Abstract:
A patch-clamp system employs a high-frequency characterization of cell wall membranes. Changes in the frequency response of a tank circuit incorporating the cell wall membrane impedance provides highly sensitive and highly time-resolved measurements of ion channel activity.

Description:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR  DEVELOPMENT 
       [0001]    — 
       CROSS REFERENCE TO RELATED APPLICATION 
       [0002]    — 
       BACKGROUND OF THE INVENTION 
       [0003]    The present invention relates to a “patch-clamp” for investigating ion transport through cellular membranes and in particular to a patch-clamp system that may provide real-time tracing of ion channel activity with a bandwidth of up to 500 MHz. 
         [0004]    The lipid bilayers that make up cell membranes include ion channels that control the flow of ions into and out of cells. Certain ion channels open in response to signaling molecules including naturally occurring signaling molecules and drug molecules. In the development of therapeutic drugs it is necessary to determine the effect of the drug on ion channels either to avoid adverse effects or to create a positive therapeutic effect for the treatment of ion-channel related diseases. 
         [0005]    Analysis of the response of ion channels may be conducted with a so-called “patch-clamp”, traditionally a micropipette adhered to the surface of a cell by a slight suction. An electrical connection across the membrane of the cell is then made by one of a number of techniques, for example, by applying a sharp suction pulse to the pipette to open a hole in the cell wall. Measurement of small electrical changes across the cell membrane made by a miniature electrode inserted into or near the opening may then be used to deduce the flow of ions through the ion channels. The small amounts of electrical current involved in these measurements require an extremely high resistance seal between the pipette and the cell wall (a giga-ohm seal). 
         [0006]    Drug screening often requires making many ion-channel measurements. Accordingly the pipette having a single opening has been replaced with a plate having multiple small pores each of which may accept a cell. The plate array allows the parallel processing of multiple cells and may be more readily integrated into automated equipment. 
         [0007]    The sensitivity of measurements of small current flows through ion channels is significantly limited by the poor electrical characteristics of a bare electrode immersed in the aqueous medium inside or outside of the cell. As a result, rapid changes in ionic transport cannot be resolved in the time domain. 
       SUMMARY OF THE INVENTION 
       [0008]    The present invention provides a high-frequency measurement of cell wall impedance changes due to ion channel activity. This differs from the typical “direct current” measurements of currents or voltages adopted in the prior art. By construction of a “tank circuit” incorporating the impedance of the cell membrane, changes in the resonance of this tank circuit may be used to accurately and quickly assess changes in the cell wall membrane. The high-frequency measurements allow electrodes to be formed as waveguides having far higher sensitivity and response rates than a bare electrode immersed in aqueous medium. 
         [0009]    Specifically the present invention provides a high frequency patch-clamp system using an electrically insulating support providing support region for holding a cellular membrane fixed with respect to the electrically insulating support. A first and second electrode on opposite sides of the support region, separated by the cellular membrane, are connected to circuitry providing a high-frequency signal across the first and second electrodes to determine changes of impedance across the cellular membrane from measurement of a change in electrical resonance. It is thus one object of at least one embodiment of the invention to employ an alternating current measurement technique to improve the sensitivity of ion channel measurements. It is another object of the invention to eliminate the need for manipulation of freestanding electrodes during the patch-clamp process. 
         [0010]    The support region may be an aperture through the electrically insulating support forming a lip on a first side of the aperture sized to accept a cellular membrane spanning the lip to form a giga-ohm seal with the electrically insulating support. 
         [0011]    It is thus an object of the invention to permit both AC and DC measurements, the latter employing the aperture. 
         [0012]    At least one of the first and second electrodes may be a waveguide attached to the electrically insulating support. 
         [0013]    It is thus an object of at least one embodiment of the invention to permit waveguide-like electrodes to provide a high-frequency response. 
         [0014]    The waveguide may be a strip-line. 
         [0015]    It is thus an object of at least one embodiment of the invention to provide a waveguide that may be readily fabricated on an insulating substrate. 
         [0016]    The circuitry may be a tank circuit incorporating a capacitance across the cellular membrane as a capacitance of the tank circuit. 
         [0017]    It is thus another object of the invention to provide a simple method of measuring impedance changes across a cellular membrane by monitoring a tank circuit resonance. 
         [0018]    The circuit includes at least one inductor in series with a capacitance. 
         [0019]    It is thus an object of at least one embodiment of the invention to provide a simple method of tuning the tank circuit for convenient measurement. 
         [0020]    The electrically insulating support may be a planar support and includes multiple apertures and lips each associated with a least one different second electrode for parallel measurements of cellular membranes at each of the multiple apertures. 
         [0021]    It is thus an object of at least one embodiment of the invention to provide a system suitable for high throughput measurement. 
         [0022]    The high-frequency signal may be in excess of one MHz. 
         [0023]    It is thus an object of at least one embodiment of the invention to permit measurement of extremely small impedance values. 
         [0024]    The impedance measured may be capacitance of the cellular membrane. 
         [0025]    It is thus an object of at least one embodiment of the invention to permit measurement of membrane capacitance in lieu of current or voltage transfer. 
         [0026]    Alternatively, the impedance measured may be resistance of the cellular membrane. 
         [0027]    It is thus an object of at least one embodiment of the invention to permit conventional current flow measurements as manifest in resistance. 
         [0028]    The first electrode may include two separated portions wherein the high-frequency signal is applied to one portion and monitored at the second portion. 
         [0029]    It is thus an object of at least one embodiment of the invention to permit either reflected or transmitted energy measurements or both to be made. 
         [0030]    These particular features and advantages may apply to only some embodiments falling within the claims and thus do not define the scope of the invention. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0031]      FIG. 1  is an elevational cross-section of a prior art patch-clamp used for whole-cell recording; 
           [0032]      FIG. 2  is an elevational cross-section of a planar patch-clamp implementing the present invention allowing reflected or transmitted energy measurement; 
           [0033]      FIG. 3  is a schematic representation of a four-port tank circuit implemented using the patch-clamp of  FIG. 2 ; 
           [0034]      FIG. 4  is the plot of a frequency resonance of the tank circuit of  FIG. 3 , measurable in the present invention to determine capacitive or resistive impedance across the cell membrane; 
           [0035]      FIG. 5  is a perspective view of a multiport planar patch-clamp system using the present invention; 
           [0036]      FIG. 6  is a perspective view of an experimental apparatus implementing the present invention and 
           [0037]      FIG. 7  is a figure similar to that of  FIG. 2  showing patch-clamp of the present invention applied to an intact cell membrane. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0038]    Referring now to  FIG. 1 , a prior art whole-cell patch-clamp  10  may employ a micropipette  12  having an aperture  14  to which a cell  16  is drawn by suction. The cell  16  may attach to the aperture  14  to create a giga-ohm seal to a lip  18  of that aperture. The cell  16  may otherwise be suspended in a liquid medium  20  providing an environment desired for a particular experiment. 
         [0039]    A sharp suction may be used to open a hole  21  in the cell wall of the cell  16  providing a low resistance path from the interior cytoplasm of the cell through a solution  24  to a microelectrode  26  within the micropipette  12 . The microelectrode  26  is typically a silver electrode coated with silver chloride for electrochemical stability. 
         [0040]    A sensitive current detector  30  may be connected between the microelectrode  26  and the liquid medium  20  to measure the passage of ions  32  through channels in the cell wall. The current detector  30  may provide for a voltage-clamping action, if desired, using a conventional voltage feedback circuit. Generally the bare microelectrode  26  provides electrical characteristics that severely limit the frequency of the measure of ionic currents. Further, only resistive impedance of the cell wall may be determined. 
         [0041]    Referring now to  FIG. 2 , the present invention provides an insulating substrate  40  having an aperture  42  providing a lip  44  on one side of the substrate  40  that may be spanned by a cellular membrane  46 , for example, using the whole cell technique described above, or by a variety of other techniques well known in the art. A first electrode  48  may be positioned on one side of insulating substrate  40  and lip  44  while a second electrode  50  may be positioned on the other side of the insulating substrate  40  and lip  44  so that the first electrode  48  and second electrode  50  are on opposite sides of the cellular membrane  46 . Each of electrodes  48  and  50  may connect via terminals  52  and  54  to measurement circuitry  70  that will apply a high-frequency signal across electrodes  48  and  50 . 
         [0042]    An additional first electrode  48 ′ may also be positioned on the same side of the substrate  40  as electrode  48  to join thereto at the lip  44  but to extend to a second terminal  52 ′ at which transmission through the network may be measured as will be described. 
         [0043]    Electrode  50  may communicate with a solution  24  supporting the underside of the cellular membrane  46  and may for example be a silver/silver-chloride electrode of a type known in the art to minimize artifacts created by oxidation-reduction reactions at the metallic surface. As such, electrode  50  will be considered ground for the purpose of discussion. 
         [0044]    Electrodes  48  and  48 ′, where they contact the liquid medium  20 , may also be silver/silver-chloride material; however, in the preferred embodiment, for most of their lengths they may be insulated from the liquid medium  20  and, in this insulated portion, electrodes  48  and  48 ′ may preferably be a micro-strip-line having a controlled and well-defined electrical characteristic such as will provide a waveguide for high frequency transmission of electrical signals. Such micro-strip-lines may, for example, provide a conductor sandwiched between a well-characterized dielectric in turn sandwiched between ground planes to provide the necessary boundary conditions for waveguide propagation. The fabrication of the micro-strip-lines may be made by using well-known integrated circuit techniques or surface coating methods. 
         [0045]    Referring now to  FIG. 3 , the device of  FIG. 2  provides a four terminal network  56  in which terminal  52  and terminal  54  provide input terminals for application of a radiofrequency signal (for example, on the order of 100 MHz) and optional measurement of reflected energy, and terminal  52 ′ and  54  provide output terminals for measurement of transmitted energy. 
         [0046]    Terminal  52  may lead to electrode  48  through an inductor  60  being preferably a discreet inductor (for example, on the order of 20 nH) but possibly being formed by the length of electrode  48  itself which may be used to tune the tank circuit as will be described. Electrode  48  may in turn connect to ground ( 50 ) through a coupling capacitive  62  through the cellular membrane  46  and through a small resistance  64  representing the giga-ohm seal between the substrate  40  and the cellular membrane  46  and a parallel resistive component through the cellular membrane  46 . 
         [0047]    The junction of electrode  48  and capacitance  62  is also connected with electrode  48  which may then join with an inductor  66  which leads to terminal  54 . 
         [0048]    The circuit so described will be recognized as a tank circuit providing for series resonance between the inductors  60  and capacitance  62 . This resonance may be measured between terminals  52  and  54  as a reflected energy by measurement circuitry  70  or between terminals  52 ′ and  54  as a transmitted energy by measurement circuitry  70 ′. Such analyzers may, for example, provide for a frequency sweep measuring reflected or transmitted energy or may provide for parallel resonance measurements by broadband excitation and frequency analysis for using the fast Fourier transform or other similar technique. 
         [0049]    Referring now to  FIG. 4 , the measured resonance  72  will show an amplitude (power, voltage, current) as a function of frequency having a peak at a center frequency  74  determined by the inductance  60  and capacitance  62 , and having a width  76  (Q=f/□f) determined by the resistance and dielectric losses  64 . As will be understood in the art, changes in the capacitance  62  will be reflected in movement of the center frequency  74  of the resonance  72  by a frequency shift whereas changes in the resistance  64  will be reflected in changes in the width  76  of the resonance  72 . 
         [0050]    Monitoring the resonance  72  will provide extremely high time resolution of ion transfer events through the cellular membrane  46 . Either or both of resistance and capacitance can be measured in this manner and it will be understood that a selection of component values of the inductors  60  and  66  and capacitance  62  (the latter which may be controlled through the selection of aperture size) can be used to accentuate one or the other of these measurements. 
         [0051]    Referring now to  FIG. 5 , the substrate  40  may include multiple apertures  42  each having a dedicated set of electrodes  48  and  48 ′ to permit high throughput analysis of multiple cells at each of the apertures  42 . The inductors  60  and  66  may be discrete inductors placed on the surface of the substrate  40  using conventional electrical assembly techniques. 
         [0052]    Referring now to  FIG. 6 , terminals  54  and  52  may be implemented through a standard high-frequency coaxial connector as may terminals  52 ′ and  54 . The impedance of the micro-strip-lines may be set to approximate a standard impedance of approximately 50 ohms to provide impedance matching with subsequent measurement circuitry  70  and with the tank circuit by proper selection of the component values and device dimensions and/or the use of matching networks (not shown). 
         [0053]    A chamber  77  of solution may be placed underneath the substrate  40  and connected to the electrodes  50  on the underside of substrate  40 . Liquid medium  20  may be limited in extent about the aperture  42  to limit its effect on the transmission of high-frequency signals through electrodes  48  and  48 ′. 
         [0054]    Referring now to  FIG. 7 , if simultaneous DC and AC measurements through the cellular membrane  46  are not required (such as can be conducted using the system of  FIG. 2 ), the present invention can be used with an intact cell  80  resting on the insulating substrate  40  without an aperture  42  (or with an optional aperture  42  only used to stabilize the cell  80  with a slight negative pressure). In this case, the measurement is an AC measurement of series capacitance  82   a  and  82   b , the first from electrode  48  through the cellular membrane  46  into the cellular fluid  84  and the second from the cellular fluid  84  through the cellular membrane  46  to electrode  48 ′. As shown in this figure, normally the electrodes  48  will be covered with a dielectric layer  86  separating them from the liquid medium  20  to eliminate resistive mode conduction. 
         [0055]    It is specifically intended that the present invention not be limited to the embodiments and illustrations contained herein, but include modified forms of those embodiments including portions of the embodiments and combinations of elements of different embodiments as come within the scope of the following claims.