U.S. patent application number 16/854786 was filed with the patent office on 2020-10-08 for rapid identification of microorganisms.
The applicant listed for this patent is Board of Regents of the Nevada System of Higher Education, on behalf of the University of Nevada, Re. Invention is credited to Qingsu Cheng, Bahram Parvin.
Application Number | 20200318165 16/854786 |
Document ID | / |
Family ID | 1000004905773 |
Filed Date | 2020-10-08 |
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United States Patent
Application |
20200318165 |
Kind Code |
A1 |
Parvin; Bahram ; et
al. |
October 8, 2020 |
Rapid Identification of Microorganisms
Abstract
Methods of labeling, identifying and differentiating
microorganisms using functionalized Buckyballs are provided herein.
The invention further provides methods for imaging or inhibiting
gene expression using functionalized Buckyballs of the invention.
The invention also provides a system for labeling, identifying and
differentiating microorganisms.
Inventors: |
Parvin; Bahram; (Reno,
NV) ; Cheng; Qingsu; (Reno, NV) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents of the Nevada System of Higher Education, on
behalf of the University of Nevada, Re |
Reno |
NV |
US |
|
|
Family ID: |
1000004905773 |
Appl. No.: |
16/854786 |
Filed: |
April 21, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15757196 |
Mar 2, 2018 |
10626469 |
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PCT/US2015/050122 |
Sep 2, 2016 |
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16854786 |
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62214687 |
Sep 4, 2015 |
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62249098 |
Oct 30, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12Q 1/6841 20130101; C12Q 1/689 20130101; A61K 31/713
20130101 |
International
Class: |
C12Q 1/689 20060101
C12Q001/689; A61K 31/713 20060101 A61K031/713; C12Q 1/6841 20060101
C12Q001/6841 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grant
number DE-AC02-05CH11231 awarded by the U.S. Department of Energy.
The government has certain rights in the invention.
Claims
1-31. (canceled)
32. A method of inhibiting gene expression in a living
microorganism, the method comprising: a) functionalizing a
Buckminsterfullerene molecule with one or more RNA oligonucleotides
complementary to one or more mRNA segments of interest
corresponding to a gene of interest; b) hybridizing the one or more
RNA oligonucleotides to one or more complementary protecting layers
comprising segments of DNA or RNA and optionally a detectable
label; and c) contacting the sample with the functionalized
Buckminsterfullerene molecule for a period of time; wherein the one
or more RNA oligonucleotides hybridize with free mRNA in the
cytoplasm, preventing transcription and gene expression; and
wherein, the detectable label, if present, is not detected when the
protecting layers are hybridized to the one or more RNA
oligonucleotides and the detectable label is detected when the
protecting layers are not hybridized.
33. The method of claim 32, wherein the one or more RNA
oligonucleotides are selected by bioinformatics analysis.
34. The method of claim 32, wherein the protecting layers are about
75% complementary to the corresponding RNA oligonucleotides.
35. The method of claim 32, wherein the one or more RNA
oligonucleotides are each independently about 80% to a 100%
complementary to the corresponding species specific signature RNA
sequences.
36. The method of claim 32, wherein the one or more RNA
oligonucleotides each independently comprise about 20 to about 50
individual nucleotides.
37. The method of claim 32, wherein the one or more RNA
oligonucleotides are siRNA oligonucleotides.
38. The method of claim 32, wherein the Buckminsterfullerene
molecule is selected from the group consisting of C60
Buckminsterfullerene, C70 Buckminsterfullerene and C60-pyrrolidine
tris acid Buckminsterfullerene.
39. The method of claim 32, wherein the detectable label is from
the group consisting of a fluorescent tag, a radioactive isotope,
an amino acid, a nucleic acid, and a peptide.
40. The method of claim 39, wherein the detectable label is
selected from the group consisting of glycine, tryptophan,
arginine, cysteine, fBSA, .sup.14C, .sup.125I, and cy3/6-FAM.
41. The method of claim 32, wherein the detectable label is
detected using a method selected from the group consisting of
autoradiography, fluorescence microscopy, X-ray fluorescence
microscopy, UV-vis spectroscopy, TEM and fluorescent
spectroscopy.
42. The method of claim 32, wherein the method does not require
sample fixation.
43. The method of claim 32, wherein the microorganism is selected
from the group consisting of bacteria, fungi, archaea and
protists.
44. The method of claim 32, wherein the microorganism internalizes
the functionalized Buckminsterfullerene.
45. The method of claim 32, wherein biological processes can be
monitored and profiled by dynamic visualization of mRNA
expression.
46-73. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/757,196, filed Mar. 2, 2018, issued as U.S.
Pat. No. 10,626,469, a 35 U.S.C. .sctn. 371 national phase
application from, and claims priority to International Application
No. PCT/US2016/050122, filed Sep.2, 2016, which claims priority
under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Application No.
62/214,687 filed Sep. 4, 2015, and U.S. Provisional Application No.
62/249,098 filed Oct. 30, 2015, all of which applications are
incorporated herein by reference in their entireties.
BACKGROUND
[0003] Soil hosts most of the biodiversity in the environment,
where each cubic centimeter of soil matrix can contain hundreds of
thousands of microorganisms that cohabitate in a complex assemblage
of mineral and organic matter. The structure and function of
microbial communities are dynamic processes that play important and
beneficial roles in productivity of ecosystems, including oxygen
production, crop growth, bioremediation, carbon sequestration,
nitrogen fixation, and water purification. Simultaneously,
microbial species may act as pathogens for living organisms. For
example, plants from hundreds of different species are killed
annually in Australia by P. cinnamomi; and grain development, in
wheat, is affected by infection of G. graminis var. tritici in
vascular tissue. Therefore, there is a need to develop the probes
and assays that enable studying microbial species in their native
environment, i.e., in situ imaging. Applications of in situ imaging
include, but are not limited to, the insights and understanding of
the (i) composition and population of a normal gut microbiome as a
function of exposure to antibiotics and/or under environmental
stress; (ii) interactions and cross talk between microbes and plant
roots in rhizosphere; (iii) localization of endophytes in healthy
plant tissues for improved yield; and (iv) profiling of the
microbial communities in soil crust for erosion control, water
retention, and nutrient cycling.
[0004] To meet the requirements of in situ imaging and
identification of microorganisms, synthesized probes must (i)
penetrate the cell wall and lipid membrane, (ii) be non-sticky to
the soil matrix, and (iii) differentiate between living and dead
microorganisms. Previously, guanidinium-rich molecular transporters
(GR-MoTrs) have been demonstrated to be internalized in different
strains of algae by crossing both the cell wall and the lipid
membrane; however, it was later discovered that these molecular
transporters were sticky to the matrix substrate. Other
polymer-based nanoparticles, such as lipofectamine, have also been
found to be sticky to the natural environment. Moreover, in some
cases, synthesized probes ideally should facilitate radiolabeling
to meet the general requirements of in situ imaging. For example,
the structure of a microbial community can be imaged with x-ray
microtomography and MRI, but these techniques are destructive and
do not report biological activities, the successful imaging of
which is highly dependent on the design of the imaging instruments.
Thus, a need still exists to develop probes that allow efficient in
situ visualization of microbial density that overcome the problems
associated with the currently available technologies.
[0005] There is thus a need in the art for probes that allow
efficient in situ visualization of microbial presence and density
that are non-destructive and non-invasive. Additionally, there
remains a need in the art for probes and methods which are capable
of differentiating microbial populations quickly, cheaply and
effectively. The present invention fulfills these needs.
BRIEF SUMMARY OF THE INVENTION
[0006] The invention provides a method of selectively labeling a
specific species of microorganism in a sample, the method
comprising: [0007] a) functionalizing a Buckminsterfullerene
molecule with one or more RNA oligonucleotides complementary to one
or more species specific signature RNA sequences of the
microorganism in the sample; [0008] b) hybridizing the one or more
RNA oligonucleotides to protecting layers comprising segments of
DNA or RNA and a detectable label; and [0009] c) contacting the
sample with the functionalized Buckminsterfullerene molecule for a
period of time;
[0010] wherein, the detectable label is not detected when the
protecting layers are hybridized to the one or more RNA
oligonucleotides and the detectable label is detected when the
protecting layers are not hybridized, thereby selectively labeling
a specific species of microorganism in the sample.
[0011] In certain embodiments, the one or more species specific
signature RNA sequences are 16S rRNA sequences or mRNA sequences.
In other embodiments, the one or more RNA oligonucleotides are
selected by bioinformatics analysis. In yet other embodiments, the
protecting layers are about 75% complementary to the corresponding
RNA oligonucleotides. In yet other embodiments, the one or more RNA
oligonucleotides are each independently about 80% to a 100%
complementary to the corresponding species specific signature RNA
sequences. In yet other embodiments, the one or more RNA
oligonucleotides each independently comprise about 20 to about 50
individual nucleotides.
[0012] In certain embodiments, two or more specific species of
microorganisms are labelled simultaneously with different
functionalized Buckminsterfullerene molecules specific for each
organism, and wherein each different functionalized
Buckminsterfullerene comprises a unique detectable label such that
each species of microorganism is labelled with a unique detectable
label corresponding to that specific species. In other embodiments,
the presence or absence of the two or more specific species of
microorganisms can be determined by detecting the presence or
absence of the corresponding unique detectable label. In other
embodiments, the relative abundance of each of the two or more
specific species of microorganism is determined by measuring the
relative intensity of the two or more unique detectable labels.
[0013] In certain embodiments, the two or more specific species of
microorganisms are contained in a single mixed sample. In other
embodiments, the microorganisms are live microorganisms. In yet
other embodiments, the microorganism is selected from the group
consisting of bacteria, fungi, archaea and protists.
[0014] In certain embodiments, the microorganism is labeled in a
medium selected from the group consisting of a solution, an organic
matrix and a soil matrix. In other embodiments, the functionalized
Buckminsterfullerene molecules are functionalized so that they do
not adhere or stick to the medium and can be removed from the
medium.
[0015] In certain embodiments, the Buckminsterfullerene molecule is
selected from the group consisting of C60 Buckminsterfullerene, C70
Buckminsterfullerene and C60-pyrrolidine tris acid
Buckminsterfullerene. In certain embodiments, the detectable label
is selected from the group consisting of a fluorescent tag, a
radioactive isotope, an amino acid, a nucleic acid, and a peptide.
In other embodiments, the detectable label is selected from the
group consisting of glycine, tryptophan, arginine, cysteine, fBSA,
.sup.14C, .sup.125I, and cy3/6-FAM.
[0016] In certain embodiments, detectable label is detected using a
method selected from the group consisting of autoradiography,
fluorescence microscopy, X-ray fluorescence microscopy, UV-vis
spectroscopy, TEM and fluorescent spectroscopy.
[0017] In certain embodiments, the microorganism internalizes the
functionalized Buckminsterfullerene.
[0018] In certain embodiments, identification of the microorganism
does not require sample fixation.
[0019] The invention further provides a method of labeling and
identifying a microorganism, the method comprising: [0020] a)
functionalizing a Buckminsterfullerene molecule with a detectable
label; [0021] b) incubating the microorganism with the
functionalized Buckminsterfullerene molecule for a period of
time.
[0022] In certain embodiments, the Buckminsterfullerene molecule is
selected from the group consisting of C60 Buckminsterfullerene, C70
Buckminsterfullerene and C60-pyrrolidine tris acid
Buckminsterfullerene.
[0023] In certain embodiments, the detectable label is selected
from the group consisting of a fluorescent tag, a radioactive
isotope, an amino acid, a nucleic acid, and a peptide. In other
embodiments, the detectable label is selected from the group
consisting of glycine, tryptophan, arginine, cysteine, fBSA,
.sup.14C, .sup.125I, and cy3/6-FAM. In other embodiments, the
detectable label is detected using a method selected from the group
consisting of autoradiography, fluorescence microscopy, X-ray
fluorescence microscopy, UV-vis spectroscopy, TEM and fluorescent
spectroscopy.
[0024] In certain embodiments, the microorganism internalizes the
functionalized Buckminsterfullerene.
[0025] In certain embodiments, more than one microorganism is
labeled and live microorganisms are differentiated from dead
microorganisms. In other embodiments, dead microorganisms
internalize more of the functionalized Buckminsterfullerene
molecules than living microorganisms.
[0026] In certain embodiments, the microorganism does not require
sample fixation.
[0027] In certain embodiments, the microorganism is selected from
the group consisting of bacteria, fungi, archaea and protists.
[0028] The invention further provides a method of detecting gene
expression in a living microorganism, the method comprising: [0029]
a) functionalizing a Buckminsterfullerene molecule with one or more
RNA oligonucleotides complementary to one or more mRNA segments of
interest corresponding to a gene of interest; [0030] b) hybridizing
the one or more RNA oligonucleotides to one or more complementary
protecting layers comprising segments of DNA or RNA and a
detectable label; and [0031] c) contacting a sample containing a
living microorganism with the functionalized Buckminsterfullerene
molecule for a period of time;
[0032] wherein, the detectable label is not detected when the
protecting layers are hybridized to the one or more RNA
oligonucleotides and the detectable label is detected when the
protecting layers are not hybridized, thereby detecting gene
expression in a living microorganism.
[0033] The invention also provides a method of inhibiting gene
expression in a living microorganism, the method comprising: [0034]
a) functionalizing a Buckminsterfullerene molecule with one or more
RNA oligonucleotides complementary to one or more mRNA segments of
interest corresponding to a gene of interest; [0035] b) hybridizing
the one or more RNA oligonucleotides to one or more complementary
protecting layers comprising segments of DNA or RNA and optionally
a detectable label; and [0036] c) contacting the sample with the
functionalized Buckminsterfullerene molecule for a period of time;
[0037] wherein the one or more RNA oligonucleotides hybridize with
free mRNA in the cytoplasm, preventing transcription and gene
expression; and
[0038] wherein, the detectable label, if present, is not detected
when the protecting layers are hybridized to the one or more RNA
oligonucleotides and the detectable label is detected when the
protecting layers are not hybridized.
[0039] In certain embodiments, the one or more RNA oligonucleotides
are selected by bioinformatics analysis. In certain embodiments,
the protecting layers are about 75% complementary to the
corresponding RNA oligonucleotides. In certain embodiments, the one
or more RNA oligonucleotides are each independently about 80% to a
100% complementary to the corresponding species specific signature
RNA sequences. In certain embodiments, the one or more RNA
oligonucleotides each independently comprise about 20 to about 50
individual nucleotides. In certain embodiments, the one or more RNA
oligonucleotides are siRNA oligonucleotides.
[0040] In certain embodiments, the Buckminsterfullerene molecule is
selected from the group consisting of C60 Buckminsterfullerene, C70
Buckminsterfullerene and C60-pyrrolidine tris acid
Buckminsterfullerene.
[0041] In certain embodiments, the detectable label is from the
group consisting of a fluorescent tag, a radioactive isotope, an
amino acid, a nucleic acid, and a peptide. In other embodiments,
the detectable label is selected from the group consisting of
glycine, tryptophan, arginine, cysteine, fBSA, .sup.14C, .sup.125I,
and cy3/6-FAM. In yet other embodiments, the detectable label is
detected using a method selected from the group consisting of
autoradiography, fluorescence microscopy, X-ray fluorescence
microscopy, UV-vis spectroscopy, TEM and fluorescent
spectroscopy.
[0042] In certain embodiments, the detection and inhibition methods
do not require sample fixation.
[0043] In certain embodiments, the microorganism is selected from
the group consisting of bacteria, fungi, archaea and protists.
[0044] In certain embodiments, the microorganism internalizes the
functionalized Buckminsterfullerene.
[0045] In certain embodiments, biological processes can be
monitored and profiled by dynamic visualization of mRNA
expression.
[0046] The invention further provides a system for labelling,
identifying and differentiating living microorganisms of different
species within a sample, the system comprising: [0047] a) one or
more source wells and one or more sink wells, wherein the source
wells and the sink wells are in fluidic communication with each
other; [0048] b) one or more functionalized Buckminsterfullerene
molecules for each species of microorganism in the sample, wherein
the Buckminsterfullerene molecules are functionalized with one or
more RNA oligonucleotides complementary to one or more species
specific signature RNA sequences of the microorganisms in the
sample, wherein the one or more RNA oligonucleotides are hybridized
to protecting layers comprising segments of DNA or RNA and a
detectable label; [0049] wherein, the detectable label is not
detected when the protecting layers are hybridized to the one or
more RNA oligonucleotides and the detectable label is detected when
the protecting layers are not hybridized; and wherein, each sink
well comprises a different type of functionalized
Buckminsterfullerene molecule, bound to a different detectable
label, each corresponding to a different microorganism species;
and
[0050] wherein the microorganisms are labeled, identified and
differentiated by:
[0051] a) placing a sample comprising one or more different species
of microorganisms in the source well;
[0052] b) allowing the microorganisms to migrate to the one or more
sink wells, coming in contact with and internalizing the one or
more functionalized Buckminsterfullerene molecules; and
[0053] wherein the microorganisms emit a signal if in contact with
a Buckminsterfullerene molecule comprising an RNA oligonucleotide
which matches a species specific signature RNA sequence within the
microorganism.
[0054] In certain embodiments, the number of sink wells is
equivalent to the number of microorganism species of interest
within the sample. In other embodiments, each sink well further
comprises a microbial attractant which attracts the microorganism
species of interest matching the functionalized
Buckminsterfullerene molecule present in that same sink well. In
other embodiments, the microbial attractant is a nutrient, mineral
or environmental condition meant to draw the microorganism of
interest to the sink well. In yet other embodiments, the microbial
attractant is one or more conditions selected from the group
consisting of a sugar gradient, a protein gradient, a metal ion
gradient, a temperature gradient, a salinity gradient, a light
gradient and a specific wavelength of light.
[0055] In certain embodiments, the functionalized
Buckminsterfullerene molecules are printed into the one or more
sink wells.
[0056] In certain embodiments, the one or more species specific
signature RNA sequences are 16S rRNA sequences or mRNA sequences.
In certain embodiments, the one or more RNA oligonucleotides are
selected by bioinformatics analysis. In certain embodiments, the
protecting layers are about 75% complementary to the corresponding
RNA oligonucleotides. In certain embodiments, the one or more RNA
oligonucleotides are each independently about 80% to a 100%
complementary to the corresponding species specific signature RNA
sequences. In certain embodiments, the one or more RNA
oligonucleotides each independently comprise about 20 to about 50
individual nucleotides. In certain embodiments, the microorganisms
are selected from the group consisting of bacteria, fungi, archaea
and protists.
[0057] In certain embodiments, the Buckminsterfullerene molecules
are selected from the group consisting of C60 Buckminsterfullerene,
C70 Buckminsterfullerene and C60-pyrrolidine tris acid
Buckminsterfullerene.
[0058] In certain embodiments, the detectable labels are selected
from the group consisting of fluorescent tags, radioactive
isotopes, amino acids, nucleic acids, and peptides. In certain
embodiments, the detectable labels are selected from the group
consisting of glycine, tryptophan arginine cysteine, fBSA,
.sup.14C, .sup.125I, and cy3/6-FAM. In certain embodiments, the
detectable labels are detected using a method selected from the
group consisting of autoradiography, fluorescence microscopy, X-ray
fluorescence microscopy, UV-vis spectroscopy, TEM and fluorescent
spectroscopy.
[0059] In certain embodiments, identification of microorganisms
does not require sample fixation.
[0060] In certain embodiments, the system further comprises an
imaging device which can observe and record the signal emitted from
each sink well.
[0061] In certain embodiments, the system can determine the
presence or absence of the one or more microorganism species of
interest. In certain embodiments, the system can determine the
relative abundance of each of the one or more microorganism species
of interest.
[0062] The invention further provides a functionalized
Buckminsterfullerene composition comprising: [0063] C60-pyrrolidine
tris acid Buckminsterfullerene; and [0064] one or more detectable
labels selected from the group consisting of a fluorescent tag, a
radioactive isotope, an amino acid, a nucleic acid, and a
peptide.
[0065] In certain embodiments, the composition further comprises
one or more non-coding RNA oligonucleotides. In certain
embodiments, the one or more RNA oligonucleotides each
independently comprise about 20 to about 50 individual
nucleotides.
[0066] In certain embodiments, the composition further comprises
protecting layers, wherein the protecting layers are segments of
DNA or RNA which can be hybridized to the one or more RNA
oligonucleotides. In certain embodiments, the protecting layers are
about 75% complementary to the corresponding RNA oligonucleotides.
In certain embodiments, the one or more detectable labels are bound
to the protecting layers.
[0067] In certain embodiments, the one or more detectable labels
are selected from the group consisting of a fluorescent tag, a
radioactive isotope, an amino acid, a nucleic acid, and a peptide.
In other embodiments, the one or more detectable labels are
selected from the group consisting of glycine, tryptophan,
arginine, cysteine, fBSA, .sup.14C, .sup.125I, and cy3/6-FAM.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0069] FIG. 1 is a schematic of C60-pyrrolidine tris acid, which
indicates that C60-pyrrolidine tris acid has three potential
decoration sites while maintaining the carbon cage structure
intact.
[0070] FIGS. 2A-2F illustrate cellular uptake of fBSA labelled
C60-pyrrolidine tris acid by E. coli and B. subtilis monitored by
Confocal Laser Scanning Microscopy. FIGS. 2A and 2B, fBSA-C60
pyrrolidine tris acid uptake by E. coli (FIG. 2A) and B. subtilis
(FIG. 2B) visualized by exciting fBSA fluorescence using the 488 nm
laser. FIGS. 2C and 2D are bright field (BF) images of the
microorganisms. FIGS. 2E and 2F are merged fluorescence and BF
images indicate that the fluorescent signals co-localize with the
presence of the microorganisms indicating interactions between fBSA
labelled C60-pyrrolidine tris acid and microorganisms.
[0071] FIGS. 3A and 3B illustrate cellular uptake of
C60-pyrrolidine tris acid by E. coli and B. subtilis monitored by
Transmission Electron Microscopy. The dark spots, pointed by the
arrows, represent C60-pyrrolidine-tris acid uptake by E. coli (FIG.
3A) and B. subtilis (FIG. 3B).
[0072] FIGS. 4A-4D illustrate cellular uptake of radiotracers
labelled C60-pyrrolidine tris acid by microorganisms monitored by
Autoradiography. FIGS. 4A and 4B illustrate .sup.14C60-pyrrolidine
tris acid uptake by E. coli (FIG. 4A) and B. subtilis (FIG. 4B).
FIGS. 4C and 4D .sup.125I-C60-pyrrolidine tris acid uptake by E.
coli (FIG. 4C) and B. subtilis (FIG. 4D).
[0073] FIGS. 5A-5D illustrate the non-stickiness of C60-pyrrolidine
tris acid to multiple matrices, with increasing weights, monitored
by Ultraviolet (UV) light absorption. The hatched regions indicate
immediate recovery following incubation and the filled regions
indicate recovery following multiple washes. FIGS. 5A-5D illustrate
recovery of C60-pyrrolidine tris acid from alumina (FIG. 5A), VWR
sand (FIG. 5B), wild sand (FIG. 5C), and natural soil (FIG. 5D).
The results indicate that C60-pyrrolidine tris acid remains
non-sticky to the various matrices. The total recovery of
C60-pyrrolidine tris acid is approximately 100%.
[0074] FIGS. 6A and 6B illustrate the non-stickiness of
C60-pyrrolidine tris acid monitored by Confocal Laser Scanning
Microscopy following several washes. Fluorescence microscopy
indicates that C60-pyrrolidine tris acid had no adherence to
alumina (FIG. 6A) and glass beads (FIG. 6B), which are clearly
present in bright field microscopy.
[0075] FIG. 7 illustrates the non-stickiness of radiotracers
.sup.14C- and .sup.125I-labeled C60-pyrrolidine tris acid, on
several matrices, monitored by Autoradiography following several
washes. The left column shows the background radiation. The middle
and right columns indicate that neither .beta.-radiation nor
.gamma.-radiation are detected by autoradiography after several
washes, indicating C60-pyrrolidine tris acid is not sticky to
multiple matrices
[0076] FIG. 8 illustrates cellular uptake of microorganisms
embedded in different matrices and monitored by autoradiography
following several washes. Left and right columns indicate uptake by
.sup.14C- and .sup.125I-labelled C60-pyrrolidine tris acid on B.
subtilis and E. coli, respectively. The data indicate a residual
signal that is presumably due to the uptake of microorganisms,
since FIG. 7 indicates non-stickiness to the same substrates.
[0077] FIG. 9 illustrates the non-stickiness of .sup.14C-labelled
C60-pyrrolidine tris acid with five different matrices, monitored
by Liquid Scintillation Count (LSC) following several washes.
Hatched columns show similar level of LSC from incubation and
washout of VWR sand, wild sand, alumina, glass beads, and natural
soil with .sup.14C labelled-C60-pyrrolidine tris acid. Filled bars
show the background LSC on the same substrates.
[0078] FIGS. 10A-10D illustrate the non-stickiness of
C60-pyrrolidine tris-Cysteine to multiple soil matrices monitored
by Ultraviolet light absorption. Recovery of C60-Cysteine from
alumina (FIG. 10A), VWR sand (FIG. 10B), Wild Sand (FIG. 10C) and
Natural Soil (FIG. 10D) of different mass indicate that the newly
synthesized C60-pyrrolidine tris-Cysteine remains non-sticky to
multiple matrices. The hatched regions indicate immediate recovery
following incubation, and the filled regions indicate recovery
following multiple washes. The total recovery of C60-pyrrolidine
tris acid is approximately 100%.
[0079] FIG. 11 indicates cellular uptake C60-Cystine for
differentiating live and dead microorganisms (E. coli and B.
subtilis), quantified by analyzing images from confocal microscopy.
Live microorganisms showed a significantly lower uptake than the
dead microorganisms. The uptake was quantified by the average pixel
intensities of micoorganisms over the background.
[0080] FIGS. 12A-12F illustrate autofluorescence of B. subtilis and
E. coli monitored with a confocal microscope indicate no signals.
FIGS. 12A and 12B illustrate autofluorescence background for B.
subtilis (FIG. 12A) and E. coli (FIG. 12B) with 488 nm excitation.
FIGS. 12C and 12D are bright field images of B. subtilis (FIG. 12C)
and E. coli (FIG. 12D) indicate presence of microorganisms. FIGS.
12E-12F are merged bright field and autofluorescence provides
additional evidence for absence of any signal.
[0081] FIGS. 13A-13D illustrate positive and negative controls for
C60-pyrrolidine tris acid monitored with transmission electron
microcopy. C60-pyrrolidine tris acid is clearly present in (FIG.
13A) DI H.sub.2O and (FIG. 13B) a tissue section from mouse as
background. The background, without C60-pyrrolidine tris acid, is
void of any signal in (FIG. 13C) E. coli and (FIG. 13D) B.
subtilis.
[0082] FIG. 14 illustrates uptake of .sup.14C-labelled
C60-pyrrolidine tris acid monitored by Liquid Scintillation Count
for E. coli and B. subtilis.
[0083] FIGS. 15A-15H illustrate time course studies for uptake of
C60-pyrrolidine tris acid-fBSA, monitored by confocal microscopy,
for E. coli (A, C, E, and F) or B. subtilis (B, D, F, and H)
indicate time-dependency.
[0084] FIGS. 16A-16B illustrate retention of C60-pyrrolidine tris
acid-fBSA, monitored by confocal microscopy, after 6 rinses for
(FIG. 16A) E. coli and (FIG. 16B) B. subtilis.
[0085] FIGS. 17A-17C provide steps in quantification of fluorescent
images captured through confocal microscopy for Tables 1 and 2:
(FIG. 17A) Enhanced image for visualization; (FIG. 17B) segmented
microbes followed by connected components; and (FIG. 17C) extracted
boundaries for each segmented microbe.
[0086] FIG. 18 provide experimental setup for measuring UV
absorption: (a) Incubate C60-pyrrolidine tris acid with the
substrate for 30 mins, (b) apply a vacuum manifold to
C60-pyrrolidine tris acid and collect filtrate, (c) add fresh water
to the substrate again and incubate for another 30 mins, and (d)
apply a vacuum manifold to wash water and collect filtrate
again.
[0087] FIGS. 19A-19C illustrate auto-fluorescence of (FIG. 19A) VWR
sand, (FIG. 19B) wild sand, and (FIG. 19C) natural soil, all
monitored by confocal microscopy with 488 nm excitation.
[0088] FIGS. 20A-20C are drawings illustrating the use of C60
molecules functionalized with rRNA reporter RNA complexes for the
labeling of specific bacterium. FIG. 20A is a schematic
illustrating an exemplary method of decorating C60 with rRNA
reporters which allows for visualization of each microbe at a
specific excitation frequency. FIG. 20B illustrates the
functionalization and internalization of the functionalized C60
molecules into a bacterium. FIG. 20C illustrates the
internalization of the functionalized C60 molecules into a
bacterium.
[0089] FIG. 21 is a series of images illustrating the
differentiation of B. subtilis and S. sanguinis by disclosed
C60-rRNA-Reporter complex. Scale bar is 10 .mu.m.
[0090] FIGS. 22A-22B are a series of images illustrating the
differentiation of B. subtilis and S. sanguinis using the
compositions of the invention as monitored by super resolution
microscopy. B. subtilis incubated with B. subtilis probe complex
shows no fluorescent signal at 488 nm excitation frequency, but
fluoresces at 568 nm excitation frequency. S. sanguinis incubated
with S. sanguinis probe complex shows fluorescent signal at 488 nm
excitation frequency, and no fluorescence signal at 568 nm
excitation frequency. Mixed bacteria and probe complexes indicate
that each bacterium can be visualized at its corresponding
excitation frequency. Scale bar is 10 .mu.m.
[0091] FIG. 23 is a schematic illustrating a method for generating
C60-siRNA complexes that can be used for regulating gene
expression.
[0092] FIG. 24 is a schematic illustrating a method for
simultaneously imaging a specific bacterium and activating or
suppressing transcription in the specific bacterium.
[0093] FIG. 25 is a schematic illustrating a method for
simultaneously imaging a specific bacterium by identifying both a
specific mRNA sequence and a specific rRNA sequence in the specific
species of bacterium
[0094] FIG. 26 is an image of a microfluidic system of the
invention capable of rapidly diagnosing and differentiating
microbial presence. The microfluidic system comprises a source well
(left side, panel a) and two sink wells (top right, panel b, and
bottom right, panel d) in fluidic communication with each
other.
DETAILED DESCRIPTION OF THE INVENTION
[0095] The invention relates to the unexpected discovery that
functionalized Buckyballs (e.g., C60-pyrrolidine tris acid) are a
versatile platform for internalizing chemical payloads into
microorganisms. In certain embodiments, functionalized Buckyballs
can be used to transport a detectable label into a living
microorganism. In certain embodiments, the Buckyballs can transport
detectable label complexes which are able to discriminate between
different microorganisms and selectively emit a signal only once
inside a specific organism. In other embodiments, the invention
includes devices and methods which utilize the functionalized
Buckyballs of the invention to determine microbial density and/or
differentiate between different species of microorganism in a
sample.
Definitions
[0096] As used herein, each of the following terms has the meaning
associated with it in this section.
[0097] As used herein, unless defined otherwise, all technical and
scientific terms generally have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Generally, the nomenclature used herein and the
laboratory procedures in cell culture, molecular genetics, organic
chemistry, and peptide chemistry are those well-known and commonly
employed in the art.
[0098] As used herein, the articles "a" and "an" refer to one or to
more than one (i.e. to at least one) of the grammatical object of
the article. By way of example, "an element" means one element or
more than one element.
[0099] As used herein, the term "about" will be understood by
persons of ordinary skill in the art and will vary to some extent
on the context in which it is used. As used herein, "about" when
referring to a measurable value such as an amount, a temporal
duration, and the like, is meant to encompass variations of .+-.20%
or .+-.10%, more preferably .+-.5%, even more preferably .+-.1%,
from the specified value, as such variations are appropriate to
perform the disclosed methods.
[0100] As used herein, the term "bacteria" means a large domain of
prokaryotic microorganisms. Typically a few micrometres in length,
bacteria have a wide range of shapes, ranging from spheres to rods
and spirals. There are broadly speaking two different types of cell
wall in bacteria, called Gram-positive and Gram-negative.
Gram-positive bacteria possess a thick cell wall containing many
layers of peptidoglycan and teichoic acids. In contrast,
Gram-negative bacteria have a relatively thin cell wall consisting
of a few layers of peptidoglycan surrounded by a second lipid
membrane containing lipopolysaccharides and lipoproteins. Most
bacteria have the Gram-negative cell wall, and only the Firmicutes
and Actinobacteria have the alternative Gram-positive
arrangement.
[0101] As used herein, the terms "bacterial pathogen" or
"pathogenic bacteria" mean a bacterium that causes disease.
Examples of pathogenic bacteria which can be detected and monitored
by the disclosed methods and compositions include, without
limitation, any one or more of (or any combination of)
Acinetobacter baumanii, Actinobacillus sp., Actinomycetes,
Actinomyces sp. (such as Actinomyces israelii and Actinomyces
naeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas
veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae),
Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Acinetobacter
baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such
as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus
thuringiensis, and Bacillus stearothermophilus), Bacteroides sp.
(such as Bacteroides fragilis), Bartonella sp. (such as Bartonella
bacilliformis and Bartonella henselae, Bifidobacterium sp.,
Bordetella sp. (such as Bordetella pertussis, Bordetella
parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such
as Borrelia recurrentis, and Borrelia burgdorferi), Brucella sp.
(such as Brucella abortus, Brucella canis, Brucella melintensis and
Brucella suis), Burkholderia sp. (such as Burkholderia pseudomallei
and Burkholderia cepacia), Campylobacter sp. (such as Campylobacter
jejuni, Campylobacter coli, Campylobacter lari and Campylobacter
fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia
trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci,
Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as,
Corynebacterium diphtheriae, Corynebacterium jeikeum and
Corynebacterium), Clostridium sp. (such as Clostridium perfringens,
Clostridium difficile, Clostridium botulinum and Clostridium
tetani), Eikenella corrodens, Enterobacter sp. (such as
Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter
cloacae and Escherichia coli, including opportunistic Escherichia
coli, such as enterotoxigenic E. coli, enteroinvasive E. coli,
enteropathogenic E. coli, enterohemorrhagic E. coli,
enteroaggregative E. coli and uropathogenic E. coli) Enterococcus
sp. (such as Enterococcus faecalis and Enterococcus faecium)
Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis),
Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella
tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella
morbillorum, Haemophilus sp. (such as Haemophilus influenzae,
Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus
parainfluenzae, Haemophilus haemolyticus and Haemophilus
parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori,
Helicobacter cinaedi and Helicobacter fennelliae), Kingella kingii,
Klebsiella sp. (such as Klebsiella pneumoniae, Klebsiella
granulomatis and Klebsiella oxytoca), Lactobacillus sp., Listeria
monocytogenes, Leptospira interrogans, Legionella pneumophila,
Leptospira interrogans, Peptostreptococcus sp., Moraxella
catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp.,
Mycobacterium sp. (such as Mycobacterium leprae, Mycobacterium
tuberculosis, Mycobacterium intracellulare, Mycobacterium avium,
Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm sp.
(such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma
genitalium), Nocardia sp. (such as Nocardia asteroides, Nocardia
cyriacigeorgica and Nocardia brasiliensis), Neisseria sp. (such as
Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella
multocida, Plesiomonas shigelloides. Prevotella sp., Porphyromonas
sp., Prevotella melaninogenica, Proteus sp. (such as Proteus
vulgaris and Proteus mirabilis), Providencia sp. (such as
Providencia alcalifaciens, Providencia rettgeri and Providencia
stuartii), Pseudomonas aeruginosa, Propionibacterium acnes,
Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii,
Rickettsia akari and Rickettsia prowazekii, Orientia tsutsugamushi
(formerly: Rickettsia tsutsugamushi) and Rickettsia typhi),
Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia,
Salmonella sp. (such as Salmonella enterica, Salmonella typhi,
Salmonella paratyphi, Salmonella enteritidis, Salmonella
cholerasuis and Salmonella typhimurium), Serratia sp. (such as
Serratia marcesans and Serratia liquifaciens), Shigella sp. (such
as Shigella dysenteriae, Shigella flexneri, Shigella boydii and
Shigella sonnei), Staphylococcus sp. (such as Staphylococcus
aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus,
Staphylococcus saprophyticus), Streptococcus sp. (such as
Streptococcus pneumoniae (for example chloramphenicol-resistant
serotype 4 Streptococcus pneumoniae, spectinomycin-resistant
serotype 6B Streptococcus pneumoniae, streptomycin-resistant
serotype 9V Streptococcus pneumoniae, erythromycin-resistant
serotype 14 Streptococcus pneumoniae, optochin-resistant serotype
14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C
Streptococcus pneumoniae, tetracycline-resistant serotype 19F
Streptococcus pneumoniae, penicillin-resistant serotype 19F
Streptococcus pneumoniae, and trimethoprim-resistant serotype 23F
Streptococcus pneumoniae, chloramphenicol-resistant serotype 4
Streptococcus pneumoniae, spectinomycin-resistant serotype 6B
Streptococcus pneumoniae, streptomycin-resistant serotype 9V
Streptococcus pneumoniae, optochin-resistant serotype 14
Streptococcus pneumoniae, rifampicin-resistant serotype 18C
Streptococcus pneumoniae, penicillin-resistant serotype 19F
Streptococcus pneumoniae, or trimethoprim-resistant serotype 23F
Streptococcus pneumoniae), Streptococcus agalactiae, Streptococcus
mutans, Streptococcus pyogenes, Group A streptococci, Streptococcus
pyogenes, Group B streptococci, Streptococcus agalactiae, Group C
streptococci, Streptococcus anginosus, Streptococcus equismilis,
Group D streptococci, Streptococcus bovis, Group F streptococci,
and Streptococcus anginosus Group G streptococci), Spirillum minus,
Streptobacillus moniliformi, Treponema sp. (such as Treponema
carateum, Treponema petenue, Treponema pallidum and Treponema
endemicum, Tropheryma whippelii, Ureaplasma urealyticum,
Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio
parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio
vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae,
Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela and Vibrio
furnisii), Yersinia sp. (such as Yersinia enterocolitica, Yersinia
pestis, and Yersinia pseudotuberculosis) and Xanthomonas
maltophilia among others.
[0102] As used herein, the terms "Buckminsterfullerene" or
"Buckyball" mean a spherical fullerene molecule with the formula
C60 or other spherical fullerene molecules (e.g., C70). These
spherical fullerene molecules have a cage-like fused-ring structure
(truncated icosahedron). For example, C60 is made of twenty
hexagons and twelve pentagons, with a carbon atom at each vertex of
each polygon and a bond along each polygon edge.
Buckminsterfullerene is the most common naturally occurring
fullerene molecule, as it can be found in small quantities in soot.
Solid and gaseous forms of the molecule have been detected in deep
space.
[0103] As used herein, the term "contacting means placement in
direct physical association, including both a solid and liquid
form. Contacting an agent with a cell can occur in vitro by adding
the agent to isolated cells or in vivo by administering the agent
to a subject.
[0104] As used herein, the term "fungus" means living,
single-celled and multicellular organisms belonging to the kingdom
Fungi. Most species are characterized by a lack of chlorophyll and
presence of chitinous cell walls, and some fungi may be
multinucleated. The methods disclosed herein can be used to detect
and identify antigens associated with particular fungi.
[0105] The term "fungal pathogen" means a fungus that causes
disease. Examples of fungal pathogens which can be detected and
monitored by the disclosed methods and compositions include,
without limitation, any one or more of (or any combination of)
Trichophyton rubrum, T mentagrophytes, Epidermophyton floccosum,
Microsporum canis, Pityrosporum orbiculare (Malassezia furfur),
Candida sp. (such as Candida albicans), Aspergillus sp. (such as
Aspergillus fumigatus, Aspergillus flavus and Aspergillus
clavatus), Cryptococcus sp. (such as Cryptococcus neoformans,
Cryptococcus gattii, Cryptococcus laurentii and Cryptococcus
albidus), Histoplasma sp. (such as Histoplasma capsulatum),
Pneumocystis sp. (such as Pneumocystis jirovecii), and Stachybotrys
(such as Stachybotrys chartarum).
[0106] As used herein, "hybridization" means to form base pairs
between complementary regions of two strands of DNA, RNA, or
between DNA and RNA, thereby forming a duplex molecule.
Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method and the composition and length of the hybridizing nucleic
acid sequences. Generally, the temperature of hybridization and the
ionic strength (such as the Na.sup.+ concentration) of the
hybridization buffer will determine the stringency of
hybridization. Calculations regarding hybridization conditions for
attaining particular degrees of stringency are discussed in
Sambrook et al., (1989) Molecular Cloning, second edition, Cold
Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11). The
following is an exemplary set of non-limiting hybridization
conditions:
[0107] Very High Stringency (Detects Sequences That Share at Least
90% Identity) [0108] Hybridization: 5.times.SSC at 65.degree. C.
for 16 hours [0109] Wash twice: 2.times.SSC at room temperature
(RT) for 15 minutes each [0110] Wash twice: 0.5.times.SSC at
65.degree. C. for 20 minutes each
[0111] High Stringency (Detects Sequences That Share at Least 80%
Identity) [0112] Hybridization: 5.times.-6.times.SSC at 65.degree.
C.-70.degree. C. for 16-20 hours [0113] Wash twice: 2.times.SSC at
RT for 5-20 minutes each [0114] Wash twice: 1.times.SSC at
55.degree. C.-70.degree. C. for 30 minutes each
[0115] Low Stringency (Detects Sequences That Share at Least 60%
Identity) [0116] Hybridization: 6.times.SSC at RT to 55.degree. C.
for 16-20 hours [0117] Wash at least twice: 2.times.-3.times.SSC at
RT to 55.degree. C. for 20-30 minutes each.
[0118] The term "label" as used herein means a detectable compound
or composition that is conjugated directly or indirectly to another
molecule, such as an antibody or a protein, to facilitate detection
of that molecule. Specific, non-limiting examples of labels include
fluorescent tags, enzymatic linkages (such as horseradish
peroxidase), radioactive isotopes (for example .sup.14C, .sup.32P,
.sup.125I, .sup.3H isotopes and the like) and particles such as
colloidal gold. In some examples, a molecule is labeled with a
radioactive isotope, such as .sup.14C, .sup.32P, .sup.125I, .sup.3H
isotope. Methods for labeling and guidance in the choice of labels
appropriate for various purposes are discussed for example in
Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current Protocols
in Molecular Biology, John Wiley & Sons, New York, 1998),
Harlow & Lane (Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York, 1988).
[0119] The term "microorganism" as used herein means a
single-celled, or unicellular, organism which include bacteria,
fungi, archaea or protists, but not viruses and prions (which are
generally classified as non-living). Microorganisms that cause
disease in a host are known as pathogens.
[0120] The term "Nanoparticle" as used herein means a microscopic
particle whose size is measured in nanometers (nm). It is defined
as a particle that does not have a dimension >1000 nm, such as
having a size between about 10 and about 1000 nm, for example,
between about 10 and about 100 nm, between 100 and about 500 nm, or
between about 500 and about 1000 nm. Nanoparticles are effectively
a bridge between bulk materials and atomic or molecular structures.
A bulk material should have constant physical properties regardless
of its size, but at the nano-scale this is often not the case.
Size-dependent properties are observed such as quantum confinement
in semiconductor particles, surface plasmon resonance in some metal
particles and superparamagnetism in magnetic materials. Semi-solid
and soft nanoparticles have been manufactured. A prototype
nanoparticle of semi-solid nature is the liposome.
[0121] At the small end of the size range, nanoparticles are often
referred to as clusters. Metal, dielectric, and semiconductor
nanoparticles have been formed, as well as hybrid structures (e.g.,
core-shell nanoparticles). Nanospheres, nanorods, and nanocups are
just a few of the shapes that have been grown. Semiconductor
quantum dots and nanocrystals are types of nanoparticles. Such
nanoscale particles are used in biomedical applications as drug
carriers or imaging agents.
[0122] Nanoparticle characterization is necessary to establish
understanding and control of nanoparticle synthesis and
applications. Characterization is done by using a variety of
different techniques, mainly drawn from materials science. Common
techniques are electron microscopy (transmission or scanning,
abbreviated TEM or SEM respectively), atomic force microscopy
(AFM), dynamic light scattering (DLS), x-ray photoelectron
spectroscopy (XPS), powder x-ray diffractometry (XRD), and Fourier
transform infrared spectroscopy (FTIR).
[0123] The term "nucleic acid" as used herein means a
deoxyribonucleotide or ribonucleotide polymer in either single or
double stranded form, and unless otherwise limited, encompassing
analogs of natural nucleotides that hybridize to nucleic acids in a
manner similar to naturally occurring nucleotides. The term
"nucleotide" includes, but is not limited to, a monomer that
includes a base (such as a pyrimidine, purine or synthetic analogs
thereof) linked to a sugar (such as ribose, deoxyribose or
synthetic analogs thereof), or a base linked to an amino acid, as
in a peptide nucleic acid. A nucleotide is one monomer in a
polynucleotide. A nucleotide sequence refers to the sequence of
bases in a polynucleotide.
[0124] A "target nucleic acid" (such as a target 16S rRNA, miRNA,
or target mRNA) is a defined region or particular portion of a
nucleic acid molecule, for example a small non-coding RNA (such as
an miRNA, siRNA, or piRNA) or mRNA of interest. Where the target
nucleic acid sequence is a target miRNA or a target mRNA, such a
target can be defined by its specific sequence or function; by its
gene or protein name; or by any other means that uniquely
identifies it from among other nucleic acids.
[0125] In some examples, alterations of a target nucleic acid
sequence (e.g., an miRNA, siRNA, piRNA, or an mRNA) are "associated
with" a disease or condition. That is, detection of the target
nucleic acid sequence can be used to infer the status of a sample
with respect to the disease or condition. For example, the target
nucleic acid sequence can exist in two (or more) distinguishable
forms, such that a first form correlates with absence of a disease
or condition and a second (or different) form correlates with the
presence of the disease or condition. The two different forms can
be qualitatively distinguishable, such as by nucleotide
polymorphisms or mutation, and/or the two different forms can be
quantitatively distinguishable, such as by the number of copies of
the target nucleic acid sequence that are present in a sample.
[0126] As used herein, the term "probe" means a nucleic acid
molecule or peptide capable of detecting a target. In some
examples, a probe includes a detectable label.
[0127] "RNA (ribonucleic acid)" as used herein is a long chain
polymer which consists of nucleic acids joined by 3'-5'
phosphodiester bonds. The repeating units in RNA polymers are four
different nucleotides, each of which comprises one of the four
bases, adenine, guanine, cytosine, and uracil bound to a ribose
sugar to which a phosphate group is attached. In general, DNA is
transcribed to RNA by an RNA polymerase. RNA transcribed from a
particular gene contains both introns and exons of the
corresponding gene; this RNA is also referred to as pre-mRNA. RNA
splicing subsequently removes the intron sequences and generates a
messenger RNA (mRNA) molecule, which can be translated into a
polypeptide. Triplets of nucleotides (referred to as codons) in an
mRNA molecule code for each amino acid in a polypeptide, or for a
stop signal.
[0128] Another form of RNA is small non-coding RNA, including
microRNA (miRNA), which are single-stranded RNA molecules that
regulate gene expression. miRNAs are generally about 18-25
nucleotides in length. microRNAs typically modulate gene expression
(e.g., increase or decrease translation) by promoting cleavage of
target mRNAs or by blocking translation of the cellular transcript.
miRNAs are processed from primary transcripts known as pri-miRNA to
short stem-loop structures called precursor (pre)-miRNA and finally
to functional, mature miRNA. Mature miRNA molecules are partially
complementary to one or more messenger RNA molecules, and their
primary function is to down-regulate gene expression. miRNA
sequences are publicly available. For example, miRBase
(mirbase.org) includes a searchable database of annotated miRNA
sequences. miRNA sequences are also available through other
databases known to one of ordinary skill in the art, including the
National Center for Biotechnology Information
(ncbi.nlm.nih.gov).
[0129] "Small non-coding RNA" means any non-coding RNA of about 60
nucleotides or less. Small (or short) non-coding RNAs include
microRNA (miRNA; above). Other small non-coding RNAs include small
interfering RNA (siRNA), which are about 19-23 nucleotides in
length. siRNAs are double-stranded nucleic acid molecules that
modulate gene expression through the RNAi pathway. siRNA molecules
generally have 2-nucleotide overhangs on each 3' end. However,
siRNAs can also be blunt ended. Generally, one strand of a siRNA
molecule is at least partially complementary to a target nucleic
acid, such as a target mRNA. siRNAs are also referred to as "small
inhibitory RNAs" or "short inhibitory RNAs." As used herein, siRNA
molecules need not be limited to those molecules containing only
RNA, but further encompasses chemically modified nucleotides and
non-nucleotides having RNAi capacity or activity. In an example, a
siRNA molecule is one that reduces or inhibits the biological
activity or expression of mRNA.
[0130] Additional small non-coding RNAs include Piwi-interacting
RNA (piRNA), which are about 25-30 nucleotides in length and bind
Piwi proteins. piRNAs are involved in germ cell development, stem
cell self-renewal, and retrotansoposon silencing. Transcription
initiation RNAs (tiRNAs) are about 18 nucleotides in length. They
are generally found downstream of transcriptional start sites and
are involved in regulating transcription of protein-coding genes by
targeting epigenetic silencing complexes. Centromere repeat
associated small interacting RNA (crasiRNA) are about 34-42
nucleotides in length and are processed from longer dsRNAs. They
are involved in recruitment of heterochromatin and/or centromeric
proteins. Another type of small non-coding RNA is telomere-specific
small RNA (tel-siRNA), which are about 24 nucleotides in length and
are 2'-O-methylated at their 3' end. They are involved in
epigenetic regulation.
[0131] The term "sample" as used herein means a biological specimen
containing DNA (for example, genomic DNA or cDNA), RNA (including
mRNA or miRNA), protein, or combinations thereof, in some examples
obtained from a subject. Examples include, but are not limited to
cells, cell lysates, chromosomal preparations, peripheral blood,
urine, saliva, tissue biopsy, surgical specimen, bone marrow,
amniocentesis samples, and autopsy material. In one example, a
sample includes RNA, such as mRNA.
[0132] The following abbreviations are used herein: [0133] CNTs
carbon nanotubes [0134] DMF dimethylformamide [0135] EDC
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride [0136]
fBSA fluorescent bovine serum albumin [0137] GR-MoTrs
guanidinium-rich molecular transporters [0138] mRNA messenger RNA
[0139] MES 2-(N-morpholino)ethaneosulfonic acid [0140] NHS
N-hydroxysuccinimide [0141] PEI poly(ethylenimine) [0142] PEG
polyethylene glycerol [0143] PI propidium iodide [0144] PLGA
poly(lactic-co-glycolic acid) [0145] rRNA ribosomal RNA [0146]
siRNA small interfering RNA [0147] TEM transmission electron
microscopy [0148] UV ultraviolet [0149] rcf relative centrifugal
force
Compositions
[0150] The invention provides compositions capable of entering a
microorganism comprising one or more functionalized hydrocarbon
macromolecules. In certain embodiments, the macromolecules are
Buckminsterfullerenes or Buckyballs. In other embodiments, the
Buckyballs are 60 carbon Buckyballs (C.sub.60) although the
invention can also include C.sub.70 Buckyballs as well as other
spherical fullerene molecules and quantum dots.
[0151] In certain embodiments, the Buckyballs are functionalized
with one or more carboxyl groups. In other embodiments, the
Buckyballs are functionalized with three carboxyl groups. In other
embodiments, the Buckyballs are C.sub.60 pyrrolidine tris-acid.
[0152] In certain embodiments, the Buckyballs possess an intact
carbon cage which retains sufficient hydrophobicity to inhibit
adherence to an organic matter matrix and/or a soil matrix.
[0153] In certain embodiments, the Buckyballs are functionalized
with one or more detectable labels. In other embodiments, the
detectable labels are one or more compounds selected from the group
consisting of fluorescent tags, radioactive isotopes, amino acids,
glycine, tryptophan, arginine and cysteine. In other embodiments,
the Buckyballs are functionalized with fluorescent bovine serum
albumin (fBSA). In yet other embodiments, the Buckyballs are
functionalized with fBSA through a EDC/NHS coupling that activates
the carboxylic group, wherein fBSA replaces the NHS ester to form a
stable conjugate. In certain embodiments, the Buckyballs are
functionalized with .sup.14C. In certain embodiments, the
Buckyballs are functionalized with .sup.125I.
[0154] In certain embodiments, the compositions comprise Buckyballs
functionalized with nucleic acids. In other embodiments, the
nucleic acids are non-coding RNA oligonucleotides. In other
embodiments, the nucleic acids specifically target distinct
components of 16S ribosomal RNA. In other embodiments, the
Buckyballs can be functionalized with specific RNA oligonucleotide
sequences targeting a 16S ribosomal RNA region that is unique to a
species of microorganism and specifically identifies that
microorganism. In yet other embodiments, the detectable labels are
one or more compounds selected from the group consisting of
fluorescent tags, radioactive isotopes, amino acids, glycine,
tryptophan, arginine and cysteine. In certain embodiments the
composition comprises a Buckyball functionalized with a
reporter/signature RNA oligonucleotide. In other embodiments, the
composition comprises signature information of a certain microbial
species. In other embodiments, the composition comprises a
Buckyball functionalized with a reporter/signature RNA
oligonucleotide and one or more detectable labels.
[0155] In certain embodiments, the Buckyball further comprises a
protective layer. In other embodiments, the protective layer is a
DNA and/or an RNA layer. In other embodiments, the protective layer
hybridizes with a nucleic acid which has been functionalized on to
the Buckyball. In other embodiments, the protective layer is
conjugated with one or more detectable labels. In yet other
embodiments, the detectable labels are one or more compounds
selected from the group consisting of fluorescent tags, radioactive
isotopes, amino acids, glycine, tryptophan, arginine and cysteine.
In yet other embodiments, the detectable labels are one or more
compounds selected from the group consisting of fBSA, .sup.14C,
.sup.125I, and cy3/6-FAM. In yet other embodiments, the one or more
detectable labels conjugated to the protective layer are silent
when the protective layer is hybridized to the functionalized
Buckyball and active once released.
[0156] In certain embodiments, the composition comprises a
Buckyball functionalized with a small non-coding RNA. In certain
embodiments, the small non-coding RNA is an siRNA. In other
embodiments, the small non-coding RNA is one that targets and
hybridizes with a specific RNA sequence. In yet other embodiments,
the small non-coding RNA is one that targets a specific mRNA. In
certain embodiments, the composition further comprises a safeguard
RNA which hybridizes to the siRNA to form a complex. In certain
embodiments, the functionalized siRNA targets and hybridizes with a
specific mRNA sequence, inhibiting its mode of action, silencing
certain cellular functions.
[0157] In certain embodiments, the composition comprises a
Buckyball functionalized with both an rRNA signature sequence as
described previously herein and a siRNA sequence as described
previously herein. In certain embodiments, the rRNA signature
sequence is selected so that the Buckyball recognizes a particular
microorganism species, such as a particular bacterium, and the
siRNA is selected so that it will hybridize with a specific target
mRNA of the particular microorganism, silencing a certain cellular
function. In certain embodiments, the composition further comprises
a detectable label and a protective layer.
[0158] In certain embodiments, the RNA oligonucleotides comprise
about 20 to about 50 nucleotides. In other embodiments, the RNA
oligonucleotides comprise about 20 to about 30 nucleotides. In
other embodiments, the RNA oligonucleotides are conjugated to the
Buckyball through an amine group at the 5'. In other embodiments,
the RNA oligonucleotides are conjugated to the Buckyball through a
covalent bond. In yet other embodiments, the RNA oligonucleotides
are conjugated to the Buckyball through an amide bond.
Methods
[0159] The invention provides methods of labeling, identifying
(e.g. recognizing), differentiating and modifying microorganisms
using functionalized Buckyball complexes.
[0160] In certain embodiments, the invention provides methods of
labeling one or more microorganisms using Buckyballs functionalized
with one or more detectable labels, the method comprising
contacting the one or more microorganisms with Buckyballs
functionalized with one or more detectable labels for a period of
time, during which the microorganisms internalize at least a
portion of the functionalized Buckyballs. In other embodiments, the
one or more detectable labels are one or more compounds selected
from the group consisting of fluorescent tags, radioactive
isotopes, amino acids, glycine, tryptophan, arginine and cysteine.
In yet other embodiments, the one or more detectable labels are
selected from the group consisting of fBSA, .sup.14C and .sup.125I.
In other embodiments, the Buckyballs are C60-pyrrolidine tris
acid.
[0161] In certain embodiments, the contacting is a period of
incubation. In other embodiments, the contacting/incubating period
of time is between 4 minutes and 120 minutes. In other embodiments,
the Buckyballs are contacted with the one or more microorganisms in
a solution. In yet other embodiments, the Buckyballs contacted in
an aqueous solution at a concentration of between about 0.01
.mu.g/mL and about 1000 .mu.g/mL (Buckyballs/solvent).
[0162] In certain embodiments, the detectable labels can be
detected by one or more methods selected from the group consisting
of autoradiography, fluorescence microscopy, X-ray fluorescence
microscopy, TEM, and fluorescent spectroscopy
[0163] In certain embodiments, the invention provides methods of
identifying and differentiating live versus dead microorganisms,
the method comprising contacting a sample of microorganisms with
Buckyballs functionalized with one or more detectable labels for a
period of time, measuring the signal from the detectable labels and
determining whether a microorganism is alive or dead based on the
localized signal emanating from the cell. In other embodiments, the
one or more detectable labels are amino acids. In certain
embodiments, dead microorganism cells emit higher signals than
living cells. Without necessarily subscribing to any single theory,
the higher signal in dead cells may be a result of a loss of
homeostasis and cell membrane/cell wall integrity, allowing for
more rapid internalization of labeled Buckyballs through the cell
membrane/cell wall.
[0164] In certain embodiments, the invention provides methods of
labeling specific species of microorganism within a sample using
the RNA functionalized Buckyballs of the invention. The method
comprises: [0165] a) selecting a species of microorganism of
interest which has a known genome containing one or more species
specific signature RNA sequences; [0166] b) functionalizing the
Buckminsterfullerene molecules with one or more RNA
oligonucleotides, wherein the one or more RNA oligonucleotides are
complementary to the one or more species specific signature RNA
sequences of the microorganism of interest; [0167] c) hybridizing
the one or more RNA oligonucleotides with o protecting layers
comprising segments of DNA or RNA and one or more detectable
labels; and [0168] d) incubating a microorganism with the
functionalized Buckminsterfullerene molecules for a period of
time;
[0169] wherein, the one or more detectable labels are silent when
the protecting layer are hybridized to the one or more RNA
oligonucleotides and active when the protecting layers are not
hybridized; and
[0170] wherein, the functionalized Buckminsterfullerene molecules
only label a microorganism in the presence of the one or more
species specific signature RNA sequences once internalized into the
species of interest and the reporting-protecting layer is
released.
[0171] In certain embodiments, the invention provides methods of
differentiating different species of microorganisms using
functionalized Buckyballs of the invention. The method
comprises:
[0172] a) synthesizing a species specific detector/reporter
Buckyball for each microorganism of interest within a sample, the
synthesis comprising; [0173] i) selecting one or more RNA
oligonucleotides which are capable of hybridizing with one or more
distinct signature regions of RNA in a microorganism of interest;
[0174] ii) conjugating the one or more RNA oligonucleotides to a
functionalized Buckyball; [0175] iii) hybridizing one or more
complementary reporting-protecting layers, which themselves have
been conjugated to one or more specific detectable labels, to the
one or more RNA oligonucleotides to form a Buckyball-RNA-protecting
layer-detectable label complex; [0176] iv) repeating the synthesis
for each microorganism of interest;
[0177] b) contacting a mixture of microorganisms with a mixture of
synthesized Buckyballs;
[0178] c) observing and measuring the signal produced within each
cell by the one or more specific detectable labels wherein each
detectable label corresponds with a different species of
microorganism.
[0179] In other embodiments, the one or more detectable labels
conjugated to the protective layer are silent when the protective
layer is hybridized to the functionalized Buckyball and active once
released.
[0180] In certain embodiments, the RNA oligonucleotides are capable
of recognizing and hybridizing with 16S rRNA which is unique to
each species of microorganism. In other embodiments, the RNA
oligonucleotides are about a 80% to 100% match for the signature
RNA region of the organism of interest. In other embodiments, the
RNA oligonucleotides comprise about 20 to about 50 nucleotides. In
other embodiments, the RNA oligonucleotides comprise about 20 to
about 30 nucleotides. In other embodiments, the RNA
oligonucleotides are conjugated to the Buckyball through an amine
group at the 5'. In other embodiments, the RNA oligonucleotides are
conjugated to the Buckyball through a covalent bond. In yet other
embodiments, the RNA oligonucleotides are conjugated to the
Buckyball through an amide bond. In certain embodiments, the RNA
oligonucleotide can be selected through any reasonable means
including bioinformatics analysis.
[0181] In certain embodiments, the reporting-protecting layer can
be a complementary RNA or DNA sequence which is bound to a
detectable label. In certain embodiments, the protecting layer is
about a 75% match to the RNA oligonucleotide. In certain
embodiments, the reporting-protecting layer prevents degradation of
the RNA nucleotide. In certain embodiments, the detectable label is
silent when the reporting-protecting layer is hybridized with the
RNA oligonucleotide but is observable when unbound from the RNA
oligonucleotide. In certain embodiments, the reporting-protecting
layer is released from the complex when it is replaced by the
signature region of RNA in the microorganism of interest.
[0182] In certain embodiments, the one or more detectable labels
are fluorescent labels, radioactive isotopes, amino acids, nucleic
acids, and peptides.
[0183] In certain embodiments, the Buckyballs are contacted with
the microorganisms in a solution. In other embodiments, the
Buckyballs are contacted with the microorganisms in an aqueous
solution at a concentration of between about 0.01 .mu.g/mL and
about 1000 .mu.g/mL (Buckyballs/solvent).
[0184] In certain embodiments, the invention provides methods of
inhibiting gene expression in a cell using the RNA functionalized
Buckyballs of the invention. The method comprises: [0185] a)
synthesizing functionalized Buckminsterfullerene molecules capable
of inhibiting an mRNA segment of interest, the synthesis
comprising; [0186] i) selecting one or more siRNA oligonucleotides
which are able to hybridize with the mRNA segment of interest;
[0187] ii) conjugating the one or more siRNA oligonucleotides to a
functionalized Buckminsterfullerene molecule; [0188] iii)
hybridizing one or more complementary protecting layers to the one
or more siRNA oligonucleotides to form a protected inhibitor
complex; [0189] b) contacting a cell containing the mRNA segment of
interest with the synthesized Buckminsterfullerene molecules; and
[0190] c) allowing the microorganisms to internalize the
Buckminsterfullerene molecules, whereby the functionalized siRNA
oligonucleotides can hybridize with free mRNA in the cytoplasm,
preventing transcription and gene expression.
[0191] In certain embodiments, the invention provides methods of
detecting and imaging gene expression in a cell using the RNA
functionalized Buckyballs of the invention. The method comprises:
[0192] a) synthesizing functionalized Buckminsterfullerene
molecules capable of binding an mRNA segment of interest
corresponding to the gene of interest, the synthesis comprising;
[0193] i) selecting one or more siRNA oligonucleotides which are
able to hybridize with the mRNA segment of interest; [0194] ii)
conjugating the one or more siRNA oligonucleotides to a
functionalized Buckminsterfullerene molecule; [0195] iii)
hybridizing one or more complementary protecting layers to the one
or more siRNA oligonucleotides to form a protected inhibitor
complex, wherein the one or more complementary protecting layers
are conjugated with a detectable label which is silent when the
protecting layer is hybridized and active when it is not
hybridized; [0196] b) contacting a cell containing the mRNA segment
of interest with the synthesized Buckminsterfullerene molecules;
and [0197] c) allowing the microorganisms to internalize the
Buckminsterfullerene molecules, whereby the functionalized siRNA
oligonucleotides can hybridize with free mRNA in the cytoplasm,
releasing the complementary protecting layer.
[0198] In certain embodiments, the siRNA oligonucleotides are
capable of recognizing and hybridizing with mRNA which is unique to
a specific species of microorganism. In other embodiments, the RNA
oligonucleotides are about a 80% to a 100% match for the mRNA
segment of interest. In other embodiments, the siRNA
oligonucleotides comprise about 20 to about 50 nucleotides. In
other embodiments, the siRNA oligonucleotides comprise about 20 to
about 30 nucleotides. In other embodiments, the siRNA
oligonucleotides are conjugated to the Buckyball through an amine
group at the 5'. In other embodiments, the siRNA oligonucleotides
are conjugated to the Buckyball through a covalent bond. In yet
other embodiments, the siRNA oligonucleotides are conjugated to the
Buckyball through an amide bond. In certain embodiments, the siRNA
oligonucleotide can be selected through any reasonable means
including bioinformatics analysis.
[0199] The invention further provides a combined method of
simultaneously differentiating different species of microorganism
and inhibiting gene expression in a specific microorganism using
the RNA functionalized Buckyballs of the invention. The method
comprises:
[0200] a) synthesizing species specific reporter/inhibitor
Buckyballs for each microorganism of interest within a sample, the
synthesis comprising; [0201] i) selecting one or more RNA
oligonucleotides which are capable of hybridizing with one or more
distinct signature regions of RNA in a microorganism of interest;
[0202] ii) selecting one or more siRNA oligonucleotides which are
capable of hybridizing with one or more distinct mRNA segments of
interest; [0203] iii) conjugating the one or more RNA
oligonucleotides and siRNA oligonucleotides to a functionalized
Buckyball; [0204] iv) hybridizing one or more complementary
reporting-protecting layers for the RNA oligonucleotides, which
themselves have been conjugated to one or more specific detectable
labels, to the one or more RNA oligonucleotides and hybridizing one
or more complementary protecting layers to the one or more siRNA
oligonucleotides to form a Buckyball-RNA/siRNA-protecting
layer-detectable label complex; [0205] v) repeating the synthesis
for each microorganism of interest;
[0206] b) contacting a mixture of microorganisms with a mixture of
synthesized Buckyballs;
[0207] c) visualizing and measuring the signal produced within each
cell by the one or more specific detectable labels wherein each
detectable label corresponds with a different species of
microorganism.
[0208] In certain embodiments, each Buckyball can be functionalized
with RNA corresponding to multiple different microorganisms. In
other embodiments, a single batch of Buckyballs may be synthesized
which have been functionalized with RNA oligonucleotides
corresponding to two or more different microorganisms and
reporting-protecting layers, each conjugated with different
detectable labels wherein once the Buckyballs have been
internalized, they will only release the appropriate
reporting-protecting layer such that the identity of the
microorganism can be determined by observing and measuring the
signal from the detectable label.
[0209] In certain embodiments, the method does not require sample
fixation. In other embodiments, the methods can be used with living
microorganisms. In yet other embodiments, the methods can be used
to visual, monitor and profile mRNA expression by dynamic
visualization.
[0210] In certain embodiments, the functionalized Buckyballs are
synthesized with substituents that eliminate stickiness (adherence)
to an organic matrix or the microbial environment.
Devices and Systems
[0211] The invention further provides devices and systems for
differentiating microorganisms within a microbial community using
the compositions and methods of the invention.
[0212] In certain embodiments, the invention comprises a system
comprising one or more source wells and one or more sink wells
wherein the one or more source wells are in fluidic communication
with the one or more sink wells. Each of the one or more sink wells
independently comprises one or more Buckyball compositions of the
invention functionalized to recognize a specific species of
microorganism and release a specific detectable label in the
presence of said microorganism and optionally a microbial
attractant specifically chosen to attract said microorganism. By
selectively releasing a specific detectable label which corresponds
to a specific species, this allows the system to determine the
presence or absence of a species of microorganism in a well. In
certain embodiments, the one or more Buckyball compositions are
printed into the one or more sink wells.
[0213] In certain embodiments of the system, a microbial sample
mixture comprising one or more microorganism species is placed in
the source well. The microbial sample mixture will then disperse,
spreading the microorganisms to the sink wells through random
diffusion, propulsion or through active locomotion by the
microorganisms. In certain embodiments, certain microorganisms will
be attracted to specific wells due to the presence of a microbial
attractant. In certain embodiments, a microorganism that has
entered a sink well will internalize one or more of the Buckyballs
of the invention. In other embodiments, if the microorganism
internalizes a Buckyball comprising an RNA oligonucleotide
corresponding to a matching sequence in the microorganism, the
Buckyball will release a reporting-protecting layer comprising a
detectable label, producing a detectable signal. In certain
embodiments, the signal emitted by the detectable label can be
measured to determine the presence or absence of the corresponding
microorganism as well as the relative abundance of said
microorganism.
[0214] In certain embodiments, the microbial attractant is a
nutrient, mineral or environmental condition which would
selectively draw a microorganism of interest to sink well. In
certain embodiments, the microbial attractant can be one or more
conditions selected from the group consisting of a sugar gradient,
a protein gradient, a metal ion gradient, a temperature gradient, a
salinity gradient, a light gradient and a specific wavelength of
light.
[0215] In certain embodiments, each sink well comprises a different
Buckyball composition of the invention capable of detecting a
different species of microorganism. In certain embodiments, one or
more sink wells comprise Buckyball compositions capable of
detecting multiple species of microorganisms.
[0216] In certain embodiments, the system comprises about 2 to
about 1,000 sink wells. In other embodiments, the system comprises
a number of sink wells equivalent to the number of identified
microbial species present in a studied microbial system. For
example, the typical human oral cavity comprises a microbial biome
which is home to hundreds of well characterized species. In certain
embodiments, a system designed to study 100 highly prevalent
microorganisms native to the human oral cavity can have about 100
sink wells, each sink well comprising a Buckyball functionalized
with an RNA oligonucleotide specifically designed to hybridize with
a signature RNA sequence unique to one of the microbial species
present in a typical human oral cavity, and each Buckyball further
comprising a unique detectable label conjugated to a
reporting-protecting layer.
[0217] In certain embodiments, the system further comprises one or
more control wells. In other embodiments, the control wells do not
comprise compositions of the invention. In other embodiments, the
control wells comprise unfunctionalized Buckyballs. In yet other
embodiments, a portion of the control wells are positive controls
wherein the wells comprise microorganisms of a known population
density which have been exposed to the corresponding Buckyballs of
the invention and emit a signal from the detectable label which can
be used as a basis of comparison.
[0218] In certain embodiments, the system further comprises one or
more imaging devices capable of rapidly recording a signal from the
one or more sink wells to determine the presence or absence of
signaling microorganisms in each well as well as the relative
signal intensities. In other embodiments, the imaging device can
report this information to a user as an estimated microbial density
for each species within the sample mixture.
[0219] Every formulation or combination of components described or
exemplified can be used to practice the invention, unless otherwise
stated. Specific names of compounds are intended to be exemplary,
as it is known that one of ordinary skill in the art can name the
same compounds differently. When a compound is described herein
such that a particular isomer or enantiomer of the compound is not
specified, for example, in a formula or in a chemical name, that
description is intended to include each isomers and enantiomer of
the compound described individual or in any combination.
[0220] Although the description herein contains many embodiments,
these should not be construed as limiting the scope of the
invention but as merely providing illustrations of some of the
presently preferred embodiments of the invention.
[0221] All references throughout this application (for example,
patent documents including issued or granted patents or
equivalents; patent application publications; and non-patent
literature documents or other source material) are hereby
incorporated by reference herein in their entireties, as though
individually incorporated by reference, to the extent each
reference is at least partially not inconsistent with the
disclosure in this application (for example, a reference that is
partially inconsistent is incorporated by reference except for the
partially inconsistent portion of the reference).
[0222] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures, embodiments, claims, and
examples described herein. Such equivalents were considered to be
within the scope of this invention and covered by the claims
appended hereto. For example, it should be understood, that
modifications in reaction conditions, including but not limited to
reaction times, reaction size/volume, and experimental reagents,
such as solvents, catalysts, pressures, atmospheric conditions,
e.g., nitrogen atmosphere, and reducing/oxidizing agents, with
art-recognized alternatives and using no more than routine
experimentation, are within the scope of the present application.
In general the terms and phrases used herein have their
art-recognized meaning, which can be found by reference to standard
texts, journal references and contexts known to those skilled in
the art. Any preceding definitions are provided to clarify their
specific use in the context of the invention.
[0223] It is to be understood that wherever values and ranges are
provided herein, all values and ranges encompassed by these values
and ranges, are meant to be encompassed within the scope of the
present invention. Moreover, all values that fall within these
ranges, as well as the upper or lower limits of a range of values,
are also contemplated by the present application.
[0224] The following examples further illustrate aspects of the
present invention. However, they are in no way a limitation of the
teachings or disclosure of the present invention as set forth
herein.
EXAMPLES
[0225] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only, and the invention is not limited to these
Examples, but rather encompasses all variations that are evident as
a result of the teachings provided herein.
Materials and Methods
Conjugation of Fluorescent Bovine Serum Albumin/Amino Acids
[0226] C60-pyrrolidine tris acid (1 mg) (Sigma) was dispersed in
0.5 mL of 2-(N-morpholino)ethanosulfonic acid (pH 5.6) (MES)
(Sigma) buffer under sonication for 30 min at ambient conditions.
0.25 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (0.4 mol/L) (EDC) (Sigma) and 0.25 ml of
N-hydroxysuccinimide (0.2 mol/L) (NETS) (Sigma) in MES solution
were added to the activated carboxylate groups. The mixture was
washed with PBS and centrifuged at 12,000 g for 30 min in a 5 KDa
molecular weight cutoff centrifugal filter (Millipore) for 5 times,
to remove EDC and NHS. 2 mg of fluorescent bovine serum albumin
(fBSA) (Sigma)/cysteine (Acros) were then added into the
C60-pyrrolidine tris acid/PBS solution at 4.degree. C. overnight
for conjugation. The mixture was finally washed with PBS and
centrifuged in a micro-centrifuge tube to remove un-conjugated
protein/cysteine in supernatant; this centrifugation was repeated 5
times at 12,000 rcf for 30 min. The C60-pyrrolidine tris
acid-fBSA/cysteine pellet was collected and stored at -20.degree.
C. until use.
[0227] For further labeling C60-pyrrolidine tris-cysteine with
Atto565 NHS Easter (Sigma),C60-pyrrolidine tris-cysteine (1 mg)
were suspended in 1 mL EDC/MES solution (0.1 mol/L); Atto 565 NHS
Easter (20 .mu.L) was then added at 4.degree. C. overnight for
conjugation. The mixture was finally washed with PBS and
centrifuged in a micro-centrifuge tube to remove un-conjugated
Atto565 in supernatant; the centrifugation was repeated 5 times at
12,000 rcf for 30 min. The C60-pyrrolidine tris
acid-cysteine-Atto565 pellet was collected and stored at
-20.degree. C. until use.
Radio-Labeling of .sup.14C and .sup.125I to C60-Pyrrolidine Tris
Acid
[0228] .sup.14C labeling was carried out on dry ice and under argon
in the fume hood. C60-pyrrolidine tris acid (100 .mu.g) was
dispersed in 1 mL dimethylformamide (DMF) (Sigma) homogeneously in
a 15 mL conical tube. Potassium carbonate (1 mg) was added as base.
Then, 50 .mu.L .sup.14C-methyl iodide (1.85 MBq) was added, and the
tube was capped tightly. Until this point, H.sub.2O was avoided.
The tube was transferred at room temperature to allow the mixture
to react for 1 hour with periodic agitation (every 10 min).
Potassium carbonate was then removed while excess DMF and unreacted
.sup.14C-methyl iodide were evaporated using an oil bath. Next,
.sup.14C labeled C60-pyrrolidine tris acid was suspended in 1 mL
DMF, and its radioactivity was counted in the liquid scintillation
counter. The radiochemical yield was calculated as .about.20% as
following:
Radiochemical Yield = Product Radioactivity Added Radioactivity
.times. 1 0 0 % Equation 1 ##EQU00001##
[0229] .sup.125I-labeling was performed under ambient conditions in
a 1.5 mL microcentrifuge tube. C60-pyrrolidine tris acid (100
.mu.g) was dispersed in 1 mL phosphate buffer (pH 8.0). 5 .mu.L of
Na.sup.125I (0.56 MBq) and 5 .mu.L of N-chlorosuccinimide (10 mg/mL
in DI H.sub.2O) were then added. The reaction was allowed to
continue for 20 min with periodic shaking (every 5 min). The
reaction was then quenched by adding 10 .mu.L of sodium bisulphite
(10 mg/mL in DI H.sub.2O) (Sigma) and 10 .mu.L of NaI (10 mg/mL in
DI H.sub.2O) (Sigma). The mixture was centrifuged at 20,000 rcf for
15 min to pellet the .sup.125I-labeled C60-pyrrolidine tris acid.
The pellet was washed with DI H.sub.2O and re-pelleted 5 times at
20,000 rcf for 15 min until the radioactivity of the wash reached
normal level. The yield was calculated to be .about.20% according
by Equation 1.
Fluorescence Microscopy
[0230] A Zeiss 710 confocal microscopy system fluorescence
microscope was utilized to (i) profile microbial and matrix
autofluorescence, and microbial uptake, (ii) differentiate live and
dead cells, and (iii) evaluate adherence to the matrix. Depending
upon the use-case, samples were imaged with either a Zeiss
Plan-Neofluar 10.times./0.30 objective or a Plan-Apochromat
63.times./1.40 Oil immersion objective. The soil matrices were
imaged with the 10.times. objective, while microbial species were
imaged and subsequently quantified with the 63.times. objective.
The excitation filters were set at 488 and 561 nm, and the emission
filters were set to receive signals between 493-556 nm and 597-700
nm, respectively. The laser intensity was set at 20% to excite fBSA
and matrix autofluorescence. A twin-gate main beam splitter with
two wheels and each wheel having 10 filter positions (e.g., 100
possible combinations) was used to separate excitation and emission
beams. The pinhole was set at 600 .mu.m to receive as many photons
as possible.
[0231] To examine the auto-fluorescence of microorganisms, 1.0 mL
of bacterial cells (OD.sub.630=0.6) were pelleted by centrifugation
at 5,000 rcf to remove the old medium, then suspended in 1.0 mL
fresh medium. 5 .mu.L of the suspension was then mounted onto a
glass slide under a coverslip, sealed with nail polish hardener. To
examine the retention of C60-pyrrolidine tris acid within the
microorganisms, 1.0 mL of bacterial cells (OD.sub.630=0.6) were
pelleted at 5,000 rcf to remove the old medium. Cells were
suspended in fresh medium in a 1.5 mL micro-centrifuge tube and
incubated in the dark at with C60-pyrrolidine tris acid-fBSA (5
.mu.g/mL) predetermined times: 5 min, 30 min, 1 hour, and 2 hours.
The tube was then centrifuged at 5,000 rcf to remove the
non-penetrated C60-pyrrolidine tris acid-fBSA, and the
microorganisms were suspended in fresh medium. 5 .mu.L of the
suspension was then mounted onto a glass slide under a coverslip,
sealed with nail polish hardener.
[0232] To examine the retention of C60-pyrrolidine tris acid within
the microorganisms after wash, the pellets were washed with DI
H.sub.2O, and the C60-pyrrolidine tris acid-fBSA internalized
microorganisms were re-pelleted at 5,000 rcf for 5 min in the dark.
This was repeated 6 times. 5 .mu.L of the suspension was then
mounted onto a glass slide under a coverslip, sealed with nail
polish hardener. The fluorescent images were taken using a Zeiss
710 confocal microscopy system.
[0233] To differentiate live and dead microorganisms, freshly
prepared C60-pyrrolidine tris-cysteine-Atto565 conjugate was used.
First, live or dead microorganisms (E. coli and B. subtilis) (dead
cells were obtained by incubating live cells at 90.degree. C. for
30 minutes) were incubated in the dark with C60-cysteine-Atto565
(10 .mu.g/mL) for 30 min. Second, both live and dead microorganisms
were pelleted at 5,000 rcf for 5 min, and the supernatant was
carefully removed. Third, the live and dead microorganism pellets
were washed with DI H.sub.2O, then re-pelleted. Finally, 5 .mu.L of
the final suspensions for live and dead microorganism were mounted
onto two respective glass slides under coverslips, sealed with nail
polish hardener. The prepared slides were imaged using a Zeiss 710
confocal microscope under identical microscopic conditions (e.g.,
laser intensity, pinhole setting, gain). The images obtained with
the Zeiss 710 confocal microscope were not modified by other
software and were analyzed by ImageJ software. The process of the
image analysis is: (I) The fluorescent signal of the microorganisms
(single cell or cluster of cells) were selected by the freehand
selection tool and the background signal was subtracted. (II) The
mean intensity of the selected area was measured by ImageJ. (III)
The average signal was then calculated by averaging the mean
intensity of multiple selected areas.
[0234] To examine the adherence of C60-pyrrolidine tris acid to the
matrix, several substrates were used. These are VWR Sand (VWR),
alumina (Acros), glass beads, wild sand (collected at the Aquatic
Park Innovation Center, Berkeley Calif.), and natural soil
(collected at the backyard garden of Aquatic Park Innovation
Center, Berkeley Calif., courtesy of the building management).
These substrates (0.2 g) were incubated in the dark with 1 mL
C60-pyrrolidine tris acid-fBSA (5 .mu.g/mL) for 30 min. Each
substrate was then washed 6 times with 1 mL DI H.sub.2O (10 min
incubation). Next, 10 grains of substrate particle were carefully
dried of liquid, and then mounted onto glass slides with nail
polish hardener as a sealant. The fluorescent images were taken by
Zeiss 710 confocal microscope.
Retention of C60-Derivatives From Multiple Substrates
[0235] To examine the retention of C60-derivatives in multiple
substrates with different masses (2 g, 4 g, 6 g, and 8 g), each
substrate (e.g., wild sand, natural soil, VWR sand) was incubated
with C60-pyrrolidine tris acid at a concentration of 5 .mu.g/mL on
a layer of filter paper (with an average pore size of 25 .mu.m).
After 30 min of incubation, vacuum suction was applied, and the
first filter-through of C60-pyrrolidine tris acid was collected.
The residue on the filter paper was washed with DI H.sub.2O (10 min
of incubation), and the filter-through of C60-pyrrolidine tris acid
was collected for the second time. Both filter-throughs were
measured by UV-vis spectrometer at 335 nm. The recovery of
C60-pyrrolidine tris acid was determined as following:
Recovery of C60-Pyrrolidine Tris Acid=Absorption of Filter-throughs
at 335 nm/Original amount of C60 at 335 nm.times.100% Equation
2
Transmission Electron Microscopy (TEM)
[0236] TEM was used to examine whether the retention of
C60-pyrrolidine tris acid was localized within the cytosol.
Microorganisms were incubated with C60-pyrrolidine tris acid (5
.mu.g/mL) for 30 min. Cells were pelleted by centrifugation at
5,000 rcf (5 min) and washed 5 times with DI H.sub.2O.
Microorganisms were fixed in 2.5% glutaraldehyde/PBS solution for
30 min. The cells were then pelleted by centrifugation at 5,000 rcf
for 5 min. The cell pellet was placed in fresh 2.5%
glutaraldehyde/PBS solution at 4.degree. C. overnight. Next, cell
pellets were dehydrated by a series of acetone treatments (30% for
15 min, 50% for 15 min, 70% for 15 min, 90% for 15 min and a final
treatment at 100% for 30 min, and repeated 3 times), embedded in
resin (2:1 mix of propylene oxide:resin for 1 hour, 1:1 mix of
propylene oxide:resin for 1 hour, 1:2 mix of propylene oxide: resin
for 1 hour, 100% resin overnight, and change to fresh resin 1 h),
incubated for 24 h at 37.degree. C., sectioned (60 nm in
thickness), and imaged using a Tecnai 12 TEM.
Liquid Scintillation Counter
[0237] To examine the retention of C60-pyrrolidine tris acid within
multiple substrates, each substrate (0.2 g) was incubated in the
dark with 1 mL of .sup.14C labeled C60-pyrrolidine tris acid (5
.mu.g/mL) for 30 min. The substrate was pelleted without
microorganisms at 5,000 rcf and washed those pellets 6 times with 1
mL DI H.sub.2O (10 min incubation). The final pellets were added to
3 mL of scintillation cocktail. The data were recorded using a
liquid scintillation counter (Perkin Elmer).
Autoradiography
[0238] To examine the retention of C60-pyrrolidine tris acid in B.
subtilis and E. coli with multiple substrates, each substrate (0.2
g) was incubated in the dark with 1 mL .sup.14C- and
.sup.125I-labeled C60-pyrrolidine tris acid (5 .mu.g/mL) for 30
min. Substrates both with and without microorganisms were pelleted
at 5,000 rcf and washed those pellets 5 times with 1 mL fresh DI
H.sub.2O (10 min incubation). The final pellet was dispersed in 0.1
mL fresh LB broth. 1/10 of the volume was carefully transferred
onto a piece of plastic wrap, under which was a phosphor imager
film. After 24 hours of sitting in complete darkness, the film was
transferred into a Cyclone Plus Phosphor Imager (Perkin Elmer) for
imaging.
Cell Culture
[0239] E. coli (Invitrogen) and B. subtilis (ATCC) were cultured in
LB Broth at 37.degree. C. with constant shaking. E. coli and B.
subtilis were not used until OD.sub.630 reached 0.6.
Statistical Analysis
[0240] Four samples were analyzed at each condition. The data in
the graphs are represented by their mean.+-.standard deviation
(SD).
Preparation of Microfluidics
[0241] Glass slides (Fisher Scientific) were washed with ethanol,
dried with air, and exposed to 4 mW/cm.sup.2 UV light (UVP, LLC)
for 2 hr. The hyrogel precursor (0.5 mL) consists of 10% (v/v) 700
MW PEG diacrylate (PEG-DA) (Sigma) and 0.5% (v/v)
2-hydroxy-2-methylpropiophenone (Sigma), and is evenly distributed
over the glass slides by a spin coater (SCK-200P). The slides were
then placed under approximately 4 mW/cm.sup.2 UV light for 15
seconds under a mask to gel. The slides were then incubated in 50
mM triethylene glycol mono-mercaptoundecyl ether (Sigma) for 15
min, rinsed in 70% ethanol for 15 min and washed with DI water.
During this process, the microfluidics is stored in humid
environment to avoid desiccation.
Example 1
Synthesis of Functionalized C60-Pyrrolidine Tris Acid
[0242] C60-pyrrolidine tris acid is a derivative of fullerene C60
(FIG. 1) and possesses three key properties: (i) containing three
carboxyl groups that allow for further decoration (e.g.,
fluorescent tagging and radioactive isotope labeling); (ii) being
extremely small (1-2 nm for a single molecule, 10-20 nm for a
cluster of molecules), which facilitates intercellular movement and
actions; and (iii) maintaining an intact carbon cage that retains
enough hydrophobicity to inhibit adherence of C60-pyrrolidine tris
acid to the soil matrix and organic matter. C60 was evaluated as
both a fluorescent and radiotracer reporter, where the rationale
for functionalization is summarized below.
[0243] (i) Functionalization of C60-pyrrolidine tris acid with fBSA
is based on EDC/NHS coupling that activates the carboxylic group,
where fBSA replaces the NHS ester to form a stable conjugate.
[0244] (ii) Functionalization of C60-pyrrolidine tris acid with
.sup.14C was based on the methylation of the carboxyl group with
potassium hydroxide/dimethyl sulfate and radioactive methyl iodide.
The final yield of .sup.14C-methylated C60-pyrrolidine tris acid
was about 20%.
[0245] (iii) Functionalization of C60-pyrrolidine tris acid with
.sup.125I uses the Finkelstein reaction. First, C60-pyrrolidine
tris acid was functionalized with Cl by using N-chlorosuccinimide.
Second, Cl was replaced by .sup.125I through the Finkelstein
reaction. The final yield of .sup.125I-labeled C60-pyrrolidine tris
acid was about 20%.
Example 2
Microbial Internalization of C60-Pyrrolidine Tris Acid
[0246] In order to visualize whether C60-pyrrolidine tris acid can
internalize within microorganisms, a three-step validation protocol
was used that involved fluorescence microscopy, transmission
electron microscopy (TEM), and autoradiography. The model organisms
included both Gram-negative and Gram-positive bacteria.
[0247] E. coli (Gram-negative) and B. subtilis (Gram-positive) were
incubated with functionalized C60-pyrrolidine tris acid with fBSA
for 30 minutes. Samples were then washed with DI H.sub.2O to remove
excess probes, and samples were then imaged by confocal microscopy.
FIG. 2 indicates a positive association of C60-pyrrolidine tris
acid with microorganisms, where in FIGS. 2A and 2B, both E. coli
and B. subtilis have fluorescent signal emission following
excitation by a 488 nm laser. It was confirmed that these
fluorescent signals are solely from C60-pyrrolidine tris acid-fBSA,
because neither E. coli nor B. subtilis has an auto-fluorescence
signal under the same conditions in the absence of fluorescent C60
(FIGS. 12A-12F). In addition, these fluorescent signals co-localize
with E. coli and B. subtilis cells by combining bright field and
fluorescent imaging, which indicates either internalization within
the cell or binding to the cell wall.
[0248] To test the hypothesis that functionalized C60-pyrrolidine
tris acid internalizes within the cell, microorganisms were imaged
with TEM. Both E. coli and B. subtilis were incubated with
C60-pyrrolidine tris acid, washed to remove excess compound,
sectioned into slices of 60 nm thickness, and then imaged by TEM.
FIGS. 3A and 3B shows that C60-pyrrolidine tris acid localizes
within the cell in both E. coli and B. subtilis, respectively. The
control study consisted of (i) C60-pyrrolidine tris acid in DI
H.sub.2O, (ii) C60-pyrrolidine tris acid on a mouse tissue section,
(iii) an E. coli section without C60-pyrrolidine tris acid
incubation, and (iv) a B. subtilis section without C60-pyrrolidine
tris acid incubation. These data are shown in FIGS. 13A-13D. These
results indicate positive cellular uptake of C60-derivatives in
microorganisms, hence providing an opportunity to monitor the
cellular activity in situ by further functionalization of C60.
[0249] It was next determined whether C60-pyrrolidine tris acid
could be radiolabeled, which would thus provide a platform for
imaging thick sections in an opaque environment. C60-pyrrolidine
tris acid was functionalized with .sup.14C and .sup.125I, and
samples are incubated as before. FIGS. 4A-4D shows .beta.-radiation
and .gamma.-radiation emission from both E. coli (FIGS. 4A and 4C)
and B. subtilis (FIGS. 4B and 4D), which are incubated with
.sup.14C- and .sup.125I-labeled C60-pyrrolidine tris acid and
imaged through autoradiography. The liquid scintillation data (FIG.
14) also shows a positive association of .sup.14C-methylated
C60-pyrrolidine tris acid by both E. coli and B. subtilis (about
12,000 counts per second). These results provide additional
confirmation that C60-pyrrolidine tris acid can be internalized by
microbes and visualized.
[0250] Finally, to investigate whether the number of washes or the
incubation time has an impact on the C60 internalization, these
parameters are changed, and the previous studies were repeated. The
rationale was that internalization might be a function of combined
physical size, electrostatics, hydrophobicity, and diffusivity.
Results, shown in FIGS. 15A-H and FIGS. 16A and 16B and Tables 1
and 2, indicate that cellular (i) uptake is correlated with
increased incubation time, and (ii) retention is not affected by
the number of washes. These results were obtained through unbiased
and automated quantitative analysis, with an example shown in FIG.
17.
TABLE-US-00001 TABLE 1 Quantified signal intensity for FIGS.
15A-15H Microorganism Background Signal Intensity Signal Intensity
Figure 15A 273 .+-. 71 40 .+-. 8 Figure 15B 345 .+-. 56 120 .+-. 10
Figure 15C 372 .+-. 103 70 .+-. 15 Figure 15D 529 .+-. 105 150 .+-.
13 Figure 15E 654 .+-. 54 224 .+-. 48 Figure 15F 860 .+-. 123 257
.+-. 32 Figure 15G 1128 .+-. 210 321 .+-. 22 Figure 15H 1323 .+-.
239 284 .+-. 41
TABLE-US-00002 TABLE 2 Signal intensity for FIGS. 16A and 16B
Microorganism Background Signal Intensity Signal Intensity Figure
16A 579 .+-. 167 40 .+-. 15 Figure 16B 942 .+-. 167 225 .+-. 34
Example 3
Non-Stickiness of C60-Pyrrolidine Tris Acid to the Substrates
[0251] Chemical staining dyes and antibodies for immunostaining
known in the art are usually sticky to the natural environment
(e.g., soil, sand), creating significant background noise during
visualization. Therefore, the non-stickiness of the C60-derivatives
of the invention were determined. The degree of non-stickiness was
evaluated using a variety of substrates (e.g., glass beads,
alumina, VWR sand, wild sand, and natural soil), with UV absorption
and imaging that includes both fluorescence microscopy and use of
an autoradiography/scintillation counter. These substrates covered
a range of synthetic and natural environments for validation, while
the readouts provided both bulk (e.g., UV absorption) and spatial
information (e.g., imaging). The results are summarized below.
[0252] The non-stickiness of the C60 derivatives was determined by
incubating functionalized C60-pyrrolidine tris acid solution in a
substrate on a layer of filter paper, applying a vacuum to remove
the solution, and running one or more H.sub.2O washes through the
substrate (FIG. 18). The stickiness of C60-pyrrolidine tris acid
was quantified with multiple substrates, by measuring the UV
absorption of filter-through at 335 nm, which is the specific
absorption wavelength of C60. FIG. 5A indicates that more than 70%
of the C60-pyrrolidine tris acid was recovered from silica without
H.sub.2O wash (red column) regardless of the mass of matrix. With
respect to non-synthetic substrates, similar recovery rates were
reported for homogenized VWR sand (>80%, FIG. 5B), Wild Sand
(>60%, FIG. 5C), and Natural Soil (>70%, FIG. 5D), all
without H.sub.2O wash (red column). The remainder of
C60-pyrrolidine tris acid is fully recovered from the matrices,
following H.sub.2O rinses (green columns in FIGS. 5A-5D). These
results indicate that C60-pyrrolidine tris acid is not sticky to
the natural environment. The rationale for requiring a second wash
is due to the meso-porous architecture of the matrices that trap
C60-derivatives.
[0253] To investigate the non-stickiness of C60-pyrrolidine tris
acid spatially, several studies were designed. (I) fBSA-labeled
C60-pyrrolidine tris acid was evaluated against
non-auto-fluorescent matrices such as glass beads and alumina
(FIGS. 6A and 6B). Using fluorescence microscopy, each substrate
emits an initial fluorescent signal after incubation with
C60-pyrrolidine tris-fBSA, with the signal being lost following
multiple H.sub.2O washes, thus providing confirmation that
C60-pyrrolidine was not sticky to the natural environment. (II)
fBSA-labeled C60-pyrrolidine tris acid was evaluated against
matrices such as VWR sand, wild sand, and natural soil (FIG. 19).
However, these matrices are auto-fluorescent and mask fluorescent
probes, making it difficult to visualize the fluorescent-labeled
C60. (III) Further validation by autoradiography and liquid
scintillation is pursued in all substrates (e.g., pretreated VWR
sand, glass beads, alumina, wild sand, and natural soil). FIG. 7
indicates that neither .beta.- nor .gamma.-radiation were detected
from incubated matrices (middle and right columns) after 6 H.sub.2O
rinses. Therefore, the disclosed C60-derivatives are non-sticky to
the natural environment and can be removed entirely by H.sub.2O
washes.
Example 4
Uptake of Functionalized C60-Pyrrolidine Tris Acid in Microbes
Embedded in Soil Matrices
[0254] Natural soil is a complicated biomaterial, hosting thousands
of microorganisms with intrinsic organic and inorganic matters that
hinder probe delivery. .sup.14C- and .sup.125I-radiolabeled
C60-pyrrolidine tris acid are incubated with a mixture of soil and
microorganisms and then washed to remove excess probes as before.
The autoradiography, shown in FIG. 8, indicates strong .beta.- and
.gamma.-radiation from the mixture of soil and microorganisms.
Comparison of this result with both (i) FIGS. 4A and 4B, which
indicated association with microorganisms, and (ii) FIGS. 7A and
7B, which indicated non-stickiness to the matrix, suggests that
radiotracers can label microbes in their native environment.
Moreover, interesting observations are made when .beta.-radiation
is quantified using a liquid scintillation counter, comparing both
control and treated matrices with .sup.14C-labeled C60-pyrrolidine
tris acid. All control matrices (e.g., background) show around
1,000 counts per second (FIG. 9, filled columns), while
.sup.14C-labeled C60-pyrrolidine tris acid incubated with glass
beads, alumina, VWR sand, wild sand, and natural soil show 2,000,
2,000, 2,000, 4,000, and 6,000 counts per second respectively (FIG.
9, hatched columns). These results indicate that the natural
microorganisms in wild sand and natural soil have successfully
taken up .sup.14C-labeled C60-pyrrolidine tris acid, which accounts
for the increased number of counts per second.
Example 5
Differentiation of Live and Dead Microorganisms
[0255] To investigate differentiation between live and dead cells,
C60 was functionalized with four different amino acids and then
screened. This approach was motivated by the fact that different
microorganisms have varying preferences for a specific amino acid.
C60-pyrrolidine tris acid was functionalized with the amino acids
glycine, tryptophan, arginine, and cysteine. In particular,
cysteine functionalized C60-pyrrolidine tris acid allowed for the
differentiation of live and dead cells in the presence of the
substrate matrix.
[0256] To validate non-stickiness to the matrix, both UV absorption
and microscopy are utilized. FIGS. 10A-10D show a trend for
recovering C60-pyrrolidine tris-cysteine from alumina (FIG. 10A),
VWR sand (FIG. 10B), wild sand (FIG. 10C), and natural soil (FIG.
10D), all of which show results comparable to those previously
reported (FIG. 5). More than 60% of C60-pyrrolidine tris-cysteine
is removed from alumina, wild sand, and natural soil, without wash
(hatched column). The rest of C60-pyrrolidine tris-cysteine was
fully recovered with one additional H.sub.2O wash (filled
column).
[0257] To differentiate and quantify live and dead microorganisms,
fluorescence microscopy was used. The results indicate a
significantly lower signal for live cells than for dead cells for
both E. coli and B. subtilis, as shown in FIG. 11. Both live E.
coli and B. subtilis showed a base line fluorescence signal of
approximately 1,000 (in pixel intensity), whereas dead E. coli and
B. subtilis showed a significantly higher signals, of approximately
6,500 and 2,000, respectively. This observation is potentially due
to the fact the dead cells have a leaky structure, which allows
more C60-pyrrolidine tris-cysteine to cross their cellular
membrane. In addition, the cell wall of dead B. subtilis
(Gram-positive, and with a thicker cell wall) may not be as leaky
as dead E. coli; thus, dead E. coli shows a higher fluorescence
signal than B. subtilis. The net result is that functionalized
C60-pyrrolidine tris acid can differentiate cellular states.
Example 6
Methods of Distinguishing Live Bacteria With C60 functionalized
With Reporter RNA Oligo
[0258] Methods were developed to distinguish different species of
live bacteria by functionalization of C60 with RNA oligonucleotides
hybridized with reporter compounds. The signature RNA
oligonucleotides are designed in accordance with the signature
information on 16s ribosome RNA, which is unique to each species
(Reischl et al., Clin Chem 52, 1985-1987 (2006) and Dresios et al.,
Journal of molecular biology 345, 681-693 (2005), each of which is
hereby incorporated by reference in its entirety). The strategy to
recognize a certain bacteria is shown in FIGS. 20A-20C and operates
as follows: [0259] (1) C60 molecules were functionalized with
signature RNA oligonucleotides containing signature information
(e.g., calling card) of a certain species. The Signature RNA
oligonucleotides were typically screened through bioinformatics
analysis. [0260] (2) A reporter sequence, which is conjugated to a
fluorophore, was hybridized with the signature sequence. The
fluorophore was silent while hybridized but fluoresced once
released. [0261] (3) The C60 complex penetrated the bacteria and
released the reporter if the bacteria contained ribosomal signature
information that matches the signature RNA oligonucleotide on the
C60 complex. The released reporter sequence emitted a fluorescent
signal and was detected while free in the cytoplasm. To validate
the protocol, functionalized C60s, with a distinct region of rRNA,
were synthesized for live imaging B. subtilis and S. sanguinis,
which are gram-positive/negative, respectively. The 16s RNA
signature sequence was reported by Gendel, et al (Gendel, Food
Microbiol 13, 1-15 (1996)), which is hereby incorporated by
reference in its entirety, with an amine group at the 5' end. A 75%
matching reporter with a cy3/6-FAM fluorophore at 5' was hybridized
with the signature RNA. The whole C60 complex (10 .mu.g/mL) was
incubated with B. subtilis for 30 minutes. The results are shown in
FIGS. 21 and 22 and are summarized as follows: [0262] (1) C60
pyrrolidine tris acid, B. subtilis and S. sanguinis have no
autofluorescence under the excitation of 568 nm laser. [0263] (2)
The C60-rRNA-Reporter has no fluorescent signal under excitation
with a 488 or 568 nm laser, indicating that the fluorophore is
silenced by hybridization. [0264] (3) The C60-rRNA-Reporter can
distinguish B. subtilis or S. sanguinis by fluorescent signal when
the reporter has been released by hybridizing with matching
ribosomal RNA. [0265] (4) Each C60-rRNA-Reporter complex is only
capable of recognizing a single bacterial species with a signature
rRNA sequence. [0266] (5) A mixture of C60-rRNA-Reporters can
further distinguish a mixture of B. subtilis and S. sanguinis in
live status.
Example 7
C60-siRNA Complexes Used for Silencing Gene Expression
[0267] mRNA conveys genetic information that is transcripted from
DNA. This genetic information can be translated into proteins, thus
carrying out specific cellular functions. In eukayotic cells,
precursor mRNA is transcribed and post edited in the nucleus, where
C60 molecules cannot reach. However, mature mRNA is transferred
into the cell cytoplasm as an template for translation. In this
state, mRNA can be targeted by the C60-rRNA complexes of the
invention.
[0268] A schematic of this process is provided in FIG. 23 and
operates as follows: [0269] (1) C60 pyrrolidine-tris acid is
functionalized with siRNA, the sequence of which is selected based
on the desired target genes. [0270] (2) A protecting RNA hybridizes
the attached siRNA, preventing degradation. Optionally, the
protecting RNA can be functionalized with a detectable label which
is silent while the protecting RNA is hybridized but detectable if
it is released. [0271] (3) The C60-siRNA complex is transported
across the cellular membrane. Once inside: [0272] (i) If the target
gene is active and the corresponding mRNA is present in the
cytoplasm, the attached siRNA releases the protecting RNA, targets
the active mRNA and shuts down the translation by competitively
hybridizing with it. If a detectable label is bound to the
protecting RNA, the label will begin emitting. [0273] (ii) If the
target gene is inactive, the siRNA would remain hybridized to the
protecting RNA.
[0274] The compositions of the invention can therefore be used to
silence multiple genes by using a specifically designed probe to
light up cells in which certain cell functions are shut down and/or
be designed to activate/stimulate certain cell functions.
[0275] In certain embodiments, a Buckyball can be functionalized to
detect 16s RNA as described in Example 6 and simultaneously inhibit
mRNA transcription as described above in Example 7 in a single
step. An illustration of this embodiment is shown in FIG. 24.
Example 8
Simultaneous Imaging of a Specific Microbe and its mRNA
[0276] mRNA can be targeted by the disclosed C60 complexes which
can be transport through the cellular membrane as demonstrated
elsewhere herein. mRNA in a specific microbe can be visualized by
delivering one set of functionalized Buckyballs, where each
Buckyball is functionalized with two different RNA
oligonucleotide/protector layer complex, each with a unique
fluorophore. The first set recognizes a specific microorganism, by
hybridizing to a unique region of 16S rRNA, and emits at one
specific wavelength. The second set targets the mRNA and emits at a
second specific wavelength. Thus, if both wavelengths are present
then mRNA is expressed in a specific microorganism. Alternatively,
two sets of Buckyballs can be synthesized, where each target either
rRNA or mRNA. An alternative embodiment comprises a variation with
only one step, as shown in FIG. 25:
[0277] (1) C60 pyrrolidine-tris acid is functionalized with an rRNA
signature sequence and an siRNA. The rRNA is selected to recognize
a specific bacterium and the siRNA is selected to bind to a
specific mRNA to silence certain cellular functions.
[0278] (2) A reporter and a protector hybridize the attached rRNA
signature and siRNA, preventing degradation. The reporter is
quenched while hybridizing with rRNA signature.
[0279] (3) C60-siRNA complex are transported across the cellular
membrane. Once inside: [0280] (i) The rRNA recognizes the bacterium
by hybridizing with a specific rRNA sequence and releases the
reporter, thereby illuminating the bacterium. [0281] (ii) The siRNA
recognizes the mRNA and shuts down translation by competitively
binding. The method can be used to visualize an organism, such as a
bacterium, and regulate gene expression, such as silencing gene
expression or activating gene expression, thus altering cell
function.
Example 9
Microfluidic System for Rapid Diagnosis of Microbial Presence
[0282] The compositions of the invention can be used in a
microfluidic system for the rapid diagnosis of microbial presence
as demonstrated in FIG. 26. The system comprises a source well
(left hand side) and two sink wells (right hand side, top and
bottom). As a proof of concept, a mixture of B. subtilis and S.
sanguinis were prelabeled with the compositions of the invention
and deposited in the source well. The sink wells contained iron
(FIG. 26, top right well) and glucose (FIG. 26, bottom right well)
media, creating a gradient in the channels, helping to draw the
microbes to their preferred medium. After 15 minutes, the mixed
microorganisms from the source well self selected into their
respective sink wells (B. subtilis into the iron sink and S.
sanguinis into the glucose sink) and separation was readily
observed due to the fluorescent labeling of the microbes using the
compositions of the invention.
[0283] In an alternative embodiment of the microfluidic system, a
mixture of microbes can similarly be added to the source well
without prelabeling using the compositions of the invention.
Functionalized Buckyballs corresponding to each microorganism of
interest can instead be printed into each sink well, wherein each
sink well also contains a medium which would attract the
microorganism of interest corresponding to the Buckyball printed
into that well. If a microorganism of interest is present in the
mixture placed in the source well, it will migrate to the sink well
containing its preferred medium, internalize the functionalized
Buckyball of the invention containing the matching RNA
oligonucleotide and emit a signal. An imaging device can be used to
rapidly image the wells of such a system and determine the presence
or absence of one or more microbial species as well as the relative
abundance of each species.
[0284] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of this invention
may be devised by others skilled in the art without departing from
the true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and
equivalent variations.
* * * * *