Kits and methods for capturing molecules using magnetic means - Patents.com

Abstract

The present invention relates to a kit and method for capturing molecules contained in a sample by at least one magnetic layer comprising a repeatable juxtaposition of at least one first and second region, wherein the first region contains magnetic particles polarized in a first direction and the second region contains magnetic particles that are either unpolarized or polarized in a second direction different from the first polarization direction of the magnetic particles in the first region, and generates a magnetic field having at least one fluctuation of intensity of at least 0.1 mT at a distance of at least 1 μm from the at least one magnetic layer, the fluctuation defining the maximum level of intensity of the magnetic field and therewith the level for capturing magnetic nanoparticles on a capture support.

Description

[Technical field]

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

[0002] ELISA tests (acronyms for "Enzyme-Linked ImmunoSorbent Assay") are generally used to quantitatively diagnose molecular markers (antigens, antibodies, etc.) present in fluids, biopsies, cultures, or any other sample.

[0003] However, although this technique is currently one of the most robust and widespread diagnostic methods, it has the drawback of being complex, requiring expensive automata and taking several hours to complete.

[0004] The ELISA test is a heterogeneous phase immunoassay technique, i.e., it requires a solid support (usually a titer plate containing multiple wells) to which a suitable molecule has been pre-bound in order to capture the molecule to be assayed.

[0005] Once the molecule of interest has been captured on the support, washing allows the remainder of the sample to be removed, allowing the molecule to proceed to detection and quantification steps.

[0006] For example, in the case of the so-called "sandwich-type" ELISA test, which makes it possible to assay an antigen in solution, the surface of the support is covered with a defined amount of a so-called capture antibody, which is suitable for binding to the desired antigen.

[0007] A solution that may contain the antigen is then applied to the support; the antigen 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 solution. A solution containing an antibody, called the detection antibody, bound to a detection means is then deposited on the support, where the detection means is adapted to bind to the antigen immobilized on the support. The detection antibody can be directly marked and emit a detectable signal, but can also be bound to an enzyme that catalyzes a substrate to emit a detectable signal.

[0009] By adding an additional washing step, the antigen bound to the detection antibody is retained on the support, and the antibody itself is bound to an enzyme.

[0010] Finally, to detect and quantify the antigen, a substrate is deposited on the support, which is converted by the enzyme into a detectable signal (e.g., a color that is analyzed spectroscopically or by fluorescence) that represents the binding between the antigen and the detection antibody.

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

[0012] Non-Patent Document 1 describes a method for capturing molecules in a sample by circulating the sample in a fluid microchannel placed under a polydimethylsiloxane (PDMS) matrix to which NdFeB magnetic particles are immobilized.

[0013] Patent document 1 describes a method for capturing molecules in a sample, the method comprising the steps of mixing the sample with magnetic particles, each of which binds to an element capable of selectively binding to the molecule to be captured to form at least one complex comprising the magnetic particles, the element, and the molecule bound to the element, and immobilizing the at least one complex on a support comprising a regular magnetic field microsource.

[0014] These ordered magnetic field microsources are distributed near the surface of the support intended to be in contact with the sample according to a predetermined pattern and have a predetermined magnetic orientation.

[0015] The method described in this document is interesting, however, the capture support is difficult to manufacture and industrialize, especially in a clean environment, which means high production costs. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] International Publication No. 2014 / 111187 [Non-patent literature]

[0017] [Non-Patent Document 1] D. Issadore et al., "Lab Chip", 2011, Volume 11, p. 147 Summary of the Invention [Problem to be solved by the invention]

[0018] The aim of the present invention is, inter alia, to overcome these drawbacks of the prior art.

[0019] More precisely, it is an object of the present invention to provide a kit and a method for capturing molecules contained in a sample by high performance magnetic means, which has a reduced economic footprint, compatible with low cost production techniques. [Means for solving the problem]

[0020] Thus, the present invention relates to a kit for capturing a molecule contained in a sample, the kit comprising: a) magnetic nanoparticles having a maximum dimension of less than 1 μm, each of the nanoparticles being bound to at least one capture element, the at least one capture element specifically binding to the molecule; b) a support for capturing said magnetic nanoparticles comprising or consisting essentially of at least one magnetic layer, said magnetic layer comprising a repeatable juxtaposition of at least first and second regions, said first regions comprising magnetic particles polarised in a first direction and said second regions comprising magnetic particles that are not polarised or are polarised in a second direction different from the first polarization direction of the magnetic particles of the first region, whereby said at least one magnetic layer generates a magnetic field having at least one variation in intensity 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 intensity of said magnetic field defining a maximum and a minimum of a level of intensity of said magnetic field and defining at said maximum of the level of said magnetic field a zone for capturing magnetic nanoparticles on the capture support; Includes.

[0021] The inventors have discovered that, contrary to all expectations, it is possible to attract nanoparticles bound to capture elements using a magnetic layer having magnetic particles with weak magnetic properties. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The magnetic layer used in the present invention is flexible and particularly compatible with magnetic tapes. The magnetic layer of the present invention is composed of a magnetic composite material such as ferrite randomly distributed in a polymer or otherwise oriented along a pre-orientation axis. Ferrite is a ferromagnetic ceramic obtained by molding iron oxide Fe2O3XO at high pressure and temperature (above 1000°C), where X can be manganese, zinc, cobalt, nickel, barium, strontium, etc.

[0023] The present invention therefore consists in repurposing the use of magnetic tapes, commonly used for robust information storage devices (audio and video cassettes, credit cards, badges, transport tickets, etc.), which are difficult to demagnetize, for application to the capture of nanometric magnetic particles in solution.

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

[0025] The magnetic layers of the present invention are "encoded," i.e., at least a portion of the magnetic grains that compose them are polarized / magnetized. Hereinafter, the terms "polarization" and "magnetization" are synonymous and will be used interchangeably.

[0026] This encoding (or this polarization) is not performed randomly, but is configured to reveal at least one juxtaposition of a first region containing magnetic grains polarized in a first direction with a second region containing magnetic grains that are not polarized or are polarized in a second direction different from the first polarization direction of the magnetic grains, thereby defining at least one junction between the first and second regions. Each region thus emits its own magnetic field (when polarized), and as a result the magnetic layer can be modeled as multiple magnetic field sources.

[0027] The polarization of the magnetic grains constituting said at least one magnetic layer is carried out in particular by means of a write head, which is well known in the field of magnetic tape encoding. Typically, a local magnetic field is applied to a region of the magnetic layer by means of a small electromagnet.

[0028] This particular juxtaposition of the first and second polarized regions allows for a variation in the intensity of the generated magnetic field at a distance of at least 1 μm from the at least one magnetic layer, thereby resulting in maxima and minima in the level of the intensity of the magnetic field. The level of the intensity of the magnetic field corresponds to the absolute value of the intensity of the magnetic field in Tesla. In the present invention, the terms "level of intensity" and "level" can be used instead. The maximum of the level of the intensity of the magnetic field attracts the suspended nanoparticles, which generate zones where their magnetic energy is minimized, so that they are called "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 level defines the trapping zone of the nanoparticles. The trapping zone and the energy sink therefore coincide at the same location.

[0030] These capture zones extend over a distance of up to 35 μm from the orthogonal projection on the surface of the magnetic layer of the maximum or each maximum of the level of magnetic field strength, where "up to 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] The magnetic energy (E) of a nanoparticle is determined by the magnetic field generated by the at least one magnetic layer according to the following formula:

[0032]

number

[0033] Nanoparticles by

[0034]

number

[0035] is equal to the inverse of the dot product of the magnetizations.

[0036]

number

[0037] where E is the magnetic energy of the nanoparticle (unit: Joules); M is the magnetization of the nanoparticle in amperes per meter, B is the strength of the magnetic field in tesla.

[0038] For the magnetic materials used in the present invention, the magnetization is a strictly increasing function of the strength of the magnetic field, so that the minimum of the magnetic energy of the nanoparticles corresponds to the maximum of the magnetic field level and therefore to the capture zone.

[0039] When the magnetic nanoparticles are magnetized by the single magnetic field generated by the at least one magnetic layer, a capture zone is located at the junction of the first and second regions.

[0040] The magnetic field generated by the at least one magnetic layer exhibits a fluctuation in intensity of at least 0.1 mT and at most 1 T, advantageously at least 0.1 mT and at most 500 mT, more advantageously at least 0.5 mT and at most 300 mT, and even more advantageously at least 1 mT and at most 200 mT.

[0041] These variations in the strength of the magnetic field make it possible to generate a strong magnetic field gradient, i.e. a magnetic field gradient sufficient to exert a significant trapping force on the Brownian motion of the nanoparticles. Such a magnetic field gradient is therefore localized. Moreover, the gradient refers to a trapping zone, which is at least 10 Tm at a distance of 10 μm from the at least one magnetic layer. -1 , advantageously 10 T m -1 ~10 5 T m -1 , and even more advantageously 500 T m -1 ~5×10 3 T m -1 In this way, the strong magnetic field gradient guides the suspended nanoparticles towards the capture zone of the at least one magnetic layer.

[0042] Once the magnetic nanoparticles are captured by the at least one magnetic layer, they are positioned in the or each capture zone, this particular positioning being of great interest for direct detection and quantification of the captured molecules, as will be explained in more detail below.

[0043] The strength of the magnetic field can be measured by a magneto-optical technique called MOIF (Magneto-Optical Imaging Film).

[0044] The MOIF technique is based on the Faraday effect. In general, it consists of immersing a flat membrane made of a material whose optical properties are affected in a known way by the magnetic field in the magnetic field of the object whose intensity is to be measured. Usually, the membrane is attached to the object. The flat membrane has a width and length at least equal to the width and length of the zone of the test object. Following this first step, the flat membrane is illuminated by a beam of light of known amplitude and polarization, which passes through the flat membrane. Analysis of the polarization and the amplitude of the light beam that has passed through the flat membrane gives a measurement of the planar component of the magnetic field present therein. An exemplary measurement of the intensity of the magnetic field of an object by the MOIF technique is given by the paper Grechishkin et al., J. Appl. Phys.120,174502 (2016).

[0045] In practice, a thin planar film, the thickness of which is typically less than 1 micrometer and which is composed of a magneto-optical material (for example, a rare earth garnet), is deposited on a transparent non-magnetic substrate (for example, glass, quartz, or silica) and then covered with a very thin reflective layer, a mirror (for example, made of gold, silver, or aluminum), the thickness of which is less than 100 nm. Thus, the film of the magneto-optical material compound is covered on one of its faces with a transparent non-magnetic substrate and on the other face with a reflective layer. This assembly is attached to an object that emits a magnetic field, such as a capture support according to the invention. The film of magneto-optical material is then illuminated with a beam of polarized light that first passes through a transparent non-magnetic layer (whose optical volume is not affected by the magnetic field of the magnetic object and therefore does not enter into the polarization of the beam), then passes through the film of optical material (where the magnetic field generated by the magnetic object affects its optical properties and therefore enters into the polarization of the beam), is reflected by a reflecting layer, passes again through the film of optical material (which again affects the polarization of the beam), then passes again through the glass (which does not affect the polarization of the beam) and ends up in a polarization analyzer. The rotation angle of the polarization of the reflected beam with respect to the incident beam is proportional to the magnetic field, the Faraday rotation coefficient of the garnet and the thickness of the magneto-optical material. The distribution of the intensity of the magnetic field generated by the at least one magnetic layer is therefore obtained by a calibration curve that represents the rotation due to the Faraday effect of the light beam as a function of the intensity of the magnetic field. This curve is specific to the magneto-optical material used.

[0046] In particular, the strength of the magnetic field can be measured by the MOIF technique using the MagView CMOS system marketed by MATESY GmbH, using DLGi5 type garnet as the film of magneto-optical material and a model C sensor as the polarization analyzer, the calibration curve of which is shown in Figure 11. In this device, the mirror is replaced by a CMOS sensor so that there is no need to reflect light.

[0047] The results can be confirmed by numerical and analytical simulations, for example, by one of the following two approaches: - a finite element approach carried out using the COMSOL Multiphysics® 5.0 modelling software; this software allows the numerical simulation to be carried out in a two-dimensional environment with a thickness of 10 mm; upstream, the thickness and width of said at least one magnetic layer are measured optically, typically using bright field microscope images, on the one hand, and its afterglow value or effect, on the other hand; see below for more details; these data are entered into the software and the magnetic field generated by said at least one magnetic layer is simulated in steady state using the MFNC (Magnetic Field No Current) toolbox, - the so-called semi-analytical approach; this is based, for example, on the approach developed in the paper by Chigirinsky S. et al, Advanced Study Center Co. Ltd., 20 (2009), 85-91; here too, upstream, the thickness and width of the at least one magnetic layer are measured on the one hand, and the remanence or coercivity 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 an analytical decomposition of the equation giving the magnetic field of each element is carried out, for example, using the Scilab® 6.02 software (publisher Scilab Enterprises); the magnetic field generated by each element of the at least one magnetic layer is added to the magnetic field generated by all the other elements of the at least one magnetic layer at each point in space; if at least one additional magnetic field source is also present (see below in more detail), the magnetic field generated by the at least one additional magnetic field 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 present invention, the second region comprises magnetic grains polarized in a second, different direction offset by at least 30°, advantageously offset by 30°-180°, with respect to the first polarization direction of the magnetic grains of the first region. In the present invention, "30°-180°" means 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°, 102°, 104°, 105°, 106°, 107°, 108°, 109°, 109°, 108°, 109°, 109°, 102°, 10 °, 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°, 147 152°, 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 includes magnetic grains polarized in a second, different direction that is at least 60° offset, more advantageously at least 90° offset, even more advantageously at least 120° offset, and even more advantageously at least 150° offset.

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

[0051] According to another embodiment, the magnetic grains of the second region are not polarized. Such a configuration also allows for the occurrence of variations in the strength of the magnetic field, thereby allowing for a maximum level of strength of the magnetic field generated by the at least one magnetic layer.

[0052] According to one embodiment of the invention, the at least one first region and the at least one second region have the same dimensions, or they have different dimensions, 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 in the range of 10 to 500 μm. In the present invention, "10 to 500 μm" means 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, 250 μm, , 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm.

[0054] Advantageously, said at least one first region and / or said at least one second region have a width in the range of 50 to 250 μm, more advantageously of 70 to 150 μm, even more advantageously of 90 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, which may in particular correspond to a tape, or they represent different patterns.

[0056] According to one embodiment, the at least one magnetic layer is coated with a protective film having a thickness of less than 1 μm. Such a film advantageously makes it possible to protect the at least one magnetic layer, but due to its very small thickness it cannot hinder its capture / attraction capabilities.

[0057] The at least one magnetic layer may itself constitute a capture support, which advantageously has a thickness of at least 5 μm, more advantageously between 10 and 20 μm.

[0058] The magnetic layer is advantageously disposed on a support member.

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

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

[0061] The support member can be a single layer of the materials listed above.

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

[0063] According to one embodiment of the present invention, the capture support comprises a capture receptor configured to receive a sample containing capture molecules and to be bounded by at least one wall comprising said at least one magnetic layer. The capture receptor has a minimum dimension of 20 μm to 1000 μm. In the present invention, "20 μm to 1000 μm" means 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, 50 ... μm, 520 μm, 540 μm, 560 μm, 580 μm, 600 μm, 620 μm, 640 μm, 660 μm, 680 μm, 700 μm, 720 μm, 740 μm, 760 μm, 780 μm, 800 μm, 820 μm, 840 μm, 860 μm, 880 μm, 900 μm, 920 μm, 940 μm, 960 μm, 980 μm and 1000 μm.

[0064] According to one embodiment of the invention, the capture support is selected from among a chamber, a parallelepiped chamber, a hollow right cylinder, a well, a right circular cone, in particular a well in the form of a truncated right circular cone or a truncated pyramid, a microfluidic channel, a titration plate, a test tube and a microtube.

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

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

[0067] In the case of a well, the at least one magnetic layer is arranged on one wall of the well, and if there are several magnetic layers, on at least one wall of the well, advantageously on the wall forming the bottom of the well.

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

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

[0070] In the case of a titration plate containing a plurality of wells, the at least one magnetic layer is arranged on one wall of the at least one well, and if there are a plurality of magnetic layers, on at least one wall of the at least one well. Advantageously, the at least one magnetic layer is arranged on the 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 there are several magnetic layers, on at least one wall of the test tube or microtube, advantageously on the circumferential wall of the test tube or microtube.

[0072] Said at least one magnetic layer can be fixed to the support. Advantageously, said fixing is irreversible. In this case, said fixing can be carried out, for example, by gluing, rolling or stamping. Alternatively, said fixing is reversible. Thus, said at least one magnetic layer can be fixed by a hook and loop system, more commonly called a Velcro system, or by a reversible adhesive, such as an animal-derived adhesive.

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

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

[0075] According to one embodiment of the invention, the capture support comprises at least two magnetic layers, advantageously arranged in the same plane.

[0076] Alternatively, the magnetic layers are arranged on different planes and the two or at least two layers are superimposed on one another.

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

[0078] The use of the magnetic layer of the invention to capture nanometric magnetic particles is generally counterintuitive insofar as these nanoparticles are captured by magnetic layers with strong magnetic properties, for example magnetic layers made of rare earth-based alloys. Indeed, it is customary to use magnetic layers with strong magnetic properties to capture nanoparticles that inherently exhibit weak magnetization due to their small volume. Examples of such "ferro" magnetic layers based on rare earths are described in particular in document WO2014 / 111187. The remanence of the ferromagnetic layer is between 0.7T and 1.5T, and the coercive field is between 600kA / m and 2400kA / m. 3 It is.

[0079] The at least one magnetic layer according to the invention exhibits 5 to 15 times lower values ​​of apparent remanence and coercivity. The coercivity of the at least one magnetic layer is between 10 and 400 kA / m 3 The range is.

[0080] "Apparent remanence" refers to the remanence of the at least one magnetic layer as a whole, and not of each of the magnetic grains that make it up.

[0081] The coercive field of a ferromagnetic material indicates the strength of the magnetic field that must be applied to the material, once it has initially reached its magnetization in a saturated state, in order to cancel the magnetization of the material.

[0082] Remanence is a material intensive variable that measures the induction or density of magnetic flux that persists in a ferromagnetic material after it has been magnetized with a strong external magnetic field. Remanence is measured in Tesla (T). A sample of a pre-magnetized 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 quantity that allows one to characterize the strength of the magnetic source. The magnetic flux produced by this sample is proportional to its moment. This magnetic flux can be measured with a vibrating sample magnetometer (VSM), or an extraction magnetometer, or a SQUID magnetometer. Usually, a sufficiently strong magnetic field (typically 4-6 Tesla) is applied to the sample along its preferred magnetization axis to saturate its magnetization, and then this so-called "saturation" magnetic field is turned off. Measurement of the magnetic flux produced by the sample under zero magnetic field (0T) after saturation gives the remanence of the sample. The remanence of the material is then obtained. The remanence of the material 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 of 0.6 T or less, and preferably has a remanence of 0.01 T to 0.6 T. In the present invention, "0.01 T to 0.6 T" refers to 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, 0.29 T , 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 preferably, the at least one magnetic layer exhibits a remanence between 0.01T and 1T, more preferably between 0.02T and 0.5T, and even more preferably between 0.05T and 0.2T.

[0085] If the thickness of the magnetic layer or all magnetic layers is too thin, i.e. if it has a width and / or length that is much larger than its thickness, at least ten times larger, it becomes difficult to determine the volume of the magnetic material and therefore to calculate its remanence. This is especially the case for commercial magnetic tapes, where a thin magnetic layer rests on a substrate layer, and moreover the boundary between these two layers is often difficult to evaluate due to industrial manufacturing processes. In this case, the coercivity of the magnetic layer or set of magnetic layers is measured instead. The coercivity is equal to the magnetic moment of the sample divided by the surface of the sample (no longer its volume). The coercivity is expressed in units of the surface density of magnetic flux, i.e. pm·Gauss. Usually, a 2 mm×2 mm sample (a typical size that fits into a laboratory magnetometer) is cut from the capture support to be tested. Its exact area is measured with an optical microscope. The procedure to be followed to obtain the resonance of a magnetic material is the same as for the remanence, i.e. 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 coercivity of 2000 to 30,000 μm·Gauss. In the present invention, "2000 to 30,000 μm·Gauss" means 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 s, 13,000 μm·Gauss, 14,000 μm·Gauss, 15,000 μm·Gauss, 16,000 μm·Gauss, 17,000 μm·Gauss, 18,000 μm·Gauss, 19,000 μm·Gauss, 20,000 μm·Gauss, 21,000 μm·Gauss, 22,000 μm·Gauss, 23,000 μm·Gauss, 24,000 μm·Gauss, 25,000 μm·Gauss, 26,000 μm·Gauss, 27,000 μm·Gauss, 28,000 μm·Gauss, 29,000 μm·Gauss, 30,000 μm·Gauss.

[0087] According to one embodiment of the invention, the at least one magnetic layer has a coercivity of 5000-20,000 μm·Gauss, advantageously 8000-14,000 μm·Gauss, more advantageously 9000-11,000 μm·Gauss. The trapped magnetic nanoparticles have as their largest dimension a dimension of less than 1 μm.

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

[0089] The term "superparamagnetic" denotes the property of particles of small size ferromagnetic or ferrimagnetic material that, under the influence of thermal agitation, randomly change direction of magnetization in the absence of an applied magnetic field.

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

[0091] In comparison to microparticles bound to capture elements, nanoparticles bound to capture elements in particular showed a higher diffusion coefficient (10-fold) and a significantly increased (10 3 times) concentration (m 3 log per unit time), they show much higher performance in terms of molecular capture. However, their respective magnetic forces are 10 3 Divided by (the number when reduced to 1 / 10).

[0092] According to one embodiment of the invention, the magnetic nanoparticles have as their largest dimension a size in the range of 50 nm to 500 nm, advantageously in the range of 50 nm to 250 nm, more advantageously in the range of 100 to 250 nm, even more advantageously in the range of 150 to 200 nm.

[0093] According to one embodiment, the magnetic nanoparticles contain 10-90% iron, advantageously 30-80% and more advantageously 50-70% iron. The more iron they contain, the greater their magnetization in the presence of an additional magnetic field, and the stronger their attraction by said at least one magnetic layer. Thus, the more iron they contain, the less they need to increase their magnetization and the faster they are attracted, as shown below.

[0094] According to one embodiment of the present invention, the nanoparticles are encapsulated. They can in particular be obtained by copolymerization of iron oxide with polystyrene. This encapsulation makes it possible to limit the release of iron from the nanoparticles, which in fact would hinder the detection and quantification of the captured molecules.

[0095] Magnetic nanoparticles can have any shape, such as parallelepipeds, tori, spheres, etc. Nanoparticles can have smooth or irregular surfaces. If they have irregular surfaces, they have a so-called "potato-like" shape.

[0096] Advantageously, the magnetic nanoparticles are spherical and therefore resemble "beads". Thus, the particles may be referred to as "beads" even if their geometry is not perfectly spherical.

[0097] Preferably, the beads are monodisperse, the dimensional uniformity of the beads giving them identical properties and thereby improving the spreading of the beads for their capture by the at least one magnetic layer. By "monodisperse" is meant that the standard deviation of the average diameter of the beads is 40 nm or less over 200 nm, advantageously 20 nm or less over 200 nm.

[0098] In some cases, the beads are commercially available dispersed in a matrix that contains little or no magnetic material, such as a plastic polymer, silica (SiO2).

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

[0100] To enable binding of the capture element to the magnetic nanoparticle, the surface of the magnetic nanoparticle is functionalized, in particular with Staphylococcus aureus protein A or G, or otherwise with a carbodiimide. When protein A or G is used, the bond between the capture element and the nanoparticle is not a covalent bond, as opposed to the use of a carbodiimide.

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

[0102] Antibodies or "immunoglobulins" consist of four amino acid chains, in which two light chains and two so-called heavy chains can be distinguished. Each heavy chain is linked to a light chain by a disulfide bridge. Furthermore, the end of a heavy chain and the end of the associated light chain together define a paratope due to the hypervariable region. Thus, an antibody contains two paratopes, each of which allows binding to an epitope of an antigen. The heavy chains are linked to each other by a so-called hinge region.

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

[0104] The Fab fragment corresponds to the entire light chain and the end of the heavy chain linked to the light chain. Thus, the Fab fragment includes the Fv fragment. There are two Fab fragments for antibodies.

[0105] An F(ab')2 fragment corresponds to an association of two Fab fragments linked together by the hinge region of the heavy chain.

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

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

[0108] Organelles are specialized compartments contained in the cytoplasm of eukaryotic cells where specific biological functions are carried out. Among the organelles are the endoplasmic reticulum, the Golgi apparatus, mitochondria, lysosomes, and peroxisomes.

[0109] Vesicles are compartments present in the cytoplasm of cells and composed of at least one lipid bilayer. They circulate within the cytoplasm and have various functions, such as storage, transport, or digestion of cellular waste products.

[0110] A cell is a compartment that constitutes an organism; it is limited 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 function.

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

[0112] Bacteria are unicellular prokaryotic microorganisms that contain a single cytoplasmic compartment that contains DNA. Therefore, this DNA is not isolated from the cytoplasm by a nucleus, as in eukaryotic cells. Bacteria reproduce by simply dividing in half through fission.

[0113] The type of capture element used is easily adapted by the skilled artisan depending on the type of capture molecule.

[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 has multiple effects on the elements of the kit, making it possible, on the one hand, to accelerate the capture of nanoparticles by the capture support and, on the other hand, to obtain a more localized capture zone, i.e., more precisely, a narrower capture zone, typically extending over a distance of less than 15 μm from the orthogonal projection on the surface of the magnetic layer.

[0116] On the other hand, applying the magnetic field of the at least one additional magnetic field source advantageously increases the magnetization of the magnetic nanoparticles, thereby making it possible to accelerate or even induce their capture by the at least one magnetic layer of the capture support.

[0117] On the other hand, the presence of an additional magnetic field at a level equal to or greater than the level 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 the energy sink of a portion of the resulting total magnetic field is greater than the amplitude of the energy sink of the only magnetic field generated by the at least one magnetic layer, which also contributes to accelerating the capture of nanoparticles.

[0118] The capture zone is strengthened on the surface of the support, where the magnetic field generated by the at least one magnetic layer above the junction between the first and second regions follows the same axis and the same direction as the additional magnetic field. This significantly increases the level of the total strength of the magnetic field. Conversely, above the first and second regions, the level of the resultant force of the additional magnetic field and the magnetic field generated by the at least one magnetic layer does not increase much, since they are not collinear. Thus, the maximum of the level of the strength of the magnetic field increases more significantly than the minimum; thereby highlighting the amplitude of certain energy sinks. On the one hand, this accelerates the capture on those capture zones that are more strongly attracted by these energy sinks, and on the other hand, reduces the extent of the capture zone, which makes it better defined geographically and more precise.

[0119] When the magnetic field generated by the at least one additional magnetic field source at the junction between the first and second regions 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, the capture zone is eliminated: in fact, the level of strength of the magnetic field generated at this level is no longer maximum and the energy of the nanoparticles is no longer minimized there.

[0120] In the particular case where the polarization of the first and second regions is opposite and parallel to the surface of the support, when the additional magnetic field is directed in the same direction as the magnetic field jointly generated by the first and second regions at their junction on the surface of the support, all other capture zones will be strengthened and all other capture zones will be weakened or cancelled out for the reasons described above.

[0121] If the additional magnetic field is equal to or greater than the magnetic field generated by the at least one magnetic field source, the capture zones are located at points where the additional magnetic field has the same orientation and is in the same direction as the magnetic field generated by the at least one magnetic tape. The capture zones can then be offset from their initial positions, as outlined below.

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

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

[0124] If some magnetic field sources are present, they may be selected from a combination of at least one permanent magnet, at least one coil, and / or at least one electromagnet. In particular, they may be selected from assemblies of permanent magnets, coils, electromagnets, and combinations of assemblies of these.

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

[0126] A permanent magnet is an object made of hard magnetic material that has acquired, either artificially or naturally, the persistent property of generating a magnetic field. The peculiarity of a permanent magnet lies in the fact that its magnetic field, once obtained, is generated continuously without the need for specific action. Hard magnetic materials are those whose remanence and coercive field are greater than 0.3 T and 250 kA / m, respectively.

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

[0128] In the following description, the term "permanent magnet" may simply be referred to as "magnet."

[0129] When the at least one additional magnetic field source is a magnet, it is advantageously coupled to a soft ferromagnetic element, otherwise called a "yoke" or "magnetic circuit". This soft ferromagnetic element extends the magnet and has a magnetic permeability of more than 100 S.I. ("International System", unitless) and a saturation of 1.6 to 2.4 T. Unlike hard ferromagnetic elements, soft ferromagnetic elements exhibit low remanence and a weak coercive field. Such soft ferromagnetic elements exhibit no magnetization in the absence of an external magnetic field and allow for the guidance of 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 a conductive wire. This winding can optionally be made around a ferromagnetic material called a core. In contrast to a magnet, a coil only emits a magnetic field when a certain action is applied to it, in this case when an electric current is applied and passes through the conductive wire. Therefore, when this current is no longer applied, no further magnetic field is generated.

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

[0132] According to one embodiment of the present invention, the at least one magnetic layer has two opposing surfaces, which are a capture surface and an opposite 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 such a case, the nanoparticles are at least partially immobilized on the planar coil in the capture zone.

[0133] An electromagnet, when supplied with an electric current, produces a magnetic field; it converts electrical energy into magnetic energy. It consists of a coil and a core and / or one or more pole pieces made of soft ferromagnetic material. Thus, an electromagnet does not emit a magnetic field when no electric current passes through it, as is the case with a coil.

[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, said at least one additional magnetic field source is fixed to the capture support. Advantageously, said fixation is reversible. Therefore, said fixation can be achieved by clipping to the capture support. Alternatively, said fixation is irreversible. Therefore, said fixation can be achieved by gluing to the support.

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

[0137] Uniform means that the gradient is greater than or equal to 100 T m along the magnetization axis of the at least one additional magnetic field source. -1and less than 150 T m along an axis perpendicular to the magnetization axis on the surface of the at least one additional magnetic field source. -1 In the present invention, "100 T m -1 "Less than 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 , 10 T·m -1 or 0T m -1 In the present invention, "150 T·m -1 "Less than 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 , 10 T·m -1 or 0T m -1 means...

[0138] The magnetic field generated by the at least one additional magnetic field source can have any direction; advantageously, its direction is perpendicular to the surface of the support, and more advantageously, its direction and orientation is the same as that generated by the 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 disposed at any position around the capture support. Thus, the at least one additional magnetic field source can be equally well positioned to face the capture surface of the at least one magnetic layer or otherwise face its second surface. Alternatively, the at least one additional magnetic field source can be positioned at a distance from the capture support and not facing either the first capture surface or the second capture surface of the at least one magnetic layer.

[0140] The at least one additional magnetic field source may have an area 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 arranged under the magnetic layer, its surface protrudes on either side of the magnetic layer.For example, when the additional magnetic field source is an assembly of magnets, one of the magnets or at least one part of the surface does not face the at least one magnetic layer, or at least one magnet does not have a surface that faces the at least one magnetic layer.

[0141] Indeed, in all cases, the significant effect brought about by this at least one magnetic field source is, on the one hand, the magnetization of the magnetic nanoparticles, and, on the other hand, the increase in the amplitude of the energy sink, 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.To avoid the risk of demagnetization of the magnetic layer, it is advantageous that the value of the coercive field of the at least one additional magnetic field source is at most 90% of the value of the coercive field of the at least one magnetic layer.

[0143] In the present invention, "1 mT to 400 mT" means 1 mT, 5 mT, 10 mT, 15 mT, 20 mT, 25 mT, 30 mT, 35 mT, 40 mT, 45 mT, 50 mT, 55 mT, 60 mT, 65 mT, 70 mT, 75 mT, 80 mT, 85 mT, 90 mT, 95 mT, 100 mT, 105 mT, 110 mT, 115 mT, 120 mT, 125 mT, 130 mT, 135 mT, 140 mT, 145 mT, 150 mT, 155 mT, 160 mT, 165 mT, 170 mT, 175 mT, 180 mT, 185 mT, 190 mT, 195 mT, 200 mT , 205mT, 210mT, 215mT, 220mT, 225mT, 230mT, 235mT, 240mT, 245mT, 250mT, 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 between 10 mT and 400 mT, more advantageously between 50 mT and 200 mT.

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

[0146] Alternatively, the at least one additional magnetic field source is configured to emit a pulsed magnetic field, advantageously with a pulse duration of 1 millisecond or more, which makes it possible to increase the magnetization of the magnetic nanoparticles for a time that is long enough for the displacement of the magnetic nanoparticles to be affected thereby, as compared to simple Brownian motion.

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

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

[0149] The presence of this non-magnetic layer is advantageous in that it makes it possible to delay the triggering of the attraction of the nanoparticles by the capture support at the desired moment, as will be seen in more 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 materials, silicone paper, adhesive tape, non-magnetic metal alloys such as dura or titanium, quartz, organic materials such as paper or bamboo, wood, gold, or a combination of these materials.

[0151] Advantageously, the polymer is chosen from the list of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), cycloolefin 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, or alternatively, the non-magnetic layer has a composition different from the composition of the support member.

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

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

[0155] According to one embodiment of the present invention, the at least one magnetic layer is covered by the non-magnetic layer over at least 1% of its capture surface, where "at least 1%" means 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%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 108%, 109%, 109%, 10 " is understood to mean 2%, 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, the at least one magnetic layer covers at least 30% of its capture surface, 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.

[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 present invention also relates to a kit as defined above, wherein the non-magnetic layer has a thickness between 1 and 300 μm. In the present invention, "1 to 300 μm" means 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, 46μm, 47μm, 48μm, 49μm, 50μm, 51μm, 52μm, 53μm, 54μm, 55μm, 56μm, 57μm , 58μm, 59μm, 60μm, 61μm, 62μm, 63μm, 64μm, 65μm, 66μm, 67μm, 68μm, 69μ m, 70μm, 71μm, 72μm, 73μm, 74μm, 75μm, 76μm, 77μm, 78μm, 79μm, 80μm, 81 μm, 82μm, 83μm, 84μm, 85μm, 86μm, 87μm, 88μm, 89μm, 90μm, 91μm, 92μm, 9 3μm, 94μm, 95μm, 96μm, 97μm, 98μm, 99μm, 100μm, 101μm, 102μm, 103μm, 1 04μm, 105μm, 106μm, 107μm, 108μm, 109μm, 110μm, 111μm, 112μm, 113μm, 114μm, 115μm, 116μm, 117μm, 118μm, 119μm, 120μm, 121μm, 122μm, 123μm , 124μm, 125μm, 126μm, 127μm, 128μm, 129μm, 130μm, 131μm, 132μm, 133μm m, 134μm, 135μm, 136μm, 137μm, 138μm, 139μm, 140μm, 141μm, 142μm, 143 μm, 144μm, 145μm, 146μm, 147μm, 148μm, 149μm, 150μm, 151μm, 152μm, 15 3μm, 154μm, 155μm, 156μm, 157μm, 158μm, 159μm, 160μm, 161μm, 162μm, 1 63μm, 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, 1 89μm, 190μm, 191μm, 192μm, 193μm, 194μm, 195μm, 196μm, 197μm, 198μm, 199μm, 200μm, 201μm, 202μm, 203μm, 204μm, 205 μm, 206μm, 207μm, 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, 2 38μm, 239μm, 240μm, 241μm, 242μm, 243μm, 244μm, 245μm, 246μm, 247μm, 248μm, 249μm, 250μm, 251μm, 252μm, 253μm, 254 μm, 255μm, 256μm, 257μm, 258μm, 259μm, 260μm, 261μm, 262μm, 263μm, 264μm, 265μm, 266μm, 267μm, 268μm, 269μm, 270μm , 271 μm, 272 μm, 273 μm, 274 μm, 275 μm, 276 μm, 277 μm, 278 μm, 279 μm, 280 μm, 281 μm, 282 μm, 283 μm, 284 μm, 285 μm, 286 μm, 287 μm, 288 μm, 289 μm, 290 μm, 291 μm, 292 μm, 293 μm, 294 μm, 295 μm, 296 μm, 297 μm, 298 μm, 299 μm or 300 μm.

[0159] Preferably, the non-magnetic layer has a thickness of 1 to 150 μm, more preferably 5 to 100 μm, even more preferably 10 to 80 μm, and even more preferably 30 to 60 μm.

[0160] The non-magnetic layer is desirably neither too thin nor too thick. In fact, if the non-magnetic layer is too thin, i.e., less than 1 μm, it does not have a significant effect on attenuating the attraction of nanoparticles by the at least one magnetic layer.

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

[0162] Typically, for a magnetic layer having a coercivity of 12000 μm·G and whose first and second regions have a width of 50 μm, the non-magnetic layer advantageously has a thickness of 20 to 60 μm. For magnetic layers with lower coercivity, the thickness of the magnetic layer must be reduced accordingly.

[0163] If the non-magnetic layer is too thick (depending on the thickness and width of the at least one magnetic layer region), strong magnetic field gradients will be hidden by this non-magnetic layer.Therefore, the magnetic field at the surface of the non-magnetic layer is uniform or non-existent in some cases.Then, nanoparticles are not immobilized or are otherwise immobilized randomly on the surface of the non-magnetic layer, not in a specific pattern.

[0164] Applying the magnetic field of the at least one additional magnetic field source advantageously makes it possible in such a case to "manifest" a strong magnetic field gradient at the surface of the non-magnetic layer by increasing the amplitude of the energy sink. 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 more detail below.

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

[0166] The present invention also relates to a method for capturing a molecule contained in a sample, the method comprising: a) contacting the sample with magnetic nanoparticles as defined above to form at least one capture complex between the molecule and the at least one capture element bound to the magnetic nanoparticle; b) attracting the at least one capture complex formed during step a) by said magnetic field generated by at least one magnetic layer of a capture support as defined above, such that the at least one capture complex is immobilized to the capture support in at least one capture zone as defined above. Includes.

[0167] The purpose of step a) of the present invention is to complex the molecules to be captured via the capture elements with nanoparticles, so that these complexed molecules can be indirectly attracted via the nanoparticles by said at least one magnetic layer in the subsequent step b) and finally captured by immobilization on a support.

[0168] In the remainder of this specification, the capture complex may simply be referred to by the term "complex."

[0169] According to one embodiment of the invention, the capture element is an antibody or an antigen, and therefore the at least one capture complex formed during step a) is an immune complex.

[0170] In the present invention, "sample" means any simple or complex fluid.

[0171] 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 (granular), liquid-gas (foams) or liquid-liquid (emulsions). Complex fluids deviate from the classical linear Newtonian relationship between stress and shear rate. They exhibit unusual mechanical responses to applied stress or strain due to the geometric constraints imposed by the coexistence of phases. This mechanical response includes transitions as well as fluctuations between solid-like and fluid-like behavior. In particular, the sample may 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 may also be a culture medium or a medium used in a bioprocess, such as a purified or clarified culture medium.

[0172] In the present invention, by "simple fluid" is meant a Newtonian fluid whose mechanical behavior is characterized by a single function of temperature, viscosity, and a measure of the "slip" of the fluid. The stress applied to a simple fluid is directly proportional to the strain rate. In particular, the sample can be deionized water. Deionized water has the advantage of significantly limiting or preventing the formation of nanoparticle clusters.

[0173] According to one embodiment of the invention, once the sample has been contacted with the nanoparticles during step a), they are placed on a capture support to allow attraction of said at least one capture complex during step b).

[0174] Thus, the contacting and attracting steps are performed separately so that the nanoparticles are not attracted by the at least one magnetic layer when they come into contact with the sample, which advantageously makes it possible to obtain a uniform distribution of the nanoparticles in the sample, thereby enabling more efficient complex formation between the capture elements and the capture molecules.

[0175] Alternatively, the sample is first placed on the support before being brought into contact with the nanoparticles during step a). Steps a) and b) are therefore carried out at the same location, i.e. at the support. This embodiment is of great interest in certain applications, especially since it does not require fluid handling via micropumps or microvalve type actuators.

[0176] According to one embodiment of the present invention, between steps a) and b), an ultrasonic treatment of the mixture containing the nanoparticle-molecule complexes in the captured suspension is carried out with the aim of breaking up any aggregates of the complexes that may have formed. Such aggregates would interfere with the quantification step described below. This ultrasonic treatment can in particular be carried out at a frequency of at least 10000 Hz. The ultrasonic treatment can be a continuous or pulsed irradiation. When the ultrasonic treatment is pulsed, the duration of each ultrasonic treatment can be between 200 and 700 ms, in particular between 300 and 600 ms, in particular 500 ms, with an interval of 1 to 6 s, in particular between 1 and 4 s, in particular 2 s. These ultrasonic treatment conditions make it possible to ensure the function of breaking up the aggregates while avoiding overheating of the medium, which may denature the capture elements, the capture molecules and the detection elements.

[0177] During step a), the concentration of nanoparticles is advantageously greater than 10 6 ~10 11 In the present invention, "10 6 ~10 11 "particles / mL" means 10 6 particles / mL, 10 7 particles / mL, 10 8 particles / mL, 10 9 particles / mL, 10 10particles / mL and 10 11 Mean particles / mL.

[0178] 10 6 The minimum concentration of particles / mL provides a sufficient concentration for efficient capture of capture molecules dispersed in the sample. 11 The maximum concentration of particles / mL makes it possible to avoid excessive aggregation of nanoparticles (clusters with a diameter of 15 μm or less), which on the one hand destroys the bond between the capture element and the capture molecule and on the other hand has a negative effect on the quantification of the captured molecule. High concentrations of nanoparticles also contribute to screening of the magnetic field, i.e. attenuating the magnetic field, which has a negative effect on the attraction of nanoparticles. In the present invention, "clusters with a diameter of 15 μm or less" means 15 μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 0.5 μm and 0.2 μm.

[0179] According to one embodiment, step a) further comprises contacting the sample with an element for detecting said molecule.

[0180] The detection element has a marker that can be fluorescent, luminescent, or colored so as to be recognized by an appropriate detection means. The marker can be an enzyme with redox properties.

[0181] During step a), a so-called "sandwich-type" complex is formed, which is formed by the magnetic nanoparticles, the capture elements, the capture molecules and the detection elements. The capture molecules are then surrounded by the capture elements and the detection elements in a "sandwich" manner.

[0182] In the formation of these "sandwich type" complexes, where the detection element is attached to the capture molecule from the first step, this is made possible by the good diffusion properties of the magnetic nanoparticles in the mixture, therefore the formation of such complexes becomes more difficult with magnetic microparticles.

[0183] The above mentioned sonication step can also be carried out under the same conditions in this embodiment to avoid any aggregation of the "sandwich type" complex.

[0184] Alternatively, the detection element can be placed in the presence of the capture molecule during step c) following immobilization of the complex formed by the nanoparticles, the capture element and the capture molecule to the support, so that such a so-called "sandwich type" complex is subsequently formed during this step c).

[0185] Finally, at the end of step a), a mixture is obtained that contains a complex of nanoparticles, capture elements and capture molecules, or a complex of nanoparticles, capture elements, capture molecules and detection elements (if such detection elements are present), in which a single captured molecule and a nanoparticle bound to a single capture element, and optionally a single detection element may remain.

[0186] The mixture may be deposited, for example, by pipetting, onto the capture support in order to immobilize the nanoparticles during step b) The mixture may also be injected into the capture support, for example if the capture support is a chamber.

[0187] When the mixture is placed on a support, all magnetic nanoparticles (complexed or uncomplexed) are attracted by the at least one magnetic layer and come to rest relative to the support, or more precisely, relative to the capture surface of the at least one magnetic layer.

[0188] During their immobilization, the magnetic nanoparticles (complexed or not) are not randomly distributed with respect to the support, or more precisely with respect to the capture surface of said at least one magnetic layer.

[0189] In practice, the nanoparticles are immobilized in the capture zones defined in the present invention, and therefore only a small fraction of the nanoparticles, less than 15%, are either immobilized outside these capture zones or are not captured.

[0190] The nanoparticles are thus distributed relative to the support, and more precisely relative to the capture surface of said at least one magnetic layer, according to a specific pattern defined by all of the junctions of the first and second regions.

[0191] This distribution is of great interest since it allows one to determine where the nanoparticles are captured, allowing direct quantification of the captured molecules without a washing step.

[0192] The organization of the first and second regions of the at least one magnetic layer allows detection and quantification to occur without washing the support immediately after immobilization of the capture complexes and binding or "sandwiching" the capture complexes with the detection elements.

[0193] To do this, it is first necessary to determine the amount of marking in the capture zone and then to determine the amount of marking outside the capture zone.

[0194] The skilled person will be able to easily adapt the means for determining the amount of marking implemented as a function of the type of marker bound to the detection element, in particular these means can be chosen from a spectrophotometer in scanning mode, an epifluorescence microscope, a confocal microscope, a two-photon microscope, measurement of the redox activity of enzymes, etc.

[0195] For example, in the context of fluorescent marking, those skilled in the art will benefit from using a fluorescent 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 presented in the "Examples" section of this specification.

[0196] The amount of marking outside the capture zone corresponds to the "background signal", in other words, this marking corresponds to the detection elements not bound to the capture complex and to a minor fraction of the complex immobilized outside the capture zone, as well as the effect of the matrix (residual signal of the medium).

[0197] To obtain the amount of a particular marking released in the capture zone, the amount of marking captured outside the capture zone is subtracted from the marking captured in the capture zone.

[0198] The present invention also relates to a capture method as defined above, wherein the attraction of said at least one capture complex during step b) is performed by the cooperation of a magnetic field generated by said at least one magnetic layer and a magnetic field generated by at least one additional magnetic field source as defined above.

[0199] As described above, the at least one additional magnetic field source makes it possible to increase or even saturate the magnetization of the magnetic nanoparticles and further increases the amplitude of the energy sink, thereby accelerating the attraction of the nanoparticles by the at least one magnetic layer.

[0200] Furthermore, as mentioned above, the additional magnetic field makes it possible to reinforce a specific capture zone, thereby promoting localized capture, thereby favoring wash-free detection of captured molecules, as mentioned above.

[0201] This cooperation also makes it possible to avoid the use of evaporation of solvent from the sample aimed at bringing the nanoparticles closer to the capture support, thereby accelerating their capture, which would in fact heat the sample or require very long waiting times, which could have a negative effect on the binding between the capture / detection elements and the captured molecules and therefore on the quantification of the captured molecules.

[0202] Thus, attraction of nanoparticles is achieved by the cooperative action of the at least one magnetic field source and the at least one magnetic layer, each of which exerts different actions and functions.

[0203] Depending on its function and operation, the at least one magnetic field source can be located anywhere with respect to the capture support, as described above.

[0204] Thus, the at least one additional magnetic field source may include: facing the capture surface of the at least one magnetic and / or non-magnetic layer, - Facing the opposite surface or supporting member, or at any other position, e.g. laterally offset, can be arranged equally well.

[0205] Significant magnetization of the nanoparticles is advantageously achieved by applying a magnetic field at a level between 1 mT and 400 mT, advantageously between 10 mT and 400 mT, more advantageously between 50 mT and 200 mT.

[0206] The action of the at least one additional magnetic field source may be carried out throughout steps a) and b).

[0207] Advantageously, this is induced during step b), so that during step a) and before triggering the action of said at least one additional magnetic field source, the magnetic field at the magnetic nanoparticles is insufficient or zero.

[0208] The triggering of the action of the at least one additional magnetic field source during step b) is ensured by generating a magnetic field at the magnetic nanoparticles between 1 mT and 400 mT, advantageously between 10 mT and 400 mT, more advantageously between 50 mT and 200 mT.

[0209] To induce the action of the at least one additional magnetic field source, the additional magnetic field source can be, according to a first alternative, closer to the capture support, for this embodiment, the at least one additional magnetic field source is advantageously a permanent magnet.

[0210] According to a second alternative, the induction is achieved by passing a current through said additional magnetic field source. In this embodiment, said at least one additional magnetic field source is advantageously a coil or an electromagnet, and the induction is achieved by passing a current through said additional magnetic field source.

[0211] Advantageously, even if the action of said at least one additional magnetic field source is stopped at the end of step b), for example by moving it away or by stopping the passage of current therethrough, the captured nanoparticles remain in a fixed position relative to the capture support in the capture zone.It is therefore possible to move the capture support to a suitable location and detect the marker of the detection element without affecting the position of these nanoparticles and therefore without directly detecting them without compromising the washing steps.

[0212] 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 the attraction of said at least one capture complex by the capture support during step b) is induced by the magnetic field of said at least one additional magnetic field source.

[0213] In this embodiment, the presence of a non-magnetic layer reduces or prevents the nanoparticles from being attracted solely by the magnetic field generated by the at least one magnetic layer.

[0214] For this reason, advantageously, - on the other hand, the magnetic field generated by all of said at least one magnetic layer and said at least one additional magnetic field 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 intensity defining a maximum and a minimum of the level of the intensity of the magnetic field and defining, at said maximum of the level of the magnetic field, a capture zone of magnetic nanoparticles on the capture support; - on the other hand, the magnetic field generated by the at least one magnetic layer does not exhibit at least one variation in its intensity of at least 0.1 mT at a distance of at least 1 μm from the surface of the non-magnetic layer, whereby the magnetic field generated exclusively by the at least one magnetic layer does not make it possible to define a capture zone of magnetic nanoparticles on the surface of the non-magnetic layer.

[0215] The surface of the support corresponds to the surface onto which the nanoparticles are immobilized.

[0216] Thus, the attraction of the magnetic nanoparticles during step b) is due to the action of said at least one magnetic field source on these nanoparticles, i.e. - a significant increase in the magnetization of these particles due to the magnetic field generated by this magnetic field source, and / or -Increased amplitude of energy sink Depends on.

[0217] This aspect of the invention is very interesting since it makes it possible to optimize the execution of steps a) and b) at the same location, i.e. the capture support, by reducing or counteracting the possibility that nanoparticles may be attracted by the action of said at least one magnetic layer alone during step a).

[0218] The presence of a non-magnetic layer results in at least a portion of the nanoparticles (complexed or not) being immobilized against this non-magnetic layer and not against the at least one magnetic layer, more precisely, a non-magnetic layer having a capture surface and an opposite surface facing the at least one magnetic layer, during step b) at least a portion of the nanoparticles being immobilized against the first capture surface of the non-magnetic layer.

[0219] The present invention also relates to a capture method as defined above, in which the sample is placed on a capture support prior to step a) of contacting with the magnetic nanoparticles.

[0220] Advantageously, the action of said at least one magnetic field source is induced during step b).

[0221] Thus, during step a), the amplitude of magnetization of the magnetic nanoparticles and / or the energy sink of the at least one magnetic layer is not sufficient to allow attraction of the nanoparticles only by the action of the at least one magnetic layer covered by the non-magnetic layer, and thus the mixing of the nanoparticles with the sample is hardly, if at all, disturbed by premature attraction by the at least one additional magnetic source.

[0222] Surprisingly, the inventors have discovered that, despite the presence of a non-magnetic layer and its effect on the attractiveness of the at least one magnetic layer on the nanoparticles, the immobilized nanoparticles remain in position relative to the support in the capture zone when the action of the magnetic field generated by the at least one additional magnetic field source is stopped at the end of step b), for example by moving away from this magnetic field source or by stopping the current.

[0223] One explanation for this phenomenon is that the gradient of the magnetic field 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 relative to the non-magnetic layer. A further explanation is that there is an adsorption, i.e. a chemical / physical bond, between the magnetic nanoparticles aggregated together on the one hand and the particles absorbed at the capture surface of the non-magnetic layer on the other hand.

[0224] Thus, again, it is possible to move the capture support to the appropriate location and detect the marker of the detection element without affecting the position of these particles and thereby directly detecting them without compromising the washing steps.

[0225] The present invention also relates to a capture method as defined above, comprising a subsequent step c) of displacing the magnetic nanoparticles captured relative to the support to a collection zone.

[0226] This displacement is performed using a technique called "connecting rod crank": - this crank is the magnetic field generated by the magnetic regions of the at least one magnetic layer; this magnetic field is called b(R) (R is a point in space); the at least one magnetic layer exhibits a variation in the intensity of the generated magnetic field in order to generate a magnetic energy minimum, - the connecting rod is a spatially homogenous magnetic field (in direction and strength) generated by said at least one additional magnetic field source; in the present application, the amplitude and orientation of said magnetic field can be modified.

[0227] The "uniform" magnetic field generated by the connecting rod is applied to the entire zone in which the magnetic field of the crank is generated, and therefore coexists in space with the magnetic field generated by said at least one magnetic layer, so that their vector values ​​are linearly superimposed in a medium of relative magnetic permeability equal to 1 (such as air or water).

[0228] Due to the fact that the magnetic field generated by the connecting rod can be modulated, during step c) it is possible to rotate the connecting rod at least 1° clockwise or counterclockwise around at least one axis of rotation and / or to amplify said magnetic field. Thus, the magnetic field of the connecting rod can be varied in time. Thus, the magnetic field of the connecting rod is denoted as B(t), where t is time.

[0229] As a result, the magnetic field of the crank varies in space but not in time, whereas the magnetic field of the connecting rod varies in time.

[0230] Therefore, in the zone where the connecting rod and crank are applied, the total magnetic field St is the vector sum of b and B, i.e., B T (R,t)=b(R)+B(t).

[0231] In this way, during step c) it is possible to vary the position of the capture zone of the magnetic nanoparticles relative to the support.

[0232] In the present invention, "1° to 360°" means 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°, 99°, 100 °, 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°, 112°, 113°, 114°, 115°, 116°, 117°, 1 18°, 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°, 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°, 2 90°, 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°, 3 26°, 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°.

[0233] Then, a vector sum of the magnetic fields of the connecting rod and the crank is obtained, which is different from the one obtained at the end of step b), and the position of maximum of the level of the magnetic field strength generated by the connecting rod and the crank assembly is moved so that the trapping zone is offset with respect to this step b), and thus the nanoparticles attracted by the generated magnetic field gradient are simultaneously moved until they are located in the new position of the trapping zone.

[0234] Advantageously, step c) is repeated in the same direction of rotation until the nanoparticles, progressively displaced in the same direction, reach a collection zone.

[0235] By repeating step c), a "conveyor belt" effect is obtained, and the nanoparticles are moved in the same direction along the surface of the capture support towards a collection zone, where they are also collected as the capture molecules form complexes with the nanoparticles.

[0236] Finally, the present invention makes it possible to recover captured molecules without applying a fluid flow to the support that would displace all of the molecules contained in the mixture.

[0237] The present invention further relates to the use of a kit as defined above for the capture of molecules contained in a sample, advantageously for the capture and detection of molecules contained in a sample.

[0238] The present invention also relates to the use of a magnetic layer for attracting nanoparticles having a maximum dimension of less than 1 μm, the magnetic layer comprising a repeatable juxtaposition of at least first and second regions, the first region comprising magnetic grains polarized in a first direction and the second region comprising magnetic grains that are not polarized or are polarized in a second direction different from the first polarization direction of the magnetic particles of the first region, whereby the one magnetic layer generates a magnetic field having at least one variation in its intensity of at least 0.1 mT at a distance of at least 1 μm from the at least one magnetic layer, the at least one variation in intensity defining a maximum and a minimum of the level of the intensity of the magnetic field, and at the maximum of the level of the magnetic field a capture zone of magnetic nanoparticles is defined on the magnetic layer, the nanoparticles being each bound to at least one capture element of a molecule.

[0239] Another kit has also been described for capturing molecules contained in a sample, the kit comprising: a) magnetic nanoparticles having a maximum dimension of less than 1 μm, each of the nanoparticles being bound to at least one capture element, the at least one capture element specifically binding to the molecule; b) a support for capturing said magnetic nanoparticles, comprising or consisting essentially of at least one magnetic layer, said magnetic layer comprising a repeatable 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 not polarized or that are polarized in a second direction different from the first polarization direction of the magnetic particles of the first region, whereby said at least one magnetic layer generates a magnetic field that does not show at least one fluctuation of intensity of at least 0.1 mT at a distance of at least 1 μm from said at least one magnetic layer; c) at least one additional magnetic field source, where the magnetic field generated by the assembly of the at least one magnetic layer and the at least one additional magnetic field 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 at least one magnetic layer; Including, The at least one variation in intensity defines a maximum and a minimum level of intensity of the magnetic field and at the maximum of the magnetic field level defines a zone for capturing magnetic nanoparticles on a capture support.

[0240] Advantageously, the additional magnetic field source is external to the at least one magnetic layer.

[0241] Further advantageously, the at least one magnetic layer has a capture surface, the at least one magnetic layer being at least partially covered by a non-magnetic layer on the capture surface, and the magnetic field generated by the at least one magnetic layer and all of the at least one additional magnetic field 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 the non-magnetic layer, the at least one variation in intensity defining a maximum and a minimum of the level of the intensity of the magnetic field and defining a zone for capturing magnetic nanoparticles on the capture support at the maximum of the level of the magnetic field.

[0242] All of the above mentioned features regarding the capture support, the additional magnetic field source and the nanoparticles associated with the kits described above apply mutatis mutandis in this kit.

[0243] The present invention also relates to a method for capturing a molecule contained in a sample, the method comprising: a) contacting the sample with magnetic nanoparticles as defined above to form at least one capture complex between the molecule and the at least one capture element bound to the magnetic nanoparticle; b) attracting the at least one capture complex formed during step a) by a magnetic field generated by the assembly of at least one magnetic layer of a capture support as defined above and at least one additional magnetic field source, such that the at least one capture complex is immobilized to the capture support in at least one capture zone as defined above. Includes.

[0244] The present invention also relates to the use of a kit as defined above for the capture of molecules contained in a sample, advantageously for the capture and detection of molecules contained in a sample.

[0245] The present invention finally relates to the use of an assembly comprising a magnetic layer and an additional magnetic field source for attracting nanoparticles having a maximum dimension of less than 1 μm, said magnetic layer comprising a repeatable juxtaposition of at least first and second regions, said first region comprising magnetic grains polarized in a first direction and said second region comprising magnetic grains that are not polarized or are polarized in a second direction different from the first polarization direction of the magnetic particles of the first region, whereby the magnetic field generated by said one magnetic layer and all of said additional magnetic field 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 magnetic layer, said at least one variation in intensity defining a maximum and a minimum of the level of the intensity of said magnetic field and defining a zone for capturing magnetic nanoparticles on said magnetic layer at said maximum of the level of the magnetic field.

[0246] Advantageously, each of said nanoparticles is bound to at least one capture element of a molecule.

[0247] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the capture of nanoparticles by a magnetic layer according to the invention. The capture system is shown in a cross-sectional view. The strength of the magnetic field emitted by the magnetic layer is defined by a color code whose scale is presented to the right of the cross-sectional view (unit: T). The arrows in the magnetic layer represent the polarization direction of the grains that compose it.

[0248] Figure 2 shows a photograph of the capture by the system shown in Figure 1. In this photograph, the white dots represent nanoparticles.

[0249] Figure 3 shows the capture of nanoparticles by a magnetic layer according to the invention, which is covered by a non-magnetic layer. The capture system is shown in a cross-sectional view. The strength of the magnetic field generated by the magnetic layer is defined by a color code whose scale is presented to the right of the cross-sectional view (unit: T). The arrows from the nanoparticles represent the direction of movement of the nanoparticles attracted by the magnetic layer. The arrows in the magnetic layer represent the polarization direction of the grains that compose it.

[0250] Figure 4 shows a photograph of the capture by the system shown in Figure 3. In this photograph the white dots represent nanoparticles.

[0251] Fig. 5a shows the capture of nanoparticles by a magnetic layer according to the invention, which is covered with a non-magnetic layer and the capture is performed in the presence of a magnetic field from an additional magnetic source. The capture system is shown in a cross-sectional view. The direction of the magnetic field generated by the additional magnetic source is indicated by a white arrow with a black border on the cross-sectional view and is perpendicular to the magnetic layer. The strength of the magnetic field generated by the magnetic layer is defined by a color code whose scale is presented to the right of the cross-sectional view (units: T). The arrows from the nanoparticles represent the direction of movement of the nanoparticles attracted by the magnetic layer. The arrows in the magnetic layer represent the polarization direction of the grains that compose it.

[0252] Fig. 5b shows the capture of nanoparticles by a magnetic layer according to the invention, which is covered with a non-magnetic layer and the capture is performed in the presence of a magnetic field from an additional magnetic field source. The capture system is shown in a cross-sectional view. The direction of the magnetic field generated by the additional magnetic field source is indicated by a white arrow with a black border on 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 magnetic field source) is defined by a color code whose scale is presented to the right of the cross-sectional view (units: T). The arrows from the nanoparticles represent the direction of movement of the nanoparticles attracted by the magnetic layer. The arrows in the magnetic layer represent the polarization direction of the grains that compose it.

[0253] Figure 6 shows a photograph of the capture by the system shown in Figure 5a, where the white dots represent nanoparticles.

[0254] Figure 7 shows the percentage (%) of nanoparticles captured (X-axis) as a function of time (Y-axis) for each capture shown in Figures 1-3. Time is expressed in minutes. Curve A corresponds to the capture kinetics shown in Figure 5a, curve B corresponds to the capture kinetics shown in Figure 1, and curve C corresponds to the capture kinetics shown in Figure 3.

[0255] Figure 8 shows an anti-mouse detection capture antibody bound to a nanoparticle complex carrying a fluorescent dye and grafted with an anti-mouse ovalbumin capture antibody, which is also an ovalbumin grafted nanoparticle.

[0256] FIG. 9 is a graph showing the calculated fluorescence (arbitrary units) as a function of the concentration (μg / mL) of anti-ovalbumin antibody.

[0257] Figure 10 shows a photograph of the complex of Figure 8 after capture. Each white dot represents a fluorescent dye carried by the capture antibody (anti-mouse) of Figure 8. For Figure 10A, a mouse anti-ovalbumin concentration of about 50 μg / mL was used; for Figure 10B, a concentration of about 25 μg / mL was used; for Figure 10C, a concentration of about 12.5 μg / mL was used; for Figure 10D, a concentration of about 6.25 μg / mL was used; Figure 10E is a negative control without anti-mouse ovalbumin antibody.

[0258] 11 is a graph showing the calibration curve of DLGi5 garnet in a MagView CMOS type MOIF system, which shows the rotation (in degrees) of the polarization of a light beam passing through the garnet as a function of the magnetic field strength (in Tesla).

[0259] Figure 12 shows photographs after trapping of nanoparticles in the microfluidic chamber. White dots represent nanoparticles. Figures 12A-12E show the trapping kinetics (A: 0 s; B: 10 s; C: 30 s; D: 80 s and E: 120 s).

[0260] Figure 13 shows photographs after capture of nanoparticles in the microfluidic chamber in the presence of an additional magnetic field source. White dots represent nanoparticles. Figures 13A-13F show the capture kinetics (A: 0 s; B: 2 s; C: 5 s; D: 12 s; E: 34 s and F: 60 s).

[0261] Figure 14 shows the percentage of trapped nanoparticles (Y-axis) as a function of time of trapping (X-axis) shown in Figure 12. Time is expressed in seconds.

[0262] Figure 15 is a spectroscopic graph showing the size of nanoparticles in solution. The size was determined using a dynamic light scattering technique. The Y-axis represents the relative frequency of nanoparticles as a percentage, and the X-axis is a logarithmic scale representing the size of the nanoparticles in nanometers.

[0263] (Example) Example 1: Capturing nanoparticles with magnetic strips The capture of nanoparticles by the magnetic strip of a magnetic card was first tested.

[0264] (Experimental Protocol) The nanoparticles used (Chemicell nanoscreenmag ARA 200nm) had an average diameter of 200nm and a mass of 1.25g / cm 3The nanoparticles have a density of 1.1 × 10 12 nanoparticles / g and 4.4×10 9 Dilute to nanoparticles / mL level.

[0265] To break up any possible nanoparticle aggregates, a solution of nanoparticles diluted in deionized water (ddH2O) was mixed using a SONIC RUPTOR 4000 sonicator. Intermittent sonication was performed in the tube at 20% of a total power of 400 W and an estimated frequency of around 20000 Hz. A total of three ultrasonic pulses were emitted with a duration of 500 ms every 2 seconds.

[0266] To verify that the captured nanoparticles are not in the form of nanoparticle clusters, we measured the size of the nanoparticles in the solution by "diffraction light scattering." The results are shown in FIG.

[0267] The capture support is a magnetic card made of a highly magnetic polymer, consisting of a PVC support member and a magnetic strip made of three magnetic layers arranged on the same plane. The support member and magnetic layers were assembled according to the ISO7811 standard. Each magnetic layer was encoded with a series of "1"s corresponding to 182 times the letter F in hexadecimal using an MSR605 encoder, which allows the magnetic field orientation to change by 180 degrees every approximately 55 μm.

[0268] The capture of nanoparticles is performed by depositing 5 μL of a solution of nanoparticles and depositing the droplets onto the magnetic layer of a magnetic card.

[0269] A schematic of this trapping is shown in Figure 1. In this figure, nanoparticles 1 are in solution and some 1' are attracted by a magnetic layer 3 of magnetic strips (represented by black arrows). The magnetic layer has a first region 5 and a second region 7 where the polarization of the grains is reversed (represented by large white arrows). The magnetic field generated by each region is represented by small white arrows that follow an arc starting on one side of the region and ending on the other. The direction of these arrows indicates the direction of the generated magnetic field. The strength of the magnetic field is represented by a color scale. This magnetic field strength was obtained by a finite element approach implemented using the COMSOL Multiphysics® 5.0 modeling software, as described above. White indicates a strong magnetic field strength. On top of the magnetic layer 3, the strength of the magnetic field generated by the first and second regions (5, 7) can be seen, each about 100 μm wide, with the magnetic field showing fluctuations. The presence of a maximum in the strength of the magnetic field 9 can be clearly seen above the junction between the first region 5 and the second region 7, with the strength of the magnetic field being lower in each region (5,7) itself. Thus, at each maximum in the level of the magnetic field 9, a capture zone 11 is defined by its orthogonal projection on the surface of the magnetic layer 3, where the nanoparticles 1' are immobilized.

[0270] Images are then captured with 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) is used. Images are captured at a total magnification of 50x with an acquisition time of 3 seconds (gain 14db). An example of image capture can be seen in Figure 2. In this figure the white dots represent the nanoparticles. The array of captured nanoparticles can be clearly seen.

[0271] The percentage of nanoparticles captured by the magnetic strip is quantified following the protocol described in the publication of Fratzl et al., Soft Matter (14) 2671 2680 (2018). Briefly, this quantification is obtained by the ratio of the area covered by nanoparticles to the area not covered by nanoparticles.

[0272] The capture kinetics results are shown in FIG.

[0273] (result) As can be seen in Figure 7, capture of magnetic nanoparticles occurs immediately after deposition of the droplets on the magnetic substrate, reaching 40% in 2 min.

[0274] Furthermore, the results shown in Figure 15 show a single peak due to the suspended beads exhibiting a mean diameter of 282 nm (9.5 nm standard deviation), corresponding to the diameter of a single nanoparticle. These results demonstrate that the nanoparticles are independent of each other and do not form clusters.

[0275] Example 2: Capturing nanoparticles by a magnetic strip covered with a non-magnetic layer In this example, the capture of nanoparticles by the magnetic strip of a magnetic card covered with a non-magnetic layer is examined.

[0276] (Experimental Protocol) The nanoparticles and capture support used are the same as those used in Example 1, except that the magnetic strip is covered by a 60 μm thick self-adhesive layer of black polymer (vinyl).

[0277] The method of trapping nanoparticles and the determination of the percentage of trapped nanoparticles are the same as in Example 1.

[0278] A diagram of this capture is shown in Figure 3, which includes the elements shown in Figure 1. In addition, a non-magnetic layer 13 is arranged on the surface of the magnetic layer 3. As can be seen in this Figure 3, only a part of the magnetic field generated by the magnetic layer 3 projects above the surface of the non-magnetic layer 13, so that the maximum of the level of the magnetic field is "hidden" by the non-magnetic layer. Therefore, at a distance of at least 1 μm from the surface of the capture support, there is no variation in the strength of said magnetic field of at least 0.1 mT, and therefore no capture zone. The nanoparticles are therefore very weakly attracted and immobilized in a random manner to the magnetic strip and remain mainly in solution.

[0279] A photograph of the resulting capture results is seen in Figure 4, where the white dots represent the nanoparticles. In this figure, the alignment seen in Figure 2 has disappeared.

[0280] The capture kinetics results are shown in FIG.

[0281] (result) As can be seen in Figure 7, the nanoparticles are captured very slowly by the magnetic strip of the magnetic card: after 10 minutes, the capture reaches only 15%.

[0282] Example 3: Capture of nanoparticles by a magnetic strip covered with a non-magnetic layer in the presence of an additional magnetic field source In this example, the capture of nanoparticles by the magnetic strip of a magnetic card covered with a non-magnetic layer is tested in the presence of an additional magnetic field source.

[0283] (Experimental Protocol) The nanoparticles and capture supports used are the same as those used in Example 2.

[0284] The additional magnetic field source is a parallelepiped (20 x 10 x 1 mm) head-to-tail assembly (vertical / horizontal magnetization) of NdFeB macro magnets (N35, adhesion 800 g) magnetized along the 1 mm axis.

[0285] The magnetic card is deposited on an additional magnetic field source such that the magnetic layer is not located on the opposite side of the magnetic field source.

[0286] The method of trapping nanoparticles and the determination of the percentage of trapped nanoparticles are the same as in Example 1.

[0287] Diagrams of this trapping are shown in Figures 5a and 5b. In Figure 5a, an additional magnetic field source (not shown) emits a magnetic field with a direction perpendicular to the magnetic layer 3, represented by a white arrow with a black border. In Figure 5b, an additional magnetic field source (not shown) emits a magnetic field with a direction parallel to the magnetic layer 3, represented by a white arrow with a black border. In these two figures it is possible to see the influence of the direction of the magnetic field generated by the additional magnetic field source on the location of the trapping zone.

[0288] These figures show the elements shown in figures 1 and 3. They clearly show, on the one hand, that the strength of the magnetic field generated at the surface of the non-magnetic layer 13 is much stronger and that the maxima of the level of strength of the magnetic field 9 are no longer hidden by the non-magnetic layer 13. Furthermore, compared to what was initially shown in figure 1, only every other maximum of the level of strength of the magnetic field 9 is present and therefore every other capture zone 11 is present. However, these maxima 9 have a greater strength than what was initially shown in figure 1.

[0289] In Figure 5a, the trapping zone 11 is located above the junction where the polarization of the adjacent regions (5,7) is oriented due to an additional magnetic field perpendicular to the magnetic layer 3. However, above the junction where the polarization of the adjacent regions moves apart, the magnetic field level is minimized.

[0290] Interestingly, in FIG. 5b, an additional magnetic field having a direction parallel to the magnetic layer 3 displaces the trapping zone 11 and the maximum of the level of intensity of the magnetic field 9 so that it is no longer located at the junction between the first and second regions (5,7), but in the first region 5 itself.

[0291] A photograph of the obtained trapping results can be seen in Figure 6, where the white dots represent nanoparticles. Unlike Figure 4, where there is no trapping sequence, a new sequence appears in Figure 6 that differs from the sequence obtained in Figure 2. Here, it can be seen that the nanoparticles form regularly arranged parallel bands, with very few nanoparticles present outside these bands.

[0292] The capture kinetics results are shown in FIG.

[0293] (result) In this example, capture of magnetic nanoparticles is triggered immediately after application of an external magnetic field, with capture approaching 100% in 2 minutes.

[0294] Combining the present results with those of Example 2, it can be concluded that when the magnetic strip is covered with a non-magnetic layer, it is possible to induce immobilization and very rapid capture (2 min) of nanoparticles by inducing the magnetic field of an additional magnetic field source.

[0295] Example 4: Capture and quantification of capture elements bound to nanoparticles Finally, detection and quantification of multiple concentrations of anti-mouse ovalbumin antibodies (capture elements) bound to magnetic nanoparticles was tested.

[0296] Each measurement is performed by keeping the detection antibody (anti-mouse antibody) and the number of total nanoparticles the same, but varying the amount of nanoparticles bound to the anti-mouse ovalbumin antibody (by supplementing with nanoparticles bound to ovalbumin).

[0297] (Experimental Protocol) The nanoparticles used (Carboxyl Adembeads 200 nm (reference number 02120-Ademtech)) have a diameter of 200 nm and a mass of approximately 2.0 g / cm 3The nanoparticles have a density of about 100 nm, a saturation magnetization of about 40 emu / g, an iron oxide content of about 70% and a solids content of 30 mg / mL (3%). The nanoparticles are covered with COOH carboxylic acid groups with a density of over 350 μmol / g.

[0298] The capture support is a magnetic card consisting of a PVC support member, on which are placed three coplanar magnetic layers consisting of high coercivity magnetic polymers. The support member and magnetic layers are assembled according to ISO 7811 standards. Each magnetic layer is encoded in successive 1s using an MSR605 encoder as shown in Example 1.

[0299] The additional magnetic field source is a parallelepiped (20 x 10 x 1 mm) head-to-tail assembly of NdFeB macro magnets (N35, adhesion 800 g) magnetized along the 1 mm axis.

[0300] The capture elements are anti-ovalbumin (IgG) antibodies produced in mice. These antibodies are grafted onto the nanoparticles at a final concentration of 10 μg / mL to 50 μg / mL.

[0301] Engraftment on nanoparticles at a final concentration of 10 μg / mL is carried out according to the following protocol: · Activate 90 μg of nanoparticles (i.e., 3 μL of Ademtech 30 mg / mL stock solution of Ademtech nanoparticles) with 25 μL of a solution containing EDC (10 mg / mL) and NHS (10 mg / mL); Incubate for 15 minutes at room temperature with agitation; Remove the supernatant by capturing the nanoparticles using a centimeter magnet; this centimeter magnet is a nickel-plated neodymium magnetic cylinder with a diameter of 10 mm and a height of 40 mm; Add 25 μL (i.e., 25 μg) of a solution of anti-mouse ovalbumin antibody at a concentration of 1 mg / mL; Incubate for 2 hours at room temperature with stirring; Removing the supernatant by capturing the nanoparticles using the centimeter magnet described above; and · Suspend the nanoparticles in 50 μL of a solution of PBS-Tween 0.05%-BSA (1 mg / mL); nanoparticles functionalized with anti-ovalbumin antibodies are obtained at a potential concentration of 500 μg / ml.

[0302] The other part of the nanoparticles is grafted with ovalbumin (OVA). The grafting is carried out following the same protocol as above, except that instead of adding 25 μL of a solution of anti-ovalbumin antibodies, 25 μL of OVA at a concentration of 1 mg / mL (i.e. 25 μg) is added.

[0303] The detection element is an antibody specifically directed against mouse antibodies (and thus anti-ovalbumin antibodies) and is coupled to the Alexa 488 fluorescent dye (excitation maximum=490 nm; emission maximum=525 nm) for detection.

[0304] Figure 8 shows an anti-mouse antibody 15 bound to a nanoparticle complex 19 carrying a fluorescent dye 17 and engrafted with an anti-ovalbumin antibody 21. Also shown are nanoparticles 19 engrafted with OVA 23 used to test the specificity of the interaction between the antibodies.

[0305] Detection and quantification of anti-mouse ovalbumin antibodies is performed by the following protocol: In a 0.5 mL tube, mix 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; · Incubate in a 0.5 mL tube at ambient temperature for 15 minutes; Remove 5 μL of the solution and deposit a droplet onto the magnetic layer of the magnetic card; The magnetic card is deposited onto the additional magnetic field source such that the PVC support member is located between the magnetic layer and the additional magnetic field source.

[0306] Five different conditions are met: 1) 4.5 μg of nanoparticles grafted with anti-mouse ovalbumin antibody (i.e., anti-ovalbumin antibody at a concentration of approximately 50 μg / mL); 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); 3) 1.125 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 12.5 μg / mL); 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); 5) 4.5 μg of nanoparticles grafted with ovalbumin (i.e., zero concentration of anti-ovalbumin antibodies).

[0307] Image capture of the magnetic layer of the magnetic card is then performed with a fluorescence microscope (Olympus BX41M) equipped with a "GFP" cube (excitation 460-490 nm) coupled to a CCD camera (Diagnostic Instruments SPOT RT monochrome digital camera). A blue excitation light source (460-490 nm) is used. Images are captured at a total magnification of 50x and with an acquisition time of 5 s (gain 1).

[0308] The images after capture are shown in Figure 10. It is clearly observed that the nanoparticles bound to the anti-mouse antibody are captured along the capture zone in the form of a band. Figures 10A-10E correspond to conditions 1)-5), respectively, as described above. The amount of anti-mouse antibody detected is gradually decreasing for conditions 1)-5), which is consistent with the amount of anti-ovalbumin antibody used in each condition. Condition 5 is a negative control, since no capture antibody is present. These results are also consistent with the results shown in Figure 9.

[0309] The fluorescence signal is quantified by calculating the area corresponding to the fluorescence peak on the capture zone measured between the capture zones minus a general "background signal". Indeed, unlike examples 1-3, where the nanoparticles were fluorescent, here all the detected fluorescence does not only correspond to the captured molecules (in this case anti-ovalbumin antibodies), but also to the detection elements that remained in solution. It is then necessary to subtract the fluorescence emitted by these "free" detection elements from the fluorescence emitted by the detection elements bound to the captured molecules.

[0310] Taking the example of capture shown in FIG. 10, the total fluorescence of the area of ​​the capture zone is measured in the form of a fluorescence band, from which the fluorescence measured between these regions is subtracted.

[0311] Fluorescence quantification is given in arbitrary units (AU).

[0312] The results obtained are shown in FIG.

[0313] (result) As shown in FIG. 9, the resulting fluorescence quantification of the capture zone is proportional to the concentration of anti-ovalbumin antibody added to the mixture (R 2 =0.97).

[0314] From the fluorescence signal quantification method used (specific signal in the capture zone and non-specific signal outside it), these results allow to conclude that the nanoparticles bound to the detection elements are indeed captured in the capture zone.

[0315] However, these results allow concluding that it is possible to quantify the number of molecules captured without a washing step between the immobilization of the nanoparticles and the detection on the detection element.

[0316] Example 5: Trapping of nanoparticles by a microfluidic chamber 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.

[0317] A sonication step is also performed.

[0318] The capture support contains 18 microfluidic chambers, each with independent inlet and outlet vents. Each microfluidic chamber is 6 mm long, 2.4 mm wide, and 240 micrometers deep. The chambers are placed adjacent to each other with a pitch of 4.5 mm to form a bar. The microfluidic chambers are glued to a magnetic layer, which is then glued to a PVC support member. The support member and magnetic layer were assembled according to the ISO 7811 standard. The magnetic layer is encoded using an MSR605 encoder with a series of "1's" corresponding to 182 times the letter F in hexadecimal, which allows the magnetic field orientation to vary by 180° every approximately 55 μm.

[0319] Six microliters of nanoparticle solution was injected into each of these microfluidic chambers. The chambers were filled one by one. After each filling, the capture was visualized with a fluorescent microscope with a 10x magnification objective. The film was generated at a frame rate of 1.12 frames / s. Images obtained at times 0, 10, 30, 80 and 120 seconds of recording are shown in FIG. 12.

[0320] (result) Despite the substantial depth of the microfluidic chamber approaching 300 μm, it can be seen in FIG. 12 that the nanoparticles are well trapped at the bottom of the microfluidic chamber (the set of aligned white dots) from 10 seconds of trapping.

[0321] Example 6: Trapping of nanoparticles by a microfluidic chamber in the presence of an additional magnetic field source The nanoparticles and capture supports used are the same as in Example 5.

[0322] In addition, the capture support is based on a head-to-tail assembly (perpendicular magnetization) of 20 NdFeB macromagnets (Supermagnete, reference number Q-10-04-02-N) of parallelepiped shape (10 x 4 x 2 mm) magnetized along a 2 mm axis and with an energy product of 50 Megagauss Oersted. The 20 magnets are arranged side by side with a pitch of 0.5 mm along a 4 mm axis, forming a bar. Eighteen of the 20 magnets are placed below each of the 18 microfluidic chambers and two on each side.

[0323] Six microliters of nanoparticle solution was injected into each of these microfluidic chambers. The chambers were filled one by one. After each filling, the capture was visualized with a fluorescent microscope with a 10x magnification objective. The film was generated using image capture at a rate of 1.12 frames per second. Images obtained at times 0, 2, 5, 12, 34 and 60 seconds are shown in FIG. 13.

[0324] The percentage of nanoparticles captured by the microfluidic chamber is quantified following the protocol described in the publication of Fratzl et al., Soft Matter (14) 2671-2680 (2018). The capture kinetics were performed in three replicates in three different chambers. The capture kinetics results are shown in Figure 14.

[0325] (result) For Example 5, the nanoparticles are fully trapped at the bottom of the microfluidic chamber. Similar to Examples 1 and 3, the trapping of nanoparticles is much faster than Example 5 without the use of an external magnetic field source. In addition, as seen in Figure 13, every other trapping zone shown in Figure 12 disappears due to the external magnetic field generated by the macro-magnet assembly (the spacing between each line of dots is doubled in Figure 13).

[0326] The capture kinetics data shown in FIG. 14 indicates that nanoparticle capture was complete after 15 seconds. [Brief description of the drawings]

[0327] [Figure 1] 1 illustrates the capture of nanoparticles by a magnetic layer according to the present invention. [Diagram 2] Photographs of captures by the system shown in FIG. 1 are shown. [Diagram 3] 1 shows the trapping of nanoparticles by a magnetic layer according to the present invention, which is covered by a non-magnetic layer, and FIGURE 2 shows a trapping system in cross section. [Figure 4] A photograph of the capture by the system shown in Figure 3 is shown, where the white dots represent nanoparticles. [Figure 5a] 1 shows the trapping of nanoparticles by a magnetic layer according to the present invention, which is covered with a non-magnetic layer, the trapping being performed in the presence of a magnetic field from an additional magnetic field source, and FIG. [Figure 5b] 1 shows the trapping of nanoparticles by a magnetic layer according to the present invention, which is covered with a non-magnetic layer, the trapping being performed in the presence of a magnetic field from an additional magnetic field source, and FIG. [Figure 6] A photograph of the capture by the system shown in Figure 5a is shown, where the white dots represent nanoparticles. [Figure 7] Figure 1 shows the percentage (%) of nanoparticles captured (X-axis) as a function of time (Y-axis) for each capture shown in Figures 1-3. Time is expressed in minutes. Curve A corresponds to the capture kinetics shown in Figure 5a, curve B corresponds to the capture kinetics shown in Figure 1, and curve C corresponds to the capture kinetics shown in Figure 3. [Figure 8] 1 shows an anti-mouse detection capture antibody bound to a nanoparticle complex carrying a fluorescent dye and grafted with an anti-mouse ovalbumin capture antibody, which is also an ovalbumin grafted nanoparticle. [Figure 9] 1 is a graph showing the amount of fluorescence (arbitrary units) calculated as a function of the concentration (μg / mL) of anti-ovalbumin antibody. [Figure 10]Photographs are shown of the complexes of FIG. 8 after capture. Each white dot represents a fluorescent dye carried by the capture antibody (anti-mouse) of FIG. 8. For FIG. 10A, a mouse anti-ovalbumin concentration of about 50 μg / mL was used; for FIG. 10B, a concentration of about 25 μg / mL was used; for FIG. 10C, a concentration of about 12.5 μg / mL was used; for FIG. 10D, a concentration of about 6.25 μg / mL was used; FIG. 10E is a negative control without anti-mouse ovalbumin antibody. [Figure 11] 1 is a graph showing a calibration curve for DLGi5 garnet in a MagView CMOS type MOIF system, the graph showing the rotation (in degrees) of the polarization of a light beam passing through the garnet as a function of the magnetic field strength (in Tesla). [Figure 12] Photographs after trapping of nanoparticles in the microfluidic chamber are shown. White dots represent nanoparticles. Figures 12A-12E show the trapping kinetics (A: 0 s; B: 10 s; C: 30 s; D: 80 s and E: 120 s). [Figure 13] Photographs after trapping of nanoparticles in the microfluidic chamber in the presence of an additional magnetic field source are shown. White dots represent nanoparticles. Figures 13A-13F show the trapping kinetics (A: 0 s; B: 2 s; C: 5 s; D: 12 s; E: 34 s and F: 60 s). [Figure 14] The percentage of trapped nanoparticles (Y-axis) as a function of time of trapping (X-axis) is shown in Figure 12. Time is expressed in seconds. [Figure 15] 1 is a spectroscopic graph showing the size of nanoparticles in solution. The size was determined using a dynamic light scattering technique. The Y-axis represents the relative frequency of nanoparticles as a percentage, and the X-axis is a logarithmic scale representing the size of the nanoparticles in nanometers.

Claims

1. A kit for capturing a molecule contained in a sample, comprising: a) magnetic nanoparticles having a maximum dimension of less than 1 μm, said nanoparticles each being bound to at least one capture element, said at least one capture element specifically binding to said molecule; b) A support for capturing said magnetic nanoparticles on its surface, comprising or consisting essentially of at least one magnetic layer, said magnetic layer comprising a repeatable 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 not polarized or are polarized in a second direction different from said first polarization direction of said magnetic particles of said first regions, whereby said at least one magnetic layer has a magnetic field strength of at least 0.1 mT at a distance of at least 1 μm from said at least one magnetic layer. a support and a magnetic field having at least one variation in intensity, the first and second directions being opposite to each other and parallel to a surface of the support, the at least one variation in intensity defining a maximum and a minimum of the level of the intensity of the magnetic field and defining a zone for capturing the magnetic nanoparticles on the capture support at the maximum of the level of the magnetic field, the at least one magnetic layer being a flexible magnetic tape comprising a magnetic composite material, the magnetic composite material being randomly distributed in a polymer or oriented along a pre-orientation axis; Including the kit.

2. 2. The kit of claim 1, wherein the at least one magnetic layer has a coercivity of 2000 to 30,000 μm·Gauss.

3. The kit of claim 1 or 2, further comprising at least one additional magnetic field source.

4. The kit of any one of claims 1 to 3, wherein the at least one magnetic layer has a capture surface, the at least one magnetic layer being at least partially covered on the capture surface by a non-magnetic layer.

5. The kit of claim 4, wherein the non-magnetic layer has a thickness of 1 to 300 μm.

6. A method for capturing a molecule contained in a sample, the method comprising: a) contacting the sample with the magnetic nanoparticles of claim 1 to form at least one capture complex between the molecule and the at least one capture element bound to the magnetic nanoparticle; b) attracting the at least one capture complex formed during step a) by the magnetic field generated by at least one magnetic layer of the capture support such that the at least one capture complex is immobilized to the capture support in at least one capture zone according to claim 1. A method comprising:

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

8. 8. The method of claim 7, wherein the capture support further comprises a non-magnetic layer according to claim 5, and wherein the attraction of the at least one capture complex by the capture support during step b) is induced by the magnetic field of the at least one additional magnetic field source.

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

10. Use of the above defined kit according to any one of claims 1 to 5 for said capture of molecules contained in a sample.

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