Molecular sieve abstract
A molecular sieve particle-based analytic chemistry system is disclosed
in which populations of encoded molecular sieve particles carrying
different chemical functionalities are distributed into wells etched
in an optical fiber bundle. The chemical functionalities are encoded
on separate shaped molecular sieve particles using luminescent dyes
and/or molecular sieve particle shapes and thus, a single sensor
array may carry thousands of chemistries. Such encoded molecular
sieve particles can provide at least a five-fold enhancement in
tunable parameters for increasing the encoding possibilities of
high throughput screening assays relative to the present dye-modified
polymeric microsphere standard.
Molecular sieve claims
What is claimed is:
1. A chemical analysis method, comprising preparing separate subpopulations
of shaped molecular sieve particles, said subpopulations carrying
chemical functionalities that change optical signatures of said
shaped molecular sieve particles in the presence of targeted analytes;
encoding optical signature of the shaped molecular sieve particles
in each subpopulation with a description of the chemical functionalities
carried by that subpopulation; combining the subpopulations to produce
a system; applying the system; detecting changes in the optical
signatures indicative of the presence of the targeted analytes,
and decoding the optical signature of the shaped molecular sieve
particles to identify the chemical functionalities.
2. The method of claim 1 wherein encoding the optical signatures
with the chemical functionalities comprises doping the shaped molecular
sieve particles with luminescent dyes.
3. The method of claim 1 wherein encoding the optical signatures
with chemical functionalities comprises attaching encoding dyes
to the shaped molecular sieve particles.
4. The method of claim 1 wherein encoding the optical signatures
with chemical functionalities comprises entrapping encoding dyes
within the shaped molecular sieve particles.
5. The method described in claim 1 further comprising: encoding
the shaped molecular sieve particles with the chemical functionalities
by entrapping dyes within or attaching dyes to the shaped molecular
sieve particles; and applying the chemical functionalities to the
shaped molecular sieve particles.
6. The method of claim 1 further comprising enabling the chemical
functionalities to produce an optically interrogatable species in
the presence of targeted analytes to change the optical signature.
7. The method of claim 1 further comprising changing the optical
signature by the presence or absence of a luminescent signal from
the shaped molecular sieve particles.
8. The method of claim 1 wherein said shaped molecular sieve particles
have pore sizes ranging from 0.5 to 50 nm.
9. The method of claim 1 wherein said shaped molecular sieve shaped
particles are selected from the group consisting of DAM-1 SBA-15
and MCM-41.
10. The method of claim 1 wherein said shaped molecular sieve
particles have a composition comprising silicon, aluminum, and oxygen
and combinations thereof.
11. An analytic chemistry sensor, comprising: a bundle of optical
fibers; and a population of shaped molecular sieve particles carrying
chemical functionalities at a terminal end of the fiber optic bundle.
12. The sensor of claim 11 wherein each of the shaped molecular
sieve particles is located within separate wells formed at terminal
ends of optical fibers of the bundle.
13. The sensor of claim 12 wherein the separate wells are formed
by anisotropic etching of the cores of the optical fibers with respect
to the cladding.
14. The sensor of claim 11 further comprising a source of electromagnetic
radiation for exciting optically interrogatable chemicals bound
to the chemical functionalities.
15. The sensor of claim 11 further comprising a source of electromagnetic
radiation for exciting optically interrogatable atoms that comprise
the shaped molecular sieve particle.
16. The sensor of claim 11 wherein the population of shaped molecular
sieve particles includes separate subpopulations, each subpopulation
carrying a different chemical functionality and an optically interrogatable
code descriptive of the chemical functionality.
17. The sensor of claim 16 further comprising a source of electromagnetic
radiation for exciting optically interrogatable chemicals bound
to the chemical functionalities.
18. The sensor of claim 16 further comprising a source of electromagnetic
radiation for exciting optically interrogatable atoms that comprise
the shaped molecular sieve particle.
19. The sensor described in claim 16 wherein the code of each
subpopulation comprises luminescent dyes.
20. The sensor described in claim 16 wherein the code of each
subpopulation comprises a unique shaped molecular sieve particle.
21. The sensor described in claim 16 further comprising a filter
and a frame capturing camera for detecting optical signatures indicative
of a status of the chemical functionalities and optical signatures
indicative of the encoding of the shaped molecular sieve particles.
22. The sensor of claim 11 wherein said shaped molecular sieve
particles have pore sizes ranging from 0.5 to 50 nm.
23. The sensor of claim 11 wherein said shaped molecular sieve
shaped particles are selected from the group consisting of DAM-1
SBA-15 and MCM-41.
24. The sensor of claim 11 wherein said shaped molecular sieve
particles have a composition comprising silicon, aluminum, and oxygen
and combinations thereof.
25. A method for constructing and using an analytic chemistry sensor,
comprising: forming wells at terminal ends of optical fibers within
a bundle; distributing shaped molecular sieve particles carrying
chemical functionalities within the wells; and, monitoring a status
of the chemical functionalities from a proximal end of the bundle.
26. The method described in claim 25 wherein forming the wells
comprises anisotropically etching of cores of the optical fibers
with respect to cladding.
27. The method described in claim 25 further comprising forming
a population of shaped molecular sieve particles in the wells from
separate subpopulations, each subpopulation carrying a different
chemical functionality and an optically interrogatable code descriptive
of the chemical functionality.
28. The method described in claim 27 further comprising randomly
distributing the subpopulations within the wells.
29. The method described in claim 27 further comprising serially
adding the subpopulations to the wells.
30. The method of claim 27 wherein said shaped molecular sieve
particles have pore sizes ranging from 0.5 to 50 nm.
31. The method of claim 27 wherein said shaped molecular sieve
shaped particles are selected from the group consisting of DAM-1
SBA-15 and MCM-41.
32. The method of claim 27 wherein said shaped molecular sieve
particles have a composition comprising silicon, aluminum, and oxygen
and combinations thereof.
33. A method for constructing and using an analytic chemistry sensor,
comprising: forming wells at terminal ends of optical fibers within
a bundle; distributing shaped molecular sieve particles carrying
chemical functionalities within the wells; and, monitoring a status
of a molecular sieve particle shape from an end face of the bundle.
34. A composition comprising a plurality of optical fibers in an
optical fiber array and a population of shaped molecular sieve particles,
wherein said optical fibers have wells at a first terminal end of
said fibers and a plurality of said wells contain at least one shaped
molecular sieve particle.
35. A composition comprising: a) a substrate; b) a population of
shaped molecular sieve particles comprising separate subpopulations,
each subpopulation comprising: i) a chemical functionality for testing
for interaction with a target analyte; and ii) an encoding optical
signature that can be used to identify said chemical functionality;
wherein said shaped molecular sieve particles are distributed on
said substrate.
36. A composition according to claim 35 wherein said substrate
is an optical fiber array comprising a plurality of individual fibers
and said shaped molecular sieve particles are located within wells
at a first terminal end of said fibers.
37. A composition according to claim 35 wherein said encoding optical
signature comprises at least one chromophore.
38. A composition according to claim 35 wherein said encoding optical
signature comprises at least one luminescent dye.
39. A composition according to claim 35 wherein said encoding optical
signature comprises a unique molecular sieve particle shape.
40. A composition according to claim 38 wherein said luminescent
dye is entrapped within said shaped molecular sieve particles.
41. A composition according to claim 38 wherein said luminescent
dye is attached to said shaped molecular sieve particles.
42. A composition according to claim 35 wherein at least a first
subpopulation of shaped molecular sieve particles is a different
pore size than a second subpopulation of shaped molecular sieve
particles.
43. A composition according to claim 35 wherein the individual
fibers of said array are of uniform size ranging from 200 nanometers
to 100 micrometers and shape including but not limited to circles
and hexagons.
44. A composition according to claim 35 wherein the individual
fibers of said array are of a plurality of sizes ranging from 200
nanometers to 100 micrometers.
45. A composition according to claim 35 wherein the shapes of
said individual fibers are selected from the group consisting of
circles and hexagons.
46. A composition according to claim 35 wherein said chemical functionality
is selected from a group consisting of nucleic acids, oligonucleotides,
peptides, proteins, enzymes, antigens, antibodies, receptors, ligands,
luminophores, fluorophores, chromophores, phosphors, pH indicators,
cation indicators, anion indicators, metal ion indicators, reactive
oxygen species indicators, nitric oxide indicators, oxygen indicators
and carbon dioxide indicators.
47. A composition according to claim 35 further comprising at least
one target analyte interacting with at least one said chemical functionality.
48. A composition according to claim 47 wherein said target analyte
is selected from a group consisting of nucleic acids, oligonucleotides,
peptides, proteins, enzymes, antigens, antibodies, receptors, ligands,
luminophores, fluorophores, chromophores, phosphors, pH indicators,
cation indicators, anion indicators, metal ion indicators, reactive
oxygen species indicators, nitric oxide indicators, oxygen indicators
and carbon dioxide indicators.
49. A composition according to claim 35 wherein said shaped molecular
sieve particles have pore sizes ranging from 0.5 to 50 nm.
50. A composition according to claim 35 wherein said shaped molecular
sieve particles are selected from a group consisting of DAM-1 SBA-15
and MCM-41.
51. A composition according to claim 35 wherein said shaped molecular
sieve particles have a composition comprising silicon, aluminum,
and oxygen and combinations thereof.
52. A method of determining the presence of a target analyte in
a sample comprising: a) contacting said sample with a composition
comprising: i) a substrate; ii) a population of shaped molecular
sieve particles comprising separate subpopulations, each subpopulation
comprising: 1) a chemical functionality for testing for interaction
with a target analyte; and 2) an encoding optical signature that
can be used to identify said chemical functionality; wherein said
shaped molecular sieve particles are distributed on said substrate;
and b) determining the presence or absence of the target analyte.
53. A method according to claim 52 wherein said substrate is an
optical fiber array comprising a plurality of individual fibers
and said shaped molecular sieve particles are located within wells
at a first terminal end of said fibers.
54. A method according to claim 52 further comprising identifying
the location of each subpopulation on said substrate.
55. A method according to claim 52 further comprising identifying
the shape of molecular sieve particles.
56. A method according to claim 52 wherein said encoding optical
signature comprises at least one chromophore.
57. A method according to claim 52 wherein said encoding optical
signature comprises at least one luminescent dye.
58. A method according to claim 52 wherein said encoding optical
signature comprises a unique shaped molecular sieve particle.
59. A method according to claim 57 wherein said luminescent dye
is entrapped within said shaped molecular sieve particles.
60. A method according to 57 wherein said luminescent dye is attached
to said shaped molecular sieve particles.
61. A method according to claim 52 wherein at least a first subpopulation
of molecular sieve particles is a different shape than a second
subpopulation of molecular sieve particles.
62. A method according to claim 52 wherein said shaped molecular
sieve particles have pore sizes ranging from 0.5 to 50 nm.
63. A method according to claim 52 wherein said shaped molecular
sieve particles are selected from a group consisting of DAM-1 SBA-15
and MCM-41.
64. A method according to claim 52 wherein said shaped molecular
sieve particles have a composition comprising silicon, aluminum,
and oxygen and combinations thereof.
65. A method according to claim 53 wherein the individual fibers
of said array are of uniform size ranging from 200 nanometers to
100 micrometers and shape including but not limited to circles and
hexagons.
66. A method according to claim 53 wherein the individual fibers
of said array are a plurality of sizes ranging from 200 nanometers
to 100 micrometers.
67. A method according to claim 53 wherein the shapes of said individual
fibers are selected from the group consisting of circles and hexagons.
68. A method according to claim 52 wherein said chemical functionality
is selected from a group consisting of nucleic acids, oligonucleotides,
peptides, proteins, enzymes, antigens, antibodies, receptors, ligands,
luminophores, fluorophores, chromophores, phosphors, pH indicators,
cation indicators, anion indicators, metal ion indicators, reactive
oxygen species indicators, nitric oxide indicators, oxygen indicators
and carbon dioxide indicators.
69. A method of making a composition comprising: a) forming wells
at a terminal end of an optical fiber array; and b) distributing
shaped molecular sieve particles within said wells, wherein said
shaped molecular sieve particles comprise separate subpopulations,
each subpopulation comprising: i) a chemical functionality for testing
for interaction with a target analyte; and ii) an encoding optical
signature that can be used to identify said chemical functionality.
70. A method according to claim 69 wherein said forming comprises
anisotropically etching the cores of the individual fibers of said
array.
71. A method according to claim 69 wherein said distributing comprises
serially adding said subpopulations to said wells.
72. A method according to claim 69 wherein said distributing comprises
randomly distributing said subpopulations to said wells.
73. A sensor comprising: a) an array of optical fibers; b) a population
of shaped molecular sieve particles comprising separate subpopulations,
each subpopulation comprising: i) a chemical functionality for testing
for interaction with a target analyte; and ii) an encoding optical
signature that can be used to identify said chemical functionality;
wherein said shaped molecular sieve particles are distributed on
a first terminal end of said array; and c) a source of electromagnetic
radiation.
74. A sensor according to claim 73 further comprising a detector
of electromagnetic radiation.
75. A sensor according to claim 73 wherein the shaped molecular
sieve particles have pore sizes ranging from 0.5 to 50 nm.
76. A sensor according to claim 73 wherein the shaped molecular
sieve particles are selected from the group consisting of DAM-1
SBA-15 and MCM-41.
77. A sensor according to claim 73 wherein said shaped molecular
sieve particles have a composition comprising silicon, aluminum,
and oxygen and combinations thereof.
Molecular sieve description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application
Serial No. 60/269909 which is fully incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field of the Invention
[0003] The present invention relates to the use of encoded molecular
sieve particles in an optical sensor analytical system.
[0004] 2. Description of the Prior Art
[0005] The use of optical fibers and optical fiber strands in combination
with light absorbing dyes for chemical analytical determinations
has undergone rapid development, particularly within the last two
decades. Many of the recent improvements employing optical fiber
sensors in both qualitative and quantitative analytical determinations
concern the desirability of depositing and/or immobilizing various
light absorbing dyes at the distal end of the optical fiber. In
this manner, a variety of different optical fiber chemical sensors
and methods have been reported for specific analytical determinations
and applications such as pH measurement, oxygen detection, and carbon
dioxide analyses.
[0006] Fiber optic sensors have been constructed that permit the
use of multiple dyes with a single, discrete fiber optic bundle.
U.S. Pat. Nos. 5244636 and 5250264 to Walt et al. disclose systems
for affixing multiple, different dyes on the distal end of the bundle,
the teachings of each of these patents being incorporated herein
by this reference. The innovation of these patents is the placement
of multiple chemical functionalities at the end of a single optical
fiber bundle sensor. This configuration yields an analytic chemistry
sensor that can be remotely monitored via the typically small bundle.
The drawback, however, is the difficulty in applying the various
chemistries associated with the chemical functionalities at the
sensor's end; and, the functionalities are built on the sensor's
end in a serial fashion. Not only is this a slow process, but in
practice, only tens of functionalities can be applied.
[0007] U.S. Pat. No. 6023540 by Walt et al. discloses a microsphere-based
analytic chemistry system and method for making the same is disclosed
in which microspheres or particles carrying bioactive agents are
combined randomly or in ordered fashion and dispersed on a substrate
to form an array while maintaining the ability to identify the location
of bioactive agents and particles within the array using an optically
interrogatable, optical signature encoding scheme. As a preferred
embodiment, U.S. Pat. No. 6023540 teaches the use of a modified
fiber optic bundle or array as a substrate to produce a high density
array. The disclosed system and method have utility for detecting
target analytes and screening large libraries of bioactive agents.
The teachings of U.S. Pat. No. 6023540 are fully incorporated
by reference herein.
[0008] In brief, the main limitation to present state-of-the-art
technology whether it be through the use of microspheres, microbeads
or particles, is the limited number of methods available to encode
the array. Currently, polymeric-based microbeads are encoded by
immobilized luminescent dyes only. In addition, there is a physical
limitation to how many ultraviolet, visible, and near-infrared dyes
can be used simultaneously to encode an array since the emission
spectra of luminescence dyes are broad. Furthermore, present state-of-the-art
technology utilizes only spherical microbeads. While an optional
encoding avenue would be the use of microbeads with different diameters,
this approach is limited by the difficulty in fabricating a large-scale
batch of microbeads with a tight and uniform bead diameter distribution
(which is the only way a plurality of spherical microbeads with
different diameters could be employed in a reliable optical size-encoding
scheme). In other words, present state-of-the-art microparticle-based
analytical systems focus on the microparticle's chemical functionality
and luminescent signature only. Therefore, while a small variety
of silica-based and polymeric microspheres materials have been utilized,
none of these microspheres offer size and shape selectivity.
[0009] The present invention represents an improvement over U.S.
Pat. No. 6023540 as well as other comparable flow cytometric and
fiber-optic sensor systems using microbeads, microspheres and/or
microparticles. The key feature of the improvement is the added
analytical performance features provided by shaped molecular sieve
particles, namely optical encoding based on the molecular sieve
particles' macroscopic geometric shapes and increased selectivity
based on the molecular sieves particles' molecular-sized pore diameters
i.e., pore sizes. Such encoded molecular sieve particles can provide
at least a five-fold enhancement in tunable parameters for increasing
the encoding possibilities of high throughput screening assays relative
to the present dye-modified polymeric microsphere, microbead or
microparticle standards.
SUMMARY OF THE INVENTION
[0010] It is an object of the present invention to provide a molecular
sieve particle-based fiber-optic microwell array sensor, wherein
a microwell array is etched onto an optical fiber bundle that is
filled with molecular sieve particles having specific morphologies
and pore sizes. The molecular sieve particles can be further modified
with guest molecules including but not limited to, dyes, peptides,
proteins, enzymes, antigens, antibodies, receptors, ligands, catalysts,
nucleic acids, and oligonucleotides to form the basis of an optical
chemical sensor or biosensor. These molecular sieve particle-based
fiber-optic microwell array sensors also form the basis for combinatorial
encoding and/or analysis. It is another object of the present invention
to provide methods for synthesizing said molecular sieve particle-based
fiber-optic microwell array sensors.
[0011] A key feature of the invention is the added analytical performance
features provided to the microwell sensors by the shaped molecular
sieve particles--namely optical encoding based on the molecular
sieve particles' macroscopic geometric shapes and increased selectivity
based on the molecular sieves particles' molecular-sized pore diameters.
The term "shaped molecular sieve particles" as used herein
encompasses the macroscopic geometric shapes of the particles as
well as their molecular-sized pore diameters. In addition to the
ability to control and tune molecular sieve particle porosity, molecular
sieve particles can be molecularly imprinted to further enhance
selectivity to include the detection of analytes such as chiral/optically
active molecules.
[0012] One key feature of the encoded molecular sieve particle-based
optical sensor analytical system is the added optical encoding possibilities
resulting from the plurality of the molecular sieve particle macroscopic
geometric shapes that can be synthesized. In addition to spheres,
molecular sieve particles can be synthesized with distinct gyroidal,
discoidal, and hexagonal cylindrical shapes to increase the parameters
by which optical shape-based encoding can be performed. Furthermore,
the high surface area of the shaped molecular sieve particles enables
encoding and detection in single microwells that is not possible
with plastic beads or amphorous silica.
[0013] Perhaps the most intriguing aspect of the molecular sieve
particle-based fiber-optic microwell array sensor approach is the
differences in the atomic compositions of molecular sieve materials
in comparison to silica-based and polymeric microsphere materials.
Specifically, silica-based and polymeric microspheres materials
are comprised mainly of low atomic weight atoms such as carbon,
hydrogen, nitrogen, oxygen, and silicon. Conversely, molecular sieve
materials can be synthesized with a variety of atoms such as aluminum,
titanium, iron, nickel, cobalt, germanium, gallium, boron, tin,
selenium and other metals, metalloids, and non-metals. A variety
of atoms can permit a number of new and alternative methods to be
utilized for array encoding such as optical encoding by spectroscopic
absorption techniques and/or energy dispersive and wavelength dispersive
x-ray fluorescence techniques. Such approaches have the advantage
of allowing the same limited number of luminescent dyes and the
same number of macroscopic geometric shapes to be used multiple
times, and thus, the total possible number of encoding combinations
could be increased substantially.
[0014] One embodiment of the invention provides a chemical analysis
method, comprising
[0015] preparing separate subpopulations of shaped molecular sieve
particles, each subpopulation carrying chemical functionalities
that change optical signatures of said shaped molecular sieve particles
in the presence of targeted analytes;
[0016] encoding optical signature of the shaped molecular sieve
particles in each subpopulation with a description of the chemical
functionalities carried by that subpopulation;
[0017] combining the subpopulations to produce a system;
[0018] applying the system;
[0019] detecting changes in the optical signatures indicative of
the presence of the targeted analytes; and
[0020] decoding the optical signature of the shaped molecular sieve
particles to identify the chemical functionalities.
[0021] Another embodiment of the invention provides an analytic
chemistry sensor, comprising:
[0022] a bundle of optical fibers; and
[0023] a population of shaped molecular sieve particles carrying
chemical functionalities at a terminal end of the fiber optic bundle.
The terminal end may be the distal end or the proximal end of the
optic fiber bundle.
[0024] Another embodiment of the invention provides the analytic
chemistry sensor further comprising a source of electromagnetic
radiation for exciting optically interrogatable chemicals bound
to the chemical functionalities or for exciting optically interrogatable
atoms that comprise the shaped molecular sieve particle.
[0025] An embodiment of the invention provides a method for constructing
and using an analytic chemistry sensor, comprising:
[0026] forming wells at terminal ends of optical fibers within
a bundle;
[0027] distributing shaped molecular sieve particles carrying chemical
functionalities within the wells; and;
[0028] monitoring a status of the chemical functionalities from
an end face of the bundle.
[0029] A Another embodiment of the invention provides a method
for constructing and using an analytic chemistry sensor, comprising:
[0030] forming wells at terminal ends of optical fibers within
a bundle;
[0031] distributing shaped molecular sieve particles carrying chemical
functionalities within the wells; and;
[0032] monitoring a status of the chemical functionalities from
a proximal end of the bundle.
[0033] Another embodiment of the invention provides a composition
comprising a plurality of optical fibers in an optical fiber array
and a population of shaped molecular sieve particles, wherein said
optical fibers have wells at a first terminal end of said fibers
and a plurality of said wells contain at least one shaped molecular
sieve particle.
[0034] Yet another embodiment of the invention provides a method
of determining the presence of a target analyte in a sample comprising:
[0035] a) contacting said sample with a composition comprising:
[0036] i) a substrate;
[0037] ii) a population of shaped molecular sieve particles comprising
separate subpopulations, each subpopulation comprising:
[0038] 1) a chemical functionality for testing for interaction
with a target analyte; and
[0039] 2) an encoding optical signature that can be used to identify
said chemical functionality;
[0040] wherein said shaped molecular sieve particles are distributed
on said substrate; and
[0041] b) determining the presence or absence of the target analyte.
[0042] An embodiment of the invention also provides a method of
making a composition comprising:
[0043] a) forming wells at a terminal end of an optical fiber array;
and
[0044] b) distributing shaped molecular sieve particles within
said wells, wherein said shaped molecular sieve particles comprise
separate subpopulations, each subpopulation comprising:
[0045] i) a chemical functionality for testing for interaction
with a target analyte; and
[0046] ii) an encoding optical signature that can be used to identify
said chemical functionality.
[0047] An embodiment of the invention further provides a sensor
comprising:
[0048] a) an array of optical fibers;
[0049] b) a population of shaped molecular sieve particles comprising
separate subpopulations, each subpopulation comprising:
[0050] i) a chemical functionality for testing for interaction
with a target analyte; and
[0051] ii) an encoding optical signature that can be used to identify
said chemical functionality;
[0052] wherein said shaped molecular sieve particles are distributed
on a first terminal end of said array; and
[0053] c) a source of electromagnetic radiation.
[0054] An embodiment of the invention provides the optical fiber
sensor further comprising a detector of electromagnetic radiation.
[0055] An embodiment of the invention provides a method for constructing
and using an analytic chemistry sensor, comprising:
[0056] forming wells at terminal ends of optical fibers within
a bundle;
[0057] distributing shaped molecular sieve particles carrying chemical
functionalities within the wells; and;
[0058] monitoring a status of a molecular sieve particle shape
from an end face of the bundle.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] The following drawings 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 drawings in combination with the detailed description
of the invention presented herein.
[0060] FIG. 1 shows a schematic representation of the molecular
sieve particle-based fiber-optic microwell array sensor fabrication
process. In Step 1 molecular sieve particles are filled with luminescent
dye; in step 2 a polished fiber-optic bundle is etched to create
a microwell array; and in step 3 dye-modified molecular sieve particles
are deposited across the microwell array's distal face.
[0061] FIG. 2 shows a scanning electron micrograph of a gyroid-shaped
DAM-1 molecular sieve particle in an individual microwell. The white
bar denotes 5 micrometers.
[0062] FIG. 3 shows a scanning electron micrograph of a hexagonal-cylinder
shaped DAM-1 molecular sieve particle in an individual microwell.
The white bar denotes 2 micrometers.
[0063] FIG. 4 shows a scanning electron micrograph of a spherical-shaped
SBA-15 molecular sieve particle in an individual microwell. The
black bar denotes 2 micrometers.
[0064] FIG. 5 represents a fluorescence image (4.times.) of the
distal face of a DAM-1 molecular sieve particle-based fiber-optic
microwell array sensor immersed in pH 8.5 phosphate buffer solution;
white represents high intensities.
[0065] FIG. 6 represents (A) white light and (B) fluorescence images
(4.times.) of the same distal face region of a DAM-1 molecular sieve
particle-based fiber-optic microwell array sensor immersed in pH
8.5 phosphate buffer solution; white represents high intensities.
[0066] FIG. 7 represents images of the same distal face region
of a DAM-1 molecular sieve particle-based fiber-optic microwell
array sensor: (A) White light image (4.times.); (B) Fluorescence
image (4.times.) with the sensor immersed in pH 8.5 phosphate buffer
solution; (C) Fluorescence image (4.times.) with the sensor immersed
in pH 4.5 phosphate buffer solution; (D) Scanning electron micrograph.
The fluorescence images in (B) and (C) were normalized to have the
same grayscale; white represents high intensities.
[0067] FIG. 8 shows a scanning electron micrograph of the same
DAM-1 molecular sieve particle-based fiber-optic microwell array
sensor as shown in FIG. 7(D). The white bar denotes 5 micrometers.
[0068] FIG. 9 represents a molecular sieve particle-based microwell
array chemical sensor (MWACS) where the gray shading denotes the
chemical sensing (polymer+dye) layer and the pores of the hexagonal-cylinder
shaped molecular sieve particle are parallel with respect to the
optical fiber/microwell's core.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0069] The present invention is generally concerned with molecular
sieve particle-based fiber-optic microwell arrays, methods for synthesizing
said arrays and practical applications of the same. The molecular
sieve particle-based fiber-optic microwell arrays of the present
invention can be efficacious in high throughput screening and combinatorial
chemistry-related applications. The ability of shaped molecular
sieve particles to provide additional selectivity to the sensor,
makes the sensor a powerful tool in combinatorial encoding and/or
analytical applications.
[0070] In general, according to one aspect, the invention concerns
an analytic chemistry system that comprises a population of shaped
molecular sieve particles. Within the population may be separate
subpopulations, each of which carries a chemical functionality which
changes the optical signature of the shaped molecular sieve particles
in the presence of targeted analytes. This signature change can
occur via many different mechanisms. A few examples include the
binding of a dye-tagged analyte to the shaped molecular sieve particle,
the production of a dye species on or near the shaped molecular
sieve particle, the destruction of an existing dye species, a change
in optical signal upon analyte interaction with dye within the shaped
molecular sieve particle, or any other optically interrogatable
event. Although the subpopulations may be randomly mixed together,
the chemical functionality associated with each shaped molecular
sieve particle is determined via an optical signature which is encoded
with a description of the chemical functionality. As a result, by
observing whether the optical signature of a particular shaped molecular
sieve particle is exhibiting a change, or not, and then decoding
the signature for the functionality of the shaped molecular sieve
particle, the presence or absence of the analyte targeted by the
functionality may be determined. Examples of chemical functionalities
associated with molecular sieve particles include, but are not limited
to, nucleic acids, oligonucleotides, peptides, proteins, enzymes,
antigens, antibodies, receptors, ligands, luminophores, fluorophores,
chromophores, phosphors, pH indicators, cation indicators, anion
indicators, metal ion indicators, reactive oxygen species indicators,
nitric oxide indicators, oxygen indicators and carbon dioxide indicators.
Examples of target analytes include, but are not limited to nucleic
acids, oligonucleotides, peptides, proteins, enzymes, antigens,
antibodies, receptors, ligands, luminophores, fluorophores, chromophores,
phosphors, acids, bases, cations, anions, metal ions, reactive oxygen
species, nitric oxide, oxygen and carbon dioxide.
[0071] In specific embodiments, the shaped molecular sieve particles
are encoded using dyes that are preferably entrapped within the
shaped molecular sieve particles, the chemical functionality being
added on to the surfaces. The dyes may be chromophores or phosphors
but are preferably fluorescent dyes, which due to their strong signals
provide a good signal-to-noise ratio for decoding. The encoding
may be accomplished by one or more dyes (using a variety of dye
concentration ratios), and more encoding dimensions may be added
in the shapes of the molecular sieve particles and pore sizes, for
example.
[0072] According to another aspect, the invention also concerns
an analytic chemistry fiber optic bundle sensor. This sensor has
a population of molecular sieve particles carrying chemical functionalities
at, on or near, a distal end of the bundle. In an embodiment of
the invention, an array of microwells are etched into the distal
face of an optical fiber bundle and filled with molecular sieve
particles having specific morphologies and pore sizes. The ability
to monitor optical signature changes associated with individual
or multiple molecular sieve particles is provided by coupling those
signature changes into separate optical fibers or groups of fibers
of the bundle for transmission to the proximal end where analysis
is performed either manually, by the user, or automatically, using
image processing techniques. When the molecular sieve particles
are imaged directly, determination of molecular sieve particles'
macroscopic geometric shapes can be performed either manually, by
the user, or automatically, using image processing techniques.
[0073] In a preferred embodiment, each one of the shaped molecular
sieve particles is located within separate wells formed at terminal
ends of optical fibers of the bundle. These microwells are formed
by anisotropic etching of the cores of the optical fibers with respect
to the cladding according to a previously detailed procedure. See
Pantano and Walt, Chem. Mater. 8: 2832 (1996).
[0074] In another embodiment, the shaped molecular sieve particles
include separate subpopulations, typically randomly distributed
in an array across the bundle end, each subpopulation carrying a
different chemical functionality and an optically interrogatable
code descriptive of the chemical functionality.
[0075] In another embodiment of the invention, said shaped molecular
sieve particles are further modified with guest molecules or chemical
functionalities including but not limited to dyes, peptides, proteins,
enzymes, antigens, antibodies, receptors, ligands, catalysts and
oligonucleotides to form the basis of an optical chemical sensor
or biosensor. In yet another embodiment of the present invention,
the molecular sieve particle-based fiber-optic microwell array sensors
form the basis for combinatorial encoding and/or analytical applications.
Useful shaped molecular sieve particles used in the synthesis of
the molecular sieve particle-based fiber-optic microwell array sensors
of the present invention include, but are not limited to, Dallas
Amorphous Material-1 (DAM-1), Mobil Composition of Matter-41 (MCM-41),
and Santa Barbara-15 (SBA-15) type materials having silica and transition
metal oxide compositions. DAM-1 molecular sieve particles can be
synthesized in spherical, gyroidal, discoidal, and hexagonal-cylindrical
shapes with pore diameters of .about.60 angstroms, SBA-15 molecular
sieve particles can be synthesized in spherical shapes with pore
diameters of .about.100 angstroms, and MCM-41 has been prepared
as hexagons, spheres, discoids, gyroids, tubules, rods, and helicoids
with pore diameters of .about.40 angstroms.
[0076] In an embodiment of the invention, fabrication of the molecular
sieve particle-based fiber-optic microwell array sensors is accomplished
by distribution of the shaped molecular sieve particles within the
microwells, which can be accomplished in a variety of ways. For
example, in particular embodiments of the invention, adsorption
or sedimentation methods are used to distribute the shaped molecular
sieve particles within the microwells followed by heating steps
to improve retention in microwells. Such heating treatments are
not practical with plastic beads. For example, in particular embodiments
of the invention, the host and/or guest surfaces of the molecular
sieve particle are chemically modified to improve retention in microwells.
In particular embodiments of the invention, the microwell interior
is chemically modified to improve molecular sieve particle retention
in microwells. In another embodiment, molecular sieve particles
can be grown in situ in the microwells. In all cases, the retention
of the molecular sieve particle in the well is an improvement over
the prior art where microbead retention in the well relies upon
microbead swelling, or electrostatic attractions, or the deposition
of a thin polymeric film across the bead array. The array of dye-modified
molecular sieve particles are optically addressed entirely or individually
by an epifluorescence microscopy/charge coupled device (CCD) imaging
system, and/or a scanning electron microscope, and/or energy dispersive
or wavelength dispersive X-ray fluorescence analyzer, and/or other
spectroscopic imaging systems known in the art.
[0077] Although each sensor is different insofar that it has a
different distribution of the subpopulations of molecular sieve
particles within its wells, only those shaped molecular sieve particles
that exhibit a positive optical response or signature change need
to be decoded. Therefore, the burden is placed on the analysis rather
than on sensor manufacture. Moreover, since the molecular sieve
particles and fibers in the array can be monodisperse, the luminescent
regions arising from signal generation are extremely uniform and
can be analyzed automatically using commercially available microscopy
analysis software, such image processing software is capable of
defining different spectral regions automatically and counting the
number of segments within each region in several seconds.
[0078] The above and other features of the invention including
various novel details of construction and combinations of parts,
and other advantages, will now be more particularly described with
reference to the accompanying drawings and pointed out in the claims.
It will be understood that the particular method and device embodying
the invention are shown by way of illustration and not as a limitation
of the invention. The principles and features of this invention
may be employed in various and numerous embodiments without departing
from the scope of the invention.
WORKING EXAMPLES
[0079] In an embodiment of the invention, the molecular sieve particle-based
fiber-optic microwell array sensor of the present invention is fabricated
by a three step process as illustrated in FIG. 1. First, molecular
sieve particles are synthesized, the excess templating molecules
used in the synthesis of the molecular sieve particles are removed,
and pH-sensitive fluorescent dye (fluorescein isothiocyanate; FITC)
is entrapped in the sieves' pores, as described in Step 1 of FIG.
1. A fiber-optic bundle is polished and its distal face reacted
in a hydrofluoric acid etching solution to create a microwell array
as described in Step 2 of FIG. 1. Finally, the dye-modified molecular
sieve particles are suspended in water and dispensed onto the distal
face of the microwell array to create a molecular sieve particle-based
fiber-optic microwell array sensor as described in Step 3 of FIG.
1.
[0080] The molecular sieve particles used in the fabrication of
the present invention are prepared according to published procedures.
The synthesis and characterization of Dallas Amorphous Material-1
(DAM-1) molecular sieve particles is described in "Preparation
of DAM-1 Materials" by Balkus, Jr., K. J., Ma, Y., and Coutinho,
D. H. published in Mater. Res. Soc. Symp. Proc, 2001 662 NN65.
SBA-15 and MCM-41 are prepared according to published procedures.
See Zhao et. al., Science, 279: 548 (1998) and Kresge et. al., Nature,
359: 710 (1992) respectively. The molecular sieve particles are
calcinated to remove the excess template used in their synthesis.
Fluorescein isothiocyanate (FITC) is incorporated into the molecular
sieve particles by soaking the particles in FITC for 1 h as described
in Step 1 of FIG. 1. The FITC-modified molecular sieve particles
are filtered and washed extensively with water before further processing.
[0081] The fabrication of a microwell array is carried out on an
optical imaging fiber's distal face, as described in Step 2 of FIG.
1. A high-resolution imaging fiber (i.e., a coherent fiber-optic
bundle) comprises thousands of micrometer-sized, coherently-fused
optical fibers. A differential core/clad etching rate leads to microwell
formation. For a given core diameter, microwell volume is controlled
by the etch reaction time which in turn controls microwell depth.
There are several methods to control the diameters of cores. One
method utilizes a standard glass pipette puller to taper the imaging
fiber before the etching step. Standard pullers are suitable for
soft-glass imaging fibers while laser-based pipette pullers are
required for all-silica imaging fibers. A second approach takes
advantage of the large variety of core shapes and diameters of commercially
available imaging fibers. This flexibility provides a method to
tailor the size of individual microwells to accommodate a variety
of different sized molecular sieve particles. The imaging fiber
used is preferably a high-resolution 3.2-mm diameter imaging fiber
from Edmund Scientific. This inexpensive .about.50000 count bundle
comprises .about.8 micron wide hexagonal cores.
[0082] Microwell array preparation is carried out by cleaving (90.degree.
scribing angle) a desired length of imaging fiber with a sapphire
scribe or a fine metal file. The roughly cleaved imaging fiber faces
are polished by hand with 320- and 600-grit sandpaper. The imaging
fiber was secured in an appropriate fiber chuck and both faces are
polished successively on lapping films of 12-, 3-, and 1-micron
abrasive sizes. Polishing is accomplished by maneuvering a secured
fiber's face in a figure eight-like fashion over each wetted abrasive
surface. Imaging fiber faces were sonicated in water between each
successive film to remove fiber and abrasive particulates. All polished
imaging fiber faces are inspected under a stereo-zoom microscope
for scratches.
[0083] The polished distal imaging fiber face is suspended in the
hydrogen fluoride (HF)etching solution for 1-4 min as described
in Step 2 of FIG. 1. The HF-etching solution (15% ammonium bifluoride
(NH.sub.4F:HF)) is prepared by mixing 2.5 parts (v/v) of a 40% aqueous
NH.sub.4F solution, 1.2 parts (v/v) of a 49% aqueous HF solution,
and 1.0 part (v/v) deionized water. This 33% (w/w) NH.sub.4F:HF
solution is diluted with deionized water to produce a 15% (w/w)
NH.sub.4F:HF solution. The fiber is removed from the HF-etching
solution and quickly quenched in deionized water. The fiber is sonicated
in methanol for 1-4 min where the sonication intensity is regulated
to .about.70% full power using a variable autotransformer. The exact
sonication time is determined by periodic visual inspection of the
microwell array; sonication is stopped after the microwells were
clear of glass salts and core residuals. This procedure yields flat
bottom wells with the same .about.8-micron side-to-side dimensions
as the original cores; the efficiency of the well formation across
the distal imaging fiber face is .about.99%. The core etch rate
is .about.1.15 microns/min and well depths of 1-14 microns can be
obtained by varying the etch time.
[0084] A 10 microliter-aliquot of a thoroughly-mixed 2.5% (v/v)
aqueous solution of FITC-modified molecular sieve particles is dispersed
onto a vertically-positioned microwell array face. The particle-covered
array is allowed to stand for 30 min and excess particles are removed
by deionized water rinsing. Scanning electron microscopy is utilized
to characterize molecular sieve particle-based fiber-optic microwell
array sensors. Scanning electron microscopy is preferably performed
at <20 keV. Samples are sputter coated with a .about.20 nm thick
gold layer. FIG. 2 shows a gyroid-shaped DAM-1 molecular sieve particle
in an individual microwell. FIG. 3 shows a hexagonal-cylinder shaped
DAM-1 molecular sieve particle in an individual microwell. FIG.
4 shows a spherical-shaped SBA-15 molecular sieve particle in an
individual microwell.
[0085] A modified epifluorescence microscope system (Labophot 1A;
Nikon) is used for white-light and luminescence imaging with the
proximal polished face of the fiber-optic bundle mounted to the
microscope stage and the etched face of the fiber-optic bundle (i.e.,
the microwell array face) immersed in buffer. White-light and luminescence
imaging can also be performed with the microwell array face mounted
to the microscope stage to optically determine molecular sieve particle
shapes. Phosphate buffer solutions (PBSs; pH 4.5-8.5) were prepared
by mixing appropriate proportions of a 5 mM dibasic sodium phosphate/100
mM KCl solution with a 5 mM monobasic sodium phosphate/100 mM KCl
solution. The collimated radiation from a 75-W xenon-arc lamp was
passed through two neutral density filters (ND2+ND4) to control
the excitation intensity. The radiation was passed through a 485
nm excitation filter, reflected by the 505 nm dichroic mirror, and
focused on the imaging fiber's proximal face by a 4.times. or a
40.times. microscope objective. The light is transmitted through
the imaging fiber to the distal imaging fiber face (i.e., the microwell
array face) where it excites the FITC-modified molecular sieve particles.
The fluorescence is collected by the same imaging fiber and microscope
objective, transmitted through the same dichroic mirror, and filtered
by a 535 nm emission filter. The filtered fluorescence image was
captured by a scientific-grade CCD camera. Imaging software and
a Pentium PC were used to control CCD functions and to process all
images. FIG. 5 shows a representative fluorescence image (4.times.)
of a DAM-1 molecular sieve particle-based fiber-optic microwell
array sensor (3.2-mm diameter) immersed in pH 8.5 PBS.
[0086] An experiment was performed to confirm that the observed
fluorescence from the molecular sieve particle-based fiber-optic
microwell array sensor emanated from a FITC-modified molecular sieve
particle. First, the 10 microliter-aliquot of a thoroughly-mixed
2.5% (v/v) aqueous solution of FITC-modified DAM-1 particles is
dispersed onto a small portion of vertically-positioned microwell
array face. FIG. 6 shows a transmitted white light image (4.times.)
and a fluorescence image (4.times.) of the same DAM-1 molecular
sieve particle-based fiber-optic microwell array sensor immersed
in pH 8.5 PBS. Both images show that DAM-1 molecular sieve particles
are distributed across a well-defined region of the microwell array
face. For example, dark intensities in the white-light image (FIG.
6A) indicate that some microwells were filled/partially-filled with
particles such that the white light projected onto the distal array
face could not be transmitted through those microwells and be detected
by the CCD camera. Similarly, the bright intensities in the fluorescence
image (FIG. 6B) indicate that some microwells were filled/partially-filled
with dye-modified molecular sieve particles whose fluorescence could
be collected in an `epi` microscopic fashion through the bundle
and detected by the CCD camera.
[0087] The next stage of this experiment was to acquire white-light
and fluorescence images of a defect on the microwell array face
so that this defect could be used for identifying the exact location
of a specific dye-modified molecular sieve particle. FIG. 7A shows
the white-light image of such a defect (i.e., a triangular point)
on the perimeter of the microwell array face. Analysis of this image
demonstrates that there are both bright regions (empty microwells)
and dark regions (filled or partially-filled microwells). Fluorescence
images of this region were acquired with the DAM-1 molecular sieve
particle-based fiber-optic microwell array sensor immersed in pH
8.5 (FIG. 7B) and pH 4.5 (FIG. 7C) PBS. These two fluorescence images
were normalized to the same grey scale and indicate that only one
microwell displayed fluorescence that followed the expected behavior
of the FITC pH-sensitive dye. Quantitatively, the fluorescence intensity
from this microwell decreased by greater than 40% when the sensor
was removed from the pH 8.5 PBS (FIG. 7B) and then immersed in the
pH 4.5 PBS (FIG. 7C). It should also be noted that this microwell
displayed dark intensities during white-light imaging (FIG. 7A)
which further supports that it was host to a molecular sieve particle.
Once the fluorescence images of this sensor region were acquired,
scanning electron microscopy was used to find and identify the microwell
that housed this particular molecular sieve particle. FIG. 7D shows
this same sensor region and the individual microwell from which
the fluorescence was generated. A higher magnification scanning
electron micrograph (FIG. 8) shows that this particular microwell
was filled with a gyroid-shaped DAM-1 molecular sieve particle.
PROPHETIC EXAMPLES
[0088] A photodeposition protocol has been developed that can partially
fill individual microwells with a chemical sensing (polymer+dye)
layer resulting in the fabrication of a microwell array chemical
sensor (MWACS). MWACSs fabrication has been demonstrated using 2-14
micron-thick, water- and organic solvent-based polymer layers where
individual microwell are <16 micron deep; specifically O.sub.2-sensitive
polysiloxane- and pH-sensitive polyvinyl alcohol-based MWACSs were
characterized. Several novel analytical methodologies can be envisioned
through the union of molecular sieve particles and MWACSs.
[0089] The molecular sieve particle-based MWACS arrangement is
depicted in FIG. 9 where the gray shading denotes the photodeposited
polymeric sensing layer. In reviewing the manner by which a hexagonal-cylinder
shaped DAM-1 molecular sieve particle can reside in an individual
microwell (see FIG. 3), a key feature of the molecular sieve particle-based
MWACS arrangement is the ability for a molecular sieve particle's
pores to be aligned with the microwell's core (i.e., the optical
channel).
[0090] In one embodiment, the molecular sieve particle is empty
and the polymeric sensing layer contains an indicator or indicator
chemistries including but not limited to, nucleic acids, oligonucleotides,
peptides, proteins, enzymes, antigens, antibodies, receptors, ligands,
luminophores, fluorophores, chromophores, phosphors, pH indicators,
cation indicators, anion indicators, metal ion indicators, reactive
oxygen species indicators, nitric oxide indicators, oxygen indicators
and carbon dioxide indicators. This arrangement increases the molecular
selectivity of the MWACS in that only appropriately-sized analytes
will be able to diffuse through the molecular sieve particle pores
and reach the polymeric chemical sensing layer.
[0091] In another embodiment, the polymeric sensing layer contains
a first type of indicator chemistry and the molecular sieve particle
pores contain a second type of indicator chemistry. This arrangement
produces a multi-analyte sensor allowing for concurrent detection
of two or more analytes. The high surface area of these molecular
sieve particles provides for enhanced indicator chemistry loading
and catalytic activity and thus improved analytical sensitivity
relative to that of plastic beads or amphorous silica.
[0092] In another embodiment, the polymeric sensing layer contains
indicator chemistry and the molecular sieve particle pores contain
a drug, pharmaceutical, enzyme, protein, antibody, or oligonucleotide
for controlled-release applications. It has been shown that proteins
can be absorbed by molecular sieve particles and that proteins can
be released from molecular sieve particles as a function of solution
pH. Several novel scenarios can now be envisioned when the molecular
sieve particle contained such (bio)molecules. 1) The polymeric layer
can contain a pH-sensitive dye (or other indicator dye) to monitor
the release process. 2) The polymeric layer can contain a photoactivatable
molecule that would produce protons following irradiation in order
to control the release process. 3) The polymeric layer can contain
a pH-sensitive dye (or other indicator dye) to monitor the release
process and a photoactivatable molecule that would produce protons
to control the release process. This is possible as long as the
spectral properties of the indicator and the photoactivatable molecule
are properly chosen. 4) The polymeric layer can contain other photosensitive
molecules (such as caged molecules or photoactivatable/photodetachable
linkers) that would photochemically control the release process.
Again, the high surface area of these molecular sieve particles
can enable enhanced loading of the drug, pharmaceutical, enzyme,
protein, antibody, or oligonucleotide in the microwell relative
to that of plastic beads or amphorous silica. |