Cell analysis using chemfet sensor array-based systems

Abstract

Various cell analysis systems taught by the present invention can measure electrical and metabolic activity of a single living cell for between about 10 cells to about 500,000 cells in a single analysis using subcellular addressable capability and simultaneous data acquisition. Individual sensor array devices taught by the present invention can have a sensor array with ChemFET sensors between 20 million and 6 billion built as a massively parallel array and can provide simultaneous measurement of cells at a data acquisition rate in the range of kilohertz (kHz). Various ChemFET sensor arrays taught in the present invention can detect chemical analytes and detect changes in cell membrane potential, and various cell analysis systems taught in the present invention also provide controlled chemical and electrical interrogation of cells. The frame rate may be improved by selecting a smaller subset of pixels to monitor (i.e., by windowing down the region of the sensor array device over which data is collected).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 730,960, filed on September 13, 2018, and U.S. Provisional Application No. 62 / 848,842, filed on May 16, 2019. Both applications named in this section are incorporated herein by reference in their entirety. Background Art

[0003] Electrophysiological and metabolic phenotyping of cell behavior remains an important avenue of research in modern cell biology, and has significant unmet needs for researchers in areas such as engineered tissues, primary cell types, and oncology discovery. Currently, successful measurement of cellular excitability is hampered by cumbersome patch clamp and microelectrode voltammetry, while interrogation of metabolic function remains hampered by technical limitations of large populations and biochemical workflows that cannot be effectively used to obtain key measures of single cell behavior.

[0004] It is becoming increasingly clear that while it is not possible or feasible to measure all possible interactions between 20,000 genes and 200,000 proteins within a cell, studies of model systems at the single cell level will provide a deeper understanding of normal cellular physiology and alterations during pathological conditions such as cancer, diabetes, and neurodegenerative diseases. Recent applications that highlight the advantages of studying single cells over cell populations include, for example, studies of transcriptomics and aging, studies of signaling pathways within cells, and bioenergetics measurements of metabolism.

[0005] Therefore, there is a need in the art for systems and methods that can provide the end user with tools for interrogating individual cells in a population and that will allow for discrete measurements of temporal responses at the cellular level in a high-throughput multiplexed manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] A better understanding of the features and advantages of the present teachings will be obtained by reference to the following detailed description which sets forth illustrative embodiments and by reference to the accompanying drawings, in which:

[0007] Figure 1 is a block diagram depicting a chemical field effect transistor (ChemFET) based cell analysis system according to various embodiments of the systems and methods taught herein.

[0008] Figure 2A is a schematic depiction of cells positioned on a cell analysis system according to the teachings of the present invention. Figure 2B is a schematic cross-sectional view of a cell positioned on a sensor device according to the teachings of the present invention.

[0009] Figure 3 is a table showing the ChemFET system properties for cell analysis across a range of cell sizes.

[0010] FIG. 4A to FIG. 4C are cross-sectional illustrations of sensors and associated structures of various designs in accordance with the teachings of the present invention.

[0011] Figure 4D is a pair of holes (such as Figure 4A and Figure 4B shown) and without holes (such as Figure 4C Comparison of U-2OS cell responses on an array device of (shown), where cells were interrogated under conditions used to depolarize the membrane potential and then return to the resting potential.

[0012] Figure 5A is a block diagram depicting various embodiments of a sensor array, array controller, and computer interface for a ChemFET-based cell analysis system in accordance with various embodiments of the systems and methods taught herein.

[0013] Figure 5B is a block diagram depicting various embodiments of a sensor array and array controller for a ChemFET-based cell analysis system in accordance with various embodiments of the systems and methods taught herein.

[0014] Fig. 6A and Figure 6B is a flow chart depicting a workflow associated with cell analysis according to various embodiments of systems and methods of the present teachings.

[0015] Fig. 7A and Figure 7B is the time response curve of a cell having a membrane at resting potential and then interrogated under conditions used to depolarize the membrane potential and return it to resting potential.

[0016] Fig. 8A is Figure 8B Electron microscopy images of cells generated from the sensor array at the indicated time points. Figure 8B yes Fig. 8A A line drawing rendering of an image.

[0017] Fig.9A U-2OS cells (C 1 and C 2 ) compared with U-2OS cells transfected with voltage-gated calcium channel subunits (C 3 and C 4 ) in which each cell type was treated with the calcium channel blocker verapamil.

[0018] Fig. 9B is with Fig.9A The image was taken at the time (1.7 seconds) corresponding to the frame 25 indicated in Fig.9A Composite electron micrographs comparing two regions of interest, where the response of U-2OS cells (left) is compared to that of transfected U-2OS cells (right).

[0019] Fig. 9C yes Fig. 9B Composite electron microscopy image in which the square of the indicated area within the cell has been removed to provide a clear image of the cell.

[0020] Fig. 10A is an electron microscopic image of rat neonatal cells subjected to a 4.5 mM KCl pulse, some of which (C 1 ) relative to other exemplary cells in the population that remain in a quiescent state (C 2 ) exhibit a depolarizing response.

[0021] Fig. 10B Comparison of the time response curves of rat neonatal cells subjected to a 4.5 mM KCl pulse, where the experiments were performed on two different apparatuses with different data acquisition rates.

[0022] Fig.11 are electron microscopic images of HEPG2 cells subjected to the mitochondrial toxin carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), indicating some exemplary cells associated with cellular efflux.

[0023] Fig. 12A Shown are the averaged background-subtracted time-dose-response curves of U-2OS cells treated with FCCP.

[0024] Fig. 12B Shown are the averaged background-subtracted time-dose-response curves of U-2OS cells pretreated with nutrient solution containing oligomycin prior to treatment with FCCP.

[0025] FIG. 13A to FIG. 13C are the averaged background-subtracted time-dose-response curves of the mixtures of different cell types subjected to FCCP.

[0026] Fig.14A Shown FIG. 13A to FIG. 13C Time dose-response curves for selected cells in the mixture of cells presented.

[0027] Fig. 14B It is taken at the time corresponding to frame 40 (2.7 seconds) Fig.14A Electron microscope image of the experiment.

[0028] Fig. 14C yes Fig. 14B Electron microscope image of Fig. 13B The squares within the cells have been removed to provide a clear image of the cells.

[0029] Fig.15A and Fig. 15B U-2OS cells transfected with each voltage-gated calcium channel subunit at consecutive time points (C 3 and C 4 ) of the electron microscope image. Fig. 15C It is for Fig.15A and Fig. 15B The time response curve of the time point in the interval containing the time points.

[0030] Fig.16 Is displayed for FIG. 7A to FIG. 15C Table of ChemFET system properties for cell lines to be analyzed.

[0031] Fig.17 is a graph of the pH response of the device. DETAILED DESCRIPTION

[0032] The systems and methods taught by the present invention relate to a cell analysis system based on chemical field effect transistor (ChemFET) sensor array technology. Each cell analysis system taught by the present invention can measure the electrical and metabolic activities of a single living cell using subcellular addressability and simultaneous data acquisition for between about 10 cells and about 500,000 cells in a single analysis. Each sensor array device taught by the present invention can have a sensor array with between 20 million and 660 million ChemFET sensors built into a massively parallel array, wherein the spacing of the sensors of the sensor array can be between about 3.36 μm and about 850 nm, respectively. Further, each cell analysis system taught by the present invention can provide simultaneous measurements of cells at data acquisition rates in the kilohertz (kHz) range. Further, the fully automated fluidic system integrated into each cell analysis system taught by the present invention allows for strictly controlled applications such as chemical agents in various studies for indicating measurements of cellular responses to various types of chemical interrogations. Because each ChemFET sensor array taught by the present invention can detect chemical analytes as well as detect changes in cell membrane potential, controlled electrical stimulation can also be used to interrogate cells in each cell analysis system taught by the present invention.

[0033] Chemical Field Effect Transistor (ChemFET) Arrays for Cellular Analysis

[0034] Figure 1 1 is a block diagram of exemplary components of a cell analysis system 100 according to the teachings of the present invention. Figure 1As depicted in , cell analysis system 100 can include various fluidic systems as well as array controller 50, user interface 60, and sensor array device assembly 115. As will be described in more detail herein, various cell analyses can be performed using sensor array device 110 of sensor array device assembly 115.

[0035] Figure 2A A graphical depiction of a cell analysis system 100A is generally shown. Figure 2A As depicted in FIG. 1 , a cell 5 having a nucleus 2 and a cell membrane 4 is positioned over a plurality of microwells in a subset of microwells, thereby defining a contact area or footprint occupied by the cell 5 over a corresponding subset of sensors. As described herein, “contact area” and “footprint” may be used interchangeably. Many features of the cell analysis system 100A are similar to those of FIG. Figure 1 The schematic depiction of the cell analysis system 100 has the same features as described. Figure 2A The cell analysis system 100A may include a reagent fluid system 10 and a washing solution fluid system 20. The reagent fluid system 10 may include a plurality of reagent containers, such as Figure 2A Each reagent container can be connected to a reagent fluid line (such as Figure 2A The flow rate of each reagent fluid line from the cell analysis system taught by the present invention can be controlled by a valve (such as Figure 2A The washing solution fluid system 20 may include a washing solution container 21, which may contain a washing solution of known electrolyte composition, a washing solution fluid line 23, a washing solution fluid line valve 25, and a reference electrode 27 in the washing solution fluid line 23. As will be described in more detail herein, the reference electrode 27 may provide a stable reference voltage 127 for the sensors in the sensor array device. Thus, Figure 2A The sensor array device 110 can be in fluid communication with the reagent fluid system 10 and the wash solution fluid system 20. Figure 2A , but additional electrodes that can be in communication with the sensor array device can be used to provide electrical current to cells on the sensor array (e.g. Figure 2A 5) provides electrical stimulation to the cells.

[0036] The sensor array device 110 may include a sensor array or pixel array 120. As described herein, the terms "sensor" and "pixel" and the terms "device" and "chip" and derivatives of these terms may be used interchangeably. In addition, "sensor array" and "ChemFET sensor array" and derivatives thereof may be used interchangeably. Although in Figure 2AThe sensor array device 110 may include a microwell array 122, which may be arranged in a variety of array geometries, such as a hexagonal closest packing geometry. Figure 2A The depiction shown in the figure depicts each micropore being cooperatively engaged with each sensor or pixel in the sensor array 120, so that each micropore 122A1 to micropore 122A6 is cooperatively engaged with the corresponding sensor 120A1 to sensor 120A6. However, for various embodiments of the sensor array device taught by the present invention, there may be more than one pixel per hole. As will be described in more detail herein, various types of sensor array devices taught by the present invention can be manufactured with defined but different micropore depths. Still other types of sensor array devices taught by the present invention may not have a micropore structure formed on the sensor array. Each sensor of the sensor array 120 may have a sensing surface that is fluidly connected to the fluid in the micropore array 122. For various embodiments of the cell analysis system 100 taught by the present invention, each sensor of the sensor array 120 may be a chemical field effect transistor (ChemFET), wherein each sensor in the sensor array 120 includes at least one chemically sensitive field effect transistor. In accordance with the present teachings, sensor array 120 may include ChemFETs that may be fabricated with sensing surfaces modified to be selective for analysis of target chemical species of interest in cell biology, such as glucose, sucrose, lactate, and urea. By way of another non-limiting example, ion sensitive field effect transistors (ISFETs) may have sensing surfaces modified to be selective for various ions of particular interest in various cell metabolism studies, such as hydrogen, potassium, calcium, and chloride.

[0037] In this regard, the inventors have recognized that the various embodiments of the cell analysis system taught by the present invention can be used to monitor changes in the cell electrophysiology and metabolism of cells subjected to any of a variety of conditions or stimuli. Moreover, the inventors have recognized that any change in the state of the cell that can cause a change in the potential of the sensing surface of the ChemFET sensor can be monitored by the sensors of the various embodiments of the ChemFET sensor array taught by the present invention. For example, the inventors have recognized that the cell capacitance is coupled to the sensing surface of the sensor so that when the potential across the cell membrane changes in response to a chemical or electrical stimulus, the changed potential across the cell membrane can be detected by the sensors of the various embodiments of the ChemFET sensor array taught by the present invention. Additionally, any change in, for example, cell metabolism that can cause a change in the potential of the sensing surface of the ChemFET sensor can be detected by the sensors of the various embodiments of the ChemFET sensor array taught by the present invention. As will be described in more detail herein, such changes can be detected locally in relation to the contact area or occupied area of ​​the cell anchored on the sensor array surface, or can be detected in an area that is not related to the cell occupied area (e.g., a cell that may flow out of the cell in response to a condition or stimulus).

[0038] The data collected in the experiment of monitoring the cellular response to various stimuli with various embodiments of the ChemFET sensor array device taught by the present invention can be presented to the end user in a variety of formats. In one format, the time response is presented as a detector count that can be easily associated with the millivolt (mV) change of the sensing surface potential over time. In another format, the spatial visualization of the cell can be presented as an electron microscope image for any selected time in the selected time within the time course of the selected application. The inventors have recognized that since electron microscope imaging is based on various responses that can be triggered for living cells, it can be used as a general tool, for example, for visualizing cells on the sensor array. For example, by viewing the electron microscope image of the cell anchored on the sensor array, the end user can select the area of ​​interest as part of the application configuration before running the experiment. As will be described in more detail herein, the data rate of the experiment is increased by downward windowing in the selected area of ​​the sensor array device. According to the teachings of the present invention, a considerable pixel coverage range on the occupied area of ​​the cell plus a high data rate can provide subcellular monitoring of the action potential of each excitable cell that may require a data rate in the sub-millisecond range, for example.

[0039] exist Figure 2B, a partial cross-sectional view of a sensor array 120 having a first sensor 120-1 and a second sensor 120-2 is depicted. In various embodiments of the sensor array device of the present teachings, the sensor array 120 may include a floating gate upper portion 130 coupled to a sensor floating gate structure 140. Alternatively, for various embodiments of the sensor array device of the present teachings, the sensor array 120 may include a sensor floating gate structure 140. As will be described in more detail herein, the floating gate upper portion 130 may include a top metal layer formed in a dielectric 135, a sensor plate 132, and a metal via 134.

[0040] The sensor floating gate structure 140 may have a metal layer 136 coupled to the sensor plate 132 through metal vias 134. The metal layer 136 is the uppermost floating gate conductor in the sensor floating gate structure 140. In the illustrated example, the sensor floating gate structure 140 includes multiple layers of conductive materials within a layer of dielectric material 150. The sensors 120-1 and 120-2 may include conductive terminals including source / drain regions 142 and source / drain regions 144 within a semiconductor substrate 160. The source / drain regions 142 and source / drain regions 144 include doped semiconductor materials having a different type of conductivity than the conductivity type of the substrate 160. For example, the source / drain regions 142 and source / drain regions 144 may include doped P-type semiconductor materials, and the substrate 160 may include doped N-type semiconductor materials. A channel region 152 separates the source / drain regions 142 and source / drain regions 144. The floating gate structure 140 is located above the channel region 146 and is separated from the substrate 160 by a gate dielectric 152. The gate dielectric 152 may be, for example, silicon dioxide. Alternatively, other suitable dielectrics may be used for the gate dielectric 152, such as materials with higher dielectric constants, such as silicon carbide (SiC), silicon nitride (Si 3 N 4 ), silicon oxynitride (Si 2 N 2 O), aluminum nitride (AlN), hafnium dioxide (HfO 2 ), tin oxide (SnO 2 ), cesium oxide (CeO 2 ), titanium oxide (TiO 2 ), tungsten oxide (WO 3 ), aluminum oxide (Al 2 O 3 ), lanthanum oxide (La 2 O 3 ), gadolinium oxide (Gd 2 O 3 ) and any combination thereof.

[0041] As will be described in greater detail herein, the sensing surface 126S of the sensor plate 132 can serve as a sensor surface for monitoring changes in cell electrophysiology and metabolism of cells subjected to any of a variety of conditions or stimuli, for example. Figure 2B The cells 5 shown in the figure are depicted as being positioned on the sensor plates 132 of the sensors 120-1 and 120-2. The cells 5 are depicted as being anchored to the sensor array 120 by the surface coating 124. The surface coating 124 can be any cell-compatible material (such as various biopolymer materials, including poly-D-lysine, laminin, fibronectin, collagen, and combinations thereof) and various extracellular matrix (ECM) preparations. The end user can use the cell analysis system taught by the present invention to run an application, which can controllably make various reagents and solutions such as Figure 2B The flow indicated by the arrows at the top of FIG. 1 passes over the surface of sensor array 120 .

[0042] Sensors 120-1 and 120-2 respond to changes in the surface potential of ion layer 128 proximate sensing surface 126S, which may result in changes in the voltage on floating gate 140. Thus, as previously described herein with respect to Figure 2A The described applied reference voltage ensures that the voltage of the floating gate exceeds the threshold voltage, provided that a small change in the floating gate voltage can cause current to flow through the channel region 146, thereby generating an output signal of the sensors 120-1 and 120-2. In this regard, changes in the surface potential of the ion layer 128 can be measured by measuring the current in the channel region 146 between, for example, the source region 142 and the drain region 144. Therefore, the sensors 120-1 and 120-2 can be used directly to provide a current-based output signal on an array line connected to the source region 142 or the drain region 144, or indirectly used with additional circuitry to provide a voltage-based output signal.

[0043] As described herein, any change in the state of the cell 5 that can change the surface potential in the ion layer 128 can be monitored by various embodiments of the ChemFET sensor array device taught by the present invention. With respect to the output signal, any cell activity that can increase the surface potential will produce an output signal of positive amplitude of the ChemFET sensor, while any cell activity that can reduce the surface potential will produce an output signal of negative amplitude of the ChemFET sensor. In this regard, any change in the state of the cell that can change the surface potential of the ChemFET sensor can produce a measurable output signal. For example, any metabolic activity that can increase the ion concentration of the cationic species for which the ISFET sensor is selective will cause an increase in the surface potential. The result will be an output signal of positive amplitude of the ISFET sensor. Conversely, any metabolic activity that can reduce the ion concentration of the cationic species for which the ISFET sensor is selective will cause a decrease in the surface potential. The result will be an output signal of negative amplitude of the ISFET sensor. In another example, the surface potential can be changed by capacitively coupling the cell 5 to the sensor array 120 so that when the potential across the cell membrane 4 changes in response to a chemical or electrical stimulus, the changing potential across the cell membrane can be detected by the ChemFET sensors 120-1 and 120-2.

[0044] Table I summarizes the properties of exemplary ChemFET sensors taught by the present invention.

[0045]

[0046] Table I: Exemplary sensor array devices

[0047] Various embodiments of the sensor array devices taught by the present invention may have between about 20M and about 660M pixels with center-to-center spacing between each sensor; or have a spacing between about 850nm and about 3.36μm. With respect to data collection, the collection of sensor output signals from all sensors in the array constitutes a data frame. For various sensor array devices taught by the present invention having between about 20 million pixels and about 660 million pixels, a frame is a data file of considerable size collected as a frame in Hertz (Hz) per second. Further, there is an inverse relationship between the selected area of ​​interest representing the number of pixels and the rate at which data can be collected, so that the frame rate can be increased by selecting a smaller subset of pixels to monitor (i.e., by windowing down the area of ​​the sensor array device over which data is collected). The impact of windowing down is demonstrated in Table I by comparing the values ​​entered in the second to last column (the values ​​are the maximum frame rate for collecting data from the entire device) with the values ​​entered in the last column (the values ​​are the maximum frame rate for collecting data from a single row of devices). Therefore, the frame rate is significantly increased by windowing down to collect data from a single row.

[0048] Additionally, as provided in Table 1, the only difference between device A and device B is the total number of sensors per device, where device A has twice as many sensors per device B. As shown in Table 1, the frame rate of device B is half the frame rate of device A, which is consistent with the inverse relationship between the number of pixels and the rate at which data can be collected. Therefore, a device can be selected that has a desirable frame rate that matches the application.

[0049] Thus, for various embodiments of the cell analysis system taught by the present invention, an end user can select a sensor array device from various sensor array devices having different properties that can match the cell analysis of interest. For example, a sensor array device used to detect electrophysiological activity of cells may have different properties than a sensor array device used to detect a specific analyte for metabolic research.

[0050] Sensor array device properties that may be varied to provide various sensor array devices in accordance with the present teachings include pixel size and the rate at which data may be collected from the sensor array device. Figure 3 An overview of the five classes of cells categorized by size is provided for four exemplary sensor array devices of varying sensor (pixel) size, as given in Table 1 for Devices B through E. The five classes of cells are identified descriptively and by mean diameter and mean occupied area.

[0051] By inspection Figure 3, for an average diameter of 5 μm and an average area of ​​20 μm anchored on the sensor array surface 2 For very small cells, the minimum contact area or footprint corresponds to about 1 row and about 2 pixels for the Chip 1 device, which increases to 6 rows and 32 pixels for the Chip 4 device. Similarly, for cells with an average diameter of 10 μm and an average area of ​​78 μm anchored on the sensor array surface, 2 For small cells with an average diameter of 25 μm and an average area of ​​491 μm, the minimum contact area or occupied area corresponds to about 3 rows and about 8 pixels for the chip 1 device, and increases to 12 rows and 126 pixels for the chip 4 device. 2 For the medium cell, the minimum contact area or occupied area corresponds to about 7 rows and about 50 pixels for the chip 1 device, and increases to 29 rows and 792 pixels for the chip 4 device. For chip 1, the average diameter of the cells anchored on the sensor array surface is 50 μm and the average area is 1,964 μm 2 The large cells of can have a minimum contact area or footprint corresponding to about 15 rows and about 201 pixels, which increases to 59 rows and 3,168 pixels for the chip 4 device. Finally, for the average diameter of 100 μm and the average area of ​​7,854 μm anchored on the sensor array surface 2 For the super-large cells, the minimum contact area or occupied area corresponds to about 30 lines and about 803 pixels for the chip 1 device, and increases to 118 lines and 12,668 pixels for the chip 4 device. Figure 3 , the trend is that pixel coverage increases with increasing cell size and decreasing pixel size.

[0052] From a pixel perspective, the Pixel Coverage Percent column is the percentage of the area of ​​the cell that is covered by a single pixel. For very small cells anchored on the sensor array surface, a single pixel for a Chip 1 device corresponds to 50% coverage, while a single pixel for a Chip 4 device corresponds to 3% coverage. Similarly, for small cells anchored on the sensor array surface, a single pixel corresponds to 12% coverage for a Chip 1 device, while a single pixel corresponds to 0.8% coverage for a Chip 4 device. For medium cells anchored on the sensor array surface, a single pixel corresponds to 2% coverage for a Chip 1 device, while a single pixel corresponds to 0.1% coverage for a Chip 4 device. Large cells anchored on the sensor array surface may have a single pixel corresponding to 0.5% coverage for a Chip 1 device, while a single pixel corresponds to 0.03% coverage for a Chip 4 device. Finally, for extra large cells anchored on the sensor array surface, a single pixel corresponds to 0.1% coverage for a Chip 1 device, while a single pixel corresponds to 0.008% coverage for a Chip 4 device. Based on the coverage of Figure 3 From inspection, the trend is that the percentage of cell coverage per pixel decreases as cell size increases and pixel size decreases.

[0053] Taking into account Figure 3 As presented in the table, the pixel coverage range of the exemplary sensor array device taught by the present invention can be selected for each average cell diameter. For example, for cells of about 5 μm to about 100 μm, the sensor array device can be selected to provide a coverage range of about 8 pixels within 3 rows of pixels to 12,668 pixels within about 118 rows of pixels for the corresponding occupied area of ​​the cells anchored on the sensor array surface. Within the range of cell sizes, the pixel size can vary so that each pixel of the selected sensor array device can cover about 12% of the cell to about 0.008% of the cell. Based on Figure 3 From the data presented in, it is clear that for any cell size, an exemplary sensor array device can be selected that can provide a substantial number of sensors relative to the contact area that a cell can occupy on the sensor array device. The spatial resolution that can be provided by each sensor array device taught by the present invention can allow subcellular discrimination of signals; thus providing subcellular analysis.

[0054] With respect to data collection, for each cell analysis system taught by the present invention, the collection of sensor output signals from all sensors in the array constitutes a data frame. Considering that each sensor array device taught by the present invention can have about 20 million pixels to about 660 million pixels, the data frame from each sensor array device taught by the present invention is a data file of considerable size. Additionally, each cell analysis system taught by the present invention includes a control circuit system coupled to a sensor array device configured to generate a considerable number of data frames from the sensor array device per second. In addition, there is an inverse relationship between the selected area of ​​interest and the rate at which data can be collected, so that the frame rate can be increased by selecting a smaller subset of pixels to monitor (i.e., by windowing down the area of ​​the sensor array device over which data is collected).

[0055] For example, referring to Table I, a sensor array device with 40 million pixels can collect data at a frame rate of about 120 frames per second (fps). Then, if a region of interest of 20 million pixels is selected, data can be collected at a frame rate of about 240 frames per second (fps), and for a region of interest of a single sensor array row, data can be collected at a frame rate of about 75,000 frames per second (fps). Specifically, with respect to Figure 3 An exemplary sensor array device according to the present teachings is presented in FIG. 1 , with the maximum frame rate for each row of sensors provided in the second to last column. In the last column, the maximum frame rate at which data can be collected for a fraction of a row covered by cells is presented, as calculated by dividing the maximum frame rate per row by the rows per cell diameter.

[0056] If you pass the inspection Figure 3As can be seen in the last column, for target regions of interest across a range of cell sizes, a fairly large number of frames per second can be collected, providing data collection comfortably in the kHz range. For very small cells anchored on the sensor array surface, data can be collected at a maximum frame rate of 75,000fps for the Chip 1 device, while data can be collected at a maximum frame rate of 27,000fps for Chip 4. Similarly, for small cells anchored on the sensor array surface, data can be collected at a maximum frame rate of 25,000fps for the Chip 1 device, while data can be collected at a maximum frame rate of 13,500fps for Chip 4. For medium cells anchored on the sensor array surface, data can be collected at a maximum frame rate of 10,714fps for the Chip 1 device, while data can be collected at a maximum frame rate of 5,587fps for Chip 4. Large cells anchored on the sensor array surface can have a maximum frame rate of 5,000fps for the Chip 1 device, while data can be collected at a maximum frame rate of 2,746fps for the Chip 4 data. For very large cells anchored on the sensor array surface, data can be collected at a maximum frame rate of 2,500 fps for the Chip 1 device, and 1,373 fps for Chip 4.

[0057] According to Figure 3 From an inspection of the , the trend is that the frame rate decreases as cell size increases and pixel size decreases, which is consistent with an inverse relationship between the selected area of ​​interest and the rate at which data can be collected. Therefore, the frame rate can be increased by selecting a smaller subset of pixels to monitor (i.e., by windowing down the area of ​​the sensor array device over which data is collected). Additionally, referring to Table 1, a device can be selected with a desirable frame rate that matches the application.

[0058] Additional sensor array device properties that may be varied relate to various types of microwell structures that may be associated with various sensor array devices taught by the present invention. Figure 2A The microwell array 122 on the sensor array 120 can be fabricated on the sensor array, for which each microwell can have at least one sensor or pixel. For various embodiments of the sensor array device taught by the present invention, each microwell can have more than one sensor or pixel.

[0059] FIG. 4A to FIG. 4C Demonstrated sensor array devices (such as Figure 2A A generally enlarged partial cross-sectional view of a top portion of a sensor array 110 . Figure 4A1 is a partial cross-sectional view of an embodiment of a sensor array according to the teachings of the present invention on which a microwell array 122A has been fabricated. An exemplary microwell 122A1 illustrating all of the microwells of the microwell array 122A may have sidewalls 124A, which are containment structures that may prevent various chemical analytes (e.g., analytes of interest released by cells proximate to the sensor) from diffusing from the sensor. Figure 4B The sensor array 120 is a partial cross-sectional view of an embodiment of a sensor array according to the teachings of the present invention on which a microwell array 122B has been formed. The exemplary microwell 122B1 has a sidewall 124B that provides a relative Figure 4A The microwell array 122A is a microwell array of substantially shallower microwells. Figure 4C is a partial cross-sectional view of an embodiment of a sensor array according to the teachings of the present invention in which no microwell array is fabricated on sensor array 120C.

[0060] Figure 4D is a pair of holes (such as Figure 4A and Figure 4B shown) and without holes (such as Figure 4C ) where the cells were interrogated under conditions designed to depolarize the membrane potential and then return it to the resting potential. Given that the measured change in cell potential decreases with the square of the cell's distance from the sensor, this can be achieved by using a sensor array with shallow microwells (e.g., Figure 4B microwell array 122B) or a sensor array with no holes at all (e.g. Figure 4C sensor array 120) to minimize the distance. Figure 4D The comparison shown in exemplifies the increase in amplitude of the response to the device without the hole.

[0061] For various types of sensor array devices, the center-to-center spacing or pitch between each sensor can be between about 850 nm and about 3.36 μm, while the width of the sensor plate 132 can be between about 600 nm and about 3.1 μm. In accordance with the teachings of the present invention, the width of the microwells at the bottom of the microwells on the sensor plate cannot be greater than about 90% of the width of the sensor plate 132. By way of non-limiting example, for a sensor plate having a width of about 600 nm such as Figure 4A-4CFor a sensor plate 132 of , each microwell array may have a width of about 540 nm at the bottom of the microwells on the sensor plate, while for a sensor plate having a width of 3.1 μ, each microwell array may have a width of about 2.8 μ at the bottom of the microwells on the sensor plate. In accordance with the teachings of the present invention, for various embodiments of the microwell array, the ratio of the width to the height of the microwells may be between about 1:2 and about 1:4. By way of non-limiting example, for each microwell array having microwells having a width of about 540 nm, the microwell height may be up to about 1.1 μ, while for each microwell array having microwells having a width of about 2.8 μ, the microwell height may be up to about 5.6 μ. By way of further non-limiting example, for each microwell array having microwells having a width of about 540 nm, the microwell height may be up to about 2.2 μ, while for each microwell array having microwells having a width of about 2.8 μ, the microwell height may be up to about 11.2 μ. Although non-limiting examples have been given, various embodiments of microwell arrays according to the present teachings may have any width to height ratio between about 1:2 and about 1:4.

[0062] for FIG. 4A to FIG. 4C , a partial cross-sectional view of an upper portion of a floating gate structure is depicted as floating gate upper portion 130. Various embodiments of sensor arrays incorporating floating gate structures have been described, for example, in US Pat. No. 9,128,044 and US Pat. No. 9,841,398, both of which are incorporated herein by reference in their entirety.

[0063] FIG. 4A to FIG. 4C The upper portion 130 of the floating gate may include a top metal layer, a sensor plate 132, and metal vias 134 and a metal layer 136, all of which are formed in a dielectric substrate 135. A passivation layer 126 may be deposited to form a continuous top layer over the surface of each microwell defining the microwell array. The sensing surface 126S is a portion of the passivation layer formed over the sensor plate 132. The metal layers 132, 134, and 136 may be suitable metal materials or alloys thereof, such as titanium, silver, gold, platinum, and tungsten. The dielectric substrate 135 may be a dielectric material such as silicon dioxide or silicon nitride. The passivation layer 126 may be a metal oxide layer such as titanium oxide, titanium nitride, and titanium oxynitride. For various embodiments of the ChemFET device, in the formation of a sensing layer (such as FIG. 4A to FIG. 4C The change in potential at the solid-liquid interface between the solution contacted by the sensing layer 126S) can cause a voltage change on the floating gate. Figure 2A The applied reference voltage described ensures that the voltage of the floating gate exceeds the threshold voltage, provided that a small change in the floating gate voltage produces, for example, a sensor (such as Figure 2B The output signals of sensors 120-1 and 120-2 of the sensor array 120.

[0064] The composition of the sensing surface can be modified in various ways to provide selectivity for various analytes of interest (e.g., analytes of interest in various cell metabolism and nutrition studies). For example, detection of various ions can be accomplished by using the passivation layer itself or by using ionophores coated onto the passivation layer. For example, the sensing surface can be modified in various ways to provide selectivity for various analytes of interest (e.g., analytes of interest in various cell metabolism and nutrition studies). For example, detection of various ions can be accomplished by using the passivation layer itself or by using ionophores coated onto the passivation layer. FIG. 4A to FIG. 4C Hydrogen ions can be detected by coating the sensing layer 126S with a modified sensing layer 126S, which can be titanium oxide, titanium nitride, or titanium oxynitride. Potassium ions can be selectively detected by coating the sensing layer with, for example, valinomycin or salinomycin. Sodium ions can be selectively detected by coating the sensing layer with monensin, nystatin, or the synthetic ionophore SQI-Pr (CAS#1022595-16-9). Calcium can be selectively detected by coating the sensing layer with, for example, ionomycin, calcimycine, or ETH 1001 (CAS#58801-34-6). Additionally, for various analyses, the selectivity of the ionophore may not need to be for a single species, but in addition be able to bind more than one ion in the ion genus. For example, by coating the sensing layer with beauvericin, calcium and barium ions can be detected, while by coating the sensing layer with nigericin, potassium, hydrogen and lead ions can be detected. Gramicidin can be coated on the sensing layer to detect hydrogen, sodium and potassium ions. According to the teachings of the present invention, various ion carriers that are selective to ion genera can be used in applications that do not require single ion specificity or are unlikely to exist or produce other ions that compound binds. Additionally, various classes of photocurable polymers can provide selectivity for various analytes (including a series of ions (such as potassium, calcium, ammonium, chloride and nitrate) and glucose, sucrose and urea) that are of concern to cell biology research (see, for example, Sensors, 2009, 9, 7097-7100).

[0065] In addition to providing detection selectivity for various analytes by changing the composition of the sensing surface portion of the passivation layer in contact with the sensor plate, the passivation layer can be treated with a material that provides cell compatibility, such as Figure 2B Alternatively, Figure 2AAs depicted in , the sensor surface of a sensor array device having a microwell array positioned above the sensor array can be treated by coating a cytocompatible material on the sensor array surface comprising the microwell array. To coat the various embodiments of the sensor array device taught by the present invention, the end user can select from a variety of cytocompatible materials a particular material that may be most suitable for the cell line of interest and a set of experiments, and the sensor array surface can be coated with the cytocompatible material before preparing the sensor array with the cell sample. Exemplary cytocompatible materials that can be coated on the sensor device surface include various biopolymer materials, such as poly-D-lysine, laminin, fibronectin, collagen, and combinations thereof, as well as various preparations of extracellular matrix (ECM).

[0066] like Figure 2A and Figure 2B As depicted in , providing a cell-compatible coating over the sensor array can provide an interface on the sensor array surface to which various types of cells can be anchored. Once the cells are stably anchored by providing a constantly renewed nutrient solution (e.g., by flowing the nutrient solution through a flow cell), the cells can grow and divide.

[0067] Therefore, each sensor array device taught by the present invention can have several properties that can provide each sensor array device that can be selected for target cell analysis.Such properties can include the presence or absence of a micropore array manufactured on the sensor array, the depth of each micropore in the micropore array of the sensor array device containing the micropore array, the nature of the surface treatment performed on the sensing surface for providing selectivity for chemical analysis, the type of surface treatment performed on the sensor array device for providing a cell-compatible coating, the pixel spacing and size, and the rate at which data can be collected.By way of non-limiting example, each sensor array device taught by the present invention can have a micropore array arranged on the sensor array, or does not have a micropore array arranged on the sensor array.Each sensor array device taught by the present invention can have a sensing surface that provides selectivity for various chemical analytes of concern in cell analysis.Such sensor array devices can be used in various cell metabolism and electrophysiology studies, for example, and can provide monitoring of various spontaneous and time cell activities.

[0068] Chemical Field Effect Transistor (ChemFET) Array-Based Systems for Cellular Analysis

[0069] As described previously in this article, Figure 1 1 is a block diagram of exemplary components of a cell analysis system 100 according to the teachings of the present invention. Figure 1 As depicted in , the cell analysis system 100 may include various fluid systems as well as an array controller 50 , a user interface 60 , and a sensor array device assembly 115 .

[0070] With respect to fluid delivery and control for performing various cell analyses on the sensor array device 110, the cell analysis system 100 may include a reagent fluid system 10, a wash solution fluid system 20, a fluid multiplexer system 30, and a valve controller 40. Additionally, because the flow cell 107 may define flow paths for various reagents and wash solutions above the sensor array device 110, the flow cell 107 is an integral part of the fluid system of the cell analysis system 100. The reagent fluid system 10 according to the teachings of the present invention may include a plurality of reagent containers, such as reagent containers 11A-11N that may be placed in controllable fluid communication with the sensor array device 110 via a flow cell inlet line 103A. The reagent fluid system 10 may include a reagent fluid line for each reagent container, such as reagent fluid lines 13A-13N corresponding to reagent containers 11A-11N, respectively. Additionally, each fluid reagent line may have a plurality of reagent containers, such as reagent containers 11A-11N, that are controlled by a valve (e.g., Figure 1 13A-13N) as inline valves for each of the reagent fluid lines 13A-13N, respectively. Wash solution fluid system 20 may include a wash solution wash solution container 21 that may be placed in controllable fluid communication with flow cell inlet line 103A via wash solution fluid line valve 25. Fluid multiplexer system 30 may include a fluid multiplexer system waste container 31 that is in fluid communication with fluid multiplexer 35 via fluid multiplexer fluid line 33.

[0071] like Figure 1 As depicted in , the reagent fluid line valves 15A-15N may be controlled by a valve controller 40. In this regard, the valve controller 40 may control the flow of fluid from each reagent container 11A-11N to the fluid multiplexer 35. Additionally, as Figure 1 As depicted in FIG. 1 , the valve controller 40 can be controlled by Figure 1 The washing solution fluid line valve 25, depicted as an inline washing solution fluid line valve 25 on the washing solution fluid line 23, controls the flow of fluid from the washing solution container 21 of the washing solution fluid system 20. Figure 1 As indicated by the control lines from the valve controller 40 to each reagent container 11A-11N, the valve controller 40 may also actuate the control of pneumatic valves on the pneumatic lines from an inert gas source (not shown) that provides a controllable head pressure to each reagent container 11A-11N and to the wash solution container 21. Thus, fluids from the various reagent and wash solution containers of the cell analysis system 100 may be controllably moved through the fluid lines using the pressure differential from the source to the outlet as a motive force.

[0072] In conjunction with the control provided by valve controller 40, Figure 1 The fluid multiplexer 35 of the sensor array device 110 can controllably perform fluid operations, including but not limited to: providing selected reagent delivery to the sensor array device 110; washing the fluid multiplexer 35 and the flow cell 107; and filling the fluid multiplexer 35 with selected reagents. Such fluid operations can provide cross-contamination-free delivery of reagents to the flow cell 107, can provide sharp transitions between reagent fluid streams, and provide a constant electrolyte fluid environment for the reference electrode 27 in the wash solution fluid line 23, thereby providing a constant stable reference voltage to the sensor array device 110. The functions of various embodiments of the fluid multiplexer 35 for directing flow in various embodiments of the fluid system of the sensor array system have been described in U.S. Patent Publication 2010 / 0137143, U.S. Patent 8,546,128, and U.S. Patent 8,673,627, all of which are incorporated herein by reference in their entirety.

[0073] For example, in combination with the valve controller 40, Figure 1 The fluid multiplexer 35 can selectively provide fluid communication between any of the reagent fluid lines 13A-13N and the flow cell inlet line 103A, thereby providing selective reagent flow through the flow cell 107 of the sensor array device assembly 115. The non-limiting illustrative reagent solution fluid paths taught by the present invention are given by a reagent delivery operation in which the wash solution fluid line valve 25 is in a closed state and one of the reagent fluid line valves 15A-15N is in an open state, provided that one of the selected reagents is in fluid communication with the fluid multiplexer 35. Under such conditions, the selected reagent can flow through the fluid multiplexer 35 and then through the fluid multiplexer fluid line 33 to the fluid multiplexer waste container 31. Additionally, a selected reagent may flow through the fluid multiplexer 35 to the flow cell inlet line 103A, where it may then flow from the inlet port 102 through the flow cell 107 to the outlet port 104, and finally through the flow cell outlet line 103B to the flow cell waste container 101.

[0074] Regarding the fluid control of the washing solution, in conjunction with the valve controller 40, Figure 1The fluid multiplexer 35 of the embodiment of the present invention can selectively provide fluid communication between the wash solution container 21 and the flow cell inlet line 103A. Therefore, with the wash solution fluid line valve 25 in an open state, the solution container 21 can be in fluid communication with the fluid multiplexer waste container 31 and the flow cell waste container 101, provided that washing of the fluid multiplexer 35 and the flow cell 107 can be performed. The non-limiting illustrative wash solution fluid path of the present teachings is given by a wash operation in which the wash solution fluid line valve 25 is in an open state and each of the reagent fluid line valves 15A-15N is in a closed state, provided that the wash solution can flow through the wash solution fluid line 23 to the T-joint with the flow cell inlet line 103A. When the flow cell inlet line 103A is in fluid communication with the fluid multiplexer 35, the wash solution can flow through the fluid multiplexer fluid line 33 to the fluid multiplexer waste container 31. When flow cell inlet line 103A is additionally in fluid communication with sensor array device assembly 115 , wash solution may pass through flow cell 107 from inlet port 102 to outlet port 104 , and may then flow through flow cell outlet line 103B to flow cell waste container 101 .

[0075] According to the teachings of the present invention, it is possible, for example, to sequentially follow a washing operation and to place the selected reagent in contact with the Figure 1 Prior to fluid communication with the flow cell 107 of the present invention, priming the fluid multiplexer 35 with the selected reagent is performed. A non-limiting illustrative reagent priming fluid path of the present teachings is given by a reagent priming operation in which the wash solution fluid line valve 25 is in an open state and one of the reagent fluid line valves 15A-15N is in an open state, provided that one of the selected reagents is in fluid communication with the fluid multiplexer 35. Under such operation, the flow rate of the wash solution relative to the flow rate of the reagent is selected so that the wash solution flows through the fluid multiplexer 35 to the fluid multiplexer waste container 31 through the fluid multiplexer fluid lines 33 in addition to the passages in the fluid multiplexer 35 (such as the reagent fluid lines 13A-13N) in fluid communication with the selected reagent fluid. Thus, when a reagent delivery operation as previously described herein is initiated, the reagent selected in the reagent priming operation is in direct flow communication with the flow cell inlet line 103A.

[0076] Therefore, various embodiments of the fluid system of the present teachings are configured to perform a series of operations that may include washing, filling, and reagent delivery. Such operations performed in sequence can avoid cross contamination of reagents in system fluid lines and compartments, provide a sharp transition between reagent fluid streams, and provide a constant electrolyte fluid environment for the reference electrode, thereby providing a constant stable reference voltage to the sensor array.

[0077] According to the teachings of the present invention, noise sources generated from the fluid line can affect the reference voltage (e.g., by a Figure 1 As previously described herein, the reference voltage from the reference electrode 27 in the wash solution fluid line 23 is Figure 1 A stable reference voltage for the reference electrode 27 of the sensor array device 110 can be provided by having a fluid system that ensures that the reference electrode 27 is in continuous contact with a wash solution of known electrolyte composition. Additionally, high frequency noise from fluid lines (e.g., reagent fluid lines 13A-13N and fluid multiplexer fluid line 33) can be filtered out by capacitively coupling electrodes positioned in the fluid lines to the reference electrode. In accordance with the teachings of the present invention, the electrode can be, for example, a hollow cylindrical structure of an inert metal, non-limiting examples of which include platinum or titanium. Such a hollow cylindrical metal structure can provide effective ohmic contact with the fluid in the flow stream. As Figure 1 As depicted in FIG. 1 , the reagent fluid line electrodes 17A-17N positioned in the reagent fluid lines 13A-13N are coupled to the reference electrode 27 through individual reagent fluid line capacitors 19A-19N, each of which is coupled to the reagent fluid line electrodes 17A-17N, respectively. In a similar manner, the fluid multiplexer fluid line electrode 37 positioned in the fluid multiplexer fluid line 33 is coupled to the reference electrode 27 through the fluid multiplexer fluid line capacitor 39.

[0078] like Figure 1 As depicted in , the cell analysis system 100 may additionally include an array controller 50 and a user interface 60. According to the teachings of the present invention, the array controller 50 may provide various power and bias voltages and control and timing signals to the sensor array device 110, and additionally provide a data and processor interface for high-speed acquisition of data from the sensor array device 110. In this regard, Figure 5A and Figure 5B Various functions of the array controller 50 are depicted in relation to various functions of the sensor array device 110 in accordance with the teachings of the present invention.

[0079] Figure 5AGenerally, various aspects of the functions between the array controller 50 and the sensor array 120 of the sensor array device 110 and between the array controller 50 and the user interface 60 are shown. According to various embodiments of the cell analysis system taught by the present invention, the array controller 50 can have multiple power supplies 52 for providing power to the sensor array device 110 and a supporting digital interface 54, which is a high-speed digital interface configured to read data generated by the sensor array device 110. In addition, the array controller 50 is configured to provide signals to the sensor array device 110, such as control signals from the signal controller 56 and timing signals from the timing generator 58. Multiple power supplies can be provided to the sensor array device 110, including analog power supplies, digital power supplies, I / O power supplies, and ground. In an exemplary embodiment, each of the power supply voltages can be controllable within the range of 1.2 to 3.3V. Each of these power supply voltages can be provided to the sensor array device 110 through a separate conductive path to promote noise isolation. These power supply voltages can be derived from corresponding power supplies, or one or more of these power supply voltages can be derived from a common source in the array controller 50. According to various embodiments of the array controller in accordance with the teachings of the present invention, the power supply may include one or more digital-to-analog converters (DACs) that may be controlled by the system processor 62 to allow any or all of the power supply voltages to be changed under software control. For example, the computer-controlled power supply may facilitate switching between a 1.6 volt power supply voltage and a 1.8 volt power supply voltage, depending on the requirements of the type of sensor array device in use as read from data stored on various embodiments of the sensor array device.

[0080] Figure 5A The hardware controller 70 shown in the figure can be implemented as one or more separate circuit boards or as a part of an interface board. One function of the hardware controller 70 is to provide pneumatic and fluid control, such as to control the timing and duration of various fluids and reagents flowing through the sensing surface of each sensor in the sensor array 120 and to read the pressure and flow sensors related to pneumatic and fluid flow control as determined by the system processor 62 software. Additionally, the hardware controller 70 can support temperature measurement and control for the sensor array device 110, as well as temperature measurement and control for the assembly and subassembly of the system. According to the teachings of the present invention, by non-limiting example, thermistors and thermocouples can be used to measure temperature. In response to temperature measurement, the system processor 62 can control the device that can include non-limiting examples such as fans, heaters and thermoelectric coolers.

[0081] The array controller 50 may be manufactured as a "stand-alone" circuit board or as one or more computer compatible "cards" that form part of a computer. In one aspect, the functions of the array controller taught by the present invention may be controlled by a system processor 62 through a processor interface 55 (e.g., a PCI bus, an Ethernet connection, etc.). In one embodiment, all or a portion of the array controller is manufactured as one or more printed circuit boards, and the sensor array device is configured to be attached to one of the printed circuit boards. External connections to the system processor 62 may include standard computer interfaces such as Ethernet and USB. Finally, the system processor 62 may include a display 64.

[0082] like Figure 5B As depicted in , various power supplies and bias voltages are provided to the sensor array device from the array controller 50. Figure 5B The sensor array device 110 may be combined with one or more analog-to-digital converters (ADCs) 114 and one or more data multiplexers and a high-speed serial output 116 to convert analog output signals of the sensor array 120 into a high-speed serial data interface 54, thereby transmitting the analog output signals from the sensor array device 110 to the system processor 62 (see FIG. Figure 5A ) provides digital data. For various embodiments of the sensor array device 110, the analog-to-digital converter (ADC) 114 may have a computer-selectable input range (eg, 200 mV, 300 mV) to facilitate compatibility with sensor output signals of different ranges.

[0083] According to the teachings of the present invention, Figure 5BIn various embodiments of the sensor array device 110, the array row selection and sequencing 112, ADC 114 and data multiplexer and high speed serial output 116 can be controlled by logic on the timing sequencer 118 of the sensor array device 110 with timing provided by the array controller 50. In an exemplary embodiment, the number of rows from which data is acquired and the sampling rate at which it is acquired can be controlled based on control data provided to the sensor array device 110 from the array controller 50. In a non-limiting example, the timing generator 58 can be implemented as a programmable clock generator integrated circuit controlled by the system processor 62 to allow control of different sensor array device types, operating modes and acquisition data rates. Control signals between the array controller 50 and the sensor array device timing sequencer 118 can be exchanged using a standard serial interface type (such as I2C or SPI). Information written to the sensor array device 110 can include values ​​stored or written to registers on the device of the sensor array device timing sequencer 118 to control the operating mode of the sensor array device 110 and read data from the sensor array device 110. The information read from the sensor array device 110 may include stored data including chip type, serial number, manufacturing date, etc.

[0084] Figure 5B The reference electrode 27 can be coupled to a power source to provide a reference potential for the output voltage (output signal) of each sensor from the sensor array 120. By way of non-limiting example, the reference electrode voltage of the reference electrode 27 can be provided by flowing a solution having a known and stable pH through the sensor array device 110 (see Figure 2A ) is set. Under such defined pH conditions, the reference electrode voltage can be adjusted until the sensors in the sensor array (e.g. Figure 2A The output signal of the sensor of the sensor array 120 indicates that the sensor has a voltage at a desired reference level. Once the reference voltage is set using a solution with a known and stable pH, subsequent changes in the sensor voltage can reflect that a local change in pH has occurred at the sensing surface of the sensor of the sensor array.

[0085] Applications and methods of systems using ChemFET-based sensor arrays for performing cell analysis

[0086] Fig. 6A Flowchart 200 presents a workflow related to embodiments of the applications and methods taught by the present invention that can be performed using the cell analysis system and related devices, components and assemblies taught by the present invention. For example, flowchart 200 can be used Figure 1 The cell analysis system 100 may include a sensor array device interfaced with an array controller and a system processor, such as Figure 1 , Figure 5A and Figure 5B The sensor array device 110, the array controller 50 and the user interface 60 are provided.

[0087] Fig. 6A The flowchart 200 may start with step 210: inoculating a ChemFET sensor array device with a cell sample. A cell suspension of known density may be flushed or otherwise drawn into a sensor array device assembly (e.g., Figure 1 After the cell sample is plated, the sensor array device assembly can be covered and placed for a target period of time for incubation to allow the cells to settle and anchor on the sensor array surface. Before the sensor array device assembly is plated with the cell sample, the flowchart 200 can additionally include steps for preparing the sensor array surface with a user-selected cell-compatible material (such as various biopolymer materials, including poly-D-lysine, laminin, fibronectin, collagen and combinations thereof) and various extracellular matrix (ECM) preparations. The sensor array surface can be prepared with a cell-compatible material before plated inoculation, and the sensor array device can be stored for future use. Alternatively, the sensor array surface can be prepared in situ with a cell-compatible material before the sensor array device assembly is plated with the cell sample.

[0088] After the cells have been stably associated with the sensor array surface, at step 212 of flowchart 200, a live cell imaging application can be performed and at step 214, the results can be displayed. Information from the live cell imaging application at step 214 can provide the end user with an overview of the cell distribution on the sensor surface, information about individual cell morphology, and an assessment of individual cell viability. For example, Figure 1 The cell analysis system 100 can provide electron microscope cell imaging for end users. For such imaging applications, any change in the state of cells anchored on the surface of the sensor array device that can cause a change in the potential of the sensing surface of the ChemFET sensor can be detected by each sensor in the sensor array that responds to the cell activity. The electron microscope image of the responsive cell at a selected time during, for example, a live cell imaging application can be displayed, for example, on a display (such as Figure 5A The sensor array surface is presented to the end user on a display 64 of the user interface 60. As previously described herein, given that electron microscopy imaging is based on a variety of responses that can be elicited for living cells, obtaining electron microscopy images of responsive cells on the sensor array surface can be used as an initial step in visualizing and evaluating cells. As an alternative or in addition to electron microscopy imaging, other methods of visualizing cells, such as optical microscopy, can be used.

[0089] At step 216 of flow chart 200, based on the information provided from the live cell image display step 214, the region of interest can be selected based on, for example, identifying the best region for the distribution and activity of the cells and taking into account the application of interest. As previously described herein, there is an inverse relationship between the rate at which data can be collected and the selection of the region of interest. For example, in order to monitor the action potential of each excitable cell, a data acquisition rate in the sub-millisecond to millisecond range may be required, while in order to monitor the lactate produced by oxidative phosphorylation, a data acquisition rate in the range of seconds to minutes may be required. At step 218, the end user can select an application from a menu presented, for example, on a graphical user interface (GUI) and then initiate a run as given in step 220. Finally, at step 222, the end user can receive the results of the experiment, for example, but not limited to, as displayed on a display (such as Figure 5A The results may be provided on the display 64 of the user interface 60, such as by sending them as a report to the system processor (such as Figure 5A The system processor 62 of the user interface 60) provides the results or both.

[0090] Alternatively, the end user may run the application without first running the live cell imaging application to select the region of interest. Figure 6B Flowchart 300 may start with step 310 of inoculating a ChemFET sensor array device with a cell sample as previously described for step 210 of 6A. In accordance with the teachings of the present invention, the inoculation of cells may be performed on a sensor array device treated with a cytocompatible material. At step 320, the end user may select an application from a menu presented, for example, on a graphical user interface (GUI) and then initiate a run as given in step 330. Finally, at step 340, the end user may receive the results of the experiment, for example, but not limited to, as displayed on a display (e.g., Figure 5A The results may be provided on the display 64 of the user interface 60, such as by sending them as a report to the system processor (such as Figure 5A When the application is run without a region of interest selected, data is collected at a frame rate associated with the sensor array device, such as shown in Table 1. Although the end user can zoom in and expand any area on the device, such as an electron microscope image, the resolution of the data ultimately presented is fixed by the frame rate associated with the device.

[0091] Various methods of in situ processing of sensor array devices to provide a coating of a cytocompatible material on the sensor array surface are suitable for use, for example, Figure 3With respect to an exemplary method for in situ modification of a sensor array surface with a cytocompatible material, the following solution of an exemplary cytocompatible material was used to coat the surface of three ISFET sensors selective for sensing hydrogen ions (pH):

[0092] 1. Phosphate buffered saline (PBS) containing 0.1% poly-D-lysine (PDL)

[0093] 2. PBS containing 0.1% poly-D-lysine (PDL) + 0.05% laminin

[0094] 3. Commercial extracellular matrix (ECM) formulations Can be used without dilution

[0095] will belong to Figure 3 The three sensor array devices of the type identified as chip 1 in the table were opened in a sterile laminar flow cell culture hood, and all manipulations were then performed under sterile conditions with sterile solutions and materials. Each sensor array device was rinsed twice with 200 μL of water containing a solution composed of 70% ethanol injected into one of the solution ports by a pipette and gently aspirated from the other solution port. Once the solution is applied to the chip surface, be careful not to suck the surface or chamber under the flow cell dry or run dry, so the vacuum line is positioned above the outlet port opposite the port for solution injection and the chip is never vacuum dried. The 70% ethanol solution was allowed to stay on the sensor array device for 30 minutes at room temperature, and then rinsed repeatedly with 200 μL of cell culture grade PBS. The sensor array device was then rinsed with 200 μL of solution as described above and allowed to stand for one hour at room temperature before being rinsed three times with 200 μL of fresh PBS, after which the sensor array device was ready for use. Alternatively, a sensor array device prepared to provide a cytocompatible surface as described can be sealed and stored for later use.

[0096] To verify sensor array functionality for a sensor array device treated as described above to provide a cytocompatible sensor surface, a cell analysis system (e.g. Figure 1 The cell analysis system 100 of the present invention is used to evaluate the processed sensor array device. The washing solution container and three reagent containers are filled with the following solutions of the following compositions:

[0097] 1. Fill the washing solution container and the first reagent container with a solution having the following composition and properties:

[0098] 20 mM HEPES, pH 7.4

[0099] 140mM NaCl

[0100] 2.5 mM KCl

[0101] 1.8 mM CaCl 2

[0102] 1.0 mM MgCl 2

[0103] Osmolarity: 300mOsm

[0104] 2. Fill the second reagent container with a solution having the following composition and properties:

[0105] 20 mM HEPES, pH 7.6

[0106] 140mM KCl

[0107] 2.5 mM NaCl

[0108] 1.8 mM CaCl 2

[0109] 1.0 mM MgCl 2

[0110] Osmolarity: 300mOsm

[0111] 3. The third reagent container is filled with a solution having the composition and properties used to fill the second reagent container but adjusted to a pH of 7.1 using a dilute hydrochloric acid solution.

[0112] The following testing protocol was performed for each sensor array device and a control having an untreated surface:

[0113] 1. Wash buffer solution is used to set the reference electrode potential and to condition the sensor array device.

[0114] 2. For each sensor device, two pixels in different sensor areas were selected to measure the potential during the test.

[0115] 3. The order of drawing the solutions through the flow cells of each device is as follows:

[0116] a. Switch from the wash solution to the solution in the first reagent container to verify that they do not have a large effect on switching between the same solutions in different containers.

[0117] b. Switch back to the wash solution and then switch to the second reagent container solution to measure the effect of changing the pH from 7.4 to 7.6.

[0118] c Switch back to the wash solution and then switch to the third reagent container solution in order to measure the effect of changing the pH from 7.4 to 7.1.

[0119] The data generated from the testing protocol verified that the sensor array device treated to provide a cytocompatible surface had a response indicating that there was no adverse effect on the sensor array device performance due to the coating of the device. Fig.17 A graph of the pH response of the device is presented in .

[0120] FIG. 7A to FIG. 8B Data related to measurements of changes in cell membrane potential over the time course of an induced depolarization followed by a recovery period are presented. The sensor array device used in this study is selective for sensing hydrogen ions such as Figure 3 The chip 1 device shown in the table and comprising a microwell array. The device is treated to provide a cell-compatible poly-D-lysine coating as previously described herein. U-2OS cells (ATCC catalog number ATCC-HB-96) are human osteosarcoma cell lines commonly used for heterologous expression studies, which are transduced with green fluorescent protein (GFP) to monitor cells on the sensor array surface using a fluorescent microscope and transduced with ion channel constructs to provide a response to depolarizing stimuli. U-2OS for plate inoculation is prepared using a standard dissociation procedure, centrifuged at 200 × g to precipitate, and resuspended in complete cell culture medium at a density of 250,000 cells / ml. Then, 200 μL of the cell suspension is aspirated through the sensor array device. Then, the sensor array device is placed in a sterile 10 cm cell culture dish and covered. Then, the sensor array device prepared in this manner is transferred to an incubator and incubated at least overnight at 37° C. before use in the cell analysis system taught by the present invention.

[0121] Fig. 7A The graphs presented in FIG. 1 are time response curves representing the time course of focusing on two cells C during the course of a cell imaging study. 1 and C 2 The data collected on the area of ​​interest were obtained using two solutions. FIG. 7A to FIG. 8B Cell imaging studies presented in:

[0122] 1. Fill the washing solution container and the first reagent container with a solution having the following composition and properties:

[0123] 20 mM HEPES, pH 7.3

[0124] 140mM NaCl

[0125] 2.5 mM KCl

[0126] 1.8 mM CaCl2

[0127] 1.0 mM MgCl 2

[0128] Osmolarity: 300mOsm

[0129] 2. The remaining reagent containers; fill the second to fourth containers with solutions having the following composition and properties:

[0130] 20 mM HEPES, pH 7.3

[0131] 140mM KCl

[0132] 2.5 mM NaCl

[0133] 1.8 mM CaCl 2

[0134] 1.0 mM MgCl 2

[0135] Osmolarity: 300mOsm

[0136] Prior to initiating a cell imaging study, the solution in the flow cell for the initial condition was a wash solution. The study was initiated when one of the second to fourth reagent containers was aspirated through the flow cell and past the sensor array. Since the predominant concentration of cations in the solution was potassium, it could provide a depolarizing stimulus to the cells. In addition to monitoring the activity of the cells, a reference response FET was also monitored in an area of ​​the sensor array where no cells were anchored. R .

[0137] About Cell C 1 and C 2 Advances in cell imaging research Figure 7B When the depolarized solution reaches the cell at stage I indicated in the figure, it passes through cell C 1 and C 2 The early indication of depolarization is confirmed by the increase in the signal of , and it continues to increase in phase II until equilibrium is reached at phase III where the response persists. When a wash solution is introduced into the flow cell and the depolarizing solution is washed out of the flow cell, recovery is indicated at phase IV of the graph and continues until cell C 1 and C 2 Equilibrate to the wash solution at stage V. In contrast to the slightly negative response of the reference curve, the response of the cell is a characteristic signature of its presence on the sensor array surface, with cells undergoing depolarization exhibiting a strong positive voltage response approximately 20 times higher than the baseline noise level.

[0138] Figure 7BThe time indicated by the hatched line at 30 frames is Fig. 8A The electron microscope image corresponds to the time. Figure 8B is Fig. 8A Line graph of the main features visualized in the electron microscopy image of . Based on electron microscopy imaging of cells on the sensor array, the size of cells across the sensor array was estimated to have a diameter of approximately 10 to 20 pixels or 30 to 60 μ given the 3 μ pixel pitch of the sensor array device used in the study. As mentioned earlier in this article, U-2OS cells were transduced with green fluorescent protein (GFP) to allow for the detection of FIG. 7A to FIG. 8B The sensor arrays prepared for the electron microscopy imaging experiments presented in can also be imaged using fluorescence microscopy. Results obtained from data collected from fluorescence microscopy imaging of the sensor arrays confirmed that the diameter of cells across the sensor array ranged from 30μ to 60μ.

[0139] Fig.9A U-2OS cells (C 1 and C 2 ) and U-2OS cells transfected with voltage-gated calcium channel α, β, and α-2-δ subunits (C 3 and C 4 ) time response curve. Fig.9A The time response curves presented in represent data collected over the region of interest for U-2OS cells compared to transfected U-2OS cells, wherein the effects on the cellular response of each type of cell treated with the calcium channel blocker verapamil are shown by cycling through an initial state of resting potential, followed by conditions for depolarizing the membrane potential and returning to the resting potential. Fig. 9B It is taken at the time corresponding to frame 25 (1.7 seconds) Fig.9A Electron microscope image of Fig. 9B The squares in the cells indicate the Fig.9A The data is sampled in pixels. Fig. 9C yes Fig. 9B Electron microscope image of Fig. 9B The squares within the cells have been removed to provide a clear image of the cells.

[0140] FIG. 9A to FIG. 9C The data from the experiments in which the sensor array device used was generated were selective for sensing hydrogen ions as Figure 3The chip 2 device summarized in the table of and comprising a microwell array. The device with a poly-D-lysine surface was prepared as previously described herein. Briefly, the sensor array device surface was soaked in water containing 70% ethanol for 30 minutes and then rinsed with PBS before adding a solution of 0.1% poly-D-lysine, allowing the sensor array device surface to stand for two hours. The device was then rinsed twice with 200 μL of cell culture medium (e.g., medium modified with McCoy's 5A containing 10% fetal bovine serum). After rinsing with cell culture medium, the device was prepared for plating with a cell suspension.

[0141] The preparation of transfected cells was carried out using a cell suspension of 1 × 10^6 cells / mL. Mammalian BacMam gene delivery particles encoding a mixture of voltage-gated calcium ion channel α, β and α-2-δ subunits were added to a fine suspension to reconstruct functional channels. Functional voltage-gated calcium ion channels require at least both α subunits and β subunits, wherein α-2-δ is used to enhance surface expression and ion flux. BacMam viral transduction is achieved by premixing particles and adding to cells with a rough ratio of ten times excess of viral particles to cell number. Control (no virus) and virus-treated cell plates were inoculated onto the sensor device and placed in a cell culture incubator overnight to allow adhesion and expression of functional ion channels before the experiment was performed the next day.

[0142] During the experiments, control or virally transduced cells were exposed to an elevated depolarizing stimulus of 30 mM KCl in the presence of 10 μM of the calcium channel blocker verapamil. Fig.9A The time response curves show the change from the control group (C 1 and C 2 ) to cells transfected with voltage-gated calcium channels (C 3 and C 4 ) to obtain discernible differences in the feature label responses. FIG. 9A to FIG. 9C The findings presented in demonstrate that the cell analysis systems and methods taught by the present invention can readily distinguish cells that do or do not express voltage-gated calcium channels based on the selective activity of blockers against cultures expressing the target protein.

[0143] Fig. 10A This is an electron microscopic image of rat neonatal hippocampal neurons subjected to a 4.5 mM KCl pulse. Fig. 10B Comparison of the time response curves of rat neonatal cells subjected to a 4.5 mM KCl pulse, where the experiments were performed on two different apparatuses with different data acquisition rates.

[0144] Reference summarizes the array device properties Figure 3 The table, in Fig. 10A and Fig. 10B The sensor array device used in the experiment in which the data was generated was a device with the size of a chip 2, for which data was collected at a frame rate of 30 samples / second. Fig. 10B The sensor array device used in the experiments for the data in Figure 1 was a device having the dimensions of Chip 1, where the active area of ​​the device was windowed down to an area of ​​interest that was 50% of the total area of ​​the device to provide a maximum frame rate of 240 samples / second. Both the Chip 1 device and the Chip 2 device were ISFET devices that were selective for sensing hydrogen ions and contained arrays of microwells. Devices with poly-D-lysine surfaces were prepared as described herein to prepare FIG. 9A to FIG. 9C The device was used in experiments with voltage-gated calcium channels. Rat neonatal hippocampal neurons were dissociated from intact rat E18 neonatal hippocampal neurons and resuspended at a density of 1×10^6 cells / mL, then plated onto the device. The device was placed in a cell culture incubator and fresh neuronal culture medium was replaced every three days on top of the plated cells for a period of ten days, which allowed the cells to adhere and mature into an excitable phenotype capable of displaying spontaneous depolarizing activity. The spontaneous activity of rat neonatal hippocampal neurons during pulses from solutions with basal potassium chloride (KCl) concentrations ranging from 2.5mM to 4.5mM was tested.

[0145] like Fig. 10A As confirmed by electron microscopy images, spontaneous depolarization of the cell activity of the preparation was observed. Fig. 10B The effect of frame rate on data resolution is demonstrated. Fig. 10B In the graph, it is observed that the temporal response curve of the sensor device for a frame rate of 240 samples / s has two peaks ( Fig. 10B -I), while the time response curve of the sensor device for a frame rate of 30 samples / s has a single peak ( Fig. 10B -II). Fig. 10A Electron microscopy data and Fig. 10B The time response curves of demonstrate the ability of the cell analysis systems and methods taught by the present invention to visualize intrinsic electrical activity from a primary cell type known to fire action potentials in culture after a ten day maturation period.

[0146] Fig.11 It is an electron microscope image of cells from an immortalized human hepatocyte line (HEPG2 cells; ATCC catalog number ATCC-HB-8065) ​​subjected to the mitochondrial toxin carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), wherein some exemplary cells are indicated to be associated with the flow of cell efflux. The HEPG2 cell line is used as a model of liver hepatocellular carcinoma in cell biology, and is additionally used for studies of LDL and cholesterol import and liver toxicology.

[0147] Fig.11 The sensor array device used in the experiments in which the data were generated is selective for sensing hydrogen ions Figure 3 A chip 2 device is prepared as described herein with a poly-D-lysine surface to prepare FIG. 9A to FIG. 9C The device was used in experiments with voltage-gated calcium channels. HEPG2 cells were resuspended at 1×10^6 cells / mL and plated onto the chip to be cultured overnight in a cell culture incubator to allow the cells to adhere to the prepared surface of the chip. The mitochondrial protonophore FCCP was tested at a concentration of 100 μM, which is a concentration known to trigger a response that leads to cell death. Electron microscopy images from the experiments are presented in Fig.11 middle.

[0148] During the application of FCCP, cellular responses were observed, demonstrating the ability of the cell analysis systems and methods taught by the present invention to visualize the outflow of cellular contents into a laminar flow system. Fig.11 The two cells in C HEPG1 and C HEPG2 The ability is described, and the efflux of the cell contents of the two cells is respectively represented by C efflux1 and C efflux2 It is worth noting that not all cells demonstrated efflux activity, as Fig.11 Targeting C HEPG3 These results indicate that the cell analysis systems and methods taught by the present invention can further distinguish acute novel phenotypes expressed in response to toxin treatment.

[0149] Fig. 12A Shown are the averaged background-subtracted time-dose-response curves of U-2OS cells treated with FCCP. Fig. 12B Shown are the averaged background-subtracted time-dose-response curves of U-2OS cells pretreated with nutrient solution containing oligomycin prior to treatment with FCCP.

[0150] Reference summarizes the array device properties Figure 3 The table, in Fig. 12A and Fig. 12B The data from the sensor array device used in the experiment was generated using a Fig. 12A The data presented in the chip 2 device and for Fig. 12B The Chip 1 device presented in FIG. Both the Chip 1 device and the Chip 2 device are ISFET devices selective for sensing hydrogen ions and include microwell arrays. Devices with poly-D-lysine surfaces were prepared as described herein to prepare FIG. 9A to FIG. 9CThe device was used in experiments with voltage-gated calcium channels. U-2OS cells were resuspended at 1×10^6 cells / mL and plated onto the chip to be cultured overnight in a cell culture incubator to allow the cells to adhere to the prepared surface of the chip. To test the dose dependence of FCCP treatment, Fig. 12A and Fig. 12B Cells were exposed to a range of concentrations of mutations during the experiment as indicated. Fig. 12A In the experiment, the experiment was performed in the absence of oligomycin and in Fig. 12B In the present embodiment, a dose-response curve of FCCP was generated in the presence of oligomycin.

[0151] Fig. 12A and Fig. 12B The data in are made up of the average cell response from the sample tested with FCCP. Data show that in the absence and presence of oligomycin, the dose-dependent FCCP response is easy to distinguish. For the current prior art of cell analysis tools, oligomycin is often used as the pre-treatment of FCCP to strengthen the FCCP response in the cell. On the contrary, the cell analysis system of the present invention does not need pharmacology to fill and can prove robust, dose-dependent FCCP time response curve.

[0152] The data for which are presented in FIG. 13A to FIG. 13C In the experiments, three different cell types were selected to test the instrument's ability to distinguish cell types based on their response to FCCP. In order to generate a data set for comparison of a wide range of different cell types, donor-derived primary human aortic smooth muscle (HASM) cells (HASMC; Gibco catalog number C0075C), human cervical cancer cells (HeLa cells; ATCC catalog number CRMCCL2), and a mouse-derived leukemia cell line (MMM cells; ATCC catalog number J774A.1) were selected for study.

[0153] FIG. 13A to FIG. 13C are the averaged background-subtracted time dose-response curves for each type of cell line tested individually on separate devices. Fig.13A is a set of dose-response curves for HASM cells. Fig. 13B is a set of dose-response curves for HeLa cells, and Fig. 13C is a set of dose-response curves for MMM cells. FIG. 14A to FIG. 14C For the data shown in , a mixture of cells was exposed to 100 μM FCCP and their responses were recorded to see if different cell types could be identified by their response to FCCP. Fig.14A Shown are synthetic time dose-response curves for selected cells in a mixture of different cell types where cells were subjected to FCCP. Fig. 14B It is taken at the time corresponding to frame 40 (2.7 seconds) Fig.14A Electron microscopy images of experiments in which the squares inside the cells indicate the Fig.14A The data is sampled in pixels. Fig. 14C yes Fig. 14B Electron microscope image of Fig. 13B The squares within the cells have been removed to provide a clear image of the cells.

[0154] Figures 13A to 14C The sensor array device used in the experiments in which the data were generated is selective for sensing hydrogen ions Figure 3 A chip 2 device is prepared as described herein with a poly-D-lysine surface to prepare FIG. 9A to FIG. 9C Device used in experiments with voltage-gated calcium channels. Each cell line was resuspended at 1×10^6 cells / mL and then mixed in a uniform ratio and then plated onto the device to culture overnight in a cell culture incubator to allow the cells to adhere to the prepared surface of the chip.

[0155] exist FIG. 13A to FIG. 13C The time response curves represent each of the three cell types of different tissue origin tested individually on separate devices, and it is noteworthy that each cell type displayed different kinetics and amplitude responses to FCCP. FIG. 13A to FIG. 13C The data presented in demonstrate the potential to identify each cell line by its characteristic signature response to pharmacological probes. Fig.14A Represents data collected from analysis of all three cell types on a single chip. Fig.14A The time response data presented in the figure were analyzed using waveform analysis, C 1 and C 2 Identified as HASM cells, C 3 For MMM cells, and C 4 and C 5 Identified as HeLa cells. Fig. 14C The electron microscope image was captured at the time when the HeLa cell (C 4 and C 5 ) compared with HASM cells and MMM cells (C 1 -C 3 ) behavioral differences. In general, FIG. 13A to FIG. 13C and FIG. 14A to FIG. 14C The data presented in demonstrate the potential of the systems and methods taught by the present invention to identify distinct cell lineages in a mixed cell population using signature-labeled cellular responses and behaviors.

[0156] FIG. 15A to FIG. 15CThe data are representative data of the experiments in which the sensor array device used is selective for sensing hydrogen ions. Figure 3 To test the ability of the various cell analysis systems taught by the present invention to record cellular signals with subcellular resolution and high temporal fidelity, data were collected at 120 frames per second for a Chip 1 device with a large number of cells plated on a cytocompatible surface. FIG. 9A to FIG. 9C Device and cell preparation were performed as described. In brief, a device with a poly-D-lysine surface was prepared and a preparation of U-2OS cells transfected with voltage-gated calcium channels was prepared. The chip 1 device was plated with transfected U-2OS cells using a cell suspension of 1×10^6 cells / mL. As previously described herein, mammalianized BacMam gene delivery particles encode a mixture of voltage-gated calcium channel α, β, and α-2-δ subunits to provide a cell suspension with a reconstructed functional channel. Functional voltage-gated calcium channels require at least both α and β subunits, with α-2-δ being used to enhance surface expression and ion flux.

[0157] Fig.15A and 15B Two electron microscope images from two exemplary time points are shown, and the data captured at intervals of about one to two seconds are separated by 345 milliseconds (ms). About 500 milliseconds before starting data collection, cells are perfused with the following isotonic agents:

[0158] 20 mM HEPES, pH 7.3

[0159] 130mM NaCl

[0160] 12.5 mM KCl

[0161] 1.8 mM CaCl 2

[0162] 1.0 mM MgCl 2

[0163] Osmolarity: 300mOsm

[0164] Fig.15A and Fig. 15Bis an electron microscope image showing an example of transient signals in subcellular domains of several cells presented in the electron microscope image. For example, for the cell designated as cell i, the signal is clearly visible at the 1577 millisecond time point, while the signal is substantially reduced at the 1922 millisecond time point. For the cell designated as cell ii, there is a clearly visible signal at the 1577 millisecond time point, while the signal is reduced at the 1922 millisecond time point to show the signal in the adjacent cell. For the cell designated as cell iii, there is no visible signal at the 1577 millisecond time point, while there is a clearly visible signal at the 1922 millisecond time point.

[0165] Fig. 15C is included in the Fig.15A and Fig. 15B Time response curves within the time interval of the electron microscopy images of cells i-iii. Fig. 15C The time response curve of also includes the signal collected and averaged for the non-responsive cell iv (i.e., the cell response control) and the signal collected and averaged for the sensor area v (i.e., the device response control). Fig. 15C As can be seen from the time response curves, both controls remained constant during the data capture period. FIG. 15A to FIG. 15C When the sampling interval of the data presented in FIG. 1 is about 8.3 milliseconds, the time response curve in the entire experiment is as follows: Fig. 15C The signal shown for an exemplary cell in FIG captures the rising phase and exponential decay of many subsequent cells. FIG. 15A to FIG. 15C In the data presented in this paper, high frame rate recordings of subcellular domains reveal Fig.15A and Fig. 15B The electron microscope images show the easy visualization of Fig. 15C The number and intensity of discrete active areas quantified in the time response curves are shown.

[0166] FIG. 15A to FIG. 15C The data presented in the present invention collected at high frame rates with subcellular resolution demonstrate that cellular activity in discrete microdomains is consistent with bioelectrical or biochemical activity. Cellular activity in such microdomain regions is consistent with previous descriptions of localized vesicle release in TIRF microscopy. Additionally, the data also indicate the potential of various embodiments of the ChemFET sensor array-based systems taught by the present invention to assay for genetic or pharmacological interventions that could potentially control the mechanisms that underlie vesicle fusion and release in normal and diseased cell function.

[0167] As previously addressed in this article Figure 3 As described, the sensor array device properties that may be varied to provide various sensor array devices in accordance with the teachings of the present invention include pixel size and the rate at which data may be collected from the sensor array device. Fig.16 A similar summary is provided for cells as described in the Applications and Methods section herein.

[0168] By inspection Fig.16 , for an average diameter of 6 μm and an average area of ​​28 μm anchored on the sensor array surface 2 For MMM cells, the minimum contact area or occupied area corresponds to about 2 rows and about 3 pixels for the chip 1 device, and increases to 7 rows and 45 pixels for the chip 4 device. Similarly, for the average diameter of 15 μm and the average area of ​​177 μm anchored on the sensor array surface, 2 For HEPG2 cells, the minimum contact area or occupied area corresponds to about 4 rows and about 18 pixels for the chip 1 device, and increases to 18 rows and 285 pixels for the chip 4 device. Similarly, for the average diameter of 18 μm and the average area of ​​254 μm anchored on the sensor array surface, 2 The average diameter of HeLa cells was 19 μm and the average area was 284 μm. 2 For U-2OS cells, the minimum contact area or occupied area corresponds to between about 5 rows and about 26 pixels or about 6 rows and about 29 pixels for the Chip 1 device, and increases to between about 21 rows and 410 pixels or about 22 rows and 458 pixels for the Chip 4 device, respectively. For the average diameter of 25 μm and the average area of ​​491 μm anchored on the sensor array surface 2 For HASM cells, the minimum contact area or occupied area corresponds to about 7 rows and about 50 pixels for the chip 1 device, and increases to 29 rows and 792 pixels for the chip 4 device. The cell body of the rat neonatal hippocampal neural (RNH) cell can be located at about 20 μm and 314 μm. 2 The average area is about 100 μm and 7,854 μm 2 For each RNH cell anchored on the sensor array surface, the minimum contact area or occupied area of ​​the RNH cell body can range from about 6 rows and about 32 pixels to about 30 rows and about 803 pixels for the Chip 1 device, and increases to about 24 rows and 506 pixels to about 118 rows and 12,668 pixels for the Chip 4 device. Fig.16 Inspections such as Figure 3 As noted, the trend is that pixel coverage increases with increasing cell size and decreasing pixel size.

[0169] From a pixel perspective, the pixel coverage percentage column is the percentage of the area of ​​the cell covered by a single pixel. For MMM cells anchored on the sensor array surface, a single pixel corresponds to 33% coverage for the Chip 1 device, while a single pixel corresponds to 2% coverage for the Chip 4 device. For HEPG2 cells anchored on the sensor array surface, a single pixel corresponds to 6% coverage for the Chip 1 device, while a single pixel corresponds to 0.4% coverage for the Chip 4 device. In contrast, for HeLa cells anchored on the sensor array surface, a single pixel corresponds to 4% coverage for the Chip 1 device, while a single pixel corresponds to 0.2% coverage for the Chip 4 device. For U-2OS cells anchored on the sensor array surface, a single pixel corresponds to 3% coverage for the Chip 1 device, while a single pixel corresponds to 0.2% coverage for the Chip 4 device. For individual RNH cells anchored on the sensor array surface, on the Chip 1 device, single pixel coverage can range from 3% for RNH cells with an average diameter of 20 μm to 0.1% coverage for RNH cells with an average diameter of 100 μm. In contrast, for the Chip 4 device, a single pixel corresponds to a coverage range of 0.2% for RNH cells with an average diameter of 20 μm to 0.008% for RNH cells with an average diameter of 100 μm. Fig.16 Inspections such as Figure 3 As noted, the trend is that the percentage of cell coverage per pixel decreases with increasing cell size and decreasing pixel size.

[0170] Taking into account Fig.16 The pixel coverage of the exemplary sensor array device of the present invention can be selected for each average cell diameter as presented in the table. The relevance of the device properties to the cell lines used in various applications of the present invention falls into the following categories: Figure 3 within the range described in . In this regard, for cells of about 5 μm to about 100 μm, the sensor array device can be selected to provide a coverage range of about 8 pixels within 3 rows of pixels to about 12,668 pixels within about 118 rows of pixels for the corresponding occupied area of ​​the cells anchored on the sensor array surface. Within the range of cell sizes described, the pixel size can vary such that each pixel of the selected sensor array device can cover from about 12% of the cell to about 0.008% of the cell. Based on Figure 3 surround Fig.16From the data presented in, it is apparent that, for any cell size, an exemplary sensor array device can be selected that can provide a substantial number of sensors relative to the contact area that a cell can occupy on the sensor array device. The spatial resolution that can be provided by each sensor array device taught by the present invention can allow subcellular discrimination of signals; thus providing subcellular analysis.

[0171] Similarly, with Figure 3 The content presented in Fig.16 It is evident from the last column that for target regions of interest across a range of cell sizes, a substantial number of frames per second can be collected, providing data collection comfortably in the kHz range. Recall that in the last column, the maximum frame rate at which data can be collected for a small fraction of the rows covered by the cells is presented, as derived by dividing the maximum frame rate per row by the rows per cell diameter.

[0172] For MMM cells anchored on the sensor array surface, data can be collected at a maximum frame rate of 37,500 fps for the Chip 1 device, while data can be collected at a maximum frame rate of 23,142 fps for Chip 4. For HEPG2 cells anchored on the sensor array surface, data can be collected at a maximum frame rate of 18,750 fps for the Chip 1 device, while data can be collected at a maximum frame rate of 9,000 fps for Chip 4. For HeLa cells anchored on the sensor array surface, data can be collected at a maximum frame rate of 15,000 fps for the Chip 1 device, while data can be collected at a maximum frame rate of 7,714 fps for Chip 4. Similarly, for U-2OS cells anchored on the sensor array surface, data can be collected at a maximum frame rate of 12,500 fps for the Chip 1 device, while data can be collected at a maximum frame rate of 7,364 fps for Chip 4. For HASM cells anchored on the sensor array surface, data can be collected at a maximum frame rate of 10,714fps for the Chip 1 device, and at a maximum frame rate of 5,586fps for Chip 4. Finally, for individual RNH cells, the body of such cells anchored on the sensor array surface, data can be collected at a maximum frame rate of between about 2,500fps to about 12,500fps for the Chip 1 device, and at a maximum frame rate of between about 1,373fps to about 6,750fps for Chip 4.

[0173] According to the teachings of the present invention, Fig.16 It is clear from the content presented in FIG. 1 that the respective maximum frame rates for the cell lines used in the respective applications according to the teachings of the present invention fall within the range Figure 3The range of about 1,250fps to about 75,000fps presented in Figure 3 and Fig.16 From an inspection of the , the trend is that the frame rate decreases as cell size increases and pixel size decreases, which is consistent with an inverse relationship between the region of interest selected and the rate at which data can be collected. Therefore, the frame rate can be improved by selecting a smaller subset of pixels to monitor (i.e., by windowing down the area of ​​the sensor array device over which data is collected) or by selecting a device with a desirable frame rate that matches the application.

[0174] What has been described herein relates to systems, applications and methods for various cell analysis systems based on chemical field effect transistor (ChemFET) sensor array technology. The various cell analysis systems taught by the present invention can measure the electrical and metabolic activities of a single living cell using subcellular addressability and simultaneous data acquisition for between about 10 cells and about 500,000 cells in a single analysis. The various sensor array devices taught by the present invention can have a sensor array with between 20 million and 660 million ChemFET sensors built into a massively parallel array, wherein the spacing of the sensors of the sensor array can be between about 3.36 μm and about 850 nm, respectively. Further, the various cell analysis systems taught by the present invention can provide simultaneous measurements of cells at data acquisition rates in the kilohertz (kHz) range. Further, the fully automated fluidic system integrated into the various cell analysis systems taught by the present invention allows for strictly controlled applications, such as chemical agents in various studies for indicating measurements of cellular responses to various types of chemical interrogations. Because each ChemFET sensor array taught by the present invention can detect chemical analytes as well as detect changes in cell membrane potential, controlled electrical stimulation can also be used to interrogate cells in each cell analysis system taught by the present invention.

[0175] Although the preferred embodiments of the present teachings have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only by way of example. Without departing from the present teachings, those skilled in the art will now expect numerous variants, changes and substitutions. It should be understood that the different alternatives of the various embodiments described herein may be used to practice what is disclosed herein. It is contemplated that the following claims define the scope of the present disclosure and thus cover systems, devices, applications and methods within the scope of these claims and their equivalents.

Claims

1. A cell analysis system, comprising: a sensor array device comprising a chemical field effect transistor (ChemFET) sensor array, wherein the ChemFET sensor array comprises at least 20 million sensors; a flow cell mounted on the sensor array device, wherein the flow cell is configured to provide a fluid interface between the sensor array device and the cell analysis system; a reference electrode in fluid communication with the flow cell, wherein the reference electrode is configured to provide a stable reference potential to the ChemFET sensor array; as well as An array controller is configured to control the number of rows from which data is acquired and a sampling rate of acquisition of the sensor array device.

2. The cell analysis system according to claim 1, wherein the ChemFET sensor is an ion selective field effect transistor (ISFET) sensor.

3. The cell analysis system according to claim 2, wherein the ISFET sensor is selective for hydrogen ions. 4 . The cell analysis system of claim 1 , wherein the cell analysis system further comprises a fluid system configured for controllable fluid delivery through the flow cell.

5. The cell analysis system according to claim 1, wherein the surface is treated with a coating.

6. The cell analysis system according to claim 5, wherein the coating is selected from the group consisting of basement membrane matrix preparations, poly-D-lysine, laminin, and combinations thereof.

7. The cell analysis system of claim 1 , wherein the array controller includes a programmable clock generator integrated circuit to provide control of different sensor array types, operating modes, and acquisition rates for detecting changes in the state of cells capacitively coupled to a sensing surface of a selected device.

8. The cell analysis system of claim 1, wherein the sensor array device further comprises an array of microwell structures disposed on the ChemFET sensor array, wherein each microwell structure is coupled to at least one sensor.

9. A method for cell analysis, the method comprising: Plating the cell sample on a sensor array surface of a chemical field effect transistor (ChemFET) sensor array device comprising a ChemFET sensor array, wherein the ChemFET sensor array comprises at least 20 million sensors; selecting a region of interest from an image of the cell sample plated on the sensor array surface, wherein selecting the region of interest defines a frame rate for data acquisition; running a cell analysis application to acquire data of a selected region of interest at a defined frame rate; as well as Results for the selected region of interest are displayed for the cell analysis application.

10. A method for cell imaging using a ChemFET sensor array-based system, the method comprising: Plating the cell sample on a sensor array surface of a chemical field effect transistor (ChemFET) sensor array device mounted in a flow cell, wherein the ChemFET sensor array comprises at least 20 million sensors, performing an experiment on the cell sample, wherein the ChemFET sensor array-based system is configured to output a signal for each sensor in the ChemFET sensor array during the experiment; as well as An electron microscope image of the experiment is displayed, wherein the electron microscope image includes output signals of each sensor in the ChemFET sensor array at selected times during the experiment.

Images