Patent Application
20220097051
The present invention relates to an apparatus for the quantification of biological, cellular and non-cellular components dispersed in a sample of biological fluids (blood, urine, saliva, sweat, etc.) or in a sample of fluids extracted from biological solids (e.g. faeces). This quantification is performed by magnetophoretic separation and concentration of the components of interest from the rest of the sample and impedimetric detection of the quantity of these components. For the purpose of the present description, “cellular biological components”, or simply “cellular components”, means biological corpuscles larger or comparable in size to that of the cells, such as for example blood corpuscles (e.g. red blood cells, white blood cells and platelets), the corpuscles contained in urine, pathogens such as bacteria and the eggs of certain parasites. “Non-cellular biological components”, or simply “non-cellular components” are understood to be those corpuscles of biological origin that are smaller in volume than cells but larger than individual molecules. Such non-cellular components may be, for example, the crystals of certain substances that develop under particular pathological conditions, such as, for example, the hemozoin crystals produced by the plasmodium of malaria within infected erythrocytes.
In general, the apparatus of the present invention is aimed at the quantification of all those biological components dispersed in a fluid that have different magnetic properties with respect to the medium in which they are suspended. For the purpose of the present description, it is specified that this category comprises those biological components which, following a specific transformation, for example due to a pathological condition, assume a peculiar magnetic behaviour that allows their magnetophoretic separation from their counterparts under physiological conditions. More particularly, the present invention relates to an apparatus that allows and foresees the spatial isolation and concentration of one or more cellular and non-cellular biological components dispersed in a biological fluid (e.g. blood, urine, sweat, saliva, etc.) or in a fluid extracted from a solid biological sample (e.g. faeces), exploiting the differences between the magnetic properties of said components and the magnetic properties of the other components of no interest. Once the separation and concentration have taken place, the apparatus of the present invention foresees, therefore, that the quantification of such cellular and non-cellular biological components takes place through the measurement of the impedance variation between two or more electrodes placed near the concentration zones.
By way of example, one of the fields of application of the apparatus of the present invention can be implemented and realised in such a way as to be used for the detection and quantification of parasite eggs (e.g. Schistosoma mansoni) in infested faeces, in that these eggs have a paramagnetic behaviour distinct from the typically diamagnetic behaviour of an aqueous solution in which they can be resuspended [S. Karl et al., PLOS Neglected Tropical Diseases, 7(5), 2219 (2013)].
Another field of application of the present invention relates to, then, the diagnosis of all those pathologies that cause an alteration of the magnetic properties of one or more types of blood cellular components and/or give rise to the formation of substances with magnetic properties different from plasma, said substances being absent or in different concentrations under physiological conditions. In particular, pathologies are known that cause the alteration of the magnetic properties of erythrocytes, or red blood cells, such as malaria and poisoning that cause an increase in methemoglobinemia. For example, in the case of malaria, it is known that the plasmodium, during the malarial pathogenesis, produces a particular substance to which mention was made above, which takes the name of hemozoin and is a paramagnetic substance. In particular, hemozoin is produced in the form of crystals that accumulate in the infected erythrocytes, making them paramagnetic with respect to the medium in which they are dispersed, that is, plasma. Furthermore, in the non-early phases of malaria, the membrane of the infected red blood cells breaks down, giving rise to the release of the hemozoin crystals in the plasma, which is, instead, diamagnetic. Pathologies are, furthermore, known in which it is not the magnetic properties of the cellular components of the blood that vary but, on the contrary, their density. An example of this type is given by sickle-cell anaemia, where, even if the diamagnetism of the red blood cells remains unchanged, their density changes. In this case, by adding a strongly paramagnetic substance to the plasma, such as gadolinium for instance, it can be thought to exploit the magnetic difference between the red blood cells and the gadolinium solution added to the plasma together with the difference in density between diseased red blood cells and healthy red blood cells to obtain the separation and, therefore, to perform the count of the pathological erythrocytes.
At the state of the art, techniques are known for the separation of cellular components of blood, based on the different magnetic behaviour of these components under physiological and pathological conditions. In particular, patent application U.S. Pat. No. 5,985,153A describes a device for the separation of cells or other magneto-sensitive biological entities comprising: a substrate, an external magnetic field generator and a microfluidic system for the loading and unloading of blood. Document US0127222A describes, instead, a generic system for the immobilisation of cells previously marked with magnetic particles so that they can be attracted by ferromagnetic structures formed on a chip and placed in an external magnetic field. In application WO2010091874 a particular ferromagnetic structure is described, composed of magnetic conduits, capable of attracting magnetic particles in particular points in which magnetic domain walls are located. In all the prior art documents mentioned above, as well as in a part of the scientific literature listed in the bibliography [S. Bhakdi et al., Optimized high gradient magnetic separation for isolation of Plasmodium-infected red blood cells, Malaria Journal 2010, 9:38]; [J. Nam et al., Magnetic Separation of Malaria-Infected Red Blood Cells in Various Developmental Stages, Anal. Chem., 85, 7316-7323 (2013)]; [Ki-Ho Han and A. Bruno Frazier, Paramagnetic capture mode magnetophoretic microseparator for high efficiency blood cell separations, Lab Chip, 6, 265-273 (2006)], only the magnetophoretic separation of the components of interest from the rest of the blood sample is described, and no mention is made of the detection of the number of such components.
In the patent application US20120003687A and in scientific publications [E. Du., et al., Electric Impedance Microflow Cytometry for Characterization of Cell Disease States, Lab Chip. 2013 Oct. 7; 13(19): 3903-3909] and [M., et al. Ibrahim, J. Claudel, D. Kourtiche and M. Nadi, Geometric parameters optimization of planar interdigitated electrodes for bioimpedance spectroscopy, J Electr Bioimp, vol. 4, pp. 13-22, 2013], techniques of impedimetric quantification of cellular components are, instead, described. These techniques have, however, never been used in association with magnetophoretic separation and concentration. Impedimetric detection requires that the volumetric fraction of the components near the electrodes is sufficiently high in order to obtain a signal-to-noise ratio in the output signal that is sufficient to ensure a correct quantification of the separated components. This concentration is usually obtained with microfluidic techniques aimed at the movement of the blood fluid, which considerably increase the degree of complexity of the system and make it unsuitable for use by a non-specialist user, for example the patient himself or herself. The proposed apparatus intends to overcome these difficulties by replacing the separation by microfluidic movement with a system of magnetic concentration and separation of the components of interest on areas of the measurement cell in which the detection electrodes are located.
Again, with reference to the diagnosis of all those pathologies that cause an alteration of the magnetic properties of one or more types of blood components, in order to carry out the measurement, the non-specialised user can dispense on the special support of said cell a drop of blood just taken, optionally diluted and treated with anticoagulant, and then place it in contact with the substrate of said cell on which the concentrator elements and electrodes are housed, in turn placed within an external magnetic field in such a way that a component of the force of gravity opposes the magnetic attraction force directed towards the concentrators. The measurement cell can also be a pre-assembled measurement cell, with substrate and support appropriately distanced, optionally integrated with a microfluidic system with preloaded fluids that implements only dilution and anticoagulation of the blood drop and transport within the measurement cell. It is obvious that the measurement cell of the apparatus of the present invention can be further integrated with a microfluidic system that carries out not only the functions of dilution and transport of the sample but also the movement of the blood functional to the same separation of the components, even if this embodiment would have a greater complexity than the embodiments that foresee that the separation takes place only by magnetophoresis and impedimetric detection. For a volume of the blood drop taken of the order of about ten microlitres and assuming that the capture of the components of interest takes place at a maximum distance from the concentrators of between 20 and 200 micrometres, the dimensions of the active area for the capture on the substrate must be of the order of a cm2 and, in particular, comprised between 0.1 and 5 cm2. The support and, therefore, the measurement cell, must also have approximately the same dimensions. On these active area values a high concentration of the components of interest is required in order to ensure an adequate signal-to-noise ratio. As will be explained more clearly here below, this concentration can be quantified by a so-called concentration factor Fc which is given, in the absence of sliding of the components parallel to the substrate and assuming that all the components are captured, by the ratio between the active area of the substrate within which the drop containing the components to be quantified is confined, and the area defined by the detection electrodes. In order to have an adequate signal-to-noise ratio in the output signal, the concentration factor Fc should preferably be at least around 100.
The object of the present invention is, therefore, to provide an apparatus that allows the quantification of the components of interest from a quantity of fluid in which they are dispersed comprised between 5-500 microL (5-50 microL, in the case wherein the fluid is blood), and produces an output signal with a signal-to-noise ratio such as to allow the detection of biological cellular and non-cellular components with a lower concentration limit up to tens of components per microlitre.
This object is achieved by the apparatus of the present invention, which comprises:
The apparatus of the present invention can, moreover, comprise a microfluidic system with preloaded fluids that implements the dilution of the biological fluid sample and the transport within the measurement cell. To this end, the microfluidic system, in turn, comprises:
The measurement cell, in turn, comprises:
Said at least one concentrator can be a cylinder or a parallelepiped or an element of another shape placed on the substrate, placed at the detection electrodes, and is made of ferromagnetic material. The concentrator generates an intense local field gradient which attracts the components to be quantified, causing them to concentrate in proximity of said concentrator and, therefore, in proximity of the detection electrodes. In this way, by suitably sizing both the concentrator and the detection electrodes, the concentration factor can increase up to the value necessary to obtain an adequate signal-to-noise ratio.
The detection electrodes are placed in proximity of the concentrator elements, while the reference electrodes are placed not in proximity of the concentrator elements. In other words, the reference electrodes are placed in areas without said concentrators. For the purpose of the present description, “detection electrodes are placed in the proximity of the concentrators” means that the detection electrodes are placed in relation to the concentrators in such a way that their distance in the direction perpendicular to the substrate does not exceed 5 μm and that the projection of the detection electrodes on the substrate plane is contained within the projection of the concentrators on said substrate plane, or, although not contained within the projection of the concentrators on the substrate plane, is not distant from the projection of the concentrators on the substrate in the direction of the width (e.g. the largest dimension) of the concentrators, for a value not exceeding twice the width of the electrodes. The expression “the reference electrodes are placed not in proximity of the concentrators” means, instead, that the detection electrodes are placed with respect to the concentrators in such a way that the distance between the projection of the concentrators on the substrate and the projection of the detection electrodes on the substrate in the direction of the width of the concentrators is equal to at least twice the characteristic width of the concentrators.
In this way, the separate components accumulate selectively on the detection electrodes but not on the reference electrodes, causing a specific change in impedance between the detection electrodes with respect to the spurious impedance that may be recorded between the reference electrodes. The output signal of the impedimetric quantification system is, therefore, proportional to the difference between the impedance variation recorded between the detection electrodes and that between the reference electrodes. From this output signal, it is then possible to estimate the number of components of interest or, equivalently, their concentration, by comparison with an appropriate calibration curve, carried out by means of a processor.
The means of generation of the magnetic field are configured to realise a modulation of the field and relative gradient at the substrate that houses the concentrator elements, that is to produce variations in time of the magnetic field intensity from a minimum value equal to about 10% of the saturation field of the concentrators to a maximum value that must be higher than the saturation field of the magnetic concentrators. As will be explained in greater detail below, if the magnetisation means are made by means of permanent magnets, this modulation can be simply obtained by a linear motion of moving towards/away from the substrate of the magnets. In particular, when the magnets are brought into proximity of the substrate, the components are captured, while when the magnets are moved away the components detach from the concentrators and from the electrodes. In the case of components with resistance greater than that of the liquid, during the approach phase there is a positive increase in resistance measured at the ends of the detection electrodes, with a characteristic time τC. Following a sudden moving away of the magnet, on the other hand, the resistance returns to its initial level for the detachment of the components, with another characteristic time TR. Selective capture and release activation by modulation of the magnetic field on the substrate allows a more accurate measurement of impedance variation associated with the components. In this way, thanks to selective capture and release activation, spurious variations, i.e. not synchronous with the moving towards/away of the magnets, can be easily identified, thus obtaining a more accurate measurement of the impedance variation effectively associated with the components, as well as an improved subtraction of the background and drifts associated therewith.
The improvement in the sensitivity of the test, linked to an increase in the ratio between the true signal and the one due to false positives, and the possibility of discriminating between different blood cells, is further guaranteed by the fact that the measurement cell can be inclined with respect to the horizontal plane, i.e. with respect to the plane perpendicular to the gravity acceleration vector.
The separation of the components from the matrix in which they are dispersed, in fact, occurs thanks to the competition between the gravitational force and the magnetic attraction force according to modalities that depend on the characteristics of the components and the angle α formed between the gravity acceleration vector and the line perpendicular to the substrate.
As will be made clearer by the example described here below, the capture efficiency of the captured components varies according to the angle α formed by the line perpendicular to the substrate and by the gravity acceleration vector. For α=0° (
The overall capture efficiency when α>0°, does not depend only on the local capture of the components present within a capture volume in proximity of each concentrator. The same downward sliding motion induced by gravity gradually causes the components above to transit in proximity of the concentrators, thus allowing an increase in the capture efficiency, defined as the number of captured components per unit of volume of liquid in which they are suspended. By shaping the containment ring, as well as the geometry of concentrators and electrodes, so as to convey geometrically the components on the active capture area during their sliding motion induced by gravity, the capture efficiency can therefore be further improved.
A second object of the present invention is, then, to provide an apparatus for the quantification of cellular and non-cellular components that is able to discriminate between different components.
For this purpose, the measurement cell can have a variable inclination instead of a fixed one, and the apparatus of the present invention can comprise a mechanical system configured to make this inclination vary between 0° and 180°.
In other words, the apparatus of the present invention can comprise a mechanical system configured to vary between 0° and 180° the angle α formed between the perpendicular to the substrate of the measurement cell in which the fluid sample is collected and the gravity acceleration vector.
In addition, even the same time modulation of the magnetic field mentioned above and the consequent measurement of capture and release times, which are characteristic of the components to be quantified, provides additional information for the identification of the nature of the same captured components.
Even if, as mentioned above, the apparatus of the present invention is applicable to the diagnosis of any pathology that is the cause of a variation in the magnetic properties of one or more biological components, among the various pathologies for the diagnosis of which it is possible to use the apparatus of the present invention, malaria is of particular interest, in that the diagnostic devices for this type of pathology, present today on the market, have some limitations that make them not always easy to use in particularly disadvantaged contexts, such as those typical of endemic areas, often located in developing countries. The most sensitive method currently available for the diagnosis of malaria is based, in fact, on gene recognition of the various plasmodium strains by PCR (Polymerase Chain Reaction). This type of method is particularly complex and delicate and, therefore, difficult to apply in non-technologically advanced contexts. Moreover, PCR is not a pan-plasmodium method, but is targeted towards specific strains and therefore subject to the problems deriving from continuous mutations of the plasmodium. The “thin smear and/or thick drop” method, on the other hand, which consists of counting the red blood cells infected by plasmodium in a drop of blood under an optical microscope, while not requiring complex instrumentation, requires highly expert personnel and involves a certain variability in the interpretation of the results, as well as long analysis times. Rapid tests (RDT) based on antibody-antigen interaction, on the other hand, are characterised by such low sensitivity as to prevent their use for early diagnosis. In addition, due to the latent presence of the antigen in patients' bodies in the endemic area, methods based on antibody-antigen interaction result in a high number of false positives.
A third object of the present invention is, therefore, to provide an apparatus that also allows the early diagnosis of malaria, whether pan-plasmodium, which has an adequate sensitivity and is simple and economical enough to be used even in those areas where the economic means available do not allow the use of complex instrumentation and specialised personnel.
This object is achieved by the apparatus of the present invention, since the latter is able to carry out both magnetic separation and quantification of infected erythrocytes and direct detection of free hemozoin crystals in plasma. The quantification of infected erythrocytes makes it possible to obtain a direct evaluation of the parasitaemia, which is normally quantified by calculating the ratio between infected erythrocytes and healthy erythrocytes, optionally also in the early phase of the disease, before the completion of the first plasmodium reproduction cycle (48-72 hours). The direct detection of hemozoin crystals is, instead, particularly useful in the non-initial phases of the disease, such as, for example, at the time of the first fever attack, since, in these phases, the erythrocytes have already undergone membrane rupture, and the only thing actually quantifiable in circulation is free hemozoin.
These and further objects of the present invention will be made clearer by the reading of the following detailed description of some preferred embodiments of the present invention by way of a non-limiting example of the more general concepts claimed, as well as the examples concerning experimental tests carried out on the present invention.
The following description refers to the accompanying drawings, in which:
Referring to
The microfluidic system, in turn, comprises
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.
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.
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 104 A/m and a macroscopic gradient of its square module of at least 5*1014 A2/m3 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.
Referring to
Referring to
Referring to
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
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.
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.
Referring to
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.
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.
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.
With such dimensions, assuming a length L of the straight electrodes shown in
of about 400 is obtained Referring to
Referring to
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+) for all detection electrodes and reference electrodes and one for a second input signal (V−) 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+, V−) can be used for each zone. Consequently, the total number of contacts is 12.
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:
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
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+, while a first electrode (64, 74′) of each pair of reference electrodes (64, 64′, 74, 74′) is connected to a second input V−. 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
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
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
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*10−4 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*10−6, 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.
Assuming a volume of red blood cells VRBC=9.1*10−11 cm-3, a density of blood cells ρRBC=1.15 g cm-3 and a density of plasma ρP=1.025 g cm−3, the sum of the weight force and the force of Archimedes on the single blood cell, Fgb=(ρRBC−ρP)VRBCg (
A similar estimate can be made for single plasma suspended hemozoin crystal. Assuming an average volume VHC=2.2*10−14 cm−3, a density ρHC=1.15 g cm−3, and a difference in susceptibility Δχ=4.1*10−4 with respect to plasma, it is obtained that for hemozoin the value of the H2 gradient necessary to balance Fgb is of the order of 1.7*1013 A2 m−3.
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*1014 A2 m−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.
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.
Under the same conditions (angle α=0) measurements on samples of red blood cells treated with NaNO2 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 (Δχ=3.6*10−6), [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*1014 A2 m−3) is very similar to that produced by magnets (7*1014 A2 m−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.
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°.
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+ and V−) 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.
Also in
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
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.
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.
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 τC, 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 τR 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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102019000000821 | Jan 2019 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/050376 | 1/17/2020 | WO | 00 |
