U.S. patent application number 10/135168 was filed with the patent office on 2002-10-24 for optical fiberless sensors.
Invention is credited to Clark, Heather, Kopelman, Raoul, Monson, Eric, Parus, Stephen, Philbert, Martin, Thorsrud, Bjorn.
Application Number | 20020155600 10/135168 |
Document ID | / |
Family ID | 25395312 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020155600 |
Kind Code |
A1 |
Kopelman, Raoul ; et
al. |
October 24, 2002 |
Optical fiberless sensors
Abstract
Fiberless optical sensors (plasticized PVC, acrylamide or gold
particles) are described having a size ranging from between
approximately 1 micrometer and 1 nanometer in diameter. The sensors
comprise ionophores useful for the detection of intracellular
analytes.
Inventors: |
Kopelman, Raoul; (Ann Arbor,
MI) ; Clark, Heather; (Middletown, CT) ;
Monson, Eric; (Ann Arbor, MI) ; Parus, Stephen;
(Ann Arbor, MI) ; Philbert, Martin; (Ann Arbor,
MI) ; Thorsrud, Bjorn; (Lima, OH) |
Correspondence
Address: |
MEDLEN & CARROLL, LLP
Suite 350
101 Howard Street
San Francisco
CA
94105
US
|
Family ID: |
25395312 |
Appl. No.: |
10/135168 |
Filed: |
April 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10135168 |
Apr 30, 2002 |
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09665789 |
Sep 20, 2000 |
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6379955 |
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10135168 |
Apr 30, 2002 |
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08889538 |
Jul 8, 1997 |
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6143558 |
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Current U.S.
Class: |
435/325 |
Current CPC
Class: |
Y10S 435/805 20130101;
G01N 33/528 20130101; A61K 49/0097 20130101 |
Class at
Publication: |
435/325 |
International
Class: |
C12N 005/00; C12N
005/02 |
Claims
We claim:
1. A composition, comprising cells containing at least one
fiberless optical sensor.
2. The composition of claim 1, wherein said cells are eukaryotic
cells.
3. The composition of claim 2, wherein said cells are mammalian
cells.
4. The composition of claim 1, wherein said sensor comprises a
solid particle ranging in size between approximately 1 micrometer
and 1 nanometer in diameter.
5. The composition of claim 4, wherein said sensor comprises a
polymer.
6. The composition of claim 5, wherein said polymer is selected
from the group consisting of poly(vinyl chloride), poly(vinyl
chloride) carboxylated and poly(vinyl chloride-co-vinyl
acetate-co-vinyl alcohol).
7. The composition of claim 6, wherein said sensor further
comprises an ionophore.
8. The composition of claim 7, wherein said sensor further
comprises a chromoionophore.
9. The composition of claim 8, wherein said sensor further
comprises an additive.
10. The composition of claim 9, wherein said sensor further
comprises a plasticizer.
11. A method, comprising: a) providing i) one or more cells, ii) a
plurality of fiberless optical sensors, and iii) a means for
detecting said sensors; b) introducing said plurality of sensors
into said one or more cells; and c) detecting said sensors in said
cells with said detecting means.
12. The method of claim 11, wherein said cells are eukaryotic
cells.
13. The method of claim 12, wherein said cells are mammalian
cells.
14. The method of claim 11, wherein said sensors comprise a solid
particle ranging in size between approximately 1 micrometer and 1
nanometer in diameter.
15. The method of claim 14, wherein said sensors comprise a
polymer.
16. The method of claim 15, wherein said polymer is selected from
the group consisting of poly(vinyl chloride), poly(vinyl chloride)
carboxylated and poly(vinyl chloride-co-vinyl acetate-co-vinyl
alcohol).
17. The method of claim 16, wherein said sensors further comprise
an ionophore.
18. The method of claim 17, wherein said sensors further comprise a
chromoionophore.
19. The method of claim 18, wherein said sensors further comprise
an additive.
20. The method of claim 19, wherein said sensors further comprise a
plasticizer.
21. A method, comprising: a) providing i) one or more cells, ii) a
plurality of fiberless optical sensors, iii) an exogenous cellular
stimulus, and iv) a means for detecting said sensors; b)
introducing said plurality of sensors into said one or more cells;
c) stimulating said one or more cells with said exogenous cellular
stimulus, and d) detecting said sensors in said cells with said
detecting means.
22. The method of claim 21, wherein said cells are eukaryotic
cells.
23. The method of claim 22, wherein said cells are mammalian
cells.
24. The method of claim 21, wherein said sensors comprise a solid
particle ranging in size between approximately 1 micrometer and 1
nanometer in diameter.
25. The method of claim 24, wherein said sensors comprise a
polymer.
26. The method of claim 25, wherein said polymer is selected from
the group consisting of poly(vinyl chloride), poly(vinyl chloride)
carboxylated and poly(vinyl chloride-co-vinyl acetate-co-vinyl
alcohol).
27. The method of claim 26, wherein said sensors further comprise
an ionophore.
28. The method of claim 27, wherein said sensors further comprise a
chromoionophore.
29. The method of claim 28, wherein said sensors further comprise
an additive.
30. The method of claim 29, wherein said sensors further comprise a
plasticizer.
31. A method comprising: a) providing i) first and second
preparations of cells, ii) a plurality of fiberless optical
sensors, iii) an exogenous cellular stimulus, and iv) a means for
detecting said sensors; b) introducing said plurality of sensors
into said first and second preparations of cells; c) stimulating
said first preparation of cells with said exogenous stimulus, d)
detecting said sensors in said cells with said detecting means, and
e) comparing the sensors in said first preparation of cells with
the sensors in said second preparation of cells.
32. The method of claim 31, wherein said cells are eukaryotic
cells.
33. The method of claim 32, wherein said cells are mammalian
cells.
34. The method of claim 31, wherein said sensors comprise a solid
particle ranging in size between approximately 1 micrometer and 1
nanometer in diameter.
35. The method of claim 34, wherein said sensors comprise a
polymer.
36. The method of claim 35, wherein said polymer is selected from
the group consisting of poly(vinyl chloride), poly(vinyl chloride)
carboxylated and poly(vinyl chloride-co-vinyl acetate-co-vinyl
alcohol).
37. The method of claim 36, wherein said sensors further comprise
an ionophore.
38. The method of claim 37, wherein said sensors further comprise a
chromoionophore.
39. The method of claim 38, wherein said sensors further comprise
an additive.
40. The method of claim 39, wherein said sensors further comprise a
plasticizer.
41. The method of claim 31, wherein said stimulus is a toxin.
42. The method of claim 41, wherein said toxin is a bacterial
toxin.
Description
FIELD OF THE INVENTION
[0001] The invention relates generally to optical fiberless
sensors, method of fiberless sensor fabrication and uses of such
sensors in cells.
BACKGROUND
[0002] The ability of cells, tissues, an organ system and an entire
organism to rapidly respond and adapt to exogenous stimuli is a
requirement for the maintenance of life. Exposure of a single cell
to such stimuli can manifest itself in a variety of ways, including
a flux of essential intracellular ions (i.e. Na+, K+, Ca++, Cl-,
H+), as well as changing oxygen and glucose levels. These changes
can trigger additional signaling cascades, ultimately resulting in
the recruitment of the appropriate cellular machinery for a
response to the stimuli.
[0003] Of course, some stimuli are pathogenic to cells. Such
stimuli cause a combination of linked and cascading biochemical
events leading up to disease and/or cell death. For example,
exposure to bacteria, viruses, toxins and toxicants may result in a
myriad of intra/extracellular responses, depending on the pathogen
or pathogenic agent in question and the route of exposure. The
determination and understanding of which of the "downstream"
biochemical signals elicited are indicators of physical, chemical
or mechanical injury are fundamental to the development of
countermeasures and therapy.
[0004] Classical biochemical investigations of the toxicologic
effects of chemicals on organs and tissues were typically performed
on homogenates. This approach reduced complex arrays of cells to a
uniform blend. While providing important new information on
fundamental mechanisms of toxicology/pharmacology, these studies
are limited in their ability to discriminate between cells which
are passively or actively involved.
[0005] More recent molecular and imaging techniques have improved
cellular resolution. However, these newer imaging techniques
frequently provide only static "snapshots" of dynamic cellular
processes. Other approaches, while more dynamic, suffer from the
fact that the approach alters the cells under study. For example,
commercially available fluorescent probes used in the detection of
calcium fluxes, chemically bind the moiety in question and
potentially alter its homeostasis in situ.
[0006] Clearly, the most extensive work done intracellularly
focused on the direct injection of dyes into the cell. While this
method has provided researchers with a simple technique to study
cellular processes, it has also proven problematic. For instance,
the dye may itself be toxic, or otherwise interfere with the cell
chemistry. Another problem is that there is no way to position the
dye once it is introduced into the cell. Often, the dye is
selectively trapped in some organelles, rather than dispersed
evenly throughout the cell.
[0007] An additional, critical limitation with the dye injection
approach is that the technology is currently limited in selectivity
to a small number of analytes. For instance, while there are good
dyes for calcium ion detection, there are none for potassium,
sodium or chloride.
[0008] Fiber optic probes, or optodes, with a polymer sensing
element, solve the above problems of dye injection. See W. Tan et
al., "Submicrometer Intracellular Chemical Optical Fiber Sensors,"
Science 258:778 (1992). These micro-fiberoptic sensors (100-1000
nm) are based on optical grade silica fibers pulled to submicron
size. The pulled fiber tips are much less fragile than those of the
electrochemical microsensors, which are made from pulled
micropipettes. Attached to the tip is a dye-polymer matrix, which
is very durable and smooth and runs tightly bound to the tip, even
during penetration of biological tissues. The matrix on the end of
the fiber often includes several components, such as a
chromoionophore, an ionophore, and appropriate ionic additives, all
trapped inside a polymer layer, so that no chemicals are free to
diffuse throughout the cell. The effects of toxicity of the dyes
are thus minimized. Also, the probe can be carefully positioned in
the cell, allowing any specific area to be imaged or monitored.
[0009] Nonetheless, the fiber optic probes have the significant
drawback of being unable to easily monitor more than one location
in the cell. For monitoring more than one location, multiple probes
are needed. Due to size constraints, it can prove difficult to
position several fibers inside a single cell. Moreover, even the
insertion of single fiber sensor can easily damage a cell or short
out the cross membrane electrical potential and having several
fibers compounds this problem.
[0010] Thus, improved methods for studying cells and intracellular
analytes are needed. Such improved methods should be amenable to
monitoring the cell at more than one location and should have
minimal toxicity.
SUMMARY OF THE INVENTION
[0011] The invention relates generally to optical fiberless
sensors, method of fiberless sensor fabrication and uses of such
sensors in cells. The sensors of the present invention are: (1)
small enough to enter a single mammalian cell relatively
non-invasively, (2) fast and sensitive enough to catch even minor
alterations in the movement of essential ions and (3) mechanically
stable enough to withstand the manipulation of the sensor to
specific locations within the cell.
[0012] Importantly, the fiberless sensors of the present invention
are non-toxic and permit the simultaneous monitoring of several
cellular processes. In one embodiment, the present invention
contemplates the use of such fiberless sensors to monitor a single
cell exposed to a variety of noxious or trophic stimuli.
[0013] The fiberless sensors of the present invention are
particularly useful for the direct, real-time, non-invasive,
intracellular studies of chemical insults and in elucidation of
subcellular mechanisms of action induced by pathogens and related
toxins. These sensors are immensely smaller, faster and more
sensitive than fiber-optic sensors currently used. The spatially
and temporarily highly resolved and highly detailed chemical
information gained from using these sensors, greatly speeds up
current protocols of research and also leads to new and improved
methodologies.
[0014] In one embodiment, the present invention contemplates a
method comprising: a) providing i) one or more cells, ii) a
plurality of fiberless optical sensors, and iii) a means for
detecting said sensors; b) introducing said plurality of sensors
into said one or more cells; and c) detecting said sensors in said
cells with said detecting means.
[0015] In another embodiment, the present invention contemplates a
method comprising: a) providing i) one or more cells, ii) a
plurality of fiberless optical sensors, iii) an exogenous cellular
stimulus, and iv) a means for detecting said sensors; b)
introducing said plurality of sensors into said one or more cells;
c) stimulating said one or more cells with said exogenous cellular
stimulus, and d) detecting said sensors in said cells with said
detecting means.
[0016] In one embodiment, the present invention contemplates a
method comprising: a) providing i) first and second preparations of
cells, ii) a plurality of fiberless optical sensors, iii) an
exogenous cellular stimulus, and iv) a means for detecting said
sensors; b) introducing said plurality of sensors into said first
and second preparations of cells; c) stimulating said first
preparation of cells with said exogenous stimulus, d) detecting
said sensors in said cells with said detecting means, and e)
comparing the sensors in said first preparation of cells with the
sensors in said second preparation of cells.
[0017] It is not intended that the present invention be limited by
the nature of the cells. Both prokaryotic and eukaryotic cells can
be monitored using the sensors of the present invention. Among
eukaryotic cells, it is specifically contemplated that the sensors
of the present invention are introduced into mammalian cells. All
types of mammalian cells are contemplated (e.g. oocytes, epithelial
cells, etc.). In some embodiments, cells such as neurons and
astrocytes in primary culture are contemplated. Thus, the present
invention contemplates generally compositions comprising mammalian
cells containing fiberless optical sensors.
[0018] In one embodiment, the fiberless sensors are used in the
eye. This readily permits monitoring of responses to agents coming
in contact with the eye (e.g. gases, aerosols, etc.). In another
embodiment, the fiberless sensors are used in the
cardiovasculature. This readily permits cardiac monitoring.
[0019] It is also not intended that the present invention be
limited by the precise composition of the fiberless sensors. The
fiberless sensors of the present invention are either solid or
semisolid particles ranging in size between approximately 5
micrometer and 1 nanometer in diameter. The ultimate small size is
attained by fine grinding and filtering or by micro-emulsion
techniques used to form mono-disperse colloidal particles (rather
than nano-fabrication). In one embodiment, the sensor is selected
from the group consisting of polymer fiberless sensors, acrylamide
fiberless sensors, and metal fiberless sensors.
[0020] In one embodiment, the polymer fiberless sensors of the
present invention comprise an ionophore, a chromoionophore and a
polymer. It is not intended that the present invention be limited
to a particular polymer. In one embodiment, the polymer is selected
from the group consisting of poly(vinyl chloride), poly(vinyl
chloride) carboxylated and poly(vinyl chloride-co-vinyl
acetate-co-vinyl alcohol). In a preferred embodiment, the polymer
fiberless sensors further comprise an additive and a
plasticizer.
[0021] In one embodiment, the acrylamide fiberless sensors of the
present invention comprise polyacrylamide and a reactive dye. In a
preferred embodiment, the acrylamide fiberless sensors further
comprise N,N-methylenebi-(acrylamide) and the mixture is
polymerized to a gel.
[0022] In one embodiment, the metal fiberless sensors of the
present invention comprise protein (or peptide) in combination with
a metal selected from the group consisting of gold, silver,
platinum and alloys thereof. In one embodiment, the protein (or
peptide) is dye-labeled (e.g. with FITC).
[0023] Regardless of the sensor type (e.g. metal or polymer), the
fiberless sensor of the present invention is contemplated to be
capable of measuring intracellular analytes, and more particularly,
capable of detecting a change in the concentration of intracellular
analytes. It is not intended that the present invention be limited
to specific analytes. Nonetheless, preferred analytes measured by
the sensors of the present invention include, but are not limited
to, intracellular ions (i.e. Na+, K+, Ca++, Cl-, H+), as well as
oxygen and glucose.
[0024] It is not intended that the present invention be limited by
the manner in which the sensors of the present invention are
introduced into cells. In one embodiment, a buffered suspension of
fiberless sensors is injected into the sample cell with a
commercially-available pico-injector. In another embodiment, the
fiberless sensors of the present invention are shot into a cell
with a commercially-available particle delivery system or "gene
gun" (such gene guns were developed and are now routinely used for
inserting DNA into cells).
[0025] In some embodiments, the fiberless sensors of the present
invention are positioned with in a cell or remotely steered into a
cell, by photon pressure or "laser tweezers". This technique uses
an infra-red laser beam which traps the particles. Alternatively,
the particles can be moved magnetically, by remotely steering
magnetic nanoparticle pebbles (commercially available) into a
cell.
[0026] It is also not intended that the present invention be
limited by the detecting means. In one embodiment, the fiberless
sensors of the present invention are addressed by laser beams
(rather than fibers), and their fluorescent signals are collected
and analyzed by procedures identical to those used for the
fiber-tip nanosensors. See U.S. Pat. Nos. 5,361,314 and 5,627,922
to Kopelman et al., hereby incorporated by reference.
DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 schematically shows several embodiments of a
detection system for detecting the fiberless optical sensors of the
present invention.
[0028] FIG. 2 schematically shows one given embodiment of a
detection system for detecting the fiberless optical sensors of the
present invention.
[0029] FIG. 3 schematically shows the introduction of the optical
fiberless sensors of the present invention into cells.
[0030] FIG. 4 shows the fluorescence spectrum of one embodiment of
a fiberless sensor of the present invention when inside and outside
a cell.
DEFINITIONS
[0031] To facilitate understanding of the invention, a number of
terms are defined below.
[0032] An "allergic reaction" is any abnormal or altered reaction
to an antigen (or "allergen"). Typically this reaction is
characterized by hypersensitivity of the body to specific
substances, whether protein, lipid or carbohydrate in nature.
Allergic reactions may be local, such as contact dermatitis, or may
be systemic, such as anaphylaxis. Among allergic diseases,
bronchial asthma is one of the most significant. In most urban
hospitals, it is the leading cause of admission of children.
Current medical practice accepts asthma in afflicted individual to
be an unavoidable, incurable illness.
[0033] The term "analyte" is intended to comprise any substance
within a cell. Analytes of particular interest include (but are not
limited to) intracellular ions (i.e. Na+, K+, Ca++, Cl-, H+), as
well as oxygen and glucose.
[0034] The term "chemical reaction" means reactions involving
chemical reactants, such as inorganic compounds.
[0035] The phrase "exogenous cellular stimulus" means a stimulus
exogenous to a cell that is capable of stimulating the cell. By
"stimulating the cell" is meant that the status of the
intracellular analytes of the cell is changed (e.g. the
concentration is changed).
[0036] Such stimuli include, but are not limited to a variety of
noxious, pathogenic and trophic stimuli. In one embodiment, the
stimulus is a toxic agent (or "toxicant"). In another embodiment,
the toxic agent is a biological toxin.
[0037] It is not intended that the present invention be limited to
particular toxins. For example, prokaryotes are a known source of a
variety of toxins. Among species of bacteria, the most notorious
toxin sources are certainly Clostridum botulinum and Clostridium
parabotulinum. The species produce the neurogenic toxin known as
botulinus toxin. While a relatively rare occurrence in the United
States, involving only 355 cases between 1976 and 1984 (K. L.
MacDonald et al., Am J Epidemiology 124, 794 (1986)), the death
rate due to the botulism toxin is 12% and can be higher in
particular risk groups. C. O. Tacket et al., Am. J. Med. 76, 794
(1984).
[0038] Many other bacteria produce protein toxins of significance
to humans, including Bacillus anthracis, Bordetella pertussis
(diptheria), Pasteurella pestis, Pseudomonas aeruginosa,
Streptococcos pyrogenes, Bacillus cereus, E. coli, Shigella,
Staphylococcus aureus, Vibrio cholerae, and Clostridium tetani.
Thorne and Gorbach, Pharmacology of Bacterial Toxins, In:
International Encyclopedia of Pharmacology and Therapeutics, F.
Dorner and J. Drews (eds.), Pergamon Press, Oxford (1986), pp.
5-16.
[0039] "Initiating a reaction" means causing a reaction to take
place. Reactions can be initiated by any means (e.g. heat,
wavelengths of light, addition of a catalyst, etc.)
[0040] The term "microorganism" as used herein means an organism
too small to be observed with the unaided eye and includes, but is
not limited to bacteria, viruses, protozoans, fungi, and
ciliates.
[0041] The term "bacteria" refers to any bacterial species
including eubacterial and archaebacterial species.
[0042] The term "virus" refers to obligate, ultramicroscopic,
intracellular parasites incapable of autonomous replication (i.e.,
replication requires the use of the host cell's machinery).
[0043] A "solvent" is a liquid substance capable of dissolving or
dispersing one or more other substances. It is not intended that
the present invention be limited by the nature of the solvent
used.
DESCRIPTION OF THE INVENTION
[0044] The invention relates generally to optical fiberless
sensors, method of fiberless sensor fabrication and uses of such
sensors in cells. The fiberless sensors allows for direct
insertion, without the need for buffering solutions, which could
change the analyte contents inside a cell. The fiberless sensors
have the advantages of fiber micro-optodes, but are much smaller,
less invasive and totally encapsulated by the cell or even by one
of its organelles. They also have the potential for multiple
analyte measurements and multiple positioning inside a single
cell.
[0045] The fiberless sensors of the present invention comprise one
ore more active sensor molecule (e.g. calcium green) that is
embedded in an inert host (e.g., acrylic polymer). These sensors
have numerous advantages compared with individual molecular tags
(e.g., calcium green), including but not limited to: (1) No
toxicity or interference with cell chemistry; (2) No selective
sequestration in subcellular organelles; (3) No lipophilicity
requirement for "smuggling" the molecule across the membrane; (4)
No ion buffering; (5) No quenching of fluorescence by heavy metals;
(6) No dye loss through leakage; (7) No alteration of fluorescence
by sample viscosity; (8) No poor resolution at cell edges. Most
importantly, (9) the fiberless sensors allow parallel processing of
multiple chemical analytes (most fluorescent tags do not allow this
due to mutual interference by spectral overlap, fluorescence
quenching and chemical interactions).
[0046] A. Optical Fiberless Sensors
[0047] The present invention contemplates fiberless sensors or
Probes Encapsulated By BioListic Embedding (PEBBLEs). While a
variety of such fiberless sensors are contemplated (including but
not limited to metal particles), the preferred fiberless sensors of
the present invention are finely ground or formed particles
comprising polymer matrices that are incorporated with fluorescent
dyes, ionophores, and/or other components. These sensors, with
sizes ranging from the submicrometer to micrometer, can be made
from any polymer matrix. A preferred polymer matrix comprises
plasticized poly(vinyl chloride).
[0048] The fiberless sensors are particularly suitable for chemical
analysis in mammalian cells, by inserting the sensors into the
cell, and monitoring remotely. The sensor particles or beads can be
dispersed in buffer solution and pico-injected into a cell. The
particles can be monitored singly, in groups located at different
positions, or several different kinds can be injected for
simultaneous measurements of several distinct intracellular ion or
small molecule concentrations.
[0049] The fiberless sensors circumvent many of the problems
associated with optical fiber sensors. While the fiberless sensors
of the present invention can be made of the same polymer matrix as
that used on the end of an optode, they do not have the fiber size
constraints and the associated consequences for the cell. A
plurality of fiberless sensors of the present invention can be
injected at one time, giving a means for simultaneous measurements.
This can even be done with a single penetration, and the cell wall
can be allowed to recover. The polymer matrices have been shown to
have very little effect on the cell itself (such as toxicity), and
can be left inside for the lifetime of the cell.
[0050] B. Sensor Fabrication
[0051] In one embodiment, the fiberless sensor of the present
invention comprises several components, such as a chromoionophore,
an ionophore, and appropriate ionic additives, all trapped inside a
polymer layer, so that no chemicals are totally free to diffuse
throughout the cell. The precise composition depends on which
analyte (or analyte ion) is sought to be measured.
[0052] In one embodiment, the particle is prepared for each analyte
by selecting one ionophore (30 mmol/kg), one chromoionophore (15
mmol/kg), one additive (15 mmol/kg), one polymer (33 wt %) and one
plasticizer (66 wt %). This mixture should then be dissolved in
solvent. The preferred solvent is freshly distilled THF (200 mg
mixture in 5 mL THF). The solution can then be coated onto
polystyrene spheres, and ground in liquid nitrogen.
[0053] It is not intended that the present invention be limited by
the nature of the ionophore and/or chromoionophore. For e.g.,
Porphyrins (from Aldrich chemical), are dyes that can be used for
oxygen sensors; Calixarenes and Cobyrinates are examples of
ionophores that can be used for sodium and nitrite ions
respectively. Other illustrative examples (allowing for mixing and
matching) are provided in the tables below (all of the chemicals
are commercially available; most are available from Fluka Chemical
Corp, Ronkonkoma, N.Y.).
[0054] C. Uses Of The Present Invention
[0055] The invention will be useful for, among other things, in the
identification of cellular and subcellular responses which lead to
disease. The fields in which the fiberless optical sensors of the
present invention will find application are vast, and include basic
research, diagnosis, and treatment of disease. Direct benefits to
humans and the environment include the development of new drugs,
understanding the synergistic response to complex mixtures of
pollutants, and prevention of
1TABLE 1 ILLUSTRATIVE IONOPHORES Analyte Ion Ionophore Alternate
Name Primary amines Amine Ionophore I Ammonium Ammonium Ionophore I
Barium Barium Ionophore I Cadmium Cadmium Ionophore I ETH 1062
Calcium Calcium Ionophore I ETH 1001 Calcium Ionophore II ETH-129
Calcium Ionophore III Calcimycin Calcium Ionophore IV ETH 5234
Carbonate Carbonate Ionophore I ETH 6010 Carbonate Ionophore II ETH
6019 Carbonate Ionophore III ETH 6022 Carbonate Ionophore IV Cesium
Cesium Ionophore I Chloride Chloride Ionophore I Chloride Ionophore
II ETH 9009 Copper (II) Copper (II) Ionophore I o-XBDiBDTC Hydrogen
Hydrogen Ionophore I Hydrogen Ionophore II ETH 1907 Hydrogen
Ionophore III Hydrogen Ionophore IV ETH 1778 Hydrogen Sulfite
Hydrogen sulfite Ionophore I ETH 5444 Lead Lead Ionophore I ETH 322
Lead Ionophore II MBDiBDTC Lead Ionophore III ETH 5435 Lead
Ionophore IV Lead Ionophore V 15-Crown-5 Lithium Lithium Ionophore
I ETH 149 Lithium Ionophore II ETH 1644 Lithium Ionophore III ETH
1810 Lithium Ionophore IV ETH 2137 Lithium Ionophore V 12-Crown-4
Lithium Ionophore VI 6,6-Dibenzyl-14- crown-4 Lithium Ionophore VII
Lithium Ionophore VIII Magnesium Magnesium Ionophore I ETH 1117
Magnesium Ionophore II ETH 5214 Magnesium Ionophore III ETH 4030
Magnesium Ionophore IV ETH 7025 Nitrite Nitrite Ionophore I *
Nitrite Ionophore II Nitrite Ionophore III Potassium Potassium
Ionophore I Valinomycin Potassium Ionophore II Potassium Ionophore
III BME-44 Silver Silver Ionophore I Silver Ionophore II MAO Silver
Ionophore III Silver Ionophore IV Sodium Sodium Ionophore I ETH 227
Sodium Ionophore II ETH 157 Sodium Ionophore III ETH 2120 Sodium
Ionophore V ETH 4120 Sodium Ionophore VI Sodium Ionophore X Uranyl
Uranyl Ionophore I ETH 295 Zinc Zinc Ionophore I
*(Cyanouqua-cobyrinic acid hepatokis 2-phenylethyl ester)
[0056]
2TABLE 2 ILLUSTRATIVE CHROMOIONOPHORES Chromoionophores Alternative
Name Chromoionophore I ETH 5294 Chromoionophore II ETH 2439
Chromoionophore III ETH 5350 Chromoionophore IV ETH 2412
Chromoionophore V chromoionophore VI ETH 7075 Chromoionophore XI
ETH 7061
[0057]
3TABLE 3 ILLUSTRATIVE ADDITIVES Cesium
tetrakis(3-methylphenyl)borate Potassium
tetrakis[3,5-bis(trfluoromethyl)phenyl]borate Potassium
tetrakis(4-chlorophenyl)borate Sodium tetrakis[3,5-bis(trfluorome-
thyl)phenyl]borate Sodium tetrakis(4-fluorophenyl)borate Dihydrate
Sodium tetraphenylborate Tetrabutylammonium tetraphenylborate
Tetradodecylammonium tetrakis(4-chlorophenyl)bo- rate
Tetraheptylammonium tetraphenylborate Tetraphenylphosphonium
tetraphenylborate
[0058]
4TABLE 4 ILLUSTRATIVE PLASTICIZERS Plasticizors Alternate Name
Benzyl ether Benzyl 2-nitrophenyl ether Bis(1-butylpentyl) adipate
Bis(1-butylpentyl) decane-1, 10-diyl diglutarate Bis(2-ethylhexyl)
adipate Bis(2-ethylhexyl) sebacate DOS 1-Chloronaphthalene
Chloroparaffin 1-Decanol Dibutyl phthlate Dibutyl sebacate
Dibutyltin dilaurate 1,2-Dimethyl-3-nitrobenzene Dioctyl
phenylphosphate Dipentyl phthalate 1-Dodecanol Dodecyl
2-nitrophenyl ether ETH 217 [12(4-Ethylphenyl)dodecyl]
2-nitrophenyl ether ETH 8045 2-Fluorophenyl 2-nitrophenyl ether
1-Hexadecanol 10-Hydroxydecyl butyrate ETH 264 2-Nitrodiphenyl
ether 2-Nitrophenyl octyl ether o-NPOE 2-Nitrophenyl pentyl ether
1-Octadecanol Octyl [2-(trifluoromethyl)phenyl] ether ETH 5406
5-Phenyl-1-pentanol 1-Tetradecanol Tetraundecyl
benzhydrol-3,3',4,4'-tetracarboxylate ETH 2112 Tetraundecyl
benzophenone-3,3',4,4'-tetracarboxylate ETH 2041 Tributyl phophate
Trioctylphosphine oxide Tris(2-ethylhexyl) phosphate
Tris(2-ethylhexyl) trimellitate
[0059] developmental and degenerative disorders. Fiberless optical
sensors will find application in any setting where current
techniques assess whole organism (including but not limited to
intraembryonic applications) or whole cell chemistry in an attempt
to elucidate specific mechanisms of toxicity. The fiberless optical
sensors will bring to light an entirely new level of detail not
previously available.
[0060] 1. Elucidate Responses to Toxicants
[0061] The responses to toxicants can be elucidated in
developmental and adult models of toxicity in vitro, in utero, in
vivo, or in slice single cell suspensions, mono or bi-layers,
colonies. The types of toxicants that can be evaluated include
bacterial, viral, prion, fungal, protozoan, plant, anthropogenic
(pesticides, complex organic compounds used in manufacturing and
their emissions and discharges, oxidants, environmental degradation
products of natural and anthropogenic chemicals), synthetic and
natural nutritional supplements, electromagnetic radiation, contact
media (ingestion, inhalation, dermal, mucosal).
[0062] The fiberless sensors of the present invention can be used
with success to assist in the identification of structure-activity
relationships and chemical nature of toxicants inside and outside
the cell. This information will provide predictive abilities for a
myriad of applications including pesticide design, understanding
the biochemical role of CFC substitutes on plants and animals prior
to their mass production and release, and ultimately even setting
regulations for toxicologically-significant levels of pollutants in
air, water, and food.
[0063] In one embodiment, the present invention contemplates
utilizing the intracellular analyte response pattern to identify
identify the pathogenic/toxic agent. That is to say, the response
to one type of toxic agent (or even a particular agent within a
class of toxic agents) can be monitored for the pattern of
intracellular analyte changes. Such a pattern becomes a
"fingerprint" for exposure to that particular agent, allowing for
more rapid treatment of the individual and/or prompt removal of the
individual from the source or "zone" of exposure.
[0064] 2. Development of Diagnostic Tools and Treatment of
Disease
[0065] As noted above, the fiberless sensors of the present
invention can be used to measure any alteration in endogenous
analytes of any cell. The present invention specifically
contemplates transcutaneous monitoring (e.g. ear, skin) as well as
continuous flow monitoring of cells in culture, organotypic
culture, organ slices, isolated perfused organs, organs in situ,
and whole animal monitoring.
[0066] The present invention contemplates that the fiberless
sensors of the present invention can be used as diagnostic tools
for earlier intervention (i.e. earlier than currently available)
and treatment of disease. Examples of where early intervention is
important include, but are not limited to, allergic responses and
septic shock.
[0067] Allergic Reponses
[0068] In one embodiment, the fiberless sensors are used to detect
allergic responses. In this regard, inhalation of allergens by
sensitized subjects typically results in an early phase response
characterized by bronchoconstriction within 10 minutes of
inhalation, reaching a maximum within 1 to 2 hours. In some
subjects, the airway narrowing recurs after 3 to 4 hours (i.e.,
late phase), reaching a maximum during the next few hours. P. M.
O'Byrne et al., Am. Rev. Respir. Dis. 136:740 (1987). This late
phase is thought to be due to the cellular phase of inflammation.
F. E. Hargreave et al., Eur. J Respir. Dis. 69 (Suppl 147): 16
(1986). P. M. O'Byrne, Chest 90:575 (1986). J. Dolovich et al., J.
Allergy Clin. Immunol. 83 (Suppl): 521 (1987).
[0069] The present invention contemplates the use of fiberless
optical sensors in the relevant cells of the allergic individual to
allow for detection of an allergic response within seconds or (at
most) minutes after exposure to an allergen. This allows for
earlier intervention and treatment.
[0070] Sepsis and Septic Shock
[0071] Sepsis is a major cause of morbidity and mortality in humans
and other animals. It is estimated that 400,000-500,000 episodes of
sepsis resulted in 100,000-175,000 human deaths in the U.S. alone
in 1991. Sepsis has become the leading cause of death in intensive
care units among patients with non-traumatic illnesses. [G. W.
Machiedo et al., Surg. Gyn. & Obstet. 152:757-759 (1981).] It
is also the leading cause of death in young livestock, affecting
7.5-29% of neonatal calves [D. D. Morris et al., Am. J. Vet. Res.
47:2554-2565 (1986)], and is a common medical problem in neonatal
foals. [A. M. Hoffman et al., J. Vet. Int. Med. 6:89-95 (1992).]
Despite the major advances of the past several decades in the
treatment of serious infections, the incidence and mortality due to
sepsis continues to rise. [S. M. Wolff, New Eng. J. Med.
324:486-488 (1991).]
[0072] Sepsis is a systemic reaction characterized by arterial
hypotension, metabolic acidosis, decreased systemic vascular
resistance, tachypnea and organ dysfunction. Sepsis can result from
septicemia (i.e., organisms, their metabolic end-products or toxins
in the blood stream), including bacteremia (i.e., bacteria in the
blood), as well as toxemia (i.e., toxins in the blood), including
endotoxemia (i.e., endotoxin in the blood). Septicemia and septic
shock (acute circulatory failure resulting from septicemia often
associated with multiple organ failure and a high mortality rate)
may be caused by a number of organisms.
[0073] The systemic invasion of microorganisms presents two
distinct problems. First, the growth of the microorganisms can
directly damage tissues, organs, and vascular function. Second,
toxic components of the microorganisms can lead to rapid systemic
inflammatory responses that can quickly damage vital organs and
lead to circulatory collapse (i.e., septic shock) and oftentimes,
death.
[0074] The present invention contemplates the use of fiberless
sensors in patients at risk for septic shock. Introduction of the
sensors in the patient's cells (e.g., lymphocytes) allows for
earlier detection of toxin exposure and thus, prompt
intervention.
[0075] 3. Imaging
[0076] The fiberless sensors of the present invention are ideal for
simultaneous real-time monitoring of many (e.g. six) chemical
analytes as well as acting as parallel processors. They may also be
used for spatially resolved information. The spatial distribution
of the fiberless sensors in a cell can be observed with Laser
Scanning Confocal Microscopy(LSCM). Of interest is whether any
segregation occurs in one location of the cell preferentially over
others. Cellular component labeling, 3-D imaging and visualization,
can be done at an Imaging laboratory. Rearrangement of fiberless
sensors following injection can be monitored by periodically
acquiring a series of 3-D LSCM images. High resolution
(100.times.NA 1.4) images can be obtained to determine analyte
concentration and changes can be determined with sub-section
imaging of sensor locations. Several (2,3, or more) individual
sensor locations can be identified within the small localized area
of the cell where the pebbles are injected. Only those locations
are then imaged confocally in order to follow in time (scale of
hours) analyte changes after a biochemical perturbation.
Significant photobleaching from such occasional scans, are not
anticipated [i.e. the extremely small (micron or submicron)
photoexcited region limits photo-damage to that region and enables
fast diffusive replenishment from the dark environment]. Also,
images can be obtained simultaneously at two or more wavelengths
using an electronically tunable filter on a time scale of
hours.
[0077] Utilizing the fiberless optical nanosensors of the present
invention, one can get a chemical video for a single analyte (e.g.
calcium), analogous to a black and white movie, or a chemical video
of a list of chemical analyte (e.g., calcium, sodium, potassium,
chloride, oxygen and pH), analogous to a color video or,
alternatively, to six single color videos, each taken with a
different narrow-band optical filter.
[0078] For such monitoring, the cell is placed under a microscope
objective lens (see FIG. 1), and may be immobilized by a
micropipette (standard technique). One or several fiberless sensors
are introduced into the cell(s) and are addressed optically by
laser beams.
[0079] Generally, each fiberless sensor is uni-functional, but six
or more kinds ("colors") of sensors can be introduced
simultaneously. The time-resolved color images are collected on a
CCD (charged coupled device) or equivalent optical detectors (see
FIG. 1) and the digital information is deposited in an optical
disk. Eventually the disk is processed and enhanced for optimal
imaging and evaluation. Data can be processed in a variety of ways
(intensity versus time, etc.).
DESCRIPTION OF PREFERRED EMBODIMENTS
[0080] One of the preferred embodiments of this invention is to use
the fiberless optical sensors of the present invention for the
measurement of solute fluxes in mammalian cell systems following
exposure to external noxious stimuli. The sensors permits
simultaneous measurement of multiple chemical species, effectively
producing a "physiogram" of chemical alterations in a single cell.
Several different approaches are contemplated to measure the solute
fluxes in mammalian cell systems following exposure to external
noxious stimuli, including but not limited to 1) the measurement of
ionic/solute concentrations in normal respiring cells in suspension
culture, 2) the measurement of ionic/solute fluxes in normal
respiring cells in suspension culture, cells on substrates and
tissue slices, and 3) the detection of specific alterations in
intracellular ionic/solute concentrations and fluxes following
exposure to pathogenic stimulus and modulation with pharmacologic
agents.
[0081] In Vivo and Ex Vivo Applications
[0082] The present invention also contemplates application of
fiberless sensors to in vivo measurements for time-resolved
monitoring of specific anatomically distinct regions of the cell
under both natural and toxic conditions. The present invention also
contemplates ex vivo measurements (e.g. using cells outside the
body on a patch) for detecting exposure to toxic conditions.
[0083] Pharmacological Agents
[0084] In some of the embodiments of this invention, pharmacologic
agents can be used for the manipulation of ionic fluxes. The
glucose kinase inhibitors, 2-dioxy-D-glucose or 2-cyclohexen-1-one
can be used to inhibit glucose utilization in tissue slices and
cells in culture (Parry and Walker, 1966; Miwa et al., 1990).
Levels of nitric oxide in solution can be manipulated by incubation
of biological specimens with the nitric oxide synthase inhibitor
NG-monomethyl-L-arginine (Mulligan et al., 1991).
[0085] Manipulating And Positioning The Sensors
[0086] In one embodiment, the present invention contemplates moving
the fiberless sensors within a cell. Movement can be achieved with
magnets or with an optical force trap ("laser tweezers").
[0087] The laser optical force trap (laser tweezers) was originally
developed to hold and manipulate small dielectric particles, and
later, atoms and molecules. The optical force trap can immobilize
and manipulate small particles, including the fiberless sensors of
the present invention, in aqueous solutions.
[0088] The operation of laser tweezers depends upon gradient forces
of radiation pressure generated in a convergent cone of laser
light. A laser beam is directed into a light microscope objective
of high numerical aperture and is focused into a
diffraction-limited spot in the field of view of the objective.
Small dielectric particles are attracted to the center of the spot
and are held there by the scattering and gradient forces. The
magnitude of the force holding the particles in the spot depends
partially upon the difference between the refractive indexes of the
particles and its surrounding environment; generally, the technique
is capable of generating a few microdynes of force. Movement of the
laser beam will tend to move any trapped particles with the light
beam. The technique is non-ablative: the wavelength of laser light
is selected for extremely low absorption by the material being
manipulated, and the small amount of energy that is absorbed by the
material is dissipated into the surrounding aqueous medium.
[0089] Experimental
[0090] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof. In the experimental
disclosure which follows, the following methodology apply.
[0091] Reagents for the PVC PEBBLEs: Poly(vinyl chloride) (PVC),
chromoionophore II (ETH 2439) and 2-nitrophenyl octyl ether
(o-NPOE) were obtained from Fluka Chemical Corp (Ronkonkoma, N.Y.).
The fluorescent label
1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyaine perchlorate
(DiIC.sub.18) was obtained from Molecular Probes, Inc. (Eugene,
Oreg.). Sterile phosphate buffer (pH 7.4) was obtained from Fisher
Chemicals (Pittsburgh, Pa.).
[0092] Preparation of the PVC PEBBLEs: Membranes contained 33% PVC
and 66% o-NPOE by weight. All components were dissolved in freshly
distilled tetrahydrofuran (THF). The fiberless sensors were
prepared by drying and crushing small (50 nm) polystyrene
nanospheres and coating with the PVC cocktail. When dry, the
nanospheres were crushed again, to break up the membrane. Due to
the nature of the PVC matrix, the membrane could not be crushed
directly into small pieces without the introduction of the
polystyrene beads. The particles were 1-5 .mu.m in size.
[0093] Optics: The complete optical path (see FIG. 2) included: Ion
Laser Technology (Salt Lake City, Utah) argon ion laser; 514.5 nm
laser band-pass filter (Newport Corp. Irvine, Calif.); Uniblitz
shutter controller (Rochester, N.Y.); fiber coupler (Newport Corp.
Irvine, Calif.); Olympus inverted fluorescence microscope, IMT-II
(Lake Success, N.Y.); Nikon 50 nm f/1.8 camera lenses; Acton 150 mm
spectrograph (Acton, Mass.); and, a Princeton Instruments
1024.times.256 LN2 cooled CCD array (Trenton, N.J.).
[0094] Biolistics Particle Delivery System: A Biolistic PDS-1000/He
System (bench-top model) from Bio-Rad (Hercules, Calif.) with grade
5 helium was used to inject the cells with the fiberless sensors of
the present invention (FIG. 3). Sample preparation for the particle
delivery system required dispersion of the polymer beads in water,
and the careful application of a thin film of beads onto the target
membrane. Low firing pressures were used, with the best results
being obtained at 1100 psi, with a vacuum of 15 torr on the system.
Repeated rinsing was performed to ensure that sensors not in the
cells were removed from the sample. The cells were analyzed within
a few hours.
[0095] Cell Lines: Stocks of human SY5Y neuroblastoma and C6 glioma
cells (American Type Cell Culture cell bank) are maintained in
culture according to recommended protocols supplied by ATCC.
EXAMPLE 1
[0096] In this example, fiberless optical sensors have been
prepared for the intracellular monitoring of ion or small molecule
concentrations. As noted previously, the sensors are made from a
pulverized matrix (plasticized PVC, acrylamide or gold), containing
fluorescent dye molecules and non-fluorescent ionophores coated
onto a polystyrene nanosphere. In this example, single sensors, on
the order of 1-5 .mu.m (see the fabrication methods described
above), were inserted into human neuroblastoma cells with a
particle delivery system.
[0097] Neuroblastoma cells (cell line SH-SY5Y) were plated-into 60
mm petri dishes and incubated in a CO.sub.2 environment. The cells
were used when they reached a confluency of about 80%. Phosphate
buffer was removed from the cells before sensor injection, then
immediately replaced to prevent the cells from dying out.
[0098] The pH sensitive dyes were found to be ideal indicators for
the location of the sensor; they changed color, depending on
whether the probe was inside or outside the cell. The system
allowed for direct insertion, without the need for buffering
solutions, which would have changed the analyte contents inside a
cell. The fiberless sensors had the advantages of fiber
micro-optodes, but were much smaller, less invasive and totally
encapsulated by the cell or even by one of its organelles. They
also had the potential for multiple analyte measurements and
multiple positioning inside single cells. The composition (ETH
2439/DiI) used was a good indicator of the location of the sensor
in the sample. Without an environmentally sensitive dye, it is
difficult to differentiate a sensor that is encapsulated inside a
cell from one that is embedded in a cell wall or even free in
solution. As apparent from the spectra in FIG. 4, the sensor
composition provided a method of determining the location of a
sensor within the cell spectroscopically.
[0099] Sophisticated equipment (cooled CCD, spectrometer, etc.) was
not necessary to obtain preliminary information from micron-sized
sensors used in this example. Indeed, the sensors could also be
illuminated with a mercury arc lamp, viewed with the aid of a
microscope, and easily photographed with a standard camera. The red
color of the sensors the cell was clearly visible inside the cell
walls. Also, the green sensors that were floating free in solution,
were easily seen. The ability to visually analyze the sensors
provided a quick way to find sensors in regions of interest and
also enabled the positioning of the sample for further spectral
analysis.
[0100] The spectra (FIG. 4) clearly showed that there was a
difference in pH between the intracellular environment and the
surrounding buffer solution. The sensor inside the neuroblastoma
cells produced a spectrum that was indicative of a relatively
acidic environment compared to that of the buffer. The buffer
solution, produced a spectrum indicating a relatively basic
environment compared to that inside the cell. The spectra taken was
that of a single sensor in a single cell, or a singe sensor
floating in the solution. This example illustrates that fiberless
optical sensors produced a fluorescence powerful enough even at a
size (1-2 .mu.m), and was small enough to fit inside a mammalian
cell without any apparent perturbation.
EXAMPLE 2
[0101] In this example, involves stimulating cells containing
fiberless sensors with an exogenous cellular stimulus and detecting
the sensors in the stimulated cells. In this example,
lipopolysaccharide (LAPS) is used as the exogenous stimulus. LAPS
is produced by a variety of pathogenic microorganisms. LAPS has
been shown to stimulate a variety of biochemical signaling pathways
in the immune system, skin, lung, brain, neuroendocrine and
neuro-immune axes, cardiac muscle and other mammalian systems.
Among other effects, LAPS induces oxidative injury and increases
cellular glutathione content. LAPS as the etiologic agent which
initiates ionic/solute cascades can be used.
[0102] Using lymphocytes, the alterations in the regional cellular
content of NO, Cl, Na+, H+ and glucose are monitored following
exposure to LAPS. Independent confirmation of changes in the levels
of ions/solute can be obtained by the use of pharmacologic agents
which are known to interact with specific channels or receptors for
the chemical species in question.
EXAMPLE 3
[0103] In this Example, fiberless sensors are introduced into
oocytes. The mouse oocyte provides a large "target" (45-80 um in
diameter) into which the fiberless optical sensors of the present
invention can be injected. The use of oocytes permits the
exploitation measurement of large ionic fluxes which may be
toxicologically and pharmacologically induced in the cell. Oocytes
possess several molecular mechanisms for the restoration of ionic
equilibria such as pH. Three major transport systems exist to
regulate the ionic strength and pH of the oocyte: (i) ATP-dependent
pumps, (ii) channels and carriers which facilitate diffusion of
ions across the plasmalemma, and (iii) exchanges which translocate
two or more ions down a gradient. In addition to these ionic
fluxes, the mouse oocyte has a number of second messenger systems
which are directly linked to maturation processes. These include
cAMP and inhibitors of cAMP signaling. The mammalian oocyte in situ
exists within a dynamic follicular environment to which it responds
with a myriad of intracellular ionic events.
[0104] Large numbers (20-30) of mouse oocytes can be obtained from
3-6 week-old mice. Briefly, 2-5 I.U. pregnant mares's serum
gonadotropin (PMSG, "Gestyl"; Organanon) and human chorionic
gonadotropin (hCG, Sigma) is administered i.p. hCG can be given
44-48 h after the priming dose of PMSG and the unfertilized ova
collected by flushing the freshly excised oviducts 12-14 h after
hCG. Oocytes collected can be used without further preparation in
Hank's Balanced Salt Solution (HBSS).
[0105] The fiberless sensors can be introduced into the oocytes
using the Biolistics Particle Delivery System (described above).
The sensors can be detected visually or spectrophotometrically.
EXAMPLE 4
[0106] In this example, measurements are made in neural cells
following an in vivo intravenous infusion of LAPS to mice. Briefly,
a solution of LAPS (50-1,000 ng) in physiological saline can be
injected into the femoral vein of the mouse. Physiological
processes are halted by irradiating the head with 10 kW microwave
for 0.5 sec (Cober Electronics Metabolic Vivostat, Conn.) and
immediate immersion in liquid nitrogen. Cerebella and hippocampi
are dissected and homogenized in deionized water for in vitro
analysis of ions using the fiberless optical sensors of the present
invention.
EXAMPLE 5
[0107] In this example, acrylamide fiberless sensors are
fabricated. The polymerization solution consisted of 0.4 mM
reactive dye, 27% acrylamide, 8% N,N-methylene-bis(acrylamide), and
in 0.1 M phosphate buffer, pH 6.5. To 1 mL of solution was added 20
.mu.L of N,N,N',N'-tetraethyldiethylenet- riamine and heated at
60.degree. C. for a few minutes. The resulting gel was allowed to
cool, and the excess liquid evaporate. The polymer was crushed with
the aid of liquid nitrogen.
[0108] A variety of reactive dyes (commercially available from
Molecular Probes, Inc.) can be used with the acrylamide sensors,
including but not limited to 5-(and 6-)-carboxynaphthofluorescein
("CNF"), 5-(and 6-)-carboxyfluorescein, SNAFL.TM.-dextran,
SNARF.TM.-dextran, HPTS-dextran, BCECF-dextran,
Fluorescein-dextran, Rhodal Green-dextran, DM-NERF-dextran, Oregon
Green-dextran, and Cl-NERF-dextran.
EXAMPLE 6
[0109] In this example, metal fiberless sensors are fabricated.
Such sensors are made by combining 0.1% protein or peptide (by
weight) in colloid solution. Spontaneous adsorption of the protein
to the metal surface takes place within minutes. A variety of
colloids can be used, including but not limited to, colloids of
gold, silver, platinum and gold/silver alloy. A variety of proteins
and peptides can also be used, including but not limited to
Cytochrome c' (isolated by Terry Meter and Michael Cusanovich,
University of Arizona), Horseradish Peroxidase (Sigma), Cytochrome
c (Sigma), Phycobiliproteins (Molecular Probes, Inc), Zinc fingers,
Dye-labeled proteins (Dyes available from Molecular Probes). Among
dye-labeled proteins, FITC, Fluorescein, Oregon
Green,Tetramethylrhodamine, Rhodamine Red and Texas Red can be
used.
[0110] From the above, it should be clear that the fiberless
optical sensors of the present invention offer a number of
advantages over existing technologies for the detection of
intracellular analytes. Importantly, the sensors permit monitoring
of the cell at more than one location with no (or minimal)
toxicity.
* * * * *