U.S. patent application number 10/355462 was filed with the patent office on 2004-01-22 for methods and apparatus for assays of bacterial spores.
Invention is credited to Bearman, Gregory H., Ponce, Adrian.
Application Number | 20040014154 10/355462 |
Document ID | / |
Family ID | 27670648 |
Filed Date | 2004-01-22 |
United States Patent
Application |
20040014154 |
Kind Code |
A1 |
Ponce, Adrian ; et
al. |
January 22, 2004 |
Methods and apparatus for assays of bacterial spores
Abstract
A sample of unknown bacterial spores is added to a test strip.
The sample of unknown bacterial spores is drawn to a first sample
region on the test strip by capillary action. Species-specific
antibodies are bound to the sample when the unknown bacterial
spores match the species-specific antibodies, otherwise the sample
is left unbound. DPA is released from the bacterial spores in the
bound sample. The terbium ions are combined with the DPA to form a
Tb-DPA complex. The combined terbium ions and DPA are excited to
generate a luminescence characteristic of the combined terbium ions
and DPA to detect the bacterial spores. A live/dead assay is
performed by a release of the DPA for live spores and a release of
DPA for all spores. The detection concentrations are compared to
determine the fraction of live spores. Lifetime-gated measurements
of bacterial spores to eliminate any fluorescence background from
organic chromophores comprise labeling the bacterial spore contents
with a long-lifetime lumophore and detecting the luminescence after
a waiting period. Unattended monitoring of bacterial spores in the
air comprises the steps of collecting bacterial spores carried in
the air and repeatedly performing the Tb-DPA detection steps above.
The invention is also apparatus for performing the various methods
disclosed above.
Inventors: |
Ponce, Adrian; (Altadena,
CA) ; Bearman, Gregory H.; (Pasadena, CA) |
Correspondence
Address: |
Daniel L. Dawes
Myers, Dawes & Andras LLP
Suite 1150
19900 MacArthur Blvd.
Irvine
CA
92612
US
|
Family ID: |
27670648 |
Appl. No.: |
10/355462 |
Filed: |
January 31, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60353268 |
Feb 1, 2002 |
|
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60395372 |
Jul 12, 2002 |
|
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60414170 |
Sep 27, 2002 |
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Current U.S.
Class: |
435/7.32 ;
435/287.2 |
Current CPC
Class: |
G01N 21/6408 20130101;
G01N 33/84 20130101; C12Q 1/04 20130101; G01N 33/54306 20130101;
G01N 33/56911 20130101 |
Class at
Publication: |
435/7.32 ;
435/287.2 |
International
Class: |
G01N 033/554; G01N
033/569; C12M 001/34 |
Claims
We claim:
1. A method for lateral flow immunoassay for species-specific
bacterial spore detection and quantification comprising: providing
a matrix including terbium ions; releasing DPA from the bacterial
spores; combining the terbium ions with the DPA in solution; and
exciting the combined terbium ions and DPA to generate a
luminescence characteristic of the combined terbium ions and DPA to
detect the bacterial spores.
2. The method of claim 1 where releasing DPA from the bacterial
spores comprises microwaving the spores.
3. The method of claim 1 where releasing DPA from the bacterial
spores comprises germinating the spores with L-alanine.
4. The method of claim 1 where releasing DPA from the bacterial
spores comprises sonicating the spores with microspheres.
5. The method of claim 1 where releasing DPA from the bacterial
spores comprises autoclaving the spores.
6. The method of claim 1 where exciting the combined terbium ions
and DPA to generate a luminescence characteristic of the combined
terbium ions and DPA comprises radiating the combined terbium ions
and DPA with ultraviolet light.
7. A method for lateral flow immunoassay for bacterial spore
detection and quantification comprising: adding a sample of unknown
bacterial spores to a test strip; drawing the sample of unknown
bacterial spores to a first sample region on the test strip;
selectively binding species-specific antibodies to the sample when
the unknown bacterial spores match the species-specific antibodies,
otherwise leaving the sample unbound; releasing DPA from the
bacterial spores in the bound sample; combining the terbium ions
with the DPA; and exciting the combined terbium ions and DPA to
generate a luminescence characteristic of the combined terbium ions
and DPA to detect the bacterial spores.
8. The method of claim 7 further comprising: adding a standard of
known bacterial spores with known concentration to the test strip;
drawing the standard of known bacterial spores to a second sample
region on the test strip; selectively binding species-specific
antibodies to the standard when the known bacterial spores match
the species-specific antibodies, otherwise leaving the standard
unbound; releasing DPA from the bacterial spores in the bound
standard; combining the terbium ions with the DPA; exciting the
combined terbium ions and DPA to generate a luminescence
characteristic of the combined terbium ions and DPA to detect the
bacterial spores of the standard; and comparing the intensity of
the excited luminescence from the sample with the standard to
derive a quantification of the spore concentration in the
sample.
9. The method of claim 8 further comprising confirming arrival of
the sample and standard in the first and second sample regions
respectively by means of a visual indicator.
10. The method of claim 8 where releasing DPA from the bacterial
spores in the sample and standard comprises microwaving the
spores.
11. The method of claim 8 where releasing DPA from the bacterial
spores in the sample and standard comprises germinating the spores
with L-alanine.
12. The method of claim 8 where releasing DPA from the bacterial
spores in the sample and standard comprises sonicating the spores
with microspheres.
13. The method of claim 8 where releasing DPA from the bacterial
spores in the sample and standard comprises autoclaving the
spores.
14. The method of claim 8 where exciting the combined terbium ions
and DPA in the sample and standard to generate a luminescence
characteristic of the combined terbium ions and DPA comprises
radiating the combined terbium ions and DPA in the sample and
standard with ultraviolet light.
15. A method for live/dead assay for bacterial spores comprising:
providing a solution including terbium ions in a sample of live and
dead bacterial spores; releasing DPA from viable bacterial spores
by germination from a first unit of the sample; combining the
terbium ions with the DPA in solution released from viable
bacterial spores; and exciting the combined terbium ions and DPA
released from viable bacterial spores to generate a first
luminescence characteristic of the combined terbium ions and DPA to
detect the viable bacterial spores; releasing DPA from dead
bacterial spores in a second unit of the sample by autoclaving,
sonication or microwaving; combining the terbium ions with the DPA
in solution released from dead bacterial spores; and exciting the
combined terbium ions and DPA released from dead bacterial spores
to generate a second luminescence characteristic of the combined
terbium ions and DPA to detect the dead bacterial spores; and
generating a ratio of the first to second luminescence to yield a
fraction of bacterial spores which are alive.
16. A method for lifetime-gated measurements of bacterial spores to
eliminate any fluorescence background from organic chromophores
comprising: providing a solution including terbium ions with a
sample of bacterial spores; labeling the bacterial spore contents
with a long-lifetime lumophore; releasing DPA from the bacterial
spores; combining the terbium ions with the DPA in solution; and
exciting the combined terbium ions and DPA for a first time period;
waiting a second time period before detecting luminescence; and
detecting a luminescence characteristic of the combined terbium
ions and DPA after the second time period during a defined temporal
window synchronized with luminescence timed from the long lifetime
lumophore to detect the bacterial spores.
17. The method of claim 16 where the first time period of
excitation is of the order of nanoseconds, where the second time
period is of the order of microseconds and where the defined
temporal window is of the order of milliseconds.
18. The method of claim 17 where the first time period of
excitation is of the order of 1-10 nanoseconds, where the second
time period is of the order of tens of microseconds and where the
defined temporal window is of the order of 1-10 milliseconds.
19. The method of claim 16 where the first time period of
excitation is of the order of nanoseconds, where the second time
period is of the order of tenths to tens of milliseconds and where
the defined temporal window is of the order of hundreds of
microseconds.
20. A method for unattended monitoring of bacterial spores in the
air comprising: collecting bacterial spores carried in the air;
suspending the collected bacterial spores in a solution including
terbium ions; releasing DPA from the bacterial spores; combining
the terbium ions with the DPA in solution; and exciting the
combined terbium ions and DPA to generate a luminescence
characteristic of the combined terbium ions and DPA; detecting the
luminescence to determine the presence of the bacterial spores; and
generating an alarm signal when the presence of bacterial spores is
detected or the concentration thereof reaches a predetermined
magnitude.
21. The method of claim 20 where collecting bacterial spores
carried in the air comprises capturing the bacterial spores with an
aerosol sampler or impactor.
22. The method of claim 20 where detecting the luminescence to
determine the presence of the bacterial spores comprises monitoring
the luminescence with a spectrometer or fluorimeter.
23. The method of claim 20 where collecting bacterial spores
carried in the air comprising continuously sampling the air and
where detecting the luminescence to determine the presence of the
bacterial spores comprises continuously monitoring the
luminescence.
24. The method of claim 23 where releasing DPA from the bacterial
spores comprising microwaving the bacterial spores to heat the
solution and where combining the terbium ions with the DPA in
solution comprises cooling the heated solution to increase the
fraction of bound Tb-DPA complex.
25. An apparatus for unattended monitoring of bacterial spores in
the air comprising: a biosampler for capturing the bacterial spores
in the air and having a collection vessel containing a solution
including terbium ions into which the captured bacterial spores are
suspended; means for releasing DPA from the bacterial spores in the
solution to allow the DPA to combine with the terbium ions to form
a Tb-DPA complex; an energy source for exciting the Tb-DPA complex
to generate luminescence; an electro-optical circuit to measure the
luminescence; and an alarm circuit coupled to the electro-optical
circuit to detect a bacterial spore concentration above a
predetermined threshold.
26. An apparatus for lateral flow immunoassay for bacterial spore
detection and quantification comprising: a strip of material for
providing lateral capillary flow of a solution including terbium
ions across the strip; an input region on the strip for receiving a
liquid sample containing terbium ions, the first zone being
provided with a first antibody for specific binding to a specific
specie of bacterial spores; a sample region of the strip laterally
displaced from the input region and communicated thereto by means
of capillary flow therebetween, the sample region being provided
with a second antibody to capture bacterial spores with the
attached first antibody and to immobilize them; means for releasing
DPA from the bacterial spores in the sample region of the strip to
then allow the terbium ions to combine with the DPA in solution;
and an energy source to excite the combined terbium ions and DPA in
the sample region of the strip to generate a luminescence
characteristic of the combined terbium ions and DPA; and a
luminescence detector to identify the presence or measure the
concentration of the bacterial spores in the sample region of the
strip.
27. The apparatus of claim 26 where the means for releasing DPA
from the bacterial spores in the sample region of the strip
comprises a microwave heater, means for adding L-alanine to the
solution, means for sonicating the spores with microspheres, or an
autoclave.
Description
RELATED APPLICATIONS
[0001] The present application is related to U.S. Provisional
Patent Application serial No. 60/353,268 filed on Feb. 1, 2002;
U.S. Provisional Patent Application serial No. 60/395,372, filed on
Feb. 1, 2002; U.S. Provisional Patent Application serial No.
60/414,170, filed on Sep. 27, 2002, and U.S. Provisional Patent
Application serial No. (CIT3770), filed on ______, each of which is
incorporated herein by reference and to which priority is claimed
pursuant to 35 USC 119.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention is directed assays of bacterial endospore
levels.
[0004] 2. Description of the Prior Art
[0005] The prior art for species-specific bacterial spore
detection, using the lateral flow immunoassay method, is based on
observing the red color of gold nanoparticles. It uses two
antibodies, in combination, to specifically detect the bacterial
spore species of interest in solution. One of the antibodies is
attached to a colloidal gold nanoparticle, and the other antibody
is immobilized on the nitrocellulose membrane downstream from the
point of sample introduction. When about 100 .mu.l of sample is
added to the test strip membrane on top of the area 30 that
contains the colloidal gold labeled antibodies, specific binding
between bacterial spores and gold labeled antibodies occurs.
Simultaneously, capillary action moves the gold labeled antibodies
(both spore bound and not bound) along the strip membrane 32. In
the sample region 34 of the test strip 32 (downstream), specific
binding of a second antibody captures bacterial spores with the
attached colloidal gold labeled antibody, which gives rise to a red
line in the sample region 34 due to the immobilized gold
nanoparticles as shown in the bottom left of FIG. 1. In the control
region 36 of the test strip 32 (further downstream), as an internal
control, a polyclonal antibody binds the gold labeled antibodies
that did not bind bacterial spores of interest, which also gives
rise to a red line. Thus, observation of two bands, one each in the
sample and control regions, indicates a positive test for the
bacterial spore of interest. The observation of only one band as
shown in the bottom right of FIG. 1 is a negative test result. The
fundamental limitation of this method is its sensitivity; a minimum
concentration of 10.sup.5 spores/ml is needed before the red color
from the gold nanoparticles becomes detectable; for reference, a
100 .mu.l sample containing 10,000 anthrax spores is lethal.
[0006] Therefore what is needed is a method for improving the
detection limit of lateral flow immunoassay based detection of
bacterial spores, which is reported to be 10.sup.5 spores/ml. This
prior art detection limit prevents detection of trace quantities of
bacterial spores. A trace quantity of 8000 anthrax spores, for
example, is enough fill a person.
[0007] The prior art the method for determining the fraction of
viable bacterial spores is based on two measurements. First, the
viable bacterial spore count is measured by colony counting, and
second, the total bacterial spore count is measured by direct
microscopic counting. The ratio of viable to total bacterial spore
count yields the fraction of spores that remain viable within a
given sample. The procedure for colony counting to determine
endospore concentration is comprised of the steps of (1) heat
shocking the sample to kill vegetative cells while bacterial spores
remain viable, (2) plating a known volume of the sample with a
known dilution factor onto a growth medium, and (3) incubating the
growth plates for 2 days. Finally, the resulting visible colonies
are counted and reported as colony forming units (CFU's). The
procedure for direct microscopic counting is comprised of the steps
of (1) placing the sample on a slide with an indentation of a known
volume. The glass surface of the slide is inscribed with squares of
known area. (2) The bacteria in each of the several squares are
counted and the average count is multiplied by an appropriate
factor to yield the number of total cells per milliliter in the
original suspension.
[0008] These methods suffer prohibitive difficulties with low
concentration samples collected in the field. First, bacterial
spores tend to attach themselves onto particulates (dust etc.) and
may easily represent the bulk of the biomass in a field sample.
Unfortunately, attached bacterial spores cannot be counted with
either colony counting or direct microscopic counting. Second,
colony-counting methods only work for cultivable bacteria, which
are in the minority in field samples (<10% of microbial species
form colonies). Finally, the traditional methods are lengthy (>2
days) and labor intensive. These problems have made quantification
of low concentration field samples extremely difficult, and have
subsequently prevented the application of these methods towards a
reliable and/or real-time bacterial spore live/dead assay.
[0009] There is a need to develop a live/dead assay for bacterial
spores, because there is a need to measure the fraction of
bacterial spores that remain viable for samples exposed to harsh
environmental conditions such as desert and arctic environments. In
terms of planetary protection, which is primarily concerned with
spacecraft sterilization, in order to improve sterilization
procedures, one must measure the fraction of viable spores after
completion of various sterilization protocols. The samples of
interest contain low bacterial spore concentration and many
particulates, for which the prior art methods useless.
[0010] Prior art methods for monitoring aerosolized bacterial
spores includes air filtering with subsequent PCR analysis of gene
segments from species of interest, and aerosol sampling with
subsequent culturing and colony counting. The PCR based method is
strongly dependent on impurities in the air, such as city
pollution, and requires specially trained technicians to perform
sample preparation prior to running the PCR reaction. The procedure
for colony counting, which is comprised of (1) heat shocking the
sample to kill vegetative cells while bacterial spores remain
viable, (2) plating a known volume of the sample with a known
dilution factor onto a growth medium, (3) incubating the growth
plates for two days, also requires the active participation of a
technician. This also assumes that the spore forming microbes are
cultivable. It is estimated that only 10% of bacterial species are
cultivable. The cost of labor, technical complexity of PCR and slow
response time of colony counting have prevented the wide spread
application of these methods for monitoring of bacterial spores in
the air.
BRIEF SUMMARY OF THE INVENTION
[0011] Lateral Flow Immunoassay
[0012] One embodiment of the invention is defined as a method for
lateral flow immunoassay for bacterial spore detection and
quantification comprising the steps of providing a matrix including
terbium ions, releasing DPA from the bacterial spores, combining
the terbium ions with the DPA in solution, and exciting the
combined terbium ions and DPA to generate a luminescence
characteristic of the combined terbium ions and DPA to detect the
bacterial spores. The matrix The detection of the spores or their
concentration above a predetermined threshold generates an alarm
signal.
[0013] The DPA is released from the bacterial spores by microwaving
the spores, germinating the spores with L-alanine, sonicating the
spores with microspheres or autoclaving the spores. These methods
by no means necessarily exhaust the ways in which the DPA can be
released from the spores and all other methods of lysing the spores
are deemed equivalent.
[0014] Exciting the combined terbium ions and DPA generates a
luminescence characteristic of the combined terbium ions and DPA.
This is achieved by radiating the combined terbium ions and DPA
with ultraviolet light. Again, any method by which luminescence can
be induced is included within the scope of the invention and is
deemed to be equivalent.
[0015] The invention can also be characterized as a method for
lateral flow immunoassay for bacterial spore detection and
quantification. The method starts with the step of adding a sample
of unknown bacterial spores to a test strip. The sample of unknown
bacterial spores is drawn to a first sample region on the test
strip by capillary action. Species-specific antibodies are bound to
the sample when the unknown bacterial spores match the
species-specific antibodies, otherwise the sample is left unbound.
DPA is released from the bacterial spores in the bound sample. The
terbium ions are combined with the DPA to form a Tb-DPA complex.
The combined terbium ions and DPA are excited to generate a
luminescence characteristic of the combined terbium ions and DPA to
detect the bacterial spores.
[0016] The method further comprises the steps of performing the
same steps with a standard of known bacterial spores with known
concentration. The sample is added to the test strip and drawn to a
second sample region on the test strip. Species-specific antibodies
are selectively bound to the standard when the known bacterial
spores match the species-specific antibodies, otherwise the
standard unbound is left unbound. DPA is released from the
bacterial spores in the bound standard and combined with the
terbium ions. The combined terbium ions and DPA are excited to
generate a luminescence characteristic of the combined terbium ions
and DPA to detect the bacterial spores of the standard. The
intensity of the excited luminescence from the sample is compared
with the excited luminescence from the standard to derive a
quantification of the spore concentration in the sample. The method
may further comprise the step of confirming arrival of the sample
and standard in the first and second sample regions respectively by
means of a visual indicator.
[0017] Live/Dead Assay for Bacterial Spores
[0018] The invention is defined in another embodiment as a method
for live/dead assay for bacterial spores comprising the steps of:
providing a solution including terbium ions in a sample of live and
dead bacterial spores; releasing DPA from viable bacterial spores
by germination from a first unit of the sample; combining the
terbium ions with the DPA in solution released from viable
bacterial spores; exciting the combined terbium ions and DPA
released from viable bacterial spores to generate a first
luminescence characteristic of the combined terbium ions and DPA to
detect the viable bacterial spores; releasing DPA from dead
bacterial spores in a second unit of the sample by autoclaving,
sonication or microwaving; combining the terbium ions with the DPA
in solution released from dead bacterial spores; exciting the
combined terbium ions and DPA released from dead bacterial spores
to generate a second luminescence characteristic of the combined
terbium ions and DPA to detect the dead bacterial spores;
generating a ratio of the first to second luminescence to yield a
fraction of bacterial spores which are alive.
[0019] Lifetime-Gated Measurements of Bacterial Spores and Imaging
Bacterial Spores Using an Active Pixel Sensor
[0020] In yet another embodiment the invention is a method for
lifetime-gated measurements of bacterial spores to eliminate any
fluorescence background from organic chromophores comprising the
steps of providing a solution including terbium ions with a sample
of bacterial spores; labeling the bacterial spore contents with a
long-lifetime lumophore; releasing DPA from the bacterial spores;
combining the terbium ions with the DPA in solution; exciting the
combined terbium ions and DPA for a first time period; waiting a
second time period before detecting luminescence; and detecting a
luminescence characteristic of the combined terbium ions and DPA
after the second time period during a defined temporal window
synchronized with luminescence timed from the long lifetime
lumophore to detect the bacterial spores.
[0021] In one embodiment the first time period of excitation is of
the order of nanoseconds, the second time period is of the order of
microseconds and the defined temporal window is of the order of
milliseconds.
[0022] In another embodiment the first time period of excitation is
of the order of 1-10 nanoseconds, where the second time period is
of the order of tens of microseconds and where the defined temporal
window is of the order of 1-10 milliseconds.
[0023] In still another embodiment the first time period of
excitation is of the order of nanoseconds, the second time period
is of the order of tenths to tens of milliseconds and where the
defined temporal window is of the order of hundreds of
microseconds.
[0024] Unattended Monitoring of Bacterial Spores in the Air
[0025] In yet another embodiment the invention is a method for
unattended monitoring of bacterial spores in the air comprising the
steps of collecting bacterial spores carried in the air; suspending
the collected bacterial spores in a solution including terbium
ions; releasing DPA from the bacterial spores; combining the
terbium ions with the DPA in solution; exciting the combined
terbium ions and DPA to generate a luminescence characteristic of
the combined terbium ions and DPA; detecting the luminescence to
determine the presence of the bacterial spores; and generating an
alarm signal when the presence of bacterial spores is detected or
the concentration thereof reaches a predetermined magnitude.
[0026] The step of collecting bacterial spores carried in the air
comprises capturing the bacterial spores with an aerosol sampler or
impactor. The step of detecting the luminescence to determine the
presence of the bacterial spores comprises monitoring the
luminescence with a spectrometer or fluorimeter.
[0027] Preferably, the step of collecting bacterial spores carried
in the air comprises continuously sampling the air and the step of
detecting the luminescence to determine the presence of the
bacterial spores comprises continuously monitoring the
luminescence.
[0028] When the step of releasing DPA from the bacterial spores
comprises microwaving the bacterial spores to heat the solution,
the step of combining the terbium ions with the DPA in solution
comprises cooling the heated solution to increase the fraction of
bound Tb-DPA complex.
[0029] The invention is also apparatus for performing the various
methods disclosed above. For example, the invention includes an
apparatus for unattended monitoring of bacterial spores in the air
comprising: a biosampler for capturing the bacterial spores in the
air and having a collection vessel containing a solution including
terbium ions into which the captured bacterial spores are
suspended; means for releasing DPA from the bacterial spores in the
solution to allow the DPA to combine with the terbium ions to form
a Tb-DPA complex; an energy source for exciting the Tb-DPA complex
to generate luminescence; an electro-optical circuit to measure the
luminescence; and an alarm circuit coupled to the electro-optical
circuit to detect a bacterial spore concentration above a
predetermined threshold.
[0030] The invention is also an apparatus for lateral flow
immunoassay for bacterial spore detection and quantification
comprising: a strip of material for providing lateral capillary
flow of a solution including terbium ions across the strip; an
input region on the strip for receiving a liquid sample containing
terbium ions, the first zone being provided with a first antibody
for specific binding to a specific specie of bacterial spores; a
sample region of the strip laterally displaced from the input
region and communicated thereto by means of capillary flow
therebetween, the sample region being provided with a second
antibody to capture bacterial spores with the attached first
antibody and to immobilize them; means for releasing DPA from the
bacterial spores in the sample region of the strip to then allow
the terbium ions to combine with the DPA in solution; an energy
source to excite the combined terbium ions and DPA in the sample
region of the strip to generate a luminescence characteristic of
the combined terbium ions and DPA; and a luminescence detector to
identify the presence or measure the concentration of the bacterial
spores in the sample region of the strip.
[0031] While the apparatus and method has or will be described for
the sake of grammatical fluidity with functional explanations, it
is to be expressly understood that the claims, unless expressly
formulated under 35 USC 112, are not to be construed as necessarily
limited in any way by the construction of "means" or "steps"
limitations, but are to be accorded the full scope of the meaning
and equivalents of the definition provided by the claims under the
judicial doctrine of equivalents, and in the case where the claims
are expressly formulated under 35 USC 112 are to be accorded full
statutory equivalents under 35 USC 112. The invention can be better
visualized by turning now to the following drawings wherein like
elements are referenced by like numerals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a diagram of a prior art lateral flow immunoassay
for bacterial spores.
[0033] FIG. 2a is a microscopic image of a spore (about 1 .mu.m in
diameter) highlighting a DPA rich spore core.
[0034] FIG. 2b is a diagram of a Tb.sup.3+ ion (shaded ball) by
itself has a low absorption cross section (<10
M.sup.-1cm.sup.-1) and consequently has low luminescence intensity.
The Tb.sup.3+ ion can bind the light harvesting DPA (absorption
cross section>10.sup.4 M.sup.-1) originating from the spore; DPA
binding gives rise to bright Tb luminescence.
[0035] FIG. 2c is a diagram of a photophysical scheme for DPA
sensitized luminescence of the Tb complex (absorption-energy
transfer-emission, AETE).
[0036] FIG. 3 is a diagrammatic illustration showing a few drops of
bacterial spore containing sample are added to the test strip
membrane.
[0037] FIGS. 4a-4c are graphs of the intensity of Tb luminescence
verses time. FIG. 4a shows the intensity during germination
starting with t=0 when L-alanine was added. FIG. 4b shows the Tb
luminescence after completion of germination corresponding to
Tb-DPA complex. FIG. 4c shows Tb luminescence induced by
autoclaving.
[0038] FIG. 5 is a diagram illustrating the active pixel sensor
imaging method as applied to Tb luminescence in bacterial
spores.
[0039] FIG. 6 is the lifetime series decay of the bacterial spores
illuminated in FIG. 5.
[0040] FIG. 7 is a simplified diagram of an unattended air monitor
for bacillus using Tb-DPA detection.
[0041] FIGS. 8a and 8b are graphs of the relative luminescence
intensity as a function of time and wavelength respectively. FIG.
8a illustrates the time course of spore monitoring and FIG. 8b
shows the spectrum just before spore release, less than 15 minutes
after spore release and 60 minutes after spore release.
[0042] The invention and its various embodiments can now be better
understood by turning to the following detailed description of the
preferred embodiments which are presented as illustrated examples
of the invention defined in the claims. It is expressly understood
that the invention as defined by the claims may be broader than the
illustrated embodiments described below.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] Lateral Flow Immunoassay
[0044] The invention is directed to lateral flow immunoassay for
bacterial spore detection and quantification using lanthanide
luminescence with both high sensitivity and selectivity in less
than five minutes. The method combines lateral flow immunoassay and
dipicolinic acid (DPA) triggered terbium (Tb) luminescence
technologies. The lateral flow immunoassay provides high
selectivity for specific bacterial spore species, and the DPA
triggered Tb luminescence method for bacterial spore detection
enables greatly improved detection limits over the prior art
detection schemes.
[0045] The new technology has significantly improved detection
limits, because it is based on Luminescence turn-on against a dark
background, which is much more sensitive than measuring the
scattered light, from gold nanoparticles against a bright
background. Based on DPA-triggered Tb luminescence experiments, we
anticipate single spore detection limits for 100 .mu.l samples
(i.e. 10 spores/ml).
[0046] The solution for developing a lateral flow immunoassay based
detection of bacterial spores with single spore detection limits is
to use DPA triggered Tb luminescence as the detection scheme. The
methodology for achieving single spore detection is more expressly
disclosed in copending U.S. patent application entitled "An
Improvement In A Method For Bacterial Endospore Quantification
Using Lanthanide Dipicolinate Luminescence," Ser. No. (CIT3405)
______, filed ______, and assigned to the same assignee as the
present invention, which application is incorporated herein by
reference.
[0047] Consider now the DPA-triggered Tb luminescence detection of
bacterial spores. Dipicolinic acid DPA, 2,6 pyridinedicarboxylic
acid) is present in high concentrations (about 1 molar or about 15%
of by weight) in the core of bacterial spores 38 as a 1:1 complex
with Ca.sup.2+ as shown in FIG. 2a. For all known lifeforms, DPA is
unique to bacterial spores and is released into bulk solution upon
germination, which is the process of spore-to-vegetative cell
transformation. Thus, DPA is an indicator molecule for the presence
of bacterial spores. Fortuitously, DPA is also a classic inorganic
chemistry ligand that binds metal ions with high affinity. DPA
binding to terbium ions triggers intense green luminescence under
UV excitation as shown in FIGS. 2b and 2c. Thus, the green
luminescence turn-on signals the presence of bacterial spores, and
the intensity of the luminescence can be correlated to the number
of bacterial spores per milliliter. Potential interferents such as
sugars, nucleic and amino acids are present in much lower
concentrations in endospores and vegetative cells and have binding
constants for Tb that are approximately six orders of magnitude
less than that of DPA (K.sub.A=10.sup.8.7 M.sup.-1). This method is
relatively immune to these interferents.
[0048] The core of bacterial spores contains 1 molar dipicolinic
acid (DPA) (.about.15% of the spore dry weight). It has been shown
that the DPA can be released into bulk solution by microwaving the
sample (germination with L-alanine, sonication with microspheres,
and autoclaving have also been used to release DPA from the spore).
When the released DPA binds terbium ions in bulk solution, bright
green luminescence is triggered under UV excitation.
[0049] The mechanism of DPA-triggered Tb luminescence is based on
the unique photophysical properties of lanthanide ions. The
luminescence of lanthanide ions is characterized by long lifetimes
(0.1 to 1 ms), small extinction coefficients (a.k.a. absorbtivity,
about 1 M.sup.-1 cm.sup.-1) and narrow emission bands. These
characteristics arise because the valence f orbitals are shielded
from the environment by the outer 5 s and 5 p electrons, and
because the transition between the emitting excited stare and
ground state is highly forbidden. Thus, direct excitation of
terbium ions leads to weak luminescence due to the small absorption
cross section. However, coordination of aromatic chromophores, like
DPA, triggers intense terbium luminescence. The juxtaposition of
DPA, which has an absorbtivity of 5000 M.sup.-1 cm.sup.-1 serves as
a light-harvesting center (e.g. antenna effect). Strong electronic
coupling and downhill energies allow the DPA centered excitation
energy to be efficiently transferred to the lanthanide ion, which
subsequently luminesces bright green.
[0050] Consider now the details of lateral flow immunoassay with
DPA-triggered Tb luminescence detection of bacterial spores 10. The
test strip 18 is comprised of a nitrocellulose membrane 12 that has
species-specific antibodies bound in the sample regions, which are
regions 26 and 22 of the strip as shown in FIG. 3. Region 26
contains antibodies for the bacterial spore species 10 of interest
(e.g. B. anthracis antibody), and region 22 contains antibodies for
B. subtilis (standard 20). First, about 100 .mu.l of sample 10 in a
liquid, such as water, and standard 20 in a solution of the same or
a different liquid are added to their respective test strip
membranes 12 and 16 in the sample port region 30. Capillary action
moves the spores 10 along the strip membrane 12 and 16. In the
sample region 14 of the test strip 12 (downstream), specific
binding of membrane-bound antibodies captures and immobilizes the
bacterial spores 10, while components of the sample 10 that do not
bind the antibody continue to flow out of the sample region 14.
Regions 24 contain an indicator, such as cobalt chloride, that
changes visible color when the liquid or solvent front arrives,
after about five minutes, which should suffice to provide adequate
separation of the specific binding components to the nonspecific
components of the sample 10. For example, where the indicator is
cobalt chloride, the color changes to blue to pink on arrival of
the liquid, which is in this embodiment is water. The choice of
liquid and indicator is a matter of design choice and many other
selections can be equivalently substituted.
[0051] In the next step, DPA is released from the core of the
spores 10 by microwaving the test strip 12. The released DPA binds
Tb dissolved in the solution and triggers green luminescence, which
signals the presence of bacterial spores. The green luminescence
can be read or measured by a conventional spectrometer or
fluorometer (not shown).
[0052] The control is performed on a parallel test strip to which
about 100 .mu.l containing a known concentration of Bacillus
subtilis is added. The standard 20 undergoes the identical
procedure as the unknown sample 10. Green luminescence in region 22
and a change in color in regions 24 indicates that the assay has
worked properly and the ratio of luminescence intensity from the
sample 10 in region 26 and standard 20 in region 22 is proportional
to the concentration of the bacterial spore of interest. The
microwaving step can be completed in less than 2 minutes. Thus the
complete assay can be performed within 7 to 10 minutes. The sample
10 and standard 20 may be processed simultaneously or sequentially
as may be desired.
[0053] Live/Dead Assay for Bacterial Spores
[0054] The invention also includes a method and apparatus to
measure the fraction of bacterial spores that remain viable or
alive, hence a live/dead assay for bacterial spores. The method
combines dipicolinic acid triggered terbium luminescence and
dipicolinic acid release from (1) viable bacterial spore through
germination, and (2) all viable and nonviable bacterial spores by
autoclaving, sonication, or microwaving. The ratio of the results
from steps (1) and (2) yield the fraction of bacterial spores that
are alive.
[0055] The invention does not suffer from the aforementioned prior
art problems of colony or microscopic counting, because it is based
on a molecular approach that (1) works whether or not a bacterial
spore is attached on a particulate, (2) does not require bacteria
to be cultivable, and (3) can be performed on the timescale of 20
minutes.
[0056] The solution for developing a live/dead assay for bacterial
spores requires a molecular approach. DPA can be released into bulk
solution by inducing germination with L-alanine or by autoclaving
the sample. In germination, only viable spores release DPA, while
autoclaving forces all spores, viable and nonviable, to release
DPA. Microwaving and sonication also releases DPA from all spores,
whether dead or alive. Again, when the released DPA binds terbium
ions in bulk solution, bright green luminescence is triggered under
UV excitation.
[0057] The luminescence intensity can be correlated to the
concentration of viable bacterial spores when germination is used
to release the DPA, and to the total bacterial spore concentration
when either autoclaving, sonication, or microwaving is used to
release DPA. Thus, these methods of DPA release allow us to
quantify both the viable and total bacterial spore count, and
subsequently the fraction of spores that are viable for a given
sample.
[0058] Since germination releases the DPA content of viable
bacterial spores, while autoclaving, sonication, and microwaving
releases the DPA content of all bacterial spores, including
non-viable bacterial spores, using the DPA triggered Tb
luminescence method in conjunction with the DPA release, induced by
(1) germination and (2) either autoclaving, sonication, and
microwaving, allows us to determine the viable and total spore
count, respectively, and subsequently the fraction of viable
bacterial spores as illustrated in FIGS. 4a, 4b and 4c. FIG. 4a
shows the time course data of endospore germination monitored by
DPA triggered Tb luminescence at 543 nm. Time zero corresponds to
L-alanine induced germination. FIG. 4b shows the spectrum of the
luminescence corresponding to Tb-DPA complex, which is induced
after completion of germination. FIG. 4c compares the spectrum for
an autoclaved sample verses a control sample which is not
autoclaved.
[0059] Lifetime-Gated Measurements of Bacterial Spores and Imaging
Bacterial Spores Using an Active Pixel Sensor
[0060] Finally, the method of the invention is amenable to
lifetime-gated measurements to eliminate any fluorescence
background from organic chromophores. It is also possible to
quantify the fraction of bacterial spores that remain viable by
inducing DPA release by germination and microwaving as described
below, and to obtain further increased sensitivity by preparing
special Tb complexes that enhance the luminescence turn-on, and DPA
binding affinity.
[0061] Consider now the problem of imaging bacterial spores. The
imaging methodology is again based on a combination of dipicolinic
acid triggered terbium luminescence (Tb luminescence assay) and
imaging using an active pixel sensor (APS), which is well known to
the art. The Tb luminescence assay enables specific detection of
bacterial spores with a current detection limit of 5,000 spores/ml
when TbCl.sub.3 is used as the analysis reagent. This assay can be
performed in 30 minutes or less depending on the DPA release
mechanism that is employed. APS is ideally suited to image the
resultant Tb luminescence when spores are present because of its
inherent ability to perform lifetime gated imaging.
[0062] In this embodiment the spores or their contents have been
labeled with a long-lifetime lumophore which fact is used to
advantage during detection. Since almost every natural fluorescent
material decays in a few nanoseconds, delayed luminescence is a
powerful discriminator against background biological or
mineralogical signals. For example, flavinoids, NADH, collagen and
many other biological and cellular components fluoresce in the
wavelength region of 300-500 nm, but all have lifetimes less than a
few tens of nanoseconds.
[0063] Jet Propulsion Laboratory has developed a true snapshot
imager, using CMOS technology in an APS that is ideally suited for
imaging and measurement of delayed luminescence probes. In this
implementation, the entire imager can be cycled off and on in a
clock cycle, typically less than a microsecond. The basic
measurement cycle is to pulse an excitation source for the
luminescence with an on time of a few nanoseconds, wait 30 .mu.s
and than turn on the imager for 2 ms, turn it off and read out the
image and the photon counts for each pixel. A unique feature of the
CMOS or Active Pixel Sensor (APS) technology is that each pixel can
contain active circuit elements and can perform signal averaging to
improve the signal to noise as well as other processing. By imaging
the collection tape, we can count the pixels that contain
luminescence signal and get a spore count.
[0064] FIG. 5a shows a diagrammatic timing sequence for excitation,
a delay .phi., and detector integration time .DELTA.. Image data
taken with the APS for an Europium probe with a lifetime of
.about.8O0 .mu.s is shown in FIG. 5b in which we applied a few
spots of the Europium probe to an APS 256.times.256 imager and
excited the fluorescence with a pulsed N.sub.2 laser at 337 nm and
a pulse width of .about.4 ns. Excitation can be performed with a
compact laser, laser diode or LED. By adjusting the timing of the
detection window, delay .phi., the decay curve of the fluorophore
can be mapped out as shown in FIG. 6, which is a graph of the
lifetime data obtained from the images of FIG. 5b. The fluorescence
signal in FIG. 6 is summed up from all the pixels on the upper spot
of the APS sensor as shown in FIG. 5b.
[0065] Unattended Monitoring of Bacterial Spores in the Air
[0066] Consider now the technology that is required to enable one
to achieve unattended monitoring of bacterial spores in the air.
The novelty of the method lies again in the combination of (1)
aerosol capture methods and (2) lanthanide luminescence detection
of bacterial spores This combination will enable an alarm for
airborne bacterial spores similar in concept to a smoke detector,
which works continuously and unattended.
[0067] The invention as described below does not suffer from the
above mentioned problems of the prior art, because it (1) does not
require cultivable bacteria, and (2) can be performed continuously
with a sampling rate of at least four readings per hour using
current instrumentation, and (3) does not require active sampling
by a trained technician.
[0068] Online monitoring of aerosolized bacterial spores, such as
Bacillus anthracis and Clostridium botulism spores, is essential in
locations such as public transportation, mail sorting, food
preparation, health care facilities and even military environments.
We have become especially motivated to develop a method of
unattended monitoring of bacterial spores in the air after the
anthrax attacks following the Sep. 11, 2002 terrorist attacks.
Another motivation was the application of the method towards
planetary protection, which is primarily concerned with spacecraft
sterilization.
[0069] A solution for unattended monitoring of airborne bacterial
spores is achieved by the combination of (1) aerosol capture
methods and (2) lanthanide luminescence detection of the bacterial
spores as described above. The luminescence intensity arising from
DPA detection can be correlated to the concentration of bacterial
spores. When this detection method is coupled to an aerosol capture
device that suspends aerosolized spores into a terbium containing
solution, unattended monitoring of bacterial spores in the air is
enabled.
[0070] In general, the method comprises the steps of capturing
aerosolized bacterial spores with an aerosol sampler or impactor of
which there are many commercial models are available. The captured
spores are then lysed using microwave radiation, autoclaving, or
other methods that release DPA from the core of the spores. The
released DPA then binds terbium ions or other chromophores that
give rise to luminescence turn-on upon DPA binding. The
luminescence turn-on is monitored by a luminescence spectrometer or
fluorimeter. Continuous sampling of the air while monitoring for
luminescence turn-on gives rise to an alarm capability for
aerosolized bacterial spores, which does not require human
participation over extended periods, such as time periods of the
order of 8 hours.
[0071] In the illustrated embodiment stock solutions of purified
Bacillis subtilis spores were purchased from Raven Biological. A
Lovelace nebulizer was used to generate an aerosol 40 of the
bacterial spore air suspensions. The spore "smoke" detector
instrument as shown in the diagram of FIG. 7, is comprised of three
components: (1) a biosampler 42 for aerosol capture, (2) a
microwave with temperature and pressure control 44 for releasing
the DPA from the spores, and (3) a lifetime-gated luminescence
spectrometer 50 for luminescence detection. The lifetime gating
works by exciting the sample with a short Xe-lamp flash 51 and
waiting several microseconds before detecting light from the sample
46, thus eliminating the background fluorescence from impurities
with 10-ns fluorescent lifetimes.
[0072] The biosampler 42, filled with 20 ml of 10 .mu.M TbCl.sub.3
glycerol solution, has a 95% transfer efficiency for
microbe-containing aerosols. Once bacterial spores are suspended in
the biosampler collection vessel 47, microwaving completely or
sufficiently releases DPA into bulk solution 46 within 8 minutes or
less. The resulting free DPA then binds Tb in bulk solution, giving
rise to luminescence turn-on under UV excitation. A fiber optic
probe 48 immersed in the sample solution transmits the Luminescence
to the spectrometer 50. Spectrometer 50 is coupled to alarm circuit
52 which then generates an appropriate alarm signal when a
predetermined detection occurs, namely a wireless or wired signal
with identification information is generated and transmitted to a
remote monitoring station. The monitoring station may monitor a
plurality of remote biosensors such as shown in FIG. 7 and
providing a continuous time, date, place and biomeasurement report
from them.
[0073] While the biosampler 42 is continually sampling the air, a
cycle comprising an 8-minute microwaving step at 140.degree. C. at
1 atmosphere, a 7 minute cooling period, and a 30 second
luminescence measurement is performed repeatedly. Cooling down to
room temperature is required because the binding constant for the
Tb-DPA complex at 140.degree. C. is much lower than at room
temperature, thus leading to near zero fraction bound at
140.degree. C. FIG. 8a shows the time course of the luminescence
intensity at 543.5 nm versus time for the online monitoring for
aerosolized bacterial spores in the device of FIG. 7. After five
data points are collected in the time interval between t=0 and 63
minutes, we initiated the nebulizer for 5 minutes to generate
aerosolized bacterial spores, which were directed to the inlet of
the biosampler 42. The sixth data point at t=81 min. clearly shows
the presence of Tb-DPA luminescence, thus signaling the presence of
bacterial spores. The luminescence intensity in the plateau region
after 130 minutes corresponds to a spore concentration of 10.sup.5
spores/ml. The luminescence increases for two more heating and
cooling cycles and then plateaus 60 minutes after the initiation of
the spore event.
[0074] FIG. 8b shows the luminescence spectra before and after the
generation of aerosolized bacterial spores. Clearly, the
signal-to-noise ratio of 10, one cycle after spore introduction,
shows that we can detect aerosolized spores with a response time of
about 15 minutes. Spore lysing methods, such as sonication with
microbeads, that do not require high temperature will lead to
increased sampling rates.
[0075] Thus, we have demonstrated quantification of aerosolized
bacterial spores with a response time of about 15 minutes or less,
a sensitivity of 10.sup.5 spores/ml, and a dynamic range of four
orders of magnitude. The sensitivity can be improved by optimizing
aerosol collection and spectrometer performance. Ultimately, the
most attractive feature we have demonstrated is the unattended
monitoring of aerosolized bacterial spores for the duration of a
workday (i.e.--8 hrs).
[0076] Many alterations and modifications may be made by those
having ordinary skill in the art without departing from the spirit
and scope of the invention. Therefore, it must be understood that
the illustrated embodiment has been set forth only for the purposes
of example and that it should not be taken as limiting the
invention as defined by the following claims. For example,
notwithstanding the fact that the elements of a claim are set forth
below in a certain combination, it must be expressly understood
that the invention includes other combinations of fewer, more or
different elements, which are disclosed in above even when not
initially claimed in such combinations.
[0077] The words used in this specification to describe the
invention and its various embodiments are to be understood not only
in the sense of their commonly defined meanings, but to include by
special definition in this specification structure, material or
acts beyond the scope of the commonly defined meanings. Thus if an
element can be understood in the context of this specification as
including more than one meaning, then its use in a claim must be
understood as being generic to all possible meanings supported by
the specification and by the word itself.
[0078] The definitions of the words or elements of the following
claims are, therefore, defined in this specification to include not
only the combination of elements which are literally set forth, but
all equivalent structure, material or acts for performing
substantially the same function in substantially the same way to
obtain substantially the same result. In this sense it is therefore
contemplated that an equivalent substitution of two or more
elements may be made for any one of the elements in the claims
below or that a single element may be substituted for two or more
elements in a claim. Although elements may be described above as
acting in certain combinations and even initially claimed as such,
it is to be expressly understood that one or more elements from a
claimed combination can in some cases be excised from the
combination and that the claimed combination may be directed to a
subcombination or variation of a subcombination.
[0079] Insubstantial changes from the claimed subject matter as
viewed by a person with ordinary skill in the art, now known or
later devised, are expressly contemplated as being equivalently
within the scope of the claims. Therefore, obvious substitutions
now or later known to one with ordinary skill in the art are
defined to be within the scope of the defined elements.
[0080] The claims are thus to be understood to include what is
specifically illustrated and described above, what is
conceptionally equivalent, what can be obviously substituted and
also what essentially incorporates the essential idea of the
invention.
* * * * *