U.S. patent application number 11/371465 was filed with the patent office on 2010-05-13 for monitoring and manipulating cellular transmembrane potentials using nanostructures.
Invention is credited to Michael J. Ignatius, Elena Molokanova, Alex Savtchenko.
Application Number | 20100116664 11/371465 |
Document ID | / |
Family ID | 36954044 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100116664 |
Kind Code |
A1 |
Ignatius; Michael J. ; et
al. |
May 13, 2010 |
Monitoring and manipulating cellular transmembrane potentials using
nanostructures
Abstract
The use of nanostructures to monitor or modulate changes in
cellular membrane potentials is disclosed. Nanoparticles having
phospholipid coatings were found to display improved responses
relative to nanoparticles having other coatings that do not promote
localization or attraction to membranes.
Inventors: |
Ignatius; Michael J.;
(Eugene, OR) ; Molokanova; Elena; (Eugene, OR)
; Savtchenko; Alex; (Eugene, OR) |
Correspondence
Address: |
LIFE TECHNOLOGIES CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Family ID: |
36954044 |
Appl. No.: |
11/371465 |
Filed: |
March 8, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60659975 |
Mar 8, 2005 |
|
|
|
Current U.S.
Class: |
204/536 |
Current CPC
Class: |
G01N 33/6872 20130101;
B82Y 15/00 20130101; B82Y 5/00 20130101; G01N 33/588 20130101 |
Class at
Publication: |
204/536 |
International
Class: |
G01N 27/26 20060101
G01N027/26 |
Claims
1. A method for assaying changes in transmembrane potential, the
method comprising: providing at least one target, wherein the
target is a cell, cellular fraction, or artificial membrane
structure; contacting the target with at least one nanostructure to
form a treated target; stimulating the treated target; assaying
emission from the nanostructure; and correlating the emission with
the change in transmembrane potential.
2. The method of claim 1, wherein the contacting step comprises
introducing the nanostructure into the target.
3. The method of claim 1, wherein the contacting step comprises
introducing the nanostructure into a cellular membrane of the
target.
4. The method of claim 1, wherein the contacting step comprises
introducing the nanostructure onto or near a cellular membrane of
the target.
5. The method of claim 1, further comprising assaying emission from
the nanostructure after the contacting step but before the
stimulating step.
6. The method of claim 1, wherein the stimulating step comprises
electrical stimulation, magnetic stimulation, chemical stimulation,
biological stimulation, contacting the target with a drug suspected
of being able to activate ion channels, contacting the target with
a drug suspected of being able to inhibit ion channels, or
combinations thereof.
7. The method of claim 6, wherein the electrical stimulation
comprises use of a patch clamp, or application of an external
electric field.
8. The method of claim 6, wherein the chemical stimulation
comprises contacting the target with a potassium salt or a sodium
salt.
9. The method of claim 6, wherein the biological stimulation
comprises contacting the target with a light-sensitive ion
channel.
10. The method of claim 1, wherein the stimulating step comprises
maintaining the target at a first membrane potential voltage,
depolarizing or hyperpolarizing the target to a second membrane
potential voltage, and returning the target to the first membrane
potential voltage.
11. The method of claim 10, wherein the second membrane potential
voltage is more positive than the first membrane potential
voltage.
12. The method of claim 10, wherein the second membrane potential
voltage is positive, and the first membrane potential voltage is
negative.
13. The method of claim 10, wherein one of the first membrane
potential voltage and the second membrane potential voltage is
about 0 mV.
14. The method of claim 10, wherein the first membrane potential
voltage is about -70 mV and the second membrane potential voltage
is about +40 mV.
15. The method of claim 1, wherein the cell is a eucaryotic cell, a
procaryotic cell, bacterial cell, a Gram-positive bacterial cell, a
Gram-negative bacterial cell, a fungal cell, an insect cell, an
avian cell, a reptilian cell, an oocyte, a fly cell, a zebrafish
cell, a nematode cell, a fish cell, an amphibian cell, or a
mammalian cell.
16. The method of claim 1, wherein the cell is a eucaryotic
cell.
17. The method of claim 1, wherein the cellular fraction is a
nucleus, a ribosome, a mitochondria, an endoplasmic reticulum, a
Golgi apparatus, a vacuole, a synaptic vesicle, or a lysosome.
18. The method of claim 1, wherein the artificial membrane
structure is a phospholipid micelle.
19. The method of claim 1, wherein the nanostructure is a
nanocrystal, a film, a nanowire, a patterned substrate, or a
mesh.
20. The method of claim 1, wherein the nanostructure is a
semiconductor nanocrystal.
21. The method of claim 1, wherein the nanostructure is a
semiconductor core-shell nanocrystal.
22. A method for assaying changes in transmembrane potential, the
method comprising: providing at least one cell; contacting the cell
with at least one semiconductor nanocrystal to form a treated cell;
stimulating the treated cell with light; assaying emission from the
nanocrystal; and correlating the emission with the change in
transmembrane potential.
23. A method for the optical control of the transmembrane potential
of a target, the method comprising: providing at least one target,
wherein the target is a cell or cellular fraction; contacting the
target with at least one nanostructure under conditions suitable
for interaction or insertion of the nanostructure with a cellular
or subcellular membrane to prepare a treated target; delivering
energy to the treated target; and detecting response of the
target.
24. The method of claim 23, wherein the conditions suitable for
interaction or insertion comprise active uptake via endocytosis,
electroporation, liposome-mediated delivery, pluronic block
copolymer-mediated delivery, cell-penetrating peptide-mediated
uptake, protein-mediated uptake, microinjection, transfection,
viral delivery, optoporation, pore-forming substrates, membrane
intercalators, or combinations thereof.
25. The method of claim 23, wherein the delivering energy step
comprises illuminating at a wavelength or wavelength range suitable
for absorption by the nanostructure.
26. The method of claim 23, wherein the delivering energy step
comprises laser illumination, mercury lamp illumination, xenon lamp
illumination, halogen lamp illumination, or LED illumination.
27. The method of claim 23, wherein the detecting step comprises
use of a camera, a digital camera, a video camera, a CCD camera, a
digital camera mounted on a fluorescent microscope, a
photomultiplier, a fluorometer, a luminometer, a microscope, or the
human eye.
28. The method of claim 23, wherein the detecting step comprises
use of a secondary detection mechanism.
29. The method of claim 23, wherein the detecting step comprises
detection at a single time point, detection at multiple time
points, or continuous detection.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Patent Application Ser. No. 60/659,975 filed Mar. 8, 2005, the
contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to compositions and methods useful for
monitoring and manipulating cellular transmembrane voltages. In
particular, nanoparticles and their use in monitoring and
manipulating transmembrane voltages is disclosed.
DESCRIPTION OF RELATED ART
[0003] All cells have phospholipid membranes that serve as
bimolecular barriers and to separate cell contents from the
extracellular environment. The purpose of the plasma membrane is to
maintain the necessary difference in composition between two
compartments by restricting or permitting the passage of materials
through the membrane as a function of intracellular signaling.
[0004] Each cell has a resting membrane potential originating from
the so-called "separation of charge" across the normally
impermeable phospholipid bilayer. Because of unequal distribution
of positively and negatively charged ions in the extracellular and
intracellular compartments, all living cells have a negative
resting membrane potential, ranging from -5 mV to -100 mV. The ion
permeability of the plasma membrane is determined by the presence
of ion channels, transmembrane proteins specialized in passive ion
transport. Membrane potential can be changed by changing membrane
permeability to a certain ion in response to an activating
stimulus, thus allowing a flux of ions down their electrochemical
gradient. Transport of ions across the membrane through ion
channels will lead to disturbance of the existing equilibrium of
ion concentrations on both sides of membrane and, thus, to changes
of electrical properties of the cell.
[0005] Cells communicate with each other through changes in
membrane potential. Therefore, monitoring the cellular membrane
potential and its changes allows monitoring of cell viability and
function.
[0006] Ion channels are transmembrane proteins present in both
excitable and non-excitable cells. Ion channels permit and regulate
movement and conduction of ions down their electrochemical
gradients across a normally ion-impermeant lipid bilayer. They
produce electrical signals leading to action potential generation
that controls a number of key processes, including neuronal
signaling, heart beat, brain function, sensory transduction and
muscle contraction. In addition to setting the resting membrane
potential and controlling cellular excitability, these
transmembrane proteins play important roles affecting the
physiological state of cells by being involved in cell
proliferation, hormone secretion and homeostasis of water and
electrolytes.
[0007] Activation of ion channels by any mechanism results in
redistribution (changes in concentrations) of
intracellular/extracellular ions and consequent change in cellular
membrane potential. Thus, recording changes in membrane potential
allows direct monitoring of ion channel activity.
[0008] A typical organism has hundreds to thousands of different
types of ion channels, while an individual cell can have ten to
twenty different types. Each ion channel exhibits high selectivity
for one or a few ion species.
[0009] Different ion channel families are classified based on their
activation stimuli, selectivity to different ions, inactivation
mechanisms and pharmacological profiles. There are
voltage-activated and ligand-activated ion channels. Superfamilies
of voltage-gated sodium, potassium, calcium, and chloride ion
channels have been defined using electrophysiological,
pharmacological and molecular techniques; they are named according
to their selective permeability for a particular cation with
reference to their voltage dependence, kinetic behavior or
molecular identity. Superfamilies of ligand-gated channels are much
less structurally related and named after their activation ligand,
i.e. cyclic nucleotide-gated channels or GABA channels.
[0010] Functionally, the opening or closing of ion channels can be
controlled or "gated" by the binding of signaling molecules
(ligand-gated channels), by a change in the membrane potential
(voltage-gated channels), or by mechanical stimulation
(mechanosensitive channels) that results in conformational changes
within ion channel structures leading to opening of a pore and
allowing a flux of ions inside or outside the cell.
[0011] For example, a voltage-gated sodium channel is closed at a
resting membrane potential below -60 mV, and opens upon
depolarization of the membrane (i.e., a shift in the membrane
potential to a less negative value).
[0012] The structures of voltage-gated sodium, calcium and
potassium channels have common functional elements. All ion
channels are transmembrane proteins comprised of several homologous
repeats arranged around a common ion-selective aqueous pore that
opens in response to an activating stimulus that allows ions to
enter or exit the cell. Each repeat consists of six transmembrane
domains (S1-S6) with the S4 domain playing the specialized role of
voltage sensor. Channel opening and closing (`gating`) is
controlled by this voltage sensitive domain of the protein
containing charged amino acids that respond to changes in the
electric field. Translocation of a voltage sensitive domain leads
to conformational changes in the structure of the channel resulting
in conducting (open/activated) or non-conducting
(closed/inactivated) states.
[0013] Although ligand-gated channels differ significantly from one
another, there are two structural elements present in every
channel: a ligand-binding domain and a pore domain. Binding of a
specific ligand triggers conformational changes leading to opening
of the pore domain and allowing the ion flux into/out of the cell,
which is reflected in a change in transmembrane potential.
[0014] The various states of ion channel activation provide unique
opportunities for more efficient drug discovery, enabling
state-dependent molecules to be developed that, for example, only
bind to non-conducting (inactivated) channels. A desirable goal is
to target drugs to tissues exhibiting abnormal electrical activity,
while leaving normal channels in active tissues unaffected. Also,
identifying new ion channels, testing their functions, and
validating them as drug targets are current efforts of many biotech
companies and academic researchers.
[0015] Ion Channels in Drug Discovery
[0016] Ion channels are of particular importance in the
pharmaceutical industry in two areas: ion channels as drug targets
and ion channel safety pharmacology. Ion channels are significant
targets in the drug discovery process, generating several billion
dollars in sales per annum. Abnormal ion channel function or ion
channel expressions have been linked to a number of therapeutic
areas (i.e. cardiac arrhythmia, hypertension, epilepsy, pain). Ion
channel modulator drugs for these have yet to be developed. Many
pharmaceutical companies have active ion channel drug development
projects or programs. Additionally, a number of biotech or
biopharmaceutical companies focus exclusively on ion channel drug
development (ChanTest, Cleveland, Ohio; BioFocus, Cambridge, UK;
Icagen, Durham, N.C.).
[0017] Ion channels are involved in many vital functions, and any
dysfunction of ion channels caused by changes in biochemical
regulation, expression levels, or structural mutations can impact
the well-being of living organisms. In humans, inherited or induced
changes in ion channel function could result in serious
complications to health. Several disease states are related to
dysfunctional ion channels. Ion channel defects produce a
clinically diverse set of disorders that vary from cystic fibrosis
and some forms of migraine to renal tubular defects and episodic
ataxias.
[0018] In particular, ion channels have been implicated in cardiac
arrhythmias, familial periodic paralyses, cystic fibrosis,
epilepsy, diabetes, asthma, angina pectoris, malignant
hyperthermia, pain, hypertension, epilepsy, etc. Ion channels
represent key molecular targets for drug discovery. Pharmaceutical
and biotechnology companies have successfully targeted ion channels
in their bid to make new more effective drugs. Now various ion
channel blockers or openers are being used and evaluated as
therapeutic drugs for a variety of diseases.
[0019] Voltage-gated calcium ion channels are involved in numerous
cellular functions, and their role in generating a defined disease
phenotype is complex. Certain types of calcium-channels may play a
role in nociception and migraine pathophysiology. In human
medicine, calcium-channel blockers are being evaluated for, among
other things, treating glaucoma, deep vein thrombosis, and
pulmonary hypertension, in renal transplantation, and for
prevention of perfusion injury.
[0020] Several voltage-dependent calcium channels blockers have
been shown to be effective in inhibiting pain. Furthermore,
blockage of so-called non-L-type calcium channels was found to
exert therapeutic effects in the treatment of severe pain and
ischemic stroke.
[0021] Dysfunction of potassium channels has been associated with
the pathophysiology of a number of neurological, as well as
peripheral, disorders (e.g., episodic ataxia, epilepsy,
neuromyotonia, Parkinson's disease, congenital deafness, long QT
syndrome).
[0022] Activation of potassium ion channels generally reduces
cellular excitability, making potassium-channel openers potential
drug candidates for the treatment of diseases related to
hyperexcitability such as epilepsy, neuropathic pain, and
neurodegeneration.
[0023] Most notably, mutations of the HERG potassium ion channels
expressed in cardiac tissues or pharmacological blockage of HERG
channels cause heart disease (long Q-T syndrome), which leads to
increased risk of ventricular tachycardia and sudden death. Several
drugs affect these channels and can lead to life threatening
cardiac arrhythmias. In this perspective, drug discovery companies
usually find it necessary to evaluate each of their drug candidates
for interference with these channels. Thus, many companies conduct
HERG testing before any further investigation is carried out.
[0024] The dynamic nature of sodium ion channel expression makes
them important targets for pharmacological manipulation in the
search for new therapies for pain. For example, mutations in the
gene encoding the alpha subunit of sodium-channels have been linked
to paroxysmal disorders such as epilepsy, long QT syndrome,
hyperkalemic periodic paralysis in humans and to motor endplate
disease and cerebellar ataxia in mice. Voltage-gated sodium ion
channel have been shown to be key mediators of the pathophysiology
of pain. One of the most frequently used anesthetic drugs used is
Lidocain, which inhibits sodium ion channels. Changes in brain
sodium-channels may be a cause of central pain, and further,
abnormal expression of sodium-channel genes and its contributions
to hyperexcitability of primary sensory neurons have been
discussed. Recently, sensory-neuron-specific (SNS) TTX-resistant
sodium-channels have been examined for their role in nociception
and pain. This study suggests that blockage of SNS expression or
function may produce analgesia.
[0025] Experimental Approaches for Ion Channel Research
[0026] The preferred method for studying ion channels is the patch
clamp method (Neher, E. and Sakmann, B., Nature 260(5554): 799-802
(1976); Hamill, O. P., et al., Pflugers Arch. 391(2): 85-100
(1981)).
[0027] This technique consists of contacting a cell with the tip of
a very clean glass micropipette (diameter of about 1 .mu.m), and
obtaining a high resistance seal (leakage resistance >1 GOhm,
GigaSeal) between the glass and the cell surface by applying gentle
suction. Next, by applying greater suction or a large voltage, it
is possible to break the intra-pipette portion of membrane and
thereby make direct electrical contact between the cell interior
and the pipette electrode (whole-cell configuration of patch clamp
method). Different voltages can then be applied to the pipette
electrode, and the currents measured represent the current through
the cell membrane, which includes the integral current through the
ion channels present.
[0028] To date, the patch-clamp method has been considered the
industry gold standard for monitoring ion channel activity. The
patch-clamp directly records ion channel activity, has
sub-millisecond temporal resolution, very high information content
and is extremely sensitive--including the ability to study "single"
ion channels. Due to its high information content,
patch-clamp-based screening has very low rate of "false negatives"
and "false positives".
[0029] Although this technique allows detailed biophysical
characterization of ion channel activation, inactivation, gating,
ion selectivity, and drug interactions, throughput is quite low and
ease-of-use of patch-clamp instrumentation is generally
unsatisfactory for effective mass screening. The demands of ion
channel high throughput screening ("HTS") include robust
instrumentation and high signal-background ratio combined with
satisfactory ease-of-use. Historically, ion channel HTS is equated
with low information content, emphasizing the need for novel rapid
and easy methods in which more useful information can be gathered
about membrane potential changes in various cell types.
[0030] Ion Channel HTS Approaches
[0031] Traditionally, HTS technologies employed for ion channel
primary screening rely on binding assays, ion-flux assays and
fluorometric approaches. Until now the most significant task for
these methods has been to generate enough HTS data with acceptable
information content and reliability.
[0032] The search for molecules that modulate ion channel function
has been hindered by the lack of direct electrical measurements in
HTS formats. Membrane excitability in cell-based assays is a
dynamic phenomenon that requires fast, precise and accurate
measurements to gather high information content data. Real-time
measurements of transmembrane potential kinetics that accurately
reflect ion channel activity are fundamental to cell physiology,
but are difficult to measure in existing HTS format methods.
[0033] Reliable and robust high HTS assays for ion channels are
important in ion-channel based drug discovery. Ion channels are
dynamic proteins, and therefore require assays that "sense" their
various functional states. Competition-binding assays, although
successfully used for other target classes, often fail to identify
ligands that modulate specific ion channel states. Cell-based
functional assays, therefore, are preferred for HTS of ion channel
targets.
[0034] Currently Available Assays
[0035] Modern technologies employed for ion channel screening
include: binding assays, ion flux assays, fluorometric imaging and
electrophysiology.
[0036] Binding assays for cell surface receptors are used in screen
development and primary screening. This type of assay is frequently
carried out using scintillation proximity assay (SPA) or
fluorescence detection techniques, which have replaced the older
radiolabelled ligands and filtration assays. The SPA technique
relies upon excitation of a scintillant microbead upon binding of a
radiolabelled ligand to a receptor immobilized on the surface of
the bead.
[0037] Fluorescence spectrometry is used to measure the binding
equilibrium between a fluorophore-labeled ligand and receptor.
Unfortunately binding assays only detect binding of compounds to
ion channels and do not reveal changes in target function, such as
modulation of ion channel kinetics.
[0038] Optical readouts of ion channel function are favorable for
HTS because they are versatile, amenable to miniaturization and
automation and potentially sensitive.
[0039] Fluorescence readouts are used widely both to monitor
intracellular ion concentrations and to measure membrane
potentials. For example, large transient increases in intracellular
calcium concentration through activation of ion channels can be
monitored using fluorescent probes such as Fluo-3 and Calcium
Green. In addition to ion-selective fluorescent indicators, there
are several fluorescent dyes that are sensitive to changes in
membrane potential, including styryl, bisoxonol, and fluorescence
resonance energy transfer-based voltage-sensitive dyes.
[0040] For example, the fluorescent dye bis-(1,3-dibutylbarbituric
acid) trimethine oxonol, or DiBAC4(3), has been the reagent of
choice for measuring membrane potential in HTS formats.
Redistribution of the dye in the cellular membrane as a result of
depolarizing or hyperpolarizing stimuli in cells causes changes in
fluorescence. However, utilization of DiBAC4(3) has several
limitations, including slow kinetics (in the seconds to minutes
range) and fluctuations in response to changes in temperature and
concentration of the dye. In addition, screening experiments using
bisoxonol dyes require multi-step procedures and take 30-60 min for
dye loading, potentially compromising the fidelity and reducing
throughput of DiBAC4(3)-based screening assays.
[0041] HTS Patch-Clamp
[0042] The patch clamp technique is widely used to study currents
through ion channels. The whole-cell patch-clamp is used today in
tertiary screening of selected lead molecules in late stages of the
drug discovery process. Whole-cell patch-clamp, however, is not
suitable for initial high throughput screening.
[0043] Although very powerful, this technique is labor-intensive
and, therefore, limited to few data point measurements per day.
This low throughput has encouraged the use of other less specific
and less sensitive technologies for high-throughput screening of
ion channel targets.
[0044] Optimal HTS Ion Channel Assay Requirements
[0045] In high-throughput screening campaigns (200,000+ samples),
binding assays remain the first choice in terms of throughput and
cost. This reflects the technical ease of performing these types of
assays and, hence, their ability to be automated. However, in
modern ion channel drug discovery screening, there is a trend
toward use of cellular-based functional assays as primary screening
tools.
[0046] Cellular functional assays are used as primary or secondary
assays to determine functionality of compounds from a binding
screen and also to assess toxicity. These types of assays are
information rich and therefore potentially of significant value in
drug discovery.
[0047] Identifying targets and putative drug candidates by
obtaining as much knowledge as possible per experiment about the
effects of each compound is the ultimate goal for initial ion
channel screening.
[0048] Voltage Sensitive Probes
[0049] Currently used fluorescent voltage sensor dyes, which
respond to potential-dependent accumulation and redistribution
across the cellular membrane, are limited to steady-state assays of
membrane potential. This is because the fluorescence response of
these dyes occurs minutes after the change in membrane potential.
Since voltage sensor dyes are charged they also interfere with the
membrane potential caused by the ionic current; to reduce this
signal-to-noise effect the dye concentration has to be kept below a
certain level. Thus, redistribution-based voltage sensor dyes are
prone to false-negatives. In addition, compound-voltage dye
interactions can show high false-positive rates.
[0050] Voltage-sensing Fluorescence Resonance Energy Transfer
(FRET) acceptors, for example coumarin-tagged phospholipids
integrated into the cell membrane ameliorate many of the problems
associated with standard voltage sensors, allowing sub-second
kinetic determination. Using high throughput screening FRET-based
voltage sensors a throughput of several 96-well plates per hour can
be performed with the Voltage Ion Probe Reader (VIPR.TM.), a
product developed by Aurora BioSciences (now Vertex
Pharmaceuticals, Inc.; Cambridge, Mass.).
[0051] Compared with results obtained with traditional patch-clamp
method, VIPR assays are less sensitive. The temporal resolution in
fluorescence-based ion-channel assays using voltage-sensor dyes
reduces the accessible kinetic range relative to patch-clamp-based
ion channel assays.
[0052] Ion-specific fluorescent probes for intracellular ions have
been shown to be useful for ion channel screening. Depending on the
application, a number of different dyes are available with
different ranges of affinities, of which fluorescent calcium
indicators are the most commonly used. A significant disadvantage
of calcium dye-based ion-channel assays is their slow kinetic
resolution of changes of intracellular calcium concentration, due
to uncontrolled or unpredictable cellular processes. This can
interfere with assay results. To achieve high throughput and low
noise, FLIPR-type fluorescent readers are commonly used in
conjunction with calcium-specific dyes. So far only assays
involving measurements of calcium channel activity or other
non-selective cation channels have proven to be robust enough for
effective HTS efforts.
[0053] In summary, an optimal HTS ion channel screening method
would have high temporal resolution, high sensitivity and high
information content, resulting in low rates of "false negatives"
and "false positives". Despite the materials and methods available
to study ion channels, there exists a need for new materials and
methods that are easy, robust, and useful.
[0054] Nanoparticles or Nanocrystals
[0055] Numerous studies have been published describing
nanoparticles and methods for their use. Semiconductor nanocrystals
are sometimes referred to as "Quantum Dots" or "QDots", although
these are registered trademarks of Quantum Dot Corporation (a
wholly owned subsidiary of Invitrogen Corp.; Carlsbad, Calif.).
Nanoparticles are typically spherical or nearly so, having a
central core, a surrounding shell, and optional capping groups,
linkers, and other surface-conjugated materials.
[0056] Semiconductor nanoparticles are nanometer-scale crystals
composed of hundreds to thousands of atoms of an inorganic
semiconductor material in which electron-hole pairs can be created
and confined.
[0057] Specific optical properties of nanoparticles are based on
the mechanism of quantum confinement. Quantum confinement is the
trapping of electrons or electron "holes" (charge carriers) in a
space small enough that their quantum (wave-like) behavior
dominates their classical (particle-like) behavior.
[0058] In nanoparticles, where motions of electrons/holes are
highly limited in three dimensional space, quantum confinement
results in a strong increase of optical excitation energies
compared to the bulk semiconductor material. For quantum
confinement to occur, the dimension of the confining device or
particle must be comparable to the electron-hole Bohr radius of the
material it is made from. After electron-hole pairs in the core of
a nanocrystal are formed upon excitation with light, they can
recombine and re-emit light having a narrow and symmetric emission
spectrum that depends directly on the size of the crystal. The
smaller the nanoparticle core, the bigger the bandgap between the
valence and conduction bands, the bluer the emitted photon; and
vice versa (redder emission) for larger nanoparticles.
[0059] Commercially available semiconductor nanocrystals are
comprised of several layers, including a core, an inorganic
lattice-matching crystalline shell (to improve the nanocrystal's
optical properties and possibly serving to minimize cytotoxicity),
and a coating or coatings (to allow water compatibility and for
effective interaction with modifiers such as
biomacromolecules).
[0060] Nanocrystals are used in information technology (the quantum
computer), light emitting diodes, lasers, and telecommunication
devices, bar coding, photodetectors, optical switching, and
thermoelectric devices. Recently, nanocrystals have been used for
cell labeling, cell tracking, in vivo imaging, DNA detection,
protein labeling, and in other detection modes.
[0061] Nanocrystals have excellent optical properties as biological
optical sensors, including size-tunable emission, narrow spectral
width, broad excitation spectrum, high quantum yields, high
two-photon cross-section, and low photobleaching rates.
[0062] However, until now biological and biotechnological
applications of nanocrystals have been mostly limited to their use
as biomarkers rather than as detectors of biological processes.
[0063] Applications of nanocrystals in industry include, for
example, nanocrystal-based electro-luminescent devices capable of
emitting light of various wavelengths in response to external
stimuli, where variations in applied voltage could result in change
of color of the light emitted by the device.
[0064] Many patents and patent publications report nanocrystal
compositions, methods for their preparation, and methods for their
use. The following collection is a sampling of the research done to
date.
[0065] U.S. Pat. No. 5,505,928 (issued Apr. 9, 1996) describes
methods of preparing III-V semiconductor nanocrystal materials.
Examples of such materials include GaAs, GaP, GaAs--P, GaSb, InAs,
InP, InSb, AlAs, AlP, and AlSb. The produced materials can be 1-6
nm in size, and are relatively monodisperse.
[0066] U.S. Pat. No. 5,990,479 (issued Nov. 23, 1999) describes
nanocrystals linked to affinity molecules. Listed affinity
molecules include monoclonal and polyclonal antibodies, nucleic
acids, proteins, polysaccharides, and small molecules such as
sugars, peptides, drugs, and ligands.
[0067] U.S. Pat. No. 6,114,038 (issued Sep. 5, 2000) describes
water soluble, functionalized nanocrystals having a capping
compound of the formula HS(CH.sub.2).sub.nX, wherein X is a
carboxylate. The nanocrystals also have a diaminocarboxylic acid
which is operably linked to the capping compound.
[0068] U.S. Pat. No. 6,207,229 (issued Mar. 27, 2001) describes a
coated nanocrystal capable of light emission includes a
substantially monodisperse nanoparticle selected from the group
consisting of CdX, where X=S, Se, or Te; and an overcoating of ZnY,
where Y=S, or Se. Methods of preparing the nanocrystals using a
first semiconductor core and a precursor capable of thermal
conversion into a second semiconductor material that forms a
coating layer over the core.
[0069] U.S. Pat. No. 6,207,392 (issued Mar. 27, 2001) describes
semiconductor nanocrystals having one or more attached linking
agents. The nanocrystals can include nanocrystals of Group II-VI
semiconductors such as MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe,
SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe,
and HgTe as well as mixed compositions thereof; as well as
nanocrystals of Group III-V semiconductors such as GaAs, InGaAs,
InP, and InAs.
[0070] U.S. Pat. Nos. 6,251,303 (issued Jun. 26, 2001), 6,319,426
(issued Nov. 20, 2001) and 6,444,143 (issued Sep. 3, 2002) describe
a water-soluble semiconductor nanocrystal. The outer layer of the
nanocrystal contains a molecule having at least one linking group
for attachment of the molecule to the overcoating shell layer, and
at least one hydrophilic group optionally spaced apart from the
linking group by a hydrophobic region sufficient to prevent
electron charge transfer across the hydrophobic region.
[0071] U.S. Pat. No. 6,274,323 (issued Aug. 14, 2001) describes a
method of detecting a polynucleotide in a sample, using a
semiconductor nanocrystal in an immunosorbent assay.
[0072] U.S. Pat. No. 6,306,610 (issued Oct. 23, 2001) describes
semiconductor nanocrystals having attached multidentate ligands.
The nanocrystals can be associated with various biological
molecules such as proteins and nucleic acids.
[0073] U.S. Pat. No. 6,322,901 (issued Nov. 27, 2001) describes
monodisperse coated nanocrystals that emit light in a spectral
range of no greater than about 60 nm full width at half max (FWHM).
The spectral range of the nanocrystals is about 470 nm to about 620
nm, and the particle size of the nanocrystal core is about 20
angstroms to about 125 angstroms.
[0074] U.S. Pat. No. 6,326,144 (issued Dec. 4, 2001) describes
semiconductor nanocrystals linked to various compounds using a
linker of structure H.sub.zX((CH.sub.2).sub.nCO.sub.2H).sub.y and
salts thereof, where X is S, N, P or O.dbd.P; n is greater than or
equal to 6; and z and y are selected to satisfy the valence
requirements of X.
[0075] U.S. Pat. Nos. 6,423,551 (issued Jul. 23, 2002) and
6,699,723 (issued Mar. 2, 2004) describe a water soluble
semiconductor nanocrystal having a linking agent capable of linking
to an affinity molecule. A list of affinity molecules includes
monoclonal and polyclonal antibodies, nucleic acids (both monomeric
and oligomeric), proteins, polysaccharides, and small molecules
such as sugars, peptides, drugs, and ligands. Examples of linking
agents include N-(3-aminopropyl)3-mercapto-benzamide,
3-aminopropyl-trimethoxysilane, 3-mercaptopropyl-trimethoxysilane,
3-maleimidopropyl-trimethoxysilane, and
3-hydrazidopropyl-trimethoxysilane.
[0076] U.S. Pat. No. 6,426,513 (issued Jul. 30, 2002) describes a
water-soluble semiconductor nanocrystal comprising a quantum dot
having a selected band gap energy; an overcoating layer comprising
a material having a band gap energy greater than the band gap
energy of the quantum dot; and an outer layer comprising a compound
having a formula, SH(CH.sub.2).sub.nX, where X is carboxylate or
sulfonate, and n is greater than or equal to 8.
[0077] U.S. Pat. No. 6,500,622 (issued Dec. 31, 2002) describes
semiconductor nanocrystals having attached polynucleotide
sequences. The nanocrystals can be used to determine the presence
or absence of a target sequence in a sample. The nanocrystal can be
identified using a spectral code.
[0078] U.S. Pat. No. 6,548,168 (issued Apr. 15, 2003) describes a
method of stabilizing particles with an insulating, semiconducting
and/or metallic coating. A particle-coating admixture containing a
bifunctional ligand is used to bind a particle to the coating.
Examples of bifunctional ligands include 3-mercaptopropyl
trimethoxysilane ("MPS"), 1,3-propanedithiol, 3-aminopropanethiol
("APT"), and 3-amino propyl trimethoxysilane ("APS").
[0079] U.S. Pat. No. 6,576,291 (issued Jun. 10, 2003) describes a
method of manufacturing a nanocrystallite, the method comprising
contacting a metal, M, or an M-containing salt, and a reducing
agent to form an M-containing precursor, M being Cd, Zn, Mg, Hg,
Al, Ga, In, or Tl; contacting the M-containing precursor with an X
donor, X being O, S, Se, Te, N, P, As, or Sb to form a mixture; and
heating the mixture in the presence of an amine to form the
nanocrystallite. The nanocrystallites can be used in a variety of
applications including optoelectronic devices including
electroluminescent devices such as light emitting diodes (LEDs) or
alternating current thin film electroluminescent devices
(ACTFELDs).
[0080] U.S. Pat. No. 6,649,138 (issued Nov. 18, 2003) describes a
water-dispersible nanoparticle comprising: an inner core comprised
of a semiconductive or metallic material; a water-insoluble organic
coating surrounding the inner core; and, surrounding the
water-insoluble organic coating, an outer layer comprised of a
multiply amphipathic dispersant molecule, wherein the dispersant
molecule comprises at least two hydrophobic regions and at least
two hydrophilic regions. The nanoparticles can be conjugated to
various affinity molecules, allowing use in applications such as
fluorescence immunocytochemistry, fluorescence microscopy, DNA
sequence analysis, fluorescence in situ hybridization (FISH),
fluorescence resonance energy transfer (FRET), flow cytometry
(Fluorescence Activated Cell Sorter; FACS) and diagnostic assays
for biological systems.
[0081] U.S. Pat. No. 6,815,064 (issued Nov. 9, 2004) describes a
nanoparticle containing a Group 2 element, a Group 12 element, a
Group 13 element, a Group 14 element, a Group 15 element, a Group
16 element, Fe, Nb, Cr, Mn, Co, Cu, or Ni in an inorganic shell
around the semiconductor core. The compositions and methods of
preparation are proposed to facilitate the overgrowth of a
high-quality, thick shell on a semiconductive core by compensating
for the mismatching of lattice structures between the core and
shell materials.
[0082] Despite the materials and methods available to study ion
channels, there exists a need for new materials and methods that
are easy, robust, and useful. Additionally, there is a need for
methods of controlling the membrane potential of cells to
facilitate studying the effects of administered materials.
SUMMARY OF THE INVENTION
[0083] The use of nanostructures to measure or modulate changes in
cellular or subcellular membrane potentials is disclosed.
Nanostructures associated with cells respond to changes in membrane
potential, and can be easily monitored. The methods can be used to
monitor the effects of added external agents on cellular membrane
potential.
DESCRIPTION OF THE FIGURES
[0084] The following figures form part of the present specification
and are included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to one or more of these figures in combination with the
detailed description of specific embodiments presented herein.
[0085] FIG. 1 shows the effect of electrophysiological stimulation
on cells containing intracellular nanoparticles. The upper trace
shows the change in fluorescence intensity of semiconductor
nanocrystals inside the cell in response to change in transmembrane
potential using the patch-clamp method. The x-axis is time in
seconds; the y-axis is Arbitrary Units. Semiconductor nanocrystals
were loaded inside cell through patch pipette. The lower trace
represents the corresponding voltage stimulation protocol along the
same time scale.
[0086] FIG. 2 shows the effect of electrophysiological stimulation
on cells containing intracellular lipid-modified nanoparticles. The
upper traces show the change in fluorescence intensity of
phospholipid-functionalized semiconductor nanocrystals in response
to change in transmembrane potential using the patch-clamp method
from three areas of interest (2 cells and background). Cell #1 was
exposed to voltage stimulation, while cell #2 was not exposed to
voltage stimulation. Semiconductor nanocrystals were applied to the
extracellular side of the cellular membrane. The lower trace
represents the corresponding voltage stimulation protocol along the
same time scale.
[0087] FIG. 3 shows the results of monitoring transient changes in
cells caused by addition of a high concentration of potassium
chloride (100 mM). The circle, diamond, triangle, and square
symbols represent different regions of interest (ROI).
DETAILED DESCRIPTION OF THE INVENTION
[0088] While compositions and methods are described in terms of
"comprising" various components or steps (interpreted as meaning
"including, but not limited to"), the compositions and methods can
also "consist essentially of" or "consist of" the various
components and steps, such terminology should be interpreted as
defining essentially closed-member groups.
[0089] Methods of Assaying Changes in Transmembrane Potential
[0090] One embodiment of the invention is directed towards methods
for assaying a change in transmembrane potential. The methods can
comprise providing at least one target, wherein the target is a
cell, cellular fraction, or artificial membrane structure;
contacting the target with at least one nanostructure to form a
treated target; stimulating the treated target; assaying emission
from the nanostructure; and correlating the emission with the
change in transmembrane potential. An optional additional step can
comprise assaying emission from the nanostructure after the
contacting step but before the stimulating step. This additional
step can act as a "control" or "blank" measurement.
[0091] The target can be one or more intact cells, can be one or
more cellular fractions, or one or more artificial membrane
structures. Examples of cellular fractions include any luminal
organelles such as nucleus, ribosomes, mitochondria, endoplasmic
reticulum, Golgi apparatus, vacuoles, synaptic vesicles and
lysosomes. Examples of the artificial membrane structures include
phospholipid micelles, micro- and nanocapsules and semi-liquid
films on supportive structures. The contacting step can comprise
introducing the nanostructure into the target. Alternatively, the
contacting step can comprise introducing the nanostructure into a
cellular membrane of the target. The nanostructure can
alternatively be introduced onto or near a cellular membrane of the
target. Nanostructures "near" the target are sufficiently close in
proximity so as to be able to detect changes in transmembrane
potential. As an example, nanostructures closer than about 100
microns are sufficiently near a target so as to have this
property.
[0092] The target can be stimulated by a wide variety of methods.
Examples of such stimulation methods include electrical
stimulation, magnetic stimulation, chemical stimulation, biological
stimulation, or combinations thereof. Examples of electrical
stimulation include the use of a patch clamp, and application of an
external electric field. Examples of chemical stimulation include
contacting the target with a potassium salt or a sodium salt, or
with different types of intramembrane pore-forming molecules.
Examples of biological stimulation include activating the target
with a light-sensitive ion channel, or contacting the target with
the chemical entities, acting as modifiers of ion channel activity.
Examples of magnetic stimulation include activating the target with
alternating electromagnetic field of the appropriate frequency and
amplitude.
[0093] Targets can be electrically stimulated by a variety of
methods. One stimulation protocol (voltage amplitudes and duration
of stimulation) is often chosen based on activation kinetics of the
ion channel of interest. For example, targets can be maintained at
a first membrane potential voltage, subjected to a depolarizing
pulse at a second membrane potential voltage, and returned to the
first membrane potential voltage. The second membrane potential
voltage is typically more positive than the first membrane
potential voltage, but it is possible that the first membrane
potential voltage is more positive than the second membrane
potential voltage. For example, the first membrane potential
voltage can be negative, while the second membrane potential
voltage can be positive. An example is -70 mV for the first
membrane potential voltage, and +40 mV for the second membrane
potential voltage. Alternatively, the first or second membrane
potential voltage can be 0 mV. Examples include -200 mV for the
first membrane potential voltage, and 0 mV for the second membrane
potential voltage. An additional example is 0 mV for the first
membrane potential voltage, and 200 mV for the second membrane
potential voltage. Specific examples of first membrane potential
voltages and second membrane potential voltages can be
independently selected from about -200 mV, about -180 mV, about
-160 mV, about -140 mV, about -120 mV, about -100 mV, about -80 mV,
about -60 mV, about -40 mV, about -20 mV, about 0 mV, about 20 mV,
about 40 mV, about 60 mV, about 80 mV, about 100 mV, about 120 mV,
about 140 mV, about 160 mV, about 180 mV, about 200 mV, and ranges
between any two of these values.
[0094] Alternatively, more complicated voltage patterns can be used
in the methods. The methods can further comprise exposing the
targets to at least one step voltage prior to subjecting them to
the depolarizing pulse at a second membrane potential voltage. The
step voltage is an intermediate voltage between the first membrane
potential voltage and the second membrane potential voltage. The
step voltage can be used to measure leak subtraction. For example,
a first membrane potential voltage of -80 mV, a step voltage of -50
mV, and a second membrane potential voltage of 20 mV can be
used.
[0095] The depolarizing pulse can generally be applied for any
length of time. For example, the depolarizing pulse can be applied
for up to about 5000 seconds. Examples of the length of time
include about 10 microseconds, about 1 milliseconds, about 10
milliseconds, about 100 milliseconds, about 1 second, about 2
seconds, about 3 seconds, about 4 seconds, about 5 seconds, about
10 seconds, about 20 seconds, about 30 seconds, about 40 seconds,
about 50 seconds, about 60 seconds, about 70 seconds, about 80
seconds, about 90 seconds, about 100 seconds, about 500 seconds,
about 1000 seconds, about 2000 seconds, about 3000 seconds, about
4000 seconds, about 5000 seconds, and ranges between any two of
these values.
[0096] The one or more cells can generally be any type of cells
which have a membrane and membrane potential. For example, the
cells can be bacterial (Gram-positive or Gram-negative),
eucaryotic, procaryotic, fungal, insect, avian, reptilian, oocyte,
fly, zebrafish, nematode, fish, amphibian, or mammalian cells. The
methods can also be used on non-cell materials such as artificial
membranes, liposomes, and phospholipid bilayers. Examples of
primary mammalian cells include human, mouse, rat, dog, cat, bear,
moose, cow, horse, pig, or Chinese hamster ovary ("CHO") cells.
Other examples of types of cells include immune system cells (e.g.,
B-cells, T-cells), oocytes, red blood cells, white blood cells,
neurons, epithelial, glia, fibroblast, cancer cells, and
immortalized cells.
[0097] The nanostructures can be introduced into the target by a
number of methods. Examples of such methods include use of a patch
pipette, passive or active uptake via endocytosis or other uptake
mechanisms, electroporation, liposome-mediated delivery, pluronic
block copolymer-mediated delivery, cell-penetrating
peptide-mediated uptake, protein-mediated uptake, microinjection,
transfection, viral delivery, optoporation, pore-forming
substrates, membrane intercalators, or combinations thereof.
[0098] Methods of nanostructures loading into the cellular membrane
(or other kinds of membranes mentioned above) include the
immobilization of the nanostructures onto the supportive structures
(for example, onto the bottom of a well in the microtiter plate)
and subsequent addition of solution containing cells to an
experimental chamber (such as a microtiter plate well).
[0099] The nanostructures can generally be any nanostructures.
Examples of nanostructures include a nanocrystal, a film, a
nanowire, a patterned substrate, and a mesh. Nanoparticles can
generally be any nanoparticles. Semiconductor nanoparticles or
nanocrystals typically have a semiconductor core, a shell, and
optionally, one or more surface treatments. Semiconductor
nanoparticles are commercially available from companies such as
Quantum Dot Corp. (a wholly owned subsidiary of Invitrogen Corp.;
Carlsbad, Calif.) and Evident Technologies (Troy, N.Y.). There also
exist many published descriptions of the preparation of
nanoparticles.
[0100] The semiconductor core and shell can independently be made
of a material of an element from Group 2 or 12 of the Periodic
Table of the Elements, and an element selected from Group 16 of the
Periodic Table of the Elements. Examples of such materials include
ZnS, ZnSe, ZnTe, CDs, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe,
CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, and BaTe.
Alternatively, the semiconductor core and shell can independently
be made of a material made of an element from Group 13 of the
Periodic Table of the Elements, and an element from Group 15 of the
Periodic Table of the Elements. Examples of such materials include
GaN, GaP, GaAs, GaSb, InN, InP, InAs, and InSb. Alternatively, the
semiconductor core and shell can independently be made of a
material made of an element from Group 14 of the Periodic Table of
the Elements. Examples of such a material include Ge, and Si.
Alternatively, the semiconductor core and shell can independently
be made of lead materials such as PbS or PbSe. The semiconductor
core and shell can be made of alloys or mixtures of any of the
above listed materials as well.
[0101] The semiconductor nanocrystal can generally be of any size
(average diameter), but typically are about 0.1 nm to 1000 nm in
size. More narrow ranges of sizes include about 0.1 nm to about 1
nm, about 1 nm to about 50 nm, and about 1 nm to about 20 nm.
Specific size examples include about 0.1 nm, about 0.5 nm, about 1
nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm,
about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about
12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17
nm, about 18 nm, about 19 nm, about 20 nm, about 25 nm, about 30
nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, and ranges
between any two of these values.
[0102] A typical single-color preparation of nanoparticles has
crystals that are preferably of substantially identical size and
shape. Nanocrystals are typically thought of as being spherical or
nearly spherical in shape, but can actually be any shape.
Alternatively, the nanocrystals can be non-spherical in shape. For
example, the nanocrystal's shape can change towards oblate
spheroids for redder colors. It is preferred that at least about
60%, at least about 70%, at least about 80%, at least about 90%, at
least about 95%, and ideally about 100% of the particles are of the
same size. Size deviation can be measured as root mean square of
the diameter, with less than about 10% root mean square being
preferred. Size deviation can be less than about 10% rms, less than
about 9% rms, less than about 8% rms, less than about 7% rms, less
than about 6% rms, less than about 5% rms, or ranges between any
two of these values. Such a collection of particles is sometimes
referred to as being "monodisperse".
[0103] It is well known that the color (emitted light) of the
semiconductor nanocrystal can be "tuned" by varying the size and
composition of the nanocrystal. Nanocrystals preferably absorb a
wide spectrum of wavelengths, and emit a narrow wavelength of
light. The excitation and emission wavelengths are typically
different, and non-overlapping. The width of emission is preferably
less than about 50 nm, and more preferably less than about 20 nm at
full width at half maximum of the emission band (FWHM). Examples of
emission widths (FWHM) include about 50 nm, about 40 nm, about 30
nm, about 20 nm, and about 10 nm. The emitted light preferably has
a symmetrical emission of wavelengths. The emission maxima can
generally be at any wavelength from about 200 nm to about 2000 nm.
Examples of emission maxima include about 200 nm, about 400 nm,
about 600 nm, about 800 nm, about 1000 nm, about 1200 nm, about
1400 nm, about 1600 nm, about 1800 nm, about 2000 nm, and ranges
between any two of these values.
[0104] Nanoparticles can also have a metal core, and in some cases,
a surrounding shell structure. The metal core can be made from
noble metals. Examples of such metals include silver, gold, and
copper.
[0105] The nanoparticles can have surface coatings adding various
functionalities. For example, the nanocrystals can be coated with
lipids, phospholipids, fatty acids, polynucleic acids,
polyethyleneglycol, primary antibodies, secondary antibodies,
antibody fragments, protein or nucleic acid based aptamers, biotin,
streptavidin, proteins, peptides, small organic molecules, organic
or inorganic dyes, precious or noble metal clusters.
[0106] Alternatively, the nanoparticles can be made from a range of
inorganic materials, including silicon, alumina, zirconia, ceria,
yttria and oxides of tin and zinc. For example, silicon
nanoparticles possess many of the advantageous features of compound
semiconductor nanocrystals, such as size-tunable luminescence
across the visible spectrum. In addition, silicon nanoparticles
also low toxicity, high biocompatibility, efficient and stable
surface functionalization, and potential low cost.
[0107] The use of nanoparticles in ion channel assays has multiple
desirable features. Since nanoparticles have rapid response times,
distinctive voltage dependencies are difficult to unintentionally
inactivate, and the nanoparticles can provide a direct optical
readout of voltage gradient changes across a membrane. The
nanoparticles also possess other desirable qualities such as low
toxicity, high photo-stability, the ability to be used in
multiplexing applications, and their ability to be targeted using
conjugated or otherwise associated materials.
[0108] Spectral characteristics of nanoparticles can generally be
monitored using any suitable light-measuring or light-accumulating
instrumentation. Examples of such instrumentation are CCD
(charge-coupled device) cameras, video devices, CIT imaging,
digital cameras mounted on a fluorescent microscope,
photomultipliers, fluorometers and luminometers, microscopes of
various configurations, and even the human eye. The emission can be
monitored continuously or at one or more discrete time points. The
photostability and sensitivity of nanoparticles allow recording of
changes in electrical potential over extended periods of time.
[0109] Additional methods of assaying the emission from the
nanostructure include measuring changes in light intensity, light
polarization, light absorption, color of the emission, emission
lifetime or half-life, or the "blinking" pattern.
[0110] An additional embodiment of the invention is directed
towards nanoparticles coated with phospholipids. An example of such
a nanocrystal is a commercially available phospholipid-coated Maple
Red-Orange EviTag-T2 nanocrystal (Evident Technologies; Troy,
N.Y.). There also exist published descriptions on preparation of
lipid coated semiconductor nanocrystal materials.
[0111] Methods for the Excitation of Cells
[0112] An additional embodiment of the invention is directed
towards the use of nanostructures to control the transmembrane
potential of cells. Optical methods are attractive for use in
biological applications due to their non-invasive nature and ease
of use. For example, photo-induced electrical excitation of
neuronal cells has been demonstrated using a film of semiconductor
material (Frohmherz, P. and Stett, A., Phys. Rev. Lett. 75(8):
1670-1673 (1995); Starovoytov, A. et al., J. Neurophysiol. 93(2):
1090-1098 (2005)). Neuronal cells were attached to a thin film of a
semiconductor material, achieving close contact of the
extracellular membrane and the semiconductor surface. Illumination
of the substrate with a laser beam has been shown to electrically
excite the cells attached to the semiconductor surface.
[0113] Nanostructures such as nanoparticles exposed to light can
act as a generator of a local electromagnetic field in their
vicinity. The effect is believed to be due to creation of free
charge carriers (electron-hole pairs upon illumination of
nanoparticles) and consecutive charge separation. The currently
proposed mechanism of action is electrostatic coupling of the
cellular membrane and the surface of semiconductor, effectively
forming a capacitor. When nanoparticles are placed in close
proximity to a cell, the cumulative electromagnetic field generated
by photo-excited nanoparticles will interact with the cellular
transmembrane electrical gradient, resulting in an electromagnetic
field that dictates the cellular membrane potential. Local
depolarization of part of cellular membrane may be sufficient to
generate depolarization in the whole cell.
[0114] In addition to use of the above described nanocrystals,
modified nanoparticles can be used to achieve a strong, stable, and
controllable local electric field. Such modifications include high
surface charge (e.g. CdTe/CdSe as core/shell combination), doping
nanoparticles with materials that would act as donors or acceptors
of one type of free charge carriers, creating nanoparticles with p-
or n-type surface traps, conjugation of molecules that would
contribute to a charge separation, and so on. Active generation of
a cellular transmembrane potential can be achieved through use of
nanoparticles that can convert light into electric power.
[0115] In conventional solar cells, electron-hole pairs are created
by light absorption in a semiconductor core, with charge separation
and collection accomplished under the influence of electric fields
within the core.
[0116] As nanoparticles are approximately the same thickness as a
cellular membrane, insertion into the membrane exposes the poles of
the nanoparticle to both the extra- and intracellular space. Upon
illumination with light, nanoparticles become a path for free
charge carrier flow through the membrane, passing an electric
current and in turn affecting the transmembrane potential. This
way, voltage control over the cell could be achieved by changing,
for example, the incident light's intensity and/or
polarization.
[0117] Nanoparticles can be synthesized in shapes of different
complexity such as spheres, rods, discs, triangles, nanorings,
nanoshells, tetrapods, and so on. Each of these geometries have
distinctive properties: spatial distribution of the surface charge,
orientation dependence of polarization of the incident light wave,
and spatial extent of the electric field. Non-uniform coating of
nanoparticles with a dielectric material (such as phospholipids)
can also help guide the free charge carriers from one side of the
membrane to the other.
[0118] In order to manipulate free charge carrier concentration and
mobility, nanoparticles can be doped with impurities such as
indium, phosphorus, boron, and aluminum, and so on. A blend of
nanoparticles and organic polymers may be advantageous for this
application as nanoparticles are highly efficient in conducting
electrons, whereas polymers are better at conducting holes.
Functionalization of semiconductor nanoparticles with chromophores
could also optimize this application by separating photon
absorption from free charge carrier transport.
[0119] Accordingly, methods for the optical control of the
transmembrane potential of a target can comprise providing at least
one target, wherein the target is a cell or cellular fraction;
contacting the target with at least one nanostructure under
conditions suitable for interaction or insertion of the
nanostructure with a cellular or subcellular membrane to prepare a
treated target; delivering energy to the treated target; and
detecting response of the target.
[0120] The cells can be any of the cells described above. The
nanostructure can be any nanostructure including any of the
nanostructures described above.
[0121] The conditions suitable for interaction or insertion can
include a variety of methods. Examples of such methods include
passive or active uptake via endocytosis, electroporation,
liposome-mediated delivery, pluronic block copolymer-mediated
delivery, cell-penetrating peptide-mediated uptake,
protein-mediated uptake, microinjection, transfection, viral
delivery, optoporation, pore-forming substrates, membrane
intercalators, or combinations thereof.
[0122] The delivering energy can include delivering light,
electrical energy, magnetic energy, and so on. The delivering
energy step can be performed by essentially any illumination
method, including laser illumination, mercury lamp illumination,
xenon lamp illumination, halogen lamp illumination, LED
illumination, and so on. An illuminating step is preferably
performed at a wavelength or wavelength range suitable for
absorption by the nanostructure.
[0123] The detecting step can be performed using a variety of
methods using any suitable light-measuring or light-accumulating
instrumentation. Examples of such instrumentation are a camera, a
digital camera, a video camera, a CMOS camera, a CCD camera, a
digital camera mounted on a fluorescent microscope, a
photomultiplier, a fluorometer, a luminometer, a microscope, and
even the human eye. The cellular response can be monitored
continuously or at one or more discrete time points.
[0124] Alternatively, the detecting step can include use of a
secondary detection mechanism. An example of such a secondary
detection mechanism is the use of fluorescence resonance energy
transfer ("FRET"). With FRET, the nanostructure can transfer its
energy to a second molecule that then emits a detectable signal.
Additional secondary detection mechanisms rely on changes in a cell
that can be independently detected. For example, the cell may
undergo lysis. Alternatively, the cell may undergo a chemical
change, increasing or decreasing the concentration of one or more
chemical or biochemical agents that can be independently
measured.
[0125] At least one additional material can be added to the at
least one cell or to the treated cell to assay the cellular
response to the additional material. For example, the cell can be
first contacted with the at least one nanoparticle, illuminated,
and the cellular response detected as a "control" sample. The
treated cell can then be contacted with the additional material to
prepare a material-treated cell, illuminated, and detected. This
second cellular response can be compared with the first (control)
cellular response. A difference between the first cellular response
and the second cellular response would indicate whether the
addition of the material had any effect on cellular behavior. A
different additional material, or an additional dose of the same
additional material can be added, followed by illumination and
detection of a third cellular response. This can be done in a
serial manner any number of times. For example, increasing dosages
of a material can be detected, resulting in a third cellular
response, a fourth cellular response, a fifth cellular response, a
sixth cellular response, and so on. These serial cellular responses
can be plotted or otherwise compared, and the effects of the serial
treatments can be determined.
[0126] Alternatively, "control" and "test" samples can be performed
in parallel. For example, a first cell can be contacted with a
nanoparticle, illuminated, and the control cellular response
detected. In parallel, either serially or simultaneously, a second
cell can be contacted with a nanoparticle and a test material,
illuminated, and the test cellular response detected. The control
cellular response and the test cellular response can be
compared.
[0127] The at least one additional material can generally be any
material. Examples of such materials include drug candidates,
modulators of cellular function, molecular moieties for enhanced
drug delivery, molecular probes candidates, and so on.
[0128] Assay Materials
[0129] An additional embodiment of the invention is directed
towards one or more containers having a layer of nanostructures
deposited on one or more surfaces. For example, the container can
be a test tube, centrifuge tube, or microtiter plate (e.g., 96 or
384 well plate). The entire inner surface of the tube or plate's
wells can be coated with the nanostructures mentioned above.
Alternatively, the lower or bottom inner surface of the tube or
wells can be coated with the nanostructures. These assay materials
can be stored for subsequent use with cells or other biological or
artificial membrane materials.
[0130] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor(s) to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the scope of the
invention.
EXAMPLES
Example 1
Preparation of Cells on Coverslips
[0131] Experiments were performed on A431 (a human cell line from
an epidermoid carcinoma) cells or CHO (Chinese hamster ovary) cells
stably expressing M1 muscarinic G.sub.q-protein coupled receptor
using nanoparticles commercially available from Quantum Dot Corp.
(a wholly owned subsidiary of Invitrogen Corp.; Carlsbad, Calif.)
and Evident Technologies (Troy, N.Y.). The intracellular (pipette)
solution (pH 7.3) was composed of 140 mM CsCl, 10 mM EGTA, 10 mM
HEPES. The extracellular solution (pH 7.4) was composed of 140 mM
NaCl, 5 mM KCl, 1.8 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM EGTA, 10
mM glucose, 10 mM HEPES.
[0132] In experiments with extracellular delivery of nanoparticles,
several types of commercially available nanocrystals were used. In
one series of experiments, streptavidin-functionalized QD605
(Quantum Dot Corp.) in the buffer solution B from the QDot.RTM. kit
were added to extracellular solution in concentrations from 25 to
500 .mu.g/ml. In another experimental series, non-functionalized
Maple Red-Orange EviTag-T2 (Evident Technologies, Troy, N.Y.) were
used in the same concentrations.
[0133] For experiments with intracellular applications of
nanoparticles, streptavidin-functionalized QD605 (Quantum Dot
Corp.) were added to the pipette solution in concentrations from 25
to 500 .mu.g/ml.
[0134] Glass 18 mm round coverslips with cells plated on the
surface were transferred into a special chamber 508SW (ALA
Scientific Instruments, Westbury, N.Y.). Control extracellular
solution was substituted with semiconductor nanocrystal-containing
extracellular solution. After 30 minutes at room temperature, the
coverslips with cells were washed with PBS solution until excess
free-floating nanocrystals were removed. To visually confirm that
washing had removed all free-floating nanoparticles, coverslips
were placed under the microscope. If excitation was seen by the
naked eye, the washing procedure was repeated two more times.
[0135] After the washing procedure was completed, the coverslip was
mounted in a microscope chamber and the cells were maintained in
buffered EBSS solution during the experiment. Only cells labeled
with nanoparticles were chosen for further experiments.
Example 2
Use of Patch Pipette
[0136] Glass micropipettes for patch-clamp experiments were pulled
from borosilicate glass capillaries (1.2 mm no-capillary glass,
Sutter Instruments; Novato, Calif.) using a Sutter 2000.TM. pipette
puller (model Sutter 2000; Sutter Instruments; Novato, Calif.)
using the prerecorded 4-step patch pipette pulling protocol. The
open diameter of the pipette tip was 1.5-2.2 .mu.m with a
resistance of 2-3 M.OMEGA.. The micropipettes were filled with
intracellular solution.
[0137] Experiments were performed at room temperature in whole-cell
patch-clamp configuration using a Axopatch200B patch-clamp
amplifier (Molecular Devices; Sunnyvale, Calif.). After successful
giga-seal formation, brief pulses of suction were used to rupture
the cellular membrane to achieve whole-cell patch-clamp
configuration.
[0138] The following test protocol was used for cell stimulation.
The membrane potential was set at -70 mV. A depolarizing pulse
necessary to take the cell to +40 mV was applied to the interior of
the cell for 2 seconds, followed by returning the membrane
potential to -70 mV.
Example 3
External loading of streptavidin-coated nanoparticles
[0139] The emission intensity of externally applied
streptavidin-functionalized nanoparticles occurring in response to
voltage stimulation of the cell (QD605-streptavidin, Quantum Dot
Corp.) was visualized using a cooled CCD Optronics Tec 470 camera
(Optronic Engineering, Goleta, Calif.) linked to a computer.
Voltage changes elicited across the cellular membrane via patch
pipette attached to a cell did not result in changes in the
emission intensity of these particular nanoparticles. Nine cells
were tested in this series, and none exhibited changes in emission
intensity to the voltage stimulation protocol described in the
previous example. The streptavidin coating of the nanoparticles
used in this example may have prevented the nanocrystals from being
strategically placed inside the cellar membrane, the site of the
highest membrane gradient. The streptavidin coating of the
nanoparticles used in this example may have prevented the
nanocrystals from associating with the cellular membrane in such a
way that they could effectively monitor the voltage gradient across
the membrane.
Example 4
Intracellular Loading of Nanoparticles
[0140] This example was designed to test the emission of
nanoparticles loaded intracellularly in response to a voltage
change across the cellular membrane.
[0141] It is preferred to position the nanoparticles in close
proximity to the cellular membrane in order to achieve modulation
of optical signal by voltage. Since the main part of the voltage
gradient exists across the cytoplasmic membrane, the nanoparticles
located close to the membrane would be exposed to a significant
portion of the total electrical gradient.
Example 5
Protocol for Intracellular Loading of Nanoparticles
[0142] Nanoparticles were added to the patch pipette solution at a
concentration of 200 .mu.g/ml. Initial experiments were performed
using streptavidin-coated nanoparticles QD605 (Quantum Dot Corp.).
A431 cells, plated on glass 18 mm round coverslips were placed into
the electrophysiology chamber mounted on a Zeiss Axiovert 100
microscope.
[0143] After establishing a whole-cell patch-clamp configuration,
several brief pulses of positive pressure were applied to the
pipette interior. These small changes of intra-pipette pressure
were used to facilitate cell perfusion with the intracellular
semiconductor nanocrystal-containing solution. Voltage stimulation
experiments on the cells were conducted after loading of
nanocrystals was achieved.
[0144] The following test protocol was used for cell stimulation.
The membrane potential was set at -70 mV. A depolarizing pulse of
+40 mV was applied to the interior of the cell for 1 to 2 seconds,
and subsequently the membrane potential was returned to -70 mV.
[0145] Emission of the nanoparticles was recorded constantly during
voltage stimulation of the cell using a CCD Optronic Tec 470 camera
(Optronic Engineering; Goleta, Calif.). The effects of the voltage
stimulation on emission intensity of nanoparticles are shown in
FIG. 1. Ten of twelve cells responded to the voltage stimulation
protocol, as evidenced by a change in semiconductor nanocrystal
emission intensity. Thus, the nanoparticles were able to respond to
the changes in transmembrane potential by changing their optical
characteristics.
Example 6
Use of Treated Semiconductor Nanoparticles as Voltage Sensors
[0146] One prospective use for semiconductor nanoparticle-based
membrane potential-sensitive assays is high throughput screening
for drug discovery. One of the major challenges for HTS assays is
the ease of voltage indicator loading into cells.
Phospholipid-coated quantum dots were selected as an example of a
surface-modified nanoparticle for these experiments.
[0147] In this example, modified nanoparticles (phospholipid-coated
EviTag-T2 (Evident Technologies, Troy, N.Y.)) were applied to A431
cells externally. Cells attached to 18 mm round coverslips were
incubated for 45-60 minutes in an extracellular solution containing
nanoparticles at 25 to 500 .mu.g/ml.
[0148] After incubation, the coverslips and attached cells were
placed into a special chamber 508SW (ALA Scientific Instruments,
Westbury, N.Y.) on an Zeiss Axiovert 100 microscope, equipped with
a CCD camera for optical recordings. After establishing a
whole-cell patch-clamp configuration as described previously,
voltage stimulation experiments were performed.
[0149] The following test protocol was used for cell stimulation.
Membrane potential was set at -70 mV. A depolarizing pulse of +40
mV was applied to the interior of the cell for 1 to 2 seconds, and
then the membrane potential was returned to -70 mV.
[0150] Emission of the nanoparticles was recorded during voltage
stimulation of the cell using a CCD Optronic Tec 470 camera
(Optronic Engineering, Goleta, Calif.). The effects of the
electrophysiological stimulation are shown in FIG. 2. Of the 8
cells tested under these experimental conditions, 6 cells responded
to the voltage stimulation protocol by transiently changing their
emission intensity.
[0151] These results suggest that nanoparticles having a
hydrophobic phospholipid coating can localize in or on the cellular
membrane, and therefore, are able to report on the cellular voltage
potential. This method of loading the voltage-sensing nanoparticles
represents an especially advantageous means to prepare cells for
high throughput screening.
Example 7
Summary of Results from Examples 1-6
[0152] These results demonstrate that nanoparticles can be used as
a self-contained fluorescent voltage indicator. The nanoparticles
can be used as a direct optical detection system for changes of the
voltage gradient across a membrane. Optimization of delivery and
surface modifications can further improve the usefulness of the
nanoparticles in the above described methods.
Example 8
Patch-Clamp Recordings from Optically Excited Cells
[0153] Cells having an expressed ion channel target can be prepared
using established cell culture preparation procedures. CHO or A431
cells, plated on round glass 18 mm coverslips will be incubated
with a solution containing nanoparticles at appropriate
concentrations for 15-60 minutes at room temperature. After the
incubation, the coverslips will be washed four times with PBS
solution.
[0154] Alternatively, glass coverslips or plate wells can be
pre-coated with the nanoparticles allowing cells to be seeded on
top of the nanoparticle layer. Wells of the plate will be filled
with nanoparticle-containing solution at the appropriate
concentration. The plate can be stored for several hours under the
sterile conditions.
[0155] After the nanoparticles-containing solution is washed away,
the coverslip will be transferred into a special microscope chamber
508SW (ALA Scientific Instruments, Westbury, N.Y.) and maintained
in buffered EBSS solution during the experiment.
[0156] Glass micropipettes for patch-clamp experiments will be
pulled from borosilicate glass capillaries (Sutter 1.2 mm
no-capillary glass) using a Sutter 2000.TM. pipette puller (model
Sutter 2000, Sutter Instruments, Novato, Calif.) using a
prerecorded 4-step patch pipette pulling protocol. The open
diameter of the pipette tip will be 1.5-2.2 .mu.m.
[0157] The micropipettes will be filled with a solution containing
140 mM potassium aspartate, 5 mM NaCl, and 10 mM HEPES (pH 7.35).
Voltages and currents will be recorded at room temperature using a
Axopatch 200B patch-clamp amplifier (Molecular Devices; Sunnyvale,
Calif.).
[0158] After establishing the successful Giga-seal, brief pulses of
suction will be used to rupture the cellular membrane to achieve
whole-cell patch-clamp configuration. The following test protocol
will be used for cell stimulation. Brief pulses of excitation light
(emitted by laser, or by other light source) will be used to
illuminate the patched cell. Voltage and current changes through
the cellular membrane will be recorded in the whole-cell
configuration.
Example 9
Optical Recordings from Optically Excited Cells
[0159] Cells having an expressed ion channel target can be prepared
using established cell culture preparation procedures. CHO or A431
cells, plated on round glass 18 mm coverslips will be incubated
with a voltage sensitive dye (e.g., a semiconductor
nanoparticles-based voltage sensor) for 15-60 minutes. After the
incubation, the coverslips will be washed four times with PBS
solution.
[0160] The second step will be an incubation of tested cells with a
solution containing nanoparticles at an appropriate concentration
for 15-60 minutes at room temperature. After the incubation, the
coverslips will be washed four times with PBS solution. After the
nanoparticle solution is washed away, the coverslip will be mounted
on a microscope chamber and maintained in buffered EBSS solution
during the experiment.
[0161] Alternatively, glass coverslips or plate wells can be
pre-coated with the nanoparticles allowing cells to be seeded on
top of the nanoparticle layer. Wells of the plate will be filled
with nanoparticle-containing solution at the appropriate
concentration. The plate can be stored for several hours under the
sterile conditions.
[0162] Alternatively, at the beginning of experiment the cell
suspension will be incubated with specially prepared suspension of
semiconductor nanoparticles. After incubating for 5-60 minutes, the
cells will be dispensed into wells of a microtiter plate (e.g., a
96, 384, or 836 well plates). The microtiter plates will be mounted
on the microscope stage for the experiment.
[0163] Voltage stimulation will be achieved by illuminating the
cell suspension with brief pulses of excitation light (emitted by
laser, or by other light source). Emission of the nanoparticles
will be recorded during voltage stimulation of the cell using a
cooled CCD camera (e.g., Optronics Tec 470 (Optronic Engineering;
Goleta, Calif.) or XR/MEGA-10Z.TM. fast camera (Stanford Photonics,
Inc.; Palo Alto, Calif.)) linked to a computer.
[0164] The emission pattern change of the nanoparticles will
indicate the cellular response to excitation by photo-activated
nanoparticles on the cell surface.
Example 10
First Preparation Method for Target Cells in Microplate Wells
[0165] A solution containing non-functionalized Maple Red EviTag-T2
(Evident Technologies, Troy, N.Y.) or streptavidin-functionalized
QD605 nanocrystals (Quantum Dot Corp.) at various concentrations
were added to the 96-well Microplates (Nunc; Denmark). The
pretreated plates were stored under sterile conditions for six
hours, allowing the solution to dry, and leaving the layer of
nanoparticles on the bottom of the wells.
[0166] Experiments were performed on CHO cells stably expressing M1
muscarinic G.sub.q-protein coupled receptor. A suspension of cells
was added to the plates and incubated for 12-24 hours at 37.degree.
C. in the presence of carbon dioxide.
[0167] After the incubation, the plates with cells were washed with
PBS solution until any excess free-floating cells and nanocrystals
had been removed. To confirm that washing had removed all
free-floating nanoparticles, plates were visually inspected with a
microscope. If excitation was seen by the naked eye, the washing
procedure was repeated two more times. After washing, the pates
were transferred into PathWay.sup.NT screening station (BD
Biosciences; San Jose, Calif.) for evaluation.
Example 11
Second Preparation Method for Target Cells in Microplate Wells
[0168] Cells were plated in 96-well plates. Plates were either
glass-bottomed or poly-L-lysine-coated (Nunc; Denmark). Maple Red
EviTag-T2 nanoparticles were added to the cell-containing solution.
Cells were incubated in the presence of nanoparticles for 15-60
minutes. Any excess nanoparticles were washed away. Plates with
nanoparticle-treated cells were placed inside an environmentally
controlled chamber of Pathway HT machine (BD Biosciences; San Jose,
Calif.).
[0169] The series of images of cells from each well were acquired
in kinetic mode from several wells consecutively. First ten images
in the series were taken as control images to ensure the stability
of a signal from labeled cells. The following step was an
application of potassium chloride solution into a well.
Concentration of potassium chloride solution was chosen to achieve
the final potassium chloride concentration of 100 mM thus shifting
the membrane potential of cells (to about 0 mV) in depolarizing
direction. The optical response of nanocrystal-labeled cells to
depolarization stimuli for each individual well was recorded using
Pathway HT machine (BD Biosciences; San Jose, Calif.).
[0170] After the assays, a series of images were processed using
MethaMorph software (Molecular Devices, Sunnyvale, Calif.). Regions
of interest were chosen either around the cellular membrane or in
extracellular space (control).
Example 12
Sensitivity of Externally Applied Nanoparticles to Changes in
Cellular Membrane Potential Detected By High Content Screening
[0171] CHO cells stably expressing M1 muscarinic G.sub.q-protein
coupled receptors were plated in 96-well microplates, either
glass-bottomed or poly-L-lysine-coated (Nunc; Denmark). Maple Red
EviTag-T2 nanoparticles were added to the cell-containing solution.
Cells were incubated in the presence of nanoparticles for 15-60
minutes. Any excess nanoparticles were washed away. Plates with
nanoparticle-treated cells were placed inside an environmentally
controlled chamber of a Pathway HT.TM. screening station (BD
Biosciences; San Jose, Calif.).
[0172] A series of images of cells from each well were acquired in
a kinetic mode from several wells consecutively. First, several
images in the series were taken as control images to ensure the
stability of a signal from labeled cells. Next, potassium chloride
solution was added into the well. The concentration of potassium
chloride solution was selected to achieve the final potassium
chloride concentration of 100 mM, thus shifting the membrane
potential of cells in a depolarizing direction. The optical
response of nanocrystal-labeled cells to depolarization stimuli for
each individual well was recorded using a Pathway HT.TM. screening
station (BD Biosciences; San Jose, Calif.).
[0173] After the experiments, the series of images was processed
using MethaMorph software (Molecular Devices, Sunnyvale, Calif.).
Regions of interest (ROI) were selected either around the cellular
membrane or in the extracellular space (control).
[0174] Depolarization of cells by extracellular application of
potassium chloride resulted in transient decrease in optical signal
from cells. It should be noted that optical signal from
extracellular space exhibited some intensity decrease as well.
However, the effect of potassium chloride application in cells was
significantly higher. For example, in one experiment, change in the
maximum response in cellular membrane from 12 cells was 349.+-.56
AU, whereas signal intensity change for extracellular space was
only 191.+-.38 AU (3 ROIs).
[0175] On average, background-subtracted signal intensity in cells
decreased 17.4.+-.5.1% (number of experiments=8). FIG. 3 represents
an example of transient changes in emission intensity from several
cells one well in response to cells' exposure to potassium chloride
in high concentration.
[0176] These results demonstrate that changes in the amplitude of
optical signal emitted by nanoparticles associated with the
cellular membrane reflects changes in membrane potential, and
confirm that nanoparticles can act as a sensor of cellular membrane
potential.
[0177] All of the compositions and/or methods and/or processes
and/or apparatus disclosed and claimed herein can be made and
executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the compositions and/or methods and/or apparatus and/or
processes and in the steps or in the sequence of steps of the
methods described herein without departing from the concept and
scope of the invention. More specifically, it will be apparent that
certain agents which are both chemically and physiologically
related may be substituted for the agents described herein while
the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those skilled in the art
are deemed to be within the scope and concept of the invention.
* * * * *