Abstract:
Among others things, techniques, systems, and apparatus are disclosed for recording electrophysiological signals. In one aspect, a microelectrode sensing device includes a printed circuit board (PCB), a chip unit electrically connected to the PCB, and a cell culture chamber positioned over the chip unit and sealed to the PCB with the chip unit between the PCB and the cell culture chamber. The chip unit includes a substrate; a conductive layer positioned over the substrate that includes one or more recording electrodes; an insulation layer positioned over the conductive layer; another conductive layer positioned over the insulation layer that includes positioning electrodes; and another insulation layer positioned over the other conductive layer. The recording and positioning electrodes are electrically independent so as to independently receive a stimulus signal at each recording electrode and positioning electrode and independently detect a sensed signal at each recording electrode.

Description:
CLAIM OF PRIORITY 
       [0001]    This application claims priority to Chinese Patent Application No. 200810115784.7, filed on Jun. 27, 2008 in the State Intellectual Property Office of the People&#39;s Republic of China, the entire contents of which are hereby incorporated by reference. 
       BACKGROUND 
       [0002]    This application related to microelectrode sensing devices. 
         [0003]    Microelectrode arrays (MEAs) can be used to facilitate electrophysiological measurements of excitable cells (e.g., neuronal cells). Electrophysiological measurements can be obtained under various conditions. For example, pharmacological studies can be performed by applying various chemical compounds onto the neuronal cells and recording the resultant electrical activities. 
         [0004]    Higher amplitude of electrophysiological signals and improved effectiveness of stimulation can be achieved by positioning neurons on or closely in the neighborhood of a recording/stimulation site. Attempts to provide such controlled positioning of neuronal cells by employing physical containment such as micro-wells and micro-channels and/or appropriate materials to obtain desirable cell patterns have been mostly unsatisfactory. 
       SUMMARY 
       [0005]    Techniques, systems and apparatus are described for performing cell manipulation and electrophysiological detection using microelectrode sensing devices. 
         [0006]    In one aspect, a microelectrode sensing device includes a printed circuit board (PCB) and a chip unit electrically connected to the PCB. The chip unit includes a substrate; a conductive layer positioned over the substrate that includes one or more recording electrodes; an insulation layer positioned over the conductive layer; another conductive layer positioned over the insulation layer that includes positioning electrodes (e.g., four or more); and another insulation layer positioned over the other conductive layer. The microelectrode sensing device includes a cell culture chamber positioned over the chip unit and sealed to the PCB with the chip unit between the PCB and the cell culture chamber. The recording and positioning electrodes are electrically independent so as to independently apply a stimulus signal at each recording electrode and positioning electrode and independently detect a sensed electrical signal at each recording electrode. 
         [0007]    Implementations can optionally include one or more of the following features. The chip unit can be electrically connected to the PCB by wire bonding. The PCB can include two rows of contact pads arranged to surround the chip unit, and the chip unit can include contact pads distributed at each edge of the chip unit. The chip unit can be electrically connected to the PCB using electrical connections between the contact pads of the PCB and the contact pads of the chip unit. The PCB can include additional contact pads located at each edge of the PCB. At least one of the two rows of contact pads of the PCB that surround the chip unit is electrically connected to the additional contact pads located at each edge of the PCB using conductive wires shaped to form an arc. The contact pads of the chip unit can be equally spaced apart at the edge of the chip unit. Each of the recording and positioning electrodes can be separately connected to a separate one of the contact pads on the chip unit. Each of the recording and positioning electrodes can be separately connected to a separate one of the contact pads on the chip unit using a conductive wire shaped to form an arc. A width of the conductive wire can be shaped to increase from the recording electrodes to the contact pads on the chip unit. The two conductive layers can include ground electrodes connected through a passage at the insulation layer that is between the two conductive layers and arranged to separate the wire connecting each of the recording and positioning electrodes to the contact pads on the chip unit. A surface of each recording electrode can include spongy material. The spongy material can include platinum black. The substrate of the chip unit can include glass wafer. Each positioning electrode can be shaped to form a ring. Each insulation layer can include two layers of Si3N4 and SiO2. 
         [0008]    In another aspect, a system includes a microelectrode sensing device that includes a printed circuit board (PCB) and a chip unit electrically connected to the printed circuit board. The chip unit includes a substrate; a conductive layer positioned over the substrate that includes one or more recording electrodes; an insulation layer positioned over the conductive layer; another conductive layer positioned over the insulation layer that includes positioning electrodes; and another insulation layer positioned over the other conductive layer. The microelectrode sensing device includes a cell culture chamber positioned over the chip unit and sealed to the PCB with the chip unit between the PCB and the cell culture chamber. The recording and positioning electrodes are electrically independent so as to independently apply a stimulus signal at each recording electrode and positioning electrode and independently detect a sensed signal at each recording electrode. The system includes a metal clamp connected to the microelectrode sensing device. The metal clamp includes a metal enclosure to receive the microelectrode sensing device; a cover plate detachably connected to the metal enclosure to hold the received microelectrode sensing device against metal enclosure; and another PCB. The other PCB on the metal clamp includes positioning structures to provide a connection to the metal enclosure, and spring-pins to provide an electrical connection to the PCB of the microelectrode sensing device. 
         [0009]    Implementations can optionally include one or more of the following features. The metal enclosure can include guide-paths corresponding to a shape of the microelectrode sensing device to restrict movement of the microelectrode sensing device along a direction of movement of the spring-pins. The cover plate can be placed on top of the microelectrode sensing device to press the microelectrode sensing device tightly against the spring pins of the other PCB to create a stable electrical connection between the PCB of the microelectrode sensing device and the other PCB of the metal clamp. The system can include a welding clamp that includes insertion structures corresponding to the spring pins of the other PCB of the metal clamp arranged in vertical alignment with the spring pins. The welding clamp can include positioning structures corresponding to the positioning structures of the metal clamp. 
         [0010]    In another aspect, a method for recording electrophysiological signals includes adding a cell suspension that includes multiple cells to a cell culture chamber of a microelectrode sensing device that includes at least one recording electrode and corresponding positioning electrodes. Experimental data is obtained by selectively applying positioning signals to one or more of the positioning electrodes to cause the cells in the cell suspension to form one or more cell patterns. The one or more recording electrodes are used to record electrophysiological signals associated with the cell patterns. Also, control data is obtained using the recording electrodes to record electrophysiological signals associated with the cells in absence of positioning signals. The obtained experimental data is compared with the obtained control data to identify an effect of selectively applying the positioning signals. 
         [0011]    Implementations can optionally include one or more of the following features. Selectively applying the positioning signals can include selectively applying the positioning signals to all positioning electrodes. Selectively applying the positioning signals includes selectively applying the positioning signals to selective positioning electrodes. Selectively applying the positioning signals can include selectively applying the positioning signals continuously. Selectively applying the positioning signals can include selectively applying the positioning signals periodically. 
         [0012]    The techniques, systems, and apparatus as described in this specification can optionally provide one or more of the following advantages. An electrode sensing device can be implemented as an array of positioning and sensing electrodes. Each electrode on the microelectrode sensing device can be operated separately to generate different cell patterns and detect an electrophysiological signal. At the same time, the microelectrode sensing device can complete other functions, such as cell electro-rotation. 
         [0013]    Also, the microelectrode sensing device can include a chip unit, a printed circuit board (PCB) and a cell culture chamber. A substrate of the chip unit can be implemented using glass, and the positioning electrodes can be arranged in a ring for easy observation of the device under an inverted microscope. In addition, the microelectrode sensing device as described in this specification can be implemented to reduce crosstalk in electrodes, reduce noise and increase signal to noise ratio. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0014]      FIG. 1  shows an example process of assembling microelectrode sensing device. 
           [0015]      FIG. 2  shows an example of an assembled microelectrode sensing device. 
           [0016]      FIG. 3   a  shows a cross-sectional view of electrodes area of an example chip unit. 
           [0017]      FIG. 3   b  shows a cross-sectional view of a wire area of an example chip unit. 
           [0018]      FIG. 4  shows an example process of chip unit microfabrication. 
           [0019]      FIGS. 5   a - e  show mask designs of each layer on a chip unit. 
           [0020]      FIG. 6   a  shows an example of a PCB design of a microelectrode sensing device. 
           [0021]      FIG. 6   b  shows an enlarged view of example pads. 
           [0022]      FIG. 7   a  is a picture captured from under an upright microscope showing electrodes of an example microelectrode sensing device. 
           [0023]      FIG. 7   b  shows an enlarged view of electrodes in an example microelectrode sensing device. 
           [0024]      FIG. 8   a  is a picture captured from under an inverted microscope that shows electrodes of an example microelectrode sensing device. 
           [0025]      FIG. 8   b  shows an enlarged view of electrodes in an example microelectrode sensing device. 
           [0026]      FIG. 9  shows an assembly of an example metal clamp. 
           [0027]      FIG. 10  shows a PCB design of an example metal clamp. 
           [0028]      FIG. 11  shows a sketch map of a guide-path of an example metal clamp. 
           [0029]      FIG. 12  shows a sketch map of an example welding clamp. 
           [0030]      FIG. 13  shows usage of an example welding clamp. 
           [0031]      FIG. 14  shows an example process for cell positioning. 
           [0032]      FIG. 15  shows an example process for cell electro-rotation. 
       
    
    
     DETAILED DESCRIPTION 
       [0033]    Micro-electrode arrays (MEAs) can be used for real-time electrophysiological measurement at the cellular tissue and system levels. A typical miniature microelectrode array can be used to monitor and record bioelectric activities of cultured cells, such as myocardial cells. 
         [0034]    Because neurons cultured in vitro can not be easily positioned at or near the electrodes, an undesired spatial distance may exist between the neurons and the electrodes. Applying electrical stimulations and detecting electrophysiological signals are sensitive to the spatial relation between the neurons and the electrodes. In other words, the amplitudes of the detected electrophysiological signals become lower as the distance become larger. 
         [0035]    Positioning cells in a microelectrode device can be accomplished by having micro-wells or micro-tunnels fabricated on the surface of MEAs through micro-fabrication techniques. However, such implementations require moving the neurons to the wells or tunnels through capillaries one by one. In addition, neurons may escape from the wells and tunnels. 
         [0036]    Also, the surface of the MEAs can be modified using the differences between the adherence ability of neurons to different substrate to achieve positioning cells. However, beside the requirement of binding biological molecule to the substrate, getting the aim of positioning accurately is difficult, and the modified MEAs can not be used again. 
         [0037]    Techniques, systems, and apparatus are provided for positioning cells at or near the recording or sensing electrodes in a microelectrode sensing device. The ability to position the cells at a desired location can enhance the detection ability of MEAs and increase the applicable fields for the MEAs. The same microelectrode sensing device includes recording electrodes for recording bioelectrical activities of the positioned cells. 
         [0038]      FIG. 1  shows an example process of assembling a microelectrode sensing device. A microelectrode sensing device  100  includes a chip unit  102 , a printed circuit board (PCB)  104 , and a cell culture chamber  106 . The chip unit  102  is assembled to the PCB  104  by wire bonding the chip unit  102  to the PCB  104 . In addition, the wire bonded chip unit  102  is inserted inside the cell culture chamber  106 . The chip unit  102  is sealed between the PCB  104  and the cell culture chamber  106 . 
         [0039]    To encapsulate the microelectrode sensing device, the chip unit  102  is assembled to the PCB  104 . Then, pads on both the chip unit  102  and the PCB  104  are connected using conductive materials such as spun gold  101  and  103 . For example, spun gold  103  is connected to outer row of pads of the PCB  104 , and spun gold  101  is connected to inner row of pads of the PCB  104 . The spun gold  103 ,  101  are protected by silica resin  115 . The chip unit  102  is located inside the cell culture chamber  106  with the cell culture chamber  106  sealed to the PCB  104 . The cell culture chamber  106  can be sealed to the PCB  104  using silica resin or similar adhesives. 
         [0040]      FIG. 2  shows an example of an assembled microelectrode sensing device. As described above, the microelectrode device  100  includes a chip unit  102 , a PCB  104  and a cell culture chamber  106 . A chip unit  102  is attached to a PCB  104  and enclosed inside a cell culture chamber  106 . 
         [0041]      FIGS. 3   a  and  3   b  show example layers of a chip unit in a microelectrode sensing device. The chip unit (e.g., chip unit  102 ) includes multiple layers, such as a substrate layer  305 , a first conductive layer  301 , a first insulation layer  302 , a second conductive layer  307 , and a second insulation layer  304 . The insulation layers can include two different layers of Si 3 N 4  and SiO 2 . 
         [0042]    The first conductive layer can include one or more recording electrodes  306 , interconnects (i.e., wires)  303  and pads. The surfaces of the recording electrodes  306  are electroplated with a layer spongy material, such as platinum black  311 . The second conductive layer includes four or more positioning electrodes  309 , wires  308 , and pads. 
         [0043]    Each of the recording and positioning electrodes is separately linked to a pad, such as a bond pad. Also, signals can be applied to each electrode independent of other electrodes. Similarly, each electrode can be used to detect bioelectric activities separate and independent of the other electrodes. 
         [0044]    To improve cell positioning effect and increase the signal to noise ratio, the microelectrode sensing device can be optimized by turning the wires  303 ,  308  using arcs. Thus, all wires have rounded corners. Also, the wires  303  and  312  (see  FIG. 5a  below) can be gradually widened from the recording electrodes  306  to the pads. Further, the ground electrodes  315  and  321  can be placed at both conductive layers  301  and  307 . The ground electrodes  315  and  321  can be connected through a passage  335  at the first insulation layer  302  to ensure that any two wires  303  and  308  can be separated by ground electrodes  315 ,  321  and  335 . 
         [0045]    In addition, to reduce attenuation of crosstalk in electrodes, reduce noise and increase signal to noise ratio, the chip unit  102  can be optimized in design. For example, many ground electrodes can be fabricated on the conductive layers, and these ground electrodes can be connected through passages in the first insulation layer. Thus, each wire that links an electrode to a pad can be separated by ground electrodes. 
         [0046]    To facilitate viewing or imaging of the microelectrode device under an inverted microscope the substrate of the chip unit  102  can be implemented to include glass. In addition, the positioning electrodes  309  can be arranged in a ring. 
         [0047]    Fabrication of a Chip Unit. 
         [0048]      FIG. 4  shows an example process  400  for fabricating a chip unit. A substrate is cleaned ( 402 ). The first conductive layer (e.g., Ti—Au—Ti) is sputtered on the cleaned substrate ( 404 ). Recording electrodes, wires, pads, and ground electrodes are created on the conductive layer using a first mask ( 406 ). For example, recording electrodes, wires, pads, and ground electrodes can be patterned by photolithography through mask A, shown in  FIG. 5a  below, and etched. A first insulation layer is deposited over the substrate including the first conductive layer ( 408 ). For example, an insulation layer of Si3N4-SiO2 can be deposited on the substrate using PECVD. The contact holes of recording electrodes, pads, and ground electrode passages are created in the first insulation layer using a second mask ( 410 ). For example, the contact holes can be patterned through mask B, shown in  FIG. 5   b  below, and etched. A second conductive layer is created over the substrate including the first conducting layer and the first insulation layer ( 412 ). For example, a conductive layer of Ti—Au—Ti can be sputtered over the first insulation layer. 
         [0049]    Positioning electrodes, wires, and pads are created on the second conductive layer using a third mask ( 414 ). For example, positioning electrodes, wires, pads, and ground electrodes can be patterned through mask C, shown in  FIG. 5   c  below, and etched. A second insulation layer is deposited over the substrate including the first conductive layer, the first insulation layer, and the second conductive layer ( 416 ). For example, an insulation layer of Si 3 N 4 —SiO 2  can be deposited on the substrate using PECVD and be in contact with the first insulation layer. The contact holes of positioning electrodes, recording electrodes, pads, and ground electrodes are created using a fourth mask ( 418 ). For example, the contact holes of positioning electrodes, recording electrode, pads, and ground electrodes can be patterned through mask D, shown in  FIG. 5   d,  and etched. The windows of electrodes for electroplating are patterned using a fifth mask, such as mask E shown in  FIG. 5E  below ( 420 ). 
         [0050]    A layer of platinum black is applied to the windows of electrodes ( 422 ). Each window patterned by the fifth mask indicates an area used to mark the electrode for applying platinum black. In other words, all electrodes located inside the patterned window are electroplated with platinum black. For example, platinum black can be electroplated to the electrodes located inside the windows by applying a constant current utilizing an ultrasonic agitation. The wafer is cut into chips and cleaned ( 424 ). 
         [0051]      FIG. 5   a,    5   b,    5   c,    5   d,  and  5   e  show various masks used to fabricate a chip unit.  FIG. 5   a  shows a mask for etching recording electrodes (e.g., recording electrodes  306 ), wires (e.g., wires  303 / 312 ), pads (not shown in this figure), and ground electrodes (e.g., ground electrodes  315 ).  FIG. 5   b  shows a mask for etching (wet and dry) recording electrodes (e.g., recording electrodes  306 ), pads, and ground electrode passages (e.g., ground electrode passages  335 ) using lithography.  FIG. 5   c  shows a mask for etching positioning electrodes (e.g., positioning electrodes  309 ), recording electrodes (e.g. recording electrodes  306 ), wires (e.g. wires  308 ), pads (not shown in this figure), and ground electrodes (e.g., ground electrodes  321 ) using lithography.  FIG. 5   d  shows a mask for etching (wet and dry) positioning electrodes (e.g. positioning electrodes  309 ), recording electrodes (e.g., recording electrodes  306 ), pads (not shown in this figure), and ground electrodes (e.g., ground electrodes  321 ) using lithography.  FIG. 5   e  shows a mask for generating lithography windows of electrodes for electroplating platinum black onto the recording electrodes for example. 
         [0052]    PCB of a Microelectrode Sensing Device 
         [0053]      FIGS. 6   a  and  6   b  show an example PCB. The PCB (e.g., PCB  104 ) includes a carved out center area  604  for facilitating observation under an inverted microscope. The chip  102  is assembled to the area indicated by  603 . The PCB also includes pads  605  and  606  that surround the carved out center area  604  near the inner region of the PCB. Also, additional pads  607 ,  608  are located near the outer edge of the PCB and surround the pads  605  and  606 . The PCB also includes wires  602  for connecting pads  605  or  606  of the PCB to the pads  607  or  608  on the same PCB. The wires  602  that connect the inner pads  605 ,  606  with outer pads  607 ,  608  are shaped as arcs. The pads  605  and  606  of the PCB can include two types of pads arranged in two pad areas. For example, the two types of pads can be arranged in alternating positions, such as two rows. For example, pads  605  represent an inner row of pads. Pads  606  represent an outer row of pads. 
         [0054]    To facilitate bonding and increase bonding efficiency, two types of pads  605  and  606  are provided on the PCB at two pad areas. For example, at one pad area (e.g., an inner row), the pads  605  are shaped as circles. At another pad area (e.g., an outer row), the pads  606  are shaped as rectangles. Other shapes and arrangement can be implemented. Also, the wires  602  connecting the pads at inner brim of PCB to the pads at outer brim of PCB have rounded corners to increase the signal to noise ratio. The pads on the chip unit can be distributed at the edge of chip equably. 
         [0055]    As described above, the microelectrode sending device can be designed and assembled for easy viewing under an inverted microscope.  FIG. 7   a  shows an example picture of a microelectrode sensing device captured under an upright microscope. The picture of the microelectrode sensing device shows all of the positioning and recording electrodes.  FIG. 7   b  shows an enlarged version of the picture shown in  FIG. 7   a.    
         [0056]      FIG. 8   a  shows another example picture of a microelectrode sensing device including all of the recording and positioning electrodes.  FIG. 8   b  shows an enlarged version of  FIG. 8   a.  The design of the microelectrode sensing device shown in  FIGS. 8   a - 8   b  provides a larger observable area under an inverted microscope. 
         [0057]    Example Device for Cell Manipulation 
         [0058]      FIG. 9  shows an example device for performing cell manipulation electrophysiological detection. The device  900  used for cell manipulation includes a microelectrode sensing device  903  connected to a metal clamp. The metal clamp includes a PCB  905  with spring pins, a metal enclosure  904  and a cover plate  901 . The PCB  905  for the metal clamp is different from the PCB attached to the chip unit. The PCB  905  with spring-pins is attached or connected to the metal enclosure  904 . The spring-pins are used to apply signals to the chip unit. Two electrical connections are implemented to apply the signals to the chip unit. Using bonding, an electrical connection is provided between the chip unit  102  and the PCB  104  attached to the chip unit. Another electrical connection is provided between PCB  104  attached to the chip unit and the PCB  905  of the clamp using the spring-pins. The PCB  905  can include at least  3  positioning holes that can be used to firmly attach the PCB  905  to the metal enclosure  904 . In use, the microelectrode sensing device  903  is placed on the spring-pins and pressed tightly by the cover plate  901  through two spanners  902 . Also, the spring-pins on the PCB  905  are used to connect the PCB  905  of the metal clamp with the PCB  104  of the microelectrode sensing device  903 . 
         [0059]    The microelectrode sensing device  903  used for cell manipulation and electrophysiological detection includes a chip unit, a PCB, and a cell culture chamber. Thus, the microelectrode sensing device  903  can be fabricated to be the same as the microelectrode sensing device  100 . 
         [0060]    The cover plate  901  is used to fix the microelectrode sensing device  903  into the metal clamp. The cover plate  901  also ensures a steady connection between the two PCBs. The metal enclosure  904  includes a guide-path (shown in  FIG. 11  below) corresponding to the microelectrode sensing device  903  to restrict the movement of the microelectrode sensing device  903  in the direction of the spring-pins. 
         [0061]      FIG. 10  shows an example PCB design for a metal clamp. The reference number  1001  represents a brim of a PCB for a metal clamp. The PCB for the metal clamp includes positioning holes  1002  used for fixing the PCB to a metal enclosure (e.g., metal enclosure  904 ). The PCB includes a window  1004  used for observing an attached microelectrode sensing device under a microscope. Also, the PCB includes pads  1005  of spring-pins. In addition, the PCB includes electrical outlets  1010  for attached cables to them. Further wires  1009  provide various interconnects in the PCB. The wires are implemented to have rounded corners, such as an arc. 
         [0062]      FIG. 11  shows an example of a guide-path on a metal enclosure of a metal clamp. As described with respect to  FIG. 9  above, a metal enclosure includes guide-paths  1101  that correspond to a microelectrode sensing device to restrict the movement of the microelectrode sensing device  903  in the direction of the spring-pins. Also, guide-paths  1101  ensure that the pads of the microelectrode sensing device match with the spring-pins of the PCB of the metal clamp. 
         [0063]    The device used for cell manipulation also includes a welding clamp that ensures the vertical and correct welding of spring-pins.  FIG. 12  shows an example of a welding clamp. A welding clamp includes inserting holes  1202  and positioning holes  1201 . The position, diameter, and quantity of inserting holes  1202  are matched to the corresponding spring-pins on a PCB of a metal clamp. For example, the inserting holes  1202  ensure that the spring-pins vertically lineup with the PCB  905  on the metal clamp. The positioning holes  1201  are matched to the positioning holes (e.g., positioning holes  1002 ) on the PCB of the metal clamp. 
         [0064]      FIG. 13  shows an example usage of a welding clamp. The positioning holes  1201  are used to fix the PCB  905  to the welding clamp through the positioning holes  1002  of the microelectrode sensing device. In addition, the positioning holes  1201  ensure that the spring-pins in the inserting holes  1202  can be inserted into the pads  1005  of the PCB  905  correctly. 
         [0065]    Cell Position and Electrophysiological Detection 
         [0066]      FIG. 14  is an experimental flow chart shown an example of cell positioning. A cell suspension, such as neurons is added to a cell culture chamber (e.g., cell culture chamber  106 ) ( 1402 ). Positioning signals are applied to different positioning electrodes to generate a dielectrophoretic force ( 1404 ). For example, positioning signals can be applied to all positioning electrodes, or applied to only certain electrodes, in order to construct different cell patterns and/or concentrations. Also, the positioning signals can be applied all the time (i.e., continuously), or applied periodically. For example, one type of the positioning signals can include a pair of 5 MHz, 2 V sinusoidal signals with the phase-angle difference of 180° applied for 30 min. Further, positioning signals are not applied to any of the positioning signals to obtained control measurements ( 1406 ). 
         [0067]    After the application of the positioning signals, the microelectrode sensing device is incubated to allow the cells to form a cell network ( 1408 ). For example, the microelectrode sensing device can be incubated at 37° C. in a humidified incubator containing 5% CO 2  atmosphere. For a cell network that includes excitable cells, such as neurons, electrophysiological signals can be detected at different recording electrodes ( 1410 ). The electrophysiological signals obtained from cell networks of different cell concentration (due to different applications of different positioning signals to different positioning electrodes) are compared ( 1412 ). Based on this comparison, the effect of different cell concentrations and patterns can be obtained ( 1414 ). In addition, the electrophysiological signals obtained from the control positioning electrodes (due to no positioning signals applied to any positioning electrodes) are compared against those obtained from different cell concentrations and/or patterns ( 1416 ). Based on this comparison, the effects of application of the positioning signals can be determined ( 1418 ). 
         [0068]      FIG. 15  shows an example process for performing cell electro-rotation. The environment of cell suspension is changed to a low conductivity environment ( 1502 ). The cell suspension in the low conductivity environment is added to a cell culture chamber of a microelectrode sensing device ( 1504 ). Different positioning signals with different phases are applied to different positioning electrodes to form an electric field of rotation ( 1506 ). For example, different pairs of sinusoidal signals having different phases can be applied to selective positioning electrodes. Different pairs of positioning signals having the same frequency and amplitude but different phases are applied to selective neighboring electrodes. For example, one or more positioning electrodes are applied with a pair of sinusoidal signals with 0° phase. One or more positioning electrodes are applied with a pair or sinusoidal signals with 90° phase. One or more positioning electrodes are applied with a pair of sinusoidal signals with 180° phase. One or more positioning electrodes are applied with a pair of sinusoidal signals with 270° phase. Cell electro-rotation is observed under a microscope under above conditions ( 1508 ). 
         [0069]    While this document contains many specifics, these should not be construed as limitations on the scope of an invention that is claimed or of what may be claimed, but rather as descriptions of features specific to particular embodiments. Certain features that are described in this document in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable sub-combination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a sub-combination or a variation of a sub-combination. Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. 
         [0070]    Only a few examples and implementations are disclosed. Variations, modifications and enhancements to the described examples and implementations and other implementations may be made based on what is disclosed.