Systems and methods for patterning and spatial electrochemical mapping of cells

Abstract

Disclosed herein are devices for electrically assessing and / or manipulating cells. One aspect relates to electrical mapping of cells on the surface of a semiconductor substrate via cross electrode impedance measurements. Additionally, according to some aspects, the electrode array enables spatially addressable electrical stimulation and / or recording of electrical signals in real time using CMOS circuitry. Some of these aspects relate to performing cell patterning through electrochemical gas generation and extracellular electrochemical mapping using the electrode array.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 040,439, filed June 17, 2020, entitled “Systems and Methods for Patterning and Spatial Electrochemical Mapping of Cells,” which is incorporated herein by reference in its entirety. Background Technology

[0003] This disclosure relates to semiconductor devices for the electrical evaluation of cells or other biological samples. Summary of the Invention

[0004] This document discloses various devices for electro-assessment and / or manipulation of cells. One aspect relates to electro-mapping of cells on the surface of a semiconductor substrate via cross-electrode impedance measurement. Additionally, according to some aspects, electrode arrays enable spatially addressable electrical stimulation and / or recording of electrical signals in real time using CMOS circuitry. Some of these aspects involve performing cell patterning using electrode arrays via electrochemical gas generation and extracellular electrochemical mapping.

[0005] Some embodiments relate to an apparatus for mapping one or more cells. The apparatus includes a semiconductor substrate. The semiconductor substrate includes: a plurality of electrodes exposed at a surface of the semiconductor substrate; and active circuitry coupled to the plurality of electrodes and configured to measure a first set of cross-electrode currents between a first electrode and some or all of the remaining electrodes, and to measure a second set of cross-electrode currents between a second electrode and some or all of the remaining electrodes. The apparatus further includes one or more processors configured to receive the measured cross-electrode currents from the active circuitry and to generate a mapping of the one or more cells based on the first set of cross-electrode currents and the second set of cross-electrode currents.

[0006] In some embodiments, the active circuitry is further configured to apply a stimulation signal at the first electrode of the plurality of electrodes and a reference voltage at the remaining electrodes from which the cross-electrode current is measured. The stimulation signal may have a frequency less than 10 kHz, preferably between 0.1 kHz and 5 kHz. The plurality of electrodes may be arranged in an array with a spacing of less than 30 μm, preferably less than 5 μm. The semiconductor substrate may include silicon. The semiconductor substrate may include a silicon substrate, and the active circuitry may include complementary metal-oxide-semiconductor (CMOS) components in the silicon substrate. The plurality of electrodes may include a plurality of pads disposed on an insulating surface of the semiconductor substrate. The active circuitry may include a plurality of recording circuits, each configured to measure the current at one of the electrodes. The plurality of recording circuits may include at least eight recording circuits, at least ten recording circuits, and preferably at least 4000 recording circuits. Each recording circuit may include a transimpedance amplifier (TIA). The TIA may include an impedance component having a resistance of at least 10 MΩ, at least 100 MΩ, or between 10 MΩ and 1 GΩ, wherein the output voltage of the TIA is proportional to the voltage across the impedance component. The impedance component may include a switched capacitor. The one or more cells may be deployed in a first hole of a porous plate, and the plurality of electrodes may be a first electrode array exposed to the first hole. The device may also include a second electrode array exposed to the surface of the semiconductor substrate and to a second hole of the porous plate. The porous plate may include at least 24, at least 96, or at least 384 holes. The size of the plurality of electrodes may be determined such that more than one electrode is configured to contact one of the one or more cells. The plurality of pads may include Au. The plurality of pads may include Pt.

[0007] Some embodiments relate to a method for mapping one or more cells in contact with an array of electrodes deployed on a surface region of a semiconductor substrate. Each electrode in the electrode array has an electrode location on the surface region. The method includes: applying a stimulation signal at each of at least one electrode in the electrode array; measuring a set of cross-electrode currents between the electrode and some or all of the remaining electrodes in the electrode array; generating representative values ​​associated with the electrode locations of the electrodes based on the set of cross-electrode currents; and generating a graph of the representative values ​​on the surface region based on the generated representative values ​​and the corresponding associated electrode locations of the at least one electrode.

[0008] In some embodiments, generating the representative value includes selecting the maximum current value from the set of cross-electrode currents as the representative value. Generating the representative value may include selecting the maximum current value from the set of cross-electrode currents as the representative value. The at least one electrode may include all electrodes in the electrode array. The spatial resolution of the graph may be 20 μm or less, and preferably 5 μm or less.

[0009] In some embodiments, the generated map is a first map generated at a first time and includes a plurality of pixels, and the method further includes: at a second time following the first time, generating a second map of the representative value on the surface region, wherein the second map includes a plurality of pixels; determining a first count of pixels in the first map having representative values ​​within a predetermined range; determining a second count of pixels in the second map having representative values ​​within the predetermined range; and determining cell adhesion characteristics based on a comparison of the first count and the second count. The map may include a plurality of pixels, each pixel being associated with a representative value. The at least one electrode may include a first electrode having a first electrode location and a second electrode having a second electrode location, the first electrode and the second electrode being adjacent to each other on the surface region, and the map may include a first pixel and a second pixel corresponding to the first electrode location and the second electrode location, respectively. Generating the map may include determining an amplified representative value associated with a third pixel between the first pixel and the second pixel. Determining the amplified representative value may include: by using a cross-electrode current I between the first electrode and the second electrode when a stimulation signal is applied at the second electrode. 12 The amplified electrode current is calculated by dividing by the product of the first current I1 and the second current I2, where I1 is the sum of the cross-electrode currents measured at all remaining electrodes when a stimulation signal is applied at the first electrode, and I2 is the sum of the cross-electrode currents measured at all remaining electrodes when a stimulation signal is applied at the second electrode. The number of pixels in the image may be greater than the number of electrodes in the electrode array. The electrode positions in the electrode array may be arranged in multiple rows and columns. The electrode array may have M rows and N columns, and the image may have at least 3M × 3N pixels.

[0010] Some embodiments relate to a system for mapping one or more cells. The system includes: a plurality of electrodes exposed at a surface region of a semiconductor substrate; circuitry deployed in the semiconductor substrate, controllable to apply a stimulation signal and / or measure a current at one or more of the plurality of electrodes; at least one non-transitory computer-readable medium storing executable instructions thereon; and at least one processor programmed by the executable instructions to perform a method. The method includes the following actions: controlling the circuitry to apply a stimulation signal at each of the plurality of electrodes; controlling the circuitry to measure a set of cross-electrode currents between the electrode and some or all of the remaining electrodes of the plurality of electrodes; generating a representative value associated with the electrode location of the electrode based on the set of cross-electrode currents; and generating a graph of the representative values ​​on the surface region based on the generated representative values ​​and the corresponding associated electrode locations of the plurality of electrodes.

[0011] In some embodiments, generating the representative value includes selecting the maximum current value from the set of cross-electrode currents as the representative value. Generating the representative value may include selecting the maximum current value from the set of cross-electrode currents; and calculating impedance based on the selected maximum current value as the representative value. The graph may include a plurality of pixels, each pixel being associated with a representative value. The plurality of electrodes may include a first electrode having a first electrode location and a second electrode having a second electrode location, the first electrode and the second electrode being adjacent to each other on the surface region. The graph may include a first pixel and a second pixel corresponding to the first electrode location and the second electrode location, respectively, and generating the graph may include determining an enlarged representative value associated with a third pixel between the first pixel and the second pixel.

[0012] Some embodiments relate to a method for providing an electrochemical reaction with a spatially positioned array of electrodes exposed on the surface of a semiconductor substrate. The method includes: selecting one or more electrodes in the electrode array; and controlling circuitry in the semiconductor substrate to apply one or more stimulation signals at the one or more electrodes to initiate an electrochemical reaction at the one or more electrodes.

[0013] In some embodiments, the electrochemical reaction may be a half-reaction that generates a gas in solution, and the one or more stimulus signals may include a potential higher than the redox potential used to generate the gas. The solution may include a plurality of cells adhered to the surface of the semiconductor substrate, and the method may further include: generating a gas at selected one or more electrodes such that at least one cell of the plurality of cells deployed on the selected one or more electrodes detaches from the surface of the semiconductor substrate. The gas may include H2, Cl2, or O2. The plurality of cells may be a plurality of cells of a first type, and the method may further include: seeding one or more cells of a second type on the surface of the semiconductor substrate at the location where at least one cell of the first type has detached. In some embodiments, the method may further include: mapping the regrowth time series of the plurality of cells on the surface at the location where at least one cell of the first type has detached; and determining the growth rate of the plurality of cells based on the mapping. The control circuit applying one or more predetermined potentials may include performing cyclic voltammetry at the selected one or more electrodes, and the method may further include: using the circuit to measure the value of an electrical characteristic at each of some or all of the remaining electrodes in the electrode array located outside the selected one or more electrodes; and generating a graph of the electrical characteristic based on the measurement results. The electrical characteristic may be a characteristic of open-circuit potential. The electrical characteristic may be a current. The current characteristic may be the maximum extent of the range of cyclic current.

[0014] In some embodiments, applying one or more predetermined potentials by the control circuit may include applying a pulsating voltage signal at one of the selected one or more electrodes. During a first portion of the pulsating voltage signal, the electrode is oxidized, and during a second portion of the pulsating voltage signal, the oxide on the electrode is reduced. The method may further include: using the circuit to measure a current signal at the electrode during the second portion of the pulsating voltage signal; determining an oxygen concentration at the location of the electrode based on the time-varying rate of change of the current signal; and generating a graph of the oxygen concentration based on the determined result. The one or more potentials may be relative to the potential of a reference electrode.

[0015] Some embodiments relate to a system. The system includes a semiconductor substrate. The semiconductor substrate includes: an electrode array comprising a plurality of independently addressable electrodes disposed on a surface of the semiconductor substrate; and circuitry controllable by one or more processors to apply one or more potentials relative to a potential of an electrode in the electrode array or a potential of a reference electrode at a group of electrodes in the electrode array to initiate an electrochemical reaction at the group of electrodes.

[0016] In some embodiments, the electrode array may include a plurality of pads deployed on an insulating surface of the semiconductor substrate. The plurality of pads may include Au or Pt. The reference electrode may be an Ag / AgCl reference electrode. The electrode array may include at least 1,000, at least 4,000, or at least 1,000,000 electrodes, and the circuitry may include a plurality of recording circuits, each configured to measure the current at an electrode in the electrode array. The plurality of recording circuits may include at least 10 or at least 4,000 recording circuits. Each recording circuit may include a transimpedance amplifier (TIA). The TIA may include an impedance component having a resistance of at least 10 MΩ, wherein the output voltage of the TIA is proportional to the voltage across the impedance component. The impedance component may include a switched capacitor.

[0017] Some embodiments relate to a system for providing spatially positioned electrochemical reactions. The system includes: an electrode array exposed at a surface region of a semiconductor substrate; circuitry deployed in the semiconductor substrate and coupled to the electrode array; at least one non-transitory computer-readable medium storing executable instructions thereon; and at least one processor programmed by the executable instructions to perform a method. The method includes actions including: selecting a pattern of electrodes in the electrode array; and controlling circuitry to apply one or more predetermined potentials at the pattern of electrodes relative to a potential of an electrode in the electrode array or a potential of a reference electrode, such that an electrochemical reaction is initiated at the pattern of electrodes. Attached Figure Description

[0018] Various aspects and embodiments will be described with reference to the following accompanying drawings. It should be understood that the drawings are not necessarily drawn to scale. Items appearing in multiple drawings are indicated by the same reference numerals in all drawings in which they appear. In the drawings:

[0019] Figure 1a This is a schematic side view of a semiconductor substrate according to some embodiments;

[0020] Figure 1b yes Figure 1a The diagram shows a two-dimensional data curve of the simulated voltage distribution in the device.

[0021] Figure 1c Is with Figure 1b The example shown in the figure corresponds to the data curve of the simulated electric field line;

[0022] Figure 2aThis is a schematic side view of an apparatus having a semiconductor substrate in the absence of cells, according to some embodiments;

[0023] Figure 2b It is illustrated in Figure 2a A schematic side view of a scene where cells are deployed above some electrodes of an electrode array;

[0024] Figure 2c It is illustrated in Figure 2a A schematic side view of a scene outside the electrode array and where cells are deployed between some electrodes;

[0025] Figure 3a and Figure 3b The illustration shows an example of cell mapping using the maximum current distribution;

[0026] Figure 4A This is a schematic diagram illustrating an example of high-resolution magnified calibration using cross-electrode current;

[0027] Figure 4B It is a schematic circuit diagram of a cell-circuit model;

[0028] Figure 5a and Figure 5b The illustration shows an example of magnified cross-electrode impedance mapping compared to a fluorescence microscope image;

[0029] Figures 6a to 6c The illustration shows an example of using cross-electrode impedance mapping to quantify cell adhesion;

[0030] Figure 7 It consists of a series of fluorescence microscopy images and normalized cross-electrode impedance maps;

[0031] Figure 8a This is the normalized impedance histogram measured without the addition of tetracycline.

[0032] Figure 8b This is a normalized impedance histogram of MDCK cells cultured in vitro for 6-7 days (DIV).

[0033] Figure 9 A series of normalized cross-electrode impedance maps are shown under stimulation signals of different frequencies;

[0034] Figure 10a The illustration shows a labeled cell and an example of its adhesion over time, measured by cross-electrode impedance.

[0035] Figure 10b The illustration shows an example of measuring cell-cell adhesion;

[0036] Figure 11This is a schematic diagram illustrating cell patterning via electrochemical gas generation;

[0037] Figure 12 Examples of cell spatial patterning and defined cocultures are illustrated;

[0038] Figures 13A to 13D These are a series of illustrations showing variations of cell patterning using an electrode array;

[0039] Figure 14 A series of fluorescence microscopy images are shown, illustrating the process of defining a co-culture via patterning and then inoculating a second cell type;

[0040] Figure 15 It is a series of schematic diagrams illustrating heterogeneous cell populations, the use of patterned electrochemical gases on selected electrodes to generate and eliminate unwanted cells, and homogeneous cultures with desired properties after subsequent cell growth.

[0041] Figure 16 An example of a wound healing test is illustrated;

[0042] Figures 17a to 17d An experiment demonstrating the permeation technology was shown;

[0043] Figures 18A to 18B The illustration shows an experiment using an electroporation protocol, in which Fluo-4 was injected into cells using Fluo-4 AM;

[0044] Figure 19 A series of schematic diagrams are shown illustrating the use of spatial addressing and serial transmission via gas generation to generate control and cross-effect transmission;

[0045] Figures 20a to 20b The illustration shows an example of extracellular electrochemical labeling;

[0046] Figure 21a The figure shows a data curve representing the selected electrode voltage over time;

[0047] Figures 21b to 21c It is a heatmap, which shows the total value of the open-circuit potential plotted within the array for one cycle;

[0048] Figures 22a to 22b The illustration shows an example of electrochemical oxygen labeling in cells;

[0049] Figure 23a A series of schematic diagrams illustrating three parameters that can be used for live-cell assessment are shown.

[0050] Figure 23b The image illustrates a fluid aperture packaged on top of a chip mounted under a microscope for simultaneous optical and electrical measurements.

[0051] Figure 23c It is a color microscope image illustrating cells and electrode arrays;

[0052] Figure 23d This is a schematic diagram illustrating electrodes connected to an exemplary pixel circuit;

[0053] Figure 24a , Figure 24b This is a schematic diagram illustrating some additional schemes for measuring cell-cell connectivity according to some embodiments;

[0054] Figure 25a This is a schematic diagram illustrating a pixel amplifier configured as a buffer for measuring metabolic state.

[0055] Figure 25b It is a series of data curves showing the results of multi-parameter measurements;

[0056] Figure 25c A pair of nuclear fluorescence images (top) and a comparison of detail region 1 (bottom) 72 hours after inoculation show that the cell density is lowest at the leading edge compared to the trailing edge;

[0057] Figure 25d This is a composite diagram showing detailed region 2 covering the cell nucleus and cell adhesion;

[0058] Figure 26a This is a series of fluorescence images illustrating the comparative study results of electrode impedance in three scenarios;

[0059] Figure 26b The diagram illustrates how PtB reduces the Z-axis of the bare electrode. te A graph of the measured values;

[0060] Figure 26c The diagram illustrates the relationship between cell barrier maps at different frequencies and a reference.

[0061] Figure 26d A graph showing cell density and connectivity extracted from the nucleus of a fluorescence image is presented;

[0062] Figure 26e The Z values ​​are shown in both reference-free and reference-based measurements. te Comparison between them;

[0063] Figure 26f Z-dimensions of cell density extracted from the control group are shown. te and Z s A comparison between them. Detailed Implementation

[0064] This disclosure relates to various devices for electro-evaluation and / or manipulation of cells. In one embodiment, the device includes a semiconductor substrate having complementary metal-oxide-semiconductor (CMOS) circuitry electrically bonded to an electrode array, which may also be fabricated on the surface of the semiconductor substrate and exposed to the cells using CMOS-compatible fabrication techniques. The inventors have recognized and understand that electrode arrays can be fabricated economically and integrated with active circuitry using semiconductor processing techniques. Furthermore, the smaller electrode size and electrode-electrode spacing of the electrodes in the electrode array compared to using electrodes larger than the cell size allows for higher spatial resolution evaluation of multiple cells. For example, individual cells can be distinguishable when using a high-density electrode array for mapping compared to a large electrode covered by a cell aggregate. Additionally, according to some aspects, the electrode array enables spatially addressable electrical stimulation and / or recording of electrical signals in real time using CMOS circuitry. Some of these aspects relate to performing cell patterning using the electrode array via electrochemical gas generation and extracellular electrochemical mapping.

[0065] One aspect involves the electrical mapping of cells on the surface of a semiconductor substrate via cross-electrode impedance measurement. The inventors have recognized and understood that the impedance, or cross-electrode impedance, measured between two electrodes can be affected by the impedance along the current path between the electrodes. Therefore, the presence of one or more cells along the current path can affect the cross-electrode impedance, making it possible to map cells using cross-electrode impedance measurement.

[0066] Figure 1a This is a schematic side view of a semiconductor substrate according to some embodiments. Figure 1a A device 100 with an electrode array 106 is shown, the electrode array including a plurality of electrodes 106_1, 106_2, 106_3...106_n deployed on a surface 104 of a semiconductor substrate 102. Figure 1a An example of cross-electrode impedance measurement is illustrated by applying a voltage excitation to a first electrode (such as 106_1) and measuring the current at a second electrode (such as 106_2). The measured current (also referred to as the cross-electrode current between electrodes 106_1 and 106_2) flows along one or more current paths 109 in a medium 108 in contact with the electrode array 106. Electrode 106_1 may be connected to a stimulation source circuit 110 and may be referred to as a stimulation electrode. Electrode 106_2 may be connected to a current measurement circuit 112 and may be referred to as a recording electrode.

[0067] The cross-electrode impedance between electrodes 106_1 and 106_2 can be obtained from the values ​​of the cross-electrode current and the stimulation voltage between the electrode pair using any suitable method known in the art, for example, by dividing the stimulation voltage value by the cross-electrode current value. A processing unit 120 can be provided that receives signals from active circuitry within the semiconductor substrate 102 and performs the cross-electrode impedance determination. It should be understood that it is not necessary to calculate the actual impedance value, and any representative measurement indicating the impedance between the two electrodes can be used. As an alternative or supplement to calculating the impedance value, if the stimulation voltage value is programmed to a known constant, the cross-electrode current can be used as an indicator of the cross-electrode impedance when comparing measurements at different electrodes.

[0068] Figure 1b yes Figure 1a The diagram shows a two-dimensional data curve of the simulated voltage distribution in the device, and shows that when a voltage is applied to the stimulation electrode 106_1, the potential in the medium 108 decreases away from the stimulation electrode 106_1 along both the vertical direction (V) and the transverse direction (L). Figure 1c Is with Figure 1b The example shown is a data curve of the simulated electric field line. Figure 1c The electric field line 114 emanating from the stimulation electrode 106_1 is shown flowing along an upward line from the electrode 106_1, bending laterally toward the recording electrode (such as the recording electrode 106_2), and then pointing downward to terminate at the recording electrode 106_2.

[0069] The presence of cells can alter the shape and distribution of the electric field lines 114 between the electrodes, thereby causing changes in the impedance of the cross electrodes, as discussed in detail below with reference to Figure 2. Figure 2a This is a schematic side view of a device 200 with a semiconductor substrate 202 in the absence of cells, according to some embodiments. Figure 2a In the electrode array 206, electrode 206_0 is configured as a stimulation electrode, wherein electric field lines 214_1 and 214_2 link the stimulation electrode 206_0 and the recording electrode 206_1. Figure 2b The illustration shows the deployment of cell 220 in... Figure 2a A schematic side view of the scene above some of the electrodes of the electrode array. Figure 2c The illustration shows the deployment of cell 230 in... Figure 2a A schematic side view of the scene outside the electrode array and between some electrodes.

[0070] The inventors have recognized and understood that biological cells possess a lipid bilayer that forms a continuous membrane barrier around the cell. Electrically, the membrane can behave as a capacitor in parallel with a high resistance and can have different impedances compared to the surrounding medium (such as a solution containing the cell). A cell with a high-impedance membrane positioned on top of an electrode array will subsequently affect the solution (such as...) Figures 2a to 2c The current distribution in solution 208. Figure 2c In the first method, suspended cells block field lines in the solution and reduce nearest-neighbor coupling between electrodes. In contrast, cells that adhere to the surface and cover both the stimulating and recording electrodes will increase cross-electrode coupling by blocking vertical field lines.

[0071] As an example of the effect of cells on the impedance of cross electrodes, and not wanting to be bound by a particular theory, the inventors recognized that if... Figure 2b The cells shown (such as cell 220) adhere to surface 204, thereby covering part or all of the stimulating electrode 206_0 and the recording electrode 206_1. Cell 220 increases cross-electrode coupling by blocking the electric field lines 214_1 and 214_2 between the two electrodes from perpendicularly passing through solution 208. As a result, compared to Figure 2 without cells, the more perpendicular field line 214_2 is suppressed and more field lines 214_1' are enhanced, leading to a decrease in impedance between electrodes 206_0 and 206_1.

[0072] On the other hand, if the cells do not adhere to surface 204, or if the cells (such as...) Figure 2c The cells (230) shown adhere to surface 204 but are deployed laterally outside and between the electrode pairs 206_0 and 206_2. These cells can block the electric field lines 214_3 between the electrode pairs and reduce the cross-electrode coupling between electrodes 206_0 and 206_2. As a result, the cross-electrode impedance between electrodes 206_0 and 206_2 can be increased.

[0073] Therefore, the presence of a cell above the electrode array and whether it adheres to the surface can be detected using cross-electrode impedance measurements. It should be understood that cells adhered to the surface can have varying degrees of non-zero spacing between the outer portion of the cell membrane and the surface. Devices according to some aspects of this application can provide detection of the degree of cell adhesion. For example, stronger adhesion will more strongly increase cross-electrode coupling because the gap distance between the cell and the surface of the semiconductor substrate along the vertical direction is smaller.

[0074] Cross-electrode measurements offer several advantages. For example, such measurements are non-invasive and can be repeated without affecting cell viability or the electrodes.

[0075] In some embodiments and as described above Figure 2b The described cross-electrode impedance technique measures the increase in cross-electrode coupling between electrode pairs due to the suppression of vertical electric field lines by the presence of cells, in contrast to techniques that measure the decrease in cross-electrode coupling (or the measured increase in impedance) due to blockage caused by the presence of cells. One advantage of using the increase in cross-electrode coupling as an indicator of cell presence is that this increase is primarily attributed to electrode pairs that are close to each other, and in some cases to the nearest neighbor coupling between electrode pairs. Therefore, the increase in cross-electrode coupling (or the measured decrease in impedance) can be separated from the total background current flowing through the stimulating electrode to the many remaining electrodes in the electrode array. As a result, the signal-to-background ratio and the sensitivity of cell detection can be improved.

[0076] Compared to cross-electrode impedance techniques, the inventors have recognized that simple impedance measurements at individual electrodes are insufficient to detect the presence of cells. When such measurements are performed on a single electrode, the sum of all returning currents is measured as the impedance signal on the electrode. That is, this measurement is merely an impedance measurement of the electrode, rather than a measurement of the change in the electric field in the solution on the electrode. As a result, the inventors have observed that the impedance of the electrode itself is insensitive to the presence of cells, even when cells are cultured directly on its surface.

[0077] Return to reference Figure 1a In some embodiments, the stimulation signal applied to the stimulation electrode 106_1 by the stimulation source circuit 110 is a low-frequency alternating current (AC) signal with a frequency less than 10 kHz, less than 5 kHz, between 0.1 kHz and 5 kHz, or between 0.1 kHz and 2 kHz. The low-frequency stimulation signal is chosen because the cell membrane acts as a parallel capacitor with high resistance, and at high frequencies, the capacitor impedance will decrease, causing the cell to have high conductivity. The inventors have recognized and understand that measuring the cross-electrode current at low frequencies can provide high signal contrast for detecting cell adhesion. An example of the frequency response of cross-electrode impedance measurement is provided in Example 4 below.

[0078] Still refer to Figure 1aThe semiconductor substrate 102 may include active circuitry 116. Active circuitry 116 may include a plurality of stimulation circuits 110 and a plurality of recording circuits 112. In some embodiments, stimulation circuitry 110 may include one or more current injectors, one or more voltage sources, or combinations thereof. Some aspects of the active circuitry design relate to current-based stimulators and related methods for electrogenic cells, as disclosed in International Application Publication No. WO 2019 / 010343 (Attorney General's File No. H0776.70105WO00), which is hereby incorporated by reference in its entirety. Some aspects may also relate to electronic circuitry and related methods for analyzing electrogenic cells, as disclosed in International Application Publication No. WO 2019 / 089495 (Attorney General's File No. H0498.70647WO00), which is hereby incorporated by reference in its entirety. In some embodiments, the active circuitry may include a programmable current injector for performing current-voltage measurements using one or more electrodes in an electrode array as working electrodes and / or counter electrodes.

[0079] In some embodiments, each recording circuit includes a transimpedance amplifier (TIA) having a switched capacitor as an impedance component. The impedance component has a resistance of at least 10 MΩ, at least 100 MΩ, or between 10 MΩ and 1 GΩ to amplify the recorded current signal at the input of the TIA, while the output of the TIA provides an output voltage proportional to the recorded current signal and the voltage across the impedance component.

[0080] Active circuitry 116 can be used to reconfigure the electrodes in electrode array 106 as stimulating or recording electrodes. In some embodiments, active circuitry 116 includes routing and switching components programmable to connect selected electrodes of electrode array 106 to excitation source circuitry 110, current measurement circuitry 112, or other circuitry to enable different functions. Depending on the application, more than one electrode can be configured as a stimulating electrode, and more than one electrode can be recorded simultaneously. For example, when using cross-electrode impedance measurement to map local cell properties, typically only one electrode acts as the stimulating electrode at a time. In some other embodiments, a subset of one or more electrodes can be selected to act as a stimulus or one or more potentials or currents can be applied to induce an electrochemical reaction at the location of one or more selected electrodes. The following embodiments will be discussed in more detail in the sections on cell-cell adhesion measurement, patterning, and spatial electrochemical mapping of cells.

[0081] In some embodiments, electrodes can be biased using a low-impedance source / loop in an active circuit. For example, a low-output-impedance voltage source can be used to provide a stimulation signal at the stimulation electrode, while a low-input-impedance transimpedance amplifier can be provided for current measurement at the recording electrode. In such embodiments, each electrode can be selectively connected to a voltage source for stimulation, a transimpedance amplifier for current measurement, a voltage source for looping, or a transimpedance amplifier for simultaneous stimulation and current measurement. The inventors understand and recognize that low-impedance sources / loops can facilitate the use of low-impedance sources / loops in applications such as... Figure 1c The example shown illustrates the formation of edge electric field lines in the solution.

[0082] Semiconductor substrate 102 may include silicon, and in such embodiments, active circuitry 116 may be an integrated circuit including CMOS components fabricated using standard CMOS processing techniques. Electrode array 106 may be deployed within semiconductor substrate 102, for example, as a conductor exposed from a surface 104 of semiconductor substrate 102 facing dielectric 108. In some embodiments, surface 104 is an insulating surface that provides mechanical support and electrical isolation to electrode array 106 while also providing a suitable surface for cell growth. Although Figure 1a The electrode array 106 is shown partially embedded in the semiconductor substrate 102, but this arrangement is merely illustrative and not required. In some embodiments, the top surface of the electrodes in the electrode array 106 may be above, perpendicularly aligned with, or below the surface 104 of the semiconductor substrate 102. Alternatively or additionally, the top surface of the electrodes may have a passivation layer or a functionalized layer. In some embodiments, holes may be patterned in the passivation layer or functionalized layer on top of the electrodes to expose the conductive surfaces of the electrodes to the dielectric.

[0083] It should be understood that the semiconductor substrate 102 can be any substrate manufactured using semiconductor processing technology, and is not limited to a silicon wafer. For example, the semiconductor substrate 102 may include group IV semiconductors, group III-V semiconductors, group II-V semiconductors, and sp... 2Hybrid carbon materials, chalcogenides, metals, metal compounds, oxides, nitrides, silicides, polymer materials, or combinations thereof. The semiconductor substrate 102 can be a single component or a composite of multiple components. Components in the semiconductor substrate 102 can include active circuit layers, wiring layers, redistribution layers, circuit boards, or combinations thereof. Component layers in the semiconductor substrate can be formed in additional processing during CMOS processing, or can be formed individually and bonded to each other using packaging techniques known in the art. Conductors are provided in the semiconductor substrate 102 that interconnect the active circuitry 116 with the electrode array 106. In some embodiments, connection points are provided at the bottom surface of the semiconductor substrate for electrically bonding components within the semiconductor substrate to the processing unit 120. Electrical connections between the processing unit 120 and the semiconductor substrate 102 can be provided via any suitable means, such as, but not limited to, controlled collapsed chip bonding or flip-chip bonding, wire bonding, flexible cables, or wireless communication.

[0084] Return to reference Figure 1a In some embodiments, apparatus 100 may operate to perform methods such as scaling or selective electrochemistry. Operation of apparatus 100 may be under program control. In some embodiments, processing unit 120 in apparatus 100 may include computer 20 with storage medium 21, memory 23, and processor 25, and such processing may be performed in computer 20 or any other computing device. Storage medium 21 and memory 23 may be any suitable non-transitory computer-readable medium, such as, but not limited to, computer memory, compact disk, optical disk, magnetic tape, flash memory, circuit configuration in a field-programmable gate array (FPGA) or other semiconductor device, or other tangible computer storage media. In some embodiments, storage medium 21 may be non-volatile memory, and memory 23 may be volatile memory. Computer-executable instructions may be loaded from storage medium 21 into memory 23 and then executed by processor 25 to perform some or all of the methods described throughout this disclosure. However, the distinction between storage medium 21 and memory 23 is not critical, and in some embodiments, either or both may be present.

[0085] Processor 25 can be any suitable processing device, such as, but not limited to, one or more processors, central processing units (CPUs), digital signal processors (DSPs), controllers, addressable controllers, general-purpose or special-purpose microprocessors, microcontrollers, addressable microprocessors, programmable processors, programmable controllers, special-purpose processors, special-purpose controllers, or any other suitable processing device. Some or all of the components within processing unit 120 can be packaged as a system-on-a-chip (SoC). Furthermore, it should be understood that... Figure 1aThis is a schematic diagram of processing unit 120. The actual implementation of processing unit 120 can feature distributed processing. For example, the host computer can control the overall process of measurement, calibration, and result analysis.

[0086] Turning now to electrode array 106. In some embodiments, as part of a semiconductor manufacturing process, electrode array 106 may be patterned on surface 104 to form active circuitry 116 within semiconductor substrate 102, and may be conductive pads comprising a metal such as Au or Pt or alloys thereof. For example, the pads may be formed from Al plated with Au as the top layer. In such embodiments, substrate 110 may additionally include conductors that vertically interconnect the exposed electrode array 14 to circuitry within substrate 110.

[0087] The electrodes in electrode array 106 can be arranged on surface 104 in any suitable manner, such as a two-dimensional array with regular pitch along the row and column directions. In some embodiments based on cross-electrode impedance mapping, the spacing of the electrode array can be selected to be approximately the size of a typical cell or smaller, such that the cell can cover at least two electrodes to increase coupling between the cell and at least two electrodes. For example, when the cell size is about 30 μm, the spacing of the electrode array can be set to less than 30 μm, less than 20 μm, less than 5 μm, or between 1 μm and 20 μm. Providing a small spacing between the electrodes allows the cell to cover two or more electrodes, thereby allowing measurement of the cell-substrate gap distance via increased cross-electrode coupling at the electrodes below the cell.

[0088] In some embodiments, when an electrode array is fabricated on top of a semiconductor substrate containing CMOS active circuitry during a CMOS-compatible manufacturing process, the spacing of the electrode array and the size of each electrode can be selected by taking into account the spacing and density of the CMOS active circuitry. For example, in some embodiments, at least eight, at least ten, or at least 4,000 recording circuits may be disposed within the semiconductor substrate, and the electrode array may have at least 1,000, at least 4,000, or at least 1,000,000 electrodes. In such embodiments, each electrode may have a lateral dimension of no more than 10 μm or no more than 5 μm, such that the overall lateral extent of the electrode array is contained within the surface of the semiconductor substrate. Electrode arrays according to various aspects of this disclosure may also be referred to as CMOS microelectrode arrays (MEAs).

[0089] Return to reference Figure 1a Medium 108 can be a cell culture medium and can be a solution containing any number of chemical and / or biological reagents in addition to cells. Although in Figure 1aNot shown, but the medium 108 may be contained in a container deployed on top of the semiconductor substrate 102. In some embodiments, the container may be a hole in a porous plate adhered to the semiconductor substrate, wherein one or more holes have an open bottom that exposes the contents of the hole to the semiconductor substrate. The semiconductor substrate may include more than one electrode array, such that electrical evaluation in multiple holes can be performed in parallel.

[0090] A CMOS-compatible wafer-level porous platform, and methods for operating it, are disclosed for use in biomedical or other applications. In some applications, circuitry is disposed beneath the porous array to electrically engage with electrodes in the holes. This platform may sometimes be referred to as a CMOS-porous platform. The inventors have recognized and understood that, in order to engage with electrodes in a large array, circuitry can be fabricated on a single silicon (Si) wafer with a size at least the same as or larger than that of the porous array. According to one aspect of this disclosure, standard CMOS fabrication processes, such as those known to be used in standard semiconductor manufacturing plants, can be used, for example, without the need for expensive custom-designed complex manufacturing processes, thereby reducing fabrication costs in some cases. The CMOS-porous platform according to some aspects of this disclosure can be used in applications including electrophysiological studies using electrical methods and general cell assessments, and / or high-throughput formats (e.g., 24, 96, and 384-well plate formats).

[0091] In some embodiments, the Si wafer is part of a semiconductor device and has an array of mask regions, some or all of which have multiple circuits of the same design. The inventors have recognized and understood that, during manufacturing, in certain circumstances, the mask regions of a wafer can reuse the same photolithographic mask design repeated for each wafer, thereby reducing processing costs and increasing wafer manufacturing throughput.

[0092] According to one aspect, when a via array is coupled on top of a wafer, digital and analog circuitry within the mask area can be arranged corresponding to one or more vias. Therefore, some embodiments can provide wafer-level integration of the electrical interface with the via array using manufacturing methods that reduce manufacturing costs by using non-diced wafers and / or standard-compatible methods that use standard CMOS-compatible technologies.

[0093] One aspect of this disclosure relates to a technique for mapping the spatial distribution and size of cells using cross-electrode impedance measurements. The mapping can further represent individual cell characteristics, such as adhesion to a semiconductor substrate surface. In some embodiments, because the presence of cells is primarily reflected locally in the cross-electrode coupling between the stimulating electrode and a nearby recording electrode, mapping is performed by first selecting a single electrode as the stimulating electrode and measuring a set of cross-electrode impedance data at locations across the entire electrode array, compared to other electrodes. Subsequently, different electrodes are selected as stimulating electrodes, and a new set of cross-electrode impedance data is measured. Cross-electrode measurements are repeated by sequentially positioning the electrodes in the electrode array to apply a stimulation signal, and the corresponding set of measured cross-electrode impedance data can then be processed to generate a value indicating the intensity of the presence or cell characteristics at each location of the stimulating electrode. The processed values ​​can then be combined to form a map of the transelectrode array region. In some embodiments, “electrochemical imaging” of live cell cultures is demonstrated through high-resolution in-situ impedance and electrochemical measurements. Some embodiments relate to using CMOS-MEA to perform label-free and non-invasive tracking of cell growth kinetics and accurate measurements of cell-substrate adhesion, cell-cell adhesion, and metabolic states.

[0094] On the other hand, it relates to providing electrochemical reactions spatially positioned using patterned electrode arrays. Using a selected number of electrodes in the electrode array, an active circuit in a semiconductor substrate can apply a potential to initiate an electrochemical reaction in a solution region directly above the selected electrodes. As a result, electrochemistry can be selectively performed at a programmed spatial pattern based on the size, shape, and distribution of selected electrodes on the surface of the semiconductor substrate.

[0095] In some embodiments, cell patterning can be performed using spatially programmed electrochemistry. For example, cells adhering to the electrode can be selectively removed from the electrode surface by electrochemically generating microbubbles on the electrode.

[0096] In some embodiments, an array of electrochemical electrodes can be used to spatially map the concentration of an analyte, which is measured using an active circuit in a semiconductor substrate. One application is the electrochemical mapping of solutions using redox electrochemistry.

[0097] The following applications are each incorporated herein by reference in their entirety: U.S. Provisional Patent Application Serial No. 63 / 040,439, filed June 17, 2020, by Park et al.; U.S. Provisional Patent Application Serial No. 63 / 040,424, filed June 17, 2020, by Ham et al.; and U.S. Provisional Patent Application Serial No. 63 / 040,412, filed June 17, 2020, by Ham et al. In addition, the following are each incorporated herein by reference in their entirety: PCT patent application filed June 16, 2021, entitled “Complementary Metal-Oxide-Semiconductor (CMOS) Multi-Well Apparatus for Electrical Cell Assessment” and PCT patent application filed June 16, 2021, entitled “Apparatuses for Cell Mapping Via Impedance Measurements and Methods to Operate the Same”.

[0098] The following examples are intended to illustrate certain embodiments of the invention, but do not represent the full scope of the invention.

[0099] Example 1: Real-time cell measurement using a CMOS microelectrode array (MEA) and imaging system

[0100] This example illustrates the use of electroimaging for three parameters that can be used for live-cell assessment. Figure 23a : Cell-substrate impedance Zs (reflects cell adhesion and cell-substrate adhesion), transmembrane impedance Zte (reflects cell-cell adhesion, cell monolayer integrity, and barrier function), and extracellular redox potential Vredox (reflects cell metabolic state and respiration).

[0101] In this example, a custom-designed CMOS IC was used, which implemented parallel impedance and electrochemical functions on a 64×64=4,096 electrode array. Figure 23b -d). Fluid pores are encapsulated on the top of the chip for cell culture and mounted below a top-down fluorescence microscope for simultaneous optical and electrical measurements. Figure 23b The electrode array is positioned at the center of the device, with resolutions tailored to single cells or a small number of cells (e.g., Figure 23c The MDCK cells were composed of 8μm diameter Pt electrodes spaced 20μm apart, resulting in a 1.26×1.26 mm electrode. 2The total sensing area is [not specified]. Silicon nitride, which behaves similarly to glass culture plates, is used to insulate the rest of the surface. No differences were observed in the growth or morphology of cells cultured on the device compared to conventional culture plates. For long-term measurements, an integrated temperature sensor and heater regulate the cells to 35–37°C, and a small incubation chamber is placed above the device to regulate CO2 to 5%.

[0102] Each electrode in the array is connected to its own pixel circuitry. Figure 23d The pixel circuit is highly configurable and programmable via a digital interface. The pixel circuit includes an operational amplifier that can be configured as a buffer for measuring electrode voltage Ve or as a transimpedance amplifier for measuring electrode current Ie. Some aspects of the pixel circuit configuration relate to current-based stimulators and related methods for electrogenic cells, as disclosed in International Application Publication No. WO 2019 / 010343 (Attorney's File No. H0776.70105WO00), which is hereby incorporated by reference in its entirety. Some aspects may also relate to electronic circuits and related methods for analyzing electrogenic cells, as disclosed in International Application Publication No. WO 2019 / 089495 (Attorney's File No. H0498.70647WO00), which is hereby incorporated by reference in its entirety.

[0103] Figure 23a This diagram illustrates three cellular parameters measured using a complementary metal-oxide-semiconductor (CMOS) integrated circuit (IC) for live cell assessment, where live cell assessment is defined as: cell-substrate impedance Z... s Assessing cell adhesion via transmembrane impedance Z te Assessing cell-cell adhesion and via extracellular redox potential V redox Assess metabolic status. Each measurement is non-invasive and rapid (<1 minute), allowing for sequential repetition every 5 to 10 minutes for real-time investigation. Figure 23b This image illustrates how a fluorescence microscope can be paired with a packaged CMOS IC to perform simultaneous optical and electrocytic measurements. A reference electrode, Pt (shown) or Ag / AgCl, can also be used in this example. Figure 23c This is a color fluorescence image of passaged canine kidney (Madin-Darby Canine Kidney, MDCK) epithelial cells cultured on top of a CMOS electrode array. 4,096 circular platinum electrodes, each 8 μm in diameter and 64 × 64, are spaced 20 μm apart. Platinum black (PtB) can be electrodeposited onto the electrodes to reduce electrode impedance, thereby achieving a higher signal-to-noise ratio. te Measurement. Figure 23dThis is a circuit diagram of an exemplary circuit for electrodes in an electrode array. Each of the 4,096 electrodes is connected to its own peripheral circuitry via shielded wiring (approximately 1 to 10 mm). The operational amplifier-based circuitry can be configured to connect via V... s Apply voltage and via feedback resistor R f (Approximately 100 MΩ) Measure current, or via I s Apply current and buffer / measure electrode voltage V e The output V of the operational amplifier amp It is routed outside the chip for analog-to-digital conversion. The switch is digitally programmed using a real-time software interface.

[0104] According to several aspects, the measurement technique in this example features high channel count (4,096), parallel current and open-circuit potential measurements, which offer unique advantages over other MEA devices. For example, the measurements described in this example are prevented in MEA devices that measure electrode capacitance, voltage using a high-pass filter to block DC signals, or current using a small number of channels (<32).

[0105] Example 1A: Cell mapping using maximum current distribution

[0106] This example describes a technique for labeling cells using a CMOS electrode array comprising a 64×64 array of 4,096 platinum electrodes with a 20 μm pitch.

[0107] The inventors have recognized and understood that alternating current (AC) impedance measurement between a pair of electrodes can detect cells by comparing an insulating cell membrane with a conductive culture medium. In classical impedance measurements, the solution path around the cell skewing the measurement and reducing detection sensitivity because the solution contribution of the measured electrode-electrode current is much greater than the small change in current due to the cell. The device disclosed herein improves detection sensitivity by alternatively measuring the change in electric field distribution due to the cell.

[0108] An AC voltage (1.9 kHz frequency, 200 mV magnitude) was applied to one electrode, and the resulting AC current was measured through the remaining 4,095 electrodes using a transimpedance amplifier. Figure 3a The results are illustrated in the figure, showing a heatmap 301 of the closest current distribution measured from 11 × 11 recording electrodes to a stimulating electrode 311 when no cells are present. In heatmap 301, each pixel corresponds to the position of an electrode. Each electrode has an electrode position or location that can be represented in various ways, such as, but not limited to, coordinates or the number of pixels. Heatmap 302 is a similar current distribution measured to heatmap 301, but with cells on top of electrode 311. Impedance measurements were performed using a signal frequency of 1.9 kHz.

[0109] Figure 3a The data curve 303, which shows the measured cross electrode current versus distance to the stimulation pixel, indicates that the cross electrode coupling with the adjacent electrode is almost an order of magnitude higher when cells are present, compared to the electrode without cells on top.

[0110] In this example, the fluorescent nuclear MDCK cell line was used for optical confirmation. Figure 3b A fluorescence microscopy image 304 spanning the entire 64×64 electrode array is shown, where brighter pixels represent fluorescence signals indicating the presence of cells. To generate a cross-electrode impedance map of the same region as image 304, the stimulating electrodes are scanned sequentially across the array. For each given stimulating electrode, cross-electrode current values ​​are measured from the remaining electrodes, which serve as recording electrodes. The recorded cross-electrode currents are collected, and a maximum value is determined, referred to as the maximum current value corresponding to a given stimulating electrode. Figure 3b A heatmap 305 of the transelectrode array is shown, generated using the maximum current value (Ie) determined from the stimulation electrode at each pixel location.

[0111] Figure 3b Figure 306, showing the overlap of selected region 1 as the maximum current signal 309 and the nuclear fluorescence signal 307, also illustrates the ability to label cell clusters at single-cell resolution. As a result, this example demonstrates the use of nuclear fluorescence labeling to confirm the presence of cells, with a strong correspondence between the maximum current map and fluorescence imaging.

[0112] The maximum current value (Ie) for each stimulation electrode location is determined using any suitable method based on the set of cross-electrode currents measured from the recording electrodes. This determination can be a simple comparison of the absolute arithmetic values ​​of the cross-electrode currents, and may additionally include data processing such as noise filtering, background cancellation, or any suitable signal processing technique known in the art prior to the comparison. This can be done after the measured current values ​​are digitized and... Figure 1a The processing unit shown (such as processing unit 120) performs the processing and comparison of current values.

[0113] Example 2: High spatial resolution mapping using cross-electrode current

[0114] This example describes a method for generating magnified views of cross-electrode couplings with a spatial resolution higher than the electrode array spacing.

[0115] According to some embodiments, the nearest cross electrode measurement can be used for each stimulation electrode. Figure 4A An example of high-resolution magnified mapping using a 3×3 impedance grid for each of electrodes 1 to 9 is shown. In some embodiments, electrodes at the edges of the electrode array may be skipped in the magnified impedance grid, as described below.

[0116] Figure 4B This is a schematic circuit model that can be used to apply an AC stimulation voltage V. A and measuring the cross electrode current I 12 And calculate the cell-substrate impedance Z s and transmembrane impedance Z te Extracting a single Z for each electrode te At that time, regarding Z s The calculations use a 3×3 impedance grid.

[0117] To measure cell-substrate adhesion, a cross-electrode field is formed. Instead of applying a bias voltage between the two electrodes, a bias voltage is applied from one electrode to all the remaining electrodes. This allows the field lines that begin at the stimulation electrode and extend far into the culture well to terminate at an electrode far from the stimulation electrode. Otherwise, these field lines would need to curl backward toward adjacent electrodes, increasing the amount of current measured that is not correlated with the immediate cell-electrode interface.

[0118] A cross-sectional type model can be used to model the interface to increase spatial resolution. If we assume Z... s < <Z te Z e,1 and Z e,2 (Based on the fact that it is found to be effective for most measurements in some respects), then:

[0119] (Formula A1)

[0120] The measured cross-electrode current can also be written and expressed in the form of (Equation A1).

[0121] (Formula A2)

[0122] To determine Z e,1 and Z e,2 When a stimulus is applied to electrode n, the sum of currents measured across the array is used.

[0123] (Formula A3)

[0124] Then, Z can be solved using all the measured currents for (A3) and (A2). s ,

[0125] (Formula A4)

[0126] To generate Z s The high spatial plot was measured using the nearest neighboring cross electrode for each stimulation electrode: a 3×3 grid was used for each electrode (except for electrodes at the edges of the electrode array). See also Figure 4A This creates an overall Z-shape of 190×190 pixels. s Image (compared to 64×64 electrodes in the array).

[0127] exist Figure 4A In the example shown, for each of the nine pixels in the 3×3 grid 405 of the center electrode 5, a normalized impedance value Z is used to fill the grid based on the measured current to its nearest neighboring electrode. Each normalized impedance value Z is calculated as follows:

[0128] (Equation 1)

[0129] Among them, V AC It is the magnitude of the applied AC voltage, I xy It is the magnitude of the AC current measured through electrode y when an AC signal is applied to electrode x, and I x [I y [ ] is the sum of the magnitudes of the AC currents measured through all other electrodes when an AC signal is applied to electrode x[y]. Then, the edge-normalized impedance value is calculated as...

[0130] (Equation 2)

[0131] The square root of 2 is determined to normalize the distance difference between the edge and corner electrodes. Then, the center normalized impedance value is calculated as follows:

[0132] (Equation 3)

[0133] Compared to using the maximum current distribution, using cross-electrode current not only increases the effective spatial resolution, but also makes it possible to map unadhesive cells that cause a decrease in cross-electrode current.

[0134] Figure 5a and Figure 5b An example of cross-electrode impedance mapping magnified compared to a fluorescence microscope image is shown. Figure 5a A fluorescence microscopy image 501 of the cross-electrode array and a thermogram 502 of the normalized cross-electrode impedance of the cell culture immediately after seeding are shown. A magnified view 504 of a portion of thermogram 502 shows the reduction in the normalized cell-substrate impedance Zs of the cross-electrode for unattached cells at single-cell resolution. The mapping immediately after cell seeding, where cells are not attached, indicates a smaller normalized impedance value compared to the cell-free electrode.

[0135] Figure 5b Fluorescence microscopy image 505 and cross-electrode impedance map 506 are shown after 24 hours of culture. Figure 5bAlso shown is magnified Figure 507, which is an overlay of a fluorescence microscopy image and a cross-electrode impedance map of the selected area. The results indicate that many cells have adhered to the surface, resulting in a sharp increase in the normalized cross-electrode impedance.

[0136] Example 3: Quantification of Cell Adhesion

[0137] This example describes a method for quantifying cell adhesion using cross-electrode impedance mapping.

[0138] Ethylenediaminetetraacetic acid (EDTA) was applied to the cells. EDTA is a calcium-dependent compound required for the removal of integrin. 2+ A calcium chelating agent was used to maintain cell adhesion. EDTA was applied, and cells rapidly detached within approximately 50 minutes. The cells were then washed with normal culture medium to remove the EDTA, during which they re-adhere within approximately 200 minutes.

[0139] Cross-electrode impedance mapping was used to capture cell detachment and re-adhesion with high spatial and temporal resolution, such as... Figure 6a The figure shown in the illustration depicts a series of normalized impedance plots of MDCK cells over time, with 5 mM EDTA applied at approximately t=5 minutes and washed at approximately t=55 minutes.

[0140] Figure 6b This is a graph showing the average normalized impedance of different regions of the cell culture over time, as specified in Figure 601. Figure 6c c is a histogram of normalized impedance values ​​before, during, and after EDTA washing across the array.

[0141] To represent a biologically relevant instance of quantifying cell adhesion, measurements were taken in a genetically modified MDCK cell line where tetracycline was used to turn the RasV12 and GFP genes on and off. Figure 7 The results are shown in the figure. Figure 7 These are a series of fluorescence microscopy images and normalized cross-electrode impedance maps of MDCK cells in 7 days of in vitro culture (DIV). Tetracycline was added after 2 DIV measurements to activate the cancer-associated gene RasV12, which also expresses GFP, allowing for imaging of gene expression. Tetracycline was then removed after 4 DIV measurements to deactivate gene expression. When the RasV12 gene was expressed, cells exhibited less adhesion to the surface, which returned to normal after it was deactivated.

[0142] RasV12 is an oncogene and is known to increase cell metabolism and decrease cell adhesion when strongly expressed, both of which contribute to cancer-like cell growth and tumors. Initially, tetracycline was excluded from the culture medium, and cells adhered normally. When tetracycline was introduced, gene expression increased, resulting in increased GFP and decreased cell adhesion. Tetracycline removal then reversed cell adhesion, resulting in stronger cell adhesion while also reducing overall GFP expression; some portions of the cell culture were not as strongly shut down as others. The effect on cell adhesion was quantitatively compared to the control culture without tetracycline, as shown in Figure 8. Figure 8b This is a normalized impedance histogram of MDCK cells in 6-7 days of in vitro culture (DIV). Tetracycline was added after 2 DIV measurements to activate the cancer-associated gene RasV12. Figure 8a This is a normalized impedance histogram of the control without tetracycline. The histogram has been normalized to the maximum number of pixels above approximately 8 kΩ for cell-free impedance. Cell adhesion was reduced compared to the control, showing a smaller decreasing trend over time.

[0143] Example 4: Frequency Response

[0144] This example illustrates the effect of the frequency used in cross-electrode impedance measurements.

[0145] The scanning mapping frequency is used to determine the optimal frequency for measuring cell adhesion using cross-electrode impedance mapping. Figure 9 A series of normalized cross-electrode impedance plots are shown for stimulation signals at different frequencies. These plots were normalized to a median of + / - 1 standard deviation. Lower frequencies exhibit [the following characteristics]... Figure 7 The high signal contrast associated with GFP fluorescence, as shown in the optical measurements, indicates that lower frequencies are better for measuring cell adhesion. The 1.9 kHz used still exhibits good contrast compared to 240 Hz, but above 10 kHz, the cell layers appear more homogeneous.

[0146] Example 5: Cell-to-cell adhesion

[0147] Previous examples involved how to characterize cells and their adhesion over time via cross-electrode impedance measurements, such as... Figure 10a As depicted in [the text]. Figure 10a In this study, an AC voltage was applied to a single electrode, and the current was measured through the remaining electrode array using a transimpedance amplifier. Adhesion varied primarily with changes in cell-substrate adhesion and the resulting gap height.

[0148] This example describes a method for measuring cell-cell adhesion, or the degree to which cells are connected to each other. Cells in a culture not only adhere to the surface but also to each other via cell-cell junctions. The tightness of these junctions defines the permeability of the cell layer and is important for epithelial tissues, which act as barriers to the body surface, internal organ linings, and other tissues. In this example, transmembrane impedance Z-strain is measured. te The barrier function is measured using a mapping technique. In this way, cell-cell connectivity can be assessed using electrodes that are only covered by cells to reduce any pores, while also enabling assessment of spatial heterogeneity.

[0149] In this example, the stimulation protocol was modified to measure the vertical field component 1014, as follows: Figure 10b As shown in the diagram. Figure 10b In this setup, electrode 1006_2 and its surrounding electrodes 1006_1 and 1006_3 are biased with an AC voltage. The current Ie,n is measured through the central electrode 1006_2. Because the central electrode 1006_2 is biased with the same signal, it will not allow current to flow to the surrounding electrodes; therefore, it will only carry current due to the impedance of the cell layer above the electrode. Outside the central and surrounding electrodes, the rest of the array is biased to ground or a reference voltage level to act as a current loop. This type of measurement is similar to measuring transmembrane resistance (TEER), which is measured using two electrodes on opposite sides of a cell culture suspended in a porous membrane. Figure 10b The technique illustrated enables cell labeling of TEER across the top of an electrode array without the need for a special suspension. Advantages include the requirement for fewer cells, the ability to assess spatial heterogeneity, and the ability to combine cell-cell and cell-substrate adhesion measurements using the same device.

[0150] Figure 24a , Figure 24b This includes schematic diagrams illustrating some additional schemes for measuring cell-cell connectivity according to some embodiments. Figure 24a , Figure 24b In the measurement, changes in the vertical field above the electrodes were used to best isolate the effects of cell-cell junctions using two circuit configurations: 1) rapid (<1 s / measurement) parallel electrode measurement of the control reference ( Figure 24a ), and 2) slow scan without reference (40 s / measurement) relative measurement ( Figure 24b Rapid measurements are ideal for scanning across multiple frequencies, and the absence of a reference during scanning contributes to greater stability of long-term measurements and is more ideal for device miniaturization. For both types of measurements, platinum black (PtB) deposition can optionally be used to apply Z... e Reduced by approximately 5 times to improve Z teSensitivity. Cross-frequency experiments showed that the mid-frequency range of approximately 2 kHz to 5 kHz is optimal for assessing cell-cell connectivity.

[0151] Now, let's discuss the use of... Figure 24a , Figure 24b The transmembrane impedance Zte is calculated using the proposed scheme.

[0152] exist Figure 24a , Figure 24b In order to measure cell-cell adhesion or the degree of cell-to-cell connectivity, the stimulation protocol can be modified to measure the vertical field component. Measurements are performed by applying an AC voltage to all electrodes, compared to a grounded reference (left side), and the transmembrane electrode current I is measured via a transimpedance amplifier. te,n (n = 1, 2, ... 4096) (measurement duration 1 s / frequency). The resulting field distribution is perpendicularly aligned with the cell connectivity, thereby reducing I. te Non-reference measurements (right side) can be performed by applying an AC voltage to electrode (n) and its adjacent electrodes to create an effective vertical field measurement with the remaining electrodes grounded. To generate the cell map, the applied signal is scanned across the array (40 s / frequency per scan).

[0153] exist Figure 24a In the parallel scheme, an AC voltage is applied to each electrode relative to a reference, and the current I of each electrode is measured. te,n This creates a vertical field in the solution, and the peripheral electrodes will also have edge fields targeting low frequencies. Since the current then needs to pass through the cell layer, the magnitude of the current will be related to the transmembrane impedance Z. te Proportional. Second scanning scheme ( Figure 24b The electrodes and their surrounding electrodes are biased with an AC voltage, and the current passing only through the center electrode is measured. Because the center electrode is biased with the same signal, it will not allow current to flow to the surrounding electrodes; therefore, it will only carry current due to the impedance of the cell layer above the electrode. Outside the center and its surrounding electrodes, the rest of the array is biased to ground to act as a current loop.

[0154] In either case, the measured vertical current I te,n This can be expressed as,

[0155]

[0156] Using (A3), Z can be solved. te ,

[0157]

[0158] For measurements, an intermediate frequency of approximately 1–5 kHz was determined to be optimally correlated with cell-cell connectivity (see Example 15 below). For PtB electrodes, Z e,n Subsequently, it is sufficiently smaller than Z. te (See also Example 15 below), which makes the estimate:

[0159]

[0160] For Z that only uses Pt electrodes te Experiment, subtracting I from cell-substrate impedance n Measured value. Z is calculated using a 3×3 set of electrodes based on the scanning array measurement. te,no ref Because the peripheral electrodes lack adjacent bias electrodes to create a vertical field, the resulting overall image is 62×62 pixels. An image of 64×64 pixels was created using measurements from a reference.

[0161] Example 5A: Via extracellular redox potential V redox Metabolic state to be labeled

[0162] In addition to impedance measurement, platinum electrodes are also used for both oxygen potential sensing and extracellular redox monitoring. This example demonstrates that the extracellular redox potential V can be in situ mapped using the adjacent location of a Pt electrode directly beneath a living cell. redox It can be used to monitor the redox environment of cells, and even O2 consumption can be used to measure the metabolic state of cell cultures.

[0163] To complete the measurement, the pixel amplifier is configured as a buffer, such as... Figure 25a As shown in the diagram.

[0164] Typically, in aerobic metabolism, cells utilize energy generated by the movement of electrons from oxidizable organic molecules (e.g., glucose) towards O2. To facilitate the regulation of these electron flows, a common reducing environment is created by the thiol-based compound glutathione (GSH), often considered a cellular redox buffer. In simplified terms, the cell's redox potential is subsequently the balance between the up-pulling potential (oxidizing) of O2 and the down-pulling potential (reducing) of GSH. The redox environment is important not only for electron transfer but also for neutralizing harmful reactive oxygen species, cell-cell signaling, and regulating cellular states. For example, across a range from negative to positive, the redox potential can determine whether a cell is in a state of proliferation, differentiation, apoptosis, or necrosis.

[0165] Figure 25bThis is a series of data plots showing the results of multi-parameter measurements. Measurements were performed at +24, +48, ​​and +72 hours after MDCK cell seeding, including cell adhesion (top), cell-cell adhesion (middle), and metabolic state (bottom). Cells exhibited growth from the lower right to the upper left, where proliferating cells at the leading edge showed the most negative Vg compared to the more dormant trailing edge. redox Z te It is also highest at the leading edge because the cell density is lowest (see detail area 1), and therefore the cell-cell connections are minimal. Figure 25c This is a pair of nuclear fluorescence images (top) and a comparison of detail region 1 (bottom) taken 72 hours after inoculation, showing the lowest cell density at the leading edge compared to the trailing edge. Figure 25d This is a composite image showing the detailed region 2 covering the cell nucleus and cell adhesion. Figure 25d It shows a good spatial correspondence with single-cell resolution.

[0166] One objective of this example is to study the effects of voltage levels close to V by pairing it with impedance techniques used to monitor cell growth. redox What information can be provided? Figure 25b In this example, a negative VV in the range of 30 mV to 80 mV was observed for electrodes with cells compared to those without cells. redox ( Figure 25b From a detailed regional comparison perspective, V redox Spatial information is distinct from and different from cell adhesion or cell barrier, among which the most negative V redox It is located at the leading edge rather than at the lowest density. Generally, a negative signal can indicate locally lower [O2] or locally higher [GSH] near the cell.

[0167] To further explore V redox Signal origin was determined by testing O2 dependence on isolated MDCK cell cultures via oxygen purging. The signal difference between areas with and without cells was eliminated after O2 removal. To complement, GSH-based reducing power was assessed via oxidation titration. Ferricyanide [Fe(CN)6] was selected. 3- Titration was performed due to its previous non-toxic use in cell cultures and its oxidative half-cell potential compared to the cellular environment. The culture medium exhibited a reducing capacity of 4 µM, while the cells had a much larger capacity of >200 µM.

[0168] In summary, these measurements show that the measured V redox This relates to both the in-situ [O2] and [GSH]-based reducing capacities of the cell. It can be inferred that under aerobic respiration, [O2] decreases from its normal dissolved concentration under atmospheric conditions (approximately 200 µM), which reduces V...redox The oxygen consumption rate decreases until it is regulated by the extracellular reduction potential of the cell. Therefore, although it is difficult to quantify the oxygen consumption rate using our technique, the Vo of the extracellular redox potential... redox The measurement results can be used to monitor the metabolic state of cells because they can show the amount of O2 used and the reducing environment of the cells. Therefore, a more negative signal at the leading edge of the cell layer ( Figure 25b This is attributed to the combination of respiration and the proliferative state (the cell's most negative redox potential state).

[0169] Example 6: Antibody-cell binding

[0170] Screening for antibody-cell binding can be low-throughput because it requires fluorescent labeling of the antibody, necessitating a washing step to remove unbound fluorescent antibodies, or special optical measurements such as surface plasmon resonance (SPR). According to one aspect, the cross-electrode impedance technique described herein provides the ability to measure antibody-cell binding events via cell-substrate or cell-cell adhesion measurements. As the antibody binds to the underside of the cell, the gap distance effectively decreases, resulting in a reduction in the amount of measured cross-electrode current. Similarly, as the antibody binds to the sideside of the cell, the cell-cell gap distance should also decrease, resulting in a reduction in the amount of measured vertical current. If this antibody binding can be performed without labeling, it allows for the sequential addition of different antibodies without the need for a washing step, thereby significantly improving throughput.

[0171] Example 7: Cell patterning generated by electrochemical gases

[0172] This example describes a method for patterning cells on top of an electrode array. The inventors have recognized and understood that tiny bubbles can be generated electrochemically to create pores in the cell membrane, thereby killing cells through depolarization. After death, the cells detach from the surface, as... Figure 11 The diagram illustrates this. Therefore, by controlling which electrodes generate gas, the spatial resolution of the electrode array can be used to pattern cells.

[0173] Unwilling to be bound by a specific theory, the inventors recognized that for most inert electrode materials (platinum, gold, etc.), the electrode potential can be adjusted to be below the hydrogen ion / hydrogen redox half-cell reduction potential (E0). 0 To produce hydrogen gas,

[0174]

[0175] Alternatively, oxygen can be generated by adjusting the electrode potential to be higher than the oxygen / water redox potential.

[0176]

[0177] Similarly, since most cell culture media contain chloride salts, chloride gas can also be generated by adjusting the electrode potential to be higher than the chlorine / chloride redox potential.

[0178]

[0179] Therefore, cell removal can be performed by selectively applying a predetermined potential higher than the redox potential to generate gas at one or more electrode sites. This can be achieved, for example, by... Figure 1a One or more stimulation source circuits 110 are connected to selected electrodes to apply a potential. The potential does not need to be the same for all selected electrodes, and programmable heterogeneity can be used when the electrodes are biased differently. The potential can be a potential relative to the potential of a reference electrode in the matrix above the electrode.

[0180] To achieve more controllable patterning, the electrode current can be used to set the electron transfer rate, which in turn sets the gas generation rate. Controlling the gas generation rate can optimize the selective electrochemical reaction because using an excessively fast gas generation rate can create large bubbles on the surface, thereby blocking the electrode from contacting the solution.

[0181] Figures 13A to 13D These are a series of diagrams illustrating the patterned changes in cells using an electrode array. Figure 13A and Figure 13B The illustration depicts an embodiment in which one or more predetermined patterning voltages are applied to a selected electrode to remove cell patterning by generating an electrochemical gas. Figure 13C and Figure 13D An embodiment is illustrated in which one or more predetermined patterning currents are applied to a selected electrode to remove cell patterning. Figure 13A and Figure 13C The illustration shows an example of voltage / current patterning with the reference electrode acting as a loop. Figure 13B and Figure 13D The illustration shows an example of differential voltage / current patterning using cross-electrode gas generation without a reference electrode, where a positive current flows through one set of electrodes and a negative current flows through a second set of electrodes (the loop).

[0182] Example 8: Cell spatial patterning and constrained coculture

[0183] This example illustrates the spatial patterning of cells and the co-culture definition using an electrode array.

[0184] In this example, use as Figure 12The CMOS electrode array, MDCK cells, and H2 gas generation are shown. In this experiment, H2 gas was generated by applying -1.25 V to a platinum electrode using a control Ag / AgCl dummy reference electrode. Figure 12 Fluorescence microscopy images are shown before (middle) and after (right) the application of the patterning voltage 80 seconds later, demonstrating the successful definition of patterns within cells based on electrode patterns. Square pores of various sizes were fabricated on a uniform cell layer with high spatial resolution at an electrode spacing of 20 µm, as confirmed using nuclear fluorescent labeling and fluorescence imaging.

[0185] Figure 14 A series of fluorescence microscopy images illustrating the process of defining a co-culture by patterning followed by inoculation with a second cell type are shown. Cell types are distinguished by different nuclear fluorescent labels. Figure 14 In the experiments, co-cultures of two different cell types were defined by seeding a second MDCK cell line with different nuclear fluorescent markers after initial patterning. The second cell type filled the resulting space, demonstrating the ability to spatially define co-cultures with high spatial resolution. Further patterning and seeding can also be performed to define multiple cell co-cultures and patterns in a bottom-up manner.

[0186] Example 9: Directed cell evolution by eliminating culture heterogeneity

[0187] This example describes a method for directed cell evolution that eliminates cells from cell cultures whose characteristics are not desired.

[0188] Figure 15 This diagram illustrates a series of schematic representations of heterogeneous cell populations, the elimination of unwanted cells using patterned electrochemical gases generated on selective electrodes, and homogeneous cultures with desired properties after subsequent cell growth. Which cells to eliminate can be selected via optical imaging or by other properties measured using the electrode array. The ability to eliminate cells from the culture without removing them from the culture plate is superior to current methods, which require cell suspension and separation using a cell sorter (with a further reseeding step for reculturing), or removal of single cells with desired properties using micropipettes followed by reseeding. Furthermore, because the spatial position of each cell remains unchanged due to cell adhesion during the process, the phylogenetic history of the cells can be preserved. This elimination process can also be used to perform further analysis on a subset of cells after the culture electrode array, where cells not intended for further measurement are killed before cell suspension and removal.

[0189] Example 10: Wound Healing Test

[0190] This example illustrates the combined application of cross-electrode impedance mapping and cell patterning in wound healing assays.

[0191] These experiments measured cell growth rate and metabolism and can be used to screen drugs that affect these parameters. Compared to the electrochemical patterning described herein, other tools mechanically create wounds in cell cultures via mechanical scraping, which is both difficult to control and limited in terms of wound patterning.

[0192] In this example, wounds were formed in MDCK cells, and growth was then measured in real time. Figure 16 A serrated pattern with varying separation distances is shown in the cells centrally defined on the device surface. These patterns were defined by applying an electrode current of -10 nA for 40 s to a control Ag / AgCl sham reference electrode. Regeneration of the cultures was then measured using impedance mapping. Typical cell cultures took approximately 3 days to fill the wound, while cultures treated with growth inhibitors showed very little regrowth. Figure 16 As illustrated by the normalized cross-electrode impedance plot, the control culture showed regrowth after 72 hours in the culture. The second culture, treated with a growth-slowing drug (cytochalasin B (1 µM)), showed very little growth over the 72-hour period, demonstrating the assay's ability to be used for drug screening.

[0193] Example 11: Molecular transport

[0194] This example illustrates a technique for membrane permeation and molecular transport using planar electrodes. Unlike electroporation, which applies a concentrated electric field to break down cell membranes, planar electrode permeation works via bubble formation, conceptually similar to the patterning techniques discussed herein. Unlike patterned cells, which kill cells to perform patterning, for molecular transport, smaller pores are formed on the cell that will reseal over time afterward.

[0195] Figures 17a to 17d The illustration shows an experiment demonstrating permeation technology using nanowire electrodes, and some aspects of this technology can also be applied using electrode arrays utilizing planar electrodes. Figures 17a to 17d In the experiment shown, an in vivo assay was performed by dissolving Fluo-4 in an extracellular solution (left figure). Figure 17a Using a pixel stimulator (middle image, Figure 17a An electroporation protocol was applied to the nanoelectrode, which was then recovered in Fluo-4. If electroporation was successful, Fluo-4 infiltrated into the cells. Following recovery, a cell death assay was performed by dissolving EthD-1 in an extracellular solution to determine whether cells had died due to irreversible electroporation (right figure). Figure 17a Cells successfully electroporated and recovered were retained in Fluo-4 for imaging. Figure 17b A heatmap is shown illustrating the average EthD-1 and Fluo-4 intensities for eight study protocols (three columns of five biphasic pulses at 20 Hz) performed using HEK 293 cells to increase voltage values. For each of the eight protocols, the CNEA array was divided into subgroups of 128 pixels, repeated across a grid across the array. Imaging was performed for each pixel, and the 128 images for each protocol were averaged together. Figure 17c This demonstrates the effect on HEK 293 cells. Figure 17b The average intensity results are as follows. Successful electroporation is considered to start at about 1.3 V, while irreversible electroporation starts at about 1.7 V. Figure 17d The results for neurons under the same testing conditions are shown. For successful electroporation, the lower threshold is <1.2 V, while for irreversible electroporation, the lower threshold is about 1.5 V.

[0196] Figures 18A to 18B The figure illustrates another embodiment of injecting Fluo-4 into cells using Fluo-4 AM. While monitoring fluorescence, an electroporation protocol was applied to the nanoelectrode using a pixel stimulator (center). If electroporation is successful, Fluo-4 is able to flow out of the cell, causing a decrease in fluorescence. For a successful protocol, the cell membrane recovers after electroporation. Figure 18A (See right image). Figure 18B The illustration shows an example using neurons and their fluorescence, along with an applied electroporation signal. During electroporation, fluorescence decreases. Immediately thereafter, the cell membrane recovers, causing fluorescence stabilization. Electroporation signals can be applied multiple times without affecting cell viability.

[0197] exist Figures 17a to 17d and Figures 18A to 18B In the two experiments shown, it was observed that the voltage signal needed to have a certain duration (at least >50 ms) to be observed for any permeation / transportation. This indicates the need for a Faraday process to generate bubbles, as the required voltage is also comparable to the water window voltage (via water splitting to produce H2 and O2 gases) using platinum electrodes. Figures 18A to 18B In this study, the permeation signal was shown to be effective by causing transient leakage of the fluorescent dye, while... Figures 17a to 17d In this process, fluorescent dyes are delivered into the cells.

[0198] This delivery capability can be readily used to screen membrane-impermeable compounds for their effects on cells and cell-cell interactions. In this latter application of cell-cell interactions, the spatial capability of the electrode array, which allows for the selection of cells for delivery, can be useful, allowing for the measurement of the delivered cells and their undelivered neighbors in relation to the compound's effects. Without such delivery capability, membrane-impermeable compounds would otherwise require chemical modification for delivery, which is expensive and time-consuming, or delivery using single-cell-based micropipettes, also expensive and time-consuming. In addition to compounds, RNA / DNA / plasmids can also be delivered for applications in synthetic biology.

[0199] Example 12: Serial transmission in cross-effects analysis

[0200] This example describes the multi-step delivery of compounds into cells using an electrode array.

[0201] Figure 19 A series of schematic diagrams illustrate the use of spatial addressing and serial delivery to generate controlled and cross-effect delivery via gas generation. Since the electrode properties are not modified during gas evolution, the spatial capabilities of addressable electrodes provide a further advantage for screening cross-compound effects. For example, if it is desired to study the effects of two compounds on cells, only two compound delivery steps are needed to form a complete drug effect matrix.

[0202] Example 13: Extracellular Electrochemical Marking

[0203] This example illustrates electrochemical scaling using redox electrochemistry on an electrode array.

[0204] Electrochemical measurements of cells using electrodes can measure the bulk concentration of an analyte in solution using a single large working electrode. Such electrochemical electrode-based measurements include Clark electrodes for measuring dissolved oxygen concentration and hydrogen ion concentration (pH). According to one aspect of this disclosure, an array of electrochemical electrodes can be used for spatial mapping of analyte concentrations measured via electronics within a CMOS integrated circuit. This electrochemical mapping can then be applied to cell analysis of cells cultured directly on top of the electrode array.

[0205] In this example, to demonstrate the ability to perform electrochemical labeling using an electrode array that utilizes CMOS integrated circuits, the common redox pair of ferricyanide / ferrous cyanide [Fe(CN)6] is used. 3- / [Fe(CN)6] 4- Perform cyclic voltammetry.

[0206]

[0207] Figure 20a This diagram illustrates a cyclic voltammetry configuration that uses a CMOS integrated transimpedance amplifier to measure the current through each Pt electrode and an external transimpedance amplifier to measure the current through the Ag / AgCl dummy reference electrode. Figure 20a In the experiment, a cyclic voltage ramp was applied using 1.5 M KCl + 5 mM K3[Fe(CN)6] at a scan rate of 35 mV / s. The sum of the currents of the 13 × 13 electrodes was used for measurement, which was equal to the sum of the currents of the reference electrodes. Figure 20b The maximum range of electrode currents (|I) associated with the diffusion of ferricyanide (the starting reactant) is shown. e,max – I e,min |; Top left) and the maximum range of currents associated with ferrocyanide diffusion (products) minus the maximum / minimum voltage current (|I e,max – I e,min | – |I e,vmax – I e,vmin The two spatial diagrams (lower left and right). With these parameters constrained, the one on the right... Figure 20b The individual electrode records for the example are shown. The non-radial diffusion of ferrocyanide is attributed to the convection effect in the solution.

[0208] In this experiment, a subset of the 64×64 electrode array, 13×13=169, was connected to the same number of corresponding transimpedance amplifiers, and a cyclic linear voltage ramp was applied, such as... Figure 20a The diagram in the figure is shown. Figure 20b A spatial plot of the current density is shown, illustrating the increase in cathode and anodic current values ​​at the electrode edges, which can be attributed to increased radial diffusion / mass transport at the edges compared to planar diffusion at the central electrode. Similarly, product formation limits the current density visualized by subtracting the maximum / minimum voltage current range from the peak current range, as illustrated in the data curves in Figure 20, plot 2001. Cyclic voltammetry data plot 2001 shows the trend of product diffusion towards the upper right corner. This spatial measurement of the current demonstrates the capability of current-based electrochemical scaling.

[0209] The open-circuit potential of an electrode can also be used to measure the concentration of a chemical substance in solution. For high concentrations of redox couples in solution, the open-circuit potential of a platinum electrode in solution can be determined by the Nernst equation. The Nernst equation correlates the reduction potential of an electrochemical reaction with the standard electrode potential, temperature, and the activity of the chemical substance undergoing reduction and oxidation.

[0210]

[0211] Among them, E HE is the electrode voltage potential relative to the standard hydrogen electrode (SHE). 0 It is the half-cell reduction potential, φ t is the thermal voltage (approximately 25.7 mV at 25°C), [Ox][Red] is the concentration of the oxidizing / reducing chemical, and n is the number of electrons transported in the cellular half-reaction. For the ferricyanide / ferrocyanide reaction, the measurement of the open-circuit potential then reflects the concentration ratio of these ions in the solution.

[0212] In this example, the potential of the remaining portion of the electrode array is measured. Specifically, the open-circuit potential is used to calibrate the generation of ferrocyanide and its transport across the CMOS electrodes.

[0213] A cyclic potential is applied to a group of 13×13 electrodes (excluding 9 electrodes within the group of 13×13 electrodes, e.g.) Figure 21b (As shown in the diagram), while simultaneously measuring the open-circuit potential of the remaining electrodes. Figure 21a This shows the selected electrode voltage V plotted over time. el The data curves show the increases and decreases related to ferricyanide / ferrousyanide concentrations. Figure 21b It is a heatmap showing the total value of the open-circuit potential (maximum value minus minimum value) plotted across the array for one cycle to represent the diffusion / mass transport trending towards the upper left corner. Figure 21c The diagram shows a heatmap and data curve of the open-circuit potential over the shortest time, plotted against the distance from the center of the 13×13 electrodes, illustrating the transient aspects of diffusion / mass transport.

[0214] In summary, for cyclic voltammetry, the open circuit around the measuring electrode indicates the flow of ferrocyanide toward the upper right corner of the device.

[0215] Example 14: Electrochemical Oxygen Labeling in Cells

[0216] This example illustrates a technique for applying electrochemical mapping to cell analysis. For instance, a platinum-based Clark electrode can be measured by applying a pulsating voltage or a sequence of voltage pulses to sequentially oxidize and then reduce platinum. Because platinum oxide blocks oxygen reduction, the current drops to zero after oxide formation. When the oxide is subsequently reduced, a negative current flows through the platinum electrode due to the presence of oxygen.

[0217]

[0218] Then, the local oxygen concentration is consumed, and the electrode waits for further oxygen to diffuse to the electrode so that current can pass through. Therefore, the rate of the equation is limited by oxygen diffusion, which is proportional to the oxygen concentration in the solution and can be measured by measuring the electrode current.

[0219] In experiments using electrode arrays, measurements were performed using a salt solution (phosphate-buffered saline) exposed to ambient air, followed by purging with nitrogen to reduce oxygen concentration. Figure 22a The diagram shows a sequence of voltage pulses 2202 applied to the stimulation electrodes of the electrode array, and a series of data curves 2204 measured using a CMOS electrode array in ambient air, after partial nitrogen purging and partial recovery (N2 purging stopped). Data curve 2204 shows the electrode current reflecting the oxygen concentration.

[0220] Compare the current I before and after purging el It showed a significant reduction. Then, experiments were performed using HEK293 cells, and the results were... Figure 22b As shown in the image. Figure 22b The maximum current I of the cross electrodes within the electrode array region is shown. max The cross-electrode impedance thermogram 2206 and the electrode current ΔI above the electrode array region. el The changes are shown in heatmap 2208. Similar oxygen measurements performed on HEK293 cells showed a decrease in oxygen concentration at the cell location, as confirmed by impedance mapping.

[0221] Cells consume oxygen as part of aerobic metabolism, therefore the oxygen concentration around cells is lower than in areas without cells. Indeed, as shown in cross-electrode impedance mapping, mapping the electrode currents across the array reveals smaller current values ​​at cell locations compared to areas without cells. Larger current values ​​are also observed at the left and bottom edges of Figure 2208, attributed to edge effects and increased diffusion / mass transport.

[0222] Example 15: The effect of platinum black and frequency on cell barrier sensitivity

[0223] In this example, platinum black (PtB) is used to reduce the electrode impedance Z. e To improve the sensitivity of cell barrier measurements. Figure 26a The results of a comparative study of electrode impedance against cells cultured for approximately 72 hours are shown, along with those against electrodes in three conditions: low density, high density, and no cells. Figure 26b The diagram illustrates the Z-axis of the bare electrode in PtB. te The measurement results are reduced by about 5 times, which makes it possible to measure data curves of cell-cell connections with two different densities at a higher signal-to-noise ratio. Figure 26c The illustration shows cell barrier maps compared to the reference at different frequencies. When compared to the density map extracted from the imaging, the lower frequency measurements show more expansion and do not capture the cell layer edges, but the 1.8 kHz measurement shows the highest contrast in cell connectivity measurements. Figure 26d The diagram shows cell density and connectivity extracted from cell nuclei in a fluorescence image. Figure 26e The Z values ​​measured at 1.8 kHz with and without a reference are shown. te Comparison between them. Z measured without a reference. te Slightly smaller, but the relationship is direct for both cellular and non-cellular regions (two clusters). This is because Z can be easily subtracted from the cell-substrate adhesion measurements. e Therefore, measurements without a reference are preferred. Figure 26f Z shows the control cell density. te and Z s A comparison between them. For this comparison, Z is interpolated via bilinear interpolation. s Perform downsampling to have the same characteristics as Z. te The measurements have the same spatial resolution. However, because cell barrier measurements are performed on cell-cell connectivity, the cell barrier becomes more dependent on cell density. The correlation between Zs and cell density is also small, which can be observed in cell-circuit models (…). Figure 4B In Z, it is considered as s High levels have an impact, and in Z... s The calculation uses information about Z s < <Z te The assumption is no longer valid.

[0224] Therefore, having described at least one embodiment and several aspects of examples of the invention, it is to be understood that various changes, modifications, and improvements will readily occur to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to fall within the spirit and scope of the invention. Furthermore, while advantages of the invention have been indicated, it should be understood that not every embodiment of the technology described herein will include every described advantage. Some embodiments may not implement any of the features described herein as advantageous, and in some cases, one or more of the described features may be implemented to achieve other embodiments. Therefore, the foregoing description and figures are merely examples.

[0225] Various aspects of the present invention can be used individually, in combination, or in various arrangements not specifically discussed in the embodiments described above; therefore, its application is not limited to the details and arrangements of the components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment can be combined in any way with aspects described in other embodiments.

[0226] Furthermore, the present invention can be implemented as a method for which examples have been provided. Actions performed as part of the method can be ordered in any suitable manner. Therefore, embodiments can be constructed in which actions are performed in a different order than illustrated, and may include performing some actions simultaneously, even those shown as sequential in the illustrated embodiments.

[0227] The use of ordinal numbers such as “first,” “second,” and “third” to modify a claim element in the claims does not imply that any priority, position, or order of a claim element exceeds that of another action or chronological order in the execution of the method. Rather, it is merely used as a label to distinguish one claim element with a certain name from another element with the same name (but using that ordinal number).

[0228] The terms “approximately” and “about” can be used to mean within ±20% of the target value in some embodiments, within ±10% of the target value in some embodiments, within ±5% of the target value in some embodiments, and within ±2% of the target value in some embodiments. The terms “approximately” and “about” can include the target value.

Claims

1. A method for providing an electrochemical reaction with a spatially positioned array of electrodes exposed on the surface of a semiconductor substrate, the method comprising: A semiconductor substrate is provided, comprising an array of electrodes exposed on the surface of the semiconductor substrate; The surface of the semiconductor substrate is contacted with a plurality of cells and a solution comprising one or more reagents; Select one or more electrodes from the electrode array; as well as The circuitry in the semiconductor substrate is controlled to apply one or more stimulation signals at one or more electrodes to initiate an electrochemical reaction at the selected one or more electrodes, thereby generating bubbles in the solution to cause at least one of the following: permeation of one or more cells in the plurality of cells, and detachment of one or more cells in the plurality of cells from the surface of the semiconductor substrate.

2. The method according to claim 1, wherein, The electrochemical reaction is a half-reaction that produces gas in solution, and wherein the one or more stimulus signals include a potential higher than the redox potential used to produce the gas.

3. The method according to claim 1 or 2, wherein, The gas in the bubbles includes H2, Cl2, or O2.

4. The method according to claim 3, wherein, The plurality of cells are of the first type, and the method further includes: On the surface of the semiconductor substrate, at a location where at least one cell of the first type has detached, one or more cells of the second type are inoculated.

5. The method according to claim 3, further comprising: On the surface, at the location where at least one cell has detached, the time sequence of the regeneration of the plurality of cells is marked; as well as Based on the calibration, the growth rate of the plurality of cells is determined.

6. The method according to claim 1 or 2, wherein, The control circuit applies one or more predetermined potentials, including performing cyclic voltammetry at the selected one or more electrodes, and the method further includes: Using the circuit, the electrical characteristics at each of some or all of the remaining electrodes in the electrode array, other than the selected one or more electrodes, are measured; An electrical characteristic map is generated based on the results of the measurements.

7. The method according to claim 6, wherein, The electrical characteristic mentioned is the characteristic of the open-circuit potential.

8. The method according to claim 6, wherein, The electrical characteristic mentioned is electric current.

9. The method according to claim 8, wherein, The characteristics of the current are the maximum extent of the range of circulating current.

10. The method according to claim 1 or 2, wherein, The control circuit applies one or more predetermined potentials, including applying a pulsating voltage signal at one of the selected electrodes, wherein... During a first portion of the pulsating voltage signal, the electrode is oxidized, and during a second portion of the pulsating voltage signal, the oxide on the electrode is reduced, and the method further includes: Using the circuit, a current signal at the electrode is measured during the second portion of the pulsating voltage signal; Based on the time-varying rate of change of the current signal, the oxygen concentration at the location of the electrode is determined; and An oxygen concentration map is generated based on the determined results.

11. The method according to claim 1 or 2, wherein, The one or more stimulation signals are potentials relative to the potential of the reference electrode.

12. A system for providing spatial positioning of an electrochemical reaction, the system comprising: Semiconductor substrate, the semiconductor substrate comprising: An electrode array, comprising a plurality of independently addressable electrodes disposed on the surface of the semiconductor substrate; and A circuit, controllable by one or more processors, to apply one or more potentials relative to the potentials of the electrodes in the electrode array or a reference electrode at an electrode group in the electrode array to initiate an electrochemical reaction at the electrode group, thereby generating bubbles in a solution comprising a plurality of cells adhered to the surface of the semiconductor substrate, resulting in at least one of the following: permeation of one or more of the plurality of cells, and detachment of one or more of the plurality of cells from the surface of the semiconductor substrate.

13. The system according to claim 12, wherein, The electrode array includes multiple pads deployed on the insulating surface of the semiconductor substrate.

14. The system according to claim 13, wherein, The plurality of pads include Au or Pt.

15. The system according to any one of claims 12 to 14, wherein, The reference electrode is an Ag / AgCl reference electrode.

16. The system according to any one of claims 12 to 14, wherein, The electrode array includes at least 1,000, at least 4,000, or at least 1,000,000 electrodes, and the circuit includes a plurality of recording circuits, each configured to measure the current at an electrode in the electrode array.

17. The system according to claim 16, wherein, The plurality of recording circuits includes at least 10 recording circuits or at least 4,000 recording circuits.

18. The system according to claim 16, wherein, Each recording circuit includes a transimpedance amplifier (TIA).

19. The system according to claim 18, wherein, The TIA includes an impedance component with a resistance of at least 10 MΩ, wherein the output voltage of the TIA is proportional to the voltage across the impedance component.

20. The system according to claim 19, wherein, The impedance component includes a switched capacitor.

21. A system for providing spatial positioning of an electrochemical reaction, the system comprising: An electrode array, wherein the electrode array is exposed at a surface region of a semiconductor substrate; A circuit, wherein the circuit is deployed in the semiconductor substrate and coupled to the electrode array; At least one non-transitory computer-readable medium having executable instructions stored thereon; as well as At least one processor, the processor being programmed by the executable instructions to perform a method including the following actions: Select the pattern of the electrodes in the electrode array; as well as The control circuit applies one or more predetermined potentials at the pattern of the electrodes relative to the potential of the electrodes in the electrode array or the potential of a reference electrode, such that an electrochemical reaction is initiated at the pattern of the electrodes, thereby generating bubbles in a solution comprising a plurality of cells adhered to the surface of the semiconductor substrate, resulting in at least one of the following: permeation of one or more of the plurality of cells, and detachment of one or more of the plurality of cells from the surface of the semiconductor substrate.

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