and then rinsed to remove any impurities, traces, and / or residues from its surface. The biological sample 20 is placed on the support 10 for contact with the first and second fluids 30 and 40, and for subsequent analysis. The composition of such a sample 20 of biological material is described at the beginning of this section and is not repeated.

[0053] As previously mentioned, the first fluid 30 comprises at least one first entity 31 capable of binding with the biological material of the sample 20. The first entity 31 may not bind with the biological material if the latter does not include molecules having an affinity or, more generally, if the latter does not include molecules capable of interacting with said first entity 31. Conversely, when the first entity 31 has an affinity or is, more generally, capable of interacting with molecules of the sample 20 of biological material, the first entity 31 is capable of forming non-covalent bonds with the molecules of the sample 20 when the first fluid 30 is in contact with said sample 20.

[0054] As previously mentioned, the second fluid 40 comprises at least one second entity 41 capable of binding with the first entity 31. Indeed, the second entity 41 is configured to interact with the first entity 31, for example, given the affinity existing between the first entity 31 and the second entity 41. In any case, the process 100 according to the invention provides for mixing the first fluid 30 and the second fluid 40 so that these interactions can take place. The advantage of these "planned" interactions will be seen in more detail in the description of the particular embodiment.

[0055] With further reference to Figure 1, a second step of the mixing process 100 according to the invention consists of providing at least one emitter 50, 52 capable of converting electrical energy into acoustic waves AW. The emitter 50, 52 is thus capable of emitting acoustic waves as soon as it is powered by an electrical energy source. This energy source can come from a battery or cell directly integrated into the emitter, or from the electrical distribution network. In the latter case, the emitter 50, 52 can be connected to this network by means of connection not shown. As will be better understood later, the emitter 50, 52 has the role of generating acoustic waves AW which, in turn, upon passing through the air, are capable of generating a discontinuity in air pressure, that is to say, a pressure wave PW intended to impact the surface of the fluids.

[0056] At this stage, it should be noted that this "second" supply step 120 of said at least one emitter can be carried out either before or after the "first" supply step 110 of the support 10 and the other elements. The order in which these steps 110 and 120 are carried out is irrelevant. Thus, Figure 1 illustrates only one embodiment of the mixing process 100 according to the invention. Advantageously, the emitter 50, 52 is capable of generating acoustic waves AW at a frequency between 40 Hz and 200 kHz. This frequency range is particularly advantageous for generating acoustic waves AW which in turn generate a pressure wave PW which is able to impact the surface of fluids without degrading biological material or altering any interactions between the first entity 31 and the biological material of sample 20, and / or the interactions between the second entity 41 and the first entity 31.

[0057] According to a first implementation variant, the emitter is a loudspeaker 50 emitting at a frequency between 40 Hz and 160 Hz, preferably between 40 Hz and 60 Hz. Loudspeakers emitting acoustic waves AW at this frequency are widely available commercially. At such frequencies, the loudspeaker 50 is particularly effective at compressing the air and generating a pressure wave PW capable of impacting the surface of fluids without degrading the sample 20 of biological material, even at distances as great as several tens of centimeters.In practice, although the optimal frequency of the speaker 50 can be chosen empirically according to the air temperature, the viscosity of the first and second fluids 30, 40 and even the power of the speaker 50, the best results, i.e. the most efficient and fastest mixtures, were obtained when the speaker 50 emits at a frequency between 40 Hz and 60 Hz, the efficiency of a mixture being able to be appreciated according to the percentage and homogeneity of the mixture of fluids obtained.

[0058] In this regard, and advantageously, the temperature of the support 10 plan and / or the first and second fluids 30, 40 is adjustable. More precisely, the temperature of the support

[0059] The viscosity of the first and second fluids 30 and 40 can be set between 20°C and 36°C, allowing the first and second fluids 30 and 40 to have a suitable viscosity, particularly for the transmitter frequency. By adjusting the viscosity via temperature,

[0060] 11. It is possible to improve the repeatability of the mixtures performed.

[0061] In the particular embodiment illustrated in Figure 3, the loudspeaker 50 is coupled to a waveguide 60. In this respect, the mixing process 100 according to the invention may optionally include a third step consisting of providing 125 a waveguide 60 capable of directing the acoustic waves AW from the loudspeaker 50 to the support 10, and thus to the first and second fluids 30, 40. As previously described, the "third" step 125 of providing the waveguide can be carried out either before the "first" step 110 of providing the support 10 and the other elements, or between this first step 110 and the second step 120 of providing at least one emitter. The order in which these steps 110, 120, and 125 are carried out is irrelevant.In the embodiment illustrated in Figure 3, the waveguide 60 comprises a single outlet nozzle 62 for the acoustic waves AW, opening opposite the support 10 and separated from the surface 47 of the thin film 45 by a distance of 1 cm or less, preferably 0.8 cm or less. The waveguide 60 further comprises a guiding portion 61 for the acoustic waves AW connected to the outlet nozzle 62. In Figure 3, the guiding portion 61 has a frustoconical shape and includes a proximal end 61a of the loudspeaker 50, abutting or connected to a diaphragm 51 of the loudspeaker 50, and a distal end 61b of the loudspeaker 50 connected to the outlet nozzle 62. The diaphragm 51 is a mesh wall of the loudspeaker 50 through which the acoustic waves AW exit the loudspeaker.

[0062] Thus, when, as illustrated in Figure 3, the loudspeaker 50, in particular its diaphragm 51, has dimensions greater than the desired impact surface, the waveguide 60 allows both to guide the acoustic waves AW towards the surface and also to focus them, that is to say to concentrate them, on a surface having a smaller area than that of the diaphragm 51 of the loudspeaker 50. The waveguide 60 therefore acts as a funnel for the acoustic waves AW.

[0063] In the embodiment illustrated in Figure 4, the waveguide 60 comprises two outlet nozzles 62a and 62b for the acoustic waves AW. These nozzles 62a and 62b are closer to the support 10 than the loudspeaker 50 is to the support 10 and open opposite the support 10. The use of two outlet nozzles 62a and 62b instead of one outlet nozzle 62 increases the proportion of the fluid surface that is mixed and accelerates the mixing process. Furthermore, the use of two outlet nozzles 62a and 62b improves the distribution of the fluids over the surface.

[0064] In this particular implementation, in addition to the fact that the waveguide 60 includes two output nozzles 62, it also includes a guiding portion 61 having a general shape different from that seen in Figure 3. The guiding portion 61 comprises a first portion 61a, proximal to the loudspeaker 50, in the shape of a U and a second portion 61b, distal to the loudspeaker, in the shape of a Y. The first portion 61a allows for effective coupling with the loudspeaker 50 when it is offset, while the second portion 61b allows for the connection of the first portion 61a with the two output nozzles 62a, 62b.

[0065] In the case of the waveguide 60 with a single nozzle 62, as in the case of the waveguide with two nozzles 62a, 62b, the shape of the guiding portion 61 is not limiting. In practice, the shape of the waveguide is designed to accommodate the placement of the loudspeakers 50 and the support 10. According to another embodiment illustrated in Figure 5, the transmitter is an ultrasonic transducer 52 emitting at a frequency between 16 kHz and 200 kHz. That being said, preferably, the ultrasonic transducer 52 emits at a frequency between 16 kHz and 40 kHz. Many types of transducers 52 emit in this frequency range: electrostatic transducers, piezoelectric transducers, micro-machined ultrasonic capacitive transducers, etc.However, it is advantageous to use an air-filled piezoelectric transducer because it is capable of generating acoustic waves in the previously mentioned preferred frequency range while being very widespread commercially and very cheap.

[0066] Furthermore, the air-filled piezoelectric transducer 52, like most ultrasonic transducers, emits acoustic waves with better directivity and therefore lower lateral dispersion than loudspeakers. This improves the spatial accuracy of the pressure wave's impact on the fluid surface, even though these transducers 52 require more power than loudspeakers 50 or must be positioned closer to the support 10. Moreover, since ultrasonic transducers 52 are generally smaller than loudspeakers 50, a waveguide 60 is usually unnecessary to focus the acoustic waves, making the installation shown in Figure 5 less bulky than those in Figures 3 and 4, which use a loudspeaker 50 coupled with a waveguide 60.For comparison, ultrasonic transducers can have a diameter close to that of the 62, 62a, and 62b nozzles of the waveguide, i.e., approximately 1 cm in diameter. That being said, there is nothing preventing them from being coupled to a 60 waveguide, even if, in practice, the attenuation is likely to be too significant.

[0067] In this regard, it should be noted that the air-filled piezoelectric transducer(s) can be located at a distance from the support 10 between A and A / 2, where A is the wavelength of the ultrasonic waves. At these distances, the piezoelectric transducer(s) can generate acoustic waves AW capable of generating a pressure wave PW as they pass through the air without having to use their full power.

[0068] Let us now return to Figure 1. The mixing process 100 according to the invention comprises a step 130 of depositing the first and second fluids 30, 40 onto the flat support 10 so as to form a thin layer 45 of fluids on the flat support 10. At the end of this deposition step 120, the two fluids, namely the first fluid 30 and the second fluid 40, are indeed on the support 10 but are not yet mixed. At this stage, the thin layer 45 may not cover the sample 20 completely, or it may at least partially cover it. However, if the sample 20 is in contact with the first entity(ies) 31 and / or the second entity(ies) 41, the interactions are not yet optimized, and mixing of the first and second fluids is necessary for this to occur.In this respect, the invention aims to improve the mixing of the first and second fluids 30, 40 so that interactions can occur quickly and efficiently without damaging the sample 20 of biological material.

[0069] The mixing process 100 according to the invention further comprises a step 140 of emitting acoustic waves AW towards the thin film 45 so as to mix the first and second fluids 30, 40. In this regard, it should be noted, although implicit in light of the foregoing, that the emitter 50, 52 is separated from a surface 47 of the thin film 45 by air, over a non-zero distance d. Consequently, the emitter 50, 52 is also separated from the support 10, the sample 20 of biological material, the first fluid 30, and the second fluid 40.

[0070] In the preceding description, in relation to Figures 3, 4, and 5, we have seen various implementations for carrying out this emission step 140. However, the mixing process 100 according to the invention is by no means limited to these particular implementations. During the emission step 140 of the acoustic waves AW, it is essential that these waves be emitted in the direction of the thin layer 45 formed by the first and second fluids. Of course, even in the particular implementations where the acoustic waves AW pass through the waveguide 60, such as the one illustrated in Figure 4, upon exiting the emitter 50, 52, the acoustic waves AW are emitted in the direction of the thin layer 45 since the waveguide 60 is itself configured and arranged so as to guide the acoustic waves in the direction of the thin layer 45.In this case, the acoustic waves AW are considered to be emitted towards the thin layer 45 since they pass through the waveguide which guides them towards the thin layer 45 even if this transmission is indirect.

[0071] Secondly, and according to an essential aspect of the invention, it is important that the acoustic waves AW propagate only through air. This means that a non-zero distance must exist between the emitter(s) 50, 52 and the surface 47 of the thin film 45 to provide an air-filled space in which the acoustic waves AW are able to propagate and generate a pressure wave PW.

[0072] Indeed, as they propagate through the air, the acoustic waves AW create a pressure variation in the air, which gives rise to a pressure wave PW. The pressure wave PW is a discontinuity in air pressure formed during the propagation of the acoustic waves AW, which moves like a slip line or an air current. Since this pressure wave PW travels towards the surface 47 of the thin film 45 only through the air, when it reaches the surface 47 of the thin film, it possesses sufficient kinetic energy to impact said surface 47 and transfer its kinetic energy to it.

[0073] Thus, the height profile of surface 47 varies with the pressure variation induced by the pressure wave PW over time. Upon reaching surface 47, the pressure wave PW propagates within the thin layer 45 and changes the shape of the thin layer 45, first on the surface at the point or impact zone, and then progressively in volume around this point or zone. This change in shape leads to the circulation of the first and second fluids 30, 40 at an average mixing velocity of between 7.6 mm / s and 29.5 mm / s. Therefore, the average distance traveled in 1 minute varies between 50.4 mm and 61.7 mm. If the support 10 has a surface area of ​​14 cm² 2 Therefore, the entire support 10 is explored in less than 2 minutes, or even less than 1 minute.

[0074] Furthermore, the acoustic waves AW do not penetrate the volume of the thin film 45, and therefore cannot use the thin film 45 as a vehicle to the biological sample 20. Consequently, during the emission of the acoustic waves AW, there is no risk of degradation of the sample 20 or of alteration of the specific reactions that may occur between the sample 20 and the first and second fluids 30, 40.

[0075] Furthermore, since the acoustic waves AW only travel through air, there is no risk of contamination of the sample 20 by these acoustic waves. In the mixing process 100 according to the invention, the mixing can be carried out on the flat support 10 without the need for specific protective measures to shield it and the sample 20 from any potential droplets. Air is the only element to impact the surface of the thin film 45, making this mixing process 100 a non-contaminating and contactless process, namely, without contact with an individual, without contact with any other material such as a fluid other than the first and second fluids 30, 40 and air, and without contact with any instrument or surface.

[0076] Preferably, during step 140, the acoustic waves are emitted towards the thin film at an angle between 85° and 95°, preferably perpendicular to the surface of the thin film, which further improves the mixing.

[0077] According to a particularly advantageous implementation, during the acoustic wave emission step 140, the emitter 50, 52 is configured so that the pressure wave PW impacts between 5% and 30% of the area of ​​a surface 11 of the flat support 10 onto which the first and second fluids 30, 40 are deposited during the deposition step 130. This particular implementation makes it possible to generate a pressure wave PW localized on the surface 47 of the thin film 45, which in turn generates a greater number of vortices in the thin film 45.

[0078] When the emitter is a loudspeaker 50, it is preferable to use a waveguide 60 which, as seen previously, allows focusing, i.e. concentrating, on a surface having a smaller area than that of the diaphragm 51 of the loudspeaker 50. In this case, the outlet nozzle(s) 62, 62a, 62b of the waveguide 60 have an outlet orifice of dimensions similar to the dimensions of the surface 11 which is intended to be impacted by the pressure wave or pressure waves PW when there are two nozzles 62a, 62b.

[0079] When the transmitter is an ultrasonic transducer 52, it is advantageous to select a transducer 52 with dimensions similar to those of the surface 11 intended to be impacted by the pressure wave PW. Indeed, ultrasonic transducers 52 generally have very good directivity and spatial accuracy, which allows for better targeting of the impact zone of the pressure wave PW.

[0080] Advantageously, the emitter 50, 52 is mobile, which is particularly suitable for large areas. Indeed, this allows the method 100 according to the invention to be implemented on large surfaces, that is to say, surfaces greater than or equal to 35 cm 2 , ensuring the homogeneity of the mixture over the entire surface. If, as will be described later, several emitters 50, 52 are mobile, this increases accordingly the area of ​​the surface 11 that can be covered by the mixture.

[0081] In the case where the emitter 50 is a loudspeaker, it can also be very advantageous for the acoustic waves AW to be emitted at a duty cycle between 5% and 20% during the emission stage 140. This deforms the surface 47 of the thin film 45, creating a depression at the point of impact of the pressure wave PW and a circular wave around the point of impact. As this wave disperses, it creates a localized vortex, but also a significant fluid displacement around the vortex and, consequently, throughout the entire volume of the thin film 45. At the point of impact, the pressure is positive, causing the thin film 45 to depress, while around the point of impact, there is an upward suction (due to the Venturi effect) which creates a column of fluid that collapses when the acoustic waves AW are stopped. This results in a uniform fluid displacement, like a moving wave.

[0082] This increases mixing efficiency, improves mixing uniformity, and enhances fluid distribution across the surface of the thin layer. The mixture reaches the edges more quickly, which is highly advantageous for large areas. A duty cycle of no more than 20% prevents the wave from lingering too long on the surface and causing undesirable effects on the elements present in the mixture.

[0083] It should be noted that for fluids with a viscosity close to that of water, the duty cycle is ideally between 5% and 20%. For fluids with a viscosity higher than that of water, the duty cycle is advantageously between 15% and 20%. For fluids with a viscosity lower than that of water, the duty cycle is advantageously between 5% and 15%. According to one embodiment, it is possible to emit ultrasound for 1 minute and then stop for 10 to 15 seconds at 60 Hz. As previously mentioned, the temperature of the support 10 and / or the first and second fluids 30, 40 can be set between 20°C and 36°C, which allows for first and second fluids 30, 40 to have a suitable viscosity.

[0084] Advantageously, the size of the mixed zone can be modified and the mixing efficiency increased by adjusting the power of the pressure waves. In this regard, the power of emitters 50 and 52 can be adjusted to increase the power of the pressure waves.

[0085] Figure 10 shows an example of a two-dimensional profile of the variation in thickness of the thin film 45 at and around the impact zone of the pressure wave PW generated by a loudspeaker 50 emitting at a frequency of 60 Hz. Figure 11 illustrates the three-dimensional topography of the surface 47 of the thin film 45 for this example. These figures provide an illustrative visualization of the shape of the surface 47 obtained without contact of the surface 47 with any instrument, fluid, or other object. In this example, the support 10 measures 36.92 cm 2 , the thickness of the thin film 45 is 71 pm.

[0086] Table 1 illustrates the minimum thickness of the thin film 45 at the point or zone of impact of the pressure wave according to the pressure exerted by the pressure wave on the surface.

[0087] Table 1: Pressure measurement applied to a 0.04 mq / ml solution of Triton X100 using a microphone

[0088] For each experiment, the pressure wave pressure (PW) was set between 18.5 and 55.6 mPa (millipascals). To achieve this, the adjustment voltage was increased in 0.5 V increments, and the pressure was then measured with a calibrated microphone before the experiment. A photograph of the surface was recorded for each experiment (e.g., Figure 11). In each case, the fluorescence intensity (green level) was lowest at the pressure wave impact zone, with a higher intensity corona surrounding this zone, and the intensity became uniform away from the nozzle. Using Equation 1 below, the variation in green level can be converted into a variation in film thickness.

[0089] W) = ^ ( 1 )

[0090] Where x is the green level and H is the thickness of the thin film 45. The thickness of the thin film 45 at the impact zone decreases from 70 pm to 9.1 pm when the pressure wave pressure PW increases from 18.5 to 55.6 mPa. This variation follows a sigmoidal law.

[0091] According to a preferred embodiment, it is advantageous for the thin film 45 to have, at rest, a thickness of between 50 µm and 200 µm, preferably between 70 µm and 100 µm, which promotes obtaining a profile such as that illustrated in Figure 10. In this respect, the first fluid 30 and the second fluid 40 preferably have a total volume of between 100 pL and 1000 pL, so that when they are spread on a support 10 having a surface area of ​​14 cm² 2Thicknesses ranging from 60 µm to 120 µm are obtained. It should be noted that the support 10, as well as the biological sample 20, can be rinsed during the process according to the invention. Naturally, the total volume can be adjusted according to the desired thickness of the thin film.

[0092] Preferably, the acoustic waves AW are emitted onto a surface representing between 10% and 25% of the deposition surface 11, which further improves the shape of the surface 47 of the thin film 45. According to one embodiment, the pressure waves PW have a diameter of 7 mm (approximately 1.54 cm). 2 ) while surface 11 of the support measures 14 cm 2 According to another embodiment, the PW pressure waves have a diameter of 10 mm (approximately 3.14 cm). 2 ) while surface 11 of the support measures 14 cm 2 .

[0093] When a waveguide 60 is used to direct the acoustic waves AW from the loudspeaker 50 to the support 10 and thus to the surface 47 of the thin film 45, the surface values ​​(acoustic wave emission surface relative to the deposition surface 11) given above apply not only to the case where a single nozzle is used as to the case where two nozzles are used.

[0094] That being said, when the waveguide 60 has a single nozzle 62, as illustrated in Figure 3, it is advantageous for the nozzle 62 to open opposite the center of the support 10. Thus, when the pressure wave PW arrives at the surface 47 of the thin film 45, it exerts a localized and focused pressure, which promotes homogeneous mixing of the fluids over a larger area. In this configuration, the mixing rate of the surface can reach 85% of the surface 47 after one minute. Indeed, the fluids are more likely to move with the same amplitude in all directions, and mixing is more likely to occur homogeneously over a high proportion of the surface 47. This also applies, analogously, when a single loudspeaker 50 is used, as illustrated by way of example in Figure 5. The same observations apply.

[0095] When the waveguide 60 has two nozzles 62a, 62b, as illustrated in Figures 4 and 6, or when two transducers 52 are used, it is preferable that they be positioned at an equal distance from the center of the support 10, so that the distance d separating the two nozzles 62a, 62b or the two transducers 52 is between 20 mm and 35 mm, preferably 30 mm. For example, if the support 10 has a length (longest dimension of the support 10) of 51 mm, then the distance d between the two nozzles 62a, 62b or the two transducers 52 can advantageously be 30 mm. The use of two nozzles 62a, 62b makes it possible to achieve a mixing rate of 92% of the surface after one minute, which is a higher mixing rate than that obtained when only one nozzle is used. Although the use of two transducers 52 in the aforementioned configuration does not allow such mixing rates to be achieved, such an arrangement is preferred.We'll come back to that later.

[0096] The distance d between the two nozzles 62a, 62b or the transducers 52 depends little or not at all on the shape of the nozzles or transducers 52, respectively. Thus, although Figures 8 and 9 illustrate mixing carried out with circular nozzles or transducers, the nozzles and transducers can be of any other shape, for example, parallelepiped-shaped. Furthermore, the distance d between the two nozzles 62a, 62b does not depend on the dimensions of the support 10. Incidentally, the larger the support 10, the more nozzles or transducers must be added to maintain the distance d specified above between each nozzle or transducer. Figure 8 illustrates mixing carried out with two transducers 52, while Figure 9 illustrates mixing carried out with two nozzles 62a, 62b.

[0097] Although there is no edge effect, it is advantageous for the support 10 to have a surface area 6 to 10 times greater than that of the nozzles or transducers. This is because the vortex phenomenon described earlier is independent of how the spreading was carried out, regardless of whether the edges of the thin film are free or not. Indeed, it is not enclosed in a cavity as is the case in some prior art microfluidic devices.

[0098] To increase the brightness of the acquired images, it is possible to overcome the support 10 flat surface of light-emitting diodes 12. The diodes 12 allow precise control of the brightness and reflections of the support 10, thus making the acquisition less dependent on the excess or deficiency of light from the environment in which the support 10 is located. The illumination can be continuous or synchronized with an image sensor 70. When the illumination is used continuously, this allows the frequency of the light-emitting diodes 12 to be decoupled from that of the CCD camera 24.

[0099] Preferably, the support 10 is a transparent microscope slide, allowing observation of the fluid mixing with an image sensor 70 such as a CCD camera. The CCD camera 70 can have an acquisition frequency suitable for observing the evolution of the mixing on the surface of the support 10. A standard CCD camera capable of acquiring a few images per second, e.g., 14 images per second, is sufficient to observe the evolution of the mixing. The mixing method 100 according to the invention can therefore include an optional step 150 of image acquisition using the image sensor 70.

[0100] In addition, the visualization of mixtures can be improved by using fluorescein or fluorescent microspheres—for example, those from Cospheric—as tracers for particle image velocimetry (PIV) analysis. Sodium fluorescein (S) has a fluorescence spectrum with an excitation peak at 494 nm and an emission peak at 521 nm, enabling fluorescence at an excitation wavelength of 390 nm.

[0101] 3. Examples of application of the present invention - Immunohistochemistry

[0102] In one application example, the present invention can be implemented in immunohistochemical analyses. In this context, the support 10 can be a microscope slide, the first entity 31 can be a primary antibody capable of binding with antigens 21 of the sample 20 of biological material, while the second entity 41 comprises a secondary antibody 41a capable of binding with the primary antibody 31.

[0103] Furthermore, the second entity 41 may advantageously comprise an enzyme 41b linked to the secondary antibody 41a, said enzyme 41b being a fluorescein capable of producing a color, distinct from the color of the first and second fluids 30, 40, when said second entity 41 binds with the first entity 31. The enzyme 41b acts as a tracer and allows monitoring of the mixing efficiency. Figure 7 illustrates such an implementation.

[0104] 3.1 - Equipment

[0105] The support 10 plan is a transparent microscope slide.

[0106] The biological sample 20 may consist of sections of the ileocecal appendix containing the most common antigenic targets, namely actin, Ki67, and CD20. The markers are, respectively: cytoplasmic, nuclear, and membrane. These sections may be pre-treated to improve the analysis.

[0107] Primary antibody 31 may be chosen, without limitation, from the antibodies in the following list:

[0108] - Mouse monoclonal antibody specific to smooth muscle actin (1 A4) (0.03 pg / mL) (Cat. No. 760-2833 / 05268303001): This is a cytoplasmic antibody.

[0109] - Specific mouse monoclonal antibody targeting the CD20 (L26) antigen (0.3 pg / mL) (Cat. No. 760-2531 / 05267099001): This is a membrane antibody.

[0110] - Specific rabbit monoclonal antibody targeting Ki67 (30-9) antigen (2 pg / mL) (Cat. No. 790-4286 / 05278384001): This is a nuclear antibody.

[0111] The use of this antibody panel makes it possible to cover all types of antigens.

[0112] The secondary antibody 41a can be the UltraView Universal HRP Multimer. This is a multimer technology involving the direct conjugation of the HRP 41b enzyme (horseradish peroxidase) to the secondary antibody produced in the horse.

[0113] To reveal the reaction, it is also possible to introduce a mixture of UltraView Universal DAB Chromogen and UltraView Universal DAB H2O2:

[0114] - UltraView Universal DAB Chromogen: DAB (3,3'-diaminobenzidine) is a chemical substance that, when catalyzed by a peroxidase, produces a brown precipitate. - UltraView Universal DAB H2O2: When exposed to the HRP enzyme, hydrogen peroxide is decomposed into water (H2O) and oxygen (O2). The DAB, when in contact with these products, is oxidized.

[0115] To perform preliminary tests, a series of solutions containing Triton X100 and glucose, between 5% and 30% by mass, is used to produce samples with viscosities ranging from 1.1 mPa / s to 2.9 mPa / s. In addition, a drop containing fluorescein (tracer) is placed on the surface of the film to monitor the mixing.

[0116] When the transmitter is a 50 speaker, it can be chosen, without limitation, from the following speakers:

[0117] - Vibrating speaker, reference Dyna VOX 207409 "EXC 25 Boost sound bodies, 25 W: used to generate vibrations on a 23 mm Teflon disc.

[0118] - Peerless speaker, reference 830986-3: wideband speaker with an overall diameter of 83 mm, with a resonance at 110 Hz and a nominal power of 25 W under 8 0.

[0119] - When the transducer is an ultrasonic transducer 52, it can be chosen, without limitation, from among the following air transducers:

[0120] - Generic TCT40-16T transducer,

[0121] - Generic TCT40-16R transducer

[0122] - Murata MA 40S4S transducer,

[0123] - Murata MA 40S4R transducer.

[0124] When the flat support 10 is fitted with light-emitting diodes 12, the diodes may be chosen, without limitation, from the following diodes:

[0125] - Chanzon warm white diodes 100F5T-YT-WH-WW, diameter 5 mm, 3-3.2 V, 20 mA, 14000-16000 pcd.

[0126] - Chanzon 50F10T-YT-WH-BL diodes, 455-465 nm, 10 mm diameter, 3-3.2 V, 20 mA, OOpcd. And Lumetheus 2001001021_50x, 465 nm, 5 mm diameter, 100 mA, 5000 pcd.

[0127] - Generic diodes emitting at 390 nm, diameter 5 mm, 3 V, 20 mA. This wavelength is at the beginning of the excitation curve, but corresponds to a compromise between the sensitivity of the camera and the color separating power used in the processing.

[0128] When an image sensor 70 is used to monitor the evolution of mixtures, it is chosen, without limitation, from among the following cameras 70:

[0129] - Logitech Webcam C925e camera: the video format used is MP4, with a resolution of 1920 x 1080 pixels.

[0130] - HEU HD PRO Webcam.

[0131] 3.2 First example of implementation: Pressure wave mixing at 40 Hz and 60 Hz Thin layer 45 containing 0.04 g / L of Triton X100 (1 OpL Triton X100 for 300 pL of glucose solution), glucose, between 5% and 30% by mass, and 2 pL of fluorescein.

[0132] Transducer: 50 loudspeaker excited by a sinusoidal wave at frequencies of 40 Hz and 60 Hz. The electrical power was pre-set so that the sound pressure was equal to 37 Pa using a calibrated microphone, with the amplifier set to 400mV.

[0133] A camera 70 was used to track the mixing of fluorescein in the thin layer 45 for 120 s. The final image, at 120 seconds, is used to evaluate the percentage of the surface 47 of the thin layer 45 that contains fluorescein relative to the total surface of the film using a homemade computer application.

[0134] After analysis by imaging, at 40 Hz and 60 Hz, the mixing rate reaches nearly 60%, at constant pressure, after 120 seconds.

[0135] 3.3 Second example of implementation: 60 Hz pressure wave mixing with a single-nozzle waveguide (i.e., single nozzle)

[0136] Thin film 45 contained 0.04 g / L of Triton X100, glucose (between 5% and 30% by mass), and 100 pL of Ventana buffer to which 2 pL of fluorescein was added before being applied to slide 10. Slide 10 and thin film 45 were placed in an incubator at the IHC reaction incubation temperature of 36°C. Slide 10 measured 76 x 26 mm. 2 and are limited by a 25 x 22 mm identification label 2 .

[0137] Transmitter: A loudspeaker 50 is driven by a sinusoidal wave at a frequency of 60 Hz. The electrical power has been pre-set to achieve a sound pressure level of 480 Pa using a calibrated microphone, with the amplifier set to 400 mV. The loudspeaker 50 is coupled to a waveguide 60 comprising at least one circular nozzle 62 opening opposite the center of the blade 10. The nozzle 62 has a diameter of 10 mm. It should be noted that mixing can be carried out with one or two nozzles 61, or even more if the film surface area is large. Furthermore, the more nozzles there are, the higher the total pressure (in pascals, Pa).

[0138] The same considerations as in the first example (section 3.2) apply with regard to monitoring the mixing by camera 70.

[0139] After analysis by imaging, at 60 Hz, the mixing rate reaches nearly 85%, at constant pressure, after 60 seconds using a waveguide 60 with a single nozzle 62 opening opposite the center of the blade 10. 3.4 Third embodiment example: Pressure wave mixing at 60 Hz with a waveguide 60 before two nozzles 62a, 62b

[0140] The thin layer 45 is of the same nature as that described in the third example 3.3 of embodiment.

[0141] Transducer: a loudspeaker 50 driven by a sine wave at a frequency of 60 Hz. The electrical power was pre-set to achieve a sound pressure level of 480 Pa using a calibrated microphone, with the amplifier set to 400 mV. The loudspeaker 50 is coupled to a waveguide 60 comprising two circular nozzles 62a and 62b spaced 30 mm apart and located approximately equidistant from the center of the blade 10. Nozzles 62a and 62b each have a diameter of 10 mm.

[0142] The same considerations as in the first and second examples (sections 3.2 and 3.3) apply with regard to monitoring the mixing by camera 70.

[0143] After analysis by imaging, at 60 Hz, the mixing rate reaches nearly 92%, at constant pressure, after 60 seconds using a waveguide 60 with two nozzles 62a, 62b spaced 30 mm apart.

[0144] 3.5 Fourth implementation example: 40 kHz pressure wave mixing with two ultrasonic transducers 52

[0145] Thin layer 45 containing 100 pL of Triton X100 and an aliquot of 7 pL of a fluorescein solution deposited on a slide 10.

[0146] Transmitter: Two TCT40-16T ultrasonic transducers 52 were excited at a frequency of 40 kHz. The two transducers 52 are separated by a center-to-center distance of 20 mm and have a diameter of 7 mm. The distance between the blade 10 and the transducers 52 is 8.5 mm. The sound pressure level of 2.4 Pa was measured using a balance on which a foam pad was placed. The grids of the transducers 52 were removed.

[0147] The same considerations as in previous examples (sections 3.2, 3.3 and 3.4) apply with regard to monitoring the mixing by camera 70.

[0148] After analysis by imaging, at 40 kHz, the mixing rate reaches nearly 56%, at constant pressure, after 60 seconds.

[0149] The configurations shown in the cited figures are only possible examples, by no means limiting, of the invention which on the contrary encompasses the variants of designs within the reach of the person skilled in the art.

Claims

DEMANDS 1. A process (100) for mixing fluids by acoustic waves, the process (100) comprising the following steps: (110) provide a flat support (10), a sample (20) of biological material deposited on the flat support (10), a first fluid (30) comprising at least a first entity (31) capable of binding with the biological material, and a second fluid (40) comprising at least a second entity (41) capable of binding with the first entity (31), (120) provide at least one transmitter (50, 52) capable of converting electrical energy into acoustic waves (AW), (130) deposit the first and second fluids (30, 40) onto the sample (20) so as to form a thin layer (45) of fluids on the flat support (10), (140) emit acoustic waves (AW) in the direction of the thin film (45) so as to mix the first and second fluids (30, 40), the emitter (50, 52) being separated from a surface (47) of the thin film (45) by air over a non-zero distance (d), the mixing process (100) being characterized in that, during the emission step (140), the acoustic waves (AW) propagate only through the air so as to generate a pressure wave (PW) capable of impacting the surface (47) of the thin film (45).

2. Mixing method (100) according to claim 1, wherein, during the acoustic wave emission step (140), the emitter (50, 52) is configured so that the pressure wave (PW) impacts between 5% and 30% of the area of ​​a surface (11) of the flat support (10) on which the first and second fluids (30, 40) are deposited during the deposition step (130).

3. Mixing method (100) according to any one of claims 1 or 2, wherein the emitter (50, 52) is mobile.

4. Mixing method (100) according to any one of the preceding claims, wherein the emitter is a loudspeaker, said loudspeaker (50) having a frequency between 40 Hz and 160 Hz, preferably between 40 Hz and 60 Hz.

5. Method according to claim 4, wherein, during the emission step (140), the acoustic waves (AW) are emitted at a duty cycle of between 5% and 20%.

6. Method according to any one of claims 4 to 5, comprising a step (125) of providing a waveguide (60) capable of directing acoustic waves (AW) from the loudspeaker (50) to the surface (47) of the thin film (45).

7. Mixing method (100) according to claim 6, wherein the waveguide (60) comprises at least one acoustic wave (AW) outlet nozzle (62, 62a, 62b) opening opposite the surface (47) of the thin layer (45) and separated from the surface (47) of the thin layer by a distance less than or equal to 1 cm.

8. Mixing method (100) according to any one of claims 1 to 3, wherein the emitter is an ultrasonic transducer (52), preferably an air piezoelectric transducer, the ultrasonic transducer (52) emitting at a frequency between 16 kHz and 200 kHz.

9. Mixing method (100) according to any one of the preceding claims, wherein, in step (120), an emitter (50, 52) is provided and, where appropriate, in step (125) a waveguide (60) comprising a nozzle (62) is provided, the emitter (50, 52) or the nozzle (62) being located opposite a center of the support (10).

10. Mixing method (100) according to any one of claims 1 to 7, wherein, in step (120), two emitters (50, 52) are provided or, where appropriate, in step (125), a waveguide (60) comprising two nozzles (62a, 62b) is provided, the two emitters (52) or the two nozzles (62a, 62b) being located at the same distance from a center of the support (10) and separated by a distance (d) between 20 mm and 35 mm, preferably equal to 30 mm.

11. Mixing method (100) according to any one of the preceding claims, wherein the emitter (50, 52) is capable of generating acoustic waves (AW) at a frequency between 40 Hz and 200 kHz.

12. Mixing method (100) according to any one of the preceding claims, wherein the thin layer (45) of fluids has, at rest, a thickness of between 50 pm and 120 pm, preferably a thickness of between 70 pm and 100 pm.

13. Mixing method (100) according to any one of the preceding claims, wherein the support (10) is transparent and comprises or is optionally surmounted by light-emitting diodes (12).

14. A mixing method (100) according to any one of the preceding claims, wherein the first entity (31) is a primary antibody capable of binding with antigens (21) of the sample (20) of biological material, the second entity (41) comprises a secondary antibody (41a) capable of binding with the primary antibody (31).

15. Mixing method (100) according to claim 14, wherein the second entity (41) comprises an enzyme (41b) linked to the secondary antibody (41a), said enzyme (41b) being capable of producing a color, distinct from the color of the first and second fluids (30, 40), when said second entity (41) binds with the first entity (31).

Images