Patent Publication Number: US-2004058423-A1

Title: Fast electrical lysis of cells and rapid collection of the contents thereof using capillary electrophoresis

Description:
CROSS-REFERENCE TO RELATED APPLICATION(S)  
     [0001] This invention claims priority to U.S. provisional patent application No. 60/377,476 filed May 3, 2002. 
    
    
     STATEMENT OF GOVERNMENT INTEREST  
     [0002] This invention was made with Government support under Grant No. CA91216, awarded by the National Institutes of Health. The Government has certain rights in this invention. 
    
    
     
       TECHNICAL FIELD  
       [0003] The invention relates to the field of lysis and collection of material from a biological cell, particularly to cell chambers and micro-collection devices configured for electrical lysis of the cell, and more particularly for systems configured to collect the contents of the lysed cell in a capillary electrophoresis tube.  
       BACKGROUND OF THE INVENTION  
       [0004] Capillary electrophoresis (CE) has found abundant applications for bioanalysis, especially for the identification and quantification of cellular analytes. The capillary&#39;s micron dimensions and picoliter to nanoliter sample volumes are ideal matches for examination of the contents of biological cells. A number of reviews describing the analysis of single cells with CE have been published [1-6]. In the past decade:, CE has demonstrated increasing utility for the quantitative analysis of single cells. New applications for the analysis of dynamic cellular properties using a variety of micro-collection devices including CE tubes demand methods of cellular lysis and collection with sufficient temporal resolution to accurately measure these processes. In particular intracellular signaling pathways involving many enzymes can he modulated on subsecond time scales.  
       [0005] One method described in U.S. Pat. Nos. 6,156,576 and 6,335,201 for combining rapid cellular lysis and collection of the contents of cells is a laser-based cell lysis system (the Laser Micropipette System [LMS]), which was developed by our lab to rapidly lyse a single cell (or cells) with rapid subsequent collection of the contents for the analysis of enzymatic activity by capillary electrophoresis The LMS, has a number of valuable advantages for performing cell-based assays. Among these are the rapid temporal resolution of the sampling method and its compatibility with optical microscopy. In the current LMS design, a capillary is mounted above a cell placed on the stage of an inverted microscope. The cell is loaded with fluorescent reporters of an enzyme activity, such as a kinase activity. At a desired time, a focused pulse from a Nd:YAG laser is used to create a shock wave which lyses the cell. The cell&#39;s contents (including the reporters) are loaded into the capillary and separated by the electrophoresis. The reporters undergo a shift in their electrophoretic mobility when acted upon by a kinase. The advantages of the LMS include i) rapid cell lysis and fast termination of cellular reactions (&lt;&lt;33 ms), ii) efficient sampling of cell contents, iii) applicability to both adherent and non-adherent cells, and iv) excellent reproducibility. We have shown that this method for rapid cell sampling prevents artifacts in the measurement of kinase activity compared to hypoosmotic lysis over several seconds. [12].  
       [0006] Unfortunately, current instrumental design and complexity limit the numbers and types of investigators that can easily utilize the laser micropipette system. One drawback is the relatively high cost of the Nd:YAG laser required for the cell lysis. Perhaps more limiting is the technical expertise required to properly align the laser through the microscope and to maintain its proper alignment over time. The current design also imposes limits on the throughput of experiments. At present, cells are cultured on a coverslip in random locations and at a low density. This is necessary because the shock wave, which rapidly lyses the cell of interest, propagates through the substrata and media lysing other nearby cells. For single-cell experiments one must search for a cell without close neighbors, which is a time-consuming process. Additionally only one measurement per cell culture chamber can be performed.  
       [0007] Other techniques for lysing cells include the use of capillaries to perform the lysis. In one method for studies of large, non-mammalian cells (200-1000 microns in size), a capillary tapered to a sharp point is inserted into the cell to remove a plug of cytoplasmic sample [7-9]. Other methods include detergent and/or hypoosmotic solutions to lyse mammalian cells and to release the cell&#39;s contents into a capillary for analysis. [1-6]. Mammalian cells, are smaller (typically less than about 100 microns) and in certain practices, the whole cell is injected into the capillary for analysis. In other methods, application of an electric field through the walls of a capillary has been used to lyse a cell that has been drawn into the capillary. [10].  
       [0008] Electrical lysis of cells has been demonstrated as a modification of various electroporation techniques. Electroporation is a phenomenon that has been studied extensively. [13-18]. Multiple electrical pulses of a defined pulse width and voltage are used to induce a potential difference across the cell. When the transmembrane potential difference is large enough, pores are formed in the membrane which rapidly seal upon removal of the electric field. This process is widely used to load exogenous molecules into cells. A number of innovative strategies have been developed to rapidly and reversibly electroporate single, adherent cells. Owar and colleagues demonstrated that an electrolyte-filled capillary or miniaturized carbon-fiber electrodes could selectively electroporate single cells or portions of adherent cells [23, 24]. Haas et al utilized a pulled glass pipette to porate single cells within the brains of living animals. [51]. In addition a number of investigators have used modified patch clamp methods to electroporate single, adherent, tissue-cultured cells while others have used microfabricated electrodes. [26-29]. Several of the above designs successfully employed single, brief (about 1 ms) DC voltage pulses to perform electroporation.  
       [0009] If the applied transmembrane voltage exceeds a critical value, pore formation becomes irreversible and cell lysis occurs. Some investigators have used this observation to move from electroporation to electrical lysis of cells. In one method, Cheng and coworkers reported the use of a pulsed, high voltage field of 20 second duration to lyse cells on a microfabricated device for a variety of applications including obtaining DNA and RNA from bacteria and the destruction of unwanted cells. [19, 20]. These studies showed that combined AC fields for the study of membrane deformation or for dielectrophoretic separation of blood cells or  E. coli  cells, could also be used for cell lysis over a range of frequencies from 20 KHz to 1 MHz with potential differences of 40 to 2 kV/cm respectively. Krueger&#39;s studies of membrane deformation by electrical fields in the context of electroporation also reported cellular lysis with field strengths in this range. [25]. Cheng et al were able to achieve lysis of  E. coli  with several hundred bursts of an AC field of 500 volts each of 50 microseconds duration across an array o electrodes on a substrate with an inter electrode gap of about 200 microns. [19].  
       [0010] Similarly, Lee and coworkers describe a micro-machined cell lysis device based on a similar theory. [21]. Ramsey and colleagues also utilized AC electrical fields for fast, high throughput lysis of cells on a microfluidic device followed by separation of the intracellular contents. [see 22 and U.S. Pat. Publication No. 20030075446]. Yeung&#39;s group lysed a lymphocyte preloaded into a capillary with a 15-20 second exposure to an electric field from a Tesla coil. Lysis of the cell required 15-20 seconds by this method and the cell was also required to stick to the inner surface of the capillary. [10].  
       [0011] In another area of endeavor, other investigators have prepared surfaces of substrates with patterned electrodes [41, 42]. Standard vapor deposition methods in the presence of a mask were used to pattern the surface of a coverslip with electrodes composed of indium tin oxide (which is transparent) or an alternative metal. Other investigators have prepared other surface molecules for cell attachment. [43].  
       [0012] While the foregoing studies have made significant contributions to the analysis of cells, subsecond electrical lysis of single adherent cells followed by rapid loading of the cellular constituents into a micro-collection device such as a capillary electrophoresis tube for separation has yet to be demonstrated. Moreover, all of the prior art methods for adherent cells require several seconds and/or several pulses to disrupt cell membranes and therefore are generally appropriate only for investigation of the properties of static or slowly changing cellular analytes, such as DNA, RNA, or protein content. Many biochemical reactions occur on time scales of seconds or less, thus the temporal resolution (i.e. the time to terminate cellular reactions) of the sampling technique becomes critical when analyzing cellular properties such as enzymatic activity. For such biochemical analyses, accurate measurement requires complete cell lysis with termination of biochemical reactions in subsecond time periods. For non-adherent cells, in a system described by Ramsey, U.S. Pat. Pub. No. 2003007544, subsecond lysis could be achieved when the cell was loaded into a microfluidic device. A drawback of such a microfluidic device, as with the internal capillary lysis system described by Xue and Yeung [10] is that the cell must be drawn within the interior of the capillary or microfluidic device, which disturbs the resting state and resulting biological activity of the cell, potentially leading to artifacts.  
       [0013] Finding an inexpensive and easy-to-use alternative for rapid cell lysis would be beneficial for the analysis of rapid cellular processes. What is needed in the art are new apparatus and methods for rapid cell lysis that maintains the subsecond temporal resolution and optical compatibility of laser-mediated lysis, but overcomes the limitations of cost and difficulty of use. Moreover, what is needed is a method to lyse a single selected cells with a single subsecond pulse of an electric field and to collect the contents thereof substantially simultaneously with the lysis of the cell.  
       SUMMARY OF THE INVENTION  
       [0014] The invention is directed apparatus and methods to rapidly lyse a biological cell using a single electrical pulse of subsecond duration followed by efficient loading of the cellular contents into a micro-collection device. Micro fabricated electrodes are described that create a maximum voltage drop across a cell&#39;s plasma membrane at a minimum inter-electrode voltage. The influence of the inter-electrode distance, pulse duration, and pulse strength on the rate of cell lysis are described. The ability to rapidly lyse a cell and collect and separate the cellular contents is demonstrated by loading cells with the exemplary flourophores Oregon Green and two isomers of carboxyfluorescein. All three fluorophores are detected from lysed with a separation efficiency comparable to that of standards.  
       [0015] In one aspect, the invention provides embodiments of cell chambers configured with electrodes and surface materials for electrical lysis of cells. In one embodiment, the cell chamber includes a substrate material having a bottom and a top surface. An electrode is layered on the top surface of the substrate material; and a cell adhesion material is layered on top of the electrode. In certain embodiments, the substrate material and the electrode material are transparent materials. In certain embodiments, the cell adhesion material is a hydrophilic region layered over the electrodes and the area between the electrodes is layered with a hydrophobic region. Together the hydrophobic and hydrophilic regions pattern the cells on the cell chamber over the electrodes. In another embodiment, the cell chamber includes an array of cell adhesion materials comprised of a plurality of hydrophilic regions above a plurality of electrodes in an array with the plurality of hydrophilic regions being separated from one another by adjacent hydrophobic regions.  
       [0016] In another embodiment, the cell chamber includes a substrate material for receiving a plurality of cells, the substrate material having a top surface and a bottom surface. The substrate includes an array of openings through the substrate material between the top surface and the bottom surface. The openings provide a conductive pathway for electrical current to flow between electrodes placed above and below the openings.  
       [0017] In another embodiment, the cell chamber includes an array of separately addressable electrode pairs on the top surface of the substrate, each of the electrode pairs comprising a first electrode and a second electrode of opposite polarity. The first and second electrodes are separated from one another by a distance of less than 200 microns, and the array of electrode pairs are separated from each other by a distance greater than 200 microns. The cell chamber also includes an array of cell adhesion materials comprised of a plurality of hydrophilic regions separated from one another by adjacent hydrophobic regions and positioned above the array of electrode pairs so that the plurality of hydrophilic regions are located above the plurality of electrode pairs.  
       [0018] In another aspect, the invention provides micro-collection devices for electrical lysis of a biological cell and collection of at least a portion of contents thereof. In one embodiment, the micro-collection device includes a micro capillary electrophoresis tube having an exterior wall encircling an interior lumen, a distal end for contacting an electrolyte and a proximal end for collecting at least a portion of the contents of a lysed cell. The proximal end preferably has an outer diameter of less than about 200 microns. The capillary electrophoresis tube has and an electrode pair attached to the proximal end where the electrode pair includes a first and a second electrode separated from one another by distance of less than 200 microns.  
       [0019] In another embodiment, the capillary electrophoresis tube has an exterior wall encircling an interior lumen, the exterior wall having a thickness of less than 200 microns. The exterior wall has a pair of electrodes including a first electrode on an exterior surface thereof and a second electrode on an interior surface thereof so that the electrodes are separated from one another by a distance of less than 200 microns. The electrode pair is attached to the proximal end of the capillary electrophoresis tube.  
       [0020] Another embodiment includes the capillary electrophoresis tube having an exterior wall encircling an interior lumen, a distal end for contacting an electrolyte and a proximal end for collecting at least a portion of the contents of a lysed cell The proximal end is tapered and has an outer diameter of less than 200 microns. The capillary electrophoresis tube further includes an electrically conductive material deposited on the proximal end of the tube and a conductive lead extending down a portion of the length of the tube contacting the electrically conductive material.  
       [0021] In another embodiment, the capillary electrophoresis tube includes a removably insertable wire within the lumen of the capillary electrophoresis tube at a distal end thereof opposite the proximal end that collects the sample. When the wire is inserted into the lumen at the distal end in the presence of an electrolyte, the wire is put into electrically conductive contact with the electrolyte, making the electrolyte in the lumen serve as a liquid electrode.  
       [0022] In another aspect, the invention includes systems that use one or more of the foregoing cell chambers or capillary electrophoresis tubes. The system includes electrical means to lyse the biological cell within 1 second or less of application of an electrical potential to the electrical means and a micro-collection means configured to collect at least a portion of the contents of the lysed cell within a period of about 1 second or less from lysing the cell. In certain embodiments the system commences collection simultaneously with lysis.  
       [0023] One embodiment of such a system includes a first electrode that is at least one of positioned on a substrate or positionable within less than 200 microns from a biological cell in contact with the substrate. It also includes a second electrode that is at least one of positioned on the substrate less than 200 microns of the first electrode or positionable less than 200 microns of the first electrode. The first and second electrodes are thereby configured for positioning a biological cell there between. The system further includes a micro-collection device having a proximal end configured to capture at least a portion of the contents of the biological cell and being at least one of positioned or positionable less than 200 microns of at least one of the first and second electrodes. The micro-collection device is configured to collect at least the portion of the contents of the biological cell within less than about 1 second of lysis, but the micro-collection device is not configured to hold the biological cell or to be in contact with the biological cell during lysis.  
       [0024] In another aspect, the invention provides methods of electrical lysis of a biological cell and collection of at least a portion of the contents thereof, that include the acts of depositing the biological cell on a substrate, providing electrical means to lyse the biological cell on the substrate within 1 second or less of application of an electrical potential to the electrical means; and collecting at least a portion of the contents of the lysed cell with a micro-collection means configured to collect the contents of the lysed cell within a period of about 1 second or less from lysing the cell.  
       [0025] In various embodiments of the methods, a single subsecond pulse of an AC or DC field is applied across the biological cell using the forgoing devices of the invention. In certain embodiments, the act of collecting is commenced simultaneously with the act of electrical lysis. In other embodiments, the act of collecting commences within 1 second, within 100 ms, within 10 ms, within 1 ms, within 100 μs within 10 μs or within 1 μs of commencing the electrical lysis. Electrical lysis is commenced by applying an electrical potential of about 5 to about 30 Kv/cm in one embodiment, about 10 to 20 Kv/cm in another embodiment, or about 15 Kv/cm in another embodiment, between the first electrode  18  and the second electrode  19 . The electrical potential is applied for a duration of about 10 ms or less in certain embodiments, or 1 ms or less, or 100 μs , or 10 μs or less in other embodiments. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0026]FIG. 1 depicts a top view of a cell chamber with an array of first electrodes for electrical lysis of a biological cell according to one embodiment of the invention.  
     [0027]FIG. 2 depicts a side view of a cell chamber with an array of first electrodes for electrical lysis of a biological cell in combination with a capillary electrophoresis tube configured with a second electrode on the proximal end.  
     [0028]FIG. 3 is a top view of an embodiment of a cell chamber configured with independently addressable electrodes.  
     [0029]FIG. 4 is a top view of another embodiment of a cell chamber configured with independently controllable electrodes.  
     [0030]FIG. 5 is a flow diagram that depicts a method of making a cell chamber with first electrodes having hydrophilic regions on top separated by hydrophobic regions between the electrodes.  
     [0031]FIG. 6 is a top view of a cell chamber configured with electrode pairs according to another embodiment of the invention.  
     [0032]FIG. 7 is a side view of another embodiment of a cell chamber and capillary electrophoresis tube with an electrode on the exterior of the proximal end of the tube in an embodiment of the invention.  
     [0033]FIG. 8 is a side view of is a side view of a cell chamber with an array of first electrodes and a capillary electrophoresis tube with a wire in the distal end of the lumen according to another embodiment of the invention.  
     [0034]FIG. 9 is a side view of a cell chamber with an array of first electrodes and capillary electrophoresis tube with the second electrode and a tapered proximal end according to another embodiment of the invention.  
     [0035]FIG. 10 is a side view of a cell chamber with a single first electrode and capillary electrophoresis tube with the second electrode and a tapered proximal end according to another embodiment of the invention  
     [0036]FIG. 11 is a side view of a cell chamber without electrodes and one embodiment of a capillary electrophoresis tube configured with both first and second electrodes on the proximal end according to another aspect of the invention.  
     [0037]FIG. 12 is a side view of a cell chamber without electrodes and a capillary electrophoresis tube configured with first and second electrodes on the proximal end according to another aspect of the invention.  
     [0038]FIG. 13 are side views of a biological chip micro-collection device in other embodiments of the invention. FIG. 13A depicts a biological chip useful with a cell chamber having an electrode pair according to the invention. FIG. 13B depicts the biological chip configured with a second electrode useful with cell chambers having a first electrode according to the invention. FIG. 13C depicts the biological chip configured with first and second electrodes.  
     [0039]FIG. 14 are side views of an optical fiber bundle micro-collection device in other embodiments of the invention. FIG. 14A depicts the optical fiber bundle useful with a cell chamber having an electrode pair according to the invention. FIG. 14B depicts the optical fiber bundle configured with a second electrode useful with cell chambers having a first electrode according to the invention. FIG. 14C depicts the optical fiber bundle configured with first and second electrodes.  
     [0040]FIG. 15 depicts a system of the invention that includes a micro delivery device in conjunction with the capillary electrophoresis tube micro-collection device.  
     [0041]FIG. 16 depicts two different placements of electrodes and fields for electrical lysis according to methods of the invention.  
     [0042]FIG. 17 are graphs depicting cellular lysis times as a function of electrical pulse height ( 17 A), pulse width ( 17 B) and distance between electrodes ( 17 C) in a method of the invention.  
     [0043]FIG. 18 are photographs depicting electrical lysis of cells according to the methods of the invention.  
     [0044]FIG. 19 shows electrophoresis charts depicting the separation of flourophores from electrically lysed cells according to methods of the invention.  
     [0045]FIG. 20 is a chart depicting similar collection efficiency of the electrical methods of the present invention in comparison to laser based methods of the prior art.  
     [0046]FIG. 21 illustrates an array of capillary electrophoresis tubes with electrodes in combination with an array of first electrodes according to another embodiment of the invention. 
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS  
     [0047] Prior to setting forth the present invention in detail, the meanings of certain terms used herein are provided to aid in an understand thereof. These definitions are not intended to be exclusive, but rather are inclusive of the ordinary meanings of the terms, except if the ordinary meaning is construed to conflict with definitions set forth below.  
     [0048] “Attached” with respect to an electrode or conductive material in reference to a component part means that the electrode or conductive material is firmly in contact with the component part. In one embodiment “attached” means that a layer of the conductive material is deposited on the component part directly or indirectly through another layer in contact with the component part. In another embodiment “attached” means that the conductive material is bonded to the component part though an adhesive, weld or other bonding means. In another embodiment “attached” means that the conductive material is fixed or fastened to the component part through a mechanical contact.  
     [0049] “Transparent” means that a substrate, coating or layer of material permits transmission of at least 10 percent of incident light having a wavelength in the ultraviolet or visible spectrum.  
     [0050] A “micro-collection device” is a device configured to collect small amounts of material from a lysed cell in a small volume. The material is collected in a volume that is less than about 10 nanoliters, less than about 1 nanoliters, less than about 100 picoliters, less than 10 picoliters or less than 1 picoliter. In typical embodiments, the material is collected from a volume of about 0.5 picoliter to about 1 nanoliter of volume.  
     [0051] A “capillary electrophoresis tube” is a micro-capillary configured on a proximal end to collect a small amount of sample material and on a distal end to be in contact with an electrophoresis buffer. In typical configurations, the capillary electrophoresis tube contains at least one window through the tube permitting analysis of sample material that is at least partially separated into zones as a result of electrophoretic and/or electo-osmotic movement in the tube. In certain embodiments, the capillary electrophoresis tube may include a gel in the interior lumen of the tube to facilitate the electrophoretic separation. Capillary electrophoresis tubes are distinguishable from ordinary micro capillaries, typically by having a length of 20 cm or greater and/or by having outer walls surrounding the lumen where the outer walls are covered by a protective coating such as polyimide.  
     [0052] A “cell chamber” means any substrate material having a surface upon which a sample including a biological cell can be applied. The term includes, but is not limited to, conventional microscope cover slips, conventional microscope glass slides, reaction vessels, tissue culture containers, dishes and the like. The cell container is preferably in a configuration that can be adapted for mounting on an instrument for detecting the position of the cell located in or on the container.  
     [0053] A “cell adhesion material” or “cell adhesion layer” is an electrically permeable biologically non-toxic material that adheres to a biological cell, specifically or non-specifically, and that is deposited on a surface above an electrode at a density that permits electrical conductivity from the electrode through the deposited material into an electrolyte above or below the cell adhesion materials. Thus, a cell adhesion material does not include ordinary solid substrate materials such as glass, polystyrene or polypropylene on which cells may adhere but that does not permit the passage of current. Example non-specific cell adhesion materials include hydrophilic molecules such as those containing anionic moieties or cationic moieties in the presence of the electrolyte at a selected pH. Polylysine is one example of a non-specific hydrophilic molecule suitable for a cell adhesion layer. Examples of specific cell adhesion materials include proteins, particularly antibodies or fragments thereof, lectins, carbohydrates, fibronectin, collagen, laminins, vitronectin, endothelial cell attachment factor, integrin ligands, Matrigel, endothelial cell attachment factor, nucleic acids, ligands that bind molecules on the surface of cells or other members of a biological binding pair.  
     [0054] Throughout the description that follows, citation is made to various references that may aid in understanding or practice of various embodiments of the invention. Each such reference is herein incorporated by reference in its entirety to the extent their teachings do not conflict with the requirements of the invention described herein.  
     [0055] Turning now to the description of exemplary embodiments of the invention, one aspect of the invention includes various embodiments of cell chambers  10  configured to facilitate rapid electrical lysis of a biological cell. FIGS.  1 - 4  and  6 - 10  show various examples of the inventive cell chambers  10  alone or in combination with micro-collection devices  15  (exemplified by capillary electrophoresis tubes  42 ) used in various configurations of the invention. A common feature of the various embodiments of the invention, is that to implement electrical lysis of cells, electrodes of 2 different polarities (ground and source in a DC configuration/alternating positive and negative in the AC configuration).  
     [0056]FIGS. 1 and 2 illustrate top and side views, respectively, of one embodiment of a cell chamber  10  of the invention. The cell chamber  10  includes a lower substrate material  12  that in certain embodiments, is a transparent material such as in a cover slip or glass slide. Suitable materials for the lower substrate material  12  include, but are not limited to glass, quartz, silicon nitride or a transparent plastic such as polydimethyl siloxane (PDMS), polystyene, polycarbonate, polyethylene, polymethyl methacrylate (PMMA) and the like. The substrate material  12  may be configured to fit onto a suitable instrument for detecting the position of biological cells  58  and  59  in the cell chamber  10 . The substrate  12  has a top surface  16  and a bottom surface  14 . A fist first electrode  18  is located on the top surface  16 . The first electrode  18  may be a transparent electrode in certain embodiments. A transparent electrode may be made for example, of indium tin oxide or a layer of one or more conductive metal less than about 300 angstroms in thickness. For a traditional metal such as gold 200-300 angstroms is the approximate upper limit of transparency. For a metal such as indium tin oxide (ITO) thickness greater than 1000 angstroms can be used with retention of transparent properties. The first electrode  18  may be a single metal, an alloy, or composite structure. In the exemplary embodiment illustrated in FIG. 1, the first electrode  18  is a composite of layers of metal that includes a lower layer  6  of titanium of about 50 angstroms thickness deposited on the top surface  16  of the substrate material  12  and an upper layer  8  of gold of about 80 angstroms in thickness deposited over the lower layer  6  of titanium.  
     [0057] In the embodiment illustrated in FIG. 1, the first electrode  18  is one of a plurality of electrodes  18  arranged in an array  30  on the top surface  16  of the substrate material  12 . The first electrode  18  is connected to a conductive lead wire  26  configured to put the first electrode  18  in conductive contact with ground or one pole of an electrical power supply  27 . In certain embodiments, the electrical power supply  27  can be a DC source while in other embodiments, the electrical power supply  27  is an AC source. AC power sources suitable for the invention provide alternating current at a frequency greater than 1 kilohertz. In certain embodiments the frequency is greater than 10 kilohertz, In other embodiments the frequency is greater than 100 kilohertz. In still other embodiments the frequency is one megahertz or greater.  
     [0058]FIG. 2 shows a side view of the cell chamber  10  of FIG. 1 as used in a system in combination with a micro-collection device  15  (capillary electrophoresis tube  42 ) configured with a second electrode  19  on a proximal end  50  thereof. The capillary electrophoresis tube  42  depicted in FIG. 2 includes an exterior wall  44  enclosing an interior lumen  46  in which the contents of lysed cell will be collected and separated by electrophoresis. The system is preferably configured with a selectable positioning device  40  to move at least one of the cell chamber  10  or the capillary electrophoresis tube  42  relative to one another, so as to place the proximal end  50  of the capillary electrophoresis tube  42  at a selectable position above a selected cell  59  and simultaneously place the second electrode  19  less than about 200 microns, less than about 100 microns, less than about 60 microns, or 20 microns or less from the first electrode  18  located beneath or adjacent to the selected cell  59 .  
     [0059] The array  30  of electrodes  18  are separated from one another by a non-conductive layer  17  of material that is preferably also a transparent material. The thickness of the non-conductive layer  17  may be a varied according to particular applications and/or manufacturing constraints. Any thickness suitable for the observation or detection instrument may be used. In advantageous embodiments, the non-conductive layer  17  is about 10 to 10,000 angstroms in thickness, in other embodiments the non-conductive layer  17  is about 50 to 1000 angstroms, in preferred embodiments about 100-500 angstroms and in typical embodiments about 200 angstroms in thickness. Suitable materials for the non-conductive layer  17  include but are not limited to, air, glass, quartz, silicon nitride, or a transparent plastic such as PDMS, silicon nitride, polystyene, polycarbonate, polyethylene or other non-conductive substance that is transparent at the thickness applied. Preferably the non-conductive layer  17  is made of material such as PDMS or silicon nitride that has silanol-reactive chemical moieties that can be used to pattern the upper surface of the non-conductive layer  17  with different types of materials, either directly, or indirectly through a binding layer  23 . The binding layer  23  may be made of any suitable material for cross linking to the different types of material used to pattern the surface of the uppermost surface of the cell chamber  10 . The binding layer  23  should preferably be deposited at a sufficiently low density to provide electrical conductivity between the first electrode  18  and the electrolyte medium  56 .  
     [0060] The different materials used to pattern the surface include at least one cell adhesion material  22  deposited above the first electrode  18  and one hydrophobic material  28   a  deposited away form the first electrode and/or between electrodes  18  in the array  30  The hydrophobic material  28   a  forms hydrophobic regions  28 . In non-specific embodiments, the cell adhesion material  22  is a hydrophilic material  24   a.  The hydrophilic material  24   a  located above the first electrode a forms a hydrophilic region  24  that is a functional part of cell adhesion layer  22 . The hydrophilic regions  24  facilitates adhesion of a biological cell  58  to the cell chamber  10  above the first electrode  18 . The hydrophobic regions  28  also facilitate patterning of the biological cells  58 ,  59  on the cell chamber  10  above the electrodes  18  because biological cells  58 ,  59  do not typically adhere to the hydrophobic regions  28  but tend to adhere very well to the hydrophilic regions  24 . Suitable materials for forming the hydrophilic regions  24  include, but are not limited to, poly amines such polylysine, self assembled monolyaers with polar or charged end groups, palladium, gold, glass, metal oxides, poly(N-isopropylacrylamide), palladium, gold, metal oxides, porous glass or other cell adhesion material, Suitable materials for forming the hydrophobic regions  28  include, but are not limited to, polyethylene glycol (PEG) monomers, self assembled monolayers with terminal carbon chains of C4 or greater in length, cellulose acetate, paraffin, agarose, sulfonate-terminated alkylsilanes, PEG-derivatized alkanethiol SAMs, interpenetrated copolymers of PEG, alkanethiol SAMs functionalized with tri(propylene sulfoxide) fluorocarbon polymers, poly(vinyl alcohol) multilayers of PEG functionalized with silicon chloride, polyethylene oxide, and the like.  
     [0061] The positioning of the second electrode  19  in close proximity to the first electrode  18  provides for separate addressing of each electrode  18  in the array. When the electrodes  18  in the array  30  are linked to a common contact lead  26  as illustrated in FIG. 1, each of the electrodes  18  in the array  30 , including those not specifically addressed by placing the second electrode  19  immediately above, will generate some unwanted amount of angular electrical field potential with the second electrode  19 . To minimize these angular field effects with non-addressed electrodes, each of the electrodes  18  in the array  30  should preferably be separated from each other by a distance greater than 200 microns in certain embodiments, greater than 300 microns in other embodiments, or greater than 500 microns in other embodiments.  
     [0062] Another way to minimize unwanted field effects is to have each electrode  18  in the array of electrodes independently connected to independently controllable switches  25  as illustrated in FIG. 3 and  4 . These switches  25  may be independently controlled by switch control module  31 . FIG. 3 illustrates a single switch control module  31  that separately controls each individual electrode  18  using separate switches within the control module. FIG. 4 illustrates a switch control module  31 , that uses row  31   a  and column  31   b  addressing modules that activate selected rows and columns of the controllable switches  25  in the array so that only one electrode  18 , having both the row and the column address switches  25  activated will be placed in conductive contact with ground (or one pole of the power source  27 ). FIG. 4 shows the switch elements  25  being located on the cell chamber  10  for illustrative purposes. In certain embodiments, standard lithographic techniques can be used to fabricate appropriate controllable switch elements  25  directly on the cell chamber  10  beneath the electrodes  18 . In other embodiments, however, the switch elements  25  may be located within the control module  31   a,    31   b  requiring only separate conductive leads  26  to each of the electrodes  18  as depicted in FIG. 3.  
     [0063]FIG. 5 illustrates an example method of forming a patterned layer of hydrophobic regions  28  adjacent of hydrophilic regions  24  in a plane on the cell chamber  10  where the hydrophilic regions are above electrodes  18 . A first overlay mask  90  is positioned above the substrate material  12  and a layer of electrode material  18   a  is deposited on the substrate through the first overlay mask  90  forming the array  30  of electrodes  18  on the top surface  16  of the substrate material  12 . A second overlay mask  91  is configured to block the surface of the electrodes  18  and the non-conductive layer  17  is deposited on the substrate  12  through the second mask  91  using a suitable process for the particular non-conductive material used for the layer, for example, by spin coating when the non conductive material  17  is PDMS. If the non-conductive material is PDMS or similar substance that is insulating if in a dense layer but can allow a current to be conducted when applied in a thin layer, the substance is first applied as a lower layer  3  having a density sufficient to provide electrical insulation between the electrodes  18 . Then it is applied as an upper layer  5  at a lower density sufficient to provide pores so that the PDMS layer will provide electrical conductivity between the electrode  18  and the electrolyte medium  56 . In that case, the upper layer of PDMS or other substance also functions as the binding layer  23  although being made of the same material as the non-conductive layer  17 .and the second mask  91  may be removed prior to depositing the upper layer so that the upper layer  5  is also deposited on the electrodes  18 . In an alternative process not illustrated, a separate binding layer  23  is applied over the non-conductive layer  17 . The non-conduct layer  17  is cured (or otherwise fixed) onto the substrate  12  in a curing step  88 . A third overlay mask  93  having a pattern corresponding to the first overlay mask is positioned over the cell chamber  10  and the hydrophilic material  24   a  is adsorbed or otherwise linked to the cell chamber  10  by being deposited through the third mask  93 . The third mask  93  is removed and the cell chamber  10  is washed, thereby establishing the hydrophilic regions  24  positioned above the electrodes  18 . The hydrophobic material  28   a  is then deposited over the exposed non-conductive layer  17  by a suitable process such as grafting when the hydrophobic material  28   a  is a polyethylene glycol monomer using a suitable process, for example as described in Hu et al, [44]. Other suitable methods for manufacturing the patterned surface of cell adhesion materials  22  and hydrophobic regions  28  include, but are not limited to methods described by Folch et al, Kane et al, Craighead et al, Jung et al, and Whitesides et al. [43, 45-48].  
     [0064] Although not illustrated in FIG. 2, it will be understood that similar masking techniques can be used to lay down the conductive contacts  26  to the electrodes  18 .  
     [0065] In this exemplary process, where the hydrophilic material  24   a  is polylysine deposited above the electrode and the hydrophobic material  28   a  is PEG, the formation of the hydrophilic region  24  blocks PEG from being grafted to the hydrophilic regions. It should be understood, however, that where a selection of different materials for the cell adhesion layer  22 , the hydrophilic region  24  and/or the hydrophobic regions  28  is used, other physical or chemical processes suitable for the selected materials are used to fabricate the layers. Moreover, other suitable lithographic masks may additionally be employed to protect from unwanted depositions of the hydrophobic material  28   a  in the same vicinity as the hydrophilic regions  24 .  
     [0066] The process illustrated in FIG. 5 is for exemplary purposes only. A variety of lithographic methods may be used to pattern the cell chambers  10  described in the present invention. Further suitable process for example, include those described by Hu et al, [44] or by Chen et al., [421 or by Cheng et al, [19 20], in combination with methods described by Folch et al, Kane et al, Craighead et al, Jung et al, and Whitesides et al. [43, 45-48].  
     [0067]FIG. 6 illustrates another embodiment of a cell chamber  10  according to the invention. In this embodiment, electrode pairs  38  are deposited on the substrate  12  rather than a single electrode or array of electrodes. The electrode pairs  38  have both the first electrode  18  and the second electrode  19  of opposing polarity located on the substrate  12 . The first electrode  18  and the second electrode  19  are separated from one another by first distance  37  that is less than about 200 microns in one embodiment, or less than 100 microns, or less than 60 microns, or less than 40 microns, or about 20 microns or less in various other embodiments. Each of the electrode pairs  38  has contact leads  26   a  and  26   b  to place the electrodes in conductive contact with opposite poles of power supply  27  and/or to ground. As with the other embodiments of the invention, this first distance  37  is sufficiently close to lyse a cell positioned between the first  18  and second  19  electrodes within less than one second in certain embodiments, within less than 300 ms in other embodiments, or within 33 ms or less in still other embodiments. Lysis occurs when an electrical potential of about 5 to about 30 Kv/cm in one embodiment, about 10 to 20 Kv/cm in another embodiment, or about 15 Kv/cm in another embodiment, is applied across the pair of electrodes  38  in a single pulse of a duration of about 10 ms or less, 1 ms or less, 100 microseconds or less, or 10 microseconds or less in various embodiments.  
     [0068] The electrode pairs  38  are separated from one another by a second distance  41 , greater than the first distance  37 , where the second distance  41  is sufficiently far to prevent electrical lysis of a cell located between pair of electrodes  38   b,    38   c,  and  38   c  when the difference of potential of about 10 to about 50 volts is applied across a selected set of electrode pairs  38   a.  In various embodiments, the second distance  41  is greater than 200 microns, greater than 300 microns, or in other embodiments, greater than 500 microns. A layer of cell adhesion material  22  is deposited over the electrode pairs  38  and a hydrophobic region  28  is deposited over the substrate material adjacent and between the electrode pairs  38 . The cell adhesion material  22  again may be formed of hydrophilic regions  24  located over the electrode pairs  38 . The lead wires  26   a  and  26   b  are used place the first  18  and second  19  electrodes of the electrode pair  38  in conductive contact with an electrical power supply or ground. In certain embodiments, the electrodes  18  and  19  of the electrode pair  38  may be transparent electrodes, however, if the substrate  12  and/or non-conductive layer  17  below the electrode pair  38  is transparent, it is not necessary to also make the electrodes  18  and  19  transparent because a cell deposited between the electrode pairs  38  will be visible through a microscope or other suitable optical viewing instrument.  
     [0069]FIG. 7 illustrates another embodiment of a cell chamber  10  of the invention. In this embodiment no electrodes  18  or electrode pairs  38  are deposited on the substrate  12 . Rather, the substrate material  12  is configured with a plurality of conductive openings  36  between the top surface  16  and the bottom surface  14  of the substrate material  12 . The conductive openings  36  are separated from one another by a plurality of adjacent non-conductive regions  33 . The size of the conductive openings is  36  is selected to be smaller than the size of the biological cell  58  to be lysed. The top surface  16  of the substrate material  12  is coated with a plurality of hydrophobic regions  28  deposited above the plurality of non-conductive regions  33 . The conductive openings  36  may optionally include hydrophilic materials  24  deposited over a porous mesh of the substrate material  12 . In these embodiments, the cells  58 ,  59  deposited on the top surface  16  of the cell chamber  10  will preferentially locate above the conductive openings  36 . The cell chamber  10  is located above a metallic layer or wire that forms the first electrode  18 . In certain embodiments, the metallic wire is separated from the bottom surface  14  and/or the conductive openings by a volume into which the electrolyte  56  is also contained below the bottom surface.  
     [0070] While the embodiments of cell containers  10  described herein before each include transparent materials for the substrates layer  12 , and/or the non-conductive layer  17 , and/or the electrodes  18  and/or the hydrophobic  28  and hydrophilic  24  layers, such transparent materials are only for preferred embodiments for use with a transmissive optical viewing instrument such as a microscope. The invention is not, however, limited to the use of such transparent materials for any of the structures described herein. In other embodiments, these structures may be made of opaque rather than transparent materials, especially when configured for use with reflective microscopes or other detection instruments that do not rely on the transmission of light through the cell containers  10 .  
     [0071] Another aspect of the invention illustrated in FIGS. 2 and 7- 12 , is system used with the various cell chambers  10  of the invention, which includes a micro-collection device  15  (i.e., capillary electrophoresis tube  42 ) configured with one or more electrodes to facilitate both rapid lysis of a single selected cell and collection of at least a portion of the contents thereof all within one second or less The micro-collection devices  15  illustrated in these Figures are capillary electrophoresis tubes  42 . Other exemplary embodiments of a micro-collection devices  15  are illustrated in FIGS. 13 and 14.  
     [0072] The capillary electrophoresis tubes  42  of FIGS. 2 and 7- 10  are configured with the second electrode  19  at the proximal end  50  thereof. The second electrode  19  has a second electrical lead  60  to put the second electrode  19  in contact with the power supply  27  (or ground source) having a polarity opposite the polarity of the first electrode  18  located on the cell chamber  10 . At least one of the cell chamber  10  and the capillary electrophoresis tube  42  is moveable relative to one another to selectively position the second electrode  19  over a selected cell  59  and above the first electrode  18 . An O-ring, gasket, or other barrier  13  that extends above the upper surface of the cell chamber  10  and surround a perimeter provides a reservoir for containing a conductive medium  56  such as a buffer or electrolyte. The conductive medium  56  is placed between the first electrode  18  and the second electrode  19  so that the proximal end  50  of the capillary electrophoresis tube  42  with the second electrode  19  attached thereto is positioned over, or in proximity to, the selected biological cell  59  by a distance of less than about 200 microns in one embodiment, or less than 100 microns, or less than 60 microns, or less than 40 microns, or about 20 microns or less in various other embodiments. A single pulse of electrical potential of less than one second is applied between the first  18  and second electrodes  19  so that electrical current flows between the first electrode  18  and the second electrode  19  causing lysis of the selected cell  59  within less than one second.  
     [0073] In the embodiment illustrated in FIG. 7, the electrical current flows between the lower electrode  18  via the conductive openings  36 , through the selected cell  59  and electrolyte medium  56 . In a modification of the embodiment shown in FIG. 7, at least one of the cell chamber  10  and the first electrode  18  are moveable relative to one another to position the first electrode  18  beneath a selected conductive opening  36  under a selected cell  59  while the position of the second electrode  19  on the capillary electrophoresis tube  42  remains fixed. This embodiment minimizes angular field potential between the first lysing electrode  18  and other conductive openings  36  not positioned directly beneath the second lysing electrode  19  on the capillary electrophoresis tube  42  located directly above the selected cell  59 .  
     [0074]FIG. 8 illustrates another embodiment of a capillary electrophoresis tube  42  configured with an electrode  19  at the proximal end. The capillary electrophoresis tube  42  is configured with a lead wire  62  at the distal end  67 . The lead wire  62  is connected to one pole of the power supply  27  at one end, and is in electrically conductive contact with the electrolyte material  56  within the lumen  46  of the capillary electrophoresis tube  42  at the other end. In this embodiment, the electrolyte material  56  within the lumen functions as the second electrode  19  in liquid form, so that when the lead wire  62  is charged with a different of potential with the first electrode  18 , current flows between the first electrode  18 , the electrolyte medium  56  within the lumen, and the lead wire  62  at the distal end causing electrical lysis of the selected cell  59  positioned beneath the proximal end  50  of the capillary electrophoresis tube  42 .  
     [0075]FIG. 9 illustrates another embodiment of a capillary electrophoresis tube  42  configured with the second electrode  19  at the proximal end  50  thereof. In this embodiment, the proximal end  50  of the capillary electrophoresis tube  42  is tapered so that at the proximal end  50  the capillary wall  44  has an outer diameter of about 40 microns or less, and the internal lumen  46  has a diameter of about 15 microns or less.  
     [0076]FIG. 9 further illustrates that any of the embodiments of the capillary electrophoresis tubes  42  configured with the second electrode  19  on the outer surface of the capillary wall  44  at the proximal end  50  thereof, the second electrode  19  can be made of a conductive layer of one or more metals deposited around the periphery of the proximal end  50  of the capillary electrophoresis tube  42  by a metal deposition processes. The conductive metal of the second electrode  19  may be attached to the lead wire  60  by bonding, soldering or adhesion though an electrically conductive adhesive  64 . In an exemplary embodiment, the second electrode  19  on the proximal end  50  of the capillary electrophoresis tube  42  is comprised of a first metal layer  81 , such as titanium, deposited by vapor deposition, sputtering, or other suitable method to form a bonding layer having a first thickness, for example, about 40 angstroms. The first metal layer  81  is overlaid with a second conductive metal  82  such as gold to a second thickness, for example about 80 angstroms or more, by a similar process. The deposition of the metal layers  81  and  82  forming the second electrode  19  is limited to the proximal end  50  by masking the outer wall  44  of the capillary electrophoresis tube  42  above the proximal end  50  during the deposition processes. Alternatively, or in addition, the capillary electrophoresis tube  42  may be oriented at an angle of 45 degrees or greater with respect to the direction of the deposition source so that only the proximal end  50  is contacted by the metal being deposited. In some embodiments, the metal(s) are deposited only on a portion of the proximal end  50  as illustrated, for example in FIG. 2 or  7 . In other embodiments, the metal layer(s) are deposited evenly around the proximal end  50  in annular configuration, as illustrated for example, in FIGS. 9, 10,  15  and  21 . The deposition of the metal layers  81  and/or  82  on the proximal end  50  of the capillary electrophoresis tube  42  can be evenly distributed around the proximal end  50  by rotating the capillary electrophoresis tube  42  during the deposition process. The capillary electrophoresis tube  42  is optionally surrounded by an insulating layer  65  near the proximal end  50 .  
     [0077] Capillary electrophoresis tubes  42  having the second electrode  19  deposited on the proximal end  50  on the outer surface of capillary wall  44  have advantages in ease of construction, the ability to uniformly focus the electrical field across the proximal end  50  end, and in subsequent operation of the capillary electrophoresis system for separating collected components of the lysed cell. Because an electrical wire  62  is not within the lumen  46  of the capillary electrophoresis tube  42  as in the embodiment shown in FIG. 8, there is no electrical disturbance in the electrophoresis of material through the capillary electrophoresis tube  42 . In addition, it is generally easier to attach the lead wire  60  to the outside surface of the capillary wall  44  than to fabricate a capillary electrophoresis tube  42  with the wire within the inner lumen  46  surface of the capillary. Finally, particularly with embodiments having a tapered proximal end  50 , the second electrode  19  surrounding the periphery thereof promotes a more equal and focused distribution of electrical field though the selected cell.  
     [0078]FIG. 10 illustrates use of a capillary electrophoresis tube  42  having the second electrode  19  configured for fast electrical lysis in another embodiment of a system of the invention. In this embodiment of the system, the first electrode  18  is a transparent electrode made of a naked layer of metal or metals deposited on the top surface  16  the substrate material  12 . The naked first electrode  18  is used directly as the surface that will contact the biological cells  58 ,  59 . This embodiment lacks the non conductive layer  17 , lacks an array of electrodes  30  and lacks the cell adhesion layer  22  or the hydrophilic regions  24  separated by hydrophobic regions  28 . The cell is selected merely by positioning the capillary electrophoresis tube  42  with the second electrode  19  at the proximal end  50 , and preferably with a tapered proximal end  50 , within about 200 microns, or within 100 microns, or within 60 microns, or within 40 microns, or within about 20 microns of the selected cell  89 . The selected cell  59  is lysed by applying a subsecond pulse of electrical field of about 10 to 15 Kv/cm (30-40 volts for a 20 micron gap) between the first  18  and second  19  electrodes located. Typically, the non selected cell  58  will not lyse with a subsecond pulse of the electrical field if the distance between the second electrode  19  and the non selected cell  58  is greater than about 200 microns or greater than about 300 microns or greater than about 500 microns.  
     [0079]FIGS. 11 and 12 illustrate yet another aspect of the present invention, which includes a capillary electrophoresis tube  42  having a proximal end  50  configured with both the first  18  and second electrodes  19  for electrical lysis of the biological cell and collection of at least a portion of the contents thereof. In these embodiments, the lysing electrodes  18  and  19  are each positioned on the proximal end  50  of capillary electrophoresis tube  42  itself preferably within about 0 to 200 microns above or below the ultimate proximal end  50  of the capillary electrophoresis tube  42 . This configuration differs in several respects from other systems for electrical lysis within a capillary as described by Xue and Yeung [10] or within a microfluidic device as described, for example, by Ramsey in U.S. Pat. Pub. No. 20030075446. With the present invention, there is no need for the cell to be in direct contact with the walls or interior of the micro-collection device  15  (i.e., capillary electrophoresis tube  42 ) prior to lysis. Lysis is conducted entirely outside the micro-collection device. In addition, there is no need for manipulations to draw the selected cell  59  into the capillary. Instead, the cell is ready for lysis merely by positioning the first electrode  18  and second electrode  19  within less than about 200 microns in one embodiment, or less than 100 microns, or less than 60 microns, or less than 40 microns, or about 20 microns or less of the selected cell Therefore, electrical lysis can be executed without perturbing the resting state of the cell by being loaded into a capillary or other microfluidic device. Moreover, there is no need to configure both of the lysing electrodes to be in conductive contact with the lumen of the microfluidic device. The capillary electrophoresis tubes  42  with both first  18  and second  19  electrodes on the bottom end are particularly usefully for selectively lysing non-adherent cells.  
     [0080] In FIG. 11, the first  18  and second  19  electrodes are in electrical contact with different poles of the electrical power supply or to ground. The electrodes  18  and  19  are mounted on the opposite sides of the exterior surface of the capillary wall  44  and separated from one another by a distance of less than about 200 microns in one embodiment, or less than 100 microns, or less than 60 microns, or less than 40 microns, or about 20 microns or less in various other embodiments. Preferably, but not necessarily, the lysing electrodes  18  and  19  extend beyond the proximal end  50  so that they can be positioned on either side of the selected cell  59 . This embodiment is most suitable for capillary electrophoresis tubes  42  having a tapered proximal  50  end where the outer diameter of the capillary wall  44  is less than the foregoing separation distance between the first  18  and second electrodes  19 .  
     [0081] In the embodiment illustrated in FIG. 12, the first lysing electrodes  18  is located on the exterior surface of the capillary wall  44  and the second lysing electrode  19  is located on the interior surface. This embodiment is more suitable for capillary electrophoresis tubes  42  lacking tapered ends, because the capillary wall  44  of most commercially available capillary electrophoresis tubes  26  typically has a thickness less than about 200 microns.  
     [0082] The close proximity of the first  18  and second  19  lysing electrodes in either of the embodiments depicted in FIGS. 11 and 12 provides the advantage of allowing lysis of a single selected cell on an ordinary cell chamber because the electric field is concentrated at the tip of the capillary electrophoresis tube  42  which is proximal to the selected cell  59  and distal from a non-selected cell  58 . The cell chamber  10  itself need not be configured with electrodes.  
     [0083] The foregoing embodiments of the invention have focused on the use of single capillary electrophoresis tube for collection of cellular contents. In other embodiments, such as depicted in FIG. 21, and array  31  of capillary electrophoresis tubes  42  may be configured to correspond to the array of electrodes  30  and/or hydrophilic regions  24  on a cell chamber  10 . In this embodiment, multiple cells  58 ,  59  may be selected for simultaneous lysis and collection in a high throughput screening system.  
     [0084] In another modification of the various apparatus of the invention, a single capillary electrophoresis tube  42  or array of capillary electrophoresis tubes  42  can be configured with adjacent micro-dispensing devices  86  such as illustrated in FIG. 15. The micro dispensing devices  86  may be a siphoning capillary, a reverse polarity capillary electrophoresis tube preloaded with a test substance, or a valve controlled capillary or nozzle for dispensing nanoliter volumes of test material into the medium  56  in the vicinity of the cell immediately adjacent to the capillary electrophoresis tube  42 . In use of such a system  22 , very small amounts of test material are introduced into the medium immediately prior to lysis of the selected cell  59  and collection of the contents thereof. Using such configurations, multiple cells on a single slide can be tested with different test materials in a high throughput assay system.  
     [0085] While a capillary electrophoresis tube  42  is a preferred type of micro-collection device  15  in various embodiments of the invention, as mentioned herein before, other micro-collection devices are also suitable for use and or configuration according to the practice of the invention. Examples of other suitable micro-collection devices include surface bound macromolecules  71  on an analytic “chips” such as described, for example, in U.S. Pat. No. 6,440,667. Still another type of suitable device includes optical fiber bundles bound to arrays of macromolecules  71  through a bead, as described for example in U.S. Pat. No. 6,429,027. Yet another type of device is a simple a carbon fiber, that is configured to detect the release of electrons from materials released from the lysed as described for example by Travis et al or Suaud-Chagnv et al [49, 50]. As illustrated in FIGS. 13A and 14A, a micro-collection device  15  such as chip  70  depicted in FIG. 13A, or fiber optic bundle  76  depicted in FIG. 14A, can be used without modification in combination with the cell chambers having an array of electrode pairs, such as illustrated for example, in FIG. 6. FIGS. 13B and 14B illustrate how these other micro-collection devices can be configured with single electrodes on their proximal ends analogous to the capillary electrophoresis tubes  42  depicted in FIGS. 2 and 7- 10 . FIGS. 13C and 14C illustrate that these micro-collection devices may also be configured to contain electrode pairs analogous to the capillary electrophoresis tubes  42  shown in FIGS. 11 and 12.  
     [0086] Another aspect of the invention is methods for lysing a biological cell using a single subsecond pulse of an AC or DC field across the biological cell using the various apparatus of the invention, and collecting at least a portion of the contents of the lysed cell thereof in a micro-collection device. In certain embodiments, the act of collecting is commenced simultaneously with the act of electrical lysis. In other embodiments, the act of collecting commences within 1 second, within 100 ms within 10 ms, within 1 ms, within 100 μs within 10 μs or within 1 μs of commencing the electrical lysis. Coordination between the timing of lysis and timing of collection can be accomplished using a timing control switch that activates the process of collection simultaneously, or within the foregoing prescribed times of commencing the electrical lysis. In preferred practices, the act of collecting uses a capillary electrophoresis tube with a proximal end positioned within the foregoing distance of the selected cell. The methods further include the act of separating at least a portion of the contents of the lysed cell in the capillary electrophoresis tube or other micro-collection device. Methods for controlling the timing between lysis, collection and separation of cellular contents using a capillary electrophoresis system, as well as configurations therewith using a microscope are described in the Examples that follow and in U.S. Pat. Nos. 6,156,576 and 6,335,201 where lysis is described using laser based lysis.  
     [0087] In preferred embodiments of methods of the invention, the selected cell  59  is positioned between the first electrode  18  and the second electrode  19 . In other embodiments the selected cell  59  need not be positioned directly between the first  18  and second electrodes  19 , but is positioned in close proximity thereto, within less than less than about 200 microns or less than 100 microns, or less than 60 microns, or less than 40 microns, or about 20 microns or from the center of the gap between the first  18  and second electrodes  19 . The inter-electrode distance is likewise less than about 200 microns in one embodiment, or less than 100 microns, or less than 60 microns, or less than 40 microns, or about 20 microns or less in various other embodiments. Electrical lysis is commenced by applying an electrical potential of about 5 to about 30 Kv/cm in one embodiment, about 10 to 20 Kv/cm in another embodiment, or about 15 Kv/cm in another embodiment, between the first electrode  18  and the second electrode  19 . The electrical potential is applied in a single pulse of an AC or DC field for a duration of about 10 milliseconds or less in certain embodiments, or 1 millisecond or less, or 100 microseconds or less in other embodiments or 10 microseconds or less in other embodiments.  
     [0088] In certain practices of the methods, the use of an AC field may provide advantages over DC fields, in that unlike DC fields, gas generation at electrodes employing an AC field (is not a significant issue. This property is important because bubbles formed at electrodes can be drawn into separation channels or into the capillary electrophoresis tube where they will interfere with electrophoresis. In addition, the use of AC fields prevents unwanted electrophoretic motion within the electrolyte during the time of lysis.  
     [0089] The devices, systems and methods of the present invention are suitable for use with both adherent cells and non-adherent cells. Adherent cells will tend to adhere to the surface of the cell container, especially over the hydrophilic regions  24 . Non-adherent cells can be urged into a desired position between the first electrode  18  and the second electrode  19  using mechanical or non-mechanical tools. One example of a non-mechanical tool for this purpose is a laser microtweezer.  
     EXAMPLES  
     Example I  
     [0090] Materials and Methods for Electrical Lysis and Micro Collection of the Contents of a Cell In a Capillary Electrophoresis Tube  
     [0091] Reagents. Oregon Green 488 carboxylic acid diacetate (6-isomer) (Oregon Green diacetate), 5-carboxyfluorescein diacetate and 6-carboxyfluorescein diacetate (carboxyfluorescein diacetates) were purchased from Molecular Probes (Eugene, Oreg.). Oregon Green free acid and carboxyfluorescein free acids were obtained by hydrolyzing the diacetate forms of the fluorophores in Na2CO3 solution (pH 10) at 37° C. overnight. Tissue culture materials were obtained from Gibco BRL (Gaithersburg, Md.). Acrylamide, ammonium persulfate and TFMED were bought from Bio-Rad Laboratories (Hercules, Calif.). Methacryloxy-propyltrimethoxysilane (MAPS), was obtained from United Chemical Technologies, Inc. All other chemicals were from Fisher Scientific (Pittsburgh., Pa.). The buffer, ECB, was composed of 135 mM NaCl, 5 mM ICI, 10 mM HEPES, 2 mM MgCl2 and 2 mM CaCl2 adjusted to pH 7.4 with NaOH.  
     [0092] Coating Capillaries with Polyacrylamide. Fused-silica capillaries (50-μ i.d., 360-μ o.d.; Polymicro Technologies, Phoenix, Ariz.) were coated with polyacrylamide. Briefly the capillary was washed with 1 M HCl (1 hr), H2O (10 min), 1 M NaOH (1 hr), and H2O (10 min). The capillary was then washed with methanol (10 min), and filled with 50% MAPS (in methanol). Both ends of the capillary were sealed and the capillary was placed at room temperature for 12-15 hrs. After cleaving the sealed ends of the capillary, it was rinsed with methanol followed by water, each for 10 min. The capillary was filled with freshly prepared reaction buffer (3% acrylamidc, 0.6% ammonium persulfate and 0.2% TEMED in water). After 3 hrs, the capillary was rinsed with water for 10 min and dried with nitrogen for 10 minutes. Prior to use the capillary was rinsed with ECB.  
     [0093] Cell Culture. Rat basophilic leukemia (RBL) cells were used as the model cell for this study. These cells were grown at 37° C. in a humidified 5% CO2 atmosphere in Dulbecco&#39;s modified eagle medium (DMEM) supplemented with fetal bovine serum (10%), and L-glutamine (584 mg/L). Penicillin (100 units/mL) and streptomycin (100 μg/mL) were added to the media to inhibit bacterial growth. The cells were grown in a cell chamber constructed by using Sylgard (Dow Corning, Midland, Mich.) to attach a silicon “O” ring ({fraction (15/16)}-in. outer diameter) to a 25-mm, round, no. 1, glass cover slip. The cover slip was coated with titanium and gold as described below. The cells were allowed to grow for 12-24 h prior to use. Cells were plated at concentrations determined empirically to produce approximately one cell per 1000× field of view on the day of the experiment. For experiments the growth media was removed front the cell chamber and replaced with ECB.  
     [0094] Preparation of Electrically Conductive Coverslips. To make the cover slips (i.e, substrate  12 ) conductive, they were coated first with 50 A of titanium followed by 80 A of gold with an E-beam vacuum evaporator. The purpose of the initial titanium layer was to provide a surface to which the gold could strongly adhere. To prepare the cover slips for coating the following procedure was used: i) clean in an ultrasonic bath containing detergent solution (Alconox, 0.5% (weight/volume)) for 15 minutes, ii) rinse with water, iii) soak in 30% HCl for 15 min, iv) rinse with water, v) dry with filtered, compressed nitrogen, and vi) bake for 30 min at 120° C. Metal-coated cover slips had a resistance of about 15-20 ohms/cm. The transparency of the cover slips was compromised slightly by the metal layers; nevertheless, imaging of the cells through the coated cover slip with an inverted microscope was easily performed. A short length of platinum wire was attached to the gold layer of the cover slip with a conductive epoxy and used to connect the metal coating on the cover slip to the pulse generator.  
     [0095] Loading Cells with Fluorescent Compounds. 6-carboxyfluorescein diacetate, 5-carboxyfluorescein, and/or Oregon Green diacetate were loaded into the cells for these experiments. These dyes are membrane permeant but are not fluorescent until they are hydrolyzed into free acids by ubiquitous intracellular esterases. The free acids are trapped within the cells, although cells do transport the dye out over time via anionic transporters and other as yet undefined mechanisms. Carboxyfluorescein diacetates and/or Oregon Green diacetate were dissolved in ECB with glucose (10 mM) and immediately added to the cell chamber. When both fluorophores were loaded into the same cell, the concentration of the two carboxyfluorescein diacetates and Oregon Green diacetate was 4, 4, and 1 μM, respectively. When Oregon Green diacetate was the only fluorophore loaded, the concentration was 0.5 μM. Cell chambers containing the diacetates were incubated in the dark for 30 min at room temperature. The cells were then washed five times in ECB and were used within −5-10 min. After loading cells with fluorophores, a constant flow of FCB (5.5 ml/min) through the cell chamber was utilized to remove fluorescent dye transported from the cells into the surrounding buffer. Since the cell chamber served as the inlet reservoir during electrophoresis, continual replacement of the chamber buffer was required to avoid contamination of the capillary with fluorophore extravasated from the intact cells remaining in the cell chamber during experiments.  
     [0096] Cell Imaging. An inverted fluorescence microscope with a 100× oil-immersion objective (Diaphot 200, Nikon, Melville, N.Y.) was used for transmitted light and fluorescence imaging (ex/cm 488/514 nm). Images were digitized by a Hitachi CCD camera (Model KP-MIAN) and recorded with a video cassette recorder. Images were collected at standard video rate (30 frames/s), thus the time interval for each frame was 33 ms.  
     [0097] Production and Metal-Coating of Tapered Capillary Tips With an Electrode at the Proximal End One end of the capillary was pulled by a capillary puller (Model P-2000, Sutter Instrument Co. Novato, Calif.) to form a sharp tip. The tip was carefully cut with a ceramic stone under a dissecting microscope to form a tip of ˜30-40μ in outer diameter. The internal diameter was 10-15 μm. The capillary was then attached to a syringe containing pressurized water so that water flowed through the capillary during subsequent steps. The capillary tip was washed with water and placed in an ultrasonic bath containing a detergent solution (Alconox, 0.5% (weight/volume)) for 5 min. After which, the capillary tip was washed with methanol and then water. The syringe of pressurized water was then detached born the capillary and the capillary was dried under a stream of nitrogen. During metal coating, the body of the capillary was coiled and covered with polyimide tape to avoid metal deposition in unwanted regions. The outer wall of the capillary tip was coated sequentially with titanium and gold by E-beam evaporation for a 5-6 cm length to form the electrode. The capillary tip was oriented at a 45° angle with respect to the metal source so that the end and one side of the capillary were coated while the opposite side of the capillary was not coated with metal. The capillary end and exposed side were coated with 50 Å of titanium followed by 80 Å of gold. A platinum wire was glued to the metal-coated portion of the capillary with conductive epoxy, and was surrounded by a small plastic pipette tip. A large plastic pipette tip was also employed to insulate the small pipette tip. The two ends of the large pipette tip were, sealed by a nonconductive epoxy to fix the electrode/capillary in place.  
     [0098] Cell Lysis By An Electric Field. The gold-coated capillary tip was mounted perpendicularly to the cover slip on a 3-axis micromanipulator (World Precision Instruments, Sarasota, Fla.). The micromanipulator enabled precise positioning of the capillary lumen over the cell selected for analysis. A pulse generator (model HP 214B, Hewlett-Packard, Palo Alto, Calif.) was used to apply a voltage pulse between the capillary and coverslip. The pulse generator provided pulses in the shape of a square wave with variable strength and width and was operated in burst mode at 1 pulse per burst. The pulse was triggered by an external voltage (5 volts) generated by an A/D board (KPCI 3100, Keithley Instruments, Cleveland, Ohio) installed in a computer (Dell Computer, Round Rock, Tex.).  
     [0099] Capillary Electrophoresis. A high-voltage power supply (CZE1000R, Spellman, Plainview, N.Y.) was used to apply a voltage across the capillary for electrophoresis. The capillary (50μ inner diameter, 380μ outer diameter) possessed a total length of 75 cm. The electrophoretic buffer was ECB and was chosen to mimic the ionic composition of a physiologic extracellular fluid. The cell chamber served as the inlet reservoir and was held at ground potential. The outlet reservoir was held at a constant voltage between +12 and +14 kV as indicated. A custom computer program written in TestPoint (Keithley Instrument, Inc., Cleveland, Ohio) was utilized to simultaneously trigger the voltage pulse for cell lysis and initiate the electrophoresis. The inlet and outlet reservoirs were at equal heights to avoid gravity flow. Free acid standards of carboxyfluorescein and Oregon Green were loaded into the capillary by gravity-induced hydrodynamic injection.  
     [0100] Fluorescence Detection. A 1 cm optical window in the capillary was created by burning off the polyimide coating 50 cm from the inlet. The capillary lumon was interrogated by the focused laser beam of an argon ion laser (488 nm, Uniphase, San Jose, Calif.). The focusing long possessed a focal length of 5.0 cm (CVI, Albuquerque, N. Mex.). Fluorescence was collected at a right angle to the capillary and laser beam with a microscope objective (40×, 0.75 n.a., Plan Fluor, Nikon, Melville, N.Y.). The light was collected and measured with a photomultiplier tube (PMT) (8928, Hammamatsu, Bridgewater, N.J.) after filtering with a 488 notch plus filter (Kaiser Optical Systems, Ann Arbor, Mich.), and a band-pass filter (535DF50, Chroma, Brattleboro, Vt.). A spatial filter was placed in front of the filters to remove the scattered laser light. The PMT current was amplified and converted to a voltage with a preamplifier (Model 1212, DL Instruments, Dryden N.Y.). The signal was digitized by a data acquisition board (KPCI 3100, Keithley Instruments, Inc., Cleveland, Ohio). The data were plotted using Origin (Microcal, Northhampton, Mass.).  
     [0101] Quantitation of the Amount of Cellular Fluorophore Loaded into a Capillary. Cells were loaded with Oregon Green as described above. Two sets of experiments were performed simultaneously. In one set of experiments, single cells were lysed and loaded into the capillary of an LMS. The area of the peak obtained from each cell was then compared to that of standards to determine the number of moles of Oregon Green collected from the cell. With the LMS, the standards were loaded by hydrodynamic flow for 7 seconds with a height difference between the inlet and outlet reservoir fluid levels of 2.5 cm. The volume loaded was calculated using Poiseulle&#39;s equation. The number of moles was then obtained from the known concentration of the sample and the volume loaded.  
     [0102] In the second set of experiments, single cells were lysed and loaded into a capillary using the electrical lysis system (1 ms pulse duration, 40 V, 20 μm inter-electrode distance). The area of the peak obtained from each cell was then compared to that of standards to determine the number of moles of Oregon Green collected from the cell. The moles of standard loaded into the capillary was calculated from the concentration and the volume loaded. In contrast to the LMS, a simple Poiseulle equation could not be used to calculate the volume of standard loaded into the capillary since the capillary lumen was tapered at one end.  
     [0103] In order to determine the volume of standard loaded under a specific set of hydrodynamic conditions, a concentrated Oregon Green sample of known concentration was hydrodynamically loaded into the capillary (used for electrical lysis). The hydrodynamic force was created by placing the capillary inlet into the sample solution and elevating the level of the inlet solution 7 cm above the level of the outlet solution for 5 s. In one instance the sample was electrophoresed and the peak area measured. In a second trial, the concentrated Oregon Green sample was loaded in an identical fashion but the sample was not electrophoresed. Instead, the loaded sample was pushed back, out of the capillary into a collection tube and diluted to a known final volume (50 μl). This diluted sample was then loaded into the capillary under conditions identical to the concentrated sample and electrophoresed. All electrophoretic and detector conditions were identical to that when the concentrated sample! of Oregon Green was electrophoresed.  
     [0104] Because the peak area is proportional to the number of electrophoresed moles and the concentrated and diluted samples possessed identical injection volumes, the ratio of the peak areas of the two samples yields the degree of dilution of the diluted Oregon Green (i.e. C c V inj /A c =C d V inj /A d  for V inj , the injection volume; C c , and C d , the concentration of the concentrated and dilute Oregon Green samples, respectively; A c  and A d , the peak area of the concentrated and dilute Oregon Green samples, respectively). The degree of dilution (C d /C c =A d /A c ) can then be used to calculate the injection volume since the final volume of the diluted sample was known (i.e. A d /A c =V inj /V final  for V final , the final volume of the diluted sample).  
     Example II  
     [0105] Results of Subsecond Electrical Lysis and Collection of Cellular Contents in a Capillary Electrophoresis Tube  
     [0106] The goal of these experiments was to determine whether fast electrical lysis of a cell in combination with electrophoresis of the cell&#39;s contents in a capillary might be compatible with the study of fast signaling reactions in cells. To be successfully used to follow rapidly changing cellular reactions, the method should preferably have the ability to terminate the cellular chemical reactions of interest on time scales less than one second, or less than 500 ms, or less than 100 ms and preferably, less than ˜33 ms. The actual time of lysis may be lower than 33 ms, which is the lower limit of measurability in this example because that is the period that elapses between frames of a video recorder. For many cellular reactions such rapid lysis can be achieved by lysis of the cell on similar time scales. [11,12]. Turbulence from the lytic process and diffusion of the reactants can then greatly slow the rate of many chemical reactions essentially terminating the reaction. A second desired requirement is that the method be capable of selecting a single cell for analysis. Since individual cells are asynchronous in their response to stimuli, loading the contents of multiple cells at once into the capillary would yield a population average which is frequently not representative of the behavior of a single cell. Since most mammalian cells grow attached to a surface and removal from that surface disrupts or alters multiple signaling pathways, the lytic method must not require removal of the cell from the surface prior to lysis nor can it disturb or stress the cell prior to the moment of lysis (since this can also activate signaling pathways). [30-34]. Lastly, the strategy for cell lysis must be compatible with capillary electrophoresis. Efficient loading of the cellular contents into a capillary electrophoresis tube without deterioration of the subsequent electrophoretic separation is most desirable.  
     [0107] Design/Fabrication of the Capillary Electrode. Lysis of cells in an electric field is due to dielectric breakdown of the membrane as the transmembrane voltage reaches a certain magnitude. In mammalian cells membrane breakdown leading to pore formation begins at a transmembrane voltage of 0.2-1.5 V depending on the conditions and the cell type. [13-18]. Limited numbers of small pores are reversible and this is the basis for electroporation. As the transmembrane voltage is increased, larger pores and greater numbers of pores form creating irreversible holes in the membrane lysing the cell.  
     [0108] In designing the electrodes the goal was to utilize an electrode geometry that leads to the fastest formation of irreversible pores at the lowest inter-electrode voltage. Two different electrode placements with respect to the adherent cell are possible as illustrated in FIG. 16. The potential across the plasma membrane of the cell depends on the cosine of the angle (α)  98  between the electric field lines  97  and a line  100  drawn perpendicular to the surface of the membrane  102  of the cell  59 . [13-18]. Thus, the potential drop across the membrane is maximal for field lines perpendicular to the cell surface and minimal for field lines nearly parallel to the cell surface. When electrodes are placed to the right and left of a cell as illustrated in FIG. 16A, the field lines  97  or direction of the electric field is parallel to the surface of the flattened, adherent cell  59 . For this geometry, the electric field is less efficient at creating pores throughout the membrane. In contrast, electrodes positioned above and-below the cell are more efficient in creating a high transmembrane voltage throughout large areas of the plasma membrane as depicted in FIG. 16B.  
     [0109] Multiple investigators have demonstrated that pores initially form in the cell membranes facing the electrodes rather than,—in the membrane regions away from the electrodes. [35-38]. For example in flattened, adherent cells with electrodes placed to the right and left of the cell as in FIG. 16A pores initially formed in the outermost tips of the cell facing the electrode.[35]. Additionally a number of investigators have also demonstrated that pores form at a lower inter-electrode voltage in the membrane facing the positive electrode (the hyperpolarized portion of the cell&#39;s membrane) than in the membrane facing the negative electrode (depolarized membrane). The reason for this phenomena is not known but suggests that the positive electrode should be positioned above the cell. Thus the largest and earliest pores will form in the cellular membrane facing the buffer solution rather than in the membrane regions tightly adhered to the surface of the cell chamber. This would yield the fastest release of the cell&#39;s contents into the buffer solution contained just below the lumen of the capillary. For these reasons the design shown in FIG. 16B was selected for testing as the geometry most likely to give fast cell lysis at the lowest possible inter-electrode voltage. However, designs with the electrodes placed adjacent to the cells as depicted in FIG. 16A are may also be effective, particularly when the cells have a spherical geometry, as occurs, for example in many blood cell types.  
     [0110] The design of FIG. 16B requires that electrodes be placed above and below the cell and that each electrode be in close proximity to the cell. Further the electrodes must be capable of lysing only one cell yet any cell in the chamber must be selectable for lysis and analysis. To meet those requirements one electrode was fabricated on the substrate on which the cell grew and the second electrode was placed on the tip of the capillary as depicted in FIG. 10. A thin layer of titanium and then gold was evaporated with a glass coverslip. The coverslip was assembled into a cell chamber and the cells cultured on the metal coated coverslip. The cells attached and grew as well on the metal-coated coverslip as! on the glass surface. Since the cover slip-based electrode extended throughout the cell chamber, the second or upper electrode needed to confer both the ability to lyse a single cell and the ability to select any cell in the chamber. To meet this need, one end of a capillary was pulled to a fine tip (30-40μ outer diameter) using a laser-based capillary puller. The tip was then coated with a thin layer of titanium and gold. A platinum wire was used to connect the metal-coated region of the capillary to a pulse generator. Placing the small capillary tip in close proximity to the cell and underlying electrode should largely confine the electric field to the region between the capillary tip and the immediately adjacent surface of the coverslip. The capillary was also mounted on a micromanipulator so that it could be positioned over any cell contained in the chamber.  
     [0111] Influence of the Magnitude of the Voltage Pulse on Cell Lysis. For this application, termination of the cellular reactions depends on the rate of lysis of the cells. For this reason, we initially determined how fast a cell could be lysed by a voltage pulse. In addition since electroporation depends on both the magnitude and the duration of the voltage pulse, we also sought to determine; how these parameters influenced the rate of cell lysis. Cells were loaded with the fluorophore Oregon Green. Oregon Green is trapped in the cytoplasm of cells as long as the plasma membrane remains intact. The fluorescence of Oregon Green in a cell was measured before during and alter application of a voltage pulse to a cell. The time for complete loss of Oregon Green fluorescence from the cell (as judged by fluorescence microscopy) was used as a measure of how fast lysis occurred, i.e., the time for leakage of the cell&#39;s contents.  
     [0112] The influence of the magnitude of the voltage pulse was investigated by varying the voltage while keeping all other parameters fixed. Four different pulse strengths between 10 and 40 V were tested with a pulse duration of 1000 μs and an inter-electrode distance of 20 μm. Six cells were lysed at each voltage, and the tune for the fluorescence to decrease to background was plotted as a function of the voltage as shown in FIG. 17. Error bars were plotted to show the standard deviation. At the lowest voltages (10-20 V), the efflux of the Oregon Green from the cells was slow requiring hundreds of milliseconds to return to the background level of fluorescence. From the transmitted-light images, the profiles of the cells could still be observed. These cells possessed the morphology of a dead cell (refractile, granular appearance) suggesting that only remnants of the cytoskeleton, plasma membrane, and some organelles remained. These findings suggest that the cells were slowly lysed following the voltage pulse. This may have been due to the formation of membrane pores of limited size and number requiring long times for complete efflux of intracellular molecules. The large error bars are due to the cell-to-cell variability in the voltage intensity required to lyse or permeabilize cells. [23,27,35]. This variability may be due to differences in the cellular morphology, cell cycle, and other factors. When the voltages were 30 V and greater, almost all cells were lysed within one image frame, i.e. 33 ms as illustrated in FIGS. 18A,B. The transmitted-light images also showed that very few remnants of the cells remained attached to the substrate as shown in FIGS. 18C,D. At these higher voltages, electrolysis of water at the electrodes was also increased as evidenced by larger numbers and sues of gas bubbles forming on the electrodes. These field strengths (15 kV/cm) are similar to that described previously to cause cellular lysis in the context of electroporation, and are approximately 10 fold greater than that needed to permeabilize cells. [13-18].  
     [0113] Influence of Pulse Duration on Cell Lysis. In a second set of experiments, the influence of the duration of the voltage pulse was investigated by fixing all parameters except the pulse length. Six different pulse durations (1 μs, 10 μs, 100 μs, 200 μs, 500 μs, and 1000 μs) were tested while the voltage was fixed at 40 V and the inter-electrode distance was again 20 microns. Six cells were lysed for each pulse length, and the time for the fluorescence to decrease to background was plotted as a function of the pulse width as shown in FIG. 17B. Error bars were plotted to show the standard derivation. Cells receiving the 1 μs and 10 μs long pulses showed a decrease in fluorescence. However, during the time that the cells were examined, the fluorescence did not approach the background fluorescence level suggesting that the cells were not lysed. Most likely the cells were reversibly electroporated during exposure to these brief voltage pulses. Since the capacitance of the system was unknown, it was possible that the peak voltage of these shorter pulses did not reach 40 V. For this reason a digital oscilliscope was used to determine the true pulse width and magnitude when the voltages were applied across the capillary and coverslip electrodes. For a selected pulse duration of 1 μs and voltage of 40 V, the true peak voltage was only 30 V with a width of 1 μs. For a selected width of 10 μs and voltage of 40 V, the actual voltage pulse was square in shape with a width of 10 μs and a magnitude of 40 V. The capacitance of the system therefore did not blunt the magnitude or duration of the applied 10 μs-voltage pulse.  
     [0114] Longer voltage pulse durations (100-1000 μs) resulted in the complete loss of cellular fluorescence over time after application of the pulse. For pulse widths of 100 and 200 μs the cellular fluorescence required hundreds of milliseconds to return to the background level as shown in FIG. 18. Cellular ghosts or remnants of the cytoskeleton, plasma membrane, and organelles remained visible after application of the voltage pulse. These results suggest that the cells were slowly lysed at these pulse duration. For longer pulse widths (≧500 μs), the cells were lysed more completely and rapidly. Nearly all cells had complete loss of fluorescence within 33 μs. Transmitted light images showed no or few cell remnants attached to the coverslip. Thus, cells could be rapidly lysed with single, brief voltage pulses.  
     [0115] Influence of Inter-Electrode Distance on Cell Lysis. The degree of electroporation of a cell depends on the Inter-electrode distance when the absolute voltage and pulse duration are maintained constant. For this reason, we sought to determine how the inter-electrode distance influenced the rate of cell lysis when both the voltage and the pulse duration were fixed. Cells were loaded with Oregon Green and the time for fluorophore efflux was measured as the interelectrode distance was varied. As seen in FIG. 17C the greater the inter-electrode distance, the slower the cell lysis. For an inter-electrode distance between 10-20 μm, the cells were nonfluorescent within 33 ms suggesting that they were very rapidly lysed. It took substantially longer (˜330 ms) for Oregon Green to exit the cell when the inter-electrode distance was 30-40 μm. At these distances the ghosts or profiles of the dead cells were visible by transmitted light microscopy. For these voltages and pulse widths, the required distance between the capillary and cell was much smaller than that required for laser-based lysis (˜60 μm) making the alignment of the capillary over the cell slightly more difficult for electrical lysis. [11] 
     [0116] Variability of the Capillary-Based Electrode. The properties of each fabricated capillary/electrode combination were slightly different. This can be seen in FIG. 17, panels A and C. In FIG. 17A, release of Oregon Green occurs within 33 ms when the average electric field between the electrodes is as low as 1.5 V/μm. However in FIG. 17C the release of the Oregon Green within 33 ms does not occur until the field reaches an average of 2 V/μm. This variability is most likely due to differences in the manufacturing process. After the capillary tip was pulled, it was manually cleaved to yield an outer diameter varying between 30 and 40 μm at the very tip. In addition to the variability in the exact tip diameter, the shape of the tip was also difficult to control with this process. Further optimization of the tip pulling protocol might eliminate the manual cleavage procedure. Nevertheless with the current manufacturing process, each capillary/electrode combination was easily tested for the minimum average electric field required to lyse all cells within 33 ms. Cell lysis in subsequent experiments was then performed with the electric field optimal for that particular metal-coated capillary tip.  
     [0117] On average the electrode fabricated on the capillary could be used ˜50 times before loss of the gold/titanium coating became significant. This was most likely due to two factors: i) electrolytic removal of gold from the capillary/electrode surface during cell lysis, and ii) electrolytic removal of titanium due to pin holes in the gold coating. Due to the relatively large voltages applied across the electrodes during cell lysis; oxidation of gold and exposed titanium is expected to occur at the anode on the capillary. [39]. With each brief voltage pulse, the anode will loose gold and/or titanium until eventually all of the metal is removed. After prolonged usage, large flakes of metal were observed detaching from the glass surface. This suggests that the underlying titanium might have undergone oxidation first releasing the gold from the surface. It may be possible to minimize metal “lilt-off” from the glass surface by depositing a thicker gold coating to cover pinholes. With the current method one side of the capillary is in a “shadow” during metal deposition and so is poorly coated or uncoated. The boundary region between this poorly coated side and the heavily-coated capillary regions (the end and opposite capillary side) may leave exposed titanium which can be electroplated from the glass undercutting the adjacent more heavily coated regions of the capillary. Rotating the capillary during metal deposition would eliminate the shadowed side of the capillary and potentially increase its lifetime.  
     [0118] Capture and Separation of a Cell&#39;s Contents. During electrical lysis of a cell, the high voltages applied across the electrodes can generate several gases. Oxidation of chloride and water at the anode can generate chlorine and oxygen, respectively, while reduction of water at the cathode can generate hydrogen gas. [39]. Consequently, a substantial amount of turbulence might occur near the electrodes as gas is generated and released. A small amount of turbulence is desirable since it promotes dilution of the cell&#39;s contents essentially terminating most cellular reactions. However, excessive mixing can lead to extreme dilution making the cellular contents undetectable or to an inability to collect the cell&#39;s contents into the capillary lumen. Further, gas generation at the electrodes might enter the capillary blocking or impairing analyte movement through the capillary during electrophoretic separation.  
     [0119] To determine whether a cell&#39;s contents could be collected and detected following electrical lysis, cells were loaded with Oregon Green. The capillary was then positioned 20 microns above the cell with the capillary lumen centered over the cell. The cell was lysed by a single electrical pulse (30-40 volts, 1 ms pulse width). A continuous high voltage was applied across the capillary ends simultaneously with the lytic voltage pulse. The fluorescence of analytes moving through the capillary was then monitored. A standard of Oregon Green was used to identify the migration time of Oregon Green in the capillary. One peak at a migration time nearly identical to the migration time of the Oregon Green standard was observed (n&gt;20) as shown in FIGS. 18A,B Since no hydrodynamic flow was present, the contents of the cell were most likely loaded into the capillary by a combination of both electrophoretic movement and electroosmotic fluid flow. The average number of theoretical plates (±standard deviation) was similar for the standard and cell-derived peaks, 214,972±30,485 (n=8) and 197,109+29,113 (n=8), respectively. The similar number of theoretical plates suggests that the sample derived from the cell was not extensively diluted during cell lysis.  
     [0120] To determine whether the contents of an electrically lysed cell could be separated, cells were loaded with Oregon Green, 5-carboxyfluorescein, and 6-carboxyfluorescein. As described above, the cell was lysed and loaded into the capillary and the contents electrophoresed through the capillary. For all cells (n&gt;5), the electropherogram displayed three peaks with migration times nearly identical to that of a standard composed of Oregon Green, 5-carboxyfluorescein, and 6-carboxyfluorescein as shown in FIGS. 19C,D. As a negative control, a RBL cell not loaded with any of these fluorophores was lysed and analyzed in the same manner. In this case, no peak was detected.  
     [0121] Collection Efficiency of the Cellular Contents. To estimate the loading efficiency of the cellular lysate into the capillary following electrical lysis, the number of moles of Oregon Green obtained from; a cell that was electrically lysed was compared to that from a cell lysed with the laser-induced shock wave. Cells treated in an identical fashion except for the method used to lyse the cells, should contain on average the same number of moles of fluorophore. Since the loading efficiency of the laser-based strategy has been measured, comparison of the numbers of moles collected from cells lysed by the two methods should yield an estimate of the collection efficiency following electrical lysis.  
     [0122] For the experiment, cells were loaded with fluorophore and then split into two portions. One group of cells was utilized in experiments on a laser micropipette system and single cell lysis was performed as described previously with a laser-generated shock wave. A second group of cells was electrically lysed. In both instances, the contents of the cells were loaded into an overlying capillary and electrophoretically separated. The area of the peak on the electrophoretic trace was then compared to that of standards to determine the number of moles of Oregon Green loaded from the cell. Eleven cells were electrically lysed while thirteen cells were analyzed with the LMS. Due to the cell-to-cell variability in the concentration of the fluorophore loaded into the cell, large standard derivations were obtained in both cases. For this reason, each single-cell measurement was plotted rather than the mean values, which is depicted in FIG. 20. The distribution in the number of moles of Oregon Green was similar for both lytic methods; therefore, the collection efficiency of the two methods was likely to be similar. Previously, Sims and colleagues demonstrated that the efficiency of collection for the LMS was close to 100%. [11] These results suggest that collection of the cellular contents into the capillary following electrical lysis was highly efficient.  
     [0123] Various embodiments of the invention have been described in particular figures and certain places in the text. The invention is not limited to these particular embodiments singularly, but also includes systems having any combination of the various embodiments that are not mutually exclusive of one another in functional operation. For example, the array of capillary electrophoresis tubes depicted in FIG. 21 may be configured without electrodes, or with any of the embodiments of electrodes described in the other Figures.  
     [0124] Insubstantial changes from the claimed subject matter as viewed by a person with ordinary skill in the art, now known or later devised, are expressly contemplated as being equivalently within the scope of the invention and its various embodiments. Therefore, obvious substitutions now or later known to one with ordinary skill in the art are defined to be within the scope of the defined elements. The invention and its various embodiments are thus to be understood to include what is specifically illustrated and described above, what is conceptually equivalent, what can be obviously substituted and also what essentially incorporates the essential idea of the invention.  
     [0125] Many alterations and modifications may be made by those having ordinary skill in the art without departing from the scope of the invention. Therefore, it must be understood that the illustrated embodiments have been set forth only for the purposes of example and that it should not be taken as limiting the invention except in accordance with the claims that follow and equivalents thereof.  
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