U.S. patent application number 13/291018 was filed with the patent office on 2012-05-31 for cell handling, electroporation and electrofusion in microfluidic systems.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Cristian Ionescu-Zanetti, Michelle Khine, Adrian Lau, Luke P. Lee, Jeonggi Seo.
Application Number | 20120135887 13/291018 |
Document ID | / |
Family ID | 38224940 |
Filed Date | 2012-05-31 |
United States Patent
Application |
20120135887 |
Kind Code |
A1 |
Lee; Luke P. ; et
al. |
May 31, 2012 |
CELL HANDLING, ELECTROPORATION AND ELECTROFUSION IN MICROFLUIDIC
SYSTEMS
Abstract
Method and systems provide improved cell handling in
microfluidic systems and devices using lateral cell trapping and
methods of fabrication of the same that allow for selective low
voltage electroporation and electrofusion.
Inventors: |
Lee; Luke P.; (Orinda,
CA) ; Seo; Jeonggi; (Albany, CA) ;
Ionescu-Zanetti; Cristian; (Berkeley, CA) ; Khine;
Michelle; (Merced, CA) ; Lau; Adrian; (San
Francisco, CA) |
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
38224940 |
Appl. No.: |
13/291018 |
Filed: |
November 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11466104 |
Aug 21, 2006 |
8058056 |
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13291018 |
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PCT/US05/08349 |
Mar 14, 2005 |
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11466104 |
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60710305 |
Aug 21, 2005 |
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60552892 |
Mar 12, 2004 |
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Current U.S.
Class: |
506/10 ; 264/219;
506/40 |
Current CPC
Class: |
G01N 33/48728 20130101;
B01L 2400/0487 20130101; B01L 3/502707 20130101; B01L 2200/0668
20130101; C12M 23/16 20130101; C12M 35/02 20130101; C12N 15/87
20130101; B01L 3/502761 20130101 |
Class at
Publication: |
506/10 ; 506/40;
264/219 |
International
Class: |
C40B 30/06 20060101
C40B030/06; B29C 33/38 20060101 B29C033/38; C40B 60/14 20060101
C40B060/14 |
Claims
1. A method of selectively introducing substances of interest into
cells comprising: placing one or more cells into an integrated
microfluidic chip providing controllable cell trapping; optical
characterizations; and simple cell loading for multiple single cell
analysis; locally electroporating a trapped cell with a focused
electric field.
2. The method of claim 1 further wherein: said integrated chip
comprises a PDMS microfluidic device with individual lateral cell
trapping sites. said field is generated using low applied voltages.
e.g., about .about.0.76V. said field is generated by electrodes
that are located at a distance from said cell, said distance
sufficient to reduce the potential of adverse products from
electrode reactions.
3. A method of fabricating an integrated device for cell
electroporation and/or electrofusion comprising: preparing a mold
by making height patterns defining narrow patch channels using deep
etching; adding patterns for wide connection regions; introducing a
settable material into the mold and curing; detaching the set
material from the mole; placing holes for connection of tubes;
connecting tubes to reservoirs, via said holes, to load cells
and/or electrolyte solutions and to apply suction to patch
channel.
4. The method of claim 3 further wherein: said mold is constructed
from silicon.
5. The method of claim 3 further wherein: said mold is constructed
from one or more of: a ceramic; a metal; metal alloy; a hardenable
polymer.
6. The method of claim 3 further wherein: said moldable material
comprises polydimethylsiloxane (PDMS) and a curing agent.
7. A Low-Voltage Single Cell Electroporation Array for
Intracellular Compound Delivery device comprising: a substrate; a
main reservoir able to hold cells in a fluidic medium; at least one
lateral opening in a side of said main reservoir; at least one
trapping channel operatively connected to said at least one lateral
opening; and at least two electrical connections, one to said main
reservoir and one to said trapping channel, such that an electric
field can be focused where a cell contacts said lateral opening.
such that a cell in said main reservoir can be selectively
immobilized at said lateral opening by negative pressure in said
trapping channel.
8. The device according to claim 7 further wherein: said substrate
is a three dimensional structure comprising a length, a width and a
thickness, said thickness being a smallest dimension; and said side
of said main reservoir is roughly parallel to said thickness.
9. The device according to claim 7 further wherein: said at least
two electrical connections can also be used measuring electrical
characteristics between said main reservoir and said trapping
channel.
10. The device according to claim 7 further wherein: said lateral
opening has effective dimensions of less than about 3 microns by 3
microns.
11. The device according to claim 7 further comprising: at least
three lateral openings in said main channel, said lateral openings
spaced less than 40 microns apart.
12. The device according to claim 11 further wherein: said lateral
openings are electrically connected to operate as independent
electroporation locations.
13. The device according to claim 12 further wherein: patch lateral
openings are in a horizontal plane with multiplexed electroporation
sites having a distance between sites of between one hundred .mu.m
and one thousand .mu.m.
14. The device according to claim 7 further comprising:
microfluidic features to move substances to appropriate positions
of said device.
15. A multiple cell electroporation/electrofusion device
comprising: a substrate; a main reservoir able to hold cells in a
fluidic medium running parallel to the largest dimensions of said
substrate; a plurality of lateral openings in sides of said main
reservoir, at least some of said openings operatively connected to
a plurality of trapping channels; a microfluidic input for
introducing cells in a fluid to said main reservoir; one or more
microfluidic trapping connections for applying negative pressure to
said lateral openings; such that cells in said main reservoir can
be selectively immobilized at said lateral openings; one or more
electrical connections for applying an electric field to a cell at
one of said openings, said field focused where a cell membrane
contacts said opening.
16. The device according to claim 15 further wherein: said
substrate is formed of an elastomer; said lateral openings have a
cross section less than about 3 microns by 3 microns; and said
lateral openings are operatively connected to trapping channels
with cross sections less than about 3 microns by 3 microns.
17. A multiple low voltage cell electroporation device comprising:
a substrate; means for holding cells in fluid suspension in a main
channel, said means running parallel to the largest dimensions of
said substrate; lateral cell trapping means formed in said
substrate and operatively connected to said means for holding cells
in fluid suspension; means for applying negative pressure to said
lateral cell trapping means in order to selectively immobilize
cells at said lateral trapping means; means for applying an
electric field between said means for holding cells and said
lateral trapping means. such that said field is focused at an area
of an immobilized cell.
18. The device according to claim 17 further comprising: means for
measuring electrical properties between said means for holding
cells and said lateral trapping means.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of utility application
Ser. No. 11/466,104 filed 21 Aug. 2006, which claims priority from
provisional patent application 60/710,305 filed 21 Aug. 2005 and is
a continuation in part of PCT/US 2005/008349, filed 14 Mar. 2005,
which claims priority from provisional patent application
60/552,892, filed 12 Mar. 2004, all incorporated herein by
reference for all purposes.
COPYRIGHT NOTICE
[0002] Pursuant to 37 C.F.R. 1.71(e), applicants note that a
portion of this disclosure contains material that is subject to
copyright protection (such as, but not limited to, diagrams, device
photographs, or any other aspects of this submission for which
copyright protection is or may be available in any jurisdiction.).
The copyright owner has no objection to the facsimile reproduction
by anyone of the patent document or patent disclosure, as it
appears in the Patent and Trademark Office patent file or records,
but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
[0003] The present invention relates to methods and/or system
and/or apparatus involving analysis and/or handling of cells and/or
other biological material and that can be adapted to other
applications. In specific embodiments, the invention involves
methods and/or system and/or apparatus effective single cell
electroporation using an efficient cell handling system or
array.
BACKGROUND OF THE INVENTION
[0004] The discussion of any work, publications, sales, or activity
anywhere in this submission, including in any documents submitted
with this application, shall not be taken as an admission that any
such work constitutes prior art. The discussion of any activity,
work, or publication herein is not an admission that such activity,
work, or publication existed or was known in any particular
jurisdiction.
[0005] Handling, characterization, and visualization of individual
cells has become increasingly valued in the fields of drug
discovery, disease diagnoses and analysis, and a variety of other
therapeutic and experimental work. However, few high-resolution
methods exist to control and manipulate the biochemical nature of a
single cell's interior; yet roughly 90% of the cell's biologically
active structures, such as intracellular proteins, are located
within the confines of the cell membrane. The cell membrane serves
as an effective barrier between the cytoplasm and the outside world
and, as such, is relatively impermeable to most ionic and polar
substances.
[0006] One way to and access the cell's interior is by temporarily
increasing the cell membrane's permeability. This can be
accomplished via electroporation, a technique using high electric
fields to induce structural rearrangements of the cell membrane.
Pores are formed when the transmembrane potential exceeds the
dielectric break-down voltage of the membrane (about 0.2-1.5V).
Material such as polar substances other-wise impermeant to the
plasma membrane (such as dyes, drugs, DNA, proteins, peptides, and
amino acids) can thus be introduced into the cell.
[0007] In the early 1980s, Eberhard Neumann et al. demonstrated the
feasibility of electroporation for delivering DNA to a population
of mammalian cells. Since then, methods of bulk electroporation has
become a standard technique routinely used to simultaneously
transfect millions of cells in culture. However, bulk
electroporation requires very high voltages (>10.sup.3V) and has
little control over the permeabilization of individual cells,
resulting in suboptimal parameters. Reversible electroporation, in
which the pores can reseal, is therefore difficult.
[0008] While single cell electroporation has been hoped to have
advantages over bulk methods, practical systems or methods for
single cell electroporation have generally been less commonly used
than bulk methods. Lundqvist et al. first demonstrated single cell
electroporation using carbon fiber microelectrodes in 1998. To
induce electroporation, they placed the microelectrodes 2-5 microns
away from adherent progenitor cells. Other single cell
electroporation techniques developed since include:
electrolyte-filled capillaries, micropipettes, and microfabricated
chips.
[0009] In 2001, Huang et al. introduced a microfabricated single
cell electroporation chip. In 2002, Nolkrantz et al. demonstrated
functional screening of intracellular proteins by using
electroporation to introduce fluorogenic enzyme substrates and
receptor ligands into single cells.
[0010] Chip-based cellular handling devices have been proposed
using silicon oxide coated nitride membranes, silicon elastomers,
polyimide films, quartz or glass substrates. Recently, three
dimensional structures more similar to patch pipettes have also
been fabricated. Some earlier chip-based devices developed to date
generally use a horizontal geometry where the patch pore is etched
in a horizontal membrane dividing the top cell compartment from the
recording electrode compartment.
[0011] The following are incorporated herein by reference to
provide background. [0012] 1. J. A. Lundqvist, F. Sahlin, M. A.
Aberg, A. Strimberg, P. S. Eriksson and O. Orwar, Proc. Natl. Acad.
Sci., 95, 10356-10360 (1998). [0013] 2. K. Nolkrantz, C. Farre, A.
Brederlau, I. D. Karlsson, C. Brennan, P. S. Eriksson, S. G. Weber,
M. Sandberg and O. Orwar, Anal. Chem., 73(18), 4469-4477(2001).
[0014] 3. K. Haas, W. C. Sin, A. Javaherian, Z. Li and H. T. Cline,
Neuron, 29, 583-591 (2001). [0015] 4. Y. Huang and B. Rubinsky,
Sens. Actuators, A, 89, 242-249 (2001). [0016] 5. T. Y. Tsong,
Biophysical Journal, 1991, 60, 297-306. [0017] 6. J. C. Weaver, J.
Cell Biochem, 2002, 51,426-435. [0018] 7. J. L. Rae, R. A. Levis.
Eur J Physiol. 2002,443,664-670. [0019] 8. I. G. Abidor, V. B.
Arakelyan, L. V. Chernomordik, Y. A Chizmadzhec, V. F. Patushenko,
M. R. Tarasevich, Bioelectrochem. Bioenerg. 1979, 6, 37-52. [0020]
9. J. C. Weaver and K. T. Powell, in "Electroporation and
Electrofusion in Cell Biology" (Neumann, E., Sowers, A., Jordan C,
eds.) pp 111-126. Plenum Press. New York. [0021] 10. Weaver, J. C.,
and Mintzer, R. A. Decreased bilayer stability due to transmembrane
potentials. Phys Lett., 1981, 86A, 57-59. [0022] 11. E. Neumann, M.
Schaefer-Ridder, Y. Wang, P. H. Hofschneider, EMBO J,
1982,1,841-845. [0023] 12. D. C. Chang, B. M. Chassy, J. A.
Saunders, A. E. Sowers,"Guide to Electroporation and
Electrofusion". Academic Press, Inc. 1992. San Diego. [0024] 13. K.
Nolkrantz, C. Farre, A. Brederlau, R., I. D. Karlsson, C. Brennan,
P. S. Eriksson, S. G. Weber, M. Sandberg, O. Orwar, O. Anal. Chem,
2001; 73(18); 4469-4477. [0025] 14. J. Seo, C. Ionescu-Zanetti, J.
Diamond, J., R. La, L. P. Lee, Applied Physics Letter, 2004,
84,11:1973-1975. [0026] 15. E. Neumann, K. Toensing, S. Kakorin, P.
Budde, and J. Frey, Biophys J, 1998, 74, 98-108. [0027] 16. D.
Needham and R. M. Hochmuth, Biophys J., 1989, 55, 100 1-1009.
[0028] 17. J. Akinlaja and F. Sachs, Biophys J., 1998, 75,
247-254.
SUMMARY
[0029] The present invention, in specific embodiments, involves
methods and or devices that provide improved cellular handling in
particular to enable single cell electroporation. In other
embodiments the invention involves systems for multiple single-cell
electroporation and/or electrofusion of cells with other cells or
vesicles. In general, the invention accomplishes successful single
cell electroporation by generally both isolating the cell and
providing a well focused electric field through the device
configuration. In specific embodiments, a device according to the
invention can selectively trap targeted cells and focus an electric
field for: reversible electroporation (in which the pores reseal),
intracellular perfusion, and/or cell fusion.
[0030] Specific embodiments involve lateral cell trapping junctions
at a micron scale, integrated with microfluidic channels wherein
cell immobilization or trapping pores generally are arranged as
openings in a sidewall or analogous structure of a main fluidic
channel. At times herein, this cell trapping structure is referred
to as a lateral pore or junction. In specific embodiments,
microfabricated devices according to the invention can be ideally
suited to both isolate single cells and focus an electric field for
cell electroporation. Microfabrication technology according to
specific embodiments of the invention also enables the
incorporation of other functionalities onto devices of the
invention. The invention thus can enable a comprehensive screening
device that can not only permeate single cells--but also introduce
materials into the cell and monitor its response. In further
embodiments, the invention involves a device or system able to
introduce otherwise impermeable compounds such as dyes, drugs, or
DNA into cells.
[0031] In further embodiments, the invention involves a PDMS based
platform able to create a transmembrane potential across a cell
using low voltages such that dielectric breakdown of the membrane
is achieved. In particular embodiments, the invention applies a low
voltage (<about 1 Volt) to create a large potential drop (of
about 750 V/cm) across the cell membrane. In response to this
transmembrane potential, dielectric breakdown of the membrane is
achieved and cell membrane phospholipids can rearrange to create
transient pores. These pores allow compounds to be delivered into
the cell, for example via an integrated capillary channels or a
backside perfusion channel. In alternative embodiments, cells can
also be fused with each other or with pre-loaded vesicles for
volume controlled intracellular delivery using electrofusion.
[0032] The present invention, in further embodiments, involves an
integrated multiple cellular handling array system or device that
utilize lateral cell trapping junctions to enable electroporation
and/or electrofusion and associated electrical measurements. In
specific example systems, the intersectional design of a
microfluidic network provides multiple cell addressing and
manipulation sites for efficient electrophysiological measurements
and cell manipulations at a number of sites.
[0033] In specific embodiments, a device of the invention provides
and also allows for visual observation of membrane deformation. In
specific embodiments, device fabrication is based on micromolding
of one or more elastomers (such as, polydimethylsiloxane (PDMS)),
allowing for inexpensive mass production of disposable
high-throughput biochips. Other embodiments can be constructed from
bonded silicon/polysilicon surfaces or injection molded
polymers.
[0034] The lateral design according to some specific embodiments of
the invention also allows for construction of systems having
efficient multiplexing of measurements, exchange of intracellular
and extracellular electrolyte while the cell is attached to a pore,
and optical observation of membrane deformation and cellular
content. Thus, in specific embodiments, the invention enables high
throughput, low cost systems allowing for cell electroporation,
electrofusion and other cellular manipulations and/or assays.
[0035] In further specific embodiments, the novel methods and
devices according to specific embodiments of the invention can be
used in various micrometer systems. Applications include BioMEMS,
lab on a chip, cell-based assays, etc.
Other Features & Benefits
[0036] The invention and various specific aspects and embodiments
will be better understood with reference to drawings and detailed
descriptions provided in this submission. For purposes of clarity,
this discussion refers to devices, methods, and concepts in terms
of specific examples. However, the invention and aspects thereof
may have applications to a variety of types of devices and systems.
It is therefore intended that the invention not be limited except
as provided in the attached claims and equivalents.
[0037] Furthermore, it is well known in the art that systems and
methods such as described herein can include a variety of different
components and different functions in a modular fashion. Different
embodiments of the invention can include different mixtures of
elements and functions and may group various functions as parts of
various elements. For purposes of clarity, the invention is
described in terms of systems that include many different
innovative components and innovative combinations of innovative
components and known components. No inference should be taken to
limit the invention to combinations containing all of the
innovative components listed in any illustrative embodiment in this
specification.
[0038] In some of the drawings and detailed descriptions below, the
present invention is described in terms of the important
independent embodiment of a biologic assay and/or array system and
components thereof. This should not be taken to limit the
invention, which, using the teachings provided herein, can be
applied to a number of other situations.
[0039] In some of the drawings and detailed descriptions below, the
present invention is described in terms of a number of specific
example embodiments including specific parameters related to
dimensions of structures, pressures or volumes of liquids, or
electrical values. Except where so provided in the attached claims,
these parameters are provided as examples and do not limit the
invention to other devices or systems with different
dimensions.
[0040] All references, publications, patents, and patent
applications cited in this submission are hereby incorporated by
reference in their entirety for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1A-D illustrate example aspects of cell manipulation
devices according to various embodiments of the invention.
[0042] FIG. 2 illustrates aspects of an example double channel
device allowing for rapid change of intracellular and extracellular
solutions according to alternative specific embodiments of the
invention.
[0043] FIG. 3 illustrates an example of a simple lateral junction
cell trapping disposable concentric device according to alternative
specific embodiments of the invention.
[0044] FIG. 4A-B illustrate geometries for an opening to a lateral
cell trapping junction particularly suited for electroporation
and/or electrofusion according to specific embodiments of the
invention.
[0045] FIG. 5A-B illustrate optional different geometries for an
opening to a lateral cell junction according to specific
embodiments of the invention.
[0046] FIG. 6A-C illustrate aspects of an example integrated
cellular manipulation array on a microfluidic platform according to
specific embodiments of the invention.
[0047] FIG. 7A-D show four frames from a micrograph movie showing a
HeLa cell being trapped at a lateral junction by applying a
negative pressure (e.g., 2 psi) to a trapping channel according to
specific embodiments of the invention.FIG.
[0048] FIG. 8 illustrates aspects of an example circuit model of a
cell according to specific embodiments of the invention
[0049] FIG. 9A-B are graphs illustrating example electrical
measurements according to specific embodiments of the invention
where (A) Short square wave voltages (inset) are applied to the
circuit and the resulting current is measured; and (B): The leak
current R.sub.leak is linear and subtracted from the measured
current to isolate the current across the cell, R.sub.cell.
[0050] FIG. 10A-B are graphs each illustrating example electrical
measurements for three different runs demonstrating reversible
electroporation according to specific embodiments of the
invention.
[0051] FIG. 11A-B illustrate electrical and optical characteristics
of a Hela (human carcinoma) cell electroporated and loaded with
propidium iodide from the intracellular perfusion channel (e.g., as
described above) according to specific embodiments of the
invention.
[0052] FIG. 12A-B are micrographs illustrating electrofusion of two
cells (A) and two vesicles (B) according to specific embodiments of
the invention.
[0053] FIG. 13A-I illustrate an example of aspects of fabrication
of a device array according to specific embodiments of the
invention.
[0054] FIG. 14A-B illustrate current response to a 20 mV voltage
pulse before (a) and after (b) cell trapping of an example device
according to specific embodiments of the invention.
[0055] FIG. 15 is a diagram showing an example of whole mammalian
cell currents recorded according to specific embodiments of the
invention.
[0056] FIG. 16 illustrates an example schematic of a hollow
electrode interface (in this example configured on a PCB) mating
with device and tubing according to specific embodiments of the
invention.
[0057] FIG. 17 is a block diagram showing a representative example
logic device in which various aspects of the present invention may
be embodied.
[0058] FIG. 18 (Table 1) illustrates an example of diseases,
conditions, or statuses that can evaluated or for which drugs or
other therapies can be tested according to specific embodiments of
the present invention.
[0059] FIG. 19 is a schematic representation of a basic design unit
for fast reagent application and removal according to specific
embodiments of the invention.
[0060] FIG. 20 is a schematic representation showing an example
methodology for the application of multiple reagents to a target
sample area according to specific embodiments of the invention. (A
state where channel n4 is on is shown). FIG. 21 is a schematic
representation showing an example methodology for applying an
arbitrary concentration to a sample area according to specific
embodiments of the invention. (A microfluidic mixer is being driven
by input pressures/flow rates in this example.)
[0061] FIG. 22 is a schematic representation showing use of
trapping channels for immobilizing cells in the target area for
fast reagent application and removal according to specific
embodiments of the invention.
DESCRIPTION OF SPECIFIC EMBODIMENTS
1. Overview
[0062] The present invention, according to various specific
embodiments as described herein, provides methods, systems, and
devices that allow for very dense integration of cell handling and
microfluidic control in an integrated platform and that allow for
inexpensive manufacture, easy of visualization, and other
advantageous as will be apparent from the descriptions herein. In
specific embodiments, the invention is involved with efficient
systems and methods for cell electroporation and/or
electrofusion.
[0063] In general, electroporation occurs when a cell's
transmembrane potential exceeds the dielectric breakdown potential
of the membrane, typically reported to be about 0.2-1.5V In
specific devices according to the invention an electric field is
applied across a cell held using a trapping pore and with Ag/AgCl
(or other conductor) electrodes electrically connected to channels
on either side of the cell. With a trapping channel made from a
good electrical insulator (e.g., polydimethysiloxane (PDMS)), the
electric field can be focused such that the greatest potential drop
occurs first across the portion of the cell membrane within the
trapping channel (e.g., 3.1 .mu.m.times.4 .mu.m in specific
embodiments). As resistance is inversely proportional to surface
area, and the surface area of the first membrane is <100.times.
that of the membrane outside the trapping site, the resistance of
the first membrane is significantly larger than the other membrane.
Therefore the critical transmembrane potential--and thus
electroporation--occurs here first. In further embodiments, after
this portion of the membrane porates and its resistance decreases,
the other side of the cell porates and cell fusion is achievable
with the abutting cell.
2. Example Device Configurations
[0064] FIG. 1A-D illustrate example aspects of cell manipulation
devices according to various embodiments of the invention. Each of
the examples shows lateral cell trapping junctions according to
specific embodiments of the invention. With the pores in the
horizontal plane, closely spaced (only about 10-20 microns apart)
multiplexed parallel sites are possible. Drugs, reagents, or other
material can therefore be administered in small volumes, while
operation of the device can be recorded and/or viewed in parallel
at a number of sites. In various example fabrications, the whole
device is fabricated using micromolding of an elastomer such as
polydimethylsiloxane (PDMS), a high-throughput, inexpensive
procedure.
[0065] More specifically, FIG. 1A is a schematic diagram showing in
cross-section two lateral cell trapping pores or junctions on the
sides of a central channel. In this and other figures herein,
specific examples are shown wherein a narrow trapping fluidic
connection operatively connects to the lateral pore. A larger
fluidic connection or reservoir is provided at the other end of the
narrow trapping connection (or channel) to allow easier fluidic
access. Such trapping connections and pores can be configured in a
circular central channel, a curved central channel, or a roughly
linear central channel, as described elsewhere herein. Spacing
between pores and the dimensions of elements can be varied, as will
be understood to those of skill in the art from the teachings
described herein.
[0066] FIG. 1B is a top view micrograph of an example device
showing a circular central channel with 14 radial cell trapping
pores. Each trapping pore is connected by a narrow trapping channel
to a fluidic reservoir that can be connected to other microfluidic
controls on the device as described elsewhere herein. In this
example, two larger channels that can be used for ingress or egress
of cells, fluids, or other materials connect to the central
channel. There are fourteen small channels illustrated in this
example (though only eight are opened in this particular chip) for
cell trapping. Operational electrical connectivity of electrodes in
one of the ingress/egress channels and three trapping connections
is shown schematically in the figure. The small circle at the lower
left indicates one of the lateral capture sites. In this design,
cell pores are radially about 200 microns apart, as shown by the
dimensional bar. This dimension is an example only, and much
smaller versions of this same design can also be constructed.
[0067] FIG. 1C is a top view micrograph of an example higher
density device showing a roughly linear main channel, and 12
trapping pores and connected trapping channels that are about 20
microns (.mu.m) apart or less. Again, optional electrical
connectivity is schematically shown. In this figure, the 12
circular diagrams indicate locations of trapped cells at the
lateral trapping junctions.
[0068] FIG. 1D is a schematic showing and alternative
cross-sectional side view. When trapped, a cell is pulled laterally
into the small trap-ping channel by applying a negative pressure.
The trapped cell acts as a high resistance component in the
circuit.
[0069] FIG. 2 illustrates aspects of an example double channel
device allowing for rapid change of intracellular and extracellular
solutions according to alternative specific embodiments of the
invention. In this alternative design, negative pressure can be
maintained in the intracellular channel with respect to the
extracellular channel to cell trapping, while the two separated
channels allow rapid exchange of fluids in either channel. This
type of trapping junction can be incorporated in various designs as
will be understood from the teachings provided herein.
[0070] FIG. 3 illustrates an example of a simple lateral junction
cell trapping disposable concentric device according to alternative
specific embodiments of the invention. This is a further
alternative configuration showing two fluidic connections for the
intracellular area, allowing for fast fluidic exchange and
providing a very simple immobilization area for cell
electroporation. In particular example embodiments, a device such
as shown in FIG. 3 or in any of the other figures can be
incorporated into various cell handling or lab-on-a-chip systems
that may incorporate many other handling steps of structures.
3. Pore Configurations
[0071] FIG. 4 and FIG. 5 are close-up micrographs showing aspects
of cell trapping pores according to specific embodiments of the
invention. In each of these figures, a different geometry opening
is presented to the cellular area for effecting cell trapping.
Either of these cell trappings junctions or other junction
configurations including semi-circular capture areas can be
incorporated into devices according to specific embodiments of the
invention as an alternative to the straight lateral capture pore
illustrated in other examples provided herein. Different
configurations may provide better trapping and/or sealing in
particular situations.
[0072] FIG. 4A-B illustrate geometries for an opening to a lateral
cell trapping junction particularly suited for electroporation
and/or electrofusion according to specific embodiments of the
invention. FIG. 4A is a micrograph illustrating an
electroporation/backside perfusion connection and a cell trapping
poor in which an electrode can be configured in one end of a Y
shaped channel. FIG. 4B illustrates a configuration wherein two
cell trapping pores are closely spaced, in this example within an
indentation off of the mail cell transport channel, to allow the
immobilization of either two cells; a cell and a vesicle; or two
vesicles in a position convenient for eletrofusion.
[0073] The lateral design according to some specific embodiments of
the invention also allows for construction of systems having
efficient multiplexing of measurements, exchange of intracellular
and extracellular electrolyte while the cell is attached to the
pore, and optical observation of membrane deformation and cellular
content. Thus, in specific embodiments, the invention enables high
throughput, low cost cell-based patch clamp measurements and other
cellular manipulations and/or assays.
[0074] According to specific embodiments of the invention, aspects
of the invention can be incorporated into one or more integrated
systems that provide simple yet elegant means for trapping multiple
cells instantaneously by pneumatic controls and allows simultaneous
electrical and optical characterizations, providing an ideal
mechanism for high throughput screening (HTS) single cells analysis
and drug discovery.
[0075] FIG. 5A-B illustrate optional different geometries for an
opening to a lateral cell junction according to specific
embodiments of the invention.
4. Example Integrated Cell Manipulation System
[0076] In further embodiments, a high-density integrated cell
handling system provides improves visualization and control of cell
position allowing the integration of whole cell electrophysiology
with easily manufactured microfluidic lab-on-a-chip devices. As
described elsewhere herein, devices of the invention can be
fabricated by micromolding of polydimethylsiloxane (PDMS) or
materials with similar or analogous properties. In specific
embodiments, holding a cell at a pore at an integrated substrate
eliminates the need for vibration isolation and allows for more
precise electric focus as compared to other methods. The present
invention in specific embodiments achieves this even with very
dense pore placement. According to specific embodiments, a cell
manipulation device of the invention also allows direct cell
visualization including of multiple cells and using standard
microscopy.
[0077] In specific embodiments of the invention, microfluidic
integration allows capillaries to be arrayed about less than 10-30
microns apart, for a total chamber volume of less than about 0.5
nanolitres. The geometry of cell pores and fluidic connection in
specific embodiments permits high quality, stable, whole-cell seals
despite the hydrophobicity of some surfaces, such as PDMS. In
particular embodiments, the lateral geometry of the trapping
junctions in combinations with the long, thin trapping channels as
shown provides great surface area allowing for a more stable
seal.
[0078] Replacing silicon micromachining with PDMS micromolding has
a number of advantages, among which, the fabrication is
sufficiently simple (requiring only molding and bonding) and
economical to enable the production of single use disposable
devices. Further, unlike silicon based devices, the PDMS device is
transparent and can be bonded to a 12 mm glass coverslip,
permitting placement on the stage of an inverted microscope and
visualization of cells during recording.
[0079] FIG. 6A-C illustrate aspects of an example integrated
cellular manipulation array on is a microfluidic platform according
to specific embodiments of the invention. As above, cell trapping
is achieved by applying negative pressure to trapping connection
capillaries which open into a main chamber containing cells in
suspension, which are represented schematically as spheres in FIG.
6A. Attached cells deform, protruding into the capillaries.
[0080] Where desired, electroporation, electrofusion, and/or
electrical monitoring is enabled by connecting electrodes (such as
the AgCl electrodes described herein) in each of the capillaries,
as well as the main chamber. Signals are fed through a multiplexing
circuit and into the data acquisition system (multiplexer setup and
microscope objective not to scale). The device can be bonded to a
glass coverslip for optical monitoring.
[0081] FIG. 6B is a scanning electron micrograph of three recording
capillary orifices as seen from a main chamber of a cell
manipulation system according to specific embodiments of the
invention. Example capillary cross-section dimensions are 4
.mu.m.times.3 .mu.m, with a pore-to-pore (or site-to-site) distance
of about 20 .mu.m.
[0082] FIG. 6C is a darkfield optical microscope image of cells
trapped at three capillary orifices. An example device having 12
capillaries arrayed six along each side of the main chamber fluidic
channel, along a 120 .mu.m distance is shown in the micrograph of
FIG. 1C.
[0083] In this example configuration, cell trapping was confirmed
by light microscopy. The cells are placed in suspension in the
central chamber and are sequentially brought to the patch pores by
applying negative pressure (28 kPa) to the patch capillaries. In an
example system, the total time required for trapping is under three
seconds per cell. Trapped cells (shown schematically in FIG. 6A)
can be visualized using dark field microscopy as seen in FIG.
6C.
[0084] In an array according to specific embodiments of the
invention, the recording capillary and the cell substrate are
mechanically bonded, eliminating the need for external positioning
devices and minimizing the effects of ambient vibration. Early
testing has confirmed that seals last for more than about 20-40 min
even without the use of vibration isolation equipment, though it is
expected that longer seal times can be achieved.
[0085] Advantageous features of this design are inherent
microfluidic integration, very high density of cell trapping sites,
and the ability to measure both cell deformation and membrane
integrity.
[0086] As a further improvement to a microfluidics based lateral
geometry for electrical measurements of cells, the average seal
resistance is increased to provide better electrical
characteristics, for example by partial cure bonding of the
elastomer, which results in improvement in cell attached seal
resistance to the gigaohm range.
[0087] FIG. 7A-D show four frames from a micrograph movie showing a
HeLa cell being trapped at a lateral junction by applying a
negative pressure (e.g., 2 psi) to a trapping channel according to
specific embodiments of the invention. In FIG. 7C, the frame is
magnified in order to show cell positioning on the pore. In FIG.
7D, real time observation of the cell membrane deformation is
shown. In this example figure, pore openings are approximately 1-3
microns in cross-section.
[0088] As can be seen in the example figures, fluorescent images of
trapped cells indicated that the cell membrane routinely protrudes
large distances (x>10 m) into the channel for a relatively low
trapping pressure of 28 kPa. Therefore, seal formation is not
restricted to the recording capillary orifice, and can occur
several micrometers along the length of the capillary. A pressure
spike leads to a membrane break, corresponding to a rise in
cellular capacitance. Cytoplasmic access can also be confirmed by
observing the diffusion of dye from the cell interior into the
recording capillary. The ability to make these types of
measurements is a unique advantage of the lateral trapping design,
because both the recording capillary and the cell are in the same
optical plane. Mechanical and electrical breakdown of the membrane
and dye diffusion out of the cell can be quantified, and such data
can be used to characterize single cell electroporation on a
similar platform.
5. Electroporation and Electrofusion Operation
[0089] In one operation example, the feasibility of electroporating
single cells using an elastomeric device with small (3.times.4
.mu.m) lateral trapping/electroporation channels was shown by
electroporating Hela cells using low applied voltages (<1V). The
average transmembrane potential required for electroporation of
Hela cells is 0.51V+0.13. Membrane permeation was assessed
electrically by measuring characteristic `jumps` in current that
correspond to drops in cell resistance, and microscopically by
recording either the escape of a cytoplasmic dye Calcein AM or the
entrance of Trypan blue stain. The device configuration focuses the
electric field, eliminating the need to manipulate electrodes or
glass pipettes and allows parallel single cell electroporation.
[0090] As described above, the invention hydrodynamically traps
individual cells at the point of largest potential drop. This
avoids the need to manually manipulate electrodes to target an
immobilized cell as is common in traditional single cell
electroporation set-ups. In specific embodiments, the electrodes or
electrical connections can be placed far from the trapping site, an
improvement over some earlier designs in which it is critical to
place the electrodes as close to the cell as possible.
[0091] In specific embodiments, the invention makes use of a
trapping channel whose height (e.g., about 3.1 .mu.m) is
approximately a third of the cell's diameter. A large inlet channel
(e.g., cross-section: 200.times.50 .mu.m) is used to introduce the
cells into the device; the cells flow freely pass the trapping
channels. A cell is hydrodynamically trapped by applying negative
pressure to the trapping channel via an attached syringe or other
suction mechanism as a cell passes by.
[0092] By sequestering individual cells in PDMS channels before
electroporation, the invention focuses the electric field such that
the greatest potential drop occurs across the first membrane of the
cell. In this way, localized electroporation can be achieved at
relatively low applied voltages.
[0093] Using one of the example chip layouts shown, a large
circular chamber allows cells to move at a relatively slow speed
(.about.20 .mu.m/s) compared to the speed of the cells in the inlet
channel (.about.100 .mu.m/s). The trapping channels are arrayed
around the circular chamber to sequester individual cells. The
cross-sectional dimensions of an example trapping channels are 4
.mu.m.times.3.1 .mu.m.
[0094] Because resistance is inversely proportional to surface
area, the small portion of the cell inside the immobilizing channel
has a much higher resistance (.about.80.times.) than the portion
outside the channel. The greatest potential drop therefore occurs
across the portion of the cell membrane inside the channel. In this
configuration, low applied voltages are sufficient to achieve
electroporation with a high electric field across that first
membrane (.about.750 kV/cm). Furthermore, the planar aspects of the
invention in specific embodiments allows for parallel trapping and
electroporation of several cells. This design also enables the
electrodes to be placed a distance away from the cell, eliminating
the potential of adverse products from reactions occuring at the
electrodes.
[0095] In further embodiments, a system of the invention uses
Ag/AgCl electrodes and a patch clamp amplifier, allowing accurate
current traces not commonly reported in electroporation
experiments. Ag/AgCl electrodes have smaller double layers and less
loss of applied potential at the solution interface. Finally, the
trans-parent PDMS, unlike opaque silicon, enables fluorescent
detection and monitoring.
[0096] FIG. 8 illustrates aspects of an example circuit model of a
cell according to specific embodiments of the invention The
potential drop across the cell is significantly greater than any
other potential drop. Therefore, other cells in close proximity to
the trapped cell would not be electroporated by the electric field.
The cell itself is modeled as a parallel combination of a resistive
R.sub.cell and a capacitive C.sub.cell element (whose effect is
transient). The R.sub.leak is the resistance of the pathway where
the current by-passes the cell, thus it is in parallel to
R.sub.cell. R.sub.leak is determined from the initial current at
low voltages, when R.sub.cell is so high such that all the current
can be assumed to go through R.sub.leak It is assumed that
R.sub.leak remains constant before and after electroporation. After
electroporation the only element that changes in the model is the
R.sub.cell, which drops because of the formation of
electropores.
[0097] Since the phospholipid membrane of the cell has much higher
resistance than both the cytosol and the extracellular medium, only
the potential drop across the membrane is significant. The
resistance of the cell can thus be further broken down into the
resistance of the membrane inside the channel
(R.sub.mem.sub.--.sub.channel) and the resistance of the membrane
outside the channel (R.sub.mem.sub.--.sub.outside). By
approximation from trapped cell images, the membrane surface area
outside of the channel is about 80 times that of the membrane
within the channel. This implies that R.sub.mem.sub.--.sub.channel
is about 80 times larger than R.sub.mem.sub.--.sub.outside. Hence
the resistance of the cell can be approximated as solely the
resistance of the membrane within the channel. This resistance
distribution gives rise to localized electroporation of the cell
membrane within the channel.
[0098] In addition to recording electrical measurements, two
different assay experiments were performed to verify poration:
recording either the escape of Calcein AM or the entrance of
[0099] Trypan blue. Calcein is membrane permeant; in live cells,
the non-fluorescent Calcein AM is converted to green-fluorescent
Calcein by intracellular esterases. The resulting fluorescent
Calcein is highly charged and therefore cannot be excised from the
cytoplasm once it has infiltrated the cell unless non-selective
electropores are introduced. The high sensitivity Calcein AM
fluorescence is useful in quantifying the diffusion of dye out of
the cell once the cell is electro-permeated. The color of the
membrane impermeant dye Trypan blue is normally undetectable at low
concentrations; once the membrane is permeabilized and the dye can
accumulate within the cell, a dark blue color becomes apparent.
Therefore, by using the Trypan blue assay, the electro-permeated
area can be readily visualized.
[0100] Thus, according to specific embodiments, devices and systems
as described herein can selectively immobilize and locally and
reversibly electroporate single cells with less than 1 V. Moreover,
the electroporated cell can be simultaneously monitored
electrically (via impedance measurements) and optically (via
fluorescence detection), enabling multiplexing for high content
screening. An improved electroporation device of the invention
provides more controlled intracellular compound delivery by either:
(1) using intracellular perfusion and (2) using preloaded vesicles
or cells to deliver compounds to the targeted cell via
electrofusion.
Example Experimental Set-Up
[0101] In an example set-up, Ag/AgCl electrodes are connected via
tubing to one of the main channels of the chip, and to one or more
of the immobilizing channels. These electrodes, which serve both to
apply the voltage and record the current, are good recording
electrodes because of their minimal electrical double layer (EDL).
The other main channel is connected to a syringe for cell loading.
The two electrodes are connected to an amplifier (PC-ONE Patch
clamp, Dagan) which provides the voltage and measures the current.
The amplifier is controlled by a custom-made Labview (National
Instruments) application through a data acquisition card
(PCI-6024E, National Instruments). The chip is monitored with an
inverted microscope (Eclipse TS 100, Nikon) with a fluorescent
module and is video captured with a camera (DXC-190, Sony) and a
video capture card (microVideo DC50, Pinnacle) on the same
computer.
[0102] To start the electroporation experiments, all channels in
the chip are filled with filtered PBS solution and extra care is
taken to expel any air bubbles in the tubing. The linear resistance
of the open channel is first measured via the amplifier to be
.about.12 M.about.. The cell/dye suspension is then introduced into
the device after incubation with a syringe; the injection is
controlled manually to allow cell trapping by applying negative
pressure on the trapping channel. Once a cell is trapped, a
`current-voltage trace` program written in Labview is run to input
a sequence of pulses with increasing amplitude (at 0.1V intervals
from 0V to 1.0V) while recording the current at a sampling rate of
10 kHz (FIG. 9A, inset). A second sequence of pulses is also
applied 60 s after the first sequence to allow time for
resealing.
[0103] With the pulse duration at about .about.6.5 ms, recording
was performed from 17 Hela cells. The characteristic `jump` in
current is observed in 15 of the 17 cells. The volt-age is varied
from 0 to 1V in 0.1V intervals. A typical resulting current trace
from one of the cells is shown in
[0104] FIG. 9A. Leak current, i.e. the cur-rent that goes around
the cell because the seal resistance is not infinite, is subtracted
(based on the circuit diagram depicted in FIG. 8) to isolate the
current across the cell (FIG. 9B). The leak resistance, R.sub.leak,
is measured for each cell from initial current traces at low
voltages and assumed to be constant (.about.35M.about.). A
significant jump in current is evident at 0.8V. The average applied
voltage of electroporation for the 15 cells that show jumps is
0.76V+0.095. As discussed before, the major potential drop is
across the membrane trapped in the channel. Hence, for this cell,
the transmembrane voltage across the electroporated membrane is
.about.0.6V, which is within the voltage range (0.2-1.5V) of
dielectric breakdown suggested by most published data [3,4]. The
average transmembrane potential for the population of cells is
0.51V+0.13.
[0105] The data also demonstrates evidence for cell resealing. FIG.
10A-B are graphs each illustrating example electrical measurements
for three different runs demonstrating reversible electroporation
according to specific embodiments of the invention. FIG. 10A
illustrates current for three consecutive pulse sequences, showing
that the current in the second run pulse sequence shows similar
characteristics of low current at low applied voltages as in the
first run, indicating that the cell cell has returned to higher
resistance and the electroporation openings have resealed. The
final run data shows a cell after multiple pulse sequences, in
which a much more linear response results, apparently illustrating
that the cell finally loses its ability to reseal after multiple
runs. Thus, after the first sequence of pulses is applied, the
membrane is permeated as the current jumps to a relatively high
level. However, when the second sequence of pulses is applied after
60 seconds, small applied voltages (<0.6 V) again result in very
low currents, similar to those of the first sequence of pulses.
This is evidence that the pores shrink after release from the
electric field. In the subsequent run, the current jump occurs
sooner than in the first run because the pores still exist;
therefore, it is easier to reopoen them with the electric field
than to create new ones. The final run in the sequence is presented
to compare the resealing capabilities with the more linear response
of a cell that has lost its ability to reseal.
[0106] FIG. 11A-B illustrate electrical and optical characteristics
of a Hela (human carcinoma) cell electroporated and loaded with
propidium iodide from the intracellular perfusion channel (e.g., as
described above) according to specific embodiments of the
invention. Valves at each end of the perfusion channel allow
compounds to be introduced after a cell is trapped. FIG. 11A shows
typical current versus time graph wherein a jump in current
correlates with electroporation and a decrease in electrical
resistance because of the pores. FIG. 11B illustrates optical
measurements of intracellular perfusion. Cell is trapped,
electroporated, and loaded with propidium iodide from the backside.
The graph is the time lapse intensity of the cell's cross
section.
[0107] FIG. 12A-B are micrographs illustrating electrofusion of two
cells (A) and two vesicles (B) according to specific embodiments of
the invention. FIG. 12A shows two Hela cells (membranes dyed with
rhodamine) trapped in the cell fusion device after the electric
field has been applied. FIG. 12B shows two vesicles trapped. For an
example electrofusion operation, 3V for 30 ms was applied across
the cells. Unlike the PBS used for electroporation, the
electrofusion media was a combination of BSA with glucose and
magnesium and chloride ions. Furthermore, the time required for
electrofusion was significantly longer (.about.20 minutes). The
membrane of the two cells stuck together and transfer of calcein
was observed. Other pulse parameters and medium for electrofusion
may be optimized in different embodiments. To demonstrate
electrofusion, one Hela cell was preloaded with calcein and the
other was not. After pulse application, the calcein slowly leaked
into the other cell.
[0108] Thus, the invention can selectively trap targeted cells and
focus the electric field for reversible electroporation (in which
the pores reseal), intracellular perfusion, and cell fusion while
simultaneously monitoring the cell electrically and optically.
[0109] In further embodiments, lateral trapping junctions of the
invention can be embodiment into larger-scale integrated systems
for high-throughput cellular analysis and previously described.
6. Example Fabrication
[0110] Systems and devices as described herein can be fabricated
using any techniques or methods familiar from the field of
photolithography, nano-fabrication, or micro-fluidic fabrication.
For completeness of this disclosure and to discuss additional and
independent novel aspects according to specific embodiments of the
invention, specifics of example fabrication methods are provided
below.
[0111] Example fabrication steps according to specific embodiments
of the invention are shown diagrammatically in FIG. 13(a-f). In
this example, a silicon or other suitable substrate mold is
prepared using surface micromachining and/or photolithography
techniques. In other examples, any other appropriate material and
any other techniques for making a mold could be used.
[0112] In a micromachining example according to specific
embodiments of the invention, first, 3.1 .mu.m height patterns are
made, defining the narrow cell trapping channels using deep
reactive ion etching (FIG. 13A). Second, 50 .mu.m high patterns are
added for wide connection regions using SU-8 negative photoresist
(FIG. 13B). After a base and a curing agent of PDMS were mixed
(1:10), the liquid mixture is poured onto the mold and cured at
80.degree. C. for 1 hour (FIG. 13C).
[0113] After the PDMS is cured, the devices are detached and can be
mechanically punched. In this specific example, the devices and the
glass substrate pre-coated with a thin PDMS layer are treated with
oxygen plasma (FIGS. 13D and E) and the devices are bonded to the
thin PDMS (FIG. 13F).
[0114] SEM images of overall device geometry before bonding (upside
down) and a closeup of the patch pore after bonding are shown in
FIGS. 13G and H. A SEM image of the mold is shown in FIG. 13I.
[0115] In this example, it was observed that the top of the orifice
is rounded. The rounding of the top of the orifice is a beneficial
result of mold fabrication, and it was observed that the channel
top is rounded next to the patch orifice in the mold (FIG. 13I).
When the SU8 is selectively polymerized in order to create the
large channels on top of the small patch channel defined in Si,
light scattering near the Si surface results in a deviation from
the intended vertical SU8 wall. The resulting rounded feature at
the bottom of the SU8 wall (FIG. 13I, arrow A) is also present on
top of the small Si wall (FIG. 13I, arrow B), resulting in rounding
of the patch orifice top. This feature is reproducible in specific
fabrication embodiments at every patch orifice.
[0116] For fluidic connections to outside tubing, 0.5 mm holes can
be punched mechanically into the cured and detached PDMS device.
The device can be subsequently bonded to a thin PDMS layer which is
spin cast and cured onto a glass substrate. Plastic or other tubes
can be connected to the reservoirs, via punched holes, to load both
cells and electrolyte solutions and to apply suction to the patch
channel.
Example Alternative Fabrication Methods
[0117] The following further example fabrication methods are
provided for completeness of this disclosure. It will be recognized
from these teachings that many alterations can be made to this
method to accommodate different materials and/or methods of
manufacturing and/or to provide different configurations as
otherwise described herein.
[0118] A specific example device according to embodiments of the
invention can be fabricated as follows, similarly as described
above. First, a mold is prepared using surface micromachining
techniques. As an example, first the narrow patch capillaries are
made with 3.1 micron high patterns using deep reactive ion etching.
Recording capillaries are 20 microns apart, which allows trapping
of, for example, 12 cells along a main channel in a volume of 0.36
nL (150.times.60.times.40 micron.sup.3 for length, width, and
height, respectively). Therefore, in the active device area, the
reagent dead volume is 30 pL per recording site. Second, 50 micron
high patterns are added for wide connection regions, for example
using negative photoresist. After the PDMS (e.g., Dow-Corning
Sylgard 184) base and curing agent were mixed (at an example ration
of 1:10), the liquid mixture is poured onto the mold and cured at
room temperature for 24 hours. For fluidic connections to outside
tubing, 0.5 mm holes were punched mechanically into the cured and
detached PDMS device. A thin PDMS layer was spin cast on a glass
substrate at 3000rpm for 30 seconds and partially cured (e.g.,
90.degree. C. for 1 min.). The device is bonded to the substrate by
gently placing the two in contact and fully curing the bottom layer
(120.degree. C. for 5 minutes). For use, plastic tubes are
connected to the reservoirs, via punched holes, to load both cells
and electrolyte solutions and to apply suction to the channel.
[0119] Partial cure bonding improves the geometry of the recording
capillary by altering the final geometry of the bonded and cured
capillary allowing for a tight seal between the cell membrane and
the capillary walls even with the hydrophobicity of the PDMS.
Partial curing is believed to affect the cross-section of the
trapping channel geometry, where instead of providing a square
cross-section provides a rounded cross section allowing for a more
stable seal.
7. Example Operation
[0120] FIG. 14A-B illustrate current response to a 20 mV voltage
pulse before (a) and after (b) cell trapping of an example device
according to specific embodiments of the invention. Disassociated
cells were suspended in PBS and injected into the main channel.
Gentle pressure (1 psi) was applied to the trapping connection
while cells were loaded into the main fluidic channel in order to
prevent contamination at the trap site. A cell can either be
trapped randomly or selectively by controlling the flow through the
main fluidic channel. In specific example experiments with an early
design, it was found that a cell within 100-200 .mu.m of the
channel opening can be trapped within a 1 s time interval by
applying 2 psi of negative pressure to the channel.
[0121] Right after trapping the cell, negative pressure was removed
and the cell was allowed to form a seal with the rim of the
channel. In specific embodiments, membrane protrusion into the
channel can be seen using standard microscopy and visualization of
trapping can be used to control pressure application. In
alternative embodiments, cell trapping can be confirmed for example
by measuring the resistance at a pore and using the change in
measured resistance to confirm the presence of cell and to reduce
pressure at that pore where desired.
[0122] The current response from the cell by a 20 mV/50 ms current
pulse is shown in FIG. 14B. By applying positive pressure to the
trapping channel, the trapped cell was expelled from the channel.
As soon as the cell was expelled, the current response returned to
that of the open channel. Subsequent cell trapping in the same
channel resulted in lower seal resistance, presumably due to
contamination at the opening of the trapping channel. In specific
embodiments, the invention proposes a single use cell-trapping
device, so this contamination does not affect overall usability. In
alternative embodiments, a variety of known cleaning techniques
could be used to remove contamination from the cell pores.
[0123] In experimental results such as with the device illustrated
in FIG. 6, electrical connection to the recording capillaries is
achieved by inserting Ag/AgCl electrodes into tubing connections
outside the active area of the device. The electrodes are in turn
connected to the inputs of a multiplexer circuit, which connects to
electric signal generates for electroporation and/or electrofusion
and may also feed into a headstage of a traditional patch clamp
amplifier (in an example system, a customized multiplexor was used
in combination with PC-ONE patch clamp components from Dagan,
Minnesota). The amplifier was driven by custom software (for
example, written in a LabView programming environment) and
interfaced via an analog to digital conversion board.
[0124] Once the device is filled, adherent cells are trypsinized,
spun down at 1000 rpm for 5 minutes, and resuspended in sterile
electrolyte solution at a concentration 5.times.10.sup.6 cells/ml.
A 3 ml syringe is used to inject cells into the main channel.
Gentle positive pressure (7 kPa) is applied to the patch channel
while cells were loaded into the main fluidic channel in order to
prevent contamination at the patch site. A cell can either be
trapped randomly or selectively by controlling the flow through the
main fluidic channel. A cell found within 100-200 microns of the
patch channel opening could be trapped within a 3 s time interval
by applying 14-21 kPa of negative pressure to the patch channel.
Immediately after trapping the cell, the negative pressure is
removed and the cell is allowed to form an electrical seal with the
patch channel orifice. Patch array measurements can be performed
without the use of vibration isolation equipment.
[0125] For patch analysis and traditional electrophysiology,
currents were sampled at 5 kHz and filtered with a 2 kHz low-pass
Bessel filter. The holding potential was -80 mV and depolarizing
stimuli were applied at an interval of 10 s, unless otherwise
specified. All signals were post-processed with automated leak
subtraction by custom written routines that subtracted a calculated
leak current (square wave) from all data traces. The leak
resistance was assumed to be constant, and obtained by dividing the
resting potential (-80 mV) by the current passed at that
voltage.
[0126] Both internal and external electrolyte solution contained
(mM): 140 KCl, 2 CaCl.sub.2, 2 MgCl.sub.2, 20 HEPES, 10 Glucose. pH
was adjusted to 7.3 with KOH and osmolality adjusted to 300 mOsms
with glucose.
[0127] Thus, in specific embodiments, the invention can be embodied
as a disposable electromanipulation microarray with integrated
microfluidics as an electrophysiological tool. Key features of
specific example designs include high electroporation site density,
built-in integration with microfluidics, and the ability to study
cells by standard microscopy techniques during handling. A
micromolded array according to specific embodiments of the
invention is capable of mammalian cell recording in a high density
format. The device contains an array of lateral capillaries which
trap cells efficiently to form tight electrical seals. This scheme
has the advantage of integrated microfluidics for compound exchange
on both the intracellular and extracellular sides of the cell
membrane. The distance between trap sites can be on the order of 20
.mu.m.
[0128] Another convenient feature of this design is the low fluidic
volumes associated with perfusion chambers and channels. In one
example design, the volume of the main chamber containing 12 cell
holding sites is 0.36 nL. By comparison, other planar technology
can require reagent volumes of 10-100 microL per site. Therefore,
the reduction in dead volume over such proposals is of order
10.sup.4. This allows rapid solution exchange to expose attached
cells to different reagents in fast succession, with very small
reagent consumption and highly uniform solution content between the
arrayed sites.
8. Array of Hollow Cylindrical Electrodes for Microfluidic and
Electric Interface
[0129] According to further specific embodiments of the invention,
an arrangement of hollow Ag/AgCl cylindrical electrodes can be used
with a microfluidic electroporation system as described above to
serve as both a fluidic interface and an electrical interface for
microfluidic chips. In specific embodiments, as fluid flows through
these hollow electrodes, electrical and fluidic connections are
established. This eliminates the need for fragile, cumbersome, and
expensive Ag/AgCl pellet electrodes that have often been used in
patch clamp applications and also minimizes microfluidic circuitry.
While polymeric devices themselves can be manufactured for just
pennies a piece, the high cost of the electrodes can be a the
cost-limiting factor. Additionally, the delicate pellet electrodes
tend to break easily, especially because they are jammed into the
fluidic tubing while the fluid must flow around them.
[0130] While electrodes made of noble metals can be easily
deposited onto a glass substrate, Ag/AgCl is more difficult.
Integrated thin film Ag/AgCl electrodes have been demonstrated but
have their drawbacks. In further specific embodiments of the
invention, low cost electrodes are provided that can efficiently
mate with various microfluidic systems, including systems described
herein. Such electrodes are critical, for example, to
electro-physiological manipulations and measurements for high
through-put screening. The invention, therefore, according to
specific embodiments, routes fluid flow through instead of around
the electrodes using a detachable Ag/AgCl array, for example built
on a printed circuit board (PCB). These electrodes, according to
specific embodiments of the invention, serve not only as electrical
connections, but fluidic conduits as well.
[0131] In specific examples, fluid flows through the hollow Ag/AgCl
electrodes that connect the device to tubing that then connects to
a syringe for sample loading. The configuration of the electrodes,
as well as additional processing capabilities, can be specifically
designed to mate with the microfluidic device.
[0132] FIG. 16 illustrates an example schematic of a hollow
electrode interface (in this example configured on a PCB) mating
with device and tubing according to specific embodiments of the
invention. In this figure, the conduit/electrodes are embedded in a
detachable PCB interface that includes passivated lateral
connectors for easy electrical connection to other equipment, as
will be understood in the art. In these example figures, a circular
arrangement of six electrode/conduits are shown, which are arranged
to mate with channel connections on a microfluidic devices and
optionally also with an external source of fluidics. This is only
an example arrangement, however, and any other convenient
arrangement is possible.
9. Diagnostic and Drug Development Uses
[0133] As described above, following identification and validation
of a assay for a particular cellular process, in specific
embodiments devices and/or systems as described herein are used in
clinical or research settings, such as to screen possible active
compounds, predicatively categorize subjects into disease-relevant
classes, text toxicity of substances, etc. Devices according to the
methods the invention can be utilized for a variety of purposes by
researchers, physicians, healthcare workers, hospitals,
laboratories, patients, companies and other institutions. For
example, the devices can be applied to: diagnose disease; assess
severity of disease; predict future occurrence of disease; predict
future complications of disease; determine disease prognosis;
evaluate the patient's risk; assess response to current drug
therapy; assess response to current non-pharmacologic therapy;
determine the most appropriate medication or treatment for the
patient; and determine most appropriate additional diagnostic
testing for the patient, among other clinically and
epidemiologically relevant applications. Essentially any disease,
condition, or status for which a cellular characteristic measurable
using cell electroporation can be evaluated.
Kits
[0134] A device according to specific embodiments of the present
invention is optionally provided to a user as a kit. Typically, a
kit of the invention contains one or more cellular electroporation
devices constructed according to the methods described herein. Most
often, the kit contains a device packaged in a suitable container.
The kit typically further comprises, one or more additional
reagents, e.g., substrates, tubes and/or other accessories,
reagents for collecting blood samples, buffers, e.g., erythrocyte
lysis buffer, leukocyte lysis buffer, hybridization chambers, cover
slips, etc., as well as a software package, e.g., including the
statistical methods of the invention, e.g., as described above, and
a password and/or account number for accessing the compiled
database. The kit optionally further comprises an instruction set
or user manual detailing preferred methods of using the kit
components for sensing a substance of interest.
[0135] When used according to the instructions, the kit enables the
user to identify disease specific cellular processes and/or to
introduce substances into a cell using electroporation. The kit can
also allow the user to access a central database server that
receives and provides expression information to the user. Such
information facilitates the discovery of additional diagnostic
characteristics by the user. Additionally, or alternatively, the
kit allows the user, e.g., a health care practitioner, clinical
laboratory, or researcher, to determine the probability that an
individual belongs to a clinically relevant class of subjects
(diagnostic or otherwise). In HTS, a kit according to specific
embodiments of the invention can allow a drug developer or
clinician to determine cellular responses to one or more treatments
or reagents, either for diagnostic or therapeutic purposes.
Embodiment in a Programmed Information Appliance
[0136] The invention may be embodied in whole or in part within the
circuitry of an application specific integrated circuit (ASIC) or a
programmable logic device (PLD). In such a case, the invention may
be embodied in a computer understandable descriptor language, which
may be used to create an ASIC, or PLD that operates as herein
described.
Integrated Systems
[0137] Integrated systems for the collection and analysis of
cellular and other data as well as for the compilation, storage and
access of the databases of the invention, typically include a
digital computer with software including an instruction set for
sequence searching and/or analysis, and, optionally, one or more of
high-throughput sample control software, image analysis software,
collected data interpretation software, a robotic control armature
for transferring solutions from a source to a destination (such as
a detection device) operably linked to the digital computer, an
input device (e.g., a computer keyboard) for entering subject data
to the digital computer, or to control analysis operations or high
throughput sample transfer by the robotic control armature.
Optionally, the integrated system further comprises an electronic
signal generator and detection scanner for probing a patch clamp
device. The scanner can interface with analysis software to provide
a measurement of the presence or intensity of the hybridized and/or
bound suspected ligand such as by measurement of electrical
characteristics of the cell membrane.
[0138] Readily available computational hardware resources using
standard operating systems can be employed and modified according
to the teachings provided herein, e.g., a PC (Intel x86 or Pentium
chip-compatible DOS,TM OS2,.TM. WINDOWS,.TM. WINDOWS NT,.TM.
WINDOWS95,.TM. WINDOWS98,.TM. LINUX, or even Macintosh, Sun or PCs
will suffice) for use in the integrated systems of the invention.
Current art in software technology is adequate to allow
implementation of the methods taught herein on a computer system.
Thus, in specific embodiments, the present invention can comprise a
set of logic instructions (either software, or hardware encoded
instructions) for performing one or more of the methods as taught
herein. For example, software for providing the data and/or
statistical analysis can be constructed by one of skill using a
standard programming language such as Visual Basic, Fortran, Basic,
Java, or the like. Such software can also be constructed utilizing
a variety of statistical programming languages, toolkits, or
libraries.
[0139] FIG. 17 is a block diagram showing a representative example
logic device in which various aspects of the present invention may
be embodied. FIG. 17 shows an information appliance (or digital
device) 700 that may be understood as a logical apparatus that can
read instructions from media 717 and/or network port 719, which can
optionally be connected to server 720 having fixed media 722.
Apparatus 700 can thereafter use those instructions to direct
server or client logic, as understood in the art, to embody aspects
of the invention. One type of logical apparatus that may embody the
invention is a computer system as illustrated in 700, containing
CPU 707, optional input devices 709 and 711, disk drives 715 and
optional monitor 705. Fixed media 717, or fixed media 722 over port
719, may be used to program such a system and may represent a
disk-type optical or magnetic media, magnetic tape, solid state
dynamic or static memory, etc. In specific embodiments, the
invention may be embodied in whole or in part as software recorded
on this fixed media. Communication port 719 may also be used to
initially receive instructions that are used to program such a
system and may represent any type of communication connection.
[0140] Various programming methods and algorithms, including
genetic algorithms and neural networks, can be used to perform
aspects of the data collection, correlation, and storage functions,
as well as other desirable functions, as described herein. In
addition, digital or analog systems such as digital or analog
computer systems can control a variety of other functions such as
the display and/or control of input and output files. Software for
performing the electrical analysis methods of the invention are
also included in the computer systems of the invention.
[0141] Optionally, the integrated systems of the invention include
an automated workstation. For example, such a workstation can
prepare and analyze samples by performing a sequence of events
including: preparing samples from a tissue or blood sample;
exposing the samples to at least one patch clamp device comprising
all or part of a library of candidate probe molecules; and
detecting cell reactions by various electrical measurements. The
cell reaction data is digitized and recorded in the appropriate
database.
[0142] Automated and/or semi-automated methods for solid and liquid
phase high-throughput sample preparation and evaluation are
available, and supported by commercially available devices. For
example, robotic devices for preparation of cells. Alternatively,
or in addition, robotic systems for liquid handling are available
from a variety of sources, e.g., automated workstations like the
automated synthesis apparatus developed by Takeda Chemical
Industries, LTD. (Osaka, Japan) and many robotic systems utilizing
robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.;
Orca, Beckman Coulter, Inc. (Fullerton, Calif.)) which mimic the
manual operations performed by a scientist. Any of the above
devices are suitable for use with the present invention, e.g., for
high-throughput analysis of library components or subject samples.
The nature and implementation of modifications to these devices (if
any) so that they can operate as discussed herein will be apparent
to persons skilled in the relevant art.
Other Embodiments
[0143] Although the present invention has been described in terms
of various specific embodiments, it is not intended that the
invention be limited to these embodiments. Modification within the
spirit of the invention will be apparent to those skilled in the
art.
[0144] All publications, patents, and patent applications cited
herein or filed with this submission, including any references
filed as part of an Information Disclosure Statement, are
incorporated by reference in their entirety.
[0145] A basic example operation unit of this approach comprises a
main channel (containing the reaction target) and an injection
channel (used for reagent delivery). A schematic is shown in FIG.
19. In operation, a generally constant flow is supplied to the main
channel (e.g., via a syringe pump) and the injection channel is
being driven by a pressure (or flow) source at the channel inlet
P(t)--pressure as a function of time. When P(t)>P0 (Po=the
pressure in the main channel), a plume of solution form in the main
channel, engulfing the sample area. If P(t)<Po, no reagent
enters the main channel, and the existing plume is removed quickly
by the existing flow velocity in the main channel. While the
configuration of channels can be varied according to specific
embodiments of the invention, one desirable configuration is a
lateral configuration where all the channels are in roughly
horizontal planes. Thus, this and subsequent related figures can
preferable be viewed as top-down view or bottom-up view of a
device, with lateral channels therein.
[0146] In specific embodiments, because flow in the main channel
follows laminar profiles, the distance between the injection
channel and the target sample area is not a critical parameter.
Therefore, multiple reagents can be applied by simply arraying a
number of injection channels. In FIG. 20, injection channels n1-5
are preloaded with relevant reagents and controlled individually by
input pressures Pi-s(t). The pressure application can be timed so
that only one channel is on at a time, or multiple channels are on
simultaneously. In FIG. 20, a situation in which channel n4 is on
is displayed. This can be achieved for example by setting
Pi-3<Po, Ps<Po, and P-4>P0 so that a plume is only present
at the outlet of channel 4.
* * * * *