Systems and methods for cell patterning and spatial electrochemical mapping

Abstract

Disclosed is a device for electrically evaluating and / or manipulating cells. One embodiment is directed to electrically mapping cells on the surface of a semiconductor substrate via cross-electrode impedance measurements. Furthermore, according to some embodiments, the electrode array enables real-time, spatially addressable electrical stimulation and / or recording of electrical signals using CMOS circuitry. Some of these embodiments are directed to using the electrode array to perform cell patterning via electrochemical gas generation and extracellular electrochemical mapping.

Description

Technical Field

[0001] (Related Application) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 040439, filed on June 17, 2020, entitled "Systems and Methods for Patterning and Spatial Electrochemical Mapping of Cells", inventors "Park, et al.", which is hereby incorporated by reference in its entirety.

Background Art

[0002] The present disclosure relates to semiconductor devices for electrically evaluating cells or other biological samples.

Summary of the Invention

Means for Solving the Problems

[0003] Disclosed herein are various devices for electrically evaluating and / or manipulating cells. One aspect is directed to electrically mapping cells on the surface of a semiconductor substrate via cross-electrode impedance measurements. Further, according to some aspects, the electrode array enables spatially addressable electrical stimulation and / or recording of electrical signals in real time using a CMOS circuit. Some of these aspects are directed to performing cell patterning by electrochemical gas generation and extracellular electrochemical mapping using the electrode array.

[0004] Several embodiments relate to an apparatus for mapping one or more cells. The apparatus comprises a semiconductor substrate. The semiconductor substrate comprises a plurality of electrodes exposed on the surface of the semiconductor substrate, and an active circuit connected to the plurality of electrodes and configured to measure a first set of cross-electrode currents between a first electrode of the plurality of electrodes and some or all of the remaining electrodes, and a second set of cross-electrode currents between a second electrode of the plurality of electrodes and some or all of the remaining electrodes. The apparatus further comprises one or more processors configured to receive the cross-electrode currents measured from the active circuit and to generate a map of one or more cells based on the first and second sets of cross-electrode currents.

[0005] In some embodiments, the active circuit is further configured to apply a stimulus signal to a first electrode of a plurality of electrodes and a reference voltage to the remaining electrodes on which the cross-electrode current is being measured. The stimulus signal may have a frequency of less than 10 kHz, preferably in the range of 0.1 to 5 kHz. The plurality of electrodes may be arranged in an array having a pitch 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 circuit may include complementary metal oxide semiconductor (CMOS) elements in the silicon substrate. The plurality of electrodes may include a plurality of pads arranged on an insulating surface of the semiconductor substrate. The active circuit includes a plurality of recording circuits, each recording circuit may be configured to measure the current at one of the plurality of electrodes. The plurality of recording circuits may include at least 8 recording circuits, at least 10 recording circuits, preferably at least 4000 recording circuits. Each recording circuit may include a transimpedance amplifier (TIA). The TIA may include an impedance element having a resistance of at least 10 MΩ, at least 100 MΩ, or between 10 MΩ and 1 GΩ, and the output voltage of the TIA is proportional to the voltage across the impedance element. The impedance element may include a switching capacitor. One or more cells may be placed in the first well of the multiwell plate, and the multiple electrodes may be a first electrode array exposed in the first well, and the apparatus may further include a second electrode array exposed on the surface of a semiconductor substrate and exposed in the second well of the multiwell plate. The multiwell plate may include at least 24, at least 96, or at least 384 wells. The multiple electrodes may be sized so that two or more electrodes are configured to contact one of the one or more cells. The multiple pads may include Au. The multiple pads may include Pt.

[0006] Several embodiments relate to a method for mapping one or more cells in contact with an electrode array disposed 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, for each electrode of at least one electrode in the electrode array, the steps of: applying a stimulus signal to the electrode; measuring a set of cross-electrode currents between the electrode and some or all of the remaining electrodes in the electrode array; generating a representative value associated with the electrode location of the electrode based on the set of cross-electrode currents; and generating a map of the representative values ​​on the surface region based on the generated representative value and the individual associated electrode locations of the at least one electrode.

[0007] In some embodiments, the step of generating a representative value may include selecting the maximum current value of a set of cross-electrode currents as the representative value. At least one electrode may include all electrodes in the electrode array. The map may have a spatial resolution of 20 μm or less, preferably 5 μm or less.

[0008] In some embodiments, the generated map may be a first map generated in a first time, comprising a plurality of pixels, and the method comprises the steps of: generating a second map of representative values ​​on a surface region in a second time following the first time, the second map comprising 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 properties based on a comparison of the first count and the second count. The map may comprise a plurality of pixels, each pixel associated with a representative value. At least one electrode comprises a first electrode having a first electrode location and a second electrode having a second electrode location, the first and second electrodes being adjacent to each other on a surface region, and the map may comprise first and second pixels corresponding to the first and second electrode locations, respectively. The step of generating the map may include determining an enlarged representative value associated with a third pixel located between the first and second pixels. The step of determining the amplified representative value is the cross-electrode current I between the first and second electrodes when the stimulus signal is applied to the second electrode. 12 The process may include the step of calculating the expanded electrode current by dividing by the product of a first current I1 and a second current I2, where I1 is the sum of the cross-electrode currents measured on all the remaining electrodes when the stimulus signal is applied to the first electrode, and I2 is the sum of the cross-electrode currents measured on all the remaining electrodes when the stimulus signal is applied to the second electrode. The number of pixels in the map may be greater than the number of electrodes in the electrode array. The electrode locations in the electrode array may be arranged in multiple rows and multiple columns. The electrode array may have M rows and N columns, and the map may have at least 3M × 3N pixels.

[0009] Some embodiments relate to a system for mapping one or more cells. The system comprises a plurality of electrodes exposed on a surface area of ​​a semiconductor substrate; circuits disposed within the semiconductor substrate and controllable to apply a stimulus signal and / or measure current in one or more of the plurality of electrodes; at least one non-temporary computer-readable medium storing executable instructions; and at least one processor programmed by the executable instructions to perform a certain method. This method considers each electrode within a set of electrodes. • Controlling the circuit to apply a stimulus signal to the electrode, • Controlling the circuit to measure the set of cross-electrode currents between the electrode and some or all of the remaining electrodes among the multiple electrodes, • An operation to generate a representative value associated with the electrode location of the electrode based on a set of cross electrode currents, This includes the operation of generating a map of representative values ​​on a surface region based on the generated representative values ​​and the individual associated electrode locations of multiple electrodes.

[0010] In some embodiments, the operation to generate representative values ​​includes selecting the maximum current value of a set of cross-electrode currents as a representative value. The operation to generate representative values ​​may also include selecting the maximum current value of a set of cross-electrode currents and calculating the impedance based on the selected maximum current value as a representative value. The map may include multiple pixels, each of which may be associated with a representative value. The multiple electrodes include a first electrode having a first electrode location and a second electrode having a second electrode location, and the first and second electrodes may be adjacent to each other on a surface region. The map may include first and second pixels corresponding to the first and second electrode locations, respectively, and the operation to generate the map may include determining an expanded representative value associated with a third pixel located between the first and second pixels.

[0011] Some embodiments relate to a method for providing a spatially positioned electrochemical reaction using an electrode array exposed on the surface of a semiconductor substrate. The method includes the steps of selecting one or more electrodes in the electrode array and controlling a circuit in the semiconductor substrate to apply one or more stimulus signals to one or more electrodes to initiate an electrochemical reaction at one or more electrodes.

[0012] In some embodiments, the electrochemical reaction may be a half-reaction that generates a gas in solution, and one or more stimulus signals may include a potential higher than the redox potential for gas generation. The solution may include a plurality of cells attached to the surface of a semiconductor substrate, and the method may further include the step of generating a gas at one or more selected electrodes so that at least one of the plurality of cells located on the one or more selected electrodes is detached from the surface of the semiconductor substrate. The gas may include H2, Cl2, or O2. The plurality of cells may be a plurality of first type cells, and the method may further include the step of seeding one or more second type cells on the surface of the semiconductor substrate at the location where at least one first type cell has detached. In some embodiments, the method may further include the steps of mapping the time sequence of regrowth of the plurality of cells on the surface at the location where at least one cell has detached, and determining the growth rate of the plurality of cells based on the mapping. The step of controlling the circuit to apply one or more predetermined potentials may include performing cyclic voltammetry on one or more selected electrodes, and this method may further include using the circuit to measure the electrical characteristic values ​​of each of the remaining electrodes in some or all of the electrode array outside the one or more selected electrodes, and generating an electrical characteristic map based on the measurement results. The electrical characteristic may be the open-circuit potential characteristic. The electrical characteristic may be the current characteristic. The current characteristic may be the maximum range of the periodic current.

[0013] In some embodiments, the step of controlling the circuit to apply one or more predetermined potentials may include applying a pulsed voltage signal to one or more selected electrodes. During the first portion of the pulsed voltage signal, the electrodes are oxidized, and during the second portion of the pulsed voltage signal, the oxides on the electrodes are reduced. The method may further include using the circuit to measure a current signal at the electrodes during the second portion of the pulsed voltage signal, determining the oxygen concentration at the electrode location based on the rate of change of the current signal over time, and generating an oxygen concentration map based on the result of the determination. The one or more potentials may be relative to the potential of a reference electrode.

[0014] Several embodiments relate to a system, which includes a semiconductor substrate. The semiconductor substrate comprises an electrode array including a plurality of individually addressable electrodes arranged on the surface of the semiconductor substrate, and a circuit controllable by one or more processors, which applies one or more potentials to an electrode group in the electrode array, relative to the potential of an electrode in the electrode array or the potential of a reference electrode, thereby initiating an electrochemical reaction in the electrode group.

[0015] In some embodiments, the electrode array may include a plurality of pads disposed on an insulating surface of a 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 1000, at least 4000, or at least 1,000,000 electrodes, and the circuit may include a plurality of recording circuits, each recording circuit configured to measure the current at the electrodes of the electrode array. The plurality of recording circuits may include at least 10 recording circuits, or at least 4000 recording circuits. Each recording circuit may include a transimpedance amplifier (TIA). The TIA may include an impedance element having a resistance of at least 10 MΩ, and the output voltage of the TIA is proportional to the voltage across the impedance element. The impedance element may include a switching capacitor.

[0016] Several embodiments relate to a system for providing a spatially positioned electrochemical reaction. The system comprises an electrode array exposed on a surface area of ​​a semiconductor substrate; a circuit disposed within the semiconductor substrate and connected to the electrode array; at least one non-temporary computer-readable medium storing executable instructions; and at least one processor programmed by the executable instructions to perform a certain method. This method includes selecting an electrode pattern in the electrode array and controlling the circuit to apply one or more predetermined potentials to the electrode pattern relative to the potential of the electrodes in the electrode array or to the potential of a reference electrode, so that an electrochemical reaction is initiated in the electrode pattern. [Brief explanation of the drawing]

[0017] Various aspects and embodiments will be described with reference to the drawings below. It should be understood that the drawings are not necessarily drawn to scale. Articles appearing in multiple drawings are indicated by the same reference number in all drawings in which they appear.

[0018] [Figure 1a] These are schematic side views of semiconductor substrates according to several embodiments. [Figure 1b] Figure 1a shows a two-dimensional data plot of the simulated voltage distribution in the device. [Figure 1c] This is a data plot of the simulated electric field line corresponding to the example shown in Figure 1b. [Figure 2a] This is a schematic side view of a device equipped with a semiconductor substrate that does not contain cells, according to several embodiments. [Figure 2b] Figure 2a is a schematic side view illustrating a scenario in which cells are placed on several electrodes of an electrode array. [Figure 2c] Figure 2a is a schematic side view illustrating a scenario in which cells are positioned outside the electrode array, between certain electrodes. [Figure 3a] An example of cell mapping using the distribution of maximum current is shown. [Figure 3b] An example of cell mapping using the distribution of maximum current is shown. [Figure 4A] This is a schematic diagram illustrating an example of high-resolution magnified mapping using cross-electrode current. [Figure 4B] This is a schematic diagram of a cellular circuit model. [Figure 5a] An example of magnified cross-electrode impedance mapping is shown in comparison to fluorescence microscope images. [Figure 5b] An example of magnified cross-electrode impedance mapping is shown in comparison to fluorescence microscope images. [Figure 6a] This example demonstrates the use of cross-electrode impedance mapping to quantify cell adhesion. [Figure 6b] This example demonstrates the use of cross-electrode impedance mapping to quantify cell adhesion. [Figure 6c] This example demonstrates the use of cross-electrode impedance mapping to quantify cell adhesion. [Figure 7] A series of fluorescence microscope images and normalized cross-electrode impedance maps. [Figure 8a] This is the normalized impedance histogram of the control measurement without tetracycline addition. [Figure 8b] This is a normalized impedance histogram of MDCK cells over a 6-7 day in vitro culture (DIV) period. [Figure 9] A series of normalized cross-electrode impedance maps under stimulus signals of various frequencies are shown. [Figure 10a] This example demonstrates mapping cells and their adhesion over time using cross-electrode impedance measurements. [Figure 10b] An example of measuring intercellular adhesion is shown. [Figure 11] This is a schematic diagram illustrating cell patterning by electrochemical gas generation. [Figure 12] An example of cell spatial patterning and co-culture delimitation is shown. [Figure 13A]This is a series of figures showing modified examples of cell patterning using electrode arrays. [Figure 13B] This is a series of figures showing modified examples of cell patterning using electrode arrays. [Figure 13C] This is a series of figures showing modified examples of cell patterning using electrode arrays. [Figure 13D] This is a series of figures showing modified examples of cell patterning using electrode arrays. [Figure 14] A series of fluorescence microscopy images are shown illustrating the process of defining co-cultures by patterning and then seeding the second cell type. [Figure 15] This is a series of schematic diagrams showing heterogeneous cell populations, the removal of unwanted cells using patterned electrochemical gas generation on a selective electrode, and the subsequent homogeneous culture with desired characteristics after cell growth. [Figure 16] An example of a wound healing assay is shown. [Figure 17a] This section presents an experiment demonstrating a transparency technique. [Figure 17b] This section presents an experiment demonstrating a transparency technique. [Figure 17c] This section presents an experiment demonstrating a transparency technique. [Figure 17d] This section presents an experiment demonstrating a transparency technique. [Figure 18A] This paper demonstrates an experiment using an electroporation protocol in which Fluo-4 is injected into cells using Fluo-4 AM. [Figure 18B] This paper demonstrates an experiment using an electroporation protocol in which Fluo-4 is injected into cells using Fluo-4 AM. [Figure 19] A series of schematic diagrams illustrating spatial addressing and serial delivery via gas generation, as well as the occurrence of cross-effect delivery, are shown. [Figure 20a] An example of extracellular electrochemical mapping is shown. [Figure 20b] An example of extracellular electrochemical mapping is shown. [Figure 21a] The data plot shows the selected electrode voltage plotted over time. [Figure 21b] This is a heatmap showing one cycle, plotted across the array as the overall amplitude of the open-circuit potential. [Figure 21c] This is a heatmap showing one cycle, plotted across the array as the overall amplitude of the open-circuit potential. [Figure 22a] An example of electrochemical oxygen mapping of cells is shown. [Figure 22b] An example of electrochemical oxygen mapping of cells is shown. [Figure 23a] A series of schematic diagrams showing electrical imaging of three parameters useful for evaluating living cells are presented. [Figure 23b] This image shows the fluid well packaged on top of the chip mounted below the microscope, for simultaneous optical and electrical measurements. [Figure 23c] This is a colorized microscope image showing cells and an electrode array. [Figure 23d] This is a schematic diagram showing electrodes connected to an exemplary pixel circuit. [Figure 24a] This is a schematic diagram illustrating several additional schemes for measuring intercellular connectivity according to several embodiments. [Figure 24b] This is a schematic diagram illustrating several additional schemes for measuring intercellular connectivity according to several embodiments. [Figure 25a] This is a schematic diagram showing a pixel amplifier configured as a buffer for measuring metabolic state. [Figure 25b] This is a series of data maps showing the results of multiparametric measurements. [Figure 25c] This image shows a pair of nuclear fluorescence images taken 72 hours after seeding (top) and a detailed region 1 comparison (bottom) showing the lowest cell density on the anterior margin compared to the posterior margin. [Figure 25d] This is a composite map showing detailed region 2 where the cell nucleus and cell adhesion are superimposed. [Figure 26a] This is a series of fluorescence images showing the results of a comparative study of electrode impedance under three different scenarios. [Figure 26b]This data plot shows that PtB reduced the Zte measurement of the bare electrode. [Figure 26c] This shows cell barrier maps against references at various frequencies. [Figure 26d] This shows cell density and connectivity maps extracted from the nuclei of the fluorescence images. [Figure 26e] This shows a comparison between Zte measured without a reference point. [Figure 26f] This shows a comparison of Zte and Zs in relation to the extracted cell density. [Modes for carrying out the invention]

[0019] This disclosure relates to various devices for electrically evaluating and / or manipulating cells. In one embodiment, the device includes a semiconductor substrate having complementary metal oxide semiconductor (CMOS) circuits electrically interfaced with an electrode array exposed to cells, which can be fabricated on the surface of the semiconductor substrate using CMOS-compatible manufacturing techniques. The inventors have recognized and understood that electrode arrays can be economically manufactured and integrated with active circuits by using semiconductor processing techniques. Furthermore, electrodes in an electrode array having a small electrode size and electrode-to-electrode pitch can enable higher spatial resolution evaluation of multiple cells compared to using electrodes larger than the size of the cells. For example, individual cells are identifiable when mapping using a high-density electrode array compared to a large electrode covered by a cell aggregate. Furthermore, according to some embodiments, the electrode array enables real-time, spatially addressable recording of electrical stimulation and / or electrical signals using CMOS circuits. Some of these embodiments aim to perform cell patterning by electrochemical gas generation and extracellular electrochemical mapping using the electrode array.

[0020] One embodiment aims to electrically map cells on the surface of a semiconductor substrate via cross-electrode impedance measurement. The inventors recognized and understood that the electrical impedance measured between two electrodes, or cross-electrode impedance, can be influenced by the impedance along the current path between the electrodes. As a result, the presence of one or more cells along the current path can affect the cross-electrode impedance, and these cells can be mapped using cross-electrode impedance measurement.

[0021] Figure 1a is a schematic side view of a semiconductor substrate according to several embodiments. Figure 1a shows an apparatus 100 having an electrode array 106 including a plurality of electrodes 106_1, 106_2, 106_3, ... 106_N arranged on the surface 104 of a semiconductor substrate 102. Figure 1a shows an example of cross-electrode impedance measurement by applying a voltage stimulus to a first electrode such as 106_1 and measuring a current at a second electrode such as 106_2. The measured current (also referred to as the cross-electrode current between electrode 106_1 and electrode 106_2) flows along one or more current paths 109 in a medium 108 that is in contact with the electrode array 106. Electrode 106_1 can be connected to a stimulus source circuit 110 and may be referred to as a stimulus electrode. Electrode 106_2 can be connected to a current measurement circuit 112 and may be referred to as a recording electrode.

[0022] The cross-electrode impedance between electrodes 106_1 and 106_2 is obtained from the values ​​of the cross-electrode current and stimulation voltage between the pair of electrodes by any suitable method known in the art, for example, by dividing the stimulation voltage amplitude by the cross-electrode current amplitude. A processing unit 120 is provided to receive a signal from the active circuit in the semiconductor substrate 102 and perform the determination of the cross-electrode impedance. 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 to or addition to calculating the impedance value, the cross-electrode current can be used as an indicator of the cross-electrode impedance when comparing measurements at various electrodes, provided that the stimulation voltage amplitude is programmed to be a known constant.

[0023] Figure 1b is a two-dimensional data plot of the simulated voltage distribution in the apparatus shown in Figure 1a, showing that when a voltage is applied to the stimulating electrode 106_1, the potential in the medium 108 decreases along both the vertical (V) and lateral (L) directions away from the stimulating electrode 106_1. Figure 1c is a data plot of the simulated electric field lines corresponding to the example shown in Figure 1b. Figure 1c shows that the electric field line 114 emanating from the stimulating electrode 106_1 flows along a line pointing upward from the electrode 106_1, curves laterally toward a recording electrode such as the recording electrode 106_2, and then points downward, terminating at the recording electrode 106_2.

[0024] The presence of cells can alter the shape and distribution of the electric field lines 114 between electrodes, resulting in a change in cross-electrode impedance, as will be discussed later in relation to Figure 2. Figure 2a is a schematic side view of an apparatus 200 with a semiconductor substrate 202 without cells, according to one of several embodiments. In Figure 2, the electrode 206_0 of the electrode array 206 is configured to be a stimulating electrode, and the electric field lines 214_1, 214_2 link the stimulating electrode 206_0 and the recording electrode 206_1. Figure 2b is a schematic side view showing a scenario in Figure 2a where cells 220 are placed on some of the electrodes of the electrode array. Figure 2c is a schematic side view showing a scenario in Figure 2a where cells 220 are placed outside the electrode array, between some electrodes.

[0025] The inventors recognized and understood that living cells have a lipid bilayer that forms a continuous membrane barrier around the cell. Electrically, the membrane can behave as a capacitor connected in parallel with high resistance and can have a different electrical impedance compared to the surrounding medium, such as a solution containing cells. Cells with a high-impedance membrane on top of an electrode array then affect the current distribution in a solution, such as solution 208, in Figures 2a-2c. In Figure 2c, suspended cells block the electric field lines in the solution and reduce nearest-neighbor coupling between electrodes. In contrast, cells attached to a surface and covering both the stimulating and recording electrodes increase cross-electrode coupling by blocking the perpendicular electric field lines.

[0026] As an example of the effect of cells on cross-electrode impedance, without wishing to be bound by any particular theory, the inventors have recognized that when cells such as cell 220, as shown in Figure 2b, adhere to the surface 204 and cover part or all of the stimulating electrode 206_0 and recording electrode 206_1, the cell 220 will increase cross-electrode coupling by preventing the electric field lines 214_1,214_2 between the two electrodes from running perpendicularly through the solution 208. As a result, compared to Figure 2a where no cells are present, more perpendicular electric field lines 214_2 are suppressed and more electric field lines 214_1' are strengthened, resulting in a lower impedance between electrode 206_0 and electrode 206_1.

[0027] On the other hand, if cells are not attached to surface 204, or if cells such as cell 230 shown in Figure 2c are attached to surface 204 but are positioned laterally outside the pair of electrodes 206_0 and 206_2, the cells may block the electric field line 214_3 between the pair of electrodes, reducing 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.

[0028] Therefore, whether or not cells are present above the electrode array and attached to the surface can be detected using cross-electrode impedance measurement. It should be understood that cells attached to the surface may have varying degrees of non-zero separation between the outer range of the cell membrane and the surface. Apparatus according to some embodiments of the present application can provide detection of the degree of cell attachment. For example, stronger adhesion will result in a stronger increase in cross-electrode coupling due to a smaller gap distance along the vertical direction between the cell and the surface of the semiconductor substrate.

[0029] Cross-electrode measurements offer several advantages. For example, these measurements are non-invasive and can be repeated without affecting cell viability or the electrodes.

[0030] In some embodiments, as described above in relation to Figure 2b, the cross-electrode impedance technique measures an increase in cross-electrode coupling between electrode pairs due to the suppression of the vertical electric field line from the presence of cells, in contrast to techniques that measure a decrease in cross-electrode coupling (or an increase in measured impedance) due to the blocking from the presence of cells. One advantage of using an increase in cross-electrode coupling as an indicator for detecting the presence of cells is that the increase is due to electrode pairs that are close to each other, and in some cases, mainly due to the nearest neighbor coupling between electrode pairs. Thus, the increase in cross-electrode coupling (or the decrease in measured impedance) can be isolated from the overall background current flowing through the stimulating electrode to many of the remaining electrodes in the electrode array. As a result, the signal-to-background ratio and sensitivity of cell detection can be improved.

[0031] In contrast to cross-electrode impedance techniques, the inventors recognized that mere impedance measured at individual electrodes cannot detect the presence of cells. In such single-electrode measurements, the sum of all return currents is measured as the impedance signal on the electrode. That is, such measurements are impedance measurements of the electrode only, and do not measure changes in the electric field in the solution on the electrode. As a result, the inventors observed that the impedance of the electrode itself is not sensitive to the presence of cells, even when cells are cultured directly on its surface.

[0032] Referring again to 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 having a frequency of less than 10 kHz, less than 5 kHz, between 0.1 and 5 kHz, or between 0.1 and 2 kHz. The reason for selecting a low-frequency stimulation signal is that the cell membrane acts as a capacitor connected in parallel with high resistance, and at high frequencies, the capacitor impedance decreases, making the cell highly conductive. The inventors have recognized and understood that measuring the cross-electrode current at low frequencies can provide high signal contrast for the detection of cell adhesion. An example of the frequency response of cross-electrode impedance measurement is provided in Example 4 below.

[0033] Referring further to Figure 1, the semiconductor substrate 102 may include an active circuit 116. The active circuit 116 may include a plurality of stimulating circuits 110 and a plurality of recording circuits 112. In some embodiments, the stimulating circuit 110 may comprise one or more current injectors, one or more voltage sources, or a combination thereof. Several aspects of the active circuit design relate to current-type stimulators and related methods for electromotive cells, as disclosed in International Patent Application Publication No. 2019 / 010343, Agent Reference No. H0776.70105WO00, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the active circuit may comprise 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.

[0034] In some embodiments, each recording circuit comprises a transimpedance amplifier (TIA) having a switching capacitor as an impedance element. The resistance of the impedance element is at least 10 MΩ (megaohms), at least 100 MΩ (megaohms), or between 10 MΩ and 1 GΩ (gigaohms) to provide amplification of 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 proportional to the voltage across the impedance element.

[0035] The electrodes in the electrode array 106 can be reconfigured using the active circuit 116 as stimulating or recording electrodes. In some embodiments, the active circuit 116 includes routing and switching components that are programmable to connect selected electrodes of the electrode array 106 to a stimulating source circuit 110, a current measurement circuit 112, or other circuit components that allow for various functions. Depending on the application, two or more electrodes may be configured as stimulating electrodes, and two or more electrodes may simultaneously be recording electrodes. For example, when mapping local cell characteristics using cross-electrode impedance measurements, typically only one electrode acts as a stimulating electrode at a time. In some other embodiments, a subset of one or more electrodes can be selected to act as stimulating electrodes or to apply one or more potentials or currents to initiate electrochemical reactions at the locations of one or more selected electrodes. The latter embodiments are discussed in more detail in the following sections with respect to cell adhesion measurements, patterning, and spatial electrochemical mapping of cells.

[0036] In some embodiments, electrodes may be biased using a low-impedance source / return within the active circuit. For example, a low-output impedance voltage source may be used to provide a stimulus signal to the stimulation electrode, while a low-input impedance transimpedance amplifier may be provided for current measurement at the recording electrode. In such embodiments, each electrode may be selectively connected to a voltage source for stimulation, a transimpedance amplifier for current measurement, a voltage source for return, or a transimpedance amplifier for simultaneous stimulation and current measurement. The inventors have understood and recognized that a low-impedance source / return facilitates the formation of fringing field lines in solution, as shown in Figure 1c.

[0037] The semiconductor substrate 102 may contain silicon, and thus in this embodiment, the active circuit 116 may be an integrated circuit comprising CMOS components manufactured using standard CMOS processing techniques. The electrode array 106 may be arranged within the semiconductor substrate 102 as a conductor exposed from, for example, the surface 104 of the semiconductor substrate 102 facing the medium 108. In some embodiments, the surface 104 is an insulating surface that provides mechanical support and electrical isolation to the electrode array 106 and provides a surface suitable for cell growth. Figure 1a shows the electrode array 106 partially embedded in the semiconductor substrate 102, but such arrangement is illustrative and not required. In some embodiments, the upper surfaces of the electrodes in the electrode array 106 may be above, vertically aligned with, or below the surface 104 of the semiconductor substrate 102. Additionally or alternatively, the upper surfaces of the electrodes may have a passivation layer or a functionalization layer. In some embodiments, holes may be patterned in the passivation or functionalization layer above the electrode to expose the conductive surface of the electrode to the medium.

[0038] It should be understood that the semiconductor substrate 102 may 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, sp2 hybrid carbon materials, chalcogenides, metals, metal compounds, oxides, nitrides, silicides, polymer materials, or combinations thereof. The semiconductor substrate 102 may be a single component or a composite of multiple components. The components within the semiconductor substrate 102 may include active circuit layers, wiring layers, redistribution layers, circuit boards, or combinations thereof. The component layers within the semiconductor substrate may be formed in an additive process during CMOS processing, or they may be formed separately and joined to each other using packaging techniques known in the art. Conductors for interconnecting the active circuit 116 and the electrode array 106 are provided on the semiconductor substrate 102. In some embodiments, connection points for electrically interface the components within the semiconductor substrate with the processing unit 120 are provided on the bottom surface of the semiconductor substrate. The electrical connection between the processing unit 120 and the semiconductor substrate 102 may be provided by any suitable method, for example, but not limited to, a C4 connection (controlled collapse chip connection), flip-chip bonding, wire bonding, flexible cable, or wireless communication.

[0039] Referring again to Figure 1a, in some embodiments, the apparatus 100 can operate to perform a certain method, such as performing mapping or selective electrochemistry. The operation of the apparatus 100 may be program-controlled. In some embodiments, the processing unit 120 within the apparatus 100 may include a computer 20 having a storage medium 21, memory 23, and a processor 25, and such processing may be performed in the computer 20 or any other computer device. The storage medium 21 and memory 23 may be any suitable non-temporary 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 medium. In some embodiments, the storage medium 21 may be a non-volatile storage medium, and the memory 23 may be a volatile storage medium. Computer-executable instructions are loaded from the storage medium 21 into the memory 23 before execution by the processor 25, and can perform some or all of the methods described throughout this disclosure. However, the distinction between the storage medium 21 and the memory 23 is not important, and in some embodiments, one or both may be present.

[0040] The processor 25 may be any suitable processing device, for example, but not limited to, one or more processors, a central processing unit (CPU), a digital signal processor (DSP), a controller, an addressable controller, a general-purpose or dedicated microprocessor, a microcontroller, an addressable microprocessor, a programmable processor, a programmable controller, a dedicated processor, a dedicated controller, or any other suitable processing device. Some or all of the components within the processing unit 120 may be packaged as a system-on-a-chip (SOC). Furthermore, it should be understood that Figure 1a is a schematic representation of the processing unit 120. Actual implementations of the processing unit 120 may have distributed processing. For example, a host computer may control the overall flow of measurement, mapping, and result analysis.

[0041] Hereinafter, we refer to the electrode array 106. In some embodiments, the electrode array 106 can be patterned on a surface 104 as part of a semiconductor manufacturing process to form an active circuit 116 within the semiconductor substrate 102, and can be a conductive pad containing a metal such as Au or Pt or an alloy thereof. For example, the pad may be formed of Al with plated Au as an upper layer. In such embodiments, the substrate 110 may further include conductors that perpendicularly interconnect the exposed electrode array 14 to the circuit within the substrate 110.

[0042] The electrodes in the electrode array 106 may be arranged on the surface 104 in any suitable arrangement, for example, a two-dimensional array having a regular pitch along the row and column directions. In some embodiments of the cross-electrode impedance mapping method, the pitch of the electrode array can be selected to be on the order of or less than the size of a typical cell, so that a cell can cover at least two electrodes and increase the coupling between the cell and at least two electrodes. For example, if the size of the cell is about 30 μm, the pitch of the electrode array can be set to less than 30 μm, less than 20 μm, less than 5 μm, or between 1 and 20 μm. By providing a small pitch between electrodes, it becomes possible for a cell to cover two or more electrodes, enabling measurement of the cell-substrate gap distance through increased cross-electrode coupling at the electrode below the cell.

[0043] In some embodiments where an electrode array is manufactured during a CMOS-compatible manufacturing process on top of a semiconductor substrate containing CMOS active circuits, the pitch of the electrode array and the size of each electrode can be selected by taking into account the pitch and density of the CMOS active circuits. For example, in some embodiments, at least 8, at least 10, or at least 4000 recording circuits may be provided in the semiconductor substrate, and the electrode array may have at least 1000, at least 4000, or at least 1,000,000 electrodes. In such embodiments, each electrode may have a lateral dimension of 10 μm or less, or 5 μm or less, so that the overall lateral range of the electrode array is contained within the surface of the semiconductor substrate. Electrode arrays according to embodiments of this disclosure are sometimes also referred to as CMOS microelectrode arrays (MEAs).

[0044] Referring again to Figure 1, the medium 108 may be a cell culture medium or a solution containing cells in addition to any number of chemicals and / or biological reagents. Although not shown in Figure 1, the medium 108 may be contained in a container positioned on top of the semiconductor substrate 102. In some embodiments, the container may be a well of a multi-well plate attached to the semiconductor substrate, with one or more wells having an open bottom that exposes the contents of the well to the semiconductor substrate. The semiconductor substrate may have multiple electrode arrays so that electrical evaluation in multiple wells can be performed in parallel.

[0045] A CMOS-compatible, wafer-scale, multi-well platform and a method for operating it, usable for biomedical or other applications. In some applications, the circuitry is located at the bottom of a multi-well array and electrically interfaces with electrodes in the wells. The platform is sometimes referred to as a CMOS-multi-well platform. The inventors have recognized and understood that, in order to interface with electrodes in a large array, the circuitry can be manufactured on a single silicon (Si) wafer having dimensions at least the same as or larger than the dimensions of the multi-well array. According to one aspect of this disclosure, a standard CMOS manufacturing process known to be used in a standard semiconductor manufacturing plant can be used, for example, without expensive customization for complex manufacturing procedures, thus reducing manufacturing costs in some cases. CMOS-multi-well platforms according to some aspects of this disclosure can be used in applications including electrophysiological studies and general cell evaluation using electrical methods, and / or in high-throughput formats (e.g., 24-well, 96-well, and 384-well plate formats).

[0046] In some embodiments, a Si wafer is part of a semiconductor device and has an array of reticle regions, some or all of which have multiple circuits of the same design. The inventors have recognized and understood that during manufacturing, the reticle regions of a wafer can, in some cases, reuse the same lithography mask design repeated across the wafer, thus reducing processing costs and increasing wafer manufacturing throughput.

[0047] According to one embodiment, the digital and analog circuits within the reticle region may be arranged to correspond to one or more wells when a multiwell array is coupled to the top of the wafer. Thus, some embodiments can provide wafer-scale integration of electrical interfaces with multiwell arrays by not dicing the wafer and / or by using a manufacturing method that conforms to standards using standard MOS-compatible techniques and reduces manufacturing costs.

[0048] One aspect of this disclosure relates to a method for mapping the spatial distribution and dimensions of cells using cross-electrode impedance measurements. The mapping can further represent the characteristics of individual cells, such as their adhesion to the surface of a semiconductor substrate. In some embodiments, since the presence of cells is primarily reflected locally in the cross-electrode coupling between a stimulating electrode and a nearby recording electrode, the mapping is performed by first selecting an individual electrode as the stimulating electrode and measuring a set of cross-electrode impedance data relative to other electrodes at multiple locations across the electrode array. Subsequently, a different electrode is selected as the stimulating electrode, and a new set of cross-electrode impedance data is measured. The cross-electrode measurements are repeated by sequentially setting electrodes in the electrode array and applying a stimulating signal, and the measured cross-electrode impedance data of the corresponding sets can be processed to generate values ​​for each location of the stimulating electrode indicating the presence or absence of cells, or the strength of the cell properties. The processed values ​​are combinatorial and can form a map across the region of the electrode array. In some embodiments, "electrochemical imaging" of living cell cultures is demonstrated by high-resolution in-situ impedance and electrochemical measurements. Several embodiments relate to using CMOS-MEA for unlabeled, non-invasive tracking of cell growth dynamics, as well as for accurate measurement of cell-substrate adhesion, cell-cell adhesion, and metabolic status.

[0049] Another aspect relates to providing spatially positioned electrochemical reactions using a patterned electrode array. 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 in a programmed spatial pattern based on the size, shape, and distribution of the selected electrodes on the surface of the semiconductor substrate.

[0050] In some embodiments, cell patterning can be performed using spatially programmed electrochemistry. For example, cells attached to an electrode can be selectively removed from the electrode surface by electrochemically generating small bubbles on the electrode.

[0051] In some embodiments, an array of electrochemical electrodes can be used to spatially map the concentration of a sample (analyte) measured using an active circuit within a semiconductor substrate. One application is the electrochemical mapping of solutions using redox electrochemistry.

[0052] The following applications, U.S. Provisional Patent Application Serial No. 63 / 040439 (filed June 17, 2020, inventors Park, et al.), U.S. Provisional Patent Application Serial No. 63 / 040424 (filed June 17, 2020, inventors Ham, et al.), and U.S. Provisional Patent Application Serial No. 63 / 040412 (filed June 17, 2020, inventors Ham, et al.), are incorporated herein by reference in their entirety. Furthermore, the following applications, PCT Patent Application (filed June 16, 2021, title "Complementary Metal-Oxide-Semiconductor (CMOS) Multi-Well Apparatus for Electrical Cell Assessment") and PCT Patent Application (filed June 16, 2021, title "Apparatuses for Cell Mapping Via Impedance Measurements and Methods to Operate the Same"), are incorporated herein by reference in their entirety.

[0053] The following examples are intended to illustrate specific embodiments of the present invention and do not exemplify the entire scope of the invention.

[0054] (Example 1: Real-time cell measurement and imaging system using a CMOS microelectrode array (MEA)) This example describes the electrical imaging of three parameters useful for evaluating viable cells (Figure 23a): cell-substrate impedance: Zs (reflecting cell adhesion and cell-substrate adhesion), transepithelial impedance: Zte (reflecting intercellular adhesion and the integrity and barrier function of the cell monolayer), and extracellular redox potential: Vredox (reflecting cellular metabolic state and respiration).

[0055] In this example, a custom-designed CMOS IC is used, which parallelizes impedance and electrochemical capabilities across a 64 × 64 = 4096 electrode array (Figures 23b-d). Fluid wells are packaged on 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 located in the center of the instrument and consists of 8 μm diameter Pt electrodes spaced at a 20 μm pitch for single or a small number of cell resolutions (e.g., MDCK cells in Figure 23c), with a size of 1.26 × 1.26 mm. 2 The total detection area is obtained. The remainder of the surface is insulated with silicon nitride, which behaves similarly to glass culture plates. No differences are 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 mini culture chamber is placed on top of the device, with the CO2 adjusted to 5%.

[0056] Each electrode in the array is connected to its own pixel circuit (Figure 23d), which is highly configurable and programmed via a digital interface. The pixel circuit includes an operational amplifier, which can be configured as a buffer for measuring electrode voltage Ve or as a transimpedance amplifier for measuring electrode current Ie. Several embodiments of the pixel circuit configuration relate to current-type stimulators and related methods for electromotive cells, as disclosed in International Publication No. 2019 / 010343, Agent Reference No. H0776.70105WO00, the disclosure of which is incorporated herein by reference in its entirety. Several embodiments may also relate to electronic circuits and related methods for analyzing electromotive cells, as disclosed in International Publication No. 2019 / 089495, Agent Reference No. H0498.70647WO00, the disclosure of which is incorporated herein by reference in its entirety.

[0057] Figure 23a is a schematic diagram showing three cellular parameters electrically measured using a complementary metal-oxide-semiconductor (CMOS) integrated circuit (IC) for viable cell evaluation: cell adhesion by cell-substrate impedance Zs, intercellular adhesion by transepithelial impedance Zte, and metabolic state by extracellular redox potential Vredox. Each measurement is non-invasive and fast (<1 minute), and measurements can be repeated continuously every 5-10 minutes for real-time investigation. Figure 23b is an image showing that a fluorescence microscope can be paired with a packaged CMOS IC for simultaneous optical and electrical cellular measurements. In this example, a reference electrode, Pt (illustrated), or Ag / AgCl can be used. Figure 23c is a colorized fluorescence image of Madin-Darby canine kidney (MDCK) epithelial cells cultured on a CMOS electrode array. 64 × 64 = 4096 circular platinum electrodes with a diameter of 8 μm are spaced at a pitch of 20 μm. Platinum black (PtB) can be electrodeposited onto the electrodes to reduce electrode impedance for higher signal-to-noise ratio Zte measurements. Figure 23d is a schematic diagram of an exemplary circuit for electrodes in an electrode array. Each of the 4096 electrodes is connected to its own peripheral circuitry via a shielding path (~1-10 mm). The operational amplifier-based circuit can be configured to apply a voltage via Vs and measure the current via a feedback resistor Rf (~100 MΩ), or to apply a current via Is and buffer / measure the electrode voltage Ve. The output of the operational amplifier Vamp is routed externally for analog-to-digital conversion. The switch is digitally programmed using a real-time software interface.

[0058] In some embodiments, the high-channel-count (4096) parallel current and open-circuit potential measurements characterized by the measurement method in this example offer unique advantages over other MEA devices. For example, measurements like those described in this example are suppressed in MEA devices that measure electrode capacitance, voltage (using a high-pass filter to block DC signals), and current (with a small number of channels (<32)).

[0059] (Example 1A: Cell mapping using the distribution of maximum current) This example describes a method for mapping cells using a CMOS electrode array containing a 64×64 array of 4096 platinum electrodes at a 20 μm pitch.

[0060] The inventors recognized and understood that alternating current (AC) impedance measurement between a pair of electrodes can detect cells using the contrast between the insulating cell membrane and the conductive culture medium. In traditional impedance measurements, the solution pathway around the cell diverts the measurement and results in lower detection sensitivity. The solution involvement of the measured electrode-electrode current is much greater than the small change in current attributable to the cell. The device disclosed herein improves detection sensitivity by instead measuring the change in electric field distribution attributable to the cell.

[0061] An AC voltage (1.9 kHz frequency, 200 mV amplitude) was applied to one electrode, and the resulting AC current was measured using a transimpedance amplifier via the remaining 4095 electrodes. The results are shown in Figure 3a, which shows a heatmap 301 of the measured current distribution of the nearest 11 × 11 recording electrodes to one stimulating electrode 311 when no cells are present. In heatmap 301, each pixel corresponds to the location of the electrode. Each electrode has an electrode location or electrode position, which can be represented in several ways, such as coordinates or pixel number, for example, but not limited to these. Heatmap 302 is a measured current distribution similar to heatmap 301, but with cells located above electrode 311. Impedance measurements were performed at a signal frequency of 1.9 kHz.

[0062] The data plot 303 in Figure 3a, showing the measured cross-electrode current versus distance to the stimulated pixel, indicates that in the presence of cells, cross-electrode coupling with adjacent electrodes is approximately an order of magnitude higher than that of electrodes without cells above them.

[0063] In this example, a fluorescent nucleus MDCK cell line was used for optical confirmation. Figure 3b shows a fluorescence microscope image 304 across the entire 64×64 electrode array, 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, stimulating electrodes were sequentially scanned across the array. For each given stimulating electrode, the cross-electrode current value from the remaining electrodes was measured as the recording electrode. The recorded cross-electrode currents were collected, and the maximum value was determined, which is referred to as the maximum current value corresponding to the given stimulating electrode. Figure 3b shows a heatmap 305 across the electrode array generated using the maximum current value (Ie) determined from the stimulating electrode at each pixel location.

[0064] Figure 3b also shows map 306, which is an overlay of selected region 1 of the nuclear fluorescence signal 307 and the maximal current signal 309, demonstrating its ability to map clusters of cells with single-cell resolution. As a result, this example demonstrates that the presence of cells was confirmed using nuclear fluorescence markers, along with a strong correspondence between the maximal current map and the fluorescence imaging.

[0065] The maximum current value (Ie) is determined for each stimulating electrode location using any suitable method based on a set of cross-electrode currents measured from the recording electrode. The determination may be a simple comparison of the absolute values ​​of the cross-electrode currents, or it may include further data processing before the comparison, such as noise filtering, background subtraction, or any suitable signal processing technique known in the art. The processing and comparison of current values ​​may be performed after digitization of the measured current values ​​using a processing unit such as the processing unit 120 shown in Figure 1a.

[0066] (Example 2: High spatial resolution mapping using cross-electrode current) This example illustrates how to generate an enlarged map of a cross-electrode coupling with a spatial resolution higher than the pitch of the electrode array.

[0067] According to some embodiments, the nearest neighbor cross electrode measurements may be used for each stimulation electrode. FIG. 4A shows an example of high-resolution enlarged mapping using a 3×3 impedance grid for each of electrodes 1-9. In some embodiments, electrodes at the edge of the electrode array may be skipped from the enlarged impedance grid, as described later.

[0068] FIG. 4B is a schematic circuit model that can be used to calculate the cell-substrate impedance Z A and the trans-epithelial impedance Z 12 for the application of an AC stimulation voltage V s and the measurement of the cross electrode current I te . The 3×3 impedance grid is used for Z s calculation, and one Zte is extracted for each electrode.

[0069] To measure cell-substrate attachment, a change in the cross electrode electric field is formed. Instead of applying a bias between two electrodes, a bias is applied from one electrode to all the remaining electrodes. Thereby, electric field lines starting from the stimulation electrode and extending far into the culture well can terminate on electrodes far from the stimulation. Otherwise, these electric field lines would have to loop back towards adjacent electrodes, increasing the amount of measurement current not related to the nearest cell-electrode interface.

[0070] The interface may be modeled using a cross-sectional model to enhance spatial resolution. Assuming Z s [[ID=2G]]<<Z te , Z e,1 , Z e,2 , these have been found to be valid for most measurements according to some aspects. That is,

[0071] [[ID=SS]]

Number

[0072] The measured cross electrode current can also be rewritten with respect to (Equation A1).

[0073]

number

[0074] Z e,1 and Z e,2 To determine this, the sum of the measured currents across the array is used when a stimulus is applied to electrode n.

[0075]

number

[0076] From equations A3) and A2, Z s This can be solved.

[0077]

number

[0078] This uses all measurement currents.

[0079] Z s To generate a high-spatial map, nearest neighbor cross-electrode measurements were used for each stimulating electrode. A 3x3 grid was used for each electrode (except those at the edges of the electrode array). See Figure 4A. This is the total Z of 190x190 pixels. s Generate an image (compared to 64 x 64 electrodes in the array).

[0080] In the example shown in Figure 4A, each of the nine pixels in the 3x3 grid 405 for the central electrode 5 is filled with a normalized impedance value Z based on the measured current to its nearest electrode. Each normalized impedance value Z is calculated as follows:

[0081]

number

[0082] Here, VAC The amplitude of the applied AC voltage, I xy I is the magnitude of the AC current measured by electrode y when an AC signal is applied to electrode x. x [I y ] is the sum of the magnitudes of the AC currents measured by all other electrodes when an AC signal is applied to electrode x[y]. The edge-normalized impedance value is then calculated as follows:

[0083]

number

[0084] The square root of 2 was determined to normalize the difference in distance between the edge electrode and the corner electrode. The center normalized impedance value is then determined as follows. Note that "median" is the median function.

[0085]

number

[0086] Using cross-electrode current not only increases effective spatial resolution compared to using the maximum value of the current distribution, but also makes it possible to map unglued cells (which cause a decrease in cross-electrode current).

[0087] Figures 5a and 5b show an example of magnified cross-electrode impedance mapping compared to fluorescence microscopy images. Figure 5a shows a fluorescence microscopy image 501 across the electrode array and a heatmap plot 502 of normalized cross-electrode impedance of cell cultures immediately after seeding. A magnified map 504 of a portion of heatmap 502 shows the decrease in cross-electrode normalized cell-substrate impedance Zs for unadhered cells at single-cell resolution. Mapping of cells immediately after seeding that are not adhered to cells shows smaller normalized impedance values ​​where cells are present compared to uncovered electrodes.

[0088] Figure 5b shows fluorescence microscopy images 505 and cross-electrode impedance maps 506 after 24 hours of culture. Figure 5b also shows an enlarged map 507, which is an overlay of fluorescence microscopy images and cross-electrode impedance maps in a selected region. The results indicate that many cells adhered to the surface, resulting in a dramatic increase in normalized cross-electrode impedance.

[0089] (Example 3: Quantification of cell adhesion) This example illustrates how cross-electrode impedance mapping can be used to quantify cell adhesion.

[0090] Ethylenediaminetetraacetic acid (EDTA) is applied to the cells. EDTA is necessary for integrin proteins to maintain cell adhesion. 2+ It is a calcium chelating agent that removes EDTA. When EDTA is applied, cells rapidly detach over a period of approximately 50 minutes. Then, the EDTA is washed away by adding a normal culture medium, and the cells reattach over a period of approximately 200 minutes.

[0091] Cell detachment and reattachment were recorded with high spatial and temporal resolution using cross-electrode impedance mapping, as shown in Figure 6a, which shows a series of normalized impedance maps over time for MDCK cells, accompanied by 5 mM EDTA application at t=~5 min and washing at t=~55 min.

[0092] Figure 6b is a data plot showing the average normalized impedance for various regions of the cell culture specified in map 601. Figure 6c is a histogram of normalized impedance values ​​before, during, and after EDTA washing across the array.

[0093] To demonstrate a biologically relevant example for quantifying cell adhesion, recombinant MDCK cell lines were measured, and the RasV12 and GFP genes were switched on and off using tetracycline. The results are shown in Figure 7. Figure 7 shows a series of fluorescence microscopy images and normalized cross-electrode impedance maps of MDCK cells over 7 days of in vitro culture (DIV). After 2 DIV measurements, tetracycline was added to turn on the cancer-related gene RasV12, which also expresses GFP, making gene expression imageable. Then, after 4 DIV measurements, tetracycline was removed to turn off gene expression. It is shown that when the RasV12 gene is expressed, cells exhibit reduced adhesion to the surface, and this returns to normal after it is turned off.

[0094] RasV12 is an oncogene known to increase cellular metabolism and, when strongly expressed, decrease cell adhesion, both of which contribute to cancer-like cell growth and tumor formation. Initially, tetracycline was removed from the medium, and cells adhered normally. Upon introduction of tetracycline, the gene was expressed, resulting in increased GFP and decreased cell adhesion. Removal of tetracycline reversed cell adhesion, causing cells to adhere more strongly, while also reducing overall GFP expression. Some portions of the cell culture were not as strongly turned off as others. The effect on cell adhesion was quantitatively compared with a control culture without tetracycline introduction, as shown in Figure 8. Figure 8b is a normalized impedance histogram of MDCK cells over 6-7 day in vitro culture (DIV). Tetracycline was added after 2 DIV measurements to turn on the cancer-related gene RasV12. Figure 8a is a normalized impedance histogram of a control measurement without tetracycline addition. The histogram is normalized to the maximum number of pixels exceeding the cell-free impedance value of ~8kΩ. Cell adhesion decreased compared to the control and showed a smaller decrease over time.

[0095] (Example 4: Frequency Response) This example illustrates the effect of frequency used in cross-electrode impedance measurements.

[0096] The mapping frequencies were scanned to determine the best frequency for measuring cell adhesion using cross-electrode impedance mapping. Figure 9 shows a series of normalized cross-electrode impedance maps under stimulus signals of various frequencies. The plots are normalized to median ± 1 standard deviation. Lower frequencies show higher signal contrast correlated with optically measured GFP fluorescence, as shown in Figure 7, indicating that lower frequencies are better for measuring cell adhesion. 1.9 kHz used shows good contrast compared to 240 Hz, but above 10 kHz, the cell sheet appears much more uniform.

[0097] (Example 5: Intercellular adhesion) The previous example relates to a method for mapping cells and their adhesion over time using cross-electrode impedance measurements, as shown in Figure 10a. In Figure 10a, an AC voltage is applied to a single electrode, and the current through the rest of the electrode array is measured using a transimpedance amplifier. Adhesion is primarily a function of cell-substrate adhesion and the height of the resulting gap.

[0098] This example illustrates a method for measuring intercellular adhesion, or how well cells connect to one another. Cells in culture not only adhere to surfaces but also to each other via cell-cell junctions. The density of these junctions defines the permeability of cell sheets and is important for epithelial tissues that function as barriers to body surfaces, internal organ linings, and other tissues. In this example, this barrier function is represented by transepithelial impedance Z te This is measured by performing mapping. In this way, cell-cell connectivity can be evaluated by reducing arbitrary pores using only electrodes covered by cells, and spatial heterogeneity can also be evaluated.

[0099] In this example, the stimulation protocol is modified to measure the vertical electric field component 1014, as shown in Figure 10b. In Figure 10b, electrode 1006_2 and its surrounding electrodes 1006_1, 1006_3 are biased with an AC voltage. Current I e,n However, this is measured via the central electrode 1006_2. The central electrode 1006_2 does not allow current to pass through the surrounding electrodes because they are biased with the same signal, and therefore only allows current due to the impedance of the cell sheet above the electrode to pass through. Outside the central electrode and its surrounding electrodes, the rest of the array is biased to ground or a reference voltage level and functions as a current return. This type of measurement is similar to measuring transepithelial electrical resistance (TEER), which is measured using two electrodes on either side of a cell culture on a suspended porous membrane. The technique shown in Figure 10b makes it possible to map TEER across cells at the top of the electrode array without requiring a special suspension. Advantages include requiring fewer cells, being able to assess spatial heterogeneity, and being able to combine cell-cell adhesion measurements and cell-substrate adhesion measurements using the same device.

[0100] Figure 24 includes schematic diagrams showing several additional schemes for measuring intercellular connectivity according to several embodiments. In Figure 24, the change in the vertical electric field above the electrodes is measured to best isolate the effects of cell-cell connectivity using two circuit configurations: 1) high-speed (<1 s / measurement) parallel electrode measurement versus reference value (Figure 24a) and 2) low-speed scanning (40 s / measurement) relative measurement without a reference value (Figure 24b). High-speed measurement is ideal for scanning across multiple frequencies, but scanned measurements do not require a reference value which helps to stabilize measurements over longer periods and are therefore more ideal for miniaturizing the device. For both types of measurements, platinum black (PtB) deposition can be optionally used, Z e By reducing it by approximately 5 times, Z te Sensitivity can be improved. Experiments across frequencies showed that the intermediate frequency range of ~2kHz to 5kHz was best for evaluating intercellular connectivity.

[0101] Here, the transepithelial impedance Z using the scheme in Figure 24 te Let's explain the calculation.

[0102] To measure intercellular adhesion or how well cells connect to each other, the stimulation protocol can be modified to measure the vertical electric field component in Figures 24a and 24b. Each transepithelial electrode current I was measured via a transimpedance amplifier (measurement period of 1 s / frequency). te,n By applying an AC voltage to all electrodes using (n=1,2,...,4096), measurements can be taken relative to a grounded reference value (left). The resulting electric field distribution is I te Cell connectivity is reduced by vertical alignment. Measurements without a reference value can be performed by applying an AC voltage to electrode (n) and its neighboring electrodes, and using the remaining grounded electrodes to generate effective vertical electric field measurements (right). To generate a cell map, the applied signal is scanned across the array (40 s per scan / frequency).

[0103] In the parallel scheme shown in Figure 24a, an AC voltage is applied to each electrode relative to a certain reference value, and the current I of each electrode te,n This is measured, generating a vertical electric field in the solution (the surrounding electrodes also have a low-frequency fringe electric field). Then, due to the current that needs to pass through the cell sheet, the magnitude of the current is the transepithelial impedance Z te It becomes proportional to this. The second scanning scheme, Figure 24b, biases the electrode and its surrounding electrodes with an AC voltage and measures the current passing only through the central electrode. Because they are biased with the same signal, the central electrode does not pass current through the surrounding electrodes, but only current due to the impedance of the cell sheet above the electrode. Outside the center and its surrounding electrodes, the rest of the array is biased to ground and acts as a current return.

[0104] In either case, the measured vertical current I te,n It can be expressed as follows:

[0105]

number

[0106] Then, using (Equation A3), Z te This can be solved.

[0107]

number

[0108] Regarding the measured values, intermediate frequencies between ~1kHz and 5kHz were determined to correlate best with cell-cell connectivity (see also Example 15 below). In the PtB electrode, Z e,n is, Z te It is considerably smaller than (see also Example 15 below), and is estimated as follows:

[0109]

number

[0110] Z using only Pt electrodes te In the experiment, from the cell-substrate impedance, I n The measurement is subtracted. Using a 3x3 set of electrodes, Z te,no ref As a result of the scanned array measurement used to calculate the overall map, the generated map is 62 × 62 pixels, and the peripheral electrodes do not have adjacent bias electrodes that form a perpendicular electric field. The measured values ​​relative to the reference value create a map containing 64 × 64 pixels.

[0111] (Example 5A: Extracellular redox potential V redox Metabolic state mapping) Beyond impedance measurements, platinum electrodes are used for both oxygen potential difference sensing and extracellular redox monitoring. This example uses the proximity of a Pt electrode directly beneath living cells to measure the extracellular redox potential V redoxWe have demonstrated that it is possible to map cells in situ, monitor their redox environment and O2 consumption, and map the metabolic state of cell cultures.

[0112] To achieve the measurement, the pixel amplifier is configured as a buffer, as shown in the schematic diagram in Figure 25a.

[0113] Generally, during aerobic metabolism, cells utilize energy derived from the transfer of electrons from oxidizable organic molecules (e.g., glucose) to O2. To help mediate these electron flows, an overall reducing environment is generated by the thiol compound glutathione (GSH), which is often considered the cellular redox buffer. Simply put, the cellular redox potential is the balance between O2, which raises the potential (oxidizes), and GSH, which lowers the potential (reduces). The redox environment is important not only for electron transfer but also for neutralizing harmful reactive oxygen species, intercellular signaling, and regulating the cellular state. For example, within the negative-to-positive range, the redox potential can determine whether a cell is in a proliferative, differentiated, apoptotic, or necrotic state.

[0114] Figure 25b is a series of data maps showing the results of multiparametric measurements. Measurements were performed at +24 hours, +48 hours, and +72 hours after MDCK cell seeding, including cell adhesion (top), cell-cell adhesion (middle), and metabolic state (bottom). Cells show growth from the lower right corner to the upper left corner, with proliferating leading edge cells showing the most negative V compared to more dormant posterior edge cells. redox This indicates Z te The highest cell density is at the anterior margin, resulting from the lowest cell density (see detail region 1), and therefore the fewest intercellular connections. Figure 25c is a pair of nuclear fluorescence images at +72 hours after seeding (top) and detail region 1 comparison (bottom) showing the lowest cell density on the anterior margin compared to the posterior margin. Figure 25d is a composite map showing detail region 2 overlaying cell nuclei and cell adhesions. Figure 25d shows good spatial correspondence with single-cell resolution.

[0115] One goal in this example is to monitor cell growth by pairing it with an impedance technique, thereby enabling close-range V redox The objective is to investigate what kind of information it can provide (Figure 25b). In this example, compared to electrodes without cells, negative V in the range of 30mV to 80mV redox This was observed in the electrode with cells (Figure 25b). From a comparison of detailed regions, V redox The spatial information is separate and distinct from cellular attachments or cell barriers, and is the most negative V redox It is located at the anterior margin and is not the lowest density. Generally, negative signals may show locally low [O2] or locally high [GSH] near the cell.

[0116] V redox To further explore the signal origin, O2 dependence was examined by oxygen purging in separate MDCK cell cultures. During O2 removal, the signal difference between cell-present and cell-absent regions was eliminated. For complementarily, the reducing capacity of the GSH system was examined by oxidative titration. Ferrocyanide [Fe(CN)6] was used for titration, due to the previous non-toxic use in the cell culture and the potential of its oxidative half-cell compared to the cellular environment. 3- The medium was selected. The medium showed a reducing power of 4 μM, while the cells had a much greater capacity of >200 μM.

[0117] In summary, these measurements are based on the measured V redox However, we show that it is related to both the situ [O2] and [GSH] reducing power of the cell. We show that in aerobic respiration, V redox Under atmospheric conditions that reduce the V2, we hypothesize that [O2] decreases from its normal solubility of ~200 μM until it is regulated by the extracellular redox potential of the cell. Therefore, although it is difficult to quantify the oxygen consumption rate using our method, the V2 of the extracellular redox potential is... redox The measurement is useful for monitoring the metabolic state of cells, as it can indicate both O2 utilization and the reducing environment of the cell. Therefore, a more negative signal at the leading edge of the cell sheet (Figure 25b) is attributed to the cell's proliferative state, respiration combined with the most negative redox potential state.

[0118] (Example 6: Antibody-Cell Binding) Antibody-cell binding screening can have low throughput because it requires fluorescent tagging of antibodies (which necessitates a washing step to remove unbound fluorescent antibodies) or specialized optical measurements such as surface plasmon resonance (SPR). In one embodiment, the cross-electrode impedance method described herein can provide the ability to measure antibody-cell binding events through either cell-substrate adhesion or cell-cell adhesion measurements. Antibody binding to the underside of cells results in a smaller gap distance, leading to a decrease in the measured cross-electrode current. Similarly, antibody binding to the sides of cells should result in smaller gap distances between cells, leading to a decrease in the measured vertical current. The ability to perform such antibody binding without labeling allows for the addition of various antibodies into the sequence without the need for a washing step, significantly improving throughput.

[0119] (Example 7: Cell patterning by electrochemical gas generation) This example illustrates a method for patterning cells on an electrode array. The inventors recognized and understood that small bubbles can be electrochemically generated, creating small pores in the cell membrane and killing the cells through depolarization. After death, the cells begin to detach from the surface, as shown in the schematic diagram in Figure 11. Therefore, by controlling which electrodes generate gas, cells can be patterned with the spatial resolution of the electrode array.

[0120] While we do not wish to be bound by any particular theory, the inventors believe that for the most inert electrode materials (platinum, gold, etc.), the electrode potential is the hydrogen ion / hydrogen gas oxidation-reduction half-cell reduction potential (E 0 We recognized that hydrogen gas could be generated by adjusting it to less than ).

[0121] [ka]

[0122] Alternatively, oxygen gas can be generated by adjusting the electrode potential above the oxygen gas / hydroxyl oxidation-reduction potential.

[0123] [ka]

[0124] Similarly, since many cell media contain chloride salts, chloride gas can also be generated by adjusting the electrode potential above the chlorine gas / chloride redox potential.

[0125] [ka]

[0126] Therefore, cell removal can be performed by selectively applying a predetermined potential above the redox potential for gas generation at one or more electrode locations. The potential can be applied, for example, by connecting one or more stimulation source circuits 110 in Figure 1a to the selected electrodes. The potential does not need to be the same across all selected electrodes; programmable heterogeneity can be used if the electrodes are biased differently. The potential may also be the potential relative to the potential of a reference electrode in the medium above the electrode.

[0127] For more controllable patterning, the electrode current can be used to set the electron transfer rate, and therefore the gas generation rate. By controlling the gas generation rate, selective electrochemical reactions can be optimized, as a gas generation rate that is too fast can be used to isolate the electrode from the solution, thereby forming large bubbles on the surface.

[0128] Figure 13 is a series of diagrams illustrating variations of cell patterning using an electrode array. Figures 13a and 13b show embodiments in which one or more predetermined patterning voltages are applied to electrodes selected for patterned removal of cells by electrochemical gas generation. Figures 13c and 13d show embodiments in which one or more predetermined patterning currents are applied to electrodes selected for patterned removal of cells. Figures 13a and 13c show an example of voltage / current patterning with a reference electrode acting as a return. Figures 13b and 13d show an example of differential voltage / current patterning using cross-electrode gas generation without a reference electrode, where one set of electrodes passes the positive current and a second set of electrodes passes the negative current (return).

[0129] (Example 8: Cellular patterning and co-culture delimitation) This example illustrates spatial patterning and co-culture delimitation of cells using electrode arrays.

[0130] In this example, a CMOS electrode array, MDCK cells, and H2 gas generation are used, as shown in Figure 12. In this experiment, H2 gas was generated by applying -1.25 V to a platinum electrode-Ag / AgCl pseudo-reference electrode. Figure 12 shows fluorescence microscope images before (center) and after (right) the patterning voltage was applied for 80 seconds, demonstrating that the intracellular pattern was successfully defined based on the electrode pattern. With an electrode pitch of 20 μm, square pores of various sizes were fabricated on a uniform cell sheet with high spatial resolution, as confirmed by nuclear fluorescence markers and fluorescence imaging.

[0131] Figure 14 shows a series of fluorescence microscopy images illustrating the process of defining a co-culture by patterning and then seeding a second cell type. Cell types were distinguished by various nuclear fluorescent markers. In the experiment in Figure 14, a co-culture of two distinct cell types was defined by seeding a second MDCK cell line with various nuclear fluorescent markers after initial patterning. The second cell type filling the resulting space demonstrates the ability to spatially define the co-culture with high spatial resolution. Further patterning and seeding can be performed to define multiple cell co-cultures and patterns using a bottom-up approach.

[0132] (Example 9: Directional cell evolution by eliminating culture heterogeneity) This example illustrates a method of targeted cell evolution for removing cells from a cell culture whose characteristics are undesirable.

[0133] Figure 15 is a series of schematic diagrams showing a heterogeneous cell population, the removal of unwanted cells using patterned electrochemical gas generation on a selective electrode, and a homogeneous culture with desired characteristics after subsequent cell growth. Selection of which cells to remove can be performed by optical imaging or by other characteristics measured using an electrode array. The ability to remove cells from a culture without the need to remove them from a culture plate is advantageous over current processes that require suspending cells, separating them using a cell sorting machine, and then re-culturing them in a further re-seeding step, or removing single cells with desired characteristics using a micropipette and then re-seeding. Furthermore, the cell history lineage is storable, the spatial location of each cell does not change, and the cells remain adherent throughout the process. These removal processes can also be used for further analysis performed on a subset of cells after culturing the electrode array, where cells undesirable for further measurement are killed first before cell suspension and removal.

[0134] (Example 10: Wound healing assay) This example illustrates how the combined application of both cross-electrode impedance mapping and cell patterning can be used in wound healing assays.

[0135] These assays are useful for measuring cell proliferation rate and metabolism and for screening drugs that affect these parameters. Compared to the electrochemical patterning described here, other tools, such as mechanical scratching, mechanically generate wounds in cell cultures, the latter being difficult to control and limited in terms of wound patterns.

[0136] In this example, wounds were fabricated in MDCK cells, and their growth was mapped in real time. Figure 16 shows intracellular sawtooth patterns defined at the center of the device surface, with varying separation distances. These patterns were defined by applying an electrode current of -10 nA for 40 s against an Ag / AgCl pseudo-reference electrode. Culture regrowth was then measured using impedance mapping. Typical cell cultures required ~3 days to fill the wound, while cultures treated with growth inhibitors showed very little regrowth. As shown in the normalized cross-electrode impedance map in Figure 16, the control culture showed regrowth after 72 hours of culture. A second culture treated with the growth-inhibiting agent, cytochalasin B (1 μM), showed very little growth over 72 hours, demonstrating the assay's capability for drug screening.

[0137] (Example 11: Molecular Delivery) This example illustrates a method using planar electrodes for membrane permeabilization and molecular delivery. Unlike electroporation, which applies a focused electric field to disrupt the cell membrane, this method utilizes planar electrode permeabilization through gas bubble formation, and is conceptually similar to the patterning technique discussed here. Unlike cell patterning, where cells die and then patterning is performed, smaller pores are created on the cell surface for molecular delivery, and then resealed over time.

[0138] Figure 17 shows an experiment demonstrating a permeabilization technique using nanowire electrodes, and this technique can also be applied to electrode arrays using planar electrodes. In the experiment shown in Figure 17, Fluo-4 and a bioassay are dissolved in extracellular solution (left panel, Figure 17a). The electroporation protocol can be applied to the nanoelectrodes using a pixel stimulator (middle panel, Figure 17a) and recovered in Fluo-4. If electroporation is successful, Fluo-4 will penetrate into the cells. After recovery, a death assay, EthD-1, is dissolved in the extracellular solution to determine whether cells are present due to irreversible electroporation (right panel, Figure 17a), and whether the cells died as a result of irreversible electroporation. Successfully electroporated cells and Fluo-4 are recovered and retained for imaging. Figure 17b shows a heatmap of averaged EthD-1 and Fluo-4 intensities for eight investigated protocols that increased voltage amplitude (three rows of five biphase pulses at 20 Hz) using HEK293 cells. The CNEA array was divided into 128-pixel subgroups for each of the eight protocols and repeated in a grid across the array. Imaging was performed for each pixel, and the 128 images for each protocol were averaged together. Figure 17c shows the average intensity results from Figure 17b for HEK293 cells. Successful electroporation started at ~1.3V, while irreversible electroporation started at ~1.7V. Figure 17d shows results using neurons under the same testing conditions, showing a lower threshold, <1.2V, for successful electroporation and ~1.5V for irreversible electroporation.

[0139] Figure 18 shows another experiment in which Fluo-4 is injected into cells using Fluo-4 AM. An electroporation protocol is applied to the nanoelectrode using a pixel stimulator (center) while monitoring fluorescence. If electroporation is successful, Fluo-4 can leach out of the cell, causing a decrease in fluorescence. In a successful protocol, the cell membrane recovers after electroporation (right panel, Figure 18a). Figure 18b shows an example using neurons with their fluorescence and applied electroporation signal. Fluorescence decreases during electroporation. Immediately thereafter, the cell membrane recovers, and the fluorescence returns to a plateau state. The electroporation signal may be applied multiple times without affecting cell viability.

[0140] In both experiments shown in Figures 17 and 18, it was observed that a voltage signal was required for a certain duration, at least >50 ms, to observe either permeabilization or delivery. This indicates the need for a Faraday process to generate bubbles, as the required voltage is comparable to the water window voltage with the platinum electrode used (H2 & O22 gas generation by water splitting). In Figure 18, it is shown that such a permeabilization signal is effective by causing transient leakage of the fluorescent dye, while in Figure 17, the fluorescent dye is delivered to the cells.

[0141] Such delivery capabilities can be easily used to screen membrane-impermeable compounds for their effects on cells and intercellular interactions. The spatial capability of electrode arrays, which allows cells to select for delivery, is useful in this latter application to intercellular interactions, allowing the effects of the compound to be measured on delivered cells and their undelivered neighboring cells. Without such delivery capabilities, membrane-impermeable compounds would need to be chemically modified for delivery, which would be expensive and time-consuming, or delivered on a single-cell basis using micropipettes, which would also be expensive and time-consuming. Beyond compounds, RNA / DNA / plasmids can also be delivered for applications in synthetic biology.

[0142] (Example 12: Serial delivery for cross-effect analysis) This example illustrates the multi-step delivery of compounds within cells using an electrode array.

[0143] Figure 19 shows a series of schematic diagrams illustrating the generation of controlled and cross-effect delivery using spatial addressing and serial delivery via gas generation. Since the electrode properties do not change during gas generation, this, combined with the spatial capability of the addressable electrode, provides further advantages for cross-compound effect screening. For example, if it is desired to investigate the effects of two compounds on cells, two compound delivery steps are required to form a complete matrix of drug effects.

[0144] (Example 13: Extracellular electrochemical mapping) This example illustrates electrochemical mapping using redox electrochemistry on an electrode array.

[0145] Electrochemical measurements of cells using electrodes can utilize a single, large working electrode to measure the bulk concentration of a sample in solution. Such electrochemical electrode 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 to spatially map sample concentrations measured via electronics in a CMOS integrated circuit. Such electrochemical mapping can be applied to cellular analysis of cells cultured directly on top of the electrode array.

[0146] In this example, cyclic voltammetry is used to demonstrate the capability of electrochemical mapping using an electrode array measured with a CMOS integrated circuit, specifically for ferricyanide / ferrocyanide [Fe(CN)6]. 3- / [Fe(CN)6] 4- This is done using general redox pairs.

[0147] [ka]

[0148] Figure 20a is a schematic diagram showing a cyclic voltammetry configuration using a CMOS integrated transimpedance amplifier to measure the current flowing through Ag / AgCl pseudo-descriptor electrodes, as well as the current of each Pt electrode and an external transimpedance amplifier. In the experiment in Figure 20a, a periodic voltage ramp was applied with a scanning speed of 35 mV / s using 1.5 M KCl + 5 mM K3 [Fe(CN)6]. The sum of the currents of the 13 × 13 electrodes was used for measurement, and it is equal to that of the reference electrode. Figure 20b shows two spatial maps (|I) of the maximum range of electrode currents. e,max -I e,min | (upper left) shows, which relates to the diffusion of ferricyanide (starting reactant), and the maximum current range - maximum / minimum voltage current (|I e,max -I e,min |-|I e,vmax -I e,vmin | (bottom left) is shown, which is related to the diffusion (product) of the ferrocyanide. Exemplary individual electrode recordings are shown on the right in Figure 20b, along with these defined parameters. The diffusion of non-radial ferrocyanides is due to convection effects in the solution.

[0149] In this experiment, a subset of 64×64 electrode arrays, 13×13=169 in total, was connected to an equal number of individual transimpedance amplifiers, as shown in the schematic diagram of Figure 20a, and a periodic linear voltage ramp was applied. Figure 20b shows a spatial map of current density, indicating the magnitude of increased cathode and anode currents at the electrode edges, which would be due to increased radial diffusion / mass transport at the edges compared to planar diffusion at the central electrode. Similarly, product generation limits the current density, visualized by the peak current range—voltage maximum / minimum current range, as shown in data plot 2001 of Figure 20. The cyclic voltammetry data plot 2001 shows a trend of product diffusion toward the upper right corner. These spatial measurements of current demonstrate the capability of current-based electrochemical mapping.

[0150] The open-circuit potential of an electrode can be used to measure the concentration of chemical species in a solution. At high concentrations of redox pairs in a solution, the open-circuit potential of a platinum electrode in the solution can be determined by the Nernst equation. The Nernst equation relates to the reduction potential of an electrochemical reaction relative to a standard electrode potential, temperature, and the activity of the chemical species undergoing reduction and oxidation.

[0151] [ka]

[0152] Here, E H This is the electrode voltage potential relative to the standard hydrogen electrode (SHE), and E 0 This is the half-cell reduction potential, and φ t [Ox] / [Red] is the thermal voltage (~25.7mV at 25°C), [Ox] / [Red] is the concentration of the oxidized / reduced chemical species, and n is the number of electrons transferred in the half-cell reaction. In the ferricyanide / ferrocyanide reaction, measuring the open-circuit potential reflects the ratio of the concentrations of these ions in the solution.

[0153] In this example, the remaining potentials of the electrode array were measured. In particular, ferrocyanide formation and transport across the CMOS electrodes are mapped using the open-circuit potential.

[0154] A periodic potential is applied to a 13×13 electrode group (9 electrodes are excluded from the 13×13 electrode group, as shown in Figure 21b), while the open-circuit potential of the remaining electrodes is measured. Figure 21a shows the selected electrode voltage V plotted over time. el Figure 21b is a data plot showing the increase and decrease associated with the ferricyanide / ferrocyanide concentration. Figure 21b is a heatmap showing one cycle, where the overall amplitude (maximum-minimum) of the open-circuit potential plotted across the array indicates diffusion / mass transport tending toward the upper left corner. Figure 21c is a heatmap and data plot showing the minimum time of the open-circuit potential plotted against the distance from the center of the 13×13 electrodes, illustrating the transient aspects of diffusion / mass transport.

[0155] In summary, cyclic voltammetry, by measuring the open circuit around the electrode, reveals the flow of ferrocyanide toward the upper right corner of the device.

[0156] (Example 14: Electrochemical oxygen mapping of cells) 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 pulsed voltage or a sequence of voltage pulses, which sequentially oxidizes and then reduces the platinum. The current drops to zero after the oxide is formed because the platinum oxide inhibits oxygen reduction. Subsequently, as the oxide is reduced, the platinum electrode allows a negative current to pass through due to the presence of oxygen.

[0157] [ka]

[0158] Subsequently, the local oxygen concentration is depleted, and the electrode awaits further diffusion of additional oxygen to allow current to flow. Thus, 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.

[0159] In experiments using electrode arrays, measurements were taken using a salt solution (phosphate-buffered saline) exposed to ambient air, followed by purging with nitrogen gas to reduce the oxygen concentration. Figure 22a shows a series of data plots 2204 of measurements using a CMOS electrode array in ambient air, along with partial nitrogen purging and partial recovery (N2 purging cessation), and the voltage pulse sequence 2202 applied to the stimulating electrodes in the electrode array. Data plot 2204 shows that the electrode current reflects the oxygen concentration.

[0160] Current I before and after purging el A significant decrease is observed when comparing the two. Experiments were then conducted using HEK293 cells, and the results are shown in Figure 22b. Figure 22b shows the maximum cross-electrode current I across the electrode array region. maxCross-electrode impedance heatmap 2206 using and electrode current ΔI across the electrode array region el A heatmap of the changes is shown in 2208. The same style of oxygen measurement using HEK293 cells shows a decrease in oxygen concentration at the location of the cell, as confirmed by the impedance map.

[0161] Because cells consume oxygen as part of aerobic metabolism, the oxygen concentration around cells is lower than in areas without cells. Indeed, mapping electrode currents across the array shows that cell locations have smaller current magnitudes than areas without cells, as can be imaged using cross-electrode impedance maps. The left and bottom edges of map 2208 show larger current magnitudes, which are attributed to edge effects and increased diffusion / mass transport.

[0162] (Example 15: Effects of platinum black and frequency on cell barrier sensitivity) In this example, platinum black (PtB) is used to determine the electrode impedance Z. e This reduced the Z of the bare electrode and improved the sensitivity of cell barrier measurement. Figure 26a shows the results of a comparative study of electrode impedance for cells cultured for ~72 hours with electrodes under three scenarios: low density, high density, and no cells. Figure 26b shows the Z of PtB with bare electrodes. te This data plot shows a reduction in measurements of approximately five times, enabling the measurement of intercellular connectivity at two different densities with a higher signal-to-noise ratio. Figure 26c shows cell barrier maps relative to a reference at various frequencies. Lower frequency measurements show greater spread and do not capture cell sheet edges, but the 1.8 kHz measurement showed the highest contrast for cell connectivity measurements compared to the concentration map extracted from imaging. Figure 26d shows cell density and connectivity maps extracted from nuclei in fluorescence images. Figure 26e shows Z measured with and without a reference at 1.8 kHz. te This shows a comparison between them. Without a reference point, Z is slightly smaller. teAlthough it is measured, the relationship between the region with cells and the region without cells (two clusters) is direct. Measurement without a reference is preferred, Z e This is because the contribution could be easily subtracted from the cell substrate adhesion measurement. Figure 26f shows the Z ratio to the extracted cell density. te and Z s This shows a comparison with Z. s It is downsampled via bilinear interpolation, Z te It has the same spatial resolution as the measurement. The cell barrier shows a strong dependence on cell density due to the measurement being adapted to intercellular connectivity. s There is also a small correlation between and cell density, which can be seen from the cell circuit model (Figure 4B), Z s High, Z s The Z used in the calculation s < <Z te This is because it has a certain effect, under the assumption that it does not last at all.

[0163] Having thus described several aspects and embodiments of at least one embodiment of the present invention, it should be understood that various changes, modifications, and improvements will readily arise for those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and to be within the spirit and scope of the invention. Furthermore, while the advantages of the present invention have been shown, it should be understood that not all embodiments of the technology described herein will include all described advantages. 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 further embodiments. Accordingly, the foregoing description and drawings are for illustrative purposes only.

[0164] Various aspects of the present invention may be used individually, in combination, or in various configurations not specifically discussed in the embodiments described above, and are therefore not limited to application to the details and arrangement of components described in the above description or shown in the drawings. For example, an aspect described in one embodiment may be combined in any way with an aspect described in another embodiment.

[0165] Furthermore, the present invention may also be embodied as a method, and examples thereof are provided. The operations performed as part of the method may be ordered in any suitable manner. Thus, embodiments may be constructed in which the operations are performed in a different order than those described, which may include performing several operations simultaneously, even though they are shown as sequential operations in the exemplary embodiments.

[0166] To modify a claim element, the use of ordinal terms in a claim, such as "first," "second," "third," etc., does not in itself imply priority, precedence, or order of one claim element over others, or a temporal order of the actions in which the method is performed, but is used simply as a label to distinguish one claim element having a particular name from other elements having the same name (except for the use of ordinal terms).

[0167] The terms “approximately” and “about” may be used in some embodiments to mean within ±20% of the target value, within ±10% of the target value, within ±5% of the target value, and within ±2% of the target value. The terms “approximately” and “about” may include the target value.

Claims

1. A method for providing a spatially positioned electrochemical reaction using an electrode array exposed on the surface of a semiconductor substrate, The steps include selecting one or more electrodes within an electrode array, The steps include controlling a circuit within a semiconductor substrate to apply one or more stimulus signals to one or more electrodes, and initiating an electrochemical reaction at one or more selected electrodes in a solution containing multiple cells attached to the surface of the semiconductor substrate, A method comprising the step of generating a gas at one or more selected electrodes so that at least one cell among a plurality of cells placed on the one or more selected electrodes is detached from the surface of a semiconductor substrate.

2. Electrochemical reactions are half-reactions that produce gas in a solution. The method according to claim 1, wherein one or more stimulus signals include a potential higher than the redox potential for gas generation.

3. Gas is H ２ , Cl ２ , or O ２ The method according to claim 1, including the method described in claim 1.

4. Multiple cells are multiple first-type cells, The method according to claim 1, further comprising the step of seeding one or more second-type cells onto the surface of a semiconductor substrate at locations on the surface where at least one first-type cell has been detached.

5. The steps include mapping the time sequence of regrowth of multiple cells on the surface at the site where at least one cell has detached, The method according to claim 3 or 4, comprising the step of determining the growth rate of a plurality of cells based on mapping.

6. The step of controlling the circuit to apply one or more predetermined potentials includes performing cyclic voltammetry on one or more selected electrodes. The steps include using a circuit to measure the electrical characteristic values ​​of some or all of the remaining electrodes in an electrode array located outside one or more selected electrodes, The method according to any one of claims 1 to 5, further comprising the step of generating a map of electrical characteristics based on the results of a measurement.

7. The method according to claim 6, wherein the electrical characteristics are those of the open-circuit potential.

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

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

10. The step of controlling a circuit to apply one or more predetermined potentials includes applying a pulse voltage signal to one or more selected electrodes, During the first part of the pulsed voltage signal, the electrode is oxidized, and during the second part of the pulsed voltage signal, the oxide on the electrode is reduced. The steps include using a circuit to measure the current signal at the electrode during the second portion of the pulse voltage signal, A step of determining the oxygen concentration at the electrode position based on the time rate of change of the current signal, A method according to any one of claims 1 to 9, further comprising the step of generating an oxygen concentration map based on the result of a decision.

11. The method according to any one of claims 1 to 10, wherein one or more stimulus signals are relative to the potential of a reference electrode.

12. A system including a semiconductor substrate, The semiconductor substrate includes an electrode array comprising a plurality of individually addressable electrodes arranged on the surface of the semiconductor substrate, A system comprising: a circuit controllable by one or more processors, which applies one or more potentials to an electrode group in an electrode array, relative to the potential of an electrode in the electrode array or the potential of a reference electrode, thereby initiating an electrochemical reaction in the electrode group in a solution containing multiple cells attached to the surface of a semiconductor substrate, so that at least one of the multiple cells arranged on the electrode group is detached from the surface of the semiconductor substrate.

13. The system according to claim 12, wherein the electrode array includes a plurality of pads arranged on an insulating surface of a semiconductor substrate.

14. The system according to claim 13, wherein multiple 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 electrode array includes at least 1,000, at least 4,000, or at least 1,000,000 electrodes. The circuit includes multiple recording circuits. The system according to any one of claims 12 to 15, wherein each recording circuit is configured to measure the current at the electrodes of the electrode array.

17. The system according to claim 16, wherein the plurality of recording circuits include 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 TIA includes an impedance element having a resistance of at least 10 MΩ. The system according to claim 18, wherein the output voltage of the TIA is proportional to the voltage across the impedance element.

20. The system according to claim 19, wherein the impedance element includes a switching capacitor.

21. A system for providing spatially positioned electrochemical reactions, An electrode array exposed on the surface region of a semiconductor substrate, A circuit placed within a semiconductor substrate and connected to an electrode array, At least one non-temporary computer-readable medium storing executable instructions, A system comprising at least one processor programmed with executable instructions to perform a certain method, The method involves selecting the pattern of electrodes within an electrode array, A system comprising the operation of controlling a circuit to apply one or more predetermined potentials relative to the potential of electrodes in an electrode array or the potential of a reference electrode in an electrode pattern, thereby initiating an electrochemical reaction in the electrode pattern in a solution containing multiple cells attached to the surface of a semiconductor substrate, so that at least one of the multiple cells arranged on the electrode pattern is detached from the surface of the semiconductor substrate.

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