Biosensor for detecting biological cells in a sample, method for producing a biosensor, method and device for operating a biosensor, and analysis system for analyzing a sample containing biological cells

Abstract

The invention relates to a biosensor (100) for detecting biological cells in a sample. The biosensor (100) comprises a plurality of first conductor strips (110) which are arranged parallel to one another and each of which can be individually connected to a first electrical potential. The biosensor (100) also comprises a plurality of second conductor strips (120) which are arranged parallel to one another and each of which can be individually connected to a second electrical potential, the first conductor strips (110) and the second conductor strips (120) crossing at a plurality of crossing points. The biosensor (100) further comprises an insulator layer structure (140), the insulator layer structure (140) being arranged between the first conductor strips (110) on one side and the second conductor strips (120) on the other side. The biosensor (100) additionally comprises a plurality of recessed portions (150) which are formed in the insulator layer structure (140) on the side of the second conductor strips (120), through the second conductor strips (120), in the region of the crossing points. Each recessed portion (150) is designed to couple to at least one biological identification element (X) capable of binding to the biological cells, in order to immobilize and detect at most one biological cell in the region of each of the recessed portions (150).

Description

[0001] R. 413359

[0002] - 1 -

[0003] Description

[0004] title

[0005] Biosensor for detecting biological cells in a sample, method for manufacturing a biosensor, method and apparatus for operating a biosensor, and analysis system for analyzing a sample containing biological cells.

[0006] State of the art

[0007] The invention relates to a device or a method according to the preamble of the independent claims. The present invention also relates to a computer program.

[0008] Biosensor technologies have revolutionized cell detection and can provide powerful tools for precise and rapid analysis in various scientific, medical, and industrial applications. In particular, such biosensors can leverage the integration of biological detection elements with advanced transducers to convert cellular interactions into measurable signals. Some well-known biosensor technologies include electrochemical biosensors, optical biosensors, and piezoelectric sensors. Electrochemical biosensors are a compact and cost-effective detection method that exploits the intricacies of biological interactions by converting them into measurable electrical signals. These biosensors typically rely on enzymatic reactions or binding events that occur at the sensor interface and cause changes in current or voltage.The inherent sensitivity and specificity of electrochemical biosensors make them advantageous instruments for applications such as disease diagnostics and environmental monitoring. R. 413359.

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[0010] However, challenges also exist regarding electrochemical biosensors. Potential interference from complex sample matrices can pose a challenge, requiring careful sample preparation and sensor optimization. Furthermore, ensuring the stability and efficacy of biological detection elements during immobilization can be another complex task. Despite these challenges, ongoing efforts are underway to optimize performance and improve the detection accuracy of electrochemical biosensors for cell identification. One challenge with electrochemical biosensor chips, for example, can be capturing a single cell and leaving it intact for subsequent experiments. Even when a cell is captured, determining its precise location can be difficult.This can be particularly problematic if all cells are important and a specific cell is to be targeted for further investigation, e.g., as part of a liquid biopsy diagnosis.

[0011] Disclosure of the invention

[0012] Against this background, the approach presented here introduces a biosensor, a method for manufacturing a biosensor, a method for operating a biosensor, a device that uses this method, and finally a corresponding computer program and an analysis system for analyzing a sample containing biological cells, according to the main claims. Advantageous further developments and improvements of the device specified in the independent claim are possible through the measures listed in the dependent claims.

[0013] According to embodiments, a multifunctional biosensor chip integration can be implemented to improve cellular diagnostics. Specifically, a sensor can be provided that can capture and test individual cells and subsequently determine their precise position. In other words, a chip array can be provided that can not only identify immobilized cells but also accurately locate their position. For example, it can also provide additional detection of R. 413359.

[0014] - 3 - cell-binding markers enable further investigations using surface plasmon resonance (SPR), fluorescence microscopy, electrical detection, or genetic analysis. For example, the biosensor can be used to measure a variety of parameters that cannot all be measured simultaneously or in combination using other isolated methods.

[0015] A biosensor for the detection of biological cells in a sample is presented, wherein the biosensor has the following features: a plurality of parallel first conductor strips made of an electrically conductive material, wherein each of the first conductor strips can be individually connected to a first electrical potential; a plurality of parallel second conductor strips made of an electrically conductive material, wherein each of the second conductor strips can be individually connected to a second electrical potential, and wherein the first conductor strips and the second conductor strips intersect at a plurality of intersection points; an insulating layer structure made of an electrically insulating material, wherein the insulating layer structure is arranged between the first conductor strips on the one hand and the second conductor strips on the other hand;and a plurality of depression sections formed in the insulator layer structure on the side of the second conductor strips in the area of ​​the intersection points, wherein each depression section is designed to couple with at least one biological recognition element capable of binding to biological cells in order to immobilize a maximum of one biological cell in the area of ​​each depression section.

[0016] The biosensor can be designed as an electrochemical biosensor, an optical biosensor, or a combination thereof. The biosensor can also be referred to as a biosensor device. (See biological R. 413359.)

[0017] - 4 -

[0018] The cells can be cancer cells or other human, animal, or plant cells. The sample can contain the biological cells and an electrically conductive buffer. The conductive buffer allows for the transport of electrical charge between the first and second conductor strips. The electrically conductive material can be gold or another metallic material. The electrically insulating material can be silicon dioxide, glass, or another insulator. The first and second electrical potentials can be different. The second conductor strips can have holes created by forming the well sections. The well sections can be blind holes.A specific well section can be individually addressed and read by applying electrical potentials to the conductor strips that intersect at the junction point where the specific well section is located. The biosensor can also have a carrier substrate, with the first conductor strips positioned between the carrier substrate and the insulating layer structure. Furthermore, the biosensor can have a protective layer on the insulating layer structure on the side of the second conductor strips outside the well sections.

[0019] In particular, this allows for the detection and, additionally or alternatively, the counting of individual cancer cells in individual wells or

[0020] The biosensor allows for the use of well sections. Compared to conventional designs, it can utilize a larger fluid volume and facilitate multiple washing cycles to capture a maximum number of cancer cells. Furthermore, each immobilized cell can be characterized using various methods, including electrochemical signals, refractive index changes with surface plasmon resonance (SPR), optical measurements with fluorescence microscopy, and subsequent genetic analysis. Another advantage is that a high-resolution optical system is not required, as it can be assumed that each well contains only one cell at most. As long as the electrical signal from each well is detectable, detection and analysis can be performed efficiently. R. 413359

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[0022] The biosensor can also feature a grid structure formed at the intersection points of the first conductor strips, designed to couple surface plasmon resonance modes into the biosensor. This enables the detection of refractive index changes or fluorescence excitation within cells immobilized in the region of the depressions. Such an approach enhances the possibilities for gaining insights into cell viability. Furthermore, with this configuration, the upper part of the cells remains accessible for various microscopy applications, including fluorescence or brightfield imaging.

[0023] Furthermore, the insulating layer structure can comprise a first layer with a first thickness and a second layer with a second thickness, the second thickness being greater than the first. The first layer can form the bottom areas of all recessed sections. The second layer can form the side walls of all recessed sections. Such a structure offers the advantage that, particularly due to the thinner first layer, the transport of electrical charge between the first and second conductor strips is facilitated, while reliable electrical insulation can still be achieved due to the thicker second layer. Sufficient volume can also be created to accommodate the recessed sections.

[0024] Furthermore, the insulator layer structure can be formed in each of the well sections to couple with the at least one biological recognition element. For this purpose, a surface of the insulator layer structure within each of the well sections can be treated and additionally or alternatively coated to facilitate the binding of the at least one biological recognition element. Such an embodiment offers the advantage that the immobilization of a single cell within or in a region of a well section can be reliably achieved and simplified. R. 413359

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[0026] According to one embodiment, each of the depression sections can have a diameter between 15 and 35 micrometers, for example, between 10 and 30 micrometers or between 15 and 20 micrometers. In this embodiment, a through-hole to the respective first conductor strip in the insulating layer structure can be formed in a base region of each depression section. The diameter of the through-hole can be a fraction of the diameter of a depression section. The through-hole allows for the transport of electrical charge. The depression sections can also be referred to as microholes. Such an embodiment offers the advantage that exactly one cell can be accommodated and immobilized within each of the depression sections.

[0027] The biosensor can also have multiple parallel third conductor strips made of an electrically conductive material. Each of these third conductor strips can be individually connected to a third electrical potential. The third conductor strips and the second conductor strips can intersect at multiple points. Furthermore, the biosensor can have a separating layer made of an electrically insulating material. This separating layer can be positioned between the third conductor strips and the second conductor strips, with the recessed sections extending through the separating layer. The third electrical potential can correspond to the first electrical potential, the second electrical potential, or another electrical potential.Such an embodiment offers the advantage that by applying the respective electrical potentials to the second and third conductor strips, a cell immobilized in a well section can be brought back to the vicinity of the well section's entrance, providing a versatile method for capturing the cell for subsequent, further analysis steps. In other words, such an embodiment offers the possibility of lifting or extracting a cell from any well section when necessary, increasing the versatility and application potential of the biosensor in various cell analysis scenarios. R. 413359.

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[0029] According to a further embodiment, each of the depression sections can have a diameter between 300 and 1000 nanometers, for example, between 200 and 600 nanometers or between 300 and 500 nanometers. In a base region of each depression section, the insulating layer structure can have a thickness between 1 and 5 nanometers or between 3 and 5 nanometers. The transport of electrical charge can be facilitated by so-called tunneling through the base region. The depression sections can also be referred to as nanoholes. Such an embodiment offers the advantage that exactly one cell can be contained within each depression section.

[0030] According to yet another embodiment, each of the depression sections can have a diameter between 300 and 1000 nanometers, between 200 and 600 nanometers, or between 300 and 500 nanometers. In this embodiment, a nanowire can be arranged in each depression section, the base of which is connected to one of the first conductor strips and extends through the insulating layer structure in a base region of each depression section and through the respective depression section, with its head protruding from the depression section. The head of each nanowire can be configured to couple with the at least one biological recognition element. The depression sections can also be referred to as nanoholes. Since the wire is located in a hole smaller than a cell, the cell cannot cause damage during cell flow, thus preventing signal loss.When a cell collides with the wire, it bounces off, which can provide statistical information about the speed and number of cells. If a cell becomes stuck to the wire, this can cause it to bend and thus permanently change its electrical voltage.

[0031] A method for manufacturing an embodiment of a biosensor mentioned herein is also presented, the method comprising the following steps: R. 413359

[0032] - 8 -

[0033] Producing an assembly comprising the insulator layer structure and the first and second conductor strips applied to it;

[0034] Formation of the depression sections in the area of ​​the intersections through the second conductor strips in the insulator layer structure on the side of the second conductor strips.

[0035] By carrying out the method, an embodiment of the biosensor mentioned herein can be advantageously produced. The production step and the shaping step can each comprise several sub-steps.

[0036] Furthermore, a method for operating an embodiment of a biosensor mentioned herein is presented, the method comprising the following steps:

[0037] Outputting an electrical signal to the conductor strips to apply the electrical potentials to the conductor strips of a junction in the area of ​​which a recessed section is arranged, to which at least one biological recognition element is coupled, to which a biological cell is bound; and

[0038] Evaluating a change in the electrical signal to characterize the biological cell.

[0039] This process can be implemented, for example, in software or hardware, or in a hybrid form of software and hardware, for example in a control unit.

[0040] The approach presented here further provides a device designed to carry out, control, and implement the steps of a variant of the method presented herein in appropriate facilities. This embodiment of the invention in the form of a device also allows the problem underlying the invention to be solved quickly and efficiently. R. 413359

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[0042] For this purpose, the device may have at least one processing unit for processing signals or data, at least one storage unit for storing signals or data, at least one interface to a sensor or actuator for reading sensor signals from the sensor or for outputting data or control signals to the actuator, and / or at least one communication interface for reading or outputting data embedded in a communication protocol. The processing unit may, for example, be a signal processor, a microcontroller, or the like, and the storage unit may be flash memory or a magnetic storage unit.The communication interface can be configured to read or output data wirelessly and / or via wired connections, whereby a communication interface that can read or output wired data can, for example, read this data electrically or optically from or output it into a corresponding data transmission line.

[0043] In this context, a device can be understood as an electrical device that processes sensor signals and outputs control and / or data signals accordingly. The device may have an interface, which can be implemented in hardware and / or software. In the case of a hardware-based interface, the interfaces can, for example, be part of a so-called system ASIC, which incorporates various functions of the device. However, it is also possible that the interfaces are separate integrated circuits or consist at least partially of discrete components. In the case of a software-based interface, the interfaces can be software modules, which, for example, are located on a microcontroller alongside other software modules.

[0044] Furthermore, an analysis system for analyzing a sample containing biological cells is presented, wherein the analysis system has the following features: an embodiment of a biosensor mentioned herein; R. 413359

[0045] - 10 - a microfluidic device configured to supply the sample to the biosensor; and an embodiment of a device referred to herein, wherein the device is electrically connected to the conductor strips of the biosensor.

[0046] In the analysis system, an embodiment of the biosensor mentioned herein and an embodiment of the device mentioned herein can advantageously work together to detect the biological cells. The microfluidic device can have at least one channel or chamber in which the biosensor can be arranged.

[0047] Also advantageous is a computer program product or computer program with program code that can be stored on a machine-readable carrier or storage medium such as a semiconductor memory, a hard disk memory or an optical memory and is used to carry out, implement and / or control the steps of the method according to one of the embodiments described above, in particular if the program product or program is executed on a computer or device.

[0048] Examples of the approach presented here are shown in the drawings and explained in more detail in the following description. It shows:

[0049] Fig. 1 shows a schematic representation of a biosensor according to an exemplary embodiment;

[0050] Fig. 2 shows a schematic representation of a biosensor according to an exemplary embodiment;

[0051] Fig. 3 shows a schematic representation of a biosensor according to an exemplary embodiment;

[0052] Fig. 4 shows a schematic representation of the production of the biosensor from Fig. 1; R. 413359

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[0054] Fig. 5 shows a schematic representation of a biosensor according to an exemplary embodiment;

[0055] Fig. 6 shows a schematic representation of an analysis system according to an exemplary embodiment;

[0056] Fig. 7 shows a flowchart of an embodiment of a method for operating a biosensor; and

[0057] Fig. 8 shows a flowchart of an exemplary embodiment of a method for manufacturing a biosensor.

[0058] In the following description of favorable embodiments of the present invention, the same or similar reference numerals are used for the elements shown in the various figures and acting similarly, without repeating these elements.

[0059] Fig. 1 shows a schematic representation of a biosensor 100 according to an exemplary embodiment. In the illustration of Fig. 1, the biosensor 100 is shown both in a top view (left in the illustration) and in a sectional view (right in the illustration). The biosensor 100 is a biosensor for the detection of biological cells in a sample, in particular cancer cells. The biosensor 100 comprises a plurality of first conductor strips 110, a plurality of second conductor strips 120, an insulating layer structure 140, and a plurality of recessed sections 150.

[0060] The first conductor strips 110 are arranged parallel to each other. The first conductor strips 110 are made of an electrically conductive material, for example, gold. Each of the first conductor strips 110 can be individually connected to a first electrical potential or is connected to one. The second conductor strips 120 are arranged parallel to each other. The second conductor strips 120 are made of an electrically conductive material, for example, gold. Each of the second conductor strips 120 is individually connected to a second R. 413359

[0061] - 12 - electrical potential connectable or connected. The first conductor strips 110 and the second conductor strips 120 cross at a plurality of intersection points. Thus, the second conductor strips 120 extend transversely to the first conductor strips 110.

[0062] In the illustration of Fig. 1, a voltage source V is shown only as an example for clarification. This voltage source is electrically connected to each of the first conductor strips 110 and each of the second conductor strips 120 in order to apply the potentials to the conductor strips 110 and 120. The voltage source V is, for example, part of a device for operating the biosensor 100 or the same.

[0063] The insulating layer assembly 140 is formed from an electrically insulating material. The insulating layer assembly 140 is arranged between the first conductor strips 110 on one side and the second conductor strips 120 on the other. In other words, the first conductor strips 110 are arranged on a first side of the insulating layer assembly 140, and the second conductor strips 120 are arranged on a second side of the insulating layer assembly 140, opposite the first side.

[0064] The recessed sections 150 are formed in the area of ​​the intersections where the first conductor strips 110 and the second conductor strips 120 cross. The recessed sections 150 extend through the second conductor strips 120 into the insulator layer structure 140 on the side of the second conductor strips 120. Each recessed section 150 is designed to couple with at least one biological recognition element X capable of binding to biological cells, in order to immobilize a maximum of one biological cell in the area of ​​each recessed section 150. In other words, at most one single cell can be immobilized via biological recognition elements X in the area of ​​at least one of the recessed sections 150, or at most of all recessed sections 150.

[0065] In the top view, it can be seen that the biosensor 100 is designed as a biosensor chip with an array of well sections 150 similar to a microtiter plate. In other words, the biosensor 100, according to R. 413359, has

[0066] - 13 - The illustrated embodiment features a microhole structure. The sectional view shows only three depression sections 150 as examples, with a cell immobilized in only two of them. Furthermore, a support substrate 105 is shown, wherein the first conductor strips 110 are arranged between the substrate 105 and the insulating layer structure 140.

[0067] According to the embodiment shown here, the insulator layer structure 140 is formed in each of the depression sections 150 to couple with the at least one biological recognition element X. In particular, the insulator layer structure 140 comprises a first layer 142 with a first thickness and a second layer 146 with a second thickness that is greater than the first thickness. The first layer 142 forms the bottom regions of all depression sections 150. The second layer 146 forms the side walls of all depression sections 150.

[0068] According to the embodiment shown here, each of the recessed sections 150 has a diameter between 15 and 35 micrometers, between 10 and 30 micrometers or between 15 and 20 micrometers.

[0069] Furthermore, in the insulating layer structure 140, more precisely in its first layer 142, a through-opening 144 to the respective first conductor strip 110 is formed in a bottom area of ​​each of the depression sections 150. The diameter of the through-opening 144 is a fraction of the diameter of a depression section 150.

[0070] Furthermore, according to the embodiment shown here, a lattice structure 115 is formed in the area of ​​the intersections on the first conductor strips 110. The lattice structure 115 is designed to couple surface plasmon resonance modes into the biosensor 100, more precisely into the recessed sections 150, in response to excitation by, for example, a laser. This is illustrated in more detail in the figure by an enlarged section of the area of ​​a recessed section 150.

[0071] In other words, the Biosensor 100 is designed as a chip array for capturing cells at microhole positions, i.e., the R. 413359

[0072] - 14 -

[0073] The biosensor 100 is designed with well sections 150. A schematic representation of the biosensor 100 is shown in Fig. 1. The biosensor 100 comprises mutually perpendicular gold strips, or first conductor strips 110, and second conductor strips 120 on two sides of the insulating layer structure 140, which is, for example, designed as a thin glass substrate. The first conductor strips 110 are located below the glass, the second conductor strips 120 above. The microholes, or well sections 150, are strategically arranged at the junctions or intersections of the first conductor strips 110 and the second conductor strips 120. By applying a voltage to each of the conductor strips 110 and 120, an electric current can flow through a conductive buffer of the sample at a single hole position or selected well section. The well sections 150 are generally larger than the cells, measuring approximatelyThe microholes are 15-20 pm in diameter and can enclose the cells. According to the embodiment shown here, biological recognition elements X, designed as so-called redox linkers, are first attached to the cell, and then the redox-labeled cell is inserted into the biosensor 100 containing a conductive buffer, e.g., salt water. As can be seen in Fig. 1, the bottom of the microholes or well sections 150 is covered with a glass coating, i.e., the first layer 142 of the insulating layer structure 140, and has only a small opening 144, for example, 1-2 pm in diameter, which connects the first conductive strip 110 to the buffer. The recognition elements X can be attached to the glass bottom of the well section 150 or, in other words, bind to the well section 150. For this purpose, a recognition strand X can, for example, consist of ssDNA, and a reverse complementary ssDNA can be immobilized in the cavity.Alternatively, in this procedure it could also suffice if cell-DNA complexes sediment into the cavities and unbound DNA is previously separated from the complexes, for example by centrifugation / washing. The cells are now located between two oppositely charged electrodes, i.e., the first conductor strip 110 and the second conductor strip 120.

[0074] By applying a voltage when the cells are immobilized, the fixed cell can be characterized using the EClS (Electric Cell-Substrate Impedance Sensing) technique. Due to the field variations near the R. 413359

[0075] - 15 -

[0076] The edges and the dielectrophoresis effect improve the ability to trap particles or cells at well section 150. This not only accelerates the immobilization process but also minimizes the risk of cells detaching during subsequent washing steps. The signal change in this method depends on the properties of the redox linker or biological recognition element X and the number of recognition elements X in the current path. By using multiple redox-labeled nucleotides (1–1000) within a DNA origami linker structure, the signal per affinity molecule can be amplified. These affinity molecules are programmed to target specific cell surface receptors. Since the number of specific cell surface receptors ranges from 10 2 and 10 6Since there can be a certain number of redox tags per cell, and most, if not all, of the receptors bind to the affinity reagent, it is likely that the molarity of the redox tag exceeds the detection limit. Because different redox tags can be electrochemically distinguished, it is possible to target different surface molecules on a cell simultaneously. Furthermore, these affinity reagent conjugates could contain biotin / streptavidin or click chemistry modalities, or reverse complementary ssDNA, which further support the immobilization of cells in the cavities or depressions.

[0077] Following cell sedimentation at the bottom of the microhole or well section 150, it is also possible to couple a surface plasmon resonance (SPR) mode into the cell by using a grating or grating structure 115 on the gold strip or first conductor strip 110 beneath the glass layer or insulating layer assembly 140. This enables the detection of refractive index changes or fluorescence excitations within the cell. Such an approach improves the possibilities for gaining insights into cell viability. Furthermore, with this configuration, the upper part of the cell remains accessible for various microscopy applications, including fluorescence or brightfield imaging.

[0078] In other words, Fig. 1 illustrates the detection of redox-labeled cells using ECIS and SPR techniques. The biosensor 100 is located at R. 413359.

[0079] - 16 - for example, in a microfluidic channel, and the sample buffer has high electrical conductivity. A lattice structure 115 on the gold strips or first conductor strips 110 helps to couple an SPR mode into the system and detect the change in the refractive index or excite fluorescent molecules. Some exemplary approximate dimensions of the biosensor 100 are a diameter of each of the well sections 150 of approximately 15–20 pm, a thickness of the first conductor strips 110 of approximately 50–100 nm, a thickness of the second conductor strips 120 of approximately 10–50 nm, and a thickness of the second layer 146 of the insulating layer structure 140 of approximately 10–20 pm.

[0080] Fig. 2 shows a schematic representation of a biosensor 100 according to an exemplary embodiment. The representation in Fig. 2 is similar to that of Fig. 1. The biosensor 100 in Fig. 2 corresponds to the biosensor from Fig. 1 except that the base of the well sections 150 is formed completely without a through-hole, and the dimensions of individual features of the biosensor 100 differ from those of the biosensor in Fig. 1. The well sections 150 form a nanohole structure. Thus, each of the well sections 150 is smaller than a cell. A cell immobilized in the region of a well section is connected to the base of the well section 150 via a biological recognition element X. In the representation of Fig. 2, the optional grid structure and the substrate or support substrate are also omitted.

[0081] According to the embodiment shown here, each of the recessed sections 150 has a diameter between 300 and 1000 nanometers, between 200 and 600 nanometers, or between 300 and 500 nanometers. In a bottom region of each of the recessed sections 150, the insulating layer structure 140 has a thickness between 1 and 5 nanometers or between 3 and 5 nanometers. In other words, the thickness of the first layer 142 of the insulating layer structure 140 is between 1 and 5 nanometers or between 3 and 5 nanometers. Furthermore, the thickness of the second layer 146 of the insulating layer structure 140 is between approximately 5 and 10 nanometers. Additionally, the thickness of the second conductor strips 120 is between approximately 5 and 10 nanometers. R. 413359

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[0083] According to the embodiment shown here, the biosensor 100 is designed as a chip array for capturing cells at well sections 150 configured as nanohole positions, while measuring the current flowing through a cell connected to the hole position or well section 150 by a redox linker or detection element X. The biosensor 100 resembles the biosensor from Fig. 1 with two sets of gold strips or conductor strips 110 and 120 and an insulator or insulating layer structure 140 between them. In this biosensor 100, the hole diameter or diameter of the well sections 150 is in the nanometer range (300-500 nm), and the insulating layer or first layer 142 is between 3-5 nm thick. Since the distance between two electrodes (in this case gold, but they can also be any conductive electrodes) orSince the distance between the first conductor strip 110 and the second conductor strip 120 is only a few nanometers, an electric current can be generated by applying a voltage to each of the first and second conductor strips 110 and 120 at a single hole position or a single depression section 150, thus tunneling through the glass and the conductive buffer. Each gold strip or conductor strip 110 and 120 is independently connected to the voltage source.

[0084] To control cell immobilization on the Biosensor 100 or chip, the Biosensor 100 is integrated into a microfluidic chamber. A redox linker, acting as a biological recognition element X and consisting of DNA origami building blocks modified with redox-active tags, a biotin tag, and an azide / alkyne tag, serves to immobilize the cells at the positions of the well sections 150 or nanohole positions. In the presence of cells, copper-free thickening (Cu) is possible. Example: Strained ring systems such as cyclooctine. The nanoholes or well sections 150 can be decorated with streptavidin to enable biotin binding. The biotin itself is bound to a DNA strand that contributes to the DNA origami structure. These DNA origami structures can only be bound to a glass surface or surface of the insulator layer assembly 140 from one side and to the target cell from the other side.Therefore, if DNA origami building blocks are introduced into the buffer and this is then inserted into the Biosensor 100, the DNA building blocks bond with the base of the R. 413359.

[0085] - 18 -

[0086] The DNA origami structure is formed in situ by connecting segment 150 (e.g., by biotin, azide-alkyne click chemistry, or an immobilized primer for hybridization that is reverse complementary). A biotin unit is located at the apex of the DNA origami structure. This biotin serves to interact with cell-bound affinity molecules modified with streptavidin to immobilize the desired cells. Cell-bound affinity molecules could be directly linked to the origami structures. Alternatively, affinity molecules could be pre-modified covalently with origami structures using click chemistry, thiol-maleimide chemistry, or primary amine NHS esters. In the subsequent phase, the cells labeled with complementary antibodies are introduced into the biosensor 100 via the microfluidic channel.Cells possessing the corresponding affinity reagent-biotin conjugate bind to the redox linker acting as recognition element X, while the remaining cells are washed away in subsequent steps.

[0087] Fig. 3 shows a schematic representation of a biosensor 100 according to an exemplary embodiment. The representation in Fig. 3 corresponds to that of Fig. 1 and / or Fig. 2. The biosensor 100 in Fig. 3 corresponds to or is similar to the biosensor from Fig. 2, except that a nanowire 360 ​​with a foot end and a head end is arranged in each of the recess sections 150, the foot end being connected to one of the first conductor strips 110, the nanowire 360 ​​extending through the insulating layer structure 140 in a bottom region of each of the recess sections 150 and through the respective recess section 150, the head end protruding from the recess section 150, and the head end of each nanowire 360 ​​being configured to couple with the at least one biological recognition element X.

[0088] An alternative method for cell detection, according to the embodiment shown here, involves the use of nanowires 360 that are slightly longer than the depth of the recessed sections 150 designed as nanoholes. In this setup, the nanowire 360 ​​protrudes through the recessed section 150 and can bend when a cell attaches to it. This approach offers advantages over conventional nanowire connections. Since the nanowire 360 ​​is located in a recessed section 150 that is smaller than the R. 413359

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[0090] Since the cell is protected, it cannot cause damage during cell flow, thus preventing signal loss. If the Nanowire 360 ​​were damaged, the signal could otherwise be affected. When a cell collides with the Nanowire 360, it rebounds, providing statistical information about cell velocity and quantity. If a cell becomes attached to the Nanowire 360, it causes a bend and consequently a permanent change in voltage. To selectively bind cells only to the tip or end of the Nanowire 360, another conductive material, such as platinum (Pt), can be introduced to which the linker binds exclusively, for example, via streptavidin-biotin or click chemistry to immobilize an affinity reagent. This nanowire sensor technology for cell detection increases both the precision and robustness of the cell detection system.

[0091] Fig. 4 shows a schematic representation of the fabrication of the biosensor 100 from Fig. 1. In other words, the fabrication of a microchip is shown schematically here. Fig. 4 illustrates nine exemplary steps A to I of the chip fabrication or fabrication of the biosensor 100. Steps A to I are carried out in connection with a fabrication process such as the one shown in Fig. 8.

[0092] First, in a first step (A), gold strips are deposited onto a resolvable surface or layer using conventional lithography techniques. Then, in a second step, the grid or lattice structure is applied to the first conductor strips using a focused ion beam (FIB) or similar methods. Next, in a third step (C), the gold or the first conductor strips are covered with the substrate. In a fourth step (D), the chip is rotated and the resolvable layer is removed. In a fifth step (E), the chip surface is coated with a thin SiO2 layer as an insulator, and a column is created at each hole or well position using lithography. Finally, in a sixth step (F), the chip is coated with a 10-20 pm thick SiO2 layer to complete the insulator layer buildup.In a seventh step G, further gold strips R. 413359 are then added for the second conductor strips.

[0093] - 20 - applied. To protect the remaining gold strips and prevent cells from adhering to any part other than the holes or depressions, the chip is coated with a thin protective layer in an eighth step (H). In a ninth step (I), the pillars are removed and the bottom of the depressions is opened by 1-2 pm to create the through-holes so that the first conductor strips can make contact with the buffer. The partial illustration depicting the ninth step (I) shows the completed BioSensor 100 in a sectional view.

[0094] Fig. 5 shows a schematic representation of a biosensor 100 according to an exemplary embodiment. The representation in Fig. 5 is similar to the sectional view of Fig.

[0095] 1. The biosensor 100 in Fig. 5 corresponds to the biosensor from Fig. 1, except that the biosensor 100 additionally has a plurality of third conductor strips 530 and a separating layer 548. The third conductor strips 530 are arranged parallel to each other. The third conductor strips 530 are formed from an electrically conductive material. Each of the third conductor strips 530 can be individually connected to a third electrical potential. The third conductor strips 530 and the second conductor strips 120 intersect at the plurality of intersection points. Thus, the third conductor strips 530 extend transversely to the second conductor strips 120 and, for example, along the first conductor strips 110. The separating layer 548 is formed from an electrically insulating material. The separating layer 548 is arranged between the third conductor strips 530 on the one hand and the second conductor strips 120 on the other.The depression sections 150 also extend through the separating layer 548. The diagram also shows an electric field E, which can be generated by applying an electrical voltage to the second conductor strips 120 and the third conductor strips 530, as well as a liquid flow or fluid flow F of the sample across the biosensor 100.

[0096] With the additional third conductor strips 530 and the separating layer 548, the biosensor 100, according to the embodiment shown here, has a lifting system or lifting function. In the diagnostic process, the ability to perform additional experiments on identified cells is of great importance. For example, if a cancer cell is detected, it is R. 413359

[0097] - 21 - important to determine the specific type of cancer or to perform drug tests on the cell. The integration of the biosensor 100 into a cytometry system according to the embodiment shown here facilitates such experiments. As shown in Fig. 5, the introduction of a third conductive layer in the form of the third conductor strips 530 on the microhole chip enables the lifting of cells. This configuration makes it possible to detach a cell from the surface and suspend it in each well section 150. By applying a voltage to the two upper conductive layers, i.e., the third conductor strips 530 and the second conductor strips 120, as shown in Fig. 5, the cell can be brought close to the entrance of the well section 150, which is a versatile method for capturing the cell for further analysis. In other words, Fig. 5 shows5. Lifting a cell in a biosensor 100 similar to a microtiter plate and passing it through the liquid stream F (cytometry) for further experiments.

[0098] Fig. 6 shows a schematic representation of an analysis system 600 according to an exemplary embodiment. The analysis system 600 is designed to perform an analysis of a sample containing biological cells. The analysis system 600 comprises the biosensor 100 from one of the figures described above, a device 670 for operating the biosensor 100, and a microfluidic device 690. The microfluidic device 690 is designed to supply the sample to the biosensor 100. The biosensor 100 is, for example, arranged in a fluid chamber of the microfluidic device 690.

[0099] The device 670 is electrically connected to the conductor strips of the biosensor 100. The device 670 comprises an output device 672 and an evaluation device 674. The output device 672 is configured to output an electrical signal to the conductor strips of the biosensor 100 in order to apply electrical potentials to the conductor strips at a junction point in the area of ​​which a recessed section is arranged, to which at least one biological recognition element is coupled, and to which a biological cell is bound. The evaluation device 674 is configured to evaluate a change in the electrical signal in order to characterize the biological cell. R. 413359

[0100] - 22 -

[0101] Fig. 7 shows a flowchart of an embodiment of method 770 for operating a biosensor. Method 770 can be implemented to operate the biosensor from any of the figures described above. Method 770 can be implemented in conjunction with the device from Fig. 6 or a similar device. Method 770 comprises a step 772 of outputting an electrical signal to the conductor strips to apply electrical potentials to the conductor strips at a junction, in the area of ​​which a recessed section is arranged to which at least one biological recognition element is coupled, to which a biological cell is bound. Method 770 also comprises a step 774 of evaluating a change in the electrical signal to characterize the biological cell.

[0102] Fig. 8 shows a flowchart of an embodiment of method 880 for manufacturing a biosensor. By carrying out method 880, the biosensor can be manufactured from one of the figures described above. Method 880 comprises a step 882 of producing an assembly that includes the insulator layer structure and the first and second conductor strips applied thereto. Method 880 also comprises a step 888 of forming the recessed sections in the region of the intersections through the second conductor strips in the insulator layer structure on the side of the second conductor strips.

[0103] If an embodiment includes an “and / or” connection between a first feature and a second feature, this is to be read as meaning that the embodiment according to one embodiment has both the first feature and the second feature, and according to another embodiment either only the first feature or only the second feature.

Claims

R. 413359 - 23 - Claims 1. Biosensor (100) for detecting biological cells in a sample, the biosensor (100) having the following features: a plurality of first conductor strips (110) arranged parallel to one another, made of an electrically conductive material, wherein each of the first conductor strips (110) can be individually connected to a first electrical potential; a plurality of second conductor strips (120) arranged parallel to one another, made of an electrically conductive material, wherein each of the second conductor strips (120) can be individually connected to a second electrical potential, the first conductor strips (110) and the second conductor strips (120) intersecting at a plurality of intersection points; an insulating layer structure (140) made of an electrically insulating material, wherein the insulating layer structure (140) is arranged between the first conductor strips (110) on the one hand and the second conductor strips (120) on the other hand;and a plurality of depression sections (150) formed in the insulator layer structure (140) on the side of the second conductor strips (120) in the region of the intersection points, each depression section (150) being designed to couple with at least one biological recognition element (X) capable of binding to biological cells in order to immobilize and detect a maximum of one biological cell in the region of each depression section (150). R. 413359 - 24 - 2. Biosensor (100) according to claim 1, comprising a grid structure (115) formed in the region of the intersection points on the first conductor strips (110), which is formed to couple surface plasmon resonance modes into the biosensor (100).

3. Biosensor (100) according to any of the preceding claims, wherein the insulator layer structure (140) comprises a first layer (142) with a first thickness and a second layer (146) with a second thickness, wherein the second thickness is greater than the first thickness, wherein the first layer (142) forms bottom areas of all depression sections (150), and wherein the second layer (146) forms side walls of all depression sections (150).

4. Biosensor (100) according to one of the preceding claims, wherein the insulator layer structure (140) is configured in each of the recessed sections (150) to couple with the at least one biological recognition element (X).

5. Biosensor (100) according to one of the preceding claims, wherein each of the depression sections (150) has a diameter between 15 and 35 micrometers, wherein in a bottom region of each of the depression sections (150) a through-hole (144) to the respective first conductor strip (110) in the insulator layer structure (140) is formed, wherein a diameter of the through-hole (144) is a fraction of the diameter of a depression section (150).

6. Biosensor (100) according to claim 5, comprising a plurality of third conductor strips (530) arranged parallel to one another, made of an electrically conductive material, wherein each of the third conductor strips (530) can be individually connected to a third electrical potential, wherein the third conductor strips (530) and the second conductor strips (120) intersect at the plurality of intersection points, and comprising a separating layer (548) made of an electrically insulating material, wherein the R. 413359 - 25 - The separating layer (548) is arranged between the third conductor strips (530) on the one hand and the second conductor strips (120) on the other hand, wherein the recessed sections (150) extend through the separating layer (548).

7. Biosensor (100) according to any one of claims 1 to 4, wherein each of the depression sections (150) has a diameter between 300 and 1000 nanometers, wherein in a bottom region of each of the depression sections (150) the insulator layer structure (140) has a thickness between 1 and 5 nanometers or between 3 and 5 nanometers.

8. Biosensor (100) according to any one of claims 1 to 3, wherein each of the depression sections (150) has a diameter between 300 and 1000 nanometers, wherein a nanowire (360) is arranged in each of the depression sections (150), the foot end of which is connected to a respective first conductor strip (110) and which extends through the insulating layer structure (140) in a bottom region of each of the depression sections (150) and through the respective depression section (150) and the head end of which protrudes from the depression section (150), wherein the head end of each nanowire (360) is configured to couple with the at least one biological recognition element (X).

9. Method (880) for producing a biosensor (100) according to any one of the preceding claims, wherein the method (880) comprises the following steps: Producing (882) an assembly comprising the insulator layer assembly (140) and the first conductor strips (110) and second conductor strips (120) applied thereto; Forming (888) the recessed sections (150) in the area of ​​the intersections through the second conductor strips (120) in the insulator layer structure (140) on the side of the second conductor strips (120). R. 413359 - 26 - 10. Method (770) for operating a biosensor (100) according to any one of claims 1 to 8, wherein the method (770) comprises the following steps: Output (772) of an electrical signal to the conductor strips (110, 120) to apply the electrical potentials to the conductor strips (110, 120) of a junction in the area of ​​which a recessed section (150) is arranged, to which at least one biological recognition element (X) is coupled, to which a biological cell is bound; and Evaluate (774) a change in the electrical signal to characterize the biological cell.

11. Device (670) configured to perform and / or control the steps of the method (770) according to claim 10 in corresponding units (672, 674).

12. Analysis system (600) for analyzing a sample containing biological cells, wherein the analysis system (600) has the following features: a biosensor (100) according to any one of claims 1 to 8; a microfluidic device (690) configured to supply the sample to the biosensor (100); and a device (670) according to claim 11, wherein the device (670) is electrically connected to the conductor strips (110, 120) of the biosensor (100).

13. Computer program configured to execute and / or control the steps of the method (770) according to claim 10. R. 413359 - 27 - 14. Machine-readable storage medium on which the computer program according to claim 13 is stored.

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