Abstract:
A system and method for non-invasively measuring the electrical potential radiated by a cell. To do this, a probe is positioned within ten microns distance from the cell for receiving the signal. Also, a reference potential is determined for the cell&#39;s environment. A sensor records the signal and compares the reference potential to the cell&#39;s signal to measure the electrical potential of the cell.

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention pertains generally to systems and methods for measuring electrical fields. More specifically, the present invention pertains to systems and methods for measuring extremely small electrical fields produced by a living cell with the nutrient medium surrounding it. The present invention is particularly, but not exclusively, useful for systems and methods that can non-invasively measure the free-space electric field potential of cells, such as biological entities from the group including animal cells, plant cells, neurons, bacterial specimens and amoebae.  
       BACKGROUND OF THE INVENTION  
       [0002]     Living cells, including neurons, exhibit electrical potentials that, although quite small, can be measured. For example, the total charge contained in a typical cell is approximately 5×10 −13 C, when measured for a voltage of −50 mV relative to the nutrient bath in which the cell is maintained. Obviously, obtaining such measurements can be difficult. Nevertheless, the effort may be worthwhile because a measurement of a cell&#39;s electrical potential contains information that can be very useful for evaluating the health and chemical composition of the cell. Such information may be particularly useful when successive measurements of a cell can be taken over extended periods of time. In addition, a cell&#39;s electrical potential may change in response to the exposure of the cell to a biological, chemical or pharmacological substance. Thus, the measurement of this change can be useful in detecting the presence of one of these substances.  
         [0003]     Existing methods for measuring the electrical potential of a cell include procedures that require the insertion of a probe into the cell, or direct contact between the probe and the membrane of the cell. Fromherz et al., however, have proposed another approach for measuring the electrical potential of a cell which uses a custom-built field effect transistor (FET). This method relies on a capacitive coupling between the cell and the transistor that is on the order of picofarads. Consequently, the distance between the cell and the transistor must be around five one-hundredths of a micron (0.05 μm). As will be appreciated by the skilled artisan, this is a very small distance, and is virtually tantamount to actual contact.  
         [0004]     In light of the above, it is an object of the present invention to provide a non-invasive system and method for measuring the electrical potential produced by a cell, in which the active electronics do not have to be in the immediate vicinity of the cell. Another object of the present invention is to provide a system and method for non-invasively measuring the electrical potential of the cell that is effective when the region of the probe that couples to the electric potential produced by the cell and passes the signal to the first stage electronics is as much as ten microns (10 μm) distant from the cell. Still another object of the present invention is to provide a system and method for measuring the electric potential produced by the cell within the nutrient bath that surrounds it, so that the cell can be maintained for extended periods of time in its required nutrient and electrolyte environment, without the cell being substantially disturbed. Yet another object of the present invention is to provide a system and method for measuring the electrical potential of a cell to determine whether the cell has been exposed to one or more biological, chemical or pharmacological substances. Another object of the present invention is to provide a system and method for measuring the electrical potential of a cell that is easy to use, is relatively simple to manufacture and is comparatively cost effective.  
       SUMMARY OF THE INVENTION  
       [0005]     A system for non-invasively measuring the electrical potential of a cell includes, in combination, a reference electrode, a probe, a sensor and a comparison means, such as a computer, or an operational amplifier. In overview, the cell radiates an electromagnetic signal that is received by the probe and subsequently recorded by the sensor. The computer (operational amplifier) then compares this signal to a reference potential that is determined by the reference electrode, to measure the electrical potential of the cell. For the present invention all varieties of cells are contemplated, including such biological entities as: animal cells, plant cells, neurons, bacterial specimens and amoebae.  
         [0006]     In use, the probe is immersed or is built into a nutrient bath wherein the cell to be evaluated is located. More specifically, the probe is positioned so that its detection surface is within a predetermined distance from the cell (e.g. within ten microns (10 μm) from the cell). This positioning allows the conducting surface of the probe to receive the signal that is radiated from the cell. Preferably, in order to optimize signal reception, the conducting surface of the probe, and the cell will have a substantially same spatial extent (i.e. they will be about the same size). After being received, the signal is electronically passed from the conducting surface to the sensor for recording.  
         [0007]     Along with the probe, a reference electrode is also immersed or is built into the nutrient bath. As the probe receives the signal from the cell, the reference electrode is used to determine a reference potential for the bath. The potential of the cell is measured relative to this reference potential. As envisioned for the present invention, when the signal that is radiated by the cell has a frequency greater than about ten Hertz (&gt;10 Hz), the sensor will be able to record the signal with a signal to noise ratio (SNR) greater than one (SNR&gt;1). It is also envisioned for the system of the present invention that the sensor may record signals that are simultaneously radiated from a plurality of cells. In this case, the sensor will use the resultant plurality of signals to measure an integrated response. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0008]     The novel features of this invention, as well as the invention itself, both as to its structure and its operation, will be best understood from the accompanying drawings, taken in conjunction with the accompanying description, in which similar reference characters refer to similar parts, and in which:  
         [0009]      FIG. 1  is a perspective schematic view of the system of the present invention being used in its intended environment;  
         [0010]      FIG. 2  is a cross sectional view of the probe of the present invention and a cell whose electrical potential is being measured by the probe, as seen along line  2 - 2  in  FIG. 1 ; and  
         [0011]      FIG. 3  is a cross sectional view of another embodiment of a probe for the present invention. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0012]     Referring initially to  FIG. 1 , a system in accordance with the present invention is shown and generally designated  10 . In detail,  FIG. 1  shows that the system  10  includes a system electronics unit  12  which has a sensor  14  that may be electronically connected to a computer  16 , if desired. Further, the system  10  is shown to include a reference electrode  18  that may be, electronically connected to the computer  16  via a line  20 , also if desired. Alternatively, the reference electrode  18  may be electronically connected to the sensor  14  via a line  20   a . The system  10  also includes a probe  22  that is electronically connected to the sensor  14  via a line  24 .  
         [0013]     Still referring to  FIG. 1  it is seen that an intended operational environment for use of the system  10  includes a tray  26 , or sealed enclosure, for holding a nutrient bath  28 . As intended for the present invention, the nutrient bath  28  is appropriately selected to provide the proper nutrients and electrolyte conditions for maintaining the particular biological entities to be evaluated. Typically, these biological entities will be cells  30  (of which the cells  30   a ,  30   b  and  30   c  are only exemplary) that are taken from the group of all cells comprising animal cells, plant cells, neurons, bacterial specimens and amoebae. Additionally, the cells  30  can be modified, for example by the insertion of protein pores or genetically modified cells  30  can be used.  
         [0014]     Referring now to  FIG. 2 , it is seen that the probe  22  includes an electrode  32  that is covered by a dielectric insulator  34  to protect the probe  22  from direct resistive contact with the nutrient bath and cell. The conducting surface  36  of the probe  22  senses the electric potential produced over its volume by the cell  30   a . As is well known by the skilled artisan, these signals are indicative of the potential within the cell  30   a . Still referring to  FIG. 2  the general structure of an exemplary cell  30   a  is shown. Specifically, the cell  30   a  is shown to have an outer membrane with a thickness  40  that is about ten nanometers (nm). Also, the cell  30   a  has an averaged radius  42  that extends from the center  44  of the cell  30  to the membrane  38  and is equal to about ten microns (10 μm).  
         [0015]     Referring now to  FIG. 3 , another embodiment of a probe (designated probe  122 ) for use in the present invention is shown. As shown, the probe  122  includes an electrode  132  that is covered by a dielectric insulator  134 . For this embodiment of the present invention, a dielectric insulator  134  is provided to prevent electrical contact between the electrode  132  and the nutrient bath  128 . A line  124  connects the electrode  132  to the sensor  14  (sensor  14  shown in  FIG. 1 ). As further shown in  FIG. 3 , the probe  122  includes a guard  50  that partially surrounds the covered electrode  132  and is connected to the sensor  14  (shown in  FIG. 1 ) by line  52 . With this cooperation of structure, an opening in the guard  50  allows the cell  130  to approach the covered electrode  132 . Typically, the opening will have approximately the same spatial extent as the cell  130 , as shown. In accordance with the present invention, the guard  50  can be maintained at substantially the same potential as the electrode  132  to prevent capacitive coupling between the electrode  132  and the nutrient bath  128 .  
         [0016]     Continuing with  FIG. 3 , it can be seen that the probe  122  further includes a conducting layer  54  (that is also insulated from the nutrient bath  128 ). An insulating layer  56  is disposed between the conducting layer  54  and the guard  50  to isolate the conducting layer  54  from the guard  50 . As further shown, line  58  is provided to connect the conducting layer  54  to the sensor  14  (shown in  FIG. 1 ). In accordance with the present invention, the potential of the conducting layer  54  can be controlled to minimize the distortion of the electric fields within the nutrient bath  128 , minimize coupling between adjacent probes  122  when multiple probes  122  are used, or to maximize the measured signal.  
         [0017]     In another embodiment of a probe  122  (not shown), a portion or all of the sensor  14  (shown in  FIG. 1 ) can be attached to the electrode  32 ,  132  for movement therewith. For this embodiment, batteries for powering the attached portion of the sensor  14  can be coupled with the electrode  32 ,  132  and the attached portion of the sensor  14  or the batteries can be positioned remotely and connected to the sensor  14  using a cable.  
         [0018]     Preferably, the electrode  32 ,  132 , line  24 ,  124  and other regions of the signal input to the sensor  14  are isolated from the outside world by an impedance of at least 1 GΩ, and more preferably 100 GΩ, at the frequency of interest. Further, the sensor  14  is preferably stable with respect to drift at its input due to the input bias current of its first stage amplifier when connected to the purely capacitive input of the electrode  132 . Additionally, it is preferable that the current and voltage noise of the first stage amplifier in the sensor  14  is sufficiently low so that when connected to the purely capacitive impedance of the electrode  32 ,  132 , the cell signal can be measured with adequate fidelity over the required bandwidth.  
         [0019]     A suitable circuit for use in the sensor  14  is disclosed in co-pending U.S. patent application Ser. No. 09/783,858 for an invention entitled “Low Noise, Electric Field Sensor,” filed on Feb. 13, 2001, which is assigned to Quantum Applied Science and Research Inc. The entire contents of U.S. application Ser. No. 09/783,858 are incorporated by reference herein. Alternatively, a circuit having an impedance of sufficiently high value at the signal of interest to provide a current path to the sensor ground without reducing the signal presented at the amplifier input to an unacceptably low level can be used in the sensor  14 .  
         [0020]     In operation, the reference electrode  18 , and the probe  22  are both immersed or are otherwise positioned into the nutrient bath  28 . When a cell  30  (e.g. cell  30   a ) comes within a distance  46  of the probe  22 , the probe  22  picks up a signal from the cell  30   a . For purposes of the system  10  of the present invention, the distance  46  will effectively be somewhere within a range from zero to approximately ten microns. Preferably, the distance  46  will be around one micron, or one-half micron. Regardless the extent of the distance  46 , the potential that is received by the conducting surface  36  of the probe  22  is applied at the input of the sensor  14 . It is noted that in order to optimize the received signal, the conducting surface  36  and the cell  30   a  should be about the same size. In any event, as the signal from cell  30   a  is being sent to the sensor  14 , a reference potential for the nutrient bath  28  is simultaneously sent from the reference electrode  18  to the computer  16 . The electronics unit  12  then compares the signal received from the cell  30   a  with the reference potential for the nutrient bath  28 . The result is a read out  48  that is indicative of the electrical potential of the cell  30   a  with respect to the bath. It will be appreciated by the skilled artisan that several cells  30  can be measured at the same time. The result in this latter case will be an integrated response. Either way, the read out can be used to evaluate the condition and activity of the cell or cells  30  in a manner well known to those skilled in the pertinent art.  
         [0021]     In accordance with the methods of the present invention, the change in a cell&#39;s electrical potential that occurs in response to the exposure of the cell to a biological, chemical or pharmacological substance can be measured. Specifically, the electrical potential of the unexposed cell  30  can first be measured as described above. Next, the cell  30  can be exposed to an agent such as a biological, chemical or pharmacological substance. In one implementation, the agent is simply added to the nutrient bath  28 . After the addition, the electrical potential of the exposed cell  30  can be continuously monitored to determine whether and when the exposure has altered the cell  30 , and the corresponding change in the cell&#39;s electrical potential. Once a cell&#39;s response to an agent has been measured, this information can be used, for example, to determine the exposure history of another cell (of the same cell type).  
         [0022]     By way of example, the strength of a signal from a cell  30 , and the general signal to noise ratio (SNR) that is received by the unit  12  can be estimated. Assuming 20% of the charge on cell  30   a  is opposite the conducting surface  36  of the probe  22 , it can be shown that a total charge on the order of 3×10 −16 C will be coupled from the cell  30   a  into the electronics unit  12 . This will give an input voltage to the sensor  14  of around one hundred micro volts (100 μV). Alternatively, the fraction of voltage from cell  30   a  that is coupled into the sensor  14  can be approximately given by the expression C electrode /(C electrode-cell +C sensor ). This expression predicts an input signal of one hundred sixty micro volts (160 μV). Thus, even for a single small cell  30   a , the SNR of over 100 is predicted at frequencies above 10 Hz. For larger cells, the signal will, of course, be increased.  
         [0023]     While the particular Circuit and Method to Non-Invasively Detect the Electrical Potential of a Cell or Neuron as herein shown and disclosed in detail is fully capable of obtaining the objects and providing the advantages herein before stated, it is to be understood that it is merely illustrative of the presently preferred embodiments of the invention and that no limitations are intended to the details of construction or design herein shown other than as described in the appended claims.