KIT AND METHOD FOR CAPTURING A MOLECULE WITH MAGNETIC MEANS

Abstract

A kit and a method for capturing a molecule contained in a sample utilizing at least one magnetic layer including a, possibly repeated, juxtaposition of at least one first and one second region, the first region including magnetic particles polarized in a first direction and the second region including magnetic particles that are non-polarized or polarized in a second direction different from the first direction of polarization of the magnetic particles of the first region, so as to generate 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 the at least one magnetic layer, the variation defining a maximum of the standard of the intensity of the magnetic field and level therewith a zone for capturing magnetic nanoparticles on the capture support.

Claims

1.-10. (canceled)

11. A kit for capturing a molecule contained in a sample comprising: a) magnetic nanoparticles having a largest dimension of less than 1 μm, said nanoparticles each being coupled to at least one capture element, said at least one capture element specifically binding to said molecule, and b) a support for capturing said magnetic nanoparticles comprising or consisting essentially of at least one magnetic layer, said magnetic layer comprising a juxtaposition, possibly repeated, of at least a first and a second region, the first region comprising magnetic particles polarized in a first direction, and the second region comprising magnetic particles that are non-polarized or polarized in a second direction different from the first direction of polarization of the magnetic particles of the first region, so that 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 defining a maximum and a minimum of the standard of the intensity of said magnetic field, so as to define, at said maximum of the standard of said magnetic field, a zone for capturing the magnetic nanoparticles on the capture support.

12. The kit according to claim 11, wherein said at least one magnetic layer has a retentivity of 2000 to 30,000 μm.Math.Gauss.

13. The kit according to claim 11, further comprising at least one additional magnetic field source.

14. The kit according to claim 11, wherein said at least one magnetic layer having a capture surface, said at least one magnetic layer is at least partially covered on said capture surface by a non-magnetic layer.

15. The kit according to claim 14, wherein said non-magnetic layer has a thickness of 1 to 300 μm.

16. A method of capturing a molecule contained in a sample, said method comprising the following steps: a) bringing said sample into contact with magnetic nanoparticles, so as to form at least one capture complex between said molecule and said at least one capture element coupled to said magnetic nanoparticles; b) the attraction of said at least one capture complex as formed during step a) by the magnetic field generated by at least one magnetic layer of a capture support, so that said at least one capture complex is immobilized against said capture support at an at least one capture zone, wherein the magnetic nanoparticles, the at least one magnetic layer of a capture support, and said at least one capture zone are as defined according to claim 11.

17. The method according to claim 16, wherein the attraction of said at least one capture complex during step b) is carried out by the joint action of the magnetic field generated by said at least one magnetic layer and by the magnetic field generated by at least one additional magnetic field source.

18. The method according to claim 17, wherein the capture support further comprises a non-magnetic layer having a thickness of 1 to 300 μm, and where the attraction of said at least one capture complex by the capture support during step b) is triggered by means of the magnetic field of said at least one additional magnetic field source.

19. The method according to claim 18, wherein the sample is placed at the capture support before step a) of bringing into contact with the magnetic nanoparticles.

20. A method of capturing a molecule contained in a sample, comprising; providing the kit of claim 11, and bringing a sample into contact into contact with magnetic nanoparticles from said kit.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0263] FIG. 1 shows a capture of nanoparticles by a magnetic layer according to the invention. The capture system is shown in sectional view. The intensity of the magnetic fields emitted by the magnetic layer is defined by a color code whose scale is presented to the right of the sectional view (in T). The arrows in the magnetic layer represent the direction of the polarization of the grains composing it.

[0264] FIG. 2 shows a photograph of a capture by the system shown in FIG. 1. In this photograph, the white dots represent the nanoparticles.

[0265] FIG. 3 shows a capture of nanoparticles by a magnetic layer according to the invention, said layer being covered with a non-magnetic layer. The capture system is shown in sectional view. The intensity of the magnetic fields generated by the magnetic layer is defined by a color code whose scale is presented to the right of the sectional view (in 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 direction of the polarization of the grains composing it.

[0266] FIG. 4 shows a photograph of a capture by the system shown in FIG. 3. In this photograph, the white dots represent the nanoparticles.

[0267] FIG. 5a shows a capture of nanoparticles by a magnetic layer according to the invention, said layer being covered with a non-magnetic layer and the capture being carried out in the presence of the magnetic field from an additional source. The capture system is shown in sectional view. The direction of the magnetic field generated by the additional source is shown by the white arrow with a black border above the sectional view and is perpendicular to the magnetic layer. The intensity of the magnetic fields generated by the magnetic layer is defined by a color code whose scale is presented to the right of the sectional view (in 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 direction of the polarization of the grains composing it.

[0268] FIG. 5b shows a capture of nanoparticles by a magnetic layer according to the invention, said layer being covered with a non-magnetic layer and the capture being carried out in the presence of the magnetic field from an additional source. The capture system is shown in sectional view. The direction of the magnetic field generated by the additional source is shown by the white arrow with a black border above the sectional view and is parallel to the magnetic layer. The intensity of the total magnetic fields (generated by the magnetic layer and the additional source) is defined by a color code whose scale is presented to the right of the sectional view (in 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 direction of the polarization of the grains composing it.

[0269] FIG. 6 shows a photograph of a capture by the system shown in FIG. 5a. In this photograph, the white dots represent the nanoparticles.

[0270] FIG. 7 shows the percentage of nanoparticles captured (X axes) as a function of time (Y axes) for each capture of the captures shown in FIGS. 1 to 3. The time expressed in minutes. Curve A corresponds to the capture kinetics shown in FIG. 5a, curve B corresponds to the capture kinetics shown in FIG. 1, and curve C corresponds to the capture kinetics shown in FIG. 3.

[0271] FIG. 8 shows anti-mouse detection capture antibodies carrying a fluorochrome and coupled to nanoparticle complexes grafted with anti-mouse ovalbumin capture antibodies. It is also a nanoparticle grafted with ovalbumin.

[0272] FIG. 9 is a graph showing the amount of fluorescence calculated (arbitrary unit) as a function of the concentration of anti-ovalbumin antibody (μg/ml).

[0273] FIG. 10 shows photographs after capture of the complexes of FIG. 8. Each white point represents a fluorochrome carried by a capture antibody (anti-mouse) of FIG. 8. For FIG. 10A, a mouse antiovalbumin concentration of approximately 50 μg/ml was used; for FIG. 10B, a concentration of approximately 25 μg/ml was used; for FIG. 10C, a concentration of approximately 12.5 μg/ml was used; for FIG. 10D, a concentration of approximately 6.25 μg/ml was used; FIG. 10E is a negative control without the use of anti-mouse ovalbumin antibodies.

[0274] FIG. 11 is a graph showing the calibration curve of a DLGi5 garnet in an MOIF system of the MagView CMOS type. This graph shows the rotation of polarization of a light beam (in degrees) having passed through said garnet as a function of the intensity of the magnetic field (in Tesla).

[0275] FIG. 12 shows photographs after capture of nanoparticles in microfluidic chambers. The white dots represent nanoparticles. FIG. 12A to 12E show capture kinematics (A: 0 seconds; B: 10 seconds; C: 30 seconds; D: 80 seconds and E: 120 seconds).

[0276] FIG. 13 shows photographs after capture of nanoparticles in microfluidic chambers in the presence of an additional magnetic field source. The white dots represent nanoparticles. FIG. 13A to 13F show capture kinematics (A: 0 seconds; B: 2 seconds; C: 5 seconds; D: 12 seconds; E: 34 seconds and F: 60 seconds).

[0277] FIG. 14 shows the percentage of captured nanoparticles (Y axes), as a function of time (X axes) for the capture shown in FIG. 12. The time is expressed in seconds.

[0278] FIG. 15 is a spectroscopic analysis graph showing the size of nanoparticles in solution. The size was determined using the 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 nanoparticles in nanometers.

EXAMPLES

Example 1: Capture of Nanoparticles by a Magnetic Strip

[0279] The capture of nanoparticles by a magnetic strip of a magnetic card was tested first.

[0280] Experimental Protocol

[0281] The nanoparticles used (Chemicell nanoscreenmag ARA 200 nm) have an average diameter of 200 nm, a density of 1.25 g/cm.sup.3, a saturation magnetization of 420,000 A/m, a mass concentration of 25 mg/ml, a length emission wavelength of 476 nm and an excitation wavelength of 490 nm. The nanoparticles are diluted in ddH.sub.2O to a level of 1.1×10.sup.{circumflex over ( )}12 nanoparticles/g and 4.4×10.sup.9 nanoparticles/ml.

[0282] In order to break up the aggregates of nanoparticles that may be present, the solution of nanoparticles diluted in deionized water (ddH.sub.2O) was mixed using a SONIC RUPTOR 4000 sonicator. Intermittent sonication was produced within a tube at 20% of the total power of 400 W and an estimated frequency around 20000 Hz. A total of 3 sonication pulses were emitted with a duration of 500 milliseconds every 2 seconds.

[0283] In order to verify that the nanoparticles to be captured are not in the form of clusters of nanoparticles, the inventors have measured the size of the nanoparticles in solution by “Diffraction Light Scattering.” The results are given in FIG. 15.

[0284] The capture support is a magnetic card made up of a PVC support member and a magnetic strip made up of three magnetic layers arranged on the same plane and made up of high coercivity magnetic polymers. The support member and the magnetic layers were assembled according to the ISO 7811 standard. The magnetic layers are each encoded with a succession of “1” corresponding to 182 times the LETTER F in Hexadecimal using an MSR605 encoder, which allows the magnetic field orientations to be varied by 180 degrees approximately every 55 μm.

[0285] The capture of the nanoparticles is carried out by depositing 5 μl of the solution of nanoparticles and depositing a drop on the magnetic layers of the magnetic card.

[0286] A schematic view of this capture is shown in FIG. 1. In this figure, nanoparticles 1 are in solution, and some 1′ are attracted by a magnetic layer 3 of the magnetic strip (represented by black arrows). The magnetic layer has first 5 and second 7 regions whose grain polarization is reversed (represented by large white arrows). The magnetic field generated by each of the regions is represented by small white arrows that follow arcs starting on one side of a region and ending on the other side. The direction of these arrows indicates the direction of the generated magnetic field. The intensity of the field is represented by a color scale. This intensity of the magnetic field was obtained by the finite element approach carried out using the COMSOL Multiphysics® 5.0 modeling software, as described above. A white color indicates a strong intensity of the magnetic field. Above the magnetic layer 3, the intensity of the magnetic field generated by the first and second regions (5, 7) is visible, each of which is about 100 μm wide, the field exhibiting variations. The presence of maximums of the intensity of the magnetic field 9 can be clearly seen above the junctions between a first region 5 and a second region 7, and a lower intensity of the magnetic field at each region (5, 7) itself. At each maximum of the standard of the magnetic field 9, a capture zone 11 is thus defined by orthogonal projection on the surface of the magnetic layer 3, and at which the nanoparticles 1′ will be immobilized.

[0287] Then, images are captured with a fluorescence microscope (Olympus BX41 M) 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 with a total magnification of 50× and with a capture time of 3 seconds (Gain 14 db). An example of an image capture can be seen in FIG. 2. In this figure, the white dots represent the nanoparticles. An ordering of the captured nanoparticles can be clearly seen.

[0288] The percentage of nanoparticles captured by the magnetic strip is quantified by following the protocol described in the publication by Fratzl et al, Soft Matter (14) 2671-2680 (2018). Briefly, this quantification is obtained by the ratio of the area covered by the nanoparticles to the area not covered by the nanoparticles.

[0289] The results of the capture kinetics are shown in FIG. 7 and represented by curve B.

[0290] Results

[0291] As can be seen in FIG. 7, the capture of the magnetic nanoparticles is triggered instantly after depositing the drop on the magnetic substrate, and reaches 40% in 2 minutes.

[0292] Furthermore, the results shown in FIG. 15 show a single peak with suspended beads exhibiting an average 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.

Example 2: Capture of Nanoparticles by a Magnetic Strip Covered with a Non-Magnetic Layer

[0293] This example tests the capture of nanoparticles by a magnetic strip of a magnetic card covered with a non-magnetic layer.

[0294] Experimental Protocol

[0295] The nanoparticles used and the capture support are the same as those used in Example 1, except that the magnetic strip is covered by a self-adhesive layer of black polymer (Vinyl) 60 μm thick.

[0296] The method for capturing the nanoparticles, as well as the determination of the percentage of nanoparticles captured, are the same as those of Example 1.

[0297] A diagram of this capture is shown in FIG. 3. This view includes the elements shown in FIG. 1. In addition, a non-magnetic layer 13 is arranged on the surface of the magnetic layer 3. As can be seen in this FIG. 3, only a part of the magnetic fields generated by the magnetic layer 3 protrudes on the surface of the non-magnetic layer 13, so that the maximums of the standard of the magnetic field are “hidden” by the non-magnetic layer. There is therefore no variation in the intensity of said magnetic field of at least 0.1 mT at a distance of at least 1 μm from the surface of the capture support, and therefore no capture zones. The nanoparticles are therefore very weakly attracted, immobilized in a random manner against the magnetic strip and very predominantly remain in solution.

[0298] A photo of the capture result obtained is visible in FIG. 4, in which the white dots represent the nanoparticles. The ordering visible in FIG. 2 has disappeared in this figure.

[0299] The results of the capture kinetics are shown in FIG. 7, and represented by curve C.

Results

[0300] As can be seen in FIG. 7, the nanoparticles are captured very slowly by the magnetic strip of the magnetic card. After 10 minutes, the capture reaches only 15%.

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

[0301] This example tests the capture of nanoparticles by a magnetic strip of a magnetic card covered with a non-magnetic layer in the presence of an additional magnetic field source.

[0302] Experimental Protocol

[0303] The nanoparticles used and the capture support are the same as those used in Example 2.

[0304] The additional magnetic field source is a head-to-tail assembly (vertical/horizontal magnetization) of NdFeB macro-magnets (N35, adhesion force of 800 g) parallelepipedal (20×10×1 mm), magnetized along the 1 mm axis.

[0305] The magnetic card is deposited on the additional magnetic field source so that the magnetic layers are not arranged opposite said source.

[0306] The method for capturing the nanoparticles, as well as the determination of the percentage of nanoparticles captured, are the same as those of Example 1.

[0307] A diagram of this capture is shown in FIGS. 5a and 5b. In FIG. 5a, the 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 FIG. 5b, the 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. These two figures make it possible to see the effect of the direction of the magnetic field generated by the additional field source on the position of the capture zones.

[0308] These views show the elements presented in FIGS. 1 and 3. These figures clearly show on the one hand that the intensity of the magnetic field generated at the surface of the non-magnetic layer 13 is much stronger and that the maximums of the standard of the intensity of the magnetic field 9 are no longer hidden by the non-magnetic layer 13. Moreover, only every other maximum of the standard of the intensity of the magnetic field 9 is present compared to those initially shown in FIG. 1, and therefore every other capture zone 11 is present. However, these maximums 9 have an intensity greater than those initially shown in FIG. 1.

[0309] In FIG. 5a, with an additional magnetic field perpendicular to the magnetic layer 3, the capture zones 11 lie above the junctions toward which the polarization of the adjacent regions (5, 7) is oriented. However, the standard of the field is minimized above the junctions for which the polarization of the adjacent regions moves away.

[0310] It is interesting to note in FIG. 5b that with an additional magnetic field with a direction parallel to the magnetic layer 3, the capture zones 11 and the maximums of the standard of the intensity of the magnetic field 9 have been displaced and no longer arranged at the junctions between first and second regions (5, 7), but at the first regions 5 themselves.

[0311] A photo of the capture result obtained is visible in FIG. 6, in which the white dots represent the nanoparticles. Unlike FIG. 4, where there was no capture ordering, a new ordering appears in FIG. 6, different from that obtained in FIG. 2. Here, the nanoparticles form regularly arranged parallel bands, and we see that there are few nanoparticles present outside these bands.

[0312] The results of the capture kinetics are shown in FIG. 7, and represented by curve A.

[0313] Results

[0314] In this example, the capture of the magnetic nanoparticles is triggered immediately upon application of the external field. The capture is close to 100% in 2 minutes.

[0315] By combining the present result with that of Example 2, it can be concluded that it is possible to trigger the immobilization and the very rapid capture (2 minutes) of the nanoparticles when the magnetic strip is covered with a non-magnetic layer, by triggering the magnetic field of the additional source.

Example 4: Capture and Quantification of a Capture Element Coupled to Nanoparticles

[0316] Finally, the detection and quantification of several concentrations of anti-mouse ovalbumin antibodies (capture element) coupled to magnetic nanoparticles were tested.

[0317] Each measurement is carried out by keeping the number of detection antibodies (anti-mouse antibodies) and total nanoparticles identical, but varying the quantity of nanoparticles coupled to the anti-mouse ovalbumin antibodies (by supplementing with nanoparticles coupled to ovalbumin).

[0318] Experimental Protocol

[0319] The nanoparticles used (Carboxyl Adem beads 200 nm (ref. 02120—Ademtech)) have a diameter of 200 nm, an approximate density of 2.0 g/cm.sup.3, an approximate saturation magnetization of 40 emu/g, an approximate iron oxide content of 70% and a solid content of 30 mg/ml (3%). The nanoparticles are covered with COOH carboxylic functions with a density greater than 350 μmol/g.

[0320] The capture support is a magnetic card composed of a PVC support member on which three magnetic layers rest that are arranged on the same plane and composed of high coercivity magnetic polymers. The support member and the magnetic layers were assembled according to the ISO 7811 standard. The magnetic layers are each encoded with a succession of 1s using an MSR605 encoder, as shown in Example 1.

[0321] The additional magnetic field source is a head-to-tail assembly of NdFeB macro-magnets (N35, Adhesion force of 800 g) parallelepipedal (20×10×1 mm), magnetized along the 1 mm axis.

[0322] The capture element is an anti-ovalbumin (IgG) antibody produced in mice. These antibodies are grafted onto the nanoparticles at a final concentration of 10 μg/ml to 50 μg/ml.

[0323] The grafting on the nanoparticles at a final concentration of 10 μg/mL is carried out by the following protocol: [0324] activation of 90 μg of nanoparticles (i.e. 3 μl of Ademtech 30 mg/ml stock solution of Ademtech nanoparticles)) with a 25 μl solution containing EDC (10 mg/ml) and NHS (10 mg/ml); [0325] incubation for 15 minutes at room temperature with stirring; [0326] removal of the supernatant by capturing the nanoparticles using a centimeter magnet. This centimeter magnet is a neodymium magnetic cylinder, nickel-plated, 10 mm in diameter and 40 mm high; [0327] addition of a solution of 25 μl of anti-mouse ovalbumin antibody at a concentration of 1 mg/ml (i.e. 25 μg); [0328] incubation for 2 h at room temperature with stirring; [0329] removal of the supernatant by capturing the nanoparticles using a centimeter magnet as mentioned above; and suspension of the nanoparticles in a solution of 50 μL of PBS-Tween 0.05%—BSA (1 mg/ml). Nanoparticles functionalized with anti-ovalbumin antibody are obtained at a potential concentration of 500 μg/ml.

[0330] The other part of the nanoparticles is grafted with Ovalbumin (OVA). The grafting is carried out according to the same protocol as that described above with the difference that instead of adding a solution of 25 μl of anti-ovalbumin antibody, 25 μl of OVA are added at a concentration of 1 mg/ml (i.e. 25 μg).

[0331] The detection element is an antibody directed specifically against mouse antibodies (therefore against the anti-ovalbumin antibody) and is coupled to an Alexa 488 fluorochrome (Max excitation=490 nm; Max emission=525 nm) for detection.

[0332] FIG. 8 shows anti-mouse antibodies 15 carrying a fluorochrome 17 and coupled to the nanoparticle complexes 19 grafted by the antiovalbumin antibodies 21. Also shown is a nanoparticle 19 grafted with OVA 23 used for testing the specificity of the interaction between the antibodies.

[0333] The detection and quantification of anti-mouse ovalbumin antibodies is carried out by the following protocol: [0334] in a 0.5 ml tube, mixture of 4.5 μg of nanoparticles previously grafted with anti-ovalbumin antibodies and/or with OVA); 2 μl of anti-mouse detection antibody (for a final concentration of 1 μg/ml) and 20 μl of PBS; [0335] incubation for 15 minutes at ambient temperature in the 0.5 mL tube; [0336] removing 5 μl of the solution and depositing a drop on the magnetic layers of the magnetic card; and [0337] depositing the magnetic card on the additional magnetic field source, so that the PVC support member is arranged between the magnetic layers and the additional magnetic field source.

[0338] Five different conditions are met: [0339] 1) 4.5 μg of nanoparticles grafted with anti-mouse ovalbumin antibodies (i.e. an approximate anti-ovalbumin antibody concentration of 50 μg/ml); [0340] 2) 2.25 μg of nanoparticles grafted with anti-mouse ovalbumin antibodies and 2.25 μg nanoparticles grafted with ovalbumin (i.e. an approximate anti-ovalbumin antibody concentration of 25 μg/ml); [0341] 3) 1.125 μg of nanoparticles grafted with anti-mouse ovalbumin antibodies and 2.25 μg nanoparticles grafted with ovalbumin (i.e. an approximate anti-ovalbumin antibody concentration of 12.5 μg/ml); [0342] 4) 0.625 μg of nanoparticles grafted with anti-mouse ovalbumin antibodies and 2.25 μg nanoparticles grafted with ovalbumin (i.e. an approximate anti-ovalbumin antibody concentration of 6.25 μg/ml); [0343] 5) 4.5 μg of nanoparticles grafted to ovalbumin (i.e. a zero anti-ovalbumin antibody concentration);

[0344] Then, image captures of the magnetic layers of the magnetic card are taken with a fluorescence microscope (Olympus BX41 M) 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 with a total magnification of 50× and with a capture time of 5 seconds (Gain 1).

[0345] FIG. 10 shows the images obtained after capture. It is clearly observed that the nanoparticles coupled to the mouse antibody, which in turn is coupled to the anti-mouse antibody, are captured along capture zones in the form of bands. FIG. 10A to 10E respectively correspond to conditions 1) to 5) as described above. The amount of anti-mouse antibody detected is degressive for conditions 1) to 5), which is consistent with the amount of anti-ovalbumin antibody used in each condition. Condition 5 is a negative control, as no capture antibody is present. These results also agree with those obtained in FIG. 9.

[0346] The fluorescent signal is quantified by calculating the respective areas corresponding to the fluorescence peaks on the capture zones from which the general “background signal” is subtracted that is measured between the capture zones. In fact, unlike Examples 1 to 3 where the nanoparticles were fluorescent, here all the fluorescence detected not only corresponding to the captured molecules (the anti-ovalbumin antibodies in the present case), 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 that emitted by the detection elements coupled to the captured molecule.

[0347] Taking the example of the capture shown in FIG. 10, the total fluorescence of the areas of the capture zones is measured in the form of fluorescent bands, from which the fluorescence measured between these areas is subtracted.

[0348] The fluorescence quantification is obtained in arbitrary units (AU)

[0349] The results obtained are shown in FIG. 9.

[0350] Results

[0351] As shown in FIG. 9, the fluorescence quantification of the capture zones obtained is proportional to the concentration of anti-ovalbumin antibodies that were added to the mixture (R.sup.2=0.97).

[0352] From the fluorescence signal quantification method used (specific signal in the capture zones and non-specific signal outside it), these results make it possible to conclude that the nanoparticles coupled to a detection element are indeed captured in the capture zones.

[0353] However, these results make it possible to conclude that it is possible to quantify the number of molecules captured without a washing step between the immobilization of the nanoparticles and the detection of the detection element.

Example 5: Capture of Nanoparticles by Microfluidic Chambers

[0354] The nanoparticles used are the same as those used in Example 1, and were diluted 500 times in deionized water (ddH.sub.2O) to achieve a concentration of 50 μg/mL.

[0355] The sonication step is also carried out.

[0356] The capture support comprises 18 microfluidic chambers each having an independent inlet and a vent at the outlet. Each microfluidic chamber is 6 mm long, 2.4 mm wide, and has a depth of 240 micrometers. The chambers are aligned next to each other with a pitch of 4.5 mm so as to form a bar. The microfluidic chambers are glued to a magnetic layer, which in turn is glued to a PVC support member. The support member and the magnetic layer were assembled according to the ISO 7811 standard. The magnetic layer is encoded with a succession of “1” corresponding to 182 times the LETTER F in Hexadecimal using an MSR605 encoder, which makes it possible to vary the magnetic field orientations by 180 degrees approximately every 55 μm.

[0357] 6 microliters of the nanoparticle solution were injected into each of these microfluidic chambers. The chambers were filled one by one. After each filling, the capture was visualized with an epifluorescence microscope with a 10× magnification objective. A film was generated with a frame rate of 1.12 frames per second. The images obtained at times 0, 10, 30, 80 and 120 seconds of the recording are shown in FIG. 12.

[0358] Results

[0359] Despite the substantial depth of the microfluidic chambers, approaching 300 μm, it can be seen in FIG. 12 that the nanoparticles are well captured at the bottom of the microfluidic chambers (sets of aligned white dots), from 10 seconds of capture.

Example 6: Capture of Nanoparticles by Microfluidic Chambers in the Presence of an Additional Magnetic Field Source

[0360] The nanoparticles and the capture support used are the same as in Example 5.

[0361] In addition, the capture support is based on a head-to-tail assembly (vertical magnetization) of 20 NdFeB macro-magnets (Supermagnete, reference Q-10-04-02-N), parallelepipedal (10×4×2 mm), magnetized along the 2 mm axis and having an energy product of 50 megaGauss Oersted. The 20 magnets are arranged side by side with a pitch of 0.5 mm along the 4 mm axis so as to form a bar. 18 of the 20 magnets are placed below each of the 18 microfluidic chambers, and 2 are placed on each side.

[0362] 6 microliters of the nanoparticle solution were injected into each of these microfluidic chambers. The chambers were filled one by one. After each filling, the capture was visualized with an epifluorescence microscope with a 10× magnification objective. A film was generated with an image capture at the rate of 1.12 frames per second. The images obtained at time 0, 2, 5, 12, 34 and 60 seconds are shown in FIG. 13.

[0363] The percentage of nanoparticles captured by the microfluidic chambers is quantified by following the protocol described in the publication by Fratzl et al, Soft Matter (14) 2671-2680 (2018). Capture kinetics were performed in triplicate in 3 different chambers. The results of the capture kinetics are shown in FIG. 14.

[0364] Results

[0365] As for Example 5, the nanoparticles are well captured at the bottom of the microfluidic chambers. By analogy with Examples 1 and 3, the capture of the nanoparticles is much faster here than in Example 5, without an external magnetic field source. In addition, as can be seen in FIG. 13, every other capture zone shown in FIG. 12 has disappeared (the spacing between each line of dots has doubled in FIG. 13), due to the external magnetic field generated by the assembly of the macro-magnets.

[0366] The capture kinetics data shown in FIG. 14 show that the capture of the nanoparticles is complete after 15 seconds.