U.S. patent application number 15/849238 was filed with the patent office on 2018-07-19 for visible detection of microorganisms.
This patent application is currently assigned to Wisconsin Alumni Research Foundation. The applicant listed for this patent is Wisconsin Alumni Research Foundation. Invention is credited to Sundaram Gunasekaran, Seokwon Lim.
Application Number | 20180202939 15/849238 |
Document ID | / |
Family ID | 46064698 |
Filed Date | 2018-07-19 |
United States Patent
Application |
20180202939 |
Kind Code |
A1 |
Gunasekaran; Sundaram ; et
al. |
July 19, 2018 |
Visible Detection of Microorganisms
Abstract
Methods of detecting very low levels of targets, such as cells,
are provided. In some embodiments, for example, the methods can
detect bacteria present in a sample at concentrations less than 25
cells/mL. The method involves detecting nanoparticle aggregation in
the absence of the target.
Inventors: |
Gunasekaran; Sundaram;
(Madison, WI) ; Lim; Seokwon; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wisconsin Alumni Research Foundation |
Madison |
WI |
US |
|
|
Assignee: |
Wisconsin Alumni Research
Foundation
Madison
WI
|
Family ID: |
46064698 |
Appl. No.: |
15/849238 |
Filed: |
December 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13300148 |
Nov 18, 2011 |
9851308 |
|
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15849238 |
|
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61415498 |
Nov 19, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 50/52 20180101;
B82Y 30/00 20130101; G01N 21/78 20130101; Y02A 50/30 20180101; G01N
33/54346 20130101 |
International
Class: |
G01N 21/78 20060101
G01N021/78; G01N 33/543 20060101 G01N033/543; B82Y 30/00 20060101
B82Y030/00 |
Claims
1. A method of determining whether a sample comprises a target,
comprising: a) contacting the sample with a linker, wherein the
linker comprises a first functionality and a plurality of second
functionalities, wherein the first functionality is capable of
binding to the target, and wherein each of the plurality of second
functionalities is capable of binding to a third functionality; b)
contacting the sample from (a) with a plurality of nanoparticles,
wherein each of the plurality of nanoparticles comprises a third
functionality that is capable of binding to the second
functionality, and wherein the linker is added in step (a) at a
concentration less than the concentration of nanoparticles and
sufficient to aggregate the nanoparticles added in step (b) in the
absence of target; and c) detecting nanoparticle aggregation in the
sample from (b), wherein the absence of nanoparticle aggregation
indicates that the sample comprises the target.
2. (canceled)
3. (canceled)
4. (canceled)
5. The method of claim 1, wherein the linker is an antibody and the
first functionality is the antigen binding region of the
antibody.
6. The method of claim 5, wherein the second functionality is
biotin and the third functionality is streptavidin.
7. The method of claim 1, wherein the nanoparticles are selected
from gold nanoparticles, silver nanoparticles, platinum
nanoparticles, magnetite nanoparticles, gold/iron alloy
nanoparticles, and latex nanoparticles.
8. The method of claim 1, wherein the target is selected from
prokaryotic cells, eukaryotic cells, and parasites.
9. The method of claim 1, wherein detecting nanoparticle
aggregation comprises determining at least one characteristic
selected from sample color, UV-VIS spectrum, UV-VIS peak
wavelength, and absorbance.
10. The method of claim 9, wherein the at least one characteristic
of the sample from (b) is compared to at least one characteristic
of a standard.
11. (canceled)
12. (canceled)
13. A method of determining whether a sample comprises a target,
comprising: a) contacting the sample with a linker, wherein the
linker comprises a first functionality and a plurality of second
functionalities, wherein the first functionality is capable of
binding to the target, and wherein each of the plurality of second
functionalities is capable of binding to a third functionality; b)
contacting the sample from (a) with a plurality of nanoparticles,
wherein each of the plurality of nanoparticles comprises a third
functionality that is capable of binding to the second
functionality, and wherein the linker is added in step (a) at a
concentration just in excess relative to the concentration of the
nanoparticles added in step (b) and the nanoparticles do not
aggregate in the absence of target; and c) detecting nanoparticle
aggregation in the sample from (b), wherein the presence of
nanoparticle aggregation indicates that the sample comprises the
target.
14. (canceled)
15. (canceled)
16. (canceled)
17. The method of claim 13, wherein the linker is an antibody and
the first functionality is the antigen binding region of the
antibody.
18. The method of claim 17, wherein the second functionality is
biotin and the third functionality is streptavidin.
19. The method of claim 13, wherein the nanoparticles are selected
from gold nanoparticles, silver nanoparticles, platinum
nanoparticles, magnetite nanoparticles, gold/iron alloy
nanoparticles, and latex nanoparticles.
20. The method of claim 13, wherein the target is selected from
prokaryotic cells, eukaryotic cells, and parasites.
21. (canceled)
22. The method of claim 13, wherein detecting nanoparticle
aggregation comprises determining at least one characteristic
selected from sample color, UV-VIS spectrum, UV-VIS peak
wavelength, and absorbance.
23. The method of claim 22, wherein the at least one characteristic
of the sample from (b) is compared to at least one characteristic
of a standard.
24. (canceled)
25. (canceled)
26. A kit for determining whether a sample comprises a target,
comprising (i) a linker, wherein the linker comprises a first
functionality and a plurality of second functionalities, wherein
the first functionality is capable of binding to the target, and
wherein each of the plurality of second functionalities is capable
of binding to a nanoparticle; (ii) a plurality of nanoparticles,
wherein each of the plurality of nanoparticles comprises a third
functionality that is capable of binding to the second
functionality; and (iii) a standard, wherein the standard is a
representation of at least one characteristic of a control reaction
that comprises the linker and the plurality of nanoparticles, but
not the target.
27. (canceled)
28. (canceled)
29. (canceled)
30. The kit of claim 26, wherein the linker is an antibody and the
first functionality is the antigen binding region of the
antibody.
31. The kit of claim 30, wherein the second functionality is biotin
and the third functionality is streptavidin.
32. The kit of claim 26, wherein the nanoparticles are selected
from gold nanoparticles, silver nanoparticles, platinum
nanoparticles, magnetite nanoparticles, gold/iron alloy
nanoparticles, and latex nanoparticles.
33. (canceled)
34. (canceled)
35. The kit of claim 26, wherein the at least one characteristic is
selected from sample color, UV-VIS spectrum, UV-VIS peak
wavelength, and absorbance.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 13/300,148, filed on Nov. 18, 2011 and issuing as U.S. Pat. No.
9,851,308 issuing Dec. 26, 2017, which application claims the
benefit of U.S. Provisional Application No. 61/415,498, filed Nov.
19, 2010, which is incorporated by reference herein in its entirety
for any purpose.
BACKGROUND
[0002] The presence of even low levels of spoilage or pathogenic
microorganisms in various products can be dangerous and even
lethal. Therefore, sensitive, rapid, and simple-to-use methods for
detecting microorganisms that can be performed in
out-of-the-laboratory settings, and by untrained persons, are
preferred over those requiring sophisticated techniques that are
laborious, time consuming, or require skilled personnel to carry
out. For example, the analysis of genetic information based on the
polymerase chain reaction (PCR), a popular method for sensitive
detection of bacteria, requires complicated sample preparation
protocols performed by skilled personnel. Some alternative methods
are known, such as detection of intact bacterial cells using
specific antibody-antigen binding, but simple, fast, and sensitive
methods are needed.
[0003] Since sol particle immunoassays (SPIA) were introduced,
target-mediated (i.e., on-target) aggregation of gold nanoparticles
(AuNPs) has been used to visually detect the occurrence of some
biological events or the presence of small biomolecules such as
proteins and DNA. However, using the principle of on-target
aggregation of AuNPs for visual detection of large targets such as
microorganisms, especially at extremely low concentrations, has
proved difficult.
SUMMARY
[0004] In some embodiments, methods of determining whether a sample
comprises a target are provided. In some embodiments, the method
comprises (a) contacting the sample with a linker, wherein the
linker comprises a first functionality and a plurality of second
functionalities, wherein the first functionality is capable of
binding to the target, and wherein each of the plurality of second
functionalities is capable of binding to a third functionality; (b)
contacting the sample from (a) with a plurality of nanoparticles,
wherein each of the plurality of nanoparticles comprises a third
functionality that is capable of binding to the second
functionality; and (c) detecting nanoparticle aggregation in the
sample from (b), wherein the absence of nanoparticle aggregation
indicates that the sample comprises the target.
[0005] In some embodiments, the method comprises (a) contacting the
sample with a linker, wherein the linker comprises a first
functionality and a plurality of second functionalities, wherein
the first functionality is capable of binding to the target, and
wherein each of the plurality of second functionalities is capable
of binding to a third functionality; (b) contacting the sample from
(a) with a plurality of nanoparticles, wherein each of the
plurality of nanoparticles comprises a third functionality that is
capable of binding to the second functionality; and (c) detecting
nanoparticle aggregation in the sample from (b), wherein the
presence of nanoparticle aggregation indicates that the sample
comprises the target. In some embodiments, the linker is present in
an excess concentration relative to the concentration of the
nanoparticles.
[0006] In some embodiments, the first functionality is selected
from an antigen binding region of an antibody, a ligand, a
receptor, a small molecule, and a lectin. In some embodiments, the
second functionality is selected from biotin, streptavidin, an
antigen, an antibody, a ligand, a receptor, a polyhistidine tag,
nickel, an aptamer, an aptamers target, trans-cyclooctene, and
tetrazine. In some embodiments, the third functionality is selected
from biotin, streptavidin, an antigen, an antibody, a ligand, a
receptor, a polyhistidine tag, nickel, an aptamer, an aptamers
target, trans-cyclooctene, and tetrazine. In some embodiments, the
second and third functionalities together form a binding pair
selected from biotin/streptavidin, ligand/receptor, polyhistidine
tag/nickel, aptamer/aptamer target, antibody/antigen, and
trans-cyclooctene/tetrazine.
[0007] In some embodiments, the linker is an antibody and the first
functionality is the antigen binding region of the antibody. In
some embodiments, the second functionality is biotin and the third
functionality is streptavidin.
[0008] In some embodiments, the nanoparticles are selected from
gold nanoparticles, silver nanoparticles, platinum nanoparticles,
magnetite nanoparticles, gold/iron alloy nanoparticles, and latex
nanoparticles. In some embodiments, the target is selected from
prokaryotic cells, eukaryotic cells, and parasites.
[0009] In some embodiments, detecting nanoparticle aggregation
comprises determining at least one characteristic selected from
sample color, UV-VIS spectrum, UV-VIS peak wavelength, and
absorbance. In some embodiments, the at least one characteristic of
the sample from (b) is compared to at least one characteristic of a
standard. In some embodiments, the standard is a control reaction
comprising the linker and the plurality of nanoparticles, but not
the target. In some embodiments, the standard is a representation
of at least one characteristic of a control reaction that comprises
the linker and the plurality of nanoparticles, but not the
target.
[0010] In some embodiments, kits for determining whether a sample
comprises a target are provided. In some embodiments, a kit
comprises (i) a linker, wherein the linker comprises a first
functionality and a plurality of second functionalities, wherein
the first functionality is capable of binding to the target, and
wherein each of the plurality of second functionalities is capable
of binding to a nanoparticle; and (ii) a plurality of
nanoparticles, wherein each of the plurality of nanoparticles
comprises a third functionality that is capable of binding to the
second functionality.
[0011] In some embodiments, the first functionality is selected
from an antigen binding region of an antibody, a ligand, a
receptor, a small molecule, and a lectin. In some embodiments, the
second functionality is selected from biotin, streptavidin, an
antigen, an antibody, a ligand, a receptor, a polyhistidine tag,
nickel, an aptamer, an aptamers target, trans-cyclooctene, and
tetrazine. In some embodiments, the third functionality is selected
from biotin, streptavidin, an antigen, an antibody, a ligand, a
receptor, a polyhistidine tag, nickel, an aptamer, an aptamers
target, trans-cyclooctene, and tetrazine. In some embodiments, the
second and third functionalities together form a binding pair
selected from biotin/streptavidin, ligand/receptor, polyhistidine
tag/nickel, aptamer/aptamer target, antibody/antigen, and
trans-cyclooctene/tetrazine. In some embodiments, the nanoparticles
are selected from gold nanoparticles, silver nanoparticles,
platinum nanoparticles, magnetite nanoparticles, gold/iron alloy
nanoparticles, and latex nanoparticles.
[0012] In some embodiments, the linker is an antibody and the first
functionality is the antigen binding region of the antibody. In
some embodiments, the second functionality is biotin and the third
functionality is streptavidin.
[0013] In some embodiments, a kit further comprises a standard. In
some embodiments, the standard is a representation of at least one
characteristic of a control reaction that comprises the linker and
the plurality of nanoparticles, but not the target. In some
embodiments, the at least one characteristic is selected from
sample color, UV-VIS spectrum, UV-VIS peak wavelength, and
absorbance.
BRIEF DESCRIPTION OF THE FIGURES
[0014] This patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawings will be provided by the
United States Patent Office upon request and payment of the
necessary fee.
[0015] FIG. 1 shows the results of on-target detection of bacteria
using antibody-conjugated gold nanoparticles (AuNPs), as described
in Example 2. FIG. 1A shows the color of the reactions containing
E. coli concentrations of 10.sup.1 to 10.sup.9 CFU/400 .mu.L (tubes
1 to 9, respectively). FIG. 1B shows the control reaction (left
tube) and the reaction containing 10.sup.9 CFU/400 .mu.L (right
tube) after centrifugation.
[0016] FIG. 2 shows the results of on-target detection of bacteria
using an AuNP concentration that is 25% of the concentration used
in the experiment shown in FIG. 1, as described in Example 3. FIG.
2A shows the control reaction (right tube) and the reaction
containing 10.sup.9 CFU/400 .mu.L (left tube) using the original
concentration of AuNPs after centrifugation. FIG. 2B shows the
control reaction (right tube) and the reaction containing 10.sup.9
CFU/400 .mu.L (left tube) using 25% of the original concentration
of AuNPs after centrifugation.
[0017] FIG. 3 shows a schematic of a proposed mechanism for
off-target cell detection, as discussed in Example 4. FIG. 3A shows
binding of AuNPs to the cell surface through a biotinylated
antibody linker. FIG. 3B shows aggregation of AuNPs following
cross-linking of multiple AuNPs through the biotinylated
antibody.
[0018] FIG. 4 shows the results of the off-target detection assay
described in Example 4. FIG. 4A shows the color difference between
samples containing E. coli (red, right tube) and not containing E.
coli (purplish, left tube) after 15 minutes. FIG. 4B shows the same
samples after one hour. FIG. 4C shows the UV-VIS spectra of samples
containing 10.sup.1 to 10.sup.7 CFU/mL E. coli, as described in
Example 4. The peak absorbance wavelength for each trace is
indicated by an arrow.
[0019] FIG. 5 shows the peak absorbance change and peak wavelength
shift in a mixture containing biotinylated BSA and
streptavidin-coated AuNPs, as described in Example 5.
[0020] FIG. 6 shows (A) a plot of .DELTA..lamda.max versus
biotinylated BSA concentration for mixtures of biotinylated BSA and
streptavidin-coated AuNPs at various time points; (B) a photograph
of the mixtures from (A) at the 3 hour time point, with an
indication of the regions exemplified in (C); and (C) a schematic
showing an exemplary mechanism explaining the phenomena observed at
different concentrations of biotinylated BSA; as described in
Example 5.
[0021] FIG. 7 is a diagram of the method involving pre-incubation
with free streptavidin, as described in Example 5.
[0022] FIG. 8 shows a plot of .DELTA..lamda.max versus biotinylated
BSA concentration for mixtures of biotinylated BSA and
streptavidin-coated AuNPs in the presence of various concentrations
of free streptavidin, as described in Example 5. The inset shows
photographs of the mixtures in the presence of 10 .mu.g
streptavidin.
[0023] FIG. 9 shows a plot of .DELTA..lamda.max versus biotinylated
antibody concentration for mixtures of biotinylated antibody and
streptavidin-coated AuNPs in the presence of various concentrations
of free streptavidin, as described in Example 5.
[0024] FIG. 10 shows an exemplary method of detecting microbial
contamination in a sample, as described in Example 6.
[0025] FIG. 11 shows photographs of assay reactions in the presence
of various concentrations of bacteria and various concentrations of
biotinylated antibody, as described in Example 6. The red boxes
indicate two exemplary biotinylated antibody concentrations that
provided sensitive detection of low levels of bacteria.
DETAILED DESCRIPTION
[0026] The present invention provides a novel strategy for
detection of targets, such as cells. In this strategy, the
aggregation of nanoparticles does not occur in the presence of the
target, but rather indicates the absence of the target. The method
uses a bifunctional linker that comprises a first functionality
that binds to a target, and a second functionality that facilitates
aggregation of nanoparticles. When the linker is bound to the
target, aggregation of the nanoparticles does not occur to a
significant extent. In the absence of target, however, the linker
is available to facilitate aggregation of the nanoparticles.
Aggregation of the nanoparticles can be detected through changes in
at least one characteristic of the sample. For example, in some
embodiments, aggregation causes the color of the sample changes.
Further, the present method is effective for detecting targets that
are much larger than the nanoparticles, such as cells, and is very
sensitive--the method can detect E. coli concentrations of less
than 25 cells/mL. In addition, the reagents used in the present
method can be designed such that they provide a desired level of
specificity for one or more targets. That is, in some embodiments,
reagents can be designed such that the method detects, for example,
a particular target (such as a particular strain of bacteria). In
some embodiments, reagents can be designed such that the method
detects, for example, two or more targets (such as two or more
strains of bacteria), e.g., by binding to a functionality that is
shared among the two or more targets, or by mixing together
reagents that are specific for each target to be detected, or a
combination of the two methods.
Definitions
[0027] The terms "bifunctional linker" and "linker" are used
interchangeably herein to refer to a molecule that comprises a
first functionality that binds to a target, and a second
functionality that is capable of binding to at least one
nanoparticle. In some embodiments, a linker comprises a plurality
of second functionalities. In some embodiments, a second
functionality is capable of binding to a plurality of
nanoparticles.
[0028] The term "nanoparticles," as used herein, refers to
particles that are less than 1 .mu.m in diameter and which cause a
detectable change in a sample when they aggregate. Nanoparticles
may be of any shape, including spherical and rod-shaped, so long as
the longest dimension is within the diameter thresholds discussed
herein. In some embodiments, a nanoparticle is less than 0.5 .mu.m,
less than 0.1 .mu.m, less than 50 nm, less than 10 nm, less than 5
nm in diameter. In some embodiments, nanoparticles are
functionalized with a moiety (referred to herein as a "third
functionality") that interacts with the second functionality of a
linker, described above. The term "nanoparticles" includes
nanoparticles that are functionalized with such a moiety.
[0029] The term "target," as used herein, refers to an entity that
is to be detected. In some embodiments, a target is greater than
0.1 .mu.m in its longest dimension. In some embodiments, a target
is greater than 0.5 .mu.m, greater than 1 .mu.m, greater than 2
.mu.m, or greater than 5 .mu.m in its longest dimension.
Exemplary Linkers
[0030] The methods described herein use a linker comprising a first
functionality that is capable of binding to a selected target, and
a second functionality that is capable of binding to at least one
nanoparticle. In some embodiments, a second functionality is
capable of binding to a plurality of nanoparticles. In some
embodiments, the linker comprises a plurality of second
functionalities. In some embodiments, the linker facilitates the
aggregation of the nanoparticles. For example, in some embodiments,
a linker comprising a plurality of second functionalities brings a
plurality of nanoparticles in close proximity such that they
aggregate. In some embodiments, the linker comprises a single
second functionality, wherein the second functionality is capable
of binding to a plurality of nanoparticles. In some such
embodiments, the second functionality brings a plurality of
nanoparticles in close proximity such that they aggregate.
[0031] The first functionality, in some embodiments, is any moiety
that is capable of binding to the target. In some embodiments,
multiple first functionalities are capable of binding to a single
target. Nonlimiting exemplary first functionalities include antigen
binding regions of antibodies, ligands, receptors, small molecules,
and lectins (such as mannose-binding lectins), etc. When the first
functionality is an antigen binding region of an antibody, the
linker may comprise the entire antibody, or just a portion of the
antibody that includes the antigen binding region. In some
embodiments, when a first functionality is an antigen binding
region of an antibody, the target comprises the antigen on its
surface. In some embodiments, the target comprises multiple copies
of the antigen on its surface such that a plurality of linkers can
bind to a single target. In some embodiments, when a first
functionality is a ligand, the target comprises the receptor on its
surface. In some embodiments a first functionality binds to
multiple targets, for example, when multiple different targets
comprise the same or a similar antigen on their surfaces. One
skilled in the art can select a suitable first functionality
depending on the particular target(s) to be detected.
[0032] The second functionality, in some embodiments, is a member
of a binding pair, wherein the nanoparticles comprise the other
member of the binding pair (in some embodiments, a third
functionality). Nonlimiting exemplary binding pairs include
biotin/streptavidin, ligand/receptor, polyhistidine tag/nickel,
aptamer/aptamer target, antibody/antigen,
trans-cyclooctene/tetrazine (see, e.g., Haun et al., Nat.
Nanotechnol. 5(9): 660-6 (2010)), etc. One skilled in the art can
select a suitable second functionality and/or third functionality
depending on the linker, the nanoparticles, and the intended
application.
[0033] In some embodiments, the linker comprises a plurality of
copies of the second functionality. In some embodiments, the linker
comprises at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9, at least 10, at least 15, or at least 20
copies of the second functionality.
[0034] In some embodiments, a second functionality is capable of
binding to multiple copies of the third functionality. In some
embodiments, a second functionality is able to bind to at least 3,
at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, or at least 10 copies of the third functionality. In some
such embodiments, a linker may comprise one or just a few copies of
the second functionality.
[0035] Nonlimiting exemplary linkers may comprise proteins,
peptides, nucleic acids, aptamers, small molecules, carbohydrates,
polymers, binding pairs, etc. In some embodiments, one or more
portions of the protein, peptide, nucleic acid, aptamer, small
molecule, carbohydrate, polymer, etc., acts as the first
functionality and/or the second functionality of the linker. For
example, in some embodiments, a linker may comprise a biotinylated
protein, wherein the protein is a ligand for a particular receptor
on the surface of a target cell, and the nanoparticles are
functionalized with streptavidin. In some such embodiments, the
first functionality of the linker is the ligand portion of the
protein (which may be the entire protein) and the second
functionality is biotin. In some embodiments, a linker is a
biotinylated antibody. In some such embodiments, the antigen
binding region is the first functionality and biotin is the second
functionality.
Exemplary Nanoparticles
[0036] Nonlimiting exemplary nanoparticles include gold
nanoparticles, silver nanoparticles, platinum nanoparticles,
magnetite nanoparticles, gold/iron alloy nanoparticles, and latex
nanoparticles.
[0037] In some embodiments, nanoparticles have an average size of
between 1 nm and 1 .mu.m. In some embodiments, the nanoparticles
are gold nanoparticles (AuNPs). In some embodiments, the gold
nanoparticles have an average size of between 1 nm and 100 nm. In
some embodiments, gold nanoparticles have an average size of
between 1 nm and 50 nm, between 5 nm and 50 nm, between 5 nm and 30
nm, between 5 nm and 20 nm, or between 10 and 15 nm.
[0038] For a particular detection method, nanoparticles are
selected that are smaller than the target to be detected. In some
embodiments, the target is at least 10-fold, at least 20-fold, at
least 30-fold, at least 50-fold, at least 100-fold, or at least
200-fold larger than the nanoparticles. As a nonlimiting example,
in a method of detecting E. coli, which is about 1 to 3 .mu.m in
its longest dimension, nanoparticles may be selected for detection
that are between 10 nm and 20 nm. One skilled in the art can select
suitably sized nanoparticles depending on the target to be
detected.
[0039] In some embodiments, nanoparticles are functionalized with
(i.e., comprise) a member of a binding pair (in some embodiments,
referred to as a third functionality). In some embodiments, the
nanoparticles are functionalized with (i.e., comprise) a member of
a binding pair that binds to the member of the binding pair
comprised in the linker (i.e., the second functionality).
Nonlimiting exemplary binding pairs are described in the "Exemplary
Linker" section, above. In some embodiments, nanoparticles are
functionalized with (i.e., comprise) streptavidin. One skilled in
the art can select a suitable binding pair depending on the
particular nanoparticles, linker, and application.
[0040] Methods of functionalizing nanoparticles are known in the
art. In some embodiments, for example, nanoparticles are
functionalized with streptavidin according to the method described
in Example 1. One skilled in the art can select a suitable method
of functionalizing nanoparticles depending on the particular
application.
Exemplary Targets
[0041] The methods described herein can be used to detect targets
that are greater than 0.1 .mu.m in their longest dimension. In some
embodiments, a target is greater than 0.2 .mu.m, greater than 0.5
.mu.m, greater than 1 .mu.m, greater than 2 .mu.m, or greater than
5 .mu.m. Nonlimiting exemplary targets include prokaryotic cells
(such as bacterial cells), eukaryotic cells (including yeast),
parasites, etc. In some embodiments, a target is a microbial food
and/or water contaminant.
[0042] Nonlimiting exemplary targets that can be detected using the
methods described herein include E. coli (including E. coli
0157:H7), Staphylococcus aureus, Salmonella species (including
Salmonella enteritidis and Salmonella typhimurium), Clostridium
botulinum, Pseudomonas aeruginosa, Campylobacter jejuni, Yersinia
enterocolitica, Yersinia psudotuberculosis, Listeria
monocyteogenes, Vibrio cholerae (both O1 and non-O1), Vibrio
parahaemolyticus, Vibrio vulnificus, Clostridium perfringens,
Bacillus cereus, Aeromonas hydrophila, Shigella, Streptococcus,
Cryptosporidium, Giardia lamblia, Entamoeba histolytica, Cyclospora
cayetanensis, Anisakis, Diphyllobothrium, Nanophyetus,
Eustrongylides, Acanthamoeba, Ascaris lumbricoides, Trichuris
trichiura, Legionella, fecal coliforms, non-fecal coliforms (such
as Enterobacter, Klebsiella, Citrobacter), etc.
[0043] As discussed above, for a particular target, nanoparticles
are selected such that the target is at least 10-fold, at least
20-fold, at least 30-fold, at least 50-fold, at least 100-fold, or
at least 200-fold larger than the nanoparticles.
Exemplary Samples
[0044] The present methods can be used to detect targets in a
variety of sample types, including water samples (such as drinking
water, water used for irrigation, and waste water); food;
biological samples (such as bodily fluids, pharmaceuticals, etc.);
cosmetics; air samples; etc. In some embodiments, a sample is
diluted, concentrated, suspended, and/or dissolved prior to, or
during, the methods described herein. In some embodiments, a sample
is at least partially separated prior to using it in the methods
described herein, such as, for example, separating blood fractions.
In some embodiments, a buffering agent is added to a sample prior
to, or during, the methods described herein. In some such
embodiments, the buffering agent is added to bring and/or maintain
the sample at a particular pH during at least a portion of the
method. One skilled in the art can prepare a sample for use in the
present methods, depending on the state of the sample (i.e.,
liquid, solid, gel, paste, etc.), the thickness of the sample, the
density of the sample, the pH of the sample, the predicted
microbial concentration in the sample, etc.
Exemplary Methods of Detecting Targets
[0045] In some embodiments, methods of determining whether or not a
sample comprises a target are provided. In some embodiments, the
method comprises contacting the sample with a linker and
nanoparticles and detecting aggregation of the nanoparticles. In
some embodiments, aggregation of the nanoparticles indicates the
absence of the target in the sample. In some embodiments, minimal
or no aggregation of the nanoparticles indicates the presence of
the target in the sample. In some such embodiments, the linker is
present at a lower concentration than the nanoparticles.
[0046] In some embodiments, the method comprises contacting the
sample with a linker and nanoparticles and detecting aggregation of
the nanoparticles. In some embodiments, aggregation of the
nanoparticles indicates the presence of the target in the sample.
In some embodiments, minimal or no aggregation of the nanoparticles
indicates the absence of the target in the sample. In some such
embodiments, the linker is present at an excess concentration
relative to the nanoparticles.
[0047] In some embodiments, the linker is added to a sample before
the nanoparticles are added. In some such embodiments, if target is
present in the sample, the linker binds to the target. If no target
is present, the linker remains in solution. In some embodiments,
nanoparticles are then added. If the linker is in solution, and not
bound to the target, in some embodiments, the linker facilitates
aggregation of the nanoparticles. If the linker is bound to target
cells, in some embodiments, its ability to facilitate aggregation
of the nanoparticles is impaired and little or no aggregation
occurs. Thus, in some embodiments, the absence of the target is
indicated by aggregation of the nanoparticles.
[0048] In some embodiments, the linker is present at an excess
concentration relative to the nanoparticles. In some such
embodiments, if no target is present, the excess linker prevents
aggregation of the nanoparticles. In some embodiments, the linker
is present at a concentration sufficient to prevent aggregation of
the nanoparticles in the absence of target. In some embodiments, if
target is present, sufficient linker is bound to the target such
that the remaining linker in solution facilitates aggregation of
the nanoparticles. Thus, in some such embodiments, the presence of
the target is indicated by aggregation of the nanoparticles.
[0049] A sample to which linker and nanoparticles have been added
is referred to, in some embodiments, as a "test sample."
[0050] In some embodiments, linker is added at a concentration such
that a small number of targets will "soak up" most or all of the
linker. In some embodiments, for example, if a linker is a
biotinylated antibody, the linker is added at a concentration of
between about 0.1 .mu.g/mL and 100 .mu.g/mL. In some embodiments,
the linker is added at a concentration of between about 0.1
.mu.g/mL and about 50 .mu.g/mL. In some embodiments, the linker is
added at a concentration of between about 0.1 .mu.g/mL and about 25
.mu.g/mL. In some embodiments, the linker is added at a
concentration of between about 0.1 .mu.g/mL and about 10 .mu.g/mL.
In some embodiments, the linker is added at a concentration of
between about 0.1 .mu.g/mL and about 3 .mu.g/mL. In some
embodiments, the linker is added at a concentration of between
about 0.5 .mu.g/mL and about 3 .mu.g/mL. An appropriate linker
concentration can be determined by one skilled in the art,
considering such factors as the identity of the linker, the
identity of the target, the number of binding sites for the first
functionality on the target to be detected, the efficiency of
binding to the target, the predicted number of targets in a sample,
etc.
[0051] In some embodiments, linker is added at an excess
concentration, such that a small number of targets will "soak up"
some of the linker, leaving sufficient linker in solution to
facilitate aggregation of the nanoparticles. In some such
embodiments, for example, if a linker is a biotinylated antibody,
the linker is added at a concentration of between about 0.1
.mu.g/mL and 100 .mu.g/mL. In some embodiments, the linker is added
at a concentration of between about 1 .mu.g/mL and about 100
.mu.g/mL. In some embodiments, the linker is added at a
concentration of between about 1 .mu.g/mL and about 50 .mu.g/mL. In
some embodiments, the linker is added at a concentration of between
about 1 .mu.g/mL and about 25 .mu.g/mL. In some embodiments, the
linker is added at a concentration of between about 1 .mu.g/mL and
about 10 .mu.g/mL. In some embodiments, the linker is added at a
concentration of between about 2 .mu.g/mL and about 10 .mu.g/mL. In
some embodiments, the linker is added at a concentration of between
about 2 .mu.g/mL and about 5 .mu.g/mL. An appropriate linker
concentration can be determined by one skilled in the art,
considering such factors as the identity of the linker, the
identity of the target, the number of binding sites for the first
functionality on the target to be detected, the efficiency of
binding to the target, the predicted number of targets in a sample,
etc.
[0052] In some embodiments, nanoparticles are added at a
concentration such that aggregation of the nanoparticles by the
available linker is detectable above the background of
non-aggregated nanoparticles. As a nonlimiting example, if gold
nanoparticles between 10 nm and 15 nm in size are used to detect,
e.g., E. coli, a concentration of nanoparticles between 1 nM to 20
nM, 5 nM to 15 nM, or between 5 nM and 10 nM may be used. An
appropriate nanoparticle concentration can be determined by one
skilled in the art, considering such factors as the type of
nanoparticle, the identity of the linker, the identities of the
second and third functionalities, the efficiency of binding of the
second and third functionalities, the number of binding sites for
the nanoparticles on the linkers, the concentration of linker in
the test sample, etc. The concentration should be selected such
that there is an observable difference between aggregated and
non-aggregated nanoparticles.
[0053] In some embodiments, detection of aggregation is carried out
by detecting a change in at least one characteristic of the test
sample. In some embodiments, the change in at least one
characteristic of the test sample is determined as a change from
time 0, immediately after the nanoparticles are added, to a time T
after incubation of the test sample. In some embodiments, time T is
5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30
minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55
minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100
minutes, or 120 minutes. Thus, in some embodiments, a
characteristic of the sample at time 0 is compared to the same
characteristic of the sample at time T.
[0054] In some embodiments, the change in at least one
characteristic of the test sample is determined by comparing the at
least one characteristic of the test sample to one or more
standards. In some embodiments, the standard is a control sample.
In some embodiments, a control sample comprises the same linker and
nanoparticles as the test sample, but does not contain the target.
In some embodiments, the control sample has undergone the same
processing as the test sample, although the processing of the
control sample may or may not have occurred contemporaneously with
the processing of the test sample.
[0055] In some embodiments, a standard is a representation of at
least one characteristic of a test sample or a control sample. Such
a representation may be of a sample that comprises the target, or a
sample that lacks the target. In some embodiments, at least one
characteristic of a test sample is compared to both a standard that
represents the at least one characteristic in a sample that
comprises the target, and a standard that represents the at least
one characteristic in a sample that lacks the target.
[0056] Various characteristics of a test sample may be used to
determine whether the nanoparticles have aggregated. Nonlimiting
exemplary characteristics include color, UV-visible spectrum,
UV-visible peak wavelength, and absorbance.
[0057] In some embodiments, aggregation of nanoparticles causes a
change in a test sample that is visible to the naked eye. In some
embodiments, aggregation of nanoparticles causes a color change in
a test sample. In some embodiments, for example, when gold
nanoparticles are used, a test sample in which the nanoparticles
have not aggregated is red in color, while a test sample in which
the nanoparticles have aggregated is purple in color. In some
embodiments, the peak wavelength of a test sample in which the
nanoparticles (such as gold nanoparticles) have not aggregated is
about 530 nm, while the peak wavelength of a test sample in which
the nanoparticles have aggregated is about 550 nm to 560 nm, with
increased absorption at wavelengths above 600 nm.
[0058] In some embodiments, the presence of aggregation in a test
sample indicates that the target is not present at levels
detectable by that particular assay. Thus, in some embodiments, if
gold nanoparticles are being used in the method, and the test
sample turns purplish over time and/or the peak UV-visible
wavelength shifts from about 530 nm to 550 nm or longer, the sample
does not contain a detectable amount of the target.
[0059] In some embodiments, the absence of aggregation in a test
sample indicates that the target is present. Thus, in some
embodiments, if gold nanoparticles are being used in the method,
and the test sample remains red over time and/or the peak
UV-visible wavelength remains at about 530 nm, the sample contains
the target.
[0060] In some embodiments, the presence of aggregation in a test
sample indicates that the target is present. Thus, in some
embodiments, if gold nanoparticles are being used with an excess
amount of antibody in the method, and the test sample turns
purplish over time and/or the peak UV-visible wavelength shifts
from about 530 nm to 550 nm or longer, the sample contains a
detectable amount of the target.
[0061] In some embodiments, the absence of aggregation in a test
sample indicates that the target is not present at levels
detectable by that particular assay. Thus, in some embodiments, if
gold nanoparticles are being used with an excess amount of antibody
in the method, and the test sample remains red over time and/or the
peak UV-visible wavelength remains at about 530 nm, the sample does
not contain the target.
[0062] In some embodiments, the absorbance of the sample at a
particular wavelength is detected with a colorimeter in order to
detect aggregation. In some embodiments, a change in the absorbance
at a particular wavelength indicates a change in the amount of
aggregation in a sample, and the presence or absence of the target.
In some embodiments, for example, for a system using gold
nanoparticles and a limiting amount of an antibody to the target,
if the absorbance at 530 nm is detected, and the absorbance
decreases after addition of the sample, the sample does not contain
a detectable amount of the target (i.e., aggregation occurs, and
the peak wavelength shifts to 550 nm or longer, reducing the
absorbance at 530 nm). In other embodiments, for example, for a
system using gold nanoparticles and an excess amount of an antibody
to the target, if the absorbance at 530 nm is detected, and the
absorbance decreases after addition of the sample, the sample
contains a detectable amount of the target (i.e., aggregation
occurs, and the peak wavelength shifts to 550 nm or longer,
reducing the absorbance at 530 nm).
[0063] In some embodiments, the color of the sample is determined
using a histogram of a color image of the sample. In some
embodiments, the color of the sample is determined using a
Turboscan.
Exemplary Standards
[0064] In some embodiments, at least one characteristic of a test
sample is compared to at least one characteristic of a standard. In
some embodiments, a standard is a control sample that has been
treated under the same conditions, and comprises the same linker
and nanoparticles, as the test sample, but lacks the target. One
skilled in the art can include a suitable control sample in a
method if desired.
[0065] In some embodiments, a standard is a representation of at
least one characteristic of a test sample. For example, in some
embodiments, if the at least one characteristic is the color of the
test sample, a standard may be a representation of one or more
colors to which the test sample can be compared. In some
embodiments, if the at least one characteristic is a UV-visible
spectrum of the test sample, a standard may be a representation of
a UV-visible spectrum that would be expected from a test sample
comprising the target and/or a representation of a UV-visible
spectrum that would be expected from a test sample that does not
comprise the target. One skilled in the art can make and use a
suitable standard according to the characteristic to be considered
and the particular method employed.
Exemplary Kits
[0066] In some embodiments, kits are provided. In some embodiments,
a kit comprises a linker and nanoparticles. In some embodiments, a
kit further comprises at least one standard. Nonlimiting exemplary
linkers, nanoparticles, and standards are described herein.
[0067] The following examples are offered by way of illustration
and are not intended to limit the invention in any way.
EXAMPLES
Example 1
Materials and Methods
[0068] Deionized water, filtered at 18 .OMEGA.cm of resistivity,
was used in all experiments. Most chemicals (hydrogen
tetrachloroaurate, trisodium citrate, streptavidin, bovine serum
albumin (BSA), borate buffer, phosphate buffered saline (PBS),
HEPES buffer, glycerine and NaOH) were purchased from Fisher
Scientific (Pittsburgh, Pa., USA) and used as received unless
indicated otherwise. Biotin was purchased from Sigma-Aldrich (St.
Louis, Mo., USA) and anti-tissue/cell preparation of E. coli
polyclonal rabbit IgG antibody (pAb) (GTX13626) and biotinylated
antibody (GTX40640) were from Genetex Inc. (Irvine, Calif., USA).
DH5.alpha. E. coli was cultured in LB plates then gathered and
diluted in PBS to obtain a 1.times.10.sup.10 CFU/mL suspension,
which was then diluted as needed.
Synthesis of Gold Nanoparticles (AuNPs)
[0069] AuNPs (13-nm average diameter) were prepared by adding 10%
v/v of trisodium citrate to a boiling solution of 1 mM hydrogen
tetrachloroaurate. After the color of the solution turned wine red,
the citrate-stabilized AuNPs were stirred until the solution cooled
down to room temperature.
Functionalizing AuNPs with Antibody
[0070] AuNPs were functionalized with antibody by charge adsorption
as follows. 500 .mu.L of freshly prepared colloidal AuNPs was mixed
with 400 .mu.L borate buffer (pH 7.4), and then 100 .mu.L of pAb
solution (400 .mu.g/mL in PBS) was added. After 10 min of
incubation, the mixture was centrifuged and washed with borate
buffer (pH 9.5) twice to remove unbound pAb. Centrifuged
pAb-conjugated AuNPs were stored in 500 .mu.L PBS/0.1% w/v sodium
azide.
Functionalizing AuNPs with Streptavidin
[0071] AuNPs were functionalized with streptavidin by charge
adsorption as follows. The streptavidin solution was prepared in
borate buffer (pH 7.4) at 50 .mu.g/mL. 400 .mu.L of the
streptavidin solution was mixed with 600 .mu.L of freshly prepared
colloidal AuNPs (estimated to be at a concentration of about 8 nM).
Following 30 min of incubation, the streptavidin-coated AuNPs were
centrifuged, the supernatant was removed, the streptavidin-coated
AuNPs were washed several times, and then resuspended in 600 .mu.L
of PBS/0.1% bovine serum albumin
[0072] The biotin-dependence of the aggregation of streptavidin
functionalized AuNPs in the presence of biotinylated antibodies was
confirmed as follows. 100 .mu.L of 10 .mu.g/mL biotin was added to
200 .mu.L of streptavidin-coated AuNPs. After 15 minutes, 100 .mu.L
of biotinylated pAb was added to the biotin-treated AuNPs. No
visible color change was observed, suggesting that the aggregation
of the AuNPs is dependent on the binding of the streptavidin on the
AuNPs to the biotinylated antibodies.
UV-Vis Absorption Spectra
[0073] Absorption spectra were measured using a UV-vis
spectrophotometer (UV-1601PC, Shimadzu, Columbia, Md., USA). For
sample preparation, PBS was added to each reaction sample to a
final volume of 1 mL.
Example 2
On-Target Detection Resulted in No Visible Color Change
[0074] To investigate the effect of attaching AuNPs on E. coli on
the overall color of the sample solution, 200 .mu.L of
pAb-conjugated AuNPs were added to 200 .mu.L of various
concentrations of E. coli in PBS, for total E. coli concentrations
of 10.sup.1 to 10.sup.9 CFU/400 .mu.L. The samples were then
incubated at room temperature for one hour. The attachment of AuNPs
on E. coli was verified by examining the color of sediment obtained
by lightly centrifuging the solution at 3000 rpm for 10 min.
[0075] The results of that experiment are shown in FIG. 1. Even at
the highest concentrations of E. coli tested, no visually
distinguishable color change was observed. See FIG. 1A. Following
centrifugation, however, the presence of a dark-red sediment
confirmed that the AuNPs were bound to the E. coli. See FIG. 1B.
However, the color of the supernatant remained unchanged. Compare
tube on left (no E. coli control) with tube on the right (10.sup.9
CFU/400 .mu.L E. coli). These results suggest that only a small
fraction of the AuNPs in the sample is bound to the cell surface,
and that this fraction is insufficient to cause a visible color
change in the system.
Example 3
On-Target Detection with Lower Concentration of AuNPs Resulted in
No Visible Color Change
[0076] Next, the effect of lowering the initial AuNPs concentration
was investigated. The AuNPs were diluted with PBS to a
concentration of 25% of its original level. In addition, in this
experiment, biotinylated antibody was first bound to the E. coli,
and then streptavidin-functionalized AuNPs were added. After
binding of the biotinylated antibodies to E. coli, the cells were
washed multiple times to remove unbound antibodies.
[0077] For direct visual comparison of the sample color, all tests
were performed under the same conditions, keeping sample volumes
and particle concentrations the same. Samples were diluted in PBS
to 200 .mu.L, and then 200 .mu.L of streptavidin-coated AuNPs was
added to bind the AuNPs to pAb. For the control, the same procedure
was followed without the biotinlyated pAb such that AuNPs would not
specifically bind to the cell surface.
[0078] The results of that experiment are shown in FIG. 2. The
binding of AuNPs to E. coli (10.sup.9 CFU/400 .mu.L) at both high
and low concentrations of AuNPs was verified by examining the color
of the centrifuged sediment. See FIG. 2. FIG. 2A shows samples
containing higher a higher concentration of AuNPs (100%
concentration). FIG. 2B shows samples containing a lower
concentration of AuNPs (25% concentration). Although the color of
the samples containing a lower AuNPs concentration was lighter,
there was still no distinguishable change in the sample color upon
binding of AuNPs to E. coli. Compare sample on left (with
biotinylated antibody) to sample of right (without biotinylated
antibody) in FIG. 2B.
[0079] The above experiments further indicate that the binding of
AuNPs directly to bacteria does not change the sample color
sufficiently for on-target visual detection of bacteria. Without
being bound by any particular theory, this result may be due to one
or more of the following reasons. First, it may be that only a
small fraction of the total AuNPs binds to the bacteria, even at a
low initial AuNP concentration. Second, considering that AuNPs bind
to bacteria at the antigen sites, uniform covering of AuNPs on the
cell surface may be unlikely because antigen sites are not equally
spaced and/or the relatively large AuNPs, compared to inter-antigen
spacing, occlude several antigen sites on the cell surface. See
FIG. 3A. Third, it may be that monolayered AuNP binding to the cell
surface cannot produce a plasmonic effect strong enough for visual
indication as would three-dimensional clusters of AuNPs that occur
off-target. See FIG. 3B.
Example 4
Observable Color Change Using Off-Target Detection
[0080] Because on-target binding of AuNPs to cells did not result
in a visible color change in the above experiments, the potential
of detecting cells using off-target aggregation of AuNPs was
investigated. According to the manufacturer, the biotinylated
antibody is labeled with 7 to 10 biotins per unit. Therefore, one
free antibody should be able to crosslink multiple
strepavidin-coated AuNPs and facilitate color-changing aggregation
of AuNPs. An unaggregated solution of AuNPs is red, and the color
shifts to purple as the AuNPs aggregate. The ability of the
biotinylated antibody to cause the color shift was confirmed by
incubating the antibody with streptavidin-coated AuNPs, with and
without excess biotin. Data not shown. In the absence of excess
biotin, addition of biotinylated antibody to streptavidin-coated
AuNPs results in a color change from red to purple. When excess
biotin is added, which blocks the biotin binding sites on the
streptavidin-coated AuNPs and prevents the biotinylated antibody
from crosslinking the AuNPs, the color change does not occur.
[0081] To test the off-target detection of cells, 100 .mu.L of E.
coli suspended in PBS at 10.sup.2, 10.sup.4, 10.sup.6, and 10.sup.8
CFU/mL was mixed with 100 .mu.L of 10 .mu.g/mL biotin-conjugated
antibody in PBS (resulting in E. coli concentrations of 10,
10.sup.2, 10.sup.3, and 10.sup.4 CFU/mL and 5 .mu.g/mL antibody).
The mixture was incubated for 30 minutes with vigorous stirring.
After the incubation, 200 .mu.L of streptavidin-coated AuNPs was
added and the color change was monitored as a function of time.
[0082] The results of that experiment are shown in FIG. 4. A
visible color change from red to purple occurs within about 15 min.
See FIG. 4A. At one hour, a more pronounced color change is
observed. See FIG. 4B. While the color change is not directly
proportional to the E. coli concentration, a shift in the UV-VIS
peak absorbance is evident when E. coli are present. See FIG. 4C.
Accordingly, at the very least, this off-target method produced a
yes/no indication of the presence of bacteria in this experiment,
even at E. coli concentrations as low as 10 CFU/mL.
[0083] Similar experiments were carried out with Pseudomonas
aeruginosa and Salmonella typhimurium, demonstrating that the
off-target detection method can detect a range of targets.
Example 5
Characteristics of AuNP Aggregation
[0084] In order to examine the color-changing characteristics of
AuNP aggregation as a function of target concentration, an assay
system was designed using streptavidin-coated AuNPs and
biotinylated BSA.
[0085] Biotinylated BSA (Sigma-Aldrich; 0.5 .mu.g in 100 .mu.l) was
mixed with 200 .mu.l streptavidin-coated AuNPs made as described in
Example 1, in a total volume of 400 .mu.l (brought up to volume
with PBS). The mixture was incubated at room temperature
(.about.10.degree. C. to 30.degree. C.). The absorption spectrum of
the mixture was taken at 0 hour, 1 hour, 2 hours, and 3 hours to
determine the absorbance and peak wavelength changes of the mixture
over time, as the streptavidin-coated AuNPs bind to the
biotinylated BSA. As shown in FIG. 5, the peak absorbance of the
mixture decreased with time, while the peak absorption wavelength
increased. At the same time that the peak absorbance decreased, the
absorbance at higher wavelengths increased (see large arrows in
FIG. 5). These changes indicate that the streptavidin-coated AuNPs
were aggregating in the presence of the biotinylated BSA.
[0086] The relationship between the concentration of biotinylated
BSA and the change in the peak absorbance over time was then
investigated. Increasing amounts of biotinylated BSA in 100 .mu.l
(0.5, 1, 1.5, 2, 2.5, and 3 ng/100 .mu.l) were incubated with 200
.mu.l of streptavidin-coated AuNPs in a total volume of 400 .mu.l
(brought up to volume with PBS), and the mixture was incubated at
room temperature. The absorption spectrum of each mixture was taken
at 30 minutes, 1 hour, 2 hours, and 3 hours, and the
.DELTA..lamda.max measured from each time point to the next, at
each concentration of biotinylated BSA. The results of that
experiment are shown in FIG. 6A. The greatest peak wavelength
changes over time occurred at the lower amounts, or concentrations,
of biotinylated BSA. At the lowest concentration of biotinylated
BSA (e.g., 0.1 .mu.g in this experiment), there is insufficient
biotinylated BSA to aggregate the AuNPs, so they remain in solution
with no wavelength change over time. At the "middle" concentrations
of biotinylated BSA (e.g., 0.5 .mu.g and 1 .mu.g in this
experiment), there is sufficient biotinylated BSA to aggregate the
AuNPs, resulting in peak wavelength changes over time. At higher
concentrations of biotinylated BSA (e.g., 1.5 .mu.g and greater in
this experiment), there is excess biotinylated BSA and aggregation
does not occur because there is sufficient biotinylated BSA to coat
each AuNP. These regions are shown in FIG. 6B (lowest
concentrations=Region A; "middle" concentrations=Region B; highest
concentrations=Region C) and illustrated in FIG. 6C.The effect of
free streptavidin on the color change observed in FIG. 6B was
determined. A schematic representation of the assay is shown in
FIG. 7. A similar experiment as described above for FIG. 6 was
carried out, but in the presence of 0 .mu.g streptavidin, 1 .mu.g
streptavidin, or 10 .mu.g streptavidin. Free streptavidin was
incubated with the biotinylated BSA prior to addition of the AuNPs.
Each mixture was incubated at room temperature for 2 hours before
the absorption spectrum was taken. The .DELTA..lamda.max was
measured from each time point to the next, at each concentration of
biotinylated BSA. The results of that experiment are shown in FIG.
8. The addition of free streptavidin caused the greatest
.DELTA..lamda.max to shift to a higher concentration of
biotinylated BSA. In other words, more biotinylated BSA was
required to aggregate the streptavidin-coated AuNPs in the presence
of free streptavidin. Compare, e.g., FIG. 8 inset to FIG. 6B.
[0087] A similar experiment was carried out using various
concentrations of biotinylated antibody, preincubated with 10
.mu.g, 1 .mu.g, or 10 .mu.g free streptavidin, and then mixed with
200 .mu.l of streptavidin-coated AuNPs, in a total volume of 400
.mu.l (brought up to volume with PBS). The results of that
experiment are shown in FIG. 9. Briefly, the antibody concentration
at which the greatest .DELTA..lamda.max shifted from 2 .mu.g
biotinylated antibody (in the presence of 0 or 1 .mu.g
streptavidin) to 3 to 4 .mu.g antibody (in the presence of 10 .mu.g
streptavidin).
Example 6
Sensitivity of Off-Target Microorganism Detection Using AuNPs
[0088] An exemplary method and model for off-target microbial
detection using AuNPs is shown in FIG. 10. Briefly, in a first
step, a sample that may or may not comprise a microorganism is
incubated with biotinylated antibodies that bind, for example, to a
marker on the surface of the microorganism. If microorganisms are
present, the biotinylated antibodies bind to the surface of the
microorganisms. In a second step, strepatavidin-coated AuNPs are
added to the sample. If there are no microorganisms in the sample,
the biotinylated antibody cross-links the biotinylated AuNPs and
large-scale aggregates form, causing a visual color change in the
sample from red to purple. If microorganisms are present, the
streptavidin-coated AuNPs bind to the biotinylated antibodies on
the surface of the microorganisms, preventing large-scale
aggregation of the AuNPs and the color change.
[0089] In order to determine the potential sensitivity of the
off-target detection assay illustrated in FIG. 10, the assay was
carried out using a range of biotinylated antibody concentrations
(0, 0.5, 1, 2, 3, 4, and 5 .mu.g in 100 .mu.l) and a range of
bacterial concentrations (0, 10.sup.2, 10.sup.4, and 10.sup.6 CFUs
in 100 .mu.l), with 200 .mu.l streptavidin-coated AuNPs (total
final volume of 400 .mu.l). The mixtures were incubated at room
temperature for 2 hours.
[0090] The results of that experiment are shown in FIG. 11. In this
experiment, it was demonstrated that two different color change
phases can be observed. At 1 .mu.g biotinylated antibody, indicated
by the first red box, the streptavidin-coated AuNPs are aggregated
in the absence of bacteria (0 CFU), and the color of the mixture is
purple. This aggregation occurs because the biotinylated antibody
concentration to streptavidin-coated AuNP concentration is such
that there is less biotinylated antibody than streptavidin-coated
AuNP, allowing multiple streptavidin-coated AuNPs to bind to each
biotinylated antibody, which results in aggregation. With just 100
CFUs of bacteria present, the biotinylated antibodies are bound to
the surface of the bacteria and no longer aggregate the
streptavidin-coated AuNPs, resulting in a red color. At 4 .mu.g
biotinylated antibody, indicated by the second red box, the AuNPs
are not aggregated in the absence of bacteria (0 CFU) because there
is an excess of biotinylated antibody (see, e.g., FIG. 6C, "Region
C"). With 10.sup.4 CFUs of bacteria present, however, aggregation
begins to occur because much of the biotinylated antibody is
"soaked up" by the bacteria, leaving enough biotinylated antibody
in solution to aggregate the streptavidin-coated AuNPs.
[0091] Thus, the present system can be fine-tuned to a very high
level of sensitivity for a particular threshold of microbial
contamination. For example, in some embodiments, for testing a
sample in which little or no microbial contamination can be
tolerated, such as food or pharmaceuticals, a lower amount of
biotinylated antibody is used, and the system will be purple in the
absence of contamination, and turn red at very low levels of
contamination. In some embodiments, for testing a sample in which
higher levels of microbial contamination are tolerated, or if the
sample has been concentrated, higher amounts of biotinylated
antibodies are used, and the system will be red in the absence of
contamination, and turn purple at higher levels of
contamination.
[0092] The foregoing description is considered as illustrative only
and is not intended to limit the claimed invention. Numerous
modifications and changes may readily occur to those skilled in the
art. The invention is not limited to the exact construction and
operation shown and described, and accordingly, all suitable
modifications and equivalents are considered to fall within the
scope of the invention.
* * * * *