METHOD FOR TRAKING AND DETECTING A MOLECULE IN A SAMPLE WITHOUT WASHING IT

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a method for capturing and detecting, particularly by immunoassay, a molecule in a sample, said molecule to be captured or detected typically being an antigen or an antibody. Also described below, but not forming part of the claimed invention, is a molecule capture kit adapted for carrying out said methods. BACKGROUND OF THE INVENTION

[0002] The ELISA test (acronym for the English term "Enzyme-Linked Immuno Sorbent Assay") is commonly used to quantitatively diagnose molecular markers (antigens or antibodies) present in fluids, biopsies, cultures or any other sample.

[0003] This test is particularly used to diagnose infections (HIV, HPV, etc.), the state of the cardiovascular and immune systems (Troponin C, specific antibodies, etc.), the presence and development of cancers (PSA, CEA, etc.).

[0004] This technique, which is currently the most robust and widespread diagnostic method, has some drawbacks, namely its complexity, the use of expensive automated equipment and its duration, which can reach several hours.

[0005] The ELISA test is a heterogeneous phase immunoassay technique, meaning that it requires a solid support (typically, a titration plate comprising a plurality of wells) to which a suitable molecule is previously attached to capture the molecule to be measured.

[0006] Once the molecule of interest has been captured on said support, a washing allows the rest of the sample to be removed and the detection and quantification step of said molecule to proceed.

[0007] For example, in the case of the so-called "sandwich" ELISA test, which allows the measurement of an antigen in a solution, the surface of the support is coated with a determined quantity of a so-called capture antibody, the said antibody being adapted to bind to the antigen sought.

[0008] Then the solution likely to contain said antigen is applied to the support; said antigen then binds to the capture antibody located on the surface of the support.

[0009] Next, the support is washed to remove any unbound antigen that may remain in the solution.

[0010] A solution containing a detection antibody, which is adapted to bind to the antigen fixed to the support and is also coupled to an enzyme, is then deposited on the support.

[0011] A new washing step is implemented, so as to retain on the support the antigen bound to the detection antibody, said antibody being itself coupled to the enzyme.

[0012] Finally, for the detection and quantification of the antigen, a substrate is deposited on the support which is converted by the enzyme into a detectable signal (for example a color or spectroscopically, i.e. by emission of fluorescence) representative of the binding between the antigen and the detection antibody.

[0013] This signal can be observed with the naked eye or by means of an instrument, such as a spectrophotometer.

[0014] The ELISA test includes different implementations, but they all include multiple grafting and incubation steps separated by washing steps.

[0015] The significant duration of the ELISA test is mainly due to the succession of different steps.

[0016] Variations of this test using magnetic microbeads have already been described.

[0017] The article by D. Issadore et al, Self-assembled magnetic filter for highly efficient immunomagnetic separation, Lab Chip, 2011, 11, 147 describes a method for capturing a molecule in a sample by circulating said sample in a fluidic microchannel arranged below a polydimethylsiloxane (PDMS) matrix in which magnetic NdFeB grains have been immobilized.

[0018] Due to the manufacturing process used for said support, the grains are randomly distributed in the thickness of the matrix and their magnetic orientation is random.

[0019] Each grain generates a magnetic field gradient which is likely to attract magnetic particles with a diameter of 1 µm coupled to the molecules to be detected to form complexes.

[0020] However, such a magnetic field gradient is less effective the smaller the size of the particles to be captured, so that this device is less efficient for capturing magnetic particles smaller than 1 µm, for example on the order of 500 nm or less, and especially nanoparticles.

[0021] On the other hand, the capture of magnetic particles is relatively slow.

[0022] This capture step is followed by washing the surface of the PDMS matrix and then fluorescence detection of the complexes immobilized on said surface. Guillaume Blaire et al., 2013 (Eur Phys JB, 86(4), 165, 1-6) describe a method for capturing and detecting a molecule in a sample by fluorescence, in which the molecule is mixed with magnetic nanoparticles, and the complexes are immobilized on a microchannel. The micro-sources of magnetic fields are ordered, and complex detection is performed by fluorescence. The method includes a washing step and uses a nanoparticle concentration of less than 10⁶ particles / ml. BRIEF DESCRIPTION OF THE INVENTION

[0023] One aim of the invention is to provide a method for detecting a molecule such as an antigen or an antibody requiring a limited number of steps, or even a single step, and which is significantly shorter than known tests.

[0024] This process must also be compatible with the instrumentation already used by biologists, such as titration plates, fluorometers, multichannel pipettes, so that it can be directly implemented in research and analysis laboratories.

[0025] According to the description, but not part of the claimed invention, a method for capturing a molecule in a sample is proposed, comprising the following steps: a) mixing said sample with magnetic particles, each of said particles being coupled with an element capable of selectively binding to said molecule to be captured, so as to form at least one complex comprising a magnetic particle b) immobilizing said at least one complex on a support comprising ordered magnetic field microsources.

[0026] According to the invention, a method for capturing a molecule in a sample is proposed, comprising the following steps: a) mixing said sample with magnetic particles, each of said particles being coupled with an element capable of selectively binding to said molecule to be captured, so as to form at least one complex comprising a magnetic particle, said element and said molecule bound to said element, said element and said molecule bound to said element, these magnetic particles having a diameter between 5 nm and 500 nm, b) immobilizing said at least one complex on a support comprising ordered microsources of magnetic field, c) detecting the immobilized molecule by fluorescence, where the spatial organization of the ordered micro magnetic field sources defines areas of strong magnetic field gradient and areas of weak magnetic field gradient, in that the detection step is implemented directly after the immobilization step, without an intermediate step of washing the support, by determining the fluorescence density on the surface of the ordered micro magnetic field sources in the areas of strong magnetic field gradient, defining a specific signal, and in the areas of weak magnetic field gradient, defining a non-specific signal, where said support is a microchannel comprising in at least one of its walls an array of orderedly arranged micro-magnetic sources, and where the concentration of magnetic particles in the mixture obtained at the end of step a) is greater than 10 6< particles / ml.

[0027] By "ordered," we mean that the micro-sources of magnetic fields are distributed near the surface of the support intended to be in contact with the sample according to a specific pattern, and that they also have a specific magnetic orientation. Such an arrangement effectively excludes a random distribution and / or orientation of the micro-sources of magnetic fields.

[0028] According to one embodiment of the description, but not part of the claimed invention, the process includes a washing step of said support to remove said sample, the complexes being retained on the support by the micro-sources of magnetic field.

[0029] According to the invention, the detection of complexes is implemented at the level of ordered micro-sources of magnetic fields, without prior washing of the support.

[0030] In this text, the term "molecule" refers to a protein, including an antibody, a nucleic acid (DNA or RNA), a virus, a bacterium, an antigen, a cell, and generally any biological object.

[0031] According to one embodiment of the description, but not part of the claimed invention, the magnetic particles have a diameter between 5 nm and 1 µm.

[0032] According to the invention, the magnetic particles have a diameter between 5 nm and 500 nm.

[0033] Preferably, the magnetic particles have a diameter between 5 and 50 nm.

[0034] According to one embodiment, the support comprises a non-magnetic matrix containing a plurality of ordered three-dimensional agglomerates of magnetized micro or nanoparticles of a hard or soft magnetic material, said agglomerates forming the micro-sources of magnetic field.

[0035] According to another embodiment, the support comprises a non-magnetic matrix containing a plurality of magnetic micro-coils arranged in a pattern determined with respect to the surface of the support.

[0036] The said non-magnetic matrix can be flexible.

[0037] Furthermore, said non-magnetic matrix may be made of a translucent or transparent material.

[0038] According to one embodiment of the invention, the molecule to be captured is an antigen and the element capable of binding to said molecule is an antibody that is a receptor for said antigen.

[0039] Optionally, a detection antibody bearing a fluorescent, luminescent, or colorimetric marker is added to the mixture. This antibody is capable of binding to the antigen bound to the antibody coupled to the magnetic particle. The detection antibody may also be labeled with an enzyme that catalyzes a redox reaction, enabling electrochemical detection.

[0040] According to another embodiment of the invention, the molecule to be captured is an antibody and the element capable of binding to said molecule is an antigen recognized by said antibody.

[0041] According to one embodiment of the description, but not part of the claimed invention, the support is a titration plate comprising a plurality of wells, the micro-sources of magnetic field being arranged in a wall of each of said wells, the mixture being deposited in at least one of said wells.

[0042] According to another embodiment of the description, but not part of the claimed invention, the support is a flat plate comprising a plurality of micro-sources of magnetic field, the mixture being deposited in the form of at least one drop on said support.

[0043] According to the invention, the support is a microfluidic channel of which at least one of the walls comprises micro-sources of magnetic field.

[0044] Another object of the invention relates to a method for detecting and, where appropriate, quantifying a molecule, in which said molecule is captured on a support by implementing the capture method described above, and then a step of detecting said molecule is implemented.

[0045] According to the description, but not part of the claimed invention, the detection of the molecule captured on the support can be carried out by luminescence, colorimetry, electrochemistry and / or radiometry.

[0046] According to the invention, the detection of the molecule captured on the support is carried out by fluorescence.

[0047] A first advantage of this process is the significant reduction in the number of steps prior to detection and, where applicable, quantification (only a mixing step and, possibly, a washing step are required), compared to known immunoassay tests (the standard sandwich ELISA test comprising at least six steps).

[0048] Indeed, the attraction of magnetic particles by micro-sources of magnetic field allows on the one hand to quickly extract the objects associated with said particles and on the other hand ensures a more efficient washing (in the cases of the description, but not part of the claimed invention, where such washing is implemented), the magnetic interaction forces firmly retaining the immobilized complexes that one wishes to preserve.

[0049] Furthermore, this process offers an alternative to all possible implementations of the ELISA test, and can be implemented to perform "sandwich" type tests as well as direct tests or competitive tests by choosing the appropriate antigens and antibodies.

[0050] This process is also adaptable for performing immunoprecipitation tests.

[0051] Another advantage of this process is its great simplicity of implementation and its low cost, combined with the absence of the need for an extreme energy source for capturing the complexes and washing.

[0052] Insofar as the washing step prior to detection can be dispensed with, the process may only include a single mixing step in contact with the support containing the ordered micro-sources of magnetic field.

[0053] Because of its simplicity, the process could be implemented in an integrated "point-of-care" type testing device.

[0054] Another object of the description, but not part of the claimed invention, relates to a molecule capture kit, comprising a support including ordered micro-sources of magnetic field and a plurality of magnetic particles.

[0055] These magnetic particles have a functionalized surface adapted for coupling an element capable of selectively binding to said molecule.

[0056] According to one embodiment, but not part of the claimed invention, the support is a titration plate comprising a plurality of wells, the micro-sources of magnetic field being arranged in at least one wall of each well.

[0057] According to one embodiment, but not part of the claimed invention, the support is a flat plate on the surface of which the micro-sources of magnetic field are arranged.

[0058] According to one embodiment, the support is a fluidic microchannel, the magnetic field microsources being arranged on the surface of at least one of the walls of said microchannel.

[0059] Another advantage of this method is that it allows for the detection of the captured molecule without washing, thanks to the experimenter's knowledge of the areas where the magnetic particles are captured. The specific signal is obtained by subtracting from the total signal a signal corresponding to the non-specific capture of molecules.

[0060] According to one embodiment, but not part of the claimed invention, the molecules are detected by fluorescence, luminescence, or colorimetry using a detection antibody. A fluorescence, luminescence, or absorbance image of the surface bearing the micromagnets allows the determination of the specific and non-specific signals. The total signal corresponds to the presence of molecules in areas of high magnetic field gradient, while the non-specific signal corresponds to the presence of molecules in areas of low magnetic field gradient.

[0061] According to another embodiment, but not part of the claimed invention, the molecules are detected electrochemically using a detection antibody coupled to an enzyme having redox properties. Electrodes are arranged on the surface of the support so as to allow the measurement of the total and non-specific signal. The total signal corresponds to electrodes arranged at the level of ordered micro-sources of magnetic fields, the non-specific signal corresponds to electrodes arranged at areas devoid of micro-sources of magnetic fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Other features and advantages of the invention will become apparent from the detailed description that follows, with reference to the accompanying drawings in which: THE Figures 1A to 1Care schematic views of the implementation steps of the detection process according to an embodiment, not forming part of the claimed invention but useful for understanding it, the figure 2 , not part of the claimed invention but useful for understanding it, schematically illustrates a magnetically structured surface designed to form micro-sources of magnetic field, the figures 3A to 3D , not forming part of the claimed invention but useful for its understanding, schematically illustrate the different stages of the thermo-magnetic imprinting process for creating a substrate comprising micro-sources of magnetic field, the Figures 4A to 4D , not forming part of the claimed invention but useful for its understanding, schematically illustrate the different stages of the so-called "Micro Magnetic Imprinting" process, the figure 5, not forming part of the claimed invention but useful for understanding it, illustrates an embodiment of the invention in which the mixture is deposited on a flat surface in the form of a drop, the figure 6 , not part of the claimed invention but useful for understanding it, presents a comparison of the percentages of beads captured on a flat support and in a titration well as a function of time, the figures 7A to 7E illustrate a method for manufacturing a fluidic microchannel comprising micro-sources of magnetic fields enabling the implementation of the method according to the invention, the figure 8 is a curve of the fluorescence signal as a function of the amount of antigen obtained during experiments, the figure 9 represents the streamlines of a fluid containing magnetic particles placed in contact with a support comprising two magnetic domains magnetized in opposite directions, the Figure 10Arepresents the distribution of fluorescent complexes captured by magnetic particles at micro-sources of magnetic fields, determined during experiments and observed by microscopy; the figure 10B illustrates the ratio between the specific signal and the non-specific signal measured on microscopy images of the type of Figure 10A . DETAILED DESCRIPTION OF METHODS OF IMPLEMENTING THE INVENTION

[0063] THE Figures 1A to 1C schematically illustrate the main steps of the detection process corresponding to a "sandwich" type immunoassay test.

[0064] In this case, the molecule to be detected and measured is an antigen.

[0065] This process involves mixing the following elements: a sample likely to contain the antigen 1 whose presence is to be detected and quantified, magnetic particles 2 previously coupled to a capture antibody 3, i.e. a receptor protein specific to the antigen to be detected, in excess of the antigen, a detection antibody 4 carrying a fluorescent, luminescent or colored marker 5, in excess of the antigen.

[0066] The said marker 5 can be carried directly by the detection antibody or indirectly, via an enzyme.

[0067] The said mixture is made in a container, in which, under the effect of the diffusion of the different constituents, the antigen 1 binds to the capture antibody 3 coupled to a magnetic particle 2, then the detection antibody 4 binds to the antigen 1.

[0068] This forms a "sandwich" complex consisting of a magnetic particle 2, the capture antibody 3, the antigen 1, the detection antibody 4 and the marker 5.

[0069] The bonds between the antigen and the capture antibody, and between the detection antibody and the antigen, are non-covalent bonds.

[0070] In contrast, the coupling between the magnetic particle and the capture antibody is a covalent bond.

[0071] The said mixture is deposited, for example by means of a pipette, onto a support comprising ordered micro-sources of magnetic field.

[0072] The effect of micro-magnetic field sources is to attract magnetic particles and consequently immobilize the aforementioned complexes on the substrate, according to a pattern closely linked to the arrangement of the micro-magnetic field sources relative to the surface of said substrate. For example, if the micro-magnetic field sources are arranged in a checkerboard pattern where two adjacent squares have magnetizations in opposite directions, the magnetic particles will immobilize at the junction between two micro-magnetic field sources, which is where the leakage field, and its gradient, is most intense.

[0073] Antigens not bound to magnetic particles and labeled antibodies not bound to the antigen are not attracted by the micro-sources of magnetic field remain in suspension in the mixture.

[0074] An optional washing step, as described but not part of the claimed invention, allows these residues to be eliminated and only the complexes to be retained on the support, these being retained by the micro-sources of magnetic field by interaction with the magnetic particles.

[0075] Indeed, the magnetic force generated on the complexes by the micro-sources of magnetic field (which are capable of generating very strong magnetic field gradients) is greater than the suction force exerted during washing.

[0076] In this way, the molecules of interest, coupled to the magnetic particles, were captured and held on the support.

[0077] However, as explained in detail below, it is also possible to dispense with the washing step. Indeed, knowledge of the ordering of the micro-sources of magnetic field relative to the surface of the support and the implementation of an appropriate detection technique make it possible to detect the particles trapped by these ordered micro-sources without the need to remove the sample or wash the surface of the support.

[0078] Once this capture has been carried out, a quantification method known to the person skilled in the art can then be implemented, depending on the nature of the markers used.

[0079] Among these quantification methods, we can note fluorescence, luminescence, colorimetry, a RedOX reaction, radioactivity, turbidimetry, magnetic detection by localized GMR micro and nano-magnetic sensors, etc. According to the invention, quantification is done by fluorescence.

[0080] The total duration of this test is typically between 10 and 20 minutes, whereas it is between 2 and 6 hours for an ELISA test implementing the classic mixing and detection steps.

[0081] Compared to the ELISA test, which, as mentioned above, is a heterogeneous phase immunoassay method, the method according to the invention can be considered a homogeneous phase immunoassay method.

[0082] Indeed, the binding between the antigen to be measured and the detection antibody occurs in the volume of the sample and not on the surface of the support, the said support being intended only to immobilize the complexes during the washing prior to detection and quantification or for direct detection without washing as envisaged above.

[0083] The presence, in said volume, of a large number of magnetic particles of dimensions which may be smaller than those used in known processes, and the high diffusion coefficient which their small size confers on these particles, increases their probability of encountering the detection antigens and antibodies.

[0084] It should be noted that, although the example given above concerns a "sandwich" type immunoassay test, which can replace the classic sandwich ELISA test, the process can be adapted to offer the same modalities as other immunoassay tests (of the direct ELISA or competitive ELISA type) or other processes involving the capture of a molecule, such as immunoprecipitation.

[0085] For example, an immunoprecipitation test is intended to purify a specific protein from a sample containing a plurality of proteins.

[0086] This test uses antibodies specifically designed to bind selectively to the protein we want to detect.

[0087] In such a test, the antibodies are coupled to a plurality of magnetic particles and the particles are introduced into the sample.

[0088] Antibodies linked to magnetic particles will then bind to proteins that are specifically recognized.

[0089] The proteins that we seek to isolate are linked to antibodies which are themselves coupled to magnetic particles, thus forming complexes.

[0090] Thanks to ordered micro-sources of magnetic field, it is then possible to immobilize said complexes on a support and then, if necessary, to proceed with a wash to remove the rest of the sample.

[0091] In contrast, in processes not using magnetic field sources, the antibodies are chemically bound to the well of the titration plate which has been previously functionalized.

[0092] Compared to the method of D. Issadore et al, the use of a support comprising ordered micro-sources of magnetic field makes it possible, for a given size of magnetic particles, to increase the range of the micro-sources of magnetic field and / or, at a given range, to increase the force exerted on the small particles.

[0093] Therefore, the invention allows for faster and more efficient capture.

[0094] On the other hand, the ordering of the micro-sources of magnetic field makes it possible to carry out the detection without necessarily washing the support, which is not possible when the micro-sources of magnetic field are arranged randomly in the support, the person skilled in the art then being unable to differentiate the captured particles from the uncaptured particles. Magnetic particles

[0095] The particles used are magnetic particles of micrometric (microparticles) or nanometric (nanoparticles) dimensions. According to the invention, the magnetic particles have a diameter between 5 nm and 500 nm.

[0096] Advantageously, these particles can be considered as beads.

[0097] In the rest of the text, the particles can therefore be referred to as "balls" even if their geometry is not a perfect sphere.

[0098] Such beads are commercially available in large quantities and at low cost, particularly for applications such as magnetic resonance imaging (MRI), which does not increase the cost of the test.

[0099] Preferably, said beads are monodisperse, the dimensional uniformity of the beads giving them identical properties and thus improving the diffusion of the beads in the mixture and their capture by the micro-sources of magnetic field.

[0100] In some cases, the beads are marketed in a dispersed form in a matrix with little or no magnetic properties, such as a polymer, silica (SiO2), etc.

[0101] The beads are preferably biocompatible.

[0102] To enable the coupling by covalent bond of an element intended to couple selectively with the molecule to be captured, the surface of the particles is functionalized, for example by A or G proteins.

[0103] In this text, the term "nanoparticle" refers to an object whose three dimensions are on the nanometric scale, that is to say, whose characteristic size, for example the average diameter if it is a ball, is typically less than 100 nm.

[0104] The term "microparticle" refers to an object whose three dimensions are on the micrometric scale, that is to say whose characteristic size, for example the average diameter if it is a ball, is between 100 nm and 100 µm.

[0105] Unless otherwise stated, the term "particle" or "bead" used in the rest of the text encompasses both nanoparticles and magnetic microparticles.

[0106] Due to their dimensions, the magnetic particles used exhibit superparamagnetic properties.

[0107] It is recalled that the term "superparamagnetic" refers to the property of small ferromagnetic or ferrimagnetic material particles to randomly change the direction of magnetization in the absence of an applied magnetic field, under the effect of thermal agitation.

[0108] The characteristic of being superparamagnetic implies that in the absence of an external excitation magnetic field, the beads have no net magnetic moment, so they do not attract each other, thus preventing their agglomeration.

[0109] Compared to microparticles, nanoparticles, once coupled to an antibody, exhibit much greater performance in terms of antigen capture.

[0110] This improvement can be explained by the reduction in scale of nanoparticles compared to microparticles.

[0111] Thus, for a diameter reduction of a factor of 100 (for example, between a microbead with a diameter of 3 µm and a nanobead with a diameter of 30 nm): the diffusion coefficient is multiplied by 100, the surface area / volume ratio is multiplied by 100, the number of beads per m³ is multiplied by 10⁶.

[0112] Since capture antibodies bind to the surface of particles, increasing the surface area / volume ratio allows, for an equal mass of particles, an increase in the number of capture antibodies and consequently improves the sensitivity of the test.

[0113] Furthermore, reducing particle size increases their diffusion rate in the mixture and therefore allows for faster complex formation.

[0114] Another advantage of nanoparticles is that they are of equivalent dimensions to the biological objects (DNA strands, cancer markers, specific proteins) that we seek to detect.

[0115] Thus, they are animated by the same Brownian motion as these objects, which increases the ability to capture them.

[0116] Finally, increasing the number of capture antibodies using nanoparticles shifts the thermodynamic equilibrium of the association reaction and could therefore allow the use of antibodies currently neglected due to their lower affinity.

[0117] Therefore, although the method according to the description, but not part of the invention, can be implemented with any size of magnetic particle, particles with a diameter between 5 nm and 1 µm will preferably be chosen, more preferably between 5 nm and 500 nm, or even between 5 nm and 100 nm or between 5 and 50 nm. Micro-sources of magnetic field

[0118] By micro-source of magnetic field we mean a magnet whose three dimensions are on the micrometric scale, that is to say whose length, width and thickness are less than 1 mm.

[0119] According to one embodiment, said micro-sources of magnetic field are made of a hard magnetic material that can be permanently magnetized.

[0120] This ensures the autonomy of the test device, i.e. avoids any need for an electrical power supply or an external magnetic field to enable the capture of superparamagnetic particles.

[0121] However, it is conceivable that said micro-sources could be made of a soft magnetic material, with an external magnetic field source (for example a permanent magnet or an electromagnet) then having to be placed in the vicinity of the support to magnetize them, temporarily or permanently.

[0122] The magnetic field gradient is generally strongest at the periphery of a micro-source of magnetic field and, where applicable, at the interface between two differently oriented micro-sources of magnetic field.

[0123] The micro-sources of magnetic field used in the invention generate magnetic fields comparable to those of currently available macroscopic magnets, i.e. of an intensity on the order of 0.1 to 1×10 6< A / m.

[0124] On the other hand, the magnetic field gradients measured above the surface of the support can reach 10< 6 T / m, that is, several orders of magnitude greater than the gradients generated by macroscopic magnets.

[0125] The magnetic forces induced on magnetic particles are much greater than those generated by macroscopic magnets (i.e., those with at least two dimensions greater than 1 mm), which can be explained by the change of scale.

[0126] Thus, reducing the magnetic field source by a factor of 1000 (for example between a 10 mm cube magnet and a 10 µm magnet) has the effect of multiplying the magnetic force exerted by the micro-source by 1000.

[0127] Therefore, such micro-sources of magnetic field are currently the only experimentally proven means of efficiently capturing nanoparticles.

[0128] To date, the inventors have experimentally demonstrated that these micro-sources of magnetic field are indeed capable of capturing nanoparticles with a diameter greater than or equal to 5 nm.

[0129] However, the interest in micro-sources of magnetic fields is not limited to superparamagnetic nanoparticles.

[0130] Indeed, even for microparticles, the increase in magnetic force compared to that of conventional macroscopic magnets facilitates the capture of complexes comprising microparticles.

[0131] Micro-sources of magnetic fields can be manufactured by one of the following processes.

[0132] According to one embodiment of the invention, the micro-sources are manufactured by magnetic structuring at the micrometric scale of a layer of a hard magnetic material.

[0133] The production of high-performance permanent magnets at the micrometer scale can involve the following two successive steps: the synthesis of high-performance thick magnetic films, with a thickness between 1 and 100 µm; the realization of micro-sources of magnetic field (which can be considered as micro-permanent magnets) by magnetic structuring of said magnetic film. Magnetic film synthesis

[0134] A physical deposition process by triode sputtering which allows the deposition of a magnetic material in thick layers (typically 1-100 µm) is described in NM Dempsey et al., App. Phys. Lett. 90, 092509 (2007).

[0135] As an example, layers of NdFeB, FePt and SmCo have been synthesized and possess remarkable magnetic properties.

[0136] Other methods of manufacturing layered magnets can be considered, in particular electrolytic deposition, sol-gel deposition, evaporation deposition, pulsed laser deposition, etc.

[0137] It is also possible to glue a layer of magnetic material onto a substrate.

[0138] The materials that can be used to form these layers are hard magnetic materials, which can then be permanently magnetized.

[0139] Among the materials suitable for this use, we can mention rare earth magnets, of formula RFeB (where R consists essentially of Nd, Pr, Tb, Dy or a mixture of several of these elements), RCo or RCoCu (crystallographic structure of type 1 / 5), RCoCuFe (crystallographic structures of type 1 / 7 or 2 / 17), RFeN (where R consists essentially of Sm), transition metal alloys of the 3d series (Fe, Co, Ni)-noble metal (Pt or Pd as the major element), ferrite magnets, MnBi, MnAl, MnGa, FeGa, AINiCo magnets.

[0140] According to an advantageous embodiment, the hard magnetic material is chosen from the NdFeB, SmCo and FePt families.

[0141] Furthermore, the generation of strong magnetic fields and magnetic field gradients requires magnetic microstructuring of the hard magnetic material layer. This structuring ensures the ordering of the micro-sources of magnetic field.

[0142] Most advantageously, said layer can be magnetically micro-structured by one of the two techniques presented below. Magnetic structuring using the so-called "topographic" method

[0143] This technique involves structuring the substrate before deposition, on which the hard magnetic material is then deposited, and / or directly structuring the magnetic layer.

[0144] The dimensions of the topographic structures determine the dimensions of the micro-magnets obtained.

[0145] Each portion of magnetic layer etched and / or deposited on a micro-plot or in a micro-cavity of the substrate can be considered as an independent magnetic element.

[0146] Note that planarization, optical lithography, and chemical etching steps may be necessary.

[0147] These micro-elements thus produced are then magnetized in a chosen direction.

[0148] They then constitute a set of independent micro-magnets, all exhibiting the same direction of magnetization [A. Walther et al., J. Magn. Mag. Mat. 321 (2009) 590].

[0149] These individual micro-magnets, possibly located at different heights, constitute systems exhibiting very strong magnetic field gradients at micrometer scales [M. Kustov et al., J. App. Phys. 108, 063914 (2010)].

[0150] There figure 2 illustrates schematically an example of topographic microstructuring.

[0151] Substrate 5 comprises a plurality of micro-cavities which, in this example, appear as parallel trenches.

[0152] The hard magnetic material is then deposited in these trenches so as to form parallelepiped magnetic regions 6.

[0153] The formation of such cavities and the deposition of magnetic material are in themselves known to those skilled in the art and will therefore not be described in detail here.

[0154] If necessary, polishing is then carried out to obtain a flat plate whose surface has an alternation of regions of the substrate 5 and regions of hard magnetic material 6.

[0155] An external magnetic field is then applied so as to permanently magnetize the regions 6 of hard magnetic material.

[0156] A person skilled in the art is able to define the intensity of the magnetic field to be applied to obtain the desired magnetization according to the materials used.

[0157] Such micro-sources of magnetic field generate a strong magnetic field gradient and are therefore suitable for the capture and immobilization of magnetic nano or microparticles.

[0158] The dimensions of micro-sources are on a sub-millimeter scale and depend on the precision of the topographic processes used.

[0159] It goes without saying that the shapes of these micro-sources are not limited to parallelepiped shapes; they can thus be arranged in the form of a checkerboard, but also have a circular, hexagonal, etc. shape. Magnetic structuring using the so-called "imprint" method thermo-magnetic (or "Thermo-Magnetic Patterning" according to Anglo-Saxon terminology)

[0160] The principle of thermo-magnetic imprint structuring is to use a heat source to locally heat certain areas of a magnetically hard layer and thus create volumes magnetized in alternating directions, constituting micro-sources of magnetic field.

[0161] As an example, a nanosecond pulsed laser can be used as a heat source according to a method whose principle is described with reference to figures 3A to 3D .

[0162] As illustrated in the figure 3A , a layer 6 of a hard magnetic material is magnetized in a given direction and sense (schematized by the arrow).

[0163] This layer 6 is then placed in a uniform external magnetic field H ext ( µ 0 H ext < µ 0 H c) in the opposite direction to the original magnetization direction, then it is locally irradiated by a pulsed L laser (in our case a KrF type excimer laser (248 nm), or an Nd-YAG laser).

[0164] As illustrated in the figure 3B said irradiation is carried out through a mask M which, in this example, is provided with a number of parallel rectangular openings.

[0165] The temperature on the surface of the irradiated areas increases very rapidly, then the heat diffuses into the material (cf. figure 3C ).

[0166] Given the reduction of the coercive scope µ 0 H c As the temperature of a material increases, magnetic reversal of the irradiated areas can be achieved by applying an external magnetic field during the laser pulse. H ext .

[0167] As illustrated in the 3D figure, the layer is ultimately made up of a network of micro-magnets with alternating magnetizations shown schematically by the arrows and dimensions defined by the dimensions of the mask used during laser irradiation.

[0168] Systems made by this process exhibit very strong magnetic field gradients at the micrometer scale [M. Kustov et al., J. App. Phys. 108, 063914 (2010)].

[0169] For more details concerning this process, see the article by F. Dumas-Bouchiat et al., App. Phys. Lett. 96, 102511 (2010).

[0170] This thermo-magnetic imprinting principle can be extended to all kinds of hard magnetic layers, including those with isotropic or in-plane magnetizations. A process known as "Micro Magnetic Imprinting"

[0171] According to another embodiment of the invention, the micro-sources 21 are in the form of ordered agglomerates of nano or microparticles of a hard magnetic material enclosed in a non-magnetic matrix and magnetized.

[0172] A particularly advantageous feature is that the matrix can be made of a flexible material, which allows for a flexible support.

[0173] THE Figures 4A to 4D illustrate steps in a method of manufacturing the support according to the invention.

[0174] With reference to the Figure 4A , a master substrate M1 is used to order nano or microparticles before enclosing them in a non-magnetic matrix.

[0175] The said master substrate M1 has a magnetically structured face, that is to say a face M10 made up of a plurality of micro-sources M10a, M10b of magnetic field distributed according to a determined pattern.

[0176] The magnetization of the different micro-sources is shown schematically by an arrow.

[0177] On the Figures 4A to 4D , the magnetically structured M10 face of the master substrate is flat.

[0178] However, in certain embodiments of the invention, it may be preferable to use a master substrate whose face is not flat but has cavities or bumps, in order to reproduce in the film formed from the master substrate complementary bumps or cavities.

[0179] The master substrate can be manufactured using various techniques known to those skilled in the art.

[0180] In particular, a thermo-magnetic imprint structuring process or a topography structuring process, as described above, can be implemented.

[0181] In a non-limiting example illustrated in the Figure 4A, the master substrate consists of a magnetically hard M11 layer of NdFeB with a thickness of 5 µm on a silicon support substrate M12 and magnetically structured by thermo-magnetic imprinting so as to form M10a regions with magnetization opposite to that of the M10b regions.

[0182] In this master substrate, the intensity of the magnetic field and the magnetic field gradient is maximum at the interfaces between the M10a, M10b regions exhibiting opposite magnetizations.

[0183] Therefore, nano or microparticles exhibiting a positive magnetic susceptibility are attracted by the magnetophoretic force towards these interfaces.

[0184] The master substrate is not limited to this particular shape, however, but can be made of a hard or soft magnetic material structured by topography.

[0185] The master substrate may optionally be an electrical conductor.

[0186] When the master substrate has a topographically structured surface, i.e., not flat, this particular topography can be used to print a complementary shape on the final film.

[0187] When it is desired to form a flat film from such a master substrate, it is necessary to planarize it beforehand, either by removing material (for example by means of a mechano-chemical polishing to remove the bumps), or by adding material (for example by means of a deposition technique to fill the cavities).

[0188] With reference to the Figure 4B , nano or microparticles P of a hard magnetic material are sent onto the magnetically structured face of the master substrate 1.

[0189] Under the effect of the attractive magnetophoretic force of the magnetically structured face of the master substrate, the P particles agglomerate at the edges of each of the micro-sources.

[0190] Since the force decreases as one moves away from the face of the master substrate, these agglomerates 21 generally have a triangular or trapezoidal cross-section, with a wider base on the side of the master substrate M1 and decreasing with distance.

[0191] To promote the ordering of particles not only in a plane but also in a direction perpendicular to the master substrate, it is possible to simultaneously apply an external magnetic field with an appropriate direction.

[0192] During particle delivery, the master substrate is advantageously agitated to optimize particle trapping at the edges of the micro-magnetic field sources.

[0193] Furthermore, during or after this sending, a jet of dry gas can be projected to promote the trapping of particles at the level of said edges and / or to eliminate untrapped particles before the deposition of the non-magnetic matrix.

[0194] The particles are made of a hard magnetic material, which can be one of the materials from the list developed above.

[0195] Therefore, applying an external magnetic field after the film has formed will permanently magnetize them and thus form a self-contained magnetically structured film.

[0196] With reference to the figure 4C , then, on the particles agglomerated on the master substrate, a matrix 22 in a non-magnetic material is deposited in the form of a thin layer.

[0197] The thickness of matrix 22 is typically between 100 nm and 5 mm

[0198] Advantageously, the matrix is ​​made of an elastomeric material, which gives the film a certain degree of flexibility.

[0199] According to preferred embodiments of the invention, the matrix is ​​made of one of the following materials: polydimethylsiloxane (PDMS), epoxy resin, rubber, bakelite, polymethyl methacrylate (PMMA), polystyrene (PS), photosensitive resin, parylene, oxides such as SiO2, Al2O3, metals such as Cu, Ag, carbon materials such as graphite, DLC, etc.

[0200] If necessary, matrix 22 is allowed to harden or crosslink for the appropriate time.

[0201] Furthermore, the matrix can advantageously be made of a transparent or translucent material.

[0202] A person skilled in the art is able to select an appropriate matrix from among the products available on the market based on the desired properties.

[0203] With reference to the figure 4D , we delaminate the film 2 consisting of the matrix 22 and the agglomerates 21 of magnetic particles with respect to the master substrate M1.

[0204] The master substrate can then be reused for the manufacture of a new film.

[0205] Thus, although the master substrate requires the implementation of microfabrication techniques and therefore presents a certain cost, it can be reused indefinitely, and the fabrication of the magnetically structured film itself, which does not involve

[0206] no such complex and expensive techniques, only inexpensive materials are used.

[0207] This process is also easily industrialized and allows for the production of large surface area films in large quantities at low cost.

[0208] According to one embodiment of the invention, a layer of a material can be deposited on the master substrate before depositing the particles, which facilitates the delamination of the film and remains attached to the master substrate during delamination.

[0209] Given the small size of the particles to be immobilized on the support, the ordered micro-sources of magnetic field are preferably arranged directly on the surface of the support - or in the immediate vicinity of it - in contact with the sample to provide maximum attractive forces. Multi-well plate

[0210] Conventionally, titration plates comprising a plurality of wells (e.g., 96 or 384 wells) are used in immunodetection tests.

[0211] To immobilize the complexes, ordered micro-sources of magnetic fields, as described above, can be integrated into such a plate.

[0212] This integration can be achieved by manufacturing the plate itself from a suitable material to form ordered micro-sources of magnetic fields, according to one of the processes described above. The micro-sources of magnetic fields are then preferably placed at the bottom of each well.

[0213] According to another embodiment, but not part of the claimed invention, it is possible to insert inside each well a support of the dimensions of said well and on which said ordered magnetic field microsources are arranged.

[0214] According to another embodiment, but not part of the claimed invention, it is possible to make a titration plate in which the wells have no bottom, and to bring under said plate a flat support containing ordered micro-sources of magnetic field arranged at the level of each well, so as to constitute the sealed bottom of said wells.

[0215] Thus, the process is compatible with the supports usually used in immunoassay, which does not require modification of existing laboratory equipment. Support plan

[0216] According to a particularly advantageous embodiment of the description, but not part of the claimed invention, the support on which the complexes are immobilized is not a titration plate but a flat support on the surface of which are arranged a plurality of ordered magnetic micro-sources.

[0217] Such a support can be achieved by the various micro-structuring processes of magnetic layers described above.

[0218] Alternatively, the said support can be produced by the "Micro Magnetic Imprinting" process described above.

[0219] This manufacturing technique has the advantage of being able to produce a large quantity of supports at low cost, only the master substrate, which is reusable at will, being expensive.

[0220] Furthermore, with this technique, the support has the advantage of being able to be made of a transparent or translucent material.

[0221] This property can in some cases facilitate detection and quantification by optical methods.

[0222] Thus, it allows the support to be illuminated from below, which makes it possible to observe the coloration from above the support with a camera and filters that a person in the trade will know how to choose.

[0223] On the contrary, this transparency can be problematic, for example in the case of intense fluorescence, where signals could interfere between contiguous locations.

[0224] Therefore, the material of the support can be chosen according to the detection method envisaged.

[0225] When such a flat support is used, the mixture of the molecule to be detected, magnetic particles, and the element capable of selectively binding to said molecule is prepared in a container, then limited volumes of the mixture are taken and deposited onto said support in droplet form, as illustrated in the figure 5 .

[0226] The droplet size depends on the volume deposited and the hydrophilicity of the support material. This size is therefore adjustable, typically ranging from 1 microliter to 200 microliters.

[0227] The ordered micro-sources of magnetic field are advantageously distributed over the surface of the support in a regular pattern, so as to allow the capture of complexes regardless of the region of the support on which the drop has been deposited.

[0228] The location for each drop is preferably marked on the flat surface, for example by local magnetic marking, or chemically (by making the surface of the support locally hydrophilic or hydrophobic), or topographically, by a shallow depression made in the surface of the support. It is also possible to visually mark the location to help the experimenter locate it.

[0229] Unexpectedly, it was shown that the capture rate of magnetic beads was significantly higher in drops deposited on such a flat support than in a solution deposited in a well of a titration plate.

[0230] There figure 6 presents the percentage of 100 nm diameter beads captured respectively in a droplet (data represented by a disk) and the quantity of beads of the same type captured in a well (data represented by a square).

[0231] After 10 minutes, 75% of the beads were captured by the ordered micro-sources of magnetic field arranged at the bottom of the titration well, while more than 90% of the beads were recovered by the ordered micro-sources of magnetic field arranged in the support receiving the drop.

[0232] This phenomenon is explained by the presence of a convection movement induced inside the fluid by the conveyance of magnetic particles towards the ordered micro-sources of magnetic field which capture them.

[0233] The rapid diffusion of the particles therefore makes it possible to do without any vibration device which is necessary in conventional tests to accelerate the mixing of the solution.

[0234] Another advantage of the flat support compared to a conventional titration plate is that it allows, for the same support footprint, to provide more molecule capture sites. Kinetics of magnetic nanoparticle capture by a support comprising ordered micro-magnetic field sources

[0235] The remarkable kinetics and capture efficiency of magnetic nanoparticles by supports comprising ordered micro-sources of magnetic fields are due to hydrodynamic coupling between the particles.

[0236] Indeed, the magnetic particles closest to the surface of the support are rapidly attracted by the ordered micro-sources of magnetic field, and their drag hydrodynamically drives the movement of more distant particles towards the micro-sources of magnetic field, which, once brought closer to the surface of the support, are in turn magnetically attracted.

[0237] This coupling between magnetic forces and hydrodynamic drive results in the appearance of a convection fluidic current which stirs the fluid above the micro-sources of magnetic field, which explains the efficiency and long-distance capture kinetics of the nanoparticles, despite their small size.

[0238] This interpretation is justified by a theoretical model and by an experiment demonstrating the existence of this coupling.

[0239] In this experiment, the inventors visualized the movement of a fluid sample using non-magnetic colored particles suspended in a fluid deposited on a linear junction between two magnetized areas.

[0240] This junction simulates an element of an ordered micro-source magnetic field matrix produced by the Thermo Magnetic Patterning technique described above.

[0241] It is at this junction that the highest magnetic field gradients are located.

[0242] Measuring the movement and speed of the balls (using a technique called "particle tracking velocimetry") made it possible to reconstruct the movement and speed of the fluid.

[0243] On the figure 9 , which illustrates the streamlines induced by the attraction of superparamagnetic nanoparticles suspended above a junction between two magnetic domains D1, D2 whose magnetization directions are represented by arrows (the z-axis designating the fluid thickness above the junction), we clearly see that two symmetrical vortices are created rapidly on either side of the junction, which stir the liquid over a distance on the order of several mm.

[0244] In the absence of magnetic microparticles, no ordered movement of the fluid is apparent.

[0245] The measured fluid velocity field corresponds, to within 10%, to what is calculated by the hydrodynamic-magnetic coupling model described above.

[0246] This coupling occurs for magnetic particle concentrations greater than 10< 6< particles / ml, which is indeed observed experimentally.

[0247] The formation of convection currents is intimately linked to the ordering of micro-sources of magnetic field. Microfluidic system

[0248] According to the invention, the support is a "lab-on-chip" type microfluidic system.

[0249] The support then comprises a microchannel and an array of magnetic micro-sources arranged in an orderly fashion in at least one wall of said channel.

[0250] The said microchannel can be produced according to the "Micro Magnetic Imprinting" process described above.

[0251] In this case, as illustrated in figures 7A to 7E, the magnetically structured face of master substrate 1 is not flat but has at least one bump or cavity whose negative will be present on the film.

[0252] In the non-limiting example illustrated in the figure 7A , the magnetically structured face 10 of the master substrate 1 has a raised bump 10', the surface of the bump being made up of a plurality of micro-sources of magnetic field 10a, 10b.

[0253] For example, the said plot may have a parallelepiped shape.

[0254] Depending on the applications targeted, the rest of the surface of the master substrate can also be magnetically structured, but it is also conceivable that only the surface of the 10' pad is magnetically structured; in the latter case, the nano or microparticles brought in will only agglomerate at the edges of the magnetic micro-sources constituting the surface of the 10' pad, the rest of the surface of the master substrate not retaining any particles.

[0255] As explained above, magnetic nano or microparticles are brought onto the master substrate, where they order and aggregate into three-dimensional structures 20 at the edges of the micro-sources of magnetic field 10a, 10b ( figure 7B ).

[0256] Next, with reference to the figure 7C , the non-magnetic matrix 3 is deposited onto the master substrate.

[0257] Following delamination, the resulting film 4 is illustrated in the figure 7D therefore presents a cavity 40 complementary to the plot 10' of the master substrate.

[0258] Depending on the shape of the boss, the film thus contains one or more wells, or one or more channels.

[0259] From such a film, a micro-fluidic channel can therefore be formed.

[0260] To that end, as illustrated in the figure 7E, it is sufficient to apply against said film 4 a flat film 4' or any other structure allowing to form the fourth wall of the cavity 40.

[0261] Advantageously, said 4' plan film can also be manufactured according to the invention and comprise on its surface three-dimensional magnetic structures 20'.

[0262] Thus, from films 4 and 4', we form a device 6 comprising a micro-channel 60 which presents, on two opposite walls 61a and 61b, a magnetically structured surface.

[0263] A surface treatment can help ensure the seal between the two films.

[0264] For example, when both films have a PDMS matrix, surface activation can be performed by an oxygen plasma.

[0265] Thanks to the presence of three-dimensional magnetic structures on both faces 61a and 61b of the micro-channel 61, the trapping of nano or microparticles in a solution circulating in said micro-channel is improved.

[0266] Naturally, the nature of the magnetic particles, as well as their distribution relative to the surface of each wall, may be identical for each of the two walls or may be different.

[0267] In such a system, the mixture is injected at the entrance of the microchannel: the solution circulates in the microchannel, the complexes being progressively immobilized on the bottom of the microchannel by the micro-sources of magnetic field.

[0268] A washing step can then possibly be carried out to remove residues from the solution, with the complexes remaining magnetically trapped by the micro-sources located on the walls of the channel. Example of coupling an antibody to a magnetic particle (commercial Dynabead protocol)

[0269] As an example, the coupling of an antibody to a magnetic bead can be carried out according to the following procedure: Resuspend the beads by vortexing a solution in which they are dispersed, take the desired quantity of beads, remove the supernatant using a pipette and a magnet, add the antibody diluted in 0.01% PBS-Tween, incubate for 10 minutes at room temperature with agitation, remove the supernatant using a pipette and a magnet, resuspend the beads coupled with the antibody in 0.01% PBS-Tween. Experimental validations

[0270] An embodiment of the invention has been validated in a "sandwich" type antigen assay test carried out under the following conditions.

[0271] For this experiment, the biotinylated "coreprotein" of the hepatitis C virus was used as the antigen to be measured, a monoclonal antibody specific to this protein (Pierce Hepatitis C Virus Core Antigen Monoclonal Antibody (C7-50)) was used, superparamagnetic beads of 100 nm in diameter, functionalized with protein A (fluidMAG-Protein A, Chemicell) as the capture antibody, and quantum dots functionalized with avidin (QD625, Invitrogen) as the detection antibody.

[0272] In this experiment, the concentrations used were 1.3 nM for the capture antibody, 10⁹ / ml for the superparamagnetic beads and 10¹⁰ / ml for the quantum dots.

[0273] Quantum dots, antibody-functionalized beads and biotinylated antigen were placed, in a single step, on a flexible substrate including micro-sources of magnetic field. The mixing / capture time was set at 7 minutes.

[0274] After this time, the supernatant was removed and two washes were carried out with 50 µl of PBS.

[0275] Finally, the amount of antigen was measured directly on the support using the fluorescence of quantum dots.

[0276] There figure 8 illustrates fluorescence (in arbitrary units) as a function of antigen concentration. The entire test was completed in about ten minutes.

[0277] The detection limit is 10 pM, which is comparable to the limit of commercially available ELISA tests performed on this protein.

[0278] Beyond 5 nM, the test was positive, but the test response was saturated because of the camera used.

[0279] In any case, such concentrations are well beyond clinical cases; moreover, it is possible to dilute the sample to repeat the test if necessary. Detection without washing

[0280] The spatial organization of ordered micro-sources of magnetic field allows for detection without washing of the support or labeling of immuno-complexes.

[0281] This is done by determining the fluorescence density on the surface of the micro-sources of magnetic field ordered in the areas of strong magnetic field gradient (specific signal) and in the areas of weak magnetic field gradient (non-specific signal).

[0282] According to the description, but not part of the claimed invention, this detection can be done by fluorescence, by focusing the image capture on the surface of the support containing the micro-sources of magnetic field, or by arranging the detectors on the surface of the support itself.

[0283] According to the invention, this detection is achieved by fluorescence, by focusing the image capture on the surface of the support containing the micro-sources of magnetic field, or by arranging the detectors on the surface of the support itself.

[0284] The following experiment demonstrates the feasibility of such detection.

[0285] Biotin-functionalized magnetic nanoparticles (50 nm, 10⁹ ml⁻¹) and non-magnetic fluorescent nanoparticles (quantum dots, 30 nm, 10⁹ ml⁻¹) were mixed with low concentrations of avidin, a protein that binds almost irreversibly to four biotin molecules.

[0286] This coupling mimics the formation of immuno-complexes (magnetic nanoparticles)-(capture antibody)-(antigen)-(detection antibody)-(fluorescent marker) or other.

[0287] After 10 minutes of incubation, a drop is deposited on the surface of a support comprising ordered micro-sources of magnetic field produced by the Micro-Magnetic Imprinting technique and observed under a fluorescence optical microscope 10 minutes later.

[0288] The microscope is focused on the support.

[0289] On the Figure 10A , the image in the upper left shows the structure of the support including the ordered micro-sources of magnetic field observed in phase contrast (the micro-sources of magnetic field being arranged according to a grid which has a darker color than the rest of the matrix) while the image in the lower left shows the distribution of biotinylated quantum dots on the surface of the support in the presence of biotinylated magnetic nanoparticles and avidin, observed in fluorescence (the quantum dots have a light color in this image).

[0290] The superimposition of the two images on the right of the Figure 10A allows us to see the quantum dots resulting from a specific interaction with the magnetic nanoparticles: we can clearly see that the quantum dots (light in color) are mainly aligned along the grid (dark in color) defined by the micro-sources of magnetic field (specific signal) and therefore reproduce the pattern defined by the micro-sources of magnetic field, with only a few quantum dots located on areas inside this grid (non-specific signal).

[0291] In the absence of avidin, no pattern is visible.

[0292] The fluorescence density in areas of strong and weak magnetic field gradient was measured on the microscopy images shown in A.

[0293] There figure 10Bshows the evolution of the ratio (specific signal) / (non-specific signal), which is equal to the ratio (specific fluorescence x area of ​​regions with low magnetic field gradient) / (non-specific fluorescence x area of ​​regions with high magnetic field gradient) as a function of avidin concentration.

[0294] If no specific interaction occurs, this ratio is 1. The shaded area corresponds to the 6-sigma confidence interval. All points outside this area are significant with a probability of error less than 10⁻⁶.

[0295] A concentration of 50 nM is therefore detectable at the significance level of 99.9997% (six sigma).

[0296] The microstructuring of ordered micro-sources of magnetic fields makes it possible to define "specific" and "non-specific" regions of interest. It can therefore be used to simplify detection.

[0297] It should be noted that randomly placed micro-sources of magnetic fields would not allow this detection without washing. REFERENCES

[0298] D. Issadore et al, Self-assembled magnetic filter for highly efficient immunomagnetic separation, Lab Chip, 2011, 11,147 NM Dempsey et al., App. Phys. Lett. 90, 092509 (2007) A. Walther et al., J. Magn. Mag. Mast. 321 (2009) 590 M. Kustov et al., J. App. Phys. 108, 063914 (2010) F. Dumas-Bouchiat et al., App. Phys. Lett. 96, 102511 (2010)

Claims

1. A method for capturing and detecting a molecule in a sample, comprising the following steps: a) mixing said sample with magnetic particles, each of said particles being coupled with an element capable of binding selectively to said molecule to be captured, so as to form at least one complex comprising a magnetic particle, said element and said molecule bound to said element, these magnetic particles having a diameter comprised between 5 nm and 500 nm, b) immobilizing said at least one complex on a support comprising ordered magnetic field micro-sources, and c) detecting the immobilised molecule by fluorescence, characterised in that spatial organization of the ordered magnetic field micro-sources defines strong magnetic field gradient areas and low magnetic field gradient areas, and in that the detection step is implemented directly after the immobilisation step, without any intermediate step of washing the support, by determining the fluorescence density at the surface of the ordered magnetic field micro-sources in the high magnetic field gradient areas, defining a specific signal, and in the low magnetic field gradient areas, defining a non-specific signal, in that said support is a micro-channel comprising in at least one of its walls a network of magnetic micro-sources arranged in an ordered manner, and in that the concentration of magnetic particles in the mixture obtained upon completion of step a) is higher than 106 particles / ml.

2. The method according to claim 1, characterised in that the mixture obtained in step a) is injected at the inlet of the microchannel, flowing in the microchannel, and in that the complexes are progressively immobilised on the bottom of the microchannel by the magnetic field micro-sources during step b).

3. The method according to claim 1 or 2, characterised in that the micro-channel comprises a nonmagnetic matrix embedding a plurality of ordered three-dimensional agglomerates of magnetised micro- or nanoparticles made of a hard or soft magnetic material, said agglomerates forming the magnetic field micro-sources.

4. The method according to one of claims 1 to 3, characterised in that the molecule to be captured is an antigen and in that the element capable of binding to said molecule is a receptor antibody of said antigen.

5. The method according to claim 4, characterised in that a detection antibody carrying a fluorescent marker is added to said mixture, said antibody being capable of binding to the antigen bound to the antibody coupled to the magnetic particle.

6. The method according to one of claims 1 to 3, characterised in that the molecule to be captured is an antibody and in that the element capable of binding to said molecule is an antigen recognized by said antibody.