WO2005007866A2

WO2005007866A2 - Biochip devices for ion transport measurement, methods of manufacture, and methods of use

Info

Publication number: WO2005007866A2
Application number: PCT/US2004/017134
Authority: WO
Inventors: Antonio Guia; Jia Xu; Lei Wu; Khachonesin Sithiphong; Maria Spassova; Huimin Tao; George Walker; Mingxian Huang; Guoliang Tao; Steven Saya; Glenn Walker; Zoya Zozulya
Original assignee: Aviva Biosciences Corporation
Priority date: 2003-05-31
Filing date: 2004-06-01
Publication date: 2005-01-27
Also published as: EP1636373A4; CA2527660A1; WO2005007866A3; EP1636373A2

Abstract

The present invention provides biochips for ion transport measurement, ion transport measuring devices that comprise biochips, and methods of using ion transport measuring devices and biochips that allow for the direct analysis of ion transport functions or properties. The present invention provides biochips, devices, apparatuses, and methods that allow for automated detection of ion transport functions or properties. The present invention also provides methods of making biochips and devices for ion transport measurement that reduce the cost and increase the efficiency of manufacture, as well as improve the performance of the biochips and devices. These biochips and devices are particularly appropriate for automating the detection of ion transport functions or properties, particularly for screening purposes.

Description

BIOCHIP DEVICES FOR ION TRANSPORT MEASUREMENT, METHODS OF MANUFACTURE, AND METHODS OF USE

TECHNICAL FIELD The present invention relates generally to the field of ion transport detection

("patch clamp") systems and methods, particularly those that relate to the use of biocbip technologies.

This application claims priority to United States patent application number 60/474,508, filed May 31, 2003, hereby incorporated by reference in its entirety. This application incorporates by reference the following applications: United

States patent application number 10/760,866, filed January 20, 2004; United States patent application number 10/428,565, filed May 2, 2003; United States patent application number 60/380,007, filed May 4, 2002; United States patent application number 10/642,014, filed August 16, 2003; United States patent application number 10/351,019, filed January 23, 2003; United States patent application number 60/351,849 filed January 24, 2002; United States patent application number 10/104,300, filed March 22, 2002; United States patent application number 60/311,327 filed August 10, 2001; and United States patent application number 60/278,308 filed March 24, 2001.

BACKGROUND

Ion transports are channels, transporters, pore forming proteins, or other entities that are located within cellular membranes and regulate the flow of ions across the membrane. Ion transports participate in diverse processes, such as generating and timing of action potentials, synaptic transmission, secretion of hormones, contraction of muscles etc. Ion transports are popular candidates for drug discovery, and many known drugs exert their effects via modulation of ion transport functions or properties. For example, antiepileptic compounds such as phenytoin and lamotrigine which block voltage dependent sodium ion transports in the brain, anti-hypertension drugs such as nifedipine and diltiazem which block voltage dependent calcium ion transports in smooth muscle cells, and stimulators of insulin release such as glibenclamide and tolbutamine which block an ATP regulated potassium ion transport in the pancreas. One popular method of measuring an ion transport function or property is the patch-clamp method, which was first reported by Neher, Sakmann and Steinback (Pflueger Arch. 375:219-278 (1978)). This first report of the patch clamp method relied on pressing a glass pipette containing acetylcholine (Ach) against the surface of a muscle cell membrane, where discrete jumps in electrical current were attributable to the opening and closing of Ach-activated ion transports.

The method was refined by fire polishing the glass pipettes and applying gentle suction to the interior ofthe pipette when contact was made with the surface of the cell. Seals of very high resistance (between about 1 and about 100 giga ohms) could be obtained. This advancement allowed the patch clamp method to be suitable over voltage ranges which ion transport studies can routinely be made.

A variety of patch clamp methods have been developed, such as whole cell, vesicle, outside-out and inside-out patches (Liem et al., Neurosurgery 36:382-392 (1995)). Additional methods include whole cell patch clamp recordings, pressure patch clamp methods, cell -free ion transport recording, perfusion patch pipettes, concentration patch clamp methods, perforated patch clamp methods, loose patch voltage clamp methods, patch clamp recording and patch clamp methods in tissue samples such as muscle or brain (Boulton et al, Patch-Clamp Applications and Protocols, Neuromethods V. 26 (1995), Humana Press, New Jersey). These and later methods relied upon interrogating one sample at a time using large laboratory apparatuses that require a high degree of operator skill and time. Attempts have been made to automate patch clamp methods, but these have met with little success. Alternatives to patch clamp methods have been developed using fluorescent probes, such as cumarin-lipids (cu-lipids) and oxonol fluorescent dyes (Tsien et al., U.S. Patent No. 6,107,066, issued August 2000). These methods rely upon change in polarity of membranes and the resulting motion of oxonol molecules across the membrane. This motion allows for the detection of changes in fluorescence resonance energy transfer (FRET) between cu-lipids and oxonol molecules. Unfortunately, these methods do not measure ion transport directly but measure the change of indirect parameters as a result of ionic flux. For example, the characteristics of the lipid used in the cu-lipid can alter the biological and physical characteristics ofthe membrane, such as fluidity and polarizability.

Thus, what is needed is a simple device and method to measure ion transport directly. Preferably, these devices would utilize patch clamp detection methods because these types of methods represent a gold standard in this field of study. The present invention provides these devices and methods particularly miniaturized devices and automated methods for the screening of chemicals or other moieties for their ability to modulate ion transport functions or properties.

BRIEF SUMMARY OF THE INVENTION

The present invention recognizes that the determination of one or more ion transport functions or properties using direct detection methods, such as patch-clamp, whole cell recording, or single channel recording, are preferable to methods that utilize indirect detection methods, such as fluorescence-based detection systems.

A first aspect of the present invention is a biochip device for ion transport measurement. A biochip device comprises an upper chamber piece that comprises one or more upper chambers and a biochip that comprises at least one ion transport measuring means. In one preferred embodiment of this aspect of the present invention, a biochip device is part of an apparatus that also comprises at least one conduit that that can be positioned to engage the one or more upper chambers, where the conduit comprises an electrode or can provide an electrolyte bridge to an electrode. A second aspect of the present invention is a biochip device having one or more flow-through lower chambers. The device comprises an upper chamber piece that comprises one or more upper chambers, a biochip that comprises at least one ion transport measuring means, and at least one lower chamber base piece that comprises one or more lower chambers and at least two conduits that connect with at least one of the one or more lower chambers.

A third aspect of the invention is biochip-based ion transport measurement devices that are adapted for microscope stages. The devices comprise an upper chamber piece that comprises one or more upper chambers, a biochip that comprises at least one ion transport measuring means, and at least one lower chamber base piece, in which the bottom surface of the lower chamber base piece is transparent. Preferably, the device also includes a baseplate adapted to a microscope stage into which a lower chamber base piece can fit. A fourth aspect ofthe invention is methods of making an upper chamber piece for a biochip device for ion transport measurement. In one preferred embodiment of this aspect of the present invention, an upper chamber piece can be molded as two pieces, an upper well portion piece and a well hole portion piece. Preferably, a well hole portion piece comprises at least one groove into which at least one electrode can be inserted. After insertion ofthe electrode, the upper well portion piece and the well hole portion piece are attached to form an upper chamber piece. In another embodiment of this aspect, an upper chamber piece can be molded as a single piece, where an electrode, such as a wire electrode, can be positioned in a mold and then the upper chamber piece can be molded around it. In yet another preferred embodiment of this aspect, an upper chamber piece can be molded as a single piece without an electrode.

A fifth aspect of the invention is methods for making chips comprising ion transport measuring holes. An ion transport measuring hole can be fabricated by laser drilling one or more counterbores, and then laser drilling a through-hole through the one or more counterbores.

A sixth aspect of the invention is an ion transport measuring device that comprises an inverted chip comprising ion transport measuring holes. A chip used in inverted orientation can comprise one or more ion transport measuring holes that are fabricated by laser drilling of one or more counterbores and a through-hole through the one or more counterbores.

A seventh aspect of the invention is methods of treating ion transport measuring chips to enhance their sealing properties. In one aspect of the present invention, the chip or substrate comprising an ion transport measuring means is modified to become more electronegative, more smooth, or more electronegative and more smooth. In some aspects of the present invention, the chip or substrate comprising the ion transport measuring means is modified chemically, such as with acids, bases, or a combination thereof. Treatment of chips of the present invention with chemical solution can be performed using treatment racks that fit into vessels that hold the chemical solutions and can hold multiple glass chips while allowing access ofthe chemical solutions to the chip surfaces.

An eighth aspect of the invention is a method to measure surface energy on a surface, such as the surface of a chemically-treated ion transport measurement biochip. The surface energy measurement can be used to evaluate the hydrophilicity of a biochip biochip of the present invention that has been chemically treated to improve its electrical sealing properties, such as, for example, at chip that has been treated with base. The method can also be used for any surface characterization purpose where a measurement of surface energy or hydrophilicity is desired.

A ninth aspect of the invention is the substrates, biochips, devices, apparatuses, and/or cartridges comprising ion transport measuring means with enhanced electric seal properties. In preferred embodiments, at least a portion of at least one chip that comprises at least one ion transport measuring means has been modified to become more electronegative. In preferred embodiments, at least a portion of at least one chip that comprises at least one ion transport measuring means has been treated with at least one base, at least one acid, or both.

A tenth aspect of the present invention is a method for storing the substrates, biochips, cartridges, apparatuses, and/or devices comprising ion transport measuring means with enhanced electrical seal properties.

An eleventh aspect of the present invention is a method for shipping the substrates, biochips, cartridges, apparatuses, and/or devices comprising ion transport measuring means with enhanced electrical seal properties.

A twelfth aspect of the invention is methods for assembling devices and cartridges of the present invention. The methods include attaching an upper chamber piece to a biochip that comprises at least one ion transport measuring means using a UN adhesive. Preferably, the chip has been chemically treated to enhance its electrical sealing properties. During UN activation of the adhesive, at least a portion of the biochip is masked to prevent UN irradiation of ion transport measuring means on the chip. A thirteenth aspect of the present invention is a method of producing biochips comprising ion transport measuring means by fabricating the biochips as detachable units of a large sheet. Ion transport measuring holes can be made by wet etching and laser drilling appropriate substrates, and the sheet can be scored with a laser such that portions ofthe sheet having a desired number of ion transport measuring holes can be separated along the score lines. In some embodiments, upper chamber pieces are attached to the substrate sheet after the fabrication of holes and before separation of sections of the sheet, hi this case, the detachable units that are separated to produce devices comprise cartridges having upper chambers attached to an ion transport measuring chip.

A fourteenth aspect ofthe invention is a method of producing high density ion transport measuring chips. The ion transport measuring chips preferably have more than 16 ion transport measuring holes, and wells can be fabricated in a chip using wet etching, followed by laser drilling of ion transport measuring holes through the bottoms of the wells.

A fifteenth aspect of the invention is a biochip device for ion transport measurement comprising fluidic channel upper and lower chambers. The fluidic channels have apertures that are aligned with ion transport measuring holes on the chip. The fluidic channels can be connected to sources for generating or promoting fluid flow, such as pumps, pressure sources, and valves. The fluidic channels preferably provide electrolyte bridges to one or more electrodes that can be used in ion transport measurement.

A sixteenth aspect of the present invention is methods of preparing cells for ion transport measurement. The methods include the use of filters that can allow the passage of single cells through their pores and monitoring of cell health parameters important for electrophysiological measurements.

A seventeenth aspect ofthe present invention is a logic and program that uses a pressure control profile to direct an ion transport measurement apparatus to achieve and maintain a high-resistance electrical seal. The logic can follow decision pathways based on information from electrical measurements made by ion transport measuring electrodes in a feedback system. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts four views of one example of an upper chamber piece ofthe present invention: A) top view; B) bottom view; C) side-on cross-sectional view; and D) end- on cross-sectional view.

Figure 2 depicts a cross-sectional view of a single ion transport measuring unit of one example of an ion transport measuring device of the present invention. Figure is not necessarily to scale.

Figure 3 provides photographs of a lower chamber piece ofthe present invention that is adapted to fit a microscope stage and has flow-through lower chambers. (A) view of a plastic lower chamber base piece with connectors for inflow and outflow tubes, B) a zoomed-in view ofthe lower chamber base piece showing inflow and outflow tubes C) the lower chamber piece installed in a base plate.

Figure 4 provides photographs of one design of a base plate for adapting a biochip device to a microscope stage. (A) Top view and (B) bottom view of a base plate cut from aluminum stock. The holes (401) are threaded except for the four holes closest to the corners of the square-cut carve-out. The four unthreaded holes (402) are sized to accept a press-in 1 mm socket connector.

Figure 5 depicts one device ofthe present invention having a lower chamber base piece fitted to a baseplate (54) by means of a clamp (53) which also attaches the upper chamber piece (51) to the lower chamber base piece (not visible). The clamp also comprises wire electrodes (55) that extend into upper wells. Electrode connectors (52) have wires extending into the fluidics of each lower chamber below.

Figure 6 depicts a lower chamber piece ofthe present invention in the form of a gasket having multiple holes (601) that form the walls of lower chambers in an assembled device. In this design, the holes are formed by O-ring structures (602). Figure 7 provides photographs of a clamp part (A) upside down and (B) viewed from the top fitted over a cartridge. Figure 8 provides photographs of a cartridge device ofthe present invention (black item) shown in relation to the rest ofthe parts of a device adapted for a microscope (A) and after assembly into a baseplate (B).

Figure 9 depicts an upper chamber piece of the present invention that is made from an upper well portion piece (91) and a well-hole portion piece (92). (A) the upper well portion piece (91) is shown above the well-hole portion piece (92). (B) the upper well portion piece (91) is shown fitted on the well-hole portion piece to form wells (93), with the groove (94) where an electrode can be inserted visible along the back of the wells (93).

Figure 10 is a graph that illustrates that a decreasing hole depth (x-axis) and widening the exit hole (as for "K-configuration" chips) decreases Re (y-axis). On the left side ("K-configuration" chips): black circles, chips having 2.5 micron diameter holes with 6 micron entrance holes; black squares, chips having 2 micron diameter holes with 5 micron entrance holes; black double triangles, chips having 1.8 micron diameter holes with 4 and 6 micron entrance holes; and X's, chips having 1.5 micron diameter holes with 6 micron entrance holes. On the right side ("S-configuration chips) black triangles, chips having 2.5 micron diameter holes with 10 micron entrance holes; black squares, chips having 2 micron diameter holes with 9 micron entrance holes; open triangles, chips having 1.8 micron diameter holes with 7 micron entrance holes; and black diamonds, chips having 1.5 micron diameter holes with 8 micron entrance holes.

Figure 11 is a graph illustrating that thinner chips (for example "K-configuration" chips ofthe present invention) have a lower Ra ("improved Ra") than those with greater hole depth. Ra also decreases as hole diameter increases, however at a cost of lower Rm. Increased Rm ("improved Rm") is found with increased hole depth.

Figure 12 gives depictions of a laser drilled chip (123) having a first counterbore

(126) and a second counterbore (127) and a through-hole (128). In A) the direction of laser drilling ofthe counterbores (126 and 127) and through-hole (128) is shown by the arrow. In B), the chip is used in inverted orientation with a cell (129) sealed to the hole (128) that connects the upper chamber (121) with the lower chamber (125) having walls formed by a gasket (124). Figure is not necessarily to scale.

Figure 13 depicts treatment fixtures for chemically treating chips and devices. (A) shows a single layer treatment fixture that can fit into a glass jar containing acid, base, or other chemical solutions. (B) shows the stacked fixture.

Figure 14 shows one design of a shipping fixture for cartridges ofthe present invention. In A), a blister pack having a plastic frame (141) and openings (142) for sealing cartridges (143) is viewed from the bottom. In B), the blister pack is viewed from the top side ofthe sealed-in cartridge (143).

Figure 15 depicts a glass chip (151) with multiple ion transport holes (152) that can be attached to a multichamber upper chamber piece to form a multiunit sheet (154). The multiunit sheet (154) comprising upper chambers and a chip (151) has mark lines or perforations in the chip (153) where the sheet can be separated into sections. Cartridges with a smaller number of units (155) can be separated from the larger multiunit sheet (154). Not to scale.

Figure 16 depicts one example of a high density array chip (161) ofthe present invention. The wells (162) ofthe chip can be made by wet etching followed by laser drilling through holes through the bottoms ofthe wells (162).

Figure 17 shows an example of a high density array having upper chambers (171) that can be formed by a well plate (172) attached to the chip (173). Wells (174) in the chip (173) having laser drilled through-holes can be oriented in inverted orientation (top alternative) or standard orientation (bottom alternative). Figure 18 depicts the general format for pressure bonding, in which a chip (183) comprising a hole (182) is attached to an upper chamber piece (181) using a gasket (184) to form a seal between the upper chamber piece (181) and chip (183) when pressure (arrow) is applied. In this highly schematized depiction, a lower chamber piece (185) is also attached to the chip (183) using a second gasket (186) to form a seal between the lower chamber piece (185) and chip (183) when pressure (arrow) is applied. Figure 19 depicts a schematic view of one design a planar patch clamping chip (193) having an upper fluid channel (191) for extracellular solution (ES) and a lower fluidic channel (195) for intracellular solutions (IS1, IS2). The upper and lower channels are interfaced at a point where the recording aperture (192) ofthe planar electrode resides. Separate fluidic pumps (P) drive the flow of fluids through the two (upper and lower) fluidic channels. Recording (196) and reference electrodes (197) external to the fluidic patch clamp chip are connected via an electrolyte solution bridge to the upper (191) and lower (195) fluidic channels. A pressure source such as a pump with pressure controller that can generate both positive and negative pressures is linked to the lower fluidic channels. A multi-way valve (194) is used to connect the lower fluidic channel (195) to different solution reservoirs (IS1, IS2, etc), and a multi-way valve (198) is used to connect the upper fluidic channel (191) to cell reservoirs, compound plate (CP), wash buffers and other solutions. (Not to scale).

Figure 20 provides graphs ofthe success rate of a test of patch clamp seals using cartridges ofthe present invention having chemically treated chips. A) gives the success duration of seals on 52 chips. B) plots the accumulative success rate of cells on 53 chips (achieved gigaseals and gave Ra<15MOhm and Rm>200MOhm throughout 15 min recording period).

Figure 21 provides graphs of results of tests performed on 52 chips. A) gives Re values ofthe chips. B) gives break-in pressures during the quality control test.

Figure 22 provides graphs of Rm (membrane resistance) and Ra (access resistance) at the beginning and at end of tests using devices ofthe present invention. A) shows Rm after break-in (wide diagonals slanting upward) and at the end ofthe test (narrow diagonals slanting downward). B) shows Ra after break-in (wide diagonals slanting upward) and at the end ofthe test (narrow diagonals slanting downward).

Figure 23 provides typical patch clamp recordings immediately after break-in using a device ofthe present invention. A) uncorrected whole-cell recording, B) corrected whole cell recording, C) plot of corrected and uncorrected recording taken during the interval denoted by the arrowheads in A) and B). Figure 24 provides typical patch clamp recordings fifteen minutes after break-in using a device ofthe present invention. A) uncorrected whole cell recording, B) corrected whole cell recording, C) plot of corrected and uncorrected recording taken during the interval denoted by the arrowheads in A) and B).

Figure 25 plots the Rm and Ra values for patch clamps ofthe experiment shown in Figures 23 and 24 beginning at break-in and continuing over a 15-minute period.

Figure 26 is a flowchart of an overview of the pressure control profile program.

Figure 27 is a flowchart of part 1 of Procedure Landing ofthe pressure control profile program.

Figure 28 shows a flowchart of part 2 of Procedure Landing ofthe pressure control profile program.

Figure 29 shows a flowchart of part 3 of Procedure Landing ofthe pressure control profile program.

Figure 30 shows a flowchart of part 1 of Procedure FormSeal ofthe pressure control profile program.

Figure 31 shows a flowchart of part 2 of Procedure FormSeal ofthe pressure control profile program.

Figure 32 shows a flowchart of part 3 of Procedure FormSeal ofthe pressure control profile program.

Figure 33 shows a flowchart of part 4 of Procedure FormSeal ofthe pressure control profile program. Figure 34 shows a flowchart of part 5 of Procedure FormSeal ofthe pressure control profile program.

Figure 35 shows a flowchart of part 1 of Procedure Breakln ofthe pressure control profile program.

Figure 36 shows a flowchart of part 2 of Procedure Breakln ofthe pressure control profile program.

Figure 37 shows a flowchart of part 3 of Procedure Breakln ofthe pressure control profile program.

Figure 38 shows a flowchart of part 4 of Procedure Breakln ofthe pressure control profile program.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the manufacture or laboratory procedures described below are well known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references. Terms of orientation such as "up" and "down", "top" and "bottom", "upper" or "lower" and the like refer to orientation of parts during use of a device. Where a term is provided in the singular, the inventors also contemplate the plural of that term. Where there are discrepancies in terms and definitions used in references that are incoφorated by reference, the terms used in this application shall have the definitions given herein. As employed throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

"Ion transport measurement" is the process of detecting and measuring the movement of charge and/or conducting ions across a membrane (such as a biological membrane), or from the inside to the outside of a particle or vice versa. In most applications, particles will be cells, organelles, vesicles, biological membrane fragments, artificial membranes, bilayers or micelles. In general, ion transport measurement involves achieving a high resistance electrical seal of a membrane or particle with a surface that has an aperture, and positioning electrodes on either side ofthe membrane or particle to measure the current and/or voltage across the portion ofthe membrane sealed over the aperture, or "clamping" voltage across the membrane and measuring current applied to an electrode to maintain that voltage. However, ion transport measurement does not require that a particle or membrane be sealed to an aperture if other means can provide electrode contact on both sides of a membrane. For example, a particle can be impaled with a needle electrode and a second electrode can be provided in contact with the solution outside the particle to complete a circuit for ion transport measurement. Several techniques collectively known as "patch clamping" can be included as "ion transport measurement". An "ion transport measuring means" refers to a structure that can be used to measure at least one ion transport function, property, or a change in ion channel function, property in response to various chemical, biochemical or electrical stimuli. Typically, an ion transport measuring means is a structure with an opening that a particle can seal against, but this need not be the case. For example, needles as well as holes, apertures, capillaries, and other detection structures ofthe present invention can be used as ion transport measuring means. An ion transport measuring means is preferably positioned on or within a biochip or a chamber. Where an ion transport measuring means refers to a hole or aperture, the use ofthe terms "ion transport measuring means" "hole" or "aperture" are also meant to encompass the perimeter of the hole or aperture that is in fact a part ofthe chip or substrate (or coating) surface (or surface of another structure, for example, a channel) and can also include the surfaces that surround the interior space ofthe hole that is also the chip or substrate (or coating) material or material of another structure that comprises the hole or aperture.

A "hole" is an aperture that extends through a chip. Descriptions of holes found herein are also meant to encompass the perimeter of the hole that is in fact a part ofthe chip or substrate (or coating) surface, and can also include the surfaces that surround the interior space of the hole that is also the chip or substrate (or coating) material. Thus, in the present invention, where particles are described as being positioned on, at, near, against, or in a hole, or adhering or fixed to a hole, it is intended to mean that a particle contacts the entire perimeter of a hole, such that at least a portion of the surface of the particle lies across the opening of the hole, or in some cases, descends to some degree into the opening of the whole, contacting the surfaces that surround the interior space ofthe hole.

A "patch clamp detection structure" refers to a structure that is on or within a biochip or a chamber that is capable of measuring at least one ion transport function or property via patch clamp methods.

A "chip" is a solid substrate on which one or more processes such as physical, chemical, biochemical, biological or biophysical processes can be carried out. Such processes can be assays, including biochemical, cellular, and chemical assays; ion transport or ion channel function or activity determinations, separations, including separations mediated by electrical, magnetic, physical, and chemical (including biochemical) forces or interactions; chemical reactions, enzymatic reactions, and binding interactions, including captures. The micro structures or micro-scale structures such as for example, channels and wells, electrode elements, or electromagnetic elements, may be incorporated into or fabricated on the substrate for facilitating physical, biophysical, biological, biochemical, chemical reactions or processes on the chip. The chip may be thin in one dimension and may have various shapes in other dimensions, for example, a rectangle, a circle, an ellipse, or other irregular shapes. The size of the major surface of chips of the present invention can

9 9 vary considerably, for example, from about 1 mm to about 0.25 m . Preferably, the 9 size ofthe chips is from about 4 mm to about 25 cm with a characteristic dimension from about 1 mm to about 5 cm. The chip surfaces may be flat, or not flat. The chips with non-flat surfaces may include wells fabricated on the surfaces.

A "biochip" is a chip that is useful for a biochemical, biological or biophysical process. In this regard, a biochip is preferably biocompatible, in that it does not negatively affect cells or cell membranes. A "chamber" is a structure that comprises or engages a chip and that is capable of containing a fluid sample. The chamber may have various dimensions and its volume may vary between 0.001 microliter and 50 milliliter. In devices ofthe present invention, an "upper chamber" is a chamber that is above a biochip, such as a biochip that comprises one or more ion transport measuring means. In the devices of the present invention, a chip that comprises one or more ion transport measuring means can separate one or more upper chambers from one or more lower chambers. During use of a device, an upper chamber can contain measuring solutions and particles or membranes. An upper chamber can optionally comprise one or more electrodes. In devices of the present invention, a "lower chamber" is a chamber that is below a biochip. During use of a device, a lower chamber can contain measuring solutions and particles or membranes. A lower chamber can optionally comprise one or more electrodes.

A lower chamber "has access to" or "accesses" an upper chamber via (or through) a hole in a chip when the chip separates or is between the upper and lower chambers and a hole in the chip provides fluid communication between the referenced lower chamber and the referenced upper chamber. An upper chamber "has access to" or "accesses" a lower chamber via (or through) a hole in a chip when the chip separates or is between the upper and lower chambers and a hole in the chip provides fluid communication between the referenced upper chamber and the referenced lower chamber. Similarly an upper chamber can be "connected to" a lower chamber (or vice versa) via a hole in a chip when the hole in the chip provides fluid communication between the referenced upper chamber and the referenced lower chamber.

A "lower chamber piece" is a part of a device for ion transport measurement that fonns at least a portion of one or more lower chambers ofthe device. A lower chamber piece preferably comprises at least a portion of one or more walls of one or more lower chambers, and can optionally comprise at least a portion of a bottom surface of one or more lower chambers, and can optionally comprise one or more conduits that lead to one or more lower chambers, or one or more electrodes. A "lower chamber base piece" or "base piece" is a part of a device for ion transport measurement that forms the bottom surface of one or more lower chambers ofthe device. A lower chamber base piece can also optionally comprise one or more walls of one or more lower chambers, one or more conduits that lead to one or more lower chambers, or one or more electrodes. As used herein, a "platform" is a surface on which a device ofthe present invention can be positioned. A platform can comprises the bottom surface of one or more lower chambers of a device.

-An "upper chamber piece" is a part of a device for ion transport measurement that forms at least a portion of one or more upper chambers ofthe device. An upper chamber piece can comprise one or more walls of one or more upper chambers, and can optionally comprise one or more conduits that lead to an upper chamber, and one or more electrodes.

An "upper chamber portion piece" is a part of a device for ion transport measurement that forms a portion of one or more upper chambers of the device. An upper chamber portion piece can comprise at least a portion of one or more walls of one or more upper chambers, and can optionally comprise one or more conduits that lead to an upper chamber, or one or more electrodes.

A "well" is a depression in a substrate or other structure. For example, in devices of the present invention, upper chambers can be wells formed in an upper chamber piece. The upper opening of a well can be of any shape and can be of an irregular conformation. The walls of a well can extend upward from the lower surface of a well at any angle or in any way. The walls can be of any shape and can be of an irregular conformation, that is, they may extend upward in a sigmoidal or otherwise curved or multi-angled fashion. A "well hole" is a hole in the bottom of a well. A well hole can be a well- within-a well, having its own well shape with an opening at the bottom.

A "well hole piece" is a part of a device for ion transport measurement that comprises one or more well holes ofthe wells ofthe device. When wells or chambers (including fluidic channel chambers) are "in register with" ion transport measuring means of a chip, there is a one-to-one correspondence of each ofthe referenced wells or chambers to each ofthe referenced ion transport measuring means, and an ion transport measuring means is positioned so that it is exposed to the interior ofthe well or chamber it is in register with, such that ion transport measurement can be performed using the chamber as a compartment for measuring cunent or voltage through or across the ion transport measuring means.

A "port" is an opening in a wall or housing of a chamber through which a fluid sample or solution can enter or exit the chamber. A port can be of any dimensions, but preferably is of a shape and size that allows a sample or solution to be dispensed into a chamber by means of a pipette, syringe, or conduit, or other means of dispensing a sample.

A "conduit" is a means for fluid to be transported into or out of a device, apparatus, or system for ion transport measurement of the present invention or from one area to another area of a device, apparatus, or system of the present invention. In some aspects, a conduit can engage a port in the housing or wall of a chamber. In some aspects, a part of a device, such as, for example, an upper chamber piece or a lower chamber piece can comprise conduits in the form of tunnels that pass through the upper chamber piece and connect, for example, one area or compartment with another area or compartment. A conduit can be drilled or molded into a chip, chamber, housing, or chamber piece, or a conduit can comprise any material that permits the passage of a fluid through it, and can be attached to any part of a device. In one prefened aspect of the present invention, a conduit extends through at least a portion of a device, such as a wall of a chamber, or an upper chamber piece or lower chamber piece, and connects the interior space of a chamber with the outside of a chamber, where it can optionally connect to another conduit, such as tubing. Some prefened conduits can be tubing, such as, for example, rubber, teflon, or tygon tubing. A conduit can be of any dimensions, but preferably ranges from 10 microns to 5 millimeters in internal diameter. A "device for ion transport measurement" or an "ion transport measuring device" is a device that comprises at least one chip that comprises one or more ion transport measuring means, at least a portion of at least one upper chamber, and, preferably, at least a portion of at least one lower chamber. A device for ion transport measurement preferably comprises one or more electrodes, and can optionally comprise conduits, particle positioning means, or application-specific integrated circuits (ASICs).

A "cartridge for ion transport measurement" comprises an upper chamber piece and at least one biochip comprising one or more ion transport measuring means attached to the upper chamber piece, such that the one or more ion transport measuring means are in register with the upper chambers ofthe upper chamber piece.

An "ion transport measuring unit" is a portion of a device that comprises at least a portion of a chip having a single ion transport measuring means and a single upper chamber, where the ion transport measuring means is in register with the upper chamber. An ion transport measuring unit can further comprise at least a portion of a lower chamber that is in register with the ion transport measuring means an upper chamber.

A "measuring solution" is an aqueous solution containing electrolytes, with pH, osmolarity, and other physical-chemical traits that are compatible with conducting function ofthe ion transports to be measured.

An "intracellular solution" is a measuring solution used in the upper or lower chamber that is compatible with the electrolyte composition and physical-chemical traits ofthe intracellular content of a living cell.

An "extracellular solution" is a measuring solution used in the upper or lower chamber that is compatible with the electrolyte composition and physical-chemical traits ofthe extracellular content of a living cell.

To be "in electrical contact with" means one component is able to receive and conduct electrical signals (for example, voltage, current, or change of voltage or current) from another component. An "ion transport" can be any protein or non-protein moiety that modulates, regulates or allows transfer of ions across a membrane, such as a biological membrane or an artificial membrane. Ion transport include but are not limited to ion channels, proteins allowing transport of ions by active transport, proteins allowing transport of ions by passive transport, toxins such as from insects, viral proteins or the like. Niral proteins, such as the M2 protein of influenza virus can form an ion channel on cell surfaces.

A "particle" refers to an organic or inorganic particulate that is suspendable in a solution and can be manipulated by a particle positioning means. A particle can include a cell, such as a prokaryotic or eukaryotic cell, or can be a cell fragment, such as a vesicle or a microsome that can be made using methods known in the art. A particle can also include artificial membrane preparations that can be made using methods known in the art. Prefened artificial membrane preparations are lipid bilayers, but that need not be the case. A particle in the present invention can also be a lipid film, such as a black-lipid film (see, Houslay and Stanley, Dynamics of Biological Membranes, Influence on Synthesis, Structure and Function, John Wiley & Sons, New York (1982)). In the case of a lipid film, a lipid film can be provided over a hole, such as a hole or capillary ofthe present invention using methods known in the art (see, Houslay and Stanley, Dynamics of Biological Membranes, Influence on Synthesis, Structure and Function, John Wiley & Sons, New York (1982)). A particle preferably includes or is suspected of including at least one ion transport or an ion transport of interest. Particles that do not include an ion transport or an ion transport of interest can be made to include such ion transport using methods known in the art, such as by fusion of particles or insertion of ion transports into such particles such as by detergents, detergent removal, detergent dilution, sonication or detergent catalyzed incorporation (see, Houslay and Stanley, Dynamics of Biological Membranes, Influence on Synthesis, Structure and Function, John Wiley & Sons, New York (1982)). A microparticle, such as a bead, such as a latex bead or magnetic bead, can be attached to a particle, such that the particle can be manipulated by a particle positioning means.

A "cell" refers to a viable or non-viable prokaryotic or eukaryotic cell. A eukaryotic cell can be any eukaryotic cell from any source, such as obtained from a subject, human or non-human, fetal or non-fetal, child or adult, such as from a tissue or fluid, including blood, which are obtainable through appropriate sample collection methods, such as biopsy, blood collection or otherwise. Eukaryotic cells can be provided as is in a sample or can be cell lines that are cultivated in vitro. Differences in cell types also include cellular origin, distinct surface markers, sizes, morphologies and other physical and biological properties. A "cell fragment" refers to a portion of a cell, such as cell organelles, including but not limited to nuclei, endoplasmic reticulum, mitochondria or golgi apparatus. Cell fragments can include vesicles, such as inside out or outside out vesicles or mixtures thereof. Preparations that include cell fragments can be made using methods known in the art.

A "population of cells" refers to a sample that includes more than one cell or more than one type of cell. For example, a sample of blood from a subject is a population of white cells and red cells. A population of cells can also include a sample including a plurality of substantially homogeneous cells, such as obtained tlirough cell culture methods for a continuous cell lines.

A "population of cell fragments" refers to a sample that includes more than one cell fragment or more than one type of cell fragments. For example, a population of cell fragments can include mitochondria, nuclei, microsomes and portions of golgi apparatus that can be formed upon cell lysis.

A "microparticle" is a structure of any shape and of any composition that is manipulatable by desired physical force(s). The microparticles used in the methods could have a dimension from about 0.01 micron to about ten centimeters. Preferably, the microparticles used in the methods have a dimension from about 0.1 micron to about several hundred microns. Such particles or microparticles can be comprised of any suitable material, such as glass or ceramics, and/or one or more polymers, such as, for example, nylon, polytetrafluoroethylene (TEFLON^TM), polystyrene, polyacrylamide, sepaharose, agarose, cellulose, cellulose derivatives, or dextran, and/or can comprise metals. Examples of microparticles include, but are not limited to, plastic particles, ceramic particles, carbon particles, polystyrene microbeads, glass beads, magnetic beads, hollow glass spheres, metal particles, particles of complex compositions, microfabricated free-standing microstructures, etc. The examples of microfabricated free-standing microstructures may include those described in "Design of asynchronous dielectric micromotors" by Hagedorn et al., in Journal of Electrostatics, Nolume: 33, Pages 159-185 (1994). Particles of complex compositions refer to the particles that comprise or consists of multiple compositional elements, for example, a metallic sphere covered with a thin layer of non-conducting polymer film.

"A preparation of microparticles" is a composition that comprises microparticles of one or more types and can optionally include at least one other compound, molecule, structure, solution, reagent, particle, or chemical entity. For example, a preparation of microparticles can be a suspension of microparticles in a buffer, and can optionally include specific binding members, enzymes, inert particles, surfactants, ligands, detergents, etc. "Coupled" means bound. For example, a moiety can be coupled to a microparticle by specific or nonspecific binding. As disclosed herein, the binding can be covalent or noncovalent, reversible or irreversible.

"Micro-scale structures" are structures integral to or attached on a chip, wafer, or chamber that have characteristic dimensions of scale for use in microfluidic applications ranging from about 0.1 micron to about 20 mm. Example of micro-scale structures that can be on chips ofthe present invention are wells, channels, scaffolds, electrodes, electromagnetic units, or microfabricated pumps or valves.

A "particle positioning means" refers to a means that is capable of manipulating the position of a particle relative to the X-Y coordinates or X-Y-Z coordinates of a biochip. Positions in the X-Y coordinates are in a plane. The Z coordinate is perpendicular to the plane. In one aspect ofthe present invention, the X- Y coordinates are substantially perpendicular to gravity and the Z coordinate is substantially parallel to gravity. This need not be the case, however, particularly if the biochip need not be level for operation or if a gravity free or gravity reduced environment is present. Several particle positioning means are disclosed herein, such as but not limited to dielectric structures, dielectric focusing structures, quadropole electrode structures, electrorotation structures, traveling wave dielectrophoresis structures, concentric electrode structures, spiral electrode structures, circular electrode structures, square electrode structures, particle switch structures, electromagnetic structures, DC electric field induced fluid motion structure, acoustic structures, negative pressure structures and the like.

A "dielectric focusing structure" refers to a structure that is on or within a biochip or a chamber that is capable of modulating the position of a particle in the X-Y or X-Y-Z coordinates of a biochip using dielectric forces or dielectrophoretic forces.

A "horizontal positioning means" refers to a particle positioning means that can position a particle in the X-Y coordinates of a biochip or chamber wherein the Z coordinate is substantially defined by gravity. A "vertical positioning means" refers to a particle positioning means that can position a particle in the Z coordinate of a biochip or chamber wherein the Z coordinate is substantially defined by gravity.

A "quadropole electrode structure" refers to a structure that includes four electrodes ananged around a locus such as a hole, capillary or needle on a biochip and is on or within a biochip or a chamber that is capable of modulating the position of a particle in the X-Y or X-Y-Z coordinates of a biochip using dielectrophoretic forces or dielectric forces generated by such quadropole electrode structures.

An "electrorotation structure" refers to a structure that is on or within a biochip or a chamber that is capable of producing a rotating electric field in the X-Y or X-Y-Z coordinates that can rotate a particle. Prefened electrorotation structures include a plurality of electrodes that are energized using phase offsets, such as 360/N degrees, where N represents the number of electrodes in the electroroation structure (see generally United States Patent Application Number 09/643,362 entitled "Apparatus and Method for High Throughput Electrorotation Analysis" filed August 22, 2000, naming Jing Cheng et al. as inventors). A rotating electrode structure can also produce dielectrophoretic forces for positioning particles to certain locations under appropriate electric signal or excitation. For example, when N=4 and electrorotation structure conesponds to a quadropole electrode structure. A "traveling wave dielectrophoresis structure" refers to a structure that is on or within a biochip or a chamber that is capable of modulating the position of a particle in the X-Y or X-Y-Z coordinates of a biochip using traveling wave dielectrophoretic forces (see generally United States Patent Application Number 09/686,737 filed October 10, 2000, to Xu, Wang, Cheng, Yang and Wu; and United States Application Number 09/678,263, entitled "Apparatus for Switching and Manipulating Particles and Methods of Use Thereof filed on October 3, 2000 and naming as inventors Xiaobo Wang, Weiping Yang, Junquan Xu, Jing Cheng, and Lei Wu).

A "concentric circular electrode structure" refers to a structure having multiple concentric circular electrodes that are on or within a biochip or a chamber that is capable of modulating the position of a particle in the X-Y or X-Y-Z coordinates of a biochip using dielectrophoretic forces.

A "spiral electrode structure" refers to a structure having multiple parallel spiral electrode elements that is on or within a biochip or a chamber that is capable of modulating the position of a particle in the X-Y or X-Y-Z coordinates of a biochip using dielectric forces.

A "square spiral electrode structure" refers to a structure having multiple parallel square spiral electrode elements that are on or within a biochip or a chamber that is capable of modulating the position of a particle in the X-Y or X-Y-Z coordinates of a biochip using dielectrophoretic or traveling wave dielectrophoretic forces.

A "particle switch structure" refers to a structure that is on or within a biochip or a chamber that is capable of transporting particles and switching the motion direction of a particle or particles in the X-Y or X-Y-Z coordinates of a biochip. The particle switch structure can modulate the direction that a particle takes based on the physical properties of the particle or at the will of a programmer or operator (see, generally United States Application Number 09/678,263, entitled "Apparatus for Switching and Manipulating Particles and Methods of Use Thereof filed on October 3, 2000 and naming as inventors Xiaobo Wang, Weiping Yang, Junquan Xu, Jing Cheng, and Lei Wu.

An "electromagnetic structure" refers to a structure that is on or within a biochip or a chamber that is capable of modulating the position of a particle in the X-

Y or X-Y-Z coordinates of a biochip using electromagnetic forces. See generally United States Patent Application Number 09/685,410 filed October 10, 2000, to Wu,

Wang, Cheng, Yang, Zhou, Liu and Xu and WO 00/54882 published September 21, 2000 to Zhou, Liu, Chen, Chen, Wang, Liu, Tan and Xu.

A "DC electric field induced fluid motion structure" refers to a structure that is on or within a biochip or a chamber that is capable of modulating the position of a particle in the X-Y or X-Y-Z coordinates of a biochip using DC electric field that produces a fluidic motion.

An "electroosomosis structure" refers to a structure that is on or within a biochip or a chamber that is capable of modulating the position of a particle in the X-

Y or X-Y-Z coordinates of a biochip using electroosmotic forces. Preferably, an electroosmosis structure can modulate the positioning of a particle such as a cell or fragment thereof with an ion transport measuring means such that the particle's seal (or the particle's sealing resistance) with such ion transport measuring means is increased. An "acoustic structure" refers to a structure that is on or within a biochip or a chamber that is capable of modulating the position of a particle in the X-Y or X-Y-Z coordinates of a biochip using acoustic forces. In one aspect ofthe present invention, the acoustic forces are transmitted directly or indirectly through an aqueous solution to modulate the positioning of a particle. Preferably, an acoustic structure can modulate the positioning of a particle such as a cell or fragment thereof with an ion transport measuring means such that the particle's seal with such ion transport measuring means is increased.

A "negative pressure structure" refers to a structure that is on or within a biochip or a chamber that is capable of modulating the position of a particle in the X- Y or X-Y-Z coordinates of a biochip using negative pressure forces, such as those generated through the use of pumps or the like. Preferably, a negative pressure structure can modulate the positioning of a particle such as a cell or fragment thereof with an ion transport measuring means such that the particle's seal with such ion transport measuring means is increased.

"Dielectrophoresis" is the movement of polarized particles in electrical fields of nonuniform strength. There are generally two types of dielectrophoresis, positive dielectrophoresis and negative dielectrophoresis. In positive dielectrophoresis, particles are moved by dielectrophoretic forces toward the strong field regions. In negative dielectrophoresis, particles are moved by dielectrophoretic forces toward weak field regions. Whether moieties exhibit positive or negative dielectrophoresis depends on whether particles are more or less polarizable than the sunounding medium.

A "dielectrophoretic force" is the force that acts on a polarizable particle in an AC electrical field of non-uniform strength. The dielectrophoretic force F_DEP acting on a particle of radius r subjected to a non-uniform electrical field can be given, under the dipole approximation, by:

EZ.--P ⁼ ^^πεm^r XDEP VErms

where E_rms is the RMS value of the field strength, the symbol V is the symbol for gradient-operation, ε_m is the dielectric permittivity of the medium, and χ_DEP is the particle polarization factor, given by:

"Re" refers to the real part ofthe "complex number". The symbol ε_x ^* = ε_x - jσ_x/2τιf is the complex permittivity (of the particle x=p, and the medium

and j = V-ϊ •

The parameters ε_p and σ are the effective permittivity and conductivity of the particle, respectively. These parameters may be frequency dependent. For example, a typical biological cell will have frequency dependent, effective conductivity and permittivity, at least, because of cytoplasm membrane polarization. Particles such as biological cells having different dielectric properties (as defined by permittivity and conductivity) will experience different dielectrophoretic forces. The dielectrophoretic force in the above equation refers to the simple dipole approximation results. However, the dielectrophoretic force utilized in this application generally refers to the force generated by non-uniform electric fields and is not limited by the dipole simplification. The above equation for the dielectrophoretic force can also be written as F_DEP = 2πε_mr³χ_DEP V² V p(x, y, z) where p(x,y,z) is the square-field distribution for a unit- voltage excitation (Voltage N = 1 V) on the electrodes, N is the applied voltage.

"Traveling-wave dielectrophoretic (TW-DEP) force" refers to the force that is generated on particles or molecules due to a traveling-wave electric field. An ideal traveling-wave field is characterized by the distribution of the phase values of AC electric field components, being a linear function ofthe position ofthe particle. In this case the traveling wave dielectrophoretic force F_m__DEP on a particle of radius r subjected to a traveling wave electrical field E = E cos 2π(fi - z / λ ₀) a_x (i.e., a x- direction field is traveling along the z-direction) is given, again, under the dipole approximation, by

4π ε„

F, TW-DEP ' TW-DEP¹-¹

4 where E is the magnitude ofthe field strength, ε_m is the dielectric permittivity ofthe medium. ζ_m__DEP is the particle polarization factor, given by

"Im" refers to the imaginary part of the "complex number". The symbol ε_x ^* = ε_x - j σ_x J2πf is the complex permittivity (of the particle x=p, and the medium x=m). The parameters ε_p and σ_p are the effective permittivity and conductivity of the particle, respectively. These parameters may be frequency dependent.

A traveling wave electric field can be established by applying appropriate AC signals to the microelectrodes appropriately arranged on a chip. For generating a traveling-wave-electric field, it is necessary to apply at least three types of electrical signals each having a different phase value. An example to produce a traveling wave electric field is to use four phase-quardrarure signals (0, 90, 180 and 270 degrees) to energize four linear, parallel electrodes patterned on the chip surfaces. Such four electrodes may be used to form a basic, repeating unit. Depending on the applications, there may be more than two such units that are located next to each other. This will produce a traveling-electric field in the spaces above or near the electrodes. As long as electrode elements are ananged following certain spatially sequential orders, applying phase-sequenced signals will result in establishing traveling electrical fields in the region close to the electrodes.

"Electric field pattern" refers to the field distribution in space or in a region of interest. An electric field pattern is determined by many parameters, including the frequency of the field, the magnitude of the field, the magnitude distribution of the field, and the distribution ofthe phase values ofthe field components, the geometry of the electrode structures that produce the electric field, and the frequency and/or magnitude modulation ofthe field.

"Dielectric properties" of a particle are properties that determine, at least in part, the response of a particle to an electric field. The dielectric properties of a particle include the effective electric conductivity of a particle and the effective electric permittivity of a particle. For a particle of homogeneous composition, for example, a polystyrene bead, the effective conductivity and effective permittivity are independent of the frequency of the electric field at least for a wide frequency range (e.g. between 1 Hz to 100 MHz). Particles that have a homogeneous bulk composition may have net surface charges. When such charged particles are suspended in a medium, electrical double layers may form at the particle/medium interfaces. Externally applied electric field may interact with the electrical double layers, causing changes in the effective conductivity and effective permittivity of the particles. The interactions between the applied field and the electrical double layers are generally frequency dependent. Thus, the effective conductivity and effective permittivity of such particles may be frequency dependent. For moieties of nonhomogeneous composition, for example, a cell, the effective conductivity and effective permittivity are values that take into account the effective conductivities and effective permittivities of both the membrane and internal portion of the cell, and can vary with the frequency of the electric field. In addition, the dielectrophoretic force experience by a particle in an electric field is dependent on its size; therefore, the overall size of particle is herein considered to be a dielectric property of a particle. Properties of a particle that contribute to its dielectric properties include but are not limited to the net charge on a particle; the composition of a particle (including the distribution of chemical groups or moieties on, within, or throughout a particle); size of a particle; surface configuration of a particle; surface charge of a particle; and the conformation of a particle. Particles can be of any appropriate shape, such as geometric or non-geometric shapes. For example, particles can be spheres, non- spherical, rough, smooth, have sharp edges, be square, oblong or the like.

"Magnetic forces" refer to the forces acting on a particle due to the application of a magnetic field. In general, particles have to be magnetic or paramagnetic when sufficient magnetic forces are needed to manipulate particles. For a typical magnetic particle made of super-paramagnetic material, when the particle is subjected to a magnetic field B , a magnetic dipole μ is induced in the particle

where V_p is the particle volume, χ_p and χ_m are the volume susceptibility of the particle and its surrounding medium, μ_m is the magnetic permeability of medium,

H_m is the magnetic field strength. The magnetic force F_magnetic acting on the particle is determined, under the dipole approximation, by the magnetic dipole moment and the magnetic field gradient: F_magnetic = -0.5 V_p(χ_p -χ_m)H_m . VB_m , where the symbols " • " and "V " refer to dot-product and gradient operations, respectively. Whether there is magnetic force acting on a particle depends on the difference in the volume susceptibility between the particle and its surrounding medium. Typically, particles are suspended in a liquid, non-magnetic medium (the volume susceptibility is close to zero) thus it is necessary to utilize magnetic particles (its volume susceptibility is much larger than zero). The particle velocity v_parttci_e under the balance between magnetic force and viscous drag is given by:

F v magnetic particle 6πrη_m

where r is the particle radius and η_m is the viscosity ofthe sunounding medium. As used herein, "manipulation" refers to moving or processing ofthe particles, which results in one-, two- or three-dimensional movement of the particle, in a chip format, whether within a single chip or between or among multiple chips. Non- limiting examples of the manipulations include transportation, focusing, enrichment, concentration, aggregation, trapping, repulsion, levitation, separation, isolation or linear or other directed motion of the particles. For effective manipulation, the binding partner and the physical force used in the method should be compatible. For example, binding partner such as microparticles that can be bound with particles, having magnetic properties are preferably used with magnetic force. Similarly, binding partners having certain dielectric properties, for example, plastic particles, polystyrene microbeads, are preferably used with dielectrophoretic force.

A "sample" is any sample from which particles are to be separated or analyzed. A sample can be from any source, such as an organism, group of organisms from the same or different species, from the environment, such as from a body of water or from the soil, or from a food source or an industrial source. A sample can be an unprocessed or a processed sample. A sample can be a gas, a liquid, or a semi- solid, and can be a solution or a suspension. A sample can be an extract, for example a liquid extract of a soil or food sample, an extract of a throat or genital swab, or an extract of a fecal sample. Samples are can include cells or a population of cells. The population of cells can be a mixture of different cells or a population ofthe same cell or cell type, such as a clonal population of cells. Cells can be derived from a biological sample from a subject, such as a fluid, tissue or organ sample. In the case of tissues or organs, cells in tissues or organs can be isolated or separated from the structure of the tissue or organ using known methods, such as teasing, rinsing, washing, passing through a grating and treatment with proteases. Samples of any tissue or organ can be used, including mesodermally derived, endodermally derived or ectodermally derived cells. Particularly prefened types of cells are from the heart and blood. Cells include but are not limited to suspensions of cells, cultured cell lines, recombinant cells, infected cells, eukaryotic cells, prokaryotic cells, infected with a virus, having a phenotype inherited or acquired, cells having a pathological status including a specific pathological status or complexed with biological or nonbiological entities. "Separation" is a process in which one or more components of a sample is spatially separated from one or more other components of a sample or a process to spatially redistribute particles within a sample such as a mixture of particles, such as a mixture of cells. A separation can be performed such that one or more particles is translocated to one or more areas of a separation apparatus and at least some of the remaining components are translocated away from the area or areas where the one or more particles are translocated to and/or retained in, or in which one or more particles is retained in one or more areas and at least some or the remaining components are removed from the area or areas. Alternatively, one or more components of a sample can be translocated to and/or retained in one or more areas and one or more particles can be removed from the area or areas. It is also possible to cause one or more particles to be translocated to one or more areas and one or more moieties of interest or one or more components of a sample to be translocated to one or more other areas. Separations can be achieved through the use of physical, chemical, electrical, or magnetic forces. Examples of forces that can be used in separations include but are not limited to gravity, mass flow, dielectrophoretic forces, fraveling-wave dielectrophoretic forces, and electromagnetic forces.

"Capture" is a type of separation in which one or more particles is retained in one or more areas of a chip, hi the methods of the present application, a capture can be performed when physical forces such as dielectrophoretic forces or electromagnetic forces are acted on the particle and direct the particle to one or more areas of a chip.

An "assay" is a test performed on a sample or a component of a sample. An assay can test for the presence of a component, the amount or concentration of a component, the composition of a component, the activity of a component, the electrical properties of an ion transport protein, etc. Assays that can be performed in conjunction with the compositions and methods of the present invention include, but not limited to, biochemical assays, binding assays, cellular assays, genetic assays, ion transport assay, gene expression assays and protein expression assays. A "binding assay" is an assay that tests for the presence or the concentration of an entity by detecting binding of the entity to a specific binding member, or an assay that tests the ability of an entity to bind another entity, or tests the binding affinity of one entity for another entity. An entity can be an organic or inorganic molecule, a molecular complex that comprises, organic, inorganic, or a combination of organic and inorganic compounds, an organelle, a virus, or a cell. Binding assays can use detectable labels or signal generating systems that give rise to detectable signals in the presence of the bound entity. Standard binding assays include those that rely on nucleic acid hybridization to detect specific nucleic acid sequences, those that rely on antibody binding to entities, and those that rely on ligands binding to receptors. A "biochemical assay" is an assay that tests for the composition of or the presence, concentration, or activity of one or more components of a sample.

A "cellular assay" is an assay that tests for or with a cellular process, such as, but not limited to, a metabolic activity, a catabolic activity, an ion transport function or property, an intracellular signaling activity, a receptor-linked signaling activity, a transcriptional activity, a translational activity, or a secretory activity.

An "ion transport assay" is an assay useful for determining ion transport functions or properties and testing for the abilities and properties of chemical entities to alter ion transport functions. Prefened ion transport assays include electrophysiology-based methods which include, but are not limited to patch clamp recording, whole cell recording, perforated patch or whole cell recording, vesicle recording, outside out and inside out recording, single channel recording, artificial membrane channel recording, voltage gated ion transport recording, ligand gated ion transport recording, stretch activated (fluid flow or osmotic) ion transport recording, and recordings on energy requiring ion transporters (such as ATP), non energy requiring transporters, and channels formed by toxins such a scoφion toxins, viruses, and the like. See, generally Neher and Sakman, Scientific American 266:44-51 (1992); Sakmann and Heher, Ann. Rev. Physiol. 46:455-472 (1984); Cahalan and Neher, Methods in Enzymology 207:3-14 (1992); Levis and Rae, Methods in Enzymology 207:14-66 (1992); Armstrong and Gilly, Methods in Enzymology 207:100-122 (1992); Heinmann and Conti, Methods in Enzymology 207:131-148 (1992); Bean, Methods in Enzymology 207:181-193 (1992); Leim et al., Neurosurgery 36:382-392 (1995); Lester, Ann. Rev. Physiol 53:477-496 (1991); Hamill and McBride, Ann. Rev. Physiol 59:621-631 (1997); Bustamante and Nananda, Brazilian Journal 31:333-354 (1998); Martinez-Pardon and Ferrus, Cunent Topics in Developmental Biol. 36:303-312 (1998); Heniess, Physiology and Behavior 69:17-27 (2000); Aston-Jones and Siggins, www.acnp.org/GA GN40100005/CH005.html (February 8, 2001); U.S. Patent No. 6,117,291; U.S. Patent No. 6,107,066; U.S. Patent No. 5,840,041 and U.S. Patent No. 5,661,035; Boulton et al., Patch-Clamp Applications and Protocols, Neuromethods V. 26 (1995), Humana Press, New Jersey; Ashcroft, Ion Channels and Disease, Cannelopathies, Academic Press, San Diego (2000); Sakmann and Neher, Single Channel Recording, second edition, Plenuim Press, New York (1995) and Soria and Cena, Ion Channel Pharmacology, Oxford University Press, New York (1998), each of which is incoφorated by reference herein in their entirety.

An "electrical seal" refers to a high-resistance engagement between a particle such as a cell or cell membrane and an ion transport measuring means, such as a hole, capillary or needle of a chip or device ofthe present invention. Prefened resistance of such an electrical seal is between about 1 mega ohm and about 100 giga ohms, but that need not be the case. Generally, a large resistance results in decreased noise in the recording signals. For specific types of ion channels (with different magnitude of recording current) appropriate electric sealing in terms of mega ohms or giga ohms can be used.

An "acid" includes acid and acidic compounds and solutions that have a pH of less than 7 under conditions of use.

A "base" includes base and basic compounds and solutions that have a pH of greater than 7 under conditions of use.

"More electronegative" means having a higher density of negative charge. In the methods of the present invention, a chip or ion transport measuring means that is more electronegative has a higher density of negative surface charge.

An "electrolyte bridge" is a liquid (such as a solution) or a solid (such as an agar salt bridge) conductive connection with at least one component ofthe electrolyte bridge being an electrolyte so that the bridge can pass current with no or low resistance. A "ligand gated ion transport" refers to ion transporters such as ligand gated ion channels, including extracellular ligand gated ion channels and intracellular ligand gated ion channels, whose activity or function is activated or modulated by the binding of a ligand. The activity or function of ligand gated ion transports can be detected by measuring voltage or current in response to ligands or test chemicals. Examples include but are not limited to GABA_A, strychnine-sensitive glycine, nicotinic acetylcholine (Ach), ionotropic glutamate (iGlu), and 5-hydroxytryptamine₃ (5-HT₃) receptors.

A "voltage gated ion transport" refers to ion transporters such as voltage gated ion channels whose activity or function is activated or modulated by voltage. The activity or function of voltage gated ion transports can be detected by measuring voltage or cunent in response to different commanding currents or voltages respectively. Examples include but are not limited to voltage dependent Na⁺ channels. "Perforated patch clamp" refers to the use of perforation agents such as but not limited to nystatin or amphotericin B to form pores or perforations in membranes that are preferably ion-conducting, which allows for the measurement of current, including whole cell current.

An "electrode" is a structure of highly electrically conductive material. A highly conductive material is a material with conductivity greater than that of surrounding structures or materials. Suitable highly electrically conductive materials include metals, such as gold, chromium, platinum, aluminum, and the like, and can also include nonmetals, such as carbon, conductive liquids and conductive polymers. An electrode can be any shape, such as rectangular, circular, castellated, etc. Electrodes can also comprise doped semi-conductors, where a semi-conducting material is mixed with small amounts of other "impurity" materials. For example, phosphorous-doped silicon may be used as conductive materials for forming electrodes.

A "channel" is a structure with a lower surface and at least two walls that extend upward from the lower surface of the channel, and in which the length of two opposite walls is greater than the distance between the two opposite walls. A channel therefore allows for flow of a fluid along its internal length. A channel can be covered (a "tunnel") or open. "Continuous flow" means that fluid is pumped or injected into a chamber of the present invention continuously during an assay or separation process, or before or after an assay or separation process. This allows for components of a sample or solution that are not selectively retained on a chip to be flushed out ofthe chamber. "Binding partner" refers to any substances that both bind to the moieties with desired affinity or specificity and are manipulatable with the desired physical force(s). Non-limiting examples of the binding partners include cells, cellular organelles, viruses, particles, microparticles or an aggregate or complex thereof, or an aggregate or complex of molecules. A "specific binding member" is one of two different molecules having an area on the surface or in a cavity that specifically binds to and is thereby defined as complementary with a particular spatial and polar organization ofthe other molecule. A specific binding member can be a member of an immunological pair such as antigen-antibody, can be biotin-avidin or biotin streptavidin, ligand-receptor, nucleic acid duplexes, IgG-protein A, DNA-DNA, DNA-RNA, RNA-RNA, and the like.

A "nucleic acid molecule" is a polynucleotide. A nucleic acid molecule can be DNA, RNA, or a combination of both. A nucleic acid molecule can also include sugars other than ribose and deoxyribose incoφorated into the backbone, and thus can be other than DNA or RNA. A nucleic acid can comprise nucleobases that are naturally occurring or that do not occur in nature, such as xanthine, derivatives of nucleobases, such as 2-aminoadenine, and the like. A nucleic acid molecule of the present invention can have linkages other than phosphodiester linkages. A nucleic acid molecule of the present invention can be a peptide nucleic acid molecule, in which nucleobases are linked to a peptide backbone. A nucleic acid molecule can be of any length, and can be single-stranded, double-stranded, or triple-stranded, or any combination thereof. The above described nucleic acid molecules can be made by a biological process or chemical synthesis or a combination thereof.

A "detectable label" is a compound or molecule that can be detected, or that can generate readout, such as fluorescence, radioactivity, color, chemiluminescence or other readouts known in the art or later developed. Such labels can be, but are not limited to, photometric, colorimetric, radioactive or moφhological such as changes of cell moφhology that are detectable, such as by optical methods. The readouts can be based on fluorescence, such as by fluorescent labels, such as but not limited to, Cy-3, Cy-5, phycoerythrin, phycocyanin, allophycocyanin, FITC, rhodamine, or lanthanides; and by fluorescent proteins such as, but not limited to, green fluorescent protein (GFP). The readout can be based on enzymatic activity, such as, but not limited to, the activity of beta-galactosidase, beta-lactamase, horseradish peroxidase, alkaline phosphatase, or luciferase. The readout can be based on radioisotopes (such as ³³P, ³H , ¹⁴C, ³⁵S, ¹²⁵1, ³²P or ¹³¹I). A label optionally can be a base with modified mass, such as, for example, pyrimidines modified at the C5 position or purines modified at the N7 position. Mass modifying groups can be, for examples, halogen, ether or polyether, alkyl, ester or polyester, or of the general type XR, wherein X is a linking group and R is a mass-modifying group. One of skill in the art will recognize that there are numerous possibilities for mass-modifications useful in modifying nucleic acid molecules and oligonucleotides, including those described in Oligonucleotides and Analogues: A Practical Approach, Eckstein, ed. (1991) and in PCT/US94/00193.

A "signal producing system" may have one or more components, at least one component usually being a labeled binding member. The signal producing system includes all of the reagents required to produce or enhance a measurable signal including signal producing means capable of interacting with a label to produce a signal. The signal producing system provides a signal detectable by external means, often by measurement of a change in the wavelength of light absoφtion or emission. A signal producing system can include a chromophoric substrate and enzyme, where chromophoric substrates are enzymatically converted to dyes, which absorb light in the ultraviolet or visible region, phosphors or fluorescers. However, a signal producing system can also provide a detectable signal that can be based on radioactivity or other detectable signals. The signal producing system can include at least one catalyst, usually at least one enzyme, and can include at least one substrate, and may include two or more catalysts and a plurality of substrates, and may include a combination of enzymes, where the substrate of one enzyme is the product of the other enzyme. The operation of the signal producing system is to produce a product that provides a detectable signal at the predetermined site, related to the presence of label at the predetermined site.

In order to have a detectable signal, it may be desirable to provide means for amplifying the signal produced by the presence ofthe label at the predetermined site. Therefore, it will usually be preferable for the label to be a catalyst or luminescent compound or radioisotope, most preferably a catalyst. Preferably, catalysts are enzymes and coenzymes that can produce a multiplicity of signal generating molecules from a single label. -An enzyme or coenzyme can be employed which provides the desired amplification by producing a product, which absorbs light, for example, a dye, or emits light upon inadiation, for example, a fluorescer. Alternatively, the catalytic reaction can lead to direct light emission, for example, chemiluminescence. A large number of enzymes and coenzymes for providing such products are indicated in U.S. Pat. No. 4,275,149 and U.S. Pat. No. 4,318,980, which disclosures are incoφorated herein by reference. A wide variety of non-enzymatic catalysts that may be employed are found in U.S. Pat. No. 4,160,645, issued July 10, 1979, the appropriate portions of which are incoφorated herein by reference.

The product ofthe enzyme reaction will usually be a dye or fluorescer. A large number of illustrative fluorescers are indicated in U.S. Pat. No. 4,275,149, which is incoφorated herein by reference.

Other technical terms used herein have their ordinary meaning in the art that they are used, as exemplified by a variety of technical dictionaries.

Introduction

The present invention recognizes that using direct detection methods to determine an ion transport function or property, such as patch-clamps, is preferable to using indirect detection methods, such as fluorescence-based detection systems. The present invention provides biochips and methods of use that allow for the direct detection of one or more ion transport functions or properties using chips and devices that can allow for automated detection of one or more ion transport functions or properties. These biochips and methods of use thereof are particularly appropriate for automating the detection of ion transport functions or properties, particularly for screening puφoses.

As a non-limiting introduction to the breath of the present invention, the present invention includes several general and useful aspects, including:

1) a biochip device for ion transport measurement that comprises at least one upper chamber piece and at least one biochip that comprises at least one ion transport measuring means. The device can comprise one or more conduits that provide an electrolyte bridge to at least one electrode. 2) a biochip ion transport measuring device having one or more flow-through lower chambers.

3) a biochip devices adapted for a microscope stage.

4) methods of making an upper piece for a biochip device for ion transport measurement . 5) methods for making chips comprising ion transport measurement holes using laser drilling techniques.

6) devices that include an inverted chip for ion transport measurement.

7) methods of treating ion transport measuring chips to enhance their sealing properties. 8) a method to measure surface energy, such as on the surface of a chemically- treated ion transport measurement biochip. 9) substrates, biochips, cartridges, apparatuses, and/or devices comprising ion transport measuring means with enhanced electric seal properties. 10) methods for storing the substrates, biochips, cartridges, apparatuses, and/or devices comprising ion transport measuring means with enhanced electrical seal properties.

11) methods for shipping the substrates, biochips, cartridges, apparatuses, and/or devices comprising ion transport measuring means with enhanced electrical seal properties.

12) methods for assembling devices and cartridges of the present invention using UN adhesives.

13) a method of producing ion transport measuring chips by fabricating them as detachable units of a larger sheet.

14) a method of producing high density ion transport measuring chips.

15) a biochip device for ion transport measurement comprising fluidic channel upper and lower chambers.

16) methods of preparing cells for ion transport measurement. 17) a software program logic that controls a pressure control profile to direct an ion transport measurement apparatus to achieve and maintain a high- resistance electrical seal.

These aspects of the invention, as well as others described herein, can be achieved by using the methods, articles of manufacture and compositions of matter described herein. To gain a full appreciation of the scope of the present invention, it will be further recognized that various aspects of the present invention can be combined to make desirable embodiments ofthe invention.

I. DEVICE FOR ION TRANSPORT MEASUREMENT

The present invention comprises devices for ion transport measurement and components of ion transport measuring devices that reduce the costs of manufacture and use and are efficient and convenient to use. The devices ofthe present invention are also designed for maximum versatility, providing for different assay formats within the same basic design.

In some aspects, the present invention contemplates devices and apparatuses that have parts that are manufactured separately and can be assembled to form ion transport measuring devices that have at least one, and preferably multiple, ion transport measuring units, each of which comprises an upper chamber and at least a portion of a biochip that comprises an ion transport measuring means that during use ofthe device can connect the upper chamber with a lower chamber. -An ion transport measuring device ofthe present invention can further comprise at least a portion of at least one lower chamber that is connected to one or more upper chambers ofthe device via an ion transport measuring means ofthe chip. These devices comprising ion channel measuring units can be assembled before the assay procedure, and pieces that make up the device can be reversibly or ineversibly attached to one another.

In many prefened aspects ofthe present invention, a device or one or more parts of a device can be removed from an apparatus and can be disposable after a single use (for example, a chip comprising ion transport measuring means; one or more upper chambers designed to contain cells), and can engage one or more parts of an ion transport measuring device or apparatus that can be permanent and reusable (for example, at least a portion of a lower chamber; one or more electrodes) For example, in some aspects ofthe present invention, devices comprising one or more upper chamber pieces and at least one biochip (called cartridges) are single-use and disposable, and lower chamber pieces, one or more electrodes, and platforms or lower base pieces are reusable. In these aspects, upper chamber pieces and biochips can be reversibly or ineversibly attached to one another during use ofthe device or apparatus, and these attached upper chamber/biochip devices can be reversibly attached to or contacted with lower chamber pieces, conduits, or electrodes.

In one embodiment, the present invention contemplates an ion transport measuring device in the form of a cartridge that comprises an upper chamber piece that comprises at least one well that is open at its upper and lower ends, and a biochip that comprises at least one ion transport measuring means. The chip is reversibly or ineversibly attached to the bottom ofthe upper chamber piece such that each ofthe one or more upper wells is in register with one ofthe one or more ion transport measuring means, providing one or more independent upper chambers each in contact with a single ion transport measuring means. The chip can be in direct or indirect contact with the upper chamber piece.

In a cartridge in which an upper chamber piece is in indirect contact with an attached chip, a spacer or gasket, for example, can be between the upper chamber piece and the chip. A chip can be in direct contact with an upper chamber piece of a cartridge if it is attached during molding ofthe cartridge, by heat sealing, or by adhesives, for example. Attachment of a chip to an upper chamber piece to make a cartridge can be performed by a machine, and can be automated.

A chip can also be intergral to an upper chamber piece in a cartridge or device ofthe present invention, where the chip forms or is part ofthe lower surface ofthe upper chamber piece that can comprise, for example, glass or one or more plastics. Preferably a biochip that is part of an ion transport measuring device of the present invention comprises multiple holes used as ion transport measuring means, and an upper chamber piece comprises multiple upper chambers such that each ofthe upper chambers is in register with one ofthe ion transport measuring means ofthe chip. For example, prefened devices and apparatuses for ion transport measurement can have two or more, four or more, eight or more, or sixteen or more ion transport measuring units and comprise upper chamber pieces comprising a coπesponding number of upper chambers. For example, ion transport measuring devices can have sixteen, twenty-four, forty-eight, ninety-six or more ion transport measuring units and comprise upper chamber pieces comprising a conesponding number of upper chambers.

The upper chambers or wells can be any shape or size. Typically, the upper chambers will be in the form of wells which can be tapered or non-tapered. The wells of an upper chamber piece that can be part of an ion transport measuring device preferably can hold a volume of between about 0.5 microliters and about 5 milhliters or more, more preferably between about 10 microliters and about 2 milhliters, and more preferably yet between about 25 microliters and about 1 milliliter. The upper diameter of a well can be from about 0.05 millimeter to about 20 millimeters or more, and is preferably between about 2 millimeters and about 10 millimeters or more. The depth, or height of a well can vary from about 0.01 to about 25 millimeters or more, and more preferably will be from about 2 millimeters to about 10 millimeters. In designs in which the upper well or wells are tapered, the well can be tapered downward at an angle of from about 0.1 degree to about 89 degrees from vertical, and preferably from about 5 degrees to about 60 degrees from vertical. The well can be tapered at one or more ends, or throughout the circumference ofthe well.

An upper chamber piece can be made of any suitable material, (for example, one or more plastics, one or more polymers, one or more ceramic, one or more waxes, silicon, or glass) but for ease of manufacturing is preferably made of a moldable plastic, such as, for example, polysulfone, polyallomer, polyethylene, polyimide, polypropylene, polystyrene, polycarbonate, cylco olefin polymer (such as, for example, ZEONOR®), polyphenylene ether/PPO or modified polyphenylene oxide (such as, for example, NORYL®), or composite polymers. In some aspects, base resistant plastics such as polystyrene, cylco olefin polymers (such as, for example, ZEONOR®), polyphenylene ether/PPO or modified polyphenylene oxide (such as, for example, NORYL®), can be prefened.

-An upper chamber piece can optionally comprise one or more electrodes. -An upper chamber piece that comprises multiple upper chambers can comprise multiple electrodes, where each well contacts an independent electrode (such as, for example, independent recording electrodes). In an alternative design, an upper chamber piece can contain or contact at least a portion of a single electrode (which can be, for example, a reference electrode) that contacts all ofthe upper chambers ofthe device. In designs in which the upper chamber piece does not comprise one or more electrodes, the upper chamber piece can optionally be used as part of an apparatus for ion transport measurement in which one or more electrodes can be introduced into one or more upper chambers (such as, for example, introduced via a conduit that can be connected to or can be inserted into one or more chambers). In an alternative configuration, conduits connected with or introduced into one or more upper chambers can, during the use ofthe apparatus, be filled with a measuring solution and provide electrolyte bridges to one or more electrodes. The chip can be reversibly or irreversibly attached the lower surface of an - upper chamber piece to form a cartridge by any feasible means that provides a fluid- impermeable seal between the chip and the upper chamber piece, such as by adhesives or by pressure mounting. The chip ofthe assembled cartridge can be in direct or indirect contact with an upper chamber piece. Preferably but optionally, the chip is ineversibly attached to the upper chamber piece, such as by one or more adhesives, to make a cartridge. Such cartridges can optionally single use and disposable. Assembly of a prefened cartridge ofthe present invention is provided in Example 1. An upper chamber piece ofthe present invention can also have features that aid in the manufacture ofthe piece or assembly ofthe cartridge. For example, the lower surface ofthe upper chamber piece can comprise one or more alignment bumps or registration edges on at least one end ofthe lower side ofthe piece that allows a chip to be positioned against the lower side ofthe upper chamber piece such that the ion transport measuring holes ofthe chip are in register with the wells. Features that facilitate manufacture of an upper chamber piece include one or more sink holes that prevent the piece from deforming through thermal contraction ofthe piece during the injection molding process, and one or more glue spillage grooves that allow for seepage of excess glue that may be used in attaching a chip to the upper chamber piece. Assembly of a cartridge can be done manually, or by a machine. Preferably but optionally, at least one ofthe steps in the assembly of a cartridge ofthe present invention by a machine is automated. For example, a machine may perform one or more ofthe steps of: picking up a chip from a rack or holder, picking up an upper chamber piece from a rack, platform, shelf, or holder, applying one or more adhesives to an upper chamber piece or a chip, positioning a chip on the bottom of an upper chamber piece so that the ion transport measuring means ofthe chip are in register with the wells ofthe upper chamber piece, and allowing or promoting attachment ofthe chip to the upper chamber piece (such as by treating with UN or heat). One design of an upper chamber piece is shown in Figure 1. Figure 1 A depicts a top view of an upper chamber piece having sixteen wells (1) and Figure IB depicts a bottom view ofthe upper chamber piece showing the lower openings ofthe sixteen wells (1), and also shows the openings of two sinkholes (3). (In an assembled cartridge or device comprising a chip, the chip preferably covers and thereby seals off, the sinkhole openings.) In this design, the wells (1) are tapered such that the upper diameters ofthe wells (1) (seen in Figure IA) are larger than the lower diameters of the wells (1) (seen in Figure IB). In Figure IC, the upper chamber piece is shown side-on in cross-section, showing the sixteen wells (1) as well as features that increase the efficiency of manufacture of a device, including an alignment bump (2) for chip positioning and sink holes (3) that prevent cave-in ofthe upper chamber piece due to contraction ofthe plastic as it cools after molding ofthe piece. Figure ID is an end- on cross-sectional view ofthe piece showing a well (1) behind a sink hole (3). In Figure ID a glue spillage groove (4) is also shown. A glue spillage groove can allow for seepage of an adhesive used to seal a chip to the lower chamber piece to make a cartridge.

A chip used in a device ofthe present invention is preferably a chip that comprises ion transport measuring means in the form of holes. A chip used in a device ofthe present invention can comprise glass, silicon, silicon dioxide, quartz, one or more plastics, one or more waxes, or one or more polymers (for example, polydimethylsiloxane (PDMS)), one or more ceramics, or a combination thereof. Methods of fabricating such chips, including methods of fabricating ion transport measuring holes in chips, are disclosed in related applications, including United States patent application number 10/760,866 (pending), filed January 20, 2004; United States patent application number 10/642,014, filed August 16, 2003; and United

States patent application number 10/104,300, filed March 22, 2002; each of which is incoφorated by reference herein.

A chip used in a device ofthe present invention is preferably a "K- configuration" chip, but this is not a requirement ofthe present invention. As described in a later section of this application and in the Examples, K-configuration chips have ion transport measuring holes that comprise a through-hole that is laser drilled through one or more counterbores. A chip used in a device ofthe present invention is preferably treated to have enhanced sealing properties. Methods of chemically treating ion transport measuring chips, for example with basic solutions, to enhance their ability to form electrical seals with particles such as cells are disclosed herein. A prefened device for ion transport measurement is a cartridge that comprises a K-configuration chip with enhanced electrical sealing properties that is reversibly or irreversibly attached to an upper chamber piece. Preferably, a chip assembled into a device ofthe present invention has one or more ion transport measuring holes that is able to seal to a cell or particle such that electrical access between the chip and the inside ofthe cell or particle (or between the chip and the inside ofthe cell or particle) has an access resistance that (Ra) is less than the seal resistance (R). Preferably, the access resistance of a whole-cell configuration seal that can be formed on the hole of a chip of a device ofthe present invention is less than 80 MOhm, more preferably less than about 30 MOhm, and more preferably yet, less than about 10 MOhm. Preferably, a chip of a device ofthe present invention can form a seal with a cell such that the seal has a resistance that is at least 200 MOhm, and more preferably, at least 500 MOhm, and more, preferably yet, about 1 GigaOhm or greater. Preferably, a chip of a device ofthe present invention comprises at least one ion transport measuring means in the form of a through-hole that has been laser-drilled through at least one counterbore, in which at least the surface ofthe ion transport measuring means has been treated to enhance its electrical sealing properties, and the chip can form a seal between at least one ion transport measuring means and a cell such that the resistance (R) ofthe seal is at least ten times the access resistance ofthe seal. More preferably, a chip of a device ofthe present invention can form a seal with a cell such that the seal resistance is at least twenty times the Ra.

Preferably, a chip comprising laser-drilled ion transport measuring holes is attached to an upper chamber piece in inverted orientation, as described in a later section of this application, such that the laser entrance hole ofthe ion transport measuring hole is exposed to the upper chambers, but this is not a requirement ofthe present invention, hi the alternative, the chip can be attached to the upper chamber in "upside up" orientation.

A cartridge comprising an upper chamber piece and at least one biochip comprising one or more ion transport measuring means can be assembled into a device that comprises one or more lower chambers in which the one or more lower chambers access at least one upper chamber via a hole in the biochip. A cartridge can engage one or more parts that make up one or more lower chambers, where the one or more lower chambers are directly or indirectly attached to the underside ofthe chip, and at least one ion transport measuring hole in the chip connects the one or more lower chambers with one or more upper chambers ofthe device.

For example, a cartridge comprising an upper chamber piece and at least one biochip comprising one or more ion transport measuring means can be assembled with a lower chamber piece that comprises at least a portion of at least one lower chamber. The cartridge can be assembled with a lower chamber piece that comprises at least a portion of a single lower chamber, such as a dish, tray, or channel that provides a common lower chamber for ion transport measuring means that connect to separate upper chambers. In one embodiment, at least a portion of a lower chamber piece can be in the form of a gasket that seals around the bottom ofthe biochip that when sealed against a lower chamber base piece or platform provides an inner space as a lower chamber

Alternatively, the device can be assembled with a lower chamber piece that comprises at least a portion of more than one lower chamber. In this case, each individual lower chamber preferably connects with a single upper chamber via an ion transport measuring hole in the biochip. The lower chamber piece can form the walls and lower surfaces of lower chambers, or the lower chamber piece can form at least a portion ofthe walls of a lower chamber and other parts can form the bottom surface of the lower chambers. In one embodiment, at least a portion of a lower chamber piece can be in the form of a gasket that seals around the bottom ofthe biochip and having openings such that when the gasket is sealed against a lower chamber base piece or platform the inner spaces ofthe gasket openings provide lower chambers.

A lower chamber piece can be ineversibly attached to a cartridge ofthe present invention, such as by the use of adhesives, but preferably, a lower chamber piece is reversibly attached to a cartridge. Reversible attachment can be by any feasible means that provides a fluid-impermeable seal between the walls ofthe lower chamber or chambers and the lower surface ofthe chip, such as pressure mounting, and can use clamps, frames, screws, snaps, etc.

In one example of attachment of a lower chamber piece to a cartridge, a lower chamber piece structure comprising a compressible material such as PDMS contains channels for fluid delivery and other channels for applying vacuum pressure that can maintain a strong seal between the biochip and the structure, where the vacuum pressure provides the means of reversible attachment ofthe lower chamber piece to the biochip. Preferably, the applied vacuum pressure also scavenges any leaks that may occur or develop between lower chambers that would otherwise result in electrical cross-talk between adjacent lower chambers.

Preferred embodiments encompass devices that comprise multiple ion transport measuring units, comprising an upper chamber piece that comprises at least two upper chambers that are open at both their upper and lower ends and a chip that comprises at least two ion transport measuring means in the form of holes through the chip that are in register with the upper chambers. The upper chamber piece and chip can be reversibly or ineversibly attached to a lower chamber piece that comprises at least a portion of at least two lower chambers that are in register with the ion transport measuring means and upper chambers. Such prefened devices comprise multiple ion transport measuring units, where each unit comprises an upper chamber and a lower chamber, each in register with a hole in the biochip, in which the hole connects the upper with a lower chamber. The interaction between the chambers and the chip are such that at least one ofthe chambers of an ion transport measuring unit can be pneumatically sealed and can withstand pressures of at least plus or minus 100 m Hg, and preferably at least plus or minusl atmosphere of pressure.

In some prefened aspects ofthe present invention, a cartridge comprises an upper chamber piece comprising multiple upper chambers ineversibly attached to a chip comprising multiple ion transport measuring holes that can be reversibly engaged with a lower chamber piece that comprises at least a portion of multiple lower wells, such that the upper wells and lower wells ofthe device are in register with one another and with the ion transport measuring holes ofthe chip.

Prefened devices and apparatuses for ion transport measurement can have two or more, four or more, eight or more, or sixteen or more ion transport measuring units. For example, ion transport measuring devices can have sixteen, twenty-four, forty- eight, or ninety-six or more ion transport measuring units.

Lower chamber pieces that comprise at least a portion of multiple lower chambers of a multiple unit ion transport measuring apparatus can be provided in a variety of designs. Lower chamber pieces can comprise complete lower chamber units, or can comprise all or a portion ofthe walls ofthe multiple chamber units, such that when the lower chamber piece is fixed to or pressed against the lower side of a biochip and attached to or pressed down on a platform or lower chamber base piece, the lower chamber piece forms the walls and the platform or lower chamber base piece forms the bottoms ofthe lower chambers. For example, a device for measuring ion transport function or activity can comprise a multiple unit device that comprises an upper chamber piece having multiple upper chambers in the form of wells that are open at both the top and bottom, and a chip attached to the upper chamber piece, where the chip comprises multiple holes for ion transport measurement that are spaced such that when the device is assembled each upper chamber is over a hole. A lower chamber piece can be held or fastened against the lower side ofthe chip ofthe device, where the lower chamber piece comprises multiple openings that fit over the biochip holes to form lower chambers. In a prefened embodiment, the lower chamber piece comprises at least one compressible plastic or polymer on its upper surface that can form a fluid- impermeable seal with the bottom ofthe biochip. The lower chamber piece can also comprise at least one compressible polymer as a gasket on its lower surface that can form a seal with a platform or a lower base piece. In this design, when the device is positioned on a lower base piece or platform so that the lower chamber piece is pressed against the lower base piece or platform, the lower base piece or platform forms the bottom ofthe lower chambers. Mechanical pressure can provide a seal between the biochip and the lower chamber piece, and between the lower chamber piece and the platform. Clamps can optionally be employed to hold the seal. The compressible plastic or polymer can comprise rubber, a plastic, or an elastomer, such as for example, polydimethylsiloxane (PDMS), silicon polyether urethane, polyester elastomer, polyether ester elastomer, olefinic elastomer, polyurethane elastomer, polyether block amide, or styrenic elastomer. Preferably, in cases where the compressible plastic or polymer contacts cells, the compressible plastic or polymer is made of a biocompatible material, such as PDMS. Portions ofthe lower chamber piece that do not form a gasket can be of any suitable material, including plastics, waxes, polymers, glass, metals, and ceramics. Portions ofthe lower chamber piece that contact measuring solutions preferably comprise materials that are not affected by electrical cunent (such as nonmetals).

For example, one prefened design of a device for ion transport measurement comprises an upper chamber piece, a chip comprising ion transport measuring holes, a lower chamber piece, and a lower base piece in the form of a platform. The chip has been chemically treated, preferably with at least one base, to enhance its sealing properties. The lower chambers that are formed by a lower chamber piece that comprises an aluminum frame having a PDMS gasket on its upper surface that fits over the lower surface of a chip. PDMS is also used to coat the inner surfaces ofthe holes that form the lower chambers, and is also used as a gasket on the bottom ofthe lower chamber piece. The lower chambers can be filled with a solution while the device is held in inverted orientation prior to positioning the device on the platform. During use ofthe device, mechanical pressure holds the lower chamber piece against the chip and against the platform.

The lower base piece can optionally comprise one or more electrodes. For example, separate individual electrodes can be fabricated on or attached to the platform so that separate lower chambers ofthe device have independent electrodes that can be attached to independent circuits and used as patch clamp recording electrodes. In an alternative design, the platform can comprise or be part of a common lower chamber with a reference electrode, or a common electrode that can be used as a reference electrode can contact all ofthe lower chambers of a device having multiple lower chambers (optionally through separate electrode extensions that meet a common connector outside ofthe chambers).

The lower base piece can optionally comprise or engage one or more conduits connected to tubing that can allow for the flow of fluids into and out of individual lower chambers. In preferred embodiments, a device ofthe present invention comprises one or more flow-through lower chambers where each ofthe one or more lower chamber connects to at least one conduit for providing solutions to the lower chamber (the inflow conduit) and at least one additional conduit for removing solutions from the lower chamber (the outflow conduit). Figure 2 depicts a single ion transport measuring unit of a device in which a gasket (24) forms the walls ofthe lower chamber (25). The upper well (21) is part of an upper chamber piece that is attached to a chip (23) having an ion transport measuring means in the form of a hole (22). An inflow conduit (27) and outflow conduit (28) connects to each lower chamber, hi this type of design the lower chambers can be filled with a measuring solution (such as an intracellular solution) after the gasket is positioned on a lower base piece. The conduits can also be used for the exchange of solutions during the use ofthe device. For example, solutions containing test compounds, ionophores, inhibitors, drugs, different concentrations or combinations of ions or compounds, etc., can be delivered into and out of a chamber during ion transport measuring assays. At least some ofthe conduits or tubing can optionally comprise or lead to electrodes (such as, for example, recording electrodes). In the design depicted in Figure 2, a lower chamber electrode (26) is situated on, fabricated on, or attached to the lower chamber piece.

The present invention also includes methods of using an ion transport measuring device ofthe present invention that comprises at least one upper chamber piece reversibly or irreversibly attached to a chip, wherein the chip comprises at least one ion transport measuring means in the form of a hole through the biochip, wherein the chip has been treated to have enhanced electrical sealing properties. The device further comprises at least one lower chamber, wherein at least one well ofthe upper chamber piece comprises, contacts, or is in electrical contact with at least one electrode, and the at least one lower chamber

In one prefened design, a lower chamber piece comprises conduits that engage each lower chamber from one side (one per chamber), and conduits that engage each lower chamber from the opposite side. Conduits on one side ofthe lower chamber piece can be used for introducing solutions, such as "intracellular solutions" that can optionally comprise test compounds, into the chambers, and conduits on the opposite side ofthe lower chamber piece can be used for flushing solutions and or air bubbles out ofthe lower chambers. At least one set ofthe conduits (such as, for example, the inflow conduits) can comprise wire electrodes that are independently connected (with respect to other ion transport measuring units) to a signal amplifier and used for ion transport activity recording.

Devices such as those described herein can be part of apparatuses that also comprise patch clamp signal amplifiers and conduits, fluid dispensing means, pumps, electrodes, or other components. The apparatuses are preferably mechanized, for automated fluid dispensing or pumping, pressure generation for sealing of particles, and ion transport recording. The apparatuses can be part of a biochip system for ion transport measurement, in which software controls the automated functions.

The present invention also includes methods of using an ion transport measuring device ofthe present invention to measure one or more ion transport properties or activities of a cell or particle (such as, for example, a membrane vesicle). The methods include using a device that comprises at least one upper chamber reversibly or ineversibly attached to a chip that comprises at least one ion transport measuring means in the form of a hole through the biochip, wherein the chip has been treated to have enhanced sealing properties. In the assembled device used in the methods ofthe present invention, the holes ofthe biochip access at least one lower chamber. In these methods, the device is reversibly or ineversibly attached to a lower chamber piece that forms all or a portion of a lower chamber. An upper chamber piece and chip can optionally additionally be reversibly or irreversibly attached to a platform or lower chamber base piece that can form at least the lower surface of one or more lower chambers. For example, a cartridge comprising an upper chamber piece and chip can be attached to at least one lower chamber piece that forms the walls and lower surfaces of one or more lower chambers, or a cartridge can be attached to at least one lower chamber piece that forms the walls of one or more lower chambers and at least one platform or lower chamber base piece that forms the lower surfaces of one or more lower chambers.

The device is assembled such that the one or more upper chambers are in register with the one or more ion transport measuring holes ofthe chip, and one or more lower chambers access the one or more upper chambers via the one or more , holes ofthe chip. In prefened embodiments, each ofthe one or more upper chambers is in register with one ofthe ion transport measuring holes ofthe chip, and each ofthe lower chambers is aligned with one upper chamber that it accesses via an ion transport measuring hole.

During use ofthe device, the one or more upper chambers comprise, contact, or are in electrical contact with at least one electrode. During use ofthe device, the one or more lower chambers comprise, contact, or are in electrical contact with at least one electrode. In one alternative, the one or more upper chambers contact, comprise, or are in electrical contact with a common reference electrode, and the one or more lower chambers contact, comprise, or are in electrical contact with a individual reference electrodes. In another alternative, the one or more upper chambers contact, comprise, or are in electrical contact with individual reference electrodes, and the one or more lower chambers contact, comprise, or are in electrical contact with a common reference electrode.

The method includes: filling at least one lower chamber ofthe device with a measuring solution; adding at least one cell or particle to one or more ofthe at least upper chambers ofthe device, wherein the one or more upper chambers is connected to one ofthe at least one lower chambers that comprises measuring solution via a hole in the ion transport measuring chip; applying pressure to at least one lower chamber, at least one lower chamber, or to an upper chamber and a lower chamber that are connected via an ion transport measuring hole to create a high-resistance electrical seal between at least one cell or particle and at least one hole; and measuring at least one ion transport property or activity ofthe at least one cell or at least one particle. Preferably, one or more cells or one or more particles are in a suspension when added to the upper chamber. Narious measuring solutions and, optionally, compounds can be provided in an upper chamber or a lower chamber.

In some prefened embodiments, the methods measure at least one ion transport activity or property of a cell in the whole cell configuration, but this is not a requirement ofthe present invention, as the devices can be used in a variety of applications on particles such as, for example, vesicles, as well as cells.

The application of pressure can be manual or automated. If pressure is applied manually (for example, by means of a syringe), preferably the user can make use of a pressure display system to monitor the applied pressure. Automated application of pressure can be through the use of a software program that is able to receive feedback from the device and direct and control the amount of pressure applied to one or more ion transport measuring units.

Narious specific ion transport assay can be used for determining ion transport function or properties. These include methods known in the art such as but not limited to patch clamp recording, whole cell recording, perforated patch whole cell recording, vesicle recording, outside out or inside out recording, single channel recording, artificial membrane channel recording, voltage-gated ion transport recording, ligand-gated ion transport recording, recording of energy requiring ion transports (such as ATP), non energy requiring transporters, toxins such a scoφion toxins, viruses, stretch-gated ion transports, and the like. See, generally Νeher and Sakman, Scientific American 266:44-51 (1992); Sakman and Νeher, Ann. Rev. Physiol. 46:455-472 (1984); Cahalan and Νeher, Methods in Enzymology 207:3-14 (1992); Levis and Rae, Methods in Enzymology 207:14-66 (1992); Armstrong and Gilly, Methods in Enzymology 207: 100-122 (1992); Heinmann and Conti, Methods in Enzymology 207:131-148 (1992); Bean, Methods in Enzymology 207:181-193 (1992); Leim et al., Νeurosurgery 36:382-392 (1995); Lester, Ann. Rev. Physiol 53:477-496 (1991); Hamill and McBride, Ann. Rev. Physiol 59:621-631 (1997); Bustamante and Nananda, Brazilian Journal 31:333-354 (1998); Martinez-Pardon and Ferrus, Cunent Topics in Developmental Biol. 36:303-312 (1998); Hemess, Physiology and Behavior 69:17-27 (2000); U.S. Patent No. 6,117,291; U.S. Patent No. 6,107,066; U.S. Patent No. 5,840,041 and U.S. Patent No. 5,661,035; Boulton et al., Patch-Clamp Applications and Protocols, Neuromethods V. 26 (1995), Humana Press, New Jersey; Ashcroft, Ion Channels and Disease, Cannelopathies, Academic Press, San Diego (2000); Sakman and Neher, Single Channel Recording, second edition, Plenuim Press, New York (1995) and Soria and Cena, Ion Channel Pharmacology, Oxford University Press, New York (1998), each of which is incoφorated by reference herein in their entirety. II. AN ION CHANNEL MEASURMENT DEVICE HAVING FLOW-THROUGH LOWER CHAMBERS

The present invention includes ion transport measurement devices and apparatuses comprising flow-through lower chambers. As used herein, a "flow- through chamber" is a chamber to which fluids can be added and from which fluids can be removed via continuous fluid flow. Thus, a flow-through chamber will preferably engage at least two conduits: at least one inflow conduit for adding fluids (such as solutions) and at least one outflow conduit for the removal of fluids (such as solutions). In the alternative, a flow-through chamber can be designed as a channel through which fluids can pass.

A flow-through lower chamber can be designed with two or more ports or openings in the wall ofthe chamber, such that at least one inflow conduit and at least one outflow conduit engage one or more walls ofthe lower chamber at the ports. In an alternative, at least one inflow conduit and at least one outflow conduit can engage ports or openings at the bottom surface of a chamber. It is also possible to have a flow-through chamber in which at least one conduit engages the wall ofthe chamber and at least one conduit engages the bottom surface ofthe chamber.

Flow-through lower chambers have several advantages for ion transport measuring devices. Because the exchange of lower chamber solutions can be performed rapidly and continuously, without the need to empty the chamber of liquid when changing from a first solution to a second solution, a single patch clamp (that is, a cell or particle sealed with a high resistance electrical seal to an ion transport measuring hole) can be used for repeated tests, using, for example, different solutions that are delivered to the chamber in sequence. Adding and removing solutions in a flow-manner via conduits also facilitates automation of an ion transport measurement device, where the addition and removal of solutions can be through the automated control of pumps and valves. Addition or removal of solutions to one or more lower chambers can preferably but optionally be performed independently ofthe fluid distribution to other chambers of a device, so that conditions of particular patch clamps can be changed without disrupting or changing the conditions of other patch clamps ofthe device.

In prefened embodiments, an ion transport measurement device comprises one or more flow-through lower chambers, at least one chip comprising ion transport measuring holes, and at least one upper chamber. Preferably, a flow-through chamber is connected to two or more conduits that can provide fluid flow to and from a lower chamber. At least one ofthe at least two conduits can be used to provide solutions to a lower chamber, and at least one other ofthe at least two conduits can be used to remove solutions from a lower chamber.

Preferably, fluid flow is directed by one or more fluid pressure sources such as a pump or pumps. The conduits, or tubing or connectors leading to the conduits, can comprise valves that can be used to control the flow of solutions into or out of a lower chamber. In some prefeπed embodiments, control ofthe flow of solutions into or out of a chamber is automated, at least in part.

Lower chambers can be formed by one or more pieces ofthe device. At least a portion ofthe upper surface of a lower chamber will be formed by a chip comprising an ion transport measuring hole. The walls and bottom surface of a lower chamber can be formed by one or more pieces ofthe device. For example, in some embodiments at least a portion ofthe walls and the bottom surface of a lower chamber can be formed by a lower chamber piece. In other prefened embodiments, at least a portion ofthe walls of a lower chamber can be formed by a lower chamber piece and the bottom surface of a lower chamber can be formed by a lower chamber base piece or a platform. In some embodiments, an ion transport measuring device with one or more flow-through lower chambers can comprise a lower chamber piece that has inflow and outflow conduits that directly or indirectly connect to the walls or bottom surfaces of the one or more lower chambers. In some designs, the device can comprise a platform or a lower chamber base piece that comprises inflow and outflow conduits that directly or indirectly connect to the bottom surface of one or more lower chambers. In an especially prefeπed embodiment ofthe present invention, a device for ion transport measurement comprises a lower chamber base piece that forms the bottom of multiple lower chambers and comprises conduits that open to the lower surfaces of the lower chambers, such that each lower chamber is accessed by an inflow conduit and an outflow conduit. In this design, the device further comprises a lower chamber piece that forms at least a portion ofthe lower chamber walls, a chip comprising ion transport measuring holes that align with the lower chambers, and an upper chamber piece that comprises multiple upper wells that align with the ion transport measuring holes ofthe chip and the lower chambers formed by the lower chamber piece and lower chamber base piece.

In prefened embodiments of ion transport measuring devices having one or more flow-through lower chambers, the devices have multiple flow-through lower chambers, each of which engages an inflow conduit and an outflow conduit, such that inflow and outflow conduits connected to different chambers are separate and independent.

Components of an ion transport measuring device having one or more flow- through lower chambers, such as a lower chamber base piece, lower chamber piece, chip, and an upper chamber piece, can be reversibly or ineversibly attached to one another. In some prefened embodiments, an upper chamber piece and chip are ineversibly attached (such as by adhesives) to one another as a cartridge, and the cartridge can be reversibly attached to a lower chamber piece and lower chamber base piece. A cartridge can be attached to a lower chamber piece by any feasible means that provides a fluid impermeable seal between the lower surface ofthe chip ofthe cartridge and the walls ofthe one or more lower chambers that are formed, at least in part, by a lower chamber piece. In designs in which the device comprises a lower chamber base piece, the lower chamber base piece can be attached to a lower chamber piece by any feasible means that provides a fluid impermeable seal between the lower chamber piece and the lower chamber base piece. The attachment of a lower chamber base piece to a lower chamber base can be iπeversible, but is preferably reversible. For example, reversible attachment can be by pressure mounting, and can use compressible materials as well as clamps, frames, screws, snaps, etc.

In prefened embodiments encompassing devices having more than one ion transport measuring unit, when a multiunit device is assembled, the two or more wells ofthe upper chamber piece are in register with the two or more holes ofthe biochip, and the two or more lower chambers formed by a lower chamber piece and lower chamber base piece are aligned with the holes with the biochip. The lower chamber base piece comprises at least two inflow conduits and at least two outflow conduits, such that each lower chamber is accessed by an inflow conduit and an outflow conduit.

In some prefened embodiments, a cartridge, lower chamber piece that comprises a compressible material and a lower chamber base piece are fastened together using a clamp. In other prefeπed embodiments, a cartridge, lower chamber piece, and, optionally, a lower chamber base piece are attached using pressure mounting and at least one gasket to form seals between the parts.

The present invention also includes a lower chamber base piece for use in a device for ion transport measurement that can optionally be used independently of a larger automated apparatus and can be used to observe cells and particles within the device using an inverted microscope. In this embodiment, at least a portion ofthe lower chamber base piece that will form the bottom surface ofthe lower chambers is transparent. Preferably, the lower chamber base piece comprises at least two conduits that extend through the lower chamber base piece such that when the lower chamber base piece is assembled into a device ofthe present invention, the conduits can be used to transfer fluid from outside the device into lower chambers, and transfer fluid from inside lower chambers to the outside ofthe device. As part of a device for ion transport measurement, the base piece forms a bottom surface of lower chambers. The conduits that extend through the base piece allow for fluids such as solutions to be delivered in and out of lower chambers of ion transport measuring devices.

In this embodiment, two or more conduits go through the base piece, with each conduit having one opening on one surface ofthe base piece, and the other opening on a different surface ofthe base piece. In prefened embodiments ofthe present invention, the conduits extend from a side ofthe base piece essentially horizontally toward the center, and then turn or curve upward to end in an opening on the top surface ofthe base piece which, in an assembled device, is the bottom surface of a lower chamber. The side opening can be the site where the conduit connects with tubing connected to solution reservoirs, pressure sources, and/or electrodes, and the top opening ofthe conduits is the site where the conduit opens into a lower chamber. In a prefeπed device ofthe present invention, each lower chamber of an ion transport measuring device is connected to two such conduits, and the conduits can provide for solutions to be delivered into and washed out of a lower chamber.

A lower chamber piece and lower chamber base piece can comprise one or more plastics, one or more polymers, one or more ceramics, silicon or glass. Preferably, the part or parts of a lower chamber base piece that will form the bottom of one or more lower chambers of an ion transport measuring device is preferably made of a transparent material that is impermeable to aqueous liquids so that cells or particles inside an ion transport measuring unit are visible using an inverted microscope. Although not a requirement ofthe present invention, to simplify manufacture ofthe base piece, the entire base piece (with the exception of separate attachments such as connectors, pins, screws, etc.) is preferably made of a single material by molding or machining. Glass and transparent polymers are prefened materials, with transparent polymers such as polycarbonate and polystyrene having the advantage of easier manufacture.

Conduits can be molded into or drilled through the base piece, and can be fitted with connectors. (Connectors can comprise glass, polymers, plastics, ceramics, or metals.) The connectors can be connected to tubing that can be used to provide inflow and outflow of solutions to a lower chamber of an ion transport measuring unit. The conduits can also be used to deliver pressure to the lower chamber and to an ion transport measuring hole of a chip exposed to the chamber. Pressure can be generated, for example, by a pump or a pressure source connected to the tubing that will be filled with an appropriate solution in at least the segment connecting the lower chamber. Preferably the pressure is regulatable and can be used for purging air bubbles and or other blocking micro-particles in the ion transport measuring hole, cell and particle positioning, sealing, and optionally, membrane rupture of an attached cell when carrying out ion transport measurement procedures.

In prefened embodiments, the conduits, or tubes leading to the conduits, can also comprise electrodes. For example, a wire electrode can be threaded through tubing that is connected to a conduit of a base piece. The wire electrode can optionally extend through the conduit to the upper surface ofthe base piece (which will be the lower surface of a lower chamber of an ion transport measuring unit).

In the alternative, the base piece can comprise one or more electrodes on its upper surface. Electrodes fabricated or attached to the upper surface ofthe base piece can be connected through leads to connectors on the outer edge ofthe base piece, and the connectors can be connected to a patch clamp amplifier.

In prefened aspects ofthe present invention, a lower chamber base piece is designed to form the bottom of more than one lower chamber of an ion transport measuring device. Preferably, a lower chamber base piece is designed to form the bottoms of all the lower chambers of an ion transport measuring device that comprises at least two ion transport measuring units, more preferably at least six ion transport measuring units, and more preferably yet, at least sixteen ion transport measuring units, i a prefened embodiment described in detail in Example 5, a lower chamber base piece forms the bottom of 16 lower chambers of a 16 unit device. In many cases (as illustrated in the example) multiple lower chambers will be ananged linearly in a row, but this is not a requirement ofthe present invention.

Thus, in prefened embodiments ofthe present invention, a flow-through lower chamber base piece will comprise multiple conduits, two for each lower chamber that will occur in the ion transport measuring device: a first conduit for inflow of solutions (the "inflow conduit"), and a second conduit for outflow of solutions (the "outflow conduit"). A schematic cross-sectional view of a single ion transport measuring unit of one design of a device ofthe present invention having one or more flow-through lower chambers is shown in Figure 2. In this depiction, the lower chamber (25) is accessed by an inflow conduit (27) and an outflow conduit (28). In this depiction the lower chamber comprises an electrode (26) positioned on the upper surface ofthe lower chamber base piece, hi an alternative design, one of each pair of conduits that leads to a single chamber of an ion transport measuring device can contain or contact an electrode. The present invention also includes devices and apparatuses for ion transport measurement that include a lower chamber base piece ofthe present invention. In one embodiment ofthe present invention, a device includes: a lower chamber base piece that comprises at least two conduits, where at least a portion ofthe lower chamber base piece is transparent; a chip comprising at least one ion transport measuring hole; and an upper chamber piece that comprises at least one chamber that attaches to said chip. Preferably, the device also includes a lower chamber piece in the form of at least one gasket that fits between the lower chamber base piece and the chip where the one or more gaskets comprise at least one opening, such that the one or more gaskets form the walls ofthe one or more lower chambers and seals the lower chamber base piece to the chip. The gasket or gaskets align with the lower surface ofthe chip such that a lower chamber formed by a gasket comprises a lower surface having the openings of two conduits, and an upper surface comprising a portion of a chip having a single ion transport measuring hole.

In prefeπed aspects ofthe present invention, a lower chamber base piece is designed to fit a base plate that is adapted to fit the stage of a microscope, such as an inverted light microscope. The dimensions can be altered to fit a microscope of choice, such as, for example, an inverted light microscope sold by Leica, Nikon, Olympus, Zeiss, or other companies. Figure 3 A provides a photograph of a prefened design of a lower chamber base piece having flow-through chambers for use in a sixteen unit device, hi Figure 3(A), connectors (302) for inflow conduits can be seen leading out from one side of the lower chamber base piece (301) and connectors (302) for outflow conduits can be seen leading out ofthe opposite side ofthe lower chamber base piece. Figure 3(B) is a close-up photograph ofthe lower chamber piece showing the areas that conespond to what will be the transparent bottom surfaces (303) ofthe lower chambers when the device is fully assembled (black areas) with the conduit openings (304) visible as lighter areas within the black areas. A transparent gasket (305) lies over the top ofthe central portion of the lower chamber piece covering the areas that will be the bottom surfaces ofthe lower chambers (303). In this design, the gasket can be aligned over the lower chamber base piece by fitting a ridge that runs lengthwise down the underside ofthe gasket into a groove the runs lengthwise down the length ofthe upper surface lower chamber base piece. The gasket depicted has two ridges running along either side ofthe gasket (on either side ofthe row of openings) and the lower chamber base piece has two coπesponding grooves on either side ofthe surface having conduit openings (not visible in the photographs). When the gasket is placed on the lower chamber base piece such that the ridges ofthe gasket fit the grooves ofthe lower chamber base piece, the openings ofthe gasket align over the areas ofthe surface of the lower chamber base piece that have conduit openings and will be the bottom surfaces ofthe lower chambers.

The lower chamber base piece can also have "cuts" between the areas that will conespond to the bottom surface of lower chambers (the cuts are peφendicular to the alignment grooves, not visible in the photographs). When the gasket is placed on top ofthe lower chamber base piece, the cuts in the lower chamber base piece are between lower chamber areas defined by the openings in the gasket. These cuts can reduce the possibility of solution seepage between lower chambers.

The three alignment dowels (306) seen in the foreground of Figure 3B at lower left are used to align an upper chamber piece or cartridge over the lower chamber base piece, such that the ends ofthe lower chamber base piece fit between and abut the three pins. The two shorter pogo pins (307) are used to prevent a clamp placed on an assembly that includes a cartridge (comprising an upper chamber piece and attached chip) a gasket, and a lower chamber base piece from pressing down on the assembly prior to fastening ofthe clamp. Holding the clamp in standoff position by these pogo pins (307) prior to fastening prevents misaligned contact ofthe cartridge with the gasket.

Also seen in Figure 3B, are inflow tubes (309) and outflow tubes (308) attached to the connectors in this view. Female pin sockets (310) that connect to the lower chamber recording electrodes can also be seen. Electrical connectors that are attached to a signal amplifier can be inserted into these socket pins.

In Figure 3C, the lower chamber base piece is seated in a base plate (312) adapted to a microscope stage. To the right ofthe base plate is a plexiglass piece (313) comprising ports (314) for the addition of lower chamber solutions and screw- down pinch valves (315) for the inflow tubing.

A baseplate can be made of any suitable material, such as glass, plastics, polymers, ceramics, or metals. Metals, such as but not limited to stainless steel, are prefened, because metal materials have high mechanical strength needed during pressure sealing ofthe lower chamber. A metal base plate can also, together with a grounded microscope stage, form an electrical noise shield around a lower chamber piece fitted to the base plate.

The base plate can be carved on the top side to catch any fluids that may leak or spill and prevent the contamination ofthe microscope with the fluids. Preferably, the base plate is sealed around the lower chamber base piece, for example, with silicone glue, silicone grease, Vaseline, etc.

The base plate is preferably drilled and tapped so as to provide a mounting point for the lower chamber base piece and for a clamp that can hold additional components ofthe ion transport measuring device together (for example, gasket, chip, upper chamber piece) to form the upper and lower chambers of ion transport measuring units. The base plate is designed to hold an ion transport measuring device within a few millimeters ofthe level ofthe top ofthe microscope stage so as to ensure that the chip function may be monitored within the focal range ofthe microscope. Figure 4 illustrates the design of a base plate as adapted for a Nikon Microscope.

Flow-through lower chamber designs described herein can be used in ion transport measurement devices ofthe present invention. In prefeπed embodiments, such devices comprise upper chamber pieces having multiple wells and chip comprising multiple ion transport measuring holes. Upper chambers of such devices can comprise one or more electrodes. Such electrodes can be fabricated, positioned, or attached on a surface of an upper chamber, such as those described in a later section of this application on two-piece molding of upper chambers, can be inserted into the upper chambers ofthe assembled device from above (for example, wire electrodes inserted into the wells), or can be provided as within a tube or part of a tube that can be placed inside the upper chamber (such as a tube that delivers solutions or cell suspensions). Preferably, electrodes of upper chambers are connected as a common reference electrode, but this is not a requirement ofthe present invention. It is also possible for each upper well to have an individual (recording) electrode, and to have the electrodes ofthe lower chambers connected as a common reference electrode. In some prefeπed embodiments, the upper piece of a device ofthe present invention comprises a common reference electrode that contacts all ofthe wells. In other prefeπed embodiments, an electrode is not within or attached to the upper piece, but during assembly ofthe device is inserted into an upper well through upper opening ofthe well. In other prefeπed embodiments, an electrode can be brought into electrical contact with an upper chamber by way of a conduit that comprises an electrode or can provide an electrolyte solution bridge to an electrode. Electrodes that are connected through electrolyte bridges can be recording electrodes, but in most prefened embodiments are reference electrodes.

Figure 5 depicts the design of a device ofthe present invention having an upper chamber piece (51) and attached chip (not visible beneath the upper chamber piece) fixed on top of a gasket (not visible beneath the upper chamber piece) and lower chamber base piece (not visible beneath the upper chamber piece) by means of a clamp (53). The clamp (53) also fixes the device to a baseplate (54) adapted to a microscope. The plexiglass piece (52) holds female pin sockets (56) that connect to electrodes inserted into lower chamber piece conduits. The clamp has a wire electrode (55) that extends into upper chamber wells.

Figure 6 shows a gasket that can fit on top of a lower chamber base piece and form the walls of lower chambers such that the openings (601) in the gasket become the lower chamber spaces.

Figure 7 provides three views of one design of a clamp that can be used in the assembly of a device ofthe present invention. In Figure 7A, the clamp (71) is shown upside down to illustrate the cutout (72) that fits a cartridge. Thumb screws (73) used to attach the clamp to the base piece are alongside the clamp (71). In Figure 7B, the top view ofthe clamp on the cartridge (74) reveals the presence of an array of top chamber electrodes (75) that reach into the cartridge wells. Figure 8 provides photographs showing the parts of an ion transport measuring device ofthe present invention including a baseplate (812), a cartridge (804) comprising an upper chamber piece with a chip attached at the bottom, lower chamber base piece (801), and clamp. In Figure 8A, the black upper chamber piece ofthe cartridge (804), transparent lower chamber base piece (801), inflow tubing (809) and outflow tubing (808) leading to the lower chamber base piece (801), and metallic clamp (802) can be seen. The transparent gasket (805) is lying over the lower chamber base piece (801) behind the upper chamber cartridge. In Figure 8B, the device is assembled, with the clamp (802) screwed into a baseplate (812).

The present invention also encompasses compositions and devices that incoφorate novel elements ofthe compositions and devices described herein, including: a transparent platform beneath the lower chambers, a baseplate adapted for microscope stage, one or more flow-through bottom chambers, reference or recording electrodes outside of upper or lower chambers and connected to chamber(s) through electrolyte bridges, and reference or recording electrodes introduced into tubing attached to upper or lower chambers. The present invention also encompasses manufacture procedures and features for enhancing efficiency or accuracy of manufacture of devices and devices disclosed herein and devices made using such methods, including tapering of upper chamber wells, geometry of holes drilled into chips, ion transport measuring holes comprising one or more counterbores in chips, treatment of chips to enhance electrical sealing of particles such as cells, etc.

The present invention also includes methods of using an ion transport measuring device ofthe present invention having one or more flow-through lower chambers to measure one or more ion transport properties or activities of a cell or particle (such as, for example, a membrane vesicle). The methods include using a device that comprises at least upper chamber reversibly or ineversibly attached to a chip that comprises at least one ion transport measuring means in the form of a hole tlirough the biochip, wherein the chip has been treated to have enhanced sealing properties, and at least one flow-through lower chamber. In the assembled devices used in the methods ofthe present invention, the holes ofthe biochip access the at least one flow-through lower chamber. In these methods, an upper chamber piece and chip are reversibly or ineversibly attached to a lower chamber piece that forms all or a portion of a flow-through lower chamber. An upper chamber piece and chip are optionally additionally reversibly attached to a lower chamber base piece that can form at least the lower surface of one or more lower chambers. Preferably, an upper chamber piece and chip are attached to at least one lower chamber piece that forms the walls of one or more lower chambers and at least one lower chamber base piece that forms the lower surfaces of one or more lower chambers and comprises conduits for the inflow and outflow of solutions.

The device is assembled such that the one or more upper chambers are in register with the one or more ion transport measuring holes ofthe chip, and one or more lower chambers access the one or more upper chambers via the one or more holes ofthe chip. In prefened embodiments, each ofthe one or more upper chambers is in register with one ofthe ion transport measuring holes ofthe chip, and each ofthe lower chambers is aligned with one upper chamber that it accesses via an ion transport measuring hole. Each ofthe lower chambers is connected to at least one inflow conduit and at least one outflow conduit.

The method includes: filling at least one flow-through lower chamber ofthe device with a measuring solution; adding at least one cell or at least one particle to one or more ofthe at least one upper chamber ofthe device, wherein the one or more upper chambers is connected to one ofthe at least one lower chambers that comprises measuring solution via a hole in the ion transport measuring chip; applying pressure to at least one flow-through lower chamber, at least one upper chamber, or to an upper chamber and a lower chamber that are connected via an ion transport measuring hole to create a high-resistance electrical seal between at least one cell or particle and at least one hole ofthe biochip; and measuring at least one ion transport property or activity ofthe at least one cell or at least one particle.

Preferably, one or more cells or one or more particles are in a suspension when added to the upper chamber. Various measuring solutions and, optionally, compounds

In some prefened embodiments, the methods measure at least one ion transport activity or property of a cell in the whole cell configuration, but this is not a requirement ofthe present invention, as the devices can be used in a variety of applications on particles such as, for example, vesicles, as well as cells. The application of pressure can be manual or automated. If pressure is applied manually (for example, by means of a syringe), preferably the user can make use of a pressure display system to monitor the applied pressure. Automated application of pressure can be through the use of a software program that is able to receive feedback from the device and direct and control the amount of pressure applied to one or more ion transport measuring units .

Various specific ion transport assay can be used for determining ion transport function or properties. These include methods known in the art such as but not limited to patch clamp recording, whole cell recording, perforated patch whole cell recording, vesicle recording, outside out or inside out recording, single channel recording, artificial membrane channel recording, voltage-gated ion transport recording, ligand-gated ion transport recording, recording of energy requiring ion transports (such as ATP), non energy requiring transporters, toxins such a scoφion toxins, viruses, stretch-gated ion transports, and the like. See, generally Neher and Sakman, Scientific -American 266:44-51 (1992); Sakman and Neher, Ann. Rev. Physiol. 46:455-472 (1984); Cahalan and Neher, Methods in Enzymology 207:3-14 (1992); Levis and Rae, Methods in Enzymology 207:14-66 (1992); -Armstrong and Gilly, Methods in Enzymology 207:100-122 (1992); Heinmann and Conti, Methods in Enzymology 207:131-148 (1992); Bean, Methods in Enzymology 207:181-193 (1992); Leim et al, Neurosurgery 36:382-392 (1995); Lester, Ann. Rev. Physiol 53:477-496 (1991); Hamill and McBride, Ann. Rev. Physiol 59:621-631 (1997); Bustamante and Vananda, Brazilian Journal 31:333-354 (1998); Martinez-Pardon and Ferrus, Cunent Topics in Developmental Biol. 36:303-312 (1998); Hemess, Physiology and Behavior 69:17-27 (2000); U.S. Patent No. 6,117,291; U.S. Patent No. 6,107,066; U.S. Patent No. 5,840,041 and U.S. Patent No. 5,661,035; Boulton et al, Patch-Clamp Applications and Protocols, Neuromethods V. 26 (1995), Humana Press, New Jersey; Ashcroft, Ion Channels and Disease, Cannelopathies, Academic Press, San Diego (2000); Sakman and Neher, Single Channel Recording, second edition, Plenuim Press, New York (1995) and Soria and Cena, Ion Channel Pharmacology, Oxford University Press, New York (1998), each of which is incoφorated by reference herein in their entirety.

During the assay, while the cell or particle maintains a high-resistance seal with the ion transport measuring hole, lower chamber solutions such as intracellular solutions can be exchanged using the inflow and outflow conduits. For example, a given patch-clamped cell can be assayed without drag, after addition of drag, and after washout of drug while maintaining a high-resistance seal. In another example, a cell or particle can be assayed for ion transport activity in the presence and absence of a particular ion by means of exchange ofthe lower chamber solution.

III. METHOD OF MAKING AN UPPER CHAMBER PIECE OF A DEVICE FOR ION TRANSPORT MEASUREMENT

In ion transport measuring devices contemplated by the present invention, an upper chamber is designed to contain the cells or particles on which ion transport measurements are to be performed. In these embodiments, an upper chamber of an ion transport measuring device can comprise or engage at least a portion of an electrode used to monitor ion transport activity. In the alternative, an upper chamber, when filled with an ion transport measuring solution, can be brought into electrical contact with at least a portion of an electrode. For example, an electrode (such as, but not limited to, a metal wire) can be inserted into the well so that electrical current from the electrode would be transmitted through the conductive measuring solution. Alternatively, a tube that comprises a measuring solution (or otherwise conductive solution) that contains or contacts an electrode or a portion thereof can be put in contact with the upper chamber solution. In the latter case, the electrode (or a portion thereof) need not be within the upper chamber at all, as long as it is electrically connected to the inner part ofthe upper chamber conductive solution (electrolyte bridge).

Typically, an upper chamber electrode will be a reference electrode, although this need not be the case. In cases in which upper chamber electrodes are reference electrodes, electrode extensions or electrolyte bridges that contact individual upper chambers can be connected with one another either outside or inside the upper chamber piece.

In many ofthe devices ofthe present invention, an upper chamber piece comprises at least one upper chamber in the form of a well. Preferably, an upper chamber piece comprises multiple upper chambers or wells that allow several ion transport assays to be performed simultaneously. The upper chamber piece can optionally comprise one or more electrodes. The present invention provides methods of making upper chamber pieces that increase the efficiency and reduce the cost of making devices that measure ion transport activity of cells and particles.

Two-Piece Molding followed by Electrode Insertion

In one aspect ofthe present invention, an upper chamber piece that comprises one or more wells is made in two pieces, an upper well portion piece and a well hole portion piece, and the well hole portion piece has a groove into which a wire electrode can fit. An upper well portion piece comprises the upper portion of one or more wells. The upper well portions are open at both ends. The well hole piece comprises one or more well holes that will form the bottom portion ofthe one or more wells. A well hole is, in effect, the lower portion of a well and can have different dimensions (height, diameter, and taper angle) than the upper well portion. The well holes are also open at their upper and lower ends. The well holes have an upper diameter that is equal or smaller than the diameter ofthe lower opening ofthe upper well portion. When the upper well portion piece is attached on top ofthe well hole piece, the upper well portions are aligned over the well holes to form upper chambers (wells) that have well holes at their lower end.

After manufacturing the upper well portion piece and the well hole piece, a wire elecfrode is inserted into the groove ofthe well hole piece, and then the upper well portion piece is attached, via, for example ultrasonic welding, to the well hole piece to form an upper chamber piece comprising one or more wells, each of which is in contact with a portion of a wire electrode.

An example of this manufacture (an upper well piece made by assembling an upper well portion piece having upper portions of wells with an upper well hole piece having well holes) is depicted in Figure 9. In Figure 9A, the upper well portion piece (91) is shown suspended above the well-hole piece (92). The groove (94) into which a wire electrode can fit is seen along the backs ofthe wells (93) in the assembled upper well piece shown in Figure 9B.

The method includes: molding a well hole portion piece of an upper chamber piece of an ion transport measuring device, wherein said well hole portion piece comprises: at least one well hole, and a groove that extends longitudinally from one end ofthe well hole portion piece toward the opposite end ofthe well hole portion piece, such that the groove contacts the one or more well holes; molding an upper well portion piece of an upper chamber piece that comprises at least one upper well; inserting a wire electrode into the groove ofthe well hole portion piece; and attaching the upper well portion piece to the well hole portion piece to form an upper chamber piece that comprises one or more wells, such that the wire electrode is exposed to the interior of said one or more wells.

In this embodiment, the upper piece is made from one or more plastics and comprises wells that are open at their upper and lower ends, and each well contacts or contains a portion of a common electrode that can be used as a reference electrode in ion transport measuring assays. This method of manufacture is particularly suited to embodiments where the upper piece comprises multiple wells (at least two) that will contact a common electrode, and wells are aπanged linearly in a row. However, this is not a requirement ofthe present invention, and the principle of two-piece molding and wire insertion can be adapted to the manufacture of device components in which multiple wells or chambers that will share a common electrode are aπanged in different geometries. In such embodiments, the path ofthe groove can be designed such that it contacts all ofthe wells or chambers that are intended to be in contact with the elecfrode. This includes embodiments where there are multiple rows of wells or chambers, aπangement of wells or chambers in concentric circles or spirals as well as other aπangements of wells or chambers.

It is also possible to adapt the methods ofthe present invention to designs in which one or more wells are to be contacted by one electrode and one or more other wells are to be contacted by a different electrode. It is also possible that one well be contacted with more that one electrode. In such cases, the well hole portion piece will comprise more than one continuous groove such that more than one wire electrode can be inserted into the lower well portion piece.

Injection molding or compression molding techniques as they are known in the art can be used to make the well hole portion piece and the upper well portion piece. In the methods ofthe present invention, the upper well portion piece comprises an upper portion of at least one well or chamber and the well hole portion piece comprises a lower portion of at least one well or chamber, such that when the upper well portion piece is attached to the well hole portion piece, the two pieces together form at least one upper well or upper chamber. The well hole portion piece comprises at least one groove whose diameter coπesponds to that of a wire electrode, and the groove contacts the well holes. Preferably, the well hole portion piece comprises a well hole whose upper diameter is equal to or smaller than the lower diameter ofthe upper portion ofthe well that is part ofthe upper well portion piece. Thus, in prefened embodiments, the well hole portion piece will have a top surface around the upper diameter ofthe well hole (see Figure 9), that, when looking down into a well after the entire top chamber piece is assembled, appears as a ledge around the top of the well hole. The groove can be in this top surface or ledge. In this way the wire elecfrode can be easily inserted into the groove, and its placement on this "ledge" ensures that it will be exposed to the interior ofthe well after attachment ofthe upper well portion piece.

The wire is easily inserted into the groove ofthe lower well portion piece, as the groove is totally accessible prior to attachment the upper and lower portion pieces. After insertion ofthe wire electrode, the upper well portion piece and well hole portion piece are fused together to form a complete upper chamber piece. Any glues appropriate to the materials and applications ofthe devices can be used for this piupose. UV glues and other fast-curing glues are prefeπed for mass production ofthe upper chamber pieces, although slow-cure glues can also be used for mass production if a high capacity production process is used. Ultrasonic welding, pressuring, heating, or other bonding methods can also be used.

Upper Chamber Pieces and Devices

The present invention includes upper chamber pieces that are made using the methods ofthe present invention, and devices that comprise such pieces. Such pieces and devices can comprise wells or chambers that are open or closed at one or both ends, can comprise other components, such as, but not limited to, membranes, microstructures, ports (optionally with attached conduits), fluidic channels, particles positioning means, specific binding members, polymers, etc., and are not limited to use as ion transport measuring devices. In fact, the same design and manufacturing principles can be used to fabricate pieces that comprise wells or chambers that need not function as "upper" pieces of devices or apparatuses. Two-piece molding, wire insertion, and attachment of two pieces can be used to make devices or components of devices that comprise wells or chambers regardless of whether the components, chambers, or wells, can be considered "upper".

Plastics that can be used in the manufacture of upper and lower pieces include, but are not limited to polyallomer, polypropylene, polystyrene, polycarbonate, cyclo olefin polymers (e.g., Zeonor®), polyimide, paralene, PDMS, polyphenylene ether/PPO or modified polyphenylene oxide (e.g., Noryl®), etc. A very large number and variety of moldable plastics and their properties are known.

Electrodes can comprise conductive materials such as metals that can be shaped into wires. Various metals, including aluminum, chromium, copper, gold, nickel, palladium, platinum, silver, steel, and tin can be used as electrode materials. For electrodes used in ion channel measurement, wires made of silver or other metal halides are prefeπed, such as Ag/AgCl wires.

The design and dimensions ofthe upper and lower well pieces, as well as the dimension ofthe upper wells and lower wells, can vary according to the preferences ofthe user and are not limiting to the present invention.

Preferred Embodiments: Upper Chamber Pieces and Devices

In prefeπed embodiments ofthe present invention, the upper chamber piece comprises one or more upper wells that can function as the upper chambers of ion transport measuring units of ion transport measuring devices. Preferably, an upper chamber piece ofthe present invention comprises more than one upper well, and more preferably more than two upper wells. Even more preferably, an upper chamber piece comprises six or more upper wells, each of which can be a part of an ion transport measuring unit of an ion transport measuring device, where all ofthe six or more upper wells ofthe manufactured upper chamber piece contact a portion of a common wire electrode that extends along the upper chamber piece. The wells of an upper chamber piece that can be part of an ion transport measuring device preferably can hold a volume of between about 5 microliters and about 5 milhliters, more preferably between about 10 microliters and about 2 milhliters, and more preferably yet between about 25 microliters and about 1 milliliter. The depth, or height of a well can vary from about 0.01 to about 25 millimeters or more, and more preferably will be from about 2 milhliters to about 10 milhliters or more in depth, hi prefeπed embodiments ofthe present invention in which an upper well portion and a lower well portion together make up the well, the upper well portion is preferably from about 1 to about 25 milhliters in depth, and the lower well is preferably from about 100 microns to about 10 milhliters in depth. A low cell or particle density is often prefened for attaining a high success rate when using the ion channel measuring device described herein, hi order to reduce the cell or particle density required for optimal cell or particle landing to the recording apertures, it is desirable to have an accurate means for delivering the cells or particles to the recording aperture. For a more accurate delivery of cells or particles to the recording aperture, the upper chamber well can have one or more tapered walls, The walls can be contoured such that the cells or particles, when delivered to the upper chamber well wall (such as by robotic dispenser), are directed to the recording aperture. In these prefeπed embodiments, the shape ofthe well can vary, and can be inegular or regular, and in many cases will be generally circular or oval at its circumference. In prefeπed embodiments, the diameter of a well at its upper end will generally be from about 2 millimeter to about 10 millimeters. In some prefeπed embodiments ofthe present invention such as those depicted in Figure 1 and Figure 9, the upper circumferences ofthe wells ofthe upper chamber piece are horseshoe- shaped, and at least a portion ofthe sides ofthe wells are tapered. Figure ID, for example, shows that the wall ofthe well (1) coπesponding to the rounded end ofthe horseshoe shape tapers toward the bottom ofthe well. In other prefeπed embodiments, the walls along entire well can taper toward the bottom ofthe upper portion ofthe well, hi some prefeπed embodiments ofthe present invention the angle ofthe taper of a portion ofthe walls ofthe well or the entire well walls (the difference from vertical) is from about one degree to about 80 degrees. More preferably, the angle ofthe taper ofthe well walls is between about 5 degrees and 60 degrees from vertical. The taper can extend down the full height ofthe well, or the well can be tapered for only a portion of its height. The upper well portion can optionally be tapered, or the well hole can optionally be tapered, or both the upper well portion and the lower well portion can be tapered. Where both are tapered, the tapering need not be to the same degree or extend around the well to the same extent. Molding of Single Upper Chamber Piece around Electrode

In another aspect ofthe present invention, an upper chamber piece with at least one wire electrode can be manufactured as a single piece by molding an upper piece around a wire electrode. In this case, the mold has a means for positioning the wire electrode such that the upper chamber piece that includes the wells can be molded around it. The method includes: positioning an electrode in a mold; and injection molding an upper chamber piece using the mold such that the electrode contacts one or more wells ofthe upper chamber piece. The electrode can be positioned in any of a number of ways, for example it can extend through the mold and be held by apertures that it is threaded through on either end ofthe mold.

The injection molded upper chamber piece can comprise one or more wells or upper chambers, preferably two or more, more preferably six or more wells. The wells can be of any dimension of size, and can comprise a well hole within the well as described in the previous section.

Molding of Single Upper Piece without Electrode

In yet another aspect ofthe present invention, an upper chamber piece can be manufactured without an electrode. In this case, an upper chamber piece with a desirable number of wells is injection molded using a suitable plastic, such as, but not limited to, polyallomer, polypropylene, polystyrene, polycarbonate, polyimide, paralene, PDMS, cyclo olefin polymers (for example, Zeonor®), or polyphenylene ether/PPO or modified polyphenylene oxides (for example, Noryl®).

When the upper chamber piece is integrated into a device for ion fransport measurement, electrodes (for example, metal wires) can be inserted into the wells. Such electrodes are preferably reference electrodes and are preferably connected outside the chambers, but inserted electrodes can also be recording electrodes connected separately to a power source/signal amplifier.

In a prefeπed embodiment ofthe present invention, an electrode connection can be provided by a conduit that can be introduced into the upper chambers during use ofthe device. The conduit can comprise an electrode, or, when the conduit is filled with a conductive solution, can be in electrical contact with an electrode. When both the upper chamber and the conduit contain a conductive solution (such as a measuring solution), the upper chamber is in electrical contact with the electrode through the "electrolyte bridge" of solution provided by the conduit. Insert Molding of Glass Chip

In yet another embodiment, a pre-diced glass chip is insert-molded together with an upper chamber piece to make a one-piece cartridge. In this process, a glass chip is inserted into a mold, and the upper chamber piece is molded around the glass chip such that it forms the bottoms of upper chambers ofthe upper chamber piece. Laser drilling ofthe recording apertures is done after the molding process, and then the cartridge is chemically treated to enhance its electrical sealing properties, hi this embodiment, materials that can be treated with acid and base (such as, for example, polyphenylene ether/PPO or modified polyphenylene oxide (e.g., NORYL®) and cylco olefin polymers (e.g., ZEONOR®) are used for the construction ofthe cartridge other than the biochip.

Additional Features In some prefened embodiments ofthe present invention, the upper chamber pieces ofthe present invention or components ofthe upper chamber pieces ofthe present invention can have additional features that can aid in the manufacture of upper chamber pieces or of ion transport measuring devices. One such feature is an alignment bump (also called a registration edge) (2) as seen on the chamber piece depicted in Figure IB. One or more alignment bumps on the lower surface of an upper chamber piece can be used during attachment of a chip that comprises ion transport measuring means to the upper chamber piece. Attachment ofthe chip and the upper chamber piece must occur such that every ion transport measuring hole in the chip is aligned with a well hole. The alignment bump or registration edge allows a person or machine assembling the device to detect the location where the edge ofthe chip must be positioned.

Another useful feature for the manufacture of ion transport measuring devices that can occur on upper chamber piece ofthe present invention is a glue spillage groove. This allows for overflow of glue that is used for the attachment of a chip, such as a chip that comprises ion transport measuring means. The glue spillage groove (4) is also shown as a notch in the bottom surface ofthe part shown in Figure ID. Yet another optional feature useful in the manufacturing process of an upper chamber piece is the presence of sinkholes. Depicted in Figure IC, these sinkholes (3) allow for appropriate expansion and contraction ofthe piece during molding.

IV. METHODS OF MAKING A CHIP COMPRISING HOLES FOR ION TRANSPORT MEASUREMENT

Fabrication of Ion Transport Measuring Holes hi a Chip For optimal quality ion transport recording, ion fransport measurement chips comprising holes for ion transport measurement ideally should have a low hole resistance (Re) across the chip. For chips having multiple holes, it is also desirable to have a high degree of uniformity of Re from recording site to recording site. It is also desirable to have ion transport measuring chips that can form seals ofthe ion transport measuring holes ofthe chip with a cell membrane such that the seal resistance (R) is high and the access resistance (Ra) is low.

Chip geometry determines hole resistance (Re) which in turn determines the lowest achievable Ra. Figure 10 shows that chips ofthe present invention having shallower holes and reduced entrance hole diameters (known as "K configuration chips" or "K chips"), have reduced Re relative to standard chips ("S configuration chips" or "S chips"). Figure 10 demonstrates that for S chips, the Re of seals (y-axis) decreases with increasing width ofthe exit hole (opening at the lower side ofthe chip), and increases with increasing hole depth (x-axis). For K chips, the same relationship holds, however the Re of seals of K chips is lower than those of comparable S chips having holes with the same exit hole diameters (comparing the K configuration chips on the left side ofthe graph with the S configuration chips on the right side ofthe graph.) A wider tapering (greater angle from vertical) ofthe hole also decreases Re.

Figure 11 also shows that the Ra of a seal on a chip decreases with decreasing depth ofthe hole in the chip and widening ofthe exit hole. Improved Ra, however, comes at the expense of reduced seal resistance (here, Rm).

The present invention includes methods of making chips that can form seals with cells and cell membranes such that the seals have low access resistance and high seal resistance. The methods ofthe present invention seek to reduce hole resistance (Re) of ion transport measuring holes of chips by reducing hole depth. This is achieved by laser drilling holes in thin substrates, such as glass, quartz, silicon, silicon dioxide, or polymer substrates.

A chip with shortened holes for ion transport measurement can be made by laser drilling one or more counterbores into a glass chip, and then laser drilling a through-hole through the one or more counterbores. While a wide counterbore is prefeπed for lower Re, increased width ofthe counterbore can weaken the chip. It is also difficult to control the drilling ofthe counterbore as the bottom ofthe counterbore gets thinner and thinner, hi addition, with increased (deeper) drilling, the peripheral areas ofthe counterbores tend to be deeper than the more central portions ofthe counterbore due to optical effects (this is sometimes called the wave guide effect). To avoid these problems, a second counterbore is laser drilled into the bottom of a first counterbore. This makes drilling to a greater depth easier control, and has the effect of reducing the thickness ofthe chip in the vicinity ofthe through-hole. Thus, prefeπed methods for synthesis of biochips for ion transport measurement include laser drilling at least one counterbore through a substrate, and then drilling a through- hole through the one or more counterbores. Preferably two counterbores are laser drilled into a substrate, such that a second counterbore is drilled through a first counterbore, that is, the counterbores are nested to form (along with a through-hole) a single hole stracture. In some embodiments ofthe present invention, three, four, or more nested counterbores can be drilled into a substrate prior to drilling a through- hole through the counterbores.

Control ofthe depth of laser drilling can be done by using a separate laser device that can measure the thickness ofthe glass. In prefened aspects of this embodiment ofthe present invention, a measuring laser is used to measure the thickness ofthe substrate before or as it is being drilled, and the laser used for drilling can be regulated by the thickness ofthe remaining substrate at the bottom surface of the counterbore. Laser-based measuring devices have been used for the determination of glass thickness to an accuracy of 0.1 micron. Such a laser measurement device is available from the Keyence Company. A laser based measurement is made to determine the exact thickness ofthe substrate. This measurement determines the number of pulses to be used by the drilling laser to drill a counterbore and thereby achieve uniformity of hole depth. To improve the consistency of through-hole depth and hole resistance, the invention contemplates the integration of a laser unit with an excimer laser drilling device, together with automated control software.

Thus, the present invention comprises methods of making chips comprising holes for ion transport measurement that can form seals having a high seal resistance and low access resistance with cells and particles. The method includes: providing a substrate; laser drilling at least one counterbore in the substrate, and laser drilling at least one hole through the counterbore in the substrate. Preferably, laser drilling is done with sequential or simultaneous measurement ofthe glass thickness at the site of the pore. In practice, a substrate made of glass, quartz, silicon, silicon dioxide, polymers, or other substrates that preferably ranges in thickness from 5 to 1000 microns, and more preferably from 10 to 200 microns, is provided. A first counterbore is laser drilled, where the entrance ofthe counterbore has a diameter from about 20 to about 200 microns, preferably from about 40 to about 120 microns. The first counterbore can be drilled to a depth ofthe thickness ofthe substrate minus the through-hole depth, with the depth depending on the thickness ofthe substrate and the number of counterbores that each ion transport measuring hole will have. Subsequent counterbores will have a smaller diameter than the first counterbore, and can be of lesser depth than the first counterbore. In general, after drilling of all ofthe counterbores that will be part of an ion transport measuring hole, the remaining thickness ofthe substrate that is to be drilled out to form the through-hole (that is, the depth ofthe through-hole) will range from about 0.5 to about 200 microns, and preferably will range from about 2 to about 50 microns, more preferably from about 5 to about 30 microns. The diameter ofthe through-hole can be from about 0.2 to about 8 microns, and preferably will be from about 0.5 to about 5 microns, and even more preferably, from about 0.5 to about 3 microns.

Counterbores can be tapered. Preferably, a counterbore is tapered at an angle ranging from about 1 degree to about 80 degrees from vertical, and more preferably from about 3 degrees to about 45 degrees from vertical. Ion transport measuring holes comprising multiple counterbores can have different taper angles for different counterbores.

Through-holes can also be tapered. The angle of taper for a through-hole can range from about 0 degree to about 75 degrees from vertical, and more preferably, where a through-hole is tapered, is from about 0 degree to about 45 degrees from vertical, hi general an exit hole of a through-hole will have a nanower diameter than an entrance hole, although this is not a requirement ofthe present invention.

The present invention includes chips made using the methods ofthe present invention having at least one counterbore and at least one through-hole drilled through the counterbore. Figure 12A depicts a chip ofthe present invention (123) having a laser drilled ion transport measuring means that comprises a first counterbore (126), a second counterbore (127), and a through-hole (128).

Preferably, the chips ofthe present invention that comprise through holes laser drilled through counterbores have electrical sealing properties such that when appropriate pressure is applied to achieve a seal, a seal between the chip and a cell or particle has a seal resistance (R) that is greater than the resistance across the hole (Re). Preferably, the chips produced by the methods ofthe present invention have ion transport measuring holes that are able to seal to cells or cell membranes such that electrical access between said chip #n the inside of said cell or particle, or between said chip and the outside of said cell or particle in the region of said hole has an access resistance (Ra) that is less than the seal resistance (R). Preferably, the seal between the ion transport measuring hole of a chip made by the methods ofthe present invention and a cell or cell membrane has a seal resistance that is at least 200 MOhm, more preferably at least 500 MOhm, and more preferably yet one gigaOhm or greater.

In prefeπed embodiments of chips ofthe present invention having at least one ion transport measuring means comprising at least one laser drilled counterbore and a through-hole laser drilled through the one or more counterbores, the chip has been treated to enhance the electrical sealing properties ofthe chip. Preferably, the chip has been treated to make the surface ofthe chip at or near the ion transport measuring hole or holes more electronegative. For example, chips ofthe present invention can be chemically treated, such as by methods described herein, to become more electronegative. Preferably, a chip made by the methods ofthe present invention can produce a seal with a cell or particle that has an access resistance that is less than 80 MOhm, more preferably less than about 30 MOhm, and more preferably yet, less than about 10 MOhm. Preferably, a chip ofthe present invention comprising at least one ion transport measuring means in the form of a through-hole that has been laser-drilled through at least one counterbore can form a seal with a cell such that the resistance of the seal is at least ten times the access resistance. More preferably, a chip ofthe present invention can form a seal with a cell such that the seal resistance is at least twenty times the access resistance. A chip produced by methods ofthe present invention can be used in any ion transport measuring device, including but not limited to those described herein.

Inverted Chip

The present invention also includes methods of using chips comprising ion transport measuring holes that are in inverted orientation for ion transport measurement, that is, using chips in which the holes (or at least a portion ofthe holes, such as a portion ofthe holes made by at least one counterbore) have a negative taper.

The method comprises: assembling a device for ion transport measurement that comprises: at least one upper chamber, wherein the one or more upper chambers comprise or are in electrical contact with at least one electrode; at least one chip that comprises an ion transport measuring hole, wherein the one or more chips are assembled in the device in inverted orientation; and at least one lower chamber, wherein the one or more lower chambers comprise or are in electrical contact with at least one electrode; connecting the electrodes with a power supply/signal amplifier; introducing at least one particle or at least one cell into at least one upper chamber, and measuring ion transport activity of at least one cell or at least one particle.

By "inverted orientation" is meant that, for a chip in which ion transport measuring holes are made by drilling, the chip is positioned such that the side ofthe chip having the laser entrance hole opening is exposed to a chamber that will contain cells or particles, instead ofthe side having the laser exit hole. This is contrary to what has previously been done in the art - the "upside- up" orientation in which the cells or particles seal against the side ofthe chip that has the laser exit hole. Thus, sealing of a cell or particles against the ion transport measuring hole occurs on the side ofthe chip opposite to the side that has smaller hole size (the "back side" ofthe chip). The inverted chip orientation has several advantages. One advantage is that the chip does not require a laser polishing step, since the laser drilling performs this function as a "side-effect". A second advantage is that sealing occurs with high efficiency due to the geometry ofthe particle-chip interaction. Yet another advantage is that a stable low Ra can be produced using larger holes (for example, from about 2 to about 5 microns in diameter), due to the position at which break-in occurs during whole cell recording.

When one or counterbores are used to reduced the through-hole depth, the through-hole can be drilled from either the same direction as the counterbores, or from the opposite direction to the counterbores. In the former case, the chips is produced just like the "normal" chips are produced, they are simply assembled up side down. Figure 12B illustrates the use of a chip with laser drilled counterbores (126, 127) and through-hole (128) used in inverted orientation. The single unit ofthe ion transport measuring device shown has an upper well (121) attached to a chip (123) comprising an ion transport measuring means in the form of a hole (122) that connects the upper chamber (121) with a lower chamber (125). hi this case, a gasket (124) forms the walls ofthe lower chamber. A cell (129) is shown sealed to the through- hole (128) ofthe chip which is being used in inverted orientation.

The present invention includes devices and apparatuses having chips comprising ion transport measuring holes that are in inverted orientation, as well as methods of using chips comprising ion transport measuring holes that are in inverted orientation for ion transport measurement.

Methods of Treating Chips Comprising Ion Transport Measuring Means to Enhance the Electrical Seal of a Particle

The present invention also includes methods of modifying an ion transport measuring means to enhance the electrical seal of a particle or membrane with the ion transport measuring means. Ion transport measuring means includes, as non-limiting examples, holes, apertures, capillaries, and needles. "Modifying an ion transport measuring means" means modifying at least a portion ofthe surface of a chip, substrate, coating, channel, or other stracture that comprises or suπounds the ion transport measuring means. The modification may refer to the surface suπounding all or a portion ofthe ion transport measuring means. For example, a biochip ofthe present invention that comprises an ion transport measuring means can be modified on one or both surfaces (e.g. upper and lower surfaces) that suπound an ion transport measuring hole, and the modification may or may not extend through all or a part of the surface suπounding the portion ofthe hole that extends through the chip. Similarly, for capillaries, pipettes, or for channels or tube stractures that comprises ion transport measuring means (such as apertures), the inner surface, outer surface, or both, ofthe channel, tube, capillary, or pipette can be modified, and all or a portion of the surface that suπounds the inner aperture and extends through the substrate (and optionally, coating) material can also be modified. Methods of modifying an ion transport measuring means to enhance the electrical seal of a particle or membrane with the ion transport measuring means are also disclosed in United States patent application number 10/760,866 filed January 20, 2004, and United States patent application number 10/642,014, filed August 16, 2003, both of which are herein incoφorated by reference in their entireties.

As used herein, "enhance the electrical seal", "enhance the electric seal", "enhance the electric sealing" or "enhance the electrical sealing properties (of a chip or an ion transport measuring means)" means increase the resistance of an electrical seal that can be achieved using an ion transport measuring means, increase the efficiency of obtaining a high resistance electrical seal (for example, reducing the time necessary to obtain one or more high resistance electrical seals), or increasing the probability of obtaining a high resistance electrical seal (for example, the number of high resistance seals obtained within a given time period).

The method comprises: providing an ion transport measuring means and treating the ion transport measuring means to enhance the electrical sealing properties of the ion transport measuring means. Preferably, freating an ion transport measuring means to enhance the electrical sealing properties results in a change in surface properties of the ion transport measuring means. The change in surface properties can be a change in surface texture, a change in surface cleanness, a change in surface composition such as ion composition, a change in surface adhesion properties, or a change in surface electric charge on the surface ofthe ion transport measuring means. In some prefeπed aspects of the present invention, a substrate or stracture that comprises an ion transport measuring means is subjected to chemical freatment (for example, treated in acid, and /or base for specified lengths of time under specified conditions). For example, treatment of a glass chip comprising a hole through the chip as an ion transport measuring means with acid and/or base solutions may result in a cleaner and smoother surface in terms of surface texture for the hole. In addition, treating a surface of a biochip or fluidic channel that comprises an ion transport measuring means (such as a hole or aperture) or treating the surface of a pipette or capillary with acid and/or base may alter the surface composition, and/or modify surface hydrophr-b' _^^and δr change surface charge density and/or surface charge polarity.

Preferably, the altered surface properties improve or facilitate a high resistance electric seal or high resistance electric sealing between the surface- modified ion transport measuring means and a membranes or particle. In prefeπed embodiments ofthe present invention in which the ion fransport measuring means are holes through one or more biochips, one or more biochips having ion transport measuring means with enhanced sealing properties (or, simply, a "biochip having enhanced sealing properties") preferably has a rate of at least 50% high resistance sealing, in which a seal of 1 Giga Ohm or greater occurs at 50% of the ion transport measuring means takes place in under 2 minutes after a cell lands on an ion transport measuring hole, and preferably within 10 seconds, and more preferably, in 2 seconds or less. Preferably, for biochips with enhanced sealing properties, a 1 Giga Ohm resistance seal is maintained for at least 3 seconds. In practice, in prefeπed aspects ofthe present invention the method comprises providing an ion transport measuring means and treating the ion transport measuring means with one or more of the following: heat, a laser, microwave radiation, high energy radiation, salts, reactive compounds, oxidizing agents (for example, peroxide, oxygen plasma), acids, or bases. Preferably, an ion transport measuring means or a structure (as nonlimiting examples, a stracture can be a substrate, chip, tube, or channel, any of which can optionally comprise a coating) that comprises at least one ion transport measuring means is treated with one or more agents to alter the surface properties of the ion transport measuring means to make at least a portion of the surface of the ion transport measuring means smoother, cleaner, or more electronegative.

An ion transport measuring means can be any ion transport measuring means, including a pipette, hole, aperture, or capillary. An aperture can be any aperture, including an aperture in a channel, such as within the diameter of a channel (for example, a narrowing of a channel), in the wall of a channel, or where a channel forms a junction with another channel. (As used herein, "channel" also includes subchannels.) In some prefeπed aspects ofthe present invention, the ion transport measuring means is on a biochip, on a planar stracture, but the ion transport measuring means can also be on a non-planar stracture. The ion transport measuring means or surface suπounding the ion transport measuring means modified to enhance electrical sealing can comprise any suitable material. Prefened materials include silica, glass, quartz, silicon, plastic materials, polydimethylsiloxane (PDMS), or oxygen plasma treated PDMS. In some prefeπed aspects ofthe present invention, the ion transport measuring means comprises SiOM surface groups, where M can be hydrogen or a metal, such as, for example, Na, K, Mg, Ca, etc. In such cases, the surface density of said SiOM surface groups (or oxidized SiOM groups (SiO^")) is preferably more than about 1%, more preferably more than about 10%, and yet more preferably more than about 30%. The SiOM group can be on a surface, for example, that comprises glass, for example quartz glass or borosilicate glass, thermally oxidized SiO₂ on silicon, deposited SiO₂, deposited glass, polydimethylsiloxane (PDMS), or oxygen plasma treated PDMS.

In prefeπed embodiments, the method comprises treating said ion transport measuring means with acid, base, salt solutions, oxygen plasma, or peroxide, by treating with radiation, by heating (for example, baking or fire polishing) by laser polishing said ion transport measuring means, or by performing any combinations thereof.

An acid used for treating an ion transport measuring means can be any acid, as nonlimiting examples, HCl, H₂SO₄, NaHSO₄, HSO₄, HNO₃, HF, H₃PO₄, HBr, HCOOH, or CH₃COOH can be. The acid can be of a concentration about 0.1 M or greater, and preferably is about 0.5 M or higher in concentration, and more preferably greater than about 1 M in concentration. Optimal concentrations for treating an ion fransport measuring means to enhance its electrical sealing properties can be determined empirically. The ion transport measuring means can be placed in a solution of acid for any length of time, preferably for more than one minute, and more preferably for more than about five minutes. Acid treatment can be done under any non-frozen and non-boiling temperature, preferably at greater or equal than room temperature.

An ion transport measuring means can be treated with a base, such as a basic solution, that can comprise, as nonlimiting examples, NaOH, KOH, Ba(OH)₂, LiOH, CsOH,or Ca(OH)₂. The basic solution can be of a concenfration of about 0.01 M or greater, and preferably is greater than about 0.05 M, and more preferably greater than about 0.1 M in concentration. Optimal concentrations for treating an ion transport measuring means to enhance its electrical sealing properties can be determined empirically (see examples). The ion transport measuring means can be placed in a solution of base for any length of time, preferably for more than one minute, and more preferably for more than about five minutes. Base treatment can be done under any non-frozen and non-boiling temperature, preferably at greater or equal than room temperature.

An ion transport measuring means can be treated with a salt, such as a metal salt solution, that can comprise, as nonlimiting examples, NaCl, KC1, BaCl₂, LiCl, CsCl, Na₂SO₄, NaNO₃, or CaCl, etc. The salt solution can be of a concentration of about 0.1 M or greater, and preferably is greater than about 0.5 M, and more preferably greater than about 1 M in concentration. Optimal concentrations for treating an ion transport measuring means to enhance its electrical sealing properties can be determined empirically (see examples). The ion transport measuring means can be placed in a solution of metal salt for any length of time, preferably for more than one minute, and more preferably for more than about five minutes. Salt solution treatment can be done under any non-frozen and non-boiling temperature, preferably at greater or equal than room temperature.

Where treatments such as baking, fire polishing, or laser polishing are employed, they can be used to enhance the smoothness of a glass or silica surface. Where laser polishing of a chip or substrate is used to make the surface suπounding an ion transport measuring means more smooth, it can be performed on the front side ofthe chip, that is, the side ofthe chip or substrate that will be contacted by a sample comprising particles during the use ofthe ion fransport measuring chip or device. Appropriate temperatures and times for baking, and conditions for fire and laser polishing to achieve the desired smoothness for improved sealing properties of ion transport measuring means can be determined empirically.

In some aspects ofthe present invention, it can be prefened to rinse the ion fransport measuring means, such as in water (for example, deionized water) or a buffered solution after acid or base treatment, or treatment with an oxidizing agent, and, preferably but optionally, before using the ion transport measuring means to perform electrophysiological measurements on membranes, cells, or portions of cells. Where more than one type of treatment is performed on an ion transport measuring means, rinses can also be performed between treatments, for example, between treatment with an oxidizing agent and an acid, or between treatment with an acid and a base. An ion transport measuring means can be rinsed in water or an aqueous solution that has a pH of between about 3.5 and about 10.5, and more preferably between about 5 and about 9. Nonlimiting examples of suitable aqueous solutions for rinsing ion transport measuring means can include salt solutions (where salt solutions can range in concentration from the micromolar range to 5M or more), biological buffer solutions, cell media, or dilutions or combinations thereof. Rinsing can be performed for any length of time, for example from minutes to hours.

Some prefened methods of treating an ion transport measuring means to enhance its electrical sealing properties include one or more treatments that make the surface more electronegative, such as treatment with a base, treatment with electron radiation, or treatment with plasma. Not intending to be limiting to any mechanism, base treatment can make a glass surface more electronegative. This phenomenon can be tested by measuring the degree of electro-osmosis of dyes in glass capillaries that have or have not been treated with base, hi such tests, increasing the electronegativity of glass ion transport measuring means coπelates with enhanced electrical sealing by the base-treated ion transport measuring means. Base treatment can optionally be combined with one or more other treatments, such as, for example, treatment with heat (such as by baking or fire polishing) or laser treatment, or treatment with acid, or both. Optionally, one or more rinses in water, a buffer, or a salt solution can be performed before or after any ofthe treatments. For example, after manufacture of a glass chip that comprises one or more holes as ion transport measuring means, the chip can be baked, and subsequently incubated in a base solution and then rinse in water or a dilution of PBS. In another example, after manufacture of a glass chip that comprises one or more holes as ion transport measuring means, the chip can optionally be baked, subsequently incubated in an acid solution, rinsed in water, incubated in a base solution, and finally rinsed in water or a dilution of PBS. In some prefeπed embodiments, the surfaces of a chip sunounding ion transport measuring means can be laser polished prior to freating the chip with acid and base.

To facilitate batch treatment of glass biochips, we have used the treatment fixtures illustrated in Figure 13. Figure 13A shows a single layer treatment fixture that can fit into a glass jar containing acid, base, or other chemical solutions. The rods (131) facilitate handling and stacking ofthe treatment fixtures. Glass pins can fit into the holes (132) and chips can be stacked lengthwise on their edges between the pins. Figure 13B shows the stacked treatment fixture. The fixture is made of acid and base resistant materials such as cyclo olefin polymers (for example, ZEONOR®), polyphenylene ether/PPO or modified polyphenylene oxide (for example, NORYL®), polytetrafluoroethylene, TEFLON™, etc. Multiple layers of these racks can be stacked up to fit into one glass container, as shown in Figure 13B. This design also allows mechanisms of moving fluid to occur such as that brought about by a rotary or reciprocal shaker or a magnetic stir bar.

In an alternative design, chips are positioned flat on a treatment fixture, and are held in a tray by a door that can open and latch closed. This facilitates manipulation ofthe chips, such as by a machine. For example, after treatment ofthe chips, a machine that assembles cartridges can pick up a treated chip from the treatment fixture in order to attach it to a cartridge.

In some aspects ofthe present invention, it can be preferable to store an ion transport measuring means that has been treated to have enhanced sealing capacity in an environment having decreased carbon dioxide relative to the ambient environment. This can preserve the enhanced electrical sealing properties ofthe ion transport measuring means. Such an environment can be, for example, water, a salt solution (including a buffered salt solution), acetone, a vacuum, or in the presence of one or more drying agents or desicants (for example, silica gel, CaCl₂ or NaOH) or under nitrogen or an inert gas. Where an ion transport measuring means or stracture comprising an ion transport measuring means is stored in water or an aqueous solution, preferably the pH ofthe water or solution is greater than 4, more preferably greater than about 6, and more preferably yet greater than about 7. For example, an ion transport measuring means or a stracture comprising an ion transport measuring means can be stored in a solution having a pH of approximately 8.

Glass chips that have been base treated and stored in water with neutral pH levels can maintain their enhanced sealability for as long as 10 months or longer. In addition, patch clamp chips bonded to plastic cartridges via adhesives such as UV- acrylic or UV-epoxy glues can be stored in neutral pH water for months without affecting the sealing properties.

We have also tested patch clamp biochips and cartridges that were stored in a dry environment with dessicant for 30 days. The chips were re-hydrated and tested for sealing. In one experiment, we got 6/7 seals for the dry-stored chips. Similarly, we stored mounted chips in dry environment and were able to obtain seals after a few weeks of storage.

Dehydration can, however, reduce the sealability of chemically treated chips. To improve the seal rate for dry-stored chips, NaOH, NaCl, CaCl₂ and other salt or basic solutions can be used to rejuvenate the chips out of dry storage to restore the sealability.

The present invention also includes methods of shipping or transporting ion transport measuring means modified by the methods ofthe present invention to have enhanced electric sealing properties and stractures comprising ion transport means that have been modified using the methods ofthe present invention to have enhance electric sealing properties. Such ion transport measuring means and stractures comprising ion transport measuring means can be shipped or transported in closed containers that maintain the ion transport measuring means in conditions of low CO or air. For example, the ion transport measuring means can be submerged in water, acetone, alcohol, buffered solutions, salt solutions, or under nitrogen (N₂) or inert gases (e.g., argon). Where the ion transport measuring means or structure comprising an ion transport measuring means is stored in water or an aqueous solution, preferably the pH ofthe water or solution is greater than 4, more preferably greater than about 6, and more preferably yet greater than about 7. For example, an ion transport measuring means or a stracture comprising an ion transport measuring means can be shipped in a solution having a pH of approximately 8.

In one method of shipping a chip that has been treated to have enhanced sealing properties, the ion transport measuring devices comprising base-treated chips are shipped such that the chips are loaded up side down. The package for commercial shipments is designed to hold cartridges up side down, although the up side up configuration can also be used for shipping. To allow easy opening and facilitate automation in sequential loading ofthe devices onto apparatuses for use, a blister pack with film sealing is designed. As illustrated in the Figure 14, a blister pack is provided in the form of a molded plastic frame (141) having (142) for positioning cartridges. One ofthe slots comprises a cartridge (143), viewed from the bottom in Figure 14A and from the top in Figure 14B. The blister pack has an opening on both top and bottom sides for film sealing. The sealing film or "lidstock" is a thin foil with temperature activated adhesive and an inert coating such as EVA (ethyl vinyl acetate) polymer. For wet (water) storage, the blister pack is first sealed from top (the opening side, flipped over, and the cartridges are loaded up side up. A preservative solution such as water is then injected into each well and the rest ofthe open space in each chamber ofthe package. Another lidstock film is then used to seal the blister package from the bottom. The blister package can be optionally sterilized with radiation for long shelf life.

Yet another aspect is related to the shipping of laser processed glass chips as finished goods between to production processes, particularly if the two processes are in different production locations. The cunent invention includes a shipping fixture allowing individual placement and securing of laser-processed glass chips for shipment. The same fixture-chips assembly is then directly used for subsequent chemical processing. To withstand strong acid and base treatment, the shipping fixtures are molded with inert materials such as polyphenylene ether/or modified polyphenylene oxide (e.g., Noryl®), Teflon, and cylco olefin polymers (e.g., Zeonor®). A stack of these fixtures can be secured in one container for chemical treatments, or for shipping in aqueous solutions such as water. The liquid shipping provides buffering for vibrations during transportation, giving maximum protection of glass chips from being damaged.

The present invention also includes ion transport measuring means treated to have enhanced electrical sealing properties, such as by methods disclosed herein. The ion transport measuring means can be any ion transport measuring means, including those disclosed herein. The present invention also includes chips, pipettes, substrates, and cartridges, including those disclosed herein, comprising ion transport measuring means treated using the methods ofthe present invention to have enhanced electrical sealing properties.

The present invention also includes methods of using ion fransport measuring means and stractures comprising ion transport measuring means, such as biochips, to measure ion transport activity or functions of one or more particles, such as cells. The methods include: contacting a sample comprising at least one particle with an ion transport measuring means that has been modified to enhance the electrical seal of a particle or membrane with the ion transport measuring means, engaging at least one particle or at least one membrane on or at the modified ion transport measuring means, and measuring at least one ion transport function or property ofthe particle or membrane. The methods can be practices using the methods and devises disclosed herein. Generally, the methods ofthe present invention provide the following characteristics, but not all such characteristics are required such that some characteristics can be removed and others optionally added: 1) the introduction of particles into a chamber that includes a biochip ofthe present invention, 2) optionally positioning particles at or near an ion transport detection stracture, 3) electronic sealing ofthe particle with the ion transport detection stracture, and 4) performing ion transport recording. Methods known in the art and disclosed herein can be performed to measure ion transport functions and properties using modified ion transport measuring means ofthe present invention, such as surface-modified capillaries, pipette, and holes and apertures on biochips and channel structures.

V. METHODS FORMEASURINGTHE SURFACEENERGY OF THE SURFACE

OF A CHEMICALLY TREATED ION TRANPORT MEASURING BIOCHIP Another aspect ofthe cunent invention originated from the need for an inexpensive, fast, and sensitive method to measure surface energy on solid/liquid surface such as, for example, that of a chemically treated ion transport measurement biochip.

The method includes: dispensing a drop of defined volume of water or an aqueous solution on a surface, measuring the time it takes for the drop to evaporate; and estimating the relative or absolute surface energy ofthe surface based on the evaporation time and the difference in evaporation time with respect to control samples.

The contact angle of a liquid drop on a solid surface is a measure ofthe surface energy, assuming constant liquid/air surface energy. Very low liquid/solid energy results in extremely small contact angles (close to 0 degrees). For that reason, contact angle measurements might not be a very sensitive method for low surface energy systems.

When a liquid drop with fixed volume is in contact with a solid surface, the air/liquid surface ofthe drop will be inversely proportional to the liquid/solid surface energy. Lower liquid/solid surface energy will result in bigger spreading ofthe drop. The evaporation ofthe drop will be proportional to the air/liquid surface area at any given moment. Thus the evaporation time will be proportional to the liquid/solid surface energy.

The method can be used to determine the hydrophilicity of any type of surface. For example, the method can be used to determine the hydrophilicity of at least a portion ofthe surface of an ion transport measuring chip. In this case, a drop of water or aqueous solution is dispensed on the surface of a biochip comprising at least one ion transport measuring means, preferably a biochip that has been chemically treated to improve its electrical sealing properties. Controls can be performed simultaneously with the hydrophilicity test, or can be performed at another time. Preferably, a range of controls are performed on surfaces of known hydrophilicity to provide a hydrophilicity scale. Evaporation ofthe drop is monitored, and the time elapsed between the time the drop contacts the chip and the time it has totally evaporated is measured. Preferably, the evaporation time ofthe test drop is compared with the evaporation times ofthe one or more controls, which can be expressed as a scale. The elapsed time is used as an index for hydrophilicity. This index can be used to determine whether a chemically treated chip is within the optimal range for achieving high resistance electrical seals.

Evaporation can be monitored by diffraction, reflectance, or interference at the surface where the drop is deposited, or simply by visual observation. Evaporation can also be monitored by measuring the change in intensity or other physical or chemical properties of a dye or tracer agent that has been used to color or label the solution.

The method is not limited to testing of biochips, but can be used to measure the hydrophilicity of a surface used for any puφose. The invention uses the evaporation time of a liquid drop on a solid surface as a measure ofthe solid/liquid surface energy. The method has very low cost (an accurate liquid dispenser is the only equipment needed). It is also very fast and accurate for low surface energy systems.

Using the drop evaporation technique, we have demonstrated that the evaporation time of a 0.25 microliter water drop is 2.5 times shorter for a highly hydrophilic glass surface (treated with base) compared to chemically untreated glass. VI. METHODS OF MANUFACTURING CHIPS FOR ION TRANSPORT MEASUREMENT DEVICES

Yet another aspect ofthe present invention is a method of making a chip for ion transport measurement devices by fabricating a chip that comprises multiple rows of ion transport measuring holes and subsequently breaking the chip into discrete segments that comprise a subset ofthe total number of ion transport measuring holes.

In this method, a glass sheet is pre-processed with a laser to create patch clamp recording apertures, and preferably treated chemically to improve sealability as described in this application. The glass sheet has also been pre-scored with a laser to produce mark lines by which sets of holes can be separated from one another.

Preferably, the mark lines are continuous slashes that go through the glass to a depth of about 30% or more ofthe thickness ofthe sheet.

In some prefened embodiments, an injection molded multi-unit well plate is bonded to the glass with adhesives so that each well ofthe plate is in register with one of the ion transport recording holes. Sections of the multi-unit welled sheet sheet comprising a portion ofthe multi-unit well plate and a portion ofthe glass chip can be separated later by two metal plates closing in from two sides ofthe scored mark lines against the glass sheet, followed by bending ofthe bonded multi-well devices along with the metal plates and pulling ofthe segments away from each other. The severed sections can comprise one or more ion transport measuring units. Figure 15 shows a glass chip (151) having ion transport measuring holes (152) and mark lines (153) created by a laser. The chip is attached to a multiwell plate that to form a multiunit sheet (154). Sections (155) that can comprise one or more ion transport measuring holes (152) can be detached from the sheet (154). This approach allows for low cost, automated assembly of single well or low- density anays, such as 16-well planar patch clamp consumables. This method of manufacture improves automation, and reduces individual unit assembly time.

VII. HIGH DENSITY ION TRANSPORT MEASUREMENT CHIPS

Another aspect ofthe present invention is a high density, high throughput chip for ion transport measurement. A high density chip for ion transport measurement comprises multiple ion transport measuring holes. The invention also encompasses methods of making high-density consumable patch clamp anays for ultra high throughput screening of ion transport function.

A high density chip for ion transport measurement comprises at least 24 ion transport measuring holes, preferably at least 48 ion transport measuring holes, and more preferably, at least 96 ion transport measuring holes. A high density, high throughput chip for ion transport measurement ofthe present invention can comprise at least 384 ion transport measuring holes, or at least 1536 ion transport measuring holes. A high density ion transport measuring chip can be made using a silicon, glass, or silicon-on-insulator (SOI) wafer. The wafer is first wet-etched to create wells on the top surface, and then laser drilling is used to form the through-holes. The dimensions ofthe wafer and the wells can vary, however, in prefeπed embodiments in which a 1536 well anay is fabricated, the thickness ofthe wafer can range from about 0.1 micron to 10 millimeters, preferably from about 0.5 micron to 2 millimeters, depending on the substrate.

For wafers in the range of 1 millimeter thick, the etching tolerance should be within 2% if the through-holes are approximately 30 microns in depth. This applies to silicon wafers etched with alkaline solutions such as KOH or glass wafers etched with buffered HF. With SOI wafers, a defined thickness of SiO covers the Si wafers, and etching ofthe wells through the Si side with KOH will stop at the SiO₂ interface. This way the thickness ofthe remaining material is consistent across the whole wafer, and even consistent among different batches of etched wafers. This permits laser drilling on these etched substrates to be more standardized, and reduces the time needed for laser measurement. In a prefeπed embodiment, the etched Si wells have a volume of approximately 2 microliters, assuming a footprint of approximately 2 millimeters x 2 millimeters for each well that extends as a prism or inverted pyramid shape through the Si substrate during anisotropic etching, leaving a distance of approximately 1 millimeter between adjacent wells. hi one design, the bottom ofthe chip can be sealed against a single common reservoir for measuring solution that is connected to a common reference electrode, while individual recording electrodes can be connected at the upper surface directly or via electrolyte bridges. Alternatively, a structure with 1536 or any prefeπed number of individual isolated chambers can be sealed against the bottom of a 1536-well (or any prefened number of well) plate so that each chamber is in register with a well. In some designs of this embodiment, the top surface ofthe SOI wafer can be a common electrode, with the conductivity of Si material being adequate to provide electrical connection; however, additional metal coating on the top surface (applied before etching as mask layer) can increase conductivity ofthe upper surface. Wet etching that creates the wells removes this metal coating from the wells themselves. Chemical treatment with acid and/or base can optionally be performed on the chip for improved sealing. Another way to make a high density chip is to use very thin wafers made of glass, SiO₂, quartz, Si, PDMS, plastics, polymers, or other materials, or a thin sheet, with thickness between about 1 micron and about 1 millimeter. Laser drilling can be performed on such sheets to create through-holes. A separate, "well plate" with 1536 or any prefeπed number of wells, manufactured by molding, etching, micro- machining or other processes, is then sealed against the holes via gluing or by using other bonding methods.

The laser drilling ofthe holes can be from the front or back side ofthe chip.For high density ion transport measuring chips, either a "standard" or inverted drilling configuration can be used as described herein. Figure 16 shows a high density anay made on a Si, glass, or SOI wafer (161).

It is made with a wet etch process, which creates the wells (162) on the top surface, followed by laser drilling through the remaining ofthe material on the bottom of each ofthe wells. Figure 17 shows the high density anay having upper chambers (171) that can be formed by a well plate (172) attached to the chip (173). Wells (174) in the chip (173) having laser drilled through-holes can be oriented in inverted (top alternative) or standard (bottom alternative) orientation.

VIII. METHODS FOR ASSEMBLING ION TRANSPORT MEASUREMENT CARTRIDGES

Use of Adhesives A prefened embodiment ofthe present invention is an ion transport measurement device cartridge comprising one or more upper chamber pieces bonded via adhesive or other means to one or more ion transport measurement chips that have been treated to have enhanced electrical sealing properties in which the chip or chips contain at least one microfabricated ion transport measurement aperture (hole), optionally but preferably drilled by a laser. The one or more ion transport measurement chips are optionally laser polished on the side ofthe small exit hole, and treated with a combination of acid and base treatment as described herein.

The present invention also includes a method of assembling ion fransport measurement cartridges by bonding the ion transport measurement chip(s) with an upper chamber piece. In one embodiment, an ion transport measurement chip containing one or more ion transport measuring apertures is bonded to an upper chamber piece via a UV-activated adhesive, such that each well ofthe upper chamber piece is in register with a recording aperture on the ion transport measurement chip, and the smaller, exit holes from laser drilling ofthe ion transport measuring holes are exposed to the wells ofthe upper chamber piece.

To facilitate efficient assembly, a registration bump can preferably be molded on the bottom ofthe upper chamber piece so that when the biochip is pressed against the bump and shoulder at the bottom ofthe upper chamber piece, the recording apertures on the ion channel measurement chip are in register with the wells ofthe upper chamber piece. An example of an upper chamber piece having alignment bumps (2) is shown in Figure IB.

Prefeπed UV adhesive include, but are not limited to, UV-epoxy, UV-acrylic, UV-silicone, and UV-PDMS.

The UN dose required to completely cure the UN adhesive can at times inactivate the treated surface ofthe chip. To avoid UN radiation to chip surface areas near the recording apertures where seals are to occur, a mask made of UN-permeate glass on which spots of size between 0.5 to 5 mm are provided by depositing a thin metal layer or paint (preferably a dark or black) layer. Pressure Mounting

As an alternative to glue-based bonding, the upper chamber piece can be designed to allow an O-ring type of gasket made with PDMS to be used as seal cushion between the upper chamber piece and a biochip during a sandwich-type pressure mounting procedure. Figure 18 depicts the general format for pressure bonding, in which a chip (183) is attached to an upper chamber piece (181) using a gasket (184) to form a seal between the upper chamber piece (181) and chip (183) when pressure (anow) is applied. In this highly schematized depiction, a lower chamber piece (185) is also attached to the chip (183) using a second gasket (186) to form a seal between the lower chamber piece (185) and chip (183) when pressure (anow) is applied. Mechanical pressure can be provided by a weight or clamp, or by any other means, including fasteners or holders.

IX. BIOCHIP DEVICE FOR ION TRANSPORT MEASUREMENT COMPRISING FLUIDIC CHANNEL CHAMBERS

A further aspect ofthe present invention is a flow-through fluidic channel ion transport measuring device that can be part of a fully automated ion transport measuring device and apparatus. This device comprises a planar chip that comprises ion transport measuring holes, and upper and lower chambers on either side ofthe chip that are fluidic channels. One or more fluidic channels is positioned above the chip and one or more fluid channels is positioned below the chip. Apertures are positioned in the fluidic channels such that an ion transport measuring hole in the chip has access to an upper fluidic channel (serving as an upper chamber) and a lower fluidic channel (serving as a lower chamber).

A chip of a fluidic channel ion transport measuring device can have multiple ion transport measuring holes, and each ofthe holes can be in fluid communication with an upper fluidic channel and a lower fluidic channel. The upper fluidic channel or channels can be connected with one another, and more than one lower fluidic channel can be independent; or the device can have two or more upper fluidic channels that can be independent while the one or more lower fluidic channels can be connected with one another. In a yet another alternative, upper fluidic channels that service different ion transport measuring holes can be separate from one another and the lower fluidic channels that service different ion transport measuring holes can also be separate from one another.

Figure 19, depicts a schematic view of one possible design of a planar patch clamping chip (193) having an upper fluid channel (191) for extracellular solution (ES) and a lower fluidic channel (195) for intracellular solutions (ISl, IS2). The upper and lower channels are interfaced at a point where the recording aperture (192) ofthe planar electrode resides. Separate fluidic pumps (P) drive the flow of fluids through the two (upper and lower) fluidic channels. Recording (196) and reference electrodes (197) external to the fluidic patch clamp chip are connected via an electrolyte solution bridge to the upper (191) and lower (195) fluidic channels. A pressure source such as a pump with pressure controller that can generate both positive and negative pressures is shown linked to the lower fluidic channels. A multi-way valve (194) can be used to connect the lower fluidic channel (195) to different solution reservoirs (ISl, IS2, etc), and a multi-way valve (198) can be used to connect the upper fluidic channel (191) to cell reservoirs, a compound plate (CP), wash buffers, or other solutions.

In some prefened aspects, the device can have a molded upper piece that comprises one or more upper channels, and a molded lower piece that comprises one or more lower channels. The channels can be drilled through or molded into the pieces, which preferably comprises at least one plastic. A chip comprising one or preferably, multiple ion transport measuring holes can be situated between the upper piece and the lower piece, such that an ion transport measuring hole through the chip connects an upper channel ofthe upper piece with a lower channel ofthe lower piece. In some prefened embodiments of these aspects, an upper conduit connects to a well that is in register with a hole ofthe chip. In addition to being accessed by the conduit, the well can be open at the top, for the addition of, for example, cell suspensions or compounds. Preferably, these prefened embodiments, the chip comprises multiple holes and the upper piece comprises multiple wells in register with the holes ofthe chip. Preferably, each well is accessed by a separated and independent channel. The lower piece can comprise one or more lower channels. Preferably, in these embodiments, the lower piece comprises at least one channel, and each ofthe at least one channel accesses two or more ion transport measuring holes in the biochip. The at least one lower channel can comprise or be in electrical contact with an electrode, such as, for example, a reference electrode. Upper chamber electrodes can be dunked into well from above, inserted into the upper channels, or otherwise brought into electrical contact with the upper wells.

Designs comprising upper chamber fluidic channels, lower chamber fluidic channels, or both upper and lower chamber fluidic channels have several advantages. The external electrodes can be of multiple use, but replaceable. This reduces the cost ofthe biochip. The flow-through fluidics of both the upper and lower chambers minimizes the generation of air bubbles. Importantly, the closed fluidic channels allow for controlled delivery of low volume fluids without evaporation.

X. METHODS OF PREPARING CELLS FOR ION TRANPORT MEASUREMENT

In a further aspect ofthe present invention, methods for isolating attached cells for planar patch clamp electrophysiology are provided. Conventional cell isolation methods by non-enzymatic, trypsin, or reagent-based methods will not produce cells that are in optimal condition for high throughput electrophysiology. Typically cells produced by available protocols are either over-digested and tend to function less than optimally in planar patch clamp studies, or under-digested and resulting in cell clumps with the cell suspension. In addition, the cells isolated by conventional methods tend to have large amoimts of debris which are a major source of contamination at the recording aperture. The cunent protocols are optimized for better cell health, single cell suspension, less debris and good patch clamp performance. The cunent protocols can be used to isolate cells for any puφose, particularly when cells in an optimal state of health and integrity are desirable, including pmposes that are not related to electrophysiology studies.

This invention was developed to produce suspension CHO and HEK cells that give high quality patch clamp recording when used with chips and devices ofthe present invention. Parameters such as cell health, seal rate, Rm (membrane resistance), Ra (access resistance), stable whole cell access, and cunent density, were among the parameters optimized. The method includes: providing a population of attached cells, releasing the attached cells using a divalent cation solution, an enzyme- containing solution, or a combination thereof; washing the cells with a buffered cell- compatible salt solution; and filtering the cells to produce suspension cells that give high quality patch clamp recordings using ion transport measuring chips. Enzyme-free Cell Preparation

Enzyme-free dissociation is desirable when an ion transport expressed on a cell surface can be digested by enzymatic methods, thereby causing a change in ion transport properties. Enzyme-free methods involve a dissociation buffer that is either Ca -chelator-based or non- Ca^-chelator-based. The former is typically a solution of EDTA, while the latter can be calcium-free PBS. In such methods, attached cells grown on plates are first washed with calcium-free PBS, and then incubated with the dissociation buffer. In case ofthe calcium chelator-based dissociation, the dissociated cells must be washed at least once with a chelator-free solution before they can be used for ion transport measurement assays. The suspended cells are then passed through a filter, such as a filter having a pore size of from about 15 to 30 microns (this can vary depending on the type of cells and their average size).

Preparation of Cells using Enzyme

In some methods (see Example 6), trypsin is used to dissociate attached cells. In such methods, the cells are typically rinsed with a solution devoid of divalent cations, and then briefly treated with trypsin. The trypsin digestion is stopped with a quench medium carefully designed to achieve the optimal divalent cation mix and concentration. In the methods provided herein, the suspended cells are then passed through a filter, such as a filter having a pore size of from about 15 to 30 microns (this can vary depending on the type of cells and their average size).

-Another enzyme-based method uses a preparation commercially available from Innovative Cell Technologies (San Diego). Accumax is an enzyme mix containing protease, collagenase, and DNAse. Example 6 provides a protocol for CHO cells using Accumax and filtration.

Some prefeπed methods ofthe present invention use a combination of enzyme-free dissociation buffer, Accumax reagent, and filtration to isolate high quality cells for patch clamping (see Example 6). XL PRESSURE CONTROL PROFILE PROTOCOL FOR ION TRANSPORT MEASUREMENT

The present invention also provides a pressure protocol control program logic that can be used by an apparatus for ion transport measurement to achieve a high- resistance electrical seal between a cell or particle and an ion transport measuring means on a chip ofthe present invention in a fully automated fashion. In this aspect, the program interfaces with a machine that can receive input from an apparatus and direct the apparatus to perform certain functions.

Typically it has required months to years of experience on the part of an experimenter to master the techniques required to achieve and maintain high quality seals during their experiments. It is an object ofthe invention to produce a pressure protocol for achieving and maintaining seal quality parameters for automated patch clamp systems. The present invention provides a logic that can direct mechanical and automated patch clamp sealing of particles and membranes. The program logic includes: a protocol for providing feedback control of pressure applied to an ion transport measuring means of an ion transport measuring apparatus, comprising: steps that direct the production of positive pressure; steps that direct the production of negative pressure; steps that direct the sensing of pressure; and steps that direct the application of negative pressure in response to sensed pressure in the form of multiple multi-layer if-then and loop logic, in which the positive and negative pressure produced is generated through tubing that is in fluid communication with an ion transport measuring means of an apparatus, and in which negative pressure is sensed through tubing that is in fluid communication with an ion transport measuring means of an apparatus. Preferably, these steps are performed in a defined order that depends on the feedback the apparatus receives. Thus, the order of steps ofthe protocol can vary according to a defined script depending on whether a seal between a particle and the ion fransport measuring means is achieved during the operation ofthe program, and the properties ofthe seal achieved.

An apparatus for ion transport measurement that is controlled at least in part by the pressure program preferably comprises: at least one ion transport measurement device comprising two or more ion transport units (each comprising at least a portion of a biochip that has an ion transport measuring means, at least a portion of an upper chamber, and at least a portion of a lower chamber, and is in electrical contact with at least one recording electrode and at least one reference electrode), tubing that connects to the device and is in fluid communication with the two or more ion transport measuring means of an apparatus, and pumps or other means for producing pressure through the tubing. Preferably, the apparatus is fully automated, and comprises means for delivering cells to upper chambers (such means can comprise tubing, syringe-type injection pumps, fluid transfer devices such as one or more automated fluid dispensors) and means for delivering solutions to lower chambers (such means can comprise tubing, syringe-type injection pumps).

Preferably, in addition to promoting and maintaining a high resistance seal, the pressure protocol program can also direct the rupture of a cell or membrane delineated particle that is sealed to an ion transport measuring means. Such rapture can be by the application of pressure after sealing, and can be used to achieve whole cell access.

In operation, the program directs the apparatus to generate a positive pressure in the range of 50 ton to 2000 ton, preferably between 500 and 1000 ton, to purge any blockage ofthe recording holes. Then the program directs the apparatus to generate a positive holding pressure between 0.1 to 50 ton, preferably between 1 to 20 ton to keep the recording aperture of an ion transport measuring chip clear of debris during the addition of cells to the upper chamber. After cell addition, the program directs the release of pressure and holds the pressure at null long enough to allow cells to approximate the aperture. The program then directs a negative pressure to be applied draw a cell onto (and partly into) the ion transport recording aperture for landing and the formation of a gigaohm seal. Additional pressure steps as described Example 7 may be required for achieving gigaohm seals if a seal does not occur upon cell landing.

To achieve whole-cell access, negative pressure is increased in progressive steps until the electrical parameters indicate the achievement of whole-cell access. Alternatively, the program can direct the application of a negative pressure to a "sealed" cell that is insufficient to gain whole-cell access, and then use a electric "zap" method to disrupt the membrane patch within the aperture and thereby achieve whole-cell access. Upon achieving whole-cell access the pressure is either released immediately, or held for a few seconds then released, depending on the cell quality. Finally, during whole-cell access procedures, the seal quality could be improved after access is achieved, then held at optimal parameters by a more complex pressure protocol.

The pressure protocol involves many branchpoints or "decisions" based upon feedback from the seal parameters. It is easiest to describe the protocol as a series of steps in programming logic, or program. A pseudocode example of such logic is provided as Example 7.

The program, also herein refeπed to as program logic, control logic or programming logic, can be illustrated and described in different manners. The procedures and processes described in this program herein are one possible embodiment ofthe program. Decision branches, loops, and other components can be performed in substantially different methods to obtain the same or substantially similar results, such as the use of an "if-then" loop in place of a "while" loop. The exemplary pseudocode and program description contained herein is not intended to be limiting, merely they are examples of one possible embodiment of encoding this program. One skilled in the art will realize that the procedures and processes of this program can be accomplished in a number of programming and encoding methods, on devices such as personal computers, chipsets, mainframe computers, and other electronic devices capable of performing and executing programmed code. Additionally, the steps described herein may be executed and performed in other step- wise processes to achieve the same or substantially similar results.

The procedures and descriptions of this program are described and illustrated across several pages. Some procedures are illustrated across several figures. This is not intended to limit the varied calculations and functions of these procedures to sub- routines separated from the rest ofthe procedure, instead it is a result of space limitations in the drawing ofthe figures. Certain aspects illustrated across several figures are intended to be connected seamlessly, and operate together as one procedure or subroutine. Off-page and on-page connectors are utilized to illustrate this continuity, and are not intended to confine the execution of certain code to specific areas ofthe illustrated figures. These illustrative connectors are additionally not intended to be additional steps in the execution ofthe program disclosed herein.

The program disclosed herein can be run and executed on a variety of systems. The program can be ran on a device such as SealChipTM from Aviva Biosciences Coφoration, the PatchXpressTM from Axon Instruments, or any other electronic patch-clamp system, as described in this present application or known in the art. Additionally, the present invention can be executed in a computer-based manner. The computer-based manner ofthe present invention includes computer hardware and software. The computer-based program can run on a personal computer ofthe traditional type, including a motherboard. The motherboard contains a central processing unit (CPU), a basic input/output system (BIOS), one or more RAM memory devices and ROM memory devices, mass storage interfaces which connect to magnetic or optical storage devices including hard disk storage and one or more floppy drives, and may include serial ports, parallel ports, and USB ports, and expansion slots. The computer is operatively connected by wires to a display monitor, a printer, a keyboard, and a mouse, though a variety of connection means and input and output devices may be substituted without departing from the invention. Additionally, the present invention can be encoded on a chipset, or be encoded on computer-like components included in other devices.

A computer used in connection with the computer program may run an IBM- compatible personal computer, running a variety of operating systems including MS- DOS®, Microsoft® Windows®, or Linux®. Alternatively, the computer program may run on other computer environments, including mainframe systems such as UNIX® and VMS®, or the Apple® personal computer environment, portable computers such as palmtops, programmable controllers, or any other digital signal processors.

All of these elements and the manner in which they are connected are well- known in the art. In addition, one skilled in the art will recognize that these elements need not be connected in a single unit such as personal computer or mainframe, but may be connected over a network or via telecommunications links. The computer hardware described above may operate as a stand-alone system, or may be part of a local area network, or may comprise a series of terminals connected to a central system. Additionally, some or all aspects ofthe logic ofthe present invention can be encoded to ran on a chipset or other elecfronic hardware. Additionally, the entire program may comprise a portion of a larger program wherein this section is called as part ofthe normal execution ofthe larger program, and all references to stopping or ending execution in this case refer to returning from this section ofthe program to the calling routine. An overview ofthe program is disclosed in Figure 26. The program comprises 4 separate procedures: Procedure Landing (2610), Procedure FormSeal (2615), Procedure Breakln (2620), and Procedure RaControl (2625). The program starts (at step 2605) by being called from a separate controlling software or as a result of a user-initiated action. The program first rans the Procedure Landing (2610) to place a cell onto (and partly into) the ion transport recording aperture. When Procedure Landing (2610) has ended, the program runs Procedure FormSeal (2615) to form a gigaohm seal. Next the program calls Procedure Breakln (2620) to achieve whole-cell access. The program then runs Procedure RaControl (2625). When completed, the control logic continues to step 2630 and ends. After the execution stops, a separate program will handle the application of voltage clamp protocols and the acquisition of data pertaining to ion channel activity. An unillustrated alternate mode of execution for this program will skip directly to Procedure RaControl (2625) to handle cells that have already been accessed but whose access resistance has increased beyond Raldeal. This provides an opportunity to improve the quality of recordings in the middle of an experiment. Once a procedure called or run by the program ends, the program returns to run or execute the next procedure illustrated by Figure 26. The individual procedures are described below.

With reference to Figures 27, 28, and 29, Procedure Landing is now described. At step 2610, the program begins Procedure Landing. The start of Procedure Landing is identified by step 2705. All ofthe counters and variables used in the program are assigned and are reset (2710), then the variable KeyPress, which traps user input instructions, is set to null (2715). The program displays (2720), through a screen or other similar display device, the message "Attempting Landing" to indicate the progress ofthe control logic. Next, the program rans a Washer (2725), a pump-driven fluid delivery system, to rinse fluidics channels, which purges any blockage ofthe recording holes and clears any particles that may be present in the chambers before they have an opportunity to block the recording hole. The program waits 5 seconds (2730) while Washer is run, then the program stops the Washer (2735). The program then applies -300 ton of pressure (2740) to clear away any left-over bubbles, waits 0.5 seconds (2745), then applies 0 ton of pressure (2750). The control logic then waits 2 seconds (2755) for the measurements to stabilize. At step 2760, the program checks to see if the variable Repeat is equal to 1. If Repeat is not equal to 1, the program adds 1 to the value for Repeat (2765), and returns to step 2740. If at step 2760 the value of Repeat is 1, the control logic continues to step 2810 of Procedure Landing (as illustrated by off-page connector 2770 pointing to its matching off-page connector 2805).

With reference to Figure 28, Procedure Landing continues. The program next nulls the junction potential (2810), waits for a stable reading (2815), then records the average Re (2820), and saves the Re to logs in a file stored on the computer (2825). Next the program requests cells (2830)from a separate program or routine not listed here, and waits until 0.5 seconds before cells would be introduced to the recording chamber (2835). The program then applies + 10 ton of pressure (2840) to keep the holes cleared during cell delivery, and then waits until the pipette has completed the cell delivery and is removed after adding cells (2845). The program then applies 0 ton (the units of ton and mmHg are interchangeable terms) of pressure (2850), waits 3 seconds (2855) to enable the cells to settle closer to the recording aperture. The program then starts a timer for Elapsed (2860), then applies -50 ton of pressure (2865) to attract a cell to the aperture. The confrol program then resets the Repeat variable to 0 (2870), and continues to step 2910 of Procedure Landing (as illustrated by off-page connector 2875 pointing to off-page connector 2905).

With reference to Figure 29, Procedure Landing continues. The program then checks at step 2910 to see whether the Seal is greater than 2 x Re for 0.5 seconds, or whether Elapsed time is greater than or equal to 5 seconds. If Elapsed time is greater than or equal to 5 seconds, the program then adds 1 to the value of stored variable Repeat (2915), then checks whether Repeat is equal to 3 (2920). If Repeat is not equal to 3, the program continues to step 2925 and applies +50 ton of pressure. The program waits 1 second (2930), then applies -50 ton of pressure (2935), then returns to step 2910. If at step 2920, the program determines that Repeat is equal to 3, the program continues to step 2940. The program aborts, records "failure to land" in its log, then ends the execution ofthe program (2945). At this point the chamber should be clean and prepared for removal.

If at step 2910 the program determines that Seal is greater than 2 x Re, the program displays the message "Landing Detected" (2950), resets the value for Elapsed (2955), and ends Procedure Landing at step 2960. As illustrated by the program overview of Figure 26, once Procedure Landing is run, the program next continues to step 2615 and runs Procedure FormSeal. Procedure FormSeal is illustrated by Figures 30, 31, 32, and 33. The program calls Procedure FormSeal at step 2615. The start of Procedure FormSeal is illustrated by step 3005. The program resets KeyPress to null, and the timer to 0:00 (3010). As used throughout this program, when the variable Timer or Elapsed is reset, it immediately starts counting time in seconds. The program then displays the message "Attempting Seal" on an output device (3015). The program then applies a negative holding potential to the electrode immediately after landing by applying HP = -80 mV (3020). The program then applies -50 ton pressure (3025). At step 3030, the program checks whether the seal between the cell and the recording aperture presents greater than or equal to 1 one gigaOhm (a "gigaseal") of resistance across the recoding aperture. If the seal is greater than or equal to 1 gigaOhm, the program proceeds to step 3310 of Procedure FormSeal (as illustrated by off-page connector 3035 pointing to off-page connector 3305). If at step 3030 the program determines that the seal is not greater than or equal to 1 gigaOhm, the program checks if the seal is increasing greater than 20 megaOhms per second (3040). If the seal is increasing greater than 20 megaOhms per second, the program continues to step 3045. If at step 3040 the program determines that the seal is not increasing greater than 20 megaOhms per second, then the program continues to step 3050. At step 3045, the program checks whether the timer has reached 10 seconds. If it has not, the program returns to step 3030. If at step 3045 the program determines that the timer is greater than 10 seconds, the program continues to step 3050.

At step 3050 the program resets the timer to 0:00, and checks whether the pressure is equal to -50 ton (3055). If pressure is -50 ton, the program applies 0 ton of pressure (3060), waits 2 seconds (3065), and returns to step 3030. If at step 3055 the program determines that pressure is not equal to -50 ton, the program continues with Procedure FormSeal (as illustrated by off-page connector 3070 pointing to off- page connector 3105). This section ofthe program ensures that a landing happens, and tests whether simple pressure steps are capable of producing a gigaOhm seal.

With reference to Figure 31, Procedure FormSeal continues by displaying the status message "Ramping Pressure" (3110). The program then optimally assigns a set of values for variables to initially be used during the pressure ramp (3115). Min is set to 0 ton, Max is set to -50 ton, Duration is set to 20 seconds, Counter is set to 0, and Timer is set to 0:00. The program then executes a pressure ramp loop. Starting with step 3120, the program ramps the pressure from Min to Max over the Duration, using the assigned values for these variables. The program then checks to see if seal is greater than 1 gigaOhm, or if "whole-cell access" has been achieved (3125). Whole- cell test is where capacitance is greater than 3.5 pF. If either ofthe conditions at step 3125 are true, the program continues with Procedure FormSeal at step 3310 (as illustrated by off-page connector 3130 pointing to off-page connector 3305).

If at step 3125 both ofthe conditions are false, the program moves to step 3135, where it checks whether Timer is greater than 20 seconds. If Timer is greater than 20 seconds, the program modifies the set of values for the variables used during the pressure ramp (3140). Min is reduced by 20 ton, Max is decreased by 30 ton, Duration is increased by 10 seconds, Counter is incremented by 1, and Timer is set to equal 0:00. The program checks whether Counter is greater than 4 (3145). If Counter is greater than 4, Procedure FormSeal continues to step 3210 (as illustrated by off- page connector 3170 pointing to off-page connector 3205). If Counter is less than 4, the program applies 0 ton of pressure (3150), waits 5 seconds (3155), then returns to the beginning ofthe pressure ramp loop that begins at step 3120.

If at step 3135 the program determines that Timer is not greater than 20 seconds, the program checks whether a user input key has been pressed (3160). If a key has been pressed, Procedure FormSeal continues with step 3205 (as illustrated by off-page connector 3170 pointing to off-page connector 3205). If at step 3160 a key has not been pressed, the program returns to the beginning ofthe pressure ramping loop that begins at step 3120.

With reference to Figure 32, Procedure FormSeal continues. At step 3210, 0 ton of pressure is applied. The program then resets the value to null whether a key has been pressed by the user (3215). The program then displays "Not sealed- Retry, Skip, Abort?" (3220). The program waits for the user to input whether to retry Procedure FormSeal, skip Procedure FormSeal, or abort the program altogether (3225). The program checks for input by the user. If the user enters "Retry" (3230), the program returns to step 3110 of Procedure FormSeal (as illustrated by off-page connector 3235 pointing to off-page connector 3105) to rerun the pressure ramp loop from its start. If the user inputs "Skip" (3240), the Procedure FormSeal ends (step 3245). Once Procedure FormSeal has run, as illustrated by the program overview of Figure 25, the program next continues to step 2620 and runs Procedure Breakln. If the user enters "Abort" (3250), the program stops executing and ends (3255). If no input has been received by step 3250, the program return to continue the input loop (as illustrated by connector 3260 pointing to connector 3265.

As illustrated by Figure 33, Procedure FormSeal continues with step 3310 and displays the message "Sealed." The program applies 0 ton pressure (3315), saves Elapsed time as time to seal in the logs (3320). The program then resets the values for Min, Max, Counter, KeyPress, and duration to null (3325). The program monitors the stability ofthe seal (3330), and continues once the seal is stable. If capacitance is not greater than 3.5 pF ("whole-cell") (3335), Procedure FormSeal ends (3340), and as illustrated by the program overview of Figure 26, the program next continues to step 2620 and rans Procedure Breakln. If at step 3335 the program determines that capacitance is greater than 3.5 pF, the program displays "Premature Access" (3345), then writes this feature to the logs (3350) and Procedure FormSeal ends (3355). The program next continues to step 2620 and runs Procedure Breakln.

With reference to Figures 34, 35, 36, and 37, Procedure Breakln is now described. The program runs Procedure Breakln at step 2620. Procedure Breakln starts, as illustrated by Figure 34, at step 3405. The program resets the value for KeyPress to null (3410), then applies holding potential that is appropriate for the assay (3415). The program displays "Attempting access" (3420), then verifies whether whole-cell access has already been achieved (3425). If whole-cell has been achieved, Procedure Breakln continues to step 3610 (as illustrated by off-page connector 3430 pointing to off-page connector 3605). If whole-cell has not been achieved at step 3425, the program nulls the chamber electrode capacitance (3435). The program then sets values for several variables (3440). Min is set to 0 ton, Max is set to -300 ton, Delta is set to -20 ton, Duration is set to 1 second, and Timer is set to 0:00. The program sets the value for Pressure to Min (3445), and then applies force equal to Pressure in the lower chamber (3450).

Procedure Breakln continues at step 3510 as illustrated by Figure 35, and as indicated by the illustrated off-page connector 3455 pointing to 3505. The program checks whether Seal is less than 200 megaOhms (3510). If yes, the program displays the message "Cell Lost" (3580), then stops execution ofthe program (3585). If at step 3510 the seal is not less than 200 megaOhms, the program checks if capacitance is greater than 3.5 pF (3515). If yes, Procedure Breakln continues to step 3610 (as illustrated by off-page connector 3520 pointing to off-page connector 3605). If capacitance at step 3515 is not greater than 3.5 pF, the program checks whether Pressure is greater than Max (3525). If yes, Procedure Breakln continues to step 3445 (as illustrated by off-page connector 3530 pointing to off-page connector 3460). If Pressure at step 3525 is not greater than Max, the program checks whether KeyPress has a value (3535). If yes, Procedure Breakln continues to step 3710 (as illustrated by off-page connector 3540 pointing to off-page connector 3705). If no KeyPress value is found at step 3535, the program checks whether Seal is decreasing by greater than 200 megaOhms per second (3545). If yes, Procedure Breakln continues to step 3445 (as illustrated by off-page connector 3590 pointing to off-page connector 3460). If at step 3545 Seal is not decreasing by greater than 200 megaOhms per second, the program checks whether Timer is greater than Duration (3550). If no, Procedure Breakln goes to step 3510 (as illustrated by connector 3555 pointing to connector 3560). If at step 3550 Timer is greater than Duration, the program resets Timer to 0:00 (3565), then the program increments Pressure by Delta (3570). The Procedure then returns to step 3510 (as illustrated by connector 3575 pointing to connector 3560).

Procedure Breakln continues as illustrated by Figure 36. The program checks whether capacitance is greater than 3.5 pF for 1 second (3610). If no, Procedure Breakln continues to step 3445 (as illustrated by off-page connector 3615 pointing to off-page connector 3460) to restart the pressure steps. If at step 3610, capacitance is greater than 3.5 pF for 1 second, the program records Break-in pressure to the log file (3620), and applies 0 ton of pressure (3625). The program then resets Elapsed to 0:00, then sets Elapsed to Global (3630). The whole cell access duration is set to the be a global variable. The program then displays the message "Whole-cell access detected" (3635), writes the time of access to the log (3640) and then Procedure Breakln ends at step 3645. As illustrated by the program overview of Figure 26, the program next continues to step 2625 and rans Procedure RaControl.

Procedure Breakln continues as illustrated by Figure 37. At step 3710, the program resets the value for KeyPress to null. Next, the program displays the message "Access not detected- Force access detect, Continue, Abort?" (3715) In step 3717, the program waits for the user to input whether to force access detect, continue or abort. The program checks for input by the user. If the users enters "Force access detect" (3720), Procedure Breakln goes to step 3610 (as illustrated by off-page connector 3725 pointing to off-page connector 3605). If the user enters "Continue" (3730), Procedure Breakln goes to step 3510 (illustrated by off-page connector pointing 3735 pointing to off-page connector 3505). If the user enters "Abort" (3740), the program stops executing (3745). Ifno input has been received by step 3740, the program returns to step 3705 and continues the input loop.

Procedure RaControl, as illustrated by Figures 38, 39, and 40, are now described. The program runs Procedure RaControl from step 2625. Procedure RaControl starts at step 3810. hi step 3815, KeyPress is set to null. Next, the program displays the message "Adjusting seal quality" (3820). The program then assigns Rmlnitial the value of Rm, and assigns Ralnitial the value of Ra (3825). The values for Cm, Rm, and Ra are recorded (3830). The program verifies if Ra is less than Raldeal (3835). RaMax and Raldeal are values that can be ascribed by the user beforehand. If yes, the procedure ends (3840). If Ra is not less than Raldeal, then the program verifies if Ra is less than Ra Max and Ra is decreasing (3845). If yes, the program returns to step 3835. If the answer at 3845 is no, the program sets Elapsed to 0 seconds (3850), then the program verifies if Ra is less than RaMax (3855). If Ra is less than RaMax, then Countdown is set to 20 seconds (3860), and Procedure

RaControl continues to step 3910 (as illustrated by off-page connector 3865 pointing to off-page connector 3905). If at step 3855 Ra is not less than RaMax, Procedure RaControl continues to step 3910 (as illustrated by off-page connector 3865 pointing to off-page connector 3905. Procedure RaControl continues as illustrated by figure 39. At step 3910, the program checks whether the user has inputted "Continue" or whether Ra is less than Raldeal. If yes, the procedure ends (3915). If the answer at step 3910 is no, the program goes to step 3920.

At step 3920, the program verifies if Ra is increasing and Rm is greater than 300 megaOhms. Ifno, the program continues to step 3945. If at step 3920 Ra is increasing and Rm is greater than 300 megaOhms, the program applies -50 ton of pressure (3925), waits 0.5 seconds (3930), applies 0 ton of pressure (3935), then waits 1.5 seconds (3940). The program then continues to step 3945. The program verifies if Ra is increasing and Rm is greater than 500 megaOhms (3945). Ifno, the program continues to step 3970. If at step 3945 Ra is increasing and Rm is greater than 500 megaOhms, the program applies -80 ton pressure (3950), waits 0.5 seconds (3955), applies 0 ton of pressure (3960), then waits 1.5 seconds (3965). The program then goes to step 3970. At step 3970, the program checks if Rm is greater than 0.8 gigaOhm. If yes, it applies -50 ton of pressure (3975). Ifno, it applies -10 ton pressure (3980). From both steps 3975 and 3980, Procedure RaControl continues to step 4006 (as illustrated by off-page connector 3985 pointing to off-page connector 4003. Procedure RaControl continues as illustrated by Figure 40. The program checks, at step 4006, if Ra is greater than Raldeal, if Rm is greater than (Rmlnitial - 25%), and if countdown is greater than 0. Ifno, the program continues to step 4084 (as illustrated by connector 4009 pointing to connector 4081). If at step 4006 the answer is yes, then the program continues to step 4012 and waits 5 seconds. Then the program tests whether Ra is less than RaMax (4015). If yes, then the program sets Countdown to 20 seconds (4018), and will time down be seconds to zero and continues to step 4021. If at step 4015 Ra is not less than RaMax, the program continues to step 4021.

At step 4021, the program checks whether Ra is less than Raldeal. If yes, the program continues to step 4084 (as illustrated by connector 4024 pointing to connector 4081). If at step 4021 Ra is not less than Raldeal, the program checks whether Ra is decreasing (4027). If Ra is decreasing, the program continues to step 4054. If at step 4027 Ra is not decreasing, the program checks if Rm is not decreasing and Rm is greater than 1 gigaOhm (4030). If yes, -10 delta ton of pressure is applied (4033), and the program continues to step 4036. If at step 4030 the value is false, the program continues to step 4036. At step 4036, the program checks whether Rm is not decreasing and Rm is less than 1 gigaOhm. If yes, -5 delta ton of pressure is applied (4039) and the program continues to step 4042. If at step 4036 the answer is no, the program continues to step 4042. At step 4042 the program tests whether Rm is decreasing and Pressure is greater than -10 ton. If yes, +5 ton of pressure is applied (4045) and the program continues to step 4048. If at step 4042 the answer is no, the program continues to step 4048. At step 4048, the program checks whether Rm is less than (Rmlnitial - 25%). If yes, 0 ton of pressure is applied (4051), and the program continues to step 4054. If at step 4048 the answer is no, the program continues to step 4054.

The program next checks whether Pressure is greater than BreaklnPressure (4054). If yes, 0 ton of pressure is applied (4057), and the program continues to step 4060. If at step 4054 Pressure is not greater than BreaklnPressure, the program continues to step 4060. The program checks whether Elapsed time is greater than 120 seconds (4060). If yes, 0 ton of pressure is applied (4063), and Procedure RaControl ends (4066). If at step 4060 Elapsed is not greater than 120 seconds, the program checks whether Rm is less than 300 megaOhms (4069). Ifno, the program continues to step 4084, as illustrated by connector 4072 pointing to connector 4081. If at step 4069 Rm is less than 300 megaOhms, pressure equal to (BreaklnPressure less 10 ton) is applied (4075). The program continues to step 4006, as illustrated by connector 4078 pointing to connector 4099.

At step 4084 the program checks whether Ra is increasing. If yes, -60 ton pressure is applied (4087) and the program continues to step 3815, as illustrated by off-page connector 4090 pointing to off-page connector 3805. If at step 4084 Ra is not increasing, 0 ton of pressure is applied (4093), and the program returns to the beginning ofthe loop at step 3910, as illustrated by off-page connector 4096 pointing to off-page connector 3905.

Once Procedure RaControl has ended, the program, in an unillustrated step, records and outputs the data, preferably to a database. These data can be recorded and outputted by a variety of means, including electronic storage media (hard disk or floppy disk), elecfronic transfer via a network (such as TCP/IP or Bluetooth), or optical storage media. Additionally, in an unillustrated step, the program may display the results on an output device, such as a LCD display or computer monitor screen. In another unillustrated step, the program may optionally generate a printout ofthe results and other collected data via a printing device such as a laser printer. The results gathered by the program may, in an unillustrated step, be collated, aggregated, or compared to other previous results, or control results. Depending upon the needs and requirements ofthe user of this present invention, the program can be configured to use one or more ofthe above-referenced output methods. Having completed these steps, and having outputted the results and/or data, the program stops execution (2630). EXAMPLES

Example 1. Device for ion transport measurement comprising upper chamber piece and biochip.

-An ion transport measuring device in the form of a cartridge known as the SEALCHIP™ (Aviva Biosciences, San Diego, CA) comprising an upper chamber piece and a chip comprising ion transport measuring holes was manufactured.

Upper chamber pieces with 16 wells having dimensions of 84.8mm(long) xl4mm(wide) x7mm(high) were injection molded with polycarbonate or modified polyphenylene oxide (NORYL®) material. The distance between centers of two adjacent wells was 4.5mm. The well wall was slanted by 16 degrees on one side and 23 degrees and contoured on the other side to allow guidance for cell delivery. The well holes had a diameter of 2 mm.

A biochip with 16 laser-drilled recording apertures had dimensions of 82 mm (long) x 4.3 mm (wide) x 155 microns (thick). The distance between the first hole and a nanow edge is 7.25mm. The holes were laser drilled to have two counterbores of 100 microns (diameter) x lOOmicrons (deep) and 25 microns (diameter) x 35 microns (deep), respectively. A final through-hole was drilled from the side ofthe counterbores and had a 7 to 9 micron entrance hole and a 2.0 micron exit hole with a total through-hole depth of 20 microns. Chemical treatment with acid and base was done as described in Example 3.

The treated chip was attached to the upper chamber using UV epoxy glue. Devices produced using this methods had anRe of ~2MOhm with standard ES and IS solutions, and an average Ra of ~6.0MOhm using RBL cells with a standard pressure protocol described herein.

Example 2. A 52-chip bench mark study.

We have conducted a bench mark study using 52 single-hole biochips tested using a CHO cell line expressing the Kvl.l potassium channel. The result demonstrated a 75% success rate as determined by the following criteria: 1) achievement of sealing of at least one gigaOhm (a "gigaseal") within five minutes of cell landing on a hole, and 2) maintenance of Ra of less than 15MOhm, and Rm of greater than 200MOhm throughout 15 minutes of whole cell access time. Chip fabrication

Patch clamp chips were designed at Aviva Biosciences and fabricated using a laser-based technology (without an on-line laser measurement device). The K-type chips were made from -150 micron thick cover glass. The ion transport measuring hole stractures had -140 micron double counterbores and final through-holes of -16.5+2 micron depth. The apertures on the recording surface had a diameter of 1.8+0.5 microns. The recording surface was further smoothed (polished) by laser.

Surface Treatment Chips were received from FedEx overnight service and were inspected for integrity and cleanness. About 5% ofthe chips were excluded from further treatment in this process. Selected chips were then treated according to Example 3. Treated chips were stored in ddH₂O for 12 to 84 hours before the tests.

Batch QCfor chips

Chips were acid and base treated in batches of 20-25. Four to six pieces of each batch were randomly picked for testing their patch clamp performance with CHO-Kvl.l cells in terms of speed to seal and stability ofthe whole cell access. Batches with <75% success rate were excluded for the 50-chip tests.

Cell passage

CHO-Kvl.l cells (CHO cells expressing the Kvl.l ion channel) between passage 47 and 54 were split daily at 1 : 10 or 1 : 15 for next-day experiments. Complete Iscove media (Gibco 21056-023) with 10% FCS, lxP/S, lxNEAA, lxGln, lxHT with 0.5mg/ml Geneticin was present in media used to passage cells and not present in media used to grow cells for next-day experiments.

Cell preparation

Cells were isolated using the protocol for CHO cell preparation described in Example 6. After isolation, cells were resuspended in PBS complete media and passed through a 20 micron polyester filter into an ultra-low cluster plate (Costar 3473). The cells were used for the study between 30 minutes and 3 hour 30 minutes after the filtration.

Cell QC

Isolated cells were quality control tested with conventional pipette patch clamp recordings for their speed to seal, break-in pressure, and Rm and Ra stability. Freshly pulled pipettes were typically used within 3 hrs. Only cell preparations that passed the pipette quality control test were used for the 50-cell tests. About 50% of the preparations out of approximately 30 cell isolations passed and were used for this study.

Solutions

Intracellular solution was made according to the following formula : 8 mM NaCl; 20 mM KCl; 1 mM MgC12; 10 mM HEPES-Na; 110 mM K-Glt; 10 mM EGTA; 4 mM ATP-Mg; pH 7.25 (IM KOH3); 285 mOsm. Aliquoted at 10ml per 15ml coming centrifuge tube, and stored at 4°C

Extracellular solution (PBS complete) was DPBS (lx), with glucose, calcium and magnesium (Gibco cat# 14287-080). This solution contained:

0.9 mM CaC12, 2.67 mM KCl, 1.47 mM KH2PO4, 0.5 mM MgC12, 138 mM NaCl, 8.1 mM Na2HPO4, 5.6 mM Glucose, 0.33 mM Na-pyravate, pH 7.2-7.3, 295 mOsm.

Chip Quality Control (QC)

For each recording, the chip was assembled into a two-piece cartridge, and the lower and upper chambers were filled with intracellular and extracellular solutions, respectively. The chip was further quality control tested by inspection under the microscope and seal-test resistance measurement. Chips that showed a dirty surface, visible cracks and or had a seal test resistance greater than 2.1 MOhm were excluded.

Experiment settings

Chips that passed quality control underwent electrode offset and the overall recordings were done with 4KHz bass filter. Cell landing was monitored on computer screen. Criteria

A simple description of a positive result is: chips that achieved gigaseals and gave Ra<15MOhm and Rm>200MOhm throughout 15 min recording period.

Results

A total of 58 chips were tested, 6 of which were excluded from final analysis. Out ofthe 52 cells included, 39 (75%) passed the test criteria. 43 (83%) achieved at least 12 minutes of continuous high quality recordings (Ra<15MOhm; Rm>200MOhm); 47 (90%) achieved gigaseals.

Success rate

Success duration is plotted in Figure 20A. Accumulative success rate is plotted in Figure 20B. Success rate was consistent throughout the tests, which suggests that most ofthe critical experimental parameters were under control. 75% is a representative success rate under the cunent controlled conditions.

Electrode Resistance (Re)

90% ofthe elecfrodes selected for the tests had Re between 1.3 to 2.0 MOhms (Figure 21 A). A total of 81 chips were mounted and tested. 23(28%) failed the quality control test, among which 15(18.5%) were due to Re>2.1 MOhms. 5(6%) chips were screened out because of their dirtiness of surface; 3(4%) had blocked or cracked holes. Chips were not screened at low Re values. The reason behind the 2.1 MOhm cut off is that historically chips with the cunent geometry (double counterbore) showed lower than 75% success rate in achieving the test criteria. Re is more or less normally distributed except for a slightly higher peak at ~1.3MOhm.

Break-in Pressure

Break-in Pressure is an important parameter for cell condition. During the tests, break-in pressures were tightly distributed between -100 to -130 tons (Figure 2 IB). Our previous findings suggest that seals with more negative break-in pressure are likely to have higher and unstable Ra, while seals with lower break-in pressure are likely to have lower and unstable Rm.

Il l Membrane Resistance (Rm)

After break-in, Rm was mostly between 0.5 to 2MOhm (Figure 22A). Ending Rm had a similar distribution, but more skewed to lower values. This is consistent with the deterioration of Rm over time. However, the amount of Rm deterioration was suφrisingly small, which suggests that the seals were very stable during the 15 minutes test periods.

Access Resistance (Ra) Initial Ra had a normal distribution centered at 7MOhm (Figure 22B). 80% of the seals had Ra starting from below 10 MOhm. In most cases, Ra increased during the 15 minutes with an ending value near 1 l~13MOhm. In order to minimize disruption ofthe seals, great effort was not made trying to maintain minimal possible Ra. It is not known what the ending Ra would be and what percentage of seals would lose Rm if such efforts were made.

Typical Recordings

Figures 23-25 demonstrate sample data from one particular cell monitored during the 52-cell test refened to above. Figure 23A demonstrates the whole-cell cunent record in response to a series of voltage steps from a holding potential of -80 mN to various potentials between -60mV and +60mV. Figure 23B shows the potassium cunent, extracted from the whole-cell cunent by P/4 leak conection ofthe same cunents, compensated for leak and capacitance. Figure 23C illustrates the cunent- voltage relationship ofthe steady-state cunent averaged from data recorded at the time-points between the anowhead indicators in Figure 23 A and Figure 23B, showing the voltage-dependence ofthe potassium cunent expressed in this cell line. The larger cunents were the uncompensated cunents (from Figure 23A) and the smaller cunents were compensated (from Figure 23B). The difference between the compensated and uncompensated cunents represents the leak cunent, which was negligible in relation to total whole-cell cunent.

Figure 24 shows data similar to those in Figure 23 but is recorded at the end of a 15-minute recording period whereas data in was Figure 23 recorded at the start ofthe recording period, where the duration ofthe recording period is relative to the time at which whole-cell access was achieved. Figure 24A demonstrates the whole- cell cunent record in response to a series of voltage steps from a holding potential of - 80 mV to various potentials between -60mV and +60mV. Figure 24B shows the potassium cunent, extracted from the whole-cell cunent by P/4 leak conection ofthe same cunents, compensated for leak and capacitance. Figure 24C illustrates the cunent- voltage relationship ofthe steady-state cunent averaged from data recorded at the time-points between the anowhead indicators in Figure 24A and Figure 24B, showing the voltage-dependence ofthe potassium cunent expressed in this cell line. Once again, in Figure 24C, the leak cunent was still a small proportion ofthe whole- cell cunent.

Figure 25 shows the time-course ofthe measured seal quality parameters during the same experiment that is represented in Figures 23 and 24. Over the 15 minute recording period, the membrane resistance (Rm) decreased (due to leak cunent) slightly from 1.4 GOhms to 1.0 GOhms, and access resistance (Ra) increased from 8 MOhms to 13 MOhms. The non-uniform time-profile ofthe traces is representative ofthe effect ofthe applied pressure control protocol used to control Ra during the experiment.

Example 3. Treatment of Ion Transport Measurement Chips to Enhance their Electrical Sealing Properties

Detailed Procedure: (referenced to step numbers below). All incubation processes were carried out in self-made Teflon or modified polyphenylene oxide (Noryl®) fixtures assembled in a glass tank while shaking (80 φm, with C24 Incubator Shaker, Edison, NJ, USA). Water was always as fresh as practical from a water purification system (NANOpure Infinity UV7UF with Organic free cartridge). Nitric acid was ACS grade (EM Sciences NX0407-2, 69-70 %). Sodium hydroxide was IO N, meeting APHA requirements (VWR VWR3247-7). When necessary, chips were inspected for QC before and after treatment. The protocol used was:

1. 3 hour shaking incubation in 6M nitric acid at 50 degrees C. 2. 6 x 2 minute rinses in DI water at room temperature.

3. 60 minute incubation in DI water (shaking)

4. 2 hour shaking incubation in 5M NaOH at 33 degrees C

5. 6 x 2 minute rinses in DI water at room temperature.

6. 30 minute incubation in DI water (shaking) at 33 degrees C

7. Chips were stored in DI water at room temperature. A vial used for storage was filled to the neck to minimize air space.

Chips treated according to this protocol demonstrated enhanced electrical sealing when tested in ion transport detection devices.

Example 4. Achieving Seals with Inverted Chips

A biochip was fabricated from Bellco D263 or Coming 211 glass of thickness of -155 micron. The 16 laser-drilled recording apertures on the chip had dimensions of 82 mm (long) x 4.3 mm (wide) x 155 microns (thick). The distance between the first hole and a nanow edge is 7.25mm. The apertures were laser drilled to have one counterbore of 100 microns (diameter) x 125microns (deep). A final through-hole was drilled from the side ofthe counterbores and had a -10 micron entrance hole and 4.5 micron exit hole with a total through-hole depth of 30 microns. After standard chemical treatment as described in Example 3, the biochip was mounted to an upper chamber piece described in Example 1 in inverted configuration such that the counterbore side faced the upper chamber piece (where RBL cells were added). Recordings were done with a device adapted to Nikon microscope as described in Example 5. Typical voltage clamp quality parameters such as Rm and Ra over time are shown in Figure 22.

Example 5. A biochip device adapted to a microscope and having flow-through lower chambers.

A device for ion transport measurement known as the "Tester" device having flow-through lower chambers was designed and constructed. The device has a lower chamber base piece that formed the bottom surfaces ofthe lower chambers and comprises conduits for the inflow and outflow of solutions, and a gasket that formed the walls ofthe lower chambers. The device also comprises a cartridge that provided upper chambers and a chip comprising holes. The device was adapted for a microscope, so that the bottom surfaces ofthe lower chambers are transparent, and the device was fitted to a baseplate adapted to a microscope stage. The following description ofthe design and manufacture ofthe device makes reference to Figures 3- 8. In this design, a biochip cartridge that has a chemically-treated glass chip sealed to an upper chamber piece can be assembled onto a microscope stage-mounted lower chamber base piece that allows simultaneous or sequential testing of all recording apertures while simultaneously observing the experiment's progression microscopically. The Tester device includes a metallic base plate, in this case made of aluminum, shaped to insert onto a microscope stage, and sculpted to support and align a multi-well perfusion lower chamber base piece. The baseplate ofthe device (as shown in Figure 4) was shaped to take advantage of an existing mounting point on the Nikon microscopes by positioning the device into an aperture within the microscope stage. It is round, with an edge intended to prevent it from falling through the hole on the stage. The depth ofthe device is intended to hold the functional portion ofthe biochips as well as the cells that are added to the biochip at testing time at a convenient focal point within the focal range ofthe microscopes, that is, at essentially the same level as the upper platform ofthe microscope stage. To assemble the device, a gasket (as shown in Figure 6) was inserted over the lower chamber base piece (301 in Figure 3A) seated in a baseplate, then the cartridge, was clamped onto the gasket by compression via a clamp assembly (shown in Figures 7A and 7B) that bolted onto the base plate using four thumb-screws (73 in Figure 7A). The lower chamber piece was made of plastic and contained an anay of 16 conduits for inflow of intracellular solution, and another 16 conduits for outflow of same. The 32 conduits emerged on the top surface ofthe lower chamber base piece in alignment with the recording apertures ofthe biochip. The gasket was made of PDMS and was situated between the lower chamber piece and the chip, and contained slits, or holes (601 in Figure 6), that aligned between the emerging holes ofthe perfusion conduits ofthe lower chamber piece and the recording apertures ofthe chip, such than intracellular "lower" chambers were formed within the anay of slits or holes in the gasket. An electrode of silver-silver chloride was introduced into each of the 16 outflow conduits along one side ofthe base piece to function as recording electrodes.

With reference to Figure 8 A, the device was made up of 1) a metallic base plate (812), specifically, but not exclusively, stainless steel, 2) a transparent lower chamber piece (801), sometimes refened to as an "inner chamber anay", made from polycarbonate (but could be any other convenient transparent substance) 3) electrodes (not visible in Figure) inserted into the outflow conduits ofthe lower chamber piece, made from wires of silver or any other conductor capable of being used as a voltage sensing and cuπent-delivering electrode, and attached to a connector on the outer side ofthe lower chamber piece, 4) inert tubing connectors (not visible in Figure 8; 302 as seen in Figure 3A) glued to the lower chamber base piece at the conduit openings (or any other means that may provide a connection for a fluid conveyance system) in this case made from glass, 5) a gasket (805) that provided a seal between the lower chamber base piece and the biochip cartridge, where the gasket (in this case made of PDMS) simultaneously comprised the inner chambers, 6) a biochip cartridge (804) mounted onto the test apparatus over the gasket, and held in place by a guidance system, in this case alignment pins inserted into the plastic bottom chamber anay body in such a way as to restrict movement ofthe biochip while simultaneously guaranteeing alignment ofthe biochip's recording surface with the inner chambers, 7) a clamp (802) assembly intended to apply sufficient pressure onto the biochip cartridge so as to generate a seal between the bottom ofthe chip and the gasket, and 8) an anay of electrodes (not visible in Figure 8, 75 in Figure 7B)attached to the clamp shaped and oriented so as to enter into the top wells ofthe biochip cartridge, all 16 at a time, and where all elecfrodes were connected together so as to provide a reference electrode in the upper chambers ofthe cartridge.

Figure 5 shows the aπangement of parts installed in the baseplate (54) schematically. The clamp (53) holds the cartridge (51) down on the gasket (not visible) and lower chamber base piece (not visible). The clamp has attached electrode wires (55) that extend into the upper wells ofthe cartridge (51). This depiction also shows the lower chamber electrode anay (52) of pin sockets (56) that connect to electrode wires that are threaded through conduits leading to lower chambers. The pin sockets (56) can be connected to the signal amplifier.

Figure 8B showed the assembled device, in which the clamp (802) is screwed into the baseplate (812). The flow-through lower chamber base piece is not visible beneath the cartridge (804). Inflow tubing (809) is attached to one side ofthe lower chamber base piece and outflow tubing (808) is attached to the opposite side ofthe lower chamber base piece.

1) Metallic Base Plate:

This base plate serves multiple functions. First, the metallic body serves as an electrical noise shield for the bottom side ofthe test chamber. It completes a type of faraday cage that is contiguous with the grounded stage ofthe microscope. Secondly, the metal base was carved on the top side so as to catch any fluids that may leak or spill and prevent the contamination ofthe microscope with said fluids. To this end, the base plate was sealed, with silicone glue or with silicone grease (vacuum grease) or with any other such viscous immiscible substance (eg: Vaseline) to the transparent lower chamber piece described in 2) (below). Third, the base plate was shaped to optimize its use with a particular microscope. Specifically, in our case it was desirable for the base plate to be cut to fit onto the 107mm circular cutout hole of a Nikon microscope. Fourth, the base plate was drilled and tapped so as to provide a mounting point for the lower chamber piece and for the clamp ofthe Tester. Its design was such that held the recording aperture ofthe cartridge within a few millimeters ofthe level ofthe top ofthe microscope stage so as to ensure that the chip function could be monitored within the focal range ofthe microscope. Figure 4 illustrates the design ofthe base plate as adapted for the Nikon Microscope.

2) Transparent Lower Chamber Base Piece (Inner Chamber Anay):

This design of a lower chamber base piece, shown as (301) in Figure 3A may also be refeπed to as an inner chamber anay, or an intracellular chamber anay. For the convenience of being able to view under a microscope the progression of an experiment, it was made of a transparent material. Polycarbonate was chosen for its ease of machining. Its shape was designed to support a cartridge over it, and provide tubing connections along the long edges of either side the cartridge, as well as to provide connections to electrodes placed inside one of each pair of conduits (holes in the base piece material that function as such) supplying each recording aperture ofthe chip. The conduits drilled into each side provided a connection between the edge of the lower chamber base piece and somewhere near the center, then another conduit was drilled peφendicularly from the top surface to connect to each conduit coming from the edge. The emerging conduits at the top surface were located so as to provide for an inflow and an outflow of solution to and from each ofthe lower chambers. The lower chamber base piece did not comprise chambers, but instead the lower chambers were created by openings within the gasket material. As seen in Figure 3B, the inflow and outflow conduit openings (304) in the areas (303) ofthe upper surface of the base piece that coπesponded to the bottom surfaces ofthe lower chambers were separated from one another so as to leave an undisturbed area of surface that could be seen through with a microscope so as to visualize the recording aperture during experimentation. To this end, the top surface that was in opposition to the chip was untouched with the exception ofthe emerging inflow and outflow conduit openings and as well the bottom surface of the lower chamber base piece was left untouched so as to not disrupt transparency ofthe part. Each conduit leading to the edges ofthe base piece had a means (such as tubing connectors) for interfacing it to inflow tubing and outflow tubing (309 and 308 in Figure 3B) (see also description of part 4) that provided for delivery of solutions, as well as for pneumatic pressure control. Tubing connectors (302) can be seen in Figure 3 A. One ofthe conduits going to the edge of the part was left longer so as to house an electrode (wire) that is introduced into the lumen ofthe conduit. The added length also allowed for a second segment to be glued onto the top surface so as to house the connectors for the electrodes. The top surface of this part was trimmed down around the periphery of area covered by the cartridge so as to provide an edge that functioned to hold the gasket in place during mounting and removal ofthe cartridge. Further, between each pair of inflow and outflow holes for each bottom well was a cut intended to prevent wetting ofthe gasket material to span from one bottom chamber to adjacent bottom chambers. This lower chamber base piece as a whole contained 6 pin holes 2mm in diameter to hold 6 pins that functioned to keep the cartridge aligned during mounting. It also contained a further 4 holes to hold 4 spring-pins (307 of Figure 3B) that functioned to provide an electrical connection for an early version ofthe cartridge. The present version ofthe cartridge does not require these contacts, however they were kept in place so as to prevent contact with the gasket before the clamp part is pressed down during the mounting. Finally, two more holes were present so as to use two screws to hold the part onto the base plate.

3) Inner Chamber Electrodes: Each lower chamber contained an electrode, which in this case is a silver wire that was periodically chlorided. The wire was inserted into the lumen ofthe longer conduit ofthe base piece and bent upward into the electrode connector anay (315 in Figure 3B). The segment of wire was sufficiently long that it remained exposed within the lumen ofthe longer conduit after the inert tubing interface parts were glued into place, and the other end was soldered to a connector, in this case an anay of 1mm female pin-connector sockets inserted into holes in the part. The connector pin sockets (310) are seen in Figure 3B.

4) Inert Tubing interface:

Into each conduit ofthe base piece an inert tubing connector (in this case made from glass) was inserted that was fixed in place with epoxy glue. Epoxy was chosen only in so much as it is prefened for bonding glass to polycarbonate. The tubing segments were sufficiently long to butt against a countersunken segment ofthe conduit drilled into the lower chamber piece and stick out ofthe part enough to hold a segment of silicone tubing that was press-fit onto the glass segment. This junction should withstand a pressure greater than two atmospheres positive pressure, and greater than 700 mmHg vacuum pressure. It was determined that 3 to 5 mm insertion into the silicone tubing was sufficient to accomplish this requirement.

5) Gasket:

For convenience the flexible gasket was molded from curing PDMS. The gasket contained a raised edge on the bottom side that suπounded the chambers as a whole and was able to hug an edge present in the same periphery on the lower chamber piece so as to hold the gasket in place. As depicted in Figure 6, the gasket had oblong holes (601) in it that aligned over the exit and entrance holes ofthe lower chamber piece for each chamber ofthe anay. On the top surface ofthe gasket was a set of squared O-rings (602) that were part ofthe gasket but raised sufficiently to form a seal onto the cartridge when pressed against it with the clamp part. 5) Biochip

The fabrication of chips having holes for ion transport measurement has been described herein . In this device, the chip was made of glass and has 16 laser drilled holes. The chip was laser polished on the top surface, and treated in acid and base prior to attaching the chip in inverted orientation to an upper chamber piece with a UV adhesive.

6) Clamp Assembly:

A clamp was made from an inflexible material so as to not allow bowing of the cartridge during compression onto the gasket while mounted on the tester. In this case it was made of stainless steel for its inertness when wetted with physiological buffers. The clamp was shaped so as to fit snugly over the cartridge and was drilled so as to accommodate and be positioned by the guide-pins sticking out ofthe lower chamber piece. Four screws were finger-tightened to the base plate at each corner of the clamp assembly so as to press down the cartridge to seal it against the gasket. This part is shown in Figure 7A and 7B.

7) Upper Chamber Electrodes:

In early development it was expected that compression pins would contact the bottom of the cartridge during testing to provide a connection to the reference electrodes built in to the cartridge. The present embodiment ofthe cartridge does not contain reference electrodes, therefore these electrodes were introduced into the top wells of the cartridge. To this end, periodically chlorided silver wires were used as electrodes. The electrodes were shaped to dip deep inside each well, and on the outside of the wells the wires were soldered to a wire running along the top of the clamp part (visible in Figure 7B). At each end of this wire was a 1mm female pin connector that was used to interface with the voltage clamp amplifier. The upper chamber electrode wires (55) are shown in Figure 5.

Method:

Before use the device should be clean and dry. A SealChip™ cartridge was removed from its carrier, and rinsed with a jet of deionized water of approximately 18 MOhms resistance. The product was them dried under a stream of pressurized dry air filtered through a 0.2 μm air filter to remove water from the recording apertures and their vicinity. The clean cartridge was then placed with top-wells upward onto the pressure contact pins ofthe tester such that movement ofthe cartridge was limited by the six alignment dowels ofthe bottom chamber piece. Prior to clamping the cartridge to the gasket and lower chamber base piece, the cartridge should be supported above the gasket but without yet touching the gasket. The clamp was them placed over the cartridge such that the four mounting holes aligned with their threaded counteφarts on the base plate. The four mounting screws were them used to press down the clamp uniformly thereby pressing the cartridge down onto the PDMS gasket with sufficient pressure to form a tight seal between the chip and the gasket and between the gasket and the lower chamber base piece. The recording aperture within each chamber ofthe cartridge should aheady be aligned with openings in the gasket that form the lower chambers.

The bottom chambers were then filled from one side with sufficient solution (analogous to intracellular solution) to fill the bottom chambers and fill enough ofthe tubing on the other side such that capacitative distension ofthe tubing on the filling side would not introduce air into the recording chamber, and would not introduce air into the area ofthe tubing that contained the bottom-chamber electrode. (For this puφose, it is best to fill the chamber starting from the side that does not contain the electrode since higher pressures will be used for vacuum pressure than for positive pressure, thereby ensuring that the electrode will remain in full contact with the solution at all times.) Once the bottom chamber was filled and was free of visible bubbles, the tubing was sealed off by a clamp (a valve or any means that ensures electrical isolation between the bottom chambers ofthe anay can also be used). Sufficient positive pressure was applied to the free end ofthe inner chamber tubing so as to cause solution to be forced into the counterbore and through the hole ofthe recording aperture ofthe chip.

Once solution was seen emerging into the top chamber, the pressure was released, and immediately the top chamber was filled with sufficient solution (analogous to extracellular solution) so as to completely immerse the top side ofthe chip without bubbles remaining on the chip surface, and to fill the top well sufficiently to provide good contact with the electrode in the top well. (It is also of benefit to fill the top well sufficiently to avoid a strong meniscus effect (60 to 70 microliters with the present version ofthe SealChip™ product) whenever it is intended to view under an inverted microscope the progression ofthe experiment (for upright microscopes it is necessary to fill with more solution, -90 microliters, to allow good contact with a coverslip that must be placed over the well to enable a good view ofthe bottom ofthe well).)

The assembled tester, now ready for testing, was placed on the microscope (and connected to the voltage clamp amplifier(s) as well as to the pressure control device(s) for testing. After the termination ofthe experiment, the tester was disconnected and removed from its testing location. The extracellular medium was suctioned from each well, and each well was rinsed once with deionized water to removed any leftover particulate (debris or cellular) material that may have been left over from the experiment. Both ends ofthe tubing ofthe bottom chambers were then opened and the solution was suctioned out of the bottom wells. Each well was well rinsed with clean deionized water, then dried completely with pressurized air. Finally the screws holding down the clamp were removed and the cartridge was disassembled from the tester. Any wetting at the gaskets was wicked away with a lint-free tissue. (If any liquid is pooled around the gasket, then the gasket should be removed, rinsed then dried, and the bottom chamber anay should be likewise rinsed and dried, ensuring that the tubing is also rinsed and completely dried.)

Quality Control/Quality Assurance of SealChip™ product:

Internally to the company, the "tester unit" device described in this example has been used for QC/QA ofthe SealChip™ product before it is sent to a customer, and before it is used internally for further research. The success rate with a product that passes the QC has been as good as that with older testers that tested a single chamber at a time.

Quality Control/Quality Assurance of Cells:

Internally to the company, the tester unit device has been used to verify the quality ofthe cells used for QC/QA using known good SealChip™ product.

Research and Development: The tester unit has been used by our company for testing variations to the SOP for the SealChip™ product. In the future it may be used for discovery and screening of compounds that require exchanging of solutions on the bottom well or where compounds or particles must be delivered to the cytosolic chamber after a seal is formed with the cell membrane.

A great number of results have been achieved on the microscope adapted device ("Tester Unit") since its development. The tester unit has been the tool of choice for perfomiing quality control experiments on the SealChip™ product. The following gives examples ofthe quality of data obtained from it. (The seal resistance is designated Rm; G refers to GigaOhms and M refers to MegaOhms.)

Table 1. SealChip™ Data

Example 6. Cell preparation for ion transport measurement. PART I. CHO wt. and CHO.Kv cells

1. Use cells @ 50%~70% confluency. (18 hrs after cells seeded 1:10-1:15)

2. Remove medium and wash x2 with PBS (extra wash might be necessary if the final cell suspension has too much small debris)

3. Treat for 2' 15" with 1:10 trypsin-EDTA, at this time the supernatant might be a little turbid due to release of cells into the buffer.

4. Rock gently, aspirate to discard supernatant. Wait for 1 '25".

5. Add 1 volume of X⁺⁺-free DMEM complete with 10% FCS, NEAA, etc, rock gently to loosen and detach cells, and spin down (do not try to blow to remove the remaining cells sticking to the bottom)

6. Wash xl with PBS complete

7. Resuspend in PBS, triturate, and pass through 15~20μm filter into nonstick plate.

Cells can be used after 10 minutes of recovery and should last for up to 4hr

PBS

2. Treat for 1' with 5 ml 1:10 trypsin-EDTA (0.5ml 0.05% trysin 0.53mM EDTA from GIBCO cat. No.25300-54 in 4.5 ml PBS)

3. Rock gently, aspirate to discard supernatant.

4. Add 0.5 ml fresh 1 : 1 trypsin-EDTA , Wait for 6 mins.

5. Add 5ml of X^-free DMEM complete with 10% FCS, NEAA, etc, rock gently to loosen and detach cells, leave cell at RT for 1 hour, and spin down (do not try to blow to remove the remaining cells sticking to the bottom)

6. Wash x2 with 1ml PBS complete

7. Resuspend in PBS, triturate, and pass through 15 to 20 micron filter into non-stick plate.

Part III. CHO-Herg cells.

1. Use cells at 50%~70% confluency in T-25 flasks (VWR, Cat. No. 29185-

2. Remove medium and wash x2 with X^-free PBS (extra wash might be necessary if the final cell suspension has too much small debris)

3. Treat for 1 ' with 2 ml frypsin-EDTA( 0.5ml 0.05% trysin 0.53mM EDTA from GIBCO cat. No.25300-54 in 1.5 ml PBS)

4. Rock gently, aspirate to discard supernatant. Wait for 2 mins.

5. Add 5ml volume of X⁺⁺-free DMEM complete with 10% FCS, NEAA, etc, rock gently to loosen and detach cells, leave cell at RT for 30min, and spin down (do not try to blow to remove the remaining cells sticking to the bottom)

6. Wash x2 with 1ml PBS complete

7. Resuspend in PBS, triturate, and pass through 15~20micron polyester filter into non-stick plate if cells still cluster together.

Part IN. Protocol for isolation of CHO 1. Use cells at 70- 80% confluences in T-25 flasks (24 hrs after seeding).

2. Remove medium and wash x2 with X⁺⁺-free PBS ( ( cell should not be leave in X⁺⁺-free PBS more than 10 mins, otherwise, the minimal digestion time will be decreased) 3. Wash once with 1 :4 AccuMax (available from Innovative Cell Technologies,

San Diego, CA) ( wait about 20 second, rocking to removed the loose attached cell)

4. Treat at 37°C w 4 ml volume of 1: 4 Accumax ( diluted with

PBS) for minimal time ( cell dissociate from the flask and floated in the Accumax ) or 1.5 times minimal time.

w/o rocking w/o rocking cking

) w/o rocking

5. Add 5ml volume of

DMEM with 10% FBS, into the flasks, and removed all cell suspension to a 15 ml centrifuge tube, spin down ~300g x 3min (do not try to blow to remove the remaining cells sticking to the bottom).

6. Discard supernatant, add 1ml 1:4 ( PBSC : PBS), gently triturate to resuspend cell, centrifuge 2000φm x lmin in an micro centrifuge tube.

7. Discard the supernatant, add 800μl to 1ml 1:4 (PBSC * : PBS) , triturate, and pass through 15-20 micron filter into non-stick plate.

Part V. Protocol for Isolation of HEK

1. Use HEK-Na cells at 70- 80% confluences in T-75 flasks (16 hrs after seeding).

2. Remove medium and wash x 2 with

PBS 3. Add 6 ml X⁺⁺-free PBS, incubate at 37°C for 5 mins, aspirate supernatant

4. Add 6 ml X⁺⁺-free PBS, incubate at 37°C for 10 mins or until all cells dissociate from flask.

5. Add 2 ml Accumax directly into flask to finalize the Accumax concentration to 1 :4, incubate cell at 37°C for 4 mins 6. Add 6 ml volume of

DMEM with 10% FCS into the flasks to stop the digestion

7. Put cell mixture into a 15 ml tube, and spin down 300g x 3min

8. Discard supernatant, gently suspend cell in 4 ml Ca^4"1" free DMEM with 10% FCS, incubate cell at 37°C incubator at least 30 mins or until use it. 9. Carefully remove the supernatant, wash xl with PBS with lOOnM Cacl₂, ImM

Mgcl₂ 10. Triturate, resuspend cell in PBS with lOOnM Cacl₂, ImM Mgcl₂, filter cell mixture through 21 μm filter into non-stick plate. Example 7. Program Logic and Pressure control profile

The following is a typical program logic for software pneumatic control. It includes procedures for cell landing, form seal, break-in, and Ra control.

#start of program Count=0

Turn off compensations

Procedure Landing:

Reset button_pressed Label window "Attempting Landing"

Run washer # deliver clean ES to top chamber Wait 5 seconds Stop washer Repeat twice:

Apply -300torr pressure # clear holes of any remaining debris after filling

Wait 0.5 seconds

Apply Otorr pressure

Wait 2 seconds End repeat

Zero junction potential Wait for stable reading

Record average Re value Save Re to logs

Initiate cell addition

Wait until 0.5 seconds before cell delivery # before pipette touches ES Apply +1 Otorr # before and during delivery Wait for pipette removal # from ES chamber

Apply 0 torr Wait 3 seconds Apply -50torr

Wait until Seal > 2Re for 0.5sec or elapsed=15 seconds If elapsed then

Count=count+1

If count >= 3 then abort test and write to log Apply +50torr Run proc Landing Endif

Run FormSeal End procedure

Reset elapsed

Procedure FormSeal

Reset button_pressed

Label window "Attempting Seal" Apply -80mV HP #negative holding immediately after landing

Apply -50torr #this may not necessarily be the same as that used for landing While Seal increasing >20MOhms/second

Wait until Seal >= 1Gohm or elapsed=10 seconds Endwhile Apply Otorr Wait 2 seconds

While seal increasing >20MOhms/second and seal<1 GOhm, Wait 1 second

Endwhile

#start ramping to attempt seal

Unless seal>1 GOhm, Apply ramp from Otorr to -50torr over 20 seconds Unless seal>1 GOhm, Wait 5 seconds

Unless sea GOhm, Apply Otorr Unless seaMGOhm, wait 5 seconds

Unless sea GOhm, Apply ramp from -30torr to -80torr over 30 seconds Unless seaM GOhm, Wait 5 seconds

Unless seaMGOhm, Apply Otorr Unless seaM GOhm, wait 5 seconds

Unless seaM GOhm, Apply ramp from -50torr to -1 OOtorr over 40 seconds Unless seaM GOhm, Wait 5 seconds

Unless seaMGOhm, Apply Otorr Unless seaMGOhm, wait 5 seconds

Unless seaM GOhm, Apply ramp from Otorr to -200torr over 120 seconds Unless seaM GOhm, Wait 5 seconds

Unless seaMGOhm, Apply Otorr Unless seaMGOhm, wait 5 seconds

If not seaMGOhm Check button_pressed

If button_pressed = "continue" then abort test and write to log

Run FormSeal Endif #Seal detected, now check stability

Stop ramping and hold last pressure Wait 1 second # let seal stabilize If seaM GOhm, Apply Otorr Record Seal value into Rseal, save to logs

Unless Seal<(Rseal-200MOhms) or Seal decreasing >200MOhms/second

Wait 5 seconds End unless

If Seal<(Rseal-200MOhms) or Seal decreasing >200MOhms/second Check button_pressed

If button_pressed = "continue", goto Procedure Breakln Run FormSeal Endif

#cell sealed Endif

End Procedure

Procedure Breakln:

Reset button_pressed Label window "Attempting break-in" Null chamber capacitance

Until capacitance > 3.5pF or Pressure>300torr or Seal<(Rseal-200MOhms) or Seal decreasing >200MOhms/second

Wait 1 second

Apply -20 delta torr End until If capacitance > 3.5pF

Record break-in pressure value

Wait 0.5 seconds

Apply Otorr

Run procedure RaControl Endif

If Pressure>300torr Apply Otorr

Wait 1 second Apply -20 delta torr Apply Zap End until If pressure>300torr then abort test and write to log

Endif

If capacitance > 3.5pF

Record break-in pressure value Wait 0.5 seconds

Apply Otorr

Run procedure RaControl Endif If Seal<(Rseal-200MOhms) or Seal decreasing >200MOhms/second

Check button_pressed

If button_pressed = "continue", goto Procedure Breakln Run FormSeal Endif

End Procedure

Elapsed = 0 Procedure RaControl: Reset button_pressed

Label window "Adjusting seal quality"

Record Cm, Rm, Ra to logs

Assign Rmlnitial = Rm, Ralnitial = Ra If Ra < Raldeal then end #Raldeal does not need adjustment

If Ra < RaMax and Ra decreasing then end #no need for adjustment If Ra < RaMax then countdown = 20 seconds else countdown = "true"

While countdown Check button_pressed If button_pressed = "continue" then end

If Ra increasing and Rm > 300MOhms Apply -50torr Wait O.δseconds # max 2 seconds

Apply Otorr Wait 1.5 seconds Endif If Ra increasing and Rm > 500MOhms

Apply -δOtorr

Wait O.δseconds # max 2 seconds

Apply Otorr

Wait 1.5 seconds Endif

If Rm>0.8GOhm then apply -50torr else apply -1 Otorr

While Ra>Raldeal and Rm>(Rmlnitial-25%) and countdown Unless Ra<Raldeal or Rm<(Rmlnitial-2δ%), wait δ seconds

If Ra<RaMax then countdown=20 seconds If Ra<Raldeal then Endwhile If Ra not decreasing

If Rm not decreasing and Rnr GOhm then Apply -10 delta torr If Rm not decreasing and Rm<1GOhm then Apply -δ delta torr

If Rm decreasing and Pressure>1 Otorr then Apply +δ delta torr If Rm<(Rmlnitial-2δ%) then apply 0 torr Endif

If pressure>BreaklnPressure then apply Otorr If elapsed > 120 seconds then apply Otorr and end

If Rm<300MOhms then apply (reaklnPressure-1 Otorr) Endwhile

If -10torr>pressure>-δ0torr Apply Otorr If Ra increasing then apply -60torr

If Ra increasing then run RaControl Procedure Endif Endwhile End Procedure

Example 8. Achieving High Resistance Seals in 52-Cell Test An operator using a syringe based pressure system employed a pressure control profile similar to that described in Example 7, except that it was performed manually rather than by computer automation. The 52-cell test described in Example 2 was performed using a syringe controlled by had while the operator viewed a pressure monitor. The criteria for the test was the achievement of at least 75% success rate, with success defined as achieving a gigaohm seal to initiate a patch clamp, then during the patch clamp membrane maintaining resistance above 200 MOhms and maintaining access resistance (or series resistance) below 15 MOhms for at least 15 minutes.

Table

Table 2: 50-cell test that demonstrates the feasibility of the pressure control protocol.

demonstrates the conclusion from this experiment, showing that the goals ofthe 52- cell test were met.

Figures 23-25 give a sample ofthe time-course of an experiment where membrane resistance and access resistance values are kept within the acceptable parameters. At many locations in the recording there are deflections in the access resistance trace (Figure 25). These deflections represent locations where the pressure protocol was applied to maintain the seal quality parameters. The success rate at achieving gigaohm seals is demonstrated in Figure 20. This data is a graphical representation ofthe data identified in Table 2, where 90% ofthe chips produced a gigaohm seal with CHO cells. Figure 22 shows a histogram ofthe parameters achievable with this pressure confrol protocol. Data shown with wide diagonal bars represents initial values for Ra and Rm, and values with narrow diagonal bars represent values for Ra and Rm after 15 minutes of continuous whole-cell access under voltage clamp conditions. These data demonstrated that overall, 75% ofthe cells achieved gigaohm seals, and then whole-cell access was attained with acceptable parameters that were well-controlled for at least 15 minutes. Example 9. Single channel recording using a biochip comprising a hole for ion transport measurement.

RBL cells were prepared for patch clamp recording by simple centrifugation.

The cells were then delivered onto an ion transport measurement device with a single recording aperture. The biochip device was assembled according to Example 2. The biochip had been freated with acid and base to improve sealability. The upper chamber solution was PBS lacking calcium and magnesium. The lower chamber solution was:

150 mM KCl, 10 mM HEPES-K, 1 mM EGTA-Na, ImM ATP-Mg pH (KOH) 7.4, the upper chamber solution was : 8 mM NaCl, 20 mM KCl, 1 mM MgCl₂, 10 mM HEPES-Na, 125 mM K-Glu , 10 mM EGTA-K, 1 mM ATP-Mg pH (KOH) 7.2.

Seal formation was achieved as provided in the previous examples, but after gigaseal formation, no break-in step was performed. Single-channel recordings were obtained from a cell-attached membrane patch on an RBL cell. An inward rectifier IRKl single channel was recorded in RBL cells. A low concentration of extracellular K⁺ which does not depolarize the cell and does not inactivate the channel was used. ATP was present in the internal solution, which prevents the rundown ofthe channel activity. The noise level ofthe recordings was reduced from 10 pA to 1 pA in order to observe single channel events, which have an amplitude of a few picoamps.

The devices and methods described herein can be combined to make additional embodiments which are also encompassed in the present invention.

All headings are for the convenience of the reader and should not be used to limit the meaning ofthe text that follows the heading, unless so specified.

All references cited herein, including patents, patent applications, and publications are incoφorated by reference in their entireties.

Claims

What is claimed is:

1. A device for ion transport measurement, comprising:

an upper chamber piece that comprises at least one well, wherein said at least one well is open at its upper and lower ends; and

a chip that comprises at least one ion transport measuring means, wherein said chip has been treated to enhance the electrical sealing properties of said at least one ion transport measuring means;

wherein said chip is attached to the bottom of said upper chamber piece such that each of said at least one ion transport measuring means is in register with one of said at least one well.

2. The device of claim 1, wherein said chip has been freated to make said at least one ion transport measuring means more electronegative.

3. The device of claim 2, wherein at least a portion of said chip has been freated with at least one base.

4. The device of claim 1, wherein said at least one ion transport measuring means is at least one hole through said chip.

5. The device of claim 4, wherein said chip is in direct or indirect contact with said upper chamber piece.

6. The device of claim 1, wherein said chip comprises glass, silicon, silicon dioxide, quartz, one or more plastics, one or more polymers, one or more ceramics, one or more waxes, polydimethylsiloxane (PDMS), or a combination thereof.

7. The device of claim 1, wherein said chip is able to form a seal with a cell or particle, wherein said seal has a resistance (R) of greater than 200 megaOhms.

8. The device of claim 7, wherein said chip is able to form a seal with a cell or particle, wherein said seal has a resistance (R) of greater than 500 MegaOhms.

9. The device of claim 7, wherein electrical access between said chip and an inside of said cell or particle, or between said chip and the outside of said cell or particle in the region of said hole has an access resistance that is less than the seal resistance (R).

10. The device of claim 9, wherein resistance of a seal between said chip and said particle is less than 80 MegaOhms.

11. The device of claim 9, wherein resistance of a seal between said chip and said particle is less than 30 MegaOhms.

12. The device of claim 9, wherein resistance of a seal between said chip and said particle is greater than 10 MegaOhms.

13. The device of claim 1, wherein said chip is attached to the bottom of said upper chamber piece in inverted orientation.

14. The device of claim 1, wherein said upper chamber piece comprises one or more plastics, or more polymers, one or more ceramics, one or more waxes, silicon, or glass.

15. The device of claim 14, wherein said one or more plastics is one or more base- resistant plastics.

16. The device of claim 15, wherein said one or more base resistant plastics is cyclo olefin polymer or polyphenylene ether/PPO or modified polyphenylene oxide.

17. The device of claim 1, wherein said at least one well has an upper diameter of from about 0.05 millimeter to about 20 millimeters.

18. The device of claim 17, wherein said at least one well has an upper diameter of from about 2 millimeter to about 10 millimeters.

19. The device of claim 1, wherein said at least one well has a depth of from about 0.01 millimeter to about 25 millimeters.

20. The device of claim 19, wherein said at least one well has a depth of from about 2 millimeters to about 10 millimeters.

21. The device of claim 1 , wherein said at least one well tapers downward at an angle of from about 0.1 degree to about 89 degrees from vertical.

22. The method of claim 21, wherein said at least one well tapers downward at an angle of from about 5 degrees to about 60 degrees from vertical.

23. The device of claim 1, wherein said upper chamber piece comprises at least one electrode.

24. The device of claim 23, wherein said upper chamber piece comprises one electrode, further wherein said one electrode contacts each of said at least one well.

25. The device of claim 23, wherein said upper chamber piece comprises at least two wells and at least two elecfrodes, wherein each of said at least two electrodes contacts one of said at least two wells.

26. The device of claim 5, wherein said chip is attached to said upper chamber piece with one or more adhesives.

27. The device of claim 5, wherein said chip is attached to said upper chamber piece by pressure mounting.

28. The device of claim 5, further comprising a lower chamber piece attached to the bottom side of said chip that can form at least a portion of at least one lower chamber.

29. The device of claim 22, wherein said lower chamber piece comprises at least one gasket.

30. The device of claim 23, wherein said at least one gasket comprises rubber or an elastomer.

31. The device of claim 24, wherein said at least one gasket comprises one or more of rubber, polydimethylsiloxane (PDMS), silicone polyether urethane, polyester elastomer, polyether ester elastomer, olefinic elastomer, polyurethane elastomer, polyether block amide, stryrenic elastomer, or composite polymers.

32. The device of claim 28, wherein said at least one lower chamber is a flow- through lower chamber.

33. The device of claim 32, wherein said device further comprises a lower chamber base piece comprising at least one inflow conduit and at least one outflow conduit.

34. The ion fransport measuring device of claim 1, wherein said at least one well is at least two wells and said at least one ion transport measuring means is at least two ion transport measuring means.

35. The ion transport measuring device of claim 34, wherein said at least two ion transport measuring means are at least 16 ion transport measuring means, and said at least two wells are at least 16 wells.

36. The device of claim 28, comprising at least one lower chamber.

37. The device of claim 36, wherein each of said at least one lower chamber accesses one of said at least one well via said hole in said biochip.

38. The device of claim 37, wherein each of said at least one well comprises, contacts, or is in electrical communication with at least one electrode, further wherein each of said at least one lower chambers comprises, contacts, or is in electrical communication with at least one electrode.

39. A method of measuring at least one ion transport activity or property of at least one cell or particle, comprising: i) filling at least one lower chamber ofthe device of claim 31 with a measuring solution; ii) adding a suspension of cells or particles to one or more of at least one well ofthe device of claim 1, wherein each ofthe one or more ofthe at least one well is connected to one ofthe at least one lower chambers that comprises measuring solution via a hole in the ion transport measuring chip; iv) applying pressure to said at least one lower chamber to create a high- resistance electrical seal between at least one cell or particle and said at least one hole; and v) measuring at least one ion transport property or activity ofthe at least one cell or at least one particle.

40. The method of claim 39, wherein said at least one cell or at least one particle is at least one cell.

41. A device for ion transport measurement, comprising:

at least one upper chamber piece comprising one or more upper chambers; a biochip comprising at least one ion transport measuring means attached to the lower side of said at least one upper chamber piece; and

at least one flow-through lower chamber, wherein said at least one flow-through lower chamber accesses said at least one ion transport measuring means of said biochip.

42. The device of claim 41, wherein each of said at least one lower chamber is connected to at least two conduits.

43. The device of claim 42, wherein at least one of said at least two conduits is an inflow conduit and at least one of said at least two conduits is an outflow conduit.

44. The device of claim 43, wherein said at least one inflow conduit and said at least one outflow conduit engage the walls of said at least one lower chamber.

45. The device of claim 44, wherein said at least one inflow conduit and said at least one outflow conduit engage the bottom surface of said at least one lower chamber.

46. The device of claim 41 , wherein said at least one lower chamber is formed at least in part by a lower chamber piece.

47. The device of claim 46, wherein said lower chamber piece is reversibly or ineversibly attached to said biochip.

48. The device of claim 46, wherein said at least one lower chamber is formed by a lower chamber piece that forms the walls of said at least one lower chamber and a lower chamber base piece that forms the bottom surface of said at least one lower chamber.

49. The device of claim 48, wherein said lower chamber piece and said lower chamber base piece are reversibly or ineversibly attached to said biochip.

50. The device of claim 49, wherein said lower chamber piece and said lower chamber base piece are reversibly attached to said biochip.

51. The device of claim 50, wherein said lower chamber piece and said lower chamber base piece are attached to said biochip by pressure mounting.

52. The device of claim 48, wherein said lower chamber base piece comprises at least one inflow conduit and at least one outflow conduit.

53. The device of claim 52, wherein said at least one lower chamber is at least two lower chambers and said lower chamber base piece comprises at least two inflow conduits and at least two outflow conduits, wherein each of said at least two lower chambers engages one inflow conduit and one outflow conduit.

54. The device of claim 48, wherein at least one or more parts of said lower chamber base piece that will form the bottom of said at least one lower chamber of an ion transport measuring device is a transparent material.

55. The device of claim 54, wherein said lower chamber base piece comprises glass, polycarbonate, or polystyrene.

56. The device of claim 55, wherein said lower chamber piece can fit a base plate that can be adapted to a microscope stage.

57. The device of claim 56, further comprising a base plate that is adapted to a microscope stage.

58. The device of claim 57, wherein said base plate comprises one or more plastics, one or more polymers, one or more ceramics, or one or more metals.

59. The device of claim 58, wherein said base plate comprises one or more metals.

60. The device of claim 48, wherein said lower chamber base piece comprises at least one electrode.

61. The device of claim 52, wherein at least one electrode is inserted through at least one of said at least one inflow conduit and said at least one outflow conduit.

62. The device of claim 52, further comprising tubing that connects to the outer openings of said of said at least one inflow conduit and said at least one outflow conduit.

63. The device of claim 51, wherein said lower chamber piece comprises a gasket.

64. The device of claim 63, wherein said at least one gasket comprises rubber or an elastomer.

65. The device of claim 64, wherein said at least one gasket comprises one or more of rubber, polydimethylsiloxane (PDMS), silicone polyether urethane, polyester elastomer, polyether ester elastomer, olefinic elastomer, polyurethane elastomer, polyether block amide, stryrenic elastomer, or composite polymers.

66. The device of claim 29, wherein said at least one ion transport measuring means is at least one hole through said biochip.

67. The device of claim 66, wherein at least the upper surface of said chip has been treated with at least one base.

68. The device of claim 41, wherein said at least one upper chamber piece comprises at least one electrode.

69. The device of claim 51, further comprising at least one clamp that reversibly attached said lower chamber piece and said lower chamber base piece to said biochip.

70. The device of claim 69, wherein said clamp comprises at least one electrode that can contact at least one upper chamber.

71. A device for ion transport measurement, comprising: an upper chamber piece comprising two or more upper chambers;

a biochip attached to the lower side of said upper chamber piece, wherein said biochip comprises at least two holes, wherein said at least two holes are in register with said two or more upper chambers;

a lower chamber base piece comprising at least two inflow conduits and at least two outflow conduits;

a gasket comprising at least two openings positioned on top of said lower base piece and below said biochip, such that said gasket forms the walls of at least two lower chambers, wherein each of said at least two lower chambers connects to one of said at least two inflow conduits and one of said at least two outflow conduits, further wherein each of said at least two lower chambers accesses one of said at least two holes of said biochip.

72. The device of claim 71, wherein either each of said at least two outflow conduits or each of said at least two inflow comprises an electrode.

73. The device of claim 72, wherein said electrode is a recording elecfrode.

74. The device of claim 72, wherein said at least two upper chambers is sixteen upper chambers, further wherein said at least two holes through said biochip are sixteen holes through said biochip; wherein said sixteen upper chambers are in register with said sixteen holes, further wherein said lower chamber piece comprises sixteen inflow conduits and sixteen outflow conduits; wherein said gasket comprises sixteen openings and can form sixteen lower chambers that align with said sixteen holes when said gasket is positioned on top of said lower base piece; wherein each of said sixteen lower chambers connects to one of said sixteen inflow conduits and one of said sixteen outflow conduits.

75. The device of claim 74, wherein said two or more upper chambers comprise, contact, or are in electrical contact with at least one electrode.

76. A method of measuring at least one ion transport activity or property, comprising:

i) filling at least one lower chamber ofthe device of claim 75 with a measuring solution; ii) adding a suspension of cells or particles to one or more of at least one upper chamber ofthe device of claim 75, wherein each ofthe one or more of at least one upper chamber is connected to one ofthe at least one lower chambers that comprises measuring solution via a hole in the ion transport measuring chip; iv) applying pressure to said at least one lower chamber to create a high- resistance electrical seal between at least one cell or particle and said at least one hole; and v) measuring at least one ion transport property or activity ofthe at least one cell or at least one particle.

77. The method of claim 76, wherein said at least one cell or at least one particle is at least one cell.

78. A method of making an upper chamber piece of a device for ion transport measurement, comprising: molding an upper well portion piece of an upper chamber piece that comprises at least one upper well;

injection molding a well hole portion piece of an upper chamber piece of an ion transport measuring device, wherein said well hole portion piece comprises: at least one well hole and a groove that extends longitudinally from one end of the well hole portion piece toward the opposite end of the well hole portion piece, wherein said groove contacts said at least one well hole;

inserting a wire electrode into the groove ofthe well hole portion piece; and

attaching the upper well portion piece to the well hole portion piece to form an upper chamber piece that comprises one or more wells, such that the wire elecfrode is exposed to the interior of said one or more wells.

79. The method of claim 78, wherein said injection molding uses a moldable plastic.

80. The method of claim 79, wherein said moldable plastic comprises polytetrafluorethylene, polyallomer, polyethylene, polyimide, polypropylene, polystyrene, polycarbonate, cylco olefin polymer, polyphenylene ether/PPO, modified polyphenylene oxide, or composite polymers.

81. The method of claim 78, wherein said upper well portion piece comprises at least two upper wells and said well hole portion piece comprises at least two well holes.

82. An upper well piece made by the method of claim 78.

83. A method of making an upper chamber piece of a device for ion transport measurement, comprising: positioning a wire electrode in a mold for an upper well piece that comprises one or more wells;

injection molding an upper chamber piece using said mold such that said wire electrode contacts the interior of said one or more wells of said upper chamber piece.

84. An upper well piece made by the method of claim 83.

85. A method of making a biochip comprising holes for ion transport measurement, comprising: providing a substrate;

laser drilling at least one counterbore in said substrate; and

laser drilling at least one through-hole through said at least one counterbore.

86. The method of claim 85, wherein said subsfrate comprises glass, quartz, silicon, silicon dioxide, PDMS, or a polymer.

87. The method of claim 88, wherein said substrate comprises glass.

88. The method of claim 85, wherein said subsfrate is from about 5 microns to about 1 millimeter thick.

89. The method of claim 88, wherein said substrate is from about 10 microns to about 200 microns thick.

90. The method of claim 85, wherein the thickness of said substrate is measured with a laser prior to drilling said at least one counterbore.

91. The method of claim 85, wherein said at least one counterbore is at least two counterbores, wherein said at least two counterbores are nested.

92. The method of claim 91 , wherein the first drilled counterbore of said at least two counterbores has an entrance diameter of from about 20 microns to about 200 microns.

93. The method of claim 92, wherein said first drilled counterbore has an enfrance diameter of from about 40 microns to about 120 microns.

94. The method of claim 91, wherein said first drilled counterbore is drilled to a depth of about the thickness of the substrate minus the depth of a subsequent counterbore plus the through- hole depth.

95. The method of claim 91, wherein the thickness of said substrate is measured with a laser prior to drilling each of said at least two counterbores.

96. The method of claim 91, wherein said through-hole has a depth of from about 0.5 microns to about 200 microns.

97. The method of claim 96, wherein said through-hole has a depth of from about 5 microns to about 30 microns.

98. The method of claim 91, wherein at least one of said at least two counterbores are tapered.

99. The method of claim 91, wherein said at least one through-hole is tapered.

100. A chip made by the method of claim 85.

101. The chip of claim 100, wherein said chip has been treated to make said at least one ion fransport measuring means more electronegative.

102. The device of claim 100, wherein said chip has been treated with at least one base.

103. A chip made by the method of claim 91.

104. The chip of claim 103, wherein said chip has been freated to make said at least one ion transport measuring means more electronegative.

105. The chip of claim 103, wherein said chip has been treated with at least one base.

106. A device for ion transport measurement, comprising:

at least one upper chamber piece comprising at least one upper chamber; and at least one chip attached to said at least one upper chamber piece, wherein said at least one chip comprises at least one laser drilled ion transport measuring hole, wherein the side ofthe chip having the one or more laser entrance hole openings is exposed to said at least one upper chamber.

107. The device of claim 106, wherem said at least one ion fransport measuring hole comprises a through-hole and at least one counterbore, wherein said at least one chip is attached to said at least one upper chamber piece in inverted orientation.

108. The device of claim 10, wherein said at least one through-hole that has been drilled from the same direction as said at least one counterbore.

109. The device of claim 107, wherein said at least one through-hole that has been drilled from the opposite direction as said at least one counterbore.

110. The device of claim 107, wherein said at least one counterbore is tapered.

111. The device of claim 107, wherein said at least one through-hole is tapered.

112. A method of treating a chip that comprises at least one ion transport measuring means to enhance its electrical sealing properties, comprising: incubating said chip in a solution of acid; washing said chip in deionized water; incubating said chip in a solution of base; and storing said chip in deionized water until use.

113. The method of claim 112, further comprising laser polishing said chip prior to said incubating said chip in said solution of acid.

114. The method of clam 112, wherein said chip comprises glass, quartz, silicon, silicon dioxide, or a polymer.

115. The method of claim 114, wherein said chip comprises glass.

116. A chip treated by the method of claim 112.

117. A method of storing the chip of claim 116, comprising: maintaining said chip in water or an aqueous solution having a pH greater than about 6.

118. The method of claim 117, wherein said pH is greater than 7.

119. A method of storing the chip of claim 117, comprising maintaining said chip in a dry environment.

120. The method of claim 119, wherein said dry environment comprises a dessicant.

121. The method of claim 119, wherein said chip is incubated in an aqueous solution before use.

122. The method of claim 121, wherein said aqueous solution is a basic solution, a salt solution, or water.

123. A method of shipping the chip of claim 116, comprising maintaining said chip in conditions of low CO₂ or air.

124. The method of claim 123, wherein said chip is submerged in water, alcohol, buffered solutions, salt solutions, or under nitrogen or one or more inert gases.

125. The method of claim 123, wherein said chip is shipped as part of a device for ion transport measurement.

126. The method of claim 125, wherein said devices are shipped in containers comprising water, alcohol, buffered solutions, or salt solutions.

127. The method of claim 126, wherein said containers are blister packs.

128. A method for determining the hydrophilicity of a surface, comprising: dispensing a drop of defined volume of water or an aqueous solution on a surface; measuring the time it takes for said drop to evaporate; calculating a surface energy ofthe surface based on the evaporation time; and using said surface energy as a measure ofthe hydrophilicity of said chip.

129. The method of claim 128, wherein said evaporation is monitored by diffraction, reflectance, or interference at the surface where the drop is deposited.

130. The method of claim 128, wherein said evaporation is monitored by measuring the intensity or color change of a dye that has been used to color the solution.

131. A method of manufacturing chips for ion fransport measurement devices, comprising: fabricating multiple rows of ion fransport measuring holes on a sheet of glass, wherein said multiple rows of ion fransport measuring holes are separated by mark lines formed by laser scoring; and breaking the chip into discrete segments that comprise a subset ofthe total number of said ion fransport measuring holes.

132. The method of claim 131, wherein said ion transport measuring holes are laser fabricated.

133. The method of claim 132, wherein said sheet of glass has been chemically treated to improve the electrical sealing properties of said ion fransport measuring holes.

134. The method of claim 131, wherein said mark lines are continuous slashes that go through said glass sheet to a depth of about 30% or more ofthe thickness of said glass sheet.

135. The method of claim 131, wherein an inj ection molded multi-unit well plate is bonded to said glass sheet with adhesives to form a multi-unit welled sheet so that each well of said plate is in register with one of the ion transport recording holes prior to detaching sections of said multi-unit welled sheet.

136. A method of making a high density chip for ion transport measurement, comprising: providing a silicon, glass, or silicon-on-insulator (SOI) wafer, wet-etching a multiplicity of wells in said wafer; and laser drilling through-holes through said multiplicity of wells.

137. The method of claim 136, wherein said multiplicity of wells is at least 24 wells.

138. The method of claim 137, wherein said multiplicity of wells is at least 48 wells.

139. The method of claim 138, wherein said multiplicity of wells is at least 96 wells.

140. The method of claim 136, wherein said wafer ranges from about 0.1 micron to 10 millimeters in thickness.

141. The method of claim 140, wherein said wafer ranges from about 0.5 micron to 2 millimeters in thickness.

142. The method of claim 135, wherein said wafer is a silicon-on-insulator (SOI) wafer.

143. A high density chip made by the method of claim 135.

144. A fluidic channel ion transport measuring device, comprising: a planar chip that comprises one or more ion fransport measuring holes; at least one upper fluidic channel chamber; and at least one lower fluidic channel chamber, wherein: apertures are positioned in the fluidic channels such that at least one ion transport measuring hole in said chip has access to said upper fluidic channel chamber and to said lower fluidic channel chamber.

145. The fluidic channel ion transport measuring device of claim 144, wherein said chip comprises multiple ion transport measuring holes, wherein each ofthe holes can be in fluid communication with an upper fluidic channel and a lower fluidic channel.

146. The fluidic channel ion transport measuring device of claim 145, wherein said upper fluidic channels are connected with one another, and said lower fluidic channels are independent.

147. The fluidic channel ion transport measuring device of claim 145, wherein said upper fluidic channels are independent and said lower fluidic channels are connected with one another.

148. The fluidic channel ion transport measuring device of claim 145, wherein said upper fluidic channels that service different ion transport measuring holes can be separate from one another and the lower fluidic channels that service different ion transport measuring holes are separate from one another.

149. The fluidic channel ion transport measuring device of claim 150, wherein at least one fluidic pump drives the flow of fluids through said upper fluidic channels and at least one pump drives the flow of fluids through said lower fluidic channels.

150. The fluidic channel ion transport measuring device of claim 145, wherein electrodes external to the fluidic patch clamp chip are connected via a electrolyte solution bridge to top fluidic channels, bottom fluidic channels or to both top and bottom fluidic channels.

151. The fluidic channel ion fransport measuring device of claim 145, wherein a pressure source can generate both positive and negative pressures is linked to the lower fluidic channels.

152. A method of preparing cells for ion fransport measurement, comprising: providing a population of attached cells; releasing the attached cells using a divalent cation solution, an enzyme- containing solution, or a combination thereof; washing the cells with a buffered cell-compatible salt solution; and filtering the cells to produce suspension cells that give high quality patch clamp recordings using ion fransport measuring chips.

153. The method of claim 152, wherein said method uses a divalent cation solution.

154. The method of claim 153, wherein said method uses an enzyme-containing 5 solution.

155. The method of claim 152, wherein said method uses a filter that allows the passage of single cells.

156. The method of claim 152, wherein said method uses a filter has a pore size of from about 15 to 30 microns.

157. A program logic providing a protocol for providing feedback confrol of pressure applied to an ion transport measuring means of an ion transport measuring apparatus, comprising: steps that direct the production of positive pressure; steps that direct the production of negative pressure; steps that direct the sensing of pressure; and steps that direct the application of negative pressure in response to sensed pressure in the form of multiple multi-layer if-then and loop logic, in which the positive and negative pressure produced is generated through tubing that is in fluid communication with an ion transport measuring means of an apparatus, and in which negative pressure is sensed through tubing that is in fluid communication with an ion fransport measuring means of an apparatus.

158. The program logic of claim 157, wherein said steps are performed in a defined order that depends on the feedback the apparatus receives.

159. A program logic wherein said pressure is produced by at least one pump that is part of said apparatus.

160. A program logic according to claim 157 further wherein said protocol directs the rapture or said cell by the application of pressure to achieve whole cell access.

161. The device of claim 28, wherein each of said at least two wells comprises, contacts, or is in electrical communication with at least one electrode, further wherein each of said at least one lower chambers comprises, contacts, or is in electrical communication with at least one electrode.

162. A method of measuring at least one ion transport activity or property, comprising: i) filling at least one lower chamber ofthe device of claim 161 with a measuring solution; ii) adding a one or more cells or particles to one or more of at least one well ofthe device, wherein each ofthe one or more of the at least one well is connected to one ofthe at least one lower chambers that comprises measuring solution via a hole in the ion fransport measuring chip; iv) applying pressure to said at least one lower chamber or at least one well to create a high- resistance electrical seal between at least one cell or particle and said at least one hole; and v) measuring at least one ion fransport property or activity ofthe at least one cell.

163. The method of claim 162, wherein said at least one cell or at least one particle is at least one cell.

164. The method of claim 162, wherein said applying pressure to said at least one lower chamber or at least one well can be under automated control.