System for capturing and detecting species present in biological fluids - Patents.com

Abstract

The present invention relates to a system for detecting 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, in particular for detecting circulating tumor cells (CTCs) present in a blood fluid, the detection system comprising a means for filtering the fluid (20), the means for filtering (20) comprising a filtration membrane (21), the filtration membrane comprising at least one pore (22) configured to retain a given type of species present in the fluid, the filtration means (20) further comprising at least one opening (23) designed to ensure continuity of circulation of the biological fluid even when the at least one pore (22) is occupied during operation in the fluid.

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, as well as any other fluid requiring analysis). The present invention specifically relates to the capture of cells of interest present in biological fluids such as blood in order to analyze said cells of interest present in said biological fluids for medical monitoring. The present invention is particularly applicable to the detection of circulating tumor cells and / or the almost complete extraction of tumor cells contained in blood for the medical monitoring of patients suffering from cancerous pathologies, for the diagnosis of said cancerous pathologies as well as for oncological treatment, the present invention makes it possible to filter / extract the tumor cells. The present invention also applies to the monitoring of the efficacy of treatment and prognosis using detectable cells. [Background technology]

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

[0003] Specifically, circulating tumor cells (CTCs) in the circulating blood are studied to aid in the diagnosis of cancer and thus enable better treatment of patients.

[0004] In fact, it has been shown that cancerous tumors release CTCs into the bloodstream, a phenomenon that appears at early stages of the disease. Biological and molecular analysis of one or more cells allows for an accurate diagnosis and provides information on the aggressiveness of the cancer and the efficacy of treatment. CTCs are therefore interesting biomarkers in all stages of cancer management, diagnosis, prognosis, and monitoring.

[0005] However, CTCs are present in the blood of cancer patients at extremely low concentrations (approximately 1 / 10 9 Thus, isolation and / or near-complete extraction of these rare cells is extremely challenging.

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

[0007] Two drawbacks are associated with this system: - A very limited blood sample is used (7.5 ml, corresponding to 0.15% of the total blood volume) and the number of CTCs in the blood sample is very low, if at low concentration. - it does not allow the detection of CTCs that have lost the EpCAM protein during the epithelial-mesenchymal transition (EMT), which represents about 2 / 3 of the total population of those cells, whereas it only allows the detection of differentiated cells that have a limited life cycle and are the least dangerous.

[0008] Other approaches to isolate CTCs in vitro from blood samples are known based on the size of CTCs. Specifically, the ISET ("Isolation by Size of Epithelial Tumor Cells") system uses filtration of treated blood samples (pre-lysis of red blood cells) on a micro-perforated polycarbonate membrane. In this system, CTCs are pre-rigidified by application of paraformaldehyde to withstand the high pressure applied.

[0009] In general, the sensitivity of in vitro detection is reduced by the small amount of sample. In fact, considering that CTCs are rare in blood, their presence in a few milliliters of sample can reach several units at already advanced cancer stages. Therefore, their detection at even earlier stages is almost impossible.

[0010] More recently, systems designed for in vivo or apheresis use have been developed to capture CTCs in a physiological medium, thereby preserving their viability as much as possible, and potentially accessing larger blood volumes than those analyzed in in vitro systems.

[0011] Although the systems have advantages over in vitro systems, neither system allows for the capture and coincidence counting of CTCs specifically for real-time use. Summary of the Invention [Problem to be solved by the invention]

[0012] In fact, 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 platforms used must be transparent to allow the use of a fluorescent microscope to count the captured cells. Furthermore, these techniques do not guarantee the integrity of the labeled cells, which are modified before their use in biological analyses or culture. [Means for solving the problem]

[0013] The present invention provides a system that overcomes the shortcomings of the prior art.

[0014] To this end, the present invention proposes, according to a first aspect, a system for detecting 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, in particular circulating tumor cells present in a blood fluid, comprising filtering means for the fluid, the filtering means comprising a filtering membrane comprising at least one pore configured to retain a given type of species present in the fluid, the filtering means further comprising at least one opening configured to ensure continuity of circulation of the biological fluid even when at least one pore is occupied during operation within the fluid.

[0015] The invention according to the first aspect is advantageously accomplished by the following features, taken alone or in any technically possible combination thereof:

[0016] - the system further comprises a plurality of electrodes arranged around the at least one pore, the electrodes forming one or more electrical circuits polarized by an AC electrical signal, thereby enabling the measurement of the variation in complex impedance between said electrodes as soon as one or more cells are accommodated in or near the pore, the measurement of the variation in the complex impedance modulus and its phase enabling the differentiation of cell types.

[0017] - The AC electrical signal applied to the electrodes has a frequency such that the electric field created by the electrodes allows the capture or release of cells within the pores due to the dielectrophoretic force generated between the electrodes.

[0018] The polarization frequency induces a positive dielectrophoretic force during trapping to center the cell between the electrodes and retain the cell within the pore.

[0019] - The polarization frequency induces a negative dielectrophoretic force that separates all trapped cells; the frequency is typically 1 MHz.

[0020] - The frequency induces a dielectrophoretic force that selectively separates certain cell types, the frequency being between 50KHz and 150KHz, preferably at 100KHz to separate tumor cells.

[0021] - The polarization frequency is increased in steps between 10kHz and 200kHz in order to separate the trapped cells at different times depending on their dielectric properties.

[0022] The system further comprises an inductor connected to the electrode so as to form an electromagnetic resonator circuit, the electrode and the inductor forming a circuit for detecting the presence of captured cells, which is preferably remotely detectable.

[0023] - the filtration membrane is made of a material selected from the group including glass or metal (nickel, gold) or polymer, ferromagnetic material, magnetic metal (NiFe), or a combination of glass and silicon or nickel and silicon, silicon nitride, silicon oxide, silicon or more generally biocompatible and non-toxic materials.

[0024] The pores have a cross-sectional dimension between 0.1 μm and 100 μm, preferably between 8 μm and 12 μm or between 8 μm and 15 μm, and / or the pores are spaced at intervals between 100 nm and 100 μm. The number of pores is between 100 and 100,000,000.

[0025] the pores are substantially circular or substantially elliptical or substantially polygonal or slit-shaped;

[0026] The pores of the membrane are arranged in several groups of pores, each group having a certain pattern, and the groups can be connected to each other via a row of pores.

[0027] - The pattern formed by a group has a certain shape, i.e. hexagon, circle.

[0028] - The membrane contains pores of several groups of pores arranged in a square or star structure.

[0029] - The pores in the membrane are randomly arranged.

[0030] the filtering means comprises a planar support including a recessed area, the filtering membrane being disposed in the recessed area, the recessed area being formed with openings, the openings being disposed around the periphery of the filtering membrane;

[0031] - the system comprises a compartment in which the filtration means is housed, the compartment comprising an inlet module and an outlet module, the inlet module and the outlet module being joined to one another to allow fluid to flow from the inlet module to the outlet module and pass through the filtration means.

[0032] - the system includes an inlet rack, an outlet rack and a blade supporting the filtration means, the inlet rack and the outlet rack being joined to one another such that the blade is between the inlet rack and the outlet rack to allow fluid to flow from the inlet rack to the outlet rack and through the filtration means.

[0033] The present invention according to a second aspect provides a capture assembly comprising a plurality of systems according to the invention arranged in series, each system comprising filtering means configured to retain one type of species.

[0034] The invention according to the second aspect is advantageously accomplished by the following features, taken alone or in any technically possible combination thereof:

[0035] - 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 outlet module and the intermediate module supporting the filtering means and the modules comprising means for fixing them to each other.

[0036] The inlet module includes a fluid inlet and the outlet module includes a fluid outlet.

[0037] - the assembly comprises an inlet rack, an outlet rack and at least one intermediate rack located between the inlet rack and the outlet rack, the outlet rack and the intermediate rack supporting filtering means, the racks comprising means for fixing them to each other to form an integral assembly.

[0038] The present invention also relates to a method of trapping cells circulating in a fluid comprising the step of circulating the fluid in a system according to the first aspect of the invention, the method comprising the step of applying an electrical signal to the electrodes having a frequency such that an electric field created by the electrodes enables the cells to be trapped or released from the pores due to dielectrophoretic forces generated between the electrodes. method.

[0039] The method according to the invention is advantageously completed by the following components, either alone or in any technically possible combination thereof:

[0040] The polarization frequency induces a positive dielectrophoretic force during trapping to center the cell between the electrodes and retain the cell within the pore.

[0041] - The polarization frequency induces a negative dielectrophoretic force that separates all trapped cells; the frequency is typically 1 MHz.

[0042] - The frequency induces a dielectrophoretic force that selectively separates certain cell types, the frequency being between 50KHz and 150KHz, preferably at 100KHz to separate tumor cells.

[0043] - The polarization frequency is increased in steps between 10kHz and 200kHz in order to separate the trapped cells at different times depending on their dielectric properties.

[0044] The capture of different species is based on the physical properties of the species circulating in the fluid, in particular their size and deformability. In the case of cells, the fluid is biological (blood, urine, lymph, and generally any fluid that circulates in a human or animal and carries the cells of interest to be analyzed). This biological fluid may or may not be diluted in a buffer. This fluid may be a culture medium.

[0045] For cells potentially present in blood, platelets have a size of 2-4 μm, red blood cells have a size of about 7 μm, and white blood cells have a size of 7-15 μm, but are highly deformable. CTCs have a size that varies from 4-25 μm, but are less deformable.

[0046] Thus, as long as the cells have low deformability, they can be captured by the filtration means while allowing other non-tumor components of the biological fluid to pass through under normal blood flow conditions as exist in vivo.

[0047] The presence of the apertures ensures continuity of circulation of biological fluids under in vivo conditions of pressure and velocity, despite filling of the filtration membrane with trapped elements.

[0048] Preferably, by coupling the filtration membrane and its ports to electrodes, the present invention combines physical trapping with detection of cells trapped in the vicinity of the pores, so that counting of trapped cells can be performed in real time.

[0049] In particular, for CTCs, they have specific dielectric properties, so that they affect the impedance of the electric circuit formed by the electrodes. They affect the electric signal in real time, thus allowing real-time detection of the rate of obstruction of the filtering means or of the cells captured by the filtering means. Detection of the obstruction rate makes it possible to determine whether the filtering means can capture a species without becoming saturated. Based on the observation that CTCs have different dielectric properties than other cells that may be captured, it is possible to distinguish them from among the captured cells.

[0050] The real-time electrical detection of CTCs based on their dielectric properties allows to avoid the steps of immunolabeling of tumor cells and the setting up of a system compatible with optical microscopy required for the characterization of the labeled cells. Thus, the present invention allows to directly characterize the presence of CTCs on the filtration membrane, avoiding subsequent manipulation of the device. Thus, medical information is delivered immediately, non-invasively, at lower cost. Real-time monitoring of capture has significant advantages for both in vivo and ex vivo applications by allowing the user to adjust the exposure time of the device to the blood fluid and tumor burden of the patient to be analyzed in order to match the richness or sparsity of the device's information and thus the accuracy of the medical information provided. In addition, the present invention also allows the removal of circulating tumor cells contained in the blood after capture, providing a therapeutic modality.

[0051] 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 the analysis of the population so captured. It should be noted that many analytical methods require minimal testing, and the present invention also makes it possible to ensure that the conditions for performing the analysis are met before incurring the often high costs. In addition, there is no need to collect, transport, or prepare samples. The quality of the information is maintained, the cells are isolated under native physiological conditions, and this information is immediately returned to the specialist at the point of consultation for rapid decision-making. The present invention also allows the generation of useful data for the treatment of patients. Since blood fluid conditions vary from patient to patient and at the patient level throughout the day, the present invention can be combined with local fluid velocity measurements to serve as a calibration or reference for building a comparable database.

[0052] Advantageously, thanks to coupling the electric circuit formed by the electrodes to a resonator circuit, the impedance variations linked to the detection of CTC can be measured contactlessly and remotely in a remote wireless mode.

[0053] It is also possible to combine physical and affinity capture by using antibodies to functionalize the surface of the system.

[0054] Release of the captured cells from the filtration device as a means of collection for analysis or recultivation can be accomplished by electrical stimulation using the same electrodes as those used for detection.

[0055] Further features, objects and advantages of the present invention will become apparent from the following description, which is purely illustrative and non-limiting and which should be read in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0056] [Figure 1] 1 shows a system for trapping species present in a fluid. [Diagram 2] 1 shows a filtration membrane according to one embodiment of the present invention. [Diagram 3] FIG. 3 shows a more detailed view of the filtration membrane of FIG. 2. [Figure 4a] 1 shows diagrams of a filtration membrane in different embodiments. [Figure 4b] 1 shows diagrams of a filtration membrane in different embodiments. [Diagram 5] 1 shows diagrams of a filtration membrane in different embodiments. [Figure 6] 1 shows diagrams of a filtration membrane in different embodiments. [Figure 7] 1 shows two embodiments of the arrangement of electrodes around the ports of a filtration membrane according to the present invention. [Figure 8] 1 shows the compartments of the system of the present invention. [Figure 9a] 3A-3D show different views of the input module of the compartment of the system of the invention; [Figure 9b] 3A-3D show different views of the input module of the compartment of the system of the invention; [Figure 9c] 3A-3D show different views of the input module of the compartment of the system of the invention; [Figure 9d] 3A-3D show different views of the input module of the compartment of the system of the invention; [Figure 9e] 3A-3D show different views of the input module of the compartment of the system of the invention; [Figure 10a] 3A-3D show different views of the output module of the compartment of the system of the invention; [Figure 10b] 3A-3D show different views of the output module of the compartment of the system of the invention; [Figure 10c] 3A-3D show different views of the output module of the compartment of the system of the invention; [Figure 10d] 3A-3D show different views of the output module of the compartment of the system of the invention; [Figure 10e]3A-3D show different views of the output module of the compartment of the system of the invention; [Figure 10f] 3A-3D show different views of the output module of the compartment of the system of the invention; [Figure 11a] 4 shows the installation of a capture system in a compartment module of a system according to the invention. [Figure 11b] 4 shows the installation of a capture system in a compartment module of a system according to the invention. [Figure 12a] 9 shows the compartment of FIG. 8 with an additional intermediate module. [Figure 12b] 9 shows the compartment of FIG. 8 with an additional intermediate module. [Figure 12c] 9 shows the compartment of FIG. 8 with an additional intermediate module. [Figure 13a] 3A-3D show different views of the intermediate module of the compartment of the system of the invention; [Figure 13b] 3A-3D show different views of the intermediate module of the compartment of the system of the invention; [Figure 13c] 3A-3D show different views of the intermediate module of the compartment of the system of the invention; [Figure 13d] 3A-3D show different views of the intermediate module of the compartment of the system of the invention; [Figure 13e] 3A-3D show different views of the intermediate module of the compartment of the system of the invention; [Figure 13f] 3A-3D show different views of the intermediate module of the compartment of the system of the invention; [Figure 14] 1 illustrates a rack and a rack combination according to an embodiment of the present invention. [Figure 15] 1 illustrates a rack and a rack combination according to an embodiment of the present invention. [Figure 16] 1 illustrates a rack and a rack combination according to an embodiment of the present invention. [Figure 17]1 illustrates a rack and a rack combination according to an embodiment of the present invention. [Figure 18] 1 shows racks assembled together according to one embodiment of the present invention. [Figure 19] 1 shows racks assembled together according to one embodiment of the present invention. [Figure 20] 1 shows racks assembled together according to one embodiment of the present invention. [Figure 21] 1 shows diagrammatically the steps of a capture and detection method according to the present invention; DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Throughout the drawings, similar elements have the same references.

[0058] Although embodiments of the invention are described below in the context of capture and detection of cells present in biological fluids, the invention applies to the capture and detection of all types of species or beads present in biological or non-biological fluids (blood and its derivatives such as plasma and serum, urine, water, air, pollutants, etc.).

[0059] It is specified that the invention applies to the capture of species, and by species it is meant tumor cells, tumor cell aggregates, blood clots (blood fluid), exosomes.

[0060] 1, a system 1 for capturing circulating cells present in a fluid includes a fluid inlet 11 and a fluid outlet 12. It should be understood that fluid circulates and passes through the systems described herein.

[0061] Such a system 1 may be connected to the bloodstream of a human being via his arm 100, but it may of course also be connected to other parts of the human or animal body. Preferably, the system is connected to a peripheral vein (for example in the elbow crease) or to a central venous line of the human or animal. Alternatively, such a system 1 may be connected, on the one hand, to a test tube 101 containing a fluid and, on the other hand, to a system 102 that collects the fluid after it has passed through the capture system 1.

[0062] For ex vivo or in vivo use, a peristaltic or pressure controlled pump 13 is connected to the fluid inlet and a flow sensor 14 allows the pump 13 to be regulated. The pump 13 is used to deliver fluid to a filtering means 20, which is described in detail below. The incoming fluid thus passes through the filtering means 20 and is reinjected into the patient or animal's body or into a fluid collection tube. The fluid inlet and outlet are connected to the circulating fluid by a catheter or by any other means known to those skilled in the art and adapted to the location where the system is to be used.

[0063] Alternatively, the system can be used in vivo and does not require a pump, in which case it is placed directly into the circulating fluid.

[0064] The filtering means 20 is advantageously coupled to a means 26 for detecting the trapped cells.

[0065] A measurement unit 15 associated with the detection means 26 measures information about the detected cells.

[0066] The system 1 also includes a wired or wireless communication interface 16 for communicating with a wired or wireless terminal 2. Such a terminal 2 includes an interface that allows a user to access various information regarding the detection.

[0067] (filtration means) Figures 2 and 3 show a possible embodiment of a filtering means 20. This monolithic filtering means comprises a planar support 24, for example circular, comprising an area 25 in which a filtering membrane 21 is arranged.

[0068] The membranes are, for example, circular and are arranged in areas 25 of the same shape.

[0069] At least one pore 22 is formed in the membrane for trapping / retaining cells present in the biological fluid in which the filtration means 20 is placed. The fluid flows through the system.

[0070] The membrane is made of biocompatible and non-toxic materials selected from the group including glass or metal (nickel, gold) or polymers, ferromagnetic materials, magnetic materials (NiFe), multi-materials (glass / silicon), silicon nitride, silicon oxide, silicon.

[0071] Obviously, in applications where the fluid is not biological, the fact that the material is biocompatible is irrelevant.

[0072] The pores advantageously have a cross-sectional dimension between 0.1 μm and 100 μm, preferably between 8 μm and 12 μm or between 8 μm and 15 μm, and typically number between 1,000 and 6,000, preferably between 100 and 100,000,000. The size and number of pores depends on the type of circulating cells to be trapped and the method in which the filtration means is used (in vivo, ex vivo, in vitro).

[0073] Complementarily, the pores are spaced between 0.1 μm and 100 μm, preferably between 8 μm and 12 μm or 8 μm and 15 μm.

[0074] The pores have various shapes: they may be substantially circular, or substantially elliptical, or substantially polygonal or slit-shaped.

[0075] Advantageously, the pores of the membrane are arranged in a plurality of groups of pores, each group having a pattern, the groups being connectable with one another via rows of pores.

[0076] For example, in FIG. 2, the pores are arranged in four groups 210, each group having a polygonal shape.

[0077] The groups can be arranged on the membrane in several patterns, i.e. in a square (Fig. 2) or in a star (Fig. 4a). For the star structure, the pores are arranged in hexagonal groups 211, which are connected to each other by groups of slit-shaped pores 212. Thus, the star structure is obtained by combining two groups of pores. The star structure extends from a central group 211, which is arranged in 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, and slots 212 connect the polygonal groups. Of course, the number of branches can be different as well as the number of groups per branch.

[0078] Alternatively, as shown with respect to Figure 4b, the pores arranged in groups 211' are not connected to each other and are in separate locations 212' from each other. This maximises the number of pores in the support 24.

[0079] Openings 23 are formed in the support surrounding the pores to allow continuity in fluid flow, regardless of the degree of occultation of the filtering region by trapped elements, to maintain undisturbed blood flow.

[0080] Referring again to Figures 2 and 3, the openings are shaped as angular sectors of the membrane periphery. Such shapes are not limiting and other shapes are conceivable. Also, fluid disrupting or fluid concentrating elements can be arranged.

[0081] The number and shape of the openings are optimized so as to compromise as little as possible the mechanical strength of the filtering means 20. In the example shown in Figures 2 and 3 there are four openings 23, whereas in the example shown in Figure 5 there are six openings 23.

[0082] Alternatively, with reference to Figure 6, the membrane may include a central aperture 27 in addition to the peripheral angular sector apertures 23. In this example, the pores are polygonal groups connected in pairs by slit-shaped groups. A set of two hexagonal groups are distributed in a star shape around the central aperture 24.

[0083] For the configuration of FIG. 4, the following parameters can be used: - Circular pore diameter 8μm, 9μm, 10μm, 11μm, 12μm - 5μm pore distance, - Number of pores: 4,246, 3,395, 2,717, 2,287, 1,880, - Average number of pores per hexagonal group: 271, 220, 169, 135, 121, - Average number of pores per slit: 54, 45, 40, 27 - Surface area occupied by pores: 0.213mm 2 , 0.216mm 2 , 0.217mm 2 , - Total surface area of ​​one opening: 0.111mm 2 , - Total area of ​​the four openings: 0.444mm 2 .

[0084] The following parameters can be used for the configuration of FIG. - Circular pore diameters of 8μm, 9μm, 10μm, 11μm, 12μm, - 5μm port spacing, - 3395, 4246, 2717, 2287, 1880 pore count, - average number of pores per hexagonal group of 220, 271, 121, 169, 135, - Average number of pores per slit: 45, 54, 40, 27, 54, - The pore size is 0.216mm 2 , 0.213mm 2 , 0.217mm 2 Surface area of - 0.111mm 2 The surface area of ​​one opening of - 6 apertures occupy 0.666mm 2 of total surface area.

[0085] The following parameters can be used for the configuration of FIG. - 8μm, 9μm, 10μm, 11μm, 12μm pore diameter, - pore distance of 5 μm, - 1227, 486, 386, 324, 271 pore count, - average number of pores per hexagonal group of 220, 169, 135, 121, - Average number of pores per slit: 45, 40, 27, - The pore size is 0.213mm 2 , 0.216mm 2 , 0.217mm 2 Surface area of - Central opening occupies 0.057mm 2 Surface area of - surface area of ​​one peripheral opening of 0.111 mm2, - 6 apertures and central aperture occupy 0.723mm 2 of total surface area.

[0086] (Capture and Detection and Measurement Units) 3 and 7, in order to detect cells trapped by the pore using the filtering means 20, the trapping and detection system advantageously includes detection means 26 constituted by electrodes arranged around the pore 22. The electrodes 26 are connected together to form one or more electric circuits characterized by a complex impedance that depends on the frequency of the electrical excitation. The value of this frequency-dependent impedance is influenced by the presence of cells in the vicinity of the electrodes that bypass the pore 22.

[0087] Specifically, an AC voltage is applied to the electrodes at a given frequency such that a complex impedance is measured between the electrodes. The real part of the measured impedance is characteristic of the electrical resistance of the medium occupying that space. As for the imaginary part, it reflects the dielectric properties of the medium, in particular, its dielectric constant. During the electrical measurements we measured the phase shift of the current with respect to the applied voltage and the modulus of the complex impedance. These two measurements probe both the real and imaginary parts of the electrical impedance.

[0088] When a cell is positioned between the electrodes, the measured electrical impedance (by its modulus and "phase") is modified. The real part of the impedance is modified as well as the imaginary part. Thus, we measure these two changes, which are reproduced by a variation in the impedance modulus as well as by a variation in the current / voltage phase shift.

[0089] The measurement of complex impedance makes it possible to distinguish between cell types, in particular neoplastic / non-neoplastic. It is in fact the changes in the imaginary part that contain the most valuable information to perform this type of detection.

[0090] In particular, CTCs have specific dielectric properties, so that they change the value of this impedance in a way that makes them distinguishable from other potentially captured cell types. Depending on the detected variations, it is possible to detect the captured cells and their type. The electrodes are, for example, gold, copper, platinum, nickel, piezoelectric materials, conductive polymers. The impedance is measured in the range of 10 Hz to 1 MHz, or even in the range of 10 Hz to 5 MHz.

[0091] These electrodes thus surround the pores of the filtration membrane in order to electrically probe the dielectric properties of the medium in the vicinity of the pores in which the trapped cells are trapped.

[0092] The imaginary part of the electrical impedance, in the above mentioned signal frequency range, is dominated by the capacitance formed by the plasma membrane of the cells. This lipid bilayer, rich in membrane proteins, is a good electrical insulator that separates two conductive media, i.e. the intracellular medium (cytoplasm) and the extracellular medium (the fluid to be analyzed that contains the cells), and therefore forms a kind of electrical capacitor described by a capacitance, i.e. by a virtual impedance of capacitive origin. This membrane capacitance is not present in the electric circuit when there is no cell between the electrodes, but when a cell is positioned between the electrodes (in the region where there is an electric field emerging from the electrodes), this membrane capacitance appears in the circuit. This change leads to a variation of the imaginary part of the measured impedance. Of course, at the same time, the real part of the impedance is also modified. Thus, both parts of the impedance modified by the presence of the cell (real and imaginary) are measured during the capture. Tumor cells circulating in the blood have a very specific cell membrane morphology, resulting in the presence of numerous protuberances that are not present in healthy cells. These protuberances significantly increase the surface area of ​​the cell membrane of tumor cells when compared to the plasma surface of healthy cells. Thus, the membrane capacitance of the cells behaves like a flat capacitor whose capacitance is proportional to the surface area of ​​the opposing conductor. This explains why the membrane capacitance of tumor cells is much larger than that of healthy cells. Thus, the detection performed makes it possible not only to detect the cells held by the capture device, due to the magnitude of the variation in the imaginary part of the electrical impedance, but also to predict the healthy or tumor nature of each captured cell.

[0093] As shown in Figure 7, the electrodes can surround the pores in various ways, i.e., by wrapping around the pores or by forming a linear track around the pores. The choice of electrode shape depends on the pore density. Alternatively, the electrodes can be located on the inner walls of the pores.

[0094] For direct (active) measurements, connection tracks 29 are needed that connect the electrodes to contact pads 28. The contact pads 28 are connected to a measurement unit 15 that allows direct measurement of the value of the impedance variations induced by the presence of a cell between the electrodes.

[0095] The measurement unit may include a coil 151 connected to the electrodes to form an electromagnetic resonator for passive and wireless measurements (interrogation of the device and receiving remote signals). The wireless terminal 2 can then measure the variations in the impedance of the detection circuit without contact. The coil 151 can also be included in a membrane, the value of whose inductance can then be measured remotely.

[0096] Alternatively, a wire connection between the electrodes and the measurement unit 15 is possible, allowing the impedance value of the detection circuit to be read directly.

[0097] Terminal 2 can also be wired to a measurement unit 15 .

[0098] Regardless of how the impedance variation is measured, the measurement is direct and in real time. It is therefore possible to determine the presence of cells on the surface of the membrane. This impedance variation induced by the presence of cells in the vicinity of the capture pores depends on the nature of the captured cells, and therefore the measurement makes it possible to distinguish which of the captured cells are tumorous. This measurement is non-invasive to the cells to be detected and does not affect their viability in any way.

[0099] (Capture and Separation) The detection system is advantageously based on the use of dielectrophoretic forces, which make it possible to retain cells in association with the pores, but also to selectively separate the cells.

[0100] In fact, a dielectric object, such as a cell, bathed in a medium in which there is a non-uniform electric field (we will next talk about the gradient of the electric field coefficient), experiences forces that can cause it to move because of its polarizability.

[0101] This force is the basis of the principle of dielectrophoresis. In the presence of a non-uniform AC electric field, the direction of this force relative to the gradient of the square of the electric field coefficient depends on the frequency of the AC electric field and the dielectric properties of the object. For an object with a given size and dielectric constant, depending on the electric field frequency, the dielectrophoretic force can be positive (the dielectric object moves towards the region with the highest electric field coefficient) or negative (the dielectric object moves towards the region with the lowest electric field coefficient).

[0102] As mentioned above, an AC voltage is applied to the electrodes for real-time detection during cell capture, and therefore the configuration of the electrodes in a planar form causes the generation of a non-uniform AC electric field above the electrodes facing the fluid to be analyzed.

[0103] Regions of high electric field are close to the pores of the capture device, and regions of low electric field exist further in the fluid above the microelectrodes. Thus, these electrokinetic phenomena related to dielectrophoretic forces indicate that cells arriving in the vicinity of the pores experience forces that can either direct them towards the capture pores (positive dielectrophoresis) or repel them (negative dielectrophoresis).

[0104] The frequency at which the dielectric force changes sign is called the cut-off frequency. A dielectric object has its own cut-off frequency depending on its shape, size and dielectric properties (its dielectric constant).

[0105] For circulating blood cells, the cut-off frequency for healthy blood cells is around 150 kHz, whereas the cut-off frequency for tumor cells is significantly lower (50 kHz). This is again a consequence of the different dielectric properties of tumor cells, which are associated with a high membrane capacitance in this frequency range. Thus, by polarizing the electrodes at intermediate frequencies between these two frequencies, it appears that the cells of interest can be preferentially and selectively oriented in a preferred direction in space.

[0106] It is therefore possible to use detection systems in several configurations that allow the combination of detection by measuring electrical impedance with the application of forces that can result either in better anchoring of the cells on the microelectrodes or in their detachment.

[0107] For capture, the fluid is considered to be flowing in one direction, whereas for collection, the fluid is considered to be flowing in a direction opposite to the direction of capture.

[0108] The first configuration is trapping. During trapping, the choice of a polarization frequency that induces a positive dielectrophoretic force allows a better localization of the cells on the electrodes, thus facilitating their electrical detection. Thus, the cells held by the pore are always positioned in the same way, resulting in a very high reproducibility of the electrical impedance measurements. However, at this stage it is important not to apply excessive forces that would retain all the cells passing through the pore.

[0109] The second configuration is the collection of cells after capture. After capture, the polarization voltage is increased (the gradient of the electric field coefficient is higher), the applied dielectrophoretic force is now much stronger, and the frequency is adjusted to a high value (1 MHz) to separate all the captured cells for collection by negative dielectrophoresis. During this electrical separation, a fluid flow (opposite to the fluid flow of the capture) is applied to collect the separated cells in a reservoir. During this step, the properties of the circulating fluid can be selected to maximize the dielectrophoretic force of separation while maintaining the viability of the cells.

[0110] The third configuration is the selective separation of cells. After trapping, the polarization voltage is increased (the gradient of the electric field coefficient is higher) and the frequency is now adjusted to selectively separate a single cell type by positioning it at an intermediate frequency between 50 kHz and 150 kHz. During this electrical separation, a fluid flow (opposite to that of the trapping) is applied to collect the separated cells in a specific reservoir. During this step, the nature of the circulating fluid can be chosen to maximize the dielectrophoretic force of separation while maintaining cell viability. It is then possible to selectively and sequentially separate different cell types. At 100 kHz, only tumor cells are separated and collected.

[0111] The fourth configuration is cell separation and classification. After trapping, the polarization voltage is increased (higher gradient of the electric field coefficient) and the frequency is increased stepwise between 10 kHz and 200 kHz to separate the cells at different times depending on their dielectric properties. During these electrical separations, a fluid flow (opposite to the fluid flow of trapping) is applied to collect the separated cells in a sequential manner. During this step, the nature of the circulating fluid can be chosen to maximize the dielectrophoretic force of separation while maintaining the cell viability rate. The separated cells are collected in a channel whose cross section allows only one cell to pass at a time. The cells are then placed in a row with a position that depends on their dielectrophoretic cutoff frequency and therefore on their dielectric properties. In this queue, the cells are thus ordered according to their membrane capacitance, so that healthy cells are at the end of the queue and tumor cells are at the other end (head of the queue), with all possible gradients between these two ends.

[0112] The fifth configuration is the collection of the lysate of interest. After trapping, the polarization voltage is strongly increased (the gradient of the electric field coefficient is much higher), the applied dielectrophoretic force is very strong, and the frequency is adjusted to a low value (10 kHz), causing cell lysis to occur and releasing the contents of the trapped cells. During this cell lysis, a fluid flow (opposite to the fluid flow of trapping) is provided to collect the cell lysate in a reservoir.

[0113] (First type of compartment) According to an embodiment, as shown in Figure 8, the capture and detection system advantageously comprises a compartment 30 in which the filtering means 20 are housed. This compartment comprises an inlet module 31 forming a female part and an outlet module 32 forming a male part 32. The inlet and outlet modules can thus be assembled by screwing them together or interlocking them.

[0114] The input module 31 can be seen in Figures 9a, 9b, 9c, 9d and 9e, and the output module 32 can be seen in Figures 10a, 10b, 10c, 10d, 10e and 10f.

[0115] The input module 31 includes a housing 311 forming a female portion 310 for receiving a male portion 321 of the output module 32. If the input module 31 and the output module 32 are screwed together, the female portion is female threaded while the male portion is male threaded.

[0116] To supply fluid into the compartment, the inlet module 31 includes a hollow rod 312 extended by a conical section 313. The hollow rod 312 and the conical section 313 are connected to the female section 310, allowing fluid to be supplied from an inlet port 314 of the inlet module 31.

[0117] The inlet port 314 is shaped to be connected to a channel supplying the fluid to be analyzed. The female part 310 has the shape of a hollow cylinder connected to a conical part 313. The conical shape allows for a large flow rate at the inlet module.

[0118] The male part 321 of the outlet module 32 includes a slot 322 configured to receive the filtering means 20, the slot 322 being shaped to match the shape of the filtering means 20, which includes tabs 40 (see FIG. 2) that allow the filtering means 20 to be inserted and held in the slot 322. The filtering means 20 are inserted in the slot 322 by inserting the tabs in complementary housings 323 provided in the slot 322, which are then guided to a position where they remain stationary by applying a rotation to the filtering means 20. The slot 322 includes grooves 324 for inserting the tabs to lock the filtering means in place after rotation. These grooves are machined on both sides of the housing 323 that receive the tabs 40 of the filtering means 20. Electrical contacts may be provided in the grooves. FIGS. 11a and 11b show the filtering means 20 placed in the slot 322.

[0119] To allow the fluid to exit the filtering means 20, the outlet module 32 includes a hollow rod 325 connected to a hollow cylinder 320. The hollow cylinder 320 is between the male portion 321 and the hollow rod 320. The hollow rod 325 has an outlet port 326 configured to be connected to a channel for discharging the fluid after analysis.

[0120] When the inlet and outlet modules are assembled, the filtering means is inside the compartment and is not visible from the outside.

[0121] An advantage of the compartment is that it can be easily disassembled in order to replace the filtering means, if required.

[0122] When the filtering means is placed within the compartment, a one-piece unit is obtained.

[0123] The compartment is preferably made from a biocompatible material, especially when used in vivo.

[0124] According to one embodiment of the present invention, as shown in Figures 12a, 12b and 12c, the filtration means may be arranged in series. Thus, there are multiple filtration membranes in series, each filtration membrane having characteristics specific to the capture of one cell type. Alternatively, the filtration membranes may be defined to capture more than one cell type. In this case, the filtration membranes contain pores of different sizes and shapes.

[0125] A series connection consists of assembling several modules together.

[0126] In Figure 12a, from left to right, there is an input module 31 as described above, one or more intermediate modules 33, and an output module 32, also as described above.

[0127] The intermediate module can be seen in figures 13a, 13b, 13c, 13d, 13e and 13f.

[0128] As can be seen in these figures, the intermediate module 33 includes a male portion 331 and a female portion 330. The intermediate module 33 may be threaded or plugged into the female portion of the input module 31 and the male portion of the output module 32, respectively. If the intermediate module 33 is threaded, it includes a male portion with an external thread and a female portion with an internal thread.

[0129] The male portion 331 includes a slot 332 for receiving the filtering means 20. The slot 332 has the same shape and characteristics as the outlet module 32 described above.

[0130] In summary, the intermediate module 33 essentially differs from the output module in that it does not include a rod for fluid outlet.

[0131] Thus, by installing one or more intermediate modules, it is possible to have several filtering means in series to capture and detect several types of cells.

[0132] Modules may also be provided to allow the fluid to be modified as required, for example a module may condense the fluid before it is filtered by the filtering means.

[0133] Thus, the result is a modular system that can be adapted to different cell types.

[0134] (Second type of compartment) According to one embodiment shown in Figures 14, 15, 16 and 17, the capture and detection system advantageously includes an input rack 41, an output rack 42 and, optionally, one or more intermediate racks 43 arranged between the input rack 41 and the output rack 42 for connecting several filtering means 40 in series as described above.

[0135] The filtering means 20 are housed in an output rack 42 and, if necessary, in an intermediate rack 43 .

[0136] The racks 41, 42, 43 are such that they can fit together to form a stack of racks (see FIG. 15).

[0137] The inlet rack 41 is parallelepipedic and has a top surface 411 that includes hollow rods 412 extending from ports 413. This allows fluid to be delivered for analysis. This inlet rack 41 is connected to an outlet rack 42 or intermediate rack 43. The input rack 41 includes flexible tabs 416, 417 on its sides 414, 415 that allow the input rack 41 to be clipped into complementary housings 421, 422, 431, 432 of the output rack 42 or intermediate rack 43 to which it is connected.

[0138] The input rack 41 is clipped onto the intermediate rack 43 or output rack 41 such that the bottom surface 418 of the input rack rests on the top surfaces 423, 433 of the intermediate or output rack.

[0139] The output rack 42 is a parallelepiped and includes in its upper face 423 a groove 424 forming a housing for a blade 50, for example of the type used for microscopes (see FIG. 16).

[0140] The housing 424 comprises two lugs 425 arranged opposite each other, which allow the blade 50 to be held in place. In this respect, the blade 50, in the form of a plate, has a slot 52 complementary to the lugs 425. The blade 50 is inserted into the housing 424 by resting on a receiving space 426 formed in the bottom of the housing 424. The blade 50 is then lowered so that it is located in the housing 424. The outlet rack 42 has a port located on its lower face 427, located in its center, for example, to pass the fluid to be analyzed. The port is extended by a rod 428 similar to the one used with the inlet rack 41. This rod can be connected to a channel that allows the fluid to be discharged after passing through the filtering means 20.

[0141] The intermediate rack 43 is a parallelepiped and includes on its upper surface 433 a groove 434 forming a housing for a blade 50, for example of the type used for microscopes (see FIG. 16). The top surface of the intermediate rack 43 is similar to the top surface of the output rack 42. As the intermediate rack 43 is intended to be placed between two racks, the lower surface of the intermediate rack includes flexible tabs 436, 437 on its sides 435, which allow the intermediate rack to be clipped into the complementary housings 421, 432 of the rack to which it is to be connected. The intermediate rack 43 includes a central port 438 that allows fluids to pass through.

[0142] When several racks are stacked, the fluid to be analyzed circulates in the several racks and therefore passes through several filtering means 20 supported by blades 50 specific to each rack. The blades 50 have slots 52 with a shape adapted to the shape of the filtering means 20, which include tabs 40 (see FIG. 2) that allow them to be inserted and held in the slots. The filtering means 20 are inserted in the slots 52 by inserting the tabs into complementary housings 53 provided in the slots 52 and then applying a rotation to the filtering means 20, the latter being set to remain stationary. The slots 52 include grooves 54, into which the tabs are inserted so as to lock the filtering means 20 in place after the rotation. These grooves are machined on both sides of the slots 52 that receive the tabs 40 of the filtering means 20. Electrical contacts may be provided in the grooves.

[0143] An advantage of the second type of compartment is that it allows the blade 50 to be easily removed from the rack.

[0144] Furthermore, as already mentioned, the use of at least one intermediate rack in addition to the output rack allows for having multiple filtration membranes in series, each with specific properties for trapping one cell type. Alternatively, the filtration membranes can be defined to trap multiple cell types, in which case the filtration membranes contain pores of different sizes and shapes.

[0145] The rack described here is a parallelepiped, but can take other shapes, in particular a cylindrical shape.

[0146] (Third type of compartment) According to the embodiment shown in figures 18, 19 and 20, a number of racks 60 may be provided, stacked on top of each other, with blades 70 similar to those described above. These racks 60 take the form of a block including slots 61 for inserting the blades 70, onto which the filtering means 20 are inserted. The blades 70 are inserted in grooves 62. A port 63 in the centre of the block allows the fluid to flow, as described above.

[0147] Unlike the described racks, instead of clipping the racks together, they are held by rods 80 forming a block of racks, to improve the mechanical strength of the assembly formed by all the racks. Preferably, four rods 80 are provided, passing through the racks 70, which include ports 64 at the corners of the racks 70. To hold the racks 70 together, foils 90 formed by metal strips hold the racks 70 in pairs. The foils 90 act as springs, allowing the racks to be easily released when they are no longer under stress. The foils are attached to the two stacked racks by arms 65 protruding from the sides of each rack 70.

[0148] In fact, there are retaining blocks 101, 192 surrounding the rack column, an upper retaining block 101 and a lower retaining block 102, to hold the racks together despite the foil.

[0149] For the second and third type compartments, each blade can optionally support multiple filtration means on the same plane to have multiple cell types captured by the same blade or a larger number of cells captured on the same blade.

[0150] (method) In one embodiment, the invention relates to a method for capturing and detecting cells circulating in a fluid with reference to FIG.

[0151] The capture method may include applying an electrical signal to the electrodes having a frequency such that an electric field created by the electrodes enables the captured cells to be captured or released from the pore due to a dielectrophoretic force generated between the electrodes.

[0152] For capture and detection, the fluid is flowed through the system described above (step E1).

[0153] The capture method may include the step of applying an electrical signal to the electrodes having a frequency such that an electric field created by the electrodes enables cells to be trapped in the pore by the dielectrophoretic force generated between the electrodes (step E2).

[0154] Advantageously, the polarization frequency induces a positive dielectrophoretic force during trapping to center the cells between the electrodes and retain the cells within the pores.

[0155] To separate cells, a fluid is flowed through the system in a direction opposite to that used for capture (step E3).

[0156] After capture, it is advantageous to be able to separate the cells.The method therefore comprises a step of applying an electric signal to the electrodes having a frequency such that the electric field created by the electrodes allows the release of the captured cells in the pores due to the dielectrophoretic force generated between the electrodes (step E4).

[0157] For this purpose, a polarization frequency induces a negative dielectrophoretic force that separates all trapped cells, the frequency being typically 1 MHz.

[0158] After capture, to separate only tumor cells, the frequency is adjusted to induce a dielectrophoretic force that selectively separates one cell type, the frequency being between 50 kHz and 150 kHz, preferably at 100 kHz to separate tumor cells.

[0159] Also, after capture, the polarization frequency is increased stepwise between 10 kHz and 200 kHz to separate cells at different times that depend on their dielectric properties.

Claims

1. 1. A system for detecting at least one species present in a fluid, comprising: a filtering means for said fluid, said filtering means comprising a support comprising an area in which a filtering membrane is arranged, said filtering membrane comprising at least one pore adapted to retain a given type of species present in said fluid, said filtering means further comprising at least one opening formed in said support around said pore adapted to ensure continuity of circulation of said fluid even when said at least one pore is occupied during operation within said fluid, system.

2. 2. The system of claim 1, further comprising a plurality of electrodes disposed around the at least one pore, the electrodes forming one or more electrical circuits polarized by an AC electrical signal, thereby enabling measurement of a complex impedance variation between the electrodes upon receipt of one or more cells within or near the pore, the measurement of the variation in the complex impedance modulus and its phase enabling differentiation of cell types.

3. 3. The system of claim 2, wherein the AC electrical signal applied to the electrodes has a frequency such that an electric field created by the electrodes enables capture or release of cells within the pore due to dielectrophoretic forces generated between the electrodes.

4. 4. The system of claim 3, wherein a frequency at which the electrodes are polarized induces a positive dielectrophoretic force during the trapping to center the cell between the electrodes and retain the cell within the pore.

5. The system of claim 3 , wherein the frequency at which the electrodes are polarized induces a negative dielectrophoretic force that separates all captured cells.

6. The system described in claim 5, wherein the frequency is 1 MHz.

7. 4. The system of claim 3, wherein the frequency induces a dielectrophoretic force that selectively separates certain cell types, and the frequency is between 50 KHz and 150 KHz.

8. 4. The system of claim 3, wherein the frequency at which the electrodes are polarized is increased stepwise between 10 kHz and 200 kHz to separate the captured cells at different times depending on their dielectric properties.

9. 10. The system of claim 8, further comprising an inductor connected to the electrode to form an electromagnetic resonator circuit, the electrode and the inductor forming a circuit to detect the presence of a captured cell.

10. 10. The system according to any one of claims 1 to 9, wherein the filtration membrane is made of a material selected from the group comprising glass or metal, a ferromagnetic material, a magnetic metal, or a combination of glass and silicon or nickel and silicon, silicon nitride, silicon oxide, silicon or a biocompatible and non-toxic material.

11. the pores have a cross-sectional dimension between 0.1 μm and 100 μm; and / or the pores are spaced at intervals between 100 nm and 100 μm; The number of pores is between 100 and 100,000,000; A system according to any one of claims 1 to 10.

12. The system according to any one of claims 1 to 11, wherein the pores are circular or elliptical or polygonal or slit-shaped.

13. 13. The system according to claim 1, wherein the pores of the filtration membrane are arranged in several groups of pores, each group having a pattern, and the groups can be connected to each other via a row of pores.

14. The system of claim 13 , wherein the pattern formed by a group comprises a hexagon or a circle.

15. 14. The system of claim 12 or 13, wherein the filtration membrane comprises several groups of pores arranged in a square or star configuration.

16. The system of any one of claims 1 to 14, wherein the pores of the filtration membrane are randomly arranged.

17. 17. The system of any one of claims 1 to 16, wherein the filtering means comprises a planar support including a recessed area in which the filtering membrane is disposed, the recessed area being formed with the apertures, the apertures being disposed around the periphery of the filtering membrane.

18. 18. A system according to any one of claims 1 to 17, comprising a compartment in which the filtering means is housed, the compartment comprising an inlet module and an outlet module, the inlet module and the outlet module being joined to one another to allow fluid to flow from the inlet module to the outlet module and through the filtering means.

19. 19. The system of any one of claims 1 to 18, comprising an inlet rack, an outlet rack, and a blade supporting the filtering means, the inlet rack and the outlet rack being joined to one another such that the blade is between the inlet rack and the outlet rack to allow fluid to flow from the inlet rack to the outlet rack and through the filtering means.

20. A system described in any one of claims 1 to 18, wherein the at least one species present in the fluid includes at least one circulating cell or cell aggregate present in a human or animal biological fluid.

21. A capture assembly comprising a plurality of systems according to any one of claims 1 to 18 arranged in series, each system comprising the filtering means according to claim 1.

22. 22. A capture assembly as described in claim 21, comprising an inlet module, an outlet module, and at least one intermediate module disposed between the inlet module and the outlet module, the outlet module and the intermediate module supporting filtering means, the modules including means for fixing them to each other.

23. 22. The capture assembly of claim 21, comprising an entrance rack, an exit rack, and at least one intermediate rack disposed between said entrance rack and said exit rack, said exit rack and said intermediate rack supporting filtering means, said racks including means for securing them to one another to form an integral assembly.

24. A method of trapping cells circulating in a fluid, comprising circulating the fluid in a system according to any one of claims 2 to 19, applying an electrical signal to the electrodes having a frequency such that an electric field created by the electrodes allows the cells to be captured or released from the pore due to a dielectrophoretic force generated between the electrodes; method.

25. 25. The method of claim 24, wherein the frequency at which the electrodes are polarized induces a positive dielectrophoretic force during the trapping so as to center the cell between the electrodes and retain the cell within the pore.

26. 25. The method of claim 24, wherein the frequency at which the electrodes are polarized induces a negative dielectrophoretic force that separates all trapped cells.

27. The method of claim 26, wherein the frequency is 1 MHz.

28. 25. The method of claim 24, wherein the frequency induces a dielectrophoretic force that selectively separates certain cell types, and the frequency is between 50 KHz and 150 KHz.

29. 25. The method of claim 24, wherein the frequency at which the electrodes are polarized is increased stepwise between 10 kHz and 200 kHz to separate the captured cells at different times depending on their dielectric properties.

Images