A microfluidic device, a microfluidic system and a method for the capture and controllable release of single biological cells

Abstract

The disclosure relates to a microfluidic device (1) a microfluidic system (2) and a method (3) for the capture of single biological cells (18) and controllable release of selected single biological cells (18), the microfluidic device (1) comprises at least one microchamber (10) having a first fluid opening (12) and a second fluid opening (14), at least one capturing device (16) inside the microchamber (10). The capturing device (16) comprises an electrically conducting capturing surface (22) electrically connectable to an external electric potential (V), the capturing surface (22) is functionalized with at least one immobilizer selected to bind at least one biological cell (18) in the microchamber (10). The at least one immobilizer is bonded to the capturing surface (22) with an electrochemically dissociable bond configured to dissociate from the capturing surface (22) on application of an external electric potential (V) greater than a threshold potential (VT), to the capturing surface (22).

Description

[0001] A MICROFLUIDIC DEVICE, A MICROFLUIDIC SYSTEM AND A METHOD FOR THE CAPTURE AND CONTROLLABLE RELEASE OF SINGLE BIOLOGICAL CELLS

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to a microfluidic device for the capture of single biological cells and controllable release of selected single biological cells. The present disclosure also relates to a microfluidic system comprising at least one such microfluidic device, and to a method for capturing single biological cells and releasing selected single biological cells in the microfluidic system.

[0004] BACKGROUND

[0005] Cells are the fundamental building blocks of life. In order to perform their functions, both prokaryotic and eukaryotic cells are extremely heterogeneous in terms of geneexpression. The differences in gene-expression are important for how a cell responds to drugs as well as environmental stimuli e.g. how efficient the immune cells clear infections or if a bacterium can be killed by an antibiotic.

[0006] The advent of single-cell techniques such as microscopy, transcriptomics, epigenetics, proteomics etc. has allowed investigations of cellular heterogeneity at an unprecedented level. However, common for these single-cell methods is that they only provide a snapshot of cellular processes, with no insight into the dynamic functional processes occurring in individual cells. Thus, what gives rise to the observed cellular heterogeneity is still a largely unsolved biological question.

[0007] Recent observations suggest that changes in the metabolism of immune cells accompany most pathologies, including cancer, autoimmunity and inflammation. Similarly, the metabolism of individual microbial cells also varies within a cell population, which could explain why subpopulations of bacteria and fungi continue to replicate when exposed to certain antibiotics and antifungals.

[0008] However, current gold standard methods to profile cellular metabolism are restricted to analysis of at least 10,000 cells. Profiling metabolism of single cells to scrutinize their dynamic functional processes has not been achieved yet. How metabolism contributes to cellular heterogeneity is therefore still uncharacterized.

[0009] Moreover, no technique today allows correlation between the metabolism of single or even groups of cells to transcriptom ic and epigenetic assessment of the same cells, which is crucial to understand how metabolic heterogeneity is developed as well as the gene-expression profiles this metabolic heterogeneity results in.

[0010] SUMMARY

[0011] Therefore, an object of the disclosure is to provide a device for capturing single biological cells for single cell analysis, and controllably releasing selected biological cells for additional assessments. More specifically, an object of the disclosure is to provide a microfluidic device, a system and a method for capturing single biological cells for single cell analysis, and controllably releasing selected single biological cells.

[0012] According to a first aspect of the present disclosure, the object is at least partly achieved by a microfluidic device according to claim 1.

[0013] Hence, there is provided a microfluidic device for capturing and controllably releasing biological cells. The microfluidic device comprises at least one microchamber having a first fluid opening and a second fluid opening for flowing a liquid sample through the microchamber. The microfluidic device further comprises at least one capturing device inside the microchamber, for capturing at least one biological cell inside the microchamber

[0014] The capturing device comprises an electrically conducting capturing surface electrically connectable to an external electric potential. The capturing surface is further functionalized with at least one immobilizer selected to bind at least one biological cell in the microchamber. The at least one immobilizer is bonded to the capturing surface with an electrochemically dissociable bond configured to dissociate from the capturing surface on application of an electric potential greater than a threshold potential to the capturing surface.

[0015] Referring to a reference potential, an absolute value of the applied electric potential is greater than an absolute value of the threshold potential. The reference potential may be a reference potential of a liquid in the microchamber.

[0016] The microfluidic device may be fabricated by conventional semiconductor manufacturing technology in combination with other techniques, such as additive manufacturing and 3D-printing, at low cost. The microfluidic device may thus be provided on a glass or semiconductor substrate or chip. The capturing device comprising the capturing surface, and any electrodes may be manufactured by conventional semiconductor technology and may be integrated in the microfluidic device by deposition and patterning by conventional means known in the art.

[0017] The microfluidic structures such as microchamber chamber walls, fluidic channels and openings may be formed of a single material on the substrate for instance by additive manufacturing on a semiconductor wafer or chip, prefabricated by semiconductor technology to comprise components, such as the capturing device. Any microfluidic structures in the present disclosure, such as walls and top cover of the microchamber and any connecting channels and openings, e.g. inlets and outlets, may be fabricated by conventional techniques using polydimethylsiloxane (PDMS), or glass, on a prepared semiconductor or glass substrate.

[0018] Fluid openings are arranged to introduce or to evacuate a fluid into the microchamber. The fluid may be a cell-containing liquid sample, i.e. containing biological cells, or a fluid comprising culturing media for nourishing biological cells. Fluid comprising culturing media may, or may not, comprise other components for performing tests on the biological cells. Fluid may also be used to evacuate biological cells from the microchamber.

[0019] That the capturing surface is functionalized is to be understood as the capturing surface being biologically / chemically prepared with a selected component, or “immobilizer”, that is configured to bind a biological cell. The immobilizer is further bonded to the capturing surface by an electrochemically dissociable bond in order to be releasable from the capturing surface on application of an external voltage of a predetermined magnitude.

[0020] To “bind” the biological cell in the microchamber is herein to be understood as the biological cell being prevented from leaving the microchamber due to a biochemical bond between the cell and the immobilizer on the functionalized capturing surface.

[0021] Herein is disclosed capturing, controllable release and optional testing on single biological cells. If a predetermined property of a biological cell is detected by monitoring the cell in the microchamber, the cell may be selected for further studies and may be released and evacuated / unloaded from the microchamber. Along with any other cells identified with the property in a microfluidic system comprising a plurality of such microfluidic devices, the released cell(s) may be transported out for further analysis. Thereby, the present disclosure allows analysis and improved understanding of cellular heterogeneity in a cell population regarding e.g. metabolism and gene-expression. In other words, after monitoring the biological cells, and optionally exposing a sample of cells to a test component, such as a predetermined antibiotic, one or more cells displaying diverging properties, such as resistance to the antibiotic, may be selected for further analysis. The monitored property, such as metabolism, of single cells may be correlated to transcriptomic and epigenetic assessment of the same single cells, which may contribute to understanding of how the property, e.g. metabolism, contributes to cellular heterogeneity and may provide an important guide to disease treatments.

[0022] Monitoring is herein to be understood as describing either optical study of properties of the biological cells, electrical measurements of properties of the biological cells, or a combination of both optical study and electrical measurements of properties of the biological cells.

[0023] Optionally, the dissociable bond is configured for a threshold potential having an absolute value in a range of 0.1 V - 25 V, or more preferably in a range of 0.1 V - 5 V. Different choices of immobilizers may result in different strengths of the electrochemical bond, requiring application of different magnitudes of external electric potentials to dissociate the bond.

[0024] Optionally, the capturing device is configured to capture a single biological cell in the microchamber. The capturing device may for instance be dimensioned to only be able to accommodate a single cell. Further cells in the microchamber would thereby not be bound by the capturing device and would be evacuated from the microchamber before commencing monitoring.

[0025] The first fluid opening, and the second fluid opening may be arranged on laterally opposite sides of the capturing device. Thereby, the capturing device may be arranged in the flow of the liquid sample.

[0026] Optionally, the capturing device comprises a cell stop located adjacent to the capturing surface and protruding in a direction normal to the capturing surface. Thereby, a cell may be mechanically slowed, or event halted, to improve the chances of capturing a single cell and forming the biochemical bond between the cell and the capturing surface.

[0027] Optionally, the cell stop is configured with a width and a height greater than a largest cell extension of the biological cell intended to be captured with the microfluidic device. The width and the height are extensions of the protruding cell stop orthogonal to a fluid flow direction through the microchamber in operation. A sufficiently large cell stop improves the chances of holding or slowing down a single biological cell.

[0028] For reference, different types of biological cells vary in size, from a few hundred nanometres to tens of micrometres. The disclosed microfluidic devices and systems may be manufactured and stored by a laboratory in different sizes intended for different ranges of biological cell diameters. Thus, when a predetermined biological cell type is to be tested, the size of the biological cell type may be determined, and a correspondingly dimensioned test system having microchambers dimensioned for the relevant range of biological cell sizes may be selected from the stored test systems. Especially, a largest and / or a smallest cell extension of the predetermined biological cell type is determined.

[0029] Alternatively, if the biological cell type is not known, a test system comprising differently dimensioned microchambers, where different microchambers are dimensioned for different predetermined ranges of cell sizes, may be selected. Thereby, time is saved in that the biological cells do not need to be studied before the tests begin.

[0030] A predetermined range of cell sizes is herein to be understood as a range of cell sizes which allow isolation / detection of a limited range of biological cells / sizes, i.e. similarly sized, but not necessarily identically sized cells. Thereby, a particular microchamber will be designed to accommodate cells of a limited range of sizes for testing. Other microfluidic devices may simultaneously be designed and used for other predetermined ranges of biological cell sizes, thereby allowing blind tests on cells of unknown types and sizes, since some microchambers in a microfluidic system of microchambers will be correctly dimensioned for the unknown cell type.

[0031] Optionally, the cell stop is configured with the width and / or the height being smaller than three times the largest cell extension of the biological cell intended to be captured with the microfluidic device. A larger cell stop may improve the chances of capturing a cell.

[0032] Optionally, the cell stop comprises a concave surface facing the first fluid opening, and arranged adjacent to, and on an opposite side of the capturing surface, as seen from the first fluid opening. As such, a biological cell being loaded into the microchamber may flow over the capturing surface and be stopped or slowed down by the cell stop on the opposite side of the capturing surface. The concave surface may improve the chances of holding the cell by the cell stop until the biochemical bond between the biological cell and the immobilizer is formed.

[0033] Optionally, the cell stop comprises a microchannel arranged through the cell stop, the microchannel having a first aperture in the concave surface and a second aperture on another surface of the cell stop, the second aperture being closer to the second fluid opening compared to the first aperture. Since part of the flowing fluid (liquid sample) may flow into a cavity formed by the concave surface, and through the microchannel, towards the second fluid opening, the fluid flow assists in pulling a cell into the cavity formed by the concave surface, thereby improving trapping properties of the cell stop.

[0034] Optionally, the electrically conducting capturing surface comprises a metal or metal alloy, such as at least 50 % of silver (Ag) or gold (Au). Silver and gold are excellent electrical conductors and are well-suited for forming a chemical bond with the immobilizer.

[0035] Optionally, the electrochemically dissociable bond between the capturing surface and the at least one immobilizer is a thiol bond, a sulphide bridge, or a sulphur bridge.

[0036] Optionally, the immobilizer comprises a linker molecule.

[0037] Optionally, the linker molecule is -S-(CH2)x-. Optionally, the capturing device comprises a passivation layer on at least a portion of the capturing surface, which passivation layer is configured to prevent undesired bonds from forming on the capturing surface. The passivation layer may prevent contamination of the capturing surface, such as preventing attachment of non-wanted molecules to the capturing surface. Contamination may adversely affect monitoring of the microchamber.

[0038] Optionally, the passivation layer comprises thiolated mercaptohexanol (MCH).

[0039] Optionally, the immobilizer comprises a cell binder chosen from nucleotides, amino acids, and / or oligosaccharides.

[0040] Optionally, the immobilizer comprises a cell binder chosen from DNA, RNA, XNA, and / or antibodies.

[0041] Optionally, the immobilizer comprises a cell binder chosen from aptamers and / or antibodies.

[0042] Optionally, the immobilizer comprises a ligand for affinity bonding to the biological cell.

[0043] Optionally, the microchamber is configured for monitoring a property of the captured biological cell.

[0044] Optionally, the microchamber is configured for optical monitoring of the captured biological cell, and the monitored property is indicative of movement and / or morphology of the captured biological cell.

[0045] The microchamber may be arranged with an optically transparent cover. The captured cell may thus be optically monitored by a microscope or by an external optical sensor.

[0046] Optionally, the microchamber is configured for electrical monitoring of the captured biological cell, and the monitored property is indicative of a metabolism, lysis, pyroptosis and / or apoptosis of the captured biological cell.

[0047] Electrical monitoring may be performed by monitoring electrically detected properties of the cell-containing liquid in the microchamber.

[0048] Optionally, the microchamber comprises an integrated sensor, the sensor being configured to monitor the property of the captured biological cell. The sensor may be an integrated semiconductor electronic sensor for real time monitoring of cell activity. The sensor being “integrated” means that it may be manufactured by conventional semiconductor processing technology on the glass or semiconductor substrate.

[0049] Optionally, the integrated sensor is an ion-sensitive field-effect transistor (ISFET), such as a potentiometric ion sensor. The small size and high sensitivity of the integrated semiconductor sensor in the microfluidic device may facilitate direct monitoring of single cells. The ion-sensitive field-effect transistor may be configured to monitor biological cell metabolism by monitoring metabolism-induced pH changes in the culturing media.

[0050] From a sensing point of view, potentiometric sensing of pH using a field effect transistor is more reliable than directly monitoring a resistance (or impedance) of the culturing media, since a contribution from a solid-liquid interface between electrodes / liquid may generate large and random errors to resistance measurements.

[0051] In addition, directly monitoring changes in physiological parameters (such as pH of the culturing media) can potentially be faster than optically monitoring cell growth, since it does not rely on a visible morphology change for signal generation. Monitoring of physiological parameters can potentially profile the drug response of biological cells in realtime, using highly sensitive and reliable field effect transistor-based ion sensors. The effect of the drug may immediately be detected from changes in electrical signals generated by the monitored property, such as cell metabolism. Testing may be all-electrical and based on current-voltage measurements using conventional equipment.

[0052] Before monitoring is performed on a cell-containing sample in the microchamber, the first fluid opening and / or the second fluid opening may be closed by a sealing fluid, such as oil in order to isolate the microchamber.

[0053] Optionally, the microchamber comprises a first electrode configured to define an electric reference potential of a liquid sample in the microchamber. If the microchamber comprises an integrated sensor, the first electrode may define the reference potential in relation to the integrated sensor.

[0054] Optionally, the first electrode to at least 50 % is made of a material chosen from silver chloride (AgCI) or silver (Ag).

[0055] Optionally, the microchamber comprises a second electrode.

[0056] Optionally, the second electrode is made of at least 50 % platinum (Pt).

[0057] Optionally, the first electrode and the second electrode form an oxygen-redox cell, for measuring oxygen content and / or oxygen concentration in the microchamber 10. A microchamber also comprising the sensor may thus be configured to monitor either one or a combination of oxygen content, oxygen concentration and acidification / metabolism.

[0058] Optionally, the microfluidic device comprises a substrate extending laterally in a plane, and the microchamber, and the capturing device are arranged on the substrate. The substrate may extend laterally in a plane comprising a second axis and a third axis orthogonal to the second axis. The plane is orthogonal to a first axis.

[0059] According to a second aspect of the disclosure, the object is at least partly achieved by a microfluidic system according to claim 31. Hence, there is provided a microfluidic system comprising a plurality of microfluidic devices according to any one of the embodiments of the first aspect of the disclosure, wherein each microchamber is fluidly connected to a first fluid channel via the first fluid opening and to a second fluid channel via the second fluid opening.

[0060] The microfluidic system may comprise at least one array of microfluidic devices interconnected by first and second fluid channels. Each array of microfluidic devices may be interconnected by respective first and second fluid channels. Each array of microfluidic devices may, during testing, be supplied with a liquid cell-containing sample. The first fluid openings and the second fluid openings of the microfluidic devices comprised in the microfluidic system may be closed by filling the first fluid channel and the second fluid channel with a sealing fluid, such as oil. The first fluid openings and the second fluid openings may alternatively be closed by closing valves to which the first fluid channel and the second fluid channel are connected.

[0061] Optionally, the microfluidic system may further comprise integrated electrical interconnects configured to electrically connect any electrodes and sensors in the microfluidic devices to external electrical units for monitoring and for selectively applying an external voltage potential to a capturing surface of a capturing device, so as to selectively dissociate an immobilizer from the capturing surface of the capturing device. The external electrical units may be exemplified by control units, voltage sources, measurement units, etc.

[0062] Optionally, the microfluidic system further comprises a control unit to control operations in the at least one microfluidic device. The control unit may be electrically connected to the capturing surface of the at least one microchamber and may be configured to selectively apply the external voltage potential to the capturing surface of a capturing device to selectively dissociate the immobilizer from the capturing surface.

[0063] The control unit may be electrically connected to the microfluidic system to receive signals from each of the sensors of the microchambers comprised in the system via the integrated electrical interconnects. The control unit may interpret measurements of the sensors and provide a rapid presentation of the biological cells’ status in the microchambers.

[0064] According to a third aspect of the disclosure, the object is at least partly achieved by a method according to claim 33.

[0065] Hence, there is provided a method of testing biological cells using the microfluidic system according to any one of the embodiments of the second aspect of the disclosure. The method comprises: loading at least one biological cell into the at least one microfluidic device by flowing a cell-containing liquid sample through the microchamber in a first direction, capturing, by the capturing device, a biological cell, monitoring a property of the captured biological cell, selecting, based on the monitored property, a captured biological cell for release, applying an external electric potential greater than a threshold potential to the capturing surface of the capturing device in which the selected biological cell is captured, thereby dissociating the chemical bond, thus releasing the selected biological cell, unloading the released biological cell from the microfluidic device by flowing a fluid through the microchamber in the first direction, or in a second direction opposite to the first direction.

[0066] A cell-containing liquid sample may thus be prepared and fluidly connected to at least one microfluidic device of the microfluidic system. The cell sample may comprise cells of the same type, or cells which have at least one characteristic in common. The liquid cellcontaining sample is loaded into the microfluidic system. Each microchamber of the microfluidic chamber comprises a capturing device which may capture and bind a single cell of the cell-containing liquid sample in the microchamber. Properties of the captured cells are thereafter monitored. Based on the monitored properties, cells may be selected for release. Any selected cells are released by a applying an external electric potential, greater than a threshold potential, to the capturing surfaces of the capturing devices of the microchambers housing selected cells, thereby dissociating the chemical bond between the immobilizer and the capturing surface. Referring to a reference potential, an absolute value of the applied electric potential is greater than an absolute value of the threshold potential. The reference potential may be a reference potential of the liquid sample in the microchamber. The selected and released cells may then be unloaded / evacuated from the microchamber(s) for further analysis, such as transcriptomic and epigenetic assessments.

[0067] Hence, the method of detecting diverging properties in single cells in a cellcontaining sample allows the measured property, such as metabolism, of the single cells to be correlated to transcriptomic and epigenetic assessment of the same single cells, which may contribute to understanding of how the property, e.g. metabolism, contributes to cellular heterogeneity and may provide an important guide to disease treatments.

[0068] The property of the captured cell in each microchamber may be measured optically and / or electrically. Optical monitoring may be performed by a microscope or an optical sensor external to the microfluidic system. Optionally, optical monitoring is performed by a microscope or an optical sensor external to the microfluidic system.

[0069] Optionally, electrical monitoring is performed by a sensor and / or by an oxygen-redox cell.

[0070] Optionally, prior to monitoring the property of the captured biological cells, the microchambers of the microfluidic system may be isolated from each other by closing the first and second openings, such as by filling the first and second channels with a sealing fluid, such as oil. In such a case, an additional step of evacuating the sealing fluid is performed prior to unloading the released biological cells. Isolating the microchambers may improve electrical measurements in the cell-containing liquid sample.

[0071] The above aspects, accompanying claims, and / or examples disclosed herein above and later below may be suitably combined with each other as would be apparent to anyone of ordinary skill in the art.

[0072] Additional features and advantages are disclosed in the following description, claims, and drawings, and in part will be readily apparent therefrom to those skilled in the art or recognized by practicing the disclosure as described herein.

[0073] BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Further objects and advantages of, and features of the disclosure will be apparent from the following description of one or more embodiments, with reference to the appended drawings, where:

[0075] Fig. 1 shows a conceptual top-down view of a microfluidic device according to an example of the first aspect of the disclosure.

[0076] Fig. 2 shows a conceptual top-down view of a microfluidic device according to an example of the first aspect of the disclosure.

[0077] Fig. 3a shows a conceptual top-down view of a cell stop according to an example.

[0078] Fig. 3b shows a conceptual view, parallel with a flow direction, of a cell stop according to an example.

[0079] Fig. 4 shows a conceptual top-down view of a microfluidic device according to an example of the first aspect of the disclosure.

[0080] Fig. 5 shows a conceptual top-down view of a microfluidic device according to an example of the first aspect of the disclosure. Fig. 6 shows a conceptual top-down view of a microfluidic system according to an example of the second aspect of the disclosure.

[0081] Fig. 7 shows a conceptual top-down view of a microfluidic system according to an example of the second aspect of the disclosure.

[0082] Fig. 8 shows a conceptual top-down view of a microfluidic system according to an example of the second aspect of the disclosure.

[0083] Fig. 9 shows a flowchart of a method according to an example of the third aspect of the disclosure.

[0084] Fig. 10 shows a flowchart of a method according to an example of the third aspect of the disclosure.

[0085] DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS OF THE INVENTION

[0086] The present disclosure is developed in more detail below referring to the appended drawings which show examples of embodiments. The disclosure should not be viewed as limited to the described examples of embodiments. Like numbers refer to like elements throughout the description.

[0087] The terminology used herein is for the purpose of describing particular aspects of the disclosure only and is not intended to limit the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0088] Fig. 1 shows a microfluidic device 1 for capturing and controllably releasing biological cells, the microfluidic device 1 comprises at least one microchamber 10 having a first fluid opening 12 and a second fluid opening 14 for flowing a liquid sample through the microchamber 10.

[0089] The microfluidic device 1 further comprises at least one capturing device 16 inside the microchamber 10 for capturing at least one biological cell 18 inside the microchamber 10. The microfluidic device 1 may comprise a substrate 30 extending laterally in a plane A. The plane may comprise a second axis x and a third axis y, orthogonal to the second axis x. The plane A may be orthogonal to a first axis z. The microchamber 10, and the capturing device 16 may be arranged on the substrate 30. The substrate 30 and the axes z, x, y and the plane A are indicated in Fig. 1 . It is to be understood that the substrate 30 may also be featured in Figs 2-5 but has been omitted from the drawings for clarity.

[0090] A lateral width W of the microchamber 10 along the third axis y of the microchamber 10 may exceed a total extension of a plurality of biological cells 18, measured along a largest cell extension of the largest biological cell size 18 expected in the cell-containing liquid sample. The lateral width W of the microchamber 10 may allow biological cells 18 to flow with the liquid sample from the first fluid opening 12 to the second fluid opening 14. At least one capturing device 16 may be arranged between the first fluid opening 12 to the second fluid opening 14.

[0091] The capturing device 16 comprises an electrically conducting capturing surface 22 electrically connectable to an external electric potential V via integrated interconnects. The external electric potential V may be connected to the capturing surface 22 via integrated electrical interconnects 36 (not shown in Fig. 1 , but to be described in conjunction with the second aspect of the disclosure below). The electrically conducting capturing surface 22 may comprise a metal or metal alloy, such as at least 50 % silver (Ag) or gold (Au). Silver and gold are excellent electrical conductors and are well-suited for forming a chemical bond with the immobilizer molecules. The capturing device 16 may optionally comprise a passivation layer on at least a portion of the capturing surface 22, which passivation layer is configured to prevent undesired bonds from forming on the capturing surface 22. The passivation layer may comprise thiolated mercaptohexanol (MCH). The passivation layer may prevent contamination of the capturing surface 22, such as preventing attachment of non-wanted molecules to the capturing surface 22. Contamination may adversely affect monitoring of the microchamber 10.

[0092] The capturing surface 22 is further functionalized with at least one immobilizer selected to bind at least one biological cell 18 in the microchamber 10. The at least one immobilizer is bonded to the capturing surface 22 with an electrochemically dissociable bond configured to dissociate from the capturing surface 22 on application of an external electric potential V greater than a threshold potential VT to the capturing surface 22. Referring to a reference potential, an absolute value of the applied electric potential V is greater than an absolute value of the threshold potential VT. The reference potential may be a reference potential of a liquid in the microchamber 10.

[0093] That the capturing surface 22 is functionalized is to be understood as the capturing surface being biologically / chemically prepared with a selected component, or “immobilizer”, that is configured to bind a biological cell 18. The immobilizer is bonded to the capturing surface 22 with an electrochemically dissociable bond in order to release it from the capturing surface on application of the external electric potential V of a predetermined magnitude in reference to the reference potential, which exceeds the threshold potential VT. The dissociable bond may be configured for an absolute value of the threshold voltage in the range of 0.1 - 25 volt, or more preferably in the range of 0.1 V - 5 V.

[0094] To “bind” the biological cell 18 in the microchamber 10 is herein to be understood as the biological cell 18 being prevented from leaving the microchamber 10 due to a biochemical bond between the cell 18 and the immobilizers of the functionalized capturing surface 22.

[0095] The dissociable bond may be a thiol bond, a sulphide bridge, or a sulphur bridge. The immobilizer may comprise a linker molecule. The linker molecule may be -S-(CH2)x-.

[0096] The immobilizer may comprise a cell binder chosen from nucleotides, amino acids, and / or oligosaccharides, or a cell binder chosen from DNA, RNA, XNA, and / or antibodies, or a cell binder chosen from aptamers and / or antibodies. The immobilizer may comprise a ligand for affinity bonding to the biological cell 18.

[0097] The substrate including any sensors, any electrodes and the cell capturing surface 22 in the microfluidic device 1 may be fabricated by conventional semiconductor manufacturing technology at low cost in conventional semiconductor chip foundries. The microfluidic device 1 may thus be provided on a glass or semiconductor substrate 30 or chip (see Fig. 8). Any microstructures of the fluidics above the substrate of the present disclosure, such as walls, and top cover of the microchamber 10 and / or any connecting channels, may be fabricated by conventional techniques of a single material using for instance polydimethylsiloxane (PDMS), or glass, on a semiconductor or glass substrate 30. The disclosed microfluidic devices 1 may facilitate direct testing of many trapped single cells.

[0098] The first and second fluid openings 12, 14 are arranged to introduce or to evacuate a fluid into the microchamber. The fluid may be a cell-containing liquid sample, i.e. containing biological cells 18, or a fluid comprising culturing media for nourishing biological cells 18. Fluid comprising culturing media may, or may not, comprise antibiotics or other components for performing tests on the biological cells 18. Culturing media may also be used to evacuate biological cells 18 from the microchamber 10.

[0099] The microchamber 1 may be configured for monitoring a property of the captured biological cell 18. The microchamber 1 may be configured for optical monitoring of the captured biological cell 18, and the monitored property may be indicative of movement and / or morphology of the captured biological cell 18. The microchamber 1 may be arranged with an optically transparent cover. The captured biological cell 18 may thus be optically monitored by a microscope or by an external optical sensor (not shown).

[0100] Alternatively, the microchamber 1 may be configured for electrical monitoring of the captured biological cell 18, and the monitored property is indicative of a metabolism, lysis, pyroptosis and / or apoptosis of the captured biological cell. Electrical monitoring may be performed by monitoring electrically detected properties of the cell-containing liquid in the microchamber 1.

[0101] As shown in Fig. 2, the capturing device 16 may comprise a cell stop 24 located adjacent to the capturing surface 22 and protruding in a direction normal to the capturing surface 22. Thereby, a biological cell 18 may be mechanically slowed, or event halted, to improve the chances of forming the biochemical bond between the biological cell 18 and the capturing surface 22.

[0102] Fig. 3a and Fig. 3b exemplify how the cell stop 24 may be configured with a width w and a height h greater than a largest cell extension of the biological cell 18 intended to be captured with the microfluidic device 1. The width w and the height h are extensions of the protruding cell stop 24 orthogonal to a fluid flow direction through the microchamber 10 in operation. The width w may extend substantially in parallel with the third axis y. The height h may extend substantially in parallel with the first axis z. The microfluidic device may further be configured for a fluid flow substantially along the second axis x.

[0103] A sufficiently large cell 24 stop improves the chances of holding or slowing down a single biological cell 18. The cell stop 24 may be configured with the width w and / or the height h being smaller than three times the largest cell extension of the biological cell 18 intended to be captured with the microfluidic device 1. A larger cell stop 24 may improve the chances of capturing a biological cell 18.

[0104] The cell stop 24 may comprise a concave surface 25 facing the first fluid opening 12, and arranged adjacent to, and on an opposite side of the capturing surface 22, as seen from the first fluid opening 12. As such, a biological cell loaded into the microchamber 10 may flow over the capturing surface 22 and may be stopped or slowed down by the cell stop 24 on the opposite side of the capturing surface 22. The concave surface 25 may improve the chances of holding the biological cell 18 by the cell stop 24 until the biochemical bond between the biological cell 18 and the immobilizer is formed.

[0105] The cell stop 24 may comprise a microchannel 27 arranged through the cell stop 24. The microchannel may along the whole height h of the cell stop 24, but may be significantly more narrow than the width w of the cell stop to prevent biological cells 18 from entering the microchannel 27. The microchannel having a first aperture 27’ in the concave surface 25 and a second aperture 27” on another surface of the cell stop 24, the second aperture 27” being closer to the second fluid opening 14 compared to the first aperture 27’. Since part of the flowing fluid (liquid sample) may flow into a cavity formed by the concave surface 25, and through the microchannel 27, towards the second fluid opening 14 of the microchamber 10, the fluid flow assists in pulling a biological cell 18 into the cavity formed by the concave surface 25, thereby improving trapping properties of the cell stop 24.

[0106] Different types of biological cells 18 may vary in size, from a few hundred nanometres to tens of micrometres. The disclosed microfluidic devices 1 and microfluidic systems 2 may be manufactured and stored by a laboratory in different sizes intended for different ranges of biological cell diameters. Thus, to test a predetermined biological cell type, the size of the biological cell type may be determined, and a correspondingly dimensioned test system having microchambers 10 dimensioned for the relevant range of biological cell sizes may be selected from the stored test systems. Especially, a largest and / or a smallest cell extension of the predetermined biological cell type is determined.

[0107] Alternatively, if the biological cell type is not known, a test system comprising differently dimensioned microchambers 10, where different microchambers 10 are dimensioned for different predetermined ranges of cell sizes, may be selected. Thereby, time is saved in that the biological cells 18 do not need to be studied before the tests / monitoring begin.

[0108] A predetermined range of cell sizes is herein to be understood as a range of cell sizes which allow isolation / detection of a limited range of biological cells / sizes, i.e. similarly sized, but not necessarily identically sized cells. Thereby, a particular microchamber 10 will be designed to accommodate cells of a limited range of sizes for testing. Other microfluidic devices 1 , in a microfluidic system 2, may simultaneously be designed and used for other predetermined ranges of biological cell sizes, thereby allowing blind tests on cells of unknown types and sizes, since some microchambers 10 in a microfluidic system 2 of microchambers will be correctly dimensioned for the unknown cell type.

[0109] As exemplified in Fig. 4, the microchamber 1 may comprise an integrated sensor 20. The sensor may be configured to monitor the property of the captured biological cell 18. The sensor 20 may be an integrated semiconductor electronic sensor 20 for real time monitoring of cell activity. The sensor 20 being “integrated” means that it may be manufactured on the substrate by conventional semiconductor processing technology. The measured property may be indicative of a metabolism, lysis, pyroptosis and / or apoptosis of the captured biological cell 18. The integrated sensor 20 may be an ion-sensitive field-effect transistor (ISFET), such as a potentiometric ion sensor, which may be configured to monitor biological cell metabolism by monitoring metabolism-induced pH changes of a cell-containing liquid sample.

[0110] From a sensing point of view, potentiometric sensing of pH using a field effect transistor is more reliable than directly measuring a resistance (or impedance) of the cellcontaining liquid sample, since a contribution from a solid-liquid interface between electrodes / liquid may generate large and random errors to resistance measurements.

[0111] In addition, directly monitoring changes in physiological parameters (such as pH of the liquid) can potentially be faster than optically monitoring cell growth, since it does not rely on a visible morphology change for signal generation. Monitoring of physiological parameters can potentially profile the drug response of biological cells 18 in real-time, using highly sensitive and reliable field effect transistor-based ion sensors 20. The effect of the drug may immediately be detected from changes in electrical signals generated by the monitored property, such as cell metabolism. Testing may be all-electrical and based on current-voltage measurements using conventional equipment.

[0112] Before monitoring of a cell-containing sample in the microchamber 10, the first fluid opening 12 and / or the second fluid opening 14 may be closed by a sealing fluid, such as oil, or by valves with which the fluid openings 12, 14 may be in fluid communication.

[0113] The capturing device 16 may be configured to capture a single biological cell 18 in the microchamber. The capturing device 16 may for instance be dimensioned to only be able to accommodate a single biological cell 18. Further cells 18 in the microchamber 10 would thereby not be bound by the capturing device 16 and would be evacuated from the microchamber with the fluid flow via the second fluid opening 14 before commencing monitoring.

[0114] The first fluid opening 12 and the second fluid opening 14 may be arranged on laterally opposite sides of the capturing device 16. Thereby, the capturing device 16 may be arranged in the flow of the liquid sample.

[0115] During monitoring, when a predetermined property of a biological cell 18 is detected in the microchamber 10, the cell having the property may be selected for further studies and may be released and evacuated / unloaded from the microchamber 10. Along with any other cells identified with the property in a microfluidic system 2 (to be described in further detail below), comprising a plurality of such microfluidic devices 1 , the released cell(s) 18 having the property may be shipped out for further assessments. Thereby, the present disclosure allows analysis and improved understanding of cellular heterogeneity of a cell population, especially metabolic heterogeneity. In other words, after exposing a sample of cells to a test component, such as a predetermined antibiotic, one or more cells displaying diverging properties, such as resistance to the antibiotic, may be selected for further tests (such as transcriptom ic and epigenetic assessments). The measured property, such as metabolism, of single cells may be correlated to transcriptom ic and epigenetic assessment of the same single cells, which may contribute to understanding of how the property, e.g. metabolism, contributes to cellular heterogeneity and may provide an important guide to disease treatments.

[0116] The sensor 20 may be an integrated semiconductor electronic sensor 20 for real time monitoring of cell activity. The sensor 20 being “integrated” means that it may be manufactured on the substrate by conventional semiconductor processing technology. The measured property may be indicative of a metabolism, lysis, pyroptosis and / or apoptosis of the captured biological cell 18.

[0117] The integrated sensor 20 may be an ion-sensitive field-effect transistor (ISFET), such as a potentiometric ion sensor, which may be configured to monitor biological cell metabolism by monitoring metabolism-induced pH changes of a cell-containing liquid sample.

[0118] From a sensing point of view, potentiometric sensing of pH using a field effect transistor is more reliable than directly measuring a resistance (or impedance) of the cellcontaining liquid sample, since a contribution from a solid-liquid interface between electrodes / liquid may generate large and random errors to resistance measurements.

[0119] In addition, directly monitoring changes in physiological parameters (such as pH of the liquid) can potentially be faster than optically monitoring cell growth, since it does not rely on a visible morphology change for signal generation. Monitoring of physiological parameters can potentially profile the drug response of biological cells 18 in real-time, using highly sensitive and reliable field effect transistor-based ion sensors 20. The effect of the drug may immediately be detected from changes in electrical signals generated by the monitored property, such as cell metabolism. Testing may be all-electrical and based on current-voltage measurements using conventional equipment.

[0120] As illustrated in Fig. 5, the microfluidic device 1 may further comprise a first electrode 26 configured to define an electric reference potential of a liquid in the microchamber. If the microchamber comprises an integrated sensor, the first electrode may define the reference potential, e.g. the reference liquid potential, for the integrated sensor. Similar to the capturing surface 22, the first electrode 26 may be electrically connected to external measurement units via the integrated electrical interconnects 36 (not shown in Fig. 5). The first electrode may, to at least 50 %, be made of a material chosen from silver chloride (AgCI) or silver (Ag).

[0121] In addition, the microchamber 10 may comprise a second electrode 28. The second electrode 28 may be made of at least 50 % platinum (Pt). The first electrode 26 and the second electrode 28 may form an oxygen-redox cell 40, for measuring oxygen content and / or oxygen concentration in the microchamber (10). The first electrode 26, the second electrode 28 and consequently the oxygen-redox cell 40, may be electrically connected to the external measurement units via integrated electrical interconnects 36 (not shown in Fig. 5). A microchamber 1 also comprising the sensor 20 may thus be configured to monitor either one or a combination of oxygen content, oxygen concentration and acidification / metabolism.

[0122] Fig. 6 and Fig. 7 illustrate examples of a second aspect of the disclosure. There is shown a microfluidic system 2 comprising a plurality of microfluidic devices 1 according to any one of the examples of the first aspect of the disclosure. A microfluidic system 2 comprising a microfluidic device 1 according to Fig. 1 is depicted in Fig. 6 and a microfluidic system 2 comprising a microfluidic device 1 according to Fig. 2 is depicted in Fig. 7, but it is to be understood that any exemplary microfluidic devices 1 of the first aspect of the disclosure, such as of Fig. 4 and / or of Fig. 5 , could be comprised in a microfluidic system 2. Each microchamber 10 is fluidly connected to a first fluid channel 32 via the first fluid opening 12 and to a second fluid channel 34 via the second fluid opening 14.

[0123] The arrows illustrate how a fluid, such as a cell-containing liquid sample, may flow through the microfluidic system 2 for shipping the selected cells out from the microchambers, from the first fluid channel 32 into the microchamber 10 via the first fluid opening 12, through the microchamber 10 into the second fluid channel 34, via the second fluid opening 14. As will be discussed below, the fluid flow may in some situations be reversed, such as shown in Fig. 7.

[0124] Fig. 6 and Fig. 7 further illustrate a released biological cell 18’. Upon identification of biological cells 18 having a pre-determined property, the external electric potential V, which could be positive or negative in reference to the reference potential, may be applied to the respective capturing surfaces 22 via the integrated electrical interconnects 36 so as to dissociate the electrochemical bond between the immobilizer and the capturing surface 22. In the examples of Fig. 6 and Fig. 7, the middle microchamber 10 is shown to contain the released cell 18’. In comparison to the biological cells 18 in the two other microchambers 10, the released cell 18’ is illustrated as drifting with the fluid flow away from the capturing surface 22 to the fluid channels 32 or 34. As shown, the reverse flow depicted in Fig. 7 is particularly advantageous when the microchamber 10 comprises the cell stop 24 in order to dislodge the cell from the cell stop 24.

[0125] As shown in Fig. 8, the microfluidic system 2 may comprise at least one array 42 of microfluidic devices 1 interconnected by first and second fluid channels 32, 34. Fig. 6 and Fig. 7 show three microfluidic devices 1 of an array 42. However, any number of microfluidic devices 1 may be comprised in the array 42. The array 42 is defined as set of microfluidic devices 1 being interconnected by the same first channel 32 and the same second channel 34. The microfluidic system 2 may further comprise a plurality of arrays 42 of microfluidic devices 1 , each array 42 of microfluidic devices 1 interconnected by respective first channels 32 and second channels 34. Each array 42 of microfluidic devices may, during testing, be supplied with a liquid cell-containing sample from an external source. The first fluid openings 12 and the second fluid openings 14 of the microfluidic devices 1 comprised in the microfluidic system 2 may be closed by closing the first fluid channel 32 and / or the second fluid channel 34. The first fluid openings 12 and / or the second fluid openings 14 may be closed by filling the first fluid channel 32 and / or the second fluid channel 34 with a sealing fluid, such as oil. Alternatively, the first fluid openings 12 and / or the second fluid openings 14 may be closed by closing valves (not shown) to which the first fluid channel 32 and the second fluid channel 34 are connected. Closing the microfluidic devices 1 and thereby isolating the microchambers 10 from each other may improve the quality of electrical monitoring of biological cell properties in each microchamber 10.

[0126] The microfluidic system 2 may further comprise integrated electrical interconnects 36 configured to electrically connect any electrodes and sensors in the microfluidic devices 1 to external electrical units for monitoring and for selectively applying an external electric potential V to a capturing surface 22 of a capturing device 16 so as to selectively dissociate an immobilizer from the capturing surface 22 of the capturing device 16.

[0127] The microfluidic system 2 may further comprise a control unit 38, electrically connected to the microfluidic system 2 via the integrated electrical interconnects 36, for monitoring biological cell activity in the at least one microfluidic device 1. The control unit 38 may be electrically connected to the sensor 20 of the at least one microchamber 10 and may be configured to apply the external electric potential V to the at least one capturing surface 22 of the capturing device 16 to selectively dissociate the immobilizer from the capturing surface 22 based on the measured property of the captured biological cell 18.

[0128] The control unit 38 may be electrically connected to the microfluidic system 2 via the integrated electrical interconnects 36 to receive signals from each of the sensors 20 of the microchambers 10 comprised in the microfluidic system 2. The control unit 38 may optionally also be connected to the first electrode 26 and to the second electrode 28 (Fig. 5) and thereby to the oxygen-redox cell 40. The control unit 38 may interpret measurements of the sensors 20 and / or of the oxygen-redox cells 40 and provide a rapid presentation of the biological cells’ 18 response in the microchambers 10. The control unit 38 may apply the electric potential V to selected capturing surfaces either autonomously, based on the measured properties, or manually by an operator, after displaying the results of the measured properties to the operator.

[0129] Fig. 9 exemplifies a third aspect of the disclosure, showing a method 3 of testing biological cells 18 using the microfluidic system 2 according to any one of the examples of the second aspect of the disclosure. The method 3 comprises: loading S1 at least one biological cell 18 into the at least one microfluidic device

[0130] 1 by flowing a cell-containing liquid sample through the microchamber 10 in a first direction, capturing S2, by the capturing device 16, a biological cell 18, monitoring S3 a property of the captured biological cell 18, selecting S4, based on the monitored property, a captured biological cell 18 for release, applying S5 an external electric potential V greater than a threshold potential VT to the capturing surface 22 of the capturing device 16 by which the selected biological cell 18 is captured, thereby dissociating the chemical bond, thus releasing the selected biological cell 18, unloading S6 the released biological cell 18 from the microfluidic device 1 by flowing a fluid through the microchamber 10 in the first direction, or in a second direction opposite to the first direction.

[0131] A cell-containing liquid sample may thus be prepared and fluidly connected to at least one microfluidic device 1 of the microfluidic system 2 via the first fluid channel 32 and / or the second fluid channel 34. The cell sample may comprise biological cells 18 of the same type, or biological cells 18 which have at least one characteristic in common. The liquid cell-containing sample is loaded into the microfluidic system 2. Each microchamber 10 of the microfluidic devices 1 of the microfluidic system 2 comprises a capturing device 16 which may capture and bind a single biological cell 18 of the cell-containing liquid sample in the microchamber 10.

[0132] The property of the captured cell in each microchamber may be measured optically and / or electrically. Optical monitoring may be performed by a microscope or an optical sensor external to the microfluidic system 2. Electrical monitoring may be performed by a sensor 20 and / or by an oxygen-redox cell 40 integrated and comprised in the microchamber 1.

[0133] Based on the measured property, cells may be selected for release. Any selected biological cells 18 are released by a applying the external electric potential V, greater than the threshold potential VT, to the capturing surfaces 22 of the capturing devices 16 of the microchambers 10 housing the selected biological cells 18, thereby dissociating the chemical bond between the immobilizer and the capturing surface 22. Referring to a reference potential, an absolute value of the applied electric potential V is greater than an absolute value of the threshold potential VT. The reference potential may be a reference potential of the liquid sample in the microchamber. The selected and released biological cells 18 may then be unloaded / evacuated from the microchamber(s) 10 for further analysis, which is illustrated and described in conjunction with Fig. 6 and Fig. 7 above.

[0134] As shown in Fig. 10, prior to monitoring S3 the property of the captured biological cells 18, the microchambers 10 of the microfluidic system 2 may alternatively be isolated from each other by closing S7 the first and second openings 12, 14, such as by filling the first and second channels 32, 34 with a sealing fluid, such as oil. In such a case, an additional step of evacuating S8 the sealing fluid is performed prior to applying S5 the external electrical potential to dissociate the bond to release the biological cells 18. Isolating the microchambers 10 may improve the accuracy of electrical measurements in the cellcontaining liquid sample. Hence, the method 3 may detect diverging properties in single biological cells 18 in a cell-containing sample allowing the measured property, such as metabolism, of the single biological cells 18 to be correlated to transcriptomic and epigenetic assessment of the same single cells, which may contribute to understanding of how the property, e.g. metabolism, contributes to cellular heterogeneity and may provide an important guide to disease treatments.

[0135] The first direction is defined as shown by the arrows in the microchambers 1 in Fig. 6, by flowing a fluid through the microchamber 10 from the first fluid channel 32 to the second fluid channel 34 via the first opening 12 and the second opening 14, respectively.

[0136] The second direction of fluid flow is defined as shown by the arrows in the microchambers 1 in Fig. 7, by flowing a fluid through the microchamber 10 from the second fluid 34 channel to the first fluid channel 32 via the second opening 14 and the first opening 12, respectively. Thus, the second direction of fluid flow for unloading S6 the microfluidic device 1 is in a reverse direction as compared to the fluid flow for loading S1 the microfluidic device 1. The reverse direction of unloading S6 may be especially advantageous if the capturing device 16 comprises a cell stop 24, as illustrated in Fig. 7, in order to dislodge the biological cell 18 from the cell stop 24.

Claims

CLAIMS1. A microfluidic device (1) for the capture of single biological cells (18) and controllable release of selected single biological cells (18), the microfluidic device (1) comprising: at least one microchamber (10) having a first fluid opening (12) and a second fluid opening (14) for flowing a liquid sample through the microchamber (10), at least one capturing device (16) inside the microchamber (10) for capturing at least one biological cell (18) inside the microchamber (10), wherein the capturing device (16) comprises an electrically conducting capturing surface (22) electrically connectable to an external electric potential (V), the capturing surface (22) further being functionalized with at least one immobilizer selected to bind at least one biological cell (18) in the microchamber (10), wherein the at least one immobilizer is bonded to the capturing surface (22) with an electrochemically dissociable bond configured to dissociate from the capturing surface (22) on application of an external electric potential (V) greater than a threshold potential (VT), to the capturing surface (22).

2. The microfluidic device (1) of claim 1 , wherein the dissociable bond is configured for an absolute value of the threshold potential (VT) in a range of 0.1 V - 25 V, or more preferably in a range of 0.1 V - 5V.

3. The microfluidic device (1) of any one of the previous claims, wherein the capturing device (16) is configured to capture a single biological cell (18) in the microchamber (10).

4. The microfluidic device (1) of any one of the previous claims, wherein the capturing device (16) comprises a cell stop (24) located adjacent to the capturing surface (22) and protruding in a direction normal to the capturing surface (22).

5. The microfluidic device (1) of claim 4, wherein the cell stop (24) is configured with a width (w) and a height (h) greater than a largest cell extension of the biological cell (18) intended to be captured with the microfluidic device (1), where the width (w) and the height (h) are extensions of the protruding cell stop (24) orthogonal to a fluid flow direction through the microchamber (10) in operation.

6. The microfluidic device (1) of claim 5, wherein the cell stop (24) is configured with the width (w) and / or the height (h) being smaller than three times the largest cell extension of the biological cell (18) intended to be captured with the microfluidic device (1).

7. The microfluidic device (1) of any one of claims 4-6, wherein the cell stop (24) comprises a concave surface (25) facing the first fluid opening (12), and arranged adjacent to, and on an opposite side of the capturing surface (22), as seen from the first fluid opening (12).

8. The microfluidic device (1) of claim 7, wherein the cell stop (24) comprises a microchannel (27) arranged through the cell stop (24), the microchannel (27) having a first aperture (27’) in the concave surface (25) and a second aperture (27”) on another surface of the cell stop (24), the second aperture (27”) being closer to the second fluid (14) opening compared to the first aperture (27’).

9. The microfluidic device (1) of any one of the previous claims, wherein the electrically conducting capturing surface (22) comprises a metal or metal alloy.

10. The microfluidic device (1) of any one of the previous claims, wherein the electrically conducting capturing surface (22) comprises at least 50 % of Ag or Au.

11. The microfluidic device (1) of any one of the previous claims, wherein the electrochemically dissociable bond between the capturing surface (22) and the at least one immobilizer is a thiol bond, a sulphide bridge, or a sulphur bridge.

12. The microfluidic device (1) of any one of the previous claims, wherein the immobilizer comprises a linker molecule.

13. The microfluidic device (1) of claim 12, wherein the linker molecule is -S-(CH2)x-.

14. The microfluidic device (1) of any one of the previous claims, wherein the capturing device (16) comprises a passivation layer on at least a portion of the capturing surface (22), which passivation layer is configured to prevent undesired bonds from forming on the capturing surface (22).

15. The microfluidic device (1) of claim 14, wherein the passivation layer comprises thiolated mercaptohexanol (MCH).

16. The microfluidic device (1) of any one of the previous claims, wherein the immobilizer comprises a cell binder chosen from nucleotides, proteins, and / or oligosaccharides.

17. The microfluidic device (1) of any one of the previous claims, wherein the immobilizer comprises a cell binder chosen from DNA, RNA, XNA, and / or antibodies.

18. The microfluidic device (1) of any one of the previous claims, wherein the immobilizer comprises a cell binder chosen from aptamers and / or antibodies.

19. The microfluidic device (1) of any one of the previous claims, wherein the immobilizer comprises a ligand for affinity bonding to the biological cell (18).

20. The microfluidic device (1) of any one of the previous claims, wherein the microchamber (10) is configured for monitoring a property of the captured biological cell (18).

21. The microfluidic device (1) of claim 20, wherein the microchamber (10) is configured for optical monitoring of the captured biological cell (18), and wherein the monitored property is indicative of movement and / or morphology of the captured biological cell (18).

22. The microfluidic device (1) of claim 20 or 21 , wherein the microchamber (10) is configured for electrical monitoring of the captured biological cell (18), and wherein the monitored property is indicative of a metabolism, lysis, pyroptosis and / or apoptosis of the captured biological cell (18).

23. The microfluidic device (1) of claim 22, wherein the microchamber (10) comprises an integrated sensor (20), the sensor (20) being configured to monitor the property of the captured biological cell (18).

24. The microfluidic device (1) of claim 23, wherein the integrated sensor (20) is an ionsensitive field-effect transistor (ISFET).

25. The microfluidic device (1) of any one of the previous claims, wherein the microchamber (10) comprises a first electrode (26) configured to define an electric reference potential of a liquid sample flowing through the microchamber (10).

26. The microfluidic device (1) of claim 25, wherein the first electrode (26) to at least 50 % is made of a material chosen from silver chloride (AgCI) or silver (Ag).

27. The microfluidic device (1) of any one of claims 25-26, wherein the microchamber (10) comprises a second electrode (28).

28. The microfluidic device (1) of claim 28, wherein the second electrode (28) is made of at least 50 % Platinum (Pt).

29. The microfluidic device (1) of any one of claims 27-28, wherein the first electrode (26) and the second electrode (28) form an oxygen-redox cell (40), for measuring oxygen content and / or oxygen concentration in the microchamber (10).

30. The microfluidic device (1) of any one of the previous claims, wherein the microfluidic device (1) comprises a substrate (30) extending laterally in a plane (A), and the microchamber (10), and the capturing device (16) are arranged on the substrate (30).

31. A microfluidic system (2) comprising a plurality of microfluidic devices (1) according to any one of claims 1-30, wherein each microchamber (10) is fluidly connected to a first fluid channel (32) via the first fluid opening (12) and to a second fluid channel (34) via the second fluid opening (14).

32. The microfluidic system (2) of claim 31 , further comprising integrated electrical interconnects (36) configured to electrically connect any electrodes and sensors in the microfluidic devices to external electrical units for monitoring and for selectively applying the external electric potential V to a capturing surface (22) of a capturing device (16) so as to selectively dissociate an immobilizer from the capturing surface (22) of the capturing device (16).

33. A method (3) for capturing and releasing biological cells (18) using the microfluidic system (2) according to any one of claims 30-32, the method (3) comprising:loading (S1) at least one biological cell (18) into the at least one microfluidic device (1) by flowing a cell-containing liquid sample through the microchamber (10) in a first direction, capturing (S2), by the capturing device (16), a biological cell (18), monitoring (S3) a property of the captured biological cell (18), selecting (S4), based on the monitored property, a captured biological cell (18) for release, applying (S5) an external electric potential V greater than a threshold potential to the capturing surface (22) of the capturing device (16) by which the selected biological cell (18) is captured, thereby dissociating the chemical bond, thus releasing the selected biological cell (18), unloading (S6) the released biological cell (18) from the microfluidic device (1) by flowing a fluid through the microchamber (10) in the first direction, or in a second direction opposite to the first direction.

34. The method (3) of claim 33, wherein the step of monitoring (S3) a property of the captured biological cell (18) is performed by optical monitoring and / or by electrical monitoring.

35. The method (3) of claim 34, wherein optical monitoring is performed by a microscope or an optical sensor external to the microfluidic system 2.

36. The method (3) of claim 34, wherein electrical monitoring is performed by a sensor (20) and / or by an oxygen-redox cell (40).

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