Cell analysis using a CHEMFET sensor array-based system

Abstract

Various cellular analysis systems of the present teachings can measure the electrical and metabolic activity of single living cells with intracellular addressability and simultaneous data acquisition of about 10 cells to about 500,000 cells in a single analysis. Various sensor array devices of the present teachings can have sensor arrays with 20 million to 660 million ChemFET sensors integrated into massively parallel arrays, providing simultaneous measurement of cells at data acquisition rates in the kilohertz (kHz) range. Because various ChemFET sensor arrays of the present teachings can detect chemical analytes and changes in cell membrane potential, various cellular analysis systems of the present teachings also provide controlled chemical and electrical interrogation of cells. Frame rates can be increased by selecting a smaller subset of pixels to monitor, i.e., by windowing down the area of ​​the sensor array device from which data is collected. [Selected Figure] Figure 6A

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 730,960, filed Sep. 13, 2018, and U.S. Provisional Application No. 62 / 848,842, filed May 16, 2019. Both applications specified in this section are hereby incorporated by reference in their entireties.

[0002] Summary The electrophysiological and metabolic phenotypes of cell behavior are important means of research in modern cell biology, and there are significant unmet needs for researchers in fields such as, for example, artificial tissues, primary battery types, and oncology discoveries. The success of measuring cell excitability is currently hindered by the cumbersome patch - clamp method and microelectrode voltammetry methods, while the investigation of metabolic functions remains hampered by the technical limitations of large - scale populations and biochemical workflows and is inefficiently deployed to access important metrics of single - cell behavior.

[0003] Measuring all possible interactions that can occur between the 20,000 genes and 200,000 proteins within a cell is impossible or not practical, but it is becoming increasingly clear that research on model systems at the single - cell level will provide deeper insights into normal cell physiology and changes between pathological states such as cancer, diabetes, and neurodegenerative diseases. Recent applications highlighting the advantages of single - cell research vs cell - population research include, for example, transcriptomics and aging research, intracellular signaling pathway research, and measurement of bioenergetics of metabolism.

[0004] Accordingly, there is a need in the art for systems and methods that can provide end - users with tools for investigating single cells within a population and enable high - throughput multiplexed discrete measurements of cell - level time responses.

[0005] A better understanding of the features and advantages of the present teachings will be obtained by reference to the following detailed description of exemplary embodiments and the accompanying drawings.

Brief Description of the Drawings

[0006]

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Mode for Carrying Out the Invention

[0007] The systems and methods of the present teachings relate to cell analysis systems based on chemical field effect transistor (ChemFET) sensor array technology. The various cell analysis systems of the present teachings can measure the electrical and metabolic activities of a single live cell with intracellular addressability and simultaneous data acquisition of about 10 cells to about 500,000 cells in a single analysis. The various sensor array devices of the present teachings can have a sensor array with 20 million to 660 million ChemFET sensors incorporated in a massively parallelized array, and the sensors of the sensor array can each have a pitch of about 3.36 μm to about 850 nm. Further, the various cell analysis systems of the present teachings can provide simultaneous measurements of cells at a data acquisition rate in the kilohertz (kHz) range. Moreover, the fully automated fluid systems integrated into the various cell analysis systems of the present teachings enable precisely controlled applications of chemicals, for example, used in various studies where measurements of cell responses to various types of chemical investigations are shown. The various ChemFET sensor arrays of the present teachings can detect chemical analytes and detect changes in cell membrane potential, so cells can also be examined in the various cell analysis systems of the present teachings using controlled electrical stimulation.

[0008] Chemical Field Effect Transistor (ChemFET) Array for Cell Analysis FIG. 1 generally shows a block diagram of exemplary components of a cell analysis system 100 according to the present teachings. As depicted in FIG. 1, the cell analysis system 100 can include various fluid systems, as well as an array controller 50, a user interface 60, and a sensor array device assembly 115. As described in more detail herein, various cell analyses can be performed using the sensor array device 110 of the sensor array device assembly 115.

[0009] Figure 2A generally shows a realistic depiction of the cell analysis system 100A. As depicted in Figure 2A, a cell 5 with a nucleus 2 and a cell membrane 4 is placed on a plurality of subsets of microwells, thereby defining the area of contact or footprint that the cell 5 occupies on the corresponding subset of sensors. As listed herein, "contact area" and "footprint" can be used interchangeably. The cell analysis system 100A shares many of the same features as described for the schematic depiction of the cell analysis system 100 of Figure 1. The cell analysis system 100A of Figure 2A can include a reagent fluid system 10 and a wash solution fluid system 20. The reagent fluid system 10 can include a plurality of reagent containers, such as reagent containers 11A - 11D of Figure 2A. Each reagent container can be in fluid communication with a reagent fluid line, such as reagent fluid lines 13A - 13D of Figure 2A. The flow from each reagent fluid line of the cell analysis system of the present teachings can be controlled by valves, such as reagent fluid line valves 15A - 15D of Figure 2A. The wash solution fluid system 20 can include a wash solution container 21 that can hold a wash solution of a known electrolyte composition, as well as a wash solution fluid line 23, a wash solution fluid line valve 25, and a reference electrode 27 within the wash solution fluid line 23. As described in more detail herein, the reference electrode 27 can provide a stable reference voltage 127 to the sensors within the sensor array device. Thus, the sensor array device 110 of Figure 2A can be in fluid communication with the reagent fluid system 10 and the wash solution fluid system 20. Although not shown in Figure 2A, additional electrodes that can communicate with the sensor array device can be utilized to provide electrical stimulation to the cells on the sensor array, such as the cell 5 of Figure 2A.

[0010] The sensor array device 110 can include a sensor array or pixel array 120. As listed herein, the terms "sensor" and "pixel", as well as the terms "device" and "chip", and derivatives of these terms can be used interchangeably. Additionally, "sensor array" and "ChemFET sensor array", and derivatives thereof can be used interchangeably. Although a two-dimensional array is depicted as a normal array in FIG. 2A, various embodiments of the sensor arrays of the present teachings can be arranged in various array shapes, such as a hexagonal close-packed shape. The sensor array device 110 can include a microwell array 122, which depicts each microwell that cooperatively engages with each sensor or pixel within the sensor array 120, as shown in FIG. 2A. As a result, each of the microwells 122A1 through 122A6 cooperatively engages with the corresponding sensors 120A1 through 120A6. However, in various embodiments of the sensor array devices of the present teachings, there can be two or more pixels per well. As described in more detail herein, various types of sensor array devices of the present teachings can be fabricated with defined but different microwell depths. Still other types of sensor array devices of the present teachings may not have a microwell structure formed on the sensor array. Each sensor of the sensor array 120 can have a sensing surface that is in fluid communication with the fluid within the microwell array 122. In various embodiments of the cell analysis system 100 of the present teachings, each sensor of the sensor array 120 can be a chemical field effect transistor (ChemFET), and each sensor within the sensor array 120 can include at least one chemically sensitive field effect transistor. According to the present teachings, the sensor array 120 can include ChemFETs that are fabricated with a sensing surface selectively modified for the analysis of target chemical species of interest in cell biology, such as glucose, sucrose, lactate, and urea, for example.As another non-limiting example, an ion-sensitive field effect transistor (ISFET) can have a sensing surface that is modified to be selective for various ions of interest, particularly for various cell metabolism studies such as hydrogen, potassium, calcium, and chloride.

[0011] In that regard, the inventors recognize that various embodiments of the cell analysis system of the present teachings can be used to monitor changes in the cellular electrophysiology and metabolism of cells subjected to, for example, any of various conditions or stimuli. Moreover, the inventors recognize that changes in the state of a cell that can cause a change in the potential of the sensing surface of a ChemFET sensor can be monitored by the sensors of various embodiments of the ChemFET sensor array of the present teachings. For example, the inventors recognize that when the potential across the cell membrane changes in response to a chemical or electrical stimulus, the change in the potential across the cell membrane can be detected by the sensors of various embodiments of the ChemFET sensor array of the present teachings, such that the cell is capacitively coupled to the sensing surface of the sensor. Additionally, any change, such as a change in cell metabolism that can cause a change in the potential of the sensing surface of a ChemFET sensor, can be detected by the sensors of various embodiments of the ChemFET sensor array of the present teachings. As described in more detail herein, such changes can be detected locally, for example, in the contact area or footprint of a cell immobilized on the sensor array surface, such as in the case of cell efflux that may flow from the cell in response to a state or stimulus, or can be detected in an area not related to the cell footprint.

[0012] In experiments monitoring cell responses to various stimuli in various embodiments of the ChemFET sensor array device of the present teachings, the data collected can be presented to an end user in a number of formats. In one format, the time response is presented as detector counts that can be readily correlated with millivolt (mV) changes in the sensed surface potential as a function of time. In another format, for any of the selected times over the course of a selected application, the spatial visualization of the cells can be presented as an electron microscope image. The inventors have recognized that electron microscopy imaging can be useful as a general tool for visualizing cells on, for example, a sensor array, since it is based on various responses that can be induced in live cells. For example, by viewing an electron microscope image of cells immobilized on a sensor array, an end user can select regions of interest as part of the application configuration prior to running an experiment. As described in more detail herein, by windowing down on selected regions of the sensor array device, the data rate of the experiment is increased. According to the present teachings, substantial pixel coverage over the cell footprint combined with a high data rate can provide, for example, intracellular monitoring of action potentials of various excitable cells where a data rate in the sub-millisecond range may be required.

[0013] In FIG. 2B, a partial cross-sectional view of the sensor array 120 is depicted with a first sensor 120-1 and a second sensor 120-2. In various embodiments of the sensor array device of the present teachings, the sensor array 120 can include a floating gate top 130 coupled to a sensor floating gate structure 140. Alternatively, in various embodiments of the sensor array device of the present teachings, the sensor array 120 can include a sensor floating gate structure 140. As described in more detail herein, the floating gate top 130 can include an upper metal layer, a sensor plate 132, and a metal via 134 formed in a dielectric 135.

[0014] The sensor floating gate structure 140 can have a metal layer 136 coupled to the sensor plate 132 through the metal via 134. The metal layer 136 is the top 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 material within a layer of dielectric material 150. The sensors 120-1 and 120-2 can include conductive terminals that include source / drain regions 142 and source / drain regions 144 within the semiconductor substrate 160. The source / drain regions 142 and source / drain regions 144 include doped semiconductor material having a conductivity type different from that of the substrate 160. For example, the source / drain regions 142 and source / drain regions 144 can include doped P-type semiconductor material, and the substrate 160 can include doped N-type semiconductor material. The channel region 152 separates the source / drain regions 142 and the source / drain regions 144. The floating gate structure 140 covers the channel region 146 and is separated from the substrate 160 by the gate dielectric 152. The gate dielectric 152 can be, for example, silicon dioxide. Alternatively, other suitable dielectrics, such as materials having a higher dielectric constant, silicon carbide (SiC), silicon nitride (Si3N4), silicon oxynitride (Si2N2O), aluminum nitride (AlN), hafnium dioxide (HfO2), tin oxide (SnO2), cerium oxide (CeO2), titanium oxide (TiO2), tungsten oxide (WO3), aluminum oxide (Al2O3), lanthanum oxide (La2O3), gadolinium oxide (Gd2O3), and any combination thereof, can be used for the gate dielectric 152.

[0015] As described in more detail herein, the sensing surface 126S of the sensor plate 132 can function as a sensor surface for monitoring changes in cell electrophysiology and metabolism of cells subjected to, for example, any of a variety of conditions or stimuli. In that regard, cells 5 shown in FIG. 2B as a partial cross-section of a cell are depicted as being disposed on the sensor plates 132 of sensors 120-1 and 120-2. Cells 5 are depicted as being fixed to the sensor array 120 via a surface coating 124. The surface coating 124 can be any cell-compatible material, such as various biopolymeric materials including poly-D-lysine, laminin, fibronectin, collagen, and combinations thereof, as well as various preparations of the extracellular matrix (ECM). An end user can perform applications using the cell analysis system of the present teachings, in which various reagents and solutions can flow controllably over the surface of the sensor array 120, as indicated by the arrows at the top of FIG. 2B.

[0016] Sensors 120-1 and 120-2 respond to changes in the surface potential of the ion layer 128 proximate to the sensing surface 126S, which can cause a change in the voltage of the floating gate 140. Thus, the applied reference voltage ensures that the voltage of the floating gate exceeds the threshold voltage, under the condition that a small change in the floating gate voltage can cause a current to flow through the channel region 146, as previously described herein with respect to FIG. 2A, resulting in an output signal of sensors 120-1 and 120-2. In that regard, changes to the surface potential of the ion layer 128 can be measured, for example, by measuring the current within the channel region 146 between the source region 142 and the drain region 144. Thus, sensors 120-1 and 120-2 can be used to directly provide a current-based output signal on an array line connected to the source region 142 or the drain region 144, or to indirectly provide a voltage-based output signal with additional circuitry.

[0017] As described herein, changes in the state of cell 5 that can vary the surface potential in ion layer 128 can be monitored by various embodiments of the ChemFET sensor array device of the present teachings. With respect to the output signal, cell activity that can increase the surface potential results in a positive amplitude output signal of the ChemFET sensor, while cell activity that can decrease the surface potential results in a negative amplitude output signal of the ChemFET sensor. In that regard, changes in the state of the cell that can vary the surface potential of the ChemFET sensor can result in a measurable output signal. For example, metabolic activity that can increase the ion concentration of a cationic species, where the ISFET sensor is selective, causes an increase in the surface potential. The result is a positive amplitude output signal of that ISFET sensor. Conversely, metabolic activity that can decrease the ion concentration of a cationic species, where the ISFET sensor is selective, causes a decrease in the surface potential. The result is a negative amplitude output signal of that ISFET sensor. In another example, the surface potential can be altered by capacitive coupling of cell 5 to sensor array 120, such that as the potential across cell membrane 4 changes in response to chemical or electrical stimuli, the change in potential across the cell membrane can be detected by ChemFET sensors 120-1 and 120-2.

[0018]

Table 1

[0019] Various embodiments of the sensor array devices of the present teachings can have from about 20M to about 660M pixels, with a center-to-center spacing, or pitch, between each sensor of from about 850 nm to about 3.36 μm. With respect to data collection, the collection of sensor output signals from all sensors in the array constitutes a frame of data. In the various sensor array devices of the present teachings at from about 20 million pixels to about 660 million pixels, the frame is a fairly large-sized data file that is collected in units of Hertz (Hz) as frames per second. Further, there is an inverse relationship between the selected region of interest representing the number of pixels and the rate at which data can be collected, such that by selecting a smaller subset of pixels to monitor, i.e., by windowing down the region of the sensor array device from which data is collected, the frame rate can be increased. The effect of windowing down is illustrated in Table I by comparison of the value entered in the penultimate column, which is the maximum frame rate for collecting data from the entire device, with the value entered in the last column, which is the maximum frame rate for collecting data from a single row of the device. Thus, by windowing down to collect data from a single row, the frame rate is substantially increased.

[0020] Additionally, as provided in Table I, the only difference between Device A and Device B is the total number of sensors per device, and the number of sensors per Device B as compared to Device A is twice as many. As shown in Table 1, the frame rate for Device B is half that of Device A, consistent with the inverse relationship between the number of pixels and the rate of data that can be collected. Thus, a device with a desired frame rate suitable for the application can be selected.

[0021] Thus, in various embodiments of the cell analysis system of the present teachings, an end user can select a sensor array device from various sensor array devices having different attributes that can be matched to a cell analysis of interest. For example, a sensor array device used for detecting the electrophysiological activity of cells can have different attributes from a sensor array device used for detecting a specific analyte for metabolic research.

[0022] Sensor array device attributes that can be varied to provide the various sensor array devices of the present teachings include pixel dimensions, as well as the rate at which data can be collected from the sensor array device. FIG. 3 provides an overview of five categories of cells by size in relation to four exemplary sensor array devices of various sensor (pixel) dimensions, as given in Table 1 for devices B through E. The five categories of cells are descriptively identified as well as by average diameter and average footprint.

[0023] Upon scrutiny of FIG. 3, for extremely small cells immobilized on a sensor array surface with an average diameter of 5 μm and an average area of 20 μm 2 the minimum contact area or footprint corresponds to approximately 1 row and approximately 2 pixels for the Chip 1 device and increases to 6 rows and 32 pixels for the Chip 4 device. Similarly, for small cells immobilized on a sensor array surface with an average diameter of 10 μm and an average area of 78 μm 2 the minimum contact area or footprint corresponds to approximately 3 rows and approximately 8 pixels for the Chip 1 device and increases to 12 rows and 126 pixels for the Chip 4 device. For medium-sized cells immobilized on a sensor array surface with an average diameter of 25 μm and an average area of 491 μm 2 the minimum contact area or footprint corresponds to approximately 7 rows and approximately 50 pixels for the Chip 1 device and increases to 29 rows and 792 pixels for the Chip 4 device. For large cells immobilized on a sensor array surface with an average diameter of 50 μm and an average area of 1,964 μm 2Large cells fixed on the surface of the sensor array can have a minimum contact area or footprint corresponding to approximately 15 rows and approximately 201 pixels in Chip 1, increasing to 59 rows and 3,168 pixels in the Chip 4 device. Finally, for giant cells fixed on the surface of the sensor array, with an average diameter of 100 μm and an average area of 7,854 μm 2 For giant cells fixed on the surface of the sensor array, the minimum contact area or footprint corresponds to approximately 30 rows and approximately 803 pixels in the Chip 1 device, increasing to 118 rows and 12,668 pixels in the Chip 4 device. From the scrutiny of Figure 3, the trend is that pixel coverage tends to increase with increasing cell size and decreasing pixel size.

[0024] From the perspective of pixels, the column of percent pixel coverage is the percentage of the cell area covered by a single pixel. For extremely small cells fixed on the surface of the sensor array, a single pixel corresponds to 50% coverage in the Chip 1 device, while in the Chip 4 device, a single pixel corresponds to 3% coverage. Similarly, for small cells fixed on the surface of the sensor array, a single pixel corresponds to 12% coverage in the Chip 1 device, while in the Chip 4 device, a single pixel corresponds to 0.8% coverage. For medium-sized cells fixed on the surface of the sensor array, a single pixel corresponds to 2% coverage in the Chip 1 device, while in the Chip 4 device, a single pixel corresponds to 0.1% coverage. Large cells fixed on the surface of the sensor array can have a single pixel corresponding to 0.5% coverage in the Chip 1 device, while in the Chip 4 device, a single pixel corresponds to 0.03% coverage. Finally, for giant cells fixed on the surface of the sensor array, a single pixel corresponds to 0.1% coverage in the Chip 1 device, while in the Chip 4 device, a single pixel corresponds to 0.008% coverage. From the scrutiny of Figure 3, the trend is that the percentage of cell coverage per pixel tends to decrease with increasing cell size and decreasing pixel size.

[0025] Considering what is presented in the table of FIG. 3, the selection of pixel coverage of the exemplary sensor array devices of the present teachings can be done for various average cell diameters. For example, for cells of about 5 μm to about 100 μm, the selection of the sensor array device can be done to provide a coverage from about 8 pixels beyond 3 rows of pixels to 12,668 pixels beyond 118 rows of pixels for the corresponding footprints of the cells fixed on the sensor array surface. Since the pixel size can vary over that range of cell sizes, 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 the data presented in FIG. 3, it is clear that for any cell size, an exemplary sensor array device can be selected that can provide a significant number of sensors related to the contact area that the cell can occupy on the sensor array device. The spatial resolution that can be provided by the various sensor array devices of the present teachings can enable intracellular discrimination of signals and, therefore, provides intracellular analysis.

[0026] Regarding data collection, in the various cell analysis systems of the present teachings, the collection of sensor output signals from all sensors in the array constitutes a frame of data. Considering that the various sensor array devices of the present teachings can have from about 20 million pixels to about 660 million pixels, the frames of data from the various sensor array devices of the present teachings are data files of a significant size. Additionally, the various cell analysis systems of the present teachings include a control circuit coupled to the sensor array device that is configured to generate a significant number of data frames per second from the sensor array device. Moreover, since there is an inverse relationship between the selected region of interest and the rate at which data can be collected, the frame rate can be increased by selecting a smaller subset of pixels to monitor, i.e., by windowing down the region of the sensor array device from which data is collected.

[0027] For example, referring to Table I, a sensor array device with 40 million pixels can collect data at a frame rate of approximately 120 frames per second (fps). Subsequently, when a region of interest of 20 million pixels is selected, data at a frame rate of approximately 240 frames per second (fps) can be collected, and when a region of interest of a single sensor array row is selected, data at a frame rate of approximately 75,000 frames per second (fps) can be collected. Specifically, for the exemplary sensor array device presented in FIG. 3, the maximum frame rate per row of the sensor is provided in the penultimate column. In the last column, the maximum frame rate at which data can be collected for the fractional part of the row covered by the cell, which is derived by dividing the maximum frame rate per row by the row per cell diameter, is presented.

[0028] As can be seen from the inspection of the last column in Figure 3, a significant number of frames per second can be collected for the target area of interest over a variety of cell sizes, providing comfortable data collection within the kHz range. For the extremely small cells fixed on the sensor array surface, data can be collected at a maximum frame rate of 75,000 fps with the Chip 1 device, while with the Chip 4, data can be collected at a maximum frame rate of 27,000 fps. Similarly, for the small cells fixed on the sensor array surface, data can be collected at a maximum frame rate of 25,000 fps with the Chip 1 device, while with the Chip 4, data can be collected at a maximum frame rate of 13,500 fps. For the medium-sized cells fixed on the sensor array surface, data can be collected at a maximum frame rate of 10,714 fps with the Chip 1 device, while with the Chip 4, data can be collected at a maximum frame rate of 5,587 fps. The large cells fixed on the sensor array surface can have a maximum frame rate of 5,000 fps with the Chip 1 device, while with the Chip 4, data can be collected at a maximum frame rate of 2,746 fps. Finally, for the extra-large cells fixed on the sensor array surface, data can be collected at a maximum frame rate of 2,500 fps with the Chip 1 device, while with the Chip 4, data can be collected at a maximum frame rate of 1,373 fps.

[0029] From the inspection of Figure 3, the trend is that the frame rate tends to decrease with the increase in cell size and the decrease in pixel size, which is consistent with an inverse proportional relationship between the selected region of interest and the speed in the data that can be collected. Therefore, by selecting a smaller subset of pixels to monitor, that is, by windowing down the area of the sensor array device where data is collected, the frame rate can be increased. Additionally, referring to Table 1, a device with a desirable frame rate matching the application can be selected.

[0030] Additional sensor array device attributes that can be varied relate to various types of microwell structures that can be associated with the various sensor array devices of the present teachings. With respect to the microwell array 122 disposed on the sensor array 120 of FIG. 2A, the microwell array can be fabricated on the sensor array such that there can be at least one sensor or pixel per microwell. In various embodiments of the sensor array devices of the present teachings, there can be multiple sensors or pixels per microwell.

[0031] FIGS. 4A through 4C show generally enlarged partial cross-sectional views of the upper portion of a sensor array device, such as the sensor array 110 of FIG. 2A. The sensor array 120 of FIG. 4A is a partial cross-sectional view of an embodiment of a sensor array of the present teachings in which a microwell array 122A is fabricated. An exemplary microwell 122A1, which illustrates all of the microwells of the microwell array 122A, has sidewalls 124A, which are containment structures that can prevent various chemical analytes from diffusing from the sensor, and can have, for example, an analyte of interest released by a cell proximal to the sensor. The sensor array 120 of FIG. 4B is a partial cross-sectional view of an embodiment of a sensor array of the present teachings in which a microwell array 122B is formed. The exemplary microwell 122B1 has sidewalls 124B that provide a microwell array having microwells that are substantially shallower than the microwell array 122A of FIG. 4A. FIG. 4C is a partial cross-sectional view of an embodiment of a sensor array of the present teachings that does not have a microwell array fabricated on the sensor array 120C.

[0032] Figure 4D is a comparison of U-2OS cell responses in a welled array device (shown in FIGS. 4A and 4B) vs a well-less array device (shown in FIG. 4C), where the cells are investigated under conditions for depolarization of the membrane potential and then return to the resting potential. Considering that the measured change in cell potential decreases as the square of the distance of the cell from the sensor, the distance can be minimized by using a sensor array with shallow microwells, such as the microwell array 122B of FIG. 4B, or by not using any sensor array such as the sensor array 120 of FIG. 4C at all. The comparison shown in FIG. 4D exemplifies an increase in the magnitude of the response of the well-less device.

[0033] In various types of sensor array devices, the center-to-center spacing, or pitch, between each sensor can be from about 850 nm to about 3.36 μm, while the sensor plate 132 can have a width from about 600 nm to about 3.1 μm. According to the present teachings, the width of the microwells at the bottom of the microwells on the sensor plate cannot exceed about 90% of the width of the sensor plate 132. As a non-limiting example, in a sensor plate with a width of about 600 nm, such as the sensor plate 132 in FIGS. 4A - 4C, various microwell arrays can have a width of about 540 nm at the bottom of the microwells on the sensor plate, while in a sensor plate with a width of 3.1 μm, various microwell arrays can have a width of about 2.8 μm at the bottom of the microwells on the sensor plate. According to the present teachings, in various embodiments of the microwell array, the ratio of the width to the height of the microwells can be from about 1:2 to about 1:4. As a non-limiting example, in various microwell arrays having microwells with a width of about 540 nm, the height of the microwells can be up to about 1.1 μm, while in various microwell arrays having microwells with a width of about 2.8 μm, the height of the microwells can be up to about 5.6 μm. As an additional non-limiting example, in various microwell arrays having microwells with a width of about 540 nm, the height of the microwells can be up to about 2.2 μm, while in various microwell arrays having microwells with a width of about 2.8 μm, the height of the microwells can be up to about 11.2 μm. Although non-limiting examples are given, various embodiments of the microwell arrays of the present teachings can have any ratio of width to height from about 1:2 to about 1:4.

[0034] In FIGS. 4A through 4C, a partial cross-sectional view of the upper portion of the floating gate structure is depicted as the floating gate upper 130. Various embodiments of sensor arrays incorporating the floating gate structure are described, for example, in U.S. Patent No. 9,128,044 and U.S. Patent No. 9,841,398, both of which are hereby incorporated by reference in their entirety.

[0035] The floating gate upper part 130 from FIGS. 4A to 4C can include an upper metal layer, a sensor plate 132, and metal vias 134 and a metal layer 136, all of which are formed on a dielectric substrate 135. The passivation layer 126 can be deposited to form a continuous top layer on the surface defining each micro well of the micro well array. The sensing surface 126S is a part of the passivation layer formed on the sensor plate 132. The metal layers 132, 134, and 136 can be appropriate metal materials or, for example, alloys of titanium, silver, gold, platinum, and tungsten. The dielectric substrate 135 can be a dielectric material such as silicon dioxide or silicon nitride. The passivation layer 126 can be a metal oxide layer such as titanium oxide, titanium nitride, and titanium oxynitride. In various embodiments of the ChemFET device, a change in potential at the solid-liquid interface formed between a sensing layer, such as the sensing layer 126S from FIGS. 4A to 4C, and a solution in contact with the sensing layer can cause a change in the voltage on the floating gate. The applied reference voltage ensures that the voltage of the floating gate exceeds the threshold voltage under the condition that a small change in the floating gate voltage results in an output signal for sensors, such as sensors 120-1 and 120-2 of the sensor array 120 in FIG. 2B, as previously described herein with respect to FIG. 2A.

[0036] Various modifications can be made to the composition of the sensing surface so as to provide selectivity for various analytes of interest, e.g., analytes of interest for various cell metabolism and nutrition studies. For example, the detection of various ions can be achieved through the use of the passivation layer itself or through the use of ionophores coated on the passivation layer. For example, hydrogen ions can be detected without changing the sensing layer, such as the sensing layer 126S from FIGS. 4A to 4C, which can be titanium oxide, titanium nitride, or titanium oxynitride. Potassium ions can be selectively detected, for example, by coating the sensing layer with valinomycin or salinomycin. Sodium ions can be selectively detected, for example, by coating the sensing layer with monensin, nystatin, or a synthetic ionophore, SQI-Pr (CAS#1022595-16-9). Calcium ions can be selectively detected, for example, by coating the sensing layer with ionomycin, calcimycin, or ETH 1001 (CAS#58801-34-6). Additionally, in various assays, the selectivity of the ionophore may not need to be directed towards a single species, but rather, it can bind to multiple species of ions within the genus of ions. 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 present teachings, various ionophores having selectivity for a genus of ions can be used in applications where single ion specificity is not required or where other ions to which the compound binds are unlikely to be present or generated.Additionally, various classes of photocurable polymers can provide selectivity for various ions such as potassium, calcium, ammonium, chloride, nitrate, and various analytes of interest in cell biology research such as glucose, sucrose, urea, etc. (see, e.g., Sensors 2009, 9, 7097-7100).

[0037] By varying the composition of the sensing surface portion of the passivation layer in contact with the sensor plate, in addition to providing detection selectivity for various analytes, the passivation layer can be treated with a material that provides cell compatibility, as depicted in FIG. 2B. Additionally, as depicted in FIG. 2A, the sensor surface of a sensor array device having a microwell array disposed on the sensor array can be treated by coating the sensor array surface including the microwell array with a cell-compatible material. To coat various embodiments of the sensor array devices of the present teachings, an end user can select a specific material from various cell-compatible materials that may be most suitable for the cell line of interest and a series of experiments, and can coat the sensor array surface with the cell-compatible material prior to preparing a sensor array with a cell sample. Exemplary cell-compatible materials that can be coated on the sensor device surface include various biopolymeric materials such as poly-D-lysine, laminin, fibronectin, collagen, and combinations thereof, as well as various preparations of the extracellular matrix (ECM).

[0038] Providing a cell-compatible coating on the sensor array can provide an interface, as depicted in FIGS. 2A and 2B, on which various types of cells can be immobilized on the sensor array surface. Once the cells are stably immobilized, the cells can grow and divide, for example, by flowing a nutrient solution through a flow cell, or by providing a continuously refreshed nutrient solution.

[0039] Accordingly, the various sensor array devices of the present teachings can have various attributes that can be selected for target cell analysis, can provide various sensor array devices, and can have various attributes. Such attributes can include the presence or absence of a microwell array fabricated on the sensor array, the depth of each microwell within the microwell array for a sensor array device that includes the microwell array, the nature of the surface treatment of the sensing surface that provides selectivity for chemical analysis, a cell-compatible coating, the type of surface treatment of the sensor array device that provides pixel pitch and size, and the rate at which data can be collected. By way of non-limiting example, the various sensor array devices of the present teachings can have a microwell array disposed on the sensor array or can not have a microwell array disposed on the sensor array. The various sensor array devices of the present teachings can have a sensing surface that provides selectivity for various chemical analytes of interest in cell analysis. Such sensor array devices can be used, for example, in various cell metabolism and electrophysiology studies and can provide for monitoring of various spontaneous and timed cell activities.

[0040] Chemical field effect transistor (ChemFET) array-based system for cell analysis As previously described herein, FIG. 1 generally shows a block diagram of exemplary components of a cell analysis system 100 according to the present teachings. As depicted in FIG. 1, the cell analysis system 100 can include various fluid systems, as well as an array controller 50, a user interface 60, and a sensor array device assembly 115.

[0041] Regarding fluid delivery and control for performing various cell assays on a sensor array device 110, a cell assay system 100 can include a reagent fluid system 10, a wash solution fluid system 20, a fluid multiplexer system 30, and a valve controller 40. Additionally, since a flow cell 107 can define flow paths for various reagents and wash solutions on the sensor array device 110, the flow cell 107 is an integral part of the fluidics system of the cell assay system 100. According to the present teachings, the reagent fluid system 10 can include a plurality of reagent containers, such as reagent containers 11A - 11N, which can be arranged in controllable fluid communication with the sensor array device 110 via a flow cell inlet line 103A. The reagent fluid system 10 can include reagent fluid lines, such as reagent fluid lines 13A - 13N, corresponding to each of the reagent containers 11A - 11N, respectively. Additionally, each fluid reagent line can have fluid flow controlled by valves, such as reagent fluid line valves 15A - 15N, depicted in FIG. 1 as in-line valves for each of the reagent fluid lines 13A - 13N. The wash solution fluid system 20 can include a wash solution container 21, which can be arranged in controllable fluid communication with the flow cell inlet line 103A via a wash solution fluid line valve 25. The fluid multiplexer system 30 can include a fluid multiplexer system waste container 31, which is in fluid communication with a fluid multiplexer 35 via a fluid multiplexer fluid line 33.

[0042] As depicted in FIG. 1, the reagent fluid line valves 15A - 15N can be controlled by the valve controller 40. In that regard, the valve controller 40 can control the fluid flow from each of the reagent containers 11A - 11N to the fluid multiplexer 35. Additionally, as depicted in FIG. 1, the valve controller 40, as the cleaning solution fluid line valve 25 on the cleaning solution fluid line 23, can control the fluid flow from the cleaning solution container 21 of the cleaning solution fluid system 20 through the control of the cleaning solution fluid line valve 25 depicted in FIG. 1. As shown in FIG. 1, by the control lines from the valve controller 40 to each of the reagent containers 11A - 11N, the valve controller 40 can also activate the control of the pneumatic valves on the pneumatic line from an inert gas source (not shown) that provides a controllable pressure head to each of the reagent containers 11A - 11N, as well as the cleaning solution container 21. Thus, the fluids from the various reagent and cleaning solution containers of the cell analysis system 100 can be controllably moved through the fluid lines using the pressure difference from the source to the outlet as the driving force.

[0043] In conjunction with the control provided by the valve controller 40, the fluid multiplexer 35 of FIG. 1 can controllably perform fluid operations including, for example, but not limited to, providing selected reagent delivery to the sensor array device 110, cleaning the fluid multiplexer 35 and the flow cell 107, and priming the fluid multiplexer 35 with a selected reagent. Such fluid operations can provide delivery of reagents to the flow cell 107 without cross-contamination, provide sharp transitions between reagent fluid streams, and provide a constant electrolyte fluid environment to the reference electrode 27 of 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 for the sensor array system are described in U.S. Patent Publication No. 2010 / 0137143, U.S. Patent No. 8,546,128, and U.S. Patent No. 8,673,627, all of which are hereby incorporated by reference in their entirety.

[0044] For example, in conjunction with the valve controller 40, the fluid multiplexer 35 of FIG. 1 can selectively provide fluid communication between any of the reagent fluid lines 13A-13N and the flow cell inlet line 103A, thereby providing a selective reagent flow through the flow cell 107 of the sensor array device assembly 115. A non-limiting, exemplary reagent solution fluid path of the present teachings is provided by a reagent delivery operation where the wash solution fluid line valve 25 is closed and one of the reagent fluid line valves 15A-15N is open, 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, the selected reagent can flow through the fluid multiplexer 35 to the flow cell inlet line 103A, where it can 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.

[0045] Regarding the fluid control of the cleaning solution, in conjunction with the valve controller 40, the fluid multiplexer 35 of FIG. 1 can selectively provide fluid communication between the cleaning solution container 21 and the flow cell inlet line 103A. Therefore, with the cleaning solution fluid line valve 25 in the open state and the solution container 21 being able to be cleaned in the fluid multiplexer 35 and the flow cell 107, it can be in fluid communication with the fluid multiplexer waste container 31 and the flow cell waste container 101. A non-limiting exemplary cleaning solution fluid path of the present teachings is provided by a cleaning operation where the cleaning solution fluid line valve 25 is in the open state and each of the reagent fluid line valves 15A - 15N is in the closed state, provided that the cleaning solution can flow through the cleaning solution fluid line 23 to a T-junction with the flow cell inlet line 103A. Since the flow cell inlet line 103A is in fluid communication with the fluid multiplexer 35, the cleaning solution can flow through the fluid multiplexer fluid line 33 to the fluid multiplexer waste container 31. Since the flow cell inlet line 103A is additionally in fluid communication with the sensor array device assembly 115, the cleaning solution can flow from the inlet port 102 to the outlet port 104 through the flow cell 107 and then through the flow cell outlet line 103B to the flow cell waste container 101.

[0046] According to the present teachings, priming of a selected reagent in the fluid multiplexer 35 can be performed in a series, for example, after a washing operation and before the selected reagent is in fluid communication with the flow cell 107 of FIG. 1. A non-limiting exemplary reagent priming fluid path of the present teachings is provided by a reagent priming operation in which the wash solution fluid line valve 25 is open and one of the reagent fluid line valves 15A-15N is open, provided that one of the selected reagents is in fluid communication with the fluid multiplexer 35. Under such an operation, the flow rate of the wash solution relative to the flow rate of the reagent is selected such that the wash solution flows through the fluid multiplexer 35 and through the fluid multiplexer fluid line 33 to the fluid multiplexer waste container 31, excluding the passage in the multiplexer 35 that is in fluid communication with the selected reagent fluid, such as one of the reagent fluid lines 13A-13N. Thus, when the reagent delivery operation as described above in this specification is initiated, the reagent selected in the reagent priming operation is in direct fluid communication with the flow cell inlet line 103A.

[0047] Therefore, various embodiments of the fluid system of the present teachings are configured to perform a series of operations that can include washing, priming, and reagent delivery. Such operations, when performed in series, avoid cross-contamination of reagents in the system fluid lines and compartments, provide sharp transitions between reagent fluid streams, and provide a constant electrolyte fluid environment to the reference electrode, thereby providing a constant stable reference voltage to the sensor array.

[0048] According to the present teachings, noise sources arising from fluid lines can affect a reference voltage, for example, provided by a reference electrode 27 disposed in the cleaning solution fluid line 23 of FIG. 1. As previously described herein, a stable reference voltage from the reference electrode 27 for the sensor array device 110 of FIG. 1 can be provided by having a fluid system that ensures the reference electrode 27 is in continuous contact with a cleaning solution of a known electrolyte composition. Additionally, high-frequency noise from fluid lines, for example, reagent fluid lines 13A - 13N and the fluid multiplexer fluid line 33, can be filtered by capacitively coupling an electrode disposed in the fluid line to the reference electrode. According to the present teachings, 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 flowing stream. As depicted in FIG. 1, reagent fluid line electrodes 17A - 17N, each disposed in reagent fluid lines 13A - 13N respectively, are coupled to the reference electrode 27 through separate reagent fluid line capacitors 19A - 19N, each of which is coupled to a respective reagent fluid line electrode 17A - 17N. In a similar manner, a fluid multiplexer fluid line electrode 37 disposed within the fluid multiplexer fluid line 33 is coupled to the reference electrode 27 through a fluid multiplexer fluid line capacitor 39.

[0049] As depicted in FIG. 1, the cell analysis system 100 can additionally include an array controller 50 and a user interface 60. According to the present teachings, the array controller 50 can provide various power supplies and bias voltages, as well as control and timing signals to the sensor array device 110, and additionally, a data and processor interface for high-speed acquisition of data from the sensor array device 110. In that regard, FIGS. 5A and 5B depict various functions of the array controller 50 in relation to various functions of the sensor array device 110 of the present teachings.

[0050] FIG. 5A generally shows various aspects of functionality between an array controller 50 associated with a sensor array 120 of a sensor array device 110, as well as between the array controller 50 and a user interface 60. According to various embodiments of the cell analysis system of the present teachings, the array controller 50 has a plurality of power supplies 52 to support a digital interface 54, which is a high-speed digital interface configured to supply power to the sensor array device 110 and to read data generated by the sensor array device 110. Additionally, the array controller 50 is configured to provide signals such as control signals from a signal controller 56 and timing signals from a timing generator 58 to the sensor array device 110. The plurality of power supplies can be provided to the sensor array device 110, including an analog power supply, a digital power supply, an I / O power supply, and ground. In one exemplary implementation, each supply voltage can be controllable in the range of 1.2 to 3.3V. Each of these power supply voltages can be provided to the sensor array device 110 via separate conductive paths to facilitate noise isolation. These supply voltages can originate from their respective power supplies, or one or more of these supply voltages can originate from a common power supply in the array controller 50. According to various embodiments of the array controller of the present teachings, the power supply can include one or more digital-to-analog converters (DACs) that are controlled by a system processor 62 to enable changing any or all of the supply voltages under software control. For example, the power supply responsive to computer control can facilitate switching between a supply voltage of 1.6 volts and a supply voltage of 1.8 volts according to the requirements of the type of sensor array device in use, as read from data stored in various embodiments of the sensor array device.

[0051] The hardware controller 70 shown in FIG. 5A can be implemented as one or more separate circuit boards or as part of an interface board. One function of the hardware controller 70 is to provide pneumatic and fluid control, for example, to control the timing and duration of various fluids and reagents flowing over the sensing surface of each sensor in the sensor array 120, and to read 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 of the sensor array device 110, as well as temperature measurement and control of the system's assemblies and sub-assemblies. According to the present teachings, temperature measurement can be performed using, as non-limiting examples, thermistors and thermocouples. In response to the temperature measurement, the system processor 62 can control devices, including non-limiting examples such as fans, heaters, and thermoelectric coolers.

[0052] The array controller 50 can be manufactured as a "standalone" 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 of the present teachings can be controlled by the system processor 62 through a processor interface 55 (e.g., a PCI bus, an Ethernet connection, etc.). In one embodiment, all or part 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 can include standard computer interfaces such as Ethernet and USB. Finally, the system processor 62 can include a display 64.

[0053] As depicted in FIG. 5B, various power supplies and bias voltages are provided from array controller 50 to the sensor array device. The sensor array device 110 of FIG. 5B can incorporate one or more analog-to-digital converters (ADCs) 114 and one or more data multiplexers and high-speed serial output 116 to convert the analog output signals of sensor array 120 to a high-speed serial data interface 54, thereby providing digital data from sensor array device 110 to system processor 62 (see FIG. 5A) via processor interface 55. In various embodiments of sensor array device 110, the analog-to-digital converter (ADC) 114 can have computer-selectable input ranges (e.g., 200 mV, 300 mV) to facilitate compatibility with different ranges of sensor output signals.

[0054] According to the present teachings, in various embodiments of the sensor array device 110 of FIG. 5B, the array row selection and sequencing 112, the ADC 114, and the data multiplexer and high-speed serial output 116 can be controlled by logic on the timing sequencer 118 of the sensor array device 110 at the timing provided by the array controller 50. In one exemplary implementation, the number of rows for which data is acquired and the sampling rate of the acquisition can be controlled based on control data provided from the array controller 50 to the sensor array device 110. In one non-limiting example, the timing generator 58 can be implemented as a programmable clock generator integrated circuit that is controlled by the system processor 62 to enable control of different sensor array device types, operating modes, and acquired data speeds. 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 in the sensor array device timing sequencer 118 so as to control the operating mode of the sensor array device 110 and to read data from the sensor array device 110. Information read from the sensor array device 110 can include stored data including chip type, serial number, date of manufacture, and the like.

[0055] The reference electrode 27 of FIG. 5B can be coupled to a power supply to provide a reference potential for the output voltage (output signal) from each sensor of the sensor array 120. As a non-limiting example, the reference electrode voltage of the reference electrode 27 can be set by flowing a solution of known, stable pH over the sensor array device 110 (see FIG. 2A). Under such defined pH conditions, the reference electrode voltage can be adjusted until the output signal of the sensors within the sensor array, e.g., the sensors of the sensor array 120 of FIG. 2A, indicates that the sensors have a desired reference level of voltage. Once the reference voltage is set using a solution of known, stable pH, subsequent changes in the sensor voltage can reflect local changes in pH occurring at the sensing surface of the sensors of the sensor array.

[0056] Applications and methods using a ChemFET sensor array-based system for cell analysis The flowchart 200 of FIG. 6A presents a workflow related to embodiments of the applications and methods of the present teachings that can be implemented using the cell analysis system of the present teachings and related devices, components, and assemblies. For example, the flowchart 200 can include a sensor array device interfaced to an array controller and a system processor, such as the sensor array device 110, array controller 50, and user interface 60 of FIGS. 1, 5A, and 5B, and can be implemented using the cell analysis system 100 of FIG. 1.

[0057] The flowchart 200 of FIG. 6A can be started at step 210, and plate a ChemFET sensor array device with a cell sample. A suspension of cells at a known density can be flushed or otherwise drawn into a sensor array device assembly, such as the sensor array device assembly of FIG. 1. Following the plating of the cell sample, the sensor array device assembly can be covered and placed in a target period of incubation so that the cells can settle and adhere on the sensor array surface. Before plating the sensor array device assembly with the cell sample, the flowchart 200 can additionally include the step of preparing the sensor array surface with a cell-compatible material selected by the user, such as various biopolymer materials including poly-D-lysine, laminin, fibronectin, collagen, and combinations thereof, as well as various preparations of the extracellular matrix (ECM). The preparation of the sensor array surface with the cell-compatible material can be performed prior to plating, and the sensor array device can be stored for future use. Alternatively, the in-situ preparation of the sensor array surface with the cell-compatible material can be performed before plating the sensor array device assembly with the cell sample.

[0058] After the cells are stably associated with the sensor array surface, in step 212 of flowchart 200, a live cell imaging application can be executed, and in step 214, the results can be displayed. The information at step 214 from the live cell imaging application can provide the end user with a general survey of the cell distribution on the sensor surface, information regarding individual cell morphology, and an assessment of individual cell viability. For example, the cell analysis system 100 of FIG. 1 can provide the end user with electron microscope cell imaging. In such an imaging application, changes in the state of the cells immobilized on the surface of the sensor array device that can cause changes in the potential at the sensing surface of the ChemFET sensor can be detected by each sensor in the sensor array responsive to the cell activity cells. For example, an electron microscope image of responsive cells at a selected time during the live cell imaging application can be presented to the end user on a display, such as display 64 of user interface 60 of FIG. 5A. Considering that, as described above herein, electron microscope imaging is based on various responses that can be induced in live cells, obtaining an electron microscope image of responsive cells on the sensor array surface can be useful as a first step for cell visualization and assessment. As an alternative to, or in addition to, electron microscope imaging, other methods of visualizing cells, such as optical microscopy, can be used.

[0059] Based on the information provided from the live cell image display step 214, at step 216 of the flowchart 200, the region of interest can be selected, for example, based on identifying an optimal region regarding cell distribution and activity, and considering 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, a data acquisition rate in the range of sub-milliseconds to milliseconds may be required to monitor the action potentials of various excitatory cells, while a data acquisition rate in the range of seconds to minutes may be required to monitor lactate production from oxidative phosphorylation. At step 218, the end user can select an application, for example, from a menu presented on a graphical user interface (GUI), and then can initiate execution as provided at step 220. Finally, at step 222, the end user can receive the results of the experiment, for example, but not limited to, as provided on a display such as display 64 of the user interface 60 of FIG. 5A, as provided by transmitting as a report to a system processor such as system processor 62 of the user interface 60 of FIG. 5A, or both.

[0060] Alternatively, the end user can run the application without first running the live cell imaging application so as to select the region of interest. The flowchart 300 of FIG. 6B can be started at step 310 and plate a ChemFET sensor array device with a cell sample, as previously described for step 210 of FIG. 6A. According to the present teachings, plating of the cells can be performed on a sensor array device treated with a cell-compatible material. At step 320, the end user can select the application, for example, from a menu presented on a graphical user interface (GUI), and then can start the execution as provided at step 330. Finally, at step 340, the end user can receive the results of the experiment, for example, provided on a display such as display 64 of user interface 60 of FIG. 5A, provided to a system processor such as system processor 62 of user interface 60 of FIG. 5A, or both, by transmitting it as a report to the system processor. When the application is run without selecting the region of interest, data is collected, for example, at the frame rate associated with the sensor array device, as shown in Table I. The end user can zoom in and out on any region on the device, for example, of an electron microscope image, but the resolution of the data ultimately presented is fixed by the frame rate associated with the device.

[0061] Various methods of in-situ treatment of a sensor array device to provide a coating of a cell-compatible material on the sensor array surface are applicable to various sensor array devices of the present teachings, as presented in the table of FIG. 3, for example. Regarding exemplary methods for in-situ modification of the sensor array surface with a cell-compatible material, the following solutions of exemplary cell-compatible materials were used to coat the surfaces of three ISFET sensors selective for sensing hydrogen ions (pH). 1. 0.1% Poly-D-lysine (PDL) in phosphate buffered saline (PBS) 2.01% Poly-D-lysine (PDL) + 0.05% laminin in PBS 3. Commercial preparation of extracellular matrix (ECM), Geltrex® used without dilution

[0062] Three sensor array devices of the type identified as Chip 1 in the table of Figure 3 were opened in a sterile laminar flow cell culture hood, and all subsequent operations were performed under sterile conditions using sterile solutions and materials. Each sensor array device was flushed twice with 200 μL of a solution consisting of 70% ethanol in water, injected into one solution port with a pipette, and gently aspirated from the other port. Care was taken so that the surface below the flow cell or the chamber was not aspirated or allowed to dry until the solution was applied to the chip surface, so a vacuum line was placed directly above the outlet port on the opposite side of the port used for solution injection and the chip was not vacuum dried. The 70% ethanol solution was left on the sensor array device at room temperature for 30 minutes and then repeatedly flushed with 200 μL of cell culture grade PBS. The sensor array device was then flushed with 200 μL of solution as described above and left to stand at room temperature for 1 hour after being prepared for use and before being flushed three times with 200 μL of fresh PBS. Alternatively, sensor array devices prepared to provide a cell - compatible surface as described can be sealed and stored for later use.

[0063] To verify the sensor array functionality of the sensor array devices processed as described above to provide a cell - compatible sensor surface, a cell analysis system, such as the cell analysis system 100 of Figure 1, is used to evaluate the processed sensor array devices. The wash solution container and three reagent containers were filled with the following solutions of the following compositions. 1. The wash solution container and the first reagent container were filled with a solution having the following composition and properties. 20 mM HEPES, pH 7.4 140 mM NaCl 2.5 mM KCl 1.8 mM CaCl2 1.0 mM MgCl2 Osmotic pressure: 300 mOsm 2. The second reagent container was filled with a solution having the following composition and properties. 20 mM HEPES, pH 7.6 140 mM KCl 2.5 mM NaCl 1.8 mM CaCl2 1.0 mM MgCl2 Osmotic pressure: 300 mOsm 3. The third reagent container was filled with a solution having the composition and properties of the solution used to fill the second reagent container, but adjusted to pH 7.1 using a dilute solution of hydrochloric acid.

[0064] Each sensor array device and a control with an untreated surface were subjected to the following test protocol. 1. The wash buffer was used to set the potential of the reference electrode and to condition the sensor array device. 2. For each sensor device, two pixels in different sensor regions were selected to measure the potential during the test. 3. The order in which the solutions were aspirated through the flow cell of each device was as follows. a. Switch from the wash solution to the solution in the first reagent container to ensure that there is no significant effect when switching the same solution in different containers. b. Switch from the wash solution to the solution in the second reagent container to measure the effect of the change from pH 7.4 to 7.6. c. Switch from the wash solution to the solution in the third reagent container to measure the effect of the change from pH 7.4 to 7.1.

[0065] Data generated from the test protocol was confirmed to have a response indicating that a sensor array device treated to provide a cell-compatible surface had no adverse effect on sensor array device performance due to the device's coating. A graph of the pH response for the device is presented in the following graph. [Table 2]

[0066] Figures 7A through 8B present data regarding the measurement of changes in cell membrane potential over time during the induced depolarization and subsequent recovery phases. The sensor array device used in this study is a Chip 1 device as shown in the table of Figure 3 that is selective for sensing hydrogen ions and includes a microwell array. The device was treated to provide a cell-compatible poly-D-lysine coating as previously described herein. U-2OS cells (ATCC catalog number ATCC-HB-96), a human osteosarcoma cell line commonly used in heterologous expression studies, were transduced with green fluorescent protein (GFP) to provide for monitoring of cells on the sensor array surface using a fluorescence microscope and were transduced with an ion channel construct to provide for response to a depolarization stimulus. U-2OS was prepared for plating using standard dissociation procedures, pelleted using centrifugation at 200 x g, and resuspended in complete cell culture medium at a density of 250,000 cells per milliliter. Thereafter, 200 μL of the cell suspension was aspirated through the sensor array device. The sensor array device was then placed in a sterile 10 cm cell culture dish and covered. The sensor array device prepared in this manner was then transferred to an incubator and incubated at 37 °C for at least overnight prior to use in the cell analysis system of the present teachings.

[0067] The graph presented in FIG. 7A is a time response curve representing data collected over the region of interest, focusing on the activities of two cells, C1 and C2, during the process of cell imaging research. For the cell imaging research presented from FIG. 7A to FIG. 8B, two solutions were used. 1. The wash solution container and the first reagent container were filled with a solution having the following composition and properties. 20 mM HEPES, pH 7.3 140 mM NaCl 2.5 mM KCl 1.8 mM CaCl2 1.0 mM MgCl2 Osmotic pressure: 300 mOsm 2. The remaining reagent containers, containers 2 to 4, were filled with a solution having the following composition and properties. 20 mM HEPES, pH 7.3 140 mM KCl 2.5 mM NaCl 1.8 mM CaCl2 1.0 mM MgCl2 Osmotic pressure: 300 mOsm

[0068] The solution in the flow cell for the initial conditions before starting the cell imaging research was the wash solution. The research was started when the solution in one of reagent containers 2 to 4 was aspirated onto the sensor array through the flow cell. Since the dominant concentration of cations in that solution is potassium, it is a solution that can provide a depolarizing stimulus for the cells. In addition to monitoring the activities of the cells, a reference response FET R was monitored in the region of the sensor array where the cells were not fixed.

[0069] Regarding the progress of the cell imaging study of cells C1 and C2 in FIG. 7B, when the depolarization solution reaches the cells in Phase I shown in the graph, the early signs of depolarization are demonstrated by an increase in the signals of cells C1 and C2, and continue to increase in Phase II until equilibrium is reached in Phase III, where the response is maintained. When the washing solution is introduced into the flow cell and the depolarization solution is washed out of the flow cell, recovery is shown in Phase IV of the graph and proceeds until cells C1 and C2 are equilibrated with the washing solution in Phase V. The response of the cells is the signature of their presence on the sensor array surface as compared to a slight negative response of the reference curve, and the cells undergoing depolarization exhibit a strong positive voltage response that is approximately 20 times higher than the baseline noise level.

[0070] The time shown by the dashed line in FIG. 7B at 30 frames corresponds to the time of the electron microscope image in FIG. 8A. FIG. 8B is a line drawing of the main features visualized in the electron microscope image of FIG. 8A. Considering that the pixel pitch of the sensor array device used in the study is 3 μm based on the electron microscope imaging of the cells on the sensor array, the size of the cells across the entire sensor array was estimated to be approximately 10 - 20 pixels, i.e., 30 μm to 60 μm. As previously described herein, since the U-2OS cells were transduced with green fluorescent protein (GFP), the sensor arrays prepared for the electron microscope imaging experiments presented in FIGS. 7A - 8B were also imaged using a fluorescence microscope. From the results of the data collected from the fluorescence microscope imaging of the sensor arrays, it was confirmed that the diameter of the cells across the entire sensor array ranges from 30 μm to 60 μm.

[0071] Figure 9A shows the time response curves of U-2OS cells (C1 and C2) and U-2OS cells (C3 and C4) transfected with the voltage-dependent calcium channel alpha, beta, and alpha-2-delta subunits. The time response curves presented in Figure 9A represent data collected over the region of interest of U-2OS cells compared to the transfected U-2OS cells, and the effect of the calcium ion channel blocker verapamil on the cell response for each type of cell was monitored through a cycle of the initial state of the resting potential, subsequent depolarization of the membrane potential, and the conditions to return to the resting potential. Figure 9B is an electron microscope image of Figure 9A taken at the time corresponding to frame 25 (1.7 seconds), and the squares within the cells in Figure 9B indicate the pixels sampled for the data in Figure 9A. Figure 9C is an electron microscope image of Figure 9B with the squares within the cells in Figure 9B removed to provide a clear image of the cells.

[0072] The sensor array device used in the experiment from which the data in Figures 9A to 9C were generated is a chip 2 device as summarized in the table of Figure 3 selected to sense hydrogen ions and includes a microwell array. The device was prepared on a poly-D-lysine surface as previously described herein. Briefly, the surface of the sensor array device was immersed in a 70% aqueous ethanol solution for 30 minutes, then rinsed with PBS and allowed to stand for 2 hours before adding a solution of 0.1% poly-D-lysine. Thereafter, the device was rinsed twice with 200 μL of cell culture medium, for example, McCoy’s 5A modified medium with 10% fetal bovine serum. After rinsing with the cell culture medium, the device was prepared for plating with the cell suspension.

[0073] The preparation of the transfected cells was performed using a cell suspension of 1x10^6 cells per mL. Mammalian BacMam gene delivery particles encoding a mixture of the alpha, beta, and alpha-2-delta subunits of the voltage-dependent calcium ion channel were added to the cell suspension to reconstitute functional channels. Functional voltage-dependent calcium ion channels require at least both the alpha and beta subunits, with an alpha-2-delta that serves to enhance surface expression and ion flux. BacMam virus transduction was achieved by pre-mixing the particles at a rough ratio of 10-fold excess virus particles relative to the cell number and adding them to the cells. Control (virus-free) and virus-treated cells were plated on the sensor device and placed in a cell culture incubator overnight to allow for the attachment and expression of functional ion channels before the experiment was performed the next day.

[0074] During the experiment, control or virus-transduced cells were exposed to an elevated 30 mM KCl depolarizing stimulus in the presence of 10 μM of the calcium ion channel blocker verapamil. The time response curves in Figure 9A show distinguishable differences in the signature responses obtained from cells in the control groups (C1 and C2) versus (vs) cells transfected with voltage-dependent calcium channels (C3 and C4). The findings presented in Figures 9A through 9C demonstrate that the cell analysis system and method of the present teachings can readily distinguish between cells that express voltage-dependent calcium ion channels and those that do not, based on the selective activity of a blocker against a culture expressing a target protein.

[0075] Figure 10A is an electron microscopy image of neonatal rat hippocampal cells subjected to a pulse of 4.5 mM KCl. Figure 10B is a comparison of the time response curves of neonatal rat cells subjected to a pulse of 4.5 mM KCl, where the experiment was performed on two different devices at different data acquisition rates.

[0076] Referring to the table in FIG. 3 that summarizes the array device attributes, the sensor array device used in the experiment in which the data of FIGS. 10A and 10B were generated was a device having the dimensions of Chip 2, in which data was collected at a frame rate of 30 samples / second. The sensor array device used in the experiment in which the data at I in FIG. 10B was generated was a device having the dimensions of Chip 1, in which the active area of the device was windowed down to the region of interest of 50% of the total device area so as to provide a maximum frame rate of 240 samples / second. The devices of both Chip 1 and Chip 2 are selective ISFET devices for sensing hydrogen ions and include a microwell array. The devices were prepared on a poly-D-lysine surface as described herein for the preparation of the devices used in the experiments on the voltage-dependent calcium channels of FIGS. 9A to 9C. Rat neonatal hippocampal cells were isolated from intact rat E18 neonatal hippocampi, resuspended at a density of 1x10^6 cells per mL, and then plated on the devices. The devices were placed in a cell culture incubator, and fresh neuronal cell culture medium was exchanged on the plated cells every 3 days for 10 days, thereby enabling the adhesion and maturation of the cells into an excitatory phenotype capable of presenting spontaneous depolarization activity. Rat neonatal hippocampal cells were tested for spontaneous activity during solution pulses from a basal potassium chloride (KCl) concentration of 2.5 mM to 4.5 mM.

[0077] The cellular activity of the preparation was observed for spontaneous depolarization, as demonstrated in the electron microscopy images of Figure 10A. Figure 10B demonstrates the effect of frame rate on data resolution. In Figure 10B, the time response curve with two peaks (Figure 10B-I) was observed with a sensor device at a frame rate of 30 samples / second, as opposed to the time response curve with a single peak (Figure 10B-II) observed with a sensor device at a frame rate of 240 samples / second. The electron microscopy data of Figure 10A and the time response curves of Figure 10B demonstrate the ability of the cell analysis system and method of the present teachings to visualize the intrinsic electrical activity from primary cell types known to fire action potentials in culture after a 10-day maturation period.

[0078] Figure 11 is an electron microscopy image of cells from an immortalized human hepatocyte cell line (HEPG2 cells; ATCC catalog number ATCC-HB-8065) subjected to the mitochondrial toxin carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), with several exemplary cells shown in relation to the stream of cell efflux. The HEPG2 cell line is used in cell biology as a model for hepatocellular carcinoma and is additionally used for the study of LDL and cholesterol import and hepatotoxicology.

[0079] The sensor array device used in the experiment in which the data of Figure 11 was generated is the chip 2 device of Figure 3 selective for sensing hydrogen ions and includes a microwell array. The device was prepared on a poly-D-lysine surface as described herein for the preparation of the device used in the experiments on voltage-dependent calcium channels of Figures 9A through 9C. The HEPG2 cells were resuspended at 1x10^6 cells / mL and plated onto the chip to incubate overnight in a cell culture incubator such that the cells could adhere to the prepared surface of the chip. The mitochondrial protonophore FCCP was tested at a concentration of 100 μM, a concentration known to induce a response leading to cell death. The electron microscopy images from the experiment are presented in Figure 11.

[0080] Among the applications of FCCP, cell responses were observed to demonstrate the ability of the cell analysis systems and methods of the present teachings to visualize the efflux of cellular contents into a laminar flow system. This is because the efflux of cellular contents is shown by C efflux1 and C efflux2 for two cells, C HEPG1 and C HEPG2 in FIG. 11. In particular, not all cells demonstrated efflux activity as depicted for C HEPG3 in FIG. 11. These results indicate that the cell analysis systems and methods of the present teachings can further distinguish acute novel phenotypes represented in response to toxin treatment.

[0081] FIG. 12A shows the average background-subtracted time-dose response curve for U-2OS cells treated with FCCP. FIG. 12B shows the average background-subtracted time-dose response curve for U-2OS cells pretreated with a nutrient solution containing oligomycin prior to treatment with FCCP.

[0082] Referring to the table of FIG. 3 that summarizes the array device attributes, the sensor array devices used in the experiments from the data of FIGS. 12A and 12B were generated using the chip 2 device for the data presented in FIG. 12A and the chip 1 device for the date presented in FIG. 12B. The devices of both chip 1 and chip 2 are ISFET devices selective for sensing hydrogen ions and include a microwell array. The devices were prepared on a poly-D-lysine surface as described herein for the preparation of the devices used in the experiments with the voltage-dependent calcium channels of FIGS. 9A - 9C. U-2OS cells were resuspended at 1x10^6 cells per mL and plated on the chips to be incubated overnight in a cell culture incubator so that the cells could adhere to the prepared surface of the chips. To test the dose-dependence of FCCP treatment, the cells were exposed to a series of concentration jumps during the experiment as shown in FIGS. 12A and 12B. In FIG. 12A, the experiment was conducted in the absence of oligomycin, and in FIG. 12B, the FCCP dose-response curve was generated in the presence of oligomycin.

[0083] The data in FIGS. 12A and 12B consist of the average cell responses from the samples tested with FCCP. The data show that the dose-dependent FCCP response is readily distinguishable in the absence and presence of oligomycin. In current state-of-the-art cell analysis tools, oligomycin is frequently used as a pretreatment of FCCP to enhance the FCCP response within the cells. In contrast, the cell analysis system of the present teachings does not require pharmacological priming to demonstrate a robust dose-dependent FCCP time response curve.

[0084] In the experiments where the data is presented in FIGS. 13A - 13C, three different cell types were selected for testing the ability of the device to identify cell types based on the response to FCCP. To generate a dataset comparing a wide variety of 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 CRM CCL2), and a mouse - derived leukemia cell line (MMM cells; ATCC catalog number J774A.1) were selected for the study.

[0085] FIGS. 13A through 13C are the mean 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. For the data shown in FIGS. 14A through 14C, cell mixtures were exposed to 100 μM FCCP to understand whether different cell types could be discriminated by their response to FCCP, and their responses were recorded. FIG. 14A shows the composite time - dose response curves of the selected cells in a mixture of different cell types where the cells were subjected to FCCP. FIG. 14B is an electron microscope image of the experiment in FIG. 14A taken at the time corresponding to frame 40 (2.7 seconds) where the squares within the cells indicate the pixels sampled for the data in FIG. 14A. FIG. 14C is an electron microscope image of FIG. 14B where the squares within the cells in FIG. 13B have been removed to provide a clear image of the cells.

[0086] The sensor array device used in the experiment in which the data from FIGS. 13A to 14C were generated is the chip 2 device of FIG. 3 that is selective for sensing hydrogen ions and includes a microwell array. The device was prepared on a poly-D-lysine surface as described herein for the preparation of the device used in the experiments on the voltage-dependent calcium channels of FIGS. 9A to 9C. Each cell line was resuspended at 1x10^6 cells per mL and then mixed in a uniform ratio before being incubated overnight in a cell culture incubator so that the cells could adhere to the prepared surface of the chip.

[0087] The time response curves of FIGS. 13A to 13C represent each of three cell types of different tissue origins that were individually tested on separate devices, so it is notable that each cell type exhibits a distinct dynamic and amplitude response to FCCP. The data presented in FIGS. 13A - 13C suggest the possibility of identifying each cell line by its signature response to pharmacological probes. FIG. 14A represents data collected from the analysis of all three cell types on a single chip. Using waveform analysis on the time response data presented in FIG. 14A, C1 and C2 were identified as HASM cells, C3 was identified as MMM cells, and C4 and C5 were identified as HeLa cells. At the time the electron microscopy image of FIG. 14C was taken, the electron microscopy image captured the behavioral differences of HeLa cells (C4 and C5) compared to HASM cells and MMM cells (C1 - C3). In summary, the data presented in FIGS. 13A - 13C and FIGS. 14A - 14C demonstrate the possibility of using signature cell responses and behaviors such that the systems and methods of the present teachings can identify distinct cell lines in a mixed cell population.

[0088] The sensor array device used in the experiment, of which FIGS. 15A through 15C are representative data, is a Chip 1 device as summarized in the table of FIG. 3 that is selective for sensing hydrogen ions and is without a microwell array. To test the ability of the various cell analysis systems of the present teachings to record cell signals with intracellular resolution and high temporal fidelity, data was collected at 120 frames per second for Chip 1 devices plated on a fairly large number of cell-compatible surfaces on cells. Device and cell preparation were performed as previously described for FIGS. 9A through 9C. Briefly, the devices were prepared on poly-D-lysine surfaces and preparations of U-2OS cells were transfected with voltage-dependent calcium ion channels. Chip 1 devices were plated with transfected U-2OS cells using a cell suspension of 1×10^6 cells per mL. As previously described herein, mammalian BacMam gene delivery particles encode a mixture of voltage-dependent calcium ion channel alpha, beta, and alpha-2-delta subunits to provide a cell suspension with reconstituted functional channels. Functional voltage-dependent calcium ion channels require both alpha and beta subunits, with alpha-2-delta, which serves to enhance surface expression and ion flux.

[0089] Shown in FIGS. 15A and 15B are two electron microscope images from two exemplary time points, separated by 345 milliseconds (ms) from data captured at approximately 1-2 second intervals. Approximately 500 ms prior to the start of data collection, the cells were perfused with the following isotonic reagent. 20 mM HEPES, pH 7.3 130 mM NaCl 12.5 mM KCl 1.8 mM CaCl2 1.0 mM MgCl2 Osmolarity: 300 mOsm

[0090] Figures 15A and 15B are electron microscope images showing examples of transient signals in the intracellular domains of various cells presented in electron microscope images. For example, in the cell designated as cell i, the signal is clearly visible at the 1577 ms time point, but the signal is substantially reduced at the 1922 ms time point. In the cell designated as cell ii, there is a significantly visible signal at the 1577 ms time point, but the signal is reduced at the 1922 ms time point so as to reveal the signal in the adjacent cell. In the cell designated as cell iii, there is no significantly visible signal at the 1577 millisecond (ms) time point, but there is a signal that is clearly visible at the 1922 ms time point.

[0091] Figure 15C is a time response curve over a time interval that includes the time points of the electron microscope images of the cells designated as cells i - iii in Figures 15A and 15B. The time response curve of Figure 15C also includes a non - responsive cell iv, i.e., the signal collected and averaged for cell response control, and a sensor region v, i.e., the signal collected and averaged for device response control. As can be seen by examining the time response curve of Figure 15C, both controls remained unchanged during data capture. Since the sampling interval of the data presented in Figures 15A - 15C was approximately 8.3 ms, the time response curve over the entire experiment captured the rising phase and was followed by an exponential decay signal for a number of cells, as shown for the exemplary cells shown in Figure 15C. In the data presented in Figures 15A - 15C, high - frame - rate recordings of the intracellular domain were readily visualized, as shown in the electron microscope images of Figures 15A and 15B, and individual active regions were revealed that were quantified in terms of number and intensity, as shown in the time response curve of Figure 15C.

[0092] The impact of the data presented in FIGS. 15A - 15C at intracellular resolution collected at high frame rates is evidence of cellular activity in individual microdomains that coincides with bioelectrical or biochemical activity. Such cellular activity in microdomain regions is consistent with previous descriptions of local vesicle release in TIRF microscopy. Additionally, the data also demonstrates the potential of various embodiments of the present disclosure's ChemFET sensor array - based system to evaluate genetic or pharmacological interventions that may potentially control the mechanisms underlying vesicle fusion and release in normal and diseased cellular functions.

[0093] As previously described herein with respect to FIG. 3, sensor array device attributes that can be varied to provide various sensor array devices of the present disclosure include pixel dimensions, as well as the rate at which data can be collected from the sensor array device. FIG. 16 provides a summary of cells similar to those described in the application and method paragraphs of this specification.

[0094] Upon examination of FIG. 16, for MMM cells immobilized on a sensor array surface with an average diameter of 6 μm and an average area of 28 μm 2 , the minimum contact area or footprint corresponds to approximately 2 rows and 3 pixels for the chip 1 device and increases to 7 rows and 45 pixels for the chip 4 device. Similarly, for HEPG2 cells immobilized on a sensor array surface with an average diameter of 15 μm and an average area of 177 μm 2 , the minimum contact area or footprint corresponds to approximately 4 rows and 18 pixels for the chip 1 device and increases to 18 rows and 285 pixels for the chip 4 device. Similarly, for HeLa cells immobilized on a sensor array surface with an average diameter of 18 μm and an average area of 254 μm 2 , or for cells with an average diameter of 19 μm and an average area of 284 μm 2In U-2OS cells, the minimum contact area or footprint corresponds to approximately 5 rows and approximately 26 pixels, or approximately 6 rows and approximately 29 pixels, respectively, in the Chip 1 device, and increases to 21 rows and 410 pixels, or 22 rows and 458 pixels, respectively, in the Chip 4 device. With an average diameter of 25 μm and an average area of 491 μm 2 In HASM cells immobilized on the surface of the sensor array, the minimum contact area or footprint corresponds to approximately 7 rows and approximately 50 pixels in the Chip 1 device and increases to 29 rows and 792 pixels in the Chip 4 device. Rat neonatal hippocampal (RNH) cells can have cell bodies that can vary from approximately 20 μm and an average area of 314 μm 2 to approximately 100 μm and an average area of 7,854 μm 2 Among them. In various RNH cells immobilized on the surface of the sensor array, the minimum contact area or footprint of the RNH cell body can range from approximately 6 rows and approximately 32 pixels to approximately 30 rows and approximately 803 pixels, respectively, in the Chip 1 device, and increases to approximately 24 rows and 506 pixels to approximately 118 rows and 12,668 pixels, respectively, in the Chip 4 device. From the examination of Figure 16, as described for Figure 3, the trend is that pixel coverage tends to increase with increasing cell size and decreasing pixel size.

[0095] From the perspective of pixels, the column of percent pixel coverage is the percentage of the cell area covered by a single pixel. For MMM cells fixed on the sensor array surface, a single pixel corresponds to 33% coverage in the chip 1 device, while in the chip 4 device, a single pixel corresponds to 2% coverage. For HEPG2 cells fixed on the sensor array surface, a single pixel corresponds to 6% coverage in the chip 1 device, while in the chip 4 device, a single pixel corresponds to 0.4% coverage. In comparison, for HeLa cells fixed on the sensor array surface, a single pixel corresponds to 4% coverage in the chip 1 device, while in the chip 4 device, a single pixel corresponds to 0.2% coverage. For U-2OS cells fixed on the sensor array surface, a single pixel corresponds to 3% coverage in the chip 1 device, while in the chip 4 device, a single pixel corresponds to 0.2% coverage. For various RNH cells fixed on the sensor array surface, the coverage of a single pixel can range from 3% for RNH cells with an average diameter of 20 μm to 0.1% for RNH cells with an average diameter of 100 μm on the chip 1 device. In comparison, in the chip 4 device, a single pixel corresponds to 0.2% coverage for RNH cells with an average diameter of 20 μm and corresponds to 0.008% coverage for RNH cells with an average diameter of 100 μm. From the scrutiny of Figure 16, as described for Figure 3, the trend is that the percentage of cell coverage per pixel tends to decrease with the increase in cell size and the decrease in pixel size.

[0096] Considering what is presented in the table of FIG. 16, the selection of pixel coverage of the exemplary sensor array devices of the present teachings can be done for various average cell diameters. The correlation between cell lines and device attributes used in various applications of the present teachings is within the ranges listed in FIG. 3. In that regard, for cells from about 5 μm to about 100 μm, the selection of the sensor array device can be done to provide a coverage from about 8 pixels to over 12,668 pixels, over 118 rows of pixels, over 3 rows of pixels for the corresponding footprint of the cells immobilized on the sensor array surface. Across that range of cell sizes, since the pixel size can vary, each pixel of the selected sensor array device can cover from about 12% to about 0.008% of the cell. Based on the data presented in FIGS. 3 and 16, it is clear that for any cell size, an exemplary sensor array device can be selected that can provide a significant number of sensors related to the contact area that the cells can occupy on the sensor array device. The spatial resolution that can be provided by the various sensor array devices of the present teachings can enable intracellular discrimination of signals and, therefore, provides intracellular analysis.

[0097] Similarly, consistent with what is presented in FIG. 3, it is clear by scrutiny of the last column of FIG. 16 that a significant number of frames per second can be collected for the target area of interest across various cell sizes, providing comfortable data collection within the kHz range. Recall that in the last column, the maximum frame rate is presented for which data can be collected for the fractional part of the rows covered by the cells, derived by dividing the maximum frame rate per row by the rows per cell diameter.

[0098] For MMM cells fixed on the surface of the sensor array, data can be collected at a maximum frame rate of 37,500 fps with the chip 1 device, while with the chip 4 device, data can be collected at a maximum frame rate of 23,142 fps. For HEPG2 cells fixed on the surface of the sensor array, data can be collected at a maximum frame rate of 18,750 fps with the chip 1 device, while with the chip 4 device, data can be collected at a maximum frame rate of 9,000 fps. For HeLa cells fixed on the surface of the sensor array, data can be collected at a maximum frame rate of 15,000 fps with the chip 1 device, while with the chip 4 device, data can be collected at a maximum frame rate of 7,714 fps. Similarly, for U-2OS cells fixed on the surface of the sensor array, data can be collected at a maximum frame rate of 12,500 fps with the chip 1 device, while with the chip 4 device, data can be collected at a maximum frame rate of 7,364 fps. For HASM cells fixed on the surface of the sensor array, data can be collected at a maximum frame rate of 10,714 fps with the chip 1 device, while with the chip 4 device, data can be collected at a maximum frame rate of 5,586 fps. Finally, for various RNH cells, which are the main bodies of such cells fixed on the surface of the sensor array, data can be collected at a maximum frame rate of approximately 2,500 fps to approximately 12,500 fps with the chip 1 device, while with the chip 4 device, data can be collected at a maximum frame rate of approximately 1,373 fps to approximately 6,750 fps.

[0099] According to the present teachings, as presented in FIG. 16, as presented in FIG. 3, it is revealed that various maximum frame rates of cell lines used in various applications according to the present teachings are in the range of about 1,250 fps to about 75,000 fps. From an examination of FIGS. 3 and 16, the trend is that the frame rate tends to decrease with an increase in cell size and a decrease in pixel size, which is consistent with an inverse proportional relationship between the selected region of interest and the speed in the data that can be collected. Therefore, by selecting a smaller subset of pixels to monitor, i.e., by windowing down the region of the sensor array device from which data is collected, or by selecting a device with a desired frame rate that matches the application, the frame rate can be increased.

[0100] Described herein are systems, applications, and methods for cell analysis systems based on ChemFET sensor array technology. The various cell analysis systems of the present teachings can measure the electrical and metabolic activities of single live cells with intracellular addressability and simultaneous data acquisition of about 10 cells to about 500,000 cells in a single analysis. The various sensor array devices of the present teachings can have a sensor array with 20 million to 660 million ChemFET sensors incorporated in a massively parallelized array, and the sensors of the sensor array can each have a pitch of about 3.36 μm to about 850 nm. Further, the various cell analysis systems of the present teachings can provide simultaneous measurements of cells at a data acquisition rate in the kilohertz (kHz) range. Moreover, the fully automated fluid systems integrated into the various cell analysis systems of the present teachings enable precisely controlled applications of chemicals, for example, used in various studies where measurements of cell responses to various types of chemical investigations are shown. The various ChemFET sensor arrays of the present teachings can detect chemical analytes and detect changes in cell membrane potential, so cells can also be examined in the various cell analysis systems of the present teachings using controlled electrical stimulation.

[0101] Preferred embodiments of the present teachings have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided only as examples. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives described herein can be used in the implementation of the various embodiments described herein. The following claims define the scope of the present invention, and it is intended that systems, devices, applications, and methods within the scope of these claims, and their equivalents, be covered thereby.

Claims

1. A cell analysis system, a device comprising an array of chemFET (Chemical Field Effect Transistor) sensors, wherein the array of sensors has a sensor pitch of 850 nm to 3.3 µm, the device has a surface treated to promote cell adhesion, and for a selected device, each ChemFET sensor provides a coating of 0.008% to 12% for cells with a footprint in the range of 100 µm to 5 µm in diameter, a device; a flow cell mounted on the device, the flow cell being configured to provide a fluid interface between the device and a fluid system provided in the cell analysis system; a reference electrode in fluid communication with the flow cell, the reference electrode being configured to provide a stable reference potential to the array of sensors; an array controller, the array controller having a programmable clock generator integrated circuit for controlling different sensor array device types and controlling the acquisition data rate for detecting changes in the state of cells capacitively coupled to the sensing surface of the selected device, and further, the array controller is configured to provide a power supply voltage and a bias voltage, as well as control signals and timing signals, to the device and to provide data acquired from the device to a system processor, a cell analysis system comprising the array controller.

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 according to claim 1, further comprising a fluid system configured for controllable liquid delivery through the flow cell.

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

6. The cell analysis system according to claim 5, wherein the coating is selected from poly-D-lysine, laminin, and combinations thereof.

7. The cell analysis system according to claim 5, wherein the coating is a basement membrane matrix preparation.

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

9. The cell analysis system according to claim 8, wherein the width of the bottom of the microwell structure is 90% or less of the width of the sensor plate of the sensors in the array of the sensors.

10. The cell analysis system according to claim 9, wherein the height of the microwell structure is 2 to 4 times the width of the microwell structure.

11. The cell analysis system according to claim 1, further comprising a depolarizing electrode in fluid communication with the device, wherein the depolarizing electrode is configured to apply an electrical stimulus to the cells.

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