Abstract:
A method of forming extremely small pores in glass or a similar substrate, useful, for example, in patch clamp applications, that employs a backer plate to contain energy of a laser-induced ablation through the front surface of the substrate so as to create a rear surface shock wave providing a fire polishing of the exit aperture of the pore such as produces improved sealing with cell membranes.

Description:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
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     CROSS REFERENCE TO RELATED APPLICATION 
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     BACKGROUND OF THE INVENTION 
     The present invention relates to “patch clamping” for investigating ionic and molecular transport through cellular membranes via ion channels and, in particular, to a substrate providing a set of nano to microscale pores that may be readily sealed to cellular membranes. 
     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. 
     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 to the interior of the cell can be made, 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 may then be used to deduce the opening or closing of particular ion channels. 
     The small amount of electrical current involved in these measurements requires an extremely high resistance seal between the pipette and the cell wall (a gigaohm seal or gigaseal). Typically a gigaohm seal should be of the order of 15-20 gigaohms and at least 5 gigaohms. 
     Drug screening can require a large number of ion channel measurements. Accordingly, in current practice, the pipette can be 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 than a pipette. 
     The production of nanoscale holes in a plate structure is relatively difficult. One technique requires irradiating a glass or quartz substrate with heavy ions which leave behind a track of molecular damage that may preferentially be etched, for example, with hydrofluoric acid. The timing of the etch is controlled so that it breaks through on the far side of the substrate to produce the correct hole size. 
     The need for access to heavy ion accelerators for the production of nano-sized holes can be avoided by a second technique which employs a laser to ablate a crater through a pre-thinned glass substrate. By controlling the duration and power of the laser, a generally conical crater may break through the opposite side of the substrate with an appropriate size of hole. 
     One disadvantage to the laser approach is that it spreads molten glass and debris on the exit of the hole. This debris impedes proper adherence between the cell membrane and the lip of the hole leading to a reduction in the electrical resistance of the hole so formed. 
     SUMMARY OF THE INVENTION 
     The present invention provides an improved technique for the generation of nanoscale-sized pores through a substrate using a laser. In the technique, the substrate is backed by a thermally expanding substance different from the substrate. In the final stages of hole formation, the thermally expanding substance, heated by the laser, produces a shock wave creating a counter-facing concave crater intersecting the front surface of a crater being created by the laser. The shock wave is such as to fire polish the pore exit, substantially increasing the ability of the pore to form high resistance seals with cell membranes. Fire polishing uses heat or flame to melt irregularities which then smooth under the influence of surface tension. 
     Specifically then, the present invention provides a method of creating nanoscale holes comprising the steps of creating a layered structure comprising a substrate material receiving the nanoscale hole backed by a second, shock wave containing backer material adjacent to a rear face of the substrate material. A focused laser is applied for a first period to a front face of the substrate material to ablate a first crater opening at the front face of the substrate material and extending into the substrate material by an amount less than a thickness of the substrate material. The application of the focused laser is continued for a second period to heat material beyond the first crater to produce a shock wave generating a second crater starting at the rear face of the substrate material and extending into the substrate material to connect with the first crater thereby creating an opening between the first and second crater having a hole diameter. 
     It is thus an object of at least one embodiment of the invention to provide nanoscale sized pores that have reduced debris onto the rear surface of the substrate as occurs during standard laser hole formation. 
     The hole diameter may be less than 1000 nm. 
     It is thus an object of at least one embodiment of the invention to produce a pore size suitable for use in patch clamp applications. 
     The material may be transparent. 
     It is thus an object of at least one embodiment of the invention to provide a substrate material allowing transmission of light permitting both electrical and optical measurements of membranes. 
     The material to be drilled is borosilicate glass. 
     It is thus an object of at least one embodiment of the invention to provide a substrate suitable for use in electrophysiology applications. 
     The method may further include the step of pre-forming pockets in a front face of the substrate material and the first crater may be substantially centered within the pockets. 
     It is thus an object of at least one embodiment of the invention to permit an arbitrary thickness of the substrate as may be required for structural integrity. 
     The invention may employ a volatilizable material between the substrate material and the backer material. 
     It is thus an object of at least one embodiment of the invention to provide an increased pressure wave for improved hole formation. 
     The volatilizable substance may be water. 
     It is thus an object of at least one embodiment of the invention to permit the use of relatively safe volatilizable substances. 
     The volatilizable substance may be a liquid held between the material and the backer material by spacers defining the thickness of the volatilizable substance. 
     It is thus an object of at least one embodiment of the invention to permit precise control of the parameters of shock wave formation. 
     The focused laser may be applied so that the second crater reaches melting temperatures to create a fire polished surface. 
     It is thus an object of at least one embodiment of the invention to provide improved sealing surfaces to the aperture for the creation of higher resistance seals to cell membranes. 
     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 
         FIG. 1  is a schematic diagram of an apparatus used for producing a planar patch clamp plate per one embodiment of the present invention; 
         FIG. 2  is a block diagram of a patch clamp produced by the machine of  FIG. 1 ; 
         FIG. 3  is a cross-section along line  3 - 3  of  FIG. 2  showing a spacing of a patch clamp substrate from a backer material by a gap filled with a volatile substance; 
         FIG. 4  is a figure similar to that of  FIG. 3  showing an initial stage of laser ablation creating a first crater and showing the scattering of molten debris onto the front surface of the substrate; 
         FIG. 5  is a figure similar to that of  FIG. 3  showing transmission of energy through the substrate into the volatile substance before eruption of the first crater through the substrate; 
         FIG. 6  is a figure similar to that of  FIG. 5  showing the creation of a shock wave by a volatile substance behind the substrate; 
         FIG. 7  is an enlarged view of the substrate of  FIG. 6  after the shock wave showing the generation of a fire polished second crater counter to the first crater; and 
         FIG. 8  is a simplified representation of the use of the substrate of  FIG. 7  in a patch clamp application. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Referring now to  FIG. 1 , the present invention may use an excimer laser  10  having collimating and focusing optics  11  to direct a narrow collimated beam  12  of light along an axis  15  toward a front surface of a substrate assembly  14  held on a mechanical stage  16 . The laser may, for example, have a frequency range of 192 to 157 nm. 
     The laser  10  and stage  16  may be controlled by an automated controller  18  of the type well known in the art providing control signals  22  to the laser  10  controlling its output power in a series of pulses as will be described and providing control signals  22  to actuator motors  24  providing x-y control of the stage  16 . 
     Referring now to  FIGS. 2 and 3 , the substrate assembly  14  may include an upper substrate  26 , for example, a borosilicate cover slip having a thickness of approximately 150 microns. A front surface of the upper substrate  26  may have a series of depressions or wells  28  formed at regular x-y grid locations  29 . The wells  28  provide a thinned portion  30  at the locations  29  measured along axis  15  having a thickness of 100 to 1000μ and may be molded, ground or etched in the substrate  26 . The diameter of the wells  28  may be relatively large, for example, 5.0 mm and serve simply to permit a generally thicker substrate  26  in regions outside of the locations  29  for structural convenience. 
     The substrate  26  may have a backer plate  32  positioned adjacent to the rear surface of the substrate  26  and spaced therefrom by optional spacer  31  formed, for example, of polydimethylsiloxane (PDMS). The PDMS may be cast on the rear surface of the substrate  26  through a mold produced using integrated circuit techniques to provide precisely controlled spacer thickness or may be spun-coated and selectively removed except at the edges of the substrate  26 . 
     The space between the substrate  26  and the backer plate  32  is filled with a volatile material  34 , preferably water, but possibly other materials including, for example, acetone. The space between the substrate  26  and backer plate  32  must be determined by experiment depending on the particular laser and material of the backer plate  32  but can, for example, be as little as the separation provided strictly by the capillary forces of water without a separate spacer  31 . In one embodiment, the substrate  26  and backer plate  32  may be substantially adjacent while nevertheless providing a thoroughly induced counter shock wave, e.g., proceeding in a direction opposite the ablation provided by the collimated beam  12 . 
     Referring now to  FIGS. 1 and 4 , the excimer laser  10  may be positioned above a first location  29  and pulsed by the controller  18  to produce a series of controlled light pulses  40  of laser beam  12 , the light pulses  40  absorbed by the material of the substrate  26  to ablate, over a first time, a first crater  42 . Molten material  44  ejected from the crater  42  will generally adhere to a front surface  46  of the substrate  26  creating substantial surface roughness. The laser ablation is continued until the deepest portion of the crater  42  reaches a distance from three to 10 microns from the rear surface of the substrate  26 . 
     Referring now to  FIG. 5 , although the Applicant does not wish to be bound by a particular theory, it is believed at this point, leakage energy from the pulses  40  passes through the remainder of the substrate  26  to heat material beyond the crater  42 , preferably the volatile material  34  but possibly air or the rear surface of the substrate  26  itself hit by reflected energy. 
     As shown in  FIG. 6 , the effect of this leakage energy  50  of  FIG. 5  is to create a rapid thermal expansion to generate a shock wave  52  starting at a point beyond the crater  42  and passing from the rear surface of the substrate  26  toward its front surface. The shock wave  52  is sufficiently powerful so as to create surface melting at the rear surface of the substrate  26  when contained by the backer plate  32 . 
     Referring to  FIG. 7 , the net result is an hourglass-shaped pore  54  passing through the substrate  26  formed by the intersection of crater  42  and a counter-facing crater  53  formed by the shock wave  52 . The hourglass-shaped pore  54  has a waist diameter  55  representing the narrowest portion of the pore  54  of 1 to 200 microns and preferably substantially less than 1 micron for example 200 nm. The rear diameter  57  of the hourglass-shaped pore  54  formed by counter-facing crater  53  will generally be much larger than the waist diameter  55 , typically at least twice as large. 
     A front portion of the hourglass-shaped pore  54  formed by the crater  42  will generally have a first small cone angle  56  to provide improved control of the waist diameter  55  by reducing the effect of the depth of the crater  53 . A second cone angle  58  of the crater  53  may be substantially greater, for example, twice the angle  56 . The diameter of the crater  53 , for example, may be on the order of 10 microns and is essentially fire polished caused by the heating effect of the shock wave  52 . 
     Referring to  FIG. 8  the substrate  26  (inverted with respect to the orientation of  FIG. 7 ) may receive a cell  60  within crater  53  to expose a portion of the cell wall  62  at the waist  55  to be accessible through crater  42 . A light suction applied by a pump  67  from the side of the substrate  26  toward crater  42  may adhere the cell wall  62  to the surface of crater  53  with a 5 to 30 gigaohm resistance between a solution  64  on the side of the substrate  26  holding the cell  60  and a solution  66  on the side of the substrate  26  opposite solution  64 . 
     A sharp suction applied by a pump  67  at the front surface  46  or other means may be used to provide electrical connection to the interior of the cell  60  by a sensitive electrical detector  70  permitting measurement of electrical differences between the exterior and interior of the cell  60  through an electrode  72  communicating with the interior of the cell  60  referenced to solution  64  outside the cell  60 . 
     As used herein “fire polishing” is used to refer to a surface melting similar to that which would be provided by fire but does not require combustion. 
     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.