DE102022203848A1 - Method and apparatus for capturing at least one nucleated cell using at least one electrode for a microfluidic device and microfluidic device - Google Patents

Abstract

The approach presented here relates to a method for capturing at least one nucleated cell using at least one electrode for a microfluidic device (100). For this purpose, the method comprises a step of outputting an application signal, which causes a sample liquid with the at least one nucleated cell to be applied to a carrier substrate (105) of the microfluidic device (100), and a step of providing a current signal to an interface to the at least one Electrode to generate an electric field on or in a microcavity (110) of the carrier substrate (105), which is designed to capture the at least one nucleated cell as a target cell in the microcavity (110).

Description

State of the art

The approach is based on a method and a device for capturing at least one nucleated cell using at least one electrode for a microfluidic device and a microfluidic device according to the species of the independent claims. The subject of the present approach is also a computer program.

Circulating tumor cells (CTCs) have emerged as promising and clinically relevant biomarkers in the management of advanced malignant tumors in recent years. Their identification and characterization in suitable body fluids, such as blood or lymph fluid, is known as liquid biopsy - one of the main research areas of modern oncology.

Disclosure of Invention

Against this background, with the approach presented here, an improved method for capturing at least one nucleated cell using at least one electrode for a microfluidic device, an improved device using this method, and finally a corresponding computer program and an improved microfluidic device according to main claims presented. Advantageous developments and improvements of the device specified in the independent claim are possible as a result of the measures listed in the dependent claims.

The approach presented here enables a cost-effective microfluidic device, or an efficient and automated microfluidic analysis, using a disposable cartridge.

A method for capturing at least one nucleated cell using at least one electrode for a microfluidic device is presented, the method comprising a dispensing step and a providing step. In the output step, an application signal is output, which causes a sample liquid with the at least one nucleated cell to be applied to a carrier substrate of the microfluidic device. In the providing step, a current signal is provided at an interface to the at least one electrode in order to generate an electric field on or in a microcavity of the carrier substrate, which is designed to capture the at least one nucleated cell as a target cell in the microcavity.

The method can be carried out, for example, in the microfluidic device as a lab-on-chip cartridge. The method can advantageously be carried out automatically. Nucleated cells are cells that have a cell nucleus that contains the genetic material. The nucleated cell can be, for example, a tumor cell or, for example, a leukocyte. Accordingly, the sample liquid can be a blood sample from a patient, for example. The carrier substrate can be based on a PCB, for example, that is to say it can be arranged on a printed circuit board, and/or contain a silicon material, for example. The microcavities can, for example, be arranged in a honeycomb, round or, for example, angular manner on or in the carrier substrate. The microcavities can be shaped, for example, in order to be able to receive and catch the sample liquid with the at least one nucleated cell in order to be able to advantageously analyze it afterwards.

According to one embodiment, the method can include a step of changing a current intensity before or after the outputting step in order to change, in particular to strengthen or weaken, the electric field. In particular, the electric field is built up and/or varied between the electrode and a counter-electrode lying opposite the electrode in or on the microcavity. The electric field can advantageously create a dielectrophoresis cage, which advantageously can be opened, closed or switched off depending on the current intensity. The "DEP cage" (opened, closed and shut down) is one aspect of two possible aspects and is very useful for capturing cells. Another aspect of the approach presented here can be used to sort cells based on positions in microcavities. Depending on the activation of the electrodes in the microcavity, a "DEP levitator" can also be created with which cells can be selectively ejected electrically from the cavities. This has a different distribution of the electric field strength than a DEP cage.

An embodiment of the approach presented here is also favorable in which, in the step of providing, the current signal is output at the interface to the at least one electrode and to at least one other electrode arranged in an adjacent microcavity, so that a different electrode is applied to the at least one other electrode electric field is generated than at the electrode, in particular where the at the electric electrode generated field differs in terms of a direction and / or intensity from the electric field that was generated at the other electrode and / or wherein the other electric field is generated at the other electrode, which is arranged in a microcavity, which is arranged in a common column or a common row with respect to the microcavity with the electrode. Such an embodiment of the approach proposed here offers the advantage of preventing or at least reducing crosstalk of an electrical signal, which is caused by an electrical field at an electrode of a first microcavity, into a second, adjacent microcavity. In this way it is possible to efficiently prevent, for example, very similar or identical potentials from occurring on electrodes of microcavities which are arranged in a common row or a common column of a matrix-shaped structure, so that a cage formed separately for each microcavity can be implemented . In this way, improved separation or improved inclusion of the cell trapped in the respective microcavity can be implemented.

The method can comprise a step of washing the sample liquid using a washing buffer after the providing step in order to wash out a suspension of the sample liquid from the microcavity and the volume of the microfluidic chamber above it. The wash buffer may advantageously be implemented as a fluid which cleans the suspension but does not damage the nucleated cell. The washing step can advantageously improve the transparency of the sample liquid so that, for example, a subsequent analysis can be carried out more easily. The washing step should be gentle enough not to flush the captured nucleated cell(s) out of their capturing positions.

According to one embodiment, in or after the providing step, a release signal can be provided to the electrode after the nucleated cell has been captured in order to release a further nucleated cell from the sample liquid as a non-target cell from the electric field. Advantageously, the enable signal can trigger an enable voltage that causes the electric field to open. This advantageously allows the nucleated cell to be isolated from the other nucleated cell.

According to one embodiment, the application signal can be output in the output step, which causes a lysate to be applied to the carrier substrate in order to obtain a cell sediment with the at least one nucleated cell and a cell suspension of a lysate. The application signal can advantageously be output to an interface to a pump device, so that the pump device can pump the lysate through the microfluidic device.

Furthermore, the method can comprise a step of identifying the nucleated cells from the sample liquid after the providing step. In particular, in the identification step, the nucleated cells can be optically detected from the cell sediment and additionally or alternatively quantified. Advantageously, the sample liquid can be lysed in or before the identification step and then analyzed in order to detect whether the lysate contains tumor cells, for example. The lysing can also be carried out as a preliminary step at the beginning of the process in the identification step.

According to a further embodiment, in the step of providing, the nucleated cell or at least one other nucleated cell can be trapped in a capture plane of the microcavity and/or the cell or at least one other nucleated cell can be released from the sample liquid from the electric field into a transport level is released. In this way, a very efficient capture and subsequent transport of the relevant cell types can be realized.

An embodiment of the approach presented here is also advantageous as a method for capturing at least one nucleated cell using at least one electrode for a microfluidic device, the method having a step of applying a sample liquid with the at least one nucleated cell to a carrier substrate of the microfluidic device . Furthermore, the method comprises a step of generating an electric field on or in a microcavity of the carrier substrate with the at least one electrode, which is designed to capture the at least one nucleated cell as a target cell in the microcavity. This results in the advantages already mentioned above.

It is advantageous here if the method includes a step of changing a current intensity in order to change the electric field, in particular to strengthen or weaken it after the step of applying the sample liquid to a carrier substrate of the microfluidic device, or before or after the step creating the electric field. In this case, the electric field is built up and/or varied, for example, between the electrode and a counter-electrode lying opposite the electrode in or on the microcavity. This results in the advantages already mentioned above.

In addition, it is advantageous if a step of washing the sample liquid using a washing buffer takes place after the step of generating the electric field in order to wash out a suspension of the sample liquid from the microcavity. This results in the advantages already mentioned above.

Furthermore, it is advantageous if, in or after the step of generating the electric field after capturing the cell containing a nucleus, another cell containing a nucleus is released from the sample liquid as a non-target cell from the electric field through the electrode. This results in the advantages already mentioned above.

In addition, it is advantageous if a lysate is applied to the carrier substrate in the step of applying the sample liquid in order to obtain a cell sediment with the at least one nucleated cell and a cell suspension of a lysate. This results in the advantages already mentioned above. It is also advantageous if the step of identifying the cells containing a nucleus from the sample liquid is provided after the step of generating the electric field. This results in the advantages already mentioned above.
It is also advantageous if the nucleated cells are optically detected and/or quantified from a cell sediment in the identification step. This results in the advantages already mentioned above.

Furthermore, it is advantageous if, in the step of generating the electric field, the nucleated cell or at least one other nucleated cell is caught in a capture plane of the microcavity and/or the cell or at least one other nucleated cell is released from the sample liquid from the electric field is released into a transport level. This results in the advantages already mentioned above.

This method can be implemented, for example, in software or hardware or in a mixed form of software and hardware, for example in a control unit.

The approach presented here also creates a device that is designed to carry out, control or implement the steps of a variant of a method presented here in corresponding devices. The task on which the approach is based can also be solved quickly and efficiently by this embodiment variant of the approach in the form of a device. The device can be implemented, for example, as a control unit or as a control device.

For this purpose, the device can have at least one computing unit for processing signals or data, at least one memory unit for storing signals or data, at least one interface to a sensor or an actuator for reading in sensor signals from the sensor or for outputting data or control signals to the Have actuator and / or at least one communication interface for reading or outputting data that are embedded in a communication protocol. The arithmetic unit can be, for example, a signal processor, a microcontroller or the like, with the memory unit being able to be a flash memory, an EEPROM or a magnetic memory unit. The communication interface can be designed to read in or output data wirelessly and/or by wire, wherein a communication interface that can read in or output wire-bound data can, for example, read this data electrically or optically from a corresponding data transmission line or can output it to a corresponding data transmission line.

In the present case, a device can be understood to mean an electrical device that processes sensor signals and, depending thereon, outputs control and/or data signals. The device can have an interface that can be configured as hardware and/or software. In the case of a hardware design, the interfaces can be part of a so-called system ASIC, for example, which contains a wide variety of functions of the device. However, it is also possible for the interfaces to be separate integrated circuits or to consist at least partially of discrete components. In the case of a software design, the interfaces can be software modules which are present, for example, on a microcontroller alongside other software modules.

A computer program product or computer program with program code, which can be stored on a machine-readable carrier or storage medium such as a semiconductor memory, a hard disk memory or an optical memory and for carrying out, implementing and/or controlling the steps of the method according to one of the embodiments described above, is also advantageous used, especially when the program product or program is run on a computer or device.

Furthermore, a microfluidic device for capturing at least one nucleated cell of a sample liquid is presented, in particular wherein the microfluidic device can be formed as a lab-on-chip cartridge. The microfluidic device has a carrier substrate for receiving the sample liquid, the carrier substrate having at least one microcavity. Furthermore, the microfluidic device has at least one electrode arranged on or in the microcavity to generate an electric field designed to trap the nucleated cell in the microcavity.

The microfluidic device can advantageously be used in connection with rapid tests. For example, it can be in the form of a disposable cartridge. The carrier substrate can be formed, for example, as a layered substrate that includes a plurality of layers. For example, the electrode or multiple electrodes can be integrated into the carrier substrate. The electrode can be arranged on or in a cavity floor or on or in a cavity wall of the microcavity.

According to one embodiment, the carrier substrate can have a plurality of microcavities, each with at least one electrode, the microcavities being arranged in a matrix on the carrier substrate. An electric field can advantageously be generated by the electrodes in the microcavities. In this way, a number of target cells can be captured in the individual microcavities, for example one target cell per microcavity.

Furthermore, the at least one electrode can be arranged on a cavity floor and additionally or alternatively in a cavity wall of at least one of the microcavities. Advantageously, a position can thereby be determined at which the nucleated cell can be trapped in the microcavity.

According to one embodiment, the at least one electrode in each of the microcavities can be individually controllable. This means that the at least one electrode can advantageously always be activated in a targeted manner in that microcavity in which a nucleated cell is actually located. In particular, a control unit can also be provided, which is designed to impress a mutually independent voltage on each of the electrodes in the different microcavities. In this way, crosstalk from electric fields over more than one microcavity can be prevented very efficiently, or such an effect can at least be reduced. The avoidance of crosstalk allows a new desired field distribution to be set at a single microcavity of interest without changing the already prevailing previous field distributions at all other microcavities.

Furthermore, the electrode can be ring-shaped, point-shaped and additionally or alternatively layer-shaped. Each of the microcavities can advantageously have at least two electrodes which are arranged concentrically, for example. In this case, one electrode can be arranged at a point on the cavity floor and another electrode can be arranged in a ring around the electrode, also on the cavity floor. Alternatively, the at least two electrodes can be arranged in layers one above the other, ie integrated into the carrier substrate.

According to one embodiment, the microcavity can have at least one further electrode, it being possible for the electrode and the at least one further electrode to be electrically insulated from one another. Advantageously, the electrodes can be electrically insulated from one another by spatial separation or, for example, by an insulating layer.

An embodiment in which at least one counter-electrode is arranged on the microcavity is particularly advantageous, with the counter-electrode being arranged opposite the electrode and/or the at least one further electrode in or on the microcavity and/or from the electrode and/or the at least one further Electrode is electrically isolated. A very flexible control of the electrical field in the microcavity can be achieved by such an embodiment, so that individual cells in the microcavity can be separated or separated quickly and easily.

• 1 eine schematische Darstellung einer mikrofluidischen Vorrichtung gemäß einem Ausführungsbeispiel;
• 2 eine schematische Darstellung einer Leiterplatte gemäß einem Ausführungsbeispiel mit einem Trägersubstrat;
• 3 eine schematische Darstellung eines Interposerbereichs gemäß einem Ausführungsbeispiel;
• 4 eine schematische Aufbaudarstellung eines Ausführungsbeispiels eines Trägersubstrats;
• 5 eine schematische Querschnittsdarstellung eines Trägersubstrats;
• 6 eine schematische Darstellung eines Trägersubstrats;
• 7 eine schematische Querschnittsdarstellung eines Ausführungsbeispiels eines Trägersubstrats;
• 8 eine schematische Darstellung eines Ausführungsbeispiels eines Trägersubstrats;
• 9 eine schematische Darstellung eines Betriebsmodus gemäß einem Ausführungsbeispiel für eine mikrofluidische Vorrichtung;
• 10 eine schematische Darstellung eines Ausführungsbeispiels eines Betriebsmodus für eine mikrofluidische Vorrichtung;
• 11 eine schematische Darstellung eines Ausführungsbeispiels eines Betriebsmodus für eine mikrofluidische Vorrichtung;
• 12 eine schematische Darstellung eines Ausführungsbeispiels eines Betriebsmodus für eine mikrofluidische Vorrichtung;
• 13 eine schematische Darstellung eines Ausführungsbeispiels eines Betriebsmodus für eine mikrofluidische Vorrichtung;
• 14 eine schematische Diagrammdarstellung eines Spannungsverlaufs gemäß einem Ausführungsbeispiel für eine mikrofluidische Vorrichtung;
• 15 eine schematische Darstellung einer Mikrokavität mit Abmessungen gemäß einem Ausführungsbeispiel;
• 16 ein Ablaufdiagramm eines Verfahrens gemäß einem Ausführungsbeispiel zum Fangen zumindest einer kernhaltigen Zelle unter Verwendung zumindest einer Elektrode für eine mikrofluidische Vorrichtung;
• 17 ein Blockschaltbild einer Vorrichtung gemäß einem Ausführungsbeispiel;
• 18a eine schematische Darstellung eines Ausführungsbeispiels eines Trägersubstrats;
• 18b eine schematische Darstellung eines Ausführungsbeispiels eines Trägersubstrats;
• 19a eine schematische Querschnittsdarstellung eines Ausführungsbeispiels einer Detektionskammer einer mikrofluidischen Vorrichtung;
• 19b eine schematische Querschnittsdarstellung eines Ausführungsbeispiels einer Detektionskammer einer mikrofluidischen Vorrichtung;
• 20a eine schematische Querschnittsdarstellung eines Ausführungsbeispiels einer Detektionskammer einer mikrofluidischen Vorrichtung; und
• 20b eine schematische Querschnittsdarstellung eines Ausführungsbeispiels einer Detektionskammer einer mikrofluidischen Vorrichtung.

Exemplary embodiments of the approach presented here are shown in the drawings and explained in more detail in the following description. It shows:

• 1 a schematic representation of a microfluidic device according to an embodiment;
• 2 a schematic representation of a circuit board according to an embodiment with a carrier substrate;
• 3 a schematic representation of an interposer area according to an embodiment;
• 4 a schematic structural representation of an embodiment of a carrier substrate;
• 5 a schematic cross-sectional view of a carrier substrate;
• 6 a schematic representation of a carrier substrate;
• 7 a schematic cross-sectional view of an embodiment of a carrier substrate;
• 8th a schematic representation of an embodiment of a carrier substrate;
• 9 a schematic representation of an operating mode according to an embodiment for a microfluidic device;
• 10 a schematic representation of an embodiment of an operating mode for a microfluidic device;
• 11 a schematic representation of an embodiment of an operating mode for a microfluidic device;
• 12 a schematic representation of an embodiment of an operating mode for a microfluidic device;
• 13 a schematic representation of an embodiment of an operating mode for a microfluidic device;
• 14 a schematic diagram representation of a voltage curve according to an embodiment for a microfluidic device;
• 15 a schematic representation of a microcavity with dimensions according to an embodiment;
• 16 12 is a flow diagram of a method according to an embodiment for capturing at least one nucleated cell using at least one electrode for a microfluidic device;
• 17 a block diagram of a device according to an embodiment;
• 18a a schematic representation of an embodiment of a carrier substrate;
• 18b a schematic representation of an embodiment of a carrier substrate;
• 19a a schematic cross-sectional representation of an embodiment of a detection chamber of a microfluidic device;
• 19b a schematic cross-sectional representation of an embodiment of a detection chamber of a microfluidic device;
• 20a a schematic cross-sectional representation of an embodiment of a detection chamber of a microfluidic device; and
• 20b a schematic cross-sectional representation of an embodiment of a detection chamber of a microfluidic device.

In the following description of favorable exemplary embodiments of the present approach, the same or similar reference symbols are used for the elements which are shown in the various figures and have a similar effect, with a repeated description of these elements being dispensed with.

1 12 shows a schematic representation of a microfluidic device 100 according to an embodiment. The microfluidic device 100 is designed in particular as a lab-on-chip cartridge which is used, for example, in connection with rapid tests in order to examine sample liquids for tumor cells, also referred to as circulating tumor cells (CTCs), for example. The sample liquid is blood from a patient, for example. In this case, the microfluidic device 100 has a carrier substrate 105 for receiving the sample liquid. The carrier substrate 105 has at least one microcavity 110, preferably a plurality of microcavities 110. At least one electrode 115 is arranged in or on the microcavity 110 in order to generate an electric field. The at least one electrode 115 is formed in a punctiform, annular and/or layered manner, for example. Layered means that the at least one electrode 115 is formed as a layer, for example. The electric field is designed to trap a nucleated cell in the microcavity 110 . According to this exemplary embodiment, the sample liquid is illuminated using an exposure unit 120, which is part of a microscope or an evaluation device, for example.

According to this exemplary embodiment, the carrier substrate 105 is arranged on a printed circuit board 125 which, in turn, is arranged on a housing element 130 of the microfluidic device 100 . According to this exemplary embodiment, the carrier substrate 105 has a width b and a length l with the same or similar values, so that it is polygonal, in particular square, according to this exemplary embodiment.

In other words, the described approach describes an all-in-one system, the microfluidic device 100, which is designed for fully automated quantification and dielectrophoretic single cell sorting tion of nucleated cells, and in particular live circulating tumor cells, from whole blood using electrified microwells 110 .

A time-resolved and standardized quantification of CTCs allows the course of a patient's disease to be tracked in real time, which is also referred to as real-time monitoring. This makes it possible to make therapy decisions (precision medicine) or even to provide prognoses about progression-free survival that are adapted to the individual disease situation. Single cell sorting enables single cell analysis of such nucleated cells at a molecular and cellular level. It is also possible to isolate detected tumor cells from a contaminating background of healthy blood cells by means of suitable single cell sorting in a high-quality manner, i.e. quickly, efficiently and remaining in a viable state, and for subsequent molecular genetic analyses, such as expression profiles, PCR, NGS, or for example subculture, stem cell and chemosensitivity testing. In such cases, the more precise information about the cancer can be obtained. This concerns, for example, the type and heterogeneity of the mutations that take place or also possible resistance to the drugs used.

Against this background, the approach presented here has microcavities 110 on the bottom of a relatively flat and large-area detection chamber. The resulting "all-in-one system" not only enables sample pre-processing and (quasi) isolation-free quantification of living circulating tumor cells from healthy blood cells, but also dielectrophoretic single-cell sorting of detected tumor cells for subsequent single-cell analysis. The system is used in microfluidic environments and allows fully automated processing of whole blood.

Core elements of the approach presented here include sample pre-processing for the quantification of CTCs and single-cell sorting for single-cell analysis of CTCs at the molecular and cellular level. According to this exemplary embodiment, the sample pre-processing and CTC quantification takes place in electrified micro-cavities 110, which can be used for single-cell sorting. According to this exemplary embodiment, the single cell sorting is based on contactless manipulation using negative dielectrophoresis. For these purposes, individual cavities 110 are defined, for example, by holes at crossing points of covered conductor tracks, which are arranged as electrically separate and exclusively addressable rows and columns within a matrix on the carrier substrate 105. The release of a single target cell or, depending on the sorting strategy, non-target cell, which has previously been sedimented in a cavity 110 and is stably captured there within a "DEP cage", even during a rinsing process, takes place by activating the associated row and column using an appropriate electrical signal to transfer the cell from the capture level to the transport level (use of a “DEP levitator”). Non-target cells or, depending on the sorting strategy, target cells in adjacent cavities 110 along the same row and column are not affected in their catch states. Released cells are transported away microfluidically, whereby they do not come into contact with the chip or are flushed into neighboring DEP cages over the entire duration of the single cell sorting due to a repelling dielectric interaction.

Since the DEP cages required for cell manipulation do not necessarily extend over the entire chamber height, but rather are formed within microcavities 110 on the chamber floor according to one exemplary embodiment, there is a decoupling between the electrical and microfluidic components. As a result, the chamber height and thus the sample volume can be selected as large as desired with an otherwise constant base area and supply voltages or achieved DEP forces. In addition, since cells within microcavities 110, compared to those above a planar substrate 105, experience (greatly) reduced Stokes forces upon agitation of the medium in which they are suspended, the presence of DEP cages within cavities 110 leads to all the more stable capture positions, efficient and lossless washing can be carried out quickly. Overall, this supports automated pre-processing of whole blood and quantification of circulating tumor cells using microfluidics.

Since static DEP cages, ie DEP cages that are fixed in terms of their position and size, can be used for sorting the individual cells, there is no need to use integrated active circuit components if they are controlled appropriately. Such a passive chip can be manufactured comparatively easily and inexpensively. A purely fluidic removal of cells enables higher transport speeds than would be possible purely dielectrically. In addition, the transport speed is independent of the load on the chip, since released cells no longer have to be guided past other cells on the same level, but can simply fly over other cells in a straight line. Under these aspects, both a positive selection, as well as a negative selection, can be implemented equally practicably.

2 shows a schematic representation of a printed circuit board 125 according to an exemplary embodiment with a carrier substrate 105. The printed circuit board 125 shown here and/or the carrier substrate 105 arranged on the printed circuit board 125 correspond or are at least similar to that in FIG 1 circuit board 125 described and/or the carrier substrate 105 described there. The circuit board 125 is also referred to as a PCB carrier, for example, which has a carrier area 200 and an interposer area 205, for example. According to this exemplary embodiment, the carrier substrate 105 is in the form of a silicon chip, which is electrically connected, for example, to the interposer area 205 with an external control circuit, for example contacted or can be contacted via a plurality of connection interfaces 210, which is also referred to as bonding. In this case, the connection interfaces 210 are formed, for example, in order to make electrical contact with the electrode(s) arranged on or in the carrier substrate 105 . The drive circuit is also referred to below as a device. The interposer area 205 has at least one adjustment hole 215, in particular two, which are designed to suitably position and fix an interposer, for example by pressing or screwing it on.

A Si chip bonded to a carrier PCB, which is referred to here as printed circuit board 125, can be integrated microfluidically into a LoC cartridge, for example, and can be electrically contacted with an external control circuit via an interposer system.

For example, the chamber height required for a chamber with a base area of 12.5 mm×12.5 mm is at least 320 μm in order to accommodate blood lysate with a total volume of 50 μl. If necessary, higher chambers can also be reached without any problems, which then lead to larger sample volumes. For example, a chamber volume of > 156 µl is obtained for a chamber with a height > 1 mm and a base area of ~ 12.5 mm × 12.5 mm.

According to this exemplary embodiment, the carrier element 105 has a plurality of layers 220, with a structure of the carrier substrate 105 being described in more detail in one of the following figures. According to this exemplary embodiment, the carrier substrate 105 has a chamber 225 in which the sample liquid is examined. According to this exemplary embodiment, the microcavities 110 are arranged in the area of the chamber 225 . Furthermore, each of the microcavities 110 has at least one electrode 230, which can be controlled individually, for example. The microcavities 110 are optionally arranged in a matrix on the carrier substrate 105 .

3 12 shows a schematic representation of an interposer area 205 according to an embodiment. The interposer area 205 shown here corresponds or at least resembles that in 2 described interposer area 205. According to this exemplary embodiment, the interposer area 205 is shown in an exploded section 300 as an exploded view and in a cross-sectional section 302 as a cross-sectional view.

The printed circuit board 125 is formed as a base element on which the interposer 303, which has an intermediate element 305 and/or a cover element 310, for example, is arranged. According to this exemplary embodiment, the interposer 303, the intermediate element 305 and the cover element 310, and the printed circuit board 125 are pressed together.

According to this embodiment, a modular structure is shown: An, seen electrically, passive sorting chip, which is here from printed circuit board 125 and applied in the 3 composed of a non-visible carrier substrate, is contacted using the interposer 303 by pressing for the duration of the individual cell sorting with an active control circuit. Each bottom electrode and the counter-electrode on the passive chip is assigned a contact pad that can be controlled exclusively. For example, to realize 10000 DEP cages in a classic matrix, 100 + 100 + 1 = 201 contact pads are necessary. An exemplary size of a contact pad is 500 μm×500 μm. In the case of electrified microcavities that are implemented using PCB technology, the necessary contact pads are provided directly on the same printed circuit board 125. If, on the other hand, Si technology is used, an additional carrier PCB on which the necessary contact pads are applied is recommended. This means that an electrical connection between the Si chip and the carrier PCB is made using bonding wires, for example.

While the passive part is located, for example, within a lab-on-a-chip microfluidic system, which is described here as a microfluidic device, the active part is only optionally located within an associated and spatially separate evaluation unit.

4 shows a schematic structural representation of an embodiment of a carrier substrate 105. The carrier substrate 105 shown here corresponds or is similar to that in one of 1 or 2 described carrier substrate 105. According to this exemplary embodiment, the carrier substrate 105 has a plurality of layers 220, which are illustrated in six partial images a) to f). According to this exemplary embodiment, the sub-images a) to f) are to be understood as building on one another.

Partial image a) shows a base material 400 of the carrier substrate 105, such as silicon, which has a first oxide layer 405 and thus serves as a base. Part b) corresponds to part a). The only difference is that the carrier substrate 105 in partial image b) according to this exemplary embodiment also has a metal layer 410, which has a plurality of metal rods 415 or, for example, metal strips. In this case, the metal layer 410 is arranged on the oxide layer 405 in such a way that it acts as the at least one electrode for each of the microcavities to be formed. According to this exemplary embodiment, the metal rods 415 are arranged at a distance from one another, so that a gap S is arranged between them in each case.

Sub-image c) shows the carrier substrate 105 which, according to this exemplary embodiment, corresponds to the carrier substrate 105 shown in sub-image b). The only difference to this is that the carrier substrate 105 according to this exemplary embodiment additionally has a second oxide layer 420 which has a multiplicity of openings 412 along the metal rods 415 of the metal layer 410 . These openings 412 are arranged on the metal rods 415 in such a way that, when the carrier substrate 105 is in the operational state, they function as at least one electrode on the cavity floors of the microcavities. A diameter d of the openings 412 corresponds to a width w of a metal rod 415.

According to this exemplary embodiment, partial image d) shows a further development of partial image c). In addition to this, the carrier substrate 105 has a further metal layer 425 which is likewise formed from a multiplicity of further metal rods 430 . The further metal rods 430 extend transversely to the metal rods 415. In the region of the openings 412 of the second oxide layer 420, the further metal rods 430 have annular sections whose size and position depend on the size and position of the openings 412 and according to this exemplary embodiment form at least one further electrode 445. According to this exemplary embodiment, the electrode 230 and the further electrode 445 are electrically isolated from one another and, furthermore, optionally arranged concentrically. Additionally or alternatively, the electrodes 230, 445 are arranged in layers relative to one another. This means that the at least one electrode 230 is arranged on the cavity floor and/or in a cavity wall of at least one of the microcavities 110.

Partial image e) corresponds to partial image d) with the exception that, according to this exemplary embodiment, a photoresist layer 450 is additionally arranged over the second metal layer 425. Like the second oxide layer 420 before it, the photoresist layer 450 has further openings which are arranged at the position of the electrodes 230,445. However, the additional metal rods 430 are covered by the photoresist layer 450, which has electrically insulating properties, for example. According to this exemplary embodiment, the electrodes 230, 445 are arranged at a distance from one another. According to this exemplary embodiment, a resulting web has the same width w as each of the metal rods 415.

The carrier substrate 105 shown in partial image f) corresponds, for example, to the carrier substrate 105 described in partial image e). The only difference is that the carrier substrate 105 in partial image f) also has a counter-electrode 455 (which is designed here as a metal layer), which is arranged over the photoresist layer 450 and is therefore implemented as a third electrode.

In other words, a layered structure of the novel DEP chip in silicon technology is shown with exemplary dimensions. The chip is composed of a number of layers.

According to this exemplary embodiment, the microcavities 110 are implemented using thin-film technology. In this case, the carrier substrate 105 is realized, for example, as a thermally oxidized silicon wafer. The bottom electrodes 230, 445, which are formed by the metal rods 415, 430, and an oxide layer 420 electrically insulating them can be imaged lithographically, for example: this can be done either in combination with a sputtering or growth process and etching or alternatively with vapor deposition and lift -Off happened. Horizontal resolutions of ~ 1 µm are easily achieved. The layer thicknesses for metal films and oxides are, for example, a few nanometers up to a maximum of ~ 3 µm. A counter electrode 455, described here as a metal layer, can be manufactured in the same way and has the same manufacturing limitations as the bottom electrodes 230, 445 as well. The walls of cavities are produced, for example, by a thick lithographically structured photoresist, which is referred to here as photoresist layer 450 with dimensions from 2 μm up to 200 μm in depth with an aspect ratio of up to ~1:10. The shape of the base of a cavity 110 can be selected as desired, for example circular, hexagonal or square. Cavities 110 can be arranged on bottom electrodes 230, 445 within a "classical matrix" or, alternatively, packed tightly.

As an alternative to this, the microcavities 110 can be implemented using printed circuit board technology (PCB) as follows. The carrier substrate 105 consists, for example, of a stiff FR4 carrier, for example >1 mm thick base material 400. The functional layers of the carrier substrate 105 are connected to one another, for example, by means of an adhesive. The metal layers or conductor tracks have, for example, thin copper foils that are structured by photolithography and etched wet-chemically and have a thickness of 6 μm to 70 μm, for example. Conductor track widths and distances, for example, can be implemented as standard from as little as 15 µm. Insulators between the conductor tracks, for example, usually have thin polyimide films with thicknesses starting at 12 µm. Different metal layers and insulators are joined together using lamination processes. Boreholes at crossing points of the bottom electrodes 230, 445, also called “unplate blind vias”, are created, for example, by ablation with an (ultra) short pulse laser. These have, for example, diameters from approx. 15 µm and an aspect ratio of ~ 1:1. Exposed metal surfaces, especially those that come into contact with the medium, can be protected by a so-called "finishing". Standard end surfaces in PCB technology are, for example, ENIG, ENEPIG, EPIG or electroplated gold. It should also be noted here that the end surface is present on all free-standing metal surfaces in this exemplary embodiment, ie also on all electrodes 230, 445 and 455. It should be noted that the finishing must not lead to electrical short circuits between different contacts. The micro cavities 110 have a diameter of 100 μm and a depth of 66 μm, for example.

5 shows a schematic cross-sectional view of a carrier substrate 105. The carrier substrate 105 shown here corresponds or at least resembles that in one of 1 , 2 or 4 described carrier substrate 105. According to this exemplary embodiment, at least one chamber 220 is arranged in or on the carrier substrate 105 in a main section 500. In addition, the carrier substrate 105 has a secondary chamber 502 in a secondary section 504, which is smaller than the chamber 220. The two chambers 220, 502 have the same height h. The chamber 220 is separated from the secondary chamber 502 by a dividing wall 505 . The chamber 220 is designed to accommodate the sample liquid. The sample liquid has at least one cell 510 containing a nucleus, but in particular also at least one further cell 515 containing a nucleus. the nucleated cell 510 is shaped as a tumor cell and the other nucleated cell 515 as a leukocyte. In a bottom area of the chamber 220, the electrodes 230, 445 are also arranged concentrically. The carrier substrate 105 has the electrically conductive metal layer 455, which is formed, for example, as a third electrode and is arranged opposite the electrodes 230, 445. Electrodes 230, 445 and 455 are configured to create an electric field 535 in cage region 540 which acts on nucleated cell 510 like a cage. This fixes the nucleated cell 510 in position. Furthermore, an electric field 535 acts on each of the nucleated cells 510, 515. It is therefore possible to transport the nucleated cell 510 past the at least one other nucleated cell 515 without these touching one another, and thereby to achieve individual cell sorting. The nucleated cell 510 is thereby transported into the sub-chamber 502 .

Conventionally, in order to use the information content of circulating tumor cells from blood samples, CTC quantification with the necessary sample pre-processing and, if followed, CTC single-cell analysis with the necessary single-cell sorting are processes that are decoupled from one another. This usually requires several devices, such as independent platforms that are interconnected as "process stages", and manual process steps using laboratory equipment such as pipettes, reaction vessels or centrifuges.

Sample preprocessing typically includes at a minimum removal of red blood cells (RBCs) and fluorescent staining (labeling) of nucleated cells for optical detection and classification of CTCs. Depending on whether a (further) sorting of CTCs from leukocytes (white blood cells, WBCs) takes place before the CTC count or not, one speaks of isolation-related or (quasi) isolation-free methods.

Single cell sorting solutions generally compromise between sorting throughput and sensitivity. The selected strategy depends largely on the specific application. This can affect the nature of the sample in the initial state as well as the requirements of the subsequent single-cell analysis.

For example, fluorescence-based flow cytometers (FACS) are traditionally used for sorting larger amounts of cells, for example with a starting amount of more than one million cells. However, these have comparatively large dead volumes and can, depending on the size and intensity of the markings to be detected on the cells, to be relatively lossy. Furthermore, the possibility of visual control (imaging) is normally not available during the sorting process. Outside of microfluidic systems, individual target cells detected and possibly enriched in a preliminary stage, such as circulating tumor cells, can be isolated from non-target cells, for example healthy blood cells such as leukocytes, using microcapillaries ("cell pickers"). For this purpose, ultra-fine capillaries usually made of glass with, for example, an inner diameter in the range of a few 10 µm are moved to the target cell using a micromanipulator. By applying a well-defined negative pressure, the target cell is sucked away from the surrounding non-target cells and, for example, transferred to a separate vessel for further analysis.

If one is interested in the efficient sorting of small biopsies with, for example, a starting quantity of several tens of thousands of cells, which in turn corresponds to the number of leukocytes in a few microliters of blood, in a microfluidic environment, the approach of sorting biological cells on the basis of their dielectric properties to manipulate, so far as promising. The underlying mechanism, the so-called dielectrophoresis (DEP), describes the movement of even uncharged polarizable particles in a spatially non-homogeneous direct or alternating electric field. The electric field applied from the outside interacts with electric multipoles, which are induced by this very external field, and a dielectrophoretic force acts on the particle.

die zeitgemittelte dielektrophoretische Kraft 1. Ordnung (der Ausdruck ist für moderat inhomogene elektrische Felder ausreichend zur Beschreibung der Dielektrophorese) im allgemeinsten Fall für ein räumlich stationäres elektrisches Feld als $$<{\overrightarrow{F}}_{DEP}>=2\pi {\epsilon }_m\cdot Re\left({\overrightarrow{ƒ}}_{CM}\right)\cdot R^3\cdot \overrightarrow{\nabla }{\left|{\overrightarrow{E}}_{RMS}\right|}^2$$

ausdrücken. Hier stehen R für den Radius des betrachteten Partikels sowie ε ^~ _p und ε ^~ _m für die absoluten komplexen elektrischen Permittivitäten von Partikel p und umgebendem Medium m, wobei gilt $$\tilde{\epsilon }=\epsilon -j\frac{\sigma }{\omega },$$

mit ε als absoluter reeller elektrischer Permittivität $\text{j}=\sqrt{-1}$

als komplexer Einheit, σ als elektrischer Leitfähigkeit und ω als Kreisfrequenz des angelegten elektrischen Feldes. Je nach Vorzeichen des (Realteils des) Clausius-Mossotti-Faktors Re(f̃_CM) - dieses ist abhängig von der Frequenz des elektrischen Feldes und der relativen Abstimmung zwischen frequenzabhängiger absoluter reeller elektrischer Permittivität ε und elektrischer Leitfähigkeit σ zwischen Medium und Partikel (-innerem) - kann zur Manipulation entweder eine anziehende (positive Dielektrophorese bzw. pDEP mit Wirkung in Richtung Maxima der elektrischen Feldstärke, Re(f̃_CM) maximal +1) oder abstoßende (negative Dielektrophorese bzw. nDEP mit Wirkung in Richtung Minima der elektrischen Feldstärke, Re(f̃_CM) minimal -1/2) Kraftwirkung auf einzelne Partikel hervorgerufen werden.In this way, biological cells are manipulated in a contact-free manner and independently of markers. Since the effect is scalable on a large scale, it also results in good compatibility with modern MEMS technologies. For the simplest case of a spherical particle - as an example representative for a living circulating tumor cell to be isolated from the cellular blood components - it can be $${\widetilde{ƒ}}_{CM}=\frac{{\tilde{e}}_p-{\tilde{e}}_m}{{\tilde{e}}_p+2{\tilde{e}}_m}$$

the time-averaged dielectrophoretic force of the first order (the expression is sufficient to describe dielectrophoresis for moderately inhomogeneous electric fields) in the most general case for a spatially stationary electric field as $$<{\overrightarrow{f}}_{DEP}>=2\pi e_m\cdot Re\left({\overrightarrow{ƒ}}_{CM}\right)\cdot R^3\cdot \overrightarrow{\nabla }{\left|{\overrightarrow{E}}_{RMS}\right|}^2$$

to express. Here R stands for the radius of the considered particle and ε ^~ _p and ε ^~ _m for the absolute complex electrical permittivities of particle p and surrounding medium m, where applies $$\tilde{e}=e-j\frac{\sigma }{\omega },$$

with ε as absolute real electric permittivity $\text{j}=\sqrt{-1}$

as a complex unit, σ as electrical conductivity and ω as angular frequency of the applied electric field. Depending on the sign of the (real part of) the Clausius-Mossotti factor Re(f̃ _CM ) - this depends on the frequency of the electric field and the relative coordination between the frequency-dependent absolute real electric permittivity ε and the electric conductivity σ between the medium and the particle (inner ) - either an attractive (positive dielectrophoresis or pDEP with an effect in the direction of the maxima of the electric field strength, Re(f̃ _CM ) maximum +1) or a repulsive (negative dielectrophoresis or nDEP with an effect in the direction of the minima of the electric field strength, Re (f̃ _CM ) minimal -1/2) forces acting on individual particles.

If the approach of manipulating cells via pDEP is followed, it should be borne in mind that this is only achievable in a low-conductivity buffer. On the one hand, this leads to (significantly) lower overall heat losses, but on the other hand, an additional, separate deionization process is associated with this measure and the forced "artificial atmosphere" can have a negative effect on the viability and proliferation behavior of cells ("leakage of the cell interior"). .
For attractive DEP forces, stable force balances always arise sterically at the field-generating electrode surfaces or, more generally, at points of maximum electric field strength on a chip surface. Although DEP traps are much easier to produce in this way and stronger, in particular the retaining force increases for increasing voltages, this means that cells can come into contact with the chip surface and thus possibly stick together and cells through too high electric field strengths can be lysed. These are some of the key disadvantages that can cause a (strongly) reduced efficiency or purity of sorting using pDEP. If one strives to circumvent the problems just mentioned, negative DEP forces can alternatively be used to sort cells, as they are “only” achieved in physiological media such as blood plasma, PBS or cell culture medium. Admittedly, a comparatively increased conversion of active electrical power into heat can then be expected, which can only be achieved by suitable additions components, such as fans or Peltier elements, can be dissipated, but there is no need for prior deionization. Furthermore, by maintaining a natural medium, cell viability and expression profiles are maximally preserved.

• Zunächst wird die vorbereitete Zellsuspension von Interesse in eine große Hauptkammer, die hier auch als Kammer 220 und im Weiteren als „Kammer 1“ bezeichnet ist, eingegeben, die benachbarte kleine Nebenkammer 502, im Weiteren als „Kammer 2“ bezeichnet, wird mit einem sauberen Waschpuffer physiologischer Zusammensetzung befüllt. Für die Befüllvorgänge stehen in den einzelnen Kammern 220, 502 jeweils ein Inlet für die Eingabe und ein Outlet für die Entlüftung bzw. Ausgabe zur Verfügung. Beide Kammern 220, 502 sind nach Befüllung fluidisch luftfrei miteinander verbunden. die Zellen 510, 515 in Kammer 1 sind zufällig, jedoch homogen, verteilt, keine Zellen gelangen in Kammer 2. In Kammer 1 werden Zielzellen rein (di-)elektrisch eingefangen und „deterministisch“, das bedeutet entlang von individuell programmierbaren Trajektorien, an ebenso eingefangen Nicht-Zielzellen in einer festen Ebene 545 kollisionsfrei vorbei bis in Kammer 2 transportiert. Beispielsweise werden die Kammern 220, 502 und deren Wände durch ein doppelseitiges Klebeband geeigneter Höhe und geeigneten Layouts definiert, welches den Boden und die Decke abdichtend verbindet. Der Transport von Zielzellen geschieht kontaktlos in und mithilfe von dreidimensionalen „permanent geschlossenen DEP-Käfigen“. Ein solcher Käfig in der „Standardkonfiguration“ besteht beispielsweise aus 3 × 3 planaren quadratischen Silizium-Elektroden 230, 445, welche sich auf dem Kammerboden befinden (je Elektrode: ~ 18,8 µm × 18,8 µm mit 1,2 µm Abstand zu benachbarten Elektroden), sowie einer transparenten Indiumzinnoxid-Gegenelektrode (ITO) 455, welche sich ganzflächig auf der Kammerdecke erstreckt. Um ein Feldminimum inmitten der Kammer zu erzeugen, in welches Zellen per nDEP stabil platziert werden, werden die zentrale Bodenelektrode 230 und die Gegenelektrode 455 auf der Kammerdecke auf ein gleiches alternierendes Potential gesetzt („In-Phase“ bzw. „+“), während die äußeren Bodenelektroden 445 hierzu gegenphasig betrieben werden („Counter-Phase“ bzw. „-“).

In the case of repulsive DEP forces, cells are always moved to locations of minimum field strength, i.e. usually away from the substrate. This avoids contact between the cells and the chip surface and counteracts the sticking and destruction of cells due to excessive field increases. In view of this, it is quite challenging to achieve a stable balance of forces within a sufficiently strong DEP trap, the restraining force of which usually decreases with increasing tension. The strategy in this approach is to isolate target cells from non-target cells in one plane but using two separate chambers. A sorting process comprises the following two steps:

• First, the prepared cell suspension of interest is introduced into a large main chamber, also referred to herein as chamber 220, hereinafter referred to as "chamber 1", the adjacent small sub-chamber 502, hereinafter referred to as "chamber 2", is filled with a clean Filled with washing buffer of physiological composition. An inlet for the input and an outlet for the venting or output are available in the individual chambers 220, 502 for the filling processes. After filling, the two chambers 220, 502 are fluidly connected to one another in an air-free manner. the cells 510, 515 in chamber 1 are distributed randomly, but homogeneously, no cells reach chamber 2. In chamber 1, target cells are captured purely (di-)electrically and "deterministically", i.e. along individually programmable trajectories, to the same captured non-target cells in a fixed plane 545 transported past to chamber 2 without collision. For example, the chambers 220, 502 and their walls are defined by double-sided tape of appropriate height and layout sealingly connecting the floor and ceiling. Target cells are transported contactlessly in and with the help of three-dimensional "permanently closed DEP cages". Such a cage in the "standard configuration" consists, for example, of 3 × 3 planar square silicon electrodes 230, 445, which are located on the chamber floor (each electrode: ~ 18.8 µm × 18.8 µm with a distance of 1.2 µm to neighboring electrodes), as well as a transparent indium tin oxide counter electrode (ITO) 455, which extends over the entire surface of the chamber ceiling. In order to generate a field minimum in the middle of the chamber, in which cells are stably placed via nDEP, the central bottom electrode 230 and the counter electrode 455 on the chamber ceiling are set to an equal alternating potential ("in-phase" or "+") while the outer bottom electrodes 445 are operated in phase opposition ("counter-phase" or "-").

In chamber 2, target cells 510 previously “ejected” from chamber 1 are released into external reaction vessels, such as Eppendorf tubes, via a final rinsing process with clean buffer. This process therefore takes place purely microfluidically and thus "non-deterministically", which means that the nucleated cells according to this variant only follow a straight trajectory.

• Um im Rahmen von einfach realisierbaren und handhabbaren Versorgungsspannungen (U_DEP ≤5 V_RMS), die noch keine Elektrolyseeffekte induzieren, eine für die Zellmanipulation ausreichend hohe elektrische Feldstärke bzw. DEP-Kraft zu generieren, ist die Höhe der Kammer 1, und damit in Verbindung stehend das maximale Probenvolumen, welches Kammer 1 fassen kann, beschränkt. Beispielsweise führen maximal ~ 100 µm Kammerhöhe für eine Grundfläche der Kammer 1 von ~ 12,5 mm × 12,5 mm zu maximal ~ 15,6 µl Kammervolumen. Im Gegensatz zum Fall in Kammer 2, ist ein effizientes Waschen in Kammer 1 ferner nicht gewährleistet oder nicht praktikabel. Aus diesen Gründen ist eine externe Konzentration mit zusätzlicher Anreicherung von Zielzellen innerhalb eines sauberen Puffers vorgesehen. Dies wird üblicherweise durch eine Zentrifuge realisiert, wobei ein manueller Probentransfer zwischen den Geräten notwendig ist und zusätzliche Zellverluste zu befürchten sind. Die für die Aktuation notwendigen „dynamischen“, das bedeutet in ihrer Position und Größe variierbaren DEP-Käfige, wie oben beschrieben, können nur durch aktive Bauelemente, beispielsweise Transistoren oder Speicherelemente realisiert werden. Diese sind innerhalb der einzelnen Silizium-Elektroden in CMOS-Technologie integriert.

The sorting process listed here ensures reproducible efficiencies and purities of 100% each, but has "consequences" that are to be interpreted as follows:

• In order to generate an electric field strength or DEP force that is sufficiently high for cell manipulation within the framework of easily realizable and manageable supply voltages (U _DEP ≤5 V _RMS ) that do not yet induce electrolysis effects, the height of chamber 1, and thus in Relatedly, the maximum sample volume that chamber 1 can hold is limited. For example, a maximum chamber height of ~100 μm for a base area of chamber 1 of ~12.5 mm×12.5 mm results in a maximum chamber volume of ~15.6 μl. Furthermore, unlike the case in chamber 2, efficient washing in chamber 1 is not guaranteed or impractical. For these reasons, external concentration with additional enrichment of target cells within a clean buffer is intended. This is usually done using a centrifuge, which means that manual sample transfer between the devices is necessary and additional cell losses are to be feared. The "dynamic" DEP cages required for the actuation, ie DEP cages that can be varied in their position and size, as described above, can only be implemented using active components, for example transistors or memory elements. These are integrated within the individual silicon electrodes using CMOS technology.

$$

mal mehr Zellen auch lediglich mit einem Muster aus 2 × 2 Elektroden erzeugt werden kann („Rekonfiguration“), doch ist für Zielzellen in diesem Fall zunächst ein „Freiräumen eines Pfades zum Ausgang“ notwendig, was mit einem Anstieg der Sortierzeit in Verbindung steht. Eine Negativselektion ist nicht vorgesehen oder nicht praktikabel.According to this variant, a maximum transport speed for target cells in chamber 1 is limited (~1 electrode width/second) since the (lateral) DEP forces used for transport are limited. Furthermore, optionally, the maximum transport speed depends on the loading in individual cases tion of the chip. This means that although, compared to the standard configuration, a closed DEP cage for housing theoretically a total of $\frac{3\times 3}{2\times 2}=\frac{9}{4}=2.25$

$$

times more cells can also be generated with just a pattern of 2 × 2 electrodes (“reconfiguration”), but in this case target cells first need to “clear a path to the exit”, which is associated with an increase in sorting time. A negative selection is not intended or not practicable.

6 shows a schematic representation of a carrier substrate 105. The carrier substrate 105 shown here corresponds, for example, to that in FIG 5 described carrier substrate 105. Only their perspective differs. This means that the carrier substrate 105 is shown from the top view. A trajectory 600 is shown purely by way of example, along which the nucleated cell 510 is transported using electric fields and changing voltage values, first into the secondary section 504 and then out of the carrier substrate 105 into a sample vessel 605, for example.

the main section 500 and the chamber 220 are square in shape here. Furthermore, the chamber 220 is optionally connected to the sub-chamber 502 via a bottleneck-like connecting section 610 . The secondary chamber 502 is essentially formed in a straight line and has an inlet 615 and an outlet 620 opposite the inlet 615 . There is a separate inlet-outlet pair IN 615 and OUT 620 for this, whereby this inlet-outlet pair is only intended for rinsing/transport/dispensing with clear buffer. The outlet 620 is designed to let the separated nucleated cell 510 out of the carrier substrate 105 into the sample vessel 605 via the secondary chamber 502 .

7 shows a schematic cross-sectional view of an embodiment of a carrier substrate 105. The carrier substrate 105 shown here can be used, for example, for a microfluidic device, as is exemplified in 1 was described. The carrier substrate 105 is also at least similar to that in one of 1 , 2 , 4 , 5 , 6 described carrier substrate 105. According to this exemplary embodiment, the microcavities 110 are shown, which are arranged as depressions in the carrier substrate 105. According to this exemplary embodiment, the carrier substrate 105 shown here also has concentrically arranged electrodes 230, 445, which are designed to form the electric field 535. In order to be able to form the electric field 535 (“DEP cage”), the flat electrode can also very advantageously be used as a counter-electrode 455 , which here forms a third electrode on the roof of the microcavity 110 and is opposite the electrode 230 . According to this exemplary embodiment, all of the microcavities 110 are constructed in the same way, so that electric fields can be generated in each of them. This makes it possible, for example, to sort individual cells on the carrier substrate 105 . Furthermore, this enables the nucleated cell 510 to be transported out of the microfluidic device along a trajectory 600 . According to this embodiment, the outlet 620 is connected to a valve 700, which is formed as a double valve, for example. The valve 700 is shaped to direct the nucleated cell 510 into the sample vessel 605 and to drain off any unwanted liquid 705, such as lysate residues of the sample liquid.

In other words, a single cell sorting is described using electrified microcavities 110, wherein the nucleated cell 510 is isolated from non-target cells, i.e. from other nucleated cells 515 in a chamber, but using two separate levels 545, 710. A corresponding sorting process provides for a pre-process and a spatial separation, as a result of which a quantification of the cell 510 containing a nucleus is made possible.

According to this exemplary embodiment, the carrier substrate 105 has electrified microcavities 110 for sorting the individual cells. Externally, a 2-way valve, also referred to herein as valve 700, is provided. The valve 700 is designed to spatially completely separate the blood lysate 705, which is to be flushed away in the preliminary process and acts as a contamination, from the cell 510 containing a nucleus, using the following clean washing buffer during the individual cell sorting. Alternatively, it is conceivable to use two different reaction vessels for separating the blood lysate 705 without a valve 700 , which are placed sequentially at the outlet, ie at the outlet 620 .

At the bottom of a single microcavity 110 is a first punctiform electrode 230 which is surrounded by a second ring-shaped electrode 445 in an electrically insulated manner. These two bottom electrodes 230, 445 are arranged concentrically and coincide with the center of gravity of the base area of the microcavity 110. Electrodes 230, which run through cavities 110 as punctiform electrodes 230, form the columns. For this purpose, electrodes 445 rotated by 90°, which run through the cavities as ring-shaped electrodes, form the rows of a matrix in which each bottom electrode 230, 445 can be controlled separately. In order to be able to generate a three-dimensional "DEP cage with release function", a third full-surface is required Gen counter electrode, which is also referred to as metal layer 455 and forms a ridge top between adjacent wells.

According to this exemplary embodiment, intact nucleated cells 510, 515, ie leukocytes and circulating tumor cells, are distributed near the ground and randomly, but evenly, with the medium at rest ( and = 0) in the course of the sedimentation without contact into stable hovering positions in the middle of the microcavities 110 if all punctiform bottom electrodes 230 and the counter-electrode 455 are set to a sufficiently high and equal alternating potential, while all ring-shaped bottom electrodes 445 are operated with the same strength and in phase opposition. At the same time, however, the voltage levels U _DEP,min should be selected to be low enough to obtain open DEP cages.

Immediately before a wash ( and → and _max ) the voltage at the bottom electrodes 230, 445 is increased to a given maximum value U _DEP,max with an otherwise unchanged signal assignment - closed DEP cages with maximum holding power are created, in which all cells 510, 515 remain stably trapped against the prevailing flow forces, without coming into contact with the walls or the bottoms of the cavities 110. As a result, intact cells located outside of a cage can no longer enter it, while all cells 510, 515 within a cage remain stably trapped. The washing process results in a chamber that is completely free of contamination and is only optional.

In the following, the process of a positive selection, ie the isolation of a target cell 510 from non-target cells 515, is optionally presented without restricting the generality. Conversely, of course, a negative selection is also achieved analogously, in that non-target cells 515 are released from microcavities 110, but target cells 510 are not.

All cells 510, 515 sedimented to the bottom of the chamber are initially in an equilibrium position within the microcavities 110, which means at a constant height starting from the bottoms of the cavities 110. This lower capture level is referred to as level 710 for the further considerations. The level of the applied DEP voltage is still maximum, the medium inside the chamber is still in motion. In order to eject a target cell 510, 515 from a stable catch state in level 710 and to transfer it to an upper transport level, which is referred to here as level 545, above a web top, the bottom electrodes 230, 445 crossed at the location of the target cell 510 are provided with suitable electrical controlled by signals. For this purpose, an enabling voltage U _F,S is applied to the associated column and an enabling voltage U _F,Z is applied to the associated row. The release stresses depend on the geometry. For 0 ≤ U _F,S ≤ U _F,Z ≤ U _DEP,max the release voltages are to be selected as follows in order to ensure a purely (di-)electrical release of an individual cell from its floating position "deterministically", but without neighboring cells affecting cells in their capture positions. All of the signals used are, for example, harmonic excitation with a constant frequency.

In net terms, a cell release requires a sufficiently large repulsive DEP force in the negative z-direction along the central axis of symmetry through the microcavity 110 (“DEP levitator”). For this purpose, for a given geometry of the microcavity 110, a suitable combination of U _F,S and U _F,Z ensures that the lines of the electric field strength from the counter-electrode 455 are all as parallel as possible to the exposed surface of the punctiform bottom electrode 230 (column). drop along a z-axis and there is still a sufficiently large field strength. Disturbances of this field progression by the ring-shaped bottom electrode 445 (line) in the edge region of the microcavity 110 near a wall must be suppressed as far as possible.

At the same time, U _F,Z should be chosen so small that a burglary of the DEP cages of non-target cells 515 along the release line, i.e. to the left and right of the cavity 110 of the target cell 510, is avoided and closed DEP cages with sufficient holding power exist stay. On the other hand, U _F,S should be chosen so large that in a temporary new state of equilibrium within lowered closed DEP cages there is no contact between non-target cells 515 and the bottoms of the associated cavities 110 along the release gap, i.e. above and below the cavity 110 of the target cell 510.

If released cells have previously found protection in the level 710 within the cavities 110 and only experience reduced Stokes forces during washing, the situation changes in the level 545. Here the (removal) transport to the outlet 620 and the output with takes place a clean (washing) buffer purely microfluidically, i.e. non-deterministically, through maximum Stokes forces and thus relatively quickly. Released cells cannot enter adjacent DEP cages as they are closed. Overall, the sorting principle allows for maximum efficiency and purity.

8th shows a schematic representation of an exemplary embodiment of a carrier substrate 105. The carrier substrate 105 shown here corresponds or is similar, for example, to that in one of FIG 1 , 2 or 4 until 7 described carrier substrate 105. According to this exemplary embodiment, the only difference is the representation perspective. This means that the carrier substrate 105 shown here is shown from above. The carrier substrate 105 also has a plurality of microcavities 110 here. Here, too, the microcavities 110 are arranged in a matrix, ie in rows and columns. A trajectory 600 for the at least one nucleated cell 510 is also shown here.

9 FIG. 9 shows a schematic representation of an embodiment of an operating mode 900 for a microfluidic device. At least the carrier substrate 105 shown here is similar to that in one of 1 , 2 or 4 until 8th described carrier substrate 105. According to this exemplary embodiment, a state in which the electrodes of the microfluidic device are activated and form the electric field 535 is referred to as the operating mode 900. According to this exemplary embodiment, the operating mode 900 is illustrated using a cross-sectional representation of a microcavity 110 of the carrier substrate 105 with electrodes 230, 445 and 455 arranged in layers. The electrodes 230 , 445 and 455 are arranged on or in each individual microcavity 110 . They are arranged in such a way that when the electric field 535 is activated, the cell 510 containing a nucleus reaches a center of the microcavity 110 . That means an electric cage is open.

In other words, according to this exemplary embodiment, an operating mode 900 of single cell sorting using electrified microcavities 110 in PCB technology is shown and described, for example a positive selection, which describes isolation of a target cell 510 from non-target cells 515 .

10 FIG. 9 shows a schematic representation of an embodiment of an operating mode 900 for a microfluidic device. The microcavity 110 shown here is similar to that in FIG 9 described microcavity 110. Only the electric field 535 is shown differently according to this embodiment. According to this embodiment, the electrical cage is shown closed such that the nucleated cell 510 is trapped in position centrally within the microcavity 110 . A liquid around the nucleated cell 510 can be rinsed out, for example, without washing the nucleated cell 510 out of the microcavity 110 (=washing process). The same applies to the nucleated non-target cells 515.

11 FIG. 9 shows a schematic representation of an embodiment of an operating mode 900 for a microfluidic device. The microcavity 110 shown here is similar to that in FIG 10 described microcavity 110. Only the electric field 535 is shown differently according to this embodiment. According to this exemplary embodiment, the electrodes 230, 445 and 455 are controlled in such a way that the nucleated cell 510, which is also referred to as the target cell, can be electrically pressed out of the microcavity 110 and then microfluidically rinsed out above the microcavity by rinsing. This is achieved, for example, by changing an electrical voltage U that is present at the electrodes.

12 FIG. 9 shows a schematic representation of an embodiment of an operating mode 900 for a microfluidic device. The microcavity 110 shown here is similar to that in FIG 11 described microcavity 110. Only the electric field 535 is shown differently according to this embodiment. According to this exemplary embodiment, the electrodes 230 , 445 and 455 are driven in such a way that a further nucleated cell 515 , which is also referred to as a non-target cell, is held in the microcavity 110 . By changing the voltage, for example, the further nucleated cell 515 is released or alternatively held in its position in the microcavity 110 . This is achieved, for example, by changing an electrical voltage U which is present at the electrodes 230, 445 and 455. The voltage U required for this optionally deviates from the voltage U required for the nucleated cell 510 . This makes it possible, for example, to transport the nucleated cell 510 past the other nucleated cell 515 .

13 FIG. 9 shows a schematic representation of an embodiment of an operating mode 900 for a microfluidic device. The microcavity 110 shown here is similar to that in FIG 12 described microcavity 110. Only the electric field 535 is shown differently according to this embodiment. According to this exemplary embodiment, the electrodes 230 , 445 and 455 are driven in such a way that a further nucleated cell 515 , which is also referred to as a non-target cell, is held in the microcavity 110 . The voltages should be selected in such a way that contact between the non-target cell and the bottom of the cavity is just avoided (contactless single cell sorting)."

14 14 shows a schematic diagram representation of a voltage course 1400 according to FIG an embodiment for a microfluidic device. The voltage curve 1400 shown here shows a behavior of a voltage U applied in the microcavity over a time t. The voltage denoted as voltage U _DEP according to this exemplary embodiment represents, for example, predetermined reference values which, for example, represent a minimum threshold value and a maximum threshold value. According to this exemplary embodiment, a plurality of differently applied voltage curves is shown, which correspond, for example, to the operating modes as shown in FIGS 9 until 13 have been described. This means that by adjusting and/or changing the voltage U in the individual microcavities and/or at the individual electrodes, the electric field is changed in such a way as it is in the 9 until 13 are shown.

According to this exemplary embodiment, a first curve 1405 and a second curve 1410 represent the 9 described operating mode in which the electric cage is open. The first curve 1405 indicates that the voltage U is applied to the electrodes 230 and 455. The second curve 1410 makes it clear that the voltage U is applied to the further electrode 445 . U _DEP,min denotes the mean value over time of curves 1405 and 1410.

A third curve 1415 and a fourth curve 1420 illustrate the example in 10 described operating mode. It becomes clear that the third curve 1415 is implemented as a voltage variant of the first curve 1405, which is also applied to the electrodes 230 and 455. Similarly, the fourth curve 1420 shows the voltage profile of the further electrode 445 as a voltage variant of the second curve 1410.

Fifth and sixth curves 1425 and 1430 continue to represent those in FIGS 11 until 13 operating modes shown. UF _,Z is the mean value over time of curve 1425, and UF,S is the mean value over time of curve 1430.

freigegeben werden.In other words, a target cell can be expressed using, for example $$0\le \frac{u_{f,S}}{u_{DEP,max}}=0.3\le \frac{u_{f,S}}{u_{DEP,max}}=0.6\le 1$$

be released.

15 11 shows a schematic representation of a microcavity 110 with dimensions according to an embodiment. The microcavity 110 corresponds or at least resembles that in one of FIG 1 , 2 or 4 until 13 described microcavity 110. According to this embodiment, the electrodes 230, 445 and 455 are further arranged concentrically. In the 15 The dimensions shown are only to be understood as examples and may differ in alternative exemplary embodiments.

In other words, according to this exemplary embodiment, a side view of an electrified microcavity 110 using silicon technology is shown with exemplary dimensions. In 15 a size comparison with a cell 510 located within the cavity 110 with a maximum (large dashed circle) and a minimum (small circle) diameter to be expected is also shown.

durch elektrifizierte Mikrokavitäten 110 dielektrophoretisch manipuliert wird. Ausreichend große negative DEP-Kräfte (Re(f _CM) ≈-0,23) in physiologischen Medien lassen sich, ohne Elektrolyse-Effekte und mit minimalen Transmembranspannungen, bei Betriebsfrequenzen zwischen ca. 1 MHz bis 10 MHz erzeugen. Der wasserähnliche Waschpuffer soll eine Dichte ρ_f =1000 kg/m³ und eine Viskosität η=1 mPa·s besitzen.According to this exemplary embodiment, the cavity 110 has, for example, a diameter of 50 μm and a depth of approximately 37.5 μm. The numerical examples given below relate to a trajectory of the lightest and smallest expected spherical CTC (ρ _p =1070 kg/m ³ and R =3 µm), which occurs in a physiological medium $\left({\mathrm{\sigma }}_{\text{m}}=1.4\frac{\text{S}}{\text{m}}\right)$

is dielectrophoretically manipulated by electrified microcavities 110. Sufficiently large negative DEP forces (Re( f _CM ) ≈-0.23) in physiological media can be generated at operating frequencies between approx. 1 MHz and 10 MHz without electrolysis effects and with minimal transmembrane voltages. The water-like wash buffer should have a density ρ _f =1000 kg/m ³ and a viscosity η=1 mPa·s.

The first part of the process manages with comparatively small volumes of whole blood (≤ 20 µl) as "input". The "output" represents an ideal starting point for the second part of the process, a single cell sorting by DEP. In the final optical detection, CTCs that need to be isolated are to be expected among fewer than 80,000 to 220,000 leukocytes in less than 100 µl blood lysate, which means that such a cell suspension can be interpreted as a relatively small biopsy. Loaded: An open DEP cage results, for example, for an operating voltage U _DEP = U _{DEP, MIN} = 1.5 VRMS.

If the 50 µl chamber volume is exchanged 30 times within 5 minutes to ensure sufficient optical transparency and cleanliness of the chamber, i.e. rinsed with a washing buffer, the result is a volume flow of 5 µl/s to be set constantly on average by the microfluidic system. For a chamber with the front entry and exit area of 12.5 mm × 320 µm, an average flow rate of and = and _max =1250 µm/s connected. Closed DEP cages, which at an operating voltage U _DEP = U _DEP,max = 5 VRMS ent have sufficient holding power to withstand the flow forces or turbulence induced within the cavities 110 by the wash.

16 16 shows a flowchart of a method 1600 for capturing at least one nucleated cell using at least one electrode for a microfluidic device, according to an embodiment. This can be a method 1600 that can be used in one of the devices of the microfluidic device described with reference to the previous figures. The method 1600 includes a step 1605 of outputting and a step 1610 of providing. In step 1605 of outputting, an application signal is output which causes a sample liquid with the at least one nucleated cell to be applied to a carrier substrate of the microfluidic device. In step 1610 of providing, a current signal is provided to an interface to the at least one electrode in order to generate an electric field on or in a microcavity of the carrier substrate, which is designed to capture the at least one nucleated cell as a target cell in the microcavity. According to this exemplary embodiment, the application signal is output in step 1605 of output, which causes a lysate to be applied to the carrier substrate in order to obtain a cell sediment with the at least one nucleated cell and a cell suspension of a lysate. This means, for example, that a certain period of time is allowed for the cell sediment to settle. Also optionally, in or after step 1610 of providing, a release signal is provided to the electrode after capturing the nucleated cell in order to release another nucleated cell from the sample liquid as a non-target cell from the electric field. The release can also be an optional new step 1630.

According to this exemplary embodiment, the method 1600 also includes a step 1615 of changing a current strength and/or a voltage after the step 1605 of outputting, before or after the step 1610 of providing to change the electric field, for example to strengthen or weaken it. In an optional step 1620 of identification after step 1610 of providing, the cells containing a nucleus are identified from the sample liquid and, in particular, optically detected and/or quantified from the cell sediment.

Further optionally, the method 1600 includes a step 1625 of washing the sample liquid. In this case, the sample liquid is washed using a washing buffer after the step of providing 1610 in order to wash out a suspension of the sample liquid from the microcavity.

The method steps presented here can be repeated and carried out in a different order than the one described.

17 17 shows a block diagram of a device 1700 according to an embodiment. Device 1700 is implemented, for example, as a control device or as a control unit that is designed to control or carry out a method for capturing at least one nucleated cell using at least one electrode for a microfluidic device, as described in 16 was described. To this end, device 1700 has an output unit 1705 for outputting an application signal 1710, which causes a sample liquid with the at least one nucleated cell to be applied to a carrier substrate of the microfluidic device, and a provision unit 1715 for providing a current signal 1720 at an interface to the at least one electrode on, in order to generate an electric field on or in a microcavity of the carrier substrate, which is designed to capture the at least one nucleated cell as a target cell in the microcavity. Furthermore, the provision unit 1715 is designed to only optionally provide a release signal 1725 after the nucleated cell has been caught on the electrode, in order to release another nucleated cell from the sample liquid as a non-target cell from the electric field. The device 1700 also has a changing unit 1730, which causes a change in current intensity in order to strengthen, weaken and/or change the electric field. According to this exemplary embodiment, the device 1700 also has a washing unit 1735 which is designed to wash the sample liquid using a washing buffer in order to wash out a suspension of the sample liquid from the microcavity. Furthermore optionally, the device 1700 has an identification unit 1740, which is designed to identify the nucleated cells from the sample liquid, in particular to optically detect and/or quantify the nucleated cells from the cell sediment.

18a shows a schematic representation of an embodiment of a carrier substrate 105. The carrier substrate 105 shown here corresponds or is at least similar to that in FIG 2 described carrier substrate 105, which is arranged or can be arranged in a microfluidic device 100, for example. Such a microfluidic device 100 has already been used in at least one of the 1 until 8th described. According to this embodiment, the microcavities 110 arranged in a grid or as a passive matrix, i.e. in rows and columns. The microcavities 110 are furthermore electrically coupled to a plurality of switching units 1800, that is to say they have corresponding electrical contacts, for example. More precisely, all microcavities 110 are coupled to a switch in rows and columns. Furthermore, this means that the microcavities 110 can also be controlled in rows and/or columns.

According to this exemplary embodiment, a microcavity 110 arranged centrally in the carrier substrate 105 is illustrated as the microcavity 110 of interest. The microcavities 110 directly connected to it are designed as critical adjacent microcavities 110 in which a voltage drop is symbolically represented by a lightning bolt 1805 in each case.
The flash 1805 represents parasitic electrical crosstalk and means that a reversal of the field distribution of the central cavity 110 of interest undesirably leads to an undefined reversal of states in the neighboring microcavities 110 .

In other words, the microcavities 110 are based on bottom electrodes with a quasi-planar layered structure, which are arranged by way of example in the form of separately controllable and mutually insulated columns with punctiform electrodes as the first, lowest metal layer and rows with ring-shaped electrodes as the second, middle metal layer. However, since a passive matrix is represented according to this exemplary embodiment, which is characterized by a simple crossing of conductor tracks, a voltage drop cannot only be applied to or read from a microcavity 110 of interest. On the other hand, in order to achieve a state of selective addressing, the passive matrix is expanded to form an active matrix with suitable integrated transistor circuits both at the crossing points of conductor tracks and at the corresponding columns and rows, as is shown in 18b is described and/or illustrated.

18b shows a schematic representation of an embodiment of a carrier substrate 105. The carrier substrate 105 shown here is similar to that in FIG 18a described carrier substrate 105, wherein the carrier substrate 105 shown here is designed according to this embodiment as an active matrix. This means that although the individual microcavities 110 are also arranged in rows and columns, they are individually coupled or can be coupled to a switch in each case. According to this exemplary embodiment, however, this is shown in simplified form for the sake of clarity. It follows from this that the microcavities 110 can each be controlled individually with their own switching unit and by means of their own control signal 1850 and electrical crosstalk and thus influencing of neighboring microcavities 110 is prevented.

In other words, the microfluidic device 100 is expanded to include active circuit elements that allow an “ideal” selective addressing of individual microcavities 110 of interest, so that the carrier substrate 105 is formed as the active matrix. Microcavities 110 of interest are, for example, those microcavities 110 in which a DEP manipulation was detected and/or an electrochemical detection took place. The active circuit elements also allow electrodes within a microcavity 110 to be used not only as manipulation elements for dielectrophoretic movement of cells, but also as sensor elements for electrochemical detection of the particles released by cells in solution. Thus, the possibilities of an on-chip analysis after a sealing of microcavities 110, as for example in the 20a until 20b is further extended to monitor, for example, environmental conditions for a cell 510 in a microcavity 110.

The circuit elements of a quasi-planar active matrix could be implemented in the form of semiconductor integrated circuit technology. A complementary metal oxide semiconductor technology (CMOS) can be used for this purpose, for example, which is manufactured monolithically in silicon. Alternatively, it is conceivable to also use other technologies, such as bipolar technology and semiconductor materials, such as gallium arsenide. The nonlinear components of the circuit required for selective addressing of individual microcavities 110, that is to say, for example, transistors, memory elements, diodes, etc., can typically be produced in a standard manner in each of these technologies. If an active matrix is implemented using semiconductor circuit technology, the full-surface counter-electrode described, which is also described as the third electrode, can be built up as the third, uppermost metal layer, for example with the aid of photoresist systems. For this purpose, SU-8 photoresist is only used as an example, which is mixed with metal particles, such as silver, in a first thick layer, for example >30 μm, unmodified to define the microcavities 110 and then in a second thin layer, for example <1 μm spun on to define the full-surface counter-electrode. After a so-called soft bake, exposure, development and hard bake of both layers, the counter-electrode is optionally coated with a chemically inert metal, such as gold, to increase the electrical conductivity. galvanized. The electrical contacting of a semiconductor chip is implemented, for example, by connecting bonding wires to a carrier printed circuit board, to which a chip has previously been glued.

If electrodes within a microcavity 110 are also to be used as sensor elements for the electrochemical detection of the particles released by the cells 510 in solution, they may be functionalized beforehand with suitable counterparticles to increase the sensitivity. The counter-particles have the task, for example, of effectively binding the particles to be detected or of intensifying the electrochemical reaction between the particle and the electrode. Sensor elements could be beneficial for numerous applications. For example, antibodies of interest (=particles) secreted by B lymphocytes could be identified in this way for drug production after the electrodes have been functionalized with the corresponding antigen (=counterparticle). Another example is cell cultivation for cell line development, in which the growth conditions, such as pH value, O ₂ , CO ₂ content and/or glucose concentration, are monitored and controlled precisely and in real time using electrodes as sensor elements.

19a 12 shows a schematic cross-sectional illustration of an exemplary embodiment of a microfluidic device 100. According to this exemplary embodiment, a cell 510 containing a nucleus is arranged in one of the microcavities 110 in each case. In this case, the lysate is arranged as an aqueous medium around the cells 510 and in an intermediate region 1900 between the carrier substrate 105 and a transparent cover 1905 . The intermediate area 1900 can also be designated, for example, as the detection area of the microfluidic device 100 . According to this exemplary embodiment, each of the microcavities 110 has a first electrode 1910 and a second electrode 1915 . The electrodes 1910, 1915 can also be referred to as bottom electrodes and are shaped in order to generate an electric field and/or a magnetic field in each case, for example, in an operating state. The fields that are generated keep the nucleated cells 510 in the respective microcavities 110 while the lysate is washed, for example. The first electrode 1910 is realized centrally and in a point-like manner on a cavity floor 1920 and the second electrode 1915 is arranged, for example, in the manner of a ring around the first electrode 1910. The electrodes 1910, 1915 do not touch each other. According to this exemplary embodiment, the first electrode 1910 is in the form of a positive pole and the second electrode 1915 is in the form of a negative pole.

According to this exemplary embodiment, the carrier substrate 105 also has a third electrode 1925 which, according to this exemplary embodiment, is arranged flat on a substrate surface. The third electrode 1925 is also designed as a positive pole and follows the in 14 illustrated voltage curve over time.

In other words, the microcavities 110 are shown as electrified microcavities 110 each having a charged nucleated cell 510 .

19b shows a schematic cross-sectional view of an embodiment of a microfluidic device 100. The microfluidic device 100 shown here is similar to that in FIG 19a described microfluidic device 100. According to this embodiment, only particles 1950 of each of the nucleated cells 510 are dome-like arranged around the corresponding cell 510 that they each affect an adjacent microcavity 110. This means that an electrical and/or microfluidic crosstalk is shown here merely as an example.

20a shows a schematic cross-sectional view of an embodiment of a microfluidic device 100. The microfluidic device 100 shown here is similar to that in FIG 19a described microfluidic device 100. Here, too, a nucleated cell 510 is arranged in each of the microcavities 110 and is held in position by electrodes 1910, 1915, 1925. According to this exemplary embodiment, however, the microfluidic device 100 is subjected to a non-aqueous medium 2000 or a non-aqueous phase, which comprises, for example, a silicone oil or air, in order to prevent microfluidic crosstalk of mutually influencing or adjacent microcavities 110, as is the case, for example, in 19b was described, to eliminate and to seal the microcavities 110, for example. That is, the microcavities 110 are sealed by a choice of non-aqueous medium 2000 and physical properties associated therewith.

According to this exemplary embodiment, the chip, ie the microfluidic device 100, is flushed with at least one medium 2000 non-aqueous phase during the on-chip single-cell analysis. This achieves sealing and thus isolation of cells 510 in the aqueous phase, which is described here as an aqueous medium with physiological properties, in microcavities 110 .

In other words, the lysate, i.e. the layer of aqueous phase, above the cavities 110 after the cells 510 have been loaded is first displaced by a medium of non-aqueous phase 2000 in order to seal the cells 510 in the aqueous phase within microcavities 110 for single-cell analyzes on to reach chip. If individual cells 510 are also to be output into collection vessels for off-chip analyses, the previously input non-aqueous phase 2000 is again displaced with an aqueous phase and the situation is thus brought to the initial state.

20b shows a schematic cross-sectional view of an embodiment of a detection chamber of a microfluidic device 100. The detection chamber shown here is similar to that in FIG 20a described detection chamber. According to this exemplary embodiment, the intermediate area 1900 is only completely filled with the nonaqueous medium 2000 with the exception of the microcavities 110, so that the microcavities 110 are isolated from one another. In other words, aqueous medium is only present in the microcavities 110 themselves.

In other words, elimination of the parasitic microfluidic crosstalk is shown. The electrified microcavities 110 with nucleated cells 510 in the aqueous phase are completely isolated from each other by the non-aqueous medium 2000 . This means that a diffusive and convective carryover of the particles released by the cells 510 in solution is no longer possible and analyzes in individual microcavities 110 take place independently of one another.

If an embodiment includes an "and/or" link between a first feature and a second feature, this should be read in such a way that the embodiment according to one embodiment includes both the first feature and the second feature and according to a further embodiment either only that having the first feature or only the second feature.

Claims (16)

A method (1600) for capturing at least one nucleated cell (510) using at least one electrode (230) for a microfluidic device (100), the method (1600) comprising the following steps: - Outputting (1605) an application signal (1710) which causes application of a sample liquid with the at least one nucleated cell (510) to a carrier substrate (105) of the microfluidic device (100); and - Providing (1610) a current signal (1720) to an interface to the at least one electrode (230) in order to generate an electric field (535) on or in a microcavity (110) of the carrier substrate (105), which is designed to capturing the at least one nucleated cell (510) as a target cell in the microcavity (110). Method (1600) according to claim 1 , having a step (1615) of changing a current strength in order to change the electric field (535), in particular to strengthen or weaken it after the step (1605) of outputting or before or after the step (1610) of providing, in particular wherein the electric field (535) between the electrode (230) and a counter-electrode (455) lying opposite the electrode (230) in or on the microcavity (110) is built up and/or varied. Method (1600) according to one of the preceding claims, wherein in the step (1610) of providing the current signal (1720) at the interface to the at least one electrode (230) and to at least one other electrode arranged in an adjacent microcavity (110) output is generated, so that a different electric field is generated on the at least one other electrode than on the electrode (230), in particular wherein the field generated on the electric electrode differs in terms of direction and/or intensity from the electric field that generated at the other electrode and/or wherein the other electric field is generated at the other electrode located in a microcavity (110) located in a common column or row with respect to the microcavity (110) with the electrode (230) is arranged. Method (1600) according to any one of the preceding claims, having a step (1625) of washing the sample liquid using a washing buffer after the step (1610) of providing to wash out a suspension of the sample liquid from the microcavity (110), and/or wherein in or after the step (1610) of providing a release signal (1725) after capturing the nucleated cell (510) is provided to the electrode to remove another nucleated cell (515) from the sample liquid as a non-target cell from the electric field ( 535) to release. Method (1600) according to one of the preceding claims, wherein in the step (1605) of outputting the application signal (1710) is output, which causes a lysate to be applied to the carrier substrate (105) in order to form a cell sediment with the at least one nucleated cell (510 ) and to obtain a cell suspension of a lysate and/or wherein a step (1620) of identifying the nucleated cells (510) from the sample liquid provided after the step (1610) of providing, in particular wherein in the step (1620) of identifying the nucleated cells (510) are optically detected and/or quantified from a cell sediment. Method (1600) according to one of the preceding claims, wherein in the step (1610) of providing the nucleated cell (510) or at least one further nucleated cell (515) are trapped in a capture plane of the microcavity (110) and/or wherein by the release signal (1725) the cell (510) or at least one further cell (515) containing a nucleus is released from the sample liquid from the electric field (535) into a transport plane. A method for capturing at least one nucleated cell (510) using at least one electrode (230) for a microfluidic device (100), the method (1600) comprising the following steps: - Applying a sample liquid with the at least one nucleated cell (510) on a carrier substrate (105) of the microfluidic device (100); and - Generating an electric field (535) on or in a microcavity (110) of the carrier substrate (105) with the at least one electrode (230), which is designed to contain the at least one nucleated cell (510) as a target cell in the microcavity (110 ) catch. Apparatus (1700) set up to carry out the steps (1605, 1610, 1615, 1620, 1625, 1630) of the method (1600) according to any one of the preceding claims in corresponding units (1705, 1715, 1730, 1735, 1740). and/or to control. Computer program set up to carry out the steps (1605, 1610, 1615, 1620, 1625, 1630) of the method (1600) according to one of Claims 1 until 7 execute and/or control. Machine-readable storage medium on which the computer program claim 9 is saved. Microfluidic device (100) for capturing at least one nucleated cell (510) of a sample liquid, in particular wherein the microfluidic device (100) is formed as a lab-on-chip cartridge, wherein the microfluidic device (100) has the following features: - A carrier substrate (105) for receiving the sample liquid, wherein the carrier substrate (105) has at least one microcavity (110); and - at least one electrode (230) arranged on or in the microcavity (110) to generate an electric field (535) adapted to trap the nucleated cell (510) in the microcavity (110). Microfluidic device (100) according to claim 11 , wherein the carrier substrate (105) has a plurality of microcavities (110) each having at least one electrode (230), wherein the microcavities (110) are arranged in a matrix on the carrier substrate (105). Microfluidic device (100) according to claim 12 , wherein the at least one electrode (230) is arranged on a cavity floor and/or in a cavity wall of at least one of the microcavities (110). Microfluidic device (100) according to any one of Claims 12 until 13 , wherein the electrodes (230) in each of the microcavities (110) can be controlled individually and/or wherein the electrode (230) is ring-shaped, point-shaped and/or layer-shaped, in particular wherein a control unit is also provided, which is designed to of the electrodes in the different microcavities (110) to impress a mutually independent voltage. Microfluidic device (100) according to any one of Claims 11 until 14 , The microcavity (110) having at least one further electrode (445), the electrode (230) and the at least one further electrode (445) being electrically insulated from one another. Microfluidic device (100) according to any one of Claims 11 until 15 , wherein at least one counter-electrode (455) is arranged on the microcavity (110), wherein the counter-electrode (455) is arranged opposite the electrode (230) and/or the at least one further electrode (445) in or on the microcavity (110) and /or is electrically insulated from the electrode (230) and/or the at least one further electrode (445).

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