U.S. patent application number 15/296810 was filed with the patent office on 2017-02-09 for combination of single-cell electroporation and electrical recording using the same electrode.
The applicant listed for this patent is The Curators of the University of Missouri. Invention is credited to Jaya Ghosh, Kevin Gillis.
Application Number | 20170038362 15/296810 |
Document ID | / |
Family ID | 46544613 |
Filed Date | 2017-02-09 |
United States Patent
Application |
20170038362 |
Kind Code |
A1 |
Gillis; Kevin ; et
al. |
February 9, 2017 |
Combination of Single-Cell Electroporation and Electrical Recording
Using the Same Electrode
Abstract
Methods for stimulating exocytosis from a cell are provided
where the same electrochemical microelectrode is used to
electroporate an adjacent cell and then measure quantal exocytosis
from the adjacent cell. Also provided are methods for stimulating
and measuring exocytosis from a select cell population arrayed on a
chip comprising addressable electrodes. Calcium independent
stimulation of exocytosis with inorganic anions such as chloride
ions is also provided. These methods can provide for specific
stimulation of a desired subset of cells without exposing other
nearby cells to the stimulus.
Inventors: |
Gillis; Kevin; (Columbia,
MO) ; Ghosh; Jaya; (Columbia, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Curators of the University of Missouri |
Columbia |
MO |
US |
|
|
Family ID: |
46544613 |
Appl. No.: |
15/296810 |
Filed: |
October 18, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13359408 |
Jan 26, 2012 |
9488637 |
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15296810 |
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61461988 |
Jan 26, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61N 1/327 20130101;
G01N 33/48707 20130101; G01N 33/487 20130101; G01N 33/48735
20130101; C12M 47/06 20130101; G01N 33/48728 20130101; C12N 13/00
20130101; G01N 33/4836 20130101; G01N 27/3275 20130101 |
International
Class: |
G01N 33/483 20060101
G01N033/483; G01N 27/327 20060101 G01N027/327; G01N 33/487 20060101
G01N033/487 |
Goverment Interests
STATEMENT REGARDING GOVERNMENTAL SUPPORT
[0002] This invention was made with Government support under
contract number NS048826 awarded by the National Institutes of
Health. The Government has certain rights in the invention.
Claims
1. A method for stimulating and measuring exocytosis from a cell
the method comprising: electrically stimulating said cell with one
or more voltage pulses while said cell resides in a drop of
solution to provide a voltage gradient in solution surrounding the
cell, said solution drop being confined within a well such that
said solution drop is sitting on a surface of an electrode located
at the bottom of said well and the cell being adjacent to the
electrode surface, wherein said electrical stimulation is induced
by passing a transient current through said electrode with a
potentiostat, the potentiostat modified to include a diode network
in parallel configuration with a feedback resistor of the
potentiostat to allow for the passage of the transient current; and
measuring, by said same electrode, a release of an
electrochemically active substance from said stimulated cell while
said solution drop is sitting on said electrode surface the
potentiostat configured to pass a pico-amp level current through
said electrode.
2. The method of claim 1, further comprising: arraying a plurality
of cells on a chip, the chip comprising a plurality of addressable
electrodes in an array, each electrode of the array having a
surface, the plurality of cells residing in a plurality of the
solution drops, each of the plurality of solution drops including
at least one of the cells, and wherein the arraying step comprises
depositing a plurality of the solution drops in a plurality of
wells to confine said deposited solution drops such that each
deposited solution drop is sitting on a surface of a different
electrode of the array; and performing the electrically stimulating
step and the measuring step for one or more of the deposited
solution drops with respect to the electrodes on which those
deposited solution drops sit.
3. The method of claim 2, wherein said stimulating step and the
measuring step are performed when the cells are adjacent to
surfaces of essentially all of said addressable electrodes.
4. The method of claim 2, wherein said electrically stimulating
step comprises addressing a subset of said electrodes with a
voltage pulse to limit the electrical stimulation to a subset of
said cells, said cell subset being those one or more cells that are
adjacent to surfaces of said addressed electrode subset.
5. (canceled)
6. The method of claim 1, wherein said electrically stimulating
step comprises electroporating said cell.
7. The method of claim 6, further comprising introducing a
membrane-impermeable substance into said cell by
electroporation.
8. The method of claim 7, wherein said membrane-impermeable
substance comprises a peptide, an antibody, a DNA molecule, or an
RNA molecule.
9. The method of claim 8, further comprising using said DNA
molecule or said RNA molecule to express a gene of interest or to
inhibit expression of a gene of interest in said select cell
population.
10. The method of claim 9, further comprising incubating said cell,
wherein said cell is incubated after said electrically stimulating
step and before said measuring step.
11. The method of claim 4, further comprising applying an agent to
said stimulated cells.
12. (canceled)
13. (canceled)
14. A method for stimulating and measuring exocytosis from a cell,
comprising: applying, through an electrode located at the bottom of
a well, a voltage pulse to an intact cell while said cell resides
in a solution to provide a voltage gradient in solution surrounding
the cell, the solution being confined within said well and sitting
on a surface of said electrode such that said cell is adjacent to
the electrode surface, the applied voltage being sufficient to
cause a stimulation of said cell, stimulation of said cell being
induced by passing a transient current through said electrode with
a potentiostat, the potentiostat modified to include a diode
network to allow for the passage of the transient current; and
recording, through said same electrode while the intact cell
remains adjacent to the electrode, a current signal indicative of a
release of an electrochemically active substance from said
stimulated cell, the potentiostat passing a pico-amp level current
through said electrode.
15. (canceled)
16. (canceled)
17. The method of claim 14, wherein said applied voltage pulse
comprises a first pulse portion and a second pulse portion, wherein
said first pulse portion causes said cell stimulation, and wherein
said recording step is performed during said second pulse
portion.
18. (canceled)
19. (canceled)
20. The method of claim 14, wherein said current signal comprises
an amperometric spike.
21. (canceled)
22. The method of claim 14, wherein said stimulation comprises a
triggering of an action potential in said cell.
23. The method of claim 14, wherein said stimulation comprises
electroporation of said cell.
24. (canceled)
25. (canceled)
26. The method of claim 14, further comprising: confining a
plurality of cells in a plurality of said wells, each of said wells
having a different electrode at its second end such that said
different electrodes define a microchip electrode array; and
performing said applying step and said recording step for a
plurality of said cells, wherein said applying step comprises
selectively addressing a subset of said electrodes with said
voltage pulse to stimulate a desired subset of said cells without
exposing other nearby cells to stimulation.
27. The method of claim 14 wherein said electrode comprises an
electro-chemical electrode.
28. (canceled)
29. The method of claim 14, wherein said cell is a neuroendocrine
cell and wherein said recorded current signal is indicative of
quantal exocytosis.
30. The method of claim 14 wherein said applying and recording
steps are performed while said cell is contacting said electrode
surface.
31. The method of claim 14 wherein said applying and recordings
step are performed while said cell is within about 3 micrometers of
said electrode surface.
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application No. 61/461,988, filed Jan. 26, 2011,
and incorporated herein by reference in its entirety.
FIELD OF INVENTION
[0003] The present invention relates to a microchip device for
measurements of substances released from a cell, more specifically,
to a microchip device capable of electropermeabilizing a cell
membrane and measurement of release of electrochemically active
substances from the cell using the same electrode.
SUMMARY OF INVENTION
[0004] The present invention provides a new and improved technology
allowing simultaneous electrochemical detection and cell
stimulation. The invention develops the on-chip methods for
stimulating exocytosis from select cell population on the chip and
uses the same electrode to electrically stimulate the adjacent cell
and subsequently measure the release of electrochemically active
substances.
[0005] The invention further provides a new and improved
microfabricated device containing arrays of electrochemical
electrodes with one possible application being to increase the
throughput of single-cell measurements of quantal exocytosis from
neuroendocrine cells. According to one embodiment of the invention,
voltage pulses may be applied to planar electrodes to promote
efficient cell electropermeabilization to trigger exocytosis upon
Ca.sup.+2 influx from Ca.sup.+2-buffered bath solutions. In certain
embodiments, voltage pulses may be applied to planar electrodes to
promote efficient cell electropermeabilization to trigger
exocytosis upon influx of other stimulatory substances. The
amplifier may be modified to allow it to transiently pass large
currents to enable electroporation, yet record pA amperometric
currents with low noise.
[0006] Methods for stimulating and measuring exocytosis from a
select cell population are provided. In certain embodiments,
methods for stimulating and measuring exocytosis from a select cell
population can comprise, for a select population of cells arrayed
on a chip comprising addressable electrodes, electrically
stimulating the select cell population with the electrodes, and
measuring release of electrochemically active substances from the
select cell population, wherein a single electrode is used to
stimulate a cell adjacent to the electrode surface and to measure
release of electrochemically active substances from the adjacent
cell are provided. In certain embodiments of any of the
aforementioned methods, the methods can further comprise arraying a
plurality of cells on the chip. In certain embodiments of any of
the aforementioned methods, the select cell population comprises
cells adjacent to surfaces of essentially all of the addressable
electrodes. In certain embodiments of any of the aforementioned
methods, the select cell population comprises a subset of cells
that are adjacent to surfaces of a subset of the electrodes. In
certain embodiments of any of the aforementioned methods, the
stimulation comprises triggering an action potential in the select
cell population with a transient electric field. In certain
embodiments of any of the aforementioned methods, the stimulation
comprises electroporating the select cell population. In certain
embodiments of any of the aforementioned methods, a
membrane-impermeable substance is introduced into the select cell
population by electroporation. In certain embodiments of any of the
aforementioned methods, the membrane-impermeable substance
comprises a peptide, an antibody, a DNA molecule, or an RNA
molecule. In certain embodiments of any of the aforementioned
methods, DNA molecule or the RNA molecule is used to express a gene
of interest or to inhibit expression of a gene of interest in the
select cell population. In certain embodiments of any of the
aforementioned methods, the select cell population is incubated
after the stimulation and then measured. In certain embodiments of
any of the aforementioned methods, an agent is applied after the
stimulation of the subset of cells. In certain embodiments of any
of the aforementioned methods, the electrode is comprised of gold,
indium tin oxide, diamond-like carbon, or combinations thereof. In
certain embodiments of any of the aforementioned methods, the cell
adjacent to the electrode surface is either: i) within less than
about 3 micrometers, about 2 micrometers, about 1 micrometers, or
about 0.5 micrometers of the electrode surface; or, ii) is
contacting the electrode surface.
[0007] Also provided herein are methods for stimulating and
measuring exocytosis from a cell. In certain embodiments, the
methods for stimulating and measuring exocytosis from a cell can
comprise: applying, through an electrode, a voltage to a cell that
is adjacent to a surface of the electrode, the applied voltage
being sufficient to cause a stimulation of the cell; and recording,
through the same electrode, a current signal indicative of a
release of an electrochemically active substance from the
stimulated cell. In certain embodiments of any of these
aforementioned methods, the applied voltage comprises a voltage
pulse. In certain embodiments of any of these aforementioned
methods, the recording step is performed while the cell remains
adjacent the electrode. In certain embodiments of any of these
aforementioned methods, the applied voltage pulse comprises a first
pulse portion and a second pulse portion, wherein the first pulse
portion causes the cell stimulation, and wherein the recording step
is performed during the second pulse portion. In certain
embodiments of any of these aforementioned methods, the applied
voltage pulse comprises a series of the voltage pulses, and the
recording step is performed during a plurality of the second pulse
portions for the series. In certain embodiments of any of these
aforementioned methods, the recording step is performed within one
second of the applying step. In certain embodiments of any of these
aforementioned methods, the current signal comprises an
amperometric spike. In certain embodiments of any of these
aforementioned methods, the current signal is not a slowly changing
background Faradaic current. In certain embodiments of any of these
aforementioned methods, the stimulation comprises a triggering of
an action potential in the cell. In certain embodiments of any of
these aforementioned methods, the stimulation comprises
electroporation of the cell. In certain embodiments of any of these
aforementioned methods, a membrane-impermeable substance is
introduced into the cell by electroporation. In certain embodiments
of any of these aforementioned methods, the membrane-impermeable
substance introduced by electroporation comprises a peptide, an
antibody, a DNA molecule, or an RNA molecule. In certain
embodiments of any of these aforementioned methods, the methods can
comprise performing the applying step and the recording step for a
plurality of cells that are adjacent to a plurality of the
electrodes, the electrodes being part of a microchip electrode
array, and wherein the applying step comprises selectively
addressing the electrodes with the voltage pulses to stimulate a
desired subset of the cells without exposing other nearby cells to
stimulation. In certain embodiments of any of these aforementioned
methods, the electrode comprises an electro-chemical electrode. In
certain embodiments of any of these aforementioned methods, the
electro-chemical electrode is comprised of gold, indium tin oxide,
diamond-like carbon, or combinations thereof. In certain
embodiments of any of these aforementioned methods, the cell is a
neuroendocrine cell and quantal exocytosis from the cell is
recorded. In certain embodiments of any of these aforementioned
methods, the adjacent cell is contacting the electrode surface or
within about 3 micrometers of the electrode surface.
[0008] Also provided are methods for stimulating with exogenous
inorganic anions and measuring exocytosis from a cell that results
from this stimulation. In certain embodiments, the methods for
stimulating and measuring exocytosis from a cell can comprise:
applying, through an electrode, a voltage to a cell that is
adjacent to a surface of the electrode in the presence of an
exogenous inorganic anion, the applied voltage and concentration of
the inorganic anion being sufficient to cause a stimulation of the
cell; and, recording, through the same electrode, a current signal
indicative of a release of an electrochemically active substance
from the stimulated cell. In certain embodiments of any of these
aforementioned methods, the applied voltage comprises a voltage
pulse. In certain embodiments of any of these aforementioned
methods, the recording step is performed while the cell remains
adjacent the electrode. In certain embodiments of any of these
aforementioned methods, the applied voltage pulse comprises a first
pulse portion and a second pulse portion, wherein the first pulse
portion causes the cell stimulation, and wherein the recording step
is performed during the second pulse portion. In certain
embodiments of any of these aforementioned methods, the applied
voltage pulse comprises a series of the voltage pulses, and the
recording step is performed during a plurality of the second pulse
portions for the series. In certain embodiments of any of these
aforementioned methods, the exogenous chloride ion is at a
concentration of at least about 10 millimolar. In certain
embodiments of any of these aforementioned methods, the exogenous
inorganic anion is at a concentration of at least about 10
millimolar or at least about 15 millimolar to any one of about 50
millimolar, about 75 millimolar, about 100 millimolar, about 125
millimolar, about 150 millimolar, about 175 millimolar, or about
200 millimolar. In certain embodiments of any of these
aforementioned methods, exogenous calcium ions are essentially
absent when the voltage pulse is applied. In certain embodiments of
any of these aforementioned methods, the recording step is
performed while the cell remains adjacent the electrode. In certain
embodiments of any of these aforementioned methods, the applied
voltage pulse comprises a first pulse portion and a second pulse
portion, wherein the first pulse portion causes the cell
stimulation, and wherein the recording step is performed during the
second pulse portion. In certain embodiments of any of these
aforementioned methods, the recording step is performed within one
second of the applying step. In certain embodiments of any of these
aforementioned methods, the current signal comprises an
amperometric spike. In certain embodiments of any of these
aforementioned methods, the current signal is not a slowly changing
background Faradaic current. In certain embodiments of any of these
aforementioned methods, the methods can comprise performing the
applying step and the recording step for a plurality of cells that
are adjacent to a plurality of the electrodes, the electrodes being
part of a microchip electrode array, and wherein the applying step
comprises selectively addressing the electrodes with the voltage
pulses to stimulate a desired subset of the cells without exposing
other nearby cells to stimulation. In certain embodiments of any of
these aforementioned methods, the electrode comprises an
electro-chemical electrode. In certain embodiments of any of these
aforementioned methods, the electro-chemical electrode is comprised
of gold, indium tin oxide, diamond-like carbon, or combinations
thereof. In certain embodiments of any of these aforementioned
methods, the cell is a neuroendocrine cell and quantal exocytosis
from the cell is recorded. In certain embodiments of any of these
aforementioned methods, the adjacent cell is contacting the
electrode surface or within about 3 micrometers of the electrode
surface. In certain embodiments of any of these aforementioned
methods, the inorganic anion is selected from the group consisting
of bromide, chloride, iodide, and sulfate. In certain embodiments
of any of these aforementioned methods, the inorganic anion is a
chloride ion.
DESCRIPTION OF DRAWINGS
[0009] FIG. 1 is the schematic illustration of electroporation. The
two basic steps of the approach are shown here. Left: Voltage pulse
applied to cell sitting on the electrode (acting as stimulating
electrode in this case). Right: Pores form on the plasma membrane
i.e. the cell is electropermeabilized. Ca.sup.2+ or other
stimulating substances in the bath solution flow into the cell and
evoke exocytosis. Resulting amperometric spikes are recorded using
the same electrode (now acting as the recording electrochemical
electrode).
[0010] FIG. 2. Electroporation leads to exocytosis. Electrical
impulse was given at time indicated by arrow. Left: [Ca.sup.2+] in
bath was buffered to .about.4 .mu.M with HEDTA and [Cl.sup.-] was
.about.18 mM. Pulse was to +6V for 0.1 ms (train of 7 pulses).
Right: [Ca.sup.2+] in bath was 2 mM and [Cl.sup.-] was .about.160
mM. Pulse was to +6V for 0.1 ms (train of 7 pulses).
[0011] FIGS. 3A and 3B. Electrical stimulation leads to
electroporation as demonstrated by dye uptake and exocytosis as
measured by the underlying electrochemical electrode. A.
Fluorescent images demonstrating propidium iodide staining of
chromaffin cell before (left) and after (right) applying a stimulus
consisting of a train of 7, 0.1 ms pulses to 6V. Right:
Amperometric current including spikes indicating exocytosis from
the underlying DLC electrode in response to the electrical stimulus
(arrow). B. Images demonstrating trypan blue staining of chromaffin
cell before (above) and after (below) applying a stimulus
consisting of a train of 7, 0.1 ms pulses to 6 V. Right:
Amperometric current including spikes indicating exocytosis from
the underlying gold electrode in response to the electrical
stimulus (arrow).
[0012] FIGS. 4A and 4B. Voltage pulses sufficient to induce
electroporation do not degrade the electrochemical electrode. A.
Cyclic voltammograms were acquired at 1 V/s for a 1 mM ferricyanide
solution in 0.1 M KCl, pH3. The DLC:N electrode was 20 .mu.m in
diameter. In between CVs the solution was exchanged to a normal
extracellular solution and voltage pulses (+10 V for 0.2 ms) were
applied. B. Cyclic voltammograms were acquired at 1 V/s for a 1 mM
ferricyanide solution in 0.1 M KCl, pH3. The Au electrode was 20
.mu.m in diameter. In between CVs the solution was exchanged to a
normal extracellular solution and voltage pulses (+10 V for 0.2 ms)
were applied.
[0013] FIG. 5. Electrochemical measurement of
electropermeabilization-induced exocytosis on a chip.
Representative amperometric spikes recorded from a single cell on a
gold (Au) microelectrode in response to electropermeabilization.
The stimulus consisted of a train of 7, 0.1 ms pulses to 6 V. The
bath solution contained 135 mM NaCl and 2 mM CaCl.sub.2.
[0014] FIG. 6. Electropermeabilization-induced exocytosis is
sensitive to [Cl.sup.-].sub.e. The frequency of release events from
a single chromaffin cell electropermeabilized on-chip was found to
vary with [Cl.sup.-].sub.e. (a) Spike frequency reflecting the rate
of exocytosis was higher in the higher-[Cl.sup.-].sub.e solution
and (b) fell dramatically with a decrease in [Cl.sup.-].sub.e. (c)
Increasing [Cl.sup.-].sub.e to 32 mM led to a subsequent increase
in spike frequency in the same cell.
DETAILED DESCRIPTION OF INVENTION
[0015] High-resolution assays of release of electroactive materials
from cells, such as hormones, transmitter, and reactive oxygen
species are needed for a number of medically related problems. The
invention discussed here enables both stimulation of individual
cells and measurement of cell release in a compact device.
[0016] Peptides, hormones and neurotransmitters are stored in
membrane-bound vesicles within endocrine cells and neurons. Upon
stimulation, a rise in intracellular Ca.sup.2+ concentration
([Ca.sup.2+].sub.i) triggers the fusion of vesicles with the plasma
membrane and release of hormone or neurotransmitter to the outside
of the cell in a process called exocytosis. Since fusion of each
vesicle discharges a discrete packet of signaling molecules,
exocytosis is inherently a quantal process. Understanding the
mechanism of vesicle fusion and transmitter release is of broad
medical significance. The Drug L-Dopa exerts its effect in treating
Parkinson's disease by increasing the size of released quanta.
Botulinum neurotoxins inhibit transmitter release via cleavage of
so-called SNARE proteins at specific sites. Botulinum toxin A is,
in addition to its use in cosmetic treatment, applied for the
treatment of strabismus, blepharospasm, focal spasms, and cervical
dystonia. Thus understanding exocytosis will not only aid in the
improvement and development of therapies for diseases where release
of neurotransmitters is compromised, but it will also advance our
understanding of treatments that modulate transmitter release.
[0017] In order to achieve this goal, high-resolution assays of
exocytosis are needed to quantify transmitter release from
individual vesicles. Recently our group and others have been
developing microfabricated devices containing arrays of
electrochemical microelectrodes to measure the release of
electroactive transmitter at the single-cell and single-vesicle
level [1-5]. The goal of these efforts is to dramatically increase
the throughput of single-cell measurements of transmitter release
from neuroendocrine cells and to develop technology that allows
simultaneous electrochemical detection and fluorescence imaging of
single fusion events. This technology will enable high throughput
discovery of drugs such as L-DOPA that affect quantal exocytosis
and screening for toxins that inhibit neurotransmitter release.
[0018] Microchip approaches to measure exocytosis currently could
benefit from specific, powerful approaches for stimulating the
cells. For example, one may want to stimulate a subset of the
cells, apply a drug for a certain time, stimulate a different
subset of cells, wash out the drug and stimulate a third subset of
cells to test for reversibility. A sample application is
determining the time course of increasing quantal size by L-Dopa
application and the recovery after withdrawal of the drug. Another
example is when carrying out fluorescent imaging using high-power
objective lenses because only a small number of cells can be imaged
at once.
[0019] A common method for selective stimulation is to apply the
secretagogue with an application pipette mounted on a
micromanipulator. This is a slow and laborious procedure, however,
and it is not always possible to specifically stimulate the desired
subset of cells without exposing other nearby cells to the agent.
Our invention includes electrical stimulation approaches, which
allow specific, addressable stimulation down to the single-cell
level, precise timing of the stimulus, and complete integration
within the microchip platform.
[0020] Electrical stimulation can elicit exocytosis in at least two
ways. First, triggering action potentials in excitable cells with a
transient electric field is a widely used technique in neuroscience
and can be implemented on microchips (e.g., [6, 7]). A second
approach is to apply a strong enough electric field to permeabilize
the cell membrane [8]. Electropermeabilizing the plasma membrane
bypasses the normal excitation pathway mediated by membrane
depolarization followed by influx of Ca.sup.2+ through
voltage-gated Ca.sup.2+ channels. Instead, Ca.sup.2+ or other
stimulating substances enter through the permeabilized membrane.
This allows one to sort out whether a drug or toxin or protein
mutation affects exocytosis through a direct effect on exocytosis
as opposed to an effect on the membrane potential mediated by ion
channels.
[0021] In certain embodiments, electroporation-induced exocytosis
that is dependent on the exogenous monovalent anion or divalent
anion concentration, but not the exogenous calcium ion (Ca.sup.2+)
concentration in the bath solution, can be obtained with the
methods provided herein. It is thus possible to stimulate and
record exocytosis from cells in bath solutions where sufficient
concentrations of exogenous monovalent anion or divalent anion are
present but exogenous calcium ions are essentially absent. In
certain embodiments, an exogenous monovalent anion or divalent
anion at a concentration of at least about 10 millimolar, or at
least about 15 millimolar is sufficient to stimulate exocytosis. In
certain embodiments of any of these aforementioned methods, an
exogenous monovalent anion or divalent anion concentration of at
least about 10 millimolar, at least about 15 millimolar, or at
least about 20 millimolar to any one of about 50 millimolar, about
75 millimolar, about 100 millimolar, about 125 millimolar, about
150 millimolar, about 175 millimolar, or about 200 millimolar is
sufficient to stimulate exocytosis. In the context of certain
embodiments, exogenous calcium ions can be considered to be
essentially absent when the bath solution is prepared without
addition of a calcium ion source compound or when the bath solution
that lacks a calcium ion source compound further comprises an agent
that chelates or otherwise removes free calcium ions from solution.
Calcium ion source compounds include, but are not limited to,
calcium chloride, calcium carbonate, calcium hydroxide, calcium
nitrate, and the like. Agents that chelate calcium include, but are
not limited to, ethylenediaminetetraacetic acid (EDTA) and ethylene
glycol tetraacetic acid (EGTA). Monovalent anions that can be used
in these methods include, but are not limited to, chloride, iodide,
and bromide anions. Divalent anions that can be used in these
methods include, but are not limited to, sulfate anion. In most but
not all embodiments, the monovalent or divalent anion used is a
small inorganic anion. The large organic ion glutamate has not been
effective in stimulating exocytosis.
[0022] In certain embodiments, electroporation-induced exocytosis
that is dependent on the exogenous chloride ion (Cl.sup.-)
concentration, but not the exogenous calcium ion (Ca.sup.2+)
concentration in the bath solution, can be obtained with the
methods provided herein. It is thus possible to stimulate and
record exocytosis from cells in bath solutions where sufficient
concentrations of exogenous chloride ion are present but exogenous
calcium ions are essentially absent. In certain embodiments, an
exogenous chloride ion at a concentration of at least about 10
millimolar or at least about 15 millimolar is sufficient to
stimulate exocytosis. In certain embodiments of any of these
aforementioned methods, an exogenous chloride ion concentration of
at least about 10 millimolar, at least about 15 millimolar, or at
least about 20 millimolar to any one of about 50 millimolar, about
75 millimolar, about 100 millimolar, about 125 millimolar, about
150 millimolar, about 175 millimolar, or about 200 millimolar is
sufficient to stimulate exocytosis. In the context of certain
embodiments, exogenous calcium ions can be considered to be
essentially absent when the bath solution is prepared without
addition of a calcium ion source compound or when the bath solution
that lacks a calcium ion source compound further comprises an agent
that chelates or otherwise removes free calcium ions from solution.
Calcium ion source compounds include, but are not limited to,
calcium chloride, calcium carbonate, calcium hydroxide, calcium
nitrate, and the like. Agents that chelate calcium include, but are
not limited to, ethylenediaminetetraacetic acid (EDTA) and ethylene
glycol tetraacetic acid (EGTA). Without seeking to be limited by
theory, it is believed that electroporation may sensitize vesicles
to Cl.sup.-, perhaps due to an osmotic effect mediated by CLC
Cl.sup.- channels or other chloride ion channels in the membranes
of secretory vesicles.
[0023] Electroporation also allows one to introduce
membrane-impermeable substances into the cell, such as peptides or
antibodies, to modulate protein function or second-messenger
cascades. DNA or RNA can also be introduced into cells via
electropermeabilization in order to express or inhibit expression
of a gene of interest. The terms "electroporation" and
"electropermeabilization" are considered to describe the same
process and are thus used interchangeably herein. Whereas devices
for electropermeabilization of individual cells on microchips
already exist, the novel feature of our invention is a compact way
of both inducing electroporation and recording the signal which
results from release of substances from the cell induced by the
electroporation-induced cell perturbation. For example,
electroporation of an array of single cells on a chip could be used
to cause uptake of DNA. Following electroporation, the array could
be returned to an incubator to allow time for expression of the
protein encoded by the DNA. Following protein expression, the
transfected cells would already be specifically located adjacent to
the recording electrodes for experimentation to determine the
effect of the expressed protein.
[0024] Measurement of cell exocytosis and electropermeabilization
of the cell using the same electrode. A drawback of
permeabilization approaches is that soluble cell components leak
out of the cell, leading to a "rundown" of exocytosis over several
minutes [9]. A desirable feature of our on-chip stimulation
approach is that we can measure exocytosis immediately (<1 s)
after electro-permeabilization, before "rundown" occurs, because
the cells are already located adjacent to the recording electrode.
Another desirable feature of our on-chip approach is that we can
distinguish bona-fide exocytosis (amperometric spikes) from release
of cytosolic transmitter or other electroactive compounds (which
result in a slowly changing background Faradaic current) [10].
[0025] A number of groups have implemented cell electroporation on
a microchip, usually by applying a high electric field in a
microfluidic channel or through a narrow aperture (reviewed in
[11]). Recently the Orwar group has studied electroporation at the
single-cell level using carbon-fiber electrodes, thus demonstrating
the feasibility of electroporation using polarizable
microelectrodes suitable for electrochemistry [12]. When a
microelectrode passes current, the voltage in the electrolyte
solution drops within a few tens of .mu.m from the electrode
surface due to the small electrode geometry and diverging electric
field [12, 13]. A voltage gradient in the solution surrounding the
cell leads to an altered potential across regions of the membrane
that are orthogonal to the electric field lines. Electroporation
results when the local transmembrane voltage exceeds .about.200 mV.
In our experiments the cell is of necessity directly adjacent to
the electrochemical electrode. In certain embodiments, a cell is
directly adjacent to the electrochemical electrode when it is
within about 3 micrometers of the electrode surface. In other
certain embodiments, a cell is directly adjacent to the
electrochemical electrode when it is within about 2 micrometers,
about 1 micrometer, or about 0.5 micrometers of the electrode
surface. In still other certain embodiments, a cell is directly
adjacent to the electrochemical electrode when it is in contact
with the electrode surface. Therefore a transient current passed by
the electrode can produce a large transient voltage gradient and
permeabilization of the membrane near the electrode. Thus the
electrochemical electrode is in the perfect position to also serve
as an electrical stimulus electrode.
[0026] A consideration with this novel stimulus-recording electrode
approach is that the dynamic range of the potentiostat be
sufficient to supply a large current to induce electroporation, yet
also resolve the picoamp-level currents associated with
amperometric events. An example of a circuit that can be employed
in this regard is described at FIG. 5 of reference [14] cited
herein. In our work, we have modified a VA-10 potentiostat in
collaboration with the manufacturer, NPI Inc. The modification adds
a diode network in parallel with the op-amp feedback resistor, as
described in FIG. 15 of reference [14]. The diodes "turn on" to
bypass the feedback resistor to allow a large transient current to
be supplied to the electrode in response to a voltage step. Once
the capacitive current settles the diodes "turn off" to allow
low-noise I-V conversion by the feedback resistor [14]. FIG. 2
presents sample recordings where we elicited exocytosis following
pulses in voltage applied to the microchip working electrode
(arrows). The left trace shows a typical response when the cell is
bathed in a solution where the Ca.sup.2+ concentration is buffered
to .about.4 .mu.M and the Cl.sup.- concentration is .about.18 mM.
The right trace depicts a typical response when the bath contains 2
mM Ca.sup.2+ and .about.160 mM Cl.sup.-. These results indicate
that electric-pulse-induced exocytosis is dependent on the
composition of the bath solution and is not a direct stimulation of
exocytosis by the electric pulse. These results are not consistent
with a mechanism whereby the voltage pulse leads to
depolarization-induced Ca.sup.2+ influx through Ca.sup.2+ channels
because brief membrane depolarization does not lead to exocytosis
lasting for tens of seconds.
[0027] Validation of approach: Occurrence of Electroporation.
Propidium iodide is a known membrane-impermeant nucleic acid stain
that is excluded from viable cells. However, the increase in
membrane permeability that results from electroporation allows the
dye to enter the cell. Once it enters the cell, the dye binds to
the nucleic acids and its fluorescence is enhanced 20- to 30-fold.
Propidium iodide staining indicates that the cell has become
receptive to entry of exogenous molecules that would otherwise be
excluded (FIG. 3A). Trypan Blue is another known
membrane-impermeant nucleic acid stain that is excluded from viable
cells. The increase in membrane permeability that results from
electroporation also allows the Trypan Blue dye to enter the cell.
Once it enters the cell, the Trypan Blue dye binds to the nucleic
acids. Trypan blue staining indicates that the electroporated cell
has become receptive to entry of exogenous, membrane impermeable
trypan blue molecules that would have been excluded in the absence
of electroporation (FIG. 3B).
[0028] Further evidence for electroporation is that large voltage
pulses lead to a precipitous drop in membrane resistance in our
patch-clamp measurements (data not shown).
[0029] The experiments depicted in FIGS. 1 and 2 demonstrate that
the electrochemical electrode can record amperometric spikes with
typical features following a voltage pulse, nevertheless, it is
possible that voltage pulses will alter the sensitivity or noise of
the electrodes. Our preliminary experiments indicate no change in
noise by brief (sub ms) pulses to +10V (data not shown). We
measured cyclic voltammograms using the test analyte ferricyanide
before and after voltage pulses to test for changes in
.DELTA.E.sub.p indicative of changes in electron-transfer kinetics.
FIG. 4 is a sample experiment of this type demonstrating little
effect of a 0.2 ms pulse to +10 V on the CV.
[0030] Optimization of stimulus protocol. The electrical
stimulation protocol consists of one or more voltage step(s) from
the normal potential used for amperometry (.about.0.6V) to a test
potential (+3 to +10 V) for a duration ranging from 0.1 or 0.2 ms.
Bubbles can form on the surface of the electrode if the voltage
pulse is too long, so we visually monitored the electrodes while
developing stimulus protocols. On the occasion when we did observe
a bubble, the cell was displaced from the electrode surface so we
could not record exocytosis. Our experience is that milder stimuli
lead to exocytosis that terminates in a few seconds whereas a
second pulse leads to another burst of release. On the other hand,
stronger stimuli lead to release lasting for minutes. Transient
electropermeabilization will leave the cell more intact to allow
probing the cell over prolonged intervals and is appropriate for
introducing exogenous substances into the cell whereas prolonged
permeabilization is necessary when the goal is to clamp the
intracellular concentration of ions to that of the bath solution.
Pore resealing times have been shown to correlate with the strength
of the electroporation stimulus [12]. We found that trains of
voltage pulses of 5-8 V of 0.1-0.5 ms duration can reliably elicit
exocytosis lasting for tens of seconds.
[0031] While the invention has been described in connection with
specific embodiments thereof, it will be understood that the
inventive device is capable of further modifications. This patent
application is intended to cover any variations, uses, or
adaptations of the invention following, in general, the principles
of the invention and including such departures from the present
disclosure as come within known or customary practice within the
art to which the invention pertains and as may be applied to the
essential features herein before set forth.
EXAMPLES
[0032] The disclosed exemplary embodiments are merely
representative of certain aspects the invention, which may be
embodied in various forms. Thus, the exemplary structural,
procedural, and functional details disclosed herein are not to be
interpreted as limiting.
Example 1
Construction of and Use of Microchip
[0033] A semi-transparent Au film was sputter deposited on top of
an adhesion promoting Ti film using an RF magnetron sputtering
system. The Au/Ti film was patterned using photolithography and wet
etching into conductive traces with connection pads arranged around
the circumference of the chip to facilitate external connections. A
thick photoresist SU-8 2025 was spin-coated on the patterned Au
substrate to fabricate working electrodes and cell docking sites as
well as to insulate non-active areas of the conductive film. A PDMS
gasket with a slit in the middle was fabricated and air
plasma-sealed to the device in order to confine the drop of
solution containing cells to the part on the device where the
working electrodes are located. Gold and diamond-like-carbon (DLC)
microelectrodes were found to be effective at eliciting exocytosis
from bovine adrenal chromaffin cells via electroporation. The
potentiostat used was modified by adding a diode network to allow
it to pass large transient currents to enable electroporation, yet
record pA amperometric currents with low noise. Parameters of the
voltage pulse protocol that were applied to electropermeabilize the
cells and optimized for maximum efficiency included voltage
applied, pulse-width, and number of pulses. While applying the
voltage pulse, the surface of the electrode was visually monitored
to detect formation of any kind of bubbles in the solution. When
the microelectrode passed current, voltage drop occurred in the
electrolytic solution and this voltage gradient surrounding the
cell led to an altered potential across regions of the membrane
that are orthogonal to the electric field lines. The electric field
being the strongest near the cell-electrode interface at the bottom
of the cell of interest, effective permeabilization occurred at the
membrane near the electrode. Successful electroporation of the
plasma membrane was specifically demonstrated by cell uptake of
trypan blue. The fluorescent Ca.sup.2+-indicator Fura-4F was used
to measure changes in intracellular Ca.sup.2+ concentration caused
by electropermeabilization.
Example 2
Electroporation Induced Exocytosis
[0034] Trains of voltage pulses, 5-7 V in amplitude and 0.1-0.2 ms
in duration, reliably triggered exocytosis in single chromaffin
cells. The bath solution contained 135 mM NaCl and 2 mM CaCl.sub.2.
Recordings from such stimulation experiments consisted of trains of
individual spikes of amperometric current, tens of pA in amplitude
and tens of ms in duration (FIG. 5). Each spike of current serves
as a "signature" of exocytosis of an individual secretory granule.
Sub-threshold or milder stimulations were found to induce momentary
bursts of spike activity, presumably due to transient
electropermeabilization, while the standardized protocol led to
reliable prolonged exocytosis. Both of these stimuli types can be
useful for studies of exocytosis--whereas transient
electropermeabilization could leave the cell more intact to allow
probing the cell over prolonged intervals, prolonged
permeabilization would be necessary to study the dependency of
exocytosis on external factors like changing concentrations of a
molecule of interest in the external solution.
[0035] Individual device electrodes could be cleaned and re-used
for multiple (.about.5) times and cyclic voltammetry tests also
confirmed that voltage pulses did not degrade the sensitivity of
the electrochemical electrodes.
[0036] On electropermeabilization of cells, uptake of trypan blue
stain was observed, verifying that the plasma membrane was
permeabilized. Modest increases in intracellular Ca.sup.2+
concentration measured using a fluorescent Ca.sup.2+ indicator were
also observed following electropermeabilization.
Example 3
Stimulation of Exocytosis by Electroporation of Chloride Ion
[0037] It is well established that exocytosis in excitable cells
such as chromaffin cells is triggered by the influx of Ca.sup.2+.
Since the concentration of Ca.sup.2+ in the extracellular solution
([Ca.sup.2+].sub.e) is usually much higher than the intracellular
solution, electropermeabilization can be expected to trigger
exocytosis by allowing Ca.sup.2+ entry into the cell. We were
surprised to observe, however, that electropermeabilization leads
to a vigorous rate of exocytosis even when the extracellular
solution contained no added Ca.sup.2+ plus the addition of 5 mM
EGTA (ethylene glycol tetraacetic acid) to thoroughly chelate any
residual Ca.sup.2+ in the water (FIG. 6A). In contrast, we found
that 32mM [Cl.sup.-].sub.e was sufficient to lead to robust
exocytosis following electropermeabilization of the cell membrane
in this experiment (FIG. 6). As seen in FIG. 6, the secretion
frequency was high when the cell was permeabilized in the first
high-[Cl.sup.-].sub.e solution, fell dramatically when exposed to a
second lower-[Cl.sup.-].sub.e solution, and then increased again
when exposed to a higher-[Cl.sup.-].sub.e solution. All three
solutions contained no added Ca.sup.2+ plus 5 mM EGTA and
[Cl.sup.-].sub.e was varied upon substitution with the anion
glutamic acid while maintaining a pH of 7.2.
Electropermeabilization in the presence of glutamic acid alone did
not yield exocytosis.
[0038] We quantified the spike frequency in different
[Cl.sup.-].sub.e solutions as summarized in Table 1. In the first
experiment, data were pooled from 5 electropermeabilized cells that
were exposed to solutions with Cl.sup.--concentrations in the
following sequence (in mM): 135.fwdarw.0.fwdarw.30. Note that the
spike frequency is highest with the first solution, dips to almost
zero in the second solution and shows an 18-fold increase as
high-Cl.sup.- is re-introduced into the external solution. The
second case deals with bracketed experiments (n=5) where
[Cl.sup.-].sub.e is varied in the following order (in mM):
135.fwdarw.0.fwdarw.135. Again, the frequency is highest in the
initial solution, near-zero in the solution with 0 Cl.sup.- and
increases by 6 times when Cl.sup.- is brought back up to the
initial concentration. It can be noted that the lower frequency in
the third solution compared to the first solution can be attributed
to a run-down of exocytosis that is commonly observed for many
types of stimuli that lead to high rates of exocytosis.
TABLE-US-00001 TABLE 1 [Cl.sup.-].sub.e in solution # of Spike
Frequency (Spikes/s) 135 mM .fwdarw. 0 mM .fwdarw. 30 5 3.80
.fwdarw. 0.10 .fwdarw. 1.77 135 mM .fwdarw. 0 mM .fwdarw. 135 5
7.82 .fwdarw. 0.17 .fwdarw. 1.06
[0039] Inspection of the spikes obtained from these experiments
indicates that they have the characteristic fast rise and
exponential fall expected for single-vesicle release events
(quantal exocytosis). Therefore, spikes induced by
electropermeabilization in Cl.sup.--containing solutions appear to
have similar properties as those elicited using other more common
methods such as cell depolarization. To quantify this, we analyzed
771 spikes from 9 individual cells stimulated in solutions with a
range of [Cl.sup.-].sub.e using the software of Segura et al. [15].
The mean.+-.standard error of the pooled data for the spike
parameters were as follows: peak spike current (I.sub.max),
time-to-peak (t.sub.p), spike charge (Q) and spike duration
(interval where the amperometric current exceeds 50% of the peak
value, t.sub.1/2) were 64.1.+-.1.4 pA, 11.0.+-.0.1 ms, 2.2.+-.0.1
pC and 31.4.+-.0.4 ms respectively. The values are comparable to
those found using different stimulus methods that depend on
Ca.sup.2+ influx [16, 17, 18, 19]. Thus it can be inferred that
chloride-stimulated, electropermeabilization-induced catecholamine
release likely occurs via bona-fide exocytosis of normal secretory
granules.
[0040] Having illustrated and described the principles of the
present invention, it should be apparent to persons skilled in the
art that the invention can be modified in arrangement and detail
without departing from such principles. Although the materials and
methods of this invention have been described in terms of various
embodiments and illustrative examples, it will be apparent to those
of skill in the art that variations can be applied to the materials
and methods described herein without departing from the concept,
spirit and scope of the invention. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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