Kit and method for capturing molecules with magnetic means

Abstract

The invention relates to a kit and a method for capturing molecules contained in a sample by at least one magnetic layer comprising a possible repetition of juxtaposition of at least one first zone containing magnetic particles polarized in a first direction and at least one second zone containing magnetic particles that are not polarized or polarized in a second direction different from the first polarization direction of the magnetic particles of the first zone, so that at a distance of at least 1 pm from the at least one magnetic layer, a magnetic field is generated having at least one intensity variation of at least 0.1 mT, the variation defining a maximum of the standard of the intensity of the magnetic field, the level of which defines a zone for capturing magnetic nanoparticles on a capture support.

Description

Technical Field

[0001] The present invention relates to a kit for capturing molecules. The present invention also relates to a method for capturing molecules. Background Art

[0002] ELISA tests (acronym for "Enzyme-Linked Immunosorbent Assay") are commonly used to quantify molecular markers (antigens, antibodies, etc.) present in diagnostic fluids, biopsies, cultures or any other sample.

[0003] However, this technique, which is currently the most robust and one of the most widespread diagnostic methods, has drawbacks, namely its complexity, the use of expensive automated machines and its duration, which can reach several hours.

[0004] The ELISA test is a heterogeneous immunoassay technique, that is, it requires a solid support (usually a titer plate comprising a plurality of wells) to which suitable molecules have pre-attached in order to capture the molecules to be assayed.

[0005] Once the molecule of interest is captured on the support, washing can remove the remaining sample and proceed to the steps of detecting and quantifying the molecule.

[0006] For example, in the case of the so-called "sandwich" ELISA test, which allows the analysis of antigens in solution, the surface of the support is covered with a defined amount of a so-called capture antibody, which is suitable for binding the desired antigen.

[0007] A solution that can contain the antigen is then applied to the support; the antigen then binds to the capture antibody located on the surface of the support.

[0008] The support is then washed to remove any unbound antigen remaining in the solution. A solution containing an antibody, called a detection antibody, coupled to a detection device, which is adapted to bind to the antigen immobilized on the support, is then deposited on the support. The detection antibody can be directly labeled and emit a detectable signal, but can also be coupled to an enzyme that catalyzes the substrate, thereby causing emission of a detectable signal.

[0009] A new washing step is performed to keep the antigen bound to the detection antibody on the support, which is itself coupled to the enzyme.

[0010] Finally, to detect and quantify the antigen, a substrate is deposited on the support, which is enzymatically converted into a detectable signal (e.g., by spectroscopic analysis of a color, or by fluorescence emission) representing the binding between the antigen and the detection antibody.

[0011] The signal can be observed with the naked eye or by an instrument such as a spectrophotometer.

[0012] The article by D. Issadore et al., Lab Chip, 2011, 11, 147 describes a method for capturing molecules in a sample by circulating the sample in a fluidic microchannel disposed beneath a polydimethylsiloxane (PDMS) matrix in which NdFeB magnetic particles are immobilized.

[0013] Document WO2014111187 describes a method for capturing molecules in a sample, comprising the following steps: mixing the sample with magnetic particles, coupling each of the particles to an element capable of selectively binding to the molecule to be captured, thereby forming at least one complex comprising the magnetic particle, the element and the molecule bound to the element, and fixing the at least one complex on a support comprising an ordered magnetic field microsource.

[0014] These ordered magnetic field microsources are distributed in a defined pattern near the surface of a support intended to come into contact with the sample and also have a defined magnetic orientation.

[0015] The method described in this document is interesting. However, the capture supports are difficult to produce and industrialize, especially in a clean environment, which means high manufacturing costs. Summary of the Invention

[0016] The object of the present invention is in particular to overcome these disadvantages of the prior art.

[0017] More precisely, the object of the present invention is to provide a kit and a method for capturing molecules contained in a sample, thanks to high-performance magnetic means and with a reduced financial footprint compatible with low-cost production technologies.

[0018] The present invention therefore relates to a kit for capturing molecules contained in a sample, comprising:

[0019] a) magnetic nanoparticles having a largest dimension of less than 1 μm, said nanoparticles being each coupled to at least one capture element, said at least one capture element specifically binding to said molecule, and

[0020] b) a support for capturing said magnetic nanoparticles, said support comprising or essentially consisting of at least one magnetic layer, said magnetic layer comprising a possibly repeated juxtaposition of at least first and second regions, said first regions comprising magnetic particles polarized in a first direction and said second regions comprising magnetic particles that are non-polarized or polarized in a second direction different from the first polarization direction of the magnetic particles of said first regions, such that said at least one magnetic layer generates a magnetic field having at least one intensity variation of at least 0.1 mT at a distance of at least 1 μm from said at least one magnetic layer, said at least one variation in the intensity of the magnetic field defining a standardized maximum and a minimum value of the intensity of said magnetic field so as to define an area for capturing magnetic nanoparticles on the capture support at said standardized maximum value of said magnetic field.

[0021] The inventors have surprisingly found that a magnetic layer of magnetic particles having weak magnetism can be used to attract nanoparticles coupled to a capture element.

[0022] The magnetic layer used in the present invention is flexible and particularly suitable for magnetic tape. It is made of a magnetic composite material, such as ferrite, randomly distributed in a polymer or oriented along a pre-oriented axis. Ferrites are ferromagnetic ceramics obtained by molding iron oxide (Fe2O3XO) under high pressure and high temperature (>1000°C), where X can be manganese, zinc, cobalt, nickel, barium, strontium, or the like.

[0023] The present invention therefore consists in repurposing magnetic tapes, which are difficult to demagnetize and are usually used for reliable information storage (audio and video cassettes, credit cards, badges, transport tickets, etc.), in order to apply them to the capture of nanomagnetic particles in solution.

[0024] For the sake of clarity in the remainder of the description, the magnetic particles constituting the magnetic layer according to the invention will be referred to as "powder" or "magnetic particles" in order to clearly distinguish them from the "magnetic nanoparticles" coupled to the capture elements.

[0025] The magnetic layers of the present invention are "encoded", that is to say at least a portion of their constituent magnetic particles are polarized / magnetized. In the following, the terms "polarized" and "magnetized" are considered synonymous and will be used uniformly.

[0026] This encoding (or this polarization) is not performed randomly, but is configured to reveal at least one juxtaposition of a first region comprising magnetic particles polarized in a first direction and a second region comprising magnetic particles that are unpolarized or polarized in a second direction different from the first polarization direction of the magnetic particles, thereby defining at least one connection between the first and second regions. As a result, each region (when polarized) emits its own magnetic field, allowing the magnetic layer to be modeled as a plurality of magnetic field sources.

[0027] Polarization of the magnetic particles constituting the at least one magnetic layer is achieved in particular with a write head known from the field of magnetic tape encoding. Typically, a local magnetic field is applied to a region of the magnetic layer by means of miniature electromagnets.

[0028] This specific juxtaposition of the first and second polarization regions makes it possible to produce variations in the intensity of the generated magnetic field at a distance of at least 1 μm from the at least one magnetic layer, and thus to produce standardized maxima and minima of the magnetic field intensity. The standard of the magnetic field intensity corresponds to the absolute value of the magnetic field intensity (in Tesla). In the present invention, the terms "intensity standard" and "standard" may be used instead. The maximum values ​​of the magnetic field intensity standard produce regions that attract the suspended nanoparticles and in which the magnetic nanoparticles minimize their magnetic energy, so that they are referred to as "local minima" or "energy sinks" of the magnetic energy of the nanoparticles.

[0029] Thus, by orthogonal projection onto the surface of the magnetic layer, the maximum of the magnetic field strength criterion will define the capture zone of the nanoparticles.The capture zone and the energy valley thus coincide at the same position.

[0030] These capture zones extend over a distance of at most 35 μm from the orthogonal projection of the normalized maximum or each maximum of the magnetic field intensity onto the surface of the magnetic layer. “At most 35 μm” means 35 μm, 34 μm, 33 μm, 32 μm, 31 μm, 30 μm, 29 μm, 28 μm, 27 μm, 26 μm, 25 μm, 24 μm, 23 μm, 22 μm, 21 μm, 20 μm, 19 μm, 18 μm, 17 μm, 16 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm and 1 μm.

[0031] According to the following formula, the magnetic energy (E) of the nanoparticle is equal to the magnetization of the nanoparticle and a magnetic field generated by the at least one magnetic layer The opposite of the scalar product of :

[0032]

[0033] where E is the magnetic energy of the nanoparticle (in joules),

[0034] M is the magnetization of the nanoparticle (in amperes per meter), and

[0035] B is the magnetic field strength (in Tesla).

[0036] In the case of the magnetic materials used in the invention, the magnetization is a strictly increasing function of the magnetic field strength, so that the minimum value of the magnetic energy of the nanoparticles corresponds to the maximum value of the magnetic field standard and therefore to the capture zone.

[0037] When the magnetic nanoparticles are magnetized by the single magnetic field generated by the at least one magnetic layer, the capture zone is located at the junction of the first and second regions.

[0038] The magnetic field generated by the at least one magnetic layer exhibits an intensity variation of at least 0.1 mT and at most 1 mT, advantageously at least 0.1 mT and at most 500 mT, more advantageously at least 0.5 mT and at most 300 mT, even more advantageously at least 1 mT and at most 200 mT.

[0039] These variations in magnetic field strength make it possible to generate a strong magnetic field gradient, that is, a magnetic field gradient sufficient to exert a significant trapping force on the Brownian motion of the nanoparticles. This magnetic field gradient is therefore localized. Furthermore, the gradient is directed towards the trapping zone and has a value of at least 10 Tm at a distance of 10 μm from the at least one magnetic layer. -1 The value is advantageously from 10T.m -1 to 10 5 Tm -1 , even more advantageously from 500T.m -1 Up to 5*10 3 Tm -1 In this way, the strong magnetic field gradient guides the suspended nanoparticles towards the capture zone of the at least one magnetic layer.

[0040] When the magnetic nanoparticles are captured by the at least one magnetic layer, they localize themselves in the or each capture zone. This specific localization is very interesting for the direct detection and quantification of the captured molecules, as will be described in more detail below.

[0041] The magnetic field strength can be measured using a magneto-optical technique called MOIF (Magneto-Optical Imaging Film).

[0042] The MOIF technique is based on the Faraday effect. Typically, the technique involves immersing a flat film made of a material whose optical properties are affected by a magnetic field in a known manner in the magnetic field of an object whose strength is to be measured. Typically, the film is attached to the object. The width and length of the flat film are at least equal to the width and length of the area of ​​the test object. After this first step, the flat film is irradiated with a light beam of known amplitude and polarization, which passes through the flat film. Analysis of the polarization and amplitude of the light beam passing through the flat film provides a measurement of the planar component of the magnetic field present therein. An exemplary measurement of the magnetic field strength of an object by means of the MOIF technique is given in the article Grechishkin et al., J. Appl. Phys. 120, 174502 (2016).

[0043] In practice, a thin flat film, typically less than one micron thick and consisting of a magneto-optical material (e.g. a rare earth garnet), is deposited on a transparent non-magnetic substrate (e.g. glass, quartz or silicon dioxide) and then covered with a very thin reflective layer, a mirror (e.g. made of gold, silver or aluminum), less than 100 nm thick. Thus, the film of the magneto-optical material compound is covered on one side with a transparent non-magnetic substrate and on the other side with a reflective layer. This assembly is attached to an object emitting a magnetic field, e.g. a capture support according to the invention. The magneto-optical material film is then irradiated with a polarized light beam, which first passes through the transparent non-magnetic layer (whose optical capacity is not affected by the magnetic field of the magnetic object and therefore does not affect the polarization of the incident light beam), then passes through the optical material film (whose optical properties are affected by the magnetic field generated by the magnetic object and therefore the polarization of the incident light beam), is reflected by the reflective layer, passes through the optical material film again (which again affects the polarization of the light beam) and then passes through the glass again (which does not affect the polarization of the light beam) before terminating in a polarization analyzer. The rotation angle of the polarization of the reflected light beam relative to the incident light beam is proportional to the magnetic field, the Faraday rotation coefficient of the garnet, and the thickness of the magneto-optical material. Thus, the calibration curve, which represents the Faraday rotation of the light beam as a function of the magnetic field strength, captures the distribution of the magnetic field strength generated by the at least one magnetic layer. This curve is specific to the magneto-optical material used.

[0044] In particular, the magnetic field intensity can be measured by the MOIF technique using the MagView CMOS system sold by the company MATESY GmBH, which has a C-type sensor as polarization analyzer and a DLGi5 type garnet as magneto-optical material film, the calibration curve of which is Figure 11 In this instrument, the mirror is replaced by a CMOS sensor, so the light does not have to be reflected.

[0045] The results can be confirmed by numerical and analytical simulations, for example by one of the following two methods:

[0046] -Using COMSOL 5.0 modeling software). This software allows for numerical simulations in a two-dimensional environment with a thickness of 10 mm. Upstream, the thickness and width of the at least one magnetic layer are optically measured, typically using brightfield microscopy images, and the afterglow value or effect is measured; see below for details. This data is input into the software, and the magnetic field generated by the at least one magnetic layer is simulated in the steady state using the MFNC (Magnetic Field Without Current) toolbox.

[0047] - so-called semi-analytical methods. The latter are based on the method developed, for example, in the article by Chigirinsky S. et al., Advanced Study Center Co. Ltd., 20 (2009), 85-91. Furthermore, upstream, the thickness and width of the at least one magnetic layer are measured on the one hand, and the remanence or retention value of the at least one magnetic layer is measured on the other hand. The at least one magnetic layer is decomposed into a sum of elements with uniform magnetization and then, for example, using 6.02 software (publisher Scilab Enterprises) analyzes and solves the equations that give the magnetic field for each element. The field generated by each element of the at least one magnetic layer is added to the fields generated at each point in space by all other elements of the at least one magnetic layer. In the presence of at least one additional magnetic field source, as described in more detail below, the magnetic field generated by the at least one additional source is added to the total magnetic field generated by the at least one magnetic layer at each point.

[0048] According to one embodiment of the invention, the second region comprises magnetic particles polarized in a second, different direction, which deviates by at least 30°, advantageously by 30° to 180°, relative to the first polarization direction of the magnetic particles of the first region. In the present invention, "30° to 180°" is understood to mean 30°, 31°, 32°, 33°, 34°, 35°, 36°, 37°, 38°, 39°, 40°, 41°, 42°, 43°, 44°, 45°, 46°, 47°, 48°, 49°, 50°, 51°, 52°, 53°, 54°, 55°, 56°, 57°, 58°, 59°, 60°, 61°, 62°, 63°, 64°, 65°, 66°, 67°, 68°, 69°, 70°, 71°, 72°, 73°, 74°, 75°, 76°, 77°, 78°, 79°, 80°, 81°, 82°, 83°, 84°, 85°, 86°, 87°, 88°, 89°, 90°, 91°, 92°, 93°, 94°, 95°, 96°, 97°, 98°, 99°, 100° °, 69°, 70°, 71°, 72°, 73°, 74°, 75°, 76°, 77°, 78°, 79°, 80°, 81°, 82°, 83°, 84°, 85°, 86°, 87°, 88°, 89°, 90°, 91°, 92°, 93°, 94°, 95°, 96°, 97°, 98°, 99°, 100°, 101°, 102°, 103°, 104°, 105°, 106°, 107°, 108°, 109°, 110° 10°, 111°, 112°, 113°, 114°, 115°, 116°, 117°, 118°, 119°, 120°, 121°, 122°, 123°, 124°, 125°, 126°, 127°, 128°, 129°, 130°, 131°, 132°, 133°, 134°, 135°, 136°, 137°, 138°, 139°, 140°, 141°, 142°, 143°, 144°, 145 , 146°, 147°, 148°, 149°, 150°, 151°, 152°, 153°, 154°, 155°, 156°, 157°, 158°, 159°, 160°, 161°, 162°, 163°, 164°, 165°, 166°, 167°, 168°, 169°, 170°, 171°, 172°, 173°, 174°, 175°, 176°, 177°, 178°, 179°, or 180°.

[0049] Advantageously, the second region comprises magnetic particles polarized in a second different direction that is deviated by at least 60°, more advantageously by at least 90°, still more advantageously by at least 120°, even more advantageously by at least 150°.

[0050] According to an advantageous embodiment of the invention, the second region comprises magnetic particles polarized in a second direction opposite to the first polarization direction of the magnetic particles of the first region, ie a polarization reversal of 180°.

[0051] According to another embodiment, the magnetic particles of the second region are not polarized.This configuration also allows variations in the magnetic field strength and thus allows a standard maximum value of the magnetic field strength generated by the at least one magnetic layer to occur.

[0052] According to one embodiment of the present invention, the at least one first region and the at least one second region have the same size, or they have different sizes, in particular different widths and / or lengths.

[0053] According to one embodiment of the present invention, the at least one first region and / or the at least one second region has a width of 10 μm to 500 μm. In the present invention, "10 to 500 μm" is understood to mean 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm. , 250μm, 260μm, 270μm, 280μm, 290μm, 300μm, 310μm, 320μm, 330μm, 340μm, 350μm, 360μm, 370μm , 380μm, 390μm, 400μm, 410μm, 420μm, 430μm, 440μm, 450μm, 460μm, 470μm, 480μm, 490μm, 500μm.

[0054] Advantageously, the at least one first region and / or the at least one second region has a width advantageously ranging from 50 μm to 250 μm, more advantageously from 70 μm to 150 μm, even more advantageously from 90 μm to 110 μm.

[0055] According to one embodiment, the at least one first area and the at least one second area form the same pattern. This pattern can in particular correspond to a strip. Alternatively, they represent different patterns.

[0056] According to one embodiment, the at least one magnetic layer is coated with a protective film having a thickness less than 1 μm. Such a film advantageously makes it possible to protect the at least one magnetic layer without hampering its capture / attraction capabilities due to its very small thickness.

[0057] The at least one magnetic layer itself may constitute such a capture support. In this case, the latter advantageously has a thickness of at least 5 μm and more advantageously of 10 to 20 μm.

[0058] Said magnetic layer is advantageously placed on a support member.

[0059] According to one embodiment of the present invention, the material for the support member is selected from the following list: glass, silicon, polymers of plastic materials, organic materials such as paper or bamboo, quartz, gold, tape, non-magnetic metal alloys such as dura mater or titanium, or combinations of these materials.

[0060] Advantageously, the polymer is selected from the following list: polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polycarbonate, polyimide, polyvinyl chloride (PVC), polyethylene, polypropylene, silicone, polyester or a combination of these materials.

[0061] The support member may be a single layer of the material described above.

[0062] The magnetic layer disposed on a support member may be stretched, for example using two wound spools, like a VHS cassette.

[0063] According to one embodiment of the present invention, the capture support comprises a capture container configured to receive a sample containing molecules to be captured and delimited by at least one wall comprising the at least one magnetic layer. The capture container has a minimum dimension of 20 μm to 1000 μm. In the present invention, “20 μm to 1000 μm” is understood to mean 20 μm, 40 μm, 60 μm, 80 μm, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm, 240 μm, 260 μm, 280 μm, 300 μm, 320 μm, 340 μm, 360 μm, 380 μm, 400 μm, 420 μm, 440 μm, 460 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540 μm, 560 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 μm, 730 μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810 μm, 820 μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 0μm, 500μm, 520μm, 540μm, 560μm, 580μm, 600μm, 620μm, 640μm, 660μm, 680μm, 700μm, 720μm, 740μm m, 760μm, 780μm, 800μm, 820μm, 840μm, 860μm, 880μm, 900μm, 920μm, 940μm, 960μm, 980μm and 1000μm.

[0064] According to one embodiment of the invention, the capture support is selected from the group consisting of chambers, parallelepiped chambers, hollow right cylinders, wells, wells in the form of right cones, in particular truncated right cones or truncated pyramids, microfluidic channels, titration plates, test tubes and microtubes.

[0065] In the case of a chamber and a parallelepiped chamber, the at least one magnetic layer is arranged on one wall of the chamber and, if a plurality of magnetic layers are present, on at least one wall of the chamber.

[0066] In the case of a hollow right circular cylinder, the at least one magnetic layer is arranged on the circumferential wall of the cylinder.

[0067] In the case of a hole, the at least one magnetic layer is arranged on one wall of the hole, and if multiple magnetic layers are present, on at least one wall of the hole. Advantageously, the at least one magnetic layer is arranged on the wall forming the bottom of the hole.

[0068] In the case of a hole in the form of a cone, in particular a truncated or truncated pyramidal right cone, the at least one magnetic layer is arranged on one wall of the hole and, if a plurality of magnetic layers are present, on at least one wall of the hole.

[0069] In the case of a microfluidic channel, the at least one magnetic layer is arranged on one wall of the channel, and if a plurality of magnetic layers are present, on at least one wall of the channel.

[0070] In the case of a titer plate comprising a plurality of wells, the at least one magnetic layer is arranged on one wall of the at least one well, and if a plurality of magnetic layers are present, on at least one wall of the at least one well. Advantageously, the at least one magnetic layer is arranged on a wall forming the bottom of the at least one well.

[0071] In the case of a test tube or microtube, the at least one magnetic layer is arranged on one wall of the test tube or microtube, and if multiple magnetic layers are present, on at least one wall of the test tube or microtube. Advantageously, the at least one magnetic layer is arranged on the circumferential wall of the test tube or microtube.

[0072] The at least one magnetic layer can be fixed to the support. Advantageously, the fixing is irreversible. In this case, the fixing can be performed, for example, by gluing, rolling or stamping. Alternatively, the fixing is reversible. Thus, the at least one magnetic layer can be fixed by a hook and loop system (more commonly known as a Velcro system) or by a reversible glue (e.g., glue of animal origin).

[0073] The capture support may comprise one or more magnetic layers of the present invention.

[0074] According to one embodiment, the at least one magnetic layer is folded upon itself such that one portion of the magnetic layer is superimposed on another portion.

[0075] According to one embodiment of the present invention, the capture support comprises at least two magnetic layers. Advantageously, the magnetic layers are arranged on the same plane.

[0076] Alternatively, the magnetic layers are arranged on different planes such that the two or at least two layers overlap each other.

[0077] According to another embodiment of the present invention, the capture support comprises at least one wall for attracting the magnetic nanoparticles, the wall comprising the at least one magnetic layer. Advantageously, the capture support comprises a plurality of attracting walls, each having at least one magnetic layer, and at least one of the walls is arranged in a plane different from the other magnetic layers; advantageously, at least one of the walls is superimposed on one or at least one other wall. Alternatively or in a complementary manner, at least one of the walls is arranged orthogonally to another wall or to at least one of the other walls.

[0078] The use of the magnetic layer of the present invention to capture nanomagnetic particles is counterintuitive, since usually these nanoparticles are captured by a magnetic layer with strong magnetism, for example a magnetic layer made of a rare earth-based alloy. In fact, in order to capture nanoparticles, which inherently exhibit a weak magnetization due to their small size, a magnetic layer with strong magnetism is usually used. An example of such a "strong" magnetic layer based on rare earths is specifically described in document WO2014111187. The strong magnetic layer exhibits a remanence of 0.7T to 1.5T and a current of 600kA / m to 2400kA / m. 3 The stubborn field.

[0079] The apparent remanence and retentivity of the at least one magnetic layer according to the invention are 5 to 15 times lower. The coercive field of the at least one magnetic layer is 10 to 400 kA / m 3 .

[0080] "Apparent remanence" refers to the remanence of the at least one magnetic layer as a whole, rather than the remanence of each magnetic particle constituting it.

[0081] The coercive field of a ferromagnetic material represents the strength of the magnetic field that must be applied to the material to initially reach its saturation magnetization in order to eliminate the material's magnetization.

[0082] Remanence is a material-intensive quantity that measures the induction or density of the magnetic flux that persists after a ferromagnetic material has been magnetized using a strong external magnetic field. Remanence is measured in Teslas (T). A previously magnetized sample of a permanent magnetic material has a magnetic moment that is proportional to its volume and the remanence of the material. The magnetic moment is a vector that can characterize the strength of a magnetic source. The magnetic flux generated by the sample is proportional to its magnetic moment. This magnetic flux can be measured in a vibrating sample magnetometer (VSM) or an extraction magnetometer or a SQUID magnetometer. Typically, a sufficiently strong magnetic field (typically 4-6 Tesla) is applied to the sample along its preferential magnetization axis to saturate its magnetization, and then this so-called "saturation" magnetic field is removed. Measuring the flux generated by the sample at zero magnetic field (0 T) after saturation gives the remanent magnetic moment of the sample. The remanent magnetization of the material is then obtained, which is equal to the moment of the sample divided by the volume of the sample.

[0083] According to one embodiment of the present invention, the at least one magnetic layer has a remanence less than or equal to 0.6 T, advantageously a remanence of 0.01 T to 0.6 T. In the present invention, "0.01 T to 0.6 T" should be understood to mean 0.01 T, 0.02 T, 0.03 T, 0.04 T, 0.05 T, 0.06 T, 0.07 T, 0.08 T, 0.09 T, 0.1 T, 0.11 T, 0.12 T, 0.13 T, 0.14 T, 0.15 T, 0.16 T, 0.17 T, 0.18 T, 0.19 T, 0.2 T, 0.21 T, 0.22 T, 0.23 T, 0.24 T, 0.25 T, 0.26 T, 0.27 T, 0.28 T , 0.29T, 0.3T, 0.31T, 0.32T, 0.33T, 0.34T, 0.35T, 0.36T, 0.37T, 0.38T, 0.39T, 0.4T, 0.41T, 0.42T, 0.43T, 0.44T , 0.45T, 0.46T, 0.47T, 0.48T, 0.49T, 0.5T, 0.51T, 0.52T, 0.53T, 0.54T, 0.55T, 0.56T, 0.57T, 0.58T, 0.59T, 0.6T.

[0084] More advantageously, the at least one magnetic layer exhibits a remanence of 0.01T to 1T, more advantageously 0.02T to 0.5T, even more advantageously 0.05T to 0.2T.

[0085] When the thickness of the magnetic layer or layers is too thin—that is, when its width and / or length is significantly greater than its thickness, at least ten times greater—it becomes difficult to determine the volume of the magnetic material and, therefore, to calculate its remanence. This is particularly true in the case of commercial magnetic tapes, where a thin magnetic layer is placed on a substrate layer, and due to industrial manufacturing processes, the boundary between these two layers is often difficult to assess. In such cases, the holding force of the magnetic layer or layer group is measured instead. This holding force is equal to the magnetic moment of the sample divided by its surface (not its volume). The holding force is expressed in units of surface density of the magnetic flux, that is, in pm. Gauss. Typically, a 2 mm x 2 mm sample (typical dimensions for a laboratory magnetometer) is cut from the capture support to be tested. Its exact area is measured under an optical microscope. The procedure for determining the resonance of a magnetic material is the same as for the remanence, that is, its magnetization is saturated to obtain its magnetic moment.

[0086] Therefore, the present invention also relates to a kit as defined above, wherein said at least one magnetic layer has a holding force of 2000 to 30,000 μm.Gauss. In the present invention, “2000 to 30,000 μm.Gauss” is understood to mean 2000 μm.Gauss, 2500 μm.Gauss, 3000 μm.Gauss, 3500 μm.Gauss, 4000 μm.Gauss, 4500 μm.Gauss, 5000 μm.Gauss, 5500 μm.Gauss, 6000 μm.Gauss, 6500 μm.Gauss, 7000 μm.Gauss, 7500 μm.Gauss, 8000 μm.Gauss, 8500 μm.Gauss, 9000 μm.Gauss, 9500 μm.Gauss, 10,000 μm.Gauss, 11,000 μm.Gauss, 12,000 μm.Gauss, m.Gauss, 13,000μm.Gauss, 14,000μm.Gauss, 15,000μm.Gauss, 16,000μm.Gauss, 17,000μm.Gauss, 18,000μm.Gauss, 19,000μm.Gauss, 20,000μm.Gauss, 21,000μm.Gauss, 22,000μm.Gauss, 23,000μm.Gauss, 24,000μm.Gauss, 25,000μm.Gauss, 26,000μm.Gauss, 27,000μm.Gauss, 28,000μm.Gauss, 29,000μm.Gauss, 30,000μm.Gauss.

[0087] According to one embodiment of the present invention, the at least one magnetic layer has a retention force of 5000 to 20,000 μm.Gauss, advantageously 8000 to 14,000 μm.Gauss, more advantageously 9000 to 11,000 μm.Gauss. The maximum size of the captured magnetic nanoparticles is less than 1 μm.

[0088] Due to their size, the magnetic particles used exhibit superparamagnetism.

[0089] The term "superparamagnetism" refers to the property of small particles of ferromagnetic or ferrimagnetic materials to randomly change the direction of magnetization under the influence of thermal agitation in the absence of an applied magnetic field.

[0090] The "superparamagnetic" property of the magnetic particles means that in the absence of an external exciting magnetic field, the magnetic particles have no net magnetic moment and therefore they do not attract each other, thus preventing their agglomeration.

[0091] Compared to microparticles coupled to capture elements, nanoparticles coupled to capture elements show better performance in molecular capture, in particular due to a higher diffusion coefficient (multiplied by 10) and a greatly increased (multiplied by 10) 3 ) concentration (log number / m 3 ). However, their respective magnetic forces are divided by 103 (The number is reduced by 1 / 10).

[0092] According to one embodiment of the present invention, the maximum size of the magnetic nanoparticles is 50 nm-500 nm, advantageously 50 nm-250 nm, more advantageously 100-250 nm, even more advantageously 150-200 nm.

[0093] According to one embodiment, the magnetic nanoparticles contain 10 to 90% iron, advantageously 30 to 80%, and more advantageously 50 to 70% iron. The greater the amount of iron contained in the nanoparticles, the greater their magnetization in the presence of an additional magnetic field, and the more strongly they are attracted to the at least one magnetic layer. Therefore, the greater the amount of iron, the less need there is to increase their magnetization in order for them to be attracted more quickly, as will be seen below.

[0094] According to one embodiment of the present invention, the nanoparticles are encapsulated. They can be obtained, in particular, by copolymerization of iron oxide and polystyrene. This encapsulation limits the release of iron from the nanoparticles. In fact, this release disrupts the detection and quantification of the capture molecules.

[0095] The magnetic nanoparticles can have any shape, such as a parallelepiped, a ring, a sphere, etc. The nanoparticles can have a smooth or irregular surface. When they have an irregular surface, they have a so-called "potato-like" shape.

[0096] Advantageously, the magnetic nanoparticles are spherical and are then likened to "beads." Therefore, even if the geometry of the particles is not a perfect sphere, they can still be referred to as "beads."

[0097] Preferably, the beads are monodisperse, the size uniformity of the beads imparting to them the same properties and thus improving the diffusion of the beads for capture by the at least one magnetic layer. "Monodisperse" means that the standard deviation of the mean diameter of the beads is less than or equal to 40 nm over a range of 200 nm, advantageously 20 nm over a range of 200 nm.

[0098] In some cases, beads are sold dispersed in a matrix with little or no magnetic material, such as plastic polymers, silicon dioxide (SiO2), etc.

[0099] The beads are preferably biocompatible, that is, they have the ability not to interfere with or degrade the biological medium in which they are used.

[0100] To couple the capture element to the magnetic nanoparticles, the surface of the magnetic nanoparticles is functionalized, in particular with proteins A or G from Staphylococcus aureus or by carbodiimide. If proteins A or G are used, the bond between the capture element and the nanoparticle is not covalent, as is the case with carbodiimides.

[0101] Various capture elements can be coupled to the nanoparticles. According to one embodiment of the present invention, the capture element is selected from an antibody, a Fab fragment, a F(ab')2 fragment or a Fv fragment of an antibody, an antigen, a nucleic acid sequence, an organelle specifically corresponding to a vesicle, a cell, an aptamer or a bacterium.

[0102] Antibodies, or "immunoglobulins," consist of four amino acid chains, of which we can distinguish two light chains and two so-called heavy chains. Each heavy chain is linked to a light chain via disulfide bonds. Furthermore, due to the hypervariable region, the end of the heavy chain and the end of the associated light chain together define the paratope. Thus, antibodies contain two paratopes, each of which allows binding to an epitope of an antigen. The heavy chains are connected to each other at the so-called hinge region.

[0103] The antibody fragment that corresponds to one of the two paratopes is called fragment Fv; it is the smallest fragment of an antibody that retains epitope recognition properties.

[0104] The Fab fragment corresponds to the entire light chain and the end of the heavy chain attached to the light chain. Therefore, the Fab fragment contains the Fv fragment. Antibodies have two Fab fragments.

[0105] The F(ab')2 fragment corresponds to the union of two Fab fragments linked together by the hinge region of the heavy chains.

[0106] Fv, Fab, and F(ab')2 fragments have the same affinity for antigen as intact antibodies.

[0107] Nucleic acids are polymers whose basic units are nucleotides. Nucleic acids can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0108] Organelles are differentiated compartments contained in the cytoplasm of eukaryotic cells and in which they carry out specific biological functions. In particular, among the organelles we find the endoplasmic reticulum, Golgi apparatus, mitochondria, lysosomes, and peroxisomes.

[0109] Vesicles are compartments found in the cytoplasm of cells and composed of at least one lipid bilayer. Vesicles circulate within the cytosol and have various functions, such as storing, transporting, or digesting cellular waste products.

[0110] The cell is the compartment that makes up an organism; it is bounded by a membrane and contains, on the one hand, the DNA necessary for its reproduction and, on the other hand, the proteins necessary for its functioning.

[0111] Aptamers are synthetic oligonucleotides, most commonly RNA, that can bind to a specific ligand and sometimes catalyze a chemical reaction on that ligand. Aptamers are typically synthetic compounds that are isolated in vitro from a large combinatorial library of random sequence compounds using an iterative selection method called "Systematic Evolution of Ligands by Exponential Enrichment" (SELEX). Further details on aptamer synthesis using the SELEX method can be found in the article "Aptamers and SELEX in Chemistry & Biology" (Chem Biol. 2014 Sep 18; 21(9): p. 1055-8).

[0112] Bacteria are single-celled prokaryotic microorganisms that contain a single cytoplasmic compartment containing their DNA. Therefore, unlike eukaryotic cells, the DNA is not separated from the cytoplasm by a nucleus. Bacteria reproduce only by splitting in two via fission.

[0113] A person skilled in the art will readily adapt the type of capture element to be used depending on the type of molecule to be captured.

[0114] The present invention also relates to a kit as defined above, further comprising at least one additional magnetic field source, said additional magnetic field source being external to said at least one magnetic layer.

[0115] The magnetic field generated by the at least one additional magnetic field source will have several effects on the elements of the kit, which make it possible, on the one hand, to accelerate the capture of the nanoparticles by the capture support and, on the other hand, to obtain a more localized capture zone, that is to say, more precise and narrower, typically extending over a distance of less than 15 μm from an orthogonal projection onto the surface of the magnetic layer.

[0116] On the one hand, applying the magnetic field of said at least one additional magnetic field source advantageously makes it possible to increase the magnetization of the magnetic nanoparticles, thereby accelerating or even triggering their capture by said at least one magnetic layer of the capture support.

[0117] On the other hand, the presence of an additional magnetic field that is greater than or equal to the standard of the magnetic field generated by the at least one magnetic layer is added to the magnetic field generated by the at least one magnetic layer, so that based on the orientation of the additional magnetic field, the amplitude of some energy depressions of the resulting total magnetic field is greater than the amplitude of the energy depressions of the only magnetic field generated by the at least one magnetic layer, which also participates in accelerating the capture of nanoparticles.

[0118] On the surface of the support, a capture zone is enhanced, wherein the magnetic field generated by the at least one magnetic layer above the connection between the first and second areas follows the same axis and the same direction as the additional magnetic field. This significantly increases the standard of the total magnetic field strength. In contrast, above the first and second areas, the standard of the resultant of the additional magnetic field and the magnetic field generated by the at least one magnetic layer does not increase as much, because they are not collinear. As a result, the maximum value of the standard of the magnetic field strength increases more than the minimum value; thus, the amplitude of certain energy depressions is enhanced. This accelerates capture on these capture zones, which are more strongly attracted to these energy depressions, and on the other hand reduces the scope of the capture zone, making it better defined and more precise geographically.

[0119] The capture zone is eliminated when the magnetic field generated by the at least one additional source at the junction between the first and second zones, at the surface of the support, is greater than the magnetic field generated by the at least one magnetic layer along the same axis but in the opposite direction. In fact, the magnitude of the magnetic field generated at this level is no longer a maximum, and the energy of the nanoparticles is no longer minimized there.

[0120] In the specific case where the polarization directions of the first and second regions are opposite and parallel to the support surface, the direction of the additional magnetic field is the same as the direction of the magnetic field jointly generated by the first and second regions at their connection with the support surface. Due to the above reasons, every other capture zone is enhanced, every other capture zone is weakened or even eliminated.

[0121] When the additional magnetic field is greater than or equal to the magnetic field generated by the at least one magnetic field source, the capture zone is located at a point where the additional magnetic field has the same orientation and the same direction as the magnetic field generated by the at least one magnetic tape. The capture zone can then be displaced from its initial position as described below.

[0122] The magnetization of an object corresponds to a vector quantity that characterizes its magnetic behavior on a macroscopic scale. It is derived from the orbital magnetic moment and the magnetic spin moment of electrons. It is measured in amperes per meter or sometimes teslas.

[0123] Advantageously, said at least one additional magnetic field source is selected from a permanent magnet, a coil or an electromagnet.

[0124] When multiple magnetic field sources are present, they can be selected from a combination of at least one permanent magnet, at least one coil and / or at least one electromagnet. In particular, they can be selected from an assembly of a permanent magnet, a coil, an electromagnet and an assembly combination of the latter.

[0125] The magnetic field sources can be arranged side by side in a plane, in particular linearly, or in a three-dimensional configuration. The magnetic field sources can be placed side by side. In the present invention, "side by side" is understood to mean that the sources are adjacent or spaced apart from one another. In particular, adjacent magnetic field sources exhibit polarization reversal.

[0126] A permanent magnet is an object made of a hard magnetic material that has been artificially or naturally endowed with the ability to generate a permanent magnetic field. The characteristic of a permanent magnet is that, once its magnetic field is established, it continues to generate without requiring any specific action. Hard magnetic materials are those with a high remanent magnetization and coercive field, exceeding 0.3 T and 250 kA / m, respectively.

[0127] The hard magnetic material can be selected from rare earth-based magnets, 3d (Fe, Co, Ni)-noble metal (Pt or Pd as the main element) series transition metal alloys, ferrite magnets, and MnBi, MnAI, MnGa, FeGa, AINiCo magnets. When the material is a rare earth magnet, it can be selected from RFeB (wherein R consists of Nd, Pr, Tb, Dy or a mixture of several of these elements), SmCo or RCoCu (1 / 5 type crystal structure), SmCoCuFe (1 / 7 or 2 / 17 type crystal structure), RFeN (wherein R consists essentially of Sm).

[0128] For the remainder of the description, the term "permanent magnet" may be represented simply by "magnet".

[0129] When the at least one additional magnetic field source is a magnet, it is advantageously coupled to a soft ferromagnetic element, otherwise known as a "yoke" or "magnetic circuit." This soft ferromagnetic element extends the magnet and has a magnetic permeability greater than 100 S.I. ("International System," unitless) with a saturation of 1.6-2.4 T. Unlike hard ferromagnetic elements, soft ferromagnetic elements exhibit low remanent magnetization and a weak coercive field. Such a soft ferromagnetic element exhibits no magnetization in the absence of an external magnetic field and makes it possible to guide the magnetic field lines of the magnet, thereby increasing the value of the magnetic field generated by the magnet.

[0130] A coil consists of a winding of wire. This winding can optionally be created around a ferromagnetic material called a core. Unlike a magnet, a coil only emits a magnetic field when a specific action is applied—in this case, an electric current is passed through the wire. Therefore, once the current is no longer applied, the magnetic field ceases to be generated.

[0131] When the at least one additional magnetic field source is a coil, it is advantageously a planar coil, ie all turns are in at least one plane, advantageously in 1 to 5 planes.

[0132] According to one embodiment of the present invention, the at least one magnetic layer has two opposing surfaces, namely a capture surface and an opposing surface, and the at least one additional magnetic field source is a planar coil adjacent to the capture surface of the at least one magnetic layer. Thus, the planar coil at least partially covers the capture surface of the at least one magnetic layer. In this case, the nanoparticles will be at least partially immobilized on the planar coil in the capture zone.

[0133] An electromagnet generates a magnetic field when supplied with an electric current: it converts electrical energy into magnetic energy. It consists of a coil of wire and a core and / or one or more pole pieces made of a soft ferromagnetic material. Thus, as with a coil of wire, it emits no magnetic field when no current is flowing through it.

[0134] The at least one additional magnetic field source may or may not be attached to the capture support.

[0135] According to one embodiment of the present invention, the at least one additional magnetic field source is fixed to the capture support. Advantageously, the fixing is reversible, thus, such fixing can be achieved by clamping to the capture support. Alternatively, the fixing is irreversible, thus, such fixing can be achieved by gluing to the support.

[0136] According to an embodiment of the present invention, the at least one additional magnetic field source is configured to emit a uniform magnetic field.

[0137] Homogeneous means that the gradient of the magnetic field along the magnetization axis of the at least one additional magnetic field source is less than 100 T.m -1 , and is less than 150 T.m along an axis orthogonal to the magnetization axis on the surface of the at least one additional magnetic field source -1 In the present invention, “less than 100T.m -1 " refers to 100T.m -1 、90T.m -1 、80T.m -1 、70T.m -1 、60T.m -1 、50T.m -1 、40T.m -1 、30T.m -1 、20T.m -1 、10T.m -1 or 0T.m -1 In the present invention, “less than 150T.m -1 " refers to 150T.m -1 、140T.m -1 、130T.m -1 、120T.m -1 、110T.m -1 、100T.m-1 、90T.m -1 、80T.m -1 、70T.m -1 、60T.m -1 、50T.m -1 、40T.m -1 、30T.m -1 、20T.m -1 、10T.m -1 or 0T.m -1 .

[0138] The magnetic field generated by said at least one additional source may take any direction; advantageously, its direction is orthogonal to the surface of the support, more advantageously its direction and its orientation are identical to those generated by said at least one magnetic layer on the surface of the capture support at the junction between the first and second regions.

[0139] The at least one additional magnetic field source can be positioned anywhere around the capture support. Thus, the at least one additional source can also be positioned facing the capture surface of the at least one magnetic layer, or facing the second surface thereof. Alternatively, the at least one additional source can be positioned at a distance from the capture support, facing neither the first nor the second capture surface of the at least one magnetic layer.

[0140] The area of ​​the at least one additional magnetic field source can be larger than the area of ​​the at least one magnetic layer that it faces. In this sense, when the at least one magnetic field source is positioned below the magnetic layer, its surface protrudes beyond either side of the magnetic layer. For example, when the additional magnetic field source is a magnet assembly, a portion of the surface of one or at least one magnet does not face the at least one magnetic layer, or at least one magnet does not have a surface facing the at least one magnetic layer.

[0141] In fact, in all cases, the significant effects brought about by the at least one magnetic field source are, on the one hand, the magnetization of the magnetic nanoparticles and, on the other hand, the increase in the amplitude of the energy dip, as described above.

[0142] Thus, the at least one additional magnetic field source is advantageously configured to generate a magnetic field of 1 mT to 400 mT at the nanoparticles when the kit is used. In order to avoid the risk of demagnetization of the magnetic layer, it is advantageous if the coercive field of the at least one additional magnetic field source has a value of at most 90% of the value of the coercive field of the at least one magnetic layer.

[0143] In the present invention, “1mT to 400mT” refers to 1mT, 5mT, 10mT, 15mT, 20mT, 25mT, 30mT, 35mT, 40mT, 45mT, 50mT, 55mT, 60mT, 65mT, 70mT, 75mT, 80mT, 85mT, 90mT, 95mT, 100mT, 105mT, 110mT, 115mT, 120mT, 125mT, 130mT, 135mT, 140mT, 145mT, 150mT, 155mT, 160mT, 165mT, 170mT, 175mT, 180mT, 185mT, 190mT, 195mT, 200mT T, 205mT, 210mT, 215mT, 220mT, 225mT, 230mT, 235mT, 240mT, 245mT, 250m T, 255mT, 260mT, 265mT, 270mT, 275mT, 280mT, 285mT, 290mT, 295mT, 300mT , 305mT, 310mT, 315mT, 320mT, 325mT, 330mT, 335mT, 340mT, 345mT, 350mT , 355mT, 360mT, 365mT, 370mT, 375mT, 380mT, 385mT, 390mT, 395mT, 400mT.

[0144] Advantageously, the at least one magnetic field source is configured to generate a magnetic field of 10 mT to 400 mT, more advantageously 50 mT to 200 mT.

[0145] According to an embodiment of the invention, the at least one additional magnetic field source is configured to emit the magnetic field continuously.

[0146] Alternatively, the at least one additional magnetic field source is configured to emit a pulsed magnetic field. Advantageously, the duration of the pulse is greater than or equal to 1 ms. Compared to simple Brownian motion, such a duration makes it possible to increase the magnetization of the magnetic nanoparticles over a sufficiently long period of time so that their displacement is affected thereby.

[0147] The invention also relates to a kit as defined above, wherein said at least one magnetic layer has a capture surface on which said at least one magnetic layer is at least partially covered by a non-magnetic layer.

[0148] When such a non-magnetic layer is present, the capture and immobilization of the nanoparticles occurs on the surface of the non-magnetic layer which is part of the at least one coated magnetic layer due to the fact that the so-called "capturing" surface of the at least one magnetic layer is coated.

[0149] The presence of this non-magnetic layer is advantageous since it makes it possible to delay triggering the attraction of the nanoparticles to the capture support at a desired moment, as will be seen in detail below.

[0150] According to one embodiment of the present invention, the material of the non-magnetic layer is selected from the following list: glass, silicon, polymer of plastic material, silicon paper, tape, non-magnetic metal alloy such as duralumin or titanium, quartz, organic material such as paper or bamboo, wood, gold or a combination of these materials.

[0151] Advantageously, the polymer is selected from the following list: polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), cyclic olefin polymer (COC / COP), polycarbonate, polyimide, polyvinyl chloride (PVC), polyethylene, polypropylene, silicone, polyester or a combination of these materials.

[0152] According to one embodiment, the non-magnetic layer has the same composition as the support member. Alternatively, the non-magnetic layer has a different composition than the support member.

[0153] Advantageously, the at least one magnetic field source does not exhibit fluorescence. Even more advantageously, it is opaque so as not to reflect light.

[0154] Advantageously, the material of the non-magnetic layer consists of or comprises an adhesive tape having a laminate of a polyvinyl chloride (PVC) layer and an adhesive layer or a polypropylene layer and an acrylic glue.

[0155] According to one embodiment of the invention, said at least one magnetic layer is covered by said non-magnetic layer over at least 1% of its capture surface. "At least 1%" is understood to mean 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111 , 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0156] Advantageously, said at least one magnetic layer has at least 30% of its capture surface covered, more advantageously at least 50% of its capture surface, even more advantageously at least 60% of its capture surface, advantageously at least 80% of its capture surface covered.

[0157] According to one embodiment of the present invention, the capture surface of the at least one magnetic layer is completely covered by the non-magnetic layer.

[0158] The invention also relates to a kit as defined above, wherein said non-magnetic layer has a thickness of 1 to 300 μm. In the present invention, “1 to 300 μm” is understood to mean 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35 μm, 36 μm, 37 μm, 38 μm, 39 μm, 40 μm, 41 μm, 42 μm, 43 μm, 44 μm, 45 μm, 46 μm, m, 47μm, 48μm, 49μm, 50μm, 51μm, 52μm, 53μm, 54μm, 55μm, 56μm, 57μm, 58μm m, 59μm, 60μm, 61μm, 62μm, 63μm, 64μm, 65μm, 66μm, 67μm, 68μm, 69μm, 70μm m, 71μm, 72μm, 73μm, 74μm, 75μm, 76μm, 77μm, 78μm, 79μm, 80μm, 81μm, 82μm m, 83μm, 84μm, 85μm, 86μm, 87μm, 88μm, 89μm, 90μm, 91μm, 92μm, 93μm, 94μm , 95μm, 96μm, 97μm, 98μm, 99μm, 100μm, 101μm, 102μm, 103μm, 104μm, 105μm m, 106μm, 107μm, 108μm, 109μm, 110μm, 111μm, 112μm, 113μm, 114μm, 115μm m, 116μm, 117μm, 118μm, 119μm, 120μm, 121μm, 122μm, 123μm, 124μm, 125μm m, 126μm, 127μm, 128μm, 129μm, 130μm, 131μm, 132μm, 133μm, 134μm, 135μm , 136μm, 137μm, 138μm, 139μm, 140μm, 141μm, 142μm, 143μm, 144μm, 145μm , 146μm, 147μm, 148μm, 149μm, 150μm, 151μm, 152μm, 153μm, 154μm, 155μm , 156μm, 157μm, 158μm, 159μm, 160μm, 161μm, 162μm, 163μm, 164μm, 165μm , 166μm, 167μm, 168μm, 169μm, 170μm, 171μm, 172μm, 173μm, 174μm, 175μm,176μm, 177μm, 178μm, 179μm, 180μm, 181μm, 182μm, 183μm, 184μm, 185μm, 186μm, 187μm, 188μm, 189μm, 190μm, 191 μm, 192μm, 193μm, 194μm, 195μm, 196μm, 197μm, 198μm, 199μm, 200μm, 201μm, 202μm, 203μm, 204μm, 205μm, 206μm, 2 07μm, 208μm, 209μm, 210μm, 211μm, 212μm, 213μm, 214μm, 215μm, 216μm, 217μm, 218μm, 219μm, 220μm, 221μm, 222μm , 223μm, 224μm, 225μm, 226μm, 227μm, 228μm, 229μm, 230μm, 231μm, 232μm, 233μm, 234μm, 235μm, 236μm, 237μm, 238 μm, 239μm, 240μm, 241μm, 242μm, 243μm, 244μm, 245μm, 246μm, 247μm, 248μm, 249μm, 250μm, 251μm, 252μm, 253μm, 254μm, 255μm, 256μm, 257μm, 258μm, 259μm, 260μm, 261μm, 262μm, 263μm, 264μm, 265μm, 266μm, 267μm, 268μm, 269μm m, 270μm, 271μm, 272μm, 273μm, 274μm, 275μm, 276μm, 277μm, 278μm, 279μm, 280μm, 281μm, 282μm, 283μm, 284μm, 28 5μm, 286μm, 287μm, 288μm, 289μm, 290μm, 291μm, 292μm, 293μm, 294μm, 295μm, 296μm, 297μm, 298μm, 299μm or 300μm. ,

[0159] Advantageously, the non-magnetic layer has a thickness ranging from 1 to 150 μm, more advantageously from 5 to 100 μm, even more advantageously from 10 to 80 μm, advantageously from 30 to 60 μm.

[0160] The non-magnetic layer should be neither too thin nor too thick. In fact, in case the non-magnetic layer is too thin, ie less than 1 μm, it has no significant effect on weakening the attraction of the at least one magnetic layer to the nanoparticles.

[0161] It should be noted that the thickness of the layer will depend on the retention of the at least one magnetic layer and the width of the first and second regions. A person skilled in the art will readily be able to adjust the thickness of the magnetic layer depending on the retention of the at least one magnetic layer and the width of the regions.

[0162] Typically, for a magnetic layer having a holding force of 12000 μm.G and a first and second region having a width of 50 μm, the non-magnetic layer will advantageously have a thickness of 20 to 60 μm. For magnetic layers with lower holding forces, the thickness of the magnetic layer should be reduced accordingly.

[0163] If the non-magnetic layer is too thick (depending on the thickness and width of the region of the at least one magnetic layer), the strong magnetic field gradient will be hidden by the non-magnetic layer. As a result, the magnetic field at the surface of the non-magnetic layer will be uniform or non-existent, depending on the situation. Then, the nanoparticles will not be fixed to the surface of the non-magnetic layer, or will not be fixed randomly in a specific pattern on the surface of the non-magnetic layer.

[0164] The application of the magnetic field of the at least one additional magnetic field source advantageously makes it possible to "reveal" the strong magnetic field gradient at the surface of the non-magnetic layer by increasing the amplitude of the energy depression. This aspect of the invention makes it possible to "trigger" the capture and immobilization of nanoparticles in the capture zone, as will be seen in detail below.

[0165] The invention also relates to a capture support comprising a number (1 to 100) of microfluidic channels having, on the one hand, an input common to at least some of the channels or an independent input for each channel and, on the other hand, a vent per channel or a vent common to at least some of the channels at the outlet. These microfluidic channels are bonded to a non-magnetic layer which in turn is deposited on a magnetic layer bonded to the support member. The magnetic layer is previously encoded with a magnetization along the horizontal plane of the magnetic layer and an orientation that varies by 180° from one area to another. A centimeter or millimeter magnet made of neodymium iron boron serves as an additional magnetic field source to apply an external magnetic field having the same orientation and direction as that of the magnetic layer at the junction between the first and second areas.

[0166] The present invention also relates to a method for capturing molecules contained in a sample, the method comprising the following steps:

[0167] a) contacting said sample with magnetic nanoparticles as defined above, thereby forming at least one capture complex between said molecule and said at least one capture element coupled to said magnetic nanoparticles;

[0168] b) attracting said at least one capture complex formed during step a) by means of a magnetic field generated by at least one magnetic layer of a capture support as defined above such that said at least one capture complex is immobilized on said capture support at at least one capture zone as defined above.

[0169] The purpose of step a) of the present invention is to complex the molecules to be captured with the nanoparticles via the capture element so that these complexed molecules can be indirectly attracted via the nanoparticles through the at least one magnetic layer in the subsequent step b) and preferably captured by being fixed on a support.

[0170] For the remainder of the specification, the capture complex may be referred to simply by the term "complex".

[0171] According to one embodiment of the present invention, the capture element is an antibody or an antigen, so that the at least one capture complex formed during step a) is an immune complex.

[0172] In the present invention, "sample" refers to any simple or complex fluid.

[0173] In the present invention, "complex fluid" refers to a mixture in which two phases coexist: solid-liquid (suspensions or solutions of macromolecules such as polymers), solid-gas (particles), liquid-gas (foam) or liquid-liquid (emulsions). Complex fluids deviate from the classical linear Newtonian relationship between stress and shear rate. They exhibit unusual mechanical responses to stresses or strains imposed by the geometric constraints imposed by the coexistence of phases. The mechanical response includes transitions between solid-like behavior and fluid-like behavior as well as fluctuations. In particular, the sample can be a biological fluid, such as blood, urine, lymph, plasma, serum, saliva, tears, semen, vaginal secretions, pus from wounds, gastric juice or cerebrospinal fluid. The sample can also be a culture medium used in a biological process, such as culture medium, or purified or clarified culture medium.

[0174] In the present invention, a "simple fluid" refers to a Newtonian fluid whose mechanical behavior is characterized by a single function of temperature, viscosity, and a measure of the fluid's "slippage." The stress applied to a simple fluid is proportional to the strain rate. In particular, the sample can be deionized water. Deionized water has the advantage of greatly limiting or even preventing the formation of nanoparticle clusters.

[0175] According to one embodiment of the invention, once the sample has been brought into contact with the nanoparticles during step a), it is placed on a capture support to allow attraction of said at least one capture complex during step b).

[0176] Thus, the contacting step and the attracting step are performed separately, so that the nanoparticles are not attracted by the at least one magnetic layer when in contact with the sample. This advantageously makes it possible to obtain a uniform distribution of the nanoparticles in the sample and thus a more efficient complexation of the capture element with the molecules to be captured.

[0177] Alternatively, the sample may first be placed on a support before contacting the sample with the nanoparticles in step a). Thus, steps a) and b) are performed at the same location, i.e., on the support. This embodiment is particularly interesting in certain applications, particularly because it does not require the handling of fluids via micropumps or microvalve-type actuators.

[0178] According to one embodiment of the invention, between step a) and step b), the mixture comprising the nanoparticle-molecule complexes in the suspension to be captured is subjected to an ultrasound treatment with the aim of destroying any aggregates of the complexes that may have formed. These aggregates do disrupt the quantitative step seen below. The ultrasound treatment can in particular be carried out at a frequency of at least 10,000 Hz. The ultrasound treatment can be continuous or pulsed. When the ultrasound treatment is pulsed, the duration of each ultrasound treatment can be 200-700 milliseconds, in particular 300-600 milliseconds, in particular 500 milliseconds, with an interval of 1-6 seconds, in particular 1-4 seconds, in particular 2 seconds. These ultrasound treatment conditions allow ensuring the function of destroying aggregates while avoiding overheating of the medium that could denature the capture element, the molecules to be captured and the detection element.

[0179] During step a), the concentration of the nanoparticles is advantageously 10 6 to 10 11 Particles / ml. In the present invention, "10 6 to 10 11 Particles / ml" refers to 10 6 Particles / ml, 10 7 Particles / ml, 10 8 Particles / ml, 10 9 Particles / ml, 10 10 Particles / ml and 10 11 Particles / ml.

[0180] 10 6 The minimum concentration of particles / ml provides sufficient concentration to effectively capture the molecules dispersed in the sample. 11The maximum concentration of particles / ml makes it possible to avoid excessive aggregation of nanoparticles (clusters with a diameter less than or equal to 15 μm), which on the one hand would destroy the bond between the capture element and the molecule to be captured and on the other hand would have a negative impact on the quantification of the captured molecules. A high concentration of nanoparticles would also be responsible for shielding the magnetic fields, i.e. weakening them, and would have a negative impact on the attraction of the nanoparticles. In the present invention, "clusters with a diameter less than or equal to 15 μm" means 15 μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 0.5 μm and 0.2 μm.

[0181] According to one embodiment, step a) further comprises contacting the sample with an element for detecting said molecule.

[0182] The detection element has a label, which can be fluorescent, luminescent or colored so as to be recognized by an appropriate detection device. The label can also be an enzyme with redox properties.

[0183] During step a), a so-called "sandwich" complex is formed consisting of the magnetic nanoparticle, the capture element, the molecule to be captured and the detection element. The capture molecule is then surrounded by the capture element and the detection element, "sandwiched".

[0184] The formation of these "sandwich" complexes, in which the detection element is attached to the molecule to be captured in the first step, is made possible by the good diffusion properties of the magnetic nanoparticles in the mixture. Therefore, such complexes are more difficult to form using magnetic microparticles.

[0185] In this embodiment, the above-mentioned sonication step can also be performed under the same conditions to avoid any aggregation of the "sandwich" complex.

[0186] Alternatively, the detection element can be placed in the presence of the molecule to be captured during step c), and the complex formed by the nanoparticle, capture element and capture molecule can then be fixed on the support. Thus, the so-called "sandwich" complex as described above is formed a posteriori during this step c).

[0187] Finally, at the end of step a), a mixture is obtained comprising a complex of nanoparticles, capture elements and molecules to be captured, or a complex of nanoparticles, capture elements, molecules to be captured and detection elements (if such detection elements are present). In this mixture, the molecules to be captured alone and the nanoparticles coupled alone to the capture elements, as well as nanoparticles optionally coupled alone to the detection elements, may remain.

[0188] The mixture can be deposited on the capture support, for example by pipetting, in order to immobilize the nanoparticles during step b).For example, in case the capture support is a chamber, the mixture can also be injected into the capture support.

[0189] Once the mixture is placed on the support, all magnetic nanoparticles (complexed or uncomplexed) will be attracted by the at least one magnetic layer and will rest on the support, more precisely on the capture surface of the at least one magnetic layer.

[0190] During their immobilization, the magnetic nanoparticles (complexed or uncomplexed) are not randomly distributed on the support, but more precisely on the capture surface of the at least one magnetic layer.

[0191] In fact, the nanoparticles are immobilized in the capture zones defined in the present invention. Consequently, only a small fraction of the nanoparticles (less than 15%) is immobilized outside these capture zones or is not captured.

[0192] The nanoparticles are thus distributed on the support, more precisely on the capture surface of the at least one magnetic layer, according to a specific pattern defined by all the connections of the first and second areas.

[0193] This distribution is very interesting because it makes it possible to determine the locations where the nanoparticles will be captured, which allows direct quantification of the captured molecules without washing steps.

[0194] The organization of the first and second areas of the at least one magnetic layer allows detection and quantification directly after immobilization of the capture complex and binding to the detection element or "sandwiching" the capture complex immobilization without washing the support.

[0195] To do this, one must first determine the amount of labeling in the capture zone and then determine the amount of labeling outside of the capture zone.

[0196] A person skilled in the art will be able to readily adapt the means for determining the amount of label to be implemented according to the type of label coupled to the detection element. In particular, these means may be selected from a spectrophotometer in scanning mode, an epifluorescence microscope, a confocal microscope, a two-photon microscope, measurements of the redox activity of an enzyme, and the like.

[0197] For example, in the context of fluorescent labeling, one skilled in the art will benefit from the use of a fluorescence microscope equipped with a "GFP" cube (excitation 460-490 nm) or a "PCR" cube (excitation 650 nm - emission 660 nm) coupled to a charge coupled device (CCD) or CMOS camera, as shown in the "Examples" section of this specification.

[0198] The amount of label outside the capture zone corresponds to the “background signal.” In other words, the label corresponds to the detection elements not coupled to the captured complexes and the small fraction of complexes immobilized outside the capture zone, as well as to the effects of the matrix (residual signal of the medium).

[0199] To obtain the amount of a particular label emitted in the capture zone, the amount of label obtained outside the capture zone is subtracted from the amount of label obtained in the capture zone.

[0200] The present invention also relates to a capture method as defined above, wherein the attraction of the at least one capture complex during step b) is performed by the combined action of a magnetic field generated by the at least one magnetic layer and a magnetic field generated by at least one additional magnetic field source as defined above.

[0201] As mentioned above, the at least one additional magnetic field source makes it possible to increase or even saturate the magnetization of the magnetic nanoparticles and also to increase the amplitude of the energy dip, thus accelerating the attraction of the nanoparticles by the at least one magnetic layer.

[0202] Furthermore, as mentioned above, the additional magnetic field makes it possible to enhance certain capture areas and thus promote local capture, which therefore facilitates detection without washing the capture molecules, as mentioned above.

[0203] This synergy also makes it possible to avoid the use of evaporating solvent from the sample in order to bring the nanoparticles closer to the capture support and thus accelerate their capture. Such evaporation would in fact require heating the sample or very long waiting times, which could negatively affect the binding between the capture element / detection element and the molecules to be captured and therefore the quantification of the molecules to be captured.

[0204] Thus, the attraction of the nanoparticles is achieved by the combined action of the at least one magnetic field source and the at least one magnetic layer, each magnetic layer playing a different role and function.

[0205] Due to its function and effect, the at least one magnetic field source may be placed anywhere relative to the capture support, as described above.

[0206] Thus, the at least one additional source may also be arranged as

[0207] - a capture surface facing said at least one magnetic and / or non-magnetic layer,

[0208] - facing an opposing surface or support member, or

[0209] - In any other position, such as sideways offset.

[0210] A significant magnetization of the nanoparticles is advantageously achieved by applying a magnetic field at the level of 1 mT to 400 mT, advantageously 10 mT to 400 mT, more advantageously 50 mT to 200 mT.

[0211] The action of the at least one additional magnetic field source may be performed throughout steps a) and b).

[0212] Advantageously, it is triggered during step b).Thus, during step a) and before triggering the action of said at least one additional magnetic field source, its magnetic field at the magnetic nanoparticles is insufficient or even zero.

[0213] The triggering of the action of the at least one additional field source during step b) is ensured by generating a magnetic field of 1 mT to 400 mT, advantageously 10 mT to 400 mT, more advantageously 50 mT to 200 mT at the magnetic nanoparticles.

[0214] In order to trigger the effect of the at least one additional magnetic field source, according to a first alternative, the latter can be brought closer to the capture support.For this embodiment, the at least one additional magnetic field source is advantageously a permanent magnet.

[0215] According to a second alternative, the triggering is achieved by passing an electric current through said additional magnetic field source.For this embodiment, said at least one additional magnetic field source is advantageously a coil or an electromagnet and the triggering is achieved by passing an electric current through said additional magnetic field source.

[0216] Advantageously, even if the action of the at least one additional magnetic field source is stopped at the end of step b), for example by moving the source away or by stopping the passage of current, the captured nanoparticles remain in their proper position on the capture support in the capture zone. Thus, the capture support can be moved to a suitable position for detecting the label of the detection element without affecting the position of the nanoparticles and thus without affecting direct detection without a washing step.

[0217] The present invention also relates to a capture method as defined above, wherein the capture support further comprises a non-magnetic layer as defined above, and wherein attraction of the at least one capture complex by the capture support during step b) is triggered by the magnetic field of the at least one additional magnetic field source.

[0218] In this embodiment, the presence of the non-magnetic layer reduces the likelihood of, or even prevents, the nanoparticles from being attracted solely by the magnetic field generated by the at least one magnetic layer.

[0219] Therefore, advantageously,

[0220] - on the one hand, the magnetic field generated by all of said at least one magnetic layer and said at least one additional source has at least one variation in its intensity of at least 0.1 mT at a distance of at least 1 μm from the capture surface of said non-magnetic layer, said at least one variation in its intensity defining a maximum and a minimum value of a magnetic field intensity criterion so as to define a capture zone of magnetic nanoparticles on the capture support at said maximum of said magnetic field criterion, and

[0221] - On the other hand, the magnetic field generated by the at least one magnetic layer does not exhibit at least one variation of its intensity of at least 0.1 mT at a distance of at least 1 μm from the surface of the non-magnetic layer, so that it is not possible for the magnetic field generated by the at least one magnetic layer alone to define a capture zone of magnetic nanoparticles on the surface of the non-magnetic layer.

[0222] The surface of the support corresponds to the surface on which the nanoparticles are immobilized.

[0223] The attraction of the magnetic nanoparticles during step b) is therefore dependent on the action of said at least one magnetic field source on these nanoparticles, that is to say by

[0224] - the magnetization of the particles is significantly increased by means of the magnetic field generated by this source, and / or

[0225] -The amplitude of the energy depression has been increased.

[0226] This aspect of the invention is very interesting because it allows optimizing the performance of steps a) and b) at the same location (i.e. at the capture support) by reducing or even eliminating the possibility that the action of the at least one magnetic layer alone could attract nanoparticles during step a).

[0227] Due to the presence of the non-magnetic layer, at least some of the nanoparticles (composite or uncomposite) will not be fixed to the at least one magnetic layer, but to the non-magnetic layer. More precisely, the non-magnetic layer has a capture surface and an opposite surface facing the at least one magnetic layer, at least some of the nanoparticles being fixed during step b) on the first capture surface of the non-magnetic layer.

[0228] The present invention also relates to a capture method as defined above, wherein the sample is placed on a capture support before step a) of contacting with the magnetic nanoparticles.

[0229] Advantageously, the action of the at least one magnetic field source is triggered during step b).

[0230] Thus, during step a), the magnetization of the magnetic nanoparticles and / or the energy dip of the at least one magnetic layer are not of sufficient magnitude to attract the nanoparticles through the action of the at least one magnetic layer covered only by the non-magnetic layer. As a result, the mixing of the nanoparticles with the sample is hardly disturbed, if at all, by the early attraction of the at least one additional magnetic source.

[0231] Surprisingly, the inventors have found that, despite the presence of the non-magnetic layer and its influence on the attractive force of the at least one magnetic layer towards the nanoparticles, the immobilized nanoparticles remain in position on the support in the capture zone if, at the end of step b), the action of the magnetic field generated by the at least one additional magnetic field source is stopped, for example by moving the source away or by stopping the current.

[0232] One explanation for this phenomenon is that the magnetic field gradient generated by the at least one magnetic layer and present on the surface of the non-magnetic layer is sufficient to hold the nanoparticles in place on the non-magnetic layer. Another explanation is that there is adsorption, i.e., chemical / physical bonding, between the magnetic nanoparticles that aggregate with each other on the one hand and the particles that are attracted to the capture surface of the non-magnetic layer on the other.

[0233] Here too, the capture support can thus be moved into a suitable position for detecting the label of the detection element without affecting the position of these particles and thus without affecting the direct detection without washing steps.

[0234] The present invention also relates to a capture method as defined above, comprising a subsequent step c) of transferring the magnetic nanoparticles that have been captured on the support to a recovery zone.

[0235] The transfer is performed using a technique called "connecting rod-crank" where:

[0236] - the crank is the magnetic field generated by the magnetic regions of said at least one magnetic layer; we denote this magnetic field as b(R) (R being a point in space). said at least one magnetic layer exhibits variations in the intensity of the magnetic field generated so as to produce a magnetic energy minimum,

[0237] The connecting rod is a spatially uniform magnetic field (in direction and in strength) generated by the at least one additional magnetic field source. In this application, the amplitude and direction of the magnetic field can be modified.

[0238] The "uniform" magnetic field generated by the connecting rod is applied over the entire area where the crank magnetic field is generated. Thus, the magnetic field generated by the connecting rod and the magnetic field generated by the at least one magnetic layer coexist spatially, such that their vector values ​​are linearly superimposed in a medium with a relative magnetic permeability equal to one, such as air or water.

[0239] Due to the fact that the magnetic field generated by the connecting rod can be modulated, it is possible to rotate it clockwise or counterclockwise by at least 1° about at least one axis of rotation and / or to amplify the magnetic field during step c). Thus, the magnetic field of the connecting rod can be temporarily modified. Therefore, we note the magnetic field B(t) of the connecting rod (t is time).

[0240] Therefore, the magnetic field of the crank varies in space but not in time, and the magnetic field of the connecting rod varies in time.

[0241] Therefore, in the area where the connecting rod and crank are applied, the total magnetic field S T is the vector sum of b and B, or B T (R,t)=b(R)+B(t).

[0242] In this way, the position of the capture zone of the magnetic nanoparticles on the support can be changed during step c).

[0243] In the present invention, “1° to 360°” refers to 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 11°, 12°, 13°, 14°, 15°, 16°, 17°, 18°, 19°, 20°, 21°, 22°, 23°, 24°, 25°, 26°, 27°, 28°, 29°, 30°, 31°, 32°, 33°, 34°, 35°, 36°, 37°, 38°, 39°, 40°, 41°, 42°, 43°, 44°, 45°, 46°, 47°, 48°, 49°, 50°, 51°, 52°, 53°, 54°, 55°, 56°, 57°, 58°, 59°, 60°, 61°, 62°, 63°, 64°, 65°, 66°, 67°, 68°, 69°, 70°, 71°, 72°, 73°, 74°, 75°, 76°, 77°, 78°, 79°, 80°, 81°, 82°, 83° 1°, 62°, 63°, 64°, 65°, 66°, 67°, 68°, 69°, 70°, 71°, 72°, 73°, 74°, 75°, 76°, 77°, 78°, 79°, 80°, 81°, 82°, 83°, 84°, 85°, 86°, 87°, 88°, 89°, 90°, 91°, 92 °, 93°, 94°, 95°, 96°, 97°, 98°, 99°, 100°, 101°, 102°, 103°, 104°, 105°, 106°, 107°, 108°, 109°, 110°, 111°, 112°, 113°, 114°, 115°, 116°, 117°, 118°, 119°, 120°, 121°, 122°, 123°, 124°, 125°, 126°, 127°, 128°, 129°, 130°, 131°, 132°, 133°, 134°, 135°, 136°, 137°, 138°, 139°, 140°, 141°, 142°, 143°, 144°, 145°, 146°, 147°, 148°, 149°, 150°, 151°, 152°, 153°, 154°, 155°, 156°, 157°, 158°, 159°, 160°, 161°, 162°, 163°, 164°, 165°, 166°, 167°, 168°, 169°, 170°, 171°, 172°, 173°, 174°, 175°, 176°, 177°, 178°, 179°, 180°, 181°, 182°, 183°, 184°, 185°, 186°, 187°, 188°, 189°, 190°, 191°, 192°, 193°, 194°, 195°, 196°, 197°, 198°, 199°, 200°, 201°, 202°, ​​203°, 204°, 205°, 206°, 207°, 208°, 209°, 210°, 211°, 212°, 213°, 214°, 215°, 216°, 217°, 218°,219°, 220°, 221°, 222°, 223°, 224°, 225°, 226°, 227°, 228°, 229°, 230°, 231°, 232°, 233°, 234°, 235°, 236°, 237°, 238°, 239°, 240°, 241°, 242°, 243°, 244°, 245°, 246°, 247°, 248°, 249°, 250°, 251°, 252°, 253°, 254° 4°, 255°, 256°, 257°, ​​258°, 259°, 260°, 261°, 262°, 263°, 264°, 265°, 266°, 267°, 268°, 269°, 270°, 271°, 272°, 273°, 274°, 275°, 276°, 277°, 278°, 279°, 280°, 281°, 282°, 283°, 284°, 285°, 286°, 287°, 288°, 289°, 290°, 291°, 292°, 293°, 294°, 295°, 296°, 297°, 298°, 299°, 300°, 301°, 302°, 303°, 304°, 305°, 306°, 307°, 308°, 309°, 310°, 311°, 312°, 313°, 314°, 315°, 316°, 317°, 318°, 319°, 320°, 321°, 322°, 323°, 324°, 325 5°, 326°, 327°, 328°, 329°, 330°, 331°, 332°, 333°, 334°, 335°, 336°, 337°, 338°, 339°, 340°, 341°, 342°, 343°, 344°, 345°, 346°, 347°, 348°, 349°, 350°, 351°, 352°, 353°, 354°, 355°, 356°, 357°, 358°, 359°, or 360°.

[0244] Then, a vector summation of the magnetic fields of the connecting rod and crank is obtained, which is different from the result obtained at the end of step b), and the position of the standard maximum value of the magnetic field strength generated by the connecting rod and crank assembly is shifted, so that the capture zone is offset relative to step b). As a result, the nanoparticles attracted by the generated magnetic field gradient move simultaneously until they are located at the new position of the capture zone.

[0245] Advantageously, step c) is repeated in the same direction of rotation until the nanoparticles, gradually displaced in the same direction, reach the recovery zone.

[0246] By repeating step c), a "conveyor belt" effect is obtained, in which the nanoparticles move in the same direction along the surface of the capture support towards a recovery zone. Since the molecules to be captured are complexed with the nanoparticles, they are also recovered in this recovery zone.

[0247] Finally, thanks to the invention, it is possible to recover the molecules to be captured without applying to the support a fluid flow that would displace all the molecules contained in the mixture.

[0248] The present invention also relates to the use of a kit as defined above for capturing molecules contained in a sample, advantageously for capturing and detecting molecules contained in a sample.

[0249] The invention also relates to the use of a magnetic layer for attracting nanoparticles having a maximum dimension of less than 1 μm, said magnetic layer comprising a possibly repeated juxtaposition of at least first and second regions, said first regions comprising magnetic particles polarized in a first direction and said second regions comprising magnetic particles that are unpolarized or polarized in a second direction different from the first polarization direction of the magnetic particles of the first regions, such that said one magnetic layer generates, at a distance of at least 1 μm from said at least one magnetic layer, a magnetic field having an intensity with at least one variation of its intensity of at least 0.1 mT, said at least one variation of its intensity defining a standard maximum and a minimum value of the intensity of said magnetic field, so as to define a capture zone for magnetic nanoparticles on said magnetic layer at said standard maximum of said magnetic field, and said nanoparticles are each coupled to at least one capture element of a molecule.

[0250] Another kit for capturing molecules contained in a sample is also described, comprising:

[0251] a) magnetic nanoparticles having a largest dimension less than 1 μm, each of said nanoparticles being coupled to at least one capture element, said at least one capture element specifically binding to said molecule, and

[0252] b) a support for capturing the magnetic nanoparticles, said support comprising or essentially consisting of at least one magnetic layer, said magnetic layer comprising a possibly repeated juxtaposition of at least first and second areas, said first areas comprising magnetic particles polarized in a first direction, and said second areas comprising magnetic particles that are unpolarized or polarized in a second direction different from the first polarization direction of the magnetic particles of the first areas, such that said at least one magnetic layer generates a magnetic field that does not exhibit at least one variation of its strength of at least 0.1 mT at a distance of at least 1 μm from said at least one magnetic layer,

[0253] c) at least one additional magnetic field source, such that the magnetic field generated by the assembly of said at least one magnetic layer and said at least one additional source exhibits at least one variation in its intensity of at least 0.1 mT at a distance of at least 1 μm from said at least one magnetic layer,

[0254] Said at least one variation of its intensity defines a normal maximum and a minimum value of the intensity of said magnetic field, so as to define a zone for capturing magnetic nanoparticles on a capture support at said normal maximum value of said magnetic field.

[0255] Advantageously, said additional source is external to said at least one magnetic layer.

[0256] Advantageously also, said at least one magnetic layer has a capture surface on which said at least one magnetic layer is at least partially covered by a non-magnetic layer, and the magnetic field generated by all of said at least one magnetic layer and said at least one additional source exhibits at least one variation in its intensity of at least 0.1 mT at a distance of at least 1 μm from the capture surface of said non-magnetic layer, said at least one variation in its intensity defining a standard maximum and a minimum value of the intensity of said magnetic field so as to define an area for capturing magnetic nanoparticles on the capture support at said standard maximum value of said magnetic field.

[0257] All features described above with respect to the capture support, the additional magnetic field source and with respect to the nanoparticles of the above kit apply mutatis mutandis to this kit.

[0258] The present invention also relates to a method for capturing molecules contained in a sample, said method comprising the steps of:

[0259] a) contacting said sample with magnetic nanoparticles as defined above, thereby forming at least one capture complex between said molecule and said at least one capture element coupled to said magnetic nanoparticles;

[0260] b) attracting the at least one capture complex formed during step a) by a magnetic field generated by an assembly of at least one magnetic layer of the capture support and at least one additional magnetic field source as defined above, such that the at least one capture complex is immobilized on the capture support at at least one capture zone as defined above.

[0261] The present invention also relates to the use of a kit as defined above for capturing molecules contained in a sample, advantageously for capturing and detecting molecules contained in a sample.

[0262] The invention finally relates to the use of an assembly comprising a magnetic layer and an additional field source for attracting nanoparticles having a maximum dimension of less than 1 μm, said magnetic layer comprising a possibly repeated juxtaposition of at least first and second regions, said first regions comprising magnetic particles polarized in a first direction and said second regions comprising magnetic particles that are non-polarized or polarized in a second direction different from the first polarization direction of the magnetic particles of said first regions, such that the magnetic field generated by all said magnetic layers and said additional sources exhibits at least one variation in its intensity of at least 0.1 mT at a distance of at least 1 μm from said at least one non-magnetic layer, said at least one variation in its intensity defining a standardized maximum and a minimum value of the intensity of said magnetic field so as to define a region for capturing magnetic nanoparticles on said magnetic layer at said standardized maximum of said magnetic field.

[0263] Advantageously, said nanoparticles are each coupled to at least one capture element for a molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0264] Figure 1 The capture of nanoparticles by a magnetic layer according to the present invention is shown. The capture system is shown in a cross-sectional view. The intensity of the magnetic field emitted by the magnetic layer is defined by a color code, the scale of which is presented to the right of the cross-sectional view (in T). Arrows in the magnetic layer indicate the polarization direction of the particles comprising the magnetic layer.

[0265] Figure 2 Shown Figure 1 A captured photograph of the system shown. In this photograph, the white dots represent nanoparticles.

[0266] Figure 3 The capture of nanoparticles by a magnetic layer covered with a nonmagnetic layer according to the present invention is shown. The capture system is shown in a cross-sectional view. The intensity of the magnetic field generated by the magnetic layer is defined by a color code, the scale of which is presented to the right of the cross-sectional view (in T). Arrows from the nanoparticles indicate the direction of movement of the nanoparticles attracted by the magnetic layer. Arrows in the magnetic layer indicate the polarization direction of the particles comprising the magnetic layer.

[0267] Figure 4 Shown Figure 3 A captured photograph of the system shown. In this photograph, the white dots represent nanoparticles.

[0268] Figure 5aThe capture of nanoparticles by a magnetic layer according to the present invention, which is covered by a non-magnetic layer, is shown, and the capture is carried out in the presence of a magnetic field from an additional source. The capture system is shown in a cross-sectional view. The direction of the magnetic field generated by the additional source is indicated by a white arrow with a black border above the cross-sectional view and is perpendicular to the magnetic layer. The intensity of the magnetic field generated by the magnetic layer is defined by a color code, the scale of which is presented to the right of the cross-sectional view (in T). The arrows from the nanoparticles indicate the direction of movement of the nanoparticles attracted by the magnetic layer. The arrows in the magnetic layer indicate the polarization direction of the particles constituting the magnetic layer.

[0269] Figure 5b The capture of nanoparticles by a magnetic layer according to the present invention, which layer is covered by a non-magnetic layer, and the capture is carried out in the presence of a magnetic field from an additional source. The capture system is shown in a cross-sectional view. The direction of the magnetic field generated by the additional source is shown by a white arrow with a black border above the cross-sectional view and is parallel to the magnetic layer. The strength of the total magnetic field (generated by the magnetic layer and the additional source) is defined by a color code, the scale of which is presented to the right of the cross-sectional view (in T). The arrows from the nanoparticles indicate the direction of movement of the nanoparticles attracted by the magnetic layer. The arrows in the magnetic layer indicate the polarization direction of the particles constituting the magnetic layer.

[0270] Figure 6 Shown Figure 5a A captured photograph of the system shown. In this photograph, the white dots represent nanoparticles.

[0271] Figure 7 Shown for Figures 1 to 3 The percentage of nanoparticles captured (X-axis) as a function of time (Y-axis) for each capture is shown in FIG. Time is expressed in minutes. Curve A corresponds to Figure 5a The capture kinetics shown, curve B corresponds to Figure 1 The capture kinetics shown, curve C corresponds to Figure 3 The capture kinetics are shown.

[0272] Figure 8 An anti-mouse detection capture antibody carrying a fluorescent dye coupled to a nanoparticle complex with an anti-mouse ovalbumin capture antibody grafted thereto is shown. This is also a nanoparticle grafted with ovalbumin.

[0273] Figure 9 It is a graph showing the calculated fluorescence amount (arbitrary unit) as a function of the anti-ovalbumin antibody concentration (μg / ml).

[0274] Figure 10 Shown Figure 8 Each white dot represents a composite image of Figure 8 The capture antibody (anti-mouse) carries the fluorescent dye. Figure 10 A, mouse anti-ovalbumin concentration of approximately 50 μg / ml was used; Figure 10 B, a concentration of approximately 25 μg / ml was used; for Figure 10 C, using a concentration of approximately 12.5 μg / ml; Figure 10 D, using a concentration of approximately 6.25 μg / ml; Figure 10 E is a negative control in which no anti-mouse ovalbumin antibody was used.

[0275] Figure 11 is a diagram showing the calibration curve of the DLGi5 garnet in a MOIF system of the MagView CMOS type. The diagram shows the polarization rotation (in degrees) of a light beam passing through the garnet as a function of the magnetic field strength (in Tesla).

[0276] Figure 12 A photograph of nanoparticles after being trapped in a microfluidic chamber is shown. White dots represent nanoparticles. Figure 12 A to 12E show the capture kinematics (A: 0 seconds; B: 10 seconds; C: 30 seconds; D: 80 seconds and E: 120 seconds).

[0277] Figure 13 A photograph showing nanoparticles trapped in a microfluidic chamber in the presence of an additional magnetic field source is shown. The white dots represent nanoparticles. Figure 13 A to 13F show the capture kinematics (A: 0 sec; B: 2 sec; C: 5 sec; D: 12 sec; E: 34 sec and F: 60 sec).

[0278] Figure 14 Shown Figure 12 The percentage of captured nanoparticles captured is shown (Y-axis) as a function of time (X-axis). Time is expressed in seconds.

[0279] Figure 15 This is a spectroscopic analysis showing the size of nanoparticles in solution. Size was determined using dynamic light scattering. The Y-axis shows the relative frequency of nanoparticles as a percentage, while the X-axis is a logarithmic scale showing the nanoparticle size in nanometers.

[0280] Examples

[0281] Example 1: Capturing Nanoparticles with Magnetic Strips

[0282] First, the magnetic stripe of the magnetic card was tested for its ability to capture nanoparticles.

[0283] Experimental plan

[0284] The nanoparticles used (Chemicell nanoscreenmag ARA 200 nm) had an average diameter of 200 nm and a mass of 1.25 g / cm 3 The nanoparticles were diluted to 1.1 x 10^ 12 Nanoparticles / g and 4.4x10 9 Levels of nanoparticles / ml.

[0285] To break up any nanoparticle aggregates, a SONIC RUPTOR 4000 sonicator was used to mix the nanoparticle solution diluted in deionized water (ddHO). Intermittent sonication was performed in a test tube at 20% of a total power of 400 W and an estimated frequency of approximately 20,000 Hz. Three ultrasonic pulses of 500 milliseconds duration were delivered every two seconds.

[0286] In order to verify that the nanoparticles to be captured were not in the form of nanoparticle clusters, the inventors measured the size of the nanoparticles in solution by "diffraction light scattering". Figure 15 Given in.

[0287] The capture support is a magnetic card consisting of a PVC support member and a magnetic stripe. The magnetic stripe consists of three magnetic layers arranged in the same plane and composed of a high-coercivity magnetic polymer. The support member and magnetic layers are assembled according to the ISO 7811 standard. An MSR605 encoder is used to encode each magnetic layer with a continuous "1" corresponding to 182 times the letter F in hexadecimal notation. This allows the magnetic field orientation to change by 180 degrees approximately every 55 μm.

[0288] The capture of nanoparticles was performed by depositing 5 μl of nanoparticle solution and depositing the droplet on the magnetic layer of the magnetic card.

[0289] Figure 1 A schematic diagram of this capture is shown. In this figure, nanoparticles 1 are in solution, and some 1' are attracted to the magnetic layer 3 of the magnetic stripe (indicated by the black arrows). The magnetic layer has a first region 5 and a second region 7 with opposite polarization of the particles (indicated by the large white arrows). The magnetic field generated by each region is represented by a small white arrow that follows an arc starting on one side of the region and ending on the other side. The direction of these arrows indicates the direction of the magnetic field generated. The strength of the field is indicated by the color scale. As described above, the magnetic field is generated by using COMSOL The magnetic field intensity was obtained using the finite element method performed with 5.0 modeling software. White indicates a strong magnetic field intensity. Above the magnetic layer 3, the magnetic field intensity generated by the first and second regions (5, 7) is visible, each region being approximately 100 μm wide, and the field exhibits variations. The presence of a maximum value of the magnetic field 9 intensity can be clearly seen above the connection between the first region 5 and the second region 7, and the magnetic field intensity is lower in each region (5, 7) itself. At each maximum value of the magnetic field 9 standard, a capture zone 11 is thus defined by an orthogonal projection onto the surface of the magnetic layer 3, and at this capture zone 11 the nanoparticles 1' will be immobilized.

[0290] Images were then captured using a fluorescence microscope (Olympus BX41M) equipped with a "GFP" cube (excitation 460-emission 490 nm) coupled to a CCD camera (Diagnostic Instruments SPOT RT monochrome digital camera). A blue excitation light source (460-490 nm) was used. Images were captured at a total magnification of 50x and a capture time of 3 seconds (gain 14 dB). Figure 2 An example of image capture can be seen in Figure 1. In this image, the white dots represent nanoparticles. The ordering of the captured nanoparticles can be clearly seen.

[0291] The percentage of nanoparticles captured by the magnetic strips was quantified by following the protocol described in the publication Fratzl et al., Soft Matter (14) 2671-2680 (2018). In short, the quantification was obtained by the ratio of the area covered by nanoparticles to the area not covered by nanoparticles.

[0292] The results of the capture kinetics are shown in Figure 7 and represented by curve B.

[0293] result

[0294] like Figure 7 As shown, the capture of magnetic nanoparticles was triggered immediately after depositing the droplet on the magnetic substrate and reached 40% within 2 minutes.

[0295] also, Figure 15 The results shown in show a single peak with suspended beads with an average diameter of 282 nm (9.5 nm standard deviation), corresponding to the diameter of a single nanoparticle. These results indicate that the nanoparticles are independent of each other and do not form clusters.

[0296] Example 2: Capture of Nanoparticles by a Magnetic Strip Covered with a Non-magnetic Layer

[0297] This example tests the capture of nanoparticles by a magnetic stripe of a magnetic card covered with a non-magnetic layer.

[0298] Experimental plan

[0299] The nanoparticles and capture supports used were identical to those used in Example 1, except that the magnetic strips were covered with a 60 μm thick self-adhesive layer of black polymer (vinyl).

[0300] The methods used to capture nanoparticles and the determination of the percentage of captured nanoparticles were the same as those of Example 1.

[0301] Figure 3 The captured image is shown in Figure 1. Figure 1 Furthermore, a non-magnetic layer 13 is arranged on the surface of the magnetic layer 3. Figure 3 As shown, only a portion of the magnetic field generated by magnetic layer 3 protrudes above the surface of non-magnetic layer 13, causing the maximum value of the magnetic field standard to be "hidden" by the non-magnetic layer. Consequently, at a distance of at least 1 μm from the capture support surface, the intensity of the magnetic field does not vary by at least 0.1 mT, and thus, no capture zone exists. Consequently, the nanoparticles are very weakly attracted, randomly immobilized on the magnetic strips, and primarily remain in solution.

[0302] The captured results are shown in the Figure 4 As can be seen in the figure, the white dots represent nanoparticles. Figure 2 The ordering visible in disappears in this figure.

[0303] The results of the capture kinetics are shown in Figure 7 and represented by curve C.

[0304] result

[0305] like Figure 7 As shown, the nanoparticles were captured very slowly by the magnetic strips of the card. After 10 minutes, the capture rate had only reached 15%.

[0306] Example 3: Capture of Nanoparticles by a Magnetic Strip Covered with a Non-Magnetic Layer in the Presence of an Additional Magnetic Field Source

[0307] This example tests the capture of nanoparticles by a magnetic stripe of a magnetic card covered with a non-magnetic layer in the presence of an additional magnetic field source.

[0308] Experimental plan

[0309] The nanoparticles and capture supports used were the same as those used in Example 2.

[0310] The additional magnetic field source was a head-to-tail assembly (vertical / horizontal magnetization) of parallelepiped (20×10×1 mm) NdFeB macromagnets (N35, 800 g adhesion) magnetized along a 1 mm axis.

[0311] The magnetic card is deposited on an additional magnetic field source such that the magnetic layer is not arranged opposite the source.

[0312] The methods used to capture nanoparticles and the determination of the percentage of captured nanoparticles were the same as those of Example 1.

[0313] Figure 5a and 5b A diagram of this capture is shown in . Figure 5a In FIG, an additional magnetic field source (not shown) emits a magnetic field perpendicular to the magnetic layer 3, which is indicated by a white arrow with a black border. Figure 5b In Figure 2, an additional magnetic field source (not shown) emits a magnetic field, indicated by a white arrow with a black border, parallel to the magnetic layer 3. These two figures make it possible to see the influence of the direction of the magnetic field generated by the additional field source on the position of the trapping zone.

[0314] These figures show Figure 1 and 3 These Figure 1 The above aspect clearly shows that the magnetic field intensity generated at the surface of the non-magnetic layer 13 is much stronger, and the standard maximum value of the magnetic field intensity is no longer hidden by the non-magnetic layer 13. Figure 1 Compared to what was originally shown in FIG, only every other standard maximum value of the magnetic field 9 strength exists, and therefore every other capture zone 11 exists. However, these maxima 9 have a greater value than originally shown in FIG. Figure 1 The intensities of those shown in .

[0315] exist Figure 5a In the case of an additional magnetic field perpendicular to the magnetic layer 3, the capture region 11 is located above the junction where the polarization of the adjacent regions (5, 7) is oriented. However, above the junction where the polarization of the adjacent regions is shifted away, the field magnitude is minimized.

[0316] Interestingly, in Figure 5b It is noted that in the case of an additional magnetic field direction parallel to the magnetic layer 3, the normal maximum of the capture zone 11 and the magnetic field 9 strength has shifted and is no longer arranged at the connection between the first and second regions (5, 7), but at the first region 5 itself.

[0317] The captured results are shown in the Figure 6 As can be seen in the figure, the white dots represent nanoparticles. Figure 4 different, Figure 6 A new ordering has appeared in Figure 2 Here, the nanoparticles form regularly arranged parallel bands, and we see that few nanoparticles exist outside these bands.

[0318] The results of the capture kinetics are shown in Figure 7 and represented by curve A.

[0319] result

[0320] In this example, the capture of magnetic nanoparticles was triggered immediately upon application of the external field, with capture reaching nearly 100% within 2 minutes.

[0321] By combining the present results with those of Example 2, it can be concluded that when the magnetic strips are covered by a non-magnetic layer, the immobilization and very rapid capture (2 minutes) of nanoparticles can be triggered by triggering the magnetic field of an additional source.

[0322] Example 4: Capture and Quantification of Capture Elements Coupled to Nanoparticles

[0323] Finally, several concentrations of anti-mouse ovalbumin antibodies coupled to magnetic nanoparticles (capture elements) were tested for detection and quantification.

[0324] Each measurement was performed by keeping the number of detection antibody (anti-mouse antibody) and total nanoparticles the same, but varying the amount of nanoparticles coupled to anti-mouse ovalbumin antibody (by supplementing the nanoparticles coupled to ovalbumin).

[0325] Experimental plan

[0326] The nanoparticles used (Carboxyl Adembeads 200 nm (reference number 02120 - Ademtech)) had a diameter of 200 nm and a mass of approximately 2.0 g / cm 3 The nanoparticles have a density of about 100 μmol / g, a saturation magnetization of about 40 emu / g, an iron oxide content of about 70% and a solid content of 30 mg / ml (3%). The nanoparticles are covered with COOH carboxyl functional groups with a density greater than 350 μmol / g.

[0327] The capture support is a magnetic card consisting of a PVC support member on which three coplanar magnetic layers composed of a high-coercivity magnetic polymer are placed. The support member and magnetic layers are assembled according to ISO 7811. As shown in Example 1, each magnetic layer is encoded with consecutive 1s using an MSR605 encoder.

[0328] The additional magnetic field source was a head-to-tail assembly of parallelepiped (20×10×1 mm) NdFeB macromagnets (N35, 800 g adhesion) magnetized along a 1 mm axis.

[0329] The capture elements were anti-ovalbumin (IgG) antibodies raised in mice. These antibodies were grafted onto the nanoparticles at a final concentration of 10 μg / ml to 50 μg / ml.

[0330] Grafting onto nanoparticles at a final concentration of 10 μg / ml was performed by the following protocol:

[0331] Activate 90 μg of nanoparticles with 25 μl of a solution containing EDC (10 mg / ml) and NHS (10 mg / ml) (i.e., 3 μl of Ademtech 30 mg / ml Ademtech nanoparticle stock solution);

[0332] Incubate at room temperature with agitation for 15 minutes.

[0333] Removal of the supernatant by capturing the nanoparticles using a centimeter magnet. The centimeter magnet is a neodymium cylinder, nickel-plated, 10 mm in diameter and 40 mm high.

[0334] Add 25 μl of anti-mouse ovalbumin antibody solution at a concentration of 1 mg / ml (i.e. 25 μg);

[0335] Incubate at room temperature with agitation for 2 hours;

[0336] Removal of the supernatant by capturing the nanoparticles using a centimeter magnet as described above; and

[0337] The nanoparticles were suspended in 50 μL of PBS-Tween 0.05%-BSA (1 mg / ml) solution. Nanoparticles functionalized with anti-ovalbumin antibodies were obtained at a potential concentration of 500 μg / ml.

[0338] The other part of the nanoparticles was grafted with ovalbumin (OVA). The transplantation was performed according to the same protocol as above, except that instead of adding 25 μl of anti-ovalbumin antibody solution, 25 μl of OVA was added at a concentration of 1 mg / ml (ie 25 μg).

[0339] The detection element is an antibody specific for mouse antibodies (and therefore for anti-ovalbumin antibodies) and is coupled to the Alexa488 fluorescent dye (maximum excitation = 490 nm; maximum emission = 525 nm) for detection.

[0340] Figure 8 Shown is an anti-mouse antibody 15 carrying a fluorescent dye 17 coupled to a nanoparticle complex 19 grafted with an anti-ovalbumin antibody 21. Also shown is a nanoparticle 19 grafted with OVA 23 for testing the specificity of the interaction between the antibodies.

[0341] Detection and quantification of anti-mouse ovalbumin antibodies were performed using the following protocol:

[0342] In a 0.5 ml tube, a mixture of 4.5 μg of nanoparticles pre-grafted with anti-ovalbumin antibodies and / or OVA, 2 μl of anti-mouse detection antibody (final concentration 1 μg / ml), and 20 μl of PBS;

[0343] Incubate in a 0.5 mL tube at room temperature for 15 minutes.

[0344] Take out 5μl and deposit a drop on the magnetic layer of the magnetic card; and

[0345] • Depositing the magnetic card onto the additional magnetic field source such that the PVC support member is arranged between the magnetic layer and the additional magnetic field source.

[0346] Five different conditions are met:

[0347] 1) 4.5 μg of nanoparticles grafted with anti-mouse ovalbumin antibodies (i.e., anti-ovalbumin antibodies at a concentration of approximately 50 μg / ml);

[0348] 2) 2.25 μg of nanoparticles grafted with anti-mouse ovalbumin antibodies and 2.25 μg of nanoparticles grafted with ovalbumin (i.e., anti-ovalbumin antibodies at a concentration of approximately 25 μg / ml);

[0349] 3) 1.125 μg of nanoparticles grafted with anti-mouse ovalbumin antibodies and 2.25 μg of nanoparticles grafted with ovalbumin (i.e., an anti-ovalbumin antibody concentration of approximately 12.5 μg / ml);

[0350] 4) 0.625 μg of nanoparticles grafted with anti-mouse ovalbumin antibodies and 2.25 μg of nanoparticles grafted with ovalbumin (i.e., anti-ovalbumin antibodies at a concentration of approximately 6.25 μg / ml);

[0351] 5) 4.5 μg of nanoparticles grafted to ovalbumin (i.e., zero concentration of anti-ovalbumin antibodies);

[0352] Then, use the fluorescence microscope (Olympus BX41M) that is equipped with " GFP " cube (exciting 460-490nm) that is coupled to CCD camera (Diagnostic Instruments SPOT RT monochrome digital camera) that the magnetic layer of magnetic card is carried out image capture.Use blue excitation light source (460-490nm).Capture image with the total magnification of 50x and 5 seconds capture time (gain 1).

[0353] Figure 10 The image obtained after capture is shown. It is clearly observed that the nanoparticles coupled to the mouse antibodies, which in turn are coupled to the anti-mouse antibodies, are captured in the form of a band along the capture zone. Figure 10A to 10E correspond to conditions 1) to 5) as described above, respectively. For conditions 1) to 5), the amount of anti-mouse antibody detected was decreasing, which is consistent with the amount of anti-ovalbumin antibody used in each condition. Condition 5 was a negative control because no capture antibody was present. These results are also consistent with Figure 9 The results obtained are consistent.

[0354] The fluorescence signal is quantified by calculating the corresponding area corresponding to the fluorescence peak on the capture area, from which the general "background signal" measured between the capture areas is subtracted. In fact, unlike Examples 1-3, where the nanoparticles are fluorescent, all the fluorescence detected here corresponds not only to the captured molecules (in this case, anti-ovalbumin antibodies), but also to the detection elements remaining in the solution. It is then necessary to subtract the fluorescence emitted by these "free" detection elements from the fluorescence emitted by the detection elements coupled to the capture molecules.

[0355] by Figure 10 Taking the capture as an example, the total fluorescence of the capture zone area is measured as a fluorescence band, from which the fluorescence measured between these areas is subtracted.

[0356] Fluorescence quantification was obtained in arbitrary units (AU)

[0357] The results are shown in Figure 9 middle.

[0358] result

[0359] like Figure 9 As shown, the fluorescence quantification of the capture zone obtained is proportional to the concentration of anti-ovalbumin antibody added to the mixture (R 2 =0.97).

[0360] Based on the fluorescence signal quantification method used (specific signal in the capture zone and nonspecific signal outside it), these results allow to conclude that the nanoparticles coupled to the detection element are indeed captured in the capture zone.

[0361] However, these results allow the conclusion that the number of captured molecules can be quantified without a washing step between the immobilization of the nanoparticles and the detection by the detection element.

[0362] Example 5: Capturing Nanoparticles in Microfluidic Chambers

[0363] The nanoparticles used were the same as those used in Example 1 and were diluted 500-fold in deionized water (ddH2O) to achieve a concentration of 50 μg / mL.

[0364] A sonication step was also performed.

[0365] The capture support comprises 18 microfluidic chambers, each with an independent inlet and a vent at the outlet. Each microfluidic chamber is 6 mm long, 2.4 mm wide, and 240 microns deep. The chambers are arranged adjacent to each other at a spacing of 4.5 mm to form a strip. The microfluidic chambers are bonded to a magnetic layer, which is in turn bonded to a PVC support member. The support member and magnetic layer are assembled according to the ISO 7811 standard. An MSR605 encoder is used to encode the magnetic layer with consecutive "1s" corresponding to 182 times the letter F in hexadecimal, which causes the magnetic field orientation to change by 180 degrees approximately every 55 μm.

[0366] 6 microliters of nanoparticle solution were injected into each of these microfluidic chambers. The chambers were filled one by one. After each filling, the capture was observed using an epifluorescence microscope with a 10x magnification objective. Film was produced at a frame rate of 1.12 frames per second. Images obtained at recording times 0, 10, 30, 80, and 120 seconds were recorded. Figure 12 Shown in.

[0367] result

[0368] Although the depth of the microfluidic chamber is quite large, close to 300 μm, Figure 12 As can be seen in FIG, from 10 s of capture onwards, the nanoparticles are well trapped at the bottom of the microfluidic chamber (group of aligned white dots).

[0369] Example 6: Capture of Nanoparticles in a Microfluidic Chamber in the Presence of an Additional Magnetic Field Source

[0370] The nanoparticles and capture supports used were the same as in Example 5.

[0371] Furthermore, the capture support is based on a head-to-tail assembly of 20NdFeB macromagnets (Supermagnete, reference Q-10-04-02-N) parallelepiped (10×4×2 mm) magnetized along a 2 mm axis and having an energy product of 50 mega-Gauss-Oersted (perpendicular magnetization). Twenty magnets are arranged side by side along a 4 mm axis with a spacing of 0.5 mm, forming a strip. 18 of the 20 magnets are placed below each of the 18 microfluidic chambers, and 2 are placed on each side.

[0372] 6 microliters of nanoparticle solution were injected into each of these microfluidic chambers. The chambers were filled one by one. After each filling, the capture was observed using an epifluorescence microscope with a 10x magnification objective. The film was generated by capturing images at a rate of 1.12 frames per second. Images were obtained at time 0, 2, 5, 12, 34, and 60 seconds. Figure 13 Shown in.

[0373] The percentage of nanoparticles captured by the microfluidic chamber was quantified by following the protocol described in the publication Fratzl et al., Soft Matter (14) 2671-2680 (2018). Capture kinetics were performed in triplicate in 3 different chambers. Figure 14 Results of the capture kinetics are shown.

[0374] result

[0375] For Example 5, the nanoparticles were well captured at the bottom of the microfluidic chamber. Similar to Examples 1 and 3, in the absence of an external magnetic field source, the capture of the nanoparticles was much faster than that of Example 5. Figure 13 As shown, due to the external magnetic field generated by the macro magnet assembly, Figure 12 Every other capture zone shown disappears ( Figure 13 Double the spacing between rows of dots).

[0376] Figure 14 The capture kinetic data shown demonstrates complete capture of the nanoparticles after 15 seconds.

Claims

1. A kit for capturing molecules contained in a sample, comprising: a) magnetic nanoparticles having a largest dimension of less than 1 μm, said nanoparticles being each coupled to at least one capture element, said at least one capture element specifically binding to said molecule, and b) a support for capturing the magnetic nanoparticles on its surface, wherein the support comprises at least one magnetic layer, - the at least one magnetic layer is a flexible tape comprising a magnetic composite material randomly distributed or oriented along a pre-oriented axis in a polymer, at least part of the magnetic composite material being polarized, - the magnetic layer comprises a juxtaposition of first and second regions, the first region comprising magnetic particles polarized in a first direction and the second region comprising magnetic particles polarized in a second direction different from the first polarization direction of the magnetic particles of the first region, the first and second directions being opposite to each other and parallel to the surface of the support, - causing said at least one magnetic layer to generate a magnetic field having at least one intensity variation of at least 0.1 mT at a distance of at least 1 μm from said at least one magnetic layer, said at least one intensity variation defining a normalized maximum and a normalized minimum value of the intensity of said magnetic field, so as to define an area for capturing magnetic nanoparticles on a capture support at said normalized maximum value of said magnetic field, and - The at least one magnetic layer has a holding force of 2,000 to 30,000 μm.Gauss.

2. The kit according to claim 1, wherein The magnetic layer comprises a repeated juxtaposition of first and second regions.

3. The kit of claim 1, further comprising at least one additional magnetic field source.

4. The kit according to any one of claims 1 to 3, wherein the at least one magnetic layer has a capture surface on which the at least one magnetic layer is at least partially covered by a non-magnetic layer. 5 . The kit according to claim 4 , wherein the non-magnetic layer has a thickness of 1 to 300 μm.

6. A method for capturing molecules contained in a sample, the method comprising the steps of: a) contacting the sample with the magnetic nanoparticles according to claim 1 , thereby forming at least one capture complex between the molecule and the at least one capture element coupled to the magnetic nanoparticles; b) attracting the at least one capture complex formed during step a) by means of a magnetic field generated by at least one magnetic layer of the capture support according to any one of claims 1 to 5, such that the at least one capture complex is immobilized on the capture support at at least one capture zone according to claim 1.

7. The method according to claim 6, wherein the attraction of the at least one capture complex during step b) is performed by the combined action of a magnetic field generated by the at least one magnetic layer and a magnetic field generated by the at least one additional magnetic field source according to claim 3.

8. The method of claim 7, wherein the capture support further comprises a non-magnetic layer, and wherein the attraction of the at least one capture complex by the capture support during step b) is triggered by the magnetic field of the at least one additional magnetic field source.

9. The method of claim 8, wherein the sample is placed on the capture support prior to step a) of contacting with the magnetic nanoparticles.

10. Use of the kit according to any one of claims 1 to 5 for capturing molecules contained in a sample.

11. The use according to claim 10, wherein the method is used for capturing and detecting molecules contained in a sample.

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