WO1996010170A1 - Capillary-based separation methods for identifying bioactive analytes in a mixture - Google Patents

Capillary-based separation methods for identifying bioactive analytes in a mixture Download PDF

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Publication number
WO1996010170A1
WO1996010170A1 PCT/US1995/012444 US9512444W WO9610170A1 WO 1996010170 A1 WO1996010170 A1 WO 1996010170A1 US 9512444 W US9512444 W US 9512444W WO 9610170 A1 WO9610170 A1 WO 9610170A1
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die
cell
capillary
biosensor
ligands
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PCT/US1995/012444
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French (fr)
Inventor
Richard N. Zare
Jason B. Shear
Harvey A. Fishman
Nancy L. Allbritton
Richard H. Scheller
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The Board Of Trustees Of The Leland Stanford Junior University
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Publication of WO1996010170A1 publication Critical patent/WO1996010170A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis

Definitions

  • the present invention relates to methods of identifying bioartive analytes using capillary-based separation means.
  • Bright, G.R., et al. "Fluorescence ratio imaging microscopy” in FLUORESCENCE MICROSCOPY OF LIVING CELLS IN CULTURE.
  • METHODS IN CELL BIOLOGY (Taylor, D.L. and Wang, Y.-L., eds.) (Academic Press, San Diego) pt. B, vol 30:157 (1989).
  • Electrophoresis Apparatus for Separating and Detecting Molecular Species
  • Tsien R.Y., Methods Cell Biol 2fl:127 (1989b).
  • Capillary-based separation means have been employed to resolve small volumes containing complex mixtures of .analytes into their constituents.
  • Methods based on the principle of capillary electrophoresis are particularly effective in this regard (Jorgenson, et al. , 1983, Gordon, et al, Ewing, et al).
  • capillary electrophoresis an electric filed is applied across a capillary, typically about 10 to 100 cm long and 20-200 ⁇ m in internal diameter, that contains a separation medium, typically an electrolyte or gel.
  • the electric field induces electroosmotic flow of the separation medium through the capillary, and causes analytes in a sample mixture to separate according to, among other factors, their charge and hydrodynamic drag.
  • Detection methods that have been used with capillary electrophoresis include optical absorption (e.g., Lauer, et al.), optical fluorescence (e.g., Gassman, et al., electrochemical (e.g., Wallingford, et al., 1987b), conduct-metric detection (e.g., Huang, et al, 1987), radioisotope based detection (e.g., Berry), refractive index changes (e.g., Bruno, et al.) and mass spectrometry (Olivares, et al.).
  • optical absorption e.g., Lauer, et al.
  • optical fluorescence e.g., Gassman, et al., electrochemical (e.g., Wallingford, et al., 1987b)
  • conduct-metric detection e.g., Huang, et al, 1987
  • radioisotope based detection e.g., Berry
  • the present invention provides a method of separating small volumes of complex mixtures of analytes using capillary-based separation means, and identifying separated constituents using a highly sensitive and highly selective detection means distinct from the above.
  • the present invention includes a method for detecting a selected ligand in a mixture of ligands.
  • the method includes separating the mixture of ligands containing the selected ligand using a separation means, delivering separated ligands to a cell biosensor having a receptor capable of binding the selected ligand, and detecting binding of the selected ligand to the receptor.
  • the separation means is a capillary-based separation means, and the separated ligands are delivered to said biosensor in a cell biosensor compatible buffer.
  • the separated ligands are preferably delivered to a cell biosensor that is adjacent the outlet of the capillary-based separation means.
  • a ligand may be any analyte that is capable of binding to a receptor and, by binding, having a measurable effect on the receptors's activity or function.
  • the mixtures of ligands is derived from a tissue extract.
  • the mixture is derived from a cell extract.
  • the mixture of ligands is obtained from a combinatorial library of ligands. Selected ligands in a mixture of ligands may include such compounds as acetylcholine, bradykinin and ATP.
  • selected ligands may include a "parent" ligand, and breakdown, proteolysis or degradation products of that parent.
  • the ligands may be a selected subset of degradation products.
  • the ligand may be an agonist of the receptor, while in another, it may be an antagonist.
  • the separation means used in methods of the present invention is one of, or a combination of, various modes of capillary electrophoresis, including capillary zone electrophoresis, capillary gel electrophoresis, capillary electrochromatography, capillary isotachophoresis, affinity capillary electrophoresis, micellar electrokinetic capillary electrochromatography, capillary isoelectric focusing.
  • the separation means may also be a variation of micro liquid chromatography, such as open tube liquid chromatography.
  • a cell biosensor used in methods of d e present invention may be a cell, including a cell recombinantly expressing a specific receptor.
  • the cell biosensor is a eukaryotic cell, such as a mammalian cell or a Xenopus oocyte.
  • the cell biosensor is a cell in contact with a voltage-recording electrode, such as an extracellular electrode, intracellular voltage-recording electrode, intracellular voltage clamp electrode(s), whole-cell patch electrode, or cell-attached patch electrode.
  • the cell biosensor is a cell having a cell condition reporter, such as a dye sensitive to voltage, calcium, cAMP, calmodulin, pH, sodium, chloride, magnesium, or the like. Such reporters would detect changes in the cell biosensor in voltage, intracellular free calcium, cAMP, activated calmodulin, pH, sodium chloride and magnesium, respectively.
  • the cell biosensor is a group of cell, such as a group of confluent or semi- confluent cells.
  • the cell biosensor is a portion of an intact cell, such as an inside-out or outside-out membrane patch, a permeabilized cell, or a cellular organelle, such as a vesicle containing membrane from, for example, the sarcoplasmic reticulum of muscle.
  • the cell biosensor is in a bathing solution containing a receptor-specific antagonist, and effects of the presence of the agonist on the cell biosensor response are evaluated.
  • a receptor-specific antagonist e.g., a receptor-specific agonist
  • specific identification of ligand-bound receptors is possible.
  • the cell biosensor is exposed to a receptor agonist while, for example, separated ligands thought to function as receptor antagonists are delivered with a capillary-based separation means.
  • the invention also includes the cell biosensor attached at the outlet end of the capillary used in the capillary-based separation means.
  • the biosensors may be cells that were transferred to the outlet tip of the capillary by touching the tip to a cell in a recording chamber.
  • the cell biosensor may be cells cultured and grown directly in the outlet tip of a capillary that is subsequently used for a capillary-based separation means.
  • the cell biosensors are, for example, cells grown on a solid support, and die solid support containing the cells is attached, preferably removably, at the outlet end of the capillary.
  • Another aspect of the invention relates to moving either the cell biosensor or the outlet tip of the capillary with respect to the other to employ a different biosensor during the course of separation.
  • This embodiment can minimize effects of receptor desensitization on cell biosensor responses or signals.
  • the movement may be step-wise, such that a rapid translation is made to a region of substrate containing a different biosensor, and d e region is recorded from for e set period of time, or the movement may be continuous, such that the cell biosensor detection field and ⁇ e capillary eluent sweep across a substrate containing cell biosensors.
  • the rate of sweep may be set such that receptors do desensitize appreciably before they are out of the biosensor detection field.
  • supplemental detection means used in conjunction with a cell biosensor to identify ligands present in a mixture of ligands.
  • peaks of .analytes are identified whether or not they have biological activity, and biological activity may be correlated widi specific peaks detectable by traditional capillary electrophoresis methods.
  • the supplemental detection means does not interfere with or disturb the separated ligands as they pass through a capillary of a capillary-based separation means.
  • an optically-clear window may be incorporated in the capillary a few centimeters upstream of the outlet end of the capillary, and used for monitoring die fluorescence or UV-absorbance of the separated ligands as they pass by the window, and just before they exit the outlet and are introduced to die cell biosensor.
  • the outputs from the various detection means, including cell biosensor signal detection means, may be routed to a computer programmed for various types of analyses, such as correlation of the peaks detected with a supplemental detection device and peaks detected widi the cell biosensor.
  • the invention further includes a kit for supplying cell biosensors for use with a capillary-based separation means of the present invention.
  • the cell biosensors are supplied on a solid support that can be attached at the outlet end of a capillary- based separation means.
  • the kit may include a plurality of such solid supports, frozen cells suitable for thawing and culturing on the solid supports, and various devices to facilitate die growing of cells on the solid supports, such as a culture chamber, adapted to receive a plurality of solid supports containing cells, for growing the cells.
  • the invention includes a device for performing capillary-based separations in conjunction with a cell biosensor.
  • the device may include a capillary-based separation means, including capillaries, a power supply, solution/electrolyte reservoirs, and die like, and cell biosensors.
  • the device may be designed to be used widi a kit as described above, and may further be automated, for example, to introduce a plurality of samples sequentially to die inlet end of d e capillary, and/or to replace cell biosensors, as needed, at die outlet end of die capillary.
  • FIGURES Figures IA and IB show schematic diagrams of a capillary electrophoresis apparatus in conjunction with a cell biosensor and an optical detector used for detecting the state or condition of a cell condition reporter in die cell biosensor.
  • Figure 1C shows a photomicrograph of the end of a capillary widi its internal channel positioned above a cell biosensor (labeled "cell”).
  • Figure 2 shows plots of fluorescence as a function of time detected from cell biosensors following capillary electrophoresis separation of ligands including acetylcholine (ACh), bradykinin (BK) and adenosine triphosphate (ATP).
  • Figure 3 shows plots of fluorescence as a function of time detected from cell biosensors following capillary electrophoresis separation of a lysate of PC 12 cells and a control including acetylcholine.
  • Figure 4 shows a schematic diagram of a two-electrode oocyte voltage clamp in conjunction with a portion of a capillary electrophoresis apparatus.
  • Figure 5 shows plots of current as a function of time passed by a two electrode voltage clamp to maintain an oocyte, recombinantly expressing a serotonin receptor, at -70 mV in the presence of delivery of separated ligands including serotonin.
  • Figures 6A, 6B, 6C and 6D show plots of fluorescence as a function of time detected from cell biosensors following capillary electrophoresis separation of pulses of bradykinin, illustrating (i) d e desensitization of bradykinin receptors expressed on a cell biosensor (Figs. 6A, 6B, 6C and 6D), (ii) normal bradykinin responses on neighboring cell biosensors (Figs. 6B, 6C and 6D), (iii) translating the substrate containing the biosensors widi respect to a stationary capillary (Fig. 6B), and (iv) translating the capillary with respect to distinct stationary biosensors (Figs. 6C and 6D).
  • Figure 7 shows plots of fluorescence as a function of time detected from a cell biosensor following capillary electrophoresis separation of a mixture of ligands containing acetylcholine and bradykinin in die presence -and absence of HOE 140 (a bradykinin receptor antagonist).
  • Figures 8A and 8B show plots of fluorescence as a function of time detected from a cell biosensor following capillary electrophoresis separation of a mixture of ligands containing acetylcholine and bradykinin in die presence (Fig. 8B) and absence (Fig. 8A) of atropine and ⁇ -bungarotoxin (acetylcholine receptor antagonists).
  • Figure 9 shows a plot of fluorescence as a function of time detected from a cell biosensor following capillary electrophoresis separation of a mixture of ligands containing acetylcholine, bradykinin and Lys-bradykinin.
  • a "capillary-based separation means” refers to any method for separating a mixture of analytes that employs a capillary as the entity in which the separation occurs. Examples include variations of capillary electrophoresis, including capillary zone electrophoresis, capillary gel electrophoresis, micellar electrokinetic capillary electrochromatography, capillary isoelectric focusing, capillary isotachophoresis, and affinity capillary electrophoresis, as well as variations of micro liquid chromatography, such as open tube liquid chromatography.
  • a “capillary” refers to a tube having an inner diameter of between about 5 ⁇ m and about 1 mm. Preferably, the inner diameter is between about 20 ⁇ m and about 200 ⁇ m.
  • the length of die capillary may be between about 1 cm and about 2 m, but is typically in the range of tens of centimeters.
  • the capillary is typically made of a glass or fused silica material, but may be made from odier materials as well, including various plastics.
  • the inner and outer surfaces of die capillary may further be coated widi any of a variety of materials to improve separation, reduce electrical noise, or alter odier selected characteristics.
  • a “cell biosensor” refers to an intact cell, a portion of an intact cell (such as a membrane patch or permeabilized cell), or an intact cell in electrical communication with electrode(s) (such as intracellular recording electrodes, whole-cell electrodes, cell-attached patch electrodes, extracellular recording electrodes, etc.).
  • electrode(s) such as intracellular recording electrodes, whole-cell electrodes, cell-attached patch electrodes, extracellular recording electrodes, etc.
  • die cell does not necessarily need to be in contact with the electrode, only that the electrode be capable of detecting changes in die electric field due to changes in die cell's transmembrane electrical potential.
  • a cell biosensor designed for use widi an optical detection system will typically contain a cell condition reporter detectable by optical methods.
  • a “cell condition reporter” is defined herein as an entity that alters its state or characteristics, preferably its optical characteristics, in response to a selected change in the cell's condition.
  • Examples of cell condition reporters include, but are not limited to dyes diat are sensitive to transmembrane voltage, intracellular calcium and selected intracellular second messengers, such as cAMP.
  • An exemplary cell condition reporter is die fluorescent dye fluo-3, which fluoresces in response to increased calcium concentrations.
  • a "receptor” is a macromolecule capable of specifically interacting with a ligand molecule.
  • Receptors may be associated widi lipid bilayer membranes, such as the extracellular, golgi or nuclear membranes, and/or be present as free or associated molecules in d e cell's cytoplasm. Further, receptors may be eidier native to die cell biosensor (normally expressed by die cell from which the cell biosensor is derived), or diey may be recombinantly expressed.
  • Recombinantly expressed refers to proteins, such as receptors, expressed in a host cell as a result of die introduction into die host cell of a nucleic acid containing a region encoding die recombinantly expressed protein.
  • recombinantly expressed may refer to receptors expressed in Xenopus oocytes from mRNA eidier isolated from a selected source, such as a tissue from an organism of choice, or transcribed from a cloned cDNA.
  • Recombinantly expressed may also refer to receptors expressed using a eukaryotic expression vector used to transform (eidier transiently or stably) a host cell line. Prior to transformation, the host cell line may or may not contain and/or express an endogenous version of diat receptor.
  • a "ligand” is a molecule capable of specifically binding to a receptor and altering die receptor's function. Binding of die ligand to die receptor is typically characterized by a high binding affinity, i.e., K m > 10 5 .
  • Ligands can act on the receptor directly as agonists or antagonists, or can act indirectly, by affecting the response of the receptor to odier agonists and/or antagonists. In eidier case, the response of the receptor results in a change in the cell's condition (e.g. a change in die transmembrane voltage potential or intracellular calcium concentration) which can be detected, for example, by a voltage-recording electrode and/or a cell condition reporter (see above).
  • a voltage-recording electrode and/or a cell condition reporter see above.
  • a "cell biosensor detection field” is an area of a cell or cells on a substrate from which, at any given time, the state of a cell condition reporter is being recorded, and which, at d e same time, is at least partially exposed to effluent from a capillary of a capillary-based separation means.
  • a cell biosensor detection field is die portion of the image in the microscope mat is directed to d e photomultiplier tube for signal quantification.
  • a cell biosensor detection field may contain only a portion of a cell (such as a large Aplysia neuron), or many cells, such as near- confluent PC12 cells).
  • the present invention provides methods for detecting minute amounts of chemical analytes separated by capillary-based separation means.
  • An important feature of the present invention is that die analyte or analytes to be detected can act as ligands, that is, that d ey can bind to a specific receptor or receptors.
  • the methods employ cell biosensors expressing specific receptors to serve as ligand analyte detectors.
  • Ligands which bind to a specific receptor result in changes in die cell condition of the cell biosensor, and this change is detected with, for example, a voltage-recording electrode or cell condition reporter, such as a calcium sensitive dye.
  • Methods of die present invention are particularly advantageous for the detection of minute amounts of ligand analytes suspended in very small volumes (typically picoliter to nanoliter volumes). Accordingly, mediods of the present invention are preferably used widi separation means iat are suitable for separating ligand analytes in such small volumes, such as capillary-based or microcolumn separation means.
  • capillary-base separation means exist which are suitable for use with the methods of d e present invention, and are detailed below. These methods share the characteristic that they are performed in capillaries, typically fused silica capillaries, having an internal diameter (I.D.) on the order of tens of microns, and an outer diameter (O.D.) on die order of hundreds of microns.
  • I.D. internal diameter
  • O.D. outer diameter
  • Capillaries are ideally-suited for delivering separated analytes or ligands to cell biosensors, because die inner diameter of a capillary may be selected such diat it is similar to die diameter of, for example, a single cell being used as a cell biosensor. Alternatively, the inner diameter of a capillary may be selected such diat it is similar to die diameter of a selected group of cells, which together comprise the cell biosensor.
  • the rigid nature and relatively compact outer diameter of capillaries facilitates the placement of die oudet, or cell end of die capillary (e.g. with an "XYZ" positioner, such as a three-axis micromanipulator), at a selected distance from the cell biosensor to achieve the desired degree of dispersion of separated analytes prior to dieir contact with die cell biosensor.
  • die cell end of die capillary may be placed in close proximity to the biosensor (e.g. 20-30 ⁇ m).
  • the capillary may be placed further from the biosensor (e.g. 30-60 ⁇ m), to allow the separated ligands to disperse over an area largely covering the biosensor.
  • the spread of separated ligands may be easily monitored, for example, by the inclusion of a suitable dye in the electrophoresis buffer.
  • XYZ positioners suitable for positioning the outlet end of capillaries of capillary-based separation means of die present invention include a variety of manipulators available from, for example, Newport Corp., (Irvine, CA) and Narashige, USA, Inc. (Greenvale, NY).
  • cell biosensors may be attached directly at die oudet end of a capillary used in a capillary-based separation means, as is described in detail under "Cell Biosensors", below.
  • these various separation means preferably employ a cell biosensor compatible buffer in the course of die separation.
  • Biosensor compatible separation buffers are those buffers that are both effective to serve as separation buffers in selected capillary-based separation means, and to maintain die cell biosensor in a viable state (i.e. d at do not substantially interfere with die ability (i) of the ligand to bind to a selected receptor, (ii) of die bound receptor to cause a change in die cell condition of d e biosensor, and (iii) to detect die change in the cell condition with an electrode or a cell condition reporter).
  • die biosensor-compatible buffer will typically be a physiologically-compatible buffer, such as an extracellular Ringer solution.
  • a physiologically-compatible buffer such as an extracellular Ringer solution.
  • Such solutions are well known in the art, and may be modified for the maintenance of specific cell types or the conduct of specific experimental manipulations.
  • the composition of an exemplary Ringer solution (capillary electrophoresis Ringer; CE Ringer) suitable for use with capillary electrophoresis-based separation methods is presented in die Materials and Mediods section, below.
  • the separation buffers should be effective when employed for separations in die various capillary-based separation means. Such considerations are addressed individually widi regard to exemplary capillary- based separation means, below.
  • Capillary electrophoresis is a technique for fractionating chemical species on the basis of differences in die ratios of electrical charge to frictional drag in solution, and is able to separate complex chemical mixtures with high resolution in a few minutes.
  • a number of review articles and books provide .an introduction to principles and applications of capillary electrophoresis (Monnig, et al., Grossman and Colburn, 1992, Christensen, et al , Chen, et al, 1988, 1989).
  • a capillary electrophoresis system consists of a narrow-bore fused silica capillary (inside diameters range form about 20 ⁇ m to about 200 ⁇ m, and lengths from about 10 cm to about 100 cm in length) filled widi an electrolyte solution.
  • the ends of the capillary are placed in electrolyte-solution reservoirs that contains either a cadiode or an anode connected to a high voltage source, typically 20 to 30 kV.
  • Analytes are introduced to die inlet of the capillary, and are detected widi a detector near die outlet end.
  • the electrolyte present in the capillary prior to sample introduction is typically die same or similar to that containing the sample. Separation times are typically in the range of about 1 minute to about 30 minutes.
  • the simple embodiment described above can be elaborated upon widi features such as autosamplers, multiple injection devices, sample/capillary temperature control, programmable power supply, multiple detectors, fraction collection and computer interfacing, among others.
  • the analytes are typically introduced into die capillary by transferring the inlet (usually from the anode reservoir) to a solution of electrolyte containing the sample for a brief period before the separation field is applied, and applying eidier an electric field (to induce electroosmotic flow), or a pressure differential, such as positive pressure at the inlet, vacuum at the outlet or gravity, across die capillary to inject the sample.
  • sample volume can be reduced to occupy less than about 1 %, preferably less than about 0.1 % of the capillary volume, provided d at die sample contains enough molecules to detect.
  • sample volumes should be small in comparison to the total volume of the capillary, typically in the picoliter to nanoliter range (Gordon, et al). For example, a 100 cm capillary with an inside diameter of 75 ⁇ has a volume of about 5 ⁇ l.
  • An preferred sample volume for this capillary may range from about 5 nl to about 50 nl.
  • joule heating in capillary electrophoresis is considerably less d an in slab-gel electrophoresis because (i) the high resistance of the narrow capillary channel results in a low current draw (usually 1 to 100 ⁇ A), and (ii) the high surface area-to-volume ratio of the capillary facilitates heat transfer.
  • the walls of fused silica capillaries have a negative charge in aqueous solution due to ionization of surface silanol groups.
  • a sheath of mobile counter ions from the electrolyte solution in the capillary accumulates along the negatively-charged capillary surface, and induces electroosmosis (bulk solution flow) when an electric field is applied across d e electrolyte-filled capillary. Under typical operating conditions, this sheath of ions is positively charged, and consequendy, drags bulk solution from die anode to die cathode.
  • die inside surface of capillaries may be treated or coated such that the fixed charges are positive and accumulated mobile counterions are negative, thus inducing electroosmosis from die cadiode to die anode.
  • a practical result of electroosmotic flow is that in free-solution capillary electrophoresis, all species — whether possessing positive, neutral, or negative charge -- can be made to migrate in die same direction past a single detector during a separation.
  • "typical" CE where electroosmotic flow is towards die cadiode, a positively- charged sample component emerges early because both die electrophoretic motion of the ion and electroosmotic motion of die electrolyte -are in the same direction. If die component is negatively-charged, but its electrophoretic mobility is less than the electroosmotic mobility, dian that component also migrated toward the cadiode, but at a slower rate.
  • Modifications to capillary electrophoresis facilitating the separation of diese and odier recalcitrant molecules have included die inclusion in die capillary electrolyte of various additives, such as partitioning agents (Terabe, et al., 1984, 1985, Otsuka, et al., Sepaniak, et al., Burton, etal., Balchunas, etal., 1987, 1988, Cohen, A.S., etal, 1987b), macromolecules (Chin, Demorest, et al.
  • capillary electrophoresis Li
  • Different modes of capillary electrophoresis include (i) capillary zone electrophoresis (CZE; Jorgenson, 1984, Altria, et al.
  • capillary gel electrophoresis CGE; Hjert ⁇ n, et al., 1985a, 1985c, 1987, Cohen, et al, 1987a, 1987c), micellar electrokinetic capillary electrochromatography (MECC; Terabe, et al., 1989, Tsuda, et al., 1982), (iv) capillary electrochromatography (CEC; Knox, Pretorius, Jorgenson, et al., 1985), (v) capillary isoelectric focusing (CIEF; Hjert ⁇ n, et al., 1985b, 1987, Mazzeo, et al.), (vi) capillary isotachophoresis (CITP; Everaerts, et al., Bocek, et al), and (vii) affinity capillary electrophoresis (ACE; Chu, et al., 1992, 1993, Avila, et al., Gom
  • capillary electrophoresis all of die above modes fall under die generic definition of capillary electrophoresis. Considerations for selecting capillary electrophoresis parameters for die separation of specific types of molecules, such as DNA oligonucleotide (Dubrow), peptides (Colburn), proteins (Wiktorowicz, et al.) .and small molecules (Demarest, et al.) have been well documented.
  • the electric field used for separations with capillary electrophoresis is typically maintained at a relatively constant level.
  • certain capillary electrophoresis embodiments may be better served by using pulsed electric fields; specifically, applications which entail electrical (as opposed to optical) measurements of transmembrane voltage. Applications of pulsed electric fields in capillary electrophoresis have been described (Cohen, A.S., et ⁇ /., 1992).
  • Detection in capillary electrophoresis is typically performed on-line — that is, widiin die separation capillary or just adjacent die outlet, while analytes migrate under the influence of die applied electric field.
  • a plot of die measured detection signal versus separation time produces an "electropherogram", which represents analytes as peaks centered at characteristic migration times.
  • the height (or area) of a peak is preferably related to die concentration of the analyte that produced d e peak.
  • UV ultraviolet
  • detection means suitable for use with capillary electrophoresis including optical detectors based on ultraviolet (UV) absorbance (Lauer, et al, Terabe, et al., 1984, 1985, Otsuka, et al., Walbroehl, et al., 1984, 1986, Fujisawa, et al.
  • UV absorbance Lauer, et al, Terabe, et al., 1984, 1985, Otsuka, et al., Walbroehl, et al., 1984, 1986, Fujisawa, et al.
  • CD circular dichroism
  • any of die above detection means may be used in combination wid die cell biosensor detection means to enhance d e identification of a selected analyte.
  • Such "supplemental" detectors, or supplemental detection means are preferably those which can detect characteristics of the sample mixture near the outlet end of die capillary widiout disturbing die mixture. Examples include in-line optical detectors, including UV, fluorescence, CD and refractive index detectors, conductivity detectors and electrochemical detectors.
  • the capillary may include input and output windows near the output end for use widi a supplemental optical detection means, as described, for example, by Johnson, et al.
  • the cell biosensor, or the supplemental detection means are adversely affected by electrical noise from the electrophoresis power supply, it may be advantageous to operate die electrophoresis power supply in pulse mode — that is, turn it on for a selerted period of time (e.g. for 1 second), and dien turn it off for a similar period of time.
  • the noise- sensitive cell biosensor signal may be collected while the power supply is in the off position ("silent"), thereby avoiding electrophoresis-induced electrical noise.
  • capillary-based separation means contemplated for use with mediods of the present invention include variations of micro liquid chromatography (micro LC; Berry, et al. , 1989), in particular, open tube liquid chromatography (OTLC).
  • micro liquid chromatography micro LC; Berry, et al. , 1989
  • OTLC open tube liquid chromatography
  • OTLC refers to the mediod by which a gradient of two solutions is generated for use in the column separation.
  • Conventional methods for generating such a gradient typically employ one of two approaches, which can be characterized as “high pressure” and “low pressure” gradient generation (Berry, et al., 1990). Both of diese are generally useful for columns having internal diameters greater diat about 1 mm, but are not particularly effective for smaller columns.
  • a method and device described by Berry, et al. , 1990 allows die generation of gradients for use in columns having internal diameters as small as 5 ⁇ m.
  • OTLC is effective at separating mixtures having volumes comparable to those used widi capillary electrophoresis (picoliter to nanoliter range), and may be used as a capillary-based separation means in methods of die present invention.
  • Capillary electrophoresis and OTLC differ in that OTLC typically does not employ an electric field across the capillary. Rather, die sample is pushed through by hydrostatic pressure, typically provided by pressurizing die sample mixture at some point before it enters the inlet end of die column or capillary.
  • intact cells and portions diereof comprise a previously unappreciated class of detectors suitable for use with capillary-based separation means, such as capillary electrophoresis.
  • a cell biosensor suitable for use with capillary-based separation means of die present invention can be an intact cell, a portion of an intact cell (such as a membrane patch or permeabilized cell), or a group of intact cells. Further, the biosensor may be in electrical communication with an electrode or electrodes, or may contain a cell condition reporter, such as a calcium-sensitive dye.
  • Cells comprising die biosensor may be single eukaryotic or prokaryotic cells, groups of such cells, or a confluent layer of such cells.
  • PC12 cells comprise exemplary cell biosensors, as demonstrated in Examples 1, 3, 4 and 5 herein.
  • the cells may be transformed to recombinantly express a selerted receptor.
  • a population of transformed or untransformed cells may be selected using, for example, fluorescence-activated cell sorting (FACS), for those cells expressing high levels of a desired receptor or low levels of an undesired receptor.
  • FACS fluorescence-activated cell sorting
  • clones expressing the highest levels of a desired receptor or clones expressing the lowest levels of an undesired receptor
  • growing diem up, and repeating die process a selected number of times a population of cell can be obtained diat express a desired receptor at very high or virtually nonexistent levels (depending upon d e selection criteria employed).
  • Fluorescence activated cell sorters are available, for example, from Becton Dickinson (Mountain View, CA).
  • the cell biosensor is preferably placed adjacent die oudet of a capillary-based separation means. This may be accomplished in a number of ways.
  • the cell biosensor may be a cell attached to or growing on die bottom of a saline- or medium-filled recording chamber, and the outlet end of d e capillary may be brought such mat it is adjacent die cell (i.e. - 5 ⁇ m to - 100 ⁇ m above the cell).
  • Such an arrangement is demonstrated in Examples 1 and 2-5 for use widi an optically-detectable cell condition reporter.
  • the cell biosensor is a cell, such as a Xenopus oocyte or Aplysia neuron, placed in a recording chamber widi die outlet end of the capillary adjacent die cell as above (e.g. Example 2).
  • the cell may contain a cell condition reporter, such as a calcium-sensitive dye, or an electrode, including an intracellular voltage-recording or voltage/patch clamp electrode.
  • the cell biosensor may be a cell, attached not to the substrate, but to the end of a whole-cell electrode, that is positioned (e.g., widi a micromanipulator holding die electrode) at the oudet end of die capillary.
  • Isolated patches of d e type formed during patch clamp recording are also suitable for use as biosensors. Patch electrodes having isolated membrane patches may be positioned, for example, using a micromanipulator, adjacent die capillary outiet as described above.
  • the cell biosensor may be attached at die oudet end of die capillary of a capillary-based separation means either directly or dirough a substrate.
  • the outlet end of die capillary may be touched to a cell in a culture dish, causing die cell to detach from die culture dish and attach to the outlet tip of the capillary.
  • the attached cell is suitable for use as a biosensor if part of the capillary eluent comes in contact with die cell.
  • cells are cultured directly in the outlet tip of the capillary.
  • the portion of the capillary in which cells are to be plated may be coated prior to die plating of die cells widi a substance that supports the attachment and/or growth of cells (e.g., poly-lysine).
  • a cell biosensor may also be attached to die outlet end of a capillary used in a capillary-based separation means via a solid support on which die cell is growing.
  • one embodiment of d e present invention provides a removable solid support on which cells can be grown, and which can be attached at d e outlet end of a capillary used in a capillary-based separation means.
  • the support may consist of, for example, a short glass or plastic tube having an inner diameter diat is slightly larger than the outer diameter of the capillary at die oudet end.
  • Cells can be grown inside die tube using standard tissue culture techniques.
  • the tube may be slipped part way over die outlet end of die capillary, dius providing a biosensor for identifying a selected ligand in a mixture of ligands separated using a capillary-based separation means.
  • the portion of the tube extending past the outlet end of the capillary may include an angle (e.g., a right angle) to facilitate contact of die stream of eluent with cells located in die tube at the outer portion of the angle.
  • die outer diameter of die capillary at the outlet may be reduced, for example, by exposure to hydrofluoric acid, to accomodate a cell culture tube of a smaller inner diameter dian the normal outer diameter of the capillary.
  • a removable solid support is a substantially planar support that includes an attachment means to attach the support at die outlet end of the capillary.
  • the attachment means may be a ring, having an inner diameter as above, that can be slipped over die outlet end of the capillary.
  • the planar support may be positioned at a selected distance from the capillary oudet such that the surface of the support containing the cells is substantially perpendicular to the stream of eluent from the capillary oudet.
  • the support surface containing die cells may be placed at an angle odier dian perpendicular to die eluent stream, such that die stream is only deflected as it passes over die cells.
  • d e cell biosensor is attached to die capillary
  • an automated device can contain a supply of tubes containing cells, as described above, and a mechanical element, such as a robotic arm, can place fresh tubes over the outlet end of die capillary as desired.
  • the outlet end of a capillary can be fitted widi a rotatable motorized shaft oriented substantially parallel to the capillary and approximately coextensive with die outlet tip of the capillary.
  • a variation of die planar substrate widi attachment means, described above, can be fashioned to comprise, for example, a circular planar substrate containing cells oriented about a central axis.
  • Such as assembly can be attached to die motorized shaft such that die substrate is perpendicular to die capillary, and diat eluent from die capillary flows onto die cells in a selerted region of die substrate.
  • die motorized shaft By rotating die motor, a different portion of the substrate, containing fresh cells, is exposed to die capillary eluent.
  • the rotation may be set up to be interrupted, allowing recording from a selected region for a selected period of time, or continuous.
  • diat provides a means of moving the capillary outlet relative to a substrate containing cell biosensors, and in which it is desirable to obtain quantitative information on the amount of ligand in die separated capillary effluent, confluent or near-confluent layers of cells, due to their relative uniformity, may constitute particularly effective biosensors .
  • receptors typically desensitize following repeated applications of agonist. This desensitization increases die difficulty of obtaining quantitative information from cell biosensors, since quantitating responses typically requires generating a standard curve of responses to a range of agonist concentrations.
  • each cell biosensor detection field should have approximately the same detectable cellular response to a given amount of ligand.
  • a confluent or near-confluent layer of cells is advantageous in this regard, because die cell biosensor constitutes the cells contained in an area that is exposed to separated ligands and that is widiin die cell biosensor detection field of a detection device.
  • any scanning technique in which die outlet end of die capillary is moved wid respect to the cell biosensor, or vice versa may be automated.
  • an approach similar to that discussed above for cell biosensors attached to the capillary may be employed widi cell biosensors present in a recording chamber.
  • the "XY" controls of the stage holding die recording chamber can be interfaced widi precision motor drives (available, for example, from Newport Instruments (Irvine, CA), which can be directed to move die chamber to selected positions widi respect to die capillary oudet (it is understood, of course, that the motor drives can be incorporated to control die movement of die capillary as well).
  • die movement can be rapid, interrupted movement, or smoodi, continuous movement.
  • the continuous scanning technique affords a significant advantage regarding receptor desensitization. That is, die cell biosensor detection field can be moved at a rate diat is faster man d e predominant time constant of receptor desensitization. In this way, the detectable cell biosensor receptors do not desensitize, since cells which had been exposed to a desensitization-inducing ligand long enough to experience significant desensitization are no longer contributing to the biosensor signal.
  • receptors recover from desensitized states and return to the states they were in prior to application of ligand, they may again be used as cell biosensors.
  • the recording dish may be perfused, either continuously or periodically, widi fresh recording solution, for example, in cases when the volume of die recording solution is such that a concentration of ligand high enough to interfere with cell biosensor responses to capillary effluent may build up over die lifetime of the separation process.
  • iat solid supports and substrates referred to above may be coated with any of a variety of agents that promote the adhesion and/or growth of cells plated on die substrate.
  • agents include coatings diat alter surface charge, such as poly-lysine and poly- ornithine, various matrixes designed to support cell growdi, and selected extracellular matrix components, including collagen, fibronectin and gelatin.
  • the binding of a selected ligand to a receptor in or on a cell biosensor may be deterted by any of several mediods diat report die condition of die cell biosensor.
  • Two exemplary mediods of detecting die condition of a cell are (i) substances, or cell condition reporters, diat alter their characteristics, preferably optical characteristics, in response to selected specific changes in a cell's condition (such as increase in intracellular calcium), and (ii) electrodes capable of detecting changes in die transmembrane potential of the cell.
  • cell condition reporters are available. They can be grouped according to the type of cellular event to which they are sensitive, or according to die mediod by which they are deterted. According to mediods of die present invention, preferred mediods for detecting die state of cell condition reporters are optical. Cell condition reporters whose state may be detected by optical mediods may be referred to herein as optically-detectable cell condition reporters.
  • Optical detection may comprise assaying changes in absorbance or detection of luminescent or fluorescent signals. Fluorescence tends to be more sensitive than absorbance, because fluorescence is deterted as an increment above a low background rather than a small decrement from the full intensity of the incident beam (Tsien, 1992). Fluorescence is also advantageous over luminescence in cases where die amount of probe is limited, because each fluorophore can emit many thousands of photons (energized by die excitation beam) as opposed to die maximum of one photon emitted by a luminescent molecule. Luminescence may be advantageous, however, in cases where background fluorescence is high.
  • optically-detectable cell-condition reporters or dyes
  • ratio imaging the optical characteristics of the dye are measured at two different wavelengdis.
  • the wavelengths are selected such diat die optical property being measured (e.g. fluorescence) is essentially independent of die cellular condition to which die dye is sensitive (e.g. calcium concentration) at die first wavelength, but is maximally-sensitive to that condition at die second wavelengdi.
  • die amplitude of d e optical signal at die second wavelengdi is divided by die amplitude of the signal at the first wavelengdi.
  • This ratiometric measurement normalizes for extraneous factors such as the optical path lengdi through die cells, the fraction of cellular volume accessible to the dye, and the fraction of the detection field occupied by active cell biosensors, and provides a more reliable estimate of die cellular condition.
  • Optically-detectable cell condition reporters have been developed for detecting a variety of conditions, including transmembrane voltage, and levels of intracellular calcium, chloride, magnesium, sodium, pH, cAMP and activated calmodulin (Slavik, Tsien, 1989a).
  • Dyes sensitive to calcium are effective as cell condition reporters for selected ligands which, by binding to a specific receptor, change (typically increase) die calcium concentration in a cell.
  • receptors effective to alter intracellular calcium are presented below. For instance, experiments detailed herein demonstrate die elevation of intracellular calcium, as deterted by die dye fluo-3, in mammalian cells expressing bradykinin or acetylcholine receptors (Examples 1, 3,4,5).
  • pH-sensitive indicators have been syndiesized (reviewed in Tanasugarn, et al., and Tsien, 1989b). Modifications of an existing dye resulted in "BCECF” (Rink, et al.), an exemplary pH indicator which is amenable to ratio imaging.
  • the dye is sensitive to pH in the range of pH ⁇ 5.6 to pH — 8.0 at an excitation wavelength of 500 nm, but is relatively insensitive to pH at 450 nm.
  • SBFI die fluorescent indicator molecule
  • Detector dyes are also available for chloride and magnesium (Tsien, 1989b).
  • Electrode-sensitive dyes are currently available (Cohen, Ross, et al., Gupta, et al.). The most effective of these were identified by synthesizing and testing more than a thousand organic dyes.
  • An exemplary voltage-sensitive dye is di-4-ANEPPS (Loew, et al.). This dye displays an increase in fluorescence combined with a redshift of the excitation spectrum upon hyperpolarization. The signal-to-noise of the potentiometric fluorescence response is among the best recorded for my dye. Further, the dye is amenable to ratio imaging, shows no appreciable signal deterioration over extended recording periods and has an excellent signal-to-noise ratio.
  • Voltage-sensitive dyes such as di-4-ANEPPS, may be used as cell condition reporters in cases where die binding of a selerted ligand to a receptor results in a change in the transmembrane voltage of a cell or patch. Examples of receptors having this effect are discussed below. In particular, experiments performed in support of the present invention demonstrate that die ligand serotonin is effective to produce an inward current in an oocyte expressing a serotonin receptor (Example 2). 5. Second Messenger Dyes
  • molecular probes have been developed diat are sensitive to the concentration of organic "second messenger” molecules, such as cyclic adenosine monophosphate (cAMP) and calmodulin.
  • cAMP cyclic adenosine monophosphate
  • the indicator for cAMP is based on a cAMP-dependent protein kinase (Taylor, S.S., et al, 1990).
  • the catalytic (C) and regulatory (R) subunits are each labelled with a different fluorescent dye, such as rhodamine and fluorescein, capable of fluorescence resonance energy transfer in the holoenzyme complex R_C 2 .
  • a different fluorescent dye such as rhodamine and fluorescein
  • diis indicator is a cell condition reporter sensitive to ligands which alter die level of cAMP in die cell. Hahn, et al , (1990) have linked a tetramediinemerocyanine fluorophore to the calcium- binding protein calmodulin.
  • the resulting conjugate, MeroCaM has a reversible calcium- induced 3.4-fold increase in excitation ratio (608/532 nm), apparently due to the binding of calcium to the high affinity sites on the calmodulin molecule.
  • MeroCaM has been used to image calmodulin activation in Swiss 3T3 fibroblasts in vivo (Hahn, et al, 1992).
  • Optical Detectors for use with Optically-Detectable Cell Condition Reporters
  • a variety of methods may be used to optically monitor signals, such as fluorescence signals, in cell biosensors.
  • An exemplary apparatus for recording optical signals from cell biosensors in response to effluent from a capillary-based separation means is illustrated in Figures IA and IB.
  • the device includes a capillary 2 for the separation of a mixture of ligands.
  • a mixture of ligands is introduced into die inlet end 4 of die capillary, for example, by placing die inlet end in a vial 6 containing the mixture.
  • the other end 8 (outlet or cell end) of the capillary is placed in proximity to a cell biosensor, such as a eukaryotic cell 10 on a glass coverslip 12 forming the bottom of a recording chamber 14.
  • the cell end 8 can be positioned at a selected location by means of an XYZ positioner 16.
  • An exemplary means of achieving positioning is shown in the diagram.
  • a portion of die capillary proximal to the cell end is direaded through a hollow needle (such as a syringe needle) 18 and is glued at the needle entrance and exit points to stabilize the position of die outlet.
  • the needle is fastened to die XYZ positioner.
  • the needle may be bent into a conformation to facilitate placement of the cell end of the capillary adjacent die biosensor.
  • a power supply is indicated at 20, and electrophoresis electrodes at 22 and 24.
  • Separated analytes 26 are delivered to the cell biosensor 10, which had been loaded widi a dye (see below) responsive to cellular changes anticipated as a result of exposure of die cell to a selected ligand analyte.
  • the response of the dye is detected by an optical detector via, for example, a microscope objective 28 placed under die coverslip.
  • the optical detector may be an inverted fluorescence microscope, such as a Zeiss AxioVert or IM35 (Carl Zeiss, Inc., Thornwood, NY) or Nikon 300 or Diaphot-TMD-EF (Nikon Inc., Melville, NY).
  • Techniques for optically monitoring the state of cell condition reporters and for analyzing resultant information are known in the art (e.g. Taylor, D.L., and Wang, Taylor, D.L., et al, 1986, 1992, Herman and Jacobson, Conn, 1990, deWeer and Salzberg, Lakowicz, Slavik, Duchen, Harootunian, 1988, 1991a, 1991b, Poenie).
  • a Fluorescence microscope a Nikon Diaphot-TMD-EF
  • Figure 1C shows a view of a cell end of a 25 ⁇ m I.D. capillary positioned above a single cell as imaged widi an objective such as shown at 28 in Figure IA.
  • the optical detector may simply be a light pipe (such as a fiber optic cable) in combination widi appropriate optical filters, directed to a photomultiplier tube. Excitation light for fluorescence imaging in such situations may be provided by an appropriately-filtered high-intensity light source, directed at die sample using, for example, a fiber optic cable.
  • the cell biosensor may be placed in a spectrophotometer, and die absorbance of die cell condition reporter monitored as eluent from a capillary-based separation means, containing separated ligands, is washed over die cell biosensor.
  • Example 1 Use of an exemplary optically-detectable cell condition reporter in conjunction widi die device described in relation to Figures IA and IB is demonstrated in Example 1.
  • Example 1 Detailed in me example is a separation of acetylcholine (ACh), bradykinin (BK) and adenosine triphosphate
  • An exemplary complex biological mixture is a lysate of PC 12 cells containing endogenous! y-produced acetylcholine.
  • An experiment performed according to mediods of the present invention utilizing PC12 lysate as a mixture of ligands is presented in Example 1.
  • PC12 cells having natively-produced ACh receptors (AChRs) are used as die biosensor for acetylcholine.
  • die present invention two advantages of die present invention demonstrated by die above experiments are (i) ability to identify a selerted ligand in a highly complex mixture of ligands, and (ii) die ability to identify a selected ligand diat is present in very low concentrations (e.g., in the femptomolar range).
  • die highest sensitivity is achieved for BK.
  • five capillary electrophoresis runs of BK are performed at each of diree concentrations, using an individual cell as die cell biosensor.
  • a different PC12 cell is selected as d e cell biosensor for each run to avoid cell biosensor desensitization, and care is taken to set die capillary outlet at the same distance (-40 ⁇ m) from the cell for each run.
  • 4/5 and 5/5 cells respectively, respond to BK after electrophoresis.
  • Higher sensitivities may be obtained by differentiating the cells (Bush, et al.) and/or selecting clones expressing high amounts of the BK receptor (e.g. using fluorescence-activated cell sorting as described above).
  • detection limits for different ligands may range from more than picomoles to a few molecules (Mori, et al.).
  • Factors diat affect the detection signal include die ligand-receptor dissociation constant, the number of receptors exposed to ligand, die amplification pad ways accessed after binding, die baseline variability in the quantity being measured, and frequently, die chemoreception history of the cell.
  • Cellular response mechanisms can be manipulated to improve sensitivity and reproducibility. Differentiation of PC 12 cells, for example, has been shown to strongly potentiate the calcium response to bradykinin (Bush, et al), and receptor mutagenesis has the potential to greatly reduce desensitization (Jackson).
  • Example 3 details another application of the above system, and further demonstrates the capacity to scan the outlet of die capillary-based separation means relative to the recording chamber to employ fresh biosensors.
  • the results, shown in Figures 6A, 6B, 6C and 6D demonstrate diat effects of receptor desensitization may be minimized by providing fresh biosensors for consecutive separations. Pulses of bradykinin are separated and applied to die cell biosensor. The responses are evicent as peaks, or "spikes". The results show that die scanning can occur by moving the capillary outlet while maintaining the dish containing the cell biosensors stationary (Figs. 6C and 6D) or by translating the substrate containing die biosensors with respect to a stationary capillary (Fig. 6B). The breaks in the records reflect the interruption due to scanning.
  • the results also provide information as to die time course of receptor desensitization (die rate at which peaks decrease in response to applications of approximately the same amount of ligand).
  • die rate at which peaks decrease in response to applications of approximately the same amount of ligand die rate at which peaks decrease in response to applications of approximately the same amount of ligand.
  • an automatic system may be designed diat continually scans a substrate containing near-confluent cell biosensors at a rate fast enough that effects of desensitization are not appreciable (see above).
  • a confluent or semi-confluent layer of cells may be used, as discussed above.
  • Example 4 illustrates selective blocking of cell biosensor responses to electrophoresed ligands by specific receptor .antagonists.
  • FIGs 7, 8A and 8B show selective inhibition of cell biosensor responses to bradykinin by bradykinin receptor antagonist HOE 140 (Fig. 7) present in die recording solution, and selective inhibition of cell biosensor responses to acetylcholine by acetylcholine receptor antagonists atropine and ⁇ -bungarotoxin present in the recording solution (Figs. 8 A and 8B).
  • the receptor-specific antagonists may be present in the recording solution, -as shown in Example 4, or they may be applied locally to die cell biosensor in the biosensor detection field.
  • a second capillary containing the antagonist can be positioned to apply antagonist eluent (e.g. by pressure ejection) onto die cell biosensor detection field during a separation run.
  • the concentration of the antagonist can be adjusted such diat it is effective to antagonize the receptors in the cell biosensor detertion field, but to fall off to levels which do not substantially interfere with receptors outside of the detection field.
  • concentration of the antagonist can be adjusted such diat it is effective to antagonize the receptors in the cell biosensor detertion field, but to fall off to levels which do not substantially interfere with receptors outside of the detection field.
  • die chamber may be perfused widi fresh solution as described. It will be understood d at more dian one source of antagonist (preferably different antagonists) may be directed at die biosensor detertion field. These sources may be independently controlled, for example, using pressure ejection, to apply different antagonist at selected times.
  • Example 5 shows mat mediods of ie present invention may be used to separate and resolve distinrt ligands, such as bradykinin and Lys-bradykinin, having similar chemical characteristics.
  • the data are shown in Figure 9. Lys-bradykinin and bradykinin are identified as two well-resolved peaks, even though diey differ by only one amino acid (American Peptide Company, Inc., Sunnyvale, CA; 1994-1995 catalog).
  • Methods of die present invention may also employ direct transmembrane voltage detection widi extracellular or intracellular microelertrodes.
  • the activation of many receptors, particularly those directly linked to ion channels, results in a change in the transmembrane voltage of die cell.
  • Such a change may be deterted, for example, with die use of voltage- sensitive dyes (as detailed above), or using voltage-recording electrodes.
  • Three general modes of voltage recording using electrodes are (i) extracellular recording, where an electrode or electrodes are placed in die vicinity of die cell, but do not penetrate die plasma membrane, (ii) intracellular recording, where the electrode is in electrical communication with the interior of die cell, and (iii) patch clamp recording.
  • Intracellular and patch recording are capable of measuring an intracellular potential widi respect to die badi, and detecting changes to diat value over time, while extracellular recording is typically useful only for detecting changes in the membrane potential.
  • intracellular and patch recording it is also possible to "voltage- clamp" the cell membrane to a desired voltage by injerting current, eidier dirough the voltage recording electrode or dirough a separate current-passing electrode.
  • d e recorded signal is typically die amount of current passed to maintain die cell at d e desired potential as a function of time.
  • Extracellular electrodes function by detecting a change in die electric field in die vicinity of an electrically active cell. They are not particularly effective at quantitating the amplitude of a transmembrane voltage change, but can qualitatively detect diat a voltage change has occurred.
  • Extracellular recording electrodes may be either built in to die substrate on which die cells are grown (Pine, Meister, et al.), or placed adjacent the cell biosensor using, for example, a micromanipulator. A variety of extracellular recording techniques have been described which may be employed in mediods of die present invention (e.g., Pine, Forda, et al, Gross, et al., Cohen, et al., Meister, et al.).
  • Intracellular recording typically utilizes a sharp glass microelertrode filled widi a high salt electrolyte solution, typically — 3M KC1.
  • a metal electrode typically silver with a coating of silver chloride, is placed in electrical contact with he electrolyte solution and connected to a voltage recorder, or voltage clamp. If a voltage clamp is used, the output is typically directed to a chart recorder or a computer for later analysis.
  • Intracellular recording and/or voltage clamp may be used, for example, widi Xenopus oocytes, as detailed in Example 2 herein.
  • Oocytes are advantageous because diey are capable of expressing injected mRNA encoding specific receptors.
  • Mediods for expressing ion channels and receptors in Xenopus oocytes, and voltage/current recording from oocytes are well known
  • Patch recording typic-ally conducted in die voltage-clamp mode, exploits the observation that under appropriate conditions, it is possible to obtain an electrically-tight seal between the tip of a blunt fire-polished pipet filled with physiological saline solution and the plasma membrane of a cell (Ha ill, et al; Sakmann and Neher).
  • Such seals introduce a resistance between the interior of d e pipet and die badi ranging from about 1 GOhm to over 100 GOhms.
  • This mode of recording is referred to as "cell-attached”.
  • die patch pipet If a brief pulse of suction is applied to die patch pipet, it is possible to rupture the membrane beneath d e pipet and establish electrical communication with die interior of die cell (whole-cell mode). It is also possible to isolate a patch of membrane by pulling die patch pipet off die cell . If the pipet is pulled off in cell-attached mode, the patch is usually an " inside-out" patch, because die cytoplasmic face of the membrane patch is exposed to die extracellular solution bathing the cell. If die pipet is pulled off in whole-cell mode, d e cell membrane typically reseals and forms a patch in an outside-out configuration (die extracellular aspect of the patch membrane is facing die bath solution).
  • Whole-cell mode is similar to intracellular recording or voltage clamp, but it allows control of die cell's intracellular solution.
  • the patch modes (cell-attached, inside-out and outside-out) enable die recording of currents through single ion channel molecules.
  • electrical communication between the pipet and die patch clamp itself is via an electrode immersed in the physiological electrolyte present in the patch pipet.
  • die electrode is typically a silver wire coated widi silver chloride.
  • FIG. 4 shows a schematic of an exemplary device useful for practicing die present invention.
  • This device includes a capillary 2 for the separation of a mixture of ligands. A mixture of ligands is introduced into the capillary inlet as above.
  • the cell end 4 of the capillary is placed in proximity to a cell biosensor, such as an oocyte 6.
  • the device further includes an elertrophoresis reservoir 8 around die capillary approximately 10 cm upstream from die cell end, which is sealed at its bottom end 10 to prevent leakage of electrophoresis buffer into the solution bathing the cell biosensor.
  • the reservoir contains an electrophoresis buffer, such as CE Ringer, in electrical communication with the electrophoresis ground 12.
  • the capillary has a fracture 14 enabling electrical communication between the interior of the capillary and die electrophoresis buffer in the reservoir.
  • the fracture serves to ground die capillary upstream from the biosensor to reduce electrical interference in voltage-clamp recording.
  • the portion of the capillary above the fracture is immobilized to a splint, for example, glued 16 to a side of die reservoir containing the elertrophoresis buffer.
  • the transmembrane voltage and/or current of die cell biosensor is measured using, for example, a two-electrode voltage clamp, which includes a current-passing electrode 18, a voltage-recording electrode 20 and a badi ground 22 in electrical communication with die solution 24 bathing the cell biosensor.
  • the cell end of die capillary can be positioned at a selected location widi respect to die cell biosensor by means of an XYZ positioner attached to, for example, the elertrophoresis reservoir 8, essentially as described above.
  • the fracture 14 in the capillary can be fashioned as described by Linhares and Kissinger.
  • the capillary is glued to a 2 cm x 1 cm microscope slide with epoxy glue (e.g. SIG Mfg. Co., Montezuma, IA) such that an exposed portion, immobilized at bod ends, rests approximately 1 mm above the surface of the slide.
  • epoxy glue e.g. SIG Mfg. Co., Montezuma, IA
  • a diamond capillary glass cutter is used to make a small scratch on the top of die exposed portion, and a pointed stylist is gently pushed between the slide and die capillary to produce a fracture.
  • the fracture assembly is placed, for example, in a 5 ml Teflon vial with a hole drilled in die bottom.
  • the outlet end of the capillary is pushed through a rubber septum that plugs the hole.
  • Several approaches may be taken to reduce electrical noise during recording. If it is desired to obtain an uninterrupted record of cell biosensor electrical activity, proper grounding of all components ne-ar the recording chamber is necessary.
  • the portion of the capillary between the fracture and die outlet (cell end) may be coated widi an electrically- conductive material and grounded. Mediods for electroplating glass or silica with electrically- conductive coatings may be found, for example, in Bard.
  • methods of die present invention may be practiced using a pulsed electrophoresis power supply (Cohen, M.L., et al., 1992).
  • the acquisition computer may be programmed to sample voltage or current records only during die period diat die electrophoresis power supply is off. This may decrease die electrical noise in die system by about an order of magnitude, and may allow die recording of ion currents through single ion channels.
  • Such a biosensor system can provide a nearly unprecedented level of sensitivity - for example, since an inotropic receptor typically requires only a few agonist molecules for opening, the method may be applicable for the detection of samples containing only minute amounts of a selerted ligand, perhaps as little as a few hundred molecules.
  • Example 2 details an application of die recording system described in Figure 4 to record responses of Xenopus oocytes to serotonin delivered using a capillary-based separation means.
  • the electropherograms are shown in Figure 5. Upward deflections indicate inward current. The current is likely due to die artivation of oocyte-endogenous calcium-artivated chloride channels, stimulated by a rise in intracellular free calcium due to die binding of serotonin to die recombinantly expressed serotonin receptor.
  • diat electrodes may be effectively employed widi cell biosensors to detect a selected ligand in a mixture of ligands separated using a capillary-based separation means.
  • the result also demonstrate mat the signal detected from a cell condition reproter or electrode may be due to a secondary response to ligand binding to a specific receptor.
  • a variety of ion channels are now known to respond to intracellular second messengers activated by die binding of an agonist to a receptor (Hille).
  • the receptor Upon binding of an appropriate agonist, the receptor alters its conformation and initiates a cellular response.
  • inotropic receptors such as die nicotinic acetylcholine receptor or the N-methyl D-aspartate ( ⁇ MDA) receptor
  • the receptor is itself an ion channel, and binding of die appropriate ligand opens die ion channel, typically on the order of milliseconds. Opening of the channel results in an increased membrane conductance for those ions which can pass through the receptor, which typically results in a change in the membrane potential.
  • the membrane potential change can be detected widi voltage-sensitive electrodes, or voltage-sensitive dyes (as described above). If the membrane is clamped to a selected voltage, the opening of die channel typically results in an ionic current flowing through the channel, which can be measured by die voltage clamp apparatus.
  • ion channels and inotropic receptors are at least partially permeable to calcium. Since the electrochemical driving force for calcium is typically inward at most physiological potentials, die opening of a channel permeable to calcium is accompanied by an increase in die intracellular calcium concentration in die vicinity of die ion channel. This increase may be deterted using, for example, a calcium-sensitive dye, such as fluo-3.
  • binding of die appropriate agonist to the receptor may cause a conformational change that typically initiates one of several well-characterized biochemical signaling cascades, many of which result from an interaction of the activated receptor widi a "G-protein" (Stryer, Teichberg and Habenicht, Kito).
  • G-protein G-protein
  • the specific cascade initiated depends, of course, on die receptor activated.
  • the intermediates in an activated cascade may involve calcium and cyclic nucleotides, such as cAMP and cGMP.
  • IP 3 inositol triphosphate
  • IP 3 pathway typically results in the release of calcium from intracellular stores, and leads to a transient increase in free intracellular calcium concentration (Hille).
  • activation of receptors which exert their effect through die IP 3 pathway is expected to result in an increase in intracellular calcium, which can be deterted using, for example, one of die calcium indicator dyes described above.
  • activation of receptors which exert their effects through cAMP may be deterted using, for example, the cAMP indicator system described above.
  • One of the advantages of die methods of die present invention is die sensitivity afforded by die amplification inherent in many metabotropic receptors.
  • the present methods may be used to detect minute amounts of a selected ligand. It will be understood tiiat because receptors were often named on die basis of what was considered to be die natural agonist, and before die molecular structure of die receptor was elucidated, certain receptor names may include a group of molecularly-distinct receptors, some of which may be inotropic, and some metabotropic.
  • acetylcholine receptors refers to both nicotinic acetylcholine receptors, which are inotropic, and muscarinic receptors, which are metabotropic.
  • transmembrane receptors may be on the extracellular portion (e.g. nicotinic acetylcholine receptors (nicotinic AChRs), intracellular portion (e.g. cAMP receptors) or either, depending on die receptor subtype.
  • extracellular portion e.g. nicotinic acetylcholine receptors (nicotinic AChRs)
  • intracellular portion e.g. cAMP receptors
  • the ATP binding site is on the extracellular side in die case of calcium-selective receptors opened by ATP, and on the intracellular side in die case of potassium-selective receptors closed by ATP (Barnard, et al., Bean, et al).
  • cell biosensors may be derived from cells which are known to express a specific receptor under known conditions.
  • PC12 cells are known to express a number of receptors and ion channels (e.g. Shafer, et al), including acetylcholine (both nicotinic (Greene, et al., 1976) and muscarinic (Jumblatt, et al.) receptors, ATP receptors (Fasolato, et al.) and bradykinin receptors (Fasolato, et al.).
  • acetylcholine both nicotinic (Greene, et al., 1976) and muscarinic (Jumblatt, et al.) receptors
  • ATP receptors Fasolato, et al.
  • bradykinin receptors Fesolato, et al.
  • the acetylcholine receptors on PC12 cells resemble neuronal ACh receptors more than muscle ACh receptors. Properties of neuronal ACh receptors are reviewed, for example, by Sargent.
  • inotropic receptors suitable for use with methods of the present invention include muscle and neuronal nicotinic acetylcholine receptors, ⁇ -aminobutyric acid A (GABA receptors, glycine receptors, glutamate receptors (including kainate-type, quisqualate-type and NMDA type), 5-HT 3 , ATP receptors, cGMP receptors and cAMP receptors.
  • GABA receptors glycine receptors
  • glutamate receptors including kainate-type, quisqualate-type and NMDA type
  • 5-HT 3 ATP receptors
  • cGMP receptors cGMP receptors
  • cAMP receptors cAMP receptors
  • the glutamate family of receptors has a particularly large number of variants, some of which are selectively sensitive to different ligands, such as NMDA (e.g. Nakanishi).
  • the family may be more appropriately called an "excitatory amino acid" receptor family, because it is possible that amino acids odier dian glutamate (such as aspartate) are the natural ligand for die receptor in some circumstances.
  • Inotropic receptors are typically comprised of 4-6 homologous subunits, typically termed , ⁇ , y, ⁇ , e and ⁇ * .
  • the pentameric muscle nicotinic acetylcholine receptor for example, is composed of 4 homologous subunits in the stoichiometry a b arranged around a central cation-permeable pore.
  • Molecular cloning has demonstrated diat cDNAs encoding most receptor subunits are each members of a family of highly homologous but distinct sequences arising from alternative splicing and/or different genes.
  • the ability to construct biosensors with specific properties is an advantage for the detertion a selected ligand in a mixture of ligands.
  • a cell biosensor may be construrted which is sensitive to a selerted ligand, and which generates a known biosensor response (such an increase in calcium coupled widi a calcium-sensitive dye) diat can be deterted.
  • a cell biosensor containing a calcium-permeable AMPA-kainate, NMDA or nicotinic acetylcholine receptor may be used in conjunction widi a calcium-sensitive dye, because these receptor types are permeable to calcium, and opening of die receptors results in an influx of calcium through die receptor channel (given that die electromotive force for calcium is inward).
  • a cell biosensor containing an inotropic GABA or glycine receptor may be used widi a chloride-sensitive cell condition reporter, such as a chloride- sensitive dye, since diose receptors are generally anion-selective channels.
  • diat inotropic receptors may be present in intracellular membranes, as well as the plasma membrane of a cell biosensor.
  • the IP 3 receptor is located in the endoplasmic reticulum (ER) and is responsible for the release of calcium from die ER in response to IP 3 (die elevation of IP 3 in the cell may, in turn, be a response to the activation of a plasma membrane receptor, such as a muscarinic acetylcholine receptor; Hille).
  • the ryanodine receptor also an intracellular calcium-release channel, was first recognized in the sarcoplasmic reticulum (SR) of muscle. It is partially-opened by ryanodine, and may be fully activated by calcium or caffeine (Hille).
  • intracellular membrane receptors may be used in conjunction widi a cell biosensor in dieir native state — that is, expressed in an intracellular membrane (e.g. ER) of the cell biosensor, or diey may be recombinantly expressed on d e plasma membrane of a host cell.
  • an intracellular membrane e.g. ER
  • Plasma membrane-associated receptors may be grouped into at least diree "superfamilies" of receptors (Hille):
  • A "classical" G-protein-coupled receptors have seven transmembrane segments, the ligand-binding site is at least partially associated with a transmembrane domain, and die site of G-protein interaction is cytoplasmic
  • B tyrosine kinase, or growth factor receptors have one transmembrane segment linking a large, glycosylated extracellular ligand-binding domain widi
  • a large intracellular portion diat usually includes a protein tyrosine kinase domain and a site for autophosphorylation
  • C guanylayl cyclase family diat is similar to the growth factor receptors, except that the intracellular portion has a guanylayl cyclase domain, rather man a tyrosine kinase domain
  • metabotropic receptors falling, for example, into each of the above three classes is known in the art. The following are a few specific examples of such receptors.
  • Epidermal growth factor (EGF) receptors are members of the tyrosine kinase family of receptors. Artivation of the receptor initiates a cascade of events (Carpenter, et al, Wells, et al.) that results in internalization of the receptor and a rise in cytosolic free calcium.
  • An atrial natriuretic peptide (APN) receptor is a member of the guanylate cyclase family of receptors (Chinkers, etal.). Artivation of this receptor by APN results in an increase in cGMP. Detertion of activation of this receptor on a cell biosensor may be accomplished, for example, by co-expressing a cGMP-sensitive ion channel in the cell biosensor.
  • the 5HTlc receptor used in experiments described herein is a member of the seven transmembrane segment G-protein-coupled receptor family. Artivation of this receptor expressed in Xenopus oocytes results in an increase in intracellular calcium, presumably through an IP 3 pathway. The calcium then presumably activates calcium-sensitive chloride channels normally present in the oocyte plasma membrane (Gunderson, et al.) and results in an inward current (Julius, et al.).
  • a receptor for substance P is also a member of the seven transmembrane domain G-protein-coupled receptor family (Hershey, et al.), as is a substance K receptor, as is the ET B receptor for endodielin (Sakurai, et al, Arai, et al.), a peptide initially identified in vascular endodielial cells.
  • Other examples of receptors in this family include an interleukin- 8 (IL-8) receptor (Murphy, et al., Holmes, et al.), and a human diromboxane A 2 receptor (Hirati, et al.). Activation of the receptors identified above results in an increase in intracellular free calcium, presumably through an IP 3 pathway.
  • IL-8 interleukin- 8
  • Hirati human diromboxane A 2 receptor
  • odorant receptors have recently been cloned (Buck, et al.). These receptors also belong to the seven tr.ansmembrane segment G-protein-coupled receptor family. Cell biosensors containing odorant receptors may be particularly advantageous for use widi mediods of die present invention.
  • the olfactory system can differentiate among diousands of different odorant molecules dirough a family of perhaps several hundred different G-protein- coupled receptors (Buck, et al.).
  • a number of different odorant receptors have already been cloned (e.g. Permentier, et al.).
  • the odorant molecules can be divided into several "odor classes", which include fruity, such as citralva and citraldymediylacetal; herbaceous, such as eugenol and ediylvanillin; minty, such as menthone and eucalyptol, floral, such as geraniol and hedione; and putrid, such as pyrrolidine and pyrazine.
  • fruity such as citralva and citraldymediylacetal
  • herbaceous such as eugenol and ediylvanillin
  • minty such as menthone and eucalyptol
  • floral such as geraniol and hedione
  • putrid such as pyrrolidine and pyrazine.
  • the molecules can also be grouped by chemical classes, which include aromates, such as coniferan and helional; aldehydes, such as lyral and lilial; amines, such as triethylamine and phenylethylamine; organic acids, such as isovaleric acid and butyric acid; sulphydryls, such as 2-mercaptoethanol and furfurylmercaptan; methoxypyrazines, such as 2-isobutyl-3-meti ⁇ oxypyrazine -and metiioxypyrazine; and alkylpyrazines, such as 2,3,5-trimethylpyrazine and 2-med ⁇ ylpyrazine.
  • aromates such as coniferan and helional
  • aldehydes such as lyral and lilial
  • amines such as triethylamine and phenylethylamine
  • organic acids such as isovaleric acid and butyric acid
  • receptors in cell biosensors diat are specific for a selected ligand may be normally or recombinantly expressed by the cell from which die cell biosensor is derived.
  • a large amount of information is available on the specific receptors expressed by selerted cell types. Examples have been provided above, in particular, the receptors expressed by PC 12 cells.
  • one of skill in die art may choose to recombinantly express any of a variety of receptors using methods known to those skilled in die art.
  • Several different methods have been used for recombinantly expressing specific receptors in host cells (Gould, Conn, 1991b). The methods typically employ standard cellular and molecular techniques (Sambrook, et al, Ausubel, et al), with a focus on protein expression techniques (Goeddel, Kriegler), and may include special considerations depending on die nature of the receptor being expressed.
  • Such considerations may include (i) targeting transmembrane receptors to express appropriately in the plasma membrane of the host cell, (ii) providing a system capable of appropriate post-translational modification, and (ii) co-expression of multiple subunits in the same cell for heteromultimeric receptors.
  • the oocytes contain - 50-100 ng of endogenous maternal cytoplasmic mRNA, but translation of this message is largely inhibited by die presence of various inhibitory proteins.
  • the in vitro translation machinery in the oocyte is capable of correct post-translational modification, proteolytic cleavage and plasma membrane insertion (Wang, et al.).
  • die mRNA(s) encoding die receptor is injected, for example, into a collagenased oocyte, and expression of die mRNA is assayed anywhere between 24 hours up to one week following injection.
  • a detailed description of die oocyte expression protocol is provided in Example 2, herein.
  • a number of references specifically review this procedure (e.g. Gurdon, et al., Leonard and Snutch, and Wang, et al.).
  • numerous groups have used the oocyte system for receptor expression (e.g.
  • Pritchett, et al. transiently co-expressed GABA A receptor ⁇ and ⁇ subunits in human embryonic kidney cells using the eukaryotic expression vector pCIS2 (Eaton, et al.).
  • the vector contained die a and ⁇ subunits in a tandem arrangement, driven by a human cytomegalovirus (HCMV) promoter-enhancer.
  • HCMV human cytomegalovirus
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Advantages of this system include (i) infection and expression of a wide variety of eukaryotic cells, (ii) the virus provides the enzymes necessary for transcription in the cytoplasm of the infected cell, eliminating die need for nuclear processing, (iii) the gene product can be correctly processed and targeted to die cell surface, and (iv) a high percentage of host cells express die recombinant DNA.
  • a similar system has also been used to express potassium channels (Leonard, et al.).
  • die binding of a selected ligand, present in a mixture of ligands, to a specific receptor may be selectively detected using a cell biosensor having die specific receptor and containing a cell condition reporter, provided mat the cell biosensor is used in conjunction with a means to separate the selected ligand from the mixture of ligands.
  • the response of the cell biosensor to ligand binding may be a change in die transmembrane voltage, intracellular free calcium concentration, or die like, which is in turn detected by a cell condition reporter.
  • a die activation or inhibition of inotropic receptors typically results in a change in the transmembrane voltage, and possibly intracellular calcium concentration.
  • Activation of a metabotropic receptor typically results in an increase in the concentration of specific second messengers. These may be deterted directly (for example, calcium or cAMP), or indirectly, for example, following binding of die second messengers to targets, such as other inotropic receptors (sensitive to intracellular agonists).
  • An example of an indirect detection scheme detailed herein is die response of oocytes to serotonin in Example 2. Activation of the expressed 5-HTlc receptor presumably mobilizes internal stores of calcium, which in turn open a calcium-sensitive chloride channel. The electrical response to die opening of die chloride channel is deterted by the cell biosensor in die example.
  • Selerted ligands separated from a mixture of ligands may be receptor agonists, antagonists, competitive inhibitors, or non-competitive inhibitors. They include endogenous ligands, such as acetylcholine, and exogenous ligands, such as toxins widi an affinity for a specific receptor (e.g. ⁇ -bungarotoxin.
  • the solution bathing the biosensor may contain additional receptor agonists, .antagonists, competitive inhibitors, non-competitive inhibitors and die like. These additional components may interact widi, enhance, or inhibit the binding of die selected ligand to a specific receptor, and accordingly, may be used to confirm die identity of a selected ligand or odierwise facilitate the interpretation of information available from the cell biosensor.
  • Ligands may bind to d e extracellular portion of a transmembrane receptor, membrane portion of a transmembrane receptor, or the intracellular portion of a transmembrane receptor. Ligands can also, of course, bind to any portion of an intracellular receptor. Ligands diat bind intracellularly need to access die intracellular portion of a cell biosensor. If the cell biosensor is a potion of an intact cell with ready access to intracellular domains (such as an inside-out patch or a permeabilized cell), access does not pose undue difficulty.
  • the cell biosensor is an intact cell or cells, and die ligand is not capable of diffusing across the plasma membrane, die ligand may need to be transported into the cell by an endogenous or exogenous transmembrane transporter molecule.
  • Ligands may be obtained from a variety of sources.
  • An exemplary source of a mixture of ligands is a cell or tissue extract. This source may be particularly advantageous when information is desired as to die identity of a putative endogenous ligand for a specific receptor.
  • the mixture may include substances to stabilize the state of ligands in the mixture, for example, protease inhibitors.
  • a mixture of ligands may be obtained from a combinatorial library, such as a peptide or chemical compound library (Houghten).
  • Combinatorial libraries are typically screened by introducing a complex mixture species to a screening preparation, and if a positive response is obtained, repeating die process with lower-complexity mixtures until the compound having die desired activity is identified.
  • the complexity of the mixture is reduce on-line, by separating the mixture using a capillary-based separation means prior to applying die components to a cell biosensor.
  • the individual species in d e library are deterted serially, as described above.
  • a mixture of receptor antagonists such as conotoxin venom (Olivera, et al.) may be separated and screened in combination widi a receptor agonist constitutively present in the vicinity of the cell biosensor.
  • die agonist may be applied using, for example, a second capillary whose effluent is also directed at die cell biosensor detection field.
  • mediods of die present invention have a variety of uses. Further, the methods provide a number of advantages over methods utilized in the prior art.
  • cell biosensors of die type described herein are not particularly useful at identifying a selected ligand in a mixture of ligands, unless they are coupled to an effective separation means. This is in large part because, as detailed above, a cell biosensor produces a similar signal (change in free intracellular calcium concentration and/or change in transmembrane voltage) in response to a wide variety of ligands. If a complex mixture of ligands is directly applied to a cell biosensor, interpretation of the response will be difficult, if not impossible.
  • such a biosensor is coupled to a means by which die complex mixture of ligand is separated, so diat die effect of individual ligands present in the mixture may be assayed in a serial fashion.
  • selerted ligands present even in a highly complex mixture containing many different ligands capable of generating a response of the cell biosensor may be identified, because the cell biosensor responses to the individual ligands are separated in time.
  • the individual responses may be identified by running standards, as shown in Figures 2 and 3, and/or by using specific receptor antagonists, as shown in Figures 7, 8A and 8B, and discussed above.
  • the present invention offers a number of other distinct advantages.
  • methods of the present invention may be particularly useful in identifying ligands which are difficult to detect by traditional separation means, such as acetylcholine (Cooper, et al).
  • die mediods may be useful for identifying die biological activity of closely-related compounds, such as bradykinin and lys-bradykinin (Example 4). This approach may be used, for example, to assess die biological activity of breakdown products of a selerted ligand, such a drug or therapeutic agent.
  • an inactive precursor for an active peptide may be present in a sample, such as a blood sample. The rate of generation and identity of active (cleaved) species may be followed using mediods of die present invention.
  • a non-invasive supplemental detector or detertion means is positioned to detect the separated ligands before diey are delivered to die cell biosensor, die rate of formation/degradation and biological identity of die precursor and various proteolysis products may be readily followed.
  • the invention may be similarly be applied to the study of other biologically-active compounds widi limited lifetimes. Because both a traditional identification (with a supplemental detector) and the biological activity (with a cell biosensor) can be assessed within seconds of one another, a correlation can be established between selected "peaks" detected with a traditional, supplemental detector, and die biological activity of those peaks as determined by a specific cell biosensor.
  • Mediods of die present invention may also be used to screen for uncharacterized endogenous agonists of known receptor, such as an opiate receptor.
  • a tissue extract of a portion of the brain thought to contain d e endogenous agonist may prepared and analyzed according to methods of die present invention using, for example, a cell expressing an opiate receptor along with an inwardly-rectifying potassium channel (Reuveny, et al. , Dascal, et al.).
  • a cell expressing an opiate receptor along with an inwardly-rectifying potassium channel (Reuveny, et al. , Dascal, et al.).
  • an inwardly-rectifying potassium channel Reuveny, et al. , Dascal, et al.
  • Another exempl-ary use of methods of die present invention is for assaying the identity, concentration and release sites of neurotransmitters and hormones from individual cells (e.g. Aplysia neurons).
  • large cells are dissected and plated in an assay dish which is placed at die inlet of a capillary-based separation means.
  • the large cell is stimulated, for example, using electrical stimulation or agonists, such as peptides, and die inlet end of die capillary (etched down to a small diameter widi, for example, hydrofluoric acid) is positioned at selected locations along die cell.
  • a separation is performed, and the separated mixture is detected widi a cell biosensor expressing appropriate receptors (i.e. receptors for substances thought to be released by die large cell).
  • the above method may also be applied to assaying die identity, concentration and release sites of ligands, such as neurotransmitters from tissues, such as hippocampal slices (Nicoll, et al).
  • ligands such as neurotransmitters from tissues, such as hippocampal slices (Nicoll, et al).
  • some vesicles contain more that one neurotransmitter.
  • a capillary-based separation means to resolve individual components at may be released by stimulated cells
  • a cell biosensor expressing receptors for anticipated components released by stimulated cells
  • a related application is die determination of the content of single cells or cell organelles.
  • individual cells and to some extent, cellular organelles, comprise complex mixtures of ligands. Identification of selected ligands in those mixtures may be desirable for die development of certain therapeutic and/or diagnostic molecular tools.
  • individual cells or organelles such as vesicles, are introduced to die inlet end on a capillary-based separation means. The contents and biological activity of the cells or organelles may then be identified using a ligand separation means coupled to a cell biosensor, or to a cell biosensor in combination with a supplemental detertion means.
  • Ligands were separated by capillary electrophoresis (CE) using standard techniques (Grossman and Colburn, Gordon, et al.) with die following parameters/modifications.
  • the experiments were typically performed using a biosensor-compatible buffer (see below) as die electrophoresis buffer.
  • Capillary lengtiis were typically between about 23 and 36 cm.
  • the power supply was typically set to provide a field of 230 to 870 V/cm, resulting in electroosmosis (bulk solution flow) delivering sep-arated positive, neutral, and negative species to the cell biosensor.
  • a cell biosensor-compatible buffer is a buffer effective to maintain the cell biosensor in a stable and functional condition for die duration of an experiment.
  • a cell biosensor-compatible buffer is analogous to a physiologically-compatible buffer - that is, a buffer that does not substantially alter the signal coming from, or disrupt die intact cell.
  • a cell biosensor-compatible buffer is one that does not compromise the ability of the permeabilized cell to generate a detectable response to a selected ligand.
  • the buffer does not compromise die ability of me patch to generate the desired signal.
  • the buffer may be similar to one used for an intact cell, such as an "extracellular" Ringer solution.
  • die buffer may be similar to an "intracellular" Ringer solution, such as one containing low calcium and high potassium.
  • composition of the Ringer used in the present experiments was as follows: 135 mM NaCl, 5 mM KC1, 10 mM glucose, 2 mM MgCl 2 , 2 mM CaCl 2 , and 10 mM HEPES (pH 7.35)
  • compositions can be made widiout appreciably affecting die separation and/or physiological characteristics of die solution.
  • components other than the selerted ligand may be present in a biosensor compatible buffer, which affect the response of die biosensor to the selerted ligand.
  • Such components may include substances, such as certain organic compounds, which facilitate die separation of a mixture of ligands using capillary-based separation means.
  • Eukaryotic Cells 1. Growth and Maintenance. Rat clonal pheochromocvtoma (PC 12) were obtained from die laboratory E. Shooter, Stanford University. NG 108 cells were obtained from the laboratory L. Stryer, Stanford University. Both cell types can be obtained from the American Type Culture Collection ATCC (Rockville, MD).
  • the cells were plated on an untreated #1 glass coverslip (Fisher Scientific, Pittsburgh, PA) constituting a portion of die bottom surface of a 35 mm tissue culture dish (Fisher) in DMEM medium (Gibco/BRL Life Technologies, Gaithersburg, MD) supplemented widi 5% heat-inactivated horse serum (HyClone Labs, Logan, UT), 10% fetal calf serum (FCS; Gibco/BRL), ⁇ % 10 mg/ml glutamine (Sigma) and 1 % Penicillin/streptomycin solution containing 10,000 U/ml penicillin and 10 mg/ml streptomycin (Sigma), and maintained in a 5% C0 2 atmosphere until use (2-4 days).
  • DMEM medium Gibco/BRL Life Technologies, Gaithersburg, MD
  • FCS 10% fetal calf serum
  • FCS fetal calf serum
  • Penicillin/streptomycin solution containing 10,000 U/ml penicillin and 10 mg/ml streptomycin
  • Xenopus oocytes were isolated essentially as described by Zaeaux, et al.
  • Female Xenopus laevis were obtained from NASCO (Fort Atkinson, WI) and maintained at room temperature (20-22 °C).
  • the frogs were anesthetized by immersing for 20 minutes in 0.1-0.2% ethyl m-aminobenzoate (tricaine; MS-222; Sigma Chemical Co., St. Louis, MO).
  • a small incision was made on die abdomen and die ovarian lobes were removed into OR2 solution (82.5 mM NaCl, 2.5 mM KC1, 1 mM MgCl 2 , 5 mM HEPES, adjusted to pH 7.6 with NaOH).
  • the follicular cell layer was removed by digestion widi collagenase (2 mg/ml, type IA,
  • stage V and VI oocytes were dien transferred to ND96 solution (96 mM NaCl, 2 mM KC1, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES adjusted to pH 7.6 widi NaOH) and maintained at 18°C until used for injection with mRNA (typically a few minutes to a few hours).
  • Plasmid pSRlc obtained from D. Julius (Julius, D.J., et al, Science 241:558, 1988) was used to transcribe 5HTlc receptor cDNA in vitro using standard mediods (Ausubel, et al).
  • the plasmid was linearized with NotI (New England Biolabs, Beverly, MA), 2-5 ⁇ g of the linearized plasmid were suspended in 50 ⁇ l of a standard transcription buffer (Ausubel, et al.) containing 10 mM didiiodireitol (DTT) , 0.5 mM each of ATP, cytidine triphosphate (CTP) and uridine triphosphate (UTP), 0.25 mM guanosine triphosphate (GTP), 0.3 mM m7G(5')ppp(3')G (all from Boehringer-Mannheim, Indianapolis, ID), 50 units "RNASIN” and 40 units T7 RNA polymerase (both from Promega, Madison, WI), and die mixture was incubated at 37°C for 1 hour. A second 40 unit aliquot of T7 polymerase was added and the mixture was incubated an additional hour.
  • DTT didiiodireitol
  • DTT didiiodire
  • DEPC diethyl pyrocarbonate
  • Oocytes prepared as above were microinjected widi -50 nl of a solution containing -0.1 ⁇ g/ ⁇ l mRNA, prepared as above, using a Drummond Nanoject (Fisher Scientific, Pittsburgh, PA) pressure injector.
  • Injection pepts were fashioned using a standard patch pipet puller (Narashige, Japan) set to produce pipets with long (- 1.5 - 2 cm) tips. The tips were broken by poking a ChemWipe, and the pipets baked at > 150°C for 4-5 hours to inactivate RNases.
  • the pipets were backfilled with a DEPC-treated saline solution and tips were filled widi solution containing the RNA to be injected.
  • Injected oocytes were maintained in ND96 solution supplemented widi 2.5 mM sodium pyruvate, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin (Pen-Strep mixture obtained from Sigma, cat # P0781) at 18°C.
  • the oocytes were suitable for use as biosensors starting —24 hours after injection.
  • the loading medium contained 18 ⁇ M fluo-3, 135 mM NaCl, 5 mM KC1, 10 mM glucose, 2 mM MgCl 2 , 2 mM CaCI 2 , and 10 mM HEPES (pH 7.35).
  • the cells were placed in the same medium without fluo-3 for 0.5 h at room temperature.
  • the cell end of the capillary was positioned about 20 to -40 ⁇ m from the cells, thereby directing capillary effluent over the cells.
  • the channel of the capillary was slightly larger than the diameter of die cell (see Figure IB), enabling delivery of die effluent over die entire cell surface.
  • a 15 ⁇ M or 25- ⁇ m I.D. capillary was used for separations, and 1 to 15 mammalian cells, such as PC 12 cells, were used as die cell biosensor.
  • the effluent may be directed over selected portions of a single cell, or over a field of a plurality of cells, by selecting capillaries with appropriate I.D.s, cells widi appropriate diameters, and positioning the cell end accordingly.
  • Effluent species diat evoke changes in [Ca 2+ ]- were detected widi -an epi-illuminated fluorescence microscope.
  • Dishes containing the cells were transferred to die stage of a Diaphot-TMD-EF inverted fluorescence microscope (Nikon, Inc., Melville, NY), where they were maintained at - 35 to 37°C by heating die 100X (1.3 N.A.) oil-immersion lens used for illumination and fluorescence collertion.
  • the cells were illuminated widi 470-490 nm light (Nikon filter block, B-1A) and fluorescence ( ⁇ > 520 nm) was imaged onto an R928 photomultiplier tube (PMT; Nikon) with a Microflex PFX photomicrographic attachment (Nikon).
  • the current from the PMT was converted to a voltage and amplified with a LF355N operational amplifier. High-frequency noise was removed by a low-pass filter widi an RC time constant of 1 second.
  • the voltage signal was digitized by a "DAS” 8 analog- to-digital board (Kiethley MetraByte, Taunton, MA) and displayed widi software written in QuickBasic (Microsoft, Redmond, WA) on an IBM AT-style personal computer.
  • DAS Digital Advanced Sensor System
  • a transmitted-light image taken through die microscope objective shows how the outlet of a 25- ⁇ m i.d. capillary can be positioned above a single cell.
  • A. Changes in Intracellular Calcium - Ach/BK/ATP Mixture A standard solution of CE Ringer containing three ligands - acetylcholine (ACh; 0.8 mM), bradykinin (BK; 20 ⁇ M), and adenosine triphosphate (ATP; 5 mM) was separated by capillary electrophoresis.
  • the ligands were introduced by 5-second gravity injections (inlet elevated 10 cm above outlet) and were separated using an electric field of — 850 V/cm in a 23 cm 15 ⁇ m I.D. capillary.
  • the capillary effluent was delivered to a group of 10 - 15 PC12 cells loaded widi fluo-3 as described above.
  • Electropherograms are offset on the vertical axis to aid visualization.
  • the negative baseline slope is caused by a decrease in d e intracellular concentration of fluo-3, which can be partially stabilized by incorporating fluo-3 AM ester in the measurement buffer.
  • the trace labeled "Mixture” shows ihe fluorescence measured as a function of time as the capillary electrophoresis effluent was washed over die cells.
  • the peaks in die signal, reflecting increased intensity of fluorescence, are due to increases in [Ca 2+ ]- following binding of ligands to dieir respective receptors.
  • the increases in [Ca 2+ ]i are caused by release of calcium from internal stores and/or by entry of calcium through ligand- or voltage-gated ion channels (Pozzan, et ⁇ l., Sands, et ⁇ l.).
  • PC12 lysate was prepared as follows. PC12 cells grown and differentiated for four days in a 50 ml culture flask with 25 ml of PC 12 growth medium (see above) further containing 25 ng/ml /S-NGF (nerve growth factor; Boehringer-Mannheim, Indianapolis, IN), were pelleted and lysed in 70 ⁇ l of 70% methanol containing 0.1 mM eserine (to inhibit cholinesterase activity).
  • lysate Approximately 500 pl of the lysate were introduced to die inlet end of a capillary by a 10-second gravity injection 10 cm above die outlet (Fishman et al.). This amount of lysate corresponds to — IO *5 of die entire lysate volume. Since the cells in die flask were differentiated at — 10% confluence (i.e., — 5 * IO 6 cells were incorporated into the lysate), the 500 pl lysate volume corresponds to approximately 50 cells.
  • the lysate components were separated by capillary electrophoresis using a field of - 400 V/cm in a 25 cm 25 ⁇ m I.D. capillary.
  • the effluent was directed over 4-5 PC 12 cells, as above.
  • Fluo-3 AM ester was included in die buffer bathing die cells to aid in maintaining a steady concentration of fluo-3 in the cells.
  • FIG. 3 The top trace shows a single peak of [Ca 2+ ]- change in the first 150 seconds of separation. This peak, which migrates to the cell biosensor in — 100 seconds, is identified as acetylcholine from comparison with the electropherogram produced by running an acetylcholine standard solution (Figure 3, bottom trace).
  • a Xenopus oocyte was microinjerted as described above widi serotonin 5HTlc receptor mRNA and allowed to recombinantly express 5HTlc receptor.
  • Die oocyte was positioned at d e outlet of a separation capillary and voltage-clamped at -70 mV using a two-electrode voltage clamp (TEV-200; Dagan Corp., Minneapolis, MN) in the virtual-current mode.
  • Intracellular Ag/AgCl electrodes were constructed with an initial input impedance of — 3 to 4 MO in 3 M KC1.
  • the oocyte recording solution contained 140 mM NaCl, 2 mM KC1, 2 mM CaCl 2, and 10 mM HEPES (pH 7.2).
  • Serotonin (5-HT; 100 ⁇ M) was introduced into to die inlet end of a fractured 40- ⁇ m i.d. CE capillary by 20-second gravity injections (+ 13 cm above outlet). The fracture was located 26 cm from the inlet end of die 36 cm capillary.
  • the ligand solution was electrophoretically separated by application of a -250 V/cm field over die 26 cm portion of the capillary between die inlet and die crack. The separated effluent was pushed dirough die remaining 10 cm portion by the electroosmotic flow induced in die 26 cm portion, and delivered to the oocyte. Mock separations were run with buffer blanks.
  • Plots of current as a function of time are shown in Figure 5.
  • the top trace shows the signal obtained in response a separation using serotonin. A peak in current is seen — 8 minutes after injection of serotonin into the capillary.
  • the center trace shows the current response for a blank (control) CE run, and die bottom trace shows a return of signal when 5- HT is separated again and directed at die same oocyte. A slight increase in the migration time of 5-HT is apparent between the top and bottom traces, presumably caused by a decrease in die electroosmotic flow rate.
  • the results exemplified are representative of 5 experiments.
  • T e experiments utilized die tendency of many receptors, including nicotinic acetylcholine and bradykinin receptors, to desensitize following repeated applications of ligand (Hille, DeLorme, et al, Briggs, et al.). Bradykinin (250 ⁇ M) in CE Ringer was introduced at 120 second intervals into the inlet end of a capillary and elertrophoresed. The oudet was placed — 50 ⁇ m above a group of PC 12 cells. Data were collected as described in Example 1.
  • Figure 6A shows successive responses to the same dose of bradykinin. It is apparent from the figure diat die bradykinin receptors desensitize - that is, repeat applications of similar amounts of agonist ligand generate successively smaller responses.
  • Figure 6B shows a similar experiment as that shown in Figure 6A, except mat the coverslip containing die cell was moved a few hundred microns between each pulse of ligand, to place a fresh batch of cells under die cell end of die capillary. A robust signal was obtained with each pulse of bradykinin. The responses differ in size somewhat with respect to one anodier, since the different groups of cells differed in the number of cells they contained, and die different cells differed somewhat in the number of receptors expressed and die strength of their responses. Electrophoresis parameters - Capillary length: 30 cm, I.D.: 20 ⁇ m, Voltage: 330 V/cm.
  • Figures 6C and 6D show experiments where a group of cells was first exposed to repeated pulses as in Fig.
  • Electrophoresis parameters Capillary length: 30 cm, I.D.: 20 ⁇ m, O.D.: 350 ⁇ m, Voltage: 10 kV, Current: -20 ⁇ A, distance of capillary outlet from cells: —40 ⁇ m.
  • HOE 140 essentially eliminated the biosensor response to bradykinin, but had no effect on die acetylcholine response.
  • Figures 8A and 8B show die effects of atropine (Sigma Chemical Co., St. Louis, MO) and ⁇ -bungarotoxin (Sigma), both acetylcholine receptor antagonist, on a cell biosensor response to a mixture of acetylcholine (8 mM) and bradykinin (25 ⁇ M).
  • Figure 8A shows the response in absence of the antagonists
  • Figure 8B shows the response after the drugs are added to die cell badiing solution to final concentrations of - 1 ⁇ M (atropine) and - 1 ⁇ m ( ⁇ - bungarotoxin).
  • Electrophoresis parameters - Capillary length: 30 cm, I.D.: 20 ⁇ m, O.D.: 350 ⁇ m, Voltage: 10 kV, Current: -20 ⁇ A.
  • the acetylcholine antagonists reduced the biosensor response to acetylcholine by approximately a factor of 10 (compare the first peak (Ach) in Fig. 8A with the first peak (Ach) in Figure 8B), but did not affect the response to bradykinin.
  • EXAMPLE 5 RESOLUTION OF RELATED COMPOUNDS Methods of die present invention may be used to separate and resolve distinct ligands having similar chemical characteristics. Data were collected as described in Example 1 from a group of PC 12 cells.
  • Electrophoresis parameters Capillary length: 30 cm, I.D.: 20 ⁇ m, O.D.: 350 ⁇ m, Voltage: 10 kV, Current: —20 ⁇ A, distance of capillary outiet from cells: —40 ⁇ m.
  • Figure 9 shows the results a capillary electrophoresis run which included acetylcholine (8 mM), bradykinin (25 ⁇ M) and Lys bradykinin (25 ⁇ M). All three ligands are effectively separated and detected, as evidenced by die diree resolved peaks.

Abstract

Methods for detecting and methods of assaying the bioactivity of compounds and ligands, separated by capillary-based separation, such as capillary electrophoresis, from a mixture, employing an intact cell biosensor loaded with a calcium dye indicator for monitoring the bioactivity of said compounds and ligands. The figure shows an exemplary apparatus for recording optical signals from cell biosensors. Labelled parts are as follows: Number (2) functions to capillary separate ligands introduced at Number (4) the inlet end from a mixture of ligands contained in Number (6), Number (20) a power supply, (22) and (24) are electrodes, Number (16) an XYZ positioner, Number (18) a hollow needle through which the capillary is threaded into the recording chamber Number (14), the location where the response of a dye is detected via an optical detector, Number (28).

Description

Capillary-Based Separation Methods for Identifying Bioactive Analytes in a Mixture
FIELD OF THE INVENTION The present invention relates to methods of identifying bioartive analytes using capillary-based separation means.
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BACKGROUND OF THE INVENTION Capillary-based separation means, or methods, have been employed to resolve small volumes containing complex mixtures of .analytes into their constituents. Methods based on the principle of capillary electrophoresis are particularly effective in this regard (Jorgenson, et al. , 1983, Gordon, et al, Ewing, et al). In capillary electrophoresis, an electric filed is applied across a capillary, typically about 10 to 100 cm long and 20-200 μm in internal diameter, that contains a separation medium, typically an electrolyte or gel. The electric field induces electroosmotic flow of the separation medium through the capillary, and causes analytes in a sample mixture to separate according to, among other factors, their charge and hydrodynamic drag.
Detection methods that have been used with capillary electrophoresis include optical absorption (e.g., Lauer, et al.), optical fluorescence (e.g., Gassman, et al., electrochemical (e.g., Wallingford, et al., 1987b), conduct-metric detection (e.g., Huang, et al, 1987), radioisotope based detection (e.g., Berry), refractive index changes (e.g., Bruno, et al.) and mass spectrometry (Olivares, et al.).
The present invention provides a method of separating small volumes of complex mixtures of analytes using capillary-based separation means, and identifying separated constituents using a highly sensitive and highly selective detection means distinct from the above.
SUMMARY OF THE INVENTION In one aspect, the present invention includes a method for detecting a selected ligand in a mixture of ligands. The method includes separating the mixture of ligands containing the selected ligand using a separation means, delivering separated ligands to a cell biosensor having a receptor capable of binding the selected ligand, and detecting binding of the selected ligand to the receptor. In a preferred embodiment, the separation means is a capillary-based separation means, and the separated ligands are delivered to said biosensor in a cell biosensor compatible buffer.
The separated ligands are preferably delivered to a cell biosensor that is adjacent the outlet of the capillary-based separation means. A ligand may be any analyte that is capable of binding to a receptor and, by binding, having a measurable effect on the receptors's activity or function. In one embodiment, the mixtures of ligands is derived from a tissue extract. In a related embodiment, the mixture is derived from a cell extract. In yet another embodiment, the mixture of ligands is obtained from a combinatorial library of ligands. Selected ligands in a mixture of ligands may include such compounds as acetylcholine, bradykinin and ATP. Further, selected ligands may include a "parent" ligand, and breakdown, proteolysis or degradation products of that parent. Alternatively, the ligands may be a selected subset of degradation products. In one embodiment, the ligand may be an agonist of the receptor, while in another, it may be an antagonist. According to another aspect of the present invention, the separation means used in methods of the present invention is one of, or a combination of, various modes of capillary electrophoresis, including capillary zone electrophoresis, capillary gel electrophoresis, capillary electrochromatography, capillary isotachophoresis, affinity capillary electrophoresis, micellar electrokinetic capillary electrochromatography, capillary isoelectric focusing. The separation means may also be a variation of micro liquid chromatography, such as open tube liquid chromatography.
In another aspect, a cell biosensor used in methods of d e present invention may be a cell, including a cell recombinantly expressing a specific receptor. In one embodiment, the cell biosensor is a eukaryotic cell, such as a mammalian cell or a Xenopus oocyte. In another embodiment, the cell biosensor is a cell in contact with a voltage-recording electrode, such as an extracellular electrode, intracellular voltage-recording electrode, intracellular voltage clamp electrode(s), whole-cell patch electrode, or cell-attached patch electrode. In a related embodiment, the cell biosensor is a cell having a cell condition reporter, such as a dye sensitive to voltage, calcium, cAMP, calmodulin, pH, sodium, chloride, magnesium, or the like. Such reporters would detect changes in the cell biosensor in voltage, intracellular free calcium, cAMP, activated calmodulin, pH, sodium chloride and magnesium, respectively. In another related embodiment, the cell biosensor is a group of cell, such as a group of confluent or semi- confluent cells. In a further embodiment, the cell biosensor is a portion of an intact cell, such as an inside-out or outside-out membrane patch, a permeabilized cell, or a cellular organelle, such as a vesicle containing membrane from, for example, the sarcoplasmic reticulum of muscle.
In still another aspect of the present invention, the cell biosensor is in a bathing solution containing a receptor-specific antagonist, and effects of the presence of the agonist on the cell biosensor response are evaluated. According to this aspect of the invention, specific identification of ligand-bound receptors is possible. In a related embodiment, the cell biosensor is exposed to a receptor agonist while, for example, separated ligands thought to function as receptor antagonists are delivered with a capillary-based separation means.
The invention also includes the cell biosensor attached at the outlet end of the capillary used in the capillary-based separation means. The biosensors may be cells that were transferred to the outlet tip of the capillary by touching the tip to a cell in a recording chamber. Alternatively, the cell biosensor may be cells cultured and grown directly in the outlet tip of a capillary that is subsequently used for a capillary-based separation means. In a related embodiment, the cell biosensors are, for example, cells grown on a solid support, and die solid support containing the cells is attached, preferably removably, at the outlet end of the capillary.
Another aspect of the invention relates to moving either the cell biosensor or the outlet tip of the capillary with respect to the other to employ a different biosensor during the course of separation. This embodiment can minimize effects of receptor desensitization on cell biosensor responses or signals. The movement may be step-wise, such that a rapid translation is made to a region of substrate containing a different biosensor, and d e region is recorded from for e set period of time, or the movement may be continuous, such that the cell biosensor detection field and Λe capillary eluent sweep across a substrate containing cell biosensors. The rate of sweep may be set such that receptors do desensitize appreciably before they are out of the biosensor detection field. Also forming part of the methods of the present invention are supplemental detection means used in conjunction with a cell biosensor to identify ligands present in a mixture of ligands. According to this aspect of the invention, "peaks" of .analytes are identified whether or not they have biological activity, and biological activity may be correlated widi specific peaks detectable by traditional capillary electrophoresis methods. Preferably, the supplemental detection means does not interfere with or disturb the separated ligands as they pass through a capillary of a capillary-based separation means. For example, an optically-clear window may be incorporated in the capillary a few centimeters upstream of the outlet end of the capillary, and used for monitoring die fluorescence or UV-absorbance of the separated ligands as they pass by the window, and just before they exit the outlet and are introduced to die cell biosensor. The outputs from the various detection means, including cell biosensor signal detection means, may be routed to a computer programmed for various types of analyses, such as correlation of the peaks detected with a supplemental detection device and peaks detected widi the cell biosensor. The invention further includes a kit for supplying cell biosensors for use with a capillary-based separation means of the present invention. In one embodiment, the cell biosensors are supplied on a solid support that can be attached at the outlet end of a capillary- based separation means. The kit may include a plurality of such solid supports, frozen cells suitable for thawing and culturing on the solid supports, and various devices to facilitate die growing of cells on the solid supports, such as a culture chamber, adapted to receive a plurality of solid supports containing cells, for growing the cells.
In another aspect, the invention includes a device for performing capillary-based separations in conjunction with a cell biosensor. The device may include a capillary-based separation means, including capillaries, a power supply, solution/electrolyte reservoirs, and die like, and cell biosensors. The device may be designed to be used widi a kit as described above, and may further be automated, for example, to introduce a plurality of samples sequentially to die inlet end of d e capillary, and/or to replace cell biosensors, as needed, at die outlet end of die capillary.
These and other objects and features of the invention will be more fully appreciated when the following detailed description of die invention is read in conjunction widi die accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES Figures IA and IB show schematic diagrams of a capillary electrophoresis apparatus in conjunction with a cell biosensor and an optical detector used for detecting the state or condition of a cell condition reporter in die cell biosensor.
Figure 1C shows a photomicrograph of the end of a capillary widi its internal channel positioned above a cell biosensor (labeled "cell"). Figure 2 shows plots of fluorescence as a function of time detected from cell biosensors following capillary electrophoresis separation of ligands including acetylcholine (ACh), bradykinin (BK) and adenosine triphosphate (ATP). Figure 3 shows plots of fluorescence as a function of time detected from cell biosensors following capillary electrophoresis separation of a lysate of PC 12 cells and a control including acetylcholine.
Figure 4 shows a schematic diagram of a two-electrode oocyte voltage clamp in conjunction with a portion of a capillary electrophoresis apparatus.
Figure 5 shows plots of current as a function of time passed by a two electrode voltage clamp to maintain an oocyte, recombinantly expressing a serotonin receptor, at -70 mV in the presence of delivery of separated ligands including serotonin.
Figures 6A, 6B, 6C and 6D show plots of fluorescence as a function of time detected from cell biosensors following capillary electrophoresis separation of pulses of bradykinin, illustrating (i) d e desensitization of bradykinin receptors expressed on a cell biosensor (Figs. 6A, 6B, 6C and 6D), (ii) normal bradykinin responses on neighboring cell biosensors (Figs. 6B, 6C and 6D), (iii) translating the substrate containing the biosensors widi respect to a stationary capillary (Fig. 6B), and (iv) translating the capillary with respect to distinct stationary biosensors (Figs. 6C and 6D).
Figure 7 shows plots of fluorescence as a function of time detected from a cell biosensor following capillary electrophoresis separation of a mixture of ligands containing acetylcholine and bradykinin in die presence -and absence of HOE 140 (a bradykinin receptor antagonist). Figures 8A and 8B show plots of fluorescence as a function of time detected from a cell biosensor following capillary electrophoresis separation of a mixture of ligands containing acetylcholine and bradykinin in die presence (Fig. 8B) and absence (Fig. 8A) of atropine and α-bungarotoxin (acetylcholine receptor antagonists).
Figure 9 shows a plot of fluorescence as a function of time detected from a cell biosensor following capillary electrophoresis separation of a mixture of ligands containing acetylcholine, bradykinin and Lys-bradykinin.
DETAILED DESCRIPTION OF THE INVENTION
I. DEFINITIONS A "capillary-based separation means" refers to any method for separating a mixture of analytes that employs a capillary as the entity in which the separation occurs. Examples include variations of capillary electrophoresis, including capillary zone electrophoresis, capillary gel electrophoresis, micellar electrokinetic capillary electrochromatography, capillary isoelectric focusing, capillary isotachophoresis, and affinity capillary electrophoresis, as well as variations of micro liquid chromatography, such as open tube liquid chromatography.
A "capillary" refers to a tube having an inner diameter of between about 5 μm and about 1 mm. Preferably, the inner diameter is between about 20 μm and about 200 μm. The length of die capillary may be between about 1 cm and about 2 m, but is typically in the range of tens of centimeters. The capillary is typically made of a glass or fused silica material, but may be made from odier materials as well, including various plastics. The inner and outer surfaces of die capillary may further be coated widi any of a variety of materials to improve separation, reduce electrical noise, or alter odier selected characteristics. A "cell biosensor" refers to an intact cell, a portion of an intact cell (such as a membrane patch or permeabilized cell), or an intact cell in electrical communication with electrode(s) (such as intracellular recording electrodes, whole-cell electrodes, cell-attached patch electrodes, extracellular recording electrodes, etc.). In die case of a cell in electrical communication with an extracellular electrode, it will be understood that die cell does not necessarily need to be in contact with the electrode, only that the electrode be capable of detecting changes in die electric field due to changes in die cell's transmembrane electrical potential. It will also be understood that a cell biosensor designed for use widi an optical detection system (see below) will typically contain a cell condition reporter detectable by optical methods. A "cell condition reporter" is defined herein as an entity that alters its state or characteristics, preferably its optical characteristics, in response to a selected change in the cell's condition. Examples of cell condition reporters include, but are not limited to dyes diat are sensitive to transmembrane voltage, intracellular calcium and selected intracellular second messengers, such as cAMP. An exemplary cell condition reporter is die fluorescent dye fluo-3, which fluoresces in response to increased calcium concentrations.
A "receptor" is a macromolecule capable of specifically interacting with a ligand molecule. Receptors may be associated widi lipid bilayer membranes, such as the extracellular, golgi or nuclear membranes, and/or be present as free or associated molecules in d e cell's cytoplasm. Further, receptors may be eidier native to die cell biosensor (normally expressed by die cell from which the cell biosensor is derived), or diey may be recombinantly expressed.
"Recombinantly expressed" refers to proteins, such as receptors, expressed in a host cell as a result of die introduction into die host cell of a nucleic acid containing a region encoding die recombinantly expressed protein. For example, "recombinantly expressed" may refer to receptors expressed in Xenopus oocytes from mRNA eidier isolated from a selected source, such as a tissue from an organism of choice, or transcribed from a cloned cDNA. "Recombinantly expressed" may also refer to receptors expressed using a eukaryotic expression vector used to transform (eidier transiently or stably) a host cell line. Prior to transformation, the host cell line may or may not contain and/or express an endogenous version of diat receptor.
A "ligand" is a molecule capable of specifically binding to a receptor and altering die receptor's function. Binding of die ligand to die receptor is typically characterized by a high binding affinity, i.e., Km> 105. Ligands can act on the receptor directly as agonists or antagonists, or can act indirectly, by affecting the response of the receptor to odier agonists and/or antagonists. In eidier case, the response of the receptor results in a change in the cell's condition (e.g. a change in die transmembrane voltage potential or intracellular calcium concentration) which can be detected, for example, by a voltage-recording electrode and/or a cell condition reporter (see above).
A "cell biosensor detection field" is an area of a cell or cells on a substrate from which, at any given time, the state of a cell condition reporter is being recorded, and which, at d e same time, is at least partially exposed to effluent from a capillary of a capillary-based separation means. For example, in the case of an optically-detectable cell condition reporter, such a calcium indicator dye, being used widi a fluorescence microscope, the cell biosensor detection field is die portion of the image in the microscope mat is directed to d e photomultiplier tube for signal quantification. Accordingly, a cell biosensor detection field may contain only a portion of a cell (such as a large Aplysia neuron), or many cells, such as near- confluent PC12 cells).
II. GENERAL OVERVIEW OF THE INVENTION The present invention provides methods for detecting minute amounts of chemical analytes separated by capillary-based separation means. An important feature of the present invention is that die analyte or analytes to be detected can act as ligands, that is, that d ey can bind to a specific receptor or receptors. The methods employ cell biosensors expressing specific receptors to serve as ligand analyte detectors. Ligands which bind to a specific receptor result in changes in die cell condition of the cell biosensor, and this change is detected with, for example, a voltage-recording electrode or cell condition reporter, such as a calcium sensitive dye. III. CAPILLARY-BASED SEPARATION MEANS
Methods of die present invention are particularly advantageous for the detection of minute amounts of ligand analytes suspended in very small volumes (typically picoliter to nanoliter volumes). Accordingly, mediods of the present invention are preferably used widi separation means iat are suitable for separating ligand analytes in such small volumes, such as capillary-based or microcolumn separation means.
A variety of capillary-base separation means exist which are suitable for use with the methods of d e present invention, and are detailed below. These methods share the characteristic that they are performed in capillaries, typically fused silica capillaries, having an internal diameter (I.D.) on the order of tens of microns, and an outer diameter (O.D.) on die order of hundreds of microns.
Capillaries are ideally-suited for delivering separated analytes or ligands to cell biosensors, because die inner diameter of a capillary may be selected such diat it is similar to die diameter of, for example, a single cell being used as a cell biosensor. Alternatively, the inner diameter of a capillary may be selected such diat it is similar to die diameter of a selected group of cells, which together comprise the cell biosensor.
Further, the rigid nature and relatively compact outer diameter of capillaries facilitates the placement of die oudet, or cell end of die capillary (e.g. with an "XYZ" positioner, such as a three-axis micromanipulator), at a selected distance from the cell biosensor to achieve the desired degree of dispersion of separated analytes prior to dieir contact with die cell biosensor. In cases where the diameter of die biosensor is similar to the inner diameter of the capillary, die cell end of die capillary may be placed in close proximity to the biosensor (e.g. 20-30 μm). In cases where the inner capillary diameter is smaller diat die biosensor, the capillary may be placed further from the biosensor (e.g. 30-60 μm), to allow the separated ligands to disperse over an area largely covering the biosensor. The spread of separated ligands may be easily monitored, for example, by the inclusion of a suitable dye in the electrophoresis buffer.
XYZ positioners suitable for positioning the outlet end of capillaries of capillary-based separation means of die present invention include a variety of manipulators available from, for example, Newport Corp., (Irvine, CA) and Narashige, USA, Inc. (Greenvale, NY). Alternatively, cell biosensors may be attached directly at die oudet end of a capillary used in a capillary-based separation means, as is described in detail under "Cell Biosensors", below. Further, for use with d e mediods of die present invention, these various separation means preferably employ a cell biosensor compatible buffer in the course of die separation. A. Cell Biosensor-Compatible Separation Buffers
Methods of die present invention preferably employ biosensor-compatible electrolyte buffers in the capillary-based separation means. Biosensor compatible separation buffers are those buffers that are both effective to serve as separation buffers in selected capillary-based separation means, and to maintain die cell biosensor in a viable state (i.e. d at do not substantially interfere with die ability (i) of the ligand to bind to a selected receptor, (ii) of die bound receptor to cause a change in die cell condition of d e biosensor, and (iii) to detect die change in the cell condition with an electrode or a cell condition reporter).
In cases where intact or substantially intact cells are used, die biosensor-compatible buffer will typically be a physiologically-compatible buffer, such as an extracellular Ringer solution. Such solutions are well known in the art, and may be modified for the maintenance of specific cell types or the conduct of specific experimental manipulations. The composition of an exemplary Ringer solution (capillary electrophoresis Ringer; CE Ringer) suitable for use with capillary electrophoresis-based separation methods is presented in die Materials and Mediods section, below.
In addition to being compatible wid die selected biosensor, the separation buffers should be effective when employed for separations in die various capillary-based separation means. Such considerations are addressed individually widi regard to exemplary capillary- based separation means, below.
B. Capillary Electrophoresis
Capillary electrophoresis (CE) is a technique for fractionating chemical species on the basis of differences in die ratios of electrical charge to frictional drag in solution, and is able to separate complex chemical mixtures with high resolution in a few minutes. A number of review articles and books provide .an introduction to principles and applications of capillary electrophoresis (Monnig, et al., Grossman and Colburn, 1992, Christensen, et al , Chen, et al, 1988, 1989).
In its simplest and most common embodiment, a capillary electrophoresis system consists of a narrow-bore fused silica capillary (inside diameters range form about 20 μm to about 200 μm, and lengths from about 10 cm to about 100 cm in length) filled widi an electrolyte solution. The ends of the capillary are placed in electrolyte-solution reservoirs that contains either a cadiode or an anode connected to a high voltage source, typically 20 to 30 kV. Analytes are introduced to die inlet of the capillary, and are detected widi a detector near die outlet end. The electrolyte present in the capillary prior to sample introduction is typically die same or similar to that containing the sample. Separation times are typically in the range of about 1 minute to about 30 minutes.
The simple embodiment described above can be elaborated upon widi features such as autosamplers, multiple injection devices, sample/capillary temperature control, programmable power supply, multiple detectors, fraction collection and computer interfacing, among others. The analytes are typically introduced into die capillary by transferring the inlet (usually from the anode reservoir) to a solution of electrolyte containing the sample for a brief period before the separation field is applied, and applying eidier an electric field (to induce electroosmotic flow), or a pressure differential, such as positive pressure at the inlet, vacuum at the outlet or gravity, across die capillary to inject the sample. A variety of injection means have been described (Demi, et al., Tsuda, et al., 1987, 1992, Rose, et al., Huang, et al., 1988a), including one suitable for direct sampling of single cells (Chien, et al, Wallingford, et al, 1987a).
Because no "dead volume" is associated wid d ese open capillary separations, the sample volume can be reduced to occupy less than about 1 %, preferably less than about 0.1 % of the capillary volume, provided d at die sample contains enough molecules to detect. In fact, in order to achieve a high degree of resolution in capillary electrophoresis, sample volumes should be small in comparison to the total volume of the capillary, typically in the picoliter to nanoliter range (Gordon, et al). For example, a 100 cm capillary with an inside diameter of 75 μ has a volume of about 5 μl. An preferred sample volume for this capillary may range from about 5 nl to about 50 nl.
Despite the use of high separation fields (typically 100 to 1000 V/cm), joule heating in capillary electrophoresis is considerably less d an in slab-gel electrophoresis because (i) the high resistance of the narrow capillary channel results in a low current draw (usually 1 to 100 μA), and (ii) the high surface area-to-volume ratio of the capillary facilitates heat transfer.
The walls of fused silica capillaries have a negative charge in aqueous solution due to ionization of surface silanol groups. A sheath of mobile counter ions from the electrolyte solution in the capillary accumulates along the negatively-charged capillary surface, and induces electroosmosis (bulk solution flow) when an electric field is applied across d e electrolyte-filled capillary. Under typical operating conditions, this sheath of ions is positively charged, and consequendy, drags bulk solution from die anode to die cathode. As is pointed out below, however, die inside surface of capillaries may be treated or coated such that the fixed charges are positive and accumulated mobile counterions are negative, thus inducing electroosmosis from die cadiode to die anode. A practical result of electroosmotic flow is that in free-solution capillary electrophoresis, all species — whether possessing positive, neutral, or negative charge -- can be made to migrate in die same direction past a single detector during a separation. Using die example of "typical" CE, where electroosmotic flow is towards die cadiode, a positively- charged sample component emerges early because both die electrophoretic motion of the ion and electroosmotic motion of die electrolyte -are in the same direction. If die component is negatively-charged, but its electrophoretic mobility is less than the electroosmotic mobility, dian that component also migrated toward the cadiode, but at a slower rate.
Many groups have investigated die use of various additives to die capillary electrolyte, and various capillary wall coatings, in part because certain types of analytes are difficult to separate using unmodified, basic capillary electrophoresis techniques. For example, uncharged molecules subjected to capillary electrophoresis do not typically separate from each other, but tend to migrate together as a group. Biomolecules, on die other hand, tend to adhere to capillary walls, causing tailing of their zones and affecting the electroosmotic flow characteristics of the capillary.
Modifications to capillary electrophoresis facilitating the separation of diese and odier recalcitrant molecules have included die inclusion in die capillary electrolyte of various additives, such as partitioning agents (Terabe, et al., 1984, 1985, Otsuka, et al., Sepaniak, et al., Burton, etal., Balchunas, etal., 1987, 1988, Cohen, A.S., etal, 1987b), macromolecules (Chin, Demorest, et al. , Grossman), gels (Shirasaki), modifiers, and sieving matrices (Hjertέn, Hjertέn, et al., 1985b, 1987a, 1987b), including polyacryl amide gels (Cohen, A.S., et al., 1987a, 1987c, Guttman), as well as coating the walls of the capillaries with agents designed to alter solute-wall interactions (Walbroehl, et al., Fujisawa, et al., Wiktorowicz).
Some of the modifications described in the above paragraph comprise distinct, recognized "modes" of capillary electrophoresis (Li). Different modes of capillary electrophoresis include (i) capillary zone electrophoresis (CZE; Jorgenson, 1984, Altria, et al. , Tsuda), (ii) capillary gel electrophoresis (CGE; Hjertέn, et al., 1985a, 1985c, 1987, Cohen, et al, 1987a, 1987c), (iii) micellar electrokinetic capillary electrochromatography (MECC; Terabe, et al., 1989, Tsuda, et al., 1982), (iv) capillary electrochromatography (CEC; Knox, Pretorius, Jorgenson, et al., 1985), (v) capillary isoelectric focusing (CIEF; Hjertέn, et al., 1985b, 1987, Mazzeo, et al.), (vi) capillary isotachophoresis (CITP; Everaerts, et al., Bocek, et al), and (vii) affinity capillary electrophoresis (ACE; Chu, et al., 1992, 1993, Avila, et al., Gomez, et al). It will be understood that all of die above modes fall under die generic definition of capillary electrophoresis. Considerations for selecting capillary electrophoresis parameters for die separation of specific types of molecules, such as DNA oligonucleotide (Dubrow), peptides (Colburn), proteins (Wiktorowicz, et al.) .and small molecules (Demarest, et al.) have been well documented. The electric field used for separations with capillary electrophoresis is typically maintained at a relatively constant level. As is detailed below, however, certain capillary electrophoresis embodiments may be better served by using pulsed electric fields; specifically, applications which entail electrical (as opposed to optical) measurements of transmembrane voltage. Applications of pulsed electric fields in capillary electrophoresis have been described (Cohen, A.S., et α/., 1992).
Detection in capillary electrophoresis is typically performed on-line — that is, widiin die separation capillary or just adjacent die outlet, while analytes migrate under the influence of die applied electric field. A plot of die measured detection signal versus separation time produces an "electropherogram", which represents analytes as peaks centered at characteristic migration times. The height (or area) of a peak is preferably related to die concentration of the analyte that produced d e peak.
A variety of detection means suitable for use with capillary electrophoresis have been described, including optical detectors based on ultraviolet (UV) absorbance (Lauer, et al, Terabe, et al., 1984, 1985, Otsuka, et al., Walbroehl, et al., 1984, 1986, Fujisawa, et al. , Tsuda, Tsuda, et al., 1983a, 1983b) or fluorescence (Jorgenson, et al, 1981a, 1981b, 1983, 1987, Gassman, et al., Gozel, et al., Roach, et al., Green, et al.) of the analytes, including circular dichroism (CD) measurements (Christensen, et al), conductivity detectors (Foret, et al, 1986, Everaerts, et al., Huang, et al., 1987, 1988b), electrochemical (amperometric) detectors (Wallingford, et al, 1987b, 1988) mass spectroscopy based detectors (Mack, et al, Whitehouse, et al. , Olivares, et al. , Smith, et al.), Raman-based detectors (Chen, et al. , 1988), refractive index detectors (Chen, et al., 1989) and radioisotope based detectors (Berry).
According to mediods of die present invention, any of die above detection means may be used in combination wid die cell biosensor detection means to enhance d e identification of a selected analyte. Such "supplemental" detectors, or supplemental detection means, are preferably those which can detect characteristics of the sample mixture near the outlet end of die capillary widiout disturbing die mixture. Examples include in-line optical detectors, including UV, fluorescence, CD and refractive index detectors, conductivity detectors and electrochemical detectors. The capillary may include input and output windows near the output end for use widi a supplemental optical detection means, as described, for example, by Johnson, et al.
When either the cell biosensor, or the supplemental detection means are adversely affected by electrical noise from the electrophoresis power supply, it may be advantageous to operate die electrophoresis power supply in pulse mode — that is, turn it on for a selerted period of time (e.g. for 1 second), and dien turn it off for a similar period of time. The noise- sensitive cell biosensor signal may be collected while the power supply is in the off position ("silent"), thereby avoiding electrophoresis-induced electrical noise.
Other capillary-based separation means contemplated for use with mediods of the present invention include variations of micro liquid chromatography (micro LC; Berry, et al. , 1989), in particular, open tube liquid chromatography (OTLC).
OTLC refers to the mediod by which a gradient of two solutions is generated for use in the column separation. Conventional methods for generating such a gradient typically employ one of two approaches, which can be characterized as "high pressure" and "low pressure" gradient generation (Berry, et al., 1990). Both of diese are generally useful for columns having internal diameters greater diat about 1 mm, but are not particularly effective for smaller columns. In contrast, a method and device described by Berry, et al. , 1990, allows die generation of gradients for use in columns having internal diameters as small as 5 μm.
Accordingly, OTLC is effective at separating mixtures having volumes comparable to those used widi capillary electrophoresis (picoliter to nanoliter range), and may be used as a capillary-based separation means in methods of die present invention. Capillary electrophoresis and OTLC differ in that OTLC typically does not employ an electric field across the capillary. Rather, die sample is pushed through by hydrostatic pressure, typically provided by pressurizing die sample mixture at some point before it enters the inlet end of die column or capillary.
IV. CELL BIOSENSORS
According to mediods of die present invention, intact cells and portions diereof comprise a previously unappreciated class of detectors suitable for use with capillary-based separation means, such as capillary electrophoresis.
As discussed in d e "Definitions", a cell biosensor suitable for use with capillary-based separation means of die present invention can be an intact cell, a portion of an intact cell (such as a membrane patch or permeabilized cell), or a group of intact cells. Further, the biosensor may be in electrical communication with an electrode or electrodes, or may contain a cell condition reporter, such as a calcium-sensitive dye.
Cells comprising die biosensor may be single eukaryotic or prokaryotic cells, groups of such cells, or a confluent layer of such cells. For example, PC12 cells comprise exemplary cell biosensors, as demonstrated in Examples 1, 3, 4 and 5 herein. Further, the cells may be transformed to recombinantly express a selerted receptor. In addition, a population of transformed or untransformed cells may be selected using, for example, fluorescence-activated cell sorting (FACS), for those cells expressing high levels of a desired receptor or low levels of an undesired receptor. By selecting a clone or clones having the highest degree of d e desired characteristic (i.e. clones expressing the highest levels of a desired receptor, or clones expressing the lowest levels of an undesired receptor), growing diem up, and repeating die process a selected number of times, a population of cell can be obtained diat express a desired receptor at very high or virtually nonexistent levels (depending upon d e selection criteria employed). Fluorescence activated cell sorters are available, for example, from Becton Dickinson (Mountain View, CA).
While the invention contemplates providing a separate delivery means to deliver effluent from the oudet of a capillary-based separation means to a cell biosensor, the cell biosensor is preferably placed adjacent die oudet of a capillary-based separation means. This may be accomplished in a number of ways. For example, the cell biosensor may be a cell attached to or growing on die bottom of a saline- or medium-filled recording chamber, and the outlet end of d e capillary may be brought such mat it is adjacent die cell (i.e. - 5μm to - 100 μm above the cell). Such an arrangement is demonstrated in Examples 1 and 2-5 for use widi an optically-detectable cell condition reporter. A similar arrangement may be used in conjunction widi a voltage recording electrode, such as an extracellular electrode present in die recording chamber, or a voltage clamp, such as whole-cell clamp (Neher and Sakmann). In a similar embodiment, the cell biosensor is a cell, such as a Xenopus oocyte or Aplysia neuron, placed in a recording chamber widi die outlet end of the capillary adjacent die cell as above (e.g. Example 2). The cell may contain a cell condition reporter, such as a calcium-sensitive dye, or an electrode, including an intracellular voltage-recording or voltage/patch clamp electrode.
Alternatively, the cell biosensor may be a cell, attached not to the substrate, but to the end of a whole-cell electrode, that is positioned (e.g., widi a micromanipulator holding die electrode) at the oudet end of die capillary. Isolated patches of d e type formed during patch clamp recording (Sakmann and Neher) are also suitable for use as biosensors. Patch electrodes having isolated membrane patches may be positioned, for example, using a micromanipulator, adjacent die capillary outiet as described above.
The cell biosensor may be attached at die oudet end of die capillary of a capillary-based separation means either directly or dirough a substrate. For example, the outlet end of die capillary may be touched to a cell in a culture dish, causing die cell to detach from die culture dish and attach to the outlet tip of the capillary. The attached cell is suitable for use as a biosensor if part of the capillary eluent comes in contact with die cell. In a related embodiment of the invention, cells are cultured directly in the outlet tip of the capillary. This may be accomplished prior to the initiation of a separation run by "plating" cells in the portion of die capillary at die outlet end using, for example, suction to draw a suspension of cells into the outlet end portion of die capillary, and allowing die cells to adhere to d e inside wall of the capillary. The portion of the capillary in which cells are to be plated may be coated prior to die plating of die cells widi a substance that supports the attachment and/or growth of cells (e.g., poly-lysine). A cell biosensor may also be attached to die outlet end of a capillary used in a capillary-based separation means via a solid support on which die cell is growing. For example, one embodiment of d e present invention provides a removable solid support on which cells can be grown, and which can be attached at d e outlet end of a capillary used in a capillary-based separation means. The support may consist of, for example, a short glass or plastic tube having an inner diameter diat is slightly larger than the outer diameter of the capillary at die oudet end. Cells can be grown inside die tube using standard tissue culture techniques. When the cells have grown to possess properties rendering diem suitable for use as cell biosensors, the tube may be slipped part way over die outlet end of die capillary, dius providing a biosensor for identifying a selected ligand in a mixture of ligands separated using a capillary-based separation means. The portion of the tube extending past the outlet end of the capillary may include an angle (e.g., a right angle) to facilitate contact of die stream of eluent with cells located in die tube at the outer portion of the angle.
It will be understood that die outer diameter of die capillary at the outlet may be reduced, for example, by exposure to hydrofluoric acid, to accomodate a cell culture tube of a smaller inner diameter dian the normal outer diameter of the capillary.
Another example of a removable solid support is a substantially planar support that includes an attachment means to attach the support at die outlet end of the capillary. The attachment means may be a ring, having an inner diameter as above, that can be slipped over die outlet end of the capillary. Widi the attachment means engaged widi die outlet end of the capillary, the planar support may be positioned at a selected distance from the capillary oudet such that the surface of the support containing the cells is substantially perpendicular to the stream of eluent from the capillary oudet. Alternatively, the support surface containing die cells may be placed at an angle odier dian perpendicular to die eluent stream, such that die stream is only deflected as it passes over die cells.
Certain embodiments in which d e cell biosensor is attached to die capillary may be amenable to automation. For example, an automated device can contain a supply of tubes containing cells, as described above, and a mechanical element, such as a robotic arm, can place fresh tubes over the outlet end of die capillary as desired. Alternatively, the outlet end of a capillary can be fitted widi a rotatable motorized shaft oriented substantially parallel to the capillary and approximately coextensive with die outlet tip of the capillary. A variation of die planar substrate widi attachment means, described above, can be fashioned to comprise, for example, a circular planar substrate containing cells oriented about a central axis. Such as assembly can be attached to die motorized shaft such that die substrate is perpendicular to die capillary, and diat eluent from die capillary flows onto die cells in a selerted region of die substrate. By rotating die motor, a different portion of the substrate, containing fresh cells, is exposed to die capillary eluent. The rotation may be set up to be interrupted, allowing recording from a selected region for a selected period of time, or continuous.
In any embodiment of die invention diat provides a means of moving the capillary outlet relative to a substrate containing cell biosensors, and in which it is desirable to obtain quantitative information on the amount of ligand in die separated capillary effluent, confluent or near-confluent layers of cells, due to their relative uniformity, may constitute particularly effective biosensors . As is detailed below, receptors typically desensitize following repeated applications of agonist. This desensitization increases die difficulty of obtaining quantitative information from cell biosensors, since quantitating responses typically requires generating a standard curve of responses to a range of agonist concentrations.
One method of avoiding die desensitization problem is to record each response widi a fresh biosensor. For this to be effective, however, the different biosensors must be relatively homogeneous in their response characteristics (i.e. if optical detection mediods are being used (see below) without ratiometric imaging, each cell biosensor detection field should have approximately the same detectable cellular response to a given amount of ligand. A confluent or near-confluent layer of cells is advantageous in this regard, because die cell biosensor constitutes the cells contained in an area that is exposed to separated ligands and that is widiin die cell biosensor detection field of a detection device. By moving the outlet of the capillary and die detector to a different region of die confluent cell layer, one may obtain a fresh biosensor (one whose receptors have not desensitized) with similar response characteristics (if the cells are relatively homogeneous) to the original.
Any scanning technique in which die outlet end of die capillary is moved wid respect to the cell biosensor, or vice versa, may be automated. For example, an approach similar to that discussed above for cell biosensors attached to the capillary may be employed widi cell biosensors present in a recording chamber. The "XY" controls of the stage holding die recording chamber can be interfaced widi precision motor drives (available, for example, from Newport Instruments (Irvine, CA), which can be directed to move die chamber to selected positions widi respect to die capillary oudet (it is understood, of course, that the motor drives can be incorporated to control die movement of die capillary as well).
As discussed above, die movement can be rapid, interrupted movement, or smoodi, continuous movement. Provided diat a substrate containing a confluent or near-confluent layer of cells is available, the continuous scanning technique affords a significant advantage regarding receptor desensitization. That is, die cell biosensor detection field can be moved at a rate diat is faster man d e predominant time constant of receptor desensitization. In this way, the detectable cell biosensor receptors do not desensitize, since cells which had been exposed to a desensitization-inducing ligand long enough to experience significant desensitization are no longer contributing to the biosensor signal. As receptors recover from desensitized states and return to the states they were in prior to application of ligand, they may again be used as cell biosensors. Based on die knowledge of die rates of receptor desensitization and recovery from desensitization, which may be readily determined by appropriately-timed applications of agonists (Hille), it is possible to construct a substrate that has a large enough area to enable sustained cell biosensor recording for the lifetime of the cell biosensor, without encountering appreciable difficulty due to receptor desensitization.
The recording dish may be perfused, either continuously or periodically, widi fresh recording solution, for example, in cases when the volume of die recording solution is such that a concentration of ligand high enough to interfere with cell biosensor responses to capillary effluent may build up over die lifetime of the separation process.
It will be understood iat solid supports and substrates referred to above may be coated with any of a variety of agents that promote the adhesion and/or growth of cells plated on die substrate. Such agents include coatings diat alter surface charge, such as poly-lysine and poly- ornithine, various matrixes designed to support cell growdi, and selected extracellular matrix components, including collagen, fibronectin and gelatin.
V. RESPONSE DETECTION METHODS The binding of a selected ligand to a receptor in or on a cell biosensor may be deterted by any of several mediods diat report die condition of die cell biosensor. Two exemplary mediods of detecting die condition of a cell are (i) substances, or cell condition reporters, diat alter their characteristics, preferably optical characteristics, in response to selected specific changes in a cell's condition (such as increase in intracellular calcium), and (ii) electrodes capable of detecting changes in die transmembrane potential of the cell.
A. Cell Condition Reporter Detection
Many types of cell condition reporters are available. They can be grouped according to the type of cellular event to which they are sensitive, or according to die mediod by which they are deterted. According to mediods of die present invention, preferred mediods for detecting die state of cell condition reporters are optical. Cell condition reporters whose state may be detected by optical mediods may be referred to herein as optically-detectable cell condition reporters.
Optical detection may comprise assaying changes in absorbance or detection of luminescent or fluorescent signals. Fluorescence tends to be more sensitive than absorbance, because fluorescence is deterted as an increment above a low background rather than a small decrement from the full intensity of the incident beam (Tsien, 1992). Fluorescence is also advantageous over luminescence in cases where die amount of probe is limited, because each fluorophore can emit many thousands of photons (energized by die excitation beam) as opposed to die maximum of one photon emitted by a luminescent molecule. Luminescence may be advantageous, however, in cases where background fluorescence is high.
In part due to the advantages of fluorescence oudined above, most optically-detectable cell condition reporters currently available are fluorescent molecules diat alter dieir fluorescence as a result of binding to a particular cell constituent or as a result of a change in the cell's pH or transmembrane voltage potential. They are discussed below in relation to the cellular event to which iey are sensitive.
Many of these optically-detectable cell-condition reporters, or dyes, are amenable to ratio imaging, or ratiometric quantitative determination of die signal (Bright, et al., 1987, 1989, Taylor, D.L., and Wang, Tanasugarn, et al.). In ratio imaging, the optical characteristics of the dye are measured at two different wavelengdis. The wavelengths are selected such diat die optical property being measured (e.g. fluorescence) is essentially independent of die cellular condition to which die dye is sensitive (e.g. calcium concentration) at die first wavelength, but is maximally-sensitive to that condition at die second wavelengdi. To obtain a measurement of the cellular condition, die amplitude of d e optical signal at die second wavelengdi is divided by die amplitude of the signal at the first wavelengdi. This ratiometric measurement normalizes for extraneous factors such as the optical path lengdi through die cells, the fraction of cellular volume accessible to the dye, and the fraction of the detection field occupied by active cell biosensors, and provides a more reliable estimate of die cellular condition.
Optically-detectable cell condition reporters have been developed for detecting a variety of conditions, including transmembrane voltage, and levels of intracellular calcium, chloride, magnesium, sodium, pH, cAMP and activated calmodulin (Slavik, Tsien, 1989a).
1. Calcium-Sensitive Dyes
Among die first classes of molecular probes to be developed were fluorescent indicator dyes sensitive to calcium. The dye quin-2 was one of the first to exhibit good selectivity for calcium over magnesium and result in usable fluorescence (Tsien, 1980). Because the dye molecule was charged at neutral pH, however, introducing it into cells required micro-injection. This imposed certain limitations on the types of experiments possible. To overcome this, uncharged ester, typically acetomethyl (AM) ester, forms of the dyes were syndiesized (Tsien 1981). The uncharged esterified forms readily diffuse through d e plasma membrane and enter die cell, where diey are cleaved by endogenous esterases to liberate active, membrane- impermeant dye. Determinations of [Ca2+]- in single cells were greatly facilitated by die syndiesis of fura-2 and indo-1 (Grynkiewicz, et al.). Both dyes have bright-enough fluorescence to image single cells and introduce only a limited amount of Ca2+ buffering to the cell. In addition, d ese dyes are amenable to ratio imaging (see above), and can dius be used to estimate absolute calcium concentrations in the cell. A more recent addition is die dye fluo-3 (Kao, et al), which was used (both in native and AM-ester forms) in experiments detailed herein in Examples 1, 3, 4 and 5.
Dyes sensitive to calcium are effective as cell condition reporters for selected ligands which, by binding to a specific receptor, change (typically increase) die calcium concentration in a cell. Examples of receptors effective to alter intracellular calcium are presented below. For instance, experiments detailed herein demonstrate die elevation of intracellular calcium, as deterted by die dye fluo-3, in mammalian cells expressing bradykinin or acetylcholine receptors (Examples 1, 3,4,5).
2. pH-sensitive Dyes
A variety of pH-sensitive indicators have been syndiesized (reviewed in Tanasugarn, et al., and Tsien, 1989b). Modifications of an existing dye resulted in "BCECF" (Rink, et al.), an exemplary pH indicator which is amenable to ratio imaging. The dye is sensitive to pH in the range of pH ~5.6 to pH — 8.0 at an excitation wavelength of 500 nm, but is relatively insensitive to pH at 450 nm.
3. Dves Sensitive to Other Inorganic Molecules
Detection of intracellular sodium is possible using die fluorescent indicator molecule "SBFI" (Minta, et al.). While the dye has certain limitations, it is suitable for detection of increases in intracellular sodium of cell biosensors.
Detector dyes are also available for chloride and magnesium (Tsien, 1989b).
4. Voltage-sensitive Dves
Many dyes which insert into die plasma membrane and alter dieir fluorescence in response to die transmembrane voltage potential (voltage-sensitive dyes) are currently available (Cohen, Ross, et al., Gupta, et al.). The most effective of these were identified by synthesizing and testing more than a thousand organic dyes. An exemplary voltage-sensitive dye is di-4-ANEPPS (Loew, et al.). This dye displays an increase in fluorescence combined with a redshift of the excitation spectrum upon hyperpolarization. The signal-to-noise of the potentiometric fluorescence response is among the best recorded for my dye. Further, the dye is amenable to ratio imaging, shows no appreciable signal deterioration over extended recording periods and has an excellent signal-to-noise ratio.
Voltage-sensitive dyes, such as di-4-ANEPPS, may be used as cell condition reporters in cases where die binding of a selerted ligand to a receptor results in a change in the transmembrane voltage of a cell or patch. Examples of receptors having this effect are discussed below. In particular, experiments performed in support of the present invention demonstrate that die ligand serotonin is effective to produce an inward current in an oocyte expressing a serotonin receptor (Example 2). 5. Second Messenger Dyes
In addition to die optically-detectable cell condition reporters described above for simple spherical inorganic ions, molecular probes have been developed diat are sensitive to the concentration of organic "second messenger" molecules, such as cyclic adenosine monophosphate (cAMP) and calmodulin.
The indicator for cAMP is based on a cAMP-dependent protein kinase (Taylor, S.S., et al, 1990). The catalytic (C) and regulatory (R) subunits are each labelled with a different fluorescent dye, such as rhodamine and fluorescein, capable of fluorescence resonance energy transfer in the holoenzyme complex R_C2. When cAMP molecules bind to die R subunits, the C subunits dissociate, diereby eliminating energy transfer. The change in shape of the fluorescence emission spectrum allows cAMP concentration to be visualized in a living cell injected widi die labelled holoenzyme (Adams, et al.) by employing ratio imaging (above). Accordingly, diis indicator is a cell condition reporter sensitive to ligands which alter die level of cAMP in die cell. Hahn, et al , (1990) have linked a tetramediinemerocyanine fluorophore to the calcium- binding protein calmodulin. The resulting conjugate, MeroCaM, has a reversible calcium- induced 3.4-fold increase in excitation ratio (608/532 nm), apparently due to the binding of calcium to the high affinity sites on the calmodulin molecule. MeroCaM has been used to image calmodulin activation in Swiss 3T3 fibroblasts in vivo (Hahn, et al, 1992).
6. Optical Detectors for use with Optically-Detectable Cell Condition Reporters A variety of methods may be used to optically monitor signals, such as fluorescence signals, in cell biosensors. An exemplary apparatus for recording optical signals from cell biosensors in response to effluent from a capillary-based separation means is illustrated in Figures IA and IB. The device includes a capillary 2 for the separation of a mixture of ligands. A mixture of ligands is introduced into die inlet end 4 of die capillary, for example, by placing die inlet end in a vial 6 containing the mixture. The other end 8 (outlet or cell end) of the capillary is placed in proximity to a cell biosensor, such as a eukaryotic cell 10 on a glass coverslip 12 forming the bottom of a recording chamber 14. The cell end 8 can be positioned at a selected location by means of an XYZ positioner 16. An exemplary means of achieving positioning is shown in the diagram. A portion of die capillary proximal to the cell end is direaded through a hollow needle (such as a syringe needle) 18 and is glued at the needle entrance and exit points to stabilize the position of die outlet. The needle, in turn, is fastened to die XYZ positioner. The needle may be bent into a conformation to facilitate placement of the cell end of the capillary adjacent die biosensor.
A power supply is indicated at 20, and electrophoresis electrodes at 22 and 24.
Separated analytes 26 are delivered to the cell biosensor 10, which had been loaded widi a dye (see below) responsive to cellular changes anticipated as a result of exposure of die cell to a selected ligand analyte. The response of the dye is detected by an optical detector via, for example, a microscope objective 28 placed under die coverslip.
The optical detector may be an inverted fluorescence microscope, such as a Zeiss AxioVert or IM35 (Carl Zeiss, Inc., Thornwood, NY) or Nikon 300 or Diaphot-TMD-EF (Nikon Inc., Melville, NY). Techniques for optically monitoring the state of cell condition reporters and for analyzing resultant information (e.g. images) are known in the art (e.g. Taylor, D.L., and Wang, Taylor, D.L., et al, 1986, 1992, Herman and Jacobson, Conn, 1990, deWeer and Salzberg, Lakowicz, Slavik, Duchen, Harootunian, 1988, 1991a, 1991b, Poenie). One method for recording a fluorescent signal using a fluorescence microscope (a Nikon Diaphot-TMD-EF) is detailed in Example 1.
Figure 1C shows a view of a cell end of a 25 μm I.D. capillary positioned above a single cell as imaged widi an objective such as shown at 28 in Figure IA.
It will be understood, however, diat die use of a microscope is not necessarily required for optical detection of die state of cell condition reporters. For example, in applications where it is not necessary to obtain an image of the cell biosensor, the optical detector may simply be a light pipe (such as a fiber optic cable) in combination widi appropriate optical filters, directed to a photomultiplier tube. Excitation light for fluorescence imaging in such situations may be provided by an appropriately-filtered high-intensity light source, directed at die sample using, for example, a fiber optic cable. Alternatively, the cell biosensor may be placed in a spectrophotometer, and die absorbance of die cell condition reporter monitored as eluent from a capillary-based separation means, containing separated ligands, is washed over die cell biosensor.
Use of an exemplary optically-detectable cell condition reporter in conjunction widi die device described in relation to Figures IA and IB is demonstrated in Example 1. Detailed in me example is a separation of acetylcholine (ACh), bradykinin (BK) and adenosine triphosphate
(ATP), and subsequent detection of diese ligands by a PC12 cell-based cell biosensor using a calcium-sensitive dye as die cell condition reporter.
The results, shown in Figure 2, demonstrate the ability of a separation-detection system employing a cell biosensor to detect and identify selected ligands in a mixture of ligands separated using a capillary-based separation means. Identification is confirmed by running standards containing only the selerted ligand.
Detection of components in complex biological mixtures is a more stringent test of analyte identification capacity than is identification of standard solutions. An exemplary complex biological mixture is a lysate of PC 12 cells containing endogenous! y-produced acetylcholine. An experiment performed according to mediods of the present invention utilizing PC12 lysate as a mixture of ligands is presented in Example 1. PC12 cells having natively-produced ACh receptors (AChRs) are used as die biosensor for acetylcholine.
The results, shown in Figure 3, demonstrate die ability of die separation/detection system described herein to separate and identify a selerted ligand (acetylcholine) in a complex mixture of ligands (PC12 lysate). The lysate peak likely corresponds to an acetylcholine content in die low femtomole range (Greene, et al., 1977), illustrating the high sensitivity of the mediods described herein. Accordingly, two advantages of die present invention demonstrated by die above experiments are (i) ability to identify a selerted ligand in a highly complex mixture of ligands, and (ii) die ability to identify a selected ligand diat is present in very low concentrations (e.g., in the femptomolar range).
Of the species examined in experiments detailed herein, die highest sensitivity is achieved for BK. To investigate die detection sensitivity and reproducibility for this species, five capillary electrophoresis runs of BK are performed at each of diree concentrations, using an individual cell as die cell biosensor. A different PC12 cell is selected as d e cell biosensor for each run to avoid cell biosensor desensitization, and care is taken to set die capillary outlet at the same distance (-40 μm) from the cell for each run. At 10 μM and 100 nM concentrations, 4/5 and 5/5 cells, respectively, respond to BK after electrophoresis. Higher sensitivities may be obtained by differentiating the cells (Bush, et al.) and/or selecting clones expressing high amounts of the BK receptor (e.g. using fluorescence-activated cell sorting as described above).
In general, detection limits for different ligands may range from more than picomoles to a few molecules (Mori, et al.). Factors diat affect the detection signal include die ligand-receptor dissociation constant, the number of receptors exposed to ligand, die amplification pad ways accessed after binding, die baseline variability in the quantity being measured, and frequently, die chemoreception history of the cell. Cellular response mechanisms can be manipulated to improve sensitivity and reproducibility. Differentiation of PC 12 cells, for example, has been shown to strongly potentiate the calcium response to bradykinin (Bush, et al), and receptor mutagenesis has the potential to greatly reduce desensitization (Jackson).
Example 3 details another application of the above system, and further demonstrates the capacity to scan the outlet of die capillary-based separation means relative to the recording chamber to employ fresh biosensors. The results, shown in Figures 6A, 6B, 6C and 6D demonstrate diat effects of receptor desensitization may be minimized by providing fresh biosensors for consecutive separations. Pulses of bradykinin are separated and applied to die cell biosensor. The responses are evicent as peaks, or "spikes". The results show that die scanning can occur by moving the capillary outlet while maintaining the dish containing the cell biosensors stationary (Figs. 6C and 6D) or by translating the substrate containing die biosensors with respect to a stationary capillary (Fig. 6B). The breaks in the records reflect the interruption due to scanning.
The results also provide information as to die time course of receptor desensitization (die rate at which peaks decrease in response to applications of approximately the same amount of ligand). Using this information, an automatic system may be designed diat continually scans a substrate containing near-confluent cell biosensors at a rate fast enough that effects of desensitization are not appreciable (see above). In order to improve die reproducibility of responses from different biosensors, a confluent or semi-confluent layer of cells may be used, as discussed above. Example 4 illustrates selective blocking of cell biosensor responses to electrophoresed ligands by specific receptor .antagonists. The data, shown in Figures 7, 8A and 8B, show selective inhibition of cell biosensor responses to bradykinin by bradykinin receptor antagonist HOE 140 (Fig. 7) present in die recording solution, and selective inhibition of cell biosensor responses to acetylcholine by acetylcholine receptor antagonists atropine and α-bungarotoxin present in the recording solution (Figs. 8 A and 8B).
These results illustrate an important aspect of the present invention — that is, die ability to use specific receptor antagonists to confirm the identity of die receptor giving rise to the cell condition reporter signal. As is discussed below, many receptors, upon activation, result in a similar signal (such as a rise in intracellular free calcium). Since most cells (and accordingly, cell biosensors) express a variety of different receptors, a respoase to a selerted ligand may not be interpretable, particularly if the identity of the selected ligand is not known. In contrast to the generality of cellular responses to receptor activation, there exist a wide variety of very specific receptor antagonists, which are often able to discriminate closely-related receptor subtypes (e.g., Hille). Accordingly, it may be desirable to perform a series of separations on the same sample, and include various specific receptor antagonists in some of die runs. As is demonstrated in Example 4 and Figures 7, 8A and 8B, such an approach is effective at confirming die identity of receptors giving rise to the signal recorded from the cell condition reporter. The receptor-specific antagonists may be present in the recording solution, -as shown in Example 4, or they may be applied locally to die cell biosensor in the biosensor detection field. For example, a second capillary containing the antagonist can be positioned to apply antagonist eluent (e.g. by pressure ejection) onto die cell biosensor detection field during a separation run. The concentration of the antagonist can be adjusted such diat it is effective to antagonize the receptors in the cell biosensor detertion field, but to fall off to levels which do not substantially interfere with receptors outside of the detection field. By applying a recording chamber perfusion as described above, a build-up of antagonist concentration can be avoided. If a build-up of antagonist does occur, die chamber may be perfused widi fresh solution as described. It will be understood d at more dian one source of antagonist (preferably different antagonists) may be directed at die biosensor detertion field. These sources may be independently controlled, for example, using pressure ejection, to apply different antagonist at selected times.
Example 5 shows mat mediods of ie present invention may be used to separate and resolve distinrt ligands, such as bradykinin and Lys-bradykinin, having similar chemical characteristics. The data are shown in Figure 9. Lys-bradykinin and bradykinin are identified as two well-resolved peaks, even though diey differ by only one amino acid (American Peptide Company, Inc., Sunnyvale, CA; 1994-1995 catalog).
These results further demonstrate mat the biological activity of closely-related compounds can be assayed using a capillary-based separation means in conjunction widi a cell biosensor, such as described herein. This application may be particularly useful in assaying breakdown products or proteolytic fragments of a parent compound. For example, if the parent compound is a pro-peptide diat gets digested down to an active species, the appearance of die active species may be monitored using mediods of die present invention, by employing a receptor for the active form of the peptide. In a related application, die biological activity of breakdown products of a drug or dierapeutic agent may be monitored.
Several observations regarding use of the system shown in Figures IA and IB may be useful. More reproducible results seem to be obtained when the capillary oudet is positioned at least about 20 μm away from die cells (reducing the likelihood of damage to die cells by die electrophoresis field). Further, it is desirable to prevent unnecessary movement of die capillary widi respect to the cell biosensor when the cell biosensor consists of a single cell or only a few cells and die capillary diameter in similar to d e cell's diameter. Standard vibration-control precautions, such as using a vibration isolation table (Newport Corp., Irvine, CA), can alleviate these potential problems.
B. Direct Transmembrane Voltage Detection
Methods of die present invention may also employ direct transmembrane voltage detection widi extracellular or intracellular microelertrodes. The activation of many receptors, particularly those directly linked to ion channels, results in a change in the transmembrane voltage of die cell. Such a change may be deterted, for example, with die use of voltage- sensitive dyes (as detailed above), or using voltage-recording electrodes. Three general modes of voltage recording using electrodes are (i) extracellular recording, where an electrode or electrodes are placed in die vicinity of die cell, but do not penetrate die plasma membrane, (ii) intracellular recording, where the electrode is in electrical communication with the interior of die cell, and (iii) patch clamp recording. Intracellular and patch recording are capable of measuring an intracellular potential widi respect to die badi, and detecting changes to diat value over time, while extracellular recording is typically useful only for detecting changes in the membrane potential. With intracellular and patch recording, it is also possible to "voltage- clamp" the cell membrane to a desired voltage by injerting current, eidier dirough the voltage recording electrode or dirough a separate current-passing electrode. In voltage-clamp mode, d e recorded signal is typically die amount of current passed to maintain die cell at d e desired potential as a function of time.
Extracellular electrodes function by detecting a change in die electric field in die vicinity of an electrically active cell. They are not particularly effective at quantitating the amplitude of a transmembrane voltage change, but can qualitatively detect diat a voltage change has occurred. Extracellular recording electrodes may be either built in to die substrate on which die cells are grown (Pine, Meister, et al.), or placed adjacent the cell biosensor using, for example, a micromanipulator. A variety of extracellular recording techniques have been described which may be employed in mediods of die present invention (e.g., Pine, Forda, et al, Gross, et al., Cohen, et al., Meister, et al.).
Intracellular recording typically utilizes a sharp glass microelertrode filled widi a high salt electrolyte solution, typically — 3M KC1. A metal electrode, typically silver with a coating of silver chloride, is placed in electrical contact with he electrolyte solution and connected to a voltage recorder, or voltage clamp. If a voltage clamp is used, the output is typically directed to a chart recorder or a computer for later analysis.
Intracellular recording and/or voltage clamp may be used, for example, widi Xenopus oocytes, as detailed in Example 2 herein. Oocytes are advantageous because diey are capable of expressing injected mRNA encoding specific receptors. Mediods for expressing ion channels and receptors in Xenopus oocytes, and voltage/current recording from oocytes are well known
(e.g. Leonard and Snutch; Wang, et al.; see below).
Patch recording, typic-ally conducted in die voltage-clamp mode, exploits the observation that under appropriate conditions, it is possible to obtain an electrically-tight seal between the tip of a blunt fire-polished pipet filled with physiological saline solution and the plasma membrane of a cell (Ha ill, et al; Sakmann and Neher). Such seals introduce a resistance between the interior of d e pipet and die badi ranging from about 1 GOhm to over 100 GOhms. When a high-resistance seal is obtained, it is often possible to visualize the activity of single ion channels opening and closing in die membrane beneadi the patch pipet. This mode of recording is referred to as "cell-attached".
If a brief pulse of suction is applied to die patch pipet, it is possible to rupture the membrane beneath d e pipet and establish electrical communication with die interior of die cell (whole-cell mode). It is also possible to isolate a patch of membrane by pulling die patch pipet off die cell . If the pipet is pulled off in cell-attached mode, the patch is usually an " inside-out" patch, because die cytoplasmic face of the membrane patch is exposed to die extracellular solution bathing the cell. If die pipet is pulled off in whole-cell mode, d e cell membrane typically reseals and forms a patch in an outside-out configuration (die extracellular aspect of the patch membrane is facing die bath solution).
Whole-cell mode is similar to intracellular recording or voltage clamp, but it allows control of die cell's intracellular solution. The patch modes (cell-attached, inside-out and outside-out) enable die recording of currents through single ion channel molecules. In all modes, electrical communication between the pipet and die patch clamp itself is via an electrode immersed in the physiological electrolyte present in the patch pipet. As is die case with voltage recording, die electrode is typically a silver wire coated widi silver chloride. The methods outlined above are well known in the art (e.g., Smith and Gage, Conn,
1991a, Wallis, Hille). Equipment and instrumentation for acquiring and analyzing data using voltage recording, voltage clamping, patch clamping, oocyte injection, oocyte recording, etc. is widely commercially-available. For example, pipet pullers maybe obtained from Narashige, USA, Inc. (Greenvale, NY) or Zeitz-Instrumente Vertriebs (Augsburg, Germany); manipulators may be obtained from Narashige, USA, or Newport Corp. (Irvine, Q^ pressure injectors may be obtained from Dagan Corporation (Minneapolis, MN) or Fisher Scientific (Pittsburgh, PA); recording chambers may be obtained from Dagan Corp.; and patch clamps, voltage clamps, computer interfaces and digital tape recorders may be obtained from Dagan Corp. or Axon Instruments (Foster City, CA).
Figure 4 shows a schematic of an exemplary device useful for practicing die present invention. This device includes a capillary 2 for the separation of a mixture of ligands. A mixture of ligands is introduced into the capillary inlet as above. The cell end 4 of the capillary is placed in proximity to a cell biosensor, such as an oocyte 6. The device further includes an elertrophoresis reservoir 8 around die capillary approximately 10 cm upstream from die cell end, which is sealed at its bottom end 10 to prevent leakage of electrophoresis buffer into the solution bathing the cell biosensor. The reservoir contains an electrophoresis buffer, such as CE Ringer, in electrical communication with the electrophoresis ground 12. The capillary has a fracture 14 enabling electrical communication between the interior of the capillary and die electrophoresis buffer in the reservoir. The fracture serves to ground die capillary upstream from the biosensor to reduce electrical interference in voltage-clamp recording. The portion of the capillary above the fracture is immobilized to a splint, for example, glued 16 to a side of die reservoir containing the elertrophoresis buffer. The transmembrane voltage and/or current of die cell biosensor is measured using, for example, a two-electrode voltage clamp, which includes a current-passing electrode 18, a voltage-recording electrode 20 and a badi ground 22 in electrical communication with die solution 24 bathing the cell biosensor.
The cell end of die capillary can be positioned at a selected location widi respect to die cell biosensor by means of an XYZ positioner attached to, for example, the elertrophoresis reservoir 8, essentially as described above.
The fracture 14 in the capillary can be fashioned as described by Linhares and Kissinger. In this mediod, the capillary is glued to a 2 cm x 1 cm microscope slide with epoxy glue (e.g. SIG Mfg. Co., Montezuma, IA) such that an exposed portion, immobilized at bod ends, rests approximately 1 mm above the surface of the slide. A diamond capillary glass cutter is used to make a small scratch on the top of die exposed portion, and a pointed stylist is gently pushed between the slide and die capillary to produce a fracture. The fracture assembly is placed, for example, in a 5 ml Teflon vial with a hole drilled in die bottom. The outlet end of the capillary is pushed through a rubber septum that plugs the hole. Several approaches may be taken to reduce electrical noise during recording. If it is desired to obtain an uninterrupted record of cell biosensor electrical activity, proper grounding of all components ne-ar the recording chamber is necessary. In particular, the portion of the capillary between the fracture and die outlet (cell end) may be coated widi an electrically- conductive material and grounded. Mediods for electroplating glass or silica with electrically- conductive coatings may be found, for example, in Bard.
If regular interruptions in the electrical recording are permissible, methods of die present invention may be practiced using a pulsed electrophoresis power supply (Cohen, M.L., et al., 1992). The acquisition computer may be programmed to sample voltage or current records only during die period diat die electrophoresis power supply is off. This may decrease die electrical noise in die system by about an order of magnitude, and may allow die recording of ion currents through single ion channels.
Such a biosensor system can provide a nearly unprecedented level of sensitivity - for example, since an inotropic receptor typically requires only a few agonist molecules for opening, the method may be applicable for the detection of samples containing only minute amounts of a selerted ligand, perhaps as little as a few hundred molecules.
Example 2 details an application of die recording system described in Figure 4 to record responses of Xenopus oocytes to serotonin delivered using a capillary-based separation means. The electropherograms are shown in Figure 5. Upward deflections indicate inward current. The current is likely due to die artivation of oocyte-endogenous calcium-artivated chloride channels, stimulated by a rise in intracellular free calcium due to die binding of serotonin to die recombinantly expressed serotonin receptor.
The results show diat electrodes may be effectively employed widi cell biosensors to detect a selected ligand in a mixture of ligands separated using a capillary-based separation means. The result also demonstrate mat the signal detected from a cell condition reproter or electrode may be due to a secondary response to ligand binding to a specific receptor. In particular, a variety of ion channels are now known to respond to intracellular second messengers activated by die binding of an agonist to a receptor (Hille).
VI. RECEPTORS AND LIGANDS
A. Receptors
Nearly all cells possess on their surface various receptors specific for appropriate agonists. Upon binding of an appropriate agonist, the receptor alters its conformation and initiates a cellular response. In the case of inotropic receptors, such as die nicotinic acetylcholine receptor or the N-methyl D-aspartate (ΝMDA) receptor, the receptor is itself an ion channel, and binding of die appropriate ligand opens die ion channel, typically on the order of milliseconds. Opening of the channel results in an increased membrane conductance for those ions which can pass through the receptor, which typically results in a change in the membrane potential. The membrane potential change can be detected widi voltage-sensitive electrodes, or voltage-sensitive dyes (as described above). If the membrane is clamped to a selected voltage, the opening of die channel typically results in an ionic current flowing through the channel, which can be measured by die voltage clamp apparatus.
In addition to detecting voltage changes, it may be possible to directly detect the influx or efflux of certain ions through open ion channels, using fluorescent dyes sensitive to die concentrations of those ions (described above). For example, many ion channels and inotropic receptors are at least partially permeable to calcium. Since the electrochemical driving force for calcium is typically inward at most physiological potentials, die opening of a channel permeable to calcium is accompanied by an increase in die intracellular calcium concentration in die vicinity of die ion channel. This increase may be deterted using, for example, a calcium-sensitive dye, such as fluo-3.
In the case of metabotropic receptors, binding of die appropriate agonist to the receptor may cause a conformational change that typically initiates one of several well-characterized biochemical signaling cascades, many of which result from an interaction of the activated receptor widi a "G-protein" (Stryer, Teichberg and Habenicht, Kito). The specific cascade initiated depends, of course, on die receptor activated. The intermediates in an activated cascade may involve calcium and cyclic nucleotides, such as cAMP and cGMP.
For example, artivation of the inositol triphosphate (IP3) pathway typically results in the release of calcium from intracellular stores, and leads to a transient increase in free intracellular calcium concentration (Hille). Accordingly, activation of receptors which exert their effect through die IP3 pathway is expected to result in an increase in intracellular calcium, which can be deterted using, for example, one of die calcium indicator dyes described above. In an analogous manner, activation of receptors which exert their effects through cAMP may be deterted using, for example, the cAMP indicator system described above. One of the advantages of die methods of die present invention is die sensitivity afforded by die amplification inherent in many metabotropic receptors. Because a single receptor activation event can result in the liberation/production of thousands of second messenger molecules, the present methods may be used to detect minute amounts of a selected ligand. It will be understood tiiat because receptors were often named on die basis of what was considered to be die natural agonist, and before die molecular structure of die receptor was elucidated, certain receptor names may include a group of molecularly-distinct receptors, some of which may be inotropic, and some metabotropic. For example, the term "acetylcholine receptors" refers to both nicotinic acetylcholine receptors, which are inotropic, and muscarinic receptors, which are metabotropic. A similar situation exists for "glutamate" (Nakanishi), "serotonin" (Peters, et al.) -and certain odier receptors. Further, it will be understood diat die agonist binding site of transmembrane receptors may be on the extracellular portion (e.g. nicotinic acetylcholine receptors (nicotinic AChRs), intracellular portion (e.g. cAMP receptors) or either, depending on die receptor subtype. For example, the ATP binding site is on the extracellular side in die case of calcium-selective receptors opened by ATP, and on the intracellular side in die case of potassium-selective receptors closed by ATP (Barnard, et al., Bean, et al).
According to mediods of die present invention, cell biosensors may be derived from cells which are known to express a specific receptor under known conditions. For example, PC12 cells are known to express a number of receptors and ion channels (e.g. Shafer, et al), including acetylcholine (both nicotinic (Greene, et al., 1976) and muscarinic (Jumblatt, et al.) receptors, ATP receptors (Fasolato, et al.) and bradykinin receptors (Fasolato, et al.).
The acetylcholine receptors on PC12 cells resemble neuronal ACh receptors more than muscle ACh receptors. Properties of neuronal ACh receptors are reviewed, for example, by Sargent.
Many different receptors can be used wim the mediods of the present invention, provided diat binding of a selerted ligand alters die function of the receptor in a way detectable by a cell condition reporter. Examples of inotropic receptors suitable for use with methods of the present invention include muscle and neuronal nicotinic acetylcholine receptors, γ-aminobutyric acid A (GABA receptors, glycine receptors, glutamate receptors (including kainate-type, quisqualate-type and NMDA type), 5-HT3, ATP receptors, cGMP receptors and cAMP receptors. The physiological properties of the above receptors, expressed in vitro, have been reviewed, for example, by Barnard, et al.
Within these and other inotropic receptors, mere may exist a considerable heterogeneity among members of one class. The glutamate family of receptors, for example, has a particularly large number of variants, some of which are selectively sensitive to different ligands, such as NMDA (e.g. Nakanishi). The family may be more appropriately called an "excitatory amino acid" receptor family, because it is possible that amino acids odier dian glutamate (such as aspartate) are the natural ligand for die receptor in some circumstances.
Inotropic receptors are typically comprised of 4-6 homologous subunits, typically termed , β, y, δ, e and {*. The pentameric muscle nicotinic acetylcholine receptor, for example, is composed of 4 homologous subunits in the stoichiometry a b arranged around a central cation-permeable pore. Molecular cloning has demonstrated diat cDNAs encoding most receptor subunits are each members of a family of highly homologous but distinct sequences arising from alternative splicing and/or different genes. Expression studies suggest diat in some cases, few if any variations on die "natural" subunit composition form functional receptors, while in other cases, functional receptors may be made from most of the different combinations possible with die clones at hand. Some of die resulting variants may have ligand- binding and/or permeation properties that differ from the native receptors, and diat may be exploited for methods of die present invention. For example, certain subunit compositions of AMPA-Kainate (non-NMDA) excitatory amino acid receptors have a low calcium permeability, while others have an increased permeability (see, for example, review by Nakanishi).
According to mediods of die present invention, the ability to construct biosensors with specific properties (i.e. biosensors having specific receptors), and couple die biosensors widi a separation means, is an advantage for the detertion a selected ligand in a mixture of ligands. In other words, by judiciously choosing cells expressing (eidier natively or recombinantly) the appropriate receptor subunits, a cell biosensor may be construrted which is sensitive to a selerted ligand, and which generates a known biosensor response (such an increase in calcium coupled widi a calcium-sensitive dye) diat can be deterted.
For example, a cell biosensor containing a calcium-permeable AMPA-kainate, NMDA or nicotinic acetylcholine receptor may be used in conjunction widi a calcium-sensitive dye, because these receptor types are permeable to calcium, and opening of die receptors results in an influx of calcium through die receptor channel (given that die electromotive force for calcium is inward). Alternatively, a cell biosensor containing an inotropic GABA or glycine receptor may be used widi a chloride-sensitive cell condition reporter, such as a chloride- sensitive dye, since diose receptors are generally anion-selective channels. It will be understood diat inotropic receptors may be present in intracellular membranes, as well as the plasma membrane of a cell biosensor. For example, the IP3 receptor is located in the endoplasmic reticulum (ER) and is responsible for the release of calcium from die ER in response to IP3 (die elevation of IP3 in the cell may, in turn, be a response to the activation of a plasma membrane receptor, such as a muscarinic acetylcholine receptor; Hille). The ryanodine receptor, also an intracellular calcium-release channel, was first recognized in the sarcoplasmic reticulum (SR) of muscle. It is partially-opened by ryanodine, and may be fully activated by calcium or caffeine (Hille).
Such "intracellular membrane" receptors may be used in conjunction widi a cell biosensor in dieir native state — that is, expressed in an intracellular membrane (e.g. ER) of the cell biosensor, or diey may be recombinantly expressed on d e plasma membrane of a host cell.
A large number of metabotropic receptors may be suitable for use with the present invention. Plasma membrane-associated receptors may be grouped into at least diree "superfamilies" of receptors (Hille): (A) "classical" G-protein-coupled receptors have seven transmembrane segments, the ligand-binding site is at least partially associated with a transmembrane domain, and die site of G-protein interaction is cytoplasmic, (B) tyrosine kinase, or growth factor receptors have one transmembrane segment linking a large, glycosylated extracellular ligand-binding domain widi a large intracellular portion diat usually includes a protein tyrosine kinase domain and a site for autophosphorylation, and (C) guanylayl cyclase family diat is similar to the growth factor receptors, except that the intracellular portion has a guanylayl cyclase domain, rather man a tyrosine kinase domain, and also contains another protein kinase domain.
A wide variety of metabotropic receptors falling, for example, into each of the above three classes is known in the art. The following are a few specific examples of such receptors.
Epidermal growth factor (EGF) receptors -are members of the tyrosine kinase family of receptors. Artivation of the receptor initiates a cascade of events (Carpenter, et al, Wells, et al.) that results in internalization of the receptor and a rise in cytosolic free calcium.
An atrial natriuretic peptide (APN) receptor is a member of the guanylate cyclase family of receptors (Chinkers, etal.). Artivation of this receptor by APN results in an increase in cGMP. Detertion of activation of this receptor on a cell biosensor may be accomplished, for example, by co-expressing a cGMP-sensitive ion channel in the cell biosensor.
The 5HTlc receptor used in experiments described herein is a member of the seven transmembrane segment G-protein-coupled receptor family. Artivation of this receptor expressed in Xenopus oocytes results in an increase in intracellular calcium, presumably through an IP3 pathway. The calcium then presumably activates calcium-sensitive chloride channels normally present in the oocyte plasma membrane (Gunderson, et al.) and results in an inward current (Julius, et al.). A receptor for substance P, considered to function as a neuropeptide or modulator in the central and peripheral nervous system, is also a member of the seven transmembrane domain G-protein-coupled receptor family (Hershey, et al.), as is a substance K receptor, as is the ETB receptor for endodielin (Sakurai, et al, Arai, et al.), a peptide initially identified in vascular endodielial cells. Other examples of receptors in this family include an interleukin- 8 (IL-8) receptor (Murphy, et al., Holmes, et al.), and a human diromboxane A2 receptor (Hirati, et al.). Activation of the receptors identified above results in an increase in intracellular free calcium, presumably through an IP3 pathway.
Several putative odorant receptors have recently been cloned (Buck, et al.). These receptors also belong to the seven tr.ansmembrane segment G-protein-coupled receptor family. Cell biosensors containing odorant receptors may be particularly advantageous for use widi mediods of die present invention. The olfactory system can differentiate among diousands of different odorant molecules dirough a family of perhaps several hundred different G-protein- coupled receptors (Buck, et al.). A number of different odorant receptors have already been cloned (e.g. Permentier, et al.). The odorant molecules can be divided into several "odor classes", which include fruity, such as citralva and citraldymediylacetal; herbaceous, such as eugenol and ediylvanillin; minty, such as menthone and eucalyptol, floral, such as geraniol and hedione; and putrid, such as pyrrolidine and pyrazine. The molecules can also be grouped by chemical classes, which include aromates, such as coniferan and helional; aldehydes, such as lyral and lilial; amines, such as triethylamine and phenylethylamine; organic acids, such as isovaleric acid and butyric acid; sulphydryls, such as 2-mercaptoethanol and furfurylmercaptan; methoxypyrazines, such as 2-isobutyl-3-metiιoxypyrazine -and metiioxypyrazine; and alkylpyrazines, such as 2,3,5-trimethylpyrazine and 2-medιylpyrazine.
The cellular response to odorants binding to odorant receptors is typically an elevation in cAMP (Breer, et al), though IP3 may also be involved (Boekhoff, et al.). It is apparent that at least some odorant receptors may exhibit a relatively "broad tuning" - being responsive to several odorant molecules, while others apparently have a much narrower range (Ra ing, et al.). Expression of selerted odorant receptors widi known tuning and selective properties in cell biosensors having, for example, a calcium indicator dye or die cAMP detector discussed above, may provide a method for specifically detecting a wide variety of "aromatic" separated ligands. B. Recombinant Expression of Receptors
According to mediods of die present invention, receptors in cell biosensors diat are specific for a selected ligand may be normally or recombinantly expressed by the cell from which die cell biosensor is derived. A large amount of information is available on the specific receptors expressed by selerted cell types. Examples have been provided above, in particular, the receptors expressed by PC 12 cells. Following die teachings disclosed herein, one of skill in die art may readily select any of a number of cells, expressing specific receptors and known to those skilled in die art, for use as cell biosensors.
Similarly, following the guidance presented herein, one of skill in die art may choose to recombinantly express any of a variety of receptors using methods known to those skilled in die art. Several different methods have been used for recombinantly expressing specific receptors in host cells (Gould, Conn, 1991b). The methods typically employ standard cellular and molecular techniques (Sambrook, et al, Ausubel, et al), with a focus on protein expression techniques (Goeddel, Kriegler), and may include special considerations depending on die nature of the receptor being expressed. Such considerations may include (i) targeting transmembrane receptors to express appropriately in the plasma membrane of the host cell, (ii) providing a system capable of appropriate post-translational modification, and (ii) co-expression of multiple subunits in the same cell for heteromultimeric receptors. Several examples of expression systems which address these considerations are reviewed below. Perhaps die most widely-used expression system for transmembrane, multimeric receptors is the Xenopus laevis oocyte. Xenopus oocytes provide a simple, rapid and transient expression system that can easily accommodate heteromultimeric receptors. The oocytes contain - 50-100 ng of endogenous maternal cytoplasmic mRNA, but translation of this message is largely inhibited by die presence of various inhibitory proteins. The in vitro translation machinery in the oocyte, however, is capable of correct post-translational modification, proteolytic cleavage and plasma membrane insertion (Wang, et al.).
To recombinantly express a message encoding a specific receptor, die mRNA(s) encoding die receptor is injected, for example, into a collagenased oocyte, and expression of die mRNA is assayed anywhere between 24 hours up to one week following injection. A detailed description of die oocyte expression protocol is provided in Example 2, herein. In addition, a number of references specifically review this procedure (e.g. Gurdon, et al., Leonard and Snutch, and Wang, et al.). Further, numerous groups have used the oocyte system for receptor expression (e.g. White, et al., Meguro, et al., Hollmann, et al., Zagotta, et al., Monyer, et al., and McEachern, et al.). It may also be desirable to construct cell biosensors derived from cell lines expressing a specific receptor. Several groups have reported mediods for transforming cells, particularly mammalian cells, with sequences encoding various receptors.
Pritchett, et al. , transiently co-expressed GABAA receptor α and β subunits in human embryonic kidney cells using the eukaryotic expression vector pCIS2 (Eaton, et al.). The vector contained die a and β subunits in a tandem arrangement, driven by a human cytomegalovirus (HCMV) promoter-enhancer. The vector was introduced using a modified
CaP0 precipitation technique (Chen, et al., 1987).
Rich, et al., and Gregory, et al., used die vaccinia virus-T7 hybrid system, developed by Fuerst, et al., and Elroy-Stein, et al., to express die cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa and JME/CF15 (a CF airway epithelial cell line) cells. Advantages of this system include (i) infection and expression of a wide variety of eukaryotic cells, (ii) the virus provides the enzymes necessary for transcription in the cytoplasm of the infected cell, eliminating die need for nuclear processing, (iii) the gene product can be correctly processed and targeted to die cell surface, and (iv) a high percentage of host cells express die recombinant DNA. A similar system has also been used to express potassium channels (Leonard, et al.).
Claudio, et al., 1987b, stably integrated me a, β, y and δ subunits of the Torpedo calif ornica nicotinic AChR into the genome of a thymidine kinase deficient (tk') adenine phosphoribosyltransferase deficient (aprt') murine fibroblast L-cell using a simian virus 40 (SV40) expression vector (Claudio, et al., 1987a) containing all four subunits.
Scheuer, et al. , stably expressed sodium channels in CHO cells by co-transfecting rat IIA sodium channel cDNA in pECE (Ellis, et al.) under control of the SV40 early promoter (pVA222) with pSV2neo. Expression vectors suitable for expression of receptors are also available from a variety of commercial sources. For example, Clontech (Palo Alto, CA) offers a baclovirus expression system which utilizes insect cells as host cells to express recombinant proteins. Standard eukaryotic expression vectors are available from Clontech, Stratagene (La Jolla, CA), Promega (Madison, WI) and other suppliers.
C. Cellular Responses to Receptor Activation
According to mediods of die present invention, die binding of a selected ligand, present in a mixture of ligands, to a specific receptor may be selectively detected using a cell biosensor having die specific receptor and containing a cell condition reporter, provided mat the cell biosensor is used in conjunction with a means to separate the selected ligand from the mixture of ligands. The response of the cell biosensor to ligand binding may be a change in die transmembrane voltage, intracellular free calcium concentration, or die like, which is in turn detected by a cell condition reporter. As discussed above, a die activation or inhibition of inotropic receptors typically results in a change in the transmembrane voltage, and possibly intracellular calcium concentration. Activation of a metabotropic receptor typically results in an increase in the concentration of specific second messengers. These may be deterted directly (for example, calcium or cAMP), or indirectly, for example, following binding of die second messengers to targets, such as other inotropic receptors (sensitive to intracellular agonists). An example of an indirect detection scheme detailed herein is die response of oocytes to serotonin in Example 2. Activation of the expressed 5-HTlc receptor presumably mobilizes internal stores of calcium, which in turn open a calcium-sensitive chloride channel. The electrical response to die opening of die chloride channel is deterted by the cell biosensor in die example.
D. Ligands
Selerted ligands separated from a mixture of ligands may be receptor agonists, antagonists, competitive inhibitors, or non-competitive inhibitors. They include endogenous ligands, such as acetylcholine, and exogenous ligands, such as toxins widi an affinity for a specific receptor (e.g. α-bungarotoxin. The solution bathing the biosensor may contain additional receptor agonists, .antagonists, competitive inhibitors, non-competitive inhibitors and die like. These additional components may interact widi, enhance, or inhibit the binding of die selected ligand to a specific receptor, and accordingly, may be used to confirm die identity of a selected ligand or odierwise facilitate the interpretation of information available from the cell biosensor.
For example, according to die teachings of the present invention, interpretation of signals from cell biosensors normally expressing a specific receptor may be complicated by die fact diat a given cell is likely to be expressing a variety of receptors other diat die selected receptor. Because many different receptors can result in a similar detectable response (i.e. a voltage change or an increase in free calcium concentration), die use of specific receptor antagonists is an important aspect of the present invention in confirming the identity of the receptor responsible for a particular cell biosensor response. As discussed above, antagonists may be applied either locally to a cell biosensor, for example, by way of an additional capillary, or simply introduced into d e recording solution. Ligands may bind to d e extracellular portion of a transmembrane receptor, membrane portion of a transmembrane receptor, or the intracellular portion of a transmembrane receptor. Ligands can also, of course, bind to any portion of an intracellular receptor. Ligands diat bind intracellularly need to access die intracellular portion of a cell biosensor. If the cell biosensor is a potion of an intact cell with ready access to intracellular domains (such as an inside-out patch or a permeabilized cell), access does not pose undue difficulty. If, on die odier hand, the cell biosensor is an intact cell or cells, and die ligand is not capable of diffusing across the plasma membrane, die ligand may need to be transported into the cell by an endogenous or exogenous transmembrane transporter molecule. Ligands may be obtained from a variety of sources. An exemplary source of a mixture of ligands is a cell or tissue extract. This source may be particularly advantageous when information is desired as to die identity of a putative endogenous ligand for a specific receptor. The mixture may include substances to stabilize the state of ligands in the mixture, for example, protease inhibitors. Alternatively, a mixture of ligands may be obtained from a combinatorial library, such as a peptide or chemical compound library (Houghten). Combinatorial libraries are typically screened by introducing a complex mixture species to a screening preparation, and if a positive response is obtained, repeating die process with lower-complexity mixtures until the compound having die desired activity is identified. Using mediods of die present invention, the complexity of the mixture is reduce on-line, by separating the mixture using a capillary-based separation means prior to applying die components to a cell biosensor. The individual species in d e library are deterted serially, as described above.
In a related aspect, a mixture of receptor antagonists, such as conotoxin venom (Olivera, et al.), may be separated and screened in combination widi a receptor agonist constitutively present in the vicinity of the cell biosensor. As described above, die agonist may be applied using, for example, a second capillary whose effluent is also directed at die cell biosensor detection field. By judiciously employing, for example, bath perfusion and continuous scanning (as described above), desensitization of die receptor may be avoided, and selerted antagonistic ligands reaching the cell biosensor may result in a decrease in the receptor response.
VII. UTILITY.
As detailed above, mediods of die present invention have a variety of uses. Further, the methods provide a number of advantages over methods utilized in the prior art. For example, according to mediods of die present invention, cell biosensors of die type described herein are not particularly useful at identifying a selected ligand in a mixture of ligands, unless they are coupled to an effective separation means. This is in large part because, as detailed above, a cell biosensor produces a similar signal (change in free intracellular calcium concentration and/or change in transmembrane voltage) in response to a wide variety of ligands. If a complex mixture of ligands is directly applied to a cell biosensor, interpretation of the response will be difficult, if not impossible. Even with the simultaneous use of a variety of receptor antagonists, interpretation will be challenging — all that is required is for one of die ligands in die mixture of ligands to activate a receptor for which no antagonist is present, and activation of which, for example, increases intracellular free calcium concentration, and die response is uniterpretable.
According to mediods of die present invention, however, such a biosensor is coupled to a means by which die complex mixture of ligand is separated, so diat die effect of individual ligands present in the mixture may be assayed in a serial fashion. In this manner, selerted ligands present even in a highly complex mixture containing many different ligands capable of generating a response of the cell biosensor may be identified, because the cell biosensor responses to the individual ligands are separated in time. The individual responses may be identified by running standards, as shown in Figures 2 and 3, and/or by using specific receptor antagonists, as shown in Figures 7, 8A and 8B, and discussed above. The present invention offers a number of other distinct advantages. For example, methods of the present invention may be particularly useful in identifying ligands which are difficult to detect by traditional separation means, such as acetylcholine (Cooper, et al). Further, die mediods may be useful for identifying die biological activity of closely-related compounds, such as bradykinin and lys-bradykinin (Example 4). This approach may be used, for example, to assess die biological activity of breakdown products of a selerted ligand, such a drug or therapeutic agent. In anodier example, an inactive precursor for an active peptide may be present in a sample, such as a blood sample. The rate of generation and identity of active (cleaved) species may be followed using mediods of die present invention. In particular, if a non-invasive supplemental detector or detertion means is positioned to detect the separated ligands before diey are delivered to die cell biosensor, die rate of formation/degradation and biological identity of die precursor and various proteolysis products may be readily followed.
The invention may be similarly be applied to the study of other biologically-active compounds widi limited lifetimes. Because both a traditional identification (with a supplemental detector) and the biological activity (with a cell biosensor) can be assessed within seconds of one another, a correlation can be established between selected "peaks" detected with a traditional, supplemental detector, and die biological activity of those peaks as determined by a specific cell biosensor.
Mediods of die present invention may also be used to screen for uncharacterized endogenous agonists of known receptor, such as an opiate receptor. A tissue extract of a portion of the brain thought to contain d e endogenous agonist may prepared and analyzed according to methods of die present invention using, for example, a cell expressing an opiate receptor along with an inwardly-rectifying potassium channel (Reuveny, et al. , Dascal, et al.). Using traditional methods, one would typically fractionate die extract, resulting in perhaps a hundred or more individual samples, and die fractionated samples would be analyzed one-by- one for activity. Using methods of the present invention, the fractionating and assaying can take place in a single run lasting as little as a few minutes.
Another exempl-ary use of methods of die present invention is for assaying the identity, concentration and release sites of neurotransmitters and hormones from individual cells (e.g. Aplysia neurons). According to diis aspect of the invention, large cells are dissected and plated in an assay dish which is placed at die inlet of a capillary-based separation means. The large cell is stimulated, for example, using electrical stimulation or agonists, such as peptides, and die inlet end of die capillary (etched down to a small diameter widi, for example, hydrofluoric acid) is positioned at selected locations along die cell. A separation is performed, and the separated mixture is detected widi a cell biosensor expressing appropriate receptors (i.e. receptors for substances thought to be released by die large cell).
The above method may also be applied to assaying die identity, concentration and release sites of ligands, such as neurotransmitters from tissues, such as hippocampal slices (Nicoll, et al). In particular, some vesicles contain more that one neurotransmitter. By coupling a capillary-based separation means (to resolve individual components at may be released by stimulated cells) widi a cell biosensor (expressing receptors for anticipated components released by stimulated cells), identification and quantitation of the relative amounts of the components is possible. Determining which ligands are released from which cells in response to specific types of stimulation, and how diese responses differ in certain diseased states (such as epilepsy) may be useful for the development and targeting of appropriate therapeutic compounds.
A related application is die determination of the content of single cells or cell organelles. As discussed above, individual cells, and to some extent, cellular organelles, comprise complex mixtures of ligands. Identification of selected ligands in those mixtures may be desirable for die development of certain therapeutic and/or diagnostic molecular tools. According to this aspect of the invention, individual cells or organelles, such as vesicles, are introduced to die inlet end on a capillary-based separation means. The contents and biological activity of the cells or organelles may then be identified using a ligand separation means coupled to a cell biosensor, or to a cell biosensor in combination with a supplemental detertion means.
The following examples illustrate, but in no way are intended to limit die present invention.
MATERIALS AND METHODS
A. Capillary Electrophoresis
Ligands were separated by capillary electrophoresis (CE) using standard techniques (Grossman and Colburn, Gordon, et al.) with die following parameters/modifications. The experiments were typically performed using a biosensor-compatible buffer (see below) as die electrophoresis buffer. Fused silica capillaries (I.D. = 15, 25 or 40 μm, O.D. = 360 μm) were obtained from Polymicro, Inc., Phoenix, AZ. Capillary lengtiis were typically between about 23 and 36 cm. The power supply was typically set to provide a field of 230 to 870 V/cm, resulting in electroosmosis (bulk solution flow) delivering sep-arated positive, neutral, and negative species to the cell biosensor.
B. Biosensor-Compatible Electrophoresis Buffers
Ligand mixture separations were carried out using cell biosensor-compatible buffers. A cell biosensor-compatible buffer is a buffer effective to maintain the cell biosensor in a stable and functional condition for die duration of an experiment.
In those instances where the cell biosensor is an intact cell, a cell biosensor-compatible buffer is analogous to a physiologically-compatible buffer - that is, a buffer that does not substantially alter the signal coming from, or disrupt die intact cell. In cases where the cell biosensor is, for example, a permeabilized cell, a cell biosensor-compatible buffer is one that does not compromise the ability of the permeabilized cell to generate a detectable response to a selected ligand. Similarly, if the cell biosensor is a portion of a cell, such as a membrane patch, the buffer does not compromise die ability of me patch to generate the desired signal. In the case of an outside-out patch, the buffer may be similar to one used for an intact cell, such as an "extracellular" Ringer solution. In the case of an inside-out patch, die buffer may be similar to an "intracellular" Ringer solution, such as one containing low calcium and high potassium.
Many examples of physiologically compatible buffers are known in the literature (e.g. Wallis). In experiments performed in support of the present invention, it was determined diat a mammalian Ringer solution can serve as an exemplary "extracellular" biosensor-compatible buffer for separations with capillary electrophoresis. The composition of the Ringer used in the present experiments (capillary electrophoresis (CE) Ringer) was as follows: 135 mM NaCl, 5 mM KC1, 10 mM glucose, 2 mM MgCl2, 2 mM CaCl2, and 10 mM HEPES (pH 7.35)
It will be understood by one skilled in die art that modifications to this composition can be made widiout appreciably affecting die separation and/or physiological characteristics of die solution. For example, according to methods of die present invention, it is not required diat the cell biosensor respond in a manner identical to the native cell from which it is derived; only that it respond in a predictable manner. Accordingly, components other than the selerted ligand may be present in a biosensor compatible buffer, which affect the response of die biosensor to the selerted ligand. Such components may include substances, such as certain organic compounds, which facilitate die separation of a mixture of ligands using capillary-based separation means.
B. Eukaryotic Cells 1. Growth and Maintenance. Rat clonal pheochromocvtoma (PC 12) were obtained from die laboratory E. Shooter, Stanford University. NG 108 cells were obtained from the laboratory L. Stryer, Stanford University. Both cell types can be obtained from the American Type Culture Collection ATCC (Rockville, MD). The cells were plated on an untreated #1 glass coverslip (Fisher Scientific, Pittsburgh, PA) constituting a portion of die bottom surface of a 35 mm tissue culture dish (Fisher) in DMEM medium (Gibco/BRL Life Technologies, Gaithersburg, MD) supplemented widi 5% heat-inactivated horse serum (HyClone Labs, Logan, UT), 10% fetal calf serum (FCS; Gibco/BRL), \% 10 mg/ml glutamine (Sigma) and 1 % Penicillin/streptomycin solution containing 10,000 U/ml penicillin and 10 mg/ml streptomycin (Sigma), and maintained in a 5% C02 atmosphere until use (2-4 days).
C. Xenopus Oocvtes
1. Isolation and Maintenance. Xenopus oocytes were isolated essentially as described by Zagotta, et al. Female Xenopus laevis were obtained from NASCO (Fort Atkinson, WI) and maintained at room temperature (20-22 °C). The frogs were anesthetized by immersing for 20 minutes in 0.1-0.2% ethyl m-aminobenzoate (tricaine; MS-222; Sigma Chemical Co., St. Louis, MO). A small incision was made on die abdomen and die ovarian lobes were removed into OR2 solution (82.5 mM NaCl, 2.5 mM KC1, 1 mM MgCl2, 5 mM HEPES, adjusted to pH 7.6 with NaOH).
The follicular cell layer was removed by digestion widi collagenase (2 mg/ml, type IA,
Sigma) in OR2 solution with gentle agitation for 2-3 hours. Large (~ 1 - 1.5 mm diameter) stage V and VI oocytes were dien transferred to ND96 solution (96 mM NaCl, 2 mM KC1, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES adjusted to pH 7.6 widi NaOH) and maintained at 18°C until used for injection with mRNA (typically a few minutes to a few hours).
2. Synthesis of Recombinant Receptor mRNA. Plasmid pSRlc, obtained from D. Julius (Julius, D.J., et al, Science 241:558, 1988) was used to transcribe 5HTlc receptor cDNA in vitro using standard mediods (Ausubel, et al). Briefly, the plasmid was linearized with NotI (New England Biolabs, Beverly, MA), 2-5 μg of the linearized plasmid were suspended in 50 μl of a standard transcription buffer (Ausubel, et al.) containing 10 mM didiiodireitol (DTT) , 0.5 mM each of ATP, cytidine triphosphate (CTP) and uridine triphosphate (UTP), 0.25 mM guanosine triphosphate (GTP), 0.3 mM m7G(5')ppp(3')G (all from Boehringer-Mannheim, Indianapolis, ID), 50 units "RNASIN" and 40 units T7 RNA polymerase (both from Promega, Madison, WI), and die mixture was incubated at 37°C for 1 hour. A second 40 unit aliquot of T7 polymerase was added and the mixture was incubated an additional hour.
The template DNA was removed by incubating with 2.2 μl RQ1 RNase-free DNase (Promega), and RNA was purified by extractions with phenol/chloroform (1:1, vol/vol) and chloroform, ethanol precipitated (widi ammonium acetate), and resuspended in 10 μl diethyl pyrocarbonate (DEPC)-treated water. Prior to injection, the RNA was diluted to die desired concentration (typically 0.1 μg/μl) with DEPC-treated water.
3. Expression of Exogenous Receptors. Oocytes prepared as above were microinjected widi -50 nl of a solution containing -0.1 μg/μl mRNA, prepared as above, using a Drummond Nanoject (Fisher Scientific, Pittsburgh, PA) pressure injector. Injection pepts were fashioned using a standard patch pipet puller (Narashige, Japan) set to produce pipets with long (- 1.5 - 2 cm) tips. The tips were broken by poking a ChemWipe, and the pipets baked at > 150°C for 4-5 hours to inactivate RNases. The pipets were backfilled with a DEPC-treated saline solution and tips were filled widi solution containing the RNA to be injected.
Injected oocytes were maintained in ND96 solution supplemented widi 2.5 mM sodium pyruvate, 100 units/ml penicillin and 100 μg/ml streptomycin (Pen-Strep mixture obtained from Sigma, cat # P0781) at 18°C. The oocytes were suitable for use as biosensors starting —24 hours after injection.
EXAMPLE 1 OPTICAL DETECTION OF BIOSENSOR SIGNAL Mammalian cells (PC12 or NG108 cells) plated on coverslips as described above were loaded with fluo-3 AM ester (Molecular Probes, Eugene, OR) at room temperature for - 0.5 h. The loading medium contained 18 μM fluo-3, 135 mM NaCl, 5 mM KC1, 10 mM glucose, 2 mM MgCl2, 2 mM CaCI2, and 10 mM HEPES (pH 7.35). After loading, the cells were placed in the same medium without fluo-3 for 0.5 h at room temperature. In many experiments, 6 μM fluo-3 was introduced to die recording medium to replenish dye lost from the cells. This manipulation typically reduces the baseline decrease in fluorescence observed, for example, in Figure 9.
The cell end of the capillary was positioned about 20 to -40 μm from the cells, thereby directing capillary effluent over the cells. The channel of the capillary was slightly larger than the diameter of die cell (see Figure IB), enabling delivery of die effluent over die entire cell surface. Typically, a 15 μM or 25-μm I.D. capillary was used for separations, and 1 to 15 mammalian cells, such as PC 12 cells, were used as die cell biosensor.
Alternatively, the effluent may be directed over selected portions of a single cell, or over a field of a plurality of cells, by selecting capillaries with appropriate I.D.s, cells widi appropriate diameters, and positioning the cell end accordingly.
Effluent species diat evoke changes in [Ca2+]- were detected widi -an epi-illuminated fluorescence microscope. Dishes containing the cells were transferred to die stage of a Diaphot-TMD-EF inverted fluorescence microscope (Nikon, Inc., Melville, NY), where they were maintained at - 35 to 37°C by heating die 100X (1.3 N.A.) oil-immersion lens used for illumination and fluorescence collertion. The cells were illuminated widi 470-490 nm light (Nikon filter block, B-1A) and fluorescence (λ> 520 nm) was imaged onto an R928 photomultiplier tube (PMT; Nikon) with a Microflex PFX photomicrographic attachment (Nikon). The current from the PMT was converted to a voltage and amplified with a LF355N operational amplifier. High-frequency noise was removed by a low-pass filter widi an RC time constant of 1 second. The voltage signal was digitized by a "DAS" 8 analog- to-digital board (Kiethley MetraByte, Taunton, MA) and displayed widi software written in QuickBasic (Microsoft, Redmond, WA) on an IBM AT-style personal computer.
A transmitted-light image taken through die microscope objective (Fig. IB) shows how the outlet of a 25-μm i.d. capillary can be positioned above a single cell.
A. Changes in Intracellular Calcium - Ach/BK/ATP Mixture. A standard solution of CE Ringer containing three ligands - acetylcholine (ACh; 0.8 mM), bradykinin (BK; 20 μM), and adenosine triphosphate (ATP; 5 mM) was separated by capillary electrophoresis. The ligands were introduced by 5-second gravity injections (inlet elevated 10 cm above outlet) and were separated using an electric field of — 850 V/cm in a 23 cm 15 μm I.D. capillary. The capillary effluent was delivered to a group of 10 - 15 PC12 cells loaded widi fluo-3 as described above. The tip of the capillary was positioned approximately 40 μm above the cells, allowing the effluent to diffuse uniformly over the cells. The results are shown in Figure 2. Electropherograms are offset on the vertical axis to aid visualization. The negative baseline slope is caused by a decrease in d e intracellular concentration of fluo-3, which can be partially stabilized by incorporating fluo-3 AM ester in the measurement buffer.
The trace labeled "Mixture", shows ihe fluorescence measured as a function of time as the capillary electrophoresis effluent was washed over die cells. The peaks in die signal, reflecting increased intensity of fluorescence, are due to increases in [Ca2+]- following binding of ligands to dieir respective receptors. The increases in [Ca2+]i are caused by release of calcium from internal stores and/or by entry of calcium through ligand- or voltage-gated ion channels (Pozzan, et αl., Sands, et αl.). Identification of the diree peaks in the " Mixture" trace of Figure 2 can be accompl ished by electrophoretically separating each ligand individually (Figure 2, traces "ACh, "BK", and "ATP"). Because of the relative charge-to-frirtional drag ratios, acetylcholine migrates fastest to the sensor, closely followed by bradykinin and dien by ATP. The position of the peaks in the individual ligand traces corresponds to die position of die peaks in the mixture trace, facilitating identification of die mixture peaks. The absolute position of die ATP peak is somewhat variable due to die slow migration velocity of ATP (Yamamato, et αl.). B. Changes in Intracellular Calcium - PC 12 Lvsate/Ach.
PC12 lysate was prepared as follows. PC12 cells grown and differentiated for four days in a 50 ml culture flask with 25 ml of PC 12 growth medium (see above) further containing 25 ng/ml /S-NGF (nerve growth factor; Boehringer-Mannheim, Indianapolis, IN), were pelleted and lysed in 70 μl of 70% methanol containing 0.1 mM eserine (to inhibit cholinesterase activity).
Approximately 500 pl of the lysate were introduced to die inlet end of a capillary by a 10-second gravity injection 10 cm above die outlet (Fishman et al.). This amount of lysate corresponds to — IO*5 of die entire lysate volume. Since the cells in die flask were differentiated at — 10% confluence (i.e., — 5 * IO6 cells were incorporated into the lysate), the 500 pl lysate volume corresponds to approximately 50 cells.
The lysate components were separated by capillary electrophoresis using a field of - 400 V/cm in a 25 cm 25 μm I.D. capillary. The effluent was directed over 4-5 PC 12 cells, as above. Fluo-3 AM ester was included in die buffer bathing die cells to aid in maintaining a steady concentration of fluo-3 in the cells.
The results, in the form of a fluorescence signal as a function of time, are shown in
Figure 3. The top trace shows a single peak of [Ca2+]- change in the first 150 seconds of separation. This peak, which migrates to the cell biosensor in — 100 seconds, is identified as acetylcholine from comparison with the electropherogram produced by running an acetylcholine standard solution (Figure 3, bottom trace).
EXAMPLE 2 ELECTROPHYSIOLOGICAL DETECTION OF BIOSENSOR SIGNAL A Xenopus oocyte was microinjerted as described above widi serotonin 5HTlc receptor mRNA and allowed to recombinantly express 5HTlc receptor. Two to four days after injection, die oocyte was positioned at d e outlet of a separation capillary and voltage-clamped at -70 mV using a two-electrode voltage clamp (TEV-200; Dagan Corp., Minneapolis, MN) in the virtual-current mode. Intracellular Ag/AgCl electrodes were constructed with an initial input impedance of — 3 to 4 MO in 3 M KC1. The oocyte recording solution contained 140 mM NaCl, 2 mM KC1, 2 mM CaCl2, and 10 mM HEPES (pH 7.2).
Serotonin (5-HT; 100 μM) was introduced into to die inlet end of a fractured 40-μm i.d. CE capillary by 20-second gravity injections (+ 13 cm above outlet). The fracture was located 26 cm from the inlet end of die 36 cm capillary. The ligand solution was electrophoretically separated by application of a -250 V/cm field over die 26 cm portion of the capillary between die inlet and die crack. The separated effluent was pushed dirough die remaining 10 cm portion by the electroosmotic flow induced in die 26 cm portion, and delivered to the oocyte. Mock separations were run with buffer blanks.
Plots of current as a function of time are shown in Figure 5. The top trace shows the signal obtained in response a separation using serotonin. A peak in current is seen — 8 minutes after injection of serotonin into the capillary. The center trace shows the current response for a blank (control) CE run, and die bottom trace shows a return of signal when 5- HT is separated again and directed at die same oocyte. A slight increase in the migration time of 5-HT is apparent between the top and bottom traces, presumably caused by a decrease in die electroosmotic flow rate. The results exemplified are representative of 5 experiments.
Control experiments utilizing oocytes that had not been injected with 5HTlc receptor mRNA showed no response for 5-HT following capillary electrophoresis.
EXAMPLE 3 LOCALIZED EFFECTS OF LIGANDS
Experiments were performed to address die spatial extent over which analytes delivered to cell biosensors exert dieir effects. T e experiments utilized die tendency of many receptors, including nicotinic acetylcholine and bradykinin receptors, to desensitize following repeated applications of ligand (Hille, DeLorme, et al, Briggs, et al.). Bradykinin (250 μM) in CE Ringer was introduced at 120 second intervals into the inlet end of a capillary and elertrophoresed. The oudet was placed — 50 μm above a group of PC 12 cells. Data were collected as described in Example 1. Figure 6A shows successive responses to the same dose of bradykinin. It is apparent from the figure diat die bradykinin receptors desensitize - that is, repeat applications of similar amounts of agonist ligand generate successively smaller responses.
Figure 6B shows a similar experiment as that shown in Figure 6A, except mat the coverslip containing die cell was moved a few hundred microns between each pulse of ligand, to place a fresh batch of cells under die cell end of die capillary. A robust signal was obtained with each pulse of bradykinin. The responses differ in size somewhat with respect to one anodier, since the different groups of cells differed in the number of cells they contained, and die different cells differed somewhat in the number of receptors expressed and die strength of their responses. Electrophoresis parameters - Capillary length: 30 cm, I.D.: 20 μm, Voltage: 330 V/cm. Figures 6C and 6D show experiments where a group of cells was first exposed to repeated pulses as in Fig. 6A, and dien die capillary was moved (during die period labeled "scan") over a new group of cells. The response of the new group was comparable to the initial response in the original group. Same experimental conditions as above. The results indicate that (i) groups of cells under the capillary are uniformly stimulated and desensitized following repeat applications of ligand, and (ii) die concentration of die ligand falls off rapidly and dissipates widi distance away from the capillary, leaving neighboring example groups of cells unaffected by repeat applications of ligand.
EXAMPLE 4
EFFECTS OF RECEPTOR ANTAGONISTS ON LIGAND-INDUCED BIOSENSOR SIGNALS Cell biosensor responses to electrophoresed ligands were selectively blocked by specific receptor antagonists. The data were collected as described in Example 1. Figure 7 shows die effects of HOE 140 (Hoechst-Roussel Pharmaceuticals, Somerville, NJ), a bradykinin receptor antagonist, on die response of PC12 cells to a mixture of acetylcholine (8 mM) and bradykinin (25 μM). The bottom trace in the figure shows the response in absence of HOE 140, the top trace shows the response after the drug is added to die cell bathing solution to a final concentration of 800 μM. Electrophoresis parameters - Capillary length: 30 cm, I.D.: 20 μm, O.D.: 350 μm, Voltage: 10 kV, Current: -20 μA, distance of capillary outlet from cells: —40 μm.
The data show that HOE 140 essentially eliminated the biosensor response to bradykinin, but had no effect on die acetylcholine response.
Figures 8A and 8B show die effects of atropine (Sigma Chemical Co., St. Louis, MO) and α-bungarotoxin (Sigma), both acetylcholine receptor antagonist, on a cell biosensor response to a mixture of acetylcholine (8 mM) and bradykinin (25 μM). Figure 8A shows the response in absence of the antagonists, and Figure 8B shows the response after the drugs are added to die cell badiing solution to final concentrations of - 1 μM (atropine) and - 1 μm (α- bungarotoxin). Electrophoresis parameters - Capillary length: 30 cm, I.D.: 20 μm, O.D.: 350 μm, Voltage: 10 kV, Current: -20 μA. The acetylcholine antagonists reduced the biosensor response to acetylcholine by approximately a factor of 10 (compare the first peak (Ach) in Fig. 8A with the first peak (Ach) in Figure 8B), but did not affect the response to bradykinin. EXAMPLE 5 RESOLUTION OF RELATED COMPOUNDS Methods of die present invention may be used to separate and resolve distinct ligands having similar chemical characteristics. Data were collected as described in Example 1 from a group of PC 12 cells.
Electrophoresis parameters - Capillary length: 30 cm, I.D.: 20 μm, O.D.: 350 μm, Voltage: 10 kV, Current: —20 μA, distance of capillary outiet from cells: —40 μm. Figure 9 shows the results a capillary electrophoresis run which included acetylcholine (8 mM), bradykinin (25 μM) and Lys bradykinin (25 μM). All three ligands are effectively separated and detected, as evidenced by die diree resolved peaks.
The data demonstrate diat compounds having similar chemical characteristics, such as bradykinin and Lys bradykinin, can be resolved and detected using methods of d e present invention.
While the invention has been described with reference to specific mediods and embodiments, it will be appreciated diat various modifications and changes may be made widiout departing from die invention.

Claims

IT IS CLAIMED:
1. A method for detecting a selected ligand in a mixture of ligands, comprising
(i) separating the mixture of ligands containing die selerted ligand using a capillary-based separation means,
(ii) delivering separated ligands to a cell biosensor having a receptor capable of binding d e selerted ligand, where die separated ligands are delivered to said biosensor in a cell biosensor compatible buffer, and
(iii) detecting binding of the selected ligand to die receptor.
2. A method of claim 1, wherein said separating includes separating a mixture of ligands derived from a tissue extract or a cell extract.
3. A method of claim 1, wherein said separating includes separating a mixture of ligands obtained from a combinatorial library of ligands.
4. A mediod of claim 1, wherein said separating includes separating a mixture of ligands containing acetylcholine.
5. A mediod of claim 1, wherein said separating includes separating a mixture of ligands containing the selerted ligand and degradation products of die selected ligand.
6. A mediod of claim 1, wherein said separation means includes a separation means selected from the group consisting of capillary electrophoresis, capillary gel electrophoresis, capillary electrochromatography, capillary isotachophoresis, and affinity capillary electrophoresis.
7. A method of claim 1, wherein said cell biosensor comprises a cell.
8. A method of claim 7, wherein said cell biosensor comprises a cell recombinantly expressing a specific receptor.
9. A mediod of claim 7, wherein said cell biosensor comprises a cell in contact with a voltage-recording electrode.
10. A method of claim 7, wherein said cell biosensor comprises a Xenopus oocyte.
11. A method of claim 7, wherein said cell biosensor comprises a mammalian cell.
12. A method of claim 7, wherein said cell biosensor comprises a cell having a cell condition reporter.
13. A mediod of claim 12, wherein said reporter is a calcium-sensitive dye.
14. A method of claim 1, wherein said cell biosensor comprises a group of cells.
15. A method of claim 1, wherein said cell biosensor comprises a portion of an intact cell.
16. A method of claim 15, wherein said cell biosensor comprises an outside-out or an inside-out patch.
17. A mediod of claim 1, wherein said selected ligand is an agonist or an antagonist of die receptor.
18. A mediod of claim 1, wherein said deterting includes detecting a change in transmembrane voltage of a cell biosensor.
19. A method of claim 1, wherein said detecting includes detecting a change in intracellular calcium concentration of a cell biosensor.
20. A method of claim 1, wherein said detecting includes detecting a change in intracellular cAMP concentration of a cell biosensor.
21. A method of claim 1 , wherein said delivering includes delivering separated ligands to a cell biosensor which is in a bathing solution containing a receptor-specific antagonist, and wherein said detecting further comprises evaluating effects of the presence of die antagonist on the binding of d e selected ligand to die receptor.
22. A method of claim 1, wherein said delivering includes delivering separated ligands to a cell biosensor which is in a bad ing solution containing a receptor-specific agonist, and wherein said detecting further comprises evaluating effects of the presence of d e agonist on die binding of die selerted ligand to die receptor.
23. A mediod of claim 1, wherein said capillary-based separation means has an outlet end, and wherein said delivering includes delivering separated ligands to a cell biosensor which is attached at die oudet end of die capillary separation means.
24. A method of claim 23, wherein said cell biosensor is on a solid support diat is removably attached at die oudet end of die capillary separation means.
25. A mediod of claim 1, further comprising repeating steps (ii) through (iii) a selected number of times, where each time said delivering is to a different cell biosensor.
26. A method of claim 1, where step (i) further comprises monitoring separated ligands using a supplemental detection means.
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