CN117500600A

CN117500600A - Method and apparatus for capturing at least one nucleated cell using at least one electrode for a microfluidic device

Info

Publication number: CN117500600A
Application number: CN202280043662.XA
Authority: CN
Inventors: T·布克; S·卡迪克; F·拉尔默; M·德雷施曼; J·霍夫曼; A·塞洛特
Original assignee: Robert Bosch GmbH
Current assignee: Robert Bosch GmbH
Priority date: 2021-04-20
Filing date: 2022-04-20
Publication date: 2024-02-02

Abstract

The aspects presented herein relate to a method of capturing at least one nucleated cell using at least one electrode of a microfluidic device (100). To this end, the method comprises an output step of applying a signal, the output step causing a sample liquid with at least one nucleated cell to be applied to a carrier substrate (105) of the microfluidic device (100); and a providing step comprising providing a current signal to the interface of the at least one electrode so as to generate an electric field at or in the microcavity (110) of the carrier substrate (105), the electric field being formed for capturing the at least one nucleated cell as a target cell in the microcavity (110).

Description

Method and apparatus for capturing at least one nucleated cell using at least one electrode for a microfluidic device

Technical Field

The present solution relates to a method and a device for capturing at least one nucleated cell using at least one electrode for a microfluidic device, and to a microfluidic device according to the generic class of independent claims. The subject matter of the present solution also relates to a computer program.

Background

In recent years, circulating Tumor Cells (CTC) have become promising and clinically relevant biomarkers in the treatment of advanced malignancies. The identification and characterization of suitable body fluids, such as blood or lymph, in their presence is known as fluid biopsy, which is one of the focus of modern oncology.

Disclosure of Invention

Against this background, the solution presented here provides an improved method for capturing at least one nucleated cell using at least one electrode of a microfluidic device, furthermore an improved apparatus using the method, and finally a corresponding computer program and an improved microfluidic device according to the main claims. The measures set forth in the dependent claims enable advantageous modifications and improvements of the device specified in the independent claims.

By means of the solution presented here, a low-cost microfluidic device, or an efficient and automated microfluidic analysis, can be achieved with the use of disposable cartridges.

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

The method can be performed, for example, in a microfluidic device as a lab-on-a-chip cartridge. The method can advantageously be performed automatically. Cells having a nucleus containing genetic material are referred to as nucleated cells. The nucleated cells may be, for example, tumor cells or, for example, white blood cells. Accordingly, the sample liquid may be, for example, a blood sample of a patient. The carrier substrate may for example be PCB-based, i.e. arranged on a circuit board, and/or contain for example a silicon material. The microcavities can be arranged on or in a carrier substrate, for example in a honeycomb-like, circular or, for example, angular manner. The microcavities can, for example, be shaped to be able to hold and capture a sample liquid with at least one nucleated cell, so that it can then be advantageously analyzed.

According to an embodiment, the method can comprise a step of varying the current intensity before or after the outputting step, in order to vary, in particular to increase or decrease, the electric field. The electric field is established and/or varied, in particular, between the electrode and a counter electrode opposite the electrode in or on the microcavity. The electric field can advantageously produce dielectrophoresis cages which can advantageously be switched on, off or switched off depending on the current intensity. Here, the "DEP cage" (open, closed, and shut) is one of two possible aspects and is very helpful in capturing cells. In order to be able to sort cells according to their location in the microcavities, another aspect of the approach presented herein can be used. Upon manipulation of the electrodes in the microcavities, a "DEP suspension" can also be generated by which cells can be selectively electrically pushed out of the cavities. The DEP suspension has a different electric field strength distribution than the DEP cage.

Furthermore, an embodiment of the solution presented here is advantageous in that, in the providing step, the current signal is output to an interface of at least one electrode and of at least one further electrode arranged in an adjacent microcavity such that an electric field different from that at the electrode is generated at the at least one further electrode, in particular in that the field generated at the electrode differs in direction and/or strength from the electric field generated at the further electrode, and/or in that a further electric field is generated at a further electrode arranged in the microcavity, the further electrode and the electrode being arranged in a common column or a common row with respect to the microcavity. This embodiment of the solution presented here provides the following advantages: cross-talk of electrical signals into an adjacent second microcavity due to an electric field on the electrode of the first microcavity is prevented or at least attenuated. In this way, it is possible to efficiently prevent very similar or identical potentials from occurring on the electrodes of the microcavities arranged in a common row or a common column of the matrix-like structure, for example, and thus it is possible to realize individually formed cages for each microcavity. In this way, a better separation or better enclosure of the cells trapped in the respective microcavities can be achieved.

The method can include the step of washing the sample liquid with a wash buffer after the providing step, so as to wash a suspension of the sample liquid from the microcavities of the microfluidic chamber and the volume above it. Advantageously, the wash buffer can be implemented as a fluid that cleans the suspension but does not damage nucleated cells. The transparency of the sample liquid can advantageously be improved by the washing step, so that for example a subsequent analysis can be performed more easily. The washing step should be gentle enough to avoid washing captured nucleated cells away from their capture locations.

According to one embodiment, in or after the providing step, a release signal can be provided to the electrode after capturing the nucleated cells in order to release the further nucleated cells in the sample liquid as non-target cells from the electric field. Advantageously, the release signal can trigger a release voltage that causes the electric field to be turned on. Thereby advantageously separating the nucleated cells from the further nucleated cells.

According to one embodiment, in the outputting step an application signal can be output, which application signal causes the lysate to be applied to the carrier substrate, so as to obtain a cell suspension having at least one cell pellet of nucleated cells and the lysate. Advantageously, the application signal can be output to an interface of the pump device, such that the pump device can pump the lysate through the microfluidic device.

Furthermore, the method can include the step of identifying nucleated cells from the sample fluid after the providing step. In this case, in particular, the nucleated cells in the cell deposit can be optically detected and additionally or alternatively quantified in the identification step. Advantageously, the sample liquid can be lysed and then analyzed during or before the identification step in order to identify whether e.g. tumor cells are contained in the lysate. In this case, the cleavage can also be carried out in the identification step as a preliminary step at the beginning of the process.

According to another embodiment, the nucleated cells or the at least one further nucleated cell can be captured in the capture plane of the microcavity in the providing step and/or wherein the cells or the at least one further nucleated cell in the sample liquid are released from the electric field into the transport plane by a release signal. In this way, very efficient capture and subsequent transport of the relevant cell type can be achieved.

Furthermore, as a method for capturing at least one nucleated cell using at least one electrode for a microfluidic device, an embodiment of the solution presented here is advantageous, wherein the method has the step of applying a sample liquid with at least one nucleated cell onto a carrier substrate of the microfluidic device. Furthermore, the method comprises the step of generating an electric field on or in the microcavities of the carrier substrate with at least one electrode configured for capturing at least one nucleated cell as a target cell in the microcavities. Thereby yielding the aforementioned advantages.

It is advantageous here that the method comprises a step of varying the current intensity after the step of applying the sample liquid to the carrier substrate of the microfluidic device, or before or after the step of generating the electric field, in order to vary, in particular to increase or decrease, the electric field. Here, an electric field is established and/or changed between, for example, an electrode and a counter electrode that is opposite to the electrode in or on the microcavity. Thereby yielding the advantages already mentioned.

It is furthermore advantageous that the step of generating an electric field is followed by a step of washing the sample liquid with a washing buffer, so as to wash the suspension of sample liquid out of the microcavity. Thereby yielding the advantages already mentioned.

It is furthermore advantageous that after trapping the nucleated cells, during the step of generating the electric field or after, the further nucleated cells in the sample liquid are released from the electric field as non-target cells by means of the electrodes. Thereby yielding the advantages already mentioned.

It is furthermore advantageous that in the step of applying the sample liquid, the lysate is applied to the carrier substrate to obtain a cell suspension having a cell deposit of at least one nucleated cell and the lysate. Thereby yielding the advantages already mentioned.

It is furthermore advantageous to arrange the step of identifying nucleated cells in the sample liquid after the step of generating an electric field. Thereby yielding the advantages already mentioned.

It is furthermore advantageous to optically detect and/or quantify nucleated cells in the cell deposit in the identification step. Thereby yielding the advantages already mentioned.

It is furthermore advantageous that during the step of generating the electric field, the nucleated cells or the at least one further nucleated cell are captured in a capture plane of the microcavity and/or wherein during the release process the cells or the at least one further nucleated cell in the sample liquid are released from the electric field into a transport plane. Thereby yielding the advantages already mentioned.

The method can be implemented, for example, in software or hardware or in a hybrid form composed of software and hardware, for example, in a controller.

The solution proposed here furthermore provides a device which is designed to carry out, control or carry out the steps of the variant solution of the method proposed here in a corresponding apparatus. Such variant embodiments of the solution in the form of a device also enable the task on which the solution is based to be quickly and efficiently solved. The device can be embodied, for example, as a control unit or controller.

To this end, the device can have at least one computing unit for processing signals or data, at least one memory unit for storing signals or data, an interface for at least one sensor or actuator for reading sensor signals from the sensor or for outputting data signals or control signals to the actuator, and/or at least one communication interface for reading or outputting data embedded in a communication protocol. The computing unit may be, for example, a signal processor, a microcontroller, etc., wherein the storage unit may be a flash memory, an EEPROM or a magnetic storage unit. The communication interface can be configured to read or output data in a wireless and/or wired manner, wherein the communication interface capable of reading or outputting wired data can read the data out of or output the data into a respective data transmission line, for example, electrically or optically.

In the present case, a device can be understood as an electrical device that processes sensor signals and outputs control signals and/or data signals accordingly. The device can have an interface, which can be constructed in hardware and/or software. In the case of a hardware configuration, the interface can be, for example, part of a so-called system ASIC, which contains the various functions of the device. However, the interface can also be an integrated circuit that is self-contained or at least partially made up of discrete components. In the case of a software configuration, the interface can be a software module, which is present, for example, on the microcontroller next to other software modules.

Also advantageous is a computer program product or a computer program with a program code which can be stored on a machine-readable carrier or storage medium (e.g. a semiconductor memory, a hard disk memory or an optical memory) and which is used to execute, implement and/or manipulate the steps of the method according to any of the embodiments described above, in particular the program product or the program being executed on a computer or device.

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

The microfluidic device can advantageously be used in combination with rapid testing. The microfluidic device can be configured, for example, as a disposable cartridge. The carrier substrate can be configured, for example, as a layer substrate comprising a plurality of layers. For example, one or more electrodes can be arranged in an integrated manner into the carrier substrate. The electrodes can be arranged at or in the cavity bottom or on or in the cavity wall of the microcavity.

According to one embodiment, the carrier substrate can have a plurality of microcavities, which each have at least one electrode, wherein the microcavities are arranged in a matrix on the carrier substrate. Advantageously, the electric field can be generated by electrodes in the microcavity. Thus, multiple target cells can be captured in each microcavity, e.g., one target cell per microcavity.

Furthermore, at least one electrode can be arranged on the cavity bottom and additionally or alternatively in the cavity wall of the at least one microcavity. Thereby, it can be advantageously determined where nucleated cells can be captured in the microcavities.

According to one embodiment, at least one electrode in each microcavity can be individually manipulated. This means that at least one electrode can advantageously be controlled in a targeted manner at all times in the microcavities in which in practice also nuclear cells are present. Furthermore, a steering unit can be provided, which is configured to apply a voltage to each electrode in the different microcavities independently of each other. In this way, cross-talk of the electric field over more than one microcavity can be prevented very efficiently, or at least reduced. Avoiding crosstalk enables setting a desired new field distribution at the only microcavity of great interest, without here changing the previous field distribution that is already prevalent at all other microcavities.

Furthermore, the electrodes may be annular, punctiform and additionally or alternatively laminar. Advantageously, each microcavity can have at least two electrodes, for example, arranged concentrically. The electrodes can be arranged in a dot-like manner on the chamber bottom, and further electrodes can also be arranged annularly around the electrodes on the chamber bottom. Alternatively, at least two electrodes can be arranged in a layered stack, i.e. in an integrated manner into the carrier substrate.

According to one embodiment, the microcavity can have at least one further electrode, wherein the electrode and the at least one further electrode can be electrically insulated from one another. Advantageously, the electrodes can be electrically insulated from each other by a spatial isolation means or, for example, by an insulating layer.

Particularly advantageous are embodiments in which at least one counter electrode is arranged on the microcavity, wherein the counter electrode is arranged opposite and/or is electrically insulated from the electrode and/or the at least one further electrode arranged in or on the microcavity. By means of this embodiment, a very flexible manipulation of the electric field in the microcavity can be achieved, so that individual cells in the microcavity can be separated or isolated quickly and easily.

Drawings

Embodiments of the solutions presented herein are illustrated in the accompanying drawings and described in detail in the following description. Wherein:

FIG. 1 shows a schematic diagram of a microfluidic device according to one embodiment;

FIG. 2 shows a schematic diagram of a circuit board having a carrier substrate according to one embodiment;

FIG. 3 illustrates a schematic view of an insert region according to one embodiment;

FIG. 4 shows a schematic structural view of an embodiment of a carrier substrate;

FIG. 5 shows a schematic cross-sectional view of a carrier substrate;

FIG. 6 shows a schematic view of a carrier substrate;

FIG. 7 shows a schematic cross-sectional view of an embodiment of a carrier substrate;

FIG. 8 shows a schematic view of an embodiment of a carrier substrate;

fig. 9 shows a schematic diagram of an operating mode according to an embodiment for a microfluidic device;

fig. 10 shows a schematic diagram of an embodiment for an operational mode of a microfluidic device;

FIG. 11 shows a schematic diagram of an embodiment for an operational mode of a microfluidic device;

fig. 12 shows a schematic diagram of an embodiment for an operational mode of a microfluidic device;

fig. 13 shows a schematic diagram of an embodiment for an operational mode of a microfluidic device;

fig. 14 shows a schematic diagram of a graph of a voltage variation curve according to an embodiment for a microfluidic device;

FIG. 15 shows a schematic view of a microcavity with dimensions according to one embodiment;

FIG. 16 illustrates a method flow diagram for capturing at least one nucleated cell using at least one electrode for a microfluidic device, according to one embodiment;

FIG. 17 shows a block diagram of an apparatus according to one embodiment;

FIG. 18a shows a schematic view of an embodiment of a carrier substrate;

FIG. 18b shows a schematic view of an embodiment of a carrier substrate;

FIG. 19a shows a schematic cross-sectional view of an embodiment of a detection chamber of a microfluidic device;

FIG. 19b shows a schematic cross-sectional view of an embodiment of a detection chamber of a microfluidic device;

FIG. 20a shows a schematic cross-sectional view of an embodiment of a detection chamber of a microfluidic device; and is also provided with

Fig. 20b shows a schematic cross-sectional view of an embodiment of a detection chamber of a microfluidic device.

In the following description of advantageous embodiments of the present solution, the same or similar reference numerals are used for elements shown in different drawings and functioning similarly, wherein repeated descriptions of these elements are omitted.

Detailed Description

Fig. 1 shows a schematic diagram of a microfluidic device 100 according to one embodiment. The microfluidic device 100 is here in particular configured as a lab-on-a-chip cartridge, which is used, for example, in conjunction with a rapid test for the examination of tumor cells in a sample liquid, which is also referred to, for example, as Circulating Tumor Cells (CTCs). The sample liquid is, for example, blood of a patient. Here, the microfluidic device 100 has a carrier substrate 105 for receiving a sample liquid. The carrier substrate 105 has at least one microcavity 110, and preferably a plurality of microcavities 110. At least one electrode 115 is disposed in or on microcavity 110 to create an electric field. The at least one electrode 115 is formed here, for example, in the form of dots, rings and/or layers. By laminar is meant that at least one electrode 115 is, for example, structured in layers. The electric field is designed to trap nucleated cells in the microcavity 110. According to this embodiment, the sample liquid is illuminated using an illumination unit 120, which is for example part of a microscope or an evaluation device.

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

In other words, an all-in-one system, microfluidic device 100, is described by the described scheme, which is configured to perform fully automatic quantification and dielectrophoretic single cell sorting of nucleated cells, and in particular viable circulating tumor cells, in whole blood by means of an electrified microcavity 110.

The time resolved and normalized quantification of CTCs enables real-time tracking of disease progression in patients, also known as real-time monitoring. Whereby therapeutic decisions can be made (accurate medical treatment), or even predictions about progression free survival can be provided that are appropriate for the individual disease. Such nucleated cells can be subjected to single cell analysis at the molecular and cellular level in single cell sorting. In this case, the detected tumor cells can also be isolated from the contaminating background of healthy blood cells in a high-quality manner, i.e. rapidly, efficiently and in a viable state, by suitable single-cell sorting, and provided for subsequent molecular genetic analysis, such as expression profiling, PCR, NGS, or, for example, subculture tests, stem cell tests and chemosensitivity tests. In this case, more accurate information about the cancer can be obtained. For example, this relates to the type and heterogeneity of mutations that occur, or also to the possible resistance to the drugs used.

In this context, the solution proposed here has microcavities 110 at the bottom of a relatively flat and large-area detection chamber. The "all-in-one system" thus formed is capable not only of sample pretreatment and (almost) isolation-free quantification of surviving circulating tumor cells from healthy blood cells, but also of dielectrophoretic single-cell sorting of detected tumor cells for subsequent single-cell analysis. The system is used in a microfluidic environment and is capable of fully automated processing of whole blood.

The key elements of the protocols presented here include sample pretreatment for CTC quantification and single cell sorting for single cell analysis of CTCs at the molecular and cellular level. According to this embodiment, sample pretreatment and CTC quantification are accomplished in the electrified microcavity 110, which can be used for single cell sorting. According to this embodiment, single cell sorting is based on a non-contact operation by negative dielectrophoresis. For this purpose, the individual cavities 110 are defined, for example, by holes at the intersections of the covered conductor circuits, which are arranged as electrically separate and exclusively (exklusiv) addressable rows and columns within the matrix on the carrier substrate 105. The release of only target cells or release of non-target cells depending on the sorting strategy, which have previously been deposited into the cavity 110 and are stably captured therein within the "DEP cage" even during the washing process, is achieved by: the associated rows and columns are manipulated by means of appropriate electrical signals as a transfer of cells from the trapping layer to the transport layer (using "DEP suspensions"). Here, non-target cells or target cells depending on the sorting strategy in adjacent chambers 110 along the same row and column are unaffected in their captured state. The released cells are transported away in a microfluidic manner, wherein they are neither contacted by the chip nor flushed into adjacent DEP cages for the entire duration of single cell sorting due to repulsive dielectric interactions.

Since the DEP cage required for cell manipulation does not necessarily extend over the entire chamber height, but is instead constructed within the microcavity 110 on the bottom of the chamber according to an embodiment, decoupling means are present between the electrical and microfluidic components. Thus, the chamber height and thus the sample volume can be chosen arbitrarily large, while in other respects the bottom area and the supply voltage or the realized DEP force remain unchanged. Since the cells within microcavities 110 also experience (greatly) reduced stokes forces as they move in the medium in which they are suspended, compared to cells above planar substrate 105, the presence of the DEP cage within cavities 110 results in a more stable capture site, and thus enables efficient, loss-free washing to be performed quickly. Thus, the automatic pretreatment of whole blood and quantification of circulating tumor cells are generally facilitated by microfluidic control.

Since the stationary, i.e. fixed in terms of its position and size, DEP cages can be used for single cell sorting, there is no need to use integrated active circuit components with proper handling. Such passive chips can be manufactured relatively easily and cost-effectively. Pure fluid transport of cells enables higher transport speeds than can be achieved with pure media. Furthermore, the transport speed is independent of the chip load, since the released cells no longer need to be guided past other cells in the same level, but can simply pass over other cells in a straight line. In these respects, both positive and negative selections can be implemented equally effectively.

Fig. 2 shows a schematic diagram of a circuit board 125 with a carrier substrate 105 according to an embodiment. The circuit board 125 and/or the carrier substrate 105 disposed on the circuit board 125 shown here are or at least are similar to the circuit board 125 and/or the carrier substrate 105 described therein depicted in fig. 1. The circuit board 125 is also referred to herein, for example, as a PCB carrier, for example, which has a carrier region 200 and an interposer region 205, for example. According to this embodiment, the carrier substrate 105 is configured as a silicon chip, which is electrically connected to an external control circuit, for example by means of the interposer region 205, for example by or accessible to a plurality of connection interfaces 210, which is also referred to as Bonding (Bonding). The connection interface 210 is designed, for example, for electrically contacting electrodes arranged on or in the carrier substrate 105. Hereinafter, the steering circuit is also referred to as a device. The insert region 205 has at least one, in particular two adjustment holes 215, which are designed to be positioned and fixed in a mating manner, for example to press or screw the insert.

The Si chip, here called circuit board 125, bonded to the carrier PCB can for example be integrated in a microfluidic manner into the LoC cassette and be in electrical contact with external control circuitry via the interposer system.

For example, a chamber having a bottom area of 12.5mm by 12.5mm requires a chamber height of at least 320 μm to accommodate a total volume of 50. Mu.l of blood lysate. If necessary, a higher chamber can also be easily realized, and accordingly a larger sample volume is obtained. For example, for a chamber having a height >1mm and a bottom area of 12.5mm by 12.5mm, a chamber volume of >156 μl is obtained.

According to this embodiment, the carrier element 105 has a plurality of layers 220, wherein the structure of the carrier substrate 105 is described in more detail in one of the following figures. According to this embodiment, the carrier substrate 105 has a chamber 225 in which the sample liquid is inspected. According to this embodiment, microcavity 110 is disposed in the region of chamber 225. Furthermore, each of the microcavities 110 has at least one electrode 230, which can be individually manipulated, for example. The microcavities 110 are optionally arranged in a matrix fashion on the carrier substrate 105.

Fig. 3 shows a schematic view of an insert region 205 according to an embodiment. The insert region 205 shown here is or at least is similar to the insert region 205 described in fig. 2. According to this embodiment, the insert region 205 is shown here in the exploded section 300 as an exploded view and in the cross-sectional section 302 as a sectional view.

The circuit board 125 is configured as a base element, on which an insert 303 is arranged, which has, for example, an intermediate element 305 and/or a cover element 310. According to this embodiment, the insert 303, the intermediate member 305 and the cover member 310 and the circuit board 125 are pressed against each other.

According to this embodiment, a modular structure is shown: the time period for the single-cell sorting using the insert 303 is brought into contact by pressing with an active control circuit an electrically passive sorting chip, which here consists of the circuit board 125 and a carrier substrate applied thereto, which is not visible in fig. 3. In this case, each bottom electrode and counter electrode on the passive chip are each provided with a contact pad, which can be actuated exclusively. For example, to implement 10000 DEP cages in a classical matrix, 100+100+1=201 contact pads are required. The dimensions of the contact pads are, for example, 500 μm by 500 μm. For electrified microcavities implemented using PCB technology, the necessary contact pads are provided directly on the same circuit board 125. In contrast, if Si technology is used, an additional carrier PCB is recommended on which the necessary contact pads are applied. This means that the electrical connection between the Si chip and the carrier PCB is made, for example, by Bonding wires ) To realize the method.

Passive components are located, for example, in lab-on-a-chip microfluidic systems, which are described here as microfluidic devices, while active components can only optionally be located in associated and spatially separated evaluation units.

Fig. 4 shows a schematic structural schematic diagram of an embodiment of the carrier substrate 105. Here, the carrier substrate 105 shown here corresponds or is similar to the carrier substrate 105 described in one of fig. 1 or fig. 2. According to this embodiment, the carrier substrate 105 has a plurality of layers 220, which are shown in six sub-images a) to f). According to this embodiment, sub-images a) to f) are to be understood as components on top of each other.

Here, the sub-image a) shows a base material 400 of the carrier substrate 105, for example silicon, which has a first oxide layer 405 and thus serves as a substrate. Here, sub-image b) corresponds to sub-image a). The only difference is that the carrier substrate 105 in the sub-image b) additionally has a metal layer 410 according to this embodiment, which has a plurality of metal strips 415 or, for example, metal strips. Here, a metal layer 410 is arranged on the oxide layer 405 to act as at least one electrode for each microcavity to be formed. According to this embodiment, the metal strips 415 are arranged spaced apart from each other such that gaps S are respectively arranged therebetween.

The sub-image c) shows a carrier substrate 105, which according to this embodiment corresponds to the carrier substrate 105 shown in sub-image b). The only difference is that the carrier substrate 105 according to the present embodiment additionally has a second oxide layer 420 having a plurality of openings 412 along the metal strips 415 of the metal layer 410. In this case, the openings 412 are arranged on the metal strip 415 in such a way that they act as at least one electrode on the cavity bottom of the microcavity in the ready-to-run state of the carrier substrate 105. Here, the diameter d of the openings 412 corresponds to the width w of the metal strips 415, respectively.

According to this embodiment, the sub-image d) shows a modification of the sub-image c). Additionally, the carrier substrate 105 has a further metal layer 425, which is also composed of a plurality of further metal strips 430. Here, the further metal strip 430 extends transversely to the metal strip 415. In the region of the opening 412 of the second oxide layer 420, the further metal strips 430 have annular sections whose size and position depend on the size and position of the opening 412 and which, according to this embodiment, constitute at least one further electrode 445. Here, according to this embodiment, the electrode 230 and the further electrode 445 are electrically insulated from each other and are furthermore optionally arranged concentrically. Additionally or alternatively, the electrodes 230, 445 are arranged in layers relative to each other. This means that at least one electrode 230 is arranged on the cavity bottom and/or in the cavity wall of at least one of the microcavities 110.

Sub-image e) corresponds to sub-image d) except that, according to this embodiment, a photoresist layer 450 is additionally arranged on the second metal layer 425. As with the second oxide layer 420 described previously, the photoresist layer 450 also has additional openings disposed at the locations of the electrodes 230, 445. However, the further metal strips 430 are covered by a photoresist layer 450 having, for example, electrically insulating properties. According to this embodiment, the electrodes 230, 445 are arranged spaced apart from each other. According to this embodiment, the spacers thus formed have the same width w as each metal strip 415.

The carrier substrate 105 shown in sub-image f) corresponds, for example, to the carrier substrate 105 described in sub-image e). The only difference is that the carrier substrate 105 in the sub-image f) additionally has a counter electrode 455 (which is configured here as a metal layer) which is arranged on the photoresist layer 450 and is thus embodied as a third electrode.

In other words, the layer structure of the novel DEP chip using silicon technology is shown in an exemplary size. The chip is composed of multiple layers.

According to this embodiment, microcavity 110 is implemented using thin-film technology. In this case, the carrier substrate 105 is realized, for example, as a thermal silicon oxide wafer. The bottom electrodes 230, 445 formed by the metal strips 415, 430 and the oxide layer 420 electrically insulating them can be patterned, for example, by photolithography: alternatively, this can be achieved either in combination with spray or epitaxial growth processes and etching, or alternatively with evaporation and stripping. Here, horizontal resolution of-1 μm can be easily achieved. The layer thickness for the metal film and the oxide is, for example, several nanometers to a maximum of-3 μm. The counter electrode 455, described herein as a metal layer, can be fabricated in the same manner as the bottom electrodes 230, 445 and has the same fabrication regulatory limitations as it. The cavity walls are created, for example, by a thick, lithographically structured photoresist, referred to herein as photoresist layer 450, having a thickness dimension of 2 μm to 200 μm, having an aspect ratio of no more than-1:10. The shape of the bottom surface of the cavity 110 can be arbitrarily selected, such as circular, hexagonal or square. The cavities 110 can be arranged on the bottom electrodes 230, 445 within a "classical matrix" or alternatively in a most densely packed manner.

Alternatively, microcavity 110 can be implemented using Printed Circuit Board (PCB) technology, as follows: the carrier substrate 105 is for example composed of a rigid FR4 carrier, for example a base material 400 >1mm thick. The functional layers of the carrier substrate 105 are connected to each other, for example by means of an adhesive material. The metal layer or the conductor circuit has, for example, a thin copper foil which is structured by photolithography and etched by wet chemical methods and comprises a thickness of, for example, 6 μm to 70 μm. For example, conductor circuit widths and spacings of from 15 μm are possible according to the standard. The insulator between the conductor circuits is, for example, a thin polyimide foil typically having a thickness of 12 μm or more. The different metal layers and insulators are bonded to each other by a lamination process. The drilling, also called "electroless blind vias", at the intersection of the bottom electrodes 230, 445 is produced, for example, by ablation with a (ultra) short pulse laser. For example, they have diameters above about 15 μm and aspect ratios of-1:1. Bare metal surfaces, especially those in contact with the medium, can be protected by so-called "finishing". The standard end surface in PCB technology is for example ENIG, ENEPIG, EPIG or gold plating. It should also be noted here that the end surfaces in this embodiment are present on all the individual metal surfaces, as well as on all the electrodes 230, 445 and 455. It should be noted that finishing is not allowed to cause electrical shorts between the different contacts. Microcavity 110 has a diameter of, for example, 100 μm and a depth of 66 μm.

Fig. 5 shows a schematic cross-sectional view of the carrier substrate 105. The carrier substrate 105 shown here corresponds or at least is similar to the carrier substrate 105 described in one of fig. 1, 2 or 4. According to this embodiment, at least one chamber 220 is arranged in the main section 500 in or on the carrier substrate 105. In addition, the carrier substrate 105 has a sub-chamber 502 in a sub-section 504 that is smaller than the chamber 220. Here, the two chambers 220, 502 have the same height h. Chamber 220 is separated from subchamber 502 by baffle 505. Here, chamber 220 is configured to hold a sample liquid. The sample liquid has at least one nucleated cell 510, in particular also at least one further nucleated cell 515. Nucleated cells 510 form tumor cells, while the additional nucleated cells 515 form white blood cells. Furthermore, the electrodes 230, 445 are arranged concentrically in the bottom region of the chamber 220. The carrier substrate 105 has an electrically conductive metal layer 455, which is embodied, for example, as a third electrode and is arranged opposite the electrodes 230, 445. Electrodes 230, 445 and 455 are configured to generate an electric field 535 in cage region 540 that acts like a cage on nucleated cells 510. Thereby fixing the nucleated cells 510 in place. In addition, an electric field 535 is applied to each of the nucleated cells 510, 515, respectively. The nucleated cells 510 can thus be transported past at least one other nucleated cell 515 without bringing them into contact with each other, thereby achieving single cell sorting. Here, nucleated cells 510 are transported into the subchamber 502.

In order to exploit the information content of circulating tumor cells in a blood sample, CTC quantification and sample pretreatment required for this and, if performed subsequently, CTC single cell analysis and single cell sorting required for this are typically separate processes from each other. Often, a plurality of devices are required for this purpose, for example separate platforms which are connected together as "processing stages", and manual processing steps which are carried out in the case of laboratory devices such as droppers, reaction vessels or centrifuges.

Sample pretreatment is typically provided with at least Red Blood Cells (RBCs) elimination and fluorescent staining (labeling) of nucleated Cells for optical detection and classification of CTCs. CTCs are (further) sorted from white blood cells (english: white Blood Cells, WBC) according to whether they are (further) performed before the CTC counting, which is called a method involving separation or a (quasi) separation-free method.

Solutions for single cell sorting typically compromise between sorting throughput and sorting sensitivity. Here, the strategy chosen depends largely on the specific application. This can be related to the nature of the sample in the initial state and the requirements of subsequent single cell analysis.

Fluorescence-based activated cell sorters (FACS) have traditionally been used, for example, for sorting of larger cell numbers, e.g., starting numbers of more than one million cells. However, they have a relatively large dead volume and may be relatively vulnerable to consumption depending on the size and strength of the markers to be validated on the cells. In addition, visual control (imaging) is often not possible during sorting. Outside of the microfluidic system, single target cells, such as circulating tumor cells, detected and possibly enriched in the primary stage can also be separated in a conventional manner from non-target cells, such as healthy blood cells like white blood cells, by microcapillaries ("cell selectors"). For this purpose, micromanipulators are generally used to move ultrafine capillaries made of glass having an inner diameter in the range of, for example, several tens of micrometers to target cells. By applying a well-defined negative pressure, the target cells are aspirated from surrounding non-target cells and transferred, for example, to a separate container for further analysis.

If one were concerned with efficient sorting of small biopsies in a microfluidic environment, the initial number of which for example has tens of thousands of cells, which in turn corresponds to the number of white blood cells in for example a few microliters of blood, a solution for manipulating biological cells based on their dielectric properties has so far presented great promise. The principle of action on which the invention is based, the so-called Dielectrophoresis (DEP), refers to the movement of even uncharged polarizable particles in a spatially non-uniform constant or alternating electric field. In this case, the externally applied electric field interacts with the electric multipole that is induced exactly by the external field and exerts dielectrophoretic forces on the particles.

In this way, biological cells can be manipulated without contact and independently of the markers. Since this effect can be extended on a large scale, it is thus also well compatible with modern MEMS technology. For the simplest case of spherical particles, which for example represent viable circulating tumor cells to be separated from the blood components of the cell composition, it is possible to use:

in the most common case of spatially stationary electric fields, the time-averaged dielectrophoresis force first order equation (which is sufficient to describe dielectrophoresis for moderately inhomogeneous electric fields) is expressed as:

Where R represents the radius of the particle under consideration; andand->Representing the absolute complex permittivity of the particle p and the surrounding medium m, where applicable:

wherein epsilon is the absolute real dielectric constant;is a complex number unit; sigma is conductivity; and ω is the angular frequency of the applied electric field. According to the factor clausius-Mo Suodi->The sign of the (real part of the) sign-depending on the frequency of the electric field and the relative coordination between the absolute real permittivity epsilon and the conductivity sigma of the frequency dependence between the medium and the particles (inside) -can create or attract (positive dielectrophoresis or pDEP acting in a direction towards the maximum of the electric field strength) on individual particles>Maximum + 1), or repulsion (negative dielectrophoresis or the action of nDEP in a direction towards the minimum of the electric field strength,minimum-1/2) force acts on the operation.

If the protocol is used to manipulate cells by pDEP, it should be considered that this can only be achieved in low conductivity buffers. While this on the one hand results in (significantly) less heat loss overall, on the other hand, this approach involves an additional, separate deionization process and the forced "artificial atmosphere" can negatively affect the viability and proliferation behavior of the cells ("leakage inside the cells").

For attractive DEP forces, a stable force balance always occurs spatially on the electrode surface of the field generation or more generally at the site of greatest electric field strength on the chip surface. In this way, although the DEP traps (DEP-Falle) are significantly easier to generate and stronger, in particular the confining force increases due to the voltage rise, this can lead to cell contact and thus perhaps cell adhesion to the chip surface and cell lysis due to too high an electric field strength. These are often critical drawbacks, which can lead to a (severe) decrease in the efficiency or purity of sorting by pDEP. If the aim is to circumvent the just-cited problems, negative DEP forces can be used instead to sort cells, as they are "per se" implemented in physiological media such as plasma, PBS or cell culture media. Although, in contrast, an increase in the conversion of active electrical energy into heat is to be considered, this heat can only be dissipated if necessary by suitable additional components, for example ventilation means or peltier elements, but no prior deionization is required for this purpose. In addition, by maintaining natural media, cell viability and cell expression profile can be maintained to a maximum.

In the case of repulsive DEP forces, the cells always move towards the position of least field strength, i.e. typically away from the substrate. This avoids contact between the cells and the chip surface and resists cell adhesion and destruction due to too high an electric field. In view of this, achieving a stable force balance within a sufficiently strong DEP trap has proven quite challenging, with the confining force generally decreasing with increasing voltage. The strategy for this approach is to separate target cells from non-target cells by means of two separate chambers in one plane. Here, the sorting process includes the following two steps:

first, the prepared cell suspension of great interest is injected into a large main chamber, also referred to herein as chamber 220 and hereinafter referred to as "chamber 1", and an adjacent small sub-chamber 502, hereinafter referred to as "chamber 2", is filled with a clean wash buffer of physiological components. For the filling process, an inlet for filling and an outlet for exhaust or output are provided in each chamber 220, 502, respectively. After filling, the two chambers 220, 502 are fluidly connected to each other without air. The cells 510, 515 in chamber 1 are randomly but uniformly distributed, with no cells reaching chamber 2. In chamber 1, the target cells are captured purely (dielectrically) and passed "definitively", i.e. collision-free, along a individually programmable trajectory past the non-target cells, which are also captured in the fixation plane 545, until they are transported into chamber 2. For example, the chambers 220, 502 and their walls are defined by a double sided tape of appropriate height and appropriate layout that sealingly connects the bottom and top covers. The transport of the target cells takes place in and by means of three-dimensional "permanently closed DEP cages". Such a cage in the "standard configuration" is composed of, for example, 3 x 3 planar square silicon electrodes 230, 445 on the bottom of the chamber (each electrode: -18.8 μm x 18.8 μm with a spacing of 1.2 μm from the adjacent electrode), and a transparent indium tin oxide-counter electrode (ITO) 455 extending across the top of the chamber. To generate a field minimum in the chamber into which the cells are stably positioned by nDEP, the central bottom electrode 230 and the counter electrode 455 on the chamber top are set to the same alternating potential ("in-phase" or "+") while the outer bottom electrode 445 runs in opposite phase ("in-phase" or "-").

In chamber 2, target cells 510 previously "expelled" from chamber 1 are output to an external reaction vessel, such as an Eppendorf centrifuge tube, during a final rinse with clean buffer. Thus, the process takes place in a purely microfluidic manner and thus "nondeterministically", which means that the nucleated cells according to this variant approach follow only a straight-line trajectory.

The sorting process set forth herein ensures 100% reproducible efficiency and purity, respectively, but the "consequences" that follow should be interpreted as follows:

in order to control the voltage of the power supply (U) _DEP ≤5V _RMS ) To generate sufficiently high electric field strength or DEP forces for cell manipulation, the height of the chamber 1 and thus, in relation, the maximum sample volume that the chamber 1 can accommodate is limited. For example, for a bottom area of chamber 1 of-12.5 mm by 12.5mm, a chamber height of maximally-100 μm results in a chamber volume of maximally-15.6. Mu.l. Effective cleaning in the chamber 1 is furthermore not guaranteed or feasible with respect to the situation in the chamber 2. For these reasons, it is provided that the external concentration is carried out in clean buffer additionally by enrichment of the target cells. This is typically accomplished by centrifuges, where manual transfer of the sample between devices is required and additional cell loss is a concern. The "dynamic" DEP cages required for actuation, i.e. the DEP cages that can vary in their position and size as described before, can only be realized by active components, such as transistors or memory elements. These components are integrated in the respective silicon electrodes by CMOS technology.

According to this variant, the maximum transport speed of the target cells is limited (1 electrode width/sec) in chamber 1, because the (lateral) DEP force for transport is limited. Further optionally, the maximum transport speed is dependent on the chip load in the individual case. This means that while the model with only 2 x 2 electrodes is also able to produce a closed DEP cage for holding cells theoretically more than 3 x 3/2 x 2 = 9/4 = 2.25 times the standard configuration in total ("reconfiguration"), it is first necessary for the target cells in this case to "leave out a path to the outlet", which is related to an increase in sorting time. Negative selection is not set or not feasible.

Fig. 6 shows a schematic view of the carrier substrate 105. The carrier substrate 105 shown here corresponds, for example, to the carrier substrate 105 described in fig. 5. They differ only in the presentation view angle. This means that the carrier substrate 105 is shown from a top view. The trajectory 600 is shown purely by way of example, along which the nucleated cells 510 are first transported into the subsections 504 and then from the carrier substrate 105 into, for example, a sample container 605 using an electric field and a varying voltage value.

Here, the main section 500 and the chamber 220 are square-shaped. Further optionally, chamber 220 is connected to sub-chamber 502 by bottleneck-shaped connection section 610. The secondary chamber 502 is essentially straight in this case and has an inlet 615 and an outlet 620 opposite the inlet 615. For this purpose, there are separate inlet-outlet-doublets IN 615 and OUT 620, which are provided only for the flushing/transport/output process with clear buffer. The outlet 620 is configured for discharging the separated nucleated cells 510 from the carrier substrate 105 into the sample container 605 via the subchamber 502.

Fig. 7 shows a schematic cross-sectional view of an embodiment of the carrier substrate 105. The carrier substrate 105 shown here can be used, for example, in a microfluidic device as exemplarily depicted in fig. 1. Furthermore, the carrier substrate 105 is at least similar to the carrier substrate 105 described in one of fig. 1, 2, 4, 5, 6. According to this embodiment, microcavities 110 are shown, which are arranged as pockets in the carrier substrate 105. According to this embodiment, the carrier substrate 105 shown here also has concentrically arranged electrodes 230, 445 configured for forming an electric field 535. In order to be able to form the electric field 535 ("DEP cage"), it is also very advantageous to use a flat electrode as counter electrode 455, which here forms a third electrode on top of the microcavity 110 and is opposite to the electrode 230. According to this embodiment, all microcavities 110 are configured in the same manner so that an electric field can be generated in each microcavity. Thereby enabling single cell sorting on the carrier substrate 105, for example. Furthermore, the nucleated cells 510 can thereby be transported out of the microfluidic device along the trajectory 600. According to this embodiment, the outlet 620 is connected to a valve 700, which is configured as a double valve, for example. The valve 700 is configured for directing nucleated cells 510 into the sample container 605 and for draining undesired liquids 705, such as the lysis residue of the sample liquid.

In other words, a single cell sorting by means of an electrified microcavity 110 is illustrated, in which nucleated cells 510 are separated from non-target cells, i.e. from other nucleated cells 515 in the chamber, in particular by means of two separate planes 545, 710. The corresponding sorting process is provided with a preliminary process and a spatial separation, whereby quantification of the nucleated cells 510 can be achieved.

According to this embodiment, the carrier substrate 105 has an electrified microcavity 110 for single cell sorting. Externally, a two-way valve, also referred to herein as valve 700, is provided. The valve 700 is configured to spatially separate the blood lysate 705 to be washed out and treated as contaminants during preparation from the nucleated cells 510 during single cell sorting using a clean wash buffer that follows. Alternatively, it is also conceivable to separate the blood lysate 705 without the valve 700 using two different reaction vessels, which are placed sequentially at the outlet, i.e. at the outlet 620.

At the bottom of the individual microcavities 110 is a first spot electrode 230, which is surrounded by a second ring electrode 445 in an electrically insulating manner. The two bottom electrodes 230, 445 are arranged concentrically and coincide with the bottom center of gravity of the microcavity 110. The electrodes 230 extending through the cavity 110 as spot electrodes 230 form columns. Furthermore, the electrodes 445, which extend through the cavity as ring electrodes, twisted by 90 ° form rows of a matrix in which each bottom electrode 230, 445 can be individually manipulated. To be able to create a three-dimensional "DEP cage with release function", a third full face counter electrode, also called metal layer 455, is finally required and a mesh top surface (Stegoberseite) is formed between adjacent grooves.

According to this embodiment, with all the punctiform bottom electrodes 230 and counter electrodes 455 set to a sufficiently high and equal alternating potential, while all the annular bottom electrodes 445 run identically and in opposite phase with respect to this intensity, near-bottom and randomly, but uniformly distributed intact nucleated cells 510, 515, i.e. white blood cells and circulating tumor cells, are in a quiescent mediumIs guided into the microcavity 110 in a contactless manner as the sedimentation process proceedsIn a fixed levitation position. However, voltage level U _DEP,min At the same time, it is chosen low enough to obtain an open DEP cage.

Directly prior to cleaningIn the case where the signal distribution at the outer bottom electrode 230, 445 remains unchanged, the voltage is increased to a set maximum value U _DEP,max A closed DEP cage is created with maximum retention force in which all cells 510, 515 remain stably captured against the primary flow motive force without contact with the walls or bottom of the cavity 110. Thus, intact cells located outside the cage are no longer able to enter the cage, while all cells 510, 515 within the cage remain stably captured. The cleaning process leaves the chamber completely uncontaminated and is only optional.

Without limiting generality, the process of forward selection, i.e., the separation of target cells 510 from non-target cells 515, is optionally described below. Conversely, negative selection is of course similarly achieved by releasing non-target cells 515 from microcavities 110, while target cells 510 are not released.

All cells 510, 515 that settle to the bottom of the chamber are initially in an equilibrium position within the microcavity 110, i.e., at a constant height from the bottom of the cavity 110. In further investigation, this lower capture plane is referred to as plane 710. The level of the DEP voltage applied is still at a maximum and the medium in the chamber is still moving. To remove the target cells 510, 515 from the stable capture state in plane 710 and transfer them to an upper transport plane above the mesh top surface, referred to herein as plane 545, the intersecting bottom electrodes 230, 445 at the location of the target cells 510 are manipulated with appropriate electrical signals. For this purpose, the voltage U will be released _F,S Applied to the belonging column and will release the voltage U _F,Z Applied to the row to which it belongs. Here, the release voltage is geometry-dependent. For 0.ltoreq.U _F,S ≤U _F,Z ≤U _DEP,max The release voltage is selected in such a way as to ensure a single cell' The pure (dielectric) release is definitely "obtained from its suspension position, especially without impeding neighboring cells at its capture position. All signals used are for example harmonic excitations with a constant frequency.

Cell release requires a sufficiently large net repulsive DEP force in the negative z-direction along the central symmetry axis through microcavity 110 ("DEP suspension"). For this purpose, for a given geometry of microcavity 110, a U is used _F,S And U _F,Z The proper combination of (a) ensures that the electric field strength lines from the counter electrode 455 all fall onto the exposed surface of the point-like bottom electrode 230 (column) as parallel as possible to the z-axis and that there is furthermore a sufficiently large field strength. In the edge region of the microcavity 110 close to the wall, the disturbance of the field strength profile by the annular bottom electrode 445 (row) should be suppressed as much as possible.

At the same time U _F,Z The selection should be so small that interference with the DEP cage along the released non-target cells 515 to the left and right next to the lumen 110 of the target cells 510 is avoided and the closed DEP cage with sufficient retention force is maintained as it is. On the other hand, U _F,S Is selected to be large enough that no contact occurs between the non-target cells 515 along the released line, i.e., above and below the cavity 110 of the target cell 510, and the bottom of the cavity 110, in the new temporary equilibrium state within the lowered closed DEP cage.

The situation in plane 545 changes if the released cells have been protected in plane 710 within cavity 110 in advance and have only been subjected to an overall reduced stokes force during the washing process. The transport (transport away) and the output towards the outlet 620 takes place here purely in a microfluidic manner, i.e. non-deterministic, by means of a maximum stokes force with the aid of a clean (washing) buffer and is therefore relatively rapid. The released cells cannot enter the adjacent DEP cages because they are closed. Thus, the sorting principle as a whole enables maximization of efficiency and purity.

Fig. 8 shows a schematic view of an embodiment of the carrier substrate 105. The carrier substrate 105 shown here corresponds or is similar to the carrier substrate 105 described in, for example, one of fig. 1, 2 or 4 to 7. According to this embodiment, the only difference is the viewing angle. That is, the carrier substrate 105 shown here is shown from a top view. Here, the carrier substrate 105 also has a plurality of microcavities 110. The microcavities 110 are also arranged here in a matrix, i.e. in rows and columns. Here, a trajectory 600 for at least one nucleated cell 510 is also shown.

Fig. 9 shows a schematic diagram of an embodiment of an operational mode 900 for a microfluidic device. The carrier substrate 105 shown here is at least similar to the carrier substrate 105 described in fig. 1, 2 or one of fig. 4 to 8. According to this embodiment, the state in which the electrodes of the microfluidic device are activated and form an electric field 535 is referred to as an operational mode 900. According to this embodiment, a cross-sectional view of microcavity 110 of carrier substrate 105 with layered arrangement of electrodes 230, 445 and 455 is used to illustrate operational mode 900. Here, electrodes 230, 445 and 455 are disposed on or in each individual microcavity 110. Here, they are arranged such that in the activated state of the electric field 535, the nucleated cells 510 reach the center of the microcavity 110. This means that the electrical cage is open.

In other words, the mode of operation 900 of single cell sorting by means of electrified microcavities 110 in PCB technology, such as forward selection, is shown and described in accordance with this embodiment: it describes the separation of target cells 510 from non-target cells 515.

Fig. 10 shows a schematic diagram of an embodiment of an operational mode 900 for a microfluidic device. The microcavities 110 shown here are, for example, similar to the microcavities 110 described in fig. 9. According to this embodiment, only the electric field 535 is shown to be different. According to this embodiment, the electrical cage is closed such that the nucleated cell 510 is captured in an intermediate position where it is located in the microcavity 110. Here, the liquid located around the nucleated cells 510 can be washed away, for example, without washing away the nucleated cells 510 from the microcavities 110 (=washing process). The same applies to nucleated non-target cells 515.

Fig. 11 shows a schematic diagram of an embodiment of an operational mode 900 for a microfluidic device. Microcavity 110 is shown here as similar to microcavity 110 described in fig. 10, for example. According to this embodiment, only the electric field 535 is shown to be different. According to this embodiment, the electrodes 230, 445 and 455 are manipulated in such a way that the nucleated cells 510, also referred to as target cells, can be electrically pushed out of the microcavities 110 and then the nucleated cells that are already above the microcavities can be microfluidically rinsed out by rinsing. This is achieved, for example, by varying the voltage U applied to the electrodes.

Fig. 12 shows a schematic diagram of an embodiment of an operational mode 900 for a microfluidic device. Microcavity 110 is shown here as similar to microcavity 110 described in fig. 11, for example. According to this embodiment, only the electric field 535 is shown to be different. According to this embodiment, electrodes 230, 445 and 455 are manipulated such that additional nucleated cells 515, also referred to as non-target cells, are held in microcavity 110. For example, additional nucleated cells 515 are released or alternatively held in their position in microcavities 110 by changing the voltage. This is achieved, for example, by varying the voltage U applied to the electrodes 230, 445 and 455. The voltage U required for this is optionally different from the voltage U required for the nucleated cells 510. Thereby, for example, the nucleated cells 510 can be transported past the further nucleated cells 515.

Fig. 13 shows a schematic diagram of an embodiment of an operational mode 900 for a microfluidic device. Microcavity 110 is shown here as similar to microcavity 110 described in, for example, fig. 12. According to this embodiment, only the electric field 535 is shown to be different. According to this embodiment, electrodes 230, 445 and 455 are manipulated such that additional nucleated cells 515, also referred to as non-target cells, remain in microcavity 110. The voltage is chosen such that contact of non-target cells with the bottom of the chamber is just avoided (non-contact single cell sorting).

Fig. 14 shows a schematic diagram of a voltage profile 1400 according to an embodiment for a microfluidic device. Here, the voltage profile 1400 shown here shows the characteristics of the voltage U applied in the microcavity during the time t. According to the present embodiment, referred to as voltage U _DEP For example, represents a predetermined reference value, which represents, for example, a minimum threshold value and a maximum threshold value. According to this embodiment, a plurality of differently applied voltage profiles are shownThe lines correspond, for example, to those modes of operation described in fig. 9 to 13. This means that the electric field is changed in the manner shown in fig. 9 to 13 by adjusting and/or changing the voltage U in the respective microcavity and/or on the respective electrode.

According to this embodiment, the first curve 1405 and the second curve 1410 represent the mode of operation depicted in fig. 9, wherein the electrical cage is open. Here, a first curve 1405 indicates the voltage U applied to the electrodes 230 and 455. The second curve 1410 shows the voltage U applied to the further electrode 445. U (U) _DEP,min The time average of curves 1405 and 1410 is shown.

Third curve 1415 and fourth curve 1420 illustrate the mode of operation depicted in fig. 10. Here, it is shown 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, a fourth curve 1420, which is a voltage variant of the second curve 1410, shows a voltage profile of the further electrode 445.

Further, fifth and sixth curves 1425 and 1430 represent the operation modes shown in fig. 11 to 13. Here U _F,Z Is the time average of curve 1425, and here U _F,S Is the time average of curve 1430.

In other words, the target cell can use, for example:

is released.

Fig. 15 shows a schematic view of a microcavity 110 having dimensions according to an embodiment. Microcavity 110 corresponds to or is at least similar to microcavity 110 described in fig. 1, 2, or one of fig. 4-13. According to this embodiment, the electrodes 230, 445 and 455 are also arranged concentrically. The dimensions shown in fig. 15 are understood to be merely exemplary and can vary in alternative embodiments.

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

According to this embodiment, the cavity 110 comprises a diameter of, for example, 50 μm and a depth of about 37.5 μm. The numerical examples listed below relate to the lightest and smallest expected spheroidal CTC trajectories (ρ _p ＝1070kg/m ³ And r=3 μm) in the physiological medium (σ) through the electrified microcavity 110 _m Approximately 1.4S/m). Capable of generating a sufficiently large negative DEP force in a physiological medium at an operating frequency between about 1MHz and 10MHz Without electrolytic effects and with a minimum transmembrane voltage. The aqueous-like wash buffer should have a density ρ _f ＝1000kg/m ³ And viscosity η=1 mpa·s.

The first process part uses a relatively small volume of whole blood (. Ltoreq.20. Mu.l) as an "input". "export" is the ideal initial state of single cell sorting by DEP in the second part of the process. In the final optical detection, CTCs to be isolated are expected to have less than 80000 to 220000 white blood cells in less than 100 μl of blood lysate, whereby such cell suspensions can be interpreted as relatively small biopsies as a whole. Load: for example, an open DEP cage generates an operating voltage U _DEP ＝U _DEP,min ＝1.5V _RMS 。

If, for reliability reasons, a larger chamber volume of 50 μl is replaced, i.e. flushed 30 times with wash buffer, for example within 5 minutes, to give the chamber sufficient optical transparency and purity, a volume flow of 5 μl/s is produced which can be set in an average constant manner by the microfluidic system. Thus, for a chamber with an end inlet area and an end outlet area of 12.5mm by 320 μm, the relevant average flow rate is At an operating voltage U _DEP ＝U _DEP,max ＝5V _RMS The resulting closed DEP cage has sufficient retention to resist flow forces or turbulence caused by cleaning within the cavity 110.

Fig. 16 illustrates a flowchart of a method 1600 for capturing at least one nucleated cell using at least one electrode of a microfluidic device, according to an embodiment. This may be a method that can be used in one of the devices of the microfluidic device described in accordance with the previous figures. Here, the method 1600 includes an outputting step 1605 and a providing step 1610. In an output step 1605, an application signal is output to apply a sample liquid having at least one nucleated cell to a carrier substrate of a microfluidic device. In a providing step 1610, a current signal is provided to an interface of at least one electrode to generate an electric field at or in a microcavity of the carrier substrate, the electric field being formed for capturing at least one nucleated cell as a target cell in the microcavity. According to this embodiment, an application signal is output in an output step 1605, which application signal causes the lysate to be applied to the carrier substrate in order to obtain a cell suspension with a cell deposit of at least one nucleated cell and the lysate. This means, for example, that a certain period of time is waited for during which the cell sediment is precipitated. Further optionally, in or after providing step 1610, a release signal is provided to the electrode after capturing the nucleated cells in order to release additional nucleated cells in the sample liquid from the electric field as non-target cells. The output may also be an optional new step 1630.

According to this embodiment, the method 1600 further comprises a step 1615 of changing the current intensity and/or voltage after the outputting step 1605, before or after the providing step 1610, in order to change, e.g. increase or decrease, the electric field. In an optional identification step 1620, following the providing step 1610, nucleated cells are identified from the sample liquid and, in particular, optically detected and/or quantified from the cell deposit.

Method 1600 further optionally includes a sample liquid cleaning step 1625. Here, after providing step 1610, the sample liquid is washed using a washing buffer so as to wash the suspension of sample liquid from the microcavity.

The process steps presented herein may be performed repeatedly and in a different order than described.

Fig. 17 shows a block diagram of a device 1700 according to an embodiment. The device 1700 is for example implemented as a controller or control unit configured for manipulating or performing the method for capturing at least one nucleated cell as shown in fig. 16 using at least one electrode for a microfluidic device. To this end, the device 1700 has an output unit 1705 for outputting an application signal 1710, which application signal 1710 applies a sample liquid with at least one nucleated cell to a carrier substrate of the microfluidic device, and a supply unit 1715 for supplying a current signal 1720 to an interface of at least one electrode, in order to generate an electric field on or in a microcavity of the carrier substrate, which electric field is formed for capturing the at least one nucleated cell as a target cell in the microcavity. Furthermore, the providing unit 1715 is configured for optionally providing a release signal 1725 only after capturing the nucleated cells to the electrode in order to release further nucleated cells in the sample liquid from the electric field as non-target cells. The device 1700 also has a changing unit 1730 that causes a change in the current strength to increase, decrease, and/or change the electric field. According to this embodiment, the apparatus 1700 also has a washing unit 1735 configured to effect washing of the sample liquid using the washing buffer so as to wash out a suspension of the sample liquid from the microcavity. Furthermore, the device 1700 optionally has an identification unit 1740 configured for identifying nucleated cells in the sample liquid, in particular for optically detecting and/or quantifying nucleated cells in a cell deposit.

Fig. 18a shows a schematic view of an embodiment of the carrier substrate 105. The carrier substrate 105 shown here corresponds or at least is similar to the carrier substrate 105 described in fig. 2, which is arranged or can be arranged in the microfluidic device 100. Such a microfluidic device 100 has been described in at least one of fig. 1 to 8. According to this embodiment, microcavities 110 are arranged in a grid or passive matrix, i.e., in rows and columns. Furthermore, the microcavity 110 is electrically coupled to a plurality of switching units 1800, so that they accordingly have, for example, electrical contacts. More precisely, all microcavities 110 are coupled to one switch each in rows and columns. This also means that microcavity 110 can also be manipulated in rows and/or columns.

According to this embodiment, the microcavity 110 arranged in the center of the carrier substrate 105 is shown as a microcavity 110 of great interest. The microcavities 110 directly connected thereto are embodied here as critically adjacent microcavities 110, wherein the voltage drops are symbolically represented by lightning 1805, respectively. Lightning 1805 represents parasitic electrical crosstalk and means that the central cavity 110 field distribution variation of significant interest undesirably causes undefined changes in the states in adjacent microcavities 110.

In other words, microcavity 110 is based on a bottom electrode having a quasi-planar layer structure, which is only exemplarily arranged in the form of columns and rows that can be handled separately and are insulated from each other, wherein the columns have a punctiform electrode as the lowermost first metal layer; the rows have ring electrodes as intermediate second metal layers. However, since a passive matrix is shown according to this embodiment featuring simple crossings of conductor circuits, the voltage drop can be applied or read not only at the microcavity 110 of great interest. However, to achieve a selectively addressed state, the passive matrix is extended to an active matrix by appropriately integrated transistor circuits at the intersections of the conductor circuits and at the corresponding columns and rows, as depicted and/or illustrated in fig. 18 b.

Fig. 18b shows a schematic view of an embodiment of the carrier substrate 105. The carrier substrate 105 shown here is similar to the carrier substrate 105 described for example in fig. 18a, wherein the carrier substrate 105 shown here is implemented as an active matrix according to this embodiment. This means that individual microcavities 110, although also arranged in rows and columns, are each coupled or couplable to a switch. However, for the sake of clarity, this is shown in a simplified manner according to this embodiment. Thereby, the microcavities 110 are each individually actuated with a respective switching unit and by means of a respective control signal 1850 and electrical crosstalk and thus influence on adjacent microcavities 110 is prevented.

In other words, the microfluidic device 100 is extended by active switching elements that enable "ideal" selective addressing of individual microcavities 110 of critical interest, such that the carrier substrate 105 forms an active matrix. The microcavities 110 of great interest are, for example, those microcavities on which DEP operation is detected and/or electrochemical detection occurs. Furthermore, the active switching element enables the electrode within microcavity 110 to be used not only as a steering element for dielectrophoresis movement, but also as a sensor element for electrochemical detection of particles released by cells in solution. The possibility of performing on-chip analysis after sealing the microcavities 110, for example as shown in fig. 20a to 20b, is thereby additionally expanded, for example in order to monitor the environmental conditions of the cells 510 in the microcavities 110.

The switching elements of the quasi-planar active matrix can be realized in the form of integrated semiconductor switching technology. For this purpose, complementary metal oxide semiconductor technology (CMOS), which is produced as a silicon monolithic, can be used, for example. Alternatively, it is conceivable to use other technologies, such as bipolar technology and semiconductor materials, such as gallium arsenide. Nonlinear components of the circuitry required for selective addressing of individual microcavities 110, that is to say for example transistors, memory elements, diodes etc., can generally be manufactured typically using any of these techniques as a standard. If the active matrix is realized using semiconductor switching technology, the entire counter electrode described, also described as the third electrode, can be structured as the uppermost third metal layer, for example by means of a photoresist system. For this purpose, SU-8 photoresist is used only by way of example, which is sprayed unmodified in a first thick layer, for example >30 μm, with respect to the definition of the microcavity 110, and then in a second thin layer, for example <1 μm, with metallic particles, for example silver, offset with respect to the definition of the entire counter electrode. After so-called soft baking, exposure, development and hard baking of the two layers, the counter electrode is optionally also electroplated with a chemically inert metal, such as gold, to increase the conductivity properties. The electrical contacting of the semiconductor chip is achieved, for example, by connecting bond wires to a carrier circuit board to which the chip is previously bonded.

If the electrodes within microcavity 110 are also to be used as sensor elements to electrochemically detect particles in solution that are output by cells 510, these electrodes are functionalized beforehand with suitable counter particles (Gegenpartikel) under certain conditions to increase sensitivity. The counter particles have the following tasks, for example: efficiently bind particles to be detected or enhance the electrochemical reaction between the particles and the electrode. The sensor element can be advantageous for a variety of applications. For example, the relevant antibodies (=particles) secreted by B lymphocytes can be recognized in this way after functionalization of the electrode with the corresponding antigen (=counter particles) for use in pharmaceutical production. Another example is cell culture for cell line development, in which the growth conditions, e.g.pH, O, are tracked and controlled precisely and in real time by means of electrodes as sensor elements ₂ Content of CO ₂ Content and/or glucose concentration.

Fig. 19a shows a schematic cross-sectional view of an embodiment of a microfluidic device 100. Here, according to this embodiment, a nucleated cell 510 is disposed in each microcavity 110. Here, the lysate is disposed as an aqueous medium around the cells 510 and in the intermediate region 1900 between the carrier substrate 105 and the transparent cover 1905. Intermediate region 1900 may also be referred to as a detection region of microfluidic device 100, for example. According to this embodiment, each microcavity 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 configured as bottom electrodes in order to generate an electric and/or magnetic field, for example, in the operating state. The nucleated cells 510 are held in the corresponding microcavities 110 by the generated field while, for example, the lysate is washed. The first electrode 1910 is realized centrally and punctiform at the cavity bottom 1920, and the second electrode 1915 is arranged annularly, for example, around the first electrode 1910. Here, the electrodes 1910, 1915 do not contact each other. According to this embodiment, the first electrode 1910 forms a positive electrode and the second electrode 1915 forms a negative electrode.

According to this embodiment, the carrier substrate 105 also has a third electrode 1925, which is arranged in a planar fashion on the substrate surface according to this embodiment. Here, the third electrode 1925 is also implemented as a positive electrode and follows the time-dependent voltage profile shown in fig. 14.

In other words, microcavities 110 are shown as electrified microcavities 110, each of which has loaded nucleated cells 510.

Fig. 19b shows a schematic cross-sectional view of an embodiment of a microfluidic device 100. The microfluidic device 100 shown here is similar to the microfluidic device 100 described in fig. 19a, for example. According to this embodiment, only the particles 1950 of each nucleated cell 510 are dome-shaped arranged around the respective cell 510 such that they each affect adjacent microcavities 110. This means that electrical and/or microfluidic crosstalk is only shown here by way of example.

Fig. 20a shows a schematic cross-sectional view of an embodiment of a microfluidic device 100. The microfluidic device 100 shown here is similar to the microfluidic device 100 described in fig. 19a, for example. Here too, one nucleated cell 510 is arranged in each microcavity 110 and is held in place by electrodes 1910, 1915, 1925. However, according to this embodiment, the microfluidic device 100 is loaded with a non-aqueous medium 2000 or a non-aqueous phase, comprising for example silicone oil or air, in order to eliminate micro-fluidic cross-talk of the mutually influencing or adjacent microcavities 110 as shown in fig. 19b and for example to seal the microcavities 110. This means that the microcavity 110 is sealed by the choice of the nonaqueous medium 2000 and thus the relevant physical properties.

According to this embodiment, during single cell analysis on the chip, i.e., the microfluidic device 100, is rinsed with at least one non-aqueous medium 2000. Thus, sealing and isolation of the cells 510 in the aqueous phase in the microcavities 110 is achieved, which is described herein as an aqueous medium having physiological properties.

In other words, after loading the cells 510, the lysate, i.e., the aqueous phase layer, above the cavity 110 is first replaced with the non-aqueous phase medium 2000 in order to achieve sealing of the cells 510 of the aqueous phase within the microcavities for single cell analysis on the chip. If, in addition, individual cells 510 are to be discharged into a collection container for analysis off-chip, the nonaqueous phase 2000 which was previously supplied is replaced by the aqueous phase again, and the situation is thus returned to the initial state.

Fig. 20b shows a schematic cross-sectional view of an embodiment of a detection chamber of the microfluidic device 100. The detection chamber shown here is similar to the detection chamber described in fig. 20 a. According to this embodiment, the middle region 1900 is completely filled with only the nonaqueous medium 2000 except for the microcavity 110, so that the microcavity 110 is isolated from each other. In other words, the aqueous medium is only present in the microcavity 110 itself.

In other words, it is shown that parasitic microfluidic crosstalk is eliminated. The electrified microcavities 110 are completely isolated from the nucleated cells 510 in the aqueous phase by the non-aqueous medium 2000. Thus, particles released into solution by the cells 510 are no longer able to diffuse and convect, and analysis in each microcavity 110 proceeds independently of each other.

If an embodiment comprises an "and/or" coupling between a first feature and a second feature, this should be understood as having the first feature and the second feature in accordance with an embodiment, and having either only the first feature or only the second feature in accordance with further embodiments.

Claims

1. A method (1600) for capturing at least one nucleated cell (510) using at least one electrode (230) for a microfluidic device (100), wherein the method (1600) comprises the steps of:

-outputting (1605) an application signal (1710) that causes a sample liquid having at least one nucleated cell (510) to be applied to a carrier substrate (105) of the microfluidic device (100); and

-providing (1610) a current signal (1720) to an interface of 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), the electric field being formed for capturing the at least one nucleated cell (510) as a target cell in the microcavity (110).

2. The method (1600) according to claim 1, having a step (1615) of varying the current intensity after the outputting step (1605) or before or after the providing step (1610) for varying, in particular increasing or decreasing, the electric field (535), in particular wherein the electric field (535) is established and/or varied between the electrode (230) and a counter electrode (455) of an electrode (230) arranged in or on the microcavity (110).

3. The method (1600) according to any of the preceding claims, wherein in the providing step (1610), the current signal (1720) is output to an interface of the at least one electrode (230) and an interface of at least one further electrode arranged in an adjacent microcavity (110) such that an electric field different from that at the electrode (230) is generated at the at least one further electrode, in particular wherein the field generated at the electrode is different in direction and/or strength from the electric field generated at the further electrode, and/or wherein the further electric field is generated at the further electrode, the further electrode being arranged in a microcavity (110) arranged in a common column or a common row with respect to the microcavity (110) with the electrode (230).

4. The method (1600) according to any of the preceding claims, having a washing step (1625) of the sample liquid, which washing step is performed after the providing step (1610) using a washing buffer, in order to wash away the suspension of the sample liquid from the microcavity (110), and/or wherein in or after the providing step (1610) a release signal (1725) is provided to the electrode after capturing the nucleated cells (510), in order to release further nucleated cells (515) in the sample liquid as non-target cells from the electric field (535).

5. The method (1600) according to any of the preceding claims, wherein in the outputting step (1605) the application signal (1710) is output, which application signal causes the lysate to be applied onto the carrier substrate (105) in order to obtain a cell suspension with at least one nucleated cell (510) and the lysate, and/or wherein after the providing step (1610) an identification step (1620) is provided for identifying nucleated cells (510) from the sample liquid, in particular wherein in the identification step (1620) of nucleated cells (510) an optical detection and/or quantification is performed from the cell sediment.

6. The method (1600) according to any of the preceding claims, wherein in the providing step (1610), nucleated cells (510) or at least one further nucleated cell (515) are captured in a capture plane of the microcavity (110), and/or wherein cells (510) or at least one further nucleated cell (515) in the sample liquid are released from the electric field (535) into a transport plane by a release signal (1725).

7. A method for capturing at least one nucleated cell (510) using at least one electrode (230) for a microfluidic device (100), wherein the method (1600) comprises the steps of:

-applying a sample liquid with at least one nucleated cell (510) onto a carrier substrate (105) of the microfluidic device (100); and

-generating an electric field (535) with the at least one electrode (230) on or in the microcavity (110) of the carrier substrate (105), the electric field being formed for capturing at least one nucleated cell (510) as a target cell in the microcavity (110).

8. A device (1700) set up for performing and/or manipulating the steps (1605, 1610, 1615, 1620, 1625, 1630) of the method (1600) according to any one of the preceding claims in a respective unit (1705, 1715, 1730, 1735, 1740).

9. Computer program set up for performing and/or manipulating the steps (1605, 1610, 1615, 1620, 1625, 1630) of a method (1600) according to any one of claims 1 to 7.

10. A machine readable storage medium on which is stored a computer program according to claim 9.

11. A microfluidic device (100) for capturing at least one nucleated cell (510) of a sample liquid, in particular wherein the microfluidic device (100) is configured as a lab-on-a-chip cartridge, wherein the microfluidic device (100) has the following features:

-a carrier substrate (105) for containing a 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) for generating an electric field (535) formed for trapping nucleated cells (510) in the microcavity (110).

12. The 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).

13. The micro fluidic device (100) according to claim 12, wherein at least one electrode (230) is arranged on a cavity bottom and/or in a cavity wall of at least one micro cavity of the micro cavity (110).

14. The microfluidic device (100) according to any one of claims 12 to 13, wherein an electrode (230) can be manipulated individually in each of the microcavities (110), and/or wherein the electrode (230) is shaped in a ring, dot and/or layer, in particular wherein a manipulation unit is further provided, which is configured for applying mutually independent voltages to each of the electrodes in the different microcavities (110).

15. The microfluidic device (100) according to any one of claims 11 to 14, wherein the microcavity (110) has at least one further electrode (445), wherein the electrode (230) and the at least one further electrode (445) are electrically insulated from each other.

16. The microfluidic device (100) according to any one of claims 11 to 15, wherein at least one counter electrode (455) is arranged on the microcavity (110), wherein the counter electrode (455) is arranged in or on the microcavity (110) opposite to each other with the electrode (230) and/or with at least one further electrode (445) and/or is electrically insulated from the electrode (230) and/or at least one further electrode (445).