U.S. patent application number 09/957116 was filed with the patent office on 2002-10-10 for sample positioning and analysis system.
Invention is credited to Schmidt, Christian.
Application Number | 20020144905 09/957116 |
Document ID | / |
Family ID | 27509097 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020144905 |
Kind Code |
A1 |
Schmidt, Christian |
October 10, 2002 |
Sample positioning and analysis system
Abstract
Systems for positioning and/or analyzing samples such as cells,
vesicles, cellular organelles, and fragments, derivatives, and
mixtures thereof, for electrical and/or optical analysis,
especially relating to the presence and/or activity of ion
channels.
Inventors: |
Schmidt, Christian;
(Epalonge, DE) |
Correspondence
Address: |
KOLISCH, HARTWELL, DICKINSON,
McCORMACK & HEUSER
Suite 200
520 S.W. Yamhill Street
Portland
OR
97204
US
|
Family ID: |
27509097 |
Appl. No.: |
09/957116 |
Filed: |
September 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09957116 |
Sep 19, 2001 |
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09581837 |
Oct 13, 2000 |
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60232365 |
Sep 14, 2000 |
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60233800 |
Sep 19, 2000 |
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60322178 |
Sep 13, 2001 |
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Current U.S.
Class: |
204/403.01 |
Current CPC
Class: |
G01N 33/48728 20130101;
B01L 3/5088 20130101; B01L 3/5027 20130101 |
Class at
Publication: |
204/403.01 |
International
Class: |
G01N 027/403 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 17, 1997 |
CH |
2903/97 |
Jul 28, 1998 |
IB |
PCT/IB98/01150 |
Claims
I claim:
1. A system for positioning and/or analyzing samples such as cells,
vesicles, and cellular organelles, and fragments, derivatives, and
mixtures thereof, comprising: a substrate having a plurality of
apertures; at least one recording fluid compartment and at least
one reference fluid compartment, arranged on opposites side of the
substrate, and in contact via the apertures; and at least one
recording electrode and at least one reference electrode, each in
contact with at least one of the fluid compartments, and adapted to
apply and/or measure an electrical potential across the
apertures.
2. The system of claim 1, further comprising a support element for
independently supporting the substrate and associated system
components.
3. The system of claim 2, where the support element includes a
carrier plate and at least one spacer sandwiched between the
substrate and the carrier plate.
4. The system of claim 3, where the space defined between the
substrate and the carrier plate includes at least one of the
compartments.
5. The system of claim 3, where the carrier plate is made of a
transparent material, such as glass, and includes a plurality of
microlenses arranged in such a way that they allow the parallel
optical observation of the samples that are positioned near the
apertures.
6. The system of claim 2, where each recording electrode contacts
the substrate or the support element at least substantially at its
border.
7. The system of claim 1, where at least two fluid compartments are
arranged on one side of the substrate, each of those compartments
being in contact, via the apertures, with a single fluid
compartment arranged on the other side of the substrate.
8. The system of claim 1, where at least one electrode is arranged
adjacent the substrate.
9. The system of claim 1, where the recording fluid compartment is
arranged above or below the substrate.
10. The system of claim 1, where the substrate includes at least
one material selected from the group consisting of silicon, silicon
derivatives, glass, and plastic.
11. The system of claim 1, where the diameter of the aperture is
less than about 15 nm.
12. The system of claim 11, where the diameter of the aperture is
between about 0.3 .mu.m and about 7 .mu.m.
13. The system of claim 1, where the number of recording fluid
compartments is selected from the group consisting of 96, 384,
1536, and 9600.
Description
CROSS-REFERENCES
[0001] This application is a continuation-in-part of the following
U.S. patent applications, which are incorporated herein by
reference: Ser. No. 09/581,837, filed Jun. 16, 2000; and Ser. No.
______ , filed Sep. 14, 2000, titled EFFICIENT METHODS FOR THE
ANALYSIS OF ION CHANNEL PROTEINS, and naming Christian Schmidt as
inventor. U.S. patent application Ser. No. 09/581,837, in turn,
claims priority from PCT Patent Application Serial No.
PCT/IB98/01150, filed Jul. 28, 1998, which claims priority from
Swiss Patent Application Serial No. 2903/97, filed Dec. 17, 1997.
Each of these patent applications is incorporated herein by
reference. U.S. patent application Ser. No. ______ , filed Sep. 14,
2000, titled EFFICIENT METHODS FOR THE ANALYSIS OF ION CHANNEL
PROTEINS, and naming Christian Schmidt as inventor, in turn, claims
priority from Ser. No. 09/581,837, with priority claims as listed
above, U.S. Provisional Patent Application Serial No. 60/232,365,
filed Sep. 14, 2000; Ser. No. 60/233,800, filed Sep. 19, 2000; and
Ser. No. ______ , filed Sep. 13, 2001, titled HIGH-THROUGHPUT PATCH
CLAMP SYSTEM, and naming Christian Schmidt as inventor. Each of
these patent applications is incorporated herein by reference.
[0002] This application is based upon and claims the benefit under
35 U.S.C. .sctn. 119 of the following U.S. provisional patent
applications, which are incorporated herein by reference: Ser. No.
60/233,800, filed Sep. 19, 2000, titled DESIGN OF HIGHLY INTEGRATED
PHARMACEUTICAL SCREENING CHIPS, and naming Christian Schmidt as
inventor; and Ser. No. ______ , filed Sep. 13, 2001, titled
HIGH-THROUGHPUT PATCH CLAMP SYSTEM, and naming Christian Schmidt as
inventor.
[0003] This application incorporates by reference in their entirety
for all purposes the following U.S. Pat. Nos. 5,355,215, issued
Oct. 11, 1994; and No. 6,097,025, issued Aug. 1, 2000.
[0004] This application incorporates by reference in their entirety
for all purposes the following patent applications: U.S. patent
application Ser. No. 90/708,905, filed Nov. 8, 2000; PCT Patent
Application Serial No. PCT/IB00/00095, filed Jan. 26, 2001; and PCT
Patent Application Serial No. PCT/IB00/00097, filed Jan. 26,
2001.
[0005] This application incorporates by reference in their entirety
for all purposes the following U.S. patent applications: Ser. No.
09/337,623, filed Jun. 21, 1999; Ser. No. 09/349,733, filed Jul. 8,
1999; Ser. No. 09/478,819, filed Jan. 5, 2000; Ser. No. 09/596,444,
filed Jun. 19, 2000; Ser. No. 09/710,061, filed Nov. 10, 2000; Ser.
No. 09/722,247, filed Nov. 24, 2000; Ser. No. 09/759,711, filed
Jan. 12, 2001; Ser. No. 09/765,869, filed Jan. 19, 2001; Ser. No.
09/765,874, filed Jan. 19, 2001; Ser. No. 09/766,131, filed Jan.
19, 2001; Ser. No. 09/767,434, filed Jan. 22, 2001; Ser. No.
09/767,579, filed Jan. 22, 2001; Ser. No. 09/767,583, filed Jan.
22, 2001; Ser. No. 09/768,661, filed Jan. 23, 2001; Ser. No.
09/768,765, filed Jan. 23, 2001; Ser. No. 09/770,720, filed Jan.
25, 2001; Ser. No. 09/770,724, filed Jan. 25, 2001; Ser. No.
09/777,343, filed Feb. 5, 2001; Ser. No. 09/813,107, filed Mar. 19,
2001; Ser. No. 09/815,932, filed Mar. 23, 2001; and Ser. No.
09/836,575, filed Apr. 16, 2001; and Ser. No. ______ , filed Aug.
20, 2001, titled APPARATUS AND METHODS FOR THE GENERATION OF
ELECTRIC FIELDS WITHIN MICROPLATES, and naming James M. Hamilton as
inventor.
[0006] This application incorporates by reference in their entirety
for all purposes the following U.S. Provisional Patent
Applications: Serial No. 60/223,642, filed Aug. 8, 2000; Ser. No.
60/244,012, filed Oct. 27, 2000; Ser. No. 60/267,639, filed Feb.
10, 2001; Ser. No. 60/287,697, filed Apr. 30, 2001; Ser. No., filed
Aug. 2, 2001, titled pH PROBES FOR CELL-BASED FLUORESCENCE ASSAYS,
and naming Zhenjun Diwu, Jesse J. Twu, Guoliang Yi, Luke D. Lavis,
and Yen-Wen Chen as inventors; and Serial No. ______ , filed Aug.
31, 2001, titled KINETIC ASSAY FOR DETERMINING CALCEIN RETENTION IN
CELLS, and naming Kelly J. Cassutt, Jesse J. Twu, and Anne T.
Ferguson as inventors.
[0007] This application incorporates by reference in its entirety
for all purposes the following publications: Richard P. Haugland,
Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.
1996); and Joseph R. Lakowicz, PRINCIPLES OF FLUORESCENCE
SPECTROSCOPY (2.sup.nd Ed. 1999).
FIELD OF THE INVENTION
[0008] The invention relates to systems for positioning and/or
analyzing samples. More particularly, the invention relates to
systems for positioning and/or analyzing samples such as cells,
vesicles, cellular organelles, and fragments, derivatives, and
mixtures thereof, for electrical and/or optical analysis,
especially relating to the presence and/or activity of ion
channels.
BACKGROUND OF THE INVENTION
[0009] A variety of important biological processes occur at or
within cell membranes. It therefore is not surprising that the
biological function of membrane proteins has become an area of
active research. Signal transduction processes in general,
including nerve conduction, and neuroreceptors in particular have
been shown to be influenced by pharmacologically active
ingredients, making them obvious targets for drug
development..sup.i Ion channels and ion transporters also have been
shown to be an important class of therapeutic targets. In fact,
interactions with ion channels have become a major potential source
of adverse effects when administering a therapeutic agent, leading
the Food and Drug Administration (FDA) and other government
regulatory agencies to require safety profiling of potential
therapeutics against certain ion channels.
[0010] This understanding of the interactions between potential
drugs and cell membrane components is beginning to play a crucial
role in modern drug development. In view of the increasing number
of known receptors and the rapidly growing libraries of potential
pharmaceutical ingredients, there clearly is a need for highly
sensitive screening methods that permit the analysis of a large
number of different substances with high assay throughput per unit
time, otherwise known as "high throughput screening" (or "HTS"). In
particular, there is a need for automated and/or high throughput
screening methods that are relevant to cell membrane
components.
[0011] At present, relatively traditional methods are used for the
screening of pharmaceutical ingredients. Such methods include
ligand binding assays and receptor function tests that are
performed separately..sup.ii Although binding assays are relatively
inexpensive, and amenable to high throughput, they require labeled
high-affinity ligands, and generally are limited to assays for
ligands that can compete effectively for labeled ligand.
Fluorescent or fluorogenic reagents generally are compatible with
high throughput assays, including the analysis of ion channels
using fluorescent calcium indicators, and the evaluation of
membrane potential effects with potential-sensitive dyes. However,
such reagents typically are not sensitive enough for single cell
measurements, and generally can provide only indirect measurements
of the membrane component of interest.
[0012] The patch clamp was introduced by Neher and Sakmann in the
early 1980s as a powerful technique for the direct study of drug
effects on single receptors. In recognition of the strength of the
method, Neher and Sakmann were awarded the Nobel prize in 1991.
Classical patch-clamp methods often are used in conjunction with
functional membrane receptor assays, including receptors coupled to
G-proteins and ion channel-forming receptors..sup.iii This method
is highly specific and extremely sensitive: it can, in principle,
be used to measure the channel activity of individual receptor
molecules. In doing so, glass micropipettes with an opening
diameter of typically 1-0.1 .mu.m are pressed on the surface of a
biological cell. The membrane surface that is covered by the
micropipette is called a "patch." If the contact between the glass
electrode and the cell membrane surface is sufficiently
electrically isolating, the ion flow over the membrane patch can be
measured electrically with the aid of microelectrodes, one placed
in the glass pipette and the other placed in the milieu opposite
the membrane..sup.iv A key advantage of this electrophysiological
method is that it makes directly accessible the function of the
corresponding channel-forming proteins or receptors coupled to
channel-forming proteins via the measured electrical
characteristics of the channel-forming proteins.
[0013] Unfortunately, several major limitations have prevented
patch-clamp technology from revolutionizing receptor science and
pharmaceutical drug development. For example, to produce high
quality results, the patch-clamp method requires a tremendous
effort in technical installation and highly qualified operators.
Moreover, in addition to being expensive, a standard patch-clamp
setup may require a long set-up time and have a high failure
rate.
[0014] Thus, there is a need for a system for positioning and/or
analyzing cells that is rapid, facile, and suitable for multiarray
analysis, such as the system provided by the invention.
SUMMARY OF THE INVENTION
[0015] The invention provides systems for positioning and/or
analyzing samples such as cells, vesicles, cellular organelles, and
fragments, derivatives, and mixtures thereof, for electrical and/or
optical analysis, especially relating to the presence and/or
activity of ion channels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic view of a system for positioning
and/or analyzing samples in accordance with aspects of the
invention.
[0017] FIG. 2 is a cross-sectional side view of a substrate chip
prepared from Si/SiO.sub.2 in accordance with aspects of the
invention.
[0018] FIG. 3 is a cross-sectional side view of a measurement
system having planar electrodes in accordance with aspects of the
invention.
[0019] FIG. 4 is a cross-sectional side view of a measurement
system having point or wire electrodes in accordance with aspects
of the invention.
[0020] FIG. 5 is a cross-sectional side view of a measurement
system having open fluid compartments in accordance with aspects of
the invention.
[0021] FIG. 6 is a top view of a measurement system having multiple
measurement sites in accordance with aspects of the invention.
[0022] FIG. 7 is a cross-sectional side view of the measurement
system of FIG. 6, taken generally along line 7-7 in FIG. 6.
[0023] FIG. 8 is a cross-sectional side view of a measurement
system having optical measurement aids in accordance with aspects
of the invention.
[0024] FIG. 9 is a partially cross-sectional partially schematic
side view of a measurement system for combined electrical/optical
detection.
[0025] FIG. 10 is a contour plot of the electric potential adjacent
an aperture in a substrate in accordance with aspects of the
invention, computed by finite element method (FEM) simulation.
[0026] FIG. 11 is a plot of current versus time showing a decrease
in current upon addition of Ca.sup.2+ to a final concentration of 4
mM following the docking of vesicles at a 7-.mu.m aperture in the
unmodified surface of a suitable substrate.
[0027] FIG. 12AB is a pair of plots of current versus time showing
the time course of vesicle binding and the subsequent development
of membranes with very high electrical insulation resistance for
(A) a 4-.mu.m aperture and (B) a 7-.mu.m aperture in a
poly-L-lysine-coated SiO.sub.2 substrate.
[0028] FIG. 13 is a plot of current versus time showing the passage
of individual vesicles through a 7-.mu.m aperture, as reflected in
fluctuations in the plot recorded at a constant clamp voltage,
V.sub.c, of -80 mV.
[0029] FIG. 14 is a plot of current versus time showing the time-
and voltage-dependent switching of alamethicin pores in a membrane
produced on the substrate (C.sub.alamethicin=0.1 .mu.g/mL in 85 mM
KCl) at negative potentials.
[0030] FIG. 15AB is a pair of plots of current versus time showing
the changes in measured membrane resistance of a membrane produced
on a Si/SiO.sub.2 carrier chip after fusion with vesicles
containing nAChR (nicotinic acetylcholine receptor). FIG. 15A shows
the membrane resistance during accidental receptor openings in the
absence of ligands at 400 mM KCl and positive potentials. FIG. 15B
shows the membrane resistance 150 seconds after the addition of the
nAChR agonist carbamylcholine (20 .mu.m final concentration), where
no receptor openings are observed due to desensitization of the
receptors.
[0031] FIG. 16 is a plot of current versus time showing the time
course of positioning, binding, and subsequent development of a
tight electrical seal for a Jurkat cell.
[0032] FIG. 17 is a series of plots of current versus time showing
the current flowing through the membrane of a Jurkat cell for the
indicated positive and negative clamp voltages.
[0033] FIG. 18 is an analysis of the current flowing through the
membrane of a Jurkat cell for a +60 mV clamp voltage showing (A) a
representative plot of current versus time, and (B) a histogram
showing the relative likelihood of the measured currents.
DETAILED DESCRIPTION
[0034] The invention provides systems such as single and
multiaperture biochips for positioning and/or analyzing
membrane-bound samples, such as cells, vesicles, cellular
organelles, and/or portions thereof. Positioning a sample, as used
here, generally comprises locating or placing the sample at a
preselected position, within the system, typically for subsequent
analysis. Analyzing the sample generally comprises detecting a
presence or activity within the sample, while it is positioned at
the preselected position, typically relating at least in part to
electrical properties of the sample.
[0035] FIG. 1 shows a representative system 30 for positioning
and/or analyzing samples in accordance with aspects of the
invention. The system includes a substrate 32, at least two fluid
compartments 34a,b, and at least two electrodes 36a,b. (In some
cases, for example, where only optical measurements are to be
performed, the system may have only a single fluid compartment and
no electrodes.) The substrate comprises a separating wall of
electrically isolating material, and may include an aperture and/or
window 38 and an associated adhesion surface 40 adjacent the
aperture to which samples 42 may be bind or be fixed using any
suitable mechanism. The fluid compartments generally comprise any
region or volume adapted to support a fluid adjacent the aperture.
The electrodes generally comprise any mechanism for applying and/or
measuring an electric potential and associated electric field
across the aperture. In most embodiments, a first side of the
substrate is used as a sample or measurement side, and a second
side of the substrate is used as a reference side, although these
roles may be interchangeable. The measurement side is used to hold
samples during positioning and/or analysis, and typically includes
adhesion surface 40, a measurement fluid compartment 34a and a
measurement electrode 36a. The reference side is used to complete
the electric circuit, and typically includes a reference fluid
compartment 34b and a reference electrode 36b. Generally, despite
their different names, the measurement electrode and the reference
electrode independently may be set to any suitable voltage,
including ground.
[0036] The system may include or be interfaced with one or more
auxiliary systems, including (1) a sample handling system 44 for
introducing, removing, and/or otherwise manipulating fluids and/or
samples, (2) an analysis system 46 for analyzing samples,
particularly by mechanisms other than direct electrical
measurement, and/or (3) an incubation system for storing samples
before and/or during assays, and/or (4) a cleaning system for
cleaning substrates and/or other system components.
[0037] The system may be used for a variety of applications. These
applications may include automated and/or high-throughput
patch-clamp analysis (e.g., for drug screening), portable biosensor
analysis (e.g., for environmental analytes), and so on. These
applications also may include the separation of cells or vesicles,
the analysis of the sizes of cells or vesicles, the direct
functional analysis of ionotropic membrane proteins, for example,
in ligand binding studies, and/or the positioning of cells or
vesicles for any suitable purpose, including purely optical
investigations and/or microinjections, among others. Typically, a
sample such as a cell or vesicle is introduced into the measurement
compartment and then is directed toward the aperture, for example,
using an electric force created by the two electrodes. The sample
contacts the adhesion surface, binds across the aperture, and forms
an electrical seal with the aperture sufficient for performing an
assay of interest. The effects of an applied voltage created by the
electrodes then may be studied, typically before and/or after
exposure to a suitable assay condition The studies may be performed
by measuring changes in electrical properties across the aperture,
such as current, resistance, or the like, and/or by measuring other
changes in the sample, such as ion levels or the like.
[0038] The assay condition generally comprises any change of
condition, optionally including a change in environmental
condition, such as sample temperature, but more typically including
the addition of one or more reagents such as candidate drug
compounds to the sample. The reagent may be a chemical reagent,
such as an acid, a base, a metal ion, an organic solvent, or other
substance intended to effect a chemical change in the sample.
Alternatively, the reagent may have or be suspected to have a
biological activity or type of interaction with a given
biomolecule. Selected assay components may include membrane-active
substances, such as pore promoters, proteoliposomes, and/or
membrane proteins. Selected assay reagents also may include
oligonucleotides, nucleic acid polymers, peptides, proteins, drugs,
and other biologically active molecules.
[0039] The system may have one or more advantages over prior
systems for measuring electrical properties of cells and vesicles.
First, the system is relatively simple, both in the production of
electrically insulating patch membranes and in the associated
measurements. Thus, the system, alone or in combination with modern
microtechnological methods, is suitable for use in automated and/or
"high throughput screening" (HTS) applications. Second, the
positioning and measuring capabilities of the system are well
suited to the combination of electrical and optical measurements,
through which, on these planar membranes, obtained by means of the
positioning process according to the invention, new, important
information concerning membrane channels and receptors may be
obtained.
[0040] The following sections describe various components and
functionalities of the system, including (A) the substrate, (B) the
apertures and windows, (C) the adhesion surfaces, including
mechanisms for achieving binding, (D) the fluid compartments, (E)
the electrodes, (F) multiaperture systems, (G) the analysis system,
(H) the sample handling system, (I) the samples, (J) the sample
positioning process, and (K) the measurement process, among
others.
[0041] A. Substrates
[0042] The substrate generally comprises any surface or set of
surfaces capable of separating two fluid compartments. The
substrate typically includes an aperture that passes through the
substrate to connect the two fluid compartments and at least one
adhesion surface adjacent the aperture for binding a sample such as
a cell or vesicle for analysis. The substrate preferably is
nonconductive (e.g., electrically insulating), thus reducing or
eliminating electrical contact between the two fluid compartments,
except through the aperture.
[0043] The substrate may be formed of any suitable material.
Preferably, the substrate is nonconductive, inert (in the system),
and no more than slightly modifiable chemically. Exemplary
materials include silicon (including silicon (Si) and silicon
derivatives, such as silicon oxide (SO.sub.2; silica) and silicon
oxinitride (SiO.sub.xN.sub.y)), glass, quartz, plastic, and so on.
Among these, the silicon-based substrates have several advantages.
First, they are commercially available. Second, they are easily
processed, for example, so that they may be provided with an
aperture and/or window, as described below in Example 1. Third,
they readily may be coated or otherwise partially or completely
covered with insulating and/or adhesion-promoting materials. Such
surface layers include layers of quartz, glass, and solid and/or
gelatinous polymers, among others. Such surface layers also include
plastomers and elastomers, such as polyimides,
polymethylmethacrylates, polycarbonates, and silica gels (e.g.
Sylgard). Such surface layers also may be homogeneous or
inhomogeneous, where, for example, in the latter case, they may be
applied as droplets.
[0044] The substrate may include two or more pieces or components,
for example, being constructed of a holder on which the material
actually relevant to membrane positioning and membrane binding is
fastened or into which this material is admitted, where this
material for the positioning, or alternatively the binding, of the
membranes has at least one aperture.
[0045] The substrate may be formed with any suitable geometry,
subject to the above limitations. However, preferably, the
substrate is at least substantially planar, and more preferably,
the substrate is microscopically flat and molecularly relatively
planar, particularly at the adhesion surface.
[0046] Examples 1-7 below, among others, describe exemplary
substrates, including materials, geometries, and relationships with
other components of the system.
[0047] B. Apertures and Windows
[0048] The aperture and window(s) are the portions of the substrate
most immediately involved in the positioning and/or analysis of
samples.
[0049] The aperture generally comprises any opening or other
passage through the substrate. This opening may include a hole, a
gap, and/or a slit, among others, and may allow fluid contact
between fluid compartments positioned at opposite sides of the
aperture. The aperture may be capable of forming an electrical seal
with a sample such as a cell or vesicle that is sufficiently
"tight" to use in a patch clamp experiment. Exemplary seals
(depending on sample type and condition) have included >10
k.OMEGA., >100 k.OMEGA., >1 M.OMEGA., >10 M.OMEGA.,
>100 M.OMEGA., >1 G.OMEGA., >10 G.OMEGA., >100
G.OMEGA., and even >1T.OMEGA.. Alternatively, or in addition,
the aperture may be capable of focusing an electric field with
sufficient strength to position a sample such as a cell or vesicle
about the aperture. The aperture may include a hole, a gap, and/or
a slit, among others.
[0050] The aperture is characterized by a length L.sub.ap and a
diameter d.sub.aperture The length is determined by the thickness
of the substrate adjacent the aperture, generally ranging between
about 3 .mu.m and about 1000 .mu.m, and preferably ranging between
about 100 nm and about 20 .mu.m. The diameter of the aperture, as
measured immediately adjacent the binding surface, is influenced by
a variety of different factors, which may urge toward either
smaller or larger apertures. First, smaller apertures generally
increase the quality of the electrical seal between the aperture
and the sample, up to a limit. In particular, to form a tight
electrical seal, the aperture should be smaller, preferably
significantly smaller, than the size of the sample (i.e.,
d.sub.aperture<<d.sub.cell, d.sub.vesicle), but larger,
preferably significantly larger, than the lipids and other
molecules present in the sample. Second, smaller apertures
generally increase the mechanical stability of the membrane across
the aperture. In particular, the force required to deflect a
portion of membrane is inversely proportional to the square of the
radius of the portion being deflected (i.e., proportional to the
value of r.sub.M.sup.-2; see, e.g., Example 12), so that the
selection of small (e.g., d.sub.aperture<5 .mu.m) apertures may
significantly increase membrane stability, particularly relative to
the typical (e.g., d.sub.aperture>100 .mu.m) apertures used in
conventional black lipid membrane (BLM) systems. Third, smaller
apertures generally increase the strength and focus of the electric
field passing through the aperture, which is especially useful when
positioning samples. Fourth, larger apertures generally reduce the
access resistance, improving the quality of the voltage (or
current) clamp, probably by easing the physical access of the
conduction ions. Based on these factors, the diameter of the
aperture generally is less than about 15 to 20 .mu.m, usually is
less than about 10 .mu.m, preferably is less than about 7 .mu.m,
and more preferably is less than about 5 .mu.m. In particular,
sizes between about 0.3 .mu.m and about 7 .mu.m may yield an
outstanding probability and quality of sealing. Stated
alternatively, the aperture preferably should have a diameter of no
more than a few tens of percent, and more preferably no more than
about 30 percent, of the sample diameter. Thus, for cellular
samples, which typically have a diameter of greater than about 20
.mu.m, the aperture preferably should have a diameter of no more
than about 5-7 .mu.m.
[0051] The window generally comprises a portion of the fluid
compartment adjacent and providing access to the aperture. The
preferred size of the window is determined by factors analogous to
those described above for determining the preferred size of the
aperture. In brief, the diameter of the window preferably is less
than about 1000 .mu.m, and more preferably is significantly
smaller, being not more than about 100 .mu.m.
[0052] Examples 1-7 below, among others, describe exemplary
apertures and windows, including geometries and relationships with
other components of the system.
[0053] C. Binding and Adhesion Surfaces
[0054] The adhesion surface generally comprises any surface or set
of surfaces adjacent the aperture to which samples such as cells
and vesicles may bind for analysis. The adhesion surface typically
is at least substantially planar over an area exceeding that of the
bound portion of the sample but in some cases may be at least
slightly concave in the direction of the sample. Thus, the adhesion
surface may have an area of at least about 25 .mu.M.sup.2 for a
cell that is about 5 .mu.m in (bound) diameter, at least about 100
.mu.m.sup.2 for a cell that is about 10 .mu.m in (bound) diameter,
and so on.
[0055] Binding, as used here, generally comprises any stable or
semi-stable association between a sample and an adhesion surface
that results in an electrical seal between the sample and one or
more apertures that is sufficiently "tight" to allow the desired
measurement. Binding may be mediated by any suitable mechanism,
direct or indirect, including electrostatic interactions, covalent
bonding, ionic bonding, hydrogen bonding, van der Waals
interactions, and/or hydrophobic-hydrophilic.sup.v interactions,
among others. In general, binding may be facilitated by the
appropriate selection, treatment, and/or modification of the
substrate, the sample, the measurement medium, or a suitable
combination thereof.
[0056] Binding may be facilitated by appropriate selection of the
substrate. Thus, preferred substrates typically include a
relatively flat or gently contoured binding surface adjacent the
aperture of interest, so that cells or vesicles may bind to form an
acceptable seal with the aperture without unwanted or unnecessary
deformation. Moreover, preferred substrates also typically include
a modifiable binding surface, so that the surface may be treated as
desired to promote binding.
[0057] Binding also may be facilitated by appropriate treatment of
the substrate, as suggested above. Thus, in some applications, the
surface may be treated or otherwise modified so that electrostatic
or, in given cases, hydrophobic, van der Waals or covalent binding
of vesicles or cells, or -the corresponding membranes or membrane
fragments, is promoted. For example, the binding surface may be
coated with an adhesion promoter, such as poly-L-lysine,
poly-D-lysine, gelatin, collagen, laminin, fibronectin,
proteoglycans, polyethylenimine, albumen, BIOMATRIX EHS (Nunc Nalge
International), BIOBOND (Electron Microscopy Services, Inc.),
and/or MATRIGEL (Becton-Dickinson), among others. Alternatively, or
in addition, the binding surface is modified in a way that promotes
molecule-specific binding, such as with avidin and/or biotin, or by
modification with immobilized lectins. Alternatively, or in
addition the binding surface (especially a silicon binding surface)
may be coated with an oxide or oxynitride layer. Alternatively, or
in addition, the binding surface may be coated with largely
hydrophobic compounds such as Tocopherol. In some embodiments, an
electrically charged surface may be generated by modification, in
particular, by means of polycations and/or silanes, for example,
aminosilanes, or the substrate may have a coating or other surface
layer with an electrically charged surface. Microstructured
silicon/silicon oxide or silicon/silicon nitride substrates are
especially suitable for providing a good electrostatic attraction,
after being coated with a substance lending the desired surface
charge..sup.vi Finally, to improve the quality and consistency of
the surface characteristics, the substrate may be subjected to
oxygen plasma cleaned and/or partially or completely hydrophylized
before the modification of its surface and/or before its immediate
use, in addition to or in lieu of the above modifications. In some
aspects of the invention, unwanted hydrophylization/modification of
the hydrophobic surface can be avoided by using silicon nitride for
the surface layer.
[0058] Binding also may be facilitated by appropriate selection
and/or treatment of the sample itself. Thus, the sample may include
unsaturated lipids or other compositions that increase the fluidity
of its membrane, potentially enhancing membrane flexibility during
binding and seal formation. Alternatively, or in addition, the
sample may include charged lipids or other compositions that
increase the charge on the sample, potentially enhancing the
ability of the sample to bind electrostatically to substrate
surfaces bearing an opposite charge. For example, for a positively
charged substrate surface, the vesicle might include negatively
charged palmitoyl-oleoyl-phosphatidylglycerol (POPG).
[0059] In some cases, binding may be facilitated by interactions
between specific binding pairs (SBPs), where one member of the pair
is associated with the sample and the other member of the pair is
associated with the substrate. The interactions between members of
a specific binding pair typically are noncovalent, and the
interactions may be readily reversible or essentially irreversible.
An exemplary list of suitable specific binding pairs is shown in
Table 1.
1TABLE 1 Representative Specific Binding Pairs First SBP Member
Second SBP Member antigen antibody biotin avidin or streptavidin
carbohydrate lectin or carbohydrate receptor DNA antisense DNA
enzyme substrate enzyme histidine NTA (nitrilotriacetic acid) IgG
protein A or protein G RNA antisense RNA
[0060] Binding also may be facilitated by appropriate selection
and/or treatment of the measurement medium. For example, the medium
may include binding mediators that participate in or otherwise
promote interactions between the sample and substrate, for example,
by forming cross-bridges between the sample and substrate and/or by
counteracting the effects of binding inhibitors associated with the
sample, substrate, or medium. The binding mediators may act
specifically, for example, by binding to specific groups or
molecules on the sample or substrate. Thus, biotin might act as a
specific binding mediator by binding to and cross-linking avidin or
streptavidin on the sample and substrate. The binding mediators
also may act less specifically, or nonspecifically, for example, by
binding to classes or categories of groups or molecules on the
sample or substrate. Thus, Ca.sup.2+ ions might act as a relatively
nonspecific binding mediator by binding to and cross-linking
negative charges on the sample and substrate. Ca2+ ions are
particularly appropriate for mediating the binding of cells or
vesicles containing negative lipids and substrates containing
negative surface charges, such as SiO.sub.2 substrates.
[0061] After binding, samples such as cells or vesicles may be
broken up, for example, by treatment with a hypotonic medium, such
as pure water.
[0062] Examples 1-7, 10, and 11 below, among others, describe
exemplary adhesion surfaces, including materials, treatments,
modifications, geometries, and the kinetics and efficacy of sample
binding.
[0063] D. Fluid Compartments
[0064] The fluid compartments generally comprise any region or
volume adapted to support a fluid adjacent the aperture. The
compartments may perform several functions, including covering the
sample, providing a medium through which the cell may be moved
during positioning, and/or providing a medium for establishing
electrical contact between the electrodes, among others. The
compartments generally may have any suitable volumes, but they
typically have volumes between about 0.1 to 40-100 .mu.L. Thus,
assays typically require only a limited amount of sample,
facilitating the analysis of effects of precious compounds.
[0065] The fluid compartments may be closed or open. A closed
compartment comprises a compartment that is at least substantially
bounded or delimited on all sides by a wall or other separating
layer, exclusive of an input and/or output port. In contrast, an
open compartment comprises a compartment that is not bounded on at
least one side (i.e., over at least some solid angle) by a
wall.
[0066] Closed compartments typically are physically confined, i.e.,
bounded by some combination of the substrate, an electrode, and one
or more spacers, being established within voids and channels
therein. The spacers are used in many embodiments, particularly
those involving planar electrodes, to establish and maintain the
relative positions of the substrate and electrodes. Such spacers
typically are formed of an electrically isolating material, like
the substrate. The spacers may include channels disposed between
the aperture and the electrode. These channels, typically filled
with a conductive solution, can serve as a sample or reference
chamber. It is beneficial if the reference chamber has such small
dimensions that the reference buffer solution may be fixed therein
by capillary forces, and/or forced therein by surface tension
(e.g., at the fluid/air interface in an open compartment).
[0067] Open compartments typically are free standing, i.e., not
bounded in at least one, typically lateral, direction. Instead, the
fluid may be fixed between the substrate and electrode without
other physical boundaries by capillary forces and/or surface
tension.
[0068] Examples 2-7 below, among others, describe exemplary fluid
compartments, including geometries, boundaries, and relationships
with other components of the system.
[0069] E. Electrodes
[0070] The electrodes generally comprise any mechanism for creating
and/or modulating an electric potential across an aperture,
particularly for use in positioning and/or analyzing samples.
[0071] The electrodes may be formed of any material capable of
inducing current flow through an aperture upon application of a
physiological potential. Suitable electrodes include silver, gold,
and/or platinum, among others. Preferred electrodes include
silver/silver chloride (Ag/AgCl) and/or platinum (Pt) redox
electrodes.
[0072] The electrodes may be formed with any suitable geometry and
be disposed in any suitable arrangement, consistent with their
performing their intended function(s). Preferred electrodes have
planar, cylindrical, or point geometries. Preferred electrode
arrangements are symmetrical, with similar electrodes positioned at
similar distances and orientations from each aperture on each side.
Symmetrical electrode arrangements generally will create
symmetrical electric fields. Typically, the electrodes are located
opposite one another across a single aperture, with each electrode
reaching into at least one compartment, or at least contacting a
surface of it. The electrodes customarily are located at a distance
of about 0.5 to 3 mm, and usually about 0.5 to 1 mm, from the
substrate, although they can be closer or farther in some
embodiments. Depending on embodiment, the electrodes may be
attached directly to a recording carrier, or to a cartridge in
which this carrier is packaged, and/or to a holder that is not in
direct contact to the substrate.
[0073] The electrodes should be capable of creating an electric
potential sufficient to perform their function, for example,
positioning and/or analyzing samples, without unduly disrupting the
samples. Preferred electric potentials give rise to electric field
intensities of greater than about 100 V/m, particularly adjacent
the aperture. In the following materials, one electrode is referred
to as a measurement electrode, p Examples 2-7 below, among others,
describe exemplary electrodes, including geometries (e.g., planar,
cylindrical, and point), materials (e.g., silver and/or platinum),
and relationships with other components of the system.
[0074] F. Multiaperture Systems
[0075] The invention provides multiaperture systems for positioning
and/or analyzing samples. These systems include two or more
apertures, which may be disposed at the same and/or separate sites.
Apertures disposed at the same site may be used to study single
samples at two or more positions on the sample. In contrast,
apertures disposed at separate sites may be used to study two or
more samples, sequentially and/or simultaneously, at one or more
positions on each sample. The production of multiaperture systems
generally is straightforward, especially using silicon substrates
and Ag/AgCl electrodes, both of which are easily microstructurable.
In particular, multiaperture systems may be produced from a single
continuous substrate having two or more apertures or by joining
together two or more smaller substrates each having one or more
apertures. The latter approach may be less expensive for substrates
such as silicon with costs that increase faster than area.
[0076] Exemplary multiaperture systems employ a multiarray layout
having a plurality of separate measurement sites. In these systems,
each site includes at least one aperture and fluid and electrical
contact with at least one fluid compartment and at least one
electrode, respectively, on each side of the aperture. The fluid
compartments and electrodes on one side of the substrate (the
measurement side) generally are separated to allow independent
recordings. However, the fluid compartments and electrodes on the
other side (the reference side) may be partially or totally
combined, because these components typically function merely to
provide a common electrical potential (e.g., ground). The sample
generally may be positioned on either the measurement or the
reference side, although typically it is positioned on the
measurement side so that each fluid compartment independently can
contain the same or different types of samples. Thus, in these
exemplary systems, several apertures may be used on one substrate,
and the measurements may be performed over at least two apertures
sequentially and/or in parallel and/or in such a manner that all or
several electrodes on one side of the substrate have a common
electrical potential, or, alternatively, are combined to form one
electrode. Similarly, more than two electrodes and more than one
aperture can be present in such a way that at least one electrode,
for example, a reference electrode, serves the measurement via more
than one aperture, or the measurement arrangement can have a
substrate with more than one aperture and twice as many electrodes
as apertures in such a way that one aperture always is located
between two electrodes.
[0077] Measurement sites may be separated using any suitable
mechanism, including hydrophilic/hydrophobic surface patterning, as
described below, and/or dividing the carrier surface into small
compartment wells (e.g., by laminating a thin polydimethylsiloxane
(PDMS) layer containing small holes to the carrier surface adjacent
the aperture).
[0078] The multiaperture system generally may include any number of
measurement sites, positioned in any suitable arrangement, with any
suitable size or footprint, all consistent with forming electric
fields within each site to position and/or analyze samples.
Preferred configurations may be selected based on utility and/or
convenience. Thus, preferred systems may include features selected
from standard microplates, so that the system may be used with
standard microplate equipment, including handlers, washers, and/or
readers, among others. These features may include a rectangular
frame, with a major dimension of about 125-130 mm, a minor
dimension of about 80-90 mm, and a height of about 5-15 mm,
although other dimensions are possible. The frame may include a
base configured to facilitate handling and/or stacking, and/or a
notch configured to facilitate receiving a cover. These features
also may include 96, 384, 864, 1536, 3456, or 9600 measurement
sites, among others, positioned on a rectangular or hexagonal
array. Three exemplary configurations that will fit as rectangular
arrays within a microplate-sized frame are listed in the following
table:
2 Arrangement of Pitch (mm) Density (/mm.sup.2) Number of Sites
Sites Between Sites of Sites 96 8 .times. 12 9 1/81 384 16 .times.
24 4.5 4/81 1536 32 .times. 48 2.25 16/81
[0079] Here, pitch is the center-to-center site-to-site spacing,
and density is the number of sites per unit area. These features
also may include the color of system components, particularly
components in the optical path in optical assays. For example, in
fluorescence applications, system components preferably are made of
opaque black plastic to reduce background photoluminescence and/or
"crosstalk," where crosstalk is the transmission of light emitted
in one site to adjacent sites where it may be detected. In
contrast, in chemiluminescence applications, system components
preferably are made of opaque white plastic to increase reflection
of emitted light out of the site by the white surfaces while still
reducing crosstalk.
[0080] Examples 5 and 6 below, among others, describe exemplary
multiaperture positioning and/or analysis systems, including
additional features such as reference fiducials not described
above.
[0081] G. Analysis System
[0082] The positioning and measurement system of the present
invention optionally may be coupled to or integrated with an
analysis system for analyzing samples and sample components. The
analysis system generally comprises any mechanism for analyzing or
otherwise characterizing samples, qualitatively or quantitatively,
other than by direct electrical measurement as used by the
positioning and measurement system. The analysis system may require
that the sample be separated from the measurement system and/or
from other sample components, as described above. Alternatively, or
in addition, the analysis system may allow the sample to be studied
in situ, without such separation. Generally, measurements made by
the positioning and measurement system and measurements made by the
analysis system may be performed simultaneously or sequentially, in
any order or in any combination, in association with or independent
of one another.
[0083] The analysis system may be based on any suitable analytical
technique, including spectroscopic, hydrodynamic, and imaging
methods, among others, particularly those adaptable to
high-throughput analysis of multiple samples. Preferred analysis
systems are based on the optical analysis of samples, particularly
luminescence-based optical analysis, but also absorption,
scattering, circular dichroism, optical rotation, and imaging,
among others. In luminescence analysis, light transmitted from the
sample is detected and analyzed, and properties of the detected
light are used to infer properties of the sample, including the
presence, size, shape, mobility, quantity, activity, and/or
association state of selected components of the sample. In
photoluminescence, including fluorescence and phosphorescence, the
emission of light from the sample is induced by illuminating the
sample with appropriate excitation light. In chemiluminescence, the
emission of light from the sample is induced by chemical reactions
occurring within the sample. The analysis may involve measuring
various properties of the detected light, including its intensity,
lifetime, polarization, quantum yield, and Stokes' shift, among
others. The analysis also may involve using one or more of these
properties in techniques such as fluorescence intensity (FLINT),
fluorescence polarization (FP), fluorescence resonance energy
transfer (FRET), fluorescence lifetime (FLT), total internal
reflection fluorescence (TIRF), fluorescence correlation
spectroscopy (FCS), fluorescence recovery after photobleaching
(FRAP), and fluorescence imaging, including confocal CCD
observation, among others.
[0084] In luminescence assays, light typically is detected from a
luminophore, i.e., a molecule or other species that emits
luminescence. The luminophore may be endogenous or exogenous.
Moreover, the luminophore may be the material of interest in the
assay but more commonly is simply a reporter that provides
information about another material that is the true material of
interest. In particular, the luminophore may be an exogenous
molecule that reports on (1) membrane potential, (2) the presence
or concentration of a target metal, such as Ca.sup.2+, Mg.sup.2+,
and Zn.sup.2+, (3) the presence or concentration of an inorganic
ion, such as Na.sup.+, K.sup.+, and Cl.sup.-, (4) pH, (5) reactive
oxygen species, including nitric oxide, (6) ion channels, including
Ca.sup.2+ channels, Na.sup.+ channels, K.sup.+ channels, and
Cl.sup.- channels, (7) signal transduction, (8) cell viability, and
(9) endocytosis and exocytosis, among others. Suitable luminophores
for reporting on this and other information are described in
Richard P. Haugland, Handbook of Fluorescent Probes and Research
Chemicals (6.sup.th ed. 1996), which is incorporated herein by
reference.
[0085] The combination of optical technologies with the patch-clamp
technologies presented here permits, for the first time, the
distinction or resolution of ligand binding events and channel
activities, among others. In this way, for example, important
information regarding the stabilization of changes in receptor
conformation through ligand binding and/or the functional variation
in ligand binding sites in receptors may be obtained..sup.vii Such
information is potentially important for understanding the
particular mode of action of individual agonists and antagonists,
and thus exhibits great promise for future drug development.
[0086] The optical analysis system typically will include a light
source, a detector, and one or more optical relay structures for
directing excitation light from the light source to the sample and
for directing emission light from the sample to the detector.
However, the light source and excitation optical relay structure
are optional during analysis utilizing chemiluminescence methods.
The optical analysis system may use epi- and/or trans-detection
schemes, involving illuminating off of and/or through the sample,
respectively.
[0087] Exemplary optical analysis systems, and components thereof,
are described below under Examples and in the various patents,
patent applications, and other materials listed above under
Cross-References and incorporated herein by reference. Preferred
optical analysis systems are described in the following materials,
which are incorporated herein by reference: U.S. Pat. No.
5,355,215, issued Oct. 11, 1994; U.S. Pat. No. 6,097,025, issued
Aug. 1, 2000; U.S. Provisional Patent Application Serial No.
60/267,639, filed Feb. 10, 2001; and Joseph R. Lakowicz, Principles
of Fluorescence Spectroscopy (2.sup.nd ed. 1999).
[0088] The combined system is suitable for simultaneous and/or
sequential electrical and optical (e.g., photoluminescence)
measurements. Suitable apparatus may include a planar, and
vertically easily realizable, optically transparent structure, for
example, with the use of planar pointed electrodes, or
alternatively point electrodes disposed outside of the vertical
lines going through the aperture. This allows not only fluorescence
or electrical analysis of single cells (or membranes) but also the
combined optical-electrical observation of cells (or membranes),
particularly in response to exposure to externally applied
substances, such as potential medical drugs. This revolutionary
approach allows significantly more efficient selection of new drugs
and the elucidation of their molecular action.
[0089] Example 7 below, among others, describes an exemplary
analysis system. Additional examples are described in the various
patents and patent applications listed above under Cross-References
and incorporated herein by reference.
[0090] H. Sample-handling System
[0091] The positioning and measurement system according to the
invention optionally may be coupled to or integrated with a
sample-handling system for adding, manipulating, exchanging, and/or
removing samples and sample components, including cells and
vesicles, sample media, and compounds and reagents, such as
candidate modulators and/or other analytes. The sample-handling
system may add samples such as cells or vesicles to arbitrary
compartments, convey liquid into and/or out of arbitrary
compartments, and/or exchange samples and/or liquid between
arbitrary compartments, among others. The sample-handling system
also may separate samples, or sample components, in particular
using capillary electrophoresis (CE) and/or high-pressure liquid
chromatography (HPLC), and serve the analysis of the separated
substances, or it can be provided with means that serve the
continuous or regular testing of the state of the liquid in the
compartments as well as with means for retroactive regulation
according to preset filling parameters. Because it is reasonable,
according to the analysis strived for, to bring the membrane into
contact with measurement solution on both sides, the addition of a
substance to be investigated obviously can be done on the side
customarily serving as the reference side. The sample-handling
system may be multiplexed to interact with several substrates
and/or with a multiaperture substrate, among others.
[0092] The sample-handling system may be based on any suitable
mechanism, including tubes, pumps, hydrostatic pressure
differentials, electro-osmotic processes, piezo drop-on-demand
processes, ink-jet processes, contact transfer processes,
temperature-controlled processes, and/or mechanical displacement,
among others. In some embodiments, fluids such as reference buffers
may be introduced into a pasty gel, whereby an exchange of the
liquid lying outside the gel is possible without changing the
composition of the reference buffer stored in the gel. Suitable
gels include agarose and polyacrylamide.
[0093] Example 2 below, among others, describes an exemplary sample
handling system. Additional examples are described in the various
patents and patent applications listed above under Cross-References
and incorporated herein by reference, including U.S. patent
application Ser. No. 09/777,343, filed Feb. 5, 2001; and U.S.
Provisional Patent Application Serial No. 60/267,639, filed Feb.
10, 2001.
[0094] I. Samples
[0095] The sample generally comprises any species having a membrane
or other surface capable of forming a seal with an aperture
sufficient for performing electrical measurements such as patch
clamp experiments. The sample may include cells, vesicles, cellular
organelles, membrane-bound viruses, and fragments, derivatives, and
mixtures thereof.
[0096] Biological samples may include or be derived from (1)
eukaryotic cells, i.e., cells with a nucleus, including cells from
plants, animals, fungi, yeast, and protozoans, or enucleated
derivatives thereof; (2) prokaryotic organisms, including bacteria
and archaebacteria; (3) viruses; (4) organelles or extracts, such
as nuclei, mitochondria, endosomes, the Golgi apparatus,
peroxisomes, lysosomes, endoplasmic reticulum, chloroplasts, axons,
and dendritic processes, among others; and (5) gametes, including
eggs and sperm. These cells and other materials may be obtained
from any suitable source, including cell cultures, patient samples,
and tissues, among others. These cells also may be subjected to any
suitable treatments to alter membrane properties, for example, to
introduce a novel or modified ion channel, among others. These
treatments may include genetic modification by any suitable method,
including chemical treatment, irradiation, transfection, infection,
and/or injection, among others.
[0097] Vesicles and other synthetic samples may include or be
derived from (1) unilamellar vesicles, (2) multilamellar vesicles,
(3) small vesicles (having diameters less than about 1000 nm), (4)
large vesicles (having diameters greater than about 1000 nm), (5)
monodisperse vesicles, and (6) polydisperse vesicles. These
vesicles may be formed from any suitable lipid(s) and/or protein(s)
using any suitable technique. Exemplary lipids include DLPC, DMPC,
DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA, DPPA, DOPA, DMPG, DPPG,
DOPG, DMPS, DPPS, and DOPS, among others.
[0098] Examples 8 and 15-17 below, among others, describe exemplary
samples, including vesicle and cell samples.
[0099] J. Sample Positioning
[0100] The electrical, optical, and/or other analysis of samples
generally is preceded by a positioning step, in which the sample is
directed to or otherwise located at the adhesion surface. Sample
positioning generally occurs in two sequential substeps: (1) a
first (prepositioning) substep, in which the sample is introduced
to the measurement compartment, and (2) a second (micropositioning)
substep, in which the sample is brought into proximity or actual
contact with the adhesion surface. These steps may be performed
robotically, at least substantially without direct human
involvement or intervention.
[0101] The prepositioning substep generally involves introducing
the sample to the measurement compartment, preferably in a manner
that facilitates subsequent binding of the sample to the adhesion
surface. Thus, the sample may be introduced generally above the
adhesion surface (or associated aperture), so that it is directly
between the electrodes, if they are symmetrically arranged, and so
that gravity will tend to pull it straight down toward the
aperture. Alternatively, or in addition, the sample may be
introduced relatively close to the adhesion surface, and/or with an
initial velocity toward the adhesion surface, among others.
[0102] The micropositioning step generally involves bringing the
sample into proximity or actual contact with the adhesion surface,
once the sample is in the measurement compartment. Generally,
samples may be micropositioned using any suitable force or other
mechanism, including sedimentation (e.g., "1g-sedimentation" under
the influence of gravity), electromagnetic forces (e.g.,
electrophoresis, electro-osmosis, and the like), optical forces
(e.g., optical tweezers), fluid-mediated forces (e.g., pressure,
vacuum, flow, diffusion, and the like), and/or manual forces.
Alternatively, or in addition, the cells may be positioned by fluid
flow from the sample compartment to the reference compartment by a
hydrostatic pressure difference or by a difference in surface
tension between the two compartments. Preferably, samples are
micropositioned using electromagnetic forces, specifically, field
focusing of an electric field created by applying a potential
across the two electrodes. In brief, this technique exploits the
field focusing that occurs adjacent the aperture, creating an
electric force that, at least adjacent the aperture, points in all
positions toward the aperture, with a strength that increases with
proximity to the aperture.
[0103] Examples 2 and 9 below, among others, describe exemplary
methods for prepositioning and micropositioning samples,
respectively.
[0104] K. Measurement Process
[0105] The measurement process provided by aspects of the invention
allows in particular the measurement of ion channel flows in a
reliable and reproducible manner, often with a high signal-to-noise
ratio. These abilities reflect the precise positioning and
electrically tight binding of cells, vesicles, cellular organelles,
and/or membranes of corresponding origin, at microstructured
apertures in a planar substrate. This electrically tight binding
may be achieved at least in part by strong interactions between the
surface of the substrate and the surface of the bound membrane,
such as strong electrostatic attractions.
[0106] The electrical characteristics of transmembrane ion channels
or ionotropic receptors may be characterized using "voltage-clamp
technologies," such as classical voltage-clamp, patch-clamp, and
oocyte voltage-clamp, among others..sup.viii Specifically, an
electrical potential difference is applied across the membrane
containing the relevant ion channel(s), and, simultaneously, the
current necessary to maintain this difference is analyzed. The
relationship between the voltage and current may be expressed
mathematically using Ohm's Law, which states that V=IR, or
equivalently, I=V/R, where V=voltage, I=current, and R=resistance.
The current provides insight into membrane electrical properties,
such as its conductivity, and therefore insight into the
conformation state of the channel-forming protein (e.g., open
(passing ions) or closed (blocking ions)). Thus, the current may be
used to analyze voltage dependencies, ligand binding events, and so
on.
[0107] The ability of current measurements to yield meaningful data
in a patch clamp or other electrophysiology experiment is dependent
on ensuring that the measured current reflects ion flow through the
sample (e.g., through ion channels in the membrane) and not through
other components of the system. In particular, to obtain acceptable
or better signal-to-noise ratios, it typically is desirable to
ensure that the measured current includes no more than a ten or
twenty percent contribution from unintended sources (e.g., that
sources of noise lie under the signals to be measure by
approximately the factor of five or ten). Unfortunately, the ion
flow through ionotropic membrane proteins with 0.1 to 50 pA at a
-60-mV membrane potential is in general very small, so that leakage
currents occurring essentially between the membrane and its
fastening quickly may become significant, representing a principal
problem in all voltage-clamp technologies.
[0108] The problem of leakage currents can be solved in a variety
of ways. For example, enlarging the aperture and thereby the patch
of membrane to be analyzed may reduce the contribution of leakage
currents to the total signal, because the area of the membrane
patch and so the intended signal will grow as the radius of the
aperture squared, while the circumference of the membrane and so
the leakage current will grow merely as the radius. Unfortunately,
increasing the size of the membrane may lead to a loss in
specificity, particularly in biological systems, because more
channels and more types of channels will be in the membrane area
analyzed. Then, in general, an unambiguous or completely
artifact-free statement, for example, in the case of the addition
of ligands, may no longer be possible.
[0109] Establishing and maintaining a very high seal between the
membrane and aperture also may reduce the contribution of leakage
currents. This invention uses this principle, at least in part. To
implement the seal, a planar substrate chip having a surface that
is strongly adhesive for cells and vesicles is used. This chip
separates the two compartments clamped at different potentials
during the measurement, where a (sub)micrometer-sized aperture is
located in its middle. This aperture is filled with reference
buffer solution and electrically tightly sealed during current
measurements by strong binding of cells and vesicles to the
surface. This electrically tight binding may permit the measurement
of very small ion flows (e.g., down to at least about 0.1 pA) and,
concomitantly, the plotting of membrane resistance with a good or
better signal-to-noise ratio.
[0110] Further (mechanical) stability may be derived from using
capillary forces to fill and store the reference and/or measurement
buffers. In particular, unbounded or open fluid compartments may
experience fewer disturbances (e.g., due to temperature
differentials) of the membrane due to hydrostatic pressure than
closed systems.
[0111] The measurement systems described here may be used for a
variety of applications, some of which are described below in
Example 16, including "perforated-patch," "whole-cell," and
"inside-out" patch clamp techniques. For example, measurement
systems of the planar type are particularly well suited, due to the
short diffusion times associated therewith, to the use of
"perforated-patch" techniques..sup.IX In these techniques, an
electrical connection to the interior of the cells (cytosol) is
achieved by permeabilization of the area of the membrane suspended
across the aperture with pore-forming antibiotics. An advantage of
this technique is that it does not require washing out the cytosol
with measurement buffer solution for simultaneous electrical
access. In particular, a pore former such as, for example,
amphotericin B or nystatin can be added to the reference
compartment, after a biological cell, or, under special
circumstances, a vesicle (if its mechanical stability is
sufficiently high) is bound to the upper side of the aperture. In
doing so, the rate of perforation of the membrane patch over the
aperture is significantly greater than in comparable standard
patch-clamp techniques.
[0112] For example, measurement systems of the planar type also are
particularly well suited to the use of "whole-cell" techniques. In
these techniques, an electrical connection to the cytosol is
achieved by destroying the membrane patch, for example, using a
voltage pulse. This destruction, in turn, may facilitate the simple
addition of larger proteins into the cytoplasm, via the reference
solution, again because the planar layout of the measurement system
allows significantly faster diffusion of large macromolecules into
the cytosol or the interior of a vesicle than comparable standard
whole-cell techniques..sup.x
[0113] The system facilitates the addition and/or exchange of
various system components, including solutions and/or substances,
as suggested above. For example, in some applications, the
measurement solution, the reference solution, or both solutions may
be replaced by another solution. Alternatively, or in addition, a
substance to be analyzed may be added to the solution on the
measurement and/or reference side. The substance may include a pore
former that can be added to one or both compartments with the aim
of increasing the electrical conductivity, or, alternatively, the
permeability of the membrane with respect to certain ions. The
substance also may include detergent-solubilized proteins or
proteoliposomes of arbitrary size, with the aim of fusing them to
the membrane over the aperture and thereby making arbitrary
membrane proteins contained therein accessible to electrical or
optical measurements. The fusion of proteoliposomes is described in
detail in U.S. patent application Ser. No. ______ , filed Sep. 14,
2001, titled EFFICIENT METHODS FOR THE ANALYSIS OF ION CHANNEL
PROTEINS, and naming Christian Schmidt as inventor.
[0114] Examples 12-16 below, among others, describe exemplary
results obtained from various electrical measurements on vesicle
and cell-derived membranes.
EXAMPLES
[0115] The following examples describe selected aspects and
embodiments of the invention. These examples are included for
illustration and should not be interpreted as restricting,
limiting, or defining the entire scope of the invention. Additional
examples are described in the following patent applications, which
are incorporated herein by reference: U.S. Provisional Patent
Application Serial No. ______ , filed Sep. 13, 2001, titled
HIGH-THROUGHPUT PATCH CLAMP SYSTEM, and naming Christian Schmidt as
inventor; and U.S. patent application Ser. No. ______ , filed Sep.
14, 2001, titled EFFICIENT METHODS FOR THE ANALYSIS OF ION CHANNEL
PROTEINS, and naming Christian Schmidt as inventor.
Example 1
[0116] Substrate Chip
[0117] This example, illustrated in FIG. 2, describes an exemplary
Si/SiO.sub.2 chip substrate 50 for use in positioning and/or
studying cells, vesicles, and the like, in accordance with aspects
of the invention.
[0118] The substrate includes a body 52, a surface layer 54, a
window 56, and an aperture 58. The body comprises an at least
substantially planar, commercially available silicon wafer. The
surface layer comprises a silicon oxide or silicon oxynitride layer
formed adjacent one or more sides of the body. In this embodiment,
the surface layer has a thickness of at least about 50 to 200 nm
and provides at least one adhesion surface 60a,b capable of binding
cells, vesicles, and/or other samples. The window and aperture
comprise openings through the body and surface layer, respectively.
These openings are at least substantially concentrically aligned,
with dimensions sufficient to allow fluid contact between opposite
sides of the substrate.
[0119] The substrate may be produced using any suitable method,
including photolithography or, for apertures having diameters of
less than about 1.5 .mu.m, electron beam lithography. These methods
may involve anisotropic etching of the silicon in a medium
containing KOH, as well as reactive ion etching of the silica
layer.
[0120] In alternative embodiments, the substrate may include a body
and/or a surface layer having a different geometry and/or formed of
different materials. In addition, the window may be absent, or the
window and the aperture both may be openings in the body,
particularly in embodiments lacking a surface layer.
Example 2
[0121] Measurement System with Planar Electrodes
[0122] This example, illustrated in FIG. 3, describes an exemplary
measurement system 70 having planar electrodes, in accordance with
aspects of the invention.
[0123] The measurement system includes a substrate 72, at least two
fluid compartments 74a,b, at least two redox electrodes 76a,b, and
optionally at least four spacers 78a-d. In this embodiment, all of
these components are at least substantially planar; however, in
other embodiments, one or more of these components may have a
different geometry. Generally, samples may be introduced into
either compartment, and measurements may be performed with the
system in any orientation. However, to simplify the description,
the top fluid compartment 74a and top electrode 76a (as drawn) are
referred to here as the measurement compartment and measurement
electrode, and the bottom fluid compartment 74b and the bottom
electrode 76b (as drawn) are referred to as the reference
compartment and the reference electrode.
[0124] The substrate is used to support cells, vesicles, and other
samples for electrical analysis. The substrate includes a body 80,
a window 82, and an aperture 84 connecting the two fluid
compartments. The substrate further includes at least one adhesion
surface 86a,b positioned adjacent one or both ends of the aperture
for binding cells, vesicles, and/or other samples. An exemplary
substrate is described in more detail in Example 1.
[0125] The fluid compartments are used to support fluids such as
electrolyte solutions or growth media in apposition to the
substrate and aperture. The compartments are formed by apertures or
voids in the substrate, spacers, and/or electrodes.
[0126] The electrodes are used to apply and/or measure an electric
potential and associated electric field across the aperture.
Measurement electrode 76a comprises a 0.8-mm thick chlorinated
square (e.g., 4.times.4 mm.sup.2) or annular (e.g., d=2 mm) silver
(Ag) plate, preferably having a frustoconical or funnel-shaped
opening 88 (e.g., d.sub.min=0.4 to 1 mm). The measurement electrode
is positioned at least substantially parallel to the surface of the
substrate, preferably at a distance of up to about 1 mm from the
surface of the substrate. The measurement electrode further is
positioned so that opening 88 is at least substantially
concentrically positioned above aperture 84. Reference electrode
76b comprises a 2-mm thick square silver plate (e.g., 20.times.20
mm.sup.2), which preferably has a purity greater than about 99.98%
silver. The preferred silver/silver chloride (Ag/AgCI) reference
electrode may be produced during manufacture of the system, for
example, (1) by exposing the reference compartment to a molecular
Cl.sub.2 gas, typically while applying a potential to the
electrode, or (2) by filling the reference compartment with 1 M
HCl, and then chlorinating the exposed silver for 90 seconds under
a 0.8-V potential. The substrate is mounted, after its underside is
wetted with buffer solution, over the reference compartment filled
with reference buffer solution. In this embodiment, the reference
electrode functions to support and maintain other components of the
system.
[0127] The spacers may be used for several functions, including (1)
separating the substrate and electrodes, (2) forming the fluid
compartments, and (3) contributing to the structure of a feed
opening 90 used to introduce samples to the sample compartment.
Specifically, first and second spacers 78a,b are positioned about
the measurement electrode. These spacers include openings 92a,b
that may be aligned concentrically with opening 88 in the
measurement electrode and aperture 84 in the substrate to form a
"feed opening" for introducing samples into the system. The feed
opening may be of arbitrary form; typically, however, it is
elliptical, in particular circular, with a preferred diameter of
about 0.2 to 2 mm, and a more preferred diameter of about 0.5 to 1
mm, to facilitate its concentric alignment with the aperture. A
third spacer 78c is positioned between the measurement electrode
and the substrate to form an insulating barrier between these two
elements. This spacer, preferably formed from silicone (e.g.,
Sylgard 184, Dow Coming, USA), includes a ring-like opening 92c
that again may be mounted concentrically about aperture 84, for
example, with a radius r of about 1 mm. The ring-like opening
forms, together with the meniscus that forms between the chip and
measurement electrode, the sample chamber (sample compartment) for
the addition of cells, vesicles, and/or measurement solution.
Finally, a fourth spacer 78d is positioned between the reference
electrode and the substrate to forming an insulating barrier
between these two elements. This spacer, preferably formed as a 0.5
to 2 mm-thick silicon rubber seal (e.g., Sylgard), includes a
channel or chamber 92d having dimensions of about 1 mm in width and
less than about 6 mm in length. The spacer may be imprinted and, if
filled with buffer solution, produce contact between the aperture,
or alternatively the membrane, and the reference electrode.
[0128] The measurement system may be configured or adapted to
facilitate the addition, positioning, and/or analysis of samples.
Thus, the setup preferably has means on one or both sides of the
substrate that make possible an addition of liquid, a storage of
liquid, and, in given cases, an exchange of liquid, as well as the
addition of cells, vesicles, or other cellular organelles, or parts
of the same, between the substrate and the electrode(s). For
example, during measurement, or membrane production, a small (e.g.,
5 to 10 .mu.L) volume of measurement or vesicle solution (e.g., a
cell suspension) may be added (e.g., by pipette) directly to the
feed opening, the window, and/or the aperture, on the measurement
side of the substrate or on the upper side of the measurement
electrode. The aperture preferably has a diameter such that, when a
voltage differential exists over the chip, an inhomogeneous
electrical field, mediated by the electrodes, is set up around the
aperture. This field may increase in magnitude near the aperture,
such that samples can be moved electrophoretically toward the
aperture. Furthermore, the substrate preferably includes at least
one surface 94a,b, on one or both sides of the aperture, that is
attractive for biological membranes, permitting the
molecule-specific and/or multivalent ion-mediated binding of cells,
vesicles, membrane fragments, and/or cellular organelles. The
surface of the substrate further may be structured to create
hydrophilic and hydrophobic areas, with the hydrophilic area
preferably positioned around the aperture.
Example 3
[0129] Measurement System with Point or Wire Electrodes
[0130] This example, illustrated in FIG. 4, describes an exemplary
measurement system 110 having point or wire electrodes, in
accordance with aspects of the invention.
[0131] The measurement system includes a substrate 112, two fluid
compartments 114a,b, and two point or wire electrodes 116a,b. The
substrate and fluid compartments are used to support samples and
fluids, respectively, as described above. The substrate includes a
body 118, a window 120, and an aperture 122 connecting the two
fluid compartments. The substrate further may be surface modified
and/or fastened to a holder, including a glass or Teflon holder.
The electrodes are used to apply an electric potential and
associated electric field across the aperture, also as described
above. Here, the electrodes comprise the chlorinated end surfaces
124a,b of two silver wires 126a,b, or, alternatively, two silver
electrodes, disposed above and below the substrate. The electrodes
preferably have diameters between about 0.1 and 2 mm and a relative
separation of about 4 mm. In some embodiments, the electrodes may
be provided with a protective outer layer 128a,b that covers and
protects the outside surface of the electrodes, except at the end
surfaces.
[0132] The measurement system may be used for positioning and/or
analyzing samples. In an exemplary approach, sample medium is added
to both sides of the substrate, and held between the substrate and
electrode by capillary forces. Next, the offset is calibrated, and
a suitable voltage is applied (typically, V=-60 to -100 mV). Then,
cells or vesicles are added to an appropriate (e.g., modified) side
of the substrate, and cell binding and/or membrane formation are
pursued with the aid of a change in the electrical parameters.
Finally, the properties of ion channels or other membrane
components are studied using suitable electrophysiology methods.
Throughout, the addition or exchange of samples and/or sample media
may be performed using a sample handling system, as described
above, such as a pipette or tube mounted near the aperture.
Example 4
[0133] Measurement System Having Open Fluid Compartments
[0134] This example, illustrated in FIG. 5, describes an exemplary
measurement system 150 having open fluid compartments, in
accordance with aspects of the invention.
[0135] The measurement system includes a substrate 152, at least
two fluid compartments 154a,b, and at least two electrodes 156a,b.
These components perform at least substantially the same functions
as their namesakes in Examples 2 and 3.
[0136] The substrate comprises an insulating silicon chip 158. The
substrate may include a groove that is closed by a thin silicon
nitride (Si.sub.3N.sub.4) 160/silicon oxide (SiO2) 162 diaphragm
containing a small aperture 164 having a diameter that usually is
less than about 20 .mu.m. The substrate further may include a
surrounding insulating layer 166, for example, thermally grown
silicon oxide, to reduce system capacitance. The surface of the
substrate may be treated to promote the tight adhesion of cell or
vesicle-associated lipid bilayers, for example, by (1)
physisorption of poly-L-lysine (with a typical molecular weight
greater than about 15,000 daltons), (2) chemical modification with
4-aminobutyl-dimethyl-methoxysilane, and/or (3) attachment of
molecules that bind (specifically or nonspecifically) to the cell
surface (e.g., lectins), among others.
[0137] The fluid compartments comprise open regions of the
substrate surface adjacent the aperture to which fluid is confined.
Here, fluid is confined by a combination of hydrophilic and
hydrophobic interactions. Specifically, fluid is attracted to the
region adjacent the aperture by hydrophilic interactions and
excluded from regions away from the aperture by surrounding layers
of hydrophobic material 168a,b attached or bound to the surface.
Consequently, the buffer compartments are delineated by the surface
of the substrate on one side and by surface tension on the opposing
side, creating dome-shaped compartments, as shown.
[0138] The electrodes comprise conductive elements such as Ag/AgCl
for generating an electric potential across the aperture. The
electrodes, which may be used for positioning and/or recording, are
immersed in the fluid compartments. The electrodes may be directly
attached to the substrate (e.g., by sputtering or printing) or to a
container that contains the substrate. Here, a first (measurement
or recording) electrode is used to apply a measurement voltage, and
a second (reference) electrode is used to apply a ground.
[0139] The measurement system may be used for positioning and/or
analyzing samples, at least substantially as described above. In
particular, upon application of a voltage between the two fluid
compartments, mediated by the redox electrodes immersed in the two
compartments, a strongly inhomogeneous field is created around the
aperture that attracts cells, vesicles, and other charged objects
towards the aperture. After these samples bind and/or form
membranes, they may be analyzed electrically and/or optically,
among others.
Example 5
[0140] Measurement System Having Multiple Measurement Sites
[0141] This example, illustrated in FIGS. 6 and 7, describes an
exemplary measurement system 190 having multiple measurement sites,
in accordance with aspects of the invention. The drawings show two
alternative embodiments, separated by break lines, that include
different carrier/electrode configurations.
[0142] The measurement system includes a plurality of measurement
sites, each capable of positioning and/or analyzing a sample, as
described above. More specifically, the measurement system includes
a substrate 192, a plurality of fluid compartments 194a,b, and a
plurality of electrodes 196a,b. The measurement sites are formed
from portions of the substrate and combinations (e.g., pairs) of
fluid compartments and electrodes. The substrate preferably
comprises (1) a silicon body 198, (2) a silicon nitride diaphragm
200 having a plurality of apertures 202, at least one per
measurement site, and (3) a hydrophobic and/or insulating surface
coat 204. The fluid compartments preferably comprise (1) a
plurality of measurement compartments 194a, and (2) at least one
reference compartment 194b. The electrodes preferably comprise (1)
a plurality of measurement electrodes 196a, at least one per
aperture or measurement site, and (2) at least one reference
(ground) electrode 196b. The measurement system further may include
additional features, such as (1) a support or carrier plate 206 to
simplify the design and/or to increase the reliability of the
system, and/or (2) one or more reference fiducials 207 for
reference and/or alignment purposes, as described below.
[0143] The components of the measurement system are described in
more detail in subsequent subsections. Briefly, on one side, the
substrate contains a patterned surface that physically separates
the measurement compartments, allowing independent measurements.
The patterned surface may be created by the patterned attachment of
hydrophilic materials at measurement sites and hydrophobic
materials at intervening positions. The measurement compartments
may be accessed independently using a separate measurement
electrode for each compartment, where each electrode is connected
independently to one or more voltage sources, such as a voltage
clamp circuit. On the other side, the substrate contains a
reference compartment that can be separated but that preferably is
unified to form a single compartment in contact with a single
(usually ground) electrode. In some embodiments, the substrate
includes a silicon chip containing grooves that are closed by a
silicon nitride/silicon oxide diaphragm. The diaphragm includes a
small aperture having a diameter of less than about 20 .mu.m. The
substrate otherwise may be surrounded by an insulating layer, for
example, a thermally grown silicon oxide layer, to reduce the
system capacitance.
[0144] Substrate
[0145] The substrate generally comprises any structure adapted to
provide two or more sites for positioning and/or analyzing samples
electrically, as described above. The substrate may be formed from
any suitable material, including silicon, plastic, and/or glass.
The substrate generally may include any number of sample sites
arranged in any suitable format, as described above. Preferred
formats include 8.times.12 (96) rectangular arrays and 16.times.24
(384) rectangular arrays, with standard microplate footprints. Some
embodiments may include additional sites, including additional rows
or columns of sites. For example, in one such embodiment, the
system includes an additional row of sites, configured as an
8.times.13 (104) rectangular array.
[0146] Fluid Compartments
[0147] The fluid compartments generally comprise any region adapted
to support fluid for bathing the sample and for providing
electrical contact between the measurement and reference
compartments.
[0148] The measurement compartments are used for positioning and/or
analyzing samples. These compartments are defined by hydrophilic
spots on the chip surface, surrounded by a hydrophobic surface
coating, for localizing fluid. The measurement compartments include
an aperture positioned within the hydrophilic spot and a
measurement electrode positioned for electrical contact with the
associated measurement fluid. The hydrophilic spot typically
includes an at least substantially planar or concave adhesion
surface, which may be selected and/or treated as described above to
promote sample binding and/or membrane formation.
[0149] The reference compartments are used for completing the
electric circuit, typically to electric ground. These "backside"
compartments may be combined to form one or more large compartments
(corresponding to two or more measurement compartments), since the
separate compartments typically would contain the same buffer
solution and each be connected to ground. In particular, a single
large backside compartment and a single backside electrode are
sufficient for spatial resolution of individual recordings, if the
measurement compartments are addressed individually. In some
embodiments, the backside electrode may be deposited directly on
the recording chip or on an embedding cartridge.
[0150] Electrodes
[0151] The electrodes generally comprise any mechanism adapted to
apply and/or measure an electric potential across the aperture,
with each measurement site in contact with at least one measurement
electrode and at least one reference electrode, as described
above.
[0152] The composition of the electrodes is selected to allow
current flow at physiological potentials. Preferred electrodes
include silver/silver chloride (Ag/AgCl) and/or platinum (Pt) redox
electrodes for both the sample and reference compartments.
Particularly preferred electrodes include silver (Ag) as the
electrode material, chlorinated within a chlorine (Cl.sub.2)
atmosphere.
[0153] The number of electrodes may vary. The upper side of the
substrate (associated with the measurement compartments) preferably
includes enough separate electrodes independently to address each
corresponding recording site, e.g., 8.times.13 electrodes on a 2.25
mm grid. In contrast, the lower side of the substrate (associated
with the reference compartment) preferably includes a single
electrode.
[0154] The electrodes may be insulated outside the measurement
compartment. Preferred insulation material preferably has a high
electrical resistance and a low dielectric constant and loss.
Particularly preferred insulation material is produced from Teflon,
silicon nitride (Si.sub.3N.sub.4), and/or Sylgard by spin coating
or chemical vapor deposition (CVD). These materials are
sufficiently hydrophobic (even after short oxygen-plasma treatment)
to confine the measurement and reference compartments. Moreover,
these materials may include or be formed to include grooves or
holes, potentially improving fluid support and/or reducing
evaporation.
[0155] The electrodes at the various measurement sites may be
connected electrically to corresponding contacts 208 for clean
and/or easy access to appropriate electronic components, such as
amplifiers, recording devices, and the like. In a preferred
embodiment, the contacts are positioned near the edge or border of
the substrate, and a bonding wire 210 joins the electrodes and
contacts, although more generally any mechanism capable of
establishing an electrical connection may be employed. In
particular, the electrodes can be bonded to contacts placed on a
plastic (e.g., polypropylene) carrier that embeds the entire
recording chip.
[0156] Support Element
[0157] The support element generally comprises any mechanism for
independently and portably supporting the substrate and associated
system components, potentially simplifying design and/or increasing
reliability. The support element may support the substrate at its
edges and/or in its interior, with the interior support potentially
reducing or preventing sagging and/or stress of the substrate. In
an exemplary embodiment, the support element includes a carrier
plate 206 and a spacer 212 sandwiched between the substrate and the
carrier plate near the edges of the substrate. The carrier plate
may be formed from glass (e.g., PYREX) and/or any other suitable
material. The substrate, spacer, and carrier plate may be joined
using any suitable mechanism, such as anodic bonding. The
separation between the substrate and the carrier plate (i.e., the
spacer thickness) preferably is chosen to be less than about 1 mm,
to allow filling of the backside (i.e., reference) compartment by
capillary forces. By extending the glass plate over the borders of
the chip, in some embodiments, it may be possible to bond the upper
electrodes to contacts placed on the glass plate.
[0158] Reference fiducials
[0159] The reference fiducials generally comprise any feature or
characteristic of the measurement system adapted to provide
information that facilitates sample handling and/or analysis, for
example, as described in U.S. Pat. No. 6,258,326, issued Jul. 10,
2001, which is incorporated herein by reference.
[0160] The reference fiducials, or a subset thereof, may be used to
encode information and/or to provide reference positions. For
example, the reference fiducials may encode information relating to
the identity of the manufacturer of the system and/or one or more
properties of the system and/or the associated samples.
Alternatively, or in addition, the reference fiducials may provide
reference positions useful to correct for cross-system drift (due
to dimensional irregularities in the system) and/or to align the
system with ancillary devices, such as an electrical device for
electrical analysis and/or an optical device for optical
analysis.
[0161] The reference fiducials may encode information using any
suitable mechanism, including electrical and/or optical mechanisms.
For example, the reference fiducials may encode information
electrically, based on the resistance, capacitance, and/or
inductance, among others, of a particular portion or portions of
the system. Alternatively, or in addition, the reference fiducials
may encode information optically, based on the size, shape,
position, color, absorptivity, reflectivity, and/or transmissivity
of a particular portion or portions of the system.
[0162] The reference fiducials may be identified and read using any
suitable mechanism, including the electrical device and/or optical
device used in sample analysis.
Example 6
[0163] Measurement System Having Optical Measurement Aids
[0164] This example, illustrated in FIG. 8, describes an exemplary
measurement system 230 having optical measurement aids, in
accordance with aspects of the invention.
[0165] The measurement system includes a substrate 232, a plurality
of fluid compartments 234a,b, a plurality of electrodes 236a,b, and
a carrier plate 238, at least substantially as described above in
Example 5. However, the system further includes an optical
measurement aid for use in conjunction with a suitable optical
analysis system, as described above. The optical measurement aid
generally comprises any element or mechanism adapted to facilitate
and/or enable optical analysis of samples such as cells or vesicles
positioned on or near the substrate. The optical measurement aid
may comprise a modification of one or more of the elements listed
above and/or a new element in addition to and/or in lieu of one or
more of the elements listed above.
[0166] The optical measurement aid may comprise a support element
that includes a short spacer and/or a thin, optically transparent
carrier plate, as described above. A short spacer and/or a thin
carrier plate may shorten the optical path length between the
optical device and sample, by reducing the separation between the
substrate and carrier plate. A thin carrier plate also may better
match the optical requirements of the optical analysis system. To
this end, the thickness of the carrier plate may be selected to
correspond to the thickness of a standard microscope cover slip,
for example, 0.08 to 0.13 mm thick (No. 0), 0.13 to 0.17 mm thick
(No. 1), 0.16 to 0.19 mm thick (No. 11/2), or 0.17 to 0.25 mm thick
(No. 2), among others. To related ends, the carrier plate may be
selected to improve overall optical transmission, for example, by
using crystal-clear, pure water-white glass or super clarity,
clear-white borosilicate glass. Alternatively, or in addition, the
carrier plate may be selected to improve transmission of polarized
light, for example, by using strain-free glass or fused silica.
Alternatively, or in addition, the carrier plate may be selected to
have uniform surface quality, exceptional flatness, and/or precise
dimensions, among others.
[0167] The optical measurement aid also may comprise a carrier
plate or other interface having an array of lenses 240 such as
microlenses that correspond in number and spacing to the array of
measurement sites. These lenses may be formed from any suitable
material (such as glass or plastic) using any suitable technique
(such as etching or molding). The lenses may be used for
high-magnification and/or high numerical aperture analysis of
samples, including the analysis of single cells positioned on
single apertures. To assist such analysis, the x-y resolution of
the recording apertures and microlenses preferably is less than a
few (e.g., about 1-4) .mu.m, which may be facilitated using a
high-precision bonding process. Moreover, the z resolution of these
components preferably is less than a few (e.g., about 1-4) .mu.m,
which may be facilitated using lenses having high numerical
apertures.
[0168] The lenses in the array generally may have any shape capable
of collecting light from the sample and/or focusing light onto the
sample. For example, the lenses may be plano-convex, meaning that
they have a flat (plano) surface and an opposed outwardly bulging
(convex) surface. The plano-convex lenses may have two
orientations. In the first orientation, exemplified by lens 240,
the convex surface 242 faces toward the sample site, and the planar
surface 244 faces away from the sample site. In the second
orientation, exemplified in FIG. 8 by lens 240', the convex surface
242' faces away from the sample site, and the planar surface 244'
faces toward the sample site. In either orientation, the lens will
collect light transmitted from the sample and direct the collected
light toward a detector, such as an imaging detector (e.g., a
charge-coupled device (CCD)) or a point detector (e.g., a
photomultiplier tube (PMT)), among others.
[0169] The optical measurement aid also may include a window 246 in
the substrate having a shape configured to match an optical sensed
volume, including the frustoconical shape through which excitation
light is directed onto the sample and/or from which emission light
is detected from the sample, for example, as described in U.S.
patent application Ser. No. 09/478,819, filed Jan. 5, 2000, which
is incorporated herein by reference. The matching may be used in
optical analysis to increase sensitivity (for example, by avoiding
detection from walls of the sample well) and/or to decrease sample
volume, among others.
Example 7
[0170] Analysis System
[0171] This example, illustrated in FIG. 9, describes an exemplary
measurement system 270 for combined electrical/optical detection,
in accordance with aspects of the invention.
[0172] The combined measurement system includes an electrical
analysis system 272 and an optical analysis system 274.
[0173] The electrical analysis system generally comprises any
system for performing electrical measurements such as patch clamp
measurements on a sample such as a cell, vesicle, or biological
organelle. The electrical analysis system may include any suitable
combination of apertures 275, substrates 276, fluid compartments
278a,b, and electrodes (not visible in this view), among other
elements, as described above. Exemplary systems are described above
in Examples 2-6.
[0174] The optical analysis system generally comprises any system
for detecting light transmitted from the sample, particularly
photoluminescence and chemiluminescence light. The optical analysis
system may include a light source 280, a detector 282, and an
optical relay structure 284 for transmitting excitation light from
the light source to the sample and emission light from the sample
to the detector. The system further may include additional
components for performing additional and/or duplicative functions,
including (1) filters 286 positioned in the excitation and/or
emission optical paths for altering the intensity, wavelength,
and/or polarization of the excitation and emission light,
respectively, (2) confocal optics elements 288 such as an aperture
positioned in an image plane for reducing detection of out-of-focus
light, and (3) a reference monitor 290 positioned to detect a
portion of the excitation light for correcting for variations
(e.g., fluctuations and/or inhomogeneities) in the excitation
light.
[0175] The light source generally comprises any mechanism for
producing light suitable for use in an optical assay, such as a
photoluminescence, scattering, and/or absorbance assay, among
others. Suitable light sources include lasers, arc lamps,
incandescent lamps, fluorescent lamps, electroluminescent devices,
laser diodes, and light-emitting diodes (LEDs), among others. The
light source may be capable of use in one or more illumination
modes, including continuous and time-varying modes, among
others.
[0176] The detector generally comprises any mechanism for detecting
light transmitted from a sample in an optical assay. Suitable
detectors include charge-coupled devices (CCDs), intensified
charge-coupled devices (ICCDs), videcon tubes, photomultiplier
tubes (PMTs), photodiodes, and avalanche photodiodes, among others.
The detector may be capable of use in one or more detection modes,
including (a) imaging and point-reading modes, (b) discrete (e.g.,
photon-counting) and analog (e.g., current-integration) modes, and
(c) steady-state and time-resolved modes, among others.
[0177] The optical relay structure generally comprises any
mechanism for transmitting light between the light source, sample,
and detector (or simply the sample and detector in a
chemiluminescence assay). Suitable optical relay structures may
include mirrors, lenses, and/or fiber optics, among others. Here,
the optical relay structure includes a beamsplitter that generally
transmits excitation light toward the sample and generally reflects
emission light toward the detector.
[0178] FIG. 9 shows an exemplary embodiment of a combined
electrical/optical measurement system, including components as
described above. Here, a parallel read-out system is used for
confocal-optical recordings, for example, using the chip substrate
system of FIG. 8. In either order, the chip substrate is placed in
an appropriate light beam that is able to excite fluorescent probes
of interest, and the sample membranes or cells are positioned at
the individual apertures of the chip. The samples are excited using
one parallel light beam, for example, using a 45-degree mirrored
beamsplitter. The fluorescent light coming from the biological
sample or any associated fluorescent probes is transmitted to an
optional filter and confocal optics element to increase the
signal-to-noise ratio before being projected onto the light
sensitive chip of a CCD camera. The confocal optics element reduces
or eliminates out-of-focus light not originating from the sample.
The spatial resolution of the CCD chip allows detection of
fluorescence from all apertures (and consequently all biological
samples) simultaneously, if desired.
[0179] More generally, the system may be configured to allow top
and/or bottom illumination and/or detection of the sample(s),
permitting the following combinations: (1) top illumination and top
detection, or (2) top illumination and bottom detection, or (3)
bottom illumination and top detection, or (4) bottom illumination
and bottom detection. Same-side illumination and detection, (1) and
(4), is referred to as "epi" and is preferred for photoluminescence
and scattering assays. Opposite-side illumination and detection,
(2) and (3), is referred to as "trans" and is preferred for
absorbance assays.
[0180] Alternatively, or in addition, the system may be configured
to allow illumination and/or detection at oblique angles. For
example, illumination light may impinge on the bottom of a sample
holder at an acute angle (e.g., about 45 degrees) relative to
detection. In comparison with a straight-on epi system (light
source and detector directed at about 90 degrees to sample holder)
or a straight-through trans system (light source directed through
sample holder directly at detector), an oblique system may reduce
the amount of excitation light reaching the detector.
[0181] Suitable systems, and components thereof, for top/bottom
and/or oblique illumination are described in the following
materials, which are incorporated herein by reference: U.S. Pat.
No. 5,355,215, issued Oct. 11, 1994; U.S. Pat. No. 6,097,025,
issued Aug. 1, 2000; and U.S. Provisional Patent Application Serial
No. 60/267,639, filed Aug. Feb. 10, 2001.
Example 8
[0182] Producing, Sizing, and Binding of Vesicles
[0183] This example describes exemplary methods for producing,
sizing, and binding lipid vesicles.
[0184] A mixture of 100 .mu.L asolectin (Fluka) or egg lecithin
(EPC), 50 .mu.L palmitoyloleylphosphatidylglycerol (POPG), and 3
.mu.L dipalmitoyl phosphatidyl-ethanolamine-rhodamine
(DPPE-rhodamine) (Molecular Probes, USA) (all 10 mg/mL in
chloroform, Avanti Polar Lipids), and 70 .mu.L methanol is dried in
a rotary vaporizer (Buchi Rotavapor R-114) at low (400 mm Hg)
pressure in a 10 mL round flask to form a film. After a 1-hour
incubation under vacuum, to the flask is added either 10 mL
H.sub.2O or 10 mL of a buffer solution having a concentration of
less than 150 mM of KCl and/or less than 600 mM of sucrose or
preferably of sorbitol. After a subsequent 16-hour incubation at
37.degree. C., the resulting lipid vesicles appear as an almost
transparent cloud. The lipid vesicles are aspirated and removed
using a 1 mL pipette and stored at 4.degree. C. until further use.
Storage of the vesicle solution may be improved by the addition of
sodium azide (NaN.sub.3) to a concentration of 0.2% by weight. This
vesicle preparation procedure yields mostly (>90%) unilamellar
vesicles, with sizes up to 250 .mu.m. However, some of the vesicles
may contain additional smaller vesicles, which are not relevant for
membrane formation.
[0185] The ability of the resulting vesicles to establish
electrically tight seals against a surface aperture is enhanced by
purification of the initial vesicle mixture to remove vesicles and
lipid impurities that are smaller than about 10 .mu.m in size.
Without such purification, the binding of such smaller vesicles in
the vicinity of the aperture may prevent electrically tight sealing
of the aperture by large (e.g., larger than 10 .mu.m) vesicles. The
vesicles may be sized by dialysis, for example, using a nylon mesh
with a 20-.mu.m pore size for at least about 20 hours. If
necessary, the membrane fluidity of the resulting vesicles may be
lowered, for example, by adjusting the lipid composition so that it
includes a higher fraction of low-fluidity lipids and/or by
lowering the temperature so that it is closer to the
phase-transition temperature of the vesicles (e.g., less than or
equal to about 4.degree. C., or more preferably less than or equal
to about 1.degree. C., for some lipids). The unilamellarity of the
resulting vesicle membranes may be demonstrated and/or verified
using any suitable analytical technique, such as microscopic
analysis using a confocal microscope..sup.xi
[0186] More generally, vesicles may be produced, sized, and bound
using any suitable methods. For example, large unilamellar vesicles
(giant unilamellar vesicles, GUVs) may be produced using the
hydration method..sup.xii Similarly, proteoliposomes may be
produced using an appropriately modified hydration method..sup.xiii
Additional vesicles may be produced using the methods described in
U.S. patent application Ser. No. ______ , filed Sep. 14, 2001,
titled EFFICIENT METHODS FOR THE ANALYSIS OF ION CHANNEL PROTEINS,
and naming Christian Schmidt as inventor.
Example 9
[0187] Electrophoretic Positioning of Vesicles
[0188] This example, illustrated in FIG. 10, describes exemplary
methods for electrophoretically positioning samples such as cells
and lipid vesicles. The positioning attainable using these methods
may exceed that attainable using gravity sedimentation, in at least
the following ways: (1) a decrease in the necessary number of
vesicles or cells, (2) an increase in the total rate of membrane
formation, and (3) an increase in the probability of a successful
membrane setup or cell binding. Vesicles and cells also may be
prepositioned prior to electrophoretic positioning to improve
performance, for example, by introducing the vesicles or cells to
the sample so that they are initially positioned above the
aperture.
[0189] The electrophoretic positioning methods make use of
inhomogeneities in the electric potential and associated electric
field surrounding the aperture. FIG. 10 shows results of a finite
element method (FEM) simulation of the electric potential
distribution around a substrate in accordance with aspects of the
invention. The substrate includes a 4-.mu.m aperture positioned
between parallel electrodes. The field distribution is shown as a
series of equipotential lines (corresponding a cross-section
through the three-dimensional equipotential surfaces), with a
spacing of 4 mV, where the potential difference between the
electrodes is 80 mV. The field-line curve is distorted in this
simulation from its normal circular form to an elliptical form to
reflect leak currents in the edge region of the carrier chips. The
following parameters were used in the simulation: C.sub.buffer=10
mM KCl, V=80 mV, d.sub.aperture=4 .mu.m, and spacing between the
aperture and each electrode=1 mm.
[0190] The electric field associated with an electric potential is
minus the spatial rate of change of the potential, i.e.,
E=-.gradient..gradient.. Thus, the electric field is perpendicular
to the equipotential surfaces at all positions, pointing in the
direction of decreasing potential. Moreover, the electric field is
stronger where the equipotential surfaces are closer together, and
weaker where these surfaces are farther apart. Consequently, from
FIG. 10, the electric field points toward the aperture (on one side
of the aperture), with a strength that increases with proximity to
the aperture.
[0191] These electrophoretic positioning methods generally may be
used in any system capable of creating and focusing an electric
field through an aperture. In preferred systems, the electrodes
used to create the field are positioned relatively close together,
for example, within about 5-10 mm, reducing the voltage required to
create an acceptable electric field. Specifically, the measurement
and reference electrodes are located, one above and one below the
substrate, at a distance of about 0.2 to 3 mm, preferably about
0.5-2 mm, and more preferably about 0.5 to 1 mm. The clamp voltage
generated by these electrodes is not critical; however, it
customarily lies in the range V.sub.c=-300 to -300 mV, preferably
lies in the range-60 to -100 mV, and more preferably lies in the
range-60 to -80 mV. The associated electrophoretic-driving force
directs vesicles and cells, following the electric field, toward
the aperture. In particular, because the electric field is strongly
inhomogeneous, increasing sharply in magnitude with proximity to
the aperture, vesicles and cells move automatically toward the
aperture. In particular, the fields are most effective near the
aperture (e.g., within about 200 .mu.m of the aperture), so that
samples preferably are brought into this range by prepositioning or
reach it convectively. For this purpose, a hole (for example,
d<1 mm) may be located in the measurement electrode with respect
to the aperture.
[0192] The following subsections describe two alternative methods
of electrophoretic positioning.
[0193] Variation 1
[0194] The offset voltage V.sub.offset between the electrodes may
be corrected before each measurement. To do so, 5 .mu.L of buffer
solution is added directly to the aperture, and the measurement
electrode is brought to within about 1 mm from the substrate
surface. After a liquid meniscus forms between the surface of the
substrate and the electrode, the offset voltage and the capacitance
of the system are adjusted to compensate.
[0195] A 10-.mu.L dispersion of lipid vesicles subsequently is
added to the upper side of the measurement electrode, where the
vesicles can sediment through the circular opening located in the
measurement electrode. Vesicles that move through the measurement
electrode opening may be accelerated directly to the aperture
opening under the influence of an electric field generated by the
applied electrode voltage, V.sub.M=-50 to -80 mV. In doing so, the
focusing achieved, measured in the number of vesicles passing
through the aperture opening with unmodified surfaces, is a
function of the size of the window (that is, the portion of the
SiO.sub.2 layer laid open by etching). Smaller SiO.sub.2 windows
(e.g., less than about 45.times.45 .mu.m.sup.2) clearly increase
vesicle throughput.
[0196] Variation 2:
[0197] The offset voltage V.sub.offset between the electrodes may
be corrected before each measurement. To do so, 5 .mu.L of buffer
solution is added between the substrate and the measurement
electrode, or alternatively between the substrate and the reference
electrode, after which the voltage is determined at which the
current flow vanishes, satisfying the expression
I(V.sub.offset)=0.
[0198] A 3-.mu.L dispersion of lipid vesicles subsequently is added
to the measurement compartment near the aperture, where, in the
case of a plane parallel electrode arrangement, the vesicles can
sediment through the circular opening located in the measurement
electrode. Vesicles that come into the vicinity (e.g., less than
about 200 .mu.m) of the aperture experience a very high field
intensity (generally but not necessarily between about 100 kV/m and
several kV/m) and are accelerated according to the electric field
curve directly to the aperture. After the vesicles bind to the
substrate and form an electrically tight seal, they are analyzed
electrically.
Example 10
[0199] Sealing of Vesicles with Unmodified Surfaces
[0200] The example, illustrated in FIG. 11, describes the sealing
of vesicles, as described above, with unmodified surfaces. In
particular, FIG. 11 shows a plot of current versus time after the
docking or binding of vesicles to a 7-.mu.m aperture in the
unmodified surface of a suitable substrate. The plot shows that the
addition of Ca.sup.2+ to a final concentration of 4 mM leads
quickly to a tight electrical seal between the vesicle membrane and
the substrate surface. Specifically, the addition of Ca.sup.2+
causes a rapid, significant drop in current, indicating that the
membrane has at least substantially blocked ion pathways through
the aperture.
Example 11
[0201] Binding and Adsorption of Vesicles on Modified SiO.sub.2
Surfaces
[0202] The example, illustrated in FIG. 12AB, describes the binding
and/or other interactions of vesicles as described above with
polylysine-modified SiO.sub.2 surfaces.
[0203] The binding between these vesicles and surfaces may be
strong and rapid, manifesting itself in less than about 0.5 seconds
after an appropriate proximity is reached. The probability of
successfully positioning a vesicle and subsequently forming an
electrically tight membrane seal is strongly dependent on the size
of the aperture, the size of the SiO.sub.2 window, and the number,
size, and size distribution of the vesicles in the vesicle
solution. If substrates having aperture diameters less than about 2
.mu.m and window sizes greater than about 40 .mu.m are used in
conjunction with suspensions of vesicles having a vesicle diameter
greater than about 40 .mu.m, the probability of binding and forming
an electrically tight seal may exceed 90% (n>15, where n is the
number of trials). In general, a decrease in the width of the
aperture and an increase in the purity of the vesicle suspensions
lead to greater reproducibility in the formation of tight aperture
seals, both substrate to substrate and vesicle preparation to
vesicle preparation.
[0204] Vesicles that bind to the surface subsequently may be drawn
out to form substantially flat, defect-free membranes. Fluorescence
microscopy studies performed using vesicles labeled with both
rhodamine (to label vesicle membranes) and carboxyfluorescein (to
label vesicle interiors) show that vesicles flatten and may burst
upon binding, forming unilamellar structures, since bound vesicles
appear flat and red, suggesting that carboxyfluorescein has been
released. These studies were conducted using polylysine-coated
glass and a confocal microscope (LSM 510, Zeiss Jena, Germany).
Electrical studies of membrane resistance, R.sub.M, demonstrate
that the bound vesicles may form substantially defect-free lipid
membranes, since very high membrane resistances (e.g.,
R.sub.M>6.4 G.OMEGA. (n=26)) are measured on the substrates in
symmetric 85 mM KCl. An analogous series of measurements in
symmetric 10 mM KCl demonstrates binding of the vesicles, after
appropriate proximity, in less than about 0.2 seconds, with a
probability greater than about 70% (n>15) and a membrane
resistance greater than about 10 G.OMEGA..
[0205] To promote strong adhesion of the vesicles, the surface of
the substrate, in given cases, is coated with an adhesion promoter,
for example, polycations..sup.xiv For physisorption, for example,
an aqueous solution of polycations (e.g., 0.1% poly-1-lysine
bromide, MW 100,000, Sigma) may be added to the substrate for about
2-5 minutes directly before the measurement and subsequently rinsed
off with measurement buffer solution. The covalent binding of
peptide polycations preferably is done using previously activated
hydroxyl groups of the quartz surface, for example by means of
tosyl chloride (triphenylchloromethane)..sup.xv Through the
modification of the substrate surface, an attraction of vesicles
with negative surface charge is achieved, which is completely
sufficient for electrically tight seals between the membrane and
the substrate surface. Alternatively, the surface also may be
modified by other compounds having cation characteristics in the
desired pH range, such as, for example,
4-aminobutyl-dimethyl-methoxysilane. Finally, treating the
substrates in O.sub.2 plasma for several minutes before surface
modification leads to more consistent surface characteristics.
[0206] FIG. 12AB shows the time course of vesicle binding and the
subsequent development of membranes with very high electrical
insulation resistance for a 4-.mu.m aperture (FIG. 12A) and a
7-.mu.m aperture (FIG. 12B) in a poly-L-lysine-coated SiO.sub.2
substrate. The measurements were performed in the presence of 10
.mu.M KCl, using a clamp voltage of -80 mV.
Example 12
[0207] Membrane Electrical Properties
[0208] This example describes electrical factors relating to
preferred aperture sizes, including characteristic electrical
properties.
[0209] The thermal noise .sigma. of a circular lipid membrane is
proportional to R.sub.M.sup.-1/2:.sup.xvi 1 = 4 kTf c R M
[0210] where R.sub.M=R.sub.spec/(.pi.r.sub.M.sup.2). It follows
therefrom that 2 = r M 4 kTf c R spec
[0211] In these formulae, .sigma. is the effective noise flow, r is
the radius, f is the frequency, k is the Boltzmann constant, R is
the resistance, and T is the temperature.
[0212] Thus, to be a usable membrane for measurement purposes,
r.sub.M/{square root}{square root over (R.sub.spec)} should be very
small. The minimization of this product can be pursued according to
the invention in two ways: (1) by minimizing the membrane radius
r.sub.M, and/or (2) by the electrically tight sealing of the
membranes used.
Example 13
[0213] Electrical Parameters of Lipid Membranes
[0214] This example describes the effects of vesicle fusion on
electrical parameters of the measurement system, as described
above.
[0215] The resistance across the sample substrate changed
significantly following vesicle fusion. Before fusion, the
resistance is up to 1 M.OMEGA. (usually <450 k.OMEGA.) in 85 mM
KCl, and similarly usually up to 1 M.OMEGA. in 1 mM KCl, depending
in both cases on the size of the aperture. Greater resistances are
interpreted as artifacts, possibly reflecting, for example, the
inclusion of air bubbles under the aperture opening. After fusion,
the resistance is greater than about 6.4 G.OMEGA. in 85 mM KCl,
greater than about 10 G.OMEGA. in 10 mM KCl, and greater than about
40 G.OMEGA. in 1 mM KCl, corresponding to four order-of-magnitude
increases in resistance. Here, the resistance R is at least
approximately related to the applied voltage V and the current I
according to Ohm's law, i.e., R=V/I.
[0216] The capacitance of the sample substrate changed only
insignificantly following fusion, by several pF in 85 mM KCl, and
by 160 to 280 pF in 1-10 mM KCl.
Example 14
[0217] Vesicle Passage through Micrometer Pores
[0218] This example, illustrated in FIG. 13, describes the passage
of vesicles through apertures in the substrate.
[0219] The passage of vesicles through an aperture in the presence
of negatively charged surfaces, such as unmodified SiO.sub.2
layers, can be observed by monitoring changes in current (or,
equivalently, resistance) across the aperture. Specifically, the
passage of vesicles will lead to a decrease in current and an
associated increase in resistance. To monitor for artifacts in the
observed values, the polarity of the voltage is reversed, whereupon
no modulation of current or resistance is observed.
[0220] The duration of observed changes in the amplitude of
resistance can be correlated with size of the vesicles passing
through the aperture being monitored. For example, modulations in
the amplitude of resistance lasting up to 18 seconds suggest the
passage of very large vesicles with sufficiently fluid membranes.
Where vesicle populations with diameters greater than about 50
.mu.m (n=4) are used in conjunction with aperture openings with
diameters of about 7 .mu.m, an almost exclusive variation of the
time of passage for fixed changes in amplitude as a function of
vesicle size is observed. It is presumed that vesicles undergoing
passage through the aperture opening are drawn out during their
passage to form tubular structures with definite diameters and
closed surfaces.
[0221] By analyzing the typical time of passage for large vesicles
(d.sub.vesicle>>d.sub.aperture) and the typical change in the
amplitude of resistance for small vesicles
(d.sub.vesicle.about.d.sub.ape- rture), the composition of the
vesicles with respect to size can be determined for a given vesicle
solution. The method of the invention therefore possesses utility
for analyzing size distributions in selected populations of
vesicles and cells.
Example 15
[0222] Observation of Alamethicin Pores and Nicotinic Acetylcholine
Receptors
[0223] This example, illustrated in FIGS. 14 and 15AB, describes
observation of electrical activity of alamethicin pores and
nicotinic acetylcholine receptors (nAChR) in fused vesicles. These
observations confirm the biological utility of the invention.
[0224] FIG. 14 shows a plot of current versus time for membranes
containing alamethicin. In these experiments, a membrane is formed
over an aperture in 85 mM KCl. Next, alamethicin is added to the
measurement compartment, to a final concentration of 0.1
.mu.g/mL..sup.xvii Finally, a potential is applied, and a plot of
current versus time is generated. The amplitude and dwell times of
the current fluctuations observed in this plot, corresponding to
600 pS conductivities of the alamethicin pores, prove the
functionality and high sensitivity of the system.
[0225] FIG. 15AB shows an analogous plot of current versus time for
membranes containing the membrane protein nAChR. In these
experiments, a membrane is formed as above. Next, nAChR is
introduced into the membrane via Ca.sup.2+-mediated fusion.
Specifically, nAChR is purified from an appropriate source, and
then reconstituted into small unilamellar vesicles..sup.xviii Next,
these vesicles are added to the measurement compartment, and then
fused to the membrane by increasing the Ca.sup.2+ concentration of
the sample chamber to greater than about 1 mM. In given cases, the
fusion is supported by the subsequent temporary setup of an osmotic
gradient..sup.xix Finally, a potential is applied, and a plot of
current versus time is generated. In the absence of agonists,
typical receptor opening events are observed (FIG. 15A), whereas,
in the presence of agonists, such as carbamylcholine (20 .mu.M
final concentration), such receptor opening events are
substantially extinguished within a short time (t<100 seconds)
(FIG. 15B).
Example 16
[0226] Analysis of Cells
[0227] This example, illustrated in FIGS. 16-18AB, describes use of
the above-described systems in the study of cells.
[0228] The methods of the invention are generally applicable to the
investigation and analysis of cells. Such cells may be positioned
and electrically characterized using procedures substantially
analogous to those described above for vesicles. However, in some
cases, the positioning and measurement methods may be modified as
desired to accommodate differences in vesicles and cells,
including, among others, the cytoskeleton, the varied lipid and
protein content of cell membranes, and the cell wall in plants and
certain algae, bacteria, and fungi. For example, the cell may be
made more flexible by disrupting the cytoskeleton, for example,
using cytochalasin and/or colchicine. Similarly, in measurements
using plant cells, the cell wall may be removed to expose a
relatively smooth membrane surface capable of forming a tighter
electrical seal. Similarly, in measurements using animal cells
derived from tissues, the extracellular matrix may be removed or
digested, for example, using one or more proteases, lipases, and/or
glycosidases, among others.
[0229] The methods may be used for a variety of patch clamp
experiments, in a variety of formats or configurations. The cell,
as initially bound and sealed, is in a "cell-attached
configuration." If the membrane patch over the aperture then is
ruptured or destroyed, for example, by applying a pulse of voltage
or suction, electrical measurements can be performed over the
entire cell membrane in a "whole-cell configuration."
Alternatively, if the membrane patch over the aperture is
permeabilized, for example, by the addition of pore formers such as
amphotericin B or nystatin to the reference compartment, electrical
measurements again may be performed over the entire cell membrane
in a "perforated-patch configuration." Alternatively, if the cell
(instead of the membrane patch over the aperture) is lysed,
electrical properties of the patch may be measured in an
"inside-out configuration." In the lattermost approach, the
cytosolic side of the membrane is exposed to the measurement
solution, and the relatively small area of membrane being analyzed
potentially makes possible the study of individual channel
events.
[0230] FIGS. 16-18 show exemplary results of positioning and
voltage clamp experiments performed using Jurkat cells. These
cells, a human mature leukemic cell line, phenotypically resemble
resting human T lymphocytes and are widely used to study T cell
physiology. Similar results (not shown) were obtained using TE 671
cells and CHO cells.
[0231] The cells were cultured and prepared using standard cell
culture techniques. These cells were maintained for 2-5 days at
37.degree. C. in 5% CO2 in RPMI with Glutamax, supplemented with
10% FCS and P/S (100 U/100 .mu.g/mL). Before use, cells were
resuspended in a physiological buffer (PB=NaCl, 140 mM; HEPES, 10
mM; KCl, 5 mM; CaCl.sub.2, 2 mM; MgCl.sub.2, 1.2 mM; pH 7.3,
osmolarity 290 mOsm) at a density of 10.sup.7 cells/mL. Lower and
upper fluid compartments were filled with 20 .mu.L and 15 .mu.L of
PB, respectively. Five .mu.L of the cell suspension was added to
the upper compartment. Positioning was made at V.sub.m=-60 or -90
mV. All experiments were performed at room temperature.
[0232] FIG. 16 shows the time course of positioning, binding, and
subsequent development of a tight electrical seal for a Jurkat
cell. The cell was positioned at -60 mV. Seal formation occurred
about 15 seconds after cell addition, quickly rising to about 1000
G.OMEGA.. The aperture size was about 3 .mu.m, and the chip
resistance was about 250 k.OMEGA..
[0233] FIG. 17 shows a series of plots of current versus time
showing the current flowing through the membrane of a Jurkat cell
at the indicated positive and negative clamp voltages in a
cell-attached configuration. The curves appear quantized, switching
largely between just two values, one low and one high, particularly
at higher voltages. This characteristic suggests that
single-channel events are being observed, corresponding to the
opening and closing of the channel.
[0234] FIG. 18 shows an analysis of the current flowing through the
membrane of a Jurkat cell for a +60 mV clamp voltage. Panel A shows
a representative plot of current versus time. This plot again has a
quantized character, like FIG. 17. Panel B shows a histogram
plotting the relative occurrence of a given current versus the
current. The histogram is bimodal, with peak values of about 1.3 pA
(the relatively smaller peak at left) and about 4.8 pA (the
relatively larger peak at right), corresponding to an average of
about 3.5 pA.
Example 17
[0235] Miscellaneous Applications
[0236] The positioning and measurement systems provided by the
invention may be used for a variety of purposes and a variety of
assays. Exemplary miscellaneous applications are described
below.
[0237] Screening of Ingredients The system may be used to screen
libraries according to any suitable criterion, such as the
identification of candidate drugs, modulators, and the like.
Suitable libraries include compound libraries, combinatorial
chemistry libraries, gene libraries, phage libraries, and the like.
The system is exceptionally well suited to probing libraries whose
members are present only in small amounts, such as (1) the large
number of potential ligands that can be produced using
combinatorial chemistry, and (2) many receptor proteins, above all
ligand-controlled and G-protein-coupled receptors (GPCRs). Owing to
the process according to the invention, or alternatively the
measurement arrangement/measurement apparatus according to the
invention, it is possible to work with very few cells, either
directly or after previous isolation and reconstitution of the
receptor proteins in vesicles or lipid membranes. By the
uncomplicated arrangements of the sensor elements in arrays,
different substances or receptors can be selected simultaneously.
There is moreover the possibility of receptor cleaning and
reconstitution in lipid vesicles microchromatographically in
on-chip containers that optionally may be integrated into the
apparatus according to the invention.
[0238] Replacement of Conventional Patch-clamp Technologies
[0239] Conventional patch-clamp technologies form the foundation of
the investigation of the functionality of membrane receptors as
well as the modification of membrane characteristics as a response
to signal and metabolic processes in cells. If isolated cells of a
homogeneous cell population serve as the object of the
investigation, as is, for example, often the case in transformed
cells, the process according to the invention serves as an at least
comparable replacement for the patch-clamp technologies. As objects
of investigation for this process, for example, dissociated neurons
and cultivated mammalian cells as well as plant protoplasts are
suitable.
[0240] Portable Biosensors/Environmental Analytics
[0241] The automation and outstanding mechanical stability of the
measurement system according to the invention permits its use in
biosensors. By using suitable transformed cells, receptors
reconstituted in vesicles, or channel-forming proteins, sensors can
be set up that are sensitive to very different substrates or
metabolites. Moreover, if sufficiently tight electric seals are
formed, which is possible using the apparatus according to the
invention, then measurement sensitivity will in principle only be
dependent on the binding constant of the receptor. This sensitivity
may lie under one nanomole for G-protein-coupled receptors, and in
the nanomolar range for ionotropic receptors (e.g., 5 HT3, nAChR,
GABA.sub.AR, glycine R, and GluR)..sup.xx
[0242] The disclosure set forth above may encompass multiple
distinct inventions with independent utility. Although each of
these inventions has been disclosed in its preferred form(s), the
specific embodiments thereof as disclosed and illustrated herein
are not to be considered in a limiting sense, because numerous
variations are possible. The subject matter of the inventions
includes all novel and nonobvious combinations and subcombinations
of the various elements, features, functions, and/or properties
disclosed herein. The following claims particularly point out
certain combinations and subcombinations regarded as novel and
nonobvious. Inventions embodied in other combinations and
subcombinations of features, functions, elements, and/or properties
may be claimed in applications claiming priority from this or a
related application. Such claims, whether directed to a different
invention or to the same invention, and whether broader, narrower,
equal, or different in scope to the original claims, also are
regarded as included within the subject matter of the inventions of
the present disclosure.
[0243] .sup.i J. P. Changeux (1993), "Chemical Signaling in the
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[0244] .sup.ii J. Hodgson (1992), Bio/Technology 9:973.
[0245] .sup.iii J. Knowles (1997), "Medicines for the New
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[0246] .sup.iv O. P. Hamill, A. Marty, et al. (1981), "Improved
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[0247] .sup.v (Radler, J., H. Strey, et al. (1995), "Phenomenology
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[0248] .sup.vi For example, polycations, such as described by
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[0249] .sup.vii J. Edelstein, O. Schaad, J. -P. Changeux (1997),
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[0250] .sup.viii See Hamill, Marty, et al. (1981), loc. cit.; J. G.
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System. Sunderland, Ma., Sinauer Associates, Inc.
[0251] .sup.ix R. Horn, A. Marty (1988), "Muscarinic Activation of
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[0252] .sup.x Z. M. Pei, J. M. Ward, et al. (1996), "A Novel
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[0253] .sup.xi See, e.g., R. B. Gennis (1989), Biomembranes:
Molecular Structure and Function. New York, Springer Verlag.
[0254] .sup.xii H. H. Hub, U. Zimmerman, et al. (1982),
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[0255] .sup.xiii M. Criado and B. U. Keller (1987), "A Membrane
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[0256] .sup.xiv See Mazia, Schatten, et al. loc. cit., 1975.
[0257] .sup.xv M. L. Williamson, D. H. Atha, et al. (1989),
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[0258] .sup.xvi B. Sakmann and E. Neher (1983), Single-channel
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[0259] .sup.xvii R. B. Gennis (1989), Biomembranes: Molecular
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[0260] .sup.xviii T. Schurholz, J. Kehne, et al. (1992),
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by CHAPS Dialysis Depends on the Concentration of Salt, Lipid, and
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[0261] .sup.xix See Eray, Dogan, et al. (1995) loc. cit.
[0262] .sup.xx R. A. North (1994), Ligand- and Voltage-gated Ion
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Subtypes-Basic and Clinical Aspects, New York, John Wiley;
Peroutka, S. J. (1994), G-protein-coupled Receptors, CRC Press; E.
C. Conley, (1996), The Ion Channel Facts Book, Academic Press.
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