U.S. patent application number 11/000491 was filed with the patent office on 2006-03-16 for system for autonomous monitoring of bioagents.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Steve B. Brown, Billy W. JR. Colston, John M. Dzenitis, Dora M. Gutierrez, Bruce D. Henderer, Benjamin J. Hindson, Ramakrishna S. Madabhushi, Anthony J. Makarewicz, Don A. Masquelier, Mary T. McBride, Thomas R. Metz, Shanavaz L. Nasarabadi, Ujwal S. Setlur, Sally M. Smith, Kodumudi S. Venkateswaran.
Application Number | 20060057599 11/000491 |
Document ID | / |
Family ID | 31891539 |
Filed Date | 2006-03-16 |
United States Patent
Application |
20060057599 |
Kind Code |
A1 |
Dzenitis; John M. ; et
al. |
March 16, 2006 |
System for autonomous monitoring of bioagents
Abstract
An autonomous monitoring apparatus for monitoring air, water,
soil, or other substance for bioagents. A collector gathers a
quantity of the air, water, soil, or other substance being
monitored. A sample preparation system prepares a sample of the
selected potential bioagent particles. The sample is analyzed by a
system for detecting said bioagents.
Inventors: |
Dzenitis; John M.;
(Danville, CA) ; Brown; Steve B.; (Livermore,
CA) ; Colston; Billy W. JR.; (San Ramon, CA) ;
Gutierrez; Dora M.; (Livermore, CA) ; Henderer; Bruce
D.; (Livermore, CA) ; Hindson; Benjamin J.;
(Belmont, AU) ; Madabhushi; Ramakrishna S.;
(Fremont, CA) ; Makarewicz; Anthony J.;
(Livermore, CA) ; Masquelier; Don A.; (Tracy,
CA) ; McBride; Mary T.; (Brentwood, CA) ;
Metz; Thomas R.; (Tracy, CA) ; Nasarabadi; Shanavaz
L.; (Livermore, CA) ; Setlur; Ujwal S.;
(Livermore, CA) ; Smith; Sally M.; (Salida,
CA) ; Venkateswaran; Kodumudi S.; (Livermore,
CA) |
Correspondence
Address: |
Eddie E. Scott;Assistant Laboratory Counsel
Lawrence Livermore National Laboratory
P.O. Box 808, L-703
Livermore
CA
94551
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
31891539 |
Appl. No.: |
11/000491 |
Filed: |
November 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10643797 |
Aug 19, 2003 |
|
|
|
11000491 |
Nov 29, 2004 |
|
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60406159 |
Aug 26, 2002 |
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Current U.S.
Class: |
435/6.18 ;
435/287.2; 435/288.5; 435/30; 435/6.1 |
Current CPC
Class: |
G01N 2001/2217 20130101;
G01N 15/1459 20130101; G01N 1/2202 20130101; Y02A 90/26 20180101;
G01N 35/08 20130101; Y02A 90/10 20180101; G01N 1/2211 20130101 |
Class at
Publication: |
435/006 ;
435/030; 435/287.2; 435/288.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Goverment Interests
[0002] The United States Government has rights in this invention
pursuant to Contract No. W-7405-ENG-48 between the United States
Department of Energy and the University of California for the
operation of Lawrence Livermore National Laboratory.
Claims
1. An autonomous monitoring apparatus for monitoring air, water,
soil, or other substance for bioagents that could be in potential
bioagent particles, comprising: collector means for gathering said
air, water, soil, or other substance being monitored, said
collector separating the potential bioagent particles from said
air, water, soil, or other substance; sample preparation means for
preparing a sample of the potential bioagent particles, said sample
preparation means operatively connected to said collector means for
preparing said sample from said air, water, soil, or other
substance gathered by said collector; detector means for detecting
said bioagents in said sample, said detector means comprising a
liquid-array based multiplex immunoassay detector and a multiplex
PCR detector.
2. The apparatus of claim 1 wherein said sample preparation means
is a means for providing an immunoassay sample.
3. The apparatus of claim 2 wherein said means for providing an
immunoassay sample is a multiplex immunoassay detector that
utilizes optically encoded microbeads.
4. The apparatus of claim 1 wherein said sample preparation means
is a means for providing a nucleic acid assay sample.
5. The apparatus of claim 1 wherein said multiplex PCR detector is
a real time PCR detector.
6. The apparatus of claim 1 wherein said multiplex PCR detector
includes means for performing PCR amplification.
7. An autonomous monitoring apparatus for monitoring air, water,
soil, or other substance for bioagents, the bioagents potentially
being in potential bioagent particles within the air, water, soil,
or other substance, comprising: a collector for gathering said air,
water, soil, or other substance being monitored, said collector
separating selected potential bioagent particles from said air,
water, soil, or other substance; a wash assay sample preparation
system for preparing a sample of said selected potential bioagent
particles, said wash assay sample preparation system operatively
connected to said collector for preparing said sample from said
air, water, soil, or other substance gathered by said collector;
said wash assay sample preparation system including
optically-encoded beads, a number of reagents, and a washing
buffer; and a detector for detecting the bioagents in said sample,
said detector operatively connected to said sample preparation
system.
8. The apparatus of claim 7 wherein said reagents include detector
antibodies.
9. The apparatus of claim 7 wherein said reagents include
fluorescent reporters.
10. The apparatus of claim 7 wherein said wash assay sample
preparation system includes a filter.
11. The apparatus of claim 7 wherein said wash assay sample
preparation system includes a filter and optically-encoded
beads.
12. An autonomous monitoring apparatus for monitoring air, water,
soil, or other substance for bioagents, the bioagents potentially
being in potential bioagent particles within the air, water, soil,
or other substance, comprising: a collector for gathering said air,
water, soil, or other substance being monitored, said collector
separating selected potential bioagent particles from said air,
water, soil, or other substance; a no-wash assay sample preparation
system for preparing a sample of said selected potential bioagent
particles, said no-wash assay sample preparation system operatively
connected to said collector for preparing said sample from said
air, water, soil, or other substance gathered by said collector;
said no-wash assay sample preparation system including
optically-encoded beads and a number of reagents; and a detector
for detecting the bioagents in said sample, said detector
operatively connected to said sample preparation system.
13. An apparatus for sampling air and collecting sample particles
of a predetermined particle size range from said air for monitoring
air, water, soil, or other substance for bioagents, the bioagents
potentially being in potential bioagent particles within the air,
water, soil, or other substance, comprising: a low pass section
having an opening for gathering said air, a pre-screen unit
positioned in said opening that prevents large particles from
blocking said opening, an impactor section operatively connected to
said low pass section, said impactor section receiving said air and
separating said air into a bypass air flow that does not contain
said sample particles and a product air flow that contains said
sample particles, and a sample preparation system for preparing a
sample of the potential bioagent particles, said sample preparation
system operatively connected to said impactor section for preparing
said sample; and a detector for detecting the bioagents in said
sample, said detector operatively connected to said sample
preparation system.
14. The apparatus of claim 13 wherein said opening in said low pass
section size is a slot and said pre-screen unit is a grill
positioned in said opening.
15. The apparatus of claim 1 wherein said opening in said low pass
section size is a slot and said pre-screen unit is a screen
positioned in said opening.
16. The apparatus of claim 13 wherein said opening in said low pass
section size is an annular slot and said pre-screen unit is an
annular screen positioned in said opening.
17. A method of monitoring air, water, soil, or other substance for
bioagents, said air, water, soil, or other substance containing
potential bioagent particles of various sizes, comprising the steps
of: gathering said air, water, soil, or other substance containing
potential bioagent particles of various sizes; separating said
potential bioagent particles by size and collecting said potential
bioagent particles of a size range that are likely to contain said
bioagents; and detecting said bioagents in said potential bioagent
particles of a size range that are likely to contain said
bioagents, said step of detecting said bioagents comprising mixing
optically encoded microbeads with said potential bioagent particles
and detecting said bioagents with said optically encoded
microbeads.
18. The method of monitoring air, water, soil, or other substance
for bioagents of claim 17 wherein said step of detecting said
bioagents comprises exposing said microbeads to said air, water,
soil, or other substance; depositing said microbeads on a filter;
exposing said microbeads to a washing buffer, and releasing said
microbeads to a detector.
19. The method of monitoring air, water, soil, or other substance
for bioagents of claim 17 wherein said washing buffer comprises
buffer solutions.
20. The method of monitoring air, water, soil, or other substance
for bioagents of claim 18 wherein steps following releasing said
microbeads to a detector includes using cleaning reagents for
reconditioning said filter.
21. The method of monitoring air, water, soil, or other substance
for bioagents of claim 20 wherein said cleaning reagents include
bleach.
22. The method of monitoring air, water, soil, or other substance
for bioagents of claim 20 wherein said cleaning reagents include
morpholine propane sulfonic acid (MOPS) citrate buffer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of U.S. patent
application Ser. No. 10/643797 filed Aug. 19, 2003 and titled,
"System for Autonomous Monitoring of Bioagents" which claims the
benefit of U.S. Provisional Patent Application No. 60/406159 filed
Aug. 26, 2002 titled "System for Autonomous Monitoring of
Bioagents." U.S. patent application Ser. No. 10/643797 filed Aug.
19, 2003 and U.S. Provisional Patent Application No. 60/406159
filed Aug. 26, 2002, both titled, "System for Autonomous Monitoring
of Bioagents," are incorporated herein by this reference.
BACKGROUND
[0003] 1. Field of Endeavor
[0004] The present invention relates to bioagents and more
particularly to monitoring bioagents.
[0005] 2. State of Technology
[0006] There exists a critical need to develop distributed
biothreat agent sensor networks that can operate in civilian
applications. To operate in "Detect to Protect/Warn" type detection
architectures, these platforms need to have several key properties.
They need to be capable of detecting pathogens within a 1-2 hour
time window, allowing for enough time to respond to an event. They
need to be extremely low cost to maintain, since continuous
monitoring is essential for many applications. These platforms need
to have sufficient sensitivity to cover a broad geographical area
(limiting the necessary number of sensors) and have sufficient
selectivity to virtually eliminate false positives. Currently
available bioweapons detection systems are designed primarily for
military use on the battlefield. These systems are often expensive
to deploy and ultimately unsuited for civilian protection.
[0007] In an article titled, "U.S. Is Deploying a Monitor System
for Germ Attacks," by Judith Miller in The New York Times on Jan.
22, 2003, it was reported, "To help protect against the threat of
bioterrorism, the Bush administration on Wednesday will start
deploying a national system of environmental monitors that is
intended to tell within 24 hours whether anthrax, smallpox and
other deadly germs have been released into the air, senior
administration officials said today. The system uses advanced data
analysis that officials said had been quietly adapted since the
September 11 attacks and tested over the past nine months. It will
adapt many of the Environmental Protection Agency's 3,000 air
quality monitoring stations throughout the country to register
unusual quantities of a wide range of pathogens that cause diseases
that incapacitate and kill . . . . The new environmental
surveillance system uses monitoring technology and methods
developed in part by the Department of Energy's national
laboratories. Samples of DNA are analyzed using polymerase chain
reaction techniques, which examine the genetic signatures of the
organisms in a sample, and make rapid and accurate evaluations of
that organism . . . . Officials who helped develop the system said
that tests performed at Dugway Proving Ground in Utah and national
laboratories showed that the system would almost certainly detect
the deliberate release of several of the most dangerous pathogens.
`Obviously, the larger the release, the greater the probability
that the agent will be detected,` an official said. `But given the
coverage provided by the E.P.A. system, even a small release,
depending on which way the wind was blowing and other
meteorological conditions, is likely to be picked up.`"
[0008] In an article titled, "Biodetectors Evolving, Monitoring
U.S. Cities," by Sally Cole in the May 2003 issue of Homeland
Security Solutions, it was reported, "The anthrax letter attacks of
2001, and subsequent deaths of five people, brought home the
reality of bioterrorism to Americans and provided a wake-up call
for the U.S. government about the need for a method to detect and
mitigate the impact of any such future attacks. Long before the
anthrax letter attacks, scientists at two of the U.S. Department of
Energy's national laboratories, Lawrence Livermore National
Laboratory (LLNL) and Los Alamos National Laboratory (LANL), were
busy pioneering a "biodetector" akin to a smoke detector to rapidly
detect the criminal use of biological agents. This technology is
now expected to play a large role in the U.S. government's recently
unveiled homeland security counter-terrorism initiative, Bio-Watch,
which is designed to detect airborne bioterrorist attacks on major
U.S. cities within hours. Announced back in January, Bio-Watch is a
multi-faceted, multi-agency program that involves the U.S.
Department of Energy, the Environmental Protection Agency (EPA),
and the U.S. Department of Health and Human Services' Centers for
Disease Control and Prevention (CDC). Many of the EPA's 3,000
air-quality monitoring stations throughout the country are being
adapted with biodetectors to register unusual quantities of a wide
range of pathogens that cause diseases that incapacitate and kill,
according to the EPA. The nationwide network of environmental
monitors and biodetectors, which reportedly will eventually monitor
more than 120 U.S. cities, is expected to detect and report a
biological attack within 24 hours. Citing security reasons, the EPA
declined to disclose further details about the program at this time
. . . . The Autonomous Pathogen Detection System (APDS) is a
file-cabinet-sized machine that sucks in air, runs tests, and
reports the results itself. APDS integrates a flow cytometer and
real-time PCR detector with sample collection, sample preparation,
and fluidics to provide a compact, autonomously operating
instrument capable of simultaneously detecting multiple pathogens
and/or toxins. The system is designed for fixed locations, says
Langlois, where it continuously monitors air samples and
automatically reports the presence of specific biological agents.
APDS is targeted for domestic applications in which the public is
at high risk of exposure to covert releases of bioagents--subway
systems, transportation terminals, large office complexes, and
convention centers . . . . APDS provides the ability to measure up
to 100 different agents and controls in a single sample,` Langlois
says. `It's being used in public buildings right now.` The latest
evolution of the biodetector, APDS-II, uses bead-capture
immunoassays and a compact flow cytometer for the simultaneous
identification of multiple biological simulants. Laboratory tests
have demonstrated the fully autonomous operation of APDS-II for as
long as 24 hours."
SUMMARY
[0009] Features and advantages of the present invention will become
apparent from the following description. Applicants are providing
this description, which includes drawings and examples of specific
embodiments, to give a broad representation of the invention.
Various changes and modifications within the spirit and scope of
the invention will become apparent to those skilled in the art from
this description and by practice of the invention. The scope of the
invention is not intended to be limited to the particular forms
disclosed and the invention covers all modifications, equivalents,
and alternatives falling within the spirit and scope of the
invention as defined by the claims. (0008) The present invention
provides an autonomous monitoring apparatus for monitoring air,
water, soil, or other substance for bioagents. A collector gathers
a quantity of the air, water, soil, or other substance being
monitored. The collector separates selected potential bioagent
particles from the air, water, soil, or other substance that is
being collected. A sample preparation system prepares a sample of
the selected potential bioagent particles. The sample is analyzed
by a system for detecting said bioagents.
[0010] One embodiment includes a wash assay sample preparation
system for preparing a sample of the selected potential bioagent
particles. The wash assay sample preparation system is operatively
connected to the collector and prepares the sample from the air,
water, soil, or other substance gathered by the collector. The wash
assay sample preparation system includes optically-encoded beads, a
number of reagents, a washing buffer, and a detector for detecting
any bioagents in the sample.
[0011] Another embodiment includes a no-wash assay sample
preparation system for preparing a sample of the selected potential
bioagent particles. The no-wash assay sample preparation system is
operatively connected to the collector for preparing the sample
from the air, water, soil, or other substance gathered by the
collector. The no-wash assay sample preparation system includes
optically-encoded beads, a number of reagents, and a detector for
detecting any bioagents in the sample.
[0012] Another embodiment of the present invention provides an
apparatus for sampling air and collecting sample particles of a
predetermined particle size range from the air for autonomous
monitoring air, water, soil, or other substance for bioagents. A
low pass section has an opening for gathering said air. A prescreen
unit is positioned in the opening that prevents large particles
from blocking the opening.
[0013] The invention is susceptible to modifications and
alternative forms. Specific embodiments are shown by way of
example. It is to be understood that the invention is not limited
to the particular forms disclosed. The invention covers all
modifications, equivalents, and alternatives falling within the
spirit and scope of the invention as defined by the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The accompanying drawings, which are incorporated into and
constitute a part of the specification, illustrate specific
embodiments of the invention and, together with the general
description of the invention given above, and the detailed
description of the specific embodiments, serve to explain the
principles of the invention.
[0015] FIG. 1 is a block diagram illustrating an embodiment of an
autonomous pathogen detection system constructed in accordance with
the present invention.
[0016] FIG. 2 is a block diagram illustrating another embodiment of
an autonomous pathogen detection system constructed in accordance
with the present invention.
[0017] FIG. 3 is a block diagram illustrating a specific embodiment
of the invention designated as an AUTONOMOUS PATHOGEN DETECTION
SYSTEM (APDS).
[0018] FIGS. 4A and 4B are illustrations that show the aerosol
collection system.
[0019] FIGS. 5A and 5B are illustrations that show the cap section
limiting the larger particulate size range entering the
collector.
[0020] FIG. 6 is an illustration that shows the virtual impactor
section.
[0021] FIG. 7 shows the multistage, wetted-wall cyclone collector
section.
[0022] FIGS. 8A, 8B, and 8C show details of a specific embodiment
of the aerosol collection system.
[0023] FIG. 9 is an illustration that shows another embodiment of
the aerosol collection system.
[0024] FIG. 10 illustrates a system for sample preparation and
detection.
[0025] FIGS. 11, 12, and 13 illustrate the liquid-array based
multiplex immunoassay detection system.
[0026] FIG. 14 is a block diagram illustrating the multiplex
amplification and detection system.
[0027] FIG. 15 illustrates one specific embodiment of the in-line
nucleic acid amplification and detection system.
[0028] FIG. 16 is a block diagram illustrating another embodiment
of an autonomous pathogen detection system constructed in
accordance with the present invention.
[0029] FIG. 17 is a block diagram illustrating another embodiment
of an autonomous pathogen detection system constructed in
accordance with the present invention.
[0030] FIG. 18 illustrates another embodiment of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0031] Referring now to the drawings, to the following detailed
description, and to incorporated materials, detailed information
about the present invention is provided including the description
of specific embodiments. The detailed description and the specific
embodiments serve to explain the principles of the invention. The
invention is susceptible to modifications and alternative forms.
The invention is not limited to the particular forms disclosed. The
invention covers all modifications, equivalents, and alternatives
falling within the spirit and scope of the invention as defined by
the claims.
[0032] Terrorists sending anthrax-contaminated packages. Militant
organizations obtaining potassium cyanide. Religious cult members
poisoning local residents to fix an election. Sadly, these
scenarios are not the plots of the three latest bestsellers, but
rather, very real incidents with a very real danger. By the
mid-1990s, the U.S. Congress began to assess the vulnerability of
the U.S. civilian population to biological terrorism and found us
considerably lacking in our ability to cope with even a small-scale
biological event. Initial thinking was that Department of Defense
technology could be readily transferred to the civilian arena.
However, upon further reflection, it was concluded that although
there was overlap between military and civilian defense needs, in
the case of a biological threat, there are marked differences: (1)
the soldier is trained and equipped with protective gear so he may
respond to a threat quickly enough to prevent a lethal dose; (2)
military intelligence usually reduces the potential threat to a
relatively small number of biological agents; and, (3) military
battlefield tactics are designed to minimize the density of
soldiers. The civilian population, however, is neither trained nor
equipped, is vulnerable to any conceivable pathogen and often
gathers in large crowds (special events, sporting venues, etc.)
where a small release could potentially infect thousands. In
response to these differences, federal agencies, including
Department of Energy, have recently begun funding directed research
efforts to reduce civilian biological terrorist
vulnerabilities.
[0033] At present there are more than 30 pathogens and toxins on
various agency threat lists. Public health personnel rarely see
most, of the pathogens so they have difficulty identifying them
quickly. In addition, many pathogenic infections aren't immediately
symptomatic, with delays as long as several days, limiting options
to control the disease and treat the patients. The lack of a
practical monitoring network capable of rapidly detecting and
identifying multiple pathogens or toxins on current threat lists
translates into a major deficiency in the United States ability to
counter biological terrorism.
[0034] Referring now to FIG. 1, an embodiment of an autonomous
pathogen detection system constructed in accordance with the
present invention is illustrated by a block diagram. The autonomous
pathogen detection system is designated generally by the reference
numeral 100. The autonomous pathogen detection system 100 provides
collection 101, sample preparation 103, and detection 105. The
collection 101 includes gathering air, water, soil or other
substance to provide an air sample, water sample, soil sample or a
sample of other substances.
[0035] After the collection 101, the sample is transferred as shown
by arrow 102 for sample preparation 103. The sample preparation 103
provides an automated sample, an immunoassay sample, and/or a
nucleic acid assay sample. In sample preparation 103 the sample may
be concentrated, purified, lysed, pulverized or otherwise made to
have smaller particulates, mixed, and/or amplified.
[0036] After sample preparation 103, the sample is transferred as
shown by arrow 104 for detection. In one embodiment of the
autonomous pathogen detection system 100, the detection is by a
multiplex immunoassay detector. In another embodiment of the
autonomous pathogen detection system 100, the detection is by a
multiplex PCR detector. The detection may also be performed in the
same location as the sample preparation, in which case the transfer
arrow 104 is not required.
[0037] The autonomous pathogen detection system 100 provides an
apparatus and method for monitoring air, water, soil, or other
substance for particles containing bioagents. The autonomous
pathogen detection system 100 comprises a collector for gathering
the air, water, soil, or other substance being monitored; sample
preparation system for preparing a sample from the air, water,
soil, or other substance gathered by the collector; and a detector
for detecting any bioagents in the sample. In one embodiment the
collector is an aerosol collector. In other embodiments the
collector gathers water, soil, or other substances. The collector
in one embodiment includes separator system for separating the
particles of interest from other particles. The particles of
interest are of a predetermined size range.
[0038] In one embodiment the collector is an aerosol collector that
collects air and includes a system for separating the air into a
bypass air flow that does not contain the particles of a
predetermined particle size range and a product air flow that does
contain the sample particles of a predetermined particle size
range. A wetted-wall cyclone collector receives the product air
flow and traps and concentrates the particles of a predetermined
particle size range in a liquid.
[0039] In one embodiment the sample preparation system is
automated. In one embodiment the sample preparation system provides
an immunoassay sample. In another embodiment the sample preparation
system provides a nucleic acid assay sample. In another embodiment
the sample preparation system includes concentration of the air,
water, soil, or other substance. In another embodiment the sample
preparation system includes purification of the air, water, soil,
or other substance. In another embodiment the sample preparation
system includes lysis of the air, water, soil, or other substance.
In another embodiment the sample preparation system includes mixing
of the air, water, soil, or other substance. In another embodiment
the sample preparation system includes amplification.
[0040] In one embodiment of the autonomous pathogen detection
system 100, the detector is a multiplex immunoassay detector. In
one embodiment of the autonomous pathogen detection system 100, the
detector is a multiplex PCR detector.
[0041] The primary focus of the autonomous pathogen detection
system 100 is the protection of civilians from terrorist attacks;
however, the system also has a role in protecting military
personnel from biological warfare attacks. The autonomous pathogen
detection system 100 also has uses in medical facilities and
research and development facilities. The autonomous pathogen
detection system 100 has uses in medical monitoring. There are a
variety of medical applications where monitoring for biological
pathogens would be useful. A good example of this is monitoring in
hospitals and clinics for highly infectious agents such as
tuberculosis or nosocomial diseases that can threaten the well
being of patients and health care professionals. The autonomous
pathogen detection system 100 also has uses in environmental
monitoring, that is, any application that would benefit from
environmental monitoring of biological species. One example is
continuous aerosol monitoring of bacterial and other pathogens that
could affect the health of livestock (such as the recent hoof and
mouth disease outbreak). Another example is continuous aerosol
monitoring of viruses that could affect the health of large
portions of the population (such as the recent SARS outbreak).
[0042] Referring now to FIG. 2, another embodiment of an autonomous
pathogen detection system constructed in accordance with the
present invention is illustrated by a block diagram. This
embodiment of the autonomous pathogen detection system is
designated generally by the reference numeral 200. The autonomous
pathogen detection system 200 provides collection 201, sample
preparation 203, detection 205, and confirmation 207. The
collection 201 includes gathering air, water, soil or other
substance to provide an air sample, water sample, soil sample or a
sample of other substances.
[0043] After the collection 201, the sample is transferred as
illustrated by arrow 202 for sample preparation 203. The sample
preparation 203 provides an automated sample, an immunoassay
sample, and/or a nucleic acid assay sample. In the sample
preparation 203 the sample may be concentrated, purified, lysed,
pulverized or otherwise made to have smaller particulates, mixed,
and/or amplified.
[0044] After sample preparation 203, the sample is transferred as
illustrated by arrow 204 for detection. In one embodiment of the
autonomous pathogen detection system 200, the detection is by a
multiplex immunoassay detector. In another embodiment of the
autonomous pathogen detection system 200, the detection is by a
multiplex PCR detector.
[0045] After sample preparation 203 and detection 205 when a
pathogen has been detected, a sample is transferred from sample
preparation 203 to the confirmation module 207. This is illustrated
by arrow 206 in FIG. 2. In one embodiment, the system for
confirmation of a bioagent in the sample is a multiplex immunoassay
detector. In one embodiment of the autonomous pathogen detection
system 200, the system for confirmation of a bioagent in the sample
is a multiplex PCR detector. In one embodiment of the autonomous
pathogen detection system 200, the system for confirmation of a
bioagent in the sample is a real-time PCR detector. The detection
may also be performed in the same location as the sample
preparation, in which case the transfer arrows 204 and/or 206 are
not required.
[0046] The autonomous pathogen detection system 200 provides an
apparatus and method for monitoring air, water, soil, or other
substance for particles containing bioagents. The autonomous
pathogen detection system 200 comprises a collector for gathering
the air, water, soil, or other substance being monitored; sample
preparation system for preparing a sample from the air, water,
soil, or other substance gathered by the collector; a detector for
detecting a bioagents in the sample; and a system for confirmation
of a bioagent in the sample. In one embodiment the collector is an
aerosol collector. In other embodiments the collector gathers
water, soil, or other substances. The collector in one embodiment
includes separator system for separating the particles of interest
from other particles. The particles of interest are of a
predetermined size range.
[0047] In one embodiment the collector is an aerosol collector that
collects air and includes system for separating the air into a
bypass air flow that does not contain the particles of a
predetermined particle size range and a product air flow that does
contain the sample particles of a predetermined particle size
range. A wetted-wall cyclone collector receives the product air
flow and traps and concentrates the particles of a predetermined
particle size range in a liquid.
[0048] In one embodiment the sample preparation system is
automated. In one embodiment the sample preparation system provides
an immunoassay sample. In another embodiment the sample preparation
system provides a nucleic acid assay sample. In another embodiment
the sample preparation system includes concentration of the air,
water, soil, or other substance. In another embodiment the sample
preparation system includes purification of the air, water, soil,
or other substance. In another embodiment the sample preparation
system includes lysis of the air, water, soil, or other substance.
In another embodiment the sample preparation system includes mixing
of the air, water, soil, or other substance. In another embodiment
the sample preparation system includes amplification of the
sample.
[0049] In one embodiment of the autonomous pathogen detection
system 200, the detector 205 is a multiplex immunoassay detector.
In one embodiment of the autonomous pathogen detection system 200,
the detector 205 is a multiplex PCR detector.
[0050] In one embodiment of the autonomous pathogen detection
system 200, the system 207 for confirmation of a bioagent in the
sample is a multiplex immunoassay detector. In one embodiment of
the autonomous pathogen detection system 200, the system 207 for
confirmation of a bioagent in the sample is a multiplex PCR
detector. In one embodiment of the autonomous pathogen detection
system 200, the system 207 for confirmation of a bioagent in the
sample is a real-time PCR detector.
[0051] Referring now to FIG. 3 through FIG. 12 a specific
embodiment of the invention designated as an AUTONOMOUS PATHOGEN
DETECTION SYSTEM (APDS) is shown. The APDS is designated generally
by the reference numeral 300. The APDS 300 integrates a flow
cytometer and PCR detector with sample collection, sample
preparation, and fluidics to provide a compact, autonomously
operating instrument capable of simultaneously detecting multiple
pathogens and/or toxins. The APDS 300 is designed for locations
where it continuously monitors air samples and automatically
reports the presence of specific biological agents. Plague and
anthrax are two of the pathogens the APDS 300 identifies, along
with a host of others. The APDS 300 includes the potential to
measure up to 100 different agents and controls in a single
sample.
[0052] The APDS 300 provides a stand-alone pathogen detection
system capable of rapid, continuous, low cost environmental
monitoring of multiple airborne biological threat agents. The
system 300 provides a "Detect to Protect/Warn" system with a number
of key properties. The system 300 is capable of detecting pathogens
within a 1-2 hour time window, allowing for enough time to respond
to an event. The system 300 is extremely low cost to maintain,
since continuous monitoring is essential for many applications. The
system 300 has sufficient sensitivity to cover a broad geographical
area (limiting the necessary number of sensors) and has sufficient
selectivity to virtually eliminate false positives.
[0053] Multiplexed assays are used to reduce reagent costs, making
long term monitoring operations possible. An orthogonal detection
section combines antibody-based and nucleic acid-based assays and
reduces false positives to a very low level. Antibody assays allow
the detector to respond to all types of bioagents, including those
without nucleic acids such as protein toxins. Nucleic acid assays
allow much more sensitive detection, reducing the number of sensors
needed to protect a given area. The fully autonomous aerosol
collection and sample preparation capabilities limit maintenance
requirements and makes integration into a central security or
monitoring network possible.
[0054] Referring again to FIG. 3, a block diagram illustrates the
APDS 300. In operation, an aerosol collector system continuously
samples the air and traps particles in a swirling liquid solution.
Particles of a given size distribution are selected by varying the
flow rate across a virtual impactor unit. The in-line sample
preparation system provides all sample preparation steps (i.e.,
mix, wash, incubation, etc.), and performs multiplex detection
using a Luminex flow cytometer.
[0055] In the "detection" sub-system, a collected sample is mixed
with optically encoded microbeads. Each color of microbead contains
a capture assay that is specific for a given bioagent. Fluorescent
labels are added to identify the presence of each agent on the
bound bead. Each optically encoded and fluorescently labeled
microbead is individually read in a flow cytometer, and fluorescent
intensities are then correlated with bioagent concentrations.
[0056] In the "confirmation" sub-system, PCR amplification and
detection of nucleic acids confirms the presence of the bioagent.
An archived sample is mixed with the TaqMan real-time PCR reagent,
and then introduced by a SIA system into a flow-through polymerase
chain reaction (PCR) system. Specific nucleic acid signatures
associated with the targeted bioagent are amplified and detected
using fluorescence generated from nucleic acid replication from the
TaqMan probes.
[0057] In the "Integrated Remote Control and Feedback" sub-system,
a central computer uses a simple serial based LabVIEW control
system to control all instrument functions. A software system
provides data acquisition, real-time data analysis, and result
reporting via a graphical user interface.
[0058] The APDS 300 is integrated into a self-contained "ATM" style
chassis. All fluids and reagents are contained in the instrument.
The ADPS 300 includes the following subsystems: Aerosol Collection
301, In-Line Sample Preparation 302, Detection--Liquid-Array Based
Multiplex Immunoassay Detection and/or Nucleic Assays Detection
303, Confirmation--In-Line Nucleic Acid Amplification and Detection
304, and Integrated Remote Control and Feedback 305. The subsystem
will be described in greater detail.
APDS Aerosol Collection--301
[0059] The first stage of the APADS 300 is "aerosol collection"
that provides collection of airborne particles that could contain
targeted bioagents. Aerosol release of bioagents is considered one
of the possible scenarios of a terrorist organization. One of the
methods of rapidly exposing a large population to a biowarfare
agent is through use of an aerosol (witness the effect of the
recent, relatively small-scale anthrax mailroom releases). The
aerosol collection system 301 continuously samples the air and
traps particles in a swirling liquid solution. Particles of a given
size distribution are selected by varying the flow rate across a
virtual impactor unit.
[0060] The aerosol collection system 301 is a multi-stage aerosol
collector that utilizes a low pass aerosol section and a virtual
impactor preconcentration that delivers the particles of interest
to a wetted wall cyclone collector. The virtual impactor
preferentially captures particles 1-10 micrometer (micron) which is
the size of particles most likely to be captured in the human lung.
In the wetted wall cyclone collector, the particles are collected
in a fluid, making downstream processing much easier. The fans and
inputs to the obtain high collection rates, up to 3000 liters of
air per minute flow through the detection system, allowing many
particles to be collected over a short period. The aerosol
collection system provides improved sensitivity and reduced
collection times. An on-board computer controls air flow rates and
the size range of particles collected.
[0061] As shown by FIGS. 4A and 5A, a very high volume flow of
aerosol particles is drawn into an annular slot 401A formed in a
cap 402A that is designed to preferentially allow the passage of
particles smaller than a pre-set size. The pre-set size can be
selected as desired. A very high volume flow of aerosol particles
(e.g., up to 3313 Lpm) can be drawn into the annular slot 401A
formed in the cap 402A that is designed to preferentially allow the
passage of particles smaller than 10 microns. The accepted
particles continue on into a dichotomous virtual impaction section
403A that preferentially returns the aerosol particles smaller than
1-micron back into the environment. The remaining particles, (1-10
microns) are known as the product flow. The product flow continues
into the next section.
[0062] As best illustrated by FIG. 5A, a high volume flow of
aerosol particles is drawn into the annular slot 401A formed in the
cap 402A. The annular slot 401A is designed to limit the upper or
larger particulate size range as they enter the collector. To
efficiently pass the smaller particulate, the cap 402A is a
"passive" device in that is has no moving parts and uses the fact
that particulate with a finite mass and moving in a flow stream (in
this case air) will not follow the streamlines exactly due to their
inertia. If the curvature of a streamline is sufficiently large and
the mass of the particulate is correspondingly high, the particle
deviates far enough from the streamline to impact with a surface.
The particles are drawn into the annular slot 401A and directed
into the transition section 409A.
[0063] Referring now to FIGS. 4B and 5B, another embodiment of an
aerosol collection system is illustrated. This embodiment is
designated generally by the reverence numeral 300B. In the
embodiment 300B, a very high volume flow of aerosol particles 405B
is drawn into an annular slot 401B formed in a cap 402B that is
designed to only allow the passage of particles smaller than a
pre-set size. The pre-set size can be selected as desired. A very
high volume flow of aerosol particles 405B can be drawn into the
annular slot 401B formed in the cap 402B that is designed to only
allow the passage of particles smaller than 10 microns. A
pre-screen 404B prevents large particles from blocking small flow
paths such as the flow path 401B. The accepted particles continue
on into a dichotomous virtual impaction section 403B that returns
all the aerosol particles smaller than 1-micron back into the
environment. The remaining particles, (1-10 microns) are known as
the product flow. The product flow continues into the next section.
(0047) The pre-screen unit 404B in one embodiment is a grill
positioned in the opening 401B. The pre-screen unit in another
embodiment is a screen positioned in the opening 401B. In another
embodiment, the opening 401B in the low pass section is an annular
slot and the pre-screen unit 404B is an annular screen positioned
in the opening 401B. In another embodiment, the pre-screen 404B is
located inside the cap 402B covering the top of the inner cylinder
shown as 409B in FIGS. 5A and 5B.
[0064] As best illustrated by FIG. 5, in the embodiment 300B a high
volume flow of aerosol particles 405B is drawn into the annular
slot 401B formed in the cap 402B. The annular slot 401B is designed
to limit the upper or larger particulate size range as they enter
the collector. The embodiment 300B is particularly suited for
operating in dirty environments such as subway stations. A
prescreen unit 404B is positioned at the entrance to the annular
slot 401B to prevent large particles from blocking the small flow
paths of the virtual impactor.
[0065] To efficiently pass the smaller particulate, the cap 402B is
a "passive" device in that is has no moving parts and uses the fact
that particulate with a finite mass and moving in a flow stream (in
this case air) will not follow the streamlines exactly due to their
inertia. If the curvature of a streamline is sufficiently large and
the mass of the particulate is correspondingly high, the particle
deviates far enough from the streamline to impact with a surface.
The particles are drawn through the pre-screen 404B into the
annular slot 401B and directed into the transition section 409B. In
operation of the embodiment 300B, nightly clean-in-place protocols
are implemented to keep the walls of the wetted-wall-cyclone clean
using reagents such as dilute bleach and surfactant solutions.
Also, the optical bubble counter is cleaned periodically with a
pipe-cleaner, catheter, or other such tool.
[0066] The APDS 300A has the capability to measure particle sizes
in the sampling environment via a built in particle counter with
four size ranges, and can store and display the results in
real-time. The system is entirely self-contained requiring only a
110 V ac power connection. The on-board computer has high-speed
communications capability allowing networks of these sampling
systems to be remotely operated.
[0067] The APDS 300A is useful for most environmental sampling. It
is particularly useful with biological material collection, but can
be used for collecting any airborne matter. The APDS 300A can be
used to sample air quality in public buildings such as convention
centers and sports arenas, for sampling in food processing
facilities, sampling animal pens (such as poultry houses), or for
use in monitoring orchards or agricultural areas for the presence
of pollens or pesticides. Because of its relatively compact size
and weight it can be used to sample in confined spaces such as
found in aircraft or subway systems.
[0068] Referring now to FIG. 6, the virtual impactor section 403 is
shown in greater detail. In the virtual impactor section 403, the
separation efficiency is determined by the ratio of the major and
minor flows (or Bypass to Product) and the physical dimensions of
the nozzle and collection probe. The key is particulate larger than
the cut size become concentrated in the minor flow. The
concentration factor is the ratio of the total flow to the minor
flow. (If the minor flow is 25% of the total flow, then the
concentration factor is 4.) The aerosol passes through an
acceleration nozzle 601. The acceleration nozzle 601 has a diameter
D.sub.0. The aerosol is directed toward a collection probe 602. The
collection probe has a diameter D.sub.1. Between the acceleration
nozzle 601 and the collection probe 602, a major portion of the
flow 603 is diverted 90.degree. away. The minor or "product" flow
604 continues axially.
[0069] The flow forms streamlines 605. Small particles with low
inertia 606 follow the flow streamlines and are carried away
radially with the major flow 603. Large particles with greater
inertia 607 deviate from the flowlines but they continue moving
axially in their forward path down the collection probe 602 with
the minor or "product" flow 604. The separation efficiency is
determined by the ratio of the major and minor flows (or Bypass to
Product) and the physical dimensions of the nozzle D.sub.0 and
collection probe D.sub.1. The key is particulate larger than the
cut size become concentrated in the minor flow. The concentration
factor is the ratio of the total flow to the minor flow.
[0070] Referring now to FIG. 7, additional details of the sample
collection operation are shown. The particles, (1-10 microns) known
as the product flow are directed into a multi-stage, wetted-wall
cyclone collection section. In this stage of the sampling system
the product particles are trapped and concentrated into a liquid,
typically water, in a volume between 2 and 7 cc. An on-board
computer monitors and controls the flow of air through the system
using built in hot wire anemometers, as well as controlling the
liquid level in the cyclone. At a selected time the computer will
stop the flow of air and turn on a built-in peristaltic pump to
deliver the sample via an external liquid sample port.
[0071] The product flow particles enter a stainless steel funnel
section into the input of a multistage, wetted-wall cyclone
collector section 700. The system includes a cyclone collector 701,
peristaltic pump 707, an air pump 704, a vent 706, wash 705, 8
liter DD-H2O (double deionized water) reservoir 702, and 1 liter
bleach reservoir 703. The reservoirs 702 and 703 are provided as
external tanks outside of the front panel interface 708. The
multistage, wetted-wall cyclone collector section 700 directs the
particles of interest to the sample preparation system 302.
[0072] The on-board computer monitors and controls the flow of air
through the system using built in hot wire anemometers that have
been mounted in the two exhaust ports of the sampler. The computer
and control software also act to control the liquid level in the
cyclone, and monitor all status indicators of the sampling system.
At a selected time the computer will stop the flow of air and turn
on a built-in peristaltic pump to deliver the collected liquid
sample via an external sample port. The system also has the
capability to measure particle sizes in the background environment
via a built in particle counter such as particle counter Biotest
APC-1000, with four size ranges, and can store and display the
results in real-time.
[0073] The system 300 is entirely self-contained requiring only a
110 vac power connection. The on-board computer has high-speed
communications capability allowing networking of multiple sampling
systems to be remotely operated. The computer has extra RS-232 or
RS-485 serial ports that can be used to control other
instrumentation. A keyboard, mouse, printer, displays, and other
peripherals can be "plugged" in at the rear of the system, or it
can be started "headless" (headless=Without a display, mouse,
etc.)
[0074] Referring now to FIGS. 8A, 8B, and 8C, the APDS Aerosol
Collection 301 and APDS In-Line Sample Preparation 302 sub systems
are shown in greater detail. The aerosol collection system 301 is
designated "High Collection Rate Aerosol Sampling System"
(HiCRASS). The HiCRASS comprises: Low Pass "Cap" 402; Transition
Section 409; Virtual Impactor 403; Funnel Section 410; Multistage,
Wetted-wall Cyclone Collector 700; Bypass Fan 412; and Control
Computer 714.
[0075] The HiCRASS system provides a very high volume flow of
aerosol particles (e.g., up to 3313 Lpm) that are drawn into the
annular slot 401 formed in the cap 402 that is designed to limit
the upper or larger particulate size range as they enter the
collector. The annular slot 401 allows the passage of particles
smaller than 10 microns. To efficiently pass the smaller
particulate, the cap 402 is a "passive" device in that is has no
moving parts and uses the fact that particulate with a finite mass
and moving in a flowstream (in this case air) will not follow the
streamlines exactly due to their inertia. The curvature of the
streamline is sufficiently large and the mass of the particulate is
correspondingly high that the particle deviates far enough from the
streamline to impact with a surface. The accepted particles
continue around the corner and onto the dichotomous virtual
impaction section 403 that returns substantially all the aerosol
particles smaller than 1-micron back into the environment.
[0076] The virtual impactor 403 works as the aerosol passes through
an accelerating nozzle 601 and is directed toward a collection
probe 602 where a major portion of the flow 603 is diverted
90.degree. away from it. The flow forms streamlines 605. Small
particles with low inertia 606 follow the flow streamlines and are
carried away radially with the major flow 603. Large particles with
greater inertia 607 deviate from the flowlines but they continue
moving axially in their forward path down the collection probe 602
with the minor or "product" flow 604. The separation efficiency is
determined by the ratio of the major and minor flows (or Bypass to
Product) and the physical dimensions of the nozzle D.sub.0 and
collection probe D.sub.1. Particulate larger than the cut size
become concentrated in the minor flow. The concentration factor is
the ratio of the total flow to the minor flow. (If the minor flow
is 25% of the total flow, then the concentration factor is 4).
[0077] The remaining particles (1-10 microns) now known as the
product 604, flow down a stainless steel funnel section into the
input of the multistage, wetted-wall cyclone collector section 700.
In this stage of the system 301 the product particles are trapped
and concentrated into a liquid, typically water, in a volume
between 2 and 7 cc. The wetted-wall cyclone collector section 700
is a system that causes the product flow particles 604 to be
collected by a liquid. The wetted-wall cyclone collector section
700 operates by forcing the air stream tangentially into a cylinder
causing the air stream to circulate around the inside of the
cylinder. Particles in the air stream having sufficient inertia
will collide with the interior wall where they are collected by the
liquid that circulates along the interior wall.
[0078] The on-board computer 714 monitors and controls the flow of
air through the system using built-in hot wire anemometers, as well
as controlling the liquid level in the cyclone 700. At a selected
time the computer 714 will stop the flow of air and turn on a
built-in peristaltic pump to deliver the sample via an external
sample port. The on-board computer 714 monitors and controls the
flow of air through the system using built in hot wire anemometers
that have been mounted in the two exhaust ports of the sampler. The
computer and control software also act to control the liquid level
in the cyclone, and monitor all status indicators of the sampling
system. At a selected time the computer will stop the flow of air
and turn on a built-in peristaltic pump to deliver the collected
liquid sample via an external sample port.
[0079] The system also has the capability to measure particle sizes
in the sampling environment via a built in particle counter such as
particle counter Biotest APC-1000, with four size ranges, and can
store and display the results in real-time. The system is entirely
self-contained requiring only a 110 vac power connection. The
on-board computer has high-speed communications capability allowing
networks of these sampling systems to be remotely operated.
[0080] Referring now to FIG. 9, another embodiment of the
collection section of the present invention is illustrated. This
collection section system is designated generally by the reference
numeral 900. The system 900 samples the air 901 and collects sample
particles of a predetermined particle size range from the air. The
system 900 is particularly useful with the latest generation of
Biological Warfare agent detection systems. An air sampling system
is a critical component in integrated biological warfare detection
system. The system 900 also has use in medical facilities and
research and development facilities.
[0081] A low pass section 902 has an opening of a preselected size
for gathering the air 901 but excluding particles larger than the
sample particles. In one embodiment, the opening of a preselected
size is an annular slot that only allows the passage of particles
smaller than 10 microns. The low pass section 902 produces a total
air flow 903 that contains the sample particles of a predetermined
particle size range. The low pass section 902 allows a very high
volume flow of air to be drawn through the preselected size
opening. In one embodiment, the very high volume flow of air is
3313 Lpm or less.
[0082] An impactor section 904 is connected to the low pass section
902 and receives the total air flow 903. The impactor section 904
separating the total air flow 903 into a bypass air flow 905 that
does not contain the sample particles and a product air flow 906
that does contain the sample particles. An accelerating nozzle and
a collection probe in the impactor section 904 diverts the bypass
air flow 90.degree. from the product air flow thereby separating
the bypass air flow and the product air flow. In one embodiment,
the bypass air flow and the product air flow separation is
determined by the ratio of the bypass air flow and the product air
flow. In one embodiment, the bypass air flow and the product air
flow separation is determined by the physical dimensions of the
accelerating nozzle and the collection probe. In one embodiment,
the bypass air flow and the product air flow separation is
determined by the ratio of the bypass air flow and the product air
flow and the physical dimensions of the accelerating nozzle and the
collection probe.
[0083] A wetted-wall cyclone collector section 907 is connected to
the impactor section 904. The wetted-wall cyclone collector section
907 receives the product air flow 906 and traps the sample
particles in a liquid. The sample particles of a predetermined
particle size range are concentrated in the liquid. In one
embodiment, the wetted-wall cyclone collector section 907 traps and
concentrates the sample particles into a liquid in a volume between
2 and 7 cc. In one embodiment, the liquid is water.
[0084] The system 900 is useful for most environmental sampling. It
is particularly useful with biological material collection, but can
be used for collecting any airborne matter. The system 900 can be
used to sample air quality in public buildings such as convention
centers and sports arenas, for sampling in food processing
facilities, sampling animal pens (such as poultry houses), or for
use in monitoring orchards or agricultural areas for the presence
of pollens or pesticides. Because of its relatively compact size
and weight it can be used to sample in confined spaces such as
found in aircraft or subway systems.
APDS In-Line Sample Preparation--302
[0085] As best illustrated in FIG. 3, the in-line sample
preparation module 302 moves the sample from the aerosol collection
module 301 to appropriate modules within the APDS 300 and provides
sample preparation. In one mode, the sample preparation module 302
prepares the sample (mixing, filtering, incubation, etc.) and
delivers the sample reaction volume to the liquid-array based
multiplex immunoassay detection system 303. In another mode, the
sample preparation module 302 prepares the sample (mixing,
filtering, incubation, etc.) and delivers the sample reaction
volume to the in-line nucleic acid detection system 304.
[0086] The prior art sample preparation instrumentation uses
robotic manipulation of micropipettes coupled to disposable filter
wells. Robotics are inherently complex and difficult to scale. The
sample preparation module 302 uses Zone fluidics. Zone fluidics is
the precisely controlled physical, chemical, and fluid-dynamic
manipulation of zones of miscible and immiscible fluids in narrow
bore conduits to accomplish sample conditioning and chemical
analysis. A zone is a volume region within a flow conduit
containing at least one unique characteristic. A unit operation in
zone fluidics comprises of a set of fluid handling steps intended
to contribute to the transformation of the sample into a detectable
species or prepare it for manipulation in subsequent unit
operations. Examples of unit operations include sample filtering,
dilution, enrichment, medium exchange, headspace sampling, solvent
extraction, matrix elimination, de-bubbling, amplifying,
hybridizing, and reacting. In current analytical practice many of
these steps are handled manually or in isolated pieces of
equipment. Integration is scant at best, and there is a high degree
of analyst involvement. In zone fluidics, sample and reagent zones
are subjected to these unit operations in a sequential manner being
transported from one unit operation to the next under fluidic
control.
[0087] Samples in zone fluidics are not limited to liquids. Rather,
gases, and suspensions containing solids or cells are also
included. Where solid samples are used, particles are limited to a
size that ensures no blockages. In most cases, reagents are
prepared and then coupled to the zone fluidics manifold. The
metering capability of the pump and mixing unit operations allow
for reagents and standards to be prepared in situ. Reagents can
therefore be presented to the zone fluidics manifold in an
appropriately designed cartridge as ready-made, reagent
concentrates, lyophilized, or crystalline form. Standards can be
plumbed to the multi-position valve as discrete reservoirs
providing the required range of concentrations. As for reagents
though, standards can also be prepared in situ or diluted to cover
a larger dynamic range.
[0088] The sample preparation module 302 uses a powerful, highly
flexible technique called sequential injection analysis (SIA).
Automation is achieved through the manipulation of small solution
zones under conditions of controlled dispersion in narrow bore
tubing. Zone fluidics makes use of a multi-position selection valve
and a high precision, bi-directional pump to construct a stack of
well-defined sample and reagent zones in a holding coil of narrow
bore tubing. By appropriate manipulation of this zone stack, a wide
range of sample handling unit operations can be accommodated. The
pump is used to move the sample from one device to the next
achieving the required sample manipulation in the process. Once a
detectable species has been formed, the zone stack is transported
to the immunoassay detector 303 and to the nucleic acid detector
304.
[0089] Various embodiments of system for sample preparation and
detection have been described in connection with FIGS. 10-13.
Applicants will now describe additional embodiments of the
immunoassay system of the present invention. These embodiments of
the immunoassay system can use either "wash assay" system or
"no-wash assay" system. The "wash assay" system and the "no-wash
assay" system use optically-encoded beads. The beads are kept in a
small (.about.15 mL) stirred tank. Bead loss from agglomeration is
reduced by using dispersing agents such as ethanol in the bead
slurry. This reduces reagent cost.
[0090] The wash assay system embodiment uses a number of reagents
in addition to the liquid sample. The reagents include detector
antibody, and fluorescent reporter (streptavidin-phycoerythrin). In
the wash assay system, a bed of beads is deposited on a filter then
exposed to the sample, washing buffer, detector antibody, washing
buffer, fluorescent reporter, washing buffer, and then the beads
are released to the detector. For the wash assay system, the bead
filter performance in releasing beads is improved by implementing
frequent clean-in-place protocols using reagents such as bleach for
cleaning and morpholine propane sulfonic acid (MOPS) citrate buffer
for reconditioning the filter. This extends the time between
servicing the instrument and thus reduces operating cost.
[0091] In the no-wash assay embodiment, the reagents are
sequentially mixed but the embodiment does not include the filter
or the wash steps. It has been shown that the wash assays are more
sensitive and selective, providing better signal-to-noise ratios in
response to bioagents.
[0092] Referring now to FIG. 14, another embodiment of a system
constructed in accordance with the present invention is
illustrated. The system is designated generally by the reference
numeral 1400. The system 1400 comprises the following: Sample
Collection 1401, Sample Preparation 1402, Multiplex Amplification
PCR 1403, and Multiplex, Liquid Array Based Detection of PCR
Amplicons 1404.
[0093] The first stage of the system 1400 is "sample collection
1401" that provides collection of particles that could contain
targeted bioagents. The sample collection 1401 gathers air, water,
soil, or other substance being monitored. The sample collection
1401 separates selected potential bioagent particles from the air,
water, soil, or other substance.
[0094] The "sample preparation 1402" moves the sample from the
sample collection to appropriate modules within the system 1400 and
provides sample preparation. In one mode, the sample preparation
1402 prepares the sample (Lysis, Concentration, Purification,
Mixing, etc.) and delivers the sample to "Multiplex Amplification
PCR 1403." One mode provides "Multiplex, Liquid Array Based
Detection of PCR Amplicons 1404." An example of a flow cytometric
detection method for DNA samples is shown in U.S. patent
application 2002/0155482 by Shanavaz Nasarabadi, Richard G.
Langlois, and Kodumudi Venkateswaran published Oct. 24, 2002. The
disclosure of U.S. patent application 2002/0155482 is incorporated
herein by reference.
[0095] Referring now to FIG. 15, one specific embodiment of the
in-line nucleic acid amplification and detection system 1500 is
illustrated. The system 1500 is capable of performing, singly or in
combination, nucleic acid amplification, and nucleic acid detection
functions. The nucleic acid assay system 1500 includes a number of
components including system for injecting/aspirating a sample,
1501, system for adding PCR reagent 1502, system for mixing sample
and reagent 1503, system for transport to PCR reactor 1504, system
for performing PCR amplification 1505, system for transport of
amplified sample from PCR reactor 1506, system for detection of PCR
amplicons 1507, and system for decontamination and conditioning of
all exposed conduits 1508.
[0096] The system 1501 for injecting and or aspirating a sample
provides injection and/or aspiration of the sample. In one
embodiment the injecting/aspirating system 1501 consists of a zone
fluidics system. In another embodiment the injecting/aspirating
system 1501 consists of an FIA system. The system 1501 for
injecting and or aspirating a sample can be, for example, a
injecting/aspirating device available under the trademark
milliGAT.TM. pump, Global FIA, Inc., Fox Island, Wash.
[0097] The system 1502 for adding PCR reagent to the sample is
operatively connected to the system 1501 for injecting and or
aspirating a sample. The system 1502 for adding PCR reagent to the
sample can be, for example, a unit for adding PCR reagent to the
sample such as an injection or multi position selection valve,
available from VICI, Houston, Tex.
[0098] The system 1503 for mixing the sample and the reagent is
operatively connected to the system 1502 for adding PCR reagent to
the sample. The mixing system 1503 mixes the sample with a PCR
reagent. In one embodiment the PCR reagent includes primers. In
another embodiment the PCR reagent includes oligos. The system 1503
for mixing the sample and the reagent can be, for example, a super
serpentine reactor, available from Global FIA, Inc., Fox Island,
Wash.
[0099] The system 1504 for transporting the sample and the reagent
to a PCR reactor is operatively connected to the system 1503 for
mixing the sample and the reagent. The system 1504 for transporting
the sample and the reagent to a PCR reactor consists of a fluidics
system. The system 1504 for transporting the sample and the reagent
to a PCR reactor can be, for example, FEP tubing available from
Cole-Parmer, Vernon Hills, Ill.
[0100] The system 1505 for performing PCR amplification is
operatively connected to the system 1504 for transporting the
sample and the reagent to a PCR reactor. This results in an
amplified sample. In one embodiment the PCR amplification system
1505 includes an embedded thermocouple calibration conduit. PCR
amplification devices are described in publications such as U.S.
Pat. No. 5,589,136 for silicon-based sleeve devices for chemical
reactions, assigned to the Regents of the University of California,
inventors: M. Allen Northrup, Raymond P. Mariella, Jr., Anthony V.
Carrano, and Joseph W. Balch, patented Dec. 31, 1996 and many are
commercially available such as ABI PRISM.RTM. 7700 Sequence
Detection System by Applied Biosystems; iCycler iQ Real-Time PCR
Detection System by Bio-Rad; and Smart Cycler.RTM. System by
Cepheid.
[0101] The system 1506 for transporting the amplified sample from
the PCR reactor is operatively connected to the system 1205 for
performing PCR amplification. The system 1506 for transporting the
amplified sample from the PCR reactor can be, for example, FEP
tubing available from Cole-Parmer, Vernon Hills, Ill.
[0102] The system 1507 for detection of PCR amplicons is
operatively connected to the system 1506 for transporting the
amplified sample from the PCR reactor. The system 1507 for
detection of PCR amplicons can be, for example, a detection system
described in publications and products produced by Cepheid and
Baltimore-based Environmental Technologies Group, Inc. (ETG), a
part of London-based Smiths Aerospace.
[0103] Conduits are included within the system 1501 for injecting
and or aspirating a sample, system 1502 for adding PCR reagent to
the sample, system 1503 for mixing the sample and the reagent,
system 1504 for transporting the sample and the reagent to a PCR
reactor, system 1505 for performing PCR amplification, system 1506
for transporting the amplified sample from the PCR reactor, and
system 1507 for detection of PCR amplicons. A system 1508 for
decontamination and conditioning the conduits is directly connected
to the system 1507 for detection of PCR amplicons. The system 1508
for decontamination and conditioning the conduits is operatively
connected to the system 1501 for injecting and or aspirating a
sample, system 1502 for adding PCR reagent to the sample, system
1503 for mixing the sample and the reagent, system 1504 for
transporting the sample and the reagent to a PCR reactor, system
1505 for performing PCR amplification, system 1506 for transporting
the amplified sample from the PCR reactor, and system 1507 for
detection of PCR amplicons. The decontamination and conditioning of
all exposed conduits can be, for example, be performed by using a
decontaminant, such as bleach, which is pumped through the exposed
conduits and then washed from the system with a suitable wash
solution.
[0104] Referring now to FIG. 16, a block diagram illustrates
another embodiment of an autonomous pathogen detection system
constructed in accordance with the present invention. This
embodiment of an autonomous pathogen detection system is designated
generally by the reference numeral 1600. The autonomous pathogen
detection system 1600 provides water sample collection 1601, sample
preparation 1602, and detection 1603 and 1604.
[0105] In operation, a water sample collection unit 1601
continuously samples a water source. Water sampling systems are
known in the art. For example, a water sampling system is shown in
U.S. Pat. No. 6,306,350 issued Oct. 23, 2001 titled "Water Sampling
Method and Apparatus With Analyte Integration." The disclosure of
U.S. Pat. No. 6,306,350 is incorporated herein by reference.
[0106] The in-line sample preparation unit 1602 concentrates the
sample in a swirling buffer solution. Particles of a given size
distribution are selected by varying the flow rate across a
separator unit. The in-line sample preparation system 1602 provides
all sample preparation steps (i.e., mix, wash, incubation, etc.),
and performing multiplex detection using a Luminex flow
cytometer.
[0107] In the "detection" sub-system 1603, a collected sample is
mixed with optically encoded microbeads. Each color of microbead
contains a capture assay that is specific for a given bioagent.
Fluorescent labels are added to identify the presence of each agent
on the bound bead. Each optically encoded and fluorescently labeled
microbead is individually read in a flow cytometer, and fluorescent
intensities are then correlated with bioagent concentrations.
[0108] In the "confirmation" sub-system 1604, PCR (nucleic acid)
amplification and detection confirms the presence of the bioagent.
An archived sample is mixed with the Taqman reagent, and then
introduced by a SIA system into a flow through polymerase chain
reaction (PCR) system. Specific nucleic acid signatures associated
with the targeted bioagent are amplified and detected using
fluorescence generated from nucleic acid replication from the
Taqman probes. In the "Integrated Remote Control and Feedback"
sub-system 1605, a central computer uses a simple serial based
LabVIEW control system to control all instrument functions. A
software system provides data acquisition, real-time data analysis,
and result reporting via a graphical user interface.
[0109] The first stage of the system 1600 is "water sample
collection 1601" that provides collection of particles from a
source of water that could contain bioagents. The water sample
collection system 1601 and in-line sample preparation 1602 provide
preconcentration and delivery of the particles of interest to a
wetted wall cyclone collector. The separator system captures
particles of interest.
[0110] In the wetted wall cyclone collector, the particles are
collected in a fluid, making downstream processing much easier. An
on board computer controls water flow rates and the size range of
particles collected. A particle counter provides real-time feedback
on the size and quantity of particles collected.
[0111] Particles are drawn into the system that is designed to only
allow the collection of particles of a pre-set size. The pre-set
size can be selected as desired. The system is designed to only
collect particles that are desired. The accepted particles continue
on into a separator section that returns all the particles that are
not of the desired size back into the environment. The remaining
particles are known as the product flow. The product flow continues
into the detection sections.
[0112] The system 1600 has the capability to measure particle sizes
in the sampling environment via a built in particle counter with
four size ranges, and can store and display the results in
real-time. The system is entirely self-contained requiring only a
power connection. The on-board computer has high-speed
communications capability allowing networks of these sampling
systems to be remotely operated.
[0113] The 1600 is useful for many application of water sampling.
The system 1600 can be used to sample water quality in public
buildings, for sampling in food processing facilities, for use in
monitoring agricultural areas for the presence of pollens or
pesticides and other water sampling uses.
[0114] Referring now to FIG. 17, a block diagram illustrates
another embodiment of an autonomous pathogen detection system
constructed in accordance with the present invention. This
embodiment of an autonomous pathogen detection system is designated
generally by the reference numeral 1700. The autonomous pathogen
detection system 1700 provides soil sample collection 1701, sample
preparation 1702, and detection 1703 and 1704.
[0115] In operation, a soil sample collection unit 1701
continuously samples a soil source. Soil sampling systems are known
in the art. For example, a soil sampling system is shown in U.S.
Pat. No. 6,363,803 titled "Vehicle Mounted Soil Sampler," invented
by Elmer Hubers, patented Apr. 2, 2002. The disclosure of U.S. Pat.
No. 6,363,803 is incorporated herein by reference.
[0116] The in-line sample preparation unit 1702 concentrates the
sample in a swirling buffer solution. Particles of a given size
distribution are selected by varying the flow rate across a
separator unit. The in-line sample preparation system 1702 provides
all sample preparation steps (i.e., mix, wash, incubation, etc.),
and performing multiplex detection using a Luminex flow
cytometer.
[0117] In the "detection" sub-system 1703, a collected sample is
mixed with optically encoded microbeads. Each color of microbead
contains a capture assay that is specific for a given bioagent.
Fluorescent labels are added to identify the presence of each agent
on the bound bead. Each optically encoded and fluorescently labeled
microbead is individually read in a flow cytometer, and fluorescent
intensities are then correlated with bioagent concentrations.
[0118] In the "confirmation" sub-system 1704, PCR (nucleic acid)
amplification and detection confirms the presence of the bioagent.
An archived sample is mixed with the TaqMan reagent, and then
introduced by a SIA system into a flow through polymerase chain
reaction (PCR) system. Specific nucleic acid signatures associated
with the targeted bioagent are amplified and detected using
fluorescence generated from nucleic acid replication from the
TaqMan probes. In the "Integrated Remote Control and Feedback"
sub-system 1705, a central computer uses a simple serial based
LabVIEW control system to control all instrument functions. A
software system provides data acquisition, real-time data analysis,
and result reporting via a graphical user interface.
[0119] The first stage of the system 1700 is "soil sample
collection 1701" that provides collection of particles from a
source of soil that could contain bioagents. The soil sample
collection system 1701 and in-line sample preparation 1702 provide
preconcentration and delivery of the particles of interest to a
wetted wall cyclone collector. The separator system captures
particles of interest.
[0120] In the wetted wall cyclone collector, the particles are
collected in a fluid, making downstream processing much easier. An
on board computer controls soil flow rates and the size range of
particles collected. A particle counter provides real-time feedback
on the size and quantity of particles collected.
[0121] Particles are drawn into the system that is designed to only
allow the collection of particles of a pre-set size. The pre-set
size can be selected as desired. The system is designed to only
collect particles that are desired. The accepted particles continue
on into a separator section that returns all the particles that are
not of the desired size back into the environment. The remaining
particles are known as the product flow. The product flow continues
into the detection sections.
[0122] The system 1700 has the capability to measure particle sizes
in the sampling environment via a built in particle counter with
four size ranges, and can store and display the results in
real-time. The system is entirely self-contained requiring only a
power connection. The on-board computer has high-speed
communications capability allowing networks of these sampling
systems to be remotely operated. The 1700 is useful for many
application of soil sampling. The system 1700 can be used to sample
soil quality in monitoring agricultural areas for the presence of
pollens or pesticides and other soil sampling uses.
[0123] Referring now to FIG. 18, another embodiment of a system for
sample preparation and detection is illustrated. The system is
generally designated by the reference numeral 1800. The system 1800
is capable of performing, singly or in combination, liquid-array
based multiplex immunoassay detection and/or in-line nucleic acid
amplification and detection. In operation of the system 1800, the
aerosol collector system 1801 samples the air, particles of a given
size distribution are trapped in a liquid, and a sample of interest
is prepared. The next step is detection of any pathogens in the
sample particles. This is accomplished by a liquid-array based
multiplex immunoassay detection system 1802 and an in-line nucleic
acid amplification and detection system 1803.
[0124] The liquid-array based immunoassay detection system 1802
measures multiple pathogen targets in the sample. The immunoassay
system 1802 can use either "wash assay" system or "no-wash assay"
system. The "wash assay" system and the "no-wash assay" system use
optically-encoded beads. The beads are kept in a small (.about.15
mL) stirred tank. Bead loss from agglomeration is reduced by using
dispersing agents such as ethanol in the bead slurry. This reduces
reagent cost.
[0125] The wash assay system embodiment uses a number of reagents
in addition to the liquid sample. The reagents include detector
antibody, and fluorescent reporter (streptavidin-phycoerythrin). In
the wash assay system, a bed of beads is deposited on a filter then
exposed to the sample, washing buffer, detector antibody, washing
buffer, fluorescent reporter, washing buffer, and then the beads
are released to the detector. For the wash assay system, the bead
filter performance in releasing beads is improved by implementing
frequent clean-in-place protocols using reagents such as bleach for
cleaning and morpholine propane sulfonic acid (MOPS) citrate buffer
for reconditioning the filter. This extends the time between
servicing the instrument and thus reduces operating cost.
[0126] In the no-wash assay embodiment, the reagents are
sequentially mixed but the embodiment does not include the filter
or the wash steps. It has been shown that the wash assays are more
sensitive and selective, providing better signal-to-noise ratios in
response to bioagents.
[0127] The PCR (nucleic acid) amplification and detection system
1803 confirms the presence of any bioagent. An archived sample is
mixed with the TaqMan reagent, and then introduced by a system into
a flow through polymerase chain reaction (PCR) system. Specific
nucleic acid signatures associated with the targeted bioagent are
amplified and detected using fluorescence generated from nucleic
acid replication from the TaqMan probes.
[0128] The PCR assays for confirming immunoassay positives are
implemented using real-time PCR and the TaqMan process. Both FAM
and TAMRA dyes are used as TaqMan reporters and TAMRA and BHQ
(Black Hole Quencher) dyes are used as TaqMan quenchers. In one
embodiment the FAM-BHQ is used for the bioagent probe and TAMRA-BHQ
for an internal control probe. This allows an internal control to
be used on a two-color detector. Internal controls are critical in
PCR due to the delicacy of the reaction; otherwise, negative
results are not definitive. In one embodiment, two color excitation
is used to give strong signals for duplexed TaqMan PCR (agent plus
internal control).
[0129] In some embodiments, the PCR reagents for TaqMan PCR are
stored on the system together as MasterMix (enzyme, buffer, dNTPs)
mixed with primers and probes. In another embodiment it was found
that storing the MasterMix in one reservoir and primers plus probes
in another reservoir made the reagents more stable, potentially
eliminating the need for cooling in the instrument. In one
embodiment, extraction of DNA onto microfabricated silica pillars
is used as a means of purifying DNA from PCR inhibitors.
[0130] Sample preparation moves the sample from the sample
collection 1801 to appropriate modules within the system. The
nucleic acid assay system 1803 includes a number of components
including system for injecting/aspirating a sample, system for
adding PCR reagent, system for mixing sample and reagent, system
for transport to PCR reactor, system for performing PCR
amplification, system for transport of amplified sample from PCR
reactor for detection of PCR amplicons. A central computer 1805
uses a simple serial based LabVIEW control system to control all
instrument functions. A software system provides data acquisition,
real-time data analysis, and result reporting via a graphical user
interface.
[0131] In operation of the pathogen detection system, the in-line
nucleic acid amplification and detection system provides nucleic
acid assay methods. The methods include a number of steps. One step
consists of automatically injecting and or aspirating a sample.
Another step consists of automatically adding PCR reagent to the
sample. Another step consists of automatically mixing the sample
and the reagent. Another step consists of automatically
transporting the sample and the reagent to a PCR reactor. The PCR
reactor consists of a fluidics system. Another step consists of
automatically performing PCR amplification resulting in an
amplified sample. Another step consists of automatically
transporting the amplified sample from the PCR reactor. Another
step consists of automatically detecting PCR amplicons. The method
is performed in a nucleic acid assay system and the nucleic acid
assay system is decontaminated and conditioned before a new sample
is analyzed.
[0132] The system includes both real-time and post-PCR detection.
The system is ideal for monitoring type systems, such as those
currently being developed to detect terrorist releases of
aerosolized bioagents. On-site detection systems for infectious
diseases under development will need to incorporate sample
preparation and analysis functions. The system allows relatively
unskilled personnel, such as early responders, to perform real-time
field or point-of-care nucleic acid assays. In various other
embodiments of the autonomous pathogen detection system, the
confirmation of bioagent(s) in the sample is provided by a
multiplex immunoassay detector, a multiplex PCR detector, and a
real-time PCR detector.
[0133] The present invention provides an Autonomous Pathogen
Detection System (APDS) for monitoring the environment to protect
the public from the release of hazardous biological agents. The
Autonomous Pathogen Detection System is a countermeasure to
bioterrorism, one of the most serious threats to the safety of
United States citizens, citizens of other countries, and the
military.
[0134] The APDS program was initiated to fill the requirement of a
distributed environmental monitoring system for civilian
applications. Multiplexed assays are used to reduce reagent costs,
making long term monitoring operations possible (e.g., U.S. Postal
Service mail screening). A unique, orthogonal detection approach
that combines antibody-based and nucleic acid-based assays reduces
false positives to a very low level. Antibody assays allow the
detector to respond to all types of bioagents, including those
without nucleic acids such as protein toxins. Nucleic acid assays
allow much more sensitive detection, reducing the number of sensors
needed to protect a given area. The fully autonomous aerosol
collection and sample preparation capabilities limit maintenance
requirements and makes integration into a central security or
monitoring network possible.
[0135] There are other environmental or clinical pathogen detection
system needs. Mobile units could be transported to suspected "sick
buildings" to test for mold or fungal spores that might be causing
tenant illnesses. Units with reagents for animal diseases could be
placed in livestock transport centers or feedlots to rapidly detect
airborne pathogens and protect against disease outbreaks. Monitors
in hospitals could be used to test for airborne spread of
contagious materials among patients. The system could be used at
high profile events such as the Olympics for short-term, intensive
monitoring or more permanent installation in major public buildings
or transportation nodes. All of the individual units can be
networked to a single command center so that a small group of
technical experts can maintain and respond to alarms at any of the
units. The system is capable of meeting all of these needs.
[0136] The primary needs describe above are directed to protection
of civilians from terrorist attacks. The system also has uses in
protecting military personnel from biological warfare attacks. The
military continues to evaluate options to their current biowarfare
detection systems and the system meets many of the needs of the
military.
[0137] While the invention may be susceptible to various
modifications and alternative forms, specific embodiments have been
shown by way of example in the drawings and have been described in
detail herein. However, it should be understood that the invention
is not intended to be limited to the particular forms disclosed.
Rather, the invention is to cover all modifications, equivalents,
and alternatives falling within the spirit and scope of the
invention as defined by the following appended claims.
* * * * *