APPARATUS FOR THE QUANTIFICATION OF BIOLOGICAL COMPONENTS DISPERSED IN A FLUID

Abstract

Apparatus (100) for the quantification of biological components (3, 3′, 3″) in a fluid comprising: a measurement cell (1) comprising detection electrodes (4, 4′, 5, 5′, 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′) and reference electrodes (7, 7,′ 8, 8′, 9, 9′, 37, 37′, 64, 64′, 74, 74′, 164, 164′, 174′); an electronic unit (201) for the generation of input signals, impedimetric measurement, amplification of output signals and communication with a user interface; means for the generation of a magnetic field (101, 102, 103) with an appropriate gradient that can be modulated in time, said means of magnetisation being configured to generate a magnetic field capable of causing, in combination with concentrators (10, 10′, 10″, 14, 14′, 14′, 15) housed in the measurement cell, the separation of the components (3, 3′, 3″) to be quantified from the rest of the solution and their concentration on the detection electrodes (4, 4′, 5, 5′, 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′).

Claims

1. An apparatus for the quantification of a plurality of biological components in a fluid, comprising: a measurement cell comprising: a plurality of detection electrodes; at least one concentrator, said at least one concentrator being configured to attract magnetically the biological components to be quantified and concentrate said biological components on the detection electrodes, said detection electrodes being placed in proximity of said at least one concentrator; at least one pair of reference electrodes said reference electrodes being placed not in proximity of said at least one concentrator; a substrate housing the detection electrodes, the reference electrodes and the concentrators, said substrate comprising a first surface facing the exterior of said cell and a second surface facing the interior of said cell; a support comprising a first surface facing the exterior of said cell and a second surface facing the interior of said cell and opposed to the second surface of the substrate; at least one spacer element, confining the sample and distancing said substrate from said support; and a mechanical housing of the cell creating the electrical contact between the electrodes and an electronic board; an electronic unit connected to said board, said electronic unit being configured for the generation of input signals, the amplification of the output signals, the measurement of the impedance between the detection electrodes, of the impedance between the reference electrodes or of their difference, and the communication with a user interface; and means for the generation of a magnetic field with time-varying intensity and time-varying gradient, characterized in that said means are configured to produce variations in time up to a maximum value of the intensity of the field, said maximum value being higher than the field of saturation of the concentrators and to generate a magnetic field able to cause, in combination with the concentrators: the separation of the components to be quantified from the rest of the solution; and the concentration on the detection electrodes of the components to be quantified.

2. The apparatus according to claim 1, wherein said measurement cell comprises at least one pair of reference electrodes for each pair of detection electrodes.

3. The apparatus according to claim 1, wherein the measurement cell is fixed in a slanting position such that the angle between the normal to the substrate of said cell and the gravity acceleration vector is between 0° and 180°.

4. The apparatus according to claim 1, comprising a mechanical system configured to vary between 0° and 180° the angle between the normal to the substrate of the measurement cell and the gravity acceleration vector.

5. The apparatus according to any one of claim 1, comprising a microfluidic system with preloaded fluids configured for the dilution of the biological sample and the transport inside the measurement cell.

6. The apparatus according to claim 5, wherein said microfluidic system comprises: means for the collection of the sample of fluid and dilution with preloaded fluids on a cartridge; and means for the transport of the sample of fluid diluted inside the measurement cell.

7. The apparatus according to claim 5, wherein said microfluidic system is configured for the anticoagulation of the biological sample, said biological sample containing blood.

8. The apparatus according to claim 4 wherein the microfluidic system is integrated with the measurement cell, said microfluidic system being at least partially contained in the mechanical housing.

9. The apparatus according to claim 1, wherein said at least one spacer element are in the form of an appropriately shaped ring so as to convey the components into the zone of the electrodes.

10. The apparatus according to claim 9, wherein said means for the generation of a magnetic field are configured to produce variations in time of said field by means of a linear motion of moving towards/moving away of said means for the generation of a magnetic field at said substrate.

11. The apparatus according to claim 1, wherein said means for the generation of a magnetic field comprise: two permanent magnets in the form of a parallelepiped with square or polygonal base magnetised perpendicularly to the bases; and a sheet of mild ferromagnetic material placed between said two permanent magnets, between the bases of said magnets having the same polarity, said sheet being configured to concentrate the field lines in the plane of symmetry of the assembly of the two magnets.

12. The apparatus according to claim 2, wherein: a first electrode of each pair of detection electrodes of the measurement cell is connected to a first input by means of a first connection track; a first electrode of each pair of reference electrodes is connected to a second input by means of a second connection track; a second electrode of each pair of detection electrodes is connected to the node wherefrom an output signal (Out) is emitted by means of a third connection track; and a second electrode of each pair of reference electrodes is connected to the node wherefrom said output signal (Out) is emitted by means of a fourth connection track; above each one of said connection tracks being placed an insulating layer with dielectric constant and thickness such as to make the impedance between said connection tracks high in such a way as to make the effect of said impedance negligible.

13. The apparatus according to claim 2, wherein the substrate is divided in at least two parts, a first part carrying the detection electrodes and the concentrators in proximity of said detection electrodes and being centered with respect to the plane of symmetry of the means for the generation of a magnetic field, and a second part carrying only the reference electrodes so that they are subject to a lower magnetic field with respect to detection electrodes.

14. The apparatus according to claim 2, wherein at least one part of the substrate is centered with respect to the plane of symmetry of the means for the generation of a magnetic field, said part carrying: the detection electrodes and the reference electrodes interdigitated in such a way that said detection and reference electrodes are subject, on average, to the same magnetic field, fluctuations and drifts; and the concentrators in proximity of the detection electrodes.

15. The apparatus according to claim 12, wherein the first electrode of said at least one pair of detection electrodes and the second electrode of said at least one pair of detection electrodes are with rectangular section, with base comprised between 10 and 300 nm and height comprised between 1 and 3 μm.

16. The apparatus according to claim 15, wherein the distance between the first electrode of said at least one pair of detection electrodes and the second electrode of said at least one pair of detection electrodes is comprised between 1 and 5 μm.

17. The apparatus according to claim 12, wherein said at least one concentrator is cylindrical in shape, the diameter of the base surface of said concentrators being comprised between 10 and 50 μm, the height of said concentrators being comprised between 10 and 50 μm and the distance between said concentrators being comprised between 50 and 150 μm.

Description

[0041] The following description refers to the accompanying drawings, in which:

[0042] FIG. 1 is a frontal view of a detail of the apparatus according to the present invention, said detail comprising the measurement cell and the means of magnetisation;

[0043] FIG. 2 is a lateral view of the apparatus according to the present invention;

[0044] FIG. 3a is a frontal view of a detail of the apparatus according to the present invention, said detail comprising the central part of the measurement cell and the means of magnetisation, with a vector diagram of the forces for α=0°;

[0045] FIG. 3b is a frontal view of a detail of the apparatus of the present invention, said detail comprising the central part of the measurement cell and the means of magnetisation, with a vector diagram of the forces for a generic angle α comprised between 0 and 90°;

[0046] FIG. 3c is a frontal view of a detail of the apparatus of the present invention, said detail comprising the central part of the measurement cell and the means of magnetisation, with a vector diagram of the forces for α=90°;

[0047] FIG. 4a is an exemplifying diagram of the measurement method implemented by the apparatus of the present invention through modulation of the field generated by the means for the generation of said field, which shows the approach of the means to the measurement cell;

[0048] FIG. 4b is an exemplifying diagram of the measurement method implemented by the apparatus of the present invention through modulation of the field generated by the means for the generation of said field, which shows the moving away of the means in relation to the measurement cell;

[0049] FIG. 5 shows the shape of the signal relative to the percentage variation of the resistive component of the impedance after the moving towards and away of the means of magnetisation;

[0050] FIG. 6 is an exemplifying diagram of the positioning of the detection and reference electrodes with respect to the concentrators, in a first embodiment of the present invention;

[0051] FIG. 7a shows a section of the measurement cell in a first embodiment of the apparatus of the present invention, said section being along a plane perpendicular to the greater dimension of said at least one concentrator;

[0052] FIG. 7b shows a detail of the section shown in FIG. 7a, relative to said at least one concentrator;

[0053] FIG. 7c shows a detail of the section shown in FIG. 7a, relative to said at least one pair of detection electrodes;

[0054] FIG. 8 is a view from above of the detail of a first embodiment of the apparatus of the present invention, said detail being constituted by the substrate of the measurement cell;

[0055] FIG. 9 is a view from above of a detail constituted by the measurement cell of a second embodiment of the present invention;

[0056] FIG. 10 shows the trend of the differential percentage variation of the resistive component of the impedance, between the detection electrodes and the reference electrodes, in a second embodiment of the present invention, for δ=40 microns and α=90° (i) as a function of the equivalent parasitaemia level of a sample of red blood cells treated in such a way as to make them paramagnetic with respect to plasma (solid symbols and unbroken line) and (ii) for a sample of only healthy red blood cells that provides an indication of the signal due to false positives (dotted line);

[0057] FIG. 11 shows the trend of the signal for an equivalent parasitaemia of 0.5% in the case of α=90° and as the height (δ) of the measurement cell varies;

[0058] FIG. 12 shows the trend of the ratio between true signal (in the case of an equivalent parasitaemia of 0.5%, δ=40 microns) and spurious signal (due to false positives or fluctuations), for different values of the angle α;

[0059] FIG. 13 is a view from above of a detail constituted by an unit of the measurement cell of a third embodiment of the present invention;

[0060] FIG. 14 is a view from above of a detail constituted by two units of the measurement cell of a third embodiment of the present invention

[0061] FIG. 15 is a view from above of a detail constituted by an unit of the measurement cell of a fourth embodiment of the present invention; and

[0062] FIG. 16 is a view from above of the detail of a third and fourth embodiment of the apparatus of the present invention, said detail being constituted by the substrate of the measurement cell.

[0063] Referring to FIGS. 1, 2, 3a, 3b and 3c, an embodiment of the apparatus of the present invention (100), intended for the quantification of the erythrocytes infected by the plasmodium of malaria and hemozoin crystals, comprises: [0064] a measurement cell (1) comprising: [0065] a plurality of detection electrodes (4, 4′, 5, 5′, 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′); [0066] at least one concentrator (10, 10′, 10, 14, 14′, 14″, 15), said at least one concentrator (10, 10′, 10″, 14, 14′, 14″, 15) being configured to magnetically attract the components (3, 3′, 3″) to be quantified and concentrate said components on the detection electrodes (4, 4′, 5, 5′, 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′), said detection electrodes (4, 4′, 5, 5′, 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′) being placed in proximity of said at least one concentrator (10, 10′, 10″, 14, 14′, 14″, 15); [0067] at least one pair of reference electrodes (7, 7′, 8, 8′, 9, 9′, 37, 37′, 64, 64′, 74, 74′, 164, 164′, 174′) for each pair of detection electrodes (4, 4′, 5, 5′ 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′), said reference electrodes (7, 7′, 8, 8′, 9, 9′, 37, 37′, 64, 64′, 74, 74′, 164, 164′, 174′) being placed not in proximity of said at least one concentrator (10, 10′, 10″, 14, 14′, 14″, 15); [0068] a substrate (11) configured for the housing of the detection electrodes (4, 4′, 5, 5′, 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′), of the reference electrodes (7, 7′, 8, 8′, 9, 9′, 37, 37′, 64, 64′, 74, 74′, 164, 164′, 174′) and of the concentrators (10, 10′, 10″, 14, 14′, 14″, 15), said substrate (11) comprising a first surface (11′) facing outside said cell (1) and a second surface (11″) facing inside said cell (1); [0069] a support (12) comprising a first surface (12′) facing outside said cell (1) and a second surface (12″) facing inside said cell (1) and opposite to the second surface (11″) of the substrate (11); [0070] at least one spacer element (13, 13′) configured to confine the sample and distance said substrate (11) from said support (12); and [0071] a mechanical housing (600) configured to house the cell (1) and to make electrical contact between the electrodes (4, 4′, 5, 5′ 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′, 7, 7′, 8, 8′, 9, 9′, 37, 37′, 64, 64′, 74, 74′, 164, 164′, 174′) and an electronic board (202); [0072] an electronic unit (201) connected to said board (202), said electronic unit (201) being configured for the generation of the input signals, amplification of the output signals, measurement of the impedance between the detection electrodes (4, 4′, 5, 5′, 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′), of the impedance between the reference electrodes (7, 7′, 8, 8′, 9, 9′, 37, 37′, 64, 64′, 74, 74′, 164, 164′, 174′) or of their difference and communication with a user interface; and means for the generation of a magnetic field (101, 102, 103) with gradient that can be modulated in time, said means being configured to generate a magnetic field capable of causing, in combination with concentrators (10, 10′, 10″, 14, 14′, 14″, 15): [0073] the separation of the components (3, 3′, 3″) to be quantified from the rest of the solution; and [0074] the concentration on the detection electrodes (4, 4′, 5, 5′, 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′) of the components to be quantified (3, 3′, 3″); [0075] a microfluidic system with preloaded fluids that implements dilution and anticoagulation of the blood and transport within the measurement cell (1), said microfluidic system being integrated with the measurement cell (1), and at least partially contained in the mechanical housing (600). [0076] a mechanical system configured to vary the angle α formed between the perpendicular to the substrate of the measurement cell in which the blood sample is collected and the gravity acceleration vector, between 0° and 180°.

[0077] The microfluidic system, in turn, comprises [0078] means for the collection of the blood sample (500) and dilution with preloaded fluids (501) on a cartridge (503); and [0079] means (504) for transporting the diluted blood sample (500) inside the measurement cell (1).

[0080] In order to be able to perform the analysis, the patient's blood drop is, therefore, placed in contact with the inlet of the microfluidic system. It is then sucked in, diluted with preloaded fluids and conveyed into the measurement cell containing the substrate with the electrodes.

[0081] The measurement cell can be made with microfabrication techniques on silicon, glass or other polymeric materials, coupled with a suitable microfluidic system made in plastic material.

[0082] In the first embodiment of the apparatus of the present invention, intended specifically for use for malaria, the means (101, 102, 103) for the generation of a magnetic field must be able to generate a field which, preferably, has an intensity of at least 10.sup.4 A/m and a macroscopic gradient of its square module of at least 5*10.sup.14 A.sup.2/m.sup.3 directed towards or leaving the substrate, respectively in the case of paramagnetic or diamagnetic components with respect to the liquid medium in which they are dispersed. Such means (101, 102, 103) may, for example, be realised with a plurality of permanent magnets (101, 102) positioned so that the field generated by said magnets (101, 102) exerts a sufficient force to counteract the resultant of the weight force and that of Archimedes acting on the components of interest at great distance from the substrate (11), thus attracting them towards the surface of the same.

[0083] Referring to FIGS. 4a and 4b, the means for the generation of the magnetic field (101, 102, 103) are, in particular, constituted by two permanent magnets (101, 102) of NdFeB N52, in the shape of a parallelepiped with a square base and dimensions 20×20×5 mm, magnetised perpendicularly to the square faces and arranged with the north poles facing. Between them there is a small sheet (103) of mild ferromagnetic material with high magnetic permeability (e.g. Mumetal) that conveys and concentrates the field lines in the symmetry plane of the two magnets (101, 102). At the interface between the small sheet (103) and the air, a symmetrical magnetic field is therefore generated with respect to the separation plane of the magnets (101, 102), with a high gradient having a prevalent component directed orthogonally to the rectangular faces of the magnets (101, 102) placed in contact with or in proximity of the back of the substrate (11). Thus, the field produced is found to have an adequate intensity and gradient to capture the components (3, 3′, 3″), in an area of rectangular shape, with a width of about 2 mm and a height of about 16 mm, in order not to include the boundary effects of the magnets (101, 102).

[0084] Referring to FIGS. 4a, 4b and 5, and as mentioned above, the means for the generation of the magnetic field (101, 102, 103) are configured to realise a modulation of the field and relative gradient at the substrate (11) which carries the concentrator elements (10, 10′, 10″, 14, 14′, 14″, 15), i.e. to produce variations in the intensity of the magnetic field over time from a minimum value not exceeding 10% of the saturation field of the magnetic concentrators, to a maximum value that must be higher than the saturation field of the magnetic concentrators (10, 10′, 10″, 14, 14′, 14″, 15). In the first embodiment of the present invention, the magnetic concentrators are made, for example, in Ni and such modulation can be obtained by a linear motion of moving towards/away from the substrate (11) of the magnets (101, 102). When the means for the generation of the magnetic field (101, 102, 103) are brought in proximity of the substrate (11), the field on the Ni concentrators (10, 10′, 10″, 14, 14′, 14″, 15) is sufficient to saturate their magnetisation and therefore allows the capture of the components (3, 3′, 3″). When the means for the generation of the magnetic field (101, 102, 103) are moved away the field becomes negligible and the concentrators (10, 10′, 10″, 14, 14′, 14″, 15) become demagnetised, so that the components (3, 3′, 3″) become detached from the concentrators (10, 10′, 10″, 14, 14′, 14″, 15) and from the detection electrodes (4, 4′, 5, 5′, 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′). As already mentioned, the trend of the resistive component of the impedance measured at the ends of the detection electrodes (4, 4′, 5, 5′, 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′) in the moving towards/away phase is shown in FIG. 5. In the approach phase there is a positive ΔR increase, in the hypothesis of components (3, 3′, 3″) with resistance greater than that of the liquid, which develops over a characteristic time of capture τ.sub.C. Following a sudden moving away of the means for the generation of the magnetic field (101, 102, 103), the resistance returns to its initial level for the detachment of the components from the detection electrodes (4, 4′, 5, 5′, 6, 6′, 34, 34′, 84, 84′, 94, 94′, 184, 184′, 194, 194′), with a characteristic time TR.

[0085] Referring to FIGS. 6, 7a, 7b and 7c, in the first embodiment of the present invention it is foreseen that each pair of detection electrodes (4, 4′, 5, 5′, 6, 6′) of the measurement cell (1) comprises a first electrode (4, 5, 6) able to receive a first input signal (V+) and a second electrode (4′, 5′, 6′). Each pair of reference electrodes (7, 7′, 8, 8′, 9, 9′) comprises a first electrode (7, 8, 9) capable of receiving a second input signal (V−) of opposite polarity to the first input signal (V+) and a second electrode (7′, 8′, 9′) connected to the second electrode (4′, 5′, 6′) of each pair of detection electrodes (4, 4′, 5, 5′, 6, 6′), in a common point from which the output signal (Out) is taken. It is also possible, with obvious modifications of the electronic unit (201), to invert the role of the electrodes and use the common point of the second detection electrode (4′, 5′, 6′) and of the second reference electrode (7′, 8′, 9′) as input signal and the first electrode (4, 5, 6) of the pair of detection electrodes as first output signal and the first electrode (7, 8, 9) of the pair of reference electrodes as second output signal. In all cases the electronic unit (201) will be realised to provide the user with the difference between the impedance of the pair of detection electrodes and the impedance of the pair of reference electrodes in a suitably processed form.

[0086] In the first embodiment of the present invention, suitable for the diagnosis of malaria, the concentrators (10, 10′, 10″) are made of ferromagnetic material, such as Ni, Fe, Co, NiFe, CoFe, etc., and have the shape of a parallelepiped with the largest dimension extending perpendicularly to the plane represented in FIG. 3a. In order to ensure a sufficient concentration factor to obtain an adequate signal-to-noise ratio, the dimensions of the concentrators (10, 10′, 10″) and of the detection electrodes (4, 4′, 5, 5′, 6, 6′) should preferably be comprised within the ranges listed in Table 1.

TABLE-US-00001 TABLE 1 Component h.sub.F (μm) w.sub.F (μm) d.sub.F (μm) h.sub.E (nm) w.sub.E (gm) d.sub.E (μm) i-RBC 10-50 20-100 20-100 10-300 2-6 2-6 HC 10-50 10-50 10-50 10-300 1-3 1-5 h.sub.F is the smallest dimension of the base of a concentrator, w.sub.F is the largest dimension of the base of a concentrator and d.sub.F is the distance between a concentrator and the adjacent concentrator. h.sub.E is the smallest dimension of the base of a detection electrode, w.sub.F is the largest dimension of the base of a detection electrode and d.sub.E is the distance between two adjacent electrodes at the same concentrator.

[0087] In the first row of Table 1, the concentrator and detection electrode dimension ranges required for correct detection of the erythrocytes infected (i-RBC) by malaria plasmodium are shown; while in the second row of Table 1, the concentrator and detection electrode dimension ranges required for correct detection of free hemozoin (HC) crystals are shown.

[0088] The substrate (11) and, therefore, the same cell (1) of the present invention, the structure of the detection electrodes (4, 4′, 5, 5′, 6, 6′) and of the reference electrodes (7, 7′, 8, 8′, 9, 9′), can be replicated in four zones into which the substrate (11) is divided, each one divided into two rectangular zones with base equal to 2 mm and height equal to 4 mm. The width of 2 mm is commensurate with the extension of the high magnetic field produced by the magnets in a direction perpendicular to the plane of symmetry. The left part carries the electrodes on the concentrators, and is centred with respect to the plane of symmetry of the magnets described, while the right part has only the reference electrodes for the subtraction of the common mode signal. The subdivision of the active area into several regions with independent readings allows an increase in the ratio between the impedance variation produced by a single component attracted on the detection electrodes and the overall impedance between the electrodes, improving the signal-to-noise ratio in case of low concentrations of components to be detected. Since for each zone an output contact is needed towards the amplifier from which the output signal (Out) is to be emitted, while all the input signals (V+) and (V−) for detection electrodes and reference electrodes require only two contacts, the minimum number of contacts to be formed on the chip is equal to 4+2=6. In order to minimise the impact of possible short circuits between the electrodes during manufacture, three separate contacts can be used for each zone. Consequently, the total number of contacts is 12.

[0089] Referring to FIGS. 8 and 9, a second embodiment of the apparatus of the present invention, again specifically intended for use for malaria, foresees that the apparatus (100) comprises all the same components described above with a single difference related to the configuration of the measurement cell (1). In the second embodiment of the present invention, the measurement cell (1) comprises, in fact, a matrix of ferromagnetic concentrators of cylindrical shape (14, 14′, 14″) uniformly distributed on the substrate (11) according to a square grid. Alternatively, the matrix of concentrators can be arranged according to a hexagonal grid that maximises the packing. In FIG. 9, in particular, six pairs of detection electrodes (34, 34′) and six pairs of reference electrodes (37, 37′) are shown. The first electrode (34) of each pair of detection electrodes (34, 34′) is connected to a first input configured to receive the first input signal (V+) via a first connection track (44). The electrodes can be parallel, as shown in FIG. 9, or ring-shaped, as in the example described here below, in order to take advantage of the capturing tendency, experimentally detected, on the edges of the cylinders, and thus maximise the occupation of the sensitive area above the electrodes. The first electrode (37) of each pair of reference electrodes (37, 37′) is connected to a second input configured to receive the second input signal (V−) via a second connection track (47). Similarly, the second electrode (34′) of each pair of detection electrodes (34, 34′) is connected to the node from which the output signal (Out) is emitted through a third connection track (44′) and the second electrode (37′) of each pair of reference electrodes (37, 37′) is connected to the node from which said output signal (Out) is emitted through a fourth connection track (47′).

[0090] Above the first connection track (44), the second connection track (47), the third connection track (44′) and the fourth connection track (47′) an insulating layer (40, 40′, 50, 50′) is laid for each track, said insulating layer (40, 40′, 50, 50′) having dielectric constant and thickness such as to make the impedance between said connection tracks (44, 44′, 47, 47′) very high so that the effect of this impedance is negligible.

[0091] The configuration of the concentrators foreseen by the second embodiment allows a concentration factor to be obtained, at least for α=0, even higher than the one obtainable with respect to the first embodiment. For this purpose the dimensions of the concentrators (14, 14′, 14″) and the detection electrodes (34, 34′, 35, 35′) must, preferably, be comprised in the ranges listed in Table 2.

TABLE-US-00002 TABLE 2 Component h.sub.F (μm) w.sub.F (μm) d.sub.F (μm) h.sub.E (nm) w.sub.E (m) d.sub.E (μm) i-RBC e HC 10-50 10-50 50-200 10-300 1-3 1-5 h.sub.F is the height of a concentrator, w.sub.F is the diameter of the base of a concentrator and d.sub.F is the distance between one concentrator and the adjacent concentrator. h.sub.E is the smallest dimension of the base of a detection electrode, w.sub.F is the largest dimension of the base of a detection electrode and d.sub.E is the distance between the first finger of a detection electrode and the second finger of said detection electrode.

[0092] In Table 2, the ranges of the dimensions of the concentrators and detection electrodes required for proper detection of both erythrocytes infected (i-RBC) by the plasmodium of malaria and free hemozoin crystals (HC) are shown.

[0093] With such dimensions, assuming a length L of the straight electrodes shown in FIG. 9, equal to 6 μm, and a capture efficiency of 100% under conditions of optimal spacing, a geometric concentration factor

[00001]$F_C=\frac{{\left(d_F+w_F\right)}^2}{L.Math.\left(2w_E+d_E\right)}$

of about 400 is obtained Referring to FIG. 8, also in the second embodiment of the present invention, the substrate (11) and, therefore, the structure of the detection electrodes (4, 4′, 5, 5′, 6, 6′) and of the reference electrodes (7, 7′, 8, 8′, 9, 9′), can be replicated in four zones into which the substrate (11) is divided, each one divided into two rectangular zones wherein the left part, or first unit, carries the electrodes on the concentrators, and is centred with respect to the plane of symmetry (800) of the magnets described, while the right part, or second unit, has only the reference electrodes for the subtraction of the common mode signal.

[0094] Referring to FIGS. 13, 14 and 16, a third embodiment of the apparatus of the present invention, again specifically intended for use for malaria, foresees that the apparatus (100) comprises all the same components described above with differences related to the configuration of the measurement cell (1) as in the following.

[0095] As in the first and in the second embodiment, the substrate (11) and, thus, the structure of the detection electrodes (84, 84′, 94, 94′) and of the reference electrodes (64, 64′, 74, 74′) can be replicated in four zones (301, 302, 303, 304) into which the substrate is divided. The minimum number of contacts to be formed on the chip is always equal to 4+2=6, among which there are four output contacts (one per zone) and two input contacts (one for a first input signal (V.sup.+) for all detection electrodes and reference electrodes and one for a second input signal (V.sup.−) for all detection and reference electrodes). However, as already mentioned relatively to the first and the second embodiment, in order to minimize the impact of possible short circuits between the electrodes during manufacture, three separate contacts (Out, V.sup.+, V.sup.−) can be used for each zone. Consequently, the total number of contacts is 12.

[0096] Differently from the first and the second embodiment, at least one part of the substrate (11) is centred with respect to the plane of symmetry (801) of the means for the generation of a magnetic field (101, 102, 103), said part carrying: [0097] the detection electrodes (84, 84′, 94, 94′) and the reference electrodes (64, 64′, 74, 74′) interdigitated, or more in general, interlaced, in such a way that said detection and reference electrodes (84, 84′, 94, 94′, 64, 64′, 74, 74′) are subject, on average, to the same magnetic field, fluctuations and drifts; and [0098] the concentrators (10, 10′, 10″, 14, 14′, 14″) in proximity of the detection electrodes (84, 84′, 94, 94′).

[0099] For the purpose of the present description the word “interdigitated” means arranged in alternating, closely packed, single or double stripes. If the detection electrodes (84, 84′, 94, 94′) and the reference electrodes (64, 64′, 74, 74′) are interdigitated the subtraction of the background is improved, thus obtaining a more accurate measurement of impedance variation associated with the components to be quantified. In FIG. 13, in particular, six pairs of detection electrodes (84, 84′, 94, 94′), six pairs of reference electrodes (64, 64′, 74, 74′) and six concentrators (14, 14′, 15) are showed. Like in the second embodiment, the concentrators are ferromagnetic and have a cylindrical shape (14, 14′, 15). The electrodes (64, 64′, 74, 74′, 84, 84′, 94, 94′) and the concentrators (14, 14′, 15) are uniformly distributed on the substrate according to a rectangular grid. The concentrators (14, 14′, 15) are placed only in proximity of the detection electrodes (84, 84, 94, 94′) and not in proximity of said reference electrodes (64, 64′, 74, 74′). The detection electrodes (84, 84, 94, 94′) and the reference electrodes (64, 64′, 74, 74′) are arranged in alternating double stripes. More particularly, in each row, both the two pairs of detection electrodes (84, 84′, 94, 94′) are interposed between a first pair of reference electrodes (74, 74′) and a second pair of reference electrodes (64, 64′), and no reference electrodes (74, 74′, 64, 64′) are interposed between the two pairs of detection electrodes (84, 84′, 94, 94′). More generally, the measurement cell comprises at least a first pair of detection electrodes (84, 84′, 184, 184′) and a second pair of detection electrodes (94, 94′, 194, 194′), both the pairs of detection electrodes (84, 84′, 184, 184′, 94, 94′, 194, 194′) being interposed between a first pair of reference electrodes (74, 74′, 174, 174′) and a second pair of reference electrodes (64, 64′, 164, 164′), and no reference electrodes (74, 74′, 174, 174′, 64, 64′, 164, 164′) being interposed between the first pair of detection electrodes (84, 84′, 184, 184′) and the second pair of the detection electrodes (94, 94′, 194, 194′). The spatial configuration and distribution of the detection electrodes (84, 84′, 94, 94′) and of the reference electrodes (64, 64′, 74, 74′) described form a unit (700) that can be replicated to form a second unit (701) and many other identical units, as shoved in FIG. 14. In this way a layout is created, where reference electrodes (64, 64′, 74, 74′) and detection electrodes (84, 84′, 94, 94′) are closely interdigitated, thus guaranteeing a better subtraction of the spurious fluctuations and, as a consequence, an improvement of the sensitivity of the tests performed using the apparatus (100) of the present invention.

[0100] A first electrode (84′, 94, 184′, 194) of each pair of detection electrodes (84, 84′, 94, 94′, 184, 184′, 194, 194′) of the measurement cell (1) is, then, connected to a first input V.sup.+, while a first electrode (64, 74′) of each pair of reference electrodes (64, 64′, 74, 74′) is connected to a second input V.sup.−. The second electrode (84, 94′) of each pair of detection electrodes (84, 84′, 94, 94′) and the second electrode (64′, 74) of each pair of reference electrodes (64, 64′, 74, 74′) are connected to the node wherefrom the output signal (Out) is emitted. The electrodes (64, 64, 74, 74′, 84, 84′, 94, 94′) are ring-shaped. In particular, the end of the one of the electrodes (64, 74, 84, 94) of each pair (64, 64′, 74, 74′, 84, 84′, 94, 94′) consists in two open concentric rings. The end of the other (64′, 74′, 84′, 94′) of the electrodes (64′, 74′, 84′, 94′) of each pair (64, 64′, 74, 74′, 84, 84′, 94, 94′) consists, instead, of a straight stretch and of an open ring that surrounds the straight stretch. The stretch is configured to be entered in the inner ring of the end of the first named electrode (64, 74, 84, 94) of the pair and the open ring that surrounds the straight stretch is configured to be entered in the space between the inner and the outer ring of the first named electrode (64, 74, 84, 94) of the pair. Referring to FIG. 15, a fourth embodiment of the apparatus of the present invention, again specifically intended for use for malaria, foresees that the apparatus (100) comprises all the same components described above. The concentrators (14, 14′, 15) have the same geometry of the concentrators of the third and of the second embodiment (i.e. cylindrical shape). The measurement cell (1) has the same relative positioning of the detection and reference electrodes (164, 164′, 174, 174′, 184, 184′, 194, 194′) detailed above relatively to the third embodiment. The fourth embodiment is characterized by the same layout of the third embodiment, with the reference electrodes (164, 164′, 174, 174′) and the detection electrodes (184, 184′, 194, 194′) closely interdigitated and, in particular, arranged in alternating double stripes. The sole difference is the geometry of the electrodes. In the fourth embodiment, the end of the one of the electrodes (164, 174, 184, 194) of each pair (164, 164′, 174, 174′, 184, 184′, 194, 194′) consists in an open ring. The end of the other (164′, 174′, 184′, 194′) of the electrodes (164′, 174′, 184′, 194′) of each pair (164, 164′, 174, 174′, 184, 184′, 194, 194′) consists, instead, of a closed ring configured to be entered in the open ring of the first named electrode (164, 174, 184, 194) of the pair. Also in the fourth embodiment, the substrate (11) and, thus, the structure of the detection electrodes (184, 184′, 194′) and of the reference electrodes (164, 164′, 174, 174′) can be replicated in four zones (301, 302, 303, 304) into which the substrate is divided. In order to minimize the impact of possible short circuits between the electrodes during manufacture, three separate contacts (Out, V.sup.+, V.sup.−) can be used for each zone with a the total number of contacts equal to 12. However, the minimum number of contacts to be formed on the chip is six: four output contacts (one per zone) and two input contacts (one for a first input signal (V.sup.+) for all detection electrodes and reference electrodes and one for a second input signal (V.sup.−) for all detection and reference electrodes.

Example

[0101] The example described here below relates to the characterisation of an apparatus according to the second embodiment described above, specifically intended for use in malaria. The substrate, according to the structure described in FIG. 8, provides for an arrangement of Ni concentrators (with a diameter of 40 microns and a height of 20 microns) arranged according to a hexagonal grid with a centre-centre distance of 160 microns. The electrodes are ring shaped, made of gold with a thickness of 300 nm, width of 3 microns and spacing of 3 microns. The external electrode has an external diameter of 40 microns and is perfectly superimposed on the Ni concentrator, in order to maximise the probability of capture of the components at the electrodes, since specifically on the edges of the cylinders there is the maximum value of the magnetic field and of its gradient. Between the electrodes and the top of the concentrators an insulating layer of SiO2 is interposed with a thickness of 3 microns. The distance between the substrate and the support is determined by a polymeric container ring (50) with a thickness variable between 40 and 500 microns.

[0102] The drop of sample containing the components to be analysed is dispensed on the support on which the containment ring is prefabricated, initially placed horizontally, face up. The substrate is made to descend until it presses on the containment ring, creating the seal that allows the fluidic cell to be defined. The cell is housed within a mechanical apparatus that allows variation of the angle α, between the perpendicular to the face of the substrate and the gravity acceleration vector, between 0 and 180°. A motorised linear motion allows the magnets to move towards and move away from the substrate in a controlled way and measure correspondingly the resistance variations that are proportional to the concentration of the components. The NdFeB magnets, configured as shown in FIG. 1, are able to produce a magnetic field H in proximity of the surface of the substrate towards the measurement cell, i.e. at a distance of 0.5 mm from the surface of the magnets, in the plane of symmetry of the two facing magnets, with module and gradient of the square module equal respectively to 6*10.sup.5 A m.sup.−1 and 7*10.sup.14 A.sup.2 m.sup.−3.

[0103] For the specific application of malaria diagnostics, the components of interest with paramagnetic properties with respect to plasma are hemozoin crystals and plasmodium-infected red blood cells, which contain some hemozoin crystals. Both hemozoin crystals and red blood cells behave as insulators for input voltage signals applied to them in the range of a few MHz, such as those considered for impedimetric detection. While hemozoin has an absolute positive volumetric magnetic susceptibility, equal to 4.1*10.sup.−4 in units of the S. I., [M. Giacometti et al. APPLIED PHYSICS LETTERS 113, 203703 (2018)], the infected red blood cells have globally a still diamagnetic behaviour. However, the volumetric susceptibility is less negative than that of plasma, so that the difference in susceptibility between infected blood cell and plasma is of the order of 1.8*10.sup.−6, lower than that of hemozoin crystals but sufficient to produce their capture in an appropriate gradient of applied magnetic field H. The difference in susceptibility between the two components makes it possible to discriminate between them, choosing appropriately the value of the magnetic field gradient and the angle α on the basis of an estimate of the forces involved.

[0104] Assuming a volume of red blood cells V.sub.RBC=9.1*10.sup.−11 cm-3, a density of blood cells ρ.sub.RBC=1.15 g cm-3 and a density of plasma ρ.sub.P=1.025 g cm.sub.−3, the sum of the weight force and the force of Archimedes on the single blood cell, F.sub.gb=(ρ.sub.RBC−ρ.sub.P)V.sub.RBCg (FIG. 3a), results equal to 1.1*10.sup.−13 N. According to the expression of the magnetic force on a 1 superparamagnetic particle, F.sub.M=½μ.sub.0Δ.sub.χ∇H.sup.2, assuming a susceptibility difference Δ.sub.χ=1.8*10.sup.−6 between blood cell and plasma, [K. Han and A. B. Frazier, J. Appl. Phys. 96(10), 5797 (2004)], the value of the H.sup.2 gradient necessary to balance F.sub.gb in the case of α=0° is of the order of 1*10.sup.15 A.sup.2 m.sup.−3.

[0105] A similar estimate can be made for single plasma suspended hemozoin crystal. Assuming an average volume V.sub.HC=2.2*10.sup.−14 cm.sup.−3, a density ρ.sub.HC=1.15 g cm.sup.−3, and a difference in susceptibility Δ.sub.χ=4.1*10.sup.−4 with respect to plasma, it is obtained that for hemozoin the value of the H.sup.2 gradient necessary to balance F.sub.gb is of the order of 1.7*10.sup.13 A.sup.2 m.sup.−3.

[0106] From these estimates it results therefore that, for angle α=0, where the magnetophoretic force is antiparallel to the resultant of the gravitational force and Archimedes force, the H2 gradient produced by the particular magnets considered (7*10.sup.14 A.sup.2 m.sup.−3 at the surface of the electrodes of the substrate in the case wherein the magnet is rested on the back of the substrate 0.5 mm thick) is able to attract the hemozoin crystals but not the red blood cells infected by malaria.

[0107] Experiments conducted with angle α=0 on suspensions of hemozoin crystals in plasma diluted 1:10 with PBS, to simulate the blood dilution expected in the diagnostic test, have in fact shown the possibility of measuring a net change in resistance between the electrodes of the substrate, distinguishable from noise, up to concentrations of hemozoin equal to 1 ng/ml. Assuming that within a red blood cell infected with malaria there are about 18 hemozoin crystals, the concentration detected corresponds to a parasitaemia (percentage ratio between sick and healthy red blood cells) of 0.2%, typical of a patient who has just had a febrile malarial attack.

[0108] Under the same conditions (angle α=0) measurements on samples of red blood cells treated with NaNO.sub.2 in order to induce the transformation of haemoglobin into paramagnetic methaemoglobin that allows a malaria-infected red blood cell model to be obtained, showed no signal distinguishable from noise. Even if the treated red blood cells (t-RBC) have a difference in magnetic susceptibility with respect to plasma that is double with respect to that of infected blood cells (Δ.sub.χ=3.6*10.sup.−6), [Nam, Jeonghun, Hui Huang, Hyunjung Lim, Chaeseung Lim, Sehyun Shin. Analytical Chemistry 85, n. 15, 7316-23 (2013)], the H2 gradient necessary to balance the gravitational and buoyancy force (5*10.sup.14 A.sup.2 m.sup.−3) is very similar to that produced by magnets (7*10.sup.14 A.sup.2 m.sup.−3), so that any non-ideality or deviation from the values tabulated for the properties of the red blood cells can amply justify the absence of capture and therefore of electrical signal. The situation changes for angles α close to 90°, where the weight and buoyancy force component that opposes the magnetic attraction is practically zero. Since there is no longer a real threshold, both hemozoin and red blood cells are attracted by the macroscopic field gradient towards the substrate, and therefore concentrated on the concentrators by the local gradient during their downward sliding motion.

[0109] For the hemozoin the values of the signals detected as a function of concentrations do not vary significantly, since the attraction and concentration were already very effective for α=0. Therefore, a detection limit very similar to that found for α=0, of the order of 1 ng/ml, is obtained for α=90°.

[0110] For the treated red blood cells, instead, much more relevant signals are obtained, which allow a limit of detection (LOD) in the order of 0.005% to be obtained, corresponding to about 250 parasites per μl of blood. At the frequency of the input signals (V.sup.+ and V.sup.−) of 1 MHz the red blood cells have an insulating behaviour and therefore the variation of the resistive component of the measured ΔR/R impedance is proportional to the volumetric fraction occupied by them above the concentrator electrodes. It is therefore evident how the greater volume of red blood cells with respect to the hemozoin can lead to signals of greater extent.

[0111] FIG. 10 shows with dots and unbroken line the percentage change in resistance measured following the moving away of the magnets from the substrate, i.e. during the release of the red blood cells attracted onto the electrodes, as a function of the equivalent parasitaemia level of the sample. Considering that the diagnostic test involves diluting the patient's blood in PBS (1:10 vol-vol), the sample consisted of a suspension of NaNO.sub.2-treated red blood cells in a solution of plasma and PBS (1:10), at decreasing concentrations that can be correlated with the parasitaemia in the following way. Since in 1:10 diluted blood the haematocrit is of the order of 4%, x referring to the percentage volumetric fraction of t-RBC in the sample, the equivalent parasitaemia is x/4.

[0112] Also in FIG. 10, the dotted line corresponds to the ΔR/R signal measured with a sample of untreated red blood cells resuspended in plasma and PBS (1:10 vol-vol) with 0.4% haematocrit. This signal is associated partly with false positives and partly with spurious fluctuations induced by the motion of the magnets, but constitutes the lower signal limit below which no significant measurements can be made. As can be seen, the ΔR/R signal measured follows a substantially linear trend with the parasitaemia of the sample over more than three decades, thus allowing a quantification of the parasitaemia itself. The detection limit, resulting from the intersection of the two curves, is of the order of 0.05%, or about 250 parasites per μl of blood, and corresponds to the LOD of current rapid diagnostic tests for malaria. In contrast to these, however, the test that is the subject of the present patent application allows a quantitative evaluation of the parasitaemia, which is useful in the phase of diagnosis and of monitoring of the disease following pharmacological treatment.

[0113] The influence of the angle α on the detection limit has been studied in experiments conducted for angles of 75°, 90° and 105°, as shown in FIG. 12. It shows the ratio between the ΔR/R signal measured at a t-RBC sample with an equivalent parasitaemia of 0.5% and that measured with a sample of untreated red blood cells resuspended in plasma and PBS (1:10 vol-vol) with 0.4% haematocrit, associated with false positives. This ratio, which can be associated with the true/non-specific signal ratio of the test, is maximum for 90°, while it decreases for greater and smaller angles. At 75° the gravity still has a component that opposes the magnetic force, so that the signal of the false positives decreases but also the true signal associable to those treated and in conclusion the ratio decreases. At 105° the gravity contributes to bringing all the healthy and treated blood cells towards the electrodes of the substrate, so that the true signal but also the non-specific one increases. Also in this case however the signal-to-noise ratio decreases. Ultimately, the analysis conducted shows that the 90° angle is preferable for maximising the ratio between true and non-specific signal.

[0114] The height of the fluidic measurement cell (δ), defined by the thickness of the spacer element, must itself be optimised to increase the ratio between true and non-specific signal. FIG. 11 shows the amplitude of the current signal measured after the second movement away of the magnets, for cell heights of 40, 80 and 500 microns. As can be seen, the net signal decreases with increasing thickness, despite the fact that more blood cells are present in the cell. This is due to the fact that, in the example shown here, the sample is loaded with the cell horizontally (α=0), so that the blood cells have time to settle on the substrate within the typical time (3 minutes) needed to close the cell, make the electrical contacts, stabilise the signal and start the measurement by moving the magnets close. Considering in fact a typical sedimentation rate of 2 micron/s, even in the case of δ=500 microns, a thickness of at least 360 microns from the substrate is emptied of blood cells. This means that, at the moment when the sample is moved close, the blood cells are further away than in the case of δ=40 microns, and therefore their capture during the sliding motion is less efficient. This analysis therefore shows that, according to the protocol adopted in this example, the best conditions for increasing the signal correspond to δ=40 microns.

[0115] The increase in the cell thickness, and therefore in the number of blood cells contained in it, could instead be exploited if the initial sedimentation of the blood cells was avoided, by appropriate agitation or by initially placing the device at an angle α=180°. In this way it would be possible to concentrate the blood cells in proximity of the substrate and exploit the sliding motion induced by gravity to move the healthy red blood cells away from the concentrators, capturing instead the diseased ones.

[0116] Finally, it should be noted how the very dynamic of the signals is rich in information that allows the nature of the signals themselves to be distinguished. For α=90° and δ=40 microns, the dynamic of the signal following the approach of the magnets, corresponding to the capture time τ.sub.C, has the value of 80 s in the case of a false positive signal associated with a sample with 4% haematocrit due to healthy (untreated) blood cells and 150 s in the case of a sample with red blood cells treated with a volumetric fraction corresponding to a parasitaemia of 0.5%. This difference also remains in the case of release τ.sub.R times of 20 s and 60 s, respectively. Knowing the characteristic dynamics it is therefore possible to identify a spurious impedance variation, e.g. due to false positives or to other fluctuations of the system.