Note: Descriptions are shown in the official language in which they were submitted.
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HIGH-DENSITY ION TRANSPORT MEASUREMENT
BIOCI~IP DEVICES AND METHODS
This application claims benefit of priority to United States utility patent
application number 10/858,339 (pending), filed June 1, 2004, and claims
benefit of
priority to United States patent application number 10/760,866 (pending),
filed January
20, 2004. This application also claims benefit of priority to United States
patent
application number 60/535,461 filed 3anuary 10, 2004 and to United States
patent
application number 601585,822 filed July 6, 2004. Each and every patent and
patent
application referred to in this paragraph is hereby incorporated by reference
herein in its
entirety.
This application also incorporates by reference United States provisional
application number 60/474,508, filed May 31, 2003 (expired); United States
provisional
patent application number 601380,007, filed May 4, 2002 (expired); United
States
provisional patent application number 60/351,849 filed January 24, 2002
(expired);
United States provisional patent application number 60/311,327 filed August
10, 2001
(expired); and to United States provisional patent application number
60/278,308 filed
March 24, 2001 (expired). This application also incorporates by reference
United States
patent application number 10/48,565, filed May 2, 2003 (abandoned), to United
States
patent application number 10/642,014, filed August 16, 2003 (pending), to
United States
patent application number 101351,019, filed January 23, 2003 (abandoned), and
to United
States patent application number 10/104,300, filed March 22, 2002 (pending).
TECHNICAL FIELD
The present invention relates generally to the field of ion transport
detection
("patch clamp") devices, systems and methods, particularly those use of
biochip
technologies.
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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 tuning
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 of the 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-Glamp Applications and Protocols,
Neuromethods
V. 26 (1995), Humana Press, New Jersey).
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These and later methods relied upon interrogating one sample at a time using
large laboratory apparatus 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 of the membrane, such as fluidity and
polarizability.
Thus, what is needed is a simple device and methods to measure ion transport
activities directly. Preferably, these devices would utilize patch clamp
detection methods
because these types of methods repres ent a gold standard in this field of
study. The
present invention provides 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 prop erties.
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-clamping,
whole cell recording, or single channel recording, are preferable to methods
that utilize
indirect detection methods, such as fluorescence-based detection systems.
The present invention provides biochips for ion transport measurement, ion
transport measuring devices that comprise biochips, fluidics designs for ion
transport
measuring devices that comprise biochips, and methods of using the devices and
biochips
that allow for the direct analysis of iorz transport functions or properties.
The present
invention provides biochips, devices, apparatuses, and methods that are
particularly
suited to high throughput ion transport measurement assays. The present
invention
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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 devic es are particularly appropriate
for
automating the detection of ion transport functi ons or properties,
particularly for
screening purposes.
A first aspect of the present invention is a biochip that comprises at least
one ion
transport measuring means in the form of a hole through the biochip, in which
at least a
portion of the surface of the particle-sealing side of the biochip is
hydrophobic.
Preferably, a hydrophilic biochip of the present invention comprises a
hydrophilic surface
that surrounds the one or more ion transport measurir~g holes at the recording
site area,
and a hydrophobic surface that in turn surrounds the one or more recording
site areas. In
some preferred embodiments of this aspect of the invention, the ion transport
measuring
holes have counterbores that are microwell upper chambers, where the surface
of the
biochip has a hydrophobic surface exclusive of the mi crowells, which have a
hydrophilic
surface.
A second aspect of the present invention is a biochip for ion transport
measurement that comprises a microchannel plate (MCP). An MCP ion transport
measurement chip comprises at least two ion transport measuring holes in the
form of
microchannels through the MCP. Preferably, an ion transport measurement MCP
also
comprises microwells in the form of counterbores surrounding the two or more
microchannels. Preferably, at least a portion of an ion transport measurement
MGP is
treated to increase the electrical sealing properties of the two or more ion
transport
measuring holes. One or more portions of an MCP ion transport measuring chip
can
optionally be coated with a hydrophobic material -to prevent fluid contact
between
microwells or holes. The present invention includes methods of making MCP ion
transport measuring chips. The present invention also comprises ion transport
measuring
devices that comprise at least one MCP ion transport measuring chip, and
methods of
using devices that comprise at least one MCP ion transport measuring chip for
measuring
one or more ion transport activities or properties of at least one particle.
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A third aspect of the present invention is a flexible ion transport
measurement
biochip that comprises at least two ion transport measuring means in the form
of holes
through the flexible biochip. Preferably, a flexible ion transport measurement
biochip
comprises at least one flexible material that can be at least partially coated
with silicon
dioxide or glass. At least a portion flexible ion transport measuring chip of
the present
invention can be treated to improve its electrical sealing properties. One or
more portions
of the surface of a flexible ion transport measuring chip can be coated with a
hydrophobic
material to prevent fluid contact between ion transport measuring holes. A
flexible ion
transport measuring chip of the present invention can optionally be stored on,
supported
by, or dispensed from, one or more spools or one or more guide structures. The
present
invention includes devices that include flexible ion transport measurement
biochips and
methods of using such devices for measuring one or more ion transport
activities or
properties.
A fourth aspect of the present invention is a method of making an ion
transport
measurement device using theta tubing segments. The method comprises drilling
holes in
the septa of two or more theta tubing segments and then fusing the two or more
theta
tubing segments to produce an ion transport measurement device that comprises
at least
two ion transport measurement means in the form of holes through the septa of
the tubing
segments. In one embodiment, the theta tubing segments are fused one on top of
another.
In an alternative embodiment, the theta tubing segments axe fused side-by-
side. In the
theta tubing-based devices of the present invention, upper and lower
compartments of the
theta tubing segments provide upper and lower chambers far ion transport
measurement
assays. In preferred embodiments, inflow and outflow conduits are attached to
the
openings on either side of the upper and lower compartmerits of each theta
segment. At
least a portion of the surface of the septum of a theta segment of an ion
transport
measurement device of the present invention can be treated to improve the
electrical
sealing properties of the ion transport measuring hole of the septum. The
present
invention includes theta tubing-based ion transport measuring devices made
using the
methods of the present invention, and methods of using such devices to measure
one or
more ion transport activities or properties of one or more particles.
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A fifth aspect of the present invention is an ion transport measurement device
that
comprises a biochip that comprises two or more ion transport measuring holes,
a common
upper chamber, and an upper chamber separator unit, wherein the upper chamber
separator unit comprises separator segments that can form the walls of
individual upper
S chamber compartments when the unit is lowered onto the top of the biochip to
divide the
common upper chamber into at least two upper chamber compartmeatzts, each of
which is
in register with one of the two or more ion transport measuring holes. The
present
invention includes methods of using ion transport measurement devices having a
biochip
and multiple upper chambers formed by an upper chamber separator unit to
measure one
or more ion transport activities or properties.
A sixth aspect of the present invention is an ion transport measurement device
that comprises a biochip that comprises two or more ion transport measuring
holes, and at
least two upper chambers, where the walls of the chamber are fabricated onto
the biochip
and comprise wax, a polymer, or an O-ring. The present invention comprises
devices for
ion transport measurement that comprises a chip having built-on upper
chambers, and
methods of using these devices for ion transport measurement assays.
A seventh aspect of the invention is an ion transport nneasurement device
comprising a biochip that comprises at least one ion transport measuring hole
and at least
one flow-through upper chamber that comprises at least one inlet and at least
one outlet.
In some embodiments, a device comprises two or more flow-through upper
chambers,
and the chip comprises two or more ion transport measuring holes, each of
which
accesses a single flow-through upper chamber. In other embodiments, the chip
comprises
two or more ion transport measuring holes that access a single flow-through
upper
chamber. A flow-through chamber can be arranged as a channel having an inlet
at one
end, two or more ion transport measuring holes positioned in a linear fashion
along the
course of the channel, and an outlet at the opposite end. In some preferred
embodiments,
a flow-through chamber can have a top that is transparent, such that particles
(such as
cells) in the chamber can be viewed microscopically. A device of the present
invention
having one or more flow-through upper chambers can further comprise one or
more
lower chambers. The present invention also includes the use of ion transport
measuring
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devices having flow-through upper channels to measure one or more ion
transport
activities or properties.
An eighth aspect of the present invention is an ion transport measuring devic
a
comprising a biochip that comprises at least two ion transport measuring holes
and at
least one flow-through upper chamber positioned above the biochip, and
furthe:~r
comprising at least two delivery conduits that can be positioned over the ion
transport
hole recording sites to deliver liquid samples, suspensions, or solutions to
ion transport
recording sites. In preferred embodiments, the upper chamber comprises
microwells
which encompass the ion transport recording sites. In preferred embodiments,
the upper
surface of the chip comprises flow retarding structures that restrict the flow
of fluids to
the recording sites. In some preferred embodiments of this aspect, the
conduits comprise
multichannel pipets that can deliver solutions to a recording site. In some
preferred
embodiments of this aspect, the conduits comprise fluidic pipes that can
deliver solutions
to a recording site. In some preferred embodiments, the fluid conduits
comprise funnel
structures, in which solutions are delivered from the tip of the funnel and
the funnel
structure acts as a flow retarding structure. In some preferred embodiments,
at least a
portion of biochip, excluding the recording sites, is hydrophobic. Devices of
the present
invention having a flow-through upper chamber with overhead fluid delivery to
multiple
recording sites can also include two or more lower chambers, where each of the
lower
chambers is in register with one of the ion transport measuring holes of the
chip. The
present invention also includes methods of using ion transport measurement
devices
having an upper chamber fluid conduit delivery system to measure one or more
ion
transport activities or properties.
A ninth aspect of the present invention is an ion transport measuring device
that
comprises 1) a biochip that comprises two or more ion transport measuring
holes, 2) at
least two upper chambers positioned above the chip where the two or more upper
chambers are in register with the two or more ion transport measuring holes,
3) a lower
chamber, 4) at least two microwells on the lower surface of the chip, in which
each of the
two or more microwells is positioned around one of the two or more ion
transport
measuring hales and connected to the lower chamber, and 5) a compound delivery
plate,
in which the compound delivery plate has two or more drug delivery sites that
can align
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with the two or more microwells of the chip. The compound delivery plate can
be
reversibly positioned under the biochip such that the two or more compound
delivery
sites are in close proximity to the two or more microwells to deliver
compounds to the
microwells. In one embodiment, the compound delivery sites are loci where
compounds
can be spotted or printed. In another embodiment, the compound delivery sites
are
apertures in the compound delivery plate through which drugs can be pumped,
inj ected,
or extruded using sonic piezo elements. Preferably, the two or more upper
chambers of
the device are connected to pneumatic devices that can be used to seal cells
in the
microwells to the underside of the chip. In some preferred embodiments, at
least a p ortion
of the lower surface of the chip that is outside of the microwells is
hydrophobic. The
present invention includes methods of using ion transport measurement devices
having
compound delivery plates for compound delivery to one or more recording sites
of .a chip
to measure one or more ion transport activities or properties.
In a related aspect, the present invention comprises a device that comprises:
1) a
biochip that comprises two or more ion transport measuring holes, 2) at least
two lower
chambers positioned below the chip where the two or more lower chambers are in
register with the two or more ion transport measuring holes, 3) an upper
chamber 4) at
least two rnicrowells on the upper surface of the chip, in which each of the
two or more
microwells is positioned around one of the two or more ion transport measuring
holcs and
connected to the upper chamber, and 5) a compound delivery plate, in which the
compound delivery plate has two or more drug delivery sites that can align
with the two
or more microwells of the chip. The compound delivery plate can be reve=rsibly
positioned over the biochip such that the two or more compound delivery sites
are in
close proximity to the two or more microwells to deliver compounds to the
micro~wells.
In one embodiment, the compound delivery sites are loci where compounds can be
spotted or printed. In another embodiment, the compound delivery sites are
apertures in
the compound delivery plate through which drugs can be pumped, pipeted, or inj
ccted.
Preferably, the two or more lower chambers of the device are connected to
pnewxnatic
devices that can be used to seal cells in the rnicrowells to the chip. In some
preferred
embodiments, at least a portion of the upper surface of the chip that is
outside of the
microwells is hydrophobic. The present invention includes methods of using ion
transport
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measurement devices having compound delivery plates for compound delivery to
one or
more recording sites of a chip to measure one or more ion transport activities
or
properties.
An eleventh aspect of the present invention is a method of shipping ion
transport
devices that comprise biochips and at least one upper chamber or at least one
lower
chamber, in which at least one upper chamber or at least one lower chamber of
the device
is filled with a measuring solution and then packaged and shipped.
An twelfth aspect of the present invention comprises a method of performing
excised patch ion transport measurement comprising: sealing a cell to an ion
transport
measuring hole in a chamber of an ion transport measuring device; adding
magnetic
beads to the chamber comprising the cell, in which the magnetic beads have
been coated
with at least one specific binding member that binds one or more molecules
present on
the surface of the cell; incubating the coated magnetic beads with the cell in
the chamber;
applying a magnet to the cell to remove the magnetic beads and a portion of
the cell from
the ion transport measuring site to leave an excised patch at the ion
transport measuring
site; and measuring ion transport activity of the excised patch.
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$RIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a cross-sectional view of a portion of a chip having a
hydrophobic
coating and microwells.
Figure 2 depicts one embodiment of an ion transport measuring chip made from
an
MCP. A) Top view. B) cross-sectional view showing etched microwells and
through-
holes.
Figure 3 depicts two embodiments of a flexible chip of the present invention.
A) the chip
extends between two spools, with the assay area localized to the extended
portion of the
chip between them. B) the assay area of the chip corresponds to a portion chip
that curves
over a spool, which can comprise or engage chambers fox ion transport assays.
Figure 4 depicts preferred embodiments of the present invention: ion channel
measuring
devices that comprises theta tubing. A) a segment of theta tubing shown "face
on" in
which the opening for laser access (used in making the hole) is shown. B) an
ion
transport measuring device comprising multiple theta units arranged
vertically. The upper
and lower chambers of each unit have separate conduit attachments for ES
(extracellular
solution) and IS (intracellular solution), respectively. C) an ion transport
measuring
device comprising multiple theta units arranged side-by-side. Although
conduits
connecting with only one of the units are shown, each of the upper and lower
chambers
of each unit have separate conduit attachments for ES and IS, respectively.
Figure 5 is a cross-sectional view of one embodiment of the present invention
comprising a chip having a flow-through upper chamber.
Figure 6 is a cross-sectional depiction of one embodiment of an ion transport
measuring
device comprising flow-through upper and lower chambers and a reservoir.
Figure 7 depicts one embodiment of an ion transport measuring device having an
upper
chamber separator unit that lowers onto the chip.
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Figure 8 depicts one embodiment of a chip of the present invention in which
wax forms
the upper chambers. A) top view. B) cross sectional view.
Figure 9 depicts a cross-sectional view of one embodiment of a chip of the
present
invention in which O-rings form the upper chambers.
Figure 10 depicts one embodiment of an ion transport measuring device having a
single
flow-through upper chamber in the form of a channel that accesses multiple ion
transport
measuring holes of a chip.
Figure 11 depicts one embodiment of an ion transport measuring device in which
compound is delivered by fluidic pipes at ion transport measuring sites.
Figure 12 depicts a device that has nozzle structures that interface with a
fluid delivery
system.
Figure 13 depicts one embodiment of an ion transport measuring device in which
compound is delivered by fluid dispensing tips at ion transport measuring
sites. In this
embodiment, an electrode traverses the surface of the chip. A hydrophobic
layer coats the
electrode, except in the immediate vicinity of microwells. A) cells have been
added to an
upper chamber channel comprising ES. B) cells seal to ion transport measuring
holes
within microwells that access the channel. C) compound drops are dispensed
directly
over the ion transport measuring sites. D) compound solution floods the
microwell, but
does not flow into neighboring microwells.
Figure 14 depicts an ion transport chip having flow-retarding structures.
Figure 15 depicts one embodiment of an ion transport measuring device having a
compound delivery plate that delivers compounds to ion transport measuring
sites.
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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 incorporated 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 of the membrane or
particle to
measure the current and/or voltage across the portion of the 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
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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 of the 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 of the 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 of the 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 of
the 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 of the
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 of
the 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.
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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,
channels and wells, electrode elements, 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 vary considerably, for example, from about 1 mmz to
about 0.25
m2. Preferably, the size of the chips is from about 4 mm~ to about 25 cm2 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.
A "recording site", "ion transport measurement recording site", or "ion
transport
recording site" is the area is the area immediately surrounding an ion
transport measuring
means (such as a hole). The area can include the bound particle and solution
surrounding
the bound particle. In devices that comprise microwells, the microwell defines
the upper
chamber recording site area.
A "microwell" in a device of the present invention is a well in a chip that
has a
small volumetric capacity. Preferably, the volumetric capacity of a microwell
is less than
about 200 microliters, and more preferably less than about 50 microliters. In
devices of
the present invention, a microwell surrounds an ion transport measuring hole
in a chip
and is prererably drilled or etched into the chip. Preferred microwells are
ion transport
measuring hole counterbores. Microwells preferably contain particles that are
sealed to
the ion transport measurement holes of a chip during use of a device that
comprises
microwells. Microwells can be in the upper or lower surface of a chip.
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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 of the 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 piece" is a part of a device for ion transport measurement
that
forms at least a portion of one or more lower chambers of the device. A lower
chamber
portion 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" is a part of a device for ion transport
measurement
that forms the bottom surface of one or more lower chambers of the 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 of the 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 of the 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.
CA 02554376 2006-07-06
WO 2005/098396 PCT/US2005/000732
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 of the wells of the 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 of the referenced wells or chambers to each of the referenced ion
transport
measuring means, and an ion transport measuring means is positioned so that it
is
exposed to the interior of the well or chamber it is in register with, such
that ion transport
measurement can be performed using the chamber as a compartment for measuring
current 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 from one area to another
area of
a device, apparatus, or system of the present invention or to another
structure, such as a
dispensation or detection device. 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,
16
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WO 2005/098396 PCT/US2005/000732
an upper chamber piece or a lower chamber piece can comprise conduits in the
fornl 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 preferred 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 preferred
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
(ASIGs).
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 axe
in register with the upper chambers of the upper chamber piece.
An "ion transport measuring unit" is a portion of a device that comprises at
least a
portion of a chip having an ion transport measuring means and an upper
chamber, where
the ion transport measuring means connects the upper chamber with a portion of
a lower
chamber.
A "measuring solution" is an aqueous solution containing electrolytes, with
pH,
osmolarity, and other physical-chemical traits that are compatible with
conducting
function of the 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
of the intracellular content of a living cell.
17
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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
of the extracellular content of a living cell.
To be "in electrical contact with" means one component is able to receive and
conduct electrical signals (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.
Viral 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 l~nown in the art. A particle
can also
include artificial membrane preparations that can be made using methods known
in the
art. Preferred 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 8~ 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 of the
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
18
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WO 2005/098396 PCT/US2005/000732
York (192)). 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 v
1
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 through 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
19
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WO 2005/098396 PCT/US2005/000732
example, nylon, polytetrafluoroethylene (TEFLONTM~, 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, Volume: 33,
Pages 159-
1~5 (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 of the 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 of the 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
CA 02554376 2006-07-06
WO 2005/098396 PCT/US2005/000732
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 arranged 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
.~-Y-Z
coordinates that can rotate a particle. Preferred 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
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WO 2005/098396 PCT/US2005/000732
excitation. For example, when N=4 and electrorotation structure corresponds 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 Syvitching 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.
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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 of the 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 i~n 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.
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"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 dielec-trophoretic forces toward
weak field
regions. Whether moieties exhibit positive or negative dielectrophoresis
depends on
whether particles are more or less polarizable than the surrounding 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:
3 2
FDEP - 2~~m~~ xDEP ~-~rms
where Erns is the RMS value of the field strength, the symbol o is the symbol
for
gradient-operation, ~n, is the dielectric permittiW ty of the medium, and
,~DEP is the
particle polarization factor, given by:
*_
~p ~m
oV DEP - Re * * ,
~p -~- ~~m
"Re" refers to the real part of the "complex number". The symbol ~c = s~ - j a-
Y /2~J' is
the complex permittivity (of the particle x=p, and the medium x=m) and j = ~ .
The
parameters ~p and 6p 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
24
CA 02554376 2006-07-06
WO 2005/098396 PCT/US2005/000732
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
3~r 2
FDEP = 29l'~ml' iL DEP V ~ p(x~ Y~ Z)
where p(x,y,z) is the square-field distribution for a unit-voltage excitation
(Voltage V = 1
V) on the electrodes, V 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 of the position of the particle. In
this case the
traveling wave dielectrophoretic force FTW-DEP on a particle of radius r
subjected to a
traveling wave electrical field E = E cos~2~( ft - ~ l ~, o )~Ca r (i. e., a x-
direction field is
traveling along the z-direction) is given, again, under the dipole
approximation, by
_ z
_ 47t ~m 3y 2
FTW-DEP - ~ ~ ~TW-DEPE 'az
0
where E is the magnitude of the field strength, ~"~ is the dielectric
permittivity of the
medium. I~TW-DEP 1S the particle polarization factor, given by
*_
~p ~,n
~TW-DEP - Im * * ,
~p -~- 2~"~
"Im" refers to the imaginary part of the "complex number". The symbol
sx = ~,r - j ~,r /2~f' is the complex permittivity (of the particle x=p, and
the medium
x=m). The parameters ~p and ~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-quardrature 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,
CA 02554376 2006-07-06
WO 2005/098396 PCT/US2005/000732
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.
A,s long as
electrode elements are arranged 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
xegion 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 of the phase values of the field components, the geometry
of the
electrode structures that produce the electric field, and the frequency and/or
rn.agnitude
modulation of the 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 Fiz
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 axe 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 additZOn,
the
dielectrophoretic force experience by a particle in an electric field is
depender~.t 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
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WO 2005/098396 PCT/US2005/000732
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
S 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
whe3n
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 ;u is induced in the particle
' l B
=~p~~'p-~nt~~
m
v ~p ~°p - ~nt lHm
where Tip is the particle volume, ,~a and ,~,n are the volume susceptibility
of the particle
and its surrounding medium, ,um is the magnetic permeability of medium, FIm is
the
magnetic field strength. The magnetic force Fntadneac acting on the particle
is determined,
under the dipole approximation, by the magnetic dipole moment and the magnetic
field
gradient:
Fmagnelic - ~.5 ~p ~~p ant ~~m ~ OBm 7
where the symbols "~ " and "O " 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 Vparticle
under the balance
between magnetic force and viscous drag is given by:
_ Fnragnetic
vparticle - 6
m
27
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WO 2005/098396 PCT/US2005/000732
where ~ is the particle radius and r~", is the viscosity of the surrounding
medium.
As used herein, "manipulation" refers to moving or processing of the
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 of the 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 preferred
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
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WO 2005/098396 PCT/US2005/000732
pathological status including a specific pathological status or complexed with
biological
or non-biological 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, traveling-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. In the methods of the present application, a capture
can be
perfornled 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.
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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. Preferred 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 scorpion 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 Enzyrnology
207:131-148 (1992); Bean, Methods in Enzymology 207:181-193 (1992); Leim et
al.,
CA 02554376 2006-07-06
WO 2005/098396 PCT/US2005/000732
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 Varranda,
Brazilian
Journal 31:333-354 (1998); Martinez-Pardon and Ferrus, Current Topics in
Developmental Biol. 36:303-312 (1998); Herness, Physiology and Behavior 69:17-
27
(2000); Aston-Jones and Siggins, www.acnp.org/GA/GN40100005/CHOOS.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), Humans 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 Gena, Ion Channel Pharmacology, Oxford University Press, New York
(1998),
each of which is incorporated 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 of the present invention. Preferred
resistance of
such an electrical seal is between about 1 mega ohm and about 100 gigs 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 gigs
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 of the
electrolyte bridge
3 O being an electrolyte so that the bridge can pass current with no or low
resistance.
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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 charnels, 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 GABAA, 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 current
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 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.
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"Continuous flow" means that fluid is pumped or inj ected into a chamber of
the
present invention continuously during the separation process. This allows for
components of a sample that are not selectively retained on a chip to be
flushed out of the
chamber during the separation process.
"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 of the 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 incorporated 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
linlced 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 morphological such as changes of
cell
33
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WO 2005/098396 PCT/US2005/000732
morphology 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 , i4C,
3sS~ izsh szP
or l3il). A label optionally can be a base with modified mass, such as, for
example,
pyrimidines modified at the CS 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 absorption 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
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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 of the 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, fox example, a dye,
or emits
light upon irradiation, 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 incorporated
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
incorporated herein by reference.
The product of the 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
incorporated 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.
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Introduction
The present invention recognizes that using direct detection methods to
deternnine
an ion transport functions or properties, such as patch-clamp techniques, 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
purposes.
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 that comprises at least one ion transport measuring means in the
form
of a hole through the biochip, in which at least a portion of the surface of
the
biochip is hydrophobic.
2) a biochip for ion transport measurement that comprises a microchannel plate
(MCP).
3) a flexible ion transport measurement biochip.
4) methods of making an ion transport measurement device using theta tubing
segments.
5) an ion transport measurement device that comprises a biochip that comprises
multiple ion transport measuring holes, a common upper chamber, and an upper
chamber separator unit.
6) an ion transport measurement device that comprises a biochip that comprises
multiple ion transport measuring holes and multiple upper chambers, where the
walls of the chambers are fabricated onto the biochip.
7) an ion transport measurement device comprising a biochip that comprises at
least one ion transport measuring hole and at least one flow-through upper
chamber.
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8) a device comprising a biochip that comprises multiple ion transport
measuring hole accessing a single flow-through upper chamber, further
comprising at least two delivery conduits that can be positioned over the ion
transport measuring hole recording sites to deliver solutions to the recording
sites.
9) an ion transport measurement device that comprises: a compound delivery
plate, in which the compound delivery plate has multiple drug delivery sites
that
can align with microwells on the underside of the chip to deliver compounds to
ion transport recording sites.
10) an ion transport measurement device that comprises a compound delivery
plate, in which the compound delivery plate has multiple drug delivery sites
that
can align with the two or more microwells on the upper surface of the chip to
deliver compounds to ion transport recording sites.
11) methods of shipping ion transport devices that comprise biochips and at
least
one upper chamber or at least one lower chamber, in which the upper
chamber or chambers or the lower chamber or chambers of the device are pre-
filled with a measuring solution.
12) a method of performing excised patch ion transport measurement comprising:
sealing a cell to an ion transport measuring hole; adding coated magnetic
beads to
the chamber, removing the magnetic beads and a portion of the cell from the
ion
transport measuring site with a magnet to leave an excised patch at the ion
transport measuring site; and measuring ion transport activity of the excised
patch.
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 of the invention_
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BIOCHIPS FOR ION TRANSPORT MEASUREMENT
The present invention includes chip-based devices for ion transport
measurement.
The ion transport measuring chips used in the present invention comprise a
substrate and
at least one hole through the substrate, where the hole serves as the ion
transport
measuring means. Preferably, the devices can be used to perform multiple ion
transport
assays at the same time (or in very rapid succession), and therefore preferred
chips used
in the methods of the present invention comprise two or more holes. For
performing ion
transport measurement assays, an ion transport measuring device of the present
invention
preferably comprises a chip, at least one upper chamber (fluid compartment)
situated
above the chip, and at least one lower chamber (fluid compartment) situated
below the
chip, in which an ion transport measuring hole through the chip provides fluid
communication between a lower chamber and an upper chamber (when there is no
particle sealed to the hole). The upper surface of the chip forms the bottom,
or at least a
portion of the bottom, of at least one upper chamber, and the lower surface of
the chip
forms the bottom, or at least a portion of the top, of at least one lower
chamber. When a
chip comprises multiple holes, upper chambers are in register with the holes
when they
are aligned over the chip such that each upper chamber is accessed by one hole
of the
chip. In the same sense, lower chambers are in register with the holes when
they are
aligned under the chip such that each lower chamber is accessed by one hole of
the chip.
The walls of upper and lower chambers can be built onto or into the chip, or
can be made
up of one or more separate pieces that reversibly or irreversibly engage the
chip. An
upper chamber may have a top or cover or may be open at the top. A lower
chamber may
have a bottom or may be open at the bottom. During use of an ion transport
device,
particles (such as cells) can be sealed to the top (or upper) surface or the
bottom (or
lower) surface of the chip. The entire surface of the chip (upper or lower) to
which
particles seal during use of the device is herein referred to as the sealing
surface of the
chip, regardless of whether particles seal to the particular area of the
surface referred to
on the "sealing surface".
The chip can comprises any solid material such as metals, ceramics, polymers,
inorganic and organic hybrid materials, plastics, silicon dioxide, or glass.
The substrate
can be from about 5 microns to more than 1,000 microns thick (thicker
substrates may
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require counterbores). A substrate of from about 10 to about 200 microns in
thickness is
preferred. Preferably, a chip used in an ion transport measuring device is
biocompatible
(does not have a deleterious effect on cells) referred to herein as a
"biochip". A
nonbiocompatible substrate material can be may biocompatible by coating with a
suitable
material.
Ion transport measuring holes can be etched, drilled, cut, punched out,
milled, or
bored into the substrate. In some preferred embodiments, the chip is a glass
chip and the
ion transport measuring holes are laser drilled. The diameter of ion transport
measuring
holes is preferably from about 0.2 micron to 10 microns, more preferably from
about 0.5
micron to 5 microns, and most preferably from 0.5 micron to 3 microns.
A chip of the present invention used for ion transport measurement can
comprise
one or more microwells that encompasses an ion transport measurement hole. A
microwell is a counterbore drilled or etched into the surface of a chip at the
site of an ion
transport measuring hole that can hold a volume of liquid (such as measurement
solution)
and can therefore serve as an upper or lower chamber. Drilling counterbores
into a chip at
the site of an ion transport measuring hole thins the chip at the site of the
ion transport
measuring hole, and thus reduces hole depth and hole resistance. The design
and
fabrication of counterbores in ion transport measuring chips is described in
parent United
States patent application number 10/858,339, herein incorporated by reference
in its
entirety for all disclosure of ion transport measuring chip and chip
fabrication methods.
In some aspects of the present invention counterbores can be used as
microwells to retain
small volumes of solution such as a measuring solution or a compound solution
at a
recording site.
Preferably, a chip of the present invention is surface-treated to enhance its
electrical sealing properties, such as by using methods described herein and
in parent
United States patent application number 10/858,339, herein incorporated by
reference in
its entirety for descriptions of treatment of chips to increase electrical
sealing properties.
In some preferred embodiments, an ion transport measuring chip is single-use
and
disposable, but this is not a requirement of the invention. In some
embodiments, for
example, a chamber that comprises a chip is washed or flushed out between
successive
uses. Depending upon the design of the device, an upper chamber piece, a lower
chamber
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piece, or both, as well as associated electrodes (which can be part of the
signal amplifier
machinery or electrodes that can be attached or connected to the wells), are
preferably but
optionally reusable. In some embodiments of the present invention chamber
electrodes
can be supplied by "adaptor plates" that reversibly engage at least a portion
of one or
more upper chambers or one or more lower chambers. Adaptor plates can be
reused by
detaching the plate from a first device used in a first set of assays and
attaching the plate
to a second device to be used in a second set of assays. Adaptor plates can
also include
one or more inflow conduits, one or more outflow conduits, or one or more
conduits that
connects to a pneumatic device such as a pump or syringe that can be used to
seal
particles to ion transport measuring means of the device.
Treating Clzips Comprisirzg Ion Transport Measuring Means to Enhance the
Electrical
Peal of a Particle
Ion transport measuring means includes, as non-limiting examples, holes,
apertures, capillaries, and needles. "Modifying an ion transport measuring
means" or
"Treating an ion transport measuring means" means modifying at least a portion
of the
surface of a chip, substrate, coating, channel, or other structure that
comprises or
surrounds the ion transport measuring means. The modification may refer to the
surface
surrounding all or a portion of the ion transport measuring means. For
example, a biochip
of the present invention that comprises an ion transport measuring means can
be modified
on one or both surfaces (e.g. upper and lower surfaces) that surround an ion
transport
measuring hole, and the modification may or may not extend through all or a
part of the
surface surrounding the portion of the hole that extends through the chip.
Similarly, for
capillaries, pipettes, or for channels or tube structures that comprises ion
transport
measuring means (such as apertures), the inner surface, outer surface, or
both, of the
channel, tube, capillary, or pipette can be modified, and all or a portion of
the surface that
surrounds the inner aperture and extends through the substrate (and
optionally, coating)
material can also be modified.
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
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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).
Preferably, treating 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 of the
ion transport measuring means. In some preferred aspects of the present
invention, a
substrate or structure that comprises an ion transport measuring means is
subjected to
chemical treatment (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 andlor base
solutions rnay
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
hydrophobicity andlor change surface charge density andJor 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 preferred embodiments of the
present
invention in which the ion transport 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 "bzochip 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
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enhanced sealing properties, a 1 Giga Ohm resistance seal is maintained for at
least 3
seconds.
In practice, in preferred aspects of the 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, ox~.dizing agents (for example,
peroxide, oxygen
plasma), acids, or bases. Preferably, an ion transport measuring means or a
structure (as
nonlimiting examples, a structure 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
preferred aspects of the present invention, the ion transport measuring means
is on a
biochip, on a planar structure, but the ion transport measuring means can also
be on a
non-planar structure.
The ion transport measuring means or surface surrounding the ion transport
measuring means modified to enhance electrical sealing can comprise any
suitable
material. Preferred materials include silica, glass, quartz, silicon, plastic
materials,
polydimethylsiloxane (PDMS), or oxygen plasma treated PDMS. In some preferred
aspects of the 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 (Si0-)) 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,
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thermally oxidized Si02 on silicon, deposited Si02, deposited glass,
polydimethylsiloxane (PDMS), or oxygen plasma treated PDMS.
In preferred 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, HCI, HZS04, NaHS04, HS04, HN03, 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
transport
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, or a chip comprising 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)Z, Li~H, CsOH,or Ca(OH)Z. The basic
solution can be of a concentration 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. 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, KCl, BaClz, LiCI,
CsCI,
Na~S04, NaN03, or CaCI, 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
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about 1 M in concentration. Optimal concentrations for treating an ion
transport
measuring means to enhance its electrical sealing properties can be determined
empirically. 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 surrounding an
ion transport
measuring means more smooth, it can be performed on the front sicLe of the
chip, that is,
the side of the chip or substrate that will be contacted by a sample c
omprising particles
during the use of the ion transport 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 of the present invention, it can be preferred to rinse the ion
transport 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 SM or more), biological buffer solutions, cell media, or
dilutions or
combinations thereof. Rinsing can be performed for any length of time, for
example
3 0 from minutes to hours.
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Some preferred 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. In such tests, increasing the electronegativity of
glass ion
transport measuring means correlates with enhanced electrical sealing by the
base-treated
ion transport measuring means. Base treatment can optionally be corr~bined
with one or
more other treatments, such as, for example, treatment with heat (suc31 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 of the
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 preferred embodiments, the surfaces of a chip surrounding ion
transport
measuring means can be laser polished prior to treating the chip with acid and
base.
Methods and protocols for treating chips to increase their electrical sealing
properties, to
increase their surface hydrophilicity, and to increase their
electronega..tivity are provided
in United States utility patent application number 101858,339, filed June 1,
2004, United
States patent application number 10/760,866, and United States pateri-t
application
number 10/760,866 all of which are hereby incorporated by reference for
methods of
treating chips to increase their electrical sealing properties.
The present invention includes chips such as biochips treated to enhance their
electrical sealing properties, and devices comprising biochips treated rto
enhance their
electrical sealing properties. The device can comprise at least one biochip
that has been
treated to enhance its electrical sealing properties, where the biochip
comprises at least
CA 02554376 2006-07-06
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one hole, at least one upper chamber accessed by the at least one hole, or at
least one
lower chamber accessed by the at least one hole. In another embodiment, the
device can
comprise at least one biochip that has been treated to enhance its electrical
sealing
properties, where the biochip comprises at least one hole, at least one upper
chamber
accessed by the at least one hole and at least one lower chamber accessed by
the at least
one hole.
In some aspects of the present invention, it can be preferable to store an ion
transport measuring means (or a chip comprising 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 of the 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 desiccants (for
example,
silica gel, CaClz or NaOH) or under nitrogen or an inert gas. Where an ion
transport
measuring means or structure comprising an ion transport measuring means is
stored in
water or an aqueous solution, preferably the pH of the 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 structure 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 W-acrylic
or LJV-
epoxy glues can be stored in neutral pH water for months without affecting the
sealing
properties.
ZS 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.
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Dehydration can, however, reduce the sealability of chemically treated chips.
To
improve the seal rate for dry-stored chips, NaOH, NaCI, CaCl2 and other salt
or basic
solutions can be used to rejuvenate the chips out of dry storage to restore
the sealability.
Hydf~ophilic Chip having Hydrophobic Modificatiofas
One aspect of the present invention is a hydrophilic biochip for ion transport
measurement that comprises a substrate comprising one or more holes, one or
more
hydrophilic recording site areas on a surface of the substrate to which
particles seal
during use of the chip (the "particle-sealing side" of the chip), and at least
one
hydrophobic area on the same surface of the substrate, in which at least one
hydrophobic
area surrounds the one or more hydrophilic recording site areas; and in which
the
hydrophobic area can maintain an aqueous solution localized to the hydrophilic
recording
site area in fluid isolation.
The surface of a "hydrophilic chip" that is to be used as the particle-sealing
surface is designed so that aqueous solutions such as, for example, measuring
solutions
used in ion transport assays, that are deposited or distributed in a
hydrophilic area of the
chip surface, will remain confined to hydrophilic areas of the particle-
sealing surface as
the solutions are repelled by hydrophobic surfaces that surround the
hydrophilic areas.
On a hydrophilic chip of the present invention, recording site areas on the
side of the chip
to be used for particle-sealing are therefore designed to be hydrophilic.
Preferably, the
substrate surface recording site areas are positively or negatively charged,
and more
preferably, the substrate surface at recording sites of a hydrophobic chip of
the present
invention is negatively charged to enhance the electrical sealing at the ion
transport
measuring hole. Negatively charged surfaces include surfaces having negative
charge that
is counterbalanced by noncovalently bound positive ions.
The hydrophobic surface surrounding the recording sites can extend over the
entire portion of the chip, exclusive of recording site areas, or can be
discontinuous. In
some apsects of the present invention, the hydrophobic area of the substrate
(chip)
surface comprises essentially all of the surface area of said chip on its
particle sealing
side, excluding the one or more hydrophilic recording areas.
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In preferred embodiments of a hydrophilic chip having hydrophobic areas, the
chip comprises two or more holes and two or more hydrophilic recording site
areas each
of which is surrounded by the hydrophobic chip surface, such that an aqueous
solution
provided in any hydrophilic recording site area is isolated from an aqueous
solution
provided in any other hydrophilic recording site area. In this way, recording
site areas
can be maintained in fluid isolation in the absence of structural barriers.
A hydrophilic ion transport measuring biochip of the present invention that
has
hydrophobic surface areas can have any number of holes, from 1 to more than
one
thousand. In preferred embodiments, a hydrophilic ion transport measuring
biochip
having hydrophobic surface areas surrounding recording sites is high-density,
and can be
used for high throughput screening (such as, but not limited to, compound
screening), and
has 384 or more ion transport measuring holes. In some embodiments, a
hydrophilic ion
transport measuring biochip can have 1536 or more ion transport measuring
holes.
Preferably, a hydrophilic recording site area of a chip of the present
invention can
hold a drop of aqueous liquid of a volume of from about 1 microliter to about
2
milliliters. A hydrophilic recording site surface area can preferably have a
diameter of
from about 25 micron to about 10 millimeters, more preferably from about 500
micron to
about 2 millimeters.
A hydrophobic biochip of the present invention can comprise microwells that
define the recording site area and preferably serve as upper or even lower
chambers (in
embodiments where cells are sealed to the bottom surface of a chip). For
example,
microwells that surround ion transport measuring holes be drilled or etched
into a
substrate of a hydrophobic chip as counterbores. The microwell surfaces of a
hydrophobic chip are hydrophilic, and the microwells can retain small volumes
of
solutions distributed in the microwells that are repelled by surrounding
hydrophobic
surfaces.
A hydrophilic biochip can comprise a hydrophilic substrate that, in areas
where
the surface is hydrophobic, is modified to be hydrophobic or is coated with a
hydrophobic material. For example, the substrate can comprise glass, silicon,
silicon
dioxide, quartz, or one or more hydrophilic polymers. The thickness of the
hydrophilic
substrate is not limiting, but can be from about 1 micron to about 2
millimeters. The
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WO 2005/098396 PCT/US2005/000732
substrate material can be modified by chemical or physical means to make it
hydrophobic. Alternatively, the hydrophilic substrate can be coated with at
least one
hydrophobic plastic or polymer, such as, for example, polyethylene,
polyacrylate,
polypropylene, polystyrene, or polysiloxane. The thickness of the coating is
also not
limiting and can be as thin as one molecular layer in thickness.
A hydrophilbic biochip having one or more hydrophobic areas can be made by
providing a substrate that comprises a hydrophilic material, such as, for
example, glass;
coating the substrate with at least one hydrophobic material (such as a
hydrophobic
polymer); making at least one hole through said substrate; and removing the
hydrophobic
substrate from the area immediately surrounding said at least one hole to
define a
hydrophilic recording site area. Preferably, the recording site is chemically
treated, such
as with a salt or base solution, to improve the electrical sealing properties
of the ion
transport measuring hole at the recording site. The hole can be laser drilled
or etched
through a substrate such as glass, for example. The hydrophobic material can
be removed
from recording site areas by chemical means, however, in preferred embodiments
a
counterbore and through-hole are laser drilled or etched into the substrate at
each
recording site. The drilling or etching of the counterbore removes the
hydrophobic
coating to produce a hydrophilic microwell at the recording site.
An alternative method of making a hydrophilic chip with hydrophobic areas is
to
provide a substrate that comprises a hydrophilic material; making one or more
holes
through said substrate; and coat at least a portion of the substrate with at
least one
hydrophobic material, while masking the recording site areas around the one or
more
holes to prevent them from receiving the hydrophobic coating.
In yet another embodiment, a chip can comprise a hydrophobic substrate
material
such as a polymer or plastic, and can be coated with a hydrophilic material at
recording
sites on the particle sealing surface of the chip. The hydrophilic chip can be
made by
providing a substrate that comprises a hydrophobic material;
making one or more holes through the substrate; and coating the recording area
surrounding the one or more holes with at least one hydrophilic material. For
example,
glass can be used to coat the chip surface at the one or more recording site
areas. The
recording site areas can preferably be chemically treated, such as with at
least one salt or
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WO 2005/098396 PCT/US2005/000732
at least one base, to improve the electrical sealing properties of the one or
more ion
transport measuring holes.
A preferred embodiment of this aspect of the present invention is a
hydrophilic
ion transport measuring biochip having one or more hydrophobic areas that
comprises ion
transport measuring means in the form of holes of from about 0.2 to 10 microns
in
diameter that are surrounded by counterbores, where the counterbores are
microwell
upper chambers.
A hydrophilic/hydrophobic chip having microwell upper chambers can be made
by providing a suitable substrate, such but not limited to a glass, quartz,
silicon, silicon
dioxide, or one or more polymers, and coating the substrate with a hydrophobic
material.
Suitable materials for providing a hydrophobic coating include plastics and
polymers,
such as, for example, polyethylene, polyacrylate, polypropylene, polystyrene,
or
polysiloxane. After coating the chip, two or more holes are made, such as by
laser drilling
into the chip. The laser drilling has the effect of melting and burning the
polymer in the
area surrounding the drilled hole, provided an uncoated (hydrophilic) surface
in the area
where a cell (or other particle) can seal. Preferably, a counterbore is also
drilled into the
chip, where the counterbore can serve as a microwell on the upper surface of
the chip.
This design provides upper microwells (made by laser drilling) that are in
liquid
fluid isolation from one another, as the hydrophobic surface between wells
repels
aqueous liquids such as buffers and measuring solutions. The ion transport
holes and
areas immediately surrounding them (such as counterbore microwells) have
hydrophilic
surfaces that have been exposed by the laser drilling and therefore will
retain buffers and
solutions.
The upper microwells can optionally be connected to a common reference
electrode that can traverse the chip surface. Preferably, the electrode is
coated with a
nonconducting (and hydrophobic) material, such as a plastic or polymer used to
coat the
chip surface and traverses the surfaces of the chip. The electrode can be
uncoated where
it contacts the microwells, so that the microwells are in electric
communication without
the possibility of solution exchange or mixing between wells.
3 0 One preferred embodiment of the electrode on a hydrophilic/hydrophobic
chip is
a metallized layer on the substrate coated by deposition, growing, condensing,
or other
CA 02554376 2006-07-06
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means. The metallized layer can be removed at and near the recording sites by
laser shots
or masking. The hydrophobic layer is then coated on top of the metallized
layer to allow
for fluidic liquid separation between two adjacent recoding sites, leaving a
ring of
metallized layer uncovered near the recording sites to allow for electrical
connection of
each recording sites (in form of a hole, or a hole and a microwell) with the
metallized
layer which services as a reference electrode. The metallized layer can be
made of any
conductive material or materials including metals, non-metals, metal
derivatives, or
combinations thereof
As alternatives, individual recording electrodes can also be physically or
electrically connected (such as through electrolyte bridges) to each of the
upper chamber
microwells. In these designs, there can be individual or common lower chambers
that
engage the chip, and the one or more lower chambers comprise or are
electrically
connected to one or more reference electrodes.
Figure 1 shows a cross-sectional schematic view of one preferred design ion
transport measuring hydrophilic/hydrophobic chip of the present invention. In
this design,
the chip comprises a substrate (11) such as glass or silicon dioxide that is
hydrophilic,
through which through holes (12) have been laser drilled to provide ion
transport
measuring means. Gounterbores that serve as upper chamber microwells (13) have
also
been laser drilled into the substrate. In this design, the upper surface of
the chip (the
surface that serves as the sealing surface) is coated with an electrode layer
(14) that
contacts the microwells (13). Outside of the microwells, the electrode layer
is coated with
a hydrophobic material (15) that promotes fluid isolation of the microwells.
The
rightmost microwell in the figure is shown containing solution (17) (such as
extracellular
solution) that is in contact with the electrode but is excluded from the
hydrophobic layer
(15). A cell (16) is depicted in the well sealed to the in transport measuring
hole (12).
Where the coating material is resistant to treatment chemicals, such as base
and/or
acid, the surface of the hole on the hydrophobic chip can be chemically
treated, such as
by using methods described herein, to enhance the electrical sealing
properties of the
chip.
The present invention also includes methods of making a hydrophilic/
hydrophobic chip, and devices comprising a hydrophobic chip, where the devices
can
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employ any feasible upper chamber, lower chamber, electrode, fluidic and
pneumatic
designs, such as but not limited to those described in the present
application. The present
invention also includes methods of using a hydrophobic ion channel measuring
chip to
measure ion channel activity or properties of one or more cells or particles.
NOVEL ION TRANSPORT MEASURING BIOCHIP DESIGNS
The present invention also includes novel methods of malting high density
and/or
multiplex ion transport measuring biochips and biochips made by these methods.
These
devices can be used to record ion transport activity of more than one particle
or cell
simultaneously or in rapid sequence. In preferred aspects, the ion transport
measuring
biochips and biochips made by these methods are designed to be high density
the ion
transport measuring biochips. By "high density" is meant that the chips
comprise a large
number of ion transport measuring means. Typically, the ion transport
measuring means
are holes through the surface of the biochip, and a high density transport
measuring
biochip has multiple ion transport recording sites via multiple holes. In this
way, multiple
assays can be conducted simultaneously, or in rapid sequence, allowing for
high-
throughput ion transport measuring assays that can facilitate, for example,
compound
assays.
As used herein, "high throughput" means high quantity of independent data
collected in a defined period of time. For example, 48 or more assays that can
be
conducted within a short time span where multiple assays are initiated
simultaneously or
in rapid succession, then share experimental time as parallel or multiplexed
recordings,
and ten completed simultaneously or independently but in parallel (less than
one hour
from loading of cells to completing ion channel recording, preferably, less
than one half
hour from loading of cells to completing ion channel recording, and more
preferably, less
than fifteen minutes from loading of cells to completing ion channel
recording). More
preferably, more than 96 high throughput ion transport measurements can be
completed
in less than one half hour, and more preferably yet, the high density ion
transport
measuring devices of the present invention are capable of performing more than
100 ion
transport assays within one half hour or less. In some preferred aspects of
these
embodiments, high density ion transport measuring devices can perform hundreds
or over
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WO 2005/098396 PCT/US2005/000732
one thousand assays within one half hour or less. For example, in some
preferred aspects
of high density ion transport measuring devices described herein, the devices
can be
designed to perform 384 assays or, for example, 1536 assays, within one half
hour or
less. For another example, 48 or more assays that can be conducted within a
time span
during which continuous and repetitive data sampling are performed for kinetic
studies
with high temporal resolution. In another example, multiple lower density
assays, such as
16-assay devices, may be utilized in parallel to result in a high density
assay.
While the devices herein can be described as high-throughput, the designs are
not
limited to high throughput uses and can be used for any number of ion
transport assays,
in assays that can last from seconds to several hours.
MCP-based Chip
One aspect of the present invention is an ion transport measuring device that
comprises a microchannel plate (MCP). Microchannel glass plates that comprise
an array
of microchannels and their fabrication are known in the art of electronics and
optics for
their use as electron multipliers and photomultipliers. Some aspects of their
fabrication
and use are described in Wiza (1979) Microchannel Plate Detectors Nuclear
Instruments
and Methods 162: 587-601 _ In brief, they can be made by providing glass
fibers that have
a core glass and a cladding that comprises lead glass. The fibers are arranged
together
side-by-side in a desirable configuration, drawn, surrounded by a glass
envelope, and
fused to produce a boule. The boule can be sliced (cutting perpendicular to
the fiber
lengths) to produce slices that are cross-sections of the boules. These slices
can be
finished, for example, by polishing. The cores of the glass fibers are then
chemically
etched away, to form the microchannel plate.
An MCP made for use as part of an ion transport measuring device can be made
by fusing from 2 to over 1,000 glass fibers. An MCP ion transport measuring
chip can,
for example, be a high-density ion transport measuring chip that comprises 48
or more
microchannels that serve as ion transport measuring holes, and preferably, 96
or more
microchannels that serve as ion transport measuring holes. The core of the
fibers (the
portion made of etchable glass) used to make an MCP chip can be as wide as 40
microns
in diameter (for chips used for ion transport assays using large cells, such
as oocytes) but
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preferably are from 0.2 to 8 microns in diameter, and are more preferably from
0.5 to 5
microns in diameter, even more preferably from 0.5 to 3 microns in diameter,
and most
preferably about 2 microns in diameter. The thickness of the lead glass
cladding around
the core can vary depending on the desired spacing of the resulting ion
transport
measuring holes. The length of the fibers used in making an MCP ion transport
measuring chip are not limiting, and can be of any feasible length.
Preferably, after fusing
the glass fibers, the boule is sliced into sections that are from about 5
microns to 5000
microns thick, most preferably from about 10 to about 50 microns thick.
The core glass fibers can be randomly arranged or configured into a pattern to
make the boule.
The boule slice or wafer may be wet-etched to etch away preferentially the
embedded fibers of a softer glass so as to produce micron-sized through-holes
in the
wafer. The softer glass fibers are more easily etched by wet etching
solutions. A higher
concentration, or higher reaction temperature, or combination of both, may
also etch the
harder glass substrate of the MCP wafer, though to a lesser degree. In this
method only
one side of the MCP wafer is exposed to a wet-etching compound, by floating it
over a
reaction chamber, or by clamping an inert gasket onto the MCP wafer such as to
produce
a reaction chamber with the MCP wafer at its bottom surface, under conditions
that also
etch the harder glass substrate. The reaction is then quenched once all of the
through-
holes have emerged on the opposite surface, leaving through holes that taper
gradually
from a larger diameter end on the etching side, to a smaller diameter emergent
hole on
the non-etching side. The opposing side of the MCP wafer that is not exposed
to the
etching compound is kept immersed in a quenching medium (such as water) that
will
dilute or inactivate any emerging etching compound and prevent etching on the
emerging
surface.
In one design, depicted in Figure 2, the area surrounding the ion transport
measuring holes on the upper side of the MCP chip (21) can be chemically wet-
etched to
produce microwells (23) at the upper ends of the holes (22) through the chip
that can be
used as upper chambers. These upper chambers can be used for measuring
solution, cells
or particles, and test compounds. The MCP chip can be bonded to a bottom piece
that
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comprises one or more lower chambers. The MCP plate can also be bonded to an
upper
piece that comprises the ES chambers.
The surface of the MCP chip can be chemically treated, such as using methods
disclosed herein, to enhance the electrical seal of a particle or membrane
with the ion
transport measuring means. The entire MCP chip or a portion thereof can be
treated to
enhance its electrical sealing properties. Preferably, at least a portion of
the surface of the
MCP chip to which cells or particles are to be sealed is treated with at least
one salt or at
least one base. One or both surfaces, or one or more portions of one or both
surfaces, of
the MCP chip can also be coated, or one or more portions of one or both
surfaces, with
one or more materials that can increase its sealing properties. In some
embodiments, one
or both surfaces, or one or more portions of one or both surfaces, of the MCP
chip can be
coated with one or more hydrophobic materials that can be used to promote
fluidic
isolation of individual microwells of the MCP chip. Designs in which
hydrophobic
surfaces are used to promote fluidic isolation of individual microwells of a
chip are
further described in the previous section of this application (above).
In designs in which the bottom piece forms individual lower chambers,
reference
electrodes can be within or electrically connected with the upper wells and
recording
electrodes can be within or electrically connected with the lower wells for
ion transport
measurement, or recording electrodes can be within or electrically connected
with the
upper wells and reference electrodes can be within or electrically connected
with the
lower wells for ion transport measurement.
In one possible design involving etched rnicrowells on the upper surface, a
common reference electrode can connect all of the upper microwells. The
electrode,
which can be a conductive material such as metal, can follow paths along the
top surface
of the MCP chip and contact measuring solution only where it contacts the
interior of the
microwells. The electrode can optionally be coated with a nonconductive
material where
it traverses the chip surface, and be exposed where it contacts the interior
of the wells.
Where a device comprising an MCP chip is configured to have a common
electrode that contacts multiple lower wells of the device, the same design
can be used.
In designs in which the bottom piece forms a bottom chamber that contacts more
than one ion transport measuring hole, the bottom chamber preferably comprises
or is in
CA 02554376 2006-07-06
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electrical contact with a reference electrode, and individual upper chambers
comprise
individual recording electrodes. Alternatively, the bottom piece can comprise
multiple
lower chambers with individual recording electrodes, and the device has a
common upper
chamber with a reference electrode. In this embodiment, compounds can be added
using
compound delivery mechanisms such as, for example, fluid block delivery,
chamber
separators, or other mechanisms described herein.
In an alternative design for an ion transport measuring device that comprises
an
MCP chip, upper chambers can be constructed by attaching a manufactured piece
that
comprises well openings such that each well of the upper chamber piece aligns
with one
of the ion transport measuring holes (the microchannels of the MCP).
Individual upper
chambers preferably have a volume of from about 0.5 microliters to about 5
milliliters,
and more preferably from about 2 microliters and about 2 milliliters, and more
preferably
yet between about 10 microliters and about 0.5 milliliter. The upper chamber
piece can be
irreversibly or reversibly attached to the MCP ion transport measuring chip
using gaskets,
clamps, adhesives, welding, or other means. The upper chamber piece can
comprise
glass, ceramics, coated metals, or (preferably) plastics or polymers. In one
preferred
embodiment, the upper chamber piece comprises a separate MCP. In this design,
the
glass fibers used to make the upper chamber piece MCP are of a wider diameter
than
those used to make the ion transport measuring chip MCP_ The glass fibers used
to make
the upper chamber piece MCP also comprise a cladding of sufficient thickness
to provide
chamber spacing over the ion transport measuring holes of the ion transport
measuring
chip MCP. Conduits can connect to the wells of the upper chamber piece for the
addition
of solutions, cells, or compounds. Alternatively, a fluid dispensing device
can interface
with the upper chamber wells to dispense solutions, cells, or compounds.
A lower chamber piece can also comprise multiple chambers that connect to
individual ion transport holes of the MCP chip. The lower chamber piece can be
constructed by attaching a manufactured piece that comprises wells spaced such
that each
well of the lower chamber piece aligns with one of the ion transport measuring
holes (the
microchannels of the MCP). The lower chamber piece can be irreversibly or
reversibly
attached to the MCP ion transport measuring chip using gaskets, clamps,
adhesives,
welding, or other means. The upper chamber piece can comprise glass, ceramics,
coated
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metals, or (preferably) plastics or polymers. In one embodiment, the lower
chamber piece
comprises a separate MCP. In this design, the glass fibers used to make the
upper
chamber piece MCP are of a wider diameter than those used to make the ion
transport
measuring chip MCP. The glass fibers used to make the lower chamber piece MCP
also
comprise a cladding of sufficient thickness to provide chamber spacing over
the ion
transport measuring holes of the ion transport measuring chip MCP. Conduits
can
connect to the wells of a lower chamber piece for the addition of solutions,
and allowing
pneumatic control. The lower well electrodes can optionally be provided by a
separate
adaptor plate that can reversibly engage the lower wells and can also
optionally comprise
connections to pneumatic devices for pressure control.
In using devices having individual upper chambers and individual lower
chambers
recording electrodes (or connections to recording electrodes) can be provided
in or
attached to upper chambers, and reference electrodes (or connections to
reference
electrodes) can be provided in or attached to lower chambers. In the
alternative, recording
electrodes (or connections to recording electrodes) can be provided in or
attached to
lovcrer chambers, and reference electrodes (or connections to reference
electrodes) can be
provided in or attached to upper chambers.
In some preferred embodiments, however, a device that comprises an MCP ion
transport measuring chip can have a single lower chamber that accesses all ion
transport
measuring holes of the MCP chip. In this case, the lower chamber can also
comprise
ceramics, coated metals, glass, plastics, or polymers, and preferably connects
to conduits
that connect to pressure sources and can deliver and remove fluids to and from
the
chamber. Pressure control may be performed from either bottom chambers or
upper
chambers, or both. In these embodiments, the lower chamber preferably
comprises or is
in electrical connection with a reference electrode during use of the device,
and each
upper chamber comprises or is in electrical connection with a recording
electrode during
use of the device.
In some other preferred embodiments, a device that comprises an MCP ion
transport measuring chip can have a single upper chamber that accesses all ion
transport
measuring holes of the MCP chip. In this case, the upper chamber can also
comprise
ceramics, coated metals, glass, plastics, or polymers and it preferably
comprises or is in
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electrical connection with a reference electrode during use of the devices. In
this case
each lower chamber comprises or is in electrical connection with a recording
electrode
during use of the device. Pressure control may be performed from either bottom
chambers or upper chambers or both.
The present invention comprises ion transport measuring devices comprising an
MCP chip having greater than two through holes, and at least one upper
chamber.
Preferably an ion transport measuring device comprising an MCP chip has
multiple upper
chambers that are reversibly or irreversibly attached to the MCP
chip.,Preferably an ion
transport measuring device that comprises an MCP chip can be reversibly or
irreversibly
attached to at least one lower chamber. The present invention also comprises
ion
transport measuring devices comprising an MCP chip having multiple
microchannel
through holes, and an MCP chip having multiple microchannel upper chambers.
The present invention also comprises methods of using MCP chips for measuring
ion transport activity and properties, as well as for other assays.
Flexible Ion Transport Measurement (ITM. chip
Another aspect of the present invention is a method of making a flexible ion
transport measuring biochip that comprises a flexible sheet of material,
preferably coated
with glass, comprising multiple ion transport measuring holes. The flexible
sheet of
material can be wound around a spool and unwound to form either a curved or an
essentially flat surface for ion transport measurement. Alternatively, the
flexible sheet of
material can be curved to form a tube, on the surface of which ion transport
measurement
assays can be performed.
The method compriscs: providing a substrate that comprises a sheet of flexible
material; creating (for example, by laser drilling, chemical etching,
micromachining,
molding, etc) at least two holes in the substrate that extend through the
substrate, and
optionally coating the substrate with SiO~ or glass to provide an ITM chip.
The substrate can comprise any material that can be provided as a thin sheet
(for
example, of within the range of between 5 and 5000 microns in thickness) and
has a
flexibility that allows the sheet to be curved completely around (such as to
make a tube)
yet is hard and rigid enough to allow manufacture of ion transport measuring
hol es
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through the substrate (that is, holes of a diameter within the range of from
about 0.2 to
about 8 microns in diameter, although larger diameters can be used depending
on the cell
type to be assayed). For example, rubber, plastics, polymers or other flexible
sheet
materials can be used. One such material is polyimide or Kapton. Kapton sheets
of from
about 5 to 5000 microns in thicl~ness, preferably from about 10 to about 200
microns in
thickness, can be laser drilled to produce through holes of within the range
of from about
about 0.2 to about 8 microns in diameter, preferably from about 0.5 to 5
microns in
diameter, and more preferably from about 0.5 to about 3 microns in diameter.
Counterbores that can be used as microwells can also optionally be drilled
into the
polyimide sheet, as described herein in parent United States utility
application
10/858,339, incorporated herein in its entirety for its disclosure of
counterbores and
fabrication of counterbores. From 2 to over 50,000,000 holes can be drilled
into a single
polyimide sheet, depending on the application, which can be further rolled
around a
spool. For example, where a flexible biochip is to be used as a "chip roll" in
which
section of the flexible biochip are used to be used sequentially, the sheet
can comprise a
very large number of holes, a subset of which are to be used in any
given~assay.
Before or after laser drilling of holes in the flexible substrate, the
substrate is
preferably treated or coated with a material that allows for efficient and
high-resistance
sealing of particles such as cells to the ion transport measuring holes. The
modification of
the surface can be any modification that promotes high-resistance sealing of
particles
such as cells to the ion transport measuring holes of the chip. Preferably,
the modification
makes at least a portion of the surface of the flexible chip to which
particles are sealed
during use of the chip more hydrophilic, and more preferably, the modification
makes at
least a portion of the surface of the chip where particles seal negatively
charged. The
modification can comprise coating the surface with organic or inorganic
molecules,
synthetic molecules (for exampl e, polymers) or naturally occurring ones, in
liquid or non-
liquid form. The coated surface can be hydrophobic or hydrophilic, charged or
noncharged, and can be linked to the substrate covalently or non-covalently.
The coating
can be further modified to make it more hydrophilic. In one preferred
embodiment, the
substrate is coated with glass and the chip is treated with at least one salt
or at least one
base to improve its electrical sealing properties.
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If the coating is a naturally rigid material, surch as glass, the coating
should be thin
enough, or physio-chemically altered to permit curu-ing of the coated flexible
sheet. The
coating thickness can range from a single molecule layer to several
micrometer. The
optimal thickness for the degree of curvature that is desirable (depending on
the
application) can be determined empirically. The degree of curvature required
in the use of
the device that comprises the flexible biochip can al so be adjusted (for
example, by
adjusting spool diameter, if the substrate is to be wound around a spool, or
by adjusting
tube diameter, if the substrate is to form a tube structure) to accommodate
the coating if
necessary.
The coating can be applied in any appropriate way: vapor deposition, dipping,
soaking, direct application, spraying, "painting", chemical grafting etc. If
the coating is a
polymer, in some cases polymerization can be prorrfoted on the substrate
surface. The
coating can be adhered to the substrate by absorptioxi or chemical bonding. A
glass
coating can be applied, for example, by vapor deposition (if the substrate
material is
resistant to the heat required, or by allowing solgel hydrolyzed siloxane) to
polymerize
to glass as it dehydrates on the substrate surface.
A flexible chip can be designed such that at least a portion of the surface of
the
chip is hydrophilic and at least a portion of the surface of the chip is
hydrophobic. For
example, a flexible chip fabricated with a hydrophobic substrate (such as a
hydrophobic
polymer) can be coated with a hydrophilic material in the area immediately
surround the
ion transport measuring means. The coating can be applied to both surfaces of
the chip,
or only the surface to which particles seal during use of the chip for ion
transport
measurement. In another example, a flexible chip fabricated with a hydrophobic
substrate
(such as a hydrophobic polymer) can be surface-nzo dified to be hydrophilic in
the area
immediately surround the ion transport measuring means, such as by heating,
oxidation,
chemical treatment, etc. In yet another example, a hydrophilic chip substrate
or coating
on a substrate can be coated with a hydrophobic material or treated to make at
least a
portion of the chip surface apart from recording site areas hydrophobic.
The surface of the flexible chip or portions thereof can optionally be
chemically
treated, such as by using the methods described herein, to improve the
electrical sealing
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properties of the chip. For example, at least a portion of the flexible chip
can be treated
with at least one salt or at least one base.
In one aspect of this embodiment of the present invention, an ion transport
measuring device can be made using a flexible ion transport measuring biochip
of the
present invention that is wound around a spool (see Figure 3). In this
embodiment, the
leading edge of the flexible biochip extends from the spool to either a second
spool, or to
a guide into which is inserted. The second spool or guide is positioned at a
particular
distance from the first spool such that an expanse of the flexible biochip is
extended to be
used for ion transport assays. The extended portion of the flexible biochip
(301) can be
essentially flat (Figure 3A) or somewhat curved (Figure 3B). Preferably, the
extended
portion of the flexible biochip comprises multiple ion transport measuring
that matches
the number of wells in mufti-well plate for compound testing. Preferably, the
extended
portion of the flexible biochip comprises at least 8 ion transport measuring
holes, more
preferably, at least 12 ion transport measuring holes, even more preferably,
at least 48 ion
transport measuring holes, and yet more preferably, at least 96 ion transport
measuring
holes. For example, the extended portion of the flexible biochip can comprise
384 or
1536 ion transport measuring holes.
The present invention includes flexible ion transport measuring biochips made
using these methods, and devices that include flexible ion transport measuring
biochips.
An upper chamber piece can engage the upper side of the flexible biochip and a
lower chamber piece can engage the lower side of the flexible biochip. In
preferred
aspects of these embodiments, the upper and lower chamber pieces are reusable,
and the
flexible biochip is single-use. In these aspects, the upper and lower chamber
pieces
reversibly engage the flexible biochip for ion transport assays. Upon
completion of a set
of assays, the upper and lower chamber pieces disengage and move away from the
flexible biochip, a new section of the flexible biochip is unwound from the
spool as the
leading edge is pulled through guides and the old portion is optionally wound
on a second
spool, similar to camera film winding (in an alternative the used section can
be pulled
through guides and clipped off, similar to use of a tape dispenser). The new
section of the
flexible biochip that is unwound from the spool is to b a used in the
subsequent assay. The
upper chamber piece and lower chamber piece (preferably one or both is
reusable, but
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this is not a requirement of the present invention) now move to engage the new
extended
portion of the flexible biochip.
In Figure 3A, the flexible biochip (301) has an extended portion between two
spools (320) that engages an upper chamber piece having multiple upper
chambers (318)
and a lower chamber piece having a single lower chamber (319). An inflow
conduit (322)
and an outflow conduit (323) engage the lower chamber for directing solutions
into and
out of the chamber, and can also connect to pneumatic devices for applying
pressure to
the lower chamber.
In aspects in which the extended portion of the flexible biochip is somewhat
curved, such as by curving against the surface of another, "chamber spool", in
which the
contact surface of the spool also comprises the upper or lower chamber pieces,
the upper
and lower chamber pieces can be adapted to fit a curved biochip.
The upper chamber piece, the lower chamber piece, or both can be part of a
chamber "wheel" in which multiple chamber pieces, each of which is used in
performing
a set of assays, can sequentially engage the flexible biochip. as shown in
Figure 3B. For
example, a first set of assays can be performed using the first exterided
portion of the
flexible biochip (301) and a first lower chamber piece (319) that is part of a
lower
chamber wheel (324) and can rotate below the surface of the flexible biochip.
Upon
completion of the first set of assays, the used portion of the flexible
biochip (301) is
pulled away from the wheel (324) as a new portion of flexible biochip (301)
comes into
proximity with the lower chamber wheel (324). During this period of time, the
lower
chamber wheel (324) rotates so that the used lower chamber piece X319) moves
away
from the assay site, and a new chamber piece comprising lower chambers
(comprising
measuring solution) also attached to the wheel engages the new extended
portion of
flexible biochip at the assay site. In the meantime, the used lower chambers
can be
washed as they turn with the wheel to be re-used with a new strip of the
flexible biochip.
In this depiction, an upper chamber wheel (325) provides upper chamber pieces
(318)
that also engage the flexible chip (301), and can rotate sequentially to
engage the chip for
ion transport assays and disengage the chip when a set of assays is complete,
preferably
to be washed and re-used in subsequent assays.
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Various other upper and lower chamber configurations can be combined with the
flexible biochip. For example, an upper chamber piece that engages the
flexible biochip
can have multiple upper chambers, such that each ion transport measuring hole
is
associated with a single upper chamber, and a lower chamber piece can also
have
multiple lower chambers, such that each ion transport measuring hole is
associated with a
single lower chamber. It is also possible to have a single lower chamber that
accesses all
of the ion transport holes used in an assay and multiple individual upper
chambers. In
other cases a single upper chamber that accesses all of the ion transport
holes used in ar.~
assay and multiple individual lower chambers. Different chamber arrangements
can have
different electrode connections, connections to fluidic channels for the
addition and
removal of solutions and cells, and connection to pneumatic devices for
sealing particles
to ion transport measuring holes by the application of pressure.
In one preferred design, both the upper chamber piece and the lower chamber
piece comprise multiple chambers that align with the extended portion of the
flexible
biochip such that each ion transport measuring hole is associated with a
single upper
chamber and a single lower chamber. In this design, cells, extracellular
solutions, and
compounds can be added to the top chambers either by individual conduits or by
fluid
dispensing systems. Pneumatic conduits connect with the lower chambers to
produce
high resistance seals. Electrodes can be provided in the reusable chamber
pieces, or can
be provided in fluid conduits or as part of the ion transport recording
machinery that can
be brought into electrical contact with the chambers through electrolyte
bridges.
In yet another aspect of a flexible ion transport measurement biochip, the
flexible
biochip can form an at least partially tubular structure. The flexible biochip
can form at
least a portion of a tube. Where the flexible biochip does not form the
complete
circumference of a tube, the same flexible substrate material or a different
material can
form the remainder of the circumference of the tube. For example, the same
flexible
substrate material or a different material can form a basin or bottom surface
of a trough-
like structure that is continuous with the curved chip but can be at least in
part flat or
have a lesser degree of curvature. In this embodiment, the interior of the
"tube" can form
a single intracellular chamber, and an "upper" chamber piece can fit around
the tube to
provide upper chambers. In this aspect, cells, measuring solution (such as
extracellular
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solution) and compounds can be added to individual upper chambers that can
also
contain, or be in electrical connection with, recording electrodes. The inner
tube chamber
can be a common chamber that has fluidic and pneumatic connections for
providing
measuring solutions and applying pressure for sealing of cells or particles to
ion transport
measuring holes. Preferably in this embodiment the lower chamber comprises or
is in
electrical connection with a reference electrode.
The present invention also includes a method of using a flexible biochip for
measuring ion transport activity or properties. The flexible biochip can be
part of a device
in which sections of the flexible biochip are sequentially unwound for
sequential sets of
assays, or can be used as an at least partly curved surface.
The flexible biochip concept can be applied to not only ion transport assays,
but
also other high-throughput tests, in which a expanse of the biochip is used
for testing at a
time, where the top and optional the bottom surface of the biochip can be
engaged in
activities such as reagent delivery, detection, separation, etc.
Theta tubing-based Chip
Another aspect of the present invention is a method of making a multiplex ion
transport measuring device using theta tubing. Either semicircular or
rectangular theta
tubing can be used, however, in some cases rectangular theta tubing can be
preferred
because the septum between the theta openings (referred to herein as
"compartments") is
typically of a more uniform thickness in rectangular theta tubing. In this
method, multiply
segments of theta tubing can be stacked on top of one another or arranged side-
by-side,
where each segment comprises an ion transport measuring means (recording
site).
The method comprises: providing at least two segments of theta tubing, each of
which comprises an upper compartment and a lower compartment, where the upper
compartment and lower compartment is separated by a glass septum; cutting an
opening
in the top of the theta tubing segments to provide access to the upper
compartment; using
the access at the top of the upper compartment to make at least one hole
through the glass
septum that separates the upper and lower compartments of each piece of theta
tubing;
and attaching the at least two segments of theta tubing one on top of another,
such that
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the bottom compartment of a second theta tubing segment is on top of the upper
compartment of a first theta tubing segment.
Preferably, openings cut in the top that are made to provide access for laser
drilling or etching of the hole are sealed prior to stacking the theta tubing
segments on top
of one another. Figure 4A depicts a theta segment having an upper compartment
(418)
and a lower compartment (419) in which a hole has been cut in the top (421)
for laser
access to drill an ion transport measuring hole (402) through the septum (420)
of the
segment. Rubber, polymers, or even glass can be used to close the opening
using
adhesives or heat for sealing. For example, the opening in the top of the top
compartment
can be sealed when the theta segments are stacked on top of one another,
preferably by
placing a gasket (such as a piece of flexible rubber, plastic, or silicone)
over the opening
and stacking the next theta segment on top of it. The gasket can be held in
place by
adhesives clamps, or f heat can also be used to attach the stacked units to
one another.
Sealing of the hole can be done such that a port is left in the top of the top
chamber. In
embodiments where the units are attached side-by-side, the port can be used
for adding
compounds or cells.
In the assembled device, each theta tubing segment comprises at least one
(preferably one) ion transport recording site, and each theta tubing segment
comprises an
ion transport recording unit, having an upper chamber (upper compartment of
the theta
tubing segment) and a lower chamber (lower compartment or opening of the theta
tubing
segment). The multiple ion transport measuring units can be arranged
vertically as
depicted in Figure 4B, with the upper chambers (418) and lower chambers (419)
of each
unit open on either side and connected to inflow conduits (422) on one side
and outflow
conduits (423) on the other side. In an alternative design, depicted in Figure
4C,
multiple ion transport measuring units can be arranged side-by-side, with the
upper and
lower chambers of each unit open each open on either side and connected to
inflow
conduits (422) on one side and outflow conduits (423) on the other side.
The open sides of each chamber are used to attach conduits for fluid flow,
cell and
compound delivery, and pneumatic control. In some preferred embodiments of the
present invention, depicted in Figures 4B and 4C, individual conduits for
providing
extracellular solution, compounds, and cells, are attached to one side of each
upper
CA 02554376 2006-07-06
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compartment of the theta structure, and individual conduits lead out of each
upper
chamber at the opposite side of the theta structure. In these designs,
individual conduits
providing intracellular solution can be attached to one side of each lower
compartment of
the theta structure, and individual conduits for outflow of intracellular
solution lead out of
each lower chamber at the opposite side of the theta structure. Pressure can
be applied
either from the intracellular inflow conduit or the intracellular outflow
conduit.
Many different arrangements are possible, for providing solutions, compounds,
cells or particles, and pressure to a theta multiplex ion transport measuring
device. For
example, cells can be introduced to the lower chamber, and pressure for
sealing of cells
can be applied to the upper chamber. Conduits can be arranged in any way that
can
provide pressure for particle sealing and fluid flow for the addition of
solutions,
compounds, and particles such as cells.
In making the device, commercially available theta tubing can be used. The
glass
tubing can be cut into segments of any size that will allow the segment to
function as ion
transport measuring unit. For example, in some preferred embodiments, the
segments can
be from about 0.1 mm to about 80 mm in width, more preferably from about 1 mm
to
about 10 mm in width. The volumes of the upper and lower chambers of the units
can be
the same or different. Preferably, the extracellular chamber has internal
measurements of
at least 20 microns by 20 microns, and the intracellular chamber has internal
measurements of at least 10 microns by 10 microns.
Dimensions of the ion transport measuring through holes that are made (for
example, by laser drilling or etching) into the theta separator segments are
preferably
from about 0.3 to about 8 microns in diameter. The ion transport measuring
holes can
also include etched or laser drilled counterbores, as described previously in
this
application.
As described herein, the surface of the theta segment can be treated or coated
to
promote sealability of the surface as described previously in this
application.
From two to 100 or more theta segments can be attached in vertical or parallel
orientation (see Figures 4B and 4C). Attachment can be through the use of
adhesives,
gaskets, and the lilce. As mentioned above, the opening in the upper chamber
can be
sealed before or during attachment of the units. Conduits for the addition of
solutions,
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cells, and compounds, and for the application of pressure can be attached both
open ends
of the chamber in any functional way, and can also use gaskets, adhesive,
adaptors, etc.
Electrodes, if provided within the chambers, can be inserted into chambers
before or after
assembling the multiplex structure.
Electrodes can be situated within upper and lower chambers of the segments.
Alternatively, for a theta multiplex device, an electrode provided external to
a chamber
can be in electronic contact with one or more upper chambers through an
electrolyte
(solution) bridge. For example, one or more electrodes can be provided in one
or more
conduits leading to one or more upper chambers of the device, or provided as
part of the
ion transport recording machinery (signal source/amplifier) such that the
electrode or
electrodes are in electrical contact with an ion transport measuring solution.
Similarly,
one or more electrodes can be provided in one or more conduits leading to one
or more
lower chambers of the device, or provided as part of the ion transport
recording
machinery (signal source/amplifier) such that the electrode or electrodes are
in electrical
contact with an ion transport measuring solution. In some preferred
embodiments of the
present invention in which a device is used for whole cell ion transport
measurement, the
upper chamber of each theta ion transport measuring unit is the "extracellular
chamber"
that comprises or is in electrical contact with a reference electrode. In this
case, multiple
upper chambers can optionally be in electrical contact (for example, through
conduits that
provide solution bridges) with a single reference electrode.
Many other electrode arrangements are possible, however, including but not
limited to a single reference electrode in electrical contact with multiple
lower chambers
(which can be "intracellular" or "extracellular" chambers of the units),
individual
reference electrodes for each lower chamber, individual reference electrodes
for each
upper chamber, etc. Recording electrodes can also be provided within chambers
or in
electrical contact (for example, through conduits that provide solution
bridges) with
chambers.
The present invention also includes ion transport measuring devices made using
the methods of the present invention. These ion transport measuring devices
comprise at
least two attached theta tubing segments, wherein the theta separator segment
of each of
the theta tubing segments comprises an ion transport measuring hole.
Preferably, the
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upper and lower chambers of each theta tubing segment comprises or is in
electrical
contact with at least one electrode. Preferably, a theta ion transport
measuring device
comprises conduits that attach to upper and lower chambers of each theta
tubing segment.
The theta ion transport measuring device can comprise any functional
arrangement of
electrodes or electrical connections to electrodes, and any functional
arrangement of
fluidic and pneumatic structures (such as conduits, valves, and can connect
systems for
controlling fluid flow and pressure (for example, pumps) , and electronic
equipment for
ion transport measurement.
For example, the lower chamber of a theta ion transport measuring unit
preferably
engages an inflow conduit on one side of the lower chamber and an outflow
conduit on
the opposite side of the lower chamber. A lower chamber can include or contact
a lower
chamber electrode that is preferably introduced into the lower chamber via an
inflow or
the outflow conduit. The lower chamber electrode in this embodiment can be,
for
example, a wire electrode inserted into the conduit. The electrode can be a
common
electrode (that contacts more than one lower chamber) or an individual
electrode, in
which each lower chamber contacts or comprises its own electrode. An
individual lower
chamber electrode can also be positioned in a lower chamber. A lower chamber
common
or individual electrode can also be part of a separate part of an apparatus
used for ion
transport measurement (such as for example, a signal amplifier) that during
use of the
device, is positioned such that it is in electrical contact with one or more
lower chambers.
This can be achieved, for example, by putting the one or more lower chamber
electrodes
in contact with a salt bridge (such as a solution-filled conduit) that engages
the lower
chamber. A lower chamber also preferably engages at least one conduit that
provide
pneumatic control of the ion transport recording unit. For example, the
outflow conduit of
a lower chamber can be connected to a pressure source such as a pump or
syringe.
The upper chamber of a theta ion transport measuring unit preferably also
engages
an inflow conduit on one side of the lower chamber and an outflow conduit on
the
opposite side of the lower chamber. An upper chamber can include or contact an
upper
chamber electrode that is preferably introduced into the lower chamber via an
inflow or
the outflow conduit. The upper chamber electrode in this embodiment can be,
for
example, a wire electrode inserted into a conduit that leads to the chamber.
The electrode
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can be a common electrode (that contacts more than one upper chamber) or an
individual
electrode, in which each lower chamber contacts or comprises its own
electrode. An
individual upper chamber electrode can also be fabricated into or positioned
in an upper
chamber. An upper chamber common or individual electrode can also be part of a
separate part of an apparatus used for ion transport measurement (such as for
example, a
signal amplifier) that during use of the device, is positioned such that it is
in electrical
contact with one or more lower chambers. This can be achieved, for example, by
putting
the one or more upper chamber electrodes in contact with a salt bridge (such
as a
solution-filled conduit) that engages the lower chamber.
The present invention also includes methods of using ion transport measuring
devices comprising at least two attached theta tubing segments to measure at
least one ion
transport activity or property of at least one particle (such as a cell).
Methods of ion
transport measurement are well known in the art and also described herein.
The present device can be used for any type of ion transport measurement,
including whole cell, single channel, outside-out patch and inside-out patch
recording.
The multiplex theta device can be used for testing the effect of known arid
unknown
compounds on ion transport activity of cells and particles.
UPPER CHAMBER DESIGNS
Flow-through Upper Chamber
Another aspect of the present invention is a device for ion transport
measurement
that comprises a chip having at least one ion transport measuring hole and at
least one
upper chamber, where the one or more upper chambers comprise at least two
openings, in
which one of the openings is on one side of the one or more ion transport
measuring
holes and another of the openings is on the other side of the one or more ion
transport
measuring holes. In this device, a simple version of which is depicted in
Figure 5, the
upper chamber (518) is a "flow-through" chamber that is accessed by at least
one ion
transport measuring hole (502) through a chip (501).
Fluids such as solutions and suspensions (for example, measuring solutions,
wash
solutions, samples comprising particles such as cells, or compound solutions)
can be
added through a first opening on one end of the flow-through chamber and
removed from
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the chamber via an opening on another end of the flow-through chamber. Fluid
flow
through the chamber can be provided by pumps or syringe mechanisms, and
preferably
the flow rate is regulable. Preferably, fluid flow into and out of the chamber
is via inflow
and outflow conduits that engage the openings at either end of the chamber.
Preferably,
fluid flow into and out of the chamber can also be controlled by valves that
can permit
flow into or out of the chamber, or close off flow into or out of the chamber.
In some embodiments of this device, one of the two or more openings is
directly
or indirectly connected to a reservoir at its end where cells and,
potentially, compounds
can be added to the upper chamber such as by a fluidic system or pipette. For
example,
the device depicted in Figure 6 has a flow-through upper chamber (618) and a
flow-
through lower chamber (619) separated by a chip (601) that comprises an ion
transport
measuring hole (602). The flow-through upper chamber (618) connects to a
conduit (623)
at one end of the chamber, and a reservoir (626) for the addition of sample at
the other
end of the chamber. The device shown in Figure 6 can be a single-unit device,
or a
device of the present invention can comprise multiple ion transport recording
units, each
comprising a flow-through upper chamber and a flow-through lower chamber
connected
by an ion transport measuring means.
The one or more upper chambers of a device of the present invention can
comprise an electrode, or, during use of the device, can be in electrical
contact with an
electrode that can be part of the signal amplifier machinery or can be
provided in tubing
leading to the chamber.
In preferred embodiments of this aspect of a device of the present invention,
a
device has a single upper chamber with two openings, one on either side of the
one or
more ion transport measuring holes, such that measuring solution buffers, or
compound
containing solutions (such as Extracellular Solution, ES) can flow through the
upper
chamber. For example, measuring solution can be pumped through the upper
chamber to
fill or wash the chamber. In embodiments in which an opening directly or
indirectly
accesses a reservoir outside the chamber, particles such as cells and
compounds can
optionally be added to the upper chamber via the reservoir. In the
alternative, solutions,
particles, or compounds can be added to the upper chamber at an opening that
does not
provide access to a reservoir. Preferably, at least a portion of the upper
surface of a flow-
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through upper chamber device is transparent, so that cells in the upper
chambers can be
viewed microscopically.
A device of the present invention can have multiple flow-through upper
chambers, each of which is accessed by a single ion transport measuring hole,
or can
have multiple flow-through upper chambers, each of which is accessed by
multiple ion
transport measuring holes, or, in some preferred embodiments, can have a
single flow-
through upper chamber that is accessed by multiple ion transport measuring
holes.
One preferred embodiment is a device having a chip that comprises two or more
ion transport measuring holes that access a single flow-through upper chamber.
In this
embodiment, the flow-through chamber can be arranged as a channel having an
inlet at
one end, two or more ion transport measuring holes positioned in a linear
fashion along
the course of the channel, and an outlet at the opposite end. An upper chamber
channel
can be straight or curved, and a chip can optionally engage more than one flow-
through
upper channel. Figure 10 depicts a device comprising a chip (101) that has
multiple ion
transport measuring holes (102) that access a flow through upper chamber
channel (118)
that engages an inflow conduit (122) at one end of the channel (118) and an
outflow
conduit (123) at the other end of the channel (118). A chip of a device that
comprises a
flow-through upper chamber channel can comprise one or more holes, preferably
four or
more holes, more preferably 16 or more holes, and more preferably yet 48 or
more holes,
all of which can access a single channel. A device comprising an upper chamber
channel
that accesses multiple ion transport measuring holes of a chip preferably also
comprises
multiple lower chambers, each of which accesses on of the ion transport
measuring holes
of the chip. Preferably, the upper chamber comprises, contacts, or, during use
of the
device, is in electrical contacts with an electrode that serves as a common
upper chamber
electrode, and each of the multiple lower chambers comprises, contacts, or,
during use of
the device, is in electrical contacts with an individual electrode that serves
as a recording
electrode. Preferably, the lower chambers have inflow and outflow conduits for
the
addition and removal of measuring solutions, and are connected to at least one
pneumatic
device for applying pressure through the lower chambers to seal particles in
the upper
chamber channel to ion transport measuring holes.
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In some preferred designs, a flow-through upper chamber ion transport
measuring
device comprises an upper chamber piece that forms at least the walls of the
one or more
flow-through upper chambers, and a chip that comprises at least one ion
transport
measuring hole that forms the bottom of the chamber. The upper chamber piece
can
reversibly or irreversibly engage the chip such that a fluid impermeable seal
is formed
between the upper chamber piece and the chip. It is also possible to have an
upper
chamber piece that forms the walls of at least one upper chamber and also
comprises
lower surfaces of the chambers. In this case, the upper chamber piece itself
comprises one
or more ion transport measuring holes that are machined, etched, or drilled
into the
bottom surface of the one or more upper chambers, where the bottom surface of
the upper
chamber piece serves as the "chip" or substrate where particle sealing takes
place.
Preferably, the upper chamber piece also forms the tops of the chambers. In
one
alternative, the upper chamber piece can reversibly or irreversibly engage a
top piece that
forms the top of the one or more upper chambers. In some preferred embodiments
at least
a portion of the top surface of the chambers is transparent, allowing cells or
other
particles being assayed to be viewed microscopically. In some embodiments,
however,
the upper chamber or chambers of a device of the present invention can be open
at the
top.
An upper chamber piece can be made of any suitable material, including but not
limited to, one or more plastics, one or more polymers, glass, one or more
ceramic
materials, coated metals, or combinations thereof. Nonlimiting examples of
plastics that
can be used in the manufacture of upper chamber pieces include, but are not
limited to
polyallomer, polypropylene, polystyrene, polycarbonate, cyclo olefin polymer,
polyimide, paralene, PDMS, polyphenylene ether/PPO, Noryl~, and Zeonor~. Glass
and
transparent polymers are preferred transparent materials, with transparent
polymers such
as polycarbonate and polystyrene having the advantage of easier manufacture.
The design and dimensions of a flow-through upper chamber piece, as well as
the
dimensions of upper chambers, can vary according to the preferences of the
user and are
not limiting to the present invention. For example, the volumetric capacity of
the one or
more flow-through upper chambers formed by the piece can vary from about ten
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microliters to about 100 milliliters or more, depending in part on the number
of ion
transport measuring holes that access an upper chamber.
The upper chamber or chambers of a device of the present invention can
optionally comprise one or more electrodes. Electrodes can be attached to or
fabricated
on the walls of the chamber or chambers, or can be attached to or fabricated
on the
bottom surface of an upper chamber, such as on the surface of a chip that
forms the
bottom of the upper chamber or chambers. In preferred embodiments in which a
flow-
through upper chamber is accessed by more than one ion transport measuring
hole, an
upper chamber can comprise a single electrode that can be used as a common
reference
electrode during ion transport measurement assays. In alternative designs, an
electrode or
electrodes can be in electrical contact with the one or more upper chambers
during use of
a device. In these designs, an electrode can be provided in a conduit leading
to an upper
chamber, or can be part of another machine or device (such as, for example, a
signal
amplifier) that is connected through a salt bridge (for example, measuring
solution in a
conduit connected to the device) to the upper chamber.
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 preferred, such as Ag/AgCI wires.
In using device of the present invention having one or more flow-through upper
chambers, the device can engage a lower chamber piece. The lower chamber piece
can be
in the form of a tray or tank, and preferably has at least one inlet and at
least one outlet
for allowing measuring solution (such as IS, intracellular solution) to flow
into the
chamber and for the application of pressure for sealing particles to the one
or more ion
transport measuring holes. In some preferred embodiments the lower chamber is
also a
single flow-through channel, with an opening at one end for the introduction
of solutions
such as measuring solution, and an opening at the other end for outflow of
solutions, and
preferably, connection to pneumatic devices for applying pressure to seal
particles to the
one or more ion transport measuring holes.
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The present invention also includes ion transport measuring devices with one
or
more flow-through upper chambers that also comprise one or more lower
chambers. In
these devices, each of the one or more lower chambers is accessed by one or
more ion
transport measuring holes of the device. In preferred designs, a chip that
comprises one or
more ion transport measuring holes forms the upper surface of the one or more
lower
chambers. Preferably, at least one pneumatic device is connected to one or
more lower
chambers of a device of the present invention, such as, for example, a pump or
syringe
that is connected to a lower chamber via a conduit. The pneumatic device can
provide
pressure control to seal a particle in an upper chamber in fluid communication
with the
lower chamber to an ion transport measuring hole of the chip.
Several designs of ion transport measuring devices that comprise flow-through
upper chambers and at least one lower chamber are possible. For example, a
device can
have multiple flow-through upper chambers, each of which is accessed by one
ion of
multiple transport measuring hole, and a single lower chamber, where the
single lower
chamber is in fluid communication with multiple upper chambers via multiple
ion
transport measuring holes. In an alternative embodiment, a single flow-through
upper
chamber (such as the upper chamber channel depicted in Figure 10) is accessed
by
multiple ion transport measuring holes, each of which accesses one of multiple
lower
chambers. In another design, a device has a chip comprising one or more ion
transport
measuring holes, one or more flow-through upper chambers, and one or more
lower
chambers (preferably also having a flow-through design). Each of the flow-
through upper
chambers is accessed by a single ion transport measuring hole that also
accesses a single
lower chamber. For example, Figure 6 depicts a biochip having a single ion
transport
measuring hole, a flow-through upper chamber and a flow-through bottom
chamber,
where the cells can be viewed through the top of the upper chamber using a
microscope.
The invention also includes devices having multiple upper chambers, and
multiple lower
chambers, where each of the multiple upper chambers is in fluid communication
with one
of the multiple lower chambers via one of multiple ion transport measuring
holes.
In preferred embodiments of aspects of the present invention having a chip
comprising one or more ion transport measuring holes and at least one flow-
through
upper chamber, an upper chamber piece that forms at least the walls of one or
more upper
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chambers is reversibly or irreversibly attached to the upper surface of a chip
that forms
the bottoms of the upper chambers, and a lower chamber piece that forms at
least the
walls of one or more lower chambers is reversibly or irreversibly attached to
the lower
surface of a chip that forms the tops of the upper chambers. The lower chamber
piece can
comprise any suitable material, including but not limited to, one or more
plastics, one or
more polymers, glass, one or more ceramic materials, coated metals, or
combinations
thereof. Nonlimiting examples of plastics that can be used in the manufacture
of lower
pieces include, but are not limited to polyallomer, polypropylene,
polystyrene,
polycarbonate, cyclo olefin polymer, polyimide, paralene, PDMS, polyphenylene
ether/PPO, Noryl~, and Zeonor~. Glass and transparent polymers are some
preferred
transparent materials, with transparent polymers such as polycarbonate and
polystyrene
having the advantage of easier manufacture.
The design and dimensions of a lower chamber piece, as well as the dimensions
of lower chambers, can vary according to the preferences of the user and are
not limiting
to the present invention. For example, the volumetric capacity of the one or
more upper
chambers formed by the piece can vary from about one microliter to about 100
milliliters
or more, depending in part on the number of ion transport measuring holes that
access a
lower chamber.
The lower chamber or chambers of a device of the present invention can
optionally comprise one or more electrodes. Electrodes can be attached to or
fabricated
on the walls or lower surface of the chamber or chambers, or can be attached
to or
fabricated on the bottom surface of a chip that forms the upper surface of a
lower
chamber or chambers. In preferred embodiments in which a flow-through lower
chamber
is accessed by more than one ion transport measuring hole, a lower chamber can
comprise a single electrode that can be used as a common reference electrode
during ion
transport measurement assays. In alternative designs, an electrode or
electrodes can be in
electrical contact with the one or more lower chambers during use of a device.
In these
designs, an electrode can be provided in a conduit leading to a lower chamber,
or can be
part of another machine or device (such as, for example, a signal amplifier)
that is
connected through a salt bridge (for example, measuring solution in a conduit
connected
to the device) to a lower chamber.
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Upper chamber separator unit
In yet another aspect of the present invention, an ion transport measuring
device comprises a chip comprising two or more ion transport measuring holes;
a
common upper chamber positioned above the chip, such that the chip forms the
bottom of
the common upper chamber and the two or more ion transport measuring holes of
the
chip access the common upper chamber, and an upper chamber separator unit that
can b a
reversibly lowered onto the chip to separate the common upper chamber into
multiple
individual upper chamber compartments that are in fluidic isolation from one
another.
The upper chamber separator unit comprises multiple separator segments that
contact the
upper surface of the chip within the upper chamber to form at least a portion
of the walls
of the multiple individual upper chamber compartments, each of which is in
register with
an ion transport measuring hole of the chip.
The physical separator can reversibly fasten on to the substrate. The upper
chamber separator can comprise any fluid-impermeable material, and preferably
comprises a compressible material such as a polymer) where the separator
segments
contact the surface of the chip to seal against the chip so that the separator
forms fluid-
impermeable separated upper chamber compartments. Preferably, the upper
chamber
separator comprises one or more top pieces attached to the separator segments
to serve as
tops or lids of the upper chamber compartments. Preferably, the one or more
top pieces
comprise openings that can be used, for example, for compound delivery to the
upper
chambers, or for electrode contact with the upper chamber compartments. At
least a
portion of a top piece can optionally be transparent so that particles such as
cells in the
upper chamber compartments can be viewed microscopically.
The devices of the present invention that comprise physical separator units
for
forming chambers can comprise ion transport chips as they are known in the art
and
described herein, including, for example, planar chips, flexible chips, and
MGP chips.
Chips used in the devices can be treated, such as using methods described
herein, to
improve their sealing properties. For example, a chip used in a device of the
present
invention can be made more electronegative by, for example, chemically
treating the chip
with at least one salt or at least one base.
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The upper chamber of a device can comprise an electrode. For example, an
electrode layer that can serve as a common upper chamber electrode can be
fabricated
onto the upper surface of the chip. In this embodiment, the device comprises
or engages
multiple lower chambers, each of which comprises or contacts an individual
electrode. In
an alternative design, the upper chamber separator unit can comprise either a
common
electrode or individual electrodes that contact each upper chamber compartment
when the
separator unit is positioned on the chip. The one or more electrodes can be
attached to the
upper chamber separator, or inserted through conduits that engage the upper
chamber
separator, or can be in electrical communication with the upper chamber
compartments
via one or more conduits that engage the upper chamber separator.
Preferably, a device with an upper chamber separator unit further comprises or
engages one or more lower chambers. For example, a device can further comprise
a
common lower chamber positioned beneath the chip, where the chip forms the top
of the
common lower chamber and the two or more ion transport measuring holes access
the
lower chamber. In an alternative embodiment, the-device can further compxise
two or
more lower chamber positioned beneath the chip, where the chip forms the tops
of the
multiple lower chambers, each of which is aligned with a single ion transport
measuring
hole. In the embodiment depicted in Figure 7, the ion transport measuring
device
comprises a chip (701) having multiple ion transport measuring holes (702),
each of
which accesses an independent lower chamber (719). The device has a common
flow-
through upper chamber (718) that is accessed by multiple ion transport
measuring holes
(702). The upper chamber engages an inflow counduit (722) and an outflow
conduit
(723). The device also comprises an upper chamber separator unit (727)
comprising
separator segments (728) that, when the separator unit is lowered onto the
chip within the
common upper chamber, form independerit upper chamber compartments, each of
which
is accessed by a single ion transport measurement hole of the chip.
Preferably, the one or more lower chambers of a device that comprises an upper
chamber separator are connected to at least one pneumatic device for the
application of
pressure to the one or more lower chambers for sealing particles in the upper
chamber to
ion transport measuring holes. For example, the one or more lower chambers can
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comprise a conduit that can connect to a pump or syringe for applying negative
pressure
to the one or more lower chambers.
Where the upper chamber separator unit provides individual electrodes, the
device
can optionally have a common lower chamber that comprises or contacts a common
electrode or multiple lower chambers that comprise or contact a common
electrode or
individual electrodes.
In embodiments where the device comprises multiple lower chambers, each of the
multiple lower chambers can each comprise or contact an individual electrode.
The lower
well electrodes can optionally be provided by a separatc adaptor plate that
can reversibly
engage the lower wells and can also optionally compris a connections to
pneumatic
devices for pressure control. In this case, a common electrode (for example,
an electrode
that traverses the upper surface of the chip) can contact or be positioned
within the upper
chamber or be brought into electrical contact with the multiple upper chamber
compartments.
The upper chamber separator unit can be lowered into the upper chamber (which
can be in the form of a tank with ion transport measuring holes in the bottom)
after
measuring solution and cells (or other particles) have been introduced into
upper chamber
and preferably after particles have been sealed to the ion transport measuring
holes.
Preferably, the upper chamber is a flow-through upper chamber that connects to
inflow
and outflow conduits that can be used for adding measuring solutions and cells
before the
separator unit is lowered onto the chip, and for washing the chamber after
assays have
been completed and the separator unit has been raised off the chip.
In embodiments in which the separator unit comprises multiple electrodes, each
of
the separate electrodes can contact a separate chamber when the separator
engages the
chip. In these embodiments, the device can also compri se or engage a common
lower
chamber that can comprise or contact a common electrode that can be used as a
reference
electrode. In an alternative, a common upper chamber electrode can be built
onto the
upper surface of the chip.
After cells have sealed to the chip and the separator unit has formed separate
upper chambers, compounds can be added to the individual upper chambers,
either by
conduits or fluid dispensing systems. In embodiments in which the separator
unit also
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forms the tops of upper chamber compartments, solution dispensing can occur
through
openings in the separator unit. Ion transport recording can then be performed
using upper
chamber recording electrodes and a bottom chamber reference electrode, or
preferably, a
common upper chamber reference electrode and recording electrodes that contact
the
lower chambers of the device.
Figure 7 depicts an ion transport measuring device of the present invention
having a common upper chamber that is divided into separate upper chamber
compartments by an upper chamber separator unit. In Figure 7A, a device is
shown
having a chip (701) comprising ion transport measuring holes (702) and a
common flow-
through upper chamber (708) with an inlet (709) and an outlet (710). T'he
device also has
multiple lower chambers (711) in register with the ion transport measuring
holes (702).
Devices comprisin , chips having built-on upper wells
In a related aspect, the present invention comprises a chip comprising at
least one
ion transport measuring hole, in which the chip comprises at least one upper
well on its
top surface surrounding an ion transport measuring hole and the upper well is
built onto
the chip. In one preferred embodiment, the well comprises a layer of wax.
The chip can comprises any hard material such as metals, ceramics, polymers,
inorganic and organic hybrid materials, plastics, silicon dioxide, or glass,
and the ion
transport measuring holes can be etched, laser drilled, cut, punched ourt, or
bored into the
material. In preferred embodiments, the chip is a glass chip and the ion
transport
measuring holes are laser drilled. Preferably, the chip is surface-treated,
such as by using
methods described herein.
Preferably, the wax on the upper surface of the chip forms individual wells,
after
ion transport measuring holes is created through the chip. Figure 8A depicts
an overhead
view of a chip (801) having two upper chambers (818). Figure 8B depicts the
chip (801)
in cross section, showing the upper chambers (818) made of wax wells (828)
surrounding
ion transport measuring holes (802).
As in the previous embodiment, during use, the chip is assembled with one or
more structures to form an ion transport measuring device. In this case,
however, the chip
comprises upper chamber wells on its surface, and the chip engages a structure
that
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preferably comprises as upper piece top surface that forms the top of the
upper wells, as
well as a lower chamber piece. The wax-formed upper chamber structures on the
upper
surface of the chip are at least somewhat compressible, allowing sealing of
the upper
chamber structures to the upper piece surface when the device is assemb led.
The upper piece top surface that engages the chip can also include conduits
and,
optionally, electrodes that can connect with the one or more upper chambers of
the device
when the device is assembled.
Preferably, the chip comprises multiple wax-formed upper chambers and the
lower chamber piece it assembles with has multiple isolated lower charn_ber
wells, but
other designs are possible. For example, the chip can have a single wax-formed
upper
chamber, and can be assembled with a structure that comprises multiple
isolated lower
chambers. In an alternative, the chip comprises multiple wax-formed upper
chambers and
the lower chamber piece has a single common lower chamber.
In preferred embodiments, the chip having wax-formed upper chambers is single-
use and disposable, and the lower chamber piece and the structure that
comprises the
upper piece top surface, as well as associated electrodes (which can be p art
of the signal
amplifier machinery or electrodes that can be attached or connected to the
wells), are
reusable.
Another material that can be used for forming wells on the surface of a
biochip is
SU-8. SU-8 is a photo-curable epoxy oligomer, commonly used for computer chip
manufacture. To make one or more wells on the surface of a chip using SU-8,
the liquid
form of the oligomer is distributed on the surface of the chip. A mask is used
to pattern
one or more wells. Light induces polymerization of SU-8 in areas not covered
by the
mask. After polymerization, the unpolymerized SU-8 is washed away to leave
chamber
walls that comprise SU-8 polymer.
Chip with O-rin~upper chambers
In a related aspect of the present invention, a chip comprising at least one
ion
transport measuring hole is provided with at least one O-ring that forms an
upper
chamber around the at least one ion transport measuring hole. Figure 9 i s a
cross-
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sectional view showing a chip (901) having an ion transport measuring hole
(902)
surrounded by an O-ring (928) that forms an upper chamber (918).
The chip having O-ring upper chambers can be assembled with at least one
structure to form an ion transport measuring device.
The chip can comprises any hard material such as metals, ceramics, polymers,
plastics, silicon dioxide, or glass, and the ion transport measuring holes can
be etched,
laser drilled, cut, punched out, or bored into the material. In preferred
embodiments, the
chip is a glass chip and the ion transport measuring holes are laser drilled.
Preferably, the
chip is surface-treated to increase its sealing properties, such as by using
methods
described herein.
To assemble an ion transport measuring device, the chip preferably engages a
structure that preferably comprises as upper piece top surface that forms the
top of the
upper wells that can be reversibly attached to the top of the chip, as well as
a lower
chamber piece that can be reversibly attached to the bottom of the chip. The
upper
chamber O-ring structures on the upper surface of the chip are at least
somewhat
compressible, allowing sealing of the upper chamber structures to the upper
piece surface
when the device is assembled. The O-ring can also be sealed to the top of chip
surface
using an adhesive.
The upper piece surface can also include conduits and, optionally, electrodes
that
can connect with the one or more upper chambers of the device when the device
is
assembled.
Preferably, the chip comprises multiple O-ring upper chambers and the lower
chamber piece has multiple isolated lower chamber wells, but other designs are
possible.
For example, the chip can have a single O-ring upper chamber, and can be
assembled
with a structure that comprises multiple isolated lower chambers. In an
alternative, the
chip comprises multiple O-rmg upper chambers and the lower chamber piece has a
single
common lower chamber well.
In preferred embodiments, the chip having O-ring upper chambers is single-use
and disposable, and the upper piece surface and lower chamber piece, as well
as
associated electrodes (which can be part of the signal amplifier machinery or
electrodes
that can be attached or connected to the wells), are reusable.
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FLUIDIC SYSTEMS
Overhead Deliver~of Solutions to Ion Transport Recording Sites in Flow-Through
Upper
Chambers
The present invention provides novel fluidic systems for delivering solutions,
compounds, and particles (such as cells) to compartments of ion transport
measuring
devices. These fluidic systems can be applied to a number of chip designs and
device
designs that may vary in their structures, electrode arrangements, or pressure
systems.
In some preferred embodiments of the novel fluidic systems of the present
invention, an ion transport measuring device comprises a biochip comprising
two or more
ion transport measuring holes, and a flow-through upper chamber positioned
above the
biochip that is accessed by the two or more ion transport measuring holes. The
two or
more ion transport recording holes access the one or more upper chambers at
ion
transport measurement recording sites. The device further comprises a fluid
delivery
system comprising two or more fluid delivery units, each of which can be
aligned directly
over one of the two or more ion transport recording sites each of which
encompasses an
ion transport measurement hole. Solutions (including test compound solutions)
can be
added to ion transport recording sites that surround the ion transport
recording holes
through the fluid delivery units that can be positioned over the ion transport
recording
sites. Preferably, the fluid delivery units can align directly over and in
close proximity to
the ion transport measuring holes of the chip. The fluid delivery units can
comprise, for
example, pipets, conduits, pipes, tips, or sonic actuators. The diameter of
the dispensing
opening of a fluid delivery unit is preferably less than half the distance
between ion
transport measuring holes of the chip. Preferably, the diameter of the
dispensing opening
of a fluid delivery unit is between about 50 microns and 5000 microns, more
preferably
between about 200 microns and about 2000 microns.
The fluid delivery units are preferably part of a fluid delivery array (for
example,
a multichannel pipet array) of a fluidics block that can be reversibly
positioned over the
upper chamber such that individual delivery units of the array align with ion
transport
measurement recording sites of the device. Preferably, positioning of the
delivery units is
automated.
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The flow-through upper chamber comprises at least one inlet and at least one
outlet that can allow for fluid flow through the chamber. Chamber solutions
(such as
measuring solutions such as ES) and cells or compounds can be added via an
upper
chamber inlet. An electrode, such as a reference electrode, can optionally be
provided
within or, during use of the device, in electrical connection with the flow-
through upper
chamber.
In some preferred embodiments, a flow-through upper chamber of a device of the
present invention comprises an upper surface that comprises two or more
openings, in
which each of the two or more openings is aligned over one of the two or more
ion
transport recording sites. The openings provide access of the fluid delivery
units to ion
transport recording sites of an upper chamber. In other embodiments, a chamber
does not
have an upper surface, and each of the two or more fluid delivery units can be
aligned
directly over the one of the two or more ion transport measuring recording
sites and
solutions can be added via fluid delivery units that are positioned over ion
transport
recording sites.
These embodiments of the present invention that include ion transport
measuring
devices having flow through upper chambers and overhead delivery systems can
comprise ion transport chips as they are known in the art and described
herein, including,
for example, planar chips, flexible chips, chips having hydrophobic
modifications, and
MCP chips. Preferably, a chip of a device having a flow-through upper chamber
and an
overhead delivery fluid system comprises two or more microwells, in which each
of the
two or more ion transport measuring holes of the chip is surrounded by a
microwell on
the upper surface of the chip. Such microwells can define ion transport
measurement
recording sites of the upper chamber of a device.
Preferably, a chip of a device having a flow-through upper chamber and an
overhead delivery fluid system comprises four or more holes, more preferably
16 or
more, more preferably yet 48 or more, and most preferably 96 or more.
Where feasible, chips used in the devices can be treated, such as using
methods
described herein, to improve their sealing properties. For example, at least a
portion of a
chip used in a flow-through, overhead fluid delivery device of the present
invention can
be treated to make the surface of an ion transport measuring means or
surrounding an ion
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transport measuring means more electronegative. For example, at least a
portion of a chip
used in a device of the present invention can be treated with at least one
salt or at least
one base.
Preferably, the ion transport measuring device further comprises one or more
lower chambers positioned below the chip in register with the two or more ion
transport
measuring holes of the chip. In preferred embodiments, the chip engages a
lower
chamber piece that comprises at least the walls of two or more individual
lower
chambers, such that each ion transport measuring hole of the biochip accesses
its own
lower chamber. The lower chambers preferably each comprise or contact an
individual
electrode, or during use of the device are in electrical connection with
individual
recording electrodes. The lower chambers also preferably are connected to one
or more
pumps or other pressure-generating devices, and engage conduits for the
addition and
removal of measuring solution. The lower well electrodes can optionally be
provided by a
separate adaptor plate that can reversibly engage the lower wells and can also
optionally
comprise connections to pneumatic devices for pressure control. In other
embodiments,
the device comprises a single common lower chamber that comprises, contacts,
or, during
use of the device, can be in electrical contact with a common electrode.
Various chamber and electrode designs can be used with these devices. For
example, the upper surface of the chip can comprise microwells at the
individual
recording sites, and the upper surface of the chip can comprise a common
reference
electrode that is coated with a hydrophobic material except where it contacts
the
microwells. In this case, the device has multiple independent lower wells,
each of which
is associated with a single ion transport measuring hole. Each lower well
comprises or
contacts an independent electrode that can be used for ion transport
recording.
In an alternative, the device can have a single bottom chamber that comprises
or
contacts a reference electrode. Individual recording electrodes can be
provided in
connection with the upper microwells. The individual upper chamber electrodes
can be
inserted into the microwells, for example. In one embodiment, the recording
electrodes
can be attached to the compound delivery system, such that positioning of the
compound
delivery system over the microwells can also serve to position and dip an
electrode into
the microwell.
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During use of a device of the present invention having at least one flow-
through
upper chamber and an overhead solution delivery system, there is continuous
flow of
chamber solution (such as a measuring solution) through the upper chamber, in
which the
chamber solution enters through a chamber inlet and exits through a chamber
outlet. A
test solution, such as a compound solution, is added to a recording site via a
fluid delivery
unit such that the downward directed flow of delivery system solution from the
overhead
delivery system to the recording site directs fluid flow down toward and then
away from
the ion transport measuring hole, opposing "lateral" fluid flow of chamber
solution to the
site, so that during the fluid delivery period, fluid flow is outward from
each recording
site, and each recording site is covered by test solution that is not
significantly diluted by
chamber solution. The proximal and relatively rapid, relatively high-volume
flow of
compound solution from the fluid delivery units above the recording sites
permits a
window of time for ion transport measurement in which each recording site
experiences
an essentially undiluted compound concentration.
At the same time, flow of test solutions away from each of the two or more
recording sites (to which test solutions are being delivered simultaneously)
is
accomplished by the continuous flow-through of chamber solution, which carries
delivery solutions away from recording sites and out of the chamber. This
provides
effective fluid isolation of recording sites during ion transport measurement.
After ion transport recording, the flow of solution from fluid delivery units
is
halted, while fluid flow through the chamber continues. This allows the
chamber,
including the recording sites, to be washed. During the chamber wash, the
fluid delivery
units, which can be part of a fluidics block, can optionally move away from
their
recording site positions over the upper chamber, optionally be washed or
flushed, and
filled with a second set of test solutions. The fluidics block comprising the
delivery units
can then position back over the upper chamber, such that the individual fluid
delivery
units are aligned over individual recording sites, and a second set of
compounds can be
delivered to the recording sites. A second set of ion transport measurement
assays can be
performed as the second set of compounds is delivered to the recording sites.
The fluid isolation of recording sites during compound solution delivery and
ion
transport measurement can optionally be promoted by the use of flow retarding
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structures, upper chamber microwells, hydrophobic modifications to at least
some
portions a chip surface, or combinations of these, as discussed below.
The present invention includes methods of using ion transport measuring
devices
that include flow-through upper chambers and overhead fluid delivery systems
to
measure ion transport function and properties. In preferred embodiments, the
methods are
high throughput.
In a preferred embodiment, measuring solution is added to the one or more
lower
chambers of a device that comprises a chip having microwells that surround the
holes of
the chip and comprise the ion transport recording sites of the upper chamber,
and
measuring solution and cells are introduced into the upper chamber through
conduits
attached to one or more inlets. Pressure is applied through the lower chamber
or
chambers to seal particles against the ion transport measuring holes of the
chip. Sealing
preferably occurs in the presence of complete solution superfusion of the
upper chamber.
After the seals have formed, solution is removed from the upper channel, with
the
exception of the microwells, which in the case of a coated surface electrode,
are in
electrical connection with a reference electrode. At this time compounds are
applied to
the microwells by positioning the compound delivery system over the biochip
and
dispensing compound drops over the microwells. Ion transport measurement can
then be
performed on the cells sealed at the microwells.
In using this type of device, a single cell type can be added to this type of
device
via an inlet in the flow through upper chamber for screening different
compound
solutions that are delivered through openings in the upper chamber over the
ion transport
recording sites. Preferably, solution such as measuring solution flows
continuously
through the chamber during compound delivery and ion transport measurement.
Alternatively, different cell types or particles comprising different ion
transports
can be added at different ion transport recording sites. Immediately after
cell addition,
which can be through the fluid delivery units, pressure applied from the
bottom of the
chip can allow the cells (or other particles) to seal at ion transport
measuring holes. In
this way, a particular ion transport recording site can have a particular type
or cell or
particle sealed to it. Compounds can optionally be added through the chamber
inlet or
through openings in the chamber that are localized over the recording sites,
and ion
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transport recordings can simultaneously measure the response of various cell
types or ion
channel types to one or more compounds. Optionally, the upper chamber channel
can be
flushed to remove the compound of interest, and a second compound can be added
by
pushing or pumping a second compound-containing solution into the channel. In
this
way, multiple compounds (or different concentrations of one or more compounds)
can be
assayed for their effects on one or more cell types or one or more ion
transport types.
Flow Retardiyag Structures
A device of the present invention that comprises a chip with multiple ion
transport
measuring holes, a flow-through upper chamber, and an overhead fluid delivery
system
that can deliver solutions to individual recording sites can also comprise two
or more
flow-retarding structures that inhibit fluid flow to the ion transport
recording sites of the
device.
During the of such a device, ion transport measurement is performed as the
upper
chamber experiences continuous flow of chamber solution (such as a measuring
solution)
through the chamber, in which the chamber solution enters through a chamber
inlet and
exits through a chamber outlet, and as delivery solution (such as a compound
solution) is
delivered directly to two or more ion transport measuring sites via the
overhead delivery
system. One or more flow-retarding structures can be constructed that restrict
the flow of
chamber solution to ion transport recording sites, while still allowing the
sites to be in
fluid communication with the chamber.
Flow-retarding structures can be of any shape or size, as long as they permit
fluid
communication between recording sites and the chamber yet restrict laminar
flow of
chamber solution to the sites. Preferably, a flow-retarding structure is
designed such that
the flow of chamber solution to a recording site is essentially eliminated
when a delivery
solution is being delivered to the recording site via the overhead delivery
system the
chamber is experiencing continuous fluid flow of chamber solution. During this
process
the design permits fluid flow out of the recording site to the chamber. Thus
structures for
retarding fluid flow of chamber solution to a recording site can be designed
and tested
empirically for their effectiveness (for example using dye solutions) under
various
conditions of overhead delivery (including flow rate, aperture size of
delivery unit,
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proximity of delivery unit to ion transport recording site, etc.) and fluid
flow through the
chamber (including flow rate, chamber dimensions, microwell dimensions, etc.).
One example of flow-retarding structures is depicted in Figure 14A. This
figure
depicts a portion of flow-through upper chamber (1418) depicting an inflow
conduit
(1422) flow-retarding structures (1425) positioned around each ion transport
measuring
recording site (1413), each of which surrounds an ion transport measuring hole
(1402).
Figures 14B and 14C are enlargements of two designs of flow-retarding
structures
(1425) surrounding a recording site (1413) that comprises an ion transport
measuring
hole (1402). The arrows show the pattern of flow of chamber fluid during
washing of the
chamber.
Fluidie pipe Delivery
In some preferred aspects of the present invention, an ion transport measuring
device comprises a biochip comprising two or more ion transport measuring
holes, and at
least one flow-through upper chamber positioned above the biochip that
comprises two or
more ion transport recording sites, each of which encompasses one of the two
or more ion
transport measuring holes of the biochip. The two or more ion transport
recording holes
access the one or more upper chambers at recording sites. The device further
comprises a
fluid delivery system comprising two or more fluid delivery units in the form
of fluidic
pipes, each of which can be aligned directly over one of the two or more ion
transport
recording sites. Solutions (including test compound solutions) can be added to
ion
transport recording sites that surround the ion transport recording holes
through the fluid
delivery units that can be positioned over the ion transport recording sites.
The flow-through upper chamber comprises at least one inlet and at least one
outlet that can allow for fluid flow through the chamber. Chamber solutions
(such as
measuring solutions such as ES) and cells or compounds can be added via an
upper
chamber inlet. An electrode, such as a reference electrode, can optionally be
provided
within or, during use of the device, in electrical connection with the flow-
through upper
chamber.
In some preferred embodiments, a flow-through upper chamber of a device of the
present invention comprises an upper surface that comprises two or more
openings, in
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which each of the two or more openings is aligned over one of the two or more
ion
transport recording sites. The openings provide access of the fluidic pipes to
ion transport
recording sites of an upper chamber. In other embodiments, a chamber does not
have an
upper surface, and each of the two or more fluidic pipes can be aligned
directly over the
one of the two or more ion transport measuring recording sites and solutions
can be
added via fluidic pipes that are positioned over ion transport recording
sites.
Some preferred devices of the present invention comprise a chip that comprises
at
least two ion transport measuring holes; at least one flow-through upper
chamber that is
accessed by the at least two ion transport measuring holes; and at least two
fluidic pipes
that can be positioned over the at least one upper chamber, in which each of
the at least
two pipes aligns directly over and in close proximity to an ion transport
measuring hole.
The pipes are connected to conduits of a fluidics system that feeds solutions,
such as test
compound solutions, through the pipes to ion transport recording sites during
ion
transport measurement assays.
Figure 11 depicts one embodiment of a fluidic pipe overhead delivery system.
In
this embodiment, a device has a common upper flow-through chamber (1118)
(inlet and
outlet not depicted) positioned over a chip (1101) that comprises multiple ion
transport
measuring holes (1102). In this embodiment, the device also comprises multiple
lower
chambers (1119), each of which is in register with a single ion transport
measuring hole
(1102). Fluidic pipes (1120) are depicted positioned over the ion transport
measuring
holes (1102). The pipes are used for delivery of solutions, such as test
compound
solutions, to ion transport recording sites during ion transport measurement
assays.
A pipe used as a fluid delivery unit comprises a conduit outlet at the
delivery end
(the end that is positioned over the ion transport recording site during use
of the device)
that can provide continuous flow of a solution to the ion transport recording
site.
Preferably, the opposite end of the pipe connects to at least one solution
reservoir. In
some preferred aspects of the invention, the pipe connects to two or more
sources of
solutions, at least one of which can be an assay solution (for example, a test
compound
solution) and at least one other or which can be a wash solution or a standard
measuring
solution (such as ES). Preferably, the pipe engages a conduit that engages a
valve that
allows switching between solutions that flow through the pipe. For example,
the valve,
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which preferably can be automatically controlled, can allow compound solution
to flow
through the pipe for a period of time, followed by wash solution, optionally
followed by a
second compound solution.
The present invention also includes methods of using ion transport measuring
devices that comprise pipe arrays for delivering compounds at ion transport
measuring
sites of upper chambers. In broad outline, such methods include: providing
measuring
solution in the lower chambers of the device; providing particles in measuring
solution in
an upper chamber of the device; sealing particles at ion transport measuring
holes;
providing continuous flow of measuring solution through the upper chamber;
positioning
an array of pipes over the upper chamber; delivering compounds continuously at
recording sites through the pipes, and measuring ion transport function or
properties. The
upper chamber of the ion transport measuring device can optionally be flushed
after ion
transport measurement, and optionally wash solution followed by a new compound
solution can be added to upper chamber recording sites using the pipe array.
The process
can be repeated multiple times.
The pipes can also be used to deliver tiny amount of compounds in drops to
cells
already sealed to the recording sites in low volume of solutions, such as, for
example,
those in the microwells of a chip that comprises a hydrophobic surface between
the
microwells. In this case, chamber solution is removed from the upper chamber,
with the
exception of the microwells, prior to overhead delivery of test solutions.
Fusion of the
test solution drop and the small volume of measuring solution at the recording
sites
allows for fast and efficient compound delivery. The fused drops in the
microwell
recording sites will not fuse together to cross-contaminate since the drops
are bounded by
hydrophobic coatings. Wash out can be achieved by flushing the entire upper
chamber
with wash solution and subsequent removal of wash solution. The recording
sites are then
ready to receive the next delivery of compounds.
Multichahrael pipet delivery of conzpouhels
In some preferred aspects of the present invention, an ion transport measuring
device comprises a biochip comprising two or more ion transport measuring
holes, and at
least one flow-through upper chamber positioned above the biochip that
comprises two or
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more ion transport recording sites, each of which encompasses one of the two
or more ion
transport measuring holes of the biochip. The two or more ion transport
recording holes
access the one or more upper chambers at recording sites. The device further
comprises a
fluid delivery system comprising two or more fluid delivery units in the form
of
multichannel pipets, each of which can be aligned directly over one of the two
or more
ion transport recording sites. Solutions (including test compound solutions)
can be added
to ion transport recording sites that surround the ion transport recording
holes through the
multichannel pipets that can be positioned over the ion transport recording
sites.
The flow-through upper chamber comprises at least one inlet and at least one
outlet that can allow for fluid flow through the chamber. Chamber solutions
(such as
measuring solutions such as ES) and cells or compounds can be added via an
upper
chamber inlet. An electrode, such as a reference electrode, can optionally be
provided
within or, during use of the device, in electrical connection with the flow-
through upper
chamber.
In some preferred embodiments, a flow-through upper chamber of a device of the
present invention comprises an upper surface that comprises two or more
openings, in
which each of the two or more openings is aligned over one of the two or more
ion
transport recording sites. The openings provide access of the pipets to ion
transport
recording sites of an upper chamber. In other embodiments, a chamber does not
have an
upper surface, and each of the two or more pipets can be aligned directly over
the one of
the two or more ion transport measuring recording sites and solutions can be
added via
pipets that are positioned over ion transport recording sites.
Some preferred devices of the present invention comprise a chip that comprises
at
least two ion transport measuring holes; at least one flow-through upper
chamber that is
accessed by the at least two ion transport measuring holes; and at least two
multichannel
pipets that can be positioned over the at least one upper chamber, in which
each of the at
least two pipes aligns directly over and in close proximity to an ion
transport measuring
hole.
A multichannel pipet used as a fluid delivery unit can dispense solution
directly to
an ion transport recording site. Preferably, the pipet is part of a fluidic
block that can
move from an uptake position for receiving compound for dispensing to the
dispensing
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position over the upper chamber. In some preferred aspects of the invention,
the pipet can
be used to sequentially dispense two or more different compound solutions that
are
dispensed in successive assays. Between assays, the chamber is washed using
the flow-
through conduits.
The present invention also includes methods of using ion transport measuring
devices that comprise dispensing pipet arrays for delivering compounds at ion
transport
measuring sites of upper chambers. In broad outline, such methods include:
providing
measuring solution in the lower chambers of the device; providing particles in
measuring
solution in an upper chamber of the device; sealing particles at ion transport
measuring
holes; providing continuous flow of measuring solution through the upper
chamber;
positioning an array of dispensing pipets over the upper chamber; dispensing
compounds
at recording sites through the pipets, and measuring ion transport function or
properties.
The upper chamber of the ion transport measuring device can optionally be
flushed after
ion transport measurement, and optionally new compound solutions can be added
to
upper chamber recording sites using the pipet array. The process can be
repeated multiple
times.
In some preferred aspects of the present invention, the chip comprises
microwells
that comprise ion transport recording sites, and the top surface of the chip,
with the
exception of the microwell surfaces, is preferably hydrophobic to aid in
maintaining fluid
separation between microwells when fluid is removed from the upper chamber.
In this embodiment, the compound delivery system can deliver compound
solution from pipets over the microwells in droplets that localize to the
microwells and do
not spread to other wells due in part to the hydrophobicity of the chip upper
surface.
Preferably, the compound drops are very large compared to the microwell
volume, so that
there is little compound dilution when it is delivered. In this case, after
particle sealing,
chamber solution is removed from the upper chamber, with the exception of the
microwells, prior to overhead delivery of test solutions.
Fusion of the compound drop and the small volume of solutions at the recording
sites allows for fast and efficient compound delivery. The fused drops will
not fuse
together to cross-contaminate recording sites since the drops are bounded by
the
hydrophobic chip surface outside the microwells.
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Wash out can be achieved by flushing the entire upper chamber with wash
solution and subsequent removal of wash solution. After washout, recording
sites are
ready to receive the next delivery of compounds.
Soraic Actuators
In a related embodiment, the fluid delivery units are sonic actuators that can
be
part of a block or plate comprising solutions in wells that is localized over
the ion
transport recording sites of a device. Activation of a sonic actuator on the
plate causes a
droplet of solution from a well associated with actuator to be ejected out of
the well to the
ion transport measuring site. In a preferred aspect of this embodiment, the
compound
delivery system can deliver compound solution from delivery wells positioned
over the
upper chamber microwells in droplets that localize to the microwells and do
not spread to
other wells. The fluid isolation of the microwells can be promoted by having a
hydrophobic chip surface outside the microwells. In this case, after particle
sealing,
chamber solution is removed from the upper chamber, with the exception of the
microwells" prior to overhead delivery of test solutions. Fusion of the
compound drop
and the small volume of solutions at the recording sites allows for fast and
efficient
compound delivery. The fused drops will not fuse together to cross-contaminate
recording sites since the drops are bounded by the hydrophobic chip surface
between
microwells.
Wash out can be achieved by flushing the entire upper chamber with wash
solution and subsequent removal of wash solution. After washout, recording
sites are
ready to receive the next delivery of compounds.
In an alternatW e, the sonic actuators can be part of a block or plate that is
positioned under the lower microwells of an ion transport measuring device. In
this case,
particles such as cells are in the lower chamber and are sealed to the lower
surface of the
chip by pneumatic devices connected to the upper wells. After particle
sealing, chamber
solution is removed from the lower chamber, with the exception of the
microwells" prior
to delivery of test solutions from below. Activation of a sonic actuator on
the plate causes
a droplet of solution from a well associated with actuator to be ejected
upward out of the
well to the ion transport measuring site of a lower well. Fusion of the
ejected compound
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drop and the small volume of solution at a recording site allows for fast and
efficient
compound delivery. In both of these variations, a droplet of fluid ejected by
a sonic
actuator fuses with the small amount of solution surrounding the sealed cell
at the ion
transport measuring site. Ion transport measurement can be performed after
compound
solution delivery, and washout of the flow-through upper or lower chamber can
be
performed after recordings have been performed.
Figure 13 depicts compound delivery from a fluid delivery unit positioned over
microwells of a flow-through upper chamber of a device of the present
invention. In
Figure 13A, the flow-through upper chamber is perfused with chamber
(measuring)
solution that comprises cells (1316). The upper chamber has microwells (1303)
surrounding ion transport measuring holes (1302) in the chip (1301). The chip
also
compises a hydrophobic coating (1315) that surrounds but does not contact the
microwells. In Figure 13B, cells (1316) are sealed to ion transport measuring
holes
(1302) in the microwells (1303). This can be accomplished by the application
of pressure
via conduits that engage the lower chambers and lead to a pneumatic device. In
Figure
13C, a drop of compound solurtion (1329) is dispensed, from, for example, a
sonic
actuator plate or multichannnel pipet. In Figure 13D, the drop of compound
solution has
fused with the solution in the rnicrowell (1303). The microwells (1303) are in
fluid
isolation from one another.
Nozzles
In some preferred embodiments of the novel fluidic systems of the present
invention, an ion transport measuring device that comprises a biochip
comprising two or
more ion transport measuring holes, and at least one flow-through upper
chamber
positioned above the biochip, and a fluid delivery system comprising two or
more fluid
delivery units can further comprise two or more nozzle structures positioned
over the ion
transport measuring sites that can engage the fluid delivery units of the
fluid delivery
system.
The outflow nozzle structures can be reversibly or irreversibly aligned over
the
chip such that a single nozzle i s positioned over each ion transport
recording site, or can
be can be a part of the piece that comprises the upper chamber walls. In
either case, for
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the dispensing of solutions to ion transport measuring sites, the nozzles are
positioned
over the ion transport measurement recording sites of the device such that the
fluid
delivery units of the overhead fluid delivery system can dispense fluid into
the nozzles
that then flows to the ion transport measurement recording sites.
Preferably, when the fluid delivery system is aligned over the chip, the
outflow
nozzles are positioned close to the surface of the measuring solution of the
upper
chamber, but not in contact with it. Preferably, the nozzle is at the end of a
funnel
structure, the nozzle diameter is greater than ten times the diameter of the
cells, and the
funnel size is large enough to allow the compound solution within it to flow
out of the
nozzle over sufficient time that more compound solution can be delivered to
the funnels
(such as by dispensing pipette tips) without creating bubbles within the
funnel or nozzle
area.
Preferably, the device comprises or engages at least two lower chambers in
register with the two or more holes of the chip. During ion transport
measurement, each
of the individual lower chambers preferably comprises or is in electrical
contact with a
recording electrode.
One design of this aspect of the pres ent invention is depicted in Figure 12.
In this
embodiment, a device has a common upper flow-through chamber (1218) (inlet and
outlet not depicted) positioned over a chip (1201) that comprises multiple ion
transport
measuring holes (1202). In this embodiment, the device also comprises multiple
lower
chambers (1219), each of which is in register with a single ion transport
measuring hole
(1202). Fluidic pipes (1120) are depicted po sitioned over the ion transport
measuring
holes (1202). The device also comprises nozzles (1221) positioned over the ion
transport
measuring holes (1202).
In using the device, lower chambers are filled with measuring solutions, and
the
the upper chamber is filled with measuring solution and cells (or other
particles) are
added. Cells (or other particles) are sealed to the ion transport measuring
holes by
applying suction to the lower chambers. By controlling fluid flow through the
upper
chamber, an even but shallow bath is produced that has continuous flow. The
fluidic
delivery units, (for example, pipets or pipes for compound addition are
positioned over
the holes on the chip such that the outflow nozzles are close to the surface
of the
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measuring solution within the upper chamber, but not in contact with it. After
control
currents are recorded, compound solutions are added to the nozzles from above,
such as
by pipets or fluidic pipes.
As compound solutions flow through the nozzles to ion transport recording
sites,
the fluid delivery system comprising an array of pipets or fluidic pipes can
move away
from the for uptake of other compound solutions (in the case of pipets) or for
flushing the
delivery units of a first compound solution prior to filling them with a
second compound
solution. The fluidics block comprising the array of fluid delivery units can
then move
back to the nozzles over the ion transport recording sites for delivery of a
second set of
compound solutions to the ion transport recording sites.
The present invention includes ion transport measuring devices that include a
biochip comprising ion transport two or more ion transport measuring holes and
a
compound delivery system that can deliver compound or solution to each ion
transport
measurement site individually, and nozzles positioned over each ion transport
recording
site. In preferred embodiments, the devices are high-throughput devices that
comprise at
least 48, at least 96, or at least 3~4 ion transport measuring sites and a
corresponding
number of nozzles for dispensing compounds over the ion transport measuring
sites.
Device having compound delivery plate
Yet another aspect of the present invention is an ion transport measuring
device
that comprises a substrate comprising at least tv~ro ion transport measuring
holes, at least
two upper chambers in register with the two or more ion transport measuring
holes; at
least two lower microwells, each of which is positioned around an ion
transport
measuring hole, and each of which is connected to a common lower chamber
channel;
and a compound delivery plate, in which the compound delivery plate has drug
delivery
sites in register with the lower microwells, where the compound delivery plate
can
reversibly come into contact with the lower microwells. In this design,
depicted in Figure
13, the two or more upper chambers are connected to a pneumatic system for
sealing cells
to the ion transport measuring holes on the lowex side of the substrate and
each of the
upper chambers comprises or is in electrical conrtact with an individual
(recording)
electrode.
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Electrical and pneumatic connection to the upper wells of the ion transport
measuring chip can optionally be provided by a separate adaptor plate.
Preferably, each
independent upper well connects to a separate recording electrode.
Figure 15 depicts a device in which the lower channel (1519) which accesses
the
lower microwells(1513)) is a flow-through chamber having fluid flow of
measuring
solution through the channel. The bottom surface of the chip (1511), with the
exception
of the microwell surfaces, is preferably hydrophobic to aid in maintaining
fluid
separation between microwells when fluid is removed from the lower chamber. A
reference electrode can preferably be provided on the lower surface of the
chip,
connected to the compound delivery plate (1520), or in electrical contact with
the lower
channel.
In some preferred designs, at the time of operation of the device, the drug
delivery
sites have compounds spotted, or printed on them in drops or dried form.
In operation, measuring solution is added to the upper chambers, and measuring
solution and cells are introduced into the lower chamber channel through
conduits.
Pressure is applied to the upper wells (either individually or commonly
connected to
pressure controls) to pull cells up from the lower channel inrto the lower
microwells and
seal them against the ion transport measuring holes of the chip. Sealing
occurs in the
presence of complete solution superfusion of the bottom chamber. After the
seals have
formed, solution is removed from the channel, with the exception of the
microwells,
which in the case of coated surface electrodes, are in electrical connection
with the
electrodes. At this time compounds are applied to the microwells as the
delivery plate is
brought into contact with the lower surfaces of the microwells. Ion transport
measurement can then be performed.
The same device can be used in inverted orientation, with cells sealing to the
top
of the chip, and the compound delivery plate is positioned above the chip to
apply
compounds from the top side of the chip.
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FURTHER EMBODIMENTS
The present invention includes chips, devices, and methods for ion transport
measurement that can be used to efficiently assay test particles such as
cells. The devices
allow ion transport assays that can be used in a variety of ion transport
measurement
applications, including but not limited to determining the effects of
compounds (such as
compounds of interest or test compounds) on ion transport activity. The assays
can also
be used to assess cell condition, "sealability", responsiveness to compounds
or treatments
before performing a battery of tests using the cells, or to rapidly determine
the effects of
growth conditions, developmental stages, hormone responsiveness on the ion
transport
activity of cells. In some embodiments, the ion transport measuring devic es
can be used
for other assays in addition to ion transport measurement assays. In some
embodiments,
the ion transport measuring devices can designed such that cells in a chamber
of the
device can be microscopically viewed before, during, and/or immediately after
an ion
transport measurement assay.
Method for performing excised patch volts a clamp recordings
Excised voltage clamp recordings such as inside-out or outside in
configurations
as known in the art of voltage clamp studies can be performed by any planar or
non-
planar electrode configurations known in the art, or described in this
application or
previous applications. This is done by adding magnetic beads labeled with
antibody(s)
against common cell surface markers after the cell is sealed to the ITM sites;
incubation
to allow for bead binding to the cell surface; and applying magnets to the
beaded sealed
cells from the open access. Magnetic forces will remove the beads, together
with
associated cell membrane, which allows the formation of "excised patch"
configuration at
the ion transport measuring sites for single channel or macropatch recordings.
Method of Shipping Ion Transport Measuring Chips
The present also provides methods for shipping ion transport measuring chip
and
devices in which the upper and lower chambers of the devices or chips are pre-
filled with
an ion transport measuring solution. For example, where the devices are
intended for use
in performing ion transport measurement assays on whole cells, the devic es
can be
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packed with upper and lower chambers filled with intracellular solution (IS).
This can
reduce the time required to perform an assay, and also can reduce the
complexity of the
machinery that interfaces with the device and provides fluidic controls and
conduits,
since the machinery does not need to add measuring solution to, for example
the lower
chambers of a device, but instead can simply flush the upper chamber with an
appropriate
measuring solution such as extracellular solution (ES) prior to adding cells.
This
increases the efficiency and reduces the time needed for assays, such as high
rthroughput
screens. (In cases such as that described in Aspect 28, where cells are
distributed in lower
chambers, the machine flushes the lower chamber with, for example, ES, prior
to adding
cells.)
The devices or chips can be shipped in blister packs that lock in the
rrieasuring
solution, and the entire assembly can optionally be kept refrigerated until
use_ The
measuring solution used can be specialized for different types of ion
transport assays,
different cell types, and the like. The measuring solution can also be
simplified for more
general use with more than one cell or assay type.
To use devices shipped in measuring solution, after flushing extracellular
solution
through and adding cells to the one or more upper chambers, a vacuum can be
applied to
the one or more intracellular chambers that already contain IS to seal cells
to ion
transport measuring holes.
The aspects of the invention disclosed herein can be combined to male new
embodiments that are also within the scope of the invention. The aspects of
the invention
disclosed herein, such as, but not limited to chip designs, chamber designs,
electrode
arrangements and connections, through-hole designs and manufacture, fluidics
arrangements, etc. can be combined with other features described herein, known
in the
art, or features that are developed in the future.
All references cited herein, including patents, patent application, and
publications,
are incorporated herein by reference in their entireties.
All headings are for the convenience of the reader and should not be used to
limit
the meaning of the text that follows the heading, unless so specified.
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