System for capturing and detecting species present in a biological fluid

Abstract

The present invention relates to a system for detecting the presence of at least one species in a fluid, preferably for detecting the presence of at least one circulating cell or cell aggregate in a human or animal biological fluid, in particular circulating tumor cells (CTC) in a blood fluid, the detection system comprising a device (20) for filtering the fluid, the filtering device (20) comprising a filtering membrane (21) comprising at least one aperture (22) designed to retain a given type of species present in the fluid, the filtering device (20) further comprising at least one opening (23) designed to ensure the continuous circulation of the biological fluid during the operation within the fluid, even when the at least one aperture (22) is occupied.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of capturing specific species or particles present in biological or non-biological fluids (blood and its derivatives such as plasma and serum, urine, water, air and any other fluid that needs to be analyzed). It particularly relates to the capture of cells of interest present in biological fluids such as blood, in order to analyze these cells for medical monitoring. It is particularly suitable for the detection of circulating tumor cells, for medical monitoring of patients with cancerous lesions and / or almost complete extraction of tumor cells contained in blood, for the diagnosis of cancerous lesions, and for tumor treatment, the present invention allowing the filtration / extraction of tumor cells. It also applies to prognosis and efficacy monitoring by detectable cells. BACKGROUND

[0002] Fluids can carry several types of species or beads of interest to be counted and analyzed. In particular, animal or human biological fluids carry several types of cells, the presence of which can allow the monitoring or detection of different pathologies.

[0003] In particular, the study of circulating tumor cells (hereinafter CTC) in circulating blood helps to diagnose cancer, thus enabling better treatment of patients.

[0004] Indeed, cancerous tumors release CTC into the bloodstream, and this phenomenon has been shown to occur at an early stage of the disease. The biological and molecular analysis of one or more cells allows accurate diagnosis and provides information on the aggressiveness of the cancer and the effectiveness of the treatment. CTC are therefore a biomarker of interest at all stages of cancer management, diagnosis, prognosis and monitoring.

[0005] However, the concentration of CTC present in the blood of cancer patients is extremely low (about 1 / 10 of normal blood cells 9 ). Isolation and / or almost complete extraction of these rare cells is therefore extremely difficult.

[0006] Various in vitro methods are known for this isolation, based on immunodetection. They are based on the presence on the surface of CTC of EpCAM (epithelial cell adhesion molecule), a membrane antigen specific to cells of epithelial origin. A 7.5 ml blood sample is centrifuged and then placed in the presence of ferromagnetic nanoparticles carrying an anti-EpCAM antibody on their surface. The CTC are then separated from the other cells by applying a magnetic field.

[0007] This system has two disadvantages:

[0008] - the very limited blood sample is used (7.5 ml, equivalent to 0.15% of the total blood volume), the number of CTC being very low due to their low concentration;

[0009] - it does not allow to detect CTCs that lose the EpCAM protein during the epithelial-mesenchymal transition (EMT), which represent about 2 / 3 of the total number of these cells; on the other hand, it only allows to detect cells with a limited life cycle and not the most dangerous differentiated cells.

[0010] Other methods for isolating CTCs in vitro, based on their size, from blood samples are known. In particular, the ISET system ("Isolation by Size of Epithelial Tumor Cells") uses a filtered blood sample (previously lysed red blood cells) on a microperforated polycarbonate membrane; in this system, CTCs are previously hardened by applying paraformaldehyde in order to withstand the high pressure applied.

[0011] Generally, the sensitivity of the in vitro detection is reduced by the small volume of the sample. In fact, considering the rarity of CTCs in blood, their presence in a few milliliters of sample is equivalent to a few units already in advanced stage of cancer. Therefore, it is almost impossible to find them at an earlier stage.

[0012] Recently, systems designed for use in vivo or through apheresis have been developed to capture CTCs in the physiological medium, thus preserving their viability as much as possible, and potentially obtaining a greater volume of blood than analyzed by in vitro systems.

[0013] Despite the advantages of these systems over in vitro systems, none of these systems allows to capture and simultaneously count CTCs, in particular for real-time applications.

[0014] In fact, the known systems use immunostaining techniques to identify the tumor nature of the captured cells. These techniques require post-capture manipulations, are expensive and do not allow direct real-time detection at the bedside. In addition, the platform used must be transparent to allow counting the captured cells using a fluorescence microscope. Furthermore, these techniques do not guarantee the integrity of the labeled cells, since the labeled cells have already been altered before biological analysis or for culture. SUMMARY

[0015] The present invention provides a system that overcomes the drawbacks of the prior art.

[0016] To this end, according to a first aspect, the application proposes a system for detecting the presence of at least one species present in a fluid, preferably at least one circulating cell or cell aggregate present in a human or animal biological fluid, and in particular a circulating tumor cell present in a blood fluid, the detection system comprising a filtration device for said fluid, said filtration device comprising a filtration membrane, said filtration membrane comprising at least one aperture, said aperture being adapted to retain a given type of species present in the fluid, said filtration device further comprising at least one opening, said opening being adapted to ensure the continuity of the circulation of the biological fluid when operating within the fluid, even when said at least one aperture is occupied.

[0017] The application according to the first aspect is advantageously completed by the following features, taken separately or in any technically possible combination thereof:

[0018] - the system comprises a plurality of electrodes arranged around said at least one aperture, said electrodes forming one or more circuits polarized by an AC electrical signal, allowing to measure the variation of the complex impedance between these electrodes once one or more cells are contained in the aperture or in the vicinity of the aperture, the measurement of the variation of the modulus of the complex impedance and of its phase allowing to discriminate the type of cell;

[0019] - the AC electrical signal applied to the electrodes has a frequency such that the electric field generated by the electrodes is able to capture or release cells in the aperture by means of the dielectrophoretic forces generated between the electrodes;

[0020] - during the capture, the polarization frequency induces a positive dielectrophoretic force, thereby concentrating the cells between the electrodes and retaining the cells in the aperture;

[0021] - the polarization frequency induces a negative dielectrophoretic force to separate all the captured cells, the frequency typically being 1 MHz;

[0022] - the polarization frequency induces a dielectrophoretic force to selectively separate cell types, the frequency being between 50 kHz and 150 kHz, preferably 100 kHz for separating tumor cells;

[0023] - the polarization frequency is progressively increased between 10 kHz and 200 kHz in order to separate the cells at different times according to their dielectric properties;

[0024] - the system further comprises an inductor connected to the electrodes, thereby forming an electromagnetic resonant circuit, the electrodes and the inductor forming a circuit for detecting, preferably remotely searching, the presence of the captured cells;

[0025] - the filtering membrane is made of a material chosen from the group comprising: glass or metal (nickel, gold) or polymer, ferromagnetic material, magnetic material (NiFe), or a combination of glass and silicon or nickel and silicon, silicon nitride, silicon oxide, silicon or more generally a biocompatible non-toxic material;

[0026] - the pores have a cross-section size between 0.1 pm and 100 pm, preferably between 8 pm and 12 pm or between 8 pm and 15 pm; and / or the pores are spaced apart by a spacing between 100 nm and 100 pm; the number of pores is between 100 and 100 000 000 000;

[0027] - the pores are substantially circular or elliptical or polygonal or slit-shaped;

[0028] - the pores of the membrane are arranged in groups of several pores, each group having a pattern, and these groups can be connected to each other via a row of pores;

[0029] - the pattern formed by a group has the following shape: hexagonal, circular;

[0030] - the membrane comprises several groups of pores arranged in a square or star structure;

[0031] - the pores of the membrane are arranged randomly;

[0032] - the filtering device comprises a planar support comprising a recessed area in which the filtering membrane is located and in which said openings are formed, said openings being arranged at the periphery of the filtering membrane;

[0033] - the system comprises a compartment in which the filtering device is housed, the compartment comprising an inlet module and an outlet module, the inlet module and the outlet module being joined together to allow the passage of fluid from the inlet module to the outlet module through the filtering device;

[0034] - the system comprises an inlet holder and an outlet holder, the blades supporting the filtering device, the inlet holder and the outlet holder being joined together so that the blades are located between the inlet holder and the outlet holder to allow the passage of fluid from the inlet holder to the outlet holder through the filtering device.

[0035] The application according to the second aspect provides a capture assembly comprising a plurality of systems according to the application arranged in series, each system comprising a filtering device adapted to retain one type of species.

[0036] The application according to the second aspect is advantageously achieved with the following features, used alone or in any technically possible combination of them:

[0037] - the assembly comprises an inlet module, an outlet module and at least one intermediate module arranged between the inlet module and the outlet module, the intermediate module and the outlet module supporting the filtering means, said modules comprising means for fixing them together;

[0038] - the inlet module comprises a fluid inlet and the outlet module comprises a fluid outlet;

[0039] - the assembly comprises an input support, an output support and at least one intermediate support arranged between the input support and the output support, the intermediate support and the output support supporting the filtering means, these supports comprising means for fixing them together to form an integral assembly.

[0040] The application also relates to a method for capturing cells circulating in a fluid, the method comprising a step of circulating the fluid in a system according to the first aspect of the application, the method comprising a step of applying an electrical signal to the electrodes, the electrical signal having a frequency such that the electric field generated by the electrodes is able to capture or release the captured cells from the pores by means of dielectrophoretic forces generated between the electrodes.

[0041] The method according to the application is advantageously done with the following features, used alone or in any technically possible combination thereof:

[0042] - during the capture, the polarization frequency induces a positive dielectrophoretic force, thereby concentrating the cells between the electrodes and retaining the cells in the pores;

[0043] - the polarization frequency induces a negative dielectrophoretic force to separate all the captured cells, the frequency typically being 1 MHz;

[0044] - the frequency induces a dielectrophoretic force to selectively separate cell types, the frequency being between 50 kHz and 150 kHz, preferably 100 kHz for separating tumor cells;

[0045] - the polarization frequency is progressively increased between 10 kHz and 200 kHz in order to separate the captured cells at different times according to the dielectric properties of the cells.

[0046] The capture of different species is based on the physical properties of the species circulating in the fluid, in particular on their size and deformability. In the case of cells, the fluid is biological (blood, urine, lymph, and generally any fluid circulating in the human or animal body and carrying the cells of interest to be analyzed). This biological liquid can or can not be diluted in a buffer solution. This liquid can also be a culture medium.

[0047] For the cells that can be present in blood, the size of platelets is 2-4 pm and the size of red blood cells is approximately 7 pm; while the size of white blood cells varies from 7 to 15 pm, but they are extremely deformable. The size of CTCs varies from 4 to 25 pm, but they are not easily deformable.

[0048] Therefore, as long as the cells have low deformability, they can be captured by the filtering device, while allowing the other non-tumoral components of the biological fluid to pass under the conditions of normal blood flow present in the body.

[0049] The presence of the openings ensures the continuous circulation of the biological fluid under the conditions of pressure and speed present in the body, regardless of whether the filtering membrane is filled by the captured components.

[0050] Preferably, by coupling the filtering membrane and its ports to the electrodes, the present application combines the physical capture with the detection of the cells captured near the pores. Therefore, the counting of the captured cells can be carried out in real time.

[0051] In particular, when it comes to CTCs having specific dielectric properties, they affect the impedance of the electrical circuit formed by the electrodes. They affect the electrical signal in real time, thus allowing the real-time detection of the cells captured by the filtering device or the occlusion rate of the filtering device. The detection of the occlusion rate makes it possible to determine whether the filtering device can capture the species without saturation.

[0052] Based on the observation that CTCs have different dielectric properties from the other cells that can be captured, it is possible to discriminate them among the captured cells.

[0053] Based on the dielectric properties of CTCs, their real-time electrical detection makes it possible to avoid the steps of immunolabeling the tumor cells necessary to characterize the labeled cells and the establishment of a system compatible with optical microscopy. Therefore, the present application makes it possible to directly characterize the presence of CTCs on the filtering membrane, avoiding subsequent operations of the device. Therefore, the medical information can be delivered in a less costly and non-invasive way, in real time. The real-time monitoring of the capture has significant advantages both for in vivo and ex vivo use, by allowing the user to adjust the time of exposure of the device to the blood fluid of the patient being analyzed and to the tumor load, in order to adapt the exposure time of the device to the richness or sparsity of the information of the device, thus to the accuracy of the medical information provided. Moreover, the present application also allows the removal of the circulating tumor cells contained in the blood after the capture, thus providing a form of therapy.

[0054] The present invention provides the clinician with immediate initial information on the number of species per unit of time or volume exposed, which can then be supplemented by analysis of the population thus captured. It should be noted that many of the analysis methods require minimal testing and the present invention also makes it possible to ensure that the conditions for performing the analysis are met before generating the often costly fees. Furthermore, there is no need to collect, transport and prepare the sample. The quality of the information is maintained, the cells are isolated under natural and physiological conditions and this information is immediately returned to the expert at the consultation site in order to make an immediate decision. The present invention also allows the production of useful data for the treatment of the patient. Since the blood fluid conditions vary from patient to patient and are different at the patient level throughout the day, the present invention can be combined with local fluid velocity measurements to be used as a calibration or reference to establish a comparable database.

[0055] Advantageously, the impedance variation associated with the detection of CTCs can be measured remotely in a remote wireless mode without contact, thanks to the coupling of the electrical circuit formed by the electrodes with the resonant circuit.

[0056] The system surface can also be functionalized with antibodies, combining physical capture and affinity capture.

[0057] The release of the captured cells from the filtering device, as a means of collection for analysis or re-cultivation, can be achieved by electrical stimulation using the same electrodes used for detection. BRIEF DESCRIPTION OF DRAWINGS

[0058] Additional features, objects, and advantages of the invention will be apparent from the following description, which is purely illustrative and not limiting, and should be read in conjunction with the appended drawings, in which:

[0059] - Figure 1 A system for capturing substances present in a fluid is shown;

[0060] - Figure 2 A filtering membrane according to one embodiment of the invention is shown;

[0061] - Figure 3 A more detailed schematic view of the mesentery is shown; Figure 2

[0062] - Figure 4a , Figure 4b , Figure 5 and Figure 6 Schematic views of filtering membranes in different embodiments are shown;

[0063] - Figure 7 Two embodiments of the arrangement of electrodes around the filtering membrane port according to the invention are shown;

[0064] - Figure 8 ​A compartment of the system of the application is shown;

[0065] - Figure 9a , Figure 9b , Figure 9c , Figure 9d , Figure 9e Different views of an input module of a compartment of the system of the application are shown;

[0066] - Figure 10a , Figure 10b , Figure 10c , Figure 10d , Figure 10e , Figure 10f Different views of an output module of a compartment of the system of the application are shown;

[0067] - Figure 11a , Figure 11b Attachment of a capture system in a compartment module of a system according to the application is shown;

[0068] - Figure 12a , Figure 12b and Figure 12c Compartment of the Figure 8 with additional intermediate modules is shown;

[0069] - Figure 13a , Figure 13b , Figure 13c , Figure 13d , Figure 13e , Figure 13f Different views of an intermediate module of a compartment of the system of the application are shown;

[0070] - Figures 14 to 17 A support and a support assembly according to an embodiment of the application are shown;

[0071] - Figures 18 to 20 A support assembled together according to an embodiment of the application is shown;

[0072] - Figure 21 The steps of a capture and detection method according to the application are schematically shown.

[0073] In all the figures, similar elements have the same reference signs. DETAILED DESCRIPTION

[0074] Embodiments of the application are described below in the context of capturing and detecting cells present in a biological fluid, but the application is applicable to capturing and detecting all types of species or beads present in a biological or non-biological fluid (blood and its derivatives such as plasma and serum, urine, water, air, pollutants, etc.).

[0075] In particular, the application applies to the capture of species, by which is meant: tumor cells; collections of tumor cells; blood clots (fluid is blood); exosomes.

[0076] With regard to Figure 1 The system 1 for capturing circulating cells present in a fluid comprises a fluid inlet 11 and a fluid outlet 12. It should be understood that the fluid is circulating and passes through the system described herein.

[0077] Such a system 1 can be connected to the blood flow of a person through the person's arm 100, but it can of course also be connected to another part of the body of a person or animal. Preferably, the system will be connected to a peripheral vein (for example, the peripheral vein at the elbow crease) or a central venous line of a person or animal. Alternatively, such a system 1 can be connected on the one hand to a test tube 101 containing a fluid and on the other hand to a system 102 for collecting the fluid after passage through the capture system 1.

[0078] In the case of ex vivo or in vitro use, a peristaltic pump or a pressure-controlled pump 13 is connected to the fluid inlet, the flow sensor 14 allowing the pump 13 to be regulated. The pump 13 is used to deliver the fluid to the filtration device 20, which will be described in detail below. The incoming fluid thus passes through the filtration device 20 and is reinjected into the patient or animal body or into a fluid collection tube.

[0079] The fluid inlet and the fluid outlet are connected to the circulating fluid by catheters or any other means known to the person skilled in the art and adapted to the location in which the system is used.

[0080] Alternatively, the system can be used in vivo and does not require a pump. In this case, it is placed directly in the circulating fluid.

[0081] The filtration device 20 is advantageously coupled to a device 26 for detecting the captured cells.

[0082] The measurement unit 15 connected to the detection device 26 measures information about the detected cells.

[0083] The system 1 also comprises a wired or wireless communication interface 16 in communication with a wired or wireless terminal 2. Such a terminal 2 comprises an interface allowing the user to access various information related to the detection.

[0084] Filtration device

[0085] Figure 2 And Figure 3 A possible embodiment of the filtration device 20 is shown. This integrated filtration device comprises a planar support 24, for example circular, comprising a zone 25 in which the filtration membrane 21 is located.

[0086] The membrane is for example circular and is located in a region 25 of the same shape.

[0087] At least one hole 22 for capturing / retaining cells present in the biological fluid is formed in the membrane, the filtering device 20 being placed in the biological fluid. The fluid flows through the system.

[0088] The membrane is composed of a biocompatible and non-toxic material chosen from the group comprising glass or metal (nickel, gold), or polymer, ferromagnetic material, magnetic material (NiFe), multi-material (glass / silicon), silicon nitride, silicon oxide, silicon.

[0089] Obviously, for applications where the fluid is not biological, the fact that the material is biocompatible is irrelevant.

[0090] The holes advantageously have a cross-sectional size of between 0.1 pm and 100 pm, preferably between 8 pm and 12 pm or between 8 pm and 15 pm, and a number typically of between 1000 and 6000, preferably between 100 and 100000000. The size and number of holes depend on the type of circulating cells to be captured and the way the filtering device is used (in vivo, ex vivo, in vitro).

[0091] In addition, the holes have a pitch of between 0.1 pm and 100 pm, preferably between 8 pm and 12 pm or between 8 pm and 15 pm.

[0092] The holes have various shapes. They can be substantially circular or substantially elliptical or substantially polygonal or slit-shaped.

[0093] Advantageously, the holes of the membrane are arranged in groups of several holes, each group having a pattern, the groups being connected to each other by a row of holes.

[0094] For example, in Figure 2 , the holes are arranged in four groups 210, each group having a polygonal shape.

[0095] The groups can be placed on the membrane in several patterns: in a square Figure 2 ) or in a star Figure 4a . As for the star structure, the holes are arranged in hexagonal groups 211, the hexagonal groups 211 being connected to each other by slit-shaped groups 212. Thus, the star structure is obtained by combining two groups of holes. The star structure extends from a central group 211 located at the center of the membrane and six branches extend from this central group. On each branch, two polygonal groups are connected to the central group, the slits 212 connecting the polygonal groups. The number of branches can of course vary, as can the number of groups per branch.

[0096] Alternatively, as Figure 4bAs shown, the holes arranged in groups 211' are not connected to each other, but are located in independent positions 212' from each other. This maximizes the number of holes on the support 24.

[0097] To allow continuity of fluid flow, the openings 23 are formed in the carrier at the periphery of the holes. To maintain undisturbed blood flow, regardless of the degree of obscuring of the filtering area by the captured components.

[0098] Referring again to Figure 2 and Figure 3 , the openings are shaped around the membrane like angular sectors. Such a shape is not limiting and other shapes can also be considered. In addition, fluid disturbing elements or fluid concentrating elements can be arranged.

[0099] The number and shape of the openings are optimized to impair the mechanical strength of the filtering device 20 as little as possible. In the example shown in Figure 2 and Figure 3 , there are four openings 23, while in the example shown in Figure 5 there are six openings 23.

[0100] Alternatively, and with reference to Figure 6 , in addition to the peripheral angular sector openings 23, the membrane can also comprise a central opening 27. In this example, the holes are a polygonal group composed of pairs of slit-shaped groups. The collection of two hexagonal groups is distributed in a star around the central opening 27.

[0101] For the configuration in figure 4, the following parameters can be used:

[0102] - diameter of the circular holes: 8 pm, 9 pm, 10 pm, 11 pm, 12 pm;

[0103] - inter-hole distance: 5 pm;

[0104] - number of holes: 4246, 3395, 2717, 2287, 1880;

[0105] - average number of holes per hexagonal group: 271, 220, 169, 135, 121;

[0106] - average number of holes per slit: 54, 45, 40, 27;

[0107] - surface area occupied by the holes: 0.213 mm 2 , 0.216 mm 2 , 0.217 mm 2 ;

[0108] - total surface area of an opening: 0.111 mm 2 ;

[0109] - Total area occupied by four openings: 0.444 mm 2 .

[0110] For the configuration in Figure 5 the following parameters can be used:

[0111] - Diameter of the circular holes: 8 pm, 9 pm, 10 pm, 11 pm, 12 pm;

[0112] - Inter-hole distance: 5 pm;

[0113] - Number of holes: 3395, 4246, 2717, 2287, 1880;

[0114] - Average number of holes per hexagonal group: 220, 271, 121, 169, 135;

[0115] - Average number of holes per slit: 45, 54, 40, 27, 54;

[0116] - Surface area occupied by holes: 0.216 mm 2 , 0.213 mm 2 , 0.217 mm 2 ;

[0117] - Surface area of one opening: 0.111 mm 2 ;

[0118] - Total surface area occupied by six openings: 0.666 mm 2 .

[0119] For the configuration in Figure 6 the following parameters can be used:

[0120] - Diameter of the holes: 8 pm, 9 pm, 10 pm, 11 pm, 12 pm;

[0121] - Inter-hole distance: 5 pm;

[0122] - Number of holes: 1227, 486, 386, 324, 271;

[0123] - Average number of holes per hexagonal group: 220, 169, 135, 121;

[0124] - Average number of holes per slit: 45, 40, 27;

[0125] - Surface area occupied by holes: 0.213 mm 2 , 0.216 mm 2 , 0.217 mm 2 ;

[0126] - the surface area occupied by the central opening: 0.057 mm 2 ;

[0127] - the surface area occupied by one peripheral opening: 0.111 mm 2 ;

[0128] - the total surface area occupied by the six openings and the central opening: 0.723 mm 2 .

[0129] Capturing and detecting and measurement unit

[0130] Concerning Figure 3 and Figure 7 And to detect the cells captured by the holes using the filtration device 20, the capturing and detecting system advantageously comprises a detecting device 26 consisting of electrodes arranged around the holes 22. The electrodes are connected together to form one or more electrical circuits, characterized by a complex impedance depending on the frequency of the electrical excitation. The value of this impedance depending on the frequency is affected by the presence of cells in the vicinity of the electrodes bypassing the holes 22.

[0131] In particular, an AC voltage is applied to the electrodes at a given frequency, so as to measure the complex impedance between the electrodes. The real part of the measured impedance is characteristic of the electrical resistance of the medium occupying this space. As for the imaginary part, it reflects the dielectric properties of the medium, in particular its permittivity. During the electrical measurements, we measure the modulus of the complex impedance and the phase shift of the current with respect to the applied voltage. These two measurements probe both the real and imaginary parts of the electrical impedance.

[0132] When cells are positioned between the electrodes, the measured electrical impedance (through its modulus and "phase") is modified. Both the real and imaginary parts of the impedance are modified. We thus measure these two changes by the change in impedance modulus and the change in current / voltage phase shift.

[0133] The measurement of the complex impedance makes it possible to discriminate between cell types, in particular tumoral / non-tumoral. To perform this type of detection, the change in the imaginary part does contain the most valuable information.

[0134] In particular, since CTCs have specific dielectric properties, they change the value of this impedance in a way that is different from other potentially trapped cell types. From the detected change, it is possible to detect the captured cells and their type. The electrodes are, for example, gold, copper, platinum, nickel, piezoelectric material, conductive polymer. The measured impedance is in the range 10 Hz to 1 MHz or even 5 MHz.

[0135] The electrodes thus surround the holes of the filtration membrane in order to electrically probe the dielectric properties of the medium in the vicinity of the holes of the captured cells.

[0136] Within the above signal frequency range, the imaginary part of the electrical impedance is mainly determined by the capacitance formed by the cytoplasmic membrane. This lipid bilayer, rich in membrane proteins, is a good electrical insulator separating two electrically conductive media (the intracellular medium (cytoplasm) and the extracellular medium (the liquid containing the cells to be analyzed), thus forming a capacitor described by a capacitance, i.e. a capacitor described by a capacitance source. This membrane capacitance is not present in the circuit in the absence of a cell positioned between the electrodes (in the region where the electric field emanating from the electrodes is present), but it appears in the circuit when a cell is positioned between the electrodes (in the region where the electric field emanating from the electrodes is present). This change causes a change in the measured impedance imaginary part. Of course, at the same time, the real part of the impedance is also modified. Thus, both parts of the impedance (real and imaginary) modified by the presence of the cell are measured during capture. Tumor cells circulating in the blood have a very particular plasma membrane morphology, resulting in the presence of a large number of protrusions that are not present in healthy cells. These protrusions greatly increase the surface area of the plasma membrane of tumor cells compared to that of healthy cells. Thus, the membrane capacitance of the cell behaves like a flat capacitor, whose capacitance is proportional to the surface area of the opposite conductor. This explains why the membrane capacitance of tumor cells is much greater than that of healthy cells. Thus, the detection implemented, due to the amplitude of the change in the imaginary part of the electrical impedance, not only allows the detection of the cells retained by the capture device, but also allows the prediction of the healthy or tumor nature of each captured cell.

[0137] As Figure 7 shown, the electrodes can surround the holes in various ways: by wrapping the holes or by forming a rectilinear trajectory around the holes. The choice of the shape of the electrodes depends on the density of the holes. Alternatively, the electrodes can be located on the inner wall of the holes.

[0138] Direct (active) measurement requires connecting the electrodes to the connection tracks 29 of the contact pads 28. The pads 28 are connected to a measurement unit 15 that allows direct measurement of the value of the impedance variation induced by the presence of a cell between the electrodes.

[0139] The measurement unit can comprise a coil 151 connected to the electrodes to form an electromagnetic resonator for passive and wireless measurement (inquiry of the device and reception of a remote signal). The wireless terminal 2 can then measure the impedance variation of the detection circuit without contact. The coil 151 can also be included in the membrane and its inductance value can then be measured remotely.

[0140] Alternatively, a wire connection between the electrodes and the measurement unit 15 is possible and allows direct reading of the impedance value of the detection circuit.

[0141] The terminal 2 can also be connected to the measurement unit 15 by wire.

[0142] The measurement is direct and real-time whatever the way the impedance variation is measured. It is thus possible to determine the presence of cells on the membrane surface. This impedance variation caused by the presence of cells near the capture well depends on the nature of the captured cells, and its measurement makes it possible to discriminate which captured cells are tumoral. This measurement is non-invasive for the cells to be detected and does not affect their viability in any way.

[0143] Capture and separation

[0144] This detection system advantageously relies on the use of dielectrophoretic forces associated with the wells, making it possible to retain the cells and also to selectively separate them.

[0145] Indeed, a dielectric object like a cell, immersed in a medium in the presence of a non-uniform electric field, which we call the gradient of the modulus of the electric field, is subjected to a force that can make it move, due to its polarizability.

[0146] This force is at the basis of the dielectrophoresis principle. In the presence of a non-uniform AC electric field, the direction of this force with respect to the gradient of the square of the modulus of the electric field depends on the frequency of the AC electric field and on the dielectric properties of the object. For an object of given size and dielectric constant, the dielectrophoretic force can be positive (the dielectric object moves towards the region with the highest modulus of the electric field) or negative (the dielectric object moves towards the region with the lowest modulus of the electric field), depending on the field frequency.

[0147] As mentioned above, during cell capture, an AC voltage is applied to the electrodes in order to detect them in real time. The planar form of the electrode configuration thus leads to the creation of a non-uniform AC field above the electrodes, which faces the fluid to be analyzed.

[0148] The high electric field regions are close to the wells of the capture device, and the low electric field regions are deeper into the fluid above the microelectrodes. These electrokinetic phenomena related to dielectrophoretic forces thus indicate that the cells that reach the vicinity of the wells will be subjected to a force that can direct them towards the capture wells (positive dielectrophoresis) or repel them (negative dielectrophoresis).

[0149] The frequency at which the sign of the dielectric force changes is called the cut-off frequency. A dielectric object has its own cut-off frequency depending on its shape, size dimensions and dielectric properties (relative permittivity).

[0150] In the case of circulating blood cells, the cut-off frequency of healthy blood cells is approximately 150 kHz, while that of tumoral cell line cells is significantly lower (50 kHz). This is also the result of the different dielectric properties of tumoral cells, which are associated with a high membrane capacitance in this frequency range. It thus appears that by polarizing the electrodes at an intermediate frequency between these two frequencies, the cells of interest can be selectively preferentially directed in a preferred direction in space.

[0151] The detection system can thus be used in several configurations that allow a combination of detection by measuring the electrical impedance and the application of a force that can cause the cells to better anchor on the microelectrodes or cause their detachment.

[0152] For capture, the fluid is considered to flow in one direction, while for collection, the fluid flows in the opposite direction to capture.

[0153] The first configuration is capture. During capture, the choice of the polarization frequency of the induced dielectrophoretic force allows a better positioning of the cells on the electrodes, thus facilitating their electrical detection. The cells retained by the pores are thus always positioned in the same way, leading to a very high reproducibility of the electrical impedance measurements. However, it is important at this stage not to apply too much force, which would retain all the cells passing through the pores.

[0154] The second configuration is the collection of the cells after capture. After capture, the polarization voltage is increased (the gradient of the modulus of the electric field is higher), the dielectrophoretic force applied is now stronger, and the frequency is adjusted to high values (1 MHz) in order to separate all the captured cells for collection by negative dielectrophoresis. During this electrical separation, a fluid flow is applied (in the opposite direction to capture) to collect the separated cells in a container. The properties of the circulating fluid can be chosen during this step to preserve cell viability while maximizing the dielectrophoretic force of separation.

[0155] The third configuration is the selective separation of the cells. After capture, the polarization voltage is increased (the gradient of the modulus of the electric field is higher), and the frequency is now adjusted in order to selectively separate single cell types by positioning the intermediate frequency between 50 kHz and 150 kHz. During this electrical separation, a fluid flow is applied (in the opposite direction to capture) to collect the separated cells in a specific container. The properties of the circulating fluid can be chosen during this step to preserve cell viability while maximizing the dielectrophoretic force of separation. Different cell types can then be selectively and sequentially separated. Only tumor cells are separated and collected at 100 kHz.

[0156] The fourth configuration is the separation and classification of cells. After capture, the polarization voltage is increased (the gradient of the modulus of the electric field is higher) and the frequency is now progressively increased between 10 kHz and 200 kHz in order to separate the cells at different times according to their dielectric properties. During these electrical separations, a fluid flow is applied (in opposition to the capture) to collect the cells separated sequentially. In this step, the nature of the circulating fluid can be chosen to preserve the cell viability while maximizing the dielectrophoretic forces of separation. The separated cells are collected in a channel that allows only one cell to pass at a time in cross section. The cells are then arranged in a row, their position depending on their dielectrophoretic cutoff frequency and therefore also on their dielectric properties. Thus, in this queue, the cells will be ordered according to their membrane capacitance and therefore the healthy cells will be at one end of the queue while the tumor cells will be at the other end (at the head of the queue) with all possible gradations between these two ends.

[0157] The fifth configuration is the collection of lysates of interest. After capture, the polarization voltage is significantly increased (the gradient of the modulus of the electric field is even higher) and the dielectrophoretic forces applied are very strong, the frequency is adjusted to low values (10 kHz) and cell lysis occurs, releasing the contents of the captured cells. During this cell lysis, a fluid flow is applied (in opposition to the capture) to collect the cell lysates in a container.

[0158] The first type of compartment

[0159] According to one embodiment, as Figure 8 illustrated, the capture and detection system advantageously comprises a compartment 30 in which the filtration device 20 is housed. This compartment comprises an inlet module 31 forming a female part and an outlet module 32 forming a male part. Thus, the inlet module and the outlet module can be assembled by screwing them together or by interlocking them.

[0160] The inlet module 31 is visible in Figure 9a , Figure 9b , Figure 9c , Figure 9d and Figure 9e . The outlet module 32 is visible in Figure 10a , Figure 10b , Figure 10c , Figure 10d , Figure 10e and Figure 10f .

[0161] The inlet module 31 comprises a housing 311 forming a female part 310 for receiving a male part 321 of the outlet module 32. If the inlet module 31 and the outlet module 32 are screwed together, the female part is tapped and the male part is threaded.

[0162] To supply fluid into the compartment, the inlet module 31 comprises a hollow rod 312 which extends from a cone 313. The hollow rod 312 and the cone 313 are connected to a concave part 310 and allow the supply of fluid from an inlet port 314 of the inlet module 31.

[0163] The inlet port 314 is shaped to be connected to a channel for supplying the fluid to be analysed. The concave part 310 has the shape of a hollow cylinder which is connected to the cone 313. The conical shape allows a large amount of flow at the inlet module.

[0164] The convex part 321 of the outlet module 32 comprises a slot 322 configured to receive the filtering device 20, the slot 322 being shaped to match the shape of the filtering device 20 comprising tabs 40 (see Figure 2 ) to allow their insertion and retention in the slot 322. The filtering device 20 is inserted in the slot 322 by inserting the tabs in complementary housings 323 provided at the slot 322, then by applying a rotation to the filtering device 20, the filtering device 20 is brought in a position of retention. The slot 322 comprises grooves 324 for the insertion of the tabs to lock the filtering device in place after rotation. These grooves are machined on either side of the housing 323 receiving the tabs 40 of the filtering device 20. Electrical contacts can be provided in the grooves. Figure 11a and Figure 11b The filtering device 20 is shown arranged in the slot 322.

[0165] To allow the fluid to exit the filtering device 20, the outlet module 32 comprises a hollow rod 325 connected to a hollow cylinder 320. The hollow cylinder 320 is located between the convex part 321 and the hollow rod 325. The hollow rod 325 has an outlet port 326 which is adapted to be connected to a channel for expelling the fluid after analysis.

[0166] When the inlet module and the outlet module are assembled, the filtering device is inside the compartment and not visible from the outside.

[0167] The compartment has the advantage that it can be easily disassembled to replace the filtering device if necessary.

[0168] When the filtering device is arranged in the compartment, a one-piece unit is obtained.

[0169] The compartment is preferably made of a biocompatible material, especially when it is used in vivo.

[0170] According to one embodiment of the application, as Figure 12a , Figure 12b and Figure 12cAs shown in figure 300, the filtering devices can be arranged in series. Thus, there are a number of filtering membranes in series, each filtering membrane having specific features that capture one cell type. Alternatively, the membranes can be defined to capture more than one cell type. In this case, the membranes comprise pores of different sizes and shapes.

[0171] The connection in series comprises assembling several modules together.

[0172] In Figure 12a From left to right, there are an input module 31 as described above, one or more intermediate modules 33 and an output module 32 as described above.

[0173] The intermediate module can be in Figure 13a , Figure 13b , Figure 13c , Figure 13d , Figure 13e and Figure 13f

[0174] As can be seen from these figures, the intermediate module comprises a male part 331 and a female part 330. The intermediate module 33 can be screwed into or plugged into the female part of the input module 31 and the male part of the output module 32 respectively / be screwed together or plugged with them. In the case where the intermediate module 33 is screwed in, it comprises a threaded male part and a female part that is constrained.

[0175] The male part 331 comprises a slot 332 for receiving the filtering device 20. The slot 332 has the same shape and features as the aforementioned output module 32.

[0176] In summary, the intermediate module 33 is intrinsically different from the output module in that it does not comprise a rod for the outlet of the fluid.

[0177] By installing one or more intermediate modules, it is thus possible to have several filtering devices in series to capture and detect several types of cells.

[0178] In addition, modules can be provided to allow the fluid to be modified as needed. For example, a module can concentrate the fluid before it is filtered by the filtering device.

[0179] The result is thus a modular system that can be adapted to different cell types.

[0180] Second type of compartment

[0181] According to Figure 14 , Figure 15 , Figure 16 and Figure 17 ​The embodiment shown, the capture and detection system advantageously comprises an input holder 41 and an output holder 42, and possibly one or more intermediate holders 43 arranged between the input holder 41 and the output holder 42, in order to cascade several filtering devices 20 as described above.

[0182] The filtering devices 20 are housed in the output holder 42, and if necessary in the intermediate holders 43.

[0183] The holders 41, 42, 43 can be such that they can be assembled together to form a stack of holders (see Figure 15 ).

[0184] The input holder 41 is a parallelepiped and has a top face 411 comprising a hollow stem 412 extending from a port 413. This allows the fluid to be fed for analysis. This input holder 41 is connected to the output holder 42 or to an intermediate holder 43. The input holder 41 comprises flexible tabs 416, 417 on its lateral faces 414, 415, which allow the input holder 41 to be clipped into the complementary housings 421, 422, 431, 432 of the output holder 42 or of the intermediate holder 43 to which it is connected.

[0185] The input holder 41 is clipped in the intermediate holder 43 or in the output holder 42, so that the bottom surface 418 of the input holder rests on the top surface 423, 433 of the intermediate holder or of the output holder.

[0186] The output holder 42 is a parallelepiped and comprises a recess on its upper face 423, forming a housing for a leaf 50 of the type for example of a microscope (see Figure 16 ).

[0187] The housing 424 comprises two lugs 425 opposite each other, which enable the leaf 50 to be held in place. In this respect, the leaf 50, which is plate-shaped, has a slot 52 complementary to the lugs 425. The leaf 50 is inserted into the housing 424 by resting on a receiving space 426 formed at the bottom of the housing 424. The leaf 50 is then lowered so as to rest in the housing 424. The output holder 42 has a port on its lower face 427, for example in its centre, to let the fluid to be analysed pass through. This port is extended by a stem similar to that used for the input holder 41. This stem can be connected to a channel allowing the fluid to be discharged after passing through the filtering device 20.

[0188] The intermediate holder 43 is a parallelepiped and comprises a recess on its upper face, forming a housing for a leaf 50 of the type for example of a microscope (see Figure 16The top face of the intermediate support 43 is similar to that of the output support 42. Since the intermediate support 43 is intended to be arranged between two supports, its lower face comprises flexible tabs 436, 437 (not shown) on its lateral faces 435, which allow it to be clipped into the complementary housing 421, 432 of the support to which it should be connected. The intermediate support 43 comprises a central port 438 to allow the passage of fluid.

[0189] When several supports are stacked, the fluid to be analysed circulates in several supports and thus passes through several filtration devices 20 supported by a blade 50 dedicated to each support. The blade 50 has a slot 52 with a shape that is adapted to that of the filtration device 20, which comprises tabs 40 that allow them to be inserted and held in the slot (see Figure 2 The filtration device 20 is inserted into the slot 52 by inserting the tabs into a complementary housing 53 provided at the slot 52, then by applying a rotation to the filtration device 20, which is set to remain stationary. The slot 52 comprises grooves 54 into which the tabs are inserted to lock the filtration device 20 in place after rotation. These grooves are machined on either side of the slot 52 that receives the tabs 40 of the filtration device 20. Electrical contacts can be provided in the grooves.

[0190] The advantage of the second type of compartment is that it allows the blade 50 to be easily removed from the support.

[0191] In addition, as already mentioned, the use of at least one intermediate support in addition to the output support makes it possible to cascade several filtration membranes, each with specific features for capturing one cell type. Alternatively, the membranes can be defined to capture several cell types. In this case, the membranes comprise pores of different sizes and shapes.

[0192] The supports described herein are parallelepipeds, but other shapes can also be adopted: in particular, cylindrical.

[0193] Third type of compartment

[0194] According to the embodiment shown in Figure 18 , Figure 19 and Figure 20 , it is possible to provide several supports 60 stacked one on top of the other, which are provided with blades 70 similar to those described above. These supports 60 take the form of blocks comprising slots 61 for the insertion of the blades 70, on which the filtration devices 20 are inserted. The blades 70 are inserted into grooves 62. As described above, the ports 63 in the centre of the blocks allow the passage of fluid.

[0195] Unlike the holder already described, instead of clamping the holder together, in order to increase the mechanical strength of the assembly formed by all the holders, the holders are held by rods 80 forming holder blocks. Preferably, four rods 80 are provided and pass through the holder, the holder comprising ports 64 located at the corners of the holder. In order to hold the holders together, foils 90 formed of metal strips hold the holders in pairs. The foils act as springs and allow the holders to be easily released once they are no longer under stress. The foils are attached to two stacked holders by arms 65 that protrude from the sides of each holder.

[0196] In fact, in order to hold the holders together, despite the foils, holding blocks 1001, 1002 are provided that surround the holder posts, the upper holding block 1001 and the lower holding block 1002.

[0197] In the case of the second and third type compartments, each blade can optionally support several filtration devices on the same plane, so as to capture several cell types by the same blade or a large number of cells on the same blade.

[0198] Method

[0199] In one aspect, the application relates to a method for capturing and detecting cells circulating in a fluid, see Figure 21 .

[0200] The method of capture can comprise a step of applying an electrical signal to the electrodes, the electrical signal having a frequency such that the electric field generated by the electrodes is able to capture or release the captured cells from the wells due to the dielectrophoretic forces generated between the electrodes.

[0201] In order to capture and detect, the fluid flows through the system described above (step E1).

[0202] The method of capture can comprise a step of applying an electrical signal to the electrodes, the electrical signal having a frequency such that the electric field generated by the electrodes is able to capture the captured cells in the wells by the dielectrophoretic forces generated between the electrodes (step E2).

[0203] Advantageously, during capture, the polarization frequency induces a positive dielectrophoretic force, thereby concentrating the cells between the electrodes and retaining the cells in the wells.

[0204] In order to isolate the cells, the fluid flows through the system described above in a direction opposite to that used for capture (step E3).

[0205] It is advantageous to be able to isolate the cells after capture. Thus, the method comprises a step of applying an electrical signal to the electrodes, the electrical signal having a frequency such that the electric field generated by the electrodes allows the release of the captured cells in the wells due to the dielectrophoretic forces generated between the electrodes (step E4).

[0206] To this end, a polarization frequency induces a negative dielectrophoretic force to separate all the captured cells, typically at 1 MHz.

[0207] After capture, in order to separate only the tumor cells, a frequency induces a dielectrophoretic force to selectively separate one cell type, between 50 kHz and 150 kHz, preferably at 100 kHz to separate the tumor cells.

[0208] Furthermore, after capture, the polarization frequency is progressively increased between 10 kHz and 200 kHz in order to separate the cells at different times according to the dielectric properties of the cells.

Claims

1. A system for detecting the presence of at least one species in a biological fluid, said species being at least one circulating cell or cell aggregate present in a human or animal biological fluid, said system comprising a filtering device (20) for said fluid, said filtering device (20) comprising a planar support (24) comprising a recessed area in which a filtering membrane (21) is located, said filtering membrane comprising a plurality of holes (22) suitable for retaining a given type of species present in said fluid, said planar support (24) comprising at least one opening (23) at the periphery of said holes, said opening (23) being suitable for ensuring the continuity of the circulation of said biological fluid through said system when operating within said fluid, even when said holes (22) are occupied.

2. The system according to claim 1, comprising a plurality of angular sector openings around said filtering membrane.

3. The system according to claim 1, comprising a central opening formed within the center of said planar support (24).

4. The system according to claim 1, further comprising a plurality of electrodes arranged around said at least one hole (22), said electrodes forming one or more circuits polarized by an AC electrical signal, allowing to measure the variation of the complex impedance between these electrodes once one or more cells are contained in or near the hole (22), the measurement of the variation of the complex impedance modulus and its phase allowing to discriminate the type of cell.

5. The system according to claim 4, further comprising a coil (151) connected to said electrodes so as to form an electromagnetic resonant circuit, said electrodes and coil forming the circuit for detection.

6. The system according to claim 1, wherein said filtering membrane (21) is made of a material selected from the group comprising: glass or metal or polymer, magnetic material, or a combination of glass and silicon or nickel and silicon, silicon nitride, silicon oxide, silicon or a biocompatible non-toxic material.

7. The system according to claim 1, wherein - said holes (22) have a cross-sectional dimension between 0.1 pm and 100 pm; and / or - said holes (22) are separated by a spacing between 100 nm and 100 pm; - the number of said holes (22) is between 100 and 100000000.

8. The system according to claim 1, wherein said holes (22) are circular or elliptical or polygonal or slit-shaped.

9. The system according to claim 1, wherein said holes (22) of said membrane are arranged in groups of several holes, each group having a pattern and said groups being connectable to each other via a row of holes.

10. The system according to claim 9, wherein said pattern formed by a group has the following shape: hexagonal, circular.

11. The system according to claim 9, wherein said membrane (21) comprises several groups of holes arranged in a square or star-shaped structure.

12. The system according to claim 1, wherein said holes (22) of said membrane are arranged randomly.

13. The system according to claim 1, comprising a compartment (30) in which the filtering device (20) is housed, the compartment (30) comprising an inlet module (31) and an outlet module (32) joined together to allow the passage of fluid from the inlet module to the outlet module through the filtering device (20).

14. The system according to claim 1, comprising an inlet support (41) and an outlet support (42), a blade (50) supporting the filtering device (20), the inlet support (41) and the outlet support (42) being joined together so that the blade (50) is located between the inlet support (41) and the outlet support (42) to allow the passage of fluid from the inlet support (41) to the outlet support (42) through the filtering device (20).

15. A capture assembly comprising a plurality of systems according to claim 1 arranged in series, each system comprising a filtering device adapted to retain one type of species.

16. The capture assembly according to claim 15, comprising an inlet module, an outlet module and at least one intermediate module arranged between the inlet module and the outlet module, the intermediate module and the outlet module supporting filtering devices, the modules comprising means for fixing them together.

17. The assembly according to claim 15, comprising an input support, an output support and at least one intermediate support arranged between the input support and the output support, the intermediate support and the output support supporting filtering devices, the supports comprising means for fixing them together to form an integral assembly.

18. A method for capturing cells circulating in a fluid, comprising the step of circulating the fluid in a system according to claim 4, the method comprising the step of applying an electrical signal to the electrodes, the electrical signal having a frequency such that the electric field generated by the electrodes is able to capture or release the captured cells from the pores by means of dielectrophoretic forces generated between the electrodes.

19. The method for capturing cells circulating in a fluid according to claim 18, wherein during capture, the polarization frequency induces a positive dielectrophoretic force, thereby concentrating the cells between the electrodes and retaining them in the pores.

20. The method for capturing cells circulating in a fluid according to claim 18, wherein the polarization frequency induces a negative dielectrophoretic force to separate all the captured cells.

21. The method for capturing cells circulating in a fluid according to claim 18, wherein the frequency induces a dielectrophoretic force to selectively separate cell types, the frequency being between 50 kHz and 150 kHz.

22. The method for capturing cells circulating in a fluid according to claim 18, wherein the polarization frequency is progressively increased between 10 kHz and 200 kHz in order to separate the captured cells at different times according to their dielectric properties.

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