Apparatus for cell mapping by impedance measurements and method of operation thereof - Patent Application 20070122997

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 Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 040424, filed June 17, 2020, entitled "APPARATUSES FOR CELL MAPPING VIA IMPEDANCE MEASUREMENTS AND METHODS TO OPERATE THE SAME," inventors Ham, et al., which is incorporated herein by reference in its entirety. [Background technology]

[0002] The present disclosure relates to semiconductor devices for the electrical evaluation of cells or other biological samples. Summary of the Invention [Means for solving the problem]

[0003] Disclosed herein are various devices 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.

[0004] Some embodiments relate to an apparatus for mapping one or more cells. The apparatus includes a semiconductor substrate. The semiconductor substrate includes a plurality of electrodes exposed on a surface of the semiconductor substrate; and active circuitry 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 to measure 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 includes one or more processors configured to receive the measured cross-electrode currents from the active circuitry and 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 circuitry is further configured to apply a stimulation signal to a first electrode of the plurality of electrodes and a reference voltage to the remaining electrodes for which cross-electrode currents are being measured. The stimulation signal may have a frequency less than 10 kHz, preferably in the range of 0.1 to 5 kHz. The plurality of electrodes may be arranged in an array with a pitch less than 30 μm, preferably less than 5 μm. The semiconductor substrate may include silicon. The semiconductor substrate may include a silicon substrate, and the active circuitry may include complementary metal-oxide semiconductor (CMOS) devices within the silicon substrate. The plurality of electrodes may include a plurality of pads disposed on an insulating surface of the semiconductor substrate. The active circuitry may include a plurality of recording circuits, each configured to measure current at an electrode 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. The one or more cells may be disposed in a first well of a multiwell plate, and the plurality of electrodes may be a first electrode array exposed in the first well. The device may further include a second electrode array exposed on the surface of the semiconductor substrate and in a second well of the multiwell plate. The multiwell plate may include at least 24, at least 96, or at least 384 wells. The plurality of electrodes may be sized such that two or more electrodes are configured to contact one of the one or more cells. The plurality of pads may include Au. The plurality of pads may include Pt.

[0006] Some 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 having an electrode location on the surface region, the method including, for each electrode of at least one electrode of the electrode array, applying a stimulation 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 representative values ​​associated with the electrode locations of the electrode based on the set of cross-electrode currents, and generating a map of the representative values ​​over the surface region based on the generated representative values ​​and the respective associated electrode locations of the at least one electrode.

[0007] In some embodiments, generating the representative value includes selecting a maximum current value of the set of cross-electrode currents as the representative value. Generating the representative value may include selecting a maximum current value of the set of cross-electrode currents as the representative value. The 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 at a first time and comprises a plurality of pixels, and the method includes generating a second map of representative values ​​on the surface region at a second time subsequent to 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 a cell adhesion characteristic based on a comparison of the first count and the second count. The map may include a plurality of pixels, each pixel associated with a representative value. The at least one electrode may include a first electrode having a first electrode location and a second electrode having a second electrode location, the first electrode and the second electrode being adjacent to each other on the surface region, and the map may include a first pixel and a second pixel corresponding to the first electrode location and the second electrode location, respectively. Generating the map may include determining an expanded representative value associated with a third pixel between the first pixel and the second pixel. The step of determining the scaled representative value is the cross-electrode current I between the first and second electrodes when a stimulation signal is applied to the second electrode. 12 The method may include calculating an expanded electrode current by dividing I by the product of the first current I and the second current I, where I is the sum of the cross-electrode currents measured at all of the remaining electrodes when the stimulation signal is applied to the first electrode, and I is the sum of the cross-electrode currents measured at all of the remaining electrodes when the stimulation 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 x 3N pixels.

[0009] Some embodiments relate to a system for mapping one or more cells, comprising: a plurality of electrodes exposed at a surface region of a semiconductor substrate; circuitry disposed within the semiconductor substrate and controllable to apply a stimulation signal and / or measure current at one or more of the plurality of electrodes; at least one non-transitory computer-readable medium having executable instructions stored thereon; and at least one processor programmed with the executable instructions to perform a method. The method includes, for each electrode in the plurality of electrodes: controlling circuitry to apply a stimulation signal to the electrodes; controlling circuitry to measure a set of cross-electrode currents between the electrode and some or all of the remaining electrodes in the plurality of electrodes; generating a representative value associated with an electrode location of said electrodes based on a set of cross-electrode currents; generating a map of the representative values ​​over the surface area based on the generated representative values ​​and associated electrode locations of each of the plurality of electrodes.

[0010] In some embodiments, generating the representative value may include selecting a maximum current value of the set of cross-electrode currents as the representative value. Generating the representative value may include selecting a maximum current value of the set of cross-electrode currents and calculating impedance based on the selected maximum current value as the representative value. The map may include a plurality of pixels, each associated with a representative value. The plurality of electrodes may include a first electrode having a first electrode location and a second electrode having a second electrode location, the first electrode and the second electrode being adjacent to each other on the surface area. The map may include first and second pixels corresponding to the first and second electrode locations, respectively, and generating the map may include determining an expanded representative value associated with a third pixel between the first and second pixels.

[0011] Some embodiments relate to a method for providing spatially positioned electrochemical reactions using an electrode array exposed on a surface of a semiconductor substrate, the method including selecting one or more electrodes in the electrode array and controlling circuitry in the semiconductor substrate to apply one or more stimulation signals to the one or more electrodes to initiate an electrochemical reaction at the one or more electrodes.

[0012] In some embodiments, the electrochemical reaction may be a half-reaction that generates a gas in the solution, and the one or more stimulation 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 the semiconductor substrate, and the method may further include generating a gas at one or more selected electrodes such that at least one cell of the plurality of cells disposed 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 cells of a first type, and the method may further include seeding one or more cells of a second type on the surface of the semiconductor substrate at the location where the at least one cell of the first type detached. In some embodiments, the method may further include mapping a time sequence of regrowth of the plurality of cells on the surface at the location where the at least one cell detached, and determining a 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 at the selected one or more electrodes, and the method may further include using the circuit to measure values ​​of an electrical property at each of some or all of the remaining electrodes in the electrode array outside the selected one or more electrodes, and generating a map of the electrical property based on the results of the measurements. The electrical property may be a property of open circuit potential. The electrical property may be a current. The property of the current may be a maximum range of a range of cyclic current.

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

[0014] Some embodiments relate to a system that includes a semiconductor substrate comprising: an electrode array including a plurality of individually addressable electrodes disposed on a surface of the semiconductor substrate; and circuitry controllable by one or more processors to apply one or more electrical potentials to groups of electrodes in the electrode array relative to the potential of an electrode in the electrode array or relative to the potential of a reference electrode to initiate electrochemical reactions at the groups of electrodes.

[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 1,000, at least 4,000, or at least 1,000,000 electrodes, and the circuit may include a plurality of recording circuits, each configured to measure current at an electrode of the electrode array. The plurality of recording circuits may include at least 10 recording circuits or at least 4,000 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] Some embodiments relate to a system for providing spatially positioned electrochemical reactions, comprising: an electrode array exposed on a surface region of a semiconductor substrate; circuitry disposed within the semiconductor substrate and connected to the electrode array; at least one non-transitory computer-readable medium having executable instructions stored thereon; and at least one processor programmed with the executable instructions to perform a method including selecting a pattern of electrodes within the electrode array and controlling the circuitry to apply one or more predetermined potentials to the pattern of electrodes relative to the potential of the electrodes within the electrode array or relative to the potential of a reference electrode, such that an electrochemical reaction is initiated at the pattern of electrodes. [Brief explanation of the drawings]

[0017] Various aspects and embodiments are described with reference to the following drawings. It should be understood that the drawings are not necessarily drawn to scale. Items that appear in more than one figure are designated by the same reference numeral in all figures in which they appear.

[0018] [Figure 1a] 1 is a schematic side view of a semiconductor substrate according to some embodiments. [Figure 1b] 1b is a two-dimensional data plot of simulated voltage distribution in the device shown in FIG. 1a. [Figure 1c] FIG. 1c is a data plot of simulated electric field lines corresponding to the example shown in FIG. 1b. [Figure 2a] FIG. 1 is a schematic side view of an apparatus with a semiconductor substrate without cells present, according to some embodiments. [Figure 2b] 2a is a schematic side view showing the scenario in which cells are placed on several electrodes of an electrode array. [Figure 2c] FIG. 2a is a schematic side view showing the scenario in which cells are placed outside the electrode array, between certain electrodes. [Figure 3a] 1 shows an example of cell mapping using the distribution of maximum current. [Figure 3b] 1 shows an example of cell mapping using the distribution of maximum current. [Figure 4A] FIG. 1 is a schematic diagram showing an example of high-resolution magnification mapping using cross-electrode currents. [Figure 4B] FIG. 1 is a schematic circuit diagram of a cellular circuit model. [Figure 5a] An example of a magnified cross-electrode impedance mapping is shown in comparison with a fluorescent microscope image. [Figure 5b] An example of a magnified cross-electrode impedance mapping is shown in comparison with a fluorescent microscope image. [Figure 6a] An example of the use of cross-electrode impedance mapping to quantify cell adhesion is shown. [Figure 6b] An example of the use of cross-electrode impedance mapping to quantify cell adhesion is shown. [Figure 6c] An example of the use of cross-electrode impedance mapping to quantify cell adhesion is shown. [Figure 7] 1 is a series of fluorescence microscopy images and normalized cross-electrode impedance maps. [Figure 8a] Normalized impedance histogram of control measurements without added tetracycline. [Figure 8b] Normalized impedance histograms of MDCK cells over 6-7 days in vitro (DIV). [Figure 9] 1 shows a series of normalized cross-electrode impedance maps under stimulation signals of various frequencies. [Figure 10a] An example of mapping cells and their adhesion over time by cross-electrode impedance measurements is shown. [Figure 10b] An example of measuring cell-cell adhesion is shown. [Figure 11] FIG. 1 is a schematic diagram showing cell patterning by electrochemical gas generation. [Figure 12] An example of cell spatial patterning and co-culture definition is shown. [Figure 13]10A-10C are a series of diagrams illustrating a variation of cell patterning using an electrode array. [Figure 14] A series of fluorescence microscopy images showing the process of defining a co-culture by patterning and seeding a second cell type are shown. [Figure 15] FIG. 1 is a series of schematic diagrams showing heterogeneous cell populations, elimination of unwanted cells using patterned electrochemical gas generation on selected electrodes, and subsequent cell growth followed by homogenous culture of desired properties. [Figure 16] An example of a wound healing assay is shown. [Figure 17a] An experiment demonstrating the permeabilization technique is shown. [Figure 17b] An experiment demonstrating the permeabilization technique is shown. [Figure 17c] An experiment demonstrating the permeabilization technique is shown. [Figure 17d] An experiment demonstrating the permeabilization technique is shown. [Figure 18A] An experiment using an electroporation protocol in which Fluo-4 AM was used to inject Fluo-4 into cells is shown. [Figure 18B] An experiment using an electroporation protocol in which Fluo-4 AM was used to inject Fluo-4 into cells is shown. [Figure 19] FIG. 1 shows a series of schematic diagrams illustrating the generation of controlled and cross-effect delivery using spatial addressing and serial delivery via gas generation. [Figure 20a] 1 shows an example of extracellular electrochemical mapping. [Figure 20b] 1 shows an example of extracellular electrochemical mapping. [Figure 21a] 1 shows a data plot showing selected electrode voltages plotted over time. [Figure 21b] A heat map showing one cycle and the overall amplitude of the open circuit potential plotted across the array. [Figure 22a] 1 shows an example of electrochemical oxygen mapping of cells. [Figure 22b] 1 shows an example of electrochemical oxygen mapping of cells. [Figure 23a]A series of schematic diagrams showing electrical imaging of three parameters useful for live cell assessment are presented. [Figure 23b] 10 is an image showing fluidic wells packaged on top of a chip mounted under a microscope for simultaneous optical and electrical measurements. [Figure 23c] 1 is a colorized microscope image showing cells and electrode arrays. [Figure 23d] FIG. 2 is a schematic diagram showing electrodes connected to an exemplary pixel circuit. [Figure 24a] 1A-1C are schematic diagrams illustrating some additional schemes for measuring cell-cell connectivity, according to some embodiments. [Figure 24b] 1A-1C are schematic diagrams illustrating some additional schemes for measuring cell-cell connectivity, according to some embodiments. [Figure 25a] FIG. 1 is a schematic diagram showing a pixel amplifier configured as a buffer for metabolic state measurement. [Figure 25b] 1 is a series of data maps showing the results of multi-parametric measurements. [Figure 25c] Nuclear fluorescence image at +72 hours after seeding (top) paired with detailed area 1 comparison (bottom) showing lowest cell density on the leading edge compared to the trailing edge. [Figure 25d] 1 is a composite map showing detailed area 2 overlaying cell nuclei and cell attachments. [Figure 26a] 1 is a series of fluorescence images showing the results of a comparative study of electrode impedance under three scenarios. [Figure 26b] 10 is a data plot showing that PtB reduced Zte measurements of bare electrodes. [Figure 26c] Cell barrier maps relative to the baseline at various frequencies are shown. [Figure 26d] Cell density and connectivity maps extracted from nuclei of fluorescent images are shown. [Figure 26e] A comparison between Zte measured without a standard is shown. [Figure 26f] A comparison of Zte and Zs on extracted cell density is shown. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present 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) circuitry fabricated on the surface of the semiconductor substrate using CMOS-compatible fabrication techniques and electrically interfaced with an electrode array exposed to cells. The inventors have recognized and appreciated that by using semiconductor processing techniques, electrode arrays can be economically manufactured and integrated with active circuitry. Furthermore, electrodes in an electrode array with 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 distinguishable when mapped using a high-density electrode array compared to large electrodes covered by a collection of cells. Furthermore, according to some aspects, the electrode array enables real-time, spatially addressable electrical stimulation and / or recording of electrical signals using CMOS circuitry. Some of these aspects are directed to using the electrode array to perform cell patterning via electrochemical gas generation and extracellular electrochemical mapping.

[0020] One aspect is directed to electrically mapping cells on the surface of a semiconductor substrate via cross-electrode impedance measurements. The inventors have recognized and appreciated that the electrical impedance measured between two electrodes, or cross-electrode impedance, can be affected 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 cells can be mapped using cross-electrode impedance measurements.

[0021] FIG. 1a is a schematic side view of a semiconductor substrate according to some embodiments. FIG. 1a illustrates a device 100 having an electrode array 106 including multiple electrodes 106_1, 106_2, 106_3, ... 106_N disposed on a surface 104 of a semiconductor substrate 102. FIG. 1a illustrates 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 a cross-electrode current between electrodes 106_1 and 106_2) flows along one or more current paths 109 within a medium 108 in contact with the electrode array 106. Electrode 106_1 may be connected to a stimulus source circuit 110 and may be referred to as a stimulating electrode. Electrode 106_2 may 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 using 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 that receives signals from the active circuitry within the semiconductor substrate 102 and performs the determination of the cross-electrode impedance. It should be understood that an actual impedance value need not be calculated; any representative measurement indicative of the impedance between the two electrodes can be used. As an alternative or in addition to calculating an impedance value, the cross-electrode current can be used as an indicator of 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 simulated voltage distribution in the device shown in Figure 1a, showing that when a voltage is applied to stimulating electrode 106_1, the potential in medium 108 drops both vertically (V) and laterally (L) away from stimulating electrode 106_1. Figure 1c is a data plot of simulated electric field lines corresponding to the example shown in Figure 1b. Figure 1c shows that electric field lines 114 emanating from stimulating electrode 106_1 flow along lines pointing upward from electrode 106_1, curve laterally toward recording electrodes such as recording electrode 106_2, and point downward, terminating at recording electrode 106_2.

[0024] The presence of cells can alter the shape and distribution of electric field lines 114 between electrodes, resulting in a change in cross-electrode impedance, as described below in connection with FIG. 2. FIG. 2a is a schematic side view of a device 200 including a semiconductor substrate 202 without cells present, according to some embodiments. In FIG. 2, electrode 206_0 of electrode array 206 is configured as a stimulating electrode, and electric field lines 214_1 and 214_2 link stimulating electrode 206_0 and recording electrode 206_1. FIG. 2b is a schematic side view of FIG. 2a illustrating a scenario in which cells 220 are placed on some electrodes of the electrode array. FIG. 2c is a schematic side view of FIG. 2a illustrating a scenario in which cells 220 are placed outside the electrode array, between some electrodes.

[0025] The inventors have recognized and understood that biological 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 a high resistance and have a different electrical impedance compared to the surrounding medium, such as the solution containing the cells. Cells with high-impedance membranes on top of an electrode array then influence the current distribution in a solution, such as solution 208 in Figures 2a-2c. In Figure 2c, floating cells block electric field lines in the solution, reducing 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 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 shown in FIG. 2b, attach to surface 204 and cover some or all of stimulating electrode 206_0 and recording electrode 206_1, cell 220 will increase cross-electrode coupling by blocking electric field lines 214_1, 214_2 between the two electrodes from running vertically through solution 208. As a result, more vertical electric field lines 214_2 are suppressed and more electric field lines 214_1′ are enhanced, resulting in a lower impedance between electrode 206_0 and electrode 206_1, compared to FIG. 2a where no cells are present.

[0027] On the other hand, if a cell is not attached to the surface 204, or if a cell, such as cell 230 shown in FIG. 2c, is attached to the surface 204 but is positioned laterally outside and between the pair of electrodes 206_0 and 206_2, the cell may block the electric field lines 214_3 between the pair of electrodes, reducing the cross-electrode coupling between the electrodes 206_0 and 206_2. As a result, the cross-electrode impedance between the electrodes 206_0 and 206_2 can increase.

[0028] Therefore, the presence or absence of cells above the electrode array and attached to the surface can be detected using cross-electrode impedance measurements. It should be understood that cells attached to the surface may have various degrees of non-zero separation between the outer extent of the cell membrane and the surface. Devices according to some aspects of the present application can provide detection of the degree to which cells are attached. 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 can offer several advantages: for example, they are non-invasive and can be performed repeatedly without affecting cell viability or the electrodes.

[0030] In some embodiments, as described above in connection with FIG. 2b, the cross-electrode impedance technique measures the increase in cross-electrode coupling between electrode pairs due to the suppression of perpendicular electric field lines from the presence of cells, as opposed to techniques that measure the decrease in cross-electrode coupling (or increase in measured impedance) due to the suppression from the presence of cells. One advantage of using the increase in cross-electrode coupling as an indicator of cell presence is that the increase is due to electrode pairs that are close to each other, and in some cases is primarily due to nearest-neighbor coupling between electrode pairs. Therefore, the increase in cross-electrode coupling (or decrease in measured impedance) can be separated from the overall background current that flows through the stimulating electrode to many of the remaining electrodes in the electrode array. This can result in improved signal-to-background ratio and sensitivity for cell detection.

[0031] In contrast to the cross-electrode impedance technique, the inventors recognized that the mere impedance measured at each electrode cannot detect the presence of cells. In such a single-electrode measurement, the sum of all return currents is measured as the impedance signal on the electrode. That is, such a measurement is an impedance measurement of the electrode only, and does not measure changes in the electric field in the solution above 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 FIG. 1a, in some embodiments, the stimulation signal applied by the stimulation source circuit 110 to the stimulation electrode 106_1 is a low-frequency alternating current (AC) signal having a frequency less than 10 kHz, less than 5 kHz, between 0.1 and 5 kHz, or between 0.1 and 2 kHz. A low-frequency stimulation signal is selected because cell membranes act as capacitors in parallel with a high resistance; at high frequencies, the capacitor impedance decreases, making the cells highly conductive. The inventors have recognized and appreciated that measuring 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 measurements is provided in Example 4 below.

[0033] 1 , the semiconductor substrate 102 can include active circuitry 116. The active circuitry 116 can include multiple stimulation circuits 110 and multiple recording circuits 112. In some embodiments, the stimulation circuitry 110 can include one or more current injectors, one or more voltage sources, or a combination thereof. Some aspects of the active circuit design are related to galvanic stimulators and related methods for electrogenic cells, as disclosed in International Application Publication No. 2019 / 010343, Attorney Docket No. H0776.70105WO00, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the active circuitry can include a programmable current injector for performing current-voltage measurements using one or more electrodes in an electrode array as working 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 which is at least 10 MΩ (megohms), at least 100 MΩ (megohms), 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] Electrodes in the electrode array 106 can be reconfigured using the active circuitry 116 as stimulating or recording electrodes. In some embodiments, the active circuitry 116 includes routing and switching components that are programmable to connect selected electrodes of the electrode array 106 to the stimulus source circuitry 110, the current measurement circuitry 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 cellular properties 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 location of one or more selected electrodes. The latter embodiment is discussed in more detail in the sections below regarding cell-to-cell adhesion measurement, patterning, and spatial electrochemical mapping of cells.

[0036] In some embodiments, electrodes may be biased using low-impedance sources / returns in the active circuit. For example, a low-output-impedance voltage source may be used to provide a stimulation signal to the stimulation electrodes, while a low-input-impedance transimpedance amplifier may be provided for current measurement at the recording electrodes. 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 appreciated that low-impedance sources / returns can facilitate the formation of fringing electric field lines in the solution, as shown in FIG. 1c.

[0037] The semiconductor substrate 102 can include silicon, and thus, in embodiments, the active circuitry 116 can be an integrated circuit comprising CMOS components fabricated using standard CMOS processing techniques. The electrode array 106 can be disposed within the semiconductor substrate 102, for example, as a conductor exposed from a 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 for the electrode array 106 and a suitable surface for cell growth. While FIG. 1a shows the electrode array 106 partially embedded in the semiconductor substrate 102, such an arrangement is an illustrative example and is not a requirement. In some embodiments, the top surfaces of the electrodes in the electrode array 106 can be above, vertically aligned with, or below the surface 104 of the semiconductor substrate 102. Additionally or alternatively, the top surfaces of the electrodes can have a passivation or functionalization layer. In some embodiments, holes may be patterned in a passivation or functionalization layer on top of the electrode to expose the conductive surface of the electrode to the medium.

[0038] It should be understood that the semiconductor substrate 102 can be any substrate fabricated using semiconductor processing techniques and is not limited to a silicon wafer. For example, the semiconductor substrate 102 can include a Group IV semiconductor, a Group III-V semiconductor, a Group II-V semiconductor, an sp2 hybridized carbon material, a chalcogenide, a metal, a metal compound, an oxide, a nitride, a silicide, a polymer material, or a combination thereof. The semiconductor substrate 102 can be a single component or a composite of multiple components. The components within the semiconductor substrate 102 can include an active circuit layer, a wiring layer, a redistribution layer, a circuit board, or a combination thereof. The component layers within the semiconductor substrate can be formed additively during CMOS processing or can be formed separately and bonded together using packaging techniques known in the art. Conductors interconnecting the active circuitry 116 and the electrode array 106 are provided in the semiconductor substrate 102. In some embodiments, connection points are provided on the bottom surface of the semiconductor substrate for electrically interfacing the components within the semiconductor substrate with the processing unit 120. Electrical connection between the processing unit 120 and the semiconductor substrate 102 may be provided by any suitable method, such as, but not limited to, a controlled collapse chip connection (C4) or flip-chip bonding, wire bonding, flexible cable, or wireless communication.

[0039] Referring again to FIG. 1a, in some embodiments, the apparatus 100 can be operated to perform a method, such as performing mapping or selective electrochemistry. The operation of the apparatus 100 can be under program control. In some embodiments, the processing unit 120 in the apparatus 100 can include a computer 20 with a storage medium 21, a memory 23, and a processor 25, and such processing can be performed on the computer 20 or any other computing device. The storage medium 21 and the memory 23 can be any suitable non-transitory computer-readable medium, such as, but not limited to, computer memory, a compact disc, an optical disc, a magnetic tape, flash memory, circuitry in a field programmable gate array (FPGA) or other semiconductor device, or other tangible computer storage medium. In some embodiments, the storage medium 21 can be a non-volatile storage medium, and the memory 23 can be a volatile storage medium. Computer-executable instructions can be loaded from the storage medium 21 into the memory 23 prior to execution by the processor 25 to perform some or all of the methods described throughout this disclosure. However, the distinction between storage medium 21 and memory 23 is not important, and in some embodiments, one or both may be present.

[0040] Processor 25 may be any suitable processing device, such as, but not limited to, one or more processors, central processing units (CPUs), digital signal processors (DSPs), controllers, addressable controllers, general-purpose or special-purpose microprocessors, microcontrollers, addressable microprocessors, programmable processors, programmable controllers, special-purpose processors, special-purpose controllers, or any other suitable processing device. Some or all of the components in processing unit 120 may be packaged as a system-on-chip (SOC). Furthermore, it should be understood that FIG. 1a is a schematic representation of processing unit 120. An actual implementation of processing unit 120 may include distributed processing. For example, a host computer may control the overall flow of measurements, mapping, and analysis of results.

[0041] Reference is now made to the electrode array 106. In some embodiments, the electrode array 106 may be patterned on the surface 104 as part of a semiconductor fabrication process to form active circuitry 116 within the semiconductor substrate 102, and may be conductive pads comprising a metal such as Au or Pt or an alloy thereof. For example, the pads may be formed of Al with plated Au as a top layer. In such embodiments, the substrate 110 may further include conductors that vertically interconnect the exposed electrode array 14 to the circuitry within the substrate 110.

[0042] The electrodes in the electrode array 106 may be arranged on the surface 104 in any suitable configuration, such as a two-dimensional array with a regular pitch along the rows and columns. In some embodiments of cross-electrode impedance mapping, the pitch of the electrode array can be selected to be on the order of or less than the size of a typical cell, thereby allowing the cell to cover at least two electrodes and increasing coupling between the cell and at least two electrodes. For example, if the cell size is approximately 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. Providing a small pitch between the electrodes allows the cell to cover more than one electrode, enabling measurement of the cell-substrate gap distance via increased cross-electrode coupling with electrodes below the cell.

[0043] In some embodiments, the electrode array is fabricated during a CMOS-compatible manufacturing process on top of a semiconductor substrate containing CMOS active circuitry, and the pitch of the electrode array and the size of each electrode can be selected by considering the pitch and density of the CMOS active circuitry. For example, in some embodiments, at least 8, at least 10, or at least 4,000 recording circuits may be provided within the semiconductor substrate, and the electrode array may have at least 1,000, at least 4,000, or at least 1,000,000 electrodes. In such embodiments, each electrode may have a lateral dimension of 10 μm or less, or 5 μm or less, such that the entire lateral extent of the electrode array is contained within the surface of the semiconductor substrate. Electrode arrays according to aspects of the present disclosure may also be referred to as CMOS microelectrode arrays (MEAs).

[0044] Referring again to Figure 1, medium 108 may be cell culture medium or a solution containing any number of chemical and / or biological reagents in addition to cells. Although not shown in Figure 1, medium 108 may be contained within a container disposed on top of 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 include multiple electrode arrays so that electrical assessments in multiple wells can be performed in parallel.

[0045] A CMOS-compatible, wafer-scale, multiwell platform and method for operating the same are disclosed. In some applications, circuitry is provided beneath a multiple-well array and electrically interfaces with electrodes within the wells. The platform is sometimes referred to as a CMOS-multiwell platform. The inventors have recognized and appreciated that, to interface with electrodes within a large array, circuitry can be fabricated on a single silicon (Si) wafer having dimensions at least equal to or larger than the dimensions of the multiple-well array. According to one embodiment of the present disclosure, standard CMOS fabrication processes known to be used in standard semiconductor foundries can be used without, for example, expensive customization for complex fabrication procedures, thereby reducing manufacturing costs in some cases. CMOS-multiwell platforms according to some embodiments of the present 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 areas, some or all of which have multiple circuits of the same design. The inventors have recognized and appreciated that during manufacturing, the reticle areas of the wafer can reuse the same lithography mask design, in some cases repeated across the wafer, thus reducing the cost of processing and increasing wafer manufacturing throughput.

[0047] According to one aspect, the digital and analog circuits in the reticle area may be positioned to correspond to one or more wells when the multiwell array is bonded on top of the wafer. Thus, some embodiments can provide wafer-scale integration of the electrical interface to the multiwell array without dicing the wafer and / or using fabrication methods that use standard MOS compatible technology to meet standards and reduce manufacturing costs.

[0048] One aspect of the present disclosure relates to a technique for mapping the spatial distribution and dimensions of cells using cross-electrode impedance measurements. The mapping can further reveal properties of individual cells, such as their adhesion to the surface of a semiconductor substrate. In some embodiments, because cell presence 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. A different electrode is then selected as the stimulating electrode, and a new set of cross-electrode impedance data is measured. The cross-electrode measurement is repeated by sequentially configuring electrodes in the electrode array and applying a stimulation signal, and the corresponding sets of measured cross-electrode impedance data can be processed to generate values ​​for each stimulating electrode location that indicate the presence or absence of a cell or the strength of a cell property. The processed values ​​can then be combined to form a map across the area of ​​the electrode array. In some embodiments, "electrochemical imaging" of live cell cultures is demonstrated using high-resolution in situ impedance and electrochemical measurements. Some embodiments relate to the use of CMOS-MEAs for label-free, non-invasive tracking of cell growth kinetics and precise measurements of cell-substrate attachment, cell-cell adhesiveness, and metabolic state.

[0049] Another aspect relates to providing spatially positioned electrochemical reactions using patterned electrode arrays. Using a selected number of electrodes in the electrode array, active circuitry in the semiconductor substrate can apply potentials that initiate electrochemical reactions in solution regions directly overlying 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, spatially programmed electrochemistry can be used to perform cell patterning, for example, cells attached to an electrode can be selectively removed from the electrode surface by electrochemically generating small gas bubbles on the electrode.

[0051] In some embodiments, an array of electrochemical electrodes can be used to spatially map analyte concentrations that are measured using active circuitry within a semiconductor substrate. One application is electrochemical mapping of solutions using redox electrochemistry.

[0052] The following applications are each incorporated herein by reference in their entirety: 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. Additionally, the following applications are each incorporated herein by reference in their entirety: PCT patent application filed June 16, 2021, entitled "Systems and Methods for Patterning and Spatial Electrochemical Mapping of Cells," and PCT patent application filed June 16, 2021, entitled "Complementary Metal-Oxide-Semiconductor (CMOS) Multi-Well Apparatus for Electrical Cell Assessment."

[0053] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0054] Example 1: Real-time cell measurement and imaging system using a CMOS microelectrode array (MEA) This example describes electrical imaging of three parameters useful for live cell assessment (Figure 23a): cell-substrate impedance: Zs (reflecting cell attachment and cell-substrate adhesiveness), transepithelial impedance: Zte (reflecting cell-cell adhesiveness and cell monolayer integrity and barrier function), 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 performance across a 64 × 64 = 4096 electrode array (Figures 23b-d). A fluidic well is packaged on top of the chip to culture cells and is 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 device and consists of 8 μm diameter Pt electrodes spaced at a 20 μm pitch for single or few cell resolution (e.g., MDCK cells in Figure 23c), measuring 1.26 × 1.26 mm. 2 The device achieves a total sensing area of ​​1000 nm. The remainder of the surface is insulated with silicon nitride, which behaves similarly to glass culture plates. No differences in growth or morphology of cells cultured on the device are observed 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 above the device, regulating CO2 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 that can be configured as a buffer for measuring the electrode voltage, V, or as a transimpedance amplifier for measuring the electrode current, I,. Some aspects of the pixel circuit configuration are related to galvanic stimulation devices and related methods for electrogenic cells, as disclosed in International Application Publication No. 2019 / 010343, Attorney Docket No. H0776.70105WO00, the disclosures of which are incorporated herein by reference in their entirety. Some aspects may also be related to electronic circuits and related methods for analyzing electrogenic cells, as disclosed in International Application Publication No. 2019 / 089495, Attorney Docket No. H0498.70647WO00, the disclosures of which are incorporated herein by reference in their 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 live cell assessment: cell attachment via cell-substrate impedance Zs, cell-cell adhesion via transepithelial impedance Zte, and metabolic state via extracellular redox potential Vredox. Each measurement is noninvasive and fast (<1 min), and measurements can be continuously repeated every 5–10 min for real-time investigations. Figure 23b shows an image demonstrating that a fluorescence microscope can be paired with a packaged CMOS IC for simultaneous optical and electrical cell measurements. In this example, a reference electrode, either Pt (as shown) 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 8 μm diameter platinum electrodes are spaced at a 20 μm pitch. Platinum black (PtB) can be electrodeposited on the electrodes to reduce electrode impedance for higher signal-to-noise Zte measurements. Figure 23d shows a schematic diagram of an exemplary circuit for the electrodes in the electrode array. Each of the 4096 electrodes is connected to its surrounding circuitry via a shielded path (~1-10 mm). The op-amp-based circuit can be configured to apply voltage via Vs and measure current via a feedback resistor Rf (~100 MΩ), or to apply current via Is and buffer / measure the electrode voltage V. The output of the op-amp Vamp is routed off-chip for analog-to-digital conversion. The switches are digitally programmed using a real-time software interface.

[0058] According to some embodiments, the high channel count (4096) parallel current and open circuit potential measurements featured in the present measurement approach provide unique advantages over other MEA devices. For example, measurements such as those described in this example are inhibited in MEA devices that measure electrode capacitance, voltage (using high-pass filters to block DC signals), and current (with a low channel count (<32)).

[0059] Example 1A: Cell Mapping Using Maximum Current Distribution This example describes a technique for mapping cells using a CMOS electrode array containing a 64x64 array of 4096 platinum electrodes with a 20 μm pitch.

[0060] The present inventors have recognized and appreciated that alternating current (AC) impedance measurements between a pair of electrodes can detect cells using the contrast between an insulating cell membrane and a conductive culture medium. In traditional impedance measurements, the solution path around the cell shunts the measurement and reduces detection sensitivity. The solution contribution to the measured electrode-to-electrode current is much larger than the small changes in current due to the cell. The device disclosed herein improves detection sensitivity by instead measuring changes in the electric field distribution due 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 via the remaining 4095 electrodes using a transimpedance amplifier. The results are shown in Figure 3a, which shows a heat map 301 of the measured current distribution of the 11 x 11 nearest recording electrodes relative to one stimulating electrode 311 when no cells are present. In heat map 301, each pixel corresponds to an electrode location. Each electrode has an electrode location or electrode position, which can be represented in several ways, such as, but not limited to, coordinates or pixel numbers. Heat map 302 is a measured current distribution similar to heat map 301, but with a cell on top of electrode 311. Impedance measurements were performed at a signal frequency of 1.9 kHz.

[0062] The data plot 303 of measured cross-electrode current versus distance to stimulated pixel in Figure 3a shows that in the presence of cells, cross-electrode coupling with adjacent electrodes is almost an order of magnitude higher compared to electrodes with no cells above them.

[0063] In this example, a fluorescent nuclear MDCK cell line was used for optical confirmation. Figure 3b shows a fluorescent microscopy image 304 across the 64 x 64 electrode array, with brighter pixels representing fluorescent 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, cross-electrode current values ​​from the remaining electrodes were measured as the recording electrodes. The recorded cross-electrode currents were collected, and the maximum value was determined, referred to as the maximum current value corresponding to the given stimulating electrode. Figure 3b shows a heat map 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 fluorescent signal 307 and the maximum current signal 309, demonstrating the ability to map clusters of cells with single-cell resolution. Consequently, this example demonstrates strong correspondence between the maximum current map and the fluorescent imaging, as well as confirmation of cell presence using a nuclear fluorescent marker.

[0065] The maximum current value (Ie) is determined for each stimulating electrode location using any suitable method based on the set of cross-electrode currents measured from the recording electrodes. The determination may be a simple comparison of the absolute values ​​of the cross-electrode currents, or may further include 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 the current values ​​may be performed using a processing unit, such as processing unit 120 shown in FIG. 1a, after digitization of the measured current values.

[0066] Example 2: High-spatial resolution mapping using cross-electrode currents This example describes how to generate a magnified map of cross-electrode coupling with spatial resolution higher than the pitch of the electrode array.

[0067] According to some embodiments, nearest-neighbor cross-electrode measurements may be used for each stimulating electrode. Figure 4A shows an example of high-resolution expanded mapping using a 3 x 3 impedance grid for each of electrodes 1-9. In some embodiments, electrodes at the edges of the electrode array may be skipped from the expanded impedance grid, as described below.

[0068] Figure 4B shows the AC stimulus voltage V A Application of and cross electrode current I 12 For the measurement of cell-substrate impedance Z s and transepithelial impedance Z te is a schematic circuit model that can be used to calculate the impedance of the 3x3 grid. s Used for calculations, one Zte is extracted for each electrode.

[0069] To measure cell-substrate attachment, a cross-electrode electric field variation is created. Instead of applying a bias between two electrodes, a bias is applied from one electrode to all remaining electrodes. This allows electric field lines that originate from the stimulating electrode and extend far into the culture well to terminate on an electrode far away from the stimulating electrode. Otherwise, these field lines would have to wind back toward the adjacent electrode, increasing the amount of measured current that is not related to the immediate cell-electrode interface.

[0070] The interface may be modeled using a cross-sectional model to increase spatial resolution. s < <Z te , Z e,1 , Z e,2 These have been found to be effective for most measurements according to several aspects:

[0071]

number

[0072] The measured cross-electrode current can also be rewritten in terms of (Equation A1).

[0073]

number

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

[0075]

number

[0076] Formula A3) and (Formula A2) to Z s can be solved.

[0077]

number

[0078] This uses all the measured currents.

[0079] Z s To generate a high-resolution spatial map of the stimulating electrodes, we used nearest-neighbor cross-electrode measurements. A 3x3 grid was used for each electrode (except those at the edge of the electrode array). See Figure 4A. This is a 190x190 pixel global Z-axis map. s Generate an image (compared to the 64x64 electrodes in the array).

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

[0081]

number

[0082] where VAC is the amplitude of the applied AC voltage, I xy is the magnitude of the AC current measured by electrode y when an AC signal is applied to electrode x, I x [I y ] is the sum of the magnitude 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 and corner electrodes, and the median normalized impedance value was determined as follows, where "median" is the median function:

[0085]

number

[0086] The use of cross-electrode current not only increases the effective spatial resolution compared to using the maximum of the current distribution, but also allows for mapping of unadhered cells (which cause a decrease in cross-electrode current).

[0087] 5a and 5b show an example of a magnified cross-electrode impedance mapping compared to a fluorescent microscope image. FIG. 5a shows a fluorescent microscope image 501 across an electrode array and a heat map plot 502 of normalized cross-electrode impedance for a cell culture immediately after seeding. A magnified map 504 of a portion of heat map 502 shows the decrease in cross-electrode normalized cell-substrate impedance Zs for unadhered cells at single-cell resolution. The immediately after seeding mapping of cells without adhered cells shows smaller normalized impedance values ​​where cells are present compared to uncoated electrodes.

[0088] Figure 5b shows a fluorescence microscope image 505 and a cross-electrode impedance map 506 after 24 hours of culture. Figure 5b also shows a magnified map 507, which is an overlay of the fluorescence microscope image and cross-electrode impedance map of a selected area. The results show that many of the cells have adhered to the surface, causing a dramatic increase in the normalized cross-electrode impedance.

[0089] Example 3: Quantification of cell adhesion This example describes how to use cross-electrode impedance mapping to quantify cell adhesion.

[0090] Cells are treated with ethylenediaminetetraacetic acid (EDTA). EDTA inhibits the Ca release required for integrin proteins to maintain cell adhesion. 2+ EDTA is a calcium chelating agent that removes calcium. Upon application, cells rapidly detach over a time course of ~50 minutes. EDTA is then washed away by adding normal culture medium, and cells reattach over a time course of ~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 of MDCK cells over time, following application of 5 mM EDTA at t = ∼5 min and washout at t = ∼55 min.

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

[0093] To demonstrate a biologically relevant example of quantifying cell adhesion, we measured a genetically engineered MDCK cell line and used tetracycline to turn on and off the RasV12 and GFP genes. 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, tetracycline was added to turn on the cancer-related gene RasV12, which also expresses GFP, making gene expression imageable. After 4 DIV, tetracycline was removed to turn off gene expression. Upon RasV12 gene expression, cells became less adhesive to the surface, and after turning it off, they returned to normal.

[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 tumorigenesis. Initially, tetracycline was removed from the medium, and cells adhered normally. Upon introduction of tetracycline, the gene was expressed, resulting in an increase in GFP and a decrease in cell adhesion. Removal of tetracycline then reversed cell adhesion, causing cells to adhere more strongly and decreasing 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 to control cultures in which tetracycline was not introduced, as shown in Figure 8. Figure 8b shows normalized impedance histograms of MDCK cells over 6–7 days in vitro (DIV). After 2 DIV, tetracycline was added to turn on the cancer-associated gene RasV12. Figure 8a shows the normalized impedance histogram of the control measurement without tetracycline. Histograms are normalized to the maximum number of pixels above a cell-free impedance value of ~8 kΩ. Cell adhesion was reduced compared to controls and showed a smaller trend toward a decrease over time.

[0095] Example 4: Frequency response This example illustrates the effect of frequency used on cross-electrode impedance measurements.

[0096] The mapping frequency was 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 stimulation signals of various frequencies. The plots are normalized to the median ± 1 standard deviation. Lower frequencies exhibit higher signal contrast, which correlates with the optically measured GFP fluorescence, as shown in Figure 7, indicating that lower frequencies are better for measuring cell adhesion. The 1.9 kHz used shows better contrast compared to 240 Hz, but above 10 kHz, the cell sheet appears much more uniform.

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

[0098] This example describes a method to measure cell-cell adhesion, or how well cells are connected to each other. Cells in culture not only adhere to surfaces, but also to each other through cell-cell connections. The compactness of these connections defines the permeability of the cell sheet and is important for epithelial tissues, which act as a barrier for body surfaces, internal organ linings, and other tissues. In this example, this barrier function is measured by the transepithelial impedance Z. te Thus, cell-cell connectivity can be assessed by mitigating any pores using only the electrodes covered by cells, while also allowing for spatial heterogeneity assessment.

[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. The current I e,n is measured via the central electrode 1006_2. The central electrode 1006_2 does not pass current to the surrounding electrodes because they are biased with the same signal, and therefore passes only current due to the impedance of the cell sheet above the electrode. Outside the central electrode and its surrounding electrodes, the rest of the array is biased at ground or a reference voltage level and serves 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 allows TEER to be mapped across cells on top of the electrode array without the need for special suspensions. Advantages include the need for fewer cells, the ability to assess spatial heterogeneity, and the ability to combine cell-cell and cell-substrate adhesion measurements using the same device.

[0100] FIG. 24 includes schematic diagrams illustrating several additional schemes for cell-cell connectivity measurements, according to some embodiments. In FIG. 24, changes in the vertical electric field above the electrodes are measured to best isolate the effects of cell-cell connectivity using two circuit configurations: 1) fast (<1 s / measurement) parallel electrode measurements versus a reference (FIG. 24a); and 2) slow scan (40 s / measurement) relative measurements without a reference (FIG. 24b). Fast measurements are ideal for scanning across multiple frequencies, while scanned measurements do not require a reference, which helps make measurements more stable over time and is more ideal for device miniaturization. For both types of measurements, platinum black (PtB) deposition is optionally used to measure Z. e Reduced by about 5 times, Z te Sensitivity can be improved. Experiments across frequencies showed that intermediate frequencies, from ~2 kHz to 5 kHz, were best for assessing intercellular connectivity.

[0101] Here, the transepithelial impedance Z using the scheme of Figure 24 te The calculation of is explained below.

[0102] To measure cell-cell adhesion, or how well cells are connected to each other, the stimulation protocol can be modified to measure the perpendicular electric field component in Figures 24a and 24b. Each transepithelial electrode current, I, 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 made relative to a grounded reference (left). The resulting electric field distribution is I te The cells are aligned vertically, decreasing connectivity. A baseline-free measurement can be performed by applying an AC voltage to electrode (n) and its neighboring electrodes, generating an effective vertical field measurement with the remaining electrodes grounded (right). To generate a cell map, the applied signal is scanned across the array (40 s per scan / frequency).

[0103] In the parallel scheme of Fig. 24a, an AC voltage is applied to each electrode relative to a reference value, and the current I te,n is measured, generating a vertical electric field in the solution (the peripheral electrodes also have low-frequency fringe fields). Then, due to the current that must pass through the cell sheet, the magnitude of the current is measured by the transepithelial impedance Z te The second scanning scheme, Figure 24b, biases the electrode and its surrounding electrodes with an AC voltage and measures the current passing through only the center electrode. Because they are biased with the same signal, the center electrode does not pass current to the surrounding electrodes, passing 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 serves as the current return.

[0104] In both cases, the measured vertical current I te,n is expressed as follows:

[0105]

number

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

[0107]

number

[0108] For measurements, intermediate frequencies from between ∼1 kHz and 5 kHz were determined to best correlate with cell-cell connectivity (see also Example 15 below). For PtB electrodes, Z e,n is Z te (see also Example 15 below), which is estimated as follows:

[0109]

number

[0110] Z using only Pt electrodes te In the experiment, the cell-substrate impedance I n Measurements are subtracted. Using 3 x 3 sets of electrodes, Z te,no ref Due to the scanned array measurements that calculate , the overall map produced is 62 x 62 pixels, and the peripheral electrodes do not have adjacent bias electrodes that create a perpendicular field. Measurements against the reference values ​​produce a map containing 64 x 64 pixels.

[0111] Example 5A: Extracellular Redox Potential V redox Metabolic state mapping by Beyond impedance measurements, platinum electrodes have been used for both potentiometric sensing of oxygen and extracellular redox monitoring. This example uses the close location of a Pt electrode directly underneath a living cell to measure the extracellular redox potential, V redoxhave demonstrated that it is possible to map the metabolic state of cell cultures by mapping in situ the redox environment and O2 consumption of cells.

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

[0113] Generally, cells use energy derived from the transfer of electrons from oxidizable organic molecules (e.g., glucose) to O2 during aerobic metabolism. To help mediate the flow of these electrons, a general reducing environment is generated by the thiol compound glutathione (GSH), often considered the cellular redox buffer. Simply put, the cellular redox potential is the balance between O2, which increases the potential (oxidizes), and GSH, which decreases the potential (reduces). The redox environment is not only important for electron transfer, but also for neutralizing harmful reactive oxygen species, intercellular signaling, and regulating cellular state. For example, ranging from negative to positive, the redox potential can determine whether a cell is in a state of proliferation, differentiation, apoptosis, or necrosis.

[0114] Figure 25b is a series of data maps showing the results of multi-parametric measurements. Measurements are performed at +24 hours, +48 hours, and +72 hours after MDCK cell seeding, including cell attachment (top), cell-cell adhesion (middle), and metabolic state (bottom). Cells show growth from the bottom right corner to the top left corner, with the proliferating leading edge cells exhibiting the most negative V compared to the more dormant trailing edge. redox Indicates Z te is highest at the leading edge, due to the lowest cell density (see detailed area 1) and therefore the fewest cell-cell connections. Figure 25c is a pair of nuclear fluorescence images at +72 hours after seeding (top) and detailed area 1 comparison (bottom) showing the lowest cell density on the leading edge compared to the trailing edge. Figure 25d is a composite map showing detailed area 2 overlaying cell nuclei and cell attachments. Figure 25d shows good spatial correspondence with single-cell resolution.

[0115] One goal of this example is to measure the close V by pairing it with impedance techniques to monitor cell growth. redox The goal is to investigate what information can be provided by a negative V in the range of 30 mV to 80 mV compared to an electrode without cells (Figure 25b). redox was observed on the electrode with cells (Fig. 25b). redox The spatial information of the V is distinct and distinct from the cell attachment or cell barrier, and the most negative V redox is at the leading edge and not at the lowest density. In general, a negative signal may indicate a locally low [O2] or a 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. Upon O2 removal, the signal difference between the cell-present and cell-absent regions was eliminated. To complement this, the GSH system reduction capacity was examined by oxidation titration. Due to its previous non-toxic use in cell cultures and the potential of its oxidation half-cell compared to the cellular environment, ferrocyanide [Fe(CN)6] was used for the titration. 3- The vehicle exhibited a reducing power of 4 μM, while the cells had a much greater capacity of >200 μM.

[0117] Taken together, these measurements indicate that the measured V redox We show that V is related to both the in situ [O2] and [GSH] reducing power of the cell. redox We theorize that under atmospheric conditions that decrease [O], [O] decreases from its normal dissolved concentration of ~200 μM until it is regulated by the extracellular redox potential of the cell. Therefore, it is difficult to quantify oxygen consumption rates using our method, but we can use the V of the extracellular redox potential to quantify [O]. redox The measurement becomes useful for monitoring the metabolic state of cells, since it can indicate both O2 use and the reducing environment of the cells. Thus, the more negative signal at the leading edge of the cell sheet (Figure 25b) is due to the proliferation state of the cells, respiration combined with the most negative redox potential state.

[0118] Example 6: Antibody-Cell Binding Screening for antibody-cell binding can be low throughput because it requires fluorescent tagging of antibodies (which requires a wash step to remove unbound fluorescent antibody) or specialized optical measurements such as surface plasmon resonance (SPR). In one embodiment, the cross-electrode impedance technique described here provides the ability to measure antibody-cell binding events through either cell-substrate or cell-cell adhesion measurements. Antibody binding on the underside of cells effectively reduces the gap distance, leading to a decrease in the amount of measured cross-electrode current. Similarly, antibody binding to the side of cells should also reduce the gap distance between cells, leading to a decrease in the amount of measured vertical current. The ability to perform such antibody binding without labeling allows various antibodies to be added in an array without the need for a wash step, significantly improving throughput.

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

[0120] While not wishing to be bound by any particular theory, the inventors believe that for most inert electrode materials (platinum, gold, etc.), the electrode potential can be calculated as the hydrogen ion / hydrogen gas redox half-cell reduction potential (E 0 ), it was found that hydrogen gas could be generated.

[0121] [ka]

[0122] Alternatively, oxygen gas can be generated by adjusting the electrode potential above the oxygen gas / water 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] Thus, cell removal can be achieved 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 stimulus source circuits 110 in FIG. 1a to selected electrodes. The potential need not be the same across all selected electrodes; programmable non-uniformity can be used where the electrodes are biased differently. The potential can be 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. Controlling the gas generation rate allows for optimization of selective electrochemical reactions, as too fast a gas generation rate can be used to cut off the electrode from the solution, causing large bubbles to form on the surface.

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

[0129] Example 8: Cell Spatial Patterning and Co-Culture Definition This example describes the spatial patterning of cells and the definition of co-cultures using electrode arrays.

[0130] In this example, we used a CMOS electrode array, MDCK cells, and H gas generation, as shown in Figure 12. In this experiment, H gas was generated by applying -1.25 V to a platinum electrode-Ag / AgCl pseudo-reference electrode. Figure 12 shows fluorescence microscopy images taken 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 holes of various sizes were fabricated with high spatial resolution in a uniform cell sheet, as confirmed by nuclear fluorescent 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 seeding a second cell type. Cell types were distinguished by different nuclear fluorescent markers. In the experiment shown in Figure 14, a co-culture of two different cell types was defined by seeding a second MDCK cell line with different nuclear fluorescent markers after initial patterning. The second cell type filling the generated space demonstrates the ability to spatially define co-cultures 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: Directed cell evolution by eliminating culture heterogeneity This example describes a method of directed cell evolution to remove cells from cell culture whose properties are undesirable.

[0133] Figure 15 is a series of schematic diagrams showing heterogeneous cell populations, the elimination of unwanted cells using patterned electrochemical gas generation on selected electrodes, and the subsequent homogenous culture of desired properties after cell growth. Selection of which cells to eliminate can be performed by optical imaging or other properties measured using the electrode array. The ability to eliminate cells from culture without having to remove them from the culture plate is advantageous over current processes that require cells to be suspended, separated using a cell sorting machine, and then re-cultured with a further reseeding step, or the removal and replated use of a micropipette of single cells with desired properties. Furthermore, the lineage of cell history can be preserved because the spatial location of each cell does not change and the cells remain adherent during the process. This elimination process can also be used for further analysis performed on a subset of cells after culturing on the electrode array, where cells undesirable for further measurement are first killed before cell suspension and removal.

[0134] Example 10: Wound healing assay This example illustrates the combined application of both cross-electrode impedance mapping and cell patterning to a wound healing assay.

[0135] These assays will be useful for measuring cell proliferation rates and metabolism and screening drugs that affect these parameters. Compared to the electrochemical patterning described here, other tools involve mechanically creating wounds in cell cultures by mechanical scratching, which is difficult to control and is limited in terms of wound pattern.

[0136] In this example, wounds were created in MDCK cells, and 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 a -10 nA electrode current for 40 s relative to an Ag / AgCl pseudo-reference electrode. Culture regrowth was then measured using impedance mapping. A typical cell culture took ~3 days to fill the wound, while cultures treated with growth inhibitors showed very little regrowth. As shown by the normalized cross-electrode impedance map in Figure 16, the control culture shows regrowth after 72 hours in culture. A second culture treated with the growth-slowing drug, cytochalasin B (1 μM), showed very little growth over the course of 72 hours, demonstrating the assay's capability for drug screening.

[0137] Example 11: Molecular Delivery This example describes a method using planar electrodes for membrane permeabilization and molecular delivery. Unlike electroporation, which applies a focused electric field to disrupt cell membranes, planar electrode permeabilization works by forming gas bubbles, which is similar in concept to the patterning method discussed here. Unlike cell patterning, in which cells are killed to perform the patterning, smaller pores are generated on the cells for molecular delivery, which then reseal over time.

[0138] Figure 17 shows an experiment demonstrating the permeabilization technique using nanowire electrodes; aspects of this technique can also be applied using electrode arrays with planar electrodes. In the experiment shown in Figure 17, Fluo-4, a live assay, is dissolved in the extracellular solution (left panel, Figure 17a). An electroporation protocol is applied to the nanoelectrodes using a pixel stimulator (middle panel, Figure 17a), allowing the Fluo-4 to be recovered. If electroporation is successful, the Fluo-4 penetrates 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). Successfully electroporated cells and Fluo-4 are recovered and retained for imaging. Figure 17b shows heatmaps depicting averaged EthD-1 and Fluo-4 intensities for the eight investigated protocols of increasing voltage amplitude (three trains of five biphasic pulses at 20 Hz) performed with HEK293 cells. The CNEA array was divided into subgroups of 128 pixels for each of the eight protocols, repeated in a grid across the array. Images were taken 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 begins at ~1.3 V, while irreversible electroporation begins at ~1.7 V. Figure 17d shows results using neurons under the same test conditions, demonstrating a lower threshold of <1.2 V for successful electroporation and ~1.5 V for irreversible electroporation.

[0139] Figure 18 shows another experiment in which Fluo-4 is injected into cells using Fluo-4 AM. The electroporation protocol is applied to the nanoelectrode using a pixel stimulator (center) while fluorescence is monitored. Upon successful electroporation, Fluo-4 can be effluxed from the cell, resulting in a decrease in fluorescence. In a successful protocol, the cell membrane recovers after electroporation (right panel, Figure 18a). Figure 18b shows an example using a neuron with its fluorescence and an applied electroporation signal. During electroporation, fluorescence decreases. Shortly thereafter, the cell membrane recovers, causing the fluorescence to plateau. The electroporation signal may be applied multiple times without affecting cell viability.

[0140] In both experiments shown in Figures 17 and 18, we observed that a voltage signal of at least >50 ms duration was required to see either permeabilization or delivery. This indicates the necessity of a Faradaic process to generate bubbles, as the required voltage is comparable to the water window voltage (H2 & O2 gas generation due to water splitting) with the platinum electrode used. In Figure 18, this permeabilization signal is shown to be effective by inducing transient leakage of the fluorescent dye, while in Figure 17, the fluorescent dye is delivered into the cells.

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

[0142] Example 12: Serial Delivery for Cross-Effectiveness Analysis This example illustrates the multi-step delivery of compounds into 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. Because the electrode properties are not altered during gas generation, the spatial capabilities of addressable electrodes, combined with the ability to deliver multiple compounds, offer the added advantage of 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 generate a complete matrix of drug effects.

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

[0145] Electrode-based cellular electrochemical measurements can use a single, large working electrode to measure the bulk concentration of an analyte in solution. Such electrochemical electrode-based measurements include Clark electrodes for dissolved oxygen concentration measurements and hydrogen ion concentration (pH) measurements. According to one aspect of the present disclosure, an array of electrochemical electrodes can be used to spatially map the analyte concentration measured via electronics in a CMOS integrated circuit. Such electrochemical mapping can be applied for cellular analysis of cells cultured directly on top of the electrode array.

[0146] In this example, cyclic voltammetry is performed on the surface of ferricyanide / ferrocyanide [Fe(CN)6] to demonstrate the capability of electrochemical mapping using an array of electrodes, measured using a CMOS integrated circuit. 3- / [Fe(CN)6] 4- This is done using the general redox couple:

[0147] [ka]

[0148] Figure 20a is a schematic diagram showing the cyclic voltammetry setup using a CMOS integrated transimpedance amplifier to measure each Pt electrode current and an external transimpedance amplifier to measure the current through the Ag / AgCl pseudo-electrode. In the experiment in Figure 20a, cyclic voltage ramps were applied at a scan rate of 35 mV / s using 1.5 M KCl + 5 mM K3[Fe(CN)6]. The sum of the 13 x 13 electrode currents was used for the measurement, which is equal to that of the reference electrode. Figure 20b shows two spatial maps of the maximum range of the electrode current (|I e,max -I e,min |, top left), which is related to the diffusion of ferricyanide (the 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), which is related to the diffusion of ferrocyanide (product). An example individual electrode recording is shown on the right in Figure 20b with these parameters defined. Non-radial ferrocyanide diffusion is due to convective effects in the solution.

[0149] In this experiment, a 13 × 13 = 169 subset of the 64 × 64 electrode array was connected to an equal number of individual transimpedance amplifiers and subjected to periodic linear voltage ramps, as shown in the schematic diagram in Figure 20a. Figure 20b shows a spatial map of current density indicating increased cathodic and anodic current magnitudes at the edges of the electrodes, likely due to increased radial diffusion / mass transport at the edges compared to planar diffusion at the center electrode. Similarly, product generation limits the current density, visualized by the peak current range versus voltage maximum / minimum current range, as shown in data plot 2001 in 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 power of current-based electrochemical mapping.

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

[0151] [ka]

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

[0153] In this example, the rest potential of the electrode array was measured. In particular, ferrocyanide generation and transport across the CMOS electrodes was mapped using the open circuit potential.

[0154] A cyclic potential is applied to a 13x13 electrode group (9 electrodes are excluded within the 13x13 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 relative increase and decrease in ferricyanide / ferrocyanide concentration. Figure 21b is a heat map showing one cycle, where the overall amplitude (max-min) of the open circuit potential plotted across the array indicates diffusion / mass transport trending toward the upper left corner. Figure 21c shows a heat map and data plot showing the time of minimum open circuit potential plotted against distance from the center of the 13x13 electrode, indicating the transient aspects of diffusion / mass transport.

[0155] In summary, in cyclic voltammetry, measuring an open circuit around the electrodes indicates a flow of ferrocyanide towards the upper right corner of the device.

[0156] Example 14: Cellular electrochemical oxygen mapping This example illustrates how electrochemical mapping can be applied to cell analysis. For example, a platinum-based Clark electrode can be measured by applying a pulsed voltage or a sequence of voltage pulses, which sequentially oxidizes and reduces platinum. Because platinum oxide blocks oxygen reduction, the current drops to zero after the oxide is formed. Then, when the oxide is reduced, the platinum electrode passes a negative current due to the presence of oxygen.

[0157] [ka]

[0158] The local oxygen concentration is then depleted and the electrode waits for further diffusion of additional oxygen to the electrode to allow current to flow. The rate of the reaction is therefore 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 an electrode array, measurements were performed using a salt solution (phosphate buffered saline) exposed to ambient air, followed by a nitrogen gas purge to reduce the oxygen concentration. Figure 22a shows a voltage pulse sequence 2202 applied to the stimulating electrodes in the electrode array, and a series of data plots 2204 of measurements using a CMOS electrode array in ambient air, with a partial nitrogen purge and partial recovery (N purge stopped). Data plots 2204 show that the electrode current reflects the oxygen concentration.

[0160] Current before and after purging I el A significant decrease is observed when comparing the cross-electrode maximum current I across the electrode array area. maxCross-electrode impedance heatmap 2206 using the el Heatmaps of changes in oxygen concentration 2208 and 2210 are shown. The same style of oxygen measurements using HEK293 cells show a decrease in oxygen concentration where the cells are located, as confirmed by the impedance maps.

[0161] Because cells consume oxygen as part of their aerobic metabolism, the oxygen concentration around cells is less than in locations without cells. Indeed, mapping electrode currents across the array shows that cell locations, as imaged using cross-electrode impedance maps, have smaller current magnitudes than locations without cells. 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: Effect of Platinum Black and Frequency on Cell Barrier Sensitivity In this example, platinum black (PtB) is used to measure the electrode impedance Z e 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 that PtB reduces the Z of bare electrodes. te Figure 26c shows cell barrier maps relative to the reference at various frequencies. While lower frequency measurements show more spread and do not capture the cell sheet edges, the 1.8 kHz measurement showed the highest contrast for cell connectivity measurements when compared to density maps extracted from imaging. Figure 26d shows cell density and connectivity maps extracted from the nuclei of the fluorescence images. Figure 26e shows Z measurements with and without a reference at 1.8 kHz. te The comparison between the two is shown below. Without the reference, the Z is slightly smaller. teis measured, but the relationship between areas with and without cells (two clusters) is direct. Measurement without a reference is preferred, and Z e This is because the contribution of the α-cells to the substrate can be easily subtracted from the cell-substrate attachment measurement. te and Z s For this comparison, Z s is downsampled via bilinear interpolation, and Z te The cell barrier exhibits a strong dependence on cell density due to the measurement being adapted to cell-cell connectivity. s There is also a small correlation between Z and cell density, as can be seen from the cell circuit model (Figure 4B). s High, Z s Z used in the calculation s < <Z te This is because it has an effect on the assumption that it does not persist at all.

[0163] Having thus described several aspects of at least one embodiment of the present invention and examples thereof, it should be understood that various changes, modifications, and improvements will readily occur to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the present invention. Furthermore, while advantages of the present invention have been illustrated, it should be understood that not all embodiments of the technology described herein include all of the 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 by way of example only.

[0164] Various aspects of the present invention may be used alone, in combination, or in various configurations not specifically discussed in the embodiments described above, and therefore are not limited in application to the details and arrangements of components set forth in the above description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

[0165] The present invention may also be embodied as a method, examples of which are provided. The operations performed as part of the method may be ordered in any suitable manner. Thus, embodiments may be constructed in which operations are performed in an order different from that described, which may include performing some operations simultaneously even though they are shown as sequential operations in the example embodiment.

[0166] The use of ordinal terminology in the claims, e.g., "first," "second," "third," etc., to modify claim elements does not in itself imply a priority, precedence, or ordering of one claim element relative to others, or a chronological order of acts in a method, but rather distinguishes one claim element having a particular name from other elements having the same name (except for the use of ordinal terminology) and is used merely as a label to distinguish the claim elements.

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

Claims

1. 1. An apparatus for mapping one or more cells, comprising: a semiconductor substrate comprising: a) a plurality of electrodes exposed on a surface of the semiconductor substrate; and b) active circuitry connected to the plurality of electrodes; the active circuitry is configured to: c) measure a first set of cross-electrode currents between a first group of electrodes of the plurality of electrodes and some or all of the remaining electrodes; and d) measure a second set of cross-electrode currents between a second group of electrodes of the plurality of electrodes and some or all of the remaining electrodes; adjustably controlling the pitch of the plurality of electrodes by applying stimulation signals to electrodes in a first electrode group and a second electrode group, each of which can independently include one or more electrodes; and - The device further comprising one or more processors configured to receive the measured cross-electrode currents from the active circuitry and generate a map of the one or more cells based on the first and second sets of cross-electrode currents.

2. The apparatus of claim 1 , wherein the active circuitry is further configured to apply a reference voltage to the remaining electrodes whose cross-electrode currents are being measured.

3. The apparatus of claim 2 , wherein the stimulation signal has a frequency of less than 10 kHz.

4. The device according to any one of claims 1 to 3, wherein the pitch of the plurality of electrodes is adjusted to be less than 30 μm.

5. The device of any one of claims 1 to 4, wherein the semiconductor substrate comprises silicon.

6. the semiconductor substrate includes a silicon substrate; An apparatus according to any one of claims 1 to 5, wherein the active circuitry comprises complementary metal oxide semiconductor (CMOS) devices in a silicon substrate.

7. The apparatus of any one of claims 1 to 6, wherein the plurality of electrodes comprises a plurality of pads disposed on an insulating surface of a semiconductor substrate.

8. the active circuitry includes a plurality of recording circuits; An apparatus according to any one of claims 1 to 7, wherein each recording circuit is configured to measure the current at an electrode of the plurality of electrodes.

9. 9. The apparatus of claim 8, wherein the plurality of recording circuits comprises at least eight recording circuits.

10. 9. The apparatus of claim 8, wherein each recording circuit includes a transimpedance amplifier (TIA).

11. the TIA includes an impedance element having a resistance of at least 10 MΩ, at least 100 MΩ, or between 10 MΩ and 1 GΩ; The apparatus of claim 10 , wherein the output voltage of the TIA is proportional to the voltage across an impedance element.

12. The apparatus of claim 11 , wherein the impedance element comprises a switching capacitor.

13. one or more cells are disposed in a first well of a multi-well plate; the plurality of electrodes is a first electrode array exposed to the first well; The device of any one of claims 1 to 12, further comprising a second electrode array exposed on the surface of the semiconductor substrate and exposed in a second well of the multiwell plate.

14. 14. The device of claim 13, wherein the multiwell plate comprises at least 24, at least 96, or at least 384 wells.

15. An apparatus described in any one of claims 1 to 14, wherein the pitch is adjustably controlled so that two or more electrodes of a first electrode group are configured to contact one of one or more cells.

16. The apparatus of claim 7 , wherein the plurality of pads comprises Au.

17. The apparatus of claim 7 , wherein the plurality of pads comprises Pt.

18. 1. 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 of the electrode array having an electrode location on the surface region; - selecting the pitch of the electrode array from among a plurality of pitch values ​​by defining a first electrode group comprising one or more electrodes of the electrode array; For each electrode of the first group of electrodes of the electrode array: - applying a stimulation signal to the electrodes; - measuring a set of cross-electrode currents between one or more electrodes of the first electrode group and some or all of the remaining electrodes in the electrode array; generating a representative value associated with an electrode location of one or more electrodes of the first electrode group based on a set of cross-electrode currents; - generating a map of the representative values ​​over the surface area based on the generated representative values ​​and respective associated electrode locations of one or more electrodes of the first electrode group.

19. 20. The method of claim 18, wherein generating the representative value comprises selecting a maximum current value of the set of cross-electrode currents as the representative value.

20. 20. A method according to claim 18 or 19, wherein the step of generating a representative value comprises selecting the maximum current value of the set of cross-electrode currents as the representative value.

21. A method described in any one of claims 18 to 20, wherein the first electrode group includes all electrodes in the electrode array.

22. The method of any one of claims 18 to 21, wherein the map has a spatial resolution of 20 μm or less.

23. The method of any one of claims 18 to 22, further comprising identifying clusters of cells adjacent to the semiconductor substrate at single cell resolution based on the generated map of representative values.

24. the generated map is a first map generated at a first time and includes a plurality of pixels; The method includes generating a second map of representative values ​​over the surface region at a second time subsequent to the first time, the second map including a plurality of pixels; determining a first count of pixels in the first map having a representative value within a predetermined range; determining a second count of pixels in the second map having representative values ​​within the predetermined range; and determining a cell adhesion property based on a comparison of the first count and the second count.

25. The map includes a plurality of pixels, A method according to any one of claims 18 to 24, wherein each pixel is associated with a representative value.

26. The first electrode group includes a first electrode having a first electrode location and a second electrode having a second electrode location; the first electrode and the second electrode are adjacent to each other on a surface region; the map includes first and second pixels corresponding to the first and second electrode locations, respectively; 26. The method of claim 25, wherein generating the map includes determining an expanded representative value associated with a third pixel that is between the first pixel and the second pixel.

27. The step of determining the expanded representative value comprises: The cross-electrode current I between the first and second electrodes when a stimulation signal is applied to the second electrode １２ the first current I １ and the second current I ２ calculating a scaled electrode current by dividing by the product of I １ is the sum of the cross-electrode currents measured at all of the remaining electrodes when a stimulation signal is applied to the first electrode, and I ２ 27. The method of claim 26, wherein {overscore (x)} is the sum of the cross-electrode currents measured at all of the remaining electrodes when a stimulation signal is applied to the second electrode.

28. 28. The method of claim 27, wherein the number of pixels in the map is greater than the number of electrodes in the electrode array.

29. A method according to any one of claims 18 to 28, wherein the electrode locations in the electrode array are arranged in a plurality of rows and a plurality of columns.

30. 30. The method of claim 29, wherein the electrode array has M rows and N columns and the map has at least 3M by 3N pixels.

31. 1. A system for mapping one or more cells, comprising: a plurality of electrodes exposed on a surface region of a semiconductor substrate; a circuit disposed in the semiconductor substrate and controllable to apply a stimulation signal and / or measure current at one or more of the plurality of electrodes; at least one non-transitory computer-readable medium having executable instructions stored thereon; at least one processor programmed with executable instructions, selecting a pitch for the electrode array from among a plurality of pitch values ​​by defining a first electrode group including one or more electrodes of the electrode array; For each electrode in the first electrode group: - controlling circuitry to apply stimulation signals to the electrodes; controlling circuitry to measure a set of cross-electrode currents between said electrode and some or all of the remaining electrodes in the plurality of electrodes; generating a representative value associated with an electrode location of said electrode based on a set of cross-electrode currents; - generating a map of the representative values ​​over the surface area based on the generated representative values ​​and associated electrode locations of each of the plurality of electrodes.

32. 32. The method of claim 31 , wherein the act of generating the representative value comprises selecting a maximum current value of the set of cross-electrode currents as the representative value.

33. The operation of generating the representative value includes selecting a maximum current value of the set of cross-electrode currents; 33. The method of claim 31 or 32, comprising: calculating the impedance based on the selected maximum current value as the representative value.

34. the generated map is a first map generated at a first time and includes a plurality of pixels; The method includes the operations of generating a second map of representative values ​​over the surface region at a second time subsequent to the first time, the second map including a plurality of pixels; determining a first count of pixels in the first map having a representative value within a predetermined range; determining a second count of pixels in the second map having representative values ​​within the predetermined range; and determining a cell adhesion characteristic based on a comparison of the first count and the second count.

35. The map includes a plurality of pixels, Each pixel is associated with a representative value, the first electrode group includes a first electrode having a first electrode location and a second electrode having a second electrode location, the first electrode and the second electrode being adjacent to each other on a surface area; the map includes first and second pixels corresponding to the first and second electrode locations, respectively; A method according to any one of claims 31 to 34, wherein the act of generating the map comprises determining an upscaled representative value associated with a third pixel that is between the first pixel and the second pixel.

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