METHOD FOR MAGNETIC DETECTION OF MICROSCOPIC BIOLOGICAL OBJECTS AND ASSOCIATED DEVICES

Description

DOMAINE TECHNIQUE DE L'INVENTION

[0001] The technical field of the invention is the identification of microscopic biological objects from their magnetic moment in order to develop an early and sensitive diagnostic device. ARRIÈRE-PLAN TECHNOLOGIQUE DE L'INVENTION

[0002] The development of so-called "early" diagnostic methods and devices, which are fast, sensitive, portable to the patient's bedside and inexpensive, is a real challenge in the field of health but also in the field of defense or the environment.

[0003] Among the easy-to-use early diagnostic methods is the migration of targets in cellulose, known as "strips." This diagnostic method using strips provides results in 30 minutes but has the disadvantage of not being suitable for large targets (such as cells and certain bacteria) and, more importantly, of not being sufficiently sensitive. Other diagnostic methods used in biological laboratories include the ELISA (enzyme-linked immunosorbent assay) and the PCR (polymerase chain reaction) methods. These methods offer better sensitivity (counts between 10³ and 10⁴ CFU / ml). However, they require 3 to 4 hours to perform and necessitate personnel trained in their complex operation.

[0004] A device commonly used for detecting biological objects in biology laboratories is a flow cytometer. It allows for the counting of biological objects, one by one. However, it is not portable and is also complex to use. It is also expensive.

[0005] Lab-on-a-chips, or "biochips," offer a solution to the problems of size and complexity. Some biochips use optical target detection. However, these biochips remain poorly suited to studying certain opaque matrices. Other biochips use electrochemical target detection. However, these biochips have the disadvantage of exhibiting too many non-specific interactions with the external environment or certain matrices, which reduces detection sensitivity.

[0006] Biochips, which implement magnetic detection using magnetoresistive sensors, have seen significant development. The biological objects to be detected are labeled with magnetic particles (or beads) functionalized with antibodies specific to the target of interest. The biochips based on magnetoresistive sensors then detect, one by one, over time, the magnetic objects flowing through a microfluidic channel. Detection can be performed in liquid matrices, and the samples studied do not require prior washing. They can also be at low concentrations. Finally, signal counting and analysis can be performed simultaneously.

[0007] Among the magnetic detection methods developed in recent years, giant magnetoresistive (GMR) or tunnel magnetoresistive (TMR) sensors are the only ones to combine high detection sensitivity, a small footprint, and mature industrial production. These sensors also offer low manufacturing and operating costs.

[0008] A biochip may include a magnetoresistive sensor (GMR) or a magnetoresistive sensor (TMR) positioned beneath the microfluidic channel. Labeled biological objects flow through this microfluidic channel. The dipole field emitted by each labeled biological object passing near the magnetoresistive sensor is measured. When the emitted dipole field exceeds a detection limit of the GMR sensor, the signal is counted.

[0009] This method, however, has some major drawbacks. The dipole field is proportional to µ / z, where µ is the magnetic moment of the detected magnetic object and zb is its height in the channel above the sensor. Thus, a signal from a small cluster of magnetic beads (for example, a cluster without any biological objects) passing close to the GMR sensor (low µ and z) can produce the same signal as a properly magnetically labeled biological object passing higher in the channel (higher µ and z). Therefore, with this system, it is not possible to differentiate between clusters of magnetic beads and labeled biological objects since it is not possible to independently determine the magnetic moment and the height from a single dipole field.The presence of magnetic bead aggregates arises from the fact that it is necessary to put an excess of magnetic beads functionalized by specific antibodies in the liquid matrix to increase the chances of correctly labeling biological objects.

[0010] Document WO 2019 / 238857 A1 discloses a magnetic detection method using a biochip comprising several pairs of magnetoresistive sensors arranged on either side of the microfluidic channel, specifically above and below the channel. The magnetoresistive sensors in a pair are perfectly aligned on either side of the channel. A labeled biological object passing between the two sensors of the same pair will therefore be detected simultaneously by both sensors. The pair of two signals emitted simultaneously by two sensors of the same pair (for example, on the channel, referred to as "TOP," and below the channel, referred to as "BOTTOM") is said to be "synchronized."

[0011] The disclosed method even proposes to determine the trajectory followed by the object from the ratio of the amplitudes of the signals measured by successive pairs of sensors arranged all along the microfluidic channel.

[0012] Therefore, there is a need to determine the height and magnetic moment of a labeled biological object from the ratio of signal amplitudes. RÉSUMÉ DE L'INVENTION

[0013] In this context, the invention relates to a method for determining the magnetic moment of a magnetic object using a biochip according to independent claim 1.

[0014] Synchronized electrical signals mean that the electrical signals correspond to the measurement of the same magnetic object, even if the sensors are not aligned (the electrical signals may then show a time lag between them, for example proportional to the speed of movement of the magnetic object in the microfluidic channel).

[0015] Thanks to the analysis of simultaneous (synchronized) signals and the determination of the passage height, it is possible to determine the magnetic moment of the detected object, which is either a biological object marked by magnetic beads, an aggregate of magnetic beads, or magnetosomes.

[0016] Calculating the penetration depth using a reference curve calibrated from a magnetic calibration object allows for the reliable determination of the magnetic object's penetration depth and thus the calculation of its magnetic moment. Consequently, the discrimination of magnetic objects to be detected is improved, enabling the differentiation of biological objects labeled with a large number of magnetic beads from aggregates consisting primarily of magnetic beads (and containing no magnetic objects). This allows for the differentiation of false positives associated with aggregates from true positives associated with labeled biological objects.

[0017] There [ Fig. 1 ] presents an example of a reference curve relating the ratio R of the amplitudes of electrical calibration signals to the passage height zb of a magnetic calibration object of radius Ro, in the microfluidic channel of height hcan. The passage height zb can therefore only vary between Ro and hcan - Ro. The ratio is represented by the continuous curve and denoted "R". Said ratio R takes a minimum value when the passage height zb of the calibration object is minimal (when zb = Ro). According to the invention, the ratio R increases non-linearly as the passage height zb of the magnetic calibration object increases until it reaches a maximum value (when zb = hcan - Ro). The [ Fig. 1 Figure 1 also shows a ratio RAA, shown as a dashed line, calculated according to prior art and in particular according to the teachings of document WO 2019 / 238857 A, the calculation being adapted to the present parameter values. The ratio RAA is linear with the passage height zb and passes through 1 when the magnetic object considered is equidistant from the magnetic field sensors. It can therefore be observed that the two ratios R and RAA are equal only for two points: zb = 0 and zb = zbm. The figure also shows a difference in absolute value E (dashed line) between the two ratios R and RAA, which can be considered an absolute error. The difference E varies according to the passage height zb and can take on significant values ​​in certain cases (particularly around zbm / 2 or when zb is greater than zbm, where R tends towards infinity).

[0018] The reference curve therefore makes it possible to improve the estimation of the height of passage of the magnetic object and thus improve the determination of its magnetic moment.

[0019] It should be added that the preliminary determination of the passage height is not only used to reduce systematic error. Indeed, once the passage height is determined, it allows us to calculate a theoretical signal for a ball whose magnetic moment is known through preliminary measurements with a magnetometer. The ratio of the experimental signal to the theoretical signal of a ball allows us to determine the number N of balls contained within the detected object (the source of the experimental signal) and consequently the magnetic moment, which is then N times the magnetic moment of a ball.

[0020] The process is also compatible with a prior art biochip (such as the one disclosed by document WO 2019-238857 A).

[0021] The method also allows for the characterization of magnetic bead aggregates (not containing biological material) developed for various applications. Indeed, macroscopic magnetization measurement techniques, such as vibrating sample magnetometers or a "SQUID" magnetometer, do not allow for the measurement of the magnetic moment of single micrometric samples.

[0022] By arranged on either side of the microfluidic channel, we mean arranged on either side of the height of the microfluidic channel.

[0023] Advantageously, the magnetic field sensors are arranged opposite each other and aligned along the normal direction.

[0024] Advantageously, the reference curve is determined from the calibration magnetic object, which has a calibration magnetic moment independent of the magnetic moment of the magnetic object. Thus, the reference curve can be established regardless of the final magnetic objects to be characterized.

[0025] Advantageously, the electrical signals from the magnetic field sensors are proportional to the dipole fields emitted by the magnetic object and perceived by the magnetic field sensors. In other words, the ratio of the amplitudes of the electrical characterization signals is equal to the ratio of the amplitudes of the dipole field emitted by the calibration magnetic object on each of the magnetic field sensors.

[0026] Advantageously, the electrical signal from each magnetic field sensor is equal to the dipole field emitted by the magnetic object multiplied by a sensitivity factor of the magnetic field sensor, the sensitivity factor of the magnetic field sensor being advantageously linear and preferably equal to 2% / mT, or even 1% / mT. Advantageously, the sensitivity factors of the two magnetic field sensors are identical.

[0027] Even more advantageously, each electrical calibration signal is equal to the dipole field emitted by the magnetic calibration object at the level of a magnetic calibration sensor multiplied by a calibration sensitivity factor, the calibration sensitivity factors being equal to the sensitivity factors of the two magnetic field sensors.

[0028] Advantageously, the process includes a step for determining the reference curve comprising the following sub-steps: For different heights of passage of the calibration magnetic object between two calibration magnetic field sensors: determine the dipolar magnetic field emitted by the calibration magnetic object and perceived by one of the calibration magnetic field sensors; determine the dipolar magnetic field emitted by the calibration magnetic object and perceived by the other of the calibration magnetic field sensors; calculate the ratio of the amplitudes of the dipolar magnetic fields perceived by the calibration magnetic field sensors as a function of the different heights of passage of the calibration magnetic object.

[0029] Advantageously, the reception stage also includes a substage for identifying synchronized electrical signals, comprising measuring the time difference between the characteristic signatures of the two electrical signals. Synchronization is identified when the time difference falls within a predetermined time range, the predetermined time range being preferably determined from the speed of movement of the magnetic object. Each characteristic signature corresponds to the measurement of the magnetic object by one of the magnetic field sensors.

[0030] Advantageously, the amplitudes of electrical signals are preferentially estimated at the level of the characteristic signatures of each electrical signal.

[0031] Advantageously, the reception stage includes a sub-stage of identifying characteristic signatures in each of the electrical signals based on criteria of the shape of the electrical signals.

[0032] Advantageously, the magnetic object is a biological object marked by means of magnetic beads.

[0033] Advantageously, the method also includes a step of calculating the number of magnetic beads associated with the labeled biological object from the calculated magnetic moment and the magnetic moment of a single magnetic bead. The magnetic moment of a single magnetic bead can be deduced from a magnetic moment measurement of an assembly of magnetic beads, the magnetic moment measurement being carried out, for example, using a vibrating sample magnetometer.

[0034] Advantageously, magnetic field sensors are magnetoresistive sensors and preferably based on the giant magnetoresistance effect (GMR) or the tunnel magnetoresistance effect (TMR).

[0035] The invention also relates to a device for determining the magnetic moment of a magnetic object according to independent claim 12.

[0036] The invention also relates to a computer program according to independent claim 14 comprising instructions which lead the aforementioned device to execute the steps of the method for determining the magnetic moment of a magnetic object according to the invention.

[0037] A computer-readable medium, on which the aforementioned computer program is recorded and which is not part of the invention, is also disclosed.

[0038] The invention also relates to a method for counting biological objects according to dependent claim 10.

[0039] The counting process thus enables the counting of labeled biological objects. For example, it allows for the generation of a histogram of the passage heights of the various detected magnetic objects. It also allows for the generation of a histogram of the magnetic moments (and therefore the number of magnetic beads) associated with the detected magnetic objects. The counting process can also enable the counting of biological objects simultaneously with the circulation of the liquid matrix containing the labeled biological objects in the microfluidic channel of the biochip.

[0040] Advantageously, the counting process includes the step of determining the threshold magnetic moment, said step comprising the following sub-steps: circulate the liquid matrix without biological object, known as "negative control", in the microfluidic channel of the biochip and determine the magnetic moment of magnetic objects passing between the magnetic field sensors by means of the magnetic moment determination method according to the invention; and fix the threshold magnetic moment from a statistical distribution of the determined magnetic moments.

[0041] The invention also relates to a biological object counting system according to dependent claim 13.

[0042] The invention also relates to a computer program according to independent claim 15 comprising instructions which cause the counting device according to the invention to perform the steps of the counting process of the invention.

[0043] A computer-readable medium, on which the aforementioned computer program is recorded and which is not part of the invention, is also disclosed.

[0044] The invention and its various applications will be better understood by reading the following description and examining the accompanying figures. BRÈVE DESCRIPTION DES FIGURES

[0045] The figures are shown for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the same element appearing in different figures has a unique reference numeral. [ Fig. 1 ] presents an example of a reference curve implemented by the determination method according to the invention. Fig. 2 [ ] schematically represents a biochip that can be implemented by the determination method according to the invention. ] Fig. 3 ] schematically represents an example of layer stacking to obtain a magnetic field sensor such as that used in the biochip of the [ Fig. 2 ]. Fig. 4 ] shows an example of the application of a magnetic field to a stack of layers as illustrated in the [ Fig. 3 ]. Fig. 5 ] represents, in top view, an example of the geometry of a magnetic field sensor as used in the biochip of the [ Fig. 2 ]. Fig. 6 ] represents an example of magnetic field sensor arrangement within a biochip as illustrated in the [ Fig. 2 ]. Fig. 7 ] represents a first example of electrical signals from a biochip as illustrated in the [ Fig. 2 ] when a magnetic object passes between its two magnetic field sensors. Fig. 8 ] represents a second example of electrical signals from a biochip as illustrated in the [ Fig. 2 ] when a magnetic object passes between its two magnetic field sensors. Fig. 9 ] represents a third example of electrical signals from a biochip as illustrated in the [ Fig. 2 ] when a magnetic object passes between its two magnetic field sensors [ Fig. 10 [ ] schematically represents one method of implementing the determination process according to the invention. ] Fig. 11 ] represents a method of implementing a step in the process of the [ Fig. 10 ]. Fig. 12 ] represents an example of a dipole field calculated at different positions and for different numbers of magnetic balls. Fig. 13 [ ] schematically represents an embodiment of a determination device according to the invention. ] Fig. 14 ] represents an example of the acquisition chain of the device of the [ Fig. 13 ]. Fig. 15 ] schematically illustrates one method of implementing a counting process according to the invention. Fig. 16 ] schematically represents a principle of marking a biological object so that it can be counted by the [ Fig. 15 ]. Fig. 17 ] represents an example of a labeled biological object 5. Fig. 18 ] represents an example of a result obtained using the counting method of the [ Fig. 15 ]. DESCRIPTION DÉTAILLÉE

[0046] There [ Fig. 2 [ ] schematically represents a biochip 3 that can be implemented by a method 1 according to the invention. The prior document WO 2019-238857 A describes another embodiment of a biochip 3 that can be implemented by method 1. The biochip 3, in the example of the [ Fig. 2 [ ], includes a microfluidic channel 31 extending in a plane P. The figure shows a portion of this microfluidic channel 31, which extends along a Y direction parallel to the plane P. The channel advantageously has an inlet and an outlet so that a fluid to be analyzed can be circulated, for example, along the Y direction, or even in the direction of increasing Y. The microfluidic channel 31 can have a rectangular cross-section, as illustrated here, or a different cross-section, provided it allows for the placement of magnetic field sensors. The cross-section of the microfluidic channel 3, for example, has a height hcan, measured along a Z direction (called the "normal direction") perpendicular to the plane P, of between 20 µm and 25 µm. It can have a width L, measured along an X direction perpendicular to the Y and Z directions, of between 100 µm and 150 µm.

[0047] The biochip 3 also includes first and second magnetic field sensors 31, 32. The sensors 32, 33 are arranged on either side of the microfluidic channel 31. The first sensor 32, also called the "top" sensor, is, for example, arranged on the channel 31. A second sensor 33, also called the "bottom" sensor, is, for example, arranged below the channel 31. In the embodiment of the [ Fig. 2 The sensors are arranged opposite each other. Specifically, they are aligned, one above the other, along the normal Z direction. "Opposite" means that the top and bottom sensors 31, 32 each have a specific orientation and are oriented in opposite directions. In other words, each sensor 31, 32 can include a sensing element 321, 331, enabling the measurement of a magnetic field. These two sensing elements 321, 331 can then be oriented towards each other. Advantageously, the sensing elements 321, 331 are oriented towards the microfluidic channel 3, or even in contact with it.

[0048] There [ Fig. 3 Figure 32 and 33 show an example of a layer stacking used to create a magnetic field sensor. This is a magnetoresistive sensor. It could also be a so-called "SQUID" sensor using a superconducting loop. The layers chosen in this example allow for a giant magnetoresistance (GMR) effect. They could also be chosen to obtain a tunnel magnetoresistance (TMR) effect. In both cases (and especially in sensor applications), the stack resistance varies depending on the orientation of the magnetization of a so-called "free" layer relative to the orientation of the magnetization of a so-called "reference" layer.

[0049] There [ Fig. 4 ] shows, for example, the effect of applying a magnetic field to a stack of layers as illustrated in the [ Fig. 3 The magnetic field is applied antiparallel to the orientation of the reference layer's magnetization (the reference layer's magnetization is considered fixed). When the two magnetizations, of the free and reference layers, are parallel, the stack resistance is low. Therefore, the measurable voltage across its terminals, for a given bias current, is low. Conversely, when the two magnetizations, of the free and reference layers, are antiparallel, the stack resistance is high. Therefore, the measurable voltage across its terminals is high.

[0050] There exists a range in which the magnetizations of the free and reference layers are neither parallel nor antiparallel, but form an acute angle. Within this range, the resistivity varies linearly with the angle between the two magnetizations. The slope, for example in % / mT (considering the rate of change of resistance) or in Ω / mT or V / mT (when considering the voltage across the stack for a predetermined current), in this range therefore corresponds to the sensitivity of the stack. This is the range that is preferentially considered for the fabrication of top and bottom sensors 32, 33 since it allows the measurement of small variations in the orientation of the free layer's magnetization, for example, when the latter is disturbed by a magnetic object such as a labeled biological object.

[0051] The free layer advantageously forms the sensitive part 321, 331 of the sensors 32, 33.

[0052] THE [ Fig. 3 ] And [ Fig. 5 ] show an example of layer stacking and stacking geometry enabling magnetoresistive sensors 32, 33 to operate in the linear range as described. The stacking shown by the [ Fig. 3 ] comprises two ferromagnetic layers separated from each other by a non-magnetic material, called a "spacer". In the case of a stacking implementing the GMR effect, as presented in the [ Fig. 3 The spacer is a layer of a non-magnetic metal such as copper. In the case of a stacking employing the TMR effect, the spacer is a dielectric layer, for example, of MgO. The ferromagnetic layer, referred to as the "reference" layer, exhibits a fixed net magnetization on one of its faces (i.e., its orientation and magnitude are constant). The reference layer may, for example, comprise two ferromagnetic sublayers coupled in an antiferromagnetic manner (for example, by exchange coupling using the 0.85 nm thick Ru sublayer) to form an apparently antiferromagnetic sublayer, known as the "synthetic antiferromagnetic" or "SyAF" sublayer. The SyAF sublayer then has an upper sublayer with a fixed net magnetization.The reference layer may also include an antiferromagnetic (natural, as opposed to synthetic) IrMn underlayer, allowing the magnetization of the SyAF underlayer to be strengthened.

[0053] The ferromagnetic layer, known as the "free" layer, exhibits free magnetization. This layer can be made from a soft magnetic material such as CoFe or NiFe, or from a stack of CoFe / NiFe type sublayers.

[0054] The stack is advantageously arranged between two metallic electrodes, for example in Ta, allowing an electric current to flow within it.

[0055] The magnetizations of the free and reference layers are preferentially parallel to the plane of the layers. The orientation of the magnetization of the reference layer is imposed, for example, by the IrMn layer. The zero-field orientation of the magnetization of the free layer can be imposed by another means, for example, by means of shape anisotropy. For this purpose, the free layer advantageously has an elongated shape along a direction in the plane of the layers. In this way, the magnetization of the free layer will spontaneously orient itself along this direction. In the embodiment of the [ Fig. 5 The free layer has a yoke shape (also called a "C" or "horseshoe" shape) with a long, narrow section located at the stack. This yoke shape allows for a single-domain magnetization of the free layer. The long, narrow section also creates a shape anisotropy that orients the magnetization of the free layer along its longer length (in this case, along the X-axis). The orientation of this longer section is advantageously chosen to be perpendicular to the orientation of the magnetization of the reference layer (which, in this case, is oriented along the Y-axis). Thus, the magnetizations of the free and reference layers are, in the absence of disturbances, perpendicular to each other. In this way, the magnetoresistive sensors 32 and 33 operate in the linear range, where sensitivity is maximum and constant.

[0056] There [ Fig. 6 Figure 1 shows an example of the arrangement of magnetic field sensors 32, 33, in this case magnetoresistive, on either side of the microfluidic channel 31. In this example, the sensors 32, 33 are aligned along the Z direction. The sensing parts 321, 331, which here include the free layers of the sensors 32, 33, are oriented towards the channel 31. They are also advantageously arranged near the microfluidic channel 3. The sensing parts 321, 331 can be in contact with the microfluidic channel 31. However, this configuration may be difficult to implement practically because technological limitations, for example related to the fabrication steps of the biochip 3, may necessitate a separation between the sensors 32, 33 and the channel 31. The top sensor 32, for example, is separated from the channel 31 by a distance espT from the channel 31, for example, between 3 µm and 7 µm (in the example of the [ Fig. 1 (the value of 5 µm is considered). This distance corresponds, for example, to a layer of material separating channel 31 from the top sensor 32 and necessary for the realization of channel 31 and / or the top sensor 32. In the same way, the bottom sensor 33 is, for example, separated by a distance espB from channel 31, for example between 0.3 µm and 3 µm, for example equal to 1.7 µm.

[0057] Height refers to a distance measured along the normal Z direction. However, this nomenclature should not be taken literally, as the technical effects remain unchanged under rotation around the Y direction. In other words, sensors 32 and 33 could be positioned on the sides of channel 31 rather than on the top and bottom of it. The height then becomes a width. However, to simplify the description, only examples involving height are considered hereafter.

[0058] In the [ Fig. 6 The magnetizations of the free layers of sensors 32 and 33 are antiparallel to each other and oriented along the X direction (specifically, transverse to the Y direction of the flow). The magnetizations of the reference layers of sensors 32 and 33 are also antiparallel to each other and oriented parallel to the Y direction of the flow.

[0059] There [ Fig. 6 Figure 2 shows a magnetic object 2 at two different positions in the microfluidic channel 3. The magnetic object 2 follows, for example, the flow of a fluid matrix in the channel 3, the flow being, for example, in the direction of increasing Y. The object is therefore first on the right and then on the left, a brief instant later. The magnetic object 2 follows the flow at a constant height z, measured along the normal Z direction.

[0060] Magnetic object 2 is, for example, a labeled biological object (such as a molecule or a cell), a magnetosome (a molecule with an intrinsic magnetic moment), or an aggregate of magnetic beads. Magnetic beads are magnetic microparticles or nanoparticles designed to be attached to a biological object. In the [ Fig. 6 ], we consider a labeled biological object. The magnetic object 2 carries a dipole moment µ. The [ Fig. 6 ] illustrates the orientation of the dipole moment µ, oriented approximately along the Z direction. The dipole moment µ of the magnetic object 2 is due to the functionalization of a biological object with magnetic beads (illustrated by the small black dots on the white circle). This is referred to as a labeled biological object (labeling principle illustrated in [ Fig. 8 ]).

[0061] The component along the Y-axis of the magnetic field H dip emitted by the magnetic object 2 is detected by the top sensors 32 and bottom sensors 33. The [ Fig. 6 [ ] shows two H-dip field lines emanating from magnetic object 2, as well as the direction of the H-dip field. Depending on whether magnetic object 2 is to the right or left of sensors 32, 33 (i.e., upstream or downstream of the sensors), the lobe of the field lines coupled to sensors 32, 33 is not the same. The perceived magnetic field is also not oriented in the same direction for the two sensors 32, 33.

[0062] When magnetic object 2 is: to the right (in the figure) of sensors 32, 33, that is to say upstream (following the flow along Y), then: the lobe of the radiated magnetic field (the one on the left in this case) tends to orient the magnetization of the free layer of the top sensor 32 to the left; and the magnetization of the free layer of the bottom sensor 33 to the right; to the left (in the figure) of sensors 32, 33, that is to say downstream (following the flow along Y), then: the lobe of the radiated magnetic field (the one on the right in this case) tends to orient the magnetization of the free layer of the top sensor 32 to the right; and the magnetization of the free layer of the bottom sensor 33 to the left.

[0063] The effect of the lobe change coupled with the free layers of sensors 32, 33 during the passage of the magnetic object 2 is perceived (and therefore measurable).

[0064] There [ Fig. 7 [ ] illustrates an example of electrical signals from the two sensors 32, 33 as a function of time, during the passage of a magnetic object 2 between the two sensors 32, 33. The TOP curve represents, for example, the electrical signal emitted by the top sensor 32 and the BOTTOM curve represents, for example, the signal emitted by the bottom sensor 33. Each signal shows a characteristic signature of the measurement of a magnetic object 2. In this case, it is a first voltage peak immediately followed by a second peak with reversed polarity.

[0065] The particular shape of this signature can be explained as follows. At point a1, the TOP curve shows a zero voltage, indicating that no magnetic object is being measured. Magnetic object 2 is upstream of sensors 32 and 33, and its radiated field is still undetectable. At point a2, a non-zero electrical signal shows that the magnetization of the free layer of sensor TOP 32 is perturbed by magnetic object 2. A first lobe of the radiated field from the magnetic object therefore influences the magnetization of the free layer. The object is thus located between sensors 32 and 33 and slightly upstream of sensors 32 and 33. At point a4, the non-zero electrical signal shows that the magnetization of the free layer of sensor TOP 32 is also perturbed by magnetic object 2. However, the signal is negative, indicating that a second lobe of the radiated field from the magnetic object influences the magnetization of the free layer in the opposite direction.The object is therefore located between sensors 32 and 33, and slightly downstream of them. At point a3, the signal is zero, indicating that the magnetization of the free layer has returned to its equilibrium direction. The magnetic object is positioned between the two sensors 32 and 33 and is exactly centered (along the flow direction) with respect to sensors 32 and 33. The two lobes of the radiated field from the magnetic object influence the magnetization of the layer equally. At point a5, the magnetic object is downstream of sensors 32 and 33 but is positioned too far from them for its field to be detected.

[0066] The extent of a characteristic signature depends solely on the speed at which the object passes the sensors. It therefore depends primarily on the flow velocity in the microfluidic channel 31.

[0067] In this example, the TOP and BOTTOM curves exhibit virtually identical characteristic signatures. When the TOP curve is positive, the BOTTOM curve is also positive. The maxima and minima of both the TOP and BOTTOM curves occur at the same times and reach comparable values. This indicates that the object is detected simultaneously by both sensors (remember that in this example, the sensors are aligned).

[0068] When the sensors 32, 33 are aligned in a direction transverse to the flow, in this case in the Z direction, the passage of the magnetic object 2 between the two sensors 32, 33 triggers a simultaneous response of similar shape but whose amplitude may depend on the height z 2 of passage of the object 2.

[0069] The response to the same magnetic object can, however, vary in two different ways, illustrated by the [ Fig. 8 ] And [ Fig. 9 ].

[0070] For example, in the [ Fig. 8 The polarity of the characteristic signature of the BOTTOM curve is reversed compared to that of the TOP curve. However, the characteristic signatures are simultaneous. This arises, for example, from the fact that the orientation of the magnetization of the free layer of the second sensor 33 is reversed compared to the orientation of the magnetization of the reference layer of the second sensor 33.

[0071] In the [ Fig. 9 [ ], the BOTTOM curve has a lower maximum amplitude than the maximum amplitude of the TOP curve. However, the polarities of the signatures are identical. Therefore, magnetic object 2 passed closer to the top sensor 32 than to the bottom sensor 33. The characteristic signatures of the two curves do not appear at the same times. The signature of the TOP curve appears before that of the BOTTOM curve. This may be due to the fact that the field sensors 32 and 33 are not aligned along the Z direction (contrary to what is shown by the [ Fig. 6 The top sensor 32 (from which the TOP curve is derived) is positioned upstream of the bottom sensor 33 (from which the BOTTOM curve is derived) relative to the flow direction in the microfluidic channel 31. Therefore, the magnetic object 2 first disturbs the top sensor 32 and then the bottom sensor 33. The time shift tSYN between these two signatures (measured at the center of the signatures, when the signal is zero) depends on the speed at which the magnetic object 2 passes through sensors 32 and 33. The time shift tSYN thus depends on the flow velocity in the microfluidic channel 31.

[0072] There [ Fig. 6 ] shows, along the normal direction Z, the passage height z 2 of the magnetic object 2 in the microfluidic channel 3. This passage height z 2 is to be taken into consideration with respect to the respective heights z 32 , z 33 of the top 32 and bottom 33 sensors. The heights z 32 , z 33 taken into account are moreover preferentially the heights, along the normal direction Z, of the free layers of each sensor.

[0073] When the passage height z2 is equidistant from the heights z32 (of the top sensor) and z33 (of the bottom sensor), the measured signals have the same amplitudes (as illustrated by the [ Fig. 7] et [Fig. 8 ]). When the passage height z2 of the magnetic object 2 is not equidistant, i.e., when the magnetic object 2 passes closer to the top sensor 32 or the bottom sensor 33, the measured signals then exhibit different amplitudes (as illustrated by the [ Fig. 9 ]).

[0074] Method 1 according to the invention allows the passage of a magnetic object 2 between two sensors 32, 33 to be detected by determining its magnetic moment µ. To this end, method 1 proposes to accurately determine the passage height z2 of the magnetic object between the two sensors 32, 33. This is because the field radiated at sensor 32 is proportional to µ / (z2 - z32)< 3. A good estimate of the passage height therefore allows the magnetic moment of the magnetic object 2 to be estimated.

[0075] Method 1 can thus be applied to the detection or counting of magnetic objects or to the sorting of magnetic objects according to their magnetic moment.

[0076] There [ Fig. 10 ] schematically represents an implementation method of process 1. According to a first step, process 1 involves receiving 11 electrical signals from the top 32 and bottom 33 sensors respectively. These are, for example, the electrical signals illustrated by TOP and BOTTOM curves in the [ Fig. 9 These electrical signals correspond to the passage of the magnetic object 2 through the microfluidic channel 31 at a passage height z2, which must be determined. The received signals are unique in that they correspond to the passage of the same object 2. They are, in fact, said to be "synchronized." For example, they exhibit signatures characteristic of the measurement of the same magnetic object 2, and the difference between these characteristic signatures coincides, for example, with the flow velocity in the microfluidic channel 31.

[0077] Method 1 includes a calculation step 12 of the passage height z2 of the magnetic object 2 from the previously received synchronized electrical signals. For this purpose, method 1 uses a reference curve R(zb) to obtain the height z2 of the magnetic object 2 in the channel 3 from the ratio of the amplitudes of the synchronized electrical signals.

[0078] To apply it to synchronized signals of the [ Fig. 9 The ratio of the amplitudes is obtained, for example, from the maximum positive amplitudes MTOP, MBOTTOM of the synchronized signals TOP, BOTTOM, by calculating, for example, MTOP / MBOTTOM. The ratio of the amplitudes could also be obtained from the maximum negative amplitudes mTOP, mBOTTOM of the synchronized signals TOP, BOTTOM, by calculating, for example, mTOP / mBOTTOM. It is also possible to calculate a linear combination of the two aforementioned ratios: a × MTOP / MBOTTOM + b × mTOP / mBOTTOM.

[0079] The ratio of the amplitudes obtained is compared to the reference curve R(Z b ), as illustrated by the [ Fig. 1 ]. There [ Fig. 1 ] illustrates an example of a reference curve R(zb). The curve R relates the passage height zb of a magnetic calibration object in the microfluidic channel 31 to a ratio R of the amplitudes of electrical calibration signals. A direct reading of the curve provides a passage height (denoted zb) of the magnetic calibration object in the [ Fig. 1 ] which is equal to the passage height z 2 of the targeted magnetic object 2 (in other words, z 2 = zb ).

[0080] Method 1 involves, in a third step, calculating the magnetic moment µ of the magnetic object 2. For this purpose, the previously determined passage height z2 is used. It is compared to the first and / or second electrical signal. Indeed, each electrical signal is proportional to the magnetic dipole field perceived at sensors 32 and 33, which itself depends on the distance of the magnetic object 2 from sensors 32 and 33 and on its magnetic moment µ.

[0081] For example, one way to do this is to consider the electrical signal V32 measured by the top32 sensor. It can be approximated by V 23 ≈ S 32 ⋅ μ / r 3 Or S 32 is the sensitivity of the top 32 sensor, µ is the magnetic moment of the targeted object 2 and r is the distance of the target object 2 from r. When the voltage measured across the first sensor is at its maximum (e.g., M TOP on the [ Fig. 9 ]), the magnetic object 2 is positioned under the top sensor 32. It is not exactly directly above the top sensor 32 (since we have seen that the measured signal there is zero, as discussed with reference to the [ Fig. 7 ]) but its distance r from the first sensor 32 is very close to the passage height z2. We can therefore estimate the voltage V32 by V 32 = S 32 ⋅ μ ⋅ z 2

[0082] We therefore obtain the magnetic moment µ of the magnetic object 2 by calculating μ = V 32 S 32 ⋅ Z 2

[0083] This first method allows us to determine the magnetic moment of the magnetic object 2. The accuracy of the determination does not depend here on the relative position of the object with respect to the sensors 32, 33.

[0084] There [ Fig. 11 Figure 13 shows another way to perform the calculation of the magnetic moment µ of the magnetic object 2. In this calculation, the electrical signals TOP, BOTTOM measured across the two sensors 32, 33 are taken into account. Two theoretical curves f TOP, f BOTTOM are fitted to the two signals TOP, BOTTOM. The temporal extent of the characteristic signatures depends largely on the speed at which the magnetic object 2 passes between the two sensors 32, 33, and therefore on the flow velocity, which is known. On the other hand, the amplitude of the TOP, BOTTOM curves at any instant depends on the magnetic moment µ carried by the magnetic object 2. The fitting of the theoretical curves f TOP , f BOTTOM therefore depends only on the magnetic moment µ of the magnetic object 2. Taking into account the values ​​measured by the two sensors 32, 33 as well as the fitting of the measured values ​​makes it possible to further improve the determination of the magnetic moment µ.

[0085] The theoretical curves f TOP , f BOTTOM take into account an estimate of the magnetic field H radiated by the magnetic object 2 and perceived by a sensor. For example, the field H 32 perceived at the top 32 sensor and radiated by a magnetic object 2 at coordinates x 2, y 2 and z 2 is: H 32 = y l q 2 2 x r r 2 − x l r 1 + y r q 4 2 x l r 4 − x r r 3 sin θ sin ψ + 1 r 1 − 1 r 2 + 1 r 3 − 1 r 4 sin θ cos ψ + h q 2 2 x l r 1 − x r r 2 + h q 4 2 x r r 3 − x l r 4 cos θ with : x r = L 32 2 − x 2 x l = − L 32 2 − x 2 y r = l 32 2 − y 2 y l = − l 32 2 − y 2 h = z 2 − z 32 r 1 = x l 2 + y l 2 + h 2 r 2 = x r 2 + y I 2 + h 2 r 3 = x r 2 + y r 2 + h 2 r 4 = x l 2 + y r 2 + h 2 q 2 = y l 2 + h 2 q 4 = y r 2 + h 2 Or L 32 and l 32 are respectively the width measured along X and the length measured along Y of the top 32 sensor, ψ is the angle between µ and X, and θ is the angle between µ and Z. When the magnetic moment µ is aligned along Z, then the field H 32 measured at the top sensor 32 becomes: H 32 = h q 2 2 x l r 1 − x r r 2 + h q 4 2 x r r 3 − x l r 4

[0086] The field H 33 perceived at the level of the second sensor 33 and emitted by the magnetic object 2 at the coordinates x 2 , y 2 andz 2 can be obtained in the same way.

[0087] Knowledge of the passage height z2 and the adjustment of the magnetic fields H 32 and H 33, represented by the curves f TOP and f OTTOM in the [ Fig. 12 ], thus allowing the magnetic moment µ to be determined precisely.

[0088] There [ Fig. 1 [ ] shows an example of a reference curve R(zb). In order for the results obtained from this curve to be accurate, it is preferable that the R curve take into account characteristics of the biochip 3. For this purpose, it is generated, for example, by considering a calibration system. The calibration system includes, for example, calibration magnetic field sensors and a calibration magnetic object. The calibration system is, for example, a model, for example, a digital one. It may include calibration sensors that are, for example, models of the top and bottom sensors 32, 33. The calibration sensors are, for example, established from the relative positions of the top and bottom sensors 32, 33 with respect to each other and with respect to the microfluidic channel 31. They may also have calibration sensitivities, which are preferably equal to the sensitivities of the top and bottom sensors 32, 33.The calibration system may also include a calibration object. This object may have the same diameter as the magnetic object 2 that is to be characterized using the biochip 3 (in which case it is called the target magnetic object). It may also have the same magnetic moment as the target magnetic object 2. However, it is advantageous for the calibration magnetic object to have characteristics independent of the characteristics of the target magnetic objects 2. In this way, the reference curve can be used for different magnetic objects, regardless of their magnetic moment, or even their diameter. The independence of the calibration object from the magnetic moment of the target magnetic object 2 is possible by considering ratios of amplitudes rather than absolute values. The amplitude ratios are independent of the magnetic moments considered.Therefore, the magnetic calibration object can have a magnetic moment equal to 1.

[0089] In the example of the [ Fig. 1 ], the reference curve R(zb) corresponds to a ratio of the maximum dipole field emitted by the calibration object (having an arbitrary magnetic moment) and perceived at the calibration sensors. In this example, the calibration sensors are separated by a distance of 26 µm, measured along the normal direction. This distance thus corresponds to a biochip 3 comprising a microfluidic channel 31 with a height hcan = 20 µm (only this range of heights is illustrated in the [ Fig. 1 Since the target object 2 cannot penetrate the walls of channel 31, and its top and bottom sensors 32 and 33 are located 5 µm and 1.7 µm from channel 31, respectively, the radius Ro of the target object 2 has been taken into account (though this is not mandatory). This limits the range of passage heights zb to be considered, which extends from Ro to hcan-Ro.

[0090] Method 1 advantageously includes a step 10 for determining the reference curve. In one embodiment, the reference curve R(zb) corresponds to a ratio of the maximum dipole field emitted by the calibration object (having an arbitrary magnetic moment) and perceived at the calibration sensors. The calculation of the perceived fields, as described previously (with reference to the fields H 32 and H33), can be considered. This refers to the calculation of the field emitted by the calibration object exhibiting an arbitrary magnetic moment at the calibration sensors.

[0091] The calculation is performed for different passage heights z of the calibration magnetic object between the calibration sensors. For each passage height z, the dipole magnetic field emitted by the calibration magnetic object and perceived by each calibration sensor is determined.

[0092] There [ Fig. 12 ] shows, for example, a calculation of the maximum magnetic fields perceived by the calibration sensors (noted H 32 and H33) and radiated by a calibration magnetic object exhibiting different magnetic moments. The magnetic moment is given as a function of the number of magnetic balls rather than in standard units (i.e., A·m²). Each magnetic ball, for example, has a magnetic moment equal to 1.56 × 10⁻¹⁴ A·m². The calculation is performed for a number of balls between 1 and 20.

[0093] The maximum magnetic field detected by the first calibration sensor is shown as solid lines, while the maximum magnetic field detected by the second calibration sensor is shown as dashed lines. The maximum magnetic field detected increases or decreases depending on the height at which the magnetic object passes.

[0094] Calculating the ratio of maximum magnetic fields to passage height allows us to obtain the reference curve, such as the one illustrated by the [ Fig. 1 The calculated ratio is constant depending on the number of magnetic balls considered.

[0095] The calculation of the [ Fig. 12 This method is doubly important because it allows us to consider a theoretical detection limit (LOD) for electronic devices. Below this theoretical detection limit, we can assume that the magnetic object will not be detected. Therefore, we can determine the minimum magnetic moment (in this case, the minimum number of beads) required for accurate characterization of the target object.

[0096] The ratio R of the amplitudes of the reference curve can be calculated from the magnetic fields detected at the calibration sensors. When the sensitivities of the top and bottom sensors 32, 33 are identical, then the ratio of the amplitudes of the synchronized electrical signals is equal to the ratio R of the reference curve. However, the (actual) top and bottom sensors 32, 33 may have different sensitivities. The ratio R then preferentially takes into account the sensitivity of the top and bottom sensors 32, 33. To do this, the magnetic field detected at each calibration sensor is multiplied by the sensitivity of the calibration sensor in question and preferably by the sensitivity of the top or bottom sensor 32, 33 that it models.

[0097] The sensitivity factors of the top and bottom sensors 32, 33 are preferentially linear and preferably between 1 / mT and 2 % / mT.

[0098] When the magnetic object is a labeled biological object, using magnetic beads, it is advantageous for process 1 to include a step 14 for calculating the number of magnetic beads associated with said biological object. The calculation is performed using the previously obtained magnetic moment µ and the magnetic moment of a single magnetic bead. For example, Dynabeads™ brand magnetic beads have a magnetic moment µs = 1.56 × 10⁻¹⁴ A·m². The magnetic moment of a single magnetic bead can be deduced from a magnetic moment measurement of an assembly of beads, for example, using a vibrating sample magnetometer (VSM). The number N of beads can then be obtained by N = µ / µB.

[0099] The reception step 11 of the electrical signals from sensors 32, 33 may include a first sub-step 11a of filtering the received signals. The filtering consists of a moving average over each signal. The moving average preferably has a window of approximately 0.1 ms.

[0100] The reception step 11 may also include a substep 11b for identifying the characteristic signatures of a magnetic object's measurement. The [ Fig. 7 ] show an example of characteristic signatures. Each signature includes a positive peak immediately followed by a negative peak. Reversing the sensitivity of one of the sensors 32, 33 can reverse this signature (which becomes a negative peak followed by a positive peak), as illustrated by the [ Fig. 8 In such a case, the signal is preferentially inverted so that the measurement signatures of an object show a positive peak followed by a negative peak.

[0101] The characteristic signatures are initially separated from the measurement noise. To do this, a selection based on shape criteria can be implemented. For example, only peaks with an absolute amplitude greater than a threshold can be considered. Indeed, a minimum amplitude, both positive and negative, is expected from the peaks of a characteristic signature of a magnetic object 2. This amounts to considering voltage ranges, for example represented by the shaded areas around 0 V in the [ Fig. 9 ], as consisting mainly of measurement noise. The peaks considered exhibit a prominence PTOP, PBOTTOM, measured outside the shaded areas, strictly greater than zero. The range of voltage bands can be determined based on the noise density of the installation (measured without the introduction of the targeted magnetic objects into the microfluidic channel 3). The noise may exhibit a peak-to-peak voltage Vb, in which case the ignored bands are equal to [-Vb; Vb]. Thanks to this condition, signals generated by magnetic beads alone are ignored (notably because the dipole field they emit results in signals whose amplitude is less than or equal to the LOD).

[0102] According to another example, consistent with the one mentioned above, only peaks exhibiting alternating polarities are considered. For instance, a positive peak followed by a negative peak, or a negative peak followed by a positive peak. The time interval between the extreme amplitudes of these two peaks is expected to be less than a predetermined duration. This predetermined duration varies depending on the size of the magnetic object and its speed (the flow velocity). For an object with a diameter of 10 µm in a flow of 8 cm / s, the predetermined duration is between 100 µs and 600 µs.

[0103] To facilitate the identification of characteristic signatures, it is preferable that the signatures of several magnetic objects do not overlap. For this reason, it is preferable that the magnetic objects 2 be sufficiently diluted in the fluid matrix that allows their circulation in the microfluidic channel 31.

[0104] Two labeled biological objects produce distinct signals when separated by more than 30 µm. Considering a spacing of z32 - z33 between the two sensors 32, 33, it is preferable that the dilution of the magnetic objects allows for a spacing between two objects greater than or equal to 1.7 × (z32 - z33). Thus, this method 1 is suitable for early diagnosis since the aggregation of the biological objects satisfies these two criteria.

[0105] The reception step 11 may also include a substep 11c of identification of the synchronized electrical signals between the characteristic signatures of the signal from the top sensor 32 and the characteristic signatures of the signal from the bottom sensor 33. Two synchronized characteristic signatures correspond to two measurements of the same magnetic object 2. The synchronization of the two characteristic signatures is then called "coincidence".

[0106] During the coincidence identification substep, a first instant is selected within a characteristic signature of the first signal, and a second instant is selected within a characteristic signature of the second signal. The time lag tSYN between these two instants is measured. When sensors 32 and 33 are not aligned, the synchronized signatures may exhibit an intrinsic time lag, which corresponds to the distance separating the two sensors 32 and 33 along the Y direction, multiplied by the flow velocity in channel 31. An acceptable time lag range WSYN can therefore be determined. For example, for a flow of 8 cm / s, the time lag range WSYN can be expected to extend over 100 µs. Characteristic signatures whose time lag falls within this range are then said to be synchronized.For example, when sensors 32, 33 are aligned (without any offset along Y), the intrinsic time offset is zero and the offset between the two signatures is then within the range of 100 µs (i.e., exhibiting a relative difference of plus or minus 50 µs).

[0107] The first and second instants advantageously correspond to comparable characteristics of each signature. For example, when the first instant corresponds to the maximum (positive) of the characteristic signature of the first signal, then the second instant advantageously corresponds to the maximum (positive) of the characteristic signature of the second signal.

[0108] There [ Fig. 13 [Figure ] schematically represents a device 6 for determining 1 the magnetic moment µ of magnetic objects 2. The device 6 comprises the biochip 3 as described above. It also includes complementary means 62, 64, 65, adapted to implement the determination method 1 according to the invention.

[0109] The device 6 includes a means 62 for applying a magnetic field H0 to the biochip 3. The means 62 includes a permanent magnet 62 configured to apply the field H0 to the biochip 3. The field H0 is preferably aligned along the Z direction. This field H0 allows, for example, the magnetic moment µ of magnetic objects to be aligned along the Z direction (as illustrated by the [ Fig. 6 ]). In this way, the detection capacity of sensors 32, 33 is maximized.

[0110] A soft iron 63 can be added to isolate the biochip from the external environment (and in particular from the external magnetic field). This shielding 63 helps to homogenize the magnetic field H0 in which the biochip 3 is immersed and also improves the signal-to-noise ratio of the sensors 32, 33.

[0111] The magnetic objects 2 to be characterized are preferentially mixed in a fluid matrix. In this way, the fluid matrix and the magnetic objects 2 can be circulated in the microfluidic channel 31 of the biochip 3. The device 6 includes a control system 65 for the flow rate of the fluid matrix in which the magnetic objects 2 are immersed.

[0112] Device 6 also includes an acquisition chain 64 responsible for polarizing sensors 32, 33 (for example in current) and receiving electrical signals from sensors 32, 33.

[0113] There [ Fig. 14 Figure 64 shows an example of a data acquisition chain. It includes, in particular, a current source 641 used to bias the sensors 32 and 33 with current. A voltmeter 642 measures the voltage across each sensor 32 and 33. The voltmeter 642 advantageously includes an amplification stage, for example, +60 dB, to provide usable voltage readings. The voltmeter may also include a high-pass filter to suppress very low-frequency noise, for example, below 150 Hz. The data acquisition chain 64 may also include an additional amplification stage 643. It may also include a low-pass filter 644 to cut off very high frequencies, for example, above 37 kHz.

[0114] The chain 64 may also include a digital converter 645 and a controller 646, responsible for processing the electrical signals from the sensors 32, 33. The sampling frequency of the converter 645 depends in part on the flow velocity. For example, for a given flow velocity, the sampling frequency is preferably greater than or equal to 200 kHz.

[0115] The 646 controller allows, for example, the calculation steps 12, 13, 14 of process 1 to be carried out.

[0116] The determination 1 of the magnetic moment µ of the magnetic objects 2 passing through the microfluidic channel 31 can be carried out in "real time", i.e., simultaneously with the passage of the magnetic objects 2 between the sensors, or with a delay. In the latter case, the signals measured TOP, BOTTOM by the sensors 32, 33 are recorded in files and processed subsequently.

[0117] There [ Fig. 15 ] schematically illustrates a process 4 for counting biological objects 5 implementing the biochip 3 and the process for determining a magnetic moment, as described previously.

[0118] Biological objects 5 are, for example, biological targets of varying sizes, such as cells, bacteria, or viruses. These include, for example, cancer cells or pathogenic bacteria: salmonella, yersinia enterocolitica, or legionella pneumophila.

[0119] Except for magnetosomes, which possess an intrinsic magnetic core, biological objects are not magnetic. It is therefore necessary to label them to impart a magnetic moment. In this way, they can be counted using a method based on measuring this magnetic moment.

[0120] There [ Fig. 16 [This diagram schematically illustrates a step in the labeling of biological objects.] The counting process 4 initially comprises a labeling step 41 for the biological objects 5. To do this, the biological objects 5, in this case cells, are mixed with a matrix containing magnetic beads. The magnetic beads are capable of labeling the biological objects 5. They are, for example, associated with antibodies that can bind to antigens carried by the biological objects 5.

[0121] Among magnetic beads (ranging in size from 200 nm to 1 µm, for example), three types of surface functionalities exist. Sulfonic ester and carboxylic acid groups react chemically with antibodies and create covalent bonds, while streptavidin beads are functionalized via biotin-streptavidin interaction by antibodies previously covalently coupled to biotin.

[0122] For example, in a first step, magnetic beads ranging in size from 200 nm to 1 µm and antibodies are mixed in a neutral solution. The protocol is specific to each batch of beads being studied. The magnetic beads are, for example, micrometric beads made of nanometer-sized iron oxide cores (maghemite or magnetite) encased in a polymer layer. The antibodies bind to the polymer layer of the magnetic beads.

[0123] Antibody-functionalized beads are added to the matrix containing the biological objects 5. They are added in excess to promote the probability of encountering the biological objects.

[0124] Some of the antibodies will bind to antigens carried by each cell 5. In this way, the cells 5 are said to be "labeled," that is, functionalized using magnetic beads. Labeled cells 5 carry, for example, between 50 and 100 magnetic beads with a diameter of 1 µm. Despite the precautions taken, a portion of the cells 5, on the order of 7%, may remain unlabeled. Moreover, the larger the labeled cells 5 are, the more likely they are to carry a high number of magnetic beads, which can facilitate their counting.

[0125] There [ Fig. 17 [ ] shows an example of a labeled biological object 5, in this case a cell containing antigens, one of which is associated with an antibody. A magnetic bead is attached to the antibody in question. The labeled biological object 5, thanks to the magnetic bead, carries a magnetic moment µ.

[0126] The counting process of the [ Fig. 15 This also includes a step for determining the magnetic moment µ of magnetic objects passing between sensors 32 and 33. During this step, the matrix containing the labeled biological objects 5 circulates in the microfluidic channel 31 of the biochip 3 (as described previously). Electrical signals from sensors 32 and 33 are acquired as the biological objects 5 circulate in the channel 31. For each coincidence, as described previously, a magnetic moment µ is calculated.

[0127] There [ Fig. 18 [ ] illustrates, for example, a distribution diagram of the detected magnetic objects 2 as a function of the number of magnetic beads possessed by the detected magnetic objects. The x-axis of the figure indicates the number of magnetic beads per characterized magnetic object rather than the value of the magnetic moment. The number of magnetic beads is determined as the ratio of the magnetic moment of the detected object to the individual magnetic moment of the magnetic bead (determined beforehand).

[0128] The white bars in the [ Fig. 18 ] correspond to a count performed in a specific sample containing labeled biological objects of interest in a complex matrix (culture medium). The black bars, on the other hand, correspond to a so-called "negative control" sample containing the same complex matrix (with the same number of functionalized magnetic beads) but without a labeled biological object (this is in this case the by-product of step 2 of the [ Fig. 17 ]).

[0129] In the latter case (black bars), magnetic objects are detected and exhibit a magnetic moment. However, these are not target biological objects (since they are absent from the matrix specifically used for these measurements). They are magnetic aggregates that can be mistaken for labeled biological objects. Therefore, they must be distinguished from labeled biological objects. These magnetic aggregates comprise between two and twenty-five magnetic beads per aggregate. They can only be detected with four or more magnetic beads when they pass through the center and with twelve or more beads across the entire channel. Below four magnetic beads, the aggregates emit a dipole field that is generally lower than the limit of detection (LOD).

[0130] To avoid counting magnetic clusters, a threshold magnetic moment is considered. In the example of the [ Fig. 18], it corresponds to a threshold number of magnetic balls, equal, in the example given, to twenty-five balls.

[0131] Therefore, counting method 4 can count each magnetic object as a labeled biological object if the magnetic moment of the magnetic object (and thus the number of magnetic beads it carries) is greater than the threshold magnetic moment (i.e., greater than the threshold number of magnetic beads NT). In this way, magnetic aggregates are not counted.

Claims

1. A method (1) for determining the magnetic moment (µ) of a magnetic object (2) by means of a biochip (3), the magnetic object being a biological object (5) marked by means of magnetic beads or a magnetosome or an aggregate of magnetic beads, the biochip comprising: a microfluidic channel (31) extending in a plane (P) and having a height (hcan), measured along a direction (Z) referred to as the "normal direction" perpendicular to the plane (P); and two magnetic field sensors (31, 32), disposed on either side of the microfluidic channel (31), the method (1) comprising the following steps of: - receiving (11) two synchronised electrical signals (TOP, BOTTOM) from the magnetic field sensors (32, 33) respectively, corresponding to the passage of the magnetic object (2) into the microfluidic channel (31) at each magnetic field sensor (32, 33); - calculating (12) the passage height (z2) of the magnetic object (2), measured along the normal direction (Z), from the ratio of the amplitudes of the synchronised electrical signals (TOP, BOTTOM) and by means of a reference curve (R(zb)) relating the passage height (zb) of a calibration magnetic object in the microfluidic channel (31) to a ratio (R) of the amplitudes of the calibration electrical signals corresponding to the passage of the calibration magnetic object at said passage height (zb), said ratio (R) of the amplitudes of the calibration electrical signals non-linearly increasing as the passage height (zb) of the calibration magnetic object increases until it reaches a maximum value, the ratio of the amplitudes of the synchronised electrical signals (TOP, BOTTOM) being equal to the ratio (R) of the amplitudes of the calibration electrical signals, - calculating (13) the magnetic moment (µ) of the magnetic object (2) from the passage height (z2) determined and one of the synchronised electrical signals.

2. The method (1) according to the preceding claim, wherein the reference curve (R(zb)) is determined from the calibration magnetic object having a calibration magnetic moment independent of the magnetic moment of the magnetic object.

3. The method (1) according to one of the preceding claims, wherein the electrical signal (TOP, BOTTOM) from each magnetic field sensor (32, 33) is equal to the dipole field (Hdip) emitted by the magnetic object (2) multiplied by a sensitivity factor of the magnetic field sensor (32, 33).

4. The method (1) according to the preceding claim, wherein each calibration electrical signal is equal to the dipole field emitted by the calibration magnetic object at a calibration magnetic field sensor multiplied by a calibration sensitivity factor, the calibration sensitivity factors being equal to the sensitivity factors of both magnetic field sensors (32, 33).

5. The method (1) according to one of the preceding claims, comprising a step (10) of determining the reference curve comprising the following sub-steps of: - for different passage heights of the calibration magnetic object between two calibration magnetic field sensors: o determining the dipole magnetic field emitted by the calibration magnetic object and perceived by one of the calibration magnetic field sensors; o determining the dipole magnetic field emitted by the calibration magnetic object and perceived by the other calibration magnetic field sensor; - calculating the ratio of the amplitudes of the dipole magnetic fields perceived by the calibration magnetic field sensors as a function of the different passage heights of the calibration magnetic object.

6. The method (1) according to one of the preceding claims, wherein the reception step (11) also comprises a sub-step (11c) of identifying synchronised electrical signals comprising measuring a time difference (tSYN) between characteristic signatures of both electrical signals (TOP, BOTTOM), each characteristic signature corresponding to the measurement of the magnetic object (2) by one of the magnetic field sensors (32, 33), synchronisation being identified when the time difference (tSYN) is within a predetermined time range (WSYN).

7. The method (1) according to one of the two preceding claims, wherein the reception step (11) comprises a sub-step of identifying (11b) characteristic signatures in each of the electrical signals from shape criteria of the electrical signals.

8. The method (1) according to one of the preceding claims, wherein the magnetic object is a biological object marked by means of magnetic beads, said method (1) also comprising a step of calculating (14) the number of magnetic beads associated with the biological object (5) marked from the magnetic moment calculated and the magnetic moment of a single magnetic bead.

9. The method (1) according to one of the preceding claims, wherein the magnetic field sensors (32, 33) are magnetoresistive sensors.

10. A method (4) for counting biological objects (5), comprising the following steps of: - marking (41) the biological objects (5) by mixing them with a liquid matrix comprising magnetic beads able to attach to receptors carried by each biological object; - circulating the liquid matrix comprising the biological objects (5) marked in the microfluidic channel (31) of the biochip (3) and determining (42) the magnetic moment (µ) of magnetic objects (2) passing between the magnetic field sensors (32, 33) by means of the method (1) according to one of claims 1 to 9, each magnetic object (2) counting as a biological object (5) marked when the magnetic moment (µ) associated therewith is greater than a threshold magnetic moment (NT).

11. The counting method (4) according to the preceding claim, comprising the step of determining (40) the threshold magnetic moment (NT), said step comprising the following sub-steps of: - circulating the liquid matrix without biological objects, referred to as the "negative control", in the microfluidic channel of the biochip (3) and determining the magnetic moment (µ) of magnetic objects (2) passing between the magnetic field sensors by means of the method (1) for determining the magnetic moment according to the invention; and - setting the threshold magnetic moment (NT) from a statistical distribution of the magnetic moments determined.

12. A device (6) for determining the magnetic moment (µ) of a magnetic object (2), the magnetic object being a biological object (5) marked by means of magnetic beads or a magnetosome or an aggregate of magnetic beads, comprising: - a biochip comprising: a microfluidic channel (31) extending in a plane (P) and having a height (hcan), measured along a direction (Z) referred to as the "normal direction" perpendicular to the plane (P); and two magnetic field sensors (31, 32), disposed on either side of the microfluidic channel (31), - a magnet (62) configured to apply a magnetic field (Ho) at the biochip, - a system for controlling flow rate of a fluid matrix (65) configured to circulate a liquid matrix comprising the biological objects (5) marked in the microfluidic channel (31) of the biochip (3), - an acquisition chain (64) responsible for polarising the sensors (32, 33) and receiving both synchronised electrical signals (TOP, BOTTOM) from said sensors (32, 33), corresponding to the passage of the magnetic object (2) into the microfluidic channel (31) at each magnetic field sensor (32, 33); and comprising a controller configured to: o calculate (12) the passage height (z2) of the magnetic object (2), measured along the normal direction (Z), from the ratio of the amplitudes of the synchronised electrical signals (TOP, BOTTOM) and by means of a reference curve (R(zb)) relating the passage height (zb) of a calibration magnetic object in the microfluidic channel (31) to a ratio (R) of the amplitudes of the calibration electrical signals corresponding to the passage of the calibration magnetic object at said passage height (zb), said ratio (R) of the amplitudes of the calibration electrical signals non-linearly increasing as the passage height (zb) of the calibration magnetic object increases until it reaches a maximum value, the ratio of the amplitudes of the synchronised electrical signals (TOP, BOTTOM) being equal to the ratio (R) of the amplitudes of the calibration electrical signals, and o calculate (13) the magnetic moment (µ) of the magnetic object (2) from the passage height (z2) determined and one of the synchronised electrical signals.

13. A system for counting biological objects (5), comprising: - means for marking (41) the biological objects (5) by mixing them with a liquid matrix comprising magnetic beads able to attach to receptors carried by each biological object; - the device for determining the magnetic moment (µ) of a magnetic object according to claim 12, wherein the controller of the acquisition chain is further configured to count a biological object (5) marked when the magnetic moment (µ) associated therewith is greater than a threshold magnetic moment (NT).

14. A computer program comprising instructions which cause the device (6) according to claim 12 to execute the steps of the determination method (1) according to one of claims 1 to 9.

15. A computer program comprising instructions which cause the counting system according to claim 13 to execute the steps of the counting method (4) according to one of claims 10 to 11.